ANNUAL REPORT - Department of Biotechnology

For utilization of Multiple Aleuron Layer (MAL) lines
for improving micronutrients, a set of seven MAL
(Multiple Aleurone Layer) lines obtained from
CIMMYT Gene Bank is being explored for kernel
micronutrient enrichment and development of
suitable mapping population for the identification of
SSR markers linked to the MAL genes.
(iii) Rice bio-fortification with enhanced iron and
zinc in high yielding non basmati cultivars
through marker assisted breeding and
transgenic approaches
Six independent integration events of ferritin
transgenic lines were developed by Agrobacteriummediated
transformation in the indica rice Oryza
sativa at the MSSRF, Chennai. Matured
nontransgenic and T 2 homozygous transgenic seeds
were used for histochemical and immunoblot
analysis. At the TNAU, the plasmid "1301FerGlu1"
has been mobilized into Agrobacterium strain
LBA4404 through triparental mating. The
transconjugants were confirmed for the presence of
binary plasmid by restriction analysis and backtransformation.
The Agrobacterium LBA4404
(1301FerGlu1) was used to mobilize the gene of
interest into ASD16 and ADT43 using mature seed
derived calli as target tissue. The cultures are in
various stages of selection and regeneration.
Southern blot analysis was carried out with genomic
DNA from nontransgenic and T 2 homozygous PCRpositive
plants. Genomic DNA digested with EcoRI
and probed with ferritin cDNA showed the expected
hybridization signal of 800 bp in all the transformed
plants. This confirmed integration of the ferritin cDNA
in the rice genome. Perl's Prussian blue staining of
ferritin transgenic rice grain sections showed the
distribution of iron accumulation throughout the
alureone and sub aleurone layers and also in the
central region of starchy endosperm. However, in
non- transgenic grains, blue colour formation of iron
accumulation was restricted only to aleurone layer
and the intensity of the colour development was also
very low. This histochemical analysis of iron in rice
specifically showed temporal and spatial deposition
of storage iron. This finding showed correlation with
the Western blot analysis of expression of exogenous
ferritin gene. In all the transgenic southern positive
plants (seeds), a 28-kDa-ferritin protein was
detected, confirming that ferritin protein accumulates
DBT Annual Report 2006-07
48
in rice seeds
At IGAU, Raipur, the rice lines with high iron and zinc
levels are grown in the field for seed multiplications
and further confirmations of mineral value. The
plants were grown in the field during wet season 2006
with different levels of fertilizer applications. The
mineral analysis of these materials is in progress. Six
rice lines showing higher level of grain iron and zinc
concentrations were crossed with the five modern
rice cultivars F1 seeds (5-35 seeds) were harvested
from each cross and are being analysed for mineral
content evaluation.
At UAS, Bangalore, a field experiment has been
conducted at Shettigere, Doddajala, Bangalore with
279 short duration, 325 medium duration and 320
long duration rice genotypes representing the
breeders' active gene pool and germplasm to know
iron, zinc and phytic acid contents in different parts of
rice plant. All genotypes have been replicated thrice,
under aerobic cultivation system at Shettigere,
Bangalore. All necessary management practices
have been implemented. Phenotypic characters of
different rice genotypes have been documented.
Samples for collection of DNA, iron and zinc have
been collected. For the development of DNA markers
based on the known metal transporter genes,
attempt has been made to design the primers for
expression analysis of these genes in elite and
modern rice lines. The c-DNA sequences of these 43
metal genes were downloaded from the rice genome
and primers were designed using Primer 3
programme.Atomic Absorption Spectrophotometers,
grain milling and polishing facilities have been
created at MSSRF and IGAU for the precise
estimation of minerals.
Generation of virus-resistant rice for India:
Diversifying transgenic resistance to popular
varieties
This network project has been implemented at three
institutions viz. University of Delhi South Campus,
New Delhi, Bidhan Chandra Krishi Viswavidyalaya,
Kalyani and Tamil Nadu Agricultural University,
Coimbatore. The salient achievements reported
during last one year are; Transgenic rice seeds
(RTBV-O-Ds2) have been multiplied in the
transgenic glasshouse at UDSC. Seed setting is