Kalanşo’da (Kalanchoe blossfeldiana Poelnn.) Petal Kültürü Yönteminin Geliştirilmesi ve In Vitro Mutasyon Islahı Çalışmalarında Potansiyel Kullanımının Araştırılması

Öz

Kalanşo (Kalanchoe blossfeldiana Poelnn.), önemli bir saksılı iç mekân bitkisi olduğu gibi son yıllarda dış mekân süs bitkisi ve kesme çiçek olarak da öne çıkmaktadır. Pazar ihtiyaçlarına uygun yeni çeşitlerin geliştirilmesi amacıyla farklı ıslah yöntemleri kullanılarak çalışmalar yürütülmektedir. Mutasyon ıslahına oldukça yatkın olan bu tür için farklı genetik varyasyonlar oluşturmak amacıyla in vivo ve in vitro mutasyon ıslah çalışmaları önem taşımaktadır. In vitro mutasyon uygulamalarında vegetatif çoğaltım yöntemleri, mutant tek bireylerin genetik stabiliteyi koruyarak kitlesel çoğaltımının yapılmasında kritik öneme sahiptir. Bu çalışma iki aşamadan oluşmaktadır. Birincisi mutant bireylerin in vitro vegetatif çoğaltımını sağlamak üzere kalanşo türünde petal kültürü yönteminin geliştirilmesi ve optimizasyonu, ikincisi ise iyonize radyasyon kullanılarak elde edilmiş M1V4 kalanşo mutant tek bitkilerden petal kültürü yoluyla çoğaltım yaparak elde edilen klonlardaki genetik stabiliteyi morfolojik olarak gözlemlemek. Kalanşo için in vitro petal kültürü koşulları belirlenmiş ve 2.0 mg L-1 thidiazuron (TDZ), 0.5 mg L-1 1-naphthalenacetic asit (NAA), 30 g L-1 sukroz, 6 g L-1 agar ve ph 5.7 içeren Murashige ve Skoog (MS) besin ortamının en iyi rejenerasyonu sağladığı saptanmıştır. Ayrıca in vitro fiziksel mutagen uygulaması sonrasında laboratuvar koşullarında M1V4 aşamasına kadar çoğaltılan ve dış koşullara aktarılan mutant bireylerde çiçekler gözlenmiştir. Bunların arasından seçilen 4 farklı mutant çiçeklere sahip bitkilerden petaller in vitro kültüre alınmış ve çoğaltılmıştır. Kontrol olarak, mikroçoğaltım için ticari bir çeşidin taç yaprakları kullanıldı. Elde edilen klonların çiçekleri, başlangıç materyali olarak kullanılan mutant çiçeklerin homojen yapılı veya kimerik görünümlü olup olmadıklarına sağlı olarak homojenlik göstermiştir. Kalanşo’da petal kültürü konusundaki ilk bulguların elde edildiği bu çalışmanın ardından, kapsamlı bir şekilde geliştirilmesi konusunda çalışmalar devam etmektedir.

Anahtar Kelimeler

• Kalanşo
• in vitro mutasyon
• petal kültürü
• TDZ
• NAA
• gama ışını

Development of Petal Culture Method in Kalanchoe (Kalanchoe blossfeldiana Poelnn.) and Investigation of its Potential Use in In Vitro Mutation Breeding Studies

Öz

Kalanchoe (Kalanchoe blossfeldiana Poelnn.), is an important potted indoor plant as well as an outdoor ornamental plant and cut flower in recent years. Studies are being carried out using different breeding methods in order to develop new varieties suitable for market needs. In vivo and in vitro mutation breeding studies are important for creating different genetic variations for this species, which is highly prone to mutation breeding. In in vitro mutation applications, vegetative propagation methods are of critical importance in mass propagation of mutant single individuals while preserving genetic stability. This study consists of two stages: First, development and optimization of petal culture method in kalanchoe to provide in vitro vegetative propagation of mutant individuals, and second, morphological observation of genetic stability in clones obtained by propagation via petal culture from M1V4 kalanchoe mutant single plants obtained by using ionizing radiation. In vitro petal culture conditions were determined for kalanchoe and it was determined that Murashige and Skoog (MS) nutrient medium containing 2.0 mg L-1 thidiazuron (TDZ), 0.5 mg L-1 1-naphthalenacetic acid (NAA), 30 g L-1 sucrose, 6 g L-1 agar and pH 5.7 provided the best regeneration. In addition, after in vitro physical mutagen application, flowers were observed in mutant individuals propagated up to M1V4 stage in laboratory conditions and transferred to external conditions. Petals from plants with 4 different mutant flowers selected from these were cultured and propagated in vitro. As control, petals from a commercial variety were used for micropropagation. The flowers of the clones obtained showed homogeneity depending on whether the mutant flowers used as starting material were homogeneous or chimeric in appearance. Following this study, in which the first findings on petal culture in kalanchoe were obtained, studies are continuing to develop it comprehensively.

Anahtar Kelimeler

• Kalanchoe
• in vitro mutation
• petal culture
• TDZ
• NAA
• gamma ray

Destekleyen Kurum

Has Biotech Research Development Agriculture Industry and Trade Inc. Co. and TENMAK

Etik Beyan

Scientific rules, ethics and citation rules were followed during the all process of the study. No application that would harm nature or the environment was carried out during the research.

Teşekkür

The research was supported by Has Biotech Research Development Agriculture Industry and Trade Inc. Co. and TENMAK provided technical consultancy. We thank them for their contributions.

Kaynakça

1. AIPH. (2019). International Statistics Flowers and Plants. Hannover, Germany: International Association of Horticultural Producers, Hannover: Institut für Gartenbauökonomie, Universität Hannover.
2. Anand, P., Vanlalruati, V., Kumar, G., & Kumar, S. (2020). In vitro isolation of a gamma ray induced mutant in chrysanthemum (Dendranthema grandiflora). The Indian Journal of Agricultural Sciences 101476. https://doi.org/10.56093/ijas.v90i3.101476.
3. Asoko, N., Ruamrungsri, S., Yoosumran, V., & Saetiew, K. (2020). Improvement of Dendranthemum grandiflora cv. Canter with colchicine in vitro. International Journal of Agricultural Technology 16(2): 237-246.
4. Balkaya A., İzgi Saraç Y., & Tütüncü M. (2021). Melezleme ıslahı. In: YY. Mendi, S. Kazaz (Eds). Süs Bitkileri Islahı Kitabı-2. Klasik ve Biyoteknolojik Yöntemler. Bölüm: 3, (s: 107-144). Gece Kitaplığı Yayınevi, Ankara.
5. Barakat, M. N., Fattah, R. S. A., Badr, M., & El-Torky, M. G. (2010). In vitro culture and plant regeneration derived from ray florets of Chrysanthemum morifolium. African Journal of Biotechnology 9(8):1151- 1158.
6. Bejaoui, R. (2022). In Vitro Micropropagation of Kalanchoe (Kalanchoe blossfeldiana Poelln.). PhD Thesis. Ankara University, Institute of Natural and Applied Sciences, Department of Horticulture. 103p. Ankara.
7. Bejaoui, R., Özdemir, G. E., & Ellialtıoğlu, Ş. Ş. (2023b). In vitro or ex vitro rooting and acclimatization of Kalanchoe blossfeldiana Poelln. shoots propagated by tissue culture technique. Turkish Journal of Agricultural and Natural Sciences (TTDB) 10(4): 843–853. https://doi.org/10.30910/turkjans.1288919
8. Bejaoui, R., Gümüş, C., Sönmez, K., Kırbay, E., & Ellialtıoğlu, Ş. Ş. (2023a). The effects of different PGR contents on in vitro organogenesis and shoot proliferation in kalanchoe (Kalanchoe blossfeldiana Poelln.). In: 11th International Conference on Agriculture, Animal Sciences and Rural Development, 03-05 March 2023, Muş Alparslan University, Muş – Türkiye. Conference Proceedings Book (Eds: Karadağ, Y., Seydoşoğlu, S.). pp: 995-1010. ISBN:978-625-7720-91-5.

9. Bejaoui, R., Ellialtıoğlu, Ş. Ş., & Yanmaz, R. (2024). The effect of different cytokinin and auxin source on micropropagation of Kalanchoe blossfeldiana. Turkish Journal of Agricultural and Natural Sciences 11 (4): 1182–1192. https://doi.org/10.30910/turkjans.1531762.
10. Bhuiyan, M.S.U., Kim, T., In, J. G., Yang, D. C., & Choi, K. S. (2006). Plant regeneration from leaf explants of Kalanchoe daigremontiana Hamet & Perrier. Korean Journal of Medicinal Crop Science 14(5): 293-298
11. Boiteau, P., & Allorge-Boiteau, L. (1995). Kalanchoe (Crassulacées) de Madagascar. Systématique, écophysiologie et phytochimie. ICSN, CNRS, 91198 Gif-surYvette, Éditions Karthala, Paris.
12. Broertjes, C., & Leffring, L. (1972). Mutation breeding of Kalanchoë. Euphytica 21(3), 415-423.
13. Carra, A., Sajeva, M., Abbate, L., Siragusa, M., Sottile, F., & Carimi, F. (2012). In vitro plant regeneration of caper (Capparis spinosa L.) from floral explants and genetic stability of regenerants. Plant Cell Tissue and Organ Culture 109: 373–381. https://doi.org/10.1007/s11240-011-0102-9
14. CPVO varieties database.2017. Web page. http://cpvo.europa.eu. (Access date: 12.04.2017)
15. Datta, S. K. (2023). Technology package for induced mutagenesis. Journal of Biology and Nature, 15(1), 70-88.
16. Datta, S. K., Misra, P., & Mandal, A. K. A. (2005). In vitro mutagenesis – a quick method for establishment of solid mutant in Chrysanthemum. Current Science 88(1): 155-158.
17. Descoings, B. (2003). Kalanchoe. In: Eggli U, Hartmann HEK (eds) Illustrated Handbook of Succulent Plants. Crassulaceae. Springer Verlag, New York, pp 143–181.
18. Descoings, B. (2006). Le genre kalanchoe structure et définition. Journal de Botanique de la Société Botanique de France, 33, 3-28.
19. Ellialtıoğlu, Ş., & Yanmaz, R. (1994). Domates ve karnabaharda çift fazlı ortam ve aktif kömürün in vitro sürgün verim ve kalitesine etkisi. KÜKEM Dergisi 16 (1): 19-27.
20. Fransz, P. F., & Schel, J. H. N. (1994). Ultrastructural studies on callus development and somatic embryogenesis in Zea mays L. In: Maize (pp. 50-65). Berlin, Heidelberg: Springer Berlin Heidelberg.
21. Fujimoto, T., Otani, M., & Nakano, M. (2022). Production of colored foliage phenotypes in Kalanchoe blossfeldiana by ectopic expression of R2R3 MYB genes. J. Plant Biochem. Biotechnol. 31: 665–672. https://doi.org/10.1007/s13562-021-00760-3.
22. Green, C. E., & Phillips, R. L. (1975). Plant regeneration from tissue cultures of maize L. Crop Science 15(3): 417-421.
23. Gümüş, C., & Ellialtıoğlu, Ş. Ş. (2018). A review on tissue culture studies of Kalanchoe blossfeldiana Poelln. Turkish Journal of Scientific Reviews 11(1): 18-26. E-ISSN: 2146-0132. (In Turkish).
24. İzgi Denli, Y., & Balkaya, A. (2023). Tulipa gesneriana türü ile yerel lale genotiplerinin (Tulipa spp.) türler arası melez performanslarının belirlenmesi ve melez tohum özelliklerinin incelenmesi. Manas Journal of Agriculture Veterinary and Life Sciences 13 (2): 164-176. https://doi.org/10.53518/mjavl.1367291
25. Jácome-Blásquez, F., & Kim, M. (2023). Meristem genes are essential for the vegetative reproduction of Kalanchoë pinnata. Frontiers in Plant Science, 14: 1157619. https://doi.org/10.3389/fpls.2023.1157619.
26. Jain, S.M., & Ochatt, S.J. (2010). Protocols for in vitro propagation of ornamental plants. Springer Protocols. Humana Press.
27. Jiménez, V. M., & Bangerth, F. (2001). Hormonal status of maize initial explants and of the embryogenic and non-embryogenic callus cultures derived from them as related to morphogenesis in vitro. Plant Science 160(2), 247-257.
28. Kahraman, M. U., & Boyacı, F. (2021). Kalanşo In: In: S. Kazaz, & Y. Mendi Yalçın (Eds.), Süs Bitkileri Islahı (Türler), (pp. 239-282). Gece Publishing. ISBN: 978-625-7478-250-2. (in Turkish).
29. Kahraman, M., U., Yalçın Mendi, Y., Karabıyık, Ş., Lütken, H. V., & Favero, B. T. (2022). Kalanchoë breeding: past, present and future. Ornamental Horticulture 28(1): 19-35. doi: https://doi.org/10.1590/2447- 536X.v28i1.2403
30. Kantoğlu, K. Y., Kuşvuran, Ş., & Ellialtıoğlu, Ş. Ş. (2021). Doku kültürü yöntemlerinin stres çalışmalarında kullanımı. In: Ş.Ş. Ellialtıoğlu, H.Y., Daşgan, & Ş. Kuşvuran (Eds.) Sebzelerde Stres Toleransı ve Islah Stratejileri. (pp. 708-741). Gece Publishing, Ankara. ISBN 978-625-8449- 01-3. (in Turkish)
31. Kantoğlu, K. Y., Kunter, B., Şenel, Ü., & Haspolat, G. (2024b). Ray-floret based rapid reproduction and detection of somatic variation in selected mutant chrysanthemum individuals. Yuzuncu Yil University Journal of Agricultural Sciences 34(4): 549-558.
32. Kantoğlu, K. Y., Sarıtoprak, O., Akyüz Çağdaş, E., Okutan, E., Aktaş, H., & Ellialtıoğlu, Ş. Ş. (2024a). In vitro mutation breeding in kalanchoe (Kalanchoe blossfeldiana Poelnn.). Düzce University Journal of Forestry (in press).
33. Kazaz, S. (2016). Dünya süs bitkileri sektöründe ürün deseni, sosyo- ekonomik ve teknoloji alanında yaşanan gelişmeler ile Türkiye’nin gelecek vizyonu. VI. Süs Bitkileri Kongresi, 19-22 Nisan, Antalya.
34. Kolodziejczyk-Czepas, J., & Stochmal, A. (2017). Bufadienolides of Kalanchoe species: an overview of chemical structure, biological activity and prospects for pharmacological use. Phytochem Review 16(6): 1155-1171. https://doi.org/10.1007/s11101-017-9525-1.
35. Krupa-Malkiewicz, M. (2010). Influence of chemical mutagens on morphological traits in kalanchoe (Kalanchoe hybrida). Folia Pomeranae Universitatis Technologiae Stetinensis. Agricultura, Alimentaria, Piscaria et Zootechnica, 15.
36. Kuligowska, K., Lütken, H., Christensen, B., Skovgaard, I., Linde, M., Winkelmann, T., & Müller, R. (2015). Evaluation of reproductive barriers contributes to the development of novel interspecific hybrids in the Kalanchoë genus. BMC Plant Biology 15, 1-15.
37. Kumar, S., & Kanwar, J. K. (2006). Regeneration ability of petiole, leaf and petal explants in gerbera cut flower cultures in vitro. Folia Horticulturae Ann. 18(2): 57-64.
38. Li, R., Fan, L., Lin, J., Li, M., Liu, D., & Sui, S. (2019). In vitro mutagenesis followed by polymorphism detection using start codon targeted markers to engineer brown spot resistance in Kalanchoe. Journal of the American Society for Horticultural Science 144(3): 193-200. https://doi.org/10.21273/JASHS04571-18
39. Linjian, F., Dongpo, J., & Xinshuan, Z. (2006). Research of different culture conditions on plantlet regeneration from leaves of Kalanchoe blossfeldiana. Chinese Agricultural Science Bulletin, 5. (Accessed date: 23.04.2024): http://en.cnki.com. cn/Article_en/CJFDTotal ZNTB200605021. htm.
40. Lütken, H. V., Jensen, L. S., Topp, S. H., Mibus, H., Müller, R., & Rasmussen, S. K. (2010). Production of compact plants by overexpression of AtSHI in the ornamental Kalanchoë. Plant Biotechnology Journal 8(2): 211-222. https://doi.org/10.1111/j.1467-7652.2009.00478.x
41. Mackenzie, K. K., Lütken, H., Coelho, L. L., Kaaber, M. D., Hegelund, J. N., & Müller, R. (2018). Kalanchoë. Ornamental Crops 453-479.
42. Malaure, R. S., Barclay, G., Power, J. B., & Davey, M. (1991). The production of novel plants from florets of Chrysanthemum morifolium using tissue culture. I. shoot regeneration from ray florets and somaclonal variation exhibited by the regenerated plants. J Plant Physiol. 139: 8-13.
43. Minas, G. J. (2007). A rapid protocol for in vitro micropropagation of three types of gardenia (Gardenia spp. Ellis) for quality produce flowering pot-plants. International Conference on Quality Management in Supply Chains of Ornamentals 755, 81-86.
44. Mohammadi-Dehcheshmeh, M., Khalighi, A., Naderi, R., Sardari, M., & Ebrahimie, E. (2008). Petal: a reliable explant for direct bulblet regeneration of endangered wild populations of Fritillaria imperialis L. Acta Physiologiae Plantarum 30: 395-399.
45. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiologia Plantarum 15: 473- 497.
46. MVD. (2024). IAEA Mutant Varietiy Database. https://nucleus.iaea.org/sites/mvd/ SitePages/Search.aspx (Access date: 20.10.2024)
47. Narumi, T., Aida, R., Niki, T., Nishijima, T., Mitsuda, N., Hiratsu, K., Ohme- Takagi, M., & Ohtsubo, N. (2008). Chimeric AGAMOUS repressor induces serrated petal phenotype in Torenia fournieri similar to that induced by cytokinin application. Plant Biotechnology 25(1): 45-53.
48. Oh, M. H., & Kim, S. G. (1994). Plant regeneration from petal protoplast culture of Petunia hybrida. Plant Cell, Tissue and Organ Culture 36, 275- 283.
49. Otani, M., Aoyagi, K., & Nakano, M. (2020). Suppression of B function by chimeric repressor gene-silencing technology (CRES-T) reduces the petaloid tepal identity in transgenic Lilium sp. PLoS One 15(8):e0237176. https://doi.org/10.1371/journal.pone.0237176.
50. Prathyusha, N., Dorajeerao, A. V. D., Ravindrakumar, K., Aparna, D., & Salomi Suneetha, D. R. (2021). In vitro propagation of chrysanthemum (Chrysanthemum morifolium) cultivars from ray floret explants. The Pharma Innovation Journal 10(10): 415-417.
51. Sanikhani, M., Frello, S., & Serek, M. (2006). TDZ induces shoot regeneration in various Kalanchoe blossfeldiana Poelln. Cultivars in the absence of auxin. Plant Cell, Tissue and Organ Culture 85:75–82 https://doi.org/10.1007/s11240-005-9050-6.
52. Stefanowicz-Hajduk, J., Hering, A., Gucwa, M., Sztormowska- Achranowicz, K., Kowalczyk, M., Soluch, A., & Ochocka, J. R. (2022). An in vitro anticancer, antioxidant, and phytochemical study on water extract of Kalanchoe daigremontiana Raym.-Hamet and H. Perrier. Molecules, 27(7): 2280. https://doi.org/10.3390/molecules27072280.
53. Van Harten, A. M. (2002). Mutation breeding of vegetatively propagated ornamentals. In Breeding for ornamentals: classical and molecular approaches (Vainstein, A. (Ed.)) (pp. 105-127). Dordrecht: Springer Netherlands. Kluwer Academic Publishiers, ISBN: 978-90-481-5975.
54. Vargas, A., Herrera, I., Nualart, N., Guézou, A., Gómez-Bellver, C., Freire, E., Jaramillo Díaz, P., & López-Pujol, J. (2022). The genus Kalanchoe (Crassulaceae) in Ecuador: From gardens to the wild. Plants 11(13): 1746. https://doi.org/10.3390/plants11131746.
55. Verma, A. K., Prasad, V., Singh, S. K., & Kumar S (2012). In vitro isolation of red coloured mutant from chimeric ray florets of chrysanthemum induced by gamma-ray. Indian J. Hort. 69(4): 562-567.
56. Vlielander, I. (2002). U.S. Patent Application No. 10/080, 611.
57. Winiarczyk, K., Czerska, D., Denisow, B., Chrzanowska, E., & Pietrusiewicz, J. (2024). Regenerative potential and its variability in different topophysical zones of Kalanchoe daigremontiana leaves in in vitro culture conditions. Acta Scientiarum Polonorum Hortorum Cultus 23(2):93-101. https://doi.org/10.24326/asphc.2023.5280
58. Yuliang, C., Xiaogang, Z., & Yanping, Z., & Zhengying, Z. (2004). Isolation leaf disks culture, and high efficient plantlet regeneration of Kalanchoe blossfeldiana. Chinese Agricultural Science Bulletin 20(5), 33.
59. Zhang, R., & Guo, X. (2005). Study on regeneration of vitro leaf of Kalanchoe blossfeldiana. Shanxi Forestry Science and Technology, 01. (Access date: 20.10.2024): http://en.cnki.com.cn/Article_en/CJFDTotal- SXLK200501003.htm.