BACTERIAL BIOTECHNOLOGY

The Production of high quality agricultural products in quantities sufficient to feed the world's people is clearly a matter of vital importance. Any general improvement in agricultural practices can have a tremendous economic impact, and the methods of biotechnology suggest at least two important strategies for bringing about widespread improvements.

1) Improvement through the use of symbiotic or potentially pathogenic microorganisms -

Plants live in intimate relationship with many microorganisms. Leaf surfaces are covered by layers of symbiotic & sometimes pathogenic bacterial & yeasts, and the soil close to the root area is populated by bacteria species very different from the species found in soil away from plants. There have been a number of attempts to modify symbiotic microorganisms so as to make them even more beneficial to plants.

2) Improvement through the production of transgenic plants -

Plants now being cultivated represent the end result of many years of pain staking efforts at improvement. Traditional plant breeding programs rely on two methods; the selection of advantageous spontaneous mutations & the introduction of desirable traits from closely related species by crossbreeding. Recombinant DNA Technology is bringing revolutionary change to these endeavous. It enables scientists to select precisely defined genes with well-characterized properties, clone them, and transfer them to a given plant. These genes can be taken from organisms unrelated to the target plant -even from bacteria or animals.

Transferring cloned genes from prokaryotes (bacteria) into higher eukaryotes, such as plants, is difficult. However, there are two species of bacteria, Agrobacterium tumefaciens & A rhizogenes, that transfer a small piece of DNA into plants as part of their normal life cycle.

I) Use of symbionts & Pathogenes

There are many Symbiotic bacterial associated with specific organs of various plants, a simple plant might be to modify such bacterial & then use such interactions with the plants to introduce the modified traits at the appropriate locations. This alternative approach is technically easier, because the engineering of bacterial DNA through recombinant DNA methods is now routine, whereas the manupulation of plant DNA is yet to be perfected.

a) Protection of plants from frost damage via engineered symbiotic bacteria

One of the earliest examples of successful modification of a symbiotic bacterium was performed in Pseudomonas syringae, which is found at high concentrations on the leaves of many plants. Many strains of this bacterium produce an ice nucleation protein that is apparently located on the surface of the bacteria cell, and the prescence of this protein cause the formation of ice at temperature only little below 00 C, inflicting significant frost damage on important crop plants & so facilitating invasion of plant tissues by the bacteria.

b) Use of nitrogen fixing bacteria to improve crop yields

All animals & plants & most bacteria depend on the availability in their enviroment of some form of "combined nitrogen" nitrate (No3), or such nitrogen containing organic compounds as amono acids. The huge amount of N2 that exist in the atmosphere are unavailable to the biological world except through the process of nitrogen fixation.

The biological process of nitrogen fixation does not require the consumption of fossil fuels or electricity does not produce environmental pollution. Thus increasing the extent of biological nitrogen fixation has been an important goal for biotechnology. According to one estimate, the biological process fixes times more nitrogen (24 x 10 17 tons/year) than is converted into chemical fertilizers by industrial processes. Thus even a small improvement in exploiting the biological process would have been a global impact.

he biological process of nitrogen fixation is complex & consumes a large number of ATP, molecules, because the enzymes involved in it must overcome the same huge activation energy barriers that faces the chemical synthesis of ammonia. Two enzymes are required :component I (nitrogenase) & component II (nitrogenase reductase). After component Ii is reduced by a strong biological reductant, 16 molecules of ATP are hydrolysed to accomplish the reduction of component I. Reduced component II alone is not capable of overcoming the ativation energy barrier. Reduced component I finally reduced N2 to two molecules of NH3. Nitrogen fixation is strongly reductive reaction, and the enzymes involved are usually irreversibly inactivated when they are exposed to oxygen.

The species of closiridium & klebsilla fix nitrogen only under angerobic conditions. Other free living baueria, however, can fix nitrogen even under aerobic conditions. The cyanobaueria, which carry out oxygen - evoluing photosynthesis, perform nitrogen fixation only in speciallized cells called heterocysts, which do not produce oxygen. Azotobactor consumes oxygen at an extremly high rate, and in doing so apparently protects its nitrogen fixing machinery. Another group of bacteria fix nitrogen only when they are in a symbiotic nitrogen fixer is Rhizobium, which invades the root tissues of teguminous plants, such as alfalfa, pea, clover and soyabean and lives in intracellular vacuoles. The vacuoles become filled with an oxygen-binding protein, leghomoglobin produced by the plants. This creates an environment low in free oxygen where Rhizobium is able to carry out nitrogen fixation.

II) Production of Transgenic Plants

To produce transgenic plants, the crucial steps are the introduction of the cloned genetic material into the plant cell nuclean & the facilitated integration of the cloned gene into the plant chromosome. It is interesting that the best method for doing so uses a system that already exists in nature, the system by which the plant pathogenic bacterium Agro-bacterium tumefaciens injucts a portion of its plasmid DNA into plants & inserts it into the plant become.

The Agro-bacterium system is an excellent system for introducing foreign genes into the chromosomes of plants. The transfer into intact plant cells occur at high frequency, the T-DNA is usually integrated into the plant chromosomes at high frequencies without undergoing structural alterations, and the cells that have received the T-DNA can be selected easily by using antibiotic resistance such as the neomycin resistance maricer. Finally the transgenic plants produced in this manner are quiet stable for at least several generations. Consequently almost all existing transgenic plants with potentially desirable traits have been obtained by this method.

a) Herbicide resistance plants -

The advantages of making crop plants resistant to herbicides are obvious. Although there is a fear that such resistant may eventually increase the use of herbiude chemicals, there are also reasons to except that these transgenic plants will promote the use of sager, more biodegradable herbicides, perhaps in smaller amounts one example involves the herbicide glyphosate which inhibits 5-enol-pyruvylshikimate 3-phophate synthase - an enzyme involved in the biosynthesis of aromatic amino acids. This enzyme has been purified from crop plants & sequenced and DNA Probes corresponding to its amino acid sequence have been synthesized. These probes were used to isolate CDNA for the enzyme from the CDNA library of a plant cell line known to overproduce 5-enol-pyruvylshikimate 3-phophate synthase. The CDNA was then cloned behind the strong CaMv promoter, and the promoter gene complex was introduced into plant cells via a disarmed Ti plasmid vector. The transgenic plants produce a much higher level of the target enzyme & thus are significantly more resistant to glyphosate. These results are encouraging because glyphosate has very low toxicity to animals is rapidly degraded in soil.

b) Insect resistant plants -

Bacitlus thuringiensis is used in the biological control of caterpillars because its sporulating cells contain toxic proteins. The gene for one toxic protein was cloned behind promoters that are effectively expressed in plants & was introduced into plant cells via a Ti plasmid vector, thus producing plants toxic to caterpillar. The major problem with this approach has been the low level of expression of the toxin protein in plants, which is presumably related to the fact that the gene come from a bacterium,. Nevertheless, the method has produced tomato & tobacco plants that proved quiet resistant to caterpillars in field tests. Cotton however, is often attacked by insects that are more resistant to the B. thuringiensis toxin and the low level of expression of this toxin in transgenic cotton plants provided the plants with little if any protection. Recently, the coding sequence of the toxin was altered extensively to replace codons that are rarely used in plants as well as to preclude the formation of a strong secondary structure in MRNA. When cotton plants were provided with this modified gene, their production of the bacterial toxin increased 100-fold and they showed impressive resistant to common bepidopteran insects than damage unmodified plants.

Some seeds contain high concentrations of protease inhibitors, which are thought to interfere with the digestive process in insects. The cloning of cowpea trypsin inhibitor genes, for example and their transfer to tobacco plants have resulted in good resistance to a wide variety of leaf-eating insects.

c) Viral resistant plants -

The method most frequency used for producing virus-resistant plants sprang from the observation that, oftentimes, plants infected by nearly avirulent virus are thereafter resistant to super infection by a related, highly virulent one. Thus, deliberate infeuction with avirulent virus strain has been used to produce protection in crop plants. The method is not totally safe, however, because the avirulent strains many mutate to produce strains that are significantly pathogenic. Most plant viruses are positive strand RNA viruses covered by coat protein sub units. When the virus enters an injured plant cell the replication process begins, starting with the progressive uncoating of the virus from the 5-end of the RNA. Tranagenic plants, whose genomes contain introduced tobacco mosalc virus (TMV) coat protein genes and which continuosly synthesize the coat proteins, show resistance to virus infection. The phenotype of these plants is consistent with the idea that resistance is a result of interference with the uncoating of the virus particle. Although the plant cells are resistant to infact TMV particles, they remain sensitive to the TMV RNA or to partially uncoated TMV particles.