Agroinfiltration-mediated transient assay for rapid evaluation of constructs in pigeonpea

Yogendra, K and Gadeela, H and Sree, K N and Tyagi, W (2025) Agroinfiltration-mediated transient assay for rapid evaluation of constructs in pigeonpea. Biotechnology Notes, 6. pp. 117-125. ISSN 2665-9069

PDF - Published Version Available under License Creative Commons Attribution Non-commercial No Derivatives. Download (6MB)

Abstract

The process of generating stable transformants is time-consuming, labor-intensive, and genotype-dependent. In contrast, transient gene expression techniques, such as agroinfiltration, offer a rapid assessment of gene function and expression. Agroinfiltration, widely employed for studying gene function, has been extensively applied in leaf tissues of Nicotiana benthamiana and various other plant species. Despite its broad utility in various plants, to our knowledge, no prior investigation has been reported in pigeonpea. In this study, we developed an agroinfiltration method for transiently expressing a green fluorescent protein (mGFP5) reporter gene in four pigeonpea genotypes using syringe infiltration at the seedling stage under greenhouse conditions. The expression of the reporter gene mGFP5 was assessed at 72-, 96-, and 120 h post-infiltration (hpi). Additionally, we assessed the effect of morphogenic genes, specifically growth-regulating factor 4 (GRF4) and GRF-interacting factor 1 (GIF1), from both rice and pigeonpea on the expression of mGFP5 in four pigeonpea genotypes. Our findings demonstrate that OsGRF4-GIF1 led to enhanced mGFP5 expression compared to CcGRF4-GIF1 in four diverse pigeonpea genotypes. Fluorescence could be detected till 120 hpi. Furthermore, PCR, RT-PCR, and fluorescence quantification confirmed the presence and expression of mGFP5 at 72 hpi. Our results highlight the efficacy of agroinfiltration in quickly evaluating candidate genes in four genetically diverse pigeonpea genotypes, thereby reducing the time required for the initial assessment of constructs suitable for diverse molecular biology analyses.

Item Type:	Article
Divisions:	Global Research Program - Accelerated Crop Improvement
CRP:	UNSPECIFIED
Uncontrolled Keywords:	Agroinfiltration, Green fluorescent protein, Growth regulating factor, GRF interacting factor, Morphogenic genes, Pigeonpea
Subjects:	Others > Biotechnology Mandate crops > Pigeonpea
Depositing User:	Mr Nagaraju T
Date Deposited:	20 May 2025 08:44
Last Modified:	20 May 2025 08:44
URI:	http://oar.icrisat.org/id/eprint/13079
Official URL:	https://www.sciencedirect.com/science/article/pii/...
Projects:	UNSPECIFIED
Funders:	Novo Nordisk Foundation, Denmark, Government of India - Department of Biotechnology
Acknowledgement:	The authors thank Dr Prakash Gangashetty, the Pigeonpea breeder at ICRISAT, for generously supplying the pigeonpea seeds required for plant transformation. We acknowledge the support given by Dr Sudhakar Reddy Palakolanu, Scientist, ACI, ICRISAT in getting the sequences of GRF4-GIF1 from rice and pigeonpea commercially synthesized. Mr Pradeep Reddy, Scientific officer, ACI, ICRISAT is acknowledged for cloning and mobilizing the constructs in Agrobacterium.
Links:	Google Scholar

View Statistics

Actions (login required)

View Item