该研究由国家农业和食品技术研究所（RFP2015-00015-00和RTA2017-00003-00）资助;Gobierno de Aragón——欧洲社会基金，欧洲联盟（Grupo Consolidado A12-17R）和Jente de Extremadura——Fondo Europeo de desarrollo Regional （FEDER），Regional de Investigación（IB16181），Grupo de Investigación（AGA001，GR18196）。B.I. Guerrero得到了墨西哥国家科学与技术委员会（CONACYT，471839）的奖学金支持。

1. 田间手工授粉
<ol><li>
花粉提取
	<ol>
<li>为了获得花粉，在阶段D24收集花蕾，根据BBCH量表25，26上的第57阶段。 注意:与其他 李 子相比，日本李子需要更多的花蕾，因为它们的花药产生的花粉较少。</li>
		<li>使用塑料网（2 mm x 2 mm孔径）取出花药，并将其在室温下放在纸上24小时，直到花药开裂。</li>
		<li>通过细网（0.26 mm x 0.26 mm孔径）筛选花粉颗粒，并将它们保存在10 mL玻璃管中，盖子在4°C直至使用。</li>
	</ol>
</li>
	<li>
阉割花的授粉
	<ol>
<li>当10%-20%的花朵开放时，选择并标记几个分支。去除开放的花朵和年轻的花蕾，只留下阶段D24的花蕾，根据BBCH等级25，26的第57阶段。</li>
		<li>每次使用指甲或镊子去除800至1，000朵花蕾的花瓣，萼片和雄蕊。</li>
		<li>阉割后24小时，用精细的画笔帮助手授粉雌蕊。一些分支含有一半的雌蕊花粉，花粉为同一品种，另一半含有来自其他栽培品种的相容花粉作为对照。注意不要用其他品种的花粉颗粒污染指尖或画笔。</li>
		<li>记录每周的花朵和发育中的果实计数，以表征果实掉落模式并量化每次授粉处理中的最终果实。</li>
	</ol>
</li>
	<li>
田间补充授粉
	<ol>
<li>在第一朵花开前几天，将选定的树木围在0.8毫米网状笼中，以避免授粉昆虫的到来。</li>
		<li>当10%-20%的花朵开放时，每次授粉处理选择并标记几个分支，每次处理留下1，000-1，500朵花。</li>
		<li>第二天，当花朵开放时，在画笔的帮助下，用相应的花粉（来自同一品种的花粉用于自花授粉，来自其他兼容品种的花粉作为交叉授粉对照）授粉。</li>
		<li>每隔一天授粉，直到所有花朵都开开。</li>
		<li>记录从花开到收获的每周花卉和发育果实的数量，以表征果实掉落模式并量化每次授粉处理中的最终果实集。</li>
	</ol>
</li>
</ol>2. 实验室中的手工授粉
<ol><li>根据BBCH等级25，26的第57阶段，在D阶段24收集50-100朵花。</li>
	<li>在实验室中，每次处理阉割30朵花（自花授粉和异花授粉）。 注意:阉割应小心进行，以避免对雌蕊造成任何损害。</li>
	<li>在水下对每个花梗的基部进行新鲜切割，然后将其放在一块湿花店泡沫上（每次授粉处理一块泡沫）。</li>
	<li>24小时后，使用精细的画笔用先前收集的花粉手工授粉每个雌蕊（见第1.1节）。用来自同一品种的花粉授粉一组雌蕊，另一组用来自兼容品种的花粉作为对照。</li>
	<li>在室温下授粉后72小时离开授粉的雌蕊。花卉泡沫应不断用水润湿。</li>
	<li>将雌蕊固定在乙醇/乙酸（3:1）的固定溶液中，在4°C下至少24小时。 用75%乙醇代替固定剂。样品可以在4°C下保存在该溶液中，直到使用27。 注:确保样品完全浸没在溶液中。</li>
</ol>3. 微观观察
<ol><li>
体外花粉萌发的评估
	<ol>
<li>为了制备花粉发芽培养基，将25克蔗糖溶解在250毫升蒸馏水中，然后加入0.075克硝酸钙[Ca（NO 3）2]和0.075克硼酸（H3BO3）。</li>
		<li>向溶液中加入2克琼脂并混合直至完全溶解28。</li>
		<li>为了对培养基进行灭菌，请在120°C下高压灭菌20分钟。冷却培养基，在固化之前，将每个无菌培养皿（55 mm x 12 mm）3 mL分配到无菌层流罩中。介质固化后，将包裹在铝箔中的培养皿保存在4°C直至使用。</li>
		<li>将先前用作花粉供体的每个品种的花粉在两个培养皿中对照授粉，并在25°C下孵育24小时。 注意:接种的培养基可以在使用后立即用显微镜观察或储存在-20°C直至使用。为此，应在显微镜观察前24小时将培养皿从冰箱更换为冰箱。</li>
		<li>为了观察花粉颗粒，准备1%（v / v）苯胺蓝溶液，使胼胼体染色。首先，通过将7.97g K3PO4溶解在1，000 mL蒸馏水中来制备0.1 N磷酸钾三元酸钾（K3PO4） 溶液。为了阐述1%（v / v）苯胺蓝溶液，将1mL苯胺蓝溶解在100mL 0.1 N K3PO4中。</li>
		<li>向每个培养皿板加入2-3滴苯胺蓝溶液，并在5分钟后使用兴奋剂过滤器BP340-390和屏障过滤器LP425在紫外落射荧光显微镜下观察。将活的和不可存活的花粉粒计算在每盘的三块田中，每块田含有100-200粒花粉粒，在每个品种的两个培养皿中。</li>
	</ol>
</li>
	<li>
花粉管生长
	<ol>
<li>用蒸馏水冲洗固定的雌蕊三次（每个1小时，总共3小时），并在4°C下转移到5%（w / v）亚硫酸钠（Na2SO3）24小时。为了制备该溶液，将5克亚硫酸钠溶解在100毫升蒸馏水中。</li>
		<li>将雌蕊在120°C下在5%（w / v）亚硫酸钠中高压灭菌8分钟以软化组织。</li>
		<li>南瓜软化雌蕊在载玻片上的盖玻片下滴1%（v / v）苯胺蓝溶液以染色胼脖。</li>
		<li>使用励磁剂过滤器BP340-390和屏障过滤器LP425在显微镜下观察花粉管沿样式的生长。</li>
	</ol>
</li>
</ol>4. 确定不相容关系
<ol><li>从叶子中提取DNA<ol>
<li>为了提取DNA，请在田间收集每个品种的3-4片嫩叶，最好在春季。 注意:DNA也可以从成熟叶中提取，但幼叶中的DNA含有较少的酚类化合物。</li>
		<li>使用商业试剂盒分离DNA，并遵循提供的实验方案试剂盒（见 材料表）。</li>
		<li>在紫外可见分光光度计中定量DNA浓度并评估每个样品在260nm处的DNA质量。将DNA浓度调节至10 ng / μL。</li>
	</ol>
</li>
	<li>片段扩增的 PCR 条件<ol>
<li>标记 0.2 mL PCR 管和盖。</li>
		<li>根据 表1 制备PCR试剂，并让它们在冰上解冻。</li>
		<li>根据反应次数加上10%的过量，在1.5mL微管中设置每对引物的预混液体积，考虑每次反应的体积为16μL。按照 表1 中的顺序加入试剂并充分混合。</li>
		<li>将16μL预混液等分到每个含有4μLDNA模板或4μL灭菌蒸馏水的0.2mL PCR管中作为阴性对照（C-）。使用已知基因型的品种的DNA作为阳性对照。轻轻混合，用盖子关闭反应管，并以2，000×g 离心30秒，以收集反应管底部的整个体积。</li>
		<li>将反应管置于热循环器中，并使用以下温度曲线设置PCR程序:在94°C下初始步骤3分钟，在94°C下循环35次1分钟，在56°C下循环1分钟，在72°C下3分钟，最后一步在72°C下7分钟20。</li>
	</ol>
</li>
	<li>电泳和片段大小的估计<ol>
<li>要制备1.7%（w / v）琼脂糖迷你凝胶，将0.68g琼脂糖和40mL 1x TBE缓冲液溶解到100 mL锥形瓶中。通过以30秒的间隔在600 W的微波炉中加热溶液来熔化溶液，以避免沸腾。</li>
		<li>让溶液留在工作台上冷却，然后加入3.5μL核酸染色溶液。</li>
		<li>将凝胶托盘放入铸造支架中，然后将选定的梳子放入凝胶模具中。确保每个PCR产物和DNA分子量标准品有足够的孔。</li>
		<li>将琼脂糖溶液倒入凝胶模具中，使其冷却直至聚合。取出聚合凝胶的梳子。将凝胶置于含有足够1x TBE缓冲液的水平电泳系统中以覆盖凝胶表面。</li>
		<li>用2μL的DNA分子量分子量标准品（1 kb DNA分子量标准品）加载第一个和最后一个孔。将PCR的每种产物的3μL加载到其他孔中。关闭腔室，打开电源，然后将凝胶在100 V下运行30分钟。</li>
		<li>使用凝胶记录系统在紫外光下观察凝胶。使用DNA分子量分子量标准品确定扩增片段的大小，并将其与阳性对照进行比较，以鉴定相应的等位基因。</li>
	</ol>
</li>
</ol>5. 监测花期
<ol><li>监测不同品种不同树种在不同年份开花时的物候。确定从第一朵（约5%）到最后一朵开花（约95%）的开花期的长度。当至少50%的花朵处于F24阶段时，根据BBCH等级25，26的第65阶段，则考虑盛开。</li>
	<li>为了比较相互兼容的品种的开花日期并确定每年开花时间的开花时间，请用几年的数据制定开花时间日历。</li>
</ol>

作者没有什么可透露的。

本文描述的日本李子品种授粉要求的方法要求通过在田间或实验室中控制授粉来确定每个品种的自（不）相容性，并随后用荧光显微镜观察花粉管的生长。不相容关系是通过分子基因分型表征 S-等位基因建立的。最后，通过监测物候学进行授粉媒介的选择，以检测每年开花时重合的品种。
由于大量授粉要求未知的新品种，并且其中大多数是自相容的，因此在日本新的李子型品种中建立授粉要求变得越来越重要3。本文所述的方法，结合物候监测、受控授粉、荧光显微镜和分子基因分型，已被证明可用于确定品种14的授粉要求。
日本李子杂交种表现出许多特点，阻碍了使用单一方法确定其他李子物种的授粉要求。首先，花朵更小，更脆弱3。花粉的提取和处理与其他李子12属相似，尽管由于花药的花粉粒量较少，因此有必要收集更多数量的花。在一些品种中，如Rubirosa，不能使用花的阉割，因为这种技术会导致胚珠变性22和随后花21的下降，这可能导致自我不相容的错误诊断。出于育种目的，用作雌性亲本的敏感品种中的花卉阉割可导致后代缺乏3。
花的数量远远高于其他种类的果树，但坐果率非常低3。这使得有必要使用更多数量的花朵进行田间和实验室授粉21，22。实验室授粉和随后对花粉管生长的微观观察使得评估自我（不）相容性比在田间监测人工授粉的花朵直到收获更准确，因为这种技术避免了环境影响，并允许分析比田间实验更多的品种。然而，花朵处理比其他李子物种（如杏12）更辛苦，因为在将雌蕊放入泡沫之前，有必要在水下切割花梗。此外，与其他物种相比，必须在显微镜下分析雌蕊的数量更多，因为在日本李子杂交种之间的兼容花粉 - 雌蕊关系中，花粉管到达卵巢的花的百分比降低3。此外，花粉管在显微镜下更难检测，到达基底的管数较低，为15、18、20、28、29、30、31、32、33、34.在花粉管到达花架基之前，花卉在实验室条件下特别脆弱和退化的品种中，应通过田间授粉来评估自（不）相容性。
对花粉活力的评估可以知道手工授粉中使用的花粉是否足够，从而在发芽百分比低或零的情况下丢弃可能的自我不相容性的错误诊断。该技术的使用已经报道了日本李子品种21，22，35之间的花粉萌发存在相当大的差异。此外，这种方法也可用于检测雄性不育22，36，37，这对于在商业果园和杂交品种中丢弃雄性不育品种作为花粉供体以进行育种非常重要。
虽然可以通过实验室授粉用于自我（不）相容性评估的相同方法来确定品种之间的不相容关系，但该技术在这方面有一些缺点。授粉只能在开花季节进行，并且需要收集具有成熟树木的果园，以收集花朵，其寿命非常短3，38。此外，每年分析的关系数量很少，因为每对品种都需要特定的异花授粉。作为替代方案，通过PCR鉴定S等位基因不需要花，因为DNA可以从任何植物组织中提取;因此，可以收集样品的时间更长。此外，与需要立即使用的花卉不同，可以储存叶子或其他植物组织，因此分析不仅限于春季的几天，而是可以全年进行39。通过使用引物集PruC2-PCER和PruT2-PCER40，41扩增第二个S-RNase内含子来鉴定每个品种的两个S-等位基因，并随后分析扩增片段的大小琼脂糖凝胶电泳20，21允许将栽培品种分配到其相应的不相容性组（I.G.3).每个I.G.包括那些具有相同两个S等位基因的自相容品种，因此它们是相互不相容的。来自不同组别的品种，携带至少一个不同的S等位基因，是相互兼容的。
该技术的局限性在于，它不允许确定日本李子品种的自（不）相容性，因为它发生在其他李子品种中，其中自相容性与特定的S等位基因相关联，例如杏仁42中的Sf和甜樱桃43中的S4'。一些S等位基因最初与日本李子的自相容性有关，如Sb20，44，S e19，20，44，S g45和St46。然而，随后的工作报告了携带这些S等位基因的自不相容品种14，20，21，22，47。因此，日本李子需要进一步进行S等位基因测序的工作，以澄清相同大小或突变的不同等位基因是否被错误地鉴定为等位基因Sb，S e，S g或St3。同时，应通过田间或实验室授粉分析日本李子的自相容性评估，并随后在显微镜下监测田间果实掉落或花粉管的行为。
通过PCR分析鉴定S-等位基因已被证明足以建立品种之间的不相容关系3。但是，要选择足够的授粉者，有必要将此信息与每个地区每个品种的开花时间数据相结合数年，因为开花期的不匹配，即使只发生在某些年份，也可能导致坐果缺乏，收成显着减少14。
在花卉生物学和栽培品种之间的农艺行为中观察到的许多差异可能与它们的起源有关，因为目前种植的所有栽培品种都是来自原始物种P. salicina与同一属的其他物种之间的杂交种，但具有不同的特征5，48。这可能是与其他水果物种不同，有必要结合不同的技术来确定授粉需求的主要原因。了解每个品种的授粉要求有助于为新果园的设计充分选择品种，并允许检测和解决与已建立果园中缺乏授粉相关的生产问题。

日本李子（Prunus salicina Lindl.）原产于中国1.在19世纪，这种作物从日本引入美国，在那里它与其他北美二倍体李子2杂交。在20世纪，其中一些杂交种被传播到世界各地的温带地区。如今，术语"日本李子"是指广泛的种间杂交种，这些杂交种来自原始水杨假单胞菌与多达15个其他二倍体李子属之间的杂交种。
与蔷薇科的其他物种一样，日本李子表现出配子体自不相容性（GSI），其由包含多个等位基因的单个高度多态性的S位点控制6。S-位点包含两个基因，它们编码在雌蕊中表达的核糖核酸酶（S-RNase）和在花粉粒7中表达的F-box蛋白（SFB）。在自不相容反应中，当花粉粒（单倍体）中表达的S-等位基因与雌蕊（二倍体）中表达的两个中的一个相同时，由于S-RNase8的作用使花粉管RNA降解，跨样式的花粉管的生长被阻止。由于该过程阻止了胚珠中雌性配子体的受精，因此GSI促进了品种之间的异交。
虽然一些日本李子品种是自相容的，但目前种植的大多数品种都是自相容的，需要来自互相容捐赠者的花粉来施肥3。在杏仁9、杏10、11、12和甜樱桃13等李属的核果品种中，可以通过不同的方法确定品种的授粉需求。自（不）相容性可以通过在田间自花授粉和随后监测坐果来确定，或者通过在实验室的受控条件下进行半体内自花授粉和在显微镜下观察花粉管来确定14，15，16，17，18.品种间的不相容关系可以通过在田间或实验室使用潜在传粉品种的花粉进行异花授粉来确定，并通过PCR分析鉴定每个品种的S-等位基因14，15，16，19，20，21，22.在甜樱桃或杏仁等物种中，也可以通过鉴定与自相容性相关的特定S等位基因来评估自身（in）相容性，如甜樱桃13中的S4'或杏仁23中的Sf。 
来自主要生产国的几个李子育种计划正在释放一些新的品种2，14，其中许多具有未知的授粉要求。本文介绍了日本李子型杂交种授粉需求测定方法。自（不）相容性由田间和实验室的自花授粉决定，然后在荧光显微镜下观察花粉管。授粉品种的选择结合了通过PCR分析鉴定S基因型和监测田间开花时间。

<table><tbody><tr><td>Acetic Acid Glacial</td><td>Panreac</td><td>131008.1611</td><td></td></tr><tr><td>Agar</td><td>iNtRON Biotechnology</td><td>25999</td><td></td></tr><tr><td>Aniline blue</td><td>Difco</td><td>8504-88</td><td></td></tr><tr><td>Boric Acid (H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Panreac</td><td>131015.1210</td><td></td></tr><tr><td>Calcium Nitrate 4-hydrate (Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Panreac</td><td>131231.1211</td><td></td></tr><tr><td>Coverglass</td><td>Deltalab</td><td>D102460</td><td>24 mm x 60 mm</td></tr><tr><td>Digital Camera</td><td>Imaging Developmet Systems</td><td>UI-1490SE</td><td></td></tr><tr><td>Digital Camera Software Suite</td><td>Imaging Developmet Systems</td><td>4.93.0.</td><td></td></tr><tr><td>DNA Oligos</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>dNTP Mix, 10 mM each</td><td>ThermoSischer Scientific</td><td>R0193</td><td></td></tr><tr><td>DreamTaq Green DNA polymerase</td><td>ThermoFisher Scientific</td><td>EP0713</td><td></td></tr><tr><td>Ethanol 96&amp;deg;</td><td>VWR-Chemicals</td><td>83804.360</td><td></td></tr><tr><td>1Kb DNA Ladder (U.S. Patent No. 4.403.036) (500pb-12Kb)</td><td>Invitrogen</td><td>15615-016</td><td>Size: 250&amp;micro;g; Conc: 1.0 &amp;micro;g/&amp;micro;l</td></tr><tr><td>Gel Documentation System</td><td>Bio-Rad</td><td>1708195</td><td></td></tr><tr><td>Hand Counter</td><td>Tamaco</td><td>TM-4</td><td></td></tr><tr><td>Image Lab Software</td><td>Bio-Rad</td><td></td><td>Image Analyse System for Gel Documentation System</td></tr><tr><td>MetaPhor Agarose</td><td>Lonza</td><td>50180</td><td></td></tr><tr><td>Microcentrifuge 5415 R</td><td>Eppendorf</td><td>Z605212</td><td></td></tr><tr><td>Microscope with UV epiflurescence</td><td>Leica</td><td>DM2500</td><td>Exciter filter BP340-390, Barrier filter LP425</td></tr><tr><td>Microslides</td><td>Deltalab</td><td>D100004</td><td>26 mm x 76 mm</td></tr><tr><td>Mini Electrophoresis System</td><td>Fisherbrand</td><td>14955170</td><td></td></tr><tr><td>Minicentrifuge</td><td>ThermoFisher Scientific</td><td>15334204</td><td></td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>ThermoFisher Scientific</td><td>ND1000</td><td></td></tr><tr><td>Petri Dishes</td><td>Deltalab</td><td>200201</td><td>55 mm x 14 mm</td></tr><tr><td>Potassium Phosphate Tribasic (K&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;1.5H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Panreac</td><td>141513</td><td></td></tr><tr><td>Primer forward &#039;Pru C2&#039;</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>Primer forward Pru T2&#039;</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>Primer reverse &#039;PCER&#039;</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>RedSafe Nucleic Acid Staining Solution</td><td>iNtRON Biotechnology</td><td>21141</td><td></td></tr><tr><td>Saccharose</td><td>Panreac</td><td>131621.1211</td><td></td></tr><tr><td>Sodium sulphite anhydrous (Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Panreac</td><td>131717.1211</td><td></td></tr><tr><td>Speedtools plant DNA extraction Kit</td><td>Biotools</td><td>21272</td><td></td></tr><tr><td>TBE Buffer (10X)</td><td>Panreac</td><td>A0972,5000PE</td><td></td></tr><tr><td>Thermal Cycler T100</td><td>Bio-Rad</td><td>1861096</td><td></td></tr><tr><td>Thermomixer comfort</td><td>Eppendorf</td><td>T1317</td><td></td></tr><tr><td>Vertical Autoclave Presoclave II</td><td>JP Selecta</td><td>4001725</td><td></td></tr><tr><td>Vortex</td><td>Fisherbrand</td><td>11746744</td><td></td></tr></tbody></table>

establishing pollination requirements in japanese plum by phenological monitoring, hand pollinations, fluorescence microscopy and molecular genotyping

通常种植的日本李子品种是种间杂交种，来源于原始 水杨 李与其他 李 子物种之间的杂交。大多数杂交种表现出配子体自不相容性，其由包含多个等位基因的单个高度多态性的 S-位点控制。大多数栽培的杂交种是自不相容的，需要来自相容供体的花粉来施肥。由于大量新品种的授粉需求未知，在日本李子中建立授粉要求变得越来越重要。本文介绍了日本李子型杂交种授粉需求测定方法。自（不）相容性是通过在田间和实验室中手工授粉来确定的，然后用荧光显微镜监测花粉管伸长率，以及监测果实在田间的成熟度。通过将PCR分析对 S基因型的鉴定与田间开花时间监测相结合，对授粉品种的选择进行评估。了解品种的授粉要求有助于选择品种用于新果园的设计，并允许早期发现与已建立果园授粉不足相关的生产力问题。

每个日本李子花蕾包含一个花序，有1-3朵花。与其他核果物种一样，每朵花由四个漩涡组成:腕骨，雄蕊，花瓣和萼片，它们在花的底部融合形成一个杯子。花的结构比其他核果小，雌蕊短而脆弱，被含有少量花粉颗粒的雄蕊包围。在盛开时，每个花序的花朵在短茎上分开，显示白色花瓣在花开前几天形成一个被绿色萼片包围的气球（D期，57 BBCH）（图1A）。花在花开时完全开放，显示花药和雌蕊（阶段F，65 BBCH）（图2A）。像其他温带 李 属一样，花蕾首先开放，叶芽在几天后发芽。这使得盛开的树木看起来很壮观，显示出大量的花朵，但没有叶子。
日本李子的手工授粉需要在花前的气球阶段收集花朵（图1A），其中雌蕊和雄蕊几乎成熟，但花药仍未脱落。这个阶段阻止了携带外部花粉的昆虫的到来，因为花瓣仍然是闭合的。虽然花的数量远远高于其他李子品种，但大多数（85%-95%）无法结出果实。因此，使用大量花朵进行授粉实验是强制性的。未脱血的花药可以很容易地在实验室中与花分离，并在一张纸上延伸（图1B），其中花药在室温下24小时后裂开，显示花粉颗粒（图1C）。然后，花粉颗粒很容易通过细网筛（图1D），并且可以立即使用或储存直至使用，用于田间（图2A-D）和/或实验室授粉（图2E，F）。
对手授授粉花阉割后果实掉落的监测（图2A，B）或未阉割花的补充授粉（图2C，D）显示处理之间存在明显差异。在两种处理中，大多数花朵在授粉后2-3周下降。所有自花授粉的花朵都掉了下来。然而，来自补充授粉处理的4%的自花授粉花一直保留在树中直到收获，表明该品种（Rubirosa）表现为自相容性（图3）。用作对照的异花授粉花的行为在处理之间也有所不同。交叉授粉的花朵与补充授粉一起下降到授粉后3周，产生7%的坐果率。然而，所有被阉割的异花授粉的花朵都与所有自花授粉花朵的下降相吻合。
对于实验室中的手工授粉，将雌蕊置于湿花店泡沫中，在水下每个花梗的基部新鲜切割后（图2E），并在24小时后借助细刷授粉（图2F）。在自花授粉的花中，自相容的栽培品种在所检查的大多数雌蕊中显示出至少一个花粉管到达样式的底部，而在自不相容的品种中，花粉管生长在上部样式中被阻止（表2）。
不同品种的体外花粉萌发差异显著（表3）。当超过20%的花粉颗粒在培养基中24小时后显示花粉管长于其长度时，考虑良好的花粉活力（图4A）。然而，当大多数花粉颗粒没有发芽（图4B）时，该品种被认为是雄性不育的，不适合作为传粉媒介。
像其他 李子一样，雌蕊由三个结构组成:柱头，样式和卵巢。卵巢有两个胚珠，其中至少一个胚珠应受精以结出果实。在授粉过程中，花粉粒被转移到柱头，在24小时内发芽发生（图4C）。每个发芽的花粉颗粒产生一个花粉管，该花粉管通过雌蕊结构生长。在自不相容的品种中，花粉颗粒具有与雌蕊的两个 S 等位基因之一重合的 S 等位基因，花粉管在样式的上三分之一停止生长（图4D），阻止到达卵巢和随后的受精。然而，当花粉粒的 S 等位基因与雌蕊的等位基因不同时，花粉管可以通过这种方式生长（图4E），到达卵巢（图4F）并使胚珠受精。
PCR分析（表1）使用来自甜樱桃和日本李子的S-RNase保守区域的引物进行（图5）。使用的引物集PruC2-PCER和PruT2-PCER（见材料表）允许确定每个品种中两个S等位基因的大小。通过电泳在琼脂糖凝胶中运行扩增的片段，并在分析的品种中鉴定出7个不同的S等位基因（Sa，Sb，Sc，Se，Sf，Sh，Sk）。使用PruC2-PCER的片段大小在393和1，580 bp之间（图5），使用PruT2-PCER的片段大小在820和1，993 bp之间。鉴定出六种S基因型（SaSb，SbSc，SbSf，ScSh，SeSh，SfSk）。
每个品种的物候学允许计算花期的长度，总共四年，考虑到大多数花朵处于F期，阶段65 BBCH的盛开。在果园条件下开花（图6）可以比较栽培品种和年份之间的开花时间，并确定哪些栽培品种在每年的开花时间重合（图7）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61897/61897fig01.jpg"> 图1:日本李子中的花粉提取。（A）花蕾在气球阶段D，根据Baggiolini24，BBCH尺度的第57阶段。（B） 去除花朵上未脱除的花药。（C） 花粉颗粒的花药脱落。（D） 使用细网筛分花药中的花粉粒。<a href="https://www.jove.com/files/ftp_upload/61897/61897fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61897/61897fig02.jpg"> 图2:手工授粉实验，以确定日本李子的自（不）相容性。田间授粉:（A）在日本李子中开花，在花序和气球阶段开花。（B）球囊阶段D处的花朵，根据Baggiolini24，阉割后BBCH尺度的第57阶段。（C）笼中树，避免昆虫的到来。（D）非阉割花的补充手授粉。实验室授粉:（E）在水下花梗的基部切开，将阉割的花朵放在浸泡的泡沫上。（F） 用细画笔手工授粉雌蕊。 <a href="https://www.jove.com/files/ftp_upload/61897/61897fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61897/61897fig03.jpg"> 图3:日本李子的果实掉落受不同授粉处理的影响。阉割和非阉割花中的自花授粉和异花授粉。在"Rubirosa"授粉后4周内，树中剩余花朵的原始数量的花朵和发育中的果实的百分比。 <a href="https://www.jove.com/files/ftp_upload/61897/61897fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61897/61897fig04.jpg"> 图4:日本李子自花授粉花的花粉萌发和花粉管生长。（A） 体外花粉萌发。（B） 非发芽花粉粒（箭头）在体外。（C） 花粉粒在柱头表面萌发。（D）花粉管在上三分之一的样式中被捕。（E） 花粉管沿样式生长。（F） 花粉管在样式的底部。比例尺，200 μm。 <a href="https://www.jove.com/files/ftp_upload/61897/61897fig04large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61897/61897fig05.jpg"> 图5:使用引物组PruC2-PCER对9个日本李子品种进行PCR扩增。鉴定七个 S等位基因（Sa，Sb，Sc，Se，Sf，Sh，Sk）和六个 S基因型（SaSb，SbSc，SbSf，ScSh，SeSh，SfSk）。不相容组（I.G.3），"财富"（F），"TC Sun"（TS），"Laroda"（LR），"Queen Rosa"（QR），"Red Beaut"（RB），"金球奖"（GG），"Kelsey"（K），阴性对照（蒸馏水）（C-），"Zanzi Sun"（ZS），"Laetitia"（LT）。1 kb:大小标准。 <a href="https://www.jove.com/files/ftp_upload/61897/61897fig05large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61897/61897fig06.jpg"> 图6:日本李子物候监测。两种日本李子品种，花期不同。C阶段24，BBCH量表55阶段（左）和阶段F24，BBCH量表65阶段（右）。 <a href="https://www.jove.com/files/ftp_upload/61897/61897fig06large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61897/61897fig07.jpg"> 图7:4个日本李子品种4年的开花时间。从第一朵到最后一朵开花的时期。黄色单元格表示大多数花朵开花的盛开天数。 <a href="https://www.jove.com/files/ftp_upload/61897/61897fig07large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">试剂</td>
			<td>单次反应体积（μL）</td>
		</tr>
<tr>
<td colspan="2">H2O</td>
			<td>11.50</td>
		</tr>
<tr>
<td colspan="2">10X缓冲液，含20 mM MgCl2
</td>
			<td>2.80</td>
		</tr>
<tr>
<td colspan="2">dNTP 混合液，每个 10 mM</td>
			<td>0.80</td>
		</tr>
<tr>
<td colspan="2">前期底漆</td>
			<td>0.40</td>
		</tr>
<tr>
<td colspan="2">PruC2 （5'-CTATGGCCAAGTAATTCAAACC-3'）40 或</td>
			<td></td>
		</tr>
<tr>
<td colspan="2">PruT2 （5'- TSTTSTTGSTTTTGCTTTCTT-3'）40
</td>
			<td></td>
		</tr>
<tr>
<td colspan="2">底漆反转</td>
			<td>0.40</td>
		</tr>
<tr>
<td colspan="2">PCER （5'-TGTTTGTTCCATTCGCCTTCCC-3'）41
</td>
			<td></td>
		</tr>
<tr>
<td colspan="2">基因模板</td>
			<td>4.00</td>
		</tr>
<tr>
<td colspan="2">Taq DNA 聚合酶， 500 U</td>
			<td>0.09</td>
		</tr>
<tr>
<td colspan="2">最终卷</td>
			<td>20.0</td>
		</tr>
</tbody></table>表1:该方案中使用的反应条件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>栽培品种 （S- 基因型）</td>
			<td>检查的雌蕊数量</td>
			<td>发芽花粉粒在柱头上（%）</td>
			<td>雌蕊，花粉管在样式的底部（%）</td>
			<td>花粉管在样式的底部（平均数）</td>
			<td>自/交叉相容反应</td>
		</tr>
<tr>
<td>TC Sun （SbSc） × Larry Ann （SbSh）</td>
			<td>22</td>
			<td>92.3</td>
			<td>27</td>
			<td>1.2</td>
			<td>+</td>
		</tr>
<tr>
<td>TC Sun （SbSc） × Blackamber （SbSc）</td>
			<td>10</td>
			<td>74.8</td>
			<td>0</td>
			<td>0.0</td>
			<td>-</td>
		</tr>
<tr>
<td>TC Sun （SbSc） 自花授粉</td>
			<td>44</td>
			<td>78.3</td>
			<td>7</td>
			<td>1.0</td>
			<td>+</td>
		</tr>
<tr>
<td>金梅 （SbSc） × 黑星 （SeSf）</td>
			<td>11</td>
			<td>64.7</td>
			<td>36</td>
			<td>1.5</td>
			<td>+</td>
		</tr>
<tr>
<td>金梅 （SbSc） × TC 太阳 （SbSc）</td>
			<td>11</td>
			<td>98.4</td>
			<td>0</td>
			<td>0.0</td>
			<td>-</td>
		</tr>
<tr>
<td>金梅（ShSk）自花授粉</td>
			<td>38</td>
			<td>85.2</td>
			<td>0</td>
			<td>0.0</td>
			<td>-</td>
		</tr>
</tbody></table>表2:两个日本李子品种自花授粉和异花授粉后花粉萌发和花粉管生长方式。 检查的手授粉雌蕊数量，柱头上发芽的花粉粒的百分比，花粉管在样式底部的雌蕊百分比，样式底部花粉管的平均数量，自相容或交叉不相容（-）和自相容或交叉相容（+）。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>栽培品种</td>
			<td>发芽率（%）</td>
			<td>标清*</td>
		</tr>
<tr>
<td>厄勒蒙</td>
			<td>50</td>
			<td>3.3</td>
		</tr>
<tr>
<td>Earliqueen</td>
			<td>30</td>
			<td>4.2</td>
		</tr>
<tr>
<td>埃尔拉多</td>
			<td>10</td>
			<td>1.3</td>
		</tr>
<tr>
<td>修士</td>
			<td>52</td>
			<td>2.0</td>
		</tr>
<tr>
<td>金色日本</td>
			<td>17</td>
			<td>3.8</td>
		</tr>
<tr>
<td>金色梅花</td>
			<td>18</td>
			<td>3.3</td>
		</tr>
<tr>
<td>拉罗达</td>
			<td>46</td>
			<td>3.6</td>
		</tr>
<tr>
<td>拉里·安</td>
			<td>20</td>
			<td>5.8</td>
		</tr>
<tr>
<td>梅斯利</td>
			<td>2</td>
			<td>0.8</td>
		</tr>
<tr>
<td>欧文 T</td>
			<td>16</td>
			<td>0.3</td>
		</tr>
<tr>
<td>黄金时段</td>
			<td>20</td>
			<td>3.3</td>
		</tr>
<tr>
<td>罗莎女王</td>
			<td>42</td>
			<td>3.9</td>
		</tr>
<tr>
<td>皇家钻石</td>
			<td>19</td>
			<td>2.2</td>
		</tr>
<tr>
<td>圣罗莎</td>
			<td>36</td>
			<td>2.0</td>
		</tr>
<tr>
<td>太阳</td>
			<td>49</td>
			<td>3.1</td>
		</tr>
<tr>
<td colspan="3">*SD = 标准偏差</td>
		</tr>
</tbody></table>表3:15个日本李子品种的体外花粉萌发百分比。 平均±SD为六个重复。

Watch this Scientific Journal Video about Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping at JoVE

Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping

本文介绍了一种测定日本李子型杂交种授粉需求的方法，该方法将田间和实验室授粉以及荧光显微镜下花粉管的观察与通过PCR鉴定 S基因型和监测开花以选择授粉者相结合。

通过物候监测、手授粉、荧光显微镜和分子基因分型确定日本李子的授粉要求

The Japanese plum cultivars commonly grown are interspecific hybrids derived from crosses between the original Prunus salicina with other Prunus species. ...

establishing-pollination-requirements-japanese-plum-phenological

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

3.0K 次观看.  Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA). 本文介绍了一种测定日本李子型杂交种授粉需求的方法，该方法将田间和实验室授粉以及荧光显微镜下花粉管的观察与通过PCR鉴定 S基因型和监测开花以选择授粉者相结合。

视频: Establishing Pollination Requirements in Japanese Plum by Phenological Monitoring, Hand Pollinations, Fluorescence Microscopy and Molecular Genotyping

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

传粉生态学的田间试验:为例<em&gt;血红石蒜</em&gt;变种。<em&gt;荪</em

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

环境科学

Forced Flowering in Mandarin Trees under Phytotron Conditions

Phytotron has been widely used to assess the effect of numerous parameters on the development of many species. However, less information is available on how to achieve fast profuse flowering in young fruit trees with this plant growth chamber. This study aimed to outline the design and performance of a fast clear methodology to force flowering in young mandarin trees (cv. Nova and cv. Clemenules) and to analyze the influence of induction intensity on inflorescence type. The combination of a short water stress period with simulated spring conditions (day 13 h, 22 &#176;C, night 11 h, 12 &#176;C) in the phytotron allowed flowers to be obtained only after 68-72 days from the time the experiment began. Low-temperature requirements were adequately replaced with water stress. Floral response was proportional to water stress (measured as the number of fallen leaves): the greater the induction, the larger the quantity of flowers. Floral induction intensity also influenced inflorescence type and dates for flowering. Details on artificial lighting (lumens), photoperiod, temperatures, plant size and age, induction strategy and days for each stage are provided. Obtaining flowers from fruit trees at any time, and also several times a year, can have many advantages for researchers. With the methodology proposed herein, three, or even four, flowering periods can be forced each year, and researchers should be able to decide when, and they will know, the duration of the entire process. The methodology can be useful for: flower production and in vitro pollen germination assays; experiments with pests that affect early fruit development stages; studies on fruit physiological alterations. All this can help plant breeders to shorten times to obtain male and female gametes to perform forced-crosses.

Here, we present a protocol to force flowering in mandarin trees under phytotron conditions. Water stress, high illuminance and a simulated spring photoperiod allowed viable flowers to be obtained in a short period of time. This methodology allows researchers to have several flowering periods in 1 year.

植物素条件下的普通话树强制开花

Phytotron has been widely used to assess the effect of numerous parameters on the development of many species. However, less information is available on ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

通过追踪阿拉伯植物双重受精中的精子核形态对受精国的评价

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.

Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

We present a methodology to establish the pollination requirements of apricot (Prunus armeniaca L.) cultivars combining the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis.

杏中自体和（内）相容关系的确定结合手授、显微镜和遗传分析

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. ...

拟南芥中细菌入侵的气孔反应自动测量

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

作者聚焦：通过自动孔径测量推进气孔研究

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

免疫与感染

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo.
A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro is&#160;often reported, which severely confounds such analyses.
Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana,&#160;which increases the receptivity of ovules. The high-throughput SIV cum septum method may be coupled with gametophyte marker lines and genetically encoded biosensors to monitor the dynamic process of fertilization.

拟 南芥 花粉管接收和半体外隔膜法 双受精的实时成像

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female ...

通过物候监测、手授粉、荧光显微镜和分子基因分型确定日本李子的授粉要求

摘要

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此视频中的章节

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

传粉生态学的田间试验:为例

植物素条件下的普通话树强制开花

通过追踪阿拉伯植物双重受精中的精子核形态对受精国的评价

杏中自体和（内）相容关系的确定结合手授、显微镜和遗传分析

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

拟南芥花粉管接收和半体外隔膜法双受精的实时成像

通过物候监测、手授粉、荧光显微镜和分子基因分型确定日本李子的授粉要求

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测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

传粉生态学的田间试验:为例

植物素条件下的普通话树强制开花

通过追踪阿拉伯植物双重受精中的精子核形态对受精国的评价

杏中自体和（内）相容关系的确定结合手授、显微镜和遗传分析

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

直接观察和自动测量对丁香假单胞菌 pv 的气孔反应。番茄拟南芥中的DC3000

拟 南芥 花粉管接收和半体外隔膜法 双受精的实时成像

拟南芥花粉管接收和半体外隔膜法双受精的实时成像