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This is to acknowledge all those without whom my project would have not been
completed.I would like to thanks my biotechnology lecturer, Mr Harsh as he had been of
great help while making this term paper. He has devoted his previous time towards this
term paper. He has also provided us a great knowledge regarding this which has been
great importance in its fulfillment.
Although range wide of information in this term paper has been taken from the internet,
books and magazines are also consulted.
INTRODUCTION
The uptake of foreign DNA or transgenes by plant cells is called transformation.A
variety of techniques have been used to introduce by plant cells ;these can be grouped
into the following two broad categories: (1)indirect-gene transfers and (2)direct gene
transfers. The type of plant cells employed for transformation would largely depend on
the objectives of the study.For studies on gene regulation ,etc., (i) the cells needs to be
competent to take up DNA and allow the expression of trangene. (ii) But for the
production of trangenic tissues, the cells,in addition, must be merestematic. Finally, (iii)
trangenic plants can be produced only when the cells are also capable of regenerating
complete plantlets.
In case of plants,stable tranformation may be either nonintegrative, or integrative. In non-
integrative stable transformation, the transgene is maintained stably in an
extrachromosomal state. But such transformation are not passed from one generation to
next. On the other hand, integrative stable transformation results when the transgene
becomes integrated into the plant genome; these intrgratios are heritable.
Gene transfer methods in plants
In the direct gene transfer methods, the foreign gene of interest is delivered into the host
plant cell without the help of a vector. The methods used for direct gene transfer in plants
are:
Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG) and
dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also
used to transfer DNA into cultured cells.
Microinjection where the DNA is directly injected into plant protoplasts or cells
(specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0
micrometerdiameter) glass needle or micropipette. This method of gene transfer is used
to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.
The selection of transformed plant cells from untransformed cells is an important step in
the plant genetic engineering. For this, a marker gene (e.g. for antibiotic resistance) is
introduced into the plant along with the transgene followed by the selection of an
appropriate selection medium (containing the antibiotic). The segregation and stability of
the transgene integration and expression in the subsequent generations can be studied by
genetic and molecular analyses (Northern, Southern, Western blot, PCR).
In animals
The art and science of producing genetically engineered animals have advanced rapidly
in the past few years. One challenge in creating transgenic animals is to ensure that the
transgene turns on at the right time and in the right tissue. To be functional, the integrated
gene must be expressed and regulated appropriately. Thus, the gene to be transferred
must be accompanied by the appropriate promoter and regulatory sequences. Some genes
require an enhancer that may be located far from the promoter.
GENE TRANSFER METHOD
•
•
Binary vectors: two from one. To prevent the T-DNA introduced into the Ti plasmid from
spreading uncontrollably, binary vectors were developed.
Normally, the Ti plasmid also contains ‘virulence genes’ (vir genes), which are necessary for
the transfer of the T-DNA. In the case of the binary vectors, the vir genes are removed and the
desired DNA can be integrated between the borders. These ‘disarmed’ binary plasmids have
several
ADVANTAGES:
• They are easy to propagate in laboratory bacteria like e.g. E. coli .
The vir genes needed for transfer to the plant are arranged on a second plasmid, from which
the T-DNA and both borders are removed. With a binary vector, the target gene (T-DNA) and
virulence gene, which are naturally joined on one plasmid ring, are split between two
plasmids. One transfers the target gene, while the other helps with the transformation through
the activity of the vir genes, without the information for the transfer itself being integrate into
the plant as well.
• First step: Integration of foreign DNA (green) into a Ti plasmid without virulence
genes (vir genes). This vector is propagated in E.coli bacteria.
• Second step: The two plasmids are joined together in an Agrobacterium strain.
• Third step: The T-DNA is transferred to plant cells. The Agrobacteria and plant leaf
pieces are cultivated together, followed by regeneration. The untransformed cells are
selected with the help of the marker gene.
DISADVANTAGES also:
• They are not very stable and are still too big.
The T-DNA sometimes contains unnecessary DNA sequences that are transferred as
well.
They are not suitable for co-transformation – the transfer and integration of two
independent genes.
The left border is ‘leaky’. It has been known for a long time now that the transfer and
integration of T-DNA starts at the RB (right border) and ends at the LB (left border).
However, the LB appears to be ‘leaky’, since sequences outside the T-DNA are often
transferred as well, sometimes even the whole plasmid. Depending on the extent and
type of the transferred sequences beyond the ‘leaky’ LB, the release or breeding of this
kind of transgenic plant is problematic, since not all transferred genes/DNA sequences
are desirable (e.g. certain antibiotic resistance genes).
Stability and size: It was possible to approximately halve the size of the vector
(smaller than 5 kb). Work is still being carried out on the suitable Agrobacterium
strain.
It was possible to remove DNA sequences that do not have to be located on the plasmid.
For this to work, these so-called partition genes must be present in the genome of the
Agrobacterium instead. The integration of these genes into the Agrobacterium genome
has, however, proved difficult and was not completed within the term of the project.
‘Leaky’ left border: Vectors were developed which were designed to achieve
more accurate integration of the T-DNA. For this, the termination sequence
(‘stop signal’) used as the left border of the T-DNA was employed twice or
even four times. So far, thirty plants transformed with this vector have been
studied. DNA sequences from beyond the left border had been transferred in
only one of these plants. There was not time to carry out segregation analyses
for statistical confirmation before the end of the project.
Bibliography-
2. www.biobasics.gc.ca./english/view