“In Vitro” Ovule Culture to Improve Genetic Variability in Hydrangea macrophylla

Abstract

In flowering plants, such as Hydrangea macrophylla, the main breeding objective is to increase genetic variability in ornamental traits. This study investigates in vitro techniques, through ovule culture, to overcome the hybridization barriers and increase the efficiency of crossing in Hydrangea macrophylla in which breeding has been hampered by a fairly long breeding cycle and lack of information about its genetic resources. Two different types of media were compared, Gamborg B5 and Murashige and Skoog basal salts, to verify the germination rate of immature ovules in different intraspecific crosses. The germination rate and viability of the seedlings were influenced by the parental genotypes in the different combinations of crossing, highlighting, in some cases, the poor compatibility between some of them. The crossing combination “Parental A × Parental B”, showed the highest germinated ovules percentage (78.3%). The media used seem to less affect the ovule germination while mainly influencing the development and growth of the young seedlings and in particular the number of leaves, the branching attitude, and root length, with the Gamborg medium determining up to a 30% increase, compared to MS medium. In addition, we tested the effectiveness of using SSR markers to assess the parentage of the putative hybrids even though only three out of twelve SSR markers showed allelism. Although the number of SSR markers was low, they were allowed to profile the parentage according to Mendelian laws.

1. Introduction

The genus Hydrangea L. includes about 75 species, mainly distributed in the American and Asiatic continents [1]. It is a very popular ornamental plant for garden and interior decoration. Recently it has been commercialized as a fresh and dried cut flower.

Interest in Hydrangeas is mainly due to the particular pink, blue, white, light purple, or dark purple colored inflorescences (corymbs or panicles). Flowers are produced from early spring to late autumn and have two inflorescence morphologies: mophead flowers are large and have spherical flower heads; in contrast, lace-cap bear round, flat flower heads with a center core of subdued, fertile flowers surrounded by outer rings of showy, sterile flowers. To expand its market, new hybrids and cultivars should be developed. In flowering plants, the main breeding objective is to increase variability in ornamental traits such as flower color, flower shape and plant architecture.

To achieve this objective, intra- and inter-specific hybridizations have been widely used in breeding strategies. Hybrids between H. macrophylla (Thunb.) Ser. and H. paniculata Sieb. were produced using embryo rescue, but the resulting plants were sterile and lacked vigor [2,3,4,5]. In vitro embryo rescue procedures have been used to facilitate the recovery of interspecific hybrids of many genera [6,7], and have recently been used to recover a putative H. macrophylla (Thunb.) Ser. × H. arborescens L. hybrid [8,9]. Hybrid embryos often resume growth and develop into normal plants when removed from the ovary and placed on an aseptic nutrient medium. In some cases, entire fertilized ovules have been placed into culture, and the hybrid plant recovered; this procedure is known as ovule culture [10].

Interspecific hybrids between H. macrophylla “Schneeball” × H. arborescens “Annabelle” via ovule culture were achieved by Cai et al. [11] and the new genotypes obtained have been subjected to morphological, cytological, and molecular analysis. In vitro germination of seeds is a common technique used to overcome incompatibility barriers in ornamental plant hybrids [12,13,14,15,16]. Since most Hydrangea seeds are so small (0.5 mm diameter) and hybrid seed production is usually low (0–5 seeds/fruit), the in vitro method for Hydrangea fertilized ovules rescuing is an important tool to obtain new variability.

To this aim, we have tested the effects of some compounds such as activated charcoal (AC) and Plant Preservative Mixture (PPM^®, Plant Cell Technology, Inc., Washington, DC, USA) to improve ovule germination. In fact, several studies have shown a positive effect of AC on plant development in in vitro culture [17]. The promoting effects of AC on morphogenesis may be mainly due to the adsorption of inhibitory compounds in the culture medium and substantially decreasing the effect of toxic metabolites and phenolic exudation. In addition to this, AC is involved in the release of substances that promote growth, alteration, and darkening of culture media and adsorption of vitamins. Moreover, some studies confirm that AC may gradually release certain adsorbed products, such as nutrients and growth regulators that become available to plants [18]. To increase seed germination, Greer and Rinehart [19] have developed an in vitro method for the cultivation and assay of H. macrophylla (Thunb.) Ser. and H. paniculata Sieb. seeds, through germination on solid media in conjunction with PPM^®, and by sterilizing seed with trichloro-s-triazinetrione (Trichlor).

To increase the efficiency of hybridization and to provide new genetic material suitable for cultivation as a cut flower or as a potted plant, a number of techniques have been developed to support genetic improvement in Hydrangea. The objective of this study was to develop techniques for hybrids recovery in Ortensia testing an ovule culture procedure and identifying the culture medium that could be used to support the growth of immature ovules. In addition, we tested the possibility of using SSRs to discriminate individuals with different parental alleles. In fact, molecular marker technology (SSR) can be used for inferring parentage or paternity of the putative hybrids by considering, for assumed parent-offspring triads the inheritance of codominant molecular markers (alleles) from parents to offspring according to the Mendelian rule [20,21]. The present paper makes an innovative contribution on how to enlarge variability for obtaining new genotypes suitable for cut flower Hydrangea cultivation starting from the main CVS used nowadays. In this way new genotypes with interesting characters such as reblooming during the whole season from late spring to the first frosts, strong and upright branches, can be obtained. Furthermore, the use of SSR markers has been used, demonstrated to be a valuable tool to greatly reduce selection time in the field.

2. Materials and Methods

2.1. Plant Material

The plant material utilized in this experiment has been obtained from the Council for Agricultural Research and Economics—Research Center for Vegetables and Ornamental Crops, in Pescia (PT) Italy (43°49′00″ N; 10°48′00″ E), and from Mansuino farm, in Sanremo (IM) Italy (43°49′ N; 7°47′ E). The plants were grown in greenhouses under 70% shading. Plants remained in the greenhouse until all ovules were collected for culture. During the summer of 2021, 800 crosses among different genotypes, belonging to H. macrophylla ssp. macrophylla (38 cultivars used for pot plants and cut flowers), were made. New genotypes were obtained by hand pollinating of the emasculated flowers with pollen collected the day before and kept at a temperature of 4 °C. The main characteristics of the selected parental lines are reported in

Table 1. Flower characteristics and origin of different cultivars of Hydrangea macrophylla used in breeding activities to obtain new hybrids.

Flower Image	Name	Description
	Amor blue® Breeding goal: pot plant	It is a deciduous shrub cultivated for its showy inflorescences, with ovate glossy toothed dark leaves. The large rounded clusters of mophed flowers are intense blue during the summer time.
	Parental CBreeding goal: Europe red	Hydrangea macrophylla with a large-leaved, deciduous shrub species that produces huge, showy pink, tipped with green gold mop-head shaped flowers during the summer. It is common for garden use.
	Endless Summer Bloom Star Breeding goal: tropical red	It is an upright, bushy deciduous shrub with broadly ovate, toothed, pointed dark green leaves and, from early summer to fall. It has an endless procession of stunning vivid purple or rose pink colored blooms that sit above strong sturdy red stems.
	Koria Breeding goal: Pot plant	It is a lovely lace-cap variety, conspicuous for its serrated bracts that surround a center of blue or pink flowers, set against apple-green foliage.
	Forever&Ever® “Light Purple” Breeding goal: Tropical red	It belongs to a group called Forever&Ever® and to Hydrangeas that have proven extended blooming time and excellent hardiness. It produces large, globular, mophead flowers on both old and new wood so do not worry about pruning or unexpected frost. It starts blooming on the first hot days of spring and will continue until the first frost. Deadheading will enhance flowering and speed up the forming of new flower buds.
	Rodeo Breeding goal: Europe red	Compact spherical shrub up to about 1–1.5 m tall. Deciduous. Large green leaves. Large spherical inflorescence. Deep pink flower color. Flowering period: July—September. Sunshade. Nourishing soil.
	Parental B Breeding goal: Tropical pink	Native to Colombia is a popular variety grown for cut flowers, which can also be used for drying.
	Verena® Breeding goal: Europe pink	Popular cut flower which can also be used to dry. Plant height: 50–70 cm. Flower color: light pink. Easy pruning; easily by coloring in autumn tones; blooming richly also at low temperatures; good variety to bluish.
	Wedding Ring Breeding goal: Europe pink/blue	Is a trade name for “Fanfare”, a new variety originating from the United States where Hydrangea is the “trend” shrub of the moment. It is a hybrid of the species Hydrangea macrophylla with ball-shaped flowers. This variety is notable for its cold hardiness in winter (it adapts well to USDA Zone 5 winters) compared to many of the commercially available cultivars of H. macrophylla today.
	Parental D Breeding goal: Tropical white	Colombian Hydrangea, old varieties imported many years ago in South America, probably from France or the USA, and never correctly identified. Recurrent blooming.
	Hybrid 1 Breeding goal: Europe pink/white	New selection tropical white/pink (patent pending material).
	Parental A Breeding goal: Europe red	These plants are very beautiful and can be expected to throw out up to 30 strong flower heads during their first flowering season. A wonderful Hydrangea variety with huge flowers often found in high-class florist bouquets, it has massive blooms.

. These parentals were chosen because they were considered among the most interesting in the Hydrangeas marketed as cut flowers, while some of them were selected by the company awaiting patent.

2.2. Crossing Procedure

Sterile flowers were removed from inflorescences to be used as females before the opening of the fertile flowers. Any open flowers, along with all extremely immature fertile flowers, were removed. After the petals and anthers of all remaining fertile flowers were removed, the inflorescence was securely covered with a TNT bag to prevent unwanted pollination.

Inflorescences to be used as males in crosses were also bagged before flower opening. Pollinations were made 1 to 4 days after emasculation by the dispersion of previously collected pollen on the top of a corymb, aided by a brush. After pollination, inflorescences were covered with TNT bags which remained on the plants until collection time. Eight hundred hand pollinations were carried out from different species of the Hydrangea genus. Each set of crosses was repeated from 10 to 12 times.

Then, the capsules obtained from controlled pollinations were collected and used as starting material in the experiments, as described below.

2.3. In Vitro Ovule Culture

The ovule culture technique has been used to obtain new Hydrangea macrophylla genetic combinations from intraspecific crosses by collecting immature capsules, from 121–138 days after pollination (DAP), as reported in

Table 2. Hydrangea macrophylla crosses used in the two trials (A and B) of ovule cultures: female and male parentals and number of days (DAP) from the hand pollination of the female parent to the immature capsule collection.

Experiment A
Female Parentals ♀ (Code)		Male Parentals ♂ (Code)	D.A.P.
Amor blue® (Amor)	x	Endless Summer Bloom Star (ESBlo)	124
Verena (Vere)	x	Koria (Kori)	123
Rodeo (Rod)	x	Wedding Ring (Wedd)	121
Light Purple (Ligh)	x	Koria (Kori)	138
Parental A (ParA)	x	Parental B (ParB)	121
Experiment B
Parental C (ParC)	x	Parental A (ParA)	134
Parental D (ParD)	x	Hybrid 1 (Hyb1)	151

.

Two experiments (Trial A and B) were carried out and different types of culture medium were compared to investigate the germination rate of immature ovules in different intraspecific crosses.

Trial A: In this experiment, the ovule culture technique was applied to five Hydrangea macrophylla hand pollinated mother plants reported in Table 2, using two different media without hormones added. Basal media were Gamborg B5 (GA) including vitamins, and Murashige and Skoog basal salts (MS) including vitamins [22,23]. Sucrose and plant agar were added in the amount of 20 and 9 g/L respectively, at pH 5.7, before autoclaving at 121 °C for 20 min. For each genotype × medium combination, five replicates (Petri dishes) containing ovules were collected. Up to 40 immature seeds were placed in each Petri dish.

The capsules were sterilized through a wash with tap water and concentrated soap for 30 min, followed by a rinse with sterile water for 10 min and then in 70% ethanol for 1 min. This was followed by washing in a 2.5% active chlorine solution + 2 drops of Tween 20 and 1 mL/L of PPM with agitation for 30 min; finally, we proceeded with washing two times in sterile water for 10 min under a flow hood (Figure 1a). Then, working under a flow hood and with the help of a binocular, we proceeded with the collection of the ovules (Figure 1b). Each ovary was dissected with a longitudinal cut using forceps and scalpels, and then with the tip of a sterile syringe needle the individual ovules were taken, counted, and transferred to Petri dishes containing the two media (Figure 1c).

Petri dishes were then maintained in a growth chamber at a temperature of 23 ± 1 °C, under a light intensity of 35 µmol m⁻² s⁻¹ and a photoperiod of 16 h. Every two weeks, the germination of the ovules was assessed and the germinated ovules were transferred into special glass tubes, still containing the same medium. The contaminated ovules were removed from the experiment, even if the contamination did not interfere with the seed germination. The parameters measured to detect the degree of fertility of each cross combination are three: sprouted ovules percentage, live plants percentage, and total live plants. After about 160 days, we proceeded to the acclimatization by transferring the total surviving plants (Figure 1c) onto a medium based on peat and perlite (1:1) that was previously sterilized in autoclave. At the same time, some morphological parameters were measured on a representative sample of 15 plants for each medium and for each cross combination: number of leaves, plant height, root length, first internode height, number of branches, and plant weight.

Trial B: In the second experiment, the genotypes Parental C (ParC) × Parental A (ParA) and Parental D (ParD) × Hybrid 1 (Hyb1) were evaluated with the technique of ovule culture using two media (GA and MS), as already described for Trial A with (C1) and without (C0) activated charcoal (AC) at 0.1%. The same protocol as previously described was used for capsule sterilization and in vitro transfer. Sampling and measures were also performed in the same way as described for Trial A.

2.4. Genetic Analysis

Putative hybrids and parental lines (Table S1) were compared using 12 three-base repeat SSR loci that have been shown to produce polymorphic data in Hydrangea macrophylla [24]. Fresh, new leaves of the putative hybrids and parents were collected to extract DNA. 50–70 mg of fresh leaf tissue was allotted into a 2 mL tube with three tungsten carbide beads, frozen in liquid nitrogen, and finely ground in a tissue homogenizer (Tissue Lyser, QIAGEN). DNA was extracted by adopting the CTAB method [20]. Unfortunately, the leaf material of a putative hybrid of the “Parental A × Parental B” cross combination was not sufficient for DNA extraction.

PCR amplifications were performed in 25 μL reaction mixtures consisting of 20 ng of genomic DNA, 5 µL of colorless GoTaq^® 5X reaction buffer, 0.2 μL 10 mM dNTPs, 0.2 μL of 10 μM of each primer, and 1 U of GoTaq^® G2 DNA polymerase enzyme (Promega). The following PCR conditions were used: initial denaturation of 4 min at 94 °C, followed by 10 cycles of 94 °C for 40 s denaturation; 0.5 °C touch down from 60 °C to 57 °C for annealing; 72 °C for 40 s for extension, followed by 25 cycles at 94 °C for 30 s, 57° for 30 s, 72 °C for 30 s; and a final extension for 7 min at 72 °C.

Fragment analysis was performed by capillary electrophoresis in a QIAxcel^® Advanced system platform (Qiagen, Hilden, Germany). The technology adopted allows the separation of fragments with dimensional differences between 2 bp, whose resolution is sufficient to separate SSR amplicons with 3 bp repeats [25].

The PCR products were inserted directly into the QIAxcel Advanced system and separated using the QIAxcel DNA High Resolution cartridge with the OM800 method setting, which involves the following electrophoresis parameters: injection of the alignment marker at 5 kV for 10 s; injection of the sample at 5 kV for 5 s; and separation at 3 kV for 800 s. The QX 15 bp/600 bp alignment marker was run at the same time as the samples, while the size marker used was the QxSize Marker 25 bp/500 bp with fragments of: 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 bp. The separation took approximately 15 min. The results were analyzed with the QIAxcel ScreenGel Software to detect the size of the SSRs and the size grid composition.

2.5. Statistical Analysis

The data collected were subjected to analysis of variance (ANOVA) using the split-plot mixed model. Medium as main plot and Genotype (cross combination) as subplot were considered fixed-effect factors, while ovary within genotype was considered as random effects factor (replication). Differences among averages found to be significant with the F-test among the genotypes, in case of Trial A, were tested with Duncan’s multiple comparisons test. In order to summarize the observed variability between the various genotypes in the two media, principal component analysis (PCA) was performed.

In the Trial B, the analysis of variance was performed considering all three factors (genotype, medium, AC) as fixed effects. The variance component: between capsules within genotype [capsules(genotype)], was used as the most appropriate error. IBM SPSS 28 and NTSYS 2.20 N software were used for statistical analysis.

3. Results and Discussion

Most of the immature ovaries taken from the evaluated cross combinations had inside ovules, which were characterized by high variability, either in the number of enlarged and still viable ovules or for their shape, especially where they were numerous (Figure 2c–f).

Indeed, the non-uniform number of ovules present inside each capsule was found in almost all cross-combinations evaluated. This was probably due to the degree of compatibility between parentals, determining a variable number of ovules for each cross combination. Therefore, the parameter germinated ovules percentage, which is not significant, can appear misleading. In fact, due to the different number of ovules rescued a high percentage value rarely corresponds to an equally high number of live plants.

For example, the cross-combinations obtained from the “Parental A × Parental B”, despite having the highest germinated ovules percentage (78.3%), produced a low number of total live plants (about 2.3 total live plants) the same as “Rodeo × Wedding Ring” cross (77.9% of sprouted ovules and 6.7 total live plants) (

Table 3. Mean effects of parental genotype, medium, and genotype × medium interactions on the different parameters measured.

Source of Variation	Sprouted Ovules	Live Plants	Total Live Plants	Leaves	Plant Height	Root Length	Height 1st Internode	Plant Weight	Branches
	%		n.		mm			g	n.
Genotype (G)	n.s.	n.s.	*	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
Amor × ESBlo	51.32	97.50	5.63 b	13.66	14.43	21.89	1.16	0.14	0.45
Ligh × Kori	65.42	78.71	23.75 a	13.14	20.09	19.37	1.53	0.23	0.46
ParA × ParB	78.33	93.75	2.25 b	7.58	7.25	9.83	0.00	0.12	0.01
Rod × Wedd	77.89	83.10	6.67 b	13.75	13.26	23.31	0.31	0.16	0.57
Vere × Kori	57.41	88.54	11.18 b	13.85	23.00	16.20	1.25	0.14	0.33
Medium (M)	n.s.	n.s.	n.s.	*	n.s.	**	n.s.	**	*
GA	61.50	87.08	13.94	13.89	17.34	21.62	0.89	0.20	0.53
MS	65.25	87.15	10.91	12.26	17.65	16.34	1.18	0.14	0.29
G × M	n.s.	n.s.	n.s.	n.s.	**	n.s.	**	n.s.	n.s.

**: significant at p ≤ 0.001; *: significant at p ≤ 0.005; n.s.: not significant. Mean values within each column followed by the same letter are not significantly different at the 5% level, according to Duncan’s multiple range test.

). Only the cross combination “Light Purple × Koria” differs significantly for the high total number of live plants (with almost 24 live plants in total), even if it showed an intermediate value of germinated ovules, equal to 65.4% (Table 3). Therefore, the low statistical significance found is most likely to be attributable to the non uniform quality of the ovules taken inside the ovaries. It was possible to highlight the various cases found during the in vitro activity (Figure 2). In particular, Figure 2b,c shows unfertilized or prematurely aborted ovules, probably due to the occurrence of postzygotic barriers incompatibility [6,26]. Figure 2d,e shows a low number (one or a few) of enlarged, white and translucent ovules; finally, Figure 2f shows a relatively high number of ovules at the optimal stage for the transfer, thus suggesting the high compatibility between the parental genotypes. As reported by Reed [10], we also observed in the ovaries a part of the ovules was large and white opaque, while some were smaller and translucent. In the first case, the embryo was still inside the ovule, while in the second there was no evidence of an embryo. The small ovules represent either unfertilized ovules or ovules in which the embryo died at a very early stage of development due to the occurrence of early incompatibility barriers (Figure 2). The plants obtained from the experiments were normal in appearance shortly after germination. Most of the plants continued to grow until the medium was exhausted, then were transferred to the same medium, but individually in glass tubes (Figure 3). All of these plants survived and developed into normal-appearing acclimatized plants.

The two cross combinations that use Koria as male parental (“Verena × Koria”, “Light Purple × Koria”) are those with the highest total number of live plants, while the other crosses “Rodeo × Wedding Ring”, “Amor × Endless Summer Bloom Star” and “Parental A × Parental B” are characterized by the lowest values of the total number of live plants (Table 3). As reported by previous studies, the presence of capsules with non-uniform number of immature ovules is frequent in intra and interspecific crossings [10]. The success of the embryo-ovule rescue relies on a number of factors, including the choice of explants, culture medium, etc. [26], but also on the crossing combination. Very important is if the same genotype is used as female or male parents, in particular in interspecific crosses the numbers of fertile progeny increases when H. macrophylla is the maternal parent [11]. The degree of reproductive barrier in hybridization was reported depending on used lines in many crops (such as Dianthus, Brassica, etc) [27].

The total live plants have evidenced statistically significant differences for the source of variation “Genotype”, where “Ligth × Koria” cross combination showed the highest number of total plants survived (23.75) compared to the others, the other parameters, even if showing a high level of difference (i.e., root length), the high variability of the samples representing the various cross combinations did not result statistically significant (Table 3). This is probably due to the power of the statistical test. It is not necessarily the case that all variables analyzed have the same power; it depends on their distribution. The medium affected the number of leaves, the branching attitude, and, above all, plant weight and root length, with the GA medium determining a 30 and 24 percent increase in the latter two variables, respectively Table 3; therefore, suggesting a better development of the plants grown on GA than those maintained on MS. In fact, as reported by Reed [10] the GA media with 2% of sugar, allowed to obtain the highest germination rate of intraspecific ovules cultured on H. macrophylla. Lazzereschi et al. [14] also on Hydrangea, but on a mature seed culture, showed how the GA media hormone-free ensured a higher germination rate (80%), against 20% of the media based on MS salts. Therefore, also for these authors, GA medium seems to be the most appropriate for the development of the in vitro germination technique of mature and immature seeds of Hydrangea.

The interaction parental genotype × medium, revealed significant differences for the variables plant height and height 1st internode, while all other variables were not significant (Table 3 and Figure 4). For both parameters, the differences emerged between “Verena × Koria” cross combination grown on MS compared to GA medium.

Multivariate analysis was performed by considering all nine measured variables, and 6 Principal Components (PCs) expressing a cumulative variance of 93.7% were extracted from the orthogonal transformation of the matrix (

Table 4. Description of the total variance by the main components before and after the Varimax rotation.

Component	Total	% of Variance	% Cumulative
1	3.408	37.864	37.864
2	1.402	15.578	53.442
3	1.249	13.873	67.314
4	0.943	10.481	77.796
5	0.844	9.375	87.171
6	0.586	6.506	93.676

).

In particular, the first three PCs accounted for 67.3% of the total variance: the first component (PC1) accounted for 37.9%, the second component (PC2) for 15.6% and the third for 13.9%. The first component (PC1) is positively associated with the following variables: plant weight (0.817), plant height (0.775), total number of leaves (0.744), first internode height (0.726), root length (0.681), and total live plants (0.676) (

Table 5. Rotated component matrix. Contribution of the original variables in determining the main components after the Varimax rotation.

	1	2	3	4	5	6
Plant weight	0.817	0.015	0.070	0.368	−0.280	−0.081
Plant height	0.775	−0.454	−0.161	−0.135	0.190	0.069
Total leaves	0.744	0.407	0.037	−0.354	0.228	−0.018
Height 1st internode	0.726	−0.476	−0.083	−0.318	0.172	0.180
Root length	0.681	0.246	−0.242	0.287	0.197	−0.506
Total live plants	0.676	−0.139	0.335	0.284	−0.458	0.219
Branches	0.356	0.748	0.222	−0.383	−0.212	0.119
% Live plants	0.096	0.387	−0.708	0.355	0.141	0.440
% Sprouted ovules	0.040	0.123	0.698	0.357	0.578	0.174

). The second component (PC2) was positively correlated with the branch number. The third (PC3) is positively associated with the percentage of germinated ovules (0.698) and negatively associated with the percentage of live plants (−0.708).

Although the analysis of the variance has evidenced a low statistical significance among the different cross combinations analyzed, it has been possible through the multivariate analysis to obtain a separation according to the cross combination × medium detectable by the 2D graphic representation (Figure 5).

The parental genotype therefore seems to have a greater weight on the separation of the accessions, higher than the type of medium used, in fact in Figure 5, it is possible to show groupings only for “Rodeo” × “Wedding Ring” and “Amor blue^®” × “Endless Summer Bloom Star” cross combinations, grown on both media. The other cross combinations seem to be affected by the interaction with the culture medium because their scattered distribution in the 2-D plot is defined by the two first principal components (Figure 4). Based on these considerations, we can therefore consider the group of crosses highlighted with the circle to be optimal.

In Trial B the aim was to evaluate the effect of the addition of activated charcoal (AC) to the two media (GA and MS) in the ovule rescue culture. The mean differences obtained by ANOVA are shown in

Table 6. Mean effects of parental genotype (G), medium (M), activated charcoal (AC), and of the different interactions on the parameters measured on Trial B.

Sources of Variation	Sprouted Ovules	Live Plants	Total Live Plants	Leaves	Plant Height	Root Length	Height 1st Internode	Plant Weight	Branches
	%		n.		mm			g	n.
Parental genotype (G)	n.s.	n.s.	**	n.s.	**	n.s.	**	*	n.s.
ParC × ParA	84.38	88.42	6.15	13.13	14.52	28.18	1.12	0.20	0.23
ParD × Hyb1	87.68	83.82	2.29	15.17	27.00	34.62	1.92	0.37	0.32
Medium (M)	n.s.	*	*	n.s.	n.s.	*	n.s.	n.s.	n.s.
GA	85.99	90.45	5.17	13.51	21.74	44.31	1.84	0.28	0.26
MS	85.81	82.39	3.63	14.58	18.84	18.66	1.16	0.27	0.29
Activated charcoal (AC)	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	**	n.s.	n.s.
C0	86.74	84.01	3.89	13.62	17.88	29.77	1.00	0.26	0.18
C1	85.10	88.49	4.84	14.49	22.49	32.44	1.95	0.29	0.36
G × M	*	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
G × AC	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
M × AC	*	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.
G × M × AC	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.

**: significant at p ≤ 0.001; *: significant at p ≤ 0.005; n.s.: not significant. Mean values within each column followed by the same letter are not significantly different at the 5% level according to Duncan’s multiple range test. C0 = without AC, C1 = with AC.

. Significant differences between the two cross combinations evaluated were found for total live plant number, plant height, height 1st internode, and plant weight (Table 6). The “Parental C” × “Parental A” shows a higher and statistically significant number of total live plants than the “Parental D × Hybrid 1” cross combination. The other three variables showed significantly higher mean values in “Parental D” × “Hybrid 1,” which is thus characterized by taller plants with a longer 1st internode and higher weight, compared to the “Parental C × Parental A” cross. However a high dissimilarity in the development of seedlings and among the ovules within the ovaries was detected [16], thus confirming what was previously shown in Trial A.

Significant differences in root length and total number of live plants were observed in the two different substrates (Table 6). Plantlets grown on GA showed higher root length (44 mm) than those grown on MS medium (19 mm), as well as higher % (90.5 vs. 82.4%) and the total number of live plants (5.2 vs. 3.6) on GA compared to MS, confirming the superiority of the former medium.

The presence of AC in the ovule culture medium significantly affected only the height of the 1st internode; its presence promoted elongation of the 1st internode, which was twice that found in plants grown in the absence of AC.

First order interactions, between the different sources of variation, were significant for the sprouted ovules percentage. This parameter was significant in parental genotype (G) × medium (M) and medium (M) × AC interactions. However AC addition showed a significant effect on the height of 1st internode, but also on plant height even if not significant (17.88 vs. 22.49 mm). AC appears to have contrasting effects depending on the cultured plant species. For example, AC promotes the development of explants in somatic embryogenesis and embryogenesis in other cultures of Anemone and Nicotiana [28]. Moreover, it cannot be excluded that some of the elements that are not present in the medium but are released in minor amounts from AC, may be beneficial to embryogenesis, as suggested by Weatherhead et al. [29]. The addition of AC in some plants can also determine inhibitory responses for its presence in the culture medium, indeed AC induced negative results were also reported in some systems. AC inhibits growth shoot formation in Cymbydium forrestii [30] and in somatic embryogenesis in Rosa Hybryda [31]. However, some beneficial effects of AC can be attributed to the removal of inhibitory substances from the medium or by absorption of toxic brown/black compounds [18]. In the paper not being significant the effect of AC is necessary for an ulterior deepening, testing a greater number of genotypes, in order to be able to assert what is the effect of AC on this species with greater safety.

Twelve primer pairs producing SSR fragments were tested for DNA amplification [32]. Only three SSR markers (STAB 305_306, STAB 317_318, and STAB 321_322) showed different profiles in the parental lines and were chosen for further analysis, while nine did not produce any polymorphic band to infer any parentage or paternity (

Table 7. Allele size range (bp) of three SSR markers used in the analysis of parental and hybrids of Hydrangea macrophylla.

Sample	Parental	SSR Markers
		STAB 305_306			STAB 317_318			STAB 321_322
Amor	♀	130					154		148	151
ESBlo	♂		133	136		148	155	137		151
Amor × ESBlo	Hybrid	130		136		148	154			151
Vere	♀	130		136			154			151
Kori	♂	130		136		148	154			151
Vere × Kori	Hybrid	130		136			154			151
Rod	♀		133	136		148	154		148	151
Wedd	♂		133	136			154			151
Rod × Wedd	Hybrid	130		136			154			151
Ligh	♀	130	133				154	136		151
Kori	♂	130		136		148	154			151
Ligh × Kori	Hybrid		133	136			154	136		151
Parental C	♀	130		136			154			151
Parental A	♂			136		148	154		148	151
ParC × ParA	Hybrid	130		136		148	154			151
ParD	♀	130		136	145		154			151
Hybr1	♂		133	136	145		154			151
ParD × Hyb1	Hybrid		133	136	145	148	154	136		151

). As reported in Table 7, SSR marker analysis conducted with the three polymorphic primer pairs, allowed us to detect the distribution of the parental alleles in the hybrids, according to the Mendelian segregation rule.

In this study, we have adopted a direct codominant interpretation of SSR loci as a reliable approach to determine the allelic configurations and then the origins of the hybrids artificially obtained. The established system, to be discriminating, requires SSR markers with polymorphic alleles different between the two parents and heterozygous in at least one parent. As for many ornamental crops, breeding is applied to Hydrangea in order to create new varieties whose primary accessions are selected in the F1 generation, followed by vegetative propagation. Thus, Hydrangea cultivars are expected to possess a distinctive, predominantly heterozygous genotype, which should be easily distinguishable using SSR molecular markers. Noteworthy, this does not seem to hold for the parental lines we have used in our experiment considering that only three out of 12 SSR markers proved to be sufficiently informative to infer parentage of the hybrids. Hempel et al. [22] in their extensive study on 120 Hydrangea macrophylla accessions genetic variability came across a low allelism among some cultivars which in few cases even resulted undistinguishable, also [33] compared 39 Hydrangea macrophylla varieties using 38 SSR markers: they also found a low allelism as resulted from the average effective number of alleles of 2.36 for all loci indicating that the experimental group of cultivars has low diversity level. Therefore the low diversity within the species may explain why in our case in which a limited number of cultivars has been analysed, the SSR marker polymorphism was so low. Therefore, even if the discriminatory power of the test is proportional to the number of loci evaluated and the polymorphism of each DNA marker, we can state that, as the parental lines were known to be used in the pollination protocol, the markers used are sufficient to prove the hybrid origin of the crosses.

4. Conclusions

In floriculture, improvements in combinations of desirable traits can be achieved through breeding approaches, such as crosses within H. macrophylla, or interspecific hybridization between H. macrophylla and closely related species. The breeding activity that the research group was carrying out within the species Hydrangea macrophylla for several years, complemented by in vitro culture of ovules, has resulted in the development of some interesting crosses. The collected data highlight how the ovule culture technique applied to Hydrangea, though laborious, has allowed us to obtain viable plants from the tested combinations of crossing, in a relatively short time. Moreover, the viability of the seedlings was affected by the parental genotypes of the crosses, highlighting, in some cases, poor compatibility. Plants obtained from intraspecific experiments were normal in appearance, survived, and developed into normal-appearing mature plants. The tested media showed significant differences with GA proving to be preferable to MS for ovule rescueing. Therefore, the ovule culture is a promising technique that may have practical applications, allowing the possibility to cross genetically distinct accessions and therefore increasing the genetic variability within the Hydrangea genus. This could lead to the development of new varieties for the flower market. According to our results, the assessment of the hybrid origin of the crosses can be effectively carried out with a limited number of informative SSR loci allowing us to compare the allelic profiles of the parent-offspring triads.

The method hereby described was able to achieve hybrids whose commercial characteristics are currently under evaluation and selection within a network of Italian and foreign companies.