Agrobacterium-mediated in planta transformation of cut coleoptile: a new, simplified, and tissue culture-independent method to deliver the CRISPR/Cas9 system in rice

Background Agrobacterium -mediated transformation and particle bombardment are the two common approaches for genome editing in plant species using CRISPR/Cas9 system. Both methods require careful manipulations of undifferentiated cells and tissue culture to regenerate the potentially edited plants. However, tissue culture techniques are laborious and time-consuming. Methods and results In this study, we have developed a simplified, tissue culture-independent protocol to deliver the CRISPR/Cas9 system through in planta transformation in Malaysian rice ( Oryza sativa L. subsp. indica cv. MR 219). Sprouting seeds with cut coleoptile were used as the target for the infiltration by Agrobacterium tumefaciens and we achieved 9% transformation efficiency. In brief, the dehusked seeds were surface-sterilised and imbibed, and the coleoptile was cut to expose the apical meristem. Subsequently, the cut coleoptile was inoculated with A. tumefaciens strain EHA105 harbouring CRISPR/Cas9 expression vector. The co-cultivation was conducted for five to six days in a dark room (25 ± 2 °C) followed by rooting, acclimatisation, and growing phases. Two-month-old plant leaves were then subjected to a hygromycin selection, and hygromycin-resistant plants were identified as putative transformants. Further validation through the polymerase chain reaction verified the integration of the Cas9 gene in four putative T 0 lines. During the fruiting stage, it was confirmed that the Cas9 gene was still present in three randomly selected tillers from two 4-month-old transformed plants. Conclusion This protocol provides a rapid method for editing the rice genome, bypassing the need for tissue culture. This article is the first to report the delivery of the CRISPR/Cas9 system for in planta transformation in rice.