In Vitro Selection Of Sethoxydim-tolerantvariants And Preliminary Studies On Genetic Transformation For Glyphosate In Zoysia Matrella

With the embryogenic calli induced from the stolons of Manilagrass, selection of sethoxydim-tolerant variants in vitro and cultivation of transgenic plants with glyphosate resistance by means of agrobacterium-mediated transformation were studied preliminarily in this paper.Effects of different concentration of sethoxydim solution on callus growth and regeneration were studied. More than seven thousand calli were suffered with10μM sethoxydim stress for at least12weeks. A small number of calli survived after long-term selection and were regenerated into plantlets. Spraying those plantlets regenerated from the survival callus with0.25%commercial sethoxydim solution, we found that the survival rate of callus in the second round selection on the subculture medium supplemented with sethoxydim solution was lower than in the first round selection. Compared to the effect of sethoxydim on the growth of callus, the regeneration of callus was more sensitive to the sethoxydim. The regeneration rate of callus was decreased to3.75%with the concentration of sethoxydim increased to3.0μM. In the first field test, three of the five plantlets subjecting to test came from sehoxydim selection and two were ordinary as controls. After foliar spraying with0.25%commercial sethoxydim solution, SV1and SV3showed less injury than CK2, while SV2showed more injury than CK2. In contrast to all the plantlets sprayed with commercial sethoxydim solution, SV3showed a higher resistance to sethoxydim than SV1.To obtain the glyphosate tolerant Manilagrass, Three groups of calli were infected by agrobacterium carrying target gene and selected on the subculture medium containing with different concentrations of glyphosate solution. Calli of group A were cultivated on mediums containing2mM glyphosate for4weeks, and then were transferred to mediums containing5mM glyphosate for4weeks. Calli of group B were cultivated on mediums containing different concentration (2mM,3mM and5mM) of glyphosate for12weeks. Group C were cultivated on mediums containing2mM glyphosate for8weeks. Then the survival calli were transferred onto the regeneration medium lacking of glyphosate solution. During the experiment we found that comparing with CK1, a few of the infected calli propagated rapidly. And the calli without infection rooted earlier than the infected calli. After16weeks, fifty-four calli were regenerated into plantlets. Besides the regenerated plantlets, a small number of the calli still have the regeneration ability. All of the calli without infection lost the regeneration ability.