Cabrera-Ponce José Luis, González-Gómez Itzel Anayetzi, León-Ramírez Claudia G, Sánchez-Arreguín José A, Jofre Y Garfias Alba E
Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, CP, Guanajuato, Mexico.
Methods Mol Biol. 2018;1815:189-206. doi: 10.1007/978-1-4939-8594-4_12.
Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to induce somatic embryogenesis (SE) under in vitro conditions. An alternative strategy to yield SE is based upon the use of a cytokinin (benzyladenine) coupled with osmotic stress adaptation instead of the auxin-inducing SE in common bean. Here we described the induction of proembryogenic masses (PEM) derived from the apical meristem and cotyledonary zone of zygotic embryos, from which secondary SE indirect embryogenesis emerged. Maturation of SE was achieved by using osmotic stress medium and converted to plants. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and biobalistics as well as basic biochemical and molecular biology research.
普通菜豆（Phaseolus vulgaris L.）已被证明是一种在体外条件下难以诱导体细胞胚胎发生（SE）的顽拗型植物。产生体细胞胚胎的另一种策略是基于使用细胞分裂素（苄基腺嘌呤）并结合渗透胁迫适应，而不是在普通菜豆中使用生长素诱导体细胞胚胎发生。在此，我们描述了从合子胚的顶端分生组织和子叶区诱导产生原胚性细胞团（PEM），由此产生了次生体细胞胚胎间接胚胎发生。通过使用渗透胁迫培养基实现体细胞胚胎的成熟并转化为植株。通过在体细胞胚胎发生早期阶段对原胚性细胞团进行增殖，证明了长期反复的体细胞胚胎发生。该方案目前正应用于通过根癌农杆菌和生物射弹法进行的稳定遗传转化以及基础生化和分子生物学研究。
