Phenology

In the rice stage, the first pair of leaves completely covered the main button in the three cultivars (Fig. 1, 2, and 3). In this stage, the precocity of a cultivar is determined. ‘Moon light’ first developed the rice point in most of its stems, followed by ‘Idilio’ and finally ‘Ilusion’.

For the pea stage, the bracts separated a little and gave way to the calyx, the sepals were not differentiated, and petals were formed internally (Fig. 1, 2, and 3). This stage presented an accumulation of 133.8 GDD (8 d) for ‘Ilusion’; the other two varieties were earlier (Tab. 2). The chickpea stage had a shorter duration in ‘Ilusion’, followed by ‘Idilio’ and finally ‘Moon light’ (Tab. 2). In the three cultivars, the petals are formed internally, are small but influence the size of the calyx, keeping their pale green color. The sepals then differentiated (Fig. 1, 2 and 3). In the barrel stage, the sepals opened and allowed the emergence of the corolla formed by the petals; the Moon light cultivar began to define its color, while the petals of ‘Idilio’, and ‘Ilusion’ remained pale green (Fig. 1, 2, and 3). However, ‘Idilio’ had the lowest accumulation of degree-days (194.1 GDD, 12 d; Tab. 2).

Figure 1. Development stages for flower buds in standard carnation (Dianthus caryophyllus L.) cultivar Idilio. A: rice; B: peas; C: chickpea; D: barrel; E: star; F: bullet; G: cut point 3; H: cut point 5. 

Figure 2. Development stages for flower buds in standard carnation (Dianthus caryophyllus L.) cultivar Ilusion. A: rice; B: pea; C: chickpea; D: barrel; E: star; F: bullet; G: cut point 3; H: cut point 5. 

The star stage saw the petals come out and the opening of the sepals with a star shape in the upper part of the calyx. The color of the petals in the three cultivars was fully-defined but they had not reached their final size (Fig. 1, 2, and 3). The duration of this stage was very different between the cultivars, where ‘Moon light’ required the lowest accumulation (129.9 GDD), and ‘Ilusion’ required the highest accumulation (211.5 GDD) (Tab. 2). ‘Idilio’ and ‘Moon light’ quickly reached the bullet stage and accumulated 63.7 GDD (Tab. 2). At this stage, the first pair of leaves separated from the main bud and defined an internode of 2 cm in ‘Idilio’ and ‘Ilusion’ and 3 cm in ‘Moon light’. The three cultivars had five fully-defined sepals (Fig. 1, 2, and 3).

Figure 3. Development stages for flower buds in standard carnation (Dianthus caryophyllus L.) cultivar Moon light. A; rice, B: peas; C: chickpea; D: barrel; E: star; F: bullet; G: cut point 3; H: cut point 5. 

In the stage of cut-off point 3, all the petals were developed and had acquired the final color, obtaining the shape of a cup. The Idilio cultivar had pistils, while ‘Ilusion’ and ‘Moon light’ did not (Fig. 1, 2, and 3). The ‘Moon light’ variety present the smallest accumulation with a value of 53.5 GDD, followed by ‘Idilio’, and finally ‘Ilusion’, which was the second shortest instar (Tab. 2). Finally, cut-off point 5 was the shortest stage in all cultivars, with only 33.7 GDD for ‘Ilusion’ (Tab. 2). Because of the action of temperature and the synthesis of ethylene, the petals took on a curvature and reached their maximum opening point (Fig. 1, 2, and 3).

Growth

The behavior of fresh mass

The accumulation of dry mass in the D. caryophyllus, as a function of the GDD, had a sigmoid-type behavior and was adjusted to a logistic model with three parameters (Tab. 3). Three growth phases were observed: phase 1 included the pea stage to the accumulation of 381.5 GDD in the barrel state for the Idilio cultivar (Fig. 4), 381.5 GDD for ‘Ilusion’ (Fig. 5), and 361.6 GDD for ‘Moon light’ (Fig. 6). Phase 2 ended with the bullet stage, where the accumulation was 294.4 GDD, 397.7 GDD, and 293.5 GDD for ‘Idilio’, ‘Ilusion’, and ‘Moon light’, respectively (Fig. 4, 5 and 6). At the end, phase 3 had 108.9 GDD ‘Idilio’, 110.8 GDD ‘Ilusion’, and 99.9 GDD ‘Moon light’, respectively (Fig. 4, 5 and 6). ‘Moon light’ was the earliest cultivar, and ‘Ilusión’ was the latest. A total fresh mass of 10.12±0.16 g was obtained for ‘Idilio’, 11.01±0.60 g for ‘Ilusion’ and 8.54±0.30 g for ‘Moon light’ (Fig. 4, 5 and 6).

Table 3. Estimated parameters of the logistic model for the dry mass of flower buds from three standard carnation (Dianthus caryophyllus) cultivars grown under greenhouse. 

Cultivar	Variable	Model	Estimated parameters*			Data criterion**
			Yasym	K	Tm	AIC	BIC
Idilio	Dry weight	Logistic	2.1059	0.1050	571.09	-26.92	-26.60
	Fresh weight		11.300	0.0098	567.41	-34.45	-34.13
Ilusion	Dry weight	Logistic	2.4568	0.0060	709.61	-28.33	-28.01
	Fresh weight		16.315	0.0054	796.23	-19.68	-19.36
Moon Light	Dry weight	Logistic	2.275	0.0068	655.10	-34.89	-34.58
	Fresh weight		12.625	0.0066	671.61	-20.71	-20.39

*Yasym: asymptotic or maximum Y value, K: inflection point of the curve. Tm: inflection point at which the growth rate is maximized, **AIC: Akaike Information Criterion, and BIC: Bayesian Information Criterion.

Figure 4. Behavior of fresh mass (A) and dry mass (B) in flower buds from Dianthus caryophyllus cultivar Idilio under greenhouse conditions. The vertical bars at each sampling point indicate the standard error (n=10). B: pea; C: chickpea; D: barrel; E: star; F: bullet; G: cut point 3; and H: cut point 5. 

The period between the barrel and star stages had the greatest gain in fresh mass with 4.18 and 3.83 g for the Idilio, and Ilusion cultivars, respectively. In ‘Moon light’ 2.25 g were gained between the chickpea and barrel stages. In the first three stages (rice, peas and chickpeas), logarithmic growth was observed, represented by little accumulation of mass (Fig. 4, 5 and 6). Phase 2 had a greater gain or accumulation of fresh mass for the three cultivars. In ‘Idilio’, 59.8% of the total fresh mass was accumulated in this phase, in ‘Ilusion’, it was 52.4%, and, for ‘Moon light’, 47.76% it was (Fig. 4, 5, and 6). Growth phase 3 represented a non-asymptotic curve for the three cultivars, however, for the curve to stabilize, the flower bud had to reach its maximum point of petal opening (Fig. 4, 5, and 6).

Figure 5. Behavior of fresh mass (A) and dry mass (B) in flower buds from Dianthus caryophyllus cultivar Ilusion under greenhouse conditions. The vertical bars at each sampling point indicate the standard error (n=10). B: pea; C: chickpea; D: barrel; E: star; F: bullet; G: cut point 3; H: cut point 5. 

The behavior of dry mass and growth rates

The accumulation of dry mass presented a simple, sigmoid-type behavior, which was adjusted to a logistic model of three parameters (Tab. 2). The first three stages rice, peas and chickpeas were characterized by a low accumulation of dry mass for the three cultivars, which corresponded to logarithmic growth. The greatest accumulation occurred in the period between the barrel and star stages for ‘Idilio’, and ‘Ilusion’, with 0.7 and 0.71 g, respectively, unlike the Moon light cultivar where it occurred between the chickpea and barrel stages with 0.37 g. The total accumulated dry mass was 1.834±0.04 g for ‘Idilio’, 1.885±0.01 g for ‘Ilusion’, and 1.54±0.01 g for ‘Moon light’.

Figure 6. Behavior of fresh mass (A) and dry mass (B) in flower buds from Dianthus caryophyllus cultivar Moon light under greenhouse conditions. The vertical bars at each sampling point indicate the standard error (n=10). B: pea; C: chickpea; D: barrel; E: star; F: bullet; G: cut point 3; H: cut point 5. 

The dry mass was related to the accumulation of degree-days for ‘Idilio’ and ‘Ilusion’. The periods of higher accumulation of dry mass did not correspond to the periods with more degree-days, unlike ‘Moon light’, where the transition from chickpea to barrel saw the highest accumulation of dry mass, coinciding with the period of highest accumulation of degree-days (Fig. 4, 5, and 6).

Figure 7. Behavior of the absolute growth rate (AGR) (A) and relative growth rate (RGR) (B) for flower buds from Dianthus caryophyllus cultivar Idilio, under greenhouse conditions. 

The AGR for the three cultivars began with a slow increase during the first phase, obtaining very similar values of accumulation of degree-days, with the following values, 380,7 GDD for ‘Idilio’, 381.5 GDD for ‘Ilusion’, and 361.5 GDD for ‘Moon light’. In phase 2, the rate of accumulation of dry mass increased, mainly in ‘Idilio’, which had the highest AGR. In this phase, the decrease in this rate also began. ‘Moon light’ and ‘Ilusion’ showed their maximum value at the end of phase 2 and then started to decline, which was less marked than in ‘Idilio’ (Fig. 7, 8, and 9).

Figure 8. Behavior of the absolute growth rate (AGR) (A) and relative growth rate (B) (below) for flower buds from Dianthus caryophyllus cultivar Ilusion, under greenhouse conditions. 

The beginning of the ‘Idilio’ rice stage presented an AGR of 0.000064 g/GDD, and, at the end of its development at cut-off point 5, there was an AGR of 0.0023 g/GDD (Fig. 7). The ‘Ilusion’ rice stage had 0.00021 g/GDD, and point 5 had an AGR of 0.0028 g/GDD (Fig. 8). Finally, ‘Moon light’ had 0.00021 g/GDD in the rice stage and, at the end of its development of the flower bud, had 0.0035 g/GDD (Fig. 9).

The RGR remained more or less stable in phase 1 (rice, pea, and chickpea stages). Idilio presented values almost double that of the other varieties, in which the RGR was similar. In phase 2 (end of chickpea, barrel, star and bullet), a decrease was observed in the 3 varieties, where the decrease was more marked in Idilio. In phase 3 (cutoff point 3 and 5), the decrease continued, and Idilio presented the lowest values. Ilusion and Moonlight presented similar trends (Fig. 7, 8, and 9).

Figure 9. Behavior of the absolute growth rate (AGR) (top) and relative growth rate (RGR) (below) for flower buds from Dianthus caryophyllus variety Moonlight under greenhouse conditions. 

Phenology

The Idilio, Ilusion, and Moonlight carnation cultivars had the eight stages of development of the carnation flower bud: rice, pea, chickpea, barrel, star, bullet, cut point 3 and cut point 5 (Fig. 1, 2 and 3). ^{Arévalo et al. (2007)} and ^{Romero-Cuervo et al. (2022)} identified seven stages in the flower bud of cvs. Nelson and Moonlight. For Japanese markets, the harvest must be done at cut point 3, while for the United States market it should be cut point 5. In this study the stage of development cut point 5 was also included to achieve the stability of the growth curve with a simple sigmoid pattern, a growth pattern characteristic of plants and its organs (^{Tessmer et al., 2013}).

The phenology of the three cultivars was evaluated to predict the approximate time of harvest, as has been reported for carnation cv. Delphi (^{López et al., 2014}) because of the importance of optimal harvests for the international markets, mainly Valentine’s Day. The phenology also is an important tool for planning and carrying out important agronomic activities sustainably, with the purpose that the carnation as a cut flower continues to have the eco-friendly profile, which is associated to low CO₂ footprints and environmental outputs (^{Darras, 2021}).

Most of the phenological changes are under genetic control, the development of the carnation flower involves an important number of genes, which would be responsible for determining each of the development stages presented in this study, accordingly, ^{Zhang et al. (2018)} reported high expression pattern of the DcaMADSs gene in carnation, five genes in sepal, other five in petals, nine in the ovary, four genes in styles, and six genes in the stamen.

Interestingly, in the first stage (rice), photoassimilates are transported mostly from the leaves to the main button, because the flower is an important sink (^{Taiz et al., 2018}). The time required for this process is largely decisive for the success of each variety because it determines the vase life. In carnations, this is an attractive trait that has been obtained with plant breeding (^{Onozaki, 2018}; ^{Lin et al., 2021}).

To obtain a fully developed flower bud from rice point to point 5 (maximum opening), 762 GDD, 889 GDD, and 745.4 GDD were required for ‘Idilio’, ‘Ilusion’, and ‘Moon light’, respectively (Tab. 2). The cultivar that required less GDD and accumulated less dry and fresh mass was ‘Moon light’; this was due to the characteristics of the flower bud, mainly because it has less number of petals (data not shown). This was previously reported by several authors (^{Trudgill et al., 2005}; ^{Parra-Coronado et al., 2016}), who stated that the accumulation of GDD to reach the phenological stages differs from cultivar to cultivar. ^{Romero-Cuervo et al. (2022)} reported an accumulation of 777.68 GDD for the Moon light cultivar, with one less growth stage (cutoff point 5).

Finally, the cultivars had very different and striking colorations. ‘Idilio’ had red petals, ‘Ilusion’ had pink petals, and ‘Moon light’ had white petals (Fig. 1, 2, and 3). In carnations, colors result from the anthocyanins in the vacuoles of the epidermis of the petals (^{Morimoto et al., 2020}, ²⁰²¹). Red and dark red carnations mainly accumulate pelargonidin 3-malylglucoside, pink carnations accumulate pelargonidin 3,5-cyclicmalyl-diglucoside (^{Morimoto et al., 2020}), and white carnations do not accumulate or accumulate very few anthocyanins or may accumulate pale yellow flavonoids (^{Morimoto et al., 2021}). For red cultivars such as ‘Idilio’, it is important to apply iron to improve the color of the petals in the bullet and point 3 stages. In this regard, ^{Guo et al. (2022)} reported a positive correlation between iron and anthocyanin content in Petunia hybrida.

Color is another highly appreciated characteristic in carnations (^{Onozaki, 2018}) since color is one of the more valuable traits in the markets (^{Morimoto et al., 2020}). Accordingly, the Moon light cultivar is mainly destined for the United States market, followed by Japan, Korea, Russia, Holland, and Canada; a significant percentage is dyed for Russian and Korean markets. ‘Moon light’ has a great track record for its postharvest characteristics such as duration in the vase and good transport and storage conditions. On the other hand, ‘Idilio’ is more appreciated in Russian markets than in the United States and Spain. Generally, the red colored varieties are sent to Russia; however, seasons such as Valentine’s Day, Mother’s Day, and Women’s Day require more in the United States. ‘Ilusion’ also has good storage characteristics, vase-life, and dyeing possibilities, the latter adds value since it opens the possibility of sending flowers to other international markets that prefer colors that have not been generated through plant breeding.

Growth

The fresh and dry masses of the three cultivars were adjusted to a logistic model (Fig. 1, 2, and 3; Tab. 2). This model has been reported in the growth of ‘Moon light’ carnations (^{Romero-Cuervo et al., 2022}), Phalaenopsis orchid (^{Chen et al., 2019}), in lulo (Solanum quitoense) plants (^{Criollo-Escobar et al., 2020}), and in grape (^{Almanza et al., 2010}) and apple fruits (^{Cepeda et al., 2021}) because the logistic model explains simple, sigmoidal growth patterns very well (^{Pinzón-Sandoval et al., 2021}).

Growth in plants or their organs is defined as the increase in dry mass, volume, length, or area that results from cell division, expansion, and differentiation (^{Lambers et al., 2008}). Thus, phase 1 in the 3 carnation cultivars consisted of a phase of low fresh and dry weight gain, which was corroborated with a slow AGR and a stable RGR (Fig. 4, 5, 6, 7, 8 and 9) possibly because this phase has high cell division with a low gain of photoassimilates (^{Tessmer et al., 2013}; ^{Azcón-Bieto and Talón, 2013}). Then, a rapid growth phase was observed, where a greater AGR and a marked decrease in RGR were observed in the three materials, which was due to processes of cell elongation and increases in photoassimilates, and water (Azcón-Bieto and Talón, 2013). Finally, growth was at a slower rate, as shown by the AGR and RGR (Fig. 4, 5, 6, 7, 8 and 9). Similar results in AGR and RGR behaviors have been previously reported (^{Tessmer et al., 2013}; ^{Romero-Cuervo et al., 2022}). The fresh mass values in the three cultivars were similar to that described for cv. Moon light (^{Romero-Cuervo et al., 2022}), where the total accumulated mass under the climatic conditions (12-14ºC mean temperature) of Ubate (Colombia) was 7.96±0.21 g.

The balance between the assimilates demand of the plant organs and the photosynthetic contribution of the plant must be considered to explain the difference in growth rates between varieties, where ‘Idilio’ had the fastest growth according to the AGR and RGR, and the greatest fresh and dry mass of the flower after ‘Ilusion’ (7, 8 and 9). The latter was similar in the behavior of the rates with ‘Moon light’ but accumulated more GDD and greater fresh and dry mass (Fig. 6, 7, 8 and 9). ^{Ahmad et al. (2017)} stated that the AGR is the simplest index of plant growth, and, when comparing similar materials, determining the AGR can be a valuable tool, as was found in the three carnation varieties. AGR is also useful for understanding stem growth in standard carnation cv. Nelson (^{Baracaldo et al., 2010}).