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Volume 5, Issue 9, 1719-1732.

Research Article

ISSN 2277 7105

NUTRIENT MEDIA USED FOR MICROPROPAGATION OF

ORCHIDS: A RESEARCH REVIEW
Jayarama Reddy*
Department of Botany, St. Josephs College, 36, Langford Road, Bengaluru, India-560027.
ABSTRACT
Article Received on
20 July 2016,

Orchids create immense interest due to their high commercial value.

Revised on 11 Aug 2016,

Accepted on 31 Aug 2016

Orchids are propagated by both in vivo and in vitro methods.

DOI: 10.20959/wjpr20169-7036

Terrestrial orchids have associations with mycorrhizal fungi that are

considered necessary for seed germination and growth. Asymbiotic
germination just uses the nutrients that the seed requires to grow. A

*Corresponding Author
Dr. Jayarama Reddy
Department of Botany, St.

perusal of literature reveals that several hundred media compositions

have been used. But the most commonly used media for the

Josephs College, 36,

propagation of orchids are MS, VW and KC. In the initial stage of

Langford Road,

research only solid media were used. Later on researchers started using

Bengaluru, India-560027.

a liquid medium either in static or in moving conditions. After 1960 a

revolution of sorts took place in the clonal propagation of orchids.

Success of biotechnological approaches is dependent on regeneration of intact plants

following genetic modification, generally by micropropagation. This paper is a review of
more than 300 protocols used for the micrpropagation of orchids. Examples of currently
employed methods of recent modern techniques are being employed for micropropagation.
They have been developed to help growers to meet the demand of the horticultural industry in
the next century. Plant tissue culture, especially practical applications of micropropagation
are also presented here. Contributions of Murashige & Skoog, Knudson, Went, White,
Gautheret and others are also reviewed in this paper.
KEYWORDS: mycorrhizal, Asymbiotic, micropropagation.
INTRODUCTION
Orchids are enchanting and exquisite creation of nature. Cryopreservation is the most safe
long term method of conservation of non-orthodox seed species and somatic embryogenesis
is the most efficient micropropagation technique (Mujib, 2016). It is interesting to look at one
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of the magnificent exotic species, or, indeed, at one of our humblest forms, and observe how
profoundly it has been modified, as compared with all ordinary flowers (Jayarama Reddy,
2008). Orchids create immense interest because, orchid of their unusual flowers which are
extremely variable in size and shape with sparkling texture. They may be solitary or in spikes
emitting the fragrance of lemons, cloves or fresh lavender oil. A few are, however, highly
malodorous. Orchids are propagated vegetatively as well as by seeds. The progeny from a
cultivated orchid will be extremely heterogenous, seldom identical to the parent material.
With vegetative propagation the progeny is identical to the parent plants. Orchid cloning in
vivo is a very slow process requiring about 10 years before a clone of suitable size is obtained
(Pierik, 1987). Thus in principle, cultivated orchids are useful if propagated vegetatively in
vitro. The micropropagation of orchids by means of tissue culture has a more complex history
that is not free of controversy and includes unusual episodes (Arditti and Ernst, 1993). The
origin of all the nutrient media is the Knops solution (see table-1).Three different orchid
hybrids were successfully cultured on VW, KC and MS media by using almost all the
explants. A comprehensive protocol was developed for commercial production and
multiplication of orchids by Jayarama Reddy (2008). This paper is a review of more than 300
protocols used for the micrpropagation of orchids (see, table-2).
MATERIALS AND METHODS
Orchids or propagated by both in vivo and in vitro methods. The plant materials are either
obtained from the orchid growers or collected from the wild. They need to be grown initially
in the green houses. Seeds, leaves, shoot tips are flower buds are most commonly used
explants. Sterilized explants are inoculated on to different types of media with varied
compositions. The cultures are maintained in the growth rooms till they showed
morphogenetic responses. Sub-culturing will be usually done as orchids require different
media for different stages of growth. The plantlets produced in vitro are later gradually
transferred to green housed pots.
RESULTS AND DISCUSSION
Seed culture of Orchids
The germination of orchid seeds for a long time has been recognized as difficult and
generally uncertain of attainment. Practical orchid growers for years have attempted to find a
method which will insure germination. The difficulty of germinating seeds of orchids is due
in part to inherent causes, but undoubtedly is due also to environmental factors. The

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extremely small size of the embryo renders it liable to death if it becomes desiccated.
Generally the seeds are sown on a substratum rich in organic matter, such as sawdust, leaf
mold, wood or bark, peat, sphagnum, or mixtures of two or three substances.
Nutrient salts
Concentration in gm/l
Potassium nitrate (KNO3)
1
Magnesium
sulfate
1
(MgSO4)
Potassium phosphate dibasic
1
(K2HPO4)
Calcium
nitrate
3
(Ca(NO3)2)
Table-1: Composition of Knops Solution: after J. A. L.
W. Knop (1891)
Terrestrial orchids have associations with mycorrhizal fungi that are considered necessary for
seed germination and growth of orchid plants. While it is possible to germinate orchid seed in
vitro utilizing asymbiotic protocols (Arditti, 1992), symbiotic germination using appropriate
fungi is generally more efficient. In situ seed germination in natural habitats requires the
presence of suitable fungi for seedling development and establishment. Under natural field
conditions some orchids have been found to have a greater specificity of fungal partners than
under laboratory conditions. The term ecological specificity, applied to terrestrial orchids,
refers to the role of plantfungus interactions in defining orchid habitats under natural field
conditions, whereas those associations occurring under laboratory conditions may be termed
potential specificity and should not be assumed to be relevant to field situations. Terrestrial
orchids may have narrow or broad potential specificity, but the specificity of their
associations with endophytes in natural habitats is still poorly understood (Batty et al, 2001).
Orchid flasking
Orchid flasking is a procedure in which orchids can be grown from seeds, and is necessary
for producing orchid seedlings, since orchid seeds do not have an endosperm and usually
require a symbiotic mycorrhizal fungus to germinate. There are two different techniques for
flasking: symbiotic germination and asymbiotic germination. Symbiotic germination requires
the isolation of a mycorrhizal fungus which is added to the agar in which the seeds are grown.
Asymbiotic germination just uses the nutrients that the seed requires to grow.
In respect of orchids the first innovative horticultural method for orchid seed germination was
developed by David Moore (Morel, 1974). Fifty years after Moore's discovery, Noel Bernard

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(Bernard, 1899, 1909) made another quantum jump when he developed a method for the
symbiotic germination of orchid seeds in vitro. This is the first method derived for the in vitro
propagation of any plant. Knudson (1921, 1922 and 1924) was the first to propose a medium
for the in vitro propagation of plants in axenic culture. He developed a method for the
asymbiotic germination of orchid seeds i.e., without fungal association. Burgeff (1936) has
listed nutritional requirements regarding seed germination in 25 orchid genera. The medium
proposed by Vacin and Went (1949a,b) is widely used even today for the germination orchid
seeds. After the successful studies of Knudson (1946) on Cymbidum were published, many
workers followed his techniques and started to grow orchid seedlings under laboratory
conditions. Various workers have demonstrated that even immature seeds can be germinated
(Ito, 1955; Withner, 1955). Perkins et al. (1995) found that Microtis parviflora has a narrow
ecological specificity, as it only formed mycorrhizas with two Epulorhiza species in the field.
Morphogenisis of orchids in seed cultures of different orchids has been studied by various
authors (Sharma and Chaturvedi, 1988; Nagaraju and Parthasarathy, 1995). Variations seen in
the germination of orchid seeds under in vitro and in vivo conditions were also documented
(Clements, 1982). Arditti et. al., (1982) showed that mature seeds of Epipactis atrorubens
germinated on Nostog medium, whereas immature seeds of Epipactis gigantia germinated on
Curtis medium. Seeds of Cymbidium elegans, Coelogyne prolifera, C. cristata, C porsecta,
Aerides multiforum, Sarcanthus pellidus. Bulbophyllum cosmosus and Thunia alba were
incubated in tubes containing KC or VW medium. Best germination for all species was
observed on KC medium and protocorms formed with root initials in 10-12 weeks (Sharma
and Tandon, 1987). Mamura and Saitou in 1987 reported that seeds of Aerides japonicum
showed best germination after storage at - 180C for 380 days. Seedlings grew well on a pH
4.5 culture containing 0.8% agar. Baker et al., (1987) achieved seed propagation in Pontiera
and Cattleya. Effect of different media and growth regulators on seed germination and
seedling growth of Cymbidium was studied by Paek and Yeung (1991).Effect of media on the
germination of seeds and growth of five species of Dendrobium was studied by Devi et al.,
(1990). Bletilla striata seeds showed 99 percent germination on MS medium.

Seed

germination was achieved on KC medium but for seedling growth MS medium was found
suitable (Stenberg and Kane, 1998; cf. Jayarama Reddy, 2008). Morphogenesis of
Cyperidium acaule was studied in vitro (Leroux et al., 1997). Selection of best medium for in
vitro propagation of Dendrobium was studied and MS medium was found to be the best
(Kaur and Sharma, 1997; cf. Jayarama Reddy, 2008). Seeds of Oncidium ceboleta were
germinated on MS medium. Addition of BAP resulted in an increased rate of germination
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and development of seedling (Alloufa et al., 1998). Embryology of orchids has been studied
by Swamy (1949). Propagation by seed germination of an endangered medicinal orchid
Vanda tessellatewas achieved by (Prakash et al, 2013; cf. Jayarama Reddy, 2008) using MS
medium.
It is now clearly evident now that almost all orchids can be propagated via through seed
culture or flasking technique. However plants developed by this method are usually showing
variations from their parents, which is not acceptable in the orchid trade. Orchid industry
demands new, unique and identical orchids. Producing them is possible only though clonal
propagation.
Nutrient media used for the clonal propagation of Orchids
A perusal of literature reveals that several hundred media compositions have been used. But
the most commonly used media for the propagation of orchids are MS, VW and KC (see;
Fig-1). Media used for orchid tissue culture and seed germination may reflect both the
special requirements of each species and the preferences of the researchers. Different media
used for the propagation of different orchids is listed in the table. Occasionally a particular
recipe is proposed for a given genus or species and improvised by modifying the medium by
adding coconut water, tomato juice, banana pulp, different fruit juices, fish emulsion, leaf
extract, potato extract and even bear (Alberts, 1953; Withner, 1959; Arditti, 1967; Chow,
1986; Kimura and Kurihara, 1991; Villalobos et al., 1994; cf. Jayarama Reddy, 2008).
Subsequent to defining the medium, other aspects are developed such as suitable pH, addition
of growth regulators, and exploration of the use of alternative carbon sources. Use of
activated charcoal and antioxidants for minimizing browning effect etc. (Yam, et al., 1989;
Paek and Jun, 1994; Kumar, and Kumar, 1998; cf. Jayarama Reddy, 2008) is an important
example in this regard.
In the initial stage of research only solid media were used. Later on researchers started using
a liquid medium either in static or in moving conditions. In the later case, a shaker was used
to provide aeration for growing tissues (Heller, 1949, 1953; Steward et al., 1958; White,
1963). A distinction was also made by some authors between the starting, standard
maintenance and rooting media based on their suitability. In one of experiments, meristems
of Cymbidium were inoculated on a liquid medium, on which PLBs were formed. The PLBs
were then transferred on to a solid medium to obtain plantlets (Wimber, 1963, 1965). The
choice of liquid or solid medium may depend on the type of the explant and the objective of
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the culture. Some workers have used liquid media with the explants supported on a filter
bridge (Hollings and Stone, 1968) above the media. A solid medium was found economically
viable, convenient as it produces consistent results (Holdgate, 1977; cf. Jayarama Reddy,
2008). Extremely complex media are usually unnecessary for general plant propagation.
Variations of the media developed by Knudson (1922), Vacin and Went (1949), Murashige
and Skoog (1962), or by white (1963) are generally effective at both macro and micronutrient
levels with sucrose as the carbon source.
Soedjono (1988) used Bayfolan medium, which is cheaper than VW medium a successful
micropropagation of Dendrobium Walter Odumae. VW liquid medium was used to culture
Dendrobium explants (Hew, 1988; cf. Jayarama Reddy, 2008); Cymbidium ensifolium,
Oncidium cv. Gower Ramsey, Dendrobium cv. Miss Hawii, were cultured on VW and
modified MS media. (Nurani and Mohammed, 1992; cf. Jayarama Reddy, 2008). Rate of
protocorm proliferation was studied in Dendrobium candicum. Optimum proliferation
occurred on even half strength MS medium (Zhang et al., 1992). MS medium was used to
produce PLBs from the explants of Dendrobium wardianum. The encapsulated PLBs were
placed on MS agar medium to obtain plantlets (Sharma et al., 1992; cf. Jayarama Reddy,
2008). New Dogashima medium was used to obtain PLBs from the explants of Phalaenopsis
and Doritaenopsis (Tokuhara and Mii, 1993; cf. Jayarama Reddy, 2008).

In Vitro

conservation through clonal propagation of a rare and endangered orchid Renantheraim

shootiana was achieved using VW medium. PLBs obtained by using agar medium were
encapsulated (Sharma, 1994; cf. Jayarama Reddy, 2008). Callus induction, PLB formation
and plantlet production were achieved through culturing the explants of Dendrobium cv.
Madame Pompadour on modified MS medium (Mujib and Jana, 1994; cf. Jayarama Reddy,
2008). PLBs were obtained from the explants of Cymbidium cv.Nativity through culturing the
explants on MS medium (Begum, et al., 1994; cf. Jayarama Reddy, 2008). Multiplication
rate of protocorms of Dendrobium cv.Madame Pompadour by culturing PLBs on liquid KC
medium was done (Sounderrajan and Lokeswari, 1994). Explants of the bamboo orchid,
Arundina bambosifolia were cultured on Hellers, MS, Raghavan, Torrey or VW medium.
Growth pattern varied from medium to medium (Nagaraju and Parthasarathy, 1995).
Induction of shoots was achieved by culturing the explants of Phalaenopsis on VW medium
(Chen and Piluek, 1995; cf. Jayarama Reddy, 2008). A rapid in vitro regeneration system was
developed for mass propagation and plant quality improvement of Spathoglottis plicata.
PLBs were induced from explants by culturing them on half strength MS medium and

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subsequently plantlets were also produced (Wheilan et al., 1997; cf. Jayarama Reddy, 2008).
Bletilla striata cultures were initiated in VW, Mitra-Prasad-Roychowdhury, lchihashi or
modified lchihashi medium. It was found that modified lchihashi medium favoured good rate
of PLB formation (Vij and Dhiman, 1997; cf. Jayarama Reddy, 2008). Clonal propagation of
Dendrobium moschatum and Cymbidium aloifolium was tested on five different media,of
which Nitsch medium was best for formation and proliferation of PLBs (Devi, 1997). Clonal
propagation procedure was developed for efficient multiplication of Vanilla planifolia.
Explants were cultured on semisolid MS medium (George and Ravishankar, 1997).
Embryogenic callus was derived from the explants of Phalaenopsis Wedding Promenade and
Phalaenopsis Hanabonshi X Phalaenopsis Equestris 'Ilocos' by culturing them on New
Phalaenopsis Medium. Subsequently, PLBs were initiated from callus. (Islam, et al., 1998;
cf. Jayarama Reddy, 2008). Callus induction and plantlet regeneration through somatic
embyogensis in PhalaenopsisRichard Shaffer Santa Cruz was achieved by using VW
medium (Ishii et al., 1998; cf. Jayarama Reddy, 2008). Plant regeneration was achieved from
callus culture of Cymbidium ensifolium by using half - strength MS medium (Chang and
Chang, 1998).Protocol for the economical micropropagation of Phalaenopsis Queen Emma
using leaf explants and in vitro organogenesis and micropropagation of the orchid hybrid,
cattleya Naomi Kerns were reported by Jayarama Reddy (2011 and 2016)
Suspension culture techniques
Singh and Prakash (1985) developed a suspension culture technique for the micropropagation
of Epidendrum radicans; Tanaka and Sakanishi (1985; cf. Jayarama Reddy, 2008) used
modified MS solid medium for the initiation of PLBs and then cultured the PLBs on modified
KC solid medium for producing plantlets in Phalaenopsis ambalis. Phalaenopsis hybrid Nos.
1603 and 2723 were cultured on modified VW medium. A medium containing Thomale's
macronutrients and Ringe and Nitsch's minor elements and organic supplements was used to
culture Phalaenopsis (Homma and Asahira, 1985; cf. Jayarama Reddy, 2008). Phalenopsis
and Doritaenopsis were cultured on VW medium. PLBs were formed within 30 days and
PLBs grew into plantlets after 60 days of culture (Lin, 1986; cf. Jayarama Reddy, 2008).
Aranda cv. Christine was cultured using VW liquid medium shaken at 80 rpm (Widiastoety
et al., 1986); PLBs were produced from the explants of Aranthera cv. James Storie using VW
liquid medium (Widiastoely, 1986; cf. Jayarama Reddy, 2008). Explants of Cymbidium were
cultured on MS liquid medium, and number of PLBs produced per explant was studied (Fujii,
1999; cf. Jayarama Reddy, 2008).

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Growth supplements or adjuvants

Cymbidium vallambrosa, C.cv. Vieux Rose, C.cv. Dell Park were cultured on Peptobak Bacutil medium. In this medium pearl barley, oak flakes or perlite, and agar were used as
substrates. This is one of the and C. goeringii were cultured on MS medium (Kraus and
Monteiro, 1989; cf. Jayarama Reddy, 2008). Phalaenopsis Celebration was cultured on
Kyoto medium and a rapid clonal propagation procedure was developed (Wang, 1989; cf.
Jayarama Reddy, 2008). Clonal propagation of Aranda Deborah was achieved by Goh and
Wang (1990; cf. Jayarama Reddy, 2008). In this case KC liquid medium was used for initial
development. Improved growth and proliferation were seen on VW medium. Plantlet
development was faster in VW solid medium. Aeridis multiflorum, Luisiatereti folia, L.
trichorhiza, Neofinetia falcata, Papilionanthe teres, Rynchostylis retusa, Satyrium nepalense,
Vanda cristata and V. testacea were grown on Mitra, MS and KC media (Vij and Pathak,
1990; cf. Jayarama Reddy, 2008). To demonstrate the compatibility of the media, cut flower
orchid Dendrobium Joannie Ostenhault was cultured on liquid and solid VW media (Sharon
and Vasundhara, 1990; cf. Jayarama Reddy, 2008). MS medium was used to culture Bletilla
striata, Cleisostoma fordi and Pholidota chinesis (Yam, and Weatherhead, 1991; cf.
Jayarama Reddy, 2008). To produce PLBs and plantlets from explants of first uses of
alternative substrates. Protocorms developed in all cases but shoot and root development was
best on medium containing pearl barley (Kukulczanka et al., 1987; cf. Jayarama Reddy,
2008).
Sub-culturing technique
Fernando (1979) used different media to produce plants using apical and axillary buds of
Dendrobium Caesar Red Lip. They used VW liquid medium for initial culture and KC solid
medium for producing plantlets from PLBs. Huang (1984; cf. Jayarama Reddy, 2008)
proposed alternative media and method for in vitro propagation of Cattleya cultivars as he
failed to get success in commercially available "Murashige Multiplication Medium". He
developed starting solution, shoot multiplication medium and rooting medium and
successfully cultured, Cattleya guttata, C. J.A. Carbone 'orbit 10', C. J.A Carbone 'Soleil', C
Percia 'Cannizano', Laeliocattleya and Potinara hybrid clones. This was one of the earliest
attempts to employ subculture technique and to use genus specific media to obtain maximum
yield.

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Revolution in Orchid Micropropagation

After 1960 a revolution of sorts took place in the clonal propagation of orchids. Morel (1960,
1964, and 1965) inoculated meristems obtained from shoot tips of Cymbidium on KC agar
media to obtain PLBs. Then he cut these PLBs and sub-cultured them several times to obtain
thousands of plants from one shoot tip in one year. Successful results were also obtained in
Dendrobium, Lycaste, Miltonia, Odontoglossum and Phaius (Rao, 1977; cf. Jayarama Reddy,
2008) by employing Morel's procedure. Wimber (1963) applied the essentials of Morel's
procedure to a liquid system for the propagation of Cymbidium. The methods of free cell
culture in liquid media developed for Asparagus was applied to Cymbidium to obtain
plantlets successfully through PLBs (Steward and Mapes, 1971). Thus the techniques
developed by Morel, Wimber, Steward and Mapes provided a foundation for the
micropropagation of orchids that is still relevant today.

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Table-2: Orchid hybrids in vitro: 1 and 2-Cattleya hybrids showing

excellent growth; 3 and 4 -Dendrobium hybrids showing good rooting; 5Cattleya hybrid showing unusual growth due to high levels of auxins in
the medium; 6-Phalaenopsis hybrid showing only the growth of roots
due to high levels of cytokinins in the medium (C ourtesy: Jayarama
Reddy, 2008)
CONCLUSION
Having nearly 30,000 species belonging to about 900 genera, orchids are renowned for the
abundance of morphotypes, with apparently everlasting compilation of extraordinary and
fantastic adaptations, and represent a highly advanced terminal line of floral evolution in the
angiosperms. These and many more attractive features make orchids important and crucial
players in the multi-billion dollar (USD 21 billion in 2013) floriculture industry. Plant tissue
culture technology is being widely used for large scale plant multiplication. Apart from their
use as a tool of research, plant tissue culture techniques have in recent years, become of
major industrial importance in the area of plant propagation, disease elimination, plant
improvement and production of secondary metabolites. Any living part of the plant can be
used to produce hundreds and thousands of plants in a continuous process. In addition, plant
tissue culture is considered to be the most efficient technology for crop improvement by the
production of somaclonal and gametoclonal variants. In itro embryogeny has immense
fundamental and practical applications. Somatic embryogenesis process is complex and is
controlled by a variety of external and internal triggers. The technique of cryopreservation in
the protection of ornamental genetic resources of orchids using embryogenic culture/embryo

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as the tissue of choice, and the respective roles of the genotype, plant growth regulator,
environment and other regulating factors in embryogenesis are very critical. In vitro cell and
organ culture offers an alternative source for the conservation of endangered genotypes.
Germplasm conservation worldwide is increasingly becoming an essential activity due to the
high rate of disappearance of plant species especially the orchids and the increased need for
safeguarding the floristic patrimony of the countries.
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