Embryo Rescue Protocol for Interspecific Hybridization in Squash

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 pumpkin^2,3,4,5. 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 resistance^6,7. 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 programs^7,8.

Early crosses between C. moschata and C. pepo were made to determine their compatibility and/or taxonomic barriers^9,10,11, whereas later studies mostly focused on transferring desirable traits^12,13,14. 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. moschata^14,15,16. 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 cultivars^17,18.

Previous studies showed that hybridization between C. moschata and C. pepo is possible but difficult^8,15. 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 rescue^15,16. 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 embryos^16,20. 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 technique^25,26. 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.

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.

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...

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

Materials

List of materials used in this article

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