两个转ipt-bar双价基因水稻农艺性状分析及外源基因清除系统（Gene-deletor）导入水稻恢复系的研究
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摘要
本研究对转ipt-bar双价基因EY105-T19水稻株系和R527-T1水稻株系在封闭条件下,进行田间比较试验,同时,以籼稻恢复系品种R527和绵恢047为材料,将外源基因清除系统表达载体,通过农杆菌介导法进行了遗传转化研究。获得结果如下：
1.以田间条件下转ipt-bar双价基因EY105-T19株系T6代和转ipt-bar双价基因R527-T1株系T3代群体为材料,研究了它们的主要农艺性状,结果表明,田间条件下,转ipt-bar双价基因EY105-T19植株的株高显著高于对照,转ipt-bar双价基因R527-T1植株的株高显著低于对照。两个稳定的转基因株系与对照相比,在穗长、有效穗数、总穗数、结实率和千粒重等农艺性状上差别不显著。随机区组比较试验证明,转ipt-bar双价基因EY105-T19产量与非转基因品种相比提高了6.95%,达到显著水平,而转ipt-bar双价基因R527-T1产量与其非转基因对照无显著差异。
2.以水稻成熟种子为外植体诱导愈伤组织,发现去除75%的胚乳有效降低愈伤组织诱导的初期污染,使供试材料R527污染率降低了4.4%,绵恢047污染率降低了6.6%。采用浓度为2.0mg·L-1和3.0mg·L的2,4-D可使R527和绵恢047愈伤组织的诱导效果达到最佳。在组织分化不定芽过程中,对愈伤组织采用吸水纸脱水处理2-3天能有效加速不定芽分化并提高不定芽分化率。在继代培养基中添加15g·L-1的山梨醇明显促进胚性愈伤组织形成。获得了绵恢047再生苗53株。以农杆菌介导法将外源基因清除系统遗传转化R527,发现以农杆菌液浓度OD值为0.4侵染愈伤组织,在共培养基上添加酚类化合物乙酰丁香酮(AS)100μmol·L-1效果较好。在获得的34株再生植株中,经GUS检测获得2株阳性植株。
In this paper, we reported the research results of agronomic traits of ipt-bar EY105-T19 and ipt-bar R527-T1 transgenic rice lines and the Gene-deletor introduced into two indica restorers R527 by Agrobacterium-mediated transformation. The main results are as follows:
1. After having analysed the main agronomic traits of two transgenic rice lines Japanic ipt-bar EY105-T19 T6 generation and Indica restorer ipt-bar R527-T1 T3 generation in the fields, we found that the plant height of the ipt-bar EY105-T19 is higher than the wild type, while that of ipt-bar R527-T1 is lower; the production of ipt-bar EY105-T19 is remarkably increased by 6.95% but that of ipt-bar R527-T1 is not.
2. We found that contamination was decreased as long as 75% rice endosperm was removed during callus inducement. The contamination rate of R527 declined 4.4% while that of other restorer Mianhui047 was 6.6%. The best medium for calli induction of R527 and Mianhui047 was N6 medium added with 2.0mg·L-1 and 3.0m g·L-1 2,4-D respectively. The rate of callus differentiation was increased after using the absorbent paper for callus dehydration by 2-3 days. The callus growning on subculture medium adding with 15 g·L-1 yamanashi alcohol tended to turn to embryogenic callus. As a result, we obtained 53 Mianhui047 regenerated plants. This study results of the Gene-deletor genetic transformation for R527 by Agrobacterium-mediated method showed transformation efficiecy was improved when the callus was immersed for 15min in the Agrobacterium suspension (OD600=0.4) with 100μmol·L-1 acetosyringone (AS). The GUS assay result indicated the Gene-deletor was integrated into rice genomes of 2 plants selected from 34 R527 regenerated plants.
1.以田间条件下转ipt-bar双价基因EY105-T19株系T6代和转ipt-bar双价基因R527-T1株系T3代群体为材料,研究了它们的主要农艺性状,结果表明,田间条件下,转ipt-bar双价基因EY105-T19植株的株高显著高于对照,转ipt-bar双价基因R527-T1植株的株高显著低于对照。两个稳定的转基因株系与对照相比,在穗长、有效穗数、总穗数、结实率和千粒重等农艺性状上差别不显著。随机区组比较试验证明,转ipt-bar双价基因EY105-T19产量与非转基因品种相比提高了6.95%,达到显著水平,而转ipt-bar双价基因R527-T1产量与其非转基因对照无显著差异。
2.以水稻成熟种子为外植体诱导愈伤组织,发现去除75%的胚乳有效降低愈伤组织诱导的初期污染,使供试材料R527污染率降低了4.4%,绵恢047污染率降低了6.6%。采用浓度为2.0mg·L-1和3.0mg·L的2,4-D可使R527和绵恢047愈伤组织的诱导效果达到最佳。在组织分化不定芽过程中,对愈伤组织采用吸水纸脱水处理2-3天能有效加速不定芽分化并提高不定芽分化率。在继代培养基中添加15g·L-1的山梨醇明显促进胚性愈伤组织形成。获得了绵恢047再生苗53株。以农杆菌介导法将外源基因清除系统遗传转化R527,发现以农杆菌液浓度OD值为0.4侵染愈伤组织,在共培养基上添加酚类化合物乙酰丁香酮(AS)100μmol·L-1效果较好。在获得的34株再生植株中,经GUS检测获得2株阳性植株。
In this paper, we reported the research results of agronomic traits of ipt-bar EY105-T19 and ipt-bar R527-T1 transgenic rice lines and the Gene-deletor introduced into two indica restorers R527 by Agrobacterium-mediated transformation. The main results are as follows:
1. After having analysed the main agronomic traits of two transgenic rice lines Japanic ipt-bar EY105-T19 T6 generation and Indica restorer ipt-bar R527-T1 T3 generation in the fields, we found that the plant height of the ipt-bar EY105-T19 is higher than the wild type, while that of ipt-bar R527-T1 is lower; the production of ipt-bar EY105-T19 is remarkably increased by 6.95% but that of ipt-bar R527-T1 is not.
2. We found that contamination was decreased as long as 75% rice endosperm was removed during callus inducement. The contamination rate of R527 declined 4.4% while that of other restorer Mianhui047 was 6.6%. The best medium for calli induction of R527 and Mianhui047 was N6 medium added with 2.0mg·L-1 and 3.0m g·L-1 2,4-D respectively. The rate of callus differentiation was increased after using the absorbent paper for callus dehydration by 2-3 days. The callus growning on subculture medium adding with 15 g·L-1 yamanashi alcohol tended to turn to embryogenic callus. As a result, we obtained 53 Mianhui047 regenerated plants. This study results of the Gene-deletor genetic transformation for R527 by Agrobacterium-mediated method showed transformation efficiecy was improved when the callus was immersed for 15min in the Agrobacterium suspension (OD600=0.4) with 100μmol·L-1 acetosyringone (AS). The GUS assay result indicated the Gene-deletor was integrated into rice genomes of 2 plants selected from 34 R527 regenerated plants.
引文
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