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Article

Study on the Flower Biology of Camellia luteoflora—A Species with an Extremely Small Population

by
Fen Liu
1,†,
Bangyou Liu
2,†,
Weicheng Yang
1,*,
YueHua Song
3,
Sheng Liang
2,
Hangdan Chen
1,
Mengqing Zhou
1 and
Gaoyin Wu
1,*
1
School of Life Sciences, Guizhou Normal University, Guiyang 550025, China
2
Chishui Alsophila National Nature Reserve Management Bureau, Zunyi 646259, China
3
School of Karst Science, Guizhou Normal University, Guiyang 550025, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(9), 2098; https://doi.org/10.3390/agronomy15092098
Submission received: 11 July 2025 / Revised: 26 August 2025 / Accepted: 28 August 2025 / Published: 30 August 2025
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

The aim of this study was to elucidate the reproductive strategy of Camellia luteoflora, an endangered evergreen endemic to karst ecosystems. We observed and recorded its flowering phenology and flower-visiting insects, observed pollen morphology, determined pollen viability, and assessed stigma receptivity. The results showed that the flowering period of C. luteoflora started from early September to late December, with the average flowering period of individual flowers being 10–12 days. The pollen morphology of C. luteoflora was subprolate and prolate, with three germinal apertures and the fossulate exine ornamentation. Pollen viability was the highest at the initial opening stage (80.30%). In the process of pollen in vitro, the order of influence on the germination rate and pollen tube length was temperature > sucrose > calcium chloride (CaCl2) > boric acid (H3BO3). The best combination for the germination rate was 24 °C, 75 g/L sucrose, 0.2 g/L CaCl2, 0.15 g/L H3BO3, while that for the pollen tube length was 24 °C, 100 g/L sucrose, 0.2 g/L CaCl2, 0.25 g/L H3BO3. Stigma receptivity was the strongest at the full blooming stage. The pollen/ovule ratio (P/O) was 2240, suggesting a facultative outcrossing breeding system. The outcrossing index (OCI) was 4, suggesting that the exogamous breeding system is the cross-pollination type, partially self-compatible and insect pollinator-dependent. The flower-visiting insects included bees, weevils, and ants. In summary, C. luteoflora exhibits an extended flowering period, with a prolonged overlap of stable pollen viability and stigma receptivity, suggesting a potential strategy to cope with pollination uncertainty. However, field observations recorded only a few species of potential pollinators, while the occurrence frequency of non-pollinating insects was relatively high. It is thus hypothesized that this apparent lack of effective pollinators may act as a potential barrier to successful fertilization and natural regeneration, which might also be one of the factors contributing to its endangered status. Future studies, particularly pollinator exclusion and hand-pollination experiments, are critically needed to verify whether pollinator limitation is indeed a key factor.

1. Introduction

Camellia luteoflora (Theaceae, Sect. Luteotlora) is a perennial evergreen shrub or small tree endemic to the China–Himalayan region. Currently, its natural distribution is restricted to Chishui city in Guizhou Province, and in Gulin and Changning Counties in Sichuan Province [1], with a current wild population of merely 545 individuals [2]. Characterized by its diminutive, golden, fragrant flowers and broad, thick leaves, it is known as the “Queen of Camellias”. C. luteoflora is rich in a variety of amino acids, sugars, lipids, and other nutrients, as well as bioactive compounds such as terpenoids, flavonoids, and polyphenols [3], which endow C. luteoflora with notable pharmacological properties, including anti-oxidation, anti-inflammatory, and anti-tumor activities. It is a plant with ornamental and economic value. However, C. luteoflora faces severe threats to its survival, such as the narrow distribution area, human interference, Exobasidium gracile (shirai) Syd, and witches broom disease [4], coupled with low seed viability and germination rates, prolonged fruiting period, asynchronous seed maturation, and its reproductive rate under wild conditions is extremely low [5]. Moreover, with the clonal expansion of bamboo surrounding C. luteoflora populations and the fragmentation of their original habitats, the population size and number of individuals have shrunk sharply, leaving the species in an extremely endangered state [2]. Therefore, conducting flowering biology research to elucidate key reproductive barrier mechanisms from a reproductive biology perspective holds significant theoretical value and practical significance for formulating scientific conservation strategies and achieving population restoration.
Flowering phenology, a pivotal aspect of plant phenophase, reflects plant growth, development, and physiological processes in response to environmental cues [6]. Systematic monitoring of flowering phenology allows for accurate analysis of the temporal window during which pollination may occur, providing a crucial temporal reference for optimizing artificial pollination protocols, screening superior germplasm resources, and protecting genetic diversity [7]. In plant cross breeding, pollen is the carrier of half of the genetic information. The morphological characteristics of pollen closely relate to pollen viability, storability, and stigma recognition specificity; they are considered as a key indicator for evaluating pollen quality and predicting plant reproductive success. Irregular abnormal pollen may be abortive and thus affect pollen fertility [8]. Pollen viability constitutes a critical determinant of fruit set. In addition to intrinsic genetic factors and environmental conditions, pollen viability is further regulated by boron (B), calcium (Ca), temperature, and other variables [9]. Notably, the characterization of pollen morphological traits and assessment of germination performance directly influence pollination efficacy, fertilization success, and germplasm conservation and utilization strategies [10]. Knowing the biological characteristics of pollen can allow for the strategic timing of pollen collection from male parents, thereby enhancing pollination efficiency and breeding success [11]. The stigma, as the receptive surface of the female reproductive organ, provides an optimal microenvironment for pollen adhesion, hydration, germination, and pollen tube elongation. The transfer of viable pollen grains onto a receptive stigma is a prerequisite for the initiation and completion of fertilization [12].
The pollen viability and stigma receptivity in plants exhibit pronounced variations across different developmental stages. For example, the pollen viability of Epimedium sagittatum can reach 99% at the sepal splitting stage, sometimes sharply declining to 13% at the withering stage [13]. Moreover, intraspecific variability is also evident, for instance, with significant differences in pollen viability and stigma receptivity among various pear cultivars [14]. Therefore, simultaneous analysis of the temporal dynamics of stigma receptivity and the decay curve of pollen viability is essential for constructing a precise pollination technology system. In pollination, flower-visiting insects are the largest and most functional group of all pollinators, which play a key role in the sexual reproduction of angiosperms [15]. Insect flower-visiting behavior is a multifaceted process encompassing parameters such as flower-visiting frequency, dwell time, and daily activity schedules. These multi-dimensional behavioral characteristics not only reflect the intricate co-evolution mechanism between insects and plants but also constitute critical determinants of pollen transfer efficiency [16].
In this study, an extremely small population in Guizhou of C. luteoflora was selected as the research object. The dynamic characteristics of flowering phenology and pollen morphological structure were systematically observed through systematic observation of the dynamic characteristics of flowering phenology and pollen morphological structure. Additionally, the temporal and spatial changes in pollen viability and stigma receptivity at different developmental stages were analyzed. Concurrently, behavioral ecology observation of flower-visiting insects was performed, and pollen germination tests in vitro were carried out. This study conducted research on the floral biological characteristics and breeding system of C. luteoflora, aiming to clarify the breeding mechanism of this species and reveal the causes of its endangerment from the perspective of reproductive biology. The research results can provide a theoretical reference for the conservation and propagation of C. luteoflora.

2. Materials and Methods

2.1. Experimental Site

C. luteoflora was collected from the China Rare Species C. luteoflora Reserve in Chishui City, Guizhou Province (longitude: 106°03′ E, latitude: 28°23′ N, 497 m). The reserve is characterized by a subtropical humid monsoon climate, with an average annual temperature of 19.3 °C, a relative humidity exceeding 84%, a slope direction of south, and a soil type of purplish soil.

2.2. Research Method

2.2.1. Flowering Phenology

The population flower period: In September to December 2024, twelve C. luteoflora individuals with normal development, robust growth, and free from pests and diseases were carefully selected as the research subjects at the experimental site; they were numbered and tagged. The total number of flower buds and open flowers per plant was recorded, and the flowering ratio was calculated. The phenological phases—including bud stage, initial blooming stage, full blooming stage, withering stage, and fallen stage—were determined based on the population/average flowering ratio. For detailed examination of single flower development, 20 flowers were carefully selected from well developed inflorescences [17]. The following formula was used to calculate the flowering ratio:
Flowering ratio = (Number of open flowers/total number of flowers) × 100%

2.2.2. Pollen Morphology

Observations of pollen structure were conducted following the method described by Chen et al. [18]. Ten pollen grains were randomly selected to measure indices including polar axis length and equatorial axis length. Pollen shape classes (P/E ratio) were classified according to Erdtman’s system [19]: oblate-spheroidal (0.89–0.99), spheroidal (1.00), prolate-spheroidal (1.01–1.14), subprolate (1.15–1.33), and prolate (1.34–2.00).

2.2.3. Pollen Viability

The method described by Aydin et al. [20] was adopted with minor modifications: ten flowers of C. luteoflora at different stages were selected from 5 healthy plants, and their anthers were isolated. The anthers were placed in a 1.5 mL centrifuge tube to prepare pollen suspension. The pollen suspension was dropped onto a glass slide, and subsequently 1% aceto carmine was added dropwise and the sample was stained at 25 °C for 5 min for examination under microscope (SOPTOP EX33, SOPTOP, Ningbo, China). The viable pollen grains were stained red, while the non-viable or aborted pollen grains were stained lightly or remained colorless. The following formula was used to calculate pollen viability:
Pollen viability = (Number of stained pollen grains in the field of view/total number of pollen grains in the field of view) × 100%

2.2.4. Estimation of Pollen/Ovule Ratio (P/O) and Outcrossing Index (OCI)

The method described by Zhang et al. [21] was used with minor modifications: ten flowers of C. luteoflora that were open but whose anthers had not yet dehisced were randomly selected from 5 healthy plants and collected indoors. After the anthers dehisced, they were suspended into a volume of 1 mL. Then, 10 μL of the suspension was taken to count all pollen grains, and finally the total pollen count per flower was calculated. The petals, sepals, corolla, and stamens of C. luteoflora were removed, and the ovary was placed under a dissecting microscope. The ovaries were dissected with a dissecting needle to count the number of ovules per flower. The mating system type was evaluated according to the criteria of Cruden [22]. The following formula was used to estimate the pollen/ovule ratio:
Pollen/ovule ratio (P/O) = Average pollen quantity per single flower/average number of ovules per single flower
A total of 30 flowers of C. luteoflora at the full blooming stage were randomly selected from 5 healthy plants to measure corolla diameter, petal length, petal width, filament length, anther length, and style length using vernier caliper. The outcrossing index (OCI) is determined by three floral traits, namely floret diameter, dichogamy, and herkogamy, and its measurement, calculation, and evaluation were conducted in accordance with the criteria proposed by Dafni [23]. The OCI was calculated as the sum of three components: (1) floret diameter, where <1 mm was assigned a value of 0, 1–2 mm a value of 1, 2–6 mm a value of 2, and >6 mm a value of 3; (2) stamen–pistil maturation synchronicity, scored as 0 when anther dehiscence and stigma receptivity were synchronous or protogynous (pistil maturation first), and as 1 when protandrous (stamen maturation first); and (3) spatial congruence of reproductive organs within a floret, assigned a value of 0 where stigmas and anthers occupied the same vertical position, and a value of 1 when positioned at differing heights.

2.2.5. In Vitro Pollen Germination

In vitro pollen culture experiments were conducted with slight modifications following the methods described by Huang et al. [24] and Zhang et al. [25]. Different concentrations of sucrose solution (50, 75, 100, 125 g/L), H3BO3 (0.10, 0.15, 0.20, 0.25 g/L), CaCl2 (0.10, 0.20, 0.30, 0.40 g/L), and temperatures (18, 24, 30, 36 °C) were set up, which were combined to form 16 medium formulations. Distilled water was used as the blank control (CK) for each group. After culturing at room temperature for 3 h, observations were made under a microscope to count the pollen germination rates and measure the pollen tube length. The following formula was used to estimate the germination rate:
Germination rate = (Number of germinated pollen grains/total number of pollen grains) × 100%

2.2.6. Determination of Stigma Receptivity

The determination of stigma receptivity followed the method described by Li et al. [26]: five flowers of C. luteoflora at different stages were randomly selected, and their stigmas were excised for stigma receptivity determination. The receptivity of the stigma was determined by the benzidine–hydrogen peroxide method (1% benzidine/3% hydrogen peroxide/distilled water = 4:11:22, v/v/v). The stigmas of flowers at different stages were collected and placed on a glass slide with the benzidine–hydrogen peroxide reaction solution, ensuring complete immersion of the stigmas. After 5 min, the stigmas were observed under a stereomicroscope (Nikon SMZ800N, Nikon, Tokyo, Japan).

2.2.7. Flower-Visiting Insects

During the flowering period, flower-visiting insects were continuously monitored for 20 h per day. Observations and photographic documentation of their visitation behavior on the plants were conducted to evaluate their potential effectiveness as pollinators. Insect species were identified using the Economic Insect Fauna of China [27] and through consultation with entomological experts.

2.2.8. Statistical Analysis

The percentages in the raw data need to be converted using the arcsin formula y = arcsin(p). One-way analysis of variance (ANOVA) with Duncan’s multiple range test was performed using SPSS 22.0 software, and the significance level was set at p < 0.05. Data visualization was conducted using Origin 19.0; images were processed using Photoshop CS 6.

3. Results

3.1. Flowering Phenology

The flowering phenology of C. luteoflora can be divided into five distinct stages: the bud stage, initial blooming stage, full blooming stage, withering stage, and fallen stage. The bud stage is initiated in early to mid-September, the initial blooming stage is between mid-September and early October, and the full-bloom phase occurs during the period from mid-to late October to early November. When the withering stage initiates in mid-November, the blossoms gradually deteriorate. The overall flowering duration of the population spans approximately four months. The average flowering period of individual flowers was 10–12 days. (Figure 1). The following floral stages were identified and will be used hereafter:
  • Bud stage: Typically lasting for 4–5 days, buds enlarge and become ellipsoidal, petals are pale yellow with a reddish tinge at the apex (Figure 2A).
  • Initial blooming stage: The corolla unfolds into a campanulate shape, with an overall pale yellow color, stamens begin to emerge, and the anthers are yellow (Figure 2B).
  • Full blooming stage: The corolla is fully expanded, petals are yellow, stamens are fully exserted, with yellow anthers (Figure 2C).
  • Withering stage: The corolla exhibits irregular morphology (elliptical or campanulate), petals are yellow with localized red pigmentation, anthers are brownish-yellow (Figure 2D).
  • Fallen stage: The corolla is umbellate, petals develop an overall red coloration, stamens are exserted, with brownish-yellow anthers (Figure 2E).

3.2. Pollen Morphology

Pollen grains of C. luteoflora exhibit polar axis lengths (P) ranging from 30.06 to 34.59 μm and equatorial axis lengths (E) of 25.40–27.85 μm, resulting in a P/E ratio of 1.13–1.35 (Table S1). Based on the pollen morphological categorization criteria, the pollen of C. luteoflora is subprolate and prolate, appearing as a trilobed subsphere in polar view and elliptical in equatorial view. The pollen grains are tricolporate and radially symmetrical, and the germinal apertures have protrusions at the equator and apertures that are constricted toward the poles. Numerous irregularly shaped and unevenly sized fossae are present on the surface of C. luteoflora’s exine ornamentation, which is fossulate (Figure 3). According to Erdtman’s NPC categorization system [28], C. luteoflora’s pollen is categorized as N3P4C5.

3.3. Pollen Viability

Aceto carmine staining revealed that the majority of C. luteoflora pollen grains stained crimson (Figure 4A,B). Pollen vitality showed a trend of first increasing and then decreasing during the flowering period, and peaking at 80.3% during the initial blooming stage. In comparison to the bud stage (74.77%) and the full blooming stage (76.77%), there was no significant difference in pollen viability at this time, although it was much higher than at the withering stage (68.71%) and the fallen stage (37.30%) (Figure 4C).

3.4. Pollen Germinability

The pollen tube length and pollen germination rate of C. luteoflora varied significantly (p < 0.05) throughout the 20 culture media groups (Table 1; Figure S1). Among them, the germination rate of M6 (75 g/L sucrose, 0.15 g/L H3BO3, 0.1 g/L CaCl2, 30 °C) was significantly higher than the other treatments. The length of the pollen tube in M9 (100 g/L sucrose, 0.1 g/L H3BO3, 0.3 g/L CaCl2, 24 °C) was significantly longer than other treatments.
Based on the range analysis of the germination rate and pollen tube length for the 16 treatments in the orthogonal test (Table 2), the influence order on the germination rate and pollen tube length was temperature > sucrose > CaCl2 > H3BO3. The optimal combination for the germination rate was 75 g/L sucrose, 0.15 g/L H3BO3, 0.2 g/L CaCl2, and 24 °C (K), while the optimal combination for the pollen tube length was 100 g/L sucrose, 0.25 g/L H3BO3, 0.2 g/L CaCl2, and 24 °C (Z).
Furthermore, the optimum combinations for the pollen tube length (Z) and germination rate (K) were further established by range analysis (Figure 5). The pollen tube length for K treatment was 366.55 μm, and the germination rate was 70.63%; for Z treatment, the pollen tube length was 467.28 μm, and the germination rate was 50.33%. However, the pollen tube length of M9 was longer than that of group Z treatment, and the germination rate of M6 was higher than that of K treatment, but the differences were not significant (p < 0.05).

3.5. Stigma Receptivity

C. luteoflora demonstrated stigma receptivity across the bud, initial blooming, full blooming, and withering stages. Furthermore, stigma receptivity showed a trend of increasing and then decreasing with the progression of floral development (Table 3). Fewer bubbles and a slight color change were present during the bud stage (Figure 6A); more bubbles and a slight color change were present during the initial blooming stage (Figure 6B); many bubbles and a deep color were found during the full blooming stage (Figure 6C); conversely, fewer bubbles and a slight color change were found during the withering stage (Figure 6D).

3.6. P/O Ratio and OCI Value

Quantitative analysis revealed that C. luteoflora flowers produced an average of 26,880 pollen grains per flower and 12 ovules, resulting in a P/O ratio of 2240 (Table S3). According to Cruden’s criteria, the breeding system type of C. luteoflora belongs to facultative outcrossing.
The average corolla diameter of C. luteoflora flowers is 13.45 mm (>6 mm, recorded as 3); anther dehiscence and stigma receptivity occur almost simultaneously (recorded as 0); the stamens are arranged in two whorls clearly, with the anthers of the outer whorl higher than the stigma and those of the inner whorl lower than or level with the stigma (Figure 7) (recorded as 1). Cumulatively, C. luteoflora’s outcrossing index comes to 4. Dafni’s criteria state that C. luteoflora is an outcrossing type, relies on pollinators, and exhibits partial self-compatibility.

3.7. Flower Visitors

The flower-visiting organisms of C. luteoflora can be categorized into two functional guilds. Floral organ consumers, which target reproductive and vegetative structures such as styles, stamens, petals, and nectar/pollen foragers, primarily access floral rewards. These guilds were predominantly represented by Hymenoptera (bees, ants) and Coleoptera (weevils, leaf beetles) (Figure 8).
Bees generally began to emerge at 15:00, and their peak activity period occurred between 16:00 and 17:00. Two flower-visiting behaviors were observed: (1) Pollen collection: Bees alighted on the corolla margin of C. luteoflora without entering the floral interior or contacting the stigma. (2) Nectar collection: Bees inserted their bodies into the corolla, with residence times ranging from 8 to 15 s. In both foraging patterns, pollen adheres to their legs and abdomens.
Weevils displayed distinct flower-visiting behaviors. They bored holes in flower buds to gain entry into the flower’s interior, where they consumed basal filaments and stigmas, leading to flower abortion; alternative entry via corolla openings was also observed, resulting in complete removal of stigmas and filaments. Leaf beetles targeted open flowers, burrowing into the flowers to consume filaments and anthers. Ants primarily foraged for nectar at filament bases, but occasionally engaged in destructive behaviors, including filament excision and consumption of petals, stigmas, and anthers.

4. Discussion

A comprehensive understanding of plant flowering biology is fundamental to studying life history strategies, serving as a critical foundation for the protection, utilization, and breeding of germplasm resources [29]. The flowering phenology of plants plays a pivotal role in pollination ecology. This is especially the case for plants in the Theaceae family, where pollination predominantly occurs during winter and early spring flowering. For instance, C. huana [30], C. oleifera [31], and C. pubipetala [32] mostly flower from January to February. Individual flowers of C. luteoflora exhibit a bloom duration of 10–12 days, while the population-level flowering period spans approximately 4 months, extending from autumn to early winter within the China Rare Species C. luteoflora Reserve. This period coincides with scarce pollinator availability and diminishing day length. The extended population flowering period likely represents an adaptation to low pollinator visitation frequency by maximizing pollination opportunities. Conversely, the moderate duration of individual flowers optimizes the trade-off between reproductive efficiency and resource allocation under cool temperatures [33]. Future research employing transcriptomics, climatic scenario modeling, and pollination network analysis could provide further validation of these adaptive mechanisms.
As the carrier of male genetic material in sexual reproduction [34], pollen contains abundant genetic information and exhibits strong genetic conservatism, making it critical for studies on plant origin, evolution, taxonomy, and species identification [35]. Pollen morphological traits are regulated by the genetic information it harbors and serve as reliable taxonomic markers. The pollen of C. luteoflora is subprolate and prolate, featuring tricolporate germination apertures and foveolate exine ornamentation. This is comparable to the pollen morphology of other species within the Theaceae family, such as C. weiningensis [36] and C. oleifera [37]. Nevertheless, there are notable disparities in parameters such as exine smoothness, the depth of germination poles, and polar axis length.
Pollen quality is an essential prerequisite for determining the success of plant cross-breeding, with pollen viability serving as a crucial parameter for evaluating and grading quality [38]. This study revealed that peak pollen viability (80.30%) was observed at the initial blooming stage. Liao et al. [39] reported viability exceeding 90% in the freshly opened anthers of C. chuongtsoensis, C. ptilosperma, and C. azalea using aceto carmine staining. This interspecific discrepancy might be attributed to biological differences among species, as well as various factors including the physiological status of the plants and environmental conditions.
Pollen viability represents an indispensable precondition for pollen germination, intricately modulated by genetic, environmental, and physiological determinants, which collectively influence plant reproductive success. During in vitro pollen germination, sucrose is commonly used as an energy substrate to initiate germination and sustain pollen tube elongation [40]. Concurrently, sucrose also serves as a regulator of osmotic equilibrium [41]. Li et al. [42] reported that an optimal sucrose concentration can significantly facilitate the germination of C. reticulata pollen. Conversely, a supraoptimal sucrose concentration induces plasmolysis within pollen grains, while a suboptimal concentration fails to provide adequate nutritional support for germination, ultimately culminating in the rupture of the pollen wall. Besides sucrose, boron (B) and calcium (Ca2+) are indispensable elements in pollen physiology. Previous studies have reported that H3BO3 can form a complex with sucrose, enhancing sugar uptake and metabolism, which facilitates the extracellular Ca2+ influx into cells. This process is pivotal for the construction of the pollen tube wall, thereby promoting pollen germination and the elongation of pollen tubes—a critical step in pollen tube wall biosynthesis [43,44]. Cytosolic Ca2+ serves as a pivotal secondary messenger within the intricate signaling cascades that govern pollen tube elongation and directional reorientation. In vivo, the growth of pollen tubes predominantly relies on extracellular Ca2+ reservoirs present in the pistil. Additionally, exogenous Ca2+ has been demonstrated to significantly enhance pollen tube elongation under in vitro conditions [45]. Furthermore, temperature is a critical regulator in in vitro pollen culture. Cui et al. [46] reported that short-term low temperatures disrupt pollen development, leading to morphological and functional changes in pollen (including epidermal abnormalities, starch overaccumulation, pollen wall alterations, and excessive ROS production in anthers), thereby reducing pollen viability and fruit set. Masoomi-Aladizgeh et al. [47] discovered that photosynthesis has a minimal impact at 40 °C, and pollen development is highly temperature-sensitive; exposure to this temperature reduces pollen size and viability by nearly 40% compared to optimal conditions (28 °C). Li et al. [48] reported that the optimal temperature for pollen germination is 30 °C in C. azalea, and 26 °C in C. rostrata [22]. In addition, the synergistic effects of sucrose, H3BO3, CaCl2, and temperature are pivotal for maintaining osmotic balance and nutrient supply during pollen germination. These findings offer valuable guidance for artificial assisted pollination practices, the facilitation of pollen germination, and the formulation of nutrient solutions tailored for promoting pollen germination [49]. Range analysis in this study revealed that temperature exerted a more pronounced influence compared to sucrose, CaCl2, and H3BO3 on C. luteoflora pollen germination, with the optimal combination identified as 75 g/L sucrose + 0.15 g/L H3BO3 + 0.2 g/L CaCl2 at 24 °C. For C. rostrata, the optimal medium was 100 g/L sucrose + 0.1 g/L H3BO3 [24]; for C. liberofilamenta, the optimal medium was 1% agar + 0.1 g/L boric acid + 200 g/L sucrose [50]; and for C. oleifera, the optimal medium was 1% agar + 150 g/L sucrose + 0.15 g/L H3BO3 + 0.07 g/L MgSO4 + 0.01 g/L IAA [51]. This indicates that there are great differences in the response of the pollen germination of different Camellia plants to different in vitro media and concentrations. This study identified a suitable medium for the in vitro pollen culture of C. luteoflora, laying the groundwork for addressing key challenges in hybrid breeding, specifically asynchronous flowering, artificial pollination, and long-term germplasm conservation.
Pollination success relays upon the encounter between highly viable pollen grains and a highly receptive stigma. This study revealed that C. luteoflora exhibited high pollen viability and strong stigma receptivity simultaneously during the flowering stage, forming a highly overlapping effective pollination period in terms of time. This strategy significantly extended the pollination window and enhanced the reproductive assurance ability of the plant to cope with the uncertainty of pollinator visits or adverse environmental conditions. For C. luteoflora, a species with extremely small populations, this characteristic reduces the risk of pollination failure caused by random factors such as small population size and fluctuating pollination environments. It provides an important ecological adaptive mechanism for maintaining population continuity and reducing extinction risks under natural conditions.
Based on the P/O ratio and the OCI, the breeding system of C. luteoflora is marked by outcrossing, partial self-compatibility, and a dependence on pollinators. Although the breeding system of C. luteoflora predominantly involves outcrossing, C. luteoflora exhibits self-compatibility when outcrossing is obstructed. Comparative analysis with C. huana reveals significantly higher pollen production per flower in C. luteoflora (26,880 vs. 1099) and a higher P/O ratio [30]. As proposed by Cruden et al. [22], a substantial quantity of pollen and high P/O values are advantageous for promoting outcrossing and enhance reproductive assurance and evolutionary potential for C. luteoflora. Nevertheless, in this study, a bagging pollination test was not carried out, and the breeding system of C. luteoflora was deduced from the P/O values and OCI, which still requires further verification.
The floral morphology is intimately linked to pollinator behavior, pollination mechanisms, and plant adaptability [21]. Insect-mediated selection has profoundly shaped floral evolution, with plants developing specialized traits that attract and facilitate efficient pollination by mutualistic partners [16]. Bee-pollinated flowers generally exhibit bilateral symmetry, alluring colors, faint aroma, and nectaries, and their reproductive organs are situated within the corolla [52]. C. luteoflora features a compact floral morphology, featuring yellow petals and anthers, radial symmetry, with the stamens and pistils positioned within the corolla. These morphological traits are favorable for bee pollination. The pollinators of C. luteoflora are relatively scarce, and the primary floral visitor is Apis cerana. A. cerana’s visits last 8 to 15 s, with peak activity between 16:00 and 17:00. However, C. osmantha attracts diverse visitors (such as Andrena camellia, Vespa velutina, Eristalis tena), with a relatively extended visitation time (from 11 to 27 s) and bimodal activity pattern (11:30–13:00 and 15:00–16:30) [53]. These interspecific differences may arise from variations in floral morphology, nectar production, habitat conditions, and pollinator community structure. Notably, while the high color saturation of C. luteoflora enhances attractiveness to flower visitors, it may also trigger destructive feeding behavior by non-target species, such as flower-eating beetles, leading to pollen robbing. Such trade-offs between attractiveness and robbing risks highlight the complex evolutionary dynamics shaping floral–pollinator interactions.
C. luteoflora is a precious Camellia resource, with its flower color containing rare golden-yellow genes and thus high ornamental value. Studying the flowering biological characteristics can lay a foundation for future cross-breeding efforts. In particular, the findings on its flower opening process, pollen viability, and stigma receptivity can directly guide breeding practices.

5. Conclusions

This preliminary study on the flowering phenology, pollen viability, stigma receptivity, and breeding system of C. luteoflora revealed an extended population flowering period of four months, with constant pollen viability and stigma receptivity. However, the species faces a scarcity of flower-visiting insect species and high prevalence of non-pollinating invasive insects, which may be a major barrier to natural regeneration, leading to one of the key factors contributing to the endangered status of C. luteoflora. These findings enhance the understanding of C. luteoflora’ s biological characteristics and provide important references for the conservation and utilization of its plant resources, laying the groundwork for further investigation into its pollination ecology and the relationship between its breeding system traits and the drivers of its endangerment.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15092098/s1. Table S1: Relevant morphological parameters of C. luteoflora pollen; Table S2: Measurements of parameters of floral organs; Table S3: Estimation of P/O ratio of C. luteoflora; Figure S1: In vitro pollen germination of C. luteoflora under different culture media.

Author Contributions

All authors contributed to the study conception and design. F.L., B.L. and G.W. designed the research. F.L., B.L., S.L., Y.S., H.C. and M.Z., performed the experiments. F.L. analyzed the data. F.L. and B.L. wrote the manuscript. G.W. and W.Y. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Science and Technology Innovation Talent Team Building Project of the Science and Technology Innovation Talent Team Building Project of Guizhou Province (Qiankehepingtairencai-CXTD [2023]010); the Camellia luteoflora Very Small Populations In Situ Con-servation Project [grant code 11904-0623033]; and the Camellia luteoflora Woodland Conservation project [grant code 11904-0623034].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The flowering phenology of C. luteoflora.
Figure 1. The flowering phenology of C. luteoflora.
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Figure 2. Morphological characteristics of C. luteoflora at different stages. (A) Bud stage; (B) initial blooming stage; (C) full blooming stage; (D) withering stage; (E) fallen stage.
Figure 2. Morphological characteristics of C. luteoflora at different stages. (A) Bud stage; (B) initial blooming stage; (C) full blooming stage; (D) withering stage; (E) fallen stage.
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Figure 3. SEM images of C. luteoflora pollen morphology. (A) Polar view; (B) equatorial view; (C) germinal apertures; (D) exine ornamentation.
Figure 3. SEM images of C. luteoflora pollen morphology. (A) Polar view; (B) equatorial view; (C) germinal apertures; (D) exine ornamentation.
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Figure 4. Pollen viability of C. luteoflora. (A) Initial blooming stage; (B) withering stage; (C) line chart of pollen viability at five stages. Different lowercase letters indicate significant differences (p < 0.05).
Figure 4. Pollen viability of C. luteoflora. (A) Initial blooming stage; (B) withering stage; (C) line chart of pollen viability at five stages. Different lowercase letters indicate significant differences (p < 0.05).
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Figure 5. Verification of orthogonal test for in vitro germination of C. luteoflora. (K) Pollen germination in K treatment; (Z) Pollen germination in Z treatment; (Q) Comparison between M6 and K, and be-tween M9 and Z. Different lowercase letters indicate significant differences (p < 0.05).
Figure 5. Verification of orthogonal test for in vitro germination of C. luteoflora. (K) Pollen germination in K treatment; (Z) Pollen germination in Z treatment; (Q) Comparison between M6 and K, and be-tween M9 and Z. Different lowercase letters indicate significant differences (p < 0.05).
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Figure 6. Stigma receptivity of C. luteoflora at different stages. (A) Stigma receptivity at bud stage; (B) stigma receptivity at initial blooming stage; (C) stigma receptivity at full blooming stage; (D) stigma receptivity at withering stage.
Figure 6. Stigma receptivity of C. luteoflora at different stages. (A) Stigma receptivity at bud stage; (B) stigma receptivity at initial blooming stage; (C) stigma receptivity at full blooming stage; (D) stigma receptivity at withering stage.
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Figure 7. Internal morphology of C. luteoflora.
Figure 7. Internal morphology of C. luteoflora.
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Figure 8. Flower-visiting insects of C. luteoflora. (A) Apis cerana; (B) Sympiezomias citri; (C) Arthrotus nigrofasciatus; (D) Tetramorium caespitum.
Figure 8. Flower-visiting insects of C. luteoflora. (A) Apis cerana; (B) Sympiezomias citri; (C) Arthrotus nigrofasciatus; (D) Tetramorium caespitum.
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Table 1. Orthogonal test for in vitro germination of C. luteoflora.
Table 1. Orthogonal test for in vitro germination of C. luteoflora.
No.Sucrose (g/L)H3BO3 (g/L)CaCl2/(g/L)Temperature (°C)Germination Rate (%)Pollen Tube Length (μm)
M1500.10.11840.81 ± 3.42 cd151.29 ± 23.42 g
M2500.150.22461.59 ± 4.20 b365.94 ± 16.41 c
M3500.20.33020.75 ± 1.07 gh206.80 ± 2.44 ef
M4500.250.4360.96 ± 0.24 j60.84 ± 12.09 i
M5750.10.2366.10 ± 1.97 j70.98 ± 12.49 hi
M6750.150.13071.96 ± 5.68 a422.55 ± 46.53 b
M7750.20.42462.33 ± 6.71 b357.86 ± 28.23 c
M8750.250.31831.68 ± 3.23 ef81.95 ± 2.22 hi
M91000.10.32442.29 ± 0.44 cd472.15 ± 7.86 a
M101000.150.41826.70 ± 4.64 dfg170.84 ± 14.92 fg
M111000.20.1362.20 ± 1.17 j55.12 ± 2.99 i
M121000.250.23056.42 ± 7.23 b337.83 ± 62.40 c
M131250.10.43018.09 ± 5.53 hi273.37 ± 13.41 d
M141250.150.3360 j0 j
M151250.20.21837.74 ± 1.96 de231.29 ± 26.09 e
M161250.250.12445.96 ± 1.85 c267.84 ± 16.81 d
CK10001842.94 ± 2.24 cd101.39 ± 9.13 h
CK20002434.08 ± 4.34 e164.57 ± 8.20 g
CK30003013.20 ± 2.12 i218.72 ± 3.60 e
CK4000360 j0 i
Note: Different lowercase letters indicate significant differences (p < 0.05).
Table 2. Range analysis of orthogonal test for in vitro germination of C. luteoflora.
Table 2. Range analysis of orthogonal test for in vitro germination of C. luteoflora.
FactorSucroseH3BO3CaCl2Temperature
Germination rate (%)K131.0326.8240.2334.24
K243.0240.0640.4653.04
K331.9030.7623.6841.81
K425.4533.7527.022.31
Range17.5713.2416.7850.73
Pollen tube length (μm)Z1196.22241.95224.20158.84
Z2233.33239.83251.51365.95
Z3381.94212.77190.23310.14
Z4193.12260.92215.7346.74
Range188.8248.1561.28319.21
Table 3. Stigma receptivity in C. luteoflora at different stages.
Table 3. Stigma receptivity in C. luteoflora at different stages.
StageStigma Receptivity
Bud stage++
Initial blooming stage+++
Full blooming stage+++++
Withering stage++
Note: “++” indicates weak stigma receptivity; “+++” indicates strong stigma receptivity; “+++++” indicates strongest stigma receptivity.
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Liu, F.; Liu, B.; Yang, W.; Song, Y.; Liang, S.; Chen, H.; Zhou, M.; Wu, G. Study on the Flower Biology of Camellia luteoflora—A Species with an Extremely Small Population. Agronomy 2025, 15, 2098. https://doi.org/10.3390/agronomy15092098

AMA Style

Liu F, Liu B, Yang W, Song Y, Liang S, Chen H, Zhou M, Wu G. Study on the Flower Biology of Camellia luteoflora—A Species with an Extremely Small Population. Agronomy. 2025; 15(9):2098. https://doi.org/10.3390/agronomy15092098

Chicago/Turabian Style

Liu, Fen, Bangyou Liu, Weicheng Yang, YueHua Song, Sheng Liang, Hangdan Chen, Mengqing Zhou, and Gaoyin Wu. 2025. "Study on the Flower Biology of Camellia luteoflora—A Species with an Extremely Small Population" Agronomy 15, no. 9: 2098. https://doi.org/10.3390/agronomy15092098

APA Style

Liu, F., Liu, B., Yang, W., Song, Y., Liang, S., Chen, H., Zhou, M., & Wu, G. (2025). Study on the Flower Biology of Camellia luteoflora—A Species with an Extremely Small Population. Agronomy, 15(9), 2098. https://doi.org/10.3390/agronomy15092098

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