| With the development of gene engineering and molecular biology, the method of transgenic transformation has been utilized in modern plant breeding, which led to the realization of so called molecular designed breeding, which the foreign useful genes could been induced and inheritance directionally and stably in the host plant cells. The reproducible and high efficient plant regeneration technique system is a prerequisite for transformation of plants to obtain abundant selective colonies of transgenic plants. So, it is very important and significant to establish a high efficient somatic embryogenesis and plant regeneration system for the studies of the gene engineering, function genomic and breeding in upland cotton. The conditions of cotton somatic culture were studied systemically in this experiment, using the cotton materials as Coker 201, Coker 312 and yzl etc., and a cotton somatic culture system with short-period, high efficiency and simple manipulation was established in our research works, which then was used to study the influences of foreign genes in the transgenic cotton as well as the pigment glands on the results of cotton somatic culture, and the main results were as follows:1. The effect of different hormones and their combination, sugar types, solidification materials and sterilizing technique on the induction of callus, somatic embryogenesis and differentiation were studied. As the result showed that the combination of 2, 4-D (0.1mg/L) and KT (0.1mg/L) was the must effective one in the callus inducing. The effects of the somatic embryogenesis were different with the different proportion of the hormones, and the proportion of IBA and KT was the best in somatic embryogeneusis, and 4-D, on the other hand, was the worst one. At the certain range, the percentage of the callus induction was enhanced with the increase of the hormone 2, 4-D concentration, but the percent of somatic embryogenesis was decreased on the contrary. So, reducing the concentration of 2, 4-D was effective in somatic embryogenesis. For the somatic embryongenesis and plant regeneration, the culture results such as the percentage and quality of the regeneration plants was better in the media with glucose as sugar source obviouslythan that with sucrose.2. Comparing to the different solidification materials and the different sterilizing techniques, the results in this experiment showed that the phytagel as solidification material in the media was much better than that of agar in the callus inducing and multiplication, embryongenetic callus inducing and production, and development and germination of the somatic embryos. In addition, the results of this experiment showed that the classical sterilizing technique was not the best one in cotton somatic culture, 14 minutes at 121°Cwas enough for the microbe sterilization, extensive sterilization will destroy more availability components of the medium. So, the improved sterilizing technique was much better than the classical one in cotton somatic culture, especially for the percentage of callus inducing and growth, the formation of somatic embryoid and plant regeneration.3. After the validation in the Coker 201, Coker 312 and yzl, the process of somatic embryogenesis and plant regeneration were developed with short-period and high efficiency, and the embryogenic callus and regeneration plants have been obtained following this improved culture process. The key techniques were as follows: (1) Basic culture medium: MS inorganic elements + B5 organic components +1.0g/L Gin + 0.5g/L Asn. For callus inducing, O.lmg/L 2, 4-D and O.lmg/L KT was added in basic medium. This medium also can be used in callus growth, as long as the decreasing of 2, 4-D from O.lmg/L to 0.05mg/L. For the differentiation culture, the hormone combination was 0.5mg/L IBA+0.15mg/L KT. (2) solidification material: 2.5g/L phytagel. (3) Sugar type: 30g/L glucose. (4) sterilizing technique: medium sterilization at 121 °C for 14 minutes. (5) explant: hypocotyls. (6) Grafting technique of regenerate seeding was adopted to transplant the test tube seeding.4. The capability of callus induction was studied in this experiment, using the materials as Zhe 905 (transgenic cotton with single insect resistance gene of Bt), GK321(transgenic cotton with double insect resistance genes of Bt+CpTi), TM-1 (CK) , Coker312 (CK) , ZMS17 (transgenic cotton with single insect resistance genes of Bt), ZMS17 (transgenic cotton with double insect resistance genes ofBt+CpTi ), ZMS12 (transgenic cotton with single insect resistance genes of Bt), ZMS12 (transgenic cotton with double insect resistance genes of Bt+CpTi) as the materials. The results showed that the capabilities of callus induction in different materials were different significantly, and Coker 312 and TM-1 were the best ones for callus inducing. Comparing to the somatic culture results between glandless isogenic materials and glanded ones in the same genomic background, the result showed that the influence of glands was significant in callus inducing, the percentage and the growth of callus from the glandless cotton isogenic materials was obviously better than that of ist corresponding glanded ones, but Coker312 was an exception, callus inducing percentage from the glanless Coker312 was much lower than its corresponding glanded one.5. The studies on the effect of foreign genes on the cotton somatic culture, using the transgenic cotton isogenic line with Bt and Bt+CpTi, and their genetic background parents, shown that the effect of foreign gene on the somatic culture was different with the difference of genetic background. To the foreign insect resistant genes, Bt and Bt +cpTi, the percentage of callus inducing from the isogenic line with single insect resistant gene was significantly higher than that from double insect resistant genes ones, and the tendency was same in different genetic background materials, which indicated that the CpTi gene might have some restrain effect on the callus induction in cotton somatic culture. |
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