ANNUAL REPORT - Department of Biotechnology

a total of 326 cotyledonary node explants
transformed with pLK3, three kanamycin-rooted
plants have been obtained. To perform RNAimediated
silencing of MYMV-Vig, we constructed a
dsRNA-AC1 construct. This construct has a 369-bp
fragment from the 3' end of the AC1 gene. The
hairpin RNA construct was made by placing the AC1
fragment in sense and antisense orientations with an
intron in between.
In another project on development of cardamom for
virus resistance, plants were regenerated after
bombardment with the NIb gene of cardamom
mosaic virus. Out of the 86 plants which were
regenerated, only three showed the amplification of
the inserted gene after transferring to the
greenhouse. The PCR products from these three
plants were cloned and one of them was sequenced.
The sequence was found to be identical to NIb gene.
The transgenic plants have to be evaluated for virus
resistance under field conditions.
Bose Institute, Kolkata
In project on engineering inositol metabolic pathway
for raising salt-tolerant rice plants, several lines of
trangenic rice plants (PB-1) have been obtained for
cytosolic(3 lines for PcINO1 and 1 line for OsINO1)
and chloroplastic (6 lines for Tp-PcINO1and 2 lines
for Tp-OsINO1) introgression of the salt-tolerant
inositol synthase from Porteresia (PcINO1). T1 plants
were raised and analyzed for the stable integration of
the gene. Photosynthetic efficiency of the plants was
measured. After standardization of an in vitro
regeneration protocol of an indica rice variety IR64,
Agrobacterium mediated transformation of the same
was carried out for cytosolic expression of PcINO1
and the OsINO1 gene. Though the transformation
and regeneration efficiency was quite low in IR64 in
comparison to the PB-1 variety, transgenics of the
same as analyzed through PCR analysis. For
PcINO1 transformed plants, gene specific primers
were used that amplified a region of approximately
430 bp (described in the earlier report) and for
OsINO1 transformed plants, hptII gene specific
primers were used that amplified a region of
approximately 1 kb.
MALDI TOF MS analysis of the salt-regulated ~ 60 kD
chloroplastic MIPS, identified the protein as rice
DBT Annual Report 2006-07
42
cytosolic MIPS. Upstream genome walking
experiment showed the Oryza sativa MIPS sequence
to possess a putative transit peptide for chloroplast
localization. Thus the gene for the chloroplastic MIPS
is identified as the same for that of the cytosolic MIPS
(OsINO1-1 ) having an extension for the transit
peptide which targets the translated product to the
chloroplast. While this gene is mapped in the
chromosome 3 in Oryza, a second gene for inositol
synthase (termed OsINOI-2 ) has been identified and
mapped in chromosome 10 after determining the
exact exon and intron boundary in the process. In
addition, upstream region of OsINO1 and
PcINO1gene was cloned from genomic DNA library
of Oryza sativa var. IR 64 and Poteresia coarctata
respectively. The PcINO1 gene remarkably
possesses some cis-acting elements found in stress
regulated genes.
Multi-institutional projects
Functional genomics programme on rice:
(i) Gene expression profiling during flower and
seed development and functional validation of
identified genes
After the completion of transcriptome profiling for
eight stages of panicle and five stages of seed
development along with four vegetative stages by
using the Affymetrix rice GeneChip microarrays, a
comprehensive bioinformatics exercise to delineate
individual expression profiles of the genes encoding
transcription factors (TF) and signal transduction
components (STC) has been initiated. Resulting from
these analyses, 50 genes have been short listed for
validation of gene function and/or promoter activity.
Vector constructs for 6 genes (both for gene silencing
and overexpression) and 15 promoters have been
made and work on plant transformation is underway.
Eight new constructs have been transferred to the
groups working on rice transformation and the work
on the functional validation of these genes initiated.
Transgenic plants for 6 promoter and 4 gene
constructs have been obtained and are being
evaluated for phenotypic aberrancies reporter gene
activities. Meanwhile, some important TF and STC
gene families viz., Aux/IAA, GH3, CDPK, MADS, Fbox,
C2H 2 Zn-finger etc., involved in flower and seed
development have being analyzed and the results