Brief description of the project
(2023-2025)
Project title: IRN AP19676481 “Study of genetic diversity and development of biotechnology for ex situ conservation of cherry species (Prunus spp.) in Kazakhstan”.
Relevance.
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 cherrн 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 Pall., Prunus erythrocarpa (Nevski) Gilli, Prunus griffithii var. tianshanica (Pojark.) Ingram, Prunus verrucosa 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 biotechnology and cryopreservation for the reliable preservation of this valuable material in an aseptic culture and a cryogenic bank at ultra-low temperature (-196°C).
The goal of the project: Phenotypic and molecular genetic study of biodiversity of cherry (Prunus spp.) species in Kazakhstan and conservation of germplasm accessions based on in vitro biotechnology and cryopreservation.
Expected results:
- Expedition trips, phenotypic description and collection of wild cherry species plant material (resting vegetative buds, fruits, leaves, young shoots). Formation of an herbarium collection.
- Plant material accessions in vitro culture initiation, creation of aseptic plants collection of wild cherry species.
- Study of the wild cherry species genetic diversity using microsatellite DNA markers and DNA barcoding.
Scientific Supervisor of the project:
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
Research group:
1) Romadanova Natalya, Leading Researcher, Candidate of Biological Sciences. h-index 5. Scopus ID 36111616700; Web of Science BBD-5948-2021;
2) Aubakirova K., Leading Researcher, h-index 1, Researcher ID Web of Science M-6628-2015, Scopus Author ID 56097194200
3) Satekov E., Senior Researcher
4) Aralbaeva M., Researcher, h-index 1, Researcher ID Web of Science GBA-8887-2022, Scopus Author ID 57195069563.
5) Rymkhanova N., Researcher, h-index 1, Researcher ID Web of Science FVM-9722-2022, Scopus Author ID 57773405800.
6) Manapkanova U., Junior Researcher
List of publications of the project’s participants (2018-2022)
1) 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. 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.
2) 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. – 2022. – Vol. 11. – P. 2624-2635. https://doi.org/10.3390/plants11192624, quartiles Q1, percentile 83, Field-weighted Citation Impact (FWCI) 1,1.
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 // Molecules. – 2018. – 23(7). – Р. 1679-1696. DOI: 10.3390/molecules23071679, quartiles Q2, percentile 78, Field-weighted Citation Impact (FWCI) 1,97.
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 “Biology”). – 2022. – № 4 (93). – С. 4-13. https://doi.org/10.26577/eb.2022.v93.i4.01.
5) М.М. Aralbayeva, N.V. Mikhailenko, S.V. Kushnarenko Development of rooting method for Juglans regia L. and Corylus avellana L. in vitro shoot // Bulletin of the Karaganda University. Series «Biology. Medicine. Geography». – 2022. – № 3(107). – С. 24-32. DOI 10.31489/2022BMG3/24-32.
6) S.V. Kushnarenko, N.V. Romadanova, Т.Т. Тurdiyev, М.М. 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. – 2022. – № 2 (71). – С. 72-80. DOI: 10.26577/EJE.2022.v71.i2.07.
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 «Biology. Medicine. Geography». – 2022. – № 1 (105). – С. 86-95. DOI 10.31489/2022BMG1/86-95.
8) S.V. Kushnarenko, N.V. Romadanova, N.P. Ogar, M.M. Aralbaeva, M.A. Verzilov Current state of Hazelnut (Corylus avellana L.) population in Kazakhstan // Bulletin of the Karaganda University. Series «Biology. Medicine. Geography». – 2019. – № 2 (94). – С. 99-104. URL: https://biollogy-medcine-geography-vestnik.ksu.kz/2019-94-2.
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). – 2018. – №3 (98). – С. 4-13. URL: http://rmebrk.kz/magazine/4540#
Patents:
10) Patent № 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
11) Patent № 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
Results for 2023:
Expeditionary trips were carried out to collect wild cherry species material. The fruits and leaves of 15 accessions of Prunus griffithii var. tianshanica were collected in the Almaty region, 7 in the Zhambyl region and 2 in the Turkestan region; 35 accessions of Prunus fruticosa in the Kostanay region; 9 accessions of Prunus erythrocarpa in Zhambyl region and 12 – in Turkestan region; 7 accessions of Prunus verrucosa Franch. in the Turkestan region. A phenotypic description of 87 samples of wild cherry species was carried out using descriptors recommended for Prunus. Herbariums have been formed for representatives of four wild cherry species.
The initiation of seeds, embryos and embryonic axes of four wild cherry species: Prunus tianshanica, Prunus fruticose, Prunus erythrocarpa, Prunus verrucosa in vitro 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.
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 μl containing a thermostable DNA polymerase buffer, 0.2 μl (10 μM) of forward and reverse oligonucleotides, 0.4 μl (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°C; 35 cycles consisting of the following steps — 30 sec. at 94°C, 30 sec. at 57°C and 1 min. at 72°C; and finally, 1 cycle of 10 min. at 72°C. 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.
Publications (2023):
1) 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 – p. 17.
