ORIGINAL ARTICLES
High Temperature After Anthesis Releases Embryo Arrest in the Mukaku Kishu–type Seedless Cultivar ‘Southern Yellow’
2022 Volume 91 Issue 3 Pages 322-328
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2022 Volume 91 Issue 3 Pages 322-328
Mukaku Kishu–type seedlessness in citrus (Citrus L.) is characterized by the formation of small and edible seeds (type A seeds) with an immature seed coat and arrest of embryo development at an early stage. We investigated the effect of high temperature on embryo and seed development in the Mukaku Kishu–type seedless cultivar ‘Southern Yellow’ over a period of four years. In outdoor-grown trees, embryos at the zygote and proembryo stages were observed at 10 weeks after pollination (WAP), and embryo development was largely arrested at 18 WAP; as a result, only type A seeds formed. In glasshouse-grown trees, globular-stage embryos were observed at 10 WAP, and globular- to cotyledon-stage embryos developed by 18 WAP. The day temperature in the glasshouse was higher than outdoors. Seeds developed in the fruit of trees transferred into a glasshouse at 0–2, 0–4, or 2–4 WAP, but not 4–8 WAP. High temperature before flowering had no effect on seed development. We conclude that high temperature at 0–4 WAP releases the arrest of embryo development after 10 WAP in ‘Southern Yellow’.
Seedlessness is an important characteristic for consumers and the agro-processing industry. Male sterility, female sterility, self-incompatibility, and parthenocarpy result in the expression of seedlessness in citrus (Iwamasa, 1966; Yamamoto et al., 1995). The male-sterility and self-incompatibility traits are often used in seedless citrus breeding, although seedy fruits may result from cross-pollination by flower-visiting insects. The female-sterility trait is reliable for the stable production of seedless fruit in citrus.
‘Kishu Mikan’ (Citrus kinokuni Tanaka) has a long history of cultivation in Japan and produces fruit with seeds. A mutant variety ‘Mukaku Kishu’ reportedly produces completely seedless fruit irrespective of the cultivation environment, flower-visiting insects, and artificial pollination (Hodgson, 1967; Nagai and Tanikawa, 1928; Tanaka, 1954). Therefore, ‘Mukaku Kishu’ is important as a breeding material for seedless citrus. Yamasaki et al. (2007, 2009) reported that ‘Mukaku Kishu’ and its seedless progeny have a specific type of seed, the so-called type A seed, characterized by a small size (< 3 mm), an immature seed coat, and an embryo arrested at an early stage of development.
‘Southern Yellow’ is a seedless citrus cultivar bred from a cross between ‘Tanikawa Buntan’ pummelo (C. maxima (Burm.) Merr.) and ‘Mukaku Kishu’ at the NARO Institute of Fruit Tree Science in Japan (Kobayashi, 1995). ‘Southern Yellow’ forms type A seeds (Yamasaki et al., 2009). Interestingly, we observed that outdoor-grown ‘Southern Yellow’ has small, edible seeds with an immature seed coat, whereas glasshouse-grown fruits have perfect seeds with a mature, hard seed coat. We hypothesized that the higher temperatures in the glasshouse facilitate seed development in ‘Southern Yellow’.
The aim of the present study was to clarify the effects of high temperature on the expression of Mukaku Kishu–type seedlessness in ‘Southern Yellow’, the cultivation conditions that affect seed formation and embryo development, and the period of high-temperature sensitivity for seed development.
Potted ‘Southern Yellow’ trees were grown outdoors and in a glasshouse at the Agricultural Technology Research Center of the Hiroshima Prefectural Technology Research Institute, Higashi-Hiroshima, Japan. The temperature in the glasshouse was maintained at > 2°C and the roof vent was opened when the temperature exceeded 25°C. Flowers were pollinated artificially on the day of anthesis with pollen collected from ‘Suisho Buntan’ pummelo (C. maxima (Burm.) Merr.).
Experimental design and temperature measurementThe experiments were conducted in 2005, 2006, 2009, and 2010 (Table 1). In 2005, three trees aged 7–11 years (A, B, and C) grown in a glasshouse were used. Three to five fruits per tree, which developed from flowers that opened on 2 May, were collected at 8, 10, 14, and 18 weeks after pollination (WAP), and embryo development was examined histologically. In 2006, tree A was grown as before, whereas tree B was transferred outdoors on 13 April. Trees A and B flowered on 14 April and 24 May, respectively. All fruits were collected at 10, 14, and 18 WAP, and embryo development was examined histologically. Three to five fruits per tree were harvested at 18 WAP to examine the number of normally developed (perfect) seeds, poorly developed (imperfect) seeds, unfertilized ovules and extremely small seeds (ovule-like seeds), and type A seeds, following the classification of Yamasaki et al. (2007).
Cultivation period and cumulative temperature.
In 2009 and 2010, 10 of the 12 pots (aged 10 and 11 years, respectively) maintained outdoors before flowering were transferred to the greenhouse to undergo high temperature treatment for different periods of time. Flowering started on 20 April in 2009, and on 6 May in 2010. In 2009, the high-temperature treatment periods in the glasshouse were (i) 0–18 WAP (20 April to 31 August), (ii) 0–4 WAP (20 April to 22 May), (iii) 0–2 WAP (20 April to 8 May), and (iv) 4–8 WAP (22 May to 19 June). In 2010, they were (i) two weeks before flowering (WBF)–0 WAP (22 April to 6 May), (ii) 0–2 WAP (6 to 20 May), and (iii) 2–4 WAP (20 May to 3 June). Fruits were harvested at 18 WAP to examine the number of normally developed (perfect) seeds, poorly developed (imperfect) seeds, unfertilized ovules and extremely small seeds (ovule-like seeds), and type A seeds, following the classification of Yamasaki et al. (2007).
The air temperatures in the glasshouse and outdoors were recorded hourly from 0 to 14 WAP in 2005, 2006, and 2009; and from 0 to 4 WAP in 2010 using a 2-channel card logger (MR 5320; Chino, Tokyo, Japan) placed at a height of 1.5 m. Cumulative temperatures were calculated from the daily mean temperature and accumulated as the total of glasshouse and outdoor periods for 0–18 WAP for 2005, 2006, and 2009, and 2 WBF–4 WAP for 2010.
Histological observationsAll seeds were fixed in FAA (formalin:glacial acetic acid:70% ethanol, 5:5:90 by volume), cleared, and stained to observe embryo development as described by Herr (1974, 1982) and Kojima et al. (1991) with a slight modification. In brief, fixed seeds were soaked in 70% ethanol for 20–30 min and then in 10% KOH for 2–3 min. The seeds were washed three times in distilled water, dehydrated in 90% ethanol for 20–30 min, and cleared in benzyl benzoate four-and-a-half (BB-4½) fluid (lactic acid:chloral hydrate:phenol:clove oil:xylene:benzyl benzoate, 2:2:2:2:1:1 by volume) overnight at 4°C. The outer and inner integuments of cleared seeds were removed by tweezers or a fine needle under a stereomicroscope. The seeds were then placed in BB-4½ fluid on a microscope slide and examined using a microscope equipped with differential interference contrast optics (BX50; Olympus, Tokyo, Japan).
Previously, we noticed, in unpublished observations, that ‘Southern Yellow’ was seedless outdoors but produced seeds in a glasshouse. To confirm this phenomenon, we examined seed formation in three glasshouse-grown ‘Southern Yellow’ trees in detail. In 2005, fruit from all three trees contained monoembryonic perfect and imperfect seeds at 18 WAP (Fig. 1A). Embryo development stages at different time points are shown in Figure 1B. All embryos were in the zygote or proembryo stage at 8 WAP, but a few developed to the globular (7.9%), heart-shaped (6.1%), or cotyledon (0.9%) stage at 10 WAP. At 14 WAP, most of the embryos remained in the zygote or proembryo stage, but some had developed into globular (5.3%) or heart-shaped stage embryos (2.6%). At 18 WAP, all embryo stages were observed, with cotyledon-stage embryos present in 18.3% of the seeds.
Seed development of ‘Southern Yellow’ trees grown in a glasshouse in experiment in 2005. (A) Percentage of different seed types per fruit at 18 weeks after pollination (WAP). (B) Percentage of seeds at different embryo developmental stages at 8, 10, 14, and 18 WAP.
In 2006, to examine the effect of environmental conditions on embryo development, we compared the seeds of a glasshouse-grown tree (A) with those of an outdoor-grown tree (B). At 18 WAP (Fig. 2A), fruit of tree B contained type A seeds and no perfect or imperfect seeds (seedless phenotype); in contrast, fruit of tree A contained perfect and imperfect seeds (seedy phenotype). The number of ovule-like seeds was significantly higher in tree B. In tree A fruit, globular embryos were observed at 10 WAP, heart- (8.8%), torpedo- (3.8%), and cotyledon-stage embryos (18.8%) were observed at 14 WAP, and almost all seeds (92.4%) contained cotyledon-stage embryos at 18 WAP (Fig. 2B). In tree B fruit, zygotes and proembryos were observed at 10 and 14 WAP, whereas only 3.7% of embryos were globular at 18 WAP (Fig. 2C). The maximum, minimum, and mean air temperatures in the glasshouse and the outdoors are shown in Figure 3. Overall, the glasshouse temperature was higher than the outdoor temperature (Fig. 3B–D). In particular, the maximum temperature was maintained at a high level compared to the mean temperature, the minimum temperature (Fig. 3A, B), and the cumulative temperature (Table 1) in both years.
Seed development of ‘Southern Yellow’ trees grown in a glasshouse in experiment in 2006. (A) Percentage of different seed types per fruit at 18 WAP. (B, C) Percentage of seeds at different embryo developmental stages at 10, 14, and 18 WAP in ‘Southern Yellow’ trees grown (B) in a glasshouse or (C) outdoors.
Maximum, mean, and minimum air temperatures in the glasshouse and outdoors. A, 2005; B, 2006; C, 2009; D, 2010.
To determine the crucial period of high temperature after pollination, we compared seed development in trees subjected to high temperature treatment in the glasshouse for 0–18, 0–4, 0–2, and 4–8 WAP in 2009. Perfect and imperfect seeds were observed in the 0–18, 0–4, and 0–2 WAP treatments, but not in the 4–8 WAP treatment (Figs. 4 and 5). The maximum air temperature from 0 to 11 WAP was consistently higher in the glasshouse than outdoors, but mean and minimum air temperatures were almost identical between the two (Fig. 3C). In 2010, perfect seeds were observed in the 0–2 and 2–4 WAP treatments, but not in the 2 WBF–0 WAP treatment (Figs. 6 and 7). There were a few exceptions in which temperatures of the outdoors exceeded or were the same as the glasshouse, but the air temperature of the glasshouse was maintained at a high level throughout 2009 (Fig. 3C) and 2010 (Fig. 3D).
Seed development of ‘Southern Yellow’ trees transferred to the glasshouse or grown outdoors for different periods in 2009. Percentage of different seed types per fruit at 18 WAP are shown.
Seeds of ‘Southern Yellow’ trees grown in a glasshouse for different periods in 2009. Potted trees were transferred to the glasshouse for (A) 0–18 WAP, (B) 0–4 WAP, (C) 0–2 WAP, or (D) 4–8 WAP, or (E) remained outdoors.
Seed development of ‘Southern Yellow’ trees transferred to the glasshouse or grown outdoors for different periods in 2010. Percentages of different seed types per fruit at 18 WAP are shown.
Seeds of ‘Southern Yellow’ trees grown in a glasshouse for different periods in 2010. Potted trees were transferred to the glasshouse for (A) 2 weeks before flowering to 0 WAP, (B) 0–2 WAP, or (C) 2–4 WAP, or (D) remained outdoors.
‘Southern Yellow’ is a cultivar of ‘Mukaku Kishu’ progeny with Mukaku Kishu–type seedlessness (Yamasaki et al., 2007). We previously demonstrated that ‘Southern Yellow’ produces type A seeds similar to those of ‘Mukaku Kishu’, and that the development of the majority of embryos is arrested at the zygote and proembryo stages at 10–12 WAP (Yamasaki et al., 2009). In the present study, we found that ‘Southern Yellow’ produces perfect seeds and some embryos develop to the cotyledon stage at 14 WAP under glasshouse conditions. Overall, the temperature inside the glasshouse remained higher than outdoors. These findings suggest that the high temperature of the glasshouse promoted seed development in ‘Southern Yellow’. Previous studies have reported the effect of high temperature on embryogenesis in citrus. Nakatani et al. (1978) reported that the mean number of nucellar embryos per seed was smaller in fruits of satsuma mandarin (C. unshiu (Swingle) Marcow.) grown in a glasshouse than in outdoor-grown fruits, which suggests that high temperature above an optimal threshold may affect seed development in citrus. Nakatani et al. (1982) showed that high-temperature treatment from shoot sprouting to the end of flowering reduced the number of embryos per seed and increased the percentage of monoembryonic seeds in polyembryonic cultivars such as ‘Minneola’ tangelo (C. paradisi Macfad. × C. reticulata Blanco) and sweet orange (C. sinensis (L.) Osbeck). Similarly, in Cairo, Egypt (Higazy and Hamouda, 1974), and in Batangas, the Philippines (Torres, 1936), polyembryonic mandarin cultivars were reported to have a low number of embryos per seed and a high percentage of monoembryonic seeds. These reports suggest that the temperature during anthesis, when nucellar embryos begin to differentiate, affects embryo development. Thus, we considered that high temperature may be a major factor affecting embryo development in ‘Southern Yellow’.
In many plant species, such as Sorghum bicolor (L.) Moench (Prasad et al., 2008), Syringa vulgaris L. (Jędrzejuk and Łukaszewska, 2008), peanut (Arachis hypogaea L.) (Prasad et al., 2001), canola (Brassica napus L.) (Polowick and Sawhney, 1988; Young et al., 2004), and flax (Linum usitatissimum L.) (Cross et al., 2003), abnormal seeds are formed in response to high temperature during flowering and are ultimately sterile. Degeneration of the embryo sac or nucellus in sweet cherry has been attributed to warm weather during flowering (Beppu and Kataoka, 2011; Hedhly et al., 2007). Normal seed formation is generally inhibited by high temperature in the early stages of fruit development. However, the present study revealed a unique phenomenon in that originally arrested embryos resumed normal development at high temperature and fully developed seeds were formed. To the best of our knowledge, this phenomenon has not been reported previously.
We found that high-temperature treatment during 0–18, 0–4, 0–2, and 2–4 WAP, but not 2 WBF–0 WAP, and 4–8 WAP, led to the development of perfect and imperfect seeds. On the other hand, type A seeds were still found in some fruits of those high-temperature treatments, meaning some instability in the effect of high temperature on seed development. The factors affecting this instability are unclear, but these results indicate that high temperature during 0–4 WAP is effective for seed formation. The stages of the embryo were zygote or proembryo at 8 WAP in a glasshouse-grown tree (Fig. 2B). This is comparable to the outdoor-grown trees of ‘Southern Yellow’ previously shown by Yamasaki et al. (2007). However, at 10 WAP or later, embryos were specifically developed in the glasshouse-grown tree. These results indicate that, even though the embryos of ‘Southern Yellow’ do not begin to develop until 8 WAP, high temperature during 0–4 WAP promotes embryo development, suggesting that high temperature during this period does not affect embryo development directly. Embryo development is affected by endosperm development, and in ‘Hiratanenashi’, a seedless Japanese persimmon (Diospyros kaki L. f.), degeneration of the endosperm arrests embryo development, causing seed abortion (Ishida et al., 1990; Sobajima et al., 1975). In ‘Southern Yellow’, exposure to high temperature during 0–4 WAP may promote free nuclear division, resulting in endosperm development, which in turn promotes embryo development starting from 8 WAP. Future experiments including more detailed histological observations, gene expression analysis, as well as more precise temperature treatments are needed to elucidate the mechanism by which temperature influences embryo development.
