Book of Abstracts - Geyseco

P05
Biotechnology
P05-001: GENETIC DIVERSITY STUDIES OF COARSE
AND FINE RICE USING RAPD MARKERS
Iqbal, M.* - Iqbal, A. - Saeed, A. - Nisar, A.
CABB, University of Agriculture, Faisalabad, Pakistan
*Corresponding author e-mail: ahsanuaf2003@yahoo.com
The availability of genetically diverse gene pool is vitally important
in the varietal development program. Molecular Markers are
being extensively utilized to explore the genetic diversity among
native and exotic germplasm.
This study was designed to reveal the genetic diversity and patterns
of relationships among the 20 accessions/genotypes representative
of Basmati and Non-Basmati Rice from the existing
rice gene pool using RAPD (Random Amplified Polymorphic
DNA) Markers.
Employing RAPD, Thirty (30) decamer oligonucleotide primers
were used to estimate the genetic diversity. Out of these thirty
primers, seventeen (17) primers give the polymorphic results
and produced a total of 116 fragments, out of which 101 were
polymorphic (87.06%) while 15 fragments were monomorphic
(12.93%). Similarity coefficients had ranged from 0.47 to 0.90.
The average genetic similarity was calculated 0.68 (68%). In this
study, the Coarse rice genotypes showed more polymorphism
(85.84%) than the Fine rice genotypes (61.76%).
Genotypes were clustered into 8 distinct groups: A, B, C, D, E,
F, G and H but two genotypes i.e. Shadab and Kangni-27 showed
divergence from all the genotypes of the groups. So, these diverse
genotypes should be included in the breeding programme.
P05-002: VARIATIONS IN BARE-1 INSERTION PAT-
TERNS IN BARLEY CALLUS CULTURES
Gozukirmizi, N.* - Evrensel, C. – Yilmaz, S. – Temel, A.
Istanbul University, Faculty of Science, Department of Molecular
Biology and Genetics
*Corresponding author e-mail: nermin@istanbul.edu.tr
Stability of aging barley calli was investigated with BARE-1
transposon specific Inter-Retrotransposon Amplified Polymorphism
(IRAP) technique.
Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were
cultured on callus induction MS (Murashige and Skoog) medium
supplemented with 3 mg/L 2,4-D and maintained on same medium
for 30, 45 and 60 days. Ten IRAP primers were used in 25
different combinations and as a result, the variation level of DNA
isolated from 30-, 45- and 60-days old calli, have been found to
change 14-25%, depending on the mature embryo material and
the age of callus.
While the similarity level between 30- days and 45-days old calli
is 84%, it is 79% between 30-days and 60-days old calli and it
is 76% between 45-days and 60-days old calli. As a result, culture
conditions cause genetic variations and there are also evident
BARE-1 transposon alterations. The findings are expected
to contribute to genetic engineering studies to get better results
and also to understand, how transposons contribute to features
like tissue culture – especially callus tissue – formation and the
ability of regeneration thereof. To our knowledge, this is the first
report of employment of IRAP technique in barley in terms of
callus development.
Keywords Hordeum vulgare L.- Transposon - Tissue culture –
BARE-1 – Inter Retrotransposon Amplified Polymorphism
P - Posters
P05-003: METABOLIC ENGINEERING OF POTATO (SO-
LANUM TUBEROSUM) FOR ENHANCED ASCORBIC
ACID (VIT C) ACCUMULATION
Upadhyaya, C.P.* - Hemavathi – Nookaraju, A. – Mayank, A.G.
– Park, S.W.
Department of Molecular Biotechnology, College of Life & Environmental
Sciences, Konkuk University, Seoul, Korea
*Corresponding author e-mail: cpupadhyay@gmail.com
Potato (Solanum tuberosum L.) is an important vegetable crop
which ranks fourth among the staple foods of mankind after
wheat, rice and maize. Unlike most of the animals, humans lack
the ability to synthesize ascorbic acid on their own due to a mutation
in the gene encoding the last enzyme of ascorbate biosynthesis.
As a result, vitamin C must be obtained from dietary sources
like plants. The potato (Solanum tuberosum L. cv. Taedong Valley)
was engineered for enhanced ascorbic acid (Vit C) ovr-accumulation
which also leads to increase in tolerance for various
abiotic stresses. A gene encoding D-galacturonic acid reductase
(GalUR) isolated from strawberry and l-gulono-γ-lactone oxidase
(GLOase gene), from rat cells under the control of CaMV 35S
promoter was introduced in potato plants resulted in 1.6-3.0 folds
increase in AsA in transgenic potato.
Integration of the GalUR and GLOase gene in the plant genome
was confirmed by PCR, RT-PCR and Southern blotting. Ascorbic
acid (AsA) levels in transgenic tubers were determined by highperformance
liquid chromatography (HPLC). The increases in
levels of AsA were positively correlated with increased GalUR
and GLOase activity in both kinds of transgenics. The transgenic
lines with enhanced vitamin C content showed enhanced tolerance
to abiotic stresses induced by methyl viologen (MV), NaCl or
mannitol as compared to control plants. The leaf disc senescence
assay showed better tolerance in transgenic lines by retaining
higher chlorophyll as compared to the untransformed control
plants. Present study demonstrated that the over-expression of
GalUR or GLOase gene enhanced the level of AsA in potato tubers
and these transgenics performed better under different abiotic
stresses as compared to untransformed control.
P05-004: ANIONIC SOYBEAN PEROXIDASE AND ITS
APPLICATION IN ENZYME IMMUNOASSAY
Sakharov, I.*
M.V.Lomonosov Moscow State University
*Corresponding author e-mail: sakharovivan@gmail
Cationic horseradish peroxidase (HRP) is usually used in
сhemiluminescent enzyme immunoassay (CLEIA), one of highly
sensitive analytical methods. However, HRP has drawbacks connecting
with quick decay of CL signal and, in some cases, insufficiently
high sensitivity. We demonstrated that anionic plant
peroxidase isolated from soybeans (SbP) is more effectively
biocatalysts than HRP-C and is able to oxidize luminol in the
absence of enhancers. In addition, unlike HRP-C, SbP produces
a long-term CL signal. Although SbP is a more potent biocatalyst
in luminol oxidation than HRP-C itself, in the presence of enhancers
HRP-C produces a higher CL intensity than SbP. On the
other hand, HRP-C enhancers do not practically increase SbPinduced
CL. At screening of some phenothiazines we showed
that 3-(10’- phenothiazinyl)propane-1-sulfonate (SPTZ) is a first
potent enhancer of CL induced by anionic SbP. Also, the simultaneous
introduction of SPTZ and 4-morpholinopyridine (MORP)
in the substrate mixture resulted in an additional increase of CL
as well as a decrease of the lower detection limit (LDL) of SbP.
The SbPcatalyzed chemiluminescent signals in presence of SPTZ
and SPTZ/MORP are long-term. At comparison of the three fully
optimized systems, SbP–SPTZ– MORP versus HRP-C–SPTZ–
MORP versus HRP-C– PIP, it demonstrated that the SbP system
possessed significantly higher sensitivity and lower LDL value.
The SbP-SPTZ-MORP system was successfully employed in
CL-EIA for determination of human thyroglobulin, one of mar-
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