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).
Markers for screening
Generally, there are three types of markers used in screening/selection:
a) Morphological marker based on visible character (phenotypic expression) e.g. flower color, seed color, height, leaf shapes, etc. Morphological markers could be dominant or recessive. There are certain constraints in using these markers as the morphological markers are easily influenced by environmental factors and thus may not represent the desired genetic variation. Some of the visible markers have not much role to play in the plant breeding programme.
b) Biochemical marker: The proteins produced by gene expression are also used as markers in plant breeding programmes. The most commonly used are isozymes, the different molecular forms of the same enzyme. Each individual variety has its own isozyme variability (profiles) which can be detected by electrophoresis on starch gel.
c) Molecular marker based on DNA polymorphism detected by DNA probes or amplified products of PCR, e.g.Restriction fragment length polymorphism (RFLP), Randomly Amplified polymorphic DNA (RAPD), variable Number Tandom Repeats (VNTR), Microsatellites, etc. Plant breeders always prefer to detect the gene as molecular marker, although it is not always possible. Molecular markers provide a true representation of the genetic make up at the DNA level. They are consistent and free from environmental factors, and can be detected much before the development of plants occur. The advantage with a molecular marker is that a plant breeder can select a suitable marker for the desired trait which can be detected well in advance. A large number of markers can be generated as per the needs. The molecular markers to be used in plant breeding programme should have the following characteristics: (a) the marker should be closely linked with the desired trait, (b) the marker screening methods should be effective, efficient, reproducible and easy to carry out, (C) the entire analysis should be cost effective.
Molecular makers are of two types:
(a) based on nucleic acid (DNA) hybridization- This involves the cloning of the DNA piece followed by the hybridization with the genomic DNA, which is later detected.
The Restriction fragment length polymorphism (RFLP) was the very first technology employed for the detection of polymorphism, based on the DNA sequence differences. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and recombinations of genomic DNA. The procedure involves the isolation of genomic DNA and it’s digestion by restriction enzymes. The fragments are separated by electrophoresis and finally hybridized by incubating with cloned and labeled probes.
(b) Molecular markers based on PCR amplification.
Polymerase chain reaction (PCR) is a novel technique for the amplification of selected regions of DNA. The most important advantage is that even a minute quantity of DNA can be amplified and the PCR- based molecular markers require only a small quantity of DNA to start with. Random amplified polymorphic DNA (RAPD) markers use PCR amplification where the DNA is isolated from the genome and is denatured. The template molecules are annealed with primers and amplified by PCR. The amplified products are separated on electrophoresis and identified. Based on the nucleotide alterations in the genome, the polymorphisms of amplified DNA sequences differ which can be identified as bends on gel electrophoresis.
Amplified fragment length polymorphism (AFLP) is a novel technique involving a combination of RFLP and RAPD. AFLP is based on the principle of generation of DNA fragments using restriction enzymes and oligonucleotide adaptors (or linkers), and their amplification by PCR.