{'fraction_non_alphanumeric': 0.03677992123356251, 'fraction_numerical': 0.024297443428342568, 'mean_word_length': 4.854630715123095, 'pattern_counts': {'":': 0, '<': 0, '<?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}}
Characterization of Male Reproductive Organs in Durian; Anther Dehiscence and Pollen Longevity

We characterized the pollen germination and longevity in durian (Durio zibethinus Murr.) under in vitro conditions and observed the morphological changes during anther dehiscence. Approximately 10 anthers aggregate on a filament; these merge with others at the base to form five phalanges in the flower. Anther dehiscence occurred at 19:00, at the same time as floral anthesis. Observation with a low-vacuum scanning electron microscope revealed pollen grains clinging to the anther even after the anther had dehisced. In vitro pollen germination was highest at a sucrose concentration of 10%. In the test of pollen germination on Brewbaker and Kwack (BK) medium, the germination rate was highest (59.1%). The germination rate was decreased (5.0% to 46.0%) when any of the minerals of B, Ca, Mg, and K was removed. The pollen germination rate at anthesis was 40.4 to 50.6% in four cultivars (‘Mon Thong’, ‘Chanee’, ‘Kradum Thong’, and ‘Phaung Manee’), and the rate was maintained at more than 40% until 24 h after anthesis and then decreased gradually. When pollen was stored under desiccating conditions with silica gel, pollen following 120-h storage could germinate after rehydration. Rehydration for 12 h achieved the best results, with 47.0% germination occurring after this rehydration period.


Introduction
The reproductive biology of durian (Durio zibethinus Murr.) is relatively unique. Durian trees are large, bearing numerous cauliflorous-flowers that form clusters. They set fruits even on branches at a higher position. The durian flower begins to open in the early evening and is fully open at night. Subsequently, all floral organs except the gynoecium abscise by the following morning (Honsho et al., 2004b). Because the flowers open at night, herbivorous giant bats are thought to be the responsible pollinators. If only a single cultivar is planted in the orchard, most of the young fruit drop by selfincompatibility two to four weeks after setting (Honsho et al., 2004a), giving a poor yield. The yield of durian in most Southeast Asian countries appears to be low and erratic, mainly due to inadequate fruit set (Subhadrabandhu et al., 1991). Somsri (1987) reported that crosspollination improved both fruit shape and size. Thus, artificial cross-pollination is recommended for commercial production. However, not all durian growers conduct artificial pollination because dangerous and laborious work for pollination has to be performed during the night with a ladder to pollinate the flowers on higher branches.
The success of artificial pollination is dependent on the viability of the pollen. However, information on the longevity of pollen and optimal condition for in vitro germination are not readily available for durian. Although flowers are hermaphroditic, self-pollination is not usually possible; this is ensured by the spatial separation between the anther and stigma owing to the difference in the lengths of the style and filament. Durian is not anemophilous (Valmayor et al., 1965); pollen should be transferred by some pollinator for successful pollination. However, if pollen longevity is short, it would reduce the opportunity for a pollinator to visit the nocturnal flower, even though bats are a possible pollinator. Thus, pollen longevity would be a limiting factor for durian productivity as well as flower receptivity. Further, the morphological structure of the anther and the time of anther dehiscence may be associated with the characteristics of durian pollination. Thus, information on the morphological features of anther dehiscence and pollen longevity is required for successful artificial pollination. In addition, a method to extend pollen longevity is required for pollination at an appropriate time as well as the transportation of pollen between orchards.
In four commercially important Thai durian cultivars, we first observed anther dehiscence using stereomicroscopy and low-vacuum scanning electron microscopy (SEM), then determined the optimal concentrations of sugar and requirement of minerals for germination in vitro. In addition, we investigated the pollen longevity, and the effects of desiccation on increasing the longevity.

Materials and Methods
Durian trees, planted in a durian collection orchard at the Chantaburi Horticultural Research Center in Thailand, were used in this study.
1. Observation of anther dehiscence by stereomicroscopy and low-vacuum SEM Anthers of 'Chanee', one of the most important durian cultivars in Thailand, were collected every six hours from 7:00 on the day before anthesis (36 h before anthesis) to 1:00 on the day after anthesis (6 h after anthesis). After collection, the anthers were immediately fixed in formalin-acetic acid-alcohol (FAA). Their entire structure was observed with a stereo-microscope. Additionally, a low-vacuum scanning electron microscope (S-3000N, Hitachi Co., Ltd., Japan) was used to observe the morphological features of the anther.

Pollen germination in vitro
Dehisced anthers containing pollen were collected at 19:00 from open flowers. Due to the difficulty in isolating pollen from the anthers, the pollen and anthers were kept together on weighing paper. The pollen was separated from the anthers with a brush, and the grains that adhered to the brush were placed on the germination media. The media were placed in a petri dish, in which a few drops of water were loaded to prevent them from drying, and they were incubated at room temperature (≈27°C) for at least 12 h. After incubation, the germinated pollen grains were counted with a hand-held tally counter under a light microscope, and the germination rate was calculated. When the length of the pollen tube was greater than the diameter of the pollen, the pollen was regarded as germinated. Three replications were performed for experiments 2.2, and four replications for experiment 2.1 and 2.3. More than 300 pollen grains were recorded in each replication. When ANOVA was performed, the percentages of pollen germination were subjected to arcsine transformation before analysis. Mean separation was performed by Tukey's test. 2.1. The optimal concentrations of sucrose and minerals for germination 'Mon Thong' pollen was used in this experiment. For the germination test, BK medium consisting of Brewbaker and Kwack, 1963) was used. The medium was solidified with 1% (w/v) agar. Four sucrose concentrations of 0, 10, 20, and 30% (w/v) were examined to find out the optimal sucrose concentration for pollen germination.
To determine the inorganic elements required for pollen germination, six kinds of medium were designed. In addition to the BK medium described above, BKLB, BKLMg, BKLCa, BKLK, and BKLA media were prepared. The first four media lacked H 3 BO 4 , and KNO 3 , respectively; BKLA eliminated all of these elements. All germination media contained 10% sucrose and were solidified with 1% agar.

Pollen longevity
Anthers were collected from 'Mon Thong', 'Chanee', 'Kradum Thong', and 'Phaung Manee' and kept on paper at room temperature. Pollen grains of each cultivar were sown on the BK media with 10% sucrose, which was the optimal condition determined in experiment 2.1, at 0, 6, 12, 24, 48, 72, or 120 h after collection. Two-way ANOVA was performed for each cultivar and time after collection in this experiment.

Desiccation of pollen in storage
'Chanee' anthers were dried with approximately 100 g of silica gel in 1 L closed columnar-containers for 120 h. Each container had anthers from 3-5 flowers. After a 120-h incubation, the anthers and pollen grains were rehydrated under normal room conditions (relative humidity above 70% and temperature of 27°C) for 0, 3, 6, 12, and 24 h. Subsequently, the germinating ability of pollens was evaluated on the BK medium containing 10% sucrose.

Results and Discussion
The anthers and filaments of durian are shown in Figure 1. Each filament has multiple reniform anthers (10.3 ± 1.0; mean ± SE, n = 10). Each stamen is polyadelphous with several filaments are joined at the base forming a phalanx (Fig. 1A). Each flower has five phalanges, which originate from five staminal primordia (Honsho et al., 2004b). The periphery of an anther, which looks like a labrum, contains the pollen sac (Fig. 1B). The sample collected at 7:00 on the day before anthesis had pollen grains in the pollen sac (data not shown). The anthers had not dehisced at 13:00 (Fig. 1C, D) but had dehisced longitudinally at 19:00 (Fig. 1E, F). Anthesis of the durian flower occurs at 19:00 (Honsho et al., 2004b); therefore, anthesis and anther dehiscence occur simultaneously. Low-vacuum SEM revealed that the pollen grains (approximately 50 µm in diameter) were still held in the pollen sac from anthesis ( Fig. 1F) to 1:00 (6 h after anthesis; data not shown), indicating that the pollen was not released at dehiscence. The size of pollen was nearly equal to that of avocado (30-50 µm, Inoue et al., 1992), mango (25-45 µm, Davenport and Núñez-Elisea, 1997), and cherimoya (50 µm, Yonemoto et al., 1999). In addition, the grains adhered to the pollen sac due to their stickiness and clump formation. Valmayor et al. (1965) stated that durian flowers are not anemophilous and require pollinators under natural conditions. Our results support this report since sticky pollen grains are characteristic of zoophily, which includes entomophily and chiropterophily.
The optimal sucrose concentration for germination in 'Mon Thong' was 10%, at which a pollen germination rate of 59.1% was observed (Table 1). At the sucrose concentrations of 0%, 20%, and 30%, the pollen germination rate was 7.1%, 19.5%, and 0.3%, respectively; each ratio was significantly lower than that of 10%. An extremely high sugar concentration has been reported to impair pollen germination in many kinds of plants. In durian, the D2 clone in Malaysia germinated optimally with 20% sucrose (Bala, 1996); this variation is probably caused by the difference of the clone and minerals used. In other tropical fruit crops, a concentration of 10% was best in cherimoya as well (Rosell et al., 1999), while 30% was optimal for dragon fruit (Macha et al., 2006). The reason for the 10% sucrose medium being optimal might be that sucrose plays an osmoregulatory as well as nutritive role (Iwanami, 1980;Rosell et al., 1999).
The pollen germination rate was nearly 60% in the BK medium, but removing some or all elements from the medium decreased the rate (Table 2). Pollen germination was the lowest (5.0%) in the BKLK medium. The germination rate was moderately decreased to 46.0% in the BKLMg medium and 34.8% in the BKLCa medium. In the BKLB medium, this rate was  relatively low (16.6%) but was higher than that in the BKLA and BKLK media. Thus, potassium was the most important nutrient for pollen germination among elements. Potassium is necessary for germination and pollen tube growth (Brewbaker and Kwack, 1963;Feijó et al., 1995;Weisenseel and Jaffe, 1976). Pollen germination at anthesis was 40.2% to 52.9% in the four cultivars (Table 3). At 24 h from anthesis, the germination ability decreased but was still maintained at over 40%. Although two-way ANOVA demonstrated significant differences in cultivar and time, there were no significant differences by Tukey's test among the four cultivars at each time except at 72 h from anthesis. At 72 h from anthesis, pollen germination in 'Kradum Thong' was significantly lower than that in the others due to a rapid loss of the germination ability. Pollen germination in all cultivars approached approximately zero at 120 h from anthesis. As a result of two-way ANOVA, there was a significant difference between 24 and 36 h from anthesis, while no significant difference was found between 0, 12, and 24 h from anthesis (data not shown). Thus, the pollen could be effective for pollination at 24 h after anthesis in terms of pollen germination. However, practical pollination tests are required to obtain the precise answer for how long the pollen is effective for pollination. The pollen grains retained their germination ability until at least 24 h from anthesis. Since the next anthesis occurs and new fresh pollen is released 24 h later, a continuous supply of germinable pollen can be possible. The androecium of durian generally abscises and drops within 12 h of the flower opening (Honsho et al., 2004b). However, our result showed that the pollen grains in the anthers are viable for at least another 12 h, indicating that they can still contribute to pollination in the daylight hours if they are successfully transferred to the stigma by natural pollinators. Cross-pollination gave a higher fruit set and better fruit quality in durian compared with selfpollination (Honsho et al., 2004a;Somsri, 1987). However, because the durian flower opens at night, artificial pollination is so far performed after sunset. The result of this study indicated that pollen is viable for at least 24 h. The stigma of durian becomes receptive before nocturnal flower opening while this receptiveness is rapidly lost by the next morning of flowering (Honsho et al., unpublished data). Thus, it is possible to perform artificial pollination before dark to avoid danger in conducting cross-pollination at night.
Desiccation is effective for the extension of pollen viability (Table 4). However, the pollen grains required rehydration to recover their germination ability. The direct transfer of dried pollen on the media resulted in no germination (0%), while rehydration for 12 h resulted in 47% pollen germination. The necessity of rehydration for the restoration of the germinability of the dry-stored pollen grains in durian is consistent with the results obtained by Akihama and Omura (1986); for the dried pollen of both peach and pear stored at room temperature (20°C), the effective period for restoration was found to be 6 to 12 h under an relative humidity of 100%, which corresponds to that observed in the present study. The desiccation method makes it possible to carry out crosspollination in an orchard where only a single cultivar is grown by transporting the pollen of different cultivars from another orchard.
In conclusion, we have elucidated the characteristics of the male organ in durian in terms of pollen release and pollen longevity. Although anther dehiscence and floral anthesis occurred simultaneously, pollen was held  in the anther sac after dehiscence. The in vitro study on pollen germination in 'Mon Thong' revealed that the optimal concentration of sucrose was 10% and potassium was the most important element for germination. The pollen grains retained their germination ability for 24 h or more, and for at least 5 days under desiccation. These results suggest a new practical method for artificial crosspollination, such as the transportation of pollen from remote orchards and pollination in daylight hours.