Morphology and germination of Citrus medica L. seeds

ABSTRACT:

This study aimed to determine seed germination characteristics of citron (Citrus medica) and improve its germination. Citron was used as materials to observe its characters. Effects of seed coat removal, seed size and fruit maturity on citron seed germination were investigated. The findings indicated that the average normal seed weight was found to be 0.110 g while small 0.037 g; citron seed have ovoid shape (93.7%), spheroid (3.3%), and semi-deltoid (3.0%); seed percentage was 1%. After seed coat was removed, days taken to initial germination of citron seed were shortened by 5.6 d while germination percentage and germination potential and germination speed index of seeds significantly increased by 4.9%, 6.0% and 0.37, respectively. No significant differences taken to initial germination and germination potential were found between small and normal seed, but germination percentage and germination speed index of small seed was significantly lower than that of normal seed by 13.0% and 10.4, respectively. Early and immature fruit harvest with greenish-yellow skin had no significant impact on seed germination. The findings of this study will serve as a valuable reference for seed propagation strategies for C. medica.

Index Terms:
citron; immature fruits; seed coat removal; small seed

RESUMO:

Este estudo teve como objetivo determinar as características de germinação de sementes de cidra (Citrus medica) e melhorar a sua germinação. Sementes de cidra foram utilizadas para observar as suas características. Foram avaliados os efeitos da remoção do tegumento, do tamanho da semente e da maturação do fruto na germinação das sementes de cidra. Os resultados indicaram que o peso médio de sementes normais foi de 0,110 g, enquanto o de sementes pequenas foi de 0,037 g; as sementes de cidra apresentaram formato ovoide (93,7%), esferoide (3,3%) e semideltoide (3%); a porcentagem de sementes foi de 1%. Após a remoção do tegumento, o tempo para o início da germinação das sementes de cidra foi reduzido em 5-6 dias, enquanto a porcentagem de germinação, o potencial de germinação e o índice de velocidade de germinação aumentaram significativamente em 4,9%, 6% e 0,37, respectivamente. Não foram encontradas diferenças significativas quanto ao tempo para o início da germinação e ao potencial de germinação entre sementes pequenas e de tamanho padrão. No entanto, a porcentagem de germinação e o índice de velocidade de germinação das sementes pequenas foram significativamente inferiores aos das sementes de tamanho normal, com reduções de 13% e 10,4, respectivamente. A colheita precoce e de frutos imaturos, com casca verde-amarelada, não teve impacto significativo na germinação das sementes. Os resultados deste estudo servirão como uma referência valiosa para estratégias de propagação de C. medica via sementes.

Termos para indexação:
cidra; frutos imaturos; remoção do tegumento; semente pequena

INTRODUCTION

Citron (Citrus medicaL.) is borne by a slow-growing shrub and is one of the ancestral species in citrus along with pomelo and mandarin (Morton, 1987; Wu et al., 2018). Different from pomelo and mandarin widely cultivated and used all over the world, citron was assigned as a neglected and underutilized plant species, presumably because of its limited commercial use (Wei et al., 2008; Goldschmidt, 2017). Recently, its new information of unique cultural, medical and sacramental history, along with rapid development of modern biotechnology, renews interest in citron (Kumar et al.,2022). Citron is a medicinal and edible homologous plant and is listed in Chinese Pharmacopoeia (2025). In traditional Chinese medicine, dried citron is used as a tonic to regulate Qi, the life force (Karp and Hu, 2018). Ethno-medicinal uses of this plant are well exploited in Ayurveda to treat nausea, excessive thirst, skin disease, acne, child labor, and other general health issues (Esakkimuthu et al., 2018). Citron can be also used in food and in cosmetics (Rajendiran, 2023). Citron could produce fruits with many seeds which are used as rootstock for fingered citrons (Karp and Hu, 2018). Finger citron is favored by many people due to its ornamental and medicinal value, so the demand of citron seedlings as its rootstocks is growing rapidly (Ying et al., 2025). Moreover, citron is tolerant of Citrus Huanglongbing (Miles et al.,2017). The status underlines the urgency of efforts on citron seedling propagation.

Citron propagation occurs through seeds, grafting or cuttings (Karp and Hu, 2018). Citron seed propagation is mainly used because it is a simple and easy method to make seedlings, with less need of labor and orchard infrastructure, easier logistics for selling and transporting seeds, and it don’t have phytopathogens that are not spread by seeds (Khalangre et al., 2024). The knowledge of seed germination characteristics is prerequisite for successful seed propagation (Jadhav et al., 2023). However, citron seed germination characteristics are little known.

Some internal and external factors to the seeds can influence the germination process. Seed coat is one of the crucial factors because itacts as barrier to radicle protrusion (Kiran et al.,2024). As observed in other species of the genus Citrus, such as in Rangpur lime and lemon, the removal of the seed coat favored germination performance, reducing the time required for germination and improving the emergency percentage of seedlings (Munywoki et al. 2022; Souza et al., 2026). No reports on effects of citron seed coat were found.

Another important factor affecting germination is fruit maturity. Usually, citrus seeds are harvested after the fruits fully mature. In the study of de Carvalho et al. (2021), the recommended fruit harvest period of three citrus rootstocks for seed germination was from October to December. However, seeds from fruits harvested in August showed no loss of germination potential. Little emphasis on early fruit harvest has been given to citron seed germination.

Esen and Soost (1973 a, b) noted that small citrus seeds that were thought to be triploids were 1/3 to 1/6 of diploids. Discovering natural triploids from monoembryonic diploid seedling offspring of small seed is an effective method to cultivate new seedless citrus cultivar (Feng et al., 2024). Direct sowing of small monoembryonic seed of citrus in soil or substrate is difficult to germinate because the stored nutrients in small seed are limited (Wang et al., 2019). There are no reports on the germination of small citron seeds yet.

In the current study, citron seed morphology was observed and measured to determine seed germination characteristics of citron. Effects of seed coat, fruit maturity and seed size on citron seed germination were also investigated to improve seed germination, increase available seed number and even possibly obtain seedless citron.

MATERIAL AND METHODS

Materials

Citron ‘Xiaoxiangyuan’ used as material was planted in Kangping Town, Jiangcheng Hani and Yi Autonomous County, Yunnan Province, China, situated at latitude N 22°33’56”‌, longitude E 101°28’28, and altitude1100-1500 m. Fruit samples were immediately taken to the laboratory for experiment after they were harvest.

Seed morphology

Fruit maturity was determined by Astiari et al. (2022). Seeds from mature fruits (Yellow skin, Figure 1) were manually extracted, cleaned using running sterile water to remove the mucilage and subsequently dried in the shade (Zhu et al., 2020). According to Descriptors for Citrus Germplasm Resources (NY/T 2930-2016), 20 seeds were randomly selected to determine seed morphological characteristics, including seed weight, seed size, seed shape, episperm color, endopleura color, cotyledon color, chalazal spot color. Small seeds are 1/3 to 1/6 times smaller in size as compared to diploid seeds (Esen and Soost, 1973 a, b).

Figure 1
Immature fruit (Left) and mature fruit (Right).

After seed coat removal, polyembryonic seed could be divided into some parts while monoembryonic seed contains only one complete embryo (Figure 2) and polyembryonic seed percentage was calculated based on 100 randomly selected seeds (Zhu et al., 2020). According to Descriptors for Citrus Germplasms Resources (NY/T 2930-2016), the seed embryo is classified into four types as follows:

• 1: Monoembryonic type: Polyembryonic seed percentage≤5%;

• 2: More monoembryonic type: 5% ＜Polyembryonic seed percentage ≤50%;

• 3: More polyembryonic type: 50% ＜Polyembryonic seed percentage ≤ 80%;

• 4: Polyembryonic type: Polyembryonic seed percentage ＞ 80%.

Figure 2
Monoembryonic seed (Left), polyembryonic seed (Middle) and parts of polyembryonic seed (Right).

Effect of seed coat removal on citron seed germination

Mature fruits were harvest and seeds were immediately collected in the late period of October. The mucigeneous layer around the seeds was washed and seed coat was removed manually (Munywoki et al., 2022). Seeds with and without seed coat were used for germination test with three replications (Figure 3). Each replication needs 100 seeds.

Figure 3
Seeds with seed coat (Left) and seeds without seed coat (Right).

Germination test procedure: seeds were placed on three-layer filter paper in a dish and covered by two-layer filter paper at 25 ± 0.5 ^oC. The filter papers were always moistened using distilled water. The seed was considered germinated when the radicle reached a length equal to or greater than 1 mm (Figure 4) (Huang et al., 2025).

Figure 4
The seeds were considered to be germinated when the radicle was 1 mm or longer.

Germination parameters included days taken to initial germination, germination percentage, germination potential and germination speed index (GSI) (Maguire, 1962).

Days taken to initial germination were the days since the first radicle protrudes.

Germination percentage was calculated as follows:

$Germinationpercentage\left(\%\right)=n/N\times 100$ (n = the number of seeds germinated at the end of the germination period (30 d); N= the total number of seeds used for testing)

Germination potential was calculated in accordance with:

$Germinationpotential\left(\%\right)=nt/N\times 100$ (n_t = the number of seeds germinated in the fifteenth day; N= the total number of seeds used for testing)

Germination speed index (GSI) was calculated according to Maguire (1962):

$GSI=\sum (nd/d)$ (n_d = the number of germinated seeds at the d^th day, counted from the first day and d is the counted day)

Effect of seed size on citron seed germination

After seed coat removal, small and normal seeds (Figure 5) were used for germination test with three replications. Each replication needs 100 seeds.

Figure 5
Small (Left) and normal seeds (Right).

Germination test procedure and parameters were shown the same as the above.

Effect of fruit maturity on citron seed germination

Fruit maturity was determined by Astiari et al. (2022). Seeds from mature (Yellow skin) and immature fruits (Greenish-yellow skin, Figure 1) were subjected to a germination test with three replications. Each replication needs 100 seeds.

Germination test procedure and parameters were shown the same as the above.

Data Analysis

The study employed a paired design in experiment of seed coat, seed size and fruit maturity. Paired-samples t-test was used to compare mean values of response variables in relation to treatments of seed coat, seed size and fruit maturity. All data were analyzed using the open source software jamovi (Navarro and Foxcroft, 2019).

RESULTS

The average normal seed weight was found to be 0.110 g while small seed weight was 0.037 g. The average normal seed length and width were 7.91±0.120 mm and 5.59±0.072 mm, respectively. The average small seed length and width were 5.32±0.086 mm and 3.44±0.091 mm, respectively. Citron seed have ovoid shape (93.7%), spheroid (3.3%), and semi-deltoid (3.0%) (Figure 6). The average ovoid seed length and width were 6.11±0.142 mm and 5.72±0.081 mm; the average semi-deltoid seed length and width were 6.10±0.212 mm and 5.63±0.065 mm; the average spheroid seed length and width were 6.03±0.014 mm and 5.91±0.373 mm. Mature seed has yellowish episperm, brown endopleura, light yellow-cream cotyledon and green chalazal spot (Figure 7).

Figure 6
Citron seed shape: Ovoid (Left), spheroid (Middle) and semi-deltoid (Right).

Figure 7
Light yellow-cream cotyledon and green chalazal spot of citron seed.

Polyembryonic seed percentage was 1%, being less than 5%, so citron seed is monoembryonic type.

After seed coat removal, days taken to initial germination were shortened by 5.6 d. Seeds with and without seed coat had 91.3% and 96.0% of germination percentage, respectively. Germination percentage of seeds without seed coat was significantly higher than that with seed coat. After seed coat was removed, germination potential and GSI was significantly increased by 30.3% and 26.5, respectively. Germination parameters without seed coat showed up prior to that with seed coat (Table 1).

Table 1
Effect of seed coat and its removal on citron seed germination.

	Seeds without seed coat	Seeds with seed coat
Days taken to initial germination	5.7 b	11.3 a
Germination percentage (%)	96.0 a	91.3 b
Germination potential (%)	50.3 a	20.0 b
Germination speed index (GSI)	71.4 a	44.9 b

• Note: Same letters in the same line showed no significant differences (p < 0.05).

Small seed germinated later than normal seed, but there was no significant difference on days taken to initial germination between small and normal seed. Germination percentage of small seed was significantly less than that of normal seed by 13.0%. Germination potential of small seed was lower than that of normal seed, but germination potential of small seed did not differ significantly from that of normal seed. GSI of small seed was significantly less than that of normal seed by 10.4 (Table 2).

Table 2
Effect of seed size on citron seed germination.

	Small seed	Normal seed
Days taken to initial germination	11.3 a	10.3 a
Germination percentage (%)	82.0 b	95.0 a
Germination potential (%)	14.0 a	24.3 a
Germination speed index (GSI)	35.7 b	46.1 a

• Note: Same letters in the same line showed no significant differences (p < 0.05).

Table 3 showed effect of fruit maturity on citron seed germination. Days taken to initial germination of seeds from immature and mature fruits were equal to or larger than 11 days. Germination percentage of seeds from immature and mature fruits was 92.3% and 92.7%, respectively. Seeds from immature and mature fruits had 22.0% and 21.0% of germination potential, respectively. GSI of seeds from immature and mature fruits were 45.5 and 43.6, respectively. There were no significant differences on days taken to initial germination, germination percentage, germination potential and germination speed index between immature and mature fruits.

Table 3
Effect of fruit maturity on citron seed germination.

	Seeds from immature fruits	Seeds from mature fruits
Days taken to initial germination	11.0 a	11.3 a
Germination percentage (%)	92.3 a	92.7 a
Germination potential (%)	22.0 a	21.0 a
Germination speed index (GSI)	45.5 a	43.6 a

• Note: Same letters in the same line showed no significant differences (p < 0.05).

DISCUSSION

We found that citron seeds had three shapes, including spheroid, semi-deltoid and ovoid shape. This finding is not consistent with the previous study that showed that citron seed had only one shape i.e. ovoid (Morton, 1987). Variations in seed shapes can probably be attributed to the factors described below. One factor is that citron has many types. The seed among types has clear differences (Karp and Hu, 2018). Further research is needed to investigate differences on seed shape through more advanced technologies such as phenomic analysis. Another factor is that there were different characterization criteria time went by. More accurate characterization criteria could be useful in class of seed monomorphism and heteromorphism (Baskin and Baskin, 2023). In many studies, seed heteromorphism can influence germination (Santos-Gally et al., 2020; Elahifard et al., 2021). In future work, selection of citron seed with special shape could improve seed germination.

In previous literatures, citron seed was monoembryonic and there was no data on polyembryonic percentage. Based on Descriptors for Citrus Germplasms Resources (NY/T 2930-2016), we firstly confirmed that there were 1% polyembryomic citron seeds. The nucellar cells in polyembryonic citrus could spontaneously double genome, resulting in tetraploid (Zhang et al., 2025; He et al., 2025). Citron seed propagation could obtain tetraploid seedlings as parents of seedless breeding (Sidhu et al., 2024).

No previous studies on citrus seed germination were involved with the removal of seed coat. The seed coat could limit gas exchange during seed germination and seed coat removal may improve oxygen availability and thereby promote germination (Domingues-Neto et al., 2024). In present study, germination parameters without seed coat showed up prior to that with seed coat. However, seed coat removal costs labor and time and require skilled-labor to avoid embryo damage (Oliveira and Scivittaro, 2007). Mechanical or chemical treat could improve efficiency of removal for large-scale seed coat removal (Souza et al., 2026). Further research is needed on the germination effect of mechanical or chemical removal of seed coat.

Presently, there were no studies on germination of small citron seed. Some researchers proposed to in vitro culture of small citrus seed (Wang et al., 2019; Xie et al., 2023). Obviously, seed coat removal is easier than in vitro culture. In our study, germination percentage of small citron seed reached over 80% after seed coat removal. It was suggested that seed coat removal is a good method to improve germination percentage of small citron seed.

Fruit maturity could affect seed germination (Sanches et al., 2023; Maligsay et al., 2025). Fruits of three citrus rootstocks can be harvested as early as August in contrast to the current procedures on October without losing germination potential (Carvalho et al., 2021). In our study, no significant difference in seed germination parameters was found between immature and mature fruits of citron. Our findings were consistent with the results of Carvalho et al. (2021). Early citron harvest will result in an increase in available seeds for nurseries.

Normal citron seeds in our study showed higher germination rate (over 90%) than that of other citrus species, including 81.11% of trifoliate orange and 58.72% of Rangpur lime (Gora et al., 2018), 68.4% of Cleopatra Mandarin and 79.6% of Rangpur lime (Sharaf et al., 2016), 87.05% of rough Lemon and 83.14% of Rangpur lime (Choudhary et al., 2023), 64.44% of sweet orange (Rawat et al., 2023) and 87.5% of acid Lime (Lunagariya et al., 2022). The reason is speculated that seed germination requirements were species-specific. For one species or cultivars, seed germination characteristics should be known to obtain large-scale seed propagation (Bhatt et al., 2022).

In addition, it is worthy of further study on the interactions between seed coat removal and fruit maturity of small citron seeds. Small citron seed germination into seedlings is another meaningful topic for breeding tetraploid in the future.

CONCLUSIONS

C. medica seed has three shapes, including spheroid, semi-deltoid and ovoid shape, is monoembryonic with 1% polyembryomic seeds. Seed coat removal could significantly promote citron seed germination. Small C. medica seed after seed coat removal had high germination percentage. Early C. medica fruit with greenish -yellow skin could increase citron seed source for nurseries.

ACKNOWLEDGMENTS

This work was supported by Fujian Provincial Natural Science Foundation of China, [grant number 2022J01473], Fujian Competitive Special Fund for Scientific Research Institutes in the Public Interest [grant number 2024R1073], the Collaborative Innovation Project from PGFP & CAAS [grant number XTCXGC2021006], and the Science and Technology Innovation Team of FAAS [grant number CXTD2021003-3].

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• DATA AVAILABILITY
  Additional data will be made available by the authors upon reasonable request.

Edited by

• Editor:
  Bárbara França Dantas

Data availability

Additional data will be made available by the authors upon reasonable request.

Publication Dates

• Publication in this collection
  10 Aug 2026
• Date of issue
  2026

History

• Received
  26 Mar 2026
• Accepted
  10 July 2026