甘薯体细胞胚胎再生及遗传转化的初步研究
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摘要
甘薯(Ipomoea batatas Lam.)是一种重要的粮食和经济作物，其块根和茎叶所含营养均十分丰富，用途极广，既可以作为人类的粮食、食品和蔬菜，又能作为工业原料和饲料。甘薯遗传上的高度杂合性和多倍性，种间、种内广泛存在的杂交不亲和性和低结实率。严重制约了甘薯生产发展，培育高产、优质、抗逆性强、无病毒、耐储藏的优良甘薯品种，仍是育种工作者的首要任务。常规育种技术在甘薯品种改良方面有其局限性，遗传转化技术在提高甘薯的产量、品质和病虫害抗性方面有很大潜力。
建立一个有效的再生体系，是遗传转化成功的必备条件。与其它植物相比，甘薯是一种再生比较困难的植物。甘薯的再生频率基因型间差异很大，尚未建立一个基因型适用性较广的植株再生系统。甘薯有1100多个品种，目前被研究过的不过数十种，报道有较高植株再生频率并成功进行遗传转化的甘薯材料仅十余种，如“Jewel”、“White Star”、“栗子香”、“高系14”等。绝大多数甘薯主栽品种的再生频率很低。因而，虽然甘薯的基因工程研究取得了一定的进展，但甘薯至今没有一例转基因植株进入商品化的应用。
向甘薯基因组中导入外源基因的方法有多种，其中农杆菌介导法最为有效，转入的外源基因多以单拷贝插入，便于育种工作的利用。川薯34，是西南地区的一个优良主栽品种。本试验建立了川薯34等品种较高频率的体细胞胚胎再生体系。在此基础上，利用GUS-intron融合基因的瞬时表达，研究了川薯34有效的遗传转化条件，为进一步通过基因工程改良其品质奠定了良好的基础。主要实验结果如下：
1．通过对不同植物激素组合及甘薯不同再生途径的比较试验，建立了3个甘薯基因型较高频率的体细胞胚胎再生体系，其中川薯34、川薯8129-4、徐薯18的最高植株再生率分别达到了49.9％、29.7％、14.7％。
2．从川薯34的体胚苗中，筛选出了L22、L8两个高频体胚发生材料，可以为遗传转化工作提供良好的受体。
3．通过GUS-intron融合基因的瞬时表达，确定了川薯34有效的遗传转化条件。优化后的遗传转化条件为：用O.D.值为0.5的农杆菌LBA4404／pIG121菌液侵染川薯34的叶片、叶柄60min，侵染时用超声波辅助处理受体材料1min，在侵染液和共培养培养基中都附
西南农业天学硕下学位论又
摘要
加200腼ol/LAS，黑暗下共培养3d。采用此转化条件GUS报告基因瞬时转化频率可达30%
左右，井可以进一步稳定表达。
Sweet potato is an important food and economic crop. There is abundant nutrition in their storage root and leaf. It is widely used for man's food and materials of industry. The nucleus type of sweet potato is homogenous hexaploid. The chromosome background is rather complex. The self-crossing is sterile. Crossing in specific and interspecific crossing are non-compatible or leading to low seed ratio in sweet potato. Conventional breeding technology has localization in variety improvement while transgenic technology has great potential to increase the production,
improve quality and resist plant diseases and pests.
An efficient regeneration system is very important for successful transformation. Comparing
with other plants, regeneration of sweet potato is very difficult. The difference of regeneration frequency is great in different genotypes. There are about 1100 varieties while only ten of them have been studied. Several varieties have been reported for high regeneration frequency and been successful transformed, such as Jewel, White Star, Lizixiang, Gaoxi14 and so on. Most of popular varieties have such a low regeneration frequency that there is not one transgenic plant that has been commercially used though the genetic engineering have made much progress.
There are several methods to introduce foreign gene into the genome of sweet potato, but only the Agrobacterium-mediated transformation is most effective. The foreign gene is transformed in single copy by this means and so convenient for breeding. Chuanshu34 is an excellent variety in southwest area. This experiment established high frequency regeneration system of Chuanshu34, Chuanshu8129-4 and Xushu18 via somatic embryogenesis, optimized the transformation conditions of Chuanshu34 by transient expression of GUS-intron gene. These work founded the base for next step to improve quality of sweet potato by gene engineering technology.
The primary results are listed as following:
1.Established 3 high frequency somatic embryo regeneration systems of different genotype by testing different plant hormones. The regeneration frequency of Chuanshu34, Chuanshu8129-4, Xushu18 was 49.9%, 29.7%, 14.7% respectively.
2.L22 and L8 with high frequent embryogenesis, the two lines were chosen from plantlets of Chuanshu34. Explants from them were good for genetic transformation.
3. Optimized the transformation factors through GUS-intron transient expression. The most effective transformation factors were detailed as below:
Inoculation bacterium was O.D.600=0.5. The explant was leaf or petiole of Chuanshu34, inoculation time was 60 min, and the explant was sonicated for 1 min to increase the tansformation frequency. Inoculation bacterium and co-cultivated medium were added 200umol/LAS. Co-culture took place in dark for 3 days. Transient expression frequency detected by reporter gene was 30% or so and transgene can steadily express in next step.
建立一个有效的再生体系，是遗传转化成功的必备条件。与其它植物相比，甘薯是一种再生比较困难的植物。甘薯的再生频率基因型间差异很大，尚未建立一个基因型适用性较广的植株再生系统。甘薯有1100多个品种，目前被研究过的不过数十种，报道有较高植株再生频率并成功进行遗传转化的甘薯材料仅十余种，如“Jewel”、“White Star”、“栗子香”、“高系14”等。绝大多数甘薯主栽品种的再生频率很低。因而，虽然甘薯的基因工程研究取得了一定的进展，但甘薯至今没有一例转基因植株进入商品化的应用。
向甘薯基因组中导入外源基因的方法有多种，其中农杆菌介导法最为有效，转入的外源基因多以单拷贝插入，便于育种工作的利用。川薯34，是西南地区的一个优良主栽品种。本试验建立了川薯34等品种较高频率的体细胞胚胎再生体系。在此基础上，利用GUS-intron融合基因的瞬时表达，研究了川薯34有效的遗传转化条件，为进一步通过基因工程改良其品质奠定了良好的基础。主要实验结果如下：
1．通过对不同植物激素组合及甘薯不同再生途径的比较试验，建立了3个甘薯基因型较高频率的体细胞胚胎再生体系，其中川薯34、川薯8129-4、徐薯18的最高植株再生率分别达到了49.9％、29.7％、14.7％。
2．从川薯34的体胚苗中，筛选出了L22、L8两个高频体胚发生材料，可以为遗传转化工作提供良好的受体。
3．通过GUS-intron融合基因的瞬时表达，确定了川薯34有效的遗传转化条件。优化后的遗传转化条件为：用O.D.值为0.5的农杆菌LBA4404／pIG121菌液侵染川薯34的叶片、叶柄60min，侵染时用超声波辅助处理受体材料1min，在侵染液和共培养培养基中都附
西南农业天学硕下学位论又
摘要
加200腼ol/LAS，黑暗下共培养3d。采用此转化条件GUS报告基因瞬时转化频率可达30%
左右，井可以进一步稳定表达。
Sweet potato is an important food and economic crop. There is abundant nutrition in their storage root and leaf. It is widely used for man's food and materials of industry. The nucleus type of sweet potato is homogenous hexaploid. The chromosome background is rather complex. The self-crossing is sterile. Crossing in specific and interspecific crossing are non-compatible or leading to low seed ratio in sweet potato. Conventional breeding technology has localization in variety improvement while transgenic technology has great potential to increase the production,
improve quality and resist plant diseases and pests.
An efficient regeneration system is very important for successful transformation. Comparing
with other plants, regeneration of sweet potato is very difficult. The difference of regeneration frequency is great in different genotypes. There are about 1100 varieties while only ten of them have been studied. Several varieties have been reported for high regeneration frequency and been successful transformed, such as Jewel, White Star, Lizixiang, Gaoxi14 and so on. Most of popular varieties have such a low regeneration frequency that there is not one transgenic plant that has been commercially used though the genetic engineering have made much progress.
There are several methods to introduce foreign gene into the genome of sweet potato, but only the Agrobacterium-mediated transformation is most effective. The foreign gene is transformed in single copy by this means and so convenient for breeding. Chuanshu34 is an excellent variety in southwest area. This experiment established high frequency regeneration system of Chuanshu34, Chuanshu8129-4 and Xushu18 via somatic embryogenesis, optimized the transformation conditions of Chuanshu34 by transient expression of GUS-intron gene. These work founded the base for next step to improve quality of sweet potato by gene engineering technology.
The primary results are listed as following:
1.Established 3 high frequency somatic embryo regeneration systems of different genotype by testing different plant hormones. The regeneration frequency of Chuanshu34, Chuanshu8129-4, Xushu18 was 49.9%, 29.7%, 14.7% respectively.
2.L22 and L8 with high frequent embryogenesis, the two lines were chosen from plantlets of Chuanshu34. Explants from them were good for genetic transformation.
3. Optimized the transformation factors through GUS-intron transient expression. The most effective transformation factors were detailed as below:
Inoculation bacterium was O.D.600=0.5. The explant was leaf or petiole of Chuanshu34, inoculation time was 60 min, and the explant was sonicated for 1 min to increase the tansformation frequency. Inoculation bacterium and co-cultivated medium were added 200umol/LAS. Co-culture took place in dark for 3 days. Transient expression frequency detected by reporter gene was 30% or so and transgene can steadily express in next step.
引文
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