Pharma Tips

Plant Cell Cultures

By: Pharma Tips | Views: 8615 | Date: 01-May-2011

The technique of plant tissue and cell culture has evolved over several decades.this technique combined with recent advances in developmental, cellular, molecular, genetics, metabolic engineering, genetic transformation and using conventional plant breeding have turned plant biotechnology into an exciting research field with a significant impact on pharmaceutical industries, agriculture, horticulture and forestry.


            The technique of plant tissue and cell culture has evolved over several decades.this technique combined with recent  advances in developmental,  cellular,  molecular,  genetics,  metabolic engineering,  genetic transformation and using conventional plant breeding have turned plant biotechnology into an exciting research field with a significant impact on pharmaceutical industries,  agriculture, horticulture and forestry.


Gottlieb haberlandt accomplished the first successful plant tissue culture at the turn of the 20th century when he reported the culture of leaf mesophyll tissue and hair cells.(22)

            Schwan (1839) expressed the view that each living cell of multicellular organism should be capable of independent development if provided with proper external conditions(22). More ever, Haberlandt  pressed the view that each living cell of multicellular organism should be capable of independent development if provided with proper external conditions(22). Hannig (1904) cultured nearly matured embryo excised from seeds of several species of crucifers.(23). More ever, haberlandts lack of success was not invain because one of his students, Kotte (1922) from Germany reported the growth of isolated root tips on a medium consisting of inorganic salts(19).At the same time Robbins (1922) reported the similar success with root and stem tips and white (1934) reported that,  not only could be cultured tomato root tips to grow, but they could be repeatedly sub cultured to fresh medium of inorganic salts supplemented with yeast extract,  which is a good source of vitamin B. White (1939) reported the growth promoting effects of thiamine isolated from tomato root tips(13).Went and Thiemann (1937) discovered the Indole Acetic Acid (IAA) , a natural auxin(10). Duhamet (1939) reported the stimulation of growth of excised roots by IAA(14).

            The avenue was now open for rapid progress in the successful culture of plant tissue cultures during 1930 with refined media,  La Rue (1936) achieved better success at culturing embryo compared to Haning (1904) Johannes Van Overbeek and his co workers (1942) reported that they were able to obtain seedlings from heart shaped embryos by enriching culture media with coconut milk in addition to the usual salts, vitamins and other nutrients(23). At the same time, Panchanan Maheshwari and co-workers from India were very active in angiosperm embryology research. Braun (1943) reported tumour induction related crown gall disease. Folke Skoog (1944) described the organ formation in cultured tissue and organs of tobacco.(19). Camus (1943) from Europe was the first to report grafting experiments in plant tissue cultures.haberlandt and his associates (1946) made improvements on the medium for the growth of tobacco and sunflower tissue and Morel (1948) was well into applying tissue technique to study of parasites associated with plant tissues(19). Street (1950) and his associates began a series of extensive studies on the nutrition of excised tomato root tips. Skoog and coworkers (1951) worked on investigations on the nutritional requirements of tobacco cells and they discovered a compound (kinetin) which promotes cells division(19). Stove and Yamaki (1957) discovered gibberellins another plant growth regulator(31). Crocker et al (1935) were the first to propose that ethylene was involved in fruit ripening.(10)   

           Murashige and Skoog (1962) developed a culture medium for the rapid growth of tobacco callus.(19) Morel (1965) reported the micropropagation of orchids.(19) Guha and Maheshwari (1966) reported the first successful culturing of haploid cells of datura.Cocking (1960) isolated the plant protoplast and began to be cultured in the media(16). This led to somatic hybridization. In early 1970’s discovery of endonuclease enzymes, which led to rapid development of gene transformation.the prospects of success with the genetics of plants have created considerable public interest. Melchers, Sacristan and Holder (1978) produced somatic hybrid plants from fusion of potato and tomato protoplasts.Several research  groups have produced transformed tobacco plants following single cell transformation (i.e. gene insertion, Chilton 1983)(13). The use of agrobacterium mediated plant transformation, and the use of the so called gene gun to shoot DNA into plant cells,  has led to the development of novel plants.The Calgene of UA is the first tocommerceialize the technique of production of transformed plants(19).

NUTRITIONAL REQUIREMENTS OF PLANT  CELL CULTURES.

Introduction
          There are three essential sources of nutrition for plants growing in nature. The mineral nutrients are obtained, along with water, from the root system .atmospheric carbon dioxide is used in the process of photosynthesis to provide carbon as a source of basic energy. Lastly,   the plant, particularly its meristematic regions and young organs such as leaves, using fixed carbon and minerals, synthesizes all of the vitamins and various plant growth substances that are critical and essential for normal growth and development of the plant.

          The requirements of plant tissues grown in vitro are similar in general to those of infact plants growing in nature .the nature of explant and the composition of the nutrient medium  generally determine the successful establishment and growth of plant cells in vitro. In the first culturing attempts, either media that were known from nutrition experiments with intact plants (knoop solution), or media consisting of juices and extracts of biological origin were used.   

Today, the only that is still commonly used is coconut milk, however only for monocotyledonous cultures. Mainly media of purely chemical composition are used. Media containing nutrients of plant origin that are chemically not precisely characterized are called complex or highly enriched media ,while those containing exclusively chemically defined compounds are called synthetic or regular media. To maintain the vital functions of a culture, the basic medium consisting of inorganic salts,organic compounds (amino acids, vitamins),growth regulators (phytohormones) and carbon sources recognized as essential.(2)

Inorganic Components

           Basic media are solutions salts in differents concentrations, called macro and micro nutrients. Cultured plant tissues required a continuous supply of certain inorganic chemicals. The macro nutrients are the compounds containing N, S, P, K, Mg, Cl  and Na added in concentrations of more than 30 ppm.in contrasts the elements added in the concentrations of less called micronutrients. The micronutrients are necessary as cofactor or enzyme synthesis e.g. nickel is essential for urease synthesis.

Macro Elements:

Sulfur
           It is primarily supplied as sulphate. Usually it is utilized for protein synthesis via sulphate respiration as soluble cysteine (99.9%) and a smaller proportion as soluble methionine .the sulphate supply is directly incorporated only beneath a minimal concentration threshold. In a medium lacking sulfur or in the presence of growth limiting factors, e.g. the nonprotein Amino acid djenkolic acid (H-COOH (NH2) -CH2-s-CH2-s-CH2-CCH(NH2) -COOH), this becomes a apparent due to a five to ten fold increase in the level of ATP-dependent sulphurylase .The sulphur requirements of a culture vary depending on the object (0.5 to 10 mm) .Inorganic sulphur may be replaced by organically bound sulphur (dl-cysteine,  dl-methionine,  dl-homocysteine and glutathione) .

Phosphorus
It is commonly added as phosphate at concentrations of 1.1-1.25mm. Due to rapid uptake and interactions with other components (Fe, k, sucrose), deficiencies may rapidly arise in a medium. In addition, its uptake is influenced by the supply of other elements. For example, boron deficiency induces in daucus carota cultures a reduction in the phosphorus uptake capacity.
   
Nitrogen
            Most standard media offer nitrogen NH4+ and NO3-.Individual cultures (cannabis sativa, ipomoea, daucus carota) prefer NH4+ under certain conditions. Utilization of NO 3- requires functioning nitrate reductase, the presence of which has by now been described in numerous callus and suspension culture.

Magnesium, Potassium, Calcium
These cations play an essential role in cell metabolism. For e.g., Mg2+ is one of the essential  factors in translation. It acts as a cofactor (e.g. Glutamine synthase, gs) and activator of various enzymes. Therefore, not least in photoautotrophic cultures,  it is of central significance+,  and especially Ca+2, inhibit enzymes such as the glycolysis enzyme pyruvate kinase, while others requires Ca+2to maintain their activity (NAD-kinase, Protein-kinase,  α-amylase) or stability (α-amylase). The  Ca+2 triggered binding of pectic acid, polyerization product of galacturonic acid, to calcium pectate is an elementary step in cell wall formation. Ca+2 are also required for deposition of phospholipids and proteins on within plasma membranes. Its importance is further demonstrated by the efforts of cells to maintain their intracellular concentration 10-6 to 10-8 mm even against a concentration gradient using specific Ca+2  pumps and Ca+2 binding proteins (calmodulin) located in the cytoplasm and/or individuals organelles. The concentration increase to a value of 10-5mm  induced by the sesquiterpenoid phytohormone  abscisic acid (ABA) , an apo-carotenoid and by light is only  temporary  and  is the basis of its signaling  effect in its function  as a second  messenger. In Nicotiana tabacum culture deficiency in nitrate reductase, an increased level of Ca+2 induces increased ammonium utilization.  Chlorine plays a role by binding to positively charged histidine residues of proteins like the enzymes of the photosystem 2 and atpases of the tonoplast and by influencing osmoregulation.

Micronutrients
            The Fe, Mn, Zn, Cu, Mo,  B,  Co,  and Ni act as cofactors and as inducers of enzyme synthesis,  as for example nickel in urease synthesis in tobacco,  rice and soybean cell suspension cultures. Boron is essential for membrane function, permeability and integrity. Therefore, membrane fixed processes like ATP-ase; membrane potential and iron flow and phytohormone metabolism are influenced. Lack of iron results in increased contents of DNA and free amino acids, as well as a reduced RNA content. In order to maintain a minimum supply of Fe it is therefore usually added in complexes with EDTA or sequestrin. This also facilitates uptake over a broad pH range, which varies depending on the content of phosphate, NO3- and NH4+ in the medium. Although iodine in the form of KI is a constituent of several media, the necessity of this element remains questionable. A trace of cobalt is found in several media and yet this element is not known to have any function in higher plants.

Organic   Components

Amino acids
           Amino acids are added for substitution or augmentation of the nitrogen supply. It is to be noted that threonine, glycine and valine reduce ammonium utilization by inactivating glutamate synthase located in chlorophasts and cytoplasm. Arginine is usually able to compensate this inactivation.

Vitamins
           Plant cells are usually autotrophic with respect to vitamins. However, in most cases, the amount of vitamins synthesized even in photosynthetically active cells and tissues is insufficient to guarantee a sufficient supply. Thiamine (vitamin B1) may be the only essential vitamin for nearly all plant tissue cultures, whereas nicotinic acid (Niacin) and Pyridoxine (vitamin B6) may stimulate growth. Thiamine is added as thiamine hydrochloride in amounts ranging from 0.1-10mg/l.
           Some other vitamins that have been used in tissue culture media include p-amino benzoic acid (PABA), ascorbic acid (vitamin C), Tocopherol (vitamin E),  Biotin (vitamin H) Choline chloride,  Cyanocobalamine (vitamin b12) ,  Folic acid,  Riboflavin (vitamin B2) and Calcium pantothenate.

Carbon source
All plant tissue culture media requires the presence of a carbon source and is added in the form of carbohydrates. Sucrose is usually added in concentration of 20-30g/l. often, myoinositol is also used. Occasionally less common carbon sources such as lactose, galactose, glycerin and unrefined natural carbon sources like coffea Arabica and daucus carota are also employed. The various carbon sources used in tissue culture medium are pentoses, uronic acid, molasses, whey, potato starch, grain starch.
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