In vitro selection of Punica granatum L. cv. Kandhari Kabuli against bacterial blight and pomegranate wilt
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Date
2015
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YSPU
Abstract
The present investigation aims at “In vitro selection of Punica granatum L cv. Kandhari Kabuli against bacterial blight and pomegranate wilt”. A valuable plant regeneration and in vitro selection protocol against bacterial blight and pomegranate wilt disease was developed for Punica granatum L. cv. Kandhari Kabuli. Indirect organogenesis form juvenile (cotyledon and hypocotyl) explants excised from 14 to 15 days old in vitro germinated seedlings and mature explant (leaf) was carried out. Callus was induced from cotyledon explant on MS medium supplemented with 4.0 mg/l NAA and 3.0 mg/l BA while in case of hypocotyl explant the best treatment for callus induction was found out to be solid MS medium supplemented with 4.0 mg/l NAA and 2.0 mg/l BA. Callus was induced from leaf explant on solid MS medium supplemented with 4.0 mg/l NAA and 2.0 mg/l Kinetin. Highest percentage of callus was obtained from cotyledon (83.33%)
explants followed by hypocotyl (72.21%) and leaf (68.25%) explants. Shoots were induced from hypocotyl and cotyledon derived calli on MS medium supplemented with 1.0 mg/l BA, 0.50 mg/l Kinetin and 0.25 mg/l NAA with 76.39 and 62.50 % shoot bud induction. Leaf derived calli responded best on MS medium supplemented with 1.5 mg/l BA, 0.50 mg/l Kinetin and 0.25 mg/l NAA with 59.72 per cent of shoot bud induction. The regenerated shoots were rooted in half strength MS medium containing 500 mg/l activated charcoal. Cotyledon explants was found to be most responsive explants for indirect regeneration method. To carry out in vitro selection for resistance development the pathogen causing bacterial blight and
pomegranate wilt disease were isolated. On the basis of morphological, biochemical features and by BLASTn analysis of sequenced 16S rRNA/ITS region of the pathogen, the pathogen causing bacterial blight was identified as Xanthomonas axonopodis pv. punicae and pathogen causing wilt disease was identified as Ceratocystis fimbriata. Cell line selection against bacterial blight was done by using culture filtrate of Xanthomonas axonopodis pv. punicae while selection against wilt was done by using culture filtrate of Ceratocystis fimbriata as a selective agent. The optimum concentration of bacterial culture filtrate at which calli selected was 25.0 per cent resulting in 12.96 per cent survival of calli. However, the optimum
concentration of fungal culture filtrate for selection against pomegranate wilt disease was 40.0 per cent resulting in 18.52 per cent survival of calli. Shoots were regenerated from the selected calli after two cycles of selection. The calli selected against BCF showed shoot regeneration only after 3rd subculture passage and increased up to fourth subculture passage resulting in 44.45 per cent shoot regeneration and thereafter start decreasing. Similarly, in calli selected against FCF shoot regeneration was observed only after 2nd subculture passage and increased up to fourth subculture passage resulting in 52.78 per cent shoot regeneration and thereafter start decreasing. In vitro selected shoots were rooted in half strength MS medium supplemented with 500 mg/l activated charcoal followed by hardening. The plants selected against BCF showed resistance development against Xanthomonas axonopodis pv. punicae and plants regenerated from callus selected against FCF showed resistance against Ceratocystis fimbriata during in vitro and ex vitro testing. Dendrogram generated using RAPD and ISSR marker separated in two clusters where mother plant and control plant always fall in one cluster showing maximum similarity with each other while the selected variants clustered separately suggesting genetic variation with mother plant, control plant and within the variants.
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Keywords
selection, diseases, planting, biological phenomena, pomegranates, regeneration, vegetative propagation, auxins, punica granatum, crops