Biotechnology

Biotechnology brings together biology and technology: it is the controlled use of living organisms, cells or their components (bacteria, fungi, yeast, plants, animals, or isolated compounds from living matter) to make useful products for human benefit. This chapter covers plant tissue culture, plant breeding, disease resistant crop production, green manure and biofertilizers, biopesticides, and the tools and applications of genetic engineering.

Plant Tissue Culture

Plant tissue culture is one of the important aspects of biotechnology. It is the method of culturing an excised plant part, called an explant, on an artificial medium under controlled and aseptic environmental conditions. It works on the principle of totipotency, the ability of a living vegetative cell to develop into a complete plant. Haberlandt developed the idea of totipotency.

Types of plant tissue culture

1. Callus culture: an undifferentiated mass of cells (callus) is used as the explant for developing a new mature plant. It can regenerate different plant parts such as root, shoot, and somatic embryos.
2. Shoot tip culture: the tip of a shoot (axillary or apical) is used as explant and grown in a suitable medium; after growth the plantlets are transferred to a rooting medium and then to soil.
3. Protoplast culture: the protoplast is the living part of the cell obtained after removing the cell wall by treating it with chemicals and enzymes that degrade the cell wall, helping fusion of protoplasts.
4. Anther culture: pollen grain bearing anthers are used as explant. Plants regenerated from anther culture are haploid; when their genome is doubled, homozygous diploid plants are produced, which is very useful in plant breeding.
5. Meristem culture: meristematic tissue of the root or shoot tip is isolated and cultured on artificial media to regenerate a plant. It is a very effective means of developing virus free plants (example: potato, banana).
6. Embryo culture: the embryo is used as explant. This is useful for overcoming seed dormancy and producing viable plants from seeds that would otherwise fail due to an immature embryo.
7. Root culture: roots of several plants are cultured in liquid medium. Infection of the cultured root by Rhizobium can be carried out to help nodule formation (nodulation).

Method of tissue culture

1. Selection of plant: the first step is selecting the plant and the plant part to be used as explant (for example, shoot tip or anther).
2. Preparation of culture medium: an artificial medium is used because the plant grown in vitro mainly depends on the nutrient content of the medium. The medium should contain the required amount of essential elements: inorganic nutrients such as N, P, sulphur, zinc, and copper; a carbon source such as sucrose; organic supplements such as vitamins and thiamine; growth regulators such as auxins and gibberellins. Agar is used as a solidifying medium and the pH is maintained around 5 to 6.
3. Sterilization: sterilization is the process of killing unwanted microbial organisms and making the material free from pathogens. In vitro plants are very susceptible to infection; if pathogens enter the culture vessel, the whole plant inside is infected and lost. Sources of infection may be the explant, instruments, glassware, the operator's hands, or the culture medium. To prevent contamination:
   • The lab is sterilized using UV rays, laminar flow, or fumigation.
   • Glassware is sterilized using a hot air oven at 160 degrees C.
   • Autoclaving is used for petri plates, pipettes, and media.
   • The explant is treated with chemicals such as sodium hypochlorite.
4. Micropropagation: by tissue culture, any part of a plant can be regenerated or multiplied in large numbers. The technique of regenerating plants inside glass vessels is called micropropagation. It has the following stages:
   • Establishment of culture (inoculation): the sterilized explant is transferred to sterilized media with the help of sterilized forceps.
   • Multiplication: depending on the type and proportion of growth hormones used, the explant produces multiple shoots or callus (undifferentiated mass of cells).
   • Rooting: after the microshoots develop in the desired numbers, they are transferred to a rooting medium that facilitates root formation.
   • Transplantation: since in vitro plants are delicate and cannot tolerate the external environment directly, they are first transplanted into a green house (where humidity and temperature are maintained) and then into soil.

Significance of tissue culture

1. Helps in producing disease free plants.
2. Embryo culture is useful in overcoming dormancy of seeds.
3. It has become an alternative means of producing industrially important natural products such as drugs and insecticides.
4. It is applied as an effective means of ex situ conservation.
5. It allows production of a large amount of plants from small tissue.

Plant Breeding

Plant breeding is a method of improving the heredity of crops so that new generations of crop varieties are far better than the original types in all aspects. Plant breeders are the people who carry out plant breeding.

Objectives of plant breeding

1. To evolve higher yielding varieties.
2. To produce crops with better taste and quality products.
3. To obtain plants whose products can be stored for a long time.
4. To obtain varieties resistant to pests, insects, and pathogens.

General methods of plant breeding

1. Introduction: a simple method in which people take a variety of plant with better qualities to a new place (habitat) where it has not been grown before.
2. Selection: the process of choosing the best plants among varieties that are fit to survive and continue their generation; it is done artificially by:
   • Mass selection: a large number of plants of similar phenotype are selected, cross pollinated, and mixed together. The selected seeds are sown, the population increased, and the process repeated to obtain a superior variety.
   • Pure line selection: individual plants that are better than others are selected and allowed to self pollinate; progeny are tested separately for superior quality, and seeds are kept and sown separately.
   • Clonal selection: a clone is the progeny of a plant produced through vegetative propagation (clones are genetically similar). Clones with superior characters are selected and multiplied vegetatively, and the process is repeated until individuals with the desired character are obtained.
3. Hybridization: crossing or mating two individuals of different genotype to produce a hybrid, that is, a new variety. Process:
   • Emasculation: the stamens are removed from the female parent before the anther ripens, to prevent self pollination.
   • Bagging: the entire flower is kept in plastic bags to prevent foreign pollen from contacting the stigma.
   • Pollination: when the stigma of the emasculated flower matures, the bag is temporarily removed and the stigma is pollinated artificially with mature pollen from the male parent.
   • Tagging: the crossed flowers are properly labelled or tagged soon after pollination, and the bag is put back.
   • Raising of hybrid plants: the seeds are harvested and sown; the plants obtained are the F1 (hybrid) plants, grown over several years.
   • Selection and testing of superior recombinants: this is the most crucial step, requiring careful evaluation of progeny. The superior hybrid is repeatedly self pollinated until homozygosity is reached, so that hybrid traits are not segregated and a pure line is obtained.
   • Testing, release and commercialization of new cultivars: selected lines are tested at research fields for yield, quality traits, and disease resistance, and verified scientifically. Field trials run for 5 to 10 years and performance is recorded; good plants are then mass produced and distributed to farmers.

Mutation breeding

Breeding done by inducing mutation to get new varieties resistant to disease, induced by mutagens, either chemicals (colchicine) or physical agents (X rays, gamma rays, beta rays, etc). Example: polyploid mutation increases seed yield and can produce seedless fruit.