Mano Hiroaki, Fujii Tomomi, Sumikawa Naomi, Hiwatashi Yuji, Hasebe Mitsuyasu
Division of Evolutionary Biology, National Institute for Basic Biology, Okazaki, Japan.
School of Life Science, Graduate University for Advanced Studies, Okazaki, Japan.
PLoS One. 2014 Feb 12;9(2):e88611. doi: 10.1371/journal.pone.0088611. eCollection 2014.
The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.
敏感植物含羞草长期以来一直吸引着研究人员的兴趣，因为它在受到触摸或其他外部刺激时会产生壮观的叶片运动。尽管通过组织学、生理学、生物化学和行为学方法已经阐明了这种感震运动的各个方面，但缺乏反向遗传工具阻碍了对这些过程中涉及的分子机制的研究。为了克服这一障碍，我们开发了一种由根癌农杆菌（农杆菌）介导的高效含羞草遗传转化方法。我们发现子叶节外植体适合农杆菌介导的转化，因为它的芽形成频率高，在含有0.5μg/ml合成细胞分裂素6-苄基腺嘌呤（BAP）的培养基上诱导效率最高。通过使用携带超级双元载体pSB111的农杆菌并使用2-（N-吗啉代）乙磺酸（MES）缓冲液稳定共培养基的pH值，子叶节细胞的转化效率从几乎为0提高到了内含子-sGFP报告基因产生的30.8个阳性信号。此外，在共培养前用去污剂Silwet L-77处理外植体，使转化芽的数量增加了两倍。通过在灌溉蛭石上培养有效地诱导了再生芽的生根。生成转基因植物的整个过程实现了18.8%的转化频率，这与其他难转化的豆科植物如大豆（Glycine max）和豌豆（Pisum sativum）获得的频率相当。转基因稳定地整合到宿主基因组中并代代相传，而不影响植物的感震或感夜运动。因此，这种转化方法为研究参与含羞草运动的基因提供了一种有效的遗传工具。
