Int.J.Curr.Microbiol.App.Sci (2017) 6(9): 1909-1927

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 9 (2017) pp. 1909-1927 Journal homepage: http://www.ijcmas.com

Review Article https://doi.org/10.20546/ijcmas.2017.609.235

A Review on: In Vitro Cloning of Orchids

Dipika Sarmah1*, Swathi Kolukunde1, Monoj Sutradhar2, Brijesh Kumar Singh2, Tapas Mandal1 and Nirmal Mandal2

1Department of Floriculture and Landscaping, 2Department of Agricultural Biotechnology, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, West Bengal-741252, *Corresponding author

ABSTRA CT

Orchids are the most fascinatingly beautiful flowers and unique group of of nature. K e yw or ds With their exotic shapes, hues and the added advantage of longevity, these flowers of rare beauty have become increasingly popular in the 21st century. They belong to the Orchid, Micro family and consisting 600-800 genera and 25,000-35,000 species. Today propagation, growing orchid is more than just a hobby, it is an international business covering around Protocorm like 10% of the world floriculture trade. Especially, some of the exquisitely rare hybrids of bodies, Cymbidium, Denrobium and orchid are among the top ten cut flowers. It has become possible by adopting in vitro tissue . culture techniques for their rapid multiplication. Since, orchids are strictly out breeders, seed propagation results in unwanted heterozygous types. So, vegetative propagation Article Info techniques require accurate regeneration protocol for obtaining in vitro cultured true to

Accepted: type plants. In fact, the technical aspect of micropropagated orchids have improvised 21 July 2017 significantly in past few years. But the loop holes in micropropagation stems from the Available Online: somaclonal variation, phenolic compound exudation explants, hardening and so on. We 10 September 2017 endeavour to include the major contribution based on orchid tissue culture starting from the pioneering work of Rotor, G. 1949 to Balilashaki et al., 2014.

Introduction

‘‘If nature ever showed her playfulness in the of the world and Cymbidium orchid in formation of flowers, this is visible in the most particular contributes 3% of the total cut striking way among the orchids.”. Most of us flower production (De and Debnath, 2011). would definitely echo this view of the seventeenth century German botanist Jakol Though orchids are grown primarily as Breyne (Davis et al., 1983). Orchids are being ornamentals, some are employed as herbal praised as one of the most favourite cut medicines and food (tubers of Cynorchis and flower and an ornamental all around the Eulophia) by many different cultures and world for their exotic beauty and long lasting tribes (Arditti, 1992). Some orchid species of shelf life. Currently, a multimillion dollar the Macodes, Ludisia, Anoectochilus and orchid industry is booming in countries like Goodyera, genera are known as jewel orchids Australia, Malaysia, Singapore, and because of their beautiful foliage. Few orchids several others. Currently, the orchids hold are also being used as spice, e.g. Vanilla. sixth position among the top ten cut flowers

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The Indian orchid diversity consisting economy of many ASEAN (Association of approximately 158 genera and 1331 species the South East Asian Nations) countries are are thriving upto an elevation of 5000 m. substantially benefitted by industrial Most of terrestrial orchids are grown in production of , Phalaenopsis, humus rich moist shady locations of North Oncidium and Cymbidium orchids (Hew, Western India whereas, hills of Western 1994; Laws, 1995). During 2005 in United Ghats harbour most of the rare small flowered States, potted orchids gained a total wholesale orchids. The North Eastern hilly regions of value of US$ 144 million (U.S. Department of India are heaven for epiphytic orchids which Agriculture, 2006) and Phalaenopsis sp. can be found to grow upto an altitude of 2000 earned 75% of all the orchids sold replacing m from sea level (Chowdhery, 2009). Some Cattleya (most popular type of 1980s) of native Indian genera like Arachnis, Griesbach, 2002. Thailand, which is the Cymbidium, Dendrobiumare, Paphiopedilum, world’s sixth largest exporter of cut flowers, are largely cultivated for cut flower earns US$ 30 million a year from orchid production. Cymbidium is popularly grown in exports, and Singapore earns US$ 16 million Arunachal Pradesh, Darjeeling and a year (Reddy, 2008). Sikkim. However, tropical orchids are found in some parts Kerala and Tamil Nadu. These The value of fresh cut orchids and buds trade native Indian orchid species rule the market during 2007-2012 is concise in table 1. In for high ornamental value and also for 2012, the total transaction of the global orchid breeding purposes. In terms of export trade reached US $ 504 million with more economy, winter and spring flowering orchids than 40 exporting and 60 importing countries of temperate regions are more preferable around the world. because quality deterioration during transport is less during colder months December to The top most orchid exporting countries are May. Netherlands (39.67%), Thailand (28.41%), Taiwan (10%), Singapore (10%) and New The status of orchid trade and industry Zealand (6%) whereas, importing countries are mainly Japan (30%), UK (12%), Italy ‘‘Orchid growing has not fully achieved the (10%), France (7%) and the USA (6%). transaction from a hobby to an industry’’ Among the total cut flower orchid species of James Shoemaker, 1957. But in twenty first world, 85% are Dendrobium, 15% are century, it would be completely unfair to Phalaenopsis and Cymbidium address orchid cultivation as a mere hobby, (Cheamuangphan et al., 2013). but it has flourished into an international industry business (Griesbach, 2002). Today According to Chugh et al., (2009), the annual orchids are dominating the cut flower and flower production of India is around 1000 potted plant commerce due to its long lasting tonnes with a miniscule international market charm, high productivity, right seasonal share of 0.01%. Even though since the last blooming, convenient packing and few years orchids have made their presence transportation (De et al., 2014). The largest felt in the Indian cut flower trade, orchid exporters of potted orchids are Taiwan, cultivation and commerce in India is still at a Brazil, Italy, Thailand, Japan, New Zealand budding stage. According to the Indian tissue and UK whereas, United States stands first in culture market survey by Biotech Consortium import (Chugh et al., 2009). Asia is the main India Limited (Department of Biotechnology) source of orchid to enter the world. The and Small Farmers’ Agri-Business

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Consortium, 2005, Dendrobium sp. (as cut already been declared as Agri Export Zone flower) and Vanilla (for spice) are the most exclusively for production of Cymbidium important plants suitable for tissue culture orchids (De et al., 2014). There is a huge propagation in India. Varied agroclimatic export potential of orchid export industry zones, cheap labour, ever growing high end from north eastern hill region especially consumer markets make it a highly profitable Sikkim with improvement in supporting proposition to grow orchids in India (Singh et industries like packaging, cold storage and al., 2008). So far, the north-eastern hill region transportation which may also generate better and few parts of Kerala and Karnataka are the employment opportunity in those only Indian regions which are being used for underprivileged states commercial orchid production. In reality, the cut flower orchid business continuously gets Dendrobium spp. hampered by lack of control in airports, huge quantities of diseased and rejected cut come under one of the most flowers, even toxic dye treated flowers get common type of orchids which are accepted dumped in Indian cities for biosafety in both cut flower and potted plant form suspicion (De, 2008). around the world. These mesmerizing orchids are known for their vast range of flower Trends in growth of some important colour, size, shape and floriferousness. The commercial orchids everlasting orchids are also available throughout the year. The most popular potted Cymbidium spp. Dendrobium growing regions in the United States are Hawaii, California and Florida. The The semi-terrestrial orchid Cymbidium is wholesale value of sales for this commodity originated from tropical and subtropical Asia. in Hawaii has been found for several decades. But, they are mainly grown in winter and In the Netherlands, production of potted spring blooming seasons and in cooler orchids is now 40 to 50 million units with climates at high elevations. The most popular Dendrobium increasing in popularity. Imports Cymbidium orchid growing countries are from Thailand, the world’s largest exporter of Philippines, Borneo islands, Japan, Malaysia, tropical cut orchids and second largest , North Australia and North Eastern supplier to the EU, accounted for 22% of India. They are highly prized as cut flowers, supplies to the EU. Thailand holds a potted plants, hanging baskets, herbal particularly strong position in Dendrobium medicines and as genetic resources. It has orchids (De et al., 2014). been considered as top commercial orchids in Europe since many years. They fetch the Phalaenopsis spp. highest price in the international markets of Singapore, Japan and the Dutch market. In Phalaenopsis is the second most important India, the hills of Sikkim, Darjeeling and cut flower and potted plant orchid of the Arunachal Pradesh with cooler nights and world. It is gaining popularity due to ease in monsoonal rain of summer set the most cultural practices and diverse flower colour, favourable climate for ideal cultivation of shape, size and delicacy. It is commercially Cymbidium. Hybrid Cymbidium orchids grown in Germany, Japan, Netherlands, (more than 350) are commercially grown in Taiwan and United States. In USA, around around 25 ha of land in Sikkim with 5 lakhs 13,500,000 Phalaenopsis were sold during spikes of annual production. East Sikkim has 2005 and currently it shares 75% of the

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Int.J.Curr.Microbiol.App.Sci (2017) 6(9): 1909-1927 purchased orchids. The export value of industry through the wide applications of Phalaenopsis from Taiwan to the United tissue culture techniques owes its credit to States increased from $8 million in 2005 to $ Morel (1960) who was the first to 13 million in 2006 (De et al., 2014). magnificently initiate Cymbidium meristem tip culture. The orchids are the symbol of the In vitro clonal propagation of orchids first successful mass micropropagated floriculture crop. The concept of in vitro culture was first proposed by Haberlandt (1902) and explicitly Factor influencing in vitro cloning verified for the first time by Steward et al., (1958). In this technique, the totipotency of Explant plant is exploited to multiply the plants from their meristematic cells in controlled invitro Explant sterilization condition. It can also be called as tissue culture, cell culture or organ culture under in Explant sterilization is most important for in vitro condition (Debergh and Read, 1991). vitro propagation. Bacterial and fungal Large scale proliferation of healthy and infections can be eliminated by proper disease free quality propagating material can immersion time of explants in the sterilization be obtained with this technique. It has also agents (Yildiz and Er, 2002). Maximum been proved to be economically viable option survival percentage of cultures of for efficient biodiversity and gene pool Dendrobium nobile var. Emma white with conservation. limited necrosis and infection was observed when sterilization of axillary buds was done The orchids are highly heterozygous and their for 8 min with 10% (v/v) (Asghar et al., vegetative propagation through division, 2011). splitting of shoots and kiekis are very slow which yields only a meagre number of plants Balilashaki et al., (2014) carried out a study even after five to six years. Although orchid to identify the best method for sterilization of seeds are produced in large number, i.e., two nodes of Phalaenopsis amabilis cv. Cool to three millions per capsule, as they lack ‘Breeze’ with different concentration of endosperm, they germinate very poorly in sodium hypochlorite and they found that best nature. assembly for sterilization is about 7% sodium hypochlorite. The propagation and cultivation of orchids was revolutionized by the discovery of Type of explant Knudson (1992), showed that orchid seeds can germinate on a relatively simple medium Rotor, 1949 first demonstrated that containing sucrose. This became the standard Phalaenopsis plantlets development from the procedure for germinating orchid seeds. The buds of inflorescence stalks through earliest report of using tissue culture micropropagation. The Morel, 1960 techniques in the clonal propagation of orchid revolutionized commercial propagation of was that of Rotor (1949). Observing that orchids by shoot meristem culture techniques. plantlets could develop from buds of However, as the techniques entails the Phalaenopsis, he cultured flower stalk nodes sacrifice of the mother plant or its entire new in vitro and obtained some plantlets. The organ growth, organ culture (leaves, roots, tremendous development made in orchid inflorescence stalks) is the fast emerging as an

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Int.J.Curr.Microbiol.App.Sci (2017) 6(9): 1909-1927 ideal method for cloning elite orchids (Arditti, growth and proliferation (Roy et al., 2007). 1977). Roy and Banerjee (2003) reported embryogenic callus induction from shoot tip Meristem/ shoot tip culture for in vitro explant of Dendrobium fimbriatum Lindl. var. propagation Oculatum in modified KC medium + 0.5 mg/L NAA + 1 mg/L BAP). However, Roy et The technique of apical meristem culture for al., (2007) observed the same response of D. virus-free production of dahlias was first chrysotoxum in modified MS medium + 2 published by Morel and Martin (1952) and mM TDZ/BAP. later on Morel, (1960) did the same for production of Cymbidium. However, Wimber It is a matter of fact that only two (Vanda and (1963) was the true pioneer to establish the Vanilla) among all of the shoot tip culture detailed protocol for in vitro production of adopted orchids are monopodial orchids Cymbidium using meristem culture as starting which have a single upright shoot with material. indeterminate growth and absence of rhizomes (Arditti, 1992). So, growth and Steward and Mapes (1971) gave an account of development retardation of mother plant is the growth and development of aseptically common in shoot tip culture of monopodial cultured cells and tissues showing that orchid. Therefore, it is not always considered Cymbidium plants could be indefinitely to be economical (Philip and Nainar, 1986). multiplied as cell cultures which gave rise to However, sympodial orchids like PLBs. Infact, the time expenditure for Dendrobium, Cymbidium, Arundina, Phaius transformation of free cells into abundant and Anoectochilus may be more adaptive protocorms were comparatively very long towards shoot tip culture. (about 9 months). But, each of these obtained protocorms was capable of giving rise to a Leaf segment culture healthy plant. Since then, shoot tips are effectively being used for the induction of Leaf segment explants are advantageous over PLBs and shoot buds of many orchids (Table other type of explants, since they are easy to 2). obtain without sacrificing the mother plant and available around the year. Wimber (1965) Vanda coerulea was rapidly multiplied by first used Cymbidium leaf tissue culture for shoot tip culture technique and clonally PLBs production. Seeni and Latha, 1992 propagated in forest segments of the Western successfully regenerated endangered Ghats (Seeni and Latha, 2000). Kalimuthu et Renanthera imschootiana Rolfe (Red Vanda) al., (2006) reported initiation, multiplication, from leaf tissue culture. The direct adventive elongation and rooting of Vanilla using MS + shoot bud formation was observed in the 1 mg/L BAP + 150 mg/L CW media under bases of the leaves. But there was no such invitro condition. response from chlorophyllous distal parts of the leaves of both flowering and in vitro Success in callus cultures in which the callus grown plants. The mature leaves of Vanda can be maintained for a prolonged period coerulea (Blue Vanda) also could not through subsequent subculture has been regenerate shoot buds or PLBs (Seeni and limited to a few orchids (Ishii et al., 1998; Latha, 2000). The influence of tissue Chang and Chang, 1998; Roy and Banerjee, juvenility in terms of donor leaf size on the 2003). It is because of the uncertainties regeneration competence (frequency of related to callus induction, necrosis, restricted response and number and nature of 1913

Int.J.Curr.Microbiol.App.Sci (2017) 6(9): 1909-1927 regenerants) was also reported in Vanda portion, orientation and age. These above Kasem’s Delight ‘Tom Boykin’ (Vij et al., factors standardization time and cost 1994) and Vanda coerulea Griff. (Vij and requirement strictly restricts mass scale Aggarwal, 2003) foliar cultures. Since, there industrial micropropagation of orchids using was variation in the response of juvenile and leaf explants (Table 3). mature leaves under same in vitro conditions, it clearly reflected the effect of explant source Inflorescence axis and flower bud culture and physiological age (Murthy and Pyati, 2001). Rotor (1949) first initiated the use of orchid inflorescence segment as explant in Chen and Chang, 2001 reported deleterious Phalaenopsis cultures using flower stalks in effect on embryo formation from Oncidium vitro. This technique is advantageous over leaf explants by auxins (IAA, IBA, NAA and others, since there is no requirement of 2, 4-D). However, they found promotive sacrificing the whole plant to obtain the effect of cytokinins like 2iP, zeatin, Kin, BAP explant. and TDZ in embryogenesis. They also found somatic embryogenesis promotion by Moth orchid Phalaenopsis is monopodial, ancymidol, paclobutrazol and demotion by which mostly was mass propagated by this GA3 and cycocel (Chen and Chang, 2003a). technique. This popular orchid genus is very They again studied with auxin transport difficult to propagate vegetatively. Out of the inhibitors (TIBA and quercetin) and auxin several techniques that have been developed antagonist (PCIB). They found somatic for in vitro propagation of this popular orchid, embryogenesis from leaf tip explant most involve culturing the dormant buds retardation by IAA and 2, 4-D (Chen and present at the basal part of the inflorescence Chang, 2004). (Griesbach, 1983).

Ethylene precursor (ACC 1-amino cyclo The in vitro cultured flower stalk buds have propane-1-carboxylic acid) was shown to three modes of growth: dormant, vegetative have a dose dependent response with lower and reproductive in most of the orchids. BA concentrations (5 mM and 10 mM) (4.40 mg/L) and NAA (1 mg/L) added to the significantly retarding and higher MS medium promoted the large number of concentrations (20-50 mM) enhancing direct shoots derived from the inflorescence stalk embryo formation. Ethylene inhibitors node of Phalaenopsis (Balilashaki et al., AgNO3 and CoCl2 retarded embryo formation 2014). Except Phalaenopsis, inflorescence suggesting that ethylene may be necessary for segment generated micropropagation of few direct somatic embryogenesis (Chen and other orchids is given table 4. Chang, 2003b). The frequency of embryogenesis and the average number of The regeneration response of orchid embryos per embryo-producing explant were inflorescence explants also depends on the both reported to be affected by explant explant age. The younger source obtained orientation. Chen and Chang (2002) observed explants of Dendrobium Miss Hawaii, higher somatic embryogenesis response with Phalaenopsis Capitola, Oncidium Gower adaxial side up orientation than abaxial side Ramsey, Ascofinetia and Ponerorchis up orientation. The use of leaf explants for graminifolia Rchb. f. have been responding effective micropropagation relies on factors well under invitro condition (Nuraini and such culture composition, growth regulators, Shaib, 1992 and Mitsukuri et al., 2009). leaf source (in vitro/in vivo), to be used 1914

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Culture medium formation from wound regions of leaf within 1 month of culture. The composition of the culture media affects the induction, regeneration, number and form Aktar et al., (2008) reported ½ MS medium to of Phalaenopsis regenerants. Kosir et al., be best suitable for in vitro regeneration of (2004) obtained Phalaenopsis sp. direct shoot Dendrobium orchid. They also observed that regeneration without callus formation from different kinds of media composition with flower stalk’s dormant buds on different organic additives significantly influence the media composition. They found rapid explant growth, development and micropropagation of a large number of regeneration. vegetative shoots without roots 160 days after inoculation in media supplemented with 2 Carbon source mg/L of 6-benzylaminopurine (BAP) and 0.5 mg/L of α –naphthalene acetic acid (NAA). The carbon sources of culture medium However, media supplemented with 4.41 supplement the low CO2 concentration, light mg/L BAP and 1 mg/L NAA ceased the energy deficiency and osmoregulation regenerants to elongate. The highest maintenance prevalent under in vitro multiplication of regenerants was observed in conditions. media containing 2 mg/L BAP and lower nitrogen concentration. Rittirat et al., (2012) used wounded protocorm segment explants of Phalaenopsis Sinha and Jahan, 2011 reported maximum cornu-cervi (Breda) Blume and Rchbf. in ½ Phalaenopsis amabilis PLBs production after MS medium supplemented with 0.1 mg/L 12 weeks of culture in media containing NAA and 0.1 mg/L TDZ for PLB production. ½MS, 2% (w/v) sucrose, 2 g/L peptone, 10% The obtained PLBs produced best quality (v/v) coconut water, 1 g/L activated charcoal, plantlets (without browning or necrotic 6-BA @ 2.0 mg/L and NAA @ 0.5 mg/L. tissues) within 5 months of culture in New Dogashima (ND) medium supplemented with Chen et al., (2002) cultured leaf explants of 0.2% AC and 4% sucrose. There was 100% Paphiopedilum phiIippinense hybrid’s (hybrid plantlet survival and those were transplanted PH59 and PH60) in modified MS medium into pots containing sphagnum moss, placed (half strength macro and full strength micro in a net house with about 60% shading and elements) free of plant growth regulator in 80% relative humidity. darkness. They observed adventitious shoot

Table.1 Value of fresh cut orchids and buds global trade (2007-2012) (Unit: Million US$)

Year 2007 2008 2009 2010 2011 2012 Import 233,734,023 252,647,645 232,568,129 251,445,523 265,702,077 267,196,847 Export 230,470,421 238,702,950 217,781,745 227,389,789 244,996,271 237,543,797 Total 464,204,444 491,350,595 450,349,874 478,835,312 510,698,348 504,740,644 (Source: De et al., 2014)

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Table.2 Micropropagation of some orchids using shoot tip explants

Regenerants Source of Orchid species Medium composition (PLB/shoot explant References bud) (in vitro/in vivo) Anacamptis pyramidalis (L.) Rich. MS+ (NAA/IBA/IAA; 0.5–1 mg/L) + CW PLBs NA Morel (1970) Anoectochilus formosanus Hay. Hyponex medium + 1mgdm-3BAP/L- Shoot buds In vivo Ketet al., (2004) 2mgdm-3 Arundina bambusifolia Lindl. Raghavan and Torrey’s (1964)medium N Shoots In vitro Nagaraju and and N medium Parthasarathy (1995) Cymbidium aloifolium (L.) Sw. VW+ 5.0 mg/L NAA PLBs In vitro Devi et al., (1997) Cymbidium atropurpureum MS+ 2.5 mg/L BAP PLBs NA Subramanium and (Lindley) Rolfe. Taha (2003) D. wardianum R. Warner VW+ 1 mg/L BAP + 1.5 mg/L NAA PLBs In vivo Sharma and Tandon (1992) Dendrobiumcv. Sonia ½ MS+ 1 mg/L BAP + 7.5%CW Shoot buds In vivo Sheelaet al., (2004 Dendrobium Joannie Ostenhault VW+ 15% CW PLBs - Sharon and Vasundhara (1990) Phaiustankervilleae (Banks ex Raghavan and Torrey’s (1964)basal PLBs In vitro Nagaraju and Aiton)Blume medium Parthasarathy (1995) Vanilla planifoliaAndr. MS+ 1 mg/L BAP + 150 ml/L CW Shoots In vivo Kalimuthuet al., (2006) (Source: Chugh, et al., 2009)

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Table.3 Micropropagation of some orchids using leaf explants.

Source of Regenerants explant Orchid species Medium composition (PLB/shoot References (in vitro/in bud) vivo) crispum L. MS+ 2.0mM BAP PLBs In vitro Sheelavanthmathet al., (2005) Aerides multiflora MPR + 2 mg/L BAP + PLBs In vitro Vijet al., Roxb. 0.5 mg/L NAA (2004a,b) Dendrobium Cheingmai ½ MS+ 18.16mM TDZ Somatic In vitro Chung et al., Pink embryos (2005) Dendrobium hybrids MS + 44.4mM BAP PLBs In vitro Martin and (Sonia 17 and 28) Madassery (2006) MicroperapallidaLindl. ½ MS+ 2 mg/L NAA+ 2 PLBs In vitro Bhadra and mg/L BAP Hossain (2004) Phalaenopsis Little ½ MS+ 4.54mM TDZ Somatic In vitro Kuoet al., (2005) Steve embryos Vanilla planifolia Andr. MS+ 4.52mM 2,4-D + Callus In vivo Janarthanam and 2.22mM BAP Shoots from Seshadri (2008) MS+ 4.52mM 2,4-D + the callus 2.22mM BAP (Source: Chugh, et al., 2009)

Table.4 Micropropagation of some orchids using inflorescence explants (Obtained from in vivo source)

Regenerants Orchid species Medium composition References (PLB/shoot bud) Aranda Deborah (Arachis KC+ 1 mg/L BAP +CW PLBs Goh and hookeriana (Rcbh. f.) Wong (1990) Rchb. f. × Vanda lamellate Lindl.) Epidendrum radicans Pav. Lindl. ½ MS+ 0.1 mg/L TDZ PLBs/shoot Chen et al., buds (2002) Oncidium Sweet Sugar ½ MS+ 5 mg/L BAP + 5 PLBs Chen and mg/L NAA Chang (2000) Ponerorchis graminifolia Rchb. f. ½ MS+ 4.44mM BAP + Shoot buds Mitsukuriet 0.54mM NAA al., (2009) (Source: Chugh, et al., 2009)

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Lim and Chong, 2014 identified autotrophic, (BAP), macro and micro elements in the mix autotrophic-heterotrophic and culture medium are major factors for heterotrophic growth in Phalaenopsis commercial Phalaenopsis micropropagation. deliciosawas seedlings with 20 g/L optimum sucrose concentration. This concentration is Niknejad et al., (2011) documented recommended when raising P. deliciosa Phalaenopsis gigantean micropropagation seedlings to retain photosynthetic activity for protocol with PLB and leaf explant. They better survival during acclimatization. observed calli and PLBs development within 6 weeks of culture in New Dogashima Novotna et al., (2007) found that best Medium (NDM) supplemented with TDZ @ seedling development of Dactylorhiza species of 0.5 mg/L NAA @ of 0.1 mg/L. with glucose and sucrose at concentration of 10 g dm-3 each. Later the improvement of Habiba et al., (2014) investigated in vitro shoot growth rate and shoot length was organogenesis of Epidendrum ‘Rouge Star enhanced by cytokinins N6-(2-isopentenyl) No.8’ PLBs with different types of adenine or N6-benzyladenine and their cytokinins. They used three types of combination with auxin indole butyric acid cytokinins including kinetin (Kin), 6- (IBA). benzylaminopurine (BA) and 2-isopentenyl adenine (2ip) which was evaluated with Jawan et al., (2010) found that Vanda dearei various concentrations of 0, 0.1, 1 and 10 protocroms prefer sucrose as compared to mg/L in modified MS medium. They reported fructose and glucose. The media containing that three cytokinins at all concentrations 1/2 MS, 2% (w/v) or 4% (w/v) sucrose is best were found significantly to enhance for seedling formation. organogenesis of PLBs in Epidendrum ‘Rouge Star No. 8’ orchid except 10 mg/L of Baque et al., (2011) reported 15 g/L sucrose BA treatment when compared with control concentration to be most suitable for and 0.1mg/L kinetin gave highest number of ‘Bukduseong’ × ‘Hyesung’, while 15 and 30 PLB. In case of shoot formation, they g/L sucrose were regarded as an optimal observed highest number of shoots per concentration for in vitro growth of the explant at 1mg/L BA but the shoot formation plantlets of ‘Chunkwang’ × ‘Hyesung’ rate (66.7%) was highest at 1mg/L kinetin. hybrid. They also observed increasing Results showed that kinetin enhanced root plantlets abnormality and root growth with induction when compared with 2ip and BA. higher sucrose concentration (60 g/L). MS medium supplemented with BA (4.40 mg/L) and NAA (1.0 mg/L) promoted Plant growth regulators maximum number of shoot formation of Phalaenopsis amabilis at (Balilashaki et al., According to Krikorian (1982) the success of 2014). an in vitro system is directly influenced by the correct growth regulator used and its optimum The Dendrobium transparens L. immature concentration. The commonly used plant embryos from 120 days old capsule were growth regulators belong to the auxin and germinated on ½ MS supplemented with 1 cytokinin groups. Tokuhara and Mii (1993) mg/L NAA + 2 mg/L BAP and same reported that the combination and appropriate hormonal combination showed highest concentration of hormones α-naphthalene number of multiple shoot while rooting with 1 acetic acid (NAA), 6-benzylamino purine mg/L IAA (Suntibala and Kisore, 2009).

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Asghar et al., (2011) reported that 2 mg/L The results also highlighted exogenous auxin BAP produced maximum number of shoots, dependent embryonic callus proliferation. while 1.5 mg/L of Kin exhibited the highest shoot length of Dendrobium nobile var. Verma et al., (2011) reported that TDZ was Emma white. IBA (2 mg/L) increased the the most effective for shoot multiplication of rooting percentage (97.5%) more efficiently Digitalis lamarckii Ivan. via direct than NAA. However, necrotic yellow shoots organogenesis than BAP, zeatin and kinetin. were observed treatments with higher Pawar et al., (2012) proved that 0.2 mg/L concentrations of BAP, Kin (3.0 mg/L) and IAA + 2.0 mg/L zeatin supplemented MS CW (300 ml). medium is the best regeneration efficient medium tomato (Solanum lycopersicum L.). Pant and Thapa (2012) reported rapid in vitro micropropagation of Dendrobium primulinum Medium supplements Lindl, an endangered epiphytic orchid. They used small shoot tip explants (0.3 to 0.5mm) There are several organic additives like which were previously obtained from in vitro banana extract (BE), carrot juice, coconut grown seedlings. They observed that BAP water (CW), peptone, potato extract, tomato (1.5 mg/L) + NAA (0.5 mg/L) supplemented juice etc. which are reported have beneficial MS medium to be most effective for the shoot effects on seedling growth in many orchid multiplication and IAA was found to be species like Aranda Deborah (Goh and Wong, effective hormone for rooting in comparison 1990), Vanda coerulea (Seeni and Latha, to IBA and NAA. 2000), Vanda spathulata (Decruse et al., 2003), Dendrobium tosaense (Lo et al., Kumari et al., (2013) developed a protocol for 2004). Shivkumar et al., (2005) first in vitro propagation of commercially suggested the use of peptone for enhancing important cut flower orchid Dendrobium plant tissues growth and development. Sonia (Fa. Orchidaceae) ‘Earsakul’ with stem nodal explants. They found ½ MS and 4 mg/L Murdad et al., (2010) determined the effects BA to be best suitable for early bud break. of three types of sugars as the carbon source However, they concluded 2.0 mg/L kinetin+ and potato homogenate (PH) on in vitro 0.1 mg/L NAA in shooting media and 0.5 growth and development in vitro derived mg/L NAA in rooting media for earliest Phalaenopsis gigantean protocorms. They growth and development. protocorms developed well within 150 days of culture on media containing either fructose Devi et al., (2013) adopted different explants or PH. However, on the media containing for rapid clonal propagation of Aerides both sugar and PH, the protocorms were odorata Lour. They used ½ MS, thidiazuron stunted yellow, pale with lower growth during (TDZ), 6-benzylaminopurine (BAP) and leaf the culture period. Surprisingly, both of the base as explant. They observed 1.0 mg/L TDZ leaf size and root length of P. gigantea medium to produce PLBs at the leaf base. seedlings were significantly enhanced when Highest frequency of calli induction from leaf the media only contained sugar. base explants after 60 days of culture was observed in media containing 2 mg/L (NAA) Sinha and Jahan (2011) documented and direct shoot regeneration with NAA (2 Phalaenopsis amabilis cv. Golden horizon's mg/L) and BAP (4 mg/L). The media in vitro mass clonal propagation. They containing ½ MS and 0.5 mg/L NAA cultured mature plant derived young leaf generated highest frequency of root induction. explants on ½ MS supplemented with α- 1919

Int.J.Curr.Microbiol.App.Sci (2017) 6(9): 1909-1927 naphthalene acetic acid (0.5 mg/L), N6- g/L) medium than control. Vijaykumar et al., benzyladenine (2.0 mg/L), 10% (v/v) coconut (2012) reported higher rate of germination, water, 2% (w/v) sucrose, 1 g/L activated more number of PLBs, shoots and root charcoal and 2 g/L peptone. formation of Dendrobium aggregatum cultured in MS medium supplemented with They observed that leafy shoots rooted on the 1.5 mg/L BAP, 3% sucrose and 15% coconut previously mentioned media without growth water (CW). supplements, where 100% explants were developed into plantlets with roots within 8 Shekarriz et al., (2014) reported that modified weeks. KC medium fortified with 2 g/L peptone without CW showed highest survival CW is cost effectively employed for the percentage of seed of Phalaenopsis hybrid micropropagation of imperative species of ‘Manchester’ and germination percentage was orchids due to its endless benefits (Peixe et observed in 15% (v/v), 2 g/L peptone and al., 2007). It is a natural growth promoter modified VW medium. They suggested that which contains higher levels of zeatin, zeatin Phalaenopsis seeds cultured on a ½ MS ribosides, 1,3-diphenylurea (contains medium containing CW and peptone can be cytokinin like activity), auxins, nitrogenous used for clone propagation. compounds, inorganic elements, organic acids, sugars and their alcohols, peptides, Anti-contaminants vitamins, amino acids and many other unknown components in its composition During micropropagation the release of (Tokuhara and Mii, 2001; Nasib et al., phenolic substances is a very common 2008).George et al., (2008) demonstrated that phenomenon and often adversely affects the physiologically active substances presents in response of the explants. Browning or CW promote the cell divisions which further blackening of cultured explants is cause only enhance shoot multiplication. Baque et al., after the wounding. This activity promoted (2011) demonstrated effective plantlets the oxidation of phenolic substances under the growth enhancement in ‘Bukduseong’ × control of polyphenol oxidase. Tanaka and ‘Hyesung’ and ‘Chunkwang’ × ‘Hyesung’ Sakanishi (1977) reported poor regeneration hybrids of Calanthe with 50 m/L coconut in Phalaenopsis tissue culture because of water application. phenolic compound exudation.

Kaur and Bhutani (2012) were successful to Good growth and development of regenerate and multiply Cymbidium pendulum Phalaenopsis plantlets in vitro were obtained using protocorms explant under invitro when culture media were supplemented with condition in M medium (Mitra medium) 0.2% (w/v) AC (Hinnen et al., 1989; Ernst, supplemented with banana homogenate, 1994). Activated charcoal reduce the phenolic coconut water and peptone. The primary exudates, the beneficial effects of AC could protocorm segments first regenerated into be due to positive stimulation of many secondary protocorms and differentiated into development processes (Van Winkle and shoots without any callus stage in between. Pullman, 2006) and phenolic compounds absorbtion. Rittirat et al., 2012 reported They observed regeneration frequency to be Phalenopsis cornu-cerve (Breda) Blume Rchb significantly higher in organic growth f. best growth in ND medium supplemented supplement enriched (banana homogenate @ with 4% sucrose and 0.2% AC without callus 50 g/L, 10%coconut water and peptone @ 2 browning or necrotic tissues. 1920

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Hardening of in vitro grown plant (historical aspect – Rotor, G. 1949 to Balilashaki et al., 2014). It is a matter of fact Orchids are grown in a special media, since that orchids are aesthetically gorgeous gifts of root needs plenty of air around them in all nature and these beauties are tremendously times. The media used should provide support efficient in holding their flowering phase for a to the plant, supply water and nutrients to the long time unlike any other cut flower present roots and enough air for the roots to breath. on earth. Fortunately, the commercial demand Puchooa (2004) suggested that high humidity of these rare breeds has been increasing day (about 90%) around the plantlets for two by day which may ensure their conservation weeks was essential for hardening of the in coming years. The advanced tissue culture orchid plantlets. He found 84% survival rate techniques with clonal in vitro of Dendrobium plantlets transferred to the micropropagation sounds potential for green house into baskets containing wood industrial production of high quality orchid charcoal as wood charcoal provides good plant material which may earn appreciable drainage and adequate aeration to the roots, level of revenue in international market and which is of primordial importance in the strengthen the national economy. culture of orchids. A huge number of research has been done on The rooted plantlet of Dendrobium several kinds of orchid species in vitro microbulbon when transferred to a thermocol micropropagation which are commercially cups containing a mixture of sand, soil, brick demanded. pieces and charcoal pieces (1:1:4:4) showed 60% survival rate and the same plantlets But all the protocols developed so far may not grown directly on the tree trunk showed 40% be successful on industrial level in terms of survival rate after six weeks of their removal economic point of view. Hence, we have from in vitro conditions (Sharma et al., 2006). attempted to put together most of the Suntibala and Kishore (2009) successfully available literature of in vitro cloning of acclimatized Dendrobium tranparens L orchid using shoot tip, leaf, inflorescence axis plantlets in a potting mixture of brick and and flower bud as explants. This may be charcoal (2:1) and more than 90% of helpful in adopting the most suitable protocol transplanted plants survived under greenhouse for its respective orchid species and also condition. improvising the currently available method for in vitro mass propagation. Pant and Thapa (2012) observed that the potting mixture containing coco peat and Author contribution statement Dipika Sarmah sphagnum moss in the ratio of 2:1 can sustain and all of the other authors planned and wrote the hardening of in vitro rooted plantlets with the review manuscript and table and 70% of plantlets survival. However, they corresponded for publication. found maximum 66.67% survival of Dendrobium Sonia ‘Earsakul’ in hardening Acknowledgements mixture charcoal and brick pieces (@1:1 proportion), transplanted in green house The authors acknowledge the Bidhan Chandra condition. Krishi Viswavidyalaya, Mohanpur, WB for permitting to access the university central This review provides an overview of what the library and for providing ‘Institutional authors consider to be the most significant Assistantship’ during preparing the literature in orchid micropropagation manuscript. 1921

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How to cite this article:

Dipika Sarmah, Swathi Kolukunde, Monoj Sutradhar, Brijesh Kumar Singh, Tapas Mandal and Nirmal Mandal. 2017. A Review on: In Vitro Cloning of Orchids. Int.J.Curr.Microbiol.App.Sci. 6(9): 1909-1927. doi: https://doi.org/10.20546/ijcmas.2017.609.235

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