The Advanced Transformation Systems for Sylosanthes guianensis cv. Ryan No.5

摘要

Stylosanthes guianensis cv. Ryan No. 5 is one of tropical pastures that belongs to perennial herb and has many high qualities, such as 7.5～15T/ha high yield, and 12%～16% protein content of dry weight. In the recent years, the researches have been focused on the methods of tissue culture in vitro for its rapid propagation, and the improvement of it s characterizes through molecular genetic breeding. Several species of Stylosanthes spp. have been reported to success in tissue culture in vitro. However the molecular genetic breeding through gene transformation to Stylosanthes spp. has few reported. In this research, we reported an advanced transformation system for Stylosanthes guianensis cv. Ryan No.5 by using npt000, hpt, bar as selection genes. The results showed that (1): the rate of callus formation was 100% among the hypocotyls, the cotyledons, the young leaves, and the young shoots; the frequency of their shoot formation from the hypocotyl and the cotyledon culli was as high as almost 100%, and the time for shoot formation from the calli was 14-18 days, however, the frequency of their shoot formation from the young leave and the young shoot calli was only 15%, the time for shoot formation from the calli was 16-20 days. (2): The calli from the hypocotyls and the cotyledons become transparency and could not form shoots on all the mediums containing Kin and 2.4-D (Kin 2.0 mg/L, 2.4-D 2.0 mg/L; Kin 2.0 mg/L, 2.4-D 1.0 mg/L; Kin 2.0 mg/L, 2.4-D 0 mg/L); but on the MS mediums containing NAA and 6-BA (NAA 1.0 mg/L, 6-BA 2.0 mg/L; NAA 1.0 mg/L, 6-BA 4.0 mg/L; NAA 2.0 mg/L, 6-BA 2.0 mg/L; and NAA 2.0 mg/L, 6-BA 4.0 mg/L), the shoots were formed from the calli only on the MS medium with NAA 1.0 mg/L, 6-BA 4.0 mg/L, meanwhile 5%～8% shoot formation from the cotyledon calli, and almost 100% shoot formation from the hypocotyl calli. (3): Generally, the plant hormone NAA is used for inducing root formation from the shoots on MS medium, however the NAA is not needed for the root formation of Stylosanthes guianensis Ryan No.S. The shoots were cultivated on the MS mediums containing NAA (0 mg/L; 1.0 mg/L; 2.0 mg/L; 3.0 mg/ L; and 4.0 mg/L), the rate of the root formation was 85% on the MS medium with 0mg/L NAA, 45% on the MS medium with 1.0 mg/L NAA, and 30% on the MS medium with 2.0 mg/L and 3.0 mg/L NAA. The Shoots only formed hah- roots on the MS medium with 4.0 mg/L NAA. No lateral root formation on the MS medium with 1.0 mg/L, 2.0 mg/L, and 3.0 mg/. On the MS mediums with 2.0 mg/L and 3.0 mg/L NAA, seedlings grew slowly, or stopped growth. Thus, the hypocotyl of Stylosanthes guianensis Ryan No. 5 was the best expand for gene transformation. The best medium of the callus formation and differentiation was MS+NAA 1.0 mg/L+6-BA 4.0 mg/L. The optimal medium for root formation from callus was MS +NAA 0 mg/L. (4): In Stylosanthes guianensis Ryan No.5, the suitable concentrations for transgene selection were 0.15 mg/L for Glufosinate; 20 mg/L for Kamamycin; 15 mg/L for Hygromycin. In the above transformation systems, when the nptⅡ, hpt and bar genes were transformed to hypocotyls through Agrobacterium GV3101, 30% calli were resistant to Kanamycin; 5% calli were resistant to Hygromycin; 50% calli were resistant t0 Glufosinate.