Somatic embryogenesis and plant regeneration of blackberry using the thin cell layer technique

Abstract

The present study is the first to report somatic embryogenesis (SE) based on a plant regeneration protocol for blackberry. It uses transverse thin cell layer technique (tTCL). Two blackberry genotypes, ‘High prickle’ (Rubus sanctus) and ‘Low prickle’ (Rubus hirtus) were used as explants. The explants were soaked in ascorbic and citric acids (60 mg l−1 each) solution prior to culture on Murashige and Skoog (MS) medium containing 2.32 μM kinetin (KIN), 2.69 μM α-naphthaleneacetic acid (NAA) and 8.88 6-benzyladenine (BA). This not only reduced the phenolic compounds (in ‘High prickle’), but also produced friable and yellow-pale green calluses. The highest level of embryogenic callus initiation in both genotypes occurred in half strength MS medium containing 60 g l−1 sucrose, 9.76 μM KIN and 7.99 μM BA. The MS medium fortified with 7.57 μM abscisic acid (ABA) and malt extract (700 mg l−1) or glutamine (400 mg l−1) encouraged the formation and development of embryos on calluses originating from dermal parts of ‘High prickle’ explants. Yasuda (YA) medium enrichd with 8.88 μM BA, 10.84 μM NAA and glycerol (2%) promoted embryo development and shoot regeneration on calluses originating from dermal parts of ‘High prickle’ and ‘Low prickle’ explants respectively. Germination of embryos and growth of normal plantlets occurred on half strength MS medium containing 4.88 μM BA, 2.02 μM gibberellic acid (GA3) and 0.05 μM NAA. Histological evaluations confirmed the successful occurrence of the different stages of embryogenesis.