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

Milestone: At least two mapping populations developed to map QTLs for salinity tolerance in pigeonpea
(VV/KBS/NM) 2009
Cajanus platycarpus, C. scarabaeoides, and C. sericeus were found to be tolerant and will be used for development
of mapping populations. The minicore collection of pigeonpea provided a large range of variation for salinity
tolerance. Contrasting parents in minicore would also be available for crossing and development of mapping
populations.
Vincent Vadez and KB Saxena
Milestones: QTLs for salinity tolerance identified in pigeonpea (VV/KBS) 2011
The screening of breeding lines has revealed a large range of variation for salinity tolerance, both in the wild and the
cultivated pigeonpea. Given the low level of polymorphism within cultivated pigeonpea, accessions are available to
develop both intra- and inter-specific RIL populations.
Vincent Vadez and KB Saxena
Output C: Annually knowledge of the improvements of the biotechnological and conventional tools designed
to facilitate bio-fortification and bio-detoxification, breeding improved germplasm, and management
strategies of mandate crops and associated capacity building made available to partners internationally
MTP Output Target 2006: Variability of Fe and Zn in 80 germplasm lines developed and quantified in sorghum
Output target 6C.1: High yielding and micronutrient dense hybrids/improved populations/varieties of
sorghum and millet, and promising transgenic events of groundnut and pigeonpea with high beta-carotene
content available for testing in national trials
Activity 6C1.1: Develop groundnut transgenic events for enhanced production of beta-carotene
Milestone: 75 transgenic events of groundnut with maize psy1 gene developed and screened for high ß-carotene
production in the contained greenhouse (KKS/SNN) 2007
Development of groundnut transgenics for enhanced β-carotene: Binary gene constructs harboring the ß-
carotene biosynthetic genes, phytoene synthases (psy1 and crtB) from maize and Erwinia herbicola, respectively,
were developed. These genes are driven by oleosin promoter for oil body seed specific expression, and are being
used in Agrobacterium-mediated genetic transformation for the enhancement of ß-carotene in groundnut seeds. Over
70 putative transgenic groundnut plants were transferred to the containment greenhouse for seed development. The
T 0 putative transgenic plants were analyzed for the integration of transgenes by using PCR with gene-specific
primers, and Southern hybridization, with gene specific probes. In molecular analysis, 70% of putative groundnut
transgenic plants showed the integration of psy1 gene. The transgene expressions were observed in the developing
pods of groundnut by RT- PCR analysis. The presence of mRNA transcripts of psy1 gene was found in 3 out 4
plants tested. The T 1 seeds from transgenic events of PSY1 are being collected for further generations to study gene
stability, inheritance, and expression of transgene. The procedure for beta-carotene extraction and analysis in
groundnut seeds was optimized. The quantification of beta-carotene in primary transgenic groundnut seeds is in
progress for the selection of best events. In a preliminary analysis, the enhancement of ß-carotene was observed in 2
out of 10 events tested that showed the levels of 10 µg g -1 of the seed. For the development of marker-free
transgenic plants, maize psy1 gene driven by oleosin promoter was sub-cloned into binary vectors pCAMBIA
2300:ϕnptII (minus kanamycin gene). Around 30 marker-free putative groundnut transgenic plants carrying the
mpsy1 genes were transferred to the containment greenhouse. Molecular analysis is being carried out for these
transgenic plants. Similarly, for the development of antibodies against phytoene synthase, the psy1 gene was cloned
into pET expression vector. The over expression of PSY1 protein in E. coli is being analyzed.
A Vanamala, KK Sharma and SN Nigam
227