Effects of Low-Temperature Accumulation on Flowering of Prunus mume

Abstract

Low-temperature accumulation is one of the essential stages in the growth process of woody ornamental plants. In this study, two different low-temperature treatments, 6 °C and 10 °C, were used to analyze the effects of different low-temperature treatments on dormancy release and flowering of the ‘Gulihong’ plant using artificial low temperatures. Based on the experimental results, four typical early-blooming Prunus mume cultivars widely planted in Yangling area of Henan Province, China, including ‘Zaoyudie’, ‘Zaohualve’, ‘Nanjing gongfen’, and ‘Gulihong’, were selected as the experimental materials. The effects of low-temperature accumulation on the flowering characteristics of different cultivars were analyzed using a 6 °C artificial low-temperature treatment. The suitable cultivation temperature for early-blooming cultivars was screened to provide a theoretical basis for further exploration of P. mume bonsai cultivation techniques. The results showed that the flowering rate, flower diameter, flowering quantity, flowering uniformity, and bud development in the 6 °C treatment were significantly better than those in the 10 °C treatment. Furthermore, under 6 °C low-temperature treatment, the flowering rate and quality of different cultivars showed an increasing trend with the accumulation of low temperature, with ‘Gulihong’ exhibiting the highest flowering rate. Therefore, chill accumulation plays a significant role in promoting flowering quality.

1. Introduction

Prunus mume Sieb. et Zucc. is a traditional, famous flower in China that blooms in late winter and early spring, making it one of the earliest flowering plants in the winter season. Due to its delicate fragrance, elegant color, and rich cultural heritage [1,2,3], it has been widely cultivated in China and has great market potential. However, since P. mume blooms in winter, it cannot meet the specific viewing needs of other periods. Therefore, regulating the flowering time of P. mume can not only meet the viewing needs of the market and the public but also promote the development of the P. mume industry.

Natural dormancy is an adaptive mechanism for deciduous fruit trees to resist low temperatures in winter [4]. Temperature is the most important climatic parameter affecting the release of the bud’s natural dormancy in deciduous fruit trees. Under natural conditions, a certain accumulation of low temperatures is a necessary condition for the release of natural dormancy [5,6,7,8]. Otherwise, plants cannot germinate and grow normally, and it can even cause flower organ deformities or severe sterility [9]. Among them, chilling requirement refers to the effective hours of low temperature required by deciduous fruit trees to break natural dormancy, and chilling accumulation is considered to be the most effective influencing factor for plants to break dormancy [4,10]. In conclusion, mastering the relationship between chilling demand and natural dormancy is critical to effectively managing and optimizing the growth, development, and flowering of deciduous fruit trees.

In recent years, many scholars have also conducted research on the effect of low temperatures on breaking the dormancy of deciduous fruit trees. Zohner et al. (2016) believed that temperature mainly controls the duration of dormancy; only a small number of woody species are sensitive to photoperiod [11]. According to the recent work of Li et al. (2020), regardless of the photoperiodic conditions, low temperature can induce growth cessation and control dormancy induction during artificial low-temperature induction of Chimonanthus praecox [12]. In addition, many studies have found that dormant buds in a low-temperature environment for a long time can promote germination faster under the same growth-promoting conditions [13,14,15]. Therefore, a sufficiently low temperature has a promoting effect on the release of flower bud dormancy, and if the accumulation of chilling is insufficient, the goal of early flowering cannot be achieved.

Currently, research on the chilling requirements of woody plants mainly focuses on ornamental peach, pear, and other plants, and there is less relevant research on P. mume. However, similar to other deciduous fruit trees, P. mume is also sensitive to temperature and is usually controlled by regulating temperature to release flower bud dormancy [16]. Therefore, in this study, four early-flowering P. mume cultivars widely cultivated in Henan Province were selected as experimental materials. Based on an artificial low-temperature treatment, the effects of different low-temperature treatments and chilling accumulations on the release of dormancy and flowering were explored in order to provide a theoretical basis for P. mume cultivation technology.

2. Materials and Methods

2.1. Experimental Materials

This experiment was conducted from September to December 2021 at the Yuling World Plum Garden in Yanling, Henan Province (34°09′ N, 114°06′ E). Four early-flowering P. mume cultivars, including ‘Gulihong’, ‘Nanjing gongfen’, ‘Zaoyudie’, and ‘Zaohualve’, were selected from the same batch of eight-year-old plants grown in pots, with 42 plants of ‘Gulihong’ and 24 plants of the other three cultivars, for a total of 114 plants. The experimental plants were robust, with a compact growth habit, no pests or diseases, uniform distribution of branches, plump and full buds, and consistent specifications, with a ground diameter of about 2.5 cm to 4.1 cm and a plant height of about 80 cm. The temperature controller of the cold storage was the XMK-7 type produced by Zhejiang Yuyao Mingxing Refrigeration Accessories Factory, with a temperature accuracy of 0.1 °C.

2.2. Experimental Method

2.2.1. Methods to Break Bud Dormancy under Different Low-Temperature Treatments

Eight-year-old potted ‘Gulihong’ plants were subjected to different artificial low-temperature induction treatments. Prior to the experiment, apical dominance was removed, and 1500 mg/L paclobutrazol was sprayed to promote flower bud differentiation. After observing that the internal differentiation of the flower buds was basically completed under a microscope, the experimental materials were gradually cooled down. On 6 September, the samples were placed in two constant temperature cold storages set at 6 °C and 10 °C, respectively, to induce an artificial low-temperature induction treatment and promote buds into deep dormancy. The experiment was conducted with a light intensity of 2000 Lx and a photoperiod of 8/16 h (light/dark), simulating the average autumn light conditions at this latitude. The relative air humidity in the experimental environment was maintained at 80% through periodic watering of the ground. Starting from 17 September, three pots were selected from each cold storage every five days. After routine cultivation in the greenhouse for 30 days, the bud sprouting rate was observed under different low-temperature treatments, and the phenology and flowering conditions were recorded.

Yang and Li (2013) indicated that the Utah model was the most suitable for estimating the chilling requirements of P. mume cultivars [17]. Therefore, the two temperatures in this study were selected based on the Utah model, and 6 °C and 10 °C were respectively located in the two temperature ranges in the model, representing the effects of different temperatures on low-temperature accumulation. In other temperature ranges, a too-high temperature will offset the accumulation of low temperature, and a too-low temperature will cause freezing damage to plants.

2.2.2. Experimental Methods for the Effect of Low-Temperature Accumulation on Flowering Traits

In this experiment, potted plants of the four early-flowering cultivars of P. mume, ‘Gulihong’, ‘Nanjing gongfen’, ‘Zaoyudie’, and ‘Zaohualve’, all aged eight years old, were used. The control group of plants was placed outdoors under natural light and temperature during the experimental period. The low-temperature refrigeration environment in the cold storage was the same as that in 1.2.1. Starting from 12 September, three pots were taken out from the cold storage every five days, and the frequency of removal was continued every five days after the sprouting rate stabilized at 50%. The plants were then moved into the greenhouse for regular management, such as watering and fertilization.

2.3. Indicators Measurement

2.3.1. Flowering Rate

After being removed from the cold storage, the plants of each cultivar were observed daily at around 8 am. Five main stems were selected for each plant, each containing approximately three flower branches (about 150 flower buds). The buds were considered to have sprouted when the top of the flower bud cracked and began to show red. Observations began 30 days after sampling, and the bud sprouting rate of each cultivar was recorded and calculated daily.

2.3.2. Flower Diameter

Ten flowers from each of the different P. mume cultivars were selected and measured three times using a caliper with the cross method to determine the diameter at full bloom (precise to 0.01 cm). The flower diameter and other morphological data were recorded.

2.3.3. Bud Morphology

Four robust and uniform cultivars in a 6 °C cold storage were selected, with three plants per cultivar. Ten plump buds from the upper part of a one-year-old branch were marked for each plant, with three replicates taken from three different plants. Growth parameters were measured at 0 d (the day before the start of low-temperature induction) and every 10 d after treatment. The horizontal (W) and vertical (L) diameters of each bud were measured with an electronic calliper (to the nearest 0.001 cm) to observe changes in bud morphology during low-temperature release from dormancy.

2.4. Statistical Analysis

All experiments were performed in triplicates. The experimental data, such as mean and standard error, were processed and plotted using Microsoft Excel (Microsoft, Redmond, WA, USA), and the One-way ANOVA and Duncan’s multiple range test were tested for significance and errors using SPSS Statistics Version 26.0 (IBM Corporation, Armonk, NY, USA).

3. Results

3.1. Effect of Different Low-Temperature Treatments on Flowering Characteristics

3.1.1. Effect of Different Low-Temperature Treatments on Flowering Rate

An experiment was conducted to investigate the effect of different low-temperature treatments on the flowering characteristics of ‘Gulihong’ plants. The plants were subjected to 6 °C and 10 °C treatments starting from 12 September and were moved into a greenhouse for regular cultivation every five days until 12 October. The flowering rates of the plants were recorded once a certain chilling requirement was met, as shown in

Table 1. Effects of low-temperature treatments at 6 °C and 10 °C on the flowering rate of P. mume ‘Gulihong’.

Temperature Treatments (°C)	Flowering Rate (%)
	Exit Date
	17 September 2021	22 September 2021	27 September 2021	2 October 2021	7 October 2021	12 October 2021
6	9.52	30.69	75.36	86.79	88.48	92.00
10	1.94	11.18	36.13	42.27	46.62	60.07

Note: Exit date means the date when the experimental plants were taken out of the cold storage after different low-temperature treatments.

. Under natural conditions, the control group did not show any signs of bud break. Under the 6 °C treatment, initial flowering was limited, with a chilling accumulation of 252 CU required for plants that were moved out on 17 September. However, the flowering rate exceeded 75% when the chilling accumulation reached 492 CU on 27 September. In comparison, under the 10 °C treatment, plants moved into the greenhouse on 12 September did not show any signs of bud break initially. On 17 September, one to several flowers bloomed prematurely, with a chilling accumulation of 126 CU, and the flowering rate increased gradually as the cold storage time extended. On 12 October, the flowering rate reached 60.07% when the chilling accumulation reached 426 CU. Overall, both low-temperature treatments were able to break bud dormancy, and the flower buds were able to grow and bloom normally. The flowering rate of the plants under both treatments increased gradually with the accumulation of chilling units, but the flowering rate of the 6 °C treatment was significantly higher than that of the 10 °C treatment.

3.1.2. Effects of Different Low-Temperature Treatments on the Flower Diameter

The effects of different low-temperature treatments on the flower diameter of ‘Gulihong’ are shown in

Table 2. Effects of different low-temperature treatments on the diameter of P.mume ‘Gulihong’.

Temperature Treatments (°C)	Flower Diameter (cm)
	Exit Date
	17 September 2021	22 September 2021	27 September 2021	2 October 2021	7 October 2021	12 October 2021
6	2.15 ± 0.12 a	2.13 ± 0.20 a	2.30 ± 0.34 a	2.51 ± 0.15 a	2.53 ± 0.26 a	2.74 ± 0.20 a
10	1.91 ± 0.22 b	2.05 ± 0.23 b	2.12 ± 0.35 b	2.30 ± 0.25 b	2.48 ± 0.13 a	2.51 ± 0.18 b

Note: Exit date means the date when the experimental plants were taken out of the cold storage after different low-temperature treatments. Different lowercase letters in the same column indicate significant differences at the 0.05 level among different treatments (p < 0.05); data in the table are mean ± standard errors (n = 3).

. After greenhouse cultivation, it was found that the control group without cold treatment did not flower, and the flower diameter under both low-temperature treatments increased to varying degrees as the cold accumulation increased. The flower diameter under the 6 °C treatment was significantly higher than that under the 10 °C treatment. In the batch moved out on October 12, the flower diameter under both the 6 °C and 10 °C treatments reached the maximum value in the corresponding treatment group, with values of 2.74 cm and 2.51 cm, respectively, which significantly increased by 27.44% and 31.41% compared with the batch moved out on September 17. On October 12, the flower diameter under the 6 °C treatment increased by 9.16% compared with that under the 10 °C treatment. After greenhouse cultivation, it was found that the flowers under the 10 °C treatment were generally sparse, irregular, small, and lightly fragrant, with poor flowering quality, slow development, and occurrence of flower drop and abortion, resulting in the low ornamental value of the plants. It could be seen that the 6 °C treatment had a significantly better effect than the 10 °C treatment in improving the flowering quality in both treatments.

3.1.3. Effects of Different Low-Temperature Treatments on the Flowering Quality

The effects of different low-temperature treatments on the flowering quality of ‘Gulihong’ are shown in Figure 1. After cultivation in the greenhouse, it was found that the overall flowering of the flowers treated at 10 °C was sparse and irregular, the flowers were small and fragrant, the flowering quality was not high, the growth was slow, there were cases of bud drop and abortion, and the ornamental value of the plants was not high. It could be seen that among the two treatments of 6 °C and 10 °C, the effect of 6 °C on improving flowering quality was significantly higher than that of 10 °C.

3.2. Effects of Different Low-Temperature Accumulations on Flowering Characteristics

3.2.1. Morphological Changes in Flower Buds under Different Low-Temperature Accumulations

Table 3. Morphological changes in flower buds of different P. mume cultivars during low-temperature-induced dormancy.

Exit Date	‘Gulihong’		‘Nanjing gongfen’		‘Zaoyudie’		‘Zaohualve’
	HD	VD	HD	VD	HD	VD	HD	VD
04/09/2021	1.16 ± 0.13 d	1.72 ± 0.21 e	1.07 ± 0.13 c	1.56 ± 0.21 d	1.00 ± 0.12 d	1.49 ± 0.21 d	1.13 ± 0.22 d	1.60 ± 0.36 d
14/09/2021	1.16 ± 0.21 d	1.97 ± 0.26 d	1.06 ± 0.11 c	1.51 ± 0.21 d	1.05 ± 0.12 d	1.49 ± 0.23 d	1.16 ± 0.23 c	1.61 ± 0.26 d
24/09/2021	1.29 ± 0.10 c	2.15 ± 0.19 c	1.12 ± 0.17 b	1.63 ± 0.27 c	1.13 ± 0.12 c	1.80 ± 0.25 c	1.17 ± 0.16 c	1.85 ± 0.25 c
08/10/2021	1.39 ± 0.15 b	2.52 ± 0.18 b	1.16 ± 0.14 b	1.88 ± 0.26 b	1.24 ± 0.12 b	2.25 ± 0.24 b	1.32 ± 0.15 b	2.51 ± 0.24 b
16/10/2021	1.60 ± 0.11 a	2.98 ± 0.14 a	1.45 ± 0.16 a	2.52 ± 0.29 a	1.45 ± 0.10 a	2.78 ± 0.27 a	1.51 ± 0.01 a	2.65 ± 0.18 a

Note: Exit date means the date when the experimental plants were taken out of the cold storage after different low-temperature treatments. Different lowercase letters in the same column indicate significant differences at the 0.05 level among different treatments (p < 0.05); data in the table are mean ± standard errors (n = 3); HD represents horizontal diameter (mm); VD represents vertical diameter (mm).

shows the morphological changes in flower buds during the cold storage dormancy period. During the low-temperature induction to break the P. mume dormancy, the morphology of the flower buds also undergoes significant changes. The horizontal and vertical diameters of the flower buds increase with the continuous accumulation of low temperature. During the dormancy period, the rate of morphological changes in flower buds in each cultivar is slow. After dormancy is released, the vertical diameter of the flower buds increases significantly. The horizontal diameter of ‘Gulihong’, ‘Nanjing gongfen’, ‘Zaoyudie’, and ‘Zaohualve’ increased by 30.24%, 35.40%, 44.59%, and 41.59%, respectively, compared to before low-temperature treatment. The vertical diameter also increased by 54.38%, 61.38%, 86.81%, and 86.09%, respectively. The horizontal diameter of ‘Gulihong’ reached the maximum value on October 16, increasing by 10.34%, 10.34%, and 5.96% compared to ‘Nanjing gongfen’, ‘Zaoyudie’, and ‘Zaohualve’, respectively. The vertical diameter of ‘Gulihong’ also increased by 18.25%, 7.19%, and 12.45% compared to ‘Nanjing gongfen’, ‘Zaoyudie’, and ‘Zaohualve’, respectively. Observation showed that the morphology of flower buds of each cultivar changed significantly before and after 24 September, and it is speculated that this period may be a critical time for the physiological changes in flower buds, possibly related to dormancy release.

3.2.2. Effects of Different Low-Temperature Accumulations on Flowering Rate of Different P. mume Cultivars

Under the constant temperature treatment of 6 °C, varying degrees of low-temperature accumulation have an impact on the flowering rate of different P. mume cultivars. The results are shown in

Table 4. Effects of different low-temperature accumulation at 6 °C on the flowering rate of different P. mume cultivars.

Exit Date	Chilling Requirement	Flowering Rate (%)
		‘Gulihong’	‘Nanjing gongfen’	‘Zaoyudie’	‘Zaohualve’
12/09/2021	132	1.89	0	0	0
17/09/2021	252	9.52	0	20.57	0
22/09/2021	372	30.69	22.80	50.39	55.21
27/09/2021	492	75.36	63.52	78.54	60.03
02/10/2021	612	86.79	72.85	81.79	70.94
07/10/2021	732	88.48	76.53	80.91	82.62
12/10/2021	852	92.00	80.93	86.50	84.49

Note: Exit date means the date when the experimental plants were taken out of the cold storage after different low-temperature treatments.

. On 6 September 2021 at 10 a.m., different cultivars were placed in a 6 °C cold storage and then moved to the greenhouse for routine cultivation when the cold accumulation reached 132 CU, 252 CU, 372 CU, 492 CU, 612 CU, 732 CU, and 852 CU, respectively. The flowering rate was recorded and observed. Through observation, it was found that the control group without a low-temperature treatment did not bloom. Until 12 September, when the required cold accumulation was 132 CU, except for ‘Gulihong’, which showed a few early blooms, the flower buds of other P. mume cultivars did not sprout. On 17 September, when the required cold accumulation reached 252 CU, flower buds began to sprout, with a flowering rate of 9.52% for each cultivar. Until 22 September, when the cold accumulation reached 372 CU, ‘Gulihong’ and ‘Nanjing gongfen’ had a lower flowering rate, but ‘Zaoyudie’ and ‘Zaohualve’ had a flowering rate of more than 50%. Until 27 September, the flowering rate remained above 60%. In conclusion, with the increase in cold accumulation, the flowering rate gradually stabilizes at more than 80%.

3.2.3. Effects of Different Low-Temperature Accumulation on Flowering Quality of Different P. mume Cultivars

To investigate the effects of different levels of cold temperature accumulation on breaking dormancy and flowering traits, outdoor-grown plants were used as controls, and the flowering status of four cultivars was observed in a greenhouse. After conducting cultivation experiments in the greenhouse, it was observed that various cultivars exhibited similar responses to different levels of chilling accumulation. This means that the different plant varieties reacted in comparable ways to varying amounts of chilling exposure. They all showed that when the low-temperature accumulation was insufficient, the flowers were small, weakly fragrant, and even deformed with the phenomenon of single-petalled or heavily petalled flowers blooming together. In addition, the flowering quantity was low, sparse, and irregular, and the ornamental value of the plants was low. However, as the cold temperature continued to accumulate, the flower bud abortion of the four cultivars under cultivation was significantly improved, and the flowering quantity and uniformity were significantly increased, with the fragrance becoming more intense and the flowering quality significantly improved. These results showed that cold temperature accumulation played a certain promoting role in the flowering quality during cultivation. The mechanism behind this phenomenon may be related to the regulation of plant hormones, gene expression, and physiological and biochemical processes.

4. Discussion

Low temperature is the key factor affecting the release of dormancy and flowering of plants [18]. Different low-temperature treatments have effects on flowering traits such as flowering rate and flowering quality. Li et al. (2020) artificially low-temperature-treated ‘Suxin’ wintersweet based on the Utah model and the 7.2 °C model. In a low-temperature environment below 12 °C, when the cooling requirement reaches 570 CU, the wintersweet flower buds can expand and open normally in advance [12]. El-Yazal (2020) conducted a study on the low-temperature conditions of breaking dormancy for 20 apricot seedlings for three consecutive years and found that cold stratification at 5 °C had the best effect on breaking seed dormancy and germination [19]. Heide and Prestrud (2005) studied the growth and dormancy of four apple cultivars and one pear cultivar and found that at least 6 weeks (about 1000 h) of chilling at 6 or 9 °C were required for the release of dormancy and recovery of growth, while chilling at 12 °C for 14 weeks treatment is almost ineffective [10]. In this study, different low-temperature treatments of 6 °C and 10 °C were carried out on ‘Gulihong’, and it was found that the plants in the control group had no signs of germination under natural conditions. After cultivation in the greenhouse, both low-temperature treatments could break the dormancy of flower buds. The flower buds could also expand and open normally, but the flowering rate, flower diameter, flowering quantity, flowering uniformity, and bud development of the 6 °C treatment group were significantly better than the 10 °C treatment group. In conclusion, different low-temperature treatments have different effects on flowering traits. The optimal temperature conditions for breaking dormancy and promoting germination also vary depending on the plant species.

The accumulation of different low temperatures will have certain effects on flowering rate, flower diameter, flowering quality, and other flowering traits of plants. According to the findings of Du Y et al. (2019), 37 subtropical woody species were observed and showed that both moderate and strong chilling treatments advanced budburst and reduced forcing requirements. These results suggest that chilling plays a more significant role in regulating budburst for the majority of species [20]. Campoy et al. (2013) found that under different cold accumulations, the temperature efficiencies of apricot tree dormancy reliefs were different. With the accumulation of cold, the germination rate under all temperature treatments continued to increase [21]. Nie et al. (2012) aimed at the problem of earlier flowering of P. mume in Kunming and adopted a low-temperature treatment for early, middle, and late-flowering cultivars in order to delay the flowering period. Low-temperature refrigeration treatment had a significant effect on delaying the flowering period of P. mume [22]. In this study, the results of greenhouse cultivation showed that the plants in the control group without the cold temperature treatment did not bloom at all. Under the constant temperature treatment at 6 °C, the flowering rate showed an upward trend with the accumulation of cold. This is consistent with previous work by Anzanello et al. (2018) on grapes [23] and Gariglio et al. (2006) on peaches [24]. In addition, this result found that different cultivars responded similarly to different chilling accumulations after cultivation in the greenhouse. The flowering quantity and flowering uniformity of each cultivar were also significantly improved, the flower fragrance was more intense, and the flowering quality was significantly improved. When the cold accumulation is insufficient, the flowers are small, the fragrance is weak, accompanied by the phenomenon of single and double petals blooming at the same time, the flowering is sparse and irregular, the flower buds are aborted in severe cases, and flower development abnormalities such as ‘leaf-wrapped flowers’ will appear, which will affect the appearance. This is similar to the results of previous studies on pear [25] and apple [26] trees. The results showed that in the process of relieving flower bud dormancy at a low temperature, the accumulation of cold energy played a certain role in promoting flowering quality; otherwise, it would affect the growth and development disorders in the later stage, and even if the management in the later stage was perfect, they would not be able to develop normally.

5. Conclusions

The results of this research have significant implications for the cultivation of P. mume. By using an artificial low temperature to release dormancy, this study found that among the widely cultivated early-flowering cultivars in Henan Province, a treatment of 6 °C can significantly improve the cultivation effect of P. mume. In addition, as the accumulated cold amount increases, the flowering rate and quality of P. mume cultivars under different temperature treatments gradually improve. These research results provide a basis for us to develop targeted cultivation management measures for P. mume and help guide precise market control of flower production in Henan Province. In the future, we can further set up more temperature gradients to explore the optimal temperature for promoting the cultivation of P. mume and analyze the dormancy mechanism of P. mume at the molecular level of the transcriptome, metabolome, and other levels in order to further study the effect of temperature on releasing dormancy of P. mume and provide a more scientific basis for the development of P. mume cultivation technology. These research achievements are expected to promote the continuous improvement and perfection of P. mume cultivation technology and make a positive contribution to the development of the P. mume industry.