Establishment Of Genetic Transformation System And Transformed ICE1 Gene Into 'Tunisia Soft-seed' Pomegranate

Pomegranate is a kind of fruit with long cultivation history widely cultivated in China.Pomegranate is favored by consumers due to its nutritional value and healthy function.The industry of pomegranate is developing rapidly in our country and abroad.‘Tunisia soft seeds' pomegranate has a good resistance to drought stress and disease and with wide suitability.However,‘Tunisia soft seeds' pomegranate is susceptible to cold stress,especially when the temperature is below-10?.The scope of cultivation and popularization was limited by the weakness of seedling.The development of modern molecular biotechnology,especially theplant tissue culture and genetic engineering provided important technical support for directional improvement and new variety breeding of pomegranate.The emerging of transgenic technology makes it possible for rapidly creating pomegranate germplasm.In our study,the GUS gene was transformed into pomegranate by Agrobacterium-mediated method of usingepicotyls,hypocotyls,cotyledons and leaves as explants.The efficient genetic transformation and regeneration system was established through optimizing the pre-culture duration,infection time,co-culture duration,and the concentration of bacterium solution and AS.Then the ICE1 gene was transferred into pomegranate using the optimized system and the resistant shoots was detected by PCR.Moreover,the micro-grafting in vitro was studied to overcome the problem that the transformation of seedling is difficult to root.The main results are as follows: 1.Establishment of genetic transformation system using four explants of pomegranateThe effects of different pre-culture duration,infection time and co-culture duration,different concentration of bacterium solution and AS,and different infection mode ontransformation efficiency were compared through GUS transient expression rate.The results showed that when using cotyledons as explants,the optimal condition was 3 d of pre-culture duration,0.5 of bacteria solution concentration OD600,25 min of infection time,3 d of co-culture duration and 30.0 mg/L of AS.In this case,the temporary expression rate of GUS gene reached 60.0%.When using leaves as explants,the optimal treatment was 3 d of pre-culture duration,0.7 of bacteria solution concentration OD600,25 min of infection time,3 d of co-culture duration and 20.0 mg/L AS.Under this condition,the temporary expression rate of GUS gene reached 73.3%.The optimal treatment of epicotyls was 2 d of pre-culture duration,0.5 of bacteria solution concentration OD600,20 min of infection time,3d of co-culture duration and 30.0 mg/L AS,the temporary expression rate of GUS gene reached 70.0%.While the optimal treatment of hypocotyls was 2 d of pre-culture duration,0.5 of bacteria solution concentration OD600,25 min of infection time,3 d of co-culture duration and 30.0 mg/L AS,the temporary expression rate of GUS gene reached 73.3%.2.Agrobacterium-mediated transformation of ICE1 into pomegranateThe ICE1 was transformed into pomegranatewith Agrobacterium-mediated transformation using epicotyls,hypocotyls,cotyledons and leaves as explants.The resistant callus and shoots were obtained and screened by PCR.However,no transgenic shoots were detected at last.The resistant callus are expanding propagation and the follow-up experiments are in pressing.3.Micro-grafting in vitro of pomegranateThe cleft grafting using scion with four leaves and the roots with hypocotyl as stock is the best combination for pomegranate and the grafting survival rate reached 70.0%.