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Candidate of agricultural sciences tells about the possibilities of biotechnology. Dmitry Kravchenko from the All-Russian Research Institute of Potato Farming.

Growth and development under control:

possibilities of potato biotechnology 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, 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 initial plants for the original seed production of potatoes. For healing by the methods of apical meristem and accelerated propagation of culturein vitro in order to obtain the largest possible amount of improved source material for further seed production, it is necessary to optimize and activate the processes of growth and development of potato plants both under conditionsin vitro , and upon receipt of improved mini-tubers.

Why do we need to control growth processes? Let's name the main areas of work with potato culturein vitro :

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

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

- micropropagation of potato varieties in the process of original seed production;

- obtaining potato microtubers;

— long-term maintenance of collections of potato genotypes;

- breeding and genetic and other research work requiring material for their implementationin vitro .

Iona Skulachev

Consider the possibilities of controlling the growth processes of plantsin vitro at the stages of recovery and microclonal reproduction.

At the stage of obtaining regenerants from meristematic explants, indicators of the survival rate of objects, the intensity of their morphogenesis processes, 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 A.N. Belozersky (Moscow State University named after M.V. Lomonosov) preparationsSkQ (Skulachev ions) are compounds of triphenyldecylphosphonium cations and analogues of chloroplast plastoquinone. 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 in an artificial nutrient medium at a nanoconcentration of 2.5 nM improves the survival 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, there is a significant increase in morphogenic activity and intensity of growth of explants, as a result of which the regeneration time of microplants from meristem tissues is reduced by 24– 30 days.

After transplanting to a new nutrient medium, microplants from regenerants obtained usingSkQ1 , were characterized by faster growth and better biometric parameters. According to the complex of indicators, the plants obtained in the media were in the lead: for the initial growth of meristems and for the further growth of plants.

Even faster

Thus, 50 days after the isolation of the meristems when using the drugSkQ1 full-fledged plants suitable for further cuttings and testing for latent infection with viruses by ELISA were obtained. And 15–20 days after cuttings, grown 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 the isolation of the meristem to the planting of healthy plants in the soil can be reduced to 65–80 days. The quantitative yield of lines also increases, which means the probability of successful recovery.

The Phytotron TF 600 automated system makes it possible 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 for obtaining initial healthy plants is reduced by 7–14 days.

In addition, studies are currently underway on the effect of light radiation of various spectral compositions on the reduction of viral infection in plants.in vitro .

Accelerating cuttings

After receiving the improved initial regenerative plants, the next important stage of reproduction is their further reproduction. The challenge is to quickly increase the volume of starting material while maintaining high potential energy of growth and productivity, as well as the status of the absence of pathogens.

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 provide the maximum multiplication factor within the time limits set 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 ground and will give a high yield of standard mini-tubers. Therefore, different chemical regulators and different physical culture conditions can be used at different stages of micropropagation.

Provide conditions

According to the results of previous studies, we recommended the use of epin for accelerating cuttings. (synthetic epibrassinolide), which accelerates stem morphogenesis of plantsin vitro and increase 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%.

A thorough study of the new generation of growth-regulating substances synthesized in recent years has revealed a drug that, when added to an artificial nutrient medium, can increase the number of leaves of potato microplants, and hence the multiplication factor, up to 9–15 pieces. depending on the variety.

However, similar results can be achieved when cultivating plants.in vitro on a standard nutrient medium Murashige-Skoog with optimization of all physical parameters of cultivation, which can be ensured by the Fitotron TF 600 installation.

Possible, but be careful

Thus, an effective tool in the arsenal of manipulations with objects in culturein vitro was the use of biologically active substances that have a directed effect on the physiological processes and mechanisms of regulation of intracellular metabolism. However, it should be borne in mind that many biologically active substances have a pronounced mutagenic effect to some extent. They should be selected with special care and caution for those areas of work with culture.in vitro where the stability of potato varietal characteristics is most at 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), makes it possible to partially solve the problem of controlling plant growth processes.in 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 in case of damage, and EPO receptors on non-erythroid cells, in particular on various populations of leukocytes, in including phagocytes, are referred to as tissue-protecting receptors. The aim of the work is to study the effect of various concentrations of EPO on the functional activity of phagocytes under experimental conditions in vitro. It was carried out on the whole blood of 20 clinically healthy people. Recombinant human EPO in the preparation "Epokrin" (international non-proprietary name: epoetin alfa, Federal State Unitary Enterprise GNII OChB FMBA of Russia, St. Petersburg) was used at 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 nitrosine 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 found that EPO in the dose range from 1.88 to 30 IU/l increases the number of actively phagocytic cells and the absorption capacity of an individual phagocyte; at doses of 3.75 and 15 IU/l, EPO increased the number of cells generating active oxygen metabolites and the intensity of generation of active oxygen metabolites by an individual phagocyte in the induced HBT 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. - S. 36-39.

2. Osikov M.V. 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, health care, physical culture. - 2007. - No. 16 (71). - S. 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. - S. 143-145.

4. Osikov M.V. 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. - S. 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. - S. 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. - S. 7-13.

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

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

9. Osikov M.V. Erythropoietin as a regulator of the expression of platelet glycoproteins / M.V. Osikov, T.A. Grigoriev, A.A. Fedosov, D.A. Kozochkin, M.A. Ilinykh // 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.

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 can be a key link in the pathogenesis of various diseases and typical changes in homeostasis. So, in chronic renal failure, activation of the innate immunity and the associated manifestation of local and systemic inflammation contribute to the development and progression of cardiovascular diseases; in 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 immunity effectors. Previously, we have demonstrated the role of biologically active substances of the endogenous nature of ceruloplasmin and alpha-1-acid glycoprotein in the regulation of phagocyte function in various pathologies. In recent years, the attention of many researchers has attracted the pleiotropic effects of erythropoietin (EPO). EPO was first 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-induced factor-1 (HIF-1). Modern ideas about the mechanisms of action of EPO at the molecular level allow us to attribute it simultaneously to hormones, growth factors, and cytokines. The main point of application for the action of EPO are cells of the erythroid series in the bone marrow: burst- and colony-forming units of granulocyte-monocyte-megakaryocytic-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 EPO receptors on cells of non-erythroid tissues such as neurons, cardiomyocytes, kidney cells, and endotheliocytes made it possible to discover new biological effects of EPO. Previously, we have shown 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 hemostasis system, etc. . We believe that the indirect implementation of the pleiotropic effects of EPO may be associated with its effect on the function of phagocytic cells. At present, the EPO-EPO receptor system at the auto- and paracrine levels is considered as a link in nonspecific protection in case of damage, and EPO receptors on non-erythroid cells are designated as tissue-protecting receptors. Goal of the work- to investigate the effect of various concentrations of EPO on the functional activity of phagocytes under experimental conditions in vitro.

Materials and methods of research

The work was performed using whole blood from 20 clinically healthy human donors. Recombinant human erythropoietin in the preparation "Epokrin" (international non-proprietary name: epoetin alfa, Federal State Unitary Enterprise GNII OCHB FMBA of Russia, St. Petersburg) was used at 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 parameters were studied after 10 minutes and 30 minutes of incubation in a thermostat at 37 °C. The function of phagocytes was studied by phagocytic capacity 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 cell suspension was mixed with 20 μl of a suspension of particles of polystyrene latex. After 30 min incubation at a temperature of 37°C, the activity and intensity of phagocytosis and the phagocytic number were evaluated. The assessment of intracellular oxygen-dependent metabolism in phagocytes was carried out using the NST-test, which is based on the formation of insoluble diformazan from nitrosine tetrazolium. Spontaneous and induced NBT-test were carried out. To perform a spontaneous NBT test, 50 µl of physiological saline and 20 µl of nitrosine tetrazolium were added to 100 µl of blood; in the induced NBT test, 50 µl of a suspension of polystyrene latex in physiological saline and 20 µl of nitrosine tetrazolium 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 estimated in relation to the area of ​​the nucleus (single dusty granules - 0; cells with deposits, not exceeding 1/3 of the nucleus area in total - 1 ; cells with diformazan deposition of more than 1/3 of the nucleus area - 2; 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, a correlation analysis was used with the calculation of the Spearman correlation coefficient. Differences were considered statistically significant at p<0,05.

Research results and discussion

The results of the effect of EPO on the functional activity of phagocytes after 10 min of incubation at 37°C are presented in Table. 1 and 2. As can be seen, we did not record statistically significant changes in the absorptive capacity and oxygen-dependent metabolism of peripheral blood phagocytes. It should be noted that, as a trend, the activity, intensity of phagocytosis, phagocytic number, indicators of spontaneous and induced NBT-test increased; The highest mean values ​​were observed with the addition of EPO at a dose of 30 IU/l (200% of the physiological level in serum). It has been established that a 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 the 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 mean value in the control group, was noted 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 correlation coefficient R=0.21; p>0.05), intensity of phagocytosis (Spearman 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 spontaneous HBT test parameters (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 a single cell, increase.

According to other researchers, receptors for EPO were found on leukocytes, for example, using flow cytometry and reverse polymerase chain reaction, the expression of the gene and mRNA of the EPO receptor in T- and B-lymphocytes and monocytes was detected. A group of researchers from the Transplant Center in Bergamo (Italy) believe that one of the targets for the immunomodulatory action of EPO is dendritic cells expressing EPO receptors, interaction with which EPO leads to the expression of CD86, CD40, TLR-4. At the same time, data on the effect of EPO on the functional activity of phagocytes are contradictory. So, 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 radical by neutrophils in vivo and ex vivo and increases the stability (life span) of neutrophils in vitro. Spaan M. et al. (2013) state the activation of the absorption capacity and the decrease in the killing ability of phagocytes in patients with viral hepatitis C after cultivation in a medium supplemented with EPO. We believe that such conflicting 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 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 (PKV) , glycogen synthase kinase-3β (GSK-3β), mitogen-activated protein kinase (MAPK) and others. Perhaps, such a variety 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 a 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 absorptive capacity and oxygen-dependent metabolism of peripheral blood phagocytes was recorded. It was found that EPO in the dose range from 1.88 to 30 IU/l increases the number of actively phagocytic cells and the absorption capacity of an individual phagocyte; at doses of 3.75 and 15 IU/l, EPO increased the number of cells generating active oxygen metabolites and the intensity of generation of active oxygen metabolites by an individual phagocyte in the induced HBT test. The effect of EPO on the functional activity of phagocytes does not depend on the dose.

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

Experiment conditions	Activity phagocytosis, %		Phagocytic number, c.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 parameters of oxygen-dependent metabolism of peripheral blood phagocytes after 10 min of incubation (M±m)

Experiment conditions	Spontaneous HCT test		Induced HCT 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 absorptive capacity of peripheral blood phagocytes after 10 min of incubation (M±m)

Experiment conditions	Activity phagocytosis, %	Intensity of phagocytosis, c.u.	Phagocytic number, c.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 parameters of oxygen-dependent metabolism of peripheral blood phagocytes after 10 min of incubation (M±m)

Experiment conditions	Spontaneous HCT test		Induced HCT 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, Chelyabinsk.

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

Bibliographic link

Osikov M.V., Telesheva L.F., Ozhiganov K.S., Fedosov A.A. INFLUENCE OF ERYTHROPOIETIN ON INDICATED IMMUNE INDICATORS UNDER IN VITRO EXPERIMENTAL CONDITIONS // Modern problems of science and education. - 2014. - No. 1.;
URL: http://science-education.ru/ru/article/view?id=12138 (date of access: 01.02.2020). We bring to your attention the journals published by the publishing house "Academy of Natural History"

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 of 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, consequently, at pO2, which is much higher than the values ​​that are normally established in the body. Usually in the incubation liquid the pO2 is close to the pO2 of the air. O2 molecules freely diffuse to the cells through a thin layer of nutrient medium in a flask, 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 to be more than suitable for studying the process of cell aging, since under these conditions it proceeds more intensively, at an accelerated pace and, which is very important, 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 puts many theories of aging into the category of secondary or purely speculative, since age-related changes occur or can occur even without the implementation of the provisions postulated in them. The fact that we attach such importance 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 quickly and with less difficulty understand the physicochemical foundations of aging and the essence of the biology of this process in general.
At present, however, there is no consensus on the commonality of the causes and mechanisms of aging of cell cultures and aging of cells in a multicellular organism, as evidenced by opposite points of view in the literature (Kapitanov, 1986). Kanungo (Kanungo, 1982), for example, although he believes that the cause of 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, they are of limited value when it comes to aging in general.” One can only partly agree with the above statement. Indeed, cell aging in vitro cannot reflect the entire complex spectrum of age-related changes occurring in the whole organism at all levels and, moreover, to a large extent determined by the system of various connections in it, including reverse ones. With regard 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. Such, in particular, are 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 the heterochrony of aging among cells of the same cultured type. In addition, under these conditions, the principles of irreversibility, uncontrollability, and continuity of cell aging should obviously manifest themselves more clearly.
The above shortcomings in the study of cell aging outside the body do not seem to be fundamental, if we keep in mind that one of the main tasks of gerontology is to establish the main primary environmental factor that determines the aging of all living organisms. We believe that such a factor is hyperoxia in the Earth's atmosphere; therefore, the life of cells under in vitro conditions can be considered a convenient experimental model for studying the effect of this particular physical factor on cell aging. The usual 18-21% content of O2 in the air and, accordingly, 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 through the oxygen-peroxide mechanism of apoptosis (see Section 7.1).
There are more than enough facts indicating the leading role of excess pO2, ROS, and LPO in reducing cell survival under in vitro conditions and the protective effect of various antioxidant factors (Branton et al., 1998; Drukarch et al., 1998; Heppner et al. , 1998). Recently, L-carnosine has also been included among 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 on ordinary media for a long time after being transferred to a carnosine-containing medium showed a rejuvenating effect. The optical isomer of D-carnosine did not possess the indicated properties (Hallyday and McFarland, 2000). At the same time, during long-term cultivation, a certain percentage of cells not only does not degrade, but, adapting to toxic oxidative conditions, “achieves” that the intracellular parameter Δ (PO - AO) does not rise to high values ​​of ΔA2 or ΔC, but can stop at a slightly lower level of ΔK, which is necessary for their malignant transformation. Cases of “spontaneous” cell malignancy in culture and its possible mechanism are discussed by us separately in Chapter 4.
1.8.2. The above considerations can be considered part of our theoretical propositions on 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 "inscribed" in the oxygen-peroxide concept of cell aging. Let's start with the fact that the above-described usual conditions for culturing cells, which are toxic for them, can be mitigated by artificially reducing the concentration of O2 in the gaseous medium. In this case, 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, in fact, turned out to be a variable value depending on the O2 content in the gaseous medium, and this limit decreases under conditions of oxidative stress, and, on the contrary, increases with a decrease in pO2 (Chen et al. al., 1995).
Indeed, the presence of a culture of fibroblasts in an atmosphere with a low content of O2 (10%) lengthens their lifespan by 20-30%. The same happens with human and mouse lung cells (Packer and Walton, 1977). The period of proliferative viability of diploid human IMR90 fibroblasts with different initial levels of population doubling increases with a decrease in the O2 content in the medium to 1.6 or 12%. This period at 1% O2 increases by 22%, and the return of cultures from the medium with 1% O2 to the medium with 20% O2 rapidly develops their aging. In a culture of diploid fibroblasts from a patient with Werner's syndrome (early aging), the duration of replicative viability also increases with a decrease in pO2 (Saito et al., 1995). Slowing down the aging of cultured chick embryo chondrocytes was shown at 8% O2 content in the atmosphere compared to the control (18%), and the experimental cells retained the signs of “young” for longer and had a higher proliferation rate (Nevo et al., 1988). Under the influence of various antioxidants, the proliferation rate of cell cultures also increases, and their aging slows down (Packer and Walton, 1977; Obukhova, 1986), which confirms what has already been said above: a clearly excessive action of oxidants suppresses cell proliferation and causes their accelerated aging.
In experiments with cell cultures, it is also relatively easy to verify the action of the O2-dependent mechanism of regulation of the amount 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, while during hypoxia, on the contrary, they should decrease. Indeed, when cultured fibroblasts are grown on a medium with a low O2 content, the concentration of cytochromes is significantly reduced (Pius, 1970). Here, of course, the objective process of adaptation of the respiratory system to the intracellular level of pO2 is involved. However, in this phenomenon, the rate of adaptation is of no less importance, on which the intensity of aging of cultured cells will also depend. It seems obvious that in the process of biological evolution the multicellular organism adapted to the gradual increase in pO2 in the earth's atmosphere also gradually. At the same time, inside the cells, the “mitochondrial” mechanism of adaptation can be considered the most effective: the number of respiratory chain enzymes and the mitochondria themselves varies by a self-organizing system so that it ensures the integrity and relatively normal functioning of cells with changes in intracellular pO2 within certain evolutionarily approved limits.
A completely different situation develops 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 spasmodic perturbing effect, for which, generally speaking, they 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 cells, and the state of the cells themselves during this period is shock. Then, some time is spent on preparing and carrying out adaptive “measures” of an antioxidant nature, which are possible under 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 of ΔC (PO – AO) to the level necessary for oxidative mitogenesis. However, even this stage in the life of primary culture cannot but be limited by the hyperoxic environment that continuously oppresses it. In this situation, the adaptive mechanism itself begins to be inactivated, and accordingly, the build-up of the antioxidant system decreases, and subsequently the latter regresses. At a high LPO level, first of all, mitochondria are damaged (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 a gaseous environment.
The inability of the adaptive mechanisms of the cell to quickly and completely neutralize sudden hyperoxia, on the one hand, and the high vulnerability of the mitochondrial link to peroxidative stress, on the other hand, determine the irreversible process of cell degeneration after the occurrence of a “critical level” of damage in them. It is important to note here again: destructive changes in mitochondria as the main consumers of O2 and, in this sense, as the main anti-oxygen defense step in the antioxidant system of the cell do not leave hope for survival for most cells under harsh conditions in vitro, since in this case adapt itself is upset. -tive mechanism for reducing intracellular pO2 and LPO levels. These considerations are fully consistent with the primary role of mitochondrial changes in the initiation of the aging mechanism, postulated, however, in relation to fibroblasts cultivated in vitro (Kanungo, 1980).
Peroxidative stress and toxic effect under in vitro conditions 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 specified concentration of copper sulfate turned out to be optimal, since the higher concentrations (90 and 180 mg/l) were too toxic for rotifers, and the lower one (30 mg/l) was ineffective.
Thus, the irreversible accelerated aging and oxidative degradation of cells during a sharp change in habitat from in vivo to in vitro are the result 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, multistage and extended in time, then it can be expected that the ability inherent in cells to adapt to gradually increasing hyperoxia in this case is 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 are significantly higher than it. In support of what has been said, we refer to the very convincing facts obtained by Welk et al. (Valk et al., 1985). As a result of gradual adaptation to increasing O2 concentration, they obtained a Chinese hamster ovary cell line that is resistant to high O2 content and capable of proliferating even at 99% O2 in the atmosphere. To such a significant hyperoxia and processes dependent on it, all stages of protection turned out to be adapted - anti-oxygen, anti-radical and anti-peroxide (for more details about these results, see Chapter 4).
1.8.3. The above considerations about the features of changes in the prooxidant-antioxidant imbalance in cultured cells as the main active factor in their aging and transformation can be conditionally represented graphically (see Fig. 11). On curve 1, which reflects the indicated changes during the rapid movement of cells into the medium in vitro, three successive stages are distinguished in time, which seem to correspond to the adaptive (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 of an aging organism (Kapitanov, 1986; Khokhlov, 1988). In particular, enzymes change during cell aging in vitro, and their aneu- and polyploidization occurs (Remacle, 1989). Like cells in vivo, cultured cells accumulate lipofuscin granules with aging (Obukhova and Emanuel, 1984), indicating the obvious occurrence of peroxide processes and oxidative disturbances in the structure of lipids and proteins. These and a number of other facts, one way or another, can be consistent with the hypothesis of the oxygen-peroxide (free radical) mechanism of aging. Most of all, this mechanism is supported by data that, with an increase in the concentration of antioxidants, the lifespan of cells in vitro is longer, and with a decrease, it is shorter than in the control. Such results were obtained, for example, by changing the content of GSH in human fibroblasts (Shuji and 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, this nature of them is explained by the fact that each small artificially created increment of the prooxidant component of the imbalance Δ (PO - AO) in the cell is followed, with some delay, by the corresponding adaptive increment of the antioxidant component in it. Repeated repetition of this action ensures the adaptation and survival of cells with a gradual, stepwise increase in the level of hyperoxia.
In both of these cases, let's pay attention to the options 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 consistently satisfy the inequality (see clause 1.1.2)
ΔP (PO - AO), or rather, taking into account "apoptotic" imbalances, to the ratio (see paragraph 7.1.1)
ΔA1 (PO - AO) With the help of such procedures, ultimately, transplantable lines of transformed and tumor cells are formed, capable of long-term existence outside the body. In the context of the problems we are considering, it is more important to determine the approach to the study of 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 telomeric theory, replicative cell aging, including in vitro conditions, is associated with the shortening of telomeres after each mitosis, up to a certain minimum length, resulting in the loss of the ability for 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. An example of this is the study of Karman et al. (Carman et al., 1998) carried out on diploid embryonic cells of the Syrian hamster (SHE). These cells stopped proliferating after 20-30 doubling cycles and lost the ability to enter the S-phase after serum stimulation. At the same time, SHE cells expressed telomerase throughout the entire replicative life cycle, and the average telomere size did not decrease. It turns out that in vitro cells can sometimes age by mechanisms that are not associated with the loss of telomeres.
It seems to us that in this case, the conditions of hyperoxia in the cultivation medium make their own adjustments. If in a state of a moderately elevated level of ROS and peroxidation often perform positive functions, activating individual stages of the passage 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 inevitable and negative consequences. 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 disturbances, up to and including cell death.
Be that as it may, the two causes of cell aging in vitro—accumulation of errors under conditions of their maintenance in culture and shortening of telomeres—remain the most probable. It is believed that in both cases, the p53 and Rb protein systems are activated, and when their function is impaired, cell transformation occurs (Sherr and DePinho, 2000). More generally, we see the following: under toxic hyperoxic conditions of cultivation, normal cells, aging, most likely undergo A1 apoptosis, and tumor cells, A2 apoptosis. In the event of malfunctions in the mechanism of apoptosis, the former undergo neoplastic transformation, while the latter undergo oxidative cytolysis (see section 7.1.1).
An additional reason contributing to the intensification of oxidative degradation processes in cells in vitro can also be heat, as a constantly acting environmental factor. Indeed, using a highly sensitive method (described by the authors of Bruskov et al., 2001), it was shown that ROS are generated in aqueous solutions under the action of heat. As a result of thermal activation of atmospheric O2 dissolved in water, a sequence of reactions occurs
O2 → 1O2 → O → HO2˙ → H2O2 → OH˙.
The formed ROS, apparently, contribute to the thermal damage of DNA and other biological molecules by their "autoxidation".
Finally, we note another way to intensify the cell aging process under in vitro conditions using the anoxia-reoxygenation procedure, the results of which, in our opinion, most clearly reflect the essence of the oxygen-peroxide model of aging. The aging mechanism in this case is based on two fundamental effects: adaptive reduction (weakening) of the mitochondrial base during anoxia or hypoxia (see above); a significant increase in lipid peroxidation 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 will be able to adapt to a low level of pO2 and the more significant will be the damage to cells after ischemia is eliminated.
The following fact can serve as an example of the implementation of cell aging according to the indicated “scenario”. Hepatocytes isolated from rats of different ages were subjected to 2-hour anoxia and 1-hour reoxygenation. It has been established that in the reoxygenation phase, hepatocytes produce a large amount of oxygen radicals responsible for damage to their membranes and other structural and functional changes associated with aging, and old cells were more sensitive to reperfusion injury (Gasbarrini et al., 1998). Similar facts are considered by us in Chapter 4 in connection with the discussion of the mechanism of aging and "spontaneous" malignancy of cells in culture.

ROOTING IS A KEY STAGE IN IN VITRO PLANT PRODUCTION

IN AND. DEMENKO, K.A. SHESIBRATOV, V.G. LEBEDEV

(Department of Fruit Growing)

Izvestia TSHA magazine, issue 1, 2010

The article presents the results of many years of research on the effect of various growth regulators, carbohydrates, passage duration, agar quality, medium structure, nutrient medium salt composition on root formation. The stages of rhizogenesis and their duration are discussed. Particular attention is paid to the problem of etiolation, physical factors, hormonal and salt composition of the nutrient medium. It contains important and reliable information about the influence of light, the composition of the environment on rhizogenesis, plant life and non-sterile conditions.

Keywords: in vitro, growth regulators, nutrient medium, juvenile, etiolation, carbohydrates, strawberry, apple tree, pear, lilac, rose, Chinese actinidia, common ash.

The process of root formation is a series of different biochemical, physiological and histological events. Proximity to vascular tissues predisposes the cells to lay down root primordia. The location of the roots affects the viability of rooted plants, especially obtained in vitro. It is possible to obtain 100% in vitro rooting and 100% plant death under non-sterile conditions. With any rooting methods, the process of adventitious root formation goes through 3-4 stages: induction, initiation, the appearance of roots outside the stem part of the cutting. The duration of the first two stages is 10-15 days; during this period, precompetent cells acquire the ability to regenerate meristematic foci, and the synthesis of root-specific proteins begins in them. The appearance of roots depends on the plant genome and rooting conditions.

One of the features of in vitro plant propagation is tissue juvenilization, which is the reason for the ease of in vitro rhizogenesis. The degree of its manifestation depends on the conditions of cultivation, and primarily on the content of ethylene in the vessels.

A prerequisite for the start of root formation in any method of reproduction is etiolation. The reason for the influence of this phenomenon is associated with anatomical and biochemical changes, as well as tissue juvenileization. In etiolated cuttings, receptor proteins with high affinity for auxin were isolated. In the dark, most of the auxin is protein bound. Etiolation increases the activity of peroxidase, IAA - oxidase in tissues, which accelerates the onset of root formation. It enhances the sensitivity of tissues to exogenous auxin, making it possible to use lower concentrations. The nature of the action of etiolation in vitro depends on the type of plant and has a prolonging effect. Rooting in vitro occurs when the entire cutting is illuminated, which affects rhizogenesis and can be partially leveled by IBA, which stimulates ethylene synthesis to a lesser extent compared to IAA. The main problem of successful in vitro rhizogenesis lies in the difficulty of separating the moment of initiation of the first root primordia from the beginning of ethylene synthesis. At the same time, very high or very low levels of ethylene negatively affect rooting. Light plays an important role in the regulation of morphogenetic processes. Irradiation of already formed roots with light suppresses root elongation by 40-50%, due to a 4-fold increase in ethylene content. For species that are difficult to root, it is recommended to use a dark period at the initial stage of rooting. Its duration depends on the culture and ranges from 3~5 days to 4 weeks. A number of authors note the complex dependence of rooting on the light regime, growth regulators, and pH of the medium. The positive effect of etiolation at the stage of shoot proliferation depends on the variety. The use of auxins and darkness in the last week of M27 shoot proliferation reduced rooting by 65%.

The action of enzymatic systems that catalyze the destruction of IAA is carried out only in the presence of oxygen. Nutrient media used for rooting shoots in vitro contain very little oxygen, which does not promote the development of root hairs.

Therefore, in vitro rooting approaches must be different from in vivo rooting. Modern industrial reproduction of plants in vitro is impossible without the use of growth regulators. As a rule, analogues of IAA are used for rooting. The attitude to exogenous auxin, time and method of its use in vitro is ambiguous. A sufficient number of established roots and a good survival rate of plants under non-sterile conditions were obtained by exposure for 7 days on a medium with auxin. Longer exposure results in callus formation. Shoots of easily rooted crops can be rooted on media without auxins. However, auxins are often not a limiting factor for species that are difficult to root.

In addition to substances from the auxin group, the stimulating effect of retardants on rooting and the nature of root development is noted. Some of them activate the transport of IAA and carbohydrates to the basal part of the cutting. The in vitro rooting technique makes it possible to control physical factors, hormonal and salt composition of the nutrient medium. At the same time, illumination of the base of the shoot, long-term exposure to auxin, heterogeneity of shoots, insignificant closed volume, absence or insufficiently intense gas exchange, its specificity, insufficient oxygen in the rooting zone, possible latent vitreousness of shoots, lack of transpiration, photosynthesis and exposure to ultraviolet radiation create problems for rooting and subsequent survival of plants in non-sterile conditions. Optimization of these factors and their interactions is the main goal of in vitro studies of rhizogenesis. In the vast majority of in vitro rooting experiments, researchers note the importance of the osmotic potential of the medium, which depends on the concentration of sucrose, salt composition, especially nitrogen and potassium. As a rule, the mineral composition of the M.S. reduced by 2-8 times or replaced with White's medium. In this case, the leading role is given to the content of nitrate and ammonia nitrogen. The absence of one form or another of nitrogen negatively affects the rooting of shoots.

Shoots of fruit crops suitable for rooting are formed only after long-term cultivation. Even at the 11th-14th passage, the rooting of hard-to-root plant species is a serious problem. However, very long passaging is undesirable, especially for species whose vegetative offspring is intended for perennial plantations.

It is considered promising to root shoots obtained in vitro in sterile substrates at high humidity or in an atmosphere of artificial fog. On the ability to root in vitro, a wide range of data is presented in the scientific literature - from easily rooted species (strawberry) to no rooting (kallarian pear). Therefore, it is not surprising that many authors consider the successful establishment of shoots in vitro as a key step in micropropagation.

Materials and Methods

The ability to root in vitro strawberry, apple, pear, lilac, roses, Chinese actinidia, common ash was studied: using various growth regulators (IAA, IMC, NAA, kultar, mival. krezatsin, sodium humate, silver nitrate); carbohydrates (sucrose, glucose, maltose, fructose, sorbitol, mannitol); Surfactant (tween-40, KEP); the influence of passage duration and quality of shoots, agar and its substitutes, the structure of the medium and the impact of physical factors, the salt composition of the nutrient medium. Plants were grown according to generally accepted methods, taking into account the specifics of the rooting stage. The beginning of root formation, callus development, the number of roots, and shoot growth were taken into account. In each variant, 10-14 plants in four repetitions.

Results and its discussion

The ability of lateral shoots to root in vitro largely determines the effectiveness of micropropagation technology. This is the most expensive item (75% of manual labor costs) in the cost of the final product. Successful passage of the rhizogenesis stage depended on the culture, variety, conditions of the proliferation stage, salt and hormonal composition of the medium, and the number of passages. Only strawberry shoots were capable of rooting in the first passage, and Robinia and weigela in the second passage. The rooting of strawberries depended on the salt composition of the medium and its shape. Fossard's medium is best for rooting strawberries. The root system on this medium was characterized by strong growth, was colored and had lateral roots. Plants on Fossord's medium were more developed, had 4-5 dark green leaves. Callus development was absent. Less concentrated media reduced rooting and inhibited the development of the root system.

Wednesday M.S. stimulated the rooting and development of callus at the base of the shoot, and the presence of callus is undesirable, since when plants are transplanted into non-sterile conditions, they die from botrytis. Fusarium and Pythium. Reduction in the environment of M.S. macronutrients or nitrogen by 1/2 or 4/5 increased the rooting of almost all studied crops, and root formation occurred at lower concentrations of auxin. On the complete MS medium containing 0.5 mg/l of IBA, thick, short roots developed. On poor media, the roots were filiform and developed from the base of the shoot.

The nature of the development of the root system depended on the type of growth regulator that induces root formation. The stimulating effect of IBA and IAA in experiments with strawberries was more pronounced on solid media, and NUK - on liquid media. A more powerful root system was formed on a liquid medium containing NAA. However, the development of the above-ground system did not take place, which is associated with the ability of NAA to be absorbed and move through plant tissues. Intensive callus development was noted on all media, especially on media with NAA. Taking into account the shortcomings of auxin at the stage of rooting in vitro, it is necessary to search for other substances that would stimulate the rapid development of root and drought resistance of above-ground systems. It is known from literary sources that such properties are possessed by a cultivar that has proven itself well on intact plants with green cuttings (Table 1).

With an increase in the cultarian concentration in the medium, the growth of the above-ground and root systems, which were characterized by intensive radial growth, was inhibited. The use of cultar in the concentration range of 0.3-0.5 mg/l also inhibited the development of the aboveground system, but to a lesser extent. Callus development was not observed in any of the variants. The concentration of the cult over 1 mg/l caused the death of the shoots. Rooting of shoots in vitro is possible only at a certain length. None of the substances tested promoted the development of roots directly from the meristematic apex, i.e. stretching growth is needed at the beginning, and then roots begin to develop. The introduction of a culture medium (0.1-0.2 mg/l) completely inhibited the growth of meristematic tops by stretching and caused the development of their root system. Some of the roots were dyed green, i.e. the root system assumed the function of a photosynthetic organ in the absence of growth of the above-ground system. The culture at a concentration of 0.5 mg/l stimulated the development of roots of the second, and in some cases, the third order in lilacs.

In order to eliminate the negative effect of auxins on the process of root formation in vitro, substances from the group of organosilicon compounds (mival, krezacin, CEP) and sodium humate were tested. The use of mival and krezacin significantly influenced the rooting and development of the root system of strawberries, actinidia sinensis, pears. The optimal concentrations of the tested substances are in the range of 0.1-2 mg/l, in which all parameters of the development of the root system change. The percentage of rooting, the number of roots and their length increase (Table 2).

The rooting rate of strawberry shoots when combined with organosilicon compounds with other root formation inducers depended on their concentrations. Rooting was completely inhibited if the concentration of mival and krezacin exceeded 2 mg/l in combination with IBA and when they were jointly introduced into the nutrient medium.

A similar effect of the combined use of BCI with organosilicon compounds was noted in experiments with a pear. The stimulating effect of krezacin is manifested in a narrower range of concentrations (0.1-0.2 mg / l), and mival at concentrations of 0.1-0.5 mg / l.

It can be assumed that mival has an auxin-like effect, but does not stimulate callus development. At low concentrations of mival and krezacin, a synergism of action with IMC on the processes of growth and development of strawberries in vitro is observed - the height of the plant increases due to the length of shoots and leaves.

The separate use of mival and cultara in low concentrations contributed to 100% rooting of strawberries; combined use at high concentrations completely inhibited root formation. Actinidia sinensis, in contrast to strawberry, maximized the development of roots from the combined use of IBA with Mival, while the concentration of IBA also exceeded the concentration of Mival. The combined action of mival and krezacin inhibited the rooting of this culture. Obviously, actinidia requires a higher concentration of auxin at the induction stage compared to strawberries. Therefore, high concentrations of IBA stimulated actinidia root formation to a greater extent if it was used separately.

Sodium humate and silver nitrate significantly accelerated the passage of the stages of rooting strawberry shoots. Their use at a concentration of 0.5-1 mg/l reduced the time of shoot rooting, accelerated the production of plants suitable for transplanting into non-sterile conditions. The data of our experiments with silver nitrate and methionine give us grounds to assert that, at the first stage of rooting, significant ethylene synthesis inhibits root formation. The introduction of methionine into the nutrient medium completely suppressed rhizogenesis in the variants with IMC and Mival and significantly in the variants of the combined action of root formation inducers. It is obvious that methionine increases the content of ethylene in the phase of root induction to an inhibitory level.

At all stages of micropropagation in nutrient media, carbohydrates are used, mainly sucrose, as the most mobile carbohydrate. Carbohydrates in tissue culture are important not only as nutrients, but also as substances that, together with the salt composition, create a certain osmotic pressure. The value of carbohydrates increases at the rooting stage due to the need to prepare the plant for autotrophic nutrition under non-sterile conditions. The concentration of carbohydrates sucrose, fructose, glucose 10 g/l is insufficient for the rooting of strawberries. Mannitol and sorbitol, which have no nutritional value, but have the ability to influence the osmotic pressure, also did not contribute to the formation of roots. The appearance of the first roots on the ash shoots was noted on the 9-10th day after planting on the medium for rooting. The effect of the tested carbohydrates depended on the growth regulators used (Table 3).

Various carbohydrates did not have a significant effect on the rooting frequency: in the variant with NAA, it was 86–93%, and in the variant with IMC, it was 55–71%. However, the number of roots and their length differed significantly: the maximum values ​​were noted on the medium with 30 g/L sucrose or 10 g/L glucose. The roots on the medium with maltose were 2.4 times shorter than those on the medium with sucrose, and 3.2 times shorter than on the medium with glucose, while the plants lag behind the plants on the media with sucrose and glucose.

Growth and development of roots in vitro depend on the aeration of the nutrient medium, which, in turn, depends on the concentration of agar. Rooting of shoots in a dense medium is difficult, the development of second-order roots does not occur. With a decrease in the concentration of agar, the rooting of shoots and the development of second-order roots increase significantly.

However, with long-term development of plants on media with a low content of agar (1.5-2.5 g/l), they show signs of vitreousness. On nutrient media containing even small concentrations of agar, root hairs never develop, which indicates an insufficient oxygen content. Taking into account the importance of oxygen content during the initiation and development of roots and root hairs, attempts were made to replace agar at the rooting stage. When using the full environment of M.S. and substitutes for agar shoots died within a few days after planting. The death of shoots and plants that began to develop roots on the medium 1/2 M.S. and agar substitutes (perlite, sand), was associated with the drying of the upper layer of the substrate.

Agar stimulated earlier root formation. Perhaps it increases the diffusion of substances from the nutrient medium, and also helps the diffusion of substances from the explant that inhibit root formation. Most of the rooted shoots in the variants with agar substitutes took root in non-sterile conditions, but their growth was then weak. Combining agar with perlite resulted in a well-developed root system of strawberries, which ensured the successful establishment of plants in non-sterile conditions. The combination of perlite with agar makes it possible to reduce the concentration of agar by 3 times. After autoclaving and solidification of the medium, two layers are formed in the rooting vessels: at the top, perlite impregnated with the medium; at the bottom, an agar medium interspersed with perlite particles (Table 4).

The ratio of perlite and nutrient medium with agar 0.5-1:1 by volume was optimal. The use of such an environment for rooting pears did not give positive results.

The existing method of rooting lateral shoots of a large number of species using auxins is possible only after a long passage. The positive effect on reproduction may be due to the juvenilization of plant material. The possibility of a stimulating effect of etiolation is not excluded, since with an increase in the number of passages, the number of side shoots increases, the base of which, as a rule, is etiolated. Spur forms of the apple tree and clonal rootstock 62-396 began to take root well from the 12th-13th passage. Their rooting depended on the salt composition of the nutrient medium. The rooting of woody plants at later passages creates biological and organizational problems, which are partly solved by storing cultures at the proliferation stage at low temperatures. However, the effect of this factor may have negative consequences on rooting (the effect of etiolation and low positive temperatures). In this regard, we studied the effect of lighting conditions on the rooting of lateral shoots of apple trees in vitro (Table 5). The results of the experiments revealed a certain relationship between the influence of light, low temperature, growth regulators and rooting of shoots.

When using IMC as an inducer of root formation, the rooting of shoots increased by 14–30% if proliferating crops were exposed to darkness, especially in combination with low positive temperatures. The absence of light at the stage of reproduction significantly reduced rooting in the variant with a cultivator and mival, especially in the variant with a cultivator. Low positive temperatures reduced the inhibitory effect of etiolation in these variants.

If the induction of rooting (15 days) took place under conditions of etiolation, then rooting decreased by 10-64%, depending on the inducer of root formation. As already noted, auxins synthesized by explants play the main role in rooting. They are necessary at the stage of induction of root formation, which lasts 6-10 days. The absence of light during storage and rooting stimulates root formation only in the presence of IMC.

Reducing the dark period to 5-10 days increased the rooting rate of shoots of rootstock 62-396 by 24% when using IMC. The storage of strawberry conglomerates at low positive temperatures before the rooting of the shoots affected the rate of passage through the stages of root formation (Table 6).

The aftereffect of low positive temperatures depended on the inductor of root formation. IMC accelerated, and cultar slowed down the passage of the stages of root formation under such conditions. Numerous experiments on green cuttings convincingly show that the rooting of cuttings is strongly inhibited if they are placed completely in darkness (it is only necessary for the base of the cutting). Taking into account this reaction of the cuttings to the absence of light, we tested various methods of shading the base of the shoot in vitro. Rootstock shoots 62-396 were planted in a foil capsule filled with a nutrient medium, or activated carbon was introduced into the nutrient medium. The highest percentage of rooting (86%) was noted in the variant using the capsule. The introduction of activated carbon into the medium reduced rooting by 35%. It is known that 1 mg of activated charcoal can absorb 100 µg of 6BAP and NAA. Activated charcoal contains phenolamides, which can adversely affect rooting. Activated carbon absorbs ethylene, which has an ambiguous effect on the processes of rhizogenesis. The use of the rooting capsule stimulated the intensive growth of the root system in length, the development of second-order roots. However, the use of the capsule is not technologically advanced. We have developed a compositional medium that significantly improves the rooting of the apple tree due to the effect of etiolation. A distinctive feature of such a medium from the standard one is the placement of a medium layer (2–3 mm) consisting of water, agar, and activated charcoal (2–5%) on a standard rooting or propagation medium. The composite medium contributed to the rooting of shoots after the 4th passage, a 2-fold increase in the total number of roots, second-order roots, and stimulated rhizogenesis on media containing 6BAP. After 1.5 months, the length of rooted shoots on a medium containing 6 mg/l 6BAP. increased by 2 times. The increase in shoots in the control was 27% of the experimental variant. Light (standard medium) inhibited the growth of roots in length and the development of second-order roots. On the composite medium, the roots developed between the main medium and the darkened layer. By the end of the passage, root hairs developed on the roots. If the walls of the vessel were darkened with foil, then the root system also developed inside the medium. Based on the data obtained, the rooting of woody plants should begin after the 4th-5th passage. Shoots longer than 2.5 cm should be used for further propagation, as they do not have prerequisites for vitreous appearance. Shoots less than 1.5 cm long must be transplanted to a medium with a reduced content of 6BAP (0.1 mg/l), and shoots 1.5-2.5 cm long should be used for rooting.

The viability of plants under non-sterile conditions is largely determined by the ability of plants in vitro and in vivo to grow rapidly. Under in vitro conditions, this property depends on the culture, growth regulators, and growing conditions. Often at this stage, the death of the tops of rooted plants is noted. In this regard, we studied the effect of an electrostatic field (10–40 kW/m) on the rooting of pear shoots against the background of various growth regulators. The electrostatic field changed the nature of rhizogenesis and the growth of rooted shoots. In the best experimental variants, the number of viable shoots was greater by 47.5%, their length exceeded the length of the control ones by 17%, the number of roots and their length increased.

The size of side shoots also influenced their rooting in vitro. With an increase in the length of lateral shoots, the number of roots, their length, plant height increases and the number of leaves decreases. The growth rate of the above-ground system was higher in shoots 0.5 cm long. The increase in the number of leaves in shoots of small size is obviously associated with the residual effect of 6BAP. It is possible that the small growth of shoots is explained by the predominance of their growth by division at the stage of shoot proliferation, which is realized by an increase in the number of leaves at the rooting stage.

The rooting rate of apple shoots also depended on their length. Short shoots of clone rootstock 62-396 were capable of rooting, but the development of the aboveground and root systems was weak. An effective technique for increasing the rooting of shoots is to injure their base, especially the longitudinal cuts of the shoots. With this method, the rooting rate increased by 27% and the number of roots of the spur form of yalon increased by 4 times.

The passage of the main stages of micropropagation is either impossible or very difficult without the inclusion of growth regulators in the nutrient medium. To obtain the desired direction of morphogenesis, it is sometimes necessary to use them in high concentrations, which can cause side undesirable reactions (formation of callus, vitreousness, premature death of individual organs). At the same time, it is known that the action of growth regulators is determined by their ability to penetrate into the cell. Surface-active substances (surfactants) can weaken some barriers to the penetration of substances into the cell. The effect of tween-40 depended on its concentration, a positive effect was noted in variants with tween-40 (0.5-1 mg/l). At this concentration, the beginning and intensity of root formation took place at a lower content of root formation inducers in the medium (0.1 mg/l IBA). Higher concentrations of tween-40 reduced the ability of strawberry shoots to rhizogenesis. More active in this process was sodium humate at a concentration of 0.5 mg/l.

Obtaining a whole plant in vitro and the nature of its development depended on the salt and hormonal composition of the medium prior to rooting.

The process of root formation of lateral shoots of roses proceeded better if the previous propagation passage took place on a medium in which the ratio of NH4:N03 was 1:2. With this ratio, the number of roots and their length increased significantly. The height of the aboveground system did not depend on the ratio of ammonia and nitrate nitrogen. The rooting of pear lateral shoots depended on the content of 6BAP in the nutrient medium for propagation (Table 7).

Despite the absence of a relationship between the concentration of 6BAP and the subsequent rooting of shoots, there is a tendency for its increase with an increase in the concentration of 6BAP. The number of plants with dead tips decreases and the ability of first-order roots to branch is inhibited. This reaction of the pear to 6BAP is consistent with its physiological properties as a cytokinin. The study of the qualitative composition of the buds in the process of passage of strawberries showed that when using high concentrations of 6BAP (1 mg/l) in the nutrient medium in all varieties (Fragineta, Haveland, Zarya, Ko-kinskaya early, Redgontlit, seedling 139-13-11) starting from the 4-5th passage, the number of buds incapable of developing full-fledged plants increases. Apparently, this is due to prolonged exposure to a high concentration of 6BAP.

Agar is the most commonly used gelling material in tissue culture. It is the most expensive component of the environment. In this regard, searches are underway for agar substitutes that have gelling properties. In our experiments we carried out a gelrite* test. The use of gelrite (0.5-2.5 g/l) during rooting of rootstock 62-396 stimulated the development of vitreousness. The number of vitreous plants was reduced when gelrite was used together with agar (2.5 g/l). Such a reaction of plant tissues may be related to its ability to absorb divalent cations.

conclusions

1. Rooting of strawberry shoots is possible at the first, robinia and weigela at the second passage, apple and pear after the 5-10th passage, depending on the hormonal and salt composition of the medium. Reduction in the environment of M.S. by 1/2 of macroelements or by 1/2 -1/3 of total nitrogen, it helps to increase the rooting of shoots of strawberries and apple trees.

2. The nature of shoot rooting in vitro depends on the root formation inducers used, their concentrations and combinations. With the introduction of sodium humate, krezacin, mival, cultara into the nutrient medium, rooting significantly increased, and callus development decreased. The culture contributed to the development of the root system directly from the meristematic top with a size of 250-300 microns, while the roots were colored green. The use of surfactants (CEP, tween-40) increased the activity of IMC by 2 times and decreased the activity of the cultarian by 6 times, and made it possible to carry out the rooting stage at lower concentrations of auxins. The combined use of IMC and AgNO3 accelerated the onset of root formation in strawberries.

3. Rooting of apple shoots depended on the method and duration of etiolation, temperature regime and growth regulators. The etiolation of proliferating cultures at low temperatures further increased the rooting of shoots on the medium with IBA and decreased on the medium with cultar and mival. The composite medium contributed to the rooting of apple tree shoots after the 4th passage, an increase in the number of second-order roots, leaf area, and stimulation of the development of the above-ground and root systems on the medium with 6 BAP (6 mg/l).

4. Structural medium, consisting of perlite and agar in a ratio of 0.5-1:1 by volume, contributes to obtaining a well-developed root system of strawberries, allows to reduce the concentration of agar by 3 times.

5. The type and concentration of carbohydrates in the medium affects the nature of plant rhizogenesis. The most acceptable should be recognized as sucrose and glucose.

Bibliographic list

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Reviewer - Doctor of Agricultural Sciences. n. A.V. Isachkin

Results of a long-term investigation into the effect of various growth regulators, carbohydrates, passing duration, agar quality, medium structure, food solution saline composition on root growth are provided in the article. Both stages and duration of rhyzogenesis questions have been discussed. Much consideration is given to the etiolation problem, physical factors, both hormonal and saline composition of nutrient medium. Both true and important data on light and medium composition influence upon rhyzogenesis and viability of plants under unsterile conditions are given in the article.

Key words: in vitro, growth regulators, nutrient medium, etiolation, carbohydrates, strawberries, apple tree, pear tree, lilac, rose, Chinese actinidia, ash tree.

Demenko Vasily Ivanovich - Doctor of Agricultural Sciences N., RGAU - MSHL named after K.L. Timiryazev. Tel. 976-21-98.

Lebedev Vadim Grigorievich - Ph.D. Sc., Institute of Bioorganic Chemistry named after academicians M.M. Shemyakina and YuA. Ovchinnikov RAS.

Shestibratov Konstantin Alexandrovich - Ph.D. Sc., Institute of Bioorganic Chemistry named after academicians M.M. Shemyakin and Yu.A. Ovchinnikov RAS.

B. V. Rogovaya, M. A. Gvozdev

FEATURES OF MICROCLONAL REPRODUCTION OF STONE CROPS UNDER IN VITRO CONDITIONS

The paper presents a review that discusses the features of the methods of micropropagation of stone fruit crops in the 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 cherry, sweet cherry, peach and apricot. The issues of healing plants from various pathogens and testing the plant material of stone fruit crops for the presence of viral infections are considered.

For the first time, micropropagation was carried out by the French scientist Georges Morel on orchids in the 50s of the twentieth century. In his works, he used the technique of cultivating the apical meristem of plants. Plants thus obtained were free from viral infection.

In our country, research on the improvement of plants by the meristem method and clonal micropropagation began in the 60s at the Institute of Plant Physiology. K. A. Timiryazev Academy of Sciences of the USSR.

Microclonal propagation - obtaining in vitro plants that are genetically identical to the original explant (the method of vegetative propagation of plants in in vitro culture). Micropropagation is based on the 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 the in vitro system are becoming increasingly important: reproduction by axillary and adventitious buds, indirect morphogenesis, somatic embryogenesis.

Using these methods makes it possible:

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

To receive in a short time a large amount of healthy, virus-free material, genetically identical to the mother plant;

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

Propagate plants practically without contact with the external environment, which eliminates the impact of adverse abiotic and biotic factors;

Get the maximum number of plants per unit area;

In a short time, to obtain a large number of plants that are difficult to propagate or vegetatively non-propagated;

When growing plants with a long juvenile phase, it is possible to accelerate the transition from the juvenile to the reproductive phase of development;

For a long time (within 1-3 years) to keep the plant material in vitro conditions (without passaging on a fresh medium),

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

Develop methods for cryopreservation of in vitro sanitized material.

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

The process of micropropagation includes several stages. The main ones are:

1st stage - introduction of the explant into the culture in vitro;

2nd stage - micropropagation;

3rd stage - the process of rooting microshoots;

4th stage - implementation of the exit of rooted plants from sterile conditions to non-sterile ones.

An important step in in vitro plant micropropagation is the cultivation of virus-free mother forms of plants in growing houses or isolated boxes in winter greenhouses, in conditions inaccessible to virus vectors. Explant donor plants for subsequent introduction into in vitro culture should be tested for the presence of viral, mycoplasmal and bacterial infections using PCR diagnostic methods, either molecular hybridization or enzyme immunoassay (ELISA) .

The ELISA method allows in a short time to detect the vast majority of viruses that infect stone fruit crops: plum dwarfism virus, stone fruit necrotic ring spot virus, plum sharka 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 found to be free from viruses and other regulated pathogens according to the results of testing are assigned the category of "viral-free" basic clones. If an infection is detected, the original plants can be rehabilitated. For the recovery of stone fruit plants from viruses, it is most expedient to combine the methods of dry-air thermotherapy and in vitro culture. If a culture of isolated apical meristems fails to get rid of the tested viruses, chemotherapy methods are used based on the introduction of chemicals into nutrient media that inhibit the development of a viral infection in plants in vitro.

Sometimes, in order to actively detect bacterial microflora, the environment is enriched with various organic additives, such as casein hydrolyzate, which provokes the development of saprophytic microorganisms. Infection is assessed visually after 7-10 days. "Clean" explants are placed on nutrient media for further cultivation. Practice at this stage and the use of environments devoid of growth substances.

Introduction to in vitro culture and micropropagation of stone fruits

In clonal micropropagation of fruit stone fruit crops, apical and lateral buds, as well as meristematic tops, 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 plant material.

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

Depending on the stage of microclonal propagation of fruit stone fruit crops, 6-benzylaminopurine (6-BAP) is added to nutrient media at concentrations of 0.2-2 mg/l. At the stage of introduction into the 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 at concentrations of 0.5-2 mg/l, plum microplants 0.5-1 mg/l BAP.

The rooting process of microshoots

The rooting stage requires special attention. The process of rooting in vitro shoots of stone fruit crops depends on varietal characteristics, on the number of passages carried out, 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 prevents the processes of rhizogenesis, is excluded from the medium, and auxins are introduced into the media, mainly β-indolyl-3-butyric acid (IMA). It has been established that the optimum concentration of IMC in the composition of the nutrient medium is in the range of 0.5-1 mg/l. The presence of IMC in the medium at a concentration of 2 mg/l causes the formation of hypertrophied roots.

The joint introduction of the ribav preparation (1 ml/l) and traditional phytohormones auxins [IMA and β-indoleacetic acid (IAA) at 0.5 mg/l each] into the medium for rooting increases the percentage of rooting shoots of a number of varieties of stone fruit crops.

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

For the effective rooting of test-tube plants of stone fruit crops, not only the type of stimulant, but also the method of its application is of great importance. In addition to the introduction of auxins into the nutrient medium, to induce rhizogenesis, preliminary soaking of the shoots in a sterile aqueous solution of IBA (25-30) mg/l at an exposure of 12-24 hours is used. The experiments performed showed that the treatment of microcuttings with an aqueous solution of IBA is more effective than the introduction of this regulator into the culture medium. The mass appearance of the first adventitious roots with the use of pre-treatment with a rhizogenesis inducer was noted on the 20-25th day. Another way to induce rhizogenesis is the treatment of shoots of stone fruit crops with talc auxin-containing IMC powder 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 talcum powder with different concentrations of auxin revealed varietal specificity in the rooting of plum microcuttings.

The process of rhizogenesis proceeds most intensively on modified MS and White media. According to other data, the best medium for root formation are Heller macronutrient media with added vitamins and half-diluted MS medium with a reduced sucrose content of 15 mg/l and with the exception of mesoinositol, which promotes the formation of callus tissue. However, in most works, Murashige and Skoog media are used to root microshoots of stone fruit crops.

Micropropagation methods

There are several ways to micropropagate plants in vitro:

Methods of reproduction by axillary buds;

Methods of propagation by adventitious buds;

Indirect morphogenesis;

somatic embryogenesis.

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

Influence of the genotype on the efficiency of micropropagation

The genotype has the most significant influence on the efficiency of micropropagation. The reaction of plants to the conditions of aseptic cultivation depends on varietal characteristics and is explained by the different regenerative capacity of varieties of fruit and berry crops. For example, when using clonal micropropagation to rapidly propagate new cherry cultivars, varietal traits have been found to be the dominant factors in the ability of plants to micropropagate.

Varietal differences were manifested both at the stage of proliferation and at the stage of root formation.

Among explants of different cultivars of the same species of fruit plants, there is often a different degree of reaction to growth regulators included in the medium, which apparently reflects, to some extent, the endogenous content of growth substances, which is a genetically determined trait of a species or variety. At the same time, the realization of the morphogenetic potential in the culture of embryos 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 micropropagation of plants is the conditions of their cultivation. The optimal conditions for the cultivation of stone fruit crops are: temperature 22-26 ° C for cherries, sweet cherries and 26-28 ° C - for plums, illumination 2000-5000 lux - for cherries, sweet 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 at the proliferation stage, an increase in the multiplication factor and an increase in the proportion of shoots suitable for rooting in cherry varieties at the proliferation stage can ensure the intake of 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 with the horizontal orientation of regenerants. To increase the multiplication factor 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 value of the multiplication factor rises 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 micropropagation is the method of plant regeneration through axillary bud development. 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 in vitro micropropagation method is realized by adding cytokinins to nutrient media, which inhibit the development of the apical bud of the stem and stimulate the formation of axillary buds.

The process of microclonal propagation of sour cherries by culture of isolated apical meristems is based on the phenomenon of removal of apical dominance, which contributes to the subsequent development of already existing meristems and ensures the genetic homogeneity of the planting material.

rial. Removal of apical dominance is achieved by adding cytokinins. Many cultivars of cherries 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 fruit stone plants is associated with the proliferation of axillary meristems. Genetic stability is an inherent property of the meristem, which can be preserved in vitro if the latter is cultivated under conditions that inhibit callus formation. If media that stimulates callus formation are used, then genetic variability can occur.

To obtain higher multiplication factors, nutrient media are often enriched, in addition to preparations of a cytokinin nature, with substances from the auxin group that 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 nutrient medium to achieve maximum cell proliferation can affect the genetic stability of the resulting material.

Method of reproduction by adventive buds and indirect morphogenesis

Adventitious buds are called buds that arose directly from the tissues and cells of plant explants, usually not forming them. Adventive (or adnexal) buds are formed from meristem zones, most often formed secondarily from callus tissues. 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 the 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.

Adventive 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 genetically unstable plants may appear in this case. Therefore, this method of plant regeneration can be used to induce genetically diverse plants.

Regenerating 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 should also be taken 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 modified shoots are much more likely to occur.

For the regeneration of shoots of stone fruit crops, such as cherry, sweet cherry, peach, apricot, from the original explants (whole leaves and their segments), various media are used: Murashige-Skoog (MB), Lloyd and Mac Cone (WPM), Driver and Kuniyuki ( DKW), Kuren and Lepuavr (QL) .

For experiments on adventitious regeneration of cherries and sweet cherries, Lloyd and McCone's medium for woody plants, Woody Plant Medium (WPM), supplemented with various growth stimulants, is most often used. From cytokinins, 6-BAP, thidiazuron (TDZ) are mainly used, from auxins - NAA, IMC, 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 leaf explants of sweet cherry (with transverse cuts made on them along the central vein of the leaf), which were placed abaxial (lower) 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 that BAP and NAA at a concentration of 2 mg/l and 1 mg/l are the optimal combination of plant growth regulators of cherry and cherries. The highest frequency of regeneration was obtained on the WPM medium, although it stimulated callusogenesis more than MS, QL, DKW. 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 in the middle leaf segments; the lowest values ​​were on the apical segments, and direct regeneration (without callus formation) was noted on the basal segments.

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

The efficiency of adventitious regeneration of wild cherries (Prunus avium L.) was significantly affected 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 the largest number of adventitious shoots. For adventitious regeneration of wild cherries, WPM supplemented with 0.54 tM NAA and 4.4 tM TDZ was used.

A specific pre-cultivation pre-treatment (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 regeneration agar medium WP supplemented with 5 mg/l TDZ increased the efficiency of adventitious sweet cherry regeneration. Young leaf explants of sweet cherry showed a higher ability to regenerate than old ones.

The significant effect of ethylene inhibitors on the adventitious regeneration of leaves of various apricot varieties should be noted. For example, it was shown in the work 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 - adaxial surface to the environment.

Italian researchers developed an adventitious regeneration method from whole peach leaves that 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 leaf cultivation - 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 preserved for several months. In these studies, peach adventitious shoots appeared by indirect morphogenesis.

Indirect morphogenesis involves secondary differentiation of kidneys from callus tissues. A variety of explants are used to form callus, from which shoots are then formed. To obtain a morphogenic callus from perennial plants, one should take the tops of the shoots or sections of meristematic tissues isolated from them. Such a 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 Great Britain, in the department of physiology of the experimental station Maidstone, the regeneration of plants from stem and leaf callus was studied in the rootstock of the Colt sweet cherry. Callus initiation was carried out on Mourasige-Skoog medium containing 2.0-10.0 mg/l NAA. The resulting callus was transferred to a regeneration medium containing BAP at a concentration of 0.5 mg/L. It was possible to carry out the regeneration of shoots from calluses in this cherry rootstock.

In the Central Genetic Laboratory named after I. V. Michurin, root formation was noted in the culture of passivated callus tissues obtained from annual shoots of cherry. When transferred to 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 germ-like structures from somatic (non-sex) cells. Somatic embryo - an independent bipolar structure, not physically attached to the tissue, from which a structure is formed, in which the stem and root apexes develop simultaneously.

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

In the work, the possibility of plant regeneration from calli obtained from the roots of cherry rootstocks was investigated. Callus was obtained either from cut roots or from whole plants grown under sterile conditions by microcloning cherry shoots. In the rootstock of the Colt cherry, callus obtained from the roots of intact plants formed shoots and embryoid-like structures. Cherry calli were cultured 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. The regenerative plants were propagated through tissue culture and transplanted into soil. Seedlings of regenerated plants obtained from cherry rootstock calli did not differ in phenotype from the original rootstocks.

The 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 a combination of 2,4-D and kinetin was used. The 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.).

To date, the most reliable way to obtain genetically identical offspring is considered to be micropropagation of fruit-stone crops with axillary buds compared with somatic embryogenesis, reproduction with 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 ofplant material testing for virus infections have been reviewed as well as certain problems of genetic stability preservation depending on propagation model.