| Kentucky bluegrass (Poa pratemis L.) is a typical cool-season perennial grass, commonly used for forage and turf in temperate zone. In northern China, it is widely maintained on parks, golf courses, playing fields and public grounds, for the characters of beautiful color, high shoot density, cold tolerance and better shear-bear. Nevertheless it is sensitive to drought and heat stress, and is easily aggrieved from diseases and insects. Due to its facultative apomictic nature of reproduction, genetic improvement by conventional breeding methods has been difficult and genetic manipulation for desirable traits may be used as an alternative method in the improvement of this species. Development of an efficient plant regeneration system for this grass is essential for genetic manipulationIn this study, Kentucky bluegrass seedling were produced in vitro, then multiple shoot clumps that regenerated plantlets vigorously were induced. Meristematic cell clumps and small callus (MCCSC) was induced from the bases of explants on initial culture medium supplemented with 2,4-D and 6-BA, then was transferred to induction medium containing 6-BA for multiple shoot clumps formation. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different hormones, duration Of initial culture, the intensity of illumination and genotypes. The optimal procedure of this protocol was that shoot segments firstly cultured on initial culture media supplemented with 1-3 mg/L 6-BA and 0.2-1.0 mg/L 2,4-D for 20 d, then transferred the swelled segments separated from explants to induction media containing 1-3 mg/L 6-BA 6-BA for the formation of multiple shoot clumps.Histological observation revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots took place in the meristematic cell regions of cultures via organogenesis on induction medium.In this study, the naked multiple shoots apical meristems of three cultivars ofKentucky bluegrass which were Award, Midnight and NuGlade were used as the receptor of Agrobacterium. mediated transformation, and transgenic plantlets were produced. The multiple shoots of Kentucky bluegrass were cultived for 8 to 10 days, then the apical meristems were exposed by cutting out the leaves for the infection by Agrobacterium.vnih 0.5 x105Pa 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 5-7 mg/L lvhuanglong (chlorsulfuron) at a 15-day interval. Survival buds were transferred to medium without herbicide for recovery growth and 15-20 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.Plants were inoculated at various infection time with different concentration of Agrobacterium LBA4404 stain suspension to test the proper transformation conditions. It is generally accepted that proper Agrobacterium concentration and infection duration were OD6000.6 and 3-5 min in Award. Moreover, vacuum infiltration could obviously increase the transformation efficiency. Under 0.5 10~5 Pa pressure the transformation efficiency of Award was higher than that without vacuum infiltration.Using this transformation system, betA (betaine) and als (acetolactate synthase) genes were introduced into these poa pratensis L. cultivars for the production of plants with improved salt-tolerance.The high effective expression vectors comprising gene betA were constituted to advance the expression of target genes in monocotyledon. |
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