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

BMC Biotechnology

Methodology Article

Manoj-Kumar Arthikala¹  Kalpana Nanjareddy¹  Miguel Lara²  Lourdes Blanco³  Elizabeth S. Arellano⁴ 

[1] Ciencias Agrogenómicas, Escuela Nacional de Estudios Superiores, Universidad Nacional Autónoma de México (UNAM), C.P.37684, León, Guanajuato, Mexico;Ciencias Agrogenómicas, Escuela Nacional de Estudios Superiores, Universidad Nacional Autónoma de México (UNAM), C.P.37684, León, Guanajuato, Mexico;Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, C.P. 04510, Coyoacan, Ciudad de México, Mexico;Ciencias Agrogenómicas, Escuela Nacional de Estudios Superiores, Universidad Nacional Autónoma de México (UNAM), C.P.37684, León, Guanajuato, Mexico;Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, C.P. 62210, Coyoacan, Ciudad de México, Mexico;Instituto Nacional de Salud Pública, Av. Universidad 655, Col. Santa Maria, 62100, Cuernavaca, Morelos, Mexico;

关键词: Agrobacterium;    Gene expression;    Overexpression;    Phaseolus vulgaris;    Protoplasts;    RNAi;    SnRK1;    Sonication;    Transient transformation;   

DOI  :  10.1186/s12896-016-0283-8

 received in 2016-04-22, accepted in 2016-06-14,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundPhaseolus 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.ResultsHerein, 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.ConclusionsWe 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.

【 授权许可】

CC BY   
© The Author(s). 2016