Clonal micropropagation of the essential oil rose cultivar “Festivalnaya”: morphology, anatomy and microshoots’ ploidy level

The genus Rosa includes more than 200 species and 18,000 cultivars, which, in addition to being used in floriculture, are of economic importance due to the presence of essential oil, a source of natural aromatic components. Products (essential oil, rose oil, rose water, etc.) obtained from essential-bearing oil rose are used in the food, perfume, cosmetic and medical industries. Traditional rose propagation is carried out by cuttings, grafting or budding, which are complex and time-consuming processes. Biotechnological methods have become an alternative to the vegetative method of rose propagation. The main studies are aimed at selecting the mineral composition of nutrient media and concentrations of growth regulators, while the issues of structural and genetic stability of the material in vitro are still discussed. Therefore, the work aimed to determine the optimal conditions for in vitro cultivation, structure, ploidy, and relative DNA content of essential oil rose microshoots. Essential oil rose cultivar “Festivalnaya” (Rosa damascena Mill × Rosa gallica L.) was used as the material. Buds were sterilized and transferred on the Murashige-Skoog modified medium supplemented with 1.5 mg/l 6-benzylaminopurine (6-BAP), 0.25 mg/l gibberellic acid (GA3), 0.15 mg/l α-naphthylacetic acid. Micropropagation of the obtained microshoots was carried out on the Murashige-Skoog culture medium containing 1.0 mg/l 6-BAP; 1.0 mg/l kinetin (Kin); 1.0 mg/l 6-BAP + 1.0 mg/l Kin; 1.0 mg/l 6-BAP + 0.5 mg/l GA3. The structure analysis was carried out according to the generally accepted methods. Ploidy and relative DNA content were determined by flow cytometry. Induction of direct organogenesis in vitro from vegetative buds of essential oil rose was carried out. At the stage of micropropagation, the maximum number of adventitious microshoots was obtained on Murashige-Skoog medium with the introduction of 1.0 mg/l 6-BAP and 1.0 mg/l Kin. Structural analysis of microshoots after subculturing for 12 months revealed qualitative and quantitative changes due to specific in vitro conditions and material rejuvenation. The ploidy level of the leaf blade nuclei in vitro did not differ from those of ex situ shoots. The relative content of DNA in the leaf nuclei of shoots ex situ and microshoots in vitro was 2.21 pg and 2.24 pg, respectively, which indicates a certain stability of the material in vitro.

1. Baydar H., Baydar N.G. The effects of harvest date, fermentation duration and Tween 20 treatment on essential oil content and composition of industrial oil rose (Rosa damascena Mill.). Ind. Crop. Prod. 2005;21(2):251–255.

2. Venkatesha K.T., Gupta A., Rai A.N., Jambhulkar S.J., Bisht R., Padalia R.C. Recent developments, challenges, and opportunities in genetic improvement of essential oil-bearing rose (Rosa damascena): A review. Ind. Crop. Prod. 2022;184(15):114984.

3. Zolotilov V., Nevkrytaya N., Zolotilova O., Seitadzhieva S., Myagkikh E., Pashtetskiy V., Karpukhin M. The essential-oil-bearing rose collection variability study in terms of biochemical parameters. Agron. 2022;12(2):529.

4. Митрофанова И.В., Митрофанова О.В., Браилко В.А., Лесникова-Седошенко Н.П. Биотехнологические и физиологические особенности культивирования in vitro ценных генотипов розы эфиромасличной. Известия вузов. Прикладная химия и биотехнология. 2015;2(13):37–48.

5. Katekar V.P., Rao A.B., Sardeshpande V.R. Review of the rose essential oil extraction by hydrodistillation: An investigation for the optimum operating condition for maximum yield. Sustain. Chem. Pharm. 2022;29:100783.

6. Марко Н.В. Биоморфобиологические особенности розы эфиромасличной сорта Фестивальная при выращивании на ЮБК и в степном Крыму. Таврический вестник аграрной науки. 2020;4(24):98–113.

7. Pati P.K., Kaur N., Sharma M., Ahuja P.S. In vitro propagation of rose. Protocols for in vitro propagation of ornamental plants. Methods in molecular biology, vol 589. Eds. S. Jain and S. Ochatt. N.Y., Dordrecht, Heidelberg, L.: Springer, Humana Press; 2010:163–176.

8. Егорова Н.А., Ставцева И.В. Микроразмножение сортов эфиромасличной розы в культуре in vitro. Вестник Удмуртского университета. Серия «Биология. Науки о Земле». 2016;2:45–52.

9. Молканова О.И., Васильева О.Г., Коновалова Л.Н. Научные основы сохранения и воспроизводства генофонда ценных и редких видов растений в культуре in vitro. Бюлл. Гл. бот. сада. 2005;2(201):78−82.

10. Kyte L., Kleyn J., Scoggins H., Bridgen M. Plants from test tubes: An introduction of micropropagation – 4th ed. Portland: Timber Press; 2013. 274 рp.

11. Семенова Е.Ф., Меженная Н.А., Преснякова Е.В. Анатомо-морфологические особенности лепестков как эфироносных структур цветков представителей рода Rosa. ИВУЗ ПР Естественные науки. 2014;3(7):5–17.

12. Шевченко С.В., Кузьмина Т.Н. Некоторые особенности эмбриологии представителей видов Rosa spinosissima L., R. canina L. и сортов R. × damascena Mill. в норме и при вирусной инфекции. Сельскохозяйственная биология. 2018;53(3):624–633.

13. Егорова Н.А., Ставцева И.В., Митрофанова И.В. Влияние сорта и факторов культивирования на клональное микроразмножение розы эфиромасличной. Бюллетень ГНБС. 2016;120:36−43.

14. Егорова Н.А., Ставцева И.В., Митрофанова И.В. Влияние сорта и состава питательной среды на укоренение розы эфиромасличной при микроразмножении in vitro. Биомика. 2018;10(1):11–15.

15. Корзина Н.В., Митрофанова И.В. Интенсивность освещения и морфогенез розы эфиромасличной в культуре in vitro. Бюллетень ГНБС. 2022;145:157–163.

16. Mitrofanova I.V., Brailko V.A., Lesnikova-Sedoshenko N.P., Ivanova N.N., Mitrofanova O.V. Structure of vegetative organs in essential oil rose under standard culture conditions and long-term conservation in vitro. Acta Hortic. 2021;1315:185–190.

17. Manokari M., Priyadharshini S., Shekhawat M. Micro-structural stability of micropropagated plants of Vitex negundo L. Microsc. Microanal. 2021;27(3):626−634.

18. Kritskaya T.A., Kashin A.S., Kasatkin M.Y. Micropropagation and somaclonal variation of Tulipa suaveolens (Liliaceae) in vitro. Russ. J. Dev. Biol. 2019;50(4):209–215.

19. Bulavin I.V., Ivanova N.N., Mitrofanova I.V. In vitro regeneration of Hyssopus officinalis L. and plant genetic similarity. Dokl. Biol. Sci. 2021;499(1):109–112.

20. Plugatar Y.V., Bulavin I.V., Ivanova N.N., Miroshnichenko N.N., Saplev N.M., Shevchuk O.M., Feskov, S.A., Naumenko, T.S. Study of the component composition of essential oil, morphology, anatomy and ploidy level of Hyssopus officinalis f. cyaneus Alef. Horticulturae. 2023;9(4):480.

21. Mitrofanova I., Tsyupka V., Jain S.M. Morphoanatomical characterization of in vitro regenerated plants. Advances in plant tissue culture. Current Developments and Future Trends. Eds. A.C. Rai, A. Kumar, A. Modi, and M. Singh. Cambridge: Academic Press; 2022:175–204.

22. Zakaria H.M., Dessoky E.S., Ismail I.A., Attia A.O., El-Hallous E.I. Analysis of genetic stability of the in vitro propagated Rosa damascena (Taif rose) plant. Int. J. Adv. Res. 2017;5(4):117–122.

23. Murashige Т.A, Skoog F. Revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 1962;15(3):473–497.

24. Novikov A., Sokolov S., Drapalyuk M., Zelikov V., Ivetić V. Performance of scots pine seedlings from seeds graded by colour. Forests. 2019;10(12):1064.

25. Loureiro J., Rodriguez E., Doležel J., Santos C. Two new nuclear isolation buffers for plant DNA flow cytometry: A test with 37 species. Ann. Bot. 2007;100(4):875–888.

26. Skaptsov M.V., Smirnov S.V., Kutsev M.G., Shmakov A.I. Problems of standartization in flow cytometry of plants. Turczaninowia. 2016;19(3):120–122.

27. Hammer Ø.H., David A.T., Paul D.R. Past: Paleontological statistics software package for education and data analysis. Palaeontol. Electron. 2001;4(1):4.

28. Pati P.K, Rath S.P, Sharma M., Sood A., Ahuja P.S. In vitro propagation of rose – a review. Biotechnol. Adv. 2006;24(1):94–114.

29. Baig M.M.Q., Hafiz I.A., Hussain A., Ahmad T., Abbasi N.A. An efficient protocol for in vitro propagation of Rosa gruss an teplitz and Rosa centifolia. Afr. J. Biotechnol. 2011;10(22):4564−4573.

30. Carelli B.P., Echeverrigaray S. An improved system for the in vitro propagation of rose cultivars. Sci Hortic. 2002;92(1):69–74.

31. Roy P.K., Manum A.N.K., Ahmed G. In vitro plantlets regeneration of rose. Plant Tissue Cult. 2004;14(2):149−154.

32. Jabbarzadeh Z., Khosh-Khui M. Factors effecting tissue culture of Damask rose (Rosa damascena Mill.). Sci. Hort. 2005;105(4):475−482.

33. Razavizadeh R., Ehsanpour A.A. Optimization of in vitro propagation of Rosa hybrida cultivar Black Red. Am. Eurasian J. Agric. Environ. Sci. 2008;3(1):96−99.

34. Мухамбетжанов С.К., Нам С.В., Вечерко Н.А., Мурсалиева В.К. Факторы, влияющие на рост и развитие роз in vitro. Биотехнология. Теория и практика. 2010;1:41−53.

35. Nikbakht A., Kafi M., Mirmasoumi M., Babalar M. Micropropagation of Damask rose (Rosa damascena Mill.) cvs Azaran and Ghamsar. Int. J. Agric. Biol. 2005;7(4):535−538.

36. Podwyszyńska M., Orlikowska T., Trojak-Goluch A., Wojtania A. Application and improvement of in vitro culture systems for commercial production of ornamental, fruit, and industrial plants in Poland. Acta Soc. Bot. Pol. 2022;91(1):914.

37. Noodezh H.M., Moieni A., Baghizadeh A. In vitro propagation of the Damask rose (Rosa damascena Mill.). In Vitro Cell. Dev. Biol. Plant. 2012;48(6):530–538.

38. Rout G.R., Debata B.K., Das P. In vitro clonal multiplication of roses. Proc. Natl. Acad. Sci. India. 1990;60:311−318.

39. Vijaya N., Satyanarayana G., Prakash J., Pierik R.L.M. Effect of culture media and growth regulators on in vitro propagation of rose. Curr. Plant Sci. Biotechnol. Agric. 1991;12:209−214.

40. Mamgain J., Mujib A., Ejaz B., Gulzar B., Malik M.Q., Syeed R. Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm. f.) Merrill. Plant Cell Tiss. Organ Cult. 2022;150(1):129–140.

41. Abdolinejad R., Shekafandeh A., Jowkar A., Gharaghani A., Alemzadeh A. Indirect regeneration of Ficus carica by the TCL technique and genetic fidelity evaluation of the regenerated plants using flow cytometry and ISSR. Plant Cell Tiss. Organ Cult. 2020;143:131–144.

42. Raji M.-R., Farajpour M. Genetic fidelity of regenerated plants via shoot regeneration of muskmelon by inter simple sequence repeat and flow cytometry. J. Saudi Soc. Agric. Sci. 2021;20(2):88–89.

43. Fritsche Y., Deola F., da Silva D.A., Holderbaum D.F., Guerra M.P. Cattleya tigrina (Orchidaceae) in vitro regeneration: main factors for optimal protocorm-like body induction and multiplication, plantlet regeneration, and cytogenetic stability. S. Afr. J. Bot. 2022;149:96–108.

44. Catalano C., Carra A., Carimi F., Motisi A., Sajeva M., Butler A., Lucretti S., Giorgi D., Farina A., Abbate L. Somatic embryogenesis and flow cytometric assessment of nuclear genetic stability for Sansevieria spp.: an approach for in vitro regeneration of ornamental plants. Horticulturae. 2023;9(2):138.

45. Yokoya K., Roberts A.V., Mottley J., Lewis R., Brandham P.E. Nuclear DNA amounts in roses. Ann. Bot. 2000;85(4):557–561.

46. Doğan E., Kazaz S., Kiliç T., Dursun H., Ünsal T.H., Uran M. A research on determination of the performance Rosa damascena Mill. as pollen source in rose breeding by hybridization. Uluslararası Tarla Bitkileri Kong-resi Özel Sayısı. 2020;14:194–201.

47. Harmon D.D., Chen H., Byrne D., Liu W., Ranney T.G. Cytogenetics, ploidy, and genome sizes of rose (Rosa spp.) cultivars and breeding lines. Ornam. Plant Res. 2023;3:10.