In the previous post, we discussed the definition and different techniques in the micropropagation, in this post we shall discuss about the different stages of micropropagation.
• Stages of Micropropagation:
The process of micropropagation can be divided into five different stages (fig 1):
-Stage-0: Identification of mother plant
-Stage-I: Selection and processing of explants.
-Stage II: Shoot proliferation.
-Stage III: Rooting.
-Stage IV: Hardening and Acclimatisation.
Each stage is distinct, but in some cases two stages can be merged. The plants produced can be marketed at different stages depending on the customer’s need. Let’s discuss about each stage, more in detail:
– Stage-0: Identification of mother plant:
In this stage, the mother plant is identified and, if required, maintained in the controlled condition of the green house. The plant with desired and commercially valuable quality is chosen, this may include overproduction of certain metabolite, good quality fruits, or other such parameter. The plant should be disease-free, healthy and able to provide good quality explants.
Stage-I: Selection of explant:
Explant is chosen as per the mode of regeneration that the micropropation will be done (see previous post). The most desired explants are the shoot tips, other explants are bud, leaf or stem (internode), anther / microspore, ovule and embryo associated seed parts.
Stage II: Proliferation of shoots or SE:
The explants are carefully excised and sterilised followed by proper washing, to remove the traces of sterilising agents.
The sterile explants are inoculated onto the culture media with appropriate hormonal combination (fig 1) and incubated in a clean culture room with controlled light, temperature and humidity (see previous post).
The hormonal combination varies depending on the explant and mode of regeneration (direct, indirect or somatic embryogenesis), however the major hormones required for shooting are the cytokinins. Cytokinins are adenine derivatives and are involved in cell division, altering apical dominance and shoot formation in the tissue culture.
In presence of appropriate hormonal combination the explant (or callus) gives rise to a multiple shoots (as in fig 2).
(Just for info: Read the interesting review paper on the role of cytokinins in abiotic stress signalling pathways.)
In case of SE, rapid somatic embryogenesis occurs in this stage (see fig 3).
Individual shoots from the multiple shoot culture can be separated and inoculated again onto fresh media for another cycle of shoot proliferation (fig 1), if more number of shoots are required. Hence, this stage takes around 4-8 weeks or more depending on the number of shoots required. However, the shoot proliferation is restricted to 4-5 cycles of the initial explant inoculation, as after that the rate of proliferation mostly declines.
As each individual shoot obtained can be inoculated again onto fresh media to obtain more shoots, the increase in the number of shoots is logarithmic, giving rise to a very large number of shoots.
~The plantlets can be marketed in the shooting stage, provided the rooting is done by the purchaser (in this case another lab).
Stage III: Rooting.
As mentioned, when enough number of shoots are obtained the single shoots are separated and inoculated on rooting media (fig 1 & 4). Rooting media is the basal agar media with the rooting hormones (fig 5). The major hormones involved in the rooting are auxins, which induces cell division.
In some cases the shoots are dipped in auxin solution and directly transferred to sterilised/autoclaved soil (with compost) and rooted alongwith hardening in the culture room, not in culture vessel’s aseptic condition.
~The rooted shoots can be marketed packed within the culture vessel or in the pot with sterilised soil with hormones, depending on the customer’s demand.
Stage IV: Hardening and Acclimatization:
Once the shoot is well rooted, it makes a complete plantlet. However, the in vitro rooted plantlets are unable to photosynthesise efficiently and are adjusted to the aseptic conditions inside the culture vessel. Hence, the plantlet is not ready for the outdoor conditions and has to be acclimatised to the outer environment.
For this the plantlet is first planted onto a sterilised mix of soil and compost in the controlled conditions of the lab’s culture room. This is called hardening. During hardening the in vitro grown plants develop their photosynthetic machinery, stomatal functioning, root system and ability to deal with the micro-organisms.
After around a week or two of hardening inside the culture room and the plantlet is then transferred to the green house for acclimatization, for further enhancing the plant survival. Once the plant is acclimatised in the green house it can be slowly moved to the outdoor conditions. Hence, the plants are exposed in stepwise manner to harsher conditions.
In some cases the hardening and the acclimatisation can be both done in single step by directly transferring the rooted shoots to the greenhouse.
~Most of the times the complete acclimatised plant is marketed, especially to the nurseries or farmers. The plants are marketed at different stages, depending on which the time for the marketing varies from 4-16 weeks.
Advantages of Micro-Propagation:
The advantages of the micro propagation are:
• The rate of multiplication is very high by micropropagation than any other propagation method. A single shoot tip can give rise to hundreds to thousands of similar plantlets within 2-4 months.
• The plantlets obtained are similar to the mother plant and are uniform in the desired characteristics.
• The explant required is very small and available in high number from a single mother plant. For eg a single mother can provide a large number of shoot tips, leaf segments, internode,anther, etc.
• The micropropagation process can be carried out continuously throughout the year.
• The plants are relatively protected and disease-free.
• It is especially important in terms of some plants like seedless plants, elite varieties, genetically modified varieties, dioecious plants, haploid (pollens/ovary explant) or plants with difficult to germinate seeds.
The micropropagation has a few disadvantages as well:
• The entire process in rather expensive than the other modes of propagation and requires a proper initial set up.
• The aseptic condition and other controlled parameters have to be maintained for the in vitro regeneration. Hence the process requires skilled labors.
• The micropropagation may not be suited for some plants which produce secondary metabolites, which are harmful for the plant cells. For e.g. some plants produce phenolic compounds.
(Just for info: Researchers are working towards ways to control the accumulation of phenolic compounds in in vitro cultures, here’s one.)
• In some cases, the in vitro plants may not be able to withstand diseases and infection, which the naturally grown mother plant can withstand.
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Read other posts by The Biotech Notes:
Protoplast Isolation and Regeneration (PTC).
High Pressure Liquid Chromatgraphy (HPLC).
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-George et al. (2008) Plant Growth Regulators II: Cytokinins, their Analogues and Antagonists. Plant Propagation by Tissue Culture. Springer, Dordrecht 205-226.
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