这项工作得到了聊城大学研究基金（318012028）和山东省自然科学基金（ZR2020MC034）的支持。

1. 植物生长条件与根茎培养 
<ol><li>播种 注：野生大豆种子采自中国聊城阳谷县;美洲南瓜和 南 瓜当地品种银苏的种子是从市场上购买的。<ol><li>收集野生大豆，美洲南瓜和当地 南瓜品种的种子，将它们播种在1厘米深的蛭石中，并彻底浇水。将 20 颗种子种植在 8 厘米 x 11 厘米 x 9 厘米的塑料盒中。在24±2°C的生长室中以约70%相对湿度进行16小时光照/ 8小时黑暗循环培养植物。 注意：播种前必须打破野生大豆种子的外套。如果研究问题集中在植物和微生物之间的相互作用上，种子、蛭石、塑料盒和水在使用前必须进行消毒。</li>
		<li>根茎曲霉K599的活化和培养 注意：根茎A. rhizogenes K599菌株有一个二元载体pRed13052 携带红色荧光报告基因 DsRed2。
		<ol><li>从-80°C冰箱中取出根 茎曲霉 菌株K599，并在固体LB培养基（用50mg / L卡那霉素和50mg / L链霉素）上在28°C下活化细菌48小时。</li>
			<li>挑选菌株K599的单个克隆，并将其在1mL含液体抗生素的LB培养基中培养12小时。</li>
			<li>将500μL细菌悬浮液均匀地散布在含有固体抗生素的LB培养基上，然后在28°C下孵育24小时。</li>
		</ol></li>
	</ol></li>
</ol>2. 一步法 A . 根茎介导的毛根转化方法
<ol><li>下胚轴切口<ol><li>7天后（将种子的播种定义为第0天），幼苗子叶刚刚展开（图1A），使用无菌的锋利手术刀切割约0.5-1厘米的下胚轴（图1B）。丢弃主根和部分下胚轴。 注意：请小心使用手术刀。</li>
	</ol></li>
	<li>K599接种<ol><li>用K599细菌涂覆下胚轴切口（图1C）。</li>
		<li>将幼苗种植在潮湿的蛭石中（图1D）。</li>
		<li>通过根 茎介导的毛根转化感染每个物种的 30 株植物。在四分之一强度（0.25x）Gamborg B-5碱性培养基中用5mL重悬的K599细菌悬浮液（OD600 = 0.5-0.6）浇灌每株植物（图1E）。</li>
		<li>用高度透明的塑料袋（图1F）盖住花盆，并将它们放入生长室中。</li>
	</ol></li>
</ol>3.毛根生产
<ol><li>接种后种植植物~12天，将在切口部位诱导并产生新的毛根。15天后，毛根长度通常达到2-5厘米（图2）。</li>
	<li>为了确定产生的毛根是否是转基因的，检查依赖于K599中转化载体的报告基因的表达。<ol><li>使用化学发光成像系统检测报告基因DsRed2的表达，绿色激发光在540nm处，发射波长为600nm（图2B，图2D和图2F）。</li>
	</ol></li>
</ol>

一步法毛状根转化方法推进根相关生物学

<ol>
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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochrome+B+inhibits+darkness-induced+hypocotyl+adventitious+root+formation+by+stabilizing+IAA14+and+suppressing+ARF7+and+ARF19.">Phytochrome B inhibits darkness-induced hypocotyl adventitious root formation by stabilizing IAA14 and suppressing ARF7 and ARF19.</a> The Plant Journal: For Cell and Molecular Biology. 105 (6), 1689-1702 (2021).</li></ol>

作者没有利益冲突需要声明。

一步法根茎介导的毛根法是比下胚轴注射法更简单、更有效的复合植物生产方法。一步法ARM方法显著提高了毛根转化效率，缩短了产生毛根的时间，增加了毛根的数量，减少了所涉及的工作量。改进的转化方案最适合研究豆科植物与根瘤菌之间以及植物与丛枝菌根真菌之间的共生关系。这可以归因于这样一个事实，即在产生毛根后不需要移植复合植物，这避免了移植过程中发生的非接种菌?...

根茎农杆菌是根茎科革兰氏阴性土壤细菌。 A. rhanzogenes可以通过伤口感染几乎所有的双子叶植物，少数单子叶植物和单个裸子植物，在受感染的植物中产生毛根。细菌携带Ri（发根）质粒，Ri质粒的T-DNA携带opine合成基因和rol基因（根位点基因）。Ri质粒的T-DNA进入植物细胞并整合到宿主染色体中后，rol基因的表达诱导毛根的产生1。携带靶基因的植物二元表达载体转化为根茎曲霉，转化后的根茎曲霉用于感染植物。转基因根可以在受感染的植物中诱导，产生含有转基因根和非转基因茎和芽的复合植物。通常，复合植物可以在14-20天内获得。根茎介导的毛根转化在双子叶植物中一般不受基因型的限制2。根茎感染植物产生的毛根具有生长速度快、遗传稳定、操作方便等特点。由根茎A. rhizogene介导的毛根转化目前广泛用于研究与根相关的生物学。此外，毛根的转化也可用于验证和优化CRISPR/Cas9系统3,4,5和蛋白质亚细胞定位的靶点编辑效率。因此，毛根转化是研究植物基因功能、代谢工程以及根与根际微生物相互作用的重要工具6,7,8。
通过毛根转化获得的含有转基因根的复合植物已在双子叶植物中广泛生产，特别是在豆科植物中。将下胚轴注射根茎曲霉的传统方法已被用于生产复合莲花9，大豆10，番茄11，甘薯12和许多其他植物5,8。下胚轴注射方法效率低下，可能导致幼小或微小的下胚轴植物死亡。因此，通过切断胚根，用根茎涂覆幼苗切口，然后将下胚轴置于无菌培养基上进行生根栽培来改进该方法13。但是，这些步骤是在无菌环境中进行的，并且操作步骤相对繁琐。特别是，由此产生的复合植物需要移植，这增加了工作量。在以前的工作中，在黄瓜、大豆、粳米莲、芥芷和番茄2,14,15,16,17中建立了一步法根茎介导的（ARM）毛根转化。去除主根和部分下胚轴，在剩余下胚轴的切口部位涂覆转化根茎，然后将幼苗种植到湿润的无菌蛭石中。栽培12天后，切口部位产生毛根。一步法 ARM 方法非常高效，产生毛根所需的时间更少。形成毛根后移植也是没有必要的。由于无需移植即可避免微生物污染，因此一步法ARM方法在研究植物与微生物之间的相互作用时特别有用，例如豆科植物与根瘤菌之间的共生固氮，以及植物与丛枝菌根真菌之间的共生。本文提供了详细的一步法根茎介导的毛根转化方案，并以野生大豆、美洲茄和南瓜中生产的复合植物为例。使用该协议，研究人员可以顺利地执行一步式ARM转换。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>kanamycin</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A506636</td>
			<td></td>
		</tr>
		<tr>
			<td>LB medium</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>B540113</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic box</td>
			<td>LiaoSu</td>
			<td></td>
			<td>8 cm x 11 cm x 9 cm</td>
		</tr>
		<tr>
			<td>pumpkin</td>
			<td></td>
			<td></td>
			<td>local variety Yinsu</td>
		</tr>
		<tr>
			<td>streptomycin</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A610494&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tanon-5200Multi machine</td>
			<td>Tanon Co., Ltd., China</td>
			<td>5200Multi</td>
			<td>chemiluminescence imaging system</td>
		</tr>
		<tr>
			<td>tomato</td>
			<td></td>
			<td></td>
			<td>local variety Zhongshu4</td>
		</tr>
		<tr>
			<td>wild soybean</td>
			<td></td>
			<td></td>
			<td>collected in Yanggu County, Liaocheng, China</td>
		</tr>
	</tbody>
</table>

an efficient and reproducible method for producing composite plants by agrobacterium rhizogenes-based hairy root transformation

使用根茎农杆菌介导的毛根转化生产具有转基因根和非转基因茎芽的复合植物是研究 根相关生物学的有力工具。毛根转化在广泛的双子叶植物和几种单子叶植物中建立，并且几乎独立于基因型。传统的下胚轴注射根 茎得到 复合植株的方法效率低下、耗时、费力，且经常导致嫩小下胚轴植株死亡。此前建立了以根 茎曲霉 介导的高效一步法毛根转化，无需在产生毛根后进行移植。本研究切除部分下胚轴和主根，下胚轴切口部位涂有 根茎曲霉， 然后将下胚轴种植在无菌蛭石中。栽培12天后，下胚轴切口扩大，诱导新的毛根。本文提供了由 根茎介导的一步转化方法的详细方案，其有效性通过生产野生大豆、 美洲茄和南瓜的复合植物来证明。

高效一步法根 茎介导的毛根转化 在接种工程K599后12天，在下胚轴切口部位产生毛根。根据二元载体中所含报告基因的表达确定转基因毛根。在自然（图2A、图2C和图2E）和绿色激发光（图2B、图2D和 图2F）下观察用复合<strong cl...

Watch this Scientific Journal Video about Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation at JoVE.com

作者聚焦：通过根瘤农杆菌介导的毛状根转化简化复合植物生产 

在这里，我们提供了由 根癌农杆菌 介导的一步转化方法的详细协议，以生产复合植物。

一种基于根 茎农杆菌毛根转化生产复合植物的高效且可重复的方法

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...

我们的团队专注于基于根茎农杆菌的毛根转化。毛茸茸的根转化对于研究植物基因功能、代谢工程以及根与根际微生物之间的相互作用至关重要。本文提供了一种由根茎农杆菌介导的一步转化方法。毛根转化在各种双子叶和单子叶植物种中建立，并且几乎与基因型独立。与下胚轴注射方法相比，根茎农杆菌介导的一步法用于诱导毛根，在创建复合植物方面更简单、更有效。下胚轴注射方法效率低下，可能导致幼小或微小的下胚轴植物死亡。一步法根茎农杆菌介导的方法非常有效，需要更少的时间来产生毛根。而且，没有必要移植过去毛根的形成。α-征调控基因和β-征合成基因作为根茎农杆菌介导的毛根转化的病毒筛选标志物。

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An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

1.8K 次观看.  Liaocheng University. 我们的团队专注于基于根茎农杆菌的毛根转化。毛茸茸的根转化对于研究植物基因功能、代谢工程以及根与根际微生物之间的相互作用至关重要。本文提供了一种由根茎农杆菌介导的一步转化方法。毛根转化在各种双子叶和单子叶植物种中建立，并且几乎与基因型独立。与下胚轴注射方法相比，根茎农杆菌介导的一步法用于诱导毛根，在创建复合植物方面更简单、更有?...

视频: 作者聚焦：通过根瘤农杆菌介导的毛状根转化简化复合植物生产 

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

科研

生物学

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

三维球体制作一个高效，灵活细胞聚集方法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

用于映射植物根系架构特征的简单协议

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the &#946;-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Because of its versatile application as a model species in various fields of study, there is a need for a genetic transformation toolkit in narrowleaf plantain (Plantago lanceolata). Here, using Agrobacterium tumefaciens-mediated transformation, a protocol is presented that results in stable transgenic lines with a transformation efficiency of 20%.

根癌农杆菌介导的窄叶车前草遗传转化

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack ...

在植物中:使用根癌农杆菌瞬时转化可可植物

Vacuum-Forced Agroinfiltration for In planta Transformation of Recalcitrant Plants: Cacao as a Case Study

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation rely on the use of Agrobacterium-mediated transformation. However, the protocols currently in use are standardized for small-sized plants due to the physical and economic constraints of submitting large-sized plants to a vacuum treatment. This work presents an effective protocol for localized vacuum-based agroinfiltration customized for large-sized plants. To assess the efficacy of the proposed method, we tested its use in cacao plants, a tropical plant species recalcitrant to genetic transformation. Our protocol allowed applying up to 0.07 MPa vacuum, with repetitions, to a localized aerial part of cacao leaves, making it possible to force the infiltration of Agrobacterium into the intercellular spaces of attached leaves. As a result, we achieved the Agrobacterium-mediated transient in planta transformation of attached cacao leaves expressing for the RUBY reporter system. This is also the first Agrobacterium-mediated in planta transient transformation of cacao. This protocol would allow the application of the vacuum-based agroinfiltration method to other plant species with similar size constraints and open the door for the in planta characterization of genes in recalcitrant woody, large-size species.

作者聚焦:大型木本植物中无组织培养的 农杆菌介导的转化

Here, we present the first protocol for localized vacuum infiltration for in vivo studies of the genetic transformation of large-sized plants. Using this methodology, we achieved for the first time the Agrobacterium-mediated in planta transient transformation of cacao.

真空强制农渗在 植物 内顽固植物转化中的应用——以可可为例

Transient in planta transformation is a fast and cost-effective alternative for plant genetic transformation. Most protocols for in planta transformation ...

农杆菌介导的植物毛状根诱导和再生

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

作者聚焦:优化基于毛状根的转化方案以提高十字花科植物的效率

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.

一种基于根 茎农杆菌毛根转化生产复合植物的高效且可重复的方法

一种基于根茎农杆菌毛根转化生产复合植物的高效且可重复的方法