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JoVE Journal Biology

Biology

Embryo Rescue Protocol for Interspecific Hybridization in Squash

Published: September 12, 2022 doi: 10.3791/64071
Automatic Translation
1, 1, 1, 1
1UF/IFAS Tropical Research and Education Center

Summary

The 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.

Abstract

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.

Introduction

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 °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 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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Protocol

1. Planting and pollination

NOTE: It is important to identify compatible genotypes whose hybridization would result in fruit set and the production of viable embryos.

  1. Planting conditions and maintenance
    1. Obtain seeds of squash genotypes (cultivars/accessions) for hybridization (Table 1).
    2. 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, 1.38 g/kg P, and 1.38 g/kg K.
    3. Sow seeds to a depth equal to their length and cover with potting medium. Water the flats without creating standing water. Afterward, keep the media moist by watering it by hand once per day.
    4. At the second true-leaf stage, transplant the seedlings into 25 cm to 30 cm diameter pots amended with a complete NPK fertilizer at 3 tablespoons/pot. Fertilize the plants once a week with 500 mL/pot of liquid fertilizer containing NPK 20:20:20 added to a concentration of 1 g per gallon of water.
    5. Maintain plants in the greenhouse at temperatures between 22-28 °C under natural light regime. For vining genotypes, provide a supporting trellis in the greenhouse (Figure 1).
  2. Performing pollinations
    1. Typically, flowering in squash starts at 6 to 8 weeks from seeding depending upon the cultivar (Figure 2A,B). Begin making controlled hybridization (crosses) as soon as the plants start flowering. Under greenhouse conditions, pollination can be done year-round.
    2. Identify male and female flowers of C. pepo and C. moschata cultivars that will be ready for pollination the following day. To identify such flowers, check for flowers whose petals have a yellow hue but are not open. Gently tape the flowers closed at the top using masking tape to prevent accidental insect pollination (Figure 2C,D).
    3. The morning of the following day, the flowers are ready to pollinate. Carry out pollination before 10:00 a.m. to improve the rate of hybridization success27.
    4. Open the female and male flowers by gently removing the taped top portion of the petals. Remove the petals from the male flower and transfer the pollen by gently rubbing the anther on the stigma of the female flower (Figure 3A).
    5. After pollination, immediately close the pollinated female flower with masking tape. Use a tag to record the date of pollination and to indicate the paternal and maternal parents used in the cross (Figure 3B).
    6. A successful cross is indicated by an expanded ovary which quickly forms a small fruit within 1 week (Figure 4A). The plant is then ready for harvest 45-55 days after pollination28 (Figure 4B).

2. Embryo rescue technique

  1. Media preparation
    1. Prepare antibiotic stocks: For cefotaxime (sodium salt), dissolve the antibiotic in 4 mL of deionized or distilled water, filter through a sterile 0.22 µm syringe filter, and make 0.5 mL aliquots. Store at -20 °C. The resulting stock solution will have a concentration of 250 mg/mL.
    2. For ampicillin (sodium salt), dissolve the powder in 10 mL of water, filter through a sterile 0.22 µm syringe filter, and aliquot in 1 mL stock with a final concentration of 100 mg/mL. Store at -20 °C.
    3. Make Murashige and Skoog (MS) medium by dissolving 2.45 g of medium (4.91 g/L concentration) in 500 mL of distilled water in a 1 L bottle. Add 1.5 g of gellan gum and autoclave at 121 °C for 20 min. The gellan gum completely dissolves during autoclaving.
    4. After autoclaving, cool the medium by placing the bottle in a water bath at 50 °C. Remove the antibiotic stocks from the freezer and thaw them in the laminar flow cabinet.
    5. Transfer the medium bottle to the laminar flow hood. Add 0.6 mL of cefotaxime stock solution (250 mg/mL) and 0.25 mL of ampicillin stock (100 mg/mL) to the medium bottle and mix well. Pour about 7 mL of the medium into a sterile Petri dish (60 mm x 15 mm).
    6. Allow the medium to solidify in the Petri dishes for approximately 15-20 min. Close the Petri dishes with sealing wrap and place them into a storage box. Store at room temperature.
  2. Embryo rescue
    1. Before starting, clean and sterilize the laminar-air-flow cabinet with 70% ethyl alcohol.
    2. Harvest the squash fruit by breaking/cutting it off the main vine and disinfect the fruit surface by washing with liquid detergent (e.g., 0.3% chloroxylenol) in the lab sink until all the loose dirt is removed (Figure 5A).
    3. Rinse with ample tap water. Dry the fruit with clean paper towels. Move the fruit to the laminar-air-flow cabinet (Figure 5B).
    4. Surface sterilize the fruit by spraying 70% ethanol on the fruit in the sterile laminar-air-flow cabinet. Bisect the fruit with a sterile knife (Figure 5C) and extract the seeds.
    5. Use sterile forceps to aseptically open the seed coat and expose the immature embryos (Figure 6). Carefully place the immature embryos in a Petri dish containing MS medium supplemented with cefotaxime and ampicillin (Figure 7A). Close the Petri dish and seal with wrapping film.
      NOTE: Depending on the size of the embryo, it is possible to fit five or six embryos in a Petri dish.
    6. Place the sealed Petri dishes with embryos in a growth chamber under 16 h photoperiod at 25 °C and 70% relative humidity. If contamination occurs, promptly subculture the uncontaminated embryos into a new plate with the same medium.
    7. The cotyledons will start expanding after 4 days and will turn green in 10 days (Figure 7B). At this point, if needed, perform subculturing into new plates containing the same medium to allow tissue expansion. The roots will start to appear at 14 days (Figure 7C), and at 21 days the plantlets will have extended roots and cotyledons (Figure 7D).
      NOTE: The culture medium used in the protocol is appropriate for differentiation into shoots and roots without supplementary growth regulators.
    8. At this stage, remove the plantlets from the Petri dishes and gently wash off the media from the roots with tap water (Figure 8). Place the plantlets in a plastic container (14 cm x 9 cm x 4 cm) and cover the roots with wet paper towels (Figure 9A). Cover the container and remoisten the paper towels as necessary.
    9. Keep the containers at room temperature (25-28 °C) and with a 16 h photoperiod. This step will acclimatize the plantlets. After acclimatization for 7-10 days in the container, the seedlings will be approximately 3-4 in long. During this period, remoisten the paper towels as needed.
    10. Transfer the seedlings into 50 cell starting flats (25 cm width x 50 cm length) amended with fertilizer as previously described and move them to the greenhouse (Figure 9B). Do not over-water seedlings to avoid rotting; add approximately 10-20 mL per cell as needed.
    11. At the second to third true-leaf stage, transplant the seedlings into 30 cm diameter pots filled with potting medium amended with fertilizer as previously described (Figure 10A). Provide trellis support for vining plants and perform controlled hybridization when the plants start flowering, as previously described (Figure 10B).
    12. Maintain the plants in the greenhouse at temperatures between 22-28 °C under natural light regime. Evaluate the plants for fruit and seed characteristics.

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Representative Results

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 >92% success in the fruit set. No mature fruits were obtained by crossing O and M and E and J, while the highest number of fruits (n = 6) were obtained by crossing F and J (Table 2). The number of flowers pollinated for different cross combinations ranged from one to 11, and the success rate of pollination ranged from 0% to 100%. The number of flowers pollinated in different cross combinations varied depending on the number of flower sets and synchronization of flowering among male and female flowers. Even though fruits were obtained from all the crosses except two, evaluation of fruits after cutting them revealed that most of the fruits had aborted embryos with no viable seeds. Fruits from most of the crosses looked normal but were devoid of seeds or consisted of seeds with rudimentary embryos. A total of 44 fruits were produced from all the cross combinations, and only one fruit, developed by crossing C and J, had poorly developed embryos that could be recovered through the embryo rescue technique.

Embryo rescue and further advancement
The F1 interspecific hybrid developed by crossing C and J had 44 seeds in total, but only 10 of those had embryos that could be rescued for generation advancement. The remaining seeds had no embryos. All 10 embryos were cultured in the embryo rescue media and checked daily for their growth and development. The size of the 10 immature embryos ranged from 3.51 mm to 8.26 mm. The success rate of embryo rescue was 80%. The F1 interspecific hybrids (bridge lines) developed by crossing C. moschata and C. pepo (C and J) contained the genomes of both species at a 1:1 (50% each) ratio. These plants were used as bridge lines for the introgression of economically important traits across the two species. For example, crossing these bridge lines with C. moschata would result in hybrids with 75% C. moschata and 25% C. pepo genetic background, respectively. The fruits obtained from these bridge lines had a mixture of unviable seeds and seeds with immature embryos that subsequently required tissue culture for regeneration. For instance, one of the fruits had a total of 54 seeds, among which 14 seeds had immature embryos that were rescued using the protocol described here.

Figure 1
Figure 1: Supporting trellis for vertically growing squash plants in the greenhouse. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Illustration of open and taped flowers. Open (A) male and (B) female squash flower in the greenhouse. (C) A taped male flower from Cucurbita moschata paternal parent. (D) A taped female flower from Cucurbita pepo maternal parent. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Illustration of pollination. (A) Transfer the pollen from the male flower by gently rubbing the anther on the stigma of the female flower. (B) After pollination, tape the female flower and use a tag to record the date of pollination and the paternal and maternal parents used in the cross. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Fruit set. (A) After pollination, the ovary will expand quickly, forming a small fruit within 1 week. (B) The fruit is ready for harvest at 45 days after pollination. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Fruit preparation. (A) Wash the fruit with detergent. Harvest and disinfect the surface of the fruit by washing it with liquid detergent in the lab sink. (B) Rinse and dry the fruit. Dry the fruit with clean paper towels, following rinsing with ample tap water, and move it to the laminar-air-flow cabinet. (C) Bisect the fruit open with a sterile knife. Please click here to view a larger version of this figure.

Figure 6
Figure 6: Extract embryo from the seeds. Use sterile forceps to aseptically open the seed coat and expose the immature embryo. Please click here to view a larger version of this figure.

Figure 7
Figure 7: Embryo regeneration in the MS media. (A) Carefully place immature embryos in a Petri dish containing MS medium. (B) The cotyledons will expand and become green within 10 days. (C) The roots will start to appear at 14 days. (D) At 21 days, the plantlets will have extended roots and cotyledons which are ready to be transferred into a plastic container for acclimatization. Please click here to view a larger version of this figure.

Figure 8
Figure 8: Wash the roots. Remove the plantlets from the Petri dishes and gently wash off the media from the roots with tap water. Please click here to view a larger version of this figure.

Figure 9
Figure 9: Acclimatize the plantlets. (A) Place the plantlets in a plastic container and cover the roots with a wet paper towel for 5 days to acclimatize them. (B) Transfer the plantlets into cell trays containing commercial potting mix amended with complete NPK fertilizer. Please click here to view a larger version of this figure.

Figure 10
Figure 10: Transplant the seedlings into pots. (A) At the second to third true-leaf stage, transplant the seedlings into 30 cm diameter pots filled with potting medium amended with fertilizer. (B) Provide trellis support for vining plants and make controlled hybridization when the plants start flowering. Please click here to view a larger version of this figure.

Lab Code Species Source
A C. moschata Local Farmers Market
B C. moschata Local Farmers Market
C C. moschata Local Farmers Market
D C. moschata Local Farmers Market
E C. moschata Local Farmers Market
F C. moschata Local Farmers Market
G C. moschata University of Florida Breeding Line
H C. moschata University of Florida Breeding Line
I C. pepo NCRPIS(North Central Regional Plant Intruduction Station)
J C. pepo  NCRPIS(North Central Regional Plant Intruduction Station)
M C. pepo NCRPIS(North Central Regional Plant Intruduction Station)
O C. moschata University of Florida Breeding Line
Q C. moschata University of Florida Breeding Line
W C. pepo University of Florida Breeding Line
Y C. moschata Burpee Seeds Co

Table 1: A total of 15 genotypes of squash, four C. pepo and 11 C. moschata, were used in the study for interspecific crosses.

Cross (Female x Male) N. of pollinated flowers  N. of  fruits Fruit set (%) N. of aborted seeds N. of immature embryos N. of rescued embryos
A (C. moschata) x I (C. pepo) 5 4 80 0 0 0
H (C. moschata) x I (C. pepo) 2 2 100 0 0 0
B (C. moschata) x J (C. pepo) 2 1 50 0 0 0
C (C. moschata) x J (C. pepo) 3 1 33.3 44 10 8
E (C. moschata) x J (C. pepo) 6 0 0 0 0 0
F (C. moschata) x J (C. pepo) 11 6 54.5 0 0 0
G (C. moschata) x J (C. pepo) 2 2 100 0 0 0
 J (C. pepo) x H (C. moschata) 7 2 28.6 0 0 0
 J (C. pepo) x O (C.moschata) 6 1 16.7 0 0 0
O (C. moschata) x J (C. pepo) 6 1 16.7 0 0 0
Q (C. moschata) x J (C. pepo) 1 1 100 0 0 0
C (C. moschata) x M (C. pepo) 4 3 75 0 0 0
D (C. moschata) x M (C. pepo) 1 1 100 0 0 0
F (C. moschata) x M (C. pepo) 9 5 55.6 0 0 0
G (C. moschata) x M (C. pepo) 1 1 100 0 0 0
O (C. moschata) x M (C. pepo) 22 0 0 0 0 0
Q (C. moschata) x M (C. pepo) 2 1 50 0 0 0
F (C. moschata) x W (C. pepo) 1 1 100 0 0 0
G (C. moschata) x W (C. pepo) 1 1 100 0 0 0
H (C. moschata) x W (C. pepo) 2 1 50 0 0 0
O (C. moschata) x W (C. pepo) 0 0 0 0 0 0
Y  (C. moschata) x W (C. pepo) 3 2 66.7 0 0 0
M (C. pepo) x H (C.moschata) 3 2 66.7 0 0 0
M (C. pepo) x O (C.moschata) 4 4 100 0 0 0
Total 44 10 8

Table 2: Cross combinations attempted with the 15 genotypes of squash and the corresponding fruit set, number of aborted seeds, immature embryos, and successful embryo rescues.

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Discussion

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. Parental genotype has a considerable influence on the compatibility of interspecific hybridization between C. moschata and C. pepo. Among the 24 cross combinations tested in the current study, only one (C and J) yielded immature embryos for embryo rescue. As such, the study was limited by the lack of biological replicates for testing the efficiency of the protocol developed. However, a regeneration efficiency of 80% was obtained for the 10 immature embryos rescued from the C and J cross. Previous studies have reported lower regeneration success from embryo rescues for C. pepo and C. moschata crosses, thus demonstrating the effectiveness of the new protocol14,15. For instance, De Oliveira et al. reported that out of 26 embryos obtained from a cross between C. pepo cv. Asmara and C. moschata cv. Piramoita, no regenerants were obtained. However, the authors reported a 16% regeneration success when a different combination of genotypes (C. pepo cv. Asmara and C. moschata cv. Duda) was used14. In another study of interspecific hybridization between different Cucurbita species, Rakha et al. reported a regeneration efficiency of 40% and 15% from the immature embryos obtained by crossing C. ficifolia x C. pepo and C. martinezii x C. pepo, respectively15. The current study utilized an MS medium devoid of supplemental growth regulators compared to previously established protocols that employed complex media for embryo rescue15,29,30. Furthermore, the addition of the antibiotics cefotaxime and ampicillin was sufficient at preventing microbial contamination. Thus, the medium offers advantages as being cheaper, being easy to prepare without the need for high-skilled personnel, and it can be adopted by smaller breeding programs with limited resources.

In the current study, fruits were harvested 45-55 days post-pollination (DPP) to maximize embryo maturation and regeneration. The DPP selected for the current study was based on a previous report that showed optimum accumulation of energy reserves (mostly lipids and proteins) for embryos occurred at 60 DPP31,32. In muskmelon, Nunez-Palenius et al. reported a positive correlation between the success of embryo rescue and the DPP30.

In the current study, the second generation of fruits developed by crossing interspecific F1 hybrids with C. moschata did not yield normal seeds. This observation indicates that it is difficult to overcome the fertility barrier between C. moschata and C. pepo in a single generation as previously reported16. However, the adoption of the current protocol will aid in the successful development of Cucurbita interspecific hybrids in breeding programs. Further studies are needed to determine cross-compatibility of a wider array of genotypes to broaden germplasm accessibility to breeders

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Disclosures

The authors declare no conflicts of interest.

Acknowledgments

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

Materials

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

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References

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Embryo Rescue Protocol Interspecific Hybridization Cucurbita Species Cucurbita Moschata Cucurbita Pepo MS Media Antibiotics Barriers To Hybridization Planting Cucurbita Plants Starting Flats Potting Medium Nitrogen Fertilizer Phosphorus Fertilizer Potassium Fertilizer Seedlings Transplanting Liquid Fertilizer
Embryo Rescue Protocol for Interspecific Hybridization in Squash
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Fu, Y., Shrestha, S., Moon, P.,… More

Fu, Y., Shrestha, S., Moon, P., Meru, G. Embryo Rescue Protocol for Interspecific Hybridization in Squash. J. Vis. Exp. (187), e64071, doi:10.3791/64071 (2022).

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