Studies On Embryogenic Suspension Culture And Genetic Transformation Of AcSERK2 Of Ananas Bracteatus

Pineapple (Ananas bracteatus) is an important new type of ornamental plants, but because of self incompatible, the bud was used for breeding. However there were some big problems, such as the low reproduction and the consistency is poor. Therefore, it is very important to study the somatic embryogenesis and the system of transgenosis. The callus (induced by the suckers) were used as raw material to establish the embryogeic suspension system. At the same time, the study has been carried out on the transformation of Ananas bracteatus callus with AcSERK2 mediated by Agrobacterium tumefaciens,and obtained th transgenic plants, to lay the foundation for further study of the function of AcSERK2.The main results were as follow:The callus which is pale yellow, granular, and fragile, were used for establishing the embryogeic suspension system.During the suspension culture, the statistics of the raw weight showed that, the growth curve of the callus conform to’s’, and contains four times: 0-4d for growth dealy,4-12d for growth slowly,12-20d for growth exponentially, after 20d for growth retardation.Therefore, it is appropriate for 20 days as the cycle for successive transfer culture.Embryogenicsuspensions system were sieved through nylon sieves with successive 840-,125-,75-μm pore sizes, and then continue to culture tiny particles, after 7-8 generations, good suspension system can be obtained. Again transferred to liquid somatic embryo induction medium MS+5.0 mg/L 2,4-D+0.5 mg/L BA, and we can obtain good dispersion and uniformity of embryonic suspension system.The study has been carried out on the influence of different ways such as plate culture, shallow layer culture and suspension culture in the single cells culture.Comparing the rate of the growth round cells and cells division of the three different ways.The results showed that suspension culture was the best way for single cells culture.In the experiments of theembryoniccallus were infected by the Agrobacterium tumefaciens (LBA4404 strain) carrying expression recombinant plasmid (pFGC5941--AcSERK2--bar), the callus which were cocultivated for 3 days were selected on selection medium. After 3 generation of selcecting for resistant callus, then remove the PPT-resistant callus to differentiating mediumto obtain PPT-resistant plant. By three times selections, 110 PPT-resistant plants transferred AcSERK2 were obtained. Detected all of them, there are 5 positive plants, the transform rate was 0.3%.In the experiments of the embryonic callus were infected by the Agrobacterium tumefaciens (EHA105strain) carrying expression recombinant plasmid (pYLRNAi--AcSERK2-hpt), the callus which were cocultivated for 3 days were selected on selection medium. After 2 generation of selcecting for resistant callus, then remove the HygB-resistant callus to differentiating mediumto obtain HygB-resistant plant.By two times selections,61 HygB-resistant plants out of 1509 callus were obtained, the transform rate was 0.3%.The relative quantity of AcSERK2 in the transgenic plants were analyzed by qRT-PCR. β-actin was used as reference gene, the non-transgenic plants was used as contrast for qRT-PCR analysis. The results show that the relative quantity of AcSERK2 in the plants with the overexpressed gene significantly higher than the non-transgenic plants, the relative quantity of AcSERK2 in the non-transgenic plants significantly higher than the interference in the plant. It shows that AcSERK2 has successfully integrated into the genome.