Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India.
Plant Methods. 2011 Dec 30;7(1):49. doi: 10.1186/1746-4811-7-49.
Rice genome sequencing projects have generated remarkable amount of information about genes and genome architecture having tremendous potential to be utilized in both basic and applied research. Success in transgenics is paving the way for preparing a road map of functional genomics which is expected to correlate action of a gene to a trait in cellular and organismal context. However, the lack of a simple and efficient method for transformation and regeneration is a major constraint for such studies in this important cereal crop.
In the present study, we have developed an easy, rapid and highly efficient transformation and regeneration protocol using mature seeds as explants and found its successful applicability to a choice of elite indica rice genotypes. We have optimized various steps of transformation and standardized different components of the regeneration medium including growth hormones and the gelling agent. The modified regeneration medium triggers production of large number of shoots from smaller number of calli and promotes their faster growth, hence significantly advantageous over the existing protocols where the regeneration step requires maximum time. Using this protocol, significantly higher transformation efficiency (up to 46%) and regeneration frequency (up to 92% for the untransformed calli and 59% for the transformed calli) were achieved for the four tested cultivars. We have used this protocol to produce hundreds of independent transgenic lines of different indica rice genotypes. Upon maturity, these transgenic lines were fertile thereby indicating that faster regeneration during tissue culture did not affect their reproductive potential.
This speedy, yet less labor-intensive, protocol overcomes major limitations associated with genetic manipulation in rice. Moreover, our protocol uses mature seeds as the explant, which can easily be obtained in quantity throughout the year and kept viable for a long time. Such an easy, efficient and generalized protocol has the potential to be a major tool for crop improvement and gene-function studies on the model monocot plant rice.
水稻基因组测序项目产生了大量关于基因和基因组结构的信息，这些信息具有巨大的潜力，可以应用于基础研究和应用研究。转基因技术的成功为准备功能基因组学图谱铺平了道路，预计这将使基因在细胞和生物体内的作用与表型相关联。然而，缺乏一种简单而有效的转化和再生方法，是限制在这种重要的谷类作物中进行这些研究的主要因素。
在本研究中，我们使用成熟种子作为外植体，开发了一种简单、快速和高效的转化和再生方案，并发现其成功适用于一系列优良的籼稻基因型。我们优化了转化的各个步骤，并对再生培养基的不同成分进行了标准化，包括生长激素和凝胶剂。改良的再生培养基可刺激少量愈伤组织产生大量芽，并促进其更快生长，因此明显优于现有的再生步骤需要最长时间的方案。使用该方案，四个测试品种的转化效率（高达 46%）和再生频率（未转化愈伤组织高达 92%，转化愈伤组织为 59%）均显著提高。我们使用该方案产生了不同籼稻基因型的数百个独立的转基因株系。成熟后，这些转基因株系具有育性，这表明组织培养期间更快的再生不会影响其生殖潜力。
该快速但劳动强度较小的方案克服了与水稻遗传操作相关的主要限制。此外，我们的方案使用成熟种子作为外植体，这些外植体可以在全年轻松获得大量，并保持长时间的活力。这种简单、高效和通用的方案有可能成为改善作物和研究单子叶模式植物水稻基因功能的重要工具。
