CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

A Temporary Immersion System Improves Regeneration of In Vitro Irradiated Recalcitrant Indica Rice (Oryza sativa L.) Embryogenic Calli.

The development of gamma ray-mutated rice lines is a solution for introducing genetic variability in indica rice varieties already being used by farmers. In vitro gamma ray (60Co) mutagenesis reduces chimeras and allows for a faster selection of desirable traits but requires the optimization of the laboratory procedure. The objectives of the present work were sequencing of matK and rbcL, the in vitro establishment of recalcitrant rice embryogenic calli, the determination of their sensitivity to gamma radiation, and optimization of the generation procedure. All sequenced genes matched perfectly with previously reported matK and rbcL O. sativa genes. Embryogenic calli induction improved using MS medium containing 2 mg L-1 2,4-D, and regeneration was achieved with MS medium with 3 mg L-1 BA and 0.5 mg L-1 NAA. The optimized radiation condition was 60 Gy, (LD20 = 64 Gy) with 83% regeneration. An immersion system (RITA®, Saint-Mathieu-de-Tréviers, France) of either 60 or 120 s every 8 h allowed systematic and homogeneous total regeneration of the recalcitrant line. Other well-known recalcitrant cultivars, CR1821 and CR1113, also had improved regeneration in the immersion system. To our knowledge, this is the first study reporting the use of an immersion system to allow for the regeneration of gamma-ray mutants from recalcitrant indica rice materials.