Optimization Of Plant Regeneration System And Primary Studies On Agrobacterium-mediated Transformation Of Zoysia Japonica

Zoysiagrass (Zoysia japonica Steud.), commonly used as one of fine quality turfgrass in the world, has many attractive characteristics for a turf, such as high stress tolerant and low water and fertilizer requirements. In addition, it has strong function of soil and water conservation. However, some varieties are susceptible to some diseases and insects such as large brown patch and hunting bill burg. Particularly, the defect of short-green period is an obstacle for application as a turfgrass. Though traditional breeding methods can improve features of this lawngrass, while seed germination of the spcies is rather difficult and pistillate flower is early maturing.In this study, a superior plant regeneration system of zoysiagrass was established, and a differentiated callus line was triumphantly maintained 17 months presently. Histological sections of long-term subculturing calli were observed and plant regeneration pathway from callus induced was analyzed. On the basis of plant regeneration, factors affecting Agrobacterium-mediated transformation of Z. japonica were studied using Agrobacterium tumefaciens strain EHA105 harboring pCAMBLA1301 containing gus and hpt gene. The Agrobacterium-mediated transformation system of Z. japonica was established preliminarily, which will be valuable for the improvement of zoysiagrass by genetic engineering. The major results as following:1. Optimization of zoysiagrass plant regeneration system. Calli were induced from Zoysia japonica seeds, and the optimum concentration range of 2,4-D was 2.0-3.0 mg/L in the induced medium. The addition of low concentration of BA (0.01-0.02mg/L) was favorable for the long-term subculture and differentiation of calli. The best medium for callus induction was basal medium â… + 2, 4-D 2.0-3.0 mg/L +BA 0.01-0.02 mg/LReduced concentration of 2,4-D appropriately and addition of KT properly were suitable for maintaining of long-term subculturing calli and maturing of differentiable calli. The best medium for callus subculture was: basal medium â…¡+2, 4-D 2.0 mg/L+ KT 0.5mg/L.The best medium for plant regeneration was: MS + NAA 0.3mg/L +KT 0.5mg/L + BA 1.0mg/L.2. By observation of histological sections, the dominant pathway of plant regeneration was through organogenesis from nodular compact callus, and at the same time the regeneration through somatic embryogenesis from this callus line take place as well in this study.3. The Agrobacterium-mediated genetic transformation system of Zoysia japonica was established using callus as inoculation material. Taken the callus induced in this study as explants, Agrobacterium twnefaciens strain EHA105 harboring pCAMBIA1301 containing gus and hpt gene was used. Active calli were immersed in Agrobacterium suspension for over 18h, then the co-cultivation was carried out on medium containing lOQumol/L acetosyringone (AS) for 5-8 days. After that the resistant calli were obtained by transferring the explants to selection medium supplemented with 45-60mg/L hygromycin (Hyg) and 400mg/L cefotaxime (Cef) and subcultured every 2 weeks. After 6 weeks, the resistant calli were transferred into differentiation medium containing 50mg/L carbenicillin (Cn) and and 9 resistant plantlets were obtained presently.