Creation of Fragrant Rice by Targeted Editing of fgr Gene Using Magnetic Nanoparticle- mediated Pollen Magnetofection in Rice

Abstract

Background CRISPR/Cas9 technology has been widely used for plant genome editing, while the delivery of gene editing components mainly depends on Agrobacterium or bombardment. They both need a long period of tissue culture process and some varieties are very difficult to regenerate from tissue culture, which seriously limits the process of gene editing. Result The CRISPR/Cas9 expression vector of fragrance gene fgr was constructed and combined with magnetic nano particles (MNPs) to form a gene carrier complex. To determine the suitable method of magnetofection, the appropriate mass ratios of MNPs / vector was explored, and the location of the complex in pollen was confirmed. The T1 plants obtained from the pollination of magnetofected pollen on wild-type rice showed decreased height and seed setting rate, with the hygromycin resistant rate (i.e. positive rate) 13.07% and 3 lined mutated. T2 plants were obtained from the self-crossing of positive T1 lines, with positive rate 58.80% and 8 lines mutated. According to the mutation of T1 and T2 generation, the genetic modes of magnetofected rice mainly included “continuous-generation editing” and “interval-generation editing”. It showed great potential that negative and homozygous mutated plants (ho/-C) could be obtained as early as in T2 generation in lines with “continuous-generation editing”. Conclusion Gene targeted editing mediated by pollen magnetofection is tissue culture independent, which shortens the breeding cycle, provides new technical support for rice breeding, and is of great significance to accelerate the breeding process of fine varieties.