Analysis of influence factors of adventitious bud induction from in vitro leaves of blackberry cultivar 'Arapaho' and establishment of regeneration system

Using in vitro leaves from aseptic seedling of blackberry(Rubus spp.) cultivar 'Arapaho' as explants,effects of basic medium type and concentrations of 6-BA and IBA,dark culture time,leaf position and inoculation mode of explant on adventitious bud induction and effect of IBA concentration on adventitious bud rooting were studied by orthogonal and single factor experiments.And on this basis,regeneration system of in vitro leaves of cultivar 'Arapaho' was preliminarily established.The orthogonal experiment result shows that influence of basic medium type on induction rate and average number of adventitious bud is the most,while influence of IBA concentration on induction rate and that of 6-BA concentration on average number of adventitious bud are both less.And the optimal medium being suitable for adventitious bud induction of cultivar 'Arapaho' is MS medium containing 2.0 mg·L-1 6-BA and 1.0 mg·L-1 IBA.The results of single factor experiments show that effects of dark culture time,leaf position and inoculation mode of explant on induction rate of adventitious bud are significant.The optimal time of dark culture is 21 d.The regeneration capacity of leaves at middle and upper parts of a plantlet is stronger,in which,induction effect of adventitious bud from leaves at the 3rd and 4th positions is the best.Inoculating with foliar facing upwards on medium is more beneficial to induce adventitious bud.Rooting rate of adventitious bud is 100.0% in MS medium containing 0.2 mg·L-1 IBA with more roots and well growth vigor.The regeneration system of in vitro leaves of cultivar 'Arapaho' as follows: using the 3rd and 4th position leaves as explants,after proper pruning,inoculating with foliar facing upwards on MS medium containing 2.0 mg·L-1 6-BA and 1.0 mg·L-1 IBA,dark culturing for 21 d then light culturing for 30 d,transferring the adventitious bud to MS medium containing 0.5 mg·L-1 6-BA and 0.3 mg·L-1 NAA for subculture,transferring to MS medium containing 0.2 mg·L-1 IBA for rooting when height of adventitious bud reaches about 2 cm,finally,obtaining complete plantlets.