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  1. 1. Shehzadi Fatima University Of Sargodha
  2. 2. Micropropagation  Plants can be propagated by sexual (through generation of seeds) or asexual (through multiplication of vegetative parts) means.  Clonal propagation refers to the process of asexual reproduction by multiplication of genetically identical copies of individual plants, where the term clone is used to represent a plant population derived from a single individual by asexual reproduction.  In vitro clonal propagation through tissue culture is referred to as micro propagation. Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.
  3. 3. Micropropagation is used to multiply noble plants such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well to vegetative reproduction. Technique of Micropropagation:  Micro propagation is a complicated process and mainly involves 5 stages (stage 0 and IV) for more comprehensive representation of micro- propagation.  Stage 0:This is the initial step in micropropagation, and involves the selection and growth of stock plants for about 3 months under controlled conditions.  Stage I: In this stage, the initiation and establishment of culture in a suitable medium is achieved. Selection of appropriate explants is important.The most commonly used explants are organs, shoot tips and axillary buds.
  4. 4.  Stage II: In this stage, the major activity of micro propagation occurs in a defined culture medium. Stage II mainly involves multiplication of shoots or rapid embryo formation from the explant.  Stage III:This stage involves the transfer of shoots to a medium for rapid development into shoots. Sometimes, the shoots are directly planted in soil to develop roots. In vitro rooting of shoots is preferred while simultaneously handling a large number of species.  Stage IV:This stage involves the establishment of plantlets in soil.This is done by transferring the plantlets of stage III from the laboratory to the environment of greenhouse. For some plant species, stage III is skipped, and un-rooted stage II shoots are planted in pots or in suitable compost mixture.
  5. 5. StageV: In this stage, the major activity of micro propagation occurs in a defined culture medium. Stage II mainly involves multiplication of shoots or rapid embryo formation from the explant.
  6. 6. Stages involved in micropropagation
  7. 7. Approaches involved in Micropropagation 1. Multiplication by axillary buds/apical shoots. 2. Multiplication by adventitious shoots. Besides the above two approaches, the plant regeneration processes namely organogenesis and somatic embryo- genesis may also be treated as micro propagation. 3. Organogenesis:The formation of individual organs such as shoots, roots, directly from an explant or from the callus and cell culture induced from the explant. 4. Somatic embryogenesis:The regeneration of embryos from somatic cells, tissues or organs.
  8. 8. 1 Multiplication by axillary buds/apical shoots. Actively dividing meristems are present at the axillary buds and apical shoots (shoot tips). =>The axillary buds located in the axils of leaves are capable of developing into shoots. =>By means of induced in vitro multiplication in micro propagation, it is possible to develop plants from meristem and shoot tip cultures and from bud cultures.
  9. 9. Shoot tip cultures- Apical meristem is a dome of tissue located at the extreme tip of a shoot.The apical meristem along with the young leaf primordia constitutes the shoot apex.
  10. 10. Meristem or shoot tip is isolated from a stem by a V- shaped cut.The size (frequently 0.2 to 0.5 mm) of the tip is critical for culture. In general, the larger the explant (shoot tip), the better are the chances for culture survival. For good results of micro propagation, explants should be taken from the actively growing shoot tips, and the ideal timing is at the end of the plants dormancy period.  The most widely used media for meristem culture are MS medium andWhite’s medium.
  11. 11.  In stage I, the culture of meristem is established. Addition of growth regulators namely cytokinins (kinetin, BA) and auxins (NAA or IBA) will support the growth and development. In stage II, shoot development along with axillary shoot proliferation occurs. High levels of cytokinins are required for this purpose. Stage III is associated with rooting of shoots and further growth of plantlet. Consequently, stage II medium and stage III medium should be different in composition.The optimal temperature for culture is in the range of 20-28°C (for majority 24-26°C). Lower light intensity is more appropriate for good micro propagation.
  12. 12. Bud Cultures- The plant buds possess quiescent or active meristems depending on the physiological state of the plant.Two types of bud cultures are used— single node culture and axillary bud culture. Single node culture : This is a natural method for vegetative propagation of plants both in vivo and in vitro conditions. A bud along with a piece of stem is isolated and cultured to develop into a plantlet. Closed buds are used to reduce the chances of infections. In single node culture, no cytokinin is added.
  13. 13. Axillary bud culture: In this method, a shoot tip along with axillary bud is isolated.The cultures are carried out with high cytokinin concentration. As a result of this, apical dominance stops and axillary buds develop. A schematic representation of axillary bud culture for a rosette plant and an elongate plantisgivenas follows.
  14. 14. For a good axillary bud culture, the cytokinin/ auxin ratio is around 10: 1. This is however, variable and depends on the nature of the plant species and the developmental stage of the explant used. In general, juvenile explants require less cytokinin compared to adult explants. Sometimes, the presence of apical meristem may interfere with axillary shoot development. In such a case, it has to be removed.
  15. 15. 2 Multiplication by adventitious shoots  The stem and leaf structures that are naturally formed on plant tissues located in sites other than the normal leaf axil regions are regarded as adventitious shoots.  There are many adventitious shoots which include stems, bulbs, tubers and rhizomes.The adventitious shoots are useful for in vivo and in vitro clonal propagation.  The meristematic regions of adventitious shoots can be induced in a suitable medium to regenerate to plants.
  16. 16. 3 Organogenesis  Organogenesis is the process of morphogenesis involving the formation of plant organs i.e. shoots, roots, flowers, buds from explant or cultured plant tissues. It is of two types i.e direct organogenesis and indirect organogenesis.  Direct organogenesis-Tissues from leaves, stems, roots and inflorescences can be directly cultured to produce plant organs. In direct organogenesis, the tissue undergoes morphogenesis without going through a callus or suspension cell culture stage.  Indirect organogenesis-When the organogenesis occurs through callus or suspension cell culture formation, it is regarded as indirect organogenesis. Callus growth can be established from many explants (leaves, roots, cotyledons, stems, flower petals etc.) for subsequent organogenesis.
  17. 17. In general, the bigger the explant the better the chances for obtaining viable callus/cell suspension cultures. It is advantageous to select meristematic tissues (shoot tip, leaf, and petiole) for efficient indirect organogenesis.This is because their growth rate and survival rate are much better. For indirect organogenesis, the cultures may be grown in liquid medium or solid medium. Many culture media (MS, B5 White’s etc.) can be used in organogenesis.The concentration of growth regulators in the medium is critical for organo- genesis.
  18. 18. 4. Embryogenesis The process of regeneration of embryos from somatic cells, tissues or organs is regarded as somatic (or asexual) embryogenesis. Somatic embryos are structurally similar to zygotic (sexually formed) embryos, and they can be excised from the parent tissues and induced to germinate in tissue culture media. Two routes of somatic embryogenesis are known — direct and indirect.  Direct Somatic Embryogenesis-When the somatic embryos develop directly on the excised plant (explant) without undergoing callus formation, it is referred to as direct somatic embryogenesis .This is possible due to the presence of pre-embryonic determined cells (PEDQ found in certain tissues of plants.
  19. 19. • Indirect Somatic Embryogenesis- In indirect embryogenesis, the cells from explant (excised plant tissues) are made to proliferate and form callus, from which cell suspension cultures can be raised. Certain cells referred to as induced embryo genic determined cells (IEDC) from the cell suspension can form somatic embryos. Embryogenesis is made possible by the presence of growth regulators (in appropriate concentration) and under suitable environmental conditions.
  20. 20.  Somatic embryogenesis (direct or indirect) can be carried on a wide range of media (e.g. MS,White’s).The addition of the amino acid L- glutamine promotes embryogenesis.The presence of auxin such as 2, 4- dichlorophenoxy acetic acid is essential for embryo initiation. On a low auxin or no auxin medium, the embryo genic clumps develop into mature embryos.  Indirect somatic embryogenesis is commercially very attractive since a large number of embryos can be generated in a small volume of culture medium.The somatic embryos so formed are synchronous and with good regeneration capability.
  21. 21. Applications of Micro propagation Micro propagation has become a suitable alternative to conventional methods of vegetative propagation of plants.There are several advantages of micro propagation.  High Rate of Plant Propagation- Through micro propagation, a large number of plants can be grown from a piece of plant tissue within a short period. Another advantage is that micro propagation can be carried out throughout the year, irrespective of the seasonal variations.  Production of Disease-free Plants- It is possible to produce disease-free plants through micro propagation. Meristem tip cultures are generally employed to develop pathogen-free plants .
  22. 22.  Cost-effective Process- Micro propagation requires minimum growing space.Thus, millions of plant species can be maintained inside culture vials in a small room in a nursery.  Production of seeds in some crops- Micro propagation, through axillary bud prolife- ration method, is suitable for seed production in some plants. This is required in certain plants where the limitation for seed production is high degree of genetic conservation e.g. cauliflower, onion.  It is the only viable method of regenerating genetically modified cells after protoplast fusion. Micropropagation often produces more robust plants, leading to accelerated growth compared to similar plants produced by conventional methods - like seeds or cuttings.
  23. 23. Disadvantages of Micropropagation  Contamination of Cultures- During the course of micro propagation, several slow-growing microorganisms (e.g. Eswinia sp, Bacillus sp) contaminate and grow in cultures. The microbial infection can be controlled by addition of antibiotics or fungicides.  Brewing of Medium- Micro propagation of certain plants (e.g. woody perennials) is often associated with accumulation of growth inhibitory substances in the medium. Chemically, these substances are phenolic compounds, which can turn the medium into dark colour. Phenolic compounds are toxic and can inhibit the growth of tissues. Brewing of the medium can be prevented by the addition of ascorbic acid or citric acid or polyvinyl pyrrolidone to the medium.
  24. 24. Disadvantages of Micropropagation GeneticVariability -When micro propagation is carried out through shoot tip cultures, genetic variability is very low. However, use of adventitious shoots is often associated with pronounced genetic variability.  Vitrification-During the course of repeated in vitro shoot multiplication, the cultures exhibit water soaked or almost translucent leaves. Such shoots cannot grow and even may die.This phenomenon is referred to as vitrification. This can be be prevented by increasing the agar concentration (from 0.6 to 1%) in the medium. However, increased agar concentration reduces the growth rate of tissues.  For some micro propagation techniques, expensive equipment, sophisticated facilities and trained manpower are needed.This limits its use.