Lecture 6: Micropropagation Techniques in Plant Tissue Culture PDF

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. Di...

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?