This document discusses the history and development of orchid micropropagation techniques. It notes that in the 1960s, scientists developed methods for inducing plantlets from dormant buds and shoot tips in vitro, allowing for mass propagation. Since then, techniques using shoot tips, meristems, leaves, and other explants have been developed for propagating many commercial orchid species. The document also outlines some best practices for orchid micropropagation, including addressing phenolic exudation and somaclonal variation. It concludes by discussing the growth of the global orchid industry and opportunities to expand commercial orchid cultivation in India through further development of low-cost micropropagation methods.

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(U.S. Department of Agriculture, 2006)

4.  In 1949, Rotor at Cornell University demonstrated that
plantlets could be induced by aseptic culturing of the
dormant buds on the basal node
of Phalaenopsis inflorescence.
 In 1960, Morel cultured shoot tips for obtaining virusfree Cymbidium clones in vitro. Possibility of obtaining
more than four million plants in a year from a single bud by
repeatedly sectioning and subculturing the protocorm-like
bodies (PLBs) created an intense interest among the orchid
growers and has revolutionized the orchid industry.
 In 1963, Wimber published the first detailed protocol for in
vitro production of Cymbidium starting with meristem
culture.

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7. Optimum requirements for Orchid
Culture
 LIGHT : Without enough light, orchids may produce lush looking growths
but no flowers. Orchids grown under sufficient light will have lighter,
somewhat yellow-green foliage and strong upright growths.
 AIR : Orchids roots, and eventually the entire plant, will die if they do not
get air and this is the reason that, with the exception of a few terrestrial
varieties, orchids do not grow in soil. Orchid potting media should be
open, with exceptionally good drainage, yet capable of holding sufficient
moisture to support the plant's needs.
 WATER : Proper watering consists of two separate components; quantity
and frequency. Water should be provided until it runs freely from the
drainage holes. Watering frequency can be controlled by the choice of pot.
 FERTILIZER : Typically plants are fertilized once a week during the summer
and every two weeks in the fall and winter.Fertilizers used on orchids
should contain little or no urea

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12. Paphiopedilums have attained increasing demand in the flower
industry but suffer from slow plant growth and difficulty of removing
bacterial and fungal infections from explants originating from
greenhouse plants had kept them in short supply.
In 1988,Huang reported that most bacteria and fungi could be
excluded by utilizing shoot tip explants that were considerably
smaller than those usually employed for mericloning other orchids.
In 2005 Huang etal. modified the concentration of BAP and NAA in an
attempt to simplify the above protocol so that shoot increase and
rooting could be accomplished in a single step, thus shortening the
time required for obtaining plants.
Out of all the reports on shoot tip culture reviewed only two
(Vanda and Vanilla) are monopodial orchids.
Shoot tip culture can be used as a more reliable technique for tissue
culture of sympodial orchids
like Dendrobium, Cymbidium,Arundina, Phaius and Anoectochilus.

13. Orchid species
Medium composition
Regenerants (PLB/shoot bud)
Source of explant (invitro/in vivo)
Authors
Anacamptis pyramidalis(L.) Rich.
MS + NAA/IBA/IAA; 0.5–1 mg/l) + CW
PLBs
NA
Morel (1970)
Anoectochilus formosanus Hay.
Hyponex medium + 1 mg dm−3BAP/1–
2 mg dm−3
Shoot buds
In vivo
Ket et al. (2004)
Arundina bambusifoliaLindl.
Raghavan and Torrey's
(1964) medium
Shoots
In vitro
Nagaraju and Parthasarathy (1995)
Cymbidium aloifolium (L.) Sw.
N&N medium
PLBs
In vitro
Devi et al. (1997)
Cymbidium atropurpureum (Lindley)
Rolfe.
VW + 5.0 mg/l NAA
PLBs
NA
Subramanium and Taha (2003)
D. wardianum R. Warner
MS + 2.5 mg/l BAP
PLBs
In vivo
Sharma and Tandon (1992)
Dendrobium cv. Sonia
VW + 1 mg/l BAP + 1.5 mg/l NAA
Shoot buds
In vivo
Sheela et al. (2004)
1/2 MS + 1 mg/l BAP + 7.5%CW
PLBs
Dendrobium Joannie Ostenhault
VW + 15% CW
PLBs
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Sharon and Vasundhara (1990)
Phaius tankervilleae(Banks ex Aiton)
Blume
Raghavan and Torrey's (1964) basal
medium
Shoots
In vitro
Nagaraju and Parthasarathy (1995)
Vanilla planifolia Andr.
MS + 1 mg/l BAP + 150 ml/l CW
Shoots
In vivo
Kalimuthu et al. (2006)

14. Leaf segment culture

15.  In 1965, Wimber pioneered leaf tissue culture and gave the first
well-documented report on production of PLBs
from Cymbidium leaves.
 The formation of calli and plants from leaf tips may merely
reflect an inherent trait of the Orchidaceae which is ‘brought
out’ or ‘turned on’ by the culture medium (Churchill etal.)
 Only leaf tips responded by forming callus and PLBs.
 In contrast to the above report in leaf explants of Vanda hybrid
(Vanda TMA × Vanda Joaquim) the leaf base was the most
amenable region for growth with over 80% of the isolated leaf
base cultures showing proliferation
 Young leaves responded better than the old leaves.
 Successful regeneration of a large number of uniform plants
from leaf tissue culture of endangered Renanthera
imschootiana , also known as the Red Vanda, has been reported
(Seeni and Latha, 1992).

16.  Regeneration competence (frequency of response and number and
nature of regenerants) in foliar cultures was markedly influenced by
the juvenility of the tissues in terms of size of the donor leaf.
 Successful micropropagation using leaf explants depends on many
factors like medium nutrient composition, the growth hormones,
source of the leaf (in vitro/in vivo), part of the leaf taken, explant
orientation and most importantly the age of the leaf.
PROBLEM :
Though maximum reports on orchid micropropagation surveyed have
used leaves as the starting material, popular use of leaf explantmediated mass scale cultivation of commercially important orchid
species in industries is restricted because of the time and costs
involved in standardizing the above factors.

17. Orchid species
Medium composition
Regenerants (PLB/shoot bud)
Source of explant (invitro/invivo)
Authors
Acampe praemorsa (Roxb.) Blatter and McCann.
MS + 0.5 mg/l NAA + 1 mg/l TDZ
Shoot buds
In vitro
Nayak et al. (1997a)
Aerides crispum L.
MS + 2.0 μM BAP
PLBs
In vitro
Sheelavanthmath et al. (2005)
Aerides maculosum Lindl.
MS + 2 mg/l BAP
PLBs
Invitro
Murthy and Pyati (2001)
Aerides multiflora Roxb.
MPR + 2 mg/l BAP + 0.5 mg/l NAA
PLBs
In vitro
[Vij et al., 2004a] and [Vij et al., 2004b]
Ascocenda Fifth State Beauty (Ascocentrum × Vanda)
MPR medium + 1 mg/l BAP
PLBs
In vitro and in vivo
Vij and Kaur (1999)
Dendrobium Cheingmai Pink
1/2 MS + 18.16 μM TDZ
Somatic embryos
In vitro
Chung et al. (2005)
Dendrobium hybrids (Sonia 17 and 28)
MS + 44.4 μM BAP
PLBs
In vitro
Martin and Madassery (2006)
Micropera pallida Lindl.
1/2 MS + 2 mg/l NAA + 2 mg/l BAP
PLBs
In vitro
Bhadra and Hossain (2004)
Mokara ‘Chark Kuan’
MS + 0.5 mg/l Kn
PLBs
In vitro
Abdul Ghani and Harris (1992)
Paphiopedilum philippinensehybrids (pH 59 and pH
60)
1/2 MS + 4.54 μM TDZ (pH 59)
Shoot buds
In vitro
Chen et al. (2004)
1/2 MS + 0.45 μM TDZ + 4.52 μM 2,4-D (pH 60)
Phalaenopsis ‘Taisuco Hatarot’, P. Tinny Sunshine
Annie, P. Taipei Gold ‘Golden Star, P. Tinny Galaxy
Annie’
MS + 88.8 μM BAP + 5.4 μM NAA
PLBs
In vitro
[Park et al., 2002a] and [Park et al., 2002b]
Phalaenopsis Little Steve
1/2 MS + 4.54 μM TDZ
Somatic embryos
In vitro
Kuo et al. (2005)
Renantanda ammani (Renanthera
storiei × Vanda Josephine van Breno)
VW liquid medium + 20% CW
PLBs
In vivo
Goh and Tan (1979)
Spathoglottis plicata Blume
1/2 MS + 0.2% activated charcoal + 5.37 μM
BAP + 0.44 μM NAA
PLBs
In vivo
Teng et al. (1997)
Vanda cristata Lindl.
MPR + 10 mg/l BAP + 5 mg/l IAA with increased
concentration of CuSO4·5H2O (2.2 mg/l)
PLBs
In vivo
Sharma and Vij (1997)
Vanilla planifolia Andr.
MS + 4.52 μM 2,4-D + 2.22 μM BAP
Callus
In vivo
Janarthanam and Seshadri (2008)
MS + 4.52 μM 2,4-D + 2.22 μM BAP
Shoots from the callus

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21. Orchid species
Medium composition
Regenerants (PLB/shoot bud)
Authors
Aranda Deborah
KC + 1 mg/l BAP + CW
(Arachishookeriana (Rcbh. f.)
Rchb. f. × Vanda
lamellata Lindl.)
PLBs
Goh and Wong (1990)
Ascofinetia (Ascocentrum ×
Neofinetia)
PLBs
Intuwong and Sagawa (1973)
Rhizomes
Shimasaki and Uemoto
(1991)
VW + CW/PE/BE
Cymbidium georingii Rchb. f. MS + 1–3 mg/l IAA
MS + 10 mg/l NAA + 1 mg/l
BAP
Shoots
Epidendrum radicans Pav.
Lindl.
1/2 MS + 0.1 mg/l TDZ
PLBs/shoot buds
Chen et al. (2002)
Oncidium Sweet Sugar
1/2 MS + 5 mg/l
BAP + 5 mg/l NAA
PLBs
Chen and Chang (2000)
Ponerorchis
graminifolia Rchb. f.
1/2 MS + 4.44 μM
BAP + 0.54 μM NAA
Shoot buds
Mitsukuri et al. (2009)

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24. Problems in orchid
micropropagation
1) Orchid cells in tissue culture exude a large
quantity of phenolics that become toxic to the cells
when oxidized.
REMEDY :
• Quick transfer of the explants to fresh media is
often recommended to avoid possible inhibitory
effects of exudates.
• Addition of activated charcoal and ascorbic acid
to the medium can help overcome inhibitory
effects of phenolics .

25. 2) TRANSPLANTATION STAGE : It continues to be a major bottleneck in the
micropropagation of many orchids. A substantial number of micro-propagated plants do not
survive transfer from in vitro conditions to greenhouse or field environment.
REMEDY :
Acclimatization of most micro-propagated plants can be hastened by in vitro hardening of
plantlets or after transplantation by decreasing the transpiration rate by applying antitranspirants including ABA or by increasing photosynthetic rate by elevated
CO2 concentration .
3) SOMACLONAL VARIATION : High concentrations of plant growth regulators and long
periods of culture are thought to be the main causes of variation in plants cultured in
vitro (George and Sherrington, 1984). Chen et al. (1998)have reported considerable
somaclonal variations in flower morphology, including colour and shape.
REMEDY :
Biochemical traits such as isozymes can help in the identification of somaclonal variations as
a complement to monitoring morphological traits.
The exact cause of mutations occurring in tissue cultured plants is not known, the available
evidence indicates that the use of pre-existing meristems (apical or axillary) as explant
tissues, which minimizes the requirement of growth regulators to induce growth and
development, may help to maintain clonal stability of plants derived in vitro to a great extent

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27. • India's annual flower production stands at around 1000 tonnes and its
floriculture industry has a miniscule 0.01% share in the international
market.
• Even though since the last few years orchids have made their presence felt
in the Indian cut-flower trade, orchid cultivation and commerce in India is
still at a nascent stage.
• The major species grown are Dendrobium , Vanda, Paphiopedilum
, Oncidium , Phalaenopsis and Cymbidium. Vanilla (for spice)
and Dendrobium sp. (as cut flower) have been recognized as two of the
priority plants for tissue culture propagation according to a report
prepared on market survey on tissue cultured plants (Biotech Consortium
India Limited for Department of Biotechnology (DBT) and Small Farmers’
Agri-Business Consortium, 2005).
• Varied agroclimatic zones, cheap labour, ever growing high end consumer
markets make it a highly profitable proposition to grow orchids in India
• Its production, however, is restricted mainly to the north-eastern hill
region and parts of Kerala and Karnataka.
• Unfortunately, due to lack of controls at airports, huge quantities of
diseased and rejected cut flowers, often coloured with toxic dyes where
biosafety is a suspect are dumped in the Indian cities.

28. • Modern propagation and production technology has made orchids
accessible to a much broader section of the society. The fact that all major
commercial tissue culture laboratories in the world are involved in orchid
micropropagation emphasizes how popular these flowers have become.
• Development of new hybrids and their commercial cultivation have now
become a lucrative industry in many countries of the world.
• The rising popularity of orchids has created a demand for high quality
plant materials for the development of orchid industry.
• Training workshops in tissue culture techniques and hybridization to
develop new novel hybrids will also help to create job opportunities. It is
felt that due to tremendous uniformity in vegetatively propagated
plants, the future mass-market orchids will most likely be explant
propagated and not seed propagated.
• Cost efficient protocols for mass propagation of rare, threatened and
endangered orchids, new hybrids, as well as transgenic orchids have to be
developed further in order to commercialize and conserve them.