Agrobacterium-mediated Ryegrass Buds Bud Tip Of Genetic Transformation And Transgenic Plant Regeneration

Ryegrass (Lolium multiflorum Lam. and Lolium perenne L.) is one of the important grass species in the world. Genetic engineering may contribute to the development of improved ryegrass cultivars for forage and turf purpose as conventional selection procedure is slow. Transgenic plants of ryegrass have been reported using microprojectile bombardment, silicon-carbide fibre-mediated and Agrobacterium-mediated transformation with embryogenic calli, suspension cells and protoplasts as recipient However, these ryegrass transformation systems were basically influenced by the regeneration potential and genotype of donor plant, long in vitro culture period, resulted in only a few transgenic ryegrass plants have been obtained successfully. Hence it is prerequisite to establish an efficient transformation system for ryegrass genetic breeding. Here we reported an efficient procedure for ryegrass genetic transformation involving bud tips of multiple bud clumps derived from etiolated shoot tips of sterile seedlings from two elite ryegrass cultivars Gulf (annual ryegrass) and Tetrilite (perennial ryegrass) and many fertile transgenic plantlets were obtained.The first step to establish the transformation system is to establish an in vitro system with higher regeneration frequency. Bud clumps were successfully induced from shoot tips of ryegrass in order to develop an efficient propagation system. The percentage of bud clumps was significantly improved on induction medium containing 2.0mg/L 6-BA and 0.5mg/L 2,4-D, and multiple bud clumps were obtained on proliferation medium containing 2.0mg/L 6-BA.The single bud (about 1mm in height) and bud clumps derived from the multiple bud clumps subcultured for 8-10 days were infected with Agrobacterium LBA4404 harboring plasmid pCAMBIA1300-betA-als with 0.5×10~5Pa pressure for 5 minutes and co-cultured for 3 days on induction medium after blotted on sterile filter paper. Then, the inoculated explants were transferred to medium supplemented with 100mg/L cefotaxime to inhibit the growth of Agrobacterium for 8 days and further 45 days subcultures on medium containing 3-5mg/L lvhuanglong (chlorsulfuron) at a 15-day interval. Survival buds were transferred to medium without herbicide for recovery growth and 20 days later these recovery buds were transferred onto rooting medium to induce root formation. Rooted plantlets were transferred to plastic pots containing medium composed of autoclaved vermiculite-soil (1/1, v/v) and transgenic plants were obtained after PCR assay to the transplants 2 months later.Factors related with the genetic transformation were investigated respectively. Higher percentage of resistant buds (chlorsulfuron-resistant buds/infected buds) was obtained under the following conditions: explants from multiple bud clumps subcultured for 8-10 days were inoculated in Agrobacterium suspension of OD6oo=0.5 supplemented with O.lmmol/L acetosyrigone for 5 min under O-SxK^Pa pressure, and the highest percentage of resistant-buds reached 32.7%. This transformation procedure used shoot tips of etiolated multiple bud clumps as target, avoiding genotype-dependence and produced transgenic plants within a short period (3-4 months) with higher transformation efficiency.PCR assay results of the "I*! plants after open pollination showed that out of 21 transgenic lines, 3 were PCR positive in Ti generation, indicating that the transformation procedure using bud tips of ryegrass as explants mediated with Agrobacterium is feasible in ryegrass genetic breeding. The seeds (Tj) and non-transgenic controls were sown in pots full of sand and irrigated with 1.0%NaCl solution for 10 days and further 20 days with 2.0%NaCl solution. The salt-tolerance of T] was improved at least 0.5%NaCl than the controls based on the results of the survival rate of the seedlings.