Kavun solgunluk etmeni Fusarium Oxysporum F.SP.Melonis ırk 1,2'e karşı doku kültürü ve mutasyon teknikleri kullanarak dayanıklı kavun tiplerinin seçilmesi üzerinde araştırmalar : (Devam eden proje)
| dc.contributor.author | Kantoğlu, K. Yaprak | |
| dc.contributor.author | Seçer, Emine | |
| dc.contributor.author | Erzurum, Kudret | |
| dc.contributor.author | Kunter, Burak | |
| dc.contributor.author | Şekerci, Süreyya | |
| dc.contributor.author | Kayabaşı, Nüket | |
| dc.contributor.author | Ozçoban, Mustafa | |
| dc.contributor.author | Tutluer, İhsan | |
| dc.contributor.author | Peşkircioğlu, Hayrettin | |
| dc.contributor.author | Sağel, Zafer | |
| dc.contributor.author | Maden, Salih | |
| dc.contributor.author | Yanmaz, Ruhsar | |
| dc.contributor.department | TAEK-SANAEM | tr_TR |
| dc.date.accessioned | 2017-04-24T11:46:17Z | |
| dc.date.available | 2017-04-24T11:46:17Z | |
| dc.date.issued | 2009 | |
| dc.description | TENMAK D.N.. 4785 | tr_TR |
| dc.description.abstract | Ülkemiz sebze üretiminde önemli bir paya sahip olan kabakgil türleri içinde yer alan kavun (Cucumis melo L.), son yıllarda zararlı ve hastalık etmenleri ve özellikle de Fusarium oxysporum f.sp. melonis'in yol açtığı solgunluk hastalığı nedeniyle ekiliş alanı ve üretim potansiyeli açısından gerileme kaydetmeye başlamıştır. Özellikle Orta Anadolu Bölgesi'nde üretimde kullanılan yerli çeşitlerimizin dayanıksız olmaları nedeniyle bitkilerin büyük bir kısmı hasat aşamasına gelemeden çökmekte bu da üretici açısından önemli bir kayıp oluşturmaktadır. Bu önemli dezavantajı ortadan kaldırmak amacıyla yerli çeşitlerimize yönelik ıslah çalışmalarına önem verilmesi gerekmektedir. Yaşadığımız bölge olan Orta Anadolu'da bu sorun nedeniyle var olan kavun alanları hızla azalmakta üreticiler yeni arayışlar içine girmektedir. Erzurum ve ark. [14]'nın yapmış oldukları araştırmada (TOGTAG 1585 no'lu proje kapsamında) bu bölgede Fusarium oxysporum f.sp. melonis’in 0, 1,2 ve 1,2 nolu ırklarının bulunduğu, bu ırklardan 1,2 nolu ırkın yaygın olduğu belirlenmiş ve bu hastalık sonucu tarlada ürünün %'ünün çöktüğü gözlenmiştir. 1998 yılından itibaren kavunda somatikembriyogenesis yoluyla bitki eldesine yönelik olarak tarafımızdan yürütülen araştırma sonucunda; yörede yoğun olarak yetiştiriciliği yapılan Kuşçular, Yuva ve Kırkağaç kavun çeşitlerinden organogenesis ve somatik embriyogenesis yoluyla bitki eldesi için gerekli besin ortamı bileşimi, büyümeyi düzenleyici madde kombinasyonları ve kültür başlangıcında kullanılacak olan bitki parçası tipleri belirlenerek gelecekte yapılması planlanan ıslah çalışmaları için gerekli olan temel veriler elde edilmiştir. Elde etmiş olduğumuz bu temel verilerden yararlanarak yürütmeyi planladığımız bu araştırmada; bölgemizde yaygın olduğu tesbit edilen Fusarium oxysporum f.sp. melonis’in 1,2 nolu ırkına ait farklı yoğunluktaki kültür filitratları kullanılarak, mutasyona uğratılmış ve herhangi bir uygulama yapılmamış bitkilere ait parçaların kallus oluşturma ve yaşama yetenekleri belirlenmeye çalışılacaktır. Yaşama yeteneğinde olan kallus ve hücre kolonilerinden bitki rejenerasyonu sağlanarak, hastalık etmenine dayanıklı /tolerans bitkisel materyal in vitro koşullarda belirlenerek klasik ıslah çalışmalarına göre çok daha kısa bir süre içinde çok sayıda materyalin seçimi sağlanarak dayanıklı bireylerin elde edilebilmesi gerçekleştirilebilinecektir. Bunun yanı sıra bu metot kullanılarak elde edilecek başarılı sonuçlar sonucunda; damak tadımıza ve tüketim alışkanlıklarımıza uygun olan yerli kavun çeşitlerimizin hastalık etmenine karşı dayanıklı hale getirilmesi sağlanarak ülke ekonomisine ve çiftçiye önemli bir katkıda bulunulacaktır. Araştırmanın metot bölümü izolatlardan filitrat eldesi, başlangıç materyalini oluşturacak in vitro bitkilerin ışınlanması, kültürde kullanılacak olan farklı dozlarda filitrat içeren besin ortamlarının hazırlanması, ışınlanmış ve ışınlanmamış bitki parçalarının bu besin ortamlarında kültüre alınması, alt kültür işlemleri, haftalık gözlem, canlı kalan hücreleri belirlemek üzere hücre boyaması ve yaşayan bitki parçalarından, kallus kolonilerinden bitki rejenerasyonu, elde edilen bitkilerin köklendirilmesi, çoğaltılması, sitolojik gözlemlerinin yapılması ve dış koşullara transfer edilen bitkilerin yeniden hastalık etmeni ile ilgili olarak testlenerek kesin sonuçların belirlenmesi aşamalarını içermektedir. Bugüne kadar elde etmiş olduğumuz verilerin regresyon analizleri sonucunda belirlenen bulgulara göre 21.75 Gy'lik ışın dozunun in vitro bitkiler için etkili olduğu araştırma boyunca bu dozun %10 alt ve üst sınırlarının kullanılması gerektiği, bunun yanı sıra filitrat uygulamasında ise %6.73'luk uygulamanın kullandığımız farklı bitki parçaları üzerinde olumlu etki yaptığı saptanmıştır. Araştırmanın bundan sonraki aşamaları bu uygulama dozları referans alınarak sürdürülecektir. | tr_TR |
| dc.description.abstract | Fusarium wilt is a vascular disease of the Cucurbitaceae family caused by the soil fungus Fusarium oxysporum f. sp. melonis (FOM), which is very detrimental to muskmelons (Cucumis melo L.). Fusarium wilt of melon is prevalent in temperate and tropical regions and causes a worldwide problem. FOM can survive in the soil for extended periods of time as chlamydospores, and is capable of colonizing crop residues and roots of most crops grown in rotation with melon. The only effective control is the use of resistant varieties. Four races of FOM have been identified, namely 0, 1, 2 and 1.2 [1,2]. Race 1.2 was further subdivided into race 1.2y and 1.2w, which cause yellowing and wilt symptoms, respectively. Two resistance genes (Fom-1 and Fom-2) have been identified in melons [2,3,4]. Fom-1 confers resistance to FOM races 0 and 2, and Fom-2 confers resistance to races 0 and 1. These two genes are extensively used in breeding programmes, which can be assisted by marker assisted selection using markers linked to these resistance genes [5,6,7]. No genes have been identified that confer resistance to race 1.2 [8,9]. However, polygenic recessive genes have been found to confer resistance to race 1.2 in Piboule genotypes [1]. Melon production in Turkey is 1,700,000 tons [10] and it is declining the year after year because of Fusarium wilt. Therefore, Fusarium wilt has a high economic importance in the cultivation of muskmelon in Turkey. In some parts of Turkey the prevalent races of this pathogen were determinated. Fantino and Zengin [11] isolated race 1.2 from wilted plants showing intensive root rot in Eastern Thrace. In the Aegean region, Yildiz [12] recovered three races of the pathogen, race 1 being the most common (57%), followed by race 1.2 (35%) and race 0 (6%). Yücel et al. [13] obtained races 0, and 1-2 in the East Mediterranean region. Erzurum et al. [14] isolated the races 0, 1.2 and 2 in Central Anatolia. Based on these results, Fusarium wilt is a wide spread disease over all regions of Turkey. FOM has caused severe losses for farmers as our native cultivars are not resistant to this disease. It is believed our native cultivars will disappear if resistance to FOM is not introduced into the cultivated material. For this reason, many scientists in Turkey are focusing on research to develop new resistant cultivars via conventional and biotechnological breeding methods. In vitro techniques became widely spread during the 20th century, and their potential to make important contributions to plant breeding was quickly understood. In vitro techniques for crop improvement first consisted of micropropagation and plant regeneration, and then in vitro methods were also found to be useful for eliminating disease and selecting for resistant cells or explants. In vitro selection using specific chemical compounds and pathogens is another useful aspect of tissue culture. Selection with phytotoxins and culture filtrates appears to be more effective than the use of the pathogen itself [15]. Researchers now use fungal culture filtrates or toxins to investigate the response of susceptible and resistant genotypes of different plant species or cultivars to disease factors. The use of in vitro methods for the evaluation of resistance is dependent upon a positive correlation between in vitro culture filtrate resistance and whole plant disease resistance. Chawla et al. [16], Gray et al. [17], Connell et al. [18], Malepsezy and El-Kazzaz [19], El-Kazzaz and Malepsezy [20] developed protocols for in vitro determination of resistance. In comparison with field screening other biotechnological methods, selection techniques are more cost and labour effective and do not require large experimental fields. More recently, in vitro techniques were combined to mutation induction for generating genetic variation, including novel disease resistant mutants. Mutation induction can be caused by chemical or physical mutagens that alter the structure of the DNA [21]. Treatment of in vitro tissues with physical or chemical mutagens may increase the frequency of genetic variation considerably. The physical mutagens most commonly used are X-rays, gamma rays and UV light, whereas Ethyl Methane- Sulphonate (EMS) is the chemical mutagen most used in crop improvement. Irradiation treatments may be a suitable choice of mutagen for a number of reasons because the application is fast and, in contrast to chemical mutagens, there is no risk that residues remain in the medium. The in vitro mutation frequencies are much higher than somaclonal variation. More over somaclonal variation is another important point for in vitro plant breeding. You can catch desirable cells or plantlets, which are resistant for disease by this method. Finally, in vitro techniques are useful as well in classical mutation breeding programs by vegetative propagation before or after mutagenic treatment, by in vitro selection or by clonal propagation of selected mutants. According to Malepsezy and El- Kazzaz [19], in vitro selection using FOM filtrates can be effectively used for the selection of cucumber and melon [22]. One of the purposes is to use this method for the selection of various mutants, which are resistant to disease. In this research we are going to determine the resistant cells, which will come from irradiated and non-irradiated explants by using races of F. oxysporum f. sp. melonis culture filtrates in vitro conditions. If we will get successful results by this method we will be able to develop new melon cultivars, which will be resistant to F. oxysporum f. sp. melonis. The present work was performed with in vitro plantlets obtained from seeds of melon cv. Yuva which is an important commercial cultivar in Turkey. Two types of explants (cotyledon and hypocotyls with leaf and cotyledon explants) were used for callus and suspension culture initiation [23]. In this research we followed the procedure developed by Megnegneau and Branchard [22]. Petri dishes containing potato dextrose agar (PDA) medium were inoculated with FOM isolates which were named (A-1)4 and (A-6)4, which obtained from Ankara University Plant Protection Department in Turkey [14]. Petri dishes were incubated at 26°C in the dark. Fifteen days later, 2 x 106 conidia were transferred to 200 ml of liquid Richard medium. Cultures were kept at 26°C in the dark. After twenty days, fungal cultures were filtered twice through filter paper to remove mycelia. The pH of the filtrate was adjusted to pH 5.7 with 1 N HCI or 0.1 N NaOH. Subsequently, cultures were sterilized through a 0.22 ^m filter unit attached to a syringe. For filtrate preparation it is very important that to avoid thermal degradation of toxic compounds in the fungal culture filtrate. Therefore, filtrates should be aliquotted and frozen for longterm storage. The working filtrate aliquot can be kept in the refrigerator. Filtrates were added to autoclaved modified MS basal medium. According to Taner [25] and Taner et al. [23] non irradiated and irradiated cotyledon and hypocotyls with leaf and cotyledon explants were transferred into petri dishes containing half strength of MS medium with basal salts [24] supplemented with 0.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l kinetin and 250mg/l casein enzymatic hydrolysate (Sigma) (for cotyledon explants) and MS basal medium, which was supplemented with 0.5 mg/l Indole-3-acetic acid (IAA), 0.5 mg/l 6-Benzylaminopurine (BAP) (hypocotyls with leaf and cotyledon explants), both mediums contain different ratio of filtrate and 15% sucrose. For both media, the pH was adjusted to 5.6 the steps for filtrate preparation are shown in Figure 2.1.1. The different proportions used in this study were 4, 6, 8, 10, 12, 14, 16, 18 and 20 % (v/v). Control plates contained only the modified MS basal medium. Figure 2.3.2 shows the steps for the initiation of the callus cultures. Seeds of melon genotypes were surface sterilized, rinsed and germinated in vitro as previously described by Çürük [26] and Taner [25]. According to the authors seeds were surface sterilized for 20 minutes in 20% sodium hypochloride solution that was involved tween 20 (Iml/I00ml) and washed tree times in sterile distilled water. Seed cover is removed after sterilization. They are planted in magenta b-cap that is contained 0.50ml solid MS medium containing 0.7% Difco agar and 15% sucrose. The cultures maintained at 25.C, under fluorescent !!luminescence with a light intensity of 10 000 lux, with 16 h photoperiod. For resistance screening, two kinds of non-irradiated and irradiated explants (i.e., cotyledons and hypocotyls containing leaves and cotyledons) were used according to Taner et al. [23]. Cotyledon explants were transferred into Petri dishes containing half strength of MS medium with basal salts [24] supplemented with 0.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l kinetin and 15% sucrose. Hypocotyls with leaf and cotyledon explants were transferred MS basal medium, which was supplemented with 0.5 mg/l Indole-3-acetic acid (IAA), 0.5 mg/l 6-Benzylaminopurine (BAP) and 15% sucrose. For both media, the pH was adjusted to 5.6 with I N HCI or 0.1 N NaOH. It is very important that the experiments contain more than 50 explants for each combination. Seven days old in vitro plantlets which are containing real leaves and cotyledons irradiated by 6.Co gamma source after seed germination. They are irradiated with 6.Co gamma rays at nine different doses. Irradiation doses and their effects on the plants are known to be genotype-dependent, so every experiment should start with the determination of the optimal irradiation dose for their genomic material. This is typically calculated as LD50, which corresponds to the 50% survival dose. Doses weaker or stronger than LDj. may also be used, but too little mutations may be observed or lethality may be too high, respectively. Even for the same genetic material, the optimal dose for mutation induction in vitro is lower than for seed irradiation. In our experiments 25 Gy gamma irradiation dose found an effective dose for in vitro plantlets of Yuva cultivar. After irradiation, it is important to transfer the explants in fresh regeneration medium contain filtrate to avoid any toxicity of the medium components due to the irradiation. The fungal culture filtrate was added to the media at different concentrations as indicated above. Cotyledon cultures were incubated at 26°C in the dark for three weeks. At the end of this three weeks period observations made on explants according to their regeneration capacity. White, yellow callus formation from cotyledon explants determined survival capacity about explants to filtrate. Hypocotyl with leaf and cotyledon explants were incubated under a light period of 16 h at 15 000 lux at 26°C Taner et al. [23]. Regenerated plantlets that showed resistance to the culture filtrate were isolated and sub-cultured every two weeks onto the same medium (these mediums have to contain filtrate for observation of explants survival). Shoot and root regeneration of explants, their growth performance and shoot number are important observation points of our research. The callus cultures obtained from hypocotyl explants with leaves and cotyledons were maintained for three weeks according to Taner and Yanmaz [27]. We transferred calluses to hormone free MS medium that includes filtrate for somatic embryo regeneration. MS medium supplemented with 1.0 mg/l Indole-3-acetic acid (IAA) is proper medium for plantlet formation from shoot cultures and somatic embryos [25]. After that period, the rate of mortality of the explants was estimated. Plantlets are then transferred to greenhouses and advanced to the M2 generation for further selection and evaluation. According to our observations filtrate and gamma ray treatment have an important effect on tolerant plantlet and callus formation. In this research, we show a method [25,27,23] for mass-selection of melon mutants resistant to Fusarium wilt. In vitro selection of resistant cells, which are come from irradiated and non-irradiated explants, is done using culture filtrates of different FOM races. According to our results we determined effective irradiation doses and filtrate treatment dose by “Linear Regression Analysis". According to our results 21.75 Gy is effective dose for in vitro Yuva cv. Explants to induce mutation and for filtrate treatment 6.73% is the proper dose to select survive calluses and plantlets. Hence we will carry out our near future experiments with these determined doses and so that this research can lead to the development of new melon cultivars that will be resistant to Fusarium wilt. | |
| dc.identifier.citation | Kantoğlu, Y. ...[ve arkadaşları]. (2009). Kavun solgunluk etmeni Fusarium Oxysporum F.SP.Melonis ırk 1,2'e karşı doku kültürü ve mutasyon teknikleri kullanarak dayanıklı kavun tiplerinin seçilmesi üzerinde araştırmalar : (Devam eden proje). Ankara : Türkiye Atom Enerjisi Kurumu. | tr_TR |
| dc.identifier.uri | http://kurumsalarsiv.tenmak.gov.tr/handle/20.500.12878/343 | |
| dc.language.iso | tur | tr_TR |
| dc.publisher | Türkiye Atom Enerjisi Kurumu | tr_TR |
| dc.relation.ispartofseries | Türkiye Atom Enerjisi Kurumu, Teknik rapor;TAEK TR 2009-5 | |
| dc.rights | info:eu-repo/semantics/openAccess | tr_TR |
| dc.subject | Mutation (Biology) -- Plants -- Turkey | tr_TR |
| dc.subject | Mutasyon (Biyoloji) -- Bitkiler -- Türkiye | tr_TR |
| dc.subject | Melon | tr_TR |
| dc.subject | Kavun | tr_TR |
| dc.subject | Filitrat of culture | tr_TR |
| dc.subject | Kültür filitratı | tr_TR |
| dc.subject | Callus culture | tr_TR |
| dc.subject | Kallus kültürü | tr_TR |
| dc.subject | Suspension culture | tr_TR |
| dc.subject | Süspansiyon kültürü | tr_TR |
| dc.subject | Fusarium oxysporum f. sp. melonis race 1,2 | tr_TR |
| dc.subject | Fusarium oxysporum f. sp. melonis ırk 1,2 | tr_TR |
| dc.title | Kavun solgunluk etmeni Fusarium Oxysporum F.SP.Melonis ırk 1,2'e karşı doku kültürü ve mutasyon teknikleri kullanarak dayanıklı kavun tiplerinin seçilmesi üzerinde araştırmalar : (Devam eden proje) | tr_TR |
| dc.type | report | tr_TR |