Name: embryo rescue protocol for interspecific hybridization in squash
Uploaded: 2022-09-12T13:00:00
Description: Watch this Scientific Journal Video about Embryo Rescue Protocol for Interspecific Hybridization in Squash at JoVE.com

这项工作得到了美国农业部国家食品和农业研究所NRS项目编号的支持。FLA-TRC-006176和佛罗里达大学食品和农业科学研究所。

1. 种植和授粉
注意：重要的是要确定相容的基因型，其杂交将导致坐果和产生可行的胚胎。
<ol><li>种植条件和维护<ol><li>获得南瓜基因型（栽培品种/种质）的种子进行杂交（表1）。</li>
		<li>用含有 1.38 g/kg N、1.38 g/kg P 和 1.38 g/kg K 的完整 NPK 肥料改良的盆栽培养基填充 50 个细胞起始平面（25 厘米宽 x 50 厘米长）。</li>
		<li>播种到与其长度相等的深度...

<ol>
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A., Etman, A. A., Dewir, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Cucurbita+interspecific+hybrids+through+cross+pollination+and+embryo+rescue+technique.">Production of Cucurbita interspecific hybrids through cross pollination and embryo rescue technique.</a> World Applied Sciences Journal. 20 (10), 1366-1370 (2012).</li><li>Zhang, Q. I., Yu, E., Medina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+advanced+interspecific-bridge+lines+among+Cucurbita+pepo,+C.+maxima,+and+C.+moschata.">Development of advanced interspecific-bridge lines among Cucurbita pepo, C. maxima, and C. moschata.</a> HortScience. 47 (4), 452-458 (2012).</li><li>Brown, R. N., Bolanos-Herrera, A., Myers, J. R., Miller Jahn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+resistance+to+four+cucurbit+viruses+in+Cucurbita+moschata.">Inheritance of resistance to four cucurbit viruses in Cucurbita moschata.</a> Euphytica. 129 (3), 253-258 (2003).</li><li>Pachner, M., Paris, H. S., Winkler, J., Lelley, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+marker-assisted+pyramiding+of+genes+for+resistance+to+zucchini+yellow+mosaic+virus+in+oilseed+pumpkin+(Cucurbita+pepo).">Phenotypic and marker-assisted pyramiding of genes for resistance to zucchini yellow mosaic virus in oilseed pumpkin (Cucurbita pepo).</a> Plant Breeding. 134 (1), 121-128 (2015).</li><li>Hayase, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cucurbita-crosses.+XV.+Flower+pollination+at+4+am+in+the+production+of+C.+pepo+x+C.+moschata+F1+hybrids.">Cucurbita-crosses. XV. Flower pollination at 4 am in the production of C. pepo x C. moschata F1 hybrids.</a> Japanese Journal of Breeding. 13 (2), 76-82 (1963).</li><li>Reed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+rescue.">Embryo rescue.</a> Plant development and biotechnology. , 235-239 (2004).</li><li>Sharma, D. R., Kaur, R., Kumar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+rescue+in+plants-a+review.">Embryo rescue in plants-a review.</a> Euphytica. 89 (3), 325-337 (1996).</li><li>Wall, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecific+hybrids+of+Cucurbita+obtained+by+embryo+culture.">Interspecific hybrids of Cucurbita obtained by embryo culture.</a> Proceedings of the American Society of Horticultural Science. 63, 427-430 (1954).</li><li>Metwally, E. I., Haroun, S. A., El-Fadly, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecific+cross+between+Cucurbita+pepo+L.+and+Cucurbita+martinezii+through+in+vitro+embryo+culture.">Interspecific cross between Cucurbita pepo L. and Cucurbita martinezii through in vitro embryo culture.</a> Euphytica. 90 (1), 1-7 (1996).</li><li>Sisko, M., Ivancic, A., Bohanec, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+size+analysis+in+the+genus+Cucurbita+and+its+use+for+determination+of+interspecific+hybrids+obtained+using+the+embryo-rescue+technique.">Genome size analysis in the genus Cucurbita and its use for determination of interspecific hybrids obtained using the embryo-rescue technique.</a> Plant Science. 165 (3), 663-669 (2003).</li><li>Giancaspro, A., 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=Optimization+of+an+in+vitro+embryo+rescue+protocol+for+breeding+seedless+table+grapes+(Vitis+vinifera+L.)+in+Italy.">Optimization of an in vitro embryo rescue protocol for breeding seedless table grapes (Vitis vinifera L.) in Italy.</a> Horticulturae. 8 (2), 121 (2022).</li><li>Warchol, M., 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=The+effect+of+genotype,+media+composition,+pH+and+sugar+concentrations+on+oat+(Avena+sativa+L.)+doubled+haploid+production+through+oat+x+maize+crosses.">The effect of genotype, media composition, pH and sugar concentrations on oat (Avena sativa L.) doubled haploid production through oat x maize crosses.</a> Acta Physiologiae Plantarum. 40 (5), 1-10 (2018).</li><li>Nepi, M., Pacini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollination,+pollen+viability+and+pistil+receptivity+in+Cucurbita+pepo.">Pollination, pollen viability and pistil receptivity in Cucurbita pepo.</a> Annals of Botany. 72 (6), 527-536 (1993).</li><li>Harvey, W. J., Grant, D. G., Lammerink, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+sensory+changes+during+development+and+storage+of+buttercup+squash.">Physical and sensory changes during development and storage of buttercup squash.</a> New Zealand Journal of Crop and Horticultural Science. 25 (4), 341-351 (1997).</li><li>Moon, P., Meru, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+rescue+of+aged+Cucurbita+pepo+seeds+using+squash+rescue+medium.">Embryo rescue of aged Cucurbita pepo seeds using squash rescue medium.</a> Journal of Horticultural Science and Research. 2 (1), 62-69 (2018).</li><li>Nu&#241;ez-Palenius, H. G., Ram&#237;rez-Malag&#243;n, R., Ochoa-Alejo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muskmelon+embryo+rescue+techniques+using+in+vitro+embryo+culture.">Muskmelon embryo rescue techniques using in vitro embryo culture.</a> Plant Embryo Culture. , 107-115 (2011).</li><li>Vining, K. J., Loy, J. B., McCreight, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+development+and+seed+fill+in+hull-less+seeded+cultigens+of+pumpkin+(Cucurbita+pepo+L).">Seed development and seed fill in hull-less seeded cultigens of pumpkin (Cucurbita pepo L).</a> Cucurbitaceae 98: Evaluation and Enhancement of Cucurbit Germplasm. , 64-69 (1998).</li><li>Vining, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Seed development in hull-less-seeded pumpkin (Cucurbita pepo L.). , (1999).</li></ol>

在C. moschata和C. pepo之间成功的种间杂交有两个主要瓶颈：交叉相容性屏障，这是由产生杂交胚胎的基因型反应性决定的，以及受精后屏障，它阻碍杂交胚胎发育为正常种子。正如之前报道的南瓜一样，当前研究中的交叉相容性测试显示，大多数水果是单性生殖的，大多数种子无法存活16。亲本基因型对莫沙塔桔梗与桔梗种间杂交的相容性有较大影响。?...

葫芦（2n = 40）是葫芦科中一个高度多样化的属，包含27个不同的物种，其中5个被驯化1。其中，葫芦葫芦，C. pepo和C. maxima是全球最具经济重要性的。在美国，C. moschata和C. pepo是农业生产中最重要的两个物种。C. pepo 由四个亚种（卵形、卵形、花苔和古马拉）组成，包含弯颈、直颈、橡子、扇贝、椰子、蔬菜骨髓、西葫芦和南瓜2，3，4，5 的夏冬瓜品种组。C. moschata主要由冬瓜市场类型组成，包括胡桃，迪金森和奶酪组1。这两个物种在形态和表型上是多种多样的，C. pepo被认为是它的产量，早熟度，灌木生长习性以及多样化的果实性状，包括果实形状，果实大小，果肉颜色和外皮图案。另一方面，C. moschata因其对热量和湿度的适应以及抗病虫害而备受推崇6，7。C. moschata和C. pepo之间的种间杂交不仅是两个物种之间理想特征渗入的重要策略，而且还允许在育种计划中扩大遗传基础7，8。
C. moschata和C. pepo之间的早期杂交是为了确定它们的相容性和/或分类障碍9，10，11，而后来的研究主要集中在转移理想的性状12，13，14。两个物种之间的种间杂交旨在转移新性状，例如灌木或半灌木生长习性，提高C. pepo的产量以及抗病性，对非生物胁迫的适应性和增加C. moschata14，15，16的活力。例如，C. pepo（P5）和C. moschata（MO3）之间的特定杂交导致更高的果实产量13，而C. moschata种质（尼日利亚本地和Menina）已被广泛用作栽培C. pepo品种中对potyvirus抗性的主要来源17，18。
先前的研究表明，C. moschata和C. pepo之间的杂交是可能的，但很困难8，15。种间杂交可能导致没有坐果（流产），没有活种子的孤雌果实（空种子），未成熟胚胎无法发育的无籽果实（狭叶植物果实），或具有少量未成熟胚胎的果实可以通过胚胎救援拯救成成熟植物15，16。例如，通过杂交C. pepo（餐桌女王，母系）与C. moschata（大奶酪，父系）没有获得可行的种子，但是，互惠杂交从134次授粉中产生了57个可行的种子9。Hayase仅在上午04：00使用在10°C下储存过夜19的花粉进行杂交时，才从C. moschata和C. pepo杂交中获得活种子。Baggett将八个不同的C. moschata品种与C. pepo（熟食）杂交，并报告说，在103次总授粉中，获得了83种看起来正常的果实，但没有一个含有活种子8。在C. pepo（S179）和C. moschata（NK）的杂交中，Zhang等人获得了15个果实和2，994个种子，但其中只有12个种子是可行的，其余的只显示出基本的发育。这些研究表明，尽管C. moschata和C. pepo之间的种间杂交是非常有益的，但从杂交中获得具有可行种子的果实需要16。
胚胎抢救被认为是克服早期流产或发育不良引起的问题的适当方法，并且是最早和最成功的体外培养技术之一，用于再生未成熟胚胎16，20。胚胎挽救涉及对发育不足/未成熟的胚胎进行体外培养，然后转移到无菌营养培养基中，以促进幼苗和最终成熟植物的恢复21。虽然胚胎抢救通常用于南瓜育种，但缺乏对允许其常规应用的适当方法的详细描述。利用胚胎抢救技术克服葫芦种种间杂交障碍早在1954年就有报道22。然而，早期研究中胚胎抢救的成功率要么没有报道，要么非常低。Metwally等人报告说，从C. pepo和C. martinezii23杂交中分离出的100个种间杂交胚胎的成功率为10%（再生为成熟植物）。Sisko等人报道了从不同交叉组合获得的胚胎中胚胎再生的成功率不同：通过杂交C. maxima（Bos. Max）和C. pepo（淘金热）获得的杂交种的再生率为15.5%，C. pepo（西葫芦）和C. moschata（北海道）的再生率为20%，而C. pepo（淘金热）和C. moschata（Dolga）为37.5%24。 除基因型外，培养基和体外培养条件是该技术成功的重要因素25，26。在目前的研究中，测试了C. moschata和C. pepo之间的各种杂交组合，并开发了一种在南瓜中利用胚胎救援技术的简单方法。开发一种简单且易于复制的胚胎拯救技术将促进南瓜育种项目中的种间杂交和种质增强。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760-5</td>
			<td></td>
		</tr>
		<tr>
			<td>autoclave</td>
			<td>Steris</td>
			<td>AMSCO LAB 250</td>
			<td></td>
		</tr>
		<tr>
			<td>balance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cefotaxime</td>
			<td>Sigma Alfrich</td>
			<td>C 7039</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge tubes (1.5 ml)</td>
			<td>Sigma Alfrich</td>
			<td>T9661</td>
			<td></td>
		</tr>
		<tr>
			<td>detergent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol, 95%</td>
			<td>Decon Labs</td>
			<td>2805HC</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>VWR</td>
			<td>82027-408</td>
			<td></td>
		</tr>
		<tr>
			<td>gellan gum</td>
			<td>Caisson Laboratories</td>
			<td>G024</td>
			<td></td>
		</tr>
		<tr>
			<td>growth chamber or illuminated shelf</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>laminar hood / biosafety cabinet</td>
			<td>The Baker Company, Inc</td>
			<td>Edgegard</td>
			<td></td>
		</tr>
		<tr>
			<td>masking tape</td>
			<td>Uline</td>
			<td>S-11735</td>
			<td></td>
		</tr>
		<tr>
			<td>media bottle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog Medium</td>
			<td>Research Products International</td>
			<td>M10200</td>
			<td></td>
		</tr>
		<tr>
			<td>NPK fertilizer (20-20-20)</td>
			<td>BWI Companies, Inc</td>
			<td>&#160;PR200</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmocote Plus fertilizer</td>
			<td>BWI Companie,s Inc</td>
			<td>OS90590</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Sigma Alfrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (60 x 15 mm)</td>
			<td>USA Scientific, Inc</td>
			<td>8609-0160</td>
			<td></td>
		</tr>
		<tr>
			<td>plant pots</td>
			<td>BWI Companies, Inc</td>
			<td>NP4000BXL</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic food containers, reused</td>
			<td>Oscar Mayer</td>
			<td>4470003330</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic hang tags</td>
			<td>Amazon</td>
			<td>B07QTZRY6T</td>
			<td></td>
		</tr>
		<tr>
			<td>potting mix</td>
			<td>Jolly Gardener</td>
			<td>Pro-Line C/B</td>
			<td></td>
		</tr>
		<tr>
			<td>seedling starter trays</td>
			<td>BWI Companies Inc</td>
			<td>GPPF128S4</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe filter (0.22 um )</td>
			<td>ExtraGene</td>
			<td>B25CA022-S</td>
			<td></td>
		</tr>
		<tr>
			<td>trellis support</td>
			<td>The Home Depot</td>
			<td>&#160;2A060006</td>
			<td></td>
		</tr>
		<tr>
			<td>water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

embryo rescue protocol for interspecific hybridization in squash

葫芦作物（南瓜）的种间杂交对于扩大遗传变异和有用等位基因的渗入是理想的。由这些宽杂交产生的未成熟胚胎必须使用适当的胚胎修复技术再生。虽然这种技术在许多作物中已经很成熟，但缺乏对允许其常规应用的适当南瓜方法的详细描述。在这里，我们描述了一种胚胎救援方案，可用于C. pepo和C. moschata的种间杂交。为了确定胚胎救援的可行组合，进行了24个种间杂交。坐果从22个杂交中获得，表明成功率为92%。然而，获得的大多数果实都是单性生殖的，种子没有胚胎（空种子）。只有一个交叉组合包含可以使用基础植物生长培养基再生的未成熟胚胎。从种间F1果实中共抢救出10个胚胎，胚胎抢救成功率为80%。这里开发的胚胎救援方案将有助于南瓜育种项目中的种间杂交。

坐果和种子活力 进行了初步测试，以确定各种交叉组合中的坐果和种子活力。共选择15个南瓜基因型，4个C. pepo 和11个 C. moschata（表1）。在尝试的24种种间交叉组合中，有22种坐果（表2），坐果总体成功率为>92%。杂交O和M以及E和J未获得成熟果实，而杂交F和J获得的果实数量最多（n = 6）（表2）。不同杂交组合授粉的花朵数量从1到1...

Watch this Scientific Journal Video about Embryo Rescue Protocol for Interspecific Hybridization in Squash at JoVE.com

Embryo Rescue Protocol for Interspecific Hybridization in Squash

本文描述了一种胚胎救援方案，用于再生源自 葫芦 和葫芦 葫芦的种间杂交的未成熟胚胎。该协议可以很容易地复制，并将成为南瓜育种计划的重要资源。

南瓜种间杂交的胚胎救援方案

Interspecific hybridization in Cucurbita crops (squash) is desirable for widening genetic variation and for the introgression of useful alleles. Immature ...

胚胎救援方案对于来自不同葫芦物种之间的种间杂交的未成熟胚胎的再生是强制性的。它最常用于葫芦和葫芦之间的杂交。这种技术的主要优点是它的简单性。该方案仅使用含有抗生素的MS培养基。因此，它可以很容易地被任何实验室复制。可以修改这种技术以研究种间或属间杂交的不同障碍。在每一步中注意细节将有助于取得成功的结果。首先种植葫芦植物，使用补充有完整氮、磷和钾肥料的盆栽培养基填充 50 个 25 x 50 厘米的细胞起始平面，每公斤含有 1.38 克氮、磷和钾。接下来，将种子播种到与其长度相等的深度，并用盆栽介质覆盖它们。在不产生积水的情况下给平房浇水，并通过每天浇水一次来保持介质湿润。在第二个真叶阶段结束时，将幼苗移植到直径30厘米的盆中，每盆补充三汤匙完全肥料。每七天后，每盆用500毫升液体肥料施肥，其中含有20，20，20氮，磷和钾，一克加入一加仑水中。在自然光照条件下，将植物保持在温室中的温度在 22 至 28 摄氏度之间。对于葡萄基因型，请在温室中提供支撑棚架。在植物播种后六到八周开始开花后立即进行受控杂交或授粉。检查花瓣上黄色的未开封的花朵，并识别葫芦和葫芦品种的雄花和雌花。为防止意外昆虫授粉，请使用遮蔽胶带轻轻地将闭合的花朵粘在顶部。第二天早上，在上午10：00之前轻轻去除雄花和雌花的胶带顶部进行授粉。将花瓣与雄花分开，并通过轻轻摩擦花药在雌花柱上来转移花粉。授粉后，立即用遮蔽胶带关闭授粉的雌花，并用标签记录授粉日期并注明十字架中使用的父父母和母父母。由扩张的子房指示的成功杂交将在一周内形成一个小果实。授粉45至55天后收获果实。准备头孢噻肟原液。通过无菌0.22微米注射器过滤器过滤。并制成每毫升头孢噻肟 0.5 毫升 250 毫克的等分试样，然后将它们储存在零下 20 摄氏度。同样，准备氨苄西林原液。通过无菌0.22微米注射器过滤器过滤，并制备一毫升每毫升氨苄青霉素100毫克的等分试样，然后将其储存在零下20摄氏度。通过将 2.45 克培养基溶解在 1 升瓶中的 500 毫升蒸馏水中来制备 Murashige 和 Skoog 或 MS 培养基。在MS培养基中加入1.5克结冷胶，并在121摄氏度下高压灭菌20分钟。通过将瓶子放入 50 摄氏度的水浴中来冷却培养基。解冻层流柜内的抗生素储备。将培养基瓶转移到层流罩中，并在适当混合的情况下向培养基中加入 0.6 毫升头孢噻肟原液和 0.25 毫升氨苄西林原液。将约7毫升培养基倒入60×15毫米的无菌培养皿中。让培养基在培养皿中凝固约 15 至 20 分钟。用密封膜关闭培养皿，并将它们放入室温下的储存盒中，直到进一步使用。通过将南瓜果实从主藤上切下来收获，并使用 0.3% 氯二酚等液体洗涤剂在实验室水槽中清洗其表面来对水果进行消毒。用充足的自来水冲洗水果，并用干净的纸巾擦干，然后将水果转移到层压板气流柜中。通过喷洒70%乙醇对柜内的水果表面进行消毒。用无菌刀将果实一分为二并提取种子。使用无菌镊子或戴无菌手套的手无菌打开种皮并暴露未成熟的胚胎，然后将其放入含有抗生素补充的MS培养基的培养皿中。使用包装膜密封培养皿，并将它们置于温度为25摄氏度，16小时光周期和70%相对湿度的生长室中。在污染的情况下，立即将未受污染的胚胎传代培养到具有相同培养基的新平板中。一旦根在 10 到 14 天后发育，子叶在 15 到 21 天后发育，从培养皿中取出小苗，然后用自来水轻轻洗掉根部周围的培养基。将清洁的幼苗放入 14 x 9 x 4 厘米的塑料容器中，并用湿纸巾覆盖根部。盖上容器，必要时重新弄湿纸巾。将塑料容器保持在 25 至 28 摄氏度，并在 16 小时内拍照，以使小苗适应环境，同时保持纸巾的湿润状态。适应环境 7 到 10 天后，将 3 到 4 厘米长的幼苗转移到 50 个包含用肥料改良的商业盆栽混合物的单元中，并将它们移动到温室中。避免过度浇水以防止腐烂，并根据需要为每个细胞添加大约 10 至 20 毫升水。在第二至第三真叶阶段，将幼苗移植到直径25厘米的盆中，盆中装满用肥料改良的盆栽培养基，并在植物开始开花时进行受控杂交。在自然光照条件下，将温室中的植物保持在 22 至 28 摄氏度。并评估植物的果实和种子特性。选择4株葫芦和11株葫芦进行种间杂交。在尝试的24个种间杂交组合中，22个杂交获得了坐果，坐果成功率超过92%。葫芦和葫芦之间的杂交涉及基因型O和M，基因型E和J，没有产生成熟的果实。而葫芦和葫芦之间的杂交涉及基因型F和J，产生了6种果实。不同杂交组合授粉的花朵数量从1到11不等。授粉成功率在0%-100%之间 在所有杂交组合产生的44个果实中，只有1个来自葫芦和葫芦杂交的果实，基因型C和J产生了可以挽救的未成熟胚胎。对于这些发育不良的胚胎，使用胚胎救援培养基进行培养，从而使胚胎救援成功率为80%。两个最重要的程序是识别两个物种之间的相容杂交，即葫芦和葫芦，以及成功的胚胎再生。由于胚胎抢救技术简单且易于复制，世界各地的南瓜育种者可以使用这种技术来拯救来自不同葫芦物种之间的种间杂交的未成熟胚胎。

Research

JoVE Journal

Biology

Double Whole Mount in situ Hybridization of Early Chick Embryos

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Assay for Neural Induction in the Chick Embryo

在鸡胚神经诱导的含量为

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

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

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Fluorescence in situ hybridization FISH Protocol in Human Sperm

荧光原位杂交（FISH）的议定书“在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

In Situ Hybridization for the Precise Localization of Transcripts in Plants

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...