Year 2020, Volume , Issue 19, Pages 449 - 459 2020-08-31

‘Kalender’ Yonca (Medicago sativa L.) Çeşidinin In vitro Çoğaltımı Üzerine Farklı Besin Ortamları, Sitokininler ve Eksplant Tiplerinin Etkisi
Effect of Different Media, Cytokinins and Explant Types on In vitro Propagation of Alfalfa (Medicago sativa L.) cultivar ‘Kalender’

Fatma Gökşin BAHAR [1] , Meltem BAYRAKTAR [2] , Aynur GÜREL [3]


Yonca (Medicago sativa L.) tüm dünyada yetiştirilen en önemli yem bitkilerinden biridir. Yonca bitkisine ait bazı çeşitlerin in vitro çoğaltımı daha önceleri çalışılmıştır. Ancak, yonca bitkisinin in vitro çoğaltımı yüksek derecede çeşide bağımlıdır ve her bir çeşit için farklı prosedür geliştirmek gereklidir. Şimdiye kadar, Kalender yonca çeşidinde bu konuda bir çalışma rapor edilmemiştir. Bu çalışmada, farklı temel besin ortamları, sitokininler ve eksplant tiplerinin Kalender çeşidinin in vitro çoğaltımı üzerine etkileri araştırılmıştır. Başlangıçta, in vitro fideciklerden alınan farklı eksplant tipleri (meristem, sürgün ucu ve nod) sürgün rejenerasyonunu uyarmak amacıyla, farklı konsantrasyonlarda (1.0 ve 2.0 mg/L) kinetin içeren Gamborg besin ortamında (B5) kültüre alınmıştır. Yüksek sürgün rejenerasyonları (%57.78 - %93.33) yanında, yüksek kallus rejenerasyonları da (%82.22 - %93.33) gözlenmiştir. Yüksek kallus oluşumundan dolayı, ikinci bir deneme kurulmuştur. In vitro fideciklerden alınan sürgün ucu eksplantları farklı konsantrasyonlarda (0, 0.125, 0.25, 0.5 ve 1.0 mg/L) 6-Benzilaminopürin (BAP) içeren B5, Murashige ve Skoog (MS) ve Woody Plant Medium (WPM) besin ortamlarına transfer edilmişlerdir. En iyi sürgün rejenerasyon oranı (%100) 0.25 veya 1.0 mg/L BAP ilave edilmiş WPM ve 0.25 mg/L BAP ilave edilmiş MS besin ortamlarından elde edilmesine rağmen, BAP içeren besin ortamlarında kültüre alınan sürgün uçları yüksek kallus rejenerasyonu üretmişlerdir. BAP içeren besin ortamlarının aksine, WPM besin ortamında, sürgün ve kök gelişimi aynı anda gerçekleşmiş ve sağlıklı ve iyi gelişmiş bitkiler elde edilmiştir. In vitro sürgünler en iyi (%71.11) WPM besin ortamında köklenmiştir. Bütün bitkicikler başarılı bir şekilde aklimatize edilmiştir. Böylece, M. sativa’nın Kalender çeşidi için etkili bir in vitro çoğaltım protokolü geliştirilmiştir.

Medicago sativa L. is one of the most important forage crops cultivated all over the world. In vitro propagation of some M. sativa cultivars has been studied earlier; however, it is highly cultivar dependent and necessary to develop different in vitro propagation procedures for each cultivar. So far, in vitro propagation of alfalfa ‘Kalender’ cultivar has not been reported. In this study, the effects of different basal media, cytokinins and explant types on in vitro propagation of alfalfa cultivar ‘Kalender’ were investigated. Initially, different explants types (meristem, shoot tip and node) excised from in vitro seedlings were culture on Gamborg medium (B5) containing different concentrations of kinetin (1.0 and 2.0 mg/L) to induce shoot regeneration. Besides high shoot regeneration rates (57.78% - 93.33%), high callus regeneration rates (82.22% – 93.33%) were also observed. Because of high callus formation, second experiment was conducted. Shoot tip explants excised from in vitro seedlings were transferred on B5, Murashige and Skoog (MS) and Woody Plant Medium (WPM) supplemented with different concentrations of 6-benzylaminopurine (BAP) (0, 0.125, 0.25, 0.5, and 1.0 mg/L). Although, the best shoot regeneration rate (100%) was obtained on WPM supplemented with 0.25 or 1.0 mg/L BAP and MS supplemented with 0.25 mg/L BAP, shoot tips cultured on BAP-containing media produced high callus regeneration. In contrast to BAP-containing media, on WPM medium, the development of shoots and roots occurred simultaneously and healthy and well-developed plantlets were obtained. In vitro shoots rooted best (71.11%) on WPM. All plantlets were successfully acclimatized. Thus, an efficient in vitro propagation protocol for M. sativa L. cultivar ‘Kalender’ was developed.

  • Kaynakça
  • Amini, M., Deljou, A., Nabiabad, H. S. (2016). Improvement of in vitro embryo maturation, plantlet regeneration and transformation efficiency from alfalfa (Medicago sativa L.) somatic embryos using Cuscuta campestris extract. Physiology and Molecular Biology of Plants, 22(3), 321–330. DOI: 10.1007/s12298-016-0374-y Atanassov, A., Brown, D. C. W. (1984). Plant regeneration from suspension culture and mesophyll protoplasts of Medicago sativa L. Plant Cell, Tissue and Organ Culture, 3, 149-162. DOI: 10.1007/BF00033736
  • Bhattarai, K., Rajasekar, S., Dixon, R. A., Monteros, M. J. (2018). Agronomic performance and lignin content of HCT down-regulated Alfalfa (Medicago sativa L.). BioEnergy Research, 11, 505–515. DOI: 10.1007/s12155-018-9911-6
  • Cheyne, V. A., Dale, P. J. (1980). Shoot tip culture in forage legumes. Plant Science Letters, 19, 303-309. DOI: 10.1016/0304-4211(80)90052-8
  • Denchev, P. D., Kuklin, A. I., Atanassov, A. I., Scragg, A. H. (1993). Kinetic studies of embryo development and nutrient utilization in an alfalfa direct somatic embryogenic system. Plant Cell, Tissue and Organ Culture, 33, 67-73. DOI: 10.1007/BF01997600
  • Ding, Y. L., Aldao-Humble, G., Ludlowa, E., Drayton, M., Lin, Y. H., Nagel, J., Dupal, M., Zhaoa, G., Pallaghy, C., Kalla, R., Emmerling, M., Spangenberg, G. (2003). Efficient plant regeneration and Agrobacterium-mediated transformation in Medicago and Trifolium species. Plant Science, 165, 1419-1427. DOI: 10.1016/j.plantsci.2003.08.013
  • Elçi, S. (2005). Baklagil ve Buğdaygil Yem Bitkileri, Tarım ve Köyişleri Bakanlığı, Ankara, Türkiye.
  • Emiroğlu, Ü., Gürel, A. (2005). Bitki ıslahında doku kültürü tekniklerinin kullanımı. Tohum Bilimi ve Teknolojisi, 1, 91-155. E. Ü. Tohum Teknolojisi Uygulama ve Araştırma Merkezi, Bornova, İzmir.
  • Faisal, M., Ahmad, N., Anis, M. (2007). An efficient micropropagation system for Tylophora indica: An endangered, medicinally important plant. Plant Biotechnology Reports, (1), 155–161. DOI: 10.1007/s11816-007-0025-4
  • Fuentes, S. I., Suárez, R., Villegas, T., Acero, L. C., Hernández, G. (1993). Embryogenic response of Mexican alfalfa (Medicago sativa) varieties. Plant Cell, Tissue and Organ Culture, 34, 299–302. DOI: 10.1007/BF00029720
  • Gençkan, M. S. (1983). Yem Bitkileri Tarımı. Ege Üniversitesi Ziraat Fakültesi Yayınları, No: 467, Sayfa: 75-123, E. Ü. Matbaası, İzmir, Türkiye.
  • Gamborg, O. L., Miller, R. A., Ojima, K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 50 (1), 151-158. DOI: 10.1016/0014-4827(68)90403-5
  • Gupta, S., Gupta, S., Bhat, V., Gupta, M. G. (2006). Somatic embryogenesis and Agrobacterium mediated genetic transformation in Indian accessions of lucerne (Medicago sativa L.). Indian Journal of Biotechnology, 5, 269-275.
  • Hadidi, M., Ibarz, A., Pagan, J. (2020). Optimisation and kinetic study of the ultrasonic-assisted extraction of total saponins from alfalfa (Medicago sativa) and its bioaccessibility using the response surface methodology. Food Chemistry, 309, 125786. DOI: 10.1016/j.foodchem.2019.125786 Hoori, F., Ehsanpour, A. A., Mostajeran, A. (2007). Comparison of somatic embryogenesis in Medicago sativa and Medicago truncatula. Pakistan Journal of Biological Science, 10(3), 481-485.
  • Kumar, S., Chandra, A., Gupta, M. G. (2008). Plantlet regeneration via multiple shoot induction in Indian cultivars of lucerne (Medicago sativa L.). Journal of Plant Biochemistry & Biotechnology, 17(2), 181-184.
  • Kumar, S., Tiwari, R., Chandra, A., Sharma, A., Bhatnagar, R. K. (2012). In vitro direct plant regeneration and Agrobacterium-mediated transformation of lucerne (Medicago sativa L.). Grass and Forage Science, 68, 459-468. DOI: 10.1111/gfs.12009
  • Li, J. J., Wu, Y., Wang, T., Liu, J. X. (2009). In vitro direct organogenesis and regeneration of Medicago sativa. Biologia Plantarum, 53, 325-328. DOI: 10.1007/s10535-009-0059-2
  • Li, D., Liu, D., Lv, M., Gao, P., Liu, X. (2020). Isolation of triterpenoid saponins from Medicago sativa L. with neuroprotective activities. Bioorganic & Medicinal Chemistry Letters, 30 (4), 126956. DOI: 10.1016/j.bmcl.2020.126956
  • Lloyd, G., McCown, B. (1980). Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture. International Plant Propagators' Society, 30, 421–427.
  • Mc Kersie, B. D., Senaratna, T., Bowley, S. R., Brown, D. C. W., Krochko, J. E., Bewley, J. D. (1989). Application of artificial seed technology in the production of hybrid alfalfa (Medicago sativa L.), In Vitro Cellular & Developmental Biology – Plant, 25, 1183-1188. DOI: 10.1007/BF02621272
  • Moltrasio, R., Robredo, C. G., Gómez, M. C., Paleo, A. H. D., Díaz, D. G., Rios, R. D., Franzone, P. M. (2004). Alfalfa (Medicago sativa) somatic embryogenesis: genetic control and introduction of favourable alleles into elite Argentinean germplasm. Plant Cell, Tissue and Organ Culture, 77, 119-124. DOI: 10.1023/B:TICU.0000016813.94341.7a
  • Mroginski, L. A., Kartha, K. K. (1984). Tissue culture of legumes for crop improvement. Plant breeding reviews, 2, 215–264.
  • Murashige, T., Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497. DOI: 10.1111/j.1399-3054.1962.tb08052.x
  • Nofouzi, F., Oğuz, M. Ç., Khabbazi, S. D., Ergül, A. (2019). Improvement of the in vitro regeneration and Agrobacterium-mediated genetic transformation of Medicago sativa L. Turkish Journal of Agriculture and Forestry, 43, 96-104. DOI: 10.3906/tar-1804-52
  • Orcen, N. (2013). In vitro organogenesis and regeneration of alfalfa (Medicago sativa L.) domestic cultivar cv. Kayseri. Fresenius Environmental Bulletin, 22(9a), 2770-2774.
  • Pokoo, R., Ren, S., Wang, Q., Motes, C. M., Hernandez, T. D., Ahmadi, S., Monteros, M. J., Zheng, Y., Sunkar, R. (2018). Genotype- and tissue-specific miRNA profiles and their targets in three alfalfa (Medicago sativa L) genotypes. BMC Genomics, 19(10), 115-131. DOI: 10.1186/s12864-018-5280-y
  • Pupilli, F., Damiani, F., Nenz, E., Arcioni, S. (1992). In vitro propagation of Medicago and Lotus species by node culture. In Vitro Cellular & Developmental Biology – Plant, 28, 167-171. DOI: 10.1007/BF02823311
  • Röck-Okuyucu, B., Bayraktar, M., Akgun, I. H., Gurel, A. (2016). Plant growth regulator effects on in vitro propagation and stevioside production in Stevia rebaudiana Bertoni. HortScience, 51, 1573–1580. DOI: 10.21273/HORTSCI11093-16
  • Sarul, P., Vlahova, M., Ivanova, A., Atanassov, A. (1995). Direct shoot formation in spontaneously occurring root pseudonodules of alfalfa (Medicago sativa L.). In Vitro Cellular & Developmental Biology – Plant, 31(1), 21-25.
  • Song, J., Sorensen, E. L., Liang, G. H. (1990). Direct embryogenesis from single mesophyll protoplasts in alfalfa (Medicago sativa L.). Plant Cell Reports, 9(1), 21-25. DOI: 10.1007/BF00232128
  • Soya, H., Avcıoğlu, R., Geren, H. (2004). Yem Bitkileri (İkinci Basım). Hasad Yayıncılık Ltd. Şti., İstanbul, Türkiye.
  • Strickland, S. G., Nichol, J. W., McCall, C. M., Stuart, D. A. (1987). Effect of carbohydrate source on alfalfa somatic embryogenesis. Plant Science, 48, 113-121. DOI: 10.1016/0168-9452(87)90138-5
  • Stuart, D. A., Strickland, S. G. (1984). Somatic embryogenesis from cell cultures of Medicago sativa L. I. the role of amino acid additions to the regeneration medium. Plant Science Letters, 34, 165-174. DOI: 10.1016/0304-4211(84)90139-1
  • Tian, L., Brown, D. C. W., Watson, E. (2002). Continuous long-term somatic embryogenesis in alfalfa. In Vitro Cellular & Developmental Biology – Plant, 38, 279–284. DOI: 10.1079/IVP2001286
  • Tucak, M., Čupić, T., Horvat, D., Popović, S., Krizmanić, G., Ravlić. M. (2020). Variation of phytoestrogen content and major agronomic traits in alfalfa (Medicago sativa L.) populations. Agronomy, 10(1) 87, 1-11. DOI: 10.3390/agronomy10010087
  • Wan, Y., Sorensen, E. L., Liang, G. H. (1988a). Genetic control of in vitro regeneration in alfalfa (Medicago sativa L.). Euphytica, 39, 3–9. DOI: 10.1007/BF00025103
  • Wan, Y., Sorensen, E. L., Liang, G. H. (1988b). The effects of kinetin on callus characters in alfalfa (Medicago sativa L.). Euphytica, 39, 249-254. DOI: 10.1007/BF00037103
  • Wu, Z., Shu, T., Zhang, M., Liu, W. (2020). Foam fractionation for effective recovery of leaf protein from alfalfa (Medicago sativa L.). Separation Science and Technology, 55(7), 1388-1397. DOI: 10.1080/01496395.2019.1586725
  • Zare, N., Valizadeh, M., Tohidfar, M., Mohammadi, S. A., Malboobi, M. A., Habashi, A. A. (2009). Selection of regenerative genotypes from Iranian alfalfa cultivars. Journal of Food, Agriculture & Environment, 7(3&4), 567 - 572.
  • Zhao, Y., Ma, W., Wei, X., Long, Y., Zhao, Y., Su, M., Luo, Q. (2020). Identification of exogenous Nitric Oxide-Responsive miRNAs from Alfalfa (Medicago sativa L.) under drought stress by high-throughput sequencing. Genes, 11 (30), 1-22. DOI: 10.3390/genes11010030
Primary Language tr
Subjects Engineering
Journal Section Articles
Authors

Orcid: 0000-0002-1750-3080
Author: Fatma Gökşin BAHAR
Institution: EGE ÜNİVERSİTESİ
Country: Turkey


Orcid: 0000-0002-7569-6925
Author: Meltem BAYRAKTAR (Primary Author)
Institution: KIRŞEHİR AHİ EVRAN ÜNİVERSİTESİ
Country: Turkey


Orcid: 0000-0002-7002-9752
Author: Aynur GÜREL
Institution: EGE ÜNİVERSİTESİ
Country: Turkey


Thanks Çalışmada kullanılan Kalender yonca çeşidine ait tohum materyalini sağlayan Neobi Tohumculuk A.Ş.’ye teşekkür ederiz.
Dates

Publication Date : August 31, 2020

APA Bahar, F , Bayraktar, M , Gürel, A . (2020). ‘Kalender’ Yonca (Medicago sativa L.) Çeşidinin In vitro Çoğaltımı Üzerine Farklı Besin Ortamları, Sitokininler ve Eksplant Tiplerinin Etkisi . Avrupa Bilim ve Teknoloji Dergisi , (19) , 449-459 . DOI: 10.31590/ejosat.723009