Creation Of Turf-type Common Bermudagrass Embryos Tissue Culture System And Development Of Transformation System By Particle Bombardment

Turf-grasses play more and more important roles in our modern lives and there are more and more people and companies run into the field of researching on them. As the limits of traditional method, using modern, advanced biotechnology to improve turf-grasses is becoming an unavoidable trend. Burmudagrass is one of the most important worm-season turf-grasses and widely used in the temperate and tropical regions of the world. As for most plant species, improvement of bermudagrass via biotechnology depends on improved tissue culture responses, especially in plant regeneration, and a successful scheme to introduce useful transgenes. Bermudagrass is a recalcitrant species in tissue culture and is highly recalcitrant with respect to the recovery of transgenic plants. This is the bottleneck of burmudagrass transformation research. In this thesis, a tissue culture system for embryogenic callus (EC) induction and plant regeneration of common bermudagrass using mature caryopses (embryos) as explants was developed. The results showed that embryogenic callus could be induced from caryopses with high frequency, in MS medium with 5 g/L phytagel, and with 2.0－6.0 mg/L 2,4-D, and the best concentration of 2,4-D was 4.0 mg/L with 88.5% induction percentage. The best method for maintaining EC and tissue differentiation was to subculture EC in MS+2,4-D 4.0 mg/L for 1－2 times, follwed by subculturing in 1/2 MS+2,4-D 2.0 mg/L for 1－2 times, then transfer EC to 1/2 MS without hormone for a 10-day-preregeneration in light, followed by transferring to MS+6-BA 3.0 mg/L for regeneration, with regeneration frequency 31.7%. The regenerated plantlets were transplanted to pots or the field and grew healthily. The morphological and micro-structural differences between EC and non-embryogenic callus (NEC) were observed by environmental scanning electron microscopy (ESEM) and transmission electron microscopy (TEM). And ultrastructrual characteristics of the EC cells are described. On the basis of above tissue culture system, a useful bermudagrass transformation system was developed. Two foreign genes—BIN2 and PSAG12-IPT(a chimeric gene) have been transfered directly into EC cells derived from common bermudagrass mature caryopses(embryos) by particle bombardment(Biolistic PDS-1000/He) following such parameters: 1 100 psi helium pressure, 27 torr vacuum, 9 cm distance between the stopping screen and target tissue, two shots per target plate, and 1.0 μm gold particles. Numerous plantlets regenerated. PCR and Northern blot results showed that the foreign genes have integrated into common bermudagrass genome and have been expressed. Some transgenic regenerated plantlets' phenotype also implicated that the foreign genes have likely played roles. This is the first time of recovery of transgenic common bermudagrass using mature caryopses as explant. The common bermudagrass transformation system developed in this thesis is easy for manipulation and the explant—mature caryopse can be obtained quite conveniently and so research can be conducted all around the year, unlike immature inflorescences which could only be collected during the blooming season (May to September).