Genetic Improvement and Development of New Varities
Trait : Trait or character is any morphological, anatomical, biochemical or behavioural feature of an organism.
Variety : A group of plant that has the same genotype, but it differs for one or more characters from other varieties of the same crop. An improved variety is superior to the other existing varieties of the same crop in one or more characters.
Plant breeding :- The branch of agricultural sciences which leads to development of new and improved variety of crop plant. It is the purposeful manipulation of plant species in order to create desired plant types that are better suited for cultivation, give better yield and have disease resistance.
It was started about 9000-11000 year ago.
1871 – Department of Agriculture Ist time organized.
1905 – Imperial Agriculture Research Institute Pusa (IARI), Bihar.
1936 – Rebuilt in Delhi with same name
1946 – Name changed to Indian Agriculture Research Institute, Pusa
Objective of plant breeding :
1. Development of high productivity crop variety.
2. Development of variety with high nutritional quality.
3. Development of variety with high water use efficiency.
4. Development of variety with high mineral use efficiency.
5. Development of abiotic stress (Drought, Salinity) tolerant variety.
6. Development of biotic stress and insect pest resistant variety.
7. Development of early maturing variety.
8. Development of variety with less post harvest loss.
Some plant breeding institute
1. I.A.R.I. Indian agricultural research institute, Pusa, New Delhi
2. C.P.R.I. Central potato research institute, Shimla
3. C.R.R.I. Central rice research institute, Cuttack
4. I.S.B.R.I. Indian sugarcane breeding research institute, Coimbatore
5. C.A.Z.R.I. Central arid zone research institute, Jodhpur
6. N.B.R.I. National botanical research institute, Lucknow
7. N.B.P.G.R. National beauro of plant genetic resources, New Delhi
8. I.I.S.R. Indian institute of spice research, Calicut
9. I.R.R.I. International rice research institute Manila
11. C.Y.M.M.I.T. International centre for wheat and maize Improvement Mexico
Some plant Breeder :
1. Normon E. Bourloug : Father of green revolution. He developed semi dwarf varieties of wheat Sonara 64 and Lerma rojo. He got Nobel Peace Prize in 1970.
2. N.I. Vavilov : Father of centre of origin concept for cultivated plants.
3. M.S. Swaminathan : Father of green revolution in India. Father of mutational breeding in India.
He developed semi dwarf varieties of wheat Sharbati Sonara and Pusa Lerma through mutation from Sonara 64 and Lerma rojo, respectively. He was the Ist Winner of World Food Prize.
4. Gurdev Singh Khush : Former director of IRRI. He is a rice breeder. He developed high yielding rice variety IR-36. He got World Food Prize.
5. S.K. Vasil : Maize Breeder. He developed biofortified maize varieties (QPM) which are having high concentration of Tryptophan and Lysine. He got World Food Prize.
1. Collection of variability.
2. Evaluation and Selection of parents.
3. Cross hybridization among the selected plant.
4. Selection and testing of superior recombinants.
5. Testing release and commercialization of new cultivar.
(1) Collection of variability: Genetic variability is the root of any breeding programme. In many crops pre-existing genetic variability is available from wild relatives of the crop. Collection and preservation of all the different wild varieties, species and relatives of the cultivated species (followed by their evaluation for their characteristics) is a pre-requisite for effective exploitation of natural genes available in the populations.
The entire collection (of plants/seeds) having all the diverse alleles for all genes in a given crop is called germplasm collection.
(2) Evaluation and selection of parents: The germplasm is evaluated so as to identify plants with desirable combination of characters. The selected plants are multiplied and used in the process of hybridisation.
(3) Cross hybridisation among the selected parents: The desired characters have very often to be combined from two different plants (parents). This is possible by cross hybridising the two parents to produce hybrids that genetically combine the desired characters in one plant. This is a very time-consuming and tedious process since the pollen grains from the desirable plant chosen as male parent have to be collected and placed on the stigma of the flowers selected as female parent.
(4) Selection and testing of superior recombinants: This step consists of selecting, among the progeny of the hybrids, those plants that have the desired character combination. The selection process is crucial to the success of the breeding objective and requires careful scientific evaluation of the progeny. This step yields plants that are superior to both of the parents. These are self-pollinated for several generations till they reach a state of uniformity (homozygosity), so that the characters will not segregate in the progeny.
(5) Testing, release and commercialization of new cultivars: The newly selected lines are evaluated for their yield and other agronomic traits of quality, disease resistance, etc. This evaluation is done by growing these in the research fields and recording their performance under ideal fertiliser application irrigation, and other crop management practices. The evaluation in research fields is followed by testing the materials in farmers’ fields, for at least three growing seasons at several locations in the country, representing all the agroclimatic zones where the crop is usually grown. The material is evaluated in comparison to the best available local crop cultivar – a check or reference cultivar. If material is superior than best crop cultivar, than it is released for cultivation by ICAR.
Wheat and Rice : During the period 1960 to 2000, wheat production increased from 11 million tones to 75 million tonnes while rice production went up from 35 million tonnes to 89.5 million tonnes.
This was due to the development of semi-dwarf varieties of wheat and rice. Nobel laureate Norman E. Borlaug, at International Centre for Wheat and Maize Improvement in Mexico, developed semi-dwarf wheat. In 1963, several varieties such as Sonalika and Kalyan Sona, which were high yielding and disease resistant, were introduced all over the wheat-growing belt of India.
Semi-dwarf rice varieties were derived from IR-8, (developed at International Rice Research Institute (IRRI), Philippines) and Taichung Native-1 (from Taiwan). The derivatives were introduced in 1966. Later better-yielding semidwarf varieties Jaya and Ratna were developed in India.
Sugar cane : Saccharum barberi was originally grown in north India, but had poor sugar content and yield. Tropical canes grown in south India Saccharum officinarum had thicker stems and higher sugar content but did not grow well in north India. These two species were successfully crossed to get sugar cane varieties combining the desirable qualities of high yield, thick stems, high sugar and ability to grow in the sugar cane areas of north India.
Millets : Hybrid maize, jowar and bajra have been successfully developed in India. Hybrid breeding have led to the development of several high yielding varieties resistant to water stress.
Methods of New Variety Development
(2) Germplasm collection & conservation
(3) Plant introduction
(6) Mutational breeding
(7) Genetic engineering
(1) Domestication :- All the present species of cultivated plants are of wild type species in origin. Process of cultivation of wild species in order to fulfill human need is called domestication of plant. Many present day crops are the result of domestication in ancient times.
Natural home :- It is the centre of origin of a crop, which often abounds in its wild relatives and maximum genetic diversity.
Secondary home :- It is the major centre of production of a crop plant, which is away from centre of origin and lacks wild relatives
Wild relatives :- Species related to cultivated plant that occur in the wild in areas of their origin.
(2) Germplasm collection & conservation :-
Germplasm is the sum total of all the alleles of the genes present in cell of a crop species and its related species. The entire collection (of plants/seeds) having all the diverse alleles for all genes in a given crop is called germplasm collection.
It is consist of –
(i) Cultivated improved varieties
(ii) Improved varieties that are no more cultivated
(iii) Old local or desi varieties.
(iv) Varieties produced by plant breeders (undistributed)
(v) Wild species related to the crop species
The sum total of different types of all the genes and their alleles present in a population is called gene pool. The gene pool of a population is not static.
Genetic erosion – The loss of genes from a gene pool is called genetic erosion.
Genetic erosion occurs due to deforestation, urban expansion, damage to ecosystem and adoption of genetically uniform modern variety of crops.
11 million hectares of tropical forest disappear every year.
There are four basic way to conserve plant germplasm.
(i) Conservation of plant in wild state i.e. in natural habitat like forests.
(ii) Conservation of plant in botanical gardens.
(iii) Introduction of plant for cultivation in agriculture and horticulture.
(iv) Preservation of plants in seed form or some other suitable form.
Method of genetic conservation :
(i) In-situ conservation – It means maintenance of biological diversity in natural habitats like forests and natural reserve like national park, wildlife sanctuaries and bio-sphere reserves. In situ conservation of wild plants help in protecting species threatened with extinction.
(ii) Ex-situ conservation – It is the conservation of selected or rare plant in a place outside the natural home. In this conservation rare or selected plant material are grown in specific gardens.
Ginkgo biloba plant is preserved by Ex-situ conservation.
Ex-situ conservation includes offsite collections and gene bank.
(a) Offsite collection : They are living collections of wild and domesticated species in botanical garden, zoo etc.
(b) Gene bank : The place or institution, where different plant material (genes) are kept or preserved, is called ''Gene bank''
In gene bank, storage of germplasm is done either in the form of seed or vegetative material, but best and convenient way is storage of seed.
Seed means, any plant part that is used to grow a crop. Thus ‘seed’ would include grains of wheat, rice, tubers of potato, stem of sugarcane etc. which are used for producing new plants.
Cryopreservation :- Preservation of germplasm at ultra low temperature at –196° C (liquid nitrogen) called cryopreservation.
Storage of dry seeds is done at low temperature (–10 to –20°C), because under these conditions the metabolic activites are minimum, prevent their germination.
Seeds are of two types-
(a) Orthodox seed :- The seeds can not be killed or damaged as a result of decrease in moisture content& temperature. e.g. seeds of wheat, rice, maize, oat barley (Cereals) and also different pulses or legumes.
(b) Recalcitrant seeds :- The seeds which can be killed or damaged as a result of drying and decrease in temperature. These can be stored for a short span. eg. seeds of rubber, tea, coconut, Jack fruit (Artocarpus), litchi, oil palm etc.
Conservation of crop with recalcitrant seed, can be made by in situ conservation method and also by tissue culture method.
Plants with recalcitrant seed are grown in orchard, where all possible strains and varieties are maintained.
Storage of germplasm by tissue culture :
The tissue culture technique for storage of germplasm is used in case where :-
(a) No seeds are produced (banana, sugar cane)
(b) Non viable seeds
(c) Crop with recalcitrant seed
(d) Specific clone is to be maintained
Best method of tissue culture for germplasm storage is ''Shoot tip culture''.
Shoot tip culture rapidly, becoming preferred material for international exchange of germplasm as they are more stable, easier to regenerate in to whole plants and produce virus free clonal plants.
Nowadays germplasm of potato, Cassava, and banana are exchanged by this method.
The main advantage of tissue culture storage of germplasm are :-
(b) Requires small area for storage of many genotype
(c) Can be used for multiplication of rare and endangered species.
Significance of germplasm of wild species :
It is very important to conserve wild species of plant as these are highly resistant to insects, pests, disease and unfavourable growth conditions, which are necessary for survival of plants.
The loss of wild plants, will reduce the genetic variability and will be a great loss to gene pool.
Potato and sugarcane has been improved by use of germplasm of wild species having many characters like disease resistance and resistance to environmental stress.
In potato (Solanum tuberosum) gene for resistance to potato virus-X and potato leaf roll virus have been obtained from a wild species ''Solanum acaule''.
Resistance to wilt fungus (Fusarium) and cyst nematode (Globodera) has been introduced from ''Solanum spegazzini''.
Potato got resistant gene to potato virus–Y from a wild relatives ''Solanum stoloniferum''
Resistance to late blight of potato (Caused by fungus Phytophthora infestans) has been derieved from ''S.demissum''
Similarly, sugarcane (Saccharum officinarum) got resistance to red rot of sugarcane and adverse environment from a wild species ''Saccharum spontaneum''.
INTERNATIONAL EFFORTS FOR UTILIZATION OF CROP - Germplasm
Cereals (rice, wheat, maize, rye, sorghum, bajra etc) are the main sources of food for human population in the world and rice alone constitutes staple food of more than 50% world's population.
Improvement in rice production :
Dwarfing gene of rice ''dee-geo-woo-gene'' was obtained from Taiwan.
This gene was incorporated to produce high yielding early maturing IR-8 and IR-24 varieties by IRRI, Manila
Gurdev S.khush and his team crossed 13 varieties of rice from six countries and wild rice Oryza nivara (from India) to develop IR-36 variety of rice.
IR-36 variety of rice is resistance to grassy stunt virus.
IR-36 is the high yielding variety of rice and has solved major food problem in Asia.
Improvement in wheat production :
Dr. N.Borlaug (Mexican wheat breeder) develop many dwarf wheat varieties like Sonora-64 & Lerma rojo by incorporating Norin-10 (Japaniease dwarf gene) gene.
(3) Plant introduction -
This is the most rapid method of crop improvement.
The process of introducing plants with specific characters from one area to a new and changed climatic condition is called ''Plant introduction''.
(a) If plants are introduced from foreign country, it is called ''exotic collection (EC).
(b) If plants are brought from same country then it is called ''indigenous collection'' (IC).
Primary introduction :- If introduced plants may be used directly for cultivation, it is called primary introduction.
Secondary introduction :- If introduced plants may be used for cultivation after subjecting to selection/hybridization, it is called secondary introduction.
It means adaptation of introduced plant material in the changed or new climatic conditions is called acclimatization.
Plant Quarantine :
Introduced plant material are subjected to ''Quarantine laws/plant protection inspection. To check the entry of pathogen, all the introduced plant materials is throughly inspected for contamination of weeds, diseases and insect pest. This testing is known as plant quarantine.
If the plant material is found suitable, phytosanitary certificates are issued and only then the plant material is introduced in our country.
Uncontrolled plant introduction in the past are responsible for introduction of hazardous diseases like late blight of potato, leaf rust of coffee, fire blight of apple, bunchy top of banana etc.
Many weeds like Argemone maxicana, Eicchornia crassipes and Parthenium argentatum etc. introduced in our country due to uncontrolled plant introduction.
New plants like potato, groundnut, coffee, rubber, guava, grapes, papaya, litchi, gulmohar, bougainvillea etc are the result of plant introduction.
Wheat variety ''Ridley was introduced in India from Australia.
Many improved varieties of different crop plants are also outcome of these introduction e.g Sonora-64 varieties of wheat, Plametto varieties of soyabeans, Kent variety in Oat, Tiachung native 1 (TN1) of rice etc.
Mating between two or more individuals or lines, which are different in genotype.
Hybridization is the most common method for creation of genetic variations. It is performed very often to combine character from two different plants.
Types of Hybridization :
Hybridization is divided in following categories.
(i) Intravarietal hybridization : The crosses between the plant of same variety.
eg., Triticum aestivum var. kalyansona × T. aestivum var. K. sona
(ii) Intervarietal hybridization : The crosses are made between the plant belonging to two different varieties of same species (also known as intraspecific hybridization).
eg., Triticum aestivum var. kalyansona × T. aestivum var. sonalika
(iii) Interspecific hybridization : The plants or two different species belonging to the same genus are crossed together
eg., Solenum tuberosum × S. acuale
(iv) Intergeneric hybridization : The crosses are made between the plant belonging to two different genera. e.g. Triticale, Raphanobrassica are the example of intergeneric cross.
DEVELOPMENT OF RAPHANO BRASSICA AND TRITICALE
Development of Rabbage :
Rabbage or Raphano brassica was developed by Karpechanko
First natural hybridization was reported in corn (maize) by Cotton Mather.
First artificial hybrid was obtained by crossing Sweet william and carnation by Thomos Fairchild (1717) and was known as Fairchild's Mule.
Hybridization was first of all practically utilized in crop improvement by kolreuter (1760).
Hybridization or crossing leads to hybrid vigour or heterosis, which is defined as superiority of hybrid over it's parents.
The term heterosis was given by G.H. Shull in 1914.
Hybrid vigour is due to heterozygosity. Heterosis is lost by inbreeding.
Vegetatively reproducing plants are most suited for maintaining hybrid vigour.
The main steps of hybridization are following-
(a) Selection of parents
(b) Selfing of parents to induce homozygosity
(c) Emasculation :- i.e. removal of anthers or male reproductive part from flower before maturity
(d) Bagging :- Female flowers are covered with bags, so that no undesirable pollen may fall on stigma
(e) Crossing of desired & selected plants.
Two main aspects of hybridization are :-
(i) To combine characters of two plants in to one
(ii) To utilize hybrid vigour
Inbreeding :- When two individuals of a species, which have common ancestry are mated together. Most extreme form of inbreeding is self pollination.
Inbreeding depression :- Loss of vigour due to inbreeding.
Cross pollinated species shows inbreeding depression, but self pollinated species do not show inbreeding depression.
Plants of cross pollinated species are highly heterozygous, therefore they contain recessive alleles of most of the genes in heterozygous state.
When these are subjected to inbreeding there is in homozygosity and many recessive harmful alleles also become homozygous. Thus productivity is
In self pollinated crops alleles, become rapidly homozygous & then harmful alleles are removed by selection. So here inbreeding depression does not occurs.
(5) Polyploidy :-
The organism (Plant) which contains more than two complete sets of chromosomes is called polyploid.
Depending up on number of chromosomal sets, the individual are given different names-monoploid, diploid, triploids, pentaploids and hexaploid (eg-wheat)
Polyploids are characterized by gigantism.
These polyploids are used in crop improvement
Triploids are present naturally in different crop plants and generally triploid crop plants are seedless.
Most of the varieties of banana are triploids, so their fruit are seedless.
Polyploidy can be induced artificially by Colchicine treatment.
Colchicine is an alkaloid obtained from Colchicum automanale (fam. Liliaceae) plant from Bulb and Seeds.
In some plants triploids are having much vigour and increased fruit size e.g. apple & pear.
(6) Mutational breeding :-
Mutation – Sudden and inheritable change in genetic material of an organism.
Mutagens – The agents, which are used to induced mutation.
Chemical mutagen – Ethyl methane sulphonate (EMS), sodium azide.
Physical mutagen – X-rays, Gamma rays, UV rays
Induced mutation – Mutation which are induced artificially with the help of mutagens.
Mutation breeding :-
Use of induced mutations in plant breeding to develop improved varieties. Induced mutations are useful in specific situations, when the desired alleles are absent in the germplasm.
Many important varieties in different crop plants have been produced by mutation breeding
In wheat : Sharbati Sonora and pusa lerma are two important varieties of wheat produced by gamma rays treatment of sonora-64 and Lerma roja.
Sharbati-sonora is amber grain coloured variety of wheat produced by Dr. M.S. Swaminathan and is responsible for green revolution in India.
In rice : Remei & Atomita-2 are developed through mutation breeding.
In barley : Erectoids and erectiferum.
In Castor : World famous variety Aruna has been produced , in which life span has been reduced from 270 days to 102 days and also having high oil content and disease resistance.
Penicillin production has been increased enormously by UV-rays treatment of Penicillium notatum & P. chrysogenum.
In mung bean, resistance to yellow mosaic virus and powdery mildew were induced by mutations.
Mutation breeding has some important limitations as :
(i) Most of the mutations are recessive
(ii) Mutation rate is extremely low.
(iii) Most of the induced mutation are useless to the breeders and many of them are lethal.
(iv) Stability of mutant is sometimes doubtful, as some mutants have tendency to revert back to original type.
Breeding for nutritional quality
A crop product should provide the optimum nutrition to human and does not contain any antinutritional factor.
Antinutritional factors : Compounds that have harmful effects on animal's/human's growth & development.
Eg. Glucosinolates (Present in oils and cakes of rapeseed and musturd), b-N oxalyl aminoalanine (BOAA) neurotoxin found in khesari dal (Lathyrus sativus)
Cereals & millet proteins are deficient in lysine and tryptophan amino acid. (eg. Maize)
Pulses are deficient in sulphur containing amino acid (i.e. cysteine & methionine)
Three varieties of maize have been developed in India which are lysine-rich. Shakti, Rattan, Protina.
Breeding crops with higher levels of vitamins and minerals or higher proteins and healthier fats is known as biofortification. Breeding for improved nutritional quality is undertaken with the objective of improving.
1. Protein content and quality
2. Oil content and quality
3. Vitamin content
4. Micronutrient and mineral content
Maize hybrid with twice the amount of amino acid, lysine and tryptophan was developed in 2000. High protein content variety Atlas 66 has been used as a donor for improving cultivated wheat. It has been possible to develop an iron fortified variety containing over five times as much iron as in commonly consumed varieties.
The IARI New Delhi has also released several vegetable crop that are rich in vitamins and minerals eg., vitamin A enriched carrot, spinach, pumpkin, Vitamin C enriched bitter gourd, bathua, mustard tomato, iron and calcium enriched spinach and bathua and protein enriched beans, lablab and garden peas.
Breeding for disease resistance :
Methods of breeding for disease resistance : Breeding is carried out by conventional hybridization method and by mutation breeding.
Conventional breeding is often constrained by the availability of limited number of disease resistance genes that are present and identified in various crop varieties or wild relatives. Thus mutation breeding technique is used.
Yellow mosaic virus resistant and powdery mildew resistant moong varieties are developed through mutation breeding.
Yellow mosaic virus resistant variety of Okra, Parbhani Kranti was developed by gene transfer from wild variety.
Plant breeding for developing resistance to insect pests :
Another major cause for large scale destruction of crop plant and crop produce is insect and pest infestation.
Insect resistance in most crop plants may be due to morphological, biochemical or physiological characteristics.
Hairy leaves in several plants are associated with resistance to insect pests, e.g, resistance to jassids in cotton and cereal leaf beetle in wheat.
In wheat, solid stems lead to non-preference by the stem sawfly and smooth leaved and nectar-less cotton varieties do not attract bollworms.
High aspartic acid, low nitrogen and sugar content in maize leads to resistance to maize stem borers.
This is the most primitive method for crop improvement.
This is practiced in crop improvement and it is selection of phenotypically superior plants from a mixed population. This step yields plants that are superior to both of the parents.
Selection acts on genetic variation, present in a population and produces a new population with improved characters. Selection is of three types-
(a) Pure line selection
(b) Mass selection
(c) Clonal selection
(a) Pure line selection :- The progeny of a single self pollinated homozygous plant is called Pure line (term by Johanson) and these pure lines are used in selection method of crop improvement, which is called pure line selection
Pure line selection is a method of crop improvement in self pollinated crops (wheat).
In this method phenotypically superior plants are selected, these are harvested separately and their produce is maintained separately.
The seeds so obtained from different plants are shown separately and selection is made for 4 to 5 generation till the desired improvement is achieved. About 10 years time is needed to develop a new variety by this method.
(b) Mass selection :
This is practiced in cross pollinated crop plants.
The first step involves selecting plants having desirable character from a given population of plants based on phenotypic characters.
The seeds of selected plants are then mixed and sown in same field to allow natural cross pollination.
The plants are selected from this field by eliminating the undesirable ones and saving the best.
It is done for 3 to 4 generations or more then desired phenotypically similarity can be achieved.
It takes about 8 yrs. time to develop a new variety by mass selection.
(c) Clonal selection :-
This type of selection is applicable to vegetatively propagated plants eg. sugarcane, banana, potato.
Clone :- Progeny of a single vagetatively propagated plant is called clone.
Here selection is made between different plant clones and not within the same plant clone.
Kuffri Red in potato and green banana of Bombay varieties are produced through clonal selection.
Limitation of clonal selection :-
(1) Only applicable for vagetatively propagated crops.
(2) Creates no new variation
Single Cell Protein : Conventional agricultural production of cereals, pulses, vegetables, fruits, etc., may not be able to meet the demand of food at the rate at which human and animal population is increasing. The shift from grain to meat diets also creates more demand for cereals as it takes 3-10 Kg of grain to produce 1 Kg of meat by animal farming. Can you explain this statement in the light of your knowledge of food chains? More than 25 per cent of human population is suffering from hunger and malnutrition. One of the alternate sources of proteins for animal and human nutrition is Single Cell Protein (SCP).
Microbes are being grown on an industrial scale as source of good protein. Microbes like Spirulina can be grown easily on materials like waste water from potato processing plants (containing starch), straw, molasses, animal manure and even sewage, to produce large quantities and can serve as food rich in protein, minerals, fats, carbohydrate and vitamins. Incidentally such utilisation also reduces environmental pollution.
It has been calculated that a 250 Kg cow produces 200 g of protein per day. In the same period, 250g of a micro-organism like Methylophilus methylotrophus, because of its high rate of biomass production and growth, can be expected to produce 25 tonnes of protein. The fact that mushrooms are eaten by many people and large scale mushroom culture is a growing industry makes it believable that microbes too would become acceptable as food.
- Strategies for Enhancement in Food Production, class 12, biology
PLANT TISSUE CULTURE
Tissue culture technique is based on totipotent nature of plant cell.
Plant tissue culture is the technique of maintaining and growing plant cells, tissues and organs in sterilized culture medium, under controlled aseptic conditions in vitro.
Plant part that is excised from its original location and used for initiating a culture. It may be root tip, shoot bud, anther, embryo, ovule etc. Normally undetermined cells of plant are used as explant.
Surface Sterilization :
The process of treatment of explant with specific antimicrobial chemicals like sodium hypochlorite, H2O2, C2H5OH, Mercuric chloride etc.
Sterilization of culture media, plastic ware and glass ware by moist heat (steam) at high pressure. It is performed at 120ºC for 15 psi pressure for 20 minute.
Culture medium or nutrient medium :
Medium, which provides nutrition to explants which is required for normal growth and development of explants.
Standard culture medium contains inorganic Salts, Vitamins, Sucrose (as a source of energy and carbon), growth regulators (2,4–D, Cytokinins, BAP-benzylaminopurine)
Growth regulators are required for cell division and organogenesis in explant.
Murashige and Skoog's culture medium is the most commonly used culture medium
Axenic culture :
Culturing of cell under complete aseptic condition is known as Axenic culture.
Callus : Group of undifferentiated or dedifferentiated cells, which are produced through invitro culture.
Types of Cultures –
(1) Callus culture & suspension culture.
(2) Meristem culture
(3) Embryo culture
(4) Anther culture
(5) Protoplast culture
(1) Callus & Suspension culture –
Callus culture – In callus culture when an explant is placed on a agar containing medium. Many of the cells become meristematic and begin to divide and giving rise to callus in 2-3 week. The agar medium contain growth regulator like auxin 2, 4 –D and cytokinin like BAP.
Suspension culture –In case of suspension culture a single cell or small group of cells placed on liquid medium. The medium normally contains the auxin 2, 4-D. These cells divide and form small groups of cells.
The suspension cultures are continuously agitated to break the cell mass in to 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 culture.
With passage of time in a culture : -
(a) Cell tissue dry matter (biomass)
(b) The level of nutrients in the medium
(c) The medium volume declines due to evaporation.
The process of transferring the cell culture into a fresh culture medium is called subculturing. It is normally done after 4-6 week when callus develops to its maximum. During subculture only a part of the culture from a vessel is transferred into the new culture vessel.
Haberlandt was first one who grow isolated leaf cells in plant tissue culture medium.
Totipotency : - The ability of a plant cell to regenerate into complete plant.
The concept of totipotency was given by ‘‘Haberlandt’’ and practical applications of totipotency was demonstrated by ‘‘Steward’’.
Steward developed a complete carrot plant from a single cell obtained from root of wild carrot.
Shoot and root formation :
The regeneration of root and shoot is controlled by two types of growth regulators.
The auxin NAA (Naphthaline Acetic Acid) promotes root regeneration whereas cytokinins BAP promotes shoot regeneration.
Callus is 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 these shoots and develop into young plant called plantlet.
(2) Embryo culture : Culturing of immature young embryo in in-vitro medium.
Significance of Embryo Culture :
(i) In some interspecific crosses or distant hybridization the endosperm of developing hybrid seeds degenerate very early or not formed so young hybrid embryo which gets devoid of nutrition also dies. In such cases the young hybrid embryo is excised and cultured in vitro to obtain hybrid seedling.
(ii) Seeds of some plants like orchid lack stored food. In such cases embryo culture allows seedling development from the embryos. This method is also used for rapid clonal propagation in orchid.
(iii) In some species seeds may remains dormant due to inhibitors present in the endosperms/seed coat. Embryo culture in such cases allows embryo development by eliminating the inhibitors responsible for dormancy
(3) Meristem Culture
Significance of Meristem Culture : -
Rapid clonal multiplication.
Production of virus free plant.
Conservation of germplasm.
Production of transgenic plant.
(4) Anther Culture
Significance of Anther Culture : -
(i) They have single set of chromosome, so even a very small change or mutation can be detected in haploids.
(ii) These haploids are used to produce homozygous diploids (by colchicine treatment) and these homozygous diploids are used as parents in crossing.s
(iii) Use of haploids in producing pure lines has reduced the period required for developing new varieties from 10 years to 5 years.
(5) Protoplast culture : -
Somatic hybrid : A hybrid produced by fusion of somatic cells of two species or varieties.
The process of production of somatic hybrid is somatic hybridization.
Protoplast : Cell wall less plant cell is called protoplast.
STEPS OF SOMATIC HYBRIDISATION
(A) Removal of cell wall → 2 method
(i) Mechanical method → Old method
(ii) Enzymatic method ® New method
In this method cell wall is digested by using pectinase & cellulase enzyme.
(B) Fusion between protoplast → 2 methods
(i) Spontaneous fusion : - During enzymatic treatment some protoplast fused together and form multinucleated structure which is called homokaryons or homokaryocytes.
This is a intraspecific fusion.
Not very useful in study.
(ii) Induced fusion : -
Protoplast of two different species are fused together by induced fusion.
Substance which induced the fusion of protoplast are called fusogen or fusogenic agent.
Fusogenic substance and condition : -
By treatment of NaNO3
By treatment of Ca+2 ions at high pH
By treatment of polyethylene glycol [PEG]
By high voltage electric shock
Culture of the fused protoplast : -
Product of fused protoplast of two different species is called heterokaryon.
Heterokaryons are mainly used in tissue culture.
When the fused protoplasts are cultured on a suitable medium they regenerate cell wall and begin to divide ultimately to produce plantlets.
Importance of somatic hybridisation :
(i) It allows the production of hybrids between different lines and species that can not be produced normally by sexual reproduction.
Pomato is a somatic hybrid between potato and tomato.
Bromato-Brinjal & tomato
(ii) Use of somatic hybrid :
For gene transfer.
Transfer of cytoplasm.
Production of useful allopolyploids.
Somatic hybridisation is also called parasexual hybridisation.
First somatic hybrids were obtained between two species of tobacco Nicotiana gluca and N.langsdorfit by Carlson et. al.