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(ADVANCES IN BOTANICAL RESEARCH, Vol 7;

H. Woolhouse, editor; Academic Press, London1979)

Aspects of the Physiology of Orchids


JOSEPH ARDITTI

Department of Developmental and Cell Biology,


University of California, Irvine, California 92717, U.S.A.

I. Introduction................................................................................................. 422

II. Seeds.............................................................................................................. 423


A. History................................................................................................ 423
B. External Morphology ....................................................................... 438
C. Structure and Ultrastructure ........................................................... 438
D. Longevity........................................................................................... 441
E. Asymbiotic Germination.................................................................. 441
F. Symbiotic Germination..................................................................... 489
G. Summation ......................................................................................... 506

III. Phytoalexins................................................................................................ 508


A. History ............................................................................................... 508
B. Chemistry Production and Distribution........................................ 510
C. Action Spectrum and Activity ....................................................... 512
D. Biological Role .................................................................................. 517

IV. Carbon Fixation........................................................................................... 519


A. History ............................................................................................... 519
B. Stomatal Rhythms ............................................................................ 521
C. Crassulacean Acid Metabolism..................................................... 521
D. C3 Photosynthesis ............................................................................ 529
E. C4 Photosynthesis ............................................................................ 529
F. Carbon Fixation by Different Plant Organs .................................. 530
G. Photorespiration............................................................................... 532
H. Summation.......................................................................................... 532
422 J. ARDITTI

V. Flowers ........................................................................................................ 533


A. History................................................................................................ 534
B. Introduction....................................................................................... 534
C. Pollination.......................................................................................... 552
D. Post-pollination Phenomena........................................................... 566
E. Induction of Phenomena ................................................................. 617
VI. Tissue Culture ............................................................................................. 634

VII. Epilogue........................................................................................................ 637


Acknowledgements.................................................................................... 637
References . . . . ............................................... 638

I. INTRODUCTION

Orchidaceae is by most estimates the largest of all flowering plant families


(although some claim this "honour" for the Compositae) with 600-800
genera and 25,000-35,000 species (Garay, 1960; Schultes and Pease, 1963).
Orchids vary in size and weight from a few millimetres and grams [the
Australian Bulbophyllum minutissimum is 1-1.5 mm across (Nicholls, 1969)
and probably weighs no more than a gram or two] to several metres and tons
[Grammatophyllum speciosum (Sumatra to the Philippines) stems can reach
three or more metres (Grant, 1895; Holttum, 1957) and mature plants can be
huge, weighing several tons]. They can be found in dense tropical jungles;
open tropical grasslands; hot and dry deserts; cold and damp areas; on trees
which hang over the water, or on rocks subject to constant sea spray; and all
other terrestrial habitats where flowering plants can grow. Orchids can be
epiphytic, lithophytic, or terrestrial and even subterranean [like Rhizanthella
gardneri, a monotypic genus from Western Australia (Nicholls, 1969)], but
none are aquatic.
The pollination mechanisms of orchids range from cleistogamy to
pseudo-copulation. Their flowers may be bizzare, insignificant or beautiful
and emit revolting stenches, have no discernible smell, or be the source of
very pleasing fragrances. After pollination, orchid flowers undergo changes
which are no less elaborate and fascinating as the "contrivances" (to quote
Darwin, 1862, 1904) which ensure pollination. Fertilization does not
immediately follow pollination in orchids and may occur days, weeks, or even
months after the flowers have been pollinated. The fruits develop slowly and
may take a year or more to ripen; they can contain from around 1000 up to
4,000,000 seeds (for reviews see Arditti, 1967a; Darwin, 1904).
Orchid seeds are tiny, most of them being 0.25-1.2 mm long, 0.09-0.75 mm
wide and weighing 0.3-14 µg (Harvais, 1974; for reviews see Arditti, 1967;
ASPECTS OF ORCHID PHYSIOLOGY 423

Burgeff, 1936). They have complex germination requirements which in nature


are provided by mycorrhizal fungi. Once infected by the appropriate fungus,
orchid seeds can germinate but may also be parasitized and destroyed. It is
not surprising, therefore, that phytoalexin production evolved in orchids
(for reviews see Arditti, 1966b, 1975; Arditti et al., 1975; Fisch et al, 1972,
1973a).
The great diversity of orchids and their different "lifestyles" have led to or
been made possible by structural and physiological adaptations. Many of
these are not very well known, or are poorly understood, because orchids
have generally been neglected as subjects for scientific investigation (Dressier
and Williams, 1970). Even when information is available it may often be
scattered and/or published in journals of interest to those few scientists who
work with orchids. Consequently, few general reviews have been written on
orchid physiology and the result is a vicious circle: since little is known or
has been put into perspective, not much is being done. The aim of this review is
to provide at least a partial remedy.
Like most reviews this one is subject to the limitations, preferences,
idiosyncracies and style of the reviewer. The topics I have covered are one
reflection of these. Another is the citations: to provide the maximum number
of relevant citations in the minimum amount of space, I will: (i) cite previous
reviews whenever they exist, even if this may lead to repetitions; (ii) hold
citations of literature mentioned in previous reviews to a minimum; (iii) list in
greater detail papers published since the latest reviews; and (iv) exercise the
usual prerogatives of a reviewer in selecting which papers to cite (and
consequently omit some recent articles or cite older ones). A third is the
nomenclature in that I have chosen in most cases to use the taxonomic
designations and spellings given in the original literature. I have not at-
tempted to select "correct" names or "proper" spelling since orchid taxonomy
is unusual, fluid and often subject to controversies.

II. SEEDS

Orchid seeds are unique in several respects. They are exceedingly small,
but produced in large numbers. The embryos have no endosperm (Savina,
1974), no cotyledons, and no root initials. To germinate in nature they require
fungal infection. It is not surprising, therefore, that orchid seeds, their nature
and germination requirements were slow to be discovered and understood.
A. HISTORY
For many years European botanists apparently failed to note and/or
appreciate the dust-like orchid seeds and realize that they were capable of
germination (Arditti, 1967a). Fanciful ideas on orchid reproduction resulted
from the presence of caproic acid in Satyrium flowers (Anonymous, 1864;
424 J. ARDITTI

Chautard, 1864) which ". . . emit a strong and unpleasant odor of goats."
(Summerhayes, 1968). Anastasius Kircher (1601-1680) suggested that these
orchids originated from ". . . escaping spermatic fluid (of animals, i.e.,
goats) that fell on the soil (and) underwent fermentation together with
mois-ture of the earth . .." (Ames, 1942; for reviews see Arditti, 1972;
Shechter and Arditti, 1973). Another idea advanced by Kircher was that
orchids can originate from ". . . rotting corpses of animals which still
contain some seminal virtue . . ." (de Wit, 1977). Tragus (Bock), another
European botanist (c. 1600), suggested that orchids originate from "..... the
'seed' of thrushes and blackbirds, who copulate in spring meadows and
pastures" (de Wit, 1977).
A later (1855-1890), more plausible, even if erroneous, belief was that
orchid seeds were not viable and incapable of germination (His, 1807).
Orchid reproduction was assumed to be through bud or gemma-like
struc -tures which underwent a series of metamorphoses leading to the
formation of mature plants (for a review see Arditti, 1967a).
Rumphius (c. 1627-1702), the blind seer of Ambon (de Wit, 1959), may
have been the first botanist to recognize orchid seeds: "The ripe [fruit] .. .
opens up readily . . . and then the yellow flour is largely shed and is blown
away on the wind; but whether this is endowed with seed-virtue, and settles to
grow on other trees, is still unknown," (de Wit, 1977). The answer became
known 150 years later when a British botanist first described germinating seeds
of Bletilla, Orchis morio and Limodorum verecundum (Salisbury, 1804). This
was followed by the description of germinating seeds and seedlings of many
other orchids (for reviews see Arditti, 1967a; Beer, 1863; Burgeff, 1909, 1911,
1932, 1936; Pfitzer, 1882; Ramsbottom, 1922; Veitch, 1887-1894). However,
even following these descriptions the germination requirements of orchid
seeds remained unknown until 1899. In that year a French botanist, Noel
Bernard, published an account of his observation that seeds of Neottia
nidus-avis require fungal infection for germination (Bernard, 1899, 1909; for
reviews see Arditti, 1967a; Burgeff, 1909, 1911, 1932, 1936; Warcup, 1975).
The genius of Bernard lies not in observing the fungus (that had been done
before without appreciation of its importance), but in recognizing its role as
being a mutualistic symbiont rather than a parasite. He succeeded in demon-
strating the role and importance of mycorrhiza before dying at an early age
in 1911. His discovery was put to practical use in a method for commercial
germination of orchid seeds. Lest the world forget, his mentor, J. Costantin,
and friend, J. Magrou (1922) described it in a paper with the pompous title
"Applications industrielles d'une grande découverte francaise". For years after
that J. Costantin inveighed against and maligned everyone who in his
(narrow and chauvinistic) mind threatened the ".. . grande découverte . . ."
even if that man happened to be Prof. Lewis Knudson (for reviews see
Arditti, 1972, b, d; Shechter and Arditti, 1972).
ASPECTS OF ORCHID PHYSIOLOGY 425

Hans Burgeff, working in Würzburg, made major contributions to the


understanding of orchid mycorrhiza, starting in 1909 (Burgeff, 1909, 1911,
1932, 1936, 1959). However, he made two errors of commission and one of
omission. The former are his assertions that: (i) all mycorrhizal fungi of
orchids belong to a separate group he called Orcheomyces; (ii) there is high
specificity between orchids and fungi. His omission was the failure to discover
asymbiotic seed germination. Consequently he was not very gracious when
Lewis Knudson at Cornell University discovered asymbiotic seed germination
and, together with Prof. John T. Curtis (working independently at Wisconsin
University), showed there was no specificity (personal communications from
several students, friends and collaborators of Knudson during that era).
Knudson was interested in the effects of carbohydrates on green plants
(for reviews see Arditti, 1967a, 1972a, b, d; Shechter and Arditti, 1972) and
this probably led him to study orchid seeds. He analysed salep (a preparation
from Ophrys tubers used by Bernard to germinate orchid seeds) and using
Bernard's report that the fungus could invert sugar concluded that the fungus
stimulated germination by breaking down starch and other large molecules.
Next, he added sugar to Pfeffer's solution and a modification of it and
germinated Cattleya, Laelia and Epidendrum on both of them. Since he was
spending 1919-1921 in Spain, his first publication on the subject was in
Spanish in a relatively obscure publication (Knudson, 1921); his next paper
was in English and a major journal (Knudson, 1922).
Costantin perceived Knudson's findings as being a threat to Bernard's
discovery ("Tout cela semblerait indiquer que toute la théorie de la symbiose
imaginée per Noel Bernard est un pur roman") and attacked, claiming that
Knudson's seedlings were not normal since they contained starch. He also
asserted that without mycorrhiza they will not flower and wrote grandly
about the teachings of nature. Knudson replied by pointing to the weakness
in Bernard's work and presenting his data which disproved Costantin's
claims. With this the debate ended.
J. Ramsbottom, the British mycologist, was also sceptical "... if these
methods should prove capable of general application . ..", unimpressed "...
no really new facts have been added to our knowledge . . .", diplomatic ". . .
such work . . . is extremely valuable in that it approaches the subject from a
physiological standpoint. . .", and erudite as well as humorous ". . . an orchid
seedling without its fungus is like Hamlet without the Prince of Denmark."
(Ramsbottom, 1922).
In subsequent years Knudson improved his initial medium. Others de-
veloped additional solutions for use with species which do not germinate well
on Knudson's media. In addition the discovery spurred a certain amount of
basic research.
Fig. 1. Orchid seeds, x 53.5 except Nos 1-8. (Plate II from Beer, 1863; seeds are listed in
alphabetical order in joint caption for Figs 1, 2, 3.)
Fig. 2. Orchid seeds, x 52. (Plate III from Beer, 1863; seeds are listed in alphabetical order
in joint caption for Figs 1, 2, 3.)
Fig. 3. Orchid seeds, x 52.5. (Plate IV from Beer, 1863; seeds are listed in alphabetical
order in joint caption for Figs 1, 2, 3.)
ASPECTS OF ORCHID PHYSIOLOGY 429
Orchid Seeds in Figs. 1, 2, 3 (Beer, 1863 unless indicated otherwise)
Fig. Seed No.
Acanthophippium bicolor, Lindl. . . . . . . 3 29
Acroclaene punctata, Bl. . . . . . . . 3 52
Acropera citrina, Hort. . . . . . . . 2 53
Acropera luteola, Hort. . . . . . . . 2 30
Acropera loddigesii, Hort. . . . . . . . 2 24
Acropera maculata, Hort. . . . . . . . 2 31
Aerides odoratum, Lour. . . . . . . . 2 15
Aerides sp. Mont. Kashia . . . . . . . 3 60
Agrostaphyllum sp. . . . . . . . . 3 32
Anaectochylus setaceus, Blume . . . . . . 3 18
Angraecum bilobum, Lindl. . . . . . . . 1 15
Apaturia senilis, Lindl. . . . . . . . 3 55
Barkeria melanocaulon, A. Rich. Gal. . . . . . 1 19
Bletia sheperdii, Hook . . . . . . . 2 6
Brassavola cordata, Lindl. . . . . . . . 3 54
Brassia cowanii, Hort. (caudata R. Br.) . . . . 2 25
Calanthe veratrifolia, Rr. Br. (Clifford and Smith, 1969). 2 2
Cattleya amethystina, Morr. . . . . . . 2 18
Cattleya bicolor, Lindl. . . . . . . . 2 26
Cattleya crispa v. purpurea, Lindl. . . . . . 3 17
Cat tleya forbesii, Lindl. . . . . . . . 3 40
Cattleya harrissonii, Bat. . . . . . . . 3 31
Cattleya lobata (Hoehne, 1949) . . . . . 1 6
Cattleya loddigesii, Lindl. . . . . . . . 3 39
Cattleya tigrina, A. Rich. . . . . . . . 2 58
Cattleya trianaei (Hoehne, 1949) . . . . . . 1 7
Cerathandra chloroleuca, Eckl. . . . . . . 3 13
Cirrhaea viridi purpurea, Lindl. . . . . . . 3 52
Coelia alba, Lindl. ? Hort . . . . . 2 1
Corycium crispum, Sw. . . . . . . . 2 38
Corycium orobanchoides, Sw. . . . . . . 2 50
Corallorrhiza innata, R. Br. . . . . . . . 1 30
Cymbidium odontorrhizon, Willd. . . . . . . 2 27
Cymbidium sinense, Lindl. . . . . . . . 3 30
Cyrtosia lindleyana, Bl. . . . . . . 3 16
Cypripedium barbatum, Lindl. . . . . . . 1 46
Dendrobium cretaceum, Lindl. . . . . . . 3 46
Dendrobium plicatile, Lindl. . . . . . . 3 12
Dicrypta bauerii, Lindl. . . . . . . . 3 56
Dicrypta glaucescens, Hort. . . . . . . . 2 59
Dicrypta glaucescens var. Hort. . . . . . . 2 19
Disa cernua, Sw. . . . . . . . . 3 14
Disa cornuta, Sw. . . . . . . . . 3 23
Disa pulchella, Sw. . . . . . . . . 3 42
Disa tenella, Sw. . . . . . . . . 2 56
Disperis villosa, Sw. . . . . . . . . 2 54
Epidendrum ciliare, L. . . . . . . . 3 21
Epidendrum cinnabarinum, S. . . . . . . 2 21
Epidendrum cochleatum, L. . 2 37
Epidendrum crassifolium, Lindl. . . . . . . 3 8
Epidendrum lancifolium, Hort. . . . . . . 2 46
Epidendrum papillosum, Batem. . . . . . . 3 37
Epidendrum ramosum, Jacq. . . . . . . 3 2
Epidendrum stamfortianum var. cornea, B. 3 45
Epidendrum stamfortianum, Batem . . . . . 1 44
Epipactis latifolia, Swartz. . . . . . . . 1 33
Epistephium parviflorum, Bl. . . . . . . 2 28
Eulophia streptopetala, Hort. . . . . . . 2 20
Gamoplexis orobanchoides, Bl. . . . . . . 3 7
Glossodia minor (Clifford and Smith, 1969) . . . . 1 1
Congora bufonia, Lindl. . . . . . . . 2 29
430 J. ARDITTI

Fig. Seed No.


Gongora maculata v. pallida, Lindl. . . . . . . 2 13
Goodyera discolor, Lood. . . . . . . . . 2 39
Goodyera procera, Hook. . . . . . . . . 2 23
Goodyera repens, R. Br. . . . . . . . . 2 3
Goodyera semipellucida, Lindl. . . . . . . . 3 4
Govenia lilacina, Lindl. . . . . . . . . 2 12
Gymnadenia conopsea, Rich. . . . . . . . 1 14
Gymnadenia longifolia, Lindl. . . . . . . . 1 45
Habenaria dilatata, Hook. . . . . . . . . 3 10
Habenaria hispidula, Lindl. . . . . . . . . 2 48
Habenaria tridentata, Hook. . . . . . . . 2 36
Haematorchis altissima, Bl. . . . . . . . . 2 34
Himantoglossum hircinum, Spr. . . . . . . . 1 26
Huntleya violacea, Lindl. . . . . . . . . 3 25
Isochilus linearis, R. Br. . . . . . . . . 3 38
Laelia anceps, Lindl. . . . . . . . . . 3 20
Laelia galeottiana, A. Rich. . . . . . . . 3 6
Laelia perinii \. major, Lindl. . . . . . . . 3 36
Leptotes bicolor, Lindl. . . . . . . . . 2 10
Listera ovata, R. Br. . . . . . . . . . 1 17
Luisia teretifolia, Gaud. . . . . . . . . 2 32
Lycaste harrissonii, Hort. . . . . . . . . 3 58
Malaxia lilifolia, Swartz . . . . . . . .3 41
Maxillaria crocea, Lindl. . . . . . . . . 1 12
Miltonia more/liana, Hort. . . . . . . . . 2 22
Mormodes buccinator, Lindl. . . . . . . . 1 18
Mormodes viridiflora, Hort. . . . . . . . 1 20
Mormodes pardina, unicolor Hook . . . . . . 1 23
Nigritella angustifolia, Rich. . . . . . . . 1 42
Neottia aestivalis, Dec. . . . . . . . . 2 51
Neottia nidus-avis, Rich. . . . . . . . . 1 41
Neottia orchioides, Sw. . . . . . . . . 3 51
Neottia pubescens, Willd. . . . . . . . . 3 5
Neottia (Stenorrhynchus} speciosi, Jacq. . . . . . 2 45
Neottia vitalis, Lindl. . . . . . . 3 43
Octomeria lancifolia, Hort. . . . . . . . . 1 11
Odontoglossum bictoniense, Lindl. . . . . . . 2 35
Odontoglossum pescatorei (Hoehne, 1949) . . . . . 1 8
Odontoglossum pulvinatum, Lindl. . . . . . . 2 41
Odontoglossum sphacelatum, Lindl. . . . . . . 1 35
Ophrys funerea, Vivian . . . . . . . . 3 26
Ornithocephalus sp. Java . . . . . . . . 1 29
Orobanche! . . . . . . . 2 38
Orchis acuminata, Desf. . . . . . . . . 2 33
Orchis brevicornu, Vivian . . . . . . . . 1 39
Orchis coriophora, Lin. . . . . . . . . 35
Orchis fragrans, Pollin. . . . . . . . . : 38
Orchis intacta, Lin. . . . . . . . . . 1 43
Orchis latifolia, Lin. . . . . . . . . . 1 40
Orchis longicornu, Poir . . . . . . . . 28
Orchis maculata, Lin. . . . . . . . . . 1 32
Orchis maculata, var. Lin. . . . . . . . . 13
Orchis secundiflora, Pertol. . . . . . . . . 1 36
Orchis speculum, Tenor. . . . . . . . . 1 37
Otochilus fusca, Lindl. . . . . . . . . 3 11
Otochilus porecta, Lindl. . . . . . . . . 3 59
Paphiopedilum curtisii (Hoehne, 1949) . . . . . . 3
Paphiopedilum specierum (Hoehne, 1949) . . . . . 1 4
Paphiopedilum parishii . . . . . . . . 1 5
Pelexia adnata, Spr . . . . . . . . . 3 49
Pyrola rotundifolia! . . . . . . . . . 1 34
Phajus albus, Lindl., Thunia alba R. fil. . . . . . 1 16
Phajus bicolor, Lindl. . . . . . . . . . 2 43
ASPECTS OF ORCHID PHYSIOLOGY 431

Fig. Seed No.


Phajus grandifolius, Lour. . . . . . . . . 2 3
Phajus maculatus, Lindl. . . . . . . . . 2 42
Phajus wallichii, Lindl. . . . . . . . . 3 34
Pholidota rubra, Bl . . . . . . . . . 3 1
Pleurothalis sessiliflorum, Lindl. . . . . . . . 3 27
Promenaea rollissonii, Lindl. . . . . . . . 2 57
Promenaea stapelioides, Lindl. . . . . . . . 2 11
Pterygodium Caffrum, Sw. . . . . . . . . 2 47
Pterygodium catholicum, Sw. . . . . . . . 3 51
Pterygodium inversum, Sw. . . . . . . . . 3 48
Pterygodium voluere, Sw. . . . . . . . . 3 50
Sarcanthus rostratus, Lindl. . . . . . . . . 2 55
Satyrium bicatlosum, Thumb. . . . . . . . 3 57
Satyrium carneum, R. Br. . . . . . . . . 3 28
Satyrium nepalense, Don. . . . . . . . . 3 22
Scaphyglotis vestita, Brong. . . . . . . . . 1 31
Scaphyglotis violacea, Brong. . . . . . . . 1 31
Selenipedium schlimmii, Rchb. fil. . . . . . . 3 35
Sobralia decora, Batem. . . . . . . . . 3 9
Sobralia liliastrum, Lindl. . . . . . . . . 2 14
Sobralia macranlha, Lindl. . . . . . . . . 3 24
Stanhopea aurea, Lodd. . . . . . . . . 1 24
Stanhopea insignis, Forst. . . . . . . . . 2 7
Stanhopea oculata, Lindl. . . . . . . . . 2 8
Stanhopea tigrina, Batem. . . . . . . . . 1 22
Stanhopea tigrina, var. superba, Batem. . . . . . . 1 17
Stanhopea violacea, Nort. . . . . . . . . 3 33
Stanhopea warczewitzii, Hort. . . . . . . . 1 21
Sturmia loeselii, Reichbeh. . . . . . . . 1 9
Tetragamestus modestus, R. fil. . . . . . .1 27
Thelymitra ixioides, Sw. . . . . . . . . 2 9
Trichocentrum fuscum, Lindl. . . . . . . . 3 44
Trichopilia albida, H. Wendl. . . . . . . 2 44
Triphora pendula, Nutt. . . . . . . . . 2 49
Vanda coerulea, Griff. . . . . . . . . 3 53
Vanilla sp. . . . . . . . . . . 1 2
Vanilla planifolia, Andr. . . . . . . . . 2 40
Xylobium squalens, Lindl. . . . . . . . . 3 19
Zygopetalum intermedium, Lodd. . . . . . . . 2 5
Zygopetalum mackaii, Hook. . . . . . . . 2 4
Fig. 4. Scanning electron microscope photograph of Pleurothallis racemiflora seed. ×
1050. (Courtesy Dr W. Barthlott.)
Fig. 5. Scanning electron microscope photograph of Vanilla planifolia seed, × 210.
(Courtesy Dr W. Barthlott.)
Fig. 6. Scanning electron microscope photograph of Cynorkis fastigiata seed coat. ×
2100. (Courtesy Dr W. Barthlott.)
Fig. 7. Scanning electron microscope photograph of Eulophia guinensis seed coat. × 525.
(Courtesy of Dr W. Barthlott.)
Fig. 8. Scanning electron microscope photograph of Graphorkis lurida seed coat, × 1059.
(Courtesy of Dr W. Barthlott.)
Fig. 9. Scanning electron microscope photograph of Calypso bulbosa seed coat, × 2036.
(Courtesy of Dr W. Barthlott.)
Fig. 10. Scanning electron microscope photograph of Limnodorum abortivum (L.) Swartz
seed coat, × 1050. (Courtesy of Dr W. Barthlott.)
Fig. 11. Scanning electron microscope photograph of a Coryanthes species seed coat
from Costa Rica. (Courtesy of Dr W. Barthlott.)
Fig. 12. Scanning electron microscope photograph of Cyrtorchis arcuata seed. × 210.
(Courtesy of Dr W. Barthlott.)
Fig. 13. Scanning electron microscope photograph of Catasetum russellianum seed. ×
210. (Courtesy of Dr W. Barthlott.)
Fig. 14. The five basic shapes into which orchid seeds have been classified (Clifford and
Smith, 1969).

TABLE 1
Weights of Orchid Seeds (Koch and Schulz, 1975)
Species Weight (µg)
Galeola sp. (lindleyana?) 14.0
Gymnadenia conopea 8.0
Sobralia macrantha 6.3
Epidendrum radicans 6.0
Limodorum abortivum 5.73
Dendrobium antennatum 5.65
Stanhopea oculata 3.0
Grammatophyllum speciosum 2.48
Goodyera repens 2.0
Cephalanthera pollens 2.0
Haemaria discolor x 0.85
H. rubrovenia
Angraecum eburneum x 0.70
A. sesquipedalea
Acanthophippium sylhetense 0.66
Didymoplexis pollens 0.45
Anguloa ruckerib 0.39
Schomburgkia undulata 0.30
a
This hybrid is known as Angraecum Veitchii.
b
3,932,948 seeds/capsule.
438 J. ARD1TTI

B. EXTERNAL MORPHOLOGY

Rumphius described orchid seeds as being dust- or flour-like. This is still


the best description if one looks at the seeds with the naked eye, except that it
is necessary to add that their colour may be white, cream, pale green,
reddish, orange or dark brown (for reviews see Arditti, 1967a; Barthlott,
1976; Beer, 1863; Burgeff, 1936). The diversity of seed shapes is cons iderable
(Figs 1-13), but it can be reduced to five basic forms (Fig. 14; Clifford and
Smith, 1969). Orchid seeds are small and may measure down to 0.75 x 0.2
mm (Calopogon pulchellus). They may vary in length from 0.3 mm (Satyrium
odorum) to 5 mm (Sobralia species, Figs 1, 2). Their weight can be 0.3 µg
(Table 1). Embryos are much smaller and may consist of as few as ten cells.
They are situated in the middle of the testa, being attached to it by several fine
strands.
Testa cells are dead, vary in size and have longitudinal and transverse walls
of different thickness (Barthlott, 1976; Clifford and Smith, 1969; Rauh et al,
1975; Wildhaber, 1972, 1974). This gives them a net-like appearance. The
cells may be isodiametric or elongated, polygonal, tetragonal, hexagonal or
have a somewhat convoluted shape (Figs 1-3). Some testa cells appear
sculptured due to variable and discontinuous thickenings on the walls (Figs 6,
9, 13). Since these characteristics can occur in a variety of combinations,
orchid seed morphology can be a useful systematic tool (Barthlott, 1976;
Clifford and Smith, 1969; Wildhaber, 1972).
An additional characteristic of orchid seeds is that many are difficult to
wet. Because of this as well as their size, shape, weight and construction
orchid seeds are very buoyant in both air and water. Consequently they can
travel long distances in air and water currents and this, of course, aids in their
dispersal (de Lehaire, 1910; Sanford, 1974).
C. STRUCTURE AND ULTRASTRUCTURE

Orchid embryos consist of relatively undiiferentiated, mostly isodiametric


cells with dense granulated cytoplasm and conspicuous nuclei. Cells of the
posterior end are large and sometimes vacuolated. A suspensor, consisting of
very large, vacuolated, dead cells is attached to the posterior (Alvarez, 1969;
Veyret, 1969; for reviews see Arditti, 1967a; Withner, 1959). The anterior,
chalazal or meristematic portion of the embryo is composed of smaller cells
which in Cattleya aurantiaca are 8-10 µm in diameter (Harrison, 1973).
There have been few ultrastructural studies of orchid seeds due to the great
difficulties encountered in their fixation and sectioning. One of the more
recent (and few) studies was carried out in my laboratory and the following
description and electron micrographs are taken from it (Harrison, 1973):

". . . The ovoid embryo of the Cattleya aurantiacaseed has an average length of
260 µm, exclusive of the suspensor which is composed of dead cells. Average
Fig. 15. Seed of Cattleya aurantiaca seed, whole.
Figs 16-18. Electron micrographs of cells in ungerminated Cattleya aurantiaca seed. 16.
Cell in ungerminated seed, x 2700. 17. Cell in ungerminated seed, X 3375. 18. Basal cell of
an ungerminated seed, x 12 375. (Explanation of symbols: CW, cell wall; L, lipid body; M,
mitochondrion; N, nucleus; P, protoplastid; PB, protein body).
Figs 19-21. Effects of galactose on cells of orchid seedlings. 19. Nuclear chromatin
appears dispersed and nuclear envelope evaginates (arrows) into cytoplasm, x 9338. 20.
Nuclear envelope evaginates into cytoplasm, x 45 080. 21. Nuclear envelope folded back on
cytoplasmic side, x 41 160. Explanati.on of symbols: N, nucleus; NE, nuclear envelope
(Ernst et al., 1971a).
ASPECTS OF ORCHID PHYSIOLOGY 441

width is 80 µm. An orange-colored, fusiform seed coat surrounds the embryo. It


is 400 µm long and 80 µm wide. Total weight of a seed is approximately 1.5
µg (Figs. 15-18)."
"Cells of the dry, ungerminated seed vary in size depending on their positions
within the embryo. The chalazal or meristematic end of the embryo is composed
of smaller cells, generally 8-10 µm in diameter. Larger cells comprise the
remainder of the embryo and will be referred to here as the basal cells (Figs.
19-21)."
"Ultrastructurally, all of the cells, regardless of their position in the embryo,
are heavily packed with food reserves (Figs 15-21). Lipids constitute the
largest portion of the reserve material and are present as lipid bodies in all
cells (Figs 16-18; Wiesmeyer and Hofsten, 1976). Protein bodies are also
found (Fig. 17) but are restricted to cells in the upper two -thirds of the embryo,
including those in the meristematic region and upper half of the basal region.
Throughout the embryo the cytoplasm is dense and stains heavily for proteins.
Aside from an occasional small grain within a proplastid (Fig. 18), no starch or
other carbohydrate reserves were found in the dry seed. Also, plasmodesmata
are not discernible in the ungerminated seed."
"Presumptive organelles are found in the cytoplasm of the dry seed but
generally cannot be classified as to type because of their immature stage of
development and poor hydration state. Most of the visible organelles are
positioned next to the cell wall or else in the cytoplasm immediately surrounding
the nucleus. Distinguishable proplastids and mitochondria do occur in the
cytoplasm adjacent to the nuclei of the larger basal cells (Fig. 18). Endoplasmic
reticulum is not discernible."
D. LONGEVITY

Depending on species or storage conditions the longevity of orchid seeds


may vary from two months to 18 years (Janke, 1915; for a review see Arditti,
1967a). Most seeds will lose their viability if stored at room temperature
(21-22°C) without desiccation. However, longevity can be extended con-
siderably if the seeds are permitted to dry and then stored over a desiccant
(calcium chloride, for example) under low temperature (0-10°C). Orchid
seeds can also survive quick freezing (Fehlandt, 1960), lyophilization in
autoclaved coconut liquid as suspending fluid (Svihla and Osterman, 1943)
and storage at -79 °C for 351, 365 and 465 days (Ito, 1965). The information
on high temperature tolerance of orchid seed is more limited, but it is known
that at least one species (Dendrobium nobile) germinates well under tempera-
tures up to 40 °C. Soaking seeds for up to four hours at 39 °C is also without
deleterious effects (for a review see Arditti, 1967a).
E. ASYMBIOTIC GERMINATION

Knudson demonstrated that at least some orchids can germinate asym-


biotically on suitable media (Knudson, 1921, 1922, 1946). Others followed and
tried to improve his media or design new ones and to germinate additional
species (for reviews see Arditti, 1967a; Withner, 1959). Knudson's media are
suitable for most epiphytic and/or tropical orchids. However, some terrestrial
442 J. ARDITTI

species, especially those from temperate regions, are more difficult to germin-
ate. Consequently, additions, modifications or new media and procedures
must be employed for terrestrial species (for review see Stoutamire, 1974),
from Europe (Borriss, 1971; Fast, 1976; Hadley, 1970; Hadley and Harvais,
1968; Harbeck, 1963, 1964; Harvais and Hadley, 1967; Mead and Bulard,
1975; Veyret, 1969; Vöth, 1976), Canada (Harvais, 1972, 1973, 1974),
Australia (Mclntyre et al, 1971, 1972a, b; Veitch and Mclntyre, 1972;
Wrigley, 1973, 1976), South Africa (Collett, 1971; Harbeck, 1968), and the
USA (Stoutamire, 1974).
In addition to attempting the germination of various species, investigators
have also studied the effects of or requirements for a variety of factors in the
germination of orchid seeds (for reviews see Arditti, 1967a; Fast, 1964,
1967; Stoutamire, 1974; Withner, 1959).

1. Mineral Nutrition
Most media used for orchid seed germination are more concentrated than
tree trunk effluates which nurture epiphytic seedlings in nature (Table 2;
Curtis, 1946; for reviews see Arditti, 1967a; Withner, 1959). Epiphytic
species germinate well on these media, but some terrestrial orchids do not.
Their germination is much better on more dilute media (Vöth, 1976; personal
communication from Robert Ernst regarding medium RE in Table 2). In
the case of Dactylorhiza purpurella germination can take place on distilled
water and a modified Pfeffer medium (Harvais, 1972). Arundina bambusifolia
(a terrestrial orchid) seeds germinate better on the more dilute Raghavan and
Torrey medium than on Vacin and Went (Table 2; Mitra, 1971). Seeds of
Vanilla planifolia germinate best on 0.1 x the normal concentration of
Knudson B medium (Lugo-Lugo, 1955); Cypripedium calceolus germinates on
a medium (Fast, 1976) which is 2.5 times as dilute as the one used for C.
reginae (Harvais, 1973), which in turn is less concentrated than Knudson C
(Table 2). Thus, it seems that the concentration of media is an important
factor. Unfortunately, the available information is limited and there are no
clear patterns. However, a guarded generalization can be made: Lady slipper
orchids (Cypripedium, Paphiopedilum) and temperate climate terrestrials seem
to germinate better on more dilute media (Fast, 1976; Harbeck, 1963;
Harvais, 1973; R. Ernst, personal communication). Variations in the propor-
tion of ions in culture media have been reported to have little effect (Wynd,
1933). The same investigator also suggested that anions may play a more
vital role in the germination of orchid seeds than cations. More recent work
with Bletilla striata seeds (Ichihashi and Yamashita, 1977) has determined the
optimal range of ions and led to the formulation of a culture medium
(Tables 1, 2, 3).
Before the advent of chelating agents, precipitation of iron presented a
problem and a variety of substances were used to ensure its availability (for
ASPECTS OF ORCHID PHYSIOLOGY 443

reviews see Arditti, 1967a; Withner, 1959). At present, EDTA or commercial


preparations of chelated iron are used widely and the problem has been
solved (Fast, 1976; L. Koch, 1973; U. Koch, 1972; Mead and Bulard, 1975;
Mitra, 1971; Miyazaki and Nagamatsu, 1965; Mukherjee et al., 1974;
Thompson, 1977 are some examples; for reviews see Arditti, 1967a, Thompson,
1977).
Comparative studies have revealed that reduction of phosphate levels can
increase germination. The reasons for this are unclear. It is possible, of
course, that orchid seeds may be sensitive to phosphate. However, it is also
possible that high phosphate levels may lead to iron deficiency since an in-
soluble complex is formed during autoclaving. On media lacking phosphorus,
Cymbidium protocorms become ". . . yellow grey-green and are covered with
black spots, but do not die even after 100 days . . ." (de Bruijne and Debergh,
1974). Excellent germination of several species has been obtained on media
low in calcium (Table 2; Fast, 1976; Ernst, personal communication;
Raghavan and Torrey, 1964; Thompson, 1977; Vacin and Went, 1949;
Wynd, 1933). This is especially true for diandrous (i.e., Lady slipper) and
terrestrial species. Omission of calcium from culture media is without notable
effect on development of the normal green colour (de Bruijne and Debergh,
1974). Precipitation of calcium as phosphate salt is also without deleterious
effects (Storey et al., 1947). Orchid tubers and seeds contain limited amounts
of calcium (Tienken, 1947; L. C. Wheeler, personal communication; Wheeler
and Ramos, 1965; for a review see Arditti, 1967a) and may, therefore, have
low requirements.
Soaking the seeds of Galeola septentrionalis, a terrestrial orchid lacking
chlorophyll, in solutions of several potassium salts increased their germina-
tion. Concentrations of KC1 at 5 x 10-1 M, or higher, in culture media were
supraoptimal and 5 x 10-4 proved to be suboptimal. Similar treatments of
Cymbidium virescens failed to improve germination (for a review regarding
the effects of potassium, lithium and sodium on orchid seeds see Arditti,
1967a). Numerous small protocorms develop on potassium-free media and
leaves are stunted (de Bruijne and Debergh, 1974).
Microelements are not always added to orchid seed germination media be-
cause enough may be present in the agar, sugar or other salts. However, in
some cases improved seedling grow th has been reported following the addi-
tion of microelements such as boron, cobalt, copper, iodine, manganese and
molybdenum. Therefore, incorporation of microelements in culture media is
often recommended, probably on the assumption that even if not beneficial
they are most probably not harmful either (Arditti, 1967b; Harrison and
Arditti, 1970; Harvais, 1972, 1973, 1974; Ichihashi and Yamashita, 1977;
Kusomoto and Furukawa, 1977; Miya/aki and Nagamatsu, 1965; Mukerjee
et al., 1974; Kaewbanrung, 1967; Koch, 1972; Thompson, 1974a, b, 1977;
Ueda and Torikata, 1972).
TABLE 2
Major Element Composition of Several Media Which Have Been Used for Orchid Seedling Culture Media
(some from Withner, 1959) and Orchid-nurturing Tree Trunk Effuate (Curtis, 1946)
[Amounts expressed in millimoles]
Murashige Sladden Knudson Ichihashi
Fast, Burgeff
Ion and modif and
Skoog 1971 Burgeff REa B C Yamashita Eg-1
Nitrate 39.4 3.96 8.40 11.57 8.48 8.40 14.38 8.40
Ammonium 20.61 7.57 10.60 7.27 7.66 7.60 3.39 3.80
Nitrate : 1.9 0.52 0.8 1.59 1.10 1.10 4.2 2.2
Ammonium ratio
Phosphate 1.24 1.84 2.94 2.20 2.16 1.80 3.39 3.20
Sulphate 1.50 4.80 6.50 1.22 4.79 4.80 0.70 2.90
Chloride 5.98
Potassium 20.03 5.79 2.94 6.16 1.83 1.80 7.38 4.60
Magnesium 1.50 1.01 1.20 0.67 1.01 1.00 0.70 1.00
Calcium 2.99 0.13 4.80 0.63 4.24 4.20 3.49 4.20
Citrate 1.89
Iron 10 mg 0.67 0.09 0.33 0.09 0.07
chelate
Manganese 0.034
Sodium
Urea
Ammonium : Urea ratio
Total concentration 93.25 6.91 39.94 30.46 30.5 29.72 33.43 28.17
Vacin
and Burgeff Raghavan Thomale Tree trunk
Ion Went N3f and Torrey GD Pfeffer effluate Thompson Wynd
Nitrate 5.19 8.40 2.00 10.06 8.73 0.0025 2.99 9.8 7.8
Ammonium 7.56 3.80 2.00 1 5.50 0.0880
Nitrate: 0.69 2.2 1.82 0.02
Ammonium ratio
Phosphate 3.14 1.40 2.76 2.20 1.46 0.0105 2.99 2.5 3.8
Sulphate 4.83 2.90 1.43 2.16 0.81 0.0052 1.49 1.23 9.7
Chloride 3.40 1.34 0.1430
Potassium 7.03 6.20 1.98 6.16 4.77 0.0770 3.99 2.5 19.4
Magnesium 1.01 1.00 0.97 0.74 0.81 0.1770 1.49 1.23 3.9
Calcium 1.95 4.20 0..85 3.38 0.0250 0.49 4.9 1.9
Citrate 0.43
Iron 0.19 0.07 0.07 0.0073
Manganese 0.04
Sodium 0.1310
Urea 8.99
Ammonium: Urea ratio 0.33
Total concentration 30.94 31.8 12.99 24.3 21.3 0.686 22.43 22.16 46.5
a
Used as given for seedling culture and half strength for seed germination.
446 J. ARDITTI

TABLE 3
Optimal Range of Ions and Levels at a Total Medium Concentration
(Ichihashi and Yamashita, 1977) of 20 mg l -1
Range Range
(% total (% total
Cations cation concentration) Anions anion concentration)
NH4+ 16-20 N03- 66-88
K+ 35-41 H2P04- 7-23
Ca2+ 34-37 SO42- 4-14
Mg2+ 10

2. Nitrogen
Nitrate, ammonia and urea can all be used as nitrogen sources by orchid
seeds. However, several species have been reported to grow better on am-
monia (for a review see Arditti, 1967a). Several species appear unable to
utilize nitrate during the early stages of germination (de Bruijne and Debergh,
1974). Cattleya seedlings can utilize nitrate only after a 60-day growing period
(Raghavan and Torrey, 1964). The ability to grow in nitrate develops in
parallel with the appearance of nitrate reductase. This suggests that seedlings
differentiate biochemically as well as morphologically (Raghavan, 1976).
Urea has given contradictory results even with species in the same genus.
Cymbidium seedlings were reported to have been inhibited (Cappelletti, 1933)
and stimulated (Burgeff, 1936) by urea. Growth of Dendrobium phalaenopsis
and Phalaenopsis was inhibited by urea (Burgeff, 1936) whereas development of
Laeliocattleya (Magrou et al., 1949), Vanilla planifolia (Lugo-Lugo, 1955),
Cattleya (Curtis, 1947), and Vanda was enhanced. A recently developed
culture medium contains only urea and ammonia nitrogen (Thompson, 1977).
Some of the reported differences in response to or requirement for nitrate,
urea and ammonia may be due to the genera and/or species used in the
experiments. Others may be a reflection of plantlet age, and the absence of
nitrate reductase in very young seedlings. Hence, the suggestion that NH4 NO3 is
the best nitrogen source (Mitra, 1970) seems reasonable. Cymbidium
proto-corms cultured on hydroxyurea, a DNA replication inhibitor, were
mal-formed (Rücker, 1975).
Almost all amino acids as well as urea and related substances have been
incorporated in orchid seed culture media either as supplements or nitrogen
sources. Arginine, ornithine and urea were capable of replacing NH4 NO3 in
Cattleya cultures. Phenylalanine, citrulline, tyrosine, aspartic acid, glutamic
acid, glutamine, asparagine and phenylurea could not. Proline and
y-aminobutyric acid were moderately good sources of nitrogen (Raghavan,
1964, 1976; Raghavan and Torrey, 1964). Glycine, a -alanine, valine,
a -aminobutyric acid, leucine, phenylglycine, hydroxyproline, canavanine and
threonine were inhibitory (Raghavan, 1964; Raghavan and Torrey, 1964).
ASPECTS OF ORCHID PHYSIOLOGY 447

Arginine and aspartic acid increased shoot formation in Cymbidium pumilum


and C. goeringii (Ueda and Torikata, 1968, 1969, 1972). Results with other
amino acids have been extremely variable (for reviews see Arditti, 1967a;
Sanford, 1974; Withner, 1959, 1974).
Casein and lactalbumin hydrolysate, peptone and tryptone are often used
in culture media (Harvais, 1972, 1973, 1974; Mead and Bulard, 1975; Pages,
1971; Rao and Avadhani, 1963; Torikata et al., 1965; Zeigler et al, 1967;
Vöth, 1976; Ueda and Torikata, 1968, to mention several reports of many;
for reviews see Arditti, 1967a; Withner, 1959, 1974) with varying results.
Since these substances are complex mixtures which may be changed chemic -
ally during autoclaving, it is difficult if not impossible to speculate regarding
the reasons for their effects. In experiments with Orchis (Mead and Bulard,
1975) casein hydrolysate could be replaced by a reconstitution of its amino
acids or glutamine only. These findings may be due to a specific requirement
by Orchis since glutamine did not have a similar effect on Cattleya (Rag-
havan and Torrey, 1964).
Nucleic acids and related compounds have had varied effects on orchid
seed germination. Their effects are as difficult to evaluate as the complex
nitrogen sources because they too may be changed by autoclaving (Arditti,
1967a).

3. Carbohydrates
The first attempt to study the suitability of a sugar as a carbon source for
germinating orchid seeds was made before the discovery of the asymbiotic
method (Bernard, 1909). However, meaningful comparisons between sugars
became possible only when defined media became available. It is not sur-
prising, therefore, that the first comparative studies were published soon after
Knudson's initial publication (La Garde, 1929; Knudson, 1916, 1921; Quednow,
1930). Numerous studies followed (Arditti, 1967a; Ernst, 1967; Ernst et al.,
1971a; Raghavan, 1976; Withner, 1959, 1974) and identified sugars and
carbohydrates which can or cannot support germination. Not all species
germinate on sugars listed as suitable, but in these cases the problems are
more complex than the availability of an appropriate carbon source. Neither
list (suitable or unsuitable in Table 4) is surprising. Higher organisms
generally use D-sugars and orchids are no exception. Galactose is toxic to
most plant tissues and this is also the case with orchids. It inhibits the growth of
orchid seedlings and other plants at levels as low as 0.69 mM (0.0125%;
Burstrom, 1948; Arditti et al, 1972a; Ernst, 1967, 1974; Ernst et al, 1971a;
Ordin and Bonner, 1957; Thimann, 1956). Its metabolic effects may be to
interfere with cellulose synthesis (Ordin and Bonner, 1957), or hexokinase
activity (Hele, 1953) or both.
Nuclear chromatin (Fig. 19) in galactose-treated cells appeared dispersed
throughout the nucleus rather than concentrated around the nuclear envelope.
TABLE 4
Suitability of Sugars, Polysaccharides, Other Carbohydrates, and Carboxylic
Acids as Carbon Sources for Germinating Orchid Seeds (Arditti, 1967a;
Ernst, 1967; Ernst et al, 1971a; Withner, 1959, 1974)
Suitable Remarks Unsuitable Remarks
Monosaccharides Monosaccharides
C-5 C-5
D-ribose In some cases D-arabinose
D-xylose L-arabinose
D-xylose
L-xylose
C-6 C-6
D-fructose D-galactose
a D-glucose L-glucose
ßD-glucose L-mannose
D-mannose L-sorbose
C-7
Sedoheptulosan
Disaccharides Disaccharides
C-12 C-12
cellobiose D-lactose Unsuitable for
lactose Marginal except most species
for Vanilla Deoxysugars
maltose C-6
melibiose D-fucose
sucrose L-fucose
trehalose 2-deoxy -D-glucose
turanose L-rhamnose
Trisaccharides Sugar alcohols
C-18 C-4
melezitose meso-erythritol
raffinose
Tetrasaccharides C-5
C-24 L-arabinitol
stachyose
Sugar alcohols C-6
C-5 galactitol
D-arabinitol myo-inositol Protocorms
ribitol survive, but fail
xylitol to differentiate
C-6 Polysaccharides
mannitol Starch
sorbitol Cellulose
Organic acids Organic acids
malate Citric Acid
pyruvate Malic acid
Oxalic acid
Pyruvic acid
Succinic acid
Tartaric acid
Figs 22-24. Ultrastructure of cells from galactose-treated orchid seedlings. 22. Unit
membrane vesicles and myelin bodies, × 33,320. 23. Amyloplasts appear normal, × 11,360.
24. Mitochondrial cristae appear slightly swollen (black triangle), × 9660. Explanation of
symbols: Am, amyloplast; MB, myelin body; V, vesicle (Ernst et al., 1971a).
450 J. ARDITTI

In many cases the nuclear envelope evaginated into the cytoplasm (Figs 19,
20, 21). This included both membranes of the nuclear envelope rather than
the more commonly observed case of the evagination of the outer membrane
only. These evaginations appear to be devoid of chromatin (Fig. 20; Ernst
et al., 1971a).
Numerous unit membrane vesicles were found in the cytoplasm of galac-
tose-treated cells (Fig. 22). In addition, myelin bodies were present in almost
all cells (Fig. 22). The unit membrane character of the vesicles and myelin
bodies was clearly visible (Fig. 22). Both of these structures may have been
derived from the tonoplast, which was invariably broken. No intact dictyo-
somes were visible in this material (Ernst et al., 1971a).
Amyloplasts appeared normal (Figs 23, 24). Membranes were intact and
showed little or no swelling. Large starch grains and small lipid droplets were
present in the amyloplasts (Figs 23, 24). Mitochondria (Fig. 24) were intact,
but their cristae appeared slightly swollen and the matrix somewhat clumped
(Ernst et al., 1971a).
The size of polysaccharide molecules poses permeability problems in
orchids as in other plants and enzymes which can break them down are
generally extracellular in nature. Permeability problems may also prevent the
utilization of organic acids. It is reasonable to expect that orchid seeds
(which under natural conditions require mycorrhizal infections for germina-
tion) germinate successfully and seedlings grow well on trehalose and mannitol,
both carbohydrates of fungal origin (Smith, 1973). Trehalose is translocated
into orchid seedlings by fungal hyphae (Smith, 1966, 1967) and may well be a
product of glucose derived from the hydrolysis of cellulose by orchid mycor-
rhiza (Hadley, 1968).
The effects of myo-inositol (Table 4) may have been due to supraoptimal
concentrations of a compound which is variously classified as a vitamin or
cytokinin and is beneficial in ol w amounts (Arditti and Harrison, 1977;
Leopold, 1964).
Glucose is very common in plants and fungi either free or as a component
of polysaccharides; it is also an important starting point in many metabolic
pathways. It is therefore not surprising that orchid seeds and seedlings can
utilize glucose (Table 5; Freson, 1969).
There are a number of reports that some orchid species germinate and
grow better on fructose (Ernst, 1967; for reviews see Arditti, 1967a; Burgeff,
1936; Withner, 1959). Phalaenopsis seedlings, for example, take up and/or
utilize fructose in preference to glucose (Ernst et al., 1971a). However,
Cattleya aurantiaca seedlings do not grow as well on fructose as they do on
sucrose or glucose (Fig. 25; Harrison, 1973; Harrison and Arditti, 1978).
Sucrose is the sugar most commonly used in orchid seed and seedling culture.
It can support growth equally well whether autoclaved or filter-sterilized
(Fig. 26) but its effects may vary depending on concentration (Fig. 27;
ASPECTS OF ORCHID PHYSIOLOGY 451

TABLE 5
Effect of Glucose on Cymbidium Protocorms (Freson, 1969)
Concentration
% mM Effects
0 0 Protocorms do not multiply and become necrotic rapidly
0.25 13.88 Poor growth, but tissues are green
0.63 34.96 Improved multiplication, rhizoid formation, poor
differentiation
1.6 88.81 Best growth, development and chlorophyll content
4 222.02 Increased production of plantlets, reduced protocorm
multiplication and chlorophyll levels
10 555.06 Protocorms do not multiply and become necrotic rapidly

Table 6; Homès, 1973; Homès et al., 1971; Homès and Vanséveren-Van


Espen, 1972, 1973a, b; Vanséveren-Van Espen, 1973). Organogenesis is
promoted at suboptimal concentrations whereas protocorm proliferation is
enhanced by supraoptimal levels (Homès and Vanséveren-Van Espen, 1973a).
Chloroplast structure is affected by the presence of sucrose (Homes and
Vanseveren-Van Espen, 1972, 1973). In a sucrose-free medium the chloro-
plasts have peripheral vesicles, thick clusters, thylakoids forming misshapen
grana, and numerous osmophilic globules, but no starch grains. On the
addition of glucose, chloroplast structure becomes normal in 24 hours and
maximum starch accumulation takes place within four days. When Cymbidium
protocorms are cultured on 0.5 % sucrose, chlorophyll content and photo-
synthetic oxygen evolution are at a maximum (Vanséveren-Van Espen and
Courtrez-Geerinck, 1974).
Analyses of filter (i.e., cold) sterilized culture media containing different
sugars (Table 7) indicate that Phalaenopsis seedlings can release extracellular
enzymes which hydrolyse a-D-glucopyranosyl-(l? 2)-a-D-fructofuranoside and
a or ß-D-galactopyranosyl-D-glucopyranose bonds. This view is supported by
the fact that hydrolysis of ß-D-fructofuranosides and a-D-galactosides de-
creases with increasing molecular weight of the sugars (Table 7); maltose
(l? 4-a-D glucosidic bond as in amylose), cellobiose (l? 4-ß-D glucosidic bond
as in cellulose) and trehalose (1? 1-a-D glucosidic bond) are apparently taken
up whole, without external hydrolysis. This may explain, at least in part, the
inability of most orchid seedlings to grow and develop on polysaccharides
such as starch and cellulose. The germination of Miltonia and Odontoglossum
seeds on 1 % corn or potato starch (Hayes, 1969) is an exception. It may be
due to the presence of impurities (i.e., hydrolysates such as glucose and
maltose) in the starch. (For more detailed discussions see Ernst et al., 1971a.)
Orchid seedlings obviously reach a stage at which they no longer require
an exogenous supply of sugars. The length of the period during which
seedlings require a source of sugar was established by growing Cattleya
TABLE 6
Effects of Sucrose Concentration on Growth and Development of Cymbidium Protocorms
(Vanséveren-Van Espen, 1973)
Sucrose concentration % Nature of growth or organ Illumination 23½ hours 16 hours
Molarity (m M) development 5800 lux 2900 lux 5800 lux 2900 lux
16 0.89 Compact masses of very
pale protocorms
leaves 0
roots 0
rhizoids 0
2.5-10 0.14-0.56 Friable masses of pale
protocorms
leaves +
roots 0
rhizoids 0
2-1.6 0.11-0.09 Masses and individual
green protocorms
leaves +++
roots 0
rhizoids +++
0.25-1.25 13.88-69.38 Green protocorms in groups
leaves +++ +++ + + +
roots +++ +++ 0 0
rhizoids +++ +++ 0 0
0 Small dark green
protocorm masses
leaves ++
roots 0
rhizoids +
Explanation of symbols: 0, no growth and/or development; + + +, maximal growth and/or development.
Fig. 25. Effects of sucrose (open circles), glucose (closed circles), fructose (triangles) and
inositol (squares) on growth of Cattleya aurantiaca seedlings (Harrison, 1973).

Fig. 26. Growth of Cattleya aurantiaca seedlings on autoclaved (solid line) and cold
sterilized (broken line) sucrose (Harrison, 1973).
454 J. ARDITTI

Fig 27 Effects of sucrose concentration on chlorophyll levels in Cymbidium protocorms.


Explanation of symbols: a, 23½ h at 5800 lux; b, 23½ h at 2900 lux; c, 16 h at 5800 lux;
d, 16 h at 2900 lux; PF, fresh weight; S, sucrose %; 2% = 0.11 M ; 4% = 0.22 M; 6% = 0.33
M (Vanséveren-Van Espen, 1973).

aurantiaca seedlings on Knudson C medium (KC) with and without sucrose


(KC-Suc) (Fig. 28) and transferring them from one to the other (Harrison,
1973, 1977; Harrison and Arditti, 1978). Seedlings on KC-Suc reached the
protocorm stage, but did not produce roots and leaves (Fig. 28, Harrison,
1973). Those on KC developed normally. Following 21 days on KC only 13 % of
swollen seeds or protocorms formed plantlets on KC-Suc. After 28-30 days
on KC, 50 % of the protocorms transferred to KC-Suc produced leaves and
developed into larger plantlets. Seedlings with one or more leaves also
continued to develop following a transfer to KC-Suc. When the transfer
from KC to KC-Suc was made after 47 days, 92 % of the protocorms de-
veloped into complete seedlings. Protocorms maintained 15, 30 or 60 days on
KC-Suc required 21-30 days on KC before 50 % formed plantlets on KC-Suc.
These findings suggest that the need for an exogenous carbohydrate is out-
TABLE 7
D-Hexose Content of Cold-sterilized Oligosaccharide Solutions Exposed for 4 Months to Phalaenopsis Seedlings
(Ernst et al., 1971a)
Monosaccharide content, Molar ratios of
Fractions detected by thin percentage of neutral fraction monosaccharides
Sugar layer chromatography Glucose Fructose Galactose Glucose Fructose Galactose
Sucrose Sucrose (s)b, fructose, glucose 33.23 28.93 — 1.0 0.87
Sucrose (C)a Sucrose (s), fructose, glucose 4.35 4.35 —
Maltose Maltotriose ? (w)b, maltose (s), glucose 2.72 — —
Maltose (C) Maltotriose ? (w), maltose (s) 0.11 — —
Cellobiose Cellobiose (s), glucose 1.09 — —
Cellobiose (C) Cellobiose (s) 0.22 — —
Trehalose Trehalose (s), glucose 1.24 — —
Trehalose (C) Trehalose (s) trace — —
Melibiose Melibiose (s), galactose, glucose 0.63 — 3.99 1.0 6.3
Melibiose (C) Melibiose (s) 0.07 — 0.06
Lactose Lactose (s), galactose, glucose 1.26 — 9.31 1.0 6.9
Lactose (C) Lactose (s) trace — 0.03
Raffinose Raffinose (s), melibiose (s), galactose, 0.92 12.19 1.92 1.0 13.3 2.1
fructose, glucose (0.08) c (1.0) (0.158)
Raffinose (C) Raffinose (s), melibiose (t)b, fructose 0.11 2.18 0.10
Melezitose Melezitose (s), turanose (w), sucrose (t), 6.53 0.10 — 1.0 0.015
glucose (65.3) (1.0)
Melezitose (C) Melezitose (s) 0.33 0.22 —
Stachyose Stachyose (s), manninotriose (s), — 4.79 0.68 7.0 1.0
raffinose (t), galactose, fructose (1.0) (0.14)
Stachyose (C) Stachyose (s) — 0.16 0.17
a
b
(C) = cold-sterilized nutrient solution not exposed to seedlings.
c
TLC intensities: s = strong; w = weak; t = trace.
Value in parentheses is based on fructose content being equal to 1.0,
456 J. ARDITTI

Fig. 28. Growth index of Cattleya aurantiaca seedlings raised on Knudson C medium
with (solid line) and without sucrose (broken line; Harrison, 1973; Harrison and Arditti,
1978).

grown either with the appearance of the first leaf or the potential to generate it
(Harrison, 1973, 1977; Harrison and Arditti, 1978; Figs 29, 30).

4. Lipids
All cells in Cattleya aurantiaca embryos are heavily packed with food
reserves in the form of lipid bodies (Figs 16-18, Harrison, 1973, 1977;
Harrison and Arditti, 1978). Protein bodies are also found, but are re-
stricted to cells in the upper two-thirds of the embryo. Aside from small
grains within proplastids there are no starch or other carbohydrate reserves
in these seeds. This confirms previous reports that the major food reserves in
orchid seeds are lipids (Anon., 1922; Poddubnaya-Arnoldi and Zinger, 1961).
Analyses of Cymbidium seeds indicate that they contain 32 % lipids (Knudson,
1929; for a review see Arditti, 1967a). Such high concentrations of reserve
materials are generally located in cotyledons and/or the endosperm of most
plants. However, since these structures are lacking in most orchids their
embryos apparently function as storage organs. This view is supported by the
ultrastructural evidence (Figs 16-18).
In fatty seeds the breakdown and utilization of lipid reserves is associated
with glyoxysomes in the cells. These bodies which are involved in the con-
version of lipids to sugars, the predominant metabolic activity during
germination of fatty seeds, could not be discerned in germinating Cattleya
Fig. 29. Percentages of Cattleya aurantiaca protocorms forming plantlets as a function
of the length of time grown on Knudson C medium with and without sucrose. Drawing
denotes transfer sequence (Harrison, 1973; Harrison and Arditti, 1978).

Fig. 30. Percentages of Cattleya aurantiaca seedlings forming plantlets after an initial
period on Knudson C without sucrose, transfer to sucrose containing medium and return
to sucrose-free medium (Harrison, 1973; Harrison and Arditti, 1978).
458 J. ARDITTI

aurantiaca seeds (Harrison, 1973, 1977). The Cattleya seeds converted only
3 % or less of the label from acetate-2-14 C into sugars (in contrast with 90 %
conversion of acetyl units in castor beans) and used their lipid reserves very
slowly. Their lipid bodies were closely associated with or enveloped by mito-
chondria (Harrison, 1973, 1977). Clearly, then, orchids have fatty seeds, in
which the appropriate metabolic pathways are severely limited. Presently,
available evidence suggests that acetyl CoA released from the lipid bodies
may be routed through the Kreb's Cycle in mitochondria where it is oxidized to
carbon dioxide with the production of energy. To some degree this may be a
reflection of the fact that most orchids do not have cotyledons or endosperms,
the organs capable of converting acetate into sugars. The dis appearance of
these structures has apparently led to the loss of some biochemical
capabilities.

5. Hormones
Experiments with auxins, cytokinins and gibberellins and orchid seedlings
have produced inconsistent and therefore inconclusive results (for reviews see
Arditti, 1967a, c; Withner, 1959, 1974). The reasons for this appear to be: (i)
physiological, in that the requirements and responses of genera and species
vary; (ii) chemical, because different forms of each hormone were used; (iii)
dosage, since a wide range of concentrations was employed; (iv) culture
conditions, which vary from report to report; (v) age, because it may

Fig. 31. Seedlings of Cymbidium madidum grown on Knudson C with (above) and
without (below) naphthaleneacetic acid (Strauss and Reisinger, 1976).
TABLE 8
Effects of Auxin on Some Orchidsa
Orchid Auxin Remarks Reference
b
Chondrorhynca discolor x Lycaste NAA , 0.1 ppm(0.54µ M) Seed germination accelerated slightly and Strauss and
aromatica seeds seedling growth and development Reisinger, 1976
enhanced. Protocorms died after 6 months
on auxin deficient media
Bletilla sp. NAAb, 0.1 ppm (0.54 µM) Seed germination accelerated slightly and Strauss and
seedling growth and development Reisinger, 1976
enhanced
Cattleya aurantiaca NAAb, 0.1 ppm (0.54 µM) Seed germination accelerated slightly and Strauss and
seedling growth and development Reisinger, 1976
enhanced
Coeloglossum viride seeds and IAAb, 1 ppm No effect Hadley, 1970
seedlings
Cymbidium sp. seeds K salt of NAAb Inhibitory Torikata et al., 1965

Cymbidium sp. seedlings NAAb, 0.1-1 ppm Stimulates growth Torikata et al, 1965

Cymbidium kanran rhizome NAAb Growth slightly promoted Sawa, 1969


Cymbidium kanran leaf-bud NAAb No differentiation Sawa, 1969
Cymbidium madidum seeds NAAb, 0.1 ppm (0.54 µM) Seed germination accelerated slightly and Strauss and
seedling growth enhanced Reisinger, 1976
Cymbidium virescens rhizomes and leaf NAAb Promotion Sawa, 1969
buds
Dactylorhiza (Orchis) purpurella seeds IAAb, 1 ppm No effect Hadley, 1970
and seedlings
continued
TABLE 8—continued
Orchid Auxin Remarks Reference
Dactylorhiza (Orchis) purpurella IAAb Results inconclusive Harvais, 1972
seeds and seedling
Dendrobium protocorms IBA b, 5 ppm; NAA, 0.5-0.8 ppm Growth enhancement Pages, 1971
Dendrobium seedlings IAA, 2 mg l-1; NAA, 2 mg l-1 Induced callus formation, reduced Mukherjee et al.,
percentage of normal seedlings ; 1973
increased number and length of leaves
Dendrobium nobile seeds and IAAb, 0.1, 1, 10 ppm Inhibition Miyazaki and
seedlings Nagamatsu, 1965
Goodyera repens seeds and seedlings IAAb, 1 ppm No effect Hadley, 1970
Miltonia spectabilis 1 g l - 1 to 1 × 10 - 8 g l - 1 Germination not "drastically stimu - Hayes, 1969
lated"; differentiation promoted
Miltonia spectabilis var. moreliana 1 g l - 1 to 1 × 10-8 g l - 1 Germination not "drastically stimu - Hayes, 1969
lated" ; differentiation promoted
Odontoglossum grande 1 g l - 1 to 1 × 10-8 g l - 1 Germination not "drastically stimu - Hayes, 1969
lated"; differentiation promoted
Odontoglossum schlieperianum 1 g l - 1 to 1 × 10-8 g l - 1 Germination not "drastically stimu - Hayes, 1969
lated" ; differentiation promoted
b
Orchis (Dactylorchis) purpurella IAA , 0.25, 0.5, or 1 ppm Germination impeded, elongation of Hadley and
seeds and seedlings protocorms Harvais, 1968
Phalaenopsis ovules NAA, 0.5-1 ppm Growth enhancement Pages, 1971
Platanthera bifolia IAAb, 1 ppm No effect Hadley, 1969
Spathoglottis plicata seeds 2, 4-Db Germination Chennaveeraiah
and Patil, 1973
Vanda cv Miss Joaquim seeds and IAAb, 25, 50, 100 ppm Germination inhibited Goh, 1971
seedlings
Vanda cv Miss Joaquim seeds and 2, 4-Db, 0.1, 0.25, 0.5, 1, 2, 5 ppm Germination inhibited Goh, 1971
seedlings
Vanda cv Miss Joaquim seeds and 2, 4-Db Callus formation after 3 months Goh, 1971
seedlings
Vanda IAAb, 1 ppm "Not very effective in the formation Rao and
of seedlings" Avadhani, 1963
a
For a table of pre-1967 findings see Arditti 1967a.
b
2, 4-D, 2, 4-dichlorophenoxyacetic acid; IAA, indoleacetic acid; IBA, indolebutyric acid; NAA, naphthaleneacetic acid.
462 J. ARDITTI

have affected seed response; and (vi) interactions, due to the fact that several
combinations of hormones with or without other substances, culture condi-
tions and seedlings were employed.
Auxin was the first plant hormone added to orchid cultures (Burgeff,
1934; for reviews see Arditti, 1967a; Withner, 1959,1974). Results varied. In
the majority of cases auxin (mostly IAA, IBA and NAA) enhanced germina-
tion and/or seedling growth to some extent (Fig. 31). Inhibitory effects were
reported in very few cases. The same was true for "no effect" reports. In only
one instance, that of excised Dendrobium ovaries, did death occur in the
absence of auxin (Israel, 1963). More recent reports are also inconclusive
(Table 8).
Orchid pollinia are a rich source of auxin which plays an important role in
the induction of postpollination phenomena (seep. 566 et seq.). However,
only traces of auxin have been found in Cypripedium seeds and none at all in
Calanthe and Dendrobium (Poddubnaya-Arnoldi, 1960; Poddubnaya-Arnoldi
and Zinger, 1961). This and the inc onsistent results obtained with the addition
of auxin to culture media suggest that for the most part germinating orchid
seeds and developing seedlings are self-sufficient with respect to these hor-
mones. Those that require or benefit from an exogenous supply in nature
(however large or small) probably obtain it from their mycorrhizal fungi
(Hayes, 1969; for a review see Arditti and Ernst, 1974). However, since at
least one such fungus is enhanced by the addition of auxin (Downie, 1943), it
is also possible that orchids supply hormone to their symbionts.
Cytokinins have been isolated from several mycorrhizal fungi, even if not
those of orchids (Crafts and Miller, 1974; Miura and Hall, 1973). Hence, it is
reasonable to assume that the mycorrhizal fungi may also produce cytokinins.
If so, it can also be assumed that should any orchid seeds or seedlings require an
exogenous supply of these hormones, in nature this need may be satisfied by
the fungi. Evidence for this in vitro would be the response of different species
or stages of growth to one of several cytokinins in the culture medium. This
indeed is the case: some species are enhanced by cytokinins, others are
inhibited and a third group is not affected (Table 9; for reviews see Arditti,
1967a, c; Withner, 1974). Benzylamino purine (BAP) may retard
development and differentiation of cells and tissues of Cymbidium protocorms
(Gailhofer and Thaler, 1975). The hormone may induce the appearance of
enlarged mitochondria in the light. BAP also brings about an increase in the
number of young chloroplasts and delays their degeneration (Gailhofer and
Thaler, 1975). In some cases the auxin : cytokinin ratio may be important
(Hadley and Harvais, 1968), but the available information is not sufficient
for generalizations.
Germinating seeds are more sensitive to higher cytokinin concentration
than protocorms. This may indicate either that the seeds have lower re-
TABLE 9
Effects of Cytokinins and Related Substances on Some Orchidsa
Orchid Cytokinin Remarks Reference
Cattleya aurantiaca, unripe seeds Benzyl adenine Formation of protocorms not affected by Pierik and
low concentrations Proliferating Steegmans, 1972
protocorms with high concentrations
Number of roots and their fresh weight and
dry weight decreased with increasing
concentrations Number of shoots increased
by high concentrations Fresh and dry
weight of protocorms and shoots decreased
at low concentrations
Coeloglossum viride seeds and Kinetin, 1-10 ppm Germination retarded, growth rate of Hadley, 1970
seedlings protocorms increased
Cymbidium goeringi (Cym. Kinetin, 10 ppm No root formation Ueda and
virescens) shoots Torikata, 1972
Cymbidium insigne shoots Kinetin, 10 ppm No root formation Ueda and
Torikata, 1972
Cymbidium cv In Memoriam Benzyl adenine 0.1 ppm — development retarded Rucker, 1974
Cyril Strauss, protocorm Benzyl adenine 1.0 ppm — formation of roots and root hairs
inhibited
N-Benzyladenosine 10 ppm — differentiation of buds inhibited.
Chlorophyll synthesis inhibited
Kinetin 50 ppm — teratogenic and toxic effects,
chlorophyll synthesis inhibited Mitotic and
endomitotic activity stimulated by all
concentrations
continued
TABLE 9—continued
Orchid Cytokinin Remarks Reference
Differentiation of cell walls occurred
earlier Effects can be reversed by
transplanting protocorms on
cytokinin-free medium
Cymbidium kanran rhizomes Adenine sulphate No effect Sawa, 1969
Cymbidium virescens rhizomes Adenine sulphate No effect Sawa, 1969
Cypripedium calceolus seeds Kinetin or benzyl adenine, 1 ppm Germination enhanced Borriss, 1969
Cypripedium reginae seeds and 6 (γ, γ -dimethylallyl-amino) Harvais, 1973
seedlings purine Kinetin riboside, zeatin Growth impeded No

morphogenetic effects
Dactylorhiza (Orchis) purpurella Kinetin, 1-10 ppm Germination impeded, growth rate of Hadley, 1970
seeds and seedlings protocorms increased
Dactylorhiza purpurella seeds and 6 (γ, γ -dimethylallyl-amino) "Shoot characters" and chlorophyll Harvais, 1972
seedlings purine formation enhanced
Kinetin riboside "Root characters" suppressed
Goodyera repens seeds and Kinetin, 1-10 ppm No response Hadley, 1970
seedlings
Orchis purpurella seeds and Kinetin Pronounced effect on growth and Hadley and
seedlings development Harvais, 1968
Adenine Little effect
Platanthera bifolia seeds and Kinetin, 1-10 ppm Retard germination. Growth rate of Hadley, 1970
seedlings protocorms increased
Spathoglottis plicata seeds and Kinetin, 1 ppm Germination Chennaveeraiah
seedlings and Patil, 1973
Vanda cv Miss Joaquim seeds Kinetin, 2 ppm Some inhibition Rao and
Avadhani, 1963
a
For tables of pre-1967 reports see Arditti, 1967a, c.
TABLE 10
Effects of Gibberellins on Some Orchidsa
Orchid Gibberellin Remarks Reference
Cypridium calceolus seeds and seedlings GA 4, 5 ppm Differentiation enhanced Borris s, 1969
Dendrobium seedlings GA Induced callus formation; reduced percen- Mukherjee et al,
tage of normal seedlings, increased length, 1973
and number of leaves
Dendrobium nobile seeds and seedlings GA, 1, 10, 100 ppm Enhanced rapid germination and plantlet Miyazaki and
formation. Some inhibition at 100 ppm Nagamatsu, 1965
Orchis purpurella seeds and seedlings GA 3, 2.5, 5, 10 ppm Enhanced protocorm survival, caused Hadley and
abnormal elongation of emergent shoots; did Harvais, 1968
not influence the growth and overall rise of
protocorms
Vanda cv Miss Joaquim GA, 500 ppm ". . . to a certain extent inhibitory to . . . Rao and
seedling formation." Avadhani, 1963
a
For a table listing pre-1967 findings see Arditti, 1967a.
466 J. ARDITTI

quirements (if any, although it is hard to imagine that developing embryos do


not need these hormones), can produce all they need, deactivate the hormone
at a slower rate, or combine these three possibilities. Protocorms, on the
other hand, may be unable to produce enough, have the capability for
faster deactivation or combine the three. In any case, the influence of cyto-
kinins on orchid seedlings is in line with their known effects on other
plants.
Basidiomycetes, a group of fungi which includes orchid mycorrhizal sym-
bionts (Smith, 1974; Strullu, 1974), contains species which produce gibber-
ellin-like substances (Pegg, 1973). Hence, it is possible to reason (as with
cytokinins) that those orchid seeds and seedlings which require gibberellins
can obtain them in nature from their mycorrhizae. If so, responses to exo-
genous GA in culture media can be expected to vary with growth stage and
species. And this indeed seems to be the case in several instances (Table 10;
Arditti, 1967a, c; Withner, 1959, 1974). However, the effects of exogenous
gibberellins tend to be negative. Altogether, it seems that with respect to
gibberellins, orchid seedlings can synthesize whatever amounts they may need.
Further, it would appear that the seeds or seedlings may have a limited
ability to deactivate the hormone. Consequently, exogenous gibberellins,
except when added in very small amounts, may raise concentrations in
culture media to supraoptimal levels for most species.
Thyroid (Withner, 1951) and "female" (Schopfer, 1943) hormones have
also been applied to germinating orchid seeds and developing seedlings, but
no growth-promoting effects have been noted.

6. Vitamins
Since the subject has been reviewed several times, twice recently (Arditti,
1967a; Arditti and Ernst, 1974; Arditti and Harrison, 1977; Withner, 1959,
1974) only a brief summary and several additions (Tables 11, 12) will be
presented here.
Ascorbic acid (Vitamin C), biotin, folic acid, inositol (this polyol is
variously classified as a vitamin, cytokinin, or growth factor), niacin, panto-
thenic acid, pyridoxine, riboflavin, thiamine, and vitamins A, B12 , D, E and T
(which can be best described as a termite extract), as well as related sub-
stances such as p-aminobenzoic acid, glutathione and other compounds have all
been tested for their effects on orchid seed germination and seedling growth.
As with hormones, results have been inconsistent (Table 11). In part this may
be due to physiological differences between species or developmental stages.
However, the inconsistencies may also be due to the presence of vitamins as
impurities in sugars or other media components. In other words, basal media
(i.e., controls) presumed to be vitamin-free were not and formulations
presumed to include certain concentrations did in fact contain more. In such
instances the results obtained would be unreliable. Proof that
TABLE 11
Effects of Vitamins on Several Orchids a
Orchid Vitamins Remarks Reference
Cypripedium reginae seeds and Pantothenic acid, 0.5 ppm Brought about increases in leaf size to Harvais, 1973
seedlings Pyridoxine, 0.5 ppm "natural proportions"
Thiamine, 5 ppm
Dendrobium seeds Pyridoxine HC1, 300 ppm Improved germination Mukherjee et al.,
Riboflavine, 300 ppm 1974
Thiamine HC1, 300 ppm
European terrestrial species Niacin, 2 ppm Improved germination Borriss, 1969
Pyridoxine, 2 ppm
Thiamine, 2 ppm
Serapias orientalis 1 tablet of Multivit B 1-1 : Enhanced germination Vöth, 1976
Adermin (= Pyridoxine), 0.5 ppm
Lactoflavin, 1 ppm
Niacin, 10 ppm
Calcium pantothenate, 1 ppm
Thiamine, 1.5 ppm
a
For more extensive tables see Arditti, 1967a.
468 J. ARDITTI

TABLE 12
Summary of Vitamin Effects on Germinating Orchid Seeds and
Developing Seedlings (from Arditti and Harrison, 1977 and Table 11)
Vitamin Effect
Ascorbic acid (Vitamin C) Increased germination and growth in Cattleya and
Oncidium. Promotes embryonic growth in Cymbidium.
Biotin No effects on Cattleya and Epidendrum. Enhances
growth and/or colour in Cattleya, Odontoglossum,
Paphiopedilum, and Cymbidium.
Folic acid Mostly without effects.
Inositol Unsuitable as sole carbon source for Dendrobium and
Phalaenopsis. No effects on Cattleya, Epidendrum, and
Goodyera. Possibly stimulates germination of Cattleya.
Niacin The only vitamin reported to consistently enhance
germination and development of several orchids.
Pantothenic acid Generally without effects.
Pyridoxine Reported to enhance germination or growth, have no
effects or be inhibitory.
Riboflavin Does not seem to stimulate germination, enhances
differentiation of plants at leaf point stage and pro -
motes embryonic growth.
Thiamine The vitamin itself or only its pyrimidine moiety can
enhance germination and growth.
Mixtures of vitamins Improved germination and growth.
(Table 11)

this may be so was obtained from early experiments with niacin (Noggle and
Wynd, 1943).
The available evidence indicates that symbiotic fungi provide orchids with
vitamins or their precursors (Harvais and Pekkala, 1975; Hijner and Arditti,
1973; Mariat, 1948, 1952; Stephen and Fung, 1971; Vermeulen, 1947; for
reviews see Arditti, 1967a; Arditti and Ernst, 1974; Arditti and Harrison,
1977; Withner, 1959, 1974) and possibly other factors (Ueda and Torikata,
1974). When incorporated in orchid culture media, folic acid has been mostly
without effect (Table 12; Downie, 1949; Withner, 1951), but in one case it
did stimulate germination (Mariat, 1948, 1952, 1954). Therefore, it appears
that most orchids are self-sufficient in respect of this vitamin. However,
some mycorrhizal fungi require folic acid or its component moiety,
p-aminobenzoic acid (Hijner and Arditti, 1973; Stephen and Fung, 1971;
Vermuelen, 1947). Arundina chinensis (Stephen and Fung, 1971), Dactylorhiza
(Vermuelen, 1947) and Epidendrum (Hijner and Arditti, 1973) seedlings
produce enough folic or p-aminobenzoic acid to satisfy these requirements.
Niacin is reported to enhance orchid seed germination and seed develop-
ment more consistently than any other vitamin (for reviews see Arditti,
1967a, b; Arditti and Ernst, 1974; Arditti and Harrison, 1977; Withner,
ASPECTS OF ORCHID PHYSIOLOGY 469

1959, 1974). This suggests that a requirement for niacin may exist. Under
natural conditions the need is probably satisfied by the mycorrhizal fungi.
Support for this suggestion is the production and release of niacin into
culture media by two Rhizoctonia isolates, one from Dactylorhiza purpurella
(Harvais and Pekkala, 1975) and another from Cymbidium (Hijner and
Arditti, 1973).
Perhaps the most interesting case is that of thiamine. Several investigators
have reported germination and growth enhancement by thiamine (Table 11,
12; Arditti, 1967a, Arditti and Harrison, 1977; Withner, 1959, 1974). In one
instance it was shown that in addition to the vitamin itself, its pyrimidine
fraction alone was capable of such enhancement (Magrou and Mariat, 1945;
Mariat, 1944, 1948, 1952). Under symbiotic conditions fungi such as Corticium
catonii (Cappelletti, 1947) and a Rhizoctonia species isolated from Cym-
bidium (Hijner and Arditti, 1973) supply orchid seeds and seedlings with
thiamine and its components. In return the fungus, which also requires
thiamine or its thiazole moiety, obtains these substances from the orchid.
This is coevolution on the half molecule level and a good example of nutri-
tionally mutualistic symbiosis.

7. Complex Additives
A large and bewildering number of complex additives have been used in
orchid seed and seedling culture media. Coconut water, yeast extract, peptone,
microbiological preparations (Kukulczanka and Sobieozczanski, 1974) and
casein hydrolysate are among the common ones. Honey, sauerkraut juice,
salep, fish emulsion and beef extracts are prosaic even if somewhat unusual.
Malaysian beer and extracts of silkworm pupae are the most exotic
additives used (Table 13; Arditti, 1967a; Ernst, 1967b; Ernst et al., 1970;
Withner, 1959, 1974). The determination of which complex additive may be
best is more important horticulturally than scientifically. Still, these findings
may be of interest for those engaged in tissue culture and working with a plant
which does not respond to normal treatments.
Fractionations of fungi (Downie, 1949), tomato (Arditti, 1966) and
banana (Arditti, 1968) indicate that in all three the active fraction is insoluble
in water or ethanol (Withner, 1974). This still leaves a large number of
substances to choose from, but at least suggests that distribution of the
active factor(s) is not limited to fungi.
The experiments with bark substrates (Frei, 1973a, b, 1976; Frei and
Dodson, 1972; Pollard, 1973) do not necessarily approximate natural condi-
tions (Sanford, 1974) for at least four reasons. First, bark samples were added
to a culture medium at a pH of 5-5 and autoclaved (Frei, 1973b; Frei and
Dodson, 1972). This may have modified the bark chemically, hydrolysed
and/or extracted substances whic h would not necessarily be soluble in
tree-trunk effluates under natural conditions. In the forest only substances
soluble
TABLE 13
Effect of Complex Additives on Some Orchids a
Orchid Additive(s) Remarks Reference
Canadian native species, seeds Casamino acids, yeast and "Intolerant" Harvais, 1974
and seedlings potato extracts
Calypso bulbosa seeds Potato dextrose agar Best germination Harvais, 1974
and seedlings
Cymbidium seeds and seedlings Banana juice; banana plus Promoted growth of protocorms Kusumoto and
apple juice; banana juice plus Furukawa, 1977
peptone
Cymbidium seeds and seedlings Tomato juice Inferior to growth on unsupplemented Torikata et al, 1965
Knudson C Torikata et al., 1965
Fish extract plus peptone Accelerated growth of protocorms Torikata et al., 1965
Extract of silkworm pupae Stimulated growth
Cypripedium reginae seeds and Casein hydrolysate, yeast "Intolerant"
seedlings extract
Potato extract Beneficial" Harvais, 1973
Dactylorhiza purpurella seeds and Casamino acids Superior germination and growth Harvais, 1972
seedlings Casamino acids plus yeast Further improvement of growth and
extract survival
Dendrobium ovules Banana homogenate plus "Best response" Pages, 1971
indolebutyric acid or NAA
Dendrobium protocorms Banana homogenate with "Best plantlets" Pages, 1971
protease peptone
Banana homogenate plus
coconut wafer and NAA
Dendrobium hybrid seeds and Banana, coconut, tomato, Most suitable Mowe, 1973
seedlings fish emulsion
Epiphytic orchids seeds and Bark from Quercus trees Toxic or inhibitory Frei and Dodson,
seedlings 1972
European native species seeds Coconut water Enhancement Borriss, 1971
and seedlings
Paphiopedilum seeds Peptone ("Fleischpeptone") Enhancement Fast, 1971
or fish meal
Paphiopedilum seedlings Banana Enhancement
Phalaenopsis protocorms Banana Most favourable Ernst, 1967b
Pineapple, fig and tomato Pronounced increase in growth
fruits
Coconut milk Strong proliferation, retarded
differentiation
Grapes and raspberries Retarded growth, toxic
Phalaenopsis ovules Coconut water plus NAA "Best supplements" Pages, 1971
Coconut water plus peptone
Serapias parviflora and S. Yeast with or without peptone "Best addition" Vöth, 1976
orientalis
Vanda cv Miss Joaquim Tomato juice or coconut milk Bigger seedlings formed Rao and Avadhani,
1963
Pollinium extract, casein "Not very effective in the formation of
hydrolysate seedling"
Yeast extract ". . . less effective and to a certain extent
inhibitory . . ."
a
For pre-1967 literature see Arditti, 1967a.
TABLE 14
Effects of Light Intensity, Quality, Photoperiods and Sources of Illumination on
Germinating Orchid Seeds and Developing Seedlingsa

Photoperiod
Orchid (h) Light intensity Light Quality Remarks Reference
Arundina bambusifolia 12 3000 lux Philips "Natural" Germination Mitra, 1971
Brassocattleya 16 Cool white, warm Wide-spectrum Gro-Lux is Halpin and
white Standard better than standard Farrar, 1965
Gro-Lux Gro-Lux or warm white.
Cattleya 16 Wide-spectrum Cool white poorest
Gro-Lux
Cymbidium 400-5000 lux Diffuse daylight Shoots and terrestrial Werkmeister,
roots develop ; forma- 1970a, b, 1971
tion of aerial roots
ceases
Cymbidium 23½ Phytor Development of Homés et al.,
protocorms 1971a, b
Cymbidium "Rhizogenesis" inhibited in Homés et al.,
the dark, but etiolated 1973
shoots are formed
Cymbidium goeringii 8200 ergs cm-2s -1 "White" (Toshiba, Root formation; no root Ueda and
73.000 ergs cm-2s -1 FL 20 SW) formation at 4400 and 4000 Torikata,
Vitalux (NEC FL ergs cm-2s -1 under these 1972
20 BR) lights or with red or blue
illumination
Cymbidium insigne 4000 ergs cm-2s -1 As above plus blue Root formation. Best root Ueda and
and red formation with Vitalux. Torikata,
Red light not as good as 1972
blue
Cypripedium calceolus Germinates in the dark Fast, 1976
Cypripedium reginae Best germination in the Harvais, 1973
dark
Paphiopedilum seedlings Improved growth on Ernst, 1974,
darkened media 1975, 1976
Phalaenopsis Better growth on Ernst, 1975,
darkened media 1976
Serapias orientalis Germination in the dark Vöth, 1976
Terrestrial species Light inhibits germination Stoutamire,
1974
Various species 3000 lux Good for germination Mukherjee
et al, 1974
a
For a review of pre-1967 literature see Arditti, 1967a.
474 J. ARDITTI

in effluates [i.e., a dilute solution of minerals and possibly sugars and amino
acids (Table 2; Curtis, 1946; Sanford, 1974) in cool usually rain, water]
would affect orchid seeds and seedlings. Hence in these experiments the
seedlings may have been cultured on media with limited or no resemblance to
natural conditions and possibly even toxic due to substances generated during
autoclaving by extraction from the bark, through hydrolysis of larger
compounds and/or alteration of existing compounds.
Second, orchid seedlings were usually found in association with lichens
and mosses (Frei, 1973a; Pollard, 1973). This raises the possibility that their
contact with the bark may be indirect.
Third, the effluate which reaches the seedlings may be modified by its
passage through the lichens and mosses. As the water percolates through (or
washes) the bark it picks up solutes (substances which would dissolve in rain
water at ambient temperature). Before reaching the orchids the effluate
passes through the mosses and lichens which may add some solutes and/or
remove others through uptake and/or by acting as ion exchangers. Con-
sequently, it is possible that not all substances leached from bark reach the
orchids.
Fourth, it is possible that in the quadripartite association the primary
relationship which could be influenced by bark substances is the one between
(i) moss, (ii) lichen and (iii) tree. The (iv) orchids may depend on or require
moss or lichen growth to establish themselves. If so, the effects of bark
substances on the mosses and lichens would be the determining factor.
Indeed, "those trees that had the most orchids had the most lichens and
mosses. In each instance when the seedling was removed from the tree, I
found mosses and lichens between the root system of the seedling and the
bark." (Frei, 1973a). Conversely, " . . . it was found that the trees which were
the strongest in inhibition, and, as a result had no orchids, also were lacking
in mosses and lichens", and "the inhibitors in the bark . . . were a factor
only in the growth or lack of growth of moss-lichens." (Pollard, 1973).

8. Light
Requirements for and response to light and/or photoperiods by orchid
seeds vary (Table 14; for a review see Arditti, 1975a). At best, generaliza-
tion can be only tentative due to insufficient information. Still, it is
possible to suggest at present that epiphytic species can germinate both in
the light and dark. However, they seem to require light for the induction or
improvement of shoot and/or root formation. Some terrestrial species behave
similarly (Ueda and Torikata, 1972; Werkmeister, 1970a, b, 1971). Others
germinate best in the dark (Fast, 1976; Harvais, 1973; Vöth, 1976; for a
review see Stoutamire, 1974).
Several species grow and develop better when cultured on darkened media.
This phenomenon was first observed with Cymbidium (Werkmeister, 1970a, b,
ASPECTS OF ORCHID PHYSIOLOGY 475

Fig. 32. Effects of glasswool and charcoal on the development of Paphiopedilum and
Phalaenopsis. (a) Paphiopedilum seedlings grown on Thomale GD basal medium, 200 days
under Gro-Lux illumination, (b) Phalaenopsis amboinensis seedlings grown on Knudson C,
basal medium, 200 days under Gro-Lux illuminations; FW, fresh weight; a, number of
leaves; b, length of leaves; c, width of leaves; d, number of roots; e, length of roots; f,
diameter of roots (Ernst, 1975).

1971) and confirmed with Paphiopedilum and Phalaenopsis (Fig. 32; Ernst,
1974, 1975, 1976). A number of factors can be invoked to explain this effect.
One is that the charcoal contributes microelements. However, an analysis of the
charcoal (Ernst, 1975) shows that this cannot be the case (Table 15). The water
extract of charcoal had no beneficial effect on seedling growth and the micro-
elements found in charcoal are present as impurities of media components.
Another explanation is that the dark medium establishes polarity which
476 J. ARDITTI

TABLE 15
A Semi-quantitative Spectroscopic Analysis of Nuchar C and Its
Water Extract (Based on Weight of Charcoal, Ernst, 1975)
Nuchar C
Element Not extracted, % Water extract, %
Na 0.35 0.344
K 0.17 0.139
Ca 0.034 Trace
Mg 0.048 0.024
Mn 0.020 0.003
Si 0.36 0.24
Al 0.11 0.063
Sn 0.006 0.003
Fe 0.041 Trace
Cu 0.001 0.0007
Zn 0.002 Trace
Mo Trace —
B 0.0004 Trace

enhances the formation of terrestrial roots, reduces or eliminates negative-,


or a-geotrophic effects and improves differentiation (Werkmeister, 1971).
This appears plausible but is not supported by findings that growth on glass
wool (which is white) is better than on darkened agar (Ernst, 1974,1975,1976).
Absorption of growth inhibitors of seedling origin by charcoal has been
proposed as a reason for the improved growth (Ernst, 1974; Werkmeister,
1970b). Again findings with seedling cultures on glasswool do not seem to
offer corroboration. Seedlings supported on glasswool in liquid media grow
better than those on charcoal-containing media. Therefore absorption cannot
be a major factor unless dilution of inhibitors in liquid media is faster than in
agar. The pH of charcoal-containing media is the same as that of controls.
Clearly, then, a more appropriate pH is not a reason for the improved
growth on charcoal-containing media.
The equally good growth on charcoal or glasswool and charcoal suggests
that a plausible explanation for the effects of both is improved aeration. The
presence of charcoal granules (each a miniature sponge as it were) increases
the amount of air in an agar medium; seedlings on a platform of glasswool
are similarly well aerated. In nature seeds germinate and seedlings grow on
rough bark, incompletely decomposed litter, rocks and other debris. Aeration
under these conditions is probably excellent and it is not surprising therefore
that growth in vitro is better when seedlings have plenty of air. Altogether it
seems that the effects of charcoal are not merely due to the exclusion of light.
The inhibitory effects of light which are more prevalent amongst terrestrial
species have been explained as being "part of. . . [a] . . . protective mechan-
ism . .. making it impossible for seedlings to develop at the soil surface
when they would be subjected to drying during the growing period" (Stouta-
ASPECTS OF ORCHID PHYSIOLOGY 477

mire, 1974). If this is so one wonders why seeds of epiphytic species, which
may be subject to the same dangers, do not appear to possess such protection
or have a lesser need for it. In any case light-inhibited species become more
tolerant to illumination as their seedlings develop and form leaves.
There are not enough comparative studies of light quality and the effects of
sources of illumination to allow for generalizations (Fast, 1967; Halpin and
Farrar, 1965; for reviews see Arditti, 1967a; Withner, 1959, 1974).

9. Temperature
The optimal temperature for seed germination of most species is 20-25 °C,
but the range may extend from 6 °C to 40 °C (Arditti, 1967a; Mukherjee et al,
1974; Thompson, 1977; Withner, 1959). Some species may require chilling
(Stoutamire, 1974) or at least tolerate cold storage (Vöth, 1976), but even
these germinate best at 25 °C (Harvais, 1973).

10. p H
All available information indicates that orchid seeds germinate best at a
pH 4.8-5.2 with the range being at least 3.6-7.6 (Arditti, 1967a). Seedlings are
tolerant of low pH and grow well even when the pH is 3.3-3.7 (Ernst, 1967a, b,
1974; Miyazaki and Nagamatsu, 1965). However, media of a pH below 4.5
should not be autoclaved since this may lead to the hydrolysis of the agar and
release of toxic substances. Concern for the maintenance of a proper pH has
led to the formulation of buffers (Surges, 1936; Harrison and Arditti, 1970) or
media (Knudson, 1951; Sideris, 1950; Vacin and Went, 1949) all of which
appear to have been unnecessary.

11. Atmosphere
There are a number of reports that seed germination in airtight containers
is equal or superior to that under conditions of ample gas exchange (for a
review see Arditti, 1967a). More recent work has shown that Cymbidium
protocorms develop roots and shoots in shallow layers of non-agitated liquid
media. If the solution layer is deep, differentiation is inhibited but protocorm
proliferation is increased (Homes et al., 1973). Under nitrogen, differentiation
and growth are inhibited. This report tends to support findings that orchid
seedlings grow better under the improved aeration provided by glasswool
platforms or charcoal incorporation in media (Ernst, 1974, 1975, 1976).
Seeds of the achlorophyllous orchid, Galeola septentrionalis, germinate
only in airtight vessels. Germination is enhanced by an air pressure of 1.8
atmospheres (Nakamura, 1962; Nakamura et al., 1975). The presence of
oxygen and carbon dioxide at concentrations of 5 % (approximately 25 % of
normal) and 8 % (24,000 % of normal) respectively is essential. Ethylene, at
levels ranging from 2 to 8 µl l-1 enhanced germination. In subsequent experi-
ments best germination occurred under 10% O2 (50 % of normal), 6 % CO2
478 J. ARDITTI

(18,200 % of normal) and 84% N (108 % of normal) at a pressure of 1.4


kg cm-2. Tolerable ranges were 5-10 % O2 , 2-10 % CO2 and 1.1-210 kg cm-2
pressure (Nakamura, 1976). Subsequent development was not influenced by
CO2 in the range of 0.8 % and pressure from 1.0 to 1.8 kg cm-2. However, O2
levels were important with the range being 5-20 % and the optimum between
10 % and 15 %.
Galeola septentrionalis is a chlorophyll-free holomyc otrophic orchid which
grows underground. Only its inflorescence appears above the soil. Hence it is
reasonable to assume that if germination occurs underground, the require-
ments would be for high CO2 and low O2 levels. Such conditions have been
reported, with CO2 easily reaching 16 % and 12 % being "quite usual".
Oxygen levels in some soils have been reported to be as low as 1 % (for a short
review see Nakamura et al., 1975). The requirement for increased pressure is
not easy to explain due to ". .. the difficulty of measuring atmospheric
pressure in the soil . . ." (Nakamura et al., 1975).
Clearly, germinating orchid seeds differ in their requirements for atmos -
pheric conditions. This is not surprising in view of the great diversity of
orchids and their adaptations to many different ecological niches.

12. Photosynthesis
Cattleya aurantiaca seedlings cultured on Knudson C (KC) medium
(Knudson, 1946) have detectable chlorophyll 15 days after the start of culture.
Levels of chlorophyll a (Chl a) and chlorophyll b (Chl b) are nearly equal at
that time and remained so for 1-1½ months (Harrison, 1972, 1973, 1977;
Harrison and Arditti, 1978). After this period concentrations of Chl a
increased, reaching a maximum by 180 days. Levels of Chl b remained
unchanged (Fig. 33). On Knudson C without sucrose (KC-Suc) chlorophyll

Fig. 33. Levels of chlorophyll a, chlorophyll b and total chlorophyll in Cattleya


aurantiaca seedlings grown on Knudson C medium with and without sucrose (Harrison,
1973; Harrison and Arditti, 1978).
ASPECTS OF ORCHID PHYSIOLOGY 479

Fig. 34. Chlorophyll a/b ratios in seedlings raised on Knudson C medium with (solid
line) and without (broken line) sucrose (Harrison, 1973; Harrison and Arditti, 1978).

Fig. 35. Ribulose-l,5-diphosphate carboxylase activity in Cattleya aurantiaca seedlings


raised on Knudson C medium with (solid line) and without (broken line) sucrose. The
asterisk denotes values found in leaves of mature plants (Harrison, 1973; Harrison and
Arditti, 1978).

became measurable after 25 days. Levels of Chl a and Chl b were the same and
remained constant (Fig. 33). As a result, the Chl a : Chl b ratio in seedlings
on KC increased whereas that of plantlets on KC-Suc was unchanged
(Fig. 34).
Another component of the photosynthetic apparatus, the enzyme ribulose-
1,5-diphosphate carboxylase (RuDPCase), increased rapidly between the
third and ninth week in seedlings grown on KC and then reached a plateau at
a level equal to that found in mature plants. On KC-Suc, concentrations of
480 J. ARDITTI

Fig. 36. Specific activity of ribulose-1,5-diphosphate carboxylase in Cattleya aurantiaca


seedlings raised on Knudson C with (solid line) and without (broken line) sucrose (Harrison,
1973).

Fig. 37. Photosynthetic and dark fixation of CO2 by Cattleya aurantiaca seedlings raised
on Knudson C medium with or without sucrose (Harrison, 1973; Harrison and Arditti,
1978).

RuDPCase rose slowly by the ninth week and increased slightly thereafter
(Fig. 35). Specific activity of the enzyme reached a maximum after 30 days on
KC and decreased thereafter. On KC-Suc the peak was reached at 60 days,
followed by a decline (Fig. 36).
RuDPCase is the major enzyme involved in the initial incorporation of
CO2 by the Calvin cycle during photosynthesis. Therefore it is not surprising
that the photosynthetic capacity (Fig. 37) and RuDPCase (Fig. 36) increase
simultaneously. Specific activity of RuDPCase rose sharply between the 15th
ASPECTS OF ORCHID PHYSIOLOGY 481

and 40th day. Enzyme levels increased as seedlings were maintained for
longer periods on KC. Hence it seems that production of this enzyme may be
one of several events which occur in seedlings when an adequate supply of
carbohydrates is available, i.e. production of RuDPCase is one biochemical
step towards the establishment of autotrophy, but it requires a source of
energy other than CO2 .

73. Miscellaneous Factors


(a) Morphactins. Protocorm-like bodies develop from shoot-tip ("meri-
stem") cultures of Cymbidium. They develop like protocorms formed during
seed germination (Arditti, 1967a, 1977; Morel, 1974). Therefore, information
obtained from such cultures can be included in this section. Morphactin IT
3456, when applied at concentrations in the range of 0.01-10 ppm to proto-
corm-like bodies stimulates proliferation, but inhibits formation of roots,
shoots and rhizoids. It also causes: (i) deformation of leaves and shoots;
(ii) formation of two to three shoots on one protocorm; (iii) appearance of
". .. secondary . . ." protocorms or leaves (Kukulczanka and Twarda-
Predota, 1973).
(b) Gamma rays. Since orchid seeds and embryos contain fewer cells than
those of most other plants they are convenient organisms for studies of radio-
sensitivity. In one study Dendrobium nobile seeds were irradiated with 10, 20,
30, 40, 60 and 80 KR gamma rays from 137 Cs and 60 Co. Germination rates of
irradiated seeds were lower than those of controls during the initial three
weeks and indirectly proportional to dosage. However within six weeks
". . . the rate in each treatment reached almost constant value . . ." (Miya-
zaki, 1968). Survival rate showed a similar tendency at 10, 20, 30 KR. At
40 KR there was an abrupt drop. Growth stopped at 60 and 80 KR. In
addition, irradiation caused many deformities.
(c) Surfactants. Most nonionic, ionic and amphoteric biodegradable sur
factants are toxic to orchid seedlings at concentrations higher than 100 ppm
(approximately 0.3-0.4 mM). Phytotoxicity cannot be correlated with the
surface tension reduction properties of surfactants. However, a coincidence
does appear to exist between interfacial tension reduction and phytotoxicity
(Ernst et al., 1971b). This is probably because surfactants affect the cyto-
membranes (Healey et al., 1971). Sodium (linear) dodecylbenzene sulphonate
caused severe damage after 4- and 48-h exposure to 1000 ppm (2.9 mM).
Chloroplasts lost membranes and underwent drastic changes in morphology
(Figs 38-40, vs. 41-44); their thylakoids were swollen and osmophilic
granules became evident (Fig. 44). Other effects are the disintegration of
polysomes into monosomes (Fig. 41); plasmolysis (Fig. 44); swelling of
mitochondria (Fig. 44); and dispersion of chromatin (Fig. 43). These ultra-
structural changes have been attributed to emulsification of membrane
lipids and precipitation and dispersion of cell proteins (Healey et al., 1971).
Figs 38-40. Ultrastructure of Phalaenopsis seedlings (Healey et a!., 1971). 38. Internal
organization of chloroplast and nucleus with well-developed internal membranes and no
starch, × 22,800. 39. Portion of cell from presumptive mesophyll with a chloroplast dis-
tended by a large starch deposit and an extensive vacuole, × 13,800. 40. Polyribosomes, ×
53,200.
Figs 41-44. Ultrastructure of Phalaenopsis cells from seedlings treated with 1000 ppm
(2.91 mmol) sodium (linear) dodecylbenzene sulphonate (Healey et a/., 1971). 41.
Polysomes dissociated into component monosomes (arrows), after a 4 h exposure, ×
38,000. 42. Chloroplast showing extensive swelling of thylakoids (T) and the indistinct
nature of the outer envelope brought about by a 4 h treatment, × 38,000. 43. After 48 h
chromatin is dispersed throughout the nucleus (N) rather than being concentrated near the
nuclear envelope; Chloroplasts (C) are cup shaped and contain osmophilic dense granules at
their margins, × 4840. 44. Plasma membrane (arrows) remains intact after 48 h treatment,
but plasmolysis is extensive and mitochondria (M) are swollen, × 14,400.
484 J. ARDITTI

14. Development
Orchid seed lacks a radicle, hypocotyl, cotyledon, epicotyl and plumule
(Rangaswamy, 1968). In most species the mature embryo is a mass of cells
without any distinct histogens except the epidermis (Rao, 1967). On agar
media seeds swell and rupture the testa within 10-60 days (Rao, 1967;
Vanséveren-Van Espen, 1971). In Arundina, Calopogon, Dendrobium and
Spathoglottis (for a review see Rao, 1967) only a few apical cells divide to
form a promeristem. Almost all cells divide in Bromheadia and Taeniophyllum.
The promeristem gives rise to a shoot apex and structures which may be
homologous to cotyledons (Goro Nishimura, unpublished work in collabora-
tion with Drs E. A. Ball and J. Arditti).
A tunica and corpus are clearly identifiable in shoot apices (for a review
see Rao, 1967). The tunica is always one-layered and the corpus may have
three to four layers. Just before development of leaf primordia the apex is
dome-shaped. As a result the embryos turn into the top-shaped structures
commonly known as protocorms. In some species (for example, Calopogon,
Cymbidium, Dendrobium, Laeliocattleya, Spathoglottis and Vanda) rhizoids
develop from epidermal cells. These may be long, short, simple or branching.
In Cattleya, leaf primordia appear following the formation of the shoot
apex. Leaves develop next and are followed by roots which are always of
endogenous origin (Vanséveren, 1969). Organogenesis may be inhibited by
immersion of the protocorm in liquid medium—i.e., in the absence of air
(Homes et al, 1971a, b).
The following is cited verbatim with permission (Harrison, 1973, 1977):

"During the first few days of germination the larger basal cells of the embryo
in seeds of Cattleya aurantiaca (Figs 45-50) begin to swell, primarily in a
transverse direction. The first cells to enlarge are not the outer (surface) cells of
the embryo but, instead, the large basal ones located inside. These cells are the
first to be activated and show the earliest ultrastructural changes. Their protein
bodies, when present, become less dense and begin to enlarge and apparently
fuse. The impression is gained that the enlarged (fused?) protein bodies give
rise directly to the cell vacuole as their protein contents are depleted.
Mitochondria are abundant in all parts of the cytoplasm of these cells by the
fifth day of germination. Elongated mitochondria are more numerous around
the nucleus (Figs 47-48). Proplastids are also present but are fewer in number
and are located near the cell wall. Neither dictyosomes nor plasrno-desmata
were observed."
"Whereas the interior basal cells are the first to swell and undergo
ultra-structural changes, adjacent cells often appear as quiescent as they did in
the dry seed. Thus, sharp contrasts in cellular structure exist between
neighboring cells." (Fig. 47).
"After 5 days of germination on KC, many mitochondria were found
adjacent to the nuclei of the cells in the meristematic and basal regions. Numer-
ous lipid bodies were also present and each one was at least partially
en-sheathed by mitochondrial membranes (Fig. 48). The same situation was
observed in 15-day-old seeds sown on KC-Suc. Many of the mitochondria
ASPECTS OF ORCHID PHYSIOLOGY 485

present were cup-shaped or had a bowl-shaped depression in which the lipid


bodies were located. Lipid bodies farther away from the nucleus were still
compressed against each other and were not enveloped by mitochondrial
membranes. Apparently, rehydration occurs first in the region immediately
surrounding the nucleus and the area just inside the cell wall. No glyoxysomes
were evident in 15-day-old seedlings and most of the lipid reserve was still
present."

Figs 45-46. Ultrastructure of orchid seedlings raised on Knudson C without sucrose


(Harrison, 1973). 45. Thick section of a 20-day-old seedling. Small starch grains are visible,
x 393. 46. Basal cell from an 86-day-old seedling, x 3000. Explanation of symbols: N,
nucleus; S, starch grain within a plastid; V, vacuole.

"Utilization of the lipid reserves was not a rapid process but took place over a
one- to two-month period depending on the culture medium used to germin ate
seeds and raise the seedlings. Most of the lipid was used up by 27 days in
seedlings grown on KC (Fig. 49). Protocorms cultured on KC-Suc required
60-65 days to use a comparable amount of reserve lipid. In both cases, lipid
bodies in the smaller cells of the meristematic region and in the interior basal
ones were the first to be used."
"Well-defined granal chloroplasts were apparent in all cells (including the
epidermal layer) of protocorms raised on KC for 15-20 days. The same was
true for seedlings cultured 20-25 days on KC-Suc. Chloroplasts in seedlings
raised on KC accumulated starch rapidly (Fig. 50). Lesser amounts were
stored by the chloroplasts of KC-Suc protocorms." (Figs 45, 46).
"By 87 days, all of the lipid reserves were utilized by protocorms growing on
KC-Suc. Chloroplasts contained small amounts of starch and most cells were
highly vacuolated. Dictyosomes were still not evident."
Figs 47-50. Infrastructure of orchid seedlings raised on Knudson C medium (Harrison,
1973). 47. Basal cell from a five-day -old Cattleya aurantiaca seedling, × 1105. 48. The area
surrounding the nucleus of a basal cell in a five-day-old seedling of Cattleya aurantiaca, ×
8265. 49. Basal cell from a 26-day -old Cattleya aurantiaca seedling grown on Knudson C.
Lipid reserves have been depleted, × 929. 50. Electron micrograph of a basal cell from a
20-day-old seedling, × 1080. Explanation of symbols: L, lipid bodies; M, mitochondria: N,
nucleus; P, proplastid; PV, presumptive vacuole; S, starch; V, vacuole.
ASPECTS OF ORCHID PHYSIOLOGY 487

Development in Vanda is similar (Ricardo and Alvarez, 1971). In this


orchid trichome initials differentiate from epidermal cells. They have few
conventional organelles and exhibit many membrane-bound structures which
contain small crystalline inclusions.

15. Enzymes
As mentioned in the section on carbohydrates (pp. 447-456), orchid seed-
lings secrete a number of enzymes into the culture medium. The production of
ribulose-1,5-diphosphate carboxylase was described in the discussion of
photosynthesis by seedlings (p. 479). There is also no doubt that many
additional enzymes are produced by germinating seeds and developing seed-
lings. Unfortunately, however, only very few of these have been studied.
Peroxidase activity in Vanda seedlings during various stages of develop-
ment was investigated histochemically. Isozymes were studied by means of
electrophoresis (Alvarez and King, 1969). Activity of the enzymes is highest
during the early stages of development and lowest during differentiation.
This is the exact reciprocal of IAA production by seedlings. Hence, the "...
temporal and spectral activity of the enzyme in the developing seedling are in
accord with expectations if this enzyme, in fact, functions to control the level of
IAA . .." (Alvarez and King, 1969). Further, the increase of peroxidase
activity by exogenous IAA indicates that the auxin ". . . is capable of eliciting
activity of an enzyme thought to be involved in its destruction." (Alvarez and
King, 1969).
Cymbidium protocorms produce an acid phosphatase which separates in
three electrophoretic zones, each containing two activity bands. Activity of
an RNase produced by the same protocorms appears in two zones which
differ in intensity. Production of both enzymes is influenced by streptomycin,
but the effects vary with the time of application (Morawiecka, et al., 1973).

16. DNA Production


Growth of Dactylorhiza (Orchis) purpurella protocorms accelerates mark-
edly following mycorrhizal infection. Cortical parenchyma cells of these
protocorms are predominantly 32C, 64C and 128C and new DNA classes are
produced. Asymbiotic protocorms enlarge and differentiate at a slower rate,
but there is no evidence that their nuclei undergo endoreplication (Williamson
and Hadley, 1969). These observations indicate that in D. purpurella
production of new DNA requires fungal infection. Autoradiography follow-
ing 3 H-thymidine incorporation showed that DNA synthesis is induced in
fully differentiated cortical root cells of Spathoglottis plicata following in-
fection by Tulasnella calospora (Williamson, 1970).
DNA content of parenchyma cells of cultured Vanda ovules increases to-
gether with that of RNA and nuclear size. DNA as revealed by Feulgen
staining in nuclei, increases to 8C (Alvarez, 1968, 1969). Hydrolysis time
488 J. ARDITTI

curves of DNA-Feulgen from senescent parenchyma cells were different from


those of meristematic and normal parenchyma cells (Alvarez, 1970). Compari-
sons of acridine orange dye binding with Feulgen measurements indicate that
no "masking" or "unmasking" of phosphate groups occur during cellular
differentiation of Vanda seedlings. Therefore, the increased acridine orange
binding is indicative of higher DNA content (Alvarez and Reyniers, 1970). In
contrast with Dactylorhiza purpurella and other temperate zone terrestrial
orchids, Vanda seeds are easy to germinate asymbiotically. Perhaps this is a
reflection of the differences in the ability of seedlings to synthesize DNA with
or without mycorrhizal infection.
Regular endopolyploidization also occurs in some parenchyma cells of
asymbiotically germinated Cymbidium seeds (Nagl, 1972). These cells show a
disproportionate increase in nuclear DNA content and volume. This dis-
appears following inhibition of DNA synthesis with hydroxyurea. Differen-
tiation of the protocorm is accompanied by DNA amplification and endo-
mitotic nuclear cycles (Nagl et al., 1972; Nagl and Rücker, 1972). Further
evidence that DNA synthesis is required for proper growth and differentiation
has been observed from experiments with hydroxyurea (OHU). Morph-
ological differences became apparent on 10-3 M OHU. At 5 × 10-7 M protocorms
were reduced in size, abnormal and necrotic (Rücker, 1975).
Effects of several plant hormones on Cymbidium protocorms can also be
explained in terms of their influence on DNA content of the cells. Cytokinins
shifted DNA replication from diploid mitotic cells to polyploid endomitotic
ones. This enhanced premature and abnormal cellular differentiation (Nagl
and Rücker, 1974). Auxins bring about an increase in the AT -rich DNA
fractions, the GC fractions are promoted by gibberellins (Nagl and Rücker,
1976). This differential replication of the AT and GC fractions undoubtedly
affected differentiation.
The AT-rich satellite DNA from Cymbidium nuclei was characterized by
thermal denaturation and ultracentrifugation. It is a rare instance of a major
AT fraction in plants, and limited to Cymbidium at present, not having been
isolated from any other orchid (Capesius, 1976; Capesius et al., 1975).
Staining with quinacrine, 4'6-diamidino-2-phenylindole and by the Giemsa
technique indicate that this DNA is located within the centromere chromatin
(Schweizer and Nagl, 1976). The available evidence suggests that the AT -rich
DNA is sensitive to hormone treatment (Capesius et al., 1975; Nagl and
Rücker, 1976). Appearance of the AT -rich satellite in Cymbidium is cor-
related with a hormone-dependent high, but variable, amount of
hetero-chromatin. However, it appears that DNA amplification is restricted
to the non-AT-rich component of the chromatin (Schweizer and Nagl, 1976).
The effects of hormone treatments and the different amplification of the two
DNAs indicate that they play important roles in the growth and differentia-
ASPECTS OF ORCHID PHYSIOLOGY 489

tion of Cymbidium protocorms. What may be happening in other orchids is


not clear at present.
F. SYMBIOTIC GERMINATION

In nature orchid seeds germinate after being infected by a fungus. This was
discovered nearly 80 years ago, but the process is still not understood very
well, despite its fundamental (Smith, 1974) and practical importance (Blowers,
1966; Blowers and Arditti, 1970).

1. History
Orchid mycorrhizae (the term was coined by Frank in 1885) were ap-
parently seen but not recognized by H. F. Link as far back as 1824. In 1840
he presented unclear graphical evidence of fungi in Goodyera repens root
cells. The "bysoid substance" in roots of Monotropa hypopitys (Ericaceae)
was recognized as being a fungus by Reissek in 1842. Four years later he
suggested the presence of a fungus in the roots of several orchids including
Neottia nidus-avis. Schleiden (of cell theory fame) saw the fungus in 1849, but
apparently failed to grasp its significance. During the next 50 years there
were several descriptions of the fungus and clump formation in roots. How-
ever its importance was not appreciated until Noel Bernard noted that
seedlings of Neottia nidus-avis were infected by fungi and perceived its
importance. (For reviews pertaining to information presented above and
below in this section see Arditti, 1967a; Bernard, 1909a; Beau, 1914; Benike,
1910; Burgeff, 1911, 1932, 1934, 1936, 1959; Hadley, 1968; Hadley and
Williamson, 1972; Harley, 1969; Nicolai, 1914; Ramsbotton, 1922a, b, 1929;
Smith, 1974; Warcup, 1975).
Bernard died 11 years after his initial discovery, but he still managed to
perform a number of critical experiments and explain the role of mycorrhizal
fungi in orchid seed germination. His findings were later extended by a
number of investigators including Hans Burgeff in Würzburg (a series of
books and papers from 1909 to 1959), Dorothy G. Downie, University of
Aberdeen (a series of articles in the Transactions and Proceedings of the
Botanical Society of Edinburgh between 1940 and 1959) and John T. Curtis,
University of Wisconsin (1936-1939). More recently laboratories which de-
vote a major effort to orchid mycorrhizae include those of Geoffrey Hadley at
the University of Aberdeen, Scotland who is studying the physiology and
ultrastructure of orchid germination and mycorrhizae; Gaetan Harvais at
Lakehead University, Canada, who is working on physiological and nutri-
tional problems; Sarah E. Smith at the University of Adelaide, investigating
physiology, ecology and translocation; and J. H. Warcup, also at Adelaide,
who is concerned with mycorrhizal fungi and factors which affect symbiotic
germination.
490 J. ARDITTI

2. The Fungi
The mycorrhizal fungi of orchids vary considerably in their identity,
physiology and ecology although they nearly all fall into a non-sporing
group known as rhizoctonias.
(a) Identity. Almost all fungi isolated from orchids have been assigned to
the form genus Rhizoctonia (Arditti, 1967a; Burgeff, 1959; Hadley, 1968;
Smith, 1974; Warcup, 1975). The first three isolates were named Rhizoctonia
repens, R. mucoroides and R. languinosa (Bernard, 1909a; Burgeff, 1959).
Subsequent isolates were designated as Mycelium radicis followed by the
orchid name (M.R. Thrixspermum arachinites, for example). For a while
these fungi were even assumed to be a separate group and named Orcheomyces
(Burgeff, 1909, 1911, 1932, 1934, 1936). However, this classification did not
persist. Another classification error which was dispelled (Gallaud, 1904)
assigned orchid mycorrhizal fungi to Fusarium. A strain isolated from Orchis
mascula was named Corticium masculi (Sprau, 1937). It was subsequently
transferred to the nomen nudum Sistotrema (Trechispora) brinkmannii, but
may not even be an orchid endophyte (see Warcup, 1975).
Species bearing clamp connections have also been isolated from orchids.
These fungi include Corticium catonii, Corticium octosperum (Sistotrema
brinkmannii), Marasmius coniatus var didymoplexis (Didymoplexis minor)
and strains from Corallorhiza innata, Epipogum aphyllum and Gastrodia
sesamoides. However, uncertainties exist regarding the Corticiaceae as orchid
endophytes. Several experiments with rhizoctonias which belong to this
group and a number of orchids have produced tentatively negative results
(Burgeff, 1959; Warcup, 1975).
Hymenomycetous mycelia without clamps have been isolated from Epipo-
gum nutans, Galeola hydra (the fungus is Fames), G. septentrionalis (the
fungus is Armillaria melled) and G. javanica (Burgeff, 1959; Warcup,
1975).
A culture isolated from Calanthe discolor was identified as belonging to the
Hymenomycetes by the characters of its mycelium (Tokunaga and Nakagawa,
1974). Other basiomycetes isolated from orchids include Marasmius coniatus
and Xerotus javanicus (Smith, 1974). Three different strains of Ascomycete
rhizoctonias have been isolated from Pterostylis species in Australia. However,
there are "... few data on whether [they] are orchid symbionts . . ." (Warcup,
1975). Recently the perfect states of several orchid fungi have been
established (Table 16).
In Hokkaido, Japan, 54 fungi were isolated from 20 orchid species. They
were Ceratobasidium cornigerum, Rhizoctonia repens, R. solani (bi- and
multi-nucleate) and other rhizoctonia. The first two were the most common
(Nishikawa and Ui, 1976).
(b) Physiology and metabolism. Appropriate carbon sources for orchid
fungi are sugars such as sucrose, glucose, fructose, maltose, mannose,
ASPECTS OF ORCHID PHYSIOLOGY 491

galactose, xylose and arabinose. Glycosides may be split and their sugars
utilized. In addition, starch, cellulose, wood, lignin and pectins can serve as
carbon sources (Burgeff, 1936, 1959; Hadley, 1969; Hadley and Perombelon,
1963; Harvais and Raitsakas, 1975; Perombelon and Hadley, 1965; Smith,
1966, 1974; Warcup, 1975). Fungi which can utilize pectin as a carbon source
"have been shown to produce endopolymethylgalacturonase, protopectinase
and endopolygalacturonase (Hadley and Perombelon, 1963; Perombelon
and Hadley, 1965). Other orchid fungi produce cellulase (Smith, 1974).
Therefore, it would not be surprising if orchid fungi were found to produc e a
variety of other hydrolytic enzymes. These enzymes would enable the fungi to
break down macromolecules found in the soil debris on which they live. Indeed,
some orchid endophytes are soil saprophytes, others may be parasitic on a
variety of hosts. Examples are Armillaria mellea, Ceratobasidium cornigerum
(Rhizoctonia goodyerae repentis) and Thanatephorus cucumeris (Rhizoctonia
solani).
Orchid endophytes require all the usual minerals and since they can take
them up from culture media there is every reason to believe that the same is
true in the soil. The addition of microelements (solution of Harrison and
Arditti, 1970) may depress growth slightly (Warcup, 1975). This may be a
concentration effect especially since other media components usually contain
some microelements as impurities.
Both ammonium and nitrate are satisfactory nitrogen sources for some
orchid fungi (Burgeff, 1936; Smith, 1974). Others do not grow well on
ammonium as the sole nitrogen source (Hadley, 1977). Fungi of tropical
holosaprophytes grow better on organic nitrogen sources such as urea, amino
acids, peptones, proteins and nucleic acids. Suggestions that some orchid
endophytes can fix or utilize atmospheric nitrogen have been disproved
(Burgeff, 1936; Stephen and Fung, 1971). Some orchid fungi can utilize or
require an exogenous source of amino acids. Two Rhizoctonia strains from
Arundina chinensis grow best on glutamic acid as a nitrogen source. Other
suitable sources are arginine and asparagine. Proline and methionine are un-
suitable (Stephen and Fung, 1971). Asparagine, glycine and urea were good
nitrogen sources for four isolates of Tulasnella calospora whereas glutamine,
arginine and alanine were less suitable (Hadley, 1977). Similarly, Rhizoctonia
repens M32, an isolate from Orchis militaris requires one of four amino
acids for growth in vitro on a denned medium (Table 17).
These reports (Hadley, 1977; Powell and Arditti, 1975; Stephen and Fung,
1971) indicate that at least some orchid fungi may have specific requirements
for certain amino acids. Hence it is possible that the lack of growth on
nitrate or ammonia is due to the absence of a specific requirement rather than
complete inability of the fungi to utilize these ions. Aspartic and glutamic
acid and glutamine have been extracted from fungi symbiotic with orchids,
grown in vitro. However, ninhydrin positive substances could not be detected
TABLE 16
Perfect States of Several Orchid Fungi (Smith, 1974; Warcup, 1975; Warcup and Talbot, 1962, 1963, 1965, 1967, 1971)

Other names or
Perfect state imperfect state Orchid sources Remarks
Ceratobasidaceae
Ceratobasidium cornigerum Rhizoctonia goodyerae repentis Goodyera repens
Pterostylis nana, P. nutans, P. pendiculata
Prasophyllum fusco-viride, P. nigr leans
C. obscurum Acianthus reniformis
C. sphaerosporum Pomatocalpa macphersonii
Robiquetia wesselli
C. sp. 0507
C. sp. E12
C. sp. 0615
C. sp. 0638 Not yet shown to
be symbiotic with
orchid seeds
C. sp. indet. Dactylorchis purpurella
C. sp. Coeloglossum virlde, Listera ovata
Pterostylis mutica
Oliveonia pauxilla Corticium pauxillum Symbiotic with orchid
Heteromyces seeds but not yet
found in plants
Thanatephorus cucumeris Rhizoctonia solani Dactylorchis purpurella Not yet shown to
Corticium solani (Orchis purpurella) be symbiotic with
T. orchidicola Orchis mascula orchid seeds
Coeloglossum viride
T. sterigmaticus Corticium sterigmaticum Thelymitra antennifera
Ceratobasidium sterigmaticum
T. sp. indet. Thelymitra grandiflora
Tremellaceae
Sebacina vermifera Acianthus reniformis Caladenia carnea, C.
dilatata, C. latifolia, C. leptochila, C.
reticulata, Glossodia major Microtis uniflora

Tulasnellales Corybas dilatatus


Tulasnella allantospom
T. asymmetrica Thelymitra luteocilium, T. aristata, T.
epipactoides, T. grandiflora, T. pauciflora,
Dendrobium tetragonum
T. calospora Gleotulasnella calospora Dactylorchis purpurella, Diuris longifolia, D.
Rhizoctonia repens maculata, Acianthus exsertus, Caladenia
reticulata, Thelymitra antennifera, Cymbidium
canaliculatum, Dendrobium sp.
T. cruciata Thelymitra fusco-lutea, T. pauciflora
Acianthus caudatus
T. viola Thelymitra aristata
T. sp. 0632
T. sp. 257 Symbiotic with orchid
seeds but not yet found
in plants
Uncertain
Corticium catonii
Thanatephorus cucumeris Corticium solani Dactylorchis purpurella
or Ceratobasidium
494 J. ARDITTI

TABLE 17
Growth of Rhizoctonia repens M32 on Selected Amino Acids
(Powell and Arditti, 1975)

Dry weight,
Amino acid na mM mg at 25 days DW mM-1 (DW mM-1) n -1
Aspartic acid 4 14.9 152 10.2 2.6
Glycine 2 31.0 158 5.1 2.6
Serine 3 31.0 183 5.9 2.0
Glutamic acid 5 14.9 141 9.5 1.9
Glycine 2 61.2 144 2.4 1.2
Glutamic acid 5 61.2 149 2.4 0.5
Arginine 6 9.2 26 2.8 0.5
Leucine 6 22.6 31 1.4 0.2
a
Number of carbon atoms per molecule of amino acid.

in the culture medium (Harvais and Raitsakas, 1975) indicating no leakage


from the fungus.
Several orchid endophytes require an exogenous supply of vitamins
(Smith, 1974). Thiamine and p-aminobenzoic acid (PABA) are required for
geographically distinct isolates of Tulasnella calospora (Hadley, 1977) and
Rhizoctonia endophytes of Arundina graminifolia (Stephen and Fung, 1971).
On the other hand, isolates of Ceratobasidium cornigerum and Thanatephorus
cucumeris (Rhizoctonia solani) were self-sufficient for vitamins (Hadley, 1977).
Therefore, at least for the present it is not possible to establish patterns for
vitamin requirements by mycorrhizal fungi of orchids.
Folic acid is the actual requirement of the Cymbidium symbiont, but
PABA, a component of the vitamin, enhances growth equally well. This may
be taken to indicate that this fungus cannot synthesize PABA but can in-
corporate it into the vitamin. The same may be true for the four Tulasnella
calospora isolates mentioned above (Hadley, 1977).
The thiamine requirement of the isolate from Cymbidium can be satisfied
by the thiazole moiety of the vitamin. This means that the fungus can produce
only one half of the vitamin molecule but is capable of combining the two
components. Interestingly, and appropriately, both PABA and thiazole are
produced by Cymbidium protocorms (Hijner and Arditti, 1973).
Cymbidium seeds or seedlings require or at least benefit from thiamine or
its pyridine component which is produced by the endophyte (Hijner and
Arditti, 1973). This is coevolution on the half molecule level. Each partner in a
symbiotic relationship produces the vitamin component needed by the
other. As a consequence both can produce the vitamin thereby satisfying
their own needs for the entire molecule and the requirements of the other
partner. Whether the same may be true for Arundina chinensis and its endo-
phyte is not clear. However, this could prove to be the case since endophytes of
Cymbidium (Hijner and Arditti, 1973) and Dactylorhiza purpurella (Harvais
ASPECTS OF ORCHID PHYSIOLOGY 495

and Pekkala, 1975) have been shown to produce niacin and thiamine and
release them into the medium (Harvais and Pekkala, 1975; Hijner and
Arditti, 1973). Both vitamins enhance the growth of orchid seedlings (Arditti
and Harrison, 1977). Therefore ". . . the symbiotized orchid, in agreement
with Harley's view (1969), would obviously derive greater benefit from a
metabolically active endophyte upon its digestion." (Harvais and Pekkala,
1975). And, indeed, at least in vitro, orchid phytoalexins inhibit but do not
kill the fungi. The fungi in turn appear capable of metabolizing the phyto-
alexins (Fisch et al., 1973a). As a consequence the fungi remain alive, for at
least a certain period, and benefit the orchid by releasing vitamins and
possibly other compounds.

TABLE 18
Interrelationships of Orchid Mycorrhizae
Partner Effect Condition
Tree May be damaged by fungus and /or Epiphytosis
orchids which become parasitic
Fungus Benefits from tree Parasite
Benefits from and contributes to Mutualistic
orchid partner
Benefits from debris Saprophyte
Orchid Benefits indirectly from tree parasitized Epiparasite
by fungus
Benefits from and contributes to Mutualistic
fungus partner
Derives indirect nourishment from Parasite on
debris saprophytic
fungus
Debris Broken down and/or utilized by Fungus is
orchid endophyte saprophytic

Orchid endophytes can grow as soil saprophytes or as parasites on other


plants (Smith, 1974). Some may even be parasitic on the supporting trees of
epiphytic orchids (Johanson, 1974, 1977) and bring about a syndrome called
epiphytosis (Ruinen, 1953). Structural connections may be established be-
tween the host plant and epiphyte (Furman, 1959). And, depending on its
state of health, the host may or may not be parasitized as a result (Johansson,
1977). Examples of epiphytosis damage are the killing of citrus and coffee tree
branches by lonopsis utricularioides and Leochilus labiatus (Cook, 1926) and
other epiphytes (Went, 1940). Thus it appears that the orchid endophytes are
both beneficial and detrimental symbionts at the same time. As a result the
tripartite relationship between host tree, orchid and fungus has several
ramifications (Table 18).
496 J. ARDITTI

3. Orchid-Fungus Specificity
Much has been written about the specificity of orchid mycorrhiza (Arditti,
1967a; Burgeff, 1936, 1959; Harley, 1969; Smith, 1974; Warcup, 1975). The
initial belief was that specificity was high (Bernard, 1909; Burgeff, 1909,
1936). However, later work suggested that such specificity does not exist
TABLE 19
Symbiotic Association of Seed of Fourteen Orchids and Strains of
Seventeen Species of Endophytic Fungi (Warcup, 1975)

Dendrobium dicuphum F. Muell

Cymbidium finlaysonianum
T. grandiflora R.D. Fitzg.
Diuris pedunculata R.Br.

Dactylorhiza purpurella
S. cernua (L.) Richard

D. incarnata (L.) Soó


Laelia lobata Veitch
Thelymitra aristata

Spiranthes sinensis

Pterostylis nutans
Cattleya trianae
D. nobile Lindl.

Orchis morio L.
Isolate
Fungus no.
Tulasnella calospora 062 S S + 0 S + S S S S S + S 0
T. calospora 0584 S S + S S + S S S S S S S 0
T. calospora 0689 S S + 0 S S S S S S S S S 0
T. asymmetrica 0497 0 S + 0 + + + 0 0 0 — — + 0
T. asymmetrica 0591 0 S S 0 S + S 0 0 + S S S 0
T. cruciata 0471 0 S + + S + S 0 0 + + + S 0
T. violea 0353 0 S + 0 + + S 0 0 0 S 0 S 0
T. sp. 0632 0 S + + S + S 0 0 Sp a S __ S 0
T. allantospora 0579 0 S 0 0 0 0 0 0 0 0 0 0 0 0
T. sp. 257 0 S —b 0 0 0 0 0 0 0 0 0 + 0
Thanatephorus
cucumeris T35 0 0 0 0 0 0 0 S S S S S — S
Th. sterigmaticus 0708 0 0 + 0 0 0 — S + __ s + + S
Th. sp. 0426 0 0 0 0 0 0 0 0 0 0 + 0 0 0
Ceratobasidium
cornigerum 0167 0 0 0 0 0 0 + S S S S S S S
Ceratobasidium
cornigerum AD14 0 0 0 0 0 0 + S S S S + + S
C. sphaerosporum 0657 0 0 0 0 0 0 0 S S S S S 0 +
C. sp. 0507 0 0 + 0 0 0 0 + S S S S + S
C. sp. 0615 0 0 0 0 0 0 0 + — S — S +p s
C. sp. E13 0 0 0 0 0 0 0 + + + + + + +
C. obscurum 08 0 0 0 0 0 0 0 0 0 +p +p 0 0 0
Oliveonia pauxilla T330 0 — 0 0 0 0 0 0 0 — 0 0 + 0
a
Pathogenic to some/all seed.
b
No test.
Explanation of symbols: +, greater germination than inoculated seed, but no shoot
differentiation; 0, no germination beyond that of inoculated seed on the medium; S, form-
ation of a shoot.
ASPECTS OF ORCHID PHYSIOLOGY 497

(Curtis, 1937; Knudson, 1929). More recent work points to different levels of
specificity (Smith, 1974). Orchids and fungi may ". . . differ markedly in the
range of partners with which they form effective symbiosis ..." (Table 19),
(Warcup, 1975). In Japan there was "... no relation among species of
orchids, rhizoctonias and localities. In the inoculation of Orchis aristata
Fish, with 35 different isolates of rhizoctonias, the symbiotic infection of the
root was observed except R. solani and multinucleate Rhizoctonia which
caused necrosis of root cells. C. cornigerum, R. solonis and binucleate
Rhizoctonia tested caused the damping off of spinach, radish and cucumber
seedlings." (Nishikawa and Ui, 1976).
Some groups of orchids may be associated with one or a limited number of
fungi. For example the allied genera Caladenia, Glossodia, Elythranthera and
Eriochilus (all from Southern Australia) are associated with Sebacina vermin-
fera. On the other hand Diuris is associated with Tulasnella calospora (Warcup,
1971). Pterostylis species were stimulated to germinate only by
Ceratobasidium cornigerum and Diuris taxa are limited to Tulasnella calospora
(Rhizoctonia repens). Thelymitra seeds germinate well with several species of
Tulasnella, but poorly when infected with Ceratobasidium cornigerum
(Warcup, 1973).
A number of Canadian species responded differently to several fungi, but
"only one good symbiotic association was established. It was between
Goodyera oblongifolia from British Columbia and Rs 10, a rice pathogen from
Malaysia" (Table 20; Harvais, 1974).
This may be interpreted to suggest lack of specificity. There was also no
specificity between 15 Rhizoctonia isolates and a number of orchids (Tokunaga
and Nakagawa, 1974). Symbiosis tests between orchids from different areas
and 32 Rhizoctonia isolates ". . . showed no evidence of any species to species
relationships between orchid and fungus." (Table 21; Hadley, 1970).
Altogether, the available information does not support the concept of
strict specificity even if it does not exclude the possibility of preferential
association. Several factors may control interaction between orchids and
fungi. Seeds or protocorms may remain uninfected or the host could contain
and/or eliminate the fungus (Fig. 51). Another possibility is that the fungus
may parasitize and eventually kill the protocorm during one of several stages
(P, sP, SP and SSP in Fig. 51). Successful symbiosis occurs when infection is
compatible (s, S, SS in Fig. 51; Hadley, 1970). This series of alternatives is
logical and to some extent supported by the available evidence. Unfortunately,
however, the basic factors which control it are not entirely clear.

4. Structure and Ultrastructure


Living epidermal hair cells (Williamson and Hadley, 1970) and basal cells
of seeds (Burgeff, 1959) are the sites of infection, the fungus penetrating by
single hyphae which may also grow out of the cells (Fig. 52). Following in-
TABLE 20
Interactions Between Rhizoctonia Isolates and the Seeds of Nine Orchid Species After 7-10 Months in
Dixenic Culture on a Mineral Dextrose Medium, at 25° C (Harvais, 1974)
Rice ?
Orchid endophytes pathogen
Pellicularia
Orchid species E3 E7 PB47 Thrl T Rs10 chordulata
Calypso bulbosa nt nt 0 P P P 0
Corallorhiza maculata nt nt 0(P) P P P 0
Cypripedium reginae P 0 0(P) P 0(P) P 0
Epidendrum obrienii nt nt p P P P 0
Goodyera oblongifolia (0)sP (0)sP 0sP (0)P 0sP s(P),(S) 0
G. repens var. ophioides 0P,0sP 0 0 P P P 0
Habenaria dilatata nt nt P P P,(0sP) P,(0P) 0(P)
H. obtusata (0)(s)P P P P P P (0)P
H. psy codes nt nt 0 P 0 P 0
NOTE: Key to the symbols used: 0—no infection, seeds or protocorms healthy; P—parasitism with no signs of control by the host; s—controlled
infection, pelotons formed, but no growth stimulation; S—cf. s but with growth stimulation, symbiosis; nt—not tested. Combined symbols indicate
that one condition follows the other, e.g. 0SP means no infection of seeds or young protocorms, followed by controlled infection with growth stimulation,
followed by parasitism. Symbols in parentheses indicate a small proportion of the population in those categories.
TABLE 21
Summary of the Interactions Between Protocorms of 10 Orchids and 32 Rhizoctonia Isolates (Hadley, 1970)
Fungi

Laeliocattleya cv. Dee Dee


Cymbidium canaliculatum
Dactylorhiza purpurella

Epidendrum ibaguense
Coeloglossum viride

Spathoglottis plicata
Goodyera repens

E. obrienianum
E. nocturnum

E. radicans
Ceratobasidium cornigerum Rgr 0 s ss s(S) 0 — 0 s 0 0
C. cornigerum Thrl p SSP sP P P P s(sP) P 0(P)
C. cornigerum 0393 (S) ss 0 s S 0 — S 0 —
C. cornigerum 0479 s s s s s — — — — —
C. obscurum 08 p P — S P s P
C. sp. indet Fl S SSS ss SS S — ss SSS — —
C. sp. indet Cs4 s* SSP s SS S — — S — —
Thanatephorus orchidicola Del sP sP P P 0 — — (P)
T. orchidicola S2 P P 0 s* — 0 — — 0
T. sterigmaticus 060 s ss s 0 0 s(S) s 0 0(s*)
T. cucumeris 0269 sP SSS S s(S) 0 0 — s 0 0
T. cucumeris Rsl s* SSP s P 0 0 — — (P)
T. cucumeris Rs6 0 sP 0 s* — 0 — — —
T. cucumeris Rsl2A 0 SP 0 0 — 0 P — — 0
T. cucumeris Rs91 s* SSP s 0 — — 0
T. cucumeris W16 p P P P — — s(sP) P s*
T. cucumeris W48 s* SSS SS s* — 0 — 0 0 0
T. cucumeris W82 S SSS s s* — — s 0 0
T. cucumeris W87 P P 0 P 0 — — 0 0 s*
continued
TABLE 21—continued

Laeliocattleya cv. Dee Dee


Cymbidium canaliculatum
Dactylorhiza purpurella

Epidendrum ibaguense
Coeloglossum viride

Spathoglottis plicata
Goodyera repens

E. obrienianum
E. nocturnum

E. radicans
Fungi
Tulasnella calospora Amo4 0 SS s S SSS — SS SSS S S
T. calospora Pb47 s SS s SS SS — SSS S SS
T. calospora RrA S SS s SS SSS S SS SSS S SS
T. calospora 0388 s(S) SS s SS S 0 SS SS 0 S
T. cruciata 0296 0 sP 0 s 0 — s 0 s
Rhizoctonia solani Rs51 s* s s s — — s 0 0
R. solani S s* sP s s* — — — — 0
R. solani Rs16 s* SSP SSP s* — 0 (s) — —
R. solani Rs94 0 SSP 0 0 — 0 — — 0 0
R. sp. Rs102 ss SSP S 0 P SP (P)
R. sp. Sp1 s* P 0 s* P — — —
R. sp. T s* SS SS s s P SS — —
R. sp. Rs10 s* sss SSP s* s — s s*
0, No infection, protocorms healthy, s, Compatible infection but no growth stimulus seen. If symbiosis does not develop, this condition is analogous to
s*. s*, Infection followed by death of hyphae, sometimes seen as hypersensitivity reaction; no growth stimulus. S, Growth stimulus; symbiosis. P,
Parasitism without any evidence of a compatible phase. sP, Parasitism following compatible phase, usually resulting in death of protocorms. SP, SSP,
Parasitism after a symbiotic phase ("breakaway parasitism"). —, Inconclusive result; protocorms often moribund or dead due to dense growth of fungus
but no infection seen.
Symbols in parentheses indicate that a small proportion of the population was in this category.
ASPECTS OF ORCHID PHYSIOLOGY 501

Fig. 51. Possible pathways in the development of the orchid-fungus interaction (Hadley,
1970).

fection the fungus forms hyphal clusters called pelotons (Fig. 53a; Hadley,
1975). Young intracellular hyphae are circular or elliptical in section and
may stain densely (Fig. 53b). Older hyphae may be empty (Hadley et al.,
1971). The hyphae are thinly enveloped by the host cytoplasm and are sub-
sequently surrounded by an encasement layer (Fig. 53c; Hadley, 1975). In
other words the hyphae are enveloped with invaginated plasmalemma which
continues to produce pectin and cellulose (Nieuwdorp, 1972). The result in
Dactylorhiza purpurella is a combination of host cytoplasm and organelles,
host plasmalemma, encasement material and the fungal hypha (Fig. 53d).
The plasmalemma of the host is usually in contact with the encasement layer
(Hadley, 1975). A similar arrangement has been reported for Corallorhiza
trifida (Nieuwdorp, 1972) and Dactylorhiza maculata (Strullu and Gourret,
1974). In Dactylorhiza purpurella well developed pelotons are dens ely packed
despite the existence of spaces between adjacent hyphae (Hadley, 1975). A
double cell wall surrounds the fungal cells in Ophrys insectifera (von Hofsten,
1973). During later stages the hyphae start to degenerate, collapse (Fig. 53e),
become disorganized and are digested by the host. The digestion of the
hyphae is accompanied by a high oxygen uptake (Blakeman et al., 1976).
Fig. 52. (a, b) Infection of epidermal hairs of Dactylorhiza purpurella by the fungus
Thanatephorus cucumeris. (c, d) Emergence of fungal hyphae from the tip of an infected
epidermal hair, (a, b) x 750; (c, d) x 1500 (Williamson and Hadley, 1970).
ASPECTS OF ORCHID PHYSIOLOGY 503

At this stage the encasement layer may thicken, become more granular and
assume a reticulate appearance (Fig. 53f; Hadley, 1975). When degeneration
of hyphae is complete or nearly so they aggregate into clumped masses of
digested material (Fig. 53f; Borriss et al., 1971; Dorr and Kollman, 1969;
Hadley, 1975). A release of acid phosphatase by both hos t and fungal cyto-
plasm may be correlated with the digestion of these hyphae (Williamson,
1971). Esterases, on the other hand, may be associated with fungal growth
and differentiation (Williamson, 1973).

5. Physiology
The relation between orchids and their fungi is nutritive conjunctive sym-
biosis (Arditti and Ernst, 1974). It has been described as ". . . balanced
symbiosis . . . [but] . . . part of the time the plant acts as a parasite on the
fungus." (Mejstrik, 1970). Infection of protocorms induces DNA synthesis
(Williamson, 1970) and increases activities of polyphenol oxidase, ascorbic
acid oxidase, peroxidase and catalase as well as higher oxygen uptake and
respiration (Blakeman et al., 1976). Starch disappears from infected regions
of seedlings (Burgeff, 1959) due to enhanced hydrolysis (Hadley and
Williamson, 1971; Arditti, 1967a, 1972a, b, c; Arditti and Ernst, 1974;
Burgeff, 1959; Hadley, 1969; Purves and Hadley, 1975; Smith, 1974). These
events simply reflect the fact that growth and development are initiated by
infection.
Without infection there is no development in the absence of soluble sugars
(for reviews see Arditti, 1967a; Withner, 1959, 1974). Protocorms can
hydrolyse some but not all glycosidic bonds (Ernst et al., 1971a). Con-
sequently, in nature the seeds (and mature plants of saprophytic species)
obviously depend on mycorrhizal fungi for carbohydrates. This has been
demonstrated experimentally (Smith, 1966, 1967; Arditti and Ernst, 1974;
Burgeff, 1959; Purves and Hadley, 1975; Smith et al., 1969; Smith, 1974).
The fungi hydrolyse polysaccharides and take up the resulting
mono-saccharides. In the fungus these sugars are converted into trehalose,
mannitol, fructose, glucose and ribose (Downie, 1949; Smith, 1967). On
being translocated into the orchid the components or trehalose are
incorporated into sucrose (Smith, 1967).
Movement of substances from orchids to fungi was suggested on the basis
of the observation that hyphae grow from infected plantlets into soil (Burgeff,
1959) or carbohydrate-free medium (Smith, 1967; Purves and Hadley, 1975).
Initial attempts to test the hypothesis showed no transport from orchid to
fungus when green seedlings of Dactylorhiza purpurella were supplied with
14
CO2 (Hadley, 1969; Purves and Hadley, 1975; Smith et al., 1969). In
subsequent experiments label from 14 CO2 showed up in the fungal symbiont
of Dactylorhiza purpurella. However, these experiments were inconclusive
because the uninfected seedlings leaked substances into the medium. This
raised the possibility that the label in the fungus may be due to uptake of
Fig. 53
ASPECTS OF ORCHID PHYSIOLOGY 505

these nutrients. Further, there was also a possibility that the label in the
fungus could have originated from dark fixation of 14 CO2 (Purves and
Hadley, 1975). Experiments carried out to resolve these problems (Hadley
and Purves, 1974; Purves and Hadley, 1975, 1976) showed: (i) very little 14 C
movement to rhizomes and none to the fungus indicating that the fungus
does not act as a metabolic sink; (ii) limited movement of 14 C from living
rhizomes to fungi, but considerable transport from killed protocorms suggest-
ing a barrier; (iii) dark fixation by the fungus pointing to the likelihood that
the label originated from this source. The obvious conclusion from these
experiments is that there is no transport from orchids to their fungi.
Several observations that infected seedlings have a higher nitrogen content
led to the suggestion that orchid fungi can fix nitrogen (Wolff, 1927, 1933)
and presumably transport it into the orchid. These suggestions were not in
agreement with more careful work (Beijerinck, 1907; Burgeff, 1909, 1936;
Cortesi, 1912; Hollander, 1932; Huber, 192la, b) and the idea was abandoned
(Harley, 1969; Stephen and Fung, 1971a). The higher nitrogen content of in-
fected seedlings is probably due to fungal transport of nitrogenous substances
into the orchid or digestion of the endophyte by the plant (Arditti and Ernst,
1974; Smith, 1974). Peptide and polypeptide digesting enzymes have been
detected in cells which digest fungal cells (Burges, 1936; Fuchs and Ziegen-
speck, 1924). Another possibility is that the infection enhances seedling
metabolism and consequently nitrogen assimilation.
In so far as is presently known orchid seedlings do not require specific
nitrogenous substances or amino acids. Some of their fungi do (Powell and
Arditti, 1975; Harvais and Raitsakas, 1975; Stephen and Fung, 197la).
Therefore it is possible that the fungi obtain these requirements at least in
part from the orchids (Arditti and Ernst, 1974).
Orchid seedlings do not seem to have absolute requirements for vitamins
but may benefit from them (Arditti, 1967a; Arditti and Harrison, 1977;
Withner, 1959, 1974). Several endophytes benefit from or require certain
vitamins and/or produce others (Harvais and Pekkala, 1975; Hijner and
Arditti, 1973; Stephen and Fung, 1971b). Hence, it is possible that exchanges
of vitamins between orchids and their fungi do take place (Arditti and Ernst,
1974; Harley, 1969; Purves and Hadley, 1975; Smith, 1974).
Germinating orchid seeds and developing seedlings appear to be largely
autotrophic with respect to most (or at least non-gaseous) hormones. With one

Fig. 53. Ultrastructure of Dactylorhiza purpurella mycorrhiza (courtesy Dr G. Hadley):


(a) cluster of hyphae (peloton), teased out from orchid cell (× 300); (b) thin section of part of
a peloton with some host cell cytoplasm (× 1500); (c) section of single hypha showing
granular encasement layer, enveloped by host cytoplasm (× 20,000); (d) section of hypha
enclosed in host cytoplasm, with host amyloplast, mitochondrion and (part of) nucleus (×
12,000); (e) collapsed hypha with a thick granular encasement layer (× 25,000); (f) partly
digested hypha with reticulate encasement, being drawn into digested hyphal clump (×
25,000).
506 J. ARDITTI

exception (Downie, 1943), the same seems to be true for endophytes. Traces
of auxin have been found only in Cypripedium, but none was detected in
Calanthe and Dendrobium (Poddubnaya-Arnoldi, 1960; Poddubnaya-
Arnoldi and Zinger, 1961). Thus it seems that interchange of most hormones
between orchids and their fungi is not an important aspect of the symbiosis if
it occurs at all (Arditti and Ernst, 1974). The situation with ethylene may be
different. Ceratobasidium cornigerum (isolated from Pterostylis vittata) and
Tulasnella calospora (from Thelymitra aristata and Caladenia reticulatd), all
fungi which enhance orchid seed germination, have recently been reported to
produce ethylene (Hanke and Dollwet, 1976). This sugges ts the interesting, but
as yet untested, possibility that the germination of at least some orchid seeds
is enhanced by the gas. It is possible even that ethylene is required for
germination as it is by other, non-orchidaceous seeds (Hanke and Dollwet,
1976). The inability of fungal extracts to enhance germination of north
temperate species tends to argue in favour of a gaseous substance (like the
hormone ethylene) which may be provided by a living fungus. Additional
support for this idea is provided by the general failure of efforts to isolate
specific factors which can promote orchid seed germination. The extraction
procedures employed in these attempts were not suitable for the trapping of
ethylene. Therefore, it is reasonable to assume that, if present, the gas prob-
ably diffused out and was not incorporated (at least in sufficient amounts) in
the seed germination media. As this is being written, we are initiating experi-
ments on the effects of ethrel on the germination of several orchid species.
Since both orchids and their fungi can grow axenically on mineral-contain-
ing media it is obvious that they can take up inorganic ions. However, infection
may provide an advantage in this respect since 32 P is translocated into
Dactylorhiza purpurella by Rhizoctonia repens (Smith, 1966; Arditti and
Ernst, 1974).
G. SUMMATION

In as much as orchids and their fungi were compared to Hamlet and the
Prince of Denmark (Ramsbottom, 1922b), it may be appropriate to start this
summation by suggesting (with apologies for the teleological and anthropo-
morphic implications) that orchids are victims of two tragedies:
a. They have fatty seeds which lack the appropriate metabolic machinery
(Harrison, 1973).
b. Their seeds do not have an endosperm and are incapable of directly
utilizing available substrates whereas those substances orchids can use
are not usually available in nature.
Therefore, orchid seeds germinate and seedlings develop only following
fungal infection. As a consequence orchids have evolved a close and intricate
relationship with their fungi and obtain from them carbohydrates and other
substances.
ASPECTS OF ORCHID PHYSIOLOGY 507

Epiphytic orchids (especially those of tropical regions) have the simplest


requirements—only sugars. And, for the most part, that is what the fungi
provide. As a result, these orchids germinate very easily on media which
contain no more than simple sugars and minerals. Examples of this category
include, but are not limited to, Cattleya,Oncidium,Phalaenopsis, Dendrobium,
Stanhopea, Coelogyne.
Orchids of tropical origin which are primarily terrestrial have more exacting
requirements. In the absence of their symbionts, Paphiopedilum seeds, for
example, germinate better on low calcium media which contain fructose as a
carbon source (R. Ernst, personal communication). Australian terrestrial
species, even if not all are of strictly tropical origin, also seem to have rela-
tively specific requirements and are not easy to germinate asymbiotically
(Mclntyre et al., 1971, 1972a, b; Veitch and Mclntyre, 1972; Wrigley,
1973, 1976).
Terrestrial orchids of north temperate origin (including those from Europe
and North America) have the most exacting and specific requirements. Many
of them germinate not at all or very poorly without infection, but the reasons
for this are not understood at present.
Orchid seeds cannot utilize their own fatty reserves, or do so very slowly.
Neither can they hydrolyse large molecules like starch or cellulose. As a
result asymbiotic germination in the absence of sugar proceeds only to the
early protocorm stage. Then the seedlings come to a "resting" stage during
which they survive by utilizing their reserves very slowly, "waiting" as it
were for a source of simple sugars and other requirements. When these are
provided (by fungal infection in nature and appropriate media in the labora-
tory) development continues. Some substances can be taken up directly by (at
least axenically-grown) orchid seeds and seedlings in vitro. Others are
transported into the seedlings by fungi in symbiotic cultures or under natural
conditions following digestion of large molecules.
Transport into seedlings by fungi may occur across living membranes in
some cases, but digestion of the pelotons could be an even more important
means by which the orchid obtains nutrients (Burgeff, 1936, 1959; Hadley,
1975; Purves and Hadley, 1975; Smith, 1974). Digestion of the hyphae is
almost complete (only the wall remains) and this undoubtedly releases all of
the contents into the orchid. Such transfer cannot be called translocation and
favours only the orchid; it has been called "non-reciprocal" (Smith, 1974).
Consequently orchids can be viewed as necrotrophic parasites on their fungus
(Lewis, 1973). The available evidence supports this view, but also provides
indications that some aspects of the relationship may be commensal or
mutualistic (Arditti and Ernst, 1974). Orchid fungi have the potential of be-
coming parasitic and sometimes do. However, in cases of successful symbiosis
they are kept under control, without being destroyed. Phytoalexins (Section III)
undoubtedly play an important role in this respect.
508 J. ARDITTI

The evolutionary origins of orchid mycorrhiza are lost in antiquity and are
now a matter of speculation (Ames, 1948). One possibility is that having lost
the ability to utilize their reserves orchids survived only by developing a
symbiotic relationship with fungi. An argument against this suggestion is the
small chance that the symbiotic relationship would have developed fortu-
itously and soon enough after loss of the metabolic machinery. Without the
development of such a relationship, almost immediately (in an evolutionary
time scale) the orchids could not have survived despite the considerable
longevity of individual plants.
A second possibility is that orchid seeds developed a symbiotic relationship
with fungi before losing the ability to utilize their reserves (i.e., produce
carbohydrates from stored fat) and store carbohydrates. The loss of these
capabilities whether they occurred simultaneously or not, would have little
or no deleterious effect on plants which already depend on or at least benefit
from a symbiotic relationship. The coevolution of orchids and their endo-
phytes (like the relationships between them and their pollinators) is clearly
very successful. If it weren't orchids would not have evolved 20,000-30,000
species in all corners of the globe and in most ecological habitats.

III. PHYTOALEXINS

Compounds which ward off pathogens and are produced by plants following
infection are called phytoalexins. The term and the theory behind it were
proposed by K. O. A. Müller around 1940 (Müller, 1966). Research in the area
gathered momentum slowly and reached the present levels of activity by 1960.
However, phytoalexins were discovered by Noel Bernard as a result of his
work on orchids approximately 35 years before Müller named them (Bernard,
1909b, 1911; for reviews see Arditti, 1968, 1975; Burges, 1939; Magrou, 1924,
1936, 1938; Nobecourt, 1923, 1938; Nüesch, 1963).
A. HISTORY

In his work with orchid mycorrhiza Noel Bernard noted that following
fungal infection of Orchis morio roots by Rhizoctonia repens the bulbs of
these orchids appeared resistant to further fungal infections (Bernard, 1911).
He later proceeded to elucidate the nature of this phenomenon: "Ces plantes
offrent au point de vue de l'immunité un problème assez particulier . . . je me
proposals de chercher la cause, a fin de voir si elle n'avait pas une origine
humorale." To test his theory of immunity he placed bulb tissue from infected
plants on agar and introduced fungi. The fungal hyphae grew in the direction of
the bulb tissue, but stopped one or two centimetres short of reaching it (Fig.
54). Bernard suggested that the inhibition of fungal growth was due to a
diffusible substance produced by the bulb tissue. He then tested the
fungicidal effect of the substance by varying the size of bulb sections in
Fig. 54. The original figures and captions from Bernard's paper which first reported on the
existence of phytoalexins.
510 J. ARDITTI

the experiments. From his data he concluded that orchid bulbs can produce a
diffusible fungal inhibitor.
After Bernard's death the investigation of phytoalexins in orchids continued
in France. His findings were confirmed (Magrou, 1924, 1936, 1938) and shown
to occur in live bulbs only (Nobécourt, 1923, 1938). The phenomenon was
compared by Magrou to the production of antibodies in animals: "Le
phénomène . . . est de tout point comparable àla formation de anticorps . . .
chez les animaux . . .". After that interest in orchid phytoalexins remained
dormant until the chemical defence reaction in Orchis militaris was described
(Gäumann and Jaag, 1945). This was followed by the isolation of an orchid
phytoalexin (before K. O. Müller named them). Orchinol, the first character-
ized compound to which the term is properly applicable, was isolated from
orchids in 1957 (Boiler et al., 1957; Gäumann, 1960, 1963-1964; Gäumann
and Hohl, 1960; Gäumann and Kern, 1959a, b; Gäumann et al., 1950, 1960,
1961; Hardegger et al., 1963a, b, c; Nüesch, 1963; Urech et al., 1963). The
pace of research on phytoalexins was relatively slow in the early 1960s, but
during that period the first conscious isolation of a phytoalexin was carried
out by Cruickshank and Perrin.
A series of investigations of phytoalexins in orchids has been carried out in
my laboratory (Arditti, 1968, 1975; Arditti et al., 1972, 1975; Fisch and
Arditti, 1972; Fisch et al., 1972, 1973a, b). In the meantime, and independ-
ently, work on the synthesis of orchinol was undertaken by A. Stoessl in
Canada (Stoessl et al., 1974). The Canadian group has since extended its
investigations to structure/activity relationships and solubility phenomena
(Ward et al., 1975a, b). Several structural problems are still being investigated
jointly (A. Stoessl, G. L. Rock, M. H. Fisch and J. Arditti, unpublished).
B. CHEMISTRY PRODUCTION AND DISTRIBUTION

Three phytoalexins have been isolated from orchids to date. All are tri-
substituted dihydrophenanthrenes. Orchinol, isolated from Orchis militaris
(Boiler et al., 1957; Gäumann and Kern, 1959a, b), is 2,4-dimethoxy-
7-hydroxy-9,10-dihydrophenanthrene (Fig. 55,1; Fisch et al., 1973a;
Hardegger et al., 1963a, b; Letcher and Nhamo, 1973). Loroglossum hir-
cinum is the source of the other two: loroglossol, 5-hydroxy-2,4dime-
thoxy-9,10-dihydro-phenanthrene (Fig. 55, II) and hircinol, 2,5-dihydroxy,4-
methoxy-9,10-dihydrophenanthrene (Fig. 55, III; Fisch et al., 1973a;
Hardegger, 1963c; Letcher and Nhamo, 1973; Urech et al., 1963). Despite
the similarity in names, loroglossin is neither a phytoalexin nor a dihydro-
phenanthrene, but a bis-p-hydroxybenzyl ester of a complex dicarboxylic acid
(Gray et al., 1976). It was isolated from Gymnadenia conopea, Orchis maculata,
O. incarnata and O. latifolia. Orchinol may be widespread in European
terrestrial orchids although genera as well as species within a genus may differ
in their ability to produce this phytoalexin (Table 22). For example, orchinol
was produced by
ASPECTS OF ORCHID PHYSIOLOGY 511

Fig. 55. Orchid Phytoalexins. I. Orchinol; II. Loroglossol; III. Hircinol; IV. Loroglossin.

all Serapias species tested but by none of the Ophrys. In the genus Loro-
glossum, L. hiricinum (L.) Rich, produced very little orchinol whereas L.
longibracteatum synthesized large amounts (Table 22).
In Orchis militaris incubated with Rhizoctonia repens orchinol production
started 36 hours after initial contact and reached a peak after eight days
(Table 23). At that time total concentration was 920 µg g-1 tissue which
corresponds to a level of at least 0.5 × 10-2 M (Nüesch, 1963). Orchinol
concentration diminishes as the distance between the point of contact be-
tween fungus and orchid tissue increases (Table 23).
Recently a number of phenanthrenes from a laboratory synthesis of
orchinol were assayed for activity (Fig. 56, I-VII; Ward et al., 1975a). The
results (Tables 24, 25) show that several phenanthrenes have high activity.
Some of them are more active at lower levels which may be due to crystalliza-
tion when concentrations are higher. These compounds caused stunting and
distortion of the tubes formed by germinating fungal spores. The cytoplasm of
affected tubes was usually withdrawn from the walls and highly granular when
compared with controls (Ward et al., 1975a).
In fungus-infected Cymbidium, production of an as yet unidentified phyto-
alexin is accompanied by increased levels of sitosterol (SI), stigmasterol (ST)
and campesterol (CA) in a 70 : 25 : 5 ratio (Arditti et al., 1975). In addition,
ergosterol peroxide (EP) was found in these extracts (but see below). This
raises the question of whether Cymbidium phytoalexins may be related
structurally or biosynthetically to sterols. Ours seem to have been only the
second isolation of sterols from orchids. The first isolation, from Cattleya and
Arundina, was of SI, ST and CA, but in different ratios (Wan et al., 1971).
Assays of SI, ST, CA and EP on TLC plates using Cladosporium cucumerinum
indicated that the first three were not very active whereas EP exhibited con-
siderable inhib ition. In liquid cultures of Candida lipolytica all four were
only slightly active, but EP was more inhibitory than the others. The EP
isolated from fungus (Rhizoctonia repens M32)-infected Cymbidium pseudo-
bulbs (Arditti et al., 1972a) is most probably an artefact of extraction and a
512 J. ARDITTI

TABLE 22
Synthesis of Orchinol and of p-Hydroxybenzyl Alcohol in the Bulbs of
Different Orchids Incubated with Rhizoctonia repens,
a Strain From Orchis militaris L.
(Gäumann et al., 1960; Nüesch, 1963)
Relative amounts of:
Host species Orchinol p-Hydroxybenzyl Alcohol
Aceras anthropophora (L.) R. Br. ++ +
Anacamptis pyramidalis (L.) Rich. ++ ++
Charmorchis alpina (L.) Rich. +++ ++
Coeloglossum viride (L.) Hart. +++ ++
Gymnadenia albida (L.) Hartm. a +++ ++
G. conopea (L.) R. Br. ? ?
G. odoratissima (L.) Rich. + +
Loroglossum hircinum (L.) Rich. b + +
L. longibracteatum (Biv.) Moris c +++ +
Nigritella nigra (L.) Rchb.d ++ +
Ophrys apifera Huds. 0 0
O. arachnites (Scop.) Murraye 0 0
Orchis coriophora L. ? ++
O. latifolia L. + ++
O. maculata L. 0 0
O. +mascula L. +++ ++
O. militaris L. ++ ++
O. morio L. +++ +++
O. sambucina L. +++ ++
O. ustulata L. 0 ?
Platanthera bifolia (L.) Rich. 0 0
Serapias lingua L. +++ ++
S. neglecta Not. ++ ++
S. vomeracea Burm. ++ ++
a
Coeloglossum albidum Hartm.
b
Himantoglossum hircinum Sprengel.
c
Barlia longibracteata Parlat = Aceras longibracteata Rchg. = Orchis longibracteata Biv.
d
Nigritella angustifolia Rich.
e
Ophrys fuciflora Crantz.

mixture consisting of 5a, 8a-endoperoxide and 9,1-dehydroergosterol (Fisch et


al., 1973b). In addition the ergosterol itself is a fungal product.
C. ACTION SPECTRUM AND ACTIVITY

Orchinol and hircinol are relatively nonspecific in their effects and can
inhibit a number of fungi and bacteria (Tables 26, 27).
As can be seen (Table 28) all fungi with the exception of Fusarium oxy-
sporum are inhibited and the ED50 is 5 × 10-5 M.
Except for Monilinia fructicola growth is less sensitive than germination.
ASPECTS OF ORCHID PHYSIOLOGY 513

TABLE 23
The Concentration of Orchinol (µg g-l) in the Tissue-cylinders from the Bulbs
of Orchis militaris Incubated with a Rhizoctonia repens Strain from O. militaris
(Nüesch, 1963 from Gäumann and Hohl, 1960)
Time of 2 mm thick discs, numbered from the bottom to the top
incubation
(days) 1 2 3 4 5 6
1 0 0 0 0 0 0
2 28 10 0 0 0 0
5 200 112 15 Traces 0 0
8 920 380 160 50 35 45
12 650 540 460 110 100 80

Fig. 56. Structural formulae of phenanthrenes which have been tested for antifungal
activity: (I) orchinol acetate; (II) dehydroorchinol; (III) dehydroorchinol acetate; (IV)
loroglossol acetate; (V) dehydroloroglossol; (VI) dehydroloroglossol acetate; (VII)
didemethylloroglossol (Ward et al., 1975a).

When tested against powdery mildew on cucumber cotyledons orchinol was


not effective at 1 × 10-3 M (Ward et al., 1975a).
Orchinol at 50 or 100 ppm is considerably more active than hircinol against
Candida lipolytica BY 17. With orchinol, growth is almost completely in-
hibited during the first six days. Following this period, growth (i.e., turbidity of
the liquid culture) increases slowly, but even after 24 days absorbance of
TABLE 24
Percentage Inhibition of Spore Germination of Monilinia fructicola by Orchinol and Related Phenanthrenes and Dihydrophenanthrenes
(Ward et al., 1975a)
Concentration (M × 104)

5 2.5 1.25 0.625 0.313 0.156 0.078 0.039


Orchinol 100r 100r 83rs 51 15 0 0 0
Orchinol acetate 84crs 81rs 98rs 81rs 20rs 13s 0 0
Dehydroorchinol 0cs 0cs 0cs 79c 69c 64rs 22rs 0s
Dehydroorchinol acetate 0c 0c 0c 0c 0c 0c 0c 0c
Loroglossol 35crs 22crs 21rs 0s 0s 0s 0 0
Loroglossol acetate 34crs 25rs 21rs 13rs 10s 0s 0 0
Dehydroloroglossol 0c 0c 0c 0c 0c 29s 10s 0
Dehydroloroglossol acetate 93s 38crs 15crs 18rs 13s 0s s 0
Didemethylloroglossol 81s 50s 14 0 0 0 0 0
NOTE: c crystals and deposit; r rupture of germ-tube tips; s stunting and distortion.
TABLE 25
Percentage Inhibition ofZoospore Germination o/Phytophthora infestans by Orchinol and Related Phenanthrenes and Dihydrophenanthrene
(Ward et al., 1975)
Concentration (M × 104)

5 2.5 1.25 0.625 0.313 0.156 0.078 0.039


r r r
Orchinol 100 100 100 81 40 21 2 0
Orchinol acetate 100cr 100cr 100cr 100cr 95r 57 13 0
Dehydroorchinol 100cr 100cr 100cr 100cr 100cr 100cr 83s 0
Dehydroorchinol acetate 100cr 100cr 100cr 100cr 78cs 0 0 0
Loroglossol 90c 100cr 93crs 92crs 20s 0 0 0
Loroglossol acetate 89c 80c 61 12 0 0 0 0
Dehydroloroglossol 0c 0c 0c 50c 59c 20 16 0
Dehydroloroglossol acetate 58cs 59cs 66 70 59c 20 16 0
Didemethylloroglossol 100 24s 21s 0 0 0 0 0
c
NOTE: crystals or deposit; r rupture of germ-tube tips; s stunting and distortion of germ tubes.
516 J. ARDITTI

TABLE 26
Effects of Orchinol on Several Soil Fungi
(After Gäumann et al., 1960)
Inhibitory concentration
Fungi of Orchinol, molar
Phycomycetes
Mucor spinosus v. Tiegh. 10-3
Pythium de Baryanum Hesse 10-2.5
Rhizopus nigricans Ehrenb. 10-4
Ascomycetes
Alternaria tenuis auct. 10-2.5
Aspergillus clavatus Desm. 10-2
A spergillus flavus Link 10-2
Aspergillus fumigatus Fres. 10-3
Aspergillus niger v. Tiegh. 0
Botrytis cinerea Pers. 10-2
Cladosporium fulvum Cke. 10-3.5
Didymella exitialis (Mor.) E. Müll. 10-3.5
Fusarium culmorum (W. G. Sm.) Sacc. 10-3.5
Fusarium lycopersici Sacc. 10-4
Fusarium martii App. et. Wr. 10-4
Fusarium solani (Mart.) App. et Wr. 10-3
Neurospora sitophila (Mont.) Shear et Dodge 10-3.5
Ophiobolus graminis Sacc. 0
Penicillium citreo-viride Biourge 10-2
Pencillium citrinum Thorn 0
Thielavia terricola (Gil. et Abb.) Emm. 0
Trichoderma viride Pers. 10-3
Basidiomycetes
Rhizoctonia crocorum DC. —
Rhizoctonia Kühn from Pinus silvestris 10-3
Rhizoctonia solani Kühn from Solanum tuberosum 10-3

orchinol-containing cultures is much lower than that of the controls (Fig. 57).
Cultures of Candida lipolytica accelerate following prolonged exposure to
orchinol or hircinol (Fig. 57). This could be indicative of adaptation by the
fungus or degradation of the phytoalexins. Reinoculation of filter-sterilized
media which had been used in a previous assay for 13 days indicate that the
latter is taking place (Fisch et al., 1973a).
Loroglossol (Fig. 55, II) is another phytoalexin isolated from Loroglossum
hirdnum (Hardegger et al., 1963a; Urech et al., 1963). It is sparingly soluble
in water and crystallizes when transferred from ethanol stock solution into
aqueous culture media (Ward et al., 1975b). This reduced its concentration in
several assays leading to reports that it was inactive (Fisch et al., 1973a;
Hardegger et al., 1963c). However, when a dilution series directly in ethanol
was used loroglossol was shown to have antifungal activity of the same order
ASPECTS OF ORCHID PHYSIOLOGY 517

TABLE 27
Minimal Inhibitory and Lethal Concentrations of Orchinol and Hircinol
(Urech et al., 1963)
(Hircinol µg ml -1) (Orchinol µg ml-1)
Ia Da Ia Da
Staph. aureus >500 50 250
Escherichia coli 250 250 500 500
Trichophyton interdigitale 25 50 10 50
Trichophyton mentagrophytes 25 100 50 50
Endomyces albicans 50 100 50 50
Epidermophyton floccosum 100 500 100 100
Microsporum audouini >500 100 250
Sporotrichum schenckii >500 >500
Aspergillus niger 500 >500 100 500
a
I, Inhibition; D, Death.

TABLE 28
Inhibition of Spore Germination and Growth of Fungi with Orchinol
(Ward et al., 1975a)
ED50 (M × 104)
Germination Growth
Fusarium oxysporum f. vasinfectum 1.5 2.4
Glomerella cingulota 0.3 2.6
Monilinia fructicola 0.6 0.6
Phytophthora infest ans 0.4 2.2
Pythium ultimum — 3.5
Thielaviopsis basicola 0.9 —
Venturia inaequalis 0.4 —
Verticillium dahliae 0.6 3.4

(minimum inhibition dose of 10-4 -10-5 M) as hircinol and orchinol (Table 29;
Ward et al, 1975b).
D. BIOLOGICAL ROLE

The orchid dihydrophenanthrenes are probably two of the most con-


vincingly demonstrated phytoalexins. They play important roles in protecting
the orchids from fungal infections and in the establishment of mycorrhiza.
Mechanical injury can induce orchinol formation in Orchis militaris
(Nüesch, 1963) even if at lower concentrations than following fungal infec-
tion. Under natural conditions such induction would render the tubers
resistant to infection by pathogenic fungi. For example, very fast growing
fungi like Fusarium solani can invade and overcome unprotected tissues in a
very short time. On the other hand, tissues which contain even small amounts
of orchinol could inhibit the fungus at least sufficiently to allow
infection-induced phytoalexin production to reach protective levels.
518 J. ARDITTI

Fig. 57. Growth of Candida lipolytica on orchinol 100 and 50 ppm, hircinol 100 and
50 ppm and ethanol 100 ppm and 50 ppm (Fisch et al, 1973a).

TABLE 29
Percentage Inhibition of Spore Germination of Monilinia fructicola and
Phytophthora infestans by Loroglossol
(Ward et al., 1975a)

Loroglossol (M × 10-4)

Fungal species 5.0a 2.5a 1.25° 0.625 0.313 0.156 0.078


c b b b b b b
Monilinia fructicola 21 22 2l 0 0 0 0b
Phytophthora infestansd 90b 100 93b 92 20b 0 0
a
b
Crystals present.
c
Distortion and stunting of germ tubes.
d
Germination was 100% in both water and ethanol controls.
Germination was 98 % in both water and ethanol controls.

Many fungi are unable to invade tissues which contain appropriate con-
centrations of phytoalexins (Nüesch, 1963) or can produce them fast enough.
A good example of this is provided by the fact that, despite a thin cortex and
limited mechanical protection, orchid storage organs rarely rot (Nüesch,
1963). This kind of protection can be overcome only by the destruction of the
phytoalexin and, indeed, fungi which deactivate orchinol rapidly (e.g.,
Rhizoctonia solani) destroy bulbs quickly.
The fungal presence in orchid roots and germinating seeds can be con-
sidered to be a localized, regulated and stabilized parasitism. This enables
orchids to coexist with mycorrhizal fungi some of which are, or may become,
ASPECTS OF ORCHID PHYSIOLOGY 519

parasitic (Kusano, 1911; Knudson, 1929; Arditti, 1967a). A slowly de-


gradable phytoalexin would be the most effective means of regulating such
symbiosis. Compounds which could not be degraded at all or only very
slowly, might inhibit the fungi to the point of destroying the as sociation. If
degradation by the fungus is too rapid the orchid would be parasitized. Indeed
such extremes have been reported (Bernard, 1909; Burgeff, 1936). However,
continuous production of (a) reasonably degradable phytoalexin(s) would
be optimal in that the infection would be kept within tolerable limits without
damaging the fungus; this is the case with orchids and their phytoalexins.
Orchis militaris tissues start to produce phytoalexins within 36 hours after
infection and continue to do so as long as they are alive (Nüesch, 1963). Fungi
(Candida lipolytica for example), on the other hand, can destroy phytoalexins
as has been shown to be the case with orchinol and hircinol (Fisch et al.,
1973a). Or, if we are to adapt Ramsbottom's analogy, it is like preventing the
Prince of Denmark from killing his uncle by restricting him to the outer rooms
of Elsinore Castle (of course this analogy stretches the point a bit since the
orchids or fungi can hardly be compared to the murderous King Claudius).
IV. CARBON FIXATION

Interest in carbon fixation and related topics has its origins in early studies of
orchid biology (Bendrat, 1929; Chatin, 1874-1875; Czapek, 1909; Griffon,
1898, 1899; Lindt, 1885; Magnus, 1890, 1891; Webber, 1920) and nutrition
(Miwa, 1937; Tsuchiya, 1935; Went, 1946). More recent ecological, bio-
chemical, and physiological research has elucidated the pathways of fixation
and their ecological importance (Avadhani and Goh, 1974; Borriss, 1967;
Coutinho, 1963, 1964, 1965, 1969, 1970; Coutinho and Schrage, 1970;
Dueker and Arditti, 1968; Erickson, 1957a, b; Esser, 1973; Goh et al., 1977;
Hew, 1976; Knauft and Arditti, 1969; Kristen, 1965; McWilliams, 1970;
Neales and Hew, 1975; Nuerenbergk, 1963; Rubenstein et al., 1976; Wong
and Hew, 1973, 1975) and provided a basis for the understanding of existing
horticultural applications (Davidson, 1967; Wright, 1967).
A. HISTORY

Neottia nidus-avis is a chlorophyll-free European terrestrial orchid. Often


described as being a saprophyte, it is actually mycotrophic, i.e., a parasite on
its ever-present (Peklo, 1906) mycorrhizal fungus. The plant is yellowish
brown, but white forms have also been reported (Magnus, 1890, 1891). Much
of the early carbon fixation research was carried out with this species.
Chlorophyll was detected in N. nidus-avis more than 100 years ago (Wiesner,
1865, 1871, 1874; Prilieux, 1874) but only chlorophyll a was detected (Henrici
and Senn, 1925; Menke and Schmid, 1976; Montfort, 1940; Montfort and
Küsters, 1940; Reznik, 1958; Senn, 1927). Chlorophyll was also found in
520 J. ARDITTI

Limodorum abortivum, a species related to Neottia nidus-avis (Chatin,


1874-1875; Griffon, 1898, 1899).
In addition to chlorophyll, Neottia nidus-avis also contains xanthophyll
(Menke, 1940; Menke and Schmid, 1976; Montfort and Küsters; Reznik,
1958). The yellow-brown colour which was the subject of several early studies
(among them Lindt, 1885; Wiesner, 1865 and more recently, Reznik, 1958)
is brought about by a shift of the carotenoid absorption into the green. This is
probably due to the binding of carotenoids to proteins as is the case in brown
algae (Menke and Schmid, 1976). Interestingly, this possibility was intimated
in an early study comparing Phaeophyceae and diatom coloration with that of
N. nidus-avis (Molisch, 1905).
Bodies which contain the pigments ("Farbstoffkörperchen") in Neottia
nidus-avis were observed in some of the earliest studies (Lind, 1885; Wiesner,
1865). In subsequent years these bodies were identified as chloroplasts
(Montfort, 1940). With the advent of electron microscopy and development
of other modern techniques these plastids were studied in greater detail. They
were found to consist of coiled, branched and swollen thylakoids with
stroma-like material (Menke and Wolfersdorf, 1968). Br anched thylakoids
have also been found in Aceras anthropophorum (Schmid et al., 1976). In
addition to chlorophyll a, they were also found to contain all plastoquinones
and carotenoids of normal chloroplasts but in different concentrations
(Reznik et al., 1969). The thylakoids were reported to be not fully functional
However, a plastid preparation from the labellum of N. nidus-avis flowers
was reported to ". . . perform a photosystem I-dependent photoreduction of
methylviologen . . . [and] . . . photosystem II-reactions . . . are not function-
ing . . . [and]. . . that no appreciable energy transfer from carotenoids to
chlorophyll occurs." (Menke and Schmid, 1976). Plastids from the labellum of
Aceras anthropophorum were found to contain high carotenoid levels but little
chlorophyll. They can carry out cyclic phenazine methosulphate mediated
photophosphorylation. However, they were not able to perform
photo-system II-dependent photophosphorylation or evolve oxygen (Schmid
et al., 1976).
The studies mentioned above inevitably led to the question of CO2 fixation
by orchids, at that time as a part of the broader aspect of carbon assimilation
by saprophytes (for an interesting, even if older, discussion see Lebedev,
1948). Mycotrophic orchids (at that time called saprophytes by some) like
Corallorhiza innata, Limodorum and Neottia were among the first to be studied
(Griffon, 1898, 1899; Reznik, 1958; Webber, 1920). Others were Listera ovata,
Goodyera repens, Epipactis latifolia, E. rubiginosa, Orchis latifolia, O. purpurea,
O. morio, O. mascula and O. bifolia (Griffon, 1899; Montfort and Kusters,
1940). These studies showed that Neottia nidus-avis and Corallorhiza innata
(mycotrophs) do not fix CO2. Limodorum abortivum (also a mycotroph) fixes
very little whereas all green orchids are capable of carbon fixation.
ASPECTS OF ORCHID PHYSIOLOGY 521

Orchids were among the plants used in the early studies on Crassulacean
acid metabolism (CAM). These studies showed that acidity increased in
thick-leaved (succulent) orchids in the dark (Bendrat, 1929; Warburg,
1886-1888). However, the significance of this observation was not realized
until a quarter of a century later. When research in this area was resumed
(Kristen, 1965; Nuerenbergk, 1961, 1963), modern methods confirmed that
some orchids can indeed take up and fix carbon in the dark by CAM or a
similar pathway (Avadhani, 1963; Hew, 1976; Knauft and Arditti, 1969;
Neales and Hew, 1975; Rubenstein et al., 1976). More recently the possibility
that C4 fixation may occur in orchids has also been investigated (Avadhani
and Goh, 1974). Altogether it seems that orchids may fix carbon by the C3 , C4
and CAM pathways (Table 30).
Orchids are relatively slow-growing plants generally maintained in an en-
closed area, i.e., greenhouses or culture tubes. Therefore, it is not surprising
that attempts to accelerate growth by means of CO2 enrichment of air were
made or suggested some time ago (Miwa, 1937; Tsuchiya, 1935; Went, 1946). A
paper (Frackowiak, 1933) often cited as reporting on CO2 enrichment
attempts, does not even mention carbon fixation. Efforts to accelerate growth
by such means have continued through the years but results have been in-
consistent (Wright, 1967). Perhaps one reason for this is that carbon fixation
pathways of the orchid being fed CO2 were not always considered despite
suggestions that this should be done (Went, 1946; Withner, 1974). Con-
sequently CAM orchids were sometimes given CO2 during the day.
B. STOMATAL RHYTHMS

An indirect method of determining which orchids fix carbon in the dark is to


study stomatal rhythms. Studies of endogenous rhythms involving the "De
Saussure effect" (Nuerenbergk, 1961) led to porometer measurements of
stomatal opening and CO2 uptake. These measurements demonstrated that a
direct relationship exists between stomatal opening and CO2 fixation in the
dark (Kristen, 1965; Nuerenbergk, 1963). Confirmation of these findings was
provided by studies of stomatal rhythms in orchids which showed that the
stomata of Archnis, Aranda and Cattleya (all thick-leaved with CAM) are
open at night. Stomata of thin-leaved (non CAM) orchids like Arundina,
Bromheadia and Spathoglottis are open during the day (Goh et al., 1977).
Information obtained from only six orchids may be considered somewhat
limited and therefore insufficient for general conclusions regarding patterns in
the Orchidaceae, a family with 600-800 genera. However, a number of other
factors suggest that these conclusions are plausible (see reviews by Goh et
al., 1977; Knauft and Arditti, 1969).
C. CRASSULACEAN ACID METABOLISM

Evidence that thick-leaved orchids have CAM (Table 30) was first presented
TABLE 30
Carbon Fixation and Photophosphorylation by Orchids

Orchid Organ Probable pathway Remarks Reference


Acer as anthropophorum Labellum Cyclic photophos- Schmid et a I., 1976
phorylation
Acropera loddigesii Pseudobulbs No CAM Warburg, 1886-1888
Angraecum sesquipedale Leaves CAM Nuerenbergk, 1963
Aplectrum hyemale Leaf No CAM Adams, 1970
Arachnis cv Maggie Oei Leaf (1.5 mm thick) CAM Gohetal., 1977; Lee,
1970; Neales and Hew,
1975
Aranda cv Deborah Leaf (1.5 mm thick) CAM Goh et al., 1977
Aranda cv Wendy Scott Leaf (1.5 mm thick) CAM Hew, 1976; Neales and
Hew, 1975
Aranthera cv James Storie Leaf (1.5 mm thick) CAM Neales and Hew, 1975
Arundina graminifolia Leaf (0.3 mm thick) No CAM; C3 Species Avadhani and Goh, 1 974 ;
photorespires Gohetal., 1977; Hew,
1976; Neales and Hew,
1975; Wong and Hew,
1975
Ascocentrum ampullaceum Leaf (1.27 mm thick) CAM McWilliams, 1970
Brassolaeliocattleya cv Maunalani Leaf Nocturnal CO2 McWilliams, 1970
assimilation and
acidification
Brassavola perrinii Leaf Nocturnal CO, Coutinho, 1964; Szarek
assimilation and Ting, 1977
Bromheadia finlaysoniana Leaf (thin) No CAM; C3 Avadhani and Goh, 1974;
Goh et al., 1977
Bulbophyllum gibbosum Leaf (1.422 mm thick) CAM McWilliams, 1970
Calanthe vestita Leaves CAM Nuerettbergk, 1963
Catasetum fimbriatum Leaves CAM Nuerenbergk, 1963;
Borris, 1967; Goh
et al, 1977
C at t ley a sp. Leaf (thick) CAM Warburg, 1886-1888
Cat t ley a sp. Roots, leaves Fixation in the light Erickson, 1957a, b
Cattleya autumnalis Leaves CAM Coutinho, 1969
Cattleya bicolor Leaves CAM Coutinho, 1969
Cattleya cv Bow Bells Leaf (0.5 mm thick) CAM Hew, 1976; Neales and
Hew, 1975
Cattleya forbesii Leaf CAM Hew, 1976; Neales and
Hew, 1975
Cattleya gigas Roots, stems, leaves Fixation in the light Dycus and Knudson,
1957
Cattleya guttata Leaves CAM Coutinho, 1969
Cattleya intermedia Leaves CAM Coutinho, 1969
Cattleya labiata Whole plants CAM Nuerenbergk, 1961, 1963
Cattleya loddigesii Whole plants CAM Nuerenbergk, 1961, 1963
Cattleya mossiae Whole plants CAM Nuerenbergk, 1961, 1963
Cattleya skinneri Whole plants CAM Nuerenbergk, 1961, 1963
Cattleya trianae Whole plants CAM Nuerenbergk, 1961, 1963
Cattleya walkeriana Whole plants CAM Nuerenbergk, 1961, 1963
Cattleya warneri Whole plants CAM Nuerenbergk, 1961, 1963
Cattleya cv White Blossom
"Stardust" ×
C. cv Bob Betts "Glacier" Whole plants CAM Knauft and Arditti, 1969
Coelogyne cristata Leaves CAM Nuerenbergk, 1961, 1963
Coelogyne massangeana Leaves (plicate) C3 Rubenstein et al., 1976
continued
TABLE 30—continued
Orchid Organ Probable pathway Remarks Reference
Coelogyne mayeriana Leaf (0.4 mm thick) C3 Species Hew, 1976; Wong and
photorespires Hew, 1975; Neales and
Hew, 1975
Coelogyne rochussenii Leaf (0.2 mm thick) C3 Species Hew, 1976; Wong and
photorespires Hew, 1975; Neales and
Hew, 1975
Cymbidium cv Chelsea Sepals, petals, leaves C3 High rate of Arditti and Dueker,
fixation in the 1968; Dueker and
dark Arditti, 1968
Cymbidium chinense Leaves (thin) CAM(?) Warburg, 1886-1888
Cymbidium hybrid Leaves (thin) C3 Rubenstein et al, 1976
Cymbidium cv Independence Day Sepals, petals, leaves C3 High rate of Arditti and Dueker, 1968;
14
C fixation in Dueker and Arditti, 1968
the dark
Cymbidium sinense Leaves C3 High rate of 14 C Wong and Hew, 1973
fixation in the dark
Cymbidium lowianum "Yorktown" Leaves CAM Nuerenbergk, 1961
Cypripedium acaule Leaves (0.406 mm thick) No CAM McWilliams, 1970
Cyrtopodium paranaensis Leaves CAM Coutinho, 1969
Dendrobium taurinum Leaf (1.5 mm thick) CAM Neales and Hew, 1975
Encyclia atropurpurea Leaves CAM Nuerenbergk, 1961, 1963
Encyclia flabellifera Leaves CAM Coutinho, 1969
Encyclia odoratissima Leaves CAM Coutinho, 1969
Epidendrum alatum Leaf (1.397 mm thick) CAM McWilliams, 1970
Epidendrum ciliare Leaves (succulent) CAM Bendrat, 1929
Epidendrum ellipticum Leaves CAM Coutinho, 1963, 1964,
1965
Epidendrum floribundum Leaves CAM Coutinho, 1969;
Nuerenbergk, 1961
Epidendrum moseni Leaves CAM Coutinho, 1969
Epidendrum radicans Leaves (2.159 mm thick) CAM McWilliams, 1970
Epidendrum schomburgkii Leaves CAM Nuerenbergk, 1961
Epidendrum xanthinum Roots, stems, leaves Fixation in the light Dycus and Knudson,
1957
Eulophia keithii Leaf C3 Species Hew, 1976; Wong and
photorespires Hew, 1975
Gomesa crispa Coutinho, 1969
Goody era pubescens Leaf (0.431 mm thick) No CAM McWilliams, 1970
Habenaria platyphylla Whole plant C3 Raghavendra and Das,
1976
Laelia cinnabarina Leaves CAM Coutinho, 1969
Laelia crispa Leaves CAM Coutinho, 1969
Laelia flava Leaves CAM Coutinho, 1969
Laelia millerii Leaves CAM Coutinho, 1969
Laelia perrinii Leaves CAM Coutinho, 1969
Laelia purpurella Leaves CAM Coutinho, 1969
Laelia xanthina Leaves CAM Coutinho, 1969
Lanium avicula Leaves CAM Coutinho, 1969
Limodorum abortivum All parts of the plant Minimal fixation in Probably Chatin, 1874-1875;
light despite presence mycotrophic Griffon, 1898, 1899
of chlorophyll
Maxillaria aromatica Leaves pseudobulb No CAM Warburg, 1886-1888;
Coutinho, 1963
Neottia nidus-avis Plastids from labellum Only PS I is operative; Mycotrophic Menke and Schmid,
whole plant no light or dark species 1976; Reznik, 1958
carbon fixation
continued
TABLE 30—continued
Orchid Organ Probable pathway Remarks Reference
Oncidium sp. Leaves (type unknown) CAM Some Oncidium Warburg, 1886-1880
species have
thick leaves
Oncidium flexuosum Leaves (0.33 mm thick) C3 Species Neales and Hew, 1975;
photorespires Wong and Hew, 1975
Oncidium lanceanum Leaves (0.33 mm thick) Q Hew, 1976
Oncidium pumilum Leaves C3 Coutinho, 1969
Oncidium sphacelatum Leaf (thin) No CAM; C3 Species Bendrat, 1929;
photorespires Wong and Hew, 1975
Ornithidium densum Leaf (thick) CAM Warburg, 1886-1888
Paphiopedilum barbatum Leaf C3 Species Hew, 1973; Wong and
photorespires Hew, 1975
Paphiopedilum insigne CAM Nuerenbergk, 1961
Paphiopedilum cv Mildred Hunter Leaf (soft, leathery) CAM at night, C3 Rubenstein et al, 1976
during day
Paphiopedilum venustum Leaf (0.381 mm thick)
Paphiopedilum villosum Leaf (soft) No CAM Bendrat, 1929
Phalaenopsis Leaf (thick) CAM Borris, 1967
Phalaenopsis Root, stem, leaves Fixation in the light Dycus and Knudson,
1957
Phalaenopsis amabilis Leaves CAM McWilliams, 1970
Phalaenopsis schilleriana Leaves Night CO2 assimilation McWilliams, 1970
and acidification
Pleurothalis ophiocephalus CAM-like δ 13C values Szarek and Ting, 1977
Schombocattleya hybrid Leaves CAM at night; C3 Rubenstein et al., 1976
during day
Schomburgkia Nuerenbergk, 1963
.
Leaves CAM Coutiriho, 1969;
Schomburgkia crispa Nuerenbergk, 1961;
Wong and Hew, 1975
Sophronitis cernua Night CO2 Coutinho, 1969
assimilation
Spathoglottis plicata Leaf (0.3 mm thick) No CAM; C3 Species Goh et al., 1977; Hew,
photorespires 1976; Neales and Hew,
1975
Spiranthes speciosa Leaf (0.508 mm thick) No CAM McWilliams, 1970
Tainia penangiana Leaf C3 Species Hew, 1976; Wong and
photorespires Hew, 1975
Thunia marshalliana CAM Nuerenbergk, 1961
Vanda Root, stem, leaves Fixation in the light Dycus and Knudson,
1957
Vanda cv Miss Joaquim Leaves CAM Khan, 1964
Vanda tesselata Whole plant C2 Raghavendra and Das,
1976
Vanilla aromatica Whole plant CAM Nuerenbergk, 1963
Vanilla fragans Leaf (0.22 mm thick) CAM McWilliams, 1970
Vanilla planifolia Leaf (thick) CAM Bendrat, 1929;
Warburg, 1886-1888
Vanilla sp. Leaves CAM Coutinho, 1969
528 J. ARDITTI

nearly 100 years ago (Warburg, 1887-1888), when it was shown that they be-
came acidified in the dark. The acid which accumulates in orchid leaves is
malate, the same one found in other CAM plants (Avadhani and Goh, 1974;
McWilliams, 1970; for reviews see Arditti and Harrison, 1971; Avadhani,
1963; Benzing, 1973; Hew, 1976; Nuerenbergk, 1963). The list of orchids
which undergo acidification has been extended since then (Table 30) (Avad-
hani and Goh, 1974; Borriss, 1967; Coutinho, 1963, 1964, 1965, 1969, 1970;
Coutinho and Schrage, 1970; McWilliams, 1970; Nuerenbergk, 1963; Szarek
and Ting, 1977).
Orchids which accumulate malate and take up CO2 in the dark are all
thick-leaved and include Cattleya labiata, Encyclia atropurpurea, Schom-
burgkia crispa, Angraecum sesquipedale, Phalaenopsis esmeralda (Nueren-
bergk, 1963), an unidentified Phalaenopsis, Cattleya bowringiana (Borriss,
1967), Vanilla fragrans, Epidendrum alatum, E. radicans, Brassolaeliocattleya
"Maunalani", Ascocentrum ampullaceum and Phalaenopsis schilleriana (Mc-
Williams, 1970). Limited uptake and slight acidification occur in Bulbophyllum
gibbosum and Spiranthes speciosa (McWilliams, 1970). CO2 uptake in the
dark has also been reported in the following thick-leaved orchids: Oncidium
lanceanum, Phalaenopsis amabilis, Aranda cv Wendy Scott, and Cattleya cv
Bow Bells (Hew, 1976).
Evidence from experiments with 14 CO2 indicate the existence of CAM in
Paphiopedilum (see below) and Shombocattleya hybrids (Rubenstein et al.,
1976) as well as Cattleya cv White Blossom "Stardust" x C. cv Bob Betts
"Glacier" (Knauft and Arditti, 1969).
There are no dark CO2 uptake and acidification in thin-leaved orchids
such as Coelogyne cristata, Cymbidium, Paphiopedilum (see below), Castasetum
fimbriatum, Calanthe vestita (Neurenbergk, 1963), Cypripedium acaule, Paphi-
opedilum venustum (see below) and Goodyera pubescens (McWilliams, 1970).
The thin-leaved orchids Arundina graminifolia, Spathoglottis plicata, Tainia
penangiana, Paphiopedilum barbatum (see below), Eulophia keithii, Coelogyne
mayeriana, and C. rochussenii take up carbon in a manner similar to that of
C3 plants (Hew, 1976).
Further confirmation for the existence of CAM in orchids was obtained
from determinations of 13 C : 12 C ratios. During photosynthesis plants take up
preferentially the lighter of the two isotopes. Therefore the 13 C : 12 C ratio
expressed as δ 13 C can be used as an indicatio n of the mechanisms involved in
carbon fixation (Hew, 1976; Lerman et al., 1974; Lerman and Quieroz, 1974;
Neales and Hew, 1975). In orchids δ13 C is positively related to leaf thickness.
The δ 13 C is in the CAM range, i.e., —15 to —16 % for thick-leaved species like
Dendrobium taurinum, Cattleya cv Bow Bells, Aranthera cv James Storie,
Aranda cv Wendy Scott and Arachnis cv Maggie Oei.
When taken together, the available evidence indicates clearly that some
orchids fix carbon via CAM. It is possible even that CAM may occur in
ASPECTS OF ORCHID PHYSIOLOGY 529

thin-leaved genera like Paphiopedilum. According to one report a Paphiopedi-


lum hybrid ". . . is a C3 plant during the day and a CAM plant at night. . ."
(Rubenstein et al., 1976). On the other hand, a Paphiopedilum plant did not
take up CO2 in the dark and was reported to "... have 'normal' photo-
synthesis." (Neurenbergk, 1963). In addition, there was no acidification in
the dark in leaves of Paphiopedilum villosum (Bendrat, 1929) and P. venustum
(McWilliams, 1970). These differences require further research and clari-
fication.
D. C3 PHOTOSYNTHESIS

Thin-leaved orchids fix carbon in the light (Table 30; Adams, 1970;
Arditti and Dueker, 1963; Bendrat, 1929; Dueker and Arditti, 1968;
Erickson, 1957a, b; McWilliams, 1970; Nuerenbergk, 1963; Warburg,
1886-1888). Gas exchange and 14 CO2 fixation patterns and δ13 C ratios have
shown that the following are C3 orchids: Spathoglottis, Arundina graminifolia,
Coelogyne massengeana, C. mayeriena, C. rochussenii, Cymbidium sinensis, a
Cymbidium hybrid, Eulophia keithii, Tainia penangiana, Oncidium flexuosum,
Bromheadia finlaysoniana, Arundina graminifolia and a Paphiopedilum hybrid
(Avadhani and Goh, 1974; Hew, 1976; Neales and Hew, 1975; Rubenstein
et al., 1976; Wong and Hew, 1973). The existence of the C3 cycle has been
interpreted as being ". . . consistent with the primitive nature of terrestrial
thin-leaved orchids . . ." (Hew, 1976). This may be so. However, since some
orchids (terrestrial and epiphytic) are "normal" mesophytes there may be no
particular significance in the fact that they fix carbon via a pathway commonly
found in such plants.
E. C4 PHOTOSYNTHESIS

Plants which fix carbon via the C4 pathway grow best under high light
intensities and warm climates. They use water efficiently, have high photo-
synthetic ratios and may tolerate arid conditions. C4 plants are less common
among dicotyledons than among monocotyledons (Edwards, 1974). Most of
the evidence presented to date suggests that thin-leaved orchids are C3 and not
C4 plants (Hew, 1976). However, some species ". . . exhibit an interesting
mosaic of different types of 14 CO2 fixation including C-3, C-4 and CAM."
(Avadhani, 1963). More specifically there are ". . . indications that young
leaves of Arundina graminifolia may photosynthesize, at least in part via the
C4 pathway" (Avadhani and Goh, 1974), and a Schombocattleya hybrid has
been reported to be "... a C4 plant during the day and a CAM plant at
night . . ." (Rubenstein et al, 1976).
Orchids are not uncommon in habitats suitable for C4 plants, and most of
them have not been studied. Therefore, it would not be surprising if addi-
tional C4 species will be discovered in the future. Such species should prove of
interest not only in terms of orchid biology, but also as subjects for C4
530 J. ARDITTI

research. This is especially so in view of the fact that intrageneric hybrids of


orchids are easy to produce. Therefore it may be possible to produce hybrids
between C3 and C4 , C3 and CAM, and C4 and CAM species.

F. CARBON FIXATION BY DIFFERENT PLANT ORGANS


As in other plants most of the carbon fixation by orchids occurs in leaves. The
rate of carbon fixation in light depends on leaf age in Cattleya (Erickson, 1957a).
However, these findings are difficult to interpret since Cattleya is a thick-leaved
orchid which probably fixes most of its carbon via CAM. Cymbidium leaves (C3 )
fix more CO2 per mg tissue than flowers [Figs. 58, 59 (see p. 549); Table 31;
Arditti and Dueker, 1968; Dueker and Arditti, 1968].
Chlorophyll-containing floral segments, stems and roots, can also photo-
synthesize (Table 31). 14 CO2 fixation by floral segments changes with age (Fig.
59). Leafless orchids like Taeniophyllum zollingeri (Wiesner, 1897), Polyrrhiza
lindenii, Dendrophylax funalis, Sarcochilus luniferus, Doritis taenialis,
Campylocentrum fasciola, C. pachyrrhizum, Harrisela porrecta and

10 6 r

Fig. 58. 14CO2 fixation by Cymbidium buds, newly and fully opened flowers and leaves.
S—sepals; P—petals; O—ovary (Dueker and Arditti, 1968).
TABLE 31
Net Carbon Fixation in the Light by Orchid Plant Organs Expressed as % Amount Fixed by Leaves
(Dueker and Arditti, 1968)

Cymbidium Epidendrum Cattleya Phalaenopsis Vanda


xanthinum gigas hybrids suavis Cattleya
Organ cv Indep. Day
cv Chelsea "Yorktown"
Bud
Sepals 13.6 1.0
Petals 5.5 0.1
Ovary 1.2 0.4
Newly opened
Flowers
Sepals 10.7 11.9
Petals 0.5 1.0
Ovary 0.1
Fully opened
Flowers
Sepals 1.4 4.9
Petals 0.6 1.1
Ovary 0.2 0.4
Lower leaves 100 100
Upper leaves 111 129
Stems and leaves 80
Stem 113
Leaf and stem 100
Root 1814 645 3689
Leaf 100 100 100
1 year old 100
2 years old 103
3 years old 103
4 years old 91.6
5 years old 70
6 years old 50
7 years old 38
532 J. ARDITTI

Taeniophyllum filiforme probably depend almost entirely on their roots


(which are green) for carbon fixation (for a review see Churchill et al., 1972).
Orchid roots are generally fleshy; however, the diurnal pattern of CO2
fixation in roots and (thick) leaves of Aranda are different (Fig. 60, see p.
549) (Hew, 1976).
G. PHOTORESPIRATION

In a systematic study, thick-leaved orchids were shown to have high CO2


compensation points which are typical of C3 plants (Wong and Hew, 1973,
1975). A high postillumination CO2 outburst was also observed in these
orchids (Fig. 60). Their photorespiratory rates are higher than those of dark
respiration (Wong and Hew, 1975).
H. SUMMATION

It is not at all surprising that orchids have evolved two (CAM, C3 ) and
possibly even three (C4) carbon fixation patterns. The family is large,
circum-global, and can be found in almost all ecological habitats (including
subterranean, but not subaquatic). To exist in so many habitats the
Orchidaceae have evolved many adaptive mechanisms including several water
conservation features (McWilliams, 1970) and carbon fixation pathways.
Among higher plants in general, differences in CO2 fixation pathways may
exist within one family. Among Orchidaceae, as in several other families,
such differences may exist within a single genus (Wong and Hew, 1973). The
possibility that this may be the case with Paphiopedilum has already been
mentioned. The same may be true for genera like Cymbidium (Wong and
Hew, 1973), Oncidium, Dendrobium and Epidendrum which have thick- and
thin-leaved species.
Many, possibly most, orchids which have thick leaves and exhibit CAM
live under what are essentially xerophytic conditions, often as epiphytes
(Czapek, 1909; Goh et al, 1977; Knauft and Arditti, 1969; Nuerenbergk,
1963). Their roots, wrapped around or spread on their host, are usually
exposed and subject to desiccation (Nuerenbergk, 1963). Therefore it is not
surprising that they have evolved: (i) thick cuticles and cuticular ledges which
cover stomatal pores and form hyperstomatic chambers as in Paphiopedilum
venustum (Haberlandt, 1928), Vanda tricolor (Gessner, 1956) and Cattleya
bowringiana (Borris, 1967); (ii) nocturnal opening of stomata (Goh et al.,
1977); (iii) CAM (Nuerenbergk, 1963); and (iv) no stomata on the upper
surfaces of leaves (Goh et al., 1977; for a review see Withner et al., 1974).
Another possible benefit of CAM for a terrestrial orchid growing among
dense vegetation or an epiphytic one located within the canopy of a tree is
the advantage of obtaining CO2 when it is most abundant. In such environ-
ments CO2 levels in the air could be greatly reduced during the day due to
photosynthesis by the surrounding vegetation. At night most of the sur-
ASPECTS OF ORCHID PHYSIOLOGY 533

rounding vegetation does not fix carbon and in fact releases CO2 due to
respiration. Consequently CO2 levels are higher than during the day and the
ability to fix it in the dark constitutes an adaptation which enhances survival.
Measurements of CO2 levels in tropical forests in Nigeria indicate that the
highest CO 2 concentration (0.052 %) occurs at sunrise and the lowest (0.035 %) is
at noon (for a review see Sanford, 1974).
Information from reports regarding culture of orchids under controlled
conditions (Krizck and Lawson, 1974), with added air (Frackowiack, 1933),
or CO2 "fertilization" and measurements of greenhouse or culture flask air
(Miwa, 1937; Tsuchiya, 1935; Wright, 1967; for reviews see Sanford, 1974;
Withner, 1974) is difficult to interpret because in most cases the carbon
dioxide was added during light periods. At the time of writing there are no
published reports of orchids being fertilized with CO2 in the dark.
The carbon fixation pathway of C3 orchids also reflects their natural
habitats. For example, Arundina graminifolia is found in open sunny places in
the lowlands and highlands of Malaya, never in shade; Bromheadia finlay
soniana grows in open scrub and light secondary forest whereas the preferred
habitat of Spathoglottis plicata is under full sun (Goh et al., 1977; Holttum,
1953).

V. FLOWERS

In 1678 Jacob Breynius wrote in his compendium "Exoticarum Aliarumque


Minus Cognitum Plantarum", on the extraordinary diversity of orchids:
If nature ever showed her playfulness in the formation of plants this is visible in
the most striking way among the orchids. The manifold shape of these
flowers arouses our highest admiration. They take the form of little birds, of
lizards, of insects. They look like a man, like a woman, sometimes like an
austere sinister fighter, sometimes like a clown who excites our laughter. They
represent the image of a lazy tortoise, a melancholy toad, an agile,
ever-chattering monkey. Nature has formed orchid flowers in such a way that,
unless they make us laugh, they surely excite our greatest admiration. The
causes of their marvelous variety are (at least in my opinion) hidden by nature
under a sacred veil (translated by Ames, 1948).
John Lindley, the British botanist who established the family Orchidaceae
and is therefore called the father of orchidology was no less eloquent:
Orchidaceae are remarkable for the bizarre figure of their multiform flower,
which sometimes represents an insect, sometimes an helmet with the visor up,
and sometimes a grinning monkey; so various are these forms, so numerous their
colours, and so complicated their combinations, that there is scarcely a common
reptile or insect to which some of them have not been likened.
However, his scientific logic prevailed:
They all, however, will be found to consist of t heir outer pieces belonging to the
calyx, and three inner belonging to the corolla .. . (Lindley, 1830).
534 J. ARD1TTI

A. HISTORY

Less than a hundred years after Breynius (in 1793) Christian Conrad
Sprengel in his "Das entdeckte Geheimnis der Natur im Bau und in der
Befruchtung der Blumen" tried to lift the veil a bit by suggesting that the
". . . marvelous variety . . ." is a method of attracting pollinators.
Seventy years later Charles Darwin removed the veil almost completely
(Darwin, 1862 or 1904) by describing the contrivances by which orchids are
pollinated by insects. The "unveiling" was completed 100 years after Darwin
by more complete descriptions of orchid pollination (Kullenberg, 1961; van
der Pijl and Dodson, 1966).
Interest in the mechanisms which control the blooming of orchids was
apparently first generated by the gregarious flowering of Dendrobium cru-
menatum (Massart, 1895; Treub, 1887; Went, 1898). Interest in ecology and
introduction of orchids into the commercial cut-flower industry added an
economic impetus to studies of flower induction (Tables 32-36).
Fritz Müller (Fig. 80D), a contemporary of Darwin was possibly the first to
become interested in the visual effects of pollination on orchid flowers
(Müller, 1868, for reviews see Arditti; 1971a, b; Möller, 1920-1921). He made
interesting observations and drew conclusions which in a strict sense were
erroneous, but were nevertheless accepted by Darwin. Because of that Müller's
ideas remained unchallenged for nearly 50 years. The man who challenged
them, Hans Fitting (Fig. 80C; Fitting 1909a, b, 1910) did suggest the involve-
ment of a hormone (thereby becoming the first to use this concept in relation
to plants), barely missed the discovery of auxins and became a great plant
physiologist (Arditti, 1971a, b). Subsequent work on post-pollination
phenomena in orchid flowers has been sporadic (Arditti 1969, 197la, b,
1976a, b; Arditti et al, 1971; Knauft et al., 1970).
B. INTRODUCTION

There are a number of reports on the flowering dates, periodicity and


rates of orchids in their natural habitats (Curtis, 1954; Dunsterville and
Dunsterville, 1967; Goh, 1973; Quisumbing, 1968; Sanford, 1971; Vacin,
1952; Zotkiewicz, 1961) as well as in botanical gardens, experimental stations
(Montgomery and Laurie, 1944) or private collections (including Hager,
1957; Hamilton, 1977; McDade, 1947; Wróbel-Streminska, 1961). Such
studies can be used to obtain information on the factors which induce
flowering in orchids and this has been done for West African species (Tables
32, 33, 34, 35, 36) (Sanford, 1971).
In other cases information on the factors which control the flowering of
orchids was obtained experimentally (Casamajor and Went, 1953) or by
observation and deduction (for reviews see Nuerenbergk, 1961b and Schlechter,
1977). Not surprisingly, since orchids can be found in many different
TABLE 32
West African Orchids Blooming During One Fairly Consistent Period of the Year (Sanford, 1971)

A. Probably photo-controlled (long-day)


Genus Species Epiphyte Terrestrial Flowering period
Aerangis biloba (Lindl.) Schltr + July, Aug.
Aerangis kotschyana (Rchb.f.) Schltr + June, July
Ancistrorhynchus metteniae (Kraenzl.) Summerh. + June, July
1
Ancistrorhynchus straussii (Schltr) Schltr + (May), June, July (Aug.)
Angraecum birrimense Rolfe + June, July
Chamaeangis vesicata (Lindl.) Schltr + July, Aug., Sept.
Cyrtorchis arcuata subsp. variabilis + May, June, July
Summerh.
Cyrtorchis hamata (Rolfe) Schltr + July, Aug.
Diaphananthe curvata (Rolfe) Summerh. + June, July (Aug.)
Diaphananthe kamerunensis (Schltr) Schltr + July, Aug., Sept.
Diaphananthe pellucida (Lindl.) Schltr + July, Aug., Sept. (Oct.)
Diaphananthe rutila (Rchb.f.) Summerh. + May, June, July
Encheiridion macrorrhynchium (Schltr) (May), June, July (Aug.)
Summerh.
Eulophia guineensis Lindl. + Aug., Sept., Oct. (Nov.)
2
Eurychone rothschildiana (O'Brien) Schltr + May, June, July
Habenaria englerana Kraenzl. + July, Aug.
Liparis caillei Finet + (May), June, July
Liparis nervosa (Thunb.) Lindl. + + (May), June, July (Aug., Sept.)
Liparis tridens Kraenzl. + (May), June, July
Malaxis katangensis Summerh. + June, July, Aug.
Plectrelminthus caudatas (Lindl.) Summerh. + (April), May, June, July (Aug.)
Polystachya modesta Rchb.f. + (July), Aug., Sept. continued
Polystachya mukandaensis De Wild. + (June, July), Aug. (Sept.)
Polystachya subulata Finet + (April), May, June, July
TABLE 32—continued
A. Probably photo-controlled (long-day)
Genus Species Epiphyte Terrestrial Flowering period
Rangaeris muscicola (Rchb.f.) Summerh. + July, Aug. (Sept.)
Solenangis clavata (Rolfe) Schltr + July, Aug., Sept., Oct.
Tridactyle bicaudata (Lindl) Schltr + (July), Aug., Sept., Oct. (Nov.)
1
Tridactyle brevicalcarata Summerh. + Aug., Sept., Oct.
Tridactyle lagosensis (Rolfe) Schltr + July, Aug., Sept.

B. Probably photo-controlled (short-day)


Genus Species Epiphyte Terrestrial Flowering period
Ancistrochilus rothschildianus O'Brien + Oct., Nov., Dec.
Ansellia africana Lindl. + Oct., Nov., Dec., Jan.
Bulbophyllum bufo (Lindl.) Rchb.f. + Oct., Nov., Dec., Jan.
Bulbophyllum falcatum (Lindl.) Rchb.f. + (Oct.), Nov., Dec. (Jan.)
Bulbophyllum fuscum Lindl. + Oct., Nov., Dec., Jan.
Bulbophyllum kamerunense Schltr + Jan., Feb., March
Bulbophyllum lupulinum Lindl. + Nov., Dec., Jan.
Bulbophyllum melanorrhachis (Rchb.f.) Rchb.f. + Nov., Dec.
Bulbophyllum rhizophorae Lindl. + Nov., Dec.
Chamaeangis lanceolata Summerh. + Nov., Dec.
Eulophia gracilis Lindl. + Jan., Feb., March (April)
Genyorchis pumila (Sw.) Schltr. + Dec., Jan., Feb.
Graphorkis lurida (Sw.) Kuntze + Dec., Jan., Feb.
Hetaeria stammleri (Schltr) Summerh. + Dec., Jan. (Feb.)
Podangis dactyloceras (Rchb.f.) Schltr + Dec., Jan., Feb. (March)
Polystachya affinis Lindl. + Jan., Feb. (March)
Polystachya golungensis RchKf. + Nov., Dec. (Jan.)
Rangaeris rhipsalisocia (Rchb.f.) Summerh. + (Dec.), Jan., Feb. (March)
Tridactyle tridactylites (Rolfe) Schlt r + (Jan.), Feb., March, April
Zeuxine elongata Rolfe + Dec., Jan., Feb.

C. Probably day-length neutral (1) Flowering at the beginning of the


growing season (on new growths)
Genus Species Epiphyte Terrestrial Flowering period
Bulbophyllum porphyroglossum Kraenzl. + April, May
Bulbophyllum winkleri Schltr + March, April, May, June, July
Corymborkis corymbosa Thou. + May
Eulophia quartiniana A. Rich. + April, May, June
Liparisa platyglossa Schltr + April, May
Liparisa suborbicularis Summerh. + May (June)
Malaxis maclaudii (Finet) Summerh. + May, June (July)
Malaxis prorepens (Kraenzl.) Summerh. + April, May (June)
Nervilia adolphii Schltr + (March), April
Nervilia fuerstenbergiana Schltr + March
Nervilia reniformis Schltr + April, May
Nervilia kotschyi (Rchb.) Schltr + March, April, May
Nervilia umbrosa (Rchb.f.) Schltr + Feb., March (April)
Polystachyaa adansoniae Rchb.f. + (April), May, June, July (Aug.)
Polystachya calluni flora Kraenzl. + April, May
Polystachya dolichophylla Schltr + (Feb.), March, April, May
Polystachya odorata Lindl. var. odorata + (March), April, May, June, July (Aug.)
continued
TABLE 32—continued
C. Possibly day-length sensitive
(2) Flowering at the beginning of the growing season (on mature growths)
Genus Species Epiphyte Terrestrial Flowering period
3
Ancistrorhynchus cephalotes (Rchb.f.) Summerh. + April, May
Ancistrorhynchus recurvas Finet + Feb., March, April, May
Bulbophyllum simonii Summerh. + Feb., March, April
Calyptrochilum emarginatum (Sw.) Schltr + April, May
Diaphananthe bidens (Sw.) Schltr + (March), April, May (June)
Diaphananthe longicalcar (Summerh.) + May, June (July)
Summerh.
Diaphananthe plehniana (Schltr) Schltr + April, May, June
Polystachya saccata (Finet) Rolfe + March, April
Tridactylea gentilii (De Wild.) Schltr + (May), June, July

C. Probably day-length sensitive via photo-control of vegetative dormancy


(3) Flowering at the end of the growing season
Genus Species Epiphyte Terrestrial Flowering period
Ancistrorhynchus capitatus (Lindl.) Summerh. + Sept., Oct., Nov. (Jan.)
Angraecopsis parviflora (Thouars) Schltr + Sept., Oct., Nov.
Angraecopsis tridens (Lindl.) Schltr + Sept., Oct.
Bolusiellaa talbotii (Rendle) Summerh. + (June), Aug., Sept., Oct., Nov. (Jan.)
Bulbophyllum buntingii Rendle + Sept., Oct.
Bulbophylluma fuerstenbergianum (De Wild.) + Oct., Nov., Dec., Jan., Feb.
De Wild.
Bulbophyllum josephii (Kuntze) Summerh. + Oct., Nov.
Bulbophyllum magnibracteatum Summerh. + Aug., Sept. (Oct.-Dec.)
Bulbophyllum nigericum Summerh. + Oct., Nov.
Bulbophyllum pavimentatum Lindl. + Sept., Oct., Nov.
Bulbophyllum pipio Rchb.f. + Oct., Nov., Dec.
Bulbophyllum porphyrostachys Summerh. + Oct., Nov.
Chamaeangis odoratissima (Rchb.f.) Schltr + Oct., Nov.
Cyrtorchis chailluana (Hook.f.) Schltr + Aug., Sept., Oct., Nov.
Cyrtorchis monteiroae (Rchb.f.) Schltr + Aug., Sept., Oct., Nov., Dec.
4
Cyrtorchis ringens (Rchb.f.) Summerh. + Sept., Oct., Nov.
Habenaria macrandra Lindl. + (May), Sept., Oct., Nov. (Dec.)
Listrostachys pertusa (Lindl.) Rchb.f. + (Sept.), Oct., Nov.
Polystachya paniculata (Sw.) Rolfe + Aug., Sept., Oct. (Nov.)
Polystachya rhodoptera Rchb.f. + + Aug., Sept., Oct., Nov., Dec.
Solenangis scandens (Schltr) Schltr + Aug., Sept., Oct., Nov., Dec., Jan., Feb.
a
Possibly photo-controlled as blooming season shifts to later in Cameroun-Equatorial Guinea.
1
Slight tendency of some clones to flower a second time in October and November.
2
Reported to flower twice yearly in Uganda.
3
Flowering very rarely in Nov., Dec.
4
A form of C. ringens vegetatively quite distinct from the lowlands form is found at high altitudes (5000-7500 feet) in Cameroun and Fernandos
Póo. This form normally flowers in May.
540 J. ARDITTI

TABLE 33
West African Orchids Blooming During One Widely Spread Period of the Year.
(Probably Day-length Neutral and Influenced by Low Temperature or
Temperature Fluctuations; Sanford, 1971)
A. At any time during the year
Genus Species Epiphyte Terrestrial
Ancistrorhynchus clandestinus (Lindl.) Schltr +
Angraecuma chevalieri Summerh. +
Angraecuma pungens Summerh. +
Calyptrochilum christyanum (Rchb.f.) Summerh. +
Microcoeliaa caespitosa (Rolfe) Summerh.
Polystachya caloglossa Rchb.f. +
Polystachya laxiflora Lindl. +
Polystachya supfiana Schltr +

B. At any time during the growing season (Feb. or April to Oct. or Jan.)
Genus Species Epiphyte Terrestrial
a
Angraecum multinominatum Rendle +
Chauliodona buntingii Summerh. +
Eulophia horsfallti (Barem.) Summerh.
Polystachya ramulosa Lindl. + +
Polystachya tessellata Lindl. +

C. From the middle to the end of the growing season (June-July to Dec.-Jan.)
Genus Species Epiphyte Terrestrial
Bulbophyllum intertextum Lindl. +
Liparis epiphytica Schltr +
Polystachya cultriformis (Thouars) Spreng. +
a
With a slight tendency to bloom twice a year, at scattered periods.

habitats, some species are long day (LD) plants; others respond to short
days (SD); a number are not affected by daylength (i.e., are neutral day
(ND) plants); several are induced by low temperature and a few appear to
have more complex requirements (Table 35). Blooming time response is
genetically controlled as suggested by observations of West African orchids
(Tables 32, 33, 34; Sanford, 1971). Comparisons between hybrids (McDade,
1947) and their parents (Table 35) can also provide information on the
inheritance of the response to stimuli which induce flowering. For example,
the hybrid of Cattleya gaskelliana (LD at 13° or 18°C, SD at 13°C) and C.
gigas (SD at 13°C), C. cv Harold flowers in early summer (McDade, 1947)
which suggests that it is SD. This implies that in this case the SD characteristic
may be dominant. On the other hand, C. cv Enid blooms in autumn (McDade,
1947), but is known to be unaffected by daylength (Table 35); yet it is a
hybrid between C. gigas and C. mossiae both of which are SD. This
TABLE 34
West African Orchids Blooming During Two Periods of the Year.
(Probably Day-length Neutral and Influenced by Low Temperature or
Temperature Fluctuation; Sanford, 1971)
A. At almost any time during the year
Genus Species Epiphyte Terrestria
Angraecum subulatum Lindl. + l

B. At the beginning and at the end of the growing season (March to


August and Sept. to Feb. on mature growths
Genus Species Epiphyte Terrestrial
Angraecopsis ischnopus (Schltr) Schltr +
Angraecum angustipetalum Rendle +
Angraecum distichum Lindl. +
Angraecum podochiloides Schltr +
Ansellia gigantea Rchb.f. var. clotica (Bak) +
Summerh.
Bulbophyllum barbigerum Lindl. +
Bulbophyllum calamarium Lindl. +
Bulbophyllum colubrinum (Rchb.f).) Rchb.f. +
Bulbophyllum congolanum Schltr +
Bulbophyllum distans Lindl. +
Bulbophyllum schimperanum Kraenzl. +
Cyrtorchis aschersonii (Kraenzl.) Schltr +
Diaphananthe obanensis (Rendle) Summerh. +
Eulophidium saundersianum (Rchb.f.) Summerh. +
Microcoelia dahomeensis (Finet) Summerh. +
Polystachya galeata (Sw.) Rchb.f. +
Tridactyle anthomaniaca (Rchb.f.) Summerh. +

C. At the beginning and at the end of the growing season (March to


August and Sept. to Feb.) on new growths
Genus Species Epiphyte Terrestrial
Bulbophyllum calyptratum Summerh. +
Bulbophyllum flavidum Lindl. +
Bulbophyllum oreonastes Rchb.f. +
Eulophia euglossa (Rchb.f.) Rchb.f. +
Eulophidium maculatum (Lindl.) Pfitz. +
Malaxis weberbauerana (Kraenzl.) Summerh. +
Polystachya albescens subsp. albescens Summerh. +
Polystachya albescens subsp. angustifolia (Summerh.) +
Summerh.)
Polystachya coriscensis Rchb.f. +
Polystachya polychaete Kraenzl. +
Stolzia repens (Rolfe) Summerh. +
TABLE 35 Control of Flowering in
Some Orchids
Species Factors which control or induce flowering Referencea
Aerangis mystacidii Bright, dry resting period Koopowitz, 1964
Aerides multiflorum Short day Bose and Mukhopadhyay, 1977
Arachnis cv Maggie Oei Salicylic acid, tri-iodobenzoic acid and coumarin. Plant growth Goh, 1973, 1976a, b
regulators : Indifferent to daylength.
Aranda cv Deborah Inhibited by actinomycin D and cycloheximide. Apical control ; Goh and Yang, 1977; Goh and
inhibited by auxin. Flowering gradient affected by growth regulators. Seetoh, 1973; Goh, 1973, 1975,
Decapitation may induce flowering, indifferent to daylength. Salicylic 1976a, b, 1977a, b, c, d
acid, tri-iodobenzoic acid, coumarin, B995, phosphon-D and CCC
enhance flowering.

Aranda cv Hilda Galistan Similar to Aranda cv Deborah Goh, 1977c


Aranda cv Lucy Laycock Similar to Aranda cv Deborah
Aranda cv Mei Ling Similar to Aranda cv Deborah
Aranda cv Nancy Similar to Aranda cv Deborah
Aranda cv Wendy Scott Indifferent to daylight. Salicylic acid, tri-iodobenzoic acid and coumarin Goh, 1973, 1976b
enhance flowering.
Blettilla striata Gibberellin treatments, 50 ppm, enhance flowering. Sano et al., 1961
Brassavola nodosa Short days (less than 14 h) enhance flowering. Brieger et al., 1977
Brassia verrucosa Vegetative growth is enhanced by 14 h days. Brieger et al., 1977
Brassocattleya hybrids More flowers under short days. Sheehan et al., 1965
Bromheadia alticola Low temperature stimulus. Holttum, 1953
B. finlaysoniana Flowering is stimulated by wet and cool days and retarded by Holttum, 1953; Jeyanayaghy
drought. Dry periods check flower bud development. and Rao, 1966; Sanford, 1971
Bulbophyllum lobii Vegetative growth is enhanced by 14 h days. Brieger et al., 1977
Calanthe rosea Vegetative growth is enhanced by 14 h days. Brieger et al., 1977
Calanthe cv Veitchii Vegetative growth is enhanced by 14 h days. Brieger et al., 1977
Calanthe vestita Vegetative growth is enhanced by 14 h days. Brieger et al., 1977
Catasetum High light intensities stimulate formation of female flowers ; plants in Brieger, 1957; Gregg, 1975;
shade produce male flowers. Vegetative growth under 14 h Brieger et al., 1977
illumination.
Cattleya Temperatures of close to 17°C are favourable for abundant flowering. Tran Thanh Van, 1974
Cattleya amabilis Short days. Urmston, 1949
C. cv Bow Bells 16½ h photoperiods delay flowering. Franklin, 1967
C. bowringiana 14 h days do not prevent flowering. Arditti, 1968b
C. cv Dupreana GA, 10 µg/sheath advanced flowering by 1-2 days. Arditti, 1966c
C. cv Enid Not affected by daylength and temperature. Arditti, 1968b
C. gaskelliana 9 h days at 13°C or 16 h days at 18°C night temperatures induce Arditti, 1966, 1967d, 1968b;
flowering. Long day plant at 18°C, non-photoperiodic at 13°C. Rotor, 1952, 1959; Haber, 1952
C. cv Geriant GA, less than 15 µg/sheath induces deformed earlier flowers. Arditti, 1966c
C. gigas See C. warscewiczii Arditti, 1966c
C. cv Jean Barrow Short days enhance flowering, but there was no peak period. Sheehan et al., 1965
C. cv Joyce Hannington See C. cv Bow Bells. Franklin, 1967
crosses
C. labiata Short days (less than 16½ h) induce flowering. Arditti, 1966c, 1967d, 1968b;
Franklin, 1967; Rotor, 1952,
1959
C. cv Los Gatos Nights 8-12 h long initiate flowering. Arditti, 1966c
C. mossiae SD at 13°C and 18°C night temperature. 9 h days at 13°C induce Arditti, 1966c, 1967d, 1968b;
flowering. Will not bloom under 16 h days at 13°C or 18°C night Rotor, 1952, 1959; Haber,
temperature; produces flowers under 9 h days at 18°C. Responds to 1952
temperature.
C. mendelii Same as C. mossiae Urmston, 1949
C. cv Oenone (C. mossiae x 16½-17 h light starting in late June discontinued 4 months before Franklin, 1967
C. labiata desired blooming date.
C. percivaliana SD (9 h) under 18°C and 18°C at night. Arditti, 1966c, 1967d, 1968b;
Haber, 1952
continued
TABLE 35—continued
Species Factors which control or induce flowering Reference a
C. cv Pinole Flowers form only on shoots 12-18 cm long. Rotor, 1952, 1959; Urmston,
1949; Johnson and Laurie,
1943, 1945
C. schroederae Same as C. mossiae. Urmston, 1949
C. skinneri Could be SD plant whose photoperiodic response may be modified by Rotor, 1952
temperature.
C. trianae SD (9 h) plant under both 13°C and 18°C SD (9 h) Haber, 1952; Rotor, 1952, 1959;
induce flowering whereas LD (16 h) delay it. Urmston, 1949; Holdson and
Laurie, 1949
C. warscewiczii SD at 13°C Rotor, 1952, 1959;
Urmston, 1949
Cycnoches High light intensities stimulate production of female flowers. Gregg, 1975
Cymbidium Three months of 13°C night temperature. Nights in the range of 7°C to Davidson, 1977; Arditti, 1966c,
13°C coupled with bright, sunny days initiate flowering. 1967d, 1968b; Rotor, 1952,
1959; Went, 1946, 1951
C. cv Desiree A'Legann Abscisic acid did not delay flowering. Brewer et al, 1969
C. cv Guelda Gibberellic acid causes earlier opening of flowers and larger blossoms. Bivins, 1970
C. lowianum Flowers formed when night temperatures were 6-10°C.
C. cv Rozette Flowers formed when plants were maintained under 14 h days, Arditti, 1968
1000 ft cdls, and 22°C and 18°C nights.
C. cv Sicily "Grandee" Gibberellic acid increased flower size and raceme and accelerated Bivins, 1968
length and flowering.
C. virescens Flower stalk elongation is enhanced by low temperatures.
Dendrobium "Daylength treatments did not affect flowering. . . ." Temperatures Sheehan et al., 1965; Van der
close to 17°C are favourable for abundant flowering. Cytokinins can Donk, 1974; Goh, 1977c
stimulate flowering. Their effect is enhanced by gibberellins.
D. cv Anne Marie Flowers when grown warm and moist. '
D. comatum Flowers, one day before D. crumenatum. Arditti, 1968
D. crumenatum Already developed flower buds are stimulated to develop and open. Holttum, 1953, 1969; also see
text
D. draconis Prefers cool climate. Kamemoto and Sagarik, 1965
D. findlayanum May require cold temperatures for flower initiation. Arditti, 1966c
D. formosum Plants flower after long dry season. Sanford, 1971
D. heterocarpum See D. findlayanum.
D. infundibulum Periods of low temperature induce flowering. Kamemoto and Sagarik, 1965
D. cv Jaquelyn Thomas Not induced by light. Sheehan, et al., 1965
D. cv Lady Fay Not induced by light. Sheehan, et al, 1965
D. cv Merlin Blooms when grown warm and mois t. Arditti, 1966c
D. nobile Flowering is induced by low temperatures. Arditti, 1966c; Kosugi, 1952;
Rotor, 1952, 1959
D. phalaenopsis Short days and 18°C or 13°C night temperature induce flowering. Rotor, 1952, 1959
D. scabrilingue See D. infundibulum.
D. cv Thwaitesiae Lowering of temperature at night may induce flowering. Arditti, 1966c
Epidendrum radicans Flowers initiated in October 1970, developed to pollen formation in Kosugi, et al, 1973
early January 1971, and flowered on 15 January 1971.
Eria Behaves like Dendrobium crumenatum. Smith, 1926, 1927
Eulophia cuculata Burning of grasslands may induce flowering. Sanford, 1971
Grammatophyllum Alternation of wet and dry seasons may regulate flowering. Holttum, 1957; Sanford, 1971
rumphianum
Laelia albida Short days may enhance flowering. Brieger et al, 1977
Laelia purpurata LD plants. Arditti, 1968b
Laeliocattleya cv Daylength of no less than 16 h induces flowering. Hampton, 1955
Canhamiana
Miltonia Temperatures close to 17°C are favourable for abundant flowering. Tran Thanh Van, 1974
Miltonia anceps Long days enhance flower formation. Brieger et al, 1977
continued
TABLE 35—continued
Species Factors which control or induce flowering Referencea
Miltonia ioezlii Long days enhance flower formation. Brieger et al., 1977
Miltonia spectabilis Long days enhance flower formation. Brieger et al., 1977
Mormodes See Catasetum and Cycnoches. Dodson, 1962
Odontoglossum bictonense Long days enhance flower formation. Brieger et al., 1977
Odontoglossum citrosmum (O. Periods of low temperature initiate flowering. Arditti, 1966c.
pendulum)
Odontoglossum hybrids ". . . daylength is an important factor in the production of spikes." Baker, 1968
Oncidium sphacelatum Floret initiation in late December in Japan. Kosugi et al, 1973
O. splendidum Flowers are initiated during short cool days. Franklin, 1967
Paphiopedilum No photoperiodic response. Night temperatures of 13°C for 2-3 weeks. Franklin, 1967; Davidson, 1977
P. insigne No photoperiodic response. Night temperatures of 13°C for 2-3 weeks. Rotor, 1952, 1959
Phajus tankervilliae Best flowering occurred under 10½ and 13½ hours. Bose and Mukhopadhyay, 1977
Phalaenopsis No photoperiodic response. Flowering induced by 3 weeks of 14°C nights and Franklin, 1967; Halperin and
20°C days. Flowering induced by low (10-1 5°C) temperatures. Floral initiation Halevy, 1974, 1975; Halevy, 1975;
takes place when night temperature varies between 12°C and 17°C and day Tran Thanh Van, 1970, 1974;
temperatures not exceed 21° C. Treatment time: 2-5 weeks. Nishimura et al., 1972, 1976

P. amabilis Flowering is induced by short days and 18°C. Rotor, 1952, 1959
P. schilleriana See P. amabilis. Flowering is stimulated by 2-3 weeks exposure to night Rotor, 1952, 1959; De Vries, 1953
temperatures below 21°C.
Polystachya cultriformis Same as Phalaenopsis schilleriana. Sanford, 1971
Renanthera imschootiana Short days (9 h) induced earlier flowering. Bose and Mukhopadhyay, 1977
Rhynchostylis gigantea Short days (8 h) at low temperature (10°C for 16 h/day) induce early flowering. Inthuwong and Watcharaphai, 1964

R. retusa See Renanthera imschootiana.


Thrixspermum A sudden drop in temperature may initiate flowering in some species. Holttum, 1957
Vanda Weekly sprays of 10 ppm gibberellins induce flower formation. O'Neill, 1958
Vanda cv Miss Joaquim Longer periods in sunlight brought about more profuse flowering; endogenous GohandWan, 1973;
gibberellins ". . . showed no obvious correlations . . . auxin levels in the shoot apex Murashige et al, 1967
may be responsible".
Zygopetalum See Vanda. O'Neill, 1958
a
Several reviews (Arditti, 1966, 1967, 1968; Rotor, 1952, 1959; Sanford, 1971) are cited for simplicity.
TABLE 36
West African Epiphytic Orchids Demonstrating Differences in Blooming Time Response Which May Be Genetically Controlled
(Sanford 1971)
Blooming period
(Normally
Genus Species Sept.-Nov.) Original source
Ancistrorhynchus capitatus (Lindl.) Jan. Fernando Póo collections (consistently 3 years after
Summerh. transplant)
Ancistrorhynchus clandestine (Lindl.) Schltr May-June Wamba, Iseyin, Upper Ogun, Gambari
March Kumba (Cameroun)
March-April Idanre, Olokemeji
June-Aug. Erin-Ode, Ilesha, Akure
Ancistrorhynchus straussii (Schltr) Schltr (Normally June ;
Aug.)
Oct.-Nov. Sapoba
Angraecum multinominatum Rendle June-Sept. Sapoba
March-May Gambari, Olokemeji, Lagos
Angraecum subulatum Lindl. April-May; Oct. Idanre, Akure, Gambari, Kumba (Cameroun)
March- April-June ;
Jan. Gindiri, Olokemeji
Ansellia gigantea Rchb.f. var.
nilotica (Bak.) Dec.-Jan. Mongu, Nubi
Summerh. March-April Mongu, Vom
Bulbophyllum distans Lindl. March-April Sapoba (consistent after 4 years)
June Fernando Póo (consistent after 2 years)
Calyptrochilum christyanum (Rchb.f.)
Summerh. Dec.-March Gambari, Akure, Olokemeji
April-July Gambari, Akure, Olokemeji
Chamaeangis lanceolata Summerh. July-Aug. Mt. Cameroun (consistent after 4 years)
Nov.-Dec. Gambari, Sapoba
continued
TABLE 36—continued

Blooming period
(Normally Sept.
Genus Species -Nov.) Original source
Cyrtorchis ringens (Rchb.f.) Summerh. May; Nov. Mt. Cameroun
Sept.-Oct. Agoi-Ibami, Sapoba
Diaphananthe plehniana (Schltr) Schltr Oct.-Nov. Sapoba (consistent after 4 years)
April-July Sapoba (consistent after 4 years)
Graphorkis lurida (Sw.) Kuntze March Mongu (consistent after 2 years)
Jan-Feb. Ibadan, Idanre, Enugu-Ikom
Microcoelia caespitosa (Rolfe) Summerh. Once blooming, Idanre (only 1 clone)
June. (Normally
twice blooming)
Microcoelia dahomeensis (Finet)
Summerh. Dec.-Feb. Gambari
June-July Gambari
Polystachya mukandaensis De Wild. June-July Erin-Ode, Olokemeji, Upper Ogun, Iseyin
Aug.-Sept. Erin-Ode, Ehor, Agoi-Ibami, Sapoba, Ijebu-Ode, Oru
Fig. 59. Net 14CO2 fixation by flowers in the light as a function of age in Cymbidium cv
Chelsea and C. cv Independence day "Yorktown". The horizontal bar, representing
14
CO2 fixation by a mature leaf, is included for comparison purposes (Dueker and Arditti,
1968). See text, p. 530.

Fig. 60. Diurnal CO 2 gas exchange of Aranda leaf and aerial root (Hew, 1976). See text, p.
532.
550 J. ARDITTl

fact would seem to indicate a more complex pattern of inheritance than in C. cv


Harold. A statement that "the blooming time of the hybrid will vary
according to the strength of the influence of the parents . . ." (McDade,
1947) may imply inheritance of dominant and recessive characters, but it is
clearly insufficient. More research is needed in this area. The existence of
numerous orchid hybrids with carefully recorded parentages holds the
promise of a fruitful undertaking. Valuable information may even be ob-
tained from careful analysis of available data and horticultural practices.
Some of the latter are unpublished having been arrived at through trial and
error by commercial growers.
One of the more interesting flowering habits among orchids is that of
Dendrobium crumenatum, the Malayan pigeon orchid (Holttum, 1953, 1969).
All plants in a certain area flower simultaneously, but the blooms last a very
short time. This gregarious flowering, which seems to ensure pollination has
interested investigators for a long time (Arens, 1923; Beumee, 1927; Burkil,
1917; Coomans de Ruiter, 1930; Coster, 1925; Kuijper, 1931, 1933; Massart,
1895; Rutgers and Went, 1915; Seifriz, 1923; Smith, 1926,1927;Treub, 1887;
Went, F.A.F.C., 1898, 1917; Went, F.A.F.C. and Rutgers, 1915; Went,
F.W. 1930; for reviews see Arditti 1966c, 1967; Holttum, 1953, 1969). The
inflorescences of Dendrobium crumenatum are found on the terminal portion
of stems covered by sheaths which are modified leaves. These inflorescences
remain very short and produce several flowers each protected by a bract.
Every one of the flower buds develop until all its parts are formed and the
anther is ". . . almost fully grown" (Holttum, 1969). Then development
ceases until reactivated by an approximately 5°C drop in temperature. In
Malaya and Indonesia such a sudden drop may be brought about by a rain-
storm and the subsequent evaporative cooling of the buds. Gradual cooling, if
it lasts long enough (about 24 hours) can also induce flowering. Exactly nine
days after the cooling (in the great majority of cases; I have also been told of
eight days and counted ten for a few flowers in one case), just before dawn the
flower (white with a tinge of pink) opens and starts emitting a delightful
fragrance. Pollinators attracted by the combination of colour, fragrance and
great masses of flowers, arrive soon after dawn. Even if the stimulus which
promotes development of the flower buds has been discovered, the
underlaying mechanism is still unknown.
An outstanding feature of flowering in Dendrobium crumenatum is the
dependence on temperature which in turn ensures an adequate water supply
(Holttum, 1953). In Singapore, for example, the absolute minimum and
maximum are 21 °C and 34 °C respectively (70 °-93 °F). The normal daily
range may be 23 °-32 °C (sunny days) and 24 °-27 °C (rainy days). In May, the
hottest month, the mean temperature is only 1.75 °C (3 °F) higher than in
December, the coolest period (all data from Holttum, 1953). Altogether
temperature fluctuations are not a major aspect of the climate. Further, drops
ASPECTS OF ORCHID PHYSIOLOGY 551

of 5 °C or more, in which the temperature does not rise above 27 °C are


associated with rain. Thus, an ample supply of water is assured before the
development of numerous blossoms because it is also the agent which brings
about the drop in temperature required for flower bud development.
Dependence on cooling in equatorial climates may appear to be a risky
evolutionary strategy, but this is not the case. Other orchids have evolved
similar mechanisms. They include Bromheadia alticola, B. finlaysoniana
(seven days from cooling to flowering) as well as several Dendrobium species
some of which flower one day before D. crumenatum and others which bloom
one or two days after it (Holttum, 1953; Smith, 1926). In addition, several
non-orchidaceous flowers also respond to cooling including the large de-
ciduous leguminous Malayan native tree Pterocarpus indicus; the Central
American member of the Cactaceae, Epiphyllum oxypetalun and the West
Indian bulbous plant Zephyranthes rosea (Holttum, 1953).
Three orchid genera, Catasetum, Cycnoches and Mormodes, can bear male,
female or hermaphrodite flowers. In Catasetum and Cycnoches a plant may
produce all three types of flowers on the same or different racemes during one
or a number of flowering seasons. At first this phenomenon led to consider-
able taxonomic confusion in that plants were assigned to different genera or
species.
The classification problems were resolved at the turn of the century when
plants in English greenhouses produced all three types of flowers. However,
the physiological enigma remains despite rather interesting recent observa-
tions (Gregg, 1973, 1975, 1979). Plants of Catasetum or Cycnoches which
are robust or grown under full sun produce female flowers whereas those that
are smaller or maintained in shade bear male blossoms (Gregg, 1975, 1979).
Production of female flowers is accompanied by increased ethylene
evolution which can be inhibited by placing the plant under shade or covering
the raceme tip with a foil cap (Gregg, 1973, 1975). This observation led to the
assumption that femaleness is induced by ethylene. However, this does not
seem to be the case since ethylene treatments and auxin applications have
failed to induce the production of female flowers (Gregg, 1973). Thus it
appears that ethylene evolution may be (i) a separate phenomenon brought
about by strong light (a stress response?), independent of female flower
induction, (ii) a byproduct of female blossom formation, or (iii) a combination
of the two.
The stimulation of female flower production in Catasetum and Cycnoches,
by high light intensities, is an important adaptive feature because it apparently
ensures sufficient photosynthesis to support seed production. Seed capsules of
Cycnoches have been reported to contain 4,000,000 seeds (Arditti, 1967a),
whereas in Catasetum estimates are as high as 2,000,000 (Gregg, 1975).
Clearly, the production of such large numbers, even of very small seeds, re-
quires considerable amounts of photosynthates. Blossoms of Catasetum and
552 J. ARDITTI

Cycnoches are green. Therefore, it is possible that like Cymbidium flowers


(Arditti and Dueker, 1968; Dueker and Arditti, 1968) they are capable of
photosynthesis and can contribute to their own energy needs. However, under
low light intensities the amount of photosynthesis may be insufficient.
Consequently, a mechanism which allows for seed production only when
there is enough energy to support it is of survival value.

C. POLLINATION
Orchid blossoms are morphologically different from other flowers in
several important details. Petals (inner whorl) and sepals (outer whorl) may be
of the same colour and often bear considerable resemblance to each other (Fig.
61). The median petal is usually modified considerably and serves as a landing
platform for pollinators. It is called the lip or labellum. Above the labellum
(Fig. 6IB, C) in the open flower of most orchids (and below it in a few) is a
structure called the gynostemium (Fig. 6ID, E) which represents a fusion of
stamens (anthers and filaments), stigmas and styles.
Monandrous orchids have only one fertile stamen near the apex of the
column. Their pollen grains are united into tetrads and compressed into 2, 4, 6
or 8 pollinia which are attached to each other by a viscid disc called the
viscidium (Fig. 6ID, E). The pollinia are located on the rostellum (a modified
stigma) below an anther cap (Schultes and Pease, 1963; van der Pijl and
Dodson, 1966).
In the unopened buds of monandrous orchids the labellum is above the
gynostemium. However, as the flower opens it twists in a process known as
resupination (Fig. 62; a, b, c, d; Arditti, 1966d; Schultes and Pease, 1963;
Ames, 1938; van der Pijl and Dodson, 1966; Zimmerman, 1932) and the
labellum becomes ventral to the gynostemium (Figs 61; B, C; 62; a, b, c, d).
But, this is not always the case. For example, Satyrium flowers do not
resupinate and blossoms of Angraecum eburneum resupinate 360°.
In diandrous orchids (Cypripedioideae, including the genus Paphiopedilum)
the flowers are nodding and the pouch-like labellum points downward (Fig.
63). Their dorsal sepal is modified and is either erect or bent slightly forward.
Anthers are located one each at either side of the staminode. The stigma is
located below the anthers (Fig. 63).
Orchid flowers have evolved highly specific, often bizarre pollination
mechanisms (Dodson, 1967, 1975; Dodson and Frymire, 1961; Dodson et al.,
1969; Kullenberg, 1961, 1973; Kullenberg and Bergstrom, 1976; Poyanne,
1917; van der Pijl and Dodson, 1966). In all cases these mechanisms involve
"manipulation" of the pollinating vector to ensure that it will carry away
pollinia and subsequently deposit them on another flower (Figs 64-73). This is
accomplished in a number of ways.
Nectar is a major attractant in orchids (Baskin and Blis, 1969; Jeffrey and
Arditti, 1968, 1969; Payne, 1964, 1968; Sunding, 1963; for reviews see
Fig. 61. Diandrous orchid flower. (A) Ground plan of an orchid flower at transitional
stage. (B) Cymbidium flower, front view. (C) Cymbidium flower, side view. (D) Cymbidium
gynostemium intact (top), and following removal of pollen (bottom). (E) Brassia, column
and part of ovary. (A, E) from Schultes and Pease, 1963; (B-D) from Arditti (1966d).
Explanation of symbols: ds, dorsal sepal; la, labellum or lip; r, rostellum.
Fig. 62. Resupination and its result, (a, b) Vanda flower resupinating so that the lip, which
is on top in the bud stage is on the bottom of the flower, (c, d) Resupinated Cattleya (c)
and Cymbidium (d) flowers (Arditti, 1966d).
ASPECTS OF ORCHID PHYSIOLOGY 555

Fig. 63. A diandrous orchid, Paphiopedilum villosum. (A) Whole flower. (B) Staminode
(right), anther (centre), and stigma (left, below staminode). (C) Staminode, view from
above (Williams, 1877). Explanation of symbols: a, anther; ds, dorsal sepal; po, pouch; s,
staminode; st, stigma.

Jeffrey et al., 1970; van der Pijl and Dodson, 1966). It is produced by many
species and attracts a variety of pollinators including bees, moths and birds.
One of the better known cases is that of Angraecum sesquipedale (Fig. 65)
because Darwin predicted the pollinator on the basis of floral characteristics
and spur length. The moth inserts its proboscis into the spur in search of nectar
and in the process picks up the pollinia. On the next visit to another flower the
pollen is deposited in the stigma.
Fragrances, often confined within structural features, are not only important
attractants for orchid pollinators, but also ensure specificity (Dodson and
Hills, 1966; Dodson, 1975; Dodson et al., 1969; Hills et a.l,
Fig. 64. Attachment of the pollinium to the pollinator. The column lies above the lip,
forming a narrow tube through which the pollinator must enter. When the pollinating bee
leaves, it must back out (a). In the process, the viscidium attaches the pollinium to its
back; the anther cap is dislodged (b, c). When the bee enters another flower, the pollinium
is pressed into the pocket -like stigma and retained thus fertilizing the flower (Stephens and
North, 1974).
Fig. 65. Angraecum sesquipedale Thou and its pollinator Xanthopan morganii praedicta (not
to scale, but proboscis length is about 30 cm and equal to that of the spur), (a, b) pollinated
and unpollinated flower showing the spur changes in sepals and lip. (c) Xanthopan morganii
praedicta with proboscis fully extended (Strauss and Koopowitz, 1973).
Fig. 66. Pollination of Catasetum. Trigger mechanism of Catasetum. (a) Frontal view;
the broken lines show the position of the pollen- bearing pollinium under the anther cap. (b)
Sectional side view of the interior of the flower. The trigger, when touched by a bee, releases
the sticky viscidium, which twists as indicated by the upper arrow and lodges against the
bee's back. Hence, the departing bee carries the pollinium. The anther cap falls away, (c)
Male bee, Eulaema cingulata, after having triggered off the pollen of Catasetum platyglossum. (d)
The bees crawling inside the hood shaped labellum of a female flower of Catasetum
platyglossum [(a, b) from Arditti, 1966b; (c and d) from van der Pijl and Dodson, 1966].
Fig. 67. Pollination of Gongora. (a) Bee gets a toboggan-like ride down the column and
strikes the viscidium, which adheres to it. When the bee flies off (b) it carries the pollinia. At
right (c) the bee, still carrying the pollinia, enters another flower and gets another ride down
the column. On this visit the pollinia adhere to the stigma and the flower is pollinated, (d) Male
bee, Euglossa viridissima pollinating Gongora armeniaca. (e) Male bee, Euglossa dodsoni
pollinating a flower of Gongora horichiana [ (a, b, c) from Arditti, 1966b; (d, e) from van
der Pijl and Dodson, 1966],
Fig. 68. Pollination of Coryanthes speciosa. (a) Dotted line, followed from right to left,
shows the path taken by the bee as it arrives at the flower and falls into the bucket, which
contains a liquid produced by the gland at top centre. The only way out for the bee is
through a narrow opening that forces the bee against the pollinia. (b) Male bee, Euglossa
superba, scratching the lip of a Coryanthes rodriguezii flower, (c) Bee, Euglossa superba, with
pollinia on its abdomen has forced up the anther cap of a Coryanthes rodriguezii and is
struggling free [ (a) from Arditti, 1966b; (b, c) from van der Pijl and Dodson, 1966],
Fig. 69. Pollination of Oncidium planilabre by a male bee, Centris geminata. (a) Bee
striking the flower, (b) Bee with pollinia attached to the front of its head, (c) Bee strikes the
flower, pollinium is attached to its head and anther cap is dislodged, (d) Pollinium bends
downward, (e) Bee strikes another flower and pollinium is forced into the stigma [ (a, b)
from van de r Pijl and Dodson, 1966; (c, d, e) from Stephens and North, 1974].
Fécondation des Ophrys fusca, lutea et speculum.

1, Ophrys fusca, fleur vue de face; 2, coupe du labelle (grossi); 3, O. lutea, fleur vue de face;
4, coupe du labelle (grossi); 5, fécondation opérée par un petit hyménoptère (coupe du
labelle); 6, l'hyménopère s'envole avec les 2 pollinies fixées a 1'abdomen; 7, O. speculum,
fleur vue de face; 8, coupe du labelle (grossi); 9, fécondation opérée par le mâle du Colpa
auren (coupe du labelle); 10, le Colpa s'envole avec les pollinies fixees sur la tête.
a, petit coussinet garni de poils courts sur lequel frotte l'abdomen de l'insecte; b, tache
bleu métallique; c, cavité correspondant a l'éperon des Orchis, dans laquelle plonge
l'abdomen de l'insecte; i, pollinies; p, pilosité fauve entourant le labelle (poils épais et
longs); t, pétales.
Fig. 70. Pseudocopulation in Ophrys. Poyanne's original drawing and caption (Ames,
1948;Poyanne, 1916).
ASPECTS OF ORCHID PHYSIOLOGY 563

Fig. 71. Pseudocopulation in Ophrys. (a) Head of a Gorytes campestris male with pollinia
of O. insectifera (left) and Listera ovata (right, without massulae). (b) Head of a Eucera
nigrilabris male with pollinia of O. tenthredinifem and probably of an Orchis sp. (c) The
common position of Eucera sp. male on the labellum of Ophrys bombyliflora (all from
Kullenberg, 1961).

1968; van der Pijl and Dodson, 1966). In Catasetum the pollinating bee is
attracted by the fragrance to a male flower and while searching for its source
triggers the pollen release mechanism (Fig. 66a, b, c).On a subsequent visit to a
female blossom it deposits the pollen (Fig. 66d). Gongora flowers also
attract pollinators with fragrance and have evolved a unique pollination
mechanism. After scratching at the base of the lip the bee backs up (and ends up
"taking" a toboggan -like "ride") and picks up the pollinia on its back. On a
subsequent visit the process results in the deposition of the pollinia into a
stigma (Fig. 67).
Flowers of Coryanthes are remarkable even for an orchid (Fig. 68a). Its
sepals and petals fold out to form sail-like structures. The top consists of a
564 J. ARDITTI

Fig. 72. Pseudocopulation in Ophrys. (a) Andrena squalida on the labellum of Ophrys
arachnitiformis Gren. et Phil, (b) Two males of Andrena squalida jostling on the labellum of O.
arachnitiformis. (c) Median longitudinal section of Ophrys bombyliflora Link; note con-
struction of distal part of labellum. (d) Ophrys bombyliflora, three flowers from different
sides. Note construction of the distal part of the labellum (photographs and drawing
courtesy Prof. B. Kullenberg).

hood shaped or globular hypochile, an elongated midsection called the


meso-chile, and at the bottom a bucket shaped epichile. Two glands at the base
of the column drip water into the bucket and partially fill it before the flower
opens. Once the flower has opened Euglossa, Euphlusia or Eulaema bees,
attracted by the fragrance produced by the hypochile land on it and scratch.
While doing so they fall into the buc ket and escape only through a tunnel
formed by the apex of the column and the epichile. As the bees crawl out the
pollinia are deposited on their abdomens (Fig. 68b, c). On a succeeding visit
the pollinia are deposited into the stigma (van der Pijl and Dodson, 1966). The
reasons why pollinating bees (which are males) collect odour sub-
tig. 73. Pseudocopulation in Ophrys. (a) Eucera oraniensis Lep male on the labellum of an
Ophrys bombyliflora Link flower, (b) Andrena sp. male on the labellum of an Ophrys fusca
Link flower, (c) Eucera longicornis L male on the labellum of an Ophrys apifera Huds flower,
(d) Colletes cunicularius infuscatus Nosk male on the labellum of an Ophrys sphecodes
provincialis Nelson flower (photographs courtesy of Professor B. Kullenberg).
566 J. ARDITTI

stances has been clarified only recently. They collect the fragrances in order to
attract other male bees of the same species and form leks. These in turn attract
females and mating takes place (Dodson, 1975).
Pollination of Oncidium planilabre by Centris bees is based on simulationof
an enemy insect by the flowers. As a consequence the blossoms are at tacked
by the bees and pollinated (Fig. 69).
"Fear" of death, "hate" for an enemy and hunger are not the only "drives"
"utilized" by orchids to ensure pollination. Sexual "passion" is also "em-
ployed". Flowers of the genus Ophrys mimic the females of certain aculeate
Hymenoptera and also produce volatile compounds which excite the males
sexually and cause them to attempt copulation with the blossoms (Figs 70-73;
Godfery, 1925; Kullenberg and Bergstrom, 1976; Poyanne, 1917). While
they do so they pick up pollen (Figs 70-73) and subsequently deposit it on
another flower. And, when all else fails orchids are capable of self pollina tion
(Fig. 74).

Fig. 74. Self-pollination is shown in Ophrys. Before pollination (a) the column stands
upright. As a result of autolysis, or self-digestion of certain tissues by the plant, the stipe
bends (b), carrying the pollinia downward towards the sticky stigmatic surface. When the
pollinia reach the stigma and adhere to it (c), pollination is complete (Arditti, 1966d).
.—

D. POST-POLLINATION PHENOMENA

Ever since Aristotle first described orchids, their flowers have fascinated
people, mostly because of their forms. They were assumed to be the origin of
some animals and to have arisen from the semen of others (Arditti, 1972d; Fig.
75). Even science fiction writers have not been immune to their charms(Adams
and Nightingale, 1976; Boyd, 1969; Clarke, 1974; Wells, 1960). The shapes of
orchid flowers, their fascinating mimicry, fragrance production, food offering
and colour combination all accomplish the same end: attraction of pollinators.
ASPECTS OF ORCHID PHYSIOLOGY 567

Fig. 75. Illustration by Lady Grey of Groby in James Bateman's "The Orchidaceae of
Mexico and Guatemala" (1843). It is captioned: "The hag came forth, broom and all,
from a flower of Cypripedium insigne; her attendant spirits are composed of Brassia Lan-
ceana, Angraecum caudatum, Oncidium Papilio, etc., etc.; two specimens of Cycnoches sail
majestically on the globe below, on the right of which crawls Megaclinium falcatum. In the
centre, stands a desponding Monachanthus; on the left a pair of Masdevallias are dancing a
minuet, while sundry Epidendra, not unlike the 'walking leaves' of Australia, complete the
group."

Relations between orchid species and their pollinators are most often very
intimate. Each orchid is pollinated only by a specific vector (Darwin, 1904;
Dodson et al, 1969; van de r Pijl and Dodson, 1966). Hence the attraction
mechanisms are as remarkable as they are varied and as fascinating as they
are complex. But, this specificity can also constitute a serious drawback since
the available pollinators may be limited in number thereby reducing the
number of pollinated flowers. Indeed, several observers have noted that often a
very small proportion of orchid flowers are pollinated. It becomes reasonable
to assume, therefore, that survival of many orchid species would require
568 J. ARDITTI

not only adaptations that attract pollinators, but also increased longevity
which would provide a longer "waiting" period. Indeed, it is not unusual for
unpollinated orchid flowers to remain alive for a long time (Table 37). For
example, it is said that flowers of Grammatophyllum multiflorum may last
nine months (Kerr, 1972); Paphiopedilum flowers can live 3-4 months and it is
not unusual for those of Cattleya, Cymbidium and Phalaenopsis to remain fresh
for several weeks. Few species have very short-lived flowers: Dendrobium
appecdiculatum is reported to last a hard-t o-believe five minutes, whereas D.
crumenatum flowers live a day or less.
Producing complex flowers and pollinator attraction systems and main -
taining them for long periods is expensive in terms of energy resources. No
wonder, then, that orchid flowers have evolved intricate mechanisms for the
conservation of energy; utilization of substances from floral segments which
have carried out their functions; photosynthesis by flower parts be fore or
after pollination; cessation of scent and nectar production immediately after
pollination and fast wilting. Therefore, flowers which can contribute to their
own upkeep have an evolutionary survival advantage. Many orchid flowers
are coloured by chlorophyll and are, therefore, green (Arditti, 1966e; Matsu-
moto, 1966). At least in one instance, green Cymbidium flowers have been
shown to be capable of photosynthesis (Arditti and Dueker, 1968; Dueker
and Arditti, 1968). This unique adaptation, which allows blossoms to con -
tribute towards their own energy needs, most probably exists in other green
flowered orchids. In these flowers pollination is the beginning of the end for the
perianth. In other flowers one or more floral segments turn green and
apparently become photosynthetic. If a generalization can be made about
pollination -induced events (i.e., post-pollination phenomena) in orchid
flowers it is that here evolution has favoured conservation.
It is difficult to pinpoint the earliest description(s) of post-pollination
phenomena. Conrad Sprengel ". . . struck a new path in botanical science . .."
(Müller, 1883) with his book "Das entdeckte Geheimnis der Natur im Bau
und in der Befruchtung der Blumen" (Sprengel, 1793) where he may have
described for the first time the post-pollination phenomena in orchids. The
next reports appeared soon thereafter (Bulliard, 1802; Wachter, 1799-1801).
Ludolf Christian Treviranus (1779-1864), the German botanist who made
early observations of the movement of cell contents also wrote about orchid
flowers. He studied structures associated with pollination and its results
(Treviranus, 1827). After that a number of studies and reports were either
directly concerned with Orchidaceae (Brongniart, 1831; Gosse, 1863; Anderson,
1863; Riviere, 1866, 1872; Veitch, 1887; Gruignard, 1886; Anonymous, 1890,
1894; Hurst, 1898; Anonymous, 1899; Malguth, 1902; Leimbach, 1911; Resvol,
1911; Morita, 1918; Pohl, 1927; for an early review see Müller, 1883) or a
number of plants including orchids (Cruger, 1851; Rossner, 1923).
One observation of post-pollination phenomena led to a major biological
569

TABLE 37
Longevity of Orchid Flowers (Fitting, 1909a; Kerr, 1972; Morita, 1918;
Poddubnaya-Arnoldi and Selezneva, 1957)

Longevity, Longevity,
Genus or species days Genus or species days
Angraecum up to 40 Megaclinium several
Calanthe up to 30 Odontoglossum up to 20 or
Calanthe discolor 14 even 80
Cattleya up to 45-60 Oncidium up to 26 or
Coelogyne up to 21 even 60
Cymbidium virens 14-25 Paphiopedilum 9-120
Cypripedium up to 60 or Peristeria elata 1-2
even 90 Phalaenopsis up to 35
Dendrobium up to 19 Phal. amabilis up to 120
Dendrobium crumenatum 1 Phal. violacea 30
Dendrobium superbum 14 Platanthera yatabei 7-10
Epipactis erecta 8-10 Rhynchostylis retusa 30
E. falcata 8-12 Sobralia macrantha 1
E. papillosa 7 Spiranthes australis 10
E. thunbergii 7-10 Stanhopea 3
Grammatophyllum multiflorum 270 Vanda up to 60 or
Gymnadenia cucullata 8-10 even 90

advance: the discovery of the cell nucleus (Brown, 1831, 1833, 1834; for a
review see Arditti, 1977b). A second barely missed discovery was that of
auxin. Reports from Brazil by Fritz Müller (Fig. 80D) that orchid pollen is
poisonous were accepted uncritically by Darwin and this resulted in general
acceptance (Hildebrand, 1868; Magnus, 1887; Möller, 1920-1921; Müller,
1868, 1869). However, a reinvestigation in Bogor using Phalaenopsis flowers
convinced Hans Fitting (Fig. 80C) that he was dealing with a special sub-
stance (Fitting, 1909a, b, 1910, 1921). Fitting called that substance Pollen-
hormon and thereby became the first person to even use the term "hormone"
relative to plants (for reviews see Arditti, 197la, b, 1975a). Others after
Fitting showed that his Pollenhormon was the auxin indoleacetic acid by
studies of post-pollination ("post-floration") phenomena (Laibach, 1930),
comparisons between pollinia and growth substances (Laibach, 1932, 1933a, b;
Laibach and Maschmann, 1933; Mai, 1934; Maschmann and Laibach, 1932),
and application of auxins to orchids (Hubert and Maton, 1939).
This has been confirmed by qualitative determinations which have shown
that orchid pollen may contain as much as 100 µg indoleacetic acid g-1
(Müller, 1953). These findings have been confirmed recently (Klass, 1964).
However, a report that pollinia of Cattleya cv Enid contain naphthaleneacetic
acid (Klass, 1964) requires careful confirmation. This auxin (NAA) is a
synthetic substance whose isolation or even tentative identification from a
570 J. ARDITTI

natural source requires more information than used to suggest its existence
(Klass, 1964).
Despite these reports, Fitting (personal communication) never accepted the
idea that Pollenhormon was IAA. He maintained that his was a separate and
different hormone. In a way his claim may have a basis because he and
everyone else who worked with pollen extracts and diffusates did not have
pure IAA. Rather, the diffusate may have contained cytokinins, gibberellins
(unpublished results from my laboratory) and possibly other substances.
Hence, results obtained from assays of extracts or diffusates might be those
that can be expec ted from a mixture of substances, not a pure hormone. The
effects of such extracts could well have been different enough from those of
pure auxin to convince Fitting that he was dealing with a different hormone.
These considerations in no way detract from Fitting's achievement. His con-
clusions were based on what was known during the period he was active, and
this was before the discovery of auxins, cytokinins and abscisic acid, re-
discovery of gibberellins and appreciation of the effects of ethylene. In fact,
Fitting's retirement preceded the important early work on these hormones all
of which are known to have effects on post-pollination phenomena of orchid
flowers (Arditti, 1976b).
A third major research area in which early studies of post-pollination
phenomena played a significant role is the effect of pollen on the initial stages
of fruit formation (i.e. ovary swelling) and ovule formation (Hildebrand,
1863a, b; 1865).

1. Phenomena
With 20,000-30,000 species and many widely different, often bizarre,
pollination mechanisms, it is only reasonable to anticipate numerous, often
unique, post-pollination phenomena. Indeed, this is the case and the phe-
nomena can be divided into a number of categories (Tables 38, 39). Un-
fortunately, however, reports, especially those on visual changes, are widely
scattered through the systematic, ecological, pollination, horticultural,
physiological and hobby literature and often difficult to trace. One must
simply search through many articles, observe flowers and trust in luck and
serendipity. In addition, no orchid has been investigated fully to determine
which and how many phenomena it may exhibit. Therefore, compilations are
in fact a list of examples (Tables 38, 39).
Angraecum sesquipedale flowers are large, star-shaped, creamy-white and
fragrant in the late afternoon and evening (Fig. 76D); on being pollinated the
perianth segments turn yellow and scent production ceases. The dorsal sepal
starts to bend down (Fig. 76E) and shortly thereafter the lateral sepals and
petals bend inwards (Fig. 76F), but the lip does not move very much. Eventually
the entire flower closes (Fig. 76F) and the perianth dies. In A. eburneum only
the lip folds (Fig. 76A, B, C), but the perianth dies as in A. sesquipedale.
TABLE 38
Post-pollination Phenomena of Orchid Flowers Classified by Physiological or Developmental Categories

Category Examples
Redifferentiation Greening of sepals,
petals, ovaries and
gynostemia as well
as changes in their
nature
Growth Swelling of ovaries Changes in pedical Closing of Swelling of Loss of Changes in
curvature stigmas columns curvature by shape of calli
columns
Nastic movement Hyponasty of Hooding of dorsal
petals and sepals sepals
Termination of Cessation of scent Cessation of nectar
activities production exudation
Morphogenesis and Ovule development Senescence and
development death of perianth
Protein synthesis Changes in protein
complement and
levels
RNA synthesis Increased RNA levels
in gynostemia
Metabolic activity Hydrolysis of storage Anthocyanin Ethylene Changes in Yellow colour Chlorophyll
and structural production or evolution respiration production by synthesis or
compounds destruction patterns perianth destruction
Transport Mobilization of Altered patterns Starch Changes in
substances into of phosphate accumulation protein
gynostemia and movements in ovaries levels
ovaries
Relation to Changes of UV light Changes in flower
environment reflection by flowers colour
TABLE 39
Post-pollination Phenomena in Orchid Flowers
Phenomenon Occurrencea, b
Cattleya Cymbidium Vanda Phalaenopsis Zygopetalum
Senescence and Arditti, 1969; Arditti, 1969, Burg and Dijkman, In some species Arditti
death of perianth Hayes, 1968 1976a, b 1967; Dijkman and pollination accelerates unpublished
segments Burg, 1970 senescence
(Arditti, 1976a, b)
Greening of all or Arditti, 1976a, b; Arditti
some perianth Curtis, 1943; unpublished
segments Duncan and Schubert,
1943; Ringstrom, 1968
Swelling of column Hayes, 1968 Arditti, 1969,
1976a, b
Swelling of ovaries Arditti, 1969 Arditti, 1969, Burg and Dijkman, Arditti, 1976a, b Arditti
1976a, b 1967; Dijkman and unpublished
Burg, 1970
Hyponasty of Arditti, 1969, Arditti, 1976a, b
sepals and petals 1976a, b
Changes in pedicel Arditti, 1969,
curvature 1976a, b
Stigmatic closure Arditti, 1969 Arditti, 1969, Arditti, 1976a, b Arditti
1976a, b unpublished
Hood formation or Arditti, 1969,
folding by dorsal 1976a, b
sepal
Greening of Arditti, 1969 Arditti, 1969, Arditti, 1976a, b Arditti
gynostemia 1976a, b unpublished
Termination of
scent production
Cessation of nectar
production
Initiation of nectar Arditti, 1969, 1976a, b
production
Production of Arditti, 1969, 1976a, b
anthocyanins or
yellow pigments by
floral segments
Destruction of Arditti et al., 1973 Burg and Dijkman,
anthocyanins 1967; Dijkman and
Burg, 1970 √
Ovule development √c Avadhani et al.,
1971
Changes in enzyme Probably occurs Arditti, 1976a, b
complements or
levels of corollas
Hydrolysis of structural Probably occurs
compounds
Mobilization of Arditti, 1969, 1976a, b; Harrison
substances from the and Arditti, 1976
perianth into columns
and ovaries
Changes in colour and Arditti, 1969, 1976a, b; Harrison
shape of calli and Arditti, 1976

Changes in UV Thien, 1971


reflection patterns
Loss of gynostemium Arditti, 1969, 1976a, b
curvature
Twisting of gynostemia

Ethylene evolution
continued
TABLE 39—continued
Phenomenon Occurrencea, b

Epidendrum Angraecum Phajus Catasetum Cycnoches Epipactis


Senescence and death of Arditti, 1976a Strauss and
perianth segments Koopowitz, 1973

Greening of all or some Arditti


perianth segments unpublished

Swelling of column
Swelling of ovaries Arditti √c √c √c √c √c
unpublished

Hyponasty of sepals and Strauss and


petals Koopowitz, 1973
Changes in pedicel Arditti, 1976a
curvature
Stigmatic closure Arditti Arditti, 1976a
unpublished
Hood formation or folding
by dorsal sepal
Greening of gynostemia Arditti Strauss and Dodson, Dodson,
Termination of scent unpublished Koopowitz, 1973 personal personal
production Strauss and communication communication
Koopowitz, 1973

Cessation of nectar Wiefelspütz,


production 1970
Initiation of nectar
production
Production of Colour changes Strauss and Colour changes
anthocyanins or yellow (Ames, 1947) Koopowitz, 1973 (Arditti, 1976a)
pigments by floral segments
Destruction of
anthocyanins
Ovule development

Changes in enzyme
complements or levels of
corollas
Hydrolysis of structural
compounds
Mobilization of substances
from the perianth into
columns and ovaries
Changes in colour and
shape of calli
Changes in UV reflection
patterns
Loss of gynostemium Thien, 1971
curvature
Twisting of gynostemia

Ethylene evolution
continued
TABLE 39—continued
a, b
Occurrence
Brazilian
Phenomenon Miltonia
Cypripedium Stanhopea Schomburgkia Menadenium species Odontoglossum
Senescence and death of Rossner, 1923 No effect on Lip dies (Arditti, Hayes, 1968 Hayes, 1968
perianth segments flower, longevity 1976a)
(Rossner, 1923)
Greening of all or some Arditti, 1976a Hayes, 1968 Hayes, 1968
perianth segments

Swelling of column Arditti, 1976a Hayes, 1968 Hayes, 1968


Swelling of ovaries √ c
√ c
√ c
√ c
√c
Hyponasty of sepals and Arditti, 1976a
petals

Changes in pedicel
curvature
Stigmatic closure Jürgen Schrenk, Hayes, 1968
1973
Hood formation or
folding by dorsal sepal
Greening of gynostemia Arditti, 1976a Hayes, 1968

Termination of scent
production
Cessation of nectar
production
Initiation of nectar
production
Production of Colour changes Hayes, 1963
anthocyanins or yellow (Arditti, 1976a)
pigments by floral
segments
Destruction of
anthocyanins
Ovule development

Changes in enzyme
complements or levels of
corollas
Hydrolysis of structural
compounds
Mobilization of
substances from the
perianth into columns
and ovaries
Changes in colour and
shape of calli
Changes in UV reflection
patterns
Loss of gynostemium
curvature
Twisting of gynostemia
Ethylene evolution

continued
TABLE 39—continued
a, b
Occurrence
Cypripedium,
Phenomenon Neottia,
Zygopetalum Dendrobium, Eria Listera Orchis Bletia
Gymnadenia,
Platanthera
Senescence and death Hildebrand, Hildebrand, Hildebrand, Hildebrand, Hildebrand,
of perianth segments 1863a, b 1863a, b 1863a, b 1863a, b 1863a, b

Greening of all or Duncan and


some perianth Schubert, 1943
segments

Swelling of column
Swelling of ovaries √c Hildebrand, Hildebrand, Hildebrand, Hildebrand, Hildebrand,
1863a, b 1963a, b 1863a, b 1863a, b 1863a, b

Hyponasty of sepals
and petals
Changes in pedicel
curvature
Stigmatic closure
Hood formation or
folding by dorsal sepal

Greening of
gynostemia
Termination of scent
production
Cessation of nectar
production
Initiation of nectar
production
Production of
anthocyanins or yellow
pigments by floral
segments
Destruction of
anthocyanins

Ovule development

Changes in enzyme
complements or levels of
corollas
Hydrolysis of structural
compounds
Mobilization of
substances from the
perianth into columns
and ovaries
Changes in colour and
shape of calli

Changes in UV reflection
patterns
Loss of gynostemium
curvature
Twisting of gynostemia

Ethylene evolution
continued
TABLE 39—continued

Occurrencea, b
Phenomenon All or most
Rhynchostylis Mormodes Arundina Prasophyllum Bulbophyllum
orchids
Senescence and death of Arditti, 1967,
perianth segments 1976a, b

Greening of all or some


perianth segments

Swelling of column

Swelling of ovaries √c √c √c Jones, 1972 Smyth, 1969 Arditti, 1967,


1976a, b

Hyponasty of sepals and


petals
Changes in psdicel
curvature
Stigmatic closure
Hood formation or
folding by dorsal sepal

Greening of gynostemia

Termination of scent
production
Cessation of nectar
production
Initiation of nectar
production
Production of Lim et al., 1975
anthocyanins or yellow
pigments by floral
segments
Destruction of
anthocyanins
Ovule development

Changes in enzyme Lim et a I., 1975


complements or levels of
corollas
Hydrolysis of structural √ c

compounds
Mobilization of substances √ c
Lim et al., 1975
from the perianth into
columns and ovaries
Changes in colour and
shape of calli
Changes in UV reflection
patterns
Loss of gynostemium
curvature
Twisting of gynostemia Arditti
unpublished
Ethylene evolution
a
The genera and phenomena listed are only examples. Similar phenomena may occur in genera which are not listed or, some phenomena which occur in
these or other genera may have been omitted.
b
Due to space limit ation reviews are cited as much as possible.
c
A check-mark indicates that the phenomenon is presumed to occur.
Fig. 76. Post -pollination phenomena in Angraecum (Arditti, 1976a). Explanation of
symbols: D or DS, dorsal sepal; G, gynostemium or column; L, labellum; LP, lateral petal; LS,
lateral sepal; Sp, spur.
ASPECTS OF ORCHID PHYSIOLOGY 583

A. cv Veitchi is a hybrid between these species and has inherited the folding
character of both (Strauss and Koopowitz, 1973). The lip starts to fold first
(Fig. 76G, H, I, J). Next, the dorsal sepal starts to bend down (Fig. 76H, I, J).
After this, the petals start to fold inward (Fig. 761). At the end just before the
perianth dies the flower looks like a well wrapped little package (Fig. 76J;
Strauss and Arditti, 1973; Strauss and Koopowitz, 1973).
In Cymbidium (Figs 77, 79A) pollination brings about hood formation by
the dorsal sepal; yellowing of petals and sepals; anthocyanin production by all
perianth segments and the gynostemium (column); colour development, and
deformation of the calli on the labella; ovule development; greening of
gynostemia; stigmatic closure; enzyme production; increases in cell size;
alteration in respiration patterns; changes in pedicel curvature; ethylene
evolution; altered phosphate transport among floral segments; ovary swelling;
water losses; changes in dry weight and RNA synthesis (Arditti and Flick,
1974, 1976a, b; Arditti et al, 197la, b; 1973; Arditti and Knauft, 1969;
Hsiang, 195la, b; Hubert and Maton, 1939; for reviews see Ard itti 1969,
1976a, b).
Flowers of Phajus tankervilliae are fragrant and borne on flower stalks
which reach two, maybe three metres. They exude a delightful fragrance and
expose a purplish lip below a white gynostemium against the backdrop of
brownish sepals and petals (Fig. 78A). Pollination probably occurs when the
vector attempts to crawl between the lip and column. Following pollinationthe
lip changes colour to a reddish-orange brown; scent production decreases and
the angle of the flower is reduced to 4 0° or less. The lip also tends to tighten
itself around the column (at least in some cases) and dies within a week with
the rest of the perianth (Fig. 78B).
A self-pollinating variety of Phajus tankervilliae also exists. Its flowers
never open fully, and the pollen drops into the stigma during the late -bud
stage. As a result, the flower stalks are usually laden with fruits. Pollinators
play no role in this. Yet, the flower is fragrant and, once pollinated, exhibits
the same phenomena as the cross-pollinated variety.
Schomburgkia tibicinis produces flower stalks which may reach three
metres or more. The flowers are reddish-purple with undulating sepals and
petals (Fig. 78C). A white column is surrounded by a reddish-purple, trans-
lucent labellum. On a bright day, the tube formed by it (and enclosing the
column) looks like a purple tunnel with alternating lighter and darker stripes.
Following pollination, the flower changes very quickly (sometimes within
24 hours). Sepals and petals turn a deeper purple and fold inward forming
tubes (Fig. 78D). The lip undergoes a similar colour change and folds over the
column (Fig. 78D) clasping it tightly.
Brassolaelia (an intergeneric hybrid) flowers retain characteristics of both
parents, but look more like Brassavola thanLaelia. Their labella are decorated
with several lines made of purple dots (Fig. 78E). In a naturally occurring
Fig. 77. Post -pollination phenomena in Cymbidium. Phenomena (small letter) and agents
or treatments which can cause them (CAPITALS) are listed in each box. Boxes are con-
nected to the organs to which they pertain.
Fig. 78. Phajus tankervilliae (A) Unpollinated. (B) Approximately five days after pollination.
Schomburgkia tibicinis (C) Unpollinated. (D) About 26-36 hours after pollination.
Brassolaelia (E) Pollinated (F) Unpollinated. Epidendrum cv O'Brienianum (G) Unpollinated,
(H) Pollinated (Arditti, 1976a). Explanation of symbols as for Fig. 76.
Fig. 79. Swelling of Cymbidium gynostemia. (A-D) view from 45° angle, (E-G) view from
below. (H) Phalaenopsis unpollinated. (I) Three to five days after pollination. (J) Seven to
nine days after pollination (Arditti, 1976a). Explanation of symbols as for Fig. 76 plus: ac,
anther cap; c, calli; ct, column tip; cw, column wings; sc, stigmatic cavity.
ASPECTS OF ORCHID PHYSIOLOGY 587

species, these lines could attract pollinators. However, in a man-made hybrid,


they cannot possibly have any such significance. Still, as the flower ages, or is
pollinated, the spots smudge and the lines eventually disappear leaving the lip
unadorned (Fig. 78E).
Before senescence or pollination, the sepals and petals of Epidendrum cv
O'Brienianum are a pleasant red colour, smooth and turgid (Fig. 78G). The lip
has a bright yellow spot in the centre. When a flower senesces or is pollinated,
the yellow spot disappears and all perianth segments shrivel (Fig. 78H). Colour
changes also take place in Bifrenaria harrisoniae following pollination. Its
sepals and petals turn yellow. The lip becomes dark red and the trichomes on
it lose turgidity.
Phalaenopsis flowers (Fig. 79) have lobed labella and large yellow and
orange calli. The column is situated above the calli, between the edges of two
labellar lobes. Once a flower is pollinated, the labellum wraps itself, but not
tightly, around the column (Fig. 79C). The lateral petals start to fold inward
and, eventually, completely enclose the lip and column (Fig. 79D). This is
hyponasty and much less common than epinasty. The dorsal sepal may bend
down slightly, but there is almost no movement by the lateral sepals.
The sepals and petals of Menadenium labiosum are greenish and its lip is
white (Fig. 80A). On pollination, the sepals and petals enlarge, become
fleshy and turn a darker green, the column swells, but the lip shrivels and
dies (Fig. SOB).
(a) Senescence. For a period there was a tendency among some workers to
use the word ageing primarily for animals and the term senescence for plants.
This is not so any more. Ageing is still applied almost exclusively to animals,
but it is also used for plants along with senescence. The two words are often
used as synonyms, but authors who wish to cover all bases often talk and
write of "senescence and ageing" whether they mean one, or the other, or
both. In other words "the term senescence has a confusing overlap in meaning
with the term ageing" (Leopold, 1975).
One possible way to remove the confusing overlap is to use the terms in the
sense that ". . . senescence refers to the deteriorative processes that are
natural causes of death and ageing refers to the wider array of processes of
accruing maturity with passage of time" (Leopold, 1975 referring to Medawar,
1957). This is attractive to me, since it makes a useful distinction between
events in the life of orchid blossoms. In addition, it generates several interesting
questions which can be answered by using orchid flowers as the experimental
organism. Another way of defining these phenomena is that ageing is ". . . the
sum total of progressive changes in a whole plant or in one of its constituent
organs . . ." whereas senescence is ". . . changes, caused by fac tors other than
harmful external conditions, which are clearly degenerative— ultimately and
irreversibly so." But, I see problems with these definitions in that (a) "harmful
external conditions" are difficult to define (is excising an
Fig. 80. Menadenium labiosum. (A) Unpollinated. (B) Pollinated. (C) Prof. Dr Hans
Fitting, the last photograph ever taken of him (by Ms B. H. Flick). (D) Dr Fritz Müller
(Arditti, 1976a). Explanation of symbols as for Fig. 76.
ASPECTS OF ORCHID PHYSIOLOGY 589

organ or part of one during the experiment harmful, and, if so, is it harmful to
the organ or to the plant from which it was excised ?); and (b) it is not clear
at present whether irreversibility should be part of the definition. These
questions, like those generated by the Medawar definition can be answered
through experiments with orchid flowers.
First, there is a question regarding the aspects of ageing (as defined above),
which may in fact be senescence. A deteriorative process which leads to the
senescence and eventual death and elimination of a vital organ, organelle or
physiological pathway would contribute to ageing. This is because the elimina-
tion contributes to the ". . . gradual accumulation of . . . changes . . ."
(Leopold, 1975) which are part of the ". . . wide array of processes of accruing
maturity.. ." (Leopold, 1975). In other words, one can ask whether senescence
is a contributing factor to ageing. To put it differently, it is possible to ask:
(a) Does ageing consist, at least in part, of steps which are senescence?
(b) Could ageing proceed without senescence? (c) Can senescence take place
without ageing? and (d) Does ageing result from lack of repair rather than
active deterioration in contrast to senescence which is a progressive sequence?
A second question is whether senescence may not be merely accelerated
ageing. A fast accruing of maturity could lead to the deteriorative processes
that are senescence. Or, stated differently, the question is whether the differ-
ences between ageing and senescence are speed and rate.
Unpollinated orchid flowers may remain fresh and alive for weeks or
months (Kerr, 1972). Ageing and/or senescence, if any occur, are imper-
ceptible for weeks. Even after becoming apparent the symptoms develop very
slowly. In the process perianth colour may change due to anthocyanin,
chlorophyll or carotenoid production or destruction (i.e., some orchids fade
and others accumulate pigments as they age). Fresh and dry weight of sepals
and petals and phosphorus levels in the perianth may increase or decrease
depending on species (Lim et al., 1975). Gynostemia (columns) turn yellow,
then grey and finally black before softening and becoming jelly -like. After
that they shrivel and/or disintegrate. The ovary turns yellow and abscises.
Pollination, disturbance of pollinia, emasculation, ethylene, auxin, damage
and air pollution alter these events (Arditti and Knauft, 1969; Duncan and
Schubert, 1943, 1947). The rate of deteriorative changes in perianth segments
is greatly acclerated (i.e., senescence is induced or ageing is speeded up) in
some species. Flowers of Phalaenopsis and Cymbidium hybrids live up to
eight weeks if unpollinated, but die within seven days after pollen deposition.
Paphiopedilum blossoms may last up to three months but die within three
weeks after pollination.
When describing the differences between pollinated and unpollinated
flowers Hans Fitting used the terms autonome Blütendauer and aitionome
Postflorationsvorgänge (Fitting, 1909a, b, 1910). The first (autonome flower
duration), referred to ageing of flowers which was unaffected by exogenous
590 J. ARDITTI

factors including pollen. All stages of ageing occurred autonomically (i.e.,


"by themselves" as it were) with death coming as the logical end of a de-
velopmental process. Aitionome post-pollination events were described as
induced (attic-cause) phenomena in which some phases were modified and
others eliminated by exogenous influences (i.e., the pollen of another flower).
Ageing and death could be induced immediately after the juvenile phase
without an intervening maturity period.
In present -day terms autonom could be equated with ageing whereas
aitionom may be parallel to senescence. However, it is also possible that in
orchid flowers pollination merely speeds up some autonome events and also
induces additional phenomena. If so the aitionome process would be a mixture
of ageing and senescence in present day terms. This raises again the question
regarding ageing v. senescence and also generates new ones.
First, are at least some of the fast post-pollination events physiologically
similar to several of the slow pre -pollination ones? Second, can pre- and/or
post-pollination ageing be reversed? Third is the question whether, in
un-pollinated blossoms, the gynostemium, ovary, and in some cases petals and
sepals age during the entire period after a flower has opened, albeit slowly. Or,
do they go through a steady-state (phase during which no ageing occurs) which
is then followed by a period of ageing. If this is so could the ageing period be
prevented or delayed? And, once initiated is the ageing process reversible?
What initiates it in the absence of pollination?
The fourth question pertains to the nature of the pre - and post-pollination
processes: the autonom ageing could be the result of lack of repair and
maintenance, if so, events would be random. Post-pollination senescence, on
the other hand, is a programmed sequence which can be characterized through
standard procedures.
A fifth possibility is that before pollination the process may be passive (i.e.,
the flowers age and die due to lack of repair or maintenance) but becomes
active (i.e., tissues and substances are hydrolysed rapidly due to a marked
increase in specific hydrolases) in pollinated flowers. Another, is that the
process may be active in both instances but is accelerated after polli-
nation.
Sixth, in orchid flowers pollination or auxin trigger the senescence and
death or transformation of the perianth, but questions remain regarding the
induction of senescence: (i) what roles do plant hormones play in the process,
and (ii) is an as yet uncharacterized hormone, like Fitting's Pollenhormon
(Fitting, 1909a, b, 1910) also involved? At present there is no need to implicate
a new or unknown plant hormone in post-pollination phenomona of orchid
flowers, but it is possible that other substances may control senescence as is the
case with serine in oat leaves (Martin and Thimann, 1972).
There are really no answers to these questions. One can only assume that the
processes of ageing and senescence in orchid flowers are no different
ASPECTS OF ORCHID PHYSIOLOGY 591

from those in other plants. This and other factors make orchid flowers very
suitable organisms for the study of ageing and senescence. The question of
reversibility can be answered by determining whether unpollinated orchid
flower segments (perianth, gynostemium, ovary) age constantly, even if
slowly, before pollination or whether they remain at a steady state for a
certain period before starting to decline. Shoud they age or senesce constantly it
is obvious that pollination can reverse the process in some species and seg-
ments (see below) and accelerate it in others. If ageing or senescence are
initiated following a steady-state phase, it would be feasible to determine the
condition under which reversibility is possible by pollinating the flower at
various times.
Another advantage is that the phenomena can be initiated at will (rather
than having to wait for a developmental stage), and separated from each
other physiologically and studied individually.
Many other studies use (or have used) excised leaves and other organs or
tissues which have been removed surgically. They may be dying rather than
ageing or senescing as they would in situ. Consequently, fine points may be
missed entirely. The problem may even be more serious: ". . . these pieces,
having been subjected to major surgery, cannot survive for more than a few
days unless battered with nutrients and growth regulators which distort
their physiology and delay their death. With such . . . experimental material,
any effects have to be observed rapidly . . ." (Milborrow, 1974). [All this
reminds me of a statement by Theophrastus in "Enquiry Into Plants", IV, XV,
2-4 (Of Various Causes of Death): "Men try to help the tree by plastering it
with mud and tying pieces of bark, reed or something of the kind about it, so
that it may not take cold nor become dried up"—I think that maybe the
treatments really killed the tree.] Or, the effects may be missed entirely and
could be noted only in more nearly normal, un"battered" tissues. Because
fruit ripening is a slower process, at times studied in whole organs under more
normal conditions, the same events are more apt to be detected. Possibly,
therefore, some reported differences between leaf senescence and fruit ripening
are not as large or as real as they appear to be. A suitable experimental system
may reconcile at least some of them.
A third advantage is the relatively slow rate at which ageing or senescence
occur in unpollinated orchid flowers. This would allow the detection of
events which may be fleeting and therefore undetectable in other systems. For
example, it is well established that during leaf senescence levels of protein,
RNA and chlorophyll drop whereas the reverse may be true in some fruits.
However, synthesis, or at least appearance of new proteins (perhaps pro-
teases which lead to proteolysis) has been reported in senescing leaves and
perianth segments (Mart in and Thimann, 1972; Tan and Hew, 1973; Trippi
and Van, 1971). The same would appear to be true for RNA. In some in stances
such synthesis, if it occurs, may be very low and could represent such
592 J. ARDITTI

a small proportion of the total protein content as to be undetectable. In orchid


flowers these compounds could be measured.
One difficult problem to answer regarding senescence, ageing and the
difference between them is whether they involve lack of synthesis and/or
maintenance, increased destruction (i.e., catabolism and hydrolysis) or both.
Data from experiments designed to provide answers to these questions can be,
and have been, interpreted in different ways. Even elegant experiments have
not always removed doubts. Cytokinins can delay the senescence of rose
petals for example and it has been suggested that they function by suppressing
protease synthesis (Martin and Thimann, 1972). However, experiments in-
volving hormones such as cytokinins and ABA have also been subject to
different interpretations (Milborrow, 1974). Some of the reasons for this
situation lie in the systems being used. Many excised tissues (leaves for
example) or specialized organs (like cotyledons) are simply dying (Arditti,
1971b; Milborrow, 1974) rather than undergoing "programmed" senescence
which is a normal aspect (even if a final stage) of development (Varner, 1965).
As a result ". . . the effect of ABA (and probably other hormones) recorded in
such assays are usually those most readily observed . . ." (Milborrow, 1974).
And, some may be missed entirely. Not that ". . . leaf disks, explants or
sections should not be used; they certainly should, but in conjunction and
comparison with the same tissues in other and less damaged forms if valid
information on the normal . . . is to be obtained." (Milborrow, 1974). This

Fig. 81. Effects of pollination and auxins on gynostemia.


A. Longitudinal sections of the gynostemia of Coelogyne speciosa. (a) Untreated, (b)
Treated, stigmatic cavity closed; ag, agar block.
B. Transverse sections of the gynostemia of an Odontoglossum hybrid, (a) Pollinated;
p, pollinium. (b) Treated with β-indolebutyric acid (cotton moistened with 50 mg l-1 );
ct, cotton, (c) Control.
C. Transverse sections of the gynostemia of an Odontoglossum hybrid.
Top row: cells and epidermis at the backside of the gynostemia.
Bottom row: cells between vascular bundle (hatched) and surface of the stigma, (a)
Pollinated, (b) Control, (c) Treated with β-indolebutyric acid.
D. Gynostemia (backside) of Cyrtopodium punctatum. (a) Control, (b) Pollinated, (c) Agar
block.
αN: treated with α-naphthylacetic acid.
agar block 2 x 2 x 1 mm, cone. 100 mg l-1
βN: treated with β-naphthylacetic acid.
agar block 2 x 2 x 1 mm, cone. 100 mg l-1
βA: treated with β-indoleacetic acid.
agar block 2 x 2 x 1 mm, cone. 100 mg l-1
βP: treated with β-indolepropionic acid.
agar block 2 x 2 x 1 mm, cone. 100 mg l-1
βB: treated with β-indolebutyric acid.
agar block 2 x 2 x 1 mm, cone. 100 mg I"1
E. Phalaenopsis amabilis. (a) before pollination, (b) six days after pollination.
F. Arachnanthe salingi. (a) before pollination, (b) after pollination.
G. Aerides odoratum. Unpollinated, front (a) and side (b) view; two days after pollination,
front (c) and side (d) view (Fig. A-D, Hubert and Maton, 1939; Fig. E-G, Fitting,
1909a).
Fig. 82. Gynostemia.
A. Transverse sections of gynostemia showing epidermis and cells of
Cymbidium tracyanum. (a) untreated, (A) treated.
Cyrtopodium punctatum. (b) untreated, (B) treated. Vanda
tricolor var. suavis. (c) untreated, (C) treated. (Hubert and
Maton, 1939).
B. Cymbidium finlaysonianum. (a) unpollinated, (b) five days after pollination (Fitting,
1909a).
C. Effects of pollination on the swelling of Cymbidium gynostemium (Arditti and Flick,
1974).
D. Effects of pollination on the swelling of the gynostemium of Cymbidium virens (plotted
from data in Morita, 1918).
TABLE 40
Effects of Pollination and Excision of the Rostellum on Gynostemium Swelling and Curvature in Cymbidium Flowers
(Arditti and Flick, 1976)

Treatments Time after Gynostemium Swelling, mm Curvature**


First Second first treatment Day Day
description description min 1 2 4 7 T∆ 1 2 4 7
None None 9.8 99 10.0 10.8 + 1.0 c c c c
Rostellum excised None 9.8 98 10.3 10.0 + 0.2 c c c c
Pollinated None 9.8 11.5 13.1 14.2 + 4.4 c c s s
Pollinated Rostellum excised 30 9.8 11.0 12.3 14.8 + 5.0 c c c sc
Pollinated Rostellum excised 60 9.8 11.3 13.0 14.3 + 4.5 c c sc sc
Pollinated Rostellum excised 150 10.3 11.2 12.3 14.3 + 4.0 c c sc sc
a
c, curved; sc, slightly curved; s, straight.
TABLE 41
Post-pollination Phenomena in Cymbidium Flowers and their Parts (Arditti and Flick, 1976)
Gynostemium
NAA in
culture Ovary
medium, Swelling, diameter, Labellum Perianth or
Number Treatment µg/3 ml mm a Curvature b Stigma c mm d calli e labellum wilting f
Whole flowers (WF)
1 Undisturbed (control) 0 9.3 Cur 0 4.23 Y NW
2 Undisturbed (control) 25 9.3 Cur O 5.23 YO VSW
3 Pollinated 0 15.7 Str Cl 5.2 OR SW
4 Pollinated 25 15.3 Str Cl 5.4 OR SW
5 Emasculated 0 9.7 Cur O 4.7 YO VSW
6 Emasculated 25 10.3 Cur 0 5.1 YO NW
7 NAA on stigma g 0 16.0 Str Cl 4.9 R SW
8 NAA on stigma 25 15.3 Str Cl 6.1 OR SW
9 NAA on lip g, h 0 9.3 Cur O 4.4 YO NW
10 NAA on lip 25 9.8 Cur O 5.4 YO SW
Flower minus gynostemium (F-G)
11 No NAA on cut i 0 4.4 YO VSW
12 No NAA on cut 25 5.1 OR SW
13 NAA on cut g, i 0 6.0 R W
14 NAA on cut 25 5.7 R SW
15 Pollen on cut i 0 4.05 OR SW
16 Pollen on cut 25 4.25 YO SW
Flower minus labellum (F-L)
17 No NAA on cut j 0 9.7 Cur 0 4.2
18 No NAA on cut 25 10.0 Cur 0 4.8
19 NAA on cut g, j 0 14.8 Str Cl 6.0
20 NAA on cut 25 14.0 Str Cl 6.4
Gynostemium and ovary
(G+O)
2.1 Undisturbed (control) 0 8.7 Cur O 4.5
22 Undisturbed (control) 25 10.0 Cur O 4.4
23 Pollinated 0 13.0 Str Cl 4.6
24 Pollinated 25 13.0 Str Cl 5.3
25 Emasculated 0 10.0 Cur O 4.3
26 Emasculated 25 9.7 Cur O 4.0
27 NAA on stigma g 0 13.3 Str Cl 4.7
28 NAA on stigma 25 13.7 Str Cl 4.2
Gynostemium only (G)
29 Undisturbed (control) 0 9.7 Cur O
30 Undisturbed (control) 25 9.3 Cur O
31 Pollinated 0 12.0 Str Cl
32 Pollinated 25 11.0 SCur Cl
33 Emasculated 0 8.7 Cur O
34 Emasculated 25 9.0 Cur O
35 NAA on stigma g 0 10.8 SCur Cl
36 NAA on stigma 25 11.0 SCur Cl
Ovary only (O)
37 No NAA on cut k 0 4.53
38 No NAA on cut 25 4.13
39 NAA on cut g, k 0 5.03
40 NAA on cut 25 5.18
41 Pollen on cut 0 5.17
42 Pollen on cut 25 4.83

continued
TABLE 41—continued
NAA in Gynostemium Ovary

medium, Swelling, diameter, Labellum Perianth or


Number Treatment µg/3 ml mm a Curvature b Stigma c mm d calli e labellum wilting f
Labellum only (L)
43 No NAA between calli 0 YBr NW
44 No NAA between calli 25 YBr NW
45 NAA between calli g 0 YBr NW
46 NAA between calli 25 YBr NW
a
Measured at the lower edge of the stigma.
b
Cur, curved; SCur, slightly curved; Str, straight.
c
Cl, closed; O, open.
d
Measured with calipers at the ovary midsection.
e
OR, orange-red; R, red; Y, yellow; YBr, yellow-brown.
f
NW, not wilted; SW , slightly wilted; VSW, very slightly wilted.
g
NAA was applied at 25 µg/flower or floral part.
h
NAA was applied between the calli.
i
Cut resulting from removal of the gynostemium.
j
Cut resulting from removal of the labellum.
k
Cut resulting from removal of all other floral segments.
TABLE 42
Nitrogen Content in Orchid Flowers Following Pollination (Abstracted From Gessner, 1948; Hsiang, 1951b). See text, p. 604.
Time after % of mg N/
Species Treatment Flower segment treatment Control Initial levels flower
Cattleya labiata Control perianth 167 hours 100 7.5
Control gynostemium 167 hours 100 2
Pollinated perianth 167 hours 63 2.2
Pollinated gynostemium 167 hours 161 7.8
NAA treated perianth 167 hours 45 1.8
NAA treated gynostemium 167 hours 177 5.3
Coelogyne cristata Pollinated gynostemium 10 days 188
Pollinated ovary 10 days 117
Pollinated perianth 10 days 24-66
Coelogyne cristata Pollinated gynostemium 10 days 165
var hololeuca Pollinated ovary 10 days 218
Pollinated perianth 10 days 2-41
Cymbidium tracyanum Pollinated gynostemium 16 days 160 2.2
cv Doris Pollinated ovary 16 days 155 1.71
Pollinated perianth 16 days 43-100 0.24-0.47
Pollinated whole flower 16 days 113 6.24
600 J. ARDITTI

problem may have arisen from the decline in the study of whole plants or
entire organs. An important advantage offered by orchid flowers is that a
whole organ can be studied in situ unaffected by treatments or influences
other than those which are a part of their normal life cycle (i.e., the flowers
are not battered). Even in vitro the extraneous influences are minimal.
Yet another advantage of orchid flowers is that the fast process (which is
probably senescence) can be initiated at will (Arditti and Knauft, 1969). This
renders the flowers suitable for studies of the earliest stages of senescence.
(b) Swelling and straightening of the gynostemium. One of the most noticeable
effects of pollination is the swelling of gynostemia (Curtis, 1943; Dolcher, 1961,
1967; Duncan and Schubert, 1943; Fitting, 1909a; Hubert and Maton, 1939;
Morita, 1918; for reviews see Arditti, 1969, 1976a, b). This has been reported
for a number of species including Coelogyne speciosa, Odontoglossum,
Cyrtopodium punctatum, Phalaenopsis amabilis, Cymbidium., Arachnanthe
sulingi, Aerides odoratum (Figs 81, 82). In Cymbidium the swelling starts on
the second day after pollination and usually reaches a maximum in seven
days (Fig. 82C). During that time the gynostemium also straightens (Fig.
82B). Removal of the rostellum has no effect on either the swelling or the
straightening (Table 40; Arditti and Flick, 1974). Auxins can also induce
straightening and swelling of the column (Tables 40, 41; Arditti and Knauft,
1969; Hubert and Maton, 1939). Excised gynostemia also swell if pollinated
or when NAA is applied to the stigma. The same is true for gynostemia
attached to ovaries with all other floral segments removed. Gynostemia on
flowers minus labella also swell when NAA is applied to the cut resulting
from removal of the lip. However, when NAA is supplied through the base
there is no swelling (Table 41; Arditti and Flick, 1976). Experiments with
sections of gynostemia have shown that the upper parts react more rapidly
and extensively than basal portions (Dolcher, 1961b).
The swelling of the gynostemium has been described as "... a character-
istic response to ethylene" (Burg and Dijkman, 1967). However, ethylene
(10 µl l-1 ) treatments of Cymbidium flowers for up to 4-5 days did not induce
swelling (Arditti et al., 1973). This fact points to auxin as the factor which
initiates the swelling. The following reports lend support to this view. First,
the swelling is due to an increase in cell size (i.e., cell enlargement), not cell
number (i.e., cell division) at least in Odontoglossum hybrids, Cymbidium
tracyanum, Cyrtopodium punctatum and Vanda tricolor var suavis (Figs 81,
82; Hubert and Maton, 1939). Second, pollination and NAA applications
bring about increases in fresh and dry weight of gynostemia (Hsiang, 1951a).
Third, osmotic pressure increases after pollination (Hsiang, 1951a). Fourth,
cut discs of pollinated columns take up more water than those from un-
treated ones (Hsiang, 195la). Fifth, pollinated columns have a greater water
holding capacity (Hsiang, 195la).
The obvious conclusion from these facts is that the swelling is due to in-
ASPECTS OF ORCHID PHYSIOLOGY 601

creased water uptake (brought about by the increased osmotic concentration


which is the probable result of the higher dry matter content) coupled with
auxin induced wall softening (for a more detailed discussion see Arditti et al.,
1971b; Arditti and Knauft, 1969).
ABA, GA 3 and kinetin do not inhibit or reverse the effects of auxin (Arditti et
al., 1971 a, b). The same is true for actinomycin D, but cycloheximide and
ethionine reduce the swelling and straightening (Arditti and Knauft, 1969 and
unpublished results from my laboratory). This may be taken as an indication
that swelling and straightening do not require the synthesis of RNA, but are
dependent to some extent on new proteins.
(c) Stigmatic closure (Figs 79A, 81 A, B, E, F; Tables 40, 41) is a very com
m o n p o s t-pollination phenomenon in orchids (which probably serves to
protect pollen deposited on the stigma). In general, stigmatic closure is
caused in a similar fashion and by the same factors which bring about
stigmatic swelling i.e., pollination and auxin treatments. The only exceptions
are full or partial closing of the stigma induced by:
1. 10 or 100 µg GA 3 per flower.
2. 0.01, 0.05 or 0.1 µg ABA plus 10 µg GA 3 per flower.
3. 1 µg GA 3 plus 10 µg kinetin per flower.
4. 0.01, 0.05 or 0.1 µg ABA plus 10 µg kinetin per flower.
5. 100 µg kinetin per flower.
These exceptions are difficult to explain except possibly in terms of increased
synthesis or sparing of auxin.
Stigmatic surfaces of Epipactis atropurpurea contain leucoplasts with con-
centric thylakoids, abundant stroma and stroma-containing vesicles which
include at least one starch grain and lipid globules (Pais, 1973-1974). If the
same is true for other orchids it is possible to speculate that gibberellins could
initiate synthesis of a -amylase which hydrolyses the starch grains, thereby
increasing glucose concentration in the cells. This would increase osmotic
concentration and subsequently water influx which can cause closure.
NAA induced stigmatic closure is inhibited by cycloheximide (unpublished
results from my laboratory), but not by actinomycin D (Arditti and Knauft,
1969). As with swelling and straightening of the column this is an indication
that closure does not require RNA synthesis but is probably dependent on
new proteins.
(d) Anthocyanins. Both production and destruction of anthocyanin have
been reported to occur following pollination (Arditti and Knauft, 1969;
Burg and Dijkman, 1967; Dijkman and Burg, 1970; Hsiang, 1951 a; Lim et
al., 1975; for reviews see Arditti, 1969, 1976a, b; Withner, 1974). In some
flowers (Cymbidium for example) destruction may follow production (Fig. 90)
(Arditti et al., 1973). Anthocyanin production (Arditti et al., 1975) or destruc
tion (Burg and Dijkman, 1967; Dijkman and Burg, 1970) can also be induced
by emasculation and ethylene. In addition, GA 3 and ABA treatments can also
602 J. ARDITTI

cause increased anthocyanin levels in Cymbidium gynostemia and labella


(Arditti et al., 1971a, b). Kinetin applications induced only marginal in -
creases in labella (Arditti et al., 1971b). When these hormones (ABA, auxin,
cytokinins, ethylene, gibberellins) are applied in pairs they tend to reduce or
inhibit each other's effects (Arditti et al., 1971b, 1973).
Applications of actinomycin D, cycloheximide, ethio nine and puromycin
after NAA inhibit anthocyanin production (Arditti and Knauft, 1969). This
can be taken to indicate that anthocyanin production requires de novo syn-
thesis of RNA and proteins. Ethionine inhibits anthocyanin synthesis while
enhancing ethy lene production. Therefore it appears that the effects of these
inhibitors on anthocyanin levels are not due to their influence on ethylene
production.
(e) Chlorophyll. As already mentioned pollination induces chlorophyll syn-
thesis in gynostemia or perianth segments (Tables 38, 39; Fig. 77). In Angrae-
cum flowers pollination induced increased chlorophyll levels in the ovaries
but a reduction in the spurs. Actinomycin D and cycloheximide applications
following pollination reduce the losses in chlorophyll content (Strauss, 1976).
(f) Nastic movements. Pollination, emasculation and NAA applications
induce hyponasty of petals and sepals in Phalaenopsis and Angraecum, as well
as downward bending of the pedicel in Phajus and sideways movement of the
column in Mormodes (Figs 78, 79; Strauss, 1976; for reviews see Arditti
1969, 1976a, b). Hyponasty is less common than epinasty. Epinasty is in-
duced by ethylene (Abeles, 1973) and the same is true for hyponasty (Strauss,
1976). In Angraecum blossoms hyponasty of petals and sepals is inhibited by
cycloheximide (Strauss, 1976).
(g) Ovaries. Ovaries of pollinated orchid flowers swell and elongate follow-
ing pollination (Fig 83; Fitting 1909a; Heslop -Harrison, 1957; Hsiang,
1951a; Hubert and Maton, 1939), but less so than gynostemia. Both the fresh
and dry weights of ovaries increase following pollination (Strauss, 1976 and
unpublished results from my laboratory). However, the hydration value
(FW-DW/DW) decreases. This is an indication that the increase of water
content in ovaries is lower, in proportion, than that of dry matter.
The swelling is due to increases in cell size rather than number (Fig. 83D).
This suggests that the mechanism of swelling is the same as in gynostemia.
When excised ovaries are inserted in auxin containing solutions they do not
swell (Table 41: 37, 38). The same is true for a number of reports regarding
excised floral segments (Table 41: 15-18, 21, 22, 25-28). In other cases ovaries
do swell under such conditions (Table 41: 2, 4, 6, 8, 10, 12-14, 19, 20, 24,
39-42; Arditti and Flick, 1976). One possible explanation for these findings is
that auxin (exogenous or from pollen)-mediated increased uptake of water (i.e.,
solution) from the medium carries auxin into the ovary and the hormone
initiates swelling. Subsequent growth of ovaries is intermittent (Duncan and
Curtis, 1942a, b; 1943).
Fig. 83. Effects of pollination and auxin on ovaries.
A. Arachnanthe sulingi. (a) unpollinated; (b) seven days after pollination.
B. Rhynchostylis retusa. (a) unpollin ated; (b) seven days after pollination.
C. Aerides odoratum. (a) unpollinated; (b) six days after pollination (Fitting, 1909a).
D. Dendrobium bronkearti. (a) unpollinated; (b) pollinated (Hubert and Maton, 1939).
E. Increase in length of the ovaries of a Cymbidium hybrid under the influence of a -naph-
thylacetic acid. Average of six ovaries (Hubert and Maton, 1939).
604 J. ARDITTI

(h) Hormone production. Pollination, emasculation and auxin application


induce autocatalytic ethylene production by orchid flowers. The same is true
for ethylene, wounding and possibly other hormones (for references, see
section on ethylene).
(i ) Movement and mobilization of substrates. As already mentioned the
fresh (FW) and dry (DW) weights of gynostemia increase following pollina tion.
The same is generally true for NAA applications, but not for emascula tion.
These increases are due to (i) increased water uptake after pollination (Hsiang,
1951a), and (ii) increased influx of dry matter due to the creation of sinks as in
citrus flowers (Goldschmidt and Huberman, 1974). The increases in FW and
DW are accompanied by consistent losses from perianth segments in
Angraecum (Strauss, 1976), Coelogyne and Cymbidium (Gessner, 1948;
Hsiang, 1951a; and unpublished data from my laboratory). FW losses in
perianth segments are due to increased transpiration (Hsiang, 195la), but the
decreases in DW are mostly the result of export of substances. Evidence for
this is available from work with several species and a number of constituents.
In unpollinated flowers of Phalaenopsis amabilis, Dendrobium nobile and
Cattleya labiata, protein content remains balanced until "normal" death of
the blossom; pollination accelerates degradation (Schumacher, 1931). Total
nitrogen content per flower changes little or not at all following pollinationof
Coelogyne cristata and Cymbidium tracyanum (Table 42, p. 599; Gessner, 1948).
After pollination of these species and Cattleya labiata (Hsiang, 1951b) the
nitrogen content of perianth segments decreases and that of gynostemia and
ovaries increases (Table 42). This suggests that increases can also be expected
in perianth segments which turn green and persist on the capsule. The
mechanisms which lead to these alterations in protein content are not clear.
However, it is reasonable to assume that hydrolysis follo wed by export occurs
in senescing segments (i.e., perianth) and mobilization occurs in the ones
which persist (e.g., ovary and gynostemium). Even more interesting are the
differences (if any) between these events and their control before and after
pollinat ion. Further, the nature of the nitrogenous substances in perianth
segments, column and labella is not known. One can only speculate that in
senescing segments hydrolysates (i.e., amino acids) may predominate whereas
in those that remain, newly synthesized proteins could be a major fraction.
In Cattleya labiata the content of reducing sugars in the perianth and
gynostemium does not change 137.5 hours after pollination. The same is
true for the perianth of Cymbidium lowianum after 222 hours (Table 43;
Hsiang, 1951b). However, the content in gynostemia increases somewhat
during that period and the same is true for total sugar in both Cymbidium and
Cattleya (Table 43). After 20 days the levels of glucose and total sugar in the
gynostemium increase considerably (Table 43; Gessner, 1948). Sucrose con-
tent of Cattleya gynostemia and perianth segments increases after pollina-
TABLE 43
Sugar and Starch Content in Orchid Flowers Following Pollination (Abstracted From Gessner, 1948; Hsiang, 1951b)
Content
Glucose, mg /organ Reducing, mg g -1 FW Sucrose, mg g -1
After After After After After
Species Treatment Floral segment Initial 20 days Initial 73.5 h 137.5h 222 h Initial 73.5 h
Cattleya bowringiana Control perianth 76 84 141
Pollinated perianth 78
Control gynostemium 109 146 20
Pollinated gynostemium 146
Control 6 whole flowers 44.5 52.1 8.2
Pollinated 6 whole flowers 53.4
Cymbidium lowianum Control perianth 48.6
Pollinated perianth 49.7
NAA treated perianth 41.9
Control gynostemium 24.3 46.9 30.2
Pollinated gynostemium 28.8 68.9 11.28
NAA treated gynostemium 26.7 22
Control 6 whole flowers 0.94
Pollinated 6 whole flowers 1.83
Cymbidium Pollinated gynostemium 7.6 24.0
tracyanum Pollinated ovary 5.4 10.8
Pollinated perianth 3.0-5.4 4.6-8.3
Pollinated whole flower 45.9 71.5
Pollinated labellum 11.8 8.6
continued
TABLE 43—continued
Content
Sucrose total, mg/organ Sucrose total, mg g -1 DW Starch, mg/organ
After After After After After After After 15
Species" 137.5 h Initial 20 days Initial 73.5 h 137.5 h 222 h 222 h Initial days
C at t ley a bowringiana 14 90 98
25 103
7 129 153
30 176
5.4 52.6 57.5
13.6 67.1
Cymbidium lowianum 66.1 66.1
59.5 59.5
57.6 57.6
54.5 67.2 67.2
40.1 80.6 80.6
48.6
1.31 1.31
2.16 2.16
Cymbidium tracyanum 12.3 32.2 8 4
7.8 255
4-8.6 6.2-15
69.3 109.7
17.1 16.0 4 1
a
For reasons of space, the "Treatment" and "Floral segment" columns could not be repeated here. They correspond exactly with those given in the first
part of the table on the previous page.
ASPECTS OF ORCHID PHYSIOLOGY 607

tion, but this is not the case in Cymbidium (Hsiang, 1951b). Starch levels in
gynostemia and labella of Cymbidium decrease (Table 43; Gessner, 1948)
but increase in ovaries of Habenaria, Cymbidium lowianum, Dendrobium
nobile and D. thrysifolium (Seshagiriah, 1941; Hsiang, 1951b). Broadly
interpreted, these observations suggest hydrolysis, export and/or utilization
in some segments (perianth) coupled with accumulation in others (ovaries). As
in the case of nitrogen content, this assumption is consistent with the
hypothesis that substances from senescing organs are mobilized into those
which undergo changes and persist.
32
P phosphate taken up by intact racemes of Cymbidium accumulates
mostly in gynostemia and ovaries. Much smaller amounts are found in
perianth segments. Phosphorus content decreases with age at least in
Arundina flowers (Lim et al., 1975). Pollination intensified the uptake and
differences (Oertli and Kohl, 1960; Hsiang, 1951b). To determine the nature
and rate of phosphate redistribution we applied 32 PO4 to dorsal sepals,
columns and labella of Cymbidium cv Jungfrau (Fig. 84; Harrison and
Arditti, 1976) flowers which were pollinated, auxin treated or left undisturbed
(control). We then determined the radioactivity in ovaries, gynostemia,
dorsal sepals, sepals, petals and labella 3, 8, 24, 48, 96 and 168 hours after
treatment (Fig. 85). Our findings indicate that there is a continuing inter-
change of phosphate between floral segments. Pollination and NAA treat -
ments increase transport into gynostemia and ovaries while reducing
movement into perianth segments (Harrison and Arditti, 1976). Again the
movement is from senescing segments to active, juvenile ones. Similarly,
senescence increases efflux rates of 36 C1-, 80 Rb + and 14 C-metabolites from
morning glory flower tissues (Hanson and Kende, 1975) and phosphate
transport from "old" to "young", from tip t o base in corn leaves (Müller and
Leopold, 1966). Movements of phosphate and water in orchid flowers are, of
course, dependent on the vasculature and the following considerations
(Harrison and Arditti, 1976) are therefore relevant: in Cymbidium, three
vascular bundles from the inflorescence axis enter each flower (Swamy, 1948;
Fig. 86). One forms the midrib trace of the bract, whereas another divides
into three, reaching the two lateral petals and one lateral sepal (Fig. 86A).
The third serves the median petal (labellum or lip), one lateral sepal and the
dorsal sepal. Therefore, only two vascular bundles give rise to the six main
traces of the ovary (Fig. 86A). Traces leading to three stigmas (Ga, G2 and G3 )
separate first at the level of perianth insertion. They originate from traces which
supply the dorsal and lateral sepals (Fig. 86B). Traces into the gyno-stemium
(or column) branch from bundles serving petals (Fig. 86B, al a2 ), sepals (Fig.
86B, A 2 , A 3 , G2 , G3 ), and the dorsal sepal (Fig. 86B, A l G1 ). Thus, the perianth
(Fig. 86C) is well interconnected with the column (Fig. 86B, a, b, c). Fusion
occurs between all traces leading into the perianth (Fig. 86C). Altogether,
every segment of a Cymbidium flower is connected to
608 J. ARDITTI

Fig. 84. Cymbidium flowers, their parts and sites of 32 P application. (A) Flower of
Cymbidium cv Jungfrau. × 0.35. (B) View of parts of Cymbidium cv flower parts showing sites
of 32P application (stars). Explanation of symbols (these also include symbols for Fig. 86): A,
anther; A1; median stamen (outer whorl of stamens) or traces leading to it; A2 , and A3 , lateral
stamens (outer whorl of stamens) or traces leading to them; a 1; a2 , and a3, median stamens (inner
whorl of stamens) or traces leading to them; Br, bract; Ca, calli; DS, dorsal (median) sepal
(outer whorl of stigmas) or trace leading to it; G ls median stigma (whorl of stigmas) or trace
leading to it; G2 and G3 , lateral stigmas (whorl of stigmas) or traces leading to them; Gy,
gynostemium (column); L, labellum (lip or median petal); LP, lateral petals or traces leading
to them; LS, lateral sepals or traces leading to them; MP, traces leading to median petal
(labellum or lip); O, ovary; Pd, pedicel; St, stigma (Harrison and Arditti, 1976).
every other part by at least one vascular bundle (Fig. 86C). Hence, it seems
likely that the vascular bundles provide an interconnecting network for the
distribution of phosphate. Still, it is possible that some movement may also
take place through parenchyma cells.
Movement from dorsal sepals to labella is very limited; transport in the
reverse direction is slightly better. Both agree with previous reports concerning
the vascular anatomy of Cymbidium flowers (Swamy, 1948). The trace leading
to the labellum (MP) originates below that of the dorsal sepal (Fig. 86A).
Phosphate movement from the dorsal sepal might be largely directed away
from the labellum trace by the transpiration stream, or, transport through
the phloem (unaffected by transpiration) may be minimal. Nevertheless, even
with the interconnection of traces, the dorsal sepal and labellum are not in
direct contact (Fig. 86B). This could explain the relatively low exchange
between dorsal sepals and petals or sepals.
Fig. 85. Phosphate transport through Cymbidium cv Jungfrau floral parts. Organ an-
alysed and the site of 32P application are listed at the top of each figure, (a) 32P applied
between calli on labella and content measured at lip bases, (b) 32P applied to dorsal sepals and
content measured at their bases, (c) 32P applied to stigmas and content measured at
gynostemium (column) bases, (d) 32P applied to labella and content measured in ovaries, (f)
32
P applied to stigmas and content measured in ovaries, (g) 32P applied between calli on labella
and content measured in gynostemia. (h) 32P applied to dorsal sepals and content measured in
gynostemia. (i) 32P applied to stigmas and content measured in petals. Measurements : amounts
are expressed as picomoles (pmoles) of phosphate per mg dry weight at the intervals listed.
Transport is expressed as concentrations calculated on the basis of radioactivity present and
the initial 32P : 31P ratio. Explanation of symbols: solid line with dots, controls (c); broken
line with triangles, NAA (n); slashed lines with squares, pollinated flowers (p). Range of
coordinates: (c, d, f) from 0 to 1000; (a, b, e, g, h) from 0 to 100; (i) from 0 to 10 (Harrison
and Arditti, 1976).
610 J. ARDITTI

Fig. 86. Vasculature of Cymbidium flower (reproduced from Swamy, 1948 with per-
mission). (A) Differentiation of the six main traces of the ovary from vascular bundle of
the inflorescence axis. (B) Vasculature of a monandrous orchid like Cymbidium; (a) trans-
verse section of gynostemium (column); (b) drawing from wire-plasticine model of
vascula-ture; (c) vascular diagram. (C) Vascular supply of perianth segments in Cymbidium.
For explanation of symbols see caption to Fig. 84.

Only two traces connect the dorsal sepal and the gynostemium (Fig. 86B,
Al5 Gi), and this is reflected in relatively low movement of phosphate in
either direction. The somewhat better transport from stigmas to dorsal sepals in
unpollinated flowers might be explained on the basis of evaporative water
losses in the perianth segments. Pollination, which causes water influx into
the gynostemium, reduces net transport into the dorsal sepal.
Transport from labella to gynostemia is much greater than that into sepals
and petals. Vasculature of the flower may again be the reason. The labellum
trace is connected through the bundle from which it originates to traces
leading into the dorsal sepal and a lateral sepal. Together, these provide four
branch traces into the gynostemium (Fig. 86B, A1 and G1 plus either G2 and A2
or G3 and A3 ). Traces leading into lateral petals do not originate from the same
bundle as the labellum trace (Fig. 86A). A vascular contact is provided only by
the fusion of traces (Fig. 86C). The labellum traces and the trace for one sepal
originate from the same bundle (Fig. 86B). This plus fusion provide a limited
connection which can be responsible, in part, for the low rates of transport.
ASPECTS OF ORCHID PHYSIOLOGY 611

Stigmas are connected with all parts of the perianth through traces A1, A2 , a1,
a2 , G1, G2 and G3 of the gynostemium (Fig. 86B). Thus, the fusion between
traces (Fig. 86B) may explain the movement of 32 P from the stigma to the
perianth, gynostemium and ovary (Fig. 85).
Another point to consider is that pollination initiates ovule development in
orchid ovaries (Müller, 1868; Poddubnaya-Arnoldi, 1964) thereby creating
sinks for a number of substances including phosphate. This seems to be
happening in Cymbidium flowers because pollination increases phosphate
transport into ovaries. Furthermore, since all vascular traces lead to bundles
which pass through the ovary (Fig. 86B) phosphate (32P) wherever applied, can
move easily into that organ (Fig. 85). During the experiments described above
no radioactivity could be detected in the culture medium.
Radioactivity decreased drastically in serial sections below the ovary. This
allows speculation as to where the transport of phosphate may be occurring. If
phosphate moves into gynostemia along with water influx, transport probably
occurs in the xylem. But, if transport takes place primarily or only in the xylem,
leakage into the medium might be expected (due to diffusion from the cut ends
of vessels which are immersed in the medium) unless tyloses are formed (as
they are in cut flowers) and block outward flow. This suggests two
possibilities. First, translocation out of flowers may occur only if they are
attached to the plant as seems to be the case with apple leaves (Spencer and
Titus, 1973). Second, it is possible that at least downward movement may be in
the phloem. This assumption is supported by the fact that transport of 32 P
from stigmas is reminiscent, in principle, of IAA movement in Vanda flowers
(see section on auxins; Burg and Dijkman, 1967; Dijkman and Burg, 1970).
The lip or labellum in orchid blossoms is a modified median petal. This
modification is morphological as well as physiological. It loses less fresh
weight and dry weight than other petals, wilts more slowly and rolls inward
forming a tube. Even when NAA causes dry weight losses in labella, there is
still no fresh weight loss and water content remains high. The combination of a
swollen gynostemium and the still turgid, often rolled, labellum could exclude
would-be pollinators from already-pollinated flowers (Arditti et al., 1971b;
Gellert, 1923).
(j) Respiration. Respiratory rates of orchid flowers decrease with age
(Fig. 87A, B; Sheehan, 1952, 1954), but rise sharply with fruit set (Fig. 87B;
Rosenstock, 1956). The increase starts within one hour after pollination or
NAA application. Respiration in gynostemia reaches an initial peak 50 hours
after pollination and a second one 170 hours later (Fig. 87C; Hsiang, 1951b).
In the perianth respiration peaks at 50 hours, drops 20 hours later, reaches
another peak after another 125 hours and then decreases to levels below those
of the controls (Fig. 87C).
A very large proportion of the measured increases in respiration may be
612 J. ARDITTI

Fig. 87. Respiration by orchid flowers. A. Respiration of Cattleya mossiae flowers in relation to
age at 15°C (Sheehan, 1954). B. Respiration of Listera ovata (1), Platanthera chlorantha (2),
Neottia nidus avis (3), Epipactis latifolia (4), and Goodyera repens (5). Ordinate: respiration as
mg CO2 released h-1 g-1 fresh weight (Rosenstock, 1956). C. Change in respiratory rate (mm3 O2
g-1 dry weight h-1 ) of Cymbidium lowianum as ratio to control following pollination or auxin
treatment (Hsiang, 1951b).

centred in the placental tissues. The second peak is reminiscent of the


climacteric rise reported for several fruits (Table 44; Avadhani et al, 1971).
This observation is particularly interesting in view of a report that "there was
no evidence that a climacteric occurred in the respiration of orchid [Cattleya
mossiae] flowers between the tight bud stage and the fully mature flower."
(Sheehan, 1954).
Respiration in Cymbidium lowianum columns increased by a factor of
three, eight hours after pollination (Hsiang, 1951b). Gynostemia of Coelogyne
mooreana and Cattleya bowringiana exhibited a two-fold increase in respira-
ASPECTS OF ORCHID PHYSIOLOGY 613

TABLE 44
Respiration Rates in Placentas of Vanda cv Miss Joaquim (From Data in
Avadhani et al., 1971)
Respiration, oxygen uptake,
Time µl g -1 fresh weight
Immediately after pollination 750
Three weeks after pollination (when ovule 400
primordia are organized)
Five weeks after pollination 575
Nine weeks after pollination (when 150
8-celled embryo is formed)
Twelve weeks after pollination 375
Unspecified time period, later than 225
12 weeks

Fig. 88. Change in catalase activity (ml O2 g-1 fresh weight min -1) in Cymbidium lowianum as
ratio to control following pollination or auxin treatment (Hsiang, 1951b).

tion. This information together with that regarding placental tissues of polli-
nated Vanda cv Miss Joaquim and 30 day old unpollinated Cattleya mossiae
flowers is yet another indication of reduced activities in senescent organs and
increased metabolism in those which become the centre of new developmental
events.
(k) Enzymes. Ageing, pollination and emasculation bring about changes in
enzyme activities (Fig. 88) and complements (including isozymes) in orchid
flowers (Table 45) (Hsiang, 1951b; Lim et al., 1975;Tan and Hew, 1973;Tran
TABLE 45
Changes in Enzyme Complements in Orchid Flowers With Age or Following Pollination (Hsiang, 1951b; Lim et al., 1975; Tan
and Hew, 1973; Tran Thanh Van and Trippi, 1970; Trippi and Tran Thanh Van, 1971)
Time after
Enzyme Floral segment Orchid pollination Changes Reference
Catalase Gynostemium, Cymbidium 0-25 hours Increase Hsiang, 1951b
perianth lowianum
Catalase Gynostemium, Cymbidium 25-50 hours Decrease Hsiang, 1951b
perianth lowianum
Catalase Gynostemium, Cymbidium 50-200 hours Increase Hsiang, 1951b
perianth lowianum
Dehydrogenases
Glu-6-P dehydrogenase Corolla Phalaenopsis 24-48 hours Disappears T. T. Van and Trippi,
amabilis 1970; Trippi and
T. T. Van, 1971
Glutamate dehydrogenase Corolla Phalaenopsis 5 days Band 1 decreased T. T. Van and Trippi,
amabilis more rapidly 1970; Trippi and
than Band 2 T. T. Van, 1971
Malate dehydrogenase Corolla Phalaenopsis 5 days Band 1 decreased T. T. Van and Trippi,
amabilis more rapidly 1970; Trippi and
than Band 2 T. T. Van, 1971
Peroxidase Corolla Phalaenopsis Unopened to Of 7 bands, 3 Trippi and T. T. Van,
amabilis fully expanded anodic, 4 cathodic, 1971
flowers 5 appear
Peroxidase Corolla Phalaenopsis 5 days Middle anodic Trippi and T. T. Van,
amabilis band 2, and 1971
cathodic band 5
increase. Cathodic
band 6 appears
Peroxidase Corolla Phalaenopsis Ageing Increase of Trippi and T. T. Van,
amabilis peroxidase 1971
Peroxidase Flowers Arundina 1, 2, 4 days 3 isozymes Lim et al, 1975
graminifolia after opening
Peroxidase Flowers Arundina 6th day after 4th band Lim et al., 1975
graminifolia opening appears
Peroxidase Flowers Arundina 4th day Activity Lim et al., 1975
graminifolia increases
Phosphatase, acid Flowers Arundina 4th day 5 bands Lim et al., 1975
graminifolia increase
Polyphenol, oxidase Corolla Arachnis cv 1 day Decrease Tan and Hew, 1973
(emasculated flower) Maggie Oei
Polyphenol oxidase Corolla Arachnis cv 1-28 days Increase Tan and Hew, 1973
(emasculated flower) Maggie Oei
Polyphenol oxidase Gynostemium Arachnis cv 1 day Decrease Tan and Hew, 1973
(emasculated flower) Maggie Oei
Polyphenol oxidase Gynostemium Arachnis cv 1-21 days Increase Tan and Hew, 1973
(emasculated flower) Maggie Oei
Polyphenol oxidase Gynostemium Arachnis cv 21-28 days Decrease Tan and Hew, 1973
(emasculated flower) Maggie Oei
Polyphenol oxidase Corolla Arachnis cv 1 day Decrease Tan and Hew, 1973
(pollinated flower) Maggie Oei
Polyphenol oxidase Corolla Arachnis cv 1-7 days Increase Tan and Hew, 1973
(pollinated flower) Maggie Oei
Polyphenol oxidase Corolla Arachnis cv 7-14 days Plateau Tan and Hew, 1973
(pollinated flower) Maggie Oei
Polyphenol oxidase Corolla Arachnis cv 14-21 days Increase Tan and Hew, 1973
(pollinated flower) Maggie Oei
Polyphenol oxidase Corolla Arachnis cv 21-28 days Decrease Tan and Hew, 1973
(pollinated flower) Maggie Oei
continued
TABLE 45—continued

Time after
Enzyme Floral segment Orchid pollination Changes Reference
Gynostemium Arachnis cv
Polyphenol oxidase (pollinated flower) Maggie Oei 1 day 1-21 Decrease Tan and Hew, 1973
Gynostemium Arachnis cv
Polyphenol oxidase (pollinated flower) Maggie Oei days 21-28 Increase Tan and Hew, 1973
Gynostemium Arachnis cv
Polyphenol oxidase (pollinated flower) Maggie Oei days Decrease Tan and Hew, 1973
ASPECTS OF ORCHID PHYSIOLOGY 617

Thanh Van and Trippi, 1970; Trippi and Tran Thanh Van, 1971). These
changes indicate that senescence in the corolla and events in other segments
originate from ". . . endogenous correlative effects inducing cytoplasmic
alterations which act as gene regulators." (Trippi and Tran Thanh Van, 1971).
The increase in peroxidase activity correlates with senescence because this
enzyme has been shown to be involved in ethylene synthesis (Lim et al.,
1975), and, senescence of orchid flowers is affected by ethylene (see sections on
senescence and ethylene). The increases of acid phosphatase in corollas can
also be correlated with senescence (Lim et al., 1975).
Flowers which senesce faster, like those of Cattleya have a lower
poly-phenol oxidase (PPO) activity than those of longer lived ones (for
example, Arachnis cv Maggie Oei). Levels of PPO in the corolla of A. cv Maggie
Oei decrease with age (Tan and Hew, 1973.) After emasculation there were no
changes in the corolla but a decrease followed by an increase in the
gyno-stemium (Table 45). An increase was also noted following pollination
(Table 45; Tan and Hew, 1973). Therefore it has been noted that "It is
tempting to suggest that the changes in polyphenol oxidase of orchid flowers
[pollinated and depollinated (emasculated)] might be correlated with the
changes in ethylene production." (Tan and Hew, 1973.) If so, the nature of the
correla tion is not clear. It is clear, however, that the changes in enzyme and
iso-enzyme levels, content and activities are correlated with the developmental
events associated with pre- and post-pollination ageing and senescence.
(l ) RNA synthesis. Much of the evidence that RNA synthesis occurs
following pollination of orchid flowers is circumstantial having been obtained
from experiments involving the application of actinomycin D (Arditti and
Knauft, 1969; Strauss, 1976). However, direct evidence from isolation of
RNA is also available (Arditti and Flick, 1976b). Similar de novo synthesis in
Petunia hybrida (Solanaceae) styles has been reported and supports the findings
with orchids. Purely theoretical considerations, based on the current state of
knowledge also support the view that post-pollination, ageing, senescence
a n d p o s t-emasculation phenomena require d e n o v o s y n t h e s i s of RNA.
(m) Miscellaneous. Nectar production may cease or be initiated following
pollination (Dodson, 1967; van der Pijl and Dodson, 1966; Wiefelspütz, 1970;
for reviews see Arditti et al., 1971b; Jeffrey et al., 1970). Production of
fragrances may also terminate following pollination, emasculation and NAA
treatments (Strauss, 1976; van der Pijl and Dodson, 1966). The mechanisms
which regulate these events are not clear.
E. INDUCTION OF PHENOMENA

Post-pollination phenomena can be induced by a number of factors


(Table 46). Pollination, of course, induces all of these phenomena in nature
(Table 46). Emasculation or the mere dislodgement of pollinia can induce
TABLE 46
Post-pollination Phenomena in Orchid Flowers as Affected by Pollination, Emasculation and Plant Hormones

Phenomena known to be induced ABA, auxin, emasculation, ABA, auxin, or Auxin, or pollinationa
by pollination only a ethylene, GA, or pollinationa pollinationa 1 . Changes in pedicel
1. Greening of sepals, petals, 1. Senescence and death of 1. Hooding of dorsal curvature
labella and gynostemia perianth sepal 2. Closing of stigma
2. Cessation of scent and 2. Anthocyanin productionb 2. Yellowing of 3. Swelling of gynostemia
nectar production 3. Anthocyanin destruction perianth segments 4. Mobilization of
3. Hydrolysis of storage and (fading) c substances from perianth
structural compounds 4. Changes in colour and into columns and ovaries
4. RNA production shape of calli 5. Loss of curvature by
5. Starch accumulation in 5. Hyponasty columns
ovaries 6. Ovule development 6. Swelling of ovaries
6. Changes in protein levels 7. Changes in enzyme 7. Ethylene evolutiond
complements 8. Altered patterns of
8. Changes in UV reflection phosphate movement
by flowers 9. Changes in fresh and
9. Folding of labella dry weight

a
All phenomena in the table are induced by pollination in one or more species. Those also brought about by other treatments or plant hormone are
listed in separate columns. As we gain more in formation on the subject, phenomena may be moved from column to column. The listing of a phenomenon in
the pollination only column indicates that no other information is available at present and not that other factors may not induce it.
b
In some orchids like Cymbidium, for example.
c
As in Vanda, for example.
d
Also induced by ethylene, emasculation and wounding.
ASPECTS OF ORCHID PHYSIOLOGY 619

some but not all symptoms. Auxins can bring about many, but not all
phenomena. Abscisic acid (ABA), gibberellins (GA 3 ) and ethylene can each
initiate fewer events. Interactions between hormones and other substances
can affect post-pollination phenomena. For example, several auxin -induced
phenomena (e.g., anthocyanin synthesis) can be inhibited or reduced in in-
tensity by protein- or RNA-synthesis inhibitors, kinetin, GA 3 or ABA; others
cannot, e.g., swelling of the column or stigmatic closure. ABA -induced
anthocyanin synthesis can be inhibited or decreased by GA 3 , auxin or kinetin
When anthocyanin production is initiated by GA 3 , it can be reduced by ABA
kinetin or auxin (Arditti et al., 1971a, b). Since emasculation (like auxin and
pollination) initiates ethylene evolution, a suggestion could be made that all
post-pollination phenomena are initiated or controlled by the gas. However,
work with Cymbidium flowers has shown that ethylene induces anthocyanin
synthesis, but not other phenomena like swelling of the column (Table 47)
(Arditti et al., 1973). Some agents (e.g., ethionine) may stimulate ethylene
production while inhibiting anthocyanin synthesis.

1. Pollination
Pollen, dead or alive, can initiate all post-pollination phenomena in all
orchid species (Table 46) (Curtis, 1943; Duncan and Schubert, 1943; Fitting,
1909b, 1910, 1921; Laibach, 1930; Morita, 1918; McCle lland, 1919 among
others; for reviews see Arditti 1969, 1976a, b; Withner, 1974). However, the
effect of dead pollen may be less pronounced. A possible reason for this maybe
the exhaustion of auxin and/or other substances in dead pollinia and con -
tinuous synthesis in germinating pollen (Hubert and Maton, 1939). Reduced
germination of Cyrtopodium punctatum pollen and complete inhibition of
Phalaenopsis grains by naphthaleneacetic acid (Curtis and Duncan, 1947)
could be taken to support this view. If one assumes that these observationsare
due to supraoptimal auxin levels it is possible that these concentrations could
result when exogenous auxin (NAA) was added to that produced by the
germinating pollen.

2. Emasculation
Removal of pollinia or merely their dis lodgement brings about the onset of
several post-pollination phenomena in Cymbidium (Arditti and Knauft, 1969;
Curtis, 1947; Knauft et al., 1970), Angraecum (Strauss, 1976) and other
orchids (Table 46). The available evidence suggests that these effects are
ethylene mediated.
Folding of the labellum in Angraecum following emasculation starts eight
hours later than in pollinated blossoms. However, after 60 hours folding in
pollinated and emasculated flowers is equal. Hyponasty of Angraecum petals
is more pronounced after emasculation than following pollination (Strauss,
1976).
TABLE 47
Effects of 10 µl-1 Ethylene on Cymbidium cv Samarkand Flowers One Week After Treatment With Ethylene and/or Auxin
(Arditti et al., 1975)
Condition of

Duration, Column width, Sepals


Treatment hours mma Stigma Column Callib and petalsc
Untreated 0 10.3 Open Curved Y NW
Untreated 164 10.6 Open Curved O NW
Pollinated 164 14.3 Closed Straight R SW
Emasculated 164 11.0 Open Curved R SW
Lanolin 164 11.0 Open Curved YO NW
NAA (25 µg/flower) 164 16.3 Closed Straight OR SW
Ethylene (10µ1 1-1) ½ 10.8 Open Curved OR VSW
Ethylene (10µ1 1-1) 1 10.5 Open Curved OR VSW
Ethylene (10µ1 1-1) 2½ 10.5 Open Curved OR VSW
Ethylene (10µ1 1-1) 5½ 10.0 Open Curved OR VSW
Ethylene (10µ1 1-1) 7 11.5 Open Curved OR VSW
Ethylene (10µ1 1-1) 16 11.0 Open Curved OR VSW
Ethylene (10µ1 1-1) 21½ 10.5 Open Curved OR SW
Ethylene (10µ1 1-1) 64 10.8 Open Curved OR SW
Ethylene (10µ1 1-1) 78 10.5 Open Curved OR SW
Ethylene (10µ1 1-1) 111 11.0 Open Curved OR SW
Ethylene (10µ1 1-1) plus
NAA (25 µg/flower) 164 15.0 Closed Straight OR SW
a
Indicative of swelling, measured along the lower edge of the stigma.
b
O, orange; R, red; Y, yellow.
c
NW, not wilted; SW, slightly wilted; VSW, very slightly wilted.
ASPECTS OF ORCHID PHYSIOLOGY 621

3. Auxin
It was inevitable perhaps that the discovery of auxin would lead to a
search for growth factors in orchid pollinia. Assays of pollinia and their
extracts with Avena coleoptiles, Vicia faba stems, Coleus petioles, Bryonia
dioica tendrils and Phaseolus multiflorus epicotyls demonstrated that they were
a rich source of what was then referred to as "Wuchstoff" and assumed to be
auxin or closely related to it (Laibach, 1930, 1932, 1933a, b; Laibach and
Maschmann, 1933; Mai, 1934; Maschmann and Laibach, 1932). Confirmation
that both Fitting's Pollenhormon and the growth substance extracted by
Laibach and Maschmann were auxin was obtained in experiments involving
the application of known auxins to orchids (Hubert and Maton, 1939).
These and subsequent experiments showed that auxin can mimic pollinia and
initiate most post-pollination phenomena (Table 46) (Arditti et al., 1971a, b;
Arditti and Knauft, 1969; Burg and Dijkman, 1967; Dijkman and Burg,
1970; Dolcher, 196la, b, 1967; Heslop-Harrison, 1957; Hubert and Maton,
1939; Hsiang, 1951a, b; Zimmerman and Hitchcock, 1939; Strauss, 1976).
Comparisons between several auxins have shown that α-naphthaleneacetic
acid is the most effective (Dolcher, 1967; Hsiang, 1951b; Huber t and Maton,
1939) and all had more pronounced effects than indoleacetic acid
(Heslop-Harrison, 1957). This auxin can also induce embryo-sac formation
(Heslop-Harrison, 1957) and cause the seed capsule to show ". . . sign of
parthenocapy . . ." (Zimmerman and Hitchcock, 1939).
In Cymbidium flowers 0.01-250 µg NAA per flower bring about straighten-
ing of the column and raise anthocyanin levels of gynostemia and labella,
Stigmatic closure is initiated by 0.05 µg per flower and the calli change from
yellow to orange-red following application of 0.001 µg per blossom (Arditti et
al., 1971b). In Vanilla 2,4-D or β −naphthoxyacetic acid can cause formation of
fruits. These develop faster than pollinated ones, but produce inferior
perfume (Bouriquet, 1954).
Stigmatic closure, loss of gynostemium curvature and changes in calli in-
duced by NAA cannot be prevented by simultaneous application of kinetin,
GA3 or ABA. However, anthocyanin content is reduced to levels below those
brought about by NAA alone. Kinetin may reduce the wilting brought about
by NAA applications (Arditti et al., 1971a, b). Anthocyanin formation and
wilting, but not swelling of the column and Stigmatic closure can be inhibited
by actinomycin D, cycloheximide, ethionine and puromycin (Arditti and
Knauft, 1969). This suggests that anthocyanin synthesis and wilting may
require de novo RNA and protein synthesis.
Auxin transport in gynostemia is polar (i.e., from stigma to ovary) and
NAA applied through the base may not reach the stigma and/or rostellum
(Arditti and Flick, 1976a). Radioactivity from 14 C-IAA has also been re-
covered from perianth segments and ovaries of Vanda and Angraecum (Fig.
89) (Burg and Dijkman, 1967; Dijkman and Burg, 1970; Hubert and Maton,
622 J. ARD1TTI

Fig. 89. Spread of 14 C-IAA from the stigmas of V. cv Petamboeran to the floral appendages.
About 20 000 cpm of carboxyl labelled 14 C-IAA (8 me mmol-1 ) at a concentration of 0-1 mM in
0-8 % agar was applied to the stigmas of each of several flowers, and after either 3, 6, or 24
hours the flowers were dissected as indicated (dotted lines). Thus the petals, sepals, and lip
were excised and cut into outer and inner halves, and a 1 mm thick cross-section was cut
from the base of the floral column. Values are total cpm radioactivity in the indicated
tissues. The figures at six hours (not shown) were only slightly higher than those at three
hours (Burg and Dijkman, 1967).

1939; Strauss, 1976). Application of 50 µg IAA per flower in addition to the


low level of radioactive auxin increased 14 C-IAA transport into perianth
segments of Angraecum whereas pollination suppressed it. However, added
IAA reduced transport into ovaries (Strauss, 1976). Cycloheximide applied
2-6 hours after 14 C IAA, reduced its transport into perianth segments but had
no effect on movement into ovaries. If applied with the auxin, cyclo -heximide
reduced transport into the ovary but after eight hours it enhanced movement
(Strauss, 1976).
Chromatograms of Angraecum and Cattleya stigma extracts following IAA
applications indicate that the auxin is conjugated with aspartic acid to form
indoleacetic acid aspartate. Cycloheximide treatments reduced the conjugate
level sharply (Strauss, 1976).
The induction of post-pollination phenomena in orchids by auxins (i.e.,
increased uptake of water, folding, wilting, cell enlargement and anthocyanin
production) can all be explained in terms of the known effects of these
hormones. Some of these effects are probably direct whereas others may be
mediated by ethylene (Burg and Dijkman, 1967; Dijkman and Burg, 1970).

4. Ethylene
What may have been one of the earliest observations on the effects of
ethylene on orchids was made as a result of a misfortune: "During the
prevalence of severe cold weather . . . in Philadelphia . . . gas [escaping]
from the main pipes under the street. . . made its way [into] the greenhouse
ASPECTS OF ORCHID PHYSIOLOGY 623

of B. A. Fahnestock . . . through the night and by the following morning . . ."


damaged or destroyed a great many plants (Fahnestock, 1858). But it seemed
"... anomalous that of the Orchidaceae . . . every individual should have
exhibited total indifference to the gaseous influence." Plants of Phajus (Bletia)
tankervilliae, Paphiopedilum (Cypripedium) venustum and Paphiopedilum
insigne and other species "... passed unscathed through the heat of the
battle." (Fahnestock, 1858). In view of our present knowledge at first glance
it is indeed surprising that a mixture containing approximately 8.59 by
volume "defiant" gas (an old name for ethylene) and hydrocarbon vapours
had no effect on orchids. However, on second thought, there may be no reason
for surprise and no anomaly because there is no mention of flowers and orchid
plants may not be very sensitive to ethylene. For example, Cattleya
lueddemanniana and Cymbidium insigne showed no epinasty when exposed to
ethylene (Crocker et al., 1932).
Unlike the plants, orchid blossoms are very sensitive to ethylene. Con-
centrations as low as 0.002 µl l-1 for 24 hours or 0.1 µl l-1 for eight hours can
damage the sepals in Cattleya flowers that are starting to open (Davidson, 1949).
The wilting of sepal tips known as "dry-sepal" injury can be brought about by
concentrations ranging from 0.1 µl l-1 for 16 hours to 0.01 µl l-1 for 48 hours
(Davidson, 1949). Dry-sepal injury which is induced by ethylene (Jester, 1952;
Saylor, 1954) has become a major cause of considerable losses by flower
producers in air polluted areas (Anonymous, 1960; Boyd and Millar, 1965;
Bracey, 1963; Clayton and Platt, 1967; Cottrell, 1968; Hetherington, 1970;
Hindawi, 1970; Kendrick et al., 1956; Middleton et al., 1956; Thompson,
1963).
Another problem caused by ethylene is damage to packaged blossoms
(Akamine, 1976; Fischer, 1949, 1950; Lindner, 1946). Brominated charcoal
(Akamine and Sakamoto, 1951), potassium permanganate-impregnated
paper, cloth and other substances as well as packaging the flowers under
nitrogen or carbon dioxide have all been used in efforts to prevent ethylene
damage to Vanda flowers, but success has not been notable. More recent
experiments with silver nitrate treatment of Phalaenopsis (Halperin and
Halevy, 1973/1974) and Cattleya (Beyer, 1976) and hypobaric storage of
Vanda (Burg, 1973) appear more promising. Ethylene oxide, an antagonist of
ethylene metabolism (Lieberman et al., 1964) which can prolong the life of
other ethylene-sensitive flowers has apparently not been tested with orchids. A
possible analogue, ethylene carbonate, has had no effects on Cattleya
flowers to date (unpublished results from my laboratory). Rhizobitoxine, a
compound isolated from Rhizobium japonicum (L-2-amino-4-(2'-amino '3'
hydroxypropoxy)-trans-3-butenoic acid) is a potent inhibitor of ethylene
effects (Owens et al., 1971). It has been applied to orchid flowers and seems to
prolong their life (M. Lieberman, personal communication).
In Cymbidium flowers ethylene (10 µl l-1 ) brought about an increase of
Fig. 90. Effects of ethylene on anthocyanin content in Cymbidium flowers, (a)
Antho-cyanin levels in Cymbidium "Samarkand" columns (gynostemia, solid line) and lips
(labella,. broken line) following exposure to 10 µl l-1 ethylene over extended periods of time.
Inasmuch as there were no marked visual changes in flowers not subjected to ethylene, none
were extracted until the end of the experiment, (b) Anthocyanin content in Cymbidium
"Samarkand" columns (gynostemia, solid line) and lips (labella, broken line) one week
after a 24 h exposure to 0.1, 1.0 and 10 µl l-1 ethylene. (c) Anthocyanin concentration in Cym-
bidium "Samarkand" flowers: fresh, untreated, emasculated, pollinated, and following
applications of NAA (25 µg per flower) plus ethylene (10 µl l-1 ) or lanolin. Closed bars,
gynostemia (columns); open bars, labella (lips); hatched bars, gynostemia corrected for
swelling. In all cases, anthocyanins were extracted and measured one week after the
start of the experiments.
ASPECTS OF ORCHID PHYSIOLOGY 625

anthocyanin content in labella and gynostemia (Fig. 90a, c). An initial in -


crease was followed by a plateau, a second rise and a decrease (Fig. 90a, c).
One week after exposure to ethylene flowers treated with 0.1 and 1.0 µl l-1
showed no appreciable increase in anthocyanin content (Fig. 90b, c). Pigment
levels increased one week after a 24 hour exposure to 10 µl l-1 ethylene (Fig.
90b, c).
Ethylene production in orchid flowers is autocatalytic (Burg and Dijkman,
1967; Dijkman and Burg, 1970). For example, fresh Cymbidium flowers e x-
posed to 0.5 µl l-1 ethylene for only two hours start to produce ". . . ab normal
endogenous . . ." amounts of the gas (Davidson, 1971). This is biologically
important since pollination, emasculation, the mere dislodgement of pollinia
and auxin also induce ethylene formation. In turn, this gas causes the
evolution of increasing amounts of ethylene and the onset of post-pollination
phenomena such as senescence and anthocyanin production or destruction.
Cattleya flowers start to produce ethylene within four hours after pollination
(Davidson, 1971). Ethylene evolution by Cymbidium blossoms becomes
noticeable within 10-1 2 hours after pollination, emasculation or NAA
treatment (unpublished results from my laboratory). In Vanda cv Miss
Joaquim ethylene evolution starts 15 hours after emasculation and after 32
hours reaches a level of 3442 µl kg -1 h -1 (Table 48). This productio n is 8.5
times greater than that of the pink passion fruit and one of ". . . the highest
reported for any plant material." (Akamine, 1963). Ethylene production by
flowers of Vanda cv Rose Marie and Vanda cv Petamboeran starts sooner after
pollination and NAA application than following emasculation (Fig. 91A, B).
Actinomycin D, cycloheximide and puromycin when applied with or
following NAA reduce but do not greatly delay the onset of ethylene evolu tion.
This suggests that initial ethylene production may depend on pre existing
proteins and RNA but subsequent evolution requires de novo synthesis.
Other post-pollination phenomena are affected similarly by these inhibitors
(Arditti and Knauft, 1969; Strauss, 1976) thereby lending support to this view.
The same is true of a report that pollination induces RNA synthesis in
gynostemia (Arditti and Flick, 1976b).
Ethionine when applied one hour before or with NAA brings about
greatly increased ethylene evolution. Even when applied alone this analogue
of methionine initiates considerable production of the gas. This suggests that
in Cymbidium flowers ethionine may substitute for methionine and act as an
ethylene precursor. An earlier report that ethylene may be formed from
ethionine (Shimokawa and Kasai, 1967) tends to support this suggestion.
However, this report has been questioned (Yang, 1974) and therefore my
suggestion is speculative. Other possibilities are that ethionine merely acti-
vates the ethylene producing system perhaps by woundingthe tissue because it
is toxic.
626 J. ARDITTI

TABLE 48
Ethylene Production in Fading Vanda cv. Miss Joaquim Blossoms
(Akamine, 1963)
Time after Ethylene Degree
removing production of
pollinia (h) fading
(µl kg h - 1 ) (%)
0 0 0
12 0 5
15 335.21 30
22 508.26 75
25 1016.53 85
28 1638.43 90
30 2582.02 95
32 3442.15 97
34 3359.00 97
36 2851.24 97a
46 1377.07 100b
49 688.53 100c
a
Slightly glassy perianth.
b
c
Glassy perianth and slimy peduncles.
Very glassy perianth, slimy and darkened peduncles, and odoriferous.

In Vanda the gynostemium produces 10.8 nl g-1 h -1 ethylene, 150 minutes


following IAA application (Burg and Dijkman, 1967) but the perianth does
not evolve any. After 24 hours sepals and petals evolve 1.2 nl g -1 h -1 ethylene, the
labellum produces 81 nl g -1 h -1 and the gynostemium releases 50 nl g -1 h -1 . This
points to the gynostemium as the earliest and major source of ethylene. The
production site in the gynostemium may well be the rostellum (Fig. 92) which
is a modified stigma. A number of observations support this suggestion. First,
stigmas of other plants (Vaccinium angustifolium, the common lowbush
blueberry) have been reported to produce ethylene following pollination (Hall
and Forsyth, 1967; Forsyth and Hall, 1969). Second, the mere dislodgement
of pollinia can bring about post-pollination changes of the type caused by
ethylene (Curtis, 1943; Duncan and Schubert, 1947).

Fig. 91. (A) Ethylene evolution by blossoms of V. cv Rose Marie after self-pollination or
removal of pollinia. Control blossoms stayed fresh for about one week and produced no ethylene
during that time. Fading of the lower petals first became evident after 8 to 12 hours in
self-pollinated blooms, and after about 24 to 30 hours in emasculated flowers (Burg and
Dijkman, 1967). (B) Ethylene evolution by blossoms of V. cv Petamboeran after pollination
(with pollinia intact), application of 5mM IAA in lanolin to the stigmas, or removal of the pollinia.
The lower petals of blossoms which were pollinated or treated with IAA began to fade after 8 to 10
hours, those of emasculated flowers after about 35 hours, and controls after about 80 hours.
Removal of the pollinia caused an initial transient production of ethylene, perhaps a wound
response, which subsided within six hours (Burg and Dijkman, 1967).
Fig. 91
Fig. 92. Rostellum of Phalaenopsis. (a) Damage to rostellum 24 hours following detach-
ment of the pollinia (Strauss, 1976). (b) Rostellum cells. CW, cell wall; M, mitochondria; V,
vacuole (Strauss, 1976).
ASPECTS OF ORCHID PHYSIOLOGY 629

Such dislodgements only disturb the rostellum-viscidium interface (Fig. 92a)


i.e., cause some, even if limited, wounding. Third, emasculation which
wounds the rostellum induces considerable ethylene evolution (Burg and
Dijkman, 1967; Dijkman and Burg, 1970) and other post-pollination
phenomena which are mediated by the gas (Burg and Dijkman, 1967;
Dijkman and Burg, 1970; Strauss, 1976; for short reviews see Arditti and
Flick, 1974; Withner, 1974). Fourth, rostellum cells contain large numbers of
mitochondria (Fig. 92b) which even if not a source of ethylene themselves may
well provide the energy needed to produce it (Strauss, 1976). Fifth, surgical
experiments involving the removal of gynostemium tips, rostella and stigmas
(Fig. 93; Table 49) have shown that excision of the rostellum reduces

Fig. 93. Cymbidium gynostemia: Structure, parts and surgical treatments, (a) Intact
gynostemium. × 2-1. (b) Gynostemium with anther cap removed, showing pollinia,
rostellum, tip and viscidium. × 2-1. (c) Gynostemium, tip excised above the rostellum. ×
1.05. (d) Self-pollination, × 1.05. (e) Emasculation, × 1.05. (f) Gynostemium following
removal of the rostellum. × 1.05. (g) Gynostemium, tip excised below the rostellum. In
some treatments pollen was placed in a drop of lanolin covering the cut surface, × 1.05. (h)
Rostellum with pollinia still attached to it. × 4.2. (i) Rostellum following removal of the
pollinia. × 4.2, a, anther cap; p, pollinia; r, rostellum; s, stigma (a cavity in Cymbidium and
most orchids); t, tip of gynostemium above the rostellum; v, viscid disc or viscidium, which
separates easily from the rostellum and attaches to pollinators.
TABLE 49
Effects of Pollination, Excision of the Rostellum, and Removal of the Gynostemium Tip on
Post-pollination Phenomena in Cymbidium Flowers (Arditti and Flick, 1974)
Gynostemium Perianth

Treatments Swelling, mm Curvaturea Callib wiltingc Stigmad


First Second first treat- Day Day Day Day Day
description description ment, min. 1 2 4 7 T∆ e 1 2 4 7 1 2 4 7 124 7 1 2 4
None None 9.8 9.9 10.0 10.8 + 1.0 c c c c y y y or nnn s o o o
Tip cut above None 9.8 10.0 10.0 9.8 o.o c c c c y y lo o nns s o o o
rostellum
Tip cut at stigma None 9.8 9.8 9.8 9.3 —0.5 c c c c y y lo o nnn s
edge
Rostellum excised None 9.8 9.8 10.3 10.0 +0.2 c c c c y y o op nn s s o o o
Pollinated None 9.8 11.5 13.1 14.2 +4.4 c c s s y o p p nsw w ¼c ½c c
Pollinated Tip cut above 30 9.8 11.0 12.7 13.0 +3.2 c c sc sc y lo o p nn s w ½c ½c c
rostellum
Pollinated Tip cut above 60 9.8 11.8 12.8 13.1 +3.3 c c sc sc y lo o p nn s w ½c ¾c c
rostellum
Pollinated Tip cut above 150 9.8 12.0 13.0 12.6 +2.8 c c sc sc y o op p nsw w ½c c c
rostellum
Pollinated Tip cut at stigma edge 30 9.8 10.0 9.8 9.5 —0.3 cc c c y yo o p n n s s
Pollinated Tip cut at stigma edge 60 9.5 9.4 9.0 8.5 —1.0 cc c c y yo yo yo n nw w
Pollinated Tip cut at stigma edge 150 9.7 9. 7 9.0 9.0 —0.7 cc c c y yo o yo n s s w
Pollinated Rostellum excised 30 9.8 11.0 12.3 14.8 +5.0 cc c sc y yo o op n sw w o ¾c c
Pollinated Rostellum excised 60 9.8 11.3 13.0 14.3 +4.5 cc sc sc y lo o op n nw w o ½c c
Pollinated Rostellum excised 150 10.3 11.2 12.3 14.3 +4.0 cc sc sc y yo o op n sw w o o c
Tip cut at stigma Pollen placed on cut less than 1 9.8 10.8 9.8 10.0 +0.2 cc c c y yo yo op n n s s
edge edgef
a
c, curved; sc, slightly curved; s, straight.
b
lo, light orange; o, orange; p, purple; r, red; y, yellow.
c
n, no wilting; s, slightly wilted; w, wilting.
d
c, closed; ¾c, three-quarters closed; ½c, half closed; ¼c, quarter closed; o, open.
e
T ∆ , total change during 7 days.
f
Pollen was placed on the cut surface immediately after removal of the tip.
ASPECTS OF ORCHID PHYSIOLOGY 631

or delays anthocyanin production in Cymbidium (Arditti and Flick, 1974)


which is an ethylene induced phenomenon (Arditti et al., 1973). However, the
excision had no effects on phenomena which are mediated by ethylene
(Table 49). Sixth, the rostellum is in close proximity to the stigma and can
therefore be affected by pollen-borne or otherwise applied auxin(s) and other
hormones or ethylene evolved by it. This does not, of course, eliminate the
possibility that the stigma itself can also produce ethylene. Despite all this
evidence, final proof that the rostellum is a major source of the gas is still
lacking since there are no reports of direct measurements of ethylene evolu-
tion by this organ. Such measurements would be easy to make but un-
fortunately no one has made them yet.
It is not surprising, perhaps, that ethylene, the only gaseous plant hormone
mediates several post-pollination phenomena. This is because as a gas it
diffuses readily and reaches all flower segments much faster than hormones
which must be transported within the plant (such as auxin). Another reason
is the fact that ethylene evolution can be induced quickly by a number of
substances as well as by mechanical means.

5. Cytokinins
Post-pollination phenomena which might be regulated to a large extent by
cytokinins may include: (i) ovule formation (for reviews see
Poddubnaya-Arnoldi, 1964; Poddubnaya-Arnoldi and Selezneva, 1957;
Veyret, 1974; Wirth and Withner, 1959) even in cases where NAA could play
an important role (Dolcher, 1967; Heslop-Harrison, 1957); (ii) protein
metabolism (Schumacher, 1931); (iii) fruit growth (Duncan and Curtis, 1942a,
b, 1943); (iv) mobilization of substrates and creation of sinks (Gessner, 1948;
Gold smith and Huberman, 1974; Harrison and Arditti, 1972, 1976; Oertli
and Kohl, 1960; Seshagiriah, 1941); (v) greening of perianth segments and
gynostemia; (vi) regulation of senescence (Arditti, 1969). Some developing
fruits and seeds are known to contain cytokinins. It is possible that they may
act as post-pollination sources of these hormones in orchid flowers. In addi-
tion it is possible that orchid pollinia may contain cytokinins (unpublished
results by G. Hayes in my laboratory). Exogenous kinetin, 0.1-10 µg per
flower, does not induce post-pollination phenomena in Cymbidium flowers.
At 100 µg per flower kinetin causes slight stigmatic closure and at 1 and 0-1
fig per flower it brings about a limited increase in anthocyanin levels. In
combination with NAA, kinetin does not prevent stigmatic closure, swelling
and straightening of the column, and wilting or changes in the calli. When
applied with GA3 and ABA, kinetin generally reduces the intensities of their
effects. However, when 10 µg kinetin per flower are combined with 1 ju,g
GA3 per flower or 0.01, 0.05 or 0.1 µg ABA per flower stigmas close without
swelling or straightening of columns (Arditti et al., 1971a, b). These observa-
tions are difficult to explain except in terms of increased auxin levels either
632 J. ARDITTI

due to increased production, reduced destruction or both. On the whole,


though, the effects of kinetin on Cymbidium flowers are in line with the
known influence of cytokinins on other systems.

6. Gibberellins
To the extent that gibberellins may be required by pollinated orchid
flowers, they are probably supplied by the young fruits and seeds (known
sources of GA3 ) and/or the pollen (according to unpublished preliminary
assays by Dr. M. S. Strauss and M. Ma, orchid pollinia may contain gibber-
ellins). Exogenous GA3 when applied alone at 0.001-1 µg per flower does not
bring about straightening or swelling of the column and stigmatic closure. At
10 and 100 µg per flower GA3 causes slight swelling and stigmatic closure.
Anthocyanin levels in gynostemia and labella increase following applica-
tions of 0.001-100 µg GA3 per flower. In combinations with NAA, GA3
reduced anthocyanin levels, but did not affect other post-pollination phe-
nomena. Combinations of ABA and GA3 have effects which are similar to
those of ABA alone, except that anthocyanin levels are reduced. Flowers
treated with GA3 plus kinetin wilted slightly in most cases but columns did not
swell and retained their curvature; calli did not develop colour and
anthocyanin content was generally equal to that of flowers given only kinetin
(Arditti et al., 197la, b).

7. Abscisic Acid
ABA, 250 and 500 ppm sprays, inhibit the development of new growths on
Cymbidium plants (Brewer et al., 1969). When applied to flowers, ABA,
0.001-1 µg per flower, induces some, but not all, post-pollination symptoms.
The hormone raises anthocyanin levels in labella, petals, sepals and gyno-
stemia; initiates wilting; brings about folding by the dorsal sepal which forms a
hood ("hooding"); and induces calli to develop colour and lose turgidity.
However, it does not cause straightening and swelling of columns or stig-
matic closure. ABA cannot inhibit most NAA induced post-pollination
phenomena, but it does lower anthocyanin levels (Arditti et al., 1971 a).

8. Interaction Between Hormones


It appears reasonable to assume that several hormones participate in
post-pollination phenomena of orchid flowers and in subsequent development
and if so, it is obvious that the system is self-sustaining. An exogenous supply
of hormones can provide some indications regarding the role played by each.
However, the system is very complex with numerous phenomena occurring
in close physical proximity and ordered succession. Therefore, the effects of
exogenously applied hormones may not be simple to explain since they may be
interacting with regulators already present in the orchid flowers. In any case,
the available data from orchid flowers indicate that all observed effects
ASPECTS OF ORCHID PHYSIOLOGY 633

are due to the treatments, but may represent effective concentrations other
than those applied.

9. Summation
Regardless of their nature, the post-pollination phenomena in orchids
serve three basic functions: (i) To protect the pollinia, ensure a close contact
between them and the stigmatic surface and provide a favourable environ-
ment for pollen germination and tube growth. This is accomplished by the
swelling of the column and stigmatic closure, (ii) To recycle substances from
senescing organs into those that become the centre of new activities. Produc -
tion and/or activation of enzymes as well as transport and/or mobilization of
substrates play important roles in this function, (iii) To render the pollinated
flowers no longer attractive to pollinators thereby conserving "pollinator
power" and increasing the likelihood that unpollinated flowers will be
visited by vectors (Arditti and Fisch, 1977). This is important because only a
small proportion of orchid flowers is pollinated as a rule. The effects of
cessation of scent and nectar production are obvious: pollinators are no
longer attracted to the flower. Folding of the perianth and movement of the
entire flower and visible colour changes also serve the same function in an
obvious manner. In addition, the visible colour changes also alter the UV
reflectance image of the flower which also renders it less attractive to pollin-
ators (Fig. 94; Kugler, 1966; Thien, 1971). This is especially true for flowers
where the UV image is an important attracting and orienting feature.
In some orchids the labellar surface markings (Figs 95, 96) and consistency
may also play important roles in orienting and attracting pollinators. Altera-
tion of these features (as for example the trichomes of Bifrenaria harrisoniae,
the calli on Cymbidium and labellar surface on other orchids), all of which are
attractants (Porsch, 1908) may serve to discourage visits by vectors to flowers
which have already been pollinated. The more rapid onset of post-pollination
phenomena in pollinated blossoms than in emasculated ones is an indication of
the extremely "fine tuning" of the system. Pollinated flowers have completed
their function, but emasculated ones have not. Their pollen has been removed
by a vector (which may deposit it on another flower) but they are still
unpollinated. Consequently a second vector, should it carry pollinia, may
pollinate such a flower. However, the vector can not obtain pollen from it and
if the vector carries no pollinia the visit will be "wasted". Hence, it would
appear that conservation of energy and survival of the species would favour
removal of emasculated blossoms, albeit at a slower rate than pollinated ones
because the "extra" time may allow pollination. This is indeed the situation
even if at first glance (on reading) the hypothesis would appear to be
teleological to some extent. In any case, this seems to be the most likely
explanation.
634 J. ARDITTI

Fig. 94. Epidendrum cochleatum flower image. Photographs taken without (left) and
with (right) a UV filter (Thien, 1971, photograph courtesy of Dr Leonard B. Thieu).
Inset, UV pattern of Orchis laxifolia (Kugler, 1966).

VI. TISSUE CULTURE

Orchids may well be the very first flowering plants of commercial value to
be propagated in vitro both through seeds and tissue cultures. Symbiotic (i.e.,
dixenic) seed germination was the first procedure to be developed (Bernard,
1909a). The second was asymbiotic germination (Knudson, 1921, 1922,
1946). Aseptic culture of flower stalk cuttings (each with only one axillary
bud) of Phalaenopsis was the third method (Rotor, 1949). Fourth, perhaps
the most dramatic, was the development of Cymbidium shoot tip (meristem)
cultures as a means of clonal propagation (Morel, 1960, 1974). This pro-
cedure, based on work with Tropaeolum and Lupinus (Ball, 1946) revolution-
ized the orchid industry and pointed to the usefulness of tissue culture for
fast clonal propagation of other plants (Murashige, 1974).
ASPECTS OF ORCHID PHYSIOLOGY 635

Fig. 95. Single epidermal cell on the upper side of the labellum of the flower of Polystachia
fallax Kraenzlin. The cell surface shows a rather complicated poly centric cuticular fold
pattern, x 2100 (Courtesy of Dr W. Barthlott).

Since the initial report, nearly twenty years ago (Morel, 1960) that Cym-
bidium can be propagated by shoot tip culture (a process now called
meri-cloning and which produces mericlones) methods have been developed
for nearly 40 genera (for reviews see Arditti 1977a; Morel, 1974; Rao, 1977).
These methods differ considerably not only as they might be applied to
separate genera and species, but also in relation to the parts of a plant. Thus,
media which are suitable for shoot tips may not supportgrowth of root or leaf
tips, and media which are suitable for leaf tips of one species may not apply to
those of another (Arditti, 1977a). Consequently, as this is being written,
development of tissue culture procedure for orchids is mostly an empirical
science (Vajrabhaya, 1976; Vajrabhaya and Vajrabhaya, 1976).
My initial intent was to review tissue culture at length in this chapter.
However, my resolve to do so weakened as this chapter increased in length
and I finally decided to present only this short account and refer the reader to
recently published reviews (Arditti, 1977a, c; Morel, 1974; Murashige, 1974;
Rao, 1977). The new information which has accumulated since these reviews
were published does not justify a new survey.
Fig. 96. (a) Labellum of Ophrys bertolonii Moretti. c. x 21. (b) Detail from (a) showing
that the omega -sign on the labellum differs not only in colour, but also in the form of the
papillate epidermal cells, x 206 (Courtesy Dr W. Barthlott).
ASPECTS OF ORCHID PHYSIOLOGY 637

VII. EPILOGUE

Through the years orchids have been considered to have magic properties,
medicinal value or magical powers (Emboden, 1974). They have been the
subject of expensive searches which even cost human lives. At one time only the
rich could own, grow, give and/or wear them. At present they are within the
reach of many as plants for the hobby trade or cut flowers and important
factors in several economies. Unfortunately, however, orchids have been
largely neglected by scientists (Dressier and Williams, 1970) despite offering a
number of advantages (Arditti, 1971c). And, only now when all orchids are
endangered or threatened are steps being taken to preserve them (for a
review see Withner, 1977).
The Orchidaceae is "... a vast family, so vast that it is beyond imagina -
tion ..." (Withner, 1977). With a staggering 20,000-30,000, orchids exceed in
number the flora of many regions. For example, they are 4 -6 times the flora of
England which has counts of 5000 species (Withner, 1977). In size orchids
range from the few millimetres (pseudobulbs are 1.1-5 mm in diameter) and
grams of the dimunitive Bulbophyllum minutissimum (Nicholls, 1969) to the
several metres (three or more) and tons of the gigantic Grammatophyllum
speciosum (Grant, 1895; Holttum, 1957). They may be soft, delicate and
herbaceous or hard and nearly (but not) woody. Their pollination ranges
from natural selfing to pseudocopulation in one and the same or different
species.
Carbon fixation patterns include C3 , C4 and CAM. The chemical diversity
and adaptive features of the orchids are enormous. They can be found in all
climatic regions and most niches populated by flowering plants (except under
water). And this list of superlatives (especially when compiled by an orchi-
dologist) can go on and on, perhaps adnauseum. Its most surprising feature is
that it can be compiled from studies of relatively few species by a limited
number of investigators. Even the most liberal of orchidologists would prob-
ably estimate the total number of workers since Theophrastus to be less than the
count of those who are currently working with E. coli.
This review has not discussed all aspects of orchid physiology. Several areas
remain untouched. However, my hope is that it contains enough of the
seductive features and mystique of the orchids to generate if not a passion at
least sufficient curiosity for these remarkable plants.

I thank Dr P. N. Avadhani, University of Singapore; Dr R. Ernst, Uni-


versity of California, Irvine; DrG. Hadley, University of Aberdeen, Scotland;
Dr A. Stoessl, Canada, Department of Agriculture, London, Ontario; J.
Michaud and Dr. M. S. Strauss, University of California, Irvine for reading
638 J. ARDITTI

and commenting on the manuscript; Dr C. R. Harrison, Saddleback College,


Mission Viejo, California for allowing me to reproduce parts of his doctoral
dissertation; Dr W. Barthlott, University of Heidelberg; Dr C. H. Dodson,
Marie Selby Botanical Gardens, Sarasota, Florida; Dr B. Kullenberg,
Uppsala University, Sweden; Dr L. B. Thien, Tulane University, New
Orleans; the editors of Scientific American for providing or allowing the use
of photographs and line drawings; and K. Thurston for photographic dark-
room work.
Our work in orchid physiology and development has been supported by
grants from the American Orchid Society; Far Best Corporation; Malihini
Orchid Society, San Jose, Ca.; Elvenia J. Slosson Fund, University of
California; Mrs Emma D. Menninger; National Science Foundation; Office
of Naval Research; Orchid Digest Corporation; Orchid Society of San
Francisco; Population Council; Public Health Service; Stanley Smith Horti-
cultural Trust; Textilana Corporation; University of California fellowships,
scholarships and research funds for several students; UCI Industrial Associ-
ates; USDA; and the South Bay Orchid Society.

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