Plant biotechnologyChemodemes: Chemically distinct population within a species and have similar phenotypes, but different genotypes and as such are identical in external appearance but differ in chemical constituents. Before the existence of chemical race certain fundamental observance are necessary. The observation of chemodemes can be confirmed by growing different plant of species in identical conditions preferably from seeds and for many generations.by this way it shows variations either in type or content of certain constituents, like the secondary metabolitesof medicinal importance. The chemical characteristics of chemodemes are hereditary. The discovery of chemodemes has enabled to select high yielding chemical strains or to eliminate chemical strains that contain toxic metabolites. The chemodemes are induced by breeding in some species so as to manipulate active constituent to enhance therapeutic efficacy. For eg Normal rape seed oil contains acyl-glycerol (20-40%eracic oil) which makes the oil unsuitable for edible purpose but varieties are now grown presently that contain no eracic oil. The oil is very useful for manufacture of lubricants and artificial fibres. Sunflower oil constitutes an important oil seed crop and its genetic variability which facilitates the breeding of variety with widely differing oil constituent proportion. High oleic varieties are used for human consumption and high linoleic comp varieties are used for industrial protein and lubricants. The above examples examples improve plants with a short life span breeding by classical method is relatively rapid procedure however this is not so with plants such as coconut ,palm , olive .in this cases modern techniques involving gene transfer is advancement for introduction of new or modified oil characteristics. Polyploidy deviation from normal genome. Hyperploidy Hypoploidy increase in number of chromosomes.vigorous growth. decrease in number of chromosomes.stunted growth. Mutation sudden changes in heritable characters. Once radiation is started amount is monitored by radiation monitor and one of the monitor is kept at the centre and portable monitor near plants. deoxy adenosine and ethoxy caffeine. Some nitroso compounds inorganic cyanides . which inturn combine with DNA forming abnormal dna. 3) Certain antibiotics like streptomycin and actinomycin bring out degradation and denaturation of DNA. They generally bring about mutational changes by interfering with dna synthesis and also sometimes by degradation & denaturation of dna. 8-ethoxy caffeine.Large number of plants can be employed and amount of radiation received is different and therefore we get different mutations from which desired character can be selected. We can select the radiation that are more beneficial.R1 plants are selected with desired characteristics and these seeds are grown further till R5. Different groups of compounds are 1) DNA PRECURSORS adenine . uridine . bring about mutation. The plants obtained are called R1 generation. Adv of ¥ radiations . amt of changes produced is less and also individual plants must be handled. Nitroso compounds combine with heavy metals . Using chemicals : The most powerful mutagenic chemicals discovered so far are the mustard gas and it s related compounds like ethylene oxide .cu. 4) Certain alkylation agents like nitrogen mustard produce abnormal dna.after irradiation these plants are planted in specific fields surrounded by control material to avoid inter crossing with non irradiated plants. 2) DNA analogues it includes thymidine . Seeds are irradiated sown similarly. They induce gene and chromosomal mutation. By that time one can know whether the characters are heritable and constituents.size of chromosomes Plants are grown in concentric circles and divided into different selections in each selection different groups of plants can be grown.Chromosomal abbreviations shape . generally these produce abnormal DNA. They are supported to interfere with enzyme synthesis. with solutions they bring about changes in chromosomes. LIMITATIONS:--Unpredictable May not be beneficial . The seeds can be sowed in these chemical or seedling itself can be treated in young condition . R6 generation.fe. piperita species produces the infection resistant varieties. Rose produced different sized flowers and oil contents. Widely used in plant improvement both for higher yield and to get resistant plants. the non-dividing and inactive cells undergo changes to achieve meristematic state. Plant tissue culture: Plant tissue culture is a collective term for the technique of growing plant cells . tissues. . land and expenses needed in this are considerably less. organs or even plantelets on a nutrient medium in a suitable container under sterile and controlled conditions.differentiation are basic components of capacity plant cells described as totipotency . The component of cells of callus have the ability to form a whole plant and this phenomenon is called re-differentiation.must be grown for 5-6 generations. When an explants from differentiated cells / tisssues is used for culture on a nutrient medium. Menthol cobalt -60 M. The technique of plant tissue culture has developed around the concept of totipotency. Time.Once canges takes place . a property . When an explants from differentiated cells/tissues is used for culture on a nutrient medium. The phenomenon of reversion of mature cells to meristemetic state leading to the formation of callus is called de-differentiation. The component of cells of callus have ability to form a whole plant and this phenomenon is called redifferentiation. Person working with radiation has to be careful. on nature for raw material . The phenomenon of reversion of mature cells to meristematic state leading to the formation of callus is called dedifferentation. the non dividing and inactive cells undergo changes to achieve meristematic state. These 2 phenomenon of re and de. ADVANTAGES:__ We can get different varities of inexhaustible variations & mutation breeding can make plant breeders free from complete dependence . labour. Work on medicinal plants Poppy seeds treated with cobalt-60has produced different varieties some with very high amount of morphine. Can be carried out at those places only where facilities are available. 1)whole fruit 2) isolated tissue Pollen mother culture Isolated ovule & embryo culture 4)protoplast culture 5) haploid culture Shoot culture. Callus culture require sub culturing at about 1 month intervals by maintaining them at 25°c +-2°. The unique feature of callus is its ability to develop normal root and shoot . Nutrient medium is supplied with auxins to induce cell division.& de differentiation are basic component of capacity of plant cells described as totipotency. Leaf culture Essentially all organs can be used as explants . Tissue culture is a clean and rapid way for genetic engineers to grow material for identify.Root culture.molecular biology. .These two phenomenon of re. Reproductive organ culture Flower organ culture Isolated ovary culture Seed culture Fruit culture.in other words a differentiated plant cells retains its capacity to give rise to a whole plant but an animal cell loses this capacity of regeneration after de-deffferentiation.hybrid development and food science.however degree of success with different tissue can vary and calluses with different morphologies are frequently obtained. forming a plant .it plays a vital role in field of botany. And provision of light at prescribed intensityand photo period as per requirement of plant species. After cell diff will occur in this stage many metabolic pathways start leading to formation of secondary metabolites.manipulate. a property found only in plant cells and not in animals.or transfer genes from one plant to another. Callus is formed through 3 stages of development 1) Induction 2) Cell division 3) Cell differentiation During induction stage metabolic rate of the cell will be increased due to this the cell accumulates organic contents and finally divides into a no of cells. Types 1) Callus culture 2) Meristem culture 3) Organ culture Vegetative organ culture . special techniques such as immobilisation. drought resistant . CALLUS CULTURE INITIATED FROM ROOTS of tuta graveolens produce root specific essential components whereas callus culture from stem produce stem specific components. however these are exceptions persist when tissues are cultured. Nothapodytes foetida & ophiorrhiza pumila among other species has been shown to accumulate in undifferentiated culture in very low or even in undectable amounts compared to root cultures in which production levels were comparable to intact plants. The yield of sec metabolites are very poor in some of the species. production of seeds. temp.hypericum perforatum. Many do serve important function in defence or protection in plants and thus considered biologically active. pH. The anticancer compounds camptothecin from camptotheca accuminta. Are often present in extremely low amounts in the plants. salt resistant and insect resistant plants. As a result tissue specific patterns of differentiate and biosynthetic do not persist when tissues are culyured . light. especially in the case of slow growing species. However there are exception for persistant tissue specific production of secondary metabolites. Culture conditions like inoculums size.in a way.or re differentiation. The significance engineering challanges is to find a means by which we can produce desired natural products .i. Un-differentiated cultures often accumulate sec metabolites to a lesser extent and sometimes not at all. Plant regeneration using callus is of great significance in species like bergenia crassifolia.due to enzymatic activity.micro propagation of aromatic and medical plants. The potential usefulness of callus in propagation lies in the possibility of greatly extending the base for adventitious shoots or embryo production.e both sustainable and financially feasible.e seletion. . agitation. Many of the industrial medicinal plants are being micro propagated through callus culture.it can be improved by strain improvement i.the scarcity can make natural harvestation impractical for bulk production. screening & med variation. Most secondary met. prod of phytopharmaceuticals. However the capacity of callus culture often differ substantially from that of fully differentiation tissue both qualitatively & quantatively. food colours and flavours etc from plant cell culture. 2) Horticulture. This can be achieved by lowering the concentration of auxins & increasing the conc of cytokines than those required for callus induction and maintainance. permeabilisation etc. Production of these products in microbial / fungal host by transferring biosynthetic pathway is possible but is limited due to the complexity and laws of complete knowledge regarding many of these pathways. Applications of plant tissue cultures 1) In agriculture production of high yielding . When natural supply is limited due to low yield adn slow growth rates invitro cultures provides an alternative method.products of metabolic pathways. Most plants can be cultivated by di. Secondary metabolites which are large varied & mysterious group of molecules while some are mostly extraneous by.Callus culture in some medicinal plant produces the selected compounds with a yield equal to or higher than that of parent plant. genetic trans. antigens. Plants cell cultures are becoming of increased importance as an exceptance material for various biochemical and physiological studies.its useful in enzyme purification. v and dead cells. ni. it is different to obtain a suspension culture consisting only free cells as the daughter cells adhere together soon after division to form aggregate. glycine.mg. But nowdays they have been successful grown on purely synthetic media. b.e. na+.o. zn+2. Till recent time . jellying and solidifying agents. Names of media::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Gautheratemedia(1942) Nitsch media (1951) Whites media Gaunbergs media N6 meddia E1 media (gamberg) Macro nutrients ca+2. Cell suspension culture actively grows in a agitated and aeration. nh4+. The cells divides in an incubation medium leading to an increase in material. The homogenate is added to liquid medium on shakers and supernatant . small cell group and large cell aggregates dispersed in a liquid medium. po32-. inositol. folic acid. The valuable preditions from transgenic . Plant tissue culture media :] Inorganic nutrients (macro & micro ) Carbon sources. computer. These workers showed the feasibility by growing plant cells in a similar way as that of m. yeast and malt extract. Nickel in 1959 studied the production of mass amounts of cell suspension by using submerged and also described it s potential use for the study of biosynthesis of sec met. etc Micronutrients fe2+. Characteristic of suspension culture: All cell suspension cultures consists of single cells . These cultures are propagated at regular intervals showing similar patterns of growth and yield . monoclonal antibodies. Its also useful for various biochemical and physiological studies. Cell suspension culture:The plant cells in agitated liquid meddis has been reported by caplin and stward in 1949.. pyridoxine. cl-1.biotin.biotransformation .vitamins. vehicles. cell susp of higher plants were grown in media containing coconut milk and water. EDTA.thiamine. nickel (1956). growth regulators.g antibiotics. no3-. k+. buffers. For the suspension culture cell homogenate are made by subjecting sterile seedlings or young embryos to a hand operated glass homoginiser consists of intact li ing cells debris . Suspension culture can be subcultured by pippeting out and by adding to fresh medium. antibiotic. kio3. edible vaccines. The medium is conditioned dddue to release of conditioned metabolites. The tubes are mounted at mid point held by clips along the periphery of the circular disk fine to horizontal central drive shaft. Sucrose is most common sugar used for carbon source Auxin/cytokinin are suitably modified and also vitamins. It is rotated at low speed of 1-2 rpm. Various mods of shaker culture and continuous culture synthesis for large batches have been used in practice. Cells suspension cultures are maintained by sequential sub culturing during early starting phase and at the time when cell aggregation reaches maximum level. In all these methods the essential nutrient in the medium diffuses through the barrier and bring about the division and growth of single cells on the uppersurface. shakers have been imparted to impart circular motion. The apparatus is placed in a temp.layers containing cell and aggregates is pipettted out into the medium and & incubated in diffused light. Controlled room. For the preparation of cell suspension. Agitation is necessary for gaseous exchange & to maintain cells in dispersed condition. refilling of fresh batches. . The ex planted tissues are introduced into these tubes on slow rotation and alternatively immersed and exposed to air.single cells could be grown in the medium for establishing single cell clone . 1)paper raftners technique 2)plating method 3) growth of isolated cells in microchambers. The shakers are fitted with variable aped is adjusted to optimum growth rate. in these cases 3 methods are generally used. Single cell clones :. These are also called as the nipple flasks or tumble tube culture vessels. TECHNIQUEs The selection of culture media for cell suspension culture is ddone on basis of optimal growth for recording growth rate. Culture apparatus: A culture apparatus by steward consists of flask of 250 -1000ml capacity fitted with 68 small tubes at the outer end and open at the base.each platform bears a dozenor more cylindrical culture bottles of varying capacities which can be held in position by clips or rubber pads. The tubes have side necks to facilitates introduction of inocolum. Recent methods of these culture apparatus for continuous culture system which makes use of inverted cylindrical culture bottles for intiment renewal of medium . The culture med for callus culture may not be suitable for suspension cultures as the nutritional requirements may vary. harvesting of cells.. Total cell yield at highest cell separation. plant growth regulators etc. friability and growth rate on different media.so media becomes highly viscous. it has shown the variation in organo genetic potential. Maximors double coverslip method and the modified micro chambers devised are used to observe the growth of cell . Single cell clone can be subcultured but it has been observed that single cell loses its totipotency when it is continuously subcultured. Applications: . Large scale culture of cells:--------------Several studies have been carried out in order to obtain accumulated products of metabolism using large scale cultures of plants cells for industrial scale. The cells in isolation grown in microchambers undergo cell division under appropriate nutritional and hormonal conditions and the calli that develops could regenerate new plant s. certain essential acids like glutamine and serine . total cell proteins. during low growth in batch cultures cells excretes polysaccharides . Growth of cells in microchambers : Single cells are grown efficiently by hanging drops in microchambers. Cells forming aggregates increases the biomass . cells aggregates embrioids becomes easy. Precise manipulation of ingredients is possible and handling off cells . no. Cell suspension cultures can be maintained at steady growth rates as homogenous system using the parameters like cell weight. air liff system and bubble column are also in use but not a single is applicable for plant cell culture.plant cells are quiet large in then microbial cells . Enhanced planting efficiency has been observed from from the use of minimal media supplements by selected metabolites. Conditioning of the nuclei is needed in order to trigger initial cell division . gases like co2. metabolic stability etc. Primarily single cell clones have also shown difference with their secondary derivatives with different conc of sugars or ar a carbon source . Single cell clone differ from another in texture .plant cells being aerobic need oxygen supply at low level. & growth regulators like cytokinines are essential factors concerned with conditioning of the media. A liquid culture sys under agitation permits the metabolic pathways to be altered to suit particulars objectives. most large scale cultures are run as batch cultures but conventional stirred tank reactors. Auspension culture offer advantages over stn culture as the cells are uniformly placed in the medium containing nutrients. Cell culture system form foams and surface adhesions but it doesn t affect cultures .In the microchambers method division of single cell is initiated as a consequence of enrichment of micromedium. Plant cells are grown as suspension culture in a wide range and bioreactors of 22000litres. An appreciation of basic technique of cell suspension culture is essential to any assement of their realised and potential application. Selection of high yielding cells . Recently it has been shown that r. Technical barriers which includes shear sensit ivity of cell oxygen aggregation. Napthoquinones ans anthraquinones sometimes accumulate in culture cells at levels for exceeding the amount found in intact plant.e increased producers of sec metabolites are achieved by visual. transfer.5% of dry mass. ait lift loop reactor. These cultures were maintained in suspension for several years without loosing their capacity for syn and accumulating rosmarinic acid.cell wall growth by addition of cells.acid increased upto 15%. These selected cell lines are mass cultivated to achieve incred level prodn. Problems of cell culture are resolved by newly developed tech surface immobilisation of plant cells which protects cells from high shear stresses optimising production. rotating drum reactor are used for for large scale production of secondary metabolites fro m plant cell culture.. chemical. There are two basic problem in such cultivation : Biological barriers that includes low growth rate & genetic instability. Eg:. Can be induced in withocapermucan erythrorirhizon and orthosiphon aristatus by elecitors such as yeast and methyl jasmonate from nearly 0-1. air liff reactor. Following a critical evaluation of present state of tech in the field where the suspension cultures have contributed the basic of physiology of plant cells in cultures. screening cells to produce sec. Esters adn amides of cinnamic acid form a major widespread group of phenyl propanoid metabolites in plants which accumulates spontaneously often at high level in cell suspension cultures. The syn of rosmarinic acid can be stimulated by increasing sucrose level to 5-8% in which content of r. Successful selection of clones i. Metabolites is the technique to develop high yielding cell lines. radio immunoassay tech. It opens up new approaches to the study of secondary products biosynthesis in plant cells culture. stirred tank reactor. growing them on suitable medium . .acid and enzyme in biosy. Different approaches are used to produce secondary m etabolites from plants tissues 1)cell cloning 2)mass cultivation 3)immobilised plant cell culture 4)fungal elicitus induced production 5)biotransformation using plants cells.cell Drought tube .rosmarinic acid from coleus blumei. ----immobilisation allows a continuous process which increases volumetric productivity and allows down stream processing compared to extraction from tissue. a definite advantage with slow blowing plant cells.separation f cells from medium the immmobilasition seperates cells from medium and desired product is extracellular which will simplify downstream processing compared to extraction from tissues. ---decoupling of growth and product formation immobilisation is compatible with non growth associated product formation reduces problems such as aggregate growth and foaming. 1)biotransformation 2)synthesis from precursors Denovo synthesis Advantages ---Retention of bio mass enables its continuous reatialisation as a product system . ---the immobilisation cells allow the use of higher bio mass level compareddd to cell suspension culture because of the limit of mass transfer and setteling Eg-bead densities of 110gm dry weight per litre have been obtained with calcium alginate entrapped cells well as 30 gmdry wt per litre in suspension culture the high density allows a reduction in contact tim e in black bed catalyst leading to an increased volumetric productivity.Immobilisation: It is the newest culture technology in plant cells and it has been defined as a technique which confines to catalytically active enzyme or to a cell within a reactor system and prevents its entry into the mobile phase which carries the product and the substrate. . Immobilisation has been subjected as strategy to enhance the over all productivity of secondary metabolites in plant cell cultu re. And the entrap catharanthus roseus and Decius carota cells in alginate weeds. The first successful immobilisation of plant cell was reported in 1979 by brodelius.papavera somnifera have remained stable and active form upto 6 months. Immobilisation of plant cells has been used for wide range of reactors which can be divided in three groups. --. Eg.. 4)may reduce biosynthetic capacity 5)product must be released from cells into the media 6) release of single cells from cell aggregate may make processing of product difficult. 7) the micro environment favouring optimal production can be unfavourable for secondary met and can cause its degradation or metabolisation. ---Bette stability granules can be used upto 60 days - Disadvantages: 1)the catalysing capacity of immobilised cells is just half of suspended cells 2)secretion of secondary metabolites require cellular transport or artificially altered membrane 3)the efficiency of production process depends on the rate of release of products rather than actual rate of biosynthesis. NEED FOR IMMOBILISATION: .The immobilisation reduces some of the physical problems associated with cultivation of plant cells such as formation of aggregrates and susceptibility to mechanical cell damage or shear stress are problems which do not affect immobilised system as compared to cell culture.