Catharanthus Roseus'un kallus kültürlerinden dejenerant eldesi
Küçük Resim Yok
Tarih
1993
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
6, ÖZETBu araştırmada Catharanthus roseus var, roseus bitkisinden kallus kültürleri aracılığıyla rejenerasyon eldesi çalışılmıştır. Bu amaçla bitkinin tohumları herhangi bir bitki büyüme maddesinin yeralmadığı Murashige-Skoog (MS) ortamında, 28±2°C sıcaklığa sahip 16:8 uzun gün periyodunun uygulandığı büyüme odacıklarında; 20 mi ortamın yeraldığı kavanozlarda ve 15 mg/L ortamın yeraldığı deney tüplerinde çimlendirilmişlerdir. Çimlenme yüzdesi, olgun tohumlarda 94,4, genç tohumlarda % 63,96 olarak hesaplanmıştır. Kallus eldesi için, deney kaplarında gelişen genç bitkiciklerden alınan yaprak ve gövde parçaları kullanılmıştır. Bu parçaların yerleştirildiği 2 mg/L NAA + 3 mg/L BA ve 2 mg/L NAA + 5 mg/L BA içeren MS ortamları ile yine 2 mg/L NAA + 5 mg/L BA içeren Philips- Collins (PC) ortamında kallus oluşumu elde edilmiştir. Yapraktan elde edilen kallusların gövdeden elde edilen kalluslara göre daha yumuşak yapıda olduğu gözlenmiştir. Gövde orijinli kalluslar daha sert ve sıkı yapı sergilemişlerdir. MS ortamındaki yaprak orijinli kalluslar kısa sürede fazla biyomasa ulaşmasıyla da dikkat çekmiştir. 1 g yaprak orijinli kallus biyoması 2 mg/L NAA + 3 mg/L BA içeren MS ortamında 15'er gün arayla yapılan 3 altkültürün sonunda 19,75 grama ulaşırken, gövde orijinli kallus biyoması 12,14 grama ulaşmıştır. Bu olay, 2 mg/L NAA + 5 mg/L BA içeren MS'te 15,14 grama karşılık 12,28 gramla ve 2 mg/L NAA + 5 mg/L BA içeren PC ortamında 6,93 gram ağırlık artışına karşılık, 3,20 gram ağırlık artışıyla ortaya çıkmıştır. Rejenerasyon eldesi için bu kallusların yanısıra, daha önce elde edilmiş olan 5 yıllık yaşlı kalluslar da kullanılmıştır. Bu kalluslar sırasıyla 2; 2,5; 5 mg/L kinetinin, 0; 0,01; 0,02 mg/L NAA ile birlikte 91bulunduğu PC ortamında ve ayrıca 0,1; 0,2; 0,4 mg/L BA'nın 0,1; 1; 2 mg/L NAA ile birlikte bulunduğu PC ortamında kültüre edilmişlerdir. 5 ay boyunca yapılan 6 altkültürleri sonunda rejenerasyon kaydedilmemiştir. Sadece 2 mg/L NAA + 0,4 mg/L BA ekli PC ortamında bir sürgün başlangıcı gözlenmiş, sonra bu sürgün gelişmesine devam etmemiştir. Denemelerimizde elde edilen 3 aylık genç konuşlardan ise öncelikle kallus eldesi için kondukları ortamda sürgün oluşumu gözlenmiştir. Ortalama sürgün oluşumu gözlenmiştir. Ortalama 1 gram kallus kütlesi üzerinde yapılan sayımlarda 2 mg/L NAA + 3 mg/L BA içeren MS ve PC ortamlarında 3-6 adet sürgün, 2 mg/L NAA + 5 mg/L içeren MS ortamında zaman zaman rastlanan çoklu sürgün oluşumunun yanısıra genellikle 5-8 adet sürgün oluşumu kaydedilmiştir. Bu üç kallus eldesi ortamında kalluslar bu sürgün verme potansiyellerini ilk 8 altkültür boyunca korumuşlardır. 8.altkültürden sonraki altkültürlerinde ise bu kalluslardan artık sürgün gelmediği gözlenmiştir. Bunun üzerine bu kalluslar 10-12. altkültürlerindeyken, sürgün oluşumunu kışkırtmak üzere 4 mg/L NAA + 2 mg/L BA içeren MS ortamına aktarıldıklarında, 40 gün sonunda sayılamayacak kadar çok sayıda sürgün oluşturdukları gözlenmiştir. Öte yandan 2 mg/L NAA + 3 mg/L BA içeren MS ortamlarında korunmaya devam edilen kalluslardan bir kısmı yine 12.altkültürlerinden sonra 1; 2; 4 mg/L Spermine, spermidine ve putrescine poliaminlerini ayrı ayrı içeren MS ortamlarında inkübe edilmişlerdir. Bu ortamlarda 3 hafta tutulan kalluslar 3 haftanın sonunda MS normal ortamına aktarılmışlardır. Kültürler bu ortamda 19.günlerindeyken 1 mg/L spermin uygulanmış kallus kütlesinde 1 sürgün oluşumu, 21.günlerindeyken de 4 mg/L spermidin uygulanmış kallus kütlesinde bir sürgün oluşumu kaydedilmiştir. 92These calli exposed 9 different combinations of NAA and Kin with several concentrations; and 9 different combinations of NAA and BA with several concentrations in PC medium. At the end of 5 months with 6 subcultures, any regeneration could observed. Only in 2 mg/L NAA + 0.4 mg/L BA added PC a shoot organogenesis beginning was observed, however this shoot didn't continoue to growth. In the young calli, those we obtained in our study, regeneration immediately appeared when calli were in callus induction medium. 1 g callus regenerated 3-6 shoots in 2 mg/L NAA + 3 mg/L BA added MS and PC media, while 1 g callus regenerated 5-8 shoots in 2 mg/L NAA + 5 mg/L BA added MS. Callus induction medium placed calli maintained their regeneration ability till 8. subculture of them. Later on it was observed that nomore shoot came through these calli. Thus, when they transferred to medium MS containing 4 mg/L NAA + 2 mg/L BA during their 10.-12. subculture noumerous shoots were regenerated at the end of 40 days. On the other hand, some callus, which were maintaining in their original induction medium, has been treated with 1; 2; 4 mg/L spermine, spermidine, putrescine poliamines. After 3 weeks in the medium MS with poliamines these calli transferred to MS basal medium that contains neither PGRs nor PAs. By 19. day 1 shoot appeared on 1 mg/L spermine treated callus, and by 21. day 1 other shoot appeared on 4 mg/L spermidine treated callus. All plantlets, those appeared on the each kind of media which mentioned above, transferred to the medium MS supplemented with 0.5; 1 or 1.5 mg/L NAA. Between this three media best growth of shoots obtained in the 1 mg/L added medium MS. Apexes of these shoots excised and cultured in the same medium. The remained shoots showed rhisogenesis 1 month later. 95
in this study, obtaining of regeneration through calli lines of Catharanthus roseus plant species has been studied, The seeds ög this plant have been germinated in Murashige-Skoog hormon free medium. The cultures placed in a grovvth chamber which has 28 + - 2 C temperature and 16:8 h photoperiod. Tha jars with 20 mi of medium and the test tubes with 15 mi of medium have been used. The percentage of germination has been calculated as 63.96 % for immature seeds, and for mature seeds it was 94.40 %. Stem and leaf explants of these germinated plantlets have been used for callus induction. Callogenesis was induced in two different medium those contain several level of plant growth regulators. Öne of this medium is MS supplemented with either 2 mg/L NAA + 3 mg/L BA ör 2 mg/L NAA + 5 mg/L BA; the other medium is PC (Philips-Collins) supplemented with 2 mg/L NAA + 5 mg/L BA. in the experiments, it has been observed that leaf origined calli were yelloWish-green and softer than those stem origined calli. Stem origined calli had compact and hard structure. it was notable that leaf origined calli reached more biomass, when they cultured at the same time with stem explants. While 1 g leaf origined callus reached 19.75 g biomass in the 2 mg/L NAA + 3 mg/L BA containing MS medium, stem origined callus 12.14 g biomass at the end of third subculture. This had the same meaning in the 2 mg/L. NAA + 5 mg/L BA containing MS medium,-and 2 mg/L NAA + 5 mg/L BA containing PC with different weight of biomass. For obtaining of regeneration, besides using of these callus lines, also some present 5 years old callus lines of C.roseus have been tested. 94These calli exposed 9 different combinations of NAA and Kin with several concentrations; and 9 different combinations of NAA and BA with several concentrations in PC medium. At the end of 5 months with 6 subcultures, any regeneration could observed. Only in 2 mg/L NAA + 0.4 mg/L BA added PC a shoot organogenesis beginning was observed.hovvever this shoot didn't continoue to grovvth. in the young calli, those we obtained in our study, regeneration immediately appeared when calli were in callus induction medium. 1 g callus regenerated 3-6 shoots in 2 mg/L NAA + 3 mg/L BA added MS and PC media, while 1 g callus regenerated 5-8 shoots in 2 mg/L NAA + 5 mg/L BA added MS. Callus induction medium placed calli maintained their regeneration ability till 8. subculture of them. Later on it was observed that nomore shoot came through these calli. Thus, when they transferred to medium MS containing 4 mg/L NAA + 2 mg/L BA during their 10.-12. subculture noumerous shoots were regenerated at the end of 40 days. On the other hand, some callus, vvhich were maintaining in their original induction medium, has been treated with 1; 2; 4 mg/L spermine, spermidine, putrescine poliamines. After 3 vveeks in the medium MS with poliamines these calli transferred to MS basal medium that contains neither PGRs nor PAs. By 19. day 1 shoot appeared on 1 mg/L spermine treated callus, and by 21. day 1 other shoot appeared on 4 mg/L spermidine treated callus. AH plantlets, those appeared on the each kind of media vvhich mentioned above, transferred to the medium MS supplemented with 0.5; 1 ör 1.5 mg/L NAA. Between this three media best grovvth of shoots obtained in the 1 mg/L added medium MS. Apexes of these shoots excised and cultured in the same medium. The remained shoots showed rhisogenesis 1 month later. 95The shoots with roots transferred to the pots contain 1/4 send + 3/4 soil with 92 % survival rate and directed to generative phase. Survived plants were mintained in Ege University, Botany Garden. 96
in this study, obtaining of regeneration through calli lines of Catharanthus roseus plant species has been studied, The seeds ög this plant have been germinated in Murashige-Skoog hormon free medium. The cultures placed in a grovvth chamber which has 28 + - 2 C temperature and 16:8 h photoperiod. Tha jars with 20 mi of medium and the test tubes with 15 mi of medium have been used. The percentage of germination has been calculated as 63.96 % for immature seeds, and for mature seeds it was 94.40 %. Stem and leaf explants of these germinated plantlets have been used for callus induction. Callogenesis was induced in two different medium those contain several level of plant growth regulators. Öne of this medium is MS supplemented with either 2 mg/L NAA + 3 mg/L BA ör 2 mg/L NAA + 5 mg/L BA; the other medium is PC (Philips-Collins) supplemented with 2 mg/L NAA + 5 mg/L BA. in the experiments, it has been observed that leaf origined calli were yelloWish-green and softer than those stem origined calli. Stem origined calli had compact and hard structure. it was notable that leaf origined calli reached more biomass, when they cultured at the same time with stem explants. While 1 g leaf origined callus reached 19.75 g biomass in the 2 mg/L NAA + 3 mg/L BA containing MS medium, stem origined callus 12.14 g biomass at the end of third subculture. This had the same meaning in the 2 mg/L. NAA + 5 mg/L BA containing MS medium,-and 2 mg/L NAA + 5 mg/L BA containing PC with different weight of biomass. For obtaining of regeneration, besides using of these callus lines, also some present 5 years old callus lines of C.roseus have been tested. 94These calli exposed 9 different combinations of NAA and Kin with several concentrations; and 9 different combinations of NAA and BA with several concentrations in PC medium. At the end of 5 months with 6 subcultures, any regeneration could observed. Only in 2 mg/L NAA + 0.4 mg/L BA added PC a shoot organogenesis beginning was observed.hovvever this shoot didn't continoue to grovvth. in the young calli, those we obtained in our study, regeneration immediately appeared when calli were in callus induction medium. 1 g callus regenerated 3-6 shoots in 2 mg/L NAA + 3 mg/L BA added MS and PC media, while 1 g callus regenerated 5-8 shoots in 2 mg/L NAA + 5 mg/L BA added MS. Callus induction medium placed calli maintained their regeneration ability till 8. subculture of them. Later on it was observed that nomore shoot came through these calli. Thus, when they transferred to medium MS containing 4 mg/L NAA + 2 mg/L BA during their 10.-12. subculture noumerous shoots were regenerated at the end of 40 days. On the other hand, some callus, vvhich were maintaining in their original induction medium, has been treated with 1; 2; 4 mg/L spermine, spermidine, putrescine poliamines. After 3 vveeks in the medium MS with poliamines these calli transferred to MS basal medium that contains neither PGRs nor PAs. By 19. day 1 shoot appeared on 1 mg/L spermine treated callus, and by 21. day 1 other shoot appeared on 4 mg/L spermidine treated callus. AH plantlets, those appeared on the each kind of media vvhich mentioned above, transferred to the medium MS supplemented with 0.5; 1 ör 1.5 mg/L NAA. Between this three media best grovvth of shoots obtained in the 1 mg/L added medium MS. Apexes of these shoots excised and cultured in the same medium. The remained shoots showed rhisogenesis 1 month later. 95The shoots with roots transferred to the pots contain 1/4 send + 3/4 soil with 92 % survival rate and directed to generative phase. Survived plants were mintained in Ege University, Botany Garden. 96
Açıklama
Bu tezin, veri tabanı üzerinden yayınlanma izni bulunmamaktadır. Yayınlanma izni olmayan tezlerin basılı kopyalarına Üniversite kütüphaneniz aracılığıyla (TÜBESS üzerinden) erişebilirsiniz.
Anahtar Kelimeler
Biyoloji, Biology, Botanik, Botany, Bitki dokuları, Plant tissues, Bitki ıslahı, Plant breeding, Catharanthus roseus, Catharanthus roseus, Doku kültürü, Tissue culture, Kemik kallusu, Bony callus
