Chloroplasts are organelles that contain genetic materials (DNA) in higher plant cells. The special genetic characteristics of chloroplasts mean that plasmid transformation has important research value, so it has become an important research direction second to nuclear transformation. Although the techniques of chloroplast genome modification have been successfully applied in tobacco and extended to other high plants, there are still many limitations. Exogenous genes are integrated into the chloroplast genome through homologous recombination. Therefore, the low efficiency of homologous recombination directly limits transformation efficiency. Gene editing with fixed-point cutting function and DNA damage repair mechanism may effectively improve the efficiency. In the present study, we aimed to use CRISPR/Cas9 to cut the site between two homologous recombinant fragments in chloroplast transformation to improve the efficiency by activating the DNA damage repair mechanism. The Cas9 gene and gRNA were added to the chloroplast transformation system of tobacco by co-transformation or integration into a transformation vector. The acquired resistant plants were screened by multiple selection of spectinomycin and chloroplast DNA was isolated for molecular detection by PCR. The results showed that the efficiency of chloroplast transformation increased by 6–10 times with the addition of gene editing technology. Although the transformation efficiency was still far below the level of nuclear transformation, this study may help to increase the efficiency of the plant chloroplast transformation system, and expand the types of plant receptors.

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.

Heterosis is a common phenomenon in biology, and fixation of heterosis can be achieved via artificially induced apomixis. Sundaresan's team used mitosis instead of meiosis (MiMe) rice mutants that ectopically expressed BBM1 in egg cells to induce the production of clonal seeds. In this study, we ectopically expressed BBM1 in MiMe mutants, and 62.50% of the seeds produced by Yongyou 4949 hybrid rice were clonal. More importantly, a high frequency of twin seedlings (33.56%) was detected. Moreover, a low frequency (1.41%) of twin seedlings was obtained only when BBM1 was ectopically expressed, and no twin seedlings were found when the genome of the MiMe mutant was edited. These findings suggest that the high frequency of twin seedlings resulted from the ectopic expression of BBM1 combined with MiMe. The embryo sacs of the twin seedlings were removed and subjected to cytological observations. One sperm combined with the polar nucleus to develop into the endosperm, and the other sperm combined with the egg cell to form a zygotic embryo. Moreover, a synergid cell near the egg cell developed into an embryo through apogamy, thus resulting in the formation of two embryos. The results showed that AtDD45-driven BBM1 could induce synergid cells to develop into embryos autonomously through normal fertilization of egg cells, resulting in the formation of twin seedlings. This study provides a theoretical reference and new ideas for research on apomixis in hybrid rice and is important for promoting the fixation of heterosis of hybrid rice.

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