Saturday, October 27, 2012

Agar for Plant Tissue culture








 Agar :

Agar is a galactose polymer (or agarose) obtained from the cell walls of some species of red algae or *seaweed (Sphaerococcus euchema) and species of **Gelidium and ***Gracilaria, chiefly from eastern Asia, Chile and California. It is also known as Kanten, Agar-Agar, or Agal-Agal (Ceylon Agar).
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*Seaweed : Seaweed is a loose colloquial term encompassing macroscopic, multicellular, benthic marine algae. The term includes some members of the red, brown and green algae. Seaweeds can also be classified by use (as food, medicine, fertilizer, industrial, etc.).

 
Taxonomy : A seaweed may belong to one of several groups of multicellular algae: the red algae, green algae, and brown algae. As these three groups are not thought to have a common multicellular ancestor, the seaweeds are a polyphyletic group. In addition, some tuft-forming bluegreen algae (Cyanobacteria) are sometimes considered as seaweeds — "seaweed" is a colloquial term and lacks a formal definition.
 
Structure :Seaweeds' appearance somewhat resembles non-arboreal terrestrial plants.
  • thallus: the algal body
    • lamina: a flattened structure that is somewhat leaf-like
      • sorus: spore cluster
      • on Fucus, air bladders: float-assist organ (on blade)
      • on kelp, floats: float-assist organ (between lamina and stipe)
    • stipe: a stem-like structure, may be absent
    • holdfast: specialized basal structure providing attachment to a surface, often a rock or another alga.
    • haptera: finger-like extensions of holdfast anchoring to benthic substrate
 
The stipe and blade are collectively known as the frond.

Ecology :Two specific environmental requirements dominate seaweed ecology. These are the presence of seawater (or at least brackish water) and the presence of light sufficient to drive photosynthesis. Another common requirement is a firm attachment point. As a result, seaweeds most commonly inhabit the littoral zone and within that zone more frequently on rocky shores than on sand or shingle. Seaweeds occupy a wide range of ecological niches. The highest elevation is only wetted by the tops of sea spray, the lowest is several meters deep. In some areas, littoral seaweeds can extend several miles out to sea. The limiting factor in such cases is sunlight availability. The deepest living seaweeds are some species of red algae.

 

A number of species such as Sargassum have adapted to a fully planktonic niche and are free-floating, depending on gas-filled sacs to maintain an acceptable depth.
Others have adapted to live in tidal rock pools. In this habitat seaweeds must withstand rapidly changing temperature and salinity and even occasional drying.

Use: Seaweed has a variety of purposes, for which it is farmed or foraged from the wild.

 
At the beginning of 2011, Indonesia produced 3 million tonnes of seaweed and surpassed the Philippines as the world's largest seaweed producer. By 2012 the production will hit 10 million tonnes.

Food :Seaweeds are consumed by coastal people, particularly in East Asia, e.g., Brunei, Japan, China, Korea, Taiwan, Singapore, Thailand, Cambodia, and Vietnam, but also in South Africa etc.

Seaweeds are also harvested or cultivated for the extraction of alginate, agar and carrageenan, gelatinous substances collectively known as hydrocolloids or phycocolloids. Hydrocolloids have attained commercial significance as food additives. The food industry exploits their gelling, water-retention, emulsifying and other physical properties. Agar is used in foods such as confectionery, meat and poultry products, desserts and beverages and moulded foods. Carrageenan is used in salad dressings and sauces, dietetic foods, and as a preservative in meat and fish products, dairy items and baked goods.
 
Herbalism :Alginates are used in wound dressings, and production of dental moulds. In microbiology research, agar - a plant-based goo similar to gelatin and made from seaweed - is extensively used as culture medium. Carrageenans, alginates and agaroses (the latter are prepared from agar by purification), together with other lesser-known macroalgal polysaccharides, also have several important biological activities or applications in biomedicine.
 
Seaweed is a source of iodine, necessary for thyroid function and to prevent goitre. However, an excess of iodine is suspected in the heightened cancer risk in Japanese who consume a lot of the plant, and even bigger risks in post-menopausal women.

Seaweeds may have curative properties for tuberculosis, arthritis, colds and influenza, worm infestations and even tumors. In Japan, seaweed eaten as nori is known as a remedy for radiation poisoning.

 
Seaweed extract is used in some diet pills. Other seaweed pills exploit the same effect as gastric banding, expanding in the stomach to make the body feel more full.

Other uses : Other seaweeds may be used as fertilizer, compost for landscaping, or a means of combating beach erosion through burial in beach dunes. Seaweed is currently under consideration as a potential source of bioethanol. Seaweed is an ingredient in toothpaste, cosmetics and paints.

Alginates enjoy many of the same uses as carrageenan, and are used in industrial products such as paper coatings, adhesives, dyes, gels, explosives and in processes such as paper sizing, textile printing, hydro-mulching and drilling.

Health risks : Rotting seaweed is a potent source of hydrogen sulfide, a highly toxic gas, and has been implicated in some incidents of apparent hydrogen-sulphide poisoning. It can cause vomiting and diarrhoea.

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** Gelidium : Gelidium is a genus of thalloid alga comprising 124 species. Its members are known by a number of common names. Specimens can reach around 2 to 40 cm in size. Branching is irregular, or occurs in rows on either side of the main stem. Gelidium produces tetraspores. Many of the algae in this genus are used to make agar. Chaetangium is a synonym.



red algae




Chemically, agar is a polymer made up of subunits of the sugar galactose; it is a component of the algae's cell walls. Dissolved in hot water and cooled, agar becomes gelatinous; its chief use is as a culture medium for microbiological work. Other uses are as a laxative, a vegetarian gelatin substitute — a thickener for soups, in jellies, ice cream and Japanese desserts such as anmitsu, as a clarifying agent in brewing, and for paper sizing fabrics.

Species
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Gelidium affine                                            Gelidium allanii
Gelidium amamiense                                   Gelidium amansii
Gelidium ambiguum                                    Gelidium americanum                         Gelidium anthonini                                      Gelidium applanatum
Gelidium arborescens                                  Gelidium arenarium
Gelidium asperum                                       Gelidium australe
Gelidium bernabei                                       Gelidium bipectinatum
Gelidium canariense                                    Gelidium cantabricum
Gelidium capense                                        Gelidium caulacantheum
Gelidium ceramoides                                  Gelidium chilense
Gelidium coarctatum                                  Gelidium concinnum
Gelidium congestum                                  Gelidium corneum
Gelidium coronadense                                Gelidium coulteri
Gelidium crinale                                         Gelidium crispum
Gelidium deciduum                                    Gelidium decompositum
Gelidium delicatulum                                 Gelidium divaricatum
Gelidium elegans                                       Gelidium elminense
Gelidium fasciculatum                               Gelidium filicinum
Gelidium flaccidum                                   Gelidium floridanum
Gelidium foliaceum                                   Gelidium foliosum
Gelidium galapagense                                Gelidium hancockii
Gelidium heterocladum                              Gelidium hommersandii
Gelidium howei                                          Gelidium hypnosum
Gelidium inagakii                                       Gelidium inflexum
Gelidium intertextum                                  Gelidium isabelae
Gelidium japonicum                                   Gelidium johnstonii
Gelidium kintaroi                                        Gelidium latiusculum
Gelidium lingulatum                                   Gelidium linoides
Gelidium longipes                                       Gelidium macnabbianum
Gelidium madagascariense                          Gelidium maggsiae
Gelidium masudae                                       Gelidium microdentatum
Gelidium microdon                                      Gelidium microdonticum
Gelidium microphyllum                              Gelidium microphysa
Gelidium micropterum                                Gelidium minusculum
 Gelidium multifidum                                  Gelidium musciforme
Gelidium nova-granatense                           Gelidium nudifrons
Gelidium omanense                                     Gelidium pacificum
Gelidium planiusculum                                Gelidium pluma
Gelidium pristoides                                      Gelidium profundum
Gelidium pseudointricatum                          Gelidium pteridifolium
Gelidium pulchellum                                    Gelidium pulchrum
Gelidium pulvinatum                                    Gelidium purpurascens
Gelidium pusillum                                        Gelidium reediae
Gelidium refugiensis                                     Gelidium regulare
Gelidium reptans                                           Gelidium rex
Gelidium rigens                                             Gelidium robustum
Gelidium samoense                                       Gelidium sclerophyllum
Gelidium secundatum                                    Gelidium semipinnatum
Gelidium serrulatum                                      Gelidium sinicola
Gelidium spathulatum                                    Gelidium spinosum
Gelidium subfastigiatum                                Gelidium tenue
 Gelidium tsengii                                            Gelidium umbricola
Gelidium usmanghanii                                   Gelidium vagum
Gelidium venetum                                          Gelidium venturianum
Gelidium versicolor                                        Gelidium vietnamense
Gelidium vittatum                                           Gelidium yamadae
Gelidium zollingeri
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*** Gracilaria : Gracilaria is a genus of red algae (Rhodophyta) notable for its economic importance as an agarophyte, as well as its use as a food for humans and various species of shellfish. Various 

  
 

species within the genus are cultivated among Asia, South America, Africa and Oceania.


 

Distribution

Gracilaria bursa-pastoris (S.G.Gremlin) Silva and Gracilaria multipartita (Clemente) Harvey have long been established in southern England and northwestern France, but confusion between Gracilaria gracilis (Stackhouse) 

gracilaria

 

Steentoft, L.Irvine & Farnham and Gracilariopsis longissima (S.G.Gmelin) Steentoft, L. Irvine & Farnham, (as Gracilaria verrucosa (Hudson) Papenfuss or Gracilaria confervoides (L.) Greville), has prevented recognition of the northern boundaries.

 


 

Use : Gracilaria is used as a food in Japanese, Hawaiian, and Filipino cuisine. In Japanese cuisine, it is called ogonori or ogo. In the Philippines, it is called gulaman or guraman.





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The nutrient broth includes complex mixture of compounds that support metabloic activity of bacteria and provide nutrients for growth and cell division.

Agar or agar-agar is a gelatinous substance derived by boiling a polysaccharide in red algae, where it accumulates in the cell walls of agarophyte and serves as the primary structural support for the algae's cell walls. Agar is a mixture of two components: the linear polysaccharide agarose, and a heterogeneous mixture of smaller molecules called agaropectin.


Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium for microbiological work. Agar (agar-agar) can be used as a laxative, an appetite suppressant, vegetarian gelatin substitute, a thickener for soups, in fruit preserves, ice cream, and other desserts, as a clarifying agent in brewing, and for sizing paper and fabrics.

The gelling agent is an unbranched polysaccharide obtained from the cell walls of some species of red algae, primarily from the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). For commercial purposes, it is derived primarily from Gelidium amansii. In chemical terms, agar is a polymer made up of subunits of the sugar galactose.

Background

Agar consists of a mixture of agarose and agaropectin. Agarose, the predominant component of agar, is a linear polymer, made up of the repeating monomeric unit of agarobiose. Agarobiose is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agaropectin is a heterogeneous mixture of smaller molecules that occur in lesser amounts.

Agar exhibits hysteresis, melting at 85 °C (358 K, 185 °F) and solidifying from 32-40 °C (305-313 K, 90-104 °F). This property lends a suitable balance between easy melting and good gel stability at relatively high temperatures. Since many scientific applications require incubation at temperatures close to human body temperature (37 °C), agar is more appropriate than other solidifying agents that melt at this temperature, such as gelatin.

The word "agar" comes from agar-agar, the Malay name for red algae (Gigartina, Gracilaria) from which the jelly is produced. It is also known as kanten, China grass, Japanese isinglass, Ceylon moss or Jaffna moss. Gracilaria lichenoides is specifically referred to as agal-agal or Ceylon agar.

Uses

Microbiology

Agar is used throughout the world to provide a solid surface containing medium for the growth of bacteria and fungi. Microbial growth does not destroy the gel structure because most microorganisms are unable to digest agar. Agar is typically sold commercially as a powder that can be mixed with water and prepared similarly to gelatin before use as a growth medium. Other ingredients are added to the agar to meet the nutritional needs of the microbes. Many specific formulations are available, because some microbes prefer certain environmental conditions over others.

Motility assays

As a gel, an agarose medium is porous and therefore can be used to measure microorganism motility and mobility. The gel's porosity is directly related to the concentration of agarose in the medium, so various levels of effective viscosity (from the cell's "point of view") can be selected, depending on the experimental objectives.

A common identification assay involves culturing a sample of the organism deep within a block of nutrient agar. Cells will attempt to grow within the gel structure. Motile species will be able to migrate, albeit slowly, throughout the gel and infiltration rates can then be visualized, whereas non-motile species will show growth only along the now-empty path introduced by the invasive initial sample deposition.

Another setup commonly used for measuring chemotaxis and chemokinesis utilizes the under-agarose cell migration assay, whereby a layer of agarose gel is placed between a cell population and a chemoattractant. As a concentration gradient develops from the diffusion of the chemoattractant into the gel, various cell populations requiring different stimulation levels to migrate can then be visualized over time using microphotography as they tunnel upward through the gel against gravity along the gradient.

Plant biology

Research grade agar is used extensively in plant biology as it is supplemented with a nutrient and vitamin mixture that allows for seedling germination in Petri dishes under sterile conditions (given that the seeds are sterilized as well). Nutrient and vitamin supplementation for Arabidopsis thaliana is standard across most experimental conditions. Murashige & Skoog (MS) nutrient mix and Gamborg's B5 vitamin mix in general are used. A 1.0% agar/0.44% MS+vitamin dH2O solution is suitable for growth media between normal growth temps.

The solidification of the agar within any growth media (GM) is pH-dependent, with an optimal range between 5.4-5.7. Usually, the application of KOH is needed to increase the pH to this range. A general guideline is about 600 µl 0.1M KOH per 250 ml GM. This entire mixture can be sterilized using the liquid cycle of an autoclave.
plants growing in agar dish
This medium nicely lends itself to the application of specific concentrations of phytohormones etc. to induce specific growth patterns in that one can easily prepare a solution containing the desired amount of hormone, add it to the known volume of GM, and autoclave to both sterilize and evaporate off any solvent that may have been used to dissolve the often-polar hormones. This hormone/GM solution can be spread across the surface of Petri dishes sown with germinated and/or etiolated seedlings.

Experiments with the moss Physcomitrella patens, however, have shown that choice of the gelling agent — agar or Gelrite - does influence phytohormone sensitivity of the plant cell culture.

Molecular biology

Agar is a heterogeneous mixture of two classes of polysaccharide: agaropectin and agarose. Although both polysaccharide classes share the same galactose-based backbone, agaropectin is heavily modified with acidic side-groups, such as sulfate and pyruvate.

The neutral charge and lower degree of chemical complexity of agarose make it less likely to interact with biomolecules, and, therefore, agarose has become the preferred matrix for work with proteins and nucleic acids. Gels made from purified agarose have a relatively large pore size, making them useful for separation of large molecules, such as proteins and protein complexes >200 kilodaltons, as well as DNA fragments >100 basepairs. Agarose has been used widely for immunodiffusion and immunoelectrophoresis, as the agarose fibers functions as an anchor for immunocomplexes. Agarose is used generally as the medium for analytical scale electrophoretic separation in agarose gel electrophoresis and for column-based preparative scale separation as in gel filtration chromatography and affinity chromatography.

Culinary

Agar-agar is a natural vegetable gelatin counterpart. White and semi-translucent, it is sold in packages as washed and dried strips or in powdered form. It can be used to make jellies, puddings, and custards. For making jelly, it is boiled in water until the solids dissolve. Sweetener, flavouring, colouring, fruit or vegetables are then added and the liquid is poured into molds to be served as desserts and vegetable aspics, or incorporated with other desserts, such as a jelly layer in a cake.

Agar-agar is approximately 80% fiber, so it can serve as an intestinal regulator.

In Philippine cuisine, it is used to make the jelly bars in the various gulaman refreshments or desserts such as sago gulaman (aka gulaman at sago), buko pandan, agar flan, halo-halo, the various Filipino fruit salads, black gulaman, and red gulaman. One use of agar in Japanese cuisine is anmitsu, a dessert made of small cubes of agar jelly and served in a bowl with various fruits or other ingredients. It is also the main ingredient in Mizuyōkan, another popular Japanese food. In Vietnamese cuisine, jellies made of flavored layers of agar agar, called thạch, are a popular dessert, and are often made in ornate molds for special occasions. In Indian cuisine, agar agar is known as "China grass" and is used for making desserts. In Burmese cuisine, a sweet jelly known as kyauk kyaw  is made from agar. In Russia, it is used in addition or as a replacement to pectin in jams and marmalades, as a substitute to gelatin for its superior gelling properties, and as a strengthening ingredient in souffles and custards. Agar-agar may also be used as the gelling agent in gel clarification, a culinary technique used to clarify stocks, sauces, and other liquids.

Agar  of various qualities

Agar and the solidifying agents in solid growth media should be well chosen, with certain criteria to consider.

Agar is available in a range of qualities dictated by the target application. For example, Fluka brand agar  is highly purified and ideal when high transparency and brightness is needed, as in nutritional studies (Vitamin Assay Media) and sensitivity testing procedures, or when high purity and good diffusion of substances is essential. For identification and differentiation  using a purified or even highly purified agar is necessary. However, when isolating a single colony in most cases a standard quality will suffice. Typical solid media has an agar concentration of 1.0 - 1.5%, to accommodate the requirements of different applications and growth habits of the target microorganisms.

Agar Products -  Agar qualities for Microbiology 

Agar, highly purified
Agar, purified
Agar, standard quality
Agar, fibers
Select Agar
Bacteriological agar
Noble Agar

Ingredients for agar

Here are the ingredients for Nutrient Broth
proportions are for 1000ml
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Peptone 10gram
  Beef/Yeast Extract  3gram
  Sodium Chloride    5gram
  Distilled Water      
1000ml
Ph 7-7.2


If you want to make agar just add
30gram agar-agar to the above fluid.
This makes Nutrient Agar.

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Tuesday, October 23, 2012

Lemon Grass Cultivation













Scientific classification
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Kingdom           : Plantae
(unranked)        : Angiosperms
(unranked)        : Monocots
(unranked)        : Commelinids
Order               : Poales
Family              : Poaceae
Subfamily         : Panicoideae
Tribe                : Andropogoneae
Subtribe           : Andropogoninae
Genus              : Cymbopogon
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Cymbopogon (lemongrass) is a genus of about 55 species of grasses, (of which the type species is Cymbopogon citratus [a natural and soft tea Anxiolytic]) native to warm temperate and tropical regions of the Old World and Oceania. It is a tall perennial grass. Common names include lemon grass, lemongrass, barbed wire grass, silky heads, citronella grass,cha de Dartigalongue, fever grass, tanglad, hierba Luisa or gavati chaha amongst many others.

Uses

Lemongrass is native to India and tropical Asia. It is widely used as a herb in Asian cuisine. It has a subtle citrus flavor and can be dried and powdered, or used fresh.

Lemongrass is commonly used in teas, soups, and curries. It is also suitable for poultry, fish, beef, and seafood. It is often used as a tea in African countries such as Togo and the Democratic Republic of the Congo and Latin American countries such as Mexico.

Lemongrass oil is used as a pesticide and a preservative. Research shows that lemongrass oil has anti-fungal properties.

Despite its ability to repel insects, its oil is commonly utilized as a "lure" to attract honey bees. "Lemongrass works conveniently as well as the pheromone created by the honeybee's nasonov gland, also known as attractant pheromones. Because of this lemon grass oil can be used as a lure when trapping swarms or attempting to draw the attention of hived bees".


 Citronella grass (Cymbopogon nardus and Cymbopogon winterianus) grows to about 2 meters (about 6.5 feet) and has red base stems. These species are used for the production of citronella oil, which is used in soaps, as an insect repellent in insect sprays and candles, and also in aromatherapy, which is famous in Bintan Island, Indonesia. Therefore it's assumed that its origin is from Indonesia. The principal chemical constituents of citronella, geraniol and citronellol, are antiseptics, hence their use in household disinfectants and soaps. Besides oil production, citronella grass is also used for culinary purposes, in tea and as a flavoring.

Lemon Grass Oil, used as a pesticide and preservative, is put on the ancient palm-leaf manuscripts found in India as a preservative. It is used at the Oriental Research Institute Mysore, the French Institute of Pondicherry, the Association for the Preservation of the Saint Thomas Christian Heritage in Kerala and many other manuscript collections in India. The lemon grass oil also injects natural fluidity into the brittle palm leaves and the hydrophobic nature of the oil keeps the manuscripts dry so that the text is not lost to decay due to humidity.

East-Indian Lemon Grass (Cymbopogon flexuosus), also called Cochin Grass or Malabar Grass  is native to Cambodia, India, Sri Lanka, Burma,and Thailand while the West-Indian lemon grass (Cymbopogon citratus) is native to maritime Southeast Asia. It is known as serai in Malaysia, serai or sereh in Indonesia, and tanglad in the Philippines. While both can be used interchangeably, C. citratus is more suited for cooking. In India C. citratus is used both as a medical herb and in perfumes. Cymbopogon citratus is consumed as a tea for anxiety in Brazilian folk medicine, but a study in humans found no effect. The tea caused a recurrence of contact dermatitis in one case.

Lemon grass is also known as Gavati Chaha  in the Marathi language  and is used as an addition to tea, and in preparations like 'kadha,' which is a traditional herbal 'soup' used against coughs, colds, etc. It has medicinal properties and is used extensively in Ayurvedic medicine. It is supposed to help with relieving cough and nasal congestion.

Part used

leaves, steam distilled without pressure

Therapeutic Properties

Anti-Inflammatory

Anti-infectious

Antifungal

Calming

Spasmolytic

Aromatherapy uses

Florame organic aromatherapy's organic lemongrass essential oil can be used in both air diffusion and in massage. When used in air diffusion the scent has a citral (lemony) and violet perfume making it a very popular essential oil amongst perfume makers. When diffused lemongrass organic essential oil
 
helps to relieve stress and creates a sense of wellbeing. Lemongrass organic essential oil also acts as a mosquito repellent when used in air diffusion. When used in massage lemongrass organic essential oil can help to relieve the symptoms of mycosis fungoides. To help treat this condition, blend two drops of lemongrass with 5 drops of tea tree organic essential oil and five drops of palmarosa organic essential oil and dilute in 10ml of hazelnut organic oil. Massage the affected area twice daily until a difference in the skin is noted. Lemongrass essential oil also has strong anti-spasmodic properties when used in massage and help to relieve tension in muscles and joints but must be diluted to at least 5%.













Blends well with Bergamot organic essential oil, bourbon geranium organic essential oil, fine lavender organic essential oil, myrrh essential oil, cineol rosemary organic essential oil, niaouli essential oil, patchouli organic essential oil, jasmine essential oil, ylang-ylang extra essential oil, palmarosa organic essential oil, tea tree organic essential oil

Safety

Keep out of reach of children.

Avoid contact with eyes.

Do not apply undiluted to the skin.

Pregnant women should seek medical advice before use.

The Benefits of Lemongrass Extract

Lemongrass, or Cymbopogon citratus, is a tall, aromatic perennial grass native to tropical Asia. The freshly cut and dried leaves of the plant have been used traditionally as a flavoring agent. The volatile oils of the plants contain a chemical called citral, which gives it medicinal value. Lemongrass supplements are available as capsules, powders, liquid extracts and oils. The individual recommended dose varies depending on your age and overall health. Talk to a doctor before using lemongrass extract for medicinal purposes.

Cancer

Lemongrass extracts can induce apoptosis, or programmed cell death, in some cancer cell lines, says the Memorial Sloan-Kettering Cancer Center. However, these benefits have not been studied in actual patients and more research is needed before lemongrass extracts can be used in cancer treatment, says MSKCC. An animal study published in the journal “Carcinogenesis” also points out that lemongrass extracts can prevent DNA changes and thereby lower the risk of colon cancer in laboratory animals.

Antioxidant Activity

A 2005 study published in the “Journal of Agricultural and Food Chemistry” revealed that lemongrass extracts exhibit significant antioxidant activity and neutralize the unstable free radicals
 
formed as a result of various metabolic processes in the body. Unstable free radicals interact with the DNA and proteins of the cells in your body and can contribute to chronic diseases such as Alzheimer’s disease and cancer.

Infection

Lemongrass extracts also inhibit the growth of candida or yeast in the laboratory, according to an article in the February 2008 issue of the “Brazilian Journal of Infectious Diseases.” Although the study indicates that lemongrass may potentially treat fungal infections, actual clinical trials are needed to prove these benefits conclusively.

Heart Disease

A study published in the December 2002 issue of the "Asia Pacific Journal of Clinical Nutrition" reveals that polyphenols of lemongrass extract help relax the walls of the blood vessels and dilate them. This may, in turn, reduce the risk of high blood pressure and other cardiovascular diseases associated with it.

Anxiety

The essential oils of lemongrass also exhibit significant anti-anxiety activity by regulating certain neuroreceptors in the brain, as per a study in the September 2011 issue of the “Journal of Ethnopharmacology.” These results were demonstrated in laboratory animals; consult a doctor before using lemongrass extracts to treat anxiety.

How To Extract the Oil From Lemongrass

Extracting oils from plant material can be done in several ways. Two popular methods, tincturing and hot infusion, result in a medicinal oil to be taken orally in hot tea or straight onto the tongue or as therapeutic, topical oil. Lemongrass is said to act as a fungicide when applied topically or to aid in digestion when taken as a tea. It can be purchased in certain markets or stores that cater to an Asian clientele, as it is used primarily as an ingredient in Thai cuisine.

1 . Break fresh lemongrass stalks and fill the canning jar halfway with them. Breaking the stalks allows the natural oils to be released from the plant and strengthen the tincture.

2 . Fill the jar half with alcohol and half with cold water. Vodka or brandy are the most common alcohols for making tinctures, but gin is used as well. Purchase the best quality liquor you can. If for whatever reason you cannot use alcohol, a half-and-half solution of white or apple cider vinegar and water is a suitable substitution. The potency of the medicine will be only slightly lessened by using vinegar and it will make the tincture safe for people who may have adverse reactions to alcohol.

3. Cover the lid and gently shake the herbs and alcohol solution. Allow the herbs to settle and look to see that all the plant material is covered by liquid. Even a small bit peeking out could mold during the tincturing process, ruining your tincture. Add more liquid if need be.

4. Place the medicine in a cool, dark room and wait three days. Then, pour the solution into a blender and blend the plant material. This will allow greater absorption between plant material and the liquid, particularly because lemongrass is such a woody plant. Put the blended liquid back into the jar and store for at least three weeks.

5. Strain the liquid from the plant material. Put a colander over a pot and lay a cheesecloth over the colander. Dump the tincture in and make a bundle with the plant material and cheesecloth. Squeeze as much liquid as you can out of the lemongrass.

6 . Pour the tincture into a clean glass jar and store until ready to use. Tinctures are commonly taken directly on the tongue but adding the medicine to a cup of warm tea or water may be a more palatable solution.
Making a Hot Oil Infusion

7. Pour 1 cup of extra virgin olive oil into the top pan of a double boiler. Do not cover the pot.

8. Crush half a stalk of lemongrass, chopping if necessary, and place it in the oil. Do not let any water get into the oil or it will ruin the infusion. Don't wash the lemongrass before you put it in the pot. If it is still wet from the market, let it dry before placing it in the oil.

9 . Heat the double boiler until the water on the bottom begins to steam. Turn it down to a gentle simmer. Infuse the lemongrass in the oil for at least an hour, but the longer the better. Be sure to check on the water in the bottom pan to make sure it hasn't all evaporated.

10. Cool the oil down and strain the herbs through a mesh strainer. Use the oil as a topical ointment or as a soothing massage oil.

How to Extract Myrcene From Lemongrass


Extract the myrcene from lemongrass to add to your homemade perfumes.

Myrcene is obtained from essential oils. It can be found in several plant species including bay, Ylang-Ylang, hops, wild thyme and lemongrass. Once the essential oil has been extracted from the plant, myrcene acts as the main ingredient in perfumes. There are several ways in which essential oils can be extracted from the lemongrass plant, including one which utilizes items you will have at home. You can extract your own oil from lemongrass and use it as an ingredient in homemade products.

Instructions

1 Wash the lemongrass stalks and cut into small pieces. Cutting the grass will help to release the oils.

2 Place the lemongrass into the wide-mouth jar. Fill the jar with oil. You can choose from several kinds of        oil. Choose an oil that has a light color and little odor. Good options include olive oil, sesame oil, safflower    oil or almond oil.

3 Put the lid on tightly and place in a sunny windowsill for 48 hours. Shake the jar every 12 hours.

4 Remove the lid and place the muslin cloth over the top of the jar. Drain the oil into the mixing bowl.                Squeeze oil from the lemongrass in the muslin cloth.

5  Refill the jar halfway with fresh, chopped lemongrass. Pour the oil back in and place the closed jar in the       windowsill for another 48 hours. Repeat this process until the oil contains enough myrcene to have the           scent   you desire. This process is known as enfleurage.





6  Expedite the process by placing the jar into a pot of cold water every day. Heat the oil until the water is         warm to the touch. Leave the jar in the pot for 10 minutes. This process is known as maceration.

7  Mix the myrcene with other ingredients to make perfume.













Tuesday, October 2, 2012

Tissue Culture























 Tissue culture is the growth of tissues or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as *broth   or **agar .

* Broth :   ( a liquid containing nutrients for culturing microorganisms- microbiology - a liquid medium containing proteins and other nutrients for the culture of bacteria: in vitro cultures -broth cultures of intestinal tissue -a liquid mixture for the preservation of tissue )

** Agar :  Agar or agar-agar is a gelatinous substance derived by boiling a polysaccharide in red algae, where it accumulates in the cell walls of agarophyte and serves as the primary structural support for the algae's cell walls. Agar is a mixture of two components: the linear polysaccharide agarose, and a heterogeneous mixture of smaller molecules called agaropectin.

Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium for microbiological work. Agar (agar-agar) can be used as a laxative, an appetite suppressant, vegetarian gelatin substitute, a thickener for soups, in fruit preserves, ice cream, and other desserts, as a clarifying agent in brewing, and for sizing paper and fabrics.

The gelling agent is an unbranched polysaccharide obtained from the cell walls of some species of red algae, primarily from the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). For commercial purposes, it is derived primarily from Gelidium amansii. In chemical terms, agar is a polymer made up of subunits of the sugar galactose.


 Tissue culture commonly refers to the culture of animal cells and tissues, while the more specific term plant tissue culture is being named for the plants.

What are Plant Hormones

Plant hormones, also know and plant growth regulator or PGRs, are signal molecules produced within the plant at extremely low concentrations. Plant hormones regulate cellular processes and growth of the plant in various ways. In tissue culture, plant hormones are added to the media to promote a certain type of growth (division, root formation, etc.) depending on what is desired by the propagator at the time.

What is a callus

A callus of cells is a mass of undifferentiated plant cells. It often starts as a lumpy growth on existing tissue and can develop into odd-looking, perhaps "hairy" masses as many tiny plantlets begin to emerge from the callus.

Plant cell culture methods



Plant cell cultures are typically grown as cell suspension cultures in a liquid medium or as callus cultures on a solid medium. The culturing of undifferentiated plant cells and calli requires the proper balance of the plant growth hormones auxin and cytokinin.

Historical usage

In 1885 Wilhelm Roux removed a section of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the basic principle of tissue culture. In 1907 the zoologist Ross Granville Harrison demonstrated the growth of frog nerve cell processes in a medium of clotted lymph.

 
In 1913, E. Steinhardt, C. Israeli, and R. A. Lambert grew vaccinia virus in fragments of guinea pig corneal tissue. In 1996, the first use of regenerative tissue was used to replace a small distance of a urethra, which led to the understanding that the technique of obtaining samples of tissue, growing it outside the body without a scaffold, and reapplying it, can be used for only small distances of less than 1 cm.

Modern usage


In modern usage, tissue culture generally refers to the growth of cells from a tissue from a multicellular organism in vitro. These cells may be cells isolated from a donor organism, primary cells, or an immortalised cell line. The term tissue culture is often used interchangeably with cell culture

The literal meaning of tissue culture refers to the culturing of tissue pieces, i.e. explant culture.

Tissue culture is an important tool for the study of the biology of cells from multicellular organisms. It provides an in vitro model of the tissue in a well defined environment which can be easily manipulated and analysed.

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation. 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.

To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture.

Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). Single cells, plant cells without cell walls (protoplasts), pieces of leaves, or (less commonly) roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.

Choice of explant

The tissue obtained from the plant to culture is called an explant. Based on work with certain model systems, particularly tobacco, it has often been claimed that a totipotent explant can be grown from any part of the plant. However, this concept has been vitiated in practice. In many species explants of various organs vary in their rates of growth and regeneration, while some do not grow at all. The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased with inappropriate explants. Thus it is very important that an appropriate choice of explant be made prior to tissue culture.

The specific differences in the regeneration potential of different organs and explants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle, the availability of or ability to transport endogenous growth regulators, and the metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic ends of the plants like the stem tip, auxiliary bud tip and root tip. These tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins.

The pathways through which whole plants are regenerated from cells and tissues or explants such as meristems broadly fall into three types:

The method in which explants that include a meristem (viz. the shoot tips or nodes) are grown on appropriate media supplemented with plant growth regulators to induce proliferation of multiple shoots, followed by rooting of the excised shoots to regenerate whole plants,

The method in which totipotency of cells is realized in the form of de novo organogenesis, either directly in the form of induction of shoot meristems on the explants or indirectly via a callus ( unorganised mass of cells resulting from proliferation of cells of the explant) and plants are regenerated through induction of roots on the resultant shoots,

Somatic embryogenesis, in which asexual adventive embryos( comparable to zygotic embryos in their structure and development) are induced directly on explants or indirectly through a callus phase.

The first method involving the meristems and induction of multiple shoots is the preferred method for the micropropagation industry since the risks of somaclonal variation (genetic variation induced in tissue culture) are minimal when compared to the other two methods. Somatic embryogenesis is a method that has the potential to be several times higher in multiplication rates and is amenable to handling in liquid culture systems like bioreactors.

Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that become problematic during the tissue culture process. Certain soil microflora can form tight associations with the root systems, or even grow within the root. Soil particles bound to roots are difficult to remove without injury to the roots that then allows microbial attack. These associated microflora will generally overgrow the tissue culture medium before there is significant growth of plant tissue.

 

Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed from the explant by gentle rinsing, and the remainder usually can be killed by surface sterilization. Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization or localized necrosis on the surface of the explant.

An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to penetration of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues.

Tissue cultured plants are clones, if the original mother plant used to produce the first explants is susceptible to a pathogen or environmental condition, the entire crop would be susceptible to the same problem, conversely any positive traits would remain within the line also.

Applications

Plant tissue culture is used widely in plant science; it also has a number of commercial applications. Applications include:

Micropropagation is widely used in forestry and in floriculture. Micropropagation 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 in bioreactors for production of valuable compounds, like plant-derived secondary metabolites and recombinant proteins used as biopharmaceuticals.

To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.
To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo Rescue).

For production of doubled monoploid (dihaploid) plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes, usually by treatment with colchicine which causes doubling of the chromosome number.

As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants.

Certain techniques such as meristem tip culture can be used to produce clean plant material from virused stock, such as potatoes and many species of soft fruit.

Micropropagation using meristem and shoot culture to produce large numbers of identical individuals.
Production of identical sterile hybrid species can be obtained.

Laboratories

Although some growers and nurseries have their own labs for propagating plants by the technique of tissue culture, a number of independent laboratories provide custom propagation services. The Plant
 
Tissue Culture Information Exchange lists many commercial tissue culture labs. Since plant tissue culture is a very labour intensive process, this would be an important factor in determining which plants would be commercially viable to propagate in a laboratory.

The following plants can be produced by using tissue culture method :

Ornamental Plants
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MARANTHA
SYNGONIUM
CALLALILY
LIMONIUM
FERNS
CORDYLINE
DRACENA
SPATHIPHYLLUM
FICUS
PHILODENDRON
VENUS FLY TRAP
ORCHIDS
ROSE - Miniature
CALADIUM
DIFFENBACHIA
YUCCA
MARIGOLD
CALATHEA
ALPINIA
GENTIANA
CARNATION
BEAUCARNIA
CACTUS
LEGULARIA
AGLOENEMA
POMEGRANATE
PINEAPPLE
WAX FLOWER
Commercial Crops

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SUGARCANE
TURMERIC
POTATO
ASPARAGUS
BITTER GUARD
TAPIOCA



Tree Crops
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CASUARINA
BAMBOO
PAULOWNIA
NEEM
EUCALYPTUS



Fruit Crops
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BANANA
PRUNUS

TOMATO
STRAWBERRY
WATERMELON


Medicinal Plants
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PATCHOULI
MUSLI
ROSEMARY
THYME
VANILA
GLORIOSA
TULSI - Ocimum
CURRY LEAF
NAGABASHANAM
GYMNEMA SYLVESTRES
NAGATHALI
ALOE VERA
AROKYAPATCHAI
GERANIUM
STEVIA


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Starting of plant tissue culture - various steps

Plant Tissue Culture Media Preparation

Plant Tissue Culture owes its origin to the ideas of the German Scientist, Haberlandt, in the beginning of the 20th century. This was just the beginning of the tissue culture; thereafter in 70’s began the commercialization of the technology. Currently the world is revolving around it due to predicated future grain shortage; green house effect and total environmental disbalance.

Various steps have been taken in this field and have been introduced an extensive range of Ready to use PTC Medium; PTC Medium Ingredients like

Plant Growth Regulators or Phytoharmones (Auxins; Cytokinins; Gibberellins; Abscisic and & Others),

Macro & Micro Nutrients (Nitrogen; Potasssium; Phosphorus; Sulphur; Magnesium; Calcium;
                                Boron; Zinc; Iron; Iodine; Vitamins; Amino acids; Carbohydrates & Others),
Gelling Agents (Gelrite; Agar Agar & Others); 

Adsorbing Agents; 
Buffering Agents;
Pure Antibiotics and range of products used in the process.

Plant Tissue Culture Media Preparation is based on the unique property of the cell-totipotency. The cell-totipotency is the ability of the plant cell to regenerate into whole plant. In this process the excised bud is transferred into a tube containing a sterile nutrient medium. The success of tissue culture depends very much on the stage of explant selected, the sterilization period and the type of culture media used; different types of plants require different sets of culture media. Plant tissues are grown in vitro on artificial media, which supply the nutrients necessary for growth. The success of plant culture as a means of plant propagation is greatly influenced by the nature of the culture medium used. The rich tissue culture media provides a good nutrient source for bacteria and fungi, therefore precautions against microbial contamination must be taken in all in vitro procedures.

Tissue culture media used for in vitro cultivation of plant cells are composed of following basic components:
  • Complex mixture or Inorganic nutrients: Essential elements, or Mineral ions
  • Organic supplement: Vitamins and/or Amino acids
  • Carbon source: Sugar Sucrose
  • Gelling agents or Solidifying agents
  • Plant Growth Regulators
  • Growth Promoters
  • Complex organics
  • Activated charcoal
  • pH
  • Antibiotics

Complex mixture of salts is Inorganic nutrients (both micro- and macro-elements : C, H, O, N, P, S, Ca, K, Mg, Fe, Mn, Cu, Zn, B, Mb). For healthy and vigorous growth, intact plants need to take up from soil: relatively large amounts of some inorganic elements (the major plant nutrients): ions of Nitrogen (N), Potassium (K), Calcium (Ca), Phosphorous (P), Magnesium (Mg) and Sulphur (S). And some other elements in small quantities (minor plant nutrients or trace elements): Iron (Fe), Nickel (Ni), Chlorine (Cl), Manganese (Mn), Zinc (Zn), Boron (B), Copper (Cu), and Molybdenum (Mo). Among the micronutrients, iron requirement is very critical. Chelated form of Iron and Copper are commonly used in culture media.

Organic supplement: Vitamins and/or Amino acids (e.g. Nicotinic acid, Thiamine, Pyridoxine and myo-Inositol (Viamin B), Amino acids (e.g. Arginine) are essential for the culture of plant cells in vitro. The plant cells in culture are able to synthesize vitamins just like natural plants, but in suboptimal quantities which does not support proper growth of cells in culture. Therefore the medium is supplemented with vitamins to achieve good growth of cells. Similarly amino acids are added to the cell cultures to stimulate the cell growth and estabilish the cell lines. The most commonly used amino acid is glycine. However, Asparagine, Aspartic acid, Alanine, Glutamic acid, Glutamine and Proline are also used. Amino acids provide a source of reduced nitrogen and, like ammonium ions, uptake causes acidification of the medium. Organic acids especially the intermediates of krebs cycle e.g. Citrate, Malate, Succinate, Pyruvate also enhances the growth of plant cells. Sometimes antibiotics (e.g. Streptomycin, Kanamycin) are also added to the medium to prevent the growth of the microorganisms.

Carbon sources: usually sugar Sucrose (usually sucrose) - However, plant cells and tissues in the culture medium are heterotrophic and therefore depend on the external carbon for energy. During the sterilization of the medium, Sucrose gets hydrolyzed to glucose and fructose and the plant cells utilize first the glucose and than the fructose. The other carbohydrates such as Lactose, Maltose, Galactose etc., have been used in culture media but with limited success.

Gelling or Solidifying agents like Agar. Plant tissue culture media can be used in either liquid or ‘solid’ forms, depending on the type of culture being grown. Generally, a gelling agent Agar (a polysaccharide obtained from red algae, Gelidium amansil) is added to the liquid medium for its solidification. The agar obtained from seaweeds provides solid surface for the growth of cells because in the liquid medium, the tissue will be submerged and die due to lack of oxygen. Cells are grown in suspension medium without agar but such cultures are aerated regularly either by bubbling sterile air or by gentle agitation. Some other less frequently used solidifying agents are Gelrite, Biogel (polyacrlyamide pellets), Phytagel, and purified Agarose, as can a variety of Gellan gums.

Growth regulators (e.g. Auxins, Cytokinins and Gibberellins) - Plant hormones play an important role in growth and differentiation of cultured cells and tissues. There are many classes of plant growth regulators used in culture media involves namely: Auxins, Cytokinins, Gibberellins, Abscisic acid, Ethylene, 6 BAP (6 Benzyladenine), I.A.A (Indole Acetic Acid), I.B.A (Indole - -3- Butyric Acid), Zeatin, Zeatin Riboside. 

  • The Auxins facilitate the cell division and root differentiation. Auxins induce cell division, cell elongation, and formation of callus in cultures. For eg., 2,4-dichlorophenoxy acetic acid is one of the most commonly added auxins in plant cell cultures.
  • The Cytokinins induce cell division and differentiation. Cytokinins, promotes RNA synthesis and stimulate protein and enzyme activities in tissues. Kinetin and benzyl-aminopurine are the most frequently used cytokinins in plant cell cultures.
  • The Gibberellins is mainly used to induce plantlet formation from adventive embryos formed in culture.
  • Abscisic acid is used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis. Abscisic acid (ABA) inhibits cell division.
  • Ethylene is associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture.
As in Plant tissue culture media the ratio of auxins and cytokinins play an important role in the morphogenesis of culture syatems. When the ratio of auxins to cytokinins is high, embryogenesis, callus initiation, and root initiation occur. For axillary proliferation and shoot proliferation, the ratio of auxins to cytokinins is kept low. GA3 enhances the callus growth and induces dwarf plantlets to elongate. 

Plant growth promoters are α - Naphthalene acetic acid, Humic acid (granular/ powder) etc. Plant growth promoters (PGP) are those substances used for better management of nutrients and plant growth. Many times some crops fail to produce optimum yields in spite of proper nutrient supply. Physiological inefficiency in plants is responsible for such effects. Therefore, these PGA play a major role in seed germination, fruit ripening, enhances uptake of nutrients, boost protein synthesis, augment immunity and helps to withstand stress conditions, reduce flowering and fruiting drop and help in better plant growth.

Complex organics like Casein hydrolysate, Coconut milk, Malt extract, Yeast extract, Tomato juice, etc. may be added for specific purposes. Casein hydrolysate has given significant success in tissue culture and potato extract also has been found useful for anther culture. However, these natural extracts are avoided as their composition is unknown and vary from lot to lot and also vary with age affecting reproducibility of results.

Activated charcoal acts both in promotion and inhibition of culture growth depending upon plant species being cultured. It is reported to stimulate growth and differentiation in orchids, carrot, ivy and tomato whereas inhibits tobacco, soybean etc. It absorbs brown-black pigments and oxidized phenolics produced during culture and thus reduce toxicity. It also absorbs other organic compounds like PGRs, vitamins etc which may cause the inhibition of growth. Another feature of activated charcoal is that it causes darkening of medium and so helps root formation and growth.

pH - affects absorption of ions and also solidification of gelling agent. An optimum pH for culture media is 5.8 before sterilization. Values of pH lower than 4.5 or higher that 7.0 greatly inhibit growth and development in vitro. The pH of culture media generally drops by 0.3 to 0.5 units after autoclaving and keeps changing through the period of culture due to oxidation and also differential uptake and secretion of substances by growing tissue. At pH higher than 7.0 and lower than 4.5, the plant cells stop growing in the medium. 


Antibiotics used as Antmicrobial agents in Plant tissue culture media. The combination of antibiotics and chemical biocides has been tested for their ability to inhibit microbial contamination in plant cultures.

Antibiotics help in suppressing the bacterial infections in plant cell and tissue culture. Also helps in suppressing mould and yeast infections in cell cultures. Whereas, helps in elimination of Agrobacterium species after the transformation of plant tissue. The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement. The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. An alternative method is agroinfiltration.
The elements listed above are - together with Carbon (C), Oxygen (O) and Hydrogen (H) - the 17 essential elements. Certain others, such as Cobalt (Co), Aluminium (Al), Sodium (Na) and Iodine (I), are essential or beneficial for some species but their widespread essentiality has still to be established.
According to Epstein (1971), elements can be considered to be essential for plant growth if :
 
  • A plant fails to complete its life cycle without it
  • Its action is specific and cannot be replaced completely by any other element
  • Its effect on the organism is direct, not indirect on the environment
  • It is a constituent of a molecule that is known to be essential.
  •  
PLANT TISSUE CULTURE MEDIUM PREPARATION

For 1liter of growth medium, this is enough to prepare about 100 growing tubes.
  • Take a required amount of Murashige and Skoog Basal Salts with minimal organics medium (MSMO).
  • For 1liter of Murashige and Skoog Basal Salts with minimal organics medium, use a 2 liter container (because the medium boils up).
  • Add the dehydrated medium to the water and stir for a minute to dissolve completely.
  • Add the desired heat stable supplements to the medium solution.
  • Weigh out 6gms of Agar.
  • Add it to the MSMO solution.
  • Heat the solution gently while stirring until all the agar has dissolved.
  • Add more distilled water to make the total volume up to 1liter.
  • Pour the still-warm medium into the polycarbonate tubes to a depth of about 15 mm.
  • Place the tubes (with lids sitting on the tubes but not tightened) in an autoclave and sterilise for 20 minutes. (Note: Higher temperature may result in poor cell growth).
  • Cool the autoclve, then remove the tubes and tighten the lids.
  • Add heat labile supplements after autoclaving.
  •  
Plant Tissue Culture Media - 
 
  • Banana Micropropagation Medium
  • Banana Multiplication Medium
  • Gamborg Medium
  • Knudson-C Orchid Medium
  • The most extensively used nutrient medium is MS medium (developed by Murashige and Skoog in 1962) and many more...
  •  
Plant Tissue Culture Media Ingredients -
Macro & Micro Nutrients
  • Ammonium chloride
  • Ammonium molybdate
  • Ammonium sulphate
  • Potassium nitrate
  • Potassium sulphate
  • Sodium nitrate
  • Urea
  • Zinc nitrate
  • and many more...
Carbohydrates
  • Dextrose
  • Fructose
  • Maltose
  • D (+) Mannose
  • D- Ribose
  • D- Sorbitol
  • Sucrose
  • Tomato powder
  • and many more...

Amino acids
  • Aspartic acid
  • Glycine
  • L- Lysine mono- HCl
  • L- Proline
  • L- Tyrosine
  • and many more...
Plant Growth Regulators
  • IAA
  • IBA
  • NAA
  • 2,4,5 -T
  • and many more...

Antibiotics
  • Amicillin Sodium
  • Ciprofloxacin
  • Kanamycin sulphate
  • Gentamycin
  • Polymixin B sulphate
  • Vancomycin
  • and many more...
Vitamins
  • L - Ascorbic acid
  • D (+) Biotin
  • Niacin
  • Riboflavin
  • Retinol acetate
  • Thiamine HCl
  • and many more...

Gelling Agents
  • Agar Agar
  • Agarose
  • Gelrite
  • Gellan Gum
  • and many more...
Organic acids
  • Citric acid
  • Casein enzymatic hydrolysate
  • Malt extract
  • Soya peptone
  • Yeast extract
  • and many more...

Buffering agents
  • ACES buffers
  • CHAPS buffers
  • HEPES buffers
  • MES buffers
  • and many more...