Embryo Rescue Breeding of New Cold-Resistant, Seedless Grapes

: Seedlessness in grapes is much appreciated by consumers and especially in cultivars consumed either as table grapes or as raisins. In many parts of the world, low temperature is the main environmental stress limiting grape production. In this study, stenospermocarpic (seed abortion) cultivars were selected as the female parents while seeded cold-resistant cultivars were selected as the male parents to develop new cold-resistant seedless grapes using embryo rescue technology, which has previously been shown to be a highly efﬁcient way of breeding seedless grapes. Here, we report optima in genotype, sampling time, and culture medium for the embryo rescue of 14 hybrid combinations. Our results indicate that the embryo development rate (39.9%) and the seedlings rate (21.5%) were highest among the 14 crosses when ‘Ruby Seedless’ was used as the female parent and ‘Beibinghong’ was used as the male parent. The best sampling times for ‘Yuehong Wuhe’, ‘Ruby Seedless’, and ‘Melissa seedless’ were 37, 55, and 52 days after ﬂowering, respectively. Embryo rescue efﬁciency was highest when the sucrose concentration for seedlings was maintained at about 1.0%. Using molecular markers, we detected 91 hybrids with seedless traits and 18 hybrids with cold resistance traits.


Introduction
The European grapevine (Vitis vinifera L.) bears abundant fruit, and thus offers high economic benefit to growers. Its fruit is in strong demand for winemaking but also for fresh consumption and as a dried fruit. Accordingly, grapes are widely grown around the world. The seedless trait, which improves the edibility of both the fresh and the dried product, is much favored by the consumer, and it is therefore a key indicator for evaluating new cultivars selected to meet the growing demand for both fresh and dried grapes. There are many high-quality seedless grapes already in production, including 'Flame seedless', 'Crimson seedless', and 'Ruby Seedless', but seedlessness remains a main focus of breeders of new table and dried grape cultivars because of their increased market value over seeded cultivars.
Prior to 1980, the conventional breeding method for seedlessness in grapes was to use a seeded grape as the female parent and a seedless grape as the male parent. The best hybrids were then backcrossed with their parents or repeatedly crossed to obtain new cultivars [1,2]. This method, in which the female parent is limited to the seeded varieties, involves much time and effort and, thus, a high cost. Meanwhile, the hybrid progeny has a low seedless rate. In vitro culture of embryos was first achieved in cherry by Tukey in 1933 [3]. Some 50 years later, embryo rescue technology was achieved in grapes when, in 1982, two grape seedlings were successfully cultivated from the ovules of a seedless grape by Ramming and Emershad using embryo rescue [4][5][6]. Experiments show that by using this technology, a seedless grape can be used as the female parent, and the breeding process is sped up by about six years.
Embryo rescue technology involves three stages: immature ovule embryo culture, embryo germination, and embryo seedling formation. Based on large numbers of experimental studies, Li et al. (2001) concluded that the main factors determining the success of embryo rescue are the sampling time, the parental genotype, the culture medium, and conditions [7].
The emergence of molecular markers further sped up the breeding process for seedless grapes [1,8]. The application of biotechnology allows a further speeding up of seedless grape breeding [9,10]. In 1998, Lahogue obtained two RAPD markers (random amplified polymorphic markers) through screening the major genes that control seedless traits [11]. It was found that seedless traits in grapes were controlled by multiple genes, and, among these, a few played major roles in regulating the seedless traits while others, called complementary recessive genes, played auxiliary roles [8,12]. Using the seedless marker SCF27-2000, Mejía and Hinrichsen carried out a preliminarily screening and identified the seedless traits of the hybrid progeny of 'Ruby Seedless' × 'Sultanina' [13], and they reported that VvAGL11 was a candidate gene related to the seedless trait of grapes and that a seedless marker named P3-VvAGL11 was obtained [14,15]. The above markers are of great significance for the initial identification of seedless grapes, and are widely used in the preliminary screening of grape hybrid progenies [16]. In China, based on the RAPD marker UBC-26945 that had been studied earlier, Wang et al. used the hybrid combination of 'Thompson seedless' and 'Muscat' in order to continue to develop a 'GLSP1 marker, which can be used to test the seedless characteristic of grapes [17].
Low temperature is one of the most severe abiotic stresses that limits grape growth and geographical distribution. Extreme low temperatures in winter can cause serious damage to both buds and branches [18,19], and the grapevines should thus be buried in soil when the temperature falls below −15 • C in order to cope with the cold environment in winter, which not only increases the production cost of the vineyard but also causes a series of problems, such as tree body damage, soil erosion, and soil horizon destruction, that considerably limit the development of grape production [20,21]. Thus, it is of great importance to breed new cold resistant cultivars. The Chinese wild grape Vitis amurensis is extremely cold tolerant, and can survive in temperatures as low as −40 to −50 • C [22]. It is therefore an excellent germplasm resource for cold-resistance breeding [23,24]. Two RAPD markers, S238-854 and S241-717, can be used to identify the cold resistant traits that were obtained by Zhang et al. in their 2010 experimental study on the cold resistance characteristics of Chinese wild grapes [25].
In our present study, based on embryo rescue technology, using high quality Chinese grape varieties with good cold resistance as male parents and seedless grapes as female parents, new germplasm combining good cold resistance with seedlessness was developed. The cold resistance and the seedless characteristics of their hybrid offspring were preliminarily screened using molecular marker-assisted selection.

Pollen Collection, Emasculation and Hybridization
The best time to collect pollen is to choose grapevines whose growth is vigorous and when the inflorescences are yellow and blooming in a small area. Emasculation, which means removing the anthers from female parents, was performed 3~4 days before the flowering of the female parents. On the second day after emasculation, pollination started when a transparent mucus was seen to be secreted by the stigma. The whole pollination procedure lasted for 2-3 days (Figure 1). in the Grape Repository of Northwest A&F University, Yangling, Shaanxi (34°27' N, 108°08' E) during 2019-2020.

Pollen Collection, Emasculation and Hybridization
The best time to collect pollen is to choose grapevines whose growth is vigorous and when the inflorescences are yellow and blooming in a small area. Emasculation, which means removing the anthers from female parents, was performed 3~4 days before the flowering of the female parents. On the second day after emasculation, pollination started when a transparent mucus was seen to be secreted by the stigma. The whole pollination procedure lasted for 2-3 days (Figure 1).

Sampling
In this experiment, three hybrid combinations were selected for continuous sampling, and different sampling times were selected according to the different phenological phases (the different phase of grape development during the whole growth period in a year) between the male parent and the female parent. 'Melissa seedless' × 'Xinyu' was sampled at 52 days after full bloom (DAF), 54 DAF and 56 DAF; 'Ruby Seedless' × 'Shine-Muscat' was sampled at 53 DAF, 55 DAF, and 57 DAF; and 'Yuehong Wuhe' × 'SP740′ was sampled at 33 DAF, 35 DAF, and 37 DAF. These hybrid fruits were put into an ice box, and were then brought to the laboratory for embryo rescue. The embryo development rate and the seedling rate for different sampling times were recorded and compared, and the best sampling times of each combination were determined.

Sampling
In this experiment, three hybrid combinations were selected for continuous sampling, and different sampling times were selected according to the different phenological phases (the different phase of grape development during the whole growth period in a year) between the male parent and the female parent. 'Melissa seedless' × 'Xinyu' was sampled at 52 days after full bloom (DAF), 54 DAF and 56 DAF; 'Ruby Seedless' × 'Shine-Muscat' was sampled at 53 DAF, 55 DAF, and 57 DAF; and 'Yuehong Wuhe' × 'SP740 was sampled at 33 DAF, 35 DAF, and 37 DAF. These hybrid fruits were put into an ice box, and were then brought to the laboratory for embryo rescue. The embryo development rate and the seedling rate for different sampling times were recorded and compared, and the best sampling times of each combination were determined.

Ovule Culture and Embryo Development
Embryo rescue. The harvested hybrid fruits were placed on a 'clean bench' for disinfection, as in a previous study [26]. In brief, the fruits ( Figure 2a) were soaked in 75% ethanol solution for 30 s, then washed with sterile water twice, and then soaked in 1% Na-ClO for 20 min. The bottle was shaken every five mins in order to make it fully soaked and disinfected. After NaClO was poured out, the bottle was cleaned with sterile water 5 times until there was no foam in order to complete the disinfection of the grape fruit. Then, the sterilized fruits were cut open in order to strip out all of the ovules aseptically ( Figure 2b) and rescue the ovules, as per our previous study [26]. The ovules were inoculated into embryo development medium (MM3 + sucrose 60 g/L + hydrolyzed casein 0.5 g/L + inositol 0.1 g/L + agar 7 g/L + activated carbon 3 g/L) (Figure 2c). Ovules were then cultured in the dark for 60 days at 25 ± 2 • C. At the end of this time, the ovules were excised under a dissecting microscope (Figure 2d,e), and they were then placed in an embryo germination medium (WPM + 0.2 mg/L 6-BA + 20 g/L sucrose (2% sucrose) + 1.5 g/L activated carbon + 0.1 g/L inositol + 7 g/L agar) (Figure 2f,g). From the records and the counts of the numbers of embryos developing and germinating and the seedlings formed, we calculated the rate of ovule development and seedling formation. For 'Ruby seedless' × 'Shine Muscart' and 'Yuehong Wuhe' × 'Sp740', we used embryo gemination medium with different concentrations of sucrose (1%, 1.5%, 2%, and 3%) in order to evaluate the effectors of sucrose concentration on embryo rescue.

Ovule Culture and Embryo Development
Embryo rescue. The harvested hybrid fruits were placed on a 'clean bench' for disinfection, as in a previous study [26]. In brief, the fruits ( Figure 2a) were soaked in 75% ethanol solution for 30 s, then washed with sterile water twice, and then soaked in 1% NaClO for 20 min. The bottle was shaken every five mins in order to make it fully soaked and disinfected. After NaClO was poured out, the bottle was cleaned with sterile water 5 times until there was no foam in order to complete the disinfection of the grape fruit. Then, the sterilized fruits were cut open in order to strip out all of the ovules aseptically ( Figure  2b) and rescue the ovules, as per our previous study [26]. The ovules were inoculated into embryo development medium (MM3 + sucrose 60 g/L + hydrolyzed casein 0.5 g/L + inositol 0.1 g/L + agar 7 g/L + activated carbon 3 g/L) (Figure 2c). Ovules were then cultured in the dark for 60 days at 25 ± 2 °C. At the end of this time, the ovules were excised under a dissecting microscope (Figure 2d,e), and they were then placed in an embryo germination medium (WPM + 0.2 mg/L 6-BA + 20 g/L sucrose (2% sucrose) + 1.5 g/L activated carbon+ 0.1 g/L inositol + 7 g/L agar) (Figure 2f,g). From the records and the counts of the numbers of embryos developing and germinating and the seedlings formed, we calculated the rate of ovule development and seedling formation. For 'Ruby seedless' × 'Shine Muscart' and 'Yuehong Wuhe' × 'Sp740', we used embryo gemination medium with different concentrations of sucrose (1%, 1.5%, 2%, and 3%) in order to evaluate the effectors of sucrose concentration on embryo rescue. Subsequent transplantation of hybrid seedlings. When the seedlings grown from the germinated embryos had grown close to the top of the bottle, a sub-culture medium was used for propagation, which was ½Ms + 30 g/L sucrose + 7 g/L agar. The developed stem segments were cut off and packed into covered transparent glass bottles to continue growing (Figure 2h). The growth temperature was controlled at about 25 °C. During this period, seedling formation was continuously observed and recorded. The tissue culture seedlings with a good root system were selected (Figure 2i) for hardening (Figure 2j,k) and were transplanted into the field after 35 days (Figure 2l). The survival rate of the hybrid plants was recorded. Subsequent transplantation of hybrid seedlings. When the seedlings grown from the germinated embryos had grown close to the top of the bottle, a sub-culture medium was used for propagation, which was 1 2 Ms + 30 g/L sucrose + 7 g/L agar. The developed stem segments were cut off and packed into covered transparent glass bottles to continue growing (Figure 2h). The growth temperature was controlled at about 25 • C. During this period, seedling formation was continuously observed and recorded. The tissue culture seedlings with a good root system were selected (Figure 2i) for hardening (Figure 2j,k) and were transplanted into the field after 35 days (Figure 2l). The survival rate of the hybrid plants was recorded.

Analysis of Seedlessness and Cold Resistance by Molecular Markers
The DNA of the test material was extracted from the young leaves of the parents and the progenies. GSLP1-569 and S241-717 were used as molecular markers for seedless trait and cold resistance markers, respectively. The primer sequence and the reaction procedure refer to a previous study [13]. Amplification products were separated on 2% agarose and photographed (Alpha imager HP, ProteinSimple, San Jose, CA, USA) in order to identify potentially seedless and cold-resistant progenies.

Data Analyses
The growth and development of the different genotype combinations, the number of ovules germinating, and the number of seedlings developing were observed and recorded. From this data, the ovule development rate and the seedling rate of hybrid progenies were calculated: The embryo formation rate = the number of embryos developed/the number of ovules cultured.
The embryo germination rate = the number of embryos germinated/the number of embryos developed.
The plant development rate = the number of seedling recovered/the number of ovules cultured.

Preliminary Screening of the Best Combination
In this study, 14 combinations were conducted. and a total of 5807 hybrid ovules and 427 hybrid seedlings were obtained, as shown in Table 1. Among them, precocious means that the ripening time is earlier than the ripening period of the same type of fruit. Among the 14 combinations, the average development rate of embryos was 19.6%, and the average seedling rate of combinations was 8.1%, as shown in Table 2.
The effects of the genotype of both parents on embryo rescue efficiency of seedless grapes were compared and analyzed, and it was found that the highest seedling rate was 21.5% when 'Beibinghong' was used as the male parent and when 'Ruby Seedless' was used as the female parent.
In the combination of 'Flame Seedless' as the female parent with different genotypes as the male parent, the embryo development rate and the seedling rate of 'Shuangyou' as the male parent were the highest, reaching 14.3 and 11.3%, respectively. In the combination of 'Wuhe Cuibao' as the female parent with different genotypes as the male parent, the embryo development rate and the seedling rate of 'Xinyu' as the male parent were the highest, reaching 21.3 and 9.8%, respectively.

Effect of Sampling Time on Embryo Rescue
The time of maturity of the different parent cultivars varied, and this affected the sampling times. For the combination 'Yuehong Wuhe' × 'SP740 , the embryo development rate and the seedling rate were highest when sampled on 37 DAF, reaching 27.5 and 7.8%, respectively. The embryo development rate and the seedling rate of 'Melissa Horticulturae 2023, 9, 992 6 of 11 seedless' × 'Xinyu' were highest on 52 DAF, reaching 42.6 and 26.2%, respectively. The embryo development rate and the seedling rate of 'Ruby Seedless' × 'Shine-Muscat' were highest on 55 DAF, reaching 67.9 and 17.9%, respectively (Table 3).

Effect of Culture Media of Different Sucrose Concentrations on Embryo Rescue
Embryo germination media that had different sucrose concentrations had different effects on the growth of the embryo rescue seedling (Table 4). In this experiment, the seedling rates of 'Ruby Seedless' × 'Shine-Muscat' and 'Yuehong Wuhe' × 'SP740 were highest (15.7 and 25%, respectively) when the sucrose concentration was 1.0%.

Assisted Selection of Seedless Molecular Markers
The hybrid parents in 2019 were screened using the molecular marker GLSP1-569. The results show that 'Ruby Seedless' and 'Heshi Seedless' had 569 bp bands (Figure 3), which indicated that GLSP1-569 can be used as the molecular marker of seedless trait in order to identify the hybrid progenies of both 'Ruby Seedless' × 'Beibinghong' and 'Heshi Seedless' × 'Shuangyou'. As shown in Figure 4, 63 and 27 hybrids had the positive band, respectively, which indicated that these progenies may possess seedless traits (Figure 4).

Assisted Selection of Seedless Molecular Markers
The hybrid parents in 2019 were screened using the molecular marker GLSP1-569. The results show that 'Ruby Seedless' and 'Heshi Seedless' had 569 bp bands (Figure 3), which indicated that GLSP1-569 can be used as the molecular marker of seedless trait in order to identify the hybrid progenies of both 'Ruby Seedless' × 'Beibinghong' and 'Heshi Seedless' × 'Shuangyou'. As shown in Figure 4, 63 and 27 hybrids had the positive band, respectively, which indicated that these progenies may possess seedless traits (Figure 4).

Preliminary Screening of Molecular Markers for Cold Resistance
S241-717 with a length of 717bp were used as the cold-resistant marker. Gel electrophoresis shows that 'Beichun' and 'Beibinghong' both have specific bands at 717 bp (Figure 5).

Preliminary Screening of Molecular Markers for Cold Resistance
S241-717 with a length of 717bp were used as the cold-resistant marker. Gel electrophoresis shows that 'Beichun' and 'Beibinghong' both have specific bands at 717 bp ( Figure 5).

Preliminary Screening of Molecular Markers for Cold Resistance
S241-717 with a length of 717bp were used as the cold-resistant marker. Gel electrophoresis shows that 'Beichun' and 'Beibinghong' both have specific bands at 717 bp (Figure 5). Of the 12 progenies of 'Sultanina Rose' × 'Beichun', 11 showed specific bands at 717 bp, which could be preliminarily screened for cold resistance (Figure 6a). Seven of the

Discussion
In traditional cross-breeding, a seedless grape cannot be selected as the female parent as the zygotic embryo aborts [27]. Fortunately, this problem can be circumvented by embryo rescue breeding, in which different grape varieties can be selected as parent material in order to make the best combination according to their characteristics. Thus, new germplasm resources expressing superior quality, higher yield, and strong resistance to a range of biotic and abiotic stressors can be produced [8,[28][29][30]. For example, 'Blush Seedless', 'Thompson Seedless', 'Crimson Seedless', 'Emerald Seedless', and 'Flame Seedless' were all used as the maternal parent, while 'Beichun' was used the male parent in order to develop the disease-resistant seedless cultivars [31]. The results indicate that the female parent genotype combination can have a great impact on the developmental rate as well as on the seedling rate. The highest rate of embryo germination and rate of seedling was obtained when the parent material was 'Emerald Seedless' [31]. In addition, a large number of experiments have shown that the embryo development rate and the seedling rate are both high when 'Flame Seedless' and 'Ruby Seedless' are used as the female parents [32][33][34]. The results of our study show that when 'Ruby Seedless' is used as the female parent, the embryo rescue efficiency is good, rising as high as 39.9%. This makes it highly suitable as female parent material. This finding is consistent with previous results.
Sampling time determines the initial state of embryo growth and development, which plays an important role in embryo rescue. Previous studies have indicated that the germination and the seedling rates of hybrid embryos increase when fruits are in the ripening to softening stages. However, for the fruit in the later stages of the ripening, the

Discussion
In traditional cross-breeding, a seedless grape cannot be selected as the female parent as the zygotic embryo aborts [27]. Fortunately, this problem can be circumvented by embryo rescue breeding, in which different grape varieties can be selected as parent material in order to make the best combination according to their characteristics. Thus, new germplasm resources expressing superior quality, higher yield, and strong resistance to a range of biotic and abiotic stressors can be produced [8,[28][29][30]. For example, 'Blush Seedless', 'Thompson Seedless', 'Crimson Seedless', 'Emerald Seedless', and 'Flame Seedless' were all used as the maternal parent, while 'Beichun' was used the male parent in order to develop the disease-resistant seedless cultivars [31]. The results indicate that the female parent genotype combination can have a great impact on the developmental rate as well as on the seedling rate. The highest rate of embryo germination and rate of seedling was obtained when the parent material was 'Emerald Seedless' [31]. In addition, a large number of experiments have shown that the embryo development rate and the seedling rate are both high when 'Flame Seedless' and 'Ruby Seedless' are used as the female parents [32][33][34]. The results of our study show that when 'Ruby Seedless' is used as the female parent, the embryo rescue efficiency is good, rising as high as 39.9%. This makes it highly suitable as female parent material. This finding is consistent with previous results.
Sampling time determines the initial state of embryo growth and development, which plays an important role in embryo rescue. Previous studies have indicated that the germination and the seedling rates of hybrid embryos increase when fruits are in the ripening to softening stages. However, for the fruit in the later stages of the ripening, the ovule adheres strongly to the pulp, making it difficult to peel off the ovule in embryo rescue, and causing contamination of the ovule with the pulp tissue. Therefore, it is suggested that sampling time should be 1~2 days before fruit color change (veraison). Here, we found that the best sampling time for 'Ruby Seedless' × 'Sunshine Rose' was 55 DAF, which is consistent with previous reports. The addition of sucrose to the medium not only provides the carbon and the energy supply for growth but also affects the medium's osmotic potential. Increasing the sucrose concentration of the medium overcomes precocious embryo germination and also contributes to ovule growth. At the time of embryo germination, Ramming considered a medium with 1.5% sucrose concentration as suitable for embryo rescue seedling growth [35]. Our results show that for sucrose concentrations of 1.0-1.5%, the embryo germination rate of the combination was highest for both 'Yuehong Wuhe' × 'Sp740' and for 'Ruby Seedless' × 'Shine-Muscat', reaching 87.5 and 31.4%, respectively.
Molecular markers not only increase the efficiency of breeding but also help to select better hybrid combinations in line with breeding objectives. In 2011, Mejía discovered the seedless marker SCF27-2000, which also accurately detects the seedless character in grapes, and this is now widely used [15]. The reports now show that the five main molecular markers for screening and identification of seedless traits in grapes are SCC8-1018, p3-VvAGL11-1200, GSLP1-569, SCF27-2000, and VNCF7f2-198 [36].
In our study, the seedless marker GLSP1-569 was selected to detect seedless trait of our hybrid combinations [34]. Our results show that the hybrid progenies of 'Ruby Seedless' × 'Beibinghong' and 'Heshi Seedless' × 'Shuangyou' were satisfactorily detected, with 91 progeny having obvious specific bands at 569 bp5. Moreover, 17 hybrids were identified to carry cold resistance using the molecular marker S241-717.

Conclusions
A total of 14 seedless embryo rescue hybrid combinations were chosen, and a total of 427 hybrids were obtained. Among these, the best combination was 'Ruby Seedless' × 'Beibinghong'. The most suitable sampling times for 'Yuehong Wuhe', 'Ruby Seedless', and 'Melissa seedless' were 37, 55, and 52 DAF, respectively. The optimum sucrose concentration for the ovule growth medium was between 1.0-1.5%. A total of 140 hybrid progenies were screened using seedless markers GLSP1-569. The results show that 91 hybrid progeny had obvious seedless traits and that 18 hybrid progenies had cold resistance, which were identified using the cold-resistant marker S241-717 to detect the progenies of the two hybrid combinations.