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Background   Phaseolus vulgaris  is one of the most extensively studied model legumes in the world. The  P. vulgaris  genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of  P. vulgaris  genes is impeded chiefly due to the non-amenable nature of  Phaseolus 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 legume  P. vulgaris .    Results  Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from  P. 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 the  P. 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 with  PvSnRK1 -RNAi and  PvSnRK1 -OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the  PvSnRK1  transcript. We also demonstrated an improved transient transformation approach, sonication-assisted  Agrobacterium -mediated transformation (SAAT), for the leaf disc infiltration of  P. 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 multiple  P. 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 for  P. vulgaris  research.    Electronic supplementary material  The online version of this article (doi:10.1186/s12896-016-0283-8) contains supplementary material, which is available to authorized users.

bmc biotechnology

Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis