Karyotype analysis of rye (Secale spp.) species by using the extracted enzyme from the terrestrial snail stomach (Helix lucorum L.)

Year 2019, Volume: 29 Issue: 4, 801 - 808, 31.12.2019

Eissa Zarifi Anakhatoon , Shakiba Shahmoradı Mohsen Mırzapour

https://doi.org/10.29133/yyutbd.570576

Abstract

The snail (Helix lucorum L.) belongs to Helicidae family, lives in forests and moist regions and feeds from leaves, wood, and vegetable containing a lot of cellulose. The digestive system of this snail has special enzymatic components for digesting cellulosic materials and has some effects on cytoplasmic and cell wall digestion. Chromosomal studies and karyotypes analysis using different methods of staining (Aceto Carmine, Aceto Orcein, Feulgen and etc.) have some problems such as chromosomal size, amount of tannins and difficulty of contrast in chromosome detection depending on species characters. We used these components that included an active mixture of Cytase and other enzymes, for easy squashing and perfect chromosome separation in two species of rye (Secale spp.). The terrestrial snails were collected from Arasbaran humid forests (North West of Iran), and extracted the components and kept in freeze condition. The root tips of germinated seeds of two species of rye were pretreated with α-Bromonaphtalin solution and stained in Aceto-Iron Haematoxylin. Then the enzymes were used for clearing cytoplasm and softening cell walls and to obtain sufficient squashing and chromosomes spread. The using the enzymatic components of snail stomach was very effective in squashing stage, and obtaining good metaphase plates, the chromosomes were counted and distinguished very clearly. The separation of all cells, without damaging the chromosomes, was achieved easily and completely. Species of rye (Secale spp.) were diploid with 2n=2x=14 chromosomes. The satellited chromosomes were seen in both two species on short arms of chromosomes.

Keywords

Snail (Helix), Secale, Rye, Karyotype, Cytase

Project Number

7-03-03-92318

Karasal Salyangoz (Helix lucorum L.) Midesinden Elde Edilen Enzim Bileşenleri Kullanılarak Çavdar (Secale spp.) Türlerinde Karyotip Analizi

Abstract

Karasal salyangoz (Helix lucorum L.), Helicidae ailesine ait, ormanlarda ve nemli bölgelerde yaşar; yaprak, ağaç kabuğu ve bol miktarda selüloz içeren bitkilerden beslenir. Karasal salyangozun sindirim sistemi, sellülözik materyali sindirebilen özel enzim bileşenlerine sahiptir. Aseto karmin, Aceto orsein, Feulgen ve farklı boyar maddeler kullanılarak yapılan kromozom ve karyotip analizi çalışmalarında, türe ait karakterlere bağlı olarak kromozom boyu, tanen miktarı ve kromozom karşılaştırmalarında sorunlar yaşanmıştır. Kara salyangozu midesinin enzimatik bileşenleri, sitoplazma ve hücre duvarı yumuşaması üzerinde etkili olmuştur. Çalışmada, çavdar (Secale spp.) bitkisine ait iki türde, en iyi ve kolay şekilde kromozomların ayrılması ve dağılması amacıyla bu bileşenler kullanılmıştır. Sitaz ve diğer enzimlerin aktif bir karışımını içeren enzimatik bileşenleri kullanmak amacıyla, karasal salyangozlar İran’ın kuzey batısındaki nemli Arasbaran Ormanları’ndan toplanmış, bileşenler elde edilmiş ve soğuk koşullarda muhafazası sağlanmıştır. İki çavdar türünün (Secale cereale L. ve S. montanum) çimlenmiş tohumlarının kök uçları α-bromonaftalin solüsyonunda ön işleme tabi tutulmuş ve aseto-iron hematoxylin boyar madde ile boyanmıştır. Daha sonra, sitoplazmayı belirginleştirmek, hücre duvarını yumuşatmak ve tatminkar bir kromozom yayılması ve ayrılması elde etmek amacıyla kara salyangozu midesinden elde edilen enzimler kullanılmıştır. Araştırma sonucunda kara salyangozu midesinin enzimatik bileşenleri kullanılarak, her iki türde kromozomlar açık bir şekilde ayırt edilmiş ve, iyi bir metafaz safhası ve dağılımı gözlemlenmiştir. Kromozomlar bozulmadan kalırken, tüm hücrelerin tamamen ayrılması kolay bir şekilde sağlanmıştır. Çavdar türleri (Secale spp.) 2n=2x=14 kromozoma sahiptir ve diploiddir. Her iki türde de satellit (uydu) kromozom vardır ve satellitler, kromozomların kısa kolları üzerinde bulunmaktadır.

Keywords

çavdar, Secale, Karyotip, Sitaz

Supporting Institution

Seed and Plant Improvement Institute (SPII), Agricultural Research & Education Organization(AREO)

Project Number

7-03-03-92318

Thanks

Bu çalışma, Genetik Araştırma ve İran Ulusal Bitki Gen Bankası (SPII) tarafından desteklenen projenin (7-03-03-92318) parçasıydı

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