In order to be able to produce a large amount of qualitatively uniform young rhubarb plants in a short period of time regardless of weather conditions, the plants are micropropagated under controlled laboratory conditions in a sterile environment. Controlled laboratory conditions refer to abiotic factors such as light intensity, light quality, day length, temperature and humidity. Micropropagation is suitable for the mass production of genetically identical pathogen-free plants from selected virus-free genotypes of desired varieties.
Preparing the plant material for establishment
The first preparatory step for the establishment of rhubarb in the laboratory is the development of the mother plants in the greenhouse. Defined, virus-tested mother plants are cultivated in expanded clay. For the in vitro establishment of rhubarb, fresh shoots are needed in the spring. These are cut off from the rhizomes with the petioles and leaves. The vegetation point with the meristem of the fresh shoots is the starting material for further processing in the laboratory. The meristem is a plant tissue consisting of undifferentiated cells which is involved in plant growth through cell division. Under laboratory conditions an entire plant with roots and leaves is generated from the undifferentiated plant cells by progressive differentiation and organogenesis. The principle of tissue culture is the ability of somatic cells to differentiate into an entire plant.
Removing adhering fungi and bacteria by sterilisation
The fresh shoots of the rhubarb plants are cut to size with a sharp knife and collected per clone (mother plant) in a glass container that is surface sterilised with the addition of a disinfectant solution (e.g. sodium hypochlorite, ethanol, hydrogen peroxide, etc.). This is done in order to kill any fungi and bacteria that may adhere. The plant material incubates for approx. 20 minutes in the sterilisation solution and is stirred during this time with a magnetic stirrer.
The sterilisation solution is removed and all subsequent sterile work is carried out in a sterile workbench (clean bench), which is used in cell culture laboratories. A clean bench consists of a work table with a housing that is ventilated sterilely. At the end of the incubation period, the sterilisation solution is discarded into a separate container and the plant material in the glass container is washed once with sterile distilled water by means of short shaking; the water and the sterilisation solution are then disposed of.
After washing the plant material, the preparation is started on autoclaved paper or filter paper (sterilisation by stretched, saturated steam). Using a binocular microscope, sterile tweezers and sterile pointed scalpel, the outer coloured layers of the vegetation point are peeled off and the white meristem is placed on a nutrient medium that has been specifically established and optimised for rhubarb. Since the vegetation point of rhubarb is in the substrate or expanded clay, the probability of contamination (impurities) is very high. The disinfection solution often does not kill all bacteria and fungi (too high a concentration would damage the plant material). As a result, only one explant per in vitro dish is used to facilitate the selection of sterile plants.
Nutrient medium for growth, differentiation and propagation
The nutrient medium supplies the plant explant with the necessary macro- and micronutrients, salts, vitamins and sugar. In addition, specific growth regulators are added to the nutrient medium in order to control the organogenesis of the in vitro plants by selection and dosage: for example, the stimulation of cell division and shoot formation (with the addition of cytokinins) or the regeneration to a complete plant with plant elongation and root growth (with the use of auxins).
On the basis of a successful in vitro establishment, the production and further propagation of sterile plants begins, which are genetically identical to the mother plant. By subculture repetitions, the newly generated plants are pruned again in a fixed rhythm after a defined number of weeks for further propagation and transferred to fresh nutrient medium until the desired plant quantity is reached.
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