Effect of Different Media, Cytokinins and Explant Types on In vitro Propagation of Alfalfa (Medicago sativa L.) cultivar ‘Kalender’

Abstract

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.

Keywords

• Medicago sativa L.,Kalender,in vitro propagation,meristem,shoot tip,node

‘Kalender’ Yonca (Medicago sativa L.) Çeşidinin In vitro Çoğaltımı Üzerine Farklı Besin Ortamları, Sitokininler ve Eksplant Tiplerinin Etkisi

Öz

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.

Anahtar Kelimeler

• Medicago sativa L.,Kalender,in vitro çoğaltım,meristem,sürgün ucu,nod

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