Aklimatizasyon Aşamasında GA₃ ve AsA Uygulamalarının Vejetatif Gelişim Üzerindeki Etkileri

Abstract

In vitro mikroçoğaltım, sağlıklı bitkiciklerin hızlı ve mevsimden bağımsız olarak üretilmesini mümkün kılmakla birlikte, uzun vadeli başarısı büyük ölçüde aklimatizasyon aşamasındaki kayıpların en aza indirilmesine bağlıdır. Kültür kaplarının yüksek nem ve serbest yüzey nemi içermesi nedeniyle bitkicikler yeterli kütikula geliştiremez ve dış koşullara aktarıldıklarında kurumaya karşı son derece duyarlı hâle gelirler. Bu nedenle, aklimatizasyon sırasında destekleyici eksojen uygulamaların yapılması, bitkiciklerin toleransını artırmak ve ölüm oranlarını azaltmak için kritik öneme sahiptir. Bu çalışma, GA₃ (giberellik asit) ve AsA (askorbik asit) uygulamalarının aklimatizasyon sürecinde karşılaşılan kayıpları azaltmadaki ve in vitro koşullarda mikroçoğaltılmış bitkiciklerin adaptasyon başarısını artırmadaki etkilerini değerlendirmek amacıyla yürütülmüştür. Çalışmada yaşama oranı (%), bitki boyu (cm), sürgün çapı (mm), yaprak sayısı (adet/bitkicik), boğum sayısı (adet/bitkicik) ve klorofil içeriği (SPAD) ölçülmüştür. Sonuçlar, en yüksek yaşama oranının (%51,33) 25 ppm GA₃ + 300 mg L⁻¹ AsA uygulamasından elde edildiğini göstermiştir. En uzun sürgünler (18,40 cm) yine 25 ppm GA₃ + 300 mg L⁻¹ AsA uygulamasından elde edilmiştir. En kalın sürgün çapı (1,88 mm) 25 ppm GA₃ + 300 mg L⁻¹ AsA uygulamasında görülmüştür. En yüksek yaprak sayısı (15,00 adet/bitkicik) 25 ppm GA₃ + 150 mg L⁻¹ AsA ve 50 ppm GA₃ + 300 mg L⁻¹ AsA uygulamalarından elde edilmiştir. En yüksek boğum sayısı (14 adet/bitkicik) 50 ppm GA₃ + 300 mg L⁻¹ AsA uygulamasından elde edilmiştir. En yüksek klorofil içeriği (43,49 SPAD) ise 25 ppm GA₃ uygulamasından elde edilmiştir. Bu çalışma, GA₃ ve AsA kombinasyonlarının aklimatizasyon kayıplarını azaltmada etkili olabileceğini ortaya koymaktadır.

Keywords

• aklimatizasyon
• iyileştirici ajan
• in vitro

Project Number

1919B012411190

The Effects of GA₃ and AsA Applications on Vegetative Growth During Acclimatization

Abstract

In vitro micropropagation enables the rapid and season-independent production of healthy plantlets, yet its long-term success largely depends on minimizing losses during the acclimatization phase. Because the culture vessels contain high humidity and free surface moisture, plantlets develop insufficient cuticles, which makes them highly vulnerable to desiccation when transferred to external conditions. Therefore, implementing supportive exogenous treatments during acclimatization is essential to enhance plantlet tolerance and reduce mortality. This study was conducted to evaluate the effects of gibberellic acid (GA₃) and ascorbic acid (AsA) applications on reducing losses encountered during the acclimatization process and improving the adaptation success of micropropagated plantlets under in vitro conditions. Survival rate (%), plant height (cm), shoot diameter (mm), number of leaves (pieces/plantlet), number of nodes (pieces/plantlet), and chlorophyll content (SPAD) were measured. The results revealed that the highest survival rate (51.33%) was obtained from 25 ppm GA₃ + 300 mg L⁻¹ AsA, the longest shoots (18.40 cm) from 25 ppm GA₃ + 300 mg L⁻¹ AsA, the thickest shoot diameter from 25 ppm GA₃ + 300 mg L⁻¹ AsA (1.88 mm), the highest number of leaves (15.00 pieces/plantlet) from 25 ppm GA₃ + 150 mg L⁻¹ AsA and 50 ppm GA₃ + 300 mg L⁻¹ AsA, the highest number of nodes (14 pieces/plantlet) from 50 ppm GA₃ + 300 mg L⁻¹ AsA, and the highest chlorophyll content (43.49 SPAD) from 25 ppm GA₃. The study demonstrates that combinations of GA₃ and AsA can be effective in reducing acclimatization losses.

Keywords

• acclimatization
• healing agent
• in vitro

Project Number

1919B012411190

Thanks

This study was supported by the TÜBİTAK 2209-A (project number 1919B012411190) University Students Research Projects Support Program.

References

1. Abbas, H. M. K., Askri, S. M. H., Ali, S., Fatima, A., Qamar, M. T. U., Xue, S. D., ... & Zhong, Y. J. (2022). Mechanism associated with brassinosteroids crosstalk with gibberellic acid in plants. In Brassinosteroids Signalling: Intervention with Phytohormones and Their Relationship in Plant Adaptation to Abiotic Stresses(pp. 101-115). Singapore: Springer Singapore.
2. Abohatem, M. A., Al-Qubati, Y., Abohatem, H., & Bakil, Y. (2024). In vitro sprouts culture, shoots multiplication and plants acclimatization for commercial production of potato minitubers. Journal of Crop Science and Biotechnology, 27(2), 187-194.
3. Ahmad, I., Basra, S. M. A., & Wahid, A. (2014). Exogenous application of ascorbic acid, salicylic acid and hydrogen peroxide improves the productivity of hybrid maize at low temperature stress. International Journal of Agriculture & Biology, 16(4), 825-830.
4. Akram, N. A., Shafiq, F., & Ashraf, M. (2017). Ascorbic acid-a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Frontiers in Plant Science 8, 613.
5. Almokar, H. M. M., & Pirlak, L. (2018). Propagation of Aronia (Aronia melanocarpa) with tissue culture. Selcuk Journal of Agriculture and Food Sciences 32(3): 549-558.
6. Al-Douri, E. F. S., & Basheer, R. A. (2021). Effect of foliar spraying with ascorbic acid and dry yeast extract on some vegetative growth traits and chemical content of bitter almond (Prunus amygdalus var. Amara) seedlings. In IOP Conference Series: Earth and Environmental Science, 761 (1), p. 012049. IOP Publishing.
7. Babu, G. A., Mosa, Christas, K., Kowsalya, E., Ramesh, M., Sohn, S. I., & Pandian, S. (2022). Improved sterilization techniques for successful in vitro micropropagation. In Commercial Scale Tissue Culture for Horticulture and Plantation Crops. Springer Nature Singapore, Singapore, pp. 1-21.
8. Banerjee, A., & Roychoudhury, A. (2019). The regulatory signaling of gibberellin metabolism and its crosstalk with phytohormones in response to plant abiotic stresses. In Plant signaling molecules (pp. 333-339). Woodhead Publishing.

9. Barth, C., De Tullio, M., & Conklin, P. L. (2006). The role of ascorbic acid in the control of flowering time and the onset of senescence. Journal of Experimental Botany 57(8):1657-1665.
10. Bayhan, N., & Yücesan, B. (2024). The impact of sucrose and 6-benzylaminopurine on shoot propagation and vitrification in Aronia melanocarpa (black chokeberry). Plant Cell, Tissue and Organ Culture (PCTOC), 156(2), 55.
11. Bhojwani, S. S., & Dantu, P. K. (2013). Micropropagation. In Plant tissue culture: An introductory text (pp. 245-274). India: Springer India.
12. Borsai, O., Clapa, D., Fira, A., Hârța, M., Szabo, K., Dumitraș, A. F., & Pamfil, D. (2017). In vitro propagation of Aronia melanocarpa (Michx.) Elliott. In II International Symposium on Fruit Culture along Silk Road Countries, 1308 (pp. 213-222).
13. Brand, M. H., Obae, S. G., Mahoney, J. D., & Connolly, B. A. (2022). Ploidy, genetic diversity and speciation of the genus Aronia. Scientia Horticulturae, 291, 110604.
14. Cacak-Pietrzak, G., Dziki, D., Gawlik-Dziki, U., Parol-Nadłonek, N., Kalisz, S., Krajewska, A., & Stępniewska, S. (2023). Wheat bread enriched with black chokeberry (Aronia melanocarpa L.) pomace: Physicochemical properties and sensory evaluation. Applied Sciences, 13(12), 6936.
15. Celi, G. E. A., Gratão, P. L., Lanza, M. G. D. B., & Dos, Reis, A. R. (2023). Physiological and biochemical roles of ascorbic acid on mitigation of abiotic stresses in plants. Plant Physiology and Biochemistry 202: 107970.
16. Chandra, S., Bandopadhyay, R., Kumar, V., & Chandra, R. (2010). Acclimatization of tissue cultured plantlets: from laboratory to land. Biotechnology Letters 32: 1199-1205.
17. Chen, M., Maodzeka, A., Zhou, L., Ali, E., Wang, Z., & Jiang, L. (2014). Removal of DELLA repression promotes leaf senescence in Arabidopsis. Plant Science 219: 26-34.
18. Chowdhury, R. S., Kumar, V., Bhattacharya, S., Mallick, P., Ghosh, A., Bhattacharjee, S., & Kothari, S. K. (2023). Effect of gibberellic acid (GA3) on vegetative and reproductive growth and yield characters of cucumber (Cucumis sativus) under costal region of west bengal, India. International Journal of Plant & Soil Science 35(21): 90-96.
19. Çelebi-Toprak, F., Alan, A. R. (2018). A successful micropropagation protocol for three aronia (Aronia melanocarpa) cultivars. In XXX International Horticultural Congress IHC2018: II International Symposium on Micropropagation and In Vitro Techniques 1285, pp. 173-176.
20. Çoban, G. A., & Aras, S. (2022). Effects of ascorbic and oxalic acids on cucumber seedling growth and quality under mildly limey soil conditions. Gesunde Pflanzen 75:1925–1932.
21. Conklin, P. L. (2001). Recent advances in the role and biosynthesis of ascorbic acid in plants. Plant, Cell & Environment, 24(4), 383-394.
22. Conklin, P. L., & Barth, C. (2004). Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant, cell & environment, 27(8), 959-970. Dias, M. C., Correia, C., Moutinho-Pereira, J., Oliveira, H., & Santos, C. (2014). Study of the effects of foliar application of ABA during acclimatization. Plant Cell, Tissue and Organ Culture (PCTOC) 117: 213-224.
23. Dinler, B. S., & Çetinkaya, H. (2020). Bitkilerde giberellik asit hormonunun sentezi, sinyal iletimi ve tuz stresi altındaki etkileri. Ziraat Fakültesi Dergisi 15(1): 56-63.
24. Duman, H., Üner, B., Sarıtaş, S., Bolat, E., Yalçıntaş, Y. M., Kalkan, A. E., ... & Oz, F. (2025). Exploring the Potential of Black Chokeberry (Aronia melanocarpa) as a Health‐Enhancing Agent: A Comprehensive Overview. Journal of Food Biochemistry, 2025(1), 8899523.
25. Ekinci, H., Saskin, N., Ak, B. E., & Dogan, B. D. (2024). Effects of different healing agents on acclimatization success of in vitro rooted Garnem (Prunus dulcis× Prunus persica) rootstock. In Vitro Cellular & Developmental Biology-Plant 60(3): 309-317.
26. Emamverdian, A., Ding, Y., & Mokhberdoran, F. (2020). The role of salicylic acid and gibberellin signaling in plant responses to abiotic stress with an emphasis on heavy metals. Plant signaling & behavior, 15(7), 1777372.
27. El-Badawy, H. E. M. (2013). Effect of some antioxidants and micronutrients on growth, leaf mineral content, yield and fruit quality of Canino apricot trees. Journal of Applied Sciences Research, 9(2), 1228-1237.
28. El-Tohamy, W. A., Dasgan, H. Y., & Gruda, N. S. (2023). Impact of gibberellic acid on water status, growth, and development of cape gooseberry in newly reclaimed sandy lands within arid regions. Horticulturae 9(12): 1283.
29. Ergın, S., Aydogan, C., Ozturk, N., & Turhan, E. (2014). Effects of ascorbic acid application in strawberry plants during heat stress. Türk Tarım ve Doğa Bilimleri Dergisi, 1(Özel Sayı-2), 1486-1491.
30. Fahad, S., Hussain, S., Matloob, A., Khan, F. A., Khaliq, A., Saud, S., ... & Huang, J. (2015). Phytohormones and plant responses to salinity stress: a review. Plant growth regulation, 75(2), 391-404.
31. Farahat, M. M., Mazhar, A. A., Mahgoub, M. H., & Zaghloul, S. M. (2013). Salt tolerance in Grevillea robusta seedlings via foliar application of ascorbic acid. Middle-East Journal of Scientific Research, 14(1), 9-15.
32. George, E. F., & Debergh, P. C., (2008). Micropropagation: Uses and Methods. George EF, Hall MA, Klerk GJ (eds.), In: Plant Propagation by Tissue Culture. Dordrecht: Springer Netherlands, pp. 29-64.
33. Gomez, K. A., & Gomez, A. A. (1984). Statistical procedures for agricultural research. John wiley & sons.
34. Grzelak, M., Pacholczak, A., & Nowakowska, K. (2024). Challenges and insights in the acclimatization step of micropropagated woody plants. Plant Cell, Tissue and Organ Culture (PCTOC) 159(3): 1-20.
35. Halliwell, B. (1987). Oxidative damage, lipid peroxidation and antioxidant protection in chloroplasts. Chemistry and Physics of lipids, 44(2-4), 327-340.
36. Hasan, S., Sehar, Z., & Khan, N. A. (2020). Gibberellic acid and sulfur-mediated reversal of cadmium-inhibited photosynthetic performance in mungbean (Vigna radiata L.) involves nitric oxide. Journal of Plant Growth Regulation 39: 1605-1615.
37. Hazarika, B. N., Teixeira, da Silva, J. A., & Talukdar A (2006). Effective acclimatization of in vitro cultured plants: methods, physiology and genetics. Floriculture, Ornamental and Plant Biotechnology 2: 427-438.
38. Iqbal, M., & Ashraf, M. (2013). Gibberellic acid mediated induction of salt tolerance in wheat plants: Growth, ionic partitioning, photosynthesis, yield and hormonal homeostasis. Environmental and experimental botany, 86, 76-85.
39. Jalili, I., Ebadi, A., Askari, M. A., KalatehJari, S., & Aazami, M. A. (2023). Foliar application of putrescine, salicylic acid, and ascorbic acid mitigates frost stress damage in Vitis vinifera cv. ‘Giziluzum’. BMC Plant Biology, 23(1), 135.
40. Kara, Z., Yazar, K., Ekinci, H., Doğan, O., & Özer, A. (2022). The effects of ortho silicone applications on the acclimatization process of grapevine rootstocks. Selcuk Journal of Agriculture and Food Sciences 36(2): 233-237.
41. Karakoyun, M., Arikan, Ş., & İpek, M. (2024). Determination of the reactions of ‘Chester’Blackberry variety to different CaCO3 applications in in vitro conditions. Applied Fruit Science, 66(6), 2203-2209.
42. Korkmaz, K., Akgün, M., Kırlı, A., Özcan, M. M., Dede, Ö., & Kara, Ş. M. (2020). Giberellik asit ve salisilik asit uygulamalarının tuz stresi altında yetiştirilen kolzanın (Brassica napus L.) bazı fiziksel ve kimyasal özellikleri üzerine etkileri. Turkish Journal of Agriculture-Food Science and Technology 8(4): 873-881.
43. Kokotkiewicz, A., Jaremicz, Z., & Luczkiewicz, M. (2010). Aronia plants: a review of traditional use, biological activities, and perspectives for modern medicine. Journal of medicinal food, 13(2), 255-269.
44. Krishna, H., Singh, S. K., Sharma, R. R., Khawale, R. N., Grover, M., & Patel, V. B. (2005). Biochemical changes in micropropagated grape (Vitis vinifera L.) plantlets due to arbuscular-mycorrhizal fungi (AMF) inoculation during ex vitro acclimatization. Scientia Horticulturae 106(4): 554-567.
45. Kumar, K., & Rao, I. U. (2012). Morphophysiologicals problems in acclimatization of micropropagated plants in- ex vitro conditions- A Reviews. Journal of Ornamental and Horticultural Plants 2(4): 271-283.
46. Li, J., Gao, H., Jiang, J., Dzyubenko, N., Chapurin, V., Wang, Z., & Wang, X. (2013). Overexpression of the Galega orientalis gibberellin receptor improves biomass production in transgenic tobacco. Plant Physiology and Biochemistry 73: 1-6.
47. Li, X., Wu, P., Lu, Y., Guo, S., Zhong, Z., Shen, R., & Xie, Q. (2020). Synergistic interaction of phytohormones in determining leaf angle in crops. International journal of molecular sciences, 21(14), 5052.
48. Mayi, A. A., Ibrahim, Z. R., & Abdurrahman, A. S. (2014). Effect of foliar spray of humic acid, ascorbic acid, cultivars and their interactions on growth of olive (Olea european L.) transplants cvs. Khithairy and Sorany. Khithairy and Sorany. J. Agric. Vet. Sci, 7, 18-30.
49. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum, 15(3), 473-497
50. Mozafar, A., & Oertli, J. J. (1993). Vitamin C (ascorbic acid): uptake and metabolism by soybean. Journal of plant physiology, 141(3), 316-321.
51. Nagar, S., Singh, V. P., Arora, A., Dhakar, R., Singh, N., Singh, G. P., ... & Shiv, Ramakrishnan, R. (2021). Understanding the role of gibberellic acid and paclobutrazol in terminal heat stress tolerance in wheat. Frontiers in Plant Science, 12, 692252.
52. Nas, Z., Eşitken, A., & Pırlak, L. (2025). ‘Viking’ Aronya çeşidinin in vitro şartlarda bitki rejenerasyon protokolünün belirlenmesi. Bahçe 54(1): 11-16.
53. Ochmian, I. D., Grajkowski, J., & Smolik, M. (2012). Comparison of some morphological features, quality and chemical content of four cultivars of chokeberry fruits (Aronia melanocarpa). Notulae botanicae horti agrobotanici cluj-napoca, 40(1), 253-260.
54. Othman, Y. A., & Leskovar, D. I. (2022). Foliar application of gibberellic acid improves yield and head phenolic compounds in globe artichoke. Scientia Horticulturae 301: 111115.
55. Parveen, S., Arfan, M., & Wahid, A. (2025). Exogenous applications of ascorbic acid improve wheat growth, physiology and yield under salinity stress via a balance in antioxidant production and ROS scavenging. New Zealand Journal of Crop and Horticultural Science, 53(5), 1384-1407.
56. Rady, M. M., Boriek, S. H., Abd, El-Mageed ,T. A., Seif, El-Yazal, M. A., Ali, E. F., Hassan, F. A., & Abdelkhalik, A. (2021). Exogenous gibberellic acid or dilute bee honey boosts drought stress tolerance in Vicia faba by rebalancing osmoprotectants, antioxidants, nutrients, and phytohormones. Plants, 10(4), 748.
57. Ritonga, F. N., Zhou, D., Zhang, Y., Song, R., Li, C., Li, J., & Gao, J. (2023). The roles of gibberellins in regulating leaf development. Plants 12(6): 1243.
58. Sabir, M., Naseem, Z., Ahmad, W., Usman, M., Nadeem, F., & Saifullah, Ahmad, H. R. (2022). Alleviation of adverse effects of nickel on growth and concentration of copper and manganese in wheat through foliar application of ascorbic acid. International Journal of Phytoremediation, 24(7), 695-703.
59. Sajid, Z. A., & Aftab, F. (2009). Amelioration of salinity tolerance in Solanum tuberosum L. by exogenous application of ascorbic acid. In Vitro Cellular & Developmental Biology-Plant 45(5): 540-549.
60. Shah, S. H., Islam, S., Mohammad, F., & Siddiqui, M. H. (2023). Gibberellic acid: a versatile regulator of plant growth, development and stress responses. Journal of Plant Growth Regulation 42(12): 7352-7373.
61. Shaki, F., Maboud, H. E., & Niknam, V. (2019). Effects of salicylic acid on hormonal cross talk, fatty acids profile, and ions homeostasis from salt-stressed safflower. Journal of plant Interactions, 14(1), 340-346.
62. Siddiqui, M. H., Alamri, S., Alsubaie, Q. D., & Ali, H. M. (2020). Melatonin and gibberellic acid promote growth and chlorophyll biosynthesis by regulating antioxidant and methylglyoxal detoxification system in tomato seedlings under salinity. Journal of Plant Growth Regulation 39(4): 1488-1502.
63. Sidor, A., & Gramza-Michałowska, A. (2019). Black chokeberry Aronia melanocarpa L.—A qualitative composition, phenolic profile and antioxidant potential. Molecules, 24(20), 3710.
64. Smirnoff, N. (2018). Ascorbic acid metabolism and functions: A comparison of plants and mammals. Free Radical Biology and Medicine 122: 116-129.
65. Smirnoff, N., & Wheeler, G. L. (2000). Ascorbic acid in plants: biosynthesis and function. Critical Reviews In Plant Sciences 19(4): 267-290.
66. Šnebergrová, J., Čížková, H., Neradová, E., Kapci, B., Rajchl, A., & Voldřich, M. (2014) Variability of characteristic components of aronia. Czech Journal of Food Sciences 32(1):25–30.
67. Sivanesan, I., Saini, R. K., & Kim, D. H. (2016). Bioactive compounds in hyperhydric and normal micropropagated shoots of Aronia melanocarpa (michx.) Elliott. Industrial Crops and Products, 83, 31-38.
68. Sutter, E. (1984). Chemical composition of epicuticular wax in cabbage plants grown in vitro. Canadian Journal of Botany, 62(1), 74-77.
69. Thakur, N., & Singh, G. (2024). Enhancing apricot growth and leaf nutrient content through antioxidant and bio-regulator applications. Indian J Ecol, 51(3), 587-592.
70. Vasar, V. (2001). Effect of ascorbic acid and citric acid on ex vitro rooting and acclimatization of Prunus avium L. microshoots. In I International Symposium on Acclimatization and Establishment of Micropropagated Plants 616, pp. 251-254.
71. Weiss, D., & Ori, N. (2007). Mechanisms of cross talk between gibberellin and other hormones. Plant physiology, 144(3), 1240-1246.
72. Yaman, M., Palaz, E. B., Isak, M. A., Demirel, S., İzgü, T., Adalı, S., ... & Popescu, M. (2025). Integrating in vitro propagation and machine learning modeling for efficient shoot and root development in Aronia melanocarpa. Horticulturae, 11(8), 886.
73. Zhang, X., Yu, H. J., Zhang, X. M., Yang, X. Y., Zhao, W. C., Li, Q., & Jiang, W. J. (2016). Effect of nitrogen deficiency on ascorbic acid biosynthesis and recycling pathway in cucumber seedlings. Plant Physiology and Biochemistry, 108, 222-230.
74. Zheng, X., Gong, M., Zhang, Q., Tan, H., Li, L., Tang, Y., ... & Deng, W. (2022). Metabolism and regulation of ascorbic acid in fruits. Plants 11(12): 1602.