Developing of transgenic glyphosate-tolerant Indica restorer line with commercial application potential

Abstract

Hybrid rice breeding offers an important strategy to improve rice production, and therefore is crucial for ensuring worldwide food security. Purity of hybrids is a key factor in keeping the high production of hybrid rice. While another important thing is mechanical weeding of hybrid rice, which could greatly release labor from laborious work and cut the production cost. Both of these two problems could be partly resolved by developing glyphosate-tolerant restorer lines. In this study, a novel glyphosate-tolerant gene I.variabilis-EPSPS* was transferred into elite indica restorer line Minghui 86 (MH86) by Agrobacterium-mediated transformation. By Southern blot and inverse PCR analysis, 3 transgenic restorer lines (MY28, MY50, and MY51) with a single copy of I.variabilis-EPSPS* integrated into the intergenic regions were selected from 72 regenerated transgenic plants. The inheritance of I.variabilis-EPSPS* in the 3 transgenic restorer lines was proved to follow Mendelian inheritance by germinating test. In the following generations, according to Southern blot, Northern blot, 3’RACE, and Western blot, I.variabilis-EPSPS* were proved to stably inherit and express in the candidate transgenic restorer lines. At the same time, glyphosate tolerance assay was performed in the field, in which the candidate transgenic restorer lines kept normal agronomic traits under glyphosate treatments with a dosage as high as 8400 g/ha. Therefore, the candidate transgenic restorer lines were highly tolerant to glyphosate. The 3 candidate transgenic restorer lines were respectively hybridized with the male sterile line II-32A, and their hybrids were respectively named as MY28H, MY50H, and MY51H. According to Southern blot and Western blot analysis, I.variabilis-EPSPS* could stably inherit to F1 hybrids and express in these hybrids, which indicated that the true hybrids could be distinguished by I.variabilis-EPSPS*. In the field test, the transgenic hybrids with I.variabilis-EPSPS* could suffer 3360 g/ha glyphosate without the damage of agronomic traits, which indicated the application potential of these hybrids in direct seeding. Collectively, the transgenic glyphosate-tolerant restore lines developed in this study were highly tolerant to glyphosate, and they had potential to be used in commercial production of hybrid rice.