Analysis of Plant Tissue Culture - Term Paper Example

Name : xxxxxx Tutor : xxxxxxx Title : PLANT TISSUE CULTURE AND THE EFFECTS OF AUXIN HORMONE ON IT Institution : xxxxxxx @2009 Table of Content Page Plant Tissue Culture 3 Introduction 3 Plant Cell Immobilisation 4 Particles of the Tissue Culture Medium 6 Preparation of Media 9 Callus Induction 10 Culture Suspension 12 Scaling Up 12 Importance 12 Summary 17 References 18 Plant Tissue Culture Introduction Plants are made up of small component such as cells, organs or tissue that can be manipulated to grow complete plants. Without molecular genetics and plant culture, it would be void to discuss genetic engineering as they are core to this. The technique of plant tissue culture involves placing a piece of plant such as embryo and dipping it in sterile nutrient median and the science behind it. This technique production of extra copies of plant, enhance maturity, enables production of plants without the use of seeds. Gottlied Haberlandt reported the culture of leaf mesophyll tissue and hair cells (Sugiyama 1999). This happed close to the end of 20th century was the first successful cell culture and plant tissue. He drew the ideas from the previous researches and knowledge on biology of plants. The cells cultured by Habertlandat did not divide and hence no fruits were achieved. It was possible that plant growth regulators (PRs) which are necessary during cell division, proliferation and embryo induction were absent in the culture medium. He was frustrated by this but his ideas have been used by other scientists. One of his students reported the growth of isolated root tips on a medium consisting medium salts (Sugiyama 1999). In the 1930s there was a progress in innovative plant tissue culture techniques after the B vitamins and natural auxin were discovered as a necessity in the growth of isolated tissues that contain meristem. Walden and Wingender (1995) reported thiamine as promoting growth on isolated tomato root tips. The first PGR, indoleacetic acid (IAA) was discovered after a series of ingenious experiments with Oat seedlings. IAA occurs naturally and is a member of auxins which is a class of PGR. The IAA stimulated growth in excised roots. Later there were several reported successful cases of plant culture but the progress was disrupted by world war two. Johannes and associates obtained seedlings by enriching culture media with milk obtained from coconut, usual salts, vitamin and other nutrients. Organs were formed from cultured tissues and organs as demonstrated by Folke Skoog. Citokinins such as adenine and kinetin were discovered to have vitro shoot-promoting effects for the rapid propagation of plants. This was so especially for the very important agronomic and horticultural cultivators. Gamborg (2002) investigated the culture of embryogenesis tissue. In order to fulfil Haberlandt’s objectives, root tips were to lead to organogenesis. Lainbch obtain hybrids after the maturity of culture medium. He isolated zygote embryos from non-viable seed of linum perenne x L austriacum and raised zygote embryos. The process of plant tissue culture includes: nutrient medium preparation, explants isolation, equipment and explants sterilisation including sterilisation of the medium, inoculation, incubation, hardening and the establishment of the plant in the field. Plant Cell Immobilisation Plant cell immobilisation is very important in that: Cells have extended viability in all stages hence biomass is maintained over a longer time period. The downstream process is simplified when the products are secreted. Differentiation is promoted and the secondary metabolism enhanced. Bioreactor size is reduced hence a higher density of the cell leading to reduced contamination risk and cost. Shear sensitivity is reduced especially when the cells are entrapped. In some cases, the secretion of secondary metabolites is promoted. Increased flow rate can be attained by use of floe-through reactors Fluid viscosity increase which leads to mixing and aeration problems in cell suspension is minimised. The immobilised system reduces production cost for phytochemicals dramatically. However, it exhibits other problems which include: If there is decoupled production from cell growth, there is limited immobilisation. Initial biomass has to be grown in suspension. There is imperative secretion of product to form extra cellular medium. There can be extra cellular degradation problems of the products if secretion occurs. There is a diffusion barrier introduced by the gel matrix if gel entrapment is in use. Various methods of immobilisation have been developed to deal with the problems above. The method include: adsorption, entrapment and covalent coupling. The aim is to acquire high productivity at a minimum cost. Over the years, the technology is being developed day by day. Plant tissue culture is today under filtered air and aseptic conditions. The surfaces of living plant materials have micro organisms. The plants are thus sterilised using chemical solutions. This can either be alcohol or bleach. These plants are then dipped in to a liquid or places on a solid medium. This medium has a combination of organic nutrients, vitamins, plant hormones and inorganic salts. A gelling agent is added to the liquid medium to form a solid medium. That means, for solid medium to be formed, a liquid medium has to be formed first. The composition of the medium affects the morphology of tissues growing from initial plant. Proliferation of roots is as a result of excess auxin while yielding of shoots may result from excess cytokinin. Unorganised cells grow if the two are in a balance. They are manipulated to acquire the desired result. Particles of the Tissue Culture Medium The tissue culture contains ninety five percent of water, sugars, vitamins, PGRs, micro and macro nutrients and organic material. A gelling agent, plant growth regulators and antibiotics are part of the culture media. The inorganic components are macro and micro salts. Their concentration is dependant on the on the species. A plant species is used to induce callus tissue. The success in the production of callus is dependant on the qualities of a plant and its species. The growth of callus from woody plants is slow. Explants materials from fresh and young explants are preferred. After the explants materials are collected, they are sterilised by use of sodium hypochlorite, ethanol and other chemicals with the aim of eliminating micro organisms. Different types of salt media which is made from inorganic compound have been designed for callus induction from explants, cultivation of the callus and suspended cells. Murashige and Skoog (MS) developed for tobacco tissue culture has a high nitrate, potassium, and ammonia concentration. Anothe4r medium is B5 medium which has lower levels of inorganic nutrients than MS medium. Instead of using different types of basal media, its preferable to use one or two types in combination of various types and concentrations of phytohormones. To obtain higher growth rate and levels of products, the medium found as most suitable is optimised. The complex salt mixtures include mineral irons as well as essential elements needed for plant nutrition including the physiological functions of these salts. They are grouped in to iron source, macronutrients and micronutrients. Carbon is very necessary in the medium. It can is got from 2 to 4% sucrose or glucose added in the medium. For production of useful metabolism efficiently, a most efficient carbon source and optimal concentration is chosen. This depends on the products that one wants to achieve and the species of the plant. Inexpensive but efficient source have much economic benefits. Sucrose is relatively stable and easily assimilates. Gelling agent commonly used is Agar. It is produced from seaweeds. Other gelling agents include purified agar or agarose (Ramage and Williams 2002).  Thiamine is an essential vitamin for most plant cells. In some cases, vitamins stimulate growth and are hence very necessary. The vitamins in medium include: nicotinic acid, myo-inosotol, pyridoxine and thiamine HCl. For callus tissue induction and growth promotion of cells, growth regulators known as phytohormones are required. Coconut milk has growth regulator and it is thus referred to as a growth regulators supplier. Plant growth regulators include auxins, cytokinins, gibberllins, abscisic acid and ethylene. Cytokinins are promoters of cell division. Gibberellins act as regulators of cell elongation, thus determining the height of plants and set of fruits. Abscisic acid promotes development of distinct pathways e.g. somatic embryogenesis. It also inhibits division of cells. Ethylene controls ripening of fruits but if it builds up in tissue culture, it inhibits culture growth and development. Antibiotics inhibit and eventually destroy other micro organism that negatively affects the growth of a plant tissue culture. This suppresses infections by bacteria, yeast and moulds in tissue culture and plant cell. After plant tissue has been transformed, they eliminate agro bacterium species (Fowler 2000). Preparation of Media Stock solutions made from the components described above are mixed to make the medium. 21.5 mg of kinetin is dissolved in 0.5 N HCl. This is achieved by slightly heating and gradually diluting the mixture using distilled water to 100 ml. Appropriate carbon and a given volume of each stock solution are added and mixed. 0.2 NKOH or 0.2 N HCl is vital in order to regulate the PH to 5.5. Deionised or distilled water is then added to the mixture till the required volume is achieved. For solid medium, 0.6 -1.0% volume or weight of Agar is added. This medium is distributed into vessels and then sterilised using an autoclave for 15 minutes. It is sterilised at 120 C but the conditions vary depending on the volume of the vessel and the amount of medium. Callus Induction Sterilisation of explants is done by use of 2% Sodium hypochlorite solution or /and 70% solution of ethanol. The plant species determines the time period after which the submerged plant is removed. Sterile water is then used to rinse it to prevent contamination. The sterilised plant part is then cut using a sterilised scalpel approximately to a length of 1cm. It is then placed on the solid medium which is in a Petri-dish or a flask. The sterilised plant is incubated under aseptic conditions on the solid medium at 25 C for a number of weeks. A callus is produced after the incubation period. Small pieces of the callus are transferred to fresh medium as subcultures. Subsequent transfers are done severally until the callus is soft and fragile. The containers are kept closed and can only be opened in a laminar flow hood. This is highly efficient especially in filtration. This is done to maintain sterility of the media and the plant species by preventing contamination with airborne organisms. To kill fungal and bacterial contaminants, nutrient medium containers are sterilized under extreme heat ant pressure. The culture is stored where there are lights and is closely monitored. The humidity level is lowered gradually in preparation of transferring plants grown in laboratory condition to the soil conditions. The soils must be selected carefully with regard to the plant species. Culture Suspension There are two categories off callus culture; friable and compact. Densely aggregated cells are found in compact callus while loosely associated cells are found in friable callus. Suspension culture cells have a higher growth rate compared to that of the solid culture. It is thus preferred on a wide scale manufacture of metabolites. A section of callus is placed in a liquid medium that is already in a vessel. Cultivation is generally at 100 r.p.m and at 25 C on rotary shaker but conditions depends on plant species among other factors. A fine cell suspension is established after sub culturing for several generations. The suspension contains single cells and small cell aggregates. The medium used depends on the time needed and the species of explants. Scaling Up Increasing the volume at each stage is necessary for commercialization to achieve the requisite bioreactor size. In industries producing useful plant chemicals, suspension of cultures is large scale. The culture should be protected from microbial contamination. Importance Tissue culture aims at producing natural secondary metabolites with characteristics of the explants. This technology is applied in increased production of new or natural compounds using cell cultures from higher plants. Transformable precursors feed in the culture medium cultures are to achieve this. The process involved is biotransformation. This skill is of importance in the medical field. Bacterial extract is sterilized and filtered and the cells are cultured in liquid medium. Genetics of cultured cells are being modified through: Selection and mutagenesis of cell lines found in cell suspension culture, Transplanting foreign genetic material found in protoplasts through of genetic engineering, and Somatic hybridization through the fusion of plants protoplasts that are distantly related with the aim of genetically diversifying of hybrids. The technology of plant tissue culture has numerous pros and cons. One advantage is its use in production of species of crops that are more drought resistant. This is vital due to changing climatic conditions. The growth of drought resistant crops is important in dealing with the issue of food insecurity. The species produced are also more resistant to diseases, chemical pesticides, and soil conditions and herbicides thus reducing losses during crop cultivation. Foreign genes can be transplanted into protoplast. Genes of different unrelated species are brought together by hybridization through the fusion of protoplast hence a hybrid or cybrid is created. Biotechnology using tissue culture techniques also aims at producing plants capable of providing own usable nitrogen. The species grown in the laboratories are important in recovery of endangered species. The growth, germination and multiplication of these species in laboratories ensure sufficient supplies for nurseries and institutions of higher learning. This reduces cases of buying wild plants. The technology is helpful in the study of genetically modified studies. Incases where a plant is infected, new plants free from infection but with similar characteristics as the infected can be produced. The time needed to generate many plants is short. Growth conditions are controlled hence making synchronization of growth and development possible. This enhances acquisition of plants that have optimal characteristics. Original tissue is in small pieces and can be taken from lateral bud, root tissue, shoot tip or the stem. Original plant is maintained during the process of micro propagation. Adventitious shoots proliferation and the proliferation of lateral buds brings about a large increase of shoots that later produce roots. Plantlets obtained or micro cuttings have responded well while being grown in the soil or cans. There is acceleration in asexual propagation and plants respond the same way as plants that are vegetative propagated from their own roots. Plants can be grown without seasonal interruption under green house conditions. Controlled manipulations in the plant lead to disarrangement, reorganization and rearrangement of plant constituents. Plants produced through the technique of plant tissue culture can be stress resistant. Physical and chemical agents who have been known to cause mutations are induced to achieve the desired changes hence resulting in a variety of species. Visible altered cellular expressions are referred to as chimeras. Plant tissue culture is unique due to its technique that is based on selection. It is thus possible to obtain, retain and maintain propagation in large quantities of plants free from pathogens. Resulting plantlets are tested for possibility of having any pathogen present in them. If the pathogens are present, the plantlets are destroyed. This is a means of eliminating diseased plants from the cycle of circulation. However, much study is required on this area. Plant tissue culture also has disadvantages. Plants produced all have the same genetic material. This means their vulnerability to infections; pests and environmental conditions are similar. In case of hazardous conditions, there is massive loss of crops as all are affected. The technique can also lead to undesirable traits in the plant resulting from melolic division. There is also a reduction in plant diversity. There can be irreversible elimination of some alleles from the pool of genes. The plants might also have undesired characteristic and these have been seen to affect human health negatively. If care is not taken, there is a possibility of developing a poisonous plant that can easily lead to death if consumed. The plant tissue culture requires a lot of attention in all processes. Non expertises are not mandated in the field. The machines involved also incur a cost though if the desired plant culture quality and quantity is achieved, the benefits outweigh the cost of production. This technique is also time consuming during the culture preparation and development period. How ever, growth of the plant is fast compared to the growth of plants from their original seedlings. The technique requires skilled techniques and hence can only be undertaken or supervised by professional in the field of biotechnology. Organic supplements such as casamino acid, yeast extracts, malt, and Coconut milk are added as growth stimulants. These components increase the growth rate of the plant tissue culture as well as that of the plant in the field (Sugiyama 1999). Tobacco plants from single cells Auxins These are hormones found in plants necessary in formation of roots and shoot. It is necessary in phototropism, fruit development, abscission, initiation and development of roots, gravitism, embryo development and providing a shade avoidance effect. The auxin gradients are necessary in guiding embryo patterning into parts that become organs from zygotes first mitotic division. Auxin plays a major role in plant tissue culture. Accumulation of auxins initialises new leaves formation in apical meristem. According to Gautheret findings, auxin enhances the proliferation of cambial cultures thus enabling preparation of subcultures. Media enriched with auxins makes it possible to cultivate plant tissues for a long time. It is a modulator of plant growth and development. The hormone auxin controls plant morphogenesis. However, the growth is as a result of an inter play of different hormones which must be in a balance. The percentage of auxin to cytokinin dictates the growth of roots or shoots. Auxins are also growth regulators. Examples of auxins are 2, 4-dichlorophenoxyacetic acid (2, 4-D) and naphthaleneaceic acid (NAA). The concentration is 0.1 to 50 µM. Cytokinins are used side by side with auxins. Optimal concentration for auxins should be determined to achieve desired results. The auxin IAA is unstable to both light and heat thus its use is limited. Amino acid conjugates of IAA are occasionally used to alleviate the problem instability as they are more stable to light and heat. Auxin is also used in increasing the fruit size of the plant the plant tissue culture. Synthetic auxins have been developed such as used in increasing fruit size in citrus flowering. However, this usually relies on several factors. These Factors include; the water level and dry matter. Auxin analogies that are promising are used in regulation of productivity in potatoes. Other auxins increase the number of tubers, activate and accelerate tuber formation Summary Tissue culture proves to be the most challenging aspect in transformational strategies in plant. However, it cannot be assumed as it is an integral part of transformational strategies in plants. Development of efficient regeneration strategies is very necessary in order to avoid numerous deleterious effects caused by somaclonal variation. Regeneration protocols vary with transformational techniques. There are crops which are only amenable to only one specific protocols while others are amenable to many transformational and regeneration strategies. The technology cannot be ignored as its benefits outweigh the cost if a cost-benefit analysis is done. Plant tissue culture has plaid a great role in reducing food insecurity, providing jobs and an overall advantage on the environment. In the countries where this is practiced, the country achieves maximum financial benefits. References Fowler, M R 2000, Plant cell culture, laboratory techniques: In Encyclopedia of cell Technology, Wiley, New York. Gamborg, O L 2002, ‘Plant tissue culture. Biotechnology. Milestones.’ In vitro Cellular and Developmental Biology—Plant, vol. 38, pp. 84–92. Ramage, C. M. and Williams, R. R. 2002, ‘Mineral nutrition and plant morphogenesis.’ In Vitro Cellular and Developmental Biology—Plant, vol. 38, pp. 116–24. Sugiyama, M 1999, ‘Organogenesis in vitro.’ Current Opinion in Plant Biology, vol. 2, pp. 61–4. Walden, R and Wingender, R 1995, ‘Gene-transfer and plant-regeneration techniques.’ Trends in Biotechnology, vol.13, pp. 324–31. Read More