Emine ATALAY, Münüre TANUR ERKOYUNCU, Semiha ERİŞEN, Mustafa YORGANCILAR

Endemik Astragalus trojanus Stev’in Nodal Kültür ile Mikroçoğaltımı

Bu araştırmada, tıbbi özelliklere sahip endemik Astragalus trojanus Stev. bitkisinin nodal kültürler ile klonal çoğaltımı amaçlanmıştır. Tohumlar Murashige ve Skoog (MS) besin ortamında çimlendirilmiş ve elde edilen steril fidelerin nodal segmentleri eksplant olarak kullanılmıştır. İlk olarak MS ve Woody Plant Medium (WPM) besin ortamlarının sürgün çoğaltımına etkileri araştırılmıştır. Eksplantlar farklı konsantrasyonlarda (0.2, 0.5, 1.0, 2.0 mg/L) 6-benzil amino purin (BAP) içeren MS ya da WPM besin ortamlarında kültüre alınmıştır. Kültürden 4 hafta sonra en fazla sürgün sayısı (2.80 adet/eksplant) 0.5 mg/L BAP içeren WPM ortamında elde edilmiştir. Çalışmanın devamında nodal segmentler, sitokinin kaynağı ve konsantrasyonlarının sürgün çoğaltımına etkisini belirlemek amacıyla BAP, thidiazuron (TDZ), kinetin (KIN)’in farklı konsantrasyonlarını (0.2, 0.5, 1.0, 2.0 mg/L) içeren WPM temel besin ortamında kültüre alınmıştır. 4 hafta sonunda elde edilen sürgün sayıları bakımından TDZ ve KIN’in benzer etki gösterdiği, en yüksek sürgün sayısının (2.09 adet/eksplant) BAP’dan elde edildiği tespit edilmiştir. Rejenere sürgünler köklenme için 1 veya 3 mg/L indol-3-butirik asit (IBA), naftalen asetik asit (NAA) veya indol-3-asetik asit (IAA) içeren WPM ortamlarına aktarılmıştır. Kültürden 4 hafta sonra en yüksek köklenme yüzdesi (%80) 3 mg/L IBA içeren WPM ortamından elde edilirken, en fazla kök sayısı (4.33 adet/eskplant) 3 mg/L IAA içeren WPM ortamından elde edilmiştir. Köklenen bitkicikler 1:1 oranında torf ve perlit içeren plastik bardaklara aktarılarak dış ortama başarıyla alıştırılmış ve tam bitki gelişimi 15 hafta sonunda tamamlanmıştır. Bu araştırma ile, Astragalus trojanus Stev. endemik türünde etkili bir klonal çoğaltım metodu tanımlanmıştır.

Anahtar Kelimeler:

Astragalus, Klonal çoğaltım, Köklenme, Nodal segment, WPM

Micropropagation of Endemic Astragalus trojanus Stev. with Nodal Culture

The clonal propagation with nodal cultures of the medicinal endemic plant species Astragalus trojanus Stev. was aimed in this study. Seeds were germinated in Murashige and Skoog (MS) medium, and the nodal segments obtained from seedlings were used as an explant. Firstly, the effects of MS and (Woody Plant Medium) WPM nutrient media on shoot multiplication were investigated. The explants were cultured in MS or WPM nutrient media containing at different concentrations (0.2, 0.5, 1.0, 2.0 mg L-1) 6-benzyl amino purine (BAP). After 4 weeks from culture, the highest number of shoots (2.80 number/explant) was obtained in WPM medium containing 0.5 mg L-1 BAP. Nodal segments were cultured in WPM medium containing different concentrations of BAP, thidiazuron (TDZ), kinetin (KIN) (0.2, 0.5, 1.0, 2.0 mg L-1) to determine the effect of cytokinin source and concentrations on shoot multiplication. At the end of 4 weeks, it was determined that TDZ and KIN had similar effect on number of shoots. The highest number of shoots (2.09 number/explant) was obtained from WPM media containing BAP. Regenerated shoots were transferred to WPM media containing 1 or 3 mg L-1 indole-3-butyric acid (IBA), naphtalene acetic acid (NAA) or indole-3-acetic acid (IAA) for rooting. After 4 weeks from culture, the most root number (4.33 number/explant) were obtained from the WPM medium containing 3 mg L-1 IAA, while the highest rooting percentage was obtained from WPM medium containing 3 mg L-1 IBA with 80%. Rooted seedlings were transferred to plastic cups containing peat and perlite in a ratio of 1:1 and successfully acclimatized. Plant culture was completed at 15 weeks. The effective clonal propagation method was described for the endemic species Astragalus trojanus Stev.

Keywords:

Astragalus, Clonal propagation, Rooting, Nodal segment, WPM,

PDF

___

Ağar G, Şahin F, Sökmen M, Güllüce M, Şengül M, Adıgüzel A, Özkan H, Barış Ö (2008). Erzurum ve çevresindeki Astragalus L. türlerinin tanılanması ve bu türlerin biyolojik aktivitelerinin saptanması. TÜBİTAK TOVAG 105O070 No’lu Araştırma Projesi. Erzurum.
Bedir E, Çalış İ, Ikhlas KA (2000). Macrophyllosaponin E: A novel compound from the roots of Astragalus oleifolius, Chem. Pharm. Bull. 48 (7) 1081–1083.
Bedir E, Tatlı I, Çalış I, Khan IA (2001). Trojanosides I-K: New cycloartane-type glycosides from the aerial parts of Astragalus trojanus, Chemical & Pharmaceutical Bulletin 49 (11), 1482-1486.
Basalma D, Uranbey S, Gurlek D, Ozcan S (2008). TDZ-induced plant regeneration in Astragalus cicer L. African Journal of Biotechnology, 7 (8), 955-959.
Blaskesley D, Constantine D (1992. Uptake and metabolism of 6- benzyadenine in shoot cultures of a range of species. Plant Cell Tissue and Organ Culture, 28: 183–186.
Cano-Castillo M, Serrano-Martínez F, Casas JL (2009). In vitro Propagation of Astragalus nitidiflorus (Leguminosae), An Endemic and Endangered Species from South-East of Spain. ISHS Acta Horticulturae 812: III International Symposium on Acclimatization and Establishment of Micropropagated Plants.
Couceiro MA, Afreen F, Zobayed SMA, Kozai T (2006). Variation in concentrations of major bioactive compounds of St. John’s wort: Effects of harvesting time, temperature and germplasm. Plant Science, 170, 128-134.
Çöcü S, Erişen S, Duran A, Hamzaoğlu H, Gürlek D, Parmaksız İ, Mirici S (2005). Lokal endemik Astragalus duranii türünün In vitro hızlı çoğaltımı. XIV. Ulusal Biyoteknoloji Kongresi, 31 Ağustos-2 Eylül, Eskişehir.
Dinç M, Aytaç Z, Doğu S (2013). A new species of Astragalus (Fabaceae) from Turkey. Turk J Bot, 37: 841-846.
Dobránszki J, Teixeira da Silva JA (2010). Micropropagation of apple: A review. Biotech. Adv. 28 (4): 462–488.
Ekici M (2000). Türkiye’nin Holoeuce Bunge ve Acmothrix Bunge (Astragalus L.) Seksiyonlarının Revizyonu, Doktora Tezi, Gazi Üniversitesi Fen Bilimleri Enstitüsü, Ankara.
Erisen S, Yorgancilar M, Atalay E, Babaoglu M (2010). Prolific shoot regeneration of Astragalus cariensis Boiss. Plant Cell Tiss Organ Cult, 100: 229–233.
Erisen S, Atalay E, Yorgancılar M (2011). The effect of thidiazuron on the in vitro shoot development of endemic Astragalus cariensis in Turkey. Turk J Bot 35, 521-526
Erkoyuncu R (2010). Endemik Astragalus schizopterus bitkisinin in vitro rejenerasyon potansiyelinin belirlenmesi. Yüksek lisans tezi, Selçuk Üniversitesi, Fen Bilimleri Enstitüsü, Tarla Bitkileri Anabilim Dalı, Konya.
George EF (2008). Plant Tissue Culture Procedure – Background. In: Plant Propagation by Tissue Culture 3rd Ed. (eds: George, E.F., Hall, M.A., Klerk, G.J.). Volume 1. p, 1-28. Published by Springer, Nedherlans.
George EF, Debergh PC (2008). Micropropagation: uses and methods. In: George EF, Hall MA, De Klerk GJ, editors. Plant propagation by tissue culture. 3rd ed. Dordrecht, Netherlands: Springer; p. 29–64.
Gübbük H, Pekmezci M (2004). In Vitro Propagation of Some New Banana Types (Musa spp.). Turk. J. Agric. For., 28, 355-361.
Hasançebi S, Turgut Kara N, Çakır Ö, Arı Ş (2011). Micropropagation and root culture of Turkish endemic Astragalus chrysochlorus (Leguminosae). Turk J Bot, 35: 203-210
Heloir MC, Fournioux JC, Oziol L, Bessis R (1997). An improved procedure for the propagation in vitro of grapevine (Vitis vinifera cv Pinot noir) using axillary bud microcuttings. Plant Cell Tissue Org. Cult. 49, 223–225.
Hou SW, Jia JF (2004). High frequency plant regeneration from Astragalus melilotoides hypocıtyl and stem explants via somatic embriyogenesis and organogenesis. Plant Cell, Tissue and Organ Culture 79: 95-100.
Jafari N, Othman RY, Khalid N (2011). Effect of benzylaminopurine (BAP) pulsing on in vitro shoot multiplication of Musa acuminata (banana) cv. Berangan African Journal of Biotechnology Vol. 10(13), pp. 2446-2450.
Kadıoğlu B, Kadıoğlu S, Turan Y (2008). Gevenlerin (Astragalus sp.) Farklı kullanım alanları ve önemi. Alınteri Zirai Bilimler Dergisi. Cilt 14, Sayı 1,17-26.
Kobayashi AK, Bespalhok JC, Pereira LFP, Vieira LGE (2003). Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments. Plant Cell, Tissue and Organ Culture, 74: 99–102.
Lloyd G, McCown B (1980). Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Proc Int Plant Prop Soc 30:421–427.
Lou JP, Jia JP (1998a). Callus induction and plant regeneration from hypocotyl explants of the forage legume Astragalus adsurgens, Plant Cell. Rep., 17, 567 – 570.
Lou JP, Jia JP (1998b). Plant regeneration from callus protoplasts of the forage legume Astragalus adsurgens, Pall. Plant Cell. Rep., 17, 313-317.
Lu MC (2005). Micropropagation of Vitis thunbergii Sieb. et Zucc., a medicinal herb, through high-frequency shoot tip culture. Scientia Horticulturae, 107: 64–69.
Machakova I, Zazimalova E, George EF (2008). Plant Growth Regulators I: Introduction; Auxins, their Analogues and Inhibitors. In: Plant Propagation by Tissue Culture 3rd Ed. (eds: George, E.F., Hall, M.A., Klerk, G.J.). Volume 1. p, 175-204. Published by Springer, Nedherlans.
Magyar-Tábori K, Dobránszki J, Teixeira da Silva JA, Bulley SM, Hudák I (2010). The role of cytokinins in shoot organogenesis in apple. Plant Cell Tiss Organ Cult, 101:251–267.
Mamedova RP, Isaev MI (2004). Triterpenoids from Astragalus Plants, Chemistry of Natural Compounds, Vol. 40, No. 4.
Mirici S (2004). Endemik geven (Astragalus polemoniacus bunge) bitkisinin yaprak sapı ve yaprak eksplantlarından yüksek oranda adventif sürgün rejenerasyonu, S.Ü. Ziraat Fakültesi Dergisi 18(34): (2004) 31-34
Mohebalipour N, Aharizad S, Mohammadi SA, Motallebiazar AR, Arefi HM (2012). Effect of plant growth regulators BAP and IAA on micropropagation of Iranian lemon balm (Melissa officinalis L.) landraces. Journal of Food, Agriculture & Environment Vol.10 (1): 280-286.
Motallebi-Azar A,. Kazemiani S (2011). Effect of Carbon Source and Hydrolyzed Casein on Callus and Shoot Induction in Hypericum perforatum cv Helos. Int. J. Med. Arom. Plants, Vol. 1, No. 3, pp. 313-318.
Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant., 15: 473-497.
Nartop P, Gürel A, Akgün İH, Bedir E (2015). Astragalaside IV and cycloastragenol productionin in vitro produced Astragalus trojanus Stev. Indian Journal of Biotechnology, Vol. 14, pp. 540-546.
Nas MN, Bölek Y, Sevgin N (2013). Shortcut to long-distance developing of a tissue culture medium: micropropagation of mature almond cultivars as a case study. Turk J Bot, 37: 1134-1144.
Oltulu B (2008). Astragalus trojanus (geven) bitkisinin farklı in vitro besin ortamlarında rejenerasyonu. Ege Üniversitesi Fen Bilimleri Enstitüsü, Biyomühendislik A.B.D. Yüksek Lisans Tezi. İzmir.
Turgut-Kara N, Arı Ş (2006). Micropropagation of Astragalus maximus Willd. Biotechnol Biotech Eq 20(1): 20-22.
Turgut-Kara N, Arı Ş (2008). In vitro plant regeneration from embryogenic cell suspension culture of Astragalus chrysochlorus (Leguminoseae). African Journal of Biotechnology Vol. 7 (9), pp. 1250-1255, 2 May, 2008
Uranbey S, Mirici S, Sancak C, Parmaksız İ, Khawar KM, Mirici S, Özcan S (2003). Adventitious shoot regeneration in Cicer milkvetch. Biotechnology & Biotechnological Equipment, 17: 33-37.
Uysal İ (1997). Astragalus trojanus endemik türünün morfolojisi, anatomisi ve ekolojisi üzerinde gözlemler, Erc. Ünv. Fen Bil. Derg. 13:1–2, 54–66.
Üçler AÖ, Yahyaoğlu Z, Güneş İ (2000). Sürgün çoğaltılması ile in vitro’da elde edilen kivi (Actinidia chinensis Planch.) sürgünlerinin farklı yöntemlerle köklendirilmesi üzerine bir çalışma. Artvin Çoruh Üniversitesi Orman Fakültesi Dergisi (AÇÜOFD), Vol 1, No 1, s 10-17.
Shilpashree HP, Ravishankar R (2009). In vitro plant regeneration and accumulation of flavonoids in Hypericum mysorence. International Journal of Integrative biology, Vol. 8, No.1, 43-49.
Yorgancilar M, Erisen S (2011). The effect of thidiazuron (TDZ) on shoot regeneration of Astragalus schizopterus. The Journal of Animal & Plant Sciences, 21(3): 519-524.