Authors: SANDEEP KUMAR BANGARWA * and ASHWANI KUMAR
Introduction
- The process of transfer, intigration and expression of transgene in the host cells is known as genetic transformation.
- In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogeneous genetic material from its surroundings through the cell membrane(s).
- Transformation in bacteria was first demonstrated in 1928 by British bacteriologist Frederick Griffith.
- Griffith discovered that a strain of Streptococcus pneumoniae could be made virulent after being exposed to heat-killed virulent strains.
- Griffith hypothesized that some "transforming principle" from the heat-killed strain was responsible for making the harmless strain virulent.
- In 1944 this "transforming principle" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain of S. pneumoniae and using just this DNA were able to make a harmless strain virulent.
- They called this uptake and incorporation of DNA by bacteria "transformation".
- The results of Avery et al.'s experiments were at first skeptically received by the scientific community and it was not until the development of genetic markers and the discovery of other methods of genetic transfer (conjugation in 1947 and transduction in 1953) by Joshua Lederberg that Avery's experiments were accepted.
- Gene Transfer is introduction of foreign genetic material, either DNA or RNA, artificially or naturally into a cell.
- It is often also referred to as transformation and is one of the foundations of molecularbiology.
- It is now possible to introduce and express DNA stably in nearly 150 different plant species.
- 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,Terminator, Selectable marker and other genes that deliver the DNA into the host plant (Ex. vir genes of Agrobacterium) .
- In general, plant transformation systems are based on the introduction of DNA into totipotent plantcells, followed by the regeneration of such cells into whole fertile plants.
- Two essential requirements forplant transformation are therefore an efficient method for introducing DNA into plant cells and theavailability of cells or tissues that can easily and reproducibly regenerate whole plants.
- For transformation to take place, the recipient bacteria must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.
- Transformation is one of three processes for horizontal gene transfer, in which exogenous genetic material passes from bacterium to another, the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium).
- In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium
- "Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "transfection".
- Various genetic transfer techniques are grouped into two main categories. 1. Vector mediated or Indirect gene transfer 2. Vectorless or Direct gene transfer
- In this approach the transgene is combined with a vector which takes it to the target cells for inteigration.
- The term plant gene vector applies to potential vectors both for transfer of genetic information between plants and the transfer of genetic information from other organisms (bactieria fungi and animals) to plants.
- The vector mediated transfer is strongly linked to regeneration capabilities of the host plant.
- Agrobacterium tumefaciens and Agrobacterium rhizogenes are natural gene transfer systems.
- Virulence is conferred by a large tumor-inducing plasmid (Ti plasmid) containing genes encoding plant hormones (auxins and cytokinins) andenzymes that catalyze the synthesis of amino acid derivatives termed opines.
- The plant hormones areresponsible for the deregulated cell proliferation that accompanies crown gall growth, while the opinesare secreted by the plant cells and used by the bacteria as food.
- These genes are contained on a specificregion of the Ti plasmid, the T-DNA (transfer DNA), so called because it is transferred to the plantnuclear genome under the control of vir (virulence) genes carried elsewhere on the Ti plasmid. Agrobactreium mediated transformation
- The Agrobacterium system was historically the first successful plant transformation system, marking the break through in plant Genetic engineering in 1983.
- The Agrobacterium is naturally occurring gram negative soil bacterium with two common species A Tumifacience and A rhizogenes There are known as natural gene engineers for their ability to transform plants.
- A tumifacience induces tubers called crown galls, where as A rhizogenes causes hairy root diseases.
- Large plasmids in these bacteria are called tumer inducing (Ti plasmil) and root inducing (Ri plasmid) respectively.
- The Ti plasmid has two major segments of interest in transformation that is T DNA and virus region.
- The T DNA region of the Ti plasmid is the part which is transferred to plant cell and incorporated into nuclear genome of cells.
- The transfer of T DNA is mediated by genes in the another region of Ti plasmid called virs genes (virulence genes).
- Modified Ti plasmid are constructed that lack of undesirable Ti genes but contain a foreign gene (resistant to a disease) and a closely linked selectable marker gene (Eg:- for antibiotic resistance).
- Within the T DNA region any gene put in T DNA region of plasmid cysts transferred to the plant genome.
- The T DNA is generally integrated in low copy number per cell.
- Transfer of gene through to wounded plant organs A. tumifacience has limited range of host.
- It can infest about 60% gymnosperms and Angiosperm. Hence Agrobacterium mediated transformation is the method of choice in dicotyledonous plant species, where plant regeneration system are well established, However, Monocotyledons could not be successfully utilized for Agrobacterium mediated gene transfer.
Introduction of DNA into plant cells without the involvement of biological agents such as Agrobacterium and leading to stable transformation is called direct gene transfer. The various methods of direct gene transfers are
1. Electroporation
2. Chemical mediated gene transfer
3. Micro injection
4. Particle bombardment
5. Lypofection / Liposome mediated gene transfer
6. Pollen transformation
1. Electroporation
- Induction of DNA into cell by exposing them for a very brief period to high voltage electrical pulses to induce transiant pores in the plasma lemma is called Electroporation.
- The electric current leads to the formation of small temporary holes in the membrane of the protoplasts through which the DNA can pass.
- After entry into the cell, the Foreign DNA gets incorporated with the host genome, resulting the genetic transformation.
- The protoplasts are then cultured to regenerate in to whole plants.
- An isolated plasmid DNA is mixed with protoplast in the presence of the poly ethylene glycol (PEG), PVA and Ca (PO4) polyvinyl alcohol which enhance the uptake of DNA by protoplast.
- After 15-20 min of incubation the protoplasts are cultured.
- The DNA solution is injected directly inside the cell using capillary glass micropipetts with the help of micromanipulators of a microinjection assembly.
- 0.5-1.0 micrometer size needle is used. DNA direct injected in the plant cell with the help of syringe.
- The process of partical acceleration (or) biolistics acceleration of DNA into cells with sufficient force such that a part of it gets integrated in to DNA of target cells.
- It contains DNA coated Tungsten (W) or Gold particle, the microscopic pellets (coated microprojectiles)
- Introduction of DNA into cells via lyposomes is known as lipofection.
- The procedure of liposome encapsulation was developed to protect the foreign DNA during the transfer process.
- Liposome are artificial spherical vesicles which contain a small volume of aqueous solution of DNA enclosed in a lipid bilayer. Fusion of liposome with protoplast.
Involves the gene transfer by soaking the pollengrains in DNA solution prior to their use for pollination.
References:
1. Singh, B.D. 2006. Plant Biotechnology. Kalyani Publishers, Ludhiana.
2. Wikipedia
About Author / Additional Info:
I am currently pursuing Ph.D. in Plant Breeding and Genetics from MPUAT-Udaipur (Raj.)