nutrient media used for micropropagation of orchids: …

www.wjpr.net Vol 5, Issue 9, 2016.

1719

Reddy. World Journal of Pharmaceutical Research

NUTRIENT MEDIA USED FOR MICROPROPAGATION OF

ORCHIDS: A RESEARCH REVIEW

Jayarama Reddy*

Department of Botany, St. Joseph’s College, 36, Langford Road, Bengaluru, India-560027.

ABSTRACT

Orchids create immense interest due to their high commercial value.

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

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

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. 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. 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

World Journal of Pharmaceutical Research SJIF Impact Factor 6.805

Volume 5, Issue 9, 1719-1732. Research Article ISSN 2277– 7105

Article Received on

20 July 2016,

Revised on 11 Aug 2016,

Accepted on 31 Aug 2016

DOI: 10.20959/wjpr20169-7036

*Corresponding Author

Dr. Jayarama Reddy

Department of Botany, St.

Joseph’s College, 36,

Langford Road,

Bengaluru, India-560027.


1720


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 Knop’s 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


1721


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

(MgSO4)

1

Potassium phosphate dibasic

(K2HPO4)

1

Calcium nitrate

(Ca(NO3)2)

3

Table-1: Composition of Knop’s 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 plant–fungus 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

http://onlinelibrary.wiley.com/doi/10.1046/j.0028-646X.2001.00277.x/full#b6


1722


(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

http://onlinelibrary.wiley.com/doi/10.1046/j.0028-646X.2001.00277.x/full#b12


1723


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


1724


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 Heller’s, 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


1725


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).

http://www.ripublication.com/ijbbr/ijbbrv2n2_5.pdf



1726


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.


1727


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.

3 4

2 1


1728


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

excellent growth; 3 and 4 -Dendrobium hybrids showing good rooting; 5-

Cattleya 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

6 5


1729


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.

REFERENCES

1. Arditti. Fundamentals of orchid biology (ed.), John Wiley & Sons Inc., NY, 1992; 691.

2. Arditti J. Factors affecting the germination of orchid seeds. Bot. Rev., 1967; 33: 1-97.

3. Arditti J. Orchid seed germination and seedling culture-a manual. Introduction, general

outline, tropical orchids (epiphytic and terrestrial) and North American terrestrial orchids.

In: Arditti J (ed.), Orchid Biology: Reviews and perspectives. Cornell Univ. Press, Ithaca,

N.Y, 1982; II: 243-293.

4. Arditti J. Storage and longevity of orchid seeds.Malay. Orch. Rev. (Singapore), 1993; 27:

59- 63: 82-87.

5. Arditti, J Factors afecting the germination of orchid seeds. Botanical Review, 1967; 33: 1-

97.

6. Batty, AQL, Dixon, KW and Shivasithamparan, K, Constraints to symbiotic germination

of terrestrial orchid seed in a mediterraneanbushland. December, 2001; 152(3): 511–520.

7. Bernard N. Sur la germination du NeottiaNidus-avis. Compt. Rend. Acad. Sci. Paris, 128:

1253-1255.

8. Bernard N. Étudessur la tubérisation. Revue Généale de Botanique, 13: 1-92. Bot. Gaz,

1902; 111(2): 174-183.

9. Burgeff H. Samenkeimung der orchidéen und Entwic klungihrer Keimpanzen. Jena,

Germany: Verlag von Gustav Fischer, 1936.

10. Clements MA. Orchid seed germination and seedling culture- a manual: Australian native

Dendrobium nobileLindl. Sci. Rep. Saikyo Univ. Agr, 1982; 7: 35-42.

11. Devi J, Nath M, Devi M, Deka PC. Effect of different media on germination and growth

of some North East Indian sp. Of Dendrobium. J. OrchadSoci. India, 4: 1-20. ed. Cell

culture and somatic cell genetics, 1990; 2: 1-59.

12. Gautheret RJ. History of plant tissue and cell culture: A personal account. In: Vasil IK,

Gaz, 1985; 77(2): 212-219.


1730


13. Ichihashi, S and M. Yamashita Studies on the media for orchid seed germination. I. The

effects of balances inside each cation and anion group for the germination and seedling

development of Bletillastriataseeds.J. Jap. Soc. Hort. Sci., 1977; 45: 407- 413.

14. Ito I. Germination of seeds from immature pods and subsequent growth of seedlings in

orchids (epiphytic and terrestrial).In: Arditti J, ed. Orchid biology: reviews and

perspectives.II. Cornell University Press, Ithaca, New York, 1955; 295-303.

15. Ito, I. Germination of seed from immature pod and subsequent growth of seedling in

Dendrobium nobile Lindl.Sci .Reports Saikyo Univ.Agr., 1955; 7: 35-42.

16. Jayarama Reddy. Biotechnology of orchids; I.K. International Pvt Ltd. New Delhi, 2008.

17. Jayarama Reddy Protocol for the Economical Micropropagation of Phalaenopsis Queen

Emma Using Leaf Explants. International Journal of Biotechnology and Bioengineering

Research (IJBBR), 2011; 2(2): 195-206.

18. Jayarama Reddy. In Vitro Organogenesis And Micropropagation of The Orchid Hybrid,

Cattleya Naomi Kerns. European Journal of Biomedical and Pharmaceutical Sciences,

2016; 3(9): 1-5.

19. Knudson C. A new nutrient solution for germination of orchid seed. Amer Orchid Soc

Bull, 1946; 15: 214-217.

20. Knudson L. La germinacion no simbibtica de lassemillas de orquideas. Bol. Real. Soc.

Espafiola Hist. Nat, 1921; 21: 250-260.

21. Knudson L. Non symbiotic germination of orchid seeds. Bot. Gaz, 1922; 73(1): 1-25.

22. Knudson L. Further observations on non-symbiotic germination of orchid seeds. Bot.

Gaz, 1924; 77(2): 212-219.

23. Knudson L. Anew nutrient solution for the germination of orchid seeds. Amer. Orch. Soc.

Bull. 1946; 14: 214-217.

24. Leroux G, Barabe D &Vieth J. Comparative morphogenesis of Cypripedium acaule

(Orchidaceae) protocorms cultivated in vitro with or without sugar. Canadian Journal of

Botany, 1995; 73: 1391–1406.

25. Morel GM. Clonal multiplication of orchids. In: Withner CL (ed.), The Orchids:

Scientific Studies, Wiley, NY, 1974.

26. Mujib, A. Somatic Embryogenesis in Ornamentals and Its Applications. Springer (India)

Pvt. Ltd. New Delhi, 2016.

27. Nagaraju V and Parthasarathy VS In vitro propagation of Phaius and bamboo orchid by

shoot tip culture. Ann. Plant Physiol, 1995; 9: 102‐104.



https://books.google.co.in/books?id=oFRECwAAQBAJ&printsec=frontcover


1731


28. Paek KY, Yeung EC. The effects of 1-naphthaleneacetic acid and N6- benzyladenine on

the growth of Cymbidium forrestii rhizomes in vitro. Plant Cell Tiss. Org. Cult., 1991; 24:

65- 71.

29. Perkins AJ, Masuhara G, McGee PA. Specificity of the associations between Microtis

parviflora (Orchidaceae) and its mycorrhizal fungi. Australian Journal of Botany, 1995;

43: 85–91.

30. Pierik, R.L.M In vitro Culture of Higher Plants Martinus Higoff Publ. Netherlands, 1987;

344.

31. Prakash, B, Bais, RT, Singh, P and Khan, S (2013). Effect of Different pH on In vitro

Seed Germination of Vandates sellata (Roxb.)Hook.Ex. Gan Endangered Medicinal

Orchid. Advances in Life Science and Technology ISSN 2224-7181 (Paper) ISSN 2225-

062X (Online)

32. Prasad, R.N. and G.C. Mitra Nutrient requirement for germination of seed and

development ofprotocorm and seedling of Cymbidium in aseptic culture .Indian J. Exp.

Biol., 1975; 13: 123-26.

33. Sharma AK, Chaturvedi HC. Clonal multiplication and germplasm preservation of

monopodial orchids through leaf and root-tip culture. In: Vij SP, Khullar SP, eds, 1988.

34. Sharma SK, Tandon P. Axenic germination of some epiphytic orchid of Meghalaya,

India. Journal of the Orchid Society of India, 1987; 1: 85–90.

35. Stenberg ML, Kane ME. In vitro seed germination and green house cultivation of

Encyclia boothiana var. erythronioides, an endangered Florida orchid. Lindleyana, 1998;

13: 101-112.

36. Steward FC, Mapes MO. Morphogenesis in Aseptic Cell cultures of Cymbidium. Bot.

Gaz, 1971; 132(1): 65-70.

37. Swamy BGL, Embryological studies in the Orchidaceae. II. Embryology. AmMidl Nat,

1949; 41: 202–232. 10.2307/2422026

38. Vacin EF, Went FW. Some pH changes in nutrient solutions. Bot. Gaz, 1949a; 110(4):

605- 613.

39. Vacin EF, Went FW. Use of tomato juice in the asymbiotic germination of orchid seeds,

1949b.

40. Vacin, E and Went, F Some pH changes in nutrient solution. Bot Gardens Conserv News,

1949; 110: 605-613.

41. Wimber DE. Clonal multiplication of Cymbidiums through tissue culture of the shoot

meristem. Amer. Orch. Soc. Bull, 1963; 32: 105-107.


1732


42. Wimber DE. Additional observations on clonal multiplication of Cymbidiums through

culture of shoot meristems. Cymbidium Soc. News, 1965; 20: 7-10.

43. Withner CL. Orchid culture media and nutrient solutions. In: Withner CL, ed. The

Orchids. The Roland Pressn Co., New York, 1959; 589-599.

nutrient media used for micropropagation of orchids: …

Documents