Plant Breeding

INTRODUCTION

About two million years ago, the early Paleolithic man was a nomad who started using weapons for hunting animals. Later, he began eating the fruits of wild plants. After that man started cultivating plants near his habitation. The earliest settlements have been found to be located in the river valleys and plains of northern India. In these areas, the soil was fertile and plenty of water was available, so it was easy to cultivate crops. As a result of successful and plentiful agricultural production, people became self-sufficient and built great civilisations.

Agriculture and Horticulture

The word ‘agriculture’ is derived from the Latin words: ager= field and cultura = cultivation. It is the science and art of production and breeding of plants and animals useful to man. Irrespective of where we live, agriculture provides us with food, clothing, shelter, and other requirements of our daily life.

Horticulture (Hortus = garden; cultura = cultivation) is a branch of agriculture. It is the science and art of growing vegetables, fruits and ornamental plants.

Agriculture and its allied sciences aim at the maximum production of plants for food, fibre, building materials, several essential drugs and fuel needed by man for his survival, comforts and health. The use of fire and agriculture formed the basis of civilization. Fire helped to cook the hard and inedible foods, the gradual development of civilisation occurred on the basis of management of an assured supply of food by planting crops followed by harvesting and storing the product. In this way, man gradually became a producer of food from a hunter-gatherer.

Origin of Agriculture

Agriculture originated about 7,000-13,000 years ago, somewhere in the then well- watered highlands of the Indus, Euphrates, and Nile and Tigris rivers. Some other prehistoric sites of ancient agricultural activity are the Tehuacan Valley in modern Mexico and the banks of Yellow River in modern China.

South-east Asia was ideal for agricultural beginning because of its diverse vegetation to Support a stable human population. In these areas, planting of vegetative parts (like rhizomes, tubers or bulbs) which is simpler than planting of seeds, formed the Basis agriculture. But, it was seed planting that led to most profound changes in the life of man. All early civilisations whOse diets are known to us were based on seed-reproduction plants such as wheat, maize or rice.

Agriculture originated independently in several-parts of the world. The fire’s conscious act of cultivation perhaps was based on the finding that seeds and also the twigs formed the new plants when struck into the ground. When a plant is raised

consciously, it is called cultivation. First, those plants were brought into cultivation that grow rapidly and produce a crop within a season. The present day cereals and their wild varieties were origınally weedy and grew in open habitats.

The cereals originated in mountainous areas of both Old World (Asia, Europe and Africa) and New World (North and South America). which have pronounced wet and dry seasons. Under such climatic conditions, weeds germinate. grow quickly with the onset of rains and produce seeds before the summer sets in. 

Annual plants have a selective advantage i.e. if they have enough food reserves in their seeds it enables them to survive drought and also to grow quickly with the onset of rains. These plants, thus, were pre adapted for agriculture. Weediness and provision of reserve food in their seeds were the two key factors in the domestication of cereals. d be

Basis of Civilisation

The use of fire and agriculture formed the basis of civilization. Fire is the basic factor for our existence. It is difficult to imagine the birth of any human culture without fire.

The foremost use of fire was in the expansion of food supply because many foods were inedible, unpalatable or unsanitary unless they were cooked. The gradual development of civilization can be traced to the management of assured food supply by deliberate planting of crops and by harvesting and storing the product. This made man’s food producing habits change slowly and in due course of time he became a producer rather than a hunter gatherer.

In South Africa, the Kuang Tribe consisting of about 1,000 individuals, living under the extremely arid and harsh environment of Kalahari Desert, depend upon two plants for their entire food. These plants are Mongongo nut (Schinziophyton rautanenii) and Marama bean (Tylosema esculentum). The seeds of these plants contain abundant amounts of proteins, fats and carbohydrates. These plants are adapted to sand dunes which periodically keep shifüng. The tribals take care of the plants with the same enthusiasm as farmers do for crops raised by them. 

This is so because the tribals depend on these plants for their survival. Marama beans contain 30-39 per cent proteins and 36-43 pC cent oil. The composition and nutritive value of these beans is more or less equal to those of peanut and soybean. Besides, it also produces underground succulent tubes which contain about 90 percent moisture. This water is an important water emergency for humans and other animals in the arid tracts of Kalahari. Present day crops like cereals, pulses (legumes) and oil seeds are mainly eaten for the purpose of food only. But early humans gave a lot of importance to plants. multiple uses such as coconut palm, date palm, mulberry, agave, hemp, etc. The coconut palm is still a lite-supporting plant for coastal people throughout the tropics.

The coconut, Cocos nucifera, is one of nature’s greatest gifts to man. It is called Kalpa Vriksha’ in India, meaning the “Tree of Heaven”. Every part of this tree is used one way or the other. But it is its fruit that is of profound economic importance.

Coconut water which is liquid endosperm is a nutritious and refreshing drink, frece of pathogens. In severe diarrhoea, coconut water is recommended for oral rehydration. Its solid endoderm, often called ‘meat’ of coconut, is eaten raw or cooked. When dried, it is called copra. It yields coconut oil on extraction. The oil is used for cooking, for applying on hair, in making shampoos and for making soaps and margarine. The other

Uses of coconut tree are :

1. Fibrous husk coir is used for making ropes which are resistant to seawater.

2. Coir is also used for making mats, rugs, filters and for stuffing. Our country is the 15 largest producer of coir.

3. Its young inflorescence when tapped, yields a sugary sap. It may be boiled to prepare jaggery or fermented to produce alcoholic beverages.

4. The main stem is used for construction and furniture making.

5. Its massive leaves are used for thatching huts and for making baskets and hats. Midribs of pinna are used for making brooms and brushes.

6. Coconut shells serve as ladles or receptacles for collecting rubber or for making activated charcoal. Powdered shells constitute filters in plastic.

Improvement of Crops

Crop improvement involves the combining of desirable and superior characters from selected parents in one plant species and then multiplying it. Plant breeders do it by selecting plants with desired characters, crossing them and then identifying the

offspring with characteristics of both the parents. It is then mulüplied and supplied to farmers, growers and planters.

PLANT BREEDING

Plant breeding is the science of improvement in the hereditary characters of crops and production of new crop varieties which are far better than original varieties in all respects.

Objectives of Plant Breeding

The aim of plant breeding is to develop a variety which combines as many of the desirable and beneficial characters of economic value as possible.

These are:

• Increase in the yield. 
• Improved quality.
• Agronomic characteristics such as plant height, tillering, branching
• branching, erect or trailing habit.
• Adaptability to new regions and climates.
• Resistance to diseases and insect pests.
• Change in duration of maturity.
• Elimination of toxic substances.
• Development of varieties for saline soils.
• Resistance to lodging

Methods of Plant Breeding

Plant breeding started in the beginning of the 20th century after the rediscovery of Mendel’s work. Today plant breeding has become a specialised technology based on genetics.

In India, plant breeding is actively carried out at Indian Agricultural Research Institute, Delhi; Central Potato Research Institute, National Rice Research Institute, Cuttack; Sugarcane Research Institute, Coimbatore and at a number of agriculture universities and research stations.

Crop improvement has following aspects:

1. Plant introduction

2. Selection

3. Hybridisation

4. Polyploid breeding

5. Induced mutations

6. Tissue culture

7. Genetic engineering

1. Plant Introduction

The process of introducing new plants from their growing place to a new one with a different climate is termed as plant introduction. The adjustment of such pia lanis locality to their new locality is called acclimatisation. The new crops or the new varic will be introduced in the form of seeds or cuttings. In vegetatively propagated crops many cuttings are imported and in sexually propagated crops, the seeds are imported in a rapid method to effect improvement with minimum efforts and cost. It is throughout the world. 

Plant introduction has been an important basis of agricultural development since the beginning of 19th century from the Philippines, Cinchona from Peru, papaya e.g. Indies, potato from South America, date-palm from Brazil. Custard apple, coffee, tea, tobacco are other plants successfully introduced in India.

2. Selection

Selection is an important step in all breeding experiments and has been practised by man since the early days of agriculture. The selection involves picking up the better ones out of the genuine crop plants. The selected plants are separated from the inferiores and are favoured by reproducing them under controlled conditions. There are three patterns of selection. This is applied for cross pollinated plants

(a) Mass selection

This is the most common method of crop selection. In this selection, a number of similarly appearing plants are selected for the desired trait and their seeds are mixed together. The mixture of seeds obtained is sown to raise the new crop. By such a selection, the general level of population is improved. The seeds of such plants are multiplied and supplied to the farmers. 

Mass selection is a quick and easy method of crop improvement but it has following drawbacks:

(a) It is not very effective in bringing about an increase in the yield which is dependent mostly on the environment.

(b) The selection is made from the female individuals and there is no check on the

male plant which contributes equally to the progeny

(C) It is not possible to know whether the superior phenotype is due to better genotype or due to environment.

(d) Selected characters of the crop exhibit segregation because of natural cross pollination.

In Spite of these drawbacks, mass selection has been successfully employed for the improvement of many important plants such as maize, watermelon, radish, grapes, apples, onion, pear, etc.

(b) Single plant or pure line selection

The single plants with desired traits or traits are selected out of the variable population in the field. Seeds from selected plants are sown in separate rows to produce a progeny. Desired plants are again selected from this progeny. This is continued for

Several generations. The inferiors are eliminated at each generation. Wheat varieties like Kalyan-227 and PV-18 have been developed at Ludhiana by this type of selection.

Pure line selection is better than mass selection because the selected plants retain desirable  characters for several generations. But the main drawback of this method is that it takes 7-12 years for raising the desired variety. This is applied for self pollinated plants.

(c) Clonal selection

It is practised in vegetatively propagated plants mke sugarcane, banana,etc. In this method the best plant based on phenotypic characters is selected . Polat multiplied vegetatively and supplied to the farmers. A population of plants raised from a single vegetatively propagated plant is called clone.

The advantage of this method is that a desired clone can be obtained with. thin one year. But this method can be applied only to vegetatively propagated plants.

3. Hybridisation

Hybridisation is the technique of introducing characters of two desirable c species into a single offspring (hybrid) by means of artificial pollination. Hybrids are first generation (F) crosses between genetically different parents

The term hybrid is used to describe the individuals that are heterozygous even for a single gene.

Hybrids are known for their vigour, growth, size and yield. As a result of hybridisation, hybrid varieties of cereals, oil seeds, pulses, sugarbeet, onion, tomato and fruits have been developed. The procedure involves the following steps

(a) Selection of desired plants from the open pollinated population.

(b) Selfing the selected plants through several generations to produce uniform homozygous inbred lines, and 

(C) Crossing the selected inbred lines to produce uniform F, population in such quantities, such that F, seeds can be grown directly.

The hybrids produced in this way are superior to the homozygous inbreds and excel the natural populations.

Technique of hybridisation

It involves the following steps:

(i) Selection and isolation of plants: First, the plants to be used as male and female are selected. These plants are induced to flower at the same time. Crossing is done  in a greenhouse under controlled conditions.

(ii) Emasculation: When the two parent plants have bisexual flowers, the stanc one plant are removed in the bud stage. This is called emasculation.

(iii) Bagging: Male and female flowers are covered with polythene or paper D so that no foreign pollen may fall on the stigma. This is called bagging $0

(iv) Pollination: Bags are opened at the time when the act of pollination is performed. After that the female cross-pollinated flower is again bagged If the

plants are dioecious, the male plants are completely eliminated from the vicinity of the female plants which are to be cross-pollinated by pollen of a desired species. In certain crops, male sterile plants have been recorded in many crop plants like maize, wheat, sorghum, barley, carrots, cucumber, tomatoes, onions and sunflower. These male sterile plants are used as female parents and are crossed by pollen of male parents with desirable characteristics. This avoids the time consuming process of emasculation and bagging. It also overcomes the chances of failure in hybridisation likely to be caused by injury to flowers during the acts of emasculation and artificial pollination.

(v) Heterosis or hybrid vigour: It refers to the exhibition of superiority of the hybrid over both of its parents in one or more traits. It is based on the ability to give higher yield and disease and pest resistance. Plant breeders have utilized the hybrid vigour in the improvement of many commercial crops such as maize, sorghum, bajra, rice, sugarbeet, tomato, Petunia, Zinnia, cabbage and cucumber. In maize, heterotic hybrids have been reported to yield 25 per cent more than their original cross-pollinated varieties from which the pure lines were derived.

Heterosis involves two steps:

(a) Plants are selected for desirable characters over several generations to get pure lines for different characters.

(b) Pure lines for different desirable characters are obtained to have the heterotic effect in the hybrids.

It is important to note that hybrid vigour is lost by inbreeding. It decreases rapidly in the F generation and diminishes in each succeeding generation. Thus to maintain optimum hybrid vigour, its seed must be produced every year by crossing the pure lines which are constantly maintained. Heterosis 1s best suited to plants which can be vegetatively propagated, e.g., sugarcane, mango, apple, guava, rose, dahlias, chrysanthemums, etc. In these plants, the heterotic hybrids retain their desirable Characters indefinitely since there is no chance of segregation as they are multiplied vegetatively.

Polyploid Breeding or Polyploidy

Most organisms possess two sets of homologous chromosomes, Such organisms called diploids. Organisms with more than two sets of homologous chromosomes and somatic cells are called polyploids and the occurrence of more than two sets of Homologous chromosomes is called polyploidy.

polyploids whose somatic cells contain an exact multiple of the haploid set of mosomes are called euploids. Plant breeders use the Following terms to indicate Polyploids chrome number of chromosome sets in plants

Plants whose somatic cells do not contain an exact multiple of the haploid set are called aneuploids. They are also called heteroploids such as heteroploids (2n+1) or hyperploidy (2n-1). Polyploidy is of two types: Autopolyploidy and allopolyploidy. Autopolyploids result from an increase in the chromosome number of the same genome. A polyploid belongs to a single species in which all the sets of chromosomes are identical. Allopolyploids are polyploids with genetically different chromosome sets that are initially derived from two different species. They arise as a result of hybridisation between genetically different species and thus involve addition of new genomes. If a diploid plant is represented by an AA complement, then the autotriploid would be AAA and autotetraploid would be AAAA. If BB represents the chromosome complement of another species, the allotetraploid would be AABB.

Polyploidy in sexually reproducing plants may arise due to fusion of the egg with more than one sperm. It may also arise due to failure of meiosis during gamete formation. Polyploidy can also be induced by using colchicine.

Triploids can be produced by crossing tetraploids with diploids Raising of triploids by crossing diploids with tetraploids has been of much significance in plants like sugar beet, apple, pear, guava, banana and watermelon. in these plants, triploids show increased vigour and larger fruit size with higher yield.

Multi Seeded fruit like watermelon produce seedless fruit of larger size when Cultivated in triploid state. Triploids do not produce seeds. Some of the cultivated varieties of banana are also sexually sterile triploids. Such banana plants are muluplied by asexual means. 

In some plants, increased vigour and fruit size are associated with triploid condition.Triploid sugar beets have larger roots and more sugar than either diploids or tetraploids.

Many apple and pear cultivars are triploids. They have apparently originated from unreduced gametes. Cultivators selected them because of their larger fruit size and other desirable characters. Recently, several apple and pear cultivars have also been produced from crosses of diploids with naturally occurring tetraploids.

Triploidy is associated with high incidence of sterility, i.e., seedless fruits. Triploidy is now in practice to produce seedless watermelons.

Origin of Bread Wheat (Triticum aestivum) (Hexaploid)

Wild ancestor of wheat is diploid einkorn wheat, Trticum boericum (2n = 14). It grew in the Near East during 10,000 to 15,000 B.C. The culüvated diploid species of that period was T.monococcum (2n = 14). The study of chromosomal complements reveals that in the early history, T.monococcum was naturally fertilised by a wild goatgrass Aegilops speltoides (also known as Triticum speltoides). Aegilops speltoides is also diploid and contains 14 chromosomes

(i.e., 2n= 14). The diploid hybrid offspring of these species were sterile. Polyploidy occurred in those sterile individuals to give rise to tetraploid form with 28 chromosomes (i.e., 4n = 28). This is known as Triticum turgidum. Its cultivated form is called emmer wheat. One of its natural mutants is 1s 1.durum. The tetraploid emmer wheat T.durum, on natural crossing with diploid wild grass, Aegilops squarrosa (2n = 14) formed triploid (3n 21). It was found to be sterile. During the course of evolution, sterile triploid hybrids involved in doubling of their chromosomes to produce the hexaploid modern bread wheat, Triticum aestivum (6n = 42). It can be illustrated as follows.

The cultivated rice, Oryza sativa, is diploid whereas several species of Onyzn are tetraploid. In potatoes, Solanum tuberosum and S. andigena, are both tetraploid. Mustard is an allotetraploid which originated by cross fertilisation between yellow mustard followed by doubling of chromosome number.

Euploidy and aneuploidy: 

Plants which contain an exact multiplication of the haploid set of chromosomes are said to be euploids. On the other hand, when the number of chromosomes is not an exact multiple of the haploid set are called aneuploids.

Many of the sugarcane varieties cultivated today are aneuploids. These have resulted from back crossing the hybrids of Saccharum officinarum with S. Spontaneum. Many of these varieties are sterile. But as they grow and perform extremely well and are vegetatively propagated, sterility is not of any advantage.

5. Induced Mutations

Phenomenon of induced mutations is one of the quickest methods of developing new breeds and varieties of plants. In India, more than hundred varieties have been developed through the use of induced mutations. Mutations can be induced by short wave electromagnetic radiations (UV irradiations, X-rays), ionising radiations (gamma rays from radio-active isotopes, cobalt-60 and 177 caesium and certain chemicals (nitromethyl urea, nitrous acid and EMS: ethyl methanesulfonate).

Some of the examples of mutation breeding are:

(a) Many artificially mutated varieties of barley are dwarf, give high yield, insensitive to day length and resistance to mildew disease.

(b) High yielding dwarf wheat varieties, Sharbati Sonora and Pusa Lerma are two amber- grain coloured mutants produced from the red-grained Sonara 64; and Mexican Lerma Rojo 64 respectively

(c) Semi-dwarf variety of rice which is resistant to lodging is the product of a mutant gene by X-ray treatment, either by direct radiation or by crossing with induced mutants

(d) In Indonesia, irradiation of rice cultivar Pelita-1 with gamma rays resulted in a high-yielding variety named Atomita-2. The latter is resistant to brown plant hopper, can tolerate salty soils and has a good palatability. In Carolina, induced mutations have resulted in groundnuts with thick fruit shells so that they are less likely to crack during transportation.

Somatic mutations (spontaneous and induced) in vegetatively propagated crops are of great importance. These are called bud sports. Since these can be detected by the same plant, they are of greater importance in quick improvement of vegetable propagated plants, e.g., colour sports in many varieties of apple and superior shrubs or coffee plants.

Limitations of mutation breeding Mutation breeding has its own limitations

(a) Most of the induced mutations are undesirable and some of them are lethal causing death of the organism.

(b) The rate of mutation is very low and a large number of plants are employed to select a certain desirable mutant.

(c) Some mutations are not stable and have a tendency to get reverted.

(d) Most of the induced mutations are recessive. They are expressed only in recessive homozygous condition otherwise they remain undetected

(e) In sexually reproducing plants, mutations are inherited only if they are induced in gametes.

Many crop plants like sugarcane, potato, tapioca and strawberries are propagated vegetatively, even though they can bear seeds. In such plants, genetic improvement is brought about by sexual reproduction, but the maintenance of improved varieties is carried out by cloning.

Examples: Potatoes are multiplied by tubers, apples by cuttings and strawberries by runners.

6. Plant Tissue Culture

Tissue culture is an important aspect of biotechnology. It is the culture of either the micro-organisms or plant and animal cells or protoplast fusion or tissues and organs in artificial media. Plant tissue culture is the technique of maintaining and growing plant cells, tissues or organs aseptically on artificial medium in suitable containers under controlled environmental conditions. The plant part which is cultured is called explant.

It has to be first disinfected with clorox water, dilute hypochlorite or merthiolate. The explants may be root, stem, shoot tip, leaf petiole, embryo, etc. If excised, differentiated tissue is placed on a nutrient medium, which forms an unorganised and undifferentiated callus. Callus may be defined as “an irregular, unorganised and undifferentiated but actively dividing mass of cells.” Darkness favours callus formation. Differentiation of morphogenesis occurs when callus is exposed to light or provided with growth regulators like auxin such as 2, 4-D (2, 4-dichlorophenoxyacetic acid) and cytokinins,

such as BAP (benzylaminopurine). The growth regulators or hormones are required for cell division and organ regeneration from the cultures.

Culture technique

The excised tissue is grown in artificial aseptic nutrient medium under controlled Conditions, the successful culturing technique requires following important steps

(i) Nutrient medium

(ii) To make the medium and explant aseptic

(iii) Acration of culture

(i) Nutrient Medium

Culture medium should provide the nutrition to the developing cell culture for the required growth and development. Most of the nutrient or culture media contain inorganic salts of essential and non-essential elements, vitamins, glycine, minerals, 2.4% Sucrose (as source of energy) and the desired growth regulators. Skoog and Miller (1957) discussed the role of auxins and cytokinins in shoot and root formation. They noticed that tobacco pith, when placed in a nutrient medium without growth hormones, showed very poor growth. If little cytokinins (BAP) were added to the medium, a callus formation occurred. In the third set, auxin (2, 4-D) was added, and roots were formed. In the fourth set they provided higher concentration of Kinetin with low concentration of auxin and only shoots were formed. Therefore, growth regulators are required for normal development of callus into a young plant called plantlet. Tissue culture can be maintained in a solid medium or a liquid medium.

Solid media are particularly suitable for callus culture and are prepared by mixing the liquid nutrient solution with a gelling agent, usually agar, at a concentration of about 1-2%, thus producing nutrient agar. The medium ordinarily contains the auxin 2, 4-D, and often a cytokinin like BAP

Liquid media are often useful for measuring population culture. They may be placed in a test tube, stopped by a plug of cotton wool or a metal cap. The medium must be sterilised before it can be used for growth of a cell culture. Adding a small quantity of cells to the medium is called inoculation. After inoculation, the medium is kept in an incubator at the optimum growth temperature. Usually the medium contains the auxin 2, 4-D.

(ii) To Make the Medium and Explant Aseptic

Culture medium is autoclaved at 120°C for 20 minutes for sterilisation. Instruments and containers which are to be used are also sterilised. The laboratory is kept dust free and irradiated with ulra-violet rays to kill microbes, this is called sterilisation. The explants are also treated with specific anti-microbial chemicals (clorox water, dil, hypochlorite and merthiolate), this procedure is called surface sterilisation.

(iii) Aeration of Culture

Sufficient supply of oxygen is necessary for proper development and growth of tissues. Tissues grown in solid or semi-solid media need no extra supply of oxygen. If the tissues are placed on liquid media then they should be agitated to ensure aeration of culture, constant mixıng of the medium, and breakage of cell aggregates into smaller cell groups. Mechanical shakers or a Magnetic stirrer can be used. Larger volumes should also have sterile air passed through them in order to maintain the Oxygen concentration throughout the medium. Commercial filters can be used to filter the air and sterilise it. Liquid cultures can be grown as suspension cultures.

Callus and suspension cultures.

In callus culture, cells divide to form an unorganised mass of cells. It is maintained on a medium gelled usually with agar. When an explant is placed on such a medium, many of the cells become meristematic and show repeated divisions. Within 2-3 weeks a mass of cells called callus is formed. The agar medium contains both types of growth regulators like auxin 2, 4-D and cytokinin like BAP

In case of suspension culture, a single cell can be isolated from cultured tissue and placed on liquid medium. The medium normally contains the auxin 2, 4-D. The isolated cells divide and form small groups of cells. The suspension cultures are continuously agitated to break the cell mass into smaller clumps and single cells, and also maintain uniform distribution of cells and cell clumps in the medium. It also allows gaseous exchange. Suspension cultures groW much faster than callus cultures.

Suspension cultures can be maintained in either of the following two forms

(i) Batch cultures are initiated as single cells in a flask and are propagated by transferring regularly small groups of suspensions to a fresh medium.

(ii) Continuous cultures are maintained in a steady state for a long period by draining out the used medium and adding fresh medium. The process of transferring the cell culture into a fresh culture medium is called subculturing. It is not done after 4-6 weeks when callus develops to its maximum. Small pieces of callus are transferred to fresh medium in different containers. 

Significance of tissue culture

1. It is mainly useful in multiplication of desirable characters.

2. lt can help in the improvement of crop plants. rome

3. Production of disease free plants. This technique has been widely used to produce virus free potatoes.

4. To shorten the breeding cycle. Los b alo oode

5. To bypass the seed dormancy. It can be achieved by embryo culture.

6. For fusion of cells by somatic hybridisation.smad s equals can

7. For the production of transgenic plants.

Formation of plantlets

Gottleib Haberlandt (1902) was the first one to grow isolated leaf cells by plant tissue culture technique. F.C. Steward provided the first evidence of cellular totipotency in a carrot experiment. Cellular totipotency is defined as “the ability of somatic cells of a plant to produce a new complete plant”. The tissue culture experiment illustrates the concept of dedifferentiation process that occurs during regeneration. Regeneration in the case of tissue culture technique can be described as the development of specific structures like root, shoot or somatic embryo from cultured cells. The two important components of a plant are root and shoot. The regeneration

Shoot-root formation

The root and shoot is controlled by two types of growth regulators. The auxin like NAA (naphthalene acetic acid) promotes root regeneration whereas cytokinins such as BAP promotes shoot regeneration. Callus cultures are first kept on medium containing BAP

which initiates shoot formation from the callus. When shoots become 2-3 cm long, the culture is transferred to a medium containing auxin. Roots develop from the lower ends of the shoots and develop into young plants called plantlets.

Somatic embryogenesis

Somatic embryos or embryoids are non-zygotic embryo-like structures that develop in vitro cultures from somatic cells of callus raised from an embryo. Embryoids Can Be produced in culture from somatic cells of any type of tissue but it is easier to raise them from culture of immature embryos. The medium having ammonium nitrogen and auxin, such as 2, 4-D favours their differentiation. Embryoids can be produced  thousands in a small space of a culture tube or flask. Steward (1964) has estimated the 100,000 embryoids can be formed in culture from an explant consisting of an embryo of carrot. When allowed to grow, each embryoid can directly form a new plantlet without special treatment. The plantlets can be shifted from culture vessels to greenhouse for undergoing hardening process.

Plantlets are exposed to reduced light and high humidity for a suitable period of time. They are finally shifted to the normal fields. The process of hardening is essential for plantlets as they adjust themselves and become capable of facing harsh climatic conditions.

Applications of plant tissue culture

Tissue culture can help in improvement of crop plants by following techniques

(i) Micropropagation

(ii) Meristem culture

(iii) Embryo culture

(iv) Haploid culture

(v) Induced mutation

(vi) Soma-donal variations,

(vii) Somatic hybridisation.

(I)Micropropagation

It is a rapid vegetative multiplication of plants in artificial media under aseptic conditions from very small pieces of plants (e.g. shoot tips, root tips, single cells, callus, embryos, stem, etc.) Iwo of the common types of micro propagation are

(a) Multiple shoot production: 

The technique is used in rapid multiplication of pathogen free rare plants, rare hybrids, etc. A shoot tip or apical bud is taken, sterilised and placed on a culture medium having high salt content and NAA (naphthalene acetic acid). When plants are required, the shoot tips are shifted to low salt medium without NAA. Small rooted plantlets develop. Multiple shootlet production can be performed in case of banana, potato, orchids, etc.

(b) Somatic embryogenesis: 

Somatic embryos or embryoids are non-zygotic embryo-like structures that develop in vitro cultures from somatic cells with Callus raised from embryo. Embryoids can be produced in thousands in a small space of a culture tube or flask. Steward (1964) has estimated that 100,000 embryoids can be formed in culture from an explant consisting of an embryo of carrot. Each embryoid can grow and directly form a new plant without special treatment.

(ii) Meristem Culture

ncase of vegetatively propagated plants, pathogen free clones can be obtained tough shoot tip culture because shoot apical meristem is usually free of the pathogens. apical meristem is sterilised and placed over culture medium under aseptic ions . In some cases thiamine or NAA is required for root initiation. When the sans grows a few leaves, it is transferred to a soil medium. Shoot tip culture is Dlr producing pathogen free plants of potato, sugarcane, sweet potato, strawberry, ,Laly, Gladiolus, etc. These explants are also cultured in a medium containing nin (BAP) as it promotes axillary branching. In case of axillary branching. shdual shoots are cultured. But when axillary branching does not occur, the single Cut into nodal segments, which are then cultured. This technique is useful in plasm conservation and production of transgenic plants. Tmplasm conse

(iii) Embryo Culture (Embryo Rescue)

The term ’embryo rescue’ is used for embryo culture. Some rare plants reproduce through seeds with great difficulty. During Înterspecific hybridisation, often that embryo dies quite early, so that no mature seeds can be obtained. This problem can be overcome, if young hybrid embryos are excised and cultured on a synthetic medium.

• These cultured embryos can directly develop into young seedlings. Embryo rescue has made hybridisation possible between

• Jute species, Corchorus olitorius and C. capsularis for better quality of fibre and
• resistance of pests as well as pathogens.

• Disease resistance and other desirable traits of Wild Bean (Phaseolus angustissimus have been transferred to common Bean (P.vulgaris).

• Resistance to brown plant hopper found in Oryza officinalis has been transferred
• to Oryza sativa, through hybridisation followed by embryo rescue.

Characteristics of improved vitamin C content, resistance to pathogens and pests have been successfully transferred from wild variety of tomato to cultivated tomato, through hybridisation followed by embryo rescue.buborg to.

(iv) Haploid Culture

Haploid culture is also known as pollen grain culture or androgenic haploid culture. Guha and Maheshwari (1964) reported culture of androgenic (of male origin) haploids of Datura innoxia. Unopened flower buds are first sterilised in 1: 3 clorox for 10-20 minutes. The anthers are removed and kept in culture medium having sucrose,

vitamins, auxins and minerals. After 4-6 weeks, the anthers produce a large number of haploid embryoids. This is also called another culture. The embryoids can grow to form haploid plants which are sexually sterile. They are changed to homozygous diplomas

by colchicine, either to young plantlets or to callus obtained from haploids on Further culturing. Ihe main importance of haploid cultures are

(a) In haploids all mutations show their effect. Therefore, they are useful for induction of mutations and also for mutation breeding.

(b) Colchicine treatment produces pure and perfect homozygous plants in one step which otherwise takes several years for other normal plants.

(c) Use of haploids in producing pure lines has reduced the period required 10r developing new varieties from 10 years to 5 years.

(d) Haploid plants are always pure because they have only one gene for each ralt.

(e) Androgenic plants have been studied in over 250 plant species including wheat, rice, maize, rubber, tomato, etc.

(f) The new varieties of plants are superior to other varieties in yield, quality and resistance.

(v) Induced Mutation

A large population of single cell is ideal for induction of mutations. In this method, a quid culture medium is used with a shaking device so that cells can multiply without Forming calluses. Two methods for induction of mutations are as follows:

(a) Chemical mutagens are added in different doses and for different durations. The cells are then washed and transferred to solid culture for growth into callus or embryoids and subsequent development of plants. The desired mutants picked up for cultivation.

(b) Addition of toxins, pollutants, salts, herbicides or pesticides against which resistance is to be developed. Most of the cells will die but a few resistant nests will be found to survive. The surviving cells are allowed to grow on solid medium for obtaining resistant plants.

(vi) Somaclonal Variations

Genetic variations produced in plants regenerated from tissue cultures involving callus formation are described as ‘somaclonal variations’. Some of the somaclonal variations are stable and useful, e.g., resistance to diseases and pests, stress tolerance. male sterility, early maturation, better yields, etc. The usefulness of this variability in crop improvement programmes, was first demonstrated through the recovery of disease resistant plants in:

• Potato to Phytophthora infestans which causes late blight disease,

• Wheat tolerant to rust and high temperature,

• Rice to leaf ripper and Tungro virus,

• High protein content of potato,

• Short duration sugarcane and resistance against eye-spot disease, Fiji disease,and downy mildew.
• Increased shelf-life of tomatoes.

For the selection of useful genetic somaclonal variation, two approaches have been followed:

(a) Selection is exercised in cells cultured for different periods and screened for the derived traits (e.g. for resistance to specific herbicides, fungal toxins, pollutants,extremes of temperatures and salinity), and from these selected cells or cultures, plants are regenerated.

(b) In the second approach selection is exercised at the phenotypic level in regenerated plants.

The former approach of selection at the cellular level has the advantage of screening millions of cells with a relatively little effort and resources.

(vii) Somatic Hybridisation

The technique of fusion of isolated protoplasts from genetically different organisms is called somatic hybridisation or parasexual hybridisation. A hybrid produced by fusion of somatic cells is called somatic hybrid. Intraspecific and interspecific fusion of protoplasts in the presence of sodium nitrate was done by Power et.al. in 1970. Somatic hybrids in plants were first obtained between two species of tobacco (Nicotiana glaeu and N. langsdorfü) by Carlson et.al. in 1972. Enzymes like pectinase and cellulase were used to isolate protoplast from cells by digesting cell walls. The plant cells without a cell wall are called protoplasts. The isolated protoplasts when placed in solutions suitable for osmotic concentration become spherical. Fusion between protoplasts or u Adherent cells is induced by a solution of polyethylene glycol (PEG), or by silver nitrate The fusion product of two different protoplasts is called heterokaryon. The fusion of protoplasts Or pollen grains by gentle tapping even in the absence of silver nitrate has been done successfully by Ito in 1973. The bodies thus formed were grown on a suitable culture medium and due to rapid division and multiplication a callus is formed. The callus mass is shifted to medium, where finally plantlets were formed. These hybrid plantlets when grown in soil formed full plants.

Somatic hybridisation occurs between sexually incompatible species, permitting transfer of desirable characters from wild or unrelated crop species to our croP Plants. A few examples of somatic hybrids are given below:

The somatic hybrid between a non-flowering clone of potato and a flowering potato clone produced fertile flowers.

Similarly, somatic hybrids were produced between rice and carrot.

Pomato is a somatic hybrid between potato and tomato that belong to two different genera. The hybrid plant bears both fruits and tubers of the two parents.

The main significance of protoplast technology are

(a) It has opened up chances for development of hybrids of even sexually reproducing plants.

(b) There is a distinct possibility of development of new crop plants, e-g, pomato.

(c) Genetic manipulations can be carried out more rapidly when plant cells are in protoplast state, e.g., production of useful allopolyploids and transgenic plants.

Single Cell Protein (SCP)

Single-cell protein (SCP) refers to the microbial cells or total protein extracted for pure microbial cell culture (monoculture) which can be used as a protein supplement for humans or animals. The word SCP Is considered to be appropriate, since most of s

The microorganisms grow as single or filamentous individuals. This is in contrast to comnet

multicellular plants and animals. If the SCP is suitable for human consumption. It is considered as food grade. SCP is regarded as feed grade, when it is used as an animal feed supplement, but not suitable for human consumption.

Single-cell protein broadly refers to the microbial biomass or protein extract used as food or feed additive. Besides high protein content (about 60-80% of dry cell weight), SCP also contains fats, carbohydrates, nucleic acids, vitamins and minerals,

Another advantage with SCP is that it is rich in certain essential amino acids (lysine, methionine) which are usually limited in most plant and animal foods. Thus, SCP is of high nutritional value for human or animal consumption.

It is estimated that about 25% of the world’s population currently suffers from hunger and malnutrition. Most of these people live in developing countries. Therefore, SCP deserves serious consideration for its use as a food or feed supplement. In addition to its utility as a nutritional supplement, SCP can also be used for the isolation of several compounds. e.g., carbohydrates, fats, vitamins, minerals. Rugs lindros

Advantages of using microorganisms for SCP production

The protein-producing capabilities of a 250 kg cow and 250 g of microorganisms are often compared. The cow can produce about 200g protein per day. On the other hand, microorganisms, theoretically, when grown under ideal conditions, could produce about 20-25 tonnes of protein. There are many  advantages of using microorganisms for SCP production:

(i) Microorganisms grow at a very rapid rate under optimal culture conditions. Some microbes double their mass in less than 30 minutes.

(ii) The quality and quantity of protein content in microorganisms is better compared to higher plants and animals.

(iii) A wide range of raw materials. which are otherwise wasted, can be fruitfully used for SCP production.

(iv) The culture conditions and the fermentation processes are very simple.

(v) Microorganisms can be easily handled and subjected, to genetic manipulations Safety, acceptability and toxicology of SCP

There are many non-technological factors that influence the production or These include the geographical, social, political and psychological factors. In SCP countries, there are social and psychological barriers to using microorganisms as food sources. It is desirable to first consider the safety, acceptability and toxicology of SCP

source particularly when it is considered for human consumption. There are several limitations for the widespread use of SCP following are those:

(i) The nucleic acid content of microbial biomass is very high (4-6%% in algae; 10 – 15% in bacteria; 5-10% in yeast). This is highly hazardous, since humans have a limited capacity to degrade nucleic acids.

(ii) The presence of carcinogenic and other toxic substances is often observed in association with SCP. These include the hydrocarbons, heavy metals, mycotoxins and some contaminants. The nature and production of these compounds depends on the raw materials, and the type of organism used.

(iii) There is a possibility of contamination of pathogenic microorganisms in the SCP

(iv) The digestion of microbial cells is rather slow. This is frequently associated with indigestion and allergic reactions in individuals

(v) Food grade production of SCP is more expensive than some other sources of proteins, e.g. soya meal. Of course, this mainly depends on the cost of raw materials. In general, SCP for human consumption is 10 times more expensive than SCP for animal feed.

For the above said reasons, many countries give low priority for the use of SCP for human consumption. In fact, mass production of SCP using costly raw materials has been discontinued in some countries e.g., Japan, Britain, Italy. However, these countries continue their efforts to produce SCP from cheap raw materials such as organic wastes.

Micro-organisms and Substrates used for Production of SCP

Several microorganisms that include bacteria, yeasts, fungi, algae and actinomycetes using a wide range of substrates are used for the production of SCP. A.

The selection of microorganisms for SCP production is based on several criteria. These include their nutritive value, non-pathogenic nature, production cost. raw materials used and growth pattern.

Biofortification

Biofortification is the method of breeding crops with higher levels of vitamins or higher protein and healthier fats and minerals.

The objectives of breeding improved quality of crops are to increase the following:

(i) Protein content and quality

(ii) Oil content and quality

(iii) Vitamin content

(iv) Micro nutrient and mineral content

Some crop varieties with increased nutrient content are:

(i) Maize varieties rich in lysine and tryptophan amino acids.

(ii) Wheat variety with high protein content, Atlas-66 has been used as a donor for improving cultüvated wheat.

(iii) Iron fortified variety of rice. 

The Indian Agricultural Research institute (IARI), New Delhi has also released Several vegetable crops that are rich in vitamins and minerals. For example,

(i) Vitamin-C enriched Bathua (chenopodium), tomato, bitter-gourd, mustard.

(ii) Variety of bathua enriched with iron and calcium and spinach varieties with iron and calcium content.

(iii) Beans like french beans, lablab beans, broad beans and garden peas enriched with protein.

(iv) Vitamin A enriched carrots, pumpkin and spinach.