Exercises
Which part of the plant is best suited for making virus-free plants and why?
Answer
The apical and axillary meristems. Even when a plant is infected with a virus, its meristems remain free of virus because the meristematic cells divide very rapidly and lack well-developed vascular connections through which viruses spread. Meristems removed and grown in vitro therefore give healthy, virus-free plants (as done for banana, sugarcane and potato).
What is the major advantage of producing plants by micropropagation?
Answer
A very large number of plants (thousands) can be produced in a very short time and in a small space. All the plants are genetically identical to the parent (somaclones), so desirable traits are preserved. In addition, virus-free plants can be obtained from meristems, and plants can be raised throughout the year independent of season; tomato, banana and apple have been produced commercially this way.
Find out what the various components of the medium used for propagation of an explant in vitro are?
Answer
The nutrient medium contains: (i) a carbon source such as sucrose; (ii) inorganic salts – macronutrients and micronutrients; (iii) vitamins; (iv) amino acids; (v) growth regulators such as auxins and cytokinins; along with water and agar (as a solidifying agent) at a suitable pH, all kept sterile.
Crystals of Bt toxin produced by some bacteria do not kill the bacteria themselves because –
(a) bacteria are resistant to the toxin
(b) toxin is immature;
(c) toxin is inactive;
(d) bacteria encloses toxin in a special sac.
Answer
(c) toxin is inactive. Bt toxin exists as an inactive protoxin in the bacterium. It becomes active only in the alkaline pH of the insect's gut, which solubilises the crystals.
What are transgenic bacteria? Illustrate using any one example.
Answer
Transgenic bacteria are bacteria into which a foreign gene has been introduced and expressed using recombinant DNA technology.
Example – production of human insulin by E. coli: Human insulin has two polypeptide chains, A and B, joined by disulphide bridges; in the body it is made as pro-insulin with an extra C-peptide. In 1983 Eli Lilly prepared two DNA sequences coding for chains A and B, introduced them into plasmids of E. coli, and the bacteria produced chains A and B separately. The chains were extracted and joined by creating disulphide bonds to form human insulin (Humulin), which does not cause the allergic reactions seen with insulin from cattle and pigs.
Compare and contrast the advantages and disadvantages of production of genetically modified crops.
Answer
Advantages: (i) more tolerant to abiotic stresses – cold, drought, salt and heat; (ii) pest-resistant crops (e.g. Bt cotton) reduce reliance on chemical pesticides; (iii) reduced post-harvest losses; (iv) more efficient mineral use, preventing early exhaustion of soil fertility; (v) enhanced nutritional value, e.g. golden rice rich in vitamin A; (vi) tailor-made plants supply starches, fuels and pharmaceuticals to industry.
Disadvantages: (i) unpredictable effects when GM organisms are released into ecosystems, e.g. harm to non-target organisms or loss of biodiversity; (ii) transgenes may spread to wild relatives by cross-pollination, possibly creating "superweeds"; (iii) pests may develop resistance to Bt toxin; (iv) possible allergic or other health effects of new proteins in food; (v) high cost of seeds and dependence of farmers on companies, along with patent and biopiracy issues.
What are Cry proteins? Name an organism that produce it. How has man exploited this protein to his benefit?
Answer
Cry proteins are insecticidal crystal proteins (Bt toxins) coded by the cry genes. They are produced by the soil bacterium Bacillus thuringiensis. The protoxin is converted into active toxin in the alkaline gut of the insect, binds to midgut epithelial cells and creates pores, causing swelling, lysis and death of the insect.
Use: specific cry genes have been isolated and put into crop plants to make them insect-resistant – e.g. proteins coded by cryIAc and cryIIAb control cotton bollworms (Bt cotton) and cryIAb controls corn borer (Bt corn). This acts as a bio-pesticide and reduces the use of chemical insecticides.
What is gene therapy? Illustrate using the example of adenosine deaminase (ADA) deficiency.
Answer
Gene therapy is a collection of methods for correcting a gene defect diagnosed in a child or embryo by inserting a normal, functional gene into the person's cells and tissues to take over the function of the non-functional gene.
ADA deficiency: The enzyme adenosine deaminase is essential for the immune system; its deficiency is caused by deletion of the ADA gene. Bone marrow transplantation and enzyme replacement therapy are not completely curative. In the first clinical gene therapy (1990, a 4-year-old girl), lymphocytes from the patient's blood were grown in culture, a functional ADA cDNA was introduced into them using a retroviral vector, and the cells were returned to the patient. As these lymphocytes are not immortal, periodic infusion is needed. If the ADA gene from marrow cells is introduced into cells at early embryonic stages, it could give a permanent cure.
Digrammatically represent the experimental steps in cloning and expressing an human gene (say the gene for growth hormone) into a bacterium like E. coli ?
Answer
Flow of steps:
Isolate human DNA (or the mRNA of growth hormone and make cDNA from it by reverse transcriptase, since bacteria cannot remove introns)
⇒ cut the DNA with a restriction enzyme (e.g. EcoRI) and amplify the growth hormone gene by PCR
⇒ cut a plasmid vector (e.g. pBR322) with the same restriction enzyme
⇒ mix the gene and vector and join them with DNA ligase to form recombinant plasmid
⇒ make E. coli competent (CaCl₂ treatment, ice, heat shock at 42 °C) and transform it
⇒ select transformants on antibiotic medium and recombinants by insertional inactivation (or blue-white screening)
⇒ grow recombinant E. coli in a bioreactor under conditions that induce expression
⇒ extract and purify growth hormone (downstream processing)
⇒ formulate and test the product.
Can you suggest a method to remove oil (hydrocarbon) from seeds based on your understanding of rDNA technology and chemistry of oil?
Answer
Oil (triglyceride) in seeds is made from glycerol and fatty acids by specific enzymes. Using rDNA technology, the gene coding for a key enzyme of fatty acid / triglyceride synthesis can be identified and silenced – for example by RNA interference (introducing a construct that produces sense and antisense RNA, forming dsRNA that silences the enzyme's mRNA) or by knocking out the gene. Without the enzyme, oil would not be synthesised and stored in the seed, giving oil-free seeds. Alternatively, a gene for an oil-degrading enzyme (lipase) could be expressed in the seed.
Find out from internet what is golden rice.
Answer
Golden rice is a genetically modified variety of rice (Oryza sativa) whose grains are enriched with β-carotene (provitamin A), giving them a golden-yellow colour. Genes for β-carotene synthesis (phytoene synthase from daffodil/maize and phytoene desaturase from the bacterium Erwinia uredovora) were introduced so that the pathway works in the endosperm. It was developed to help overcome vitamin A deficiency, which causes night blindness, especially in countries where rice is the staple food.
Does our blood have proteases and nucleases?
Answer
Blood plasma has some proteases (e.g. the enzymes of the clotting and complement systems such as thrombin and plasmin) which act on specific substrates, and only very low levels of nucleases. It does not have digestive proteases and nucleases that freely break down proteins and nucleic acids, otherwise plasma proteins such as albumin, antibodies and hormones would be destroyed. This is why protein drugs like insulin can be given by injection into the blood rather than by mouth.
Consult internet and find out how to make orally active protein pharmaceutical. What is the major problem to be encountered?
Answer
The major problem is that proteins taken orally are digested by proteases (pepsin, trypsin) and the acidic pH of the digestive tract before they can be absorbed, and large protein molecules are poorly absorbed through the intestinal wall. Methods to make them orally active: enclosing the protein in protective carriers such as liposomes, nanoparticles or enteric-coated capsules that dissolve only in the intestine; adding protease inhibitors and absorption enhancers; chemically modifying the protein (e.g. PEGylation) to make it stable; or producing the protein in edible transgenic plants so that the plant cell wall protects it.
Important Extra Questions
What is somatic hybridisation? Give an example.
Answer
Fusion of naked protoplasts from two different plant varieties, each with a desirable character, and growing the hybrid protoplast into a new plant. Example: pomato (tomato + potato).
How was tobacco protected from the nematode Meloidogyne incognita?
Answer
By RNA interference: nematode-specific genes were introduced into tobacco using Agrobacterium vectors so that both sense and antisense RNA were made, forming dsRNA that silenced the nematode's specific mRNA; the parasite could not survive.
Name two techniques used for early molecular diagnosis.
Answer
PCR, which amplifies very low amounts of pathogen nucleic acid (e.g. detecting HIV), and ELISA, based on antigen–antibody interaction.
What was special about the transgenic cow Rosie?
Answer
Rosie (1997) produced milk enriched with human alpha-lactalbumin (2.4 g per litre), which was nutritionally more balanced for human babies than natural cow milk.
What is biopiracy? Give an example.
Answer
Use of bio-resources and traditional knowledge by multinational companies or organisations without authorisation from, or compensation to, the countries and people concerned. Example: the 1997 US patent on Basmati rice derived from Indian farmers' varieties.