Tissue Culture of Rubus sp. by Different Methods and Assessment of Genetic Fidelity of Regenerated Plants Using RAPD

Different methods employed in plant tissue culture can provide a valuable source of plants for the horticultural industry and novel germplasm for breeding programs, but the genetic fidelity and lack of somaclonal variation of regenerated plants needs to be verified. In this study, the genetic fidelity of blackberry (Rubus hirtus Waldst. & Kit.) plants regenerated in vitro was assessed with 11 randomly amplified polymorphic DNA (RAPD) markers. Three routes were assessed: callus induction on Yasuda medium with 6-benzyladenine (BA, 8.88 μM), 1-naphthaleneacetic acid (NAA, 10.84 μM) and glycerol (2%, v/v), somatic embryogenesis on Murashige and Skoog (MS) supplemented with 7.57 μM abscisic acid (ABA), and micropropagation from single nodes on MS basal medium containing NAA (0 and 2.86 μM) and BA (0, 4.44, 8.88 and 17.76 μM). MS medium with 2.86 μM NAA and 8.88 or 17,76 μM BA was the most effective medium for axillary shoot multiplication of R. hirtus and Rubus sanctus Schreb. from nodal segments. A total of 618 fragments were successfully generated by RAPD and the maximum of loci was observed in primer 1204-209 that show 10 and 69 bands. Genetic similarity exceeded 86% when regenerated plants were compared to mother plants. Based on the RAPD data profile, almost true-to-type plants were produced by different methods of plant regeneration (direct shoot regeneration, somatic embryogenesis and organogenesis).