Asha S. Nayar
Molecular Evaluation of Genomic Stability of Banana Plants Developed by in Vitro Clonal Propagation - Vellayani Department of Horticulture, College of Agriculture 2001
Attempts were made for evaluating the genomic stability of in vitro
propagated Peel banana plantlets at molecular level, during 1999- 2000, at the
Plant Molecular Biology and Biotechnology Centre, College of Agriculture,
Vellayani. Efforts were made to standardise the DNA isolation method and PCR
amplification conditions, to identify the primer producing reproducible
polymorphic bands and to compare the banding patterns characteristic to the
subcultures and the mother plant. The emerging leaves of the Rxl banana plants
before they fully unfurl, gave the highest DNA yield of2825 ng! III whereas, the
in vitro leaves gave the highest optical density (OD) ratio of 1.76. The purity of
DNA was the highest (0. D. ratio 1.81) while CTAB method (Scott et al., 1994)
was adopted. The DNA quantity was the highest in the Walbot's method, viz.
3000 ng! Ill. The OD ratio increased from 1.33 to 1.46 on addition of proteinase
k. Additional purification step increased the OD ratio from 0.93 to 1.18. One per
cent polyvinyl pyrrolidone (PVP) and 1.5 per cent J3-mercaptoethanol, when
added in the extraction buffer produced transparent DNA pellet. 0.9 per cent and
1.4 per cent of agarose concentration were found to be the best for the genomic
DNA and RAPD banding patterns respectively. The optimum PCR programme
Wdenaturation at 95° C for 1.0 minute, annealing at 36° C for 1.0 minute and 30
seconds, and extension at 72° C for 2.0 minutes. The synthesis step was extended
further by 6.0 minutes. A total of 134 RAPDs were generated when PCR
amplification was done of which 130 were polymorphic. OPA- 06, OPB-IO and
OPB- 14 produced no amplification. No difference was found in the banding
pattern of the three subcultures and the mother plant of Fed banana, when
amplification reaction was carried out using OPA-20. A total of five intense
bands and three faint bands were obtained with OPA-20.
635 / ASH/MO
Molecular Evaluation of Genomic Stability of Banana Plants Developed by in Vitro Clonal Propagation - Vellayani Department of Horticulture, College of Agriculture 2001
Attempts were made for evaluating the genomic stability of in vitro
propagated Peel banana plantlets at molecular level, during 1999- 2000, at the
Plant Molecular Biology and Biotechnology Centre, College of Agriculture,
Vellayani. Efforts were made to standardise the DNA isolation method and PCR
amplification conditions, to identify the primer producing reproducible
polymorphic bands and to compare the banding patterns characteristic to the
subcultures and the mother plant. The emerging leaves of the Rxl banana plants
before they fully unfurl, gave the highest DNA yield of2825 ng! III whereas, the
in vitro leaves gave the highest optical density (OD) ratio of 1.76. The purity of
DNA was the highest (0. D. ratio 1.81) while CTAB method (Scott et al., 1994)
was adopted. The DNA quantity was the highest in the Walbot's method, viz.
3000 ng! Ill. The OD ratio increased from 1.33 to 1.46 on addition of proteinase
k. Additional purification step increased the OD ratio from 0.93 to 1.18. One per
cent polyvinyl pyrrolidone (PVP) and 1.5 per cent J3-mercaptoethanol, when
added in the extraction buffer produced transparent DNA pellet. 0.9 per cent and
1.4 per cent of agarose concentration were found to be the best for the genomic
DNA and RAPD banding patterns respectively. The optimum PCR programme
Wdenaturation at 95° C for 1.0 minute, annealing at 36° C for 1.0 minute and 30
seconds, and extension at 72° C for 2.0 minutes. The synthesis step was extended
further by 6.0 minutes. A total of 134 RAPDs were generated when PCR
amplification was done of which 130 were polymorphic. OPA- 06, OPB-IO and
OPB- 14 produced no amplification. No difference was found in the banding
pattern of the three subcultures and the mother plant of Fed banana, when
amplification reaction was carried out using OPA-20. A total of five intense
bands and three faint bands were obtained with OPA-20.
635 / ASH/MO