葡萄遗传转化体系与转化抗病相关基因的研究
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摘要
本研究以华东葡萄“白河-35-1”试管苗的叶片和茎段(无腋芽),“佳丽酿“的花药、子房和花蕾为试材,通过器官发生途径建立了葡萄的再生体系;研究了不同培养基及激素浓度组合在葡萄花器愈伤的诱导分化中的作用;并将携带有中国野生葡萄乙二醛氧化酶相关基因的植物干扰载体pHellsgate8通过农杆菌介导法转入‘白河-35-1’,同时研究了卡那霉素的筛选浓度和筛选方式。试验结果如下:
1.以华东葡萄“白河-35-1”试管苗的叶片和茎段(无腋芽)为材料,研究不同激素浓度组合对叶片和茎段(无腋芽)不定芽再生的影响。结果表明:诱导华东葡萄“白河-35-1”叶片和茎段的最佳培养基配方为:MS+TDZ 1mg/L+NAA 0.5 mg/L+蔗糖30 g/L +琼脂0.65%+ CH 200 mg/L + pvp500 mg/L,再生率分别为12.8%和3.3%。
2.以“佳丽酿”试管苗的花药、子房、花蕾为材料,研究了不同培养基及激素浓度对花器愈伤组织诱导分化的影响。结果表明:诱导欧洲葡萄“佳丽酿”子房的最佳培养基配方为:B5+ BAP 0.5mg/L+2,4-D 0.5mg/L +蔗糖30 g/L +琼脂0.65%+ CH 200 mg/L + pvp500 mg/L,诱导欧洲葡萄“佳丽酿”花蕾的最佳培养基配方为:B5+BAP 4mg/L+2,4-D 0.5mg/L+蔗糖30 g/L +琼脂0.65%+ CH 200 mg/L + pvp500 mg/L,诱导率分别为10%和43%。
3.以华东葡萄“白河-35-1”试管苗的叶片为材料,研究了几种不同试剂处理对诱导愈伤过程中褐化现象影响的研究。结果表明:在培养基中添加100mg/L抗坏血酸,防褐化效果最好。
4.以华东葡萄“白河-35-1”单芽茎段为试材,研究了卡那霉素对葡萄不定芽再生及植株生长的影响,确定了卡那霉素的筛选方式和筛选浓度。结果表明:在葡萄遗传转化后,采用延迟筛选,起始浓度为5mg/L逐步增加到16mg/L,可减少对再生的抑制。
5.采用农杆菌介导法,侵染“白河-35-1”单芽茎段,经卡那霉素筛选,获得11个抗性芽。
The objective of this study was to research in regeneration systems of Vitis pseudoreticulata clone‘Baihe-35-1’in Organogenesis, investigating the effect of auxins and explants on callus induced from flower organ of Vitis viniferal L.‘carignane’, grape transformation by Agrobacterium infection introducing the novel Glyoxal Oxidase related gene(GLOXrg) which was carried in the EHA105 binary vector into Baihe-35-1’. The main results are as follow:
(1) Regeneration system from leaf and stem of Vitis pseudoreticulata clone‘Baihe-35-1’using TDZ and NAA was Studied. The results were: Regeneration from stem was easier than from leaf. The combination 1mg/L TDZ with 0.5 mg/L NAA resulted in the highest frequency of adventitious bud production from leaf and stem which the rate is 3.3% and 12.8%.
(2) Callus induction from flower organ of‘carignane’using BAP and 2,4-D was studied.The results were: Callus induction from bud is easier than from ovary. The combination 0.5 mg/L BAP with 0.5 mg/L 2,4-D resulted in the highest frequency of callus induction from ovary which the rate is 10%. And when 4mg/L BAP combinates with 0.5 mg/L 2,4-D, callus induction reaches its highest frequency which is 43%.
(3) Effects of different additaments on anti-browning were studied which leaf of Vitis pseudoreticulata clone‘Baihe-35-1’was taken for test material. The result showed that Vc could effectively prevent the leaf of‘Baihe-35-1’from browning. The optimal concentration of Vc is 100mg/L.
(4) Effect of Kan on bud regeneration from stem with bud of Vitis pseudoreticulata clone‘Baihe-35-1’was studied. Retarding selection was better for transformation of grape. It can reduced restraining of Kan on grape that adding Kan firstly with 4mg/L and gradually increasing to 16mg/L.
(5) 11 resistant-Kan buds of Vitis pseudoreticulata clone‘Baihe-35-1’were obtained after selection of Kan.
1.以华东葡萄“白河-35-1”试管苗的叶片和茎段(无腋芽)为材料,研究不同激素浓度组合对叶片和茎段(无腋芽)不定芽再生的影响。结果表明:诱导华东葡萄“白河-35-1”叶片和茎段的最佳培养基配方为:MS+TDZ 1mg/L+NAA 0.5 mg/L+蔗糖30 g/L +琼脂0.65%+ CH 200 mg/L + pvp500 mg/L,再生率分别为12.8%和3.3%。
2.以“佳丽酿”试管苗的花药、子房、花蕾为材料,研究了不同培养基及激素浓度对花器愈伤组织诱导分化的影响。结果表明:诱导欧洲葡萄“佳丽酿”子房的最佳培养基配方为:B5+ BAP 0.5mg/L+2,4-D 0.5mg/L +蔗糖30 g/L +琼脂0.65%+ CH 200 mg/L + pvp500 mg/L,诱导欧洲葡萄“佳丽酿”花蕾的最佳培养基配方为:B5+BAP 4mg/L+2,4-D 0.5mg/L+蔗糖30 g/L +琼脂0.65%+ CH 200 mg/L + pvp500 mg/L,诱导率分别为10%和43%。
3.以华东葡萄“白河-35-1”试管苗的叶片为材料,研究了几种不同试剂处理对诱导愈伤过程中褐化现象影响的研究。结果表明:在培养基中添加100mg/L抗坏血酸,防褐化效果最好。
4.以华东葡萄“白河-35-1”单芽茎段为试材,研究了卡那霉素对葡萄不定芽再生及植株生长的影响,确定了卡那霉素的筛选方式和筛选浓度。结果表明:在葡萄遗传转化后,采用延迟筛选,起始浓度为5mg/L逐步增加到16mg/L,可减少对再生的抑制。
5.采用农杆菌介导法,侵染“白河-35-1”单芽茎段,经卡那霉素筛选,获得11个抗性芽。
The objective of this study was to research in regeneration systems of Vitis pseudoreticulata clone‘Baihe-35-1’in Organogenesis, investigating the effect of auxins and explants on callus induced from flower organ of Vitis viniferal L.‘carignane’, grape transformation by Agrobacterium infection introducing the novel Glyoxal Oxidase related gene(GLOXrg) which was carried in the EHA105 binary vector into Baihe-35-1’. The main results are as follow:
(1) Regeneration system from leaf and stem of Vitis pseudoreticulata clone‘Baihe-35-1’using TDZ and NAA was Studied. The results were: Regeneration from stem was easier than from leaf. The combination 1mg/L TDZ with 0.5 mg/L NAA resulted in the highest frequency of adventitious bud production from leaf and stem which the rate is 3.3% and 12.8%.
(2) Callus induction from flower organ of‘carignane’using BAP and 2,4-D was studied.The results were: Callus induction from bud is easier than from ovary. The combination 0.5 mg/L BAP with 0.5 mg/L 2,4-D resulted in the highest frequency of callus induction from ovary which the rate is 10%. And when 4mg/L BAP combinates with 0.5 mg/L 2,4-D, callus induction reaches its highest frequency which is 43%.
(3) Effects of different additaments on anti-browning were studied which leaf of Vitis pseudoreticulata clone‘Baihe-35-1’was taken for test material. The result showed that Vc could effectively prevent the leaf of‘Baihe-35-1’from browning. The optimal concentration of Vc is 100mg/L.
(4) Effect of Kan on bud regeneration from stem with bud of Vitis pseudoreticulata clone‘Baihe-35-1’was studied. Retarding selection was better for transformation of grape. It can reduced restraining of Kan on grape that adding Kan firstly with 4mg/L and gradually increasing to 16mg/L.
(5) 11 resistant-Kan buds of Vitis pseudoreticulata clone‘Baihe-35-1’were obtained after selection of Kan.
引文
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