For the successful establishment of new plant species in our laboratory, various parameters have to be worked out: suitable starting material, a disinfectant with appropriate concentration and incubation time, a nutrient solution for good plant quality as well as a cutting technique and cultivation methods.
The use of suitable raw material
Leaf pieces, shoot tips, axillary buds, nodes, flowers and root tips can be used as starting material for the initiation of the culture. The choice of plant tissue depends on the habit of the plant to be established and the existing vegetation points with active cell division. If possible, material above the substrate should be used, as this reduces the likelihood of contamination.
Surface sterilisation and preparation of the plant material.
The selected starting material is used to test the appropriate sterilising agent which is compatible with the plant species and at the same time effectively removes microorganisms such as sodium hypochlorite, ethanol, silver nitrate, hydrogen peroxide, etc. The optimal concentration and incubation time are worked out in order to kill possibly adhering fungi and bacteria, but without damaging the plant tissue.
The type of preparation of the sterilised plant material is tested. Leaves are cut into small square pieces. The juvenile tips are separated from the shoots. Nodes can be inserted completely, or the axillary buds are prepared; depending on the age and condition, the outer tissue layers are also peeled off. Flowers are divided vertically, peeled or placed completely in the nutrient solution.
Establishing the correct nutrient solution
The decisive point for a successful establishment, besides surface sterilisation and preparation, is the composition of the nutrient solution. The first nutrient solution used after sterilisation of the plant material is usually the same. It shows whether the material is sterile and vital. The culture-specific medium then plays an important role in establishing the new plant species.
Orientation at the natural site of the plant species
The natural location of the plant species can serve as an orientation for the production of a nutrient solution: the pH values of the soil types (sand, loam, loess, clay or bog soil) are relevant. Nutrients are more or less readily available at different pH values: iron is found in the neutral range, magnesium and phosphorus rather in the alkaline range, manganese predominantly in the acid range. On this basis, the composition of the macro- and micronutrients in the sterile nutrient solution is selected. Water availability also plays a role, which can be controlled by the amount of gelling agent used (agar, Gelrite, etc.).
Published nutrient solutions of closely related species
Specific compositions of nutrient media for plant species are published in scientific publications. These published nutrient media can serve as a basis for establishing another plant species by preparing the nutrient solution identically or by genotype-specific adaptation. Even between phylogenetically distant species the transfer of a nutrient solution can be successful, e.g. across genera within the same plant family. Or the composition is at least an indication of how the nutrient solution should be designed regarding the nitrogen content, salt content, etc.
Adaptation and optimisation of already established nutrient solutions
If different nutrient solutions for established cultures are already available in the laboratory, the explants of the new plant species can be tested on these media according to the heuristic trial-and-error principle. If the plants develop well on one of these nutrient media, it can be used as a final medium or as a basic medium which is adapted and optimised for the respective plant species.
The concentration of the growth regulators used is a sensitive adaptation to the respective plant species. Sometimes even different varieties of a plant species require a specifically established nutrient solution. Growth regulators can be natural phytohormones or synthetic substances. On the one hand, there are cytokinins which stimulate cell division and thus promote plant reproduction. Depending on the chosen concentration, the new shoot formation can be controlled. Every plant species has its cytokinin optimum, which leads to a good plant quality with an appropriate propagation rate. Plant quality and propagation rate must be tested for consistency over several subcultures using the established nutrient solution. On the other hand, there are auxins that are used when the plants are not rooted directly in natural substrate after propagation and a sterile rooting medium is required. Auxins promote cell growth, plant elongation (apical dominance) and root formation – they induce the entire development of the plant.
Furthermore, the analysis of the cutting technique, subculture duration, temperature, day length and light quality plays a decisive role in the successful establishment of in vitro methods for new plant species.
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