Development Of Agrobacterium-mediated Transient Transformation System In Paeonia Lactiflora Pall.

Paeonia lactiflora Pall.is a traditional Chinese famous flower with good medicinal value and ornamental value.At the same time,P.lactiflora has strong adaptability to the external environment and it is an ideal material for excavating plants resistance genes.However,because the seeds of P.lactiflora has double dormancy-hypocotyls,it is difficult to reproduce.There are still unsolved problems in tissue culture,so the mature genetic transformation system has not been established,which hinders the study of molecular biology of P.lactiflora.Therefore,it is necessary to establish an efficient and rapid method for gene identification.In this study,an Agrobacterium-mediated transient transformation system for P.lactiflora was established.Several transient transformation conditions,including germination time,Agrobacterium tumefaciens concentration,infection time,acetosyringone(AS)concentration,co-culture time,negative pressure intensity,Tween-20 concentration and receptor material,were screened.In addition,Pl GPAT,Pl DHN2 and Pl HD-Zip stress resistance genes of P.lactiflora were transient overexpressed by this system,and the feasibility of the transient transformation system was verified by detecting their expression levels and physiological indexes under low temperature,high temperature and drought stress.This study provided ideas and basis for the functional research of some key genes in P.lactiflora.The main results of this study are showed below:(1)Taking the tissue culture and seedlings of P.lactiflora as transient transformation receptor materials,the plant expression vector p CAMBIA1301 containing GUS reporter gene was transferred into P.lactiflora seedlings by Agrobacterium-mediated method.Transient transformation conditions were screened by GUS histochemical staining.The results showed that when the tissue culture of P.lactiflora was used as the receptor materials,the highest transient transformation efficiency could be obtained that the concentration of Agrobacterium tumefaciens was cultured to 1.2,the 30-day-old tissue culture seedlings were transformed with 200 μmol/L AS and 0.01% Tween-20 Agrobacterium tumefaciens resuspension for 24 h,the negative pressure intensity was 10,and co-culture for 3 days in darkness.When the seedlings with 2-3 cm buds of P.lactiflora was used as the receptor materials,under the condition of Agrobacterium tumefaciens suspension infection for 12 h and other transient transformation conditions remained unchanged,the transient transformation efficiency of P.lactiflora was the highest,reaching 93.3%,which was more suitable for the study on the function of P.lactiflora resistance genes in the later stage.(2)Constructing overexpression vectors of cold resistance gene Pl GPAT,high temperature resistance gene Pl DHN2 and drought resistance gene Pl HD-Zip and transformed into P.lactiflora seedlings respectively by Agrobacterium-mediated transient transformation system.Through GUS histochemical staining,it was found that obvious blue patches appeared in transient transient transgenic P.lactiflora seedlings.The results showed that the recombinant overexpression vectors were successfully transformed into P.lactiflora seedlings by transient transformation system.(3)Under low temperature,high temperature and drought stress,the stress resistant genes in the seedlings of transient transgenic P.lactiflora were induced by abiotic stress,and gene expression level and each physiological indexe were significantly increased,among which the activity of antioxidant enzymes and the content of organic osmotic regulators were significantly higher than the control group.SOD activity,POD activity,CAT activity,soluble sugar content and proline content were about 1.46 times,1.37 times,1.22 times,1.2 times,1.15 times,respectively of the control group.MDA content was significantly lower than the control group,about 0.9 times that of the control group.The results showed that the P.lactiflora seedlings with transient transformation of stress resistant genes played their stress resistant function,which verified that the Agrobacterium-mediated transient transformation system could be used to study the stress resistant gene function of P.lactiflora.