Plant Tissue Cultur1

Plant tissue culture

Plant tissue culture

Plant tissue culture is a practice used to propagate plants under sterile conditions, often to produce clones of a plant. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:

The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits.

To quickly produce mature plants.

The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.

The regeneration of whole plants from plant cells that have been genetically modified.

The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens.

The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and nepenthes.

Plant tissue culture, the growth of plant cells outside an intact plant, is a technique essential in many areas of the plant sciences. Cultures of individual or groups of plant cells, and whole organs, contribute to understanding both fundamental and applied science.

It relies on maintaining plant cells in aseptic conditions on a suitable nutrient medium. The culture can be sustained as a mass of undifferentiated cells for an extended period of time, or regenerated into whole plants.Plant tissue culture is the aseptic (free from microorganism) culture of any plant part in vitro. Tissue culture is utilized in the field of Biotechnology. Micro-propagation is the rapid vegetative propagation of plants via tissue culture techniques. Micro-propagation permits the manipulation of physical and chemical conditions in the production of large numbers of high quality plant material within a short period of time. Plant tissue cultures can be initiated from almost any part of a plant. The physiological state of the plant does have an influence on its response to attempts to initiate tissue culture. The parent plant must be healthy and free from obvious signs of disease or decay. The source, termed explant, may be dictated by the reason for carrying out the tissue culture. Younger tissue contains a higher proportion of actively dividing cells and is more responsive to a callus initiation programme.

Tissue culture is a technique of growing plant cells by culturing explant aseptically on a suitable nutrient medium.Plant cell culture is viewed as a potential means of producing useful plant products such that conventional agriculture, with all its attendant problems and variables, can be circumvented. These problems include: environmental factors (drought, floods, etc.), disease, political and labour instabilities in the producing countries (often Third World countries), uncontrollable variations in the crop quality, inability of authorities to prevent crop adulteration, losses in storage and handling.In tissue culture cells, tissues, and organs of a plant are separated. These separated cells are grown especially in containers with a nutrient media under controlled conditions of temperature and light. The cultured plant requires a source of energy from sugar, salts, a few vitamins, amino acids, etc. that are provided in the nutrient media. From these cultured parts, an embryo or a shoot bud may develop, which then grows into a whole new plantlet. Similarly, portions of organs or tissues can be cultured in a culture media. Generally, these give rise to an unorganized mass of cells called callus (soft tissue that forms over a cut surface). In tissue culture cells, tissues, and organs of a plant are separated. These separated cells are grown especially in containers with a nutrient media under controlled conditions of temperature and light. The cultured plant requires a source of energy from sugar, salts, a few vitamins, amino acids, etc. that are provided in the nutrient media. From these cultured parts, an embryo or a shoot bud may develop, which then grows into a whole new plantlet. Similarly, portions of organs or tissues can be cultured in a culture media. Generally, these give rise to an unorganized mass of cells called callus (soft tissue that forms over a cut surface).

Pieces of plant tissue will slowly divide and grow into a colorless mass of cells if they are kept in special conditions. These are:

initiated from the most appropriate plant tissue for the particular plant variety

presence of a high concentration of auxin and cytokinin growth regulators in the growth media

a growth medium containing organic and inorganic compounds to sustain the cells

aseptic conditions during culture to exclude competition from microorganisms

Tissue culture ProcessSpecies are selected for tissue culture on the following basis. Species that have regeneration problems, specially because of poor seed set or germination (as in Anogeissus and bamboo). In these cases, seeds collected from superior trees are used for initiating cultures.

Species that vary markedly in their desirable traits, i.e. Eucalyptus. The selected trees are marked from the variant population for the desirable trait such as disease resistance, straight bole, higher productivity, etc. in consultation with officials from state forest department or growers.

Species where plants of any one particular sex is of commercial importance, for example female plants of papaya and male plants of asparagusApplication of tissue culture Micropropagation

Rapid vegetative multiplication of valuable plant material for agriculture, horticulture, and forestry.Production of disease-free plants

When the apex of shoot is used for multiplication by tissue culture, we get disease free plants because the shoot apical meristem, a group of dividing cells at the tip of a stem or root, is free from pathogens. Plant breeding

Tissue culture has also been successfully used in plant breeding programmes. Production of disease-

and pest-resistant plants, Plants grown from tissue culture usually pass trough callus phase and show many variations. These show some agronomic characteristics like tolerance to pests, diseases, etc.Cloning

Genetically identical plants derived from an individual are called clones. Processes that produce clones can be put under the term cloning. This includes all the methods of vegetative propagation such as cutting, layering, and grafting. Propagation by tissue culture also helps in producing clones. Using the shoot tip, it is possible to obtain a large number of plantlets. This technique is used extensively in the commercial field for micro propagation of ornamental plants like chrysanthemum, gladiolus, etc. and also crops such as sugar cane, tapioca, and potato.

Micro propagation is widely used in forestry and in floriculture. Micro propagation can also be used to conserve rare or endangered plant species.

A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance.

Large-scale growth of plant cells in liquid culture inside bioreactors as a source of secondary products, like recombinant proteins used as biopharmaceuticals.

To cross distantly related species by protoplast fusion and regeneration of the novel hybrid. HistoryHistory of tissue culture

In 1965, French botanist George Morel was attempting to obtain a virus-free orchid plant when he discovered that a millimeter-long shoot could be developed into complete plantlets by micro propagation. This was the beginning of tissue culture. Thereafter, in the 1970s developed countries began commercial exploitation of this technology.

in the 1980s. It was earlier used to develop ornamental plants and flowering plants for export. With tree species, the technique of tissue culture remained confined for many years to the laboratory stage and had generally invited only academic interest. But in most developing countries, the shortage of biomass and the ever-increasing energy requirements created the need to explore possibilities of mass propagation of trees by tissue culture.

As seen in many reviews studies on the production of plant metabolites by callus and cell suspension cultures have been carried out on an increasing scale since the end of the 1950's. The large scale cultivation of tobacco and a variety of vegetable cells was examined from the late 1950's to early 1960's by Tulecke and Nickell at Pfizer Inc., Mandels et at. at the Natick Laboratories in the U.S. Army , Street et al. at the University of Leicester and Martin et al. at the National Research Council of Canada Their results stimulated more recent studies on the industrial application of plant cell culture in many countries.

The knowledge of plant tissue culture started from the days of Hoberlonft (1972), R.J. Gautheret and P.Nobcourt (1939) carried in callus culture on the combat tissue of MonocotsIn 1982, the 5th International Congress of Plant Tissue and Cell Cultures was held in Japan and about 70 out of 372 papers presented there related to production of secondary metabolites in cultured cells and several papers seemed to be commercially promising such as production of shikonin by Fujita et al. of Mitsui Petrochemical and that of several antitumor compounds by Misawa et al. of Kyowa Hakko.Sanguinarine production was also studied by Kurz et al. (82) in Canada since this alkaloid has a market in the denatal care field.

The recent biotechnology boom has triggered increase interest in plant cell cultures, for example, a number of firms and academic institutions in the U.S., Japan, Canada, and Europe have been investigating intensively the production of a very promising anti-tumor compound, taxol, using this technology. given the following.

-F.Skoog and C.O.Metter (1975) formulated the idea that the proportion of auxin and cytokinin determines the roots and shoots from the callus. Then only successful plant regeneration have been achieved from the callus tissue.

M.S. tissue culture medium was first by Murashige and Skoog in 1962.

-E.C. cookling (1960) isolated the plants protoplast from the cultured cells by using enzymes like isozymes.

S.G.Guba and S.C.Maheshwari (1966) culture haploid plants from pollen grain and anther.

J.P. Nitisch (1974) brought about the double of chromosome number is haploid tissue obtained from the pollen culture.

G.Melcher (1928) established somatic hybrid by using protoplast fusion technique.

K.A. Banton, W.J.Brill., J.h.Dodds and Bengochea (1983) experienced with the transfer of

genus into the prptoplast by using plasmids as vectors.

Designing a strategy to culture cells from a plant for the first time can still seem like a matter of trial and error, and luck. However, the commercial production of valuable horticulture crops by micro propagation, which relies on tissue culture, shows that it exists in the routine, as well as experimental, world.

In the School of Biological Sciences at the University of Liverpool, we have experience over many years with the techniques and applications of plant cell culture.

Plant cells can be grown in isolation from intact plants in tissue culture systems. The cells have the characteristics of callus cells, rather than other plant cell types. These are the cells that appear on cut surfaces when a plant is wounded and which gradually cover and seal the damaged area.

Aim

Aim

Horticulture and floriculture presents huge opportunity for commercial production of fruits, vegetables , ornamental plants. Cultivation of floriculture has been identified as key activity that can generate significant revenues and growth opportunity for farmers.

The benefit s of plant tissue culture are extensive in the agriculture world. Micro propagation is favourable to traditional crop breeding method in many respects, the first being that it allows for the production of huge number of plant in a very short time.

The gerbera is very valuable ornamental species grown as a potted and for the cut flowers. The breeding potential for new flower colures and patterns such as resistance to biotic or a biotic stresses is also limited.

Gerbera is very popular and widely used as a decorative garden plant or as cut flowers. The domesticated cultivars are mostly a result of a cross between Gerbera jamesonii and another South African species Gerbera viridifolia. The cross is known as Gerbera hybrida. Thousands of cultivars exist. They vary greatly in shape and size. Colors include white, yellow, orange, red, and pink. The center of the flower is sometimes black. Often the same flower can have petals of several different colors.Gerbera is also important commercially. It is the fifth most used cut flower in the world (after rose, carnation, chrysanthemum, and tulip[citation needed]. It is also used as a model organism in studying flower formation. Gerbera contains naturally occurring coumarone derivativesPlant preparation .

Gerbera is one of the most important cut flower, successfully grown under different conditions in several parts of the world and meeting the requirements of various markets. This success is primarily due to its wide range of colors and shapes. Gerbera flowers comes in vibrant colours adding beauty to your garden. It has around 40 species spreading from Africa across to Madagascar into tropical Asia and South America. Gerbera are plants with a height up to 18 to 24 inch and 4 to 10 inch diameter flowers.

Cultured plant cells often produce reduced quantities and different profiles of secondary metabolites when compared with the intact plant and these quantitative and qualitative features may change with time. The poor product expression is often attributed to a lack of differentiation in cultures . On the other hand, there are cases of cultures that over-produce metabolites compared with the whole plantMaterials

And

Methods

Lab requirement and facilities

Laboratory :There are many textbooks describing very sophisticated laboratory systems for plant cell cultures. However, it is not always necessary to design special laboratories for this technology, but general microbiology laboratories can be used, although aseptic conditions are a prerequisite for incubation of plant cells as well as microbial cultures.A standard tissue culture lab should provide facilities:

Washing and storage of glass wares, plastic ware and other lab wares.

Preparation, sterilization and storage of nutrient medium

Aseptic manipulation of plant material.

Maintenance of culture under controlled condition of temperature, light, and if possible humidity.

Observation of culture.Laboratory Space :The organization of a tissue culture laboratory depends mainly on the nature and the scale of Activity. In general space for the following is needed---- Washing, drying and storage of vessels.

Preparation , sterilization and storage of media.

Aseptic handling of explants and culture. maintenance of culture and observation of cultureMedium room :The washing area in the media room should be provided with the brushes of various sizes and shape, a washing machine, a large sink and running hot and cold tap water. It should also have steel or plastic buckers to soak the labour to be washed, oven or a hot air cabinet to dry the washed lab wares and a dust proof cupboard to store them.The growth room and transfer room should be adequately insulted to conserve energy. A tissue culture facility require large quantity of good quality water and provision for waste water disposal but for that disposal water and sewer facility are not available. Washing area : An area with large sinks and a draining area is necessary since the conventional method for cleaning laboratory glass ware involves acid soak followed by subsequent rinsing with dist. water. Glassware should be soaked in a 2% detergent cleaner for 16hour followed by washing with 60-70 C hot tap water and dist. water. The glassware is first air dried and then kept in an oven for two hours. At 160C excise 3-dry sterilization.a washing area (vessels and planting material are cleaned, plantlets may be weaned) a media preparation room ( preparation of media, storage and sterilization)a aseptic transfer area (initiation and sub-culturing of plantlets) an incubator or a culture room (provide plantlets in culture with temperature and light requirement)The following laboratory equipment are require for tissue culture are :

Laminar air flow cabinet : Laminar air flow cabinet is the most suitable, convenient and reliable instrument for septic work. It allow one to for a longer period which is not possible inside the inoculation chamber. Laminar air flow has a number of small blower motor to blow air which passes through a number of HEPA ( high efficiency particular air) filter. Such filter remove lager then 0.3 m.

This sterilized air blow through the cabinet at 1.8 km/hr. which is sufficient to keeps the enclosed working area aseptic.

Laminar flow (tissue culture work in sterilized conditions)Oven for dry sterilization : Although autoclaves can be used for dry sterilization, an oven is useful for sterilization of scalpels and glass-wares such as petri-dishes, pipets and others.

Equipment for sterilization by filtration : The medium containing carbon sources and growth regulators are simultaneously sterilized using a autoclave but sometimes aseptic filtration is favorable to avoid decomposition of unstable chemicals. The equipment is also commercially available.

Water distillation apparatus or pure water demineralizer : To prepare media, distilled water or deionized water is generally used although tap water can be used particularly in large-scale cultivation of plant cells in a large fermentor from an economical point of view.

Culture rooms and/or Cabinets : To cultivate the plant cells, culture rooms under different temperature or and/or cabinet-type incubators are essential facilities. Temperature and light intensity as well as a duration of lighting in the room and/or in the cabinet are controlled under the optimal conditions.

Autoclave : Culture vessels, (both empty and containing media) are generally sterilized by heating in an autoclave or a pressure cooker to 121C at 15 p.s.i.(pound per square inch) for 15 (20 25 ml medium) min. the time being longer for larger medium volume.

Plant tissue culture media are generally sterilized by autoclaving at 121 C and 1.05 kg/cm2 (15-20 psi). The time required for sterilization depends upon the volume of medium in the vessel.

Media

preparation

Culture media :

This is a very crucial step for the experiment to be successful. While making the media taking individual constituents, each ingredient is separately weighed and dissolved before putting them together. After making up volume by water, pH is adjusted and then medium is autoclaved. Preferably, following four stock solutions are prepared: Major salts (20X concentration)

Minor salts (200X concentration)

Iron (200X concentration)

Organic nutrients (200X concentration)The type and composition of culture media very strongly govern the growth and morphogenesis of plant tissues. The choice of tissue culture medium largely depends upon the species to be cultured. For e.g. some species are sensitive to high salts or have different requirements for PGRs. Some tissues show better response on solid medium while others prefer a liquid medium. Therefore, development of culture medium formulations is result of systematic trial and experimentation considering specific requirements of a particular culture system.

The composition of the cultural media is one of the most important factors in determining the growth and morphology of in vitro plants. Culture media used for the in vitro cultivation of plant cells are composed of three basic components:

essential elements, or mineral ions, supplied as a complex mixture of salts;

an organic supplement supplying vitamins and/or amino acids; and

a source of fixed carbon; usually supplied as the sugar sucrose.One of the most commonly used media for plant tissue cultures is that developed by Murashige and Skoog (MS) for tobacco tissue culture. The significant feature of the MS medium is its very high concentration of nitrate, potassium and ammonia. The B5 medium established by Gamborg et al. is also being used by many researchers.composition of culture media (M.S. media)

The composition of the cultural media is one of the most important factors in determining the growth and morphology of in vitro plants. Plants grown in vitro require similar nutrients to plants grown in the soil. The basic components of any cultural medium are: The composition of the cultural media is one of the most important factors in determining the growth and morphology of in vitro plants. Plants grown in vitro require similar nutrients to plants grown in the soil.To induce a callus from an explant and to cultivate the callus and cells in suspension, various kinds of media (inorganic salt media) have been designed. Agar or its substitutes is added into the media to prepare solid medium for callus induction.

One of the most commonly used media for plant tissue cultures is that developed by Murashige and Skoog (MS) for tobacco tissue culture (28). The significant feature of the MS medium is its very high concentration of nitrate, potassium and ammonia. The B5 medium established by Gamborg et al. (29) is also being used by many researchers. The levels of inorganic nutrients in the B5 medium are lower than in MS medium.The composition of the cultural media is one of the most important factors in determining the growth and morphology of in vitro plants. Plants grown in vitro require similar nutrients to plants grown in the soil. The basic components of any cultural medium are1. Macro-elements (or macronutrients),

2. Micro-elements (or micronutrients), and

3. an Iron source.

Essential elementConcentration in

stock solution (mg/l)Concentration

in medium (mg/l)

Macroelements

NH4NO3

KNO3

CaCl2.2H2O

MgSO4.7H2O

KH2PO433000

38000

8800

7400

34001 650

1 900

440

370

170

Microelements

KI

H3BO3

MnSO4.4H2O

ZnSO4.7H2O

Na2MoO4.2H2O

CuSO4.5H2O

CoCl2.6H2O166

1240

4460

1720

50

5

5

0.83

6.2

22.3

8.6

0.25

0.025

0.025

Iron source

FeSO4.7H2O

Na2EDTA.2H2O5560

7460

27.8

37.3

Organic supplement

Vtamines

Myoinositol

Nicotinic acid

Pyridoxine-HCl

Thiamine-HCl ,Glycine20000

100

100

100

400

100

0.5

0.5

0.5

2

Carbon sourced

SucroseAdded as solid

30 000

Composition culture media

Preparation of plant tissue culture media (M.S.Media)

Measure approximately 90% of the required volume of the

Deionised-distilled water in flask/container of double the size of the required volume.

Add the dehydrated medium into the water and the stir to

Dissolve the medium completely. Gentle heating of the solution may be required to bring powder into solution.

Add desired heat stable supplement to the medium solution.

Add additional deionized-distiled water to the medium solution to obtain the final required volume.

Set the desired PH with NaoH or HCL.

Dispense the medium into culture vessels.

Sterilize the medium by autoclaving at at (121c) for appropriate time period. Higher temp. may result in poor cell growth.

Add heat libile supplements after autoclaving.

Components of Tissue Culture MediumA -Inorganic NutrientsIn vitro growth of plants also requires combination of macro and micronutrients like in vivo growth.

Macronutrients are classified as those elements which are required in concentration greater than 0.5 mM/l. They include nitrogen, potassium, phosphorus, calcium, magnesium and sulphur in form of salts in media. Nitrogen is usually supplied in form of ammonium (NH4+) and nitrate (NO3-) ions. Nitrate is superior to ammonium as the sole N source but use of NH4+ checks the increase of pH towards alkalinity. Culture media should contain atleast 25mM/l nitrogen and potassium. Other major elements are adequate in concentration range of 1-3mM/l.

Essential Element*

RoleSymbolForm absorbedDeficiency symptoms

Hydrogen Component of organic compounds and water; chemiosmotic synthesis of ATP in mitochondria and chloroplasts

H H2O

Carbon Component of organic compounds

C CO2

Oxygen Component of organic compounds and water; electron acceptor in respiration

O H2O

Nitrogen nucleic acids, some hormones, and nucleic acids, some hormones, and chlorophyll

N NO3, NH4+ Plants stunted; foliage light green, roots long and slender

Potassium Enzyme activator, involved in starch formation; regulates osmotic balance and movement of guard cells

K K+ Stems slender, numerous small necrotic spots form near the margins of leaves

Calcium Component of middle lamella (Capectate); controls activity of many enzymes; maintains membrane integrity; 2nd messenger

Ca Ca2+ Plants stunted; terminal bud dies; young leaves hooked; root tips die

Magnesium Component of chlorophyll; component of middle lamella (Mg-pectate); activates many enzymes

Mg Mg2+ Leaves with chlorotic spots; tips and margins of leaves turned upward

Phosphorus Component of nucleic acids, phospholipids, coenzymes; involved in sugar metabolism

P H2PO4HPO4 2- Plants stunted, foliage purple/dark green

Sulphur Components of the amino acids cysteine and methionine; component of coenzyme A

S SO42 Young leaves light green, no necrosis

Macronutrients

Micronutrients:Micronutrients are those elements essential for plant growth which are needed in only very small (micro) quantities . These elements are sometimes called minor elements or trace elements, but use of the term micronutrient is encouraged by the American Society of Agronomy and the Soil Science Society of America. The micronutrients are boron (B), copper (Cu), iron (Fe), chloride (Cl), manganese (Mn), molybdenum (Mo) and zinc (Zn). Recycling organic matter such as grass clippings and tree leaves is an excellent way of providing micronutrients (as well as macronutrients) to growing plants.

Micronutrients for plants

There are about eight nutrients essential to plant growth and health that are only needed in very small quantities. These are manganese, boron, copper, iron, chlorine, cobalt, molybdenum, and zinc. Some consider sulfur a micronutrient, but it is listed here as a macronutrient. Though these are present in only small quantities, they are all necessary.

Boron is believed to be involved in carbohydrate transport in plants; it also assists in metabolic regulation. Boron deficiency will often result in bud dieback.

Chlorine is necessary for osmosis and ionic balance; it also plays a role in photosynthesis.Cobalt is essential to plant health. Cobalt is thought to be an important catalyst in nitrogen fixation. It may need to be added to some soils before seeding legumes.

Copper is a component of some enzymes and of vitamin A. Symptoms of copper deficiency include browning of leaf tips and chlorosis.

Iron is essential for chlorophyll synthesis, which is why an iron deficiency results in chlorosis.Manganese activates some important enzymes involved in chlorophyll formation. Manganese deficient plants will develop chlorosis between the veins of its leaves. The availability of manganese is partially dependent on soil pH.Molybdenum is essential to plant health. Molybdenum is used by plants to reduce nitrates into usable forms. Some plants use it for nitrogen fixation, thus it may need to be added to some soils before seeding legumes.Zinc participates in chlorophyll formation, and also activates many enzymes. Symptoms of zinc deficiency include chlorosis and stunted growth.Micronutrients:Essential Element*RoleSymbolForm absorbedDeficiency symptomsLeaves affected

Chlorine Involved in water balance; possibly involved in photosynthetic reactions in which O2 is released ClCl Leaves wilted, chlorotic, ultimately necrotic; roots thickened Whole plant affected

Iron Component of cytochromes ferredoxin and nitrogenase; cofactor of peroxidase; involved in chlorophyll synthesis FeFe2+, Fe3+ Stunted growth; interveinal chlorosis of young leaves Young

Boron May be involved in sugar transport; regulates enzyme function BH2BO4 Terminal bud dies; leaves may be twisted, base of young leaves chlorotic; root tips discolored Young

Manganese Activator of enzymes; involved in electron transfer, chlorophyll synthesis, and the photosynthetic evolution of O2 MnMn2+ Interveinal necrosis of young leaves Young

Zinc Activates many enzymes; involved in the formation of pollen ZnZn2+ Stems with short internodes; leaves thick; leaf margins distorted Old

Copper Component of plastocyanin; present in lignin of xylem elements; activates enzymes CuCu+, Cu2+ Young leaves permanently wilted; foliage dark green; terminal branches unable to stand erect Young

Molybdenum Involved in nitrogen reduction MoMoO42- Young leaves twisted, chlorotic

Young

Some other nutrients essential for plantNutrients

5-HTP - higher bioavailable form of tryptophan, precursor to the neurotransmitter serotonin, promotes relaxed poise and sound sleep. Alpha-GPC (L-alpha glycerylphosphorylcholine, Choline alfoscerate) - most effective choline precursor, readily crosses the blood-brain barrier.

Caffeine - improves concentration, idea production, but hinders memory encoding. Also produces jitters.

Acetyl-L-carnitine (ALCAR) - Amino acid. Transports fatty acids through cellular membranes and cytosol into cells' mitochondria, where the fats undergo oxidation to produce ATP, the universal energy molecule. Synergistic with lipoic acid. Precursor of acetylcholine (donating the acetyl portion). Inhibits lipfuscin formation. CDP-Choline (Cytidine Diphosphate Choline) - choline precursor, a more economical alternative to Alpha GPC.

Chondroitin sulfate

Coenzyme q-10- increases oxygen transport through the mitocondria of the cells. Appears to slow age-related dementia. Creatine - increases brain energy levels via ATP production. DMAE - approved treatment for ADD/ADHD, precursor of acetylcholine, cholinergic agent, removes lipofuscin from the brain, anti-depressant

Ephedrine

Flavonoid - thought to have antioxidant effects, but recentCarbon Sources

Sucrose or glucose at 2 to 4% are suitable carbon sources which are added to the basal medium. Fructose, maltose and other sugars also support the growth of various plant cells. However, the most suitable carbon source and its optimal concentration should be chosen to establish the efficient production process of useful metabolites.The most preferred carbon or energy source is sucrose at a concentration of 20-60g/l. While autoclaving the medium, sucrose is hydrolysed to glucose and fructose which are then used up for growth. Fructose, if autoclaved is toxic. Other mono or disaccharide and sugar alcohols like glucose, sorbitol, raffinose etc may be used depending upon plant species. Carbohydrates also provide osmoticum and hence in anther culture higher concentration of sucrose (6-12%) is used.

B- Organic Supplements

a- VitaminsVitamins are organic substances required for metabolic processes as cofactors or parts of enzymes. Hence for optimum growth, medium should be supplemented with vitamins. Thiamine (B1), nicotinic acid (B3), pyridoxine(B6), pantothenic acid(B5) and Myo-Inositol are commonly used vitamins of which thiamine (0.1 to 5mg/l) is essentially added to medium as it is involved in carbohydrate metabolism. Rest vitamins are promontoryB -Amino acids

Addition of amino acids to media is important for stimulating cell growth in protoplast cultures and also in inducing and maintaining somatic embryogenesis. This reduced organic nitrogen is more readily taken up by plants than the inorganic nitrogen. L-glutamine, L-asparagine, L-cystein, L-glycine are commonly used aminoacids which are added to the culture medium in form of mixtures as individually they inhibit cell growth. C -Complex organics

Complex rganics are group of undefined supplements such as casein hydrolysate, coconut milk, yeast extract, orange juice, tomato juice etc. These compounds are often used when no other combination of known defined components produce the desired growth. Casein hydrolysate has given significant success in tissue culture and potato extract also has been found useful for anther culture. 5-PGRs: [plant growth regulators]

stimulate cell division and hence regulate the growth and differentiation of shoot and roots on explants and embryos in semisolid or in liquid medium cultures. The four major PGRs used are auxins, cytokinin, gibberellins and abscissic acid and their addition is must to the culture medium. Phytohormones or growth regulators are required to induce callus tissues and to promote the growth of many cell lines .

Auxins

induce cell division, cell elongation, apical dominance, adventitious root formation, somatic embryogenesis. When used in low concentration, auxins induce root initiation and in high, callus formation occurs. Commonly used synthetic auxins are 1-naphthaleneacetic acid (NAA). 2,4 dichlorophenoxyacetic acid (2,4-D), indole-3 acetic acid (IAA), indolebutyric acid (IBA) etc. Both IBA and IAA are photosensitive so the stock solutions must be stored in the dark. 2,4-D is used to induce and regulate somatic embryogenesisAs an auxin, 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneaceic acid (NAA) is frequently used. The concentration of auxins in the medium is generally between 0.1 to 50 M.

auxins effect given the followings.1. Increase the rate of transcription.

2. Control the activity of certain enzymes, and

3. Have an influence on the ion pumps within the membraneCytokinins

promote cell division and stimulate initiation and growth of shoots in vitro. Zeatin, 6- benzylaminopurine (BAP), kinetin, 2-iP are the frequently used cytokinins. They modify apical dominance by promoting axillary shoot formation. When used in high concentration, CK inhibits root formation and induces adventitious shoot formation.Cytokinins are usually produced in roots, young fruits, and in seeds. They enter the shoot organs via the xylem. Organs that are cut off from a continuous cytokinin supply like cut shoots age faster than those that are connected to their roots. The addition of kinetin can stop senescence. The development of adventitious roots and thus the new supply with cytokinins restores the old state.

GibberellinsGibberellins are plant hormones that regulate growth and influence various developmental processes, including stem elongation, germination, dormancy, flowering, sex expression, enzyme induction and lea Gibbrellins and abscissic acid are lesser used PGRs. Gibbrellic acid(GA3) is mostly used for internode elongation and meristem growth. Abscissic acid (ABA) is used only for somatic embryogenesis and for culturing woody species.f and fruit senescence.Gibberellins are involved in the natural process of breaking dormancy and various other aspects of germination. Before the photosynthetic apparatus develops sufficiently in the early stages of germination, the stored energy reserves of starch nourish the seedling.Functions of Gibberellins Stimulate stem elongation by stimulating cell division and elongation. Stimulates bolting/flowering in response to long days. Breaks seed dormancy in some plants which require stratification or light to induce germination. Stimulates enzyme production (a-amylase) in germinating cereal grains for mobilization of seed reserves. Induces maleness in dioecious flowers (sex expression). Can cause parthenocarpic (seedless) fruit development. Effects of Gibberellin: Juvenility -- juvenile stages of some plants have different shaped leaves than the adult stage. Gibberellins help determine whether a particular part of a plant is juvenile or adult.Seed Germination -- breaks dormancy of certain seedsFlowering -- Biennial + Gibberellin ---> Annual. Biennials when treated with gibberellin give flowers and fruits in their first year instead of the usual two years. Parthenocarpic -- stimulates pollen germination and growth.Fruit formation -- increases size of fruits.

Abscisic Acid

Abscisic acid (ABA) inhibits cell division. It is most commonly used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis.Abscisic acid (ABA) is identical with a substance that causes bud dormancy in wooden perennial plants. It was therefore at first also called dormin. In maple and birch buds causes the change from long-day to short-day conditions a marked increase in the activity of dormin (=ABA) and consequently stops the growth of buds.

Ethylene

Ethylene is a gaseous, naturally occurring, plant growth regulator most commonly associated with controlling fruit ripening, and its use in plant tissue culture is not widespread. It does, though, present a particular problem for plant tissue culture. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture. Ethylene has evolved as the central regulator of cell death programs in plants. In roots and in some plants in stems, lack of oxygen induces formation of intercellular spaces, the so-called parenchyma. Low oxygen conditions in waterlogged roots, for instance, result in lysogenous parenchyma formation through programmed death of cells in the cortex. This process is controlled by ethylene, as is programmed death of endosperm cells during cereal seed development. Applications

When fruits are stored in closed rooms, ethylene that is released from ripening and mature fruits accumulates and stimulates late-ripening fruits to premature ripening ("One rotten apple can ruin the whole basket"). For fruit storage, it is favorable to avoid formation or spread of ethylene. Therefore, fruits are often stored under hypobaric conditions to remove ethylene that is released. Conversely, banana are harvested, transported, and stored at an immature stage. Before being shipped to stores, they are treated with ethylene to induce synchronous ripening.

Sterilization

Sterilization techniqueSterilization is the freeing of an article from all living organisms, including bacteria and their spores. Sterilization of culture media, containers and instruments is essential in microbiological work for isolation and maintenance of microbes.

This is achieve by one of the following methods :

1. Dry heat

2. Flame sterilization

3. Autoclaving

4. Filter sterilization

5. wiping with 70% ethanol

6. Surface sterilizationSterilization technique in Plant Tissue CultureTechnique

Materials sterilized

Steam sterilization/Autoclaving (121C at 15 psi for 20-40 min) Nutrient media, culture vessels,

glasswares and plastic wares

Dry heat (160-180C for 3h) Instruments (scalpel, forceps, needles etc.),

glassware, pipettes, tips and other plastic wares

Flame sterilization Instruments (scalpel, forceps, needles etc.),

mouth of culture vessel

AutoclavingMedia ,culture vessels( glass ware & plastic ware)

Filter sterilization (membrane filter

made of cellulose nitrate or cellulose

acetate of 0.45- 0.22m pore size)

Thermo labile substances like growth factors,

amino acids, vitamins and enzymes.

Alcohol sterilization

Workers hands, laminar flow cabinet

Surface sterilization (Sodium

hypochlorite, hydrogen peroxide,

mercuric chloride etc)

Explants

Dry heat :

Glass ware and Teflon plastic ware (empty vessels), and instruments may be sterilized by dry heat in an oven at 160 - 180C for 3 hrs. but most worker prefer to auto clave glass ware and plastic ware .

Flame sterilization :

Instrument like forceps, scalpels, needles etc. are ordinary flame sterilized by dipping them in 95% alcohol followed by flaming. These instrument are repeatedly sterilized during the operation to avoid contamination.

Autoclaving :

Culture vessels, (both empty and containing media) are generally sterilized by heating in an autoclave or a pressure cooker to 121C at 15 p.s.i.(pound per square inch) for 15 (20 25 ml medium) min. the time being longer for larger medium volume.

Autoclaving in media sterilization

Minimum autoclaving time forplant tissue culture medium

Volume of Medium per Vessel (ml)Minimum Autoclaving (min)*

2520

5025

10028

25031

50035

100040

Plant tissue culture media are generally sterilized by autoclaving at 121 C and 1.05 kg/cm2 (15-20 psi). The time required for sterilization depends upon the volume of medium in the vessel. The minimum times required for sterilization of different volumes of medium are listed below..Filter sterilization :

some growth regulators like GA3, zeatin, ABA, urea, certain vitamins, and enzymes are heat liable. Such compounds are filter sterilized by passing their solution through membrane filter of 0.45 or lower pore size. Filter sterilized heat liable compound are filter is sterilized by autoclaving before use.Laminar air flow cabinet : Laminar air flow cabinet is the most suitable, convenient and reliable instrument for septic work. It allow one to for a longer period which is not possible inside the inoculation chamber. Laminar air flow has a number of small blower motor to blow air which passes through a number of HEPA ( high efficiency particular air) filter. Such filter remove lager then 0.3 m.

This sterilized air blow through the cabinet at 1.8 km/hr. which is sufficient to keeps the enclosed working area aseptic.

Wiping with 70% Ethanol :

the surface that can not be sterilized by other techniques, eg ,platform of the laminar flow cabinet, hands of the operators ,etc., are sterilized by wiping them thoroughly with 70% ethyl alcohol and alcohol is allowed to dry. Surface sterilization :Surface sterilization will depend mainly on the source and the type of tissue of the explant, which will determine the contamination load and tolerance to the sterilizing agents. See the table.

Sterilizing agentConcentration (%)Duration

(min)Effectiveness

Calcium hypochlorite9 - 105- 30Very good

Sodium hypochlorite2 %5 30Very good

Hydrogen peroxide 10 125 15 Good

Mercuric chloride 0.1- 12 10Satisfactory

Antibiotics4- 50 mg/l30 60Fairly good

Some other sterilization method

Infrared radiations

Methods of sterilization exist using radiation such as electron beams, X-rays, gamma rays, or subatomic particles.

Gamma rays are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets.Chemical agent

ethylene oxide : Ethylene oxide gas kills bacteria (and their endospores), mold, and fungi,chlorine bleach :used in the form of gas or in the form of compressed gas in the form of liquid(water purification plant).Hydrogen peroxide : is another chemical sterilizing agent. It is relatively non-toxic once diluted to low concentrations (although a dangerous oxidizer at high concentrations).

Phenol and phenolic compounds:- Phenolic substances may be either bactericidal or bacteriostatic,depending upon the concentration used.Factors influenzing sterilization by heat

1. The temperature and time: they are inversely related, shorter time is sufficient at high temperatures.

2. Number of microorganisms and spores: The number of survivors diminished exponentially with the duration of heating

3. Depends on the species, strains and spore forming ability of the microbes.

4. Thermal death point is the lowest temperature to give complete killing in aqueous suspension within 10 minutes

5. Depends on the nature of material: a high content of organic substances generally tends to protect spores and vegetative organisms against heat.

Temperature, pH, Light and Oxygen

The effects of temperature, pH, light and oxygen are all parameters that must be examined in the studies secondary metabolites production.

A temperature of 17- 25C is normally used for induction of callus tissues and growth of cultured cells. But, each plant species may favor a different temperature. Toivonen (49) found that lowering the cultivation temperature increased the total fatty acid content per cell in dry weight.

The medium pH is usually adjusted to between 5 and 6 before autoclaving and extremes of pH are avoided. The optimum pH is determined and controlled using a small scale bioreactor or a jar fermentor with pH control equipment.

Present scale-up technology dictates the use of stainless steel tanks for growth of plant cells on an industrial scale, thus in general, eliminating the use of light. However, since there are cases of light-stimulated secondary metabolites production (2), this factor should be investigated as it could help elucidate regulatory factors. Modification of fermentors with lighting facilities have also been carried out.

Plant material

Plants

Initially the shoot tips, immature inflorescences (0.6-0.8cm in diameter), leaf sections, capitulums explants, axillarys buds, receptacle explants from the field were taken.The explants of shoot tips, immature inflorescences, leaf sections, capitulums explants, axillarys buds and receptacles explants from field grown plants had contamination problems. Another trouble was

slow growth of explant cultures as they were treated with sterilizing chemicals which damage their growing regions.

In theory, any part obtained from any plant species can be employed to induce callus tissue, however the successful production of callus depends upon plant species and their qualities. Dicotyledons are rather amenable for callus tissue induction, as compared to monocotyledons; the callus of woody plants generally grow slowly. Stems, leaves, roots, flowers, seeds and any other parts of plants are used, but younger and fresh explants are preferable as explant materials.

Explants obtained must be sterilized using ethanol, sodium hypochlorite and/or other chemicals to remove all microorganisms from the materials and a typical sterilization procedure will be described later as an example.Gerbera plant material must first be surface sterilized to remove any bacteria or fungal spores that are present. We aim to kill all microorganisms, but at the same time not cause any adverse damage to the plant material. 1. Gerbera should be cut into small sections of florets about 1 cm across. If using a rose or other cuttings, cut the shoots into about 5 to 7 cm lengths. Whole African violet leaves can also be used. 2. Wash the prepared plant material in a detergent-water mixture for about 20 minutes. If trying hairy plant material scrub with a soft brush (toothbrush). This will help remove fungi etc., and the detergent will help wet the material and remove air bubbles that may be trapped between tiny hairs on a plant. 3. Transfer the washed plant material to the sterilizing chlorox solution. Shake the mixture for 1 minute and then leave to soak for 10-20 minutes.Introduction about plant

Gerbera

Scientific NameGerbera jamesonii

Family Asteraceae /Compositae (Daisy Family)

Common namesGerbera ,Africadaisy, Transvaal, daisy, Barberton daisy

Flowering Period All year round

Colourwhite, red, cream, orange,pink, purple & yellow

Gerbera (Gerbera jamesonii) belongs to sunflower family Asteraceae and is popularornamental of commercial importance used as a decorative garden plant, container plant, or mostly as cut flowers.The gerbera plant was first discovered by Robert Jameson in South Africa, where it grows spontaneously in shady areas, Gerbera flowers comes in vibrant colours adding beauty to your garden. It has around 40 species spreading from Africa across to Madagascar into tropical Asia and South America. Gerbera are plants with a height up to 18 to 24 inch and 4 to 10 inch diameter flowersGerbera is very popular and widely used as a decorative garden plant or as cut flowers. The domesticated cultivars are mostly a result of a cross between Gerbera jamesonii and another South African species Gerbera viridifolia. The cross is known as Gerbera hybrida. Thousands of cultivars exist. They vary greatly in shape and size. Colors include white, yellow, orange, red, and pink. The center of the flower is sometimes black. Often the same flower can have petals of several different colors.Gerbera jamesonii can be grown from seed or crown divisions. Seeds should be germinated within 1 to 2months of collection, at about 20 to 25C, and will flower after a year. Clumps can also be divided in spring. Plants require full sun and moderate watering. Rot will occur if the crowns are buried or the drainage is poor. Plants do best with frequent feeding, especially in summer, to promote flowering. Remove dead flowers regularly to encourage further flowering. Slugs and snails are partial to the leaves, and Gerbera are prone to some viral, bacterial and fungal diseases.

The genus Gerbera consists of about 30 species which are found in Africa, Madagascar, tropical Asia and South America.

Gerbera cultivars for greenhouse production have been developed in different plant sizes to accommodate a wide range of container sizes. Groups of cultivars have been bred for 3-10lt containers. These are large plants with 10-15cm diameter flowers on 45-60cm stems. Several different flower types have been developed in gerberas. Soil cultivation

Soil:Gerbera requires a deep and well drained soil with 1-3% organic substances for heavy and light soil correspondingly.

Planting layoutDensity is 6-8 plants per m2 or 60000-80000 plants per hectare. The young plants are planted in raised flower beds, 2 lines per bed with spacing of 30-40cm and 20-25cm between plants.

Planting Propagation may be achieved through seeds, basal cuttings or through dividing. Basal shoots or cuttings from the parent plant should be taken in summer (March- April). Seeds are sown or cuttings can be inserted in sandy soil until the saplings become an inch tall or the cuttings form roots. Plants grown from seeds can differ from the parent plant and seeds which do not germinate within about twenty days are likely not to germinate at all.

Some other Species of gerbera Gerbera aberdarica

Gerbera abyssinica

Gerbera ambigua

Gerbera anandria: Ghostly Daisy Gerbera tissue

cultureExplant sterilizationInitially the shoot tips, immature inflorescences (0.6-0.8cm in diameter), leaf sections, capitulums explants, axillarys buds, receptacle explants from the field were taken.

Explants obtained must be sterilized using ethanol, sodium hypochlorite and/or other chemicals to remove all microorganisms from the materials and a typical sterilization procedure will be described later as an example.Commonly used disinfectantsfor plant tissue culture

DisinfectantProduct No.Concentration (%)Exposure (min)

Calcium hypochlorite21,138-99-105-30

Sodium hypochlorite*42,504-40.5-55-30

Hydrogen peroxideH 10093-125-15

Ethyl alcoholE714870-950.1-5.0

Silver nitrateS 727615-30

Mercuric chlorideM 11360.1-1.02-10

Benzalkonium chlorideB 13830.01-0.15-20

Explant Sterilization procedures may be enhanced by: Placing the material in a 70% ethyl alcohol solution prior to treatment with another disinfectant solution. The use of a two-step (two-source) sterilization procedure has proven beneficial with certain species.

Explant dip IPA (isopropyl alcohol 70%) for 30 sec.in laminar.

Wash with DL water 2-3 times.

Hgcl2 (.05 gm) into 250ml distilled water for 5 min.

Explant wash 2- time with distilled water.

Inoculation in MS medium.

Gerbera

Inoculation

methodGerbera inoculation method

Take Gerbera & cut in suitable size.

Cut the ex-plant in sutable size.

Sterile the ex-plant with Detol solution & laballon solution for 20 min.

Wash the ex-plant tap water for three min.

Then the ex-plant sterile with in fungicide (zen solution) for 30 min.

Wash the ex-plant with sterile distill water.

Then ex-plant container transfer in Laminar Air Flow.

Wash the ex-plant with sterilized water at 2-3 times.

Ex-plant sterilized with IPA (isopropyl alcohol) for 30 second.

Wash the ex-plant with distill water at 2 times.

Then ex-plant dip in mercuric chloride (HgCl2 0.12 ) for 12 min.

Then ex-plant washed the sterilized distill water for 2 times.

Dip the ex-plant in Ascorbic acid.

Cut of blackish mark on ex-plant on sterilized plate of paper by blade.

Then inoculate ex-plant in different hormonal effect in MS media

Gerbera inoculation method

Transfer in Environmental control room Transfer in environment control roomEnvironmental factor greatly influence the process of growth and differentiation of tissues in culture. All type of plant are therefore incubated under well controlled temperature, humidity, illumination, and circulation.

A typical culture room shood have booth light and temperature programmable for 24-hr period. Usually air conditioned and room heaters are used to maintain the temperature around 25 + 2* C

Lightening is adjusted in terms of quantities and photoperiod duration by using automatic clocks (culture are generally grown in diffused light less then 1 kls*) further the humidity range varies from 20 98 % controllable to + 3* C. and a uniform fords air ventilation is necessary.

Specially designed shelves made of glass rigid wire meshor wood are provided in culture room for storing culture. Each shelf is illuminated separately by a separate set of fluorescent tubes.

Individual shelf may also be ventilated by fitting a small fan at one end of the shelf the culture vessel can be placed directly on the shelves or trays of suitable size where as culture tubes require metallic racks to hold them.

A label is provide having the details of the experiment (name of the plant, explants , medium, date of culture and other information ) is stuck on each trayto ensure identity and recording results.

Ti is the major precaution to taken against power break down. Failure of electricity may run important experiment and suspension culture may ruin due to stoppage of shakers. A generator should be kept stand by and emergency power points in the culture rooms, incubators and growth chambers in oder to maintain the necessary light and temperature condition.

Stages of gerbera tissue culture

Initiation (stage 1)The innermost tissue of surface sterilised plant in dissected aseptically and put an to the medium of growth, Medium contains major and miner elementssame vitamins. Amino acids and growth promoting hormones, solidified by agar. Preparation and establishment of explant on suitable culture medium (3-24 months) (usually shoot tips and axillary buds used)

Multiplication (Stage II) :

When the tissue starts growth in stage I and forms a shoot it is transferred to another medium containing growth promoting hormones (enhancing cell division). Multiplication is the taking of tissue samples produced during the first stage and increasing their number. Following the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds or thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and media. If the plant material grown is callus tissue, it can be placed in a blender and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue. If the tissue is grown as small plants called plantlets,The growing shoot multiplies and forms a dump of 3-4 shoots. Those are transferred to another medium for shooting and rooting after optimum growth.

Stage 3: (Rooting)Rooting of regenerated shoots/ somatic embryo in vitro (1-6 weeks).Once a sufficient number of shoots have been generated, portions of explantthat contain one or more shoots could be transferred to a medium that contains ahigher concentration of the hormone auxin, resulting in root production.

Stage 4: (Acclimatization/hardening )Once roots are visible, plantlets need to be moved from the medium.

Transfer of plantlets to sterilized soil for hardening under greenhouse environment. It involves acclimatization of bottle grown plants to the natural environment in Green House. The plants are taken out of the bottle and the media adhering to the root system in washed fully.Transfer of plantlets to sterilized soil for hardening under greenhouse environment

Observation

Observation Table

No. of

days

Date of

InoculationNo. of Ex-plant

Date of observationContamination

No.of Dead

ex-plant Result

FugalBacterial

024/03/200925

1

2

327/03/20091 0 0

4

5

6

731/3/2009 1 1 1 yellow

8

9

10

114/4/2009000Green

12

13

147/4/2009101Yellow

15

16

17

18

1912/4/2009010Yellow

20

21

22

2316/4/2009000Green

24

25

26

27

2821/4/2009010Yellow

29

29

30

31

Note : Yellow - For dead cell Green for live cellNo. of

days

Date of

InoculationNo. of Ex-plant

Date of observationContamination

No.of Dead

ex-plant Result

FugalBacterial

024/03/200925

1

2

327/03/2009

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

29

30

31

Note : Yellow - For dead cell Green for live cellNo. of

days

Date of

InoculationNo. of Ex-plant

Date of observationContamination

No.of Dead

ex-plant Result

FugalBacterial

024/03/200925

1

2

327/03/2009

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

29

30

31

Note : Yellow - For dead cell Green for live cellNo. of

days

Date of

InoculationNo. of Ex-plant

Date of observationContamination

No.of Dead

ex-plant Result

FugalBacterial

024/03/200925

1

2

327/03/2009

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

29

30

31

Note : Yellow - For dead cell Green for live cellResultResult

in vitro technique s offer new possibilities in commercial propagation of plant .the present study was also undertaken to propagate commercially important cultivate of Gerbera. For shoot formation both apical and nodal meristem were used.

In this study the shoot tip explant of Gerbera jamesonii , was placed on MS medium for a period of 6 weeks and in this period the explant showed good response of growth and multiplication.

In the present study it was also observe apical meristem respond earlier compared to nodal meristem this potential effect of explant in also discussed by Bresan et al..(1981).

Similarly, the leaf of explant also showed growth response when they are not properly remover5 before inoculation. There is an increase in the length of the leaf instead of the shoot elongation.

Conclusion

SummaryTissue culture and plant regeneration are an integral part of most plant transformation strategies, and can often prove to be the most challenging aspect of a plant transformation protocol. Key to success in integrating plant tissue culture into plant transformation strategies is the realization that a quick (to avoid too many deleterious effects from somaclonal variation) and efficient regeneration system must be developed. However, this system must also allow high transformation efficiencies from whichever transformation technique is adopted.

Not all regeneration protocols are compatible with all transformation techniques. Some crops may be amenable to a variety of regeneration and transformation strategies, others may currently only be amenable to one particular protocol. Advances are being made all the time, so it is impossible to say that a particular crop will never be regenerated by a particular protocol. However, some protocols, at least at the moment, are clearly more efficient than others. Regeneration from immature embryo-derived somatic embryos is, for example, the favored method for regenerating monocot species.There are still a number of commercially important products which are being extracted from mass produced field-plants. In order to circumvent various problems caused from these processes as the author indicated in the Introduction, plant cell culture technology has been expected to be an efficient and useful tool.

For more than 30 years, many researchers have investigated plant cell cultures for production of a variety of phytochemicals; however, in spite of their many efforts only two products such as shikonins and ginseng cells are so far being manufactured commercially. The reasons why this technology has scarcely been applied in industry are; low yield of plant metabolites, unstable producing ability of cultured cells and their slow growth rate. Therefore, at present the plant cell culture is not a cost-effective technology. In particular, any process using plant cell cultures is not favorable if the desirable products can be easily manufactured by chemical- or microbial fermentation methods.

However, a variety of scientific strategies, as described in this review, have been investigated for improving the production ability of cultured cells and substantial progress has been made. Undoubtedly, some plant metabolites are likely to be manufactured through plant cell cultures. In addition to the approaches described in this review, several research groups have paid an attention to using recombinant DNA technology as a tool for improvement of cultured cells although it is not an easy task in secondary metabolism.ReferencesReferences1.Goldstein, W. et al., In "Plant Tissue Culture as a Source of Biochemicals" Ed. Staba, E.J., 191-234 (1980) C.R.C. Press. Boca Raton, Florida.

10. Tulecke, W. and Nickell L.G., Science 130 863 (1959).

11. Mandel, M., Adv. Biochem. Eng., 2 201 (1972).

12. Street, H.E. (ed.), In "Plant Tissue and Cell Culture", Blackwell Scientific Publ., London (1973).

13. Martin, S.M., In "Plant Tissue Culture as a Source of Biochemicals" Ed. Staba, E.J., 149-166 (1980). C.R.C. Press. Boca Raton, Florida.

14. Misawa, M., In "Plant Tissue Culture and Its Bio-technological Application", Eds. Barz, W., Reinhard, E., Zenk, M.H., p. 17 (1977), Springer-Verlag, Berlin, Heidelberg, New York.

15. Misawa, M. and Samejima, H., In "Frontiers of Plant Tissue Culture 1978", Ed. Thorpe, T.A., 353-362, (1978) Univ. of Calgary.

16. Barz, W., Reinhard, E., Zenk, M.H. (Eds.), "Plant Tissue Culture and Its Bio-technological Application" Springer-Verlag, Berlin, Heidelberg, New York.

17. Zenk, M.H., In "Plant Tissue Culture and Its Bio-technological 2. Whitaker, R.J. et al., Am. Chem. Soc. Symp. Ser. 317 347-362 (1986).

3. Fukui, H., Yamazaki, K., Tabata, M., Phytochem. 23 2398-2399 (1984).

4. Deus, B. and Zenk, M.H., Biotech. Bioeng., 24 1965-1974 (1982).

5. Zenk, M.H., In "Frontiers of Plant Tissue Culture 1978", Ed. Thorpe, T.A., p.1 (1978). Univ. of Calgary.

6. Kruz, W.G.W., Adv. Appl. Microbiol. 25 209 (1979).

7. Fowler, M.W., In "Biotechnology of Higher Plants", Ed. Russel, G.E., 107-133 (1988) Intercept Ltd.

8. Misawa, M., In "Adv. in Biotechem. Eng./Biotech.", Ed. Fiechter, A., 59-88 (1985) Springer-Verlag. Berlin, Heidelberg, New York.

9. Staba, E.J., In "Proc. 5th Int'l. Cong. Plant Tissue & Cell Culture", Ed. Fujiwara, A., p. 25 (1982) Maruzen Co., Tokyo.

Application", Eds. Barz, W., Reinhard, E., Zenk, M.H., p. 27 (1977), Springer-Verlag, Berlin, Heidelberg, New York.

18. Fujiwara, A. (Ed), Proc. 5th Int'l. Cong. Plant Tissue and Cell Culture, Maruzen Co., (1982).

19. Fujita, Y. et al., In "Proc. 5th Int'l. Cong. Plant Tissue and Cell Culture", Ed. Fujiwara, A., 399-400 (1982).

20. Misawa, M. et al., ibid., 279-280 (1982).

21. Alfermann, A.W. and Reinhard, E., ibid., 401-402 (1982).

22. Misawa, M. et al., Phytochem., 27 1355 (1988).

23. Ulbrich, B., Weisner, W., Arens, H., In "Primary and Secondary Metabolism of Plant Cell Cultures", Eds. Neumann, K.H. and Reinhard, E., p.293-303 (1985), Springer-Verlag, Berlin.

24. Sahai, O. and Knuth, M., Biotechn. Prog., 11 9 (1985).

25. Fowler, M.W., In "Plant Biotechnology", Eds. Mantell, S.H., and Smith, H., p.3-37 (1983), Cambridge Univ. Press, Cambridge, U.K.

26. Scragg, A.H., in "Secondary Metabolism in Plant Cell Cultures" Eds. Morris, A.H. et al., p.202-207 (1986). Cambridge Univ. Press, Cambridge, U.K.

27. Drapeau, D. et al., Biotech. Bioeng. 30 946-953 (1984).

28. Murashige, T. and Skoog, F., Physiol. Plant., 15 473-497 (1962).

29. Gamborg, O.L. et al., Exp. Cell Res., 50 151-158 (1968).

30. Wetter, L.R. and Constabel, F., (Eds), Plant Tissue Culture Methods. (1982) Nat'l Research Council of Canada, Saskatoon.

31. Lamport, D.T.A., Exp. Cell Res., 33 195 (1964).

32. Veliky, I. and Martin, S.M., Can. J. Microbiol., 16 223 (1970).

33. Martin, S.M. and Rose, D., Can. J. Bot., 54 1264 (1976).

34. Kato, A. et al., J. Ferment. Technol., 54 82 (1976).

35. Martin, S.M., In "Plant Tissue Culture as a Source of Biochemicals" Ed. Staba, E.J., 149-166 (1980) C.R.C. Press. Boca Raton, Florida.36. Wagner, F. and Vogelmann, H., In "Plant Tissue Culture and Its Bio-technological Application", Eds. Barz, W., Reinhard, E., Zenk, M.H., p.245 (1977), Springer-Verlag, Berlin, Heidelberg, New York.

37. Tanaka, H. et al., Biotech. Bioeng., 24 2359 (1983).

38. Ten Hoopen, H.J.G. et al., In "Prog. in Plant and Molecular Biology", Eds. Nijkamp, H.J.J. et al., 673-681 (1990). Kluwer Acad. Publ., Dordorecht, Boston, London.

39. Westphal, K., ibid., 601-608 (1990).

40. Ushiyama, K., In "Plant Cell Culture in Japan", Eds. Komamine, A., Misawa, M., DiCosmo, F., p.92-98 (1991). CMC Co. Ltd., Tokyo.

41. Takahashi, S. and Fujita, Y., ibid., p.72-78 (1991). CMC Co. Ltd. Tokyo.

42. Rokem, J.S. et al., Plant Cell Rep., 3 159-160 (1984).

43. Matsumoto, T. et al., Agr. Biol. Chem., 84 967 (1980).

44. Whitaker, R.J., Hashimoto, T., Evans, D.A., Ann. NY Acad. Sci., 435 364 (1984).

Observation Table

No. of

days

Date of

InoculationNo. of Ex-plant

Date of observationContamination

No.of Dead

ex-plant Result

FugalBacterial

024/03/200925

1

2

327/03/2009

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

29

30

31

Note : Yellow - For dead cell

Green for live cell

No. of

days

Date of

InoculationNo. of Ex-plant

Date of observationContamination

No.of Dead

ex-plant Result

FugalBacterial

024/03/200925

1

2

327/03/2009

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

29

30

31

Other Equipment

Refrigerator, deep-freeze.

Automatic dish-washer.

Glass atomizer.

Dispensing devices (e.g., wire-mesh baskets, trolleys witb trays and metal racks for holding test-tubes or culture vials in the autoclave).

Acid proof baths for cleansing glassware.

Microscopes (e.g., compound, inverted) with micro photographic equipment. .

Markers, labels and vita film (or similar material for wrapping culture vessels, glass wares,tiles & lab ware

Balances: Manual-single pan, capacity 100-200g, electronics-top loading. For ( for weighting nutrient media).

pH meter: 230V, 50Hz, single phase supply with combined pH electrode, range 0-14Ph/0-1400mv, temp. compensation 0-100C.

Other Equipment

Refrigerator, deep-freeze.

Automatic dish-washer.

Glass atomizer.

Dispensing devices (e.g., wire-mesh baskets, trolleys witb trays and metal racks for holding test-tubes or culture vials in the autoclave).

Acid proof baths for cleansing glassware.

Microscopes (e.g., compound, inverted) with micro photographic equipment. .

Markers, labels and vita film (or similar material for wrapping culture vessels, glass wares, tiles & lab ware

Balances: Manual-single pan, capacity 100-200g, electronics-top loading. For (weighting, nutrient media).

pH meter: 230V, 50Hz, single phase supply with combined pH electrode, range 0-14Ph/0-1400mv, temp. compensation 0-100C .electrode, range 0-14Ph/0-1400mv, temp. compensation 0-100C

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