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Centaurea zeybekii Wagenitz’nin in vitro çimlenmesi ve mikroçoğaltımı üzerine araştırmalar Skip to content Tez Künye Durumu Centaurea zeybekii Wagenitz’nin in vitro çimlenmesi ve mikroçoğaltımı üzerine araştırmalar / Research on in vitro germination and micropropagation of Centaurea zeybekii Wagenitz Yazar:SERAP KURT Danışman: Y.DOÇ.DR. BENGİ ERDAĞ Yer Bilgisi: Adnan Menderes Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı Konu:Biyoloji = Biology Dizin: Onaylandı Yüksek Lisans Türkçe 2006 80 s. ÖZETBu çalışmada endemik bir bitki olan Centaurea zeybekii Wagenitz’nin in vitroçimlenmesi ve mikroçoğaltımı üzerine araştırmalar yapılmıştır. Türünkapitulumlarından çıkarılan tohumlar (aken meyve) denemelerde başlangıç materyaliolarak kullanılmıştır. Çalışma iki aşamada planlanmıştır. Birinci aşamada, farklı invitro çimlendirme ortamlarının, gibberellik asitin, ışığın ve sıcaklığın çimlenmeüzerine etkisi araştırılmıştır. Bu denemelerde Centaurea zeybekii tohumlarınınoptimum in vitro çimlenme koşullarının belirlenmesi ve çimlenen fideciklerin dışortamlara aktarılarak populasyonun ex situ elde edilmiş bireyler kullanılarakgüçlendirilmesi hedeflenmiştir. kinci aşamada ise, in vitro koşullarda çimlenentohumlardan steril fide gelişimi için uygun ortamların belirlenmesinin yanı sıra, bufidelerin çeşitli kısımlarının eksplant olarak kullanılması ile aksiller ve adventifsürgün oluşumu teşvik edilmeye çalışılmıştır.Kapitulumlardan çıkarılan tohumlara öncelikle canlılık testi yapılmış ve canlılıkyüzde olarak belirlenmiştir. Temmuz ve Ağustos aylarında toplanan tohumlarauygulanan tetrazolium testinin sonuçlarına göre, Temmuz ayında toplanantohumların çok az bir kısmının boyandıkları (%30) ve bu boyanmanın da çok açıkrenkte olduğu gözlenmiştir. Ağustos ayında toplanan tohumların ise % 98’ininboyandıkları ve çok net olarak kırmızı renk aldıkları görülmüştür. TTC testisonuçlarına göre Ağustos ayında toplanan tohumların hemen hepsinin canlı olduklarıve uzun bir süre (20şC sıcaklık, %50-60 nemde) canlılıklarını koruduklarıbelirlenmiştir.lk denemede, GA3 destekli ve desteksiz değişik in vitro ortamlar kullanılmış, C.zeybekii türünün in vitro çimlenmesi için en uygun ortam araştırılmıştır. Tohumlarakan çeşme suyu altında yarım saat yıkandıktan sonra, 10 dakika boyunca % 70’liketil alkolde, ardından 15 dakika % 4.5’luk sodyum hipoklorit solusyonunda tutulupsterilizasyonları sağlandıktan sonra üç defa distile su ile yıkanmıştır. Steril edilentohumlar farklı konsantrasyonlarda (1, 2 ve 3 mg/L) gibberellik asit içeren veiçermeyen çimlendirme ortamlarına (Sıvı MS, White, B5, vitamin destekli distile su)alınarak, 24±2 şC’de 16/8 saat fotoperiyot koşulları altında kültüre edilmişlerdir. Budenemenin sonunda en yüksek çimlenme yüzdesinin vitamin destekli distile suya 1mg/L GA3 ilavesi ile hazırlanmış ortamda olduğu görülürken (% 80), bunu sırasıyla 2mg/L GA3 ilaveli White (%45) ve B5 (%45) ortamları izlemiştir. Denemenin bundansonraki aşamalarında maksimum çimlenmenin elde edildiği 1 mg/L GA3 ilavelidistile su ortamı temel ortam olarak kullanılmıştır.kinci denemede çimlenme üzerine ışığın etkisi araştırılmıştır. 1 mg/L GA3 ilavelidistile su ortamlarına ekilen bir grup tohum 16/8 saat fotoperiyoda tabi tutulurken,diğer bir grup karanlıkta 24 ±2 şC’de inkübasyona bırakılmıştır. Denemenin sonundaC. zeybekii tohumları karanlık koşullarda %80 çimlenme gösterirken, aydınlıkta buoran %78 olarak belirlenmiş ve çimlenme yüzdeleri karşılaştırıldığında ışık vekaranlık uygulamaları arasında önemli bir farklılık bulunmamıştır.Üçüncü denemede tohum çimlenmesi üzerine sıcaklığın etkisi araştırılmıştır.Sterilize edilmiş tohumlar 1 mg/L GA3 ilaveli distile su ortamlarına aktarıldıktansonra 15, 20, 25, 30 ve 35 şC’lik sıcaklıklarda kültüre edilmişlerdir. 24±2 şC’deinkube edilen tohumlar en yüksek çimlenme yüzdesine sahipken (% 79), bu değerinaltında ve üstündeki sıcaklıklarda çimlenme yüzdesinin düştüğü görülmüştür.In vitro koşullarda çimlendirilen (kotiledonları belirmiş) tohumların bir kısmıbahçe toprağı içeren saksılara alınmış ve iklim odası koşullarında yaklaşık 8 ayboyunca yaşatılabilmişlerdir. Çimlenen tohumların diğer bir kısmı ise en uygungelişim gösterecekleri in vitro ortamın belirlenmesi amacı ile B5, MS ve Whiteortamlarına aktarılmışlardır. Yaklaşık 8 hafta sonra (dört haftada bir alt kültüredilmiştir) fideciklerin gelişimleri (sürgün boyu, sürgün sayısı, yaprak sayısı, yaprakgenişliği, kök boyu, kök sayısı) incelenerek sonuçlar değerlendirilmiştir.Denemelerimizde White ortamına aktarılan fidelerin hemen hepsinin bir ay sonraöldükleri gözlenmiştir. B5 ve MS ortamlarındaki fideler yaklaşık 8 hafta sonramorfolojik kriterler açısından incelendiğinde fide gelişimi için en uygun ortamın MSolduğu sonucuna varılmıştır.Bitki gelişimi için en uygun ortam olarak belirlenen MS ortamında gelişenyaklaşık 4 haftalık steril fideler aksiller sürgün çoğaltımı için, primer köklerindenayrılarak farklı sitokinin tip ve konsantrasyonlarını (0.2, 0.5, 1 ve 2 mg/L BA, 0.2,0.5, 1 ve 2 mg/L KIN ve 0.001, 0.005, 001 ve 0.02 mg/L TDZ) içeren MS temelbesi ortamlarına aktarılmışlar ve 24±2 şC’de 16/8 saat fotoperiyot koşullarınınsağlandığı kültür odasına alınmışlardır. 4 haftalık süre sonunda artan sürgün sayılarıbelirlenerek aksillar sürgün çoğaltımı için en uygun sitokinin tip ve konsantrasyonu,sürgün sayısı ve sürgün boyu açısından değerlendirilmiştir. TDZ içeren ortamlaraaktarılan fidelerde aksiller sürgün çoğaltımı teşvik edilmesine rağmen, sürgünlerinkısa boylu kümeler halinde belirdiği ve hemen hepsinin hiperhidrik olduklarıgörülmüştür. En yüksek çoğalma oranı eksplant başına 14.85 (sürgün/eksplant)sürgün sayısı ile 1 mg/L BA ilaveli MS ortamında elde edilmiştir. Maksimum sürgünboyunun en yüksek ortalaması ise bitki büyüme düzenleyicisi içermeyen ve 2 mg/LKIN içeren MS ortamlarında elde edilmiştir. KIN ve BA içeren ortamlarda artansürgün boyuna karşılık, sürgün sayısında bir azalma yani konsantrasyona bağlı olaraksürgün sayısı artışı ile sürgün boyu arasında negatif bir ilişki olduğu gözlenmiştir.Direkt adventif sürgün rejenerasyonu denemelerinde in vitro koşullardaçimlendirilmiş steril fidelerin yaprakları eksplant olarak kullanılmıştır. ki aylık sterilfidelerin yaprakları aksiller tomurcukları olmaksızın abaksial yüzleri ortamla temasedecek şekilde BA (0.2, 0.5, 1 ve 2 mg/L), KIN (0.2, 0.5, 1 ve 2 mg/L) ve TDZ(0.001, 0.005, 0.01ve 0.02 mg/L) içeren ve içermeyen MS ortamlarında kültüreedilmişlerdir. TDZ ilaveli ortamlarda yüksek oranda kalluslaşma görülmüştür.Kalluslaşma oranları 0.001 mg/ L TDZ’de % 90, 0.005 mg/ L ve 0.01 mg/L TDZ’de% 100’ü bulurken, bu oran 0.02 mg/L TDZ’de %47.5′ e düşmüştür. BA ilaveliortamlarda ise kalluslaşma oranları; 0.5 mg/L BA’da %52.5, 1mg/L BA’da %27.5 ve2 mg/L BA’ da ise %35 oranındadır. 0.2 mg/L BA ilaveli ortamlarda kalluslaşmaoranı düşük (% 5) olmasına rağmen, organogenik cevabın nodüler kallus halindebelirmesi ve alt kültürlemeler ile bu durumun değişmemesi nedeniyle bu sitokinintipi de direk adventif sürgün rejenerasyonu için etkisiz bulunmuştur. KIN ilaveliortamlarda ise düşük oranlarda kalluslaşma görülmüştür. Kalluslaşma oranları 0.2mg/L KIN’de % 5, 0.5 mg/L KIN’de % 2.5, 1 mg/L KIN’de % 4 iken, 2 mg/L KINiçeren ortamda kalluslaşma görülmemiştir. Denenen tüm konsantrasyonlardayaklaşık iki hafta sonra yaprakların yüzeylerinde orta damara yakın yerlerdesürgünler belirmeye başlamış (küçük yaprak çıkışı) ve iyi gelişmiş sürgünler 6.haftanın sonunda gözlenmiştir. Direkt sürgün rejenerasyonu gözlenen ortamlardaeksplantların ortamla temas ettiği kısımlarında kalluslaşma görülmüş ancak bukalluslar zamanla karararak etkinlik göstermemişlerdir. Eksplant başına en yükseksürgün sayısı 1 mg/L KIN içeren ortamda (6.2 sürgün/eksplant) elde edilmiştir.Maksimum sürgün boyunun en yüksek ortalaması da (4.17 cm) yine bu ortamdagözlenmiştir. Sonuç olarak C. zeybekii türünün in vitro elde edilmiş steril fideyapraklarının eksplant olarak kullanılması ile direkt adventif sürgün rejenerasyonuiçin 1 mg/L KIN ilaveli MS ortamının eksplant başına en yüksek sürgün sayısı vemaksimum sürgün boyu açısından en iyi ortam olduğunu söylemek mümkündür.Aksiller ve adventif sürgün oluşumu denemelerinden elde edilen sürgünler stokkültürden ayrılarak köklenmeleri için 0.5, 1, 2 ve 5 mg/L IAA, IBA, NAA içeren MSve ½ MS ortamlarına alınmıştır. MS ve ½ MS ortamları arasında köklenme teşvikiaçısından belirgin bir farklılık görülmemiştir. Ancak köklenme son derece düşüktürve sürgünlerin sadece %15’i 0.5 mg/L IBA ilaveli ortamlarda köklendirilebilmiştir.Köklenen bitkicikler dış ortamlara kademeli bir şekilde aktarılarak aklimatizeedilmiştir. SUMMARYIn this study, in vitro germination and micropropagation of Centaurea zeybekiihad been investigated. Achenes from the capitula were used as initial explants in theexperiments. The experiments were divided into two steps. At the first, effects ofdifferent in vitro media with gibberellic acid, illumination, temperature ongermination were investigated. In these experiments, determination of optimum invitro germination conditions for seeds of C. zeybekii and reinforcement of naturalpopulation with a support from seedlings acclimated to natural conditions have beenaimed. At the second step, determination of appropriate medium for sterile seedlingsfrom in vitro germinated seeds and induction of axillary and adventitous shootformation by using some parts of these seedlings have been strived out.Viability tests were applicated to the seeds and results were shown as % of totalnumber. According to tetrasolium test results of seeds which were collected in Julyand August, seeds of July were less (pale red) tinged (some parts were not tinged)and the percentage was 30 %. The seeds of August were almost completely tinged(red) and the percentage was 98 %. According to TTC tests, seeds collected inAugust were almost completely alive (survive long time under 20ºC temperature 50-60 % humidity conditions)In the first experiment, two different in vitro media (with GA3 and not) were usedto describe the most proper medium for the germination. Achenes were sterilized byusing a procedure of 70 % ethanol for 10 min. 4,5 % hypochlorite for 15 min. andthen they were washed three times with distilled water after the first washing undertap water for 30 minutes. Sterilized seeds were cultured under 24±2 ºC 16/8 hphotoperiod conditions by transferring them to germination media (liquid MS, B5,vitamine supported distilled water) containing gibberellic acid in differentconcentrations and without gibberellic acid. At the result of this experiment, thehighest germination rate (80 %) was in distilled water containing 1 mg/L GA3 andfollowed by White (45 %) containing 2 mg/L GA3 and B5 (45 %). In the later stepsof the experiment, distilled water containing 1 mg/L GA3 presented the maximumgermination rate was used as main medium.In the second experiment, effect of illumination on germination was investigated.A group of seeds cultured in media containing 1 mg/L GA3 was exposed to 16/8 hphotoperiod while the another group was kept in dark under 24±2 ºC condition.Finally, germination rate of C. zeybekii seeds was 80 % under dark condition and itwas 78 % under photoperiod condition showing no important difference between thegroups.At the third experiment, effect of temperature on germination was investigated. Itwas found that the highest germination rate was held (79 %) in seeds incubated under24±2 ºC and germination rate was decreased below and above this temperature.Some of in vitro germinated seeds were transferred to pots and they survived 8months under growth chamber conditions. Remaining germinated seeds weretransferred to B5, MS and White media to determine the in vitro medium foroptimum development. Approximately 8 weeks later (subculturing in 4 weeksinterval), development of seedlings (shoot length, shoot number, leaf number, leafwidth, root length, root number) were evaluated. All of the seedlings which weretransferred to White medium died one month later. MS was selected as the mostproper medium for seedling development depending on a comparison with B5medium using morphological criteria after 8 weeks.4 weeks old seedlings, that were developed on MS media which was determinedas the optimum media for seedling development, were separated from primary rootsand transferred to MS basal media with different types of cytokinin andconcentrations (0.2, 0.5, 1 ve 2 mg/L BA, 0.2, 0.5, 1 ve 2 mg/L KIN and 0.001,0.005, 001 and 0.02 mg/L TDZ) for axillary propagation. At the end of 4 week-period, increase in shoot number and shoot length were determined and optimumcytokinin type and concentration for axillary shoot propagation were determined.Inspite of induction of axillary shoot regeneration in seedlings growing in TDZmedia, the seedlindgs were developed in short groups having hyperhydricity (nearlyin all). The highest propagation rate was obtained in the medium containing 1 mg/LBA with 14.85 shoot per explant. The highest average of the maximum shoot lengthwas obtained in MS media without plant growth regulators but containing 2 mglKIN. There were decrease in shoot number which is negatively correlated withincrease in shoot length in MS media containing KIN and BA.Leaves of in vitro germinated steril seedlings were used as explants in directadventitous shoot regeneration experiments. Leaves of sterile seedlings (two monthsold) without axillary buds were cultured in the media with or without BA (0.2, 0.5, 1and 2 mg/L), KIN (0.2, 0.5, 1 and 2 mg/L) and TDZ (0.001, 0.005, 0.01 and 0.02mg/L) as their abaxial sides down and contact with the medium.. The highest callusformation rate was observed in the media containin TDZ. Although callus formationrates were 90 % in 0.001 mg/ L TDZ, 100 % in 0.005 mg/ L and 0.01 mg/L TDZ , itwas decreased as 48 % in 0.02 mg/L TDZ. In BA containing media, callus formationrates were 52,5 % in 0.5 mg/L, 27,5 % in 1mg/L BA and 35 % in 2 mg/L BA.Although the low percentage of callus formation in 0.2 mg/L BA added medium (5%), this cytokinin type is found non-efficient for direct adventitious shootproliferation as organogenic answer appears with only shoot buds and this casecontinues in the subcultures. Callus formation was also observed in mediacontaining KIN. Callus formation rates were %5 in 0.2 mg/L KIN, 2,5 % in 0.5 mg/LKIN, 4 % in 1 mg/L KIN and no callus formation was observed in the mediumcontaining 2 mg/L KIN. In all concentrations, initiations of shoots (small leafinitiations) close to midrib and well developed shoots were observed at the end ofsixth week. In the media having direct shoot regeneration, calli formation in explantson their surfaces having contact with the medium were observed but these callibecame brownish in time and showed no activity. The highest number of shoots perexplant (6,2 shoot per explant) was obtained from the medium containing 1 mgl KIN.The highest average of maximum shoot length was also observed in this medium.Finally, it is possible to stress that medium containing 1 mgl KIN is the best mediumby obtaining maximum shoot length and highest number of shoots per explant fordirect adventitous shoot regeneration with explants from in vitro germinated shootleaves.The shoots from axillary and adventitous shoot formation experiments weretransferred to MS and ½ MS media containing 0.5, 1, 2 and 5 mg/L IAA, IBA, NAAfor rooting. There was no difference between MS and ½ MS media in rooting but itwas very low and only 15 % of total shoots could rooted in medium containing 0.5mg/L IBA. Rooted plantlets were gradually acclimated to the natural conditions.
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