V. V. Rogovaya, M. A. Gvozdev

FEATURES OF MICROCLONAL PROPAGATION OF STONE CULTURES IN IN VITRO CONDITIONS

The paper presents a review that examines the features of methods for microclonal propagation of stone fruit crops in an in vitro system. Particular attention is paid to the method of propagation by axillary buds and the method of regeneration of adventitious shoots from leaf explants of cherries, sweet cherries, peach and apricot. The issues of plant health from various pathogens and testing of plant material of stone fruit crops for the presence of viral infections are considered.

For the first time, microclonal propagation was carried out by the French scientist Georges Morel on orchids in the 50s of the twentieth century. In his work, he used the technique of cultivating the apical meristem of plants. The plants obtained in this way were free from viral infection.

In our country, research on plant health using the meristem method and clonal micropropagation began in the 60s at the Institute of Plant Physiology named after. K. A. Timiryazev USSR Academy of Sciences.

Microclonal propagation is the production in vitro of plants that are genetically identical to the original explant (a method of vegetative propagation of plants in in vitro culture). Micropropagation is based on a unique property of a somatic plant cell - totipotency - the ability of cells to fully realize the genetic potential of the whole organism.

Currently, various methods of microclonal propagation of agricultural crops (primarily vegetatively propagated) in an in vitro system are becoming increasingly relevant: propagation by axillary and adventitious buds, indirect morphogenesis, somatic embryogenesis.

Using these methods makes it possible to:

Accelerate the selection process, as a result of which the time for obtaining marketable products is reduced to 2-3 years instead of 10-12;

Receive in a short period of time a large amount of healthy, virus-free material that is genetically identical to the mother plant;

Work in laboratory conditions and maintain actively growing plants all year round;

Propagate plants with virtually no contact with the external environment, which eliminates the impact of unfavorable abiotic and biotic factors;

Obtain the maximum number of plants per unit area;

In a short time, obtain a large number of plants that are difficult to propagate or cannot be propagated vegetatively;

By growing plants with a long juvenile phase, the transition from the juvenile to the reproductive phase of development can be accelerated;

Preserve plant material for a long time (for 1-3 years) in vitro (without passaging to fresh medium),

Create banks for long-term storage of valuable forms of plants and their individual organs;

Develop methods for cryopreservation of in vitro treated material.

Stages of micropropagation of stone fruit crops and testing for the presence of viral infections

The micropropagation process involves several stages. The main ones are:

Stage 1 - introduction of the explant into in vitro culture;

Stage 2 - micropropagation;

Stage 3 - the process of rooting microshoots;

Stage 4 - the transition of rooted plants from sterile to non-sterile conditions.

An important step in the method of micropropagation of plants in vitro is the cultivation of virus-free uterine forms of plants in growing houses or isolated boxes in winter greenhouses, under conditions inaccessible to virus carriers. Explant donor plants for subsequent introduction into in vitro culture must be tested for the presence of viral, mycoplasma and bacterial infections using PCR diagnostic methods or molecular hybridization or enzyme-linked immunosorbent assay (ELISA).

The ELISA method makes it possible to quickly detect the vast majority of viruses that infect stone fruits: plum dwarf viruses, necrotic ring spot viruses of stone fruits, plum Sharki potyvirus, non-poviruses of cherry leaf curl. Clones found to be free of contact viruses by ELISA are then subjected to basic testing, which includes serological tests in combination with a test on indicator plants. Plants that are tested free of viruses and other regulated pathogens are assigned the category of “virus-free” basic clones. If an infection is detected, the original plants can be rehabilitated. To improve the health of stone fruit plants from viruses, it is most advisable to combine methods of dry-air thermotherapy and in vitro culture. If using a culture of isolated apical meristems it is not possible to get rid of the tested viruses, chemotherapy methods are used based on the introduction of chemicals into the nutrient media that inhibit the development of viral infection in plants in vitro.

Sometimes, to actively detect bacterial microflora, the media is enriched with various organic additives, for example casein hydrolyzate, which provokes the development of saprophytic microorganisms. Infestation is assessed visually after 7-10 days. “Clean” explants are placed on nutrient media for further cultivation. At this stage, it is also practiced to use media devoid of growth substances.

Introduction to in vitro culture and micropropagation of stone fruit crops

In clonal micropropagation of stone fruit crops, apical and lateral buds, as well as meristematic tips, are usually used as a source of explants. Isolation of the apical meristem is carried out according to generally accepted methods after stepwise sterilization of the plant material.

For micropropagation of stone fruits, various media are used: for micropropagation of cherries - Pierik, Gautre, White, Heller, for cherries and plums - Rosenberg's medium, modified for fruit crops and for plums - Lepoivre and B5 medium. But the most suitable for microclonal propagation of cherries, sweet cherries and plums is the Murashige-Skoog (MS) nutrient medium.

Depending on the stage of microclonal propagation of stone fruit crops, 6-benzylaminopurine (6-BAP) is added to the nutrient media in concentrations of 0.2-2 mg/l. At the stage of introduction into in vitro culture, a lower concentration of cytokinin is used - 0.2 mg/l BAP. To induce the proliferation of axillary buds in order to obtain the maximum number of shoots, cherry microplants are cultivated with the addition of BAP, in concentrations of 0.5-2 mg/l, plum microplants - 0.5-1 mg/l BAP.

The process of rooting microshoots

The rooting stage requires special attention. The process of in vitro rooting of shoots of stone fruit crops depends on the varietal characteristics, on the number of passages performed, on the concentration and type of auxin, and on the method of its application. To obtain fully formed microplants of stone fruit crops, 6-BAP, which interferes with rhizogenesis processes, is excluded from the medium, and auxins, mainly β-indolyl-3-butyric acid (IBA), are introduced into the media. It has been established that the optimum concentration of IBA in the nutrient medium is in the range of 0.5-1 mg/l. The presence of IBA in the medium at a concentration of 2 mg/l causes the formation of hypertrophied roots.

The joint introduction of the drug ribav (1 ml/l) and traditional phytohormones auxins [IBA and β-indolylacetic acid (IAA) 0.5 mg/l each] into the rooting medium increases the percentage of shoot rooting of a number of varieties of stone fruit crops.

In a comparative study of root formation inducers: IAA, IAA and a-naphthylacetic acid (NAA), the high efficiency of IAA at a concentration of 6.0 mg/l was revealed. The largest number of rooted cherry microcuttings were obtained on a medium containing NAA. However, at the same time, intensive growth of callus occurred in the basal area of ​​the shoots, which made it difficult to transfer test tube plants with roots to non-sterile conditions.

For effective rooting of test tube stone fruit plants, not only the type of stimulator, but also the method of its application is of great importance. In addition to introducing auxins into the nutrient medium, to induce rhizogenesis, preliminary soaking of shoots in a sterile aqueous solution of IBA (25-30) mg/l is used with an exposure of 12-24 hours. The experiments showed that treating microcuttings with an aqueous solution of IBA is more effective than introducing this regulator into the culture medium. The massive appearance of the first adventitious roots when pre-treatment with a rhizogenesis inducer was applied was noted on days 20-25. Another way to induce rhizogenesis is to treat the shoots of stone fruit crops with talc auxin-containing powder IBA with a concentration of 0.125%, 0.25% and IAA with a concentration of 0.25%, 0.5%. When using hormonal powder, high efficiency and manufacturability of the use of rhizogenesis inducers were noted. But the use of IMC talc powder with different auxin concentrations revealed varietal specificity in the rooting of plum microcuttings.

The process of rhizogenesis occurs most intensively on modified MS and White media. According to other data, the best medium for root formation is media with macroelements according to Heller with the addition of vitamins and a half-diluted MS medium with a reduced sucrose content of 15 mg/l and with the exception of meso-inositol, which promotes the formation of callus tissue. However, most studies use Murashige and Skoog media to root microshoots of stone fruit crops.

Micropropagation methods

There are several methods for microclonal propagation of plants in vitro:

Methods of propagation by axillary buds;

Methods of propagation by adventitious buds;

Indirect morphogenesis;

Somatic embryogenesis.

For any type of regeneration in vitro, four groups of factors that determine its success can be distinguished: genotype and condition of the original parent plant; cultivation conditions and methods; composition of nutrient media; features of introducing an explant into a sterile culture.

The influence of genotype on the efficiency of micropropagation

The genotype has the most significant influence on the efficiency of micropropagation. The response of plants to aseptic cultivation conditions depends on the varietal characteristics and is explained by the different regenerative abilities of varieties of fruit and berry crops. For example, when clonal micropropagation was used to accelerate the propagation of new cherry varieties, varietal traits were found to be the dominant factors in the plants' ability to micropropagate.

Varietal differences appeared both at the proliferation stage and at the root formation stage.

Among explants of different varieties of the same type of fruit plants, different degrees of response to growth regulators included in the medium are often observed, which apparently reflects, to some extent, the endogenous content of growth substances, which is a genetically determined characteristic of the species or variety. At the same time, the realization of morphogenetic potential in embryo culture in vitro, in hybrids between the species Cerasus vulgaris, C. maackii, C. fruticosa, Padus racemosa, was mainly determined by the genotype and to a lesser extent depended on the composition of the nutrient medium.

Cultivation conditions

Another factor determining the success of plant micropropagation is their cultivation conditions. The optimal conditions for cultivating stone fruit crops are: temperature 22-26 °C for cherries and 26-28 °C for plums, illumination of 2000-5000 lux for cherries and 3500 lux for plums with a 16-hour photoperiod. Microplants should be grown in climate chambers or controlled rooms.

It should be noted that in cherry varieties at the proliferation stage, an increase in the reproduction coefficient and an increase in the proportion of shoots suitable for rooting can be ensured by alternating mineral compositions of nutrient media and the use of blue light lamps (LP 1). A large number of shoots of stone fruit crops - up to 30 - can be formed when the regenerants are horizontally oriented. To increase the multiplication rate in the first passages, conglomerates of buds and shoots of stone fruit crops can not be divided into separate units, but transferred entirely to a fresh nutrient medium. When using this technique, the multiplication factor increases sharply and can reach 40-70 per passage, depending on the variety.

Method of propagation by axillary buds: indirect morphogenesis

The most reliable method of microclonal propagation is the method of plant regeneration through the development of axillary buds. The advantage of this method is the relatively rapid reproduction of the original genotype, while ensuring the highest phenotypic and genotypic stability. The potential of this method of in vitro micropropagation is realized by adding cytokinins to the nutrient media, which suppress the development of the apical bud of the stem and stimulate the formation of axillary buds.

The process of microclonal propagation of cherries using the method of culture of isolated apical meristems is based on the phenomenon of removing apical dominance, which promotes the subsequent development of existing meristems and ensures the genetic homogeneity of the planting material.

rial. Removal of apical dominance is achieved by adding cytokinins. Many cherry varieties are characterized by high mitotic activity of the apex, which contributes to the formation of a branched conglomerate of buds and lateral microshoots.

The genetic stability of the material obtained in vitro depends on the reproduction model. The process of reproduction of stone fruit plants is associated with the proliferation of axillary meristems. Genetic stability is an integral property of the meristem, which can be preserved in vitro if the latter is cultivated under conditions that inhibit callus formation. If media that stimulate callus formation are used, genetic variability may occur.

To obtain higher reproduction rates, nutrient media are often enriched, in addition to drugs of a cytokinin nature, with substances from the auxin group, which stimulate the development of callus tissue. Combinations of these two drugs are used to induce organogenesis in callus tissues. In the callus-shoot system, the organized structure of the shoot can influence the processes of organogenesis, stimulating the meristematization of callus cells, which can give rise to organs with altered properties. Simply varying the content of growth regulators added to the culture medium to achieve maximum cell proliferation can affect the genetic stability of the resulting material.

Method of propagation by adventitious buds and indirect morphogenesis

Adventitious buds are called buds that arise directly from the tissues and cells of plant explants, which usually do not form them. Adventitious (or adventitious) buds are formed from meristem zones, most often formed secondarily from callus tissue. Adventitious buds can arise from the meristem and non-meristem tissues (leaves, stems). The formation of adventitious buds in many plant species is induced by a high ratio of cytokinins to auxins in the nutrient medium.

Regeneration of shoots, roots or embryoids from somatic plant cells of the explant can occur through indirect regeneration - callus formation and shoot formation, or through "direct" regeneration, when explant cells become capable of regeneration without the formation of callus tissues.

Adventitious shoots can form on explants of leaves, petioles, roots and other plant organs of various types of stone fruit and fruit crops. Obtaining shoots directly from explants is in some cases used for plant cloning, but this may result in genetically unstable plants. Therefore, this plant regeneration method can be used to induce genetically diverse plants.

Regenerative shoots can be induced from various parts of the leaf blade, but tissues have the greatest ability to regenerate

the base of the leaf, since the most active meristematic cells are located in this zone of the leaf blade. It is also necessary to take into account that the morphogenetic potential of leaves increases as they are located towards the top of the stem. Adventitious shoots regenerate better from the young meristematic tissue of developing leaves. However, when older leaves are used, genetically altered shoots are much more likely to occur.

To regenerate shoots of stone fruit crops, such as cherries, sweet cherries, peach, apricots, from initial explants (whole leaves and their segments), various media are used: Murashige-Skoog (MB), Lloyd and McCown (WPM), Driver and Kuniyuki ( DKW), Kuren and Lepoiv-ra (QL).

For experiments on adventitious regeneration of cherries, Lloyd and McCown's medium for woody plants - Woody Plant Medium (WPM), supplemented with various growth stimulants, is most often used. Of the cytokinins, 6-BAP, thidiazuron (TDZ) are mainly used, and of auxins - NAA, IBA, 2,4-dichlorophenoxyacetic acid (2,4-D).

It is important to note that among foreign researchers there is no consensus on the effectiveness of using TDZ in shoot regeneration compared to BAP, on the type of explant (whole leaves, with transverse cuts applied to them or segmented) and on the method of cultivating explants (abaxial or adaxial surface up).

A high percentage of regeneration was observed in whole cherry leaf explants (with transverse cuts along the midrib of the leaf), which were placed abaxial (bottom) surface up on WPM medium supplemented with 2.27 or 4.54 |M TDZ + 0.27 | M NUK.

On the other hand, the work shows that BAP is more effective than TDZ in the regeneration of plants from cherry and sweet cherry leaves, and also that BAP and NAA at a concentration of 2 mg/l and 1 mg/l are the optimal combination of cherry plant growth regulators and cherries. The highest frequency of regeneration was obtained in the WPM medium, although it stimulated callusogenesis more than in the MS, QL, and DKW media. The dependence of the efficiency of callus formation on the type of leaf segments was revealed. Thus, the highest rates of callus formation were noted on the middle leaf segments; the lowest - on the apical segments and direct regeneration (without callus formation) was noted on the base segments.

Adventitious regeneration of black cherry (Prunus serótina Ehrh.) occurred more often when leaf explants were cultivated on WPM medium supplemented with TDZ compared to the modified DKW medium.

The efficiency of adventitious regeneration of wild cherry (Prunus avium L.) was significantly influenced by the size of the explant. The results showed that the size of the leaf explant is critical for the formation of adventitious shoots; leaves 3-5 mm long formed greatest number adventitious shoots. For adventitious regeneration of wild cherries, WPM medium supplemented with 0.54 tM NAA and 4.4 tM TDZ was used.

A special pre-treatment prior to cultivation (soaking with 5 mg/L 2,4-D for one day) was effective in inducing adventitious shoots from cherry leaf explants. Subsequent cultivation of leaf explants on WP regeneration agar medium supplemented with 5 mg/l TDZ increased the efficiency of cherry adventitious regeneration. Young cherry leaf explants showed a higher ability to regenerate than old ones.

It is necessary to note the significant effect of ethylene inhibitors on the adventitious regeneration of leaves of various apricot varieties. For example, the work showed that the use of ethylene inhibitors (silver thiosulfate or aminoethoxyvinylglycine) together with a low content of kanamycin increases adventitious regeneration by more than 200%. The use of pure agar also improved regeneration from apricot leaves compared to the use of agar gel or agarose. In this work, studies were carried out on LQ, DKW media, supplemented with TDZ and NUK. The method of cultivating leaves is with the adaxial surface to the medium.

Italian researchers have developed a method of adventitious regeneration from whole peach leaves, which were incubated in the dark on media supplemented with 6-BAP and NAA. The studies used combinations of macrosalts and microsalts of various media according to MS, Quoirin, Rugini and Muganu, both cytokinins - 6-BAP and TDZ, as well as the method of cultivating leaves - adaxial surface in contact with the regeneration medium. Callus developed at the base of the leaf petioles. Adventitious shoots appeared on this callus after transfer to an auxin-free medium and cultivation in the light. The morphogenetic ability of the callus was maintained for several months. In these studies, peach adventitious shoots appeared through indirect morphogenesis.

Indirect morphogenesis involves secondary differentiation of buds from callus tissues. To form callus, from which shoots are then formed, a variety of explants are used. To obtain morphogenic callus from perennial plants, shoot tips or sections of meristematic tissue isolated from them should be taken. This system is not recommended for in vitro micropropagation of plants due to genetic instability. Indirect morphogenesis is important for studying somaclonal variability and obtaining somaclonal variants.

In the UK, in the Department of Physiology at the Maidstone Experimental Station, plant regeneration from stem and leaf callus on Colt cherry rootstock was studied. Callus initiation was carried out on Mu-rasige-Skoog medium containing 2.0-10.0 mg/l NAA. The resulting callus was transferred to a regeneration medium that contained BAP at a concentration of 0.5 mg/l. It was possible to regenerate shoots from calli on this cherry rootstock.

In the Central Genetic Laboratory named after I.V. Michurin, root formation was observed in the culture of passaged callus tissues obtained from annual cherry shoots. When reseeding on a medium with growth regulators, the appearance of meristematic formations was observed.

Somatic embryogenesis

Another method of microclonal propagation of plants in vitro is somatic embryogenesis - the process of formation of embryo-like structures from somatic (non-reproductive) cells. A somatic embryo is an independent bipolar structure, not physically attached to tissue, from which a structure is formed in which the stem and root apexes simultaneously develop.

The formation of somatic embryos in the culture of cells, tissues and organs can occur directly or indirectly. Direct somatic embryogenesis is the formation of a vegetative embryo from one or several explant tissue cells without the stage of intermediate callus formation. Indirect embryogenesis consists of several stages: placing the explant in culture, subsequent stimulation of callus growth and the formation of pre-embryos from callus cells, transfer of callus to a nutrient medium without growth factors for the formation of bipolar embryos from pre-embryos.

The work investigated the possibility of plant regeneration from calli obtained from the roots of cherry rootstocks. Callus was obtained either from cut roots or from whole plants grown under sterile conditions during microcloning of cherry shoots. In the Colt cherry rootstock, callus obtained from the roots of intact plants formed shoots and embryoid-like structures. Cherry calli were cultivated on Murashige-Skoog medium supplemented with BAP, HA and NAA. The frequency of shoot formation was higher than that of the apple tree analyzed in parallel. Regenerated plants were propagated through tissue culture and transplanted into soil. Seedlings of regenerated plants obtained from calli of cherry rootstock did not differ in phenotype from the original rootstocks.

Induction of somatic embryogenesis in cherry varieties (Prunus cerasus L.) was observed when explants were cultivated on Murashige-Skoog medium supplemented with various combinations of auxins and cytokinins. Somatic embryogenesis mainly occurred when the combination of 2,4-D and kinetin was used. Induction of somatic embryogenesis was also noted when 0.1 mg/l IBA was added to the inductive medium. The use of NAA or 6-BAP reduced the induction of somatic embryogenesis and increased the frequency of indirect regeneration in cherry varieties (Prunus cerasus L.).

Today, the most reliable way to obtain genetically identical offspring is considered to be microclonal propagation of stone fruit crops by axillary buds in comparison with somatic embryogenesis, propagation by adventitious buds and indirect morphogenesis.

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V. Rogovaia, M. Gvozdev

IN VITRO CLONAL MICROPROPAGATION OF STONE-FRUIT CULTURES

The review is focused on principal stages and methods of in vitro clonal micropropagation of stone-fruit cultures. Special emphasis is laid on auxiliary bud propagation technique and method of adventitious shoot regeneration from leaf explants of sour cherry, cherry, peach and apricot. Some aspects of plant material testing for virus infections have been reviewed as well as certain problems of genetic stability preservation depending on propagation model.

As a manuscript

Khapova Svetlana Aleksandrovna

IN VITRO CULTIVATION CONDITIONS AND SUBSEQUENT PRODUCTIVITY OF STRAWBERRY PLANTS

Specialty 01/06/07 - fruit growing

Moscow 1997

The work was carried out at the All-Russian Selection and Technological Institute of Horticulture and Nursery Growing.

Scientific supervisor - Candidate of Agricultural Sciences V.A. Vysotsky.

Official opponents: Doctor of Agricultural Sciences F.Ya. Polikarpova; Candidate of Agricultural Sciences T.A. Nikitochkina.

The leading enterprise is the Main Botanical Garden of the Russian Academy of Sciences. M.N. Tsitsina.

The defense will take place... ............ 1992

at...... o'clock at the meeting of the dissertation council D

020.20.01 at the All-Russian Selection and Technological Institute of Horticulture and Nursery Growing at the address: 115598, Moscow, Zag^b^sh st., 4, VSTISP. Academic Council

The dissertation can be found in the library of the All-Russian Selection and Technology Institute of Horticulture and Nursery Growing.

Scientific Secretary of the Specialized Council, Candidate of Agricultural Sciences

L.A. PRINEVA

GENERAL DESCRIPTION OF WORK

Relevance of the topic. In our country, strawberries are the most popular berry crop. It is valued for its early ripening.

The constant and high demand of the population for fresh strawberries and their processed products is determined by their high taste. Strawberries have a wonderful taste, delicate pulp texture and pleasant aroma, a balanced combination of sugars and acids - this makes them a dessert product.

In the eighties, the area under strawberries was 24 thousand hectares with a tendency to increase the share of this crop to 40% of the area occupied by all berries. In the Moscow region, strawberries occupy 45% of the area of ​​industrial berry plantings.

Currently, strawberry culture in the Non-Black Earth Region, as well as in Russia as a whole, requires serious attention due to the sharp reduction in fruit-bearing areas after plants freeze in harsh winters, and the spread of a number of dangerous fungal and viral diseases. The establishment of new strawberry plantings is hampered by the lack of sufficient quantities of healthy planting material of promising varieties. In particular, biotechnical methods are widely used in fruit growing practice to obtain and accelerate the propagation of healthy strawberry planting material.

Several years ago, observations were made showing that plants regenerated from somatic cells in tissue culture are not homogeneous, but exhibit considerable genetic variability. This variability is referred to as somaclonal variability. The question arises whether somaclonal variability is the result of the implementation of genetic differences in changes already existing in somatic cells or whether this is disrupted by the components of the nutrient medium.

By changing the composition of the nutrient medium on which various types of plants are cultivated, it is possible to change their physiological state, enhance and slow down their growth and photosynthesis, and increase resistance to adverse influences.

For each species and even variety of cultivated plant, the composition of the nutrient medium must be selected individually. In addition, to ensure active growth, one environment is needed, for reproduction - another, to preserve plants - a third, to accelerate - a fourth. Consequently, for each plant in agdelyyusgi, taking into account the goals, it is necessary to develop a special composition of the nutrient medium, maintaining a certain balance of its components. This fact indicates the importance and necessity of expanding research work to study the conditions of the influence of the nutrient medium in vitro on the behavior of regenerated strawberry plants.

Growing plants in vitro makes it possible to control many environmental factors: temperature, humidity, duration and intensity of daylight hours.

One of the main directions for increasing the productivity and sustainability of crop production and horticulture at the present stage is the use of intensive cultivation technologies. In most cases, as a mandatory method for weed control, such technologies include the use of new generation herbicides, which must be highly effective and, at the same time, be harmless to humans and the environment.

The system for producing strawberry plants using the in vitro method is becoming increasingly relevant, since the value of planting material is immeasurably higher than that of ordinary plants.

Research tasks also sang. The purpose of this research was to study the influence of cultivation conditions on the ability

strawberries of different groups (regular, remontant, day-neutral) for in vitro propagation and subsequent plant productivity.

To achieve this goal, the following tasks were solved:

1. To study the effect of lighting duration at the stages of micropropagation on biometric indicators.

2. Determine the degree of influence of different compositions of nutrient media on the reproduction rate of strawberry explants.

3. Determine the threshold concentrations of herbicides introduced into the environment for the subsequent selection of somaclonal variants and transformants based on herbicide resistance.

4. To study the influence of the timing of transfer of test tube plants to non-sterile conditions on survival.

5. Conduct a comparative assessment of the development of strawberries obtained using the method

in vitro with conventionally grown plants under field conditions.

Scientific novelty of research results. A significant effect of the lighting period on the number of shoots and their length, the number of leaves, as well as on the number and length of roots developing in explants of tested strawberry varieties in vitro was revealed.

The influence of nitrogen nutrition elements on the development of strawberry explants at the stages of proliferation during reproduction was studied. The possibility of combined use of 6-benzyl-aminopurine and kinetin to stimulate lateral branching in strawberry explants was experimentally demonstrated. "

For the first time, the influence of herbicides on the biometric parameters and pigment system of developing explants was studied in strawberry tissue culture.

The most favorable periods for planting test-tube strawberry plants in soil substrates in winter heated greenhouses have been determined.

Practical value of the work. The results obtained make it possible to optimize the process of clonal micropropagation of strawberries, reduce labor costs and production costs compared to conventional technology.

Using optimal timing for transferring plants to non-sterile conditions makes it possible to increase the yield of adapted plants by 20% or more. The identified selective concentrations of herbicides can be used to create herbicide-resistant forms and obtain transgenic plants.

Approbation of work. The main provisions of the dissertation work were reported at the All-Russian meeting “Young Scientists in Russian Horticulture” (Moscow, 1995); at the IV International Conference "Problems of dendrology, floriculture, fruit growing, viticulture and winemaking" (Yalta, 1996); on "The 18th International group training on plant protection services" (Thailand, Bangkok, 1996); at the IV International Scientific and Practical Conference "Non-traditional crop production, ecology and health" (Simferopol, 1997); at the VII International Conference "Biology of Plant Cells in Vitro, Biotechnology and Conservation of the Gene Pool" (Moscow, 1997); at the meetings of the sections of berry crops of the Academic Council of the All-Russian Scientific and Technical Society (1994-1997).

Publication of research results. Seven scientific articles have been published based on the dissertation materials.

Scope and structure of the dissertation. The dissertation consists of an introduction, three chapters, conclusions and recommendations, and a list of references. The text of the dissertation is presented on 133 sheets of typewritten text, contains 26 tables, 20

drawings. List of used literature in:<лючает 237 наименований, в том числе 120 иностранных.

MATERIAL AND METHODS OF RESEARCH

Research site. The experiments were carried out in the laboratory of the Faculty of Biology of Yaroslavl State University. Demidova P.G. The starting material for the experiments was obtained using the standard method of clonal micropropagation in the biotechnology laboratory of the department of propagation of fruit and berry crops of VSTISP.

Objects of research. The objects of the study were the following strawberry varieties: Mount Everest, Dukat, Geneva, Zenga-Zengana, Profuzhen, Rapella, Redgauntlit, Tribute, Tristar, Holiday.

Cultivation conditions. The vessels with explants were covered with foil and shrink film and cultured under 3 thousand light, temperature 24-26°C and relative humidity in the room 70-75%. Lighting sources: lamps of the LDTs-20 type in the lighthouse, incandescent lamps with a power of 200, 500 W in the greenhouse. The illumination in the greenhouse was 2.5 thousand lux in the morning and in the afternoon, and 4-5 thousand lux in the middle of the day.

In special experiments, when studying the influence of the photoperiod on regenerated plants, cultivation was carried out under 8, 12, 16, 24-hour daylight hours.

The influence of the mineral and hormonal composition of nutrient media on the subsequent productivity of micropropagated plants was studied with a photoperiod of 16 hours of daylight and 1=25°C. Lighting intensity 2500 lux.

Planting, transplantation, and division of conglomerates into individual shoots were carried out in sterile conditions in a KGO-1 laminar box according to generally accepted methods.

The condition of the explants was assessed using a specially developed five-point scale.

Herbicides were introduced into the nutrient medium before autoclaving in the following concentrations, selected based on the results of preliminary experiments: 2CH0*M, 10"5M, 2*10"5M, KIM, 2*1(NM, 10-ZM.

a) simazine, which inhibits photosynthetic electron transport and inhibits the release of oxygen during photosynthesis;

b) rouvdap, which inhibits the synthesis of aromatic amino acids.

A nutrient medium containing no herbicides was used as a control.

Planting of test tube strawberry plants in non-sterile conditions was carried out in two stages:

1, First, the rooted test tube plants were transplanted into perlite, and covered with glass vessels to maintain high humidity. The glass vessels were gradually opened.

2. Then, after about a month, such strawberry plants were transplanted into an autoclaved soil mixture, which consisted of soil, peat and sand in a ratio of 1:1:1 and transferred to a heated greenhouse.

In May of each year, the plants were transferred to open ground. The plants were planted in soil covered with black SUFMK-60 mulching material.

Accounts and observations. During the in vitro experiments, the following indicators were taken into account:

1) reproduction factor;

2) regeneration of vegetative organs (leaves, buds, shoots, roots), taking into account their number on the explant and the number of explants that showed morphogenetic reactions;

3) rooting of buds (or shoots).

The following indicators were measured in regenerated plants under field conditions:

1) the number of whiskers;

2) the number and area of ​​leaves, the number of horns, peduncles, flowers,

3) weight of fruits;

4) output of rooted sockets;

5) changes in the morphology of leaves and stolons;

6) the presence of chlorophyll anomalies.

RESULTS AND DISCUSSIONS

1. Reaction of strawberry explants of different varieties to the duration of illumination. In the course of our work, we determined the effect of micropropagation regimes (8, 12, 16 and 24 hours) on the productivity of plants of different groups of varieties. Explants grown under a 24-hour lighting period had the greatest number of shoots. The average number of shoots in varieties of the usual group (Zgnga-Zengana, Dukat, Redgauntlit) for the entire period of the experiment was 8.9 - 7.0 - 7.4 pcs/explant, in varieties of the remontant group (Mount Everest, Rapella) - 8 - 2 .9 pcs/explant, for varieties of the neutral day group (Tristar, Tribute) - 8.2 - 7.9 pcs/explant. The shoot-forming ability of explants of the Rapella variety turned out to be very low at all periods of illumination (Table 1).

An assessment of the condition of plants formed by explants of different varieties of strawberries depending on the light regime of cultivation showed that the plants developed best with a 16-hour lighting period, for example, the condition of explants grown with a 12-hour lighting period was 4.2 points, with 16- hourly - 4.7 points, and with 24-hour - 3.6 points.

Table 1

Average number of shoots formed by explants of different strawberry varieties depending on the duration of illumination (pcs.)

Varieties Light duration

8 hours/day 12 hours/day 16 hours/day 24 hours/day

Mount Everest 4.0 5.3 8.0 15.0

Rapella 2.0 2.8 3.4 3.6

Dukat 4.3 5.0 7.8 11.0

Zenga-Zengana 4.5 6.3 9.1 14.8

Redgauntlit 3.9 5.4 8.0 12.3

Tristar 5.4 6.7 8.5 14.2

Tribute 5.6 6.8 9.2 12.1

X 4.0 5.1 7.7 N.9

NSR05 interaction 1.2

Plants obtained under 12 and 16 hour light conditions were adapted to non-sterile conditions.

The observation results showed that strawberry plants grown under a 16-hour lighting period in vitro produced significantly more stolons than when cultivated under a 12-hour day, and also had a larger leaf area (Tables 2, 3).

The action of light depends on the formation of photoreceptors and the transformation of light energy in leaf cells, photostimulation of biosynthetic processes in them.

As our studies have shown, the lower amount of pigment content is compensated by plants due to a larger leaf surface area.

table 2

Average number of stolons in different strawberry varieties propagated under different lighting durations (pcs/plant) (1995)

Varieties Duration of lighting during micropropagation

12 hours/day 16 hours/day

Mount Everest 33 ±3.6 40 ±2.8

Rapeala 10 ±2.6 19 ±3.1

Zenga-Zengana 26 ±3.1 41 ±4.2

Redgauntlite 37 ± 5.2 57 ±4.6

Ducat 14 ±3.7 24 ±3.5

Trisgar 18 +1.8 21 ±4.1

Tribes from 19 ± 1.6 25 ± 4.2

Table 3

Effect of day length during in vitro cultivation on leaf area of ​​strawberry plants under field conditions (August 1, 1995)

Varieties Leaf area on 1 plant (cm2)

12 hours/day 16 hours/day

Ducat 240 360

Zenga-Zengana 550 720

Redgauntlit 450 576

Tristar 320 420

HCPos: light mode 24.1 grade 16.4

interaction 18.2 2. Development of strawberry explants depending on the concentration of different forms of nitrogen in the nutrient medium. As is known, the process of in vitro reproduction occurs on nutrient media, of which the Murashige-Skoog medium is the most widely used. However, this environment

contains some components (especially nitrogen) in excess concentrations.

Among mineral salts, nitrogen-containing salts are important for the growth and development of plants.

We examined the effect of reducing the composition of the Murashige-Skoog nutrient medium by two and four times. Our experiments showed that during the reproduction stages it is not advisable to reduce the salt concentration in the basic Murashige-Skoog medium. Therefore, we tried to evaluate the concentrations of different forms of nitrogen in the nutrient medium on the development of strawberry explants. It turned out that reducing ammonia nitrogen by half did not affect the reproduction rate (Table 4).

Table 4

Development of strawberry explants of the Tristar variety depending on the concentration of ammonium nitrogen in the nutrient medium

Kvdo concentration. Number of shoots, TTGT Length of shoots, w Number of roots, ptg Length of roots, mm Number of leaves, tttt.

Koshrol 1650 mg/l 4.3 3.5 2.1 0.5 12.2

0,5. 4,0 3,2 0 0 14,1

0,25 3,9 3,2 0 0 12,2

0,125 3,8 3,0 0 0 11,0

0,5 4,3 3,2 0 0 12,1

0,253 3,9 3,2 0 0 12,1

0,125 3,8 3,2 0 0 10,3

NSRo5 - number of shoots

concentration No. ШОз Russia< Роз

A two-fold decrease in nitrate nitrogen negatively affected the shoot-forming ability of plants. The reproduction rate decreased, compared to the control, by 3-4 units (Table 5).

Table 5

Development of strawberry explants of the Tristar variety depending on the concentration of nitrate nitrogen in the nutrient medium

Concentration Qty Length

UTOe escapes, escapes,

(part) pcs. cm.

Control 1900 mg/l 5.5 2.8

NSRo5 - number of shoots

concentration QOL)z = 1.3

Perhaps this effect is associated with the absorption mechanism

nitrate nitrogen. It is known that the use of keto acids for the synthesis of amino acids occurs more intensively in the presence of ammonium nitrogen in the nutrient medium; Nitrate nitrogen is used to a lesser extent for the synthesis of amino acids.

Based on the data obtained, we can conclude that a decrease in the concentration of ammonia nitrogen by 825 mg/l in the Murashige-Skoog medium does not lead to a decrease in the reproduction coefficient, which can be implemented in practice.

3. The effect of itokinins and auxins on the development of strawberry explants. Also one of the important components of the nutrient medium are growth regulators. Careful selection and identification of optimal concentrations can improve the efficiency of the clonal propagation method.

We studied the combination of 6-BAP and kinetin on the development of explants. Combinations of 6-BAP and kinetin at concentrations of 0.25 mg/l + 0.25 mg/l and 0.25 mg/l + 0.5 mg/l, respectively, slightly stimulated bud growth and leaf unfolding (Table 6).

Table 6

Dependence of the multiplication coefficient on the concentration of 6-BAP and kinetin in the nutrient medium (Zenga-Zengana variety)

Concentration of cygokinins, g/l Number of shoots formed, pcs. Length of shoots, cm.

6-BAP Kinetin

Control 6-BAP 1 mg/l 10.0 2.5

0,25 0,25 3,8 2,0

0,75 0,25 9,2 2,5

1,0 0,25 14,4 2,5

NSRosis 4.6 1.2

The best results were achieved with a combination of 6-BAP-1 mg/l and kinetin - 0.25 mg/l. In this case, the number of shoots formed was greater than in the control variant.

The most developed explants were transplanted onto rooting medium. The use of auxin-based growth regulators in our experiments ensured high rooting of strawberry shoots on the 20th day of cultivation. The Zenga-Zengtsh variety rooted equally well when using IBA and IUK at concentrations of 0.5-1 mg/l. Varieties Tristar and Dukat on a medium containing IAA - 1 mg/l compared to the control had a greater number of rooted explants.

4. Sensitivity of proliferating strawberry crops to herbicides in vitro. In recent years, more attention has been paid to the possibility of creating new forms of plants using biotechnological methods, in particular through the selection of somaclonal variants with economically valuable traits. Selection of somaclonal variants based on herbicide resistance can be carried out only after identifying lethal and sublethal concentrations of selective agents for cell, tissue and organ cultures.

We did not find such data for strawberries in the literature, so the next stage of the work was devoted to studying the sensitivity of tissues and organs of various strawberry varieties cultivated in vitro to the presence of two types of herbicides in the nutrient medium.

It should be noted that the herbiid effect of the tested drugs was maintained in in vitro culture.

In the case of using simazine in the concentration range 2 * 10-5 - 2ХУ 4М, an inhibitory effect was observed on the growth and development of explants. A concentration of 10-3M initially caused signs of chlorosis in explants of all varieties up to complete discoloration followed by death.

The Geneva variety turned out to be the most sensitive to simazine, for whose explants the concentration of 1SIM turned out to be lethal (Table 7).

Table 7

State of explants of various strawberry varieties (in points) depending on the presence of herbicides in the nutrient medium (1.5 months)

Herbicide concentration (M)

Variety Herbicide type Control (without herbicide) O 2" 10* Yu-" 24 O-5 10-4 2*10-"

Zenga-Simazin 5.0 4.8 4.6 3.8 3.6 2.2

Zengana Roundup 5.0 4.6 4.2 3.0 0 0

Dukat Simazin 5.0 4.8 4.5 4.2 3.8 2.5

Rauvdap 5.0 4.4 4.2 3.0 0 0

Redgoshlig Simazin 4.9 4.6 4.4 4.1 3.6 2.4

Roundup 4.9 4.5 4.0 2.6 0 0

Mount Simazin 4.9 4.5 4.4 3.9 3.8 1.5

Everest Roadup 4.9 4.5 3.7 2.4 0 0

Geneva Simazin 4.8 4.5 4.0 4.2 0 0

Roundup 4.8 4.5 3.3 1.9 0 0

Trisgar Simazin 4.9 4.6 4.3 4.2 3.8 0

Roundup 4.9 4.5 3.4 2.7 0 0

Tribeot Simazin 4.9 4.7 4.2 4.1 3.8 1.5

Roundup 4.9 4.5 3.6 3.0 0 0

When studying the effect of the presence of Roundup in the nutrient medium, it was revealed that the three highest concentrations (U-4, 2*1 (KM, 10"3M) led to the complete death of explants.

To confirm the reduced sensitivity of the selected explants, they were replanted on nutrient media containing sublegal concentrations of the corresponding herbicides. During subsequent cultivation of selected conditionally tolerant explants of various strawberry varieties, the death of a significant part of the crops was observed. This indicates that in the overwhelming majority of cases we are not dealing with herbicide-resistant organs and tissues, but with explants that did not have time to die in the previous subcultivation.

It is known that herbicides suppress many metabolic processes in plants, in particular, they have a significant effect on photosynthetic activity.

Simazine suppresses photosynthesis in plants by binding to the so-called 32K protein, which is part of the thylakoid membrane. Taking into account the mechanism of herbicidal action, which is based on inhibition of the Hill reaction, we conducted a series of experiments on its effect on photosynthetic activity.

Glyphosate affects the synthesis of very important aromatic amino acids, the point of its application is the enzyme 3 - phosphoshikimate -1 carboxyvinyl transferase. Probably, the suppression of this stage of metabolism causes a deficiency of aromatic amino acids, the accumulation of shikimate and, as a result, upon contact with glyphosate at a concentration of 10-3M, the death of strawberry explants.

Thus, we have determined the selective concentrations of two herbicides: simazine - KIM, roundup - 10-5M.

5. Effect of herbicides simazine and roundup on photosynthesis of isolated strawberry shoots in vitro. In the process of work, we studied the effect of pigment content in strawberry leaves when cultivated with Roundup and simazine (Fig. 1). We noted the high sensitivity of explants of the Geneva variety. This increased sensitivity is also reflected in the reaction of the photosynthetic apparatus to the content of pigments in strawberry leaves.

At the eighth week of cultivation in the variant with a concentration of Roundup 2X106M, a stimulating effect of this drug on the content of the amount of pigments in explants was noted.

6. Adaptation of microshoots to non-sterile conditions depending on the timing of<зсадки. Для выявления лучших сроков приживаемости растения земляники каждый месяц переносили в нестерильные условия. Наблюдения, проведённые за дальнейшем развитием таких растений, выявили, что самым благоприятным сроком выведения пробирочных растений был период с мая по август. Например, в 1996 году высокий процент приживаемости был у сортов Тристар - 96%, Трибьют -93%. У сорта Редгонтлит в июле 1996 года на 20% повысилась приживаемость растений по сравнению с 1994 годом этого же месяца. Эксперименты показали, что растение, высаженное в мае-июне-июле быстро развивалось. Так, растения сорта Зенга-Зенгана (Рис.2), перенесённое в нестерильные условия 11 мая с длиной побегов 3,5 см, через 1,5 месяца имело длину побегов 9 см, крупные листья; ещё через месяц растения высаживали в полевые условия. При выведении эксплантов в нестерильные условия в марте, растениям требуется 3,5 месяца для высаживания в полевые условия, а это на один месяц больше, чем в первом варианте.

and 80 -60 -40 -20 -

chlorophyll a

chlorophyll b

carotenoids

Figure 1. Pigment content (%) in strawberry leaves when cultivated on media with Roundup and simazine for two weeks.

a) variety Geneva - control (without herbicides) on media with Roundup I - concentration 2 10"6 M

b) variety Geneva "CCR - concentration 10"5 M on media with simazine p! - concentration 10"3 M

Variety Zenga-Zvngannz

Fig. 2 Survival rate of strawberry plants depending on the time of transplantation into non-sterile conditions (Zenga-Zengana variety)

When strawberry explants were transferred to non-sterile conditions in autumn and winter, the survival rate was low. For example, in the Zenga-Zengana variety in December (1996), there were 70% fewer rooted plants compared to the results of May (1996); the Dukat variety has fewer rooted plants in January: 55% compared to May (1996). Based on data for three years (1994-1996), we can note that explants transplanted into non-sterile conditions in the autumn-winter period had a low survival rate.

Apparently, the phenomenon we noted is associated primarily with the impossibility of maintaining the same conditions (illumination, spectral composition of light) at the same level throughout the year in industrial cultural premises (winter greenhouses). It is forbidden

also exclude the influence of internal biological reasons in the behavior of plants associated with the rhythms of plant growth and development.

Thus, the survival rate of plants when transferred to non-sterile conditions depended on the method and time of planting. Using optimal timing for transferring plants to non-sterile conditions makes it possible to increase the yield of adapted plants by up to 30%. 7. Economic and biological assessment of plants of different varieties of strawberries obtained by in vitro and conventional methods. Assessing the economic and biological importance of strawberry varieties, we compared the effect of two methods of propagation of plants of different varieties on yield, number of rosettes, weight of berries, number of fruits affected by rot (Table 8).

Table 8

Economic and biological assessment of strawberry plants obtained by different methods of propagation____

Varieties Method of propagation Average yield per plant for 1 year, g Average number of rosettes per plant per year of growing season Average number of rosettes per plant per year of growing season

Zenga-Zengana in vitro 126 37 38

standard 125 30 37

Redgoitlit in vitro 108 57 52

standard 105 40 53

Ducat in vitro 95 18 19

standard 99 14 18

Tristar invito 199 21 21

standard 183 18 21

Tribute invito 186 24 26

standard 178 23 25

Geneva invito 177 20 19

standard 173 21 19

NSR05: varieties 26.5 years 8.9

interaction 5.6

Experiments have shown that in 1 year of cultivation, in some strawberry varieties, the number of rosettes depends on the propagation method; for example, in the Zenga-Zengaia variety, the number of rosettes from the reference plant was 8 more. Compared to plants obtained using the conventional method. In the second year, this effect smoothed out. The percentage of fruits affected by rot was higher in varieties that had two waves of fruiting: firstly, the sharp fluctuations in night and day temperatures in the fall in the Yaroslavl region affect; secondly, it affects the accumulation of the pathogen in plants over the entire growing season. There were no significant differences in yield and berry weight.

Economic issues of strawberry propagation in vitro.

The method of clonal micropropagation is quite labor-intensive and requires a large amount of material costs. At the same time, the high profitability of the in vitro method is due to the saving of cultivation space, a reduction in the period of plant cultivation, an increase in the reproduction rate, an improvement in product quality (SSE), as well as work in the autumn-winter period.

An assessment of the economic efficiency of the production of planting material using the in vitro method was carried out using the example of strawberries of the Zenga-Zengana variety. Production costs were calculated based on the methodological recommendations of VSTISP (Table 9).

Calculations have shown that with the number of initial explants - 5 pieces, the reproduction coefficient - 8, the number of subcultivations - 3, taking into account a number of coefficients (coefficient of survival of explants, yield of shoots suitable for rooting, rooting rate, survival rate during adaptation) within six months using generally accepted technology you can get 5000 pcs. Shoots. The cost of one explant grown using conventional technology will be 1.22 rubles.

An important aspect of the economic efficiency of in vitro technology was the reduction in labor costs for growing 1 thousand pieces. strawberry plants in vitro.

Table 9

Economic assessment of the production of strawberry planting material with

using the method of clonal micropropagation (1 thousand pieces)

Indicators In vitro method

1. Cost of 1 piece, rub. 1.22

2. Cost and overhead costs (180%), rub. 1.68

3. Sales price of 1 piece, rub. 7.2

4. Profit from 1 thousand units, rub. 5.52

5. Number of microshoots per 1 m2, pcs. 1000

6. Profit per 1 m2, rub. 11.04

6. Profitability level, % 328

Thus, the method of clonal micropropagation gives

significant economic effect, which shows the advantage of using it in production.

1. The duration of the lighting period has a significant impact on the development of strawberry explants of the tested varieties. Among the studied cultivation regimes, lighting durations of 12 and 16 hours turned out to be the most favorable in terms of the reproduction coefficient, the length of the shoots formed, the number of leaves, and at the rooting stage, the number and length of roots.

2. Strawberry plants grown in vitro under 16-hour illumination produced significantly more stolons than in the case of cultivation under a 12-hour period, therefore such plants can be used as starting plants for planting queen cells. Plants grown in vitro under 12-hour light were characterized by a high content of chlorophylls

a, b and carotenoids compared to plants obtained under a 16-hour day by 10%-30%.

3. With clonal propagation of tested different varieties of strawberries, it is possible to reduce the concentrations of ammonium nitrogen by half compared to the basic environment without deteriorating such a development indicator as the multiplication factor

4. To stimulate lateral branching in explants of tested strawberry varieties, the best results are obtained by combining 6-BAP at a concentration of 1 mg/l with kinetin 0.25 mg/l.

5. The inclusion of herbicides of different spectrum of action - Roundup and simazine in concentrations of 2X106M - 10"3M - into the nutrient media had a significant effect on the growth processes of cultivated strawberry explants. Roundup had a more phototoxic effect and caused the death of the bulk of the explants at a concentration of 2*1 ( IM: The selective concentration of simazine is 10^M, roundup 10~5M for the tested seven strawberry varieties.These studies can become the basis for the selection of strawberry forms with increased resistance to herbicides.

6. The presence of the herbicides Roundup and Simazine in the nutrient media at a concentration level of 105.10~3M inhibited the synthesis of chlorophyll a, chlorophyll b and carotenoids. The concentration of 2X10"6M Roundup at the eighth week of cultivation led to an increase in the quantitative content of chlorophyll a and chlorophyll b.

7.0 the most favorable periods for transferring micropropagated plants to non-sterile conditions have been determined from May to August, which makes it possible to increase the yield of adapted plants by 20% or more.

8. An assessment of the condition of plants in field conditions showed that in the first year of cultivation in plants of the varieties Zenga-Zengana, Dukat, Profyuzhen, Khomdey, Redgauntlit, the number of rosettes depends on the propagation method. In plants grown in vitro, the number of rosettes was approximately 1.3 more

times. In the second year of the growing season, the differences in the number of rosettes formed in all studied strawberry varieties were smoothed out. There were no significant differences in the yield of the tested varieties depending on the method of propagation.

When propagating strawberry plants using the in vitro method, we recommend reducing the concentration of ammonia nitrogen in the Murashige-Skoog nutrient medium to 825 mg/l. To ensure mass propagation of shoots, the optimal combination of growth regulators 6-BAP and kinetin is 1 mg/l, 0.25 mg/l, respectively.

Transplantation of test tube plants into non-sterile conditions must be carried out from May to August.

To select somaclonal variants and transgenic specimens with increased herbicide resistance, use the following concentrations of herbicides in the nutrient medium: rauwdapa - 2X10"5, Yu"5M; simazine - 2M0"4, 1(NM.

1. Khapova S. A. The influence of cultivation conditions on the clonal micropropagation of strawberries and the processes of its adaptation to non-sterile conditions // Abstracts of the All-Russian meeting "Young Scientists for Russian Horticulture". - M. -1995. - P.156-158

2. Khapova S.A. Effect of herbicides on photosynthesis and development of explants

strawberries in vitro // Materials of the IV International Conference "Problems of dendrology, floriculture, fruit growing, viticulture and winemaking". -Yalta, -1996.-T.2.-P.61-63

3. Khapova S. Tissue-culture strawbeny no virus // The 18 th International group training on

plant protection services - Thailand. -1996. - T. 1. - P.M-M3

4. Vysotsky V.A., Khapova S.A. Sensitivity of isolated strawberry organ cultures to herbicides / Sat. work. VSTISP. - M.; -1997. - P.83-89

5. Khapova S.A. Influence of substrate composition and timing of transplantation into non-sterile

conditions for the survival rate of test-tube strawberry plants // Abstracts of the scientific conference of YarSKhA. - Yaroslavl. -1997.

6. Khapova S.A. Reaction of isolated strawberry shoots to the presence of herbicides in the nutrient medium // Abstracts of the VII International Conference "Biology of Plant Cells in Vitro, Biotechnology and Preservation of the Gene Pool". -1997. - P.382-383

7. Khapova S.A. The influence of light conditions on the morphology and synthesis of pigments

strawberry plants in field conditions // Materials of the VI International Scientific and Practical Conference "Non-traditional crop production, ecology and health". - Simferopol. -1997. - T. 1-2. - pp. 140-141

Candidate of Agricultural Sciences talks about the possibilities of biotechnology. Dmitry Kravchenko from the All-Russian Research Institute of Potato Farming.

Growth and development under control:

Potato biotechnology opportunities in vitro

Regulation of plant growth and development is an important task of modern biology. The study of regulatory mechanisms at the cellular level that control the basic vital functions of a plant, ways to control physiological processes, and regulatory mechanisms of a plant cell opens up broad prospects for using potential opportunities.

Why do you need a job? in vitro ?

Biotechnological methods associated with cultivation under conditionsin vitro , have become an integral part of the technological process of reproduction of source plants for original potato seed production. For health improvement using apical meristem methods and accelerated culture propagationin vitro In order to obtain the largest possible amount of healthy starting material for further conducting the seed production process, it is necessary to optimize and intensify the processes of growth and development of potato plants under both conditionsin vitro , and when receiving healthy mini-tubers.

Why do you need to manage growth processes? Let us name the main directions of work with potato cropsin vitro :

— improvement of potato varieties from viral and other infections (apical meristem method in various combinations and modifications);

— introduction to culturein vitro explants obtained from a completely healthy potato plant;

— microclonal propagation of potato varieties in the process of original seed production;

— obtaining potato microtubers;

— long-term maintenance of collections of potato genotypes;

— selection-genetic and other research work that requires material for its implementationin vitro .

Iona Skulachev

Let's consider the possibilities of controlling plant growth processesin vitro at the stages of healing and microclonal propagation.

At the stage of obtaining regenerants from meristematic explants, the indicators of the survival rate of objects, the intensity of the processes of their morphogenesis and the time of regeneration are of decisive importance. To improve these parameters, we recommend using a new class of nanoproducts with geroprotective properties - Skulachev ions. Synthesized at the Research Institute of Physical and Chemical Biology named after A.N. Belozersky (MSU named after M.V. Lomonosov) preparationsSkQ (Skulachev ions) are compounds of triphenyldecylphosphonium cations and chloroplast plastoquinone analogues. They regulate the intracellular balance of reactive oxygen species and differ in penetrating ability and the ratio of anti- and prooxidase activity.

Adding a drugSkQ1 into an artificial nutrient medium at a nanoconcentration of 2.5 nM improves the survival rate of meristem explants of varieties with a shorter growing season by 16–43% and varieties with a longer growing season by 7–13%.

At the same time, a significant increase in morphogenic activity and growth intensity of explants is observed, as a result of which the time of regeneration of microplants from meristem tissues is reduced by 24– 30 days.

After transplantation into a new nutrient medium, microplants from regenerants obtained usingSkQ1 , were characterized by faster growth and better biometric parameters. In terms of a set of indicators, plants obtained in media were in the lead: for the initial growth of meristems and for further plant growth.

Even faster

Thus, 50 days after the isolation of meristems when using the drugSkQ1 Full-fledged plants were obtained, suitable for further cuttings and testing for latent infection with viruses using the ELISA method. And another 15–20 days after cuttings, the regrown plants could be planted in the soil of a greenhouse or open ground to assess varietal typicality and obtain mini-tubers. Consequently, the total time from isolating the meristem to planting healthy plants in the ground can be reduced to 65–80 days. The quantitative output of the lines also increases, which means the likelihood of successful recovery.

The automated system Fitotron TF 600 allows you to obtain similar results without the use of additional growth-stimulating substances, and in combination with them, the morphogenic activity of regenerants increases by 12–21%, the time to obtain initial healthy plants is reduced by 7–14 days.

In addition, research is currently being conducted on the effect of light radiation of various spectral compositions on reducing viral infection in plantsin vitro .

Accelerating cuttings

After receiving healthy initial regenerated plants, the next important stage of reproduction is their further propagation. The challenge is to rapidly increase the volume of starting material while maintaining high potential growth energy and productivity, as well as pathogen-free status.

The process of microclonal propagation should be divided into 2 stages: accelerating cuttings and the last cutting before planting in conditionsin vivo . Accelerating cuttings should really ensure the maximum multiplication rate within the time frame specified by the seed production program. In the last passage, it is necessary to form plants that will subsequently be best adapted to growing conditions in the soil and will produce a high yield of standard mini-tubers. Therefore, at different stages of micropropagation, different chemical regulators and different physical cultivation conditions can be used.

Provide conditions

Based on the results of previous studies, we recommended using the drug epin for accelerating cuttings (synthetic epibrassinolide), which accelerates plant stem morphogenesisin vitro and increases the reproduction rate. At the last stage of cuttings, the drug fumar (aminofumaric acid dimethyl ester) was recommended, which stimulated rhizogenesis in potato plants and had a prolonged positive effect in the aftereffect, increasing potato yields in field nurseries by 9–15%.

Through a careful study of the new generation of growth-regulating substances synthesized in recent years, a drug was identified that, when added to an artificial nutrient medium, increases the number of leaves of potato microplants, and therefore the reproduction coefficient, to 9–15 pieces. depending on the variety.

However, similar results can be achieved by cultivating plantsin vitro on a standard Murashige-Skoog nutrient medium while optimizing all physical parameters of cultivation, which can be provided by the Fitotron TF 600 installation.

It's possible, but be careful

Thus, an effective tool in the arsenal of manipulation of objects in culturein vitro began the use of biologically active substances that have a targeted effect on physiological processes and mechanisms of regulation of intracellular metabolism. However, it should be taken into account that many biologically active substances have a more or less pronounced mutagenic effect. They should be selected with special care and caution for those areas of work with culturein vitro , in which the stability of potato varietal traits is at greatest risk.

On the other hand, the introduction of modern technological solutions into the practice of original potato seed production, such as chambers with controlled physical conditions (Phytotron TF 600 and its analogues), allows us to partially solve the problem of controlling plant growth processesin vitro without the use of chemical regulation.

1

Over the past 20 years, information has been accumulated on the pleiotropic, non-erythropoietic functions of erythropoietin (EPO), the EPO system - the EPO receptor at the auto- and paracrine levels is considered as a link in non-specific protection during damage, and EPO receptors on non-erythroid cells, in particular on various populations of leukocytes, in including phagocytes, are designated as tissue-protecting receptors. The purpose of the work is to study the effect of various concentrations of EPO on the functional activity of phagocytes under experimental conditions in vitro - implemented on the whole blood of 20 clinically healthy people. Recombinant human EPO as part of the drug "Epocrine" (international nonproprietary name: epoetin alfa, Federal State Unitary Enterprise GNII OCHB FMBA of Russia, St. Petersburg) was used in concentrations of 1.88 IU/l; 3.75 IU/l; 7.5 IU/l; 15 IU/l; 30 IU/l, which corresponds to 12.5, 25, 50, 100, 200% of the average physiological level of EPO in the blood, the indicators were studied after 10 and 30 minutes of incubation in a thermostat at 37 °C. The function of phagocytes was studied by the ability to absorb particles of monodisperse polystyrene latex and oxygen-dependent intracellular metabolism in a spontaneous and induced test with nitroblue tetrazolium (NBT test). It was found that a 10-minute contact of EPO with whole blood does not have a statistically significant effect on the function of phagocytes; after a 30-minute incubation of EPO with whole blood, activation of the absorption capacity and oxygen-dependent metabolism of peripheral blood phagocytes was recorded. It was revealed that EPO in the dose range from 1.88 to 30 IU/l increases the number of actively phagocytic cells and the absorptive capacity of an individual phagocyte; at doses of 3.75 and 15 IU/l, EPO increases the number of cells generating active oxygen metabolites and the intensity of generation of active oxygen metabolites by an individual phagocyte in the induced NBT test. The effect of EPO on the functional activity of phagocytes does not depend on the dose.

phagocytosis

innate immunity

erythropoietin

1. Osikov M.V. Analysis of the efferent properties of ceruloplasmin and alpha-1-acid glycoprotein in experimental peritonitis / M.V. Osikov, L.V. Krivokhizhina, A.V. Maltsev // Efferent therapy. – 2006. – T. 12, No. 4. – P. 36-39.

2. Osikov M.V. The influence of hemodialysis on the processes of free radical oxidation in patients with chronic renal failure / M.V. Osikov, V.Yu. Akhmatov, L.V. Krivokhizhina // Bulletin of the South Ural State University. Series: Education, healthcare, physical education. – 2007. – No. 16 (71). – pp. 95-97.

3. Osikov M.V. Hemostasiological effects of alpha-1-acid glycoprotein in experimental septic peritonitis / M.V. Osikov, E.V. Makarov, L.V. Krivokhizhina // Bulletin of Experimental Biology and Medicine. – 2007. – T. 144, No. 8. – P. 143-145.

4. Osikov M.V. The influence of alpha-1-acid glycoprotein on the processes of free radical oxidation in experimental liver failure // Bulletin of Experimental Biology and Medicine. – 2007. – T. 144, No. 7. – P. 29-31.

5. Osikov M.V. The role of erythropoietin in the correction of disorders of vascular-platelet hemostasis in patients with end-stage chronic renal failure / M.V. Osikov, T.A. Grigoriev // Fundamental research. – 2011. – No. 9-3. – pp. 462-466.

6. Osikov M.V. Efferent and antioxidant properties of erythropoietin in chronic renal failure / M.V. Osikov, T.A. Grigoriev, Yu.I. Ageev // Efferent therapy. – 2011. – T. 17, No. 4. – P. 7-13.

7. Osikov M.V. The influence of erythropoietin on the functional activity of platelets / M.V. Osikov, T.A. Grigoriev, A.A. Fedosov, D.A. Kozochkin, M.A. Ilyinykh // Modern problems of science and education. – 2012. – No. 6. - URL: www..02.2014).

8. Osikov M.V. Modern ideas about the hemostasiological effects of erythropoietin / M.V. Osikov, T.A. Grigoriev, A.A. Fedosov // Fundamental research. – 2013. – No. 5-1. – pp. 196-200.

9. Osikov M.V. Erythropoietin as a regulator of platelet glycoprotein expression / M.V. Osikov, T.A. Grigoriev, A.A. Fedosov, D.A. Kozochkin, M.A. Ilyinykh // Modern problems of science and education. – 2013. – No. 1. - URL: www..02.2014).

10. Broxmeyer H.E. Erythropoietin: multiple targets, actions, and modifying influences for biological and clinical consideration // J. Exp. Med. – 2013. – Vol. 210(2). – P. 205-208.

The cellular mechanisms of innate immunity are associated with the implementation of the functional activity of phagocytic cells, primarily neutrophils and monocytes/macrophages. Changes in the function of phagocytes may be a key link in the pathogenesis of various diseases and typical changes in homeostasis. Thus, in chronic renal failure, activation of the innate immune system and the associated manifestation of local and systemic inflammation contributes to the development and progression of cardiovascular diseases; in case of thermal injury, changes in the function of phagocytes are associated with the dynamics and successful completion of reparative processes. One of the key tasks of modern medical science is the search for regulators of the functional activity of innate immune effectors. Previously, we demonstrated the role of biologically active substances of the endogenous nature ceruloplasmin and alpha-1-acid glycoprotein in the regulation of phagocyte function in various pathologies. In recent years, the pleiotropic effects of erythropoietin (EPO) have attracted the attention of many researchers. EPO first became known as hematopoietin, a factor that stimulates the formation of red blood cells de novo, thanks to the pioneering work of Carnot and Deflandre, published in 1906. The main site of EPO synthesis is the peritubular and tubular cells of the kidneys, in which the EPO gene is expressed in response to a decrease in the partial pressure of oxygen with the participation of hypoxia-inducible factor-1 (HIF-1). Modern understanding of the mechanisms of action of EPO at the molecular level allows it to be classified simultaneously as hormones, growth factors and cytokines. The main point of application for the action of EPO are the cells of the erythroid series in the bone marrow: burst and colony-forming units granulocyte-monocyte-megakaryocyte-erythrocyte, erythrocyte, as well as erythroblasts and pronormoblasts, which have specific receptors. EPO is responsible for proliferation, differentiation and inhibition of apoptosis in these cells. The discovery of receptors for EPO on cells of non-erythroid tissues, such as neurons, cardiomyocytes, kidney cells, and endothelial cells, made it possible to discover new biological effects of EPO. Previously, we showed the protective role of EPO in chronic renal failure in clinical and experimental conditions in relation to affective status, psychophysiological status, functional state of the hemostatic system, etc. We believe that the indirect implementation of the pleiotropic effects of EPO may be associated with its influence on the function of phagocytic cells. Currently, the EPO-EPO receptor system at the auto- and paracrine level is considered as a link in nonspecific protection during damage, and EPO receptors on non-erythroid cells are designated as tissue-protecting receptors. Goal of the work- study the effect of different concentrations of EPO on the functional activity of phagocytes under experimental conditions in vitro.

Materials and research methods

The work was performed using whole blood from 20 clinically healthy human donors. Recombinant human erythropoietin as part of the drug "Epocrine" (international nonproprietary name: epoetin alfa, Federal State Unitary Enterprise GNII OCHB FMBA of Russia, St. Petersburg) was used in concentrations of 1.88; 3.75; 7.5; 15; 30 IU/l, which corresponds to 12.5, 25, 50, 100, 200% of the average physiological level of EPO in the blood, the indicators were studied after 10 minutes and 30 minutes of incubation in a thermostat at 37 °C. The function of phagocytes was studied by phagocytic ability and oxygen-dependent intracellular metabolism. The phagocytic ability of leukocytes was assessed by the absorption of particles of monodisperse (diameter 1.7 μm), polystyrene latex, for which 200 μl of a cell suspension was mixed with 20 μl of a suspension of polystyrene latex particles. After 30 min of incubation at a temperature of 37 °C, the activity and intensity of phagocytosis and the phagocytic number were assessed. Intracellular oxygen-dependent metabolism in phagocytes was assessed using the NBT test, which is based on the formation of insoluble diformazan from nitroblue tetrazolium. Spontaneous and induced NBT tests were performed. To perform a spontaneous NBT test, 50 μl of physiological solution and 20 μl of nitroblue tetrazolium were added to 100 μl of blood; in the induced NBT test, 50 μl of a suspension of polystyrene latex in physiological solution and 20 μl of nitro blue tertrazolium were added to 100 μl of blood. The number of diformazan-positive cells (NBT test activity) was taken into account; to calculate the NBT test index, the area of ​​granules was assessed in relation to the area of ​​the nucleus (single dust-like granules - 0; cells with deposits not exceeding 1/3 of the nucleus area in total - 1 ; cells with deposition of diformazan more than 1/3 of the nuclear area - 2; cells exceeding the size of the nucleus - 3). Statistical analysis was carried out using the Statistica for Windows 8.0 application package. Statistical hypotheses were tested using Friedman's rank analysis of variance and the Wilcoxon test. To assess the dependence of the effect of EPO on the function of phagocytes on the dose, correlation analysis was used to calculate the Spearman correlation coefficient. Differences were considered statistically significant at p<0,05.

Research results and discussion

The results of the influence of EPO on the indicators of functional activity of phagocytes after 10 minutes of incubation at 37 °C are presented in table. 1 and 2. As can be seen, we have not recorded statistically significant changes in the absorption capacity and oxygen-dependent metabolism of peripheral blood phagocytes. It should be noted that, as a trend, the activity, intensity of phagocytosis, phagocytic number, and indicators of spontaneous and induced NBT tests increased; the highest mean values ​​were observed with the addition of EPO at a dose of 30 IU/l (200% of the physiological serum level). It was found that 30-minute incubation of EPO with whole blood leads to a change in the functional activity of peripheral blood phagocytes (Tables 3 and 4). EPO in the concentration range from 1.88 to 30.00 IU/l leads to activation of the absorption capacity of phagocytes: activity, intensity of phagocytosis and phagocytic number increase. The maximum increase in the number of actively phagocytic cells, by 49.9% of the average value in the control group, was observed with the addition of EPO at a dose of 7.5 IU/l (50% of the physiological level of EPO in serum). The effect of EPO does not depend on the dose when assessing the activity of phagocytosis (Spearman's correlation coefficient R=0.21; p>0.05), the intensity of phagocytosis (Spearman's correlation coefficient R=0.17; p>0.05), phagocytic number (coefficient Spearman correlation R=0.13; p>0.05). The effect of EPO on oxygen-dependent metabolism in phagocytes is ambiguous. Thus, there was no effect of EPO at all doses used on the spontaneous NBT test (Table 4). It was noted that EPO increases the generation of oxygen metabolites by phagocytes after induction with latex particles only at doses of 3.75 and 15.00 IU/l (25 and 100% of the physiological level of EPO in serum); both the number of active cells and the NBT test index, which reflects the intensity of generation of oxygen metabolites by an individual cell, increases.

According to other researchers, receptors for EPO were found on leukocytes; thus, using flow cytometry and reverse polymerase chain reaction, expression of the EPO receptor gene and mRNA was detected in T- and B-lymphocytes and monocytes. A group of researchers from the Transplant Center in Bergamo (Italy) believe that one of the targets for the immunomodulatory effect of EPO are dendritic cells expressing EPO receptors, interaction with which EPO leads to the expression of CD86, CD40, TLR-4. However, data on the effect of EPO on the functional activity of phagocytes are contradictory. Thus, Kristal B. et al. (2008) provide evidence that EPO in patients with chronic renal failure, with an initial increase, causes a decrease in the production of superoxide anion radicals by neutrophils in vivo and ex vivo and increases the stability (lifespan) of neutrophils in vitro. Spaan M. et al. (2013) state the activation of the absorption and decrease in the killing ability of phagocytes in patients with viral hepatitis C after cultivation in a medium with the addition of EPO. We believe that such contradictory data are associated with the regulatory effect of EPO on the functional activity of phagocytes; the effect of EPO is determined by the initial level of functional activity of the cells. It is known that intracellular signal transduction after EPO binding to the receptor is provided by numerous Jak-2-dependent signaling pathways: signal transducers and transcription activators (STAT-5, STAT-3), phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB) , glycogen synthase kinase-3β (GSK-3β), mitogen-activated protein kinase (MAPK), etc. Perhaps this diversity of signaling pathways explains the ambiguous, modulating nature of the effect of EPO on the functional activity of cellular effectors of innate immunity.

Thus, the results of the study made it possible to establish that 10-minute contact of EPO with whole blood does not have a statistically significant effect on the function of phagocytes. Under experimental conditions in vitro, after a 30-minute incubation of EPO with whole blood, activation of the absorption capacity and oxygen-dependent metabolism of peripheral blood phagocytes was recorded. It was revealed that EPO in the dose range from 1.88 to 30 IU/l increases the number of actively phagocytic cells and the absorptive capacity of an individual phagocyte; at doses of 3.75 and 15 IU/l, EPO increases the number of cells generating active oxygen metabolites and the intensity of generation of active oxygen metabolites by an individual phagocyte in the induced NBT test. The effect of EPO on the functional activity of phagocytes does not depend on the dose.

Table 1. Effect of EPO on the absorption capacity of peripheral blood phagocytes after 10 min of incubation (M±m)

Experimental conditions	Activity phagocytosis, %		Phagocytic number, a.u.
Control (+ physical solution) (n=10)
EPO 1.88 IU/l (n=10)
EPO 3.75 IU/l (n=10)
EPO 7.5 IU/l (n=10)
EPO 15 IU/l (n=10)
EPO 30 IU/l (n=10)

Table 2. Effect of EPO on indicators of oxygen-dependent metabolism of peripheral blood phagocytes after 10 min of incubation (M±m)

Experimental conditions	Spontaneous NBT test		Induced NBT test
	Activity,		Activity,
Control (+ physical solution) (n=10)
EPO 1.88 IU/l (n=10)
EPO 3.75 IU/l (n=10)
EPO 7.5 IU/l (n=10)
EPO 15 IU/l (n=10)
EPO 30 IU/l (n=10)

Table 3. Effect of EPO on the absorption capacity of peripheral blood phagocytes after 10 min of incubation (M±m)

Experimental conditions	Activity phagocytosis, %	Intensity of phagocytosis, a.u.	Phagocytic number, a.u.
Control (+ physical solution) (n=10)
EPO 1.88 IU/l (n=10)
EPO 3.75 IU/l (n=10)
EPO 7.5 IU/l (n=10)
EPO 15 IU/l (n=10)
EPO 30 IU/l (n=10)

* - statistically significant (p<0,05) различия с группой контроля.

Table 4. Effect of EPO on indicators of oxygen-dependent metabolism of peripheral blood phagocytes after 10 min of incubation (M±m)

Experimental conditions	Spontaneous NBT test		Induced NBT test
	Activity,		Activity,
Control (+ physical solution) (n=10)
EPO 1.88 IU/l (n=10)
EPO 3.75 IU/l (n=10)
EPO 7.5 IU/l (n=10)
EPO 15 IU/l (n=10)
EPO 30 IU/l (n=10)

* - statistically significant (p<0,05) различия с группой контроля.

Reviewers:

Kurenkov E.L., Doctor of Medical Sciences, Professor, Head of the Department of Human Anatomy, South Ural State Medical University, Ministry of Health of Russia, Chelyabinsk.

Tseylikman V.E., Doctor of Biological Sciences, Professor, Head of the Department of Biological Chemistry, South Ural State Medical University, Ministry of Health of Russia, Chelyabinsk.

Bibliographic link

Osikov M.V., Telesheva L.F., Ozhiganov K.S., Fedosov A.A. INFLUENCE OF ERYTHROPOIETIN ON INDICATORS OF CONGENITAL IMMUNITY IN EXPERIMENTAL CONDITIONS IN VITRO // Modern problems of science and education. – 2014. – No. 1.;
URL: http://science-education.ru/ru/article/view?id=12138 (access date: 02/01/2020). We bring to your attention magazines published by the publishing house "Academy of Natural Sciences"

Cells age not only in vivo, but also in vitro. Moreover, under in vitro conditions, the role of hyperoxia, a natural and, apparently, the only factor in their aging under these conditions, is especially clearly manifested.
1.8.1. As is known, the cultivation of cells outside the body is carried out in special vessels (flasks) at atmospheric pressure and, therefore, at pO2, significantly exceeding the values ​​that are normally established in the body. Typically, the pO2 in the incubation liquid is close to the pO2 of air. O2 molecules freely diffuse through a thin layer of nutrient medium in the vial to the cells and a high pO2 is established inside them, which is impossible in vivo or, in any case, exceeds the permissible values.
From the point of view of the oxygen-peroxide concept of aging, in vitro conditions seem more than suitable for studying the process of cell aging, since under these conditions it occurs more intensely, at an accelerated pace and, very importantly, in a “pure” form, i.e. in the complete absence of any influence of body systems, which occurs during aging in vivo. This circumstance immediately places many theories of aging in the category of secondary or purely speculative, since age-related changes occur or can occur without the implementation of the provisions postulated in them. The importance we attach to the phenomenon of cell aging in vitro is due to the fact that it is under these “simple” conditions that it will be possible to understand the physicochemical foundations of aging and the essence of the biology of this process in general more quickly and with less difficulty.
At present, however, there is no consensus on the common causes and mechanisms of aging of cell cultures and aging of cells in a multicellular organism, as evidenced by opposing points of view in the literature (Kapitanov, 1986). Kanungo (1982), for example, although he believes that the reason for the aging of an organism is the aging of its cells, at the same time he believes: “in vitro conditions do not correspond to physiological ones and the properties of cells may be changed. While in vitro studies provide some useful information about the cell itself, it is of limited value when it comes to the aging of the organism as a whole.” One can only partially agree with the above statement. Indeed, cell aging in vitro cannot reflect the entire complex spectrum of age-related changes that occur in the whole organism at all levels and, moreover, are largely determined by the system of various connections in it, including feedback ones. When applied to in vitro conditions, a number of principles of aging that manifest themselves at the organismal level lose their meaning (see section 1.1.2), but some of them continue to operate in cell cultures. These are, in particular, the multifocal nature of the aging process, i.e. the development of damage in different parts of the cell or in its different molecular cycles, and heterochrony of aging among cells of the same cultured type. In addition, under these conditions, the principles of irreversibility, unregulated and continuous cellular aging should obviously manifest themselves more clearly.
The above shortcomings in the study of cell aging outside the body do not seem fundamental, if we keep in mind that one of the main tasks of gerontology is to establish the main primary environmental factor that predetermines the aging of all living organisms. Such a factor, we believe, is hyperoxia in the earth’s atmosphere, so the life of cells in vitro can be considered a convenient experimental model for studying the effect of this particular physical factor on their aging. The usual 18-21% O2 content in the air and correspondingly high levels of imbalance Δ (PO - AO) and peroxygenase processes have a depressing effect on subcellular elements, on normal physiological and metabolic processes. As a result, the latter gradually fade, and most cells die due to oxidative cytolysis or the oxygen-peroxide mechanism of apoptosis (see section 7.1).
There is more than enough evidence indicating the leading role of excess pO2, ROS and LPO in reducing cell survival in vitro and the protective effect of various antioxidant factors (Branton et al., 1998; Drukarch et al., 1998; Heppner et al. , 1998). L-carnosine has recently been classified as one of the latter. Adding physiological concentrations of it to standard media increases the lifespan of human fibroblasts in vitro and slows down the processes of physiological aging in them. Cells passaged for a long time in regular media after transferring them to a carnosine-containing medium exhibited a rejuvenating effect. The optical isomer D-carnosine did not have these properties (Halliday and McFarland, 2000). At the same time, during long-term cultivation, a certain percentage of cells not only do not degrade, but also, adapting to toxic oxidative conditions, “achieve” that the intracellular parameter Δ (PO - AO) does not increase to high values ​​of ΔA2 or ΔC, but may stop at a slightly lower level of ΔK, necessary for their malignant transformation. Cases of “spontaneous” malignancy of cells in culture and its possible mechanism are discussed separately in Chapter 4.
1.8.2. The above considerations can be considered part of our theoretical principles about the causes and consequences of cell aging in vitro. To confirm and develop these provisions, it is natural to draw on some already known facts, the content and meaning of which can easily be “fitted” into the oxygen-peroxide concept of cell aging. Let's start with the fact that the above-described conventional conditions for culturing cells, which are toxic for them, can be mitigated by artificially reducing the O2 concentration in the gas environment. At the same time, the inhibitory effect of hyperoxia and the rate of cell aging should decrease. It should also be borne in mind that such a well-known biological constant as the Hayflick limit actually turned out to be a variable value depending on the O2 content in the gas environment, and under conditions of oxidative stress this limit decreases, and with a decrease in pO2, on the contrary, it increases (Chen et al., 1995).
Indeed, keeping a fibroblast culture in an atmosphere with a low O2 content (10%) extends their lifespan by 20-30%. The same thing happens with human and mouse lung cells (Packer and Walton, 1977). The period of proliferative viability of human diploid fibroblasts IMR90 with different initial levels of population doubling increases when the O2 content in the medium decreases to 1.6 or 12%. This period at 1% O2 increases by 22%, and the return of cultures from an environment with 1% O2 to an environment with 20% O2 quickly develops their aging. In a culture of diploid fibroblasts from a patient with Werner syndrome (early aging), the duration of replicative viability also increases with a decrease in pO2 (Saito et al., 1995). A slowdown in the aging of cultured chick embryo chondrocytes was shown at an 8% O2 content in the atmosphere compared to the control (18%), and the experimental cells retained the characteristics of “young” longer and had a higher proliferation rate (Nevo et al., 1988). Under the influence of various antioxidants, the rate of proliferation of cell cultures also increases, and their aging slows down (Packer and Walton, 1977; Obukhova, 1986), which confirms what has already been said above: the clearly excessive effect of oxidants suppresses cell proliferation and causes their accelerated aging.
In experiments with cell cultures, it is also relatively easy to test the effect of the O2-dependent mechanism for regulating the number of respiratory enzymes (Murphy et al., 1984; Suzuki et al., 1998) and mitochondria (Ozernyuk, 1978). According to this mechanism, with a smooth and slow increase in the level of hyperoxia, the content of such enzymes and the number of mitochondria should gradually increase, and with hypoxia, on the contrary, they should fall. Indeed, when a fibroblast culture is grown in a medium with a reduced O2 content, the concentration of cytochromes decreases significantly (Pius, 1970). Here, of course, the objective process of adaptation of the respiratory system to the intracellular pO2 level is involved. However, in this phenomenon, the speed of adaptation is no less important, on which the intensity of aging of cultured cells will depend. It seems obvious that in the process of biological evolution, a multicellular organism adapted to the gradual increase in pO2 in the earth's atmosphere also gradually. At the same time, inside cells, the “mitochondrial” adaptation mechanism can be considered the most effective: the number of enzymes of the respiratory chain and the mitochondria themselves vary by a self-organizing system so that it ensures the integrity and relatively normal functioning of cells when intracellular pO2 changes within certain evolutionarily proven limits.
A completely different situation arises when cells are rapidly transferred from a living organism to in vitro conditions. A sharp transfer of them into a state of hyperoxia is tantamount to inflicting on them a significant abrupt disturbance, for which they, generally speaking, are not prepared. How does the primary cell culture react to such a disturbance? Apparently, during a certain initial period, the culture medium is “stressful” for the cells, and the state of the cells themselves during this period is shock. Then some time is spent on preparing and carrying out adaptive “events” of an antioxidant nature that are possible in these extreme conditions. Probably, due to the latter, at first it is possible not only to avoid oxidative degradation, but also to create conditions for stimulating the proliferative process, reducing the initially high, clearly “cytotoxic” intracellular imbalance ΔC (PO - AO) to that necessary for oxidative mitogenesis. However, this stage in the life of a primary culture cannot but be limited by the hyperoxic environment that continuously depresses it. In this situation, the adaptive mechanism itself begins to be inactivated, the growth of the antioxidant system decreases accordingly, and subsequently the regression of the latter occurs. At a high level of LPO, mitochondria are damaged first of all (see section 1.3), the number of which would continue to increase as an adaptive act in the event of a gradual increase in pO2 in the gas environment.
The inability of the cell's adaptive mechanisms to quickly and completely neutralize suddenly occurring hyperoxia, on the one hand, and the high vulnerability of the mitochondrial unit under peroxidative stress, on the other, determine the irreversible process of cell degeneration after the occurrence of a “critical level” of damage in them. It is important to note here once again: destructive changes in mitochondria as the main consumers of O2 and in this sense as the main, anti-oxygen level of protection in the cell’s antioxidant system leave no hope of survival for most cells under harsh conditions in vitro, since in this case the adapt itself is disrupted -active mechanism for reducing intracellular pO2 and LPO levels. The above considerations are fully consistent with the primary role of changes in mitochondria in the initiation of the aging mechanism, postulated, however, in relation to fibroblasts cultured in vitro (Kanungo, 1980).
Peroxidative stress and the toxic effect in vitro can be further enhanced if LPO catalysts, such as Fe2+ or Cu2+ ions, are introduced into the culture medium. Indeed, the addition of copper sulfate at a concentration of 60 mg/l to the cultivation medium led to a significant decrease in the average lifespan of rotifers by 9%, as well as to a significantly more noticeable increase in the amount of MDA than in the control. The authors of this experiment (Enesco et al., 1989) logically believe that the reduction in life expectancy occurs due to the acceleration of free radical generation processes by copper ions. The indicated concentration of copper sulfate turned out to be optimal, since higher concentrations (90 and 180 mg/l) were too toxic for rotifers, and lower concentrations (30 mg/l) were ineffective.
Thus, irreversible accelerated aging and oxidative degradation of cells during a sharp change in the environment from in vivo to in vitro are a consequence of their insufficient readiness to accept such a steep increase in oxygen exposure without serious negative consequences. If such a sharp transition to new conditions is replaced by a “soft” one, for example, multi-stage and extended over time, then we can expect that the inherent ability of cells to adapt to gradually increasing hyperoxia in this case will be fully realized. Moreover, in principle, in this way it is possible to achieve cell adaptation not only to the usual 18-21% O2 level in the atmosphere, but also to artificially created hyperoxic environments that significantly exceed it. In support of what has been said, we refer to very convincing facts obtained by Welk et al. (Valk et al., 1985). As a result of gradual adaptation to increasing O2 concentrations, they obtained a line of Chinese hamster ovary cells that is resistant to high O2 content and capable of proliferating even at 99% O2 in the atmosphere. All stages of defense - anti-oxygen, anti-radical and anti-peroxide - turned out to be adapted to such significant hyperoxia and the processes dependent on it (for more details on these results, see Chapter 4).
1.8.3. The presented considerations about the peculiarities of changes in the prooxidant-antioxidant imbalance in cultured cells as the main active factor in their aging and transformation can be roughly represented graphically (see Fig. 11). Curve 1, reflecting these changes during the rapid movement of cells into an in vitro medium, distinguishes three time-sequential stages, which seem to correspond to the adaptation (latent) phase, the logarithmic growth phase, and the stationary phase known in the literature. In this case, the aging of cell cultures is usually associated with processes in the stationary phase, where over time they undergo various changes similar to those observed in cells within an aging organism (Kapitanov, 1986; Khokhlov, 1988). In particular, during cell aging in vitro, enzymes change and their aneu- and polyploidization occurs (Re-macle, 1989). Like cells in vivo, cultured cells accumulate lipofuscin granules as they age (Obukhova and Emanuel, 1984), indicating the obvious occurrence of peroxidation processes and oxidative disorders in the structure of lipids and proteins. These and a number of other facts can one way or another be consistent with the hypothesis about the oxygen-peroxide (free radical) mechanism of aging. Most of all, this mechanism is supported by the data that when the concentration of antioxidants increases, the lifespan of cells in vitro is longer, and when it is decreased, it is shorter than in the control. Such results were obtained, for example, by changing the content of GSH in human fibroblasts (Shuji, Matsuo, 1988), catalase and SOD in cultured neurons (Drukarch et al., 1998).
As for the flat and relatively smoothly increasing curves 2 in Fig. 11, then their such nature is explained by the fact that each small artificially created increment in the pro-oxidant component of the imbalance Δ (PO - AO) in the cell is followed, with some delay, by a corresponding adaptive increment in the antioxidant component in it. Repeated repetition of this action ensures adaptation and survival of cells with a gradual, stepwise increase in the level of hyperoxia.
In both of these cases, let us pay attention to the variants leading to the so-called “spontaneous” malignancy of cells (see Chapter 4). This phenomenon, from our point of view, can be realized only in those cells where the imbalance reaches ΔK values ​​that stably satisfy the inequality (see section 1.1.2)
ΔP (PO – AO), or more precisely, taking into account “apoptotic” imbalances, – the ratio (see paragraph 7.1.1)
ΔA1 (PO - AO) With the help of such procedures, continuous lines of transformed and tumor cells are ultimately formed, capable of long-term existence outside the body. In the context of the problems we are considering, it is more important to decide on an approach to studying the relationship between aging and carcinogenesis. One of them, namely the study of the very process of the appearance of tumor cells during the aging of normal cell cultures (Witten, 1986), seems to be the most natural and therefore preferable

approach. When an imbalance of Δ (PO - AO) is established in the interval between ΔK and ΔC, cells can undergo type A2 apoptosis (see section 7.1.1).
According to the telomere theory, replicative aging of cells, including in vitro, is associated with shortening of telomeres after each mitosis, down to a certain minimum length, which results in the loss of the ability of such cells to divide (see sections 1.4.3 and 1.4 .4). An analysis of the known literature on this issue shows that this postulate is not confirmed in some cases. This is exemplified by the studies of Carman et al. (Carman et al., 1998), carried out on diploid Syrian hamster embryonic (SHE) cells. These cells, after 20-30 doubling cycles, stopped proliferation and lost the ability to enter the S-phase after stimulation with serum. At the same time, SHE cells expressed telomerase throughout the replicative life cycle, and the average telomere size did not decrease. It turns out that in vitro cells can sometimes age by mechanisms not related to telomere loss.
It seems to us that in this case, hyperoxia conditions in the cultivation environment make their own adjustments. If in a state of moderately increased levels, ROS and peroxidation often perform positive functions, activating individual stages of the mitogenic signal, replication, transcription and other processes (this was discussed in a number of previous paragraphs and is mentioned in some subsequent ones), then in the case of intense oxidative stress Negative consequences are also inevitable. For example, some macromolecules, including those involved in mitogenesis, can be modified, which, regardless of telomerase activity and telomere length, should inhibit proliferation and/or induce some other disorders, even leading to cell death.
Be that as it may, two reasons for cell aging in vitro - the accumulation of errors under conditions of keeping them in culture and shortening of telomeres - still remain the most probable. It is believed that in both cases the p53 and Rb protein systems are activated, and when their function is disrupted, cell transformation occurs (Sherr and DePinho, 2000). In a more general sense, we see the following: under toxic hyperoxic cultivation conditions, normal cells, aging, most likely undergo A1 apoptosis, and tumor cells undergo A2 apoptosis. In case of problems in the apoptosis mechanism, the former undergo neoplastic transformation, while the latter undergo oxidative cytolysis (see section 7.1.1).
An additional reason contributing to the intensification of the processes of oxidative destruction in cells in vitro can be considered heat, as a constantly operating environmental factor. Indeed, using a highly sensitive method (described by the authors Bruskov et al., 2001), it was shown that ROS are generated in aqueous solutions under the influence of heat. As a result of thermal activation of atmospheric O2 dissolved in water, a sequence of reactions occurs
О2 → 1О2 → О → HO2˙ → H2O2 → OH˙.
The resulting ROS apparently contribute to thermal damage to DNA and other biological molecules through their “auto-oxidation.”
Finally, we note another way to intensify the process of cell aging in vitro using the procedure of anoxia - reoxygenation, the results of which, in our opinion, most clearly reflect the essence of the oxygen-peroxide model of aging. The basis of the aging mechanism in this case is made up of two fundamental effects: adaptive reduction (weakening) of the mitochondrial base during the period of anoxia or hypoxia (see above); a significant increase in LPO and other processes of oxidative destruction during subsequent reoxygenation due to a sharp increase in pO2 (relative to the state of anoxia) and the impossibility of rapid utilization of excess O2 by “anoxic” mitochondria. The degree of peroxidative stress and, consequently, the rate of cell aging will depend on the duration of their stay in a state of anoxia: the longer this period, the better the mitochondrial base can adapt to low pO2 levels and the more significant the cell damage will be after the ischemia is eliminated.
An example of the implementation of cell aging according to the specified “scenario” is the following fact. Hepatocytes isolated from rats of different ages were subjected to 2-hour anoxia and 1-hour reoxygenation. It was found that in the reoxygenation phase, hepatocytes produce a large amount of oxygen radicals, which are responsible for damage to their membranes and for other structural and functional changes involved in aging, and old cells were more sensitive to reperfusion injury (Gasbarrini et al., 1998). We also consider similar facts in Chapter 4 in connection with the discussion of the mechanism of aging and “spontaneous” malignancy of cells in culture.