Tissue Culture

Cloning of entire plants from tissue cells

Tissue culture is the growth of a tissue in an artificial liquid culture medium. In 1902 German botanist Haberlandt speculated entire plant could be produced by tissue culture. He said that plant cells are totipotent i.e. each cell has the full genetic potential of the organism and therefore a single cell could become a complete plant. In 1958 F.C. Steward, a plant physiologist successfully generated an entire carrot plant from a single cell derived from a tiny piece of phloem from root. He provided the cells with sugars, minerals, vitamins and coconut milk (later it was known that coconut milk contains cytokinin). When the cultured cells began dividing, they produced a callus, an undifferentiated group of cells. Then the callus differentiated into shoot, roots and developed into a complete plant.

Micropropagation is propagation or cloning of plants by tissue culture. The three methods for micropropagation are:

(1) Meristem Culture (2) Anther Culture (3) Suspension Culture.

Meristem Culture

A meristem is a region where cell division is still taking place. If correct proportion of hormones auxin and cytokinin are added to a liquid medium many shoots develop from a single shoot tip. The shoots are called clonal plants as they are genetically identical and as meristem is virus free portion of the plant so meristem culture produces virus free plants.

Anther Cloning

Anthers are cultured in a medium containing vitamins and growth regulators. The haploid tube cells within the pollen grains divide, producing proembryos consisting of as many as 20 to 40 cells. The pollen grain rupture releasing the haploid embryos. Then chemical is added for chromosomal doubling and the plants produced are diploid but homozygous for all their alleles. This technique is a direct way to produce plants that express recessive alleles.

Suspension Culture

Rapidly growing cultures are cut into small pieces and shaken in liquid nutrient medium so that single cells or small clumps of cells break off and form a suspension. These cells will produce the same chemicals as the entire plants. For example cell suspension culture of Cinchona ledgeriana produce quinine.

Genetic Engineering of Plants

Since 1980, a plasmid of the bacterium Agrobacterium tumefaciens, (which cause a tumor like disease called crown gall in plants) has been main vehicle used to introduce foreign genes into broad leaf plants such as tomatoes, tobacco and soybean. A technique known as disarming is used first to remove disease causing gene from the bacterium. After the bacterium has been disarmed, it can be used as vector to shuttle desired genes into plant cells. A part of its plasmids integrates into the plant DNA carrying the foreign genes (as shown in the figure). New plants can be grown from these transformed cells in tissue culture. The cells develop into plantlets, which can be grown in conventional ways.

Many plant species are not natural host of the Agrobacterium. The other commonly used method, shoots microscopic metal pellets coated with DNA into plant cell (see figure) then the cells are cultured and propagated.

In 1986 the gene for the enzyme luciferase, which cause the production of light in fireflies, was inserted into tobacco plants. The plants expressed the gene and glowed in the dark.

Since then the luciferase gene has been transferred to a variety of other species including frog, fish, and the bacterium that cause T.B. if the bacteria are resistant to antibiotics they express firefly gene and glow in the dark. If the bacteria are not resistant they sicken or die. As a result doctors can prescribe appropriate antibiotic for the T. B. patient.

Bioengineered Tobacco Plant

Uses and Applications of Biotechnology

(A)In Agriculture

(I)           Introduction to foreign genes to form plants having resistance against insecticides, virus, herbicides.

(II)         Introduction of foreign genes for nitrogen fixation in crop plants.

(III)       Transgenic plants have been produced.

(IV)       Use of crops to produce drugs instead of food.

(V)         Salt tolerant plant Arabidopsis has been produced

(VI)       Improved agriculture traits include:

(a) Herbicide resistant e.g. wheat, rice, sugar beet, canola etc. (b)

(b) Salt tolerant e.g. cereals, rice, sugar etc. (c)

(c) Drought tolerant includes cereals, rice, and sugarcane. (d)

(d) Cold tolerant cereals, rice, sugarcane etc. e)

(e) Improved yield e.g. cereals, rice, corn, cotton etc.

(f)  Modified wood pulp e.g. trees.

(VII)   Improved food quality traits include.

(a) Fatty acid contents e.g. corn, soybean

(b) Protein and starch contents e.g. cereals, potatoes, soybean rice, etc.

(c) Amino acid content include corn, soybean

(d) Disease protected include wheat, corn, potatoes.

(B) In Medicine

(I) Single gene transfers have allowed to produce various products such as human hormones, clotting factors and antibiotics.

(II) A type of antibody produced by corn can deliver radioisotopes to tumor cells. A type of antibody made by soybean can be used as treatment for genital herpes.

(III) Tobacco mosaic virus have been used as a vector to introduce human gene in adult tobacco plant. Tobacco plants have been used to produce

(a) 10 grams a galactosidase an enzyme that can be used to treat a human lysosome storage disease

(b) To produce antigens to treat Non-Hodgkin's lymphoma (abnormal proliferation of lymphocytes) after being sprayed with a genetically engineered virus.

(IV) The human growth hormone, which is used to treat people who are growing more slowly than normal, was previously extracted from the pituitary glands of cadavers (a human corpse, used for organ transplant or dissection) and it took 50 glands to obtain enough for one dose. Now human growth hormone is produced in large quantity by biotechnology. Insulin, to treat diabetics, was previously extracted from-the pancreatic glands of slaughtered cattle and pigs; it was expensive and sometimes caused allergic reactions in recipients. And few of us knew of tPA (tissue plasminogen activator) a protein present in minute quantities that activates an enzyme to dissolve blood clots. Now tPA produced by biotechnology is used to treat heart attack victims by dissolving blood clots that are blocking the flow of blood in the coronary arteries of the heart. Clotting factor VIII treats hemophilia; human lung surfactant (surface proteins secreted by the lung cells) treats respiratory distress syndrome in premature infants; atrial natriuretic factor helps control hypertension. Several hormones produced by biotechnology are for use in animals. Farm animals can now be given growth hormone instead of steroids. Such animals produce a leaner meat that is healthier for humans.

(V) Transgenic animals are used to relatively large quantities of rare and expensive proteins for use in medicine. Bacteria and viruses have surface proteins, and a gene for just one of these can be used to genetically engineer bacteria. The copies of the surface protein that result can be used as a vaccine. A vaccine for hepatitis B is now available, and potential vaccines for malaria, and AIDS are in experimental stages. (As seen in this Table)

Safety and Ethical Problem Raised by Biotechnology

As our knowledge of human genetics increases, we must face a number of difficult ethical questions. On one hand, as many people argue, the potential benefit for fighting disease provide a strong ethical reason for obtaining this knowledge as fast as possible. On the other hand the potential for abusing the knowledge makes people wary (cautious) of proceeding. Most people will agree that there was no ethical conflict in altering the DNA of a bacterium or a virus, does that mean that there is no dilemma associated with altering the DNA of a plant, a dog, a chimpanzee or a human.

The use of DNA technology also involves risks for example, many a crop plants made and produce offspring with closely related species. If a crop plant is genetically engineered to be resistant to an herbicides, the potential exits that the resistance gene will be transferred by mating from the crop to wild species.

The use of DNA technology raises ethical questions and possess risks. There is a fine line between acceptable and unacceptable changes to the DNA of humans and other species. Benefits and risks of DNA technology must be considered carefully in evaluations of the potential impact to human society of a particular use of DNA technology.