Nile University Lecture Notes on Plant Tissue Culture PDF

Sugar Nutrient salts contribute approximately 20–50% to the osmotic potential of the medium, and sucrose is responsible for the remainder. The contribution of sucrose to the osmotic potential increases as it is hydrolyzed into glucose and fructose during autoclaving. This may be an importa...

Sugar Nutrient salts contribute approximately 20–50% to the osmotic potential of the medium, and sucrose is responsible for the remainder. The contribution of sucrose to the osmotic potential increases as it is hydrolyzed into glucose and fructose during autoclaving. This may be an important consideration when performing osmotic- sensitive procedures such as protoplast isolation and culture. Vitamins Vitamins are organic substances that are parts of enzymes or cofactors for essential metabolic functions. Only thiamine (vitamin B1) is essential in a culture, as it is involved in carbohydrate metabolism and the biosynthesis of some amino acids. Activated Charcoal Activated charcoal is useful for absorption of the brown or black pigments and oxidized phenolic compounds. It is also useful for absorbing other organic compounds, including PGRs, such as auxins and cytokinins, and other materials such as vitamins, and iron and zinc chelates. Activated Charcoal Carryover effects of PGRs are minimized by adding activated charcoal when transferring explants to media without PGRs. It changes the light environment by darkening the medium so it can help with root formation and growth. It may also promote somatic embryogenesis and enhance the growth and organogenesis of woody species. Gelling Agents Agar is used to solidify tissue culture media into a gel (0.6 – 0.8%). It enables the explant to be placed in precise contact with the medium (on the surface or embedded) but to remain aerated. It is derived from seaweed. If a lower concentration of agar is used (0.4%) or if the pH is low, the medium will be too soft and will not gel properly. Typical tissue culture agar melts easily at ~65°C and solidifies at ~45°C. Gelling Agents Agarose is often used when culturing protoplasts or single cells. Agarose is a purified extract of agar that leaves behind agaropectin and sulphate groups. Gellan gums like GelriteTM and PhytagelTM are alternative gelling agents. They are clear, so it is much easier to detect contamination. they cannot be reliquified by heating and gelled again, and the concentration of divalent cations like calcium and magnesium must be within a restricted range or gelling will not occur. Types of Plant Medium Cultures Murashige and Skoog (MS) medium (1962) is the most suitable and most commonly used basic tissue culture medium for plant regeneration from tissues and callus. It was developed for tobacco and based primarily upon the mineral analysis of tobacco tissue. This is a “high salt” medium, due to its content of K and N salts. Types of Plant Medium Cultures To counteract salt sensitivity of some woody species, Lloyd and McCown (1980) developed the woody plant medium (WPM). Gamborg’s B-5 medium was devised for soybean callus culture, and has lesser amounts of nitrate and particularly ammonium salts than the MS medium. It was mainly used for callus or suspension culture. Schenk and Hildebrandt (1972) developed the SH medium for the callus culture of monocots and dicots. White’s medium which was designed for the tissue culture of tomato roots, has a lower concentration of salts than the MS medium. What is the take-home message from today’s lecture. Let’s discuss. Any more questions?  Lecture 6 a Micropropagation Techniques in Plant Tissue Culture Prepared and presented by : Dr. Abdelaziz Mohamed Nasr Lesson Objectives Understand the concept and significance of micropropagation. Learn various micropropagation techniques. Discuss the applications and advantages of micropropagation. Introduction Plants can be propagated through their two developmental life cycles; the sexual, or the asexual. In the sexual cycle new plants arise after fusion of the parental gametes and develop from zygotic embryos contained within seeds or fruits. By contrast, in the vegetative (asexual) cycle the unique characteristics of any individual plant selected for propagation (termed the mother plant, stock plant) are usually perpetuated. A group of such asexually reproduced plants are termed clones. Seed propagation advantages They are often produced in large numbers so that the plants regenerated from them are individually inexpensive. Many may usually be stored for long periods without loss of viability. They are easily distributed. Most often plants grown from seed are without most of the pests and diseases which may have afflicted their parents. Seed propagation disadvantages Some plants do not produce viable seeds. Some plants produce seeds only after a long juvenile period. Some seeds lose their viability after long storage. Micropropagation techniques Micropropagation is the mass vegetative production of plants in vitro for the purpose of commercial plant production. The propagation could happen through terminal or axillary buds, or by the propagation of adventitious shoots or embryos from somatic cells. Micropropagation advantages Cultures are started with very small pieces of plants (explants), so only a small amount of space is required to maintain plants or to greatly increase their number. Methods are available to free plants from specific virus diseases, and certified virus-tested plants can be produced in large numbers. A more flexible adjustment of factors influencing vegetative regeneration is possible. It may be possible to produce clones of some kinds of plants that are otherwise slow and difficult (or even impossible) to propagate sexually. Micropropagation advantages Plants may acquire a new temporary characteristic through micropropagation which makes them more desirable to the grower than conventionally-raised stock. A bushy habit (in ornamental pot plants) and increased runner formation (strawberries) are two examples. Production can be continued all the year round. Plant material needs little attention between subcultures. Micropropagation disadvantages A specialized and expensive production facility is needed. Explants and cultures have to be grown on a medium containing sucrose or some other carbon source. As they are raised within glass or plastic vessels in a high relative humidity, and are not usually photosynthetically self-sufficient, the young plantlets are more susceptible to water loss in an external environment. Stages of Micropropagation Stage 0: Mother plant selection and preparation. They must be typical of the variety or species, and free from any symptoms of disease. It may be advantageous to treat the chosen plant (or parts of it) in some way to make in vitro culture successful. Stages of Micropropagation Stage I: Establishing an aseptic culture The customary second step in the micropropagation process is to obtain an aseptic culture of the selected plant material. Success at this stage firstly requires that explants should be transferred to the cultural environment, free from obvious microbial contaminants; and that this should be followed by some kind of growth (growth of a shoot tip, or formation of callus). Usually, a batch of explants is transferred to culture at the same time. After a short period of incubation, any container found to have contaminated explants or medium is discarded. Stages of Micropropagation Stage II: The production of suitable propagules The production of new plant outgrowths or propagules, which, when separated from the culture are capable of giving rise to complete plants. Newly derived axillary or adventitious shoots, somatic embryos. They can also be used as the basis for further cycles of multiplication. Stages of Micropropagation Stage III: Preparation for growth in the natural environment Steps are taken to grow individual or clusters of plantlets, capable of carrying out photosynthesis, and survival without an artificial supply of carbohydrates. It includes the in vitro rooting of shoots prior to their transfer to soil. Stages of Micropropagation Stage IV: Transfer to the natural environment Plantlets are transferred from the in vitro to the ex vitro external environment is extremely important. If not carried out carefully, the transfer can result in a significant loss of propagated material. Shoots developed in culture have often been produced in high humidity and a low light ‘intensity’. This results in there being less leaf epicuticular wax or wax with an altered chemical composition, than on plants raised in growth chambers or greenhouses. In some plants, the stomata of leaves produced in vitro may also be atypical and incapable of complete closure under conditions of low relative humidity. Stages of Micropropagation When supplied with sucrose (or some other carbohydrate) and kept in low-light conditions, micropropagated plantlets are not fully dependent on their own photosynthesis. They need to change to be fully capable of producing their own requirements of carbon and reduced nitrogen. In practice, plantlets are removed from their Stage III containers, then transplanted into an adequate rooting medium (such as a peat:sand compost) and kept for several days in high humidity and reduced light intensity. The change only occurs after the plants have spent a period of several days ex vitro. Shoot (or shoot tip) culture The term shoot culture is now preferred for cultures started from explants bearing an intact shoot meristem, whose purpose is shoot multiplication by the repeated formation of axillary branches. Establishment of shoot tip culture of male P. vera after 3 days of culture on the CIM containing 1.0 mgl -1 BA Shoot culture Shoot cultures are conventionally started from the apices of lateral or main shoots, up to 20 mm in length, dissected from actively-growing shoots or dormant buds. Larger explants are also sometimes used with advantage: Better survive the transfer to in vitro conditions. More rapidly commence growth. Contain more axillary buds. However, the greater the size of the explant, the more difficult it may be to decontaminate from micro-organisms. Shoot culture The shoot tip used are usually macerated from shoots originating from meristem tip culture. According to some researchers, there is a competition between cell proliferation, and the formation of the virus particles in meristem region of plant. Nucleic acid production capacity in meristematic tissue during cell division is used for cell division and this situation prevents the reproduction of virus. According to other researchers, transportation of viruses to the meristem region of the plant is prevented due to lack of transport system in meristem. Shoot culture The growth and proliferation of axillary shoots in shoot cultures is usually promoted by incorporating growth regulators (usually cytokinins) into the growth medium. In some plants, pinching out the main shoot axis is used as an alternative, or an adjunct, to the use of growth regulators for decreasing apical dominance. Current applications Conventional shoot culture continues to be the most important method of micropropagation, although node culture is gaining in importance. It is very widely used by commercial tissue culture laboratories for the propagation of many herbaceous ornamentals and woody plants. What is the take-home message from today’s lecture. Let’s discuss. Any more questions?  Lecture 7 a Micropropagation Techniques in Plant Tissue Culture Prepared and presented by : Dr. Abdelaziz Mohamed Nasr Lesson Objectives Learn various micropropagation techniques. Discuss the applications and advantages of micropropagation. Node culture Single node culture is another in vitro technique that can be used for propagating some species of plants from axillary buds. In this method, intact individual shoots may be placed on a fresh medium in a horizontal position. It has been termed ‘in vitro layering’. Each shoot may be cut into single-, or several node pieces which are sub-cultured. Leaves are usually trimmed so that each second stage explant consists of a piece of stem bearing one or more lateral buds. Each approach can be reiterated to propagate during stage II. Node culture Unfortunately, in vitro layering doesn’t result in several axillary shoots of equal length. Methods of rooting are the same as those employed for the micro-cuttings derived from shoot culture. Media for single node culture are intrinsically the same as those suitable for shoot culture. Current application Node culture is of value for propagating species that produce elongated shoots in culture (e.g. potato and Alstroemeria). Nowadays the technique is becoming more and more popular in commercial micropropagation. The main reason is that it gives more guarantee for clonal stability. There is less likelihood of associated callus development. Multiple shoots from seeds (MSS) It initiates multiple shoot cultures directly from seeds. Seeds are sterilized and then placed onto a basal medium containing cytokinin. As germination occurs, clusters of axillary and/or adventitious shoots (‘multiple shoots’) grow out and may be split up and serially subcultured on the same medium. It is likely that multiple shoots can be initiated from the seeds of many species, particularly dicotyledons. The technique is effective in both herbaceous and woody species: soybean, sugar beet, and almonds. Somatic embryogenesis Somatic embryos are often initiated directly upon explanted tissues. One of the most common is during the in vitro culture of explants associated with, or immediately derived from, the female gametophyte (Ovaries and ovules). Somatic embryogenesis Somatic embryos are structurally similar to the embryos found in true seeds. Such embryos often develop a region equivalent to the suspensor of zygotic embryos and, unlike shoot or root buds, come to have both a shoot and a root pole. Somatic embryos are embryos produced from cells or tissues of the plant body Embryogenesis  B) The megaspore develops All seed plants produce into a megagametophyte. It two types of is slightly larger than a gametophytes. (A) microgametophyte and has Microspores (pollen seven cells, one of which grains) develop into has two nuclei. One of the microgametophytes (also cells is the egg called pollen grains). After one sperm cell fertilizes the egg cell, the new egg cell nucleus is diploid, and the cell is a zygote. It develops, by mitosis, into a new sporophyte. The suspensor of the embryo pushes the developing embryo deep into the developing endosperm to obtain nutrition for embryo growth. Somatic embryogenesis The nucellus tissue of many plants has the capacity for direct embryogenesis in vitro. Adventitious embryos are commonly formed in vitro directly upon the zygotic embryos of monocotyledons, dicotyledons and gymnosperms, upon parts of young seedlings (especially hypocotyls and cotyledons) and upon somatic embryos at various stages of development. Protoplasts isolated from embryogenic suspensions, may give rise to somatic embryos directly, without any intervening callus phase. Arabidopsis ovule containing the embryo sac at about 4 hours after double fertilization. Current application From a quantitative point of view, indirect embryogenesis does provide an efficient method of micropropagation; the same is not true of direct embryogenesis when it is unaccompanied by the proliferation of embryogenic tissue. To increase the number of somatic embryos formed directly on immature zygotic embryos of sunflowers, the larger zygotic embryos were cut into four equal pieces. The commercial application of this technology remains limited except, perhaps, where embryos arise directly from parental tissue. Methods of Stage of culture Micropropagatio I. Initiating a culture II. Increasing propagules III. Preparation for soil transfer n Shoot Cultures Transfer of disinfected shoot Induce multiple (axillary) shoot Elongation of buds formed at Stage tips to solid or liquid media and formation and growth of the shoots to a II to uniform shoots. Rooting the the commencement of shoot sufficient size for separation, either as shoots in vitro or outside the culture growth to ca. 10mm. new Stage II explants or for passage to vessel III. Meristem Transfer of very small shoot tips Growth of shoots to ca. 10mm, As for shoot tip cultures. (length 0.2-0.5mm) to culture. then as shoot tip culture, or as culture Longer shoot tips (1-2mm) can shoot multiplication omitted and be used as explants if obtained shoots transferred to Stage III. from heat treated plants. Node culture Propagation by inducing the axillary As for shoot tip culture but bud at each node to grow into a single shoots grown longer to show As for shoot tip cultures. shoot. Subculturing can be repeated clear internodes. indefinitely. Direct Establishing suitable Growth of the embryos into The direct induction of somatic embryogenic tissue explants or plantlets which can be embryogenesis previously-formed somatic embryos on the explants without transferred to the outside prior formation of callus. embryos. environment. What is the take-home message from today’s lecture. Let’s discuss. Any more questions?  Lecture 8 a Callus Culture in Plant Tissue Culture Prepared and presented by : Dr. Abdelaziz Mohamed Nasr Lesson Objectives Understand the concept and significance of callus culture. Learn the process of callus induction and organ formation. Discuss the applications and challenges of callus culture. Callus culture Callus is a coherent and amorphous tissue, formed when plant cells multiply in a disorganized way. It is often induced in or upon parts of an intact plant by wounding, by the presence of insects or microorganisms, or as a result of stress. Callus can be initiated in vitro by placing small pieces of the whole plant (explants) onto a growth-supporting medium under sterile conditions. During this process, cell differentiation and specialization, which may have been occurring in the intact plant, are reversed, and the explant gives rise to new tissue, which is composed of meristematic and unspecialized cell types. Pale yellow (p-y) callus developed from cambium of root discs, Dark orange (d-o) callus line established from p-y callus by visual selection and subsequent subculture. Callus culture During dedifferentiation, storage products typically found in resting cells tend to disappear. Although most experiments have been conducted with the tissues of higher plants, callus cultures can be established from gymnosperms, ferns, and mosses. Callus cultures are more easily established from some organs than others. Young meristematic tissues are most suitable, but meristematic areas in older parts of a plant, such as the cambium, can give rise to callus. Callus culture The choice of tissues from which cultures can be started is greatest in dicotyledonous species. In most cereals, for example, callus growth can only be obtained from organs such as zygotic embryos, germinating seeds, seed endosperm or the seedling mesocotyl, and very young leaves, but so far never from mature leaf tissue. The callus formed on an original explant is called ‘primary callus’. Secondary callus cultures are initiated from pieces of tissue dissected from primary callus. Callus culture is often performed in the dark as the light can encourage differentiation of the callus. Monocot seedling Callus culture Callus tissue is not of one single kind. Strains of callus differing in appearance, color, degree of compaction and morphogenetic potential commonly arise from a single explant. Sometimes the type of callus obtained, its degree of cellular differentiation and its capacity to regenerate new plants, depend upon the origin and age of the tissue chosen as an explant. Variability is more likely when callus is derived from an explant composed of more than one kind of cell. Types of callus Compact callus Shows as large, densely aggregated cells. Friable callus Shows as loosely associated cells, and the callus becomes soft and breaks apart easily Mostly used to start cell suspension cultures. Stages of callus culture Induction phase Division phase Differentiation phase Induction Cells in explant dedifferentiate and begin to divide. Division phase Rapid cell division. In case of long-term culture, the requirements of auxin and/or cytokinin are reduced, which is called habituation. Differentiation phase Differentiation and formation of organized structure. Organogenesis Somatic embryogenesis Notes on callus culture Explants grown in agar medium with appropriate nutrients with suitable proportion of auxin and cytokinin exhibit callusing at cut ends, which gradually extends to the entire surface of the tissue. Callus cultures need to be subcultured every 3 – 5 weeks. Repeated subculture on an agar medium improves the friability of the callus. Notes on callus culture Subculture is the transfer of cultures with or without dilution from one culture vessel to another containing fresh culture medium. It is also known as passage. The callus tissue shows an eventual loss of organogenic response as subculture proceeds. Callus cultures may show some changes of chromosomal structure or number such as aneuploidy, polyploidy. Current applications Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots, roots or somatic embryos from which whole plants can subsequently be produced. Callus cultures can also be used to initiate cell suspensions. To study nutrition requirement of plants. Current application To study cell and organ differentiation and morphogenesis. Somaclonal variations and its exploitation. Genetic transformation The production of secondary metabolites. What is the take-home message from today’s lecture. Let’s discuss. Any more questions?