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)/CRISPR associated (Cas) system offers vast potential in the improvement of crop plants. For CRISPR/Cas9 mediated editing in rice, transformation protocols should be standardized for specific genotypes. We standardized the Agrobacterium-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, different days old calli and bacterial culture of different optical density were evaluated. Callus induction with IWP-CI-medium-1 followed by callus proliferation for one month in IWP-CI-medium-2 produced transformable calli. Infection with 0.3 OD Agrobacterium culture for 10 min was less damaging and successful than higher density cultures. The transformation efficiency varied between the Improved White Ponni and WP-22-2 emphasizing the need for standardizing tissue culture protocols for every genotype. Transformation efficiency of 9.46 per cent and 3.53 per cent respectively were observed for Improved White Ponni and WP-22-2 which were higher than previous reports.

Keywords

• agrobacterium
• transformation
• rice