Next Article in Journal
EDI-YOLO: An Instance Segmentation Network for Tomato Main Stems and Lateral Branches in Greenhouse Environments
Previous Article in Journal
An Explainable Machine Learning Framework for the Hierarchical Management of Hot Pepper Damping-Off in Intensive Seedling Production
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 11
Issue 10
10.3390/horticulturae11101259
horticulturae-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
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

A Diploid–Tetraploid Cytochimera of Dashu Tea Selected from a Natural Bud Mutant

by
Chi Zhang
1,†,
Sulei She
1,2,†,
Haiyan Wang
1,
Jiaheng Li
1,
Xiao Long
1,
Guolu Liang
1,
Qigao Guo
1,
Songkai Li
3,
Ge Li
4,
Lanyan Qian
4,
Di Wu
1,* and
Jiangbo Dang
1,*
1
Key Laboratory of Agricultural Biosafety and Green Production of Upper Yangtze River (Ministry of Education), Chongqing Key Laboratory of Forest Ecological Restoration and Utilization in the Three Gorges Reservoir Area, College of Horticulture and Landscape Architecture, Southwest University, Beibei, Chongqing 400715, China
2
Wuxi Commission of Agriculture and Rural Affairs in Chongqing, Wuxi, Chongqing 405800, China
3
Mengzi Agricultural Comprehensive Service Center, Honghe 661100, China
4
Honghe Economic Crop Technology Promotion Station, Honghe 661100, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(10), 1259; https://doi.org/10.3390/horticulturae11101259
Submission received: 24 July 2025 / Revised: 20 August 2025 / Accepted: 2 September 2025 / Published: 18 October 2025
(This article belongs to the Topic Plant Breeding, Genetics and Genomics, 2nd Edition)
Browse Figures
Versions Notes

Abstract

Polyploids play significant roles in tea production due to their strong tolerance to adverse environmental conditions and their high levels of certain chemical components. Tetraploid can be used to produce more polyploid tea plants, but there have been only a handful of tetraploids found in tea plants. In spite of the extremely low probabilities, bud mutant selection is an effective way to obtain polyploid tree crops. In the present study, a Dashu tea, cytochimera, derived from a bud mutation was identified by using flow cytometry and chromosome observation. The morphology and photosynthetic characteristics of leaves were investigated briefly. Some chemical components were determined. Finally, the pollen viability and ploidy of progeny were detected. The results show that tetraploid cells account for 71.48 ± 3.88%–72.19 ± 2.80% of the leaf tissue in this cytochimera. Compared with the original diploid, the cytochimera exhibited broader, longer, and thicker leaves. Its net photosynthetic rate (high to 41.77 ± 0.38 μmol CO2·m−2·s−1) was higher than that of the original diploid (peak value 28.00 ± 2.29 μmol CO2·m−2·s−1) for most of the day when measured in September. Notably, the total content of 19 free amino acids in the tender spring shoots of cytochimera was 22.96 ± 0.58 mg/g, approximately twice of that of the diploid materials analyzed. The contents of 10 free amino acids, including theanine, were significantly higher than those in diploids, with some free amino acid contents reaching up to seven times those observed in diploids. In addition, the cytochimera produced larger pollen grains than the original diploid, although the in vitro germination rate was lower (14.63 ± 1.11%). Three open-pollinated progenies of cytochimera were identified as triploids. To sum up, cytochimera has larger and thicker leaves, a higher photosynthetic rate, and higher content of total free amino acids and some free amino acids, especially theanine, than the original diploid. Moreover, cytochimera has a certain level of fertility and can produce triploids. These findings suggest the potential for selecting polyploid tea plants from bud mutants and for developing new tea germplasms with enhanced amino acid contents.

1. Introduction

Polyploids exhibit a larger organ size, higher concentrations of active compounds, and enhanced stress tolerance, making them widely utilized in various crop species [1,2,3,4]. Common polyploid crops including wheat, tobacco, sugarcane, sweet potatoes, and potatoes play key roles in agricultural production worldwide [5].
Polyploidy has long been utilized in tea production, where it plays a significant role in enhancing yield, improving quality, and increasing stress resistance [6,7]. Polyploid tea plants exhibit traits such as larger leaves, thicker buds, and greater cold resistance [8,9,10], which make them particularly suitable for cultivation in high-altitude and high-latitude regions. The primary harvestable product of tea plants is the tender bud. Due to the generally low fertility of polyploids, more nutrients are allocated to vegetative growth, potentially leading to higher levels of secondary metabolites. Therefore, the practical significance of polyploidy in tea production and application is arguably greater than in other crops. Among polyploids, triploids demonstrate notable advantages: Tao [11] reported that certain chemical components in triploid tea varieties are higher than those in diploids. Additionally, a study by Das et al. [12] revealed that triploids derived from crosses between tetraploids and diploids exhibited heterosis for caffeine and catechin content, with some triploid hybrids showing higher levels than their parents. Based on these advantages, countries such as Japan and the Soviet Union began to explore and utilize naturally occurring polyploids in tea plants in the early 1930s [6]. Subsequently, countries including China, India, Sri Lanka, Kenya, and Russia also adopted ploidy breeding as a primary technique in tea plant breeding programs [10,13,14,15]. Therefore, polyploid is of high value in tea plant breeding.
In nature, nearly all polyploid tea plants originate from seeds derived from 2n gametes, which explains why most naturally occurring polyploid tea plants are triploids [6,7,10]. Triploids are often hypofertile, making it difficult to obtain further polyploids from them. Tetraploids can produce many triploids and tetraploids and thus may play more important roles in polyploid breeding of tea plants. However, only a handful of tetraploids have been reported in tea plants [4,13]. This makes progress in polyploid breeding very slow. Cytochimeras containing tetraploid cells have been obtained from natural bud mutants [16,17]. This suggests that although it is infrequent and uncontrollable, natural bud mutant selection is a practical pathway for obtaining tetraploids. Natural bud mutants are important resources for crop breeding in species such as apple, grape, orange, and peach [18,19,20,21]. However, only a few nature mutants have been used in tea production [13,22], and reports of polyploidy in tea-plant bud mutants remain scarce.
Tea contains many chemical components, including polyphenols, free amino acids, alkaloids, and terpenes, which account for rich taste, flavor and health benefits [23]. Free amino acids are reported to be responsible for the umami taste in tea, and the taste intensity increases with the amino acid concentration [24]. Theanine is the most abundant free amino acid (0.02 mg/g to 26.46 mg/g), comprising about 60–70% of the total free amino acids content [25,26]. It was reported that the total free amino acid content in triploid tea was higher than that in diploids [11]. This suggests that polyploidy may affect amino acid content in tea.
During a field survey, a diploid + tetraploid (2x + 4x) cytochimera that developed from a bud mutant was found. We hypothesized that this cytochimera might possess novel characteristics and differ significantly from the original diploid. Therefore, this study aimed to provide the first comprehensive characterization of this cytochimera by systematically evaluating its leaf morphology, photosynthetic characteristics, key chemical components, pollen viability, and the ploidy level of their progeny. These findings offer fundamental data crucial for the utilization of cytochimeras and polyploids in tea plant breeding.

2. Materials and Methods

2.1. Plant Materials

Two hundred ‘Dashu’ tea plants with large leaves were selected for ploidy analysis using flow cytometry. Among these, one plant exhibited a branch with notably larger and thicker leaves. All plants were collected from Delong town, Nanchuan district, in Chongqing, China.

2.2. Methods

2.2.1. Ploidy Analysis by Using Flow Cytometry

Ploidy analysis was performed according to the protocol described by Dang et al. [17]. Fresh fully expanded leaves were finely chopped with a razor blade directly into 100 μL of lysis buffer solution (Chinese patent: ZL 201610578478.1) and the resulting mixture was filtered. Subsequently, DAPI (4′,6-diamidino-2-phenylindole) (Macklin, Shanghai, China) was added to the filtrate, and the solution was incubated at 4 °C. The samples were then analyzed using a CyFlow® Ploidy Analyser (Sysmex Partec GmbH, Goerlitz, Germany).

2.2.2. Chromosome Preparation

Root tips from cutting-propagated seedlings of the cytochimera were used for chromosome counting. The method of Dang et al. [17] was followed with minor modifications. Approximately 0.5 cm root tips were pretreated in a 0.002 mol/L 8-hydroxyquinoline (Macklin, Shanghai, China) solution for 5 h at room temperature, and then fixed overnight in Carnoy’s solution. The approximately 0.2 cm root segments of the root tips were excised, rinsed thoroughly with distilled water, and subsequently digested in a mixed enzyme solution (3% cellulose + 1% macerozyme) (Yakult, Tokyo, Japan) for 1.5 h at 37 °C.

2.2.3. Leaf Morphology and Anatomical Structure Investigation

For morphological analysis, a total of 10 fully expanded leaves were randomly selected from the spring shoots of the cytochimera and the original diploid plants. The length and width of each leaf were measured to calculate the leaf shape indices (length/width). For anatomical analysis, leaf samples of the cytochimera and the original diploid were prepared using the paraffin sectioning technique. The resulting 5 µm thick cross-sections were stained with safranin-fast green and observed using a microscope.

2.2.4. Photosynthetic Character Analysis

Mature leaves from spring shoot were used as materials to extract chlorophyll and carotenoids, according to the method reported by Gao [27]. The contents of chlorophyll a, chlorophyll b and carotenoids were calculated using the formulas provided in the book edited by Gao [27]. Three biological replicates were prepared for each line, with each replicate consisting of 5 leaves.
Ca (mg/L) = 12.72 × A663 − 2.59 × A645,
Cb (mg/L) = 22.88 × A645 − 4.67 × A663,
Ccar (mg/L) = (1000 × A470−3.27Ca − 104Cb)/229.
The diurnal variation of the net photosynthetic rates (Pn) was measured on attached, mature leaves on sunny days in September. Measurements were conducted hourly from 7:00 to 18:00 using an LC pro-SD photosynthesis system (ADC, England) under natural light conditions. Three biological replicates were prepared for each line, with each replicate consisting of 3 leaves. Measurements were repeated 5 times on the same leaf at each time point.

2.2.5. Detection of Main Components Contents

Tender spring shoots containing one bud and two fully expanded leaves were collected, fixed using a microwave oven, dried in a drying oven at 80 °C, and then ground and sieved (600–1000 µm). The catechin components and caffeine content were analyzed by HPLC (high-performance liquid chromatography) following the procedure outlined in the Chinese national standard GB/T 8313-2018. The contents of free amino acids including Aspartic acid (Asp), Glutamic acid (Glu), Asparagine (Asn), Glutamine (Gln), Serine (Ser), Threonine (Thr), Proline (Pro), Theanine (Thea), γ-aminobutyric acid, Arginine (Arg), Valine (Val), Methionine (Met), Cysteine (Cys), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Tyrosine (Tyr), Glycine (Gly) and Alanine (Ala) were determined using the method reported by Tan et al. [28]. Three repetitions were set for every material; at least 20 tender shoots were used in every repetition.

2.2.6. Pollen Viability Observation

Anthers from the cytochimera and the original diploid were collected on a sunny day and placed in a shaded, cool, and well-ventilated room. Approximately 5 days later, the anthers dehisced and released pollen, which were then collected and observed. To measure pollen diameter, the pollen was suspended in 15% sucrose solution and examined under a microscope (×1000 magnification; ×63, Olympus, Tokyo, Japan). Images were captured using a charge-coupled device (CCD) camera. Pollen diameters were measured using a micrometer integrated into the corresponding microscope software. 10 view fields were observed and at least 10 pollens were measured in every field.
To assess pollen viability, pollen grains were incubated in a germination medium consisting of 15% sucrose solution supplemented with 100 mg/L boric acid and 0.5% agar. After approximately 2 h, pollen germination rates were recorded. Pollen grains were considered viable if the length of the pollen tube was equal to or greater than the pollen grain diameter. Ten repetitions were set for every material.

2.2.7. Statistical Analysis

All data were analyzed by using Excel 2013 (Microsoft), and the difference between the cytochimera and the original diploid was tested by using a t-test. Multiple comparisons were carried out using SPSS Statistics 25 (IBM) according to the F-test.

3. Results

3.1. Plant Screening and Cytochimera Identification

In 2022, a survey of ‘Dashu’ tea plants in Delong town, Nanchuan district, Chongqing, China, was conducted based on morphologic observation. A total of 200 trees with large and thick leaves were selected for analysis using a flow cytometer. One plant was observed to have a main branch bearing leaves that were visibly thicker and wider than those on other branches (Figure 1A,B). This branch was identified as a 2x + 4x cytochimera. The leaves from this chimeric branch were composed of a mix of 2x and 4x cells, with 4x cells accounting for an average of 71.48 ± 3.88% (65.93–76.67%) of the total cell population (Figure 1C). To assess the stability of the chimera, cuttings were propagated from its secondary branches. All 10 resulting seedlings were confirmed to be 2x + 4x cytochimeras, with a consistent 4x cell frequency of 72.19 ± 2.80% (68.10–76.58%). No cells with other ploidy levels were detected besides 2x cells (Figure 2). This indicated that the chimera was relatively stable.
To further confirm the ploidy level, chromosome counts were performed on root tips from cutting seedlings. Cells with both diploid (2n = 2x = 30) and tetraploid (2n = 4x = 60) chromosome numbers were observed (Figure 1C). An examination of 93 mitotic cells revealed that 33 were diploid and 60 were tetraploid.

3.2. Morphology and Anatomical Structure of Leaves

The length and width of mature leaves (from spring shoots) of the cytochimera and the original diploid were measured, and their leaf anatomical structures were analyzed based on microscopic observations of paraffin sections. These observations showed that the leaves of the cytochimera were significantly longer and wider than those of the original diploid (p < 0.01), Moreover, the leaf shape index of the cytochimera was also significantly higher (p < 0.01) (Table 1).
Paraffin section analysis revealed that the leaves of the cytochimera (347.60 ± 7.58 µm) were significantly thicker than those of the original diploid (295.66 ± 8.24 µm) (p < 0.01) (Figure 3, Table 2). Both plant types possessed a single-layered upper and lower epidermis, with no significant differences in epidermal thicknesses. While the transverse length of upper epidermal cells was similar between the two, the lower epidermal cells of the cytochimera were significantly smaller in transverse length than those of the diploid (p < 0.01). The palisade parenchyma in both plant types consisted of one or two layers of cells. Notably, the cells in the palisade parenchyms of the cytochimera were visibly larger than those of the original diploid, resulting in a significantly thicker palisade parenchyma layer (p < 0.01). Similarly, the spongy parenchyma cells of the cytochimera were larger, leading to a relatively modest but statistically significant increase in the thickness of the spongy parenchyma (p < 0.01).

3.3. Photosynthetic Characteristics of Leaves

The contents of photosynthetic pigments in the leaves were measured (Table 3). The contents of chlorophyll a (0.53 ± 0.02 mg/10 cm2), chlorophyll b ( 0.30 ± 0.01 mg/10 cm2) and total chlorophyll in the cytochimera leaves were all significantly higher than those in the original diploid (p < 0.01).
The diurnal variation in the net photosynthetic rates (Pn) was measured on sunny days in autumn (September) (Figure 4). Both the cytochimera and the original diploid exhibited bimodal variation curves. However, the timing of the peaks differed between the two plant types. The cytochimera reached its first peak (36.32 ± 2.65 μmol CO2·m−2·s−1) at 10:00, while the diploid peaked an hour later at 11:00. The second peak of the cytochimera (41.77 ± 0.38 μmol CO2·m−2·s−1) occurred at 15:00, whereas the diploid’s second peak was observed at 14:00. Notably, the Pn of the cytochimera was significantly higher than that of the original diploid at 8:00, 9:00, 10:00, 13:00, 14:00, 15:00, and 16:00 (p < 0.01, t-test).

3.4. Contents of Main Components

The contents of gallic acid, catechins, caffeine, and amino acids in fixed tender spring shoots of the cytochimera and the original diploid were analyzed (Table 4 and Table 5). Additionally, two high-performing Dashu tea lines (D1 and D2, both diploids) from the same region were used as contrasts. As shown in Table 4, gallic acid and catechin gallate levels were low across all four materials. Gallocatechin gallate and catechin were found at slightly higher levels compared to gallic acid and catechin gallate. Epigallocatechin gallate was the most abundant catechin compound (>86.80 ± 3.18 mg/g), followed by caffeine (48.67 ± 2.58 mg/g–58.5 ± 1.96 mg/g). Gallocatechin, epigallocatechin, epicatechin, and epicatechin gallate were present at moderate levels.
Among the catechins, only the gallocatechin content was significantly higher in the cytochimera compared to the original diploid (p < 0.01). The contents of most other catechins did not differ significantly between the cytochimera and its diploid counterpart. Interestingly, the caffeine content in the cytochimera (58.5 ± 1.96 mg/g) was significantly higher than that in the fixed tender spring shoots of the original diploid and was comparable to the levels in controls (D1 and D2).
The contents of all 19 amino acids were detected in the fixed tender spring shoots of 4 materials (Table 5). Notably, the total amino acids content in the cytochimera (22.96 ± 0.58 mg/g) was the highest among all tested materials, significantly exceeding the other three. The levels of Ile and Leu were below the detection limit in all samples and Tyr was below the detection limit in three of the materials. Asn, Pro, Val and Gly were present at low levels (lower than 0.10 mg/g) across all 4 samples. Asp, Glu, Ser, Thr, Met, Cys, Lys and Ala were found at moderate levels (higher than 0.10 mg/g but less than 1.00 mg/g). In contrast, Gln and Thea were present at high levels, exceeding 1.00 mg/g.
The cytochimera exhibited higher concentrations of Asp, Glu, Asn, Ser, Pro, Thea, γ-aminobutyric acid, Arg, Val, and Cys compared to the other three materials. Specifically, Thea content reached 13.71 ± 0.35 mg/g. The Ser and Arg contents in the cytochimera were approximately 4-fold and 7-fold higher, respectively, than in the other materials. Furthermore, the γ-aminobutyric acid content was approximately 7-fold higher than that of the original diploid.

3.5. Pollen Viability and Ploidy of Progeny

The pollen grains of the cytochimera were significantly larger, with an average diameter of 35.59 ± 1.65 µm (n = 10), compared to of the original diploid (30.07 ± 0.84, n = 10) (p < 0.01, t-test). In vitro pollen viability was also assessed for both the cytochimera and the original diploid. The pollen germination rate of the cytochimera was 14.63 ± 1.11%, markedly lower than that of the original diploid at 64.08 ± 4.77% (p < 0.01, t-test). Three triploid plants were obtained from open-pollinated seeds of the cytochimera, although only one survived to maturity (Figure 5).

4. Discussion

Polyploids can arise through various natural pathways, including 2n gamete fusion [29], polyspermy [30], and endopolyploids [31]. Certain environmental stresses, such as high temperature [32,33], low temperature [29], and radiation [34], may also play a role in the formation of polyploids. However, pathways involving sexual reproduction, like 2n gamete or polyspermy, may not produce 2x + 4x cytochimera, as the resulting zygotes are typically polyploid. Endopolyploidy is common in angiosperms, and it is usually regulated by programmed gene expression. But it is high in cotyledons, leaf stalks, lower leaves and fruits, lower in flower organs and roots, and very low in upper leaves [35,36,37]. Therefore, endopolyploid was not stable in plants, and endopolyploid cells may not develop into a complete tree branch without artificial intervention. Moreover, radiation is not commonly intense enough in natural environments to induce polyploidy. The cytochimera identified in the present study was discovered in a mountainous region at an altitude exceeding 1000 m, where temperature can drop to −5 °C during spring snowfall and freezing conditions. Given that intense natural radiation is rare, it is plausible that the cytochimera originated from somatic genome reduplication within the meristematic cells of a lateral bud, and might be induced by low temperatures.
The stability of the cytochimera was confirmed by the consistent 67.53–76.37% frequency of tetraploid cells in leaves of 10 cutting-propagated seedlings. This stability is crucial for its potential application. Cytochimeras have been identified in other plants species [16,17]. Nukaya et al. [38] correlated ploidy levels with cell size to confirm a periclinal ploidy chimaera in meiwa kumquat. Dang et al. [17] confirmed the presence of a citrus cytochimera based on variations in cell size within the leaf tissue. Based on the analysis of leaf anatomy in this study, we propose that this plant is a periclinal chimera. The leaf epidermis, derived from the L1 meristematic layer, exhibited cell sizes comparable to those of the original diploid. In contrast, the internal tissue (palisade and spongy mesophyll), derived from L2 and L3 layers, displayed significantly larger cell sizes than the original diploid. Therefore, this 2x (L1)-4x (L2, L3) structure is consistent with previous findings in other chimeric species like meiwa kumquat [38] and citrus [17], where ploidy levels were correlated with cell size in different tissue layers.
Chemical components in many polyploid plants were found to be higher than those in diploid [2]. For instance, elevated levels of citrulline and other amino acids have been reported in polyploid watermelon [39,40,41]. In tea, triploid varieties showed increased levels of amino acids, polyphenol, and soluble carbohydrate, although the total amino acid content in polyploids was only 7.4% higher than that in diploids [11]. In the present study, the most striking finding was the dramatic increase in free amino acids in fixed buds of the cytochimera, with the total content being nearly twice that of diploid and other local lines. While most catechin levels were unchanged, the caffeine content was also significantly higher in the cytochimera. This significant enhancement of amino acids suggests that the processed tea products from this cytochimera could possess a stronger umami flavor than that of the original diploid. Although its total free amino acid content in tender shoots of the cytochimera is at a medium level among various tea plants accessions [25,26], the substantial improvement over its original diploid highlights the potential of using somatic polyploidization as a strategy for breeding high-amino-acid tea resources.
From an agronomic perspective, the wider, longer and thicker leaves of the cytochimera suggest a potential for higher shoot yield. Furthermore, the production of viable pollen of cytochimera and the successful generation of three triploids from open-pollinated seeds of the cytochimera are highly important. This indicates that the cytochimera can be used not only for its traits but also as a promising intermediate parent in triploid tea breeding programs.

5. Conclusions

In this study, a stable 2x + 4x periclinal cytochimera of ‘Dashu’ tea, consisting of 70% tetraploid cells, was identified and characterized. Compared to the original diploid, the cytochimera exhibits superior agronomic traits, including larger and thicker leaves, a higher net photosynthetic rate, and dramatically enhanced content of some free amino acids. The ability of the cytochimera to produce viable pollen and triploid offsprings was also confirmed. These findings indicate that the selection of polyploid bud mutants may be a shortcut to develop elite germplasm. The identified cytochimera is a promising resource both as potential new cultivar for producing high-amino-acid tea and as a valuable parent for breeding novel triploid tea varieties.

Author Contributions

Conceptualization, C.Z., S.S., G.L. (Guolu Liang) and J.D.; Methodology, C.Z., S.S., H.W. and J.D.; Investigation, C.Z., S.S., H.W., J.L., X.L. and J.D.; Resources, G.L. (Guolu Liang) and Q.G.; Data Curation, C.Z., S.S. and J.D.; Writing—Original Draft Preparation, C.Z., S.S. and J.D.; Writing—Review and Editing, C.Z., S.S., J.D., D.W., S.L., G.L. (Ge Li) and L.Q.; Supervision, G.L. (Guolu Liang) and Q.G.; Project Administration, J.D.; Funding Acquisition, D.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Chongqing Science and Technology-driven Forestry Project, grant number YB2023-5 and Chongqing Key R&D Program for Technological Innovation and Application Demonstration (Social and People’s Livelihood), grant number cstc2018jscx-mszdX0050.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors are grateful to Huarong Tong at the Collage of Food Science, Southwest University, for providing technical assistance during chemical compositions detection, and Ke Wu at Chongqing Jinshanhu Agricultural Development Co., LTD for assisting with the sample collection.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
DAPI4′,6-diamidino-2-phenylindole.
HPLCHigh-performance liquid chromatography.

References

  1. Van de Peer, Y.; Mizrachi, E.; Marchal, K. The evolutionary significance of polyploidy. Nat. Rev. Genet. 2017, 18, 411–424. [Google Scholar] [CrossRef]
  2. Trojak-Goluch, A.; Kawka-Lipińska, M.; Wielgusz, K.; Praczyk, M. Polyploidy in industrial crops: Applications and perspectives in plant breeding. Agronomy 2021, 11, 2574. [Google Scholar] [CrossRef]
  3. Rice, A.; Šmarda, P.; Novosolov, M.; Drori, M.; Glick, L.; Sabath, N.; Meiri, S.; Belmaker, J.; Mayrose, I. The global bio-geography of polyploid plants. Nat. Ecol. Evol. 2019, 3, 265–273. [Google Scholar] [CrossRef]
  4. Liu, S.Y.; Wang, Y. Analysis and prospect of current research status on polyploidy in tea plants. China Tea 2025, 47, 40–48. (In Chinese) [Google Scholar]
  5. Salman-Minkov, A.; Sabath, N.; Mayrose, I. Whole-genome duplication as a key factor in crop domestication. Nat. Plants 2016, 2, 16115. [Google Scholar] [CrossRef]
  6. Zhai, X.M.; Tang, M.; Luo, H.Y.; Wu, X.C.; Hou, Y.J. Research progress of tea polyploidy breeding. Chin. Agric. Sci. Bull. 2018, 34, 8–12. (In Chinese) [Google Scholar]
  7. Liu, Q.; Lin, K.Q.; Wei, J.; Chen, W.J.; Chen, Z.W. Summary of research progress and prospects on tea polyploid species. Mol. Plant Breed. 2023, 4, 1272–1278. (In Chinese) [Google Scholar]
  8. Wachira, F.N. Triploidy in tea (Camellia sinensis): Effect on yield and yield attributes. J. Horticul. Sci. 1994, 69, 53–60. [Google Scholar] [CrossRef]
  9. Alam, H.; Razaq, M.; Salahuddin. Induced polyploidy as a tool for increasing tea (Camellia sinensis L.) production. J. Northeast Agric. Univ. 2015, 22, 43–47. [Google Scholar] [CrossRef]
  10. Zakir, M.; Dawid, J. Polyploidy and it’s applications in tea (Camellia sinensis L.) breeding, review. Int. J. Res. Stud. Sci. Eng. Technol. 2020, 7, 30–34. [Google Scholar]
  11. Tao, H. The influence of chromosomal multiple changes in tea plants on the content of major chemical components. J. Tea Busi. 1988, 11–14. (In Chinese) [Google Scholar]
  12. Das, S.K.; Sabhapondit, S.; Ahmed, G.; Das, S. Biochemical evaluation of triploid progenies of diploid × tetraploid breeding populations of Camellia for genotypes rich in catechin and caffeine. Biochem. Genet. 2013, 51, 358–376. [Google Scholar] [CrossRef] [PubMed]
  13. Chen, L.; Apostolides, Z.; Chen, Z.M. Global Tea Breeding; Springer: Berlin/Heidelberg, Germany, 2012. [Google Scholar]
  14. Samarina, L.S.; Matskiv, A.O.; Shkhalakhova, R.M.; Koninskaya, N.G.; Hanke, M.-V.; Flachowsky, H.; Shumeev, A.N.; Manakhova, K.A.; Malyukova, L.S.; Liu, S.; et al. Genetic diversity and genome size variability in the Russian genebank collection of tea plant [Camellia sinensis (L.) O. Kuntze]. Front. Plant Sci. 2022, 12, 800141. [Google Scholar] [CrossRef] [PubMed]
  15. Gunasekara, M.T.K.; Ranatunga, M.A.B. Polyploid in tea (Camellia sinensis L.) and its application in tea breeding: A review. Int. J. Res. Stud. Sci. Eng. Technol. 2003, 68, 14–26. [Google Scholar]
  16. Tang, Z.P.; Gao, X.; Qin, R.Y.; Sun, N.J.; Lan, H.G.; Wei, R.J.; Deng, G.Z.; Liu, B.H. A new Fortunella crassifiolia cultivar ‘Cuimi Kumquat’. J. Fruit Sci. 2018, 35, 131–134. [Google Scholar]
  17. Dang, J.B.; Li, C.; Sun, D.N.; Guo, Q.G.; Liang, G.L. A tetraploid-dominated cytochimera developed from a natural bud mutant of the nonapomictic mandarin variety ‘Orah’. Mol. Breed. 2024, 44, 20. [Google Scholar] [CrossRef]
  18. Ban, S.; Jung, J.H. Somatic mutations in fruit trees: Causes, detection methods, and mo-lecular mechanisms. Plants 2023, 12, 1316. [Google Scholar] [CrossRef]
  19. Kumar, A.; Sharma, D.P.; Kumar, P.; Sharma, G.; Suprun, I.I. Comprehensive insights on apple (Malus × domestica Borkh.) bud sport mutations and epigenetic regulations. Sci. Hortic. 2022, 297, 110979. [Google Scholar] [CrossRef]
  20. Wang, L.; Huang, Y.; Liu, Z.; He, J.; Jiang, X.; He, F.; Lu, Z.; Yang, S.; Chen, P.; Yu, H.; et al. Somatic variations led to the selection of acidic and acidless orange cultivars. Nat. Plants 2021, 7, 954–965. [Google Scholar] [CrossRef]
  21. Wei, T.L.; Wan, Y.T.; Liu, H.N.; Pei, M.S.; He, G.Q.; Guo, D.L. CHH hypermethylation con-tributes to the early ripening of grapes revealed by DNA methylome landscape of ‘Kyoho’ and its bud mutant. Hortic. Res. 2024, 12, uhae285. [Google Scholar] [CrossRef]
  22. Luo, F.Q. Bud mutation tea cultivar—Yanling Silver Side Tea. J. Tea Commun. 2015, 42, 60–61. (In Chinese) [Google Scholar]
  23. Ji, H.G.; Lee, Y.R.; Lee, M.S.; Hwang, K.H.; Kim, E.H.; Park, J.S.; Hong, Y.S. Metabolic phe-notyping of various tea (Camellia sinensis L.) cultivars and understanding of their intrin-sic metabolism. Food Chem. 2017, 233, 321–330. [Google Scholar] [CrossRef]
  24. Yu, Y.S.; Feng, X.X.; Blank, I.; Wang, H.L.; Zhu, Y.W.; Liu, Z.B.; Ni, L.; Lin, C.; Wang, K.Q.; Liu, Y. A review of umami taste of Tea: Substances, perception mechanism, and physiological measurement prospects. Trends Food Sci. Technol. 2025, 162, 105082. [Google Scholar] [CrossRef]
  25. Fang, R.; Redfern, S.P.; Kirkup, D.; Porter, E.A.; Kite, G.C.; Terry, L.A.; Berry, M.J.; Simmonds, M.S.J. Variation of theanine, phenolic, and methylxanthine compounds in 21 cultivars of Camellia sinensis harvested in different seasons. Food Chem. 2017, 220, 517–526. [Google Scholar] [CrossRef]
  26. Huang, R.; Wang, Z.; Wen, W.; Yao, M.; Liu, H.; Li, F.; Zhang, S.; Ni, D.; Chen, L. Comprehensive dissection of variation and accumulation of free amino acids in tea accessions. Hortic. Res. 2023, 11, uhad263. [Google Scholar] [CrossRef] [PubMed]
  27. Gao, J.F. Guidance for Plant Physiology Experiments; Higher Education Press: Beijing, China, 2006; pp. 74–77. (In Chinese) [Google Scholar]
  28. Tan, F.; Tan, C.; Zhao, A.; Li, M. Simultaneous determination of free amino acid content in tea infusions by using high-performance liquid chromatography with fluorescence detection coupled with alternating penalty trilinear decomposition algorithm. J. Agric. Food Chem. 2011, 59, 10839–10847. [Google Scholar] [CrossRef]
  29. de Storme, N.; Copenhaver, G.P.; Geelen, D. Production of diploid male gametes in Arabidopsis by cold-induced destabilization of postmeiotic radial microtubule arrays. Plant Physiol. 2012, 160, 1808–1826. [Google Scholar] [CrossRef] [PubMed]
  30. Toda, E.; Okamoto, T. Polyspermy in angiosperms: Its contribution to polyploid formation and speciation. Mol. Reprod. Dev. 2020, 87, 374–379. [Google Scholar] [CrossRef]
  31. Lang, L.; Schnittger, A. Endoreplication—A means to an end in cell growth and stress response. Curr. Opin. Plant Biol. 2020, 54, 85–92. [Google Scholar] [CrossRef]
  32. Guo, L.; Xu, W.; Zhang, Y.; Zhang, J.; Wei, Z. Inducing triploids and tetraploids with high temperatures in Populus sect. Tacamahaca. Plant Cell Rep. 2017, 36, 313–326. [Google Scholar] [CrossRef]
  33. Wang, J.; Li, D.; Shang, F.; Kang, X. High temperature-induced production of unreduced pollen and its cytological effects in Populus. Sci. Rep. 2017, 7, 5281. [Google Scholar] [CrossRef]
  34. Shi, F.L.; Shi, F.L.; Zhao, M.; Wang, L.; Jia, H. The germination and chromosome mutagenesis effect analysis of 60Co-γ rays on Medicago falcata L. Seed 2016, 35, 40–43. (In Chinese) [Google Scholar]
  35. Scholes, D.R.; Paige, K.N. Plasticity in ploidy: A generalized response to stress. Trends Plant Sci. 2015, 20, 165–175. [Google Scholar] [CrossRef] [PubMed]
  36. Barow, M. Endopolyploidy in seed plants. Bioessays 2006, 28, 271–281. [Google Scholar] [CrossRef]
  37. Tourdot, E.; Mauxion, J.P.; Gonzalez, N.; Chevalier, C. Endoreduplication in plant organogenesis: A means to boost fruit growth. J. Exp. Bot. 2023, 74, 6269–6284. [Google Scholar] [CrossRef]
  38. Nukaya, T.; Sudo, M.; Yahata, M.; Ohta, T.; Tominaga, A.; Mukai, H.; Yasuda, K.; Kunitake, H. The confirmation of a ploidy periclinal chimera of the meiwa kumquat (Fortunella crassifolia Swingle) induced by colchicine treatment to nucellar embryos and its morphological characteristics. Agronomy 2019, 9, 562. [Google Scholar] [CrossRef]
  39. Liu, W.G.; Zhao, S.J.; Cheng, Z.Q.; Wan, X.S.; Yan, Z.H.; King, S.R. Lycopene and citrulline contents in watermelon (Citrullus lanatus) fruit with different ploidy and changes during fruit development. Acta Hortic. 2010, 871, 543–550. [Google Scholar] [CrossRef]
  40. Zheng, F.; Zan, Y.F.; Dang, X.M.; Yang, Y. Comparative study on lycopene and citrulline content in different ploidy mini-watermelons fruit. Chin. J. Trop. Agric. 2012, 32, 7–10. [Google Scholar]
  41. Chen, Z.H.; Lu, X.Q.; He, N.; Yang, D.D.; Zhu, H.J.; Liu, W.G. Changes of main amino acid contents in citrulline biosynthesis pathway and expression analysis of related genes during fruit development of watermel-on with different ploidy. Chin. Cucurbits Veg. 2024, 37, 18–26. [Google Scholar]
Horticulturae 11 01259 g001
Figure 1. Identification of the cytochimera. (A) The cytochimera branch (indicated by the red frame); (B) shoots, leaves, flowers, and young fruits of the cytochimera and the original diploid. Scale bar = 2 cm; (C) DNA content analysis of the cytochimera using flow cytometry.
Figure 1. Identification of the cytochimera. (A) The cytochimera branch (indicated by the red frame); (B) shoots, leaves, flowers, and young fruits of the cytochimera and the original diploid. Scale bar = 2 cm; (C) DNA content analysis of the cytochimera using flow cytometry.
Horticulturae 11 01259 g001
Horticulturae 11 01259 g002
Figure 2. Identification of cutting seedlings of the cytochimera. (A) Cutting seedlings of the cytochimera; (B) DNA content analysis of cytochimera seedlings using flow cytometry (diploid and tetraploid chromosomes were exhibited near the fluorescence peaks). M1, diploid; M2, tetraploid.
Figure 2. Identification of cutting seedlings of the cytochimera. (A) Cutting seedlings of the cytochimera; (B) DNA content analysis of cytochimera seedlings using flow cytometry (diploid and tetraploid chromosomes were exhibited near the fluorescence peaks). M1, diploid; M2, tetraploid.
Horticulturae 11 01259 g002
Horticulturae 11 01259 g003
Figure 3. Leaf sections of the cytochimera (A) and the original diploid (B) were taken perpendicular to the main veins.
Figure 3. Leaf sections of the cytochimera (A) and the original diploid (B) were taken perpendicular to the main veins.
Horticulturae 11 01259 g003
Horticulturae 11 01259 g004
Figure 4. Diurnal variation in the net photosynthetic rates of the cytochimera and the original diploid. n = 3. “T” represents leaf surface temperature; “Pn” indicates the net photosynthetic rate.
Figure 4. Diurnal variation in the net photosynthetic rates of the cytochimera and the original diploid. n = 3. “T” represents leaf surface temperature; “Pn” indicates the net photosynthetic rate.
Horticulturae 11 01259 g004
Horticulturae 11 01259 g005
Figure 5. Triploid progeny derived from the cytochimera. (A) Surviving triploid progeny; (B) flow cytometry analysis of DNA content in seedlings of the surviving progeny. M1, diploid; M2, triploid.
Figure 5. Triploid progeny derived from the cytochimera. (A) Surviving triploid progeny; (B) flow cytometry analysis of DNA content in seedlings of the surviving progeny. M1, diploid; M2, triploid.
Horticulturae 11 01259 g005
Table 1. Leaf morphology of the cytochimera and the original diploid (n = 10).
Table 1. Leaf morphology of the cytochimera and the original diploid (n = 10).
MaterialLengthWidthLeaf Shape Index
2x10.87 ± 0.553.61 ± 0.323.04 ± 0.38
2x + 4x14.18 ± 0.815.74 ± 0.422.48 ± 0.12
p-value (t-test)3.19 × 10−92.16 × 10−102.66 × 10−4
Table 2. Leaf anatomical structure of cytochimera and original diploid (n = 10).
Table 2. Leaf anatomical structure of cytochimera and original diploid (n = 10).
MaterialThickness of LeavesThickness of Upper EpidermisTransverse Length of Upper Epidermis CellThickness of Palisade ParenchymaThickness of Spongy ParenchymaThickness of Lower EpidermisTransverse Length of Lower Epidermis Cell
2x295.66 ± 8.2421.99 ± 1.6320.64 ± 2.2577.64 ± 8.11177.06 ± 13.3018.96 ± 1.4128.16 ± 1.57
2x + 4x347.60 ± 7.5821.93 ± 1.4420.95 ± 1.80110.15 ± 11.46197.77 ± 12.3417.75 ± 2.5222.19 ± 2.50
p-value (t-test)1.86 × 10−110.940.818.42 × 10−72.00 × 10−30.21.94 × 10−3
Table 3. Contents of pigment related to photosynthesis (mg/10 cm2) (n = 3).
Table 3. Contents of pigment related to photosynthesis (mg/10 cm2) (n = 3).
MaterialChlorophyll AChlorophyll BTotal ChlorophyllCarotenoid
2x0.31 ± 0.060.20 ± 0.030.51 ± 0.090.13 ± 0.01
2x + 4x0.53 ± 0.020.30 ± 0.010.83 ± 0.030.14 ± 0.01
p-value (t-test)0.0040 0.0030 0.0035 0.1824
Table 4. Contents of gallic acid, catechins and caffeine in fixed tender spring shoots of different materials (mg/g) (n = 3).
Table 4. Contents of gallic acid, catechins and caffeine in fixed tender spring shoots of different materials (mg/g) (n = 3).
MaterialsGallic acidGallocatechinEpigallocatechinCatechinEpicatechin
D10.06 ± 0.01 Bb22.85 ± 2.41 Cd24.81 ± 2.91 Aa3.98 ± 0.47 Aa12.52 ± 1.01 Aa
D20.08 ± 0.01 Aa41.97 ± 1.51 Ab22.43 ± 0.26 ABab1.73 ± 0.00 Bc10.62 ± 1.29 ABb
2x0.08 ± 0.00 Aa33.53 ± 1.20 Bc20.83 ± 1.10 ABb1.88 ± 0.17 Bbc9.17 ± 0.70 Bbc
2x + 4x0.08 ± 0.00 Aa46.53 ± 1.44 Aa19.85 ± 0.38 Bb2.13 ± 0.18 Bb8.76 ± 0.31 Bc
MaterialEpigallocatechin gallateGallocatechin gallateEpicatechin gallateCatechin gallateCaffeine
D193.20 ± 11.93 Bb1.06 ± 0.08 Ab19.67 ± 1.82 Aab0.00 ± 0.00 Cc56.57 ± 5.24 ABab
D2111.48 ± 2.16 Aa 1.43 ± 0.15 Aa21.37 ± 0.88 Aa0.09 ± 0.00 Bb51.28 ± 2.34 ABbc
2x98.51 ± 3.53 Bb1.38 ± 0.19 Aa18.14 ± 0.90 Ab0.14 ± 0.01 Aa48.67 ± 2.58 Bc
2x + 4x86.80 ± 3.18 Bc1.21 ± 0.04 Aab20.77 ± 0.75 Aa0.08 ± 0.00 Bb58.50 ± 1.96 Aa
Note: Different lowercase letters within the same column represent significant differences (p < 0.05); different uppercase letters within the same column represent extremely significant differences (p < 0.01).
Table 5. Contents of amino acids in fixed tender spring shoots of different materials (mg/g) (n = 3).
Table 5. Contents of amino acids in fixed tender spring shoots of different materials (mg/g) (n = 3).
MaterialsAspGluAsnGlnSerThrProTheaγ-Aminobutyric AcidArg
D10.14 ± 0.01 B b0.67 ± 0.05 Bb0.04 ± 0.00 Bb1.45 ± 0.12 Aa0.44 ± 0.05 Bc0.14 ± 0.02 Aa0.01 ± 0.00 Dd7.49 ± 0.65 Cc0.55 ± 0.01 Bb0.05 ± 0.01 Cd
D20.08 ± 0.00 D d0.47 ± 0.01 Cc-1.23 ± 0.03 ABb0.47 ± 0.03 Bc0.1 ± 0.00 Bb0.03 ± 0.00 Cc7.62 ± 0.17 Cc0.16 ± 0.02 Cc0.14 ± 0.01 Bb
2x0.10 ± 0.00 C c0.42 ± 0.04 Cc-1.26 ± 0.05 ABb0.53 ± 0.02 Bb0.1 ± 0.00 Bb0.04 ± 0.00 Bb9.08 ± 0.29 Bb0.14 ± 0.02 Cc0.11 ± 0.00 Bc
2x + 4x0.28 ± 0.01 A a1.33 ± 0.01 Aa0.09 ± 0.00 Aa1.14 ± 0.07 Bb2.21 ± 0.08 Aa0.15 ± 0.00 Aa0.05 ± 0.00 Aa13.71 ± 0.35 Aa1.06 ± 0.10 Aa0.73 ± 0.02 Aa
MaterialsValMetCysIleLeuLysTyrGlyAlaTotal
D10.07 ± 0.00 Bb0.74 ± 0.07 Aa0.25 ± 0.00 Dd--0.69 ± 0.04 A b0.03 ± 0.000.02 ± 0.00 Bc0.15 ± 0.00 Cd12.92 ± 0.95 Bc
D20.07 ± 0.00 Bb0.66 ± 0.05 ABa0.35 ± 0.02 Cc--0.77 ± 0.04 A a-0.05 ± 0.01 Ab0.24 ± 0.01 Ab12.41 ± 0.27 Bbc
2x0.07 ± 0.00 Bb0.47 ± 0.06 Cb0.54 ± 0.02 Bb--0.71 ± 0.05 A ab-0.05 ± 0.01 Aab0.25 ± 0.00 Aa13.82 ± 0.43 Bb
2x + 4x0.08 ± 0.00 Aa0.55 ± 0.03 BCb0.63 ± 0.05 Aa--0.72 ± 0.01 A ab-0.05 ± 0.00 Aa0.20 ± 0.00 Bc22.96 ± 0.58 Aa
Note: Different lowercase in the same column represent significant differences (p < 0.05). Different capital letters in the same column represent extremely significant differences (p < 0.01). “-“ indicates the content is below detection limits.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhang, C.; She, S.; Wang, H.; Li, J.; Long, X.; Liang, G.; Guo, Q.; Li, S.; Li, G.; Qian, L.; et al. A Diploid–Tetraploid Cytochimera of Dashu Tea Selected from a Natural Bud Mutant. Horticulturae 2025, 11, 1259. https://doi.org/10.3390/horticulturae11101259

AMA Style

Zhang C, She S, Wang H, Li J, Long X, Liang G, Guo Q, Li S, Li G, Qian L, et al. A Diploid–Tetraploid Cytochimera of Dashu Tea Selected from a Natural Bud Mutant. Horticulturae. 2025; 11(10):1259. https://doi.org/10.3390/horticulturae11101259

Chicago/Turabian Style

Zhang, Chi, Sulei She, Haiyan Wang, Jiaheng Li, Xiao Long, Guolu Liang, Qigao Guo, Songkai Li, Ge Li, Lanyan Qian, and et al. 2025. "A Diploid–Tetraploid Cytochimera of Dashu Tea Selected from a Natural Bud Mutant" Horticulturae 11, no. 10: 1259. https://doi.org/10.3390/horticulturae11101259

APA Style

Zhang, C., She, S., Wang, H., Li, J., Long, X., Liang, G., Guo, Q., Li, S., Li, G., Qian, L., Wu, D., & Dang, J. (2025). A Diploid–Tetraploid Cytochimera of Dashu Tea Selected from a Natural Bud Mutant. Horticulturae, 11(10), 1259. https://doi.org/10.3390/horticulturae11101259

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