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AP 09258985 (superviser Mursaliyeva V.)

Brief description of the project

(Project’s duration is 36 months, 2021-2023 г.)

  Project title: ИРН АРО9258985 «Biotechnology development of adventitious root culture of turkestan soaproot Allochrusa gypsophiloides for production of commercially valuable secondary metabolites in vitro».

  Allochrusa gypsophiloides (Regel) Schischk. (family Caryophyllaceae) or Turkestan soaproot (TSR) is an endemic Central Asian species listed in the Red Book of Kazakhstan since 1981. TSR is a valuable medicinal plant of the natural flora of Kazakhstan, a super-producer of triterpene saponins with high surface-active properties and a wide range of pharmacological activity (bronchodilator, antiviral, immunostimulating, etc.). At present, natural reserves depletion and degrading state of endemic species natural populations because of economic activity in its growth area necessitates the search for alternative ways to obtain secondary metabolites from TSR.

The project idea is to develop and obtain adventitious root cultures (AR) of TSR, to assess their growth and biosynthetic activity in vitro for the production of commercially valuable saponins by a biotechnology and to preserve Allochrusa gypsophiloides endemic species.

  The Project goal: Development of scientific and biotechnological basis for in vitro secondary metabolites production in adventitious root culture of Allochrusa gypsophiloides, a valuable source of medical and technical saponins.

To achieve the goal, the main trophic and hormonal factors of the nutrient medium that regulate growth and biosynthetic potential of TSR in vitro will be studied; morphological, histological and biochemical features of differentiation and growth of adventitious roots have been revealed, the metabolic productivity of clonal root cultures of TSR has been assessed, quantitative and qualitative characteristics of triterpene saponins extracts obtained by the biotechnological method have been given.

  Expected research results: 1) the scientific and biotechnological basis for in vitro secondary metabolites production of the endemic species Allochrusa gypsophiloides based on clonal adventitious root cultures; 2) laboratory protocol of adventitious roots culture of TSR; 3) in vitro clonal adventitious root cultures of TSR with a high growth index and biosynthetic potential; 4) scientific and methodological recommendations for the adventitious root cultures of TSR.

  Scientific Supervisor of the project:

  Valentina Mursaliyeva, leading researcher of the Laboratory of Plant Physiology and Biochemistry of IPBB, Candidate of biol. Sc. (Ph.D.), chief specialist in the field of plant tissue culture and micropropagation of valuable plant species. Mursaliyeva V. K. was scientific supervisor of 7 applied projects (2000 — 2017), principal investigator within the framework of the scientific-technical programs of the IRPH “Phytochemistry” (2012-2014), Mangyshlak Experimental Botanical Garden (2018-2020). The author has published: 5 methodological recommendations, 11 innovative patents of the Republic of Kazakhstan, more than 80 publications, including 4 articles in referred international journals listed in Web of Science and Scopus database (https://app.webofknowledge.com/author/record/12173077,41802049, H-index 2, Scopus Author ID: 55428004100, ORCID iD: 0000-0003-4803-9169).

  Research group:

  Dr. Bakdaulet Usenbekov – Head of the laboratory, Candidate of biol. Sc. (Ph.D.), associated professor, has more than 70 publications, including 9 foreign articles in rating journals listed in Web of Science (ResearcherID: AAQ-3706-2020) or/and Scopus database (H-index 2, Scopus Author ID: 56447130000).

  Dr. Asil Nurzhanova, leading research scientist, Doctor of biol. Sci., professor, chief specialist in the field of cytogenetics and ecological biotechnology, has more than 12 articles in indexed journals listed in Web of Science (H-index 6, ResearcherID: M-6739-2015) or/and Scopus database (Scopus Author ID: 15029299700).

  Dr. Balakyz Yeskaliyeva, senior researcher, Candidate of chem. Sci. (Ph.D.), associated professor, leading specialist in the field of chemistry of natural compounds, has 18 foreign publications in rating journals listed in Web of Science (H-index 5, ResearcherID: B -2119-2015) or/and Scopus database (Scopus Author ID: 6505763988).

  Dr. Daria Satybaldiyeva, senior researcher, Ph.D., 8-years of research experience, co-author of 22 publications, including 2 indexed in Web of Science (ResearcherID: E-4942-2015, H-index 1), co-author of scientific-methodological recommendation and 2 patents for utility model.

  Ms. Tlek Mukchanov, junior researcher, 10-years of research experience, co-author of 5 publications in rating republic journals, scientific-methodological recommendation and patent for utility model.

  Ms. Alisher Kasymov, junior researcher, 12-years of research experience, has more than 12 publications, including 5 articles in CCES, co-author of scientific-methodological brochure.

  Foreign scientists: Dr. Ramazan Mammadov, Herbal products and Biotechnology Laboratory, Muğla Stkı Koçman University, Turkey. Dr. R.Mammadov has  more than 56 papers indexed in Web of Science (ResearcherID: F-6984-2017) or/and Scopus database (H-index 11, https://www.scopus.com/authid/detail.uri?authorId=16245554900).

  List of publications of the project’s participants (2015-2020)

  1. Mursaliyeva V., Imanbayeva A., Parkhatova R. (2020). Seed germination of Allochrusa gypsophiloides (Caryophyllaceae), an endemic species from Central Asia and Kazakhstan. Seed Science and Technology, (IF=0,5, Q4, SJR=0,209, percentile: 45). 48(2):289-295. DOI: https://doi.org/10.15258/sst.2020.48.2.15
  2. Mursaliyeva V., Imanbayeva A. (2020). Micropropagation of Turkestan Soap Root Allochrusa gypsophiloides – natural source of saponins. International Journal of Secondary metabolite, 7(1):1-7. https://doi.org/10.21448/IJSM.627140
  3. Satybaldiyeva D.N., Mursaliyeva V.K., Mammadov R., Zayadan B.K. (2016). Phenolic profiles and brine shrimp cytotoxicity of the ethanolic extract from the aerial part of Crocus alatavicus // Inter. J. of Biology and Chemistry (KazNU), 9(1):38-41. DOI: https://doi.org/10.26577/2218-7979-2016-9-1-38-41.
  4. Satybaldiyeva D., Mursaliyeva V., Rakhimbayev I., Zayadan B., Mammadov R. (2015). Preliminary Phytochemical Analysis and Antioxidant, Antibacterial Activities of Crocus alatavicus from Kazakhstan. Notulae Botanicae Horti Agrobotanici Cluj-Napoca (IF=1,168, Q3, SJR=0,35, percentile: 48). 43(2):343-348. DOI: https://doi.org/10.15835/NBHA43210089
  5. Mursaliyeva V.K., Kozhebaeva Zh.S., Rakhimbayev I., Gemejiiyeva N.G. (2016). Qualitative and quantitative analysis of saponins of Allochrusa gypsophiloides (Regel) Schischk. KazNU bulletin. Biology series, №3(68):114–123. DOI: https://doi.org/10.1901/kbs2016.68.3 (in Russian).
  6. Salehi B., Armstrong L., Rescigno A., Yeskaliyeva B., Seitimova G., Beyatli A., Sharmeen J., Mahomoodally M.F., Sharopov F., Durazzo A., Lucarini M., Santini A., Abenavoli L., Capasso R., Sharifi-Rad J. (2019). Lamium Plants — A Comprehensive Review on Health Benefits and Biological Activities. Molecules (IF=3,267, Q2, SJR=0,698, percentile: 71), 24 (10):1913-1936, DOI: https://doi.org/10.3390/molecules24101913
  7. Eskalieva B.K., Burasheva G. Sh, Adhikari A., Aisa H., Choudhary M. (2016). Saponins from Climacoptera subcrassa. Chemistry of Natural Compounds, 52(2):363-364. DOI:  https://doi.org/10.1007/s10600-016-1646-z
  8. Muratova A.Yu., Lyubun Y.V., Sungurtseva I.Yu., Nurzhanova A.A., Turkovskaya O.V. (2019). Physiological and biochemical responses of Miscanthus × giganteus to soil contamination with heavy metals. Ecobiotechnology, 2 (4):482-493. DOI: 10.31163/2618-964X-2019-2-4-482-493. RISC (in Russian).
  9. Patent for utility model No. 2969. A method for seed germination increasing of Turkestan soap root Allochrusa gypsophiloides (2017). Mukhanov T.M., Gemedzhieva N.G., Nam S.V., Mursaliyeva V. K.
  10. Patent for utility model No. 2274. The micropropagation of Turkestan soap root Allochrusa gypsophiloides (2016). Mursaliyeva V.K., Kozhebaeva Zh.S.

   Results obtained for 2021

  In vitro establishment, micropropagation of the initial material were carried out. The clonal adventitious root culture (AR) of TSR from 15 highly productive lines collected from the natural populations in the south of Kazakhstan were obtained.

An optimal schedule of micropropagation of the original seeds and the vegetative initial material during 7 months’ cultivation has been developed for obtaining of the primary aseptic culture of TSR.  The clonal AR of TSR from the root tips from seedlings in vitro and from aseptic rooted shoots (micro clones) with the optimal cultivation cycle duration of 1.5 months has been received. The general dynamics of biomass accumulation of AR in vitro has been established:  increasing to 40-50 cultures’ days with a further decrease. The stimulating effect of auxins (IBA and NAA) on the root growth index was 214% compared to control medium (without auxin) with the earlier inducing IBA action. It was revealed that the characteristic accumulation order of metabolites (carbohydrates> saponins> phenols> flavonoids) in vitro was preserved. The influence of the exogenous phytohormones (auxins) on the metabolite levels in AR culture was established.

The saponins content excess 2 times (in control) and 3 times (for medium with NAA or IBA) in relation to their level in native root extracts was revealed. The introduction of IBA into the medium stimulated the maximum saponins accumulation in the AR biomass with their active release into the medium and caused a decrease of flavonoids content in the biomass due to their exudation into the medium. The selective accumulation of phenols and flavonoids in vitro compared their level in native roots extracts was revealed: in NAA medium — the phenol increase in 3 times, in the control — flavonoids in 4.5 times.

 Results for 2022:

The influence of trophic and hormonal factors of cultivation on the growth, differentiation of adventitious roots (AR), and biosynthetic potential AR culture in vitro was studied. A laboratory procedure for obtaining a culture of AR of TSR has been developed. Optimal physical conditions for the growth activity of the culture: a constant temperature regime of 25-28 ° C, aeration of the culture by continuous mixing at a speed of 100 rpm, and darkness. The optimal ratio of the number of explants to the volume of the culture flask with nutrient medium: 10/250 (50 ml of medium) and 20/300 (100 ml of medium). The optimal duration of the AR cultivation cycle is 50-60 days, the optimal medium is the liquid basic MS medium with half the concentration of macro-micro salts, 30 g/l of sucrose, and 1 mg/l of IBA or NAA.

Histological study of rhizogenesis in vitro showed that the stimulating effect of exogenous auxins is associated with callus induction on primary explants, followed by mass differentiation of root apexes in the callus, mass development of adventitious roots with the formation of an extensive root network. The type of auxin (NAA or IBA) affected the duration of the induced processes but had no significant effect on the growth index and the saponins content in the culture. The accelerated stimulating effect of IBA led to a reduction in the duration of the slow growth phase, to an increase in the duration of the log-growth phase with a maximum growth index of Kr 426, and to a longer stationary growth of the AR culture. Prolonged action of NAA was noted in the lengthening of the slow growth phase, a reduction in the duration of the log-growth phase with a maximum increase in Kr 439. Induced rhizogenesis on auxin-containing medium led to a twofold accumulation of root biomass compared to the control medium and a threefold increase of saponins content in vitro relative to their level in native roots.

  Publications (2022):

  1. Mursaliyeva V., Algazy A., Mukhanov T., Mammadov R., Gemejiyeva N. Quantitative determination of metabolites of Turkestan soaproot (Allochrusa gypsophiloides Regel Schischk.) grown in Kazakhstan // International Journal of Nature and Life Sciences. 2022, vol. 6(2), pp. 129-141. https://doi.org/10.47947/ijnls.1180197
  2. Atli B., Ozcakir B., Isik B., Mursaliyeva V., & Mammadov R. Secondary Metabolites in Fungi. Natural Products and Biotechnology, 2022, 2(2), рр. 114–138. Retrieved from https://natprobiotech.com/index.php/natprobiotech/article/view/41
  3. Algazy A.T., Mukhanov T.M., Sarsenbek B.T., Usenbekov B. N., Mursaliyeva V.K. In vitro establishment of Turkestan soaproot Allochrusa gypsophioloides // Abst. of the 3rd Inter. scient. and Pract. Confer. dedicated to the 85th anniversary of the Tashkent Pharmaceutical Institute «Modern pharmaceutics: actual problems and prospects», November 25-26, 2022, Toshkent, pp. 62-63.

  Patent

Patent RK for utility model №7649. Method for obtaining adventitious roots culture of Turkestan soaproot Allochrusa gypsophiloides in vitro.  Mursaliyeva V., Algazy A., Mukhanov T., Sarsenbek B. Application 2022/0918.2 from 11.10.2022. Bull. N49 from 09.12.2022.

  Scientific internships:

Mugla Sitki Kocman University (г. Мугла, Турция), internship on mastering methods for determining antioxidant and antibacterial activity, November 21-28, 2022