An efficient in vitro propagation method has been developed for the first time for Musa acuminata (AAA) cv. Vaibalhla, an economically important banana cultivar of Mizoram, India. Immature male flowers were used as explants. Murashige and Skoog's (MS) medium supplemented with plant growth regulators (PGRs) were used for the regeneration process. Out of different PGR combinations, MS medium supplemented with 2 mg L(-1) 6-benzylaminopurine (BAP) + 0.5 mg L(-1) α-naphthalene acetic acid (NAA) was optimal for production of white bud-like structures (WBLS). On this medium, explants produced the highest number of buds per explant (4.30). The highest percentage (77.77) and number (3.51) of shoot formation from each explants was observed in MS medium supplemented with 2 mg L(-1) kinetin + 0.5 mg L(-1) NAA. While MS medium supplemented with a combination of 2 mg L(-1) BAP + 0.5 mg L(-1) NAA showed the maximum shoot length (14.44 cm). Rooting efficiency of the shoots was highest in the MS basal medium without any PGRs. The plantlets were hardened successfully in the greenhouse with 96% survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro regenerated plantlets of M. acuminata (AAA) cv. Vaibalhla. Eight RAPD and 8 ISSR primers were successfully used for the analysis from the 40 RAPD and 30 ISSR primers screened initially. The amplified products were monomorphic across all the regenerated plants and were similar to the mother plant. The present standardised protocol will find application in mass production, conservation and genetic transformation studies of this commercially important banana.
Agrobacterium-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA) was performed using Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA2301AtNHX1 plasmid. For the transformation efficiency assay, the un-and pre-cultured immature male flower explants were subjected to various methods of injury such as hypodermal needle injury, with and without sonication and vacuum infiltration. The explants were then inoculated in bacterial suspension by occasional shaking for 30 minutes. After inoculation, explants were co-cultivated for 3 days in dark condition at 22° C in a liquid MS basal medium supplemented with 100 µM acetosyringone. For selection of transformants, the treated explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP (2 mg/L), NAA (0.5 mg/L) and ascorbic acid (75 mg/L) along with antibiotics kanamycin (100 mg/L), cefotaxime (300 mg/L) and augmentin (300 mg/L).The putative transformation was analyzed using histochemical GUS assay. 14 and 21 days pre-cultured male flower explants subjected to 4-5 needle point injury resulted in 93.33% and 100% putative transformation respectively. 30s sonication combined with 5 minutes of vacuum infiltration for 7, 14 and 21 days pre-cultured immature male flower explants gave 73.33%, 66.66% and 71.42% putative transformation respectively.
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