Effect of antibiotics on shoot regeneration and suppression of Agrobacterium growth in apple rootstock
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Date
2011
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NAUNI,SOLAN,UHF
Abstract
ABSTRACT
The present investigation aims at “Studies on cell culture for genetic transformation in
Punica granatum L.”. Genetic transformation experiment was conducted with cotyledonary
explants excised from in vitro raised seedlings of Punica granatum L. cv. Kandhari Kabuli.
Explants were preconditioned on standardized callus induction medium (solid MS medium
supplemented with 4.0 mg/l BA and 3.0 mg/l NAA) for 48 hours followed by 48 hours of cocultivation
with Agrobacterium tumefaciens. Friable putative transgenic were used for initiation
of cell suspension culture. Cells of various shapes such as oval, elongated, columnar, elliptical
and isodiametric were observed in the suspension culture. Cells proliferated best on selective MS
medium supplemented with 4.0 mg/l BA + 3.0 mg/l NAA and 50 mg/l kanamycin along with 500
mg/l cefotaxime with average cell doubling time of 5 days. Combination of a cytokinin (BA) and
an auxin (NAA) was found to be better for cell proliferation as compared with the combination of
two cytokinins (BA and TDZ). Growth curve of cells in suspension culture consisted of lag phase
(5-6 days), exponential phase (7 days) and stationary phase (4-5 days) followed by cell death.
Plating efficiency of 0.7 % was obtained by plating of cell aggregates (5-6 cells) on selective MS
medium supplemented with 4.0 mg/l BA and 3.0 mg/l NAA utilizing modified Bergman
technique. Putative transgenic macrocalli did not show shoot regeneration. Positive PCR results
were observed in transgenic calli obtained through cell suspension culture. The protocol, thus
offer an opportunity to produce the putative transformed tissues/ plantlets with single cell origin.
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