Studies On High Frequency Regeneration And Genetic Transformation Of Ornamental Pineapple

Ornamental pineapple including its flower and leaf, with high value, is a Bromeliaceae flower plant. But in china, there are few wonderful varieties, most of which are imported from abroad, in addition to its weak germination of seeding, low traditional propagation coefficient, long breeding cycle, so, building the tissue culture . and regeneration system is very urgent to protect and propagate seedlings presently, it is necessary to culture a good transgenic variety by making use of genetic engineering technology. In this paper, we studied the effects of different phytohormone combination on embryogenic callus (EC) induction and plant regeneration; the Morphological and ultrastructure differences between EC and Non-embryogenic callus (NEC) during proliferation and differentiation phases using the ornamental pineapple ('Vriesea Tiffany') stem, as explants. Also, the factors on genetic transformation of ornamental pineapple by Agrobacterium tumefaciens transfection were studied as well as the OsCYP2 genetic transformation plant. The main results are as follows:1,The EC induction and high frequent plant regeneration system of ornamental pineapple were developed. Callus induction was induced on Murashige-Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (6-BA), a-naphthaleneacetic acid (NAA) and 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Plantlet differentiation was induced on MS medium supplemented with NAA and 6-BA. The results showed that the best treatment in terms of response quality and mean number of EC for explants was 0.5 mg/L NAA with 2.0 mg/L 6-BA. The best medium for EC induction was MS+2.0 mg/L6-BA+0.5 mg/L NAA+2.0 mg/L 2,4-D with the induction frequency 80%. The best shooting regeneration of EC was obtained on MS supplemented with 0.1 mg/L NAA and 0.5 mg/L 6-BA with the maximum reaching 100%. Morphological and ultrastructure differences between EC and Non-embryogenic callus (NEC) were observed by electron microscopy during proliferation and differentiation phases. Structural cell organelles are abundant in the EC cells, neither NEC cells.2,A genetic transformation system was developed by Agrobacterium tumefaciens transfection, which lay the foundation of future genetic engineering breeding of ornamental pineapple. GFP was transformed into the EC cell of Ornamental pineapple by the vector of Pbin438-GFP and the gene has expressed in the EC cell. The GFP protein was detected in the EC cell of Ornamental pineapple by confocal laser scanning microscope.3,OsCYP2 trans genic plants were obtained. The expression of transgene was confirmed by Hygromycin B resistance and PCR analysis. One of immunophilin gene from rice, OsCYP2, was proved to enhance the ability to resistant the stress conditions.-It is possible to obtain the stress resistance ornamental pineapple. Therefore, this study has some practical significance.