In this meristem tissue culture method post we have briefly explained about what is meristem?, isolation of meristem-tips, process, meristem tissue culture maintenance, applications, advantages, and disadvantages.
Meristem Tissue Culture
Meristem tissue culture focuses on the shoot apical meristem in particular. This method is also known as shoot tip culture or apical meristem culture. It was first reported in 1952 by Morel and Martin, two scientists. Morel used this culture to microproduce orchid cymbidium in 1965.
Similarly, meristem tissue culture follows steps similar to micropropagation: (i) initiation of culture, (ii) shoot multiplication, (iii) rooting of the developed shoots, and (iv) transfer of the plantlets to the pots or soil
Explants of meristem are then placed on Murashige and Skoog’s (MS) medium, which is considered to be an effective medium for the majority of species. Lower salt concentration is suitable for some species. Fungicides (bavistin) or antibiotics (chloramphenicol/streptomycin) can be added to the medium during growth to remove the endophytic contamination. In this context, you will learn about the definition, process, applications, advantages, and limitations of the meristem tissue culture method.
Meristem tissue culture is a tissue culture technique that uses an apical meristem with 1-3 leaf primordia to prepare clones of a plant through vegetative propagation. The isolation of meristem is accomplished primarily through the use of a V-shaped cut in the stem. In this method, adventitious roots can be regenerated by culturing shoot meristem. The preferred size of the shoot tip for the generation of the virus-free plant is 10 mm, but the size of the shoot tip does not matter in vegetative propagation.
What is Meristem?
Meristem Tissue Culture Method
A meristem is a site in the plant body where new cells form and the complex processes of growth and differentiation are initiated.
Growth means the irreversible increase in size that comes from both cell division and cell enlargement. Cell differentiation refers to the changes that a cell undergoes structurally and biochemically so that it can perform a specialized function. Since cells and tissues are derived from meristems, we do not consider meristems themselves to be tissues.
There are different categories of meristems, each with a specific function. Shoot and root apical meristems are at the tips of branches and roots; they are the ultimate sources of all cells in the plant.
Meristem is the shoot tip found at the shoot and root apical regions. It is dome-shaped and measures about 0.1 mm wide by 0.25-0.3 mm long. The apical shoot meristem is the portion of a stem that elongates and is free of pathogens, as demonstrated in 1949 prior to the introduction of meristem tissue culture.
Limmaset and Cornuet were the two scientists who noticed the virus’s decreased growth towards the apical meristem. Thus, among other techniques for producing plants free of viruses and other related organisms, meristem tissue culture is widely used.
Isolation of Meristem-tips
The procedure for isolation of sterile, healthy meristems is divided into 2 steps: (1) preparation and disinfection of donor plant material, and (2) excision.
Because plant material carries many superficial organisms, a surface sterilization procedure must be used to reduce the number of microorganisms. It reduces the risk of transferring contaminants into the culture tube along with the meristem tip. The procedure detailed below and shown has been found to reduce contamination to less than 5% on meristem-tips taken from glasshouse-grown plant material.
Select healthy, rapidly growing parent plants from which to remove buds. Remove the terminal bud along with 2-3 cm of the stem. Be careful not to allow the stems to wilt after removal. Remove all but the smallest leaves from the stems. Subsequent steps should be conducted using sterile containers and utensils.
Move the stems to the sterile working area and place them in a 10% solution of commercial bleach (0.525% sodium hypochlorite) for 15 minutes. Add a few drops of ‘Tween 20’ to the bleach solution to reduce the surface tension of the water and allow better penetration of the sterilants. Remove the stems from the bleach solution and rinse for 2 minutes in sterile water. Repeat the rinse a second time.
Excision of the meristem-tip (‘apical dome’) requires a great deal of patience and skill; it is recognized that every person will develop their own technique. Some aspects of the excision process are critical. Among these is the maintenance of sterile conditions, including the prevention of virus transmission on the excision tools, and the removal of the apical dome without excessive damage in order to ensure its survival.
Meristem Tissue Culture
Rinse the explants thoroughly four times with distilled water. After that, place each explant in a sterilised Petri plate. Dissect the shoot tip’s outer leaves. After the outer leaves have been dissected, the apex region will be exposed and separated with a sharp scalpel. Transfer the apical meristem to MS (Murashige and Skoog’s) nutrient medium. Incubate the culture in a light for 16 hours at 25 degrees Celsius.
Transfer the shoots to a hormone-free medium after they have developed a single or multiple shoots. Then, transfer the plants to the compost-filled pots and keep them in the greenhouse for hardening.
One week after excision, the meristem-tip cultures should be inspected under the dissecting microscope and contaminated and dead cultures discarded. If contamination exceeds 5-10%, the disinfection and excision procedures should be reviewed. Normally, 25 – 40% of the cultures can be expected to die as a result of dissection damage and/or too small a size of explant. Dead cultures can be recognized by their black appearance and lack of new growth.
The usual sequence of events preceding regeneration is (1) swelling of the meristem-tip, (2) callus production, (3) shoot development and, finally (4) root development. This sequence can take from 1-4 months. However, nutrient deficiency, medium dehydration and accumulation of secondary metabolites may cause the growth of meristems to slow down after 4 – 6 weeks. Consequently, it is necessary to transfer the meristems to fresh medium at intervals of no more than 4 weeks.
The meristem-tips are maintained on the Excision and Induction Medium until the first signs of shoot development appear. They are then transferred immediately to Regeneration Medium.
1. Plants are frequently infected with multiple types of viruses, some of which are unknown. Commercial horticulturists use the term virus-free to refer to plants that have been virus-free.
2. Micropropagation is the sexual or vegetative propagation of whole plants using tissue culture techniques. Many plant species’ shoot tips or meristem cultures can be successfully used for micropropagation.
3. Many plants produce seeds that are highly heterozygous or recalcitrant in nature. Such seeds are not permitted for the storage of genetic resources. As a result, such plants’ meristems can be stored in vitro.
4. Many plant breeding experiments result in the production of abortive or nonviable seeds by hybrid plants. As a result, it creates a barrier to crossability in plants by preventing non-viable seeds from developing into mature plants. To expedite the breeding process, shoot tips or meristems from such hybrid plants can be cultured.
5. Unless and until they are made homozygous diploid, haploid plants derived from anther or pollen culture are always sterile. Meristem or shoot-tip culture of haploid plants can be used for propagation, allowing for detailed genetic analysis based on morphological characteristics and biochemical assays.
6. Plantlets derived from shoot-tip or meristem cultures are easily accepted for international exchange by quarantine authorities and are not subject to any checks. As a result of this technique, crop plants can be easily exchanged in crop improvement programs based on materials from all over the world.
1. Apical shoot culture also contributes to the production of virus-free plants.
2. In-vitro or cryopreservation can be used to preserve germplasm or seeds.
3. Meristem contains a high concentration of auxin, which promotes plant growth.
1. Isolation of meristem is quite difficult.
2. Explants take more regeneration time to grow.