The authors would like to acknowledge funding support for aspect of the work through Linkage Grants LP0776563 (GB, AS) and LP0211919 (GB) and industry partners Sappi and Mondi as well as Australian Postgraduate Award (EM) from the Australian Research Council and the Young Innovators and Scientist Award through the Australian Department of Agriculture (LT). We also like to thank the Zander Myburg, Qing Wang, Colleen MacMillan and Simon Southerton for the many discussions and ideas they put forward during the development of this protocol and to Martin Ranik, Minique De Castro, Julio Najera, Valerie Frassiant, Angelique Manuel and Noemie Defaix for assistance in laboratory related work.

1.植物材料的制备<ol><li>之前的实验，提高从种子或切割的优选树种新的幼苗和生长树/秒，直到在用于实验的区域中的杆的直径是直径约1厘米。 注:需要可能会有所不同，由于植物的生长速度，因此三至九个月这一步之间允许的时间。 </li></ol> 2.二进制向量创建<ol><li>从此部分进行工作，以在实验室或温室，其中根癌农杆菌可以处理和规定为实验室适当的个人防护设备部分7被使用。 </li><li>制备含有任何兴趣击倒或过表达盒和一个β葡萄糖醛酸酶（GUS）报道基因盒的一个基因或融合于取决于研究的类型GUS基因的感兴趣的启动子的二元载体。 注意:有一些二元载体BAC的可商购或可来源通过提供给插入基因和感兴趣的启动子进二元载体研究网络以及许多技术kbones。在这个协议的情况下达到实验者就如何创建一个二进制向量的决定。 </li><li>变换二元载体导入A的解除武装应变利用农杆菌电或热休克14，直到需要进行实验适当存放。 </li><li>重复步骤2.2为任何阳性和/或阴性对照。 注意:阴性对照应包括含有无目的基因进行兴趣研究的基因和感兴趣研究的启动子的启动子的GUS二元载体。利息研究的启动子阳性对照应包括融合到GUS记者花椰菜花叶病毒35S启动子（CaMV35S启动）。 </li></ol> 3. A.制备农杆菌接种<ol><li>至少一周之前EXPERimentation，拿A.少量（约1微升）步2.2和2.3 农杆菌中，并准备与琼脂独立LB培养基上显得捉襟见肘含有细菌选择适当的抗生素板块14。在孵化器成长在28°C，形成小菌落，然后在4个商店 直到需要°以下。 </li><li>之前的实验48小时后，转移A的一个菌落从各板成的50ml 农杆菌螺杆含有5ml预热（28℃）的LB培养基包含用于细菌选择适当的抗生素和在200rpm搅动振荡器上孵化约48小时，在28℃直至14的顶管混合物非常浑浊。 </li><li> 4-6之间茎小时之前，工厂的接种（第4）加1毫升LB / A的农杆菌混合成一个新的容量50mL的螺纹含有新鲜温热（28°C）24毫升（1:20稀释）顶管LB培养基与适当的抗生素细菌选择。如果在600nm（OD 600，如通过光谱学测定）的光密度（OD）大于0.1，直到这实现进一步用温水的LB稀释。 </li><li>将稀释LB / A.农杆菌混合重上摇动孵化和动摇在相同条件下（200转，28°C），直到OD 600为0.4-0.6，并删除之间。 </li><li>离心机LB / A.农杆菌混合物在1000 xg离心15分钟，4℃。 </li><li>倒出液体，并立即重悬A.农杆菌沉淀在1ml的冷却MS媒体15，转移〜2毫升离心管，直到需要接种（第4）冰上存储。这种溶液被称为接种媒体。 </li></ol> 4.接种植物与干A.农杆菌 <ol><li>春末或初夏使用具有积极而快速增长形成层在第1中创建植物在开始实验。一个好的诊断为活性和快速增长的形成层是韧皮部可从木质部容易地剥离。 </li><li>发现茎植物的基地附近一个清晰的直线部分，并移除任何树叶和树枝。 </li><li>用锋利的手术刀（最好是第11号）或刀片，通过引入两个垂直平行的切口，通过20毫米的长度和1个5毫米相隔韧皮部再创建茎明确的部分1厘米2&#39;形成层窗口"横向切断连接在其底端的两条垂直切割。 </li><li>通过剥离切口向上露出木质部发展组织及步骤3.6补充足够的接种媒体湿用吸管（通常是5-10微升）露出木质部开发的表面产生的韧皮部条。立即重新插入韧皮条。 </li><li>绑定韧皮部地带茎紧密封口膜。 </li><li>重复步骤4.2至4.5为任何额外形成层窗户兴趣创建的基因（S）或子（S）NY新形成层窗口至少1cm上方或下方等形成层的窗口，并在90°的偏移。 </li><li>完成步骤4.2至4.5的阳性和阴性对照载体（步骤2.4）确保每个向量被添加到在实验中使用的各植物的相同干。 </li><li>定期监测树干直径的生长和收获GUS检测时至少5 mm径向增长茎中被观察到（第5）。 注意:所需要的径向生长的量依赖于组织的用于下游分析所需的量。 </li></ol> 5.收获了GUS的组织学分析<ol><li>消费税从阀杆"形成层窗口&#39;移动任何组织不能形成层窗口中新的增长点的一部分。 <ol><li>对于与成熟的木质部和韧皮部的细胞/组织形态的研究，从剥离木质部韧皮部。 </li><li>对于需要在形成层区保持完整或启动子的表达模式的研究是要评估版，切片形成层窗口横向使用刀片或其他锋利的刀刃成厚0.5和1之间的光盘。 </li></ol></li><li>放置加工形成层窗口组织在14毫升圆底管和在pH 7 14 0.1M磷酸盐缓冲液冲洗两次。确保组织被完全淹没，保留在溶液中至少五分钟，所有过量的溶液在末端被除去第二漂洗。 </li><li>添加5毫升GUS试剂（0.5毫米×-GLUC（5-溴-4-氯-3-吲哚 - β-D-葡糖醛酸），10毫摩尔EDTA，0.5％的Triton X-100 V / V，0.5毫钾铁氰化物（III），0.05毫亚铁氰化钾（II）中，由于最终体积为0.1mM磷酸盐缓冲液pH 7;参见Hawkins 等 16）到每个管中。如果组织没有完全淹没，添加额外的GUS试剂。 </li><li>在黑暗中在55℃孵育管10分钟。 </li><li>在黑暗中在37℃孵育管用于在摇床培养箱进一步12-16小时使用（30至60转）温和搅拌以允许混合。 </li><li>当从摇床培养箱除去随机检查GUS试剂的pH值使用石蕊试纸pH范围0-7管的一小部分。 <ol><li>如果任何管子的pH值低于6再贴上标签。继续检查管子的其余部分，以确认pH和标签如果pH为6或更低。 注意:用pH值低于6的样品可能不适合用于分析。这些样品必须从进一步分析中排除。 </li></ol></li><li>滗GUS试剂并有足够的70％的乙醇代替以覆盖组织。 </li><li>直到需要储存于4℃。 </li></ol> 6.确定转基因组织<ol><li>使用镊子，取出全部来自于小托盘或培养皿管和地点形成层窗口组织允许微观可视化。 </li><li>使用1X和4X放大率之间的解剖显微镜，鉴定并计算细胞或组织的数量有一个明亮的蓝色染色。蓝染色的细胞或组织被称为从这时开始扇区。理货他们在以下几个方面。 <ol><li>对于其中韧皮部已被删除（步骤5.1.1）的样品，计算在显影木质部（形成层扇区， 图1a）的表面上发现形成层组织的扇区数。 </li><li>对于已经被切割成盘（步骤5.1.2）的样品，计算在不同的细胞或组织类型中发现的扇区数（ 图1B，1C，1D）。扇区可以在以下的组织类型中找到:周皮（周皮扇区， 图1E），韧皮部（韧皮部扇区， 图1F），形成层组织（形成层扇区， 图1G，1H，1I），伤口实质（伤口薄壁扇区， 图1J）和侵填体（TYLOSE部门， 图1K）。 <ol><li>在显微镜下一个ssessment确保盘的两侧已经被浏览和跨两个盘出现的行业相匹配，以使数字不被高估。 </li></ol></li></ol></li><li>放置仅含有组织部门放回含有70％的乙醇和存储，直到需要该管。 </li><li>重复步骤6.1至6.3为任何额外形成层窗口包括正和/或阴性对照。 </li><li>除以扇区的总数乘以1厘米2形成层窗口的总数来推导每形成层的cm 2的转化事件的平均数计算每个扇区类型为每个基因或启动子和控制平均转化效率分别接种（ATScm -2）。 </li><li>继续执行步骤7.2和8的启动子表达模式的分析和步骤7.1，7.2或7.3的技术评估组织形成层细胞和组织的形态。 </li></ol> 7.分析在形成层组织细胞和组织形态注:下面是已成功地用于ISSA的分析技术的选择衍生的样品。 <ol><li>分析木质部小区区域，管腔面积，细胞壁厚度和细胞壁面积使用扫描电子显微镜（SEM）的。 注:此协议需要最少的样品制备和允许相对高吞吐量与概括的形态性状行业分析。 <ol><li>从这一步开始，请确保白大褂，保护眼睛和木本时组织被处理，处理后用于手套。 </li><li>识别用于分析的形成层部门和使一个切口大约0.5mm至其任一侧使用单个边缘刀片创建光盘（ 图2a）。 </li><li>修剪掉多余木质部组织留下木质部组织约1mm到扇区的切向一侧（ 图2b）。 </li><li>小心LY使用新鲜双刃刀片（ 图2c）横向切割通过该扇区的中心。 </li><li>使在横向平面任扇区侧的两个附加浅径向切口划定转基因组织（ 图2d）。如GUS染色不会深入渗透到组织沿着细胞的径向文件遵循在形成层表面发现染色的组织的两侧。 </li><li>附上准备形成层界面到的SEM销存根的阶段使用SEM导电性带（ 图2e）装入和保持在干燥器直到需要用于SEM可视化。 </li><li>重复步骤7.1.2至7.1.6为任何附加扇区包括来自阴性对照。 </li><li>在低真空模式使用SEM（能源5千伏，斑3.0纳米），可视化和取该扇区内的细胞/组织的照片，以及将细胞/组织直接相邻的扇区（ 图2F）。倍率的量依赖于感兴趣的特征。对于木质部纤维，是2000倍适宜（ 图2G）。 </li><li>一旦形成层部门已经显现，并在适当的放大倍率拍摄的照片，用图像测量软件测量感兴趣木质部细胞/组织形态特征。 </li><li>进行统计分析，并进行多成对分析使用配对t检验来比较在转基因部门和相邻的非转基因的对照细胞/组织用于每个扇区确定的p（内从扇区边缘0.5毫米测量）测量形态之间的差-值。 </li><li>重复步骤7.1.10为阴性对照。 <ol><li>在阳性对照示出了低的p值（α&lt;0.05）的情况下，计算出转基因和相邻的非转基因细胞/组织的形态学性状之间的差异。在非配对t检验使用这个"差分值"与negat比较感兴趣的基因的作用香港专业教育学院控制以确定p值。 </li></ol></li></ol></li><li>使用光学显微镜的干细胞/组织的形成层细胞/组织形态或启动子表达模式组织化学分析。 注:虽然耗费更多的时间，这种方法允许更多的分析选项，包括形成层动态， 如形成层宽度以及启动子表达模式的各个方面。此外，如果无法访问的SEM，该协议可被用作用于步骤7.1的替代品。 <ol><li>使用剃刀刀片兴趣消费部门和直接与螺丝帽在至少2天的样品小瓶1-3毫升100％的乙醇于4℃一些周围组织的小块（不大于1mm 3）和地点在摇床上。的方式，将允许感兴趣的表面，以用切片机进行访问准备小块。 </li><li>重复为任何附加扇区包括来自阴性对照。 </li><li>除去液体并用2取代5％乙醇:75％的LR白混合物2天维持的条件。 </li><li>重复步骤7.2.3用50％乙醇:50％的LR白，25％乙醇:75％的LR用100％LR白白然后最后两次。 </li><li>将小块与对齐到短端利率表面嵌入模具，并用新鲜LR的白色仔细覆盖和聚合按照制造商的说明。 </li><li>切割在旋转切片机从感兴趣的表面5μm的部分，并安装在载玻片。用番红O（0.01％）染色可视化细胞壁和/或其它特征。 </li><li>添加安装媒体，放置盖玻片，并允许设置过夜。 </li><li>查看100X和600X放大倍率和捕捉图像的光学显微镜下。 </li><li>从捕获使用图像测量软件图像形态特征。 <ol><li>对于定量形态性状的统计分析，从步骤7.1.10遵循。 </li><li>对于形态性状的定性分析，比较和描述了基因或感兴趣的启动子和负极和/或阳性对照观察到的模式。 </li></ol></li></ol></li><li>在浸软纤维使用光学显微镜微纤丝角（MFA）分析。 <ol><li>消费转基因木质部直接从一个扇区以及从邻近它的非转基因组织的组织（在0.5毫米， 图2h），并发生在分开1.5毫升管中。重复为任何附加扇区包括来自阴性对照。 </li><li>完成步骤7.3.3 7.3.5在通风橱。 </li><li>添加过氧化氢的250微升和250微升冰醋酸至每个管。 </li><li>放置管在加热块在90℃下，在通风橱2小时。 </li><li>从管中取出组织并用蒸馏水仔细漂洗至少两次。 </li><li>使用的水溶性安装介质，安装在载玻片组织并与前固定用盖玻片尖锐的镊子挑逗分开。 </li><li>视图下，在放大倍数超过400倍较大的个体纤维的光镜和捕捉图像。 </li><li>为了确定纤维的微纤丝角，测量坑孔和/或细胞壁条纹和利用图像测量软件的纤维（ 图2I）的长轴之间的角度。 </li><li>进行统计分析，从步骤7.1.10遵循。 </li></ol></li></ol> 8.启动子的分析表达模式<ol><li>分析专干组织中启动子表达模式。 <ol><li>相符的每个扇区类型感兴趣的启动子，以及阳性和阴性对照的频率如在步骤6.2.2说明。 </li><li>比较不同扇区类型的频率的使用卡方检验，建立p值的阳性对照感兴趣的启动子。细胞/组织特异性的进一步分析可以使用步骤7.2根据需要进行。 <ol><li>如果多于一个的启动子的兴趣正在调查然后重复所有感兴趣的启动子和阳性对照之间的这种分析作出比较。 </li><li>如果阴性对照观察到的部门或蓝染色，然后将它添加到分析，或根据程度或内源性染色怀疑（见讨论 ）抛弃。 </li></ol></li></ol></li><li>启动子表达模式的分析过程中形成层衍生发展与分化。 <ol><li>使用解剖显微镜，可视化所有在步骤6.2中确定的形成层的扇区（图1G，1H，1I），并判断在从形成层的三种组织类型的存在/不存在蓝染色的;韧皮部（P），显影木质部（X1）或开发的木质部组织（X2）（参见图3a）。 </li><li>按照步骤8.1.2，比较存在/不存在GUS染色的频率中的int启动子的P，X1和X2的区域用卡方检验，建立p值阳性对照erest。细胞/组织特异性的进一步分析可以使用步骤7.2根据需要进行。 <ol><li>欲了解更多考虑的步骤8.1.2.1 8.1.2.2和。 </li></ol></li></ol></li></ol>

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<li>Spokevicius, A. V., Van Beveren, K., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agrobacterium-mediated+transformation+of+dormant+lateral+buds+in+poplar+trees+reveals+developmental+patterns+in+secondary+stem+tissues%5BTitle%5D)+AND+%22Funct+Plant+Biol%22%5BJournal%5D)">Agrobacterium-mediated transformation of dormant lateral buds in poplar trees reveals developmental patterns in secondary stem tissues.</a> Funct Plant Biol. 33 (2), 133-139 (2006).</li>
<li>Spokevicius, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((beta-tubulin+affects+cellulose+microfibril+orientation+in+plant+secondary+fiber+cell+walls%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">beta-tubulin affects cellulose microfibril orientation in plant secondary fiber cell walls.</a> Plant J. 51 (4), 717-726 (2007).</li>
<li>MacMillan, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+fasciclin-like+arabinogalactan+protein+family+of+Eucalyptus+grandis+contains+members+that+impact+wood+biology+and+biomechanics%5BTitle%5D)+AND+%22New+Phytol%22%5BJournal%5D)">The fasciclin-like arabinogalactan protein family of Eucalyptus grandis contains members that impact wood biology and biomechanics.</a> New Phytol. 206 (4), 1314-1327 (2015).</li>
<li>Creux, N. M., Bossinger, G., Myburg, A. A., Spokevicius, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+somatic+sector+analysis+of+cellulose+synthase+%28CesA%29+promoter+regions+in+woody+stem+tissues%5BTitle%5D)+AND+%22Planta%22%5BJournal%5D)">Induced somatic sector analysis of cellulose synthase (CesA) promoter regions in woody stem tissues.</a> Planta. 237 (3), 799-812 (2013).</li>
<li>Hussey, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SND2%2C+a+NAC+transcription+factor+gene%2C+regulates+genes+involved+in+secondary+cell+wall+development+in+Arabidopsis+fibers+and+increases+fiber+cell+area+in+Eucalyptus%5BTitle%5D)+AND+%22BMC+Plant+Biology%22%5BJournal%5D)">SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibers and increases fiber cell area in Eucalyptus.</a> BMC Plant Biology. 11, (2011).</li>
<li>Melder, E., Bossinger, G., Spokevicius, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overexpression+of+ARBORKNOX1+delays+the+differentiation+of+induced+somatic+sector+analysis+%28ISSA%29+derived+xylem+fiber+cells+in+poplar+stems%5BTitle%5D)+AND+%22Tree+Genet.+Genomes%22%5BJournal%5D)">Overexpression of ARBORKNOX1 delays the differentiation of induced somatic sector analysis (ISSA) derived xylem fiber cells in poplar stems.</a> Tree Genet. Genomes. 11 (5), (2015).</li>
<li>Baldacci-Cresp, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PtaRHE1%2C+a+Populus+tremula+x+Populus+alba+RING-H2+protein+of+the+ATL+family%2C+has+a+regulatory+role+in+secondary+phloem+fiber+development%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">PtaRHE1, a Populus tremula x Populus alba RING-H2 protein of the ATL family, has a regulatory role in secondary phloem fiber development.</a> Plant J. 82 (6), 978-990 (2015).</li>
<li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSambrook%2C+J+%22Molecular+cloning%3A+A+laboratory+manual%22">Molecular cloning: A laboratory manual</a>. , 3rd edn, Cold Springs Harbour Press. (2001).</li>
<li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+revised+medium+for+rapid+growth+and+bio-assays+with+tobacco+tissue+cultures%5BTitle%5D)+AND+%22Physiol.+Plant%22%5BJournal%5D)">A revised medium for rapid growth and bio-assays with tobacco tissue cultures.</a> Physiol. Plant. 15 (3), 473-497 (1962).</li>
<li>Chaffey, N. The use of GUS histochemistry to visualise lignification gene expression in situ during wood formation. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHawkins%2C+S+%22Wood+formation+in+trees%3A+Cell+and+Molecular+Biology+Techniques%22">Wood formation in trees: Cell and Molecular Biology Techniques</a>. , Taylor and Francis. 271-295 (2002).</li>
<li>Hodal, L., Bochardt, A., Nielsen, J. E., Mattsson, O., Okkels, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection%2C+expression+and+specific+elimination+of+endogenous+beta-glucuronidase+activity+in+transgenic+and+nontransgenic+plants%5BTitle%5D)+AND+%22Plant+Sci%22%5BJournal%5D)">Detection, expression and specific elimination of endogenous beta-glucuronidase activity in transgenic and nontransgenic plants.</a> Plant Sci. 87 (1), 115-122 (1992).</li>
<li>Hansch, R., Koprek, T., Mendel, R. R., Schulze, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+protocol+for+eliminating+endogenous+beta-glucuronidase+background+in+barley%5BTitle%5D)+AND+%22Plant+Sci%22%5BJournal%5D)">An improved protocol for eliminating endogenous beta-glucuronidase background in barley.</a> Plant Sci. 105 (1), 63-69 (1995).</li>
</ol>

The authors declare that they have no competing financial interests.

在ISSA协议是在几个月的基因和所涉及的木材感兴趣的启动子的分析在空间中树种创造转基因茎组织的相对简单和有效的方法和茎的形成。举手之劳，除了保持植物活，需要成长接种后其鲜明的对比，其中需要大量的培养，以维持组织或植物，其中木材生产可能需要长达几年开始或者一个真正的体外方法转基因茎组织专干不创建1。 ISSA也被证明是适用于广泛范围的树种，包括杨树...

树茎包括行星生物量的显著量是巨大的生物，文化和商业上的重要性。二级茎由许多其他生命形式提供资源和庇护所创造的栖息地。他们以他们居住，并充当生产木材，纸浆和造纸等木材和非木材产品的可再生资源的生态系统提供多项服务。二次干发展并且更具体木材形成通过调节特定细胞类型的发展复杂的分子体系的约束，其细胞壁的生化成分，以及他们如何被布置为形成组织和器官。解剖专干的开发和木材形成的分子基础是由许多因素，包括木材和干性内部和之间的茎，长代次，异交交配系统，高杂合度，高遗传负荷，季节性休眠，长的可变性混淆成熟特点建立期和成熟的树木纯粹的物理尺寸。其结果是，干的发展相对于植物发育的分子控制的其它方面的详细知识次要的认识，仍处于起步阶段。 许多体外技术已被用于研究和了解二次干发展，尤其是木材和二级细胞壁的形成。这些协议包括使用整个植物或植物部分的系统，其中任一转基因植物产生或特定辅助细胞或组织转化为木材和/或仲干发展1的具体方面的研究。转基因植物可以从各种各样的植物组织和细胞类型的回收后的遗传转化，但是，进展缓慢，特别是在分析木纤维性状由于长期再生时和干成熟时间（年的顺序），高的技术和劳动德曼DS，可以通过低的候选基因，以及在传播一些木本植物物种困难。类似的技术已在非木质模型系统被开发，如拟南芥，成功地克服了这些限制，但不是所有的二次干细胞类型中在这些本茎和相关的季节性或长寿性状不能在这些物种2进行研究。备选地，植物部分系统，例如Pinu酒店小号辐射愈伤组织培养物3减少相关的时限。然而这些方法被限制于单个细胞类型的研究，并如所指出用于体外实验遭受类似的限制。同样，涉及整个茎段根尖干文化4显示的承诺，但目前还没有适用于特定基因或发起人的研究。最近，涉及毛状根替代协议已被开发用于桉树并已成功施加5，但是，这种方法仍需要在体外培养，涉及二次根而非茎和迄今它仅限于单一树种。 诱导的体部门分析（ISSA），如这里描述的，被开发来克服的一些问题，提供用于基因和启动子的高通量功能筛选工具的培养基中，在木材形成和二次茎组织发育疑似角色。 ISSA是被开发，以减少所产生的转基因细胞和组织在一个完整的辅助阀杆同时克服劳动时间在体内转化和筛选系统，体外方法中使用的常规技术和吞吐量的限制。这里所描述的协议允许用于同时建立数百独立地转化组织扇区和小区的一个短的时间期间内的次级茎，在树种和感兴趣组织而不克相对较短的时间框架和低廉的劳动力成本中enetic和/或环境的变化。 ISSA 体内技术首先被用于辅助阀杆6和芽7组织以来，通过参与形成层分化和基因和/或促进研究二次茎组织了细化说明，并有包括: 微管蛋白 （TUB）8，fasciclin样阿拉伯半乳聚糖 （ FLA）9， 纤维素酶 （CESA）10， 次生壁相关NAC域 （SND 2）11，ARBORKNOX（ARK1）12和非常有趣的新基因 （RING）H2蛋白13。在次级进行了这些研究茎杨树和桉树植物和提供见解细胞形态，细胞壁化学和基因表达。 这里描述的协议的目的是汇集经验第二，通过知识的ISSA从一系列在过去十年中发表和未发表的研究开发和应用获得的。他们专注于专干组织6 在体内转化，集中力量对涉及新疆杨"锥状&#39;克隆， 蓝桉以及11个桉×赤桉克隆研究。本文通过从植物和细菌，茎组织，生长和组织的收获，转基因细胞和组织的鉴定，为收集和分析数据的表型评估和方法编制转型的种植协议进行研究。虽然技术已成功应用于测量细胞壁的单糖组成也9,11，由于篇幅所限，本文集中用于二次ST测量细胞和组织的形态和理解基因表达模式技术只有EMS。因此，如所概述的协议是适合于那些希望获得进一步深入了解的基因连接于次级的作用和/或表达的茎使用成本低，在技术上容易，中到高通量方法。

<table><tbody><tr><td>Plants</td><td>NA</td><td>NA</td><td>Please consult local nursery suppliers for plants as needed</td></tr><tr><td>Agrobacterium strain</td><td>NA</td><td>NA</td><td>There are many possible avenues to obtain Agrobactrium strains. We suggest you follow up within your local research community as there may be restrictions in obtaining the bacteria in your country and region.</td></tr><tr><td>Binary vector (gene and promoter)</td><td>NA</td><td>NA</td><td>We have developed a range of vectors to suite the ISSA protocol using a the Gateway Recombinase system. This include overexpression, RNAi knockouts and promoter fusion vectors based on modified pCAMBIA vectors and happy to provide as needed. In addition, there are many vectors avialable to the research community.</td></tr><tr><td>LB media</td><td>Sigma</td><td>L3022</td><td>The same product could be sourced from another company</td></tr><tr><td>LB media with agar</td><td>Sigma</td><td>L2897</td><td>A like product could be sourced from another company</td></tr><tr><td>Antibiotics</td><td>Sigma</td><td>NA</td><td>The catalog number will be dependent on the antibiotic you require as a range of antibiotic are used for bacterial selection in binary vectors. This product could be sourced from a&amp;nbsp; range of companies</td></tr><tr><td>50 ml Screw top tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td>The same product could be sourced from another company</td></tr><tr><td>2 ml Microtube</td><td>Watson Bio Lab</td><td>132-620C</td><td>The same product could be sourced from another company</td></tr><tr><td>MS Media</td><td>Sigma</td><td>M9274</td><td>The same product could be sourced from another company</td></tr><tr><td>Scalpel blade no. 11</td><td>Sigma</td><td>S2771</td><td>The same product could be sourced from another company</td></tr><tr><td>Parafilm &quot;M&quot;</td><td>Bemis</td><td>PM996</td><td>This is the best product to use to bind the cambial window post creation&amp;nbsp;</td></tr><tr><td>14 ml round bottom tubes</td><td>Thermo Scientific</td><td>150268</td><td>The same product could be sourced from another company</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td>The same product could be sourced from another company</td></tr><tr><td>Triton</td><td>Sigma</td><td>X100</td><td>The same product could be sourced from another company</td></tr><tr><td>X-Gluc</td><td>X-GLUC direct</td><td></td><td>You will need to go to the website to order - http://www.x-gluc.com/index.html</td></tr><tr><td>Potassium Ferricyanide(III)</td><td>Sigma</td><td>244023</td><td>The same product could be sourced from another company</td></tr><tr><td>Potassium Ferrocyanide(II)</td><td>Sigma</td><td>P9387</td><td>The same product could be sourced from another company</td></tr><tr><td>Litmus paper</td><td>Sigma</td><td>WHA10360300</td><td>The same product could be sourced from another company</td></tr><tr><td>Single edge razor blade</td><td>ProSciTech</td><td>L055</td><td>The same product could be sourced from another company</td></tr><tr><td>Double edge razor blade</td><td>ProSciTech</td><td>L056</td><td>The same product could be sourced from another company</td></tr><tr><td>SEM Pin Stub</td><td>ProSciTech</td><td>GTP16111</td><td>The same product could be sourced from another company</td></tr><tr><td>Sample vial with screw cap</td><td>ProSciTech</td><td>L6204</td><td>The same product could be sourced from another company</td></tr><tr><td>Ethanol</td><td>sigma</td><td>E7023</td><td>The same product could be sourced from another company</td></tr><tr><td>LR white</td><td>ProSciTech</td><td>C025</td><td>The same product could be sourced from another company</td></tr><tr><td>Embedding Mould</td><td>ProSciTech</td><td>RL090</td><td>We recommend this variety, however there are plenty of options available</td></tr><tr><td>Water Soulable mounting media</td><td>ProSciTech</td><td>IA019</td><td>One example of a mounting media that could be used however other options do exist and could be explored.</td></tr><tr><td>Hydrogen peroxide</td><td>Sigma</td><td>216763</td><td>A like product could be sourced from another company</td></tr><tr><td>Glacial acetic acid</td><td>Sigma</td><td>A9967</td><td>A like product could be sourced from another company</td></tr><tr><td>Safranin O</td><td>ProSciTech</td><td>C138</td><td>A like product could be sourced from another company</td></tr><tr><td>Quanta Environmental Scanning Electron Microscope</td><td>FEI</td><td></td><td>This is the instrument used at part of this study but any other SEM that has a low vacuum mode could be utilised</td></tr><tr><td>ImageJ imaging software</td><td></td><td></td><td>can be sourced from the following URL http://rsbweb.nih.gov/ij/</td></tr></tbody></table>

the use of induced somatic sector analysis (issa) for studying genes and promoters involved in wood formation and secondary stem development

在树上二级茎生长和相关的木材形成都来自生物和商业的角度显著。然而，相对知之甚少的支配其发展的分子控制。这部分是由于经常与二次生长过程的研究相关的物理，资源和时间的限制。许多体外技术已被用于涉及两个木质和非木质植物物种或植物部分或整个植物系统。然而，关于它们的适用性进行二次茎的生长过程的研究问题，某些物种和劳动强度的顽抗往往望而却步中高吞吐量的应用。此外，在寻找专干发展，木材形成，当被调查的具体特征可能只历经数年的增长成为衡量一棵树的生命周期的后期。在解决体内 p这些挑战替代rotocols被开发出来，命名为诱导体部门分析，涉及直接在工厂的专干转基因体组织部门的创建。该协议的目的是提供一种有效的，简单和相对快速的方法来创建用于基因和启动子的功能分析，可在一个范围内树种可以利用转基因植物次生组织中。这里提出的结果表明，转基因次级干扇区可以在所有活的组织和细胞类型中创建二次茎的各种树种的木材形态特征的以及在次要启动子表达模式茎可容易地评估促进中到高通量功能特性。

使用该协议的所有活二次干细胞和组织类型已被证明是容易A.农杆菌转化并已确定为根据最初转化的细胞类型行业类型和其后续发展的增长模式。行业类型包括周皮，韧皮部，形成层，伤口实质和纤基乙酸钠（ 图1b，1C，1D）和一致的位置将在本段的剩余部分描述可以发现。一个周皮扇区包括发现只在周皮和从不延伸到所述韧皮?...

Watch this Scientific Journal Video about The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development at JoVE.com

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

诱导体细胞行业分析（ISSA）的为研究木材形成和二次开发茎相关基因和推动者使用

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

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Research

JoVE Journal

Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

8.8K 次观看.  The University of Melbourne. Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

视频: The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

现代树轮研究技术的角度 - 如何克服Dendroecological和木解剖挑战

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

科研

环境科学

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

遗传学

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

非放射性原位挪威云杉杂交议定书适用的植物种类范围

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

生物学

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

发育生物学

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

使用浸泡珠测定分析鸡胚胎发生过程中的细胞分化，形态发生和图案化

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

植物启动子分析: 菜豆根瘤特异启动子的鉴定与表征

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

激光捕获微分块RNA-测序空间和时间组织特异性基因表达分析

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

植物细胞类型的分离和转录组分析

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...