{'fraction_non_alphanumeric': 0.03422782037239869, 'fraction_numerical': 0.02610441767068273, 'mean_word_length': 5.167464114832536, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Curd Initiation and Transformation in Tropical Cauliﬂower Cultivars under Different Temperature Treatments

. This study investigated the effects of different temperature treatments (18, 24, and 30 8 C) on apex development in tropical cauliﬂower cultivars of varying maturity types. Two commercial cultivars, H-37 (early maturity) and H-80 (mid–late maturity), were used as the testing materials. ‘H-37’ reached the curd-initiation phase earlier than ‘H-80’ and showed superior growth during the curd’s initial development phase under all temperature treatments. Analysis of variance revealed signiﬁcant effects regarding main temperature and cultivar as well as their interaction. ‘H-37’ at a temperature of 18 8 C demonstrated the optimal transformation of apex development from the vegetative to reproductive stage. A temperature of 24 8 C promoted the apex development of ‘H-37’ at the curd initial development phase. Gene expression analysis results indicated that the BoFLC2 expression of ‘H-37’ was signiﬁcantly down-regulated than that of ‘H-80’ after curd initiation and advanced growth. A temperature 30 8 C accelerated the ending of juvenile stage and forward to curd initiation in ‘H-80’ and declined with temperature decreased. Moreover, expression of the BoFLC2 transcript level of both tropical cauliﬂower cultivars nearly disappeared at the high temperature of 30 8 C following curd initiation, suggesting that heat stress hinders curd formation. The results of this study also indicate that the number of leaves required to induce curd initiation is less than nine in tropical cauliﬂower

Cauliflower (Brassica oleracea var. botrytis) is a prominent commercial vegetable worldwide. The aesthetic appearance of cauliflower curd is the primary factor determining its market value. The curd is composed of undifferentiated inflorescences and an inflorescence meristem that continuously generates replicas (Smyth, 1995). Many Brassica species have chilling requirements and flowering induced by vernalization. After vernalization, cauliflower will exhibit normal curd development under suitable environmental conditions and then begin flowering (Labate et al., 2006). However, unsuitable temperatures produce malformed curds, such as buttoning curds, riceyness curds, or bolting in the small plant stage. Fern andez et al. (1997) indicated that low temperature is crucial for the apical growth of cauliflower when transitioning from the vegetative to reproductive stage. In addition, curd initia-tion and growth are related to effective temperature (Pearson et al., 1994). Therefore, retardation or lack of curd initiation is attributable to insufficiently low temperature (Liptay, 1981). The success of curd initiation is determined by temperature (Rahman et al., 2007) and maturity type (Nowbuth and Pearson, 1998). Wurr and Fellows (2000) suggested that the optimal temperature for curd initiation is lower in cauliflower types that mature in the early summer (9 to 13°C) than in those that mature in winter (13 to 14°C). One study demonstrated that climate change postpones curd initiation and that more leaves are produced when the temperature is increased by 2.9°C to reach 17°C (Wurr et al., 1996). Curd earliness is critical to marketability because less time for harvest corresponds to reduced cost of pest control and increased market value. A high correlation between maturity type and season must exist to meet these targets. The season factor obviously affects early cultivars when they were planted late in autumn-winter (Augustine et al., 1980). Cebula et al. (2005) suggested that the period before curd formation is shorter in early-maturity cultivars than in late-maturity cultivars in moderate climates. Another study reported lower temperature requirements for curd growth in late-maturity varieties (Wurr et al., 2004).
Relevant studies have revealed that curd initiation is correlated with maturity, including duration in the juvenile period and number of leaves at curd initiation (Booij and Struik, 1990;Wurr and Fellows, 2000). The number of leaves is determined at the end of the juvenile period and is also a stable characteristic (Hand and Atherton, 1987). However, curd initiation sometimes does not occur under high-temperature conditions, even with a sufficient number of leaves. Optimal temperature is a critical factor for curd initiation and development. Williams and Atherton (1990) indicated that curd initiation occurred earlier with fewer leaves at a low temperature (5°C) and that more leaves were required at a warm temperature (20°C). However, the environment changes the number of leaves that develop at curd initiation phases, and another study suggested that the apex diameter could be used as an index for the end of juvenility (Fellows et al., 1999). Curd initiation is determined through visual observation when the apex diameter reaches the initiation point at 0.6 mm (Olesen and Grevsen, 2000;Pearson et al., 1994;Rahman et al., 2013;Wurr, 1981). Tissue dissection is performed under a binocular microscope to accurately determine the amount of time required for curd initiation, with a vertical apex diameter greater than 0.04 mm indicating initiation (Fellows et al., 1999;Wurr et al., 2004). A previous study indicated that BoFLCs polymorphisms existed in Brassica genus crops. The expression of Flowering Locus C (FLC) in Brassica oleracea includes BoFLC3 in broccoli (Lin et al., 2018) and BoFLC4 in cabbage (Lin et al., 2005). However, in Chinese kale and broccoli, BoFLC5 does not contribute to flowering time because of premature stop codons (Razi et al., 2008). More reports demonstrated that BoFLC2 was associated with cauliflower crops (Matschegewski et al., 2015;Okazaki et al., 2007;Ridge et al., 2015). However, the majority of research was on curd initiation, and development generally has focused on cauliflowers with relatively low ambient temperatures. No study has investigated the effect of temperature on curd initiation and development of tropical cauliflowers. Thus, in this experiment, we investigated the influence of temperatures (18 to 30°C) by performing tissue dissection and evaluating BoFLC2 expression during the transformative period of curd initiation for two tropical cauliflower cultivars under high temperatures. In addition, we evaluated the apical changes in response to different temperature treatments of tropical cultivars. This research can provide insight into curd initiation and formation in tropical cauliflower species and serve as a reference for cauliflower production in tropical regions.

Materials and Methods
Two cauliflower F 1 cultivars, cv. H-37 and cv. H-80, obtained from Ching Long Seed Co. Ltd., Tainan City, Taiwan, were used as the testing materials. The commercial culture guide identifies 'H-37' as an earlymaturity and heat-tolerant type and 'H-80' as a mid-to late-maturity type. Seeds were sown in a 128-plug tray filled with peatmoss and grown in a greenhouse. On day 21, seedlings were transplanted to plastic pots with diameters of 9 cm containing peatmoss with 0.5 g of organic fertilizer No. 1 (5.0% N, 2.5% P 2 O 5 , 2.5% K 2 O, and 65.0% organic material) from Taiwan Fertilizer Co. Ltd. (Taipei, Taiwan). On day 26, we transferred the seedlings of two cauliflower cultivars to a growth chamber and exposed them to 18, 24, and 30°C treatments with a 16/8-h day/night photoperiod. The light intensity was 5150 Lux and relative humidity was 56% to 61% in growth chamber. The experimental design was a randomized complete block split-plot design with temperature treatment as the main plot and cultivar as the subplot.
Nine plants per cultivar were successively sampled for each temperature treatment. The first investigation began 3 d after the seedlings had been transferred to a growth chamber; we evaluated nine plants of each cultivar under different temperature treatments at 7-d intervals. In our experiment, apex and leaf samples were matched and originated from the same plant, and immature leaf is near apex. The apex was used as the dissected observation and the immature leaf used to detect BoFLC2.We performed tissue dissection to assess the apex initiation and development. It can determine whether the apex and leaf originated from the same sampling. Moreover, we sampled cauliflower apex tissue and fixed them in an FAA solution (70% ethanol, formalin, and glacial acetic acid at a volume ratio of 18:1:1). A frozen section machine was used to dissect apical tissue with a thickness of 0.45 mm. We assessed the morphology of apical tissue sections using a microscope at different sampling stages. The apical growth characters (apex elongation and diameter) were investigated during the juvenile, curd initiation, and curd initial development phases. The BoFLC2 transcription levels of immature leaf samples were detected at the same phases for which apical tissue dissections were performed. For the quantification of BoFLC2 transcript analysis, 100 mg of immature leaf samples from each treatment were collected and stored at -80°C. RNA from homogenized tissue samples was extracted using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA). Total RNA (500 ng) quantified with the Qubit RNA BR kit (Thermo Fisher Scientific) was reverse transcribed using random primers and a fast reverse-transcription kit (Topgen Biotech, Kaohsiung City, Taiwan). The following specific BoFLC2 primers were selected based on a report by Ridge et al. (2015): forward 5#-CGAAGTATGGTTCACACCAT-GAGC-3# and reverse 5#-CGGAGAGGG-CAGTCTCAAGGTGGTT-3#. In addition, we used a SYBR Green Master Mix (Topgen Biotech, TW) to amplify cDNA. Quantitative real-time polymerase chain reaction was performed on the StepOne Plus Real Time PCR System (Applied Biosystems, Foster City, CA) in accordance with manufacturer's instructions. We used SAS-EG (SAS Enterprise Guide 7.1 system; SAS Institute, Inc., Cary, NC) for data analysis. We performed mean separation using the least significant difference at P < 0.05 when significance among treatments was detected.

Results and Discussion
Apex change during curd initiation. Curd initiation was defined as an apex elongation greater than 0.04 mm (Fellows et al., 1999;Wurr et al., 2004). On the basis of this definition, we distinguished the transition of tested materials from vegetative to reproductive growth according to apex dissection measurements (Fig. 1). Curd initiation occurred on day 17 for 'H-37' under all three temperature conditions, which was significantly  earlier than 'H-80', which exhibited curd initiation at day 38 for 18 and 24°C and day 31 for 30°C ( Figs. 1 and 2). Analysis of variance on curd development revealed that temperature, cultivar, and interaction showed a significant effect on the apex at the phases of curd initiation and curd initial development. Apical growth at 18°C demonstrated significantly greater apex elongation during curd initiation than at 24 and 30°C. Both apex elongation and diameter increased more quickly at 18 and 24°C than at 30°C at both stages of curd initiation and curd initial development (Table 1). As expected, the early-maturity type 'H-37' exhibited quicker apex elongation and growth, also demonstrating that tropical cauliflower cultivars at nonvernalized temperatures (18 to 30°C) can induce curd initiation. These temperatures clearly exceeded the low-temperature requirement of cauliflower cultivars grown in temperate zones, which are characteristically early-summer maturity types and undergo optimal curd initiation at a temperature of 9 to 13°C (Wurr and Fellows, 2000). Most simulative models evaluated the temperature elevation effect in the temperate climate and indicated that the optimum temperature was 14°C for curd initiation in summer/ autumn cauliflower. When the temperature was greater than 14°C, significant reduction on the growth rate of curd diameter could be observed (Pearson et al., 1994). Leaf area and dry matter were correlated with curd diameter growth and temperature. Reports also have indicated that a mean temperature of 14.3°C showed a minimum duration less than 20 d for the requirement of curd initiation (Olesen and Grevsen, 2000). Wurr et al. (2004) indicated that juvenility at an apex diameter of 0.2 mm switched to induction and turned to curd growth after curd initiation at 0.6 mm. In the United Kingdom, these phases were shorten by increased temperatures. Uptmoor et al. (2008) used thermal time required to flowering to predict flowering time at 6 to 18°C conditions. It indicated that curd initiation was accelerated under noninducing temperatures. The aforementioned studies provided the best direction for the cultivation and breeding for vernalizated induction condition. However, global warming and climate change could severely affect tropical cauliflower production. In our study, the temperature requirements for the curd initiation and development phases of tropical cultivars differed significantly from those of cultivars in temperate zones. Table 2 presents the effect of temperature and cultivar response interactions on apex growth in different phases. A cooler temperature (18°C) promoted faster growth in early-maturity cultivars (both in terms of apex elongation and diameter) when the apex transitioned from the juvenile to curd-initiation phase. After curd initiation, 'H-37' presented the best apex elongation and diameter growth at 24°C followed by 18°C. The curd-development phase differed between early-maturity and mid-to latematurity types. Temperature treatments at 18°C and 24°C resulted in favorable apical growth. Nowbuth and Pearson (1998) suggested that no curds would initiate in tropical cauliflower cultivars at temperatures greater than 25°C and 29°C. Zenkteler et al. (2012) indicated that devernalized temperatures affected the structure of the shoot meristem during transformation from the vegetative to generative phase and delayed meristem enlargement. The research assuming apex change cause by high temperature, but in our study showed that both 'H-37' and 'H-80' can reach curd initiation at 18 to 30°C, which was beyond vernalization-regulated in cauliflowers. Expression of BoFLC2. FLC represses flowering expression and has been demonstrated to have a critical role in vernalizationinduced flowering (Okazaki et al., 2007). Ridge et al. (2015) indicated that BoFLC2 was the main controller of flowering time in Brassica oleracea and demonstrated that a decrease in BoFLC2 transcript level results in curd formation. Most relevant studies have focused on vernalization-induced flowering and the regulation of FLC expression. However, Matschegewski et al. (2015) indicated that BoFLC2 was present in temperate cauliflower species during curd induction under nonvernalized temperatures. With the exception of the vernalization regulative pathway, the autonomous pathways are preset and down-regulate the floral repressor FLC to induce flowering (Marquardt et al., 2006;Reeves and Coupland, 2000). The model and pathway are displayed in Fig. 4. In this study, tropical cauliflower cultivars could induce curd under nonvernalized temperatures, which suggested that the autonomous pathways were performed and downregulated the floral repressor FLC then induced curd development. This is different to vernalization induced in Brassica genus crops. Gan et al. (2014) illustrated that the thermosensory qualities of Arabidopsis were modified by regulating FLC at an increased temperature of 29°C. The autonomous pathways of FLC is for response to environmental change (Shea et al., 2018). This research suggests that warm temperatures affect FLC expression and that floral induction was not only caused by the vernalized effect.
In this study, BoFLC2 expression in 'H-37' demonstrated an increasing tendency before curd initiation (from day 3 to 17) under all temperature treatments. BoFLC2 expression exhibited a decreasing tendency during days 17 to 24 after curd initiation (Fig. 3A), and visual curd growth subsequently was observed. Furthermore, a sharp decrease was observed under 30°C conditions compared with 18 and 24°C, which suggested that high-temperature conditions caused a significant decrease in BoFLC2, with an abnormal minimum quantity and delayed apex development. BoFLC2 expression showed a different pattern in 'H-80' (Fig. 3B) and increased from days 17 to 31. However, BoFLC2 expression abnormal decreased at day 31 to 38 under 30°C conditions, and BoFLC2 expression continued to increase at day 38 to 45 under 18 to 24°C conditions. These results coincided with the curd growth of this cultivar. Thus, the aforementioned results suggest that temperatures (18 to 24°C) can induce curding in early- Curd initial development phase was followed by curd initiation after 1 week. y Mean separation, within the column and followed by different letters, showed significant differences according to Fisher's least significant difference test at the 5% level. NS , *, **Nonsignificant or significant at P < 0.05 or 0.01, respectively. Curd initial development phase was followed by curd initiation after 1 week. y Mean separation, within columns and followed by different letters, showed significant differences according to Fisher's least significant difference test at 5% level. maturity cultivars of tropical cauliflower but that mid-to late-maturity cultivars exhibit a different pattern under high temperatures. Irwin et al. (2016) used two Arabidopsis genotypes and detected that BoFLC2 repressed dynamics at warm conditions. The vernalized and nonvernalized plants showed different environmental sensitivity. Lin et al. (2018) described a BoFLC3 expressive tendency in subtropical broccoli, which showed a nonvernalized response. Sun et al. (2018) also indicated that high temperatures delayed curd development but did not postpone the BoFLC1 expression in cauliflower, and the gene also showed a down-regulation pattern. In our study, the BoFLC2 expressive pattern of the early-maturity cultivar could be differentiated into two sections during curd-transformation periods. Before curd initiation, the BoFLC2 level significantly increased and then BoFLC2 showed positive regulation after reach curd initiation.
The results confirmed the BoFLC2 was acted and apex was amplified at a nonvernalizated temperature. The apical morphology and molecular information strongly suggested that BoFLC2 still acted in tropical early maturity cauliflower and did not perform the vernalization pathway. Development of leaves and stem. The number of leaves developed was significantly greater in 'H-80' than in 'H-37' at all three phases of curd development (Table 3). Wurr (1981) proposed that the duration of the juvenile phase was related to the characteristics of different cultivars. In 'H-80', when comparing both cultivars at 24°C conditions, temperature treatment produced a greater number of leaves at the end of the juvenile phase and the curd-initiation phase. Conversely, 'H-37' produced fewer leaves at both phases at a temperature of 24°C (Table 4). Wurr et al. (1994) predicted that individual curd initiation occurred between 17 and 21 leaves in four summer-autumn types but also explained that leaf number was affected by the environment. The number of leaves required for curd formation increased from 25 to 43 when the temperature increased from 5 to 20°C (Williams and Atherton, 1990). In this study, despite producing fewer than nine leaves at 24°C, tropical cauliflowers were able to induce curd initiation, indicating that the number of leaves required at curd initiation is a far lesscritical factor for tropical cauliflower cultivars than it is for temperate cultivars. This result also suggested that the leaf number required for curd induction varies depending on the temperature and type of tropical cultivar.
In the comparison of cultivars, analysis of variance revealed that the stem diameter of 'H-80' was significantly longer than that of 'H-37' at the three phases. Cebula et al. (2005) indicated that late-maturity types of cauliflower grew thicker stems than early-maturity types. Temperature at 24°C advanced stem growth and longer than 18°C and 30°C. Guo et al. (2004) indicated that stem diameter was associated with vernalization, which suggested that a temperature of 24°C was perhaps not an effective vernalization temperature for mid-to latematurity types because more stem growth substituted for apex growth. When both cultivars were subjected to high-temperature conditions (30°C), apex elongation underwent significant retardation and was accompanied by the continuous growth of stem and leaves. These results also revealed the coexistence of vegetative and reproductive growth during the transformative period of curd initiation, suggesting that stem diameter could not be used as a screening index for curd initiation.
This study provides information concerning apical change in tropical environments. Observation of apex elongation before and after the curd-initiation phase produced an advanced index. At the end of the juvenile phase, the early-maturity cultivar was more responsive to the cooler temperature of  18°C, which accelerated the transition from vegetative stage to reproductive stage. However, a greater temperature contributed to shortened juvenile periods for mid-to late-maturity cauliflower but delayed curd development. The curd-initiation phase was a dividing phase for both aforementioned cultivars, and a suitable temperature was advantageous for converting apex to curd development. Curd initial development phase was followed by curd initiation after 1 week. y Mean separation, within each column and followed by different letters, showed significant differences according to Fisher's least significant difference test at the 5% level. NS , *, **Nonsignificant or significant at P < 0.05 or 0.01, respectively.