Recombinant DNA technology

Recombinant DNA technology is a procedure by which geneticists remove segments of DNA from one
organism and insert them into the DNA of another.

Introduction:
• In recombinant DNA technology, genes are removed from one organism, and then spliced into
another of the same or a different species.
• Enzymes are used to excise segments of genes and insert them into foreign DNA molecules.
• After a segment of DNA is transplanted, a second enzyme, DNA ligase, is used to seal it
in place.
• At this point, geneticists have formed a recombinant DNA molecule—a molecule that
contains DNA from two different organisms.

• Plasmids from bacteria can be used to clone DNA fragments for mass production.
• Foreign genes can be spliced into plasmids.
• The plasmid carrying a foreign gene can be reinserted into bacteria or yeast cells.
• In culture, the plasmids are rapidly taken up by the new hosts. As these cells divide, the
plasmids replicate.
• This produces multiple copies of the gene.
• Genes can be mass produced on strands of mRNA.
• Messenger RNA is extracted from cells and used to make DNA; this is called reverse
transcription.

• Genes can also be mass produced using the polymerase chain reaction.
• Part of the DNA double helix is extracted, heated to split the strands, and then cooled.
Enzymes are added to catalyze new DNA formulation.
Plant and Agricultural Recombinant DNA technology

Recombinant DNA technology has a wide application in the field of agriculture. The development of
methodology to maintain individual cells in tissue culture, coupled with the technology for regenerating
whole plants from such an individual cell, has led to the beginning of a revolution in plant breeding. It is
possible today to carry out, in weeks, a breeding step that would previously have taken an entire growing
season.
A. Transgenic plants are plants that carry a foreign gene
B. Use Ti plasmid from Agrobacterium tumefaciens as a vector: The Ti plasmid is a plasmid carried
by the bacterium A. tumefacien. This bacterium causes crown gall disease in plants due to the
insertion of part of the Ti plasmid into the plant genome. Ti has been engineered so that it can
deliver genes to a plant without causing disease.

C. Examples of uses for transgenic plants

1. Reporter genes for basic research
2. Flavrsavr tomatoes (Calgene Inc.)
a) Normally tomatoes are picked when they are unripe so that they will not bruise during transit. Prior to
marketing ethylene is provided which initiates the ripening process; however, although the tomatoes appear
to ripen, the flavor is poorer than vine-ripened tomatoes. Thus, researchers at Calgene tried to block only
the enzyme that causes softening of the fruit (polygalacturonase or PG) so that the tomatoes could remain
on the vine longer. They did this by constructing an antisense RNA that binds to the normal sense PG
mRNA to block translation. The gene encoding the antisense RNA was inserted into the plant chromosome
along with a kanamycin resistance marker gene. Their approach worked: the tomatoes can remain on the
vines longer and are less susceptible to bruising in transit.
b) Concerns
1. Kn resistance gene in the tomatoes may be picked up by soil bacteria.
2. Toxic compounds from genetic manipulation
3. Unnatural method of food production
c) Depending upon the source you get your info from, the FlavrSavr tomato was/was not a financial success.
3. Other examples of transgenic plants
a) Herbicide resistant plants
b) Cotton plants that are resistant to pests due to incorporation of a bacterial gene that is toxic to insects
c) Soybeans that produce more healthful combinations of fatty acids
d) Flowers that stays fresher longer by inhibition of genes that are involved in senescence.
4. Ultimately the success/failure of genetic engineered crops will depend on the consumer.
Salt and drought tolerant crops:
Recombinant DNA technology is being used to develop salt and drought tolerant crops. Salt tolerance
breeding programmes are underway throughout the world for crops including barely, corn, grapes and
tomatoes, and genetic engineering is effectively involved in the development of such crops.

Genetically engineered nitrogen fixers:

The ability of some bacteria to fix atmospheric nitrogen into biological makes them fixers. The most
common example of symbiotically nitrogen fixing bacteria are the species of genus Rhizobium. They
contain in their DNA a cluster of genes, called nif genes that are responsible for the formation of
nitrogenase and nitrogen fixation. The genetic engineers have transferred the nif genes to E.coli by sexual
conjugation. The advantage of nitrogen fixing E.coli is that it can fix nitrogen for nonleguminous plant
also.
To make plants self sufficient in nitrogen fixation, methods have been designed to clone bacterial genes
into plants. One such method exploits the natural infection process of the plant pathogen Agrobacterium
tumefaciens. This bacterium causes crown gall disease. The bacterium contains a plasmid, a portion of
which will integrate with the host cell chromosome. The cells from the tumors may be then cultured to
produce nitrogen fixing plants.

Frost-ban
One of the most important developments in the agricultural arena, employing recombinant DNA
techniques, is the creation of defrost bacteria. These genetically engineered bacteria Pseudomonas
fluorescens and Pseudomonas syringae are resistant to frost. When allowed to grow on the leaves of
strawberry plants, these bacteria resist the formation of frost and thus protect the crop from severe
damage caused by frost formation.
It is widely believed that the genetic engineering has a remarkable potential to influence the current trends
in industry and medicine. It holds great promise in revolutionizing every sphere of human life by creating
amazing technology and products. It is just dawn of a gene revolution!

References:
http://ntur.lib.ntu.edu.tw/bitstream/246246/2006092815540060/1/00ch8.ppt
http://www.scribd.com/document_downloads/3953889?extension=ppt
http://nsdl.niscair.res.in/bitstream/123456789/674/1/Biotechnology_Introduction.pdf