Advertisement
Transgenic pigeonpea (Cajanus cajan) for resistance to legume pod borer Helicoverpa armigera
Upcoming SlideShare
Cataloging computationally putative resistance genes in grape andCataloging computationally putative resistance genes in grape and
Loading in ... 3
1 of 1
Advertisement

More Related Content

Similar to Transgenic pigeonpea (Cajanus cajan) for resistance to legume pod borer Helicoverpa armigera(20)

Advertisement

More from ICRISAT(20)

Advertisement

Transgenic pigeonpea (Cajanus cajan) for resistance to legume pod borer Helicoverpa armigera

  1. Transgenic pigeonpea (Cajanus cajan) for resistance to legume pod borer Helicoverpa armigera Oct 2012 Introduction `` Pigeonpea, an important pulse crop of the semi-arid tropics (SAT) including the Indian subcontinent is highly vulnerable to legume pod borer resulting in losses of over US$300 million annually. `` Pod borer feeds mainly on the leaves, reproductive organs and pods affecting the yield of the crop. `` Conventional breeding methods for insect resistance have not been successful due to the limited genetic variability in the cultivated germplasm. `` Genetic engineering approaches involving effective gene based alternatives will be important for sustainable resistance against insect pests in this crop. Material and methods `` The selected pigeonpea varieties have been used for genetic transformation using synthetic crystalline (CRY) genes encoding the δ-endotoxins with different promoters for efficient gene expression. `` Semi-quantitative double antibody sandwich enzyme linked immunosorbent assay (ELISA) is performed for the detection of the CRY protein before subjecting them to insect bioassays. `` Detached leaf, inflorescence and pod bioassays are performed against neonate larvae, first instar and third instar larvae respectively. Results B B -- C C -- 1 2 3 4 5 6 7 8 9 10 11 -- 100bp P 100bp 5 7 11 12 14 15 17 18 20 23 25 C B P 500bp 500bp 500bp 500bp PCR amplification of the transgenic plants indicating the integration of the transgene. Inverse PCR analysis of transgenic events in T1 generation to determine the copy number of nptII gene. RT-PCR analysis for CRY1Ac transcript of the transformants. 1 2 3 4 5 6 7 8 9 P 10 C P 100bp 1 2 3 4 5 6 7 8 9 10 11 12 C P Southern blot of DNA fron T1 transgenics plants using the Alkphos® non-radioactive labeling and detection system. ELISA of the transgenics confirming the expression of the CRY1Ac protein (+ve represents the CRY1Ac protein used as positive control and –ve represents the protein extract of the untransformed wild type parent). Detached leaf bioassay from (A) the wild type parent (B) the transformed plant (C & D) inflorescence (flower twig) against the neonate larvae of H. armigera. Pod bioassay of (A) Untransformed Parent ICPL 88039 & (B) Resistant Check : Untransformed Control ICPL 87 (C to E) transgenic pigeonpea events against third instar larvae of H.armigera. Leaf bioassay of T2 transgenic events of pigeonpea varieties ICPL 88039 against neonate larvae of H. armigera Genotype/Event Larval feed score Larval survival (%) Larval weight 3 DAI (mg) Z9-4 3.77 68.15 0.60 Z9-2 4.63 86.25 0.99 R4-5 5.04 66.66 0.81 R4-2 5.64 75.50 0.82 R39-6 5.7 90 1.79 R39-4 4.39 61.9 0.82 R39-2 5.57 80 0.99 C8-3 4.88 70.82 0.94 C6-1 3.38 69.28 0.61 C20-1 4.56 74.40 0.51 C15-4 5.81 65.78 0.55 C13-1 4.38 60.00 0.65 C10-3 2.25 67.28 0.58 Untransformed parent - ICPL 88039 7.50 85.39 1.93 Conclusions `` Over 300 transgenic events of pigeonpea have been produced using 6 different gene constructs. `` Transgenics analyzed for integration of the transgenes by PCR and RT-PCR, and selected based on expression in ELISA. `` A considerable reduction in larval weights and survival was observed in 13 tested transgenic pigeonpea events in detached leaf bioassays. `` Pod bioassays indicate a significant larval mortality (up to 100%) and up to 45-fold reduction in larval weight. `` Based on insect bioassays, 10 events with low leaf and flower damage, larval survival and weight were selected and advanced for further evaluations. Pod bioassay of T3 transgenic events of pigeonpea against second to third instar larvae of H. armigera. Genotype/Event Unit Larval Weight (mg) Damage score (0-10 scale) Larval Mortality 3-2-2 2.00 0.75 78.00 3-2-4 All larvae dead 0.83 100.00 4-2-6-5 2.70 0.75 78.00 4-2-6-8 1.00 0.50 25.00 9-4-7-8 2.30 0.67 66.00 9-4-7-9 1.93 0.80 40.00 9-4-13-3 6.20 0.67 66.00 39-4-9-6 4.05 0.75 74.00 9-4-13-5 1.80 0.50 25.00 39-4-9-7 11.38 2.10 20.00 39-4-9-1 All larvae dead 1.83 100.00 Untransformed parent - ICPL 88039 46.69 9.30 0.00 Resistant Check-ICPL 87 138.24 8.36 0.00 (A) (B) A B C D (C) (D) (E)
Advertisement