Developing high frequency in vitro plant regeneration protocol of Brassica napus

Various factors like plant growth regulator combinations, explant type, explant age were examined for establishing a convenient protocol for high frequency plant regeneration of Brassica napus. Cotyledon and hypocotyl explants of B. napus cv. BARI sarisha-8 were cultured on Murashige and Skoog (MS) medium fortified by various strengths of 6-Benzyl Amino Purine (BA), 2,4-Dichlorophenoxy Acetic Acid (2,4-D) and α-Napthalene Acetic Acid (NAA) to determine the suitable callus induction and shoot regeneration media. MS medium supplemented with 0.5 mg/L NAA and 3.0 mg/L BA was the best regeneration medium because of showing highest frequency for both callus (80% for cotyledon and 53.33% for hypocotyl explants) and shoot initiation (73.33% for cotyledon and 40% for hypocotyl explants). Four days old cotyledon explants showed the highest (73.33%) frequency of shoot regeneration and highest shoot number to each explant (3.13) when 3-7 days old cotyledon cultured. Among the four tested genotypes of B. napus, BARI sarisha-8 showed the highest shoot regeneration frequency (73.33%) with maximum shoot number to each explant (3.13) while the lowest frequency of shoot regeneration was found in BINA sarisha-4 (46.66%) with minimum shoot number per explant (1.66). The ideal rooting medium was MS media comprised with 0.1 mg/L NAA that offered the maximum frequency (100%) of rooting. The regenerated plantlets were shifted to pot soil, acclimatized and grown until maturity in natural conditions. All plants were fertile and morphologically identical with the source plants.