Efficient in vitro plant regeneration via leaf base segments of indica rice (Oryza sativa L.)

A reliable and reproducible protocol has been developed for high frequency plant regeneration from 4-5 mm long leaf base segments of 4 days old in vitro germinated seedlings of indica rice (Oryza sativa) cultivar Rasi. The effect of age of seedlings, position of leaf base segments and optimum concentration of 2,4-D on callus induction frequency was investigated with a future aim to use leaf bases for biolistic and Agrobacterium-mediated transformation experiments. Friable, nodular and white to pale yellow embryogenic callus cultures (206 mg fresh weight /explant) were obtained from the first basal segments of rice seedlings on Linsmaier and Skoog (LS) medium supplemented with 2,4-D (11.3 microM) and 3.0 microM thiamine-HCL. Plant regeneration was achieved after the transfer of 54 days old embryogenic callus cultures to Murashige and Skoog (MS) medium supplemented with BAP (2.2 microM) and NAA (0.27 microM). In vitro regenerated plants with multiple shoots and roots transferred to sterile soil in growth chamber and maintained in greenhouse exhibited normal growth and were phenotypically similar to plants germinated from seeds.