Title of Thesis
In vitro regeneration and Agrobacterium mediated transformation
of soybean (Glycine max L.)
Department of Biotechnology / Microbiology,
Faculty of Biological Sciences / Quaid-i-Azam University, Islamabad
|Number of Pages|
|Keywords (Extracted from title, table of contents and
abstract of thesis)|
Agrobacterium, Vitro, Soybean,
Cultivars, Morphology, Transforments, Mediated, Glycine, Shrubby,
Optimized, Regeneration, Protocol
mediated transformation of soybean has successfully been achieved.
However, the efficiency is usually low indicating that Agrobacterium
mediated transformation of soybean demands optimization of more
suitable conditions for transfer of T-DNA from plasmid to plant
cell; beside these constraints; transformation is also dependent
upon plant genotype, Agrobacterium strain and type of plasmid.
Present investigation was aimed to find suitable regeneration
protocol for two soybean cultivars, NARC-4 and NARC-7 and to
standardize some transformation conditions. Based on optimized
regeneration and transformation scheme, LFY gene, for early
flowering and rol genes (A, B & C) for enhanced rooting were
introduced in soybean genome. For transfer of LFY gene EHA105
harboring pROKIILFYGUSintnptII and for rol gene, LBA4404 harboring
pLBR were used.
Cotyledonary node method was found suitable for direct organogenesis
of soybean NARC-4 and NARC-7 cultivars. However, presence of
different plant growth regulators in media resulted in variation in
number of shoot produced,shoot length and in percentage response.
Statistical analysis reveals that BAP resulted in higher frequency
of shoot regeneration and number of shoots per explant while mean
shoot length was found higher when ZTR was used in the medium. Out
of both soybean cultivars, NARC-4 showed better response than
For standardization of transformation conditions, different
parameters were studied. It was observed that explant cutting in
Agro-suspension culture and infection for 1hr resulted in the
highest GUS expression (48.3% and 55.9%, respectively). While 5 days
co-cultivation resulted in 55.17% GUS response and washing for 2hr
in washing medium containing 1g/L cefotaxime was better. It was
found that 30mg/L kanamycin was sufficient in selection medium. At
this concentration number of GUS positive shoots were maximum (63.6%
response). The overall percentage transformation efficiency of both
soybean cultivars NARC- 4 and NARC-7 was 24.16% and 15.71%
respectively, when tested at best conditions.
Soybean cultivar NARC-4 was used for transformation of LFY and rol
genes. EHA 105 containing LFY gene along with GUS as reporter and
nptII as selectable marker showed transformation efficiency 2.2%.
Only 4 plants were recovered and confirmed for presence of LFY gene
by PCR. These plants were highly dwarfed with small leaves and short
nodal distance. Early flowering was observed by these transforments.
The flowers turned brown and died after few days. Few of them turned
into pods but no seed formation was observed.
All the rol transforments produced enhanced rooting as compared to
control plants. However, plants morphology varied depending upon rol
gene. RolA transforments were small in size and mildly shrubby with
ovate to elliptical leaf shape while rolC transforments were also
dwarf with divided stem at the base with ovate to slightly globular
leaf shape. Soybean rolB transformed plants showed variation in
morphology. These plants were dwarf to shrubby with variation in
leaf shape. The shrubby plants had reduced nodal distance with a
little more zigzag pattern as compared to non-transformed plants.
Rol transforments produced flowers in less time period as compared
to control plants. These flowers converted into pods and set seeds.
PCR analysis confirmed presence of respective gene in these plants.
Southern blot analysis confirmed insertion of T-DNA in soybean
genome as single copy number to multiple copies in rol gene
In this study rol and LFY genes were efficiently introduced in
soybean cultivar NARC-4 after optimization of regeneration and some