Res. Highlights 2014-15

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 Orchids
Pakyong‐737106, Sikkim, India
Research Highlights
Copyright © 2015 National Research Center for Orchids,
Pakyong-737 106, Sikkim, India
Chief Editor
Dr. D. R. Singh
Technical Editor
Dr. 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 Printers
Mumbai
Foreword
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 signicant
achievements made by the centre during 2014-15. During this year
signicant progress has been made in molecular characterization, DNA
barcoding, cpDNA isolation protocol, indemnication 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.
ICAR-National Research Centre for Orchids
Research Highlights 2014-15
Executive Summary
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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 specic 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 notied for registration by Plant Authority during
October, 2014.
ICAR-National Research Centre for Orchids
Research Highlights 2014-15
Ongoing Research Projects
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 Renement of Production and Protection
Technologies for Improved Productivity, Marketing, and
Utilization of Orchids
3.1
Development of integrated oriculture enterprise
3.2
Renement 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
ICAR-National Research Centre for Orchids
Research Highlights 2014-15
Research Highlights
Progress and Achievements
ICAR-National Research Centre for Orchids
Research Highlights 2014-15
A. Institute Funded Projects
Project1: Conservation Characterization and Sustainable Use of
Diversity in Orchids
1.1 DNA ngerprinting of commercially important orchids
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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 highquality chloroplast DNA.
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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.
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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.
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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.
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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 reecting that ISSR proling a powerful molecular method to
determine the genetic relationship and diversity.
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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 efciently. The
UPGMA dendrogram grouped all four species into two distinct clusters.
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Microsatellite markers were developed from Himalayan orchid species
Paphiopedilum fairrieanum
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Intra specic variability analysis of Coelogyne ovalis, Smitinandia micrantha
and Vanda cristata has been completed
ICAR-National Research Centre for Orchids
Research Highlights 2014-15
1.2 A comparative study of asymbiotic and symbiotic germination in selected
species and hybrids of Calanthe, Cymbidium and Pahiopedilum
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Various orchid species along with substratum were collected from natural
habitats. The seed packets were placed in the substrate for ex vitro symbiotic
germination.
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The mature seeds of Paphiopedilum species and crosses were cultured on
modied 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.
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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
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Fifty orchid species having medicinal importance were listed out by gathering
information from different sources of literature
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Information on local distribution, owering period and medicinal value was
collected and summarised
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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
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Identication of NAGS collections, NOAC#324 (Dendrobium ovatum),
NOAC#757 (Eria tomentosa) & NOAC#1188 (Micropera rostrata)
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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.
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Characterization of new Cymbidium breeding line (Cym. 'September Sunset' x
Cym. 'Palkibaris').
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Multiplication of Aranda 'Kunga Gyatso' and initiated State & AICRP trial (NorthEastern Plain & Hill Zone).
ICAR-National Research Centre for Orchids
Research Highlights 2014-15
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In-vitro plantlets of Vanda cross, PBX-12-169 (V. 'Bernice Miller' x V.
coerulescens) developed.
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Characterization of Paphiopedilum clones-I&II and ex-vitro hardening of 3
hybrid progenies (PBX-11-150, 11-155 & 12-169).
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More than 100 NAGS accessions were supplied to Forest Department,
Government of Nicobar Islands.
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Major focus given to generating intergeneric crosses to strengthen breeding
programmes and > 200 crossings were done. Promising breeding line, PBX-0529/31 derived from Cym. 'Red Beauty' x Cym. 'Golden Elf' with attractive ower
colour and early owering traits characterized.
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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.
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1st year AICRP trial for Aranda 'Kunga Gyatso' was completed in North-Eastern
Plain & Hill zone.
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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
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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
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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
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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 Renement of Production and Protection
Technologies for Improved Productivity, Marketing and
Utilization of Orchids
3.1 Development of integrated oriculture enterprise
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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.
ICAR-National Research Centre for Orchids
Research Highlights 2014-15
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The cost benet ratio was found to be 0.60
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700 plants were propagated by treating back bulbs with Thiourea 5000 ppm
3.2 Renement of production technology in Cymbidium
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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).
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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 signicant
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 orchids
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The blooms of 50 species and hybrids oven dried at 600C 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
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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
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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.
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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.
ICAR-National Research Centre for Orchids
Research Highlights 2014-15
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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 leaet 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
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Phalaenopsis plants were grown under High Light Intensity (HLI) 7400 Lux and
Low Light Intensity (LLI) 1320 Lux and the Day temperature was set at 25 0C
±20C and night temperature 200C ±20C . 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.
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Emma White were grown under High Light Intensity (HLI) 11200 Lux and Low
Light Intensity (LLI) 1800 Lux Day temperature was set at 25 0C ±20C and night
temperature 20 0C ±20C. 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
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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.
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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.
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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.
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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.
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Microbial biopesticides, Metarhizium anisopliae @2ml/Litre and Verticillium
lecanii @2ml/Litre gave effective management of scale insect, Diaspis
biosduvalii under polyhouse conditions.
ICAR-National Research Centre for Orchids
Research Highlights 2014-15
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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.
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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.
B. Externally Funded Project
Project 1: Preparation for Plant Variety Protection and Conducting Test for
Distinctness, Uniformity and Stability for Orchids

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
notied for registration by Plant Authority during October, 2014
ICAR-National Research Centre for Orchids
Research Highlights 2014-15