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Today,gradually running out of fossil fuels, the destruction of ecosystems, effortsto avoid being dependant on foreign countries for energy and to increase energydiversity have increased the importance of fuels such as bioethanol. Biomassproduction of bioethanol from sugar beet molasses will lead to open a newmarket for beet producers, to widespread the crop rotation, to create an energyfarming culture and lead to an increase in sugar beet growing areas. Bioethanolalso contributes favorable support to theecology by providing diversity in agricultural production and it is importantfor creating sustainable agricultural structure and supporting ruraldevelopment. Bioethanol in general is obtained by fermentation of plantscontaining sugar and starch. Sugar beet molasses is used for the production ofbioethanol from sugar beet. Production of bioethanol from molasses is carriedout at three stages such as yeast reproduction, fermentation and distillation.The alcohol obtained in %96 purity during ethanol production process is notused as biofuel. In order to be used as biofuel it should be in %99.5 purity.Therefore, in alcohol plants the purification and distillation units are neededin addition to fermentation unit. Attention should be paid to alternativeenergy sources such as bioethanol since the oil reserves are decreasing andenvironment problems are increasing., Günümüzde fosil yakıtların giderek tükeniyor olması, ekosistemin tahripedilmesi, ülkelerin enerjide dışa bağımlı olmaktan kurtulma ve enerjiçeşitliliğini arttırma çabaları biyoetanol gibi yakıtların öneminiarttırmıştır. Biyokütle olarak şeker pancarı melasından biyoetanol üretimipancar üreticileri için yeni bir piyasanın açılmasına, ekim nöbetininyaygınlaşmasına, enerji tarımı kültürünün oluşması ve şeker pancarı ekimalanlarının artmasına sebep olacaktır. Biyoetanol aynı zamanda tarımsalüretimde çeşitliliği sağlayarak ekolojiye olumlu katkıda bulunması,sürdürülebilir tarımsal yapı oluşturması, kırsal kalkınmayı desteklemesibakımından da önemlidir. Biyoetanol, genel olarak şeker ve nişasta içerenbitkilerden fermantasyon yoluyla elde edilir. Şeker pancarında biyoetanolüretiminde melas kullanılmaktadır. Melastan biyoetanol üretimi; mayanınçoğaltılması, fermantasyon, distilasyon olmak üzere üç aşamadagerçekleştirilmektedir. Etanol üretiminde elde edilen alkol %96 saflıkta olupyakıt alkolü olarak kullanılamaz. Etil alkolün yakıt olarak kullanabilmesi içinen az %99.5 saflıkta olması gerekir. Bu nedenle alkol fabrikalarındafermantasyon ünitesinden sonra saflaştırma ve susuzlaştırma ünitelerine ihtiyaçvardır. Günümüzde petrol rezervlerinin azalması ve çevre sorunları yüzündenbiyoetanol gibi alternatif enerji kaynaklarına önem verilmelidir.

The establishment of cell suspension culture from the callus of sugar beet (Beta vulgaris L.) breeding lines and plant regeneration from suspension-derived callus are described in this study. Using different concentrations and combinations of BAP and 2,4-D, the growth patterns of cell suspension cultures were examined during a range of culture periods (0, 3, 5, 7, 9, 11, 13 and 15 days). In all lines, the growth rates of cells were initially slow but as the culture proceeded, they increased significantly and accumulated great amounts of biomass over a period of 15 days. Medium containing high BAP (0.25 mg/l) and 2,4-D (0.25 mg/l) induced higher rates of cell division than the medium containing low BAP (0.1 mg/l) and 2,4-D (0.1 mg/l) or the control. A genotypic variation was evident when the mean growth rates of different lines were compared, with lines M114, ELK345, ÇBM315 and M1017 producing a mean rate of 34.8%, 21.5%, 19.2% and 13.4%, respectively. In all lines, approximately 50% of the suspension-derived callus formed shoots when cultured on medium containing BAP and IAA, or TDZ alone. Rooting was readily achieved when shoots were incubated on medium containing 3.0 mg/l IBA.

In this study, attempts were made to fuse the suspension-derived protoplasts of a sugar beet breeding line (ÇBM315) with the mesophyll-derived protoplasts of three breeding lines (M114, ELK345 and M1017), which were obtained from our previous experiments. Combinations of three different PEG concentrations (20%, 25% or 30%) and three different treatment durations (15, 20 or 25 min) were tested. The fusion efficiency was determined by measuring the viability, heterokaryon frequency and plating efficiency (PE) of the fused protoplasts after four weeks of culture. In general, 25% PEG combined with 20 min treatment duration produced the highest fusion frequency, but the highest rate of viability was obtained at 20% PEG with 25 min treatment duration. The capacity of heterokaryons to form cell colonies was highest when lower PEG (20 or 25%) was used for shorter treatment durations (15 or 20 min). No macrocallus or regeneration from the fused protoplasts was achieved.

In this study the possibilities of in vitro vegatitive propagation of almond ( Amygdalus communis L.) cv. Texas and cv. Nonpareil by shoot-tip culture were investigated. Different levels of IBA (0.0, 0.1 and 0.5 mg/l) and BAP (0.0, 0.5, 1.0, 2.0 and 3.0 mg/l) were tested by observing shoot development and growth during three successive stages, namely initiation, transplantation and multiplication. During the initiation stage, hormone-free medium or medium with low IBA (0.1 mg/l) only seemed favourable for shoot growth. During both the transplantation and multiplication stages, the combination of 0.1 mg/l IBA and 1.0 mg/l BAP was found to be the most effective in terms of new shoot production and shoot growth rate. In general, BAP was shown to be essential for shoot development during the last two stages but high levels (2.0 or 3.0 mg/l BAP) caused vitrification and callus formation which subsequently reduced the viability of the shoots.

For callus production, hypocotyl, cotyledon, petiole and leaf explants from seedlings of different sugar beet ( Beta vulgaris L.) breeding lines were cultured on MS medium containing BAP or KIN in combination with NAA or 2,4-D at 0.0, 0.5 or 1.0 mg/l. The use of both auxins in combination with 0.5 mg/l BAP or 0.5 mg/l KIN resulted in greater amounts of callus from all types of explants. However, hypocotyl and cotyledon explants produced significantly more callus than petiole and leaf explants when the means of all lines were taken into account. Two types of callus were usually obtained, white and friable callus with large cells (Type I) and green and compact callus with smaller cells (Type II). For shoot induction, Type I callus was transferred to MS medium containing 2.5 mg/l TDZ or 1.0 mg/l BAP in combination with 0.3 mg/l IAA, together with or without a cold pre-treatment at 4¡C for two weeks. More shoots developed from the pre-treated callus and elongated shoots could be readily rooted on MS medium containing 3.0 mg/l IBA.

In this study the possibilities of in vitro vegatitve propagation of almond ( Amygdalus communis L.) cv. Texas and cv. Nonpareil by shoot-tip culture were investigated. Different levels of sucrose, agar and pH were tested and shoot proliferation, shoot development and growth were observed during three successive stages, namely initiation, transplantation and multiplication. During the initiation stage, proliferation was achieved only with 5 and 6% sucrose and the best explant development occured with 0.5% agar and pH 5.5. During the second stage, again 5 and 6% sucrose and pH 5.5 gave the best results with respect to shoot production and growth. Although the medium with low agar concentrations (0.5%) was favourable for shoot formation and growth, 0.7% agar was determined to be the most appropriate level for this stage since low agar levels (0.5 and 0.6%) caused vitrification. During the multiplication stage, the highest rate of shoot production was achieved with 3 and 4% sucrose, 0.7% agar and pH5.5, and higher sucrose and agar levels increased callus formation at the base of the shoots.

VI oz KALLUS, OVUL, SÜSPANSİYON VE PROTOPLAST KÜLTÜRLERİ İLE ŞEKER PANCARININ {BETA VULGARIS L.) İN VİTRO KOŞULLARDA BÜYÜME VE GELİŞMESİ Gürel, Songül Doktora, Biyoloji Bölümü Danışman: Prof. Dr. Zeki Kaya Mayıs, 2000, 174 sayfa Şeker pancarında (Beta vulgaris L.) in vitro koşullarda kallus, övül, süspansiyon ve protoplast kültürleri ile yapılan büyüme ve gelişme çalışmalarında Türkiye'de ıslah edilen değişik ıslah hatları kullanılmıştır. Hipokotil ve kotiledon eksplantlan, yaprak ve yaprak sapı eksplantlanna göre daha çok kallus üretmişlerdir. Naftalen asetik asit veya 2.4-diklorofenoksi asetik asit'in 0.5 mg/1 benzilaminopürin veya kinetin ile kombinasyonu yüksek oranda kallus üretmiştir. Sadece yaprak sapı ve yaprak eksplantlan doğrudan organogenesis yoluyla sürgün meydana getirmiş, hem kinetin hem de benzilaminopürin sürgün oluşumunu artırmıştır.vu Ön soğuk muamelesi (4 °C) ve aktif kömür kullanımı, haploid embriyo üretimini artırmış, buna karşın gümüş nitrat embriyo başlangıcım azaltmış veya tamamen engellemiştir. Kolhisin ve trifluralin, kromozom katlanması bakımından benzer etkiler göstermiş, fakat her iki kimyasal da sıvı ortamda kullanıldığında daha etkili olmuşlardır Süspansiyon kültüründe, en yüksek hücre büyüme oram Mİ 14 ıslah hattından elde edilmiştir. Hücre bölünme oranlan ise, 0.25 mg/1 benzilaminopürin ve 2,4- diklorofenoksi asetik asit içeren ortamlarda en yüksek olmuştur. Protoplastlar, süspansiyon hücreleri ile yaprak mezofıl dokusundan izole edilmişlerdir. % 1.0 selülaz, % 0.1 hemiselülaz, % 1.0 pektinaz ve % 0.1 driselaz içeren enzim karışımı, hem verim hem de canlılık bakımından en uygun kombinasyon olarak tesbit edilmişlerdir. Yüksek başlangıç yoğunluğuna sahip protoplastlar (105, 2x 1 05 ve 3 x 105), daha çok hücre kolonisi oluşturmuşlardır. Kallus sadece ıslah hattı ÇBM315'in süspansiyon protoplastlanndan elde edilebilmiştir. Protoplast füzyonu işleminde sonra, en yüksek canlılık oram % 20 PEG ile 25 dakika işlem süresi kombinasyonundan elde edilmiştir. Genel olarak, % 25 PEG ile 20 dakika işlem süresi kombinasyonu en yüksek füzyon oranını vermiştir. Düşük PEG (% 20 veya 25) ile kısa işlem süresi (15 veya 20 dakika) birlikte uygulandığında, heterofüzyonlann hücre kolonisi oluşturma kapasiteleri en yüksek olmuştur. Füzyona uğratılan protoplastlardan kallus elde edilememiştir. W. VtaSEKÖÜLKrtM KURUL»vuı Kallus veya explantlardan sürgün oluşumu şeker pancannda gen transferi çalışmalarında kullanılabilir ve ovüllerden elde edilen kromozom sayılan ikiye katlanmış haploid bitkiler ise saf hatların elde edilmesine olanak sağlar. Protoplast izolasyonu ve füzyonu çalışmalarında metod geliştirilmesi doğal olarak var olan melezleme engelini ortadan kaldırmakta ve klasik ıslah metodlanna göre daha hızlı bir alternatif olmaktadır. Ancak sürgün oluşturma çalışmaları için gelecekte bu tür çalışmalara devam edilmelidir. Anahtar Kelimeler: Beta vulgaris L., ikiye-katlanmış haploid bitki üretimi, kallus kültürü, süspansiyon kültürü, protoplast kültürü, protoplast füzyonu, polietilen glikol (PEG). uı ABSTRACT IN VITRO GROWTH AND DEVELOPMENT OF SUGAR BEET (BETA VULGARIS L.) FROM CALLUS, OVULE, SUSPENSION CELLS AND PROTOPLASTS Gürel, Songül Ph.D., Department of Biology Supervisor: Prof. Dr. Zeki Kaya May, 2000, 174 pages To study in vitro growth and development of sugar beet from callus, ovule, suspension cells and protoplasts, explants from different Turkish breeding lines of sugar beet were utilized. Hypocotyl and cotyledon explants produced more callus than leaf and petiole explants. The use of naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid in combination with benzylaminopurine and kinetin at 0.5 mg/1 increased callus formation. Only petiole and leaf explants formed shoots via direct organogenesis, and both kinetin and benzylaminopurine promoted shoot formation.

A. method is described for plant regeneration from unfertilized ovaries isolated from a diploid male sterile sugar beet (Beta vulgaris L.) breeding line that was developed at the Sugar Institute, Ankara, Turkey. Ovary explants were cultured on Murashige & Skoog (MS) medium containing 2.0 mg/l benzylaminopurine (BAP). Two treatments were tested by incubating all of the explants in darkness for 15 days, and then transferring one half to light and keeping the other half in darkness throughout the culture. Callus formation occurred in both treatments more or less at similar rates: however, there was a significant difference between the treatments with regard to the shoot-forming capacity of the explants, those transferred to light after an initial incbation in darkness producing more shoots (14.6%) than those kept in darkness continuously (4.2%). Root induction was readilyachieved within two weeks when shoots were transferred to MS medium supplemented with 2.0 mg/l naphthaleneacetic acid (NAA) and 2.0 mg/l silver nitrate (AgNO3). An inverse relationship between the callus and shoot-forming capacity of the individual explants was apparent. The determination of ploidy levels of the regenerants was performed by chromosome counting in leaf samples of regenerated plants and the results revealed that all of the regenerants were diploid. Bu çalışmada, Ankara Şeker Enstitüsünde geliştirilen diploid erkek kısır bir şeker pancarı (Beta vulgaris L.) ıslah hattından alınan döllenmemiş yumurtalıklardan elde edilen bitki rejenerasyonuna ait yöntem tanımlanmıstır. Yumurtalık eksplantları, 2.0 mg/l benzilaminpürin (BAP) içeren Murashige & Skoog (MS) ortamında kültüre alınmştır. İki farklı muamele denenmiş olup; eksplantaların tamamı 15 gün karanlıkta tutulduktan sonra, yarısı normal (i) ısık ortamına aktarılırkan, diger yarısı da bütün kültür boyunca (ii) karanlık ortamda bırakılmıstır. Her iki ortamda kültüre alınan eksplantlarda aşağı yukarı aynı oranlarda kallus oluşmuştur. Ancak, muameleler arasında eksplantaların sürgün-oluşturma kapasiteleri bakımından önemli farklar meydana gelmis olup; karanlıktaki ön inkübasyon döneminden sonra ışığa aktarılanlar, sürekli karanlıkta tutulanlara göre daha fazla sayıda sürgün oluşturmuştur (% 14.6’ya karsın % 4.2). Ayrıca, eksplantların kallus ve sürgün-oluşturma kapasiteleri arasında ters orantılı bir ikili gözlenmiştir. Sürgünler 2.0 mg/l naftelenasetik asit (NAA) ve 2.0 mg/l gümüs nitrat (AgNO3) içeren MS ortamına aktarıldıklarında, iki hafta içerisinde köklenme başlanmıştır. Elde edilen bitkilerin ploidi seviyeleri rejenerantların yaprak örneklerinde yapılan kromozom sayımları ile belirlenerek, elde edilen bütün bitkilerin diploid olduğu gözlenmiştir.

Beet necrotic yellow vein virus (BNYVV) is the cause of rhizomania, an important disease of sugar beet around the world. The multipartite genome of the BNYVV contains four or five single-stranded RNA that has been used to characterize the virus. Understanding genome composition of the virus not only determines the degree of pathogenicity but also is required to development of resistant varieties of sugar beet. Resistance to rhizomania has been conferred to sugar beet varieties by conventional breeding methods or modern genome engineering tools. However, over time, viruses undergo genetic alterations and develop new variants to break crop resistance. Here, we report the occurrence of genetic reassortment and emergence of new variants of BNYVV among the isolates of Thrace and Asia Minor (modern-day Turkey). Our findings indicate that the isolates harbor European A-type RNA-2 and RNA-3, nevertheless, RNA-5 is closely related to East Asian J-type. Furthermore, RNA-1 and RNA-4 are either derived from A, B, and P-types or a mixture of them. The RNA-5 factor which enhance the pathogenicity, is rarely found in the isolates studied (20%). The creation of new variants of the virus emphasizes the necessity to develop new generation of resistant crops. We anticipate that these findings will be useful for future genetic characterization and evolutionary studies of BNYVV, as well as for developing sustainable strategies for the control of this destructive disease.

Doubled haploid induction is one of the available methods normally used for sugar beet breeding. Gynogenic haploid explants of sugar beet induced with 1 or 2 mg L-1 6-benzylaminopurine were treated with 5 g L-1 colchicine, then subcultured on a solidified MS medium plus 0.2 mg L-1 kinetin. Colchicine doubled the chromosome number of 27.7% of the treated haploid explants. With the aim of increasing the number of doubled haploid explants, the effects of five levels of proline (0.0, 0.1, 0.2, 0.3, or 0.4 mM) on the explants' proliferation, propagation, and shoot length were compared. With a large effect size (ES), proline at 0.3 mM induced the highest amount of proliferation, while proline-free medium resulted in the lowest amount of it. The highest propagation rates were observed for the explants treated on media with 0.2 and 0.3 mM proline (very large ES). Proline at 0.3 mM induced the shortest shoots (medium ES). A very strong positive correlation between proliferation and propagation, a moderate negative correlation between proliferation and length, and a strong negative correlation between propagation and length were observed. For the first time our results show beneficialeffects of proline on in vitro proliferation and propagation of sugar beet.