Summary Chemical catalysts are being replaced by biocatalysts in almost all industrial applications due to environmental concerns, thereby increasing their demand. Enzymes used in current industries are produced in microbial systems or plant seeds. We report here five newly launched leaf‐enzyme products and their validation with 15 commercial microbial‐enzyme products, for detergent or textile industries. Enzymes expressed in chloroplasts are functional at broad pH /temperature ranges as crude‐leaf extracts, while most purified commercial enzymes showed significant loss at alkaline pH or higher temperature, required for broad range commercial applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, chloroplast enzymes as a leaf powder can be stored up to 16 months at ambient temperature without loss of enzyme activity. Chloroplast‐derived enzymes are stable in crude‐leaf extracts without addition of protease inhibitors. Leaf lipase/mannanase crude extracts removed chocolate or mustard oil stains effectively at both low and high temperatures. Moreover, leaf lipase or mannanase crude‐extracts removed stain more efficiently at 70 °C than commercial microbial enzymes (<10% activity). Endoglucanase and exoglucanase in crude leaf extracts removed dye efficiently from denim surface and depilled knitted fabric by removal of horizontal fibre strands. Due to an increased demand for enzymes in the food industry, marker‐free lettuce plants expressing lipase or cellobiohydrolase were created for the first time and site‐specific transgene integration/homoplasmy was confirmed by Southern blots. Thus, leaf‐production platform offers a novel low‐cost approach by the elimination of fermentation, purification, concentration, formulation and cold‐chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.

RNA interference (RNAi) is a sequence-specific down-regulation in the expression of a particular gene, induced by double-stranded RNA (dsRNA). Feeding of dsRNA either directly or through transgenic plants expressing dsRNA of insect genes has been proven successful against lepidopteran and coleopteran pests, establishing an additional alternative to control insect pests. Lepidopteran crop pests including Spodoptera litura (Fabricius) (Noctuidae), Chilo partellus (Swinhoe) (Crambidae), Plutella xylostella (Linnaeus) (Plutellidae), and Maruca vitrata (Fabricius) (Pyralidae) are the devastating pests of a variety of crops. To tap the potential of RNAi against insect pests, a gene coding for the key enzyme in chitin biosynthesis in arthropods, the chitin synthaseA (CHSA), has been targeted through an exogenous delivery of dsRNA and plantmediated RNAi. The introduction of dsCHSA caused "Half ecdysis" and "Black body" type lethal phenotypes and a significant reduction in larval body weight. Subsequent RT-qPCR analysis demonstrated the down-regulation of CHSA gene transcripts from 1.38-to 8.33-fold in the four target species. Meanwhile, when S. litura larvae fed with leaves of transgenic tobacco plants expressing dsSlCHSA, the mRNA abundance of CHSA gene was significantly decreased resulting in lethal phenotypes like "Double head formation," "Half ecdysis," and "Black body." In addition, abnormalities in pupal-adult and adult stage were also documented, strongly suggesting the RNAi effect of CHSA gene at late developmental stages. Overall, the results demonstrated that CHSA gene expression in Lepidopteran crop pests could be suppressed by application of dsRNA either as feeding or through transgenic crop plants.

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Mormon crickets are a major rangeland pest in the western United States and are currently managed by targeted applications of non-specific chemical insecticides, which can potentially have negative effects on the environment. In this study, we took the first steps toward developing RNAi methods for Mormon crickets as a potential alternative to traditional broad-spectrum insecticides. To design an effective RNAi-based insecticide, we first generated a de novo transcriptome for the Mormon cricket and developed dsRNAs that could silence the expression of seven housekeeping genes. We then characterized the RNAi efficiencies and time-course of knockdown using these dsRNAs, and assessed their ability to induce mortality. We have demonstrated that it is possible to elicit RNAi responses in the Mormon cricket by injection, but knockdown efficiencies and the time course of RNAi response varied according to target genes and tissue types. We also show that one of the reasons for the poor knockdown efficiencies could be the presence of dsRNA-degrading enzymes in the hemolymph. RNAi silencing is possible in Mormon cricket, but more work needs to be done before it can be effectively used as a population management method.