Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene.

KEY MESSAGE: Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ). Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T1 generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T0 lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.

Elimination of a closed population of the yellow fever mosquito, Aedes aegypti, through releases of self-limiting male mosquitoes.

Establishment of novel mosquito control technologies such as the use of genetically engineered insects typically involves phased testing to generate robust data-sets that support its safe and effective use as a vector control tool. In this study, we demonstrate the ability of the transgenic self-limiting OX513A Aedes aegypti strain to suppress a wild type Ae. aegypti population in an outdoor containment facility in India. OX513A is a genetically engineered Ae. aegypti strain with a repressible dominant self-limiting gene. When male adult OX513A mate with wild female adults, a single copy of the self-limiting gene is inherited by all the progeny, leading to death of >95% of progeny during larval/pupal development. A wild-type population of Ae. aegypti was established and stabilized during a 14 week period in five paired field cage units, each consisting of control and treatment cages, followed by weekly releases of OX513A male adults to suppress the target population. The successive introductions of OX513A male adults led to a consistent decline in wild type numbers eventually resulting in the elimination of Ae. aegypti from all treated cages within 10 to 15 weeks of release. This study demonstrates that Ae. aegypti elimination may be a realistic and achievable target in relatively isolated environments.

Importance and genetic diversity of vegetable-infecting tospoviruses in India.

A survey for Peanut bud necrosis virus (PBNV), Watermelon bud necrosis virus (WBNV), Capsicum chlorosis virus (CaCV), and Iris yellow spot virus (IYSV) was conducted between 2002 and 2009 in the major vegetable-growing areas in India. PBNV was documented widely in tomato and chili peppers in 14 states representing southern, north-western, north-eastern, and central regions and WBNV was predominantly detected in watermelons and cucurbits in all except north-eastern regions. In addition, the expanded host range of PBNV to watermelons and other cucurbits and WBNV to tomato and chili peppers was observed leading to natural mixed infection of the two viruses. IYSV was found in onion in southern, central, and north-eastern regions and CaCV in tomato and chili peppers in northern and southern regions, respectively. Phylogenetic analysis of the nucleocapsid gene revealed segregation of field isolates of PBNV and WBNV into two distinct subclades, whereas isolates of CaCV and IYSV each clustered into a single clade. A proposal for establishing WBNV as a distinct tospovirus species is made based on the molecular characterization of small- (S) and medium- (M) RNA segments.

Biological and molecular characterization of Capsicum chlorosis virus infecting chilli and tomato in India.

Two isolates of Capsicum chlorosis virus (CaCV, genus Tospovirus) from tomato (CaCV-To-Ind) and chilli (CaCV-Ch-Pan), collected from Haryana and Uttar Pradesh states of northern India respectively, were compared. A comparison of the amino acid sequences of their N genes revealed more than 96% identity, confirming that the virus isolates in India have a high degree of sequence conservation and are closely related to Australian isolates. Analysis of the host range of CaCV revealed no biological difference between the isolates, but they differed from CaCV-Australia. The nucleotide sequences of S, M and L RNA of CaCV-Ch-Pan were determined. The S RNA contains 3,105 nucleotides (nt), with NSs and N genes of 1,320 and 828 nt, respectively. The M RNA consists of 4,821 nt, with an NSm gene of 927 nt and a Gn/Gc gene of 3,366 nt. The intergenic regions of S and M RNA contain 824 and 425 nt, respectively. The L RNA consists of 8,912 nt, with an RNA-dependent RNA polymerase gene of 8,634 nt.

Mating competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L.

BACKGROUND: OX513A is a genetically engineered strain of Aedes aegypti carrying a repressible, dominantly inherited transgene that confers lethality in immature heterozygous progeny. Released male OX513A adults have proven to be effective for the localised suppression of wild Ae. aegypti, highlighting its potential in vector control. Mating and life-table assessments were used to compare OX513A with reared Ae. aegypti strains collected from New Delhi and Aurangabad regions in India. RESULTS: Mating proportions of New Delhi females versus males of OX513A or New Delhi strains were 0.52 and 0.48 respectively, indicating no discrimination by females against either strain, and males of both strains were equally competitive. Developmental time from first instar to adult emergence was significantly longer for OX513A (10.7 ± 0.04 days) than for New Delhi (9.4 ± 0.04 days) and Aurangabad strains (9.1 ± 0.04 days). Differences in mean longevities, female reproductive parameters and population growth parameters between the strains were non-significant. CONCLUSIONS: The laboratory study demonstrates that only minor life-table variations of limited biological relevance exist between OX513A and Indian Ae. aegypti populations, and males had equal potential for mating competitiveness. Thus, results support the OX513A strain as a suitable candidate for continued evaluation towards sustainable management of Ae. aegypti populations in India.

Genietic and molecular characterization of Candystripel transposition events in sorghum.

In sorghum, the Candystripe1 (Cs1) transposable element causes a variegated pericarp phenotype due to its excision activity from the yl (yellow seed1) locus. The Y1 is a transcription regulator which is required for the biosynthesis of red 3-deoxyflavonoid pigments. Somatic variability in the transposition behavior of Cs1 was observed via biochemical analysis of 3-deoxyflavonoids in the leaf tissues of the Y1-cs alleles. Using somatic excisions of Cs1 as a tool, we establish that the Cs1 is active in young seedlings and the y1 locus is also functional in these tissues. Several somatic and germinal excision events were characterized and sequence analysis of independent events predominantly showed 2-bp footprints. Further, with the goal of ur.derstanding the properties of Cs1 that would facilitate the development of a transposon tagging system in sorghum, germinal excisions of Cs1 from y1 were used as a marker. Transposition of Cs1 was followed by characterization of putative insertion events. Genetic linkage between mutant phenotypes and the cosegregating restriction fragments of Cs1 provided additional evidence that Cs1 is an active transposable element in sorghum.