Genetic Transformation Of Drought-resistance And Salt-resistance Gene In Kentucky Bluegrass(Poa Pratensis L.)

Callus induction of Kentucky bluegrass(Poa pratensis L.) was studied by using the orthogonal and single-factor test.The high regeneration systems of three cultivars were establishmented. Embryonic callus of Kentucky bluegrass were used as material for genetic transformation with the DREB1A gene, BADH-CMO double-gene and CMO gene by particle bombardment.The transgenic plants were obtained. At the same time, partial transgenic plants were treated with drought and salt stress to check the actual results on drought and salt resistance. Through the hard efforts, conclusions are showed as following.I. Different explants, inoculation method and culture condition were studied in building plant regeneration system. The suitable factors were determined as follows. Mature seeds are selected as explants and rinsed by the whisk, then they are cultured in the mdium with 30mg/L sucrose in the dark. Selecting compact, friable and growing rapidly callus for subculture, can always provid good plant material for gene transfer all long.II. Plant regeneration system of Kentucky bluegrass cutivars'Baron','Mardona'and'Midnight'were established by single-factor and orthogonal test. P2, MS salt supplmented with 1mg/L 2,4-D, 0.1mg/L 6-BA, 3mg/L CuSO4,and 1g/L Casein acids Hydrolysate(CH) is the best callus induction medium for'Baron'. P5, MS salt supplmented with 2mg/L 2,4-D, 0mg/L 6-BA, 3mg/L CuSO4,and 1g/L CH is the best callus induction medium for'Md'.KM, MS salt supplmented with 2mg/L 2,4-D, 0.2mg/L 6-BA, 3mg/L CuSO4,and 1g/L CH is the best callus induction medium for'M'.The callus induction rates are high up to 38.19%,28.66% and 31.67% respectively.AK2, MS salt supplemented with 0.2mg/L KT, 1mg/L 6-BA is the suitable regeneration medium for cultivar'B'. AL2, MS salt supplemented with 1mg/L6-BA, 0.5g/L Lactalbumin Hyfrolysate (LH) is the suitable medium for cutivar'Md'.KBN, MS salt supplemented with 0.2mg/L KT, 1mg/L 6-BA, 0.5mg/L NAA is the suitable medium for cutivar'M'.The differentiation rates are high up to 53.33%,53.67% and 60.00% respectively.III. Parameters of biolistic bombardment were studied on base of regeneration systems. The optimal methods showed as follows.Plasmid DNA are coated by Ca(NO3)2 and PEG4000. 1μm golds are the vetor of plasmid DNA. 6 cm bombardment height distance, 1 time and without osmotic treatment are favorable to transgenic efficency. The concentration hygromycin(Hy) for cultivar'Md'is 130mg/L. The concentration Hy for'B'is 100mg/L.