Orchids Newsletter

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Vol. 14(2) July- December, 2013 Contents

Research Highlights 01 Training Imparted 08 Institutional Activities 07 Education and Training 08 Independance Day Celebration 07 Publications 09 Participation of scientists in Symposia/Seminar etc. 08

RESEARCH HIGHLIGHTS in primer sets DR- 12 and DR-30 with an average of 0.76 for all primer sets. The cluster analysis based on Diversity analysis of some medicinal Dendrobium UPGMA method grouped all species into three orchids using Microsatellite markers different clusters. Simple Sequence Repeat (SSR) markers were used to determine the genetic relationship among 20 species of medicinal Dendrobium orchids collected from different geographical regions of India. 4 SSR primers sets had amplified a total of 149 loci and produced total 75 polymorphic bands. The number of bands per primer ranged from 10-30 with an Fig. 1. Banding pattern generated by SSR markers average of 18.75 and size ranged from 70-2900 bp. The average Rp value was 3.75 which indicates that Diversity analysis of some fragrant Dendrobium orchids using ISSR markers the primers are able to distinguish the genotypes. The PIC value ranged from 0.56 to 0.95 with an Twenty-one species of fragrant Dendrobium orchids average of 0.72 which also indicates that all primers were collected from different geographical regions were capable of detecting polymorphism efficiently. of India and were used to determine the genetic Maximum Discrimination power (D) 0.99 was relationship and diversity using Inter Simple found in primer set DR-49 and least was 0.59 found Sequence Repeat (ISSR) markers to contribute in

NRCO, News Letter (July - Dec., 2013) 01 efficient identification, conservation and sustainable female parents. These newly developed scented utilization for improvement through conventional as varieties have a potential to create a new trend in well as molecular breeding approach. 20 selected market with a value addition of natural fragrance, ISSR primers produced clear and reproducible apart from early to mid flowering trait that fulfils the patterns of multiple bands and showed a high level of demand during off-season. The amateur visitors polymorphism among the species under study. The expressed the usefulness of these hybrids for UPGMA dendrogram and the principal coordinate intermediate nature, those prefers flowers to be fresh analysis revealed a clear differentiation between the in their garden in the segment of domestic market. species which reflects that ISSR profiling is a Two clones were selected from PBX-05-772 (Fig. 3a powerful molecular method to determine the genetic & 3b.) with florets range of 5 – 8 and another clone relationship and diversity among Dendrobium (PBX-05-751) (Fig. 3c.) derived from cross, suitable species. for cut flower cultivation with 12-13 florets.

Fig. 2. Banding pattern generated by ISSR markers

Development of scented Cymbidium hybrids

Scented orchid hybrids in Cymbidium group were the latest development at NRC for Orchids (Sikkim). Fig. 3a. PBX-05-772/2013-65 Fig. 3b. PBX-05-772/2013-66 Unlike native species of orchids, all the commercial Cymbidium hybrids are generally non-fragrant in nature. The developed new hybrid is moderately scented, similar to its male parent Cymbidium iridiodes used in hybridization programmes. The native species, Cymbidium iridiodes D. Don a threatened orchid native to North-East India, Myanmar, Nepal and South West China found at an elevation of 1000 to 2800 MSL. It is an epiphytic growing in warm to cool regions in tropical moist forests. In Sikkim, the species is reported in Fig. 3c. PBX-05-751/2013-01 Fig. 3d. Floral comparison Dickchu, Rakdong-Tintek & Rongli and maintained of variants of PBX-05-772/ 2013-66 & 65 at germplasm collection (NAGS) at NRCO under ex- situ conservatory. The PBX-05-772 (C. 'Concerto' x Report on pseudobulbs in-vitro in Coelogyne C. iridioides) and PBX-05-751 (C. 'Nonina Paleface' flaccida x C. iridioides) were the pedigree lines, identified Coelogyne flaccida is a sympodial epiphytic orchid during flowering season. For genetic improvement species with beautiful starry flowers and of native species, two exotic hybrids were used as

02 NRCO, News Letter (July - Dec., 2013) heteroblastic pseudobulb, often readily marketed in Survey, collection, evaluation and ex-situ baskets or pots with peat and moss on account of conservation of wild orchids of Kodagu, drooping character of their scented spikes. The . information on pseudobulb formation under in-vitro A survey was conducted in the district of Kodagu of conditions is less in Coelogyne sp. Seeds of matured Karnataka, in collaboration with the Central pods of Coelogyne flaccida were geminated and Horticultural Experiment Station, Chettalli for seedlings were obtained on Gamborg B5 media by documentation, conservation and evaluation so far the method of in-vitro culture. Six months grown as their commercial potentials are concerned and at seedlings were inoculated in Gamborg B5 media the same time to create an awareness towards their with 0.2 mg/l BAP (6-Benzyl aminopurine) existence. The district of Kodagu of Karnataka, is incorporated with 20 gm/l sucrose, 1.5 gm/l known for its wide biodiversity and nurture many activated charcoal and 4 gm/l agar. The plantlets wild orchids of the Western Ghats in its natural developed pseudobulbs within 3 months after forests and plantations. The survey was carried out in inoculation. It took 68 days for pseudobulb initiation all the three taluks of Kodagu. The regions of till complete development (Fig. 4a.). The hairy roots , Bhagmandala, Appangla, were tremendously long and slender with a single Galibeddu, Bettakeri, Napklu and Kakabe were pseudobulb. The pseudobulbs produced were 1-2.5 covered under survey. To the north in cm in length, 0.5-0.8 cm girth and weighing around taluk, Santahalli, Kotti reserve forest, Abbematta 0.3-0.4 gm (Fig. 4b.). As per falls, lower reach of Pushpagiri hills, Malahalli falls, available literature the auxin Abborkatte, Kushalnagar, Kauverinishargadham, regulation could be the Suntikoppa, Chettalli, Rangasamudra, Anekadu reason for the pseudobulb or forest area were surveyed. In the taluk rhizome formation under in- places like Ponnampet, Pollibetta, were vitro conditions. These included in survey. The natural forest is the main pseudobulbs, similar to home for wild orchids, but several species are found rhizome formation may help in the coffee plantations which have old forests trees in decreasing the mortality as shade plants. 33 species belonging to 20 genera under hardening conditions were collected during the survey and the passport and induction of somatic Fig. 4b. In-vitro plant of data of the samples were recorded. The embryogenesis in-vitro. Coelogyne flaccida morphological characters of the plant samples were described and evaluated for their horticultural traits. The samples were conserved ex-situ at Central Horticultural Experiment Station, Chettalli and NRC for Orchids, Pakyong. The species collected are Aerides crispa, Aerides cylindricum, Aerides ringens, Arachnis sp., Bulbophyllum fimbriatum, Bulbophyllum mysorense, Bulbophyllum neilgherrense, Coelogyne breviscapa, Cymbidium aloifolium, Cymbidium bicolor, Dendrobium aqueaum, Dendrobium herbaceum, Dendrobium barbatulum, Dendrobium crepidatum, Eria Fig. 4a. Coelogyne flaccida in-vitro pseudo bulbs

NRCO, News Letter (July - Dec., 2013) 03 mysorensis, Flickingeria nodosa, Goodyera pubescens (Fig. 5a.), Liparis viridiflora, Luisia zeylanica, Malaxis rheedei, Oberonia brunoniana, Oberonia denticulate, Oberonia ranganniana (Fig. 5b.), Phiaus luridus, Pholidota pallida, Polystachya flavens, Rhynchostylis retusa, Trias stokskii (Fig. 5c.), Vanda tessellata, Vanda testacea and Xenikophyton smeeanum (Fig. 5d.). Among the species collected Aerides crispa, Dendrobium aqueaum, Dendrobium herbaceum, Dendrobium barbatulum, Dendrobium crepidatum, Pholidota Fig. 6. Mesophyll cell collapse of Phalaenopsis pallida, Phiaus luridus, Rhynchostylis retusa and Liparis viridiflora can be exploited for horticultural Production management of Tropical and Subtropical Orchids uses for cut flower or potted plants. Species like Eria mysorensis, Phiaus luridus, and Rhynchostylis In Cattleya hyb. 'Queen Sirikhit', cocochips + brick piece retusa are fragrant and Vanda tessellata, Vanda + leaf mould (1:1:1) and cocochips + brick pieces + leaf testacea, Flickingeria nodosa have medicinal fern (1:1:1) both hav been equally found as best potting properties and used in ethno-medicines. Goodyera mixture. Out of sixteen orchid species underground pubescens is a new report from Kodagu. c u l t u r e , f l o w e r i n g p e r i o d r e c o r d e d w e r e Tainiahookeriana (Feb-April), Phaius tankervilleae (March to May), Phaius flavus (April-May), Dendrobium nobile (April to May), Epidendrum xanthimum (January- continuing), and Vanda teres (April-July).

Post harvest technology of orchids

Fig. 5a. Goodyera pubescens Fig. 5b. Oberonia ranganniana In Phalaenopsis, among 11 hybrids evaluated, 'Manchester' had highest vase life (62.2 days). In Phalaenopsis 'Detroit', among five impregnation

treatments CoCl2 (1000 ppm) for 45 minutes had

maximum vase life (75.6 days) followed by NiCl2 (1000 ppm) for 45 minutes (62 days) over control (45 days). In Phaius tankervillea, out of seven Fig. 5c. Trias stokskii Fig. 5d. Xenikophyton smeeanum harvest stages, cut flowers of loose bud stage had maximum vase life (33 days) and minimum at 9 buds Mesophyll cell Collapse of Phalaenopsis opened stage (20 days) and in Epidendrum, cut flowers harvested at all florets opened stage except 5 The mesophyll cell collapse of Phalaenopsis caused buds showed maximum vase life (28 days) followed by low temperature was observed in January- by all florets opened stage except top 10 buds (22 February when the air temperature goes down below days.) 10oC and water temperatureranges within 4-5oC. The initial symptom is localized water sunken spot, later it became blackish (Fig. 6.).

04 NRCO, News Letter (July - Dec., 2013) Report of new diaspidid scale insect, New report of an aphelinid wasp, Coccophagus Lepidosaphes pinnaeformis (Bouche) on ceroplastae (Howard) as parasitoid of soft brown Cymbidium species and hybrids from this centre scales, Coccus hesperidum infesting Orchids from for the first time Sikkim, India.

The daispidid Scale, Lepidosaphes pinnaeformis The aphelinid wasp, Coccophagus ceroplastae (Bouche) has been found to infest the leaves, leaf sheath (Howard) was found to parasitize the soft brown and pseudobulbs of many Cymbidium species and scales especially the younger stages. The females of hybrids round the year under mid altitude Sikkim this wasp deposited egg into the body of the scales Himalayas. The adult female cover is oyster-shell and the larval and pupal development took place shaped, usually curved, moderately convex, brown within the body (Fig.8a &8b). The adults black in often with lighter periphery; shed skins marginal, colour emerged by making a circular hole on the orange or tan (Fig.7a. I). Adult males with one pair of scale cover (Fig.8c). The confirmation of the species wings, male cover shorter, narrower than female was done by Dr. J. Poorani (Principal Scientist) of cover, same colour and texture; shed skin marginal National Bureau of Agriculturally Important Insects, orange or tan (Fig.7a.II ). Body of adult female white . to light violet; eggs and crawlers probably white to pinkish colour. The newly hatched crawlers move about for a short while, select a suitable place and starts feeding. The nymphs and adults insert the needle like stylets and suck the sap and devoid the cell of its contents which results in yellowing of the infested portions (Fig.7b). The confirmation of the species was done by Dr. Sunil Joshi (Principal Scientist) of National Bureau of Agriculturally Important Insects, Bangalore. Fig. 8a. Developing larvae inside Fig. 8b. Developed pupa the scale body inside the scale body

Fig. 7a. (I) Female scale (II) Male scale insect with shell cover

Fig. 8c. Adult of C. ceroplastae New report of Aphytis sp. as parasitoid of Cymbidium Scale, Lepidosaphes pinnaeformis (Bouche) infesting Cymbidium Orchids

The aphelinid wasp, Aphytis sp. was found to parasitize the cymbidium scale insect, Lepidosaphes Fig. 7b. Scale infested leaves

NRCO, News Letter (July - Dec., 2013) 05 pinnaeformis infesting Cymbidium orchids under Darjeeling Centre polyhouse conditions throughout the year. The adults of Aphytis sp. were found foraging on the Mass multiplication protocol for propagation of leaves. The females deposit their eggs on the body of Paphiopedilum villosum(Lindl.) Pfitz. through seeds developed the scale; the larval and pupal development taking place by consuming the body contents of the scale Paphiopedilum orchids or the lady's slipper orchids insect (Fig. 9a. & 9b.). The adults emerged by are among a few orchids that are widely admired for making a circular hole on the shell (Fig. 9c. & 9d.). their beauty. The genus Paphiopedilum consists of The parasitized scales appeared flat and darker in some 80 species distributed from India to colour. The activity of this parasitoid was found from Philippines. Eight species of this genus inhabit India April to first week of December. However, its out of which seven are found in northeastern states. activity decreased and it suffered high mortality due Worldwide the population of slipper orchids in to reduction in temperature during winter. The natural habitats has reduced to a critical level due to confirmation of the species was done by Dr. J. ongoing over-exploitation and destruction of natural Poorani (Principal Scientist) of National Bureau of habitats. As a result, Govt. of India declared Agriculturally Important Insects, Bangalore. destruction, collection, possession and trade of these species as a punishable offence under Wildlife Protection Act. CITES, Convention on International Trade in Endangered Species of Wild Fauna and Flora prohibited transborder trade of these species by placing them under Appendix –I. Despite of various laws in place, the population of these orchids Fig. 9a. Developing larva on body Fig. 9b. Pupa of Aphytis sp. in their natural habitats is depleting very fast. P. of scale (scale cover removed) (Scale cover removed) villosum, a beautiful orchid inhabiting in the forest of Assam and Meghalaya shares similar fate like many other species of this genus. Making these plants easily available at affordable rates would reduce pressure and would allow them to perpetuate in their natural home. To achieve afore mentioned objective the flowers of P. villosum were sib-mated, Fig. 9c. Adult of Aphytis sp and mature green capsules were harvested after 300 days. The capsules were surface sterilized using mercuric chloride (0.1%) for 15 min. Then the capsules were cut open longitudinally, and the seeds were scooped out. The seeds obtained from the capsules were mixed so that a representative sample could be obtained. The seeds were cultured on six Fig. 9d. Emergence holes on scale cover different culture media namely Thomale GD (TGD) Burgeff (BF), Mitra et al. (M), Murashige and Skoog (MS), Knudson C (KC) and Nistch (NC) and were incubated in dark. The earliest seed germination was

06 NRCO, News Letter (July - Dec., 2013) observed on Burgeff medium. The splitting of seed Thomale GD (TGD), (SH), Mitra and Mitra (MM), coat occurred in 55-60 days, and the protocorms and Burgeff (BF) medium. Seed germination and were formed in 82-90 days and for developing protocorm development was highest in Mitra & complete plant it required nearly 120- 140 days. The Mitra medium (92.4 %) followed by NC (80.4 %), germination was delayed on all other media. Further, GM (74.7 %) and SH (73.9 %) media after 36 days. the seedlings of P. villosum were cultured on NC For seedling growth, these were transferred to NC medium supplemented with various concentrations medium supplemented organic additives and of BAP (0, 0.2, 0.4, 0.6, 0.8 & 1.0 mg/l) for three activated charcoal. Addition of activated charcoal months. It was observed that increasing 0.5-2.0 mg/lit increased the plant height, leaf concentration of BAP increased the fresh weight of number root and leaf length over control. The the seedlings but reduced drastically when the seedlings were successfully acclimatized on potting concentration of BAP was increased beyond 0.4 medium consisting of leaf mould, coco peat and mg/l. The highest mean fresh weight per plant was coco chips. recorded from the seedlings cultured on medium supplemented with 0.4 mg/l BAP. The plantlets INSTITUTIONAL ACTIVITIES obtained from these experiments were acclimatized successfully. Further studies for increasing germination percentage, reducing germination period, and induction of multiple shoots are continuing in order to develop a reproducible protocol for commercial exploitation.

Asymbiotic seed germination, seedling growth and acclimation of Zygopetalum intermedium for commercial cultivation

Zygopetalum intermedium inhabit in tropical and subtropical regions of South America, Brazil, One day farmer's training programme on Production Technology th Paraguay, Argentina, Peru and Bolivia. It is well of Orchids on 18 November,2013 at Rongli, East Sikkim under Mini Mission -I known for its fragrance and comfort in cultivation. T h e r e a r e e n o r m o u s p o s s i b i l i t i e s o f commercializing this species for scented flowering potted plant as well as extraction of essential oils. However, successful commercialization of this species requires effective propagation techniques that would allow for mass propagation. An in vitro plant regeneration protocol was successfully established for Z. intermedium by culturing seeds from green capsules. Immature seeds were germinated on eight basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC), Gamborg's B5 (GM), Nitsch (NC), Vacin and Went (VW), Twenty Two days on job training for students of vocational stream (Horticulture) from 20th Nov. to 11th Dec., 2013 at NRCO

NRCO, News Letter (July - Dec., 2013) 07 4th International Conferences on 'Impact of Landscape Horticulture on Development of Urban economy with Green Environment' at Kolkata, West Bengal from 12th-14th September, 2013.

D. Barman, L. C. De & A. M. Khan

Meet of Ministers of Agriculture-Horticulture of North East India at Gangtok, Sikkim on 28th November, 2013.

L. C. De Five day farmer's training programme on Production Technology th of Orchids and Lilium from 22-26 November,2013 at NRCO Exhibition on Orchids from NRCO at CAE & PHT, Ranipool, Sikkim on 30th November, 2013

L. C. De

Agribusiness Camp as ITMU In-Charge at NRC Yak, Dirang on 17th December, 2013

S. Chakrabarti

Agribusiness Campaign as ITMU In-Charge at ICAR Research Complex for NEH Region, Barapani, Meghalaya on 21st December, 2013

S. Chakrabarti Five day farmer's training programme on Protected Cultivation of High Value Cut Flowers from 10-12th December,2013 at ICAR Res. Complex for NEH Region, Nagaland centre, Jharnapani Education and Training organized by NRCO under Mini Mission -I Scientist

Participation of Scientists in Conference, Three months attachment training on “DNA Meetings, Workshop, Symposia, Seminar Barcoding on Insect Pest of Orchids” under the etc in India and Abroad. guidance of Dr. G. T. Behere (Sr. Scientist) at ICAR Complex for NEH Region, Barapani, Meghalaya Agribusiness Camp as ITMU In-Charge at CARI, from 13th May-16th August, 2013. Port Blair from 24-30th July, 2013. Rumki H. Ch. Sangma S. Chakrabarti Three month attachment training programme for th NEH Program for 12 Five Year Plan (Collaboration ARS at IIHR, Bangalore 14th May – 14th August, th with IIHR) at Rani, Guwahati on 25 July, 2013. 2013.

L. C. De A. M. Khan

08 NRCO, News Letter (July - Dec., 2013) Five days MDP training on “Biotechnology and Administrative Intellectual Property Rights” at NAARM, th Five days training on “management Development Hyderabad from 8-12 July, 2013 Programme on Financial Issues at National Institute S. Chakrabarti of Financial management” at Faridabad (U.P.) from 16 - 20th December, 2013. Seven days Management Development Programme in Consultancy Project Management at NAARM, Rishi Kant Singh Hyderabad from 1-7th August, 2013. Publications R. Devadas Research Papers: General Management Training for Scientist sponsored by DST at Administrative Staff College of 1. Barman, D.; Devadas, R. (2013) Impact of India (ASCI), Hyderabad from 26th August to 6th Climate Change on Orchid Population and September 2013. Conservation Strategies – A Review. Journal of Crop and Weed, 9(2):1-12. Rampal Paper presented in Seminars/Symposia: Training and awareness Programme on Climate Resilient Agriculture at ICAR Research Complex 1. Barman, D.; Bharathi, T. U.; Khan, Arpita for NEH Region on 4th October 2013. Mandal.; Medhi, R. P. (2013) Impact of Landscape Horticulture on Development of A. M. Khan and Rumki H. Ch. Sangma Urban Economy with green Environment and presented paper on “Landscaping with fragrant Basic Molecular techniques in DNA fingerprinting Orchids” and “Wild Orchids: A Boon to at National Bureau of Agriculturally Important Landscapers (Poster). In: 4th International th Insects (NBAII), Bangaluru from 8-14 September, Conference on Landscape and Urban 2013. Horticulture at Kolkata from 12-14th Sept., Rampal 2013.

NAIP-NABG Subject Matter Training on 2. Chakrabarti, Syamali.; Jain, Sudeep Kumar.; Bioinformatics: In vitro to In Silico Approaches In Sinha, Debasree.; Medhi, R. P. (2013) Paper on Entomology at National Bureau of Agriculturally Diversity of fragrant Orchids of North eastern th India and sustainable utilization for food Important Insects, Hebbal, Bangalore from 18-30 security” pp:26-30. (accepted for oral November, 2013. presentation) In: International conference of Rumki H. Ch. Sangma World Biodiversity Congress (WBC) at Chiang Awareness cum training on Application of Mai, Thailand. Novemner, 2013. Information and Communication Technology from 19th to 21st December 2013, at Pakyong, NRCO. 3. Chakrabarti, Syamali.; Jain, Sudeep Kumar.; Medhi, R. P. (2013) Paper on Molecular All Scientists/ Admin./ Technical staff of NRCO markers for assessment of genetic diversity and relatedness of some important Dendrobium

NRCO, News Letter (July - Dec., 2013) 09 orchid species of India. (accepted for oral Transfer presentation) In: International Conference on Agriculture and Biotechnology at Kuala Scientific Lumpur, Malaysia from 29-30th December, ? Mr. Raj Kumar, Scientist 2013. (Floriculture) transferred 4. De, L. C.; Medhi, R. P.; Mitra, S. K. (2013) from CAZRI, Jodhpur, Orchid-A diversified flower crop. In: R a j a s t h a n Proceedings of 4th International Conferences on and joined this centre on Landscape and Urban horticulture held at 8th November, 2013. th Kolkata, West Bengal from 12-14 September, Administrative 2013. ? Shri. Davis Joseph, 5. Khan, A. Mandal.; Barman, D.; R. P. Medhi Administrative Officer (2013) Wild Orchids a Boon to Landscapers transferred to CTCRI, presented In: International Conference on Thiruvananthapuram, 'Impact of Landscape Horticulture on K e r a l a o n 1 3 t h Development of Urban Economy with Green September, 2013. Environment at Kolkata from 12-14 t h September, 2013

Published by Editorial Committee Dr. R. P. Medhi Ms. Rumki H.Ch. Sangma Director Mr. N. G. Debnath National Research Centre for Orchids (ICAR) Pakyong 737 106, East Sikkim Tel. No.03592-257954 Telefax: 03592-257289/257282

10 NRCO, News Letter (July - Dec., 2013)