RESUMO
Agrobacterium-mediated genetic transformation has been optimized in indica rice susceptible to sap-sucking insects, viz., brown planthopper (BPH) and green leafhopper (GLH). Snowdrop lectin gene (gna) from Galanthus nivalis, driven by phloem-specific rice-sucrose-synthase promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was employed for genetic transformation. Embryogenic calli--after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-gna--were selected on the medium containing phosphinothricin. PCR and Southern blot analyses confirmed the stable integration of both the genes into genomes of transgenic (T0) rice plants. Northern and Western blot analyses revealed the expression of gna in the transgenic plants. In the T1 and T2 generations, the gna and bar transgenes showed co-segregation at a ratio of 3 : 1. Plant progenies expressing gna, in T1 and T2, exhibited substantial resistance against BPH and GLH pests. This is the first report dealing with transgenic indica rice exhibiting high resistance to both insects.
RESUMO
Stem borer resistant transgenic parental lines, involved in hybrid rice, were produced by Agrobacterium-mediated gene transfer method. Two pSB111 super-binary vectors containing modified cry1Ab/cry1Ac genes driven by maize ubiquitin promoter, and herbicide resistance gene bar driven by cauliflower mosaic virus 35S promoter were, used in this study. Embryogenic calli after co-cultivation with Agrobacterium were selected on the medium containing phosphinothricin. Southern blot analyses of primary transformants revealed the stable integration of bar, cry1Ab and cry1Ac coding sequences into the genomes of three parental lines with a predominant single copy integration and without any rearrangement of T-DNA. T1 progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. Furthermore, the co-segregation of bar and cry genes in T1 progenies suggested that the transgenes are integrated at a single site in the rice genome. In different primary transformants with alien inbuilt resistance, the levels of cry proteins varied between 0.03 and 0.13% of total soluble proteins. These transgenic lines expressing insecticidal proteins afforded substantial resistance against stem borers. This is the first report of its kind dealing with the introduction of Bacillus thuringiensis (Bt) cry genes into the elite parental lines involved in the development of hybrid rice.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Endotoxinas/metabolismo , Melhoramento Genético/métodos , Controle de Insetos/métodos , Insetos/patogenicidade , Oryza/genética , Oryza/parasitologia , Plantas Geneticamente Modificadas/parasitologia , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Quimera/genética , Quimera/parasitologia , Endotoxinas/genética , Proteínas Hemolisinas , Imunidade Inata/genética , Controle Biológico de Vetores/métodosRESUMO
We have developed transgene pyramided rice lines, endowed with enhanced resistance to major sap-sucking insects, through sexual crosses made between two stable transgenic rice lines containing Allium sativum (asal) and Galanthus nivalis (gna) lectin genes. Presence and expression of asal and gna genes in pyramided lines were confirmed by PCR and western blot analyses. Segregation analysis of F2 progenies disclosed digenic (9:3:3:1) inheritance of the transgenes. Homozygous F3 plants carrying asal and gna genes were identified employing genetic and molecular methods besides insect bioassays. Pyramided lines, infested with brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH), proved more effective in reducing insect survival, fecundity, feeding ability besides delayed development of insects as compared to the parental transgenics. Under infested conditions, pyramided lines were found superior to the parental transgenics in their seed yield potential. This study represents first report on pyramiding of two lectin genes into rice exhibiting enhanced resistance against major sucking pests. The pyramided lines appear promising and might serve as a novel genetic resource in rice breeding aimed at durable and broad based resistance against hoppers.
Assuntos
Genes de Plantas/genética , Imunidade Inata/imunologia , Insetos/fisiologia , Lectinas de Ligação a Manose/genética , Oryza/genética , Oryza/parasitologia , Doenças das Plantas/genética , Lectinas de Plantas/genética , Animais , Cruzamentos Genéticos , Comportamento Alimentar , Galanthus/genética , Alho/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Homozigoto , Imunidade Inata/genética , Padrões de Herança/genética , Lectinas de Ligação a Manose/metabolismo , Oryza/imunologia , Controle Biológico de Vetores , Doenças das Plantas/parasitologia , Exsudatos de Plantas , Lectinas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/crescimento & desenvolvimento , TransgenesRESUMO
Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T1 to T5 generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper.