Download PLANT GENETIC ENGINEERING (Genetic Transformation)

PLANT GENETIC ENGINEERING (Genetic Transformation)
Transfer of foreign DNA that codes for specific genetic information from a donor species
into a recipient plant species by means of a bacterial plasmid, virus or other vector.
The inserted gene is refered to as transgene. Genetic Engineering and genetically
modified organism (GMO) refere to plant transformation and generally not to other.
biotechnologies.
Genetic Engineering is somewhat similar to backcrossing in conventional plant breeding
because in both cases a specific gene is transferred from donor to recipient plant but the
difference is that in backcrossing donor source is plant from same species whereas in
genetic engineering donor may be from any species such as plant, bacteria, fish etc.
A number of plant species have been genetically transformed with foreign DNA. It
includes corn, alfalfa, orchard grass, potato, cauliflower, soybean, lettuce, sunflower,
carrot, canola, cotton, tomato and many others.
BRIEF HISTORY
The foundations of modern plant biotechnology can be traced back to the Cell Theory of
Schleiden (1838) and Schwann (1839), which recognized the cell as the primary unit of
all living organisms. The concept of cellular totipotency, which was inherent in the Cell
Theory and forms the basis of plant biotechnology, was further elaborated by Haberlandt
(1902), who predicted the production of somatic embryos from vegetative cells.
People didn't know where genes lived until DNA, or deoxyribonucleic acid, was
"discovered" or understood in the early 1950s. British scientist Rosalind Franklin's DNA
research formed the foundation for James Watson and Francis Crick's 1953 discovery of
the structure of DNA, the ladder-like double helix. Watson and Crick perfected the DNA
structural model that Franklin explored earlier.
Understanding DNA was essential to the exploration of biotechnology. With
biotechnology, scientists could express favorable traits by lending DNA from one
organism to another. From the beginning, scientists saw the potential for new drugs
designed to help the body do what it couldn't do on its own, or crops able to protect
themselves from disease. For example, through biotechnology-developed built-in
protection, researchers have developed corn plants resistant to rootworm.
In 1973, researchers Stanley Cohen and Herbert Boyer were the first to apply this
technique. Working to help people living with diabetes, they lifted genetic materials from
one organism's DNA and copy them into another's. It's the story of insulin.
Biotechnology continues to develop. In the 1980s, testing of biotechnology-derived foods
began, and after its FDA approval in 1994, the FlavrSavr® tomato gave consumers a
more flavorful tomato that stays fresh longer. Three years after the FlavrSavr tomato's
introduction, 18 biotechnology-derived crops were approved by the U.S. government, and
research and development continues to improve agricultural productivity and enhance
foods' nutritional value.
COMMON PLANT TRANSFORMATION METHODS
A number of mechanisms are available to transfer DNA into plant cells:
1. Agrobacterium tumefaciens - transfer of DNA from bacteria to plants
2. Biolistics/ Particle bombardment - rapidly propelled tungsten or gold
microprojectiles coated with DNA are blasted into cells
3. Electroporation - electrical impulses are used to increase membrane and cell
wall permeability to DNA contained in the surrounding solution
4. Microinjection - injection of DNA directly into the cell nucleus using an ultrafine
needle
5. Poly-ethelyne-glycol - plant cell protoplasts treated with PEG are momentarily
permeable, allowing uptake of DNA from the surrounding solution.
6. Viral transformation (transduction): Package the desired genetic material into a
suitable plant virus and allow this modified virus to infect the plant. If the genetic
material is DNA, it can recombine with the chromosomes to produce
transformant cells. However genomes of most plant viruses consist of single
stranded RNA which replicates in the cytoplasm of infected cell. For such
genomes this method is a form of transfection and not a real transformation,
since the inserted genes never reach the nucleus of the cell and do not integrate
into the host genome. The progeny of the infected plants is virus free and also free
of the inserted gene.
GENETIC TRANSFORMATION IN PLANTS
Agrobacterium mediated gene transfer
It is the most widely used transformation technique in plants. Agrobacterium
tumefaciens, a soil bacteria, contains a Ti plasmid (tumor-inducing) which normally
infects dicotyledonous plant cells, making the bacteria an excellent vector for the transfer
of foreign DNA. By removing the tumor inducing genes and replacing them with the
genes of interest, efficient transformation can occur.
A.tumefaciens naturally infects the wound sites in dicotyledonous plant causing the
formation of the crown gall tumors. Later it was evident that A. tumefaciens is capable to
transfer a particular DNA segment (T-DNA) of the tumor-inducing (Ti) plasmid into the
nucleus of infected cells where it is subsequently stable integrated into the host genome
and transcribed.
The T-DNA contains two types of genes:
1. Oncogenic genes, encoding for enzymes involved in the synthesis of auxins and
cytokines and responsible for tumor formation;
2. Genes encoding for the synthesis of opines, a product resulted from condensation
between amino acids and sugars, which are produced and excreted by the crown
gall cells and consume by A. tumefaciens as carbon and nitrogen sources.
Outside the T-DNA, are located the genes for the opine catabolism. These genes are
involved in the process of T-DNA transfer from the bacterium to the plant cell and for
the bacterium-bacterium plasmid conjugative transfer genes.
Adaptation of the bacterial model for foreign gene transfer to crop plant
requires the following steps.
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Introduction of the foreign gene into the T-DNA of the bacteria
Introduction of the bacteria containing the foreign gene into cells of host plant.
Integrartion of the foreign gene into the genome of the host cell.
Expression of the foreign gene in the regenerated crop plant
Transmission of the foreign gene and its expression through normal sexual
processes to plants in succeeding generation in seed reproduced species, or
through normal sexual propagation in vegetatively reproduced species.
Agrobacterium mediated transformation is the easiest and most simple plant
transformation. Plant tissue (often leaves) is cut into small pieces, eg. 10x10mm, and
soaked for 10 minutes in a fluid containing suspended Agrobacterium. Some cells along
the cut will be transformed by the bacterium that inserts its DNA into the cell. Placed on
selectable rooting and shooting media, the plants will regrow. Some plants species can be
transformed just by dipping the flowers into suspension of Agrobacterium and then
planting the seeds in a selective medium.
Unfortunately, many plants are not transformable by this method so in such cases other
transformation methods such as Particle bombardment, Electroporation, Microinjection,
Poly-ethelyne-glycol are used to genetically transform protoplast.