Micro Propagation

By: Pharma Tips | Views: 12397 | Date: 01-May-2011

Micro propagation is a field dealing with the ability to regenerate plants directly from explants. It is defined as “true-to-type propagation of selected genotypes using in vitro culture techniques”. Unlike animals where differentiation is generally irreversible, in plants (due to an intact membrane system and a variable nucleus) even highly mature and differentiated cells retain the ability to regress to a meristematic state. The phenomenon of a mature cell reverting to the meristematic state and forming u

Micro propagation is a field dealing with the ability to regenerate plants directly from explants. It is defined as “true-to-type propagation of selected genotypes using in vitro culture techniques”. Unlike animals where differentiation is generally irreversible, in plants (due to an intact membrane system and a variable nucleus) even highly mature and differentiated cells retain the ability to regress to a meristematic state. The phenomenon of a mature cell reverting to the meristematic state and forming undifferentiated callus tissue is termed ‘dedifferentiation’. The degree of regression a cell can undergo would depend on the cytological and physiological state it had reached in situ. The ability of plant cells to be cultured indefinitely on fully defined medium and their capacity to regenerate (cellular totipotency) into the whole plant via organogenesis or embryogenesis (redifferentiation) have helped to study some of the problems haunting plant scientists.
The vegetative method of propagation plants is termed as micropropagation or cloning tissue culture or growing in vitro. Morel (1965) used the technique for the first time for orchids.(26) This propagation is now commonly accepted and adopted widely with lot of significance in commercial horticulture. It has important agro economic applications in medicinal plant preservation, floriculture and forestry.
Hundreds of commercial laboratories in different parts of the world are currently involved in micropropagation   some of them producing over 20 million plants a year.(1)

      The various advantages of micropropagation are:
1. Useful for plants that are difficult to propagate by conventional methods (e.g. plants producing little viable seeds )
2. In relatively short time (due to increased multiplication rate ) and space,  a large number of plants that is
(a) Genetically stable and true-to-type progeny (by rapid clonal propagation )
(b) Virus-free.
(c) Insect-resistant, disease-resistant, herbicide-resistant. With special phenotypic characteristic (i.e.) changed genotypes (tetraploids, haploids, and hybrids) can be produced from single individual.
1. Conservation of genetic resources of species and threatened medicinal plants (by axillary bud proliferation)
2. Plant improvement by regeneration technique in conjuction with in vitro cell manipulation and
3. To solve some theoretical problems connected with the pathway of biogenesis of chemical compounds in in plants and the relation between organogenesis and production of metabolites.

Though, there are several advantages, there are also certain disadvantages with  
the system. They are:
1. Micropropagation methods through use of tissue culture involve capital intensive expensive materials like autoclave, laminar airflow bench, controlled culture rooms etc.
2. This is a technically skilled work, knowledge about material, techniques and decisions making (during subculture and multiplication of propagule) are required in the personnel.
3. Contamination is a serious threat and cause severe damage to material and adds substantively lot to the cost of production, affects time schedule delivery of the material.
4. Specific conditions of micropropagation, rooting and hardening may be required. Therefore, each material requires separate research methods.
5. Small delicate plantlets are produced, which take longer initial time to grow.
6. Genetic stability is doubtful in certain methods.
7. It is a capital-intensive industry, if plants are produced in small number, they cost too much.
8. Otherwise also cost is a major factor for the production and sale of tissue culture raised plants.

 Factors that influence micro propagation:
 The factors that important roles in the degree of success achieved in a given micropropagation or plant regeneration system include

1. The genotype of the donor,
2. The physiological conditions of the donor material,
3. The explants source,
4. The orientation and size of explants in culture,
5. The culture medium composition (s),
6. Interactions of endogenous hormones with exogenously supplied growth regulators,
7. The incubation conditions (including light quality and Intensity, Temperature,  Relative humidity and Air quality) and
8. The timing of the subculture interval/changes in medium / incubation treatment.

      Cells must be  physiologically receptive to the hormone induction  signals before they can be induced to regenerate a plant or organ .thus ,  the timing of the application of induction signals can be critical . This physiology receptiveness to be induced is termed ‘competence’ (graham and wearing 1984, Christianson and warnic 1987). Komalavalli and Rao (2000) reported that the nature of the explant ,  seedling age ,  medium type, plant growth regulators, complex extracts (casein hydrolysate ,  coconut milk, malt extract and yeast extract ) and antioxidants (activated charcoal ,  ascorbic acid, citric acid and polyvinylepyrrolidone ) markedly influence in- vitro propagation of gymnema sylvestre  .

The other problems commonly encountered in the micropropagation industry are
1. Vitrification: in repeated cycles of in-vitro shoot multiplication of culture show water –soaked (almost translucent) leaves and may eventually die.
2. High price of propagated plants: plant propagation by tissue culture techniques is encumbered by the intensive labour requirement for the multiplication  process; thus scaling-up systems and automation of unit operations (i.e.) increased mechanization step or by placing them in ‘low vage ’ countries are necessary to cut down the production coasts (Aitken- Christiie 1991 , Vasil 1991) .
3. Somaclonal variation; it is defined, as the variation, which occurs in culture of cells and tissues that may be either genetic or epigenetic. By selecting somaclonal variants, disease resistant as well as herbicide resistant plants could be achieved. This phenomenon has not yet been used to improve medicinal plants but several examples of changes in the colour of flowers of ornamental plants indicate that secondary metabolism can be affected. The factors that frequency of somaclonal variation among regenerated plants are
1. Length of time in culture.
2. Culture stresses (Improper media components or mutagens, certain growth regulator treatments, delayed subculture intervals leadings to nutrient stress, or extreme or highly variable incubation conditions) and
3. Explant source (Non-Meristematic explants that do not orderly mitoses, used in adventitious or de novo regeneration systems).(18)

General technique of micro propagation:

Stage 1:
In stage 1, suitable media (MS/modified MS media), plant growth regulator levels and their combinations are selected in order to promote explant establishment and shoot growth. Physiological stabilization may require (3-24) months and (4-6) sub cultures on stage 1 medium. Failure to do so before transfer to stage 2 medium containing higher cytokinin level (to disrupt apical dominance of shoot tip ) may result in diminished shoot multiplication rates or production of undesirable basal callus and adventitious shoots. In many commercial labs, stabilized cultures verified as having specific pathogen tested and free of cultivable contaminants, are often maintained on media that limit shoot production to maintain genetic stability. These cultures called “mother blocks” serve as sources of shoot tips or nodal segments for initiation of new stage 2 cultures. The following factors may affect successful stage 1 establishment of meristem explants:
Explantation time: (beginning of growing seasons generally gives best results),
Position of explant on the stem,

Explant size:

Polyphenol oxidation (tissue/medium browning). Excision of explant promotes release of polyphenol, which stimulates the activity of polyphenol oxidase. Tissue browning can be reduced by the use of liquid medium with frequent transfer, the addition of antioxidants (ascorbic acid, PVP) or culturing in reduced light intensity/darkness.
Cytokinins and/or Auxins are most frequently added to stage 1 media to enhance explant survival and shoot development. BA, NAA and IBA are most widely used plant growth regulators. 2, 4-D is more effective in somatic embryogenesis.

Stage 2:
           This is characterized by repeated enhanced formation of axillary shoots from shoot tips or lateral buds cultured on medium supplemented with a relatively higher cytokinin level to disrupt apical dominance of the shoot tip. Subcultures inoculated with explants that had been shoot apices in the previous subculture often exhibit higher multiplication rates than lateral bud explants. Inverting shoot explants in the medium can double/triple the number of axillary shoots produced on vertically oriented explants per culture period in some species. Selecting only terminal shoots of axillary origin for subculture, instead of shoot bases, decrease the frequency of off-types including the preclinal chimeras. Addition of auxin, often mitigates the inhibitory effect of cytokinin on shoot elongation, (but May from callus) thus increase the number of usable shoots of sufficient length for rooting. Selection of stage 2 cytokinin type and concentration is based on shoot multiplication rate; shoot length, frequency of genetic variation and possibility of adverse carry over effects on the survivability and rooting of plantlets in stage 4. Using liquid shake cultures has the special advantage that the shoots broke apart as they multiplied and the manual cutting of shoot cultures is not required. Media with reduced salt levels are used if necessary. The nature of organogenetic differentiation is determined by the relative concentration of Auxins and Cytokinins.(19)

Stage 3:
            This may involve elongation of shoot prior to rooting (GA3 may be added), rooting of individual shoots/shoot clumps, fulfilling dormancy requirements of storage organs by cold treatment and prehardening cultures to increase survival. Where possible, commercial labs have developed procedures to transfer stage 2 micro cuttings to soil, thus by-passing stage 3 rooting. A low salt medium with optimum auxin concentration is determined based upon percentage of rooting, root number and root length (upto max. 5 mm, approx. 15 days) to prevent root damage during transplanting.(19)

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