Department of Biological Sciences (LCB), Escola Superior de Agricultura "Luiz de Queiroz" (ESALQ), Universidade de São Paulo (USP), Av, Pádua Dias, 11, CP 09, Piracicaba, SP, 13418-900, Brazil.
Plant Methods. 2010 Oct 7;6:23. doi: 10.1186/1746-4811-6-23.
The cultivar Micro-Tom (MT) is regarded as a model system for tomato genetics due to its short life cycle and miniature size. However, efforts to improve tomato genetic transformation have led to protocols dependent on the costly hormone zeatin, combined with an excessive number of steps.
Here we report the development of a MT near-isogenic genotype harboring the allele Rg1 (MT-Rg1), which greatly improves tomato in vitro regeneration. Regeneration was further improved in MT by including a two-day incubation of cotyledonary explants onto medium containing 0.4 μM 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 5 μM 6-benzylaminopurine (BAP), a cytokinin 100 times less expensive than zeatin. The use of MT-Rg1 and NAA pre-incubation, followed by BAP regeneration, resulted in high transformation frequencies (near 40%), in a shorter protocol with fewer steps, spanning approximately 40 days from Agrobacterium infection to transgenic plant acclimatization.
The genetic resource and the protocol presented here represent invaluable tools for routine gene expression manipulation and high throughput functional genomics by insertional mutagenesis in tomato.
微型番茄（MT）因其生命周期短、体型小而被视为番茄遗传学的模式系统。然而，为了提高番茄的遗传转化效率，人们已经开发出了依赖于昂贵的激素玉米素，并结合了过多步骤的方案。
在这里，我们报告了一种携带有 Rg1 等位基因（MT-Rg1）的 MT 近等基因系的开发，该等位基因极大地提高了番茄的体外再生能力。通过在含有 0.4 μM 1-萘乙酸（NAA）的培养基上对子叶外植体进行两天孵育，然后再用细胞分裂素处理，进一步提高了 MT 的再生能力。这两种策略都允许使用 5 μM 6-苄基氨基嘌呤（BAP），这是一种比玉米素便宜 100 倍的细胞分裂素。使用 MT-Rg1 和 NAA 预孵育，然后用 BAP 再生，导致了高的转化频率（接近 40%），该方案步骤更少，时间更短，从农杆菌感染到转基因植物适应大约需要 40 天。
本研究提供的遗传资源和方案代表了番茄中通过插入诱变进行常规基因表达操作和高通量功能基因组学的宝贵工具。
