One-line hybrid rice with high-efficiency synthetic apomixis and near-normal fertility.

KEY MESSAGE: High-frequency clonal seeds and near-normal fertility were obtained by engineering synthetic apomixis in hybrid rice. The one-line strategy, with the advantage of unnecessary seed production, is the final stage for the hybrid rice development and can be achieved through the fixation of heterosis via artificially inducing apomixis. Recently, synthetic apomixis has been generated in rice by combining MiMe (Mitosis instead of Meiosis) with either the ectopic expression of BABY BOOM (BBM1 or BBM4) or mutation of MATRILINEAL (MTL), resulting in over 95.00% of clonal seeds. However, the frequency of clonal seeds was only 29.20% when AtDD45 promoter was used to drive BBM1. In addition, achieving both a high frequency of clonal seeds and near-normal fertility simultaneously had been elusive in earlier strategies. In this study, using AtDD45 promoter to drive BBM1 expression in combination with the MiMe mutant resulted in the apomixis frequency as high as 98.70%. Even more, employing fusion promoters (AtMYB98_AtDD1_OsECA1-like1) to drive WUS expression in combination with pAtDD45:BBM1 and MiMe could produce clonal seeds at rates of up to 98.21%, the highest seed setting rate reached to 83.67%. Multiple-embryos were observed in clonal lines at a frequency ranging from 3.37% to 60.99%. Transmission of the high frequency of apomixis through skipped generations (atavism) was identified in two clonal lines, even though it remained stable in the majority of clonal lines. These findings significantly advance the pursuit of fixed heterosis in rice through synthetic apomixis, edging closer to its agricultural application.

Double-seedlings and embryo-free seeds generated by genetic engineering.

Apomixis can fix the heterosis of Hybrid F1, by maintaining its heterozygous genotype, and is an ideal way for the development of hybrid rice. In this paper, we designed an engineering strategy for realizing apomictic reproduction of hybrid rice in the way of induce adventitious embryos. An embryogenesis gene, AtWUS, controlled by the ovule-specific promoter, a ribonuclease gene Barnase driven by the egg cell-specific promoter pDD45, and an inactivation gene ZmAA1 driven by the pollen-specific promoter pG47 were simultaneously integrated into one T-DNA, and co-transformed with the second T-DNA carrying a Barstar gene. Double-seedlings were observed in transgenic line. Whole-genome sequencing and ploidy levels confirmed by flow cytometry showed that one of the double-seedlings was heterozygous diploid and the other seedling was homozygous haploid, which confirmed that embryogenesis in one of the double-seedlings arises from the zygote after fertilization and the other derived from an unfertilized gamete. Meanwhile we obtained embryo-free seeds at frequencies of 2.6% to 3.8% in T1 generation, and 0.75% to 3% in T2 generation. Though we did not obtained adventitious embryos in hybrid rice in this study, the phenomenon of double-seedlings and embryo-free seeds in transgenic line was informative and strongly suggested that endosperm development is an autonomously organized process in rice, independent of egg cell fertilization and embryo-endosperm communication. This provides novel insights into the induction of haploid embryos and lends theoretical support to successful clonal propagation using synthetic apomixis.

Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote.

In prokaryotes, few studies have applied the flippase (FLP)/P1-flippase recombination target (LoxP-FRT) recombination system to switch gene expression. This study developed a new method for switching gene expression by constructing an FLP/LoxP-FRT site-specific recombination system in Escherichia coli. To this end, we placed the Nos terminator flanked by a pair of LoxP-FRT in front of enhanced green fluorescent protein (eGFP). The Nos terminator was used to block the expression of the eGFP. When a plasmid expressing FLP was available, deletion of the Nos terminator would allow expression of eGFP. The regulatory effect was demonstrated by eGFP expression. The efficiency of the gene switch was calculated as high as 89.67%. The results showed that the FLP/LoxP-FRT recombinase system could be used as a gene switch to regulate gene expression in prokaryotes. This new method for switching gene expression could simplify the gene function analysis in E. coli and other prokaryotes, as well as eukaryotes.