Biotechnology and its Applications — Notes

Plant tissue culture: whole plants can be regenerated from explants (any part of a plant) grown in sterile conditions on special nutrient media — the capacity called totipotency. The medium provides a carbon source (sucrose), inorganic salts, vitamins, amino acids and growth regulators such as auxins and cytokinins.

• Micropropagation produces thousands of plants in a short time; each plant is genetically identical to the original — a somaclone (here, 5 shoots per subculture give 625 plantlets after 4 rounds). Used for tomato, banana, apple and others.
• Meristem culture: although a plant is infected with a virus, its apical and axillary meristems are free of virus; culturing them gives virus-free plants (banana, sugarcane, potato).
• Somatic hybridisation: single cells are isolated, their walls digested by cellulase and pectinase to give naked protoplasts, and protoplasts of two varieties or species are fused to give hybrid protoplasts, which are grown into somatic hybrids — for example the pomato (tomato + potato), though it did not combine all the desired traits.

Genetically modified (GM) crops — plants, bacteria, fungi and animals whose genes have been altered by manipulation are GMOs. GM plants: (i) are more tolerant to abiotic stresses (cold, drought, salt, heat); (ii) reduce reliance on chemical pesticides (pest-resistant crops); (iii) reduce post-harvest losses; (iv) increase efficiency of mineral use (preventing early exhaustion of soil fertility); (v) have enhanced nutritional value (golden rice); (vi) can provide alternative resources — starches, fuels and pharmaceuticals.

Bt crops: some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). The bacterium forms protein crystals containing a toxic insecticidal protein that exists as an inactive protoxin. When an insect ingests it, the alkaline pH of the gut solubilises the crystals and converts the protoxin to the active toxin, which binds to the surface of midgut epithelial cells and creates pores, causing swelling and lysis and death of the insect. The toxin is coded by cry genes: cryIAc and cryIIAb control cotton bollworms; cryIAb controls the corn borer. Bt cotton, Bt corn, rice, tomato, potato and soybean have been developed.

RNA interference against nematodes: the nematode Meloidogyne incognita infects the roots of tobacco and reduces yield. Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant so that it produced both sense and complementary antisense RNA. These form double-stranded RNA (dsRNA), which starts RNAi and silences the specific mRNA of the nematode; the parasite cannot survive in the transgenic host. RNAi is a method of cellular defence in all eukaryotes.

Golden rice: a GM variety containing β-carotene (provitamin A), giving grains a golden colour, to combat vitamin A deficiency and night blindness.

Genetically engineered insulin: insulin used for diabetes was earlier extracted from the pancreas of slaughtered cattle and pigs, and some patients developed allergies to the foreign protein. Insulin consists of two short polypeptide chains, A and B, linked by disulphide bridges. In mammals it is synthesised as a pro-hormone (proinsulin) with an extra stretch, the C peptide, which is removed during maturation. In 1983, the American company Eli Lilly prepared two DNA sequences corresponding to chains A and B of human insulin and introduced them into plasmids of E. coli; the chains were produced separately, extracted and combined by creating disulphide bonds to form human insulin.

Vaccines: recombinant DNA technology allows production of antigenic polypeptides in bacteria or yeast; for example the hepatitis B vaccine produced in yeast. Such vaccines are safer because they contain no live pathogen.

Stem cells are undifferentiated cells that can renew themselves by division and differentiate into specialised cells. Embryonic stem cells (from the inner cell mass of an early embryo) are pluripotent and can form almost any cell type. Adult stem cells (bone marrow, umbilical cord blood) are multipotent. Applications: bone-marrow transplants for leukaemia and other blood disorders, replacing damaged tissues, and research into spinal-cord injuries, diabetes and other diseases; cord-blood banking stores stem cells for future use.

Gene therapy is a collection of methods that allows correction of a gene defect diagnosed in a child or embryo: genes are inserted into a person’s cells and tissues to treat a disease, a normal gene compensating for the non-functional one. The first clinical gene therapy was given in 1990 to a four-year-old girl with adenosine deaminase (ADA) deficiency. ADA is crucial for the immune system; its deletion causes severe combined immunodeficiency (SCID). Treatment can be by bone-marrow transplantation or enzyme-replacement therapy (injection of functional ADA), but these are not completely curative. In gene therapy, lymphocytes from the patient’s blood are grown in culture, a functional ADA cDNA is introduced using a retroviral vector, and the cells are returned to the patient. As these cells are not immortal, periodic infusion is needed. If the gene isolated from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

Molecular diagnosis: conventional methods (serum and urine analysis) cannot detect a pathogen early. Recombinant DNA technology, PCR and ELISA allow early diagnosis:

• PCR detects very low concentrations of a pathogen by amplifying its nucleic acid — used to detect HIV in suspected AIDS patients and mutations in genes of suspected cancer patients.
• A single-stranded DNA or RNA probe, tagged with a radioactive molecule, is allowed to hybridise to its complementary DNA in a clone of cells, followed by autoradiography; a clone with a mutated gene does not appear on the film, because the probe is not complementary to it.
• ELISA is based on antigen–antibody interaction; infection can be detected by the presence of antigens (proteins, glycoproteins) or of antibodies synthesised against the pathogen.

Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are transgenic animals. Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, though over 95 percent of all existing transgenic animals are mice.

Uses:

• Normal physiology and development: studying how genes are regulated and how they affect body functions (for example, insulin-like growth factor).
• Study of disease: models for human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer’s disease, so that new treatments can be investigated.
• Biological products: medicines can be expensive to make; transgenic animals that produce useful products are introduced with the gene coding for the product — for example human protein α-1-antitrypsin, used to treat emphysema; products for phenylketonuria (PKU) and cystic fibrosis are being tried. In 1997 the first transgenic cow, Rosie, produced human protein-enriched milk (2.4 g per litre) containing human α-lactalbumin, nutritionally more balanced for human babies than natural cow milk.
• Vaccine safety: transgenic mice are used to test the safety of vaccines (for example polio vaccine) before use on humans.
• Chemical safety testing (toxicity testing): transgenic animals carrying genes that make them more sensitive to toxic substances give results in less time.

Manipulation of living organisms raises ethical and biosafety concerns: GM organisms may have unpredictable effects when introduced into an ecosystem. The Indian Government has set up organisations such as GEAC (Genetic Engineering Appraisal Committee), which makes decisions regarding the validity of GM research and the safety of introducing GM organisms for public services.

Biopatents are patents granted for biological entities and for products derived from them. Some companies have been granted patents on products and technologies that use genetic materials, plants and other biological resources long identified, developed and used by farmers and indigenous people of a country.

Biopiracy is the term used to refer to the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned and without compensatory payment.

• Basmati rice: India has about 27 documented varieties of basmati, distinct for unique aroma and flavour. In 1997 an American company obtained patent rights on basmati rice through the US Patent and Trademark Office, for a “new” variety derived from Indian farmers’ varieties crossed with semi-dwarf varieties.
• Attempts have also been made to patent uses, products and processes based on Indian traditional herbal medicines such as turmeric and neem. Most industrialised nations are rich financially but poor in biodiversity and traditional knowledge, while developing nations are rich in both. The Indian Parliament has cleared the second amendment of the Indian Patents Bill to take such issues into consideration, including emergency provisions and research and development initiatives.