Molecular analysis of genetic stability in tissue culture raised plants of broccoli (Brassica oleracea L. var. italica)
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Date
2012
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ABSTRACT
The present investigation was undertaken with an objective of enhancing the frequency of plant
regeneration in broccoli (Brassica oleracea L. var. italica) and assessing their genetic fidelity. A high
efficiency plant regeneration protocol has been developed from hypocotyl, cotyledon, leaf and petiole
explants in broccoli (Brassica oleracea L. var. italica). The green house grown seedlings from 18-20 days
old leaf and petiole explants were excised. The explants were surface sterilized and cultured on shoot
induction medium. A high efficiency of shoot regeneration was observed in leaf (62.96%) and petiole
(91.11%) explants on MS medium supplemented with 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP +
0.019 mg/l NAA, respectively. The hypocotyl and cotyledon explants were excised from the in vitro grown
seedlings and cultured on shoot regeneration on MS medium supplemented with 2.0 μM TDZ + 0.5 μM IAA
and 2.0 μM TDZ + 0.59 mM adenine, respectively. The hypocotyl explants showed high frequency of shoot
regeneration (96.09%) as compare to cotyledon explants (88.88%). MS medium supplemented with 0.20
mg/l NAA was found to be best for root regeneration (93.99%) from in vitro developed shoots. The
regenerated plantlets were acclimatized successfully on cocopeat. A protocol for high frequency plant
regeneration has been standardized. Genetic fidelity of the regenerated plantlets were studied using RAPD.
Genomic DNA was isolated from the leaves of randomly selected 20 in vitro raised plantlets and from
mother plant of broccoli using CTAB method with some modifications. The quantified DNA was then
subjected to PCR and a total of 21 primers were used for genetic fidelity studies. Among the 21 primers
initially screened, 15 produced clear and scorable amplification products. A total of 66 fragments were
amplified by 15 random primers out of which 56 were found to be monomorphic and a high degree of
monomorphism (88.45%) was observed among in vitro raised plantlets and from mother plant of broccoli.
On an average, 2.96 amplified fragments were observed per primer. Thus the technique of RAPD-PCR was
found to be reliable to assess the genetic fidelity of tissue culture raised plantlets and from mother plant of
broccoli (Brassica oleracea L. var. italica).