ANNUAL REPORT - Department of Biotechnology

showed sign of spoilage even after 30 days of harvest
whereas wild type fruits were rotten completely.
Detailed post-harvest ripening data is being
collected.
Transgenic tomato plants expressing LeMADS-RIN
gene under 35S promoter were produced with the
aim of producing tomato with delayed ripening as well
as prepare material to find out genes controlled by
LeMADS-RIN transcription factor at UDSC, New
Delhi. However, though these lines showed
substantial reduction of transcript level, all the
antisense transgenic lines were found to have no
significant delay in fruit ripening. This suggested that
stronger suppression of LeMADS-RIN gene may be
required for delay in ripening. Therefore, 3 RNAi
vectors designed to suppress transcript level of
LeMADS-RIN gene have been constructed and used
for transformation of tomato plants. These vectors
are likely to have stronger suppression of LeMADS-
RIn gene, but the degree of suppression may vary.
Transformation experiments have been initiated with
all three vectors.
Six different ethylene responsive factors have been
cloned from tomato cDNA using degenerate primers
and full-length sequences of three have been
obtained. Besides, transcript pattern for three genes
during the course of ripening and in different stress
conditions have been obtained. Sense and antisense
constructs for three ESTs have been prepared.
Molecular Taxonomy
Molecular Taxonomy was identified as R&D priority
for those taxa which could not be segregated
morphologically at family, genera, species and subspecies
level. Some of the achievements reported
during the year are as follows:
Studies have also been initiated on genetic diversity
assessment in wild species of Citrus and Atlantia at
NBRI, Lucknow. Sixty eight accessions of three
species of Atlantia (A. monophylla, A. racemosa; and
A wightii) were collected from different locations of
the Western Ghats of Maharashtra, Tamil Nadu and
Kerala and 20 accessions of Citrus from Khasi hills
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(Meghalaya), Eastern UP, Western Himalayas
(Uttranchal) for morphometric and molecular
taxonomic studies. Further work on DNA profiling of
Atlantia accessions using RAPD and microsatellite
markers is in progress. Besides, essential oils have
been extracted from five accessions of four different
varieties of Citrus using solvent extraction and
hydrodistillation method. The chemical constituents
of oil were analysed using different chromatographic
techniques. Identification of essential oil constituents
from other accessions of Citrus viz. C. jhambhiri, C.
karma and Kaithari nimboo is in progress.
At the University of Calcutta, Kolkata accessions of
Phyllanthus from different parts of West Bengal have
been collected and RAPD analysis is being carried
out to segregate the populations. In another study at
the same institute, DNA fingerprinting studies have
been done for different species of Amaranthus and
some members of Chenopodiaceae. Dendogram
analysis showed that members of Amaranthaceae
and Chenopodiaceae form broadly two clusters.
Further work is going on to identify AmA gene on the
chromosome and comparative study is on to see
variation in AmA gene in some special of
Amaranthus.
A study has been supported at CIMAP, Lucknow to
segregate different species of Phyllanthus using
molecular tools. The fragments in the RAPD profiles
generated by MAP-9 primer were cloned for
Phyllanthus amarus, P. fraternus, P. debilis and P.
urinaria. These fragments are being sequenced to
generate SCAR (Sequence Characterized Amplified
Regions) markers for differentiation of these species.
The genetic relationship among Phyllanthus
accessions of various species has been determined.
Polymorphism rates were high indicating a
substantial amount of molecular variation and
potential genetic diversity. Subsequent analysis
yielded eight groups out of which three major clusters
are of P. debilis, P. amarus and P. urinaria. Least
variation was detected among the P. amarus
accessions, close to the group of P. amarus falls the
cluster of P. fraternus. On the basis of molecular
techniques (AFLP), it is possible to predict that four