| VvSUC11 and VvSUC12 are sucrose transporters from grape berries. Studies showed that their function was the loading of sucrose from the apoplast into the parenchyma cells. They play a key role in the accumulation of sugar during this process of ripening of grapes.In agricultural production, in order to obtain high yield, people not only try to improve the biological yield of photosynthesis, but also to adopt certain measures to improve economic production. The use of sucrose transporters orientation to improve the economics of production is an effective mean to regulate the relationship between the sources to the library.To study whether sucrose transporters can improve the yields of sugar of sugar beet, we have recombined genes VvSUC11,VvSUC12 from the grapes, and root-specific promoters of sweet potato storage protein gene from sweet potato, named as SP1 and SP2. We have constructed a bivalent plant expression vector PCAMBIA2301-SP1-VvSUC11-SP2-VvSUC12 using PBI121 and pCAMBIA2301 as original vectors.Sugar beet is a 2-year-old, out crossing herbaceous plant. It is a poor seed resource because of shorter time of domestication cultivation and narrow genetic background. In particular, its high incompatibility in self-fertilization makes it easily to lose good single plant genotype causing lots of difficulties in breeding and preservation of the fine varieties. However, the development of modern biotechnology, particularly, the development of genetic engineering provides a new way in the breeding of sugar beet.. We can precede molecule breeding by the way of combining plant tissue culture and genetic engineering. At present, the large numbers of sugar beet tissue culture studies have shown that regeneration plants from the direct organogenesis (not through callus phase) and plant regeneration was a more suitable receptor plant for receiving foreign gene. Studies have shown that the rates regeneration of petioles of sugar bee as explants is higher, and after transformation, their rate of transformation is also higher.We have stetted up a high efficient direct regeneration system of its petioles. Through planting free vaccine seedlings, their pre-cultivating, induction of differentiation cultivating of petioles, adventitious buds obtained and rooting, regenerated plants obtained, a rapid propagation system of sugar beet was established. The results showed that sugar beet seeds were sterilized and germinated in the1/2MS for 10 d, then their seedlings were transferred to pre-culture medium (MS +6-BA 0.5mg/L+NAA0.05mg/L) for a 50～60 d pre-culture, and then their petioles were cut as explants and were transferred to the shoot-inducing medium(MS+6-BA0.5mg/L+NAA0.05mg /L) for induction of adventitious buds, induction rate of adventitious buds was more than 50%. Adventitious buds were placed onto the rooting medium (MS+IBA2.0mg/L) for rooting. Induction rate of rooting was more than 90%.We transformed the vector PCAMBIA2301-SP1-FvSUC11-SP2-VvSUC12 into sugar beet of KWS-9103 breeding line with the methodologies of Agrobacterium-mediated transformation. We have established the optimal genetic transformation protocol of sugar beet as followed:the explants pre-cultured for 4 days were immersed in Agrobacterium suspension of OD600= 0.5, supplemented with 0.005% Silwet L-77, and followed by 4-day culture on medium containing cefotaxime, then the buds were selected on medium containing kanamycin and cefotaxime. The percentage of kanamycin -resistant buds was as high as 42%. Results of PCR and RT-PCR proved that the aim genes had integrated into sugar beet genome and expressed. It will lay a solid foundation for studying whether sucrose transporters can improve the yields of sugar of sugar beet. |
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