Studies On The Induction Of Calli From Isolated Microspores And The Exogenous DNA Transfer By Electroporation Of The Semi-protoplasts Of Wheat

Since 1985, the technique of DNA transfer by electroporation has been used to the genetic transformation of plant cells and protoplasts. Now, with the development of biotechnology, this method is widely used in almost all kinds of plants. As to the application of this technique to the transformation of the semi-protoplasts of wheat's microspores', no relevant report can be found at present. Because the semi-protoplasts of wheat's microspores'can be easily cultured than the protoplasts of wheat's embryo calli, and the systems for the culture of the wheat's isolated microspores are increasingly completed, so the semi-protoplasts of wheat's microspores are the excellent receptor for genetic transformatims by electroporation, In view to this, this paper is mainly explored from the researches of the following 3 aspects: ①Through the culture and activity detection of the microspores of 11 different wheat and octploid rye cultivars, the optimum medium and conditions for culture, the combination of the parameters for enzyme treatment, the optimum carbohydrate source and the variety which microspores can be most easily cultured are selected; ②Through the experiments of enzyme treatment to the cell walls of the microspores, the optimum combination of the parameters for enzyme treatment is selected, which is a preparation for the experiments of electroporation; ③Through the transduction of coumarin-5-dUTP by electroporation to the receptor, namely the semi-protoplasts of the variety which microspores can be most easily cultured, the optimum combination of the parameters for electroporation is selected, The main results of this experiment are as followings: After 3 days'4℃low temperature pretreatment of wheat's immature spike, the microspores isolated have the highest average activity of 6.6%. After 30℃high temperature pretreatment of wheat's immature spike, the microspores isolated have the highest average activity of 6.7%. After 3 day's high temperature pretreatment of wheat's immature spike the microspores isolated have the highest average activity of 6.5%. If the density of anther implanting is 1000 anthers/10mL culture liquid,the microspores cultured in 4 days will have the highest average activity which amount to 5.28%. The microspores cultured in the media which carbohydrate source is maltose have the highest average activity amounting to 4.35% in 4 days, while the microspores cultured in the media which carbohydrate source is sucrose have the lowest average activity. So, maltose is the best carbohydrate source for the culture of wheat's microspores while sucrose is not suitable for their culture. Through the comparison and selection of different methods for the isolation of microspores, the method of stirring is selected as the most suitable method for isolation. Through the experiments of the destruction of microspores'cell wall under different complex enzyme treatments, the combination of the parameters for enzyme treatment which can create microspores with the thickest cell walls (not including protoplasts) is selected. Such combination's parameters are 1.5%'s concentration of complex enzymes (including 1.4%'s concentration of cellulase and 0.1%'s concentration of pectinase), 28 ℃and 8h for the average thickest cell wall of 0.83μm and CK (3.08μm) is 3.7 times of it. In addition, through the experiments of enzyme treatment, 2%'s concentration of complex enzymes (including 1.9%'s concentration of cellulose and 0.1%'s concentration of pectinase) is found to be extremely suitable for the culture of protoplasts because under the treatment of such enzyme concentration, large amount of protoplasts create. Through the experiments of the induction of calli from microspores, Cui2B medium is found to be most suitable for the induction and its distribution of additional compositions is 1.5mg/L 2,4-D:0.5mg/L KT:62g/L maltose. Through the experiments of the culture of wheat's microspores, 9 days'low temperature pretreatment of wheat's immature spikes, implanting density of 900 anther/ 10mL culture liquid and 27±1 ℃for dark culture are all found to be advantage to the induction of calli from wheat's isolated microspores. The microspores isolated from water-dependent cultivars can be more easily cultured than the microspores from drought-resistant cultivars and outlaid rye cultivars, and the microspores isolated from octploid rye cultivars are the most difficult group to be cultured, while the water-dependent variety 7015 is found to be the variety which microspores can be most easily cultured among all the 11 varieties. So, this variety's semi-protoplasts of microspores'are selected as the receptors for the experiments of electroporation. Through the experiments of transformation by electroporation, the optimum combination of the parameters for electroporation is selected and the parameters of such combination are 300V's voltage value, 1500μF's capacitance value and 3 times'pulse. Under the treatment of this combination, the transferring rate of coumarin-5-dUTP is 9.6% and the rate of undamaged cells'is 78.9%, and the product of them is 0.07574.