According to Murashige of California University, micro-propagation is distinctly divided into five stages.
Stage 0 — Mother Plant Selection:
Before initiation of micro-propagation, selection of suitable mother plant is crucial in the whole exercise of propagation. Generally, disease free mother explant is selected for the micro-propagation to reduce contamination of cultures. Certain growth parameters of mother plant can be improved by pretreatment of mother explant before initiation of cultures.
Stage I — Establishment of Aseptic Culture:
Micro-propagation begins with successful establishment of cultures of selected plant material. The initial steps of micro-propagation are generally associated with several hurdles such as rate of contamination and phenolic exudation. Plant tissues are commonly associated with bacteria and fungus.
In tissue culture media, presence of sucrose as a carbon source encouragies their growth and consequently kills inoculated tissues. Therefore, adequate sterilization methods are employed to eliminate microorganisms from explant. Several sterilizing agents or surface disinfectants such as sodium hypochlorite (2%), calcium hypochlorite and saturated chlorine water are used.
Sodium hypochlorite is often used as 5-6% commercial bleach. Examples are chlorox or javex. The plant segments are presoaked in 10-15% commercial bleach for 10-12 minutes. High concentration is minimised to avoid tissue damage or cell death. To increase wettability, a small amount of (0.1%) of surfactents such as tween 20, or teepol is added to the disinfecting chlorite (0.1 to 1%) treatment.
In addition, a quick dipping (5-30 sec) of explant in 70% ethanol is frequently performed prior to bleach soaking. In certain cases, ethanol dipping may be followed by brief flaming. In the final treatment, sterile water is employed to remove traces of mercuric chloride before inoculating explant.
Beginning of micro-propagation encounters another barrier—phenolic exudation. During propagation of explant, high polyphenol oxidases are responsible for synthesis and release of phenolics, which can eventually kill plant tissues. Several chemicals can be employed to check exudation of phenolics. Adsorption property of activated charcoal can effectively reduce the problem.
The most potential antioxidant chemical polyphenyl pyrrolidine (PVP) can effectively check phenolics exudation. Adding some other antioxidants like ascorbic acid into culture media and incubating the initial period of primary culture in citric acid, reduced light or dark incubation of culture can minimize the damage. In addition, liquid media and frequent sub culturing in liquid media are some of the promising methods in controlling phenolics.
Stage II — Multiplication of shoots:
The main objective of the stage II is the multiplication of organs like shoot and increasing their numbers considerably. They are able to give rise to new individual plant.
Multiplication of shoots through tissue culture involves four routinely used methods like:
(a) Callus mediated multiplication
(b) Adventitious shoots mediated multiplication
(c) By apical or axillary shoots
(d) Direct or indirect embryogenesis.
Callus Mediated Shoot Multiplication:
Innumerable number of plants can be accomplished by callus culture. Auxins in general favours callus induction in cultures. Ratio of auxins to cytokinins could play a decisive role in re-differentiation of shoots from the callus. The callus system can be maintained for several weeks by sub culturing every 4 weeks (passage). Differentiation in Crotolaria callus culture induced from roots stem and leaf explant, leaf lamina, stem segment.
Successful regeneration of plant lets from callus culture of ginsing (Panaxginsing) was carried out under optimum conditions. In contrast, Wang (1990), regenerated ginseng plant from friable embyrogenic callus of american ginseng (Panax Guiniplia). Callus mediated shoot multiplication involves certain drawbacks.
There might be gradual decline or total loss of regeneration potential of callus cells. In addition, callus mediated regenerated plants generally exhibit genetic variations like euploids, aneuploidy, polyploid and other chromosomal abberation, which reflects on variations of morphological characters.
Invitro Multiplication of Adventitious Shoots:
In this method, adventitious shoots arise directly from the tissues of the explant and does not involve callus mediated regeneration. The regeneration by direct organogenesis via adventitious shoot initiation refers to initiation of shoots directly from stem, tuber, bulb, leaf tissues other than leaf axils remains.
Most of the commercial practical propagation involves adventitious bud formation. The main advantage of micro-propagation by direct adventitious shoot regeneration simple to transfer several pieces of leaf petiole aseptically to culture media than to isolate same number of shoot meristems.
The propagation ratios are very high, if numerous small shoots arise rapidly from each explant. In nature, several examples of initiation of adventitious buds from leaf or stem cutting which are not propagated vegetatively as in case of flax and brassica members. Plantlet obtained directly from several explant like leaf.
Immature panicle Propagation potential of grapes exhibit production of thousands of plants from single explant. For example, in grape, shoot tip explant on the culture media produced more than eight hundred plants within few months. Similarly, petiole culture of Santapanlia ionantha produced nearly 20,000 plantlets from single petiole explant.
The plants obtained from adventitious bud induction maintain genetic uniformities which is advantageous for commercial ornamental industry. Rapid multiplication of Bowie volubilis was carried out by using leaves of bulbs, and rhizomes.
Axillary Shoot Proliferation:
Axillary shoots developed from axillary buds present in the axils of each leaf. In leaf axils unsprouted status of axillary buds is due to apical dominance exhibited by growing shoot tip region at the top. Synthesis of auxin in apical shoot meristem is probably responsible for apical dominance.
However, apical dominance can be reversed by synthesis of cytokinin in axillary buds or entry of cytokinin into the buds. In micro-propagation strategy mass multiplication of plants can be accomplished by enhanced axillary branching, which involves placing nodal explant containing 1 to 2 axillary buds on the media in horizontal position (in vitro layering) for enhanced branching.
The culture media are generally enriched with very high concentration of cytokinin. Synthetic cytokinin like BAP and kinetin play a prominent role in releasing unsprouted axillary buds and its further proliferation. The concentration of BAP or kinetin can be used between 2 and 10 mg/L or some cases upto 40 mg/L yield desirable results.
High cytokinin overcomes apical dominance and release axillary buds to develop into new shoots. Thus, mass of miniature, shoots are produced in the culture vessels. These miniature shoots are separated and transferred to fresh set of media. The shoot multiplication cycle can be repeated for several times until millions of shoots are produced.
Stage III — In Vitro Rooting:
Shoots or plantlets obtained during stage II are very small and do not contain roots enable to grow in soil, even fails to utilize soil nutrients. Therefore, adequate steps are taken in stage III to grow individual plantlets that can carry out photosynthesis and survive without external supply of carbohydrate. Therefore, in vitro grown shoots must be transferred to a rooting media.
There is a clear distinction between rooting media from shooting media. In vitro rooting can be accomplished by adding auxins to the culture media. Generally, addition of cytokinin is totally restricted due to its total inhibition role. Root induction takes place readily in many herbaceous species, but it can be very difficult or recalcitrant in most woody species.
Invitro rooting can be both in vivo and in vitro. In addition to the favourable role of auxins in root induction, strength of media is very important. Media containing half or quarter strength of the salts can be useful in the induction of roots in vitro. Supplying riboflavin and other adjuvants enhances root induction process in stage III (Fig. 7.1).
Stage IV — Transplantation or Hardening:
Once in vitro rooting process completes, plantlets are ready to be transferred from the aseptic tissue culture container into the soil. Immediate transfer of tissue culture plants into soil is detrimental for survival of regenerated plants due to desiccation, infection, and light temperature shock.
Tissue culture plants generally lack protective cuticle layer and hairy roots, therefore are subjected for light and temperature shock. Excess of water loss was noticed from the leaves of plants immediately after transplantation. High rate of water loss due to lack of protective cuticle layer and root hairs, therefore every chances of exposure of seedlings to light and temperature shock.
Plantlets are not developed on their own. The photosynthetic minimum period (7 days) is required for them to be capable of producing their own source of carbon. In addition to these problems, invitro grown roots are highly tender and their vascular systems are poorly developed.
In order to compound these problems regenerated plants are subjected to hardening and is carried out by removing test tube grown plantlets and washed under distilled water to remove media sticking to the basal portion of the shoots, plantlets are then placed in sterilized soil or humus soil plus sand (1: 1) or vermiculate soil.
These are completely covered by plastic sheet or directly placed in green house to provide 90 to 100 percent humidity for upto 15 days. Humidity is gradually reduced to 90% then to 80% and finally to 70%. The acclimatized plants are then transferred to the field.