Effect of Different Sowing Dates and Densities on Individual Morphological Development of Super Short-season Insect-resistant Cotton

[Objective] The paper was to explore the effect of different sowing dates and densities on individual morphological development of super short-season insect-resistant cotton,confirm their effects on vegetative and reproductive growth of cotton,so as to provide theoretical and practical guidance for sowing date and density management of cotton planting in Jidong cotton growing region in Yellow River Basin.[Method] With super short-season insect-resistant cotton＂546＂as materials,the effects of different sowing dates（sowing dateⅠ：May 20;sowing dateⅡ：June 2;sowing date Ⅲ：June 14）and densities（low density：120 000 plants/hm2;middle density：150 000 plants/hm2;high density：180 000 plants/hm2）on individual morphological development of super short-season insect-resistant cotton were explored.[Result] Different sowing dates and density treatments significantly affected the individual morphological development of super short-season insect-resistant cotton＂546＂.The effectiveness of sowing date was higher than the effectiveness of density,and the effectiveness of sowing date on development of number of individual fruit branches was higher than that on plant height and stem diameter.[Conclusion] The regulation of sowing date and density during the cultivation process of super short-season insect-resistant cotton ＂546＂ in Jidong cotton growing region in Yellow River Basin could effectively promote vegetative and reproductive growth of cotton,strengthening its production base.

Three decades and beyond:Breeding,biotech breakthroughs and future of China's GM insect-resistant cotton

Cotton(Gossypium spp.)is a pivotal crop in the global textile industry,providing essential natural fibers.Additionally,cottonseed offers significant value as a source of oil and as feed for livestock(Huang et al.2021;Wen et al.2023).The sector,dependent on cotton,features a comprehensive value chain extending from the processing of fibers to the production of finished textiles,and it employs tens of millions of individuals(Dorward et al.1970).

Study on Spatial-temporal Dynamics of Bt Toxic Protein Expression in Insect-resistant Transgenic Cotton and Its Degradation in Soil

[Objective] This study aimed to investigate the spatial-temporal dynamics of Bt toxic protein expression in insect-resistant transgenic cotton and its degradation in soil. [Method] Btcry1Ac toxic protein expression in roots, stems and leaves of transgenic cotton Guoshen GK45 at different developmental stages and the annual average content of BtCry1Ac toxin protein in the topsoil, rhizosphere soil and following cotton-growing area were explored and analyzed by using enzyme linked immuno sorbed assay (ELISA). [Result] The content of exogenous BtCry1Ac toxin protein decreased during the growth process of insect-resistant transgenic cotton; to be specific, the content of BtCry1Ac toxin protein in cotton stems and leaves decreased more slowly and always maintained a high level, while that in roots decreased rapidly and reached a minimum level to the following plant growth and development stage. BtCry1Ac toxin protein was detected in topsoil of both non-transgenic and transgenic cotton-growing areas, and the content of BtCry1Ac toxin protein increased in topsoil of following cotton-growing area, which was very low in rhizosphere soil. [Conclusion] Determination of Btcry1Ac toxic protein provides scientific basis for the risk assessment of the cultivation of genetically modified crops and the safety evaluation of soil ecosystem.

Commercial production of transgenic Bt insect-resistant cotton varieties and the resistance management for bollworm (Helicoverpa armigera Hubner)

There are currently three kinds of transgenic Bt insect-resistant cotton germplasm lines, Shanxi 94-24, Zhongxin 94 and R19, in China. They showed high resistance to the neonate larvae of bollworm (Helicoverpa armigera). Transgenic Bt insect-resistant cotton varieties or hybrids have been bred using the three kinds of germplasm lines as parents. Our researches reveal that there exist different expressions in resistant level at different developmental stages in the three categories of germplasm lines. When neonate larvae are fed with leaves of cotton plant at the seeding stage with less than 10 leaves on the main stem, the mortality of the neonate larvae is 100%, but the resistance level will decline at later season. When Bt gene has been transferred to the cotton genome, it can be steadily transferred to the progeny, the level of resistance to bollworm keeps fundamentally uniform. Such insects as tobacco budworm (Heliothis virencens) in laboratory directive selection are very apt to produce resistance

Selection of Homozygous Cotton Lines Transformed with Two Insect-Resistant Genes

A plant expression vector containing a chimeric Bt29K gene coding for the activated Cry1Ac protein and the arrowhead proteinase inhibitior gene API B were introduced into the cotton cultivar Jihe321 mediated by Agrobactertium tumefaciens. Based on the results of kanamycin resistant testing, PCR detection for both foreign genes and insect bioassay using Heliethis armigera , nine transgenic homozygous cotton lines with insect resistance of more than 90% and better agronomic traits were bred through six generations from the original transgenic plants. Results from insect bioassay and sequence analysis of the PCR products of plants from some homozygous lines indicated that the chimeric Bt29K gene was stably inherited in these transgenic cotton lines. The main agronomic characters of these homozygous cotton lines, such as boll productivity and fibre strength, were better than that of the original cotton cv. Jihe321.

Bt protein expression in the transgenic insect-resistant cotton in China

According to statistics of International Service for the Acquisition of Agri-biotech Applications(ISAAA),18million farmers in 28 countries planted more than 181million hectares of genetically modified(GM)crops in2014,at an annual growth rate of between 3%–4%,with6.3 million ha more than 2013[1].Among these counties,China ranks the sixth with 3.9 million ha.Globally,27kinds of transgenic crops have been approved.The four main food groups for which GM varieties are

Study on Insect-resistant Transgenic Cotton Harbouring Double-gene and Its Resistance to Insect Pests

By using the method of pollen tube pathway,the synthesized GFM CryIA gene and modified CpTI gene were transfered into the elite cotton(Gossypium hirsutun L.)varieties(lines).Through the field and lab identifications,the insect-resistant transgenic plants were obtained.PCR analysis indicated that both the synthesized GFM CryIA gene and modified CpTI gene presented positive reaction.In R1 the boliworm resistance of each transformant was different,and the insect-resistance of R3 of ZGK9708 was stable.

Effects of insect-resistant transgenic cotton on ground-dwelling beetle assemblages(Coleoptera)

We compared the ground-dwelling beetle assemblages under four scenarios in which transgenic Bt（Cry 1Ac） cotton（33B）,transgenic Bt（Cry 1Ac）＋CpTI cotton（SGK321）,conventional cotton（33）,conventional cotton（Shiyuan 321） in North China.During the survey in two years（2009-2010）,24 ground beetle species were captured with pitfall traps in 20 plots which included five replicates for each cotton type.No significant difference was observed in the number of ground beetle species captured,activity density,evenness and Shannon-Wiener diversity among the four cotton varieties.Chlaenius posticalis was less abundant in transgenic Bt＋CpTI cotton（SGK321） fields than its conventional cotton（Shiyuan 321）,but more abundant in transgenic Bt cotton（33B） fields compared with its conventional cotton（33）.There was no significant difference for other abundant species between in transgenic cotton and in conventional cotton fields.Based on non-metric multidimensional scaling（NMDS） analysis,ground-dwelling beetle assemblages were similar in transgenic and conventional cotton over the two years,but the ground-dwelling beetle assemblages in transgenic cotton 33 B significantly differed from that in the conventional cotton（strain 33） in 2010.No strong evidence that the transgenic cotton effect on ground-dwelling beetle assemblages was found in this study.

Research on Frequency of Exogenous Gene Flow from Marber-free Insect-resistant Transgenic Rice to Conventional Rice Varieties

[Objective] This study aimed to investigate the frequency of exogenous gene flow to non-transgenic conventional rice cultivars and assess the potential risks of marker-free of insect-resistant transgenic rice to agricultural ecological environment. [Method] Insect-resistant transgenic rice variety HUAHUI No.1 was planted as the experimental material and surrounded by several non-transgenic conventional rice cultivars. F1 non-transgenic rice seeds were collected according to different distances and identified by using PCR technology, the frequency of exogenous gene flow from insect-resistant transgenic rice to non-transgenic conventional rice cultivars was counted and analyzed. [Result] The average frequency of exogenous Bt gene flow to P13381 and CHUNJIANG063 was 0. Transgene flow occurred to varying degrees from insect-resistant transgenic rice HUAHUI No.1 to several non-transgenic rice lines including HEX122-2, TIANXlANG, MINGHUI63 and Pl157, with the maximum average gene flow frequency of 0.875%. The frequency of gene flow was gradually reduced with the increase of distance, and the average transgene flow frequency de- creased to 0 in all the sampling points 7 m away from transgenic rice material. [Conclusion] This study revealed that the exogenous gene flow frequency of insect-re- sistant transgenic rice variety HUAHUI No.1 was very low, leading to very small risk to the eco-environment. Rational distribution in the field for physical isolation, keeping the appropriate distance and scientific farming arrangement to avoid the synchronization of flowering can effectively control the exogenous gene flow from transgenic rice and reduce he ecological risks caused by transgene escape.

Strigolactones modulate cotton fiber elongation and secondary cell wall thickening

Cotton is one of the most important economic crops in the world,and it is a major source of fiber in the textile industry.Strigolactones(SLs)are a class of carotenoid-derived plant hormones involved in many processes of plant growth and development,although the functions of SL in fiber development remain largely unknown.Here,we found that the endogenous SLs were significantly higher in fibers at 20 days post-anthesis(DPA).Exogenous SLs significantly increased fiber length and cell wall thickness.Furthermore,we cloned three key SL biosynthetic genes,namely GhD27,GhMAX3,and GhMAX4,which were highly expressed in fibers,and subcellular localization analyses revealed that GhD27,GhMAX3,and GhMAX4 were localized in the chloroplast.The exogenous expression of GhD27,GhMAX3,and GhMAX4 complemented the physiological phenotypes of d27,max3,and max4 mutations in Arabidopsis,respectively.Knockdown of GhD27,GhMAX3,and GhMAX4 in cotton resulted in increased numbers of axillary buds and leaves,reduced fiber length,and significantly reduced fiber thickness.These findings revealed that SLs participate in plant growth,fiber elongation,and secondary cell wall formation in cotton.These results provide new and effective genetic resources for improving cotton fiber yield and plant architecture.

Pectin methylesterase inhibitors GhPMEI53 and AtPMEI19 improve seed germination by modulating cell wall plasticity in cotton and Arabidopsis

The germination process of seeds is influenced by the interplay between two opposing factors,pectin methylesterase(PME)and pectin methylesterase inhibitor(PMEI),which collectively regulate patterns of pectin methylesterification.Despite the recognized importance of pectin methylesterification in seed germination,the specific mechanisms that govern this process remain unclear.In this study,we demonstrated that the overexpression of GhPMEI53is associated with a decrease in PME activity and an increase in pectin methylesterification.This leads to seed cell wall softening,which positively regulates cotton seed germination.AtPMEI19,the homologue in Arabidopsis thaliana,plays a similar role in seed germination to GhPMEI53,indicating a conserved function and mechanism of PMEI in seed germination regulation.Further studies revealed that GhPMEI53 and AtPMEI19 directly contribute to promoting radicle protrusion and seed germination by inducing cell wall softening and reducing mechanical strength.Additionally,the pathways of abscicic acid(ABA)and gibberellin(GA)in the transgenic materials showed significant changes,suggesting that GhPMEI53/AtPMEI19-mediated pectin methylesterification serves as a regulatory signal for the related phytohormones involved in seed germination.In summary,GhPMEI53 and its homologs alter the mechanical properties of cell walls,which influence the mechanical resistance of the endosperm or testa.Moreover,they impact cellular phytohormone pathways(e.g.,ABA and GA)to regulate seed germination.These findings enhance our understanding of pectin methylesterification in cellular morphological dynamics and signaling transduction,and contribute to a more comprehensive understanding of the PME/PMEI gene superfamily in plants.

Genetic Engineering of Cotton (Gossypium hirsutum L. ) for Insect-resistance

In order to improve insect-resistance of cottonand cultivate new cotton varieties,tissue cultureand plant regeneration of cotton(Gossypiumhirsutum L.)were studied with Xinluzao 4,Xi550,Jizi 492,Hengwu 89-30,Han 93-2 and Jizi123.A system of cotton tissue culture for

GbLMI1 over-expression improves cotton aboveground vegetative growth

Leaves are the main organ for photosynthesis and organic synthesis in cotton.Leaf shape has important effects on photosynthetic efficiency and canopy formation,thereby affecting cotton yield.Previous studies have shown that LMI1(LATE MERISTEM IDENTITY1)is the main gene regulating leaf shape.In this study,the LMI1 gene was inserted into the 35S promoter expression vector,and cotton plants overexpressing LMI1(OE)were obtained through genetic transformation.Statistical analysis of the biological traits of the T_(1) and T_(2) populations showed that compared to the wild type(WT),OE plants had significantly larger leaves,thicker stems and significantly greater dry weight.Furthermore,plant sections of the main vein and petiole showed that the numbers of cells in those tissues of OE plants were significantly greater.In addition,RNA-seq analysis revealed the differential expression of genes related to gibberellin synthesis and NAC gene family(genes containing the NAC domain)between the OE and WT plants,suggesting that LMI1 is involved in secondary wall formation and cell proliferation,which promotes stem thickening.Moreover,Gene Ontology(GO)analysis revealed enrichment in the terms of calcium ion binding,and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed enrichment in the terms of fatty acid degradation,phosphatidylinositol signal transduction system,and c AMP(cyclic adenosine monophosphate)signal pathway.These results suggested that LMI1 OE plants are responsive to gibberellin hormone signals,and have altered messenger signals(c AMP,Ca^(2+))which amplify this function,to promote stronger aboveground vegetative growth.This study found the LMI1 greatly increased the vegetative growth in cotton,which is the basic requirement for higher yield.

Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

Bacillus thuringiensis(Bt)cotton production is challenged by two main problems,i.e.,the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs.Therefore,increasing the Bt protein concentration at the boll stage,especially in bolls,has become the main goal for increasing insect resistance in cotton.In this study,two protein degradation inhibitors(ethylene diamine tetra acetic acid(EDTA)and leupeptin)were sprayed on the bolls,subtending leaves,and whole cotton plants at the peak flowering stage of two Bt cultivars(medium maturation Sikang 1(SK1))and early maturation Zhongmian 425(ZM425)in 2019 and 2020.The Bt protein content and protein degradation metabolism were assessed.The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8%and 25.0 to 38.6%in the treated bolls of SK1 and ZM425 respectively,while they were decreased in the subtending leaves of these treated bolls.In the treated leaves,the Bt protein concentrations increased by 7.6 to 23.5%and 11.2 to 14.9%in SK1 and ZM425,respectively.The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves.The Bt protein concentrations in bolls were higher,increasing by 22.5 to 31.0%and 19.6 to 32.5%for SK1 and ZM425,respectively.The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents,protease and peptidase activities and significant enhancements in soluble protein contents.These results indicated that inhibiting protein degradation could improve the protein content,thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants.Therefore,the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

Cotton pads-derived carbon materials/reduced graphene oxide modified with polypyrrole for electrode of supercapacitors

This study investigates the influence of electropolymerization conditions on the deposition of polypyrrole(PPy)onto cotton-derived carbon fiber(CF)modified with reduced graphene oxide(rGO)for supercapacitors applications using an experimental/theorical approach.The surface modification of CF by rGO and/or by PPy electrodeposited at 10,25 and 50 mV s^(-1) was thoroughly examined physicochemical and electrochemically.Composite electrodes comprising CF-rGo-PPy,synthesized via electropolymerization at 25 mV s^(-1),demonstrated a remarkable increase in capacitance,showcasing~742 F g^(-1) compared to 153 F g^(-1) for CF.SEM,N_(2)-surface area,XPS,and TD-DFT approach revealed that the higher capacitance observed in CF-rGo-PPy electrodes underscores the influence of morphology and charged nitrogen species on the electrochemical performance of these modified electrodes.Notably,this electrode material achieves a specific capacitance retention of~96%of their initial capacitance after 10000 cycles at 0.5 A g^(-1) measured in a two-electrodes cell configuration.This work also discusses the influence of the scan rate used for pyrrole electropolymerization on the pseudocapacitance contribution of PPy and its possible effect on the porosity of the material.These results highlight the importance of appropriate electropolymerization conditions that allow obtaining the synergistic effect between CF,rGO and PPy.

Exogenous melatonin improves cotton yield under drought stress by enhancing root development and reducing root damage

The exogenous application of melatonin by the root drenching method is an effective way to improve crop drought resistance.However,the optimal concentration of melatonin by root drenching and the physiological mechanisms underlying melatonin-induced drought tolerance in cotton(Gossypium hirsutum L.)roots remain elusive.This study determined the optimal concentration of melatonin by root drenching and explored the protective effects of melatonin on cotton roots.The results showed that 50μmol L-1 melatonin was optimal and significantly mitigated the inhibitory effect of drought on cotton seedling growth.Exogenous melatonin promoted root development in drought-stressed cotton plants by remarkably increasing the root length,projected area,surface area,volume,diameter,and biomass.Melatonin also mitigated the drought-weakened photosynthetic capacity of cotton and regulated the endogenous hormone contents by regulating the relative expression levels of hormone-synthesis genes under drought stress.Melatonin-treated cotton seedlings maintained optimal enzymatic and non-enzymatic antioxidant capacities,and produced relatively lower levels of reactive oxygen species and malondialdehyde,thus reducing the drought stress damage to cotton roots(such as mitochondrial damage).Moreover,melatonin alleviated the yield and fiber length declines caused by drought stress.Taken together,these findings show that root drenching with exogenous melatonin increases the cotton yield by enhancing root development and reducing the root damage induced by drought stress.In summary,these results provide a foundation for the application of melatonin in the field by the root drenching method.

Enhancing cotton resilience to challenging climates through genetic modifications

Cotton is one of the most important fiber crops that plays a vital role in the textile industry.Its production has been unstable over the years due to climate change induced biotic stresses such as insects,diseases,and weeds,as well as abiotic stresses including drought,salinity,heat,and cold.Traditional breeding methods have been used to breed climate resilient cotton,but it requires a considerable amount of time to enhance crop tolerance to insect pests and changing climatic conditions.A promising strategy for improving tolerance against these stresses is genetic engineering.This review article discusses the role of genetic engineering in cotton improvement.The essential concepts and techniques include genome editing via clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(CRISPR-Cas9),overexpression of target genes,downregulation using RNA interference(RNAi),and virus-induced gene silencing(VIGS).Notably,the Agrobacterium-mediated transformation has made significant contributions to using these techniques for obtaining stable transgenic plants.

Identification of candidate genes for early-maturity traits by combining BSA-seq and QTL mapping in upland cotton(Gossypium hirsutum L.)

Cotton breeding for the development of early-maturing varieties is an effective way to improve multiple cropping indexes and alleviate the conflict between grains and cotton in the cultivated fields in China.In the present study,we aimed to identify upland cotton quantitative trait loci(QTLs)and candidate genes related to early-maturity traits,including whole growth period(WGP),flowering timing(FT),node of the first fruiting branch(NFFB),height of the node of the first fruiting branch(HNFFB),and plant height(PH).An early-maturing variety,CCRI50,and a latematuring variety,Guoxinmian 11,were crossed to obtain biparental populations.These populations were used to map QTLs for the early-maturity traits for two years(2020 and 2021).With BSA-seq analysis based on the data of population 2020,the candidate regions related to early maturity were found to be located on chromosome D03.We then developed 22 polymorphic insertions or deletions(InDel)markers to further narrow down the candidate regions,resulting in the detection of five and four QTLs in the 2020 and 2021 populations,respectively.According to the results of QTL mapping,two candidate regions(InDel_G286-InDel_G144 and InDel_G24-InDel_G43)were detected.In these regions,three genes(GH_D03G0451,GH_D03G0649,and GH_D03G1180)have nonsynonymous mutations in their exons and one gene(GH_D03G0450)has SNP variations in the upstream sequence between CCRI50 and Guoxinmian 11.These four genes also showed dominant expression in the floral organs.The expression levels of GH_D03G0451,GH_D03G0649 and GH_D03G1180 were significantly higher in CCRI50 than in Guoxinmian 11 during the bud differentiation stages,while GH_D03G0450 showed the opposite trend.Further functional verification of GH_D03G0451 indicated that the GH_D03G0451-silenced plants showed a delay in the flowering time.The results suggest that these are the candidate genes for cotton early maturity,and they may be used for breeding early-maturity cotton varieties.

GhWRKY75 positively regulates GhPR6-5b via binding to a W-box TTGAC(C/T)to orchestrate cotton resistance to Verticillium dahliae

Verticillium dahliae is an important fungal pathogen affecting cotton yield and quality.Therefore,the mining of V.dahlia-resistance genes is urgently needed.Proteases and protease inhibitors play crucial roles in plant defense responses.However,the functions and regulatory mechanisms of the protease inhibitor PR6 gene family remain largely unknown.This study provides a comprehensive analysis of the PR6 gene family in the cotton genome.We performed genome-wide identification and functional characterization of the cotton GhPR6 gene family,which belongs to the potato protease inhibitor I family of inhibitors.Thirty-nine PR6s were identified in Gossypium arboreum,G.raimondii,G.barbadense,and G.hirsutum,and they were clustered into four groups.Based on the analysis of pathogen-induced and Ghlmm transcriptome data,Gh PR6-5b was identified as the key gene for V.dahliae resistance.Virus-induced gene silencing experiments revealed that cotton was more sensitive to V.dahliae V991after PR6-5b silencing.The present study established that GhWRKY75 plays an important role in resistance to Verticillium wilt in cotton by positively regulating GhPR6-5b expression by directly binding to the W-box TTGAC(T/C).Our findings established that GhWRKY75 is a potential candidate for improving cotton resistance to V.dahliae,and provide primary information for further investigations and the development of specific strategies to bolster the defense mechanisms of cotton against V.dahliae.

Cotton ethylene response factor Gh ERF91 is involved in the defense against Verticillium dahliae

Verticillium dahliae causes significant losses in cotton production.To reveal the mechanism of the defense response to V.dahliae in cotton,transcriptomic analyses were performed using cotton cultivars M138(V.dahliae-resistant)and P2(V.dahliae-susceptible).The results revealed 11,076 and 6,640 differentially expressed genes(DEGs)in response to V.dahliae,respectively.The weighted gene co-expression network analysis of 4,633 transcription factors(TFs)indicated a“MEblue”module containing 654 TFs that strongly correlate with resistance to V.dahliae.Among these TFs,the ethylene response factor Ghi_A05G10166(GhERF91)was identified as a putative hub gene with a defense response against V.dahliae.A virus-induced gene silencing assay and exogenous application of ethephon showed that GhERF91 is activated by ethylene and positively regulates the response to V.dahliae exposure in cotton.This study provides fundamental transcriptome data and a putative causal gene(GhERF91)associated with resistance to V.dahliae,as well as genetic resources for breeding V.dahliae-resistant cotton.