Bacteria

loop

or wire across the

agar

you are removing

bacteria

from the loop. The reason it is essential is because every little bacterium that comes off that loop will grow like crazy on the agar, creating a colony. When you have an individual colony, after many times of streaking, you know, almost certainly, that you have genetically identical bacteria, barring mutations, without contamination.

So, simply put, to ensure your bacterial specimen is, to the best of your ability, without contamination.

extreme halophiles: LOVE salt, use the salt to generate ATP, and are found in the Dead Sea and Great Salt Lake

thermocidophiles: LOVE high acidity and temperatures,found in hot springs and volcanic vents

INTRODUCTION

Induction of crown gall and hairy root diseases in several dicot plants by the common soil borne Gram-negative bacteria Agrobacterium tumefaciens and A.rhizogenes, respectively , are example of natural transformation of plants wherein the bacterial genes are stably introduced into the genome of higher plants. This system represents the only known natural case in which a prokaryotic organism transfers genetic information to a eukaryotic host. This capability underlies the biotechnological uses of Agrobacterium , mostly employed for the genetic transformation of numerous plants species. Recent discoveries have expanded the potential biotechnological uses of Agrobacterium; indeed , under laboratory condition , Agrobacterium is able to transfer DNA and proteins to numerous non.plant species , including several species of yeast and other fungi as well as sea urchin embryos , human cells in culture

Agrobacterium-mediated transformation is the easiest and most simple plant transformation. Plant tissue are cut into small pieces , eg. 10×10 mm, and soaked for 10 min. 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 suspension of Agrobacterium and then planting the seeds in a selective medium. Unfortunately , many plants are not transformable bt this method .

1. DETAIL DESCRIPTION

Recombinant DNA technology is based on the insertion of a DNA fragment ( Gene) of interest into a suitable cloning vector and then its introduction into a suitable host to propagate the recombinant DNA.

Fig: generalized method of gene transfer in cells

1.1. GENE CARRIER VEHICLE -

1. If a gene is to be introduced into a host cell, a carrier molecule

that can transport the gene into the host cell is required Such

a molecule is called a cloning vehicle , carrier molecule or a

vector.

1.2. FOLLOWING ARE A FEW GENE CARRIER VEHICLES

1.Plasmids

2. Bacteriophages

3. Viruses

2.1.AGROBACTERIUM TUMEFACIENS

1. Agrobacterium tumecaiens is a soil borne gram negative

bacterium.

2 .It invades many dicot plants when they are injured at the soil

level and causes grown gall disease

3.The bacterium enters the plant through a fresh wound and

attaches itself to the wall of the intact cell.

4.This cell is genetically transformed by bacterium .

5. This transformation result in a tumour which synthesizes

OPINES :

A. The Tumors develops only at site of the wound.

B. Such tumours can be removed from the plant and cultered

in-vitro where they continue to grow indefinitely.

C. Continued presence of agrobacterium is not required for tumor

Profileration.

D. Agrobacterium induced tumours synthesize a variety of unusal

Compound called opines

E. Opines are of 3 types -

a). Octapine

b). Nopaline

c). Agropine

These opines are catabolised by Agrobacterium to obtain energy,

AT genectically engineers the plant cell for its won purpose.

3.1. TUMOUR INDUCING PRINCIPLE

1. The tumour inducing principle of AT is a plasmid calles tumour

inducing plasmid or Ti Plasmid .

a). 200 kb long.

b). Has two essential regions : T-DNA and vir region.

c). These two regions are essential for the transformation process.

3.2. TRANSFER OF TUMOUR INDUCING PRINCIPLE

1.T-DNA(transferred DNA ) is excised from the Ti-Plasmid and

transferred to the nucleus of the plant cell.

2. Here the T-DNA gets integrated into the DNA which is stable.

3. The T-DNA can be passed on to daughter cells as an integral

Part of the plant chromosome.

Figure. Induction of crown gall on a dicot plant by agrobacterium

tumefaciens.

4.1 Ti PLASMIDS

1. An extra chromosomal double stranded circular DNA molecule.

2. Tumour inducing .

3. 200 kb in size and conjugative type.

4. Encodes enzymes responsible for the synthesis and catabolism of certain opnies.

5. One of the opines is nopaline.

6. pTiC58 is present in Agrobacterium strain C58. it is 192 kb long

7. Only a small segment of the Ti Plasmid is transferred to the host plant cell and gets integrated with the grnome . This is the T-DNA.

Figure. Ti plasmid pTic 58 having 192 kb

5.1. T-DNA

1.Only a small segmented of the Ti Plasmid is transferred to the

host plant cell and gets integrated with the genome.

2.This is the T-DNA

3.It contains gene for tumour formation (Tum) and nopaline

biosynthesis (Nos).

4.The genes encodes enzymes that catalyse the synthesis of

phtohormones like the IAA and the cytokinin , isopentenyl

adenosine that cuse tumerous growth of cells in crown galls.

5.The T-DNA is bordered by 25 bp repeats, required for the

excision and transfer of T-DNA.

6.1. NOPALINE Ti PLASMID pTiC58

1. The Vir region of the Ti-plasmid contains the genes required for

the T-DNA transferprocess . the genes in this region encode the

DNA processing enzymes required for excision , transefer and

integration of the T- DNA segmented.

7.1. TUMOUR INDUCTION BY AGROBACTERIUM

1. Recognition of susceptible wounded plant cell:

a). Plant exudates: act as signal by inducing genes in the Vir Genes

of the Ti Plasmid.

b). Acetosyringone (as) , alfa- hydroxy acetosyringone (OH-HS)

2. Binding to wound cells : controlled by two chromosomal genes of

agrobacterium : chv-A and chv-B.

3. Excision , transfer and integration :

a). The border repeats of T- DNA play an important role .

b). Any DNA sequence located between the border repeats is

transferred to the host plant.

c). The T-DNA region is excised from the plasmid by the enzymes

encoded by the vir- region.

4.These enzymes specifically recognize the T-DNA borders.

5.The T-DNA enters the plant cell and integrates into the host

genome , mediated by host enzymes.

8.1. Ti-PLASMID AS A VECTOR

1. The Ti- plasmid has an innate ability to transmit bacterial DNA

into plant cells .

2. This potential is explited by the genetic engineers to use as a

vector.

3.The gene of a donor organism can be introduces into the Ti-

plasmid at the T-DNA region

4. This plasmid now becomes a recombinant plasmid.

5. By agrobacterium infection , the donor genes can be transferred

from the recombinant Ti-Plasmid and integrated into the genotype

of the host plant.

6. This results in the production of transgenic plant.

Pic. Ti-Plasmid mediated transfer of gene into a plant

9.1. DISARMED Ti PLASMID

1.Disarmed Ti-plasmid

a).Deletion of T-DNA region

b). PGV3850 is constructed from pTiC58.

2. It has pBR 322 with AmpR

3.It border repeats and NOS genes

4. Agrobacterium having this PGV3850 can transfer the modified

T-DNA into plant cells.

5. But the recipient cell will not produce tumour , but could produce

nopaline.

6. This can be used as a efficient vector for introducing foreign gene

into plants.

10.1. CONTRUCTION OF A COINTEGRATE

1. A foreign gene cloned into an appropirate plasmid (pBR322) can

be integrated with the disarmed Ti-Plamid by a homologous

recombination

2. A compound plasmid called a cointegrate is formed.

Figure. Cointegrate Plasmid .

11.1. TRANSFORMATION OF TISSUE EXPLANTS BY

CO-CULTIVATION WITH AGROBACTERIUM

1. A co-integrate plasmid derived by recombination of pGV3850 and pBR322 loaded with foreign gene is now used to transfer the foreign gene into many crop plants.

2. Small disc (a few mm diameter ) are punched from leaves of petunia, tobacco, tomato or other dicot plants.

3. These disc are incubated in a medium containing Agrobacterium carrying the recombinant disarmed T-DNA as a co-ingerate .The cointegrate plasmid has the foreign gene and also the gene for resistance to kanamycin (KmR).

4. The disc are cultered for two days . the agrobacterium infects the cut edges of the disc.

5. The disc are then transferred to a shoot inducing solid medium (high cyokinin ) containing kanamycin to select the transferred kanamycin to select the transferred kanamycin gene. Corbenicillin in the medium kills agrobacterium.

6. After 2-4 weeks the shoot develops.

7. The callus having the shoot is transferred to root inducing solid medium (high in auxin content).

8. After 4-7 weeks roots appear.

9. The rooted plantlets are transferred to soil.

Pic. Transformation of leaf disc explants by co-cultivation with agrobacterium

having the cointegrate Ti plasmid

REVIEW OF LITERATURE

Agrobacterium mediated transformation has been a method of choice in dicotyledonous plant species where plant regeneration system are well established (Van Wordragen and Dons , 1992 ; dale et al. 1993 ). The host range of this pathogen includes about 60% of gymnosperms and dicotyledonous angiosperms. Besides , transformation success has also been achieved in some monocots like Asparagus officinalis , Chlorophytum , Narcissus (hernalstees et al. 1984, hookyaasvan slogteren et al. 1984 ) .It was believed that monocots lack wound response i.e. factors that are required to initiate of `vir` genes. (Schafer et al. 1987) achieved success in transforming another monocot ,yam (Diosscorea bulbifera) from potato tubers .likewise , the use of acetosyringone (synthetic phenolic compound ) either during bacterial growth or during co-cultivation has been found to be beneficial in the transformation of other monocots. There are several reports on successful transformation of rice using Agrobacterium (Hiei et al. 1994 ; Li et al. 1994 ; vijaychandra et al. 1995 ). This method has also been extended to barley, wheat, maize and sugarcane.

APPLICATION

Genetically modified plants have been developed commercially to improve shelf life, disease resistance, herbicide resistance and pest resistance. Plants engineered to tolerate non-biological stresses like drought, frost and nitrogen starvation or with increased nutritional value (e.g. Golden rice) were in development in 2011. Future generations of GM plants are intended to be suitable for harsh environments, produce increased amounts of nutrients or even pharmaceutical agents, or are improved for the production of bioenergy and biofuels. Due to high regulatory and research costs, the majority of genetically modified crops in agriculture consist of commodity crops, such as soybean, maize, cotton and rapeseed. However, commercial growing was reported in 2009, of smaller amounts of genetically modified sugar beet, papayas, squash sweet pepper, tomatoes, petunias, carnations, roses and poplars. Recently, some research and development has been targeted to enhancement of crops that are locally important in developing countries, such as insect-resistant cowpea for Africa and insect-resistant brinjal (eggplant) for India. In research tobacco and Arabidopsis thaliana are the most genetically modified plants, due to well developed transformation methods, easy propagation and well studied genomes.They serve as model organisms for other plant species. Genetically modified plants have also been used for bioremediation of contaminated soils. Mercury, selenium and organic pollutants such as polychlorinated biphenyls (PCBs) have been removed from soils by transgenic plants containing genes for bacterial enzymes.

PRESENT STATUS

Today , agrobacterium mediated gene transfer method used in various

field like

1. Agriculture: Crops having larger yields, disease- and drought- resistancy; bacterial sprays to prevent crop damage from freezing.

2. Temperatures; and livestock improvement through changes in animal traits.

3. Industry: Use of bacteria to convert old newspaper and wood chips into sugar; oil- and toxin-absorbing bacteria for oil spill or toxic waste clean-ups; and yeasts to accelerate wine fermentation.

4. Medicine: Alteration of human genes to eliminate disease (experimental stage); faster and more economical production of vital human substances to alleviate deficiency and disease symptoms (but not to cure them) such as insulin, interferon (cancer therapy), vitamins, human growth hormone ADA, antibodies, vaccines, and antibiotics.

5. Research: Modification of gene structure in medical research, especially cancer research. Food processing: Rennin (enzyme) in cheese aging.

FUTURE PROSPECTUS

The field of Agrobacterium research is increasing our understanding of the bacterium itself; in particular, the mechanism by which the T-DNA is translocated into the plant cell and exactly which bacterial virulence proteins accompany it. This understanding is not only vital for the biotechnological application of Agrobacterium, the plant factors used by Agrobacterium to ensure transport across the plant wall, membrane, and cytoplasm, nuclear import, and finally integration of the T-DNA. The application of this knowledge to improve transformation rates will bring gene technology to species that, at present, are recalcitrant to Agrobacterium and not transformation competent using a biolistic approach. The improvement of transformation protocols is a practical advance, but the really exciting advances will be in the types of modification and the application therein. Current research foci include biodegradable plastics in plants (Mittendorf et al., 1998), vaccination against common human diseases administered by eating the plant (Staub et al., 2000), and plants as indicators of environmental toxins (Kovalchuk et al., 2003). With so much promise, Agrobacterium could be the key to future agricultural progress. It can only be hoped that regular, constructive debate can lead to legislative solutions for the ethical, health, and political issues that are likely to play such an influential role in the development of our society.

CONCLUSION

Transformation is an important topic in plant biology and transgenic plants have become a major focus in plant research and breeding programs. Agrobacterium-mediated transformation as a practical and common method for introducing specific DNA fragments into plant genomes is well established and the number of transgenic plants produced using this method is increasing. Despite the popularity of the method, low efficiency of transformation is a major challenge for scientists. Modification of different genetic and environmental aspects of transformation method may lead to better understanding of the system and result in high efficiency transformation. In this review, we deal with recent genetic findings as well as different environmental factors which potentially influence Agrobacterium-mediated transformation.