| Syler_91 | Date: Sunday, 09.11.2014, 08:38 | Message # 1 |
 Генералиссимус
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| To achieve genetic transformation in plants, we need the construction of a vector (genetic vehicle) which transports the genes of
interest, flanked by the necessary controlling sequences i.e. promoter
and terminator, and deliver the genes into the host plant. The two kinds
of gene transfer methods in plants are:
Vector-mediated or indirect gene transfer
Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used. This bacteria is known as “natural genetic
engineer” of plants because these bacteria have natural ability to
transfer T-DNA of their plasmids into plant genome upon infection of
cells at the wound site and cause an unorganized growth of a cell mass
known as crown gall. Ti plasmids are used as gene vectors for delivering
useful foreign genes into target plant cells and tissues. The foreign
gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted
sequences.
To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected and infected with Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The infected tissue is
then cultured (co-cultivation) on shoot regeneration medium for 2-3
days during which time the transfer of T-DNA along with foreign genes
takes place. After this, the transformed tissues (leaf discs/calli) are
transferred onto selection cum plant regeneration medium supplemented
with usually lethal concentration of an antibiotic to selectively
eliminate non-transformed tissues. After 3-5 weeks, the regenerated
shoots (from leaf discs) are transferred to root-inducing medium, and
after another 3-4 weeks, complete plants are transferred to soil
following the hardening (acclimatization) of regenerated plants. The
molecular techniques like PCR and southern hybridization are used to
detect the presence of foreign genes in the transgenic plants.
Vectorless or direct gene transfer
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.
Electroporation involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient
(temporary) pores in the plasma membrane which facilitates the uptake of
foreign DNA.
The cells are placed in a solution containing DNA and
subjected to electrical shocks to cause holes in the membranes. The
foreign DNA fragments enter through the holes into the cytoplasm and
then to nucleus.
Particle gun/Particle bombardment - In this method, the foreign DNA containing the genes to be transferred is
coated onto the surface of minute gold or tungsten particles (1-3
micrometers) and bombarded onto the target tissue or cells using a
particle gun (also called as gene gun/shot gun/microprojectile gun).The
microprojectile bombardment method was initially named as biolistics by
its inventor Sanford (1988). Two types of plant tissue are commonly used
for particle bombardment- Primary explants and the proliferating
embryonic tissues.
Transformation - This method is used for introducing foreign DNA into bacterial cells e.g. E. Coli. The
transformation frequency (the fraction of cell population that can be
transferred) is very good in this method. E.g. the uptake of plasmid DNA
by E. coli is carried out in ice cold CaCl2 (0-50C) followed by heat
shock treatment at 37-450C for about 90 sec. The transformation
efficiency refers to the number of transformants per microgram of added
DNA. The CaCl2 breaks the cell wall at certain regions and binds the
DNA to the cell surface.
Conjuction - It is a natural microbial recombination process and is used as a method for gene transfer. In
conjuction, two live bacteria come together and the single stranded DNA
is transferred via cytoplasmic bridges from the donor bacteria to the
recipient bacteria.
Liposome mediated gene transfer or Lipofection - Liposomes are circular lipid molecules with an aqueous interior that
can carry nucleic acids. Liposomes encapsulate the DNA fragments and
then adher to the cell membranes and fuse with them to transfer DNA
fragments. Thus, the DNA enters the cell and then to the nucleus.
Lipofection is a very efficient technique used to transfer genes in
bacterial, animal and plant cells.
Selection of transformed cells from untransformed cells
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).
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