The following points highlight the three main factors affecting micro-propagation. The factors are: 1. Genotype 2. Media 3. Cultural Conditions.
Factor # 1. Genotype:
Physiological conditions of the donor plants influence considerably in the process of morphogenesis. Larger sizes of the meristematic tissues are advantageous. Selecting juvenile parts of the plants yield better results than older ones.
The most actively growing plants are suitable for the selection of explants. In addition, excision of explant tissues is also important, for example, bulb scale of lilium regenerated freely when taken from plants during spring but not when excised during summer or winter.
Factor # 2. Media:
Salt formulation of Murashige and Skoog (1962), Gamborg (B5), Linsmayer and Skoog (1965) are some of the balanced media employed for the propagation of wide array of species. Each medium is supplemented with growth regulators of appropriate concentration can influence different natural morphogenesis. Addition of auxin and cytokinin and their role in different stages of micro-propagation have been well documented.
The stage II in micro-propagation requires comparatively high level of cytokinin. In axillary shoot proliferation, cytokinin is indispensable to overcome the apical dominance of shoots and initiates continuous branching of lateral buds from leaf axils. Almost all the media used in stage II are invariably supplied with cytokinin.
Although exogeneous auxins may not support axillary shoot proliferation but their presence in media influence the culture growth. Wide array of low auxin and high cytokinin effect may exist in different species. Hormonal factors like presence of thiauron, NAA and BA influence adventitious shoot regenerated from leaf explant.
In an extended work, regeneration percentage was improved significantly by maintaining sucrose concentration above three percent. Certain evidences have also shown that shoot production takes place only when auxin concentration is lowered. Presence of high concentration of auxin does not inhibit axillary bud proliferation and adventitious bud formation but also induce callus especially when 2, 4-D is used.
Synthetic cytokinin like BAP and kinetin are the most powerful in shoot induction and proliferation at stage II. Although the role of GA3 in shoot propagation, often employed in the culture media is not evidenced but its requirement for elongation of in vitro grown shoots has been evidenced.
In stage III of micro-propagation, priority is given to auxins for induction of roots. Adventitious root formation could be evidenced readily in herbaceous plants, but could be difficult in tree species. Cytokinin concentration is completely reduced or eliminated to ensure rooting process. Sometimes roots are unable to initiate in high salt strength regardless the types of hormones in the media.
Hence, reducing salt strength in the media to one half, one third or one forth of the standard strength facilitates rooting process.
In several cases, in vitro developed shoots becomes habituated for exogenous auxin and supply of auxins for root induction is unnecessary.
Factor # 3. Cultural Conditions:
Providing adequate light intensity for inducing morphogenesis is essential in micro-propagation. The most suitable light conditions for most of the micro-propagation work are between 1000 and 3000 lux. White fluorescence diffused light favours better proliferation of shoots. However, low light intensity results in taller green shoots, while brighter light intensity induces better rooting.
Inductions of adventitious shoots were increased in Allium cepa tissue culture in 15 hrs a day than 8 hrs a day. However, the most commonly used photoperiod is 16 hr day vs. 8 hr night. Providing high bright intensity of light might stimulate phenolic exudation leading to tissue browning. Thus, it is advisable to reduce light intensity below kilolux or incubate in complete dark conditions.
Most of the culture requires constant incubation temperature ranging between 22°C and 28°C. When the incubation temperature is above 28°C, water tends to condense on the plants and retard the growth. The cultures are generally maintained at 25°C but vary according to initiation of shoots and roots. Asparagus shoot tip shows better growth at 27°C, whereas other explants like petiole in Begonia produced enhanced shoot induction at 18°C.