Suppression of Malformed Petals in Cut Rose ‘Yves Piaget’ Flowers Caused by Pulse-Uptake Treatment of Methyl Jasmonate

Abstract

The most popular fragrant cut rose cultivar in Japan is ‘Yves Piaget’, but its petals often become malformed after harvest. These malformed flowers, referred to as “incurved flowers”, are characterized by petals curving toward the adaxial side which prevents normal flowering and significantly decreases the cut flower quality. It has been reported that jasmonic acid (JA) affects petal growth, and could suppress the emergence of incurved flowers. Therefore, in this study, we investigated the use of exogenous JA, methyl jasmonate (MeJA), applied to cut flowers of ‘Yves Piaget’ by 24 h pulse-uptake treatment. Results showed that 500 μM MeJA 24 h pulse-uptake treatment was effective at suppressing the number of incurved petals. Then, salicylic acid (SA), an antagonist of JA, was applied to see if the number of incurved petals increased. We compared three treatments: deionized water uptake treatment (control), 500 μM MeJA pulse-uptake treatment, and 500 μM SA continuous-uptake treatment. Vase life did not differ significantly between the three treatments, but the 500 μM MeJA pulse-uptake treatment produced a better-preserved flower shape due to only slight petal bluing and discoloration. In contrast, the control and 500 μM SA continuous-uptake treatments produced cut flowers with major petal wilting, bluing, and discoloration. The 500 μM MeJA pulse-uptake treatment tended to extend the number of days from flower bud to full bloom and decrease the number of incurved petals due to the continued high-water absorption. The commercial value of roses lies in the blooming process from bud until full bloom; the increased number of days from bud to full bloom after 500 μM MeJA pulse-uptake treatment improved the cut flower quality. In contrast, there was less water absorption and more incurved petals in the days after the 500 μM SA continuous-uptake treatment, indicating an antagonistic response to the 500 μM MeJA pulse-uptake treatment. In conclusion, treatment of ‘Yves Piaget’ cut flowers with 500 μM MeJA pulse-uptake could decrease the number of incurved petals and improve the cut flower quality.

Introduction

Commercial production of roses is the highest of all flowering plants worldwide. In Japan, roses are mainly used as cut and potted flowers. Approximately 194 million cut flowers were produced in fiscal year 2021 and this was the third highest in Japan following chrysanthemum and carnation (Ministry of Agriculture, Forestry, and Fisheries, 2020). Recently, the import of inexpensive cut roses has been increasing, and this means that domestic cut roses require improved quality to compete with the imported cut roses. Freshness is an important quality of domestic rose production and is an important factor in the growing demand for fragrant cut roses.

In Japan, the most popular fragrant cut rose cultivar is ‘Yves Piaget’. It has gorgeous peony-like double flowers comprising around 50 pink petals, but it often produces malformed flowers with petals that curve toward the adaxial side (Fig. 1). These malformed petals prevent normal flowering and weaken the flower fragrance, which significantly decreases the quality of this cultivar. This type of malformed flower is called an “incurved flower” (Kaneeda et al., 2019).

Fig. 1

Appearance of ‘Yves Piaget’ flowers. Normal flower (A, B) and incurved flower (C, D). Arrows represent incurved petals. The scale bar represents 25 mm.

Incurved flowers are thought to be related to the activity of carbohydrate-metabolizing enzymes in flower petals around the time of flowering. For example, strong activity of both vacuolar and cell wall invertases, which are carbohydrate-metabolizing enzymes, has been reported in rose petals at the time of flowering (Horibe et al., 2013; Yamada et al., 2007). Also, another recent study found that the activities of both vacuolar and cell wall invertases in incurved flowers of ‘Yves Piaget’ were detectable at the stage just before petal development in flower buds (Kaneeda et al., 2019), but that these activities decreased rapidly during flower opening. Therefore, it is possible that incurved flowers could be avoided by enhancing invertase activity at the time of flower bud maturation.

Phytohormones, such as auxin and gibberellins, are key factors for controlling cell proliferation and expansion, but their precise functions in flower development are unclear because of their pleiotropic roles and complex signaling interactions (Weiss et al., 2005). The lipid-derived phytohormone jasmonic acid (JA), including free-acid conjugates, regulates a wide range of biological processes, such as biotic or abiotic plant stress responses, and developmental processes (e.g., root growth, seed germination, anther development, and senescence) (Browse, 2005; Devoto and Turner, 2003). However, little is known about the role played by JA in petal development (Brioudes et al., 2009). Horibe et al. (2013) reported that the postharvest addition of α-naphthalene acetic acid (NAA) and methyl jasmonate (MeJA), a derivative of JA, affected invertase activity in cut rose flowers. Ochiai et al. (2013) reported that the promotion of early flower opening by MeJA treatment was due to petal cell wall loosening by accelerated expression of expansin and xyloglucan endotransglycosylase/hydrolase. Additionally, it has been reported that while treatment of cut rose flowers with MeJA slowed down flower opening and extended the vase life of the flowers (Horibe and Makita, 2019), treatment with NAA promoted flower opening and petal growth (Horibe and Makita, 2021). Furthermore, Singh et al. (2022) reported that JA treatment delayed flower opening, as well as petal abscission, by more than 24 h in the fragrant rose, Rosa × bourboniana.

Salicylic acid (SA) is an antagonist of JA that acts by inhibiting the conversion of 13S-hydroperoxy linolenic acid to 12-oxo-phytodienoic acid, thereby inhibiting the signaling pathway by blocking JA synthesis (Doares et al., 1995; Pieterse et al., 2012). Generally, SA exerts a negative crosstalk over the JA pathway in Arabidopsis (Proietti et al., 2013). On the other hand, SA is widely used as a natural, cheap, safe, and biodegradable anti-bacterial agent to increase the longevity of cut flowers in vases (e.g., Alstroemeria peruviana, Gerbera jamesonii, Lilium asiaticum, R. × hybrida, Polianthes tuberosa, Dianthus caryophyllus, Dendranthema × grandiflorum) (Bayat and Animifard, 2017; Kazemi and Ameri, 2012; Vahdati-Mashhadian et al., 2012). Therefore, we wanted to investigate whether the presence of exogenous SA could increase the generation of malformed flowers.

In this study, we set the following two objectives A) investigate whether the use of exogenous jasmonate, MeJA, after harvest could reduce the number of incurved petals in ‘Yves Piaget’ cut flowers (Experiment 1); B) assess whether the use of exogenous SA could generate malformed flowers with more incurved petals in ‘Yves Piaget’ cut flowers (Experiment 2).

Materials and Methods

Plant materials

Rosa × hybrida ‘Yves Piaget’ cut flowers were obtained at the commercial harvest stage from Ota floriculture auction market in Tokyo. Cut flowers were soaked in water with 0.001% (w/v) Rose 100 (an antibacterial agent and pH adjuster; FloraLife^®; Smithers-Oasis Company, Kent, OH, USA) after harvest at the farm. Flowers were kept in this water and transported to the laboratory from the market within 2 h. Promptly after transport, flowers were cut at their stem bases in deionized water to remove air embolism and cavitation then kept in a refrigerator at 4 ± 2°C with 90 ± 10% relative humidity in the dark for 2 h until they were used in experiments.

Treatment and experimental design

A preliminary experiment was conducted to compare the effects MeJA pulse treatment or continuous uptake treatment, and the effect of MeJA concentration. The preliminary results showed that 24 h 100 μM MeJA pulse treatment resulted in a lower number of incurved petals than the 100 μM MeJA continuous-uptake treatment (data not shown). Also, there was a lower number of incurved petals with 500 μM MeJA pulse-uptake treatment compared with 100 μM or 1000 μM pulse-uptake treatments (data not shown). As a result, in the subsequent experiments a 500 μM MeJA 24 h pulse-uptake treatment was used.

In Experiment 1, flowers were cut to a length of 30 cm and all leaves were removed to avoid the effects of leaf transpiration and translocated sugar. The flower stem bases were placed in deionized water with 0.005% (w/v) Kathon^TM CG (an antibacterial agent; Rohm and Hass company, Philadelphia, PA, USA) as a control water, or in the control water with 500 μM MeJA (Wako Pure Chemical Industries Ltd., Osaka, Japan) added for the pulse-uptake MeJA treatment. To complete pulse-uptake of MeJA, flowers were placed in 500 μM MeJA solution at 24 ± 2°C with 60 ± 10% relative humidity for 24 h and then transferred to the control water. The control flowers were placed under the same conditions as 500 μM MeJA treatment and the experiment was started at the same time. The solution did not contain soluble sugars to avoid the effect of exogenous carbohydrates on flowering. The solution was freshly prepared at the beginning of the experiments and was not replaced or refilled throughout the experiments. Flowers were placed individually in graduated cylinders with 50 mL solutions and held in a plant growth incubator (LH-80LED-DT; Corp. Nihon Ika Kikai Seisakusho, Tokyo, Japan) at 24 ± 2°C with 60 ± 10% relative humidity and a 16 h light photoperiod [photosynthetic photon flux density (PPFD): 20–40 μmol·m⁻²·s⁻¹]. The mouth of the graduated cylinders was sealed with parafilm to prevent any evaporation of the solution. The experimental design was completely randomized with eight stems per treatment.

Another preliminary experiment was conducted prior to Experiment 2 to determine the concentration of SA that produced the greatest number of incurved petals during continuous-uptake treatment. More incurved petals were generated with 500 μM SA than 100 μM or 1000 μM SA (data not shown). Therefore, in the second experiment, 500 μM SA was used as the JA antagonist. In Experiment 2, the flowers were prepared in the same way as in the first experiment and then placed individually in graduated cylinders filled with 50 mL with one of the following three treatment solutions: (1) deionized water with 0.005% (w/v) Kathon^TM CG (Rohm and Hass Company) as a control water, (2) pulse-uptake treatment of 500 μM MeJA with 0.005% (w/v) Kathon^TM CG, and (3) continuous-uptake treatment of 500 μM SA with 0.005% (w/v) Kathon^TM CG. The salicylic acid was applied as a continuous treatment because we were trying to inhibit the biosynthesis of jasmonates in the flowers until the end of their vase life. The flowers were kept in a plant growth incubator at 24 ± 2°C with 60 ± 10% relative humidity, and 16 h light photoperiod (PPFD: 20–40 μmol·m⁻²·s⁻¹). The experimental design was completely randomized, with five stems per treatment.

Determination of days to full bloom and vase life

Full bloom was determined as the day when the stamens and pistils became visible in the center of the flower. Vase life was determined by petal wilting, petal bluing, and petal discoloration, according to the 2020 version of the Japan Flower Promotion Center Foundation manual for evaluating the longevity of cut roses (Japan Flower Promotion Center Foundation, 2020). Each item was evaluated from grade A to D, with the end of vase life determined when there were at least two C-grade items, or one D-grade item. Petal wilting was evaluated by the resilience of the petals when touched to avoid damaging them, with a C-grade given when the petals were soft without resilience and a D-grade given when the petals were clearly dropping visually. The petals of certain cultivars of red rose sometimes become bluish at a later stage of flowering. This phenomenon has been dubbed “bluing” (Yasuda, 1970). Bluing was evaluated by fading of the petals, and a C-grade was given when visually clear bluing was observed, and a D-grade was given when more significant bluing was observed. Petal discoloration was evaluated by looking at the degree of browning and necrosis, with a C-grade for slight discoloration and a D-grade for clear discoloration and necrosis. The end of vase life was recorded by the condition of petals in each treatment, and the proportion of each factor at the end of vase life was determined by dividing the total number of cut flowers from the number of cut flowers corresponding to each factor in Experiment 2.

Evaluation of the number of incurved petals

The number of incurved petals was evaluated as the number of petals curving towards the adaxial side, up to the third day after treatment in Experiment 1, and the fourth day after treatment in Experiment 2.

Measurement of water absorption

Water absorption was only measured in Experiment 2. Water absorption was measured daily until the fourth day after treatment by checking the water level against the scale markings on the graduated cylinder.

Statistical analysis

Statistical analyses were performed using SPSS version 28.0 (IBM, Somers, NY, USA). The Student’s t-test and Tukey-Kramer test were used to make comparisons between treatments. Pearson’s correlation analysis was used to correlate water absorption and the number of incurved petals. Values are means of all replicates ± SE. The significance level was 5%.

Results

Methyl jasmonate pulse-uptake treatment to suppress incurved petals (Experiment 1)

1)  Days to full bloom and vase life

In Experiment 1, there were no significant differences in the number of days to full bloom and vase life among the tested treatments; however, the 500 μM MeJA pulse-uptake treatment tended to increase the number of days to full bloom (Table 1).

Table 1

Days to full bloom and vase life in cut flowers of ‘Yves Piaget’ treated with methyl jasmonate (MeJA) or salicylic acid (SA).

2)  Number of incurved petals

In Experiment 1, the number of incurved petals was lower in the 500 μM MeJA pulse-uptake treatment than in the control plants, although on day 3 the difference was not significant (Fig. 2). The number of incurved petals in the control on day 3 varied greatly among individuals, with some individuals showing a large increase in the number of incurved petals and others showing a slight decrease. On the other hand, the 500 μM MeJA pulse-uptake treatment showed a consistent decrease in the number of incurved petals on day 3.

Fig. 2

The number of incurved petals in cut flowers of ‘Yves Piaget’ treated with MeJA. Error bars indicate standard errors (n = 8). *, P < 0.05; NS, no significant difference (Student’s t-test, comparing treatments on given days).

Incurved petals are reduced by methyl jasmonate and increased by salicylic acid, an antagonist of jasmonate (Experiment 2)

1)  Days to full bloom and vase life

In Experiment 2, there was no significant difference in vase life between any of the treatments (Table 1); however, the 500 μM MeJA pulse-uptake treatment tended to increase the number of days to full bloom compared with the control and the 500 μM SA continuous-uptake treatment.

The most common petal condition at the end of vase life in all treatments was petal bluing, but the degree of bluing varied between treatments (Fig. 3). Major petal bluing was the most common petal condition in the 500 μM SA continuous-uptake treated plants, but in the 500 μM MeJA pulse-uptake treatment, only slight bluing occurred. The other major petal condition in the 500 μM MeJA pulse-uptake treatment was slight petal discoloration, with only a few wilted or severely discolored petals. On the other hand, wilting was much more common in the control and 500 μM SA continuous-uptake treatments. The control showed the highest ratio of wilting among all treatments.

Fig. 3

Rates of factors at the end of vase life in cut flowers of ‘Yves Piaget’ treated with MeJA or SA (n = 5).

2)  Number of incurved petals

Flowers subjected to 500 μM MeJA pulse-uptake treatment had the fewest incurved petals, whereas flowers subjected to 500 μM SA continuous-uptake treatment had the most (Fig. 4). The number of incurved petals in the control was not significantly different from that in the 500 μM MeJA pulse-uptake treatment or 500 μM SA continuous-uptake treatments, but tended to be intermediate between the two treatments. As in Experiment 1, there were large individual differences in the control, with some individuals showing a large increase in the number of incurved petals, while others showed a slight decrease.

Fig. 4

The number of incurved petals in cut flowers of ‘Yves Piaget’ treated with MeJA or SA. Error bars indicate standard errors (n = 5). Different lowercase letters indicate a significant difference at P < 0.05 (Tukey-Kramer test for comparisons among treatments).

3)  Rate of change of flower stem fresh weight and water absorption

The fresh weight of flower stems changed over the duration of Experiment 2, but there were differences between the treatments (Fig. 5A). The rate of increase in the flower stem fresh weight of the control and 500 μM SA continuous-uptake treatments fell steadily over the four days, from a sizeable increase between day 0 and day 1 (0–1), to actual declines in fresh weight between days 2–3 and 3–4. In contrast, the flower stems treated with 500 μM MeJA pulse-uptake maintained a small positive uptake rate for all four days.

Fig. 5

(A) Rate of change in flower stem fresh weight and (B) water absorption in cut flowers of ‘Yves Piaget’ treated with MeJA or SA. Error bars indicate standard errors (n = 5). NS indicates no significant difference at P < 0.05 (Tukey-Kramer test for comparisons between treatments).

The rate of water absorption in the cut flower stems was consistently higher in the 500 μM MeJA pulse-uptake treatment than in the 500 μM SA continuous-uptake treatment, even though the differences were not statistically significant (Fig. 5B).

4)  Correlation between water absorption and the number of incurved petals per flower

Correlation coefficients for water absorption and the number of incurved petals between days 1–2 in Experiment 2 were −0.5374, with a P-value of 0.0388, indicating a negative correlation (Fig. 6). Correlation coefficients among the other days were low, but tended to show weak negative correlations (data not shown). Based on the above, the number of incurved petals can be expected to increase due to a decrease in water absorption in cut flowers.

Fig. 6

Correlation coefficients for water absorption and the number of incurved petals between days 1–2 in Experiment 2 (n = 5). *, P < 0.05; (Pearson correlation coefficient).

Discussion

In this study we investigated if the quality of ‘Yves Piaget’ rose cut flowers could be improved by treatment with exogenous JA. Treatment with MeJA did not extend the vase life of the cut flowers significantly; however, it tended to increase the days to full bloom by about half a day, although this was not a statistically significant increase compared with the control (Table 1). Previous studies have also shown that MeJA treatment can delay flower opening in cut roses (Horibe and Makita, 2019, 2021). They found that the optimal concentration varied depending on the rose cultivar, but that it was typically about 500 μM, which is the same concentration used in our experiment. They also found that MeJA treatment could extend vase life, which was different from our results. This may have been due to insufficient soluble sugars, energy or osmolytes in our study, which are all required for petal growth.

In Experiment 2, treatment with MeJA was also compared against treatment with SA, which has been shown to be a JA antagonist (Pieterse et al., 2012; Proietti et al., 2013). We found no significant difference in the vase life between these two treatments and the control (Table 1). Again, this may have been due to insufficient soluble sugars, energy or osmolytes in our study. However, as in Experiment 1, the 500 μM MeJA pulse-uptake treatment did tend to increase the days to full bloom, even though the difference was not statistically significant. Despite this, the increase in days to full bloom with MeJA treatment in both experiments suggests that MeJA did slow down the flowering process in the cut roses, as also reported by Horibe et al. (2013). Therefore, MeJA treatment could improve the quality of cut roses because for ornamental plants, especially roses, their value lies in the blooming process from bud to full bloom (Horibe and Makita, 2019).

In both experiments, the 500 μM MeJA pulse-uptake treatment generated significantly fewer incurved petals compared with the control (Figs. 2 and 4). Kaneeda et al. (2019) showed that incurved petals were associated with a sharp reduction in invertase activity during early flowering relative to that in normal flowers. Also, Horibe et al. (2013) reported that MeJA could maintain invertase activity at a higher level in cut rose flowers. Thus, in our experiments, it is likely that the beneficial effect of MeJA on reducing numbers of incurved petals was because it could suppress any sharp decrease in invertase activity during early flowering.

In addition, roses in the 500 μM MeJA pulse-uptake treatment maintained a stronger scent than the control and 500 μM SA continuous-uptake treatment (data not shown). Therefore, MeJA may have improved cut flower quality in ‘Yves Piaget’. In this study, the 500 μM MeJA pulse-uptake treatment did not prevent petal bluing (Fig. 3), suggesting that sucrose is necessary to further improve cut flower quality. The 500 μM SA continuous-uptake treatment did not extend flower longevity (Table 1), which is different to the results in a previous study showing that the use of SA as an antibacterial agent could extend the vase life of five types of cut flowers, including roses (Bayat and Aminifard, 2017). However, in this study, we had already added Kathon^TM CG as an antibacterial agent in all treatments, so it is likely that all treatments would have had sufficient antibacterial activity, and this could explain why the 500 μM SA continuous-uptake treatment did not expand vase life as in the previous study (Bayat and Aminifard, 2017). The 500 μM MeJA pulse-uptake treatment ended with slight petal bluing and slight petal discoloration, whereas the control and 500 μM SA continuous-uptake treatment ended with petal wilting (Fig. 3).

In the 500 μM MeJA pulse-uptake treatment, there was a positive rate of change in flower stem fresh weight over all four days, whereas for the control and 500 μM SA continuous-uptake treatment the flower stem fresh weight change became negative after two days (Fig. 5A). In addition, water uptake was highest in the MeJA treatment and lowest in the SA treatment (Fig. 5B). Although the differences were not statistically significant, they followed a similar pattern to the numbers of incurved petals, which were lower in the MeJA treatment than in the control SA treatment (Figs. 2 and 4). Moreover, water uptake was negatively correlated with the number of incurved petals (Fig. 6). Thus, water uptake seems to be an important factor related to the emergence of incurved petals. Also, treatment with SA increased the number of incurved petals compared to the control. It is known that SA inhibits the conversion of 13S-hydroperoxy linolenic acid to 12-oxo-phytodienoic acid, thereby inhibiting the signaling pathway by blocking JA synthesis (Doares et al., 1995; Pieterse et al., 2012). Also, it has been shown that SA exerts a negative crosstalk over the JA pathway in Arabidopsis (Proietti et al., 2013). Thus, it is possible that the biosynthesis of JA in cut flowers in our study was inhibited by the application of SA.

Application of MeJA has been reported to induce normal flowering in malformed petal mutants of Arabidopsis thaliana (Brioudes et al., 2009; Reeves et al., 2012) and Brassica campestris ssp. pekinensis (Huang et al., 2015), so it is thought that JA is involved in the normal growth of petals. Therefore, in future studies it will be important to further investigate invertase activity and the number of incurved petals associated with MeJA treatment to clarify the relationships between these factors. Also, further work is needed to study the effect of simultaneous addition of phytohormones and sugar on the emergence of incurved flowers.

In conclusion, a 500 μM MeJA pulse-uptake treatment improved cut flower quality by extending the number of days to full bloom and decreasing the number of incurved malformed petals in ‘Yves Piaget’. In contrast, the 500 μM SA continuous-uptake treatment lowered cut flower quality by increasing the number of incurved malformed petals in ‘Yves Piaget’. These results suggest that MeJA and SA have antagonistic effects on the development of incurved malformed petals in the cut rose cultivar ‘Yves Piaget’.

Acknowledgements

The author would like to thank Professor Iain McTaggart for English proofreading.

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