| Kentucky blue grass is an important forage and turf grass with facultative apomictic mode of reproduction. As a kind of important conventional forage with good quality and high yield, bluegrass has a long cultivating history in China and has been planted universally. In addition, bluegrass is a very good turf grass. It plays an important role in afforesting city, turf establishment, clearing air and retaining soil. However, bluegrass has a lot of shortcomings, such as scarce leaf, non-resistance of disease and weak resistance of extreme temperature. As the main cool-season cultivars in north China, it is necessary to breed excellent bluegrass species. Gene transformation is a speedy and efficient method. New cultivars could be obtained through gene transformation. In this paper, a high frequency bluegrass' regeneration system was established, providing a good foundation for bluegrass' gene transformation.Mature seeds of bluegrass were applied as explant material in all experiments. The effects of different medium, different concentrations combination of phytohormones on callus inducing, shoots differentiation and roots inducing were studied. The optimum conditions were found, providing a basis for bluegrass' gene transformation. The main results of this research were listed as following:I. Disinfection of explant: the clean seeds were dehusked by agitation in 50% H2SO4 for 30 minutes, before puting in 70% ethanol for 30~40s. Then the seeds were surface-disinfected for 3-4 minutes in HgCl2(0.1%). Seeds were placed on callus induction medium. This kind of treatment can achieve good disinfection effect.II. Genetype selection: Poa sphendyLodes L., Kentucky bluegrass, Fylking bluegrass and Baron bluegrass were tested in this reseaerch, among which,Kentucky bluegrass was the optimum genotype in medium inducing. As a result, Kentucky bluegrass was selected for gene transformation.III. Callus induction: The effects of different medium and phytohormones on callus induction were studied, which show that MS+2,4-D(3.0mg-L-1)+6-BA(0.1mg-L-1)+sucrose (30g-L-1) +agar(7g-L-1) can give the highest rate of callus induction(78.5%).IV. The best medium for shoot differentiation is SH+2,4-D(0.1mg-L-1)+Sucrose (30g-L-1)+agar (7g-L-1). The mean number of shoots differentiated from one callus is up to 8.3.V. Roots induction: The suggested medium is SH+2,4-D(0.1mg-L-1)+sucrose (30g-L-1)+agar (7g-L-1), which can give 74.9 percent inducing rate.VI. Tube-plants transfer: The strong tube-plants with more roots were selected and acclimated in green house for 15~20days, before transferred into the field. The surviving rate is up to 90%.The above results manifest different bluegrass' gene type has high deviation in establishing regeneration system. Kentucky bluegrass was selected as gene type in this research. MS medium contained 3mg-L-12,4-D and 0.1mg-L-16-BA was applied for inducing callus culture. SH medium contained 0.lmg-L-1 2 ,4-D was used for shoot induction and roots were induced on 1/2MS medium contained 1.0 mg-L-1NAA. Under these conditions, high frequent regeneration system of bluegrass was obtained.Agrobacterium LBA4404 was conserved. And the concentration of Km and different bacteriophages were studied. Results were obtained from experiments. When the concentration of Km is 50mg-L-1, the growth of bluegrass' callus culture will be restrained rightly, or the shoots are albino. When the Carbenicillin Na2 is up to 300mg-L-1, the growth of Agrobacterium LBA4404 will be restrained thoroughly(OD600:=0.027)... |
