Abstract
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.
Highlights
Being fixed in the soil, rooted plants have evolved a variety of strategies to survive stressful environments
We optimized the concentrations of two kinds of phytohormones, the cytokinin BAP and the auxin 1-naphthaleneacetic acid (NAA), both of which affect the number of shoots formed on the cotyledonary node in other leguminous species [41,42,43]
The cotyledonary node was selected as the target tissue for Agrobacterium infection, and explants were cultured in medium supplemented with 0.5 mg/ml BAP in subsequent experiments
Summary
Being fixed in the soil, rooted plants have evolved a variety of strategies to survive stressful environments. Individual ‘‘motor cells’’ in the extensor half of the pulvinus shrink following outflow of intracellular water [9,10], which is accompanied by a large efflux of K+ and Cl- ions [11,12,13,14] These rapid movements of water and ions are difficult to explain by a simple diffusion model [12,15], suggesting that special mechanisms, such as solute-water co-transporters or contractile proteins, are involved in this process [15]. Mechanoreceptor cells in M. pudica have long been enigmatic; a recent study identified such cells on the tertiary pulvinus [26]
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