Вестник Томского государственного университета. Биология. 2019; : 22-39
Влияние длительного субкультивирования на клональное микроразмножение Melissa officinalis L. и Origanum vulgare L.
https://doi.org/10.17223/19988591/47/2Аннотация
Изучена динамика морфометрических показателей развития эксплантов при длительном субкультивировании трех сортов Melissa officinalis L. (Цитронелла, Соборная, Крымчанка) и селекционного образца Origanum vulgare L. на втором этапе клонального микроразмножения. В качестве эксплантов на этом этапе использовали сегменты стебля с одним узлом, которые культивировали на питательной среде Мурасиге и Скуга, дополненной 0,5 мг/л БАП. Анализ морфогенеза эксплантов показал активное множественное побегообразование, при котором у мелиссы формировалось от 2,2 до 5,7, а у душицы - от 9,5 до 54,8 побега на эксплант. При микроразмножении наблюдали образование корней у микропобегов (у мелиссы с частотой 44,4-92,7%) и формирование оводненных микропобегов (у душицы с частотой 16,7-44,5%). Частота этих процессов зависела от вида или сорта и количества субкультивирований. Максимальные коэффициенты размножения у мелиссы сорта Цитронелла (до 12,0) отмечены в 3-5, а у Крымчанки (17,6) и Соборной (14,2) - в пятом субкультивировании. У O. vulgare наибольшее увеличение коэффициента размножения (до 74,1) выявлено в пятом пассаже, а в более поздних 12-13-м пассажах показано снижение этого показателя почти в 4раза. Полученные данные свидетельствуют о возможности длительного микроразмножения душицы и мелиссы in vitro (как минимум, в течение 1-1,5 года), при этом максимальная эффективность этого процесса отмечена в течение пятого субкультивирования.
Список литературы
1. Паштецкий В.С., Невкрытая Н.В., Мишнев А.В., Назаренко Л.Г. Эфиромасличная отрасль Крыма. Вчера, сегодня, завтра. Симферополь : ИТ «Ариал», 2018. 320 с.
2. Атлас лекарственных растений России / под ред. А.В. Быкова. М. : Щербинская типография, 2006. 345 с.
3. Moradkhani H., Sargsyan E., Bibak H., Naseri B., Sadat-Hosseini M., Fayazi-Barjin A., Meftahizade H. Melissa officinalis L., a valuable medicine plant: A review // Journal of Medicinal Plants Research. 2010. Vol. 4, № 25. PP. 2753-2759.
4. Garcia-Beltran J.M., Esteban M.A. Properties and application of plants of Origanum sp. genus // SM Journal of Biology. 2016. № 2 (1). 1006. PP. 1-10.
5. Derwich E., Benziane Z., Manar A., Boukir A., Taouil R. Phytochemical analysis and in vitro antibacterial activity of the essential oil of Origanum vulgare from Morocco // American-Eurasian Journal of Scientific Research. 2010. Vol. 5, № 2. PP. 120-129.
6. Chishti Sh., Kaloo Z.A., Sultan Ph. Medicinal importance of genus Origanum: A review // Journal of Pharmacognosy and Phytotherapy. 2013. Vol. 5 (10). PP. 170-177. doi: 10.5897/ JPP2013.0285
7. Невкрытая Н.В., Аметова Э.Д., Марченко М.П. Итоги работы по созданию нового сорта Melissa officinalis L. // Ученые записки Таврического национального университета имени В.И. Вернадского. Серия: «Биология, химия». 2014. Т. 27 (66), № 5. С. 110-118.
8. Davey M.R., Anthony P. Plant cell culture: essential methods. Singapore : Markono Print Media Ptc. Ltd, 2010. 335 p.
9. Кушшр Г.П., Сарнацька В.В. Мжроклональне розмноження рослин. Teopiя i практика. К. : Наукова думка, 2005. 270 с.
10. Основы создания генобанка in vitro видов, сортов и форм декоративных, ароматических и плодовых культур / под ред. И.В. Митрофановой. Симферополь : АРИАЛ, 2018. 260 с.
11. Ghiorghita G.I., Maftei D. E. St., Nicuta D.N. Investigations on the in vitro morphogenetic reaction of Melissa officinalis L. species // Analele Stiintifice ale Universitatii “Alexandra loan Cuza’’, Genetica si Biologie Moleculara. 2005. Vol. 5. PP. 119-126.
12. Meftahizade H., Lofti M., Moradkhani H. Optimization of micropropagation and establishment of cell suspension culture in Melissa officinalis L. // African Journal of Biotechnology. 2010. Vol. 9, № 28. PP. 4314-4321. doi: 10.5897//AJB10.208
13. Mohebalipour N., Aharizad S., Mohammadi S.A., Motalibiazar A.R., Arefi H.M. Effect of plant growth regulators BAP and IAA on micropropagation of Iranian lemon balm (Melissa officinalis L.) landraces // Journal of Food, Agriculture & Environment. 2012. Vol. 10 (1). PP 280-286.
14. Goleniowski M.E., Flamarique C., Bima P. Micropropagation of Oregano (Origanum vulgare x applii) from meristem tips // In vitro Cellular and Developmental Biology - Plant. 2003. Vol. 39. PP. 125-128. doi: 10.1079/IVP2002361
15. Bracamonte M.A., Bima P., Bongiovanni G., Golenowski M. Nutrition and micropropagation of Origanum vulgare x applii // Molecular Medicinal Chemistry. 2006. Vol. 11. PP. 6-7.
16. Nanova Zh., Slavova Y Mass vegetative propagation of winter marjoram (Origanum vulgare ssp. Hirtum (Link) jetswaart) // Bulgarian Journal of Agricultural Science, National Centre for Agrarian Sciences. 2006. Vol. 12. PP. 531-536.
17. Oluk E.A., Cakir A. Micropropagation of Origanum sipyleum L., an endemic medicinal herb of Turkey // African Journal of Biotechnology. 2009. Vol. 8 (21). PP. 5769-5772.
18. Ozkum D. In vitro shoot regeneration of oregano (Origanum minutiflorum O. Schwarz & Davis) // Hacettepe Journal of Biology and Chemistry. 2007. Vol. 35 (2). PP. 97-100.
19. Shakeri S.M., Kazemitabar S.K., Sinaki J.M. In vitro culture of Melissa officinalis without the use of hormones // International Journal of Agriculture and Crop Sciences. 2013. Vol. 6 (20). PP. 1382-1387.
20. Meftahizade H., Moradkhani H., Naseri B., Lofti M, Naseri A. Improved in vitro culture and micropropagation of different Melissa officinalis L. genotypes // Journal of Medicinal Plants Research. 2010. Vol. 4, № 3. PP. 240-246.
21. Oana C.T., Falticeanu M., Prisecaru M. Considerations regarding the effects of growth regulators over the in vitro morphogenetic reaction at Origanum vulgare L. // Journal Plant Development. 2008. Vol. 15. PP. 133-138.
22. El Beyrouthy M., Elian G., Abou Jaoudeh C., Chalak L. In vitro propagation of Origanum syriacum and Origanum ehrenbergii // Acta Horticultural. 2015. № 1083. PP. 169-172. doi: 10.17660/ActaHortic.2015.1083.19
23. Fokina A.V., Satarova T.M., Smetanin V.T., Kucenko N.I. Optimization of microclonal propagation in vitro of oregano (Origanum vulgare) // Biosystems Diversity/ 2018. Vol. 26, № 2. PP. 98-102. doi: 10.15421/011815
24. Sevindik B., Izgu T., §im§ek O., Tutuncu M., Curuk P., Yilmaz O., Kaynak G., Kacar Y.A., Silva J.A.T., Mendi Y.Y In vitro culture of turkish Origanum sipyleum L. // American Journal of Plant Biology. Special Issue: Plant Molecular Biology and Biotechnology. 2017. Vol. 2, № 5-1. PP. 32-36. doi: 10.11648/j.ajpb.s.2017020501.16
25. Korkor A.M., Mohamed S.A., Abd El-kafie O.M., Gohar A.A. Adaptation of the in vitro culture of Origanum majorana L. for production of phenolic acids // IOSR Journal of Pharmacy and Biological Sciences. 2017. Vol. 12, № 2. PP. 30-38. doi: 10.9790/3008-1202013038
26. Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures // Physiologia Plantarum. 1962. Vol. 15, № 3. PP. 473-479.
27. Якимова О.В., Егорова Н.А. Влияние состава питательной среды, типа экспланта и генотипа на клональное микроразмножение Melissa officinalis L. // Ученые записки Крымского федерального университета имени В.И. Вернадского. Биология. Химия. 2018. Т 4 (70), № 1. С. 158-167.
28. Якимова О.В., Егорова Н.А. Влияние состава питательной среды и генотипа на клональное микроразмножение душицы in vitro // Труды Кубанского государственного аграрного университета. 2015. Вып. 4 (55). С. 304-309.
29. Liu M., Jiang F., Kong X., Tian J., Wu Z., Wu Zh. Effects of multiple factors on hyperhydricity of Allium sativum L. // Scientia Horticulturae. 2017. Vol. 217. PP. 285-296. doi: 10.1016/j.scienta.2017.02.010
30. Поливанова О.Б., Чередниченко М.Ю. Пути преодоления витрификации многоколосника фенхельного Agastache foeniculum (Pursh) Kuntze (Lamiaceae) в культуре in vitro // Известия Тимирязевской сельскохозяйственной академии. 2017. Вып. 5. С. 17-28. doi: 10.26897/0021-342X-2017-5-17-28
31. Yegorova N.A., Mitrofanova I.V., Brailko V.A., Grebennikova O.A., Paliy A.E., Stavtseva I.V Morphogenetic, physiological, and biochemical features of Lavandula angustifolia at longterm micropropagation in vitro // Russian Journal of Plant Physiology. 2019. Vol. 66, № 2. РР. 326-334. https://link.springer.com/artide/10.1134/S1021443719010060
32. Егорова Н.А., Ставцева И.В., Митрофанова И.В. Влияние сорта и факторов культивирования in vitro на клональное микроразмножение розы эфиромасличной // Бюллетень государственного Никитского ботанического сада. 2016. № 120. С. 36-43.
33. Zagorskaya М., Yegorova N. Effect of prolonged cultivation on the micropropagation in vitro of mint cultivars and breeding samples // BIO Web of Conferences. 2018. Vol. 11. Article Number 00049. doi: 10.1051/bioconf/20181100049
34. Егорова Н.А., Ставцева И.В., Якимова О.В., Каменек Л.И., Кривохатко А.Г. Некоторые аспекты клонального микроразмножения и сохранения in vitro эфиромасличных растений // Таврический вестник аграрной науки. 2015. № 1 (3). С. 18-24.
Tomsk State University Journal of Biology. 2019; : 22-39
The effect of long-term subcultivation on clonal micropropagation of Melissa officinalis L. and Origanum vulgare L.
https://doi.org/10.17223/19988591/47/2Abstract
In recent years, along with the use of essential oil and medicinal plants traditional for the south of Russia, there have been an active introduction of new species, including representatives of Lamiaceae family such as Melissa officinalis and Origanum vulgare, into production. These plant species are widely used in perfumery, cosmetics and food industries, as well as in medicine, because of the number of biologically active compounds. Therefore breeding work is being carried out to create cultivars with a high content of essential oil and valuable components in its composition. Currently, biotechnological techniques are used to address a wide range of challenges of ecology, plant breeding and seed production. One of the most popular methods is clonal micropropagation. Biotechnologies of micropropagation in vitro have been developed for many agricultural and flowering ornamental plants. However, for lemon balm and oregano, many issues relating to propagation in tissue and organ culture have not been studied enough. In particular, this applies to the study of long-term propagation in vitro. The aim of our investigation was to study the morphometric parameters of explant development during long-term subcultivation of cultivars and breeding samples of M. officinalis and O. vulgare at the second stage of clonal micropropagation.
For studies, we used three cultivars of Melissa officinalis L. (Tsitronella, Sobornaya, Krymchanka) and breeding sample № 10 of Origanum vulgare L. When introduced in vitro, axillary meristems with 2 leaf primordia were isolated from plants. At the second stage (micropropagation itself), stem segments (5-8 mm) with one node, obtained by microcutting of shoots developed from meristems, were used as explants. Explants were cultivated on Murashige and Skoog (MS) culture medium (which we modified earlier for oregano and lemon balm) supplemented with BAP and GA3. The meristems and shoots were cultivated at 26±2°С, 70% air humidity, 2-3 klx light intensity and 16-h photoperiod. After 30-35 days of cultivation, we determined the frequency of multiple shoot formation, the number and length of shoots, the number of nodes on the shoot, the frequency of rhizogenesis and hyperhydric shoots. The multiplication index was calculated as the number of microcuttings that were obtained after one subculture. To do this, the number of shoots formed on explants was multiplied by the number of nodes on a shoot. Hyperhydric microshoots were not taken into account. Then, we divided the shoots into microcuttings and transferred to a fresh culture medium. Experiments were repeated three times, and at least 20 explants were analyzed in each variation. The confidence of differences was assessed using Student’s t-test, p<0.05. Mean values and standard errors are shown in tables and graphs.
As a result of the research, we showed that with the introduction of M. officinalis meristems to the MS culture medium with 1.0 mg/l BAP and 0.5 mg/l GA3, the development of the main and sometimes adventitious shoots occurred on the 7th-10th day of cultivation. After 35 days of cultivation, 1.3 (Tsitronella cultivar) to 4.2 (Sobornaya cultivar) shoots were formed from one explant, and their length varied from 5.8 to 8.2 mm. For further micropropagation of lemon balm, microshoots obtained at the first stage were divided into one node segments and transferred to the MS medium with 0.5 mg/l BAP. Cultivation of microcuttings during seven passages led to the formation of axillary and adventitious shoots in the studied cultivars (See Fig. 1, A). The frequency of multiple shoot formation, depending on the cultivar and passage, varied from 32.0% to 93.3% (See Table 1). In some variants of the experiment, hyperhydric microshoots were formed with a frequency of 15.1-23.4%. The morphometric parameters of Sobornaya and Krymchanka cultivars were 1.3-2.5 times higher than those of Tsitronella. During successive subcultivations in all cultivars, we revealed an increase not only in multiple shoot formation frequency but also in the length of shoots in some variants. It should be noted that at the second stage of micropropagation on a culture medium with BAP, many shoots developed roots (See Fig. 1, А). The rhizogenesis frequency, depending on the cultivar and passage, reached 44.4-92.7%. The revealed spontaneous rooting of M. officinalis cultivars on a nutrient medium for micropropagation allows, at a high rhizogenesis frequency, excluding the third stage of propagation and immediately transferring microplants with roots for adaptation ex vitro. When comparing the morphometric parameters of the microcuttings development at different passages, we found that in the studied cultivars the number of shoots varied from 2.2 to 5.7 pcs./explants, and the number of nodes varied from 2.2 to 4.1 pcs./shoot. The length of the shoots reached 55.4-73.8 mm. In Tsitronella cultivar, the maximum value of the studied parameters (See Table 1) and the multiplication index (up to 12.0) (See Fig. 2) were noted in the 3rd-5th subcultivations. For Sobornaya and Krymchanka cultivars - in the fifth passage, in which the multiplication index was 14.2 and 17.6, respectively. In the seventh subcultivation, the multiplication indexes of all studied cultivars decreased to 6.3-8.9, depending on the genotype.
When meristems of O. vulgare were placed on MS culture medium, containing 1.0 mg/l BAP, after a month of cultivation, they developed up to 4.5 shoots per explant of 17.0 mm long. The frequency of multiple shoot formation was 66.6%. At the second stage of micropropagation, explants (stem segments with one node) isolated from microshoots, developed from meristem, were cultured on MS medium with 0.5 mg/l BAP. The growth of the main shoot started on the 5th-7th day, and the development of adventitious shoots - in 2-3 weeks (See Fig. 1 В). On the 35th day of cultivation, the frequency of multiple shoot formation varied from 75.0 to 95.2%, and the number of shoots ranged from 9.5 to 54.8 per explant depending on the number of subcultures (See Table 2). During micropropagation, individual shoots (from 5.2 to 28.5%, in different passages) formed 2-3 roots up to 2-3 cm in length. Sometimes, when oregano was cultivated, the hyperhydric microshoots with thickened vitreous anomalous stems and leaves were noted; they cannot be used for further propagation. The frequency of vitrified microshoots was 16.7-25.8%. However, it reached 44.5% in the fifth passage. When studying the effect of cultivation duration on micropropagation of O. vulgare in vitro, 13 passages were conducted. The maximum number of shoots (54.8 pcs / explants) and the multiplication index (74.1) were obtained in the fifth subcultivation (See Fig. 3). With further subcultivations, the multiplication index decreased in the 10th passage up to 43.0. The lowest values of this parameter (16.9-18.8) were noted in the late 12th and 13th passages. Thus, the conducted studies indicate the possibility of long-term micropropagation of O. vulgare and M. officinalis in vitro (at least for 1-1.5 years), while the maximum efficiency of this process was observed during the fifth subcultivation.
The paper contains 3 Figures, 2 Tables and 34 References.
References
1. Pashtetskii V.S., Nevkrytaya N.V., Mishnev A.V., Nazarenko L.G. Efiromaslichnaya otrasl' Kryma. Vchera, segodnya, zavtra. Simferopol' : IT «Arial», 2018. 320 s.
2. Atlas lekarstvennykh rastenii Rossii / pod red. A.V. Bykova. M. : Shcherbinskaya tipografiya, 2006. 345 s.
3. Moradkhani H., Sargsyan E., Bibak H., Naseri B., Sadat-Hosseini M., Fayazi-Barjin A., Meftahizade H. Melissa officinalis L., a valuable medicine plant: A review // Journal of Medicinal Plants Research. 2010. Vol. 4, № 25. PP. 2753-2759.
4. Garcia-Beltran J.M., Esteban M.A. Properties and application of plants of Origanum sp. genus // SM Journal of Biology. 2016. № 2 (1). 1006. PP. 1-10.
5. Derwich E., Benziane Z., Manar A., Boukir A., Taouil R. Phytochemical analysis and in vitro antibacterial activity of the essential oil of Origanum vulgare from Morocco // American-Eurasian Journal of Scientific Research. 2010. Vol. 5, № 2. PP. 120-129.
6. Chishti Sh., Kaloo Z.A., Sultan Ph. Medicinal importance of genus Origanum: A review // Journal of Pharmacognosy and Phytotherapy. 2013. Vol. 5 (10). PP. 170-177. doi: 10.5897/ JPP2013.0285
7. Nevkrytaya N.V., Ametova E.D., Marchenko M.P. Itogi raboty po sozdaniyu novogo sorta Melissa officinalis L. // Uchenye zapiski Tavricheskogo natsional'nogo universiteta imeni V.I. Vernadskogo. Seriya: «Biologiya, khimiya». 2014. T. 27 (66), № 5. S. 110-118.
8. Davey M.R., Anthony P. Plant cell culture: essential methods. Singapore : Markono Print Media Ptc. Ltd, 2010. 335 p.
9. Kushshr G.P., Sarnats'ka V.V. Mzhroklonal'ne rozmnozhennya roslin. Teopiya i praktika. K. : Naukova dumka, 2005. 270 s.
10. Osnovy sozdaniya genobanka in vitro vidov, sortov i form dekorativnykh, aromaticheskikh i plodovykh kul'tur / pod red. I.V. Mitrofanovoi. Simferopol' : ARIAL, 2018. 260 s.
11. Ghiorghita G.I., Maftei D. E. St., Nicuta D.N. Investigations on the in vitro morphogenetic reaction of Melissa officinalis L. species // Analele Stiintifice ale Universitatii “Alexandra loan Cuza’’, Genetica si Biologie Moleculara. 2005. Vol. 5. PP. 119-126.
12. Meftahizade H., Lofti M., Moradkhani H. Optimization of micropropagation and establishment of cell suspension culture in Melissa officinalis L. // African Journal of Biotechnology. 2010. Vol. 9, № 28. PP. 4314-4321. doi: 10.5897//AJB10.208
13. Mohebalipour N., Aharizad S., Mohammadi S.A., Motalibiazar A.R., Arefi H.M. Effect of plant growth regulators BAP and IAA on micropropagation of Iranian lemon balm (Melissa officinalis L.) landraces // Journal of Food, Agriculture & Environment. 2012. Vol. 10 (1). PP 280-286.
14. Goleniowski M.E., Flamarique C., Bima P. Micropropagation of Oregano (Origanum vulgare x applii) from meristem tips // In vitro Cellular and Developmental Biology - Plant. 2003. Vol. 39. PP. 125-128. doi: 10.1079/IVP2002361
15. Bracamonte M.A., Bima P., Bongiovanni G., Golenowski M. Nutrition and micropropagation of Origanum vulgare x applii // Molecular Medicinal Chemistry. 2006. Vol. 11. PP. 6-7.
16. Nanova Zh., Slavova Y Mass vegetative propagation of winter marjoram (Origanum vulgare ssp. Hirtum (Link) jetswaart) // Bulgarian Journal of Agricultural Science, National Centre for Agrarian Sciences. 2006. Vol. 12. PP. 531-536.
17. Oluk E.A., Cakir A. Micropropagation of Origanum sipyleum L., an endemic medicinal herb of Turkey // African Journal of Biotechnology. 2009. Vol. 8 (21). PP. 5769-5772.
18. Ozkum D. In vitro shoot regeneration of oregano (Origanum minutiflorum O. Schwarz & Davis) // Hacettepe Journal of Biology and Chemistry. 2007. Vol. 35 (2). PP. 97-100.
19. Shakeri S.M., Kazemitabar S.K., Sinaki J.M. In vitro culture of Melissa officinalis without the use of hormones // International Journal of Agriculture and Crop Sciences. 2013. Vol. 6 (20). PP. 1382-1387.
20. Meftahizade H., Moradkhani H., Naseri B., Lofti M, Naseri A. Improved in vitro culture and micropropagation of different Melissa officinalis L. genotypes // Journal of Medicinal Plants Research. 2010. Vol. 4, № 3. PP. 240-246.
21. Oana C.T., Falticeanu M., Prisecaru M. Considerations regarding the effects of growth regulators over the in vitro morphogenetic reaction at Origanum vulgare L. // Journal Plant Development. 2008. Vol. 15. PP. 133-138.
22. El Beyrouthy M., Elian G., Abou Jaoudeh C., Chalak L. In vitro propagation of Origanum syriacum and Origanum ehrenbergii // Acta Horticultural. 2015. № 1083. PP. 169-172. doi: 10.17660/ActaHortic.2015.1083.19
23. Fokina A.V., Satarova T.M., Smetanin V.T., Kucenko N.I. Optimization of microclonal propagation in vitro of oregano (Origanum vulgare) // Biosystems Diversity/ 2018. Vol. 26, № 2. PP. 98-102. doi: 10.15421/011815
24. Sevindik B., Izgu T., §im§ek O., Tutuncu M., Curuk P., Yilmaz O., Kaynak G., Kacar Y.A., Silva J.A.T., Mendi Y.Y In vitro culture of turkish Origanum sipyleum L. // American Journal of Plant Biology. Special Issue: Plant Molecular Biology and Biotechnology. 2017. Vol. 2, № 5-1. PP. 32-36. doi: 10.11648/j.ajpb.s.2017020501.16
25. Korkor A.M., Mohamed S.A., Abd El-kafie O.M., Gohar A.A. Adaptation of the in vitro culture of Origanum majorana L. for production of phenolic acids // IOSR Journal of Pharmacy and Biological Sciences. 2017. Vol. 12, № 2. PP. 30-38. doi: 10.9790/3008-1202013038
26. Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures // Physiologia Plantarum. 1962. Vol. 15, № 3. PP. 473-479.
27. Yakimova O.V., Egorova N.A. Vliyanie sostava pitatel'noi sredy, tipa eksplanta i genotipa na klonal'noe mikrorazmnozhenie Melissa officinalis L. // Uchenye zapiski Krymskogo federal'nogo universiteta imeni V.I. Vernadskogo. Biologiya. Khimiya. 2018. T 4 (70), № 1. S. 158-167.
28. Yakimova O.V., Egorova N.A. Vliyanie sostava pitatel'noi sredy i genotipa na klonal'noe mikrorazmnozhenie dushitsy in vitro // Trudy Kubanskogo gosudarstvennogo agrarnogo universiteta. 2015. Vyp. 4 (55). S. 304-309.
29. Liu M., Jiang F., Kong X., Tian J., Wu Z., Wu Zh. Effects of multiple factors on hyperhydricity of Allium sativum L. // Scientia Horticulturae. 2017. Vol. 217. PP. 285-296. doi: 10.1016/j.scienta.2017.02.010
30. Polivanova O.B., Cherednichenko M.Yu. Puti preodoleniya vitrifikatsii mnogokolosnika fenkhel'nogo Agastache foeniculum (Pursh) Kuntze (Lamiaceae) v kul'ture in vitro // Izvestiya Timiryazevskoi sel'skokhozyaistvennoi akademii. 2017. Vyp. 5. S. 17-28. doi: 10.26897/0021-342X-2017-5-17-28
31. Yegorova N.A., Mitrofanova I.V., Brailko V.A., Grebennikova O.A., Paliy A.E., Stavtseva I.V Morphogenetic, physiological, and biochemical features of Lavandula angustifolia at longterm micropropagation in vitro // Russian Journal of Plant Physiology. 2019. Vol. 66, № 2. RR. 326-334. https://link.springer.com/artide/10.1134/S1021443719010060
32. Egorova N.A., Stavtseva I.V., Mitrofanova I.V. Vliyanie sorta i faktorov kul'tivirovaniya in vitro na klonal'noe mikrorazmnozhenie rozy efiromaslichnoi // Byulleten' gosudarstvennogo Nikitskogo botanicheskogo sada. 2016. № 120. S. 36-43.
33. Zagorskaya M., Yegorova N. Effect of prolonged cultivation on the micropropagation in vitro of mint cultivars and breeding samples // BIO Web of Conferences. 2018. Vol. 11. Article Number 00049. doi: 10.1051/bioconf/20181100049
34. Egorova N.A., Stavtseva I.V., Yakimova O.V., Kamenek L.I., Krivokhatko A.G. Nekotorye aspekty klonal'nogo mikrorazmnozheniya i sokhraneniya in vitro efiromaslichnykh rastenii // Tavricheskii vestnik agrarnoi nauki. 2015. № 1 (3). S. 18-24.
