Callus Induction And Establishment Of Transformation System On Leaf Of Peach

Peach is well loved in China because of its longevity and beautiful auspicious meaning.And we can use its stem and root as pharmaceutical.Traditional breeding process of woody plants will take at least 8 years,which also largely increased the workload of breeders.The rapid development of bioengineering has improved gardening products,created new germplasm resources and propagation of shortcuts.However,the constructions of regeneration system and genetic transformation system have not been studied deeply.Based on current situtation,we explored the effects of different breeds,types and plant growth regulators on the frequency of callus formation and relative growth of callus from the leaves of Maotao,‘Qiumihong' and ‘Zhong Youtao 14',which could determine the suitable medium for callus induction.In addition,the Agrobacterium-Mediated transformation was conducted on the leaves of plantlets of Maotao and ‘Zhong Youtao 14' aiming to study the efficiency affected by several factors such as the concentration of bacterium and Acetosyringone(AS),infecton time,co-culture time and the way of infection were compared.The transformation system of leaves from peach was established initially,which would lay a foundation for the study of genefunction.The main results as follows:1.Tissue cultured plantets of peachThe stem segments with apical bud or axillary bud were used as explants from Maotao,‘Qiumihong' and ‘Zhong Youtao 14' respectively.After the sterilization by mercuric chloride for 6 min,the germination rate of apical bud and axillary bud were85.01% and 72.88%(Maotao),80.02% and 69.02%(‘Qiumihong')and 85.01% and72.88%(‘Zhong Youtao 14').The optimal medium for Maotao and ‘Zhong Youtao14' were composed by MS + 6-BA 1.0 mg·L-1+ IBA 0.1 mg·L-1+ Ag NO3 0.5 mg·L-1and the optimal medium for ‘Qiumihong' included MS,6-BA 1.0 mg·L-1,NAA 0.1mg·L-1 and Ag NO3 0.5 mg·L-1.2.Callus inductioned of leaves from plantlets of different Maotao resources.(1)Callus inductioned from leaves of MaotaoThe optimal medium for the leaves from plantlets of Maotao was MS + Ag NO30.5 mg·L-1 + 2,4-D 1.2 mg·L-1 + 6-BA 1.0 mg·L-1,The frequency of callus and relative callus growth were 88.09% and 3,respectively;while for the the leaves of seedlings form embryo culture in Maotao was MS + Ag NO3 0.5 mg·L-1+ TDZ 1.0 mg·L-1+NAA 1.5 mg·L-1,The frequency of callus formation and relative callus growth were69.77% and 4,respectively.The callus from tissue-cultured plantets and embryo were yellow-green and compact,which mean it have good regeneration ability.(2)Callus inductioned of leaves from plantlets of ‘Qiumihong' and ‘Zhong Youtao14'The optimal medium for ‘Qiumihong' was MS + Ag NO3 0.5 mg·L-1+ 2,4-D 1.2mg·L-1 + TDZ 1.0 mg·L-1,and the frequency of callus formation and relative callus growth were 64.55% and 4,respectively;while for ‘Zhong Youtao 14' was MS +Ag NO3 0.5 mg·L-1+ 2,4-D 1.2 mg·L-1+ 6-BA 1.0 mg·L-1,the frequency of callus formation and relative callus growth were 61.15% and 2,respectively.In short,well performed callus from the leaves in this three types peach,the callus were yellow green,compact and have high leavel regeneration ability.The culture medium for the leaves of Maotao and ‘Zhong Youtao 14' are the same medium.The further studies can be studied on this phenomenon.3.The construction of transformation system for the leaves derived from peach(1)Genetic transformation system of the leaves derived from different MaotaoresourcesIn present study,the optimal transformation system for the leaves of plantlets of Maotao included the concentration of bacteria in OD600 = 0.5,conventional infect time for 15 min,co-culture for 4d,co-culture medium with 20 mg·L-1 AS.The highest GUS transient expression rate was 65.29%.While the optimal transformation system for the leaves of seedlings form embryo culture in Maotao was the concentration of bacteria in OD600 = 0.5,10 min of vacuum assisted infection time,2 d of co-culture duration and co-culture medium with 30 mg·L-1AS.The highest GUS transient expression rate was 76.45%.(2)Genetic transformation system of the leaves from plantlets of ‘Zhong Youtao 14'The optimal treatment was the concentration of bacteria in OD600 = 0.5,10 min of sanction-assisted time,co-culture for 3 d and co-culture medium 40 mg·L-1 AS and with an ultrasonic invasion.The highest GUS transient expression rate was 72.01%.In all,the highest GUS transient expression rates of the leaves in this research were over 65%,but there also have some differentiae.The gene types and different resources of peach can effectively affect the GUS transient expression rate.