BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 3. CELL ENGINEERING

Micropropagation of Plants

Micropropagation is a modern method of vegetative plant propagation that makes it possible to obtain whole plants from individual Cells. Micropropagation involves producing plants in vitro asexually that are genetically identical to the original specimen.

The term "clone" was coined in 1903 by Webber (from the Greek klon meaning "twig or slip suitable for plant propagation").

The pioneer of micropropagation was the French scientist Georges Morel, who obtained the first orchid plantlets in the 1950s. In the USSR, under the guidance of R.G. Butenko, conditions for the micropropagation of potatoes, sugar beets, carnations, and gerberas were thoroughly investigated. The earliest studies on woody plant tissue culture were published in the mid-1920s, during which Gautheret demonstrated that cambial Tissues of certain plants are capable of callus formation in vitro. In the 1960s, Mathes obtained the first aspen plantlets brought to soil culture. To date, over 200 species of woody plants from 40 families have been propagated in vitro.

The efficiency of plant micropropagation depends on the PHYSIOLOGICAL CHARACTERISTICS OF the PLANTS AND THEIR cultivation conditions: Temperature (18 — 26°С), lighting, and air humidity (65 — 70%). Source mother plants must be healthy (free from fungal, viral, and bacterial damage) and in a state of active growth. Young, poorly differentiated tissues containing meristem (such as young leaves and buds) are best used as explants. Explants are pre-treated with fungicides and Antibiotics to prevent fungal and bacterial infections.

Stages of micropropagation:

- Selection of the donor plant, isolation of explants, and establishment of a sterile Cell culture;

- micropropagation proper—maximizing the yield of meristem clones;

- rooting of the propagated shoots followed by their adaptation to soil conditions;

- cultivation of plants in greenhouse environments and preparing them for transplanting into open soil.

Tissue cultivation at each stage requires a nutrient medium of a specific composition, which is tailored to each individual plant species. At The First stage, antibiotics at a concentration of 100 — 200 mg/L are added to the nutrient medium to establish a sterile culture, along with growth stimulators (Auxins, Cytokinins), antioxidants (ascorbic acid — 1 mg/L, Glutathione — 4 — 5 mg/L), and other BIOLOGICALLY ACTIVE SUBSTANCES. The duration of the first stage is 1-2 months.

At the Second Stage, The ratio of cytokinins to auxins in the nutrient medium plays a crucial role in selecting optimal conditions for explant cultivation.

At the Third Stage, the concentration of mineral salts in the nutrient medium is reduced by a factor of 2 to 4, carbohydrate content is lowered to 0.5— 10 %, cytokinins are eliminated, and auxins are retained. As is well known, auxins stimulate ROOT formation.

At the Fourth Stage, plants are grown in a greenhouse in preparation for soil transplantation. The most favorable time for transplanting plantlets into the substrate is spring and early summer (using plants with 2 — 3 leaves and a well-developed root system). Peat, sand (3:1), soddy soil, and perlite are used as substrates. The substrate is sterilized at 85 — 90°C for 1 — 2 hours. Fertilizers are applied 20 — 30 days after transplanting into the soil. The process of adapting plants to soil conditions is the most expensive and labor-intensive operation.

Compared to traditional propagation Methods, micropropagation offers the following advantages:

- production of genetically uniform material;

- plant rejuvenation and virus elimination through The Use of meristem culture;

- high propagation coefficient: 10⁵- 10⁶ for herbaceous and flowering plants, 10⁴- 10⁵ for shrubs, and 10⁴ for conifers;

- shortening the breeding cycle;

- accelerating the transition of plants from the juvenile to the reproductive developmental phase;

- propagation of plants that are difficult to propagate vegetatively (woody species);

- The ability to carry out operations year-round;

- thousands of plants can grow in a small laboratory space (up to 100,000 clone plants can be grown in a short period on 1 m² of laboratory area);

- the possibility of automating the cultivation process.

It is most economically advantageous to propagate selection varieties of flowers in tissue culture: orchids, agaves, begonias, chrysanthemums, cyclamens, dracaenas, irises, lilies, daffodils, and phloxes. In one year, up to 1 million clonal plants can be obtained from a single gerbera plant. A new area of application for cloning in a sterile environment has been the propagation of shrub species, fruit crops, and pineapples.