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AP 19676010 (supervizer Romadanova N.V.)

Brief description of the project

(2023-2025)

 

Project title: IRN АР19676010 «Development of biotechnology for ex situ conservation of rare, endangered Rosaceae species, for the restoration of natural populations».

 

Relevance. The aim of the project is to develop a biotechnology for the preservation in cryogenic bank and in vitro a germplasm of rare, endangered Rosaceae species, listed in the Red Book of Kazakhstan, the production of planting stocks for the restoration of natural populations.

The problem that the project is aimed at is that some Rosaceae species are endangered in Kazakhstan: Amygdalus ledebouriana Schlecht., Crataegus ambigua C.A. Mey. ex A. Beck, Cotoneaster karatavikus Pojark., Louiseania ulmifolia (Franch.) Pachom, Rosa pavlovii Chrshan., Sorbus persica Hedl., Malus sieversii (Ledeb.) M. Roem. and others. The main reason for the disappearance of plants is human economic activity. The death of plants in large quantities leads to their extinction and to global changes in ecosystems. The most vulnerable are rare species of flora – relics and endemics, the distribution of which depends on the boundaries of a particular area.

Many countries provide protection for endangered plants. The Red Book has been created in Kazakhstan, which is the main legislative document summarizing materials on the current state of rare and endangered species of plants and animals. In 2014, the second edited and supplemented edition of the Red Book of Plants was published, including 387 species, which is 81 species more than in the first edition of 1981. Replenishment of the list is the result of increasing human economic activity in recent years, as a result of which a further reduction of these species or even their disappearance is obvious.

Basic approaches to research. It is required to use scientific approaches to preserve the genetic material of endangered Rosaceae plants, including biotechnological methods, such as micropropagation, long-term storage in cryogenic banks, as well as restoration of natural populations by biotechnologically obtained plants.

Practical significance of research results. The studied plants have a valuable medicinal, food, decorative value, which are important objects for breeding, as well as sources of the gene pool. The preservation of these plants in vitro and in a cryobank, as well as the restoration of the ecology of their natural habitats, is of practical importance for solving urgent problems of the socio-economic, scientific and technical development of Kazakhstan. The planting stocks obtained during the implementation of the project will be in demand in breeding practice, and can also be sent to mass production for the implementation of the commercialization project. The created cryogenic collection and the in vitro collection can be used for the international exchange of genetic resources.

 

The goal of the project: is to develop a biotechnology for the preservation in cryogenic bank and in vitro a germplasm of rare, endangered Rosaceae species, listed in the Red Book of Kazakhstan, the production of planting stocks for the restoration of natural populations.

 

Expected results:

  1. Collection of plant material (annual shoots and fruits) of plants of the Rosaceae listed in the Red Book of Kazakhstan in their natural habitats. Studying the state of endangered populations using geobotanical methods. Selection of promising forms for breeding.
  2. Initiation to in vitro culture of shoots germinated from seeds and annual shoots. Diagnostics of test tube plants for the presence of endophytic contamination on a specialized medium 523 for the detection of fungi and bacteria. Optimization of nutrient media for micro propagation and for in vitro shoot rooting. Creation of an in vitro collection of aseptic plants as source material and medium-term storage of in vitro shoots at the for degrees of 4°C, long-term preservation of shoot tips at -196°C and planting stocks production.
  3. Optimization of methods for cryopreservation of shoot tips of some endangered Rosaceae species. Creation of a cryogenic seed bank, isolated embryonic axes, shoot tips and DNA at -196°C, long-term preservation of seeds in a freezer at a temperature of -20°C.

 

Scientific Supervisor of the project: Assoc. Prof., Ph.D. Leading research scientist of the Laboratory Germplasm Cryopreservation Romadanova N.V. More than 110 scientific papers have been published, of which 11 publications are included in the Web of Science database, 10 in the Scopus database. H-index 5. Scopus ID 36111616700; Web of Science ID BBD-5948-2021; ORCiD 0000-0003-1052-2753

 

Research group: 

1) Leading researcher, PhD, professor Kushnarenko S.V. More than 200 scientific papers have been published, of which 18 publications are included in the Web of Science database, 21 in the Scopus database. H-index 9. Scopus ID 6507153914; Web of Science ID M-6616-2015

2) Senior Researcher, PhD Aleksandrova A.M. 9 publications are included in the Web of Science database, H-index 3; Web of Science ID AGJ-1822-2022

3) Researcher Tolegen A.B. 12 scientific papers have been published, of which 2 publications are included in the Web of Science database, 2 in the Scopus database. H-index 2; Web of Science ID IPO-2780-2023; Scopus ID 57567940600.

4) Researcher Altayeva N.A. 54 scientific papers have been published, of which 4 publications are included in the Web of Science database, 3 in the Scopus database. H-index 3. Scopus ID 57216750088; Web of Science ID EKZ-9456-2022; ORCiD 0000-0002-5039-8999.

5) Junior researcher Zemtsova A.S. 5 scientific papers have been published.

6) Laboratory assistant Mikhailenko N.V. 2 scientific papers have been published

7) Laboratory assistant Artimovich N.A.

 

List of publications of the project’s participants (2018-2022)

List of main publications of the project leader included in the WoS database, Scopus and in the peer-reviewed journals of Kazakhstan Republic

  1. Kushnarenko S.V., Romadanova N.V., Aralbayeva M.M. Current state and in vitro conservation of the only endangered population of Corylus avellana in Kazakhstan // Res. on Crops. – 2020. – Vol. 21 (4). – P. 681-686. IF 0.413 DOI: 10.31830/2348-7542.2020.106 SCI 0, percentile 18.
  2. Romadanova N.V., Karasholakova L.N., Eshbakova K.A., Özek G., Özek T., Yur S., Kushnarenko S.V. Phytochemical analysis and antioxidant activity of Berberis iliensis M. Pop and Berberis integerrima Bunge fruits pulp // Res. on Crops. – 2021. – Vol. 22(4). – P. 940-947. IF 0.84 DOI: 10.31830/2348-7542.2021.154 SCI 0, percentile 38.
  3. Romadanova N.V., Tolegen A.B., Koken T.E., Nurmanov M.M., Kushnarenko S.V. Chemotherapy of in vitro apple shoots as a method of viruses eradication // International Journal of Biology and Chemistry. – 2021. – Vol. 14, № 1. – P. 48-55. WoSSCI 1. DOI: https://doi.org/10.26577/ijbch.2021.v14.i1.04
  4. 4. Romadanova N.V., Tolegen A.B., Kushnarenko S.V. Effect of Plant Preservative MixtureTM on Endophytic Bacteria Eradication from In Vitro-Grown Apple Shoots // Plants. – 2022. – Vol. 11, Issue19, 2624. IF. 4.658, Q 1, percentile 97. SCI 1, WoSSCI 1. DOI: https://doi.org/10.3390/plants11192624
  5. Romadanova N.V., Kushnarenko S.V. Biotechnology for obtaining virus-free Malus sp. planting stocks // Bulletin of Karaganda University. Series “Biology. The medicine. Geography». – 2021. – No. 3 (103). – P. 102-118. DOI 10.31489/2021BMG3/102-118 (in Russian)
  6. Romadanova N.V., Aralbaeva M.M., Rymkhanova N.K., Baigaraev D.Sh., Ramazanov A.K., Ishmuratova M.Yu., Kushnarenko S.V. Cryopreservation as a way to increase laboratory germination and germination energy of seeds // Bulletin of Karaganda University. Series “Biology. The medicine. Geography». – 2022. – No 1 (105). – P. 86-95. DOI: 10.31489/2022BMG1/86-95 (in Russian)
  7. Kushnarenko S.V., Romadanova N.V., Turdiev T.T., Aralbaeva M.M., Kalybaev K.R. Preservation in a cryobank of Juglans regia L. accession from several populations in the Sairam-Ugam State National Natural Park // Bulletin of KazNU. Ecological Series». – 2022. – No 2 (71). – С 72-80. DOI: 10.26577/EJE.2022.v71.i2.07 (in Russian)

Publications of key staff of the research team:

  1. Alexandrova A.M., Karpova O.V., Nargilova R.M., Kryldakov R.V., Nizkorodova A.S., Zhigaylov A.V., Yekaterinskaya E.M., Kushnarenko S.V., Akbergenov R.Zh., Iskakov B.K. Distribution of potato (Solanum tuberosum) viruses in Kazakhstan // International Journal of Biology and Chemistry. – 2018. – 1 (11). – P. 33-40. DOI: https://doi.org/10.26577/ijbch-2018-1-311. WoSSCI 2, Q 4.
  2. Terletskaya N.V., Zorbekova A.N., Altayeva N.A., Bari G.T., Erezhetova U. Effect of drought on growth parameters and pigment complex of wheat lines obtained from interspecific crosses // Vestnik KazNU. Al-Farabi. Biological series. — №3 (76). — 2018. – p.130 – 139.
  3. Terletskaya N.V., Altayeva N.A., Erezhetova U. The influence of drought on the functioning of the photosynthetic apparatus of the flag leaf in alloplasmic lines obtained as a result of interspecific crossings of wheat // Bulletin of the L.N. Gumilev Eurasian National University. Biological Sciences Series — No. 4 (129). – 2019. – p.58-68.
  4. Karpova O., Alexandrova A., Yeriskina E., Kryldakov R., Gritsenko D., Galiakparov N., Iskakov B. Andean and Ordinary Strains of Potato Virus S Infecting Potatoes in Southern Kazakhstan // Plant Disease. – 2020. – 104 (2). – P. 599. DOI: 10.1094/pdis-09-19-1822-pdn. IF. 4.614. SCI 0, percentile 75; WoSSCI 0, Q 1.
  5. Terletskaya N.V., Altayeva N.A., Erezhetova U., Zorbekova A.N. Morphophysiological aspects of studying drought resistance of interspecific wheat hybrids // Bulletin of the Kazakh National University named after. al-Farabi, ser. environmental. — 2020. — No. 1 (62). — P.36-44.
  6. Terletskaya N.V., Shcherban A.B., Nesterov M.A., Perfil’ev R.N., Salina E.A., Altayeva N.A., Blavachinskaya I.V. Drought Stress Tolerance and Photosynthetic Activityof Alloplasmic Lines T. dicoccum x T. Aestivum. May 2020. International Journal of Molecular Sciences21(9):3356. DOI: 10.3390/ijms21093356. SCI 12, percentile 66; WoSSCI 9, Q 1
  7. Terletskaya N.V., Lee T.E., Altayeva N.A., Kudrina N.O., Blavachinskaya I.V., Erezhetova U. Some Mechanisms Modulating the Root Growth of Various Wheat Species under Osmotic-Stress Conditions. Plants 2020, 9, 1545; doi:10.3390/plants9111545. SCI 11, percentile 72; WoSSCI 10, Q 1.
  8. Kushnarenko S., Utegenova G., Danaeva G., Aralbaeva M. IoT-based fully automatic smart providing aseptic materials for in vitro and cryogenic collections of wild Juglans regia L. for genetic resources preservation // International Journal of Agricultural Resources, Governance and Ecology/ — 2021. – Vol. 17, Issue 2-4. – P. 238 – 246. DOI: 10.1504/IJARGE.2021.121663. IF 0.744. SCI 0, percentile 25; WoSSCI 0, Q 3.
  9. Karpova O., Alexandrova A., Nargilova R., Ramazanova M., Kryldakov R., Iskakov B. AtDREB2A gene expression under control of the inducible promoter and virus 5’-untranslated regions improves tolerance to salinity in Nicotiana tabacum // International Journal of Biology and Biomedical Engineering (by NAUN). – 2021. – 15. – P. 260-274. DOI: 10.46300/91011.2021.15.32. IF. 0.49. SCI 0, percentile 17.
  10. Kushnarenko S., Aralbayeva M., Rymkhanova N., Reed B.M. Initiation pretreatment with Plant Preservative MixtureTM increases the percentage of aseptic walnut shoots // In Vitro Cellular & Developmental Biology – Plant. – 2022. IF 2.347. SCI 1, percentile 74; WoSSCI 1, Q 2. https://doi.org/10.1007/s11627-022-10279-4
  11. Terletskaya N.V., Stupko V.Yu., Altayeva N.A., Kudrina N.O., Blavachinskaya I.V., Kurmanbayeva M.S., Erezhetova U. Photosynthetic activity of Triticum dicoccum × Triticum aestivum alloplasmic lines during vegetation in connection with productivitytraits under varying moister conditions. Photosynthetica 59 (1): 74-83, 2021. DOI 10.32615/ps.2021.003. SCI 3, percentile 49; WoSSCI 3, Q 2.
  12. Alexandrova A., Karpova O., Kryldakov R., Golyaev V., Nargilova R., Iskakov B., Pooggin M.M. Virus elimination from naturally infected field cultivars of potato (Solanum tuberosum) by transgenic RNA interference // International Journal of Molecular Sciences. – 2022. – Vol. 23(14). DOI: 10.3390/ijms23148020. IF. 5.542. SCI 0, percentile 85; Q 2.

Patents:

  1. Pat. 2019/0952. The method of common hazel propagation / Kushnarenko S.V., Aralbaeva M.M., Romadanova N.V. // No. 4998; Declared Oct. 30, 2019/0952.2., publ. 06.05.20. (in Russian) https://gosreestr.kazpatent.kz/Utilitymodel/Details?docNumber=320230
  2. Pat. No. 34902. Method for obtaining apple planting stocks recovered from viruses by chemotherapy / Romadanova N.V., Nurmanov M.M., Kushnarenko S.V. // No. 34902; application 01/31/2020/0056.1., publ. 02/26/21. (in Russian) https://gosreestr.kazpatent.kz/Invention/Details?docNumber=323672

 

Results for 2023: populations were described using geobotanical methods; descriptors from 35 to 49 indicators have been developed for plants, depending on the species. JPS coordinates were recorded at accessions collection sites.

In vitro initiation of S. schrenckianus and P. tenella shoots has begun. Seeds without stratification were germinated on Knop nutrient medium (viability — 20%). The seeds of all collected plants were placed into plastic containers for stratification for 8 weeks at 4°C. Seeds from 6 S. schrenckianus accessions were stratified in moist perlite for 8 weeks at 4°C. No endophytic contamination was detected in shoots of S. schrenckianus germinated on Knop medium after diagnosing test tube plants for the presence of endophytic contamination on specialized medium 523. The optimization of nutrient media for micropropagation of S. schrenckianus and the creation of its in vitro collection were being carried out.

The creation of a cryogenic bank of seeds and DNA at -196°C has begun. Seeds from 12 S. schrenckianus accessions were placed in liquid nitrogen for long-term storage. Total DNA was isolated from leaves of M. sieversi, S. persica and L. ulmifolia, and DNA quality was tested using electropherograms. As a result, 20 DNA accessions of S. persica, 16 accessions of M. sieversi, and 6 accessions of L. ulmifolia were placed in a cryogenic bank for long-term storage.

 

Publications (2023):

  • V. Romadanova, A.S. Zemtsova, A.M. Alexandrova, N.A. Altayeva, M.M. Aralbayeva, K.T. Abidkulova, N.V. Mikhailenko, A.B. Tolegen, S.V. Kushnarenko. Development of biotechnology for the ex-situ conservation of rare, endangered Rosaceae species , for the restoration of natural populations //November 27-29, 2023 — satellite events dedicated to the problems of preserving the biodiversity of rare, endangered plant species; growing fruit and berry crops, potatoes and perennial forage crops, including in the northern regions (in print).
  • S. Zemtsova, N.V. Romadanova, N.A. Altayeva, M.M. Aralbayeva, S.V. Kushnarenko. Conservation of rare and endangered plant species biodiversity of the Rosaceae: Spiraeanthus schrenkianus (Fisch. & C.A. Mey.) Maxim. and Sibiraea altaiensis (Laxm.) Schneid. //November 27-29, 2023 – satellite events dedicated to the problems of preserving the biodiversity of rare and endangered plant species; growing fruit and berry crops, potatoes and perennial forage crops, including in the northern regions (in print).
  • V. Romadanova, A.S. Zemtsova, A.M. Aleksandrova, N.A. Altayeva, M.M. Aralbayeva, K.T. Abidkulova, N.V. Mikhailenko, A.B. Tolegen, S.V. Kushnarenko. Development of biotechnology for ex situ conservation of rare, endangered Rosaceae species, for the restoration of natural populations //36th International Conference on Ecological Agriculture, Biodiversity &Waste Management (EABWM)/ Dec. 20-22, 2023 Bangkok (Thailand). P 16

 

Results for 2024:

Five expeditions were conducted in 3 regions of Kazakhstan to collect plant material in natural habitats.

In the Turgen gorge of the Almaty region, Enbekshikazakh district, 10 accessions of Malus sieversii (annual shoots with wintering buds) were collected. The apple population is described according to international descriptors for 49 indicators. The population density is low — 62.0%. The population is represented mainly by sparse forest — 89.4%. Dominant plant species at the collection site: Crataegus dsungarica, Spiraea hypericifolia, Rosa platyacantha and others. A high susceptibility of trees — 34.8% by various pests and diseases was noted, dry dying trees were identified. The condition of the trees at the collection site is mainly adult viable — 67.4%. The trees of the population are often of medium height (5-8 m) — 52.7%.

Eight accessions of Prunus ulmifolia (annual shoots with wintering buds) were collected in the Koksu gorge (Dzungarian Alatau). The population of three-lobed almond is described by international descriptors for 49 indicators. The population density is low — 100%. The incidence of shrubs is ˂ 10%. The population is represented by medium — 50% and low — 50% in height adult viable shrubs. In the Turkestan region, Tyulkubas district, 39 accessions of Cotoneaster karatavikus were collected: 1) 8 accessions at the Kulan reservoir; 2) 21 accessions in the Sairam-Ugam National Park; 3) 8 accessions in the Baizhansai gorge. The population is described by 48 indicators. In the first place of growth, the population density is low, mainly adult viable shrubs (75%). The shrubs are not affected, fruiting is weak. In the second growing place, the population density is average, young non-fruiting shrubs are more common (42.9%) and adult viable shrubs (38.1%). The shrubs are low affected (10.5% necrosis), 61.9% of shrubs do not bear fruit. In the third growing place, the population density is average, half of the shrubs are young non-fruiting, half are adult viable. The shrubs are not affected, 50% of the shrubs have abundant fruiting, 25% have average fruiting, and 25% have weak fruiting.

Three accessions of Malus niedzwetzkyana were collected: 1) Almaty region, Uygur district, Bolshoy Kyrgyzsay gorge — 2 accessions (an adult diseased with weak fruiting and a young fruiting with average fruiting). High susceptibility of trees (65.7%) was noted – dry branches, damaged leaves and fruits. Among the pests identified were: Cydia pomonella, Hyphantria cunea, Tetranychidae donnadieu. In the Almaty region, Ili district, the outskirts of the village of Boraldai, 1 accessions. This is a young, abundantly fruiting tree. Susceptibility is 20% – fire blight (Erwinia amylovora) and codling moth (Cydia pomonella).

15 accessions of Malus sieversii were collected: 1) 1 accessions in the Almaty region, Uyghur district, foothills of the Bolshoy Kyrgyzsay gorge. This is a young, abundantly fruiting tree. Susceptibility was not detected; 2) 13 accessions in the Almaty region, Uyghur district, Bolshoy Kyrgyzsay gorge. The population density is average, represented by adult diseased trees. The incidence is 65.7%. Most trees have dry branches, damaged leaves and fruits, there are a lot of cocoons on the trees. Among the pests identified: C. pomonella, H. cunea, T. donnadieu. Fruiting: weak — 35.7%, average — 42.8%; 3) 1 accessions in the Almaty region, Uygur district, Chundzha-Kolzhat highway, 97th kilometer. This is a young tree with average fruiting and no signs of infestation.

1 accessions of Prunus tenella was collected in the East Kazakhstan region, southeastern outskirts of Ridder. This is an adult viable shrub with average fruiting, infestation of 5% (necrosis), pests were not found.

Expedition to the East Kazakhstan region, to the Kalbinsky ridge, floodplain of the river. The expedition was unsuccessful, not a single Rosa cinnamomea bush was found, all 40 accessions collected were identified as Rosa laxa. In 2025, an expedition is planned to the East Kazakhstan region, the Irtysh region, the middle reaches of the Irtysh River, where another population has been noted to grow.

21 s accessions of Sibiraea laevigata were collected in the East Kazakhstan region, in the vicinity of the city of Ridder, the floodplain of the Gromotukha River. These are mainly adult viable (90.5%) abundantly fruiting (80.1%) shrubs. Susceptibility to rust diseases and leaf beetle (Syneta albida) is 12.3%.

In total, 97 accessions of plants of the Rosaceae listed in the Red Book were collected in 2024.

In vitro initiation of shoots, germinated from seeds and one-year-old shoots, was continued. Diagnostics of test-tube plants for the presence of endophytic contamination on specialized medium 523 for the detection of fungi and bacteria was continued. Optimization of nutrient media for micropropagation and for rooting in vitro shoots was continued. The creation of a collection of in vitro aseptic plants as a source material for medium-term storage of in vitro shoots at 4°C, long-term preservation of shoot tips at -196°C and planting stocks production was continued. The collected cuttings of Malus and P. ulmifolia, were germinated in laboratory conditions at 23-25°C in water. Shoots sprouted from dormant buds (1-2 cm) were sterilized in 0.1% HgCl2 for 5-7 min and placed in test tubes with Murashige and Skoog (MS) medium containing 30 g/l sucrose, 0.5 mg/l 6-benzylaminopurine (BAP), 0.01 mg/l indolebutyric acid (IBA), pH 5.7. The percentage of in vitro initiation of apple shoots averaged 62.3±10.5a, P. ulmifolia – 51.7±38.5a.

Fruits of 8 species of the Rosaceae plants collected during expeditions were used for in vitro initiation. Fruits were sterilized in 50% commercial bleach «Belizna» for 5 minutes, after which 5 methods of seeds in vitro initiation were used:

1) seeds were stratified in perlite at 4°C (8 weeks) with a light intensity of 10 µE m-2 s-1, 8-hour photoperiod (acclimatization in cold acclimation conditions (CA);

2) seeds were stratified (8 weeks) in plastic containers under CA conditions;

3) the seeds were germinated in perlite at 23-25°C, under illumination of 40 µE m-2 s-1, photoperiod of 16 hours (conditions of a light culture room (LCR);

4) the seeds or embryonic axes were germinated under LCR conditions on Knop medium: 1 g/l Ca(NO3)2, 0.25 g/l MgSO4*7H2O, 0.25 g/l KH2PO4, 0.125 g/l KCI, 27.8 mg/l FeSO4*7H2O, 37.3 mg/l Na2EDTA*2H2O, 1.75 g/l Gelrite, 4 g/l agar, pH 5.7;

5) seeds or embryonic axes were germinated under LCR conditions on the same composition of the MS medium as for apple and P. ulmifolia. It should be noted that for all accessions, stratification at 4°C increased the percentage of seed germination. Germination of seeds in vitro on the MS medium gave consistently low germination results in all variants. Shoots (1-2 cm) obtained from seeds, germinated in perlite, were sterilized in 0.1% HgCl2 for 3-5 min and placed in test tubes with the MS medium with different concentrations of phytohormones; the composition of the nutrient media is currently being optimized. As a result, the percentage of in vitro initiation of Cotoneaster karatavikus seeds (3 forms) averaged 47.9%, Crataegus ambigua — 45.3% (5 forms), Malus niedzwetzkyana — 52.4% (2 forms), M. sieversii — 60% (8 forms), Prunus tenella — 45% (2 forms), P. triloba — 56.7% (5 forms), Sibiraea laevigata — 40% (3 forms), Spiraeanthus schrenckianus — 88.3% (6 forms). Diagnostics of test-tube plants for the presence of endophytic contamination was continued on specialized medium 523 for detecting fungi and bacteria: 10 g/l sucrose, 8 g/l casein hydrolysate, 4 g/l yeast extract, 2 g/l KH2PO4, 0.15 g/l MgSO4 7H2O, 6 g/l gelwright, pH 6.9. As a result, no endophytic contamination was detected in the shoots of S. Schrenckianus – 0±0a and S. laevigata – 0±0a, for M. sieversii the percentage of contamination on average was – 51.7±38.5b, for C. ambigua – 45.0±22.2b, for P. tenella – 25.4±20.1b, for P. triloba – 28.4±26.4b. Optimization of the composition of nutrient media for micropropagation and creation of an in vitro collection have begun. At present, the maximum multiplication rate (MR) – 2.9 for S. schrenckianus was obtained on the MS medium with 0.1 mg/l BAP, 0.01 mg/l IBA; For S. laevigata MR 4.6 was obtained on the following medium: MS with the addition of 1.0 mg/l BAP, 0.01 mg/l IBA, 0.1 ml/l GA; for M. sieversii MR 3.9 on the following medium: MS with the addition of 0.5 mg/l BAP, 0.01 mg/l IBA; for C. ambigua MR 2.0 on the following medium: MS with 0.2 mg/l BAP and 0.01 mg/l IBA; for P. tenella MR 2.8 on the MS medium with 1.0 ml/l BAP, for P. riloba MR 3.9 on the MS medium with 1.0 ml/l BAP. The transfer of the in vitro collections of M. sieversii and P. riloba to medium-term storage at 4°C has begun.

The creation of a cryogenic seed bank has begun. 10-100 seeds without preliminary treatment were placed in laminated aluminum foil bags and frozen at -20°C or the seeds were placed in cryotubes and stored at -196°C. As a result, 46 seed accessions were placed in a freezer at -20°: C. ambigua (19 forms), M. sieversii (5), S. laevigata (2), S. schrenckianus (20). 246 accessions were placed in a cryogenic bank at -196°C: C. ambigua (40 forms/120 accessions), M. sieversii (19/57), S. laevigata (3/9), S. schrenckianus (20/60). The creation of a cryogenic DNA bank has begun. To select the optimal method for extracting genomic DNA from leaves, three standard protocols widely used for plant objects were tested. For all accessions, the protocol developed by Doyle and Doyle was optimal. The quality and quantity of the isolated genomic DNA was tested by electrophoresis in 1.2% agarose gel. As a result, 552 samples of total DNA were isolated from 7 species of the Rosaceae plants (C. ambigua (40 forms/120 accessions), M. sieversii (26/78), P. tenella (12/36), P. ulmifolia (3/9), S. laevigata (11/33), S. persica (24/72), S. schrenckianus (13/39). DNA was placed in a cryogenic bank at (-196°C) for long-term storage. For C. ambigua, M. sieversii, P. ulmifolia S. laevigata, S. schrenckianus, a protocol for transferring in vitro shoots to a soil substrate was developed. For this, the shoots were in vitro rooted in LRC on a nutrient medium: ½MS, with 30 g/l sucrose, 0.5 mg/l BAP, 0.25 mg/l IBA, pH 5.7. Rooting in vitro ranged from 56.7 to 100.0%. In vitro rooted shoots (root length 2.5-8.0 cm) were transferred to 250 ml polyethylene containers with soil of the following composition: a mixture of black soil, peat, and perlite in the percentage ratio: 50:40:10. The containers were covered with a transparent plastic lid to retain the moisture necessary for shoot adaptation. The planting stocks were adapted in a greenhouse (temperature from 18°C to 24°C, humidity 40% without additional lighting. After a week, the plastic cap was removed for 10-15 minutes to ventilate the plants, in the following days the ventilation duration was increased to 8 hours. The adaptation duration was from 3 to 6 weeks. For subsequent adaptation, the plastic caps were removed. As a result, the survival rate of planting material in the greenhouse was from 76.4 to 94.2%.

After 7 months of storage at 4°C, seeds of 3 forms of C. ambigua, 3 forms of M. sieversii, 4 forms of S. laevigata, and 4 forms of S. schrenckianus were planted in polyethylene containers with peat. The resulting germination percentage was 69.7±22.1a for C. ambigua, 76.7±32.2a for M. sieversii, 29.7±17.3b for S. schrenckianus, and 38.4±23.6b for S. laevigata, which is significantly lower than the germination percentage of seeds immediately after collection or after stratification. Accordingly, seed germination after 7 months of storage at 4°C decreases by almost 3 times for S. laevigata, and by more than 2 times for S. schrenckianus, by 1.3 times for M. sieversii, and by 1.7 times for C. ambigua. The resulting planting stocks and saplings will be grown and introduced into natural conditions to restore degrading populations.

Figure 1. General view of the growing families of the Rosaceae plant territory, PLO is not a hero. Getting settled) Cotoneaster karatavicus (Population 1 (N1); B) Crataegus ambigua (N3); C) Malus sieversii (N2); d) Prunus tenella (N1); D) sibiraea laevigata (N1); F) Spiraeanthus schrenckianus (N1)

Figure 2. Introduction to in vitro culture of plants of the Rosaceae family (seeds. Cuttings, in vitro shoots, Testing of in vitro shoots on the medium 523). A) Prunus ulmifolia; Б) Spiraeanthus schrenckianus; ) Malus sieversii; В) Malus sieversii; Г) Sibiraea laevigata; Д) Crataegus ambigua; Е) Prunus tenella