Development of Efficient, Reproducible and Stable Agrobacterium-Mediated Genetic Transformation of Five Potato Cultivars
Development of Efficient, Reproducible and Stable Agrobacterium-Mediated Genetic Transformation of Five Potato Cultivars
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DOI：
10.17113/ftb.58.01.20.6187
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发表时间：
2020-01-01
影响因子：
2.4
通讯作者：
Bakhsh, Allah
中科院分区：
文献类型：
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作者：
Bakhsh, Allah
转化技术的发展使科学家们能够整合、突变或替代基因，从而产生特定的特征；把它推进到只剩下很少的技术限制的地步。基因型依赖性和外植体类型是影响马铃薯转化效率的重要因素。在本研究中，通过不同植物生长调节剂的组合，建立了一种快速、可重复和稳定的农杆菌介导的马铃薯转化过程。在355 CaMV启动子控制下，用含有pBIN19 -葡糖苷酶gusA基因表达载体的根癌农杆菌LBA4404感染了奥林匹亚女士（Lady Olympia）、格兰诺拉（Granola）、Agria、Desiree和Innovator 5个马铃薯品种的叶盘和节间外植体。以卡那霉素作为植物选择标记，筛选浓度为100 mg/L的初代转化子。两个外植体均有积极响应；为提高转化效率，节间为较合适的外植体。GUS组化分析结果表明，在奥林匹亚女士、格兰诺拉、Agria、Desiree和Innovator中，节间外植体的转化效率分别为22.2%、20%、18.6%、15%和10%，叶片外植体的转化效率分别为15.12%、17.8%和6%。此外，PCR检测证实再生植株中存在gusA和nptll基因。对后代的分子分析表明，这两个基因都得到了适当的整合和表达。结果表明，奥林匹亚女士是今后转化的最佳品种。总体而言，该方案持续时间短、快速、重复性好，适合在转基因马铃薯植株上推广应用。
The developments in transformation technology have enabled the scientists to incorporate, mutate or substitute gene(s) leading to a particular trait; advancing it to a point where only few technical limitations remain. Genotype dependency and explant types are important factors affecting transformation efficiency in potato. In the present study, a rapid, reproducible and stable Agrobacterium-mediated transformation procedure in potato was developed by a combination of different plant growth regulators. Leaf discs and internodal explants of five cultivars of potato, i.e. Lady Olympia, Granola, Agria, Desiree and Innovator were infected with Agrobacterium tumefaciens strain LBA4404 containing pBIN19 expression vector with beta-glucuronidase gusA gene under the control of 355 CaMV promoter. Kanamycin was used as plant selectable markerfor screening of primary transformants at concentration of 100 mg/L. Both explants responded positively; internode being more suitable explant for better transformation efficiency. Based on GUS histochemical assay, the transformation efficiency was 22, 20, 18.6, 15 and 10% using the internodal explant, and 15, 12, 17, 8 and 6 % using leaf discs as explant in Lady Olympia, Granola, Agria, Desiree and Innovator respectively. Furthermore, PCR assays confirmed the presence of gusA and nptll genes in regenerated plants. The molecular analysis in succeeding progeny showed proper integration and expression of both genes. The results suggest Lady Olympia as the best cultivar for future transformation procedures. Overall, the short duration, rapidity and reproducibility make this protocol suitable for wider application of transgenic potato plants.