{"id":24424,"date":"2024-01-09T16:09:37","date_gmt":"2024-01-09T10:09:37","guid":{"rendered":"http:\/\/ipbb.kz\/eng\/?page_id=24424"},"modified":"2025-12-24T17:55:08","modified_gmt":"2025-12-24T11:55:08","slug":"ap-19676481-superviser-kushnarenko-s","status":"publish","type":"page","link":"https:\/\/ipbb.kz\/eng\/ap-19676481-superviser-kushnarenko-s\/","title":{"rendered":"AP 19676481 (superviser Kushnarenko S.)"},"content":{"rendered":"

Brief description of the project <\/strong><\/p>\n

(2023-2025)<\/p>\n

\u00a0<\/strong><\/p>\n

Project title<\/strong>:<\/strong> IRN AP19676481 \u201cStudy of genetic diversity and development of biotechnology for ex situ<\/em> conservation of cherry species (Prunus <\/em>spp.) in Kazakhstan\u201d.<\/p>\n

Relevance.\u00a0<\/strong><\/p>\n

Cherry is one of the most popular stone fruit crops in the world. According to the Food and Agriculture Organization of the United Nations (FAO), in 2020 the global production of cherr\u043d was about 4.0 million tons, with almost continuous growth in global production and consumption every year. According to the report of the Agency of Statistics of the Republic of Kazakhstan, the cultivation area cherry in Kazakhstan is about 1,500 hectares, which are mainly concentrated in private households, cherry is used very little for commercial cultivation, as a result of which Kazakhstan has to import cherry fruits annually in the amount of about 25 thousand tons. There are about 150 cherry species in the world, 4 wild cherry species grow in Kazakhstan: Prunus fruticosa<\/em> Pall., Prunus erythrocarpa<\/em> (Nevski) Gilli, Prunus griffithii<\/em> var. tianshanica<\/em> (Pojark.) Ingram, Prunus verrucosa<\/em> Franch. The biodiversity of wild fruit species provides an inexhaustible genepool for breeding. Wild cherry species of Kazakhstan are a source of genes for high drought resistance, frost resistance, have all the qualities that intensive fruit growing currently presents to the rootstock of stone fruit. Within the framework of the project, it is planned to carry out a phenotypic and molecular-genetic description of wild species in Kazakhstan, to develop in vitro<\/em> biotechnology and cryopreservation for the reliable preservation of this valuable material in an aseptic culture and a cryogenic bank at ultra-low temperature (-196\u00b0C).<\/p>\n

The goal of the project<\/strong>: <\/strong>Phenotypic and molecular genetic study of biodiversity of cherry (Prunus <\/em>spp.) species in Kazakhstan and conservation of germplasm accessions based on in vitro<\/em> biotechnology and cryopreservation.<\/p>\n

Expected results<\/strong>:<\/p>\n

    \n
  1. Expedition trips, phenotypic description and collection of wild cherry species plant material (resting vegetative buds, fruits, leaves, young shoots). Formation of an herbarium collection.<\/li>\n
  2. Plant material accessions in vitro<\/em> culture initiation, creation of aseptic plants collection of wild cherry species.<\/li>\n
  3. Study of the wild cherry species genetic diversity using microsatellite DNA markers and DNA barcoding.<\/li>\n<\/ol>\n

    Scientific Supervisor of the project<\/strong>:<\/strong><\/p>\n

    Head of the Laboratory of Germplasm Cryopreservation, Candidate of Biological Sciences, Professor Kushnarenko S.V. Published more than 200 scientific papers, 22 publications in international peer-reviewed scientific journals in the Web of Science indexed database, 20 articles included in the Scopus database. H- index 9, Scopus ID 6507153914; Web of Science ID M-6616-2015<\/p>\n

    Research group<\/strong>:\u00a0 <\/strong><\/p>\n

    1)\u00a0 Romadanova Natalya, Leading Researcher, Candidate of Biological Sciences. h-index 5. Scopus ID 36111616700; Web of Science BBD-5948-2021;<\/p>\n

    2)\u00a0 Aubakirova K., Leading Researcher, h-index 1, Researcher ID Web of Science M-6628-2015, Scopus Author ID 56097194200<\/p>\n

    3)\u00a0 Satekov E., Senior Researcher<\/p>\n

    4)\u00a0 Aralbaeva M., Researcher, h-index 1, Researcher ID Web of Science GBA-8887-2022, Scopus Author ID 57195069563.<\/p>\n

    5)\u00a0 Rymkhanova N., Researcher, h-index 1, Researcher ID Web of Science FVM-9722-2022, Scopus Author ID 57773405800.<\/p>\n

    6)\u00a0 Manapkanova U., Junior Researcher<\/p>\n

    List of publications of the project\u2019s participants (<\/strong>2018-2022) <\/strong><\/p>\n

    1)\u00a0 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 \u2013 Plant. \u2013 2022. https:\/\/doi.org\/10.1007\/s11627-022-10279-4, IF 2,6; WOS quartiles Q2, Scopus percentile 72, Field-weighted Citation Impact (FWCI) 0,43.<\/p>\n

    2)\u00a0 Romadanova N.V., Tolegen A.B., Kushnarenko S.V., Zholdybayeva E.V., Bettoni J.C. Effect of Plant Preservative MixtureTM on endophytic bacteria eradication from in vitro-grown apple shoots \/\/ Plants. \u2013 2022. \u2013 Vol. 11. \u2013 P. 2624-2635. https:\/\/doi.org\/10.3390\/plants11192624, quartiles Q1, percentile 83, Field-weighted Citation Impact (FWCI) 1,1.<\/p>\n

    3) Utegenova G., Pallister K., Kushnarenko S., Ozek G., Ozek T., Abidkulova K., Kirpotina L., Schepetkin I., Quinn M., Voyich J. Chemical composition and antibacterial activity of essential oils from Ferula L. species against methicillin-resistant Staphylococcus aureus<\/em> \/\/ Molecules. \u2013 2018. \u2013 23(7). \u2013 \u0420. 1679-1696. DOI: 10.3390\/molecules23071679, quartiles Q2, percentile 78, Field-weighted Citation\u00a0 Impact (FWCI) 1,97.<\/p>\n

    4) A.S. Zemtsova, S.V. Kushnarenko, N.V. Romadanova Creation of in vitro collection and seeds cryogenic bank of Berberis Heteropoda \/\/ Experimental Biology (KazNU journal. Series \u201cBiology\u201d). \u2013 2022. \u2013 \u2116 4 (93). \u2013 \u0421. 4-13. https:\/\/doi.org\/10.26577\/eb.2022.v93.i4.01.<\/p>\n

    5) \u041c.\u041c. Aralbayeva, N.V. Mikhailenko, S.V. Kushnarenko Development of rooting method for Juglans regia L. and Corylus avellana L. in vitro<\/em> shoot \/\/ Bulletin of the Karaganda University. Series \u00abBiology. Medicine. Geography\u00bb. \u2013 2022. \u2013 \u2116 3(107). \u2013 \u0421. 24-32. DOI 10.31489\/2022BMG3\/24-32.<\/p>\n

    6) S.V. Kushnarenko, N.V. Romadanova, \u0422.\u0422. \u0422urdiyev, \u041c.\u041c. Aralbayeva, K.R. Kalybayev Preservation in a cryobank of Juglans regia L. accession from several populations in the Sairam-Ugam State National Natural Park \/\/ KazNU journal. Ecology series. \u2013 2022. \u2013 \u2116 2 (71). \u2013 \u0421. 72-80. DOI: 10.26577\/EJE.2022.v71.i2.07.<\/p>\n

    7) N.V. Romadanova, M.M. Aralbaeva, N.K. Rymkhanova, D.Sh. Baigaraev, A.K. Ramazanov, M.Iu. Ishmuratova, S.V. Kushnarenko Cryopreservation as a way to improve laboratory germination and germination energy of seeds \/\/ Bulletin of the Karaganda University. Series \u00abBiology. Medicine. Geography\u00bb.\u00a0 \u2013 2022. \u2013 \u2116 1 (105). \u2013 \u0421. 86-95. DOI 10.31489\/2022BMG1\/86-95.<\/p>\n

    8) S.V. Kushnarenko, N.V. Romadanova, N.P. Ogar, M.M. Aralbaeva, M.A. Verzilov Current state of Hazelnut (Corylus avellana<\/em> L.) population in Kazakhstan \/\/ Bulletin of the Karaganda University. Series \u00abBiology. Medicine. Geography\u00bb. \u2013 2019. \u2013 \u2116 2 (94). \u2013 \u0421. 99-104. URL: https:\/\/biollogy-medcine-geography-vestnik.ksu.kz\/2019-94-2<\/a>.<\/p>\n

    9) N.V. Romadanova, M.M. Nurmanov, I.A. Makhmutova., S.V. Kushnarenko Production of super-elite planting stocks of apple varieties and clonal rootstocks \/\/ Bulletin of Science of the S.Seifullin Kazakh Agrotechnical Research University (interdisciplinary). \u2013 2018. \u2013 \u21163 (98). \u2013 \u0421. 4-13. URL: http:\/\/rmebrk.kz\/magazine\/4540#<\/p>\n

    Patents:<\/p>\n

    10) \u00a0Patent \u2116 34902 for the invention dated 02.2021. N.V. Romadanova, Nurmanov M.M., S.V. Kushnarenko, A chemotherapy method for obtaining virus-free apple plant material.https:\/\/gosreestr.kazpatent.kz\/Invention\/Details?docNumber=323672<\/p>\n

    11) Patent \u2116 4998 for utility model dated 05.06.2020. The method of hazelnut propagation. S.V. Kushnarenko, M.M. Aralbaeva, N.V. Romadanova, https:\/\/gosreestr.kazpatent.kz\/Utilitymodel\/Details?docNumber=320230<\/p>\n

    Results for 2023:<\/strong><\/p>\n

    Expeditionary trips were carried out to collect wild cherry species material. The fruits and leaves of 15 accessions of Prunus griffithii var. tianshanica<\/em> were collected in the Almaty region, 7 in the Zhambyl region and 2 in the Turkestan region; 35 accessions of Prunus fruticosa<\/em> in the Kostanay region; 9 accessions of Prunus erythrocarpa<\/em> in Zhambyl region and 12 \u2013 in Turkestan region; 7 accessions of Prunus verrucosa<\/em> Franch. in the Turkestan region. A phenotypic description of 87 samples of wild cherry species was carried out using descriptors recommended for Prunus<\/em>. Herbariums have been formed for representatives of four wild cherry species.<\/p>\n

    The initiation of seeds, embryos and embryonic axes of four wild cherry species: Prunus tianshanica,<\/em> Prunus fruticose, Prunus erythrocarpa, Prunus verrucosa in vitro<\/em> culture to obtain aseptic plants has begun. The fruits were freed from mesocarp, washed in a soap solution for 5 minutes, then with running water. After removing endocarp, the seeds (kernels) were disinfected in a 0.1% mercuric chloride for 5 minutes under a laminar box. After washing in sterile distilled water, the explants were placed in test tubes with Murashige-Skoog and Woody Plant Medium with various combinations of phytohormones: BAP, IBA and GA. This disinfection regimen was quite effective for these explants, and no fungal or bacterial contamination was detected. A collection of wild four cherry species aseptic plants has been created.<\/p>\n

    DNA isolation and purification, barcoding (amplification and sequencing of 3 chloroplast regions and 1 nuclear genome region) have begun. Genomic DNA from dried cherry leaves was isolated using a Doyle and Doyle modified method. DNA was isolated from 87 accessions of four cherry species. DNA barcoding of wild cherry representatives has begun using markers of three plastid DNA genes (rbcLa, ITS and matK) and one spacer region (trnH2\/psbA). PCR was performed using the following primers: rbcLa (5′-ATGTCACCACAAACAGAGACTAAAGC-3′, 5′-GTAAAATCAAGTCCACCRCG-3′); matK (5′- ACCCAGTCCATCTGGAAATCTTGGTTC-3′), (5′-GTACAGTACTTTTGTGTTTACGAG-3′); trnH-pcbA (5′-GTTATGCATGAACGTAATGCTC-3′), (5′-CGCGCATGGTGGATTCACAATCC-3′); ITS (5′-ACGAATTCATGGTCCGGTGAAGTGTTCG -3′), (5′-TAGAATTCCCCGCTTCGCTCGCCGTTAC-3′). PCR was carried out in a volume of 20 \u03bcl containing a thermostable DNA polymerase buffer, 0.2 \u03bcl (10 \u03bcM) of forward and reverse oligonucleotides, 0.4 \u03bcl (10 mM) of nucleotide solutions, 40 ng of DNA and 0.4 units of DNA polymerase. Amplification was carried out according to the following program: one cycle of 4 min. at 94\u00b0C; 35 cycles consisting of the following steps — 30 sec. at 94\u00b0C, 30 sec. at 57\u00b0C and 1 min. at 72\u00b0C; and finally, 1 cycle of 10 min. at 72\u00b0C. The resulting amplification products were purified for subsequent sequencing. Sequencing is performed using BigDye v3.1 according to the manufacturer’s recommendations (Thermofisher) on a 3500 Genetic Analyzer.<\/p>\n

    Publications (2023):<\/strong><\/p>\n

    1)\u00a0 S. Kushnarenko, M. Aralbayeva, N. Rymkhanova, U. Manapkanova, A. Tolegen, E. Satekov, N. Romadanova, T. Turdiyev Ex situ preservation of Prunus spp. diversity in Kazakhstan. \/\/ 36th International Conference on Ecological Agriculture, Biodiversity and Waste Management (EABWM-23), December 18-22, Bangkok, Thailand \u2013 p. 17.<\/p>\n

     <\/p>\n

    Project results for 2024 AP19676481 <\/strong><\/p>\n

    \u201cStudy of genetic diversity and development of biotechnology for ex situ conservation of cherry species (Prunus<\/em> spp.) in Kazakhstan\u201d<\/strong><\/p>\n

    Expeditionary trips to the Almaty, Turkestan and Kostanay regions were continued to collect plant material (leaves and fruits) of four species of wild cherry. Flowering and fruiting periods of four cherry species was recorded, and mixed forms between the species were noted. Fruits and leaves were collected (for in vitro<\/em> culture initiation and molecular genetic analysis) from 25 accessions of Prunus griffithii<\/em> var. tianshanica<\/em> in the Uyghur district and 2 accessions in the Zhetysu district of the Almaty region, 18 accessions in the Turkestan region; 36 accessions of Prunus verrucosa<\/em> Franch. in the Turkestan region; 19 accessions of Prunus erythrocarpa<\/em> (Nevski) Gilli in the Turkestan region and 31 accessions of Prunus fruticosa<\/em> Pall. in the Kostanay region. In addition, an expedition was conducted to the Republic of Uzbekistan to analyze the species differences of wild cherry species, plant material (leaves and fruits) of three cherry species were collected in the Tashkent region from three different locations: 6 accessions of P<\/em>. <\/em>griffithii<\/em> var. tianshanica<\/em>, 14 accessions of P<\/em>.<\/em> erythrocarpa<\/em> and 14 accessions of P<\/em>. <\/em>verrucosa<\/em> (Figure 1).<\/p>\n

    \"\"<\/a>Figure 1 \u2013 Expeditionary trips, phenotypic description and collection of plant material of wild cherry species<\/p>\n

    A \u2013 Collection of Prunus griffithii<\/em> var. tianshanica<\/em> in Almaty region<\/p>\n

    B \u2013 Fruits of Prunus erythrocarpa <\/em>(Nevski) Gilli<\/p>\n

     <\/p>\n

    Isolated embryos and embryonic axes were used as explants for in vitro<\/em> culture initiation. Further observations showed that the most effective explant for shoot formation initiation is embryo, and plant development began in 5-7 days. It was found that the highest yield of healthy plants during in vitro<\/em> culture initiation is observed on the Murashige-Skoog (MS) medium with double iron chelate and phytohormones: 0.5 mg \/ l 6-benzylaminopurine (BAP), 0.01 mg \/ l indolyl-3-butyric acid (IBA) and 0.75 mg \/ l gibberellic acid (GA), 30 g \/ l sucrose, pH 5.7. The percent of green plants on this medium for four types of cherry averaged 64%, fungal contamination was 14.7% and necrosis 21.3%. The obtained plants were grown in a light culture room at a temperature of 24\u00b0C, illumination of 25 \u00b5mol\u2022m-2<\/sup>\u2022s-1<\/sup>, a 16-hour photoperiod, with subculturing onto fresh nutrient media every 4 weeks. Currently, 2300 aseptic shoots of four cherry species are maintained in vitro<\/em> culture, which will be used to develop a cryopreservation protocol and establishing a cryocollection (Figure 2).<\/p>\n

    \"\"<\/a><\/p>\n

    Figure 2 \u2013In vitro<\/em> culture initiation of plant material.<\/p>\n

    A \u2013 In vitro<\/em> culture initiation of Prunus fruticosa<\/em>.<\/p>\n

    B \u2013 Establishing an in vitro<\/em> collection of wild cherry species<\/p>\n

    Work continued on DNA extraction and purification, barcoding, and sample analysis using SSR markers. Sequencing (barcoding) of three plastid DNA fragments (rbcLa, trnH2\/psbA, and matK) was performed. PCR products were purified and sequenced in both directions using forward and reverse primers, with the BigDye v3 sequencing kit on an ABI3500 genetic analyzer. Barcoding was conducted on 3\u20135 samples from each population. A total of 23 accessions collected from various regions of Kazakhstan were analyzed.<\/p>\n

     <\/p>\n

    Results for 2025<\/strong><\/p>\n

    For the first time, a comprehensive study was conducted, including phenotypic assessment, analysis of genetic diversity and population structure of four wild cherry species collected in various regions of Kazakhstan and Uzbekistan. Field trips were conducted in three regions of Kazakhstan (Almaty, Turkestan and Kostanay) and two regions of Uzbekistan (Tashkent and Jizzakh). A total of 163 accessions of four species of wild cherry were collected. The following plant material was collected: fruits, leaves, young shoots, and dormant vegetative buds. The collected material was used for DNA extraction and in vitro<\/em> culture initiation.<\/p>\n

    Genetic diversity was assessed based on 13 SSR markers and three chloroplast markers. Inter-species differences in the level of genetic diversity were identified. Of the 163 accessions analysed, 143 (87.7%) were identified as pure accessions. These 143 accessions were used for further analysis and included: 33 accessions of Prunus griffithii<\/em> var. tianshanica<\/em>, 29 of P. erythrocarpa<\/em>, 31 of P. fruticosa<\/em>, and 51 of P. verrucosa<\/em>. The remaining 20 accessions were identified as hybrids and excluded from further genetic studies. Hybrid accessions were detected in three species (P. griffithii<\/em> var. tianshanica<\/em>, P. erythrocarpa<\/em>, and P. verrucosa<\/em>), while no hybrids were found in P. fruticosa<\/em>. The highest proportion of hybrids was observed in P. griffithii<\/em> var. tianshanica<\/em> (34.4%), followed by P. erythrocarpa<\/em> (18.5%) and P. verrucosa<\/em> (8.0%).<\/p>\n

    The results obtained demonstrate significant differences in the level of genetic diversity between the studied species of the genus Prunus<\/em>. P. verrucosa <\/em>was characterised by the highest genetic diversity, while P. fruticosa<\/em> showed the lowest level of genetic variability. Assessment of inter-species differences showed that P. fruticosa<\/em> is the most isolated cluster. Samples of P. griffithii<\/em> var. tianshanica<\/em> collected from an isolated population also formed a clearly distinct independent cluster.<\/p>\n

    \"\"<\/a>

    Figure 1 \u2013 Clusters were derived from structural analysis of 123 cherry accessions based on 13 SSR markers. Each column corresponds to each individual divided into three genetic clusters (K = 3). Each color represents the estimated proportion of membership in the three genetic clusters (cluster 1 = blue; cluster 2 = purple, cluster 3 = orange).<\/p><\/div>\n

    For the first time, an in vitro<\/em> collection of four wild cherry species of Kazakhstan has been established. The composition of medium for micropropagation and shoot rooting has been optimised.<\/p>\n

    An effective protocol for cryopreservation of shoot tips and isolated embryonic axes has been developed, and a cryobank of four wild cherry species has been obtained at a temperature of -196\u00baC. To optimize the cryopreservation protocol for shoot tips, the cold acclimation effects on in vitro<\/em> plant and shoot tips treatment time to the PVS2 cryoprotectant were investigated. It was found that cold acclimation of aseptic plants under alternating temperature conditions (8 h at 22\u00b0C with illumination of 10 \u00b5Mm\u207b\u00b2s\u207b\u00b9 \/ 16 h at -1\u00b0C in the dark) had a significant impact on cryopreservation efficiency. The regeneration rate of cryopreserved shoot tips increased with longer precultures durations. The highest regeneration rate (ranging from 59.9% to 67.8%) was observed after four weeks of cold acclimation for all accessions.<\/p>\n

    C \u2013 control (without liquid nitrogen exposure), LN \u2013 after liquid nitrogen exposure<\/p>\n

    \"\"<\/a>

    Figure 2 \u2013 Isolated shoot tips of Prunus fruticosa Pall. at the time of isolation (a) and after PVS2 vitrification in liquid nitrogen (LN) (b) (scale bar = 1 cm)<\/p><\/div>\n

    The conditions for the adaptation of aseptic plants when transferred to soil substrate have been optimised. Container cultures of all four wild cherry species have been obtained. As a result of the conducted research, planting material of four cherry species was obtained in container culture. This material can be used for further physiological, biochemical, molecular-genetic, and cryobiological studies, as well as for subsequent use in breeding and restoration programs.<\/p>\n

    \"\"<\/a>

    Figure 3 \u2013 Rooted shoots of Prunus fruticosa Pall.in soil substrate<\/p><\/div>\n

     <\/p>\n

    As a result of research conducted under the project \u201cStudy of genetic diversity and development of biotechnology for ex situ<\/em> conservation of cherry species (Prunus<\/em> spp.) in Kazakhstan\u201d the work has been completed in full.<\/p>\n

    Information for potential users<\/strong><\/p>\n

    The established in vitro<\/em> collection and plants with a closed root system of four wild cherry species can be used in scientific research, incorporated into breeding programs, and applied for the restoration of disturbed ecosystems in Kazakhstan.<\/p>\n

    Publications 2025<\/strong><\/p>\n

    List of publications in peer-reviewed scientific journals indexed in the Web of Science and (or) Scopus databases<\/em><\/strong><\/p>\n