The Establishment Of Plant Regeneration And Protoplast Culture System Of Kentucky Bluegrass With Drought Resistantance

Kentucky bluegrass (Poa pratensis L.) is a typical perennial herb, with a strongoccupation force in the competition populations, and it is a pioneer grass inreconstruction and rehabilitation of degraded grassland. It is also the first choice ofturfgrass in north of China. The drought makes kentucky bluegrass abovegroundturn brown, which threatens the survival of seedlings and has a certain impact on thequality and turf effect. In the study, the drought resistant materials (Longxi, Anding)Which regarded as parent materials of somatic hybridization were selected by theevaluation of drought resistance under drought stress. The plant regeneration systemof drought resistant materials (Longxi, Anding, Qinzhou)were successfullyestablished by tissue culture. And the protoplast culture system of two kinds ofmaterials (Longxi, Anding)were established. Which provided basic data to somatichybridization of wide kentucky bluegrass.1. Drought resistant materials were Selected and evaluated.The wild germplasm materias of kentucky bluegrass, collected from GansuProvince (Longxi, Anding, Xihe, Qingshui, Qinzhou), were used as doner materials,and the Midnight Ⅱ, a commercial variety, regarded as control. Physiologicalindicators and endogenous hormone contents of kentucky bluegrass exposed todrought stress were determined. The study focused on analyzing droughtadaptability of wide Kentucky bluegrass at the Physiological and hormones level,and the drought resistant materials (Longxi,Anding) were selected.(1) Physiological response and evaluation of drought resistance under droughtstress: During water stress, relative water content and chlorophyll content declined,MDA and soluble protein content increased, POD and SOD activity increased inmild drought (75-85%), while declined in severe drought (35-45%). Droughtresistance was evaluated by membership function method. The membership functionvalue of Qingshui, Qinzhou, Longxi, Xihe, Anding and MidnightⅡwas0.470,0.512,0.554,0.523,0.532and0.525respectively, So the ability to drought resistance ofdoner materials was ranked order as: Longxi> Anding> Midnight Ⅱ> Xihe>Qinzhou> Qingshui at physiological levels.(2) Changes of Plant phenotypic under drought stress: Qingshui and Qinzhouwild kentucky bluegrass wilted and died after suffering from drought stress. However, Longxi wild kentucky bluegrass and midnightⅡgrew slowly and had less yellowleaves. In addition, it had lower Leaf Chlorosis and wilting rate and could adapt todrought. The extent of the damage of doner materials exposed to drought was:Qingshui> Qinzhou> Xihe> MidnightⅡ>Anding> Longxi.(3) Changes of endogenous hormone level under drought stress: ZT content ofall the materials was significantly decreased and the descent range was59.25%-66.02%under the drought stress. Both ABA and IAA content increasedfirstly and then decreased, compared with the control, ABA content significantlyincreased and the rising range was23.63-73.95%, while IAA content significantlydecreased, and the descent range was45.13-53.35%. In addition, the ZT/ABA、ZT/IAA and (ZT+IAA)/ABA decreased, So the ability to drought resistance of donermaterials was ranked order as Longxi> Anding> MidnightⅡ> Xihe> Qinzhou>Qingshui. It was done at the evaluation standard of hormones level.2Regeneration system of doner materials was established.The callus induction, differentiation and rooting transplanting from Gansu wildkentucky bluegrass (Longxi, Anding, Qinzhou) were studied. In this study, the matureseeds were used as explants. The results showed that:(1) The best medium of callus induction from Longxi was MS+1.0mg/L2,4-D,and the callus induction rate was41.40%, the optimal differentiation medium was MS+0.5mg/L NAA+2mg/L6-BA, the green bud rate was25.38%and thedifferentiation rate was22.88%.(2) The best medium of callus induction of Anding was MS+1.0mg/L2,4-D+0.2mg/L6-BA, and the callus induction rate was67.00%, the optimal differentiationmedium was MS+0.5mg/L NAA+2mg/L6-BA, the green bud rate was63.29%and the differentiation rate was54.00%.(3) The best medium of callus induction from Qinzhou was MS+2.0mg/L2,4-D+0.1mg/L6-BA, and the callus induction rate was61.40%, the optimaldifferentiation medium was MS+0.1mg/L2,4-D+2mg/L6-BA, the green bud ratewas62.94%, the differentiation rate of51.06%.(4) The rooting medium of doner materials was the same as differentiationmedium respectively and the rooting rate was100%. 3The Protoplast culture system of Gansu wild kentucky bluegrass wereestablished.the granular callus of Gansu wild Kentucky bluegrass (Longxi, Anding)wereused as materials, and the optimum enzymatic conditions were studied.(1) The optimal protoplast isolation conditions of Longxi was1.5%CellulaseOnozukaR-10+0.5%PectolaseY-2+0.3%Driselase+1.0%Macerozyme R-10, enzymedigestion time was16h, concentration of mannitol was0.60mol/L, and the yield andvigor of protoplasts highest were6.79×106/g and89.64%.(2) The optimal protoplast isolation conditions of Anding was1.5%CellulaseOnozukaR-10+0.5%PectolaseY-2+0.3%Driselase+1.0%Macerozyme R-10, enzymedigestion time was16h, concentration of mannitol was0.50mol/L, and the yield andvigor of protoplasts highest were5.95×106/g and85.28%.