Optimization Of Wheat (Triticum Aestivum L.) Mature Embryo Regeneration System And Genetic Transformation Of WK Genes

Transgenic technology is an important means of directional genetic improvement of wheat.The gene gun transformation method is one of the main methods of wheat genetic transformation,and the wheat regeneration system is the most important factor affecting its transformation efficiency.Among the various explants of wheat,the callus induction and plant regeneration ability of immature embryos are the higher,but the extraction time is short,and it is difficult to ensure and control the consistency of their growth period,while the mature embryo of wheat is not limited by the time of harvesting,which ensure the consistency of physiological conditions among different individuals,but the regeneration ability of mature embryos is lower.Therefore,it is significant to optimize the wheat mature embryo regeneration system by using mature embryos as receptor materials for wheat genetic transformation.Trace elements of Cu²⁺,Zn²⁺and Mn²⁺in vitro regeneration of plants have an important effect on callus formation and differentiation,and the appropriate addition of trace elements can improve the ability of callus induction and differentiation,but there are few related researches on wheat at present.The research content of this thesis is to use gene gun to mediate the genetic transformation of wheat immature embryos by WK and other genes,in order to create new transgenic anti-reverse materials.Due to the limited number of transformations of immature embryos in one year,the embryo inoculation method,concentrations and ratios of Cu²⁺,Zn²⁺and Mn²⁺were optimized during the regeneration of mature embryos.Antioxidant enzyme activity and chlorophyll content under different test conditions were determined.The results were as follows:1.The callus healing time of treatment 2 was the earliest,and the callus induction rate and differentiation rate were the highest,and reached 70.00%and 18.84%,which was the suitable wheat inoculation method.2.The suitable concentration of Cu²⁺in the medium was 1 and 10μmol/L,the induction rate reached 64.63%-80.1%and the differentiation rate was 18.48%-21.83%.The suitable concentration of Zn²⁺was 10 and 30μmol/L,the induction rate was 57.82%-77.8%and the differentiation rate was 16.44%-23.52%.The suitable concentration of Mn²⁺was 100and 300μmol/L,the induction rate was 57.82%-78.91%and the differentiation rate was16.44%-21%.3.When the concentrations of Cu²⁺,Zn²⁺and Mn²⁺were 10,10 and 100μmol/L,the callus induction rate was 84.90%,and the differentiation rate was 30.09%,significantly higher than that of control and individual Cu²⁺,Zn²⁺and Mn²⁺concentration treatment,which is suitable concentration ratio.4.The results showed that with the prolongation of culture time,the activity showed an increasing tendency,and then decreased after reaching the maximum.In the whole development process,when Cu²⁺,Zn²⁺and Mn²⁺were added to the medium,the SOD,POD and CAT activity of callus were higher than that of non-added treatment.Therefore,the increase of antioxidant enzyme activity can improve the regeneration ability of callus.5.WK,Lec RLK and bar were co-transformed into wheat immature embryo callus by gene gun bombardment technique,and 333 strains and 36 strains of resistant transgenic strains were obtained respectively.PCR results of T0 generation resistant plants showed that there were 16 WK positive transgenic plants,10 Lec RLK positive transgenic plants,49Lec RLK-RNAi positive transgenic plants.PCR detection of T1 transgenic plants showed that there were 11 WK positive transgenic plants,5 Lec RLK positive transgenic plants,and 11Lec RLK-RNAi positive transgenic plants.The above research results in the creation of wheat transgenic new materialsand the establishment of high-frequency regeneration system of wheat mature embryos.