title: Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Ciencias Agrogenómicas, Escuela Nacional de Estudios Superiores, Universidad Nacional Autónoma de México (UNAM), León, C.P.37684, Guanajuato, Mexico.
authors.forename: Kalpana
authors.initials: K
authors.lastname: Nanjareddy
authors.affiliation: Ciencias Agrogenómicas, Escuela Nacional de Estudios Superiores, Universidad Nacional Autónoma de México (UNAM), León, C.P.37684, Guanajuato, Mexico.
authors.forename: Manoj-Kumar
authors.initials: MK
authors.lastname: Arthikala
authors.affiliation: Ciencias Agrogenómicas, Escuela Nacional de Estudios Superiores, Universidad Nacional Autónoma de México (UNAM), León, C.P.37684, Guanajuato, Mexico.|Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, Coyoacan, Ciudad de México, C.P. 62210, Mexico.
authors.forename: Lourdes
authors.initials: L
authors.lastname: Blanco
authors.affiliation: Instituto Nacional de Salud Pública, Av. Universidad 655, Col. Santa Maria, Cuernavaca, Morelos, 62100, Mexico.
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Arellano
authors.affiliation: Ciencias Agrogenómicas, Escuela Nacional de Estudios Superiores, Universidad Nacional Autónoma de México (UNAM), León, C.P.37684, Guanajuato, Mexico. mlara@unam.mx.|Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, Coyoacan, Ciudad de México, C.P. 04510, Mexico. mlara@unam.mx.
authors.forename: Miguel
authors.identifier: 0000-0002-5885-7950
authors.initials: M
authors.lastname: Lara
mesh_terms: D060054:Agrobacterium / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D058503:Mesophyll Cells / Q000502:physiology; D027805:Phaseolus / Q000235:genetics* / Q000382:microbiology; D018515:Plant Leaves / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000382:microbiology; D011523:Protoplasts / Q000166:cytology / Q000502:physiology*; D014169:Transformation, Bacterial / Q000235:genetics
keywords: Agrobacterium infiltration; Gene expression; Overexpression; Phaseolus vulgaris; Protoplasts; RNAi; SnRK1; Sonication; Transient transformation