Cotton (Gossypium hirsutum L.) is of great economic importance as a cultivated crop in many parts of the world. In addition to being a pillar of the textile industry, cotton and its byproducts are used for livestock feed, seed oil, and other products. Bacillus thuringiensis crystal toxin (Bt) expression in cotton provides effective protection against chewing insects but does not defend plants from piercing/sucking insect pests. With the aim to create transgenic plants with resistance against piercing/sucking pests, we used Agrobacterium-mediated genetic transformation of cotton cultivar Coker 312 to express the Allium sativum leaf agglutinin (ASLA) gene from the phloem-specific rolC promoter. The ASLA transgene was stably inherited and showed Mendelian segregation in the T1 generation. Transgenic lines, expressing the ASLA gene, showed explicit resistance against major sap-sucking pests. Green peach aphid (Myzus persicae Sulzer) choice assays showed that 75% of aphids preferred untransformed cotton plants relative to those expressing the ASLA gene. In detached leaf bioassays, plants expressing ASLA caused 82% aphid mortality and 44-53% reduction in fecundity. Clip cage bioassays with whiteflies (Bemisia tabaci Gennadius) showed 74-82% mortality and 44-60% decrease in fecundity due to ASLA gene expression. In whole plant bioassays, whiteflies showed 77% mortality and a 54% decrease in fecundity on ASLA transgenics. Importantly, we did not observe a negative effect of the ASLA gene on ladybugs (Coccinella septempunctata) that consumed these whiteflies. Together, our findings demonstrate the potential of ASLA-transgenic cotton for providing protection against two devastating insect pests, whiteflies and aphids. The ASLA-transgenic cotton appears promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.

Globally, aphid, Myzus persicae is an economically significant, polyphagous crop pest that feeds on more than 400 plant species and transmits more than 100 plant viruses. Aphid infestation is mostly managed by insecticides that cause heavy environmental contamination and insect resistance. Cloning of plant derived insecticidal genes to develop transgenic plants under suitable promoter is a promising technology. In the present study, ASAL (MN820725) was isolated from native garlic and cloned in plant transformation vector, pGA482 through Agrobacterium mediated tobacco transformation. PCR of genomic DNA of transgenic tobacco plants using gene specific primers confirmed the presence of asal gene of 546 bp. To detect the integration of gene Southern blot analysis was conducted that revealed stable integration of asal gene while, gene expression was analyzed through qRT-PCR that showed variable expression of asal gene in transgenic tobacco plants. Efficacy of asal gene was evaluated through aphid bioassay. Aphid bioassay revealed that transgenic tobacco lines LS-17, LS-20, LR-1, and LR-7 exhibited 100% aphid mortality and significantly reduced the aphid population. These findings suggested the potential of ASAL against aphids that can be further used against other notorious sap sucking pests.

Background: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plant-derived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 355 promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. Results: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP-9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. Conclusions: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloem-specific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops. (C) 2020 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. All rights reserved.

Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan. Bt cotton expressing a Cry1Ac gene is grown over a large area in Pakistan, however, there is a major concern that bollworms may develop resistance. Here we have used a durable resistance strategy against bollworms by developing a double gene construct containing Cry1Ac and Cry2Ab (pGA482-12R) for cotton transformation. Both Cry toxin genes have been cloned in the same T-DNA borders and transferred successfully into cotton via Agrobacterium-mediated transformation. Both genes are expressed in transgenic cotton plants and is likely to help breeders in developing new cotton cultivars by incorporating these genes in cotton lines having no Bt genes or expressing Cry1Ac gene (Mon 531). Positive transgenic cotton was identified by PCR using specific primers for the amplification of both Cry1Ac and Cry2Ab genes. Cry1Ac and Cry2Ab expression was confirmed with an immunostrip test and quantified using ELISA that showed significant spatio-temporal expression of Cry2Ab ranging from 3.28 to 7.72 µg/g of the tissue leaf. Insect bioassay with army worm ( Spodoptera litura ) was performed to check the efficacy of NIBGE (National Institute for Biotechnology and Genetic Engineering) double gene transgenic cotton plants and up to 93% insect mortality was observed.

Gut-expressed aphid genes, which may be more easily inhibited by RNA interference (RNAi) constructs, are attractive targets for pest control efforts involving transgenic plants. Here we show that expression of cathepsin L, a cysteine protease that functions in aphid guts, can be reduced by expression of an RNAi construct in transgenic tobacco. The effectiveness of this approach is demonstrated by up to 80% adult mortality, reduced fecundity, and delayed nymph production of Myzus persicae (green peach aphids) when cathepsin L expression was reduced by plant-mediated RNAi. Consistent with the function of cathepsin L as a gut protease, M. persicae fed on the RNAi plants had a lower protein content in their bodies and excreted more protein in their honeydew. Larvae of Coccinella septempunctata (seven-spotted ladybugs) grew more slowly on aphids having reduced cathepsin L expression, suggesting that prey insect nutritive value, and not just direct negative effects of the RNAi construct, needs to be considered when producing transgenic plants for RNAi-mediated pest control. Highlights Silencing expression of cathepsin L by RNA interference reduces protein content of Myzus persicae (green peach aphid) bodies. Honeydew of aphids with cathepsin L silenced contains elevated protein. Cathepsin L is required for efficient protein uptake from phloem sap. Aphids with cathepsin L expression silenced have increased mortality and fewer offspring. Coccinella septempunctata (seven-spotted ladybugs) grow more slowly on aphids with expression of cathepsin L silenced.