Plant tissue culture and its implications

m Regeneration Pathways…continued from last week…
Shoot regeneration efficiency in tissue culture is usually a quantitative trait that often varies between plant species and within a plant species among subspecies, varieties, cultivars, or ecotypes.
Therefore, tissue culture regeneration can become complicated especially when many regeneration procedures have to be developed for different genotypes within the same species.
The three common pathways of plant tissue culture regeneration are propagation from preexisting meristems (shoot culture or nodal culture), organogenesis and non-zygotic embryogenesis.
The propagation of shoots or nodal segments is usually performed in four stages for mass production of plantlets through in vitro vegetative multiplication.
However, organogenesis is a common method of micropropagation that involves tissue regeneration of adventitious organs or axillary buds directly or indirectly from the explants.
Non-zygotic embryogenesis is a noteworthy developmental pathway that is highly comparable to that of zygotic embryos and it is an important pathway for producing somaclonal variants, developing artificial seeds, and synthesising metabolites.
Due to the single cell origin of non-zygotic embryos, they are preferred in several regeneration systems for micropropagation, ploidy manipulation, gene transfer, and synthetic seed production.
Nonetheless, tissue regeneration via organogenesis has also proved to be advantageous for studying regulatory mechanisms of plant development.
Through plant tissue culture, Nari micropropagates more than 200,000 high health diseases free potato clone plantlets for the PNG potato industry in a year for further seed multiplication and distribution to seed potato growers.

Choice of explant

The tissue obtained from a plant to be cultured is called an explant.  Explants can be taken from different many parts of a plant. These include portions of shoots, leaves, stems, flowers, roots, single undifferentiated cells and from many types of mature cells provided are they still contain living cytoplasm and nuclei and are able de-differentiate and resume cell division.
This has given rise to the concept of totipotentency of plant cells. However this is not true for all cells or for all plants. In many species explants of various organs vary in their rates of growth and regeneration, while some do not grow at all.
The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased with inappropriate explants.
The first method involving the meristems and induction of multiple shoots is the preferred method for the micropropagation industry since the risks of somaclonal variation (genetic variation induced in tissue culture) are minimal when compared to the other two methods.
Somatic embryogenesis is a method that has the potential to be several times higher in multiplication rates and is amenable to handling in liquid culture systems like bioreactors.
Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that become problematic during the tissue culture process.
Certain soil microflora can form tight associations with the root systems, or even grow within the root.
Soil particles bound to roots are difficult to remove without injury to the roots that then allows microbial attack. These associated microflora will generally overgrow the tissue culture medium before there is significant growth of plant tissue.
Some cultured tissues are slow in their growth. For them there would be two options: (i) Optimising the culture medium; (ii) Culturing highly responsive tissues or varieties. Necrosis can spoil cultured tissues. Generally, plant varieties differ in susceptibility to tissue culture necrosis. Thus, by culturing highly responsive varieties (or tissues) it can be managed.
Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed from the explant by gentle rinsing, and the remainder usually can be killed by surface sterilisation.
Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorisation or localised necrosis on the surface of the explant.
An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilised seeds.
The hard surface of the seed is less permeable to penetration of harsh surface sterilising agents, such as hypochlorite, so the acceptable conditions of sterilisation used for seeds can be much more stringent than for vegetative tissues.
Tissue cultured plants are clones. If the original mother plant used to produce the first explants is susceptible to a pathogen or environmental condition, the entire crop would be susceptible to the same problem.
– Nari