Next Article in Journal
The Role of Radiation in the Modelling of Crop Evapotranspiration from Open Field to Indoor Crops
Previous Article in Journal
Transcriptome Analysis of ‘Kyoho’ Grapevine Leaves Identifies Heat Response Genes Involved in the Transcriptional Regulation of Photosynthesis and Abscisic Acid
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 12
Issue 11
10.3390/agronomy12112592
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparative Study on Pollen Viability of Camellia oleifera at Four Ploidy Levels

by
Rui Zhao
1,2,
Linjie Xu
1,2,
Xiangshuai Xu
1,2,
Yanmin Li
1,2,
Shixin Xiao
1,2,* and
Deyi Yuan
1,2,*
1
Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education, Central South University of Forestry and Technology, Changsha 410004, China
2
Key Laboratory of Non-Wood Forest Products of State Forestry Administration, Central South University of Forestry and Technology, Changsha 410004, China
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(11), 2592; https://doi.org/10.3390/agronomy12112592
Submission received: 3 October 2022 / Revised: 17 October 2022 / Accepted: 18 October 2022 / Published: 22 October 2022
Browse Figures
Review Reports Versions Notes

Abstract

:
Oil tea (Camellia oleifera Abel.) is one of the most important woody edible oil tree species in China, with intraspecific polyploid. In order to study the variation in pollen size and vigor of C. oleifera at ploidy level, four ploidy covers a total of 32 types of Camellia pollens as the material for the experiment. The results showed that the pollen sizes of diploid, tetraploid, hexaploidy, and octaploid were positively correlated with the ploidy level. Pollen viability of C. oleifera was determined by fluorescein diacetate (FDA) dye solution staining and medium containing 10% sucrose, 0.01% boric acid, and 1% agar germination in vitro, which indicated that the pollen viability and germination rate of the hexaploid were relatively high among the four ploidy levels, at 79.69% and 71.78% respectively. The pollen vigor of diploid NR-3, tetraploid DP43, hexaploid CJ-12, and octoploid YNYC-1 was higher than that of other materials with the same ploidy level. Knowledge of different ploidy pollen sizes and pollen viability provides basic information for formulating pollen breeding plans and pollination methods of C. oleifera.

1. Introduction

Oil tea (Camellia oleifera Abel.) belongs to the genus Camellia in the family Theaceae, which, together with oil palm (Elaeis guineensis Jacq.), olive tree (Olea europaea L.), and coconut palm (Cocos nucifera L.), is considered to be one of the four major woody oil crops in the world [1,2]. Tea oil, extracted from camellia seeds, is known as “oriental olive oil” because of its high nutritional value and health benefits [3]. It is rich in unsaturated fatty acids (more than 80% of the total oil content), mainly containing monounsaturated fatty acids (oleic acid, accounting for more than 68% of the total oil content) and part of polyunsaturated fatty acids (linoleic acid and linolenic acid) [3,4]. Studies have shown that camellia oil intake has health benefits, such as helping to lower blood fats and prevent cardiovascular disease [5]. In order to meet the rapidly growing demand for healthy vegetable oil, the Chinese government planned to increase the planting area of C. oleifera to more than 4 million hectares and the annual output of C. oleifera to 2.5 million tons [6]. With the rapid development of the camellia oil industry, a large area of C. oleifera is planted in the red soil hilly region of south China. Jiangxi, Hunan, and Guangxi provinces are the main cultivation areas, accounting for 76% of the national total output [1,7]. At present, however, the key problem in C. oleifera cultivation is to accelerate and improve the selection and breeding of different regions and improve the yield and quality of C. oleifera [6].
Pollen grains, which are produced in the anthers of higher plants, as sexual reproductive units and carriers of male genetic material, play an important role in plant breeding [8,9]. To support breeding programs, pollen fertility analysis is an indispensable prerequisite for field crossbreeding. Considering that fertilization is achieved by cross-pollination, during which the stigma is in a receptive state, the success of cross-pollination is also related to the ability of pollen germination [10]. Understanding the floral characteristics of existing germplasm, such as pollen viability and germination ability, is critical to the selection of donors for hybridization [11]. According to Nogueira et al. [12], in order to assess in vivo pollen germination, field hybridization must be performed and the results must be monitored. However, this process is time-consuming and expensive, so to reduce cost and time, as well as to aid decision-making, methods of assessing pollen viability by staining or in vitro germination are effective alternatives.
Polyploidy, also known as genome-wide replication, is a major mechanism of plant adaptation and speciation [13,14]. It is widespread in plant families and an estimated 47–70% of angiosperm species are polyploid [13]. Polyploidy can serve as a bridge for interspecific hybridization between two species previously separated by ploidy differences [15]. It has been reported that ploidy levels affect pollen tube growth and seed viability; ploidy crossing of fertile offspring may produce new, desirable traits in established varieties or allow the transfer of traits between varieties [16]. The species of oil tea not only vary greatly in traits, but also have different ploidy among some varieties [17]. Breeding improvement using mixed ploidy levels requires cytological methods to evaluate the fertility of potential parents and the suitability of potential hybrid combinations [16]. Pollen fertility can be evaluated by pollen viability, which is staining with nuclear stains and in vitro germination [18]. At present, there is no report on the pollen fertility of C. oleifera with different ploidy. Therefore, vigor staining and in vitro germination methods were used in this study to evaluate the fertility of some types of C. oleifera pollens at four ploidy levels. What is more, studies have shown that, in addition to pollen vitality, the size of pollen grains also has an impact on pollen fertility [19], and pollen size can be influenced by ploidy levels [20], so we measured pollen size in this experiment. The results of this paper will shed light on the study of pollen fertility of new hybrids and guide the hybrid breeding strategies of different varieties for C. oleifera.

2. Materials and Methods

2.1. Plant Materials and Pollen Collection

The unopened buds of C. oleifera were collected from a planted forest called the state-owned Dongfeng Forest Farm, Liping, Guizhou Province, China. The gathering time was about 10 a.m. every day, and the flowering period did not reach the peak. The anthers of each bud are picked off with tweezers in a small carton that had been folded in advance and placed at room temperature for about 4 h to disperse the pollen. Then, they were placed in a 1.5 mL centrifuge tube and stored in a −80 °C refrigerator for temporary storage.

2.2. Pollen Ploidy

Flow cytometry (Sysmex CyFlow Space, Sysmex, Kobe, Japan) was used to detect the ploidy of oil tea pollens of different types [21,22,23]. First, we added an appropriate amount of sterile water to the small centrifugal tube containing the pollen and soaked the pollen for about 20 min. The pollen immersed in the bottom of the tube was sucked out into a new small centrifugal tube and the pollen of each sample was incubated in 20 μL of 40% polyvinyl pyrrolidone and 400 μL of nuclei extract buffer (CyStain®UV PRECISE P, Sysmex, Kobe, Japan) for 5 min in the dark. The mixture was then filtered through a 30 μm CellTrics®filter into a sample tube. Then, 1600 μL of staining buffer (CyStain®UV PRECISE P) was added and it was left for 5 min [24]. Finally, flow cytometry analysis was performed using ultraviolet excitation (λ = 355–375 nm) and blue fluorescence emission parameters (λ = 435–500 nm) (rate, nuclei per second). The analysis of each sample was performed with three replications according to the metology requirements [23].

2.3. Pollen Size

Pollen diameter was measured after Carbopol fuchsin dye staining. Appropriate amount of pollens were placed on the slide and stained with 50 uL Carbopol fuchsin dye solution for 10 s, then covered immediately with a cover glass [23], and then respectively observed and photographed with an Olympus BX51 compound microscope (Olympus Corp., Tokyo, Japan). The polar and equatorial lengths of pollens were observed by scanning electron microscope (SEM-6380LV, JEOL, Tokyo, Japan) [25,26]. Pollen size was measured using image processing software Image J (National Institutes of Health, Bethesda, Rockville, MD, USA) [27].

2.4. Pollen Viability

In this study, we used fluorescein diacetate (FDA) staining to evaluate pollen viability [28]. According to the criteria for the estimation of pollen efficiency given by Rotreklová and Krahulcová [29], pollen that emits yellow-green fluorescence was considered as active. Each treatment consisted of three replicates.

2.5. Pollen Germination

The in vitro germination method was used to determine the germination rate of pollens at four ploidy [30]. The culture medium for in vitro germination of C. Oleifera pollens obtained by previous screening was 1% agar + 10% sucrose + 0.01% boric acid [31].

2.6. Statistical Analysis

SPSS 19.0 software (IBM, New York, NY, USA) was used for data analysis and one-way analysis of variance (ANOVA) was used to test the differences in pollen size, pollen viability, and pollen germination rate of different ploidy. When p ≤ 0.05, we used the Duncan multiple comparison method to evaluate the significant difference between the means. The origin pro8.5 software (origin lab company, Northampton, MA, USA) was used to draw graphs [32].

3. Results

3.1. Pollen Size Measured after Staining and Scanning Electron Microscopy

In this experiment, we first determined the ploidy of 32 types of C.oleifera pollen by flow cytometry (Figure S1), and through statistical analysis and comparison, the diameters of the 32 types of pollen grains of four ploidy vary widely, ranging from 35.88 μm (NR-2, diploid) to 58.16 μm (YNYC-3, octaploid). According to the test, the pollen diameters at four ploidy levels were significantly different (p < 0.001). The average diameter of C. oleifera pollen grains was in the order of octaploid (50.54 μm) > hexaploid (49.40 μm) > tetraploid (41.92 μm) > diploid (37.85 μm) (Table 1 and Figure 1). In terms of diploid, NR-4 had the largest pollen grain diameter (39.30 μm); DF24 (44.24 μm) had the largest pollen grain diameter among the tested tetraploid pollens. For hexaploid, LE38 had the largest pollen diameter (50.87 μm), about 3.38 μm larger than the smallest pollen TXP-14 (47.49 μm). Among the four ploidy pollens of C. oleifera, the octoploid pollen had the largest mean diameter (Table 1; Figure 1), and the pollen grain diameter fluctuated between 46.31 μm and 58.16 μm, among which the pollen grain diameter of YNYC-3 was the largest (58.16 μm) (Table 1). The average polar axis lengths of diploid, tetraploid, hexaploidy, and octaploid measured by scanning electron microscopy were 36.68 μm, 44.27 μm, 47.93 μm, and 58.51 μm, respectively. The average equatorial axis lengths of diploid, tetraploid, hexaploid, and octoploid were 22.05 μm, 27.00 μm, 29.97 μm, and 30.84 μm, respectively (Table 2). The pollen size at four ploidy levels was ordered from largest to smallest as follows: octaploid > hexaploid > tetraploid > diploid (Table 1 and Table 2; Figure 1 and Figure 2).

3.2. Pollen Viability Assessed by Staining

In hexaploid, the average pollen viability ranged from 74.28% to 88.24%, among which CJ-12 had the highest pollen viability (88.24%). The pollen vigor of each type was sorted from high to low: CJ-12 (88.24%) > XH097 (85.38%) > TXP-14 (83.95%) > LE3 (79.33%) > LE38 (76.34%) > GTDB02 (75.29%) > CJ07 (74.70%) > ASX0902 (74.28%). As for tetraploid, the pollen vigor ranks were as follows: DP43 (80.25%) > DF24 (78.24%) > DB(1) (75.71%) > DP47 (72.76%) > DBH (71.52%) > MX-5 (67.12%) > CJ-03 (65.60%) > ZX0907 (56.40%). For diploids, NR-3 had the highest pollen viability among all pollens, which was 74.61%, and the NR-1 was the lowest (45.89%). Pollen vitality was sorted as follows: NR-3 (74.61%) > NR-9 (72.04%) > NR-10 (68.91%) > NR-6 (63.77%) > NR-4 (63.56%) > NR-5 (63.17%) > NR-2 (62.83%) > NR-1 (45.89%). Observing octoploids, the pollen vigor of the eight types was quite different, between 12.10% and 77.56%, among which, for pollen vigor, the highest was YNYC-1 and the lowest was YNYC-8. In addition, the pollen vigor of YNYC-7, YNYC-8, and YNYC-9 was not high (<30%). The order of pollen vigor from high to low was as follows: YNYC-1 (77.56%) > YNYC-5 (75.11%) > YNYC-4 (74.76%) > YNYC-2 (65.45%) > YNYC-3 (63.57%) > YNYC-8 (27.87%) > YNYC-7 (24.06%) > YNYC-9 (12.10%) (Table 3). For the pollens of C. oleifera at four ploidy levels, the order of average pollen viability was as follows: hexaploid (79.69%) > tetraploid (70.95%) > diploid (64.35%) > octaploid (52.56%) (Table 4; Figure 3).

3.3. Pollen Germination Rate Assessed by In Vitro Germination

In terms of hexaploid, the pollen germination rate fluctuated from 57.03% to 84.59%, among which CJ-12 had the highest pollen germination rate (84.59%). The order of pollen germination rate was as follows: CJ-12 (84.59%) > XH097 (83.98%) > GTDB02 (80.12%) > ASX0902 (77.04%) > LE38 (64.70%) > CJ-07 (63.58%) > LE3 (63.11%) > TXP-14 (57.03%). With regards to tetraploid, the pollen germination rate fluctuated between 65.56% and 76.45%, while the pollen types with the highest and lowest pollen germination rates were still DP43 and ZX0907. The order of their pollen germination rate was as follows: DP43 (76.45%) > DBH (75.47%) > DB(1) (74.05%) > DP47 (72.06%) > MX-5 (68.15%) > DF24 (66.28%) > CJ-03 (66.22%) > ZX0907 (65.56%). For diploids, NR-9 had the highest pollen germination rate among all pollens, which was 61.00%, and the pollen germination rate of NR-1 was the lowest (34.52%). The pollen germination rate was ranked from high to low as follows: NR-9 (61.00%) > NR-10 (55.64%) > NR-3 (53.71%) > NR-5 (50.75%) > NR-4 (50.65%) > NR-6 (49.21%) > NR-2 (40.17%) > NR-1 (34.52%). Considering octoploids, the pollen germination rate of the eight types tested was quite different, which fluctuated between 8.89% and 69.79%, among which, for pollen germination, the highest rate was YNYC-1 and the lowest was YNYC-8. Besides, the pollen germination rates of YNYC-7, YNYC-8, and YNYC-9 were not high, with all being less than 20%. The germination rates of the eight pollen species were as follows: YNYC-1 (69.79%) > YNYC-5 (68.87%) > YNYC-4 (65.10%) > YNYC-2 (63.25%) > YNYC-3 (25.71%) > YNYC-8 (19.68%) > YNYC-7 (14.86%) > YNYC-9 (8.89%) (Table 3). The order of pollen germination rate from high to low was hexaploid (71.78%) > tetraploid (70.53%) > diploid (49.46%) > octoploid (42.02%) (Table 4; Figure 4 and Figure 5), and the differences were very significant (p = 0.001) (Table 5).

4. Discussion

4.1. Pollen Size Related to the Ploidy Level

Pollen, as the carrier of male heredity, has all the gene types of a variety and is an important material for cross breeding [33]. In higher plants, polyploid plants generally have larger organs compared with diploid plants [34,35] and pollen size is often positively correlated with ploidy [36]. Pollen size is often used as a biological parameter to estimate the ploidy and viability of mature plants. In general, it is quantified by image-based diameter measurements [37]. For example, in the study of Avena [38] and Rumex [39], ploidy had a significant effect on pollen grain size and pollen grain size increased with the increase in ploidy level. As reported by Jan et al. [40], there is a clear tendency for polyploid pollen grains to be larger than diploid pollen grains. For some species, the size of mature pollen grains has been found to be positively correlated with the level of somatic ploidy in donor plants. In the report of Cui et al. [41], pollen size was positively correlated with ploidy and the size of tetraploid, triploid, and diploid pollens decreased successively. Besides, from Nico et al. [37], for Arabidopsis thaliana diploid-, tetraploid-, and octaploid-times body sequence of observation, they decided on pollen size analysis based on volume at different times to distinguish the level of sex of pollen plants having a very strong discrimination ability, in order to quickly determine the cell ploidy level of the plant or plant group, providing a simple and reliable method. In this experiment, by measuring and analyzing the pollens of C. oleifera of diploid, tetraploid, hexaploid, and octoploid, we found that the average pollen size increased with the increase in ploidy; that is, from diploid to octoploid, the average pollen grain size showed an increasing trend and the pollen size of four ploidy showed very significant differences, which is consistent with the results of Souza-Pérez et al. [20], who found that pollen grain size was heterogeneous and the pollen grain size of octoploid plants was larger, thus pollen size can be influenced by ploidy levels.

4.2. Pollen Viability and Pollen Germination at Different Ploidy Levels

Cross breeding is an important way to improve the character of plants and the quality of parents directly affects the efficiency of breeding [42]. Pollen quality is evaluated according to the viability and germination of pollen grains [43]. Pollen viability determines the reproductive success of a species to a certain extent and high viability pollen will increase the rate of seed setting, which will lead to the highest yield [42,44]. In vitro pollen germination tests are used to determine the germination rate of pollen and can also be used to assess pollen viability by monitoring the germination rate over time [45]. There were significant differences in the pollen viability of different cultivars [33]. Studies have shown that the level of ploidy affects pollen viability and pollen viability varies with the change in ploidy [16,41]. In our study, the pollen viability (p = 0.008) and germination rate (p = 0.001) at four ploidy levels showed significant differences. The average pollen viability fluctuated between 52.56% and 79.69% and the pollen germination rate ranged from 42.02% to 71.78%. Indeed, the vigor and germination rate of the hexaploid were the highest (79.69% and 71.78%, respectively), followed by tetraploid and diploid, and the average vigor and germination rate of the octaploid were the lowest. Specifically observing each pollen type selected and tested for ploidy, we found that pollen viability and pollen germination rate varied in the range of 12.10–88.24% and 8.89–84.59%, respectively, while hexaploid CJ-12 and octoploid YNYC-9 had the highest and lowest pollen viability and pollen germination rate, respectively. The results of Souza-Pérez et al. [20] showed that pollen viability in octoploid was low and there were differences between both intra and ploidies. The effect of pollen morphology on pollen viability depends on plant ploidy. Further, we observed that, in octoploids, not all types had low pollen viability and germination rate. For example, the pollen viability and germination rates of YNYC-7, YNYC-8, and YNYC-9 were less than 30%, while those of YNYC-1 could go up to about 70%. Therefore, we cannot assume that the pollen vigor of all octuploid camellia species is low. To sum up, differences in pollen grain viability and pollen size found in C. oleifera suggest that ploidy levels can affect these properties to varying degrees. Our study on pollen viability is of great significance for interspecific hybridization and variety management of C. oleifera.

4.3. Comparison of Two Vigor Measurement Methods

In plant breeding, simple methods are often needed to determine the viability of fresh pollen [46]. The common method for determining pollen viability is staining [47] and the germination test is considered to be the best indicator of pollen viability, which is a very effective and convenient method to study the biological application of pollen [46]. Some studies have suggested that the pollen viability measured by the staining method and germination method is quite different [41]. From their study, the pollen viability of the same variety measured by the germination method in the same time period was lower than that measured by the staining method, and the low degree was related to the variety and the time of pollen collection. In this experiment, we found that, although the pollen viability of YNYC-3 could reach more than 60%, its pollen germination rate was only about 25%. This may be in line with the findings of Cui et al. [41], who found that a small amount of pollen viability could be observed by the staining method, but the germination method showed that they do not have the ability to germinate. We speculated that the reason for this phenomenon may be that more aborted pollens were produced; the stained pollen grains were not necessarily germinable; and though some pollens had vitality, their vigor was not high and they could not germinate further. In addition, we found that the average germination rate of pollen of some types was greater than the percentage of vigor; we also observed such research results in the study of Guo et al. [9]. Cui et al. [41] reported that the germination rate of pollen grains is faster or slower, and the actual germination strength of pollen grains may be greater than the observed value. In a word, although the results measured by the two methods may be different, it is undeniable that both methods are simple and effective methods to measure the pollen viability. Therefore, in this study, we used these two methods to measure the pollen viability of different ploidy levels of C. oleifera pollen to improve the accuracy of the results.

5. Conclusions

In our study, the pollen size of Camellia oleifera increased with the increase in ploidy. The pollen viability and germination rate of hexaploid were higher than those of diploid, tetraploid, and octoploid, among which the pollen viability and germination rate of hexaploid CJ-12 were both high. The four ploidy pollens showed significant differences in size, pollen viability, and pollen germination rate. The results of this experiment will provide more consistent results within and between laboratories, and will be easily integrated into existing C. oleifera breeding programs.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy12112592/s1, Figure S1. Flow cytometric histogram of pollen of C. oleifera at four ploidy. “A, B, C, and D” represent diploid, tetraploid, hexaploid, and octaploid, respectively; the X-axis is relative DAPI fluorescence and the Y-axis is the number of cells.

Author Contributions

Conceptualization, R.Z. and S.X.; methodology, R.Z., L.X. and Y.L.; resources, X.X.; writing—original draft preparation, R.Z.; writing—review and editing, R.Z., S.X. and D.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Special Funds for Construction of Innovative Provinces in Hunan Province (2021NK1007), the National Natural Science Foundation of China (31500553), and National Key R&D Program of China (2018YFD1000603).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Wu, L.L.; Li, J.A.; Li, Z.; Zhang, F.H.; Tan, X.F. Transcriptomic Analyses of Camellia oleifera ‘Huaxin’ Leaf Reveal Candidate Genes Related to Long-Term Cold Stress. Int. J. Mol. Sci. 2020, 21, 846. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Zhuang, R.L. Oil-Tea Camellia in China, 2nd ed.; China Forestry Publishing House: Beijing, China, 2011. [Google Scholar]
  3. Ma, J.; Hang, Y.; Rui, Y.; Chen, G.; Zhang, N. Fatty acid composition of Camellia oleifera oil. J. Für Verbrauch. Und Lebensm. 2011, 6, 9–12. [Google Scholar] [CrossRef]
  4. Yao, X.H.; Wang, Y.P.; Wang, K.L.; Ren, H.D. Effects of geographic latitude and longitude on fat and its fatty acid composition of oil-tea camellia seeds. China Oils Fats. 2011, 36, 31–34. [Google Scholar]
  5. He, L.; Zhou, G.Y.; Zhang, H.Y.; Liu, J.A. Research progress on the health function of tea oil. J. Med. Plants Res. 2011, 5, 485–489. [Google Scholar]
  6. Chen, J.M.; Yang, X.Q.; Huang, X.M.; Duan, S.H.; Long, C.; Chen, J.K.; Rong, J. Leaf transcriptome analysis of a subtropical evergreen broadleaf plant, wild oil-tea camellia (Camellia oleifera), revealing candidate genes for cold acclimation. BMC Genom. 2017, 18, 211. [Google Scholar] [CrossRef] [Green Version]
  7. Qin, S.Y.; Rong, J.; Zhang, W.J.; Chen, J.K. Cultivation history of Camellia oleifera and genetic resources in the Yangtze River Basin. Biodivers. Sci. 2018, 26, 384–395. [Google Scholar] [CrossRef]
  8. Boavida, L.C.; Vieira, A.M.; Becker, J.D.; Feijo, J.A. Gametophyte interaction and sexual reproduction: How plants make a zygote. Int. J. Dev. Biol. 2005, 49, 615–632. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Guo, J.R.; Dong, X.X.; Li, Y.; Wang, B.S. NaCl treatment markedly enhanced pollen viability and pollen preservation time of euhalophyte Suaeda salsa via up regulation of pollen development-related genes. J. Plant Res. 2020, 133, 57–71. [Google Scholar] [CrossRef] [PubMed]
  10. Hine, A.; Rojas, A.; Suarez, L.; Murillo, O.; Espinoza, M. Optimization of Pollen Germination in Tectona grandis (Teak) for Breeding Programs. Forests 2019, 10, 908. [Google Scholar] [CrossRef] [Green Version]
  11. Chagas, E.A.; Pio, R.; Chagas, P.C.; Pasqual, M.; Bettiol, J.E.N. Composição do meio de cultura e condições ambientais para germinação de grãos de pólen de porta-enxertos de pereira. Ciência Rural. 2010, 40, 261–266. [Google Scholar] [CrossRef]
  12. Nogueira, P.V.; Silva, D.F.; Pio, R.; Silva, P.A.O.; Bisi, R.B.; Balbi, R.V. Germinação de pólen e aplicação de ácido bórico em botões florais de nespereiras. Bragantia 2015, 74, 9–15. [Google Scholar] [CrossRef] [Green Version]
  13. Grant, V. Plant Speciation, 2nd ed.; Columbia Univ. Press: New York, NY, USA, 1981. [Google Scholar]
  14. Ramsey, J.; Schemske, D.W. Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annu. Rev. Ecol. Syst. 1998, 29, 467–501. [Google Scholar] [CrossRef]
  15. Lim, K.B.; Chung, J.D.; Van Kronenburg, B.C.E.; Ramanna, M.S.; De Jong, J.H.; Van Tuyl, J.M. Introgression of Lilium rubellum Baker chromosomes into L. longiflorum Thunb: A genome painting study of the F1 hybrid, BC1 and BC2 progenies. Chromosome Res. 2000, 8, 119–125. [Google Scholar] [CrossRef] [PubMed]
  16. Alexander, L. Ploidy Level Influences Pollen Tube Growth and Seed Viability in Interploidy Crosses of Hydrangea macrophylla. Front. Plant Sci. 2020, 11, 100. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Hembree, W.G.; Ranney, T.G.; Jackson, B.E.; Weathington, M. Cytogenetics, ploidy, and genome sizes of Camellia and related genera. HortScience 2019, 54, 1124–1142. [Google Scholar] [CrossRef] [Green Version]
  18. Novara, C.; Ascari, L.; Morgia, V.L.; Reale, L.; Genre, A.; Siniscalco, C. Viability and germinability in long term storage of Corylus avellana pollen. Sci. Hortic. 2017, 214, 295–303. [Google Scholar] [CrossRef] [Green Version]
  19. Mccallum, B.; Chang, S.M. Pollen competition in style: Effects of pollen size on siring success in the hermaphroditic common morning glory. Ipomoea Purpurea. Am. J. Bot. 2016, 103, 460–470. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Souza-Pérez, M.; Mourelle, D.; Tr Ujillo, C.; Borges, C.; Speroni, G. Pollen grain performance in Psidium cattleyanum (Myrtaceae): A pseudogamous polyploid species. Flora 2021, 281, 151863. [Google Scholar] [CrossRef]
  21. Kolarčik, V.; Vašková, D.; Mirková, M.; Mártonf, P. Pollen morphology in natural diploid polyploid hybridogeneous complex of the genus Onosma (Boraginaceae–Lithospermeae). Plant Syst. Evol. 2018, 305, 151–168. [Google Scholar] [CrossRef]
  22. Han, C.X.; Zhou, T.L.; Li, Y.M.; Ye, T.W.; Hu, X.; Zhao, J.R.; Yuan, D.Y.; Xiao, S.X. Regeneration of different ploidy callus from anther culture of Camellia oleifera. Mol. Plant Breed. 2022, 20, 12. [Google Scholar]
  23. Li, Y.M.; Ye, T.W.; Han, C.X.; Ye, Z.H.; Zhang, J.; Xiao, S.X.; Yuan, D.Y. Cytogenetic analysis of interspecific hybridization in oil-tea (Camellia oleifera). Euphytica 2021, 217, 28. [Google Scholar] [CrossRef]
  24. Vrána, J.; Cápal, P.; Bednářová, M.; Doležel, J. Flow cytometry in plant research: A success story. Appl. Plant Cell Biol. 2014, 22, 395–430. [Google Scholar]
  25. Hu, Z.; Zhao, Y.; Zhao, C.; Liu, J. Taxonomic importance of pollen morphology in Veratrum L. (Melanthiaceae) using microscopic techniques. Microsc. Res. Tech. 2020, 83, 865–876. [Google Scholar] [CrossRef] [PubMed]
  26. Taylor, M.; Coope, R.; Schneider, E.; Osborn, J. Pollen structure and development in Nymphaeales: Insights into character evolution in an ancient angiosperm lineage. Am. J. Bot. 2015, 102, 1685–1702. [Google Scholar] [CrossRef] [PubMed]
  27. Bispo, R.; Santos, L.; Santos, J.; Karsburg, I. Viability estimation and morphological characterization of pollen Datura suaveolens Willd (Solanaceae). Sci. Electron. Arch. 2018, 11, 61–64. [Google Scholar]
  28. Ascari, L.; Novara, C.; Dusio, V.; Oddi, L.; Siniscalco, C. Quantitative methods in microscopy to assess pollen viability in different plant taxa. Plant Reprod. 2020, 33, 205–219. [Google Scholar] [CrossRef] [PubMed]
  29. Rotreklová, O.; Krahulcová, A. Estimating paternal efficiency in an agamic polyploid complex: Pollen stainability and variation in pollen size related to reproduction mode, ploidy level and hybridogenous origin in Pilosella (Asteraceae). Folia Geobot. 2016, 51, 175–186. [Google Scholar] [CrossRef]
  30. Xiong, H.; Zou, F.; Yuan, D.Y.; Zhang, X.; Tan, X.F. Orthogonal test design for optimizing the culture medium for in vitro pollen germination of feijoa (Acca sellowiana cv. Unique). New Zealand J. Crop Hortic. Sci. 2016, 44, 192–202. [Google Scholar] [CrossRef]
  31. Yuan, D.Y.; Tan, X.F.; Zou, F.; Cui, M.J.; Liang, Y.Q. Comparative study on pollen viability detection methods of Camellia Oleifera. J. Southwest For. Univ. 2009, 29, 10–12. [Google Scholar]
  32. Deng, Z.Y.; Xiong, H.; Yuan, D.Y.; Niu, G.H.; Zou, F. Orthogonal Test Design for Optimizing Culture Medium for in vitro Pollen Germination of Chinese Chinquapin (Castanea henryi). Taiwan J. Sci. 2018, 33, 185–195. [Google Scholar]
  33. Ren, R.F.; Li, Z.D.; Zhou, H.; Zhang, L.L.; Jiang, X.R.; Liu, Y. Changes in apoptosis-like programmed cell death and viability during the cryopreservation of pollen from Paeonia suffruticosa. Plant Cell Tissue Organ Cult. (PCTOC) 2020, 140, 357–368. [Google Scholar] [CrossRef]
  34. Retagnolle, F.; Thompson, J.D.; Lumaret, R. The influence of seed size variation on seed germination and seedling vigour in diploid and tetraploid Dactylis glomerat L. Ann. Bot. 1995, 76, 607–615. [Google Scholar] [CrossRef]
  35. Qiang, S. Polyploidization, one of the driving forces for weed origin and evolution. J. Nanjing Agric. Univ. 2012, 35, 64–76. [Google Scholar]
  36. Shi, Q.H.; Liu, P.; Liu, M.J. Advances in Ploidy Breeding of Fruit Trees. Acta Hortic. Sin. 2012, 39, 1639–1654. [Google Scholar]
  37. Nico, D.S.; Linda, Z.; Martin, M.; Sharbel, T.F.; Danny, G. Volume-based pollen size analysis: An advanced method to assess somatic and gametophytic ploidy in flowering plants. Plant Reprod. 2013, 26, 65–81. [Google Scholar]
  38. Katsiotis, A.; Forsberg, R.A. Pollen grain size in four ploidy levels of genusAvena. Euphytica 1995, 83, 103–108. [Google Scholar] [CrossRef]
  39. Den Nijs, J.C.M.; Hooghiemstra, H.; Schalk, P.H. Biosystematic studies of the Rumex acetocella complex (Polygonaceae). IV. Pollen morphology and the possibilities of identification of cytotypes in pollen analysis. Phyton 1980, 20, 307–323. [Google Scholar]
  40. Jan, F.; Schüler, L.; Behling, H. Trends of pollen grain size variation in C3 and C4 Poaceae species using pollen morphology for future assessment of grassland ecosystem dynamics. Grana Palynol. 2015, 54, 129–145. [Google Scholar] [CrossRef]
  41. Cui, G.F.; Du, W.W.; Duan, Q.; Jia, W.J.; Wang, X.N. Analysis on Pollen Morphology and Variation of Viability of Lilium Asiatic Hybrids with Different Ploidy. Acta Agric. Boreali-Sin. 2015, 30, 55–60. [Google Scholar]
  42. Thuzar, M.; Puteh, A.; Abdullah, N.A.P.; Lassim, M.M.; Jusoff, K. The effects of temperature stress on the quality and yield of soya bean (Glycine max L.). J. Agric. Sci. 2010, 2, 172. [Google Scholar]
  43. Sulusoglu, M.; Cavusoglu, A. In Vitro Pollen Viability and Pollen Germination in Cherry Laurel (Prunus laurocerasus L.). Sci. World J. 2014, 7, 657123. [Google Scholar]
  44. Ferreira, M.D.S.; Soares, T.L.; Costa, E.M.R.; Silva, R.L.D.; Souza, F.V.D. Optimization of culture medium for the in vitro germination and histochemical analysis of Passiflora spp. pollen grains. Sci. Hortic. 2021, 288, 110298. [Google Scholar] [CrossRef]
  45. Shivanna, R.K.; Mohan Ram, Y.H. Pollination biology: Contributions to fundamental and applied aspects. Curr. Sci. 1993, 65, 226–233. [Google Scholar]
  46. Luo, S.; Zhang, K.; Zhong, W.P.; Chen, P.; Fan, X.M.; Yuan, D.Y. Optimization of in vitro pollen germination and pollen viability tests for Castanea mollissima and Castanea henryi. Sci. Hortic. 2020, 271, 109481. [Google Scholar] [CrossRef]
  47. Atlagić, J.; Terzić, S.; Marjanović-Jeromela, A. Staining and fluorescent microscopy methods for pollen viability determination in sunflower and other plant species. Ind. Crops Prod. 2012, 35, 88–89. [Google Scholar] [CrossRef]
Agronomy 12 02592 g001 550
Figure 1. Microscopic observation of C.oleifera pollen after carbofuchsin dye solution. Capital letters (A–D) and lowercase letters (a–d) represent the microscopic images magnified at 20× and 40× of the dyed pollen with ploidy 2, 4, 6, and 8, respectively. Bar = 50 μm.
Figure 1. Microscopic observation of C.oleifera pollen after carbofuchsin dye solution. Capital letters (A–D) and lowercase letters (a–d) represent the microscopic images magnified at 20× and 40× of the dyed pollen with ploidy 2, 4, 6, and 8, respectively. Bar = 50 μm.
Agronomy 12 02592 g001
Agronomy 12 02592 g002 550
Figure 2. Scanning electron microscopy of C.oleifera pollen, capital letters (A–D) represent “diploid, tetraploid, hexaploid, and octaploid”, respectively, and numbers “(1–3)” represent magnification of “2000× (individual), 1000× (population), and 500× (population)”, respectively.
Figure 2. Scanning electron microscopy of C.oleifera pollen, capital letters (A–D) represent “diploid, tetraploid, hexaploid, and octaploid”, respectively, and numbers “(1–3)” represent magnification of “2000× (individual), 1000× (population), and 500× (population)”, respectively.
Agronomy 12 02592 g002
Agronomy 12 02592 g003 550
Figure 3. Light field and fluorescence magnification (10×) image of C.oleifera after staining with FDA dye. Capital letters (A–D) and lowercase letters (a–d) represent microscopic observations of diploid, tetraploid, hexaploid, and octoploid pollens in bright field and fluorescence, respectively. Bar = 50 μm.
Figure 3. Light field and fluorescence magnification (10×) image of C.oleifera after staining with FDA dye. Capital letters (A–D) and lowercase letters (a–d) represent microscopic observations of diploid, tetraploid, hexaploid, and octoploid pollens in bright field and fluorescence, respectively. Bar = 50 μm.
Agronomy 12 02592 g003
Agronomy 12 02592 g004 550
Figure 4. Pollen germination after 2 h in culture medium of C. oleifera. (A–D) represent the pollens of C. oleifera at four ploidy levels of 2, 4, 6, and 8, respectively. Bar = 100 μm.
Figure 4. Pollen germination after 2 h in culture medium of C. oleifera. (A–D) represent the pollens of C. oleifera at four ploidy levels of 2, 4, 6, and 8, respectively. Bar = 100 μm.
Agronomy 12 02592 g004
Agronomy 12 02592 g005 550
Figure 5. Comparison of the average diameter, pollen viability, and pollen germination rate of four ploidy C. oleifera pollens in the column chart; 1, 2, 3, and 4 on the abscissa represent diploid, tetraploid, hexaploid, and octoploid, respectively.
Figure 5. Comparison of the average diameter, pollen viability, and pollen germination rate of four ploidy C. oleifera pollens in the column chart; 1, 2, 3, and 4 on the abscissa represent diploid, tetraploid, hexaploid, and octoploid, respectively.
Agronomy 12 02592 g005
Table
Table 1. Average diameters of pollens at four ploidy levels of C. oleifera after carbopol fuchsin dye staining.
Table 1. Average diameters of pollens at four ploidy levels of C. oleifera after carbopol fuchsin dye staining.
Ploidy
Level		Pollen
Type		Pollen
Diameter (μm)		Mean Pollen
Diameter (μm)		Ploidy
Level		Pollen
Type		Pollen
Diameter		Mean Pollen
Diameter (μm)
diploidNR-138.85 ± 3.11 ab37.85 ± 3.00 dtetraploidDP4342.81 ± 2.36 ab41.92 ± 2.73 c
NR-235.88 ± 3.18 dDP4740.89 ± 3.67 bc
NR-336.23 ± 3.63 bcDB(1)40.23 ± 2.78 cd
NR-439.30 ± 2.92 aDF2444.24 ± 2.56 a
NR-539.04 ± 3.41 bZX090740.94 ± 1.87 bc
NR-638.31 ± 2.38 cDBH42.98 ± 3.70 b
NR-937.60 ± 3.09 bcMX-542.31 ± 2.60 c
NR-1037.61 ± 2.31 bcCJ-0340.92 ± 2.28 bc
hexaploidCJ-1248.46 ± 3.71 ab49.40 ± 3.42 aboctaploidYNYC-149.47 ± 3.19 e50.54 ± 3.63 a
ASX090250.75 ± 2.87 bYNYC-253.92 ± 3.39 b
TXP-1447.49 ± 2.77 cYNYC-358.16 ± 6.19 a
CJ-0748.68 ± 5.43 abYNYC-452.40 ± 2.81 c
XH09750.06 ± 2.86 abYNYC-550.11 ± 3.35 d
LE3850.87 ± 2.73 aYNYC-747.64 ± 3.58 de
LE348.80 ± 3.37 abYNYC-846.31 ± 3.74 ef
GTDB
02		50.06 ± 3.63 abYNYC-946.34 ± 2.79 ef
Note: Two hundred pollen grains of each pollen type were selected to measure the pollen diameter and calculate their mean value and standard deviation. Different letters in the column of the table indicate significant differences.
Table
Table 2. Average polar and equatorial lengths of Camellia oleifera pollen measured by scanning electron microscopy at four ploidy levels.
Table 2. Average polar and equatorial lengths of Camellia oleifera pollen measured by scanning electron microscopy at four ploidy levels.
Ploidy LevelPolar Axis Length (μm)Equatorial Length (μm)
diploid36.68 ± 2.87 d22.05 ± 1.13 c
tetraploid44.27 ± 3.06 c27.00 ± 1.42 b
hexaploid47.93 ± 2.39 b29.97 ± 1.71 ab
octaploid58.51 ± 2.88 a30.84 ± 2.08 a
Note: Polar axis lengths and equatorial lengths were determined for 200 pollen grains at each ploidy level. Different letters in the column of the table indicate significant differences.
Table
Table 3. Pollen viability and germination rate of C. oleifera at four ploidy levels.
Table 3. Pollen viability and germination rate of C. oleifera at four ploidy levels.
Ploidy LevelPollen
Type		Pollen
Viability
(%)		Pollen
Germination
Rate (%)		Ploidy LevelPollen TypePollen
Viability
(%)		Pollen
Germination
Rate (%)
diploidNR-145.89 b34.52 dtetraploidDP4380.25 a76.45 a
NR-262.83 ab40.17 cDP4772.76 cd72.06 bc
NR-374.61 a53.71 abDB(1)75.71 c74.05 bc
NR-463.56 ab50.65 bcDF2478.24 b66.28 bc
NR-563.17 ab50.75 bZX090756.40 e65.56 c
NR-663.77 ab49.21 bcDBH71.52 cd75.47 b
NR-972.04 ab61.00 aMX-567.12 d68.15 bc
NR-1068.91 ab55.64 abCJ-03 65.60 de66.22 bc
hexaploidCJ-1288.24 a84.59 aoctaploidYNYC-177.56 a69.79 a
ASX090274.28 cd77.04 bYNYC-265.45 b63.25 ab
TXP-1483.95 ab57.03 dYNYC-363.57 bc25.71 b
CJ-0774.70 cd63.58 bcYNYC-474.76 ab65.10 ab
XH09785.38 ab83.98 abYNYC-575.11 ab68.87 ab
LE3876.34 bc64.70 cYNYC-724.06 cd14.86 bc
LE379.33 b63.11 bcYNYC-827.87 c19.68 c
GTDB
02		75.29 c80.12 abYNYC-912.10 d8.89 cd
Note: Eight types of pollen grains of C. oleifera were selected for each ploidy, and no less than 500 pollen grains of each variety were selected for pollen viability testing. The pollen germination rate was measured 2 h after pollen germination in vitro. Different letters in the column of the table indicate significant differences.
Table
Table 4. Mean pollen diameter, pollen viability, and germination rate of C. oleifera at four ploidy levels.
Table 4. Mean pollen diameter, pollen viability, and germination rate of C. oleifera at four ploidy levels.
Ploidy
Level		Mean Pollen
Diameter (μm)		Mean Pollen
Viability (%)		Mean Pollen
Germination Rate (%)
diploid37.85 ± 3.00 d64.35 ± 8.90 b49.46 ± 9.30 b
tetraploid41.92 ± 2.73 c70.95 ± 8.33 ab70.53 ± 7.00 ab
hexaploid49.40 ± 3.42 ab79.69 ± 8.58 a71.78 ± 10.00 a
octaploid50.54 ± 3.63 a52.56 ± 6.14 c42.02 ± 9.00 c
Note: The average diameter, pollen viability, and pollen germination rate of diploid, tetraploid, hexaploid, and octoploid of C. oleifera pollens were determined using eight pollen species at each ploidy level. Different letters in the column of the table indicate significant differences.
Table
Table 5. Variance analysis for the pollen diameter, pollen viability, and pollen germination rate of C. oleifera at four ploidy levels.
Table 5. Variance analysis for the pollen diameter, pollen viability, and pollen germination rate of C. oleifera at four ploidy levels.
FactorSSDFFPSignificance
pollen diameter885.142353.9360.000*
pollen viability3136.83834.7740.008*
pollen germination rate5396.71037.7040.001*
Note: SS is equal to the sum of squares between groups. DF = degree of freedom. F is equal to mean squared between groups/within groups. The p-value indicates a significant difference. * was significant at the 0.01 level.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Zhao, R.; Xu, L.; Xu, X.; Li, Y.; Xiao, S.; Yuan, D. Comparative Study on Pollen Viability of Camellia oleifera at Four Ploidy Levels. Agronomy 2022, 12, 2592. https://doi.org/10.3390/agronomy12112592

AMA Style

Zhao R, Xu L, Xu X, Li Y, Xiao S, Yuan D. Comparative Study on Pollen Viability of Camellia oleifera at Four Ploidy Levels. Agronomy. 2022; 12(11):2592. https://doi.org/10.3390/agronomy12112592

Chicago/Turabian Style

Zhao, Rui, Linjie Xu, Xiangshuai Xu, Yanmin Li, Shixin Xiao, and Deyi Yuan. 2022. "Comparative Study on Pollen Viability of Camellia oleifera at Four Ploidy Levels" Agronomy 12, no. 11: 2592. https://doi.org/10.3390/agronomy12112592

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop