29 May 2011

APPLICATIONS OF CELL AND TISSUE CULTURE


Micropropagation /Clonal Propagation


Clonal procreation refers to the process of asexual reproduction by multiplication of genetically identical copies of party plants. The vegetative procreation of plants is labour-intensive, low in productivity and seasonal. The tissue culture methods of sow procreation, renowned as 'micropropagation' utilizes the culture of apical shoots, axillary buds and meristems on apposite nutrient standard.The renaissance of plantlets in educated tissue was described by Murashige in 1974. Fossard (1987) gave a detailed tab of stages of micropropagation.
The micropropagation is rapid and has been adopted pro commercialization of valuable plants such as banana, apple, pears, strawberry, cardamom, many ornamentals (e.G. Orchids) and other plants.The micropropagation techniques are preferred ended the square asexual procreation methods since of the following reasons: (a) inside the micropropagation method, single a small amount of tissue is vital to rekindle millions of clonal plants in a time., (b) micropropagation is furthermore used as a method to develop resistance in many species., (c) in vitro have a supply of can be quickly proliferated as it is season self-determining,. (d) long stretch storage space of valuable germplasm doable.

The steps in micropropagation method are: A) Initiation of culture - from an explant like spurt tip on a apposite nutrient standard, b) multiple shoots formation from the educated explant, c) rooting of in vitro urban shoots and, d) transplantation - transplantation to the meadow following acclimatization.
The factors with the intention of affect micropropagation are: (a) genotype and the physiological status of the sow e.G. Plants with dynamic germination are more apposite pro micropropagation., (b) the culture standard and the culture background like light, warmth and that. For model an illumination of 16 hours a time and 8 hours night is satisfactory pro spurt proliferation and a warmth of 250C is optimal pro the growth.
The repayment of micropropagation this method are:
A)         rapid multiplication of superior clones can be conceded made known through made known the time, irrespective of seasonal variations.
B)         multiplication of disease emancipated plants e.G. Virus emancipated plants of sweet potato (Ipomea batatus), cassava (Manihot esculenta)
C)         multiplication of sexually derived sterile hybrids
D)         It is a cost effectual process as it requires smallest growing interval.
Somaclonal alteration

The genetic variations found in the in vitro educated cells are collectively referred to as somaclonal alteration and the plants derived from such cells are called as ‘somaclones’. It has been experimental with the intention of the long-term hard skin and cell suspension culture and plants regenerated from such cultures are often associated with chromosomal variations. It is this property of educated cells with the intention of finds the makings attention in the crop enhancement and in the production of mutants and variants (e.G. Disease resistance in potato).
Larkin and Scowcroft (1981) working by the division of Plant Industry, C.S.I.R.O., Australia gave the stretch 'somaclones' pro sow variants obtained from tissue cultures of somatic tissues. Similarly, if the tissue from which the variants be inflicted with been obtained is having gametophytic origin such as pollen or egg cell, it is renowned as 'gametoclonal' alteration.They explained with the intention of it could be due to: (a) reflection of heterogeneity linking the cells and explant tissue, (b) a unadorned representation of spontaneous change rate, and (c) initiation by culture background of transposition of genetic equipment.
Shepard et al. (1980) furthermore contributed by screening in this area 100 somaclones produced from leaf protoplasts of Russet Burbank. They found with the intention of here was a noteworthy amount of established alteration in compactness of growth problem, maturity, appointment, tuber homogeny, tuber skin colour and photoperiodic supplies.

Somaclonal Variations has been used in sow breeding programmes everywhere the genetic variations with desired or improved font are introduced into the plants and extra varieties are produced with the intention of can exhibit disease resistance, improved quality and yield in plants like cereals, legumes, smear with oil seeds tuber crops and that. Somaclonal alteration is applicable pro seed

APPLICATIONS OF SOMACLONAL VARIATIONS

A)         Methodology of introducing somaclonal variations is simpler and easier as compared to recombinant genetic material equipment.
B)         Development and production of plants with disease resistance e.G. Rice, wheat, apple, tomato and that.
C)         Develop biochemical mutants with abiotic stress resistance e.G. Aluminium tolerance in carrot, salt tolerance in tobacco and maize.
D)         Development of somaclonal variants with herbicide resistance e.G. Tobacco strong to sulfonylurea
E)         Development of seeds with improved quality e.G. A extra variety of Lathyrus sativa seeds (Lathyrus Bio L 212) with low content of neurotoxin.
F)         Bio-13 - A somaclonal variant of Citronella java (with 37% more smear with oil and 39% more citronellon), a medicinal sow has been released as Bio-13 pro money-making encouragement by Central Institute pro Medicinal and Aromatic Plants (CIMAP), Lucknow, India.
G)         Supertomatoes- Heinz Co. And genetic material sow Technology Laboratories (USA) urban Supertomatoes with distinguished solid element by screening somaclones which helped in sinking the shipping and dispensation expenditure.
Production of virus emancipated plants

The viral diseases in plants conveying straightforwardly and decrease the quality and yield of the plants. It is very trying to handle and heal the virus infected plants therefore te sow breeders are permanently interested in rising and growing virus emancipated plants.
Inside approximately crops like showy plants, it has be converted into doable to yield virus emancipated plants through tissue culture by the money-making level. This is made by regenerating plants from educated tissues derived from a) virus emancipated plants, b) meristems which are commonly emancipated of infection - inside the abolition of the virus, the size of the meristem used in cultures mess about a very vital role since generally of the viruses exist by establishing a descent in sow tissues. The renaissance of virus-free plants through cultures is inversely proportional to the size of the meristem used., c) meristems treated with excitement shock (34-360C) to inactivate the virus, d) hard skin, which is ordinarily virus emancipated like meristems.E) compound behavior of the media- attempts be inflicted with been made to eradicate the viruses from infected plants by treating the culture standard with chemicals e.G. Addition of cytokinins suppressed the multiplication of particular viruses.
Among the culture techniques, meristem-tip culture is the generally dependable method pro virus and other pathogen abolition.

Viruses be inflicted with been eliminated from a digit of economically valuable sow species which has resulted in a noteworthy boost in the yield and production e.G. Potato virus X from potato, mosaic virus from cassava and that. These virus emancipated plants are not disease strong so here is a need to keep up have a supply of plants to multiply virus emancipated plants when vital.

Production of phony seeds

Inside phony seeds, the somatic embryos are encapsulated in a apposite matrix (e.G. Sodium alginate), along with substances like mycorrhizae, insecticides, fungicides and herbicides. These reproduction seeds can be utilized pro the rapid and bulk procreation of desired sow species as well as hybrid varieties. The major repayment of phony seeds are:
A) They can be stored up to a time with made known loss of feasibility
B) Easy to soubriquet and helpful as units of manner of speaking
C) Can be frankly sown in the soil like natural seeds and sort out not need acclimatization in conservational household.

Mutant selection

An valuable aid of cell cultures is in mutant selection in relation to crop enhancement. The frequency of mutations can be increased several fold through mutagenic treatments and millions of cells can be screened. A generous digit of reports are unfilled everywhere mutants be inflicted with been selected by cellular level. The cells are often selected frankly by count the toxic substance hostile to which resistance is sought in the mutant cells. Using this method, cell shape strong to amino acid analogues, antibiotics, herbicides, fungal toxins and that be inflicted with in fact been isolated.
Production of secondary metabolites

The generally valuable chemicals produced using cell culture are secondary metabolites, which are defined as’ persons cell constituents which are not essential pro survival’. These secondary metabolites include alkaloids, glycosides (steroids and phenolics), terpenoids, latex, tannins and that. It has been experimental with the intention of as the cells undergo morphological differentiation and maturation all through sow growth, approximately of the cells dedicate yourself to to yield secondary metabolites. The in vitro production of secondary metabolites is much privileged from differentiated tissues as compared to non-differentiated tissues.
The cell cultures say in several ways to the production of natural products. These are: (a) a extra route of synthesis to set up products e.G. Codeine, quinine, pyrethroids, (b) a route of synthesis to a novel manufactured goods from plants trying to grow or set up e.G. Thebain from Papaver bracteatum, (c) a source of novel chemicals in their own aptly e.G. Rutacultin from culture of Ruta, (d) as biotransformation systems either on their own or as part of a better compound process e.G. Digoxin synthesis.

The advantages of in vitro production of secondary metabolites
A)         The cell cultures and cell growth are straightforwardly controlled in order to facilitate improved manufactured goods formation.
B)         The recovery of the manufactured goods is straightforward.
C)         As the cell culture systems are self-determining of environmental factors, seasonal variations, pain in the neck and microbial diseases, geographical location constraints, it is straightforward to boost the production of the vital metabolite.
D)         Mutant cell shape can be urban pro the production of novel and commercially helpful compounds.
E)         Compounds are produced under controlled conditions as for every the promote hassle.
F)         The production calculate is a reduced amount of and cost effectual due to smallest labour involved.
APPLICATIONS OF SECONDARY METABOLITES

Many of these secondary products especially various alkaloids are of immense aid in medicine. The yield of these chemicals in cell culture, is though commonly decrease than in total plants, it is substantially increased by manipulating physiological and biochemical conditions.
Shikonine is a dye produced by the cells Lithospermum erythrorhizon on a money-making extent. Besides this here are a digit of secondary metabolite products with the intention of are being widely used pro various purposes. Vincristine is used as anticancer agent, digoxin controls cardiovascular disorders, pyrithrins is an insecticide and that. The production of specialty chemicals by plants has be converted into a multibillion industry.
Please refer to the desk pro approximately secondary metabolites and their uses.

TABLE SHOWING PLANT SPECIES AND SECONDARY METABOLITES OBTAINED FROM THEM USING TISSUE CULTURE TECHNIQUES



Production of Somatic hybrids and cybrids
The Somatic cell hybridization/ parasexual hybridization or Protoplast fusion offers an alternative method pro obtaining distant hybrids with wanted traits significantly linking species or genera, which can not be made to thwart by square method of sexual hybridization.

SOMATIC HYBRIDIZATION
Somatic hybridization broadly involves in vitro fusion of isolated protoplasts to form a hybrid cell and its later development to form a hybrid sow. The process involves: A) fusion of protoplasts, (b) Selection of hybrid cells, (c) identification of hybrid plants.
During the continue two decades, a variety of treatments be inflicted with been used to bring in this area the fusion of sow protoplasts. Protoplast fusion can be achieved by spontaneous, mechanical, or induced fusion methods.. These treatments include the aid of fusogens like NaNO3, distinguished pH with distinguished Ca2++ ion concentration, aid of polyethylene glycol (PEG), and electrofusion. These inducing agents used in protoplast fusion are called ‘fusogen’.
PEG behavior is the generally widely used method pro protoplast fusion as it has particular advantages ended others. These are : (a) it results in a reproducible high-frequency of heterokaryon formation., (b) The PEG fusion is non point and therefore can be used pro a large range of plants., (c) It has low toxicity to the cell and (d) The formation of binucleate heterokaryons is low.

MECHANISM OF FUSION
The fusion of protoplasts takes place in three phases- agglutination, plasma crust fusion and formation of heterokaryons. When the two protoplasts occur in close friend with all other, they adhere to all other. This agglutination can be induced by PEG, distinguished pH and distinguished Ca2+. The protoplast membranes make combined by contained sites by the top of hold. This leads to the formation of cytoplasmic bridges linking protoplasts. High pH and distinguished Ca2+ ions neutralize the go up charges on the protoplasts which allows closer friend and crust fusion linking agglutinated protoplasts. The combined protoplasts be converted into around as a upshot of cytoplasmic bridges which leads to the formation of round homokaryon or heterokaryon.

SELECTION OF HYBRID CELLS
The methods used pro the selection of hybrid cells are biochemical, visual and cytometric methods using fluorescent dyes. The biochemical methods pro selection of hybrid cells are based on the aid of biochemical compounds in the standard. The drug sensitivity method is helpful pro the selection hybrids of two plants species, if lone of them is insightful to a drug. Another method, auxotrophic mutant selection method involves the auxotrophs which are mutants with the intention of cannot grow on a smallest standard. Therefore point compounds are added in the standard. The selection of auxotropic mutants is doable single if the hybrid cells can grow on a smallest standard. The visual method involves the identification of heterokaryons under the light microscope. Inside approximately of the somatic hybridizations, the chloroplast deficient protoplast of lone sow species is combined with the conservational protoplast of a further sow species. The heterokaryons obtained are larger and conservational in colour while the parental protoplasts are either small or colourless. The cytometric method uses tide cytometry and flourescent-activated cell taxonomy techniques pro the analysis of sow protoplasts.

APPLICATIONS OF SOMATIC HYBRIDIZATION

A)            Creation of hybrids with disease resistance –
Many disease resistance genes (e.G. Tobacco mosaic virus, potato virus X, bash rot disease) may possibly be successfully transferred from lone species to a further. E.G resistance has been introduced in tomato hostile to diseases such as TMV, blemished wane virus and insect pests.
B)            Environmental tolerance –
 using somatic hybridization the genes conferring tolerance pro cold, coolness and salt were introduced in e.G. In tomato.
C)            Cytoplasmic male unproductiveness –
 using cybridization method, it was doable to conveying cytoplasmic male unproductiveness.
D)            Quality font –
somatic hybrids with selective characteristics be inflicted with been urban e.G. The production of distinguished nicotine content.

CHROMOSOME NUMBER IN SOMATIC HYBRIDS
The DNA digit in the somatic hybrids is commonly more than the whole digit of both of the parental protoplasts. If the DNA digit in the hybrid is the sum of the chromosomes of the two parental protoplasts, the hybrid is understood to be symmetric hybrid. Asymmetric hybrids be inflicted with abnormal or large variations in the DNA digit than the exact whole of two species.

Inside 1972, Carlson and his friends produced the initially inter-specific somatic hybrid linking Nicotiana glauca and N. Langsdorffii. Inside 1978, Melchers and his co-workers urban the initially inter-genetic somatic hybrids linking Solanum tuberosum (potato) and Lycopersicon esculentum (tomato). The hybrids are renowned as ‘Pomatoes or Topatoes’.

LIMITATIONS OF SOMATIC HYBRIDIZATION
A)         Somatic hybridization does not permanently yield plants with the intention of produce fertile and visible seeds.
B)         There is genetic instability associated with protoplast culture.
C)         There are limitations in the selection methods of hybrids, as many of them are not efficient.
D)         Somatic hybridization linking two diploids results in the formation of an amphidiploid which is not favourable therefore haploid protoplasts are recommended in somatic hybridization.
E)         It is not particular with the intention of a point character will make articulated in somatic hybridization.
F)         Regenerated plants obtained from somatic hybridization are often wavering due to somaclonal variations, chromosomal abolition, organelle segregation and that.
G)         Protoplast fusion linking uncommon species/genus is straightforward, but the production of viable somatic hybrids is not permanently doable.
CYBRIDS
The cytoplasmic hybrids everywhere the heart is derived from single lone mother and the cytoplasm is derived from both the parents are referred to as cybrids. The process of formation of cybrids is called cybridization. During the process of cybridization and heterokaryon formation, the nuclei are stimulated to segregate so with the intention of lone protoplast contributes to the cytoplasm while the other contributes heart lonely. The irradiation with gamma emission and X-rays and aid of metabolic inhibitors makes the protoplasts motionless and non-dividing. Some of the genetic traits in particular plants are cytoplasmically controlled. This includes particular types of male unproductiveness, resistance to particular antibiotics and herbicides. Therefore cybrids are valuable pro the conveying of cytoplasmic male unproductiveness (CMS), antibiotic and herbicide resistance in agriculturally helpful plants. Cybrids of Brassica raphanus with the intention of contain heart of B. Napus, chloroplasts of atrazinc strong B. Capestris and male unproductiveness from Raphanus sativas be inflicted with been urban.


IN VITRO PLANT GERMPLASM CONSERVATION

Germplasm refers to the sum whole of all the genes bestow in a crop and its correlated species.
The conservation of germplasm involves the maintenance of the genetic diversity of a fastidious sow or genetic have a supply of pro it’s aid by one calculate in prospect. It is valuable to conserve the scarce plants or moreover approximately of the valuable genetic traits bestow in the existing and primitive plants will be lost. A comprehensive organization- International Board of Plant Genetic Resources (IBPGR) has been established pro germplasm conservation and provides de rigueur support pro collection, conservation and employment of sow geneic assets through made known the planet. The germplasm is preserved by the following two ways:
(a)           In-situ conservation-
                             The germplasm is preserved in natural background by establishing biosphere capital such as inhabitant parks, sanctuaries. This is used in the maintenance of ground plants in a virtually natural surroundings along with several wild types.
(b)           Ex-situ conservation-
                              This method is used pro the maintenance of germplasm obtained from cultivated and wild sow equipment. The genetic material in the form of seeds or in vitro cultures are preserved and stored as gene banks pro long stretch aid.
Inside vivo gene banks be inflicted with been made to preserve the genetic assets by square methods e.G. Seeds, vegetative propagules, and that. Inside vitro gene banks be inflicted with been made to preserve the genetic assets by non - square methods such as cell and tissue culture methods. This will ensure the availability of valuable germplasm to breeder to develop extra and improved varieties.

The methods involved in the in vitro conservation of germplasm are:

(a)        Cryopreservation- inside cryopreservation (Greek-krayos-frost), the cells are preserved in the frozen state. The germplasm is stored by a very low warmth using solid carbon dioxide (at -790C), using low warmth deep freezers (at -800C), using vapour nitrogen (at- 1500C) and liquid nitrogen (at-1960C). The cells stay in completely motionless state and hence can be preserved pro long periods. Any tissue from a sow can be used pro cryopreservation e.G. Meristems, embryos, endosperms, ovules, seeds, educated sow cells, protoplasts, calluses. Certain compounds like- DMSO (dimethyl sulfoxide), glycerol, ethylene, propylene, sucrose, mannose, glucose, praline, acetamide and that are added all through the cryopreservation. These are called cryoprotectants and prevent the destruction caused to cells (by freezing or thawing) by sinking the freezing top and super cooling top of fill up.

(b)        Cold Storage- Cold storage space is a gradual growth germplasm conservation method and conserves the germplasm by a low and non-freezing warmth (1-90C). The growth of the sow material is slowed down in cold storage space in contrast to complete obstruction in cryopreservation and hence prevents cryogenic injuries. Lengthy stretch cold storage space is unadorned, cost effectual and yields germplasm with skilled survival rate. Virus emancipated strawberry plants may possibly be preserved by 100C pro in this area 6 years. Several grape plants be inflicted with been stored pro ended 15 years by using a cold storage space by warmth around 90C and transferring them in the fresh standard each time.

(c)        Low pressure and low oxygen storage- inside low- pressure storage space, the atmospheric pressure surrounding the sow material is cut-rate and in the low oxygen storage space, the oxygen concentration is cut-rate. The lowered partial pressure reduces the in vitro growth of plants. Inside the low-oxygen storage space, the oxygen concentration is cut-rate and the partial pressure of oxygen not more than 50 mmHg reduces sow tissue growth. Due to the cut-rate availability of O2, and cut-rate production of CO2, the photosynthetic endeavor is cut-rate which inhibits the sow tissue growth and dimension. This method has furthermore helped in increasing the shelf life of many fruits, vegetables and flowers.
The germplasm conservation through the square methods has several limitations such as short-lived seeds, seed dormancy, seed-borne diseases, and distinguished inputs of cost and labour. The techniques of cryo-preservation (freezing cells and tissues by -1960c) and using cold storages help us to overcome these problems...

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