The Influence of Genotype, Pre-Chilling and Nutrient Media on Callus Induction and Plantlet Regeneration in Rye (Secale cereale L.) Anther Culture

Abstract

The present study investigated the effects of genotypes (diploid and tetraploid), of pre-treatment (coldtreatmentwithin one week at 4°C and non-cold treatment) and and nutrient media (MS, N6 and NN) on anther response ratio,callus induction and green plantlet regeneration in anther culture of rye. In our experiment, the MSmedium (consisting of 10 g/L of agar), NN medium (consisting of 8 g/L agar) and N6 medium (consisting of 5 g/L of agar)were used for callus formation, MS and NN media supplemented with 2.4 µmol/L of KIN, 4.4 µmol/L of BAP and 2.7 µmol/L of NAA as hormone and 30.3 g/L of sucrose as carbon source. Additionaly, N6 medium supplemented with 2 mg/L 2.4-D, 0.5 mg/L KIN, 1 mg/L IAA as hormone and 90 g/L sucrose as carbon source. Two media (MS and NN) used for callus formation were the same for plantlet regeneration, however N6 media was modified and included 2 mg/L KIN, 2 g/L activated charcoal, 20 g/L sucrose and 5 g/L agar. Our study demonstrated the negativeeffect of pre-chilling on anther response ratio and callus size, but it also showed that the positive effect on callus ratio and root formation.On the other hand, effect of pre-chilling on investigated characters changed according to rye genotypes and was significant (except for anther response ratio). While effect of nutrient media on callus ratio and size was significant, it was not significant on anther response ratio. The highest root and plantlet regeneration ratio were obtained as a result of cultured on MS media of pre-chilled anthers in diploid rye.

Keywords

• Anther culture
• in vitro
• callus
• rye

Çavdarda (Secale cereale L.) Anter Kültürü ile Kallus Oluşumu ve Bitki Rejenerasyonu Üzerine Genotip, Soğuk Uygulama ve Besi Ortamlarının Etkisi

Abstract

Bu çalışmada, çavdarda genotipin (diploid ve tetraploid), ön işlem (+4 ^oC’de 1 hafta süre ön işlem uygulanmış ve uygulanmamış) ve besin ortamlarının (MS, N₆ ve NN) anter kültüründe anter tepki oranı, kallus oluşumu ve bitki rejenerasyonu üzerine etkileri incelenmiştir. Denemede kallus oluşum ortamı olarak MS ortamında 10 g/L agar, NN ortamında 8 g/L agar kullanılmış; 2,4 µmol/L Kinetin, 4,4 µmol/L BAP, 2,7 µmol/L NAA ve karbon kaynağı olarak 30,3 g/L sükroz her iki ortama ilave edilmiştir. N₆ ortamına ise 5 g/L agar, 2 mg/L 2,4-D, 0,5 mg/L Kinetin, 1 mg/L IAA ve 90 g/L sükroz ilave edilmiştir. Rejenerasyon ortamı olarak modifiye edilmiş N₆ ortamına 2 g/L aktif karbon, karbon kaynağı olarak 20 g/L sükroz, hormon olarak 2 mg/L kinetin, katılaştırıcı madde olarak da 5 g/L agar eklenmiştir. Araştırmada soğuk ön işlem uygulamasının anter tepki oranı ve kallus büyüklüğü üzerine olumsuz, kallus oranı ve bitki rejenerasyonu üzerine olumlu etkisi belirlenmiştir. Diğer taraftan soğuk ön uygulamasının incelenen özellikler üzerine etkisi çavdar genotiplerinde farklılık göstermiştir. Ayrıca anter tepki oranı hariç, incelenen diğer özellikler üzerine ön işlem uygulamasının etkisi önemli, genotipin etkisi ise önemsiz çıkmıştır. Besin ortamlarının etkisi ise kallus oranı ve büyüklüğü üzerinde önemli, anter tepki oranı üzerine ise önemsiz bulunmuştur. En fazla kök oluşumu ve bitki rejenerasyonu soğuk uygulamalı diploid çavdar anterlerinin MS ortamında kültüre alınması ile elde edilmiştir.

Keywords

• Anter kültürü
• in vitro
• kallus

References

1. Afza, R., Shen, M., Zapata-Arias, F. J., Xie, J., Fundi, H. K., Lee, K. S, Bobadilla-Mucino, E. ve Kodym, A. (2000). Effect of spikelet position on rice anther culture efficiency. Plant Science, 153(2), 155-159.
2. Akgün, İ. ve Altındal, N. (2015). Effects of carbohydrate sources, pre-chilling application and ploidy levels on anther culture in rye (Secale cereale L.). Academia Journal of Biotechnology, 3(6), 111-116.
3. Başer, İ., Korkut, K. Z., Bilgin, O. ve Balkan, A. (2016). Anter kültürü tekniği ile ekmeklik buğday (Triticum aestivum L.) melez populasyonlarından doubled haploid bitkilerin elde edilmesi. Tarla Bitkileri Merkez Araştırma Enstitüsü Dergisi, 25(Özel sayı-1), 232-236.
4. Bazylewska, J., Broda, Z., Mikołajczyk, S. ve Pluta, M. (2015). Induction of haploids in the genus Secale L. via androgenesis. Session I: Plant differentiation processes in the culture of somatic cells and cells of generative pathwa, BioTechnologia, 96(1), 55.
5. Begheyn, R. F., Lübberstedt, T. ve Studer, B. (2016). Haploid and doubled haploid techniques in perennial ryegrass (Lolium perenne L.) to advance research and breeding. Agronomy, 6, 60.
6. Cho, M. S. ve Zapata, F. J. (1990). Plant regeneration from ısolated microspores of indica rice. Plant and Cell Physiology, 31, 881-885.
7. Çeliktaş, N. ve Hatipoğlu, R. (1997). Arpa (Hordeum vulgare L.) anter kültüründe genotip, soğuk uygulama süresi ve besi ortamı 2,4-D içeriğinin etkisi. Çukurova Üniversitesi Ziraat Fakültesi Dergisi, 12(2), 63-172.
8. Daniel, G. (1993). Anther culture in rye: improved plant regeneration using modified MS-media. Plant Breeding, 110, 259-261.

9. De Buyser, J., Henry, Y., Lonnet, P., Hertzog, R. ve Hespel, A. (1987). Florin: a doubled haploid wheat variety developed by anther culture method. Plant Breeding, 98, 53-56.
10. Dedicova, B., Obert, B., Zofajova, A., Matusik, M. ve Pretova, A. (1999). Anther cultures of barley. Biologia-Bratislava, 54(1), 91-99.
11. Dodds, J. H. ve Roberts, L. W. (1985). Experiments in plant tissue culture. Cambridge University Press, New York.
12. Eudes F. ve Amundsen, E. (2005). Isolated microspore culture of Canadian 6 x triticale cultivars. Plant Cell, Tissue and Organ Culture, 82, 233-241.
13. Finnie, S. J., Powell, W. ve Dyer, A. F. (1989). The effect of carbohydrate composition and concentration on anther culture response in barley (Hordeum vulgare L.). Plant Breeding, 103, 110-118.
14. Flehinghaus, T., Deimling, S. ve Geiger, H. H. (1991). Methodical ımprovements in rye anther culture. Plant Cell Reports, 10, 397-400.
15. Flehinghaus-Roux, T., Deimling, S. ve Geiger, H. H. (1995). Anther-culture ability in Secale cereale L. Plant Breeding, 114, 259-261. Guo, Y. D. ve Pulli S. (2000). Isolated microspore culture and plant regeneration in rye (Secale cereale L,). Plant Cell Reports, 19, 875-880.
16. Gürel, A., Tosun, M. ve Demir, İ. (1993). Bazı makarnalık ve ekmeklik buğday genotiplerinin anter kültürüne reaksiyonları. Anadolu, J. of AARI, 2, 98-111.
17. Gürel, A. (1994). Susam (Sesamum indicum L.)’da anter kültürü. Tarla Bitkileri Kongresi, Ege Üniversitesi Ziraat Fakültesi, 25-29 Nisan, Bornova.
18. González, J. M. ve Jouve, N. (2000). Improvement of anther culture media for haploid production in triticale. Cereal Research Communications, 28, 65-72.
19. Hatipoğlu, R. ve Sakin, M. A. (1996). Tütün (Nicotiana tabacum) anter kültüründe genotip ve besi ortamının haploid bitki oluşumuna etkileri üzerinde araştırma. Çukurova Üniversitesi Ziraat Fakültesi Dergisi, 11(4), 107-116.
20. Immonen, S. ve Anttila, H. (1996). Success in anther culture of rye. Vorträge für Pflanzenzüchtung, 35, 237- 244.
21. Immonen, S. ve Anttila, H. (1998). Impact of microspore developmental stage on ınduction and plant regeneration in rye anther culture. Plant Science, 139(2), 213-222.
22. Immonen, S. (1999). Androgenetic green plants from winter rye, Secale cereale L., of diverse origin. Plant Breeding, 118, 319-322.
23. Immonen, S. ve Anttila, H. (1999). Cold pretreatment to enhance green plant regeneration from rye anther culture. Plant Cell, Tissue and Organ Culture, 57, 121-127.
24. Islam, S. M. S. ve Tuteja, N. (2012). Enhancement of androgenesis by abiotic stress and other pretreatments in major crop species. Plant Science, 182, 134-144.
25. Jäger, K., Bartók, T., Ördög, V. ve Barnabás, B. (2010). Improvement of maize (Zea mays L.) anther culture responses by algae-derived natural substances. South African Journal of Botany, 76(3), 511-516.
26. Khanna, H. K. ve Raina, S. K. (1998). Genotype x culture media interaction effects on regeneration response of three indica rice cultivars. Plant Cell, Tissue and Organ Culture, 52, 145-153.
27. Ma, R., Guo, Y. D. ve Pulli, S. (2004). Comparison of anther and microspore culture in the embryogenesis and regeneration of rye (Secale cereale L.). Plant Cell, Tissue and Organ Culture, 76, 147-157.
28. Mandal, A. B. ve Bandyopadhyay, A. K. (1997). In vitro anther culture response in indica rice hybrids. Cereal Research Communications, 25(4), 891-896.
29. Mikołajczyk, S., Broda, Z. ve Weigt, D. (2012). The effect of cold temperature stress on the viability of rye (Secale cereale L.) microspores. BioTechnologia, Journal of Biotechnology, Computational Biology and Bionanotechnology, 93(2), 139-142.
30. Mishra, V. K. ve Goswami, R. (2014). Haploid production in higher plant. International Journal of Chemical and Biochemical Science, Review Paper,1, 25-45.
31. Oleszczuk, S., Zimny, J., Makowska, K., Zimny, A., Czaplicki, A., Kozdój, J. ve Sowa, S. (2013). Using androgenesis in studies on single cell-derived embryo development. Modern Phytomorphology, 4, 17-20.
32. Plamenov, D., Belchev, I., Daskalova, N., Spetsov, P. ve Moraliyski, T. (2013). Application of a low dose of gamma rays in wheat androgenesis. Archives of Biological Sciences, 65(1), 291-296.
33. Ponitka, A. ve Ślusarkiewicz-Jarzina, A. (2004). Cleared-ovule technique used for rapid access to early embryo development in Secale cereale × Zea mays crosses. Acta Biologica Cracoviensia, Series Botanica, 46, 133-137.
34. Powell, W. (1998). The influence of genotype and temperature pre-treatment on anther culture response in barley (Hordeum vulgare L.). Plant Cell, Tissue and Organ Culture, 12, 291-297.
35. Qi, Y., Ye, Y. ve Bao, M. (2011). Establishment of plant regeneration system from anther culture of Tagetes patula. African Journal of Biotechnology, 10(75), 17332- 17338.
36. Rakoczy-Trojanowska, M., Śmiech, M. ve Malepszy, S. (1997). The ınfluence of genotype and medium on rye (Secale cereale L.) anther culture. Plant Cell, Tissue and Organ Culture, 48, 15-21.
37. Saidi, N., Cherkaoui, S., Chlyah, A. ve Chlyah, H. (1997). Embryo formation and regeneration in Triticum turgidum ssp. durum anther culture. Plant Cell, Tissue and Organ Culture, 51, 27-33.
38. Silva, T. D. (2010). Indica rice anther culture: can the impasse be surpassed?. Plant Cell, Tissue and Organ Culture, 100, 1-11.
39. Szarejko, I. ve Kasha, K. J. (1991). Induction of anther culture derived doubled haploids in barley. Cereal Research Communications, 19, 219-237.
40. Targońska, M., Hromada-Judycka, A., Bolibok-Bragoszewska, H. ve Rakoczy-Trojanowska, M. (2013). The specificity and genetic background of the rye (Secale cereale L.) tissue culture response. Plant Cell Reports, 32(1), 1-9.
41. Tenhola-Roininen, T., Tanhuanpää P. ve Immonen S. 2005. The effect of cold and heat treatments on the anther culture response of diverse rye genotypes. Euphytica, 145, 1-9.
42. Tenhola-Roininen, T. (2009). Rye Doubled Haploids. Production and use in mapping studies. Jyväskylä Studies in Biological and Environmental Science, University of Jyväskylä, 198, 93.
43. Trejo-Tapia, G., Amaya, U. M., Morales, G. S., Sánchez, A. D. J., Bonfil, B. M., Rodríguez-Monroy, M. ve Jiménez-Aparicio, A. (2002). The Effects of cold-pretreatment, auxins and carbon source on anther culture of rice. Plant Cell, Tissue and Organ Culture, 71, 41-46.
44. Weigt. D., Kiel, A., Nawracała, J. ve Pluta, M. (2015). The androgenic response in anther culture of spring wheat is greater in genotypes with solid stem in contrast to genotypes with hollow stem. BioTechnologia, 96(1), 90.
45. Weigt, D., Kiel, A., Nawracała, J., Tomkowiak, A., Kurasiak-Popowska, D., Idzı Siatkowski, I. ve Ługowska, B. (2016). Obtaining doubled haploid lines of the Lr19 gene using anther cultures of winter wheat genotypes. BioTechnologia. Journal of Biotechnology, Computational Biology and Bionanotechnology, 97(4), 285-293.
46. Wenzel, G., Hoffmann, F. ve Thomas, E. (1977). Increased ınduction and chromosome doubling of androgenetic haploid rye. Theoretical and Applied Genetics, 51, 81-86.
47. Zimny, J. ve Michalski, K. (2019). Development in vitro culture techniques of advancement rye (Secale cereale L.) breeding. Acta Biologıca Cracoviensia, Series Botanica, DOI: 10.24425/abcsb.2019.127735.