Kumar Sanjeev, Prakash Sakshi, Kumari Priti, Sanan-Mishra Neeti
Plant RNAi Biology Group, International Center for Genetic Engineering and Biotechnology, New Delhi, 110067, India.
BMC Plant Biol. 2025 May 29;25(1):724. doi: 10.1186/s12870-025-06718-0.
Mungbean is one of the most economically important grain legume crops in Asia. Functional genomics studies in mungbean are necessary to understand the molecular mechanisms behind agronomic traits, to advance the crop improvement. However, this progress is significantly impeded by the absence of effective and extensive genetic analysis tools. Agrobacterium rhizogenes-mediated hairy root transformation has become a powerful tool for studying gene function and an efficient alternative for investigating root-specific interactions and processes in different species, due to its quick and simple methodology. Agrobacterium-mediated plant transformation, however, is known to be difficult in legumes, especially in mungbean.
In this report, we developed an Agrobacterium rhizogenes-mediated mungbean transformation system using both in-vitro and ex-vitro approaches, with RUBY employed as a reporter gene. We optimized various parameters, including mungbean genotypes, explant age, optical density of the bacterial culture, co-cultivation medium, and acetosyringone concentration. Our findings indicated that in-vitro transformation was more efficient than ex-vitro in terms of hairy root induction percentage and the proportion of transformed hairy roots expressing the RUBY reporter gene. However, the ex-vitro transformation technique was faster and less complex than the in-vitro method. The highest transformation efficiency for RUBY expression was achieved using 5-day-old cotyledonary nodal explants of cv. K-851, inoculated for 30 min with A4 Agrobacterium cells resuspended in full-strength MS medium at an OD₆₀₀ of 0.5 and supplemented with 100 µM acetosyringone. A total of 60 composite plants were generated and evaluated through PCR, resulting in a transformation efficiency of 6.13%. These optimized parameters also led to the highest percentage of RUBY expression using the two-step ex-vitro hairy root transformation method.
We have developed a simple, rapid, low-cost, and labor-efficient Agrobacterium rhizogenes-mediated mungbean transformation protocol using both in-vitro and ex-vitro approaches, with RUBY as a reporter gene. This method enables the generation of composite mungbean plants that are easier to handle, exhibit higher transformation efficiency, and can be effectively used for root specific functional genomics studies. We expect this technology to be widely adopted for investigating root-related processes in mungbean and other plant species.
绿豆是亚洲经济上最重要的豆类作物之一。开展绿豆功能基因组学研究对于理解农艺性状背后的分子机制、推动作物改良十分必要。然而，有效的广泛的遗传分析工具的缺失严重阻碍了这一进展。发根农杆菌介导的毛状根转化因其方法快速简便，已成为研究基因功能的有力工具，也是研究不同物种根特异性相互作用和过程的有效替代方法。然而，农杆菌介导的植物转化在豆科植物中，尤其是在绿豆中，是出了名的困难。
在本报告中，我们开发了一种发根农杆菌介导的绿豆转化系统，采用体外和离体两种方法，使用红宝石（RUBY）作为报告基因。我们优化了各种参数，包括绿豆基因型、外植体年龄、细菌培养物的光密度、共培养基和乙酰丁香酮浓度。我们的研究结果表明，就毛状根诱导率和表达红宝石报告基因的转化毛状根比例而言，体外转化比离体转化更有效。然而，离体转化技术比体外方法更快且更简单。使用5日龄cv. K - 851子叶节外植体，用OD₆₀₀为0.5的A4农杆菌细胞重悬于全强度MS培养基中，并添加100µM乙酰丁香酮，接种30分钟，实现了红宝石表达的最高转化效率。共产生了60株复合植株并通过PCR进行评估，转化效率为6.13%。这些优化参数在两步离体毛状根转化方法中也导致了最高的红宝石表达百分比。
我们开发了一种简单、快速、低成本且省力的发根农杆菌介导的绿豆转化方案，采用体外和离体两种方法，使用红宝石作为报告基因。该方法能够产生更易于处理、具有更高转化效率且可有效用于根特异性功能基因组学研究的复合绿豆植株。我们期望这项技术能被广泛应用于研究绿豆及其他植物物种中与根相关的过程。
