In this article we will discuss about:- 1. Definition of Micro-Propagation 2. Pathway of Regeneration 3. Stages 4. Techniques 5. Problems 6. Advantages 7. Disadvantages.
Definition of Micro-Propagation:
The term micro-propagation is used specifically to refer to the application of tissue culture techniques to the propagation of plants starting with very small plant parts grown aseptically in a test tube or other container.
Tissue culture helps in rapid multiplication of true-to-type plants throughout the year. Large number of plants can be produced in culture tubes in small space with uniform growth and productivity instead of growing them in large areas in nursery. Tissue culture plants especially raised through meristem are free from any diseases. Tissue culture coupled with somatic hybridization helps in evolving new cultivars in a short time.
The meristem explant used for micro-propagation is about 0.1 – 0.5 mm size having one or two leaf primordia.
Pathway of Regeneration:
There are number of pathways for the regeneration of whole plantlet from excised plant parts.
The three main pathways are:
1. Regeneration from Existing Meristems:
This is also known an axillary shoot proliferation. The existing meristems such as shoot tip or nodal bud is cultured on the medium, containing cytokinins. The shoot proliferation depends on the cytokinin used. The commonly used cytokinins are Benzyl aminopurine (BAP) and Kinetin. The regenerates are considered to be genetically stable, as compared to regeneration from adventitious meristems.
2. Regeneration from Adventitious Meristems:
Shoot multiplication either directly or by callus formation can be obtained by inducing adventitious shoots on mature plant organs such as leaves, stems and roots. For initiation of adventitious meristems, a proper balance of auxin and cytokinin is needed in culture medium. In general, shoots are formed when a high ratio of cytokinin to auxin is present and reverse is true for root formation. The plants regenerated via this method are not always genetically stable, due to accumulation of aneuploid and polyploid cells with continuous sub-culturing of callus causing mixoploids. The repeated subculture of callus also reduces its morphogenetic potential/regenerative capacity.
3. Regeneration by Somatic Embryogenesis:
The induction of somatic embryos is the best technique for rapid and true to type multiplication of plants. The somatic embryos originate from somatic or vegetative cells, and are bipolar structures, which possess both shoot and root meristem. The induction of embryo requires a high level of auxin in culture medium, followed by low auxin and cytokinin medium. Somatic embryos may arise in culture directly on explants or via callus formation or liquid suspension cultures. The somatic embryos can be encapsulated, thus producing artificial or synthetic seeds, which is an attractive alternative for propagation of plants.
Among two methods i.e. hydrated or desiccated, for artificial seed production, the production of hydrated seeds is more popular. In this method, the individual somatic embryo is encapsulated in a water based gel (hydrogel such as calcium alginate). Embryos developed through tissue culture technique are mixed with sodium alginate and dropped with pipette into a calcium chloride solution to form calcium alginate capsules. The capsules are washed in water and then placed on culture medium for germination. Artificial seeds have been produced in banana, mango, citrus, apple, olive and kiwi.
Stages of Micro-Propagation:
Four main stages are involved in micro-propagation of plants, such as explant establishment, shoot proliferation, rooting of shoots, hardening and transfer to soil/field.
1. Explant Establishment:
The establishment of explants depends on several factors such as the source of explant/genotype, type of explants such as leaf, root, surface sterilants (Table 1) and the in vitro culture conditions such as culture media composition, temp, humidity, light, etc. The explants showing growth are considered established.
2. Shoot Multiplication:
The established explants are sub-cultured after 2-3 weeks, on shoot multiplication medium. The medium is designed in such a way to avoid the formation of callus, which is undesirable for true to type multiplication of plants. Thus, the careful use of auxins like NAA, 2, 4-D and cytokinins like BAP, Kinetin is done in culture medium. It is well-established fact that cytokinins enhance shoot multiplication.
3. Rooting of Shoot:
The in vitro regenerated shoots are rooted in the medium containing auxins like NAA, IBA. The rooting can also be induced when in vitro shoots are exposed to stress conditions. The rooting should also be preferably without formation of callus, thus avoiding somaclonal variants.
4. Hardening and Transfer to Soil/Field:
The in vitro obtained plants are hardened/acclimatized before transfer to the field. The hardening is necessary as the tissue culture derived plants grow under high humidity conditions, have open stomata, lower epicuticular wax, thus leading to increased transpiration losses and resulting in mortality of plants.
Techniques of Micro-Propagation:
Meristem culture and micro-grafting techniques have been standardized in different fruit plants to produce virus free plants. The success varies with the plant species, variety and the culture environment.
(1) Meristem Tip Culture:
This technique is widely used in carnation, dahlia, orchids and in potato. The meristem tip consisting of one or two pairs of leaf primordia are cultured in a medium. The plantlets are regenerated after a few weeks. After hardening of plantlets, these are transplanted in the soil under natural environmental conditions.
Meristem tip cultured plants give rise to polyploid plants instead of diploid plants. This tip culture method is very useful for the elimination of viruses from infected plant material. Rapid multiplication of plants is possible through meristem culture. The plants produced are free from pathogens and can be stored for longer period.
(2) Micro-Grafting:
The various steps in micro-grafting include scion preparation, rootstock preparation, in vitro grafting and acclimatization/hardening of the plants. The in vitro raised nuclear seedlings are used as rootstocks. The scion (meristem 0.1 – 0.4 mm) is obtained either from young growth of field grown trees, defoliated glass house grown plants or in vitro proliferated nodal segments obtained from mature trees.
The grafting is done with the help of stereomicroscope, under aseptic conditions. Many viruses have been eliminated via micro-grafting in fruit plants e.g. citrus tristeza virus, peach latent mosaic viroid, pear vein yellow virus.
Micro-propagation has been successfully adopted in many horticultural crops. Strawberry was the first fruit to be propagated commercially by this technique. The tissue culture technique has been used in banana, grape and papaya in India.
In banana, shoot tip excised from rhizomes of wood sucker are suitable explants and MS medium supplemented with 3 per cent sucrose, gelite (0.25%) is the best. In grapes, shoot tips and two nodel micro cuttings are highly suitable explants for faster and disease free production of plants through tissue culture. In papaya, shoot tip culture technique has been demonstrated to produce female plants in the desired ratio
Problems Encountered during Micro-Propagation:
The success of micro-propagation in several instances is hampered by the following problems:
1. Microbial Contamination:
Bacterial/fungal contamination in the culture does not allow the propagates to grow. This problem can be overcome by growing donor plants in growth chambers, systemic fungicide spray prior to explant removal, effective sterilization of explants, performing inoculations in laminar air flow cabinets fitted with HEPA filters (0.2 µm) and using sterilized surgical instruments. Fumigation of inoculation room using dilute formaldehyde solution also helps to minimize this problem.
2. Browning of Cultures:
The cultured explants of certain plant species secrete phenolic substances into the medium, which cause browning due to oxidation of phenols and formation of quinones, the toxins which effect the growth of cultured explants. The use of antioxidants such as activated charcoal (1-2%), citric acid or ascorbic acid (50-100 mg/1) and polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP) in the culture medium helps to check the browning.
3. Variability in Tissue Cultured Regenerated Plants:
Variability is highly in-desirable in the micro-propagated plants. It may occur due to callusing and regeneration of plants from callus instead of direct shoot induction and proliferation. Moreover, the plants regenerated through adventitious meristems as compared to axillary meristem are susceptible to mutations, as it is derived from either a single cell or a small group of cells.
This leads to variation in regenerated plants. The variation occurred due to callusing can be overcome by addition of growth substances which inhibit callusing such as TIBA (triidobenzoic acid), phloroglucinol and phloridzin and also by reduction of inorganic salt concentration in the culture medium.
4. Hardening of Plants:
Apart from these problems the tissue culture regenerated plants have abnormal leaf morphology, poor photosynthetic efficiency, malfunctioning of stomata(open), reduced epicuticular waxes and thus are amenable to transplantation shock. Hardening of such plants is thus must before transplantation under field conditions.
Transplanting of tissue culture derived plants on appropriate soil media (Table 2), the conservation of moisture by creating high humidity around the plants, partial defoliation and application of anti-transparent are useful for hardening of in vitro raised plants. To prevent mortality, it is must to harden or acclimatize tissue cultured plants.
Advantages of Micro-Propagation:
This method has many advantages over the conventional propagation methods:
(i) The plants can be produced round the year irrespective of seasonal constraints.
(ii) Little space is required to maintain and multiply large number of plants.
(iii) A small tissue is required as an explant, thus saves the scion wood to great extent.
(iv) Such produced plants exhibit vigorous growth and higher yield.
(v) The plants are usually free from virus.
(vi) It is highly beneficial in dioecious fruit plants like date palm and papaya, where large production of female plants is possible.
(vii) This method helps in reducing the breeding cycle.
(viii) It is very usefully in plants in which vegetative propagation is not possible e.g. papaya and date palm.
(ix) It is possible to produce homozygous plants under in vitro conditions.
Disadvantages of Micro-Propagation:
The following are the main disadvantages of micro-propagation of plants:
(i) The facilities required for micro-propagation are usually costly.
(ii) Technical skill is required to carry out different procedures in the laboratory.
(iii) Pathogens once appeared in the system, they multiply at a faster rate in a short time.
(iv) The plants having high level of phenols, usually do not respond to this propagation technique.
(v) Establishment of plants (hardening) in the field conditions is very difficult. Success in Hardening of Plants
To have success in hardening of micro-propagated plants, the following approaches are needed to be adopted:
(a) Balanced proportion of roots and shoots in micro-propagated plantlets.
(b) There should be appropriate rooting media for establishment of plants ex vitro.
(c) Balanced nutrition for survival of rooted plantlets.
(d) Simultaneous rooting and acclimatization is required.
(e) Cleaning of gelling agents from roots before transfer of rooting media.
(f) Moisture content or humidity around transferred plantlets.