农杆菌介导的草坪草遗传转化研究
摘要
本实验以高羊茅和多年生黑麦草成熟种子为材料，研究了影响其植株再生的若干因素，建立了农杆菌介导的遗传转化体系。获得的主要研究结果如下：
1 不同生长激素种类和浓度的诱导培养基，对多年生黑麦草和高羊茅的组织培养产生不同影响，结果发现，dicamba作为生长激素的效果要好于2，4-D；以2，4-D为生长激素时，在9 mg l~(-l)的浓度范围内，随着2，4-D浓度的增加，植株再生率呈上升的趋势：在5 mg l~(-1)dicamba和5 mg l~(-1)2，4-D的处理中加入1 mg l~(-1) NAA对植株再生率的影响不大。
2 低浓度的6-BA(0.1mg l~(-1))可以提高高羊茅的植株再生率，而高浓度的6-BA显著抑制愈伤组织的产生，而使高羊茅植株的再生率降低。
3 在诱导培养基中加入2mg l~(-1)ABA显著抑制芽的生长和愈伤组织的产生，对高羊茅的植株再生产生负面影响。
4 比较了三种基本培养基有机营养成分对高羊茅组织培养的影响，结果发现，B5有机营养成分效果最佳，M8有机营养成分次之，而MS有机营养成分效果最差。
5 低浓度的脯氨酸、谷氨酰胺和水解酪蛋白(100 mg l~(-1))有利于提高高羊茅的植株再生率，而高浓度则起反作用。
6 在多年生黑麦草的组织培养中发现，蔗糖代替麦芽糖作为碳源，有利于提高多年生黑麦草的植株再生率。
7 在遗传转化中发现，在分化培养基中加入50 mg l~(-1)的潮霉素，可以提高对转化细胞的筛选效率。
8 建立了多年生黑麦草和高羊茅的遗传转化体系，通过这套体系获得了46株多年生黑麦草和7株高羊茅的再生植株，其中6株多年生黑麦草和1株高羊茅为PCR阳性。
In the present study, some factors influencing plant regeneration from mature seed in tall fescue and perennial ryegrass were evaluated. A system for Agrobacterium tumefaciens- mediated genetic transformation of Festuca arundinacea and Lolium perenne was established. The primary results could be summarized as follows.-
1. Wide differences were observed among the different auxins and concentrations in callus induction medium in relation to tissue culture of Festuca arundinacea and Lolium perenne. Dicamba was superior to 2, 4-D. From 0 to 9mg/l, the stepwise increase of the 2,4-D concentration in maltose containing callus induction medium obviously increased overall plant regeneration frequency. The supplement of 1 mg l-1 NAA in both the callus induction medium with 5mg l-12, 4-D and that with 5mg l-1 dicamba didn't improve plant regeneration frequency.
2. inclusion of a low concentration of 6-BA(0.1 mg l-1) in the callus induction medium were effective for plant regeneration of Festuca arundinacea. However, higher concentrations of 6-BA obviously inhibited callus formation and exhibited lower regeneration rate.
3. 2 mg r'ABA was tested in the callus induction medium. The results showed that the growth of shoot was suppressed and less calli were observed. There was a decrease in plant regeneration frequency of Festuca arundinacea.
4. Among MS, B5 and M8 vitamins, the B5 vitamins were the most efficient for callus formation and plant regeneration of Festuca arundinacea and the MS vitamins were least.
5. Proline, glutamine and casein hydrolysate at a low concentration(100mg l-1) improved plant regeneration of Festuca arundinacea. However, higher concentrations proved to be detrimental rather than beneficial.
6. The replacements of maltose by sucrose in the callus induction medium significantly improved overall plant regeneration frequency of Lolium perenne.
7. 50mg l-1 hygromycin supplemented in the callus differentiation medium resulted in
higher selection frequency.
8. An Agrobacterium fa/we/ac/ens-mediated genetic transformation system for Festuca arundinacea and Lolium perenne was established. 46 Lolium perenne and 7 Festuca arundinacea plants were regenerated under selection. PCR analysis showed that 6 Lolium perenne and 1 Festuca arundinacea PCR-positive plants were obtained.
1 不同生长激素种类和浓度的诱导培养基，对多年生黑麦草和高羊茅的组织培养产生不同影响，结果发现，dicamba作为生长激素的效果要好于2，4-D；以2，4-D为生长激素时，在9 mg l~(-l)的浓度范围内，随着2，4-D浓度的增加，植株再生率呈上升的趋势：在5 mg l~(-1)dicamba和5 mg l~(-1)2，4-D的处理中加入1 mg l~(-1) NAA对植株再生率的影响不大。
2 低浓度的6-BA(0.1mg l~(-1))可以提高高羊茅的植株再生率，而高浓度的6-BA显著抑制愈伤组织的产生，而使高羊茅植株的再生率降低。
3 在诱导培养基中加入2mg l~(-1)ABA显著抑制芽的生长和愈伤组织的产生，对高羊茅的植株再生产生负面影响。
4 比较了三种基本培养基有机营养成分对高羊茅组织培养的影响，结果发现，B5有机营养成分效果最佳，M8有机营养成分次之，而MS有机营养成分效果最差。
5 低浓度的脯氨酸、谷氨酰胺和水解酪蛋白(100 mg l~(-1))有利于提高高羊茅的植株再生率，而高浓度则起反作用。
6 在多年生黑麦草的组织培养中发现，蔗糖代替麦芽糖作为碳源，有利于提高多年生黑麦草的植株再生率。
7 在遗传转化中发现，在分化培养基中加入50 mg l~(-1)的潮霉素，可以提高对转化细胞的筛选效率。
8 建立了多年生黑麦草和高羊茅的遗传转化体系，通过这套体系获得了46株多年生黑麦草和7株高羊茅的再生植株，其中6株多年生黑麦草和1株高羊茅为PCR阳性。
In the present study, some factors influencing plant regeneration from mature seed in tall fescue and perennial ryegrass were evaluated. A system for Agrobacterium tumefaciens- mediated genetic transformation of Festuca arundinacea and Lolium perenne was established. The primary results could be summarized as follows.-
1. Wide differences were observed among the different auxins and concentrations in callus induction medium in relation to tissue culture of Festuca arundinacea and Lolium perenne. Dicamba was superior to 2, 4-D. From 0 to 9mg/l, the stepwise increase of the 2,4-D concentration in maltose containing callus induction medium obviously increased overall plant regeneration frequency. The supplement of 1 mg l-1 NAA in both the callus induction medium with 5mg l-12, 4-D and that with 5mg l-1 dicamba didn't improve plant regeneration frequency.
2. inclusion of a low concentration of 6-BA(0.1 mg l-1) in the callus induction medium were effective for plant regeneration of Festuca arundinacea. However, higher concentrations of 6-BA obviously inhibited callus formation and exhibited lower regeneration rate.
3. 2 mg r'ABA was tested in the callus induction medium. The results showed that the growth of shoot was suppressed and less calli were observed. There was a decrease in plant regeneration frequency of Festuca arundinacea.
4. Among MS, B5 and M8 vitamins, the B5 vitamins were the most efficient for callus formation and plant regeneration of Festuca arundinacea and the MS vitamins were least.
5. Proline, glutamine and casein hydrolysate at a low concentration(100mg l-1) improved plant regeneration of Festuca arundinacea. However, higher concentrations proved to be detrimental rather than beneficial.
6. The replacements of maltose by sucrose in the callus induction medium significantly improved overall plant regeneration frequency of Lolium perenne.
7. 50mg l-1 hygromycin supplemented in the callus differentiation medium resulted in
higher selection frequency.
8. An Agrobacterium fa/we/ac/ens-mediated genetic transformation system for Festuca arundinacea and Lolium perenne was established. 46 Lolium perenne and 7 Festuca arundinacea plants were regenerated under selection. PCR analysis showed that 6 Lolium perenne and 1 Festuca arundinacea PCR-positive plants were obtained.
引文
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