Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Nanjareddy, Kalpana; Arthikala, Manoj-Kumar; Blanco, Lourdes; Arellano, Elizabeth; Lara, Miguel
• Is Part Of: BMC biotechnology. Volume 16:Issue 1(2016)
• Page Start: 1
• Page End: 14
• Publication Date: 2016-12
• Subjects: Agrobacterium infiltration -- Gene expression -- Overexpression -- Phaseolus vulgaris -- Protoplasts -- RNAi -- SnRK1 -- Sonication -- Transient transformation; Biotechnology -- Periodicals; 660.605
• DOI: 10.1186/s12896-016-0283-8
• Languages: English
• ISSNs: 1472-6750

Abstract

Abstract Background Phaseolus vulgaris is one of the most extensively studied model legumes in the world. TheP. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization ofP. vulgaris genes is impeded chiefly due to the non-amenable nature ofPhaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legumeP. vulgaris . Results Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts fromP. vulgaris . The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used theP. vulgaris SNF1-related protein kinase 1 (PvSnRK1 ) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed withPvSnRK1 -RNAi andPvSnRK1 -OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of thePvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assistedAgrobacterium -mediated transformation (SAAT), for the leaf disc infiltration ofP. vulgaris . Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60–85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. Conclusions We present simple and efficient methodologies for protoplast isolation from multipleP. vulgaris tissues. We also provide a high-efficiency and amenable method for leaf mesophyll transformation for rapid gene functional characterization studies. Furthermore, a modified SAAT leaf disc infiltration approach aids in validating genes and their functions. Together, these methods help to rapidly unravel novel gene functions and are promising tools forP. vulgaris research.