Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature and is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation of transgene-free genome-edited plants in a short period and has the advantages of simplicity, high efficiency, high specificity, and low production costs, which greatly facilitate the study of gene functions. In plant molecular breeding, the gene-editing efficiency of the CRISPR-Cas9 system has proven to be a key step in influencing the effectiveness of molecular breeding, with improvements in gene-editing efficiency recently becoming a focus of reported scientific research. This review details strategies and methods for improving the efficiency of CRISPR/Cas9 gene editing in plant molecular breeding, including Cas9 variant enzyme engineering, the effect of multiple promoter driven Cas9, and gRNA efficient optimization and expression strategies. It also briefly introduces the optimization strategies of the CRISPR/Cas12a system and the application of BE and PE precision editing. These strategies are beneficial for the further development and optimization of gene editing systems in the field of plant molecular breeding.

Background: ‘Regal Splendour’ (Hosta variety) is famous for its multi-color leaves, which are useful resources for exploring chloroplast development and color changes. The expressions of chlorophyll biosynthesis-related genes (HrHEMA, HrPOR and HrCAO) in Hosta have been demonstrated to be associated with leaf color. Herein, we isolated, sequenced, and analyzed HrHEMA, HrPOR and HrCAO genes. Subcellular localization was also performed to determine the location of the corresponding enzymes. After plasmid construction, virus-induced gene silencing (VIGS) was carried out to reduce the expressions of those genes. In addition, HrHEMA-, HrPOR- and HrCAO-overexpressing tobacco plants were made to verify the genes function. Changes of transgenic tobacco were recorded under 2000 lx, 6000 lx and 10,000 lx light intensity. Additionally, the contents of enzyme 5-aminolevulinic acid (5-ALA), porphobilinogen (PBG), chlorophyll a and b (Chla and Chlb), carotenoid (Cxc), superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), proline (Pro) and catalase (CAT) under different light intensities were evaluated. Results: The silencing of HrHEMA, HrPOR and HrCAO genes can induce leaf yellowing and chloroplast structure changes in Hosta. Specifically, leaves of Hosta with HrCAO silencing were the most affected, while those with HrPOR silencing were the least affected. Moreover, all three genes in tobacco were highly expressed, whereas no expression was detected in wild-type (WT). However, the sensitivities of the three genes to different light intensities were different. The highest expression level of HrHEMA and HrPOR was detected under 10,000 lx of illumination, while HrCAO showed the highest expression level under 6000 lx. Lastly, the 5-ALA, Chla, Cxc, SOD, POD, MDA, Pro and CAT contents in different transgenic tobaccos changed significantly under different light intensities. Conclusion: The overexpression of these three genes in tobacco enhanced photosynthesis by accumulating chlorophyll content, but the influential level varied under different light intensities. Furthermore, HrHEMA-, HrPOR- and HrCAO- overexpressing in tobacco can enhance the antioxidant capacity of plants to cope with stress under higher light intensity. However, under lower light intensity, the antioxidant capacity was declined in HrHEMA-, HrPOR- and HrCAO- overexpressing tobaccos.

AP3 has been studied and is reported to affect structural changes in floral organs in various plants. However, the function of the soybean AP3 genes in flower development is unknown. Here, the full-length cDNA sequence of GmAP3 was obtained by RACE and it was verified that it belongs to the MADS-box subfamily by a bioinformatics analysis. The expression of GmAP3 is closely related to the expression of essential enzyme genes related to flower development. Yeast two-hybrid assays demonstrated that GmAP3 interacts with AP1 to determine the identity of flower organ development. A follow-up analysis showed that overexpression of the GmAP3 gene advanced flowering time and resulted in changes in floral organ morphology. The average flowering time of overexpressed soybean and tobacco plants was 6–8 days earlier than that of wild-type plants, and the average flowering time of gene-edited soybean and tobacco plants was 6–11 days later than that of wild-type plants. In conclusion, GmAP3 may directly or indirectly affect the flower development of soybean. The results of this study lay the foundation for further research on the biological functions of MADS transcriptional factors in soybeans.

Combined elemental deficiencies are more complex and insidious physiological metabolic responses than single elemental stresses. To determine the effects of phosphorus (P), iron (Fe), zinc (Zn), and their deficient combinations on photosynthetic characteristics of rice seedlings, we investigated their effects on dry weight, chlorophyll (Chl) content, rapid photosynthetic carbon assimilation CO₂ responses, and Chl fluorescence in four-week-old rice (CB9 and BJ1 cultivars) seedlings. The results showed that the dry matter, maximum carboxylation efficiency (V_c,max), and maximum electron transfer efficiency (J_max) of seedlings were all reduced to different degrees under the element deficiency treatments. JIP-test analysis showed that the decrease in the concentration of active PSII reaction centers (RC/ABS) under -Zn treatment was the main reason for the inhibition of performance index PI_ABS. The -P treatment reduced RC/ABS and inhibited electron transfer (ψ_Eo). Primary photochemical reactions (φ_Po) of -P-Zn treated seedlings were also inhibited compared to the -P treatment. The -Fe and -Fe-Zn treatments inhibited photosynthesis most severely, which not only reduced RC/ABS but also severely inhibited φ_Po and ψ_Eo. Notably, the -P-Fe and -P-Fe-Zn treatments of the CB9 improved the RC/ABS, alleviating the limitation of Fe deficiency. These results help enhance the understanding of the complex relationship between nutrient balance and photosynthesis, especially for P, Fe, Zn, and their combined deficiency.

Background: Hosta plantaginea (Lam.) Aschers (HPA), a species in the family Liliaceae, is an important landscaping plant and herbaceous ornamental flower. However, because the flower has only two colors, white and purple, color matching applications are extremely limited. To date, the mechanism underlying flower color regulation remains unclear. Methods: In this study, the transcriptomes of three cultivars—H. plantaginea (HP, white flower), H. Cathayana (HC, purple flower), and H. plantaginea ‘Summer Fragrance’ (HS, purple flower)—at three flowering stages (bud stage, initial stage, and late flowering stage) were sequenced with the Illumina HiSeq 2000 (San Diego, CA, USA). The RNA-Seq results were validated by qRT-PCR of eight differentially expressed genes (DEGs). Then, we further analyzed the relationship between anthocyanidin synthase (ANS), chalcone synthase (CHS), and P450 and the flower color regulation by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Eukaryotic Orthologous Groups (KOG) network and pathway enrichment analyses. The overexpression of CHS and ANS in transgenic tobacco petals was verified using qRT-PCR, and the petal colors associated with the overexpression lines were confirmed using absorbance values. Results: Over 434,349 transcripts were isolated, and 302,832 unigenes were identified. Additionally, through transcriptome comparisons, 2098, 722, and 606 DEGs between the different stages were found for HP, HC, and HS, respectively. Furthermore, GO and KEGG pathway analyses showed that 84 color-related DEGs were enriched in 22 pathways. In particular, the flavonoid biosynthetic pathway, regulated by CHS, ANS, and the cytochrome P450-type monooxygenase gene, was upregulated in both purple flower varieties in the late flowering stage. In contrast, this gene was hardly expressed in the white flower variety, which was verified in the CHS and ANS overexpression transgenic tobacco petals. Conclusions: The results suggest that CHS, ANS, and the cytochrome P450s-regulated flavonoid biosynthetic pathway might play key roles in the regulation of flower color in HPA. These insights into the mechanism of flower color regulation could be used to guide artificial breeding of polychrome varieties of ornamental flowers.

Anthocyanins are natural colorants are synthesized in a branch of the flavonoid pathway. Dihydroflavonol-4reductase (DFR) catalyzes dihydroflavonoids into anthocyanins biosynthesis, which is a key regulatory enzyme of anthocyanin biosynthesis in plants. Hosta ventricosa is an ornamental plant with elegant flowers and rich colorful leaves. How the function of HvDFR contributes to the anthocyanins biosynthesis is still unknown. In this study, the DFR homolog was identified from H. ventricosa and sequence analysis showed that HvDFR possessed the conserved NADPH binding and catalytic domains. A phylogenetic analysis showed that HvDFR was close to the clade formed with MaDFR and HoDFR in Asparagaceae. Gene expression analysis revealed that HvDFR was constitutive expressed in all tissues and expressed highly in flower as well as was positively correlated with anthocyanin content. In addition, the subcellular location of HvDFR showed that is in the nucleus and cell membrane. Overexpression of HvDFR in transgenic tobacco lines enhanced the anthocyanins accumulation along with the key genes upregulated, such as F3H, F3ʹH, ANS, and UFGT. Our results indicated a functional activity of the HvDFR, which provide an insight into the regulation of anthocyanins content in H. ventricosa.