农杆菌介导gagal基因转入大豆和大豆体细胞胚培养的初步研究
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摘要
本研究用南方地区的大豆作为受体材料，进行子叶节培养再生植株的研究，探讨了本地区大豆子叶节器官发生以及农杆菌介导遗传转化的主要影响因素。选出3个南方大豆材料，以子叶节为受体材料，初步建立适合的大豆遗传转化体系，获得3株gaga1基因PCR检测阳性的转基因大豆植株。对大豆体细胞胚发生途径也进行了初步研究，筛选到几个较易诱导体细胞胚的基因型。
本研究的主要结论如下：
1、子叶节器官发生途径的再生频率在不同大豆基因型之间差异较大。筛选出10个再生性较好的基因型，其中M02的再生率最高，为80％；其次是常熟散子黄豆，为73％。
2、不同大豆基因型对农杆菌菌株的易感性差异较大，筛选出南农18-6、南农88-1和常熟散子黄豆这3个易感性较好的基因型。
3、通过试验农杆菌介导的转化获得5株转gaga1基因烟草植株，测试gaga1基因对烟草花器官发育的影响，发现烟草花器官产生变异，并且其T_1代遗传稳定。
4、获得3株转gaga1基因PCR检测阳性的大豆植株，其中南农18-6有1株，转化率为0.20％；常熟散子黄豆有2株，转化率为0.24％。
5、通过体细胞胚胎发生途径，筛选到肥西黑豆和云霄乌子豆2个体细胞胚诱导较好的基因型，体细胞胚胎发生率分别是19％和13％。初步建立适合南方大豆材料的体细胞胚诱导、增殖和成熟的组织培养体系。
Soybean is one of the recalcitrant crops for in vitro culture. In the present study, the cotyledonary node regeneration system was tested, and three genotypes planted in southern China with high regeneration frequency were found. The major factors influencing the regeneration and Agrobacterium-mediated transformation were investigated.
A reliable transformation system through the cotyledonary node regeneration was set up for the tested genotypes after the studies. A gene, gagal, regulating flower development were introduced into soybean through getting three PCR-positive transformed plants.
Soybean somatic embroygenesis has been studied, and two genotypes were found with high embryogenetic potential.
The main results can be summarized as below:
1. In the cotyledonary node regeneration approach, regeneration frequencies are very different among genotypes. We have found ten genotypes with high regeneration frequency. M02 is the highest among them whose frequency is up to 80% while Changshusanzihuangdou takes the second position with frequency 73%.
2. The susceptibility to Agrobacterium is very different among the tested genotypes. In the experiment, three genotypes with good susceptibility to Agrobacterium were found.
3. Checking the effect of the construct of gaga1, we gained five transgenic tobacco plants, and their tobacco floral structure changed slightly.
4. Among the fifteen survival soybean plants after transfering to soil, three plants gave positive PCR reaction. The genotype of one of the plants is Nannong 18-6 and that of the other two is Changshusanzihuangdou.
5. Somatic embroygenesis was induced successfully from immature cotyledon. Two genotypes, Feixiheidou and Yunxiaowuzidou, were found with high embryogenesis potential. The procedure to induce secondary somatic embryos, propogate somatic embyos, and regenerate plantlets from somatic embryos has been completed.
本研究的主要结论如下：
1、子叶节器官发生途径的再生频率在不同大豆基因型之间差异较大。筛选出10个再生性较好的基因型，其中M02的再生率最高，为80％；其次是常熟散子黄豆，为73％。
2、不同大豆基因型对农杆菌菌株的易感性差异较大，筛选出南农18-6、南农88-1和常熟散子黄豆这3个易感性较好的基因型。
3、通过试验农杆菌介导的转化获得5株转gaga1基因烟草植株，测试gaga1基因对烟草花器官发育的影响，发现烟草花器官产生变异，并且其T_1代遗传稳定。
4、获得3株转gaga1基因PCR检测阳性的大豆植株，其中南农18-6有1株，转化率为0.20％；常熟散子黄豆有2株，转化率为0.24％。
5、通过体细胞胚胎发生途径，筛选到肥西黑豆和云霄乌子豆2个体细胞胚诱导较好的基因型，体细胞胚胎发生率分别是19％和13％。初步建立适合南方大豆材料的体细胞胚诱导、增殖和成熟的组织培养体系。
Soybean is one of the recalcitrant crops for in vitro culture. In the present study, the cotyledonary node regeneration system was tested, and three genotypes planted in southern China with high regeneration frequency were found. The major factors influencing the regeneration and Agrobacterium-mediated transformation were investigated.
A reliable transformation system through the cotyledonary node regeneration was set up for the tested genotypes after the studies. A gene, gagal, regulating flower development were introduced into soybean through getting three PCR-positive transformed plants.
Soybean somatic embroygenesis has been studied, and two genotypes were found with high embryogenetic potential.
The main results can be summarized as below:
1. In the cotyledonary node regeneration approach, regeneration frequencies are very different among genotypes. We have found ten genotypes with high regeneration frequency. M02 is the highest among them whose frequency is up to 80% while Changshusanzihuangdou takes the second position with frequency 73%.
2. The susceptibility to Agrobacterium is very different among the tested genotypes. In the experiment, three genotypes with good susceptibility to Agrobacterium were found.
3. Checking the effect of the construct of gaga1, we gained five transgenic tobacco plants, and their tobacco floral structure changed slightly.
4. Among the fifteen survival soybean plants after transfering to soil, three plants gave positive PCR reaction. The genotype of one of the plants is Nannong 18-6 and that of the other two is Changshusanzihuangdou.
5. Somatic embroygenesis was induced successfully from immature cotyledon. Two genotypes, Feixiheidou and Yunxiaowuzidou, were found with high embryogenesis potential. The procedure to induce secondary somatic embryos, propogate somatic embyos, and regenerate plantlets from somatic embryos has been completed.
引文
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