Clonal micropropagation and production of virus-free nursery plants of ornamental crops

Why implement clonal micropropagation in production

Clonal micropropagation (growing isolated tissues in culture) allows for the rapid production of millions of plants that are genetically identical to the original specimen. For a flower grower, this method solves three crucial tasks: sanitizing nursery plants from dangerous viruses, rapid multiplication of rare cultivars without trait segregation, and accelerating the breeding process. The technology has proven its effectiveness in growing gerbera, carnation, iris, orchids, freesia, chrysanthemum, and bulbous crops.

The method is based on the property of totipotency — the ability of any somatic cell to regenerate into a full-fledged plant organism. In artificial conditions (in vitro), cells of isolated tissues dedifferentiate and form callus mass. Meristematic cells of the ovary, ovule, apex, endosperm, anther, pollen, stem, root, or leaf are best suited for triggering this process.

The process of obtaining completely healthy nursery plants consists of strictly sequential stages:

1. Preparation and sterilization of nutrient media.
2. Preparation and sterilization of the initial plant material.
3. Isolation and planting of tissue pieces onto the nutrient medium.
4. Cultivation of tissue in an incubator until regenerant plants are obtained.
5. Micro-cuttings and rooting of micro-cuttings on the nutrient medium.
6. Transplanting the obtained plants into greenhouse substrate and growing mother plants in virus-isolated conditions.
7. Testing plants for the presence of viral infections and selecting healthy specimens.
8. Obtaining virus-free nursery plants from the selected mother plants.

Technology: from meristem in a test tube to a finished nursery plant

To work with this technology, the farm will need a specialized laboratory and greenhouse complex. It consists of a washing room, a sterilization room, manipulation boxes, and an incubator room. For the final stage of adaptation and growing mother plants, equipped greenhouses are required.

A universal Murashige and Skoog nutrient medium is used as a base for growing tissues of ornamental crops. Its composition includes mineral macro- and micronutrients, carbohydrates, vitamins, amino acids, and growth regulators. By adjusting the ratio of auxins and cytokinins, the agronomist manages cell division and the development of the future plant's organs.

In a sterile box under a microscope, virus-free meristem cells are isolated from the growth point and transferred into test tubes. Cell growth and the development of young plants are regulated in incubators, with strict control of microclimate parameters. For successful meristem development, it is necessary to maintain stable external conditions.

• Multiplication factor for micro-cuttings — up to 1 million
• Temperature in the incubator — 20–29 °C
• Relative air humidity — 70%
• Crop illumination — 2 thousand lx
• Average cell survival rate — 70–80%
• Plant height for transplanting — 3–5 cm

The development period of a full-fledged plant from the meristem depends on the biological characteristics of the specific flower crop. The difference in timelines can be several weeks. Data for major ornamental plants are presented in the table:

When young regenerants reach the required height, they are carefully removed from the test tubes. The plants are transplanted into pots with a sterile substrate of peat or perlite. At the final stage, mandatory diagnostics for viruses are performed to propagate only guaranteed healthy mother plants.