Effects of exogenous paclobutrazol and sampling time on the efficiency of in vitro embryo rescue in the breeding of new seedless grape varieties

XU Teng-fei ,GUO Yu-rui ,WANG Wen-yuan ,YUAN Xiao-jian ,CHU Yan-nan ,WANG Xiao-wei ,HAN Yu-lei,WANG Yue-jin,SONG Rui,FANG Yu-lin,WANG Lu-jun,XU Yan

1 State Key Laboratory of Crop Stress Biology for Arid Areas,College of Horticulture,Northwest A&F University,Yangling 712100,P.R.China

2 College of Plant Science,Tibet Agricultural and Animal Husbandry University,Nyingchi 860000,P.R.China

3 Institute for China Wine Industry Technology,Yinchuan 750000,P.R.China

4 Institute for Weinan Grape Research,Weinan 714099,P.R.China

Abstract Embryo rescue technology plays an important role in seedless grape breeding. However,the efficiency of embryo rescue,including the embryo formation,germination,and seedling rates,is closely related to the parental genotypes,degree of abortion,growth medium,and plant growth regulators. In this study,we investigated the effects of different concentrations of paclobutrazol (PAC),a plant growth regulator,and embryo collection times on the embryo formation,germination,and seedling rates for different hybrid combinations of grape breeding varieties used for their aroma and cold-resistance traits.The results showed that the different PAC concentrations had varying impacts on the development of ovules and embryos from the different grape varieties. The embryo formation rates of the ‘Sultanina Rose’בBeibinghong’ and ‘Kunxiang Seedless’בTaishan-2’ crosses were the highest under the 5.1 μmol L-1 PAC treatment. The 1.0 μmol L-1 PAC treatment was optimal for the germination and seedling development of the ‘Sultanina Rose’בBeibinghong’ embryos,whereas the 0.2 μmol L-1 PAC treatment induced the highest germination rate for the ‘Sultanina Rose’בKunxiang Seedless’ cross. The optimal sampling times for each cross varied as 39 d after pollination (DAP) for the ‘Flame Seedless’בMuscat Hamburg’cross,46 DAP for the ‘Kunxiang Seedless’בBeibinghong’ cross,and 41 DAP for the ‘Ruby Seedless’בBeibinghong’ and‘Fantasy Seedless’בShuangyou’ crosses. Moreover,the medium modified with 0.5 g L-1 of indole-3-butyric acid allowed the malformed seedlings to develop into plantlets and achieve larger progenies. This study provides a useful basis for further studies into grape embryo rescue and could improve breeding efforts for new seedless grape varieties.

Keywords:Vitis,grape embryo rescue,paclobutrazol,malformed seedlings

1.lntroduction

Grapes (Vitis viniferaL.) are one of the most commonly consumed fruits worldwide,and their popularity is growing.Although seeds are the products of sexual reproduction in angiosperms,they are not produced by many woody crops,such as grapevines. Furthermore,the presence of seeds can affect the taste and quality of the fruit.The reduction of the seed content in grapes is a major breeding goal,as it increases the appeal of the fresh fruit or raisins for consumers (Ledbetter and Ramming 1989),and the global demand for seedless grapes is gradually increasing. Thus,meeting this demand is essential for the industry and will require improvements in table grape breeding technologies. However,the abortion of seedless grape zygote embryos currently limits the efficiency of traditional breeding methods (Tanget al.2009).

Since Cain (1983) first germinated embryos and obtained plantlets through the culture of stenospermic grape ovulesinvitro,embryo rescue has been widely used for seedless grapes as it allows hybridization between two seedless cultivars and enlarges the parental selectivity range. Similarly,the quality levels of many other crops,such as citrus,persimmon,avocado,and watermelon,have been improved by embryo rescue (Leng and Yamamura 2006;Sánchez-Romeroet al.2007;Huet al.2013;Ta?k?net al.2013;Xieet al.2019). Although the embryo rescue technique has been approved by breeders worldwide,many obstacles affecting its development remain,such as parental selection,sampling times,culture media,and plant growth regulators.

Seedless grapes are producedviatwo main mechanisms:stenospermocarpy and parthenocarpy(Stout 1936). In stenospermocarpy,plants can pollinate,fertilize,and develop embryos normally;however,seed development is terminated at an early stage (Stout 1936;Ledbetter and Ramming 1989). In contrast,in parthenocarpy,fruits grow without fertilization,producing small,completely seedless berries,and the process is associated with impaired meiosis (Royoet al.2016).Not all seedless grapes are suitable as female parents during parent selection,particularly for parthenocarpic seedless grapes (Liet al.2015). Stenospermocarpic grapes are typically selected as the female parents of seedless table grape cultivars as they can form and then abort embryos with little impact on the berry size (Cainet al.1983;Ramming 1990;Singhet al.2011). This may be due to the presence of seminal rudiments or seed traces that promote fruit growth (Stout 1936;Nitschet al.1960). The embryo abortion process is similar to that of the early stage of seeded cultivars;thus,inoculation times for culturing ovules can be selected to achieve the highest number of embryos prior to abortion (Liuet al.2008;Liet al.2015). However,only a small number of zygotic embryos can grow to maturity,while some may germinate but become malformed plantlets (Wanget al.2007;Haoet al.2010). Therefore,it is necessary to carefully select the parents and determine the proper sampling time.Furthermore,a method to convert malformed seedlings into normal seedlings is required.

Previous research that aimed to improve the efficiency of in-ovulo embryo rescue mainly focused on different aspects of the optimization techniques (Liuet al.2008).Common plant growth regulators,such as paclobutrazol(PAC),naphthaleneacetic acid,and abscisic acid,differentially affect embryo rescue (Agüeroet al.1995).PAC belongs to the triazole chemical group (Seesangboonet al.2018) and hinders plant vegetative growth by blocking the synthesis of gibberellins (Chenet al.2020),which restricts plant vegetative growth and induces flowering in plants such as mango,citrus,andJatropha curcas(Rehmanet al.2018;Seesangboonet al.2018).The efficacy of PAC varies between different species but mainly depends on the dosage and the physiological and environmental conditions of the plants (Blaikieet al.2004). PAC was also found to promote adventitious root formation in plant tissue cultures (Vai?iukyn?et al.2018),increase the scavenging of reactive oxygen species to alleviate the negative effects of biotic and abiotic stresses on the plant calli (Hajihashemi and Ehsanpour 2014;Hajihashemiet al.2018),and promote direct somatic embryogenesis in plants (Moradiet al.2017). However,the application of PAC in the embryo rescue of fruit trees has not been reported thus far.

Environmental factors,such as cold stress,negatively affect the productivity and quality of grapes. Originating from northeastern China,an extremely cold area,the Chinese wild grapevine (VitisamurensisRupr.) is one of the most northerly grapevine species in the world,and is more cold-resistant than ordinary cultivated grapevines(V.viniferaL.). Moreover,V.amurensishas been hybridized withV.viniferaorV.amurensisto produce new hybrid progenies with superior cold resistance,such as ‘Beibinghong’ and ‘Shuangyou’,which were selected to meet the demands of breeding (Weiet al.2019). When table grape cultivars were crossed with cold-resistant wild grape species and varieties with two desirable characteristics were selected,the facility costs could be reduced and there would be no need to cover the grapes before the winter,so this would also significantly decrease labor and production costs (Grahamet al.2002). Aroma is another important quality trait in table grapes. The seeded grape species ‘Beibinghong’used in this study exhibits compound honey and almond aromas,while ‘Muscat Hamburg’ grapes have a subtle sweetness and aroma.

The effects of the exogenous gibberellin GA3on embryo rescue have previously been reported;however,it is unclear whether PAC,as an inhibitor of GA biosynthesis,could play a role in the embryo rescue of seedless grapes. This study aimed to generate new cold-resistant and aromatic seedless grapesviaembryo rescue and identify the optimal PAC concentrations for ovule and embryo culture media. Additionally,we analyzed the optimal sampling times of the different crosses and the media that enabled the malformed seedlings to develop into normal plantlets in order to efficiently improve the probability of hybrid survival. The overall goal was to use Chinese wild and hybrid grapes to further enrich seedless grapes with desirable properties for the table grape market.

2.Materials and methods

2.1.Plant materials

We used seven hybrid crosses to test the effects of the various sampling times on the embryo rescue of seedless grapes,all of which were collected from the Xinjiang Development and Research Centre of Grapes and Melons,Shanshan County,Xinjiang Uygur Autonomous Region,China (42°53′N,90°13′E) during 2017-2018.The selected parents are shown in Table 1. Theinvitroexperiments for the grape embryos were conducted at Northwest A&F University,Yangling,Shaanxi,China(34°17′N,108°04′E).

Table 1 Main parental characteristics of Vitis hybrid combinations

2.2.Process of embryo rescue

SamplingIn late April of both 2017 and 2018,various pollen samples were collected and stored in a refrigerator at 4°C (Fig.1-A). Artificial emasculation and hybridization(Fig.1-B-D) were conducted in May at the Xinjiang Development and Research Centre of Grapes and Melons. Immature fruits were collected several weeks after pollination based on the sampling time (Fig.1-E).For each hybrid combination,30 inflorescences and their fruits were analyzed as one replicate. All experiments were conducted in triplicate.

Grape embryo and culture isolationThe fruit granules were cut and rinsed under a tap for 3-4 h and then placed in jars,classified as ‘super-clean’ and all further steps were conducted under aseptic conditions. The fruits were then soaked in 70% alcohol for 30 s,rinsed twice with sterile distilled water,shaken in a 1% sodium hypochlorite solution for 15-16 min,and then rinsed with sterile water three or four times. After sterilization,the ovules were isolated and placed in disposable Petri dishes. Approximately 15 ovules were evenly distributed in each Petri dish,containing Minimal Methanol Medium(MM3) supplement with 0.05% (w/v) complex protein hydrolysate,0.01% (w/v) inositol,6% (w/v) sucrose,0.3% (w/v) activated charcoal (AC),and 0.7% (w/v) agar,pH 5.8-6.0 (Fig.1-F).

All Petri dishes were incubated for 8 weeks at (25±1)°C under darkness. The developed embryos were then cut from the ovules using a scalpel under a stereoscope and carefully inoculated into solid wood plant medium (WPM;Lloyd and McCown 1980) supplemented with 7.0 g L-1agar,1.5 g L-1AC,0.2 mg L-16-benzylaminopurine (6-BA),and 20 g L-1sucrose. Each embryo was then placed in a covered transparent glass bottle,and all embryos were grown at (25±1)°C under 16 h of white fluorescent light(40 μmol m-2s-1;Fig.1-G).

Transplantation The germinated embryos and developed plantlets were counted after 1 and 2 months,respectively (Fig.1-H). When the plantlets grew close to the mouth of the glass tubes,the stems were cutat a clean bench and placed into cans containing the subculture medium of WPM supplemented with 30.0,1.0,7.0,0.2,and 0.2 g L-1of sucrose,activated carbon,agar,6-BA,and IBA,respectively. After 2 months,when the plantlets were larger and stronger (Fig.1-J),the residual medium around the roots was removed and the plantlets were transplanted to nutrition bowls (Fig.1-K).To maintain warm and moist conditions,each plantlet was covered with a plastic cup and grown in an artificial climate room. After 2-3 weeks,the plastic cups were removed,and the plantlets were allowed to grow for 1 month before being transplanted to the fields (Fig.1-L).

Fig.1 The embryo rescue process. A,pollen collection. B,emasculation. C,artificial pollination. D,bagging after pollination.E,grape collection. F,ovule inoculation. G,embryo excision. H,plantlet growth from embryo. I and J,secondary culture. K and L,acclimatization and transplantation.

2.3.Application of PAC to the medium for ovule and embryo formation

PAC was added to the ovule culture medium (MM3 medium supplement with 0.05% (w/v) complex protein hydrolysate,0.01% (w/v) inositol,6% (w/v) sucrose,0.3% (w/v) AC,and 0.7% (w/v) agar,pH 5.8-6.0) at concentrations of 0,1.7,3.4,5.1,and 6.8 μmol L-1,and to the embryo culture medium (WPM with 20 g L-1sucrose,1.5 g L-1activated carbon,7.0 g L-1agar,0.2 mg L-16-BA,and 0.1 g L-1inositol) at concentrations of 0,0.2,0.6,and 1.0 μmol L-1.

2.4.Sampling time

Th e nu mbe r of da ys afte r po l l ina ti on (D AP)determined the sampling time (also known as the embryo rescue time). The ‘Flame Seedless’בMuscat Hamburg’,‘Kunxiang Seedless’בBeibinghong’,‘Fantasy Seedless’בShuangyou’,and ‘Ruby Seedless’בBeibinghong’ fruit crosses were collected at different times to explore the effects of sampling time on embryo rescue. To define the early embryo abortion time in the seedless varieties,we collected samples at 25 DAP,observed embryo abortion under a microscope,and determined the embryo abortion phases. The starting times of embryo abortion for these four female parents (‘Flame Seedless’,‘Kunxiang Seedless’,‘Fantasy Seedless’,and ‘Ruby Seedless’) were at 37,42,38,and38 DAP,respectively. Therefore,the embryos from the‘Flame Seedless’בMuscat Hamburg’ cross were collected at 37,38,39,40,41,and 42 DAP;those from the ‘Kunxiang Seedless’בBeibinghong’ cross were collected at 42,43,44,45,46,and 47 DAP;and those from the other two crosses were collected at 38,39,40,and 41 DAP.

2.5.Medium composition for malformed seedling development

After ovule development,the embryos were stripped and cultured on WPM supplemented with plant growth regulators. Due to the different degrees of injury experienced by the embryos during excision or for other reasons,some deformed seedlings were produced during this process. To improve the utilization of these malformed seedlings,we used the ‘Kunxiang Seedless’בBeibinghong’ and ‘Flame Seedless’בMuscat Hamburg’ crosses to identify a suitable medium that could improve the growth of malformed seedlings into plantlets.The media which were tested are described in Table 2.

Table 2 The composition of the three media1)

2.6.Data analysis

All experiments were designed randomly with three replicates per treatment. Percent embryo formation=Number of embryos formed/Number of ovules×100. Percent embryo germination=Number of embryos germinated/Number of embryos formed×100. Percent seedling rate=Number of seedlings/Number of embryos germinated×100. Percent transformation rate=Number of plantlets/Number of seedlings malformed×100. Data management and statistical analysis were performed using IBM SPSS Software (13.0) with one-way ANOVA.

3.Results

3.1.Effects of the different PAC concentrations on embryo rescue

Several crosses were selected to study the impacts of different PAC concentrations on the ovules. After 60 dof dark cultivation,there were no notable changes in color,size,or shape between the ovules treated with the different PAC concentrations,although some ovules were brown (Fig.2-A and B). The embryo formation rates of the ‘Flame Seedless’בBeibinghong’ and ‘Kunxiang Seedless’בTaishan-2’ crosses were the highest at a PAC concentration of 5.1 μmol L-1,with values of approximately 25 and 53%,respectively (Fig.2-C and D). The PAC concentrations that resulted in the lowest embryo formation rates in ‘Flame Seedless’בBeibinghong’and ‘Kunxiang Seedless’בTaishan-2’ were 3.4 and 0 μmol L-1,respectively.

Fig.2 Effects of the different paclobutrazol (PAC) concentrations on the grape ovules. A and B,ovules of the ‘Flame Seedless’בBeibinghong’ cross (A) and ‘Kunxiang Seedless’בTaishan-2’ cross (B),respectively,after 65 d of cultivation under darkness. C and D,embryo formation rates for the ‘Flame Seedless’בBeibinghong’ cross (C) and ‘Kunxiang Seedless’בTaishan-2’cross (D),respectively. Data are mean±SD (n=3). The lowercase letters indicate statistically significant differences with P＜0.05,as determined using the Duncan’s multiple range test.

We investigated the effects of PAC on embryo development in the ‘Sultanina Rose’בBeibinghong’are and ‘Sultanina Rose’בKunxiang Seedless’crosses. The results indicated that embryos treated with different concentrations of PAC exhibited different embryo germination and seedling rates (Fig.3). The highest seedling rates of 35 and 40% for ‘Sultanina Rose’בBeibinghong’ and ‘Sultanina Rose’בKunxiang Seedless’,respectively,were observed at a PAC concentration of 1.0 μmol L-1. The lowest germination and seedling rates for the ‘Sultanina Rose’בBeibinghong’ cross occurred in the medium without PAC,while those for the‘Sultanina Rose’בKunxiang Seedless’ cross occurred in the medium containing 0.6 μmol L-1PAC (Fig.3-C and D).

Fig.3 Effects of different paclobutrazol (PAC) concentrations on the embryos. A and B,embryos develop into plantlets for the‘Sultanina Rose’בBeibinghong’ cross (A) and ‘Sultanina Rose’בKunxiang Seedless’ cross (B),respectively. C and D,germination and seedling rates for the ‘Sultanina Rose’בBeibinghong’ cross (C) and ‘Sultanina Rose’בKunxiang Seedless’ cross (D),respectively.Data are mean±SD (n=3). The lowercase letters indicate statistically significant differences with P＜0.05,as determined using the Duncan’s multiple range test.

3.2.Effects of sampling time on embryo rescue

The sampling time significantly affected embryo rescue efficiency,because the embryos were difficult to save or even aborted if the sampling time was inappropriate.Therefore,the best sampling times for each cultivar must be identified to cultivate new seedless grapes through embryo rescue techniques. Here,we observed differences in the optimal sampling times between the female seedless grapes. The optimal sampling time for the‘Ruby Seedless’בBeibinghong’ cross was 40 DAP,which resulted in the highest embryo formation,germination,and seedling rates,although the seedling rate did not significantly differ between 40 and 41 DAP (Fig.4-A).For the ‘Flame Seedless’בMuscat Hamburg’ cross,the highest embryo formation,germination,and seedling rates were achieved at 39 DAP (Fig.4-B). The optimal sampling time for the ‘Kunxiang Seedless’בBeibinghong’cross was 46 DAP (Fig.4-C),while that for the‘Fantasy Seedless’בShuangyou’ cross was 38 DAP(Fig.4-D). Notably,the sampling time exerted a greater effect on the ‘Kunxiang Seedless’בBeibinghong’ and‘Flame Seedless’בMuscat Hamburg’ crosses than on the ‘Ruby Seedless’בBeibinghong’ and ‘Fantasy Seedless’בShuangyou’ crosses.

Fig.4 Effects of sampling time on the different crosses. Embryo formation,germination,and seedling rates for the ‘Ruby Seedless’בBeibinghong’ (A),‘Flame Seedless’בMuscat Hamburg’ (B),‘Kunxiang Seedless’בBeibinghong’ (C),and ‘Fantasy Seedless’בShuangyou’ (D) crosses. DAP,days after pollination. Data are mean±SD (n=3). The lowercase letters indicate statistically significant differences with P＜0.05,as determined using the Duncan’s multiple range test.

3.3.Effects of plant growth regulators on malformed seedling development

During the embryo rescue process,malformed seedlings were often produced due to either damage while handling or unsuitable growth conditions. Different types of malformed seedlings grew in media with various growth regulator concentrations,which affected their development into normal plantlets (Fig.5-A and B). The malformed plantlets from the ‘Kunxiang Seedless’בBeibinghong’ and ‘Flame Seedless’בMuscat Hamburg’ crosses were inoculated in three types of media (media A,B,and C),and exhibited different development rates (Fig.5-C). There were no clear differences for the ‘Kunxiang Seedless’בBeibinghong’cross among these three media combinations;however,the transformation rate of the ‘Flame Seedless’בMuscat Hamburg’ cross on medium C was 40.91%,which was the highest among the three treatments (Fig.5-D).

Fig.5 Transformation and utilization of the malformed seedlings. A,malformed seedlings. B,malformed seedlings grown into normal plantlets. C,transformation rate of the ‘Flame Seedless’×’Muscat Hamburg’ cross. D,transformation rate of the ‘Kunxiang Seedless’×’Beibinghong’ cross. Data are mean±SD (n=3). The lowercase letters indicate statistically significant differences with P＜0.05,as determined using the Duncan’s multiple range test.

4.Discussion

Embryo rescue techniques are widely used by plant breeders,and have been used in many fruit crops to date,including apple (Dantaset al.2006),banana (Umaet al.2011),citrus (Xieet al.2014),mango (Krishna and Singh 2007),muskmelon (Nu?ez-Paleniuset al.2006),peach(Andersonet al.2002),persimmon (Leng and Yamamura 2006;Huet al.2013),and watermelon (Ta?k?net al.2013),for purposes such as seedless breeding,triploid breeding,and interspecific breeding. Ramming and Emershad (1982)first obtained their new seedless grape cultivars from seedless female parents through embryo rescue,and this technique has been widely applied by plant breeders to rescue inherently weak,immature,and/or abortive embryos and to overcome the failure of endosperm development in interspecific,intergeneric,and interploid hybridizations (Zhuet al.2013;Liet al.2015;Yanet al.2019).

Recent studies have revealed that the influences of female and male parents on embryo formation and seedlings are noteworthy. Furthermore,embryo formation,germination,and plantlet development have been found to vary greatly between the different female parents. Cainet al.(1983) observed that genotype clearly influences the capacity of seedless variants to form viable embryos in culture. In this study,seven crosses between five seedless and five seeded cultivars were selected,and the results showed significant differences in the ovule germination and embryo formation capacities of the six seedless grapes. When ‘Flame Seedless’ was used as the female parent,the rates of embryo germination and seedling formation from the ovule culture were much higher than those obtained when Perlette or Sultanina were used (Spiegel-Royet al.1985). Both female and male parent genotypes can greatly affect the embryo formation,embryo germination,and plant development capacities in the context of grape embryo rescue(Spiegel-Royet al.1985;Liuet al.2008;Ji and Wang 2013). We also found that crosses with different male parents produced significantly different numbers of viable embryos when crossed with the same seedless cultivar.Different male parents can significantly affect embryonic development and plant recovery at certain sampling times(Grayet al.1990),and even cause ovule blackening and germination. Essentially,crossing with different female parents will influence the seeded male parents (Ebadiet al.2004). Previous studies suggested that female parents,such as ‘Ruby Seedless’,‘Blush Seedless’,‘Sultanina Rose’,‘Perlette’,and ‘Flame Seedless’,were superior to ‘Thompson’,‘Crimson Seedless’,and ‘Pink Seedless’ (Liet al.2015;Liuet al.2016;Jiaoet al.2017). Furthermore,the ripening season and seed trace size also influenced the number of ovules with normal embryos,embryo germination,and plant transportation(Pommeret al.1995).

In addition to parental selection,the sampling time,media,culture methods,and plant growth regulators largely determined the success of the embryo rescue in grape (Spiegel-Royet al.1985;Grayet al.1990;Ledbetter and Shonnard 1990;Sharmaet al.1996).Plant growth regulators such as 6-BA,GA3and IAA(Liet al.2014) are important for embryo rescue.Only a few previous studies have considered the effects of PAC on grape embryo rescue. However,the effects of several other plant growth regulators,such as 2,4-dichlorophenoxyacetic acid,kinetin,benzylaminopurine,naphthoxyacetic acid,and putrescine,on embryo rescue have been tested (Singhet al.1992),and were found to behave differently during embryo rescue by impacting embryo formation and germination,as well as polyembryos and normal plantlets (Ponceet al.2002). In this study,we found that applying exogenous PAC,which acts as an inhibitor of GA biosynthesis,could promote embryo formation during grape embryo rescue for most of the crosses. Therefore,PAC has significant application potential in grape embryo rescue breeding. Previous studies have demonstrated that differences in endogenous GAs between seeded and seedless grapes were associated with early seed abortion (Iwahoriet al.1968),and high GA levels caused seed abortion during the first stages of grape seed development (Agüeroet al.2000). Accordingly,as an inhibitor of GA biosynthesis,PAC supplementation could decrease the endogenous GA level during seed development and improve the rate of embryo growth into plantlets. However,the optimal concentration of the PAC application varied for different hybrid crosses,which may be related to the GA levels or the degree of embryo abortion. This will cause confusion when selecting the best PAC concentration. Based on our results,a 5.1-μmol L-1concentration of PAC may be suitable for grape embryo rescue,so it can promote embryo formation and development for most grape crosses. However,the impact of PAC on the development of grape embryos requires further in-depth investigation.

Managing the embryo abortion process and determining the optimal time to culture ovules are both essential for embryo rescue and ensuring the highest chance of mature embryo development. Therefore,many previous studies have investigated the importance of sampling time and confirmed that the sampling time was influenced by many factors,such as the embryo developmental stage (Spiegel-Royet al.1985;Grayet al.1990;Guoet al.2011),onset of berry ripening(Notsukaet al.2001),and genotype (Xuet al.2005;Yanget al.2007). In this study,the optimal sampling time differed among the four crosses. The most suitable sampling time for the ‘Flame Seedless’בMuscat Hamburg’ cross was 39 DAP,based on the embryo formation and seedling rate,while that for the ‘Kunxiang Seedless’בBeibinghong’ cross was 46 DAP,and that for the ‘Ruby Seedless’בBeibinghong’ and ‘Fantasy Seedless’בShuangyou’ crosses was 41 DAP. Jiet al.(2013)determined the optimal sampling times for 11 different hybrid combinations,which ranged from 39 to 63 DAP,to identify the highest germination rate. The sampling times were also found to be flexible if the crosses had the same female parents but different male parents. For example,when ‘Ruby Seedless’ was selected as the female parent and crossed with Black Olympia,the suitable sampling time was 63 DAP (Ji and Wang 2013);however,when crossed with ‘Flame Seedless’,the optimal sampling time changed to 44 DAP (Jiaoet al.2017). Our results are consistent with this finding. When ‘Beibinghong’ was selected as the male parent and ‘Kunxiang Seedless’ was selected as the female parent,the suitable sampling time was 46 DAP;however,when ‘Ruby Seedless’ was the female parent,the suitable sampling time was 41 DAP.Overall,significant differences were identified between the different hybrid combinations,and the influence of the female parent on the sampling time was greater than that of the male parent. Therefore,in practice,earlier sampling could be more beneficial to the embryo formation and seedling rates of the female parent variety with early embryo abortion when conducting grape seedless embryo rescue breeding.

Only a small number of zygotic embryos was obtained from each cross,and the probability of these embryos having normal embryo growth was extremely low,with only a few growing into plantlets normally.Previous studies found that many malformed seedlings were produced after embryo germination,which is not conducive to grape breeding (Wanget al.2007;Tanget al.2009;Haoet al.2010). Currently,the limiting factors for deformed seedlings remain unknown. We investigated the effect of media type when recovering malformed seedlings into plantlets,and found that medium C,with 0.5 g L-1of IBA,induced the highest transformation rate.Previous studies have investigated the influences of factors such as genotype,embryo type,and pre-chilling on the malformation rates and attempted to improve the breeding efficiency of seedless grapes by reducing the number of deformed seedlings during the embryo rescue process (Jiet al.2013). Our results provide some new data for the investigation of grape embryo rescue technologies and may aid in reducing the number of abnormal plantlets.

5.Conclusion

In this study,we tentatively concluded that the PAC concentration affects the embryo formation rate (1.5 μmol L-1PAC) and seedling rate (1.0 μmol L-1PAC). The different cross combinations exhibited various suitable sampling times,ranging from 39 to 46 DAP.Medium C,which contained 0.5 g L-1of IBA,was the most suitable transformation medium for the crosses investigated in this study. Grape embryo rescue will be vital for obtaining new cultivars that are resistant to various biotic and abiotic stresses,as well as highquality seedless grapes. Chinese wild grape species have many related advantages and should be used as the male parents in hybridizations withV.viniferacultivars.

Acknowledgements

This research was supported by the Key Industry Chain of Innovation Project in Shaanxi Province,China(2021ZDLNY04-08),the Fundamental Research Funds for the Central Universities,China (2452019016),the China Agriculture Research System of MOF and MARA(CARS-30-yz-7),and the Ningxia Hui Autonomous Region Flexible Introduction of Scientific and Technological Innovation Team,China (2020RXTDLX08).

Declaration of competing interest

The authors declare that they have no conflict of interest.