Construction Of Pecan Genetic Transformation System

The pecan,Carya illinoinensis,is a member of Carya genus,Juglandaceae family,also known as pecan nut and pecan hickory.Its large trunk makes it difficult for manual picking and daily maintaining management,thus pecan dwarfing has become a significant benefit to pecan breeding and nut popularization.As the development of modern molecular biology,the cultivation of new dwarfed and high resistance cultivar which based on plant transgenic technology has a profound meaning.There have been few reports concerning genetic modification of Carya so far,most of which simply introduce target gene into Arabidopsis Thaliana to verify its function.But Arabidopsis Thaliana and Carya have little common in genetic relationship,this cannot reflect the really function of the target gene in pecan.Somatic embryo based transgene receptor has many advantages,such as easy operation,short period,high transformation efficiency,etc.Therefore,this paper adopts somatic embryo as transgene receptor to construct pecan transformation system.In this study,firstly the maturation and germination of pecan somatic embryo conditions were optimized,then dwarfing-functioned gibberellin gene GA20 ox was introduced into pecan somatic embryo using agrobacterium-mediated transformation to explore the conditions of constructing transformation system,which lays the foundation for further study concerning introducing target gene.The primary research results are as follows:1.The optimized dehydration time of pecan somatic embryo is 5 days,and after the dehydration the germination rate reach peak which is 66.3%.2.The concentration of SOD,POD,CAT and MDA were adjusted to accelerate the maturation of pecan somatic embryo.The concentration of POD,CAT and MDA rises to some extent after 0-5 days' dehydration and they all decline at 6 days.The activity of SOD rapidly decreases after 0-6 days' dehydration.3.ELISA was adopted to determine the variation of endogenous hormone concentration during somatic embryo maturation.The concentration of ABA and IAA outweigh those of GA3 and ZR in this process,overall the latter ones play little role in pecan maturation and germination.The concentration of ABA in cotyledon embryo reach a peak of 110.75 ng/g·fw at day 5 while IAA concentration reach maximum value of 101.78 ng/g·fw at day 3.4.The concentration of Cerb and Hyg were studied to determine the optimized selective pressure.As somatic embryo has greater resistance to Cerb comparing to Hyg,the concentration gradient of Cerb was set to 0mg·L-1,200mg·L-1,250mg·L-1,300mg·L-1 and 350mg·L-1 while Hyb's was 0mg·L-1,60mg·L-1,80mg·L-1,100mg·L-1 and 120mg·L-1.The results indicate that it is obvious the growth of pecan somatic embryo was hindered and the proliferation was ceased in minimal media with 300mg·L-1Cerb or 100mg·L-1 Hyg after 2 weeks' cultivation,which is the reason they were chosen as the optimum concentration to select pecan somatic embryo with resistance.5.The concentration of bacterial liquid was selected in agrobacterium EHA 105 mediated pecan transformation system.The concentration of bacterium suspension A concentration gradient of agrobacterium liquid(OD600=0.05,0.2,0.5,0.8)was added into bacterium suspension respectively,and the transformation efficiency was compared under the condition of 15 min's infection and 3 days' co-cultivation.The non-antibiotic somatic embryo rate is 30.49% and the transient expression rate of GUS is 58.27% when the microbial concentration is OD600=0.5.6.A concentration gradient of infection time(0min,10 min,15min)was set under the condition of OD600=0.5 and 3 days' co-cultivation to estimate optimum infection time.The results show that though15 min's infection has higher non-antibiotic somatic embryo rate than 10 min's,the transient expression rate of GUS peak at 61.52%.7.GA20 ox corresponded RNA interference vector was transformed into pecan somatic embryo,molecular detection was conducted after the screen and differentiation of resistant somatic embryo.PCR of transformed somatic embryo was conducted with GA20 ox primer,a band of approximately 232 bp can be amplified using the c DNA of transformed somatic embryo,this result preliminary demonstrate foreign gene and pecan genome have been integrated.However,negative results were found in certain transformed somatic embryo,which indicate the very existence of false transformed somatic embryo.Hence,further optimization and adjustment of single factor should be made,meanwhile the interaction of all aspects should be taken into consideration to find the best combination,finally to increase the transformation rate.8.q RT-PCR was conducted to detect transformed somatic embryo,the result shows a downtrend expression of GA20 ox in positive somatic embryo.