Cotton is among the earliest adopted transgenic crops. Cotton producing insecticidal d-endotoxins (Cry proteins) from Bacillus thuringiensis (Bt) has been planted worldwide. Bt cotton was approved in 1997 in China for commercial use to control CBW and resulted in increased yields (Lu et al. 2012; Wu et al. 2008). However, Bt cotton only suppresses populations of a target pest with a narrow host range, and concerns with its widespread use include the potential for insects to evolve resistance (Bagla 2010), changing of the dominant pests and the impact on nontarget insect pests. Populations of mirid bugs (Heteroptera: Miridae) (Lu et al. 2010) and the common cutworm (Spodoptera litura) (Wan et al. 2008) have increased in cotton and other crops in association with regional increases in Bt cotton adoption and drops in insecticide use in that crop. The fall armyworm can be a significant cotton pest in certain years, is not the main target of Bt cotton technology (Hardke et al. 2015), and broke out in China in 2019. This situation requires the next generation of insect-resistant cotton to address potential pest outbreaks. Herbicide tolerance is an important direction in transgenic cotton breeding, as use of herbicide tolerance genes can reduce the labour cost in cotton production. Here, we report a novel transgenic cotton that is resistant to the common cutworm, fall armyworm, and CBW and tolerant to glyphosate and glufosinate, developed by transforming one construct containing the Lepidoptera insect-resistance gene Cry1Ab-vip3Aa, isolated from Bacillus thuringiensis, and the glyphosate and glyphosate herbicide-tolerance genes cp4-epsps and bar, respectively isolated from Agrobacterium tumefaciens strain CP4 and Streptomyces hygroscopicus. These genes are respectively under the control of the Ubi-10 and H4A748 promoters and the CaMV 35S promoter. The transgenic vector was constructed on the pCambia1300 backbone containing three target genes (Figure 1a) and introduced into Chinese cotton cultivar Y668 by Agrobacterium-mediated transformation. The resulting transgenic plants were tested for positive transformation by target-sequence-specific PCR, Southern blotting and bioassay tests (Figure S1, S2 and S4). Through multiple generations of testing resistance to Lepidoptera insect pests and herbicides, insertion copy variation and target gene expression, we screened out one clone line AHS10 for further study in the present research and potential utilisation. In this line, two copies of T-DNA inserted at one position were identified through resequencing and Southern blotting (Figure 1b; Figures S2 and S3). AHS10 with the fused Cry1Ab-vip3Aa genes showed good resistance to pests and tolerance to herbicides. Laboratory bioassays for Lepidoptera pest resistance were conducted in a culture room under 26°C, 14 h of light and 10 h of darkness (Figure 1d). In the T5 generation, self-pollinated pure AHS10 plants demonstrated extreme resistance to the common cutworm, reaching a mortality rate of 100% within 6 days (Figure 1e). The resistance to fall armyworm and CBW was also good (Figure 1e). Glyphosate herbicide resistance was evaluated in laboratory and field trials at concentrations of one (1.75 g/L), four (7 g/L) and eight (14 g/L) times the recommended application concentration. All bioassays supported that AHS10 plants at the seedling stage can tolerate at least eight times the recommended concentration of glyphosate (Figure 1c and Figure S4). Field test results similarly showed that AHS10 seedlings could tolerate the recommended concentration (1.5 g/L) of glufosinate and eight times the recommended concentration of glyphosate (Figures S4 and S5). Expression of cp4-epsps and Cry1Ab proteins was measured in AHS10 plants. For a given part of the plant, cp4-epsps content was consistently much higher than Cry1Ab content (Figure 1f). Bt content was low in the upper leaves, but high in the lower leaves, and the cp4-epsps content is almost the same at these two kinds of leaves (Figure 1f). Target protein contents were higher in leaves than in flower organs. Since Lepidopteran pests attack cotton bolls and flowers in addition to leaves, we quantified Cry1Ab protein in these two tissues. The content was low in flowers, but considerable in leaves and cotton bolls (Tables S1 and S2). Differences in expression most likely resulted from the different promoters used to control target gene expression. Current strategies for breeding the next generation of insect-resistant cotton are as follows: a pyramid of multiple Bt proteins (Carriere et al. 2015), developing genes for other insect-killing proteins (Badran et al. 2016), and RNA interference (Ma and Zhang 2019). As the pyramid strategy has been demonstrated to have a good pest control effect, we chose this strategy to achieve the breeding goal of improving cotton pest resistance. The AHS10 cotton plants are highly resistant to three kinds of Lepidopteran pests and tolerant to glyphosate and glufosinate, and have great potential for commercial utilisation. Given that AHS10 is to be used in agricultural production, we characterised its key agronomic traits. A10 did not differ significantly from the wild type (WT) in most agronomic traits except boll number, indicating production potential (Table S6). Promotion of pyramid strategy plants as the next generation of transgenic pest-resistant cotton could be an effective, environmentally friendly pest management measure. T.Z. designed research; J.Z. performed transgenic experiments; Q.M. and Z.S. conducted all field and laboratory bioassays, and pure-line development and analysed the data; S.J. conducted the identification of T-DNA border sequences; W.Z. conducted some field evaluation; Q.M. and T.Z. wrote the manuscript. We thank Prof. Zhicheng Shen at Zhejiang University for kindly providing the transformed vector for this research. This work was supported by the STI 2030-Major Projects (2023ZD04040-4), National Key Research and Development Program of China (2022YFF1001400), Fundamental Research Funds for the Central Universities (226-2022-00100) and Xinjiang Jinghua Seed Industry Co. Ltd. The authors declare no conflicts of interest. The data that supports the findings of this study are available in the Supporting Information of this article. Data S1: pbi70617-sup-0001-Supinfo.docx. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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