Sexual transfer of the CRISPR/Cas genome-editing system to targeted cotton cultivars could bypass their recalcitrance to regeneration from tissue culture. We used sexual hybridization to transmit a CRISPR/LbCas12a system from a regenerable Gossypium hirsutum donor to a nonregenerable G. barbadense recipient. We knocked out the GbCLA and GbPGF genes in the recipient, generating respectively albino and glandless phenotypes. Focusing on GbPGF, we detected novel mutations in the progeny across generations, and developed a set of nearly isogenic lines. The average editing efficiency of the target gene at crRNA1 exceeded 70% in the BC₃F₁ generation, yielding plants with agronomic traits or fiber quality nearly identical to those of the recurrent parent but lacking glands or gossypol. We introduced the CRISPR/LbCas12a system into three other nonregenerable G. hirsutum genotypes and one diploid cotton by hybridization and edited three more genes in two recipients.

Cotton production faces significant challenges from insect pests, with chemical pesticide use becoming increasingly limited by resistance and environmental concerns. This study explores the potential use of caffeine, a natural plant alkaloid, as an environmentally friendly insect resistance strategy in cotton. Exogenous caffeine application demonstrated potent insecticidal effects against cotton bollworm (Helicoverpa armigera) larvae, with concentrations ≥ 2 mg mL⁻¹ causing near-complete feeding cessation and up to 70% larval mortality. Building on this, we engineered transgenic cotton (Gossypium hirsutum cv. Jin668) for heterologous caffeine biosynthesis by introducing three key N-methyltransferase genes (CaXMT1, CaMXMT1, CaDXMT1) by multiple gene transformation. Transgenic lines expressing all three genes showed remarkable caffeine accumulation (up to 3.59 mg g⁻¹ dry weight), whereas two-gene combinations exhibited wild-type-level production. Feeding preference assays revealed that caffeine-enriched cotton strongly deterred feeding by H. armigera. Non-choice feeding trials demonstrated reduced leaf consumption and reduced larval growth in H. armigera fed on caffeine-producing cotton. The study highlights the effectiveness of synthetic biology approaches using the TGSII-UNiE multigene stacking system, despite challenges in transgene stability. This work advances plant-derived insect resistance research and provides a sustainable framework for reducing chemical pesticide reliance in cotton production, while underscoring unique potential of cotton as a synthetic biology platform for secondary metabolite engineering.

Global agriculture is facing severe challenges, and breeding technology is the foundation and key to the development of the seed industry. Gene editing technology refers to the precise modification of target genes to achieve deletion, insertion, and replacement of specific target gene fragments. It can precisely modify target genes or introduce certain excellent genes into crops to produce crops with excellent agronomic traits, which has great potential in molecular design breeding and is of great significance to ensuring food security. Weed damage has a huge impact on the yield and quality of crops. To control weed damage efficiently, safely and sustainably has always been a hot research topic. Currently, more than 200 types of chemical herbicides have emerged in the global market. Using chemical methods to control weeds has become an important part of modern agriculture, and the cost of weed control has been significantly reduced by promoting herbicide-resistant crops. However, with the large-scale promotion of herbicide-resistant crops and the long-term use of single herbicides, environmental safety problems such as weed resistance and escape of resistant genes have gradually been discovered. Currently, the development of functional genomics, bioinformatics and genetic engineering technology (especially the widespread application of gene editing technology in plants) has created conditions for the creation of herbicide-resistant crops and new efficient weed control systems. In this article, the main target genes of herbicides that inhibit amino acid biosynthesis, lipid metabolism, carotenoid, plastoquinone and tocopherol biosynthesis pathways and their action mechanisms are introduced at first. Secondly, two methods for mining new herbicide resistance genes and herbicide systems are introduced, including the directed mutation method of herbicide resistance genes within crops based on CRISPR/Cas system and the resistance gene guidance method based on the co-evolution theory of natural product and organisms in nature. Moreover, the research progress of three breeding methods for herbicide resistant crops was reviewed, including conventional breeding, transgenic breeding and CRISPR/Cas genome editing based breeding. Among them, the research progress of CIRSPR/Cas system, base editing technology, and prime editing system in cultivating herbicide resistant crops were highlighted. The main challenge faced by chemical control of weeds and herbicide resistant crops is resistant weeds and environmental safety issues, and gene escape, respectively. At present, the rapid development of genome editing technology provides new solutions and new opportunities for the development of herbicide resistant crops in the post genome era. Finally, the prospects for the future of herbicide-resistant crops were provided.

An efficient genome editing tool, the CRISPR/Cas12a system, has been used in research on plant functional genomics and the improvement of agronomic traits. In this study, the CRISPR/Cas12a system was optimized by using the endogenous pGhαGloA promoter in cotton. With this system, crRNAs were driven by the Pol Ⅱ pGhaGloA promoter to construct the pGhRBE3-pGhαGloA-GhPGF vector and carry out genetic transformation. The vector worked efficiently in all positive transgenic plants and the editing efficiencies at the crRNA1 and crRNA2 target sites were up to 93.37 and 88.24%, respectively. This system had significantly higher editing efficiency than the pGhRBE3 system with a Pol Ⅲ promoter-Ubi 6.7 promoter, indicating that the Pol Ⅱ promoter is more suitable for expressing multiple sgRNAs or crRNAs than the Pol Ⅲ promoter in cotton. The vector mainly generated the editing type of fragment deletion, and the deletion sizes were in the range of 3–12 bp with the editing sites spanning the 14th to 29th bases downstream of the protospacer adjacent motif (PAM). All the targeted mutation loci were stably inherited from the T₀ to T₂ generations, and three transgene-free lines with target site mutations in the GhPGF gene were obtained. These glandless and gossypol-free (or low content) cotton germplasms will play a key role in healthy cottonseed oil/cake production. Therefore, the CRISPR/Cas12a system driven by the pGhαGloA promoter can efficiently edit target genes in cotton, so it can provide a powerful tool for cotton functional genomics and genetic improvement.

Jute (Corchorus capsularis L.) is the second most important natural plant fiber source after cotton. However, developing an efficient gene editing system for jute remains a challenge. In this study, the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4, an elite jute variety widely cultivated in China. The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation (BiFC) assays. The CHLOROPLASTOS ALTERADOS 1 (CcCLA1) gene, which is involved in the development of chloroplasts, was targeted for editing at two sites in Meifeng 4. Based on this hairy root transformation, the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and -GhU6.9 promoters, respectively, to assess the efficiency of gene editing. Results indicated the 50.0% (GhU6.7) and 38.5% (GhU6.9) editing events in the target 2 alleles (gRNA2), but no mutation was detected in the target 1 allele (gRNA1) in transgenic-positive hairy roots. CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation. Two CcCLA1 mutants were albinic, with a gene editing efficiency of 5.3%. These findings confirm that the CRISPR/Cas9 system, incorporating promoter GhU6.7, can be used as a gene editing tool for jute.

Cotton (Gossypium hirsutum) is an important fiber crop worldwide. Insect attack causes cotton yield and quality losses. However, little is known about the mechanism of cotton response to insect attack. We simulated insect feeding by applying insect oral secretions (OS) to wounds, and combined transcriptome and metabolome analysis to investigate how OS from two major pest species (Helicoverpa armigera and Spodoptera litura) affect cotton defense responses. We found that respectively 12,668 and 13,379 genes were differentially expressed in comparison with wounding alone. On addition of OS, the jasmonic acid signaling pathway was rapidly and strongly induced, whereas genes involved in salicylic acid biosynthesis were downregulated. On constructing a coexpression gene network, we identified a hub gene encoding a leucine-rich repeat receptor kinase that may play an important role in early signal recognition and transduction. OS from the two insect species altered the abundance of flavonoid-related compounds in different patterns. Gossypol remained in lower concentration after OS application than after wounding alone, suggesting a suppressive effect of OS on cotton defense response. This study illustrated transcriptional and metabolic changes of cotton in responding to OS from two chewing insect species, identified potential key response genes, and revealed evidence for OS inhibition of wounding-induced cotton defense response.

Genes encoding reporter proteins are used as visual marker-assisted tools in genetic transformation as well as plant breeding. In this study, the red fluorescent protein identified in Discosoma sp. coral (DsRed2) was successfully used as a visual marker for cotton genetic engineering. DsRed2 was successfully expressed in two cotton cultivars, JIN668 and YZ1, driven by the CaMV-35S promoter via the Agrobacterium-mediated transformation. Our results suggest that DsRed2 expression provides an early-stage selection tool for the transgenic calli via visual observation. Red fluorescence can be detected not only in callus and somatic embryos but also in most tissues and organs of mature plants. The transgenic line Yz-2-DsRed2 was crossed with four different cotton cultivars to assess the transgene heritability and stability in different genetic backgrounds. The heritability of the red color was highly stable when Yz-2-DsRed2 was used as a male parent. The DsRed2 gene expressed 100% in the F₁ hybrids. To investigate the relationship between DsRed2 transcription and DNA methylation, a methylation-specific PCR approach was applied to the CaMV-35S promoter region. The results showed a negative association between DNA methylation level in the promoter region and the transgene transcription. Taken together, these findings suggest DsRed2 a visual reporter gene for cotton genetic transformation and molecular breeding programs.