Construction And Application Of Genetic Transformation System For Axillary Bud Of Dioscorea Alata L.

Greater yam（Dioscorea alata L.）is an important tropical tuber crop that has high yield,strong adaptability,and rich nutritional value.However,the tuber shape of greater yam is highly variable,as some genotypes of greater yam have underground tubers longer than one meter,which is extremely difficult to dig and use mechanized mining.In this case,we need to breed greater yam with straight and thick tubers for higher planting efficiency.The previous study has found that protein Dioscorin can affect the tuber shape of greater yam,while Da-dio5 gene is the key gene copy that accounts for the synthesis of protein Dioscorin and affects the development of tubers.In order to further study the function of Da-dio5 gene in tuber development,this study aimed to construct a genetic transformation system for greater yam by using axillary buds as explants,to construct vectors that over-express Da-dio5 and knockout Da-dio5,and transform into greater yam to validate its gene function.The main results are as follows:1.Construction of a stable genetic transformation system in greater yam with axillary buds as explants,optimization of which in three aspects:selection of antibiotic screening agents,rooting conditions,and Agrobacterium infection conditions.Growth performance of axillary buds on MS medium containing kanamycin or hygromycin indicated that the axillary buds had high tolerance to kanamycin,which could grow normally when the kanamycin concentration was high（100-700 mg/L）;whareas they were highly sensitive to hygromycin,as they had a total lethal ratio when hygromycin concentration was 40 mg/L.In this case,hygromycin was suitable as a screening agent for genetic transformation of Dioscorea alata L.axillary buds.The best medium formula of inducing axillary bud to root and germinate was obtained by orthogonal experiment.The formula was MS medium+0.1%PVP+NAA（0.2 mg/L）+6-BA（1.0 mg/L）,which resulted in a rooting and germination rate of axillary buds as 94.44%.Taking the survival rate of axillary buds after infection by Agrobacterium EHA105 as the index,we determined the optimal infection conditions as follows:the OD₆₀₀of Agrobacterium tumefaciens was 0.6,and the infection time was 20min.2.We successfully constructed the over-expressed vector of Da-dio5 as p CAM BIA1300-p2×35S-Da-dio5-flag and the knock-out vector as Da-dio5-4-CRISPR-Cas9.Then we transferred the vectors into the EHA105 strain and transformed into greater yam buds.After getting the plants from the axillary buds that had resistance to hygromycin,we tested these transgenic candidates by PCR,q RT-PCR,and Western blot.PCR assays using Hyg gene-specific primer pairs showed that 11%the resistant plant had positive bands for the Da-dio5 over-expressed vector and 5%plant had positive results for the knock-out vector.Testing Da-dio5 gene expression levels via RT-q PCR demonstrated the relative increases of expression levels in the resistant seedling transformed with the Da-dio5over-expressed vector.Western blot with anti-flag antibody that fused with Da-dio5 protein in the over-expressed vector illustrated one resistant seedling having positive band.Sequencing the target sequences of Da-dio5 in the resistant plant transformed with the CRISPR vector showed there are sequence variations flanking the target sequence of Da-dio5.