Abstract:Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube… Show more
“…The amount of expressed Cry1Ab/Ac protein ranged from 0.16 ng/mg to a highest of 0.35 ng/mg of leaf. Similar range of expression has been reported in cotton with cry1la12 under CaMV35S promoter showing 40% morality and growth reduction in fall armyworm ( Spodoptera frugiperda ) and cotton boll weevil ( Anthonomus grandis ) whereas cry1Ac + cry2A showed 60–100% mortality against Heliothis larvae ( Puspito et al, 2015 ; Oliveira et al, 2016 ). In some samples, detection of Cry1Ab/Ac protein by ELISA was hinderer due to the high mucilage content in jute plants ( Yamazaki et al, 2009 ; Kundu et al, 2011 ).…”
Jute (Corchorus sp.) is naturally occurring, biodegradable, lignocellulosic-long, silky, golden shiny fiber producing plant that has great demands globally. Paper and textile industries are interested in jute because of the easy availability, non-toxicity and high yield of cellulosic biomass produced per acre in cultivation. Jute is the major and most industrially used bast fiber-producing crop in the world and it needs protection from insect pest infestation that decreases its yield and quality. Single locus integration of the synthetically fused cry1Ab/Ac gene of Bacillus thuringiensis (Bt) in Corchorus capsularis (JRC 321) by Agrobacterium tumefaciens-mediated shoot tip transformation provided 5 potent Bt jute lines BT1, BT2, BT4, BT7 and BT8. These lines consistently expressed the Cry1Ab/Ac endotoxin ranging from 0.16 to 0.35 ng/mg of leaf, in the following generations (analyzed upto T4). The effect of Cry1Ab/Ac endotoxin was studied against 3 major Lepidopteran pests of jute- semilooper (Anomis sabulifera Guenee), hairy caterpillar (Spilarctia obliqua Walker) and indigo caterpillar (Spodoptera exigua Hubner) by detached leaf and whole plant insect bioassay on greenhouse-grown transgenic plants. Results confirm that larvae feeding on transgenic plants had lower food consumption, body size, body weight and dry weight of excreta compared to non-transgenic controls. Insect mortality range among transgenic feeders was 66–100% for semilooper and hairy caterpillar and 87.50% for indigo caterpillar. Apart from insect resistance, the transgenic plants were at par with control plants in terms of agronomic parameters and fiber quality. Hence, these Bt jutes in the field would survive Lepidopteran pest infestation, minimize harmful pesticide usage and yield good quality fiber.
“…The amount of expressed Cry1Ab/Ac protein ranged from 0.16 ng/mg to a highest of 0.35 ng/mg of leaf. Similar range of expression has been reported in cotton with cry1la12 under CaMV35S promoter showing 40% morality and growth reduction in fall armyworm ( Spodoptera frugiperda ) and cotton boll weevil ( Anthonomus grandis ) whereas cry1Ac + cry2A showed 60–100% mortality against Heliothis larvae ( Puspito et al, 2015 ; Oliveira et al, 2016 ). In some samples, detection of Cry1Ab/Ac protein by ELISA was hinderer due to the high mucilage content in jute plants ( Yamazaki et al, 2009 ; Kundu et al, 2011 ).…”
Jute (Corchorus sp.) is naturally occurring, biodegradable, lignocellulosic-long, silky, golden shiny fiber producing plant that has great demands globally. Paper and textile industries are interested in jute because of the easy availability, non-toxicity and high yield of cellulosic biomass produced per acre in cultivation. Jute is the major and most industrially used bast fiber-producing crop in the world and it needs protection from insect pest infestation that decreases its yield and quality. Single locus integration of the synthetically fused cry1Ab/Ac gene of Bacillus thuringiensis (Bt) in Corchorus capsularis (JRC 321) by Agrobacterium tumefaciens-mediated shoot tip transformation provided 5 potent Bt jute lines BT1, BT2, BT4, BT7 and BT8. These lines consistently expressed the Cry1Ab/Ac endotoxin ranging from 0.16 to 0.35 ng/mg of leaf, in the following generations (analyzed upto T4). The effect of Cry1Ab/Ac endotoxin was studied against 3 major Lepidopteran pests of jute- semilooper (Anomis sabulifera Guenee), hairy caterpillar (Spilarctia obliqua Walker) and indigo caterpillar (Spodoptera exigua Hubner) by detached leaf and whole plant insect bioassay on greenhouse-grown transgenic plants. Results confirm that larvae feeding on transgenic plants had lower food consumption, body size, body weight and dry weight of excreta compared to non-transgenic controls. Insect mortality range among transgenic feeders was 66–100% for semilooper and hairy caterpillar and 87.50% for indigo caterpillar. Apart from insect resistance, the transgenic plants were at par with control plants in terms of agronomic parameters and fiber quality. Hence, these Bt jutes in the field would survive Lepidopteran pest infestation, minimize harmful pesticide usage and yield good quality fiber.
“…The cotton transformation technique associated with Imazapyr herbicide resistance permits high efficient GM plant selection (Aragão et al ., ; Rech et al ., ). The biolistic method could effectively transform the cotton BRS 372 variety with a high transformation rate (0.5%) compared to other techniques, such as the pollen‐tube pathway, which provides very low transformation rates (approximately 0.01%; Oliveira et al ., ).…”
Section: Discussionmentioning
confidence: 97%
“…() and Oliveira et al . () reported GM cotton plants with Cry1IA12 transgene levels of 2.7 and 2.26 μg g −1 leaf fresh weight, respectively. The highest Cry10Aa expression level observed in the present P#008 GM cotton line (~14.0 μg g −1 fresh tissue in both leave and flower buds) clearly shows that Cry10A GM cotton plants can produce toxin at higher levels than usual (Figure ).…”
Section: Discussionmentioning
confidence: 99%
“…Although various characterized Cry toxins are active against lepidopteran insects, far fewer Cry proteins present toxicity to coleopteran species (Donovan et al ., ; James, ; Maagd, ; Pathak et al ., ; Shah et al ., ). Some Cry proteins, such as Cry1Ba6, Cry8Ka and Cry1Ia12, have recently been described as somewhat entomotoxic against the coleopteran pest A. grandis (Aguiar et al ., ; Grossi‐de‐Sa et al ., ; Martins et al ., ; Oliveira et al ., , ; Silva et al ., ). In this way, Cry‐independent technologies could be used as effective alternatives against coleopteran insect pest, such as recently described to control the hemipteran Bemisia tabaci (whitefly).…”
SummaryGenetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination‐related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR‐based 2−ΔΔCt analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g−1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g−1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.
“…In the past decade, Cry proteins from Bacillus thuringiensis (Bt) have been applied in the development of genetically modified (GM) cotton for CBW control [ 15 – 17 ]. An alternative approach for making GM cotton resistant to CBW is the application of RNA interference (RNAi) technology.…”
RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.
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