•
•
•
•
•
(U.S. Department of Agriculture, 2006)
 In 1949, Rotor at Cornell University demonstrated that
plantlets could be induced by aseptic culturing of the
dormant bu...
•

•
•
•

•

•
Optimum requirements for Orchid
Culture
 LIGHT : Without enough light, orchids may produce lush looking growths
but no fl...
•
•
•
•
•
•
•
•

•
•

μ
Paphiopedilums have attained increasing demand in the flower
industry but suffer from slow plant growth and difficulty of ...
Orchid species

Medium composition

Regenerants (PLB/shoot bud)

Source of explant (invitro/in vivo)

Authors

Anacamptis ...
Leaf segment culture
 In 1965, Wimber pioneered leaf tissue culture and gave the first
well-documented report on production of PLBs
from Cymbi...
 Regeneration competence (frequency of response and number and
nature of regenerants) in foliar cultures was markedly inf...
Orchid species

Medium composition

Regenerants (PLB/shoot bud)

Source of explant (invitro/invivo)

Authors

Acampe praem...













Orchid species

Medium composition

Regenerants (PLB/shoot bud)

Authors

Aranda Deborah
KC + 1 mg/l BAP + CW
(Arachishook...







Problems in orchid
micropropagation
1) Orchid cells in tissue culture exude a large
quantity of phenolics that become toxi...
2) TRANSPLANTATION STAGE : It continues to be a major bottleneck in the
micropropagation of many orchids. A substantial nu...
•
•

•
•

•
• India's annual flower production stands at around 1000 tonnes and its
floriculture industry has a miniscule 0.01% share ...
• Modern propagation and production technology has made orchids
accessible to a much broader section of the society. The f...
Micropropagation in Orchids
Micropropagation in Orchids
Micropropagation in Orchids
Micropropagation in Orchids
Micropropagation in Orchids
Micropropagation in Orchids
Micropropagation in Orchids
Micropropagation in Orchids
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Micropropagation in Orchids

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Nazish Nehal,
M. Tech (Biotechnology),
University School of Biotechnology (USBT),
GGSIP University,
New Delhi (INDIA)

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Micropropagation in Orchids

  1. 1. • • • • • (U.S. Department of Agriculture, 2006)
  2. 2.  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.
  3. 3. • • • • • •
  4. 4. 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
  5. 5. • • • • • •
  6. 6. • • • • μ
  7. 7. 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.
  8. 8. 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 – 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)
  9. 9. Leaf segment culture
  10. 10.  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).
  11. 11.  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.
  12. 12. 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
  13. 13.   
  14. 14.      
  15. 15. 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)
  16. 16.    
  17. 17. 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 .
  18. 18. 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
  19. 19. • • • • •
  20. 20. • 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.
  21. 21. • 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.
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