Optimization Of Agrobacterium-mediated Genetic Transformation Conditions Of Maize Shoot Tip And Phenotypes Analysis Of Drought Tolerance Of ZmCPP2 Overexpression In Maize

CPP（Cystein-rich polycomb-like protein or Tesmin/TOS1-like）is a member of a small family of transcription factors.Previous studies have shown that CPP could participate in stress response process in plants.Our previous studies showed that the maize CPP transcription factor,ZmCPP2,could enhance the antioxidant protection of maize via the protoplast transient expression system.In order to further understand the function of ZmCPP2 in response to abiotic stresses,it is necessary to construct the ZmCPP2 transgenic maize.Recent years,the Agrobacterium-mediated genetic transformation of maize shoot tip has received more and more attention.However,the transformation rate of this method was low.Therefore,we firstly optimized the conditions for Agrobacterium-mediated genetic transformation of maize shoot tip by constructing ZmCPP2 overexpressing maize.Then,we preliminary analyzed the drought tolerance of ZmCPP2 overexpressors.The results are as followed:Previous studies have shown that the concentration of bacteria solution（OD600）,vacuum osmotic pressure（MPa）,infection time（min）,and acetylsyringone（AS）concentration（μM）clould influence the efficiency of maize shoot tip genetic transformation.We used a single variable method to optimize these four factors separately within a certain range.We found that the concentration of bacterial liquid OD600（0.65）,or the vacuum osmotic pressure（0.05 MPa）,or the infection time（3 min）,or the AS concentration（150μM）could significantly improve the genetic transformation efficiency.When the four optimized factors were integrated for genetic transformation of maize shoot tip,the final germination rate was 10.6%and the transformation efficiency was 8.7%.Although the final genetic transformation efficiency did not show an exponential increase,it still had higher genetic transformation efficiency.Therefore,the optimal genetic transformation conditions were OD600 of 0.65,vacuum osmotic pressure of 0.05 MPa,infection time of 3 min,and AS concentration of 150 μM.The maize（three-leaf stage）was sprayed with different concentrations of the herbicide Basta,and we found that the optimal lethal concentration of Basta was 75 mg/L.In order to determine whether the maize overexpression plants were successfully obtained,genetically transformed maize plants were screened by spraying with Basta and PCR ZmCPP2 overexpressed maize plants in the To generation and ZmCPP2 overexpressed maize seeds in the T1 and T2 generations were successfully obtained.The expression levels of ZmCPP2 gene and ZmCPP2 protein were determined by qRT-PCR and western blot.The ZmCPP2 overexpressed maize was subjected to drought stress,then the phenotype was observed and the physiological and biochemical indexes were measured.Transgenic maize and the wild type were grown to three-leaf stage in soils and treated by withholding water for 7 days and recovered for 2 days.The survival rate of ZmCPP2 overexpressed maize was significantly higher than that of the wild type,and the MDA content and electrolyte leakage rate of ZmCPP2 overexpressed maize were significantly lower than those of wild type under drought stress.The above results indicate that overexpression of ZmCPP2 significantly enhances the tolerance of maize to drought stress.