Improvement of Agrobacterium-mediated transformation for tannin-producing sorghum
Improvement of Agrobacterium-mediated transformation for tannin-producing sorghum
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DOI：
10.5511/plantbiotechnology.19.0131a
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发表时间：
2019-03-01
影响因子：
1.6
通讯作者：
Matsui, Minami
中科院分区：
文献类型：
--
作者：
Kuriyama, Tomoko;Shimada, Setsuko;Matsui, Minami
高粱(Sorghum biolor L.)是世界上种植面积第五大的谷类作物，被用作食品和生产乙醇的生物质植物。利用TX430非单宁高粱品种促进了农杆菌介导的高粱转化。然而，这些方案不能应用于其他单宁生产高粱品种，如具有高粱全基因组信息的高粱模式品种BTx623。在这里，我们报道了一种改进的农杆菌介导遗传转化单宁高粱品种BTx623的方法。我们成功地对BTx623转基因植株的再生条件进行了改良。我们用携带pMDC32-35S-GFP的根癌农杆菌EHA105菌株接种未成熟胚胎，获得转基因植株。在根再生步骤中，我们发现单宁对转化愈伤组织的再生有影响。在生根方面，出现的新芽不是转移到琼脂平板上，而是转移到塑料豆荚中的蛭石上。将再生芽直接种植到蛭石中可以防止单宁的毒害作用。以蛭石为外植体的愈伤组织生根效率为78.57%。通过聚合酶链式反应证实了转基因植株基因组中存在sGFP基因，并证实了sGFP在转基因植株中的表达。这一改进的农杆菌介导法转化单宁高粱BTx623可能成为利用这种植物进行功能基因组学研究的有用工具。
Sorghum (Sorghum bicolor L.) ranks as the fifth most widely planted cereal in the world and is used for food as well as a biomass plant for ethanol production. Use of the TX430 non-tannin sorghum variety has enhanced Agrobacterium-mediated sorghum transformation. These protocols could not be applied, however, to other tannin producing sorghum varieties such as the BTx623 model cultivar for sorghum with full genome information of sorghum. Here we report an improved protocol for Agrobacterium-mediated genetic transformation of tannin-producing sorghum variety BTx623. We successfully developed modification of root regeneration condition for generation of transgenic plant of BTx623. We inoculated immature embryos with Agrobacterium tumefaciens strain EHA105 harboring pMDC32-35S-GFP to generate transgenic plants. In the root regeneration step, we found that regeneration from transformed calli was affected by tannin. For root regeneration, shoots that appeared were not transferred to agar plate, but instead transferred to vermiculite in a plastic pod. Direct planting of regenerated shoots into vermiculite prevented the toxic effect of tannin. Root regeneration efficiency from calli emerged shoots in vermiculite was 78.57%. Presence of sGFP transgene in the genome of transgenic plants was confirmed by PCR and sGFP expression was confirmed in transgenic plants. This improved protocol of Agrobacterium-mediated transformation for tannin-producing sorghum BTx623 could be a useful tool for functional genomics using this plant.