Name: semi-high throughput screening for potential drought-tolerance in lettuce (lactuca sativa) germplasm collections
Uploaded: 2015-04-17T13:00:00.000+00:00
Duration: 6 min 35 s
Description: Watch this Scientific Journal Video about Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections at JoVE.com

The authors would like to thank California Department of Food and Agriculture for funding of the project which led to the development of this protocol. CDFA SCB11019.

1. Dikim <ol><li> Fiş tepsileri doldurun (128 hücre; 3 cm kare hücreleri ile 28 x 54 cm ve 5 cm derinliğinde) fiş toprak karışımı ile. Hücrelerin düzgün dolum yardımcı olmak için boş bir fiş tepsisini kullanarak her tepsiye toprak sıkıştırmak. </li><li> Bitki marul tohumu ¼ derinlemesine hücre başına 2-3 tohum inç. Bitki Çoğaltılmış tepsiler tüm deneysel hatları kuraklık hem stresli ve kuraklık deneme için kontrol tepsiler sağlamak. </li><li> Deliksiz bir taban tepsisine fiş tepsilerini yerleştirin. </li><li> Ters tepsiye veya plastik kubbe Su tepsiler ve kapak. Çimlenme normal olarak 48-72 saat içinde, gerçekleşir. Çimlenme sonra kapağı çıkarın ve sera içine tepsileri taşımak. NOT: Bu protokol, aynı zamanda büyüme odalarına gerçekleştirilebilir. </li></ol> 2. Büyüme ve Bitki Bakımı <ol><li> Çimlenme sonrası 1-4 hafta gerektiğinde su tepsiler, normalde bu toprak alt tepsiyi doldurarak ve izin vererek, 2-3 kez haftalık uzun süre oturmak (~ 1 saat) ışınları doymuş hale içinBu gibi suda s olumsuz bitki sağlığı ve kök gelişimini etkileyebilecek ve kuraklık ekranı etkileyebilir. </li><li> 1.5 tk / galon bir oranda karıştırılmış ticari çözünür 20-20-20 gübresi ile haftada bir kez tepsileri döller. </li><li> Hücre 1-2 hafta başına bir bitki İnce tepsiler çimlenme sonrası. </li></ol> 3. başlatılıyor Kuraklık Stres <ol><li> Bitkiler 2 gruba dört haftalık ayrı tepsiler zaman. Şiddetli su stresine tabi bir grup. Yanı sıra kontrolleri sulanan diğer tepsileri kullanın. Deney boyunca adım 2.1 gibi kontrol grubu sulayın. Kuraklık stresi başlangıcında deney kalanı için tüm tepsileri gübreleme askıya alma. </li><li> Deneysel kuraklık dönemi bir hafta içinde oluşur. Kuraklık stresi deneme (Gün 0) ilk gününde su ile düşük tepsileri doldurmak ve fiş tepsilere toprak sonra tam doymuş hale kasetlerden suyu tahliye için izin verir. T başlamasından hemen önce toprak doyurulmasıHe stres dönemi fiş tepsiler bireysel hücreler arasında toprak nem herhangi bir varyasyonu en aza indirmek için yardımcı olacaktır. </li></ol> Parametreler ile Temel Kuraklık Stres Ekranı yapılması 4. Ölçülen edilecek NOT: Aşağıdaki adımlar Tablo 1 kuraklığa stres deneme süresi her gün alınan ölçümlerin bir program sağlarken kuraklık ekran boyunca kaydedilecektir üç fizyolojik ölçümler toplamak için nasıl özetlemektedir. <ol><li> Gün 0, 2, deneme 4 Yaprak Oransal Su İçeriği (OSK) ölçün. <ol><li> Tepsiden bir marul bitki Klip ve 2 yaprak çıkarın. </li><li> Bir 9. mantar delici (~ 1.6 cm çapında) kullanarak her yapraktan 3 diskleri Punch. Bir örnek olarak yaprak yumruklar birleştirin. </li><li> Yaprak diskleri tartılır, bu RWC hesaplanmasında kullanılan taze ağırlığı (FW), bir değerdir, daha sonra da bir petri plakasında diskleri yerleştirin. </li><li> Tüm plaka için yeterli Damıtılmış su ekleyinow tüm yaprak diskleri float. Bu tamamen RWC analiz için şiş ağırlıkları toplamak için yaprak diskleri hidrat etmektir. Yaprak diskleri, oda sıcaklığında 24 saat boyunca kalmasına izin verin. </li><li> Plakalardan artık tamamen hidrate yaprak diskleri çıkarın ve yavaşça diskleri tartı önce kağıt havlu ile yaprak diskleri dış kurulayın. RWC hesaplama için abartılı ağırlık (TW) olarak bu ağırlıkları kaydedin. </li><li> Bir kağıt laboratuarda yerleştirin yaprağı diskleri, 24 saat boyunca 55 ° C&#39;de inkübatör içinde silme ya da açık bir petri filtre kağıdı diski ve kurutulur. NOT: Kağıt kurutma sırasında petri yapışmasını yaprak diskleri engeller. </li><li> Kuru yaprak diskleri ve RWC hesaplama için rekor olarak kuru ağırlıkları (DW) tartılır. </li><li> Smart ve Bingham 11,12 tarafından tarif edildiği gibi OSK Weatherley formülü kullanılarak hesaplayın. OSK = (FW-DW / TW-DW) x 100 FW taze ağırlık, TW = şiş ağırlık, DW = kuru ağırlığı = </li><li> Deneme her germplazma ve tedavi için yineleyin 4.1. </li></ol></li><li> Kaydedin bitki solgunluk kuraklık stresi deneme gün 1 ve bitkilerin% 100 normal günde 4 ya da 5, soldu zaman biten günde bir kez bir başlangıç. </li><li> Kurtarma fazı ve büyüme diferansiyel <ol><li> 6. günde, kuraklığa stres dönemi sonuçlandırmak ve stresli bitkiler deneme toparlanma sürecine girmek. Kontrol tepsiler gibi, 2.1 adım, su ile düşük tepsiler doldurun ve kuraklık vurguladı tepsiler standart sulama takvimi başlamadan önce 24 saat bekletin. </li><li> Tüm bitkileri, 10 gün süre ile iyileşmeye izin verin. </li><li> 10 gün post kuraklığa stres, her bitkinin toprak bölümü tüm yukarıdaki hasat ve taze ağırlığı kaydetmek. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Ekran için örnek sayısının Hususlar. gerekli numune sayısı bu ekrandan verilerin istenen kullanıma bağlı olmalıdır. Yayın kalitesi sonuçları istenirse her satırından 3 ayrı bitkiler (3 biyolojik çoğaltır) hasat ve kalite istatistiksel analiz için yeterli puan vermek için 2 deneysel çoğaltır en az gerçekleştirmek için tavsiye edilir. İstenilen sonuç ise sadece hızlı daha sıkı veya karmaşık su stres deneyleri, daha az örnekleri gerçekleştirmek için aday germplazmı büyük bir havuz daraltmak için ve ya çoğaltır gerekli olabilir. Gerekli örnek sayısı ekim sırasında karar verilmelidir. Dikim düzeni ve tepsiler toprak kurutma hususlar. Bir çok önemli bir göz fiş tepsinin kenarına hücrelerinde toprak iç hücreleri ve bakımı daha hızlı kurur olduğunuSadece tepsi iç (Şekil 2) bitkileri hasat sonuçların önyargı önlemek için alınmalıdır. Sadece büyük olasılıkla tepsinin dikim düzeni etkileyecek iç hücrelerden bitki hasat. 3 ve 6 benzersiz genetik materyaller bir kontrol marul çeşidi ile birlikte fiş tepsi başına dikildi arasındaki bu protokol geliştirilmiş olan ekran sırasında. Bir özel ekin kuraklık toleransı için tarama zorluklardan biri genellikle pozitif ya da negatif kontroller kullanmak için hiçbir teknik stres toleranslı veya duyarlı jermplazmanın vardır olduğunu. Pozitif veya negatif kontrol, bu protokolün geliştirilmesi gibi mevcut olduğunda tüm çalışmalarda boyunca standart germplazmı kullanımı her ekranın boyunca koşulların değişkenliği iç kontrol temel düzeyde sağlar. doğrudan tohum genetik materyalinin toplama taranabilir hızına katkıda dikilebilir ancak EAC için gereken bitkilerin sayısı ile sınırlıdır, farklı tohum genetik materyalinin sayısıh ölçüm yargılama sırasında alınan. Tüm marul 3 ila 6 her tepsiye tek germplazmı sayısını sınırlayarak hiçbir marul sadece dış hücrelerde ekili olduğunu güvence her tepsi içinde iki yerde dikilmiş olabilir. Başka bir göz germplazmında arasında büyüme oranı ve bitki büyüklüğü değişkenlik küçük çeşitleri içeren hücrelerde toprak kurutma oranlarının azalmasına neden olabilir olmasıdır. Bu olabilir potansiyel olarak bu ekrandan çekti potansiyel kuraklığa dayanıklı germplazma arasında temsil, ancak bu gözlenen sonuçlara göre durum değildi üzerinde olan küçük çeşitlerinin yönünde önyargı sonuçları. Ayrıca, bu protokol aday jermplazmalarını incelemek için sıkı ikincil yöntemlerinin kullanılması onaylamak veya bu sonuçları çürütmek gerekir ya. Solgunluk gözlenmesi tutarlılık Önemi. solgunluk için bitki izleme aynı yapılmalıdırBitki stres görünüm küçük dalgalanmaları önlemek için günlük zaman, gibi solgunluk olasılıkla bir sera gibi stoma sirkadiyen yönetmelikte mevcut sıcaklık ve nem dalgalanmalardan kaynaklanan gözlemledik. Ayrıca, soldu veya değil bitkiler puanlama yaparken tutarlı olmak emin olun. Birden bireyler tutarlı sonuçlar sağlamak için ekranı sahne olacak eğer puanlama solgunluk rehberlerin bir dizi oluşturulması özellikle önemlidir. Bu protokol için kullanılan eşik bitkinin tüm yaprakları solmuş olan gibi bitki puan soldu olması gerekir. Parametrelerinin seçimi ölçüldü. Bu protokolde ölçülen parametreler az kullanılabilir emek kullanan bir yarı-yüksek verimli sistemine ölçümlerin uyum ile birlikte su sıkıntısı belirlenmesinde onların kullanışlılığı dayalı seçildi. Birçok diğer ölçümler (kuraklığa stres belirlenmesi, örneğin., Fotosentez aktivitesinin çok yararlı olabilirKök büyümesi, ağıza ait iletkenlik ...), ve gerçekleştirilecek ekranın doğasına göre (örneğin, tohum genetik materyalinin sayısı diğer parametreler, bu ekran içine dahil edilebilir), kopya sayısı, istenen iş verimine oranını ... kullanılabilir. Protokol sonuçlarının kullanımına ilişkin yorumlar. USDA koleksiyonları tüm germplazmı pratik içeren alan denemeleri yapan bir sayıdır 4.000 bireysel marul germplazmında üzerinde içerir. Bu protokol, potansiyel kuraklık toleransı USDA koleksiyonlarını taramak için araştırmacıların çok az sayıda (1-2 kişi) izin veren tek amacı ile geliştirilmiştir. Bu yöntemlerin kullanımı sayesinde USDA koleksiyonu sonra daha yakından kuraklığa stres koşullarını çoğaltabilirsiniz saha denemelerinde kullanılan 200 çeşit daraltılmıştır. Bu protokol ayrıca alan tria kullanılmak üzere 40 400 ıspanak germplazmı bir nüfusa daraltmak için kullanılanls. Tek başına bu protokol alan su stres koşullarında dayanıklı kuraklığa tolerans içeren kesinlik germplazmı yüksek derecesi ile tanımlamak için yeterli kabul edilmemelidir, ama sadece hızlı bir su açığı koşullarında potansiyel stres toleransı taranması için bir araç olarak hizmet verebilir germplazmı çok sayıda kullanarak hızlı ve verimli bir ekran.

Water availability for irrigation has been a concern across much of the United States and globally for decades, but research into the response to drought-stress, along with other abiotic stresses, has lagged behind work in the areas of disease and insect resistance, at the industrial, academic, and governmental levels largely due to a lack of funding. Water availability for agriculture has historically been only an afterthought at the level of policy makers. Recently, due to several severe droughts in important agricultural production regions in both the United States and Australia1,2 fresh water availability has been thrust into the spotlight at both the national and international levels leading to many more resources being directed towards research into developing drought-tolerant cultivars of the major grain crops. While this shifting focus toward the development of drought-tolerant major crops is beneficial, like many areas of plant research specialty crops have largely been left behind. 
A severe drought is currently limiting vegetable production in California, the largest production region for Lettuce (Lactuca sativa L.) in the United States3. These short-term weather patterns coupled with legislative and judicial action along with long-term climate change4,5 have combined to reduce water available for agriculture in many of the most productive regions of California. Lettuce production in California represents a 1.5 billion dollar industry accounting for nearly 80% of lettuce production in the United States3. Leafy vegetables have high leaf water content and lettuce, in particular, has a shallow root system6,7 which leaves the crop vulnerable to water-stress. In lettuce, as in all crops the development of drought-tolerant varieties will become increasingly important as fresh water supplies for irrigation become more constrained8.
This protocol lays out a method by which an initial screen was performed on a large collection of lettuce germplasm in order to identify a pool of potentially drought-tolerant candidate lines for use in physiological, molecular, and genetic studies to identify specific drought-tolerance traits. These candidates can also be used directly as sources of drought-tolerance in breeding programs to improve water use efficiency in commercial cultivars. This protocol was developed specifically to address the challenges that arise during the course of any screen of an extensive germplasm collection, especially issues of space availability and labor. Also, the protocol as presented was developed for use in lettuce, but has been successfully adapted for use in the screening of a germplasm collection of spinach (Spinacia oleracea) for potential drought-tolerance and could be modified simply to screen any leafy vegetable crop. 
An important consideration before initiating a drought-tolerance screen is to understand what this method is and is not. This protocol is meant to represent a rapid method by which a large germplasm pool can be quickly and efficiently narrowed to a manageable number of candidate germplasm for use in more focused and thorough studies to identify individual tolerance traits. This protocol subjects the plants to a rapidly induced severe water-stress in contrast to a more natural slowly-induced moderate continuous drought-stress that would be observed under field conditions. The type of response induced within the plant during these two types of water-stress events (rapid dehydration versus natural drought) is not identical9 which could lead to the exclusion of some materials from future trials. These two stress events are not without overlap though10 and this protocol should serve as an effective way to identify potentially drought-tolerant germplasm contained within a large collection. This protocol alone should not be considered sufficient to identify with a high degree of certainty germplasm that contains durable drought-tolerance under field water-stress conditions, but it represents a significant step forward in the rapid screening of leafy green vegetables for potential drought-tolerance traits.

<table><tbody><tr><td>plug tray 128</td><td>T.O. Plastics&lt;br/&gt; Hummert International</td><td>11-8595-1</td><td>Any brand plug tray will work, but use the same style of trays for all trials.</td></tr><tr><td>lower tray (Display tray)</td><td>T.O. Plastics&lt;br/&gt; Hummert International</td><td>11-3305-1</td><td></td></tr><tr><td>plug/planting mix (Sunshine Mix #5)</td><td>Sunshine&lt;br/&gt; Hummert International</td><td>10-0467-1</td><td>A different mix may need to be substituted if adapting this protocol to a different crop.&amp;nbsp; Sunshine mix #4 was used in spinach trials.</td></tr><tr><td>fertilizer (20-20-20)</td><td>Jack&#039;s: Professional water-soluble fertilizer&lt;br/&gt; Hummert International</td><td>07-5915-1</td><td>Any fertilizer can be used, adjust type as needed for adapting this protocol to specific crop needs.</td></tr></tbody></table>

semi-high throughput screening for potential drought-tolerance in lettuce (lactuca sativa) germplasm collections

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely drought-tolerance traits. Fresh water availability for agricultural use is a growing concern across the United States as well as many regions of the world. Short-term drought events along with regulatory intervention in the regulation of water availability coupled with the looming threat of long-term climate shifts that may lead to reduced precipitation in many important agricultural regions has increased the need to hasten the development of crops adapted for improved water use efficiency in order to maintain or expand production in the coming years. This protocol is not meant as a step-by-step guide to identifying at either the physiological or molecular level drought-tolerance traits in lettuce, but rather is a method developed and refined through the screening of thousands of different lettuce varieties. The nature of this screen is based in part on the streamlined measurements focusing on only three water-stress indicators: leaf relative water content, wilt, and differential plant growth following drought-stress. The purpose of rapidly screening a large germplasm collection is to narrow the candidate pool to a point in which more intensive physiological, molecular, and genetic methods can be applied to identify specific drought-tolerant traits in either the lab or field. Candidates can also be directly incorporated into breeding programs as a source of drought-tolerance traits.

Bu protokol kuraklığa stres yanıtı halinde, istenilen deneysel özellik bir nüfusa ayırmak için büyük bir ekran yaparken, buna çok çeşitlidir olacak oluşturulan veriler muhtemel kuraklık toleranslı ve aradaki tüm noktalarda duyarlı stres. Şekil 1, bu protokol beklenen sonuçlara türünü temsil eden grafikler içeriyor. Üç farklı marul türlerine (marul (COS), marullarda ve Butterhead) temsili çeşidi sunulan veriler dahil edilmiştir. Sadece üç marul türleri temsil eden veriler burada gösterilir iken bu protokol çoğu türleri arasında marul ve ıspanak germplazmı binlerce tarama sırasında geliştirilmiştir. Marul üç tür ek olarak daha önce bu protokol başarıyla le ilgisiz yanı sıra yakından ilişkili Lactuca serriola türlerinden germplazmında birlikte tipi marul, kırmızı ve yeşil yaprak ekran ve kök kullanılmıştır sözafy yeşil sebze ıspanak. Yazarlar tarla denemeleri için 200 marul çeşitleri aday havuzuna 4.000 bir marul koleksiyonu daraltmak için bu protokolü kullanılarak oluşturulan veriler kullanılır. <img alt="Şekil 1," src="/files/ftp_upload/52492/52492fig1highres.jpg" /> Gösterilen Şekil 1. Temsilcisi Sonuçlar Veriler üç fizyolojik ölçümlerin temsilcisi romaine (cos, ROM), marullarda (CRSP), ve Butterhead (AMA) olmak üzere üç marul türleri, bu protokolde yer olduğunu. (A) bitkilerin yüzdesi o Tüm bitkiler% 100 soldu ulaşana kadar günde 1 başlayarak, deneme kuraklığa stres döneminin her gününde soldu edilir, (B) Yaprak oransal su içeriği günlerde 0, 2 ölçülür. kaydedilir ve 4. D : kuraklığa stres, -C:. nedeniyle büyüme farkını gösteren kuraklık stres döneminden sonra bitki ve 10 gün iyileşme kontrolü (C) Ağırlıkkuraklığa stres. Hata çubukları standart sapmayı temsil etmektedir. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52492/52492fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/52492/52492fig2highres.jpg" /> Şekil 2. Temsilcisi tepsiler ve örnek dikim diyagramlar (A ve B) Örnek tepsiler tepsiler, kontrol ile karşılaştırıldığında bu protokolü kullanan bir deneme günde 3 kuraklığa stres yanıtı göstermektedir. (C ve D) Bu tepsi şemaları bu protokolde kullanılan iki dikim yöntemleri gösteren kullanılan 128 fiş tepsileri temsil eder. tepsilerine (gölgeli hücreleri gri) dış kenarları dikilmiş, ancak daha hızlı bir iç hücrelerden daha kuruması nedeniyle kenar hücrelerinin eğilimi, deneysel örnekler için kullanılmaz edilmelidir. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Gün </td><td> 0 </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td></tr><tr><td> OSK-FW </td><td> X </td><td></td><td> X </td><td></td><td> X </td><td></td><td></td></tr><tr><td> Wilt </td><td></td><td> X </td><td> X </td><td> X </td><td> X </td><td> (X) </td><td> (X) </td></tr><tr><td> Su kesintisi </td><td> X </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Sulama Özgeçmiş </td><td></td><td></td><td></td><td></td><td></td><td></td><td> X </td></tr></tbody></table> Kuraklığa stres dönemi Tablo 1. Zaman Çizelgesi. Deneysel tevkif edilir Suyaprak oransal su içeriği ölçümleri için 6. Taze örnekleri davanın günde 0 başlayan ve gün özgeçmişlerini sulama tekneler günlerde 0, 2, 4 alınır, ve bitkilerin yüzde 1 gün günlük başlangıç ​​kaydedildi ve 100 yılına kadar devam ediyor wilted Bitkilerin% normalde günde 4, soldu, ancak günde 5 ya da 6 uzanabilir.

Watch this Scientific Journal Video about Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections at JoVE.com

Marul Lactuca sativa Germplazm Koleksiyonlarında Potansiyel Kuraklığa Tolerans için Yarı Yüksek Verimli Tarama | Çevre | Video

Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

Marul Potansiyel Kuraklığa tolerans Yarı-Yüksek Verim Tarama (<em&gt; Lactuca sativa</em&gt;) Germplazm Koleksiyonlar

This protocol describes a method by which a large collection of the leafy green vegetable lettuce (Lactuca sativa L.) germplasm was screened for likely ...

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Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce (Lactuca sativa) Germplasm Collections

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9.0K Görüntüleme.  United States Department of Agriculture. This protocol was developed to screen a large germplasm collection of the leafy vegetable lettuce (Lactuca sativa L.) for drought-tolerance, in order to identify a small candidate pool of lettuce for use in physiological, molecular, and genetic studies to identify underlying drought-tolerance traits along with breeding programs.

Video: Semi-High Throughput Screening for Potential Drought-tolerance in Lettuce Lactuca sativa Germplasm Collections

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Kök-ur Nematod ile Yüksek ve Düşük Throughput Ekranlar Meloidogyne spp.</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

İmmünoloji ve Enfeksiyon

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

Küçük moleküllere steril koşullarda bitki yanıt-e doğru değerlendirmek için Hydroponic sistemi esnek bir düşük maliyetli

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Çevre

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or lethality. However, the probability of a synthetic small molecule being bioactive is low; therefore, thousands of molecules must be tested in order to find those of interest. Liquid handling robotics systems are designed to handle large numbers of samples, increasing the speed with which a chemical library can be screened in addition to minimizing/standardizing error. To achieve a high-throughput forward chemical genetics screen of a library of 50,000 small molecules on Arabidopsis thaliana (Arabidopsis), protocols using a bench-top multichannel liquid handling robot were developed that require minimal technician involvement. With these protocols, 3,271 small molecules were discovered that caused visible phenotypic alterations. 1,563 compounds induced short roots, 1,148 compounds altered coloration, 383 compounds caused root hair and other, non-categorized, alterations, and 177 compounds inhibited germination.

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

A high throughput screen of synthetic small molecules was conducted on the model plant species, Arabidopsis thaliana. This protocol, developed for a liquid handling robot, increases the speed of forward chemical genetics screens, accelerating the discovery of novel small molecules affecting plant physiology.

Sıvı işleme bir yüksek üretilen iş ileri kimyasal genetik ekran Arabidopsis thaliana yapmak için Robot kullanımı en iyi duruma getirme

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or ...

Biyomühendislik

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Pseudomonas syringae pv Direnci yüksek-Throughput Tanımlama. Fide Sel Asası Kullanarak Domates

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

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 ...

Biyoloji

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. P. nicotianae is well adapted to high temperatures; therefore, research on this pathogen is gaining importance in agriculture worldwide because of global warming. P. nicotianae-resistant varieties of tobacco plants are commonly screened by inoculation with oat grains colonized by P. nicotianae and monitoring for the disease symptoms. However, it is difficult to quantify the inoculation intensity since accurate inoculation is crucial in this case. This study aimed to develop an efficient and reliable method for evaluating the resistance of tobacco to infection with P. nicotianae. This method has been successfully used to identify resistant varieties, and the inoculation efficiency was confirmed by real-time PCR. The resistance evaluation method presented in this study is efficient and practical for precision breeding, as well as molecular mechanism research.

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. This is a practical approach for precision breeding, as well as molecular mechanism research.

Phytophthora nicotianae Direnci için Tütün Genotiplerinin Taranması

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. ...

High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

The protocol introduces a high-throughput method for measuring the relaxation of non-photochemical quenching by pulse amplitude modulated chlorophyll fluorometry. The method is applied to field-grown Glycine max and can be adapted to other species to screen for genetic diversity or breeding populations.

Darbe Genliği Modülasyonlu Klorofil Florometrisi Kullanılarak Mahsullerde Fotokimyasal Olmayan Söndürmenin Yüksek Verimli Analizi

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of ...

Arabidopsis thaliana'da Polen Hidrasyon Profillerini Ölçmek İçin Geliştirilmiş Bir Yöntem

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Yazar Spot Işığı: Arabidopsis thaliana için Yüksek Çözünürlüklü, Tek Taneli, İn Vivo Polen Hidrasyon Biyotahlili  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Arabidopsis thaliana için Yüksek Çözünürlüklü, Tek Taneli, In Vivo Polen Hidrasyon Biyotahlili

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...

Marul Potansiyel Kuraklığa tolerans Yarı-Yüksek Verim Tarama (

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Kök-ur Nematod ile Yüksek ve Düşük Throughput Ekranlar Meloidogyne spp.

Küçük moleküllere steril koşullarda bitki yanıt-e doğru değerlendirmek için Hydroponic sistemi esnek bir düşük maliyetli

Sıvı işleme bir yüksek üretilen iş ileri kimyasal genetik ekran Arabidopsis thaliana yapmak için Robot kullanımı en iyi duruma getirme

Pseudomonas syringae pv Direnci yüksek-Throughput Tanımlama. Fide Sel Asası Kullanarak Domates

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

Phytophthora nicotianae Direnci için Tütün Genotiplerinin Taranması

Darbe Genliği Modülasyonlu Klorofil Florometrisi Kullanılarak Mahsullerde Fotokimyasal Olmayan Söndürmenin Yüksek Verimli Analizi

Arabidopsis thaliana için Yüksek Çözünürlüklü, Tek Taneli, In Vivo Polen Hidrasyon Biyotahlili

Arabidopsis thaliana için Yüksek Çözünürlüklü, Tek Taneli, In Vivo Polen Hidrasyon Biyotahlili

Arabidopsis thaliana için Yüksek Çözünürlüklü, Tek Taneli, In Vivo Polen Hidrasyon Biyotahlili