Optimization of explants in vitro sterilization protocol of some deciduous tree species

Authors

DOI:

https://doi.org/10.31548/forest2021.03.007

Abstract

One of the methods of obtaining planting material of deciduous plants, in particular common ash (Fraxinus excelsior L.), broad-leaved linden (Tilia platyphyllos Scop.) and silver birch (Betula pendula Roth) is microclonal propagation. Asepticity of explants is a prerequisite for microclonal plant propagation. Chemical sterilization with liquid substances is mostly used for this purpose. The mode of decontamination is influenced by a number of factors, in particular the genotype of the plant. The purpose of the study was to optimize the sterilization protocol of F. excelsior, T. platyphyllos and B. pendula explants for microclonal propagation. For research, 20–30 cm of shoots isolated from 12-year-old T. platyphyllos, 10-year-old B. pendula, and 15-year-old F. excelsior in February-March 2021 were used. Plant material was cultured according to conventional methods on a nutrient medium MS (Murashige & Skoog, 1962). Biotechnological methods were used (plant tissue culture in vitro, microclonal propagation). MS Excel software package was used to process the experimental data, the mean and its standard error were calculated. One-way analysis of variance (ANOVA) was performed to analyze the effect of explant sterilization on asepsis. The expediency of keeping plant material during the day in 0.1 % solution of «Samshit» - F. excelsior and 0.3 % solution «Fundazole» - T. platyphyllos and B. pendula is shown. The sterilization protocol of experimental plants (efficiency over 50 %) was optimized by using a stepwise method using 70 % ethyl alcohol, 1.0 % and 2.0 % AgNO3 and 2.5 % and 5.0 % NaClO. The effect of the sterilization regime of experimental plants on asepsis is statistically significant at the level of 5%. To initiate the explants, a culture medium according to the MS prescription was used with the addition of 0.25 mg/L kinetin and 2.0 g/L activated carbon. Further studies are aimed at developing a protocol for direct regeneration of microshoots of F. excelsior, T. platyphyllos and B. pendula under the action of components of the culture medium in vitro.

Keywords: in vitro plant tissue culture, Fraxinus excelsior L., Tilia platyphyllos Scop., Betula pendula Roth, sterilization, explants, regenerative ability, culture medium, microclonal propagation.

References

Buckley, P. M., Reed, B. M. (1994). Antibiotic susceptibility of plant associated bacteria. HortScience, 29 (5), 434. https://doi.org/10.21273/HORTSCI.29.5.434c

Butenko, R. G. (1964). Culture of Isolated Tissues and Physiology of Plant Morphogenesis. Moscow, Russia: Science, 272 [in Russian].

Delgadillo-Díaz de León José Silvestre, José Francisco Morales-Domínguez, María del Socorro Santos-Díaz, Eugenio Pérez-Molphe-Balch (2013). In vitro Propagation of Mexican Oaks (Quercus spp.). Polibotánica, 35, 85−97.

Gaidamashvili, M., Khurtsidze, E., Barblishvili, T. (2015). Micropropagation of Threatened Betula Species for in vitro Conservation. International Conference on Plant, Marine and Environmental Sciences (PMES-2015). Kuala Lumpur (Malaysia), Jan. 1−2, 12−15. http://iicbe.org/upload/7805C0115056.pdf).

Kakimzhanova, A. A., Zhagipar, F. S., Naziran, F., Karimova, V. K., Nurtaza, A. S. (2019). Optimization of Microclonal Propagation Conditions for Increasing the Multiplication Factor of Poplar Microshoots. Bulletin of L.N. Gumilyov Eurasian National University. Bioscience Series, 1 (126), 57-65 [in Russian]. https://doi.org/10.32523/2616-7034-2019-126-1-57-65

Kalinin, F. L., Sarnatskaya, V. V., Polishchuk, V. E. (1980). Methods of Tissue Culture in Plant Physiology and Biochemistry. Kyiv: Naukova dumka, 488 [in Ukrainian].

Kärkönen, A., Santanen, A., Iwamoto, K., Fukuda, H. (2011). Plant Tissue Cultures. The Plant Cell Wall: Methods and Protocols. Methods in Molecular Biology, 715, 1-20. https://doi.org/10.1007/978-1-61779-008-9_1

Kataeva, N. V., Butenko, R. H. (1983). Clonal Micropropagation of Plants. Moscow, Russia: Science, 96 [in Russian].

Lebedev, V., Shestibratov, K. (2016). Large-Scale Micropropagation of Common Ash. Biotechnology, 15 (12), 19. https://doi.org/10.3923/biotech.2016.1.9

Lutova, L. A. (2003). Biotechnology of higher plants. SPb.: Publ house of -Peterb. UN, 228 [in Russian].

Murashige, T. A., Skoog, F. (1962). A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Plant Physiology, 15 (3), 473−497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Payamnour, V., Ghasemi Bezdi, K., Mehrdad, M., Ahmadi, A. (2014). Optimization of in vitro sterilization protocol for obtaining contamination-free cultures of Tilia platyphyllos. Nusantara Bioscience, 6 (1), 712. https://doi.org/10.13057/nusbiosci/n060102

Podhaietskyi, A. A., Mackiewicz, V. V., Wroblewski, S. A. (2016). Use of PPM biocide as an additional decontaminant in the process of microсloneal reproduction of vegetable substances. Bulletin of Sumy National Agrarian University. The series: Agronomy and Biology, 9 (32), 156160 [in Ukrainian].

Shabanova, E. A., Mashkina, O. S. (2015). Clonal micropropagation of economically valuable forms of poplar. Forest selection and genetics, 4, 74-81 [in Russian].

Shakhov, V. V., Fedotova, I. E., Tashmatova, L. V., Matsneva, O. V., Khromova, T. M. (2019). Comparative analysis of sterilizers on the basis of periodization of their use. Vestnik KrasGAU, 12, 38-42 [in Russian]. https://doi.org/10.36718/1819-4036-2019-12-38-42

Shakhov, V. V., Tashmatova, L. V., Matsneva, O. V., Khromova, T. M. (2018). The effectiveness of sterilizing agents in the introduction of cherry varieties to in vitro culture. Contemporary horticulture, 4, 32-37 [in Russian]. https://doi.org/10.24411/2312-6701-2018-10405

Skálová, D., Navrátilová, B., Richterová, L., Knitl, M., Sochor, M., Vašut, R. J. (2012). Biotechnological Methods of in vitro Propagation in Willows (Salix spp.). Central European Journal of Biology, 7 (5), 931−940. https://doi.org/10.31255/978-5-94797-319-8-1244-1247

Smith, R. H. (2012). Plant Tissue Culture: Techniques and Experiments. Burlington: Elsevier Science, 55.

Downloads

Published

2021-09-30

Issue

Vol. 12 No. 3 (2021)

Section

BIOLOGY OF FOREST AND URBAN ECOSYSTEMS

License

Relationship between right holders and users shall be governed by the terms of the license Creative Commons  Attribution – non-commercial – Distribution On Same Conditions 4.0 international (CC BY-NC-SA 4.0):https://creativecommons.org/licenses/by-nc-sa/4.0/deed.uk

Authors who publish with this journal agree to the following terms:

  • Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
  • Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
  • Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).