Plant Physiology - Musienko M. M. 2001

Plant Physiology and Biotechnology: Achievements and Development Prospects
Cell and Meristem Cryobank as a Way to Preserve Biological Diversity of Species

In the context of the global ecological crisis, preserving the Gene pool of living systems is of paramount importance, and this issue has been integrated into numerous international programs. The only means for the indefinitely long-term preservation of Cell strains, Tissues, and micro-Organs is cryopreservation. The term «cryopreservation» refers to a complex, multi-stage process conducted by researchers to maintain living Cells, tissues, and organs in a state of anabiosis for an indefinitely long period while preserving their stability (Fig. 213).

Structure/19.html">The Importance of reliably preserving actively metabolizing plant cells in an unchanged state can hardly be overstated. Cell strains are already frequently replacing plant raw Materials in biotechnological plants; tissue culture allows The production of somaclonal variants—a novel source of genetic diversity for plant breeding; and embryos, particularly apical Meristems, ensure the regeneration of plants that are exact copies of the originals. For this reason, cryopreservation is becoming a method for preserving the gene pool not only of cell cultures, but also of vegetatively propagated plants and others. This process is relatively simple only for sufficiently dehydrated objects (pollen, orthodox seeds) that can be directly plunged into liquid nitrogen followed by thawing in air under standard conditions. In the case of hydrated plant cells, tissues, and organs, cryopreservation is an extremely harsh process; it typically begins with a preparation stage (special pre-culturing) and concludes with recultivation, which ensures intensive Cell Division. Such a process is considered successful if the culture's growth is fully restored.

Deep cooling with liquid nitrogen (-196 °C), storage, and thawing constitute a significant stress factor for The Cell. The primary role here is played by ice crystal formation and cellular dehydration, which necessitates special preparation of cells and meristems, The Use of effective cryoprotectants, and an appropriate freezing protocol.

The most effective cryoprotectants are dimethyl sulfoxide, glycerol, Proline, and sucrose. Optimization of conditions is required not only for a specific type of cells, but often for specific strains as well. It has been proven that recultivated cell cultures of ginseng, dioscorea, and carrot are largely identical post-preservation and suitable for biotechnology. They retain their growth rate and morphogenic potential, while in the case of potato meristems, researchers have successfully regenerated plants capable of producing yields. Successful cryopreservation of in vitro cell and tissue cultures has currently been achieved for 60-65 species, of which about 30 are meristem cultures.

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Fig. 213. MAIN STAGES OF the Biotechnological Process of cryopreservation of plant genetic resources

Preserving genetic resources through the cryopreservation of meristems offers many advantages over other in vitro Methods. This is because meristems develop directly into plants, ensuring high genetic stability. Furthermore, they easily withstand freezing due to their small cells, which lack large vacuoles.

Unlike traditional storage methods, cryopreservation has significant advantages because it precludes such processes as the loss of nutrient reserves, accumulation of toxins, breakdown and inactivation of enzymatic complexes, lipid auto-oxidation, degradation of functional and genetic systems, as well as a range of other processes associated with cellular Aging.

Consequently, efforts aimed at preserving the genetic Water/126.html">Diversity of the biosphere through the cryopreservation of plant genetic resources are undergoing intensive development.

Recently, studies on cell Selection during tissue culture have begun utilizing the method of cryopreservation of immature plant embryos. The Need for such cryopreservation stems from the fact that embryos in mature seeds are quite stable and resistant to mutagenic influences. Therefore, in Introduction/32.html">Genetic Engineering research, Mutagenic Factors are used to affect immature embryos. The cryopreservation method ensures their long-term storage and, consequently, the application of cell selection, which significantly accelerates the breeding process.

The establishment of a cell and meristem cryobank will ensure the conservation of the existing gene pool, but in the long term, profound PHYSIOLOGICAL AND BIOCHEMICAL studies are required to optimize deep freezing for specific strains, varieties, and species.

The main challenges are associated with the Specific characteristics of both plant cells themselves (large sizes—up to 2000 µm, significant vacuolation) and their in vitro populations. Cells in such populations are genetically heterogeneous, asynchronous, and a portion of them exit the mitotic cycle into the G₀ phase. In every culture, cells exhibit physiological and morphological variations that affect their cryotolerance; hence, far from all cells survive cryopreservation. This physiological and morphological diversity increases even further among cells of different strains, and even more so among cells of different species.

In the case of meristematic cultures, the situation is additionally complicated. Each meristem is essentially a micro-organ averaging 500 µm in size, in which, alongside small

actively dividing meristematic cells proper, there are cells of other tissues at various stages of differentiation, often elongated and vacuolated to varying degrees. The cryotolerance of such cells is much lower than that of true meristematic cells.

Cellular interaction, particularly between cells determined for the primordium and true meristems, is of critical importance for the entire meristem. If this interaction is preserved during cryopreservation—meaning the relevant cells survive—the meristem regenerates a plant; if not, unorganized growth occurs, resulting in The formation of a callus. A plant can also be regenerated from a callus, but this pathway is undesirable for meristem cryopreservation because regeneration via callus does not guarantee an exact Replication of the initial genotype. Therefore, special studies of regenerated plants are necessary to establish their identity with the original ones.

Thus, the objective of apical meristem cryopreservation is not limited to preserving meristematic cells alone. The primary target of cryoinjury is the Plasmalemma, and its alterations are linked to the disruption of the continuity of its lipid bilayer. Therefore, during cryopreservation, along with the natural mechanisms of plant cell cryotolerance, the full arsenal of preparation and artificial cryoprotection is employed. It is precisely cells, tissues, and apical meristems cultured in vitro that make cryopreservation a universal method for preserving the gene pool, extending it to plant species with recalcitrant seeds and those that reproduce exclusively vegetatively.