Transgenic A. hypochondriacus and A. hybridus roots were generated. Further, a distinct plant regeneration program via somatic embryos produced from hairy roots was established. Work was implemented to develop an optimized protocol for root genetic transformation of the three grain amaranth species and A. hybridus, their presumed ancestor. Transformation efficiency was species-specific, being higher in A. hypochondriacus and followed by A. hybridus. Amaranthus cruentus and A. caudatus remained recalcitrant. A reliable and efficient Agrobacteruim rhizogenes-mediated transformation of these species was established using cotyledon explants infected with the previously untested BVG strain. Optimal OD600 bacterial cell densities were 0.4 and 0.8 for A. hypochondriacus and A. hybridus, respectively. Hairy roots of both amaranth species were validated by the amplification of appropriate marker genes and, when pertinent, by monitoring green fluorescent protein emission or β-glucuronidase activity. Embryogenic calli were generated from A. hypochondriacus rhizoclones. Subsequent somatic embryo maturation and germination required the activation of cytokinin signaling, osmotic stress, red light, and calcium incorporation. A crucial step to ensure the differentiation of germinating somatic embryos into plantlets was their individualization and subcultivation in 5/5 media containing 5% sucrose, 5 g/L gelrite, and 0.2 mg/L 2-isopentenyladenine (2iP) previously acidified to pH 4.0 with phosphoric acid, followed by their transfer to 5/5 + 2iP media supplemented with 100 mg/L CaCl2. These steps were strictly red light dependent. This process represents a viable protocol for plant regeneration via somatic embryo germination from grain amaranth transgenic hairy roots. Its capacity to overcome the recalcitrance to genetic transformation characteristic of grain amaranth has the potential to significantly advance the knowledge of several unresolved biological aspects of grain amaranths.

中文翻译：

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发根农杆菌介导的谷物 (Amaranthus hypochondriacus) 和多叶 (A. hybridus) 苋菜的转化。

产生了转基因A. hypochondriacus 和A. hybridus 根。此外，建立了通过从毛状根产生的体细胞胚进行的独特植物再生计划。实施工作以开发优化的方案，用于三种籽粒苋属物种和它们的假定祖先 A. hybridus 的根遗传转化。转化效率是物种特异性的，在 A. hypochondriacus 中较高，其次是 A. hybridus。Amaranthus cruentus 和 A. caudatus 仍然顽固。使用感染了先前未经测试的 BVG 菌株的子叶外植体建立了可靠且有效的发根农杆菌介导的这些物种的转化。A. hypochondriacus 和 A. hybridus 的最佳 OD600 细菌细胞密度分别为 0.4 和 0.8。两种苋菜的毛状根通过扩增适当的标记基因进行验证，并在相关时通过监测绿色荧光蛋白发射或β-葡萄糖醛酸酶活性进行验证。胚性愈伤组织由 A. hypochondriacus rhizoclones 产生。随后的体细胞胚胎成熟和萌发需要激活细胞分裂素信号、渗透压、红光和钙掺入。确保发芽的体细胞胚分化为小植株的关键步骤是在含有 5% 蔗糖、5 g/L 凝胶和 0.2 mg/L 2-异戊烯基腺嘌呤 (2iP) 的 5/5 培养基中进行个体化和继代培养，该培养基预先酸化至 pH 4.0磷酸，然后将其转移到补充有 100 mg/L CaCl2 的 5/5 + 2iP 培养基中。这些步骤严格依赖于红灯。该过程代表了一种可行的方案，用于通过谷物苋菜转基因毛状根的体细胞胚萌发进行植物再生。其克服籽粒苋菜遗传转化特性的顽固性的能力有可能显着提高对籽粒苋菜几个未解决的生物学方面的认识。