Agrobacterium-mediated Transformation Of Chinese Cabbage

The Brassica genus contains many important vegetable and oil crops,among which are cabbage?B.rapa?,kale,broccoli,cauliflower?B.olearcea?,and rapeseed?Brassica napus?are extremely important in human life and agricultural production.The cabbage crops?B.rapa?including Chinese cabbage,turnip,Choy sum,Bok choy are originated in China,and are currently widely grown in China,Japan,South Korea and other Asian countries.Bok choy?B.rapa L.ssp.chinensis?,one of the representatives of the cabbage crop,is an important vegetable mainly planted in northern China.Conventional breeding is the main method to obtain new cultivars with good quality and yield.However,conventional breeding technology has many limitations such as high cost,difficulty in genetic crossing because of hybridization incompatibility and other factors.Genetic engineering can avoid the above-mentioned drawbacks,speed up breeding,and obtain good cultivars easily.Genetic transformation is based on the establishment of a regeneration system.There are many factors that affect regeneration efficiency:the genotype of the plant,the type of explant,the type and concentration ratio of hormones and other chemicals used in the induction process.The success rate of genetic transformation is also influenced by various factors such as the best seedling age,Agrobacterium species,infection patterns,types and concentration of adapted antibiotics.To study the function of miRNA395 in cabbage,a miRNA395 silencing construct?STTM395?was used to transform Bok choy,a variety of the Chinese cabbage.A high efficient stable genetic transformation system was established,and transgenic plants were successfully produced.This study pave the way for future studies of the miRNA395function in Bok choy through microRNA silencing by STTM technology and provide an efficient genetic transformation method for genetic improvement of Bok choy.Major results:1.Using the cotyledon petiole of Bok choy cultivar”Chunyou 3”as explant,different kinds and concentrations of hormones were tested.The result showed that adventitious shoots were best induced from 4-day-old cotyledon petiole in medium MS+4mg/L 6-BA+0.6 mg/L NAA+4 mg/L AgNO₃+0.25mg/L ABA,and roots were best induced on MS medium containing 0.6mg/L IBA from healthy adventitious shoots.The induction rates for adventitious shoots and roots were 57.6%and 50%,respectively.2.An efficient Bok choy genetic transformation system was established by optimizing various factors including seedling ages,hygromycin concentrations,different antibiotics and their concentrations,and the time of pre-culture.Transgenic plants were regenerated and screened by PCR.The transformation rate was 1.75%.