Chapter 10: Biotechnology and its Applications (Biotechnology)

The meristem (apical or axillary). Even when a plant is infected with a virus, its meristems are usually free of virus, because the cells divide rapidly and the virus does not reach them quickly (meristems have no vascular connection for the virus to travel through). Plants grown from meristem culture are therefore virus-free (done for banana, sugarcane, potato).

The meristem, because it is usually free of virus.

A very large number of plants can be produced in a short time from a small piece of tissue, and all of them are genetically identical to the parent (somaclones), so useful traits are kept. It also allows virus-free plants and year-round production in little space. Used for tomato, banana, apple, etc.

Thousands of genetically identical plants quickly from a small explant.

The nutrient medium (e.g. Murashige and Skoog medium) contains:

• a carbon source, such as sucrose;
• inorganic salts (macro- and micronutrients);
• vitamins and amino acids;
• growth regulators such as auxins and cytokinins;
• agar to solidify it (for solid medium), with a suitable pH.

Sucrose, inorganic salts, vitamins, amino acids, auxins and cytokinins, and agar.

(c) toxin is inactive. The Bt toxin is made as an inactive protoxin; it becomes active only in the alkaline gut of the insect, where it is solubilised.

(c) The toxin is inactive (protoxin) in the bacterium.

Transgenic bacteria are bacteria into which a foreign gene has been inserted, so they produce the protein coded by that gene.

Example: human insulin. The insulin chains A and B are coded by two separate DNA sequences. In 1983, Eli Lilly introduced these into plasmids of E. coli. The bacteria produced the A and B chains separately; these were extracted and joined by disulphide bonds to form human insulin (Humulin).

Bacteria carrying a foreign gene, e.g. E. coli making human insulin chains A and B.

Advantages:

• More tolerant to abiotic stresses (cold, drought, salt, heat).
• Less reliance on chemical pesticides (pest-resistant crops like Bt cotton).
• Less post-harvest loss.
• Better use of minerals by plants, preventing early exhaustion of soil fertility.
• Higher nutritional value (e.g. golden rice rich in vitamin A).
• Tailor-made plants supplying starch, fuels and pharmaceuticals.

Disadvantages and concerns:

• Possible allergies or toxicity to humans and animals.
• Transgenes may spread to wild relatives, creating "superweeds".
• Harm to non-target organisms and biodiversity.
• Pests may develop resistance.
• High cost of seeds and dependence on companies; issues of biopiracy and patents.

They give stress tolerance, pest resistance, less pesticide and better nutrition, but raise concerns of allergy, gene escape, biodiversity loss, resistant pests and costs.

Cry proteins are insecticidal proteins (Bt toxins) coded by the cry genes. They are produced by the soil bacterium Bacillus thuringiensis.

The toxin, inactive in the bacterium, becomes active in the alkaline gut of an insect, binds to the midgut cells, creates pores and kills the insect. Man has inserted cry genes into crop plants to make pest-resistant (Bt) crops: e.g. cryIAc and cryIIAb control cotton bollworms and cryIAb controls corn borer. This reduces the use of chemical insecticides.

Insecticidal Bt toxins from Bacillus thuringiensis; their genes are put into crops (Bt cotton, Bt corn) to make them resistant to pests.

Gene therapy is the correction of a genetic defect by delivering a normal gene into the cells of a person, to replace or make up for a faulty gene.

ADA deficiency: the gene for the enzyme adenosine deaminase is deleted, so the immune system fails (severe combined immunodeficiency).

• Lymphocytes from the patient's blood are grown in culture.
• A functional ADA cDNA is introduced into them using a retroviral vector.
• The cells are returned to the patient.

Because these lymphocytes are not immortal, the treatment must be repeated periodically. A permanent cure may be achieved if the ADA gene is introduced into bone-marrow cells at an early embryonic stage. (The first clinical gene therapy, in 1990, was for a 4-year-old girl with ADA deficiency.)

Inserting a normal gene to correct a defect; for ADA deficiency, the patient's lymphocytes receive a working ADA gene (retroviral vector) and are returned to the body periodically.

A flow diagram of the steps:

• Isolate the human growth hormone gene (or make its cDNA from mRNA by reverse transcriptase, since bacteria cannot remove introns).
• Cut the gene and a plasmid vector (e.g. pBR322) with the same restriction enzyme, producing matching sticky ends.
• Join the gene into the plasmid with DNA ligase, forming recombinant DNA.
• Introduce the recombinant plasmid into E. coli (transformation, e.g. heat shock with calcium chloride).
• Select transformed cells using the selectable marker (antibiotic resistance or blue/white screening).
• Culture the recombinant bacteria in a bioreactor; they express the gene and make growth hormone.
• Downstream processing: extract and purify the hormone.

Isolate gene (cDNA) → cut gene and plasmid with the same restriction enzyme → ligate → transform E. coli → select → grow in a bioreactor (expression) → purify the hormone.

Oil in seeds is stored as triglycerides made from fatty acids. One could use rDNA technology to silence or block the genes for the enzymes of oil (fatty acid / triglyceride) synthesis in the seed, e.g. by RNA interference (inserting a gene that makes antisense RNA or dsRNA for those genes). The seed would then make little or no oil. (Alternatively, a gene for a lipase that breaks down the stored oil could be expressed in the seed.)

Silence the genes for oil-synthesising enzymes in the seed (e.g. by RNAi) so oil is not made.

Golden rice is a genetically modified variety of rice that produces β-carotene (provitamin A) in its grains, giving them a golden-yellow colour. Genes for β-carotene synthesis (from daffodil and a soil bacterium) were inserted into rice. It is meant to fight vitamin A deficiency, which causes night blindness, especially in countries where rice is the staple food.

A GM rice that makes β-carotene (provitamin A), to fight vitamin A deficiency.

Yes. Blood contains proteases (e.g. thrombin and plasmin of the clotting and clot-dissolving systems) and nucleases (e.g. DNases and RNases). This is why protein and nucleic acid drugs injected or taken by mouth can be broken down; they must be protected or delivered suitably.

Yes, blood contains proteases (e.g. thrombin, plasmin) and nucleases (DNases, RNases).

Protein drugs given by mouth are destroyed by stomach acid and digestive enzymes (proteases) before they can be absorbed, and large proteins are poorly absorbed through the gut wall. Methods to make them orally active:

• coating or encapsulating the protein (e.g. in liposomes, nanoparticles or enteric coatings) to protect it from acid and enzymes;
• adding protease inhibitors or absorption enhancers;
• making edible vaccines in transgenic plants.

Major problem: digestion of the protein by proteases in the gut (and its low absorption), so it does not reach the blood intact.

Protect the protein (encapsulation, coatings, protease inhibitors); the main problem is its breakdown by digestive proteases and poor absorption.