Chinese Cabbage Soaked An Transgenic Technology Research

Cabbage (including Chinese cabbage and pak-choi) in Asia, especially in China, Japan andSouth Korea is the most important vegetables. Cabbage plants need a lot of waterrequirement during its growth stages, and there’s a certain lack of insect-resistantgermplasm characteristics for cabbage breeding. By means of introduction of foreign genesinto the plants, it may create a new germplasm cabbage, and radically change the geneticresources of cabbage.Agrobacterium-mediated floral-dip transformation is a relatively new and simpleprocedure which was developed in recent years, not relating to tissue culture. The greateststrengths of this method are that transformed seeds can be directly produced, getting rid ofrounded plant tissue culture or regeneration. Compared with vacuum infiltration genetictransformation method, floral-dip method could decrease the equipment investment. So thismethod is designed for the plants which were difficult to in vitro cultivate and regenerate.In the present study, the floral-dip method mediated by Agrobacterium（C58strain bypBBB-intron-gus）was used for transformation of both Chinese cabbage and pak-choi(including Chinese cabbage08-1083-2，08-1084-2，02-311，02-507，06-940and pak-choisijiu choi sum). This provides theoretical references for transferring other gene (such asinsect-resistant gene) into cabbage. The fourteen Basta-resistant plants were screened fromthe Chinese cabbage08-1083-2in this experiment, and identified as non-transformationplants by a reporter gene, β-glucuronidase (GUS) assay. The four Basta-resistant plantswere obtained from sijiu choi sum, and they were proved to be non-transformation plants.In order to explain why it is so difficult for Agrobacteria to enter the flower buds ofcabbage, studies were performed on the differences in the development pattern andstructure of floral organ of pakchoi. Routine investigations and histological techniqueswere employed for alabastrum in different developmental stages. The results of paraffinsections and stereo microscope revealed that the young ovaries of these samples were openin the apex like a cup，and became closed with the growth of alabastrum; When alabastrumwas lower than0.21mm and0.5mm in diameter for Arabidopsis and sijiu choi sum,respectively, the carpel was open. The percent of flower buds with open carpels ininflorescence are37.5%and25%for Arabidopsis and sijiu choi sum, respectively; On thepart of the whole florescence, the percent of the newborn buds in inflorescence are25%and62.6%for sijiu choi sum and Arabidopsis, respectively. By combining the diameter,development stages, location of flower buds, it was deduced that about15d and3d forsijiu choi sum and Arabidopsis were spent respectively on he flower buds with unopenedcarpels to bloom. Recent studies have shown that Agrobacteria may get into the material through their opening carpels，and most of the transformation occurred in the singlestemmed plant with a central cluster of immature floral buds by floral-dip. We come to theconclusion that the procedure can not be used to introduce Agrobacteria into sijiu choi sum,probably owing to its premature closed carpels. And the low percentage of the newbornbuds in inflorescence in sijiu choi sum may cause its low transformation activity viafloral-dip method.To explore a low efficiency of the floral-dip transformation technique and explain whyit is difficult of success in Chinese cabbage, a parallel study of floral-dip transformation ofpakchoi and Arabidopsis thaliana was taken. Harvested seeds from both dipped flowerswere sowed, seedlings were sprayed by Basta solution containing0.225g/L PPT. Herbicideresistant plants were screened from the Arabidopsis plants, and the plant transformationfrequency was about20%, but none from the cabbage pakchoi plants. The expression of theforeign transgene in Arabidopsis was proved by GUS assay. The existence and viabilitydifferences of the Agrobacteria in flowers of the two species were investigated by bacteriaplating method. Results showed that during6days after floral-dip, there were60-900Agrobacteria clones (s/g) formed in the cultures from the extract of Arabidopsis flowerbuds, while no one formed from the pakchoi flower buds. The results suggested that it isdifficult for Agrobacteria entering the flower buds of pakchoi, which may explain to acertain extent the reason of difficulties in floral-dip transformation in pakchoi comparedwith Arabidopsis.