RESUMO
BACKGROUND: Cotton is continuously exposed to sucking and chewing insect pest pressure since emergence to harvesting. Pink bollworm (Pectinophora gossypiella) has become major chewing insect pest to reduce the cotton yield and results in bad lint quality even in transgenic crops. The efficiency of insecticidal genes has been compromised due to extensive utilization of transgenic crops. METHODS AND RESULTS: The present study was conducted to evaluate the efficacy of an alternate cry1Ia12 insecticidal gene against pink bollworm (PBW) in cotton. Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA2300 expression vector containing cry1Ia12 gene under the control of 35S CaMV was used to transform a local cotton cultivar GS-01. The various molecular analyses revealed the transgene integration and expression in primary transformants. Among five selected transgenic plants, tcL-08 showed maximum (16.06-fold) mRNA expression of cry1Ia12 gene whereas tcL-03 showed minimum (2.33-fold) expression. Feeding bioassays of 2nd and 3rd instar pink bollworm (PBW) larvae on immature cotton bolls, flowers and cotton squares revealed up to 33.33% mortality on tcL-08 while lowest mortality (13.33%) was observed in tcL-03 and tcL-15. Furthermore, the average weight and size of survived larvae fed on transgenic plants was significantly lesser than the average weight of larvae survived on non-transgenic plants. CONCLUSIONS: The present study suggests the cry1Ia12 gene as an alternate insecticidal gene for the resistance management of cotton bollworms, especially PBW.
Assuntos
Inseticidas , Lepidópteros , Mariposas , Animais , Lepidópteros/genética , Toxinas de Bacillus thuringiensis , Inseticidas/farmacologia , Proteínas Hemolisinas/genética , Endotoxinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mariposas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Larva/genética , Larva/metabolismo , Gossypium/genética , Gossypium/metabolismo , Controle de Pragas , Resistência a Inseticidas/genéticaRESUMO
Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the ßC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vß, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vß transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vß-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vß-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.
Assuntos
Begomovirus/genética , Técnicas de Silenciamento de Genes , Gossypium/genética , Gossypium/virologia , Doenças das Plantas/virologia , Interferência de RNA , Vírus Satélites/genética , DNA Satélite/genética , DNA Viral/genética , Resistência à Doença/genética , Engenharia Genética/métodos , Hibridização in Situ Fluorescente , Filogenia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/virologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cells. RNAi technology was used to silence V2 gene for resistance against these viruses in transgenic plants. The transformation of the RNAi-based gene construct targeting V2 gene of CLCuKoV-Bur, cloned under 35S promoter, was done in two elite cotton varieties MNH-786 and VH-289 using shoot apex cut method of gene transformation. The transformation efficiency was found to be 3.75 and 2.88 % in MNH-786 and VH-289, respectively. Confirmation of successful transformation was done through PCR in T 0, T 1, and T 2 generations using gene-specific primers. Transgenic cotton plants were categorized on the basis of the virus disease index in T 1 generation. Copy number and transgene location were observed using FISH and karyotyping in T 2 generation which confirmed random integration of V2 RNAi amplicon at chromosome 6 and 16. Real-time quantitative PCR analyses of promising transgenic lines showed low virus titer compared to wild-type control plants upon challenging them with viruliferous whiteflies in a contained environment. From the results, it was concluded that amplicon V2 RNAi construct was able to limit virus replication and can be used to control CLCuV in the field.