Devising an Innovative, Genotype-free Transformation Protocol for Recalcitrant Indica Rice Genotypes: Samba Mahsuri and Sundarbans’ Salt-tolerant Indica Rice Cultivars White Getu and Hamilton to Establish an Ingenious Platform for CRISPR/Cas9-mediated Genome Editing

Abstract BackgroundPlant genetic transformation involves in vitro callus induction & regeneration strategies that are quintessential for introduction of novel agronomical traits employing CRISPR/Cas9-based genome editing. However, lack of effective regeneration and transformation techniques for indica rice cultivars pose as the foremost hurdle towards genetic improvement in rice crop. We devised an astounding road-map to genotype-independent and efficacious in vitro callus induction, transformation and shoot regeneration protocol that emerges as an optimal therapy, universally adaptable to invariably any rice cultivar, in order to establish an ingenious CRISPR/Cas9-based genome editing platform in this crop.ResultsWe developed a genotype-independent regeneration and transformation protocol employing mature seed-derived calli for indica rice (one mega variety- Samba Mahsuri and two salt tolerant wild genotypes- White Getu & Hamilton) genotypes to introduce important agronomical traits via CRISPR/Cas9-based genome editing system. MS- and N6-salt based media reinforced with 2,4-D (2.5 mg/L); dicamba (1.5 mg/L); TDZ (0.1 mg/L), proline (1000 mg/L), and glutamine (2.5 mg/L) exhibited highest percentage (95-98%) of embryogenic calli initiation and development. Employing this novel protocol, we achieved unparalleled regeneration efficiencies within untransformed calli (90-94%) and transformed calli (81-86%) in these recalcitrant indica genotypes and significantly enhanced number of shoots (18-20) on MS medium containing BAP (1.5 mg/L), NAA (0.5 mg/L), TDZ (1.0 mg/L), zeatin (0.2 mg/L) and proline (500 mg/L). We successfully transformed rice calli with pCAMBIA1300-based marker- free NICTK-1_pCRISPR-Cas9 vector harbouring the cassette of plant codon optimized Cas9 via biolistic approach that exhibited notably enhanced transformation efficiencies (67-69%). The integration of Cas9 gene into rice genome was validated by PCR, Southern blotting and Sanger sequencing analyses. The transgenic lines were phenotypically indistinguishable from the wild type as no significant differences in phenotypic performances were revealed between transgenic and wild type lines. ConclusionWe devised a promising, time-efficient, universally adaptable, optimal hormonal-media therapy for triggering enhanced embryogenic callus formation, regeneration and transformation efficiencies, across recalcitrant indica rice genotypes.