Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency
Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency
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DOI：
10.1590/s1415-47572004000200022
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发表时间：
2004-01-01
影响因子：
2.1
通讯作者：
Axtell, John D.
中科院分区：
文献类型：
--
作者：
Carvalho, Carlos Henrique S.;Zehr, Usha B.;Axtell, John D.
这项工作的结果支持这样的假设，即农杆菌介导的高粱转化是可行的，类似于已经证明的对其他谷物如水稻、玉米、大麦和小麦的转化。对转化影响最大的四个因素是：高粱幼胚对农杆菌侵染的敏感性、供体植株的生长条件、外植体类型和共培养条件。在我们的方案开发过程中的一个主要问题是在共培养后外植体中出现的坏死性反应。未成熟的高粱胚对农杆菌的侵染非常敏感，我们发现共培养后的胚死亡水平是提高转化效率的限制步骤。在共培养基中添加椰子水，使用生长旺盛、生长活跃的幼胚，去除多余的细菌，显著提高了高粱胚成活率，是转化成功的关键。潮霉素磷酸转移酶(HPT)是一个很好的高粱选择标记。我们还发现，在大多数转基因植物组织中，β-葡萄糖醛酸苷酶(GUS)活性很低，尽管在未成熟的花序中GUS活性很高。尽管前景看好，但该方案的整体转化效率仍然很低，进一步的优化需要特别注意接种物中的农杆菌数量，以及对农杆菌感染不敏感的高粱基因型和外植体的选择。
The results presented in this work support the hypothesis that Agrobacterium-mediated transformation of sorghum is feasible, analogous to what has been demonstrated for other cereals such as rice, maize, barley and wheat. The four factors that we found most influenced transformation were: the sensitivity of immature sorghum embryos to Agrobacterium infection, the growth conditions of the donor plant, type of explant and co-cultivation medium. A major problem during the development of our protocol was a necrotic response which developed in explants after co-cultivation. Immature sorghum embryos proved to be very sensitive to Agrobacterium infection and we found that the level of embryo death after co-cultivation was the limiting step in improving transformation efficiency. The addition of coconut water to the co-cultivation medium, the use of vigorous and actively growing immature embryos and the removal of excess bacteria significantly improved the survival rate of sorghum embryos and was critical for successful transformation. Hygromycin phosphotransferase (hpt) proved to be a good selectable marker for sorghum. We also found that beta-glucuronidase (GUS) activity was low in most of the transgenic plant tissues tested, although it was very high in immature inflorescences. Although promising, the overall transformation efficiency of the protocol is still low and further optimization will require particular attention to be given to the number of Agrobacterium in the inoculum and the selection of sorghum genotypes and explants less sensitive to Agrobacterium infection.