![Stages of Micropropagation
Stage 0: Preparation of the mother plant
Stage 1: Initiation and establishment of cultures
Stage 2: Multiplication [shoot or rapid somatic embryo formation]
Stage 3: In-vitro germination of somatic embryo
and rooting of shoots
Stage 4: Transfer of plantlets to sterilized soil for Hardening
under greenhouse/ field conditions (transplantation)](https://image.slidesharecdn.com/micropropagation-200904052317/85/Clonal-Propagation-Introduction-Techniques-Factors-Applications-and-Disadvantages-13-320.jpg)
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Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
The document discusses clonal propagation techniques, particularly focusing on micropropagation through tissue culture as a method for rapid plant multiplication. It details the advantages of clonal propagation, including the creation of genetically identical plants and the avoidance of juvenile phases, and outlines various methods of achieving this, such as organogenesis and somatic embryogenesis. Furthermore, it addresses the stages involved in micropropagation, from explant preparation to transplantation, and highlights the commercial significance of this technique in the horticultural industry.
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Clonal Propagation: Introduction, Techniques, Factors, Applications and Disadvantages
- 1. Clonal Propagation: Techniques, Factors,Applications and Disadvantages DR. DIVYA SHARMA ASSISTANT PROFESSOR A Biodiction (A Unit of Dr. Divya Sharma)
- 2. Micropropagation Plant TissueCulture technique: Increasingly popular alternative means -- Plant vegetative propagation Plants can be propagated by sexual (through generation of seeds) or asexual (through multiplication of vegetative parts). Plant tissue culture involves – o Asexual method of propagation o Primary goal -- crop improvement In-vitro selection, genetic manipulation techniques -- Higher plant
- 3. Clonal Propagation Clonal propagation------- In-vitro selection Micropropagation Greek word --- Clone --- Twig ---- Identical copies The process of multiplication of genetically identical copies of individual plants by asexual reproduction is called Clonal Propagation The term clone is used to represent a plant population derived from a single individual by asexual reproduction. Process -- Asexual reproduction --- Multiplication occur ----- Individual plants genetically identical copies
- 4. Multiplication ofgenetically, identically individual by asexual reproduction Apomixes (seed development without meiosis and fertilization) and vegetative propagation (regeneration of new plants from vegetative parts) Short time Rapid process -- Commeralization of improvement plants. Eg.: Apple, pear etc. Asexual reproduction through multiplication of vegetative parts is the only method for the in-vivo propagation of certain plants, as they do not produce viable seeds. Eg.: Banana, Grape, Fig and Chrysanthemum. Clonal propagation has been successfully applied for the propagaton. Eg.: Apple, Potato, Tuberous and several Ornamental plants
- 5. Advantages of VegetativePropagation Faster multiplication --- Large number of plants can be produced from a single individual in a short period. Produce genetically identical plants Sexually ---- Sterile hybrids can be propagated. Seed --- Raised plants pass through an undesirable juvenile phase which is avoided in asexual propagation. Gene banks can be more easily established by clonally propagated plants
- 6. Micropropagation Aseptic methodof clonal propagation is called as Micropropagation Synonyms -- tissue culture Rapid multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods Widely used for Orchids, Ferns, many interior foliage plants etc.
- 7. Features of Micropropagation Relatively short time Easy to manipulate production cycles Disease free plants can be produced In-vitro production can be planned Recycled many times to produce an unlimited number of clones Micropropagated plants may acquire new desirable traits, like bushy habit of ornamental plants and increased number of runners in strawberry Commercially viable method for horticultural crops Eg.: Orchids, Ferns, many interior foliage plants etc.
- 8. In-vitro Clonal Propagation In-vivoclonal propagation --- Tedious, expensive and frequently unsuccessful In-vitro clonal propagation --- Through tissue culture --- Micropropagation From cells, tissue or organs Cultured aseptically on defined media Contained in culture vessels Maintained under controlled conditions of light and temperature
- 9. Commercialization of Micropropagation Broadcommercial applications ------ 1970s and 1980s Murashige (1974)
- 10. In-vitro Clonal Propagation Use oftissue culture technique for micropropagation was first started by Morel (1960) for propagation of orchids, and is now applied to several plants. Micropropagation is a handy technique for rapid multiplication of plants
- 11. Selection of PlantMaterial Part of plant Genotype Physiological condition Season Position on plant Size of explants
- 12. Explants used inMicropropagation Different kinds of explants used in micropropagation: Eg.: In case of orchids, shoot tips (Anacamptis pyramidalis, Aranthera, Calanthe, Dendrobium); Axillary bud (Aranda, Brassocattleya, Cattleys Laelia); Inflorescence segment (Aranda, Ascofinetia, Neostylis, Vascostylis); Lateral bud (Cattleya, Rhynocostylis gigantean); Leaf base (Cattleya); Nodal segment (Dendrobium; Flower stalk segment (Dendrobium, Phalaenopsis); and Root tips (Neottis, Vanilla)
- 13. Stages of Micropropagation Stage0: Preparation of the mother plant Stage 1: Initiation and establishment of cultures Stage 2: Multiplication [shoot or rapid somatic embryo formation] Stage 3: In-vitro germination of somatic embryo and rooting of shoots Stage 4: Transfer of plantlets to sterilized soil for Hardening under greenhouse/ field conditions (transplantation)
- 14. Stages of Micropropagation Maintained light, temperature and moisture regimes under which the mother plants Loosely covering growing branches with polythene Application of growth regulators Watering of plant from the base Stage 0: Preparatory stage Involves the preparation of mother plants to provide quality explants for better establishment of aseptic cultures
- 15. Stages of Micropropagation Explantselection Sterilization treatment Choice of growth medium Stage 1: Initiation of Cultures
- 16. Stage 2: Multiplication ThroughCallusing Initiation of culture on high Auxin media Development of unorganized mass of cells called callus Transfer small piece of callus on regeneration media Shoot bud regeneration Somatic embryo
- 17. Stage 2: Multiplication AdventitiousBud Formation Transfer to proliferation media Shoots can be constantly divided
- 18. Small budpresent Apical dominance is removed Cytokinins Auxins Cluster formation Off types Stage 2: Multiplication Enhanced Axillary Branching
- 19. Transfer ofshoots to a medium for rapid development into shoots Sometimes, the shoots are directly planted in soil to develop roots In-vitro of shoots is preferred while simultaneously handling a large number of species Stage 3: Rooting of shoots Rooting & Hardening
- 20. Somatic embryoscarry a preformed radical and may develop directly into plantlet. Embryos sow very poor conversion into plantlets, especially under in-vitro conditions Require an additional step of maturation to acquire the capability for normal germination Adventitious and axillary shoots developed in cultures in the presence of a cytokinins generally lack roots Obtain full plants the shoots must be transferred to a rooting medium which is different from the shoot multiplication medium, particularly in its hormonal and salt compositions For rooting, individual shoots measuring 2 cm in length are excised and transferred to the rooting medium Stage 3: Rooting of shoots Rooting & Hardening
- 21. Stage 4: Transplantation& Accilimitization Transferring the plantlets of stage 3 from the laboratory to the environment f green house. Some plant species, stage 3 skipped, and unrooted stage 2 shoots are planted in pots or in suitable compost mixture Tissue culture raised plants are characterized by: Low photosynthetic rates Non-functional stomata Low cuticular wax Abnormal leaf morphology Accilimitization Green house 2 weeks Temperature (±10º)
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- 23. In-vitro regenerative protocolof V. reitzii Nodule cluster culture formation and shoot regeneration in V. reitzii; a). Donor plant (inflorescence of adult plant); b). Yellow nodular cultures induced in MS medium free of PGR; c). Nodule cluster subcultureed in MS culture medium supplemented with GA3 resulted in high proliferation rate and the subsequent development of adventitious microshoots; d). Elongation and growth of shoot in MS culture medium free of PGR; e). Acclimatization of plantlets; f). Growth in green house
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- 26. Methods of ClonalMicropropagation Multiplication by axillary buds/apical shoots Multiplication by adventitious shoots Beside the above two approaches, the plant regeneration processes namely Organogenesis and Somatic embryogenesis may also be treated as micropropagation Organogenesis (Formation of individual organs such as shoots, roots, directly from an explant or from callus and cell culture induced from the explant) Somatic embryogenesis (Regeneration of embryos from somatic cells, tissue or organs)
- 27. Methods of Micropropagation Organogenesis Organogenesis via callus formation Direct adventitious organ formation Embryogenesis Direct embryogenesis Indirect embryogenesis Microcutting Meristem culture (Mericloning) Bud culture
- 28. 1. Multiplication byAxillary Buds and Apical Shoots Quiescent or actively dividing meristems are present at the axillary and apical shoots tips. The axillary buds located in the axils of leaves are capable of developing into shoots. In in-vivo state, only limited number of axillary meristems can form shoots. Induced in-vitro multiplication in micropropagation, possible to develop plants from meristems and shoot tip culture and form bud cultures
- 29. a. Meristem andShoot 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. For the development of disease-free plants, meristem tips should be cultured. 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.
- 30. Shoot tip (orMeristem) Culture in Micropropagation Stage 1, 2,and 3 represent in Micropropagation
- 31. a. Meristem andShoot Tip Cultures Stage 1: Establishment of meristem on culture. Addition of growth regulators like Cytokinins (Kinetin, BAP) and Auxins (NAA or IBA) Stage 2: Shoot development along with axillary shoot proliferation occurs. High levels of cytokinins required Stage 3: The root formation is facilitated by low cytokinins and high auxin concentration. This is opposite to shoot formation since high level of cytokinins is required (in stage II). 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
- 32. b. Bud Cultures The plant buds possess quiescent or active meristems depending on the physiological state of the plant. Two types of bud cultures involves: o Single node culture o Axillary bud culture
- 33. Single Node Cultures Natural method for vegetative propagation of plants both in-vivo and in-vitro conditions. The bud found in the axil of leaf is comparable to the stem tip, for its ability in micro propagation. 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 Cytokinins is added.
- 34. Single Node Techniquefor Micropropagation
- 35. Axillary Bud Cultures Isolated shoot tip along with axillary bud. The cultures are carried out with high cytokinins concentration. As a result of this, apical dominance stops and axillary buds develop. Eg.: Rosette plant and an elongate plants For a good axillary bud culture, the cytokinin/auxin ratio is around 10:1. 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.
- 36. Micropropagation of plantsby axillary bud (or shoot tips) method: (A) Rosetle plant; (B) Elongate plant
- 37. 2. Multiplication byAdventitious 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. 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.
- 38. 3. Organogenesis Organogenesisis the process of morphogenesis involving the formation of plant organs like shoots, roots, flowers, buds from explant or cultured plant tissues. Two types: o Direct organogenesis o Indirect organogenesis
- 39. a. 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. The term direct adventitious organ formation is also used for direct organogenesis. Induction of adventitious shoot formation directly on roots, leaves and various other organs of intact plants is a widely used method for plant propagation. This approach is particularly useful for herbaceous species. For appropriate organogenesis in culture system, exogenous addition of growth regulators (Auxin and Cytokinin) is required. The concentration of the growth promoting substance depends on the age and nature of the explant, besides the growth conditions.
- 40. b. 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. The explants for good organogenesis should be mitotically active immature tissues. 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 organogenesis.
- 41. Micropropagation of plantsby organogenesis: (A) Direct organogenesis; (B) Indirect organogenesis through callus; (C) Indirect organogenesis though cell culture
- 42. By varying theconcentrations of Auxin and Cytokinin, In- vitro organogenesis can be manipulated Low Auxin and low Cytokinin concentration will induce callus formation. Low Auxin and high Cytokinin concentration will promote shoot organogenesis from callus. High Auxin and low Cytokinin concentration will induce root formation.
- 43. 4. Somatic Embryogenesis Somatic embryogenesis may result in non-zygotic embryos or somatic embryos (directly formed from somatic organs), parthogenetic embryos (formed from unfertilized egg) and androgenic embryos (formed from male gametophyte). In a general usage, when the term somatic embryo is used it implies that it is formed from somatic tissues under in vitro conditions. 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. Development of somatic embryos can be done in plant cultures using somatic cells, particularly epidermis, parenchymatous cells of petioles or secondary root phloem. Somatic embryos arise from single cells located within the clusters of meristematic cells in the callus or cell suspension. Firstly, proembryo is formed which then develops into an embryo, and finally a plant. Two routes of somatic embryogenesis — Direct and Indirect Embryogenesis
- 44. a. Direct Embryogenesis When the somatic embryos develop directly on the excised plant (explant) without undergoing callus formation, it is referred to as direct somatic embryogenesis. Due to the presence of pre-embryonic determined cells (PEDQ found in certain tissues of plants. The characteristic features of direct somatic embryogenesis is avoiding the possibility of introducing somaclonal variations in the propagated plants.
- 45. b. Indirect 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. 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.
- 46. Artificial Seeds fromSomatic Embryo Artificial seeds can be made by encapsulation of somatic embryos. The embryos, coated with sodium alginate and nutrient solution, are dipped in calcium chloride solution. The calcium ions induce rapid cross-linking of sodium alginate to produce small gel beads, each containing an encapsulated embryo. These artificial seeds (encapsulated embryos) can be maintained in a viable state till they are planted
- 47. Micropropagation of plantsby embryogenesis: (A) Direct embryogenesis; (B) Indirect embryogenesis
- 48. Factors Affecting Micropropagation Genotype of the plant Physiological status of the explants Culture media Culture environment (Light, Temperature, Composition of gas phase )
- 49. Factors Affecting in-vitroRooting For efficient in vitro rooting during micropropagation, low concentration of salts (reduction to half to one quarter from the original) is advantageous. Induction of roots is also promoted by the presence of suitable Auxin (NAA or IBA).
- 50. Applications of ClonalPropagation Production of disease and virus free plantlets Production of seeds in some crops High rate of plant propagation Cost effective process Automated micropropagation
- 51. Advantages of ClonalPropagation From one to many propagules rapidly Multiplication in controlled lab conditions Continuous propagation year round Potential for disease free propagules Inexpensive per plant once establishment
- 52. Disadvantages of ClonalPropagation Contamination of cultures Brewing of medium Genetic cultures Vetrification Cost factor
- 53. Horticultural uses forplant tissue culture Clonal mass propagation Difficult or slow to propagate plants Introduction of new cultivars Vegetative propagation of sterile hybrids Pathology – eliminate viruses, bacteria, fungi etc Storage of germplasm
- 54. Production of Disease-freePlants Many plant species are infected with pathogens — viruses, bacteria, fungi, mycoplasma and nematodes that cause systemic diseases. Although these diseases do not always result in the death of plants, they reduce the quality and yield of plants. The plants infected with bacteria and fungi frequently respond to chemical treatment by bactericides and fungicides. However, it is very difficult to cure the virus-infected plants. Further, viral disease are easily transferred in seed- propagated as well as vegetatively propagated plant species. Plant breeders are always interested to develop disease-free plants, particularly viral disease-free plants. This have become a reality through tissue cultures.
- 55. Apical Meristems withLow Concentration of Viruses: Absence of vascular tissue in the meristems through which viruses readily move in the plant body. Rapidly dividing meristematic cells with high metabolic activity do not allow viruses to multiply. Virus replication is inhibited by a high concentration of endogenous auxin in shoot apices. Tissue culture techniques employing meristem-tips are successfully used for the production of disease-free plants, caused by several pathogens — viruses, bacteria, fungi, mycoplasmas.
- 56. List of theplants with virus elimination by meristem culture
- 57. Merits and Demeritsof Disease-Free Plant Production Among the culture techniques, meristem-tip culture is the most reliable method for virus and other pathogen elimination. However, requires good knowledge of plant pathology and tissue culture. Virus-free plants exhibit increased growth and vigor of plants, higher yield (E.g., Potato), increased flower size (E.g., Chrysanthemum), and improved rooting of stem cuttings (E.g., Pelargonium) Limitation: Virus-free plants are more susceptible to the same virus when exposed again. Reinfection of disease-free plants can be minimized with good knowledge of greenhouse maintenance.
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