Here is a compilation of term papers on ‘Micro-Propagation’ for class 11 and 12. Find paragraphs, long and short term papers on ‘Micro-Propagation’ especially written for school and college students.
Term Paper on Micro-Propagation
Term Paper Contents:
- Term Paper on the Introduction to Micro-Propagation
- Term Paper on the Procedures of Micro-Propagation
- Term Paper on the Factors Affecting Micro-Propagation
- Term Paper on the Applications of Micro-Propagation
- Term Paper on the Advantages of Micro-Propagation
- Term Paper on the Disadvantages of Micro-Propagation
Term Paper # 1. Introduction to Micro-Propagation:
The classical methods of in vivo vegetative propagation often fall short of that required (too slow and difficult, too expensive) or are absolutely impossible. In vitro propagation technology can overcome all such problems. It has now become possible to clone species by in vitro culture techniques that are impossible to clone in vitro. One of the aspects of plant tissue culture is the ability to produce a large number of identical individuals via in vitro cloning, and it is popularly called as ‘micro-propagation’.
Micro-propagation in tissue culture is achieved by three ways:
(i) Axillary Budding:
Development of shoots from preexisting meristems on nodal region ensures genetic stability of the re-generants and is termed as axillary budding. About 90% of the current production of the micro-propagated plants is through axillary budding and apical shoots. Propagation of long (life) cycle plants such as forest and fruit trees is multiplied mainly through this method. This approach has been used in case of strawberry, potato, asparagus, pineapple, banana, etc. Apical shoots (1-5 mm) are normally cultured on culture media having mixtures of auxin (0.01 – 0.1 mg l-1) and cytokinin (0.05 – 0.5 mg l-1).
(ii) Adventitious Shoots:
These are stem, leaf structure, bulbs, rhizomes, tubers, etc. The meristematic regions regenerate multiple shoots on a suitable culture medium.
(iii) Somatic Embryogeny:
It is the most efficient method. It does not ensure genetic stability. However, somatic embryo in cell cultures is very useful for developing scale-up technology using bio-reactor and in the production of synthetic seeds through encapsulation. Somatic embryogeny has been done in about 200 species of different groups of plants.
Term Paper #
2. Procedures of Micro-Propagation:
The two main approaches to micro-propagation are:
(i) Multiplication of shoot meristem, and
(ii) Serial subculture of stem nodes.
In vitro clonal propagation requires many stages:
Stage I-III are done under in vitro conditions, whereas Stage IV is achieved in a greenhouse environment. An additional Stage 0 has been suggested for various micro-propagating systems.
Stage 0:
Selection and maintenance of stock plants for culture initiation including correct pre-treatment of starting materials.
Stage I:
Explant isolation, sterilization and establishment on a suitable culture medium.
Stage II:
Multiplication of shoots using an appropriate nutrient medium (i.e., propagation phase).
Stage III:
Rooting of in vitro regenerated shoots, i.e., preparation of shoots and plants for transfer into soil. This involves stopping the axillary shoot formation and initiating shoot elongation, subsequently root formation is to be induced.
Stage IV:
Transfer of plantlets to sterilized soil for hardening under greenhouse environment.
The detailed methodology/procedure for micro-propagation is given below:
(1) Shoot tip/nodal stem portion is the ideal starting explant for micro-propagation.
(2) The explant is surface sterilized, then trimmed, and the remaining material is then placed on a variety of nutrient media, under aseptic conditions.
(3) This is the phase for acclimatization of the inoculated tissue to the in vitro conditions that requires about 3 or 4 months and varies from species to species.
(4) Under suitable laboratory conditions, the shoot tip grows giving rise to a small un-rooted shoot in culture.
(5) This in vitro grown shoot, after being on the initiation medium for a suitable period, is sub-cultured onto a multiplication medium. Multiplication medium has higher levels of cytokinins.
Sub-culturing can be essential for various reasons:
(i) The nutrient medium is exhausted
(ii) The nutrient medium dries out, resulting in higher salt and sugar concentrations
(iii) Brown/black colouring appears in the agar due to secretion of toxic substances by plant tissues, and
(iv) The medium becomes liquid as a result of lowering of pH by the plant.
Usually the period between sub-cultures is 3 weeks; by this period number of shoot tips increases many-folds owing to breaking of nodal axillary buds. This again provides materials for further sub-culturing.
(6) Rooting of these cultured (sub-cultured) shoots is possible by transferring to auxin-rich media that induce formation of in vitro roots. Rooting is followed by weaning to a normal humidity level. Rooting requires low light, and hence plants must not be exposed for the first time to full sunlight. Such hardening/acclimatization of in vitro grown plants, before transferring in pots in the field, can also be achieved by keeping them in plant growth cabinets where the environmental conditions can be manipulated from lower to higher degree of exposure to light, temperature and humidity.
(7) All these procedures help develop small photosynthesizing plants.
Hundreds of plants have been micro-propagated (Table 3.1):
Use of a phenolic compound called phloroglucinol (PG) found in xylem sap of apple has been reported to be of significance for shoot multiplication and rooting in a number of rosaceous fruit cultivars. Fig. 3.1 shows various steps involved in micro-propagation of a plant.
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3. Factors Affecting Micro-Propagation:
(1) Plant Health and Physical Status of Plant Explant:
Explants are to be used from a good healthy plant; plant material should be tested for bacterial and virus indexing prior to micro-propagation. Senescing tissues are avoided for culturing. Plant materials isolated from recently produced parts of a plant are more regenerative than those from older regions. Physiological state of plant has an important bearing on in vitro cell division and regeneration.
(2) Culture Nutrient Medium:
A medium which does not favour callus formation is considered to be suitable as the genetic instability of callus will lead to a high degree of genetically aberrant plants. Whereas standard tissue culture media are more suitable for achieving stages I and II of micro-propagation, only stage III needs certain modifications. The mineral components as well as two types of growth regulators (auxin and cytokinins) influence the success of micro-propagation of a culture.
The amount of NH4+ and K+ in the medium governs the number and size of shoots produced from explants. Auxin is preferred in nutrient medium when the intension is to obtain adventitious roots, whereas gibberellins and ABA are reported to inhibit root formation. Activated charcoal in the culture medium helps in induction of adventitious roots, removes the secondary metabolites from culture media which otherwise inhibit the explant. Media having a low concentration of salts favour rooting of shoots micro-propagated at stage II.
(3) Culture Environment:
The quality of light controls the organogenic differentiation and shoots growth in vitro. Morphogenesis during micro-propagation is influenced by red/far red light exposure. The light illumination of 16 h day and 8 h dark gives the best result in terms of multiplication and proliferation of shoots, though there are some exceptions to it. This photoperiod provides the average requirements of white light for shoot induction, whereas shorter photoperiods are needed to stimulate rooting. Temperature is usually maintained constant at 25-27°C.
Sometimes, depending on species a lower temperature (e.g. 18°C for bulbous species), or higher temperature (28-29°C for tropical species) is selected. Very special temperature requirements are also followed, for organogenesis. Alternating temperature conditions may also be needed, as in case of Helianthus sp., where rooting is favoured by a day temperature of 26°C and night temperature of 15°C.
Oxygen supply in culture tubes is maintained by use of metal caps, apolar inoculation and use of liquid media. Frequent shaking of culture media on a shaker, and orchid wheels – fitted with flasks having liquid media help facilitate aeration. Each morphogenic response needs an optimal oxygen concentration for a particular organ and species. Carbon dioxide in excess (up to 20%) may induce dwarfing or albinism in the shoots of Cordyline terminalis, Ficus sp.
Ethylene is known to affect different morphogenic responses in plant tissue cultures, like callogenesis, embryogenesis and rhizogenesis. It can affect potato tuberization in vitro, and shoot elongation in rose. However, ethylene can bring about positive effect as well. For instance, it can suppress apical dominance, increased axillary shoot production, increased bulb primordia development in regenerated shoots of tulips, etc.
(4) Genotypes:
Dicots can regenerate more effectively than monocots, and gymnosperms have little regenerative capacity. Dicot plants from Solanaceae, Crassulaceae, Cruciferae regenerate much easily.
(5) Position of Explant within the Plant:
Higher the shoot isolated from a tree, the lower is probability of adventitious root formation.
(6) Method of Inoculation:
Explants are placed on nutrient medium in various ways – polar (physiological base in the medium) or apolar (physiological base out of medium and top in medium). The latter way of inoculation facilitates better regeneration of roots and shoots.
(7) Contamination by Pathogens:
Bacteria (Pseudomonas sp., Bacillus sp. and Erwinia sp.) are major contaminants during micro-propagation. Addition of antibiotics (like ampicillin to kill bacteria or fungicides like Bevistin to kill fungal contaminants) may control the infection by these microbial contaminants. These anti-biotics are added to the medium by filter sterilization. However, the addition of antibiotics often leads to phytotoxic effect.
(8) Browning of the Culture Media:
It is the most frequent problem associated with micro-propagation of woody perennials. This is caused by accumulation of inhibiting compounds (phenolics) in growth medium, which turn the medium dark brown. These substances are toxic to tissues and inhibit their growth.
This can be prevented by:
(i) Adding activated charcoal (0.2 – 3.0%, w/v) to the medium. Activated charcoal is obtained after wood is carbonised at high temperature in presence of steam. It has minute pores with large inner surface area on which gases and solid compounds can be adsorbed and thus removed.
(ii) Adding polyvinylpyrrolidone (PVP) to the medium at concentration of 250 – 1000 mg l-1.
(iii) Adding anti-oxidants like citric acid, ascorbic acid, L–cystine.
(iv) Adding glutamine, arginine and asparagine.
(v) Frequent sub-culturing onto a fresh medium at short intervals.
(vi) Soaking explants in water before placing in culture – this helps reduce exudation of toxic substances from explants into the culture medium.
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4. Applications of Micro-Propagation:
(1) Micro-Propagation of Tree Species:
Due to problems associated with the conventional methods of propagation (like non-availability of seeds, infestation of seeds by pests and pathogens, and recalcitrant seeds), quality planting materials in tree species are not available. The tissue culture techniques, however, offer means for mass multiplication as well as conservation of fruit and forest trees and other elite and rare trees. Micro-propagation in vitro has been done successfully in many Himalayan plants.
For instance – (i) proliferation of shoots from terminal and axillary buds as obtained in Camellia sinensis. (ii) axillary shoots raised from nodal segments of Bauhinia variegata, Dendrocalamus brandisii and Dendrocalamus strictus. Use of BAP (10 µM) and IBA (1 µM) to cultured nodal segments enhances shoot multiplication at rapid speed (about 12 – 27 folds) within two months.
(2) Micro-Propagation of Medicinal Plants:
The use of micro-propagation techniques for medicinal plants gives many benefits to the breeders due to various reasons:
(a) It leads to mass multiplication of those plants whose seeds have low regeneration or which fail to produce seeds in a given climate,
(b) It makes available plants throughout the year,
(c) It enables the availability of uniform clones from highly heterozygous plants, and
(d) It helps conserve the plant germplasms which are on the verge of being endangered. Various explants like stem and leaf segments, shoot buds, hypocotyl, root, anthers etc., have been tried successfully to regenerate medicinal plants in vitro (Table 3.2).
(3) Micro-Propagation of Ornamental Plants:
Micro-propagation of various ornamental plants have been achieved, for instance, Chrysanthemum, Hedera, Iris, Cymbidium, Vanda, Dendrobium, Agave, Gladiolus, Dianthus, etc., and the explants used for in vitro culture in regeneration of these plants include axillary buds, apical shoots, inflorescence, leaves and roots.
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5. Advantages of Micro-Propagation:
(1) Small surface needed.
(2) Production can be maintained without seasonal variation. As a result of perfect conditioning of nutrient media and physical conditions, enabling precise timing of cutting production, the effect of seasons can be eliminated and year round production can be achieved.
(3) Stock plants can be stored in vitro.
(4) Plants difficult to be propagated in vivo can be propagated in vitro. This is sometimes due to rejuvenation, which is only possible in vitro.
(5) No attention is required between sub-cultures (e.g., watering, weeding, etc.).
(6) Virus free plants can be produced.
(7) Propagation in vitro is more rapid than in vivo.
(8) Growth of in vitro propagated plants is often stronger than those cloned in vivo.
(9) Micro-propagation can produce great savings in fuel costs, greenhouse space, etc.
(10) The propagation in vitro on its own root system makes grafting and budding onto root stocks unnecessary – it has importance for species like Rose, Lilac, etc.
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6. Disadvantages of Micro-Propagation:
(1) Experience and specialized facilities needed.
(2) Skilled operation required.
(3) If fungal/bacterial contamination occurs during early multiplication many potential propagules may be lost.
(4) Propagation technique can introduce genetic instability.
(5) Initial plantlets are small.
(6) An occasional problem with repeated cycles of in vitro shoot is the development of swollen, distorted leaves which become irreversibly translucent and eventually necrotic, a condition that may lead to death of shoot cluster. This is called ‘water soaking by Hussey, and later on described as “Vitrification” by Debergh et al. (1981).
In literature, vitrification is termed as translucency, glauciness, hyperhydration, water logging. This is particularly common if the plant has excess water available, or if the medium has a low agar concentration. This phenomenon has been described in Malus, in carnation and artichoke.
Its occurrence and degree is governed by many factors, notable amongst them are:
(i) Increasing the concentration of agar and/or sugar lessens the vitrification, as observed in case of carnation and artichoke.
(ii) It can be avoided in some cases by better gaseous exchange in culture vessels.
(iii) It is associated with high cytokinin levels.
(iv) It is induced at low light irradiance and high temperatures.
(v) Young plant explants (i.e., softer) are more susceptible to vitrification.
(vi) It may be reduced by changing the mixture of macro salts.
(7) Plants produced through this technology may show troublesome effects in. vivo; repeated production of side shoots and completes reversal to juvenile phase.
(8) Rooting in case of woody plants is often difficult.
(9) Transfer of some plants from test tube (laboratory) to soil is very difficult to achieve.
(10) Regenerative capacity may lost by repeated culture of callus and cell suspensions.
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