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

Milestone: Three mapping populations for LLS and two for confectionery traits developed (RA/SNN) 2009
Three mapping populations (ICG 11337 x JL 24, ICG 13919 x JL 24, and ICG 11337 x ICG 13919) involving
diverse parents for reaction to LLS have been developed and the material is in F 4 stage. Two populations (ICGV
01393 x Chico and ICGV 02251 x Chico) for confectionery traits have also been developed and the material is in F 3
stage.
SN Nigam and R Aruna
Output target 6A.2: Promising transgenic events of groundnut for resistance to TSV and PBNV available for
commercialization and introgression in locally adapted germplasm
Activity 6A.2.1: Develop transgenic events of groundnut for resistance to TSV and evaluate their
performance under contained greenhouse and field conditions
Milestone: 100 transgenic events of groundnut with TSVcp gene developed and screened in contained greenhouse
(KKS/PLK/SNN) 2007
Stem necrosis disease caused by the Tobacco streak virus (TSV) has emerged as a serious problem on groundnut in
Andhra Pradesh, and Karnataka, India. All the currently grown groundnut varieties are susceptible to the virus.
Research has been initiated to incorporate resistance to TSV in groundnut by using TSV coat protein (TSVcp) gene
through Agrobacterium tumefaciens-mediated genetic transformation of popular groundnut cultivars JL 24, TMV 2,
and ICGV 91114. Twenty events, with six plants per event, were planted in the P 2 greenhouse. A cotyledonous
leaflet was collected from 8 - 10 day old plants and analyzed for transgene by PCR assay, which revealed TSVcp
transgene in 68 of 118 plants tested (58% transformation rate).
At the 3-leaf plant growth stage (8 - 10 day old plants), all the 118 test plants [alongwith susceptible control (JL 24)]
were inoculated by standard mechanical sap inoculation procedure using 1: 30 (w/v) TSV-affected French bean leaf
sap extracts. All the inoculated plants developed necrotic symptoms on the inoculated leaves 7-days post inoculation
and tested positive to TSV with ELISA. These plants were monitored at weekly intervals for systemic virus
infection by testing newly emerged apical leaves by ELISA, and symptoms were recorded. All the non-transgenic
plants (TSVcp gene negative in PCR assay) were susceptible to TSV and showed symptoms typical of TSV infection
within 2-weeks post inoculation. Of the 68 transgenic plants (TSVcp gene positive), 24 plants (35.2%) showed
negative reaction to TSV (no symptoms) or delayed expression of symptoms compared to controls. Delayed
symptom expression (by 2 - 3 weeks compared to controls) was also observed in 42 plants (61.7%). After
appearance of initial symptoms, disease progress was rapid leading to premature death of the plants. It is likely that
these plants may have some tolerance at early growth stages offering resistance to virus spread, but this mechanism
is not sufficient to protect plants at later stages. All the symptomatic plants, either transgenic or controls, tested
positive to TSV with ELISA, and all asymptomatic plants were negative, indicating a correlation between virus
presence and the stem necrosis disease.
Eight plants from six events (1B, 1F, 3E, 4B, and 9C) did not show any systemic symptoms and non-inoculated
leaves were TSV negative, indicating putative TSV resistance in these plants. Considering the fact that TSV was
detected in the inoculated leaves of these plants and the lack of virus in the subsequently emerged leaves suggests a
blockage in the systemic spread of virus, which seems to be responsible for the virus resistance. Delayed symptom
expression (at the time of flowering) was observed on one or two branches in events 9B, 19B, and 22B. These plants
apparently had normal growth pattern. It is likely that these plants may also have some resistance amounting to the
protection. Further evaluation of these events is being continued. Recently, genetic transformation of groundnut cv.
ICGV 91114 has also been undertaken to incorporate transgenic resistance to TSV using TSVcp gene.
KK Sharma, Lava Kumar and Farid Waliyar
Progress reported towards the achievement of milestone for 2007 above will contribute towards achievement of the
milestones listed below.
Milestone: At least 10 promising TSVcp transgenic events identified and the disease resistance characterized under
contained green house conditions (KKS/PLK/SNN) 2008
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