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Establishment of a highly efficient transformation system for eucalyptus(Eucalyptus urophylla)and study on relevant mechanism

【摘要】:正Hypocotyls were used as explants to establish a regeneration protocol for E.urophylla. N-phenyl-N'-[6-(2-chlorobenzothiazol)yl]urea(PBU),one kind of di-substituted urea,was useful to regeneration of E.urophylla.The hypocotyls incubated on a modified MS(SPCa) medium,supplemented with PBU 6.6 μmol·L~(-1)and IAA 0.57 μmol·L~(-1),could dedifferentiate and form calluses at 100% after 7 days.30days later,the callus form 3 types,brown callus,white callus and blue one.Compared several physiological indexes,the activities of SOD,POD and CAT of blue callus were the highest;And the content of H_2O_2,MDA and soluble protein of brown callus was the highest.When inoculated at the bud induction medium,the rate of bud induction of blue callus was the highest,the rate of bud induction of brown callus was the lowest.Compared with TDZ or the combination of BA and 2,4-D,PBU stimulated more vigorous callus and restrained callus from turning to darkness.What's more,the calluses induced by PBU showed high frequency on adventitious buds formation with percent 57.Eucalyptus callus was sensitive to kanamycin and was insensitive to carbenicillin.The kanamycin content of 15 mg-L-l was suitable to choice positive transformated callus.And the carberticillin content of 150 mg·L~(-1)was suitable to restrain Agrobacterium tumefaciens.The result of polymerase chain reaction(PCR)showed the transformation rate of eucalyptus callus was percent 18.8.

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