The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of β-pore-forming toxins (β-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin\'s mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

UNASSIGNED: Genetic engineering has revolutionized agriculture by transforming biotic and abiotic stress-resistance genes in plants. The biosafety of GM crops is a major concern for consumers and regulatory authorities.
UNASSIGNED: A 14-week biosafety and toxicity analysis of transgenic cotton, containing 5 transgenes ((Cry1Ac, Cry2A, CP4 EPSPS, VIP3Aa, and ASAL)), was conducted on albino mice. Thirty mice were divided into three groups (Conventional, Non-transgenic, without Bt, and transgenic, containing targeted crop) according to the feed given, with 10 mice in each group, with 5 male and 5 female mice in each group.
UNASSIGNED: During the study, no biologically significant changes were observed in the non-transgenic and transgenic groups compared to the control group in any of the study\'s parameters i.e. increase in weight of mice, physiological, pathological, and molecular analysis, irrespective of the gender of the mice. However, a statistically significant change was observed in the hematological parameters of the male mice, while no such change was observed in the female study group mice. The expression analysis, however, of the TNF gene increases many folds in the transgenic group as compared to the non-transgenic and conventional groups.
UNASSIGNED: Overall, no physiological, pathological, or molecular toxicity was observed in the mice fed with transgenic feed. Therefore, it can be speculated that the targeted transgenic crop is biologically safe. However, more study is required to confirm the biosafety of the product on the animal by expression profiling.

Verticillium wilt is a soil-borne vascular disease caused by the fungal pathogen Verticillium dahliae. It causes great harm to upland cotton (Gossypium hirsutum) yield and quality. A previous study has shown that Hen egg white lysozyme (HEWL) exerts strong inhibitory activity against V. dahliae in vitro. In the current study, we introduced the HEWL gene into cotton through the Agrobacterium-mediated transformation, and the exogenous HEWL protein was successfully expressed in cotton. Our study revealed that HEWL was able to significantly inhibit the proliferation of V. dahlia in cotton. Consequently, the overexpression of HEWL effectively improved the resistance to Verticillium wilt in transgenic cotton. In addition, ROS accumulation and NO content increased rapidly after the V. dahliae inoculation of plant leaves overexpressing HEWL. In addition, the expression of the PR genes was significantly up-regulated. Taken together, our results suggest that HEWL significantly improves resistance to Verticillium wilt by inhibiting the growth of pathogenic fungus, triggering ROS burst, and activating PR genes expression in cotton.

The expression of a gene encoding peroxisomal Cu-Zn superoxide dismutase from Saussurea involucrata Kar. et Kir. was induced by low temperature, PEG6000 treatment, and NaCl stress. To investigate the role of SikCuZnSOD3 in the mitigation of abiotic stress, we used Agrobacterium-mediated transformation to create transgenic cotton that overexpressed SikCuZnSOD3. Phenotypic analysis of T4 generation transgenic lines showed that they generally grew better than wild-type cotton under low temperature, PEG6000 treatment, and NaCl stress. Although there were no significant differences under control conditions, transgenic plants exhibited greater survival, fresh weight, and dry weight than wild-type plants under all three stress treatments. Additional physiological analyses demonstrated that the transgenic cotton had higher relative water content, proline and soluble sugar contents, and activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase), as well as lower relative conductivity, malondialdehyde content, and H2O2 and O2- accumulation. More importantly, overexpression of SikCuZnSOD3 increased the yield of cotton fiber. Our results confirm that the overexpression of SikCuZnSOD3 can improve the abiotic stress resistance of cotton by increasing the activity of antioxidant enzymes, maintaining ROS homeostasis, and reducing cell membrane damage.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s11032-021-01217-0.

Cotton is a significant industrial crop, playing an essential role in the global economy that suffers several setbacks due to biotic and abiotic adversities. Despite such problems, biotechnological advances in cotton are limited because of genetic transformation and regeneration limitations. Here, we present a detailed protocol optimized based on previously published papers, along with our modifications. These involve changes in Agrobacterium concentration, co-cultivation time and temperature, hormones used for regeneration, media manipulation for embryogenic callus production, and efficient rescue of deformed embryos. Further, this protocol has been used in genetic studies on biotic and abiotic stress in cotton. This protocol assures a reproducible stable transgenic cotton development procedure via somatic embryogenesis that can be used by researchers worldwide. This protocol was validated in: Nat Biotechnol (2016), DOI: 10.1038/nbt.3665.

Methanol is noxious to insect pests, but most plants do not make enough of it to shield themselves from encroaching insects. Methanol emission is known to increase in the instance of herbivory. In the current study, we showed that Aspergillus niger pectin methylesterase over-expression increases methanol emission and confers resistance to polyphagous insect pests on transgenic cotton plants by impeding the possible methanol detoxification pathways. Transgenic plants emitted ∼11 fold higher methanol displaying insect mortality of 96% and 93% in Helicoverpa armigera and Spodoptera litura, respectively. The larvae were unable to survive and finish their life cycle and the surviving larvae exhibited severe growth retardation. Insects try to detoxify methanol via catalase, carboxylesterase and cytochrome P450 monooxygenase enzymes, amongst which cytochrome P450 plays a major role in oxidizing methanol to formaldehyde and formaldehyde to formic acid, which is broken down into carbon dioxide and water. In our study, catalase and esterase enzymes were found to be upregulated, but cytochrome P450 monooxygenase levels were not much affected. Leaf disc assays and In-planta bioassays also showed 50-60% population reduction in the sap sucking pests, such as Bemisia tabaci and Phenacoccus solenopsis. These findings imply that elevated methanol emissions confer resistance in plants against chewing and sap-sucking pests by tampering the methanol detoxification pathways. Such mechanism will be useful in imparting expansive resistance against pests in plants.

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) control some important insect pests. However, evolution of resistance by pests reduces the efficacy of Bt crops. Here we review resistance to Bt cotton in the pink bollworm, Pectinophora gossypiella, one of the world\'s most damaging pests of cotton. Field outcomes with Bt cotton and pink bollworm during the past quarter century differ markedly among the world\'s top three cotton-producing countries: practical resistance in India, sustained susceptibility in China, and eradication of this invasive lepidopteran pest from the United States achieved with Bt cotton and other tactics. We compared the molecular genetic basis of pink bollworm resistance between lab-selected strains from the U.S. and China and field-selected populations from India for two Bt proteins (Cry1Ac and Cry2Ab) produced in widely adopted Bt cotton. Both lab- and field-selected resistance are associated with mutations affecting the cadherin protein PgCad1 for Cry1Ac and the ATP-binding cassette transporter protein PgABCA2 for Cry2Ab. The results imply lab selection is useful for identifying genes important in field-evolved resistance to Bt crops, but not necessarily the specific mutations in those genes. The results also suggest that differences in management practices, rather than genetic constraints, caused the strikingly different outcomes among countries.

CONCLUSIONS: This study demonstrates the combinatorial management of multiple pests through a trans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy. Transgenic cotton events demonstrated improved efficacy against cotton leaf curl disease, cotton leaf hopper and root-knot nematode. Cotton (Gossypium hirsutum L.), an important commercial crop grown worldwide is confronted by several pests and pathogens, thus reiterating interventions for their management. In this study, we report, the utility of a novel Arabidopsis miRNA173-directed trans-acting siRNA (tasiRNA)-based micro RNA-induced gene silencing (MIGS) strategy for the simultaneous management of cotton leaf curl disease (CLCuD), cotton leaf hopper (CLH; Amrasca biguttula biguttula) and root-knot nematode (RKN, Meloidogyne incognita). Cotton transgenics were developed with the MIGS construct targeting a total of 7 genes by an apical meristem-targeted in planta transformation strategy. Stable transgenics were selected using stringent selection pressure, molecular characterization and stress-specific bio-efficacy studies. We identified 8 superior events with 50-100% resistance against CLCuD, while reduction in the root-knot nematode multiplication factor in the range of 35-75% confirmed resistance to RKN. These transgenic cotton events were also detrimental to the growth and development of CLH, as only 43.3-62.5% of nymphs could survive. Based on the corroborating evidences obtained by all the bioefficacy analyses, 3 events viz., L-75-1, E-27-11, E-27-7 were found to be consistent in tackling the target pests. To the best of our knowledge, this report is the first of its kind demonstrating the possibility of combinatorial management of pests/diseases in cotton using MIGS approach. These identified events demonstrate immense utility of the strategy towards combinatorial stress management in cotton improvement programs.

CONCLUSIONS: Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression. Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.

BACKGROUND: The pest Aphis gossypii Glover globally causes considerable economic losses on various crops by its feeding damage and disease transmission. Transgenic plants that produce double-stranded RNA (dsRNA) targeted to insect genes are being developed as a pest control strategy. In this study, we evaluated the effects of transgenic cotton-mediated RNA interference (RNAi) on the growth and detoxification ability of A. gossypii after the transgenic cotton lines expressing dsAgCYP6CY3-P1 (the TG cotton lines) were obtained on the basis of exploring the functions of CYP6CY3 in our previous research.
RESULTS: The developmental time of third- and fourth-instar nymphs which fed on the TG cotton lines were significantly prolonged. Life table parameters showed that the fitness of cotton aphids from the TG cotton lines decreased. Additionally, the relative expression level of CYP6CY3 in cotton aphids which fed on the TG cotton lines was significantly reduced by 47.3 % at 48 h compared with that from the nontransgenic cotton (the NT cotton). Bioassay showed that silencing of CYP6CY3 increased mortality of the nymphs to imidacloprid by 28.49 % (at 24 h) and to acetamiprid by 73.77 % (at 48 h), respectively.
CONCLUSIONS: These results indicated that the TG cotton lines delayed the growth and development of A. gossypii, but also decreased population density and increased its sensitivity to imidacloprid and acetamiprid, respectively. The results provide further support for the development and application of plant-mediated RNAi. © 2022 Society of Chemical Industry.