Micropropagation

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MICROPROPAGATION

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Methods of Plant Propagation Asexual (By multiplication of Vegetative parts) Sexual (Germination of Seeds) Sexually propagated plants show higher degree of heterogeneity Asexually propagated plants give rise to genetically identical plants

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Clonal Propagation Multiplication of genetically identical copies of cultivar. Micropropagation Clonal propagation through tissue culture technique Regeneration of whole plant through tissue culture. OR

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Micropropagation can be achieved in shorter space and time Produce plant in large number starting from single individual. Use of tissue culture for micropropagation was first initiated by G. Morel (1960) In vitro micropropagation is complicated process and requires many steps/stages Murashige (1978) proposed 4 distinct stages Stages I to III in vitro and IV in in vivo Debergh & Maene (1981) suggested an additional stage ‘0’

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Stage ‘0’ Initial step, stock plant grown three months prior to initiation of culture. Plant grown relatively low humidity Measures to be adopted for reduction of surface and systemic microbial contaminants.

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Stage ‘I’ Murashige (1974) – initiation and establishment of culture. Preparation of explants and establishment on suitable culture medium. Shoot tips and axillary buds- commercial micropropagation Procedure- surface sterilize the explant and estb. on culture medium. Control microbial contamination within explant. Stage ‘I’ last for 3 months to 2 years, requires at least 4 sub-culture.

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Explants carrying performed vegetative bud suitable for enhanced axillary branching. Virus free plant- sub millimeter portion of shoot-tip Stock plant virus free- nodal cutting Disadvantage of using small sized explant for micropropagation Shoot tip explant- low survival rate and slow initial growth. Therefore, sub-terminal/slightly older segments are used. Meristem tip culture- result in loss of horticultural traits.

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Starting material for micropropagation Leaf Tip bud Internode Axillary bud Root

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[Drew et al 1991]

Stage I:

Stage I

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Freedom from contamination in Stage I is important

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Stage II Takes bulk of micropropagation. Stimulates maximum proliferation of shoot. Shoot harvested from multiplying cultures can be either sold as stage II product or carried on to stage III. Lasts for 10-36 months with large number of sub-cultures.

Stage II:

Stage II

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Shoot Regeneration

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Stage III Shoot proliferated during stage II carried to rooting medium. Sometimes shoot are established in soil as microcuttings to develop roots. Generally shoots are rooted in vitro . When shoots/plantlets are prepared for soil it may be necessary to evaluate the survival factors such as Stage III- 1 to 6 weeks. Shoots/roots dividing individually. Hardening shoot to increase their resistance to moisture stress. Rendering plant capable of autotrophic development.

Stage III:

Stage III

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Root Initiation

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Stage IV Transfer of plantlet from stage III to stage IV in GH. Unrooted stage II acclimatize in suitable compost mixture or soil in pots under controlled condition light, temp. & humidity in GH. Supplying bottom heat aids to pots with plantlet or cutting and maintenance of dense fog system in GH enhances rooting process. Complete plant can be established in artificial growing media such as soilless mixes, rockwood plug or even sponges. Take 4 to 16 weeks to obtain finished product.

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Grading of Plantlets before transfer to pots

Stage IV:

Stage IV

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Factors affecting shoot multiplication 1. Physiological status of plant material Explants isolated from more recently produced parts of plant are more regenerative. 2. Culture Media Standard tissue culture media – suitable for achieving stage I & II micropropagation. GA & ABA inhibit root formation Stage III – requires some modification To induce adventitious root formation after axillary shoot propagation- cytokinin omitted & auxin added.

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3. Culture environment In vitro regenerated shoot are- heterotrophs Optimum light intensity for shoot multiplication is 100 lux. Blue light- Shoot; Red light- Root Micropropagation- culture – 25 0 C Light absorbed by photosynthetic pigments in cultured tissue plays important role in inducing morphogenesis, Diurnal illumination 16 hrs day & 8 hrs night generally found satisfactory for multiplication and proliferation of shoot.

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4. Genotype Genotype differ for regeneration capabilities 5. In vitro rooting Medium- low concentration of salts suitable for satisfactory rooting of shoots. Micropropagation system developed for one particular cultivar will not automatically applicable to another even within spp. Roots induce in presence of suitable auxin

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6. Acclimatization of plants transferred to soil Micropropagation successful- plant transfer from culture to soil show high survival rate and cost involved in the process is low. Transplanted plantlets/ shoot immediately irrigated with inorganic nutrient solution and maintained under high humidity for initial 10-15 days. Tissue culture plant show structural abnormalities The structural abnormalities, hetetrotrophic mode of nutrition & poor mechanism for control of transpiration loss make pl. further vulnerable to transplantation shock

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Advantages of Micropropagation Requires relatively small growing area Gives propagules such as minitubers/microcorms for plant multiplication throughout the year. Enhancing rate of multiplication Shoot multiplication –short cycle (2-6 weeks) Small size propagules- storage on large scale, easily transported. Pathogen free plant raised and maintained.

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Clonal propagation in dioecious plant is extremely important. Sophisticated instruments required. Stock of germplasm is maintained for many years. Limitations Demand greater- requires skill in handling and maintenance Shoot tip derived plant show genetic instability.

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