Micropropagation clonalpropogation,

Micropropagation

• Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods. 
• Micropropagation is used to multiply plants such as those that have been genetically modified or bred through conventional plant breeding methods. 
• In-vitro it is a complicated process requiring many steps or stages. 

• Murashige (1978) proposed distinct stages that can be adopted from overall production technology of clones under in-vitro culture conditions. 

Sterlization of Explant
This process includes 3 steps:
1. Washing under running water using liquid detergent and sodium hypochloride.
2. Two rinsing and washing with distilled water.
3. Explant sterilization with aqueous mercuric chloride in the laminar air flow bench.

Inoculation of Explant
Inoculation is carried out in the laminar air flow in the nutrient medium. Keeping all the sterile culture conditions the explant is inoculated on the medium, using a pair of sterile forceps.

• last for 3 months to 2 years and requires at least 4 passages of the subculture usually explant carrying a performed vegetative bud are suitable for enhanced axillary branching. 
• If stock plants are tested virus free than the most suitable explants are nodal cuttings. 
• The disadvantages of using small size explant are that they have a low survival rate and show slow initial growth.

Multiplication of Shoots or Somatic Embryo formation
Plant tissue culture technique also leads to the development of shoots and somatic embryos in vitro. The process includes various stages which are described below:

Stage 1

• This stage involves the maximum proliferation of regenerated shoot using a defined culture medium. Various approaches for micropropagation include–

1. Multiplication growth and proliferation of meristems excised from apical and axillary shoot of the parent plant.
2. Induction and multiplication of adventitious meristems through process of organogenesis or somatic embryogenesis on direct explants.
3. Multiplication of calli derived from organ tissue, cell etc. and the subsequent expression of either organogenesis or somatic embryogenesis in serial subcultures.

     Fig.  Schematic representation of Somatic Embryogenesis in carrot

Germination of Somatic Embryo and Rooting of Regenerated Shoot-

Stage 2- 

Shoots proliferated from stage 1 is transferred to a rooting (storage) medium. Sometimes shoots are directly established in soil as microculturing to developed roots. The shoots are generally rooted in vitro. When the shoots or plantlet are prepared for soil it may be necessary to evaluate several factors such as:

(i) Dividing the shoots and rooting them individually.

(ii) Hardening the shoots to increase their resistance to moisture stress and disease.

(iii) Rendering plants capable of autotrophic development in contrast to heterotrophic state induced by culture.

(iv) Fulfilling requirements of breaking dormancy.

Stage 2 - Requires 1-6 weeks Transfer of pellets to sterilized soil or green house.

Stage 3- 

Steps taken to ensure successful transfer of the plantlets of stage 2 from the aseptic environment of the laboratory to the environment of the green house comprises 

stage 4. 

This is known as acclimatization. It takes 4-16 weeks for the finished product (plant size in the range of 3-6 inches) to be ready for sale.

Proliferation of Shoots in the multiplication medium

in-vitro multiplication of shoot involves 3 main approaches.

(a) Multiplication of axillary and apical shoots

Axillary and apical shoots contain quiescent or active meristem depending upon the physiological state on the plant. The axillary buds are treated with hormones to break dormancy and produce shoot branches. The shoot are then reported and rooted to produce plants division. Generally the technique of proliferation by axillary shoots is applicable to any plant that produces regular axillary shoots are respond to cytokinins such as BAP and Zeatin. Apical shoots (1-5 mm) are normally cultured on media containing mixture of auxin and cytokinin. The presence of cytokinin in the media inhibits root

development, cultured material is transferred in stage 3 to a rooting medium which contain either no or reduce levels of cytokinin.

(b) Multiplication by adventitious shoots:

Adventitious shoots are stem and leaf structures that arise naturally on plant tissues located inside other than at normal leaf axil region. These structures include stems, bulbs, corns, tubers and rhizomes. Almost every one of these organs can be used as culturing in conventional propagation e.g. (leaves of Begonia) in culture similar type of shoot formation can be induced by using appropriate condition of growth regulators in media I 25°C some cultures may require initial low temperature for morphogenic resistance. Genotype screening and selection of genotypes among segregating populations could be fruitful approaches in the improvement of micropropagation capabilities of plant which are recalcitrant in tissue culture.

Acclimatization of Plant transferred to Soil

Micropropagation on a large scale can be successful only when plants after transfer form culture to soil show high survival rates and the cost involved in the process is low. Plants are transferred to the soil usually after the in-vitro rooting stage it is essential that the dark parts of tissue culture plants or shoots be washed thoroughly before their transfer to the potting mixture. Transplanted plantlets or shoots are immediately irrigated with an inorganic nutrient solution and maintained under high humidity for the initial 10-15 days. This is required between plantlets, dividing culture all adopted to almost 90-100% humidity attempts have also been made to harden the shoot system by inducing

anatropism and development of surface wax on in-vitro leaves. During large scale micropropagation of some plants certain (bacterial) contamination persists even after critical surface sterilization of explants. Addition of antibiotics or fungicides to the culture medium may control the contamination.

Browning of the Medium

Explants from the adult tissue of some woody species often produce excessive amount of phenolic substances which turn the medium dark brown. Such a medium is toxic to issues and inhibits their growth. Browning may be prevented by incorporating ascorbic acid or citric acid in culture media or by repeated subculturing.

Advantages of Micropropagation product development

1. Rapid multiplication: Micropropagation provides a method for rapid increase in the both asexually propagated and sexually propagated materials many flowers and vegetable used.
2. Product uniformity: The resulting product can have a high degree of phenotypic uniformity hence the crops can be artificially manipulated in the laboratory to yield a large plant population at the same growth.
3. High volume: Large population can be produced in relative by smaller growing space and in a reduced time frame.
4. Elite selection: It is possible to effectively capitalize on the selections of one desirable plant and then micropropagate it into large number and release as superior selection.
5.  Germplasm stage: Storage methods for effective preservation of valuable selection can be accomplished by combing micropropagation with cold storage and even cryopreservation in liquid N2.
6. Diseased induced plants: Technique to index or eliminate specific diseases particrularly viruses can readily be incorporated into micropropagation procedure.
7. Non-Specific production: Micropropagation gives propagation uses such as minitubers or minicorms for plants multiplication throughout the year irrespective of the season.
8. Cloning of dioecious species: Multiplicaiton by cloning of dioecious species is extremely important when the seed progeny yield 50% males and 50% females and plants of one of series are desired commercialized
9. 30°C is optimum temperature for cellulose formation.
10. Shoot tip culture and virus free plant

Selection of explant

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Surface sterilization and washing

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Establishment on growth medium

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Transfer to proliferation medium

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Shoot and rooting formation

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Transfer of shoots and plantlets to sterilized soil

Major stage of micropropagation

Morel and Martin, 1952, developed the technique of meristem culture for in vivo

virus eradicate of Dahlia.

We use the axillary bud or meristem tissue:

 Because the high concentration of auxin, virus is not able to survive.

 The cell division is too fast so virus is not able to replicate in this region.

Shoot tip or meristem culture

Cultivaiton of axillary or apical shoot meristem via meristem culture

Explant: Shoot apical meristem lies in the shoot tip beyond the youngest leaf and

first leaf primodium.

• It measure upto about 100 m in diameter and 250 m in length.
• Thus a shoot tip of 100-500 m in m contains 1-3 leaf primodia in addition to the apical meristem.
• 1 mm – shoot tip used for virus elimination 1 cm for clonal propagation.
• Shoot tip may be cut into five pieces to obtain more than one plantlet from each shoot tip.
• Meristem of shoot tip is cut or isolated from stem by applying a U shaped cut with a sterilized knife.