ICRISAT Archival Report 2006 - The seedlings of success in the ...

Activity 6C.2.1: Develop and evaluate groundnut transgenic plants for enhanced resistance to Aspergillus
flavus
Milestone: Performance of the nine promising groundnut transgenic events expressing RChit gene for A. flavus
resistance evaluated in contained on-station trials at ICRISAT, and best performing events identified
(KKS/FW/PLK/SNN) 2007
Aspergillus flavus and A. parasiticus, with the ability to produce aflatoxins in groundnut, present a great human and
animal health hazard globally. Extensive efforts for developing resistance to A. flavus/A. parasiticus infection and
aflatoxin contamination in cultivated groundnut have resulted in the identification of partially resistant genotypes.
Genetic engineering approach was therefore initiated to develop groundnut germplasm with durable resistance to A.
flavus/A. parasiticus invasion in groundnut. The rice chitinase (RChi) gene under the control of the CaMV 35S
promoter was introduced into a popular groundnut variety JL 24, using Agrobacterium tumefaciens-mediated
genetic transformation by using the cotyledon explants from mature seeds. Thirty transgenic events were selected
that were positive for the RChi gene integration. These events were assessed for resistance against A. flavus seed
colonization by in vitro seed inoculation with A. flavus spores. Fifteen RChit transgenic events that showed
promising resistance during T 2 generation were evaluated for post-harvest seed resistance to A. flavus by in vitro
seed inoculation assay by blotter plate method. Seeds (10 - 15% moisture) were surface sterilized with 0.1% (v/v)
mercuric chloride and inoculated with A. flavus (strain AF 11-4) spore suspension (1 × 106 spores ml -1 ) and placed
on a moist blotter paper in a petridish, and incubated in a humid chamber maintained at 80% relative humidity and
28°C for six days. The percent seed infection was recorded at the end of 6th day. Seeds of progenies with less than
10% infection were advanced to next generation by planting uninfected seed only. This procedure was continued up
to T 5 generation. Post-harvest infection in the transgenic groundnut seed ranged between 0 to 100%, compared to 60
to 100% in controls. A few progenies of nine events numbers 12, 23, 24, 27, 29, 30, 31, 36, and 44 showed
consistently low seed infection (