| Wheat(Triticum aestivum L.)is one of the most important crops in food supply system in our country. The wheat seeds have plenty of starch(65-70%), much protein(7-22%), a small quantity of lipid(2-4%), and multi-mineral elements and vitamine B etc. which are the main nutritions for human beings and animals. Therefore, the wheat breeding, especially in quality improvement by genetic engineering, is always one of the most important subjects in foundational research and applied research. In this paper, studies and analyses on tissue culture conditions for 6 wheat cultivars, heritability and genetic mechanism of progenies of 100 transgenic lines were carried out by using the modern technique of tissue culture and molecular biology individually and respectively. The main work, results and conclusions from these studies are as follows. 1. The high efficient plant regeneration systems have been established by optimizing tissue culture conditions using inflorescence explants, immature and mature embryo scutella as the target materials. ①Genotype effect: the highest frequency of the plant regeneration genotype is cultivar EM12 which has an average plant regeneration frequency of 22.0% and the highest frequency of 83.0% in one of the treatments basis on the results of tissue culture from 4,430 individual explants of six genotypes. ②Explant effect: the average plant regeneration frequencies are 29.6%, 30.1% and 5.0% from inflorescence, immature embryo and mature embryo cultures, respectively. The plant regeneration frequencies of 46.7%, 63.3% and 13.3% were recorded from inflorescence, immature embryo and mature embryo cultures, and the highest plant regeneration frequencies of 60.0%, 83.0% and 30.0% were achieved in one of the treatments under the optimalized culture conditions. The plant regeneration capacities among explant sources ranged as follows: immature embryo≥inflorescence>mature embryo. ③Hormone effect: the overall induction frequencies are both 100% with the induction medium containing 0.5~4.5mg/l 2,4-D or 1~6mg/l Picloram; while the average plant regeneration frequencies are 23.5% and 11.9% with the regeneration medium containing 0.01mg/l 2,4-D and 0.1mg/l 2,4-D, respectively; the optimalized hormone concentrations of induction media are 4.5mg/l 2,4-D, 0.5mg/l 2,4-D and 1mg/l Picloram for inflorescence, immature embryo and mature embryo respectively. ④Subculture effect: the callus subculture has no significant effects on plant regeneration from our experiments. ⑤Disinfector(GB9985) effect: the overall plant regeneration frequencies are 22.1% and 25.8%, respectively, with white cat domestic detergent of 10.0% diluted concentration and mercuric chloride as the disinfectors by using inflorescence and immature embryo as the target materials, and the frequencies of contaminated cultures are the frequencies of 8.3% and 0.0% by above-using both disinfectors respectively. ⑥Co-relation analysis of regenerated organs: the green spots regenerated and the number of plants regenerated per callus have no co-relation from each other; the root differentiation from the cultures has a negative co-relation with the plant regeneration frequency, while a significant positive co-relation with the frequency of green spot regenerated; then a clear positive co-relation was observed between the frequency of plant regeneration and the number of regenerated plants per callus. 2. Analyses of 1Dx5 transgene expression were carried out. ①Tranformant cultivation: the 140 individual plants were examined in these experiments, and the results indicated that temperature and the moisture can affect on the growth of transgenic wheat dramatically; the equation of y=1.413+0.050x1+0.149x2-0.867x3-0.024x4 is induced by measuring parameters of the length of spikes, plant height, number of nodes, length of top nodes and sword leaf marked by x1, x2, x3 and x4 respectively, the results of plant surviving frequency, number of tillering, plant height, number of node, length of top node, length of spikes, length of sword leaf and number of seeds cultivated in the greenhouse were all higher than that of the plants growing in the mess chamber. ②Optimization of the DNA extraction method: from leaf samples, the average yield of 116ng is obtained in the DNA extraction experiments by using 7 cm long leaf materials, and the highest yield of 250ng is obtained from one of 117 individual plants with the three-leaf-stage young leaf as the target materials. The best CTAB condition for leaf sample DNA extraction is 30 minutes for sample incubation and 7 minutes for sample centrifugation. ③Progeny of transgenic wheat analysis by PCR and SDS-PAGE: the 102 target materials of transformants were tested by using the modern technique of PCR, 1Dx5 transgene fragments are not detectedfrom the control lines, all of the B73-6-1 transgenic lines and 26 individual lines from 48 transgenic individual lines of B72-8-11b tested are 1Dx5-positive; Protein analysis by SDS-PAGE, the 78 transgenic individual lines with 1Dx5 trsnsgene are all found to have the correct expression of transgene, the frequencies of over-expression and low-expression are 59.0% and 7.7% respectively among tested lines; By analysing next progeny, the stable expression of the 1Dx5 transgene is observed in all seeds collected from the same line and no diversities are found, indicating that all tested transgenic lines are homozygotes. |
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