This work was supported by Biotech Risk Assessment Grant Program competitive grand no. 2006-39454-17438 to A. Snow, K. Mercer, and H. Alexander from the United States Department of Agriculture, National Institute of Food and Agriculture. Experiments using this method were conducted at and supported by the University of Kansas Field Station, a research unit of the Kansas Biological Survey and the University of Kansas. The authors would like to thank P. Jourdan and E. Regnier for helpful reviews on earlier versions of this manuscript. Additionally, this work was aided by the contributions of the staff at the University of Kansas Field Station, Waterman Farm at the Ohio State University (OSU), the USDA Ornamental Plant Germplasm Center at OSU, and the Seed Biology Lab in the Department of Horticulture and Crop Science at OSU, especially E. Renze, S. Stieve, A. Evans, and E. Grassbaugh, for technical support.

1. Birden fazla Türlerin veya Tek Türlerin Kontrollü çarpılar gelen Seed toplayın Not: Anne (tohum üreten) ebeveyn olarak vahşi, karma ve mahsul türlerini kullanarak türler Helianthus annuus (ayçiçeği) içinde 15 çapraz türlerinden Bu örnek kullanılan tohum. <ol><li> büyüme mevsiminin sonunda, etiketli torbalarda olgun tohum kafaları toplamak. Standart formatta (yani anne x baba) ebeveyn çapraz tip etiketli zarf içerisinde saman ve yer tohumdan temiz bir tohum. </li><li> aynı ebeveyn çapraz türlerinin eşit sayıda tohumun eşit miktarda kullanarak büyük zarf içinde birlikte toplu tohum. Örneğin, aynı anne ve baba anne ile 10 arası türlerinden 100 tohum. belirlenmesi (çapraz tip) bilgilerle zarfları etiketleyin. Bu büyük zarflar Bölüm 2 mezar şeritler doldurulmasında kullanılacak tohum ustalar vardır. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody&gt; <tr><td></td><td colspan="3"> anne Veli </td></tr><tr><td> babalık Veli </td><td> Düzensiz: WXW </td><td> F 1: WXC </td><td> Mahsul: CXC </td></tr><tr><td> Düzensiz: WXW </td><td> 0% WXW </td><td> % 25 F 1 XW † </td><td> % 50 CXW † </td></tr><tr><td> BC: WxF1 veya F1xW </td><td> % 12.5 WxBC </td><td> % 37,5 F 1 XBc </td><td> % 62.5 CxBC </td></tr><tr><td> F 1: WXC </td><td> % 25 wxf 1 † </td><td> % 50 F 1 xf 1 </td><td> % 75 CXF 1 † </td></tr><tr><td> F 2: F 1 xf 1 </td><td> % 25 wxf 2 </td><td> % 50 F 1 xf 2 </td><td> % 75 CXF 2 </td></tr><tr><td> Mahsul: CXC </td><td> % 50 WXC † </td><td> % 75 F 1 xC † </td><td> % 100 CXC </td></tr></tbody></table> Tablo 1. Ebeveyn çapraz tEl polinasyon. Ayçiçeği bitki vahşi hibrid çapraz türlerinden imal Dosya türleri tohum gömülme deneyde kullanım için elde tozlaşma üretilmiştir. Tüm çapraz tipleri için, anne ebeveyn ilk olarak listelenir ve baba ebeveyn ikinci listelendi. † ile işaretlenmiş Çapraz tip aynı% kırpma allel ancak farklı anne anne ile karşılıklı çapraz tip çiftleri bir parçasıdır. Pace, Ba ve diğ: Tablo daha önce yayınlanmıştır. (2015) 15. 2. Özel Tohum Defin Şeritler oluşturun NOT: Bu örnekte, üç kaldırma tarih tedaviler ve 15 çoğaltır vardı, bu yüzden toplam 45 şeritler gerekli. Bu örnek şerit başına 15 bölme yüzden, biz 15, 7 x 10 cm bölmeleri ev için yeterli ince örgülü polyester kumaş ya da sivrisinek örgü gereklidir kullanır. Bkz: Şekil 1. <ol><li> Her tohum şerit için kumaş 20 x 105 cm şerit keserek başlayın. </li><li> Daha sonra ikiye kumaşı katlayın, ölçün ve bölmeleri işaretleyin. Bir HIG kullanarakh-sıcaklık tutkal tabancası, bir kısa (10 cm) ucunu mühür. Tutkal biraz, fakat tamamen soğuduktan sonra, bir yalıtım oluşturmak için bir araya kumaş tarafı basın. DİKKAT: Cilde temas uzamış ise Yüksek sıcaklık tutkal ve yapıştırıcı tabancası uçları yanıklara neden olabilir. </li><li> Her bölme işareti açık kısa sonuna kadar katlanmış kenarından Tutkal hatları. bölmeleri arasında deliksiz mühürler sağlamak için tutkal çizgisinde birlikte iki kumaş tarafı basın. bölmeleri arasında göç tohumları deneysel hata katkıda bulunur. NOT: Şimdi bölmeleri oluşturulmuş olduğunu, katlanmamış kenar tohum ve onların kimlik bilgilerini (çapraz tip adı) almak için açık ve hazır kalmalıdır. </li><li> Rastgele her şerit için bölmelere tohum çapraz türleri atayın. <ol><li> tohum muntazam sayısı (her bölüm için 20 Bu örnekte kullanılan) tohum bölmeleri doldurun. Onlar edebiliyoruz böylece etiketler için, cut-to-fit plastik bahçe dart endüstriyel kalıcı işaretleri kullanmakmezar dayanabilir. NOT: Alternatif etiketleme yöntemleri kullanılabilir. </li><li> Her bölme tohum ve etiket ile doldurulduktan sonra, yüksek sıcaklık tutkal tabancası ile hemen bölmesini kapatın. tüm tohum şeridi tamamen kapalı kadar devam edin. tekrarlanan, blok ve / veya tedavi bilgileri birinci bölme genel şerit etiketleyin. NOT: Biz zamansal giderim, blok vardı ve blok içinde şeritler çoğaltmak beri çalışma için, bu bilgilerin tümünü dahil. </li><li> Bir sonraki şerit geçmeden önce gerektiği gibi bölmeleri ve spot tutkal arasındaki boşlukların kontrol edin. </li></ol></li></ol><img alt="Şekil 1" src="/files/ftp_upload/54663/54663fig1.jpg" /> Ayrı bölmelere için çapraz tip kimlik gösteren mezar şeridi Şekil 1. Defin tohum şerit şematik. Örnek. Anne ebeveyn ikinci listelenen baba ebeveyn ile ilk sırada listelenir. Renkler farklı anne ebeveynlik belirtmekürün için F 1 melez için mavi, vahşi sarı ve kırmızı ile. <a href="http://ecsource.jove.com/files/ftp_upload/54663/54663fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> 3. Alanında Tohum Şeritler Bury <ol><li> Evin şeritler belirtilen derinliğe kadar araziler Dig. analizde subplot gibi (bu örnekte, çapraz tip) ana arsa ve bölme kimlik olarak kullanılmak üzere şeritler rasgele kaldırma tarihi sağlamak için düzenlenmiş olan tek tek blok şeritler atama. </li><li> toprakla şeritler örtün. Kesim metal örgü donanım bez gömülü şeritler üzerinde araziler ve yeri 10 cm&#39;den büyük olması. toprak kenarları ve kapak bez kenarları korumak için U-işaretçilerine kullanın. NOT: Bu kaplama Burrowing tohum yırtıcı hariç içindir. Başka bir seçenek olarak, Mercer ve ark., 13 inşa edilmiş ve saha loca yoğun gopher basınç nedeniyle tohum şeritleri çevrili donanım bez kutuları gömülüyon.) </li><li> şerit konumu ve kaldırma tarihleri ​​açık yazılı kayıtları tamamlayacak ayırt renkli haritacılara ait bayrakları yerleştirin. </li></ol> 4. Şeritler ÇıkardıSARAYBOSNA ve Seed değerlendirin <ol><li> Belirli bir kaldırma bugüne kadar atanan tohum şeritleri Kazıp. onlar tohum değerlendirme yer alacak laboratuvar ulaşana kadar serin, nemli ortamda şeritler koruyun. Bu ıslak gazete şeritleri ambalaj ve taşıma veya gece boyunca nakliye için bir soğutucu yerleştirerek gerçekleştirilebilir. </li><li> şeritler yeterli olduğunu, dış enkaz olacak karanlık değil tohumlar temizlenene kadar su içinde şeritlerden çamur ve toprak durulayın. </li><li> Bir anda açık bir bölme kesmek için makas kullanın. çimlenmiş tohum sayın ve bunları kaldırın. Açıkça ölü tohumlar (örneğin, çürüyen ya da çürümüş tohumlar) sayın. ayrıca, Filizlenmemiş uyuyan ve ölü tohumları ayırmak için etiketli, ıslak Petri boyutlu kurutma kağıdı üzerine Filizlenmemiş tohumları yerleştirin. <ol><li> Filizlenmemiş ile Petri kapları yerleştirin25 ° C / 10 ° C 12 saat gündüz / gece: Standart çimlenme koşulları için bir büyüme odası setine tohumları. Tohumlar bir hafta çimlenme izin verin. , Canlı olarak bu saymak, ancak saha koşullarında çimlenme yoktu çünkü Filizlenmemiş anlamına herhangi. </li></ol></li><li> Büyüme odası işlemden sonra deneme tetrazolyum klorit (TZ) 13 kullanılarak canlılık açısından Filizlenmemiş tohumları kalmıştır. Bunu başarmak için, AOSA Tetrazolium Test Handbook 14 kullanılarak çalışma türleri için TZ uygun bir yüzde çözelti hazırlayın. Bu örnek için, bir% 1 çözeltisi kullanılır. <ol><li> yarısında Filizlenmemiş tohumları kesin ve bir yarısını yerleştirin embriyo ve endosperm tohum karşılamak için yeterli TZ çözeltisi içeren bir Petri kabındaki görünür idi (tohumlar, bu tamam yüzer olabilir). </li><li> 30 ° C&#39;ye ayarlanmış bir inkübatöre TZ Petri kapları yerleştirin ve 3 saat beklemek. arzu edildiği takdirde daha yüksek sıcaklıklar, daha hızlı bir reaksiyon neden olur, ancak bir risk overstaining: Not. </li></ol></li> &lt;li&gt; inkübatör Petri kutularına çıkarın ve değerlendirir. lekesiz tohumlar ölü iken kendi embriyolar kırmızı lekeli tohumlar, germinable bulunmaktadır. ayırt edici marjinal durumlarda ve özellikle türler üzerinde daha fazla ayrıntı için AOSA Tetrazolium Test El Kitabı danışın. </li></ol>

<ol>
	<li>Walck, J. L., Baskin, J. M., Baskin, C. C., Hidayati, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+transient+and+persistent+seed+banks+in+species+with+pronounced+seasonal+dormancy+and+germination+patterns.">Defining transient and persistent seed banks in species with pronounced seasonal dormancy and germination patterns.</a> Seed Sci Res. 15 (3), 189-196 (2005).</li><li>Alexander, H. A., Schrag, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+soil+seed+banks+and+newly+dispersed+seeds+in+population+dynamics+of+the+annual+sunflower.+Helianthus+annuus.">Role of soil seed banks and newly dispersed seeds in population dynamics of the annual sunflower. Helianthus annuus.</a> J Ecol. 91, 987-998 (2003).</li><li>Burnside, O. C., Wilson, R. G., Weiseberg, S., Hubbard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+longevity+of+41+weed+species+buried+17+years+in+eastern+and+western.">Seed longevity of 41 weed species buried 17 years in eastern and western.</a> Weed Sci. 44 (1), 74-86 (1996).</li><li>Baskin, C. C., Baskin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Seeds: Ecology, biogeography, and evolution of dormancy and Germination. , (2001).</li><li>Finch-Savage, W. E., Leubner-Metzger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+dormancy+and+the+control+of+germination.">Seed dormancy and the control of germination.</a> New Phytol. 171, 501-523 (2006).</li><li>Baskin, J. M., Baskin, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+classification+system+for+seed+dormancy.">A classification system for seed dormancy.</a> Seed Sci Res. 14 (1), 1-16 (2004).</li><li>Donohue, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+and+genetic+influences+on+the+germination+of+Arabidopsis+thaliana+in+the+field.">Environmental and genetic influences on the germination of Arabidopsis thaliana in the field.</a> Evolution. 54 (4), 740-757 (2005).</li><li>Roach, D. A., Wulff, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+effects+in+plants.">Maternal effects in plants.</a> Ann Rev Ecol Syst. 18, 209-235 (1987).</li><li>Pace, B. A., Alexander, H. M., Emry, J. D., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+fates+in+crop-wild+hybrid+sunflower:+crop+allele+and+maternal+effects.">Seed fates in crop-wild hybrid sunflower: crop allele and maternal effects.</a> Evol Appl. 8 (2), 121-132 (2015).</li><li>Alexander, H. M., Emry, D. J., Pace, B. A., Kost, M. A., Sparks, K. A., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+maternal+effects+and+nuclear+genetic+composition+change+across+the+life+cycle+of+crop-wild+hybrids.">Roles of maternal effects and nuclear genetic composition change across the life cycle of crop-wild hybrids.</a> Am J Bot. 101 (7), 1176-1188 (2014).</li><li>Stewart, N. C., Matthew, J., Halfhill, D., Warwick, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgene+introgression+from+genetically+modified+crops+into+their+wild+relatives.">Transgene introgression from genetically modified crops into their wild relatives.</a> Genetics. 4, 806-817 (2003).</li><li>Mercer, K. L., Shaw, R. G., Wyse, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+germination+of+diverse+crop-wild+hybrid+sunflower+seeds.">Increased germination of diverse crop-wild hybrid sunflower seeds.</a> Ecol Appl. 16, 845-854 (2006).</li><li>Delouche, J. C., Still, T. W., Rapset, M., Lienhard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tetrazolium+test+for+seed+viability.">The tetrazolium test for seed viability.</a> Mississippi Sta Uni Ag Exp Sta Techn Bull. 51, 1-63 (1962).</li><li>Association of Official Seed Analysts. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tetrazolium Testing Handbook. , (2010).</li><li>Alexander, H. M., Emry, D. J., Pace, B. A., Kost, M. A., Sparks, K. A., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+maternal+effects+and+nuclear+genetic+composition+change+across+the+life+cycle+of+crop-wild+hybrids.">Roles of maternal effects and nuclear genetic composition change across the life cycle of crop-wild hybrids.</a> Am J Bot. 10 (7), 1176-1188 (2014).</li><li>Weiss, A. N., Primer, S. B., Pace, B. A., Mercer, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+effects+and+embryo+genetics:+germination+and+dormancy+of+crop-wild+sunflower+hybrids.">Maternal effects and embryo genetics: germination and dormancy of crop-wild sunflower hybrids.</a> Seed Sci Res. 23, 241-255 (2013).</li></ol>

The authors have nothing to disclose.

Burada alanında önceden seçilen zaman aralıklarında tohum çimlenmesi, dormansi ve çeşitli tohum stoklarının mortalite gözlemlemek için tohum mezar şeritleri kullanmak için yöntemler sunuyoruz. tek tek paketler, paketlerin oluşturulması üzerinde şerit ve kompartman yapı (1) hız yalan yerine şeritler kullanmanın avantajları; ve (2) kullanım kolaylığı ve bir paket atlama veya istem dışı bir kaldırma tehlikesi olmadan bir hareket birden fazla bölme kaldırma hızı. Toprak zor ve saha koşul...

Bu yöntemin genel amacı güvenilir tarla koşullarında zamanla tohum sağkalım değerlendirmektir. Toprak tohum bankaları geçici veya uzun yıllar 1,2 sürebilir ya toprak yüzeyinde, yüzey çöp içinde veya toprak profilinde içinde dağıtılan dağınık, canlı henüz Filizlenmemiş tohum, bir rezerv bulunmaktadır. Burada sunulan benzer tohum mezar yöntemleri birkaç düzine türleri kullanılarak 17 yıllık çalışmanın uygulandığı zaman, canlı tohumlar 3 test edilen türlerin birçoğu bulundu. Tohum dormansi hayatta fide koşullarının uygun kombinasyonu 4 ortaya çıkana kadar çimlenme tohum bir bloktur. uyuşukluk-serbest bırakılması için harici tetikleme çimlenme sağlar kadar uykuda kalan, tohumlar gibi düşük kış sıcaklıkları, besin sınırlama veya mevsimlik kuraklık gibi zor koşullarda hayatta kalmak için izin verebilirsiniz. uyku hali serbest bırakılması için tetikler f bıraktığı uzatılmış soğuk bileşiklere maruz değişebilirire, ya da hayvan mide asitleri 5 ile aşınmaya veya temas yoluyla tohum ceket fiziksel saldırı. çimlenme ipuçları cins veya belirli bir tür olması ve sık sık geçmiş doğal seçilim sonucu gibi, uyumsuz tohum çimlenme uygunsuz bir zamanda ortaya çıkar, ve tohum ya da fide ölüm veya kötü fide büyümesi neden olabilir hangi olmasıdır. Uyuşukluk dormansi sürümü (örneğin, fiziksel dinlenme, fizyolojik dormansi) mekanizmaları dayalı türde bir dizi ayrılır olsa da, 6 tohum dinlenmesi en az bir bitki biyolojisi konuları anlaşılamamıştır. Böylece, bireysel tohumlar veya ilgili ekolojik koşullarında tohum grupların durumunun değerlendirilmesi için izin alan çalışmaları sadece laboratuarda standart çimlenme testleri güveniyor daha yüksek açıklayıcı güce sahip. Bilinen tohum özelliklerinin Sömürü uyuşukluk mekanizmaları içgörü sağlayabilir. Tohum dormansi Kontrol eşfizyolojik ve morfolojik faktörler genetik kontrolü de dahil olmak üzere mplex. Uyuşukluk mekanizmalarının genişliği tam bir anlayış henüz aydınlatılamamıştır edilecek olsa da, genel bir model iki bitki hormonları Gibberellik Asit (GA) ve Absisik Asit (ABA) 7 arasında bir geri besleme ilişkisi içeren ortaya çıkmıştır. ABA uykuda durumunu korumak için hizmet ederken onların dinlenmesi için bir fizyolojik bileşeni ile tohum bu genelleştirilmiş modelde, GA, dormansi serbest bırakılması için bir sinyal olarak hizmet vermektedir. Maternal genetik etkiler yanı sıra anne oluşturulan doku ve gelişim sinyalleri 8 ile dinlenmesi ve boyut gibi diğer tohum özellikleri, etkileyebilir anne büyüme ortamı. Anne fizyolojik ipuçları ile birlikte zaman zaman, dormansi muhafaza edebilir harici yapılar (veya tohum kaplamaları) oluşturulur. maternal tohum kaplamaları anne bitkinin genler tarafından kontrol edildiğinden, bunlar tohumun gerçek nükleer genetik makyaj yansıtmayabilir. Bizi vared kırpma vahşi melez bir diziden Helianthus annuus achenes 9,10. Böylece, farklı türler, çapraz tip, veya genotipleri dahil çalışma tasarımları hakkında bilgi toplamaktadır tohum özelliklerine bu anne vs embriyo genetik etkileri didiklemek için haçlar ekoloji ve tohum dinlenmesi, çimlenme ve hayatta kalma genetiği. Tohum çimlenme ve hayatta kalma fenotipleri nüfus dinamiklerini nasıl etkilediğini önemli bir örnek kırpma vahşi hibrid bölgelerinde görülebilir. kültür bitkilerinin evcilleştirme sırasında Seçimi en uyuşukluk ortadan kaldırır ve büyüme mevsiminin dışında hayatta kalmak için tohumun yeteneğini azaltır. Oysa kırpma vahşi hibrid bölgelerinde ekili ve vahşi türleri arasında gen akışı, ya da melezleme, tohum bankası dinamikleri üzerindeki potansiyel etkileri ile, vahşi bir nüfus haline ekin allel (ya da genetik varyantları) reintroduce olabilir. ekili ve yabani akrabaları arasındaki melezleri potansiyel sahip olabilir kırpma vahşi hibrid bölgelerinde bulunduekimi (örneğin, kış aylarında) 11 dışında koşullarında hayatta beklenen sadece birkaç fenotipleri ile ara dinlenmesi fenotipleri çeşitli. Bu yazının amacı, biz saha koşullarında doğal varyasyonu araştırmak için farklı zaman dilimlerinde çimlenme, dormansi ve tohum türlerinin bir dizi hayatta değerlendirebilir tohum mezar şerit yöntemi kullanılarak, nasıl göstermektir. Biz tohum özelliklerine anne ve embriyo genetik etkileri ile ilgilenen beri örnekte, 15 kırpma vahşi melez çapraz türlerinden ayçiçeği tohumu kullandı.

<table><tbody><tr><td>Small coin envelopes</td><td>Any</td><td></td><td></td></tr><tr><td>Large coin envelopes</td><td>Any</td><td></td><td></td></tr><tr><td>fine meshed polyester mosquito netting</td><td>Any</td><td></td><td></td></tr><tr><td>high-temperature glue gun</td><td>Any</td><td></td><td></td></tr><tr><td>high-temperature glue stick refills</td><td>Any</td><td></td><td></td></tr><tr><td>Industrial permenant markers</td><td>Any</td><td></td><td></td></tr><tr><td>plastic garden labels</td><td>Any</td><td></td><td></td></tr><tr><td>scissors</td><td>Any</td><td></td><td></td></tr><tr><td>Shovel</td><td>Any</td><td></td><td></td></tr><tr><td>Metal mesh hardward cloth</td><td>Any</td><td></td><td></td></tr><tr><td>Surveyor&#039;s flags, multiple colors</td><td>Any</td><td></td><td></td></tr><tr><td>Wet newspaper</td><td>Any</td><td></td><td></td></tr><tr><td>cooler</td><td>Any</td><td></td><td></td></tr><tr><td>blotter paper</td><td>Any</td><td></td><td></td></tr><tr><td>petri dishes</td><td>Any</td><td></td><td></td></tr><tr><td>Temp. controlled growth chamber</td><td>Any</td><td></td><td></td></tr><tr><td>razor blades</td><td>Any</td><td></td><td></td></tr><tr><td>Petri dishes</td><td>Any</td><td></td><td></td></tr><tr><td>Tetrazolium chloride</td><td>Any</td><td></td><td></td></tr><tr><td>water</td><td>Any</td><td></td><td></td></tr><tr><td>heat incubator</td><td>Any</td><td></td><td></td></tr></tbody></table>

reliable method for assessing seed germination, dormancy, and mortality under field conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Çeşitli anne ebeveynlik ve kırpma alel yüzdesi (Tablo 1) ile çapraz tip yüzde Filizlenmemiş çimlenmiş çıkarma tarihleri arasında farklılık ve ölü tohum (Şek. 2 ve 3). Üçüncü kaldırma (yay) tarafından Filizlenmemiş tüm tohumlar gerçekten atıl olduğu tespit edildi ise Filizlenmemiş tohumların TZ testini kullanarak, biz, ikinci kaldırma (erken ilkbahar) (Tablo 2) bazı gerçekten uyuyan tohuml...

Watch this Scientific Journal Video about Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions at JoVE.com

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

Burada gömülü, etiketli tohum şeritler ve tetrazolyum klorür (TZ) canlılık testi kullanılarak tarla koşullarında tohum hayatta kalma, çimlenme ve dormansi değerlendirmek için bir protokol mevcut.

Tarla Şartlarında Tohum çimlenme, dormansi ve Ölümlerini değerlendirilmesi için güvenilir yöntem

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

reliable-method-for-assessing-seed-germination-dormancy-mortality

Research

JoVE Journal

Environment

10.5K Görüntüleme.  Ohio State University.  Burada gömülü, etiketli tohum şeritler ve tetrazolyum klorür (TZ) canlılık testi kullanılarak tarla koşullarında tohum hayatta kalma, çimlenme ve dormansi değerlendirmek için bir protokol mevcut. 

Video: Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

Farklı Ot Türlerinin Sağlam Herbisit Direnci Testi için Protokoller

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

Çevre

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

Çekirdek biyoanalizlerin kullanarak Fungal Kolonizasyon, Sporogenesis ve Mikotoksin Üretimi Kantitasyonu

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

İmmünoloji ve Enfeksiyon

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

Drosophila Melanogaster'In Embriyodan Yetişkine Canlılığını Değerlendirmek Için Doğrudan ve Basit Bir Yöntem

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Gelişim Biyolojisi

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

Genetiği keşfedin A Tohum Kabuğunun Yatak Deneyi<em&gt; In Vitro</em&gt; Nasıl Endosperminde Denetimler Çimlenme içinde<em&gt; Arabidopsis thaliana</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Biyoloji

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Yüksek işlem hacmi sterilizasyon Arabidopsis tohum için standart yöntem

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Microrespiration analizi ve mikrodiseksiyon kullanarak soruşturma Teliospore çimlenme

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Helianthus Annuus'ta Büyük Ölçekli Termal Görüntüleme Taraması ile Bitki Transpirasyonunun Yeni Regülatörlerinin Belirlenmesi

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

Nohutta Kuru Kök Çürüklüğü Hastalığı Tahlilleri: Detaylı Bir Metodoloji

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Arabidopsis Tohumlarının Yüzey Sterilizasyonu için Yüksek Verimli, Sağlam ve Son Derece Esnek Yöntem

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Mikoheterotrofik Bitki Dokularında Mantar Kolonizasyonunu Yorumlamak ve Tohumların Simbiyotik Çimlenmesi için Mikroskopi Teknikleri

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...