Establishment of regeneration and transformation system of ground-cover chrysanthemum Yuhuaxunzhang

CUI Xin-li,CHEN Fa-di,CHEN Su-mei(College of Horticultural,Nanjing Agricultural University,Nanjing 210095,China)

ground-cover chrysanthemum;regeneration;Agrobacterium tumefaciens;transformation

Using leaf disks of ground-cover chrysanthemum Yuhuaxunzhang as explants,an efficient and reliable regeneration and transformation system was established.The results showed that the content and proportion of different plant regulators affected greatly the regeneration of leaves.The optimum medium for the highest differentiation rate amounting to 91.1% was MS medium with supplement of 1.0 mg·L-1 naphthalene-acetic acid(NAA)and 1.0 mg·L-1 6-benzylamino purine(6-BA).A period of culture in dark could effectively prevent callus browning and promote shoot regeneration rate.The transformation was performed,with the optimized pre-culture duration 3 d,the suitable agroinfection duration 10 min,and the durations for appropriate co-culture and postponed antibiotics selecting 3 d.Browning of callus was accelerated when acetosytingone(AS)was used in the transformation experiments.The leaf was very sensitive to hygromycin(Hyg),and the optimal concentration of it for callus selecting was 6-8 mg·L-1,while 7 mg·L-1 of Hyg is appropriate for rooting selecting.For the control of agrobacterium growth,the optimal concentration of carbenicillin(Carb)was 250-350 mg·L-1.Several putative transformants were obtained,and PCR analysis indicated that the exogenous gene had been integrated into the genome of transformed chrysanthemum.