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