Development of an in vitro regeneration system and validation of genetic stability in phalaenopsis hybrid winter spot with molecular marker
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Date
2016
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
Abstract
Phalaenopsis “Moth Orchids” are among the most beautiful flowers in
the world. This genus has economic value for pot plant and cut flower production
and is distributed throughout Southeast Asia. Most popular method of propagation
for orchid is through in vitro propagation, as it produces large number of clones in
relatively short duration. Despite its potential to produce numerous plants from a
single leaf segment, it is liable to unpredictable mutations or somaclonal variation
during the process of multiplication. Variation can arise due to many reasons such
as type of media, plant growth regulators and its concentration, type of explants
and number of subculture cycles. The percentage of the variation can range from
0-100% depending on varieties with an average of 10% among Phalaenopsis
(Tokuhara and Mii, 1993).
So the present investigation on “Development of an in vitro
regeneration system and validation of genetic stability in Phalaenopsis hybrid
Winter Spot with molecular marker” was taken up at the Center for Plant
Biotechology and Molecular Biology, College of Horticulture from 2013-2016.
Flowering mother plants of Phalaenopsis hybrid Winter Spot were used
as explant source. Among the explants namely inflorescence node, transverse thin
cell layer of leaf and root segments used for tissue culture study in this orchid,
inflorescence node was the best with respect to culture response.
The best surface sterilization treatment for leaf explants identified was
treatment 0.1% bavistin + prill 2 drops (30 min) and 0.1 per cent HgCl 2 ( 8 min)
which give maximum per cent of culture survival and minimum contamination
rate. The best surface sterilization treatment for inflorescence node identified was
treatment with 0.1% bavistin + 2 drops prill (30 min) , one minute dip in 70 per
cent ethanol and 0.1% HgCl 2 (7 min).From different basal media (full MS and 1⁄2 MS) tried, response was
observed only in the medium of Full MS for inflorescence node. Among the
different growth regulators tried, MS medium supplemented with BA and TDZ
was found to give good shoot regeneration from inflorescence node explants. MS
+2mgl -1 TDZ recorded highest percentage (80%) of culture establishment,
followed by MS + 4.5 mgl -1 of BA (55%) per cent of sprouting. Among the
explants tried, only inflorescence node responded with sprouting. Root segment
remained as such without any change, whereas leaf explants remained green up to
2 weeks, thereafter started drying in all the growth regulators combination.
For induction of multiple shoot, MS medium supplemented with 4.5
mgl -1 BA resulted in the highest average number of multiple shoot (4.15).
Elongation and rooting was observed in MS medium supplemented with BA
4.5mgl-1 +IAA 1mgl -1 with 80 % rooting. Root initials were observed 50 days
after inoculation. The potting media, charcoal, brick pieces and sphagnum moss in
the ratio of 1:1:1 was found ideal for hardening of Phalaenopsis hybrid winter
spot with 100% survival.
Genetic stability studies using RAPD marker were carried out with the
mother plants along with three regenerants each. Six primers were selected based
on DNA amplification pattern. In RAPD assay, M1 mother plant recorded the
highest average polymorphism of 19.7% and M3 mother plant recorded the least
average polymorphism of 8.18%.
Using NTSYS software, the similarity coefficients for first, second and
third plant between M1 mother plant, M2 mother plant and M3 mother plant and
corresponding regenerants were 0.91, 0.92 and 0.93 respectively. In fourth plant,
the similarity coefficient exhibited 100% similarity between mother plant, the first
clone C1 and third clone C3.
The established micropropagation protocol can be used with suitable
modification for large scale production of other Phalaenopsis varieties.
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173756