Hormone Concentrations Transgenic Calli Production during Tissue Culture Regeneration of Eucalyptus urophylla × E.grandis DH32-28
A tissue culture regeneration system was explored to enable a stable genetic transformation system of Eucalyp-tus urophylla × E. grandis DH32-28 to be established with the objective of providing a benchmark reference for regener-ation of transgenic Eucalyptus and enable studies of gene function and molecular breeding in Eucalyptus. Leaves and stems were used in this study as explants,and the plant growth regulators thidiazuron (TDZ),6-benzylaminopurine (6-BA),1-naphthalene acetic acid (NAA),and indole-3-butyric acid (IBA) were used to induce callus,adventitious shoot differentiation,and rooting. The critical concentration of kanamycin for screening in Agrobacterium-mediated genetic transformation experiments was determined by assays of the resistance of the cultured materials to kanamycin at the stages of callus and root induction. The results showed that using MS as the basic culture medium,the hormone combina-tions of 0.025 mg·L-1 TDZ+0.15 mg·L-1 NAA and 0.05 mg·L-1 TDZ+0.15 mg·L-1 NAA were the most suitable for callus induction from leaves and stem segments of DH32-28,respectively,and provided a callus induction rate of 98.3％. The induced calli from leaves and stem segments were transferred to an adventitious shoot differentiation medium con-taining 0.5 mg·L-16-BA+0.1 mg·L-1 NAA,and the adventitious shoot differentiation rates were 44.4％ and 96.7％,re-spectively. The highest induction rate of adventitious roots was 72.8％ in DH32-28 regenerated shoots with 1/2 MS as the basic medium and 0.5 mg·L-1 IBA. The optimal screening concentration of kanamycin was 5 mg·L-1 at the stage of callus and adventitious bud induction,and 20 mg·L-1 at the rooting stage. In this study,an efficient regeneration system for DH32-28 was successfully established. The Agrobacterium-mediated genetic transformation method was successfully used to introduce the red fluorescent protein reporter gene,DsRed2,into the recipient DH32-28 stem segments,and transgenic calli expressing DsRed2 were obtained.
Eucalyptus urophylla × E. grandis DH32-28plant regenerationgenetic transformationDsRed2
万方数据
维普
