In Vitro Direct Shoot Regeneration of Kaman 1 Walnut Variety and Kaman 5 Walnut Genotype (Juglans regia L.)

Year 2025, Volume: 8 Issue: 5, 646 - 652, 15.09.2025

Ebru Şirin , Sevil Sağlam Yılmaz , Kenan Yıldız

https://doi.org/10.47115/bsagriculture.1722353

Abstract

In this study, a simple and effective clonal micropropagation protocol was developed from axillary buds of Kaman 1 walnut variety and Kaman 5 walnut genotype. For surface sterilization of explants, they were first treated in 70% ethanol for 1 minute and then treated with 0.2% HgCl2 containing two drops of Tween 20 for 5 minutes. To prevent browning of the explants, 100 mg/l Ascorbic Acid, 100 mg/l Citric Acid and 100 mg/l Ascorbic Acid+100 mg/l Citric Acid were added to the MS nutrient medium. In addition, explants were subcultured at 3, 24 and 48 hour intervals. It was observed that the most effective treatment in both Kaman 1 walnut variety and Kaman 5 walnut genotype was 100 mg/l AA + 100 mg/l SA + 48 hours subculture. Within the scope of the shoot regeneration study, six different combinations of 0.5, 1.0 and 1.5 mg/l doses of BAP and 0.5 and 1.0 mg/l doses of IBA were tried. The highest average number of shoots per explant 2.67 was obtained from the Kaman 1 variety on MS medium supplemented with 0.5 mg/l BAP and 1.0 mg/l IBA. On the other hand, in the medium containing 0.5 mg/l BAP and 0.5 mg/l IBA, the average number of shoots was 2.00 for the Kaman 1 cultivar and 0.67 for the Kaman 5 genotype. After the shoots were kept in a solution containing 4 mg/l IBA for 10 minutes, they were planted in pots containing a peat: perlite (1:1) mixture, but rooting couldn’t be achieved from the shoots.

Keywords

Walnut , İn vitro , Tissue culture , Micropropagation , Antioxidant , Subculture

Ethical Statement

Since no studies involving humans or animals were conducted, ethical committee approval was not required for this study.

Supporting Institution

Tokat Gaziosmanpaşa Üniversitesi, BAP Birimi

Project Number

2013/35

Thanks

This study is derived from the master’s thesis of Ebru Şirin and was supported by the Scientific Research Projects Commission of Gaziosmanpaşa University (Project No: 2013/35).

References

• Akça Y. 2009. Ceviz Yetiştiriciliği. 3. baskı, pp: 329-335.
• Babaoğlu M, Gürel E, Özcan S. 2002. Bitki biyoteknolojisi-I, doku kültürü ve uygulamaları. Selçuk Üniversitesi Basımevi, Konya, Türkiye, ss: 162.
• Çördük N, Akı C. 2011. Inhibition of Browning Problem During Micropropagation of Sideritis trojana Bornm., an Endemic Medicinal Herb of Turkey. Romanian Biotechnol Lett, 16(6): 6760-6765
• D’Amato F 1977. Cytogenetics of differentiation in tissue and cell cultures. In: Reinert J, Bajaj YPS (Eds), Plant Cell, Tissue and Organ Culture, Narosa Publ House, New Delhi, pp: 343-357
• Dirlik C, Kandemir H, Çetin N, Şen S, Güler B, Gürel A. 2022. Effects of Different Culture Media Compositions on In Vitro Micropropagation from Paradox Walnut Rootstock Nodes. GU J Sci Part A, 9(4): 500-515
• Ellialtıoğlu Ş. 1999. Doku Kültürü Yoluyla Vegetatif Çoğaltmada Doku Kararması Sorunu, Nedenleri ve Çözüm Yolları. Biyoteknoloji Kükem Dergisi, 24(1): 37-47
• Emiroğlu Ü, Gürel A. 2005. Bitki ıslahında doku kültürü tekniklerinin kullanımı. Tohum Bilimi ve Teknolojisi, Cilt 1, s. 91-155, EÜ Tohum Teknolojisi ve Araştırma Merkezi, Bornova, İzmir, Türkiye
• Gruselle R, Badia N, Boxus P. 1987. Walnut micropropagation: first results. Acta Hort, 212: 511-516
• Güleryüz M. 1982. Bahçe ziraatında büyütücü ve engelleyici maddelerin kullanılması ve önemi. Atatürk Üniversitesi Yayınları, No: 279
• Kepenek K, Kolağası Z, 2016. Micropropagation of Walnut (Juglans regia L.). Acta Phys Pol A, 130(1): 150-156
• Leslie C, McGranahan G. 1992. Micropropagation of Persian walnuts (Juglans regia L). In: Bajaj YPS (Ed), Biotechnology in Agriculture and Forestry, Vol. 18, High-Tech and Micropropagation, Springer, Berlin, pp: 136-150
• Murashige G, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant, 15: 473-497
• Myrselaj M, Sota V, Kongjika E. 2020. Reducing Oxidative Stress on zygotic Embryos of Walnut (Juglans regia L.) Under In Vitro Conditions by Their Pretreatment With Ascorbic acid. Eur J Biotechnol Gen Eng, 7(1)
• Navatel J, Bourrain L. 2001. Production of walnut (Juglans regia L.) by in vitro multiplication. Acta Hort, 544: 465-471
• Penuela R, Gravito C, Sanchez-Tames R, Rodriquez R. 1988. Multiple shoot-bud stimulation and rhizogenesis induction of embryonic and juvenile explants of walnut. Acta Hort, 227: 457-459
• Revilla MA, Majada I, Rodriquez R. 1989. Walnut (Juglans regia L) micropropagation. Ann Sci For (Paris), 46(Suppl): 149-151
• Rios-Leal D, Sanchez-Olate M, Aviles F, Materan ME, Uribe M, Hasbun R, Rodriquez R. 2007. Micropropagation of Juglans regia L. In: Jain SM, Hüggman H (Eds), Protocols for Micropropagation of Woody Trees and Fruits, pp: 381-390
• Rodriquez R, Lopez C, Diaz-Sala C, Berros B. 1993. Simultaneous shoot-bud development on walnut tissues of different ages; macromorphological and histological analyses. Acta Hort, 311: 141-152
• Rout GR, Samantaray S, Mottley J, Das P. 1999. Biotechnology of the rose: a review of recent progress. Sci Hortic, 81: 201-228
• Saadat YA, Hennerty MJ. 2002. Factors affecting the shoot multiplication of Persian walnut (Juglans regia L.). Scientia Hortic, 95: 251-260
• Şen SM. 2011. Ceviz Yetiştiriciliği, besin değeri folklörü
• Tekinsoy B, Gülşen Y. 1993. Cevizin (Juglans nigra L.) doku kültürü tekniği ile çoğaltılmasında kahverengileşme sorununun çözüm yolları. 8. Kükem Kongresi, Kükem Dergisi, 16(2), Ankara
• Tuan PN, Meier-Dinkel A, Höltken AM, Wenzlitschke I, Winkelmann T. 2016. Paving the way for large-scale micropropagation of Juglans × intermedia using genetically identified hybrid seed. Plant Cell Tiss Organ Cult, 126: 153-166
• Türen T, Savalan Ş, Pehlivan EC. 2024. FeSO4.7H2O, indol-3-bütirik asit ve farklı besin ortamlarının in vitro ceviz (Juglans regia L.) fidanı çoğaltımı ve mikroaşılma üzerine etkisi. Tekirdağ Ziraat Fak Derg, 21(3): 748-758
• Vahdati K, Leslie CA, Zamani Z, McGranahan G. 2004. Rooting and acclimation of in-vitro grown shoots from mature tree of three Persian walnut cultivars. HortScience, 39: 324-327
• Vahdati K, Najafianashrafi E, Ebrahimzadeh H, Mirmasoumi M. 2009. Improved Micropropagation of Walnut (Juglans regia L.) on Media. Acta Hort, 839: 117-124
• Yıldırım HT. 2018. Bazı Ceviz Çeşitlerinde In Vitro Mikroçoğaltım. Yüksek Lisans Tezi, Tekirdağ Namık Kemal Üniversitesi, Fen Bilimleri Enstitüsü, Tekirdağ, Türkiye
• Zamir R, Shah ST, Ali N, Khattak GSS, Muhammad T. 2004. Studies on in vitro Surface Sterilization and Antioxidants on Guava Shoot Tips and Nodal Explants. Pak J Biotechnol, 1(2): 12-16.