Biotechnology and Its Applications

CHAPTER Ten

BIOTECHNOLOGY AND ITS APPLICATIONS

Biotechnology, as you would have learnt from the previous chapter, essentially deals with industrial scale production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants and animals. The applications of biotechnology include therapeutics, diagnostics, genetically modified crops for agriculture, processed food, bioremediation, waste treatment, and energy production. Three critical research areas of biotechnology are:

(i) Providing the best catalyst in the form of improved organism usually a microbe or pure enzyme.

(ii) Creating optimal conditions through engineering for a catalyst to act, and

(iii) Downstream processing technologies to purify the protein/organic compound.

Let us now learn how human beings have used biotechnology to improve the quality of human life, especially in the field of food production and health.

Ten point one BIOTECHNOLOGICAL APPLICATIONS IN AGRICULTURE

Let us take a look at the three options that can be thought for increasing food production

(i) agro-chemical based agriculture;

(ii) organic agriculture; and

(iii) genetically engineered crop-based agriculture.

The Green Revolution succeeded in tripling the food supply but yet it was not enough to feed the growing human population. Increased yields have partly been due to the use of improved crop varieties, but mainly due to the use of better management practices and use of agrochemicals (fertilisers and pesticides). However, for farmers in the developing world, agrochemicals are often too expensive, and further increases in yield with existing varieties are not possible using conventional breeding.

As traditional breeding techniques failed to keep pace with demand and to provide sufficiently fast and efficient systems for crop improvement, another technology called tissue culture got developed. What does tissue culture mean? It was learnt by scientists, during nineteen fifties, that whole plants could be regenerated from explants, i.e., any part of a plant taken out and grown in a test tube, under sterile conditions in special nutrient media. This capacity to generate a whole plant from any cell/explant is called totipotency. You will learn how to accomplish this in higher classes. It is important to stress here that the nutrient medium must provide a carbon source such as sucrose and also inorganic salts, vitamins, amino acids and growth regulators like auxins, cytokinins etc. By application of these methods it is possible to achieve propagation of a large number of plants in very short durations. This method of producing thousands of plants through tissue culture is called micro-propagation. Each of these plants will be genetically identical to the original plant from which they were grown, i.e., they are somaclones. Many important food plants like tomato, banana, apple, etc., have been produced on commercial scale using this method. Try to visit a tissue culture laboratory with your teacher to better understand and appreciate the process.

Another important application of the method is the recovery of healthy plants from diseased plants. Even if the plant is infected with a virus, the meristem (apical and axillary) is free of virus. Hence, one can remove the meristem and grow it in vitro to obtain virus-free plants. Scientists have succeeded in culturing meristems of banana, sugarcane, potato, etc.

Scientists have even isolated single cells from plants and after digesting their cell walls have been able to isolate naked protoplasts (surrounded by plasma membranes). Isolated protoplasts from two different varieties of plants - each having a desirable character - can be fused to get hybrid protoplasts, which can be further grown to form a new plant. These hybrids are called somatic hybrids while the process is called somatic hybridisation. Imagine a situation when a protoplast of tomato is fused with that of potato, and then they are grown - to form new hybrid plants combining tomato and potato characteristics. Well, this has been achieved - resulting in formation of pomato; unfortunately this plant did not have all the desired combination of characteristics for its commercial utilisation.

Is there any alternative path that our understanding of genetics can show so that farmers may obtain maximum yield from their fields? Is there a way to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced? Use of genetically modified crops is a possible solution.

Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO). GM plants have been useful in many ways. Genetic modification has:

(i) made crops more tolerant to abiotic stresses (cold, drought, salt, heat).

(ii) reduced reliance on chemical pesticides (pest-resistant crops).

(iii) helped to reduce post harvest losses.

(iv) increased efficiency of mineral usage by plants (this prevents early exhaustion of fertility of soil).

(v) enhanced nutritional value of food, e.g., golden rice, i.e., Vitamin 'A' enriched rice.

In addition to these uses, GM has been used to create tailor-made plants to supply alternative resources to industries, in the form of starches, fuels and pharmaceuticals.

Some of the applications of biotechnology in agriculture that you will study in detail are the production of pest resistant plants, which could decrease the amount of pesticide used. Bt toxin is produced by a bacterium called Bacillus thuringiensis (Bt for short). Bt toxin gene has been cloned from the bacteria and been expressed in plants to provide resistance to insects without the need for insecticides; in effect created a bio-pesticide. Examples are Bt cotton, Bt corn, rice, tomato, potato and soyabean etc.

Bt Cotton: Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). Bacillus thuringiensis forms protein crystals during a particular phase of their growth. These crystals contain a toxic insecticidal protein. Why does this toxin not kill the Bacillus? Actually, the Bt toxin protein exist as inactive protoxins but once an insect ingest the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut which solubilise the crystals. The activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death of the insect.

Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into the several crop plants such as cotton. The choice of genes depends upon the crop and the targeted pest, as most Bt toxins are insect-group specific. The toxin is coded by a gene cryIAc named cry. There are a number of them, for example, the proteins encoded by the genes cryIAc and cryIIAb control the cotton bollworms, that of cryIAb controls corn borer.

Pest Resistant Plants: Several nematodes parasitise a wide variety of plants and animals including human beings. A nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a great reduction in yield. A novel strategy was adopted to prevent this infestation which was based on the process of RNA interference (RNAi). RNAi takes place in all eukaryotic organisms as a method of cellular defense. This method involves silencing of a specific mRNA due to a complementary double-stranded RNA molecule that binds to and prevents translation of the mRNA (silencing). The source of this complementary RNA could be from an infection by viruses having RNA genomes or mobile genetic elements (transposons) that replicate via an RNA intermediate.

Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant. The introduction of DNA was such that it produced both sense and anti-sense RNA in the host cells. These two RNAs being complementary to each other formed a double stranded RNA that initiated RNA interference and thus, silenced the specific messenger RNA of the nematode. The consequence was that the parasite could not survive in a transgenic host expressing specific interfering RNA. The transgenic plant therefore got itself protected from the parasite.

The recombinant DNA technological processes have made immense impact in the area of healthcare by enabling mass production of safe and more effective therapeutic drugs. Further, the recombinant therapeutics do not induce unwanted immunological responses as is common in case of similar products isolated from non-human sources. At present, about thirty recombinant therapeutics have been approved for human use worldwide. In India, twelve of these are presently being marketed.

Ten point two point one Genetically Engineered Insulin

Management of adult-onset diabetes is possible by taking insulin at regular time intervals. What would a diabetic patient do if enough human insulin was not available? If you discuss this, you would soon realise that one would have to isolate and use insulin from other animals. Would the insulin isolated from other animals be just as effective as that secreted by the human body itself and would it not elicit an immune response in the human body? Now, imagine if bacterium were available that could make human insulin. Suddenly the whole process becomes so simple. You can easily grow a large quantity of the bacteria and make as much insulin as you need.

Think about whether insulin can be orally administered to diabetic people or not. Why?

Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and pigs. Insulin from an animal source, though caused some patients to develop allergy or other types of reactions to the foreign protein. Insulin consists of two short polypeptide chains: chain A and chain B, that are linked together by disulphide bridges.

In mammals, including humans, insulin is synthesized as a pro-hormone (like a pro-enzyme, the pro-hormone also needs to be processed before it becomes a fully mature and functional hormone) which contains an extra stretch called the C peptide. This C peptide is not present in the mature insulin and is removed during maturation into insulin. The main challenge for production of insulin using recombinant DNA techniques was getting insulin assembled into a mature form. In nineteen eighty-three, Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.

Ten point two point two Gene Therapy Sala If a person is born with a hereditary disease, can a corrective therapy be taken for such a disease? Gene therapy is an attempt to do this. Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child or embryo. Here genes are inserted into a person's cells and tissues to treat a disease. Correction of a genetic defect involves delivery of a normal gene into the individual or embryo to take over the function of and compensate for the non-functional gene.

The first clinical gene therapy was given in nineteen ninety to a four-year old girl with adenosine deaminase deficiency. This enzyme is crucial for the immune system to function. The disorder is caused due to the deletion of the gene for adenosine deaminase. In some children adenosine deaminase deficiency can be cured by bone marrow transplantation; in others it can be treated by enzyme replacement therapy, in which functional adenosine deaminase is given to the patient by injection. But the problem with both of these approaches that they are not completely curative. As a first step towards gene therapy, lymphocytes from the blood of the patient are grown in a culture outside the body. A functional adenosine deaminase cDNA (using a retroviral vector) is then introduced into these lymphocytes, which are subsequently returned to the patient. However, as these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. However, if the gene isolate from marrow cells producing adenosine deaminase is introduced into cells at early embryonic stages, it could be a permanent cure.