2018 Volume 87 Issue 3 Pages 413-420
This study was initiated to investigate the effects of wet treatment at 10°C (WT10°C) and subsequent re-drying treatment (RDT) on the germination and growth of Eustoma grandiflorum seeds. Both treatments, WT10°C and RDT, were applied at 10°C under dark conditions. In all the experiments, ‘King of Snow’, which is one of the major Eustoma cultivars in Japan, was used. When the total number of days exposed to WT10°C and RDT was set to 35 days, the prevention of rosettes and the promotion of growth were dependent on the number of days of WT10°C, and these effects were maintained after RDT. When RDT was applied to the seeds after WT10°C for 35 days, the growth promotion due to WT10°C was maintained regardless of the length of re-drying time from 7 to 28 days; however, the germination rate decreased with RDT. On the other hand, applying RDT after WT10°C for 14 days had no effect on the germination rate; however, the growth promotion was insufficient compared with WT10°C for 28 days. This problem was solved by carrying out a further 14 days of WT10°C after RDT. When the seeds were exposed to WT10°C for 14 days, then re-dried for 7 days, and exposed to WT10°C for a further 14 days, the same growth promotion was achieved as for seeds exposed to WT10°C for 28 days continuously. The above results suggest that the promotion of growth depends on the total number of days of WT10°C regardless of whether these days are continuous or not. Moreover, the growth promotion was maintained after RDT. The results also suggest the possibility that when both the processes of WT10°C and the RDT are carried out by seed companies, growers who have no cooling equipment can produce cut flowers using seedlings grown in the high-temperature season.
In Japan, Eustoma grandiflorum is one of the major cut flowers and it is horticulturally treated as an annual plant. In the warm regions of Japan, the seeds are sown from June to September in order to flower from October to April in a heated greenhouse. However, the high temperatures from June to September inhibit the bolting of almost all seedlings (Azuma and Inubushi, 1988; Harbaugh et al., 1992; Ohkawa et al., 1991; Sato et al., 2004). This problem is considered to be the cause of the decrease in cut-flower production from fall to spring. To overcome this serious problem, various methods have been developed. One of them is the wet treatment of seeds at low temperature, which is reported to promote bolting of Eustoma seedlings (Kageyama et al., 1990; Ohkawa et al., 1993; Pergola et al., 1992; Tanigawa et al., 2002). Unfortunately, this treatment is limited to growers who have large refrigerators. We think that if seed companies can conduct the wet treatment at low-temperature in advance, many growers would be able to ship cut flowers from fall to spring. To achieve this goal, the seeds need to be re-dried after the wet treatment at low temperature. Also, these treated seeds must exhibit higher bolting rates and greater promotion of growth than non-treated seeds in the high-temperature season. In priming seeds for the purpose of promoting germination, it has been reported that the improvement in germination is maintained after re-drying. In spinach, the germination rate of primed seeds stored for 10 months in a desiccator with silica gel at room temperature is greater than non-treated seeds (Masuda et al., 2005). We considered that if Eustoma grandiflorum seeds were re-dried after a wet treatment at 10°C, it would be utilized more and more by growers. Therefore, we investigated the effect of re-drying, both during and after the wet treatment of seeds at low temperature, on the growth and flowering of Eustoma grandiflorum.
In all the experiments, Eustoma ‘King of Snow’ (SAKATA SEED Corporation, Japan) was used. The seeds were soaked in 100 mL of tap water inside a glass bottle in Experiment 1. In Experiments 2 and 3, the seeds were placed on two sheets of filter paper moistened with about 5 mL of distilled water in 9-cm petri dishes. These were stored at 10°C under dark conditions (WT10°C). After each WT10°C, the seeds were moved onto two sheets of dry filter paper in 9-cm petri dishes. Then, these were re-dried (RDT) at 10°C under dark conditions in the same chamber in which the low-temperature wet treatment was performed.
In order to test the germination, some of the seeds were sown on two sheets of filter paper moistened with about 5 mL of distilled water in a 9-cm petri dish. The germinated seeds were discarded after the germination test. The remaining seeds were sown in 288-cell trays (the volume of each cell is 8.2 mL) filled with a substrate (Metro-Mix 350; HYPONeX JAPAN Corporation, LTD, Japan). The cell trays were moved into a glasshouse where the minimum temperature was maintained at more than 25°C (Experiment 1) or at ambient temperature (Experiments 2 and 3). Irrigation during the raising of the seedlings was done by mist spraying.
The seedlings derived from the non-treated and treated seeds were transplanted into a greenhouse. The planting density was 30 seedlings·m−2. Preplanting fertilizer (N:P2O5:K2O = 7:6:6, Iihana Tsukuro 766; Hiroshima Prefectural Fertilizer Product Industry Corporation, Japan) was applied at a covering of 150 g·m−2. After transplanting, photoperiods of 18 h (Experiment 1) or 20 h (Experiments 2 and 3) by incandescent light bulbs (K-RD100V75W; Panasonic Corporation, Japan) were applied. The minimum temperatures were maintained at more than 15°C (Experiment 1) or 10°C (Experiments 2 and 3) by heating.
The plants were defined as having germinated when the cotyledon opened. They were also defined respectively as bolting, flower budding, and flowering when the internode was longer than 5 mm, when the main stem had a flower bud over 5 mm long, and when more than 4 flowers had bloomed on the plant.
The rates of germination, bolting, flower budding, and flowering were measured. These values were analyzed after arc-sine transformation.
Experiment 1: Effect of a combination of WT10°C and RDT on growth after plantingThe seeds were exposed to WT10°C for 0, 5, 10, 15, 20, 25, 30, and 35 days, after which RDT was applied. The total number of days exposed to WT10°C and RDT was set to 35 days.
All the seeds were sown on September 14, 2005. The seedlings were transplanted to a greenhouse on October 14. The formation of rosettes and the growth after transplanting were investigated, and the experiment was discontinued 37 weeks after planting. Seedlings that did not bolt in the 60 days after transplantation were defined as rosettes. Each treatment consisted of 30 plants with three replications.
Experiment 2: Effect of RDT after WT10°C on germination and growthThe seeds were exposed to WT10°C for 35 days, and then re-dried for 0 (non-RDT), 7, 14, and 28 days, respectively. Some seeds were not exposed to WT10°C (non-WT10°C). In order to sow all the seeds on September 5, 2011, the start dates of WT10°C were varied depending on the number of days of RDT.
Some of the seeds were used in order to investigate the germination rate 15 days after sowing. Each treatment consisted of 100 seeds with two replications. The remaining seeds were sown in the 288-cell trays filled with substrate, and they were moved into a glasshouse to raise the seedlings. The seedlings were transplanted into a greenhouse on October 17. The leaf blade lengths of these seedlings, both the first and second true leaf, were investigated when transplanting. There were 10 plants in three replications in each treatment. After transplanting, the dates when bolting, flower budding, and flowering occurred were recorded. Each treatment consisted of 30 plants with three replications. This experiment was discontinued 33 weeks after transplanting.
Experiment 3: Effect of dividing WT10°C into parts on germination and growthIn this experiment, the following four treatments were compared. The first treatment was a control in which neither WT10°C nor RDT were applied; the second treatment was 28D in which WT10°C was applied for 28 days with no RDT; the third treatment was 14D in which WT10°C was applied for 14 days and then RDT for the next 21 days; the final treatment was 14D×2 in which WT10°C was applied for 14 days then RDT for the next 7 days, and WT10°C for the final 14 days.
The seeds were sown on September 12, 2011. Some of the seeds were used to investigate the germination rate in the first 15 days after sowing. Each treatment consisted of 100 seeds with two replications. The remaining seeds were sown in the 288-cell trays filled with substrate, and then they were moved into a glasshouse to raise the seedlings. The seedlings were transplanted into a greenhouse on October 24. The dates when bolting, flower budding, and flowering occurred were recorded. Each treatment consisted of 30 plants with three replications. This experiment was discontinued 35 weeks after planting.
The maximum temperatures changed within a range of 26 to 38°C for 30 days after sowing in Experiment 1, and the average maximum temperature was 32.2°C. Similarly, the minimum temperatures changed within a range of 23 to 27°C, and the average value was 24.8°C (Fig. 1).
Changes of maximum (●) and minimum (○) temperatures for 30 days from sowing to transplanting in Experiment 1.
The rosette rates of the 60 days after transplantation for WT10°C for 0 and 5 days were 92.2% and 91.0%, respectively (Fig. 2). However, the rosette rates for WT10°C longer than 15 days were less than those for 0 or 5 days.
Effect of a combination of WT10°C and RDT of seeds on rosette rate in Eustoma ‘King of Snow’. WT10°C and RDT are abbreviations for wet treatment at 10°C and re-drying treatment, respectively. Rosette rate indicates the rate of plants not bolted at 60 days after transplanting. Values are the mean of three replicates. Different letters indicate a significant difference at the 5% level by Tukey’s HSD test after arc-sine transformation.
Most transplanted seedlings bolted and bloomed within the experimental period (Table 1). Compared with WT10°C for 0 days (non-WT10°C), bolting and flower budding were quicker for seeds exposed to WT10°C for more than 10 days; moreover, the flowering date was earlier with WT10°C for more than 15 days. Bolting and flower budding tended to be quicker as the number of days of WT10°C increased. The number of nodes at bolting and flower budding decreased significantly with WT10°C for more than 10 or 15 days, in comparison with non-WT10°C. These numbers decreased as the number of days of WT10°C increased and was smallest with WT10°C for 30 days.
Effect of a combination of WT10°C and RDT of seeds on the bolting, flower budding, and flowering in Eustoma ‘King of Snow’.
The maximum temperatures changed within a range of 22 to 39°C for 42 days after sowing in Experiment 2, and the average maximum temperature was 32.4°C. Similarly, the minimum temperatures changed within a range of 14 to 28°C, and the average value was 19.1°C (Fig. 3).
Changes of maximum (●) and minimum (○) temperatures for 42 days from sowing to transplanting in Experiment 2.
The germination rate of seeds with non-WT10°C and RDT for 0 days (non-RDT) were 90.5% and 89.0%, respectively, 15 days after sowing (Fig. 4). There is no significant difference between these germination rates; however, the germination rates with RDT for 7, 14, and 28 days were 74.5%, 68.5%, and 70.5%, respectively. These germination rates are significantly less than those for non-RDT.
Effect of RDT after WT10°C for 35 days on germination rate 15 days after sowing in Eustoma ‘King of Snow’. WT10°C and RDT are abbreviations for wet treatment at 10°C and re-drying treatment, respectively. Values are the means of two replications. ns and ** indicate a non-significant difference and significant differences at the 1% level compared with RDT 0 day by t-test after arc-sine transformation, respectively.
The leaf blade lengths of the first true leaf were not significantly different among the treatments (Table 2). However, the leaf blade length of the second true leaf for 0 days RDT was significantly longer than those receiving other treatments. The leaf blade lengths of the second true leaf with 7, 14, and 28 days RDT were equal to or greater than that for the non-WT10°C seeds.
Effect of RDT after WT10°C for 35 days on length of true leaf blade of transplanting seedlings in Eustoma ‘King of Snow’.
No rosette plants were found for all the treatment conditions in Experiment 2, and most seedlings bolted and bloomed during the experimental period.
The bolting times with RDT for 7, 14, and 28 days were the same or longer than for seeds with no RDT; however, all these times were significantly quicker than that for seeds with no WT10°C (Table 3). The node numbers at bolting and at flower budding, and the flower budding times for RDT of 0, 7, 14, and 28 days were significantly less than those for non-WT10°C, and there were no significant differences among these four treatments. The flowering times with RDT for 0, 7, 14, and 28 days were faster than or equal to that of the non-WT10°C seeds, and again there were no significant differences among these four treatments.
Effect of RDT after WT10°C for 35 days on bolting, flower budding, and flowering in Eustoma ‘King of Snow’.
The maximum temperatures changed within a range of 22 to 39°C for 42 days after sowing in Experiment 3, and the average maximum temperature was 32.0°C. Similarly, the minimum temperatures changed within a range of 12 to 25°C, and the average value was 18.3°C (Fig. 5).
Changes of maximum (●) and minimum (○) temperatures for 42 days from sowing to transplanting in Experiment 3.
The germination rate in the early days of 28D was significantly faster than those for the other three treatments (Fig. 6). The germination rate 10 days after sowing for 14D×2 was the same as for 28D, being significantly greater than those for the control and 14D. The germination rate for 14D was significantly greater than that for the control 10 days after sowing; however, this difference became less significant on the days after this. The final germination rates for the control, 28D, 14D, and 14D×2 were 83.0%, 94.0%, 87.5%, and 91.0%, respectively. Thus, there were no significant differences among these four treatments.
Cumulative germination rate of Eustoma cultivar ‘King of Snow’ seeds at ambient temperatures in a glasshouse. ● is control in which neither WT10°C nor RDT were applied; ○ is 28D in which WT10°C was applied for 28 days with no RDT; □ is 14D in which WT10°C was applied for 14 days and then RDT for the next 21 days; ▲ is 14D×2 in which WT10°C was applied for 14 days then RDT for the next 7 days, and WT10°C for the final 14 days. WT10°C and RDT are abbreviations for wet treatment at 10°C and re-drying treatment, respectively. Values are the means of 2 replications. ns, *, and ** indicate a non-significant difference and significant differences at the 5% or 1% level compared with 28D by t-test after arc-sine transformation.
No rosette plants were found for all the treatments in Experiment 3, and most seedlings bolted and bloomed during the experimental period.
The bolting date for the control was significantly later than those for the other three treatments, in which WT10°C was applied (Table 4). The bolting date for 14D was significantly later than those for 28D and 14D×2; however, the bolting date for 14D×2 was the same as that for 28D. The number of nodes at bolting was about four regardless of treatment. The number of nodes at flower budding was not significantly different among the four treatments. The flower budding and flowering dates for 28D and 14D×2 were significantly earlier than those for the control. The flower budding and flowering dates for 14D were between those for the control and 28D, there were no significant differences for either the control or 28D.
Effect of dividing WT10°C into parts on growth in Eustoma ‘King of Snow’.
This study was conducted to investigate whether promoting the growth of Eustoma seeds by a wet treatment at 10°C (WT10°C) could be maintained even after re-drying, and whether the same growth promotion could be achieved by applying WT10°C in parts rather than continuously.
According to Tanigawa et al. (2002), the bolting rate of plants transplanted in July 28, 1998, was 100% when WT10°C was applied for 35 days. However, when WT10°C was applied for 21 or 49 days, the bolting rates decreased to 87.5% and 68.8%, respectively. This suggests that the number of days of WT10°C is the most important factor in promoting bolting. In Experiment 1, the number of days of WT10°C before RDT affected the rosette rate. The rosette rate for WT10°C of more than 15 days was significantly less than WT10°C for 0 and 5 days (Fig. 2). In this study, the seedlings that did not bolt within 60 days after transplanting are defined as rosettes. However, most rosettes bolted and then bloomed during the experimental period. According to Ohkawa et al. (1991), from the time of sowing until two pairs of true leaves had expanded, high temperatures above 25°C induced rosettes, and exposing the germinating seedlings to high temperatures for more than 14 days completely inhibited bolting in ‘Fukushihai’. Moreover, Ohkawa et al. (1994) reported that, in those seedlings with two pairs of true leaves, the rosettes induced by high temperatures were broken by exposure to temperatures from 5 to 20°C and that the most effective temperature regime to break rosettes was 15°C for 28 days. These reports suggest the possibility that, in Experiment 1, the high temperatures applied while raising the seedlings induced the formation of rosettes, and that the rosettes were then broken by the low temperatures applied during the experiment. In Experiments 2 and 3, no rosette plants were found in no-WT10°C. This is considered to be because the night temperatures after sowing were low in Experiments 2 and 3 compared with Experiment 1 (Figs. 1, 3, and 5).
Fukushima et al. (2017) reported on the effects of several WT10°C methods on the growth of Eustoma. They investigated the effect of WT10°C before sowing and after sowing on the growth, as well as the effect of wetting or watering during WT10°C. As a result, the authors pointed out that when WT10°C was applied for 35 days, the different WT10°C methods had no effect on the growth and cut-flower characteristics. However, the effect on the growth of different seed moisture conditions derived from several different WT10°C methods was not clarified. Table 1 shows that the effect of combining WT10°C with RDT on bolting, flower budding, and flowering. WT10°C applied for more than 10 days promoted bolting and flower budding, and reduced the number of nodes at bolting compared with WT10°C applied for 0 and 5 days. Moreover, with WT10°C for more than 15 days the number of nodes at flower budding was less than those in which WT10°C was applied for 0 and 5 days. These results indicate that WT10°C for less than 10 days is an insufficient chilling period to promote growth. It is considered that when the seeds were re-dried at 10°C during WT10°C, this did not count as low-temperature exposure. Therefore, we believe that if growers want stable cut-flower production of Eustoma by treating seeds with WT10°C, they must avoid re-drying during WT10°C in order to have a sufficiently long chilling period.
An interesting point in Experiment 1, is that the promotion of growth by WT10°C was maintained even after RDT, in dependent of the number of days of WT10°C (Table 1). However, in this experiment, the effects of WT10°C and RDT on growth were unable to be completely separated due to the different number of days WT10°C was applied. For this reason, in Experiment 2, the effect of RDT on germination and growth after WT10°C for 35 days was investigated. As shown in Table 3, for the four treatments with different numbers of re-drying days after WT10°C, bolting and flower budding were significantly faster than without WT10°C. Moreover, the growth after RDT for 28 days was not significantly different to that without RDT (non-RDT). Therefore, it was considered that the promotion of growth by WT10°C was maintained regardless of the number of days of RDT.
On the other hand, RDT decreases the germination rate significantly compared with non-RDT (Fig. 4). This decrease in the germination rate was also observed in Experiment 1 (data not shown). Tanigawa et al. (2002) pointed out that WT10°C of seeds accelerated germination. The decrease in the germination rate after RDT may be caused by seeds in which radicle extension has started during WT10°C for 35 days. The second true leaf lengths for seedlings derived from re-dried seeds were equal to or greater than non-WT10°C, but significantly smaller than non-RDT (Table 2). We assumed that this was due to the delay in germination by RDT after WT10°C for 35 days. RDT of seeds after WT10°C for 35 days caused two problems; a decrease in the final germination rate and germination delay.
In Experiment 3, we attempted to solve these problems by applying WT10°C in two parts. The germination rate 15 days after sowing was equivalent regardless of the treatment; however, the start of germination was earliest for WT10°C applied continuously for 28 days (28D). The start of germination for seeds exposed to WT10°C for 14 days (14D) was roughly equivalent to the control (non-WT10°C); however, that for seeds exposed in two parts (14D×2) was roughly the same as for 28D (Fig. 6).
The priming technique has been used for improving seed germination when the environmental conditions are unfavorable. The number of days for priming for some plants is shorter than the number of days of WT10°C for Eustoma seeds. The numbers of days of priming for onion seeds (Dearman et al., 1986), spinach seeds (Masuda et al., 2005), and parsley seeds (Dursun and Ekinci, 2010) were 10, 7, and 2 days, respectively. On the other hand, in Eustoma, the effective number of days of WT10°C applied to seeds to promote growth was 35 days under dark conditions (Tanigawa et al., 2002). It seems that the difference in duration of WT10°C before RDT may affect the germination rate after RDT. In fact, the germination rate decreased when RDT was applied after WT10°C for 35 days in Experiment 2 (Fig. 4). However, the final germination rate did not decrease when RDT was applied after WT10°C for 14 days in Experiment 3 (Fig. 3). Hence, we believe that the number of days of WT10°C before RDT is an important factor to consider in order to prevent a decrease in the germination rate.
In Experiment 3, the seeds of the control and 14D were in a dry state and the seeds of 28D and 14D×2 were in a wet state before sowing. As a result, germination of the control and 14D was slow compared with 28D and 14D×2 (Fig. 6). This suggests that the water absorption state of the seeds at sowing is important in order to accelerate the initiation of germination. Therefore, it seems that applying RDT after WT10°C inevitably delays germination compared with non-RDT.
Table 4 shows the effect of dividing WT10°C into two parts on the growth of Eustoma. Compared with the control (non-WT10°C), applying RDT for 21 days after WT10°C for 14 days (14D) promoted growth. This is supported by the result in Experiment 2 showing that the promotion of growth by WT10°C was maintained after RDT. However, the promotion of growth by 14D was less than by 28D, for which no RDT was applied after WT10°C. This reaction of Eustoma to 14D is considered to be due to insufficient exposure to low temperatures as confirmed in Experiment 1.
Ohkawa et al. (1993) pointed out that, in Eustoma grandiflorum, if seeds were matured at 23/18°C (day/night) temperature, the formation of rosettes decreased compared with seeds matured at 33/28°C. In addition, they pointed out the possibility of completely preventing the formation of rosettes irrespective of the cultivar by combining low-temperature ripening of immature seeds with wet treatment of the seeds at low-temperature. Imamura and Suto (2002) studied the effects of low temperatures on detached immature capsules and imbibed seeds on the formation of rosettes in Eustoma. Compared with mature seeds on the mother plants, the bolting rate increased by ripening immature capsules under artificial low-temperature conditions (2 months at 11.5 ± 1.5°C). Moreover, the bolting rate of mature seeds under artificial low-temperature conditions was further improved by wet treatment of the seeds at low temperature (5 weeks at 11.5 ± 1.5°C). The results of their experiment suggest that dividing the low-temperature treatment acts to promote growth in Eustoma.
For 14D×2 in Experiment 3, the seeds were exposed to WT10°C for 14 days, then re-dried for 7 days, and exposed, finally, to WT10°C for a further 14 days. The growth promotion following this treatment was similar to that for 28D in which WT10°C was applied continuously for 28 days. This result suggests that even for purchased seeds, WT10°C has the same effect on promoting growth irrespective of whether it is applied continuously or in parts.
In conclusion, the effect on the growth promotion of seeds by WT10°C depends on the total number of days of exposure to low-temperature, and the promotion of growth was maintained even if seeds were re-dried after WT10°C. However, RDT after WT10°C for 35 days decreased the germination rate. The growth promotion achieved by 14D×2 was equivalent to the case where WT10°C was applied continuously for 28 days. Moreover, the germination rate was unaffected by applying 14D×2. For these reasons, when treating Eustoma with WT10°C, we believe it is better to divide the WT10°C treatment, which maintains the germination rate and growth promotion.
Further study is needed on the storage method, the duration after RDT, and on different cultivars. It is hoped that the re-drying treatment of seeds after the wet treatment at 10°C may contribute to stable cut-flower production using seedlings grown in the high-temperature season.
