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Production of seedless watermelon using soft-X-irradiated pollen
Scientia Horticulturae 84 (2000) 255±264
Production of seedless watermelon using soft-X-irradiated pollen Keita Sugiyama*, Masami Morishita Kurume Branch, National Research Institute of Vegetables, Ornamental Plants and Tea, Kurume, Fukuoka 839-8503, Japan Accepted 13 August 1999
Abstract A new method for producing seedless watermelon (Citrullus lanatus) in diploid plants using softX-irradiated pollen is described. Fruit set at almost the same rate despite soft-X-irradiation. Empty seeds were produced in the watermelon cultivars `Benikodama' and `Fujihikari TR' following hand pollination using soft-X-irradiated pollen. Soft-X-irradiation doses of 800±1000 Gy resulted in small empty seeds in `Fujihikari TR', whereas 400±1000 Gy doses gave the best results for `Benikodama', although empty seeds in the latter were many and conspicuous. Soft-X-ray treatments did not affect fruit weight, shape, rind thickness or days to maturation compared to controls. However, female ¯owers treated with soft-X-irradiated pollen produced fruits with slightly higher sugar content compared to controls in `Benikodama'. Therefore, this new method is an effective technique to produce seedless watermelon. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Citrullus lanatus; Parthenocarpy; Pseudogamy
1. Introduction Currently a majority of seedless watermelon are produced by triploid plants. Seeds of triploid hybrids are obtained from crossing a diploid male with an autotetraploid female induced by colchicine treatment (Terada and Masuda, 1943; Kihara and Nishiyama, 1947; Kihara, 1958) or spontaneous chromosome * Corresponding author. Tel.: 81-942-43-8271; fax: 81-942-43-7014. E-mail address: [email protected] (K. Sugiyama).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 0 4 - 1
256
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
doubling during in vitro culture (Compton et al., 1996). However, the breeding of triploid watermelons requires more time than breeding diploid cultivars, and in Japan the cultivation of the triploid plants is dif®cult. Seedless watermelon fruits have also been produced using plant growth regulators, e.g. IAA (Terada and Masuda, 1940), NAA (Wong, 1938; Terada and Masuda, 1941), NAA, 4-CPA, GA3 (Kondou and Murozono, 1975), BA (Yamamuro, 1978) or CPPU (Hayata et al., 1995). Using plant growth regulators has occasionally resulted in deformed fruits (Kondou and Murozono, 1975) because the female ¯owers (ovary) were injured by directly spraying or rubbing growth regulators on them. This method also poses food safety problems, and therefore, has not been used. Seedless watermelons may also be produced by inducing reciprocal translocations of chromosomes (Nishimura and Sakaguchi, 1960; Oka et al., 1967; Shimotuma, 1968). However, this method is not utilized because the development of chromosome translocation lines and commercial cultivars based on these lines is dif®cult, and fruits are not completely seedless. Triploid watermelon is not distributed internationally due to high seed cost and dif®culties associated with developing new cultivars. Therefore, it is desirable to produce seedless watermelon from commercially acceptable diploid cultivars, using a scienti®c method that is chemically safe. The purpose of this study was to investigate the practicality of inducing seedlessness in two Japanese watermelon cultivars by pollinating female ¯owers using pollen treated with varying doses of Ê ). soft-X-ray (X-rays which have a wave length of about 0.1 A 2. Materials and methods In 1997: Watermelon cultivars (F1 hybrid) of `Fujihikari TR' and `Benikodama' were sown on 4 April 1997. The seedlings were transplanted 50 cm apart in a bed (2.3 m 35 m) in a greenhouse on 8th May. The bed was covered with black polyethylene mulch. Fertilization involved a pre-plant broadcast application of 10 N±10 P±10 K (kg ha 1). Plants were topped at the ®ve-leaf stage, and three lateral vines were allowed to grow. Treatment commenced on ¯owers at about the 15th node of the lateral branch. Vines were topped at the 25th node. Treatments were performed between 31 May and 7 June. Male ¯owers were harvested at random from all plants in the morning during anthesis. Pollen was retained in the male ¯owers, and their petals and sepals were not removed. A soft-X-ray machine (Soft-X-ray Unit OM-60R, OHMIC Ltd.) was set up in the greenhouse. The prepared male ¯owers were put into a plastic case with holes and a dose of soft-X-irradiated at 11.1 Gy min 1. Whole ¯owers were irradiated with a dose of 0 (unirradiated, as the control), 100, 200, 400, 800 or 1000 Gy.
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
257
Fig. 1. Normal seed and partially developed seeds: (a) normal seed, (b) and (c) empty seed (c) was not categorized as empty seed because it was very small and thin). Vertical bar 2 mm.
Female ¯owers were covered with cellophane bags before anthesis. Upon anthesis, they were arti®cially self-pollinated with treated pollen on the same day and then re-covered to prevent contact with insect-borne pollen for 3±5 days. One male ¯ower was used for three female ¯owers. Mature fruits were harvested at about 35 days in `Benikodama' and 38 days after pollination in `Fujihikari TR'. Immediately after harvesting, fruit weight, fruit shape, rind thickness, sugar content (soluble solid contents) and seed normality (maturity or emptiness) were recorded for each fruit. Fruit shape index was expressed as the ratio of height to width. Empty seeds with a hard coat were counted, but those with a thin coat were not (Fig. 1). The six treatments were arranged in a randomized complete-block design with ®ve single-plant replications. Five fruits were harvested for each treatment. Data were subjected to analysis of variance and mean separation according to Duncan's multiple range test using p 0.05 on statistical software. In 1998: `Fujihikari TR' and `Benikodama' were sown on 13 November 1997 and 26 February 1998, respectively. The seedlings of the former were transplanted 50 cm apart in two beds in a greenhouse on 23 January and of the latter were done on 24 March 1998. The blooming male ¯owers were irradiated with 800 Gy soft-X-ray and immediately used to pollinate each watermelon plant. Unirradiated pollen was used as the control. Treatments were performed between 20 February and 1 March in `Fujihikari TR', and in `Benikodama', treatments were done between 18 and 29 March. One week after treatment, fruit set was recorded. Mature fruits were harvested at about 36 days in `Benikodama' 36 and 48 days after pollination
258
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
in `Fujihikari TR'. Methods of cultivation and pollination were the same as in 1997. The treatments were arranged in a randomized block design with two replications. Twenty plants in `Fujihikari TR' and ten plants in `Benikodama' were used. Data were expressed as means SE. Mean differences between treatments and control were determined by t test. 3. Results and discussion 3.1. Relationship between soft-X-ray irradiation dose to pollen and number of seeds There were no observable differences in fruit setting between soft-X-irradiated pollen and unirradiated pollen in both `Benikodama' and `Fujihikari TR' (Table 1). We have already observed that the germination rate of pollen treated with 800 Gy of soft-X-ray was almost the same as that of the control (Sugiyama and Morishita, 1998). These results indicate that irradiated pollen presents no practical problem for producing seedless watermelon fruit. The number of normal seeds was signi®cantly reduced when female ¯owers were pollinated with irradiated pollen. In `Benikodama', normal seeds were not observed at 400±1000 Gy (Fig. 2). 200±400 empty seeds per watermelon were observed at 100±1000 Gy. In `Fujihikari TR' irradiated with the 800±1000 Gy, empty seeds alone were produced (Fig. 3). The numbers of empty seeds were approximately 50±150 at 800±1000 Gy. In `Fujihikari TR' fruits produce with soft-X-irradiated pollen, most empty seeds were as much small and thin (Fig. 4). However, they were very conspicuous in `Benikodama'. Varietal differences in number of empty seeds were observed in triploid watermelon (Kihara, 1958). In our study, `Benikodama' had more empty seeds than `Fujihikari TR'(Table 2, Fig. 2) which indicates that the number of empty seeds is determined by the characteristics of the ovule in each cultivar. Producing seedless fruits of watermelon by hormonal agents is a parthenocarpic phenomenon in which fertilization is not needed for growth. In order for Table 1 Effect of soft-X-irradiation on fruit set of watermelon Cultivar
X-ray dose (Gy)
Treated flowers (number)
Fruit set (%)
Benikodama
0 800
51 46
39.2 43.5
Fujihikari TR
0 800
54 60
64.8 66.7
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
259
Fig. 2. Relationship between dose of soft-X-irradiation and number of seeds in cultivar `Benikodama'. Vertical bars indicate SE.
Fig. 3. Relationship between dose of soft-X-irradiation and number of seeds in cultivar `Fujihikari TR'. Vertical bars indicate SE.
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Fig. 4. Watermelons fruits after pollination by soft-X-irradiation pollen compared to a normal fruit in `Benikodama' (upper photograph) and `Fujihikari TR' (lower photograph).
parthenocarpically developed watermelon to reach normal size, pollination and pseudogamy are necessary. Generally, after pollination, growth of the ovary is induced by auxin derived from pollen grain and by auxin induced by stimulation from the pollen tube in the pistil. Further ovary development is promoted by auxin derived from the developing embryo after fertilization (Saitou, 1974). However, a watermelon fruit which is produced only by hormonal treatment is smaller than that produced by pollination (Kondou and Murozono, 1975; Yamamuro, 1978; Hayata et al., 1995). Triploid watermelons produced by parthenocarpy develop to full size because parthenocarpy is induced by growth hormones provided by diploid pollen grains and provided from the ovule by
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261
Table 2 Effect of soft-X-irradiation on fruit characteristics (1997) Cultivar
X-ray dose (Gy)
Fruit weight (kg)
Fruit shapea
Benikodama
0 100 200 400c 800c 1000c
2.6ab 2.4ab 1.9b 2.0b 1.7b 2.3ab
1.14ab 1.14ab 1.08b 1.14ab 1.16a 1.17a
Fujihikari TR
0 100 200 400 800c 1000c
3.8a 4.0a 4.8a 4.8a 4.1a 3.8a
1.05a 1.05a 1.07a 1.07a 1.09a 1.05a
Thickness of rind (cm)
Sugar content (%)
Maturation (days)
6.8a 6.2a 6.4a 6.2a 6.4a 6.7a
11.8b 12.2b 12.0b 12.5ab 12.5ab 13.1a
36.0 34.6 34.5 35.6 36.8 35.3
9.9b 11.6a 11.4ab 11.3ab 10.1b 11.0ab
10.7b 11.4ab 11.0b 10.6b 11.2ab 12.2a
36.8 37.5 37.3 36.5 37.4 37.3
a
Fruit shape is expressed as the ratio of height to width. Mean separation within columns by Duncan's multiple range test, at 5% level. c Seedless watermelon fruit. b
pseudogamy. Thus endogenous auxins derived from the ovule are needed to develop normal size fruit. Soft-X-irradiated pollen germinate on a stigma, and their tubes elongate into an embryo sac. Subsequent abortion of embryos after pollination by soft-Xirradiated pollen is still under investigation, but may result from soft-X-rayinduced chromosomal abnormalities in the generative nucleus which prevent normal fertilization from occurring. However, Sari et al. (1994) reported that haploid embryos are obtained through pollination with g-irradiated pollen, which indicates that embryo formation occurs as a pseudogametic phenomenon. Therefore, it is possible for an ovary growing after pollination with soft-Xirradiated pollen to be promoted by auxin derived from the ovule. Fruit characteristics: In `Benikodama', fruit weight in control averaged 2.6 kg, slightly heavier than in irradiated groups in 1997 (Table 2). However, in 1998, `Benikodama' fruit weight in irradiation treatment was similar to that of control (Table 3). In `Fujihikari TR', there was no signi®cant difference between fruit weight in irradiated groups and that in controls. Thus, a relationship between the fruit weight and the irradiation dose was not recognized. `Benikodama' fruits were elliptical in most cases except for those treated with 200 Gy (1997 group). The shape of `Fujihikari TR' fruits were spherical at all doses of irradiation. In `Benikodama' fruits, there was no signi®cant in rind thickness between irradiated groups and controls; in `Fujihikari TR' fruit rind in irradiated groups except the 800 Gy (1997 group) was also similar to that in controls. We found no consistent relationship between dose of soft-X-irradiation and rind thickness. `Benikodama'
262
Cultivar
X-ray dose (Gy)
Number of normal seed
Number of empty seed
Fruit weight (kg)a
Fruit shapea,b
Thickness of rind (cm)a
Sugar content (%)a
Maturation (days)
Benikodama
0 800
173 0
51 198
1.5 0.18 1.6 0.16 NSc
1.11 0.02 1.12 0.02 NSc
5.5 0.14 5.4 0.35 NSc
11.0 0.40 12.5 0.35 NSc
35.8 36.0 NSc
0 800
201 0
44 149
6.2 0.23 6.4 0.27 NSc
1.03 0.13 1.03 0.02 NSc
13.4 0.60 13.0 0.58 NSc
10.8 0.23 11.3 0.24 NSc
48.0 48.0 NSc
Significanced Fujihikari TR Significanced a
Data are means standard errors of 10-fruits in `Benikodama', 20-fruits in `Fujihikari TR', respectively. Fruit shape is expressed as the ratio of height to width. c NS: non-signi®cant. d Signi®cant between control and irradiation at P < 0.05. b
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
Table 3 Comparison of seedless watermelon fruits produced by soft-X-irradiated pollens and control (1998)
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263
fruits from irradiation treatment showed similar or higher sugar content than in controls. In `Fujihikari TR', there was no signi®cant difference in sugar content of irradiated groups except the 1000 Gy (1997 group) and that of controls. All fruits of both `Benikodama' and `Fujihikari TR' matured within almost the same period regardless of soft-X-irradiation. Seedless watermelon fruits induced by hormonal agents had a tendency to be small and deformed compared with normal fruits (Hayata et al., 1995; Kondou and Murozono, 1975). In contrast, seedless watermelon fruits produced by softX-irradiated pollen were normal in size and shape. Previous studies (Kihara, 1951; Kondou and Murozono, 1975) on the production of parthenocarpic seedless watermelon using triploid or plant growth regulators showed a tendency to form a thick rind. In the present experiment, there was no relationship between rind thickness and soft-X-irradiation. In our study, the sugar content of seedless watermelon fruits produced by soft-X-irradiated pollen was similar to or higher than that of controls. These results show that soft-X-irradiation did not affect fruit weight, shape or rind thickness, and that the quality was equal to or better than that of controls. In Japan, the main reason triploid watermelons are not popular is that triploids have a longer growing period and a later maturation date than diploid ones. Occasionally, there have been some developmental defects such as hollow hearts in triploid watermelon fruits (Kihara, 1951, 1958). In contrast, in our experiment, these defects were not present in seedless watermelon produced by pollination with soft-X-irradiated pollen. In conclusion, seedless watermelon produced by pollination with soft-Xirradiated pollen can be produced by ordinary applied cultivation due to the use of common diploids. Therefore, this new technique established in our study has practical value and should be used to produce a good seedless watermelon fruit. Acknowledgements The authors are thankful to Prof. T. Harada of Hokkaido University and Prof. H. Inden of Miyazaki University for valuable suggestions and critical reading of the manuscript. References Compton, M.E., Gray, D.J., Elmstrom, G.W., 1996. Identi®cation of tetraploid regenerants from cotyledons of diploid watermelon cultured in vitro. Euphytica 87, 165±172. Hayata, Y., Niimi, Y., Iwasaki, N., 1995. Synthetic Cytokinin-1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU) Ð promotes fruit set and induces parthenocarpy in watermelon. J. Am. Soc. Hort. Sci. 120, 997±1000.
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Kihara, H., 1951. Triploid watermelon. Proc. Am. Soc. Hort. Sci. 58, 217±230. Kihara, H., 1958. Breeding of seedless fruits. Seiken Ziho 9, 1±7. Kihara, H., Nishiyama, I., 1947. An application of sterility of autotriploids to the breeding of seedless watermelons. Seiken Ziho 3, 93±103 (in Japanese with English summary). Kondou, Y., Murozono, M., 1975. Study of arti®cial fruit setting watermelon. Abstr. Jpn. Soc. Hort. Sci. Autumn Meeting 44, 178±179 (in Japanese). Nishimura, Y., Sakaguchi, S., 1960. Studies on reciprocal translocations of chromosomes in watermelon. Bull. Fac. Agric. Niigata Univ. 12, 22±29. Oka, H., Watanabe, T., Nishiyama, I., 1967. Reciprocal translocation as a new approach to breeding seedless watermelon. Can. J. Genetics Cytol. 9, 482±489. Saitou, T., 1974. Physiology and ecology of fruit set in cucumber. In: The System of Agricultural Technology, Cucumber. Soc. Cult. Agr. VILL. Press, Tokyo, p. 125±126 (in Japanese). Sari, N., Abak, K., Pitrat, M., Rode, J.C., Dumas de Vaulx, R., 1994. Induction of parthenogenetic haploide embryos after pollination by irradiation pollen in watermelon. Hort. Sci. 29, 1189± 1190. Shimotuma, M., 1968. Synthesis of some multiple interchange strains of watermelons induced by X-rays. Seiken Ziho 20, 47±53. Sugiyama, K., Morishita, M., 1998. Storage ability of soft-X-ray irradiated-pollens and effects of storage pollens on fruit characteristics in watermelon. J. Jpn. Soc. Hort. Sci. 67 (suppl. 2), 283 (in Japanese). Terada, J., Masuda, K., 1940. Parthenocarpy of watermelon by heteroauxin. Agric. Hort. 15, 458± 468 (in Japanese). Terada, J., Masuda, K., 1941. Parthenocarpy of watermelon by single or complex application of plant hormones. Agric. Hort. 16, 1915±1917 (in Japanese). Terada, J., Masuda, K., 1943. Parthenocarpy of triploid watermelon. Agric. Hort. 18, 15±16 (in Japanese). Wong, C.Y., 1938. Induced parthenocarpy of watermelon cucumber and pepper by the use of growth promoting substances. Proc. Am. Soc. Hort. Sci. 36, 632±636. Yamamuro, K., 1978. Effect of growth regulators on fruit setting of watermelon. Bull. Ibaraki Hort. Expt. Sta. 7, 1±15.
Production of seedless watermelon using soft-X-irradiated pollen Keita Sugiyama*, Masami Morishita Kurume Branch, National Research Institute of Vegetables, Ornamental Plants and Tea, Kurume, Fukuoka 839-8503, Japan Accepted 13 August 1999
Abstract A new method for producing seedless watermelon (Citrullus lanatus) in diploid plants using softX-irradiated pollen is described. Fruit set at almost the same rate despite soft-X-irradiation. Empty seeds were produced in the watermelon cultivars `Benikodama' and `Fujihikari TR' following hand pollination using soft-X-irradiated pollen. Soft-X-irradiation doses of 800±1000 Gy resulted in small empty seeds in `Fujihikari TR', whereas 400±1000 Gy doses gave the best results for `Benikodama', although empty seeds in the latter were many and conspicuous. Soft-X-ray treatments did not affect fruit weight, shape, rind thickness or days to maturation compared to controls. However, female ¯owers treated with soft-X-irradiated pollen produced fruits with slightly higher sugar content compared to controls in `Benikodama'. Therefore, this new method is an effective technique to produce seedless watermelon. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Citrullus lanatus; Parthenocarpy; Pseudogamy
1. Introduction Currently a majority of seedless watermelon are produced by triploid plants. Seeds of triploid hybrids are obtained from crossing a diploid male with an autotetraploid female induced by colchicine treatment (Terada and Masuda, 1943; Kihara and Nishiyama, 1947; Kihara, 1958) or spontaneous chromosome * Corresponding author. Tel.: 81-942-43-8271; fax: 81-942-43-7014. E-mail address: [email protected] (K. Sugiyama).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 0 4 - 1
256
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
doubling during in vitro culture (Compton et al., 1996). However, the breeding of triploid watermelons requires more time than breeding diploid cultivars, and in Japan the cultivation of the triploid plants is dif®cult. Seedless watermelon fruits have also been produced using plant growth regulators, e.g. IAA (Terada and Masuda, 1940), NAA (Wong, 1938; Terada and Masuda, 1941), NAA, 4-CPA, GA3 (Kondou and Murozono, 1975), BA (Yamamuro, 1978) or CPPU (Hayata et al., 1995). Using plant growth regulators has occasionally resulted in deformed fruits (Kondou and Murozono, 1975) because the female ¯owers (ovary) were injured by directly spraying or rubbing growth regulators on them. This method also poses food safety problems, and therefore, has not been used. Seedless watermelons may also be produced by inducing reciprocal translocations of chromosomes (Nishimura and Sakaguchi, 1960; Oka et al., 1967; Shimotuma, 1968). However, this method is not utilized because the development of chromosome translocation lines and commercial cultivars based on these lines is dif®cult, and fruits are not completely seedless. Triploid watermelon is not distributed internationally due to high seed cost and dif®culties associated with developing new cultivars. Therefore, it is desirable to produce seedless watermelon from commercially acceptable diploid cultivars, using a scienti®c method that is chemically safe. The purpose of this study was to investigate the practicality of inducing seedlessness in two Japanese watermelon cultivars by pollinating female ¯owers using pollen treated with varying doses of Ê ). soft-X-ray (X-rays which have a wave length of about 0.1 A 2. Materials and methods In 1997: Watermelon cultivars (F1 hybrid) of `Fujihikari TR' and `Benikodama' were sown on 4 April 1997. The seedlings were transplanted 50 cm apart in a bed (2.3 m 35 m) in a greenhouse on 8th May. The bed was covered with black polyethylene mulch. Fertilization involved a pre-plant broadcast application of 10 N±10 P±10 K (kg ha 1). Plants were topped at the ®ve-leaf stage, and three lateral vines were allowed to grow. Treatment commenced on ¯owers at about the 15th node of the lateral branch. Vines were topped at the 25th node. Treatments were performed between 31 May and 7 June. Male ¯owers were harvested at random from all plants in the morning during anthesis. Pollen was retained in the male ¯owers, and their petals and sepals were not removed. A soft-X-ray machine (Soft-X-ray Unit OM-60R, OHMIC Ltd.) was set up in the greenhouse. The prepared male ¯owers were put into a plastic case with holes and a dose of soft-X-irradiated at 11.1 Gy min 1. Whole ¯owers were irradiated with a dose of 0 (unirradiated, as the control), 100, 200, 400, 800 or 1000 Gy.
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
257
Fig. 1. Normal seed and partially developed seeds: (a) normal seed, (b) and (c) empty seed (c) was not categorized as empty seed because it was very small and thin). Vertical bar 2 mm.
Female ¯owers were covered with cellophane bags before anthesis. Upon anthesis, they were arti®cially self-pollinated with treated pollen on the same day and then re-covered to prevent contact with insect-borne pollen for 3±5 days. One male ¯ower was used for three female ¯owers. Mature fruits were harvested at about 35 days in `Benikodama' and 38 days after pollination in `Fujihikari TR'. Immediately after harvesting, fruit weight, fruit shape, rind thickness, sugar content (soluble solid contents) and seed normality (maturity or emptiness) were recorded for each fruit. Fruit shape index was expressed as the ratio of height to width. Empty seeds with a hard coat were counted, but those with a thin coat were not (Fig. 1). The six treatments were arranged in a randomized complete-block design with ®ve single-plant replications. Five fruits were harvested for each treatment. Data were subjected to analysis of variance and mean separation according to Duncan's multiple range test using p 0.05 on statistical software. In 1998: `Fujihikari TR' and `Benikodama' were sown on 13 November 1997 and 26 February 1998, respectively. The seedlings of the former were transplanted 50 cm apart in two beds in a greenhouse on 23 January and of the latter were done on 24 March 1998. The blooming male ¯owers were irradiated with 800 Gy soft-X-ray and immediately used to pollinate each watermelon plant. Unirradiated pollen was used as the control. Treatments were performed between 20 February and 1 March in `Fujihikari TR', and in `Benikodama', treatments were done between 18 and 29 March. One week after treatment, fruit set was recorded. Mature fruits were harvested at about 36 days in `Benikodama' 36 and 48 days after pollination
258
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
in `Fujihikari TR'. Methods of cultivation and pollination were the same as in 1997. The treatments were arranged in a randomized block design with two replications. Twenty plants in `Fujihikari TR' and ten plants in `Benikodama' were used. Data were expressed as means SE. Mean differences between treatments and control were determined by t test. 3. Results and discussion 3.1. Relationship between soft-X-ray irradiation dose to pollen and number of seeds There were no observable differences in fruit setting between soft-X-irradiated pollen and unirradiated pollen in both `Benikodama' and `Fujihikari TR' (Table 1). We have already observed that the germination rate of pollen treated with 800 Gy of soft-X-ray was almost the same as that of the control (Sugiyama and Morishita, 1998). These results indicate that irradiated pollen presents no practical problem for producing seedless watermelon fruit. The number of normal seeds was signi®cantly reduced when female ¯owers were pollinated with irradiated pollen. In `Benikodama', normal seeds were not observed at 400±1000 Gy (Fig. 2). 200±400 empty seeds per watermelon were observed at 100±1000 Gy. In `Fujihikari TR' irradiated with the 800±1000 Gy, empty seeds alone were produced (Fig. 3). The numbers of empty seeds were approximately 50±150 at 800±1000 Gy. In `Fujihikari TR' fruits produce with soft-X-irradiated pollen, most empty seeds were as much small and thin (Fig. 4). However, they were very conspicuous in `Benikodama'. Varietal differences in number of empty seeds were observed in triploid watermelon (Kihara, 1958). In our study, `Benikodama' had more empty seeds than `Fujihikari TR'(Table 2, Fig. 2) which indicates that the number of empty seeds is determined by the characteristics of the ovule in each cultivar. Producing seedless fruits of watermelon by hormonal agents is a parthenocarpic phenomenon in which fertilization is not needed for growth. In order for Table 1 Effect of soft-X-irradiation on fruit set of watermelon Cultivar
X-ray dose (Gy)
Treated flowers (number)
Fruit set (%)
Benikodama
0 800
51 46
39.2 43.5
Fujihikari TR
0 800
54 60
64.8 66.7
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
259
Fig. 2. Relationship between dose of soft-X-irradiation and number of seeds in cultivar `Benikodama'. Vertical bars indicate SE.
Fig. 3. Relationship between dose of soft-X-irradiation and number of seeds in cultivar `Fujihikari TR'. Vertical bars indicate SE.
260
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Fig. 4. Watermelons fruits after pollination by soft-X-irradiation pollen compared to a normal fruit in `Benikodama' (upper photograph) and `Fujihikari TR' (lower photograph).
parthenocarpically developed watermelon to reach normal size, pollination and pseudogamy are necessary. Generally, after pollination, growth of the ovary is induced by auxin derived from pollen grain and by auxin induced by stimulation from the pollen tube in the pistil. Further ovary development is promoted by auxin derived from the developing embryo after fertilization (Saitou, 1974). However, a watermelon fruit which is produced only by hormonal treatment is smaller than that produced by pollination (Kondou and Murozono, 1975; Yamamuro, 1978; Hayata et al., 1995). Triploid watermelons produced by parthenocarpy develop to full size because parthenocarpy is induced by growth hormones provided by diploid pollen grains and provided from the ovule by
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
261
Table 2 Effect of soft-X-irradiation on fruit characteristics (1997) Cultivar
X-ray dose (Gy)
Fruit weight (kg)
Fruit shapea
Benikodama
0 100 200 400c 800c 1000c
2.6ab 2.4ab 1.9b 2.0b 1.7b 2.3ab
1.14ab 1.14ab 1.08b 1.14ab 1.16a 1.17a
Fujihikari TR
0 100 200 400 800c 1000c
3.8a 4.0a 4.8a 4.8a 4.1a 3.8a
1.05a 1.05a 1.07a 1.07a 1.09a 1.05a
Thickness of rind (cm)
Sugar content (%)
Maturation (days)
6.8a 6.2a 6.4a 6.2a 6.4a 6.7a
11.8b 12.2b 12.0b 12.5ab 12.5ab 13.1a
36.0 34.6 34.5 35.6 36.8 35.3
9.9b 11.6a 11.4ab 11.3ab 10.1b 11.0ab
10.7b 11.4ab 11.0b 10.6b 11.2ab 12.2a
36.8 37.5 37.3 36.5 37.4 37.3
a
Fruit shape is expressed as the ratio of height to width. Mean separation within columns by Duncan's multiple range test, at 5% level. c Seedless watermelon fruit. b
pseudogamy. Thus endogenous auxins derived from the ovule are needed to develop normal size fruit. Soft-X-irradiated pollen germinate on a stigma, and their tubes elongate into an embryo sac. Subsequent abortion of embryos after pollination by soft-Xirradiated pollen is still under investigation, but may result from soft-X-rayinduced chromosomal abnormalities in the generative nucleus which prevent normal fertilization from occurring. However, Sari et al. (1994) reported that haploid embryos are obtained through pollination with g-irradiated pollen, which indicates that embryo formation occurs as a pseudogametic phenomenon. Therefore, it is possible for an ovary growing after pollination with soft-Xirradiated pollen to be promoted by auxin derived from the ovule. Fruit characteristics: In `Benikodama', fruit weight in control averaged 2.6 kg, slightly heavier than in irradiated groups in 1997 (Table 2). However, in 1998, `Benikodama' fruit weight in irradiation treatment was similar to that of control (Table 3). In `Fujihikari TR', there was no signi®cant difference between fruit weight in irradiated groups and that in controls. Thus, a relationship between the fruit weight and the irradiation dose was not recognized. `Benikodama' fruits were elliptical in most cases except for those treated with 200 Gy (1997 group). The shape of `Fujihikari TR' fruits were spherical at all doses of irradiation. In `Benikodama' fruits, there was no signi®cant in rind thickness between irradiated groups and controls; in `Fujihikari TR' fruit rind in irradiated groups except the 800 Gy (1997 group) was also similar to that in controls. We found no consistent relationship between dose of soft-X-irradiation and rind thickness. `Benikodama'
262
Cultivar
X-ray dose (Gy)
Number of normal seed
Number of empty seed
Fruit weight (kg)a
Fruit shapea,b
Thickness of rind (cm)a
Sugar content (%)a
Maturation (days)
Benikodama
0 800
173 0
51 198
1.5 0.18 1.6 0.16 NSc
1.11 0.02 1.12 0.02 NSc
5.5 0.14 5.4 0.35 NSc
11.0 0.40 12.5 0.35 NSc
35.8 36.0 NSc
0 800
201 0
44 149
6.2 0.23 6.4 0.27 NSc
1.03 0.13 1.03 0.02 NSc
13.4 0.60 13.0 0.58 NSc
10.8 0.23 11.3 0.24 NSc
48.0 48.0 NSc
Significanced Fujihikari TR Significanced a
Data are means standard errors of 10-fruits in `Benikodama', 20-fruits in `Fujihikari TR', respectively. Fruit shape is expressed as the ratio of height to width. c NS: non-signi®cant. d Signi®cant between control and irradiation at P < 0.05. b
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
Table 3 Comparison of seedless watermelon fruits produced by soft-X-irradiated pollens and control (1998)
K. Sugiyama, M. Morishita / Scientia Horticulturae 84 (2000) 255±264
263
fruits from irradiation treatment showed similar or higher sugar content than in controls. In `Fujihikari TR', there was no signi®cant difference in sugar content of irradiated groups except the 1000 Gy (1997 group) and that of controls. All fruits of both `Benikodama' and `Fujihikari TR' matured within almost the same period regardless of soft-X-irradiation. Seedless watermelon fruits induced by hormonal agents had a tendency to be small and deformed compared with normal fruits (Hayata et al., 1995; Kondou and Murozono, 1975). In contrast, seedless watermelon fruits produced by softX-irradiated pollen were normal in size and shape. Previous studies (Kihara, 1951; Kondou and Murozono, 1975) on the production of parthenocarpic seedless watermelon using triploid or plant growth regulators showed a tendency to form a thick rind. In the present experiment, there was no relationship between rind thickness and soft-X-irradiation. In our study, the sugar content of seedless watermelon fruits produced by soft-X-irradiated pollen was similar to or higher than that of controls. These results show that soft-X-irradiation did not affect fruit weight, shape or rind thickness, and that the quality was equal to or better than that of controls. In Japan, the main reason triploid watermelons are not popular is that triploids have a longer growing period and a later maturation date than diploid ones. Occasionally, there have been some developmental defects such as hollow hearts in triploid watermelon fruits (Kihara, 1951, 1958). In contrast, in our experiment, these defects were not present in seedless watermelon produced by pollination with soft-X-irradiated pollen. In conclusion, seedless watermelon produced by pollination with soft-Xirradiated pollen can be produced by ordinary applied cultivation due to the use of common diploids. Therefore, this new technique established in our study has practical value and should be used to produce a good seedless watermelon fruit. Acknowledgements The authors are thankful to Prof. T. Harada of Hokkaido University and Prof. H. Inden of Miyazaki University for valuable suggestions and critical reading of the manuscript. References Compton, M.E., Gray, D.J., Elmstrom, G.W., 1996. Identi®cation of tetraploid regenerants from cotyledons of diploid watermelon cultured in vitro. Euphytica 87, 165±172. Hayata, Y., Niimi, Y., Iwasaki, N., 1995. Synthetic Cytokinin-1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU) Ð promotes fruit set and induces parthenocarpy in watermelon. J. Am. Soc. Hort. Sci. 120, 997±1000.
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Kihara, H., 1951. Triploid watermelon. Proc. Am. Soc. Hort. Sci. 58, 217±230. Kihara, H., 1958. Breeding of seedless fruits. Seiken Ziho 9, 1±7. Kihara, H., Nishiyama, I., 1947. An application of sterility of autotriploids to the breeding of seedless watermelons. Seiken Ziho 3, 93±103 (in Japanese with English summary). Kondou, Y., Murozono, M., 1975. Study of arti®cial fruit setting watermelon. Abstr. Jpn. Soc. Hort. Sci. Autumn Meeting 44, 178±179 (in Japanese). Nishimura, Y., Sakaguchi, S., 1960. Studies on reciprocal translocations of chromosomes in watermelon. Bull. Fac. Agric. Niigata Univ. 12, 22±29. Oka, H., Watanabe, T., Nishiyama, I., 1967. Reciprocal translocation as a new approach to breeding seedless watermelon. Can. J. Genetics Cytol. 9, 482±489. Saitou, T., 1974. Physiology and ecology of fruit set in cucumber. In: The System of Agricultural Technology, Cucumber. Soc. Cult. Agr. VILL. Press, Tokyo, p. 125±126 (in Japanese). Sari, N., Abak, K., Pitrat, M., Rode, J.C., Dumas de Vaulx, R., 1994. Induction of parthenogenetic haploide embryos after pollination by irradiation pollen in watermelon. Hort. Sci. 29, 1189± 1190. Shimotuma, M., 1968. Synthesis of some multiple interchange strains of watermelons induced by X-rays. Seiken Ziho 20, 47±53. Sugiyama, K., Morishita, M., 1998. Storage ability of soft-X-ray irradiated-pollens and effects of storage pollens on fruit characteristics in watermelon. J. Jpn. Soc. Hort. Sci. 67 (suppl. 2), 283 (in Japanese). Terada, J., Masuda, K., 1940. Parthenocarpy of watermelon by heteroauxin. Agric. Hort. 15, 458± 468 (in Japanese). Terada, J., Masuda, K., 1941. Parthenocarpy of watermelon by single or complex application of plant hormones. Agric. Hort. 16, 1915±1917 (in Japanese). Terada, J., Masuda, K., 1943. Parthenocarpy of triploid watermelon. Agric. Hort. 18, 15±16 (in Japanese). Wong, C.Y., 1938. Induced parthenocarpy of watermelon cucumber and pepper by the use of growth promoting substances. Proc. Am. Soc. Hort. Sci. 36, 632±636. Yamamuro, K., 1978. Effect of growth regulators on fruit setting of watermelon. Bull. Ibaraki Hort. Expt. Sta. 7, 1±15.