Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Dawei Xu; Palash  Chandra Mondol; Muhammad Uzair; Matthew R. Tucker; Dabing Zhang

doi:10.3791/61665

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Method Article

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

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DOI:

10.3791/61665

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October 6th, 2020

Dawei Xu^*¹^,² , Palash Chandra Mondol^*¹ , Muhammad Uzair¹ , Matthew R. Tucker³ , Dabing Zhang¹^,³

¹Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University-University of Adelaide Joint Centre for Agriculture and Health, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, ²College of Agronomy, Anhui Agricultural University, Heifei, China, ³School of Agriculture, Food and Wine, University of Adelaide

^* These authors contributed equally

Summary

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Abstract

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2–3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I₂-KI) staining semi-thin cross-sectioning of developing anthers.

Introduction

Rice is the most important food crop, particularly in developing countries, and represents a staple food for over half of the world’s population. Overall, the demand for rice grain is growing and is projected to increase 50% by 2030 and 100% by 2050¹^,². Future improvements in rice yield will need to capitalize on diverse molecular and genetic resources that make rice an excellent model for monocotyledonous plant research. These include an efficient transformation system, advanced molecular map, and publicly accessible database of expressed sequence tags, which have been generated over many years