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I. Introduction................................................................................................. 422
I. INTRODUCTION
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
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
". . . 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
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
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
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
Fig. 26. Growth of Cattleya aurantiaca seedlings on autoclaved (solid line) and cold
sterilized (broken line) sucrose (Harrison, 1973).
454 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
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
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
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
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. 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).
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. 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 .
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
"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
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).
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
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.
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
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)
Cymbidium finlaysonianum
T. grandiflora R.D. Fitzg.
Diuris pedunculata R.Br.
Dactylorhiza purpurella
S. cernua (L.) Richard
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.
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
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
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
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.
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).
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
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)
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
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
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
Evidence that thick-leaved orchids have CAM (Table 30) was first presented
TABLE 30
Carbon Fixation and Photophosphorylation by Orchids
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 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
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)
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
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
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
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
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
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).
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
Ethylene evolution
continued
TABLE 39—continued
Phenomenon Occurrencea, b
Swelling of column
Swelling of ovaries Arditti √c √c √c √c √c
unpublished
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
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
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
Swelling of column
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
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
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
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
continued
TABLE 41—continued
NAA in Gynostemium Ovary
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
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).
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
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
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).
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
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.
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
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
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
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).
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).
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.
REFERENCES
Kukulczanka, K. and Twarda-P redota, B. (1973). Acta Soc. Bot. Poloniae 42,
281-294.
Kullenberg, B. (1961). "Studies in Ophrys Pollination". Almquist and Wiksells,
Boktryckeri, Uppsala.
Kullenberg, B. (1973). Zoon. Suppl. 1, 9-14.
Kullenberg, B. and Bergstrom, G. (1976). Bot. Notiser 129, 11-19.
Kusano, E. (1911). J. Coll. Agr. Tokyo 4, 1-66.
Kusumoto, M. and Furukawa, J. (1977). J. Japan. Soc. hort. Sci. 45, 421-426.
LaGarde, R. V. (1929). Ann. Missouri Bot. Card. 16, 499-514.
Laibach, F. (1930). Planta 9, 341-387.
Laibach, F. (1932). Ber. dt. hot. Ges. 50, 383-390.
Laibach, F. (1933a). Ber. dt. hot. Ges. 51, 336-340.
Laibach, F. (1933b). Ber. dt. hot. Ges. 51, 386-392.
Laibach, F. and Maschmann, E. (1933). Jb. wiss. Bot. 78, 399-430.
Lebedev, A. F. (1948). Am. Rev. Soviet Med. 5, 15-27.
Lee, Y. T. (1970). "Carbon dioxide fixation in orchid leaves". B.Sc. Hons Disserta-
tion, Botany Dept, University of Singapore.
Leimbach, G. (1911). Deutsche Bot. Monatschrift 22, 114-118, 138-142, 155-
158.
Leopold, A. C. (1964). "Plant Growth and Development". McGraw-Hill Book Co.,
New York.
Leopold, A. C. (1975). Bio Science 25, 659-662.
Lerman, J. C., Deleens, E., Nato, A. and Moyse, A. (1974). Pl. Physiol. 53, 581-584.
Lerman, J. C. and Queiroz, O. (1974). Science 183, 1207-1209,
Letcher, R. M. and Nhamo, L. R. (1973). J. Chem. Soc. 12, Perkin Trancations 1,
1263-1265.
Lewis, D. H. (1973). Biol Rev. 48, 261-278.
Lieberman, M., Asen, S. and Mapson, L. W. (1964). Nature 204, 765-758.
Lim, S. L., Chin, T. Y. and Hew, C. S. (1975). Proc. Seminar Singapore Inst. Biol.
and Singapore Nat. Acad. Sci., pp. 18-26.
Lindley, J. (1830). "An Introduction to the Natural System of Botany", Longman,
London.
Lindner, R. E. (1946). Hawaii Agr. Exp. Sta. Prog. Notes 49, 1-5.
Lindt, O. (1885). Bot. Z. 43, 825-839.
Lugo-Lugo, H. (1955). Am. J. Bot. 42, 679-684.
Magnus, P. (1887). Verhandlungen Bot. Vereins Proviz Brandenburg 28, IV.
Magnus, P. (1890). Deutsche Bot. Monatschrift 8, 97-98.
Magnus, P. (1891). Deutsche Bot. Monatschrift 9, 49-51.
Magrou, J. (1924). Ann. Sci. nat. (hot.) Ser. 10, Vol. VI, 256-276.
Magrou, J. (1936). Third Int. Congr. Comp. Path., 73-80.
Magrou, J. (1938). Ann. Inst. Pasteur 60, 565-600.
Magrou, J. and Mariat, F. (1945). Ann. Inst. Pasteur 71, 49.
Magrou, J., Mariat, F. and Rose, H. (1949). C. R. hebd. Séanc. Acad. Sci. Paris
229, 665-688.
Mai, G. (1934). Jb. wiss. Bot. 79, 681-713.
Malguth, R. (1902). "Biologische Eigentümlichkeiten der Früchte epiphytischer
Orchideen". Inaugural Dissertation, Univ. Breslau.
Mariat, F. (1944). Rev. Hort. 29, 68-69.
Mariat, F. (1948). Rev. Gen. Bot. 55, 229-243.
Mariat, F. (1952). Rev. Gen. Bot. 59, 324-377.
Mariat, F. (1954). Action de vitamines sur les germinations d'orchidées. In "Le
ASPECTS OF ORCHID PHYSIOLOGY 649
Wheeler, L. C. and Ramos, L. J. (1965). Mimeogr., Dep. Biol. Sci., Univ. So. Cal.,
Los Angeles.
Wiefelspütz, E. (1970). Die Orchidee (Special Issue).
Wiesmeyer, H. and Hofsten, A. V. (1976). Electron microscopy of orchid seedlings.
In "First Symposium on the Scientific Aspects of Orchids" (H. H. Szmant and
J. Wemple, Eds), pp. 16-26, Chem. Dep., Univ. Detroit.
Wiesner, J. (1865). Jb. wiss. Bot. 8, 575-594.
Wiesner, J. (1871). Bot. Ztng. 29, 619.
Wiesner, J. (1874). Flora 57, 73-77.
Wiesner, J. (1897). Sitzb. Math.-Naturwiss. Kl. Acad. wiss. Vienn. 56, 77-98.
Wildhaber, O. J. (1972). J. Ber. Naturwiss. Verein Wuppertal 25, 61-66.
Wildhaber, O. J. (1974). Die Orchidee 25, 225-230.
Williams, B. S. (1877). "Orchid Grower's Manual", Victoria and Paradise
Nurseries, London.
Williamson, B. (1970). Planta 92, 347-354.
Williamson, B. (1971). Abstr. First Int. Mycol. Conf., Exeter VII, 2A.
Williamson, B. (1973). Planta 112, 149-158.
Williamson, B. and Hadley, G. (1969). Nature 222, 582-583.
Williamson, B. and Hadley, G. (1970). Phytopathology 60, 1092-1096.
Wirth, M. and Withner, C. L. (1959). Embryology and development in the orchi-
daceae. In "The Orchids, A Scientific Survey" (C. L. Withner, Ed.), pp. 155-
188, Ronald Press, New York.
Withner, C. L. (1951). Am. Orch. Soc. Bull. 20, 276-278.
Withner, C. L. (1959). Orchid physiology. In "The Orchid, A Scientific Survey"
(C. L. Withner, Ed.), pp. 315-360, Ronald Press, New York.
Withner, C. L. (1974). Developments in orchid physiology. In "The Orchids,
Scientific Studies", pp. 129-168, Wiley-Interscience, New York.
Withner, C. L. (1977). Threatened and endangered species of orchids. In "Extinc-
tion is Forever" (G. T. Prance and T. S. Elias, Eds), New York Botanical
Garden, Bronx.
Withner, C. L., Nelson, P. K. and Wejksnora, P. J. (1974). The anatomy of orchids.
In "The Orchids, Scientific Studies" (C. L. Withner, Ed), pp. 267-374, Wiley-
Interscience, New York.
Wolff, H. (1927). Jb. wiss. Bot. 66, 1-34.
Wolff, H. (1933). Jb. wiss. Bot. 77, 657-684.
Wong, S. C. and Hew, C. S. (1973). J. Singapore Acad. Sci. 3, 150-157.
Wong, S. C. and Hew, C. S. (1975). Am. Orch. Soc. Bull. 44, 902-906.
Wright, D. (1967). Orch. Rev. 75, 120-122.
Wrigley, J. W. (1973). Mimeogr. Canberra Bot. Gdns.
Wrigley, J. (1976). Proc. Eighth World Orch. Conf., Frankfurt, 397-399.
Wróbel-Sterminska, W. (1961). Biul. Ogorod. Bot. 5, 163-165.
Wynd, F. L. (1933). Ann. Missouri Bot. Card. 20, 223-237.
Yang, S. F. (1974). Recent Advances in Phytochem. 7, 131-164.
Ziegler, A., Sheehan, T. and Poole, R. (1967). Am. Orch. Soc. Bull. 36, 185-202.
Zimmerman, P. W. and Hitchcock, A. E. (1939). Contr. Boyce Thompson Inst. Pl.
Res. 10, 481-508.
Zimmermann, W. (1932). Jb. wiss. Bot. 3, 393-506.
Zotkiewicz, R. (1961). Biul. Ogorod. Bot. 5, 102-104.