Cytogenetic Study of Five Varieties of Callisia repens (Jacq.) L. (Commelinaceae) from Laos

: Cytogenetical studies were carried out on ﬁve varieties of Callisia repens , i.e., turtle vine, green, pink lady, gold, and Bianca. The morphological characteristics of all ﬁve varieties differed in leaf shape and color of the plant. All ﬁve varieties have the same chromosome number, 2n = 12, and the fundamental number (NF) = 24. The number of metacentric (m), submetacentric (sm), and subtelocentric (st) chromosomes was related to the discrepancies between the various karyotypes that were found. The formula for each of the karyotypes was 2m + 2sm + 8st (turtle vine), 2m + 10st (green), 8m + 4st 14 (pink lady), 2m + 4sm + 6st (gold), and 2m + 2sm + 8st (Bianca). Therefore, all ﬁve strains had asymmetrical karyotypes. The chromosome number of C. repens has been reported previously, but this is the ﬁrst report of karyotype variation among the varieties. Furthermore, principal component analysis (PCA) of the karyotype formula was able to distinguish C. repens ‘pink lady’, C. repens ‘green’, and C. repens ‘gold’, but it was unable to differentiate between C. repens ‘Bianca’ and C. repens ‘turtle vine’. Additionally, PCA conducted on the centromeric index (CI) and the leaf colors of the ﬁve varieties of C. repens successfully separated all of them. Therefore, the prominent morphological traits and karyotype information of the ﬁve varieties of C. repens from Laos can be used to distinguish between them.

The genus Callisia Loefl.includes 41 accepted species of perennial plants with their native range in tropical and subtropical America [1,17].Some species are used as ornamentals and have been introduced to many countries, i.e., Azores, Bangladesh, southeast China, Hawaii, Louisiana, Morocco, Norfolk Is., Taiwan, Thailand, and Laos.Callisia repens (Jacq.)L. is a well-known tiny succulent creeping plant that has the common name turtle vine.It is distributed throughout Central and South America [1,2], but has become naturalized in various other parts of the world due to its attractive foliage and ease of cultivation.Furthermore, the species is usually used as an ornamental plant, as medicine [15], and has environmental uses, such as a green roof cooling of canopies and soil temperatures [18].Additionally, C. repens has single leaves with variation in them.The colors of the upper and lower surfaces of the leaves is different, with the top of the leaves being green and the bottom purple.The color on the underside is purple on all parts of the stem.When the stems stretch out to touch the soil, roots can form, and it is currently a popular ornamental plant.Therefore, C. repens is a popular ornamental plant in Thailand [7,8] and Laos.
Previous cytogenetic studies on C. repens have reported chromosome numbers 2n = 12 [15,[19][20][21], n = 6 [21], NF = 24 [15,21], and a karyotype formula of 2m + 2sm + 6st + 2t [21], 4sm + 8st [15].Thus, C. repens exhibits variations in its karyotype formula; however, there is no mention of variations based on the morphology of C. repens.In previous studies of C. repens, researchers mainly focused on presenting the chromosome number.Only two research papers [15,21] have provided karyotype formula and their results were different from each other.Therefore, the experimental setup of this focuses on the differences in the morphology of C. repens and whether these differences correlate with its karyotype.To address this question, we divided C. repens into five varieties (including green, pink lady, Bianca, gold, and turtle vine from Vientiane province, Laos) and conducted a comprehensive study of their karyotypes.So, this study aims to obtain cytological information, including the chromosome number, NF, and karyotype, in five varieties of C. repens with leaf variations from Laos.These data contribute to identifying differences among the varieties and support further studies on C. repens in other fields such as plant breeding.

Sample Collection
Experimental plant material from five varieties of C. repens, namely green (collected from Vientiane province, coll.no. A. Sengthong 402), pink lady (collected from Vientiane province, coll.no. A. Sengthong 403), Bianca (collected from Vientiane province, coll.no. A. Sengthong 404), gold (collected from Vientiane province, coll.no. A. Sengthong 405), and turtle vine (collected from Vientiane province, coll.no. A. Sengthong 406), was collected from Laos (Table 1) and voucher specimens were kept in the Faculty of Forest Science Herbarium (FOF), National University of Laos, and the Mahasarakham University Herbarium, Thailand.All fresh cultivars were grown in a nursery at the Walai Rukhavej Botanical Research Institute, Mahasarakham University, Maha Sarakham Province, Thailand.

Mitotic and Karyotype Analyses
For the purpose of studying somatic chromosomes, the root tips (about 1 cm long) were taken from living specimens.These root tips had a paradichlorobenzene (PDB) pretreatment for 6 h at 4 • C, fixed in ethanol-acetic acid 3:1 (v/v) for 30 min at room temperature, and after that stored at 4 • C or immediately used.All root tips were cleaned with distilled water, hydrolyzed in 1N HCl for 5 min at 60 • C, and then cleaned once more with distilled water.All root tips were dyed and squashed in 2% aceto-orcein.The chromosomes were observed by using a light microscope, Zeiss: Axiostar plus, to take the micrographs.Adobe Photoshop CS3 Extended was used to quantify the chromosome length and to rearrange the chromosomes.The study of chromosome count followed, [10][11][12].The chromosomal morphology nomenclature was developed using the techniques described in [5].Therefore, the chromosomes of all varieties were classified to be metacentric (m), submetacentric (sm), subtelocentric (st), or acrocentric (a).For each variety, 20 metaphase cells were used to determine the chromosomal count, chromosome structure, and karyotype.For the karyotyping, the parameters for the average length of Ls (the short arm), average length of Ll (the long arm), LT of each chromosome (total length), average relative length (RL = LT/∑LT, formula for relative length), and chromosome index (CI = Ll/LT, formulae for chromosome index are 0.500-0.599(metacentric), 0.600-0.699(submetacentric), 0.700-0.899(subtelocentric), and 0.900-1.000(acrocentric)) [15,16,[22][23][24][25].

Statistical Analysis
The means of five varieties of C. repens, including length of short arm chromosome, long arm chromosome, total arm chromosome, relative length, and centromeric index, for all variables plus one standard deviation (SD), were used to represent all results, and they were calculated based on the methods of [15,16,[22][23][24][25], and principal component analysis (PCA) [26] was performed using SPSS version 29.

Results
C. repens 'turtle vine' has ovate and small leaves.The color of the leaves on the top is green.While the color of the leaves on the bottom is purple (Figure 1a).C. repens 'green' has large ovate leaves, which are larger than in the other variants.The color of the leaves is a green color on both sides (Figure 1b).C. repens 'pink lady' has ovate and small leaves.Leaves are elongated with different colors from green to pink and cream stripes, and the underside of the leaf is magenta (Figure 1c).C. repens 'gold' has ovate and small leaves.The leaves are golden green.The bottom of the leaf is magenta (Figure 1d).C. repens 'Bianca' is characterized by small and oval shaped leaves, which are either pure green, spotted, or pink color on both surfaces (Figure 1e).

Mitotic and Karyotype Analyses
For the purpose of studying somatic chromosomes, the root tips (about 1 cm long) were taken from living specimens.These root tips had a paradichlorobenzene (PDB) pretreatment for 6 h at 4 °C, fixed in ethanol-acetic acid 3:1 (v/v) for 30 min at room temperature, and after that stored at 4 °C or immediately used.All root tips were cleaned with distilled water, hydrolyzed in 1N HCl for 5 min at 60 °C, and then cleaned once more with distilled water.All root tips were dyed and squashed in 2% aceto-orcein.The chromosomes were observed by using a light microscope, Zeiss: Axiostar plus, to take the micrographs.Adobe Photoshop CS3 Extended was used to quantify the chromosome length and to rearrange the chromosomes.The study of chromosome count followed, [11,12] and [10].The chromosomal morphology nomenclature was developed using the techniques described in [5].Therefore, the chromosomes of all varieties were classified to be metacentric (m), submetacentric (sm), subtelocentric (st), or acrocentric (a).For each variety, 20 metaphase cells were used to determine the chromosomal count, chromosome structure, and karyotype.For the karyotyping, the parameters for the average length of Ls (the short arm ), average length of Ll (the long arm), LT of each chromosome (total length), average relative length (RL = LT/∑LT, formula for relative length), and chromosome index (CI = Ll/LT, formulae for chromosome index are 0.500-0.599(metacentric), 0.600-0.699(submetacentric), 0.700-0.899(subtelocentric), and 0.900-1.000(acrocentric)) [15,16,[22][23][24][25].

Statistical Analysis
The means of five varieties of C. repens, including length of short arm chromosome, long arm chromosome, total arm chromosome, relative length, and centromeric index, for all variables plus one standard deviation (SD), were used to represent all results, and they were calculated based on the methods of [15,16,[22][23][24][25], and principal component analysis (PCA) [26] was performed using SPSS version 29.

Results
C. repens 'turtle vine' has ovate and small leaves.The color of the leaves on the top is green.While the color of the leaves on the bottom is purple (Figure 1a).C. repens 'green' has large ovate leaves, which are larger than in the other variants.The color of the leaves is a green color on both sides (Figure 1b).C. repens 'pink lady' has ovate and small leaves.Leaves are elongated with different colors from green to pink and cream stripes, and the underside of the leaf is magenta (Figure 1c).C. repens 'gold' has ovate and small leaves.The leaves are golden green.The bottom of the leaf is magenta (Figure 1d).C. repens 'Bianca' is characterized by small and oval shaped leaves, which are either pure green, spotted, or pink color on both surfaces (Figure 1e).The diploid chromosomes of the five varieties of C. repens in this study were observed as having 12 chromosomes (Figure 2, Table 1).The characteristics of the karyotypes of the five varieties of C. repens are provided in Figure 3 and Table 1.The diploid chromosomes of the five varieties of C. repens in this study were observed as having 12 chromosomes (Figure 2, Table 1).The characteristics of the karyotypes of the five varieties of C. repens are provided in Figure 3 and Table 1.The somatic chromosome number of turtle vine was 2n = 12 and NF = 24 (Figure 2a).The karyotype formula was 2m + 2sm + 8st.The arm ratio caused the karyotype to be asymmetrical.The measured lengths of the short and long arms ranged from 0.89 ± 0.01 to 5.79 ± 0.02 µm, 4.38 ± 0.02 to 8.43 ± 0.03 µm, respectively, while the total arm length ranged from 7.28 ± 0.02 to 14.21 ± 0.05 µm, the RLs ranged from 12.49 ± 0.01 to 24.38 ± 0.01, and the CIs from 0.55 ± 0.01 to 0.88 ± 0.01, as shown in Table 2 and Figure 3a.
Gold had somatic chromosomes 2n = 12 and NF = 24 (Figure 2d).It was 2m + 4sm + 6st in the karyotype formula.The arm ratio caused the karyotype to be asymmetrical.The total arm length varied between 6.63 ± 0.02 and 13.56 ± 0.05 µm, the long arm length varied between 3.65 ± 0.02 and 8.22 ± 0.03 µm, and the short arm length varied between 0.90 ± 0.02 and 5.34 ± 0.02 µm.RLs ranged from 11.92 ± 0.01 to 24.40 ± 0.01 and CIs from 0.55 ± 0.02 to 0.88 ± 0.01 (see Table 2 and Figure 3d).The sizes and karyotypes of the five varieties look similar to each other, but they differed slightly.Therefore, we used PCA to help classify the five of them.The results of the PCA are shown below.
The sizes and karyotypes of the five varieties look similar to each other, but they differed slightly.Therefore, we used PCA to help classify the five of them.The results of the PCA are shown below.

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PCA score plot for karyotype formula The PCA score plot for five varieties of C. repens, based on their karyotype formula, revealed that component 1 accounted for 66.57% of the total variance, while component 2 accounted for 31.34%.Pink lady and green were in the same stage, while Bianca, turtle vine, and gold were in the same stage.This indicates that the karyotype formulae of pink lady and green had the same chromosome types but a different number of each chromosome type.Additionally, Bianca and turtle vine had the same chromosome types and numbers, indicating that they could not be separated (Figure 4a).Therefore, when only using the karyotype formula, it is not sufficient to separate all five varieties of C. repens.It is necessary to include other information, such as CI and leaf color, to distinguish between different variations.

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PCA score plot for CI The PCA score plot for the five varieties of C. repens based on CI showed that component 1 accounted for 58.66% of the total variance, while component 2 accounted for 37.23%.Bianca was in a different stage compared to the other variations, as indicated by its wider range of CI closely related to component 1.On the other hand, the CIs of green and turtle vine were similar to each other and different from the other CI variations (Figure 4b).This result aligns with the findings of the karyotype formula, indicating that the CIs of Bianca and turtle vine can be used to separate them into different stages, although they remain closely related.

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PCA score plot for leaf color The PCA score plot for the five varieties of C. repens based on leaf color successfully separated all variations.Component 1 accounted for 72.25% of the total variance, while component 2 accounted for 22.66%.Green, Bianca, turtle vine, and gold were in the same stage, whereas only pink lady was in a different stage, exhibiting distinct leaf color compared to the other variations (Figure 4c).In conclusion, the karyotype formula alone is insufficient to separate the five varieties of C. repens.However, when combined with CI and leaf color, it becomes possible to differentiate between the variations successfully.
The PCA score plot for the five varieties of C. repens based on leaf color successfully separated all variations.Component 1 accounted for 72.25% of the total variance, while component 2 accounted for 22.66%.Green, Bianca, turtle vine, and gold were in the same stage, whereas only pink lady was in a different stage, exhibiting distinct leaf color compared to the other variations (Figure 4c).In conclusion, the karyotype formula alone is insufficient to separate the five varieties of C. repens.However, when combined with CI and leaf color, it becomes possible to differentiate between the variations successfully.

Discussion
This study revealed that the chromosome number in all varieties of C. repens was 2n = 12, which is consistent with previous reports [1,4,6,8] (Figure 2, Table 1).The NF of all varieties was determined to be 24, which aligns with previous findings [4,8] (Table 1).Notably, all varieties of C. repens exhibited an asymmetrical karyotype, as they comprised not only metacentric and submetacentric chromosomes, but also other types, which is in line with previous studies [4,8] (Table 1).The karyotype characteristics of the five varieties in this study differed from those reported in [4,8] (Table 1).In fact, the karyotypes of the five varieties of C. repens (Figure 3, Tables 1 and 2) displayed variations in terms of chromosome length, arm length, total arm length, and karyotype asymmetry.The chromosomal features allowed for the identification of the five varieties of C. repens, as they differed from one another in terms of short arms, long arms, relative lengths (RL), and centromeric index (CI).Therefore, the karyotype data, along with dominant morphological characteristics, can be utilized to differentiate between the five varieties of C. repens from Laos.
Among the five varieties of C. repens, four exhibited different karyotype formulas.Specifically, pink lady showed a karyotype formula of 8m + 4st, green had 2m + 10st, C. repens 'gold' displayed 2m + 4sm + 6st, while both Bianca and turtle vine had a karyotype formula of 2m + 2sm + 8st.However, it is noteworthy that Bianca and turtle vine shared the same karyotype formula, indicating that the karyotype formula alone cannot be used to distinguish between these two varieties.In fact, the CI can differentiate between the variations.Although the CI values appear to be in the same direction, Bianca does not fit within the accepted range.Furthermore, the leaf color of C. repens also serves as a distinguishing factor among the varieties.The leaf color can differentiate between them, as they may appear similar, but pink lady is not considered to be part of the same varieties.Following the results in this study, it is possible to classify all five varieties of C. repens by using the PCA of the karyotype formula, CI, and leaf color.

Conclusions
This study focused on five varieties of C. repens, which included turtle vine, green, pink lady, gold, and Bianca.All of these varieties share the same chromosome number, 2n = 12, and NF = 24.The karyotype formulae for turtle vine and Bianca are 2m + 2sm + 8st, for green it is 2m + 10st, for pink lady it is 8m + 4st, and for gold it is 2m + 4sm + 6st.The data regarding chromosome length, arm length, total arm length, and karyotype asymmetry represents the first report of these findings for C. repens in Laos.The sizes of the chromosomes are quite similar among all the varieties, but PCA can be used to classify them.This is the first published cytogenetic study in Laos.It provides valuable data and information for researchers interested in studying this field.This is because there are many varieties of Callisia repens that have different external characteristics.The research results suggest that plant species can be classified by studying their chromosomes.Therefore, this serves as fundamental information that can be further explored in the field of genetic editing.This exploration can potentially enhance the beauty and economic value of these plants in the future.

Figure 4 .
Figure 4. PCA score plots for five varieties of Callisia repens (a) based on karyotype formula, (b) based on CI, and (c) based on leaf color (TV: turtle vine; Gr: green; PL: pink lady; Go: gold; B: Bianca).

Figure 4 .
Figure 4. PCA score plots for five varieties of Callisia repens (a) based on karyotype formula, (b) based on CI, and (c) based on leaf color (TV: turtle vine; Gr: green; PL: pink lady; Go: gold; B: Bianca).

Table 1 .
Information about five Callisia repens species and their corresponding references, including chromosomal numbers, NF = 24, and karyotype formula.