MICROPROPAGATION IN ORCHIDS

“If nature ever showed her playfulness in the formation of flowers, this is visible in the most striking way among the orchids”.

It was rightly said in the 17th century by century German botanist Jakol Breyne.

During the last forty-five years tissue culture techniques have been extensively exploited, not only for the rapid and large-scale propagation of orchids but also for their ex situ conservation. Different protocols have been developed for the large-scale propagation of a number of orchid species through in vitro culture.

• Orchids are prized for their beautiful long lasting flowers exhibiting an incredible range of diversity in size, shape and colour.

• It is an international business covering around 8% of the world floriculture trade and has the potential to alter the economic landscape of a country.

• Large-scale multiplication of exquisite and rare hybrids using tissue culture techniques has helped orchids occupy a position as one of the top ten cut flowers.

• At present orchids are a million dollar industry in several countries like Thailand, Australia, Singapore, Malaysia and several others.

• Orchids are currently the second most valuable potted crop in the United States with a total wholesale value of US$ 144 million in 2005 (U.S. Department of Agriculture, 2006)

WHY DO WE NEED TO HAVE A RAPID AND LARGE SCALE

PRODUCTION IN ORCHIDS?

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 virus-free 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.

Some eminent works done on ORCHID Tissue

Culture…..

• As orchids are outbreeders, their propagation using seeds leads to the production of heterozygous plants. Hence, protocols providing regeneration from various vegetative parts of the plants are needed.

• The plantlets produced are usually identical to their parents (clones).

• This is of great advantage to the cut-flower industry in production of uniform blossoms during predictable periods to meet market demands.

• Moreover, orchids are inherently slow growers; their growth and development are markedly influenced by specialized microclimatic conditions and protective canopy of the floristics in their natural habitats.

• In the tropics, it may take more than two years for the orchid plantlets to reach the flowering stage. Orchids, particularly those with an epiphytic origin are, notoriously slow growing probably due to their mode of carbon acquisition. Incidentally, the most economically important orchids for cut flowers are epiphytic in origin .

• 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.

Advantages of Clonal Propagation in ORCHIDS….

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

• Shoot tip culture for in vitro propagation

• Leaf segment culture• Inflorescence axis and flower bud

culture• Rhizome segment culture• Root segment culture• Micropropagation via thin cell layer

culture

Micropropagation of orchids using various explants….

Shoot tip culture for in vitro propagation

• Wimber in 1963, published the first detailed protocol for in vitro production of Cymbidium starting with meristem culture.

• This micropropagation protocol utilizes two media, the first one is MS + 1 mg/l BAP + 3% sucrose in which the explants are initiated and subcultured for 3–4 months at a four week subculture cycle and the second one is N69 (Nitsch basal medium; + 0.5 mg/l BAP + 0.5 mg/l biotin + 0.5 mg/l folic acid and 2% sucrose in which proliferating shoots are obtained.

• From a single explant it is possible to regenerate as many as 100,000 plants in about 15 subcultures.

• Induction of embryogenic callus from shoot-tip explants of Dendrobium fimbriatu  (modified KC medium + 0.5 mg/l NAA + 1 mg/l BAP) and

 D. chrysotoxum (modified MS medium + 2 μM TDZ/BAP;have been reported by Roy and Bannerjee and Roy etal in 2007.

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.

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)

Leaf segment culture

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).

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 explant-mediated mass scale cultivation of commercially important orchid species in industries is restricted because of the time and costs involved in standardizing the above factors.

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

Inflorescence axis and flower bud culture

Inflorescence segments have emerged as effective donor organs for micropropagating orchids, ever since Rotor (1939) initiated Phalaenopsis cultures using flower stalks in vitro.

This technique has been found to have maximum application for mass propagation of the monopodial orchid Phalaenopsis, also called the moth orchid, which is fast becoming one of the most popular orchid genera but is very difficult to propagate vegetatively.

Phalaenopsis flower stalk buds cultured in vitro show three modes of growth: dormant, vegetative and reproductive [Koch, 1974]. The in vitro propagation procedures involve exposing a bud to either high auxin levels (Zimmer and Peiper, 1977), high cytokinin levels (Tanaka and Sakanishi, 1978) or high anti-auxin levels (Reisinger et al., 1976). Proliferation can be increased either by damaging the bud prior to the initial treatment or by changing growth regulator ratios after shoot formation. Effect of position of the bud on the stalk, temperature and BAP on the mode of bud growth of P. amabilis hybrid has been extensively investigated (Tanaka and Sakanishi, 1978).

Shoot production, number of leaves per shoot and their length increased when light intensity was decreased from 2.34 to 0.25 W/m2.

ThoughPhalaenopsis is most commonly propagated using inflorescence stalks, protocols for micropropagation using inflorescence segment explants have been worked out for other orchids also .

Developmental stage and age of flower stalk have been reported to have a marked influence on the frequency of PLBs formed during in vitro culture of flower stalk internodes as reported in Phalaenopsis andDoritaenopsis (Lin, 1986). The highest frequency of PLB induction occurred on sections inoculated on VW medium supplemented with 1 mg/l BAP taken after the flower stalk was formed and before the first flower became visible.

Age of the explant is another factor which affects regeneration response of orchid inflorescence explants and explants from younger sources have given better results in Dendrobium .

[Mitsukuri et al., 2009]. Younger scape nodes (a stalk from the base of the plant bearing flowers, not leaves) proved to be better explants for clonal propagation than older ones as the percentage of shoot formation was higher with younger scapes compared to scapes from full bloom flower stalks .

. Although mature inflorescences give poor shoot formation, the availability of cut flowers is in plenty and with improved techniques the shoot formation can be increased

PROBLEM : difficult sterilization procedures, phenolic exudations and recalcitrance of the tissue.

Orchid species Medium composition Regenerants (PLB/shoot bud) Authors

Aranda Deborah (Arachishookeriana (Rcbh. f.) Rchb. f. × Vanda lamellata Lindl.)

KC + 1 mg/l BAP + CW PLBs Goh and Wong (1990)

Ascofinetia (Ascocentrum × Neofinetia)

VW + CW/PE/BE PLBs Intuwong and Sagawa (1973)

Cymbidium georingii Rchb. f. MS + 1–3 mg/l IAA Rhizomes Shimasaki and Uemoto (1991)

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)

Automation of orchid mass propagation

One of the recent advances in the field of plant tissue culture is the culture of cells, somatic embryos, organogenetic propagules like bulblets, corms, microtubers or shoots in liquid suspension in bioreactors. The use of bioreactors for micropropagation helps in scaling-up of production and decreases the cost of production (Park et al., 2000). Aitken-Christie et al. (1995) suggested the possibility of mass production of protocorms in bioreactors.

About 18,000 PLBs were harvested from 20 g of inoculum in 2 l Hyponex medium in a temporary immersion culture with attached charcoal filter after eight weeks of inoculation.

It can also be used for other orchids also with little modification thereby reducing the space, labour and cost of mass propagation of orchids.

Mass production of orchid plantlets using bioreactors is very beneficial for orchids like Anoectochilus which have secondary metabolites with medicinal properties and are collected from the wild by both common man and pharmaceutical companies.

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 .

Problems in orchid micropropagation

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 anti-transpirants 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

• The largest exporters of potted orchids are Taiwan, Thailand, UK, Italy, Japan, New Zealand and Brazil while the largest importer of potted orchids is the United States.

• Today orchids such as Cymbidium, Dendrobium , Oncidium  and Phalaenopsis are marketed globally and the orchid industry has contributed substantially to the economy of many ASEAN (Association of the South East Asian Nations) countries. Orchids are currently the second most valuable potted crop in the United States with a total wholesale value of US$ 144 million.

• In 2005, 18 million potted orchids were sold at wholesale, with an average unit value of US$ 8.00

• Over 75% of all the orchids sold in the U.S. at present are Phalaenopsis sp. which had replaced Cattleya in the 1980s as the most popular orchid (Griesbach, 2002).

• Thailand, which is the world's sixth largest exporter of cut flowers, earns US$ 30 million a year from orchid exports, and Singapore earns US$ 16 million a year .

The status of orchid trade and industry overseas…..

• 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.

Orchid industry in INDIA

• 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.

CONCLUSION

THANK-YOU

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