黄瓜离体再生和遗传转化体系的建立
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摘要
黄瓜是国内外广泛种植的一类重要蔬菜作物,在蔬菜周年化供应中具有重要作用,实际生产栽培中常因各种病虫的危害造成巨大的经济损失。由于传统病虫害防治方法的种种不利因素,因此,选育抗性品种从根本上提高黄瓜抗病虫能力,是防治的最佳途径。但是黄瓜种内抗性材料缺乏,传统育种很难获得突破。本研究以不同材料的黄瓜子叶、子叶节作外植体,研究了基因型、激素对黄瓜离体分化的影响,建立了高效的离体再生体系;同时研究了AS、侵染时间、PH值对黄瓜遗传转化的影响,优化了转化体系;在此基础之上,通过根癌农杆菌介导法,将GUS基因导入黄瓜,对转化体系进行了初步探索,并进一步将抗线虫基因MiMPK1导入黄瓜,为黄瓜遗传转化的相关研究提供理论依据。
主要研究结果如下:
1.基因型是植物离体再生能力高低的重要影响因素之一,不同基因型之间的再生能力差异很大。黄瓜子叶再生体系建立所用的6份材料(中农8号、吉林旱瓜选系、四川白瓜选系、Gy14、65G、9110Gt)中,华南类型材料吉林旱瓜的再生能力最强,在培养基MS+1.5 mg/L 6-BA+0.5 mg/L ABA+2 mg/L AgNO_3上,再生频率和每外植体再生芽数分别为96.7 %和5.2;黄瓜子叶节再生体系的建立所用的8份材料(吉林旱瓜选系、四川白瓜选系、0513、228、Gy14、65G、185、K2148)之中,华南类型材料四川白瓜的再生能力最强,在培养基MS+0.5 mg/L 6-BA+2 mg/L AgNO_3上,再生频率和每外植体再生芽数分别为100 %和6.4。
2.植物激素是植物离体再生所必需,6-BA是黄瓜离体分化中最常用的植物激素之一。对于黄瓜子叶外植体的分化而言,6-BA是不定芽发生的主导因素,而ABA的添加能显著提高不定芽数目。美国加工类型材料Gy14和欧洲华北混合类型材料9110Gt的最佳诱芽培养基为MS+1.0 mg/L 6-BA+1.0 mg/L ABA+2 mg/L AgNO_3;华南类型材料吉林旱瓜和四川白瓜的最佳诱芽培养基为MS+1.5 mg/L 6-BA+0.5 mg/L ABA+2 mg/L AgNO_3;欧洲温室类型材料65G的最佳诱芽培养基为MS+1.5 mg/L 6-BA+1.0 mg/L ABA+2 mg/L AgNO_3;华北类型中农8号最佳诱芽培养基为MS+2.0 mg/L 6-BA+0.5 mg/L ABA+2 mg/L AgNO_3。对于黄瓜子叶节外植体的分化而言,培养基中仅附加6-BA即可得到很好的再生效果,不同材料对激素的敏感程度不同。材料228、185、65G的最佳诱导培养基为MS+2.0 mg/L 6-BA+2 mg/L AgNO_3;材料四川白瓜、吉林旱瓜、K2148、0513的最佳幼芽培养基为MS+0.5 mg/L 6-BA+2 mg/L AgNO_3;GY14的最佳幼芽培养基为MS+1.0 mg/L 6-BA+2 mg/L AgNO_3。
3.在黄瓜再生体系建立的基础之上,研究了子叶节对Km和Hyg的耐受性和敏感度,确定了含不同抗性基因载体的筛选剂浓度,Km的选择压为75 mg/L,Hyg的选择压浓度为10 mg/L。
4.黄瓜遗传转化体系优化的结果表明,在预培养基、菌液、共培养基中添加AS能显著提高抗性芽数量,最佳浓度分别为250 ul/L、500 ul/L、250 ul/L;侵染时间为15 min时,外植体再生频率和抗性芽数最高;选择培养基pH较低时有利于外植体分化,以PH=5.40最佳。
5.采用根癌农杆菌介导法将GUS基因转化黄瓜,对转化体系作初步探索。得到了Km抗性植株,取幼嫩叶片,经GUS组织化学染色,共有14株显示蓝色,表明GUS基因已整合到黄瓜基因组,并实现表达,初步统计阳性率为7.4 %。
6.在遗传转化体系的优化和探索的基础之上,将抗线虫基因MiMPK1导入黄瓜,筛选获得了Hyg抗性植株。提取抗性植株的基因组DNA,经PCR检测,共有28株扩增出500 bp左右的特异条带,初步统计阳性率为5.3 %,为黄瓜的抗线虫病育种提供了理论依据。
Cucumber (Cumcumis sativus L.) is one of the important vegetable crops widely cultivated around the world. It plays a very significant role to the annual supply of vegetables. But cucumber often suffers from a number of diseases and pests, and has caused huge economic losses in actual cultivation and production. Because the traditional methods of insect prevention and control have many disadvantageous factors, selection and breeding resistant variety is the best way to improve the resistance to disease and pect of cucumber. However, conventional breeding is difficult to obtain breakthrough for the lack of resistant materials. In this study, the effects of genotype and phytohormone on regeneration in vitro were studied by using different cucumber materials cotyledon and cotyledon node as explants, a highly efficient regeneration was established. The effects of AS, infection time, PH value on cucumber genetic transformation was studied as well. Then, the GUS gene and MiMPK1 gene were transferred into cucumber, which medied by Agrobacterium tumefaciens. It provided theoretical basis for the study of cucumber genetic transformation.
The main results were as follow:
1. Genotype was one of the influence factors on regeneration. The regeneration capacity of different genotypes explants was very significantly. In the study of cucumber cotyledon regeneration, Jilinhangua had the highest differentiation ability among the six materials (Zhongnong No.8, Jilinhangua, Sichuanbaigua, Gy14, 65G, 9110Gt). Its regeneration frequency and shoots per explants were 96.7% and 5.2 in the medium MS+1.5mg/L6-BA+0.5mg/LABA+2mg/LAgNO_3. In the study of cucumber cotyledon node regeneration, Sichuanbaigua had the highest differentiation ability among the eight materials (Jilinhangua, Sichuanbaigua, Gy14, 65G, 0513, 228, 185, K2148), its regeneration frequency and shoots per explant were 100 % and 6.4 in the medium (MS+0.5mg/L6-BA+2mg/LAgNO_3).
2. Plant hormone is necessary for explants regeneration. 6-BA is one of the most common hormones in cucumber differentiation in vitro . In the study of cucumber cotyledon regeneration, 6-BA was the dominant factor of adventitious buds regeneration, but the addition of ABA significantly increased the number of adventitious buds. The best inducing medium of shoots for Gy14 and 9110Gt was MS+1.0mg/L6-BA+1.0mg/LABA+2mg/LAgNO_3, the best inducing medium for Jinlinhangua and Sichuanbaigua was MS+1.5mg/L6-BA+0.5mg/LABA+2mg/LAgNO_3, the best inducing medium for 65G was MS+1.5mg/L6-BA+1.0mg/LABA+2mg/LAgNO_3, and the best inducing medium for Zhongnong No.8 was MS+2.0mg/L6-BA+0.5mg/LABA+2mg/LAgNO_3. In the study of cucumber cotyledon node regeneration, The best inducing medium of shoots for 228,185 and 65G was MS+2.0 mg/L 6-BA+2 mg/L AgNO_3, The best inducing medium for Sichuanbaigua, Jilinhangua, K2148 and 0513 was MS+0.5mg/L6-BA+2mg/LAgNO_3, and in the medium MS+1.0 mg/L 6-BA+2 mg/L AgNO_3, Gy14 had the best regeneration frequency.
3. The tolerance and sensitivity of cucumber cotyledon to Km and Hyg was studied. The selection pressure concentration of Km and Hyg were 75 mg/L and 10 mg/L.
4. The results of transgenic system improvement were that the number of resistant shoots significantly increased, while AS added to the pre-culture medium, bacteria liquid and co-culture medium, the best concentration was 250 ul/L, 500 ul/L and 250 ul/L. The regeneration frequency and resistant shoots per explants were highest when the infection time was 15 min. The lower PH value selective medium promoted explants differentiation while it was 5.4.
5. The GUS gene was transferred into cucumber medied by Agrobacterium tumefaciens. There were 14 resistant plantlets showed blue via gus histochemical identification. This indicated that GUS gene had been integrated into the cucumber genome, and expressed in tissues, the preliminary positive rate was 7.4 %.
6. The MiMPK1 gene was transferred into cucumber medied by Agrobacterium tumefaciens. PCR analysis showed that a total of 28 resistant plantlets amplified were specific bands about 500 bp, the the preliminary positive rate was 5.3 %.
主要研究结果如下:
1.基因型是植物离体再生能力高低的重要影响因素之一,不同基因型之间的再生能力差异很大。黄瓜子叶再生体系建立所用的6份材料(中农8号、吉林旱瓜选系、四川白瓜选系、Gy14、65G、9110Gt)中,华南类型材料吉林旱瓜的再生能力最强,在培养基MS+1.5 mg/L 6-BA+0.5 mg/L ABA+2 mg/L AgNO_3上,再生频率和每外植体再生芽数分别为96.7 %和5.2;黄瓜子叶节再生体系的建立所用的8份材料(吉林旱瓜选系、四川白瓜选系、0513、228、Gy14、65G、185、K2148)之中,华南类型材料四川白瓜的再生能力最强,在培养基MS+0.5 mg/L 6-BA+2 mg/L AgNO_3上,再生频率和每外植体再生芽数分别为100 %和6.4。
2.植物激素是植物离体再生所必需,6-BA是黄瓜离体分化中最常用的植物激素之一。对于黄瓜子叶外植体的分化而言,6-BA是不定芽发生的主导因素,而ABA的添加能显著提高不定芽数目。美国加工类型材料Gy14和欧洲华北混合类型材料9110Gt的最佳诱芽培养基为MS+1.0 mg/L 6-BA+1.0 mg/L ABA+2 mg/L AgNO_3;华南类型材料吉林旱瓜和四川白瓜的最佳诱芽培养基为MS+1.5 mg/L 6-BA+0.5 mg/L ABA+2 mg/L AgNO_3;欧洲温室类型材料65G的最佳诱芽培养基为MS+1.5 mg/L 6-BA+1.0 mg/L ABA+2 mg/L AgNO_3;华北类型中农8号最佳诱芽培养基为MS+2.0 mg/L 6-BA+0.5 mg/L ABA+2 mg/L AgNO_3。对于黄瓜子叶节外植体的分化而言,培养基中仅附加6-BA即可得到很好的再生效果,不同材料对激素的敏感程度不同。材料228、185、65G的最佳诱导培养基为MS+2.0 mg/L 6-BA+2 mg/L AgNO_3;材料四川白瓜、吉林旱瓜、K2148、0513的最佳幼芽培养基为MS+0.5 mg/L 6-BA+2 mg/L AgNO_3;GY14的最佳幼芽培养基为MS+1.0 mg/L 6-BA+2 mg/L AgNO_3。
3.在黄瓜再生体系建立的基础之上,研究了子叶节对Km和Hyg的耐受性和敏感度,确定了含不同抗性基因载体的筛选剂浓度,Km的选择压为75 mg/L,Hyg的选择压浓度为10 mg/L。
4.黄瓜遗传转化体系优化的结果表明,在预培养基、菌液、共培养基中添加AS能显著提高抗性芽数量,最佳浓度分别为250 ul/L、500 ul/L、250 ul/L;侵染时间为15 min时,外植体再生频率和抗性芽数最高;选择培养基pH较低时有利于外植体分化,以PH=5.40最佳。
5.采用根癌农杆菌介导法将GUS基因转化黄瓜,对转化体系作初步探索。得到了Km抗性植株,取幼嫩叶片,经GUS组织化学染色,共有14株显示蓝色,表明GUS基因已整合到黄瓜基因组,并实现表达,初步统计阳性率为7.4 %。
6.在遗传转化体系的优化和探索的基础之上,将抗线虫基因MiMPK1导入黄瓜,筛选获得了Hyg抗性植株。提取抗性植株的基因组DNA,经PCR检测,共有28株扩增出500 bp左右的特异条带,初步统计阳性率为5.3 %,为黄瓜的抗线虫病育种提供了理论依据。
Cucumber (Cumcumis sativus L.) is one of the important vegetable crops widely cultivated around the world. It plays a very significant role to the annual supply of vegetables. But cucumber often suffers from a number of diseases and pests, and has caused huge economic losses in actual cultivation and production. Because the traditional methods of insect prevention and control have many disadvantageous factors, selection and breeding resistant variety is the best way to improve the resistance to disease and pect of cucumber. However, conventional breeding is difficult to obtain breakthrough for the lack of resistant materials. In this study, the effects of genotype and phytohormone on regeneration in vitro were studied by using different cucumber materials cotyledon and cotyledon node as explants, a highly efficient regeneration was established. The effects of AS, infection time, PH value on cucumber genetic transformation was studied as well. Then, the GUS gene and MiMPK1 gene were transferred into cucumber, which medied by Agrobacterium tumefaciens. It provided theoretical basis for the study of cucumber genetic transformation.
The main results were as follow:
1. Genotype was one of the influence factors on regeneration. The regeneration capacity of different genotypes explants was very significantly. In the study of cucumber cotyledon regeneration, Jilinhangua had the highest differentiation ability among the six materials (Zhongnong No.8, Jilinhangua, Sichuanbaigua, Gy14, 65G, 9110Gt). Its regeneration frequency and shoots per explants were 96.7% and 5.2 in the medium MS+1.5mg/L6-BA+0.5mg/LABA+2mg/LAgNO_3. In the study of cucumber cotyledon node regeneration, Sichuanbaigua had the highest differentiation ability among the eight materials (Jilinhangua, Sichuanbaigua, Gy14, 65G, 0513, 228, 185, K2148), its regeneration frequency and shoots per explant were 100 % and 6.4 in the medium (MS+0.5mg/L6-BA+2mg/LAgNO_3).
2. Plant hormone is necessary for explants regeneration. 6-BA is one of the most common hormones in cucumber differentiation in vitro . In the study of cucumber cotyledon regeneration, 6-BA was the dominant factor of adventitious buds regeneration, but the addition of ABA significantly increased the number of adventitious buds. The best inducing medium of shoots for Gy14 and 9110Gt was MS+1.0mg/L6-BA+1.0mg/LABA+2mg/LAgNO_3, the best inducing medium for Jinlinhangua and Sichuanbaigua was MS+1.5mg/L6-BA+0.5mg/LABA+2mg/LAgNO_3, the best inducing medium for 65G was MS+1.5mg/L6-BA+1.0mg/LABA+2mg/LAgNO_3, and the best inducing medium for Zhongnong No.8 was MS+2.0mg/L6-BA+0.5mg/LABA+2mg/LAgNO_3. In the study of cucumber cotyledon node regeneration, The best inducing medium of shoots for 228,185 and 65G was MS+2.0 mg/L 6-BA+2 mg/L AgNO_3, The best inducing medium for Sichuanbaigua, Jilinhangua, K2148 and 0513 was MS+0.5mg/L6-BA+2mg/LAgNO_3, and in the medium MS+1.0 mg/L 6-BA+2 mg/L AgNO_3, Gy14 had the best regeneration frequency.
3. The tolerance and sensitivity of cucumber cotyledon to Km and Hyg was studied. The selection pressure concentration of Km and Hyg were 75 mg/L and 10 mg/L.
4. The results of transgenic system improvement were that the number of resistant shoots significantly increased, while AS added to the pre-culture medium, bacteria liquid and co-culture medium, the best concentration was 250 ul/L, 500 ul/L and 250 ul/L. The regeneration frequency and resistant shoots per explants were highest when the infection time was 15 min. The lower PH value selective medium promoted explants differentiation while it was 5.4.
5. The GUS gene was transferred into cucumber medied by Agrobacterium tumefaciens. There were 14 resistant plantlets showed blue via gus histochemical identification. This indicated that GUS gene had been integrated into the cucumber genome, and expressed in tissues, the preliminary positive rate was 7.4 %.
6. The MiMPK1 gene was transferred into cucumber medied by Agrobacterium tumefaciens. PCR analysis showed that a total of 28 resistant plantlets amplified were specific bands about 500 bp, the the preliminary positive rate was 5.3 %.
引文
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