Standardisation of Agrobacterium-mediated transformation in WP-22-2 – a semi-dwarf and early maturing rice mutant of improved white ponni

Abstract

Genome engineering using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPRassociated (Cas) system offers vast potential in the improvement of crop plants. For CRISPR/Cas9 mediateediting in rice, transformation protocols should be standardized for specific genotypes. We standardized theAgrobacterium-mediated transformation protocols for two genotypes – Improved White Ponni (IWP) and WP-22-2,a gamma-ray induced mutant of Improved White Ponni. Three different modifications of MS basal medium, differentdays old calli and bacterial culture of different optical density were evaluated. Callus induction with IWP-CI-medium1 followed by callus proliferation for one month in IWP-CI-medium-2 produced transformable calli. Infection with0.3 OD Agrobacterium culture for 10 min was less damaging and successful than higher density cultures. Thetransformation efficiency varied between the Improved White Ponni and WP-22-2 emphasizing the need forstandardizing tissue culture protocols for every genotype.  Transformation efficiency of 9.46 per cent and 3.53 percent respectively were observed for Improved White Ponni and WP-22-2 which were higher than previous reports.