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Article

Study on the Compatibility of Distant Hybridization Between Rhododendron Subgenus Tsutsusi and R. moulmainense, a Fragrant Rhododendron from China

by
Hongling Li
1,†,
Jing Qi
1,†,
Lele Wang
1,
Jie Song
2,
Yan Zhao
1,
Yefang Li
1,* and
Wenling Guan
1,*
1
Faculty of Landscape and Horticulture, Yunnan Agricultural University, Kunming 650201, China
2
Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650201, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(9), 1116; https://doi.org/10.3390/horticulturae11091116
Submission received: 22 July 2025 / Revised: 5 September 2025 / Accepted: 10 September 2025 / Published: 14 September 2025
(This article belongs to the Section Floriculture, Nursery and Landscape, and Turf)
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Abstract

Fragrant rhododendron varieties remain relatively limited in current germplasm resources, constraining the enhancement of ornamental and aromatic characteristics in rhododendron breeding—this limitation has emerged as a critical bottleneck in the genetic improvement of rhododendrons. This research takes fragrant flower breeding as the breeding objective and conducts hybridization between varieties of the subgenus Tsutsusi, which can flower in multiple seasons and exhibit relatively strong resistance, and the fragrant R. moulmainense. Parallel intraspecific hybridizations within the subgenus Tsutsusi were implemented as experimental controls to quantify hybridization affinity. This study combines floral tube ontogeny histomorphological analysis, ovary paraffin sectioning, and optimized pollination protocols to address hybridization constraints, providing new insights for rhododendron intersubgeneric distant hybridization to create fragrant varieties. The results showed varying fertility among combinations, with some showing sterility or weak fertility due to low pollen germination and abnormal embryo development. Both pre- and post-fertilization reproductive barriers were observed, and different pollination methods significantly influenced ovary expansion and fruit set rates. Regarding limitations, this study lacks an in-depth analysis of reproductive isolation mechanisms, only describing phenotypic characteristics through morphological and histological methods, and it does not employ molecular techniques. The fundamental causes of reproductive isolation between subgenera therefore remain unclear. Additionally, there was no long-term monitoring of seedling emergence rates, hybrid plant growth potential, or flowering traits. This limits the ability to comprehensively evaluate the breeding value and genetic stability of distant hybrids.

1. Introduction

The genus Rhododendron is the most species-rich genus within the family Ericaceae, comprising approximately 1000 species distributed across Asia, Europe, and North America, with two species native to Australia. China harbors 571 species, including 409 endemic taxa [1,2]. Due to their extremely high ornamental value and diverse flower colors, Rhododendron plants have emerged as world-famous horticultural flowers and potted plants and are extensively utilized in global landscape greenery and home gardening [3]. Among them, the subgenus Azalea is noted for its vivid flower colors, strong adaptability, and ease of cultivation and maintenance. It constitutes the group with the greatest variety and the widest application within the Rhododendron genus. Nevertheless, the vast majority of its species and varieties do not possess a notable fragrance [4]. On the contrary, the subgenus Azaleastrum, represented by R. moulmainense, is a rare fragrant wild rhododendron [5]. Conducting hybridization using these two as parents holds promise for obtaining new rhododendron germplasms with fragrance.
Hybridization breeding represents one of the most widely adopted and effective methodologies in rhododendron research, enabling the integration of superior traits of parental lines via hybridization [6]. Presently, a significant proportion of commercially available rhododendron varieties are derived through hybridization breeding [7]. According to the statistics provided by the International Rhododendron Society and authoritative institutions of various countries, as of 2023, the number of registered hybrid rhododendron varieties worldwide is approximately 28,000 to 30,000 [8]. Although some fragrant rhododendrons have been developed through early hybridization breeding [9], the number of fragrant rhododendron varieties is still insufficient to meet the demands of consumers. It is necessary to explore the existing wild fragrant rhododendron germplasm resources and obtain fragrant rhododendron varieties through distant hybridization technology.
Distant hybridization constitutes an essential process in plant evolution. Presently, it is extensively employed in research on ornamental plants such as Dendranthema morifolium [10], Lilium brownii [11,12], Rosa chinensis [13,14], and Paeonia suffruticosa [15,16]. The literature includes a series of research works on the distant hybridization of the genus Rhododendron, with scholars discovering that the hybridization incompatibility of Rhododendron has brought significant challenges to Rhododendron breeding. Owing to the presence of incompatibility, after the hybridization of R. impeditum × R. williamsianum, pollen tubes have difficulty entering the ovary [17], and the hybrid offspring of R. kiusianum × R. eriocarpum exhibit abnormal albino seedlings [18]. Hybrid incompatibility is a crucial factor restricting the advancement of rhododendron breeding, and its hybridization obstacles are mainly categorized into pre- and post-fertilization obstacles.
Pre-fertilization obstacles encompass the failure of pollen germination and the abnormal behaviors of pollen tubes that impede fertilization [19,20]. After pollination, a succession of recognition processes takes place between the pollen of different genera or species and the stigma, namely, pollen–pistil interactions [21,22]. After the hybridization of R. ‘XXL’ and R. delavayi, the abnormal growth of pollen tubes occurred mainly on the stigma and style. The apical part of the pollen tubes presented a wide-diameter, balloon-like or curled shape, and there were abnormal callose plugs, along with the occurrence of folding back [20]. When R. impeditum and R. williamsianum are hybridized, the pollen on the stigma can germinate normally and grow normally in the style, but most pollen tubes become distorted and swollen at the end after entering the ovary and are unable to reach the ovules [17]. Previous exploration studies have suggested that when overcoming the distant hybridization barriers of rhododendrons, emphasis should be placed on hybridization approaches involving the rational selection of parents, the physical and chemical treatment of pollen and stigmas [23], the improvement of pollination methods [24,25], in vitro fertilization in test tubes [26,27], somatic cell fusion [28], the use of bridge parents [29], etc.
Post-fertilization obstacles mainly manifest as hybrid embryo abortion or abnormal growth of endosperm, thereby interfering with the normal development of seeds [30,31], leading to situations such as seeds being unable to germinate and the occurrence of albino seedlings in the progeny. Following the hybridization of R. pulchrum × R. schlippenbachi, although the fruit bearing rate was relatively high, no hybrid seeds were acquired. However, two months after the hybridization of R. simsii × R. delavayi and R. simsii × R. hancockii, no ovary enlargement was witnessed; nonetheless, during the research course, it was still discovered that pollen tubes successfully entered the ovules [32]. Reproductive barriers often exist in the crosses between Dendranthema grandiflorum (Ramat.) Kitamura and its wild species and can result in a significantly low fruit bearing rate, consequently reducing breeding efficiency [33]. Currently, effective salvage can be accomplished by means of male sterility, chromosome ploidy regulation, embryo culture, promotion of pollen tube growth, genetic engineering technology, etc. [34,35].
In this research, distant hybridization was performed between cultivars of the subgenus Tsutsusi and R. moulmainense. The study purposes were to explore the cross-compatibility between them and to clarify the key barriers restricting gene flow between the subgenus Tsutsusi and R. moulmainense. Simultaneously, the pollination strategies were optimized, and it is anticipated that this approach could integrate the ornamental advantages of the plants within the subgenus Tsutsusi with the distinctive aromatic characteristics of R. moulmainense. By doing so, concern regarding the long-standing shortage of diverse fragrant varieties in rhododendron horticulture can be addressed, providing novel theoretical bases and practical guidance for the cultivation of new fragrant rhododendron cultivars.

2. Materials and Methods

2.1. Plant Material

The experiment was performed at the experimental site of the College of Landscape and Horticulture, Yunnan Agricultural University, from August 2021 to December 2022. In this trial, four well-growing varieties of the subgenus Tsutsusi were selected as the female parents (Figure 1): Rhododendron hybrid ‘Red Tiara’, Rhododendron hybrid ‘Pink Ribbons’, Rhododendron hybrid ‘Carnation’, and Rhododendron hybrid ‘Fuchsia Parasol’. The pollen of Rhododendron moulmainense was harvested in Yangbi Yi Autonomous County, Dali Bai Autonomous Prefecture, Yunnan Province, China (25°40′24.4″ N, 99°57′25.5″ E). After the flowers were picked, they were brought back to the laboratory, and the pollen was extracted and dried with silica gel for 48 h and then stored at −20 °C for further analysis. Species Identifier: Professor Guan Wenling of Yunnan Agricultural University. This specimen is deposited at the National Rhododendron Germplasm Bank of Yunnan Agricultural University. Its accession number is YAU No. 20210315.

2.2. Hybridization Pollination

A pre-experiment for testing the pollen viability of the paternal parent was conducted. It was discovered that the pollen viability was above 65% and could be utilized for pollination. Prior to hybrid pollination, enlarged flower buds that are on the verge of opening are selected, and then the stamens and petals are removed. The pollination time is from 8:00 to 11:30 a.m. For each hybrid combination, in the case of different pollination methods, the number of pollinated flowers is no fewer than 50. Following pollination, a label is attached. The modes of pollination are as follows:
The conventional pollination method: Pollination is conducted when the stigma secretes a considerable amount of mucus.
The pollen heating method: The pollen is treated at a constant temperature of 40 °C for 1 h and then used for pollination.
The early-pollination method: Pollination is performed when the stigma has not secreted mucus.
The delayed-pollination method: Pollination is conducted 8–12 days after the flower blossoms (the number of delayed days is chosen according to the length of the flowering period of different maternal plants).
The repeated pollination method: Pollination is performed continuously for three days when the stigma secretes a considerable amount of mucus.

2.3. Fluorescence Observation of Pollen Tubes

Three to five pistils were collected at eight time points, namely, 4 h, 8 h, 1 day, 2 days, 4 days, 7 days, 10 days, and 14 days after pollination; immersed in the FAA fixative solution (Vformaldehyde/Vglacial acetic acid/V70% ethanol = 1:1:18) for at least 24 h; and stored at 4 °C for further analysis. The pistils were washed stepwise in 70% alcohol, 50% alcohol, 30% alcohol, and pure water and then softened in 4 mol/L NaOH at 60 °C for 4 h. After softening, they were washed with pure water until no color fading occurred. Finally, they were stained with 0.1% aniline blue solution (containing 1.36% potassium dihydrogen phosphate) in the dark for 24 h and then placed on a microscope slide. One to two drops of glycerol were added for squash preparation and observation.

2.4. Observation of Paraffin Sections of the Ovary

Three to five ovary samples were collected from pollinations of ‘Carnation’ × R. moulmainense and ‘Red Tiara’ × R. moulmainense at 15 days, 30 days, and 45 days post-pollination. After collection, they were immersed in FAA fixative and stored in a 4 °C refrigerator for further analysis. ‘Carnation’ × ‘Fuchsia Parasol’ and ‘Red Tiara’ × ‘Red Tiara’ were used as controls, respectively. After undergoing dehydration via gradient alcohol, transparency processing with xylene, embedding in paraffin, sectioning into thin slices and adhering them to slides, dewaxing, and rehydration were performed. The samples were stained and observed under a microscope, with images captured.

2.5. Statistical Indicators of Hybridization Compatibility

Two months post-pollination, the ovary swelling rate was statistically analyzed. After another 6 months, the fruit setting rate was determined. Once the fruits reached maturity, 3 fruits were randomly selected to enumerate the number of capsule seeds and calculate the average value. One hundred seeds were randomly sampled for weighing, with this operation repeated in three groups, thereby enabling the calculation of the weight of one thousand seeds. Ninety hybrid seeds were randomly chosen each time. Each batch of 30 seeds was wrapped in tea bags and immersed in a 1 g/L GA3 solution for 24 h before aseptic sowing (Growth Medium: WPM medium + 2 mg/L 6-BA + 0.1 mg/L NAA + 7.5 g/L agar + 30 g/L sucrose). This operation was replicated three times. After 30 days, the germination rate and the green seedling rate were statistically determined. The calculation formula is presented as follows:
Ovary swelling rate (%) = the number of hybrid fruit expansions/the number of hybrid flowers × 100
Fruit bearing rate (%) = the number of hybrid fruits with seeds/the number of hybrid flowers × 100
Germination rate (%) = the number of germinating seeds/the number of seeds for testing × 100
Green seedling rate (%) = Green seedling count from germinating seeds/the number of seeds for testing × 100
Green seedling coefficient = green seedling rate/germination rate
Number of fertile seeds per unit = Average seed count per capsule × green seedling rate
1000 − seed weight of hybrid seeds = seed weight per 100 seeds × 10
The classification approach for each indicator grade refers to the method proposed by Zhuang Ping [36]. The total score is computed through the assignment method (Table 1), and the fertility is categorized into four grades: sterile type (0 ≤ score < 2.5), weakly fertile type (2.5 ≤ score < 5), fertile type (5 ≤ score < 8), and highly fertile type (score ≥ 8).

2.6. Statistical Analysis

Microsoft Excel 2016 (Redmond, WA, USA) was used to perform routine statistical analyses on the experimental data. Data are expressed as means ± standard deviation (n = 3). Multiple comparisons were performed using SPSS 26.0 (IBM, Inc., Armonk, NY, USA) to calculate the levels of significance. p < 0.05 was adopted as the criterion for statistical significance. Different letters indicate significant differences at p < 0.05 among the different treatments.

3. Results

3.1. Effect of Pollination Methods on Ovary Swelling Rate and Fruit Bearing Rate

Table 2 shows that among the four Rhododendron hybrid combinations, the combination of ‘Red Tiara’ × R. moulmainense had the highest ovary swelling rate and fruit bearing rate under the conventional pollination method, at 20.00% and 16.67%, respectively. Except for the early-pollination method, the other three pollination methods all led to fruit bearing, and both the ovary swelling rate and fruit bearing rate were higher than those under the conventional pollination method. The ovary swelling rate of ‘Carnation’ × R. moulmainense under the repeated pollination method was marginally higher than that under the conventional pollination method, and the fruit bearing rate was significantly higher than that under the conventional pollination method. In contrast, the ovary swelling rate and the fruit bearing rate of the pollen heating method and the early-pollination method were both lower than those of the conventional pollination method. The ovary swelling rate and fruit bearing rate of ‘Fuchsia Parasol’ × R. moulmainense under the conventional pollination method were both 0. Apart from the advanced pollination method, the other three pollination methods all led to fruit setting: the delayed-pollination method had the highest ovary swelling rate and fruit bearing rate, at 35.29% and 33.33%, respectively. The ovary swelling rate and fruit bearing rate of ‘Pink Ribbons’ × R. moulmainense under the conventional pollination method were 13.46% and 7.69%, respectively. The fruit bearing rates under the pollen heating method, early-pollination method, and delayed-pollination method were all lower than that under conventional pollination. The fruit bearing rate under repeated pollination was significantly higher than that under conventional pollination. The ovary swelling rate and fruit bearing rate of the control groups in these four hybrid combinations were both higher than those of the experimental groups, all exceeding 70%.

3.2. Indices of Hybrid Compatibility and Evaluation of Fertility

It is evident from Table 3 that the number of capsule seeds in the four groups of distant hybridization was significantly lower than that of their corresponding control groups. In the experimental groups, the number of capsule seeds was ranked as follows: ‘Red Tiara’ × R. moulmainense > ‘Fuchsia Parasol’ × R. moulmainense > ‘Pink Ribbons’ × R. moulmainense > ‘Carnation’ × R. moulmainense. Among the four control groups, the number of capsule seeds of ‘Red Tiara’ × ‘Red Tiara’ was significantly lower than that of ‘Carnation’ × ‘Fuchsia Parasol’, ‘Fuchsia Parasol’ × R. rivulare, and ‘Pink Ribbons’ × ‘Fuchsia Parasol’. In the experimental group, the sequence of the 1000-seed weight from the largest to the smallest was as follows: ‘Fuchsia Parasol’ × R. moulmainense, ‘Red Tiara’ × R. moulmainense, ‘Pink Ribbons’ × R. moulmainense, and ‘Carnation’ × R. moulmainense. The 1000-seed weights of the first two hybridization combinations were significantly higher than those of the latter two. There was no significant difference in the 1000-seed weight among the four control groups, all of which were above 0.04 g.
As depicted in Table 4, the seed germination rates and green seedling rates of ‘Carnation’ × R. moulmainense, ‘Red Tiara’ × R. moulmainense, and ‘Pink Ribbons’ × R. moulmainense were significantly lower than those of their corresponding control groups. The seed germination rate of ‘Fuchsia Parasol’ × R. moulmainense showed no significant difference from that of the control group, while its green seedling rate was significantly lower than that of the control group. The seed germination rates and green seedling rates of ‘Red Tiara’ × R. moulmainense and ‘Fuchsia Parasol’ × R. moulmainense were significantly higher than those of ‘Carnation’ × R. moulmainense and ‘Pink Ribbons’ × R. moulmainense. The green seedling coefficients were the highest at 1.00 for ‘Carnation’ × ‘Fuchsia Parasol’, ‘Red Tiara’ × ‘Red Tiara’, and ‘Pink Ribbons’ × ‘Fuchsia Parasol’, while the green seedling coefficient was the lowest at 0 for ‘Carnation’ × R. moulmainense.
The aseptic seeding and the growth status of hybridized seedlings are presented in Figure 2. Different degrees of albinism and chlorosis occurred in the seedlings of ‘Red Tiara’ × R. moulmainense, ‘Fuchsia Parasol’ × R. moulmainense, and ‘Pink Ribbons’ × R. moulmainense. Overall, the growth of the seedlings in the control group was superior to that in the experimental group. The ranking in terms of number of fertile seeds per unit, in descending order, was ‘Pink Ribbons’ × ‘Fuchsia Parasol’, ‘Carnation’ × ‘Fuchsia Parasol’, ‘Fuchsia Parasol’ × R. rivulare, ‘Red Tiara’ × ‘Red Tiara’, ‘Red Tiara’ × R. moulmainense, ‘Fuchsia Parasol’ × R. moulmainense, ‘Pink Ribbons’ × R. moulmainense, and ‘Carnation’ × R. moulmainense.
Based on a comprehensive assessment of the eight combinations in Table 1, the results indicate that ‘Carnation’ × ‘Fuchsia Parasol’, ‘Red Tiara’ × ‘Red Tiara’, ‘Fuchsia Parasol’ × R. rivulare, and ‘Pink Ribbons’ × ‘Fuchsia Parasol’ are of high fertility; ‘Red Tiara’ × R. moulmainense and ‘Fuchsia Parasol’ × R. moulmainense are fertile; ‘Pink Ribbons’ × R. moulmainense is of weak fertility; and ‘Carnation’ × R. moulmainense is infertile.

3.3. Analysis of Pollen Tube Growth

Fluorescence observations of pollen tubes were performed following different pollination methods. Conventional pollination: Four hours after conventional pollination of ‘Carnation’ × R. moulmainense, an extremely small quantity of pollen grains germinated on the stigma, and a small amount of callose was present. One day later, many pollen grains germinated, and some pollen tubes grew towards the style, with distinct callose inside. Four days later, the pollen tubes became distorted and intertwined, and a few pollen tubes extended towards the ovary. Seven days later, an extremely small number of pollen tubes came into contact with the ovules (Figure 3A). Pollen heating: Eight hours after pollination via the pollen heating method, many pollen grains germinated, and a few pollen tubes extended towards the style, with callose emerging. One day later, the pollen tubes elongated to one-fourth of the style length, and intense callose reactions occurred within the ovary. Two days later, the pollen tubes twisted and intertwined with each other, and by the seventh day, they contacted the ovules (Figure 3B). Early pollination: Eight hours after pollination via the early-pollination method, pollen germination commenced, and callose emerged within the pollen tubes. One day later, many pollen grains germinated on the stigma, and a few pollen tubes extended towards the style. Two days later, the pollen tubes intertwined with each other and exhibited reverse growth. Ten days later, a small quantity of pollen tubes contacted the ovules (Figure 3C). Delayed pollination: One day after pollination via the delayed-pollination method, many pollen grains germinated. Four days later, a considerable number of pollen tubes grew in bundles, and some of the pollen tubes exhibited twisted and entangled phenomena. A small quantity of pollen tubes extended towards the ovary. Seven days later, they contacted the ovules (Figure 3D). Repetitive pollination method: Four hours after performing the repetitive pollination method, many pollen grains germinated. One day later, callose reactions occurred to varying extents in both the stigma and the pollen tubes. Two days later, the pollen tubes grew in bundles. Four days later, intense callose reactions occurred within the ovary. Ten days later, the pollen tubes grew into the ovary and extended towards the ovules (Figure 3E).
Following conventional pollination, the progression of pollen tube growth in the interspecific hybrid Dianthus ‘Carnation’ × ‘Fuchsia Parasol’ was monitored using fluorescence microscopy. Representative observations are documented in Figure 4. Four hours post-pollination, a limited number of pollen grains initiated germination, followed by a significant increase in germination at 8 h. By 24 h, the pollen tubes had penetrated the style. At 48 h, the pollen tubes extended to one-third of the style length, with detectable callose deposition within the tubes. By day 4, the tubes had progressed to two-thirds of the style length. Finally, on day 7, some pollen tubes successfully entered the ovary and established contact with the ovules.
The fluorescence observations of the pollen tubes in ‘Red Tiara’ × R. moulmainense (presented in Figure 5) are as follows. Conventional pollination: Four hours post-conventional pollination, a limited number of pollen grains initiated germination, followed by a significant increase in germination eight hours later. One day post-pollination, distinct callose deposition was observed within the pollen tubes, which had successfully extended into the style. Two days later, callose reactions were detected in the ovary, and four days later, callose deposition occurred within the style. During this period, the pollen tubes exhibited swelling, and some ceased growth entirely. By day 14, a small proportion of pollen tubes established contact with the ovules (Figure 5A). Pollen heating: Eight hours post-pollination using the pollen heating method, a substantial proportion of pollen grains initiated germination. One day later, callose deposition was observed within the pollen tubes. Seven days later, a significant number of pollen tubes exhibited distortion and localized swelling. Ten days later, many pollen tubes successfully established contact with the ovules (Figure 5B). Early pollination: Following the early-pollination method, the pollen began to initiate the germination process 8 h post-pollination. One day later, a large quantity of successfully germinated pollen was observed on the stigma, accompanied by notable twisting phenomena. Four days later, a substantial amount of callose deposition was detected within the style. Fourteen days later, a remarkable callose response occurred in the ovary; however, no successful entry of pollen tubes into the ovary was observed (Figure 5C). Delayed pollination: Following the delayed-pollination method, pollen gradually initiated the germination process 8 h post-treatment. Four days later, a small but detectable amount of callose deposition was observed within the pollen tubes, with a limited number of pollen tubes successfully penetrating the ovary and establishing contact with the ovules. Seven days after treatment, an increased number of pollen tubes were observed to have successfully established contact with the ovules (Figure 5D). Repeated pollination: After repeated pollination treatment, pollen germination commenced 4 h post-treatment. By day 2, the pollen tubes had extended to approximately half the length of the style. By day 4, a substantial proportion of pollen tubes had successfully penetrated the ovary and established contact with the ovules (Figure 5E).
As depicted in Figure 6, the outcomes of the fluorescence observation of the pollen tubes in ‘Red Tiara’ × ‘Red Tiara’ demonstrated that pollen initiated germination four hours post-pollination; the pollen tubes began to grow towards the stigma eight hours post-pollination; two days post-pollination, the pollen tubes grew to one-third of the style length; four days post-pollination, the pollen tubes grew to between two-thirds and three-quarters of the style length; and seven days post-pollination, a considerable number of pollen tubes entered the ovary and contacted the ovules.
The fluorescence observation results of the pollen tubes of ‘Pink Ribbons’ × R. moulmainense (presented in Figure 7) are as follows: Eight hours after conventional pollination, a considerable number of pollen grains initiated germination; two days later, the pollen tubes became distorted and intertwined with each other; four days later, the pollen tubes grew to two-thirds of the style length; and seven days later, the pollen tubes successfully penetrated the ovary and established contact with the ovules (Figure 7A). Four hours after pollination by the pollen heating method, the pollen began to germinate, and a small quantity of callose deposition was detected within the pollen tubes, which grew towards the style; two days later, a small amount of callose deposition was also detected within the style; and seven days later, a few pollen tubes established contact with the ovules (Figure 7B). Eight hours after early pollination, the pollen began to germinate; one day later, callose deposition was detected within the pollen tubes, which entered the style; and ten days later, the pollen tubes successfully entered the ovary (Figure 7C). Four hours after delayed pollination, the pollen began to germinate; eight hours later, the pollen tubes grew towards the style; two days later, callose deposition was detected within the style; and seven days later, the pollen tubes successfully established contact with the ovules (Figure 7D). Four hours after repeated pollination, the pollen tubes grew in bundles to one-fifth of the style length, accompanied by callose deposition; one day later, the pollen tubes extended to one-third of the style length, and a callose reaction was detected within the ovary simultaneously; two days later, the pollen tubes grew to one-half of the style length; and four days later, the pollen tubes successfully penetrated the ovary and established contact with the ovules (Figure 7E).
Eight hours after pollination of ‘Pink Ribbons’ × ‘Fuchsia Parasol’, pollen grains initiated germination on the stigma (Figure 8A). By day 2, the pollen tubes had grown to one-third of the style length (Figure 8B). By day 4, they exhibited bundled growth and extended to two-thirds of the style length, with detectable callose deposition observed within the pollen tubes. Seven days later, a substantial number of pollen tubes successfully penetrated the ovary and extended toward the ovules (Figure 8C).

3.4. Analysis of the Ovary’s Development

The paraffin section analysis of the ovary from ‘Carnation’ × R. moulmainense at 15, 30, and 45 days post-pollination is illustrated in Figure 9A. At 15 days post-pollination, the ovules were arranged in an orderly manner and exhibited intact structural integrity, with spherical embryos and endosperm tissues clearly observable. At 30 days post-pollination, the ovules remained largely well-organized; however, a minor proportion displayed developmental abnormalities, whereas most embryos and endosperm continued to develop normally. At 45 days post-pollination, most ovules demonstrated abnormal development, characterized by rod-shaped embryos lacking endosperm or other irregularly shaped embryos. Additionally, embryonic degeneration was evident, with some embryos undergoing premature degradation. Only an extremely small fraction of ovules maintained normal developmental progression. The paraffin section analysis of the ovary from the control group ‘Carnation’ × ‘Fuchsia Parasol’ at 15, 30, and 45 days post-pollination is presented in Figure 9B. At 15 days post-pollination, the ovules were well-organized and plump, with embryos and endosperm clearly discernible in some cases. At 30 days post-pollination, a minor proportion of ovules exhibited morphological abnormalities; however, most embryos developed normally into spherical embryos. By 45 days post-pollination, some ovules displayed abnormal morphology and structural features, while most embryos gradually matured. Notably, an extremely limited number of embryos underwent abnormal degradation. Approximately 30 days following the pollination of ‘Carnation’ × R. moulmainense, certain ovules commenced to manifest atrophy and anomalous shapes; in contrast, in the control group, despite most ovules displaying abnormalities during the development process, a small number of abnormal embryos and some undeveloped ovules were still observed approximately 45 days after pollination.
At 15 days following pollination of ‘Red Tiara’ × R. moulmainense, the ovules were arranged in an orderly manner and exhibited regular morphology, with embryos and endosperm clearly observable in some ovules (Figure 10A-15d). At 30 days post-pollination, the ovules developed normally, forming spherical embryos with distinct structures; however, a minor proportion of embryos and endosperm showed uncoordinated development (Figure 10A-30d). At 45 days following pollination, the morphology of some ovules became irregular, and a limited number of embryos initiated abnormal degradation (Figure 10A-45d). In the case of ‘Red Tiara’ × ‘Red Tiara’, the ovules were orderly arranged and developed normally 15 days after pollination (Figure 10B-15d); by 30 days post-pollination, the ovules had developed into typical spherical embryos (Figure 10B-30d); and at 45 days post-pollination, the embryos gradually matured, with only a few ovules exhibiting abnormal development (Figure 10B-45d).

4. Discussion

4.1. Analysis of Indicators for Hybrid Compatibility

The ovary swelling rate, fruit bearing rate, and seed germination rate are commonly employed as indirect indicators for the systematic evaluation of hybrid compatibility [36]. Notably, hybrid progeny frequently exhibit typical challenges, such as significantly reduced germination rates and elevated incidences of albino or chlorotic seedlings. Consequently, the seed germination rate and green seedling rate have emerged as crucial parameters for assessing hybrid compatibility [37,38]. Research has demonstrated a significant positive correlation between the average number of seeds per capsule and the fertilization success rate. Moreover, the 1000-seed weight can serve as a reliable quantitative metric for evaluating endosperm development [25]. The findings of this study indicate that both the ovary swelling rate and the fruit set rate of the four hybrid combinations are below 20%. In studies on the distant hybridization of Azalea × R. decorum, relatively low rates of ovary enlargement and fruit set have also been observed. For example, the fruit set rate of the ‘Sima’ × R. decorum combination is 5.88%, and the ovary enlargement rate is 1.96% [39]. In the hybridization combination of R. farrarae × R. moulmainense, the fruit bearing rate was 5.11%, and no seed germination was detected [40]. This result is analogous to the hybridization outcome of ‘Carnation’ × R. moulmainense presented in this study, where the fruit set rate was 4.08% and no seed germination occurred. In this study, a multi-dimensional evaluation framework was constructed, incorporating the ovary swelling rate, fruit bearing rate, number of capsule seeds, and green seedling rate. By calculating the green seedling coefficient and unit-based fertile seed count, combined with the modified Zhuangping fertility assessment model [41], the compatibility of four hybrid combinations was systematically analyzed. The results indicate that ‘Carnation’ × R. moulmainense exhibited complete sterility; ‘Pink Ribbons’ × R. moulmainense demonstrated weak fertility; and ‘Red Tiara’ × R. moulmainense and ‘Fuchsia Parasol’ × R. moulmainense achieved fertile-type status.

4.2. Overcoming Pre-Fertilization Barriers in Rhododendron Fertilization

Pre-fertilization barriers are the predominant cause of natural reproductive isolation in most cases of distant hybridization among plants [42]. During the hybridization process in flowering plants, interactions between pollen exine proteins and stigma papilla cytoplasmic membrane proteins play a critical role. Existing studies have demonstrated that, under conditions of incompatibility, the excessive accumulation of callose can hinder the normal elongation of pollen tubes, often accompanied by phenomena such as pollen tube distortion, entanglement, and failure to penetrate the ovary and ovule [43]. These features collectively represent one of the key cytological mechanisms underlying pre-fertilization barriers in distant hybridization incompatibility [44]. One day after pollination, on the stigma of the alpine R. ‘XXL’, concomitant with the germination of R. bureavii pollen, multi-point callose depositions occurred in the stigma cells, while the pollen tubes exhibited abnormal manifestations such as local expansion and long callose embolisms [45]. Furthermore, after pollination among subgenera of Nymphaea, abnormal situations of pollen tube growth also emerged, such as tip curvature, swelling, and inability to penetrate the ovule [46,47]. Analogously, in the distant hybridization between Paeonia suffruticosa and Paeonia lactiflora, the occurrence of pre-fertilization barriers was attributed to phenomena like distorted, entangled, and branched pollen tubes and callose depositions [48].
This experiment investigated pollen tube growth dynamics in three distant hybridization combinations (‘Carnation’ × R. moulmainense, ‘Red Tiara’ × R. moulmainense, and ‘Pink Ribbons’ × R. moulmainense) through systematic observation of post-pollination developmental processes. The results indicated that the germination rate of pollen on the stigma was relatively low, and some pollen tubes failed to successfully extend into the style. With the further growth of pollen tubes, the stigma, pollen tubes, style, and ovary all demonstrated callose reactions to varying degrees. Moreover, during the growth process of pollen tubes, abnormal phenomena such as twisting and swelling at the end emerged, leading to only a very few pollen tubes being capable of successfully entering the ovary, thereby significantly lowering the fruit setting rate. During the growth process of pollen tubes in the three control groups, although a minor amount of callose was observed to be produced, its influence on the growth of pollen tubes was relatively insignificant. The outcomes of this study are in accordance with those from previous research, suggesting that excessive accumulation of callose, distortion of pollen tubes, and terminal expansion, among others, are the principal factors contributing to pre-fertilization barriers.
Overcoming pre-fertilization barriers typically involves the utilization of diverse pollination approaches, such as early pollination, delayed pollination, mentor pollination, and gibberellin application to the stigma [49]. Hao employed the delayed-pollination approach, elevating the ovary swelling rate of ‘Yin Taohong’ × R. decorum from 13.16% to 52.83% and the fruit set rate from 5.26% to 22.64% [39]. In this research, four hybrid combinations were pollinated using four pollination methods, namely, pollen heating, early pollination, delayed pollination, and repeated pollination, with conventional pollination serving as the control. The research findings demonstrate that repeated pollination significantly improves the ovary swelling rate and fruit bearing rate in the two hybrid combinations ‘Carnation’ × R. moulmainense and ‘Pink Ribbons’ × R. moulmainense. Furthermore, the pollen heating method, delayed pollination, and repeated pollination partially alleviate pre-fertilization barriers in the hybrid combinations ‘Red Tiara’ × R. moulmainense and ‘Fuchsia Parasol’ × R. moulmainense. Based on these results, it is evident that different hybrid combinations require distinct strategies to overcome pre-fertilization barriers. In practical applications, it is therefore essential to develop tailored pollination approaches for specific hybrid combinations.

4.3. Overcoming Post-Fertilization Barriers in Rhododendron Fertilization

During the distant hybridization process of rhododendrons, a combined effect of pre- and post-fertilization barriers might exist. In this study, two distant hybridization combinations, namely, ‘Carnation’ × R. moulmainense and ‘Red Tiara’ × R. moulmainense, were taken as the research subjects. Paraffin sections of the ovaries were observed on the 15th, 30th, and 45th days after pollination, and a comprehensive analysis was conducted in combination with relevant indicators, such as the thousand-grain weight and germination rate of the hybrid seeds. The results indicated that, compared with the control group, the distant hybridization combinations presented uncoordinated development of the embryo and endosperm during the ovary development process, and the trend of abnormal degradation or even degeneration of the embryo was more pronounced. This finding was consistent with the research results of Deng Yanming et al. [50]. Green seedlings were successfully obtained during reciprocal crosses between the subgenus Tsutsusi and subgenus Hymenanthes, yet many of the seedlings exhibited extremely low growth vitality and gradually manifested albino phenomena [51]. Interspecific hybridization research among varieties of the subgenus Tsutsusi, subgenus Azaleastrum, and subgenus Pentanthera has previously analyzed intersubgeneric hybridization compatibility from multiple perspectives, including the ratio of stigma length, pollen germination rate, pollen tube growth status, ovary development, and embryo development. The authors discovered that one of the main causes of the hybridization barriers among the three subgenera lies in the embryo development stage [52]. Similar phenomena have also been witnessed in distant hybridization of other woody ornamental plants. For example, in the hybridization pollination experiment of V.tinus and V. × bodnantense, pollen tubes reached the ovary 2 to 7 days after pollination; however, after 4 months, it was found that V. × bodnantense failed to form viable embryos, suggesting that abnormal embryo development or premature death was the principal cause of a low fruit bearing rate and seed abortion [53]. Furthermore, in the interspecific hybridization research of H.macrophylla and H. serrata, it was discovered that the variation in salicylic acid content might be the crucial factor resulting in embryo abortion [54]. The in vitro culture of hybrid embryos before their abortion through the adoption of embryo rescue technology constitutes an effective means to overcome the incompatibility of distant hybridization and has been utilized in the hybrid breeding of Rhododendron. Embryos of different ages after the hybridization of R.simsii, R.ovatum, and wild rhododendrons were placed on the culture medium for cultivation, and the younger embryos were more prone to form callus [55]. The abortion of hybrid embryos typically occurs more than 5 months after pollination, and consequently, 4 to 5 months after pollination is regarded as the optimal period for embryo rescue. Nevertheless, in some particular combinations, for instance, the hybridization combination of Rhododendron Sect. Vireya × subgenus Tsutsusi, embryo collection can be implemented 1.5 to 2 months after pollination [31].
This study reveals that in the ‘Carnation’ × R. moulmainense hybrid combination, a few ovules started to exhibit abnormal development 30 days after pollination, and 45 days after pollination, most ovules developed abnormally and gradually atrophied. Hence, 30 days after pollination is the optimal period for conducting embryo rescue in this combination. In the ‘Red Tiara’ × R. moulmainense hybrid combination, the ovules remained in a normal developmental state 30 days after pollination and had formed distinct spherical embryos, yet a very small number of embryos presented developmental disharmony with the endosperm; 45 days after pollination, the morphology of some ovules became irregular, and some embryos began to show abnormal degradation. This indicates that for this particular hybrid combination, 30 to 45 days after pollination might be the most appropriate period for embryo rescue. Within 30 to 45 days following hybridization pollination of rhododendrons, the embryos remain immature, and their morphology is extremely minute. Consequently, undertaking embryo rescue operations during this phase is highly challenging. To increase the success rate of embryo rescue, it is necessary to select an appropriate period for embryo rescue based on the embryo abortion period and the development rate of each hybrid combination. Furthermore, the selection of the most suitable medium for the aseptic sowing of different hybrid seeds still demands further in-depth investigation.
This experiment merely conducted a preliminary analysis of the compatibility of distant hybridization between the subgenus Tsutsusi and subgenus Azaleastrum, with a focus on fruiting characteristics and cytology. Existing studies have disclosed the mechanism of hybridization barriers in rhododendrons via molecular biological approaches [56,57,58]. To further illuminate the specific mechanism of hybrid incompatibility between these two subgenera of rhododendrons, in-depth investigations need to be conducted from multiple dimensions, such as physiology and genetics, in the hope of providing novel theoretical support for cross-subgenus hybridization of rhododendrons and laying a scientific basis for molecular breeding of fragrant rhododendrons.

5. Conclusions

This study targeted fragrant flower breeding and conducted distant hybridization between the subgenus Tsutsusi varieties, featuring strong resistance and multi-seasonal flowering, and R. moulmainense, with its fragrance trait. It provides a novel theoretical foundation for future breakthroughs in reproductive isolation among subgenera of Rhododendron, achieving distant hybridization among subgenera and obtaining new germplasm with aromatic properties. The results demonstrated that fruits could be obtained when ‘Carnation’, ‘Red Tiara’, ‘Fuchsia Parasol’, and ‘Pink Ribbons’ were utilized as female parents and crossed with R. moulmainense. Among them, the combination of ‘Red Tiara’ × R. moulmainense under repeated pollination had the highest fruit setting rate, reaching 35.56%, followed by ‘Fuchsia Parasol’ × R. moulmainense under delayed pollination, with a fruit setting rate of 33.33%. This research indicated that different pollination methods exerted a significant influence on the ovary enlargement rate and fruit setting rate of different hybrid combinations. The cross of ‘Carnation’ × R. moulmainense is of the sterile type, and ‘Pink Ribbons’ × R. moulmainense is of the weakly fertile type, while ‘Red Tiara’ × R. moulmainense and ‘Fuchsia Parasol’ × R. moulmainense are of the fertile type. Through fluorescence observation and ovary paraffin sectioning techniques, the causes of the hybridization incompatibility between the subgenus Tsutsusi and subgenus Azaleastrum were initially analyzed. It was discovered that different hybrid combinations all presented varying degrees of pre-fertilization and post-fertilization obstacles.
In this study, the outcomes of hybridization between subgenera were analyzed solely from the perspectives of morphology and histology. Long-term surveillance of the subsequent emergence rate of surviving ovules, the growth potential of hybrid plants, and their flowering characteristics has not been conducted. Consequently, a comprehensive assessment of the breeding potential and genetic stability of distant hybrid progeny remains unattainable at present. Simultaneously, reproductive isolation mechanisms have not been explored in sufficient depth. Indeed, no research has been conducted using molecular biology techniques to investigate the gene regulatory networks involved in gamete recognition, the hormone signaling pathways, and the underlying molecular mechanisms. Elucidating the fundamental causes of reproductive isolation between subgenera therefore still poses a substantial challenge. In future research endeavors, we intend to establish a long-term tracking system for hybrid progeny. Additionally, we will integrate molecular biology techniques to systematically elucidate the molecular mechanisms underlying reproductive isolation between the two subgenera. The aim is to achieve a more comprehensive and in-depth theoretical and technical foundation for overcoming the reproductive barriers among subgenera within the genus Rhododendron.

Author Contributions

This study was conceived and completed by H.L. and J.Q. under the guidance of Y.L. and W.G. H.L. and J.Q. were responsible for the experimental design and paper writing. H.L., J.Q. and L.W. jointly conducted the experiments and collected the relevant data. J.S. and Y.Z. were responsible for the preparation and drawing of the charts. The revision and finalization of the manuscript were completed by Y.L. and W.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Technologies Research for the Germplasm of Important Woody Flowers in Yunnan Province (Grant No. 202302AE090018).

Institutional Review Board Statement

The species of Rhododendron moulmainense does not fall within the category of endangered wild plant species, and the collection of materials is in accordance with both Chinese and international guidelines. The authors obtained the consent of the landowner to collect materials.

Data Availability Statement

All the data used in this research are presented completely herein.

Conflicts of Interest

The authors declare no conflict of interest.

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Horticulturae 11 01116 g001
Figure 1. Hybridization parents. (A) R. hybrid ‘Red Tiara’; (B) R. hybrid ‘Fuchsia Parasol’; (C) R. hybrid ‘Carnation’; (D) R. hybrid ‘Pink Ribbons’; (E) R. moulmainense.
Figure 1. Hybridization parents. (A) R. hybrid ‘Red Tiara’; (B) R. hybrid ‘Fuchsia Parasol’; (C) R. hybrid ‘Carnation’; (D) R. hybrid ‘Pink Ribbons’; (E) R. moulmainense.
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Figure 2. The germination and growth of hybrid seeds. (A) ‘Carnation’ × R. moulmainense; (B) ‘Red Tiara’ × R. moulmainense; (C) ‘Fuchsia Parasol’ × R. moulmainense; (D) ‘Pink Ribbons’ × R. moulmainense; (E) ‘Carnation’ × ‘Fuchsia Parasol’; (F) ‘Red Tiara’ × ‘Red Tiara’; (G) ‘Fuchsia Parasol’ × R. rivulare; (H) ‘Pink Ribbons’ × ‘Fuchsia Parasol’. The image in (F) shows growth after 30 d of aseptic seeding, and the rest of the images show growth after 100 d.
Figure 2. The germination and growth of hybrid seeds. (A) ‘Carnation’ × R. moulmainense; (B) ‘Red Tiara’ × R. moulmainense; (C) ‘Fuchsia Parasol’ × R. moulmainense; (D) ‘Pink Ribbons’ × R. moulmainense; (E) ‘Carnation’ × ‘Fuchsia Parasol’; (F) ‘Red Tiara’ × ‘Red Tiara’; (G) ‘Fuchsia Parasol’ × R. rivulare; (H) ‘Pink Ribbons’ × ‘Fuchsia Parasol’. The image in (F) shows growth after 30 d of aseptic seeding, and the rest of the images show growth after 100 d.
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Figure 3. Fluorescence observations of ‘Carnation’ × R. moulmainense pollen tubes. Picture scale bar is 200 μm. (A) Conventional pollination method; (B) pollen heating pollination method; (C) early-pollination method; (D) delayed-pollination method; (E) repeat pollination method (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; ZT: stigma; HZ: stylet; ZF: ovary).
Figure 3. Fluorescence observations of ‘Carnation’ × R. moulmainense pollen tubes. Picture scale bar is 200 μm. (A) Conventional pollination method; (B) pollen heating pollination method; (C) early-pollination method; (D) delayed-pollination method; (E) repeat pollination method (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; ZT: stigma; HZ: stylet; ZF: ovary).
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Figure 4. Fluorescence observations of ‘Carnation’ × ‘Fuchsia Parasol’ pollen tubes. The picture scale bar is 200 μm. (A) A small number of pollen grains germinate; (B) a large number of pollen grains germinate; (C) pollen tubes enter the flower style; (D) there is a small amount of callose in the pollen tube; (E) pollen tubes grow to two-thirds of the style; (F) pollen tubes are in contact with the ovules (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; ZT: stigma; HZ: stylet).
Figure 4. Fluorescence observations of ‘Carnation’ × ‘Fuchsia Parasol’ pollen tubes. The picture scale bar is 200 μm. (A) A small number of pollen grains germinate; (B) a large number of pollen grains germinate; (C) pollen tubes enter the flower style; (D) there is a small amount of callose in the pollen tube; (E) pollen tubes grow to two-thirds of the style; (F) pollen tubes are in contact with the ovules (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; ZT: stigma; HZ: stylet).
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Figure 5. Fluorescence observations of ‘Red Tiara’ × R. moulmainense pollen tubes. Picture scale bar is 200 µm. (A) Conventional pollination method; (B) pollen pollination by heating; (C) early-pollination method; (D) delayed-pollination method; (E) repeat pollination method (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; HZ: stylet; ZF: ovary).
Figure 5. Fluorescence observations of ‘Red Tiara’ × R. moulmainense pollen tubes. Picture scale bar is 200 µm. (A) Conventional pollination method; (B) pollen pollination by heating; (C) early-pollination method; (D) delayed-pollination method; (E) repeat pollination method (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; HZ: stylet; ZF: ovary).
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Figure 6. Fluorescence observations of ‘Red Tiara’× ‘Red Tiara’ pollen tubes. Picture scale bar is 200 µm. (A) Pollen grains begin to sprout in 4 h; (B) pollen tube bundles grow in 4 days; (C) pollen tubes are in contact with the ovules in 7 days (Pg: pollen grain; Pt: pollen tube; Ou: ovule; ZT: stigma; HZ: stylet).
Figure 6. Fluorescence observations of ‘Red Tiara’× ‘Red Tiara’ pollen tubes. Picture scale bar is 200 µm. (A) Pollen grains begin to sprout in 4 h; (B) pollen tube bundles grow in 4 days; (C) pollen tubes are in contact with the ovules in 7 days (Pg: pollen grain; Pt: pollen tube; Ou: ovule; ZT: stigma; HZ: stylet).
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Figure 7. Fluorescence observations of ‘Pink Ribbons’ × R. moulmainense pollen tubes. Picture scale bar is 200 µm. (A) Conventional pollination method; (B) pollen pollination by heating; (C) early-pollination method; (D) delayed-pollination method; (E) repeat pollination method (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; ZT: stigma; HZ: stylet; ZF: ovary).
Figure 7. Fluorescence observations of ‘Pink Ribbons’ × R. moulmainense pollen tubes. Picture scale bar is 200 µm. (A) Conventional pollination method; (B) pollen pollination by heating; (C) early-pollination method; (D) delayed-pollination method; (E) repeat pollination method (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; ZT: stigma; HZ: stylet; ZF: ovary).
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Figure 8. Fluorescence observations of ‘Pink Ribbons’ × ‘Fuchsia Parasol’ pollen tubes. Picture scale bar is 200 µm. (A) Pollen grains begin to sprout in 8 h; (B) pollen tube bundles grow in 4 days; (C) pollen tubes are in contact with the ovules in 7 days (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; ZT: stigma; HZ: stylet).
Figure 8. Fluorescence observations of ‘Pink Ribbons’ × ‘Fuchsia Parasol’ pollen tubes. Picture scale bar is 200 µm. (A) Pollen grains begin to sprout in 8 h; (B) pollen tube bundles grow in 4 days; (C) pollen tubes are in contact with the ovules in 7 days (Pg: pollen grain; Pt: pollen tube; Cp: callose; Ou: ovule; ZT: stigma; HZ: stylet).
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Figure 9. The ovary was observed in paraffin sections of ‘Carnation’ × R. moulmainense and ‘Carnation’ × ‘Fuchsia Parasol’. (A) ‘Carnation’ × R. moulmainense; (B) ‘Carnation’ × ‘Fuchsia Parasol’. The first column scale is 1000 µm; the second column scale is 100 µm; the third column scale is 50 µm (Ou: ovule; Em: embryo; En: endosperm).
Figure 9. The ovary was observed in paraffin sections of ‘Carnation’ × R. moulmainense and ‘Carnation’ × ‘Fuchsia Parasol’. (A) ‘Carnation’ × R. moulmainense; (B) ‘Carnation’ × ‘Fuchsia Parasol’. The first column scale is 1000 µm; the second column scale is 100 µm; the third column scale is 50 µm (Ou: ovule; Em: embryo; En: endosperm).
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Figure 10. The ovary was observed in paraffin sections of ‘Red Tiara’ × R. moulmainense and ‘Red Tiara’ × ‘Red Tiara’. (A) ‘Red Tiara’ × R. moulmainense; (B) ‘Red Tiara’ × ‘Red Tiara’. The first column scale is 1000 µm; the second column scale is 100 µm; the third column scale is 50 µm (Ou: ovule; Em: embryo; En: endosperm).
Figure 10. The ovary was observed in paraffin sections of ‘Red Tiara’ × R. moulmainense and ‘Red Tiara’ × ‘Red Tiara’. (A) ‘Red Tiara’ × R. moulmainense; (B) ‘Red Tiara’ × ‘Red Tiara’. The first column scale is 1000 µm; the second column scale is 100 µm; the third column scale is 50 µm (Ou: ovule; Em: embryo; En: endosperm).
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Table 1. Fertility index grades and weight allocations for hybridization.
Table 1. Fertility index grades and weight allocations for hybridization.
IndexSterileWeakly FertileFertile
ThresholdScoreThresholdScoreThresholdScore
Green seeding rate0 < Gs < 10110 ≤ Gs < 503Gs ≥ 505
Green seeding coefficient0 < Gc < 0.60.50.6 ≤ Gc < 0.91.5Gc ≥ 0.92
Percentage of fertile fruit0 < St < 200.520 ≤ St < 401St ≥ 401.5
Unit number of fertile seeds0 < Sf < 200.520 ≤ Sf < 2001Sf ≥ 2001.5
Table 2. Effect of pollination methods on the ovary swelling and fruit bearing rates.
Table 2. Effect of pollination methods on the ovary swelling and fruit bearing rates.
Hybrid Combination Conventional PollinationPollen Heating PollinationEarly PollinationDelayed Pollination Repeat Pollination
‘Carnation’ × R. moulmainenseOvary expansion rate10.23.73.929.811.63
Fruit setting rate4.083.71.965.829.3
‘Carnation’ × ‘Fuchsia Parasol’ (ck)Ovary expansion rate81.25
Fruit setting rate72.92
‘Red Tiara’ × R. moulmainenseOvary expansion rate2035.14026.1944.44
Fruit setting rate16.6732.43021.4335.56
‘Red Tiara’ × ‘Red Tiara’ (ck)Ovary expansion rate97.73
Fruit setting rate97.73
‘Fuchsia Parasol’ × R. moulmainenseOvary expansion rate04.26035.2923.53
Fruit setting rate04.26033.3321.57
‘Fuchsia Parasol’ × R. rivulare (ck)Ovary expansion rate85.71
Fruit setting rate83.67
‘Pink Ribbons’ × R. moulmainenseOvary expansion rate13.46807.6924.07
Fruit setting rate7.69603.8516.67
‘Pink Ribbons’ × ‘Pink Ribbons’ (ck)Ovary expansion rate95.83
Fruit setting rate95.83
Table 3. Average number of seeds and 1000-seed weight of hybrid and selfing capsules.
Table 3. Average number of seeds and 1000-seed weight of hybrid and selfing capsules.
Hybrid and Selfing CombinationNumber of Capsule Seeds (Grain)1000-Seed Weight
‘Carnation’ × R. moulmainense33.33 ± 7.51 d0.0260 ± 0.0026 c
‘Carnation’ × ‘Fuchsia Parasol’ (ck)360.67 ± 10.97 a0.0487 ± 0.0012 a
‘Red Tiara’ × R. moulmainense105.33 ± 10.21 c0.0387 ± 0.0032 b
‘Red Tiara’ × ‘Red Tiara’ (ck)296.00 ± 25.51 b0.0540 ± 0.0036 a
‘Fuchsia Parasol’ × R. moulmainense98.33 ± 18.04 c0.0390 ± 0.0070 b
‘Fuchsia Parasol’ × R. rivulare (ck)365.33 ± 66.71 a0.0477 ± 0.0021 a
‘Pink Ribbons’ × R. moulmainense83.33 ± 14.29 cd0.0303 ± 0.0067 c
‘Pink Ribbons’ × ‘Pink Ribbons’ (ck)377.33 ± 53.95 a0.0487 ± 0.0012 a
Note: The different lowercase letters show significant differences at the 0.05 level.
Table 4. Hybridization compatibility indicators and comprehensive scores.
Table 4. Hybridization compatibility indicators and comprehensive scores.
Hybrid and Selfing CombinationGermination RateGreen Seeding RateGreen Seeding CoefficientUnit Number of Fertile Seeds (Grain)Comprehensive Score
‘Carnation’ × R. moulmainense0.00 ± 0.00 d0.00 ± 0.00 e000
‘Carnation’ × ‘Fuchsia Parasol’48.48 ± 2.99 b48.48 ± 2.99 b1360.678
‘Red Tiara’ × R. moulmainense23.96 ± 4.77 c21.02 ± 3.03 d0.8892.696
‘Red Tiara’ × ‘Red Tiara’63.54 ± 6.51 a63.54 ± 6.51 a129610
‘Fuchsia Parasol’ × R. moulmainense22.86 ± 2.86 c20.00 ± 4.95 d0.8785.555.5
‘Fuchsia Parasol’ × R. rivulare28.15 ± 3.58 c27.07 ± 1.74 c0.96350.728
‘Pink Ribbons’ × R. moulmainense3.09 ± 0.05 d2.05 ± 1.78 e0.66554
‘Pink Ribbons’ × ‘Pink Ribbons’53.20 ± 0.23 b53.20 ± 0.23 b1377.3310
Note: The different lowercase letters show significant differences at the 0.05 level.
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MDPI and ACS Style

Li, H.; Qi, J.; Wang, L.; Song, J.; Zhao, Y.; Li, Y.; Guan, W. Study on the Compatibility of Distant Hybridization Between Rhododendron Subgenus Tsutsusi and R. moulmainense, a Fragrant Rhododendron from China. Horticulturae 2025, 11, 1116. https://doi.org/10.3390/horticulturae11091116

AMA Style

Li H, Qi J, Wang L, Song J, Zhao Y, Li Y, Guan W. Study on the Compatibility of Distant Hybridization Between Rhododendron Subgenus Tsutsusi and R. moulmainense, a Fragrant Rhododendron from China. Horticulturae. 2025; 11(9):1116. https://doi.org/10.3390/horticulturae11091116

Chicago/Turabian Style

Li, Hongling, Jing Qi, Lele Wang, Jie Song, Yan Zhao, Yefang Li, and Wenling Guan. 2025. "Study on the Compatibility of Distant Hybridization Between Rhododendron Subgenus Tsutsusi and R. moulmainense, a Fragrant Rhododendron from China" Horticulturae 11, no. 9: 1116. https://doi.org/10.3390/horticulturae11091116

APA Style

Li, H., Qi, J., Wang, L., Song, J., Zhao, Y., Li, Y., & Guan, W. (2025). Study on the Compatibility of Distant Hybridization Between Rhododendron Subgenus Tsutsusi and R. moulmainense, a Fragrant Rhododendron from China. Horticulturae, 11(9), 1116. https://doi.org/10.3390/horticulturae11091116

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