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

This work was supported by the USDA&#160;National Institute of Food and Agriculture, NRS Project No. FLA-TRC-006176 and the University of Florida Institute of Food and Agricultural Sciences.

1. Planting and pollination
NOTE: It is important to identify compatible genotypes whose hybridization would result in fruit set&#160;and the production of viable embryos.
<ol>
	<li>Planting conditions and maintenance
	<ol>
		<li>Obtain seeds of squash genotypes (cultivars/accessions) for hybridization (Table 1).</li>
		<li>Fill 50 cell starting flats (25 cm width x 50 cm length) with potting medium amended with complete NPK fertilizer containing 1.38 g/kg N...

<ol>
	<li>Paris, H. S., Grumet, R., Katzir, N., Garcia-Mas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Resources+of+Pumpkins+and+Squash,+Cucurbita+spp.">Genetic Resources of Pumpkins and Squash, Cucurbita spp.</a> Genetics and Genomics of Cucurbitaceae. Plant Genetics and Genomics: Crops and Models. 20, (2016).</li><li>Gong, L., Stift, G., Kofler, R., Pachner, M., 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=Microsatellites+for+the+genus+Cucurbita+and+an+SSR-based+genetic+linkage+map+of+Cucurbita+pepo+L.">Microsatellites for the genus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepo L.</a> Theoretical and Applied Genetics. 117 (1), 37-48 (2008).</li><li>Paris, H. S., 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=Assessment+of+genetic+relationships+in+Cucurbita+pepo+(Cucurbitaceae)+using+DNA+markers.">Assessment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using DNA markers.</a> Theoretical and Applied Genetics. 106 (6), 971-978 (2003).</li><li>Robinson, R. W., Decker-Walters, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cucurbits. , (1997).</li><li>Teppner, 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+pepo+(Cucurbitaceae)-history,+seed+coat+types,+thin+coated+seeds+and+their+genetics.">Cucurbita pepo (Cucurbitaceae)-history, seed coat types, thin coated seeds and their genetics.</a> Phyton (Horn). 40 (1), 1-42 (2000).</li><li>Hazra, P., Mandal, A. K., Dutta, A. K., Ram, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+pumpkin+(Cucurbita+moschata+Duch.+Ex+Poir.)+for+fruit+yield+and+other+characters.">Breeding pumpkin (Cucurbita moschata Duch. Ex Poir.) for fruit yield and other characters.</a> International Journal of Plant Breeding. 1 (1), 51-64 (2007).</li><li>Paris, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+the+cultivar-groups+of+Cucurbita+pepo.">History of the cultivar-groups of Cucurbita pepo.</a> Horticultural Reviews-Westport Then New York. 25, 71 (2001).</li><li>Baggett, 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=Attempts+to+cross+Cucurbita+moschata+(Duch.)+Poir.+'Butternut+and+C.+pepo+L.+'Delicata'.">Attempts to cross Cucurbita moschata (Duch.) Poir. 'Butternut and C. pepo L. 'Delicata'.</a> The Cucurbit Genetics Cooperative. 2, 32-34 (1979).</li><li>Erwin, A. T., Haber, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+and+Varietal+Crosses+in+Cucurbits.">Species and Varietal Crosses in Cucurbits.</a> Agricultural Experiment Station, Iowa State College of Agriculture and Mechanical Arts. , (1929).</li><li>Whitaker, T. W., Bohn, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+taxonomy,+genetics,+production+and+uses+of+the+cultivated+species+of+Cucurbita.">The taxonomy, genetics, production and uses of the cultivated species of Cucurbita.</a> Economic Botany. 4 (1), 52-81 (1950).</li><li>Bemis, W. P., Nelson, 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=Interspecific+hybridization+within+the+genus+Cucurbita+I,+fruit+set,+seed+and+embryo+development.">Interspecific hybridization within the genus Cucurbita I, fruit set, seed and embryo development.</a> Journal of the Arizona Academy of Science. 2 (3), 104-107 (1963).</li><li>Washek, R. L., Munger, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybridization+of+Cucurbita+pepo+with+disease+resistant+Cucurbita+species.">Hybridization of Cucurbita pepo with disease resistant Cucurbita species.</a> The Cucurbit Genetics Cooperative. 6, 92 (1983).</li><li>Davoodi, S., Olfati, J. A., Hamidoghli, Y., Sabouri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+heterosis+in+Cucurbita+moschata+and+Cucurbita+pepo+interspecific+hybrids.">Standard heterosis in Cucurbita moschata and Cucurbita pepo interspecific hybrids.</a> International Journal of Vegetable Science. 22 (4), 383-388 (2016).</li><li>De Oliveira, A. C. B., Maluf, W. R., Pinto, J. E. B., Azevedo, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+papaya+ringspot+virus+in+summer+squash+Cucurbita+pepo+L.+introgressed+from+an+interspecific+C.+pepo&#215;+C.+moschata+cross.">Resistance to papaya ringspot virus in summer squash Cucurbita pepo L. introgressed from an interspecific C. pepo&#215; C. moschata cross.</a> Euphytica. 132 (2), 211-215 (2003).</li><li>Rakha, M. T., Metwally, E. I., Moustafa, S. 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>

There are two main bottlenecks for successful interspecific hybridization between C. moschata and C. pepo: cross-compatibility barrier, which is determined by genotype responsiveness to produce hybrid embryos, and post-fertilization barriers, which hinder the development of hybrid embryos to normal seeds. As previously reported for squash, the cross-compatibility test in the current study revealed that most of the fruit developed parthenocarpically, with most of the seeds unviable16</s...

Cucurbita (2n = 40) is a highly diverse genus in the Cucurbitaceae family that contains 27 different species, of which five are domesticated1. Among these, Cucurbita moschata, C. pepo, and C. maxima are the most economically important worldwide. In the U.S., C. moschata and C. pepo are the two most important species in agricultural production. C. pepo consists of four subspecies (ovifera, pepo, fraternal, and gumala) that contain both summer and winter squash cultivar groups of crookneck, straightneck, acorn, scallop, cocozelle, vegetable marrow, zucchini, and pumpkin2,3,4,5. C. moschata primarily consists of winter squash market types including butternut, Dickinson, and cheese group1. The two species are morphologically and phenotypically diverse, with C. pepo regarded for its yield, earliness, bush growth habit, and diverse fruit traits including fruit shape, fruit size, flesh color, and rind pattern. On the other hand, C. moschata is prized for its adaptation to heat and humidity, as well as disease and pest resistance6,7. Interspecific hybridization between C. moschata and C. pepo is not only an important strategy for introgression of desirable characteristics between the two species, but also allows for the broadening of the genetic base in breeding programs7,8.
Early crosses between C. moschata and C. pepo were made to determine their compatibility and/or taxonomic barriers9,10,11, whereas later studies mostly focused on transferring desirable traits12,13,14. Interspecific hybridization between the two species has targeted the transfer of novel traits such as a bush or semi-bush growth habit and improved yield from C. pepo along with disease resistance, adaptability to abiotic stress, and increased vigor from C. moschata14,15,16. For example, specific crosses between C. pepo (P5) and C. moschata (MO3) have resulted in higher fruit yield13, while C. moschata accessions (Nigerian Local and Menina) have been widely used as the primary source of resistance to potyviruses in cultivated C. pepo cultivars17,18.
Previous studies showed that hybridization between C. moschata and C. pepo is possible but difficult8,15. The interspecific crosses could result in no fruit set (abortion), parthenocarpic fruits devoid of viable seeds (empty seeds), seedless fruits where the immature embryos fail to develop (stenospermocarpy), or fruits with few immature embryos that can be rescued into mature plants through embryo rescue15,16. For instance, no viable seeds were obtained by crossing C. pepo (table queen, maternal) with C. moschata (large cheese, paternal), however, the reciprocal cross yielded 57 viable seeds from 134 pollinations9. Hayase obtained viable seeds from C. moschata and C. pepo crosses only when crosses were made at 04:00 a.m. using pollen stored at 10 &#176;C overnight19. Baggett crossed eight different C. moschata varieties with C. pepo (delicata) and reported that out of 103 total pollinations, 83 fruits were obtained that appeared normal, but none of them contained viable seeds8. In a cross between C. pepo (S179) and C. moschata (NK), Zhang et al. obtained 15 fruits with 2,994 seeds, but only 12 of those seeds were viable while the remaining displayed only rudimentary development. These studies suggest that even though interspecific crossing between C. moschata and C. pepo is highly beneficial, obtaining fruits with viable seeds from the crosses is demanding16.
Embryo rescue has been suggested as an appropriate method to overcome problems arising from early aborting or poorly developed embryos and is one of the earliest and most successful in vitro culture techniques for regeneration of immature embryos16,20. Embryo rescue involves the in vitro culture of under-developed/immature embryos followed by transfer to a sterile nutrient medium to facilitate recovery of seedlings and ultimately mature plants21. Although embryo rescue is commonly used in squash breeding, a detailed description of the appropriate methodology that would allow its routine application is lacking. Using embryo rescue technique to overcome interspecific hybridization barriers in Cucurbita species was reported as early as 195422. However, the success of embryo rescue in the early studies was either unreported or very low. Metwally et al. reported a 10% success rate (regeneration into mature plants) among 100 interspecific hybrid embryos rescued from a cross between C. pepo and C. martinezii23. Sisko et al. reported a variable success rate of embryo regeneration among embryos obtained from different cross combinations: the regeneration rate of hybrids obtained by crossing C. maxima (Bos. Max)and C. pepo (Gold Rush) was 15.5%, for C. pepo (Zucchini) and C. moschata (Hokaido) was 20%, while for C. pepo (Gold Rush) and C. moschata (Dolga) it was 37.5%24. In addition to genotype, media and in vitro culture conditions are important factors for the success of the technique25,26. In the current study, various cross combinations between C. moschata and C. pepo were&#160;tested, and a simple methodology for utilizing the embryo rescue technique in squash was developed. The development of a simple and easily reproducible embryo rescue technique will facilitate interspecific hybridization and germplasm enhancement in squash breeding programs.

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		<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

Interspecific hybridization in Cucurbita crops (squash) is desirable for widening genetic variation and for the introgression of useful alleles. Immature embryos generated from these wide crosses must be regenerated using appropriate embryo rescue techniques. Although this technique is well established for many crops, a detailed description of the appropriate methodology for squash that would allow its routine application is lacking. Here, we describe an embryo rescue protocol useful for interspecific hybridization of C. pepo and C. moschata. To identify viable combinations for embryo rescue, 24 interspecific crosses were performed. Fruit set was obtained from twenty-two crosses, indicating a 92% success rate. However, most of the fruits obtained were parthenocarpic, with seeds devoid of embryos (empty seeds). Only one cross combination contained immature embryos that could be regenerated using basal plant growth media. A total of 10 embryos were rescued from the interspecific F1 fruit, and the success rate of embryo rescue was 80%. The embryo rescue protocol developed here will be useful for interspecific hybridization in squash breeding programs.

Fruit set and seed viability 
An initial test was conducted to determine fruit set and seed viability in a variety of cross-combinations. A total of 15 squash genotypes, four C. pepo and 11 C. moschata, were chosen (Table 1). Out of the 24 interspecific cross combinations attempted, a fruit set was obtained for 22 (Table 2), representing an overall &#62;92% success in the fruit set. No mature fruits were obtained by crossing O and M and E and J, wh...

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Embryo Rescue Protocol for Interspecific Hybridization in Squash

The&#160;article describes an embryo rescue protocol for the regeneration of immature embryos derived from the interspecific hybridization of Cucurbita pepo and Cucurbita moschata. The protocol can be easily replicated and will be an important resource for squash breeding programs.

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

The embryo rescue protocol is mandatory for regeneration of immature embryos derived from interspecific crosses between different Cucurbita species. It is most commonly used for crosses between Cucurbita moschata and Cucurbita pepo. The main advantage of this technique is its simplicity.The protocol uses only MS media with antibiotics. Hence, it can be easily replicated by any lab. It would be possible to modify this technique to investigate different barriers to interspecific or intergeneric hybridization.Paying attention to the details during every step will be helpful for a successful outcome. Begin with planting the Cucurbita plants by filling 50 cell starting flats of 25 by 50 centimeters using potting medium supplemented with complete nitrogen, phosphorus and potassium fertilizer, containing 1.38 grams per kilogram of nitrogen, phosphorus, and potassium each. Next, sow the seeds to a depth equal to their length and cover them with the potting medium.Water the flats without creating standing water and keep the media moist by watering them once per day. At the end of the second true leaf stage, transplant the seedlings into 30-centimeter diameter pots supplemented with three tablespoons of complete fertilizer per pot. After every seven days, fertilize each pot with 500 milliliters of liquid fertilizer containing 20, 20, 20 nitrogen, phosphorus and potassium in one gram added to a gallon of water.Maintain plants in the greenhouse at temperatures between 22 to 28 degrees Celsius under the natural light regime. For vining genotypes, provide a supporting trellis in the greenhouse. Perform the controlled hybridization or pollination as soon as the plants start flowering at six to eight weeks from the seeding.Check the unopened flowers having a yellow hue on the petals, and identify male flowers and female flowers of Cucurbita pepo and Cucurbita moschata cultivars. To prevent accidental insect pollination, gently tape the closed flower at the top using masking tape. The following morning, carry out the pollination before 10:00 AM by gently removing the taped top portion of the male and female flower.Separating the petals from the male flower, and transferring the pollens by gently rubbing the anther on the female flower stigma. After pollination, immediately close the pollinated female flower with masking tape, and use a tag to record the date of pollination and to indicate the paternal and maternal parents used in the cross. A successful cross indicated by an expanded ovary will form a small fruit within one week.Harvest the fruit after 45 to 55 days of pollination. Prepare a cefotaxime stock. Filter it through a sterile 0.22 micron syringe filter.And make 0.5 milliliter aliquots of 250 milligrams per milliliter cefotaxime before storing them at minus 20 degrees Celsius. Similarly, prepare ampicillin stock. Filter it through a sterile 0.22 micron syringe filter and prepare one milliliter aliquots of 100 milligrams per milliliter ampicillin before storing them at minus 20 degrees Celsius.Prepare Murashige and Skoog, or MS medium by dissolving 2.45 grams of the medium in 500 milliliters of distilled water in a one liter bottle. Add 1.5 grams of gellan gum to the MS medium and autoclave it at 121 degrees Celsius for 20 minutes. Cool down the medium by placing the bottle in a water bath at 50 degrees Celsius.Thaw the antibiotic stocks inside the laminar flow cabinet. Transfer the medium bottle to the laminar flow hood and add 0.6 milliliters of cefotaxime stock and 0.25 milliliters of ampicillin stock to the medium with proper mixing. Pour about seven milliliters of the medium into a 60 by 15 millimeters sterile Petri dish.Allow the medium to solidify in the Petri dishes for approximately 15 to 20 minutes. Close the Petri dishes with sealing wrap and place them into a storage box at room temperature until further use. Harvest the squash fruit by cutting it off the main vine and disinfect the fruit by washing its surface in the lab sink using a liquid detergent like 0.3%chloroxylenol.Rinse the fruit with ample tap water and dry it with clean paper towels before transferring the fruit to the laminate airflow cabinet. Sterilize the fruit surface inside the cabinet by spraying 70%ethanol. Bisect the fruit with a sterile knife and extract the seeds.Use sterile forceps or hands with sterile gloves to aseptically open the seed coat and expose the immature embryos before placing them in a Petri dish containing antibiotic-supplemented MS Medium. Seal the Petri plates using wrapping film and place them in a growth chamber having 25 degrees Celsius temperature, 16-hour photo period, and 70%relative humidity. In case of contamination, immediately subculture the uncontaminated embryos into a new plate having the same medium.Once roots are developed after 10 to 14 days, and the cotyledons are developed after 15 to 21 days, remove the plantlets out of the Petri dishes, and gently wash off the media present around the roots using tap water. Place cleaned plantlets in a 14 by 9 by 4 centimeter plastic container and cover the roots with wet paper towels. Cover the container and re-moisten the paper towels when necessary.Keep the plastic containers at 25 to 28 degrees Celsius, and in a 16-hour photo period to acclimatize the plantlets while maintaining the moist condition of paper towels. After 7 to 10 days of acclimatization, transfer the three to four centimeter long seedlings into 50 cell flats containing commercial potting mix amended with fertilizer and move them to the greenhouse. Avoid overwatering to prevent rotting and add approximately 10 to 20 milliliters of water per cell as needed.At the second to the third true leaf stage, transplant the seedlings into 25 centimeter diameter pots filled with potting medium amended with fertilizer and perform controlled hybridization when the plants start flowering. Maintain the plants in the greenhouse at 22 to 28 degrees Celsius under the natural light regime. And evaluate the plants for fruit and seed characteristics.Four Cucurbita pepo and 11 Cucurbita moschata were chosen for interspecific hybridization. Out of 24 interspecific cross combinations attempted, a fruit set was obtained for 22 crosses representing more than 92%success in the fruit set. The crosses between Cucurbita moschata and Cucurbita pepo involving genotypes O and M, and genotypes E and J, produced no mature fruit.Whereas the cross between Cucurbita moschata and Cucurbita pepo involving genotypes F and J, produced six fruits. The number of flowers pollinated for different cross combinations ranged from 1 to 11. And the success rate of pollination ranged from 0%to 100%Out of 44 fruits produced from all the cross combinations, only one fruit from a cross between Cucurbita moschata and Cucurbita pepo, genotypes C and J produced immature embryos that could be rescued.For the development of these poorly developed embryos, they were cultured using the embryo rescue media, which resulted in an 80%success rate for embryo rescue. The two most important procedures are identifying compatible crosses between two species namely, Cucurbita moschata and Cucurbita pepo, and successful embryo regeneration. Since the embryo rescue technique is simple and easily replicable, squash breeders worldwide can use this technique for rescuing immature embryos derived from interspecific crosses between different Cucurbita species.

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