Revisiting rice transformation for a fail-safe protocol and its application for various gene functional and molecular studies

Abstract

An efcient transformation and regeneration system is prerequisite for gene functional studies in rice, the staple food crop of the Asian subcontinent. Despite the availability of a large number of rice transformation protocols, it is hard to fnd a simple and minimum input based, but fail-safe protocol that ensures zero number of escapes (or the non-transformants). The fear of obtaining an unpredictable percentage of escapes at the end of rice tissue culture prompts carrying out multiple batches of transformation which not only consumes costly resources and time but also burdens one with screening of a large number of tissue culture derived plants. In the present study, we have described a simple but fail-safe rice transformation protocol for functional validation of the genes by overexpression and CRISPR-Cas9 mediated gene knockout systems. By taking the advantage of high transformability of japonica rice tissues, we have also revealed that the present rice transformation protocol can be efectively employed to visualize protein localization in various subcellular compartments of rice root and callus tissues, an alternative to conventional tobacco/onion peel infltration or protoplast transformation which are either time consuming or tricky. Additionally, the paper also discusses the importance of Southern blotting in gene overexpression studies, utility of non-conventional antibiotic selection approaches and signifcance of sgRNA designing for gene knockout studies in rice. Various troubleshooting advice are also being presented. Overall, the present protocol might serve as an excellent guide for functional validation of several genes present in the rice genome waiting to be explored.