IN VITRO SELECTION AND REGENERATION OF SALIX SP. AGAINST CANKER DISEASE
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Date
2019-05
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UHF,NAUNI
Abstract
ABSTRACT
In the present investigations,in vitro regeneration of commercially importantSalix sp.
was carried out and an attempt was made for in vitro selection of this species against Cytospora
along with molecular studies. Indirect organogenesis form leaf and inter nodal segment
was achieved. Treatment of 12.5% NaOCl for 10 minutes to both explants along with 0.2% bavistin
was found to be the best as it gave maximum number of uncontaminated cultures and per cent
survival. Callus was induced from leaf and inter nodal explant on MS medium supplemented with 0.5
mg/l NAA with 1.5 mg/l BA and 0.5 mg/l NAA with 0.5 mg/l BA respectively. Highest percentage of
callus was obtained from leaf (96.67%) explants followed by inter nodal segment (93.34%). Percent
shoot induction from leaf and inter nodal segment derived calli on 0.5 mg/l BA, 0.50 mg/l Kinetin
with 0.25 mg/l GA3 and 0.5 mg/l BA with 0.25 mg/l GA3 was 83.33% and 75.00% respectively. Leaf
explants was found to be most responsive explants for indirect regeneration. Highest multiplication
rate (1:4) was obtained on MS medium fortified with 1.50 mg/l BA with 0.25 mg/l GA3. Shoot
number was elevated with the increase in subculturing passages upto third passage thereafter there
had been a decline in it. Activated charcoal brings about elongation of shoots. The regenerated shoots
were rooted in half strength MS medium containing 500 mg/l activated charcoal. During hardening it
was observed that plantlets showed highest survival of 30% in a potting mixture containing sand, soil
and cocopeat. To carry out in vitro selection for resistance development, the isolation of canker
causing pathogen was done. On the basis of morphological features and BLASTn analysis of ITS
region of pathogen, it was identified as Cytospora chrysosperma. The optimum concentration of
fungal culture filtrate for selection against canker disease was 30.00 per cent resulting in 12.96 per
cent survival of calli. However no shoots were regenerated after selection of calli. For molecular
characterization dendrograms were generated to assess the variations among mother plant, control
calli and 30 per cent FCF treated calli, using RAPD and ISSR markers which separated them in two
clusters where mother plant and control plant always fall in one cluster showing 70 and 79 percent
similarity with each other while the selected variants clustered randomly suggesting genetic variation
with mother plant, control plant and within the variants.
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