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Research Highlights

2014-15

भा.क. अन.प.‐ रा��य आ�क� �स अनस�धान क� �ु ुृ

पा�य�ग ‐७३७ १०६, �सि�कम, भारत

ICAR-National Research Centre for OrchidsPakyong‐737106, Sikkim, India

Research HighlightsCopyright © 2015 National Research Center for Orchids, Pakyong-737 106, Sikkim, India

Chief Editor Dr. D. R. Singh

Technical EditorDr. Ram Pal

Printed:April, 2015

Published by:Dr. D. R. Singh, Director, National Research Center for Orchids, Pakyong-737 106, Sikkim, India

Printed by:Comandaa Designers and PrintersMumbai

Orchids are the precious gift of nature to the human beings. They are

adored and loved for their mystique beauty, therapeutic value and long

shelf life. The popularity of orchids as cut owers has increased such

that these have now become part of each and every important public,

private and social function. At present, a bulk of the demand for orchid

cut owers is met through imports. The import of orchid owers has

risen from 299.9 lakhs during 2008-09 to 2985.19 lakhs in 2013-14.

Import of orchid cut owers has registered the growth of 159.69 percent

over the previous year. There is nearly tenfold increase in import of cut

owers in the last six years, and this demand is further expected to

increase. These gures indicate huge and expanding market for orchid

cut owers in India. It is only a tip of the iceberg in the sea of oriculture

market. The orchid industry does not rest on the supply of cut owers of

Dendrobium orchids. It includes cut owers and pot plants of other

commercially cultivated genera like Cymbidium, Vanda, Oncidium etc.,

micropropagated plants, dry owers, biomolecules, natural dyes,

perfumes, etc.

India has vast genetic resources of orchids and great potential for

utilization in oriculture and herbal industry. Since its inception, NRCO

has been playing a vital role in conservation of genetic resources,

breeding of new varieties, molecular characterization of genetic

resources, developing package of practices for cultivation and post

harvest management, plant tissue culture and micropropagation. This

document, Research Highlights outlines the most signicant

achievements made by the centre during 2014-15. During this year

signicant progress has been made in molecular characterization, DNA

barcoding, cpDNA isolation protocol, indemnication of parasitoids and

emerging pests of orchids and drying of owers, new cultivar

development and formulation DUS test guidelines for Paphiopedilum

orchids. I am sure that this publication would be useful for the

researcher and enthusiasts in comprehending the research activities for

the year 2014-15 in a nutshell.

Foreword

ICAR-National Research Centre for Orchids Research Highlights 2014-15

A simple improved protocol for Isolation of Chloroplast DNA was developed

78 DNA barcode sequences of native Vanda species were submitted in

GenBank

Molecular Diversity analysis of native Otochilus species using RAPD,ISSR and

SSR molecular markers was completed

Molecular Diversity analysis of native 24 medicinal Dendrobium species using

RAPD and ISSR molecular markers was completed

Molecular Analysis of native 21 Fragrant Dendrobium Orchids of India Using

ISSR Markers was completed

Assessment of Molecular Diversity of Native 04 Pleione species using RAPD

and ISSR Markers was completed

Microsatellite markers developed from Himalayan orchid species

Paphiopedilum fairrieanum

Intra specic variability analysis of Coelogyne ovalis ,Smitinandia micrantha

and Vanda cristata was completed

In-vitro plantlets of Vanda cross, PBX-12-169 (V. 'Bernice Miller' x V.

coerulescens) developed.

Characterization of Paphiopedilum clones-I&II and ex-vitro hardening of 3

hybrid progenies (PBX-11-150, 11-155 & 12-169).

A New report of an encyrtid wasp, Anagyrus sp. (Howard) as a parasitoid of

long-tailed mealy bug, Psuedococcus longispinus and Mealy bug,

Pseudococcus sp. infesting Orchids from Sikkim, India.

DUS Test Guidelines of Oncidium published in PVJ of India under PPV & FRA in

April 2014 issue and notied for registration by Plant Authority during

October, 2014.

Executive Summary


A. Institute Funded Projects

Project1: Conservation Characterization and Sustainable Use of

Diversity in Orchids

1.1 DNA ngerprinting of commercially important orchids

1.2 A comparative study of asymbiotic and symbiotic germination in selected

species and hybrids of Calanthe, Cymbidium and Paphiopedilum

1.3 Application of Geographic Information System in medicinal orchid

conservation of Sikkim

Project 2: Genetic Improvement of Orchids for Yield, Quality, and

Resistance to Biotic and Abiotic Stresses

2.1 Hybrid/varietal development of important orchids

2.2 Genetic improvement of Cymbidium orchids for cut ower and pot plant

Project 3: Development and Renement of Production and Protection

Technologies for Improved Productivity, Marketing, and

Utilization of Orchids

3.1 Development of integrated oriculture enterprise

3.2 Renement of production technology in Cymbidium

3.3. Flower drying in orchids

3.4 Post harvest management in orchids

3.5 Development of protocol for mass multiplication of rare, endangered and

threatened orchid species

3.6 Flower induction in orchids

3.7 Eco-friendly pest management

Project 4: Improvement of Knowledge and Skill of Stakeholders for

Improving Production of Orchids

B. Externally Funded Project

Preparation for Plant Variety Protection and Conducting Test for

Distinctness, Uniformity and Stability for Orchids

Ongoing Research Projects



Research Highlights

Progress and Achievements

Project1: Conservation Characterization and Sustainable Use of Diversity in Orchids

1.1 DNA ngerprinting of commercially important orchids

A simple, improved and inexpensive method to obtain chloroplast DNA from orchid species was developed by modifying the existing protocols. The developed method does not require an ultracentrifuge and produced high-quality chloroplast DNA.

78 DNA barcode sequences of native Vanda species were submitted in GenBank. Among them, there are total 31 chloroplast DNA sequences of RNA polymerase C (rpoC1) gene, 27 sequences of RNA polymerase beta subunit (rpoB) gene of chloroplast DNA, 7 matK sequences and 13 sequences of Internal Transcribe Spacer (ITS) of nuclear DNA.

Molecular Diversity analysis of native Otochilus species using RAPD, ISSR and SSR molecular markers has been done and the cumulative analysis resembles with the results of ISSR analysis suggesting high potential of ISSR primers in the area of ngerprinting and diversity analysis.

Molecular Diversity analysis of native 24 medicinal Dendrobium species using RAPD and ISSR molecular markers has been completed. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustered all the species into three different major clusters.

Molecular Analysis of native 21 fragrant Dendrobium Orchids of India Using ISSR Markers was completed. The cluster analysis using UPGMA method grouped Dendrobium Species into two major Clusters. The UPGMA dendrogram and the principal coordinate analysis revealed a clear differentiation between the species reecting that ISSR proling a powerful molecular method to determine the genetic relationship and diversity.

Assessment of Molecular Diversity of Native 04 Pleione species using RAPD and ISSR Markers was done. The average resolving power (Rp) and Polymorphism Information Content (PIC) value were very high indicating all the primers used in this study were capable of detecting polymorphism very efciently. The UPGMA dendrogram grouped all four species into two distinct clusters.

Microsatellite markers were developed from Himalayan orchid species Paphiopedilum fairrieanum

Intra specic variability analysis of Coelogyne ovalis, Smitinandia micrantha and Vanda cristata has been completed


A. Institute Funded Projects

1.2 A comparative study of asymbiotic and symbiotic germination in selected species and hybrids of Calanthe, Cymbidium and Pahiopedilum

Various orchid species along with substratum were collected from natural habitats. The seed packets were placed in the substrate for ex vitro symbiotic germination.

The mature seeds of Paphiopedilum species and crosses were cultured on modied Nitsch & Nitsch medium. The stages of seed germination and seedling development were recorded. The splitting of testa was observed 15-20 days after culture. The protocorm formation and initiation of primordial leaf varied between 28-46 days and 60-68 days, respectively.

The mature seeds of Cymbidium and its crosses (10) were cultured on Mitra et al. medium and the stages of development were recorded. The swelling of embryos occurred 15-20 and the formation rhizoids were observed 28-42 days after culture. The formation of protocorm occured 45-60 days after the culture.

1.3 Application of Geographic Information System in medicinal orchid conservation of Sikkim

Fifty orchid species having medicinal importance were listed out by gathering information from different sources of literature

Information on local distribution, owering period and medicinal value was collected and summarised

The medicinal orchids facing various kinds of threats to their natural habitats were listed out for analysing nature and cause of threats

2. Genetic Improvement of Orchids for Yield, Quality, and Resistance to Biotic and Abiotic Stresses

2.1 Hybrid/varietal development of important orchids

Identication of NAGS collections, NOAC#324 (Dendrobium ovatum), NOAC#757 (Eria tomentosa) & NOAC#1188 (Micropera rostrata)

Working collection of NAGS germplasm developed from NAGS base collections, Vanilla sp collected from CPCRI, Mohitnagar and re-introduced Calanthe triplicata for clonal multiplication in germplasm management.

Characterization of new Cymbidium breeding line (Cym. 'September Sunset' x Cym. 'Palkibaris').

Multiplication of Aranda 'Kunga Gyatso' and initiated State & AICRP trial (North-Eastern Plain & Hill Zone).


In-vitro plantlets of Vanda cross, PBX-12-169 (V. 'Bernice Miller' x V. coerulescens) developed.

Characterization of Paphiopedilum clones-I&II and ex-vitro hardening of 3 hybrid progenies (PBX-11-150, 11-155 & 12-169).

More than 100 NAGS accessions were supplied to Forest Department, Government of Nicobar Islands.

Major focus given to generating intergeneric crosses to strengthen breeding programmes and > 200 crossings were done. Promising breeding line, PBX-05-29/31 derived from Cym. 'Red Beauty' x Cym. 'Golden Elf' with attractive ower colour and early owering traits characterized.

Two scented lines (PBX-05-772) and ve progeny lines of Cym. lowianum based crosses were characterized. Characterized 16 progeny lines of Paphiopedilum cross, Pl x Pw for station trial. Selected early lines were displayed at White Hall Exhibition Hall, Gangtok for selection by farmers & entrepreneurs.

st 1 year AICRP trial for Aranda 'Kunga Gyatso' was completed in North-Eastern

Plain & Hill zone.

Seven selected breeding lines (Cymbidium-3 no & Paphiopedilum-4 no.) were given denominations for institutional release.

2.2 Genetic improvement of Cymbidium orchids for cut ower and pot plant

Prepared a list of Cymbidium parental lines on the basis of breeding history, important horticultural traits, species composition and compatibility types and submitted to ICAR-Hort-PGR platform for introduction

Species composition analysis of 32 parental lines showed that majority of their genome is constituted of four species namely Cymbidium insigne, C. lowianum, C. eburneumand C. hookerianum

A total of 40 different crosses/self attempted, 35 were incompatible. The incompatible crosses/self senesced and fell within 2 months of crossing. In some cases, capsules developed without the seed setting

Project 3: Development and Renement of Production and Protection Technologies for Improved Productivity, Marketing and Utilization of Orchids

3.1 Development of integrated oriculture enterprise

Seven Cymbidium hybrids evaluated for owering behavior, ower quality and productivity. The longest ower spikes were obtained in Valley Legend Ste whereas the Korean-4 produced spikes with shortest length.


The cost benet ratio was found to be 0.60

700 plants were propagated by treating back bulbs with Thiourea 5000 ppm

3.2 Renement of production technology in Cymbidium

Seven potting mixture and three types of pots were tested for growing of Cym Levis Duke 'Bella Vista'. Plastic pot with a mixture of Coco chips + Cocopeat + Brick pieces + Slow release fertilizer (3: 1: 1: 1g) / dry leaf fern (3:1:1:1) showed maximum pseudobulb diameter (6.00cm), longest leaf length (71cm) and maximum number of bulbs (5).

The vegetative and oral traits of fteen monopodial orchid genotypes of Aranda, Aranthera, Kagawara, Mokara, Renanthera, and Vanda were correlated based on historical data. The results showed a signicant correlation between different parameters studied. Regression analysis revealed that shoot thickness has a curvilinear i.e. second degree polynomial relationship with the leaf length whereas the vase life has a linear relationship with fullness value within the given range.

3.3. Flower drying in orchids0

The blooms of 50 species and hybrids oven dried at 60 C by embedding them in sand. Of these, the drying of the blooms of Papilionanthe teres, Vanda tessellata, Cattleya, 'Guanamiau City', Phalaenopsis 'Casablanca, Detroit, Vienna, Taida S. Red, Dendrobium moschatum, Dendrobium, 'Lervia', 'Madam Pink', 'A. Abraham', Oncidium, 'Wildcat Bobcat', 'Taka Yellow', 'Sweet Sugar', Arundina bamboosifolia, Epidendrum spp., were found successful.

3.4 Post harvest management in orchids

The longevity of orets of various orchid species at different stages owering was tested in Cellophane packing. The packing of orets in cellophane enhanced the longevity of orets as compared to control. However, it differed with the genotypes.

3.5 Development of protocol for mass multiplication of rare, endangered and threatened orchid species

Eight culture media supplemented with different concentration various plant growth regulator were tested for seed germination of Red Vanda. The swelling in embryo was recorded in four media namely Gamborg +1.5 mg AC +0.25 mg BAP, Gamborg +1.5 mg AC + 0.50 mg BAP, MS +1.5 mg AC + 0.50 mg BAP and NC +1.5 mg AC + 0.50 mg BAP after ve months.

The best seed germination in Phap. primulinum was recorded on Gamborg Media + AC 1.5 mg + 0.25 mg BAP, Nitsch media + AC 1.5 mg + 0.25 mg BAP and MS media + AC 1.5 mg + 0.25 mg BAP.


The explants viz. leaf, root and ower stalk Phalaenopsis mannii were cultured on MS media containing various concentrations of BAP, NAA and IBA. Single node ower stalk explant showed shoot proliferation and leaet initiation on media with or without BAP (1, 2 and 3mg/l) within 10 to 20 days of culture.

3.6 Flower induction in orchids

Phalaenopsis plants were grown under High Light Intensity (HLI) 7400 Lux and 0Low Light Intensity (LLI) 1320 Lux and the Day temperature was set at 25 C

0 0 0±2 C and night temperature 20 C ±2 C . It was recorded that LLI plants had longer and wider but lesser leaves, which record 31% longer as compared to HLI. 1st Flower opening was 27 days earlier in HLI and also spike length was 48% longer and number of oral bud recorded was 38% Higher than LLI.

Emma White were grown under High Light Intensity (HLI) 11200 Lux and Low 0 0Light Intensity (LLI) 1800 Lux Day temperature was set at 25 C ±2 C and night

0 0temperature 20 C ±2 C. LLI plants had longer and wider but lesser leaves, which record 9% longer as compared to HLI. 1st Flower opening was 17 days earlier in HLI and also spike length was 25% longer, and number of oral bud recorded was 44% Higher than LLI

3.7 Eco-friendly pest management

The species and hybrids conserved at the centre were weekly monitored for insects and pest incidence. A new pest, long-tailed mealy bugs (Pseudococcus longispinus) was recorded infesting Dendrobium mbriatum and Dendrobium chrysanthum.

A New report of an encyrtid wasp, Anagyrus sp. (Howard) as a parasitoid of long-tailed mealy bug, Psuedococcus longispinus and Mealy bug, Pseudococcus sp. infesting Orchids from Sikkim, India.

Botanical products from neem viz. azadirachtin (neem oil 0.03% EC) @ 5ml/L and Allium sativum gave effective management of mites under polyhouse conditions with mean population reduction of 86.80 % and 91.16 %, respectively.

Microbial biopesticides, Metarhizium anisopliae and Verticillium lecanii @ 2 ml/Litre of water was found effective in reducing the populations of thrips, Dichromothrips nakahari on Cymbidium, Levis Duke 'Bella Vista' with mean number of 3.00 and 2.67 thrips/plant after 14 days of treatment respectively.

Microbial biopesticides, Metarhizium anisopliae @2ml/Litre and Verticillium lecanii @2ml/Litre gave effective management of scale insect, Diaspis biosduvalii under polyhouse conditions.


Effective management of long-tailed mealy bug, Pseudococcus longispinus was achieved successfully with Nirma detergent powder and Surf excel detergent powder @ 2gm/L with a mean mortality of about 98.61% and 93.71% respectively.

Effective management of biosduval scale, Diaspis biosduvalii was achieved with Nirma detergent powder and Surf excel detergent powder @ 2gm/L with a mean percent population reduction of about 81.03% and 77.09% respectively.


30 hybrids of Cymbidium, 23 hybrids of Vanda, 14 hybrids of Dendrobium, 10

hybrids of Oncidium, 9 hybrids of Cattleya and 10 hybrids of Phalaenopsis

maintained and multiplied under DUS project on Orchids.

Morphological descriptors of Paphiopedilum (76) nalized and preparation of

DUS Test Guidelines of Paphiopedilum is going on. DUS Test Guidelines of

Oncidium published in PVJ of India under PPV & FRA in April 2014 issue and

notied for registration by Plant Authority during October, 2014

B. Externally Funded Project

Project 1: Preparation for Plant Variety Protection and Conducting Test for

Distinctness, Uniformity and Stability for Orchids

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