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Influence of repeated pollination on seed number and fruit shape of ‘Fuji’ apples
Scientia Horticulturae 137 (2012) 131–137
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Influence of repeated pollination on seed number and fruit shape of ‘Fuji’ apples Shogo Matsumoto a,∗ , Junichi Soejima b , Tsutomu Maejima c a b c
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan Apple Research Station, NARO Institute of Fruit Tree Science (NIFTS), Shimokuriyagawa, Morioka 020-0123, Japan Nagano Fruit Tree Experiment Station, Nagano 382-0072, Japan
a r t i c l e
i n f o
Article history: Received 18 September 2011 Received in revised form 20 January 2012 Accepted 25 January 2012 Keywords: Malus × domestica Lopsided fruits S-RNase Pollinizer
a b s t r a c t Lopsided apple fruits were found in an orchard consisting of a single commercial cultivar Fuji and pollinizers of crab-apples under conditions of natural pollination. Speculating that it was caused by inadequate partial pollination, we devised an appropriate model. We artificially pollinated one out of five pistils of apple gynoecium, and then investigated the correlations among inadequate partial pollination, seed number, distribution, and fruit shape. It was speculated that a decrease in seed number and an increase in the rate of lopsidedness were caused by inadequate partial pollination. A deviation in seed distribution was also a factor in the occurrence of lopsided fruits. Since many of the main pollinator Osmia cornifrons visited a flower repeatedly at an orchard consisting of a single cultivar and pollinizers, we analyzed the effect of repeated pollination on increasing and improving seed number in fruits and fruit shape, respectively, using artificial pollination of a pollinizer and cultivars with various S-genotypes. Repeated pollination at 24-h intervals did not contribute to fertilization, but those at 4-h intervals contributed significantly as pollen parents of seeds in fruits. The ability of pistil fertilization was maintained until four days after flowering regardless of pollination, but once fertilization occurred, this pistil was not considered to contribute to seed production 24 h after initial pollination. Those results suggested that pollinizers in full bloom be shifted more than a day seemed unnecessary at an orchard with single commercial cultivar, and repeated artificial pollination must be done within one day. © 2012 Elsevier B.V. All rights reserved.
1. Introduction A new apple (Malus × domestica Borkh.) cultivation system consisting of single commercial cultivars with pollinizers, such as crab-apples, is increasing in Japan because of the sensitive issue of agrochemical sprays for late- or mid-season cultivars drifting to early-season cultivars at harvest time (Matsumoto, 2010). The system contributes to reducing apple production costs through simplification of cultivation management, and optimization of pest control. As apples have gametophytic self-incompatibility controlled by the multi-allelic S-locus (De Nettancourt, 1977; Kobel et al., 1939), we selected pollinizers suitable for commercial cultivars according to their S-genotypes, flowering period synchronization, and so on (Matsumoto et al., 2003, 2007, 2009a). We also selected Osmia cornifrons as a pollinator, and devised a suitable arrangement, such as the proper distance between pollinizers (crab apples) and commercial cultivars, in an orchard for stable commercial apple production (Matsumoto et al., 2008, 2009b).
∗ Corresponding author. Tel.: +81 52 789 4025; fax: +81 52 789 4025. E-mail address: [email protected] (S. Matsumoto). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2012.01.033
We applied the new apple cultivation system to some private apple orchards consisting of ‘Fuji’ and pollinizers. Although it proved the usefulness of this system for reducing pesticide and labor, one issue has remained, i.e., the occurrence of lopsided (asymmetriae) fruits. Many factors influence lopsided ‘Fuji’ fruits, such as the state of fruits borne, the number of keeping inflorescence, the nutrients of tree body storing, and light conditions (Noé and Eccher, 1996). In the new cultivation system, as the rate of ‘Fuji’ fruit produced by the pollinizers decreased to 47% and 39%, respectively, when ‘Fuji’ was 10 m from ‘Maypole’ and ‘Dolgo’ (Matsumoto et al., 2008) and the main pollinator, O. cornifrons, gathered pollen mainly from the area close to the nestiny side, the fruit set was actually significantly reduced at ‘Seirin Spur’ (a ‘Fuji’ sport) Trees 33 m away from the nestiny side and pollinizers ‘Maypole’ (Matsumoto et al., 2009b; Matsumoto and Maejima, 2010; unpublished results). It was suggested that lopsided fruits observed on the trees of low fruit sets might be due to inadequate pollination. Higher levels of pollen density improved the fruit set and size in Japanese pear (Pyrus pyrifolia), and endogenous gibberellin (GA3 and GA4 ) concentrations increased in pollen tubes soon after germination were positively correlated to final fruit size (Zhang et al., 2010). However, the negative effect of reducing pollen load on the apple fruit and seed set was ambiguous (Janse and Verhaegh, 1993).
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On the other hand, the number and distribution of seeds within a developing apple affect its shape and weight (Brault and de Oliveira, 1995; Keulemans et al., 1996). The index of asymmetry increased curvilinearly with decreases in the number of well-formed seeds and of fertilized ovules in ‘McIntosh’ apples (Brault and de Oliveira, 1995). The same influence of seed sets on fruit shape, in which seed is asymmetrically distributed resulted in asymmetrical fruit, and was observed in lopsided ‘Granny Smith’ apples (Drazeta et al., 2004). From the results, it was speculated that the hormone released from seeds in fruits rather than that from pollen tube might be critical for lopsided fruit generation in apple. The five stigmas of ‘Fuji’ flowers are moving away from each other, and inadequate pollination by O. cornifrons, such as pollination of a part of the stigmas might occur since O. cornifrons are passing flowers one after another. To determine whether or not the lopsided apple fruits were caused by such inadequate partial pollination, we planned the artificial pollination experiments, such as pollination of one out of five stigmas of ‘Fuji’ with ‘Maypole’ pollen. Although O. cornifrons showed strong flower constancy during one pollen-nectar foraging trip, the bees seemed to forage for different types of flowers during their 16–22 pollen-nectar foraging trips based on the S-RNase allele and simple sequence repeat analyses of pollen loaves made during those trips (Matsumoto et al., 2009b). As pollination of ‘Fuji’ flowers with different pollinizers by O. cornifrons seemed to occur, we also planned repeated pollination experiments to assess its effect for reducing lopsided fruits, and to apply the information to reduce the lopsided fruit generation. ‘Maypole’ crab apples flowered earlier than did ‘Orin’, and ‘Orin’ flowered earlier than other commercial cultivars, so we used pollen of ‘Orin’ and ‘Chouka 19’ as the 2nd and 3rd pollen sources (1st is ‘Maypole’). In this paper, we investigated the correlation between inadequate partial pollination and the occurrence of lopsided ‘Fuji’ fruits. Increasing the number of seeds in a fruit leading to a decrease in lopsided fruits was observed by repeated pollination experiments. Until now, an effective way to reduce lopsided fruits remains
unknown, despite its attendant economic loss to apple growers. We show the way to produce normal apple fruits efficiently by the careful selection of pollinizers and the timing of repeated pollination. 2. Materials and methods 2.1. Experimental area Our research was carried out from 2007 to 2010 in a 9.0-ha apple orchard of the Nagano Fruit Tree Experiment Station, in Nagano, Japan (Matsumoto et al., 2008). We mainly used two ‘Fuji’ trees grafted onto a JM7 rootstock in 1988, and planted block no. 17 (2000 m2 ) in 1989. 2.2. Pollination Anthers of flowers at the popcorn stage of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ were collected on paper for wrapping powdered medicine and allowed to dehisce for 24–48 h at 25 ◦ C. After dehiscence, the anthers in the paper were placed in a glass bottle with dry silica gel and kept in a cool, dry place. Two ‘Fuji’ trees, now 24 years old, were used as female parents. Flowers randomly chosen at the “popcorn” stage were emasculated and cross-pollinated. We only used terminal, or ‘king’ flower, which is the first to open; flower clusters in lateral buds that open later than those in terminal buds were removed. As for inadequate partial pollination, we cut two or four out of five stigmas with scissors at approx. 3–5 mm below the stigmatic surface and cauterized them with a heated forceps. The remaining stigmas were cross-pollinated with undiluted pollen, then wrapped with paper bags to avoid natural pollination. After 4 or 24 h, repeated pollination was carried out in the order of 1st ‘Maypole’, 2nd ‘Orin’, and 3rd ‘Chouka 19’ pollen.
Fig. 1. Mean seed number in a ‘Fuji’ apple (A), and mean rate of lopsidedness in ‘Fuji’ fruits (B) for each pollination treatment. One out of five or five stigmas were pollinated artificially by ‘Maypole’ pollen. The symbols a and b above the columns of (A) and (B) denote a significant difference at a 5% risk.
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Table 1 Rate of lopsided fruits in a commercial apple orchard consisting of ‘Fuji’ (S1 S9 ) with pollinizer ‘Neville Corpman’ (S24 S26 ). Tree number
Year
Fruit set (%)
No of. lopsided fruits
No. of normal fruits
Rate of lopsided fruits (%)
B-7 S-9 S-2 B-5
2008 2009 2008 2009
37 37 72 99
19 12 6 3
31 19 44 30
38.0 a 38.7 a 12.0 b 9.1 b
Values followed by a different letter within the same column differ significantly at the 1% level by Chi-square test.
2.3. Fruit set, measurement and statistical analyses We judged that the flowers with standing sepals and a tendency toward swollen ovaries and surrounding receptacle tissues had succeeded in fertilizing and setting fruit. Hand-fruit-thinning was performed to achieve a possible rate of one fruit per 50–60 mature leaves (approximately one fruit per five terminal buds) at the end of June. In mid-October, we measured the fruit weight and length of longitudinal diameters using electric vernier calipers. The diameters of seven samples from 2008 and nine samples from 2009 were measured at the end of July. Rates of lopsidedness (%) were obtained by the formula: (maximum longitudinal diameter/minimum longitudinal diameter − 1) × 100. The rate of lopsided fruits (%) denotes the rate of those fruits showing ≥15% lopsidedness. Chi-square test was applied to determine the difference between the rate of lopsided fruits on low and high fruit set trees, and between partial (used one out of five pistils for pollination) and normal (five pistils for pollination) pollinations (Tables 1 and 2). Student’s t-test was applied to test for the difference of seed number in fruits between partial and normal pollinations (Fig. 1A), and in fruit weights between lopsided and normal fruits (Fig. 4). We used Mann–Whitney’s U test to determine the difference in the rate of lopsidedness between partial and normal pollinations (Fig. 1B), since the normality of partial pollination data showed a deviation. The scatter diagram shown in Fig. 2 was obtained using simple regression analysis. The rates of pollen germination among the pollen of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ were compared using a Bartlett test and one-factor ANOVA. 2.4. S-allele-amplification and allele-specific PCR digestion Total DNA was isolated from the leaves of individual plants as described by Thomas et al. (1993). The primers and conditions used for S-allele-specific PCR-RFLP analyses were those described by Broothaerts et al. (1995; for S2 and S3 ), Janssens et al. (1995; for S7 ),
Kitahara and Matsumoto (2002; for S10 ), Matsumoto et al. (1999; for S5 and S7 ), and Verdoodt et al. (1998; for S16(=27) )). 3. Results and discussion 3.1. ‘Fuji’ fruits of oblique shape in an apple orchard, and influence of inadequate pollination on fruit shape O. cornifrons have been used as pollinators for apple cultivar Fuji production at a 9.0-ha apple orchard of the Nagano fruit tree station since its foraging behavior is superior to that of the honey bee (Matsumoto et al., 2009b). The orchard was partitioned into 49 blocks, and no severely lopsided fruits were observed at the block consisting of a single cultivar Fuji and ‘Maypole’ or ‘Dolgo’ pollinizers. Though that was probably due to the pollen flow from commercial cultivars planted outside the block, lopsided fruits were observed in an orchard consisting of commercial cultivar Fuji and pollinizer ‘Neville corpman’ at Shiwa-cho, Iwate Prefecture, Japan. As shown in Table 1, the rate of lopsided fruits on low pollination trees was significantly higher than that on high pollination trees. As ‘Fuji’ is a solely commercial cultivar at that location, we speculated that the high rate of lopsided fruits must have been produced by inadequate pollination. To confirm that such a high rate of lopsided fruits observed at the orchard consisting of ‘Fuji’ and ‘Neville Corpman’ was due to inadequate pollination, we carried out an artificial partial pollination, i.e., we pollinated one out of five stigmas of ‘Fuji’ with ‘Maypole’ pollen, and then investigated the rate of lopsidedness. That rate was significantly higher than that of normal pollination and three out of five stigmas used for pollination in 2007 (Table 2). As the rates of lopsided fruits between the normal and partial pollinations of three out of five pistils used for pollination were not significantly different, we continued the experiments (except for the partial pollination of three out of five stigmas) for more than two years, and confirmed the results (Table 2). Those results suggested that the high rates of lopsided fruits were caused by inadequate partial pollination. 3.2. Correlation between seed number with distribution in fruit and fruit shape, and influence of inadequate partial pollination on fruit weights
Fig. 2. Correlation between seed number and lopsidedness of ‘Fuji’ apple fruits. (䊉) Partial pollination in 2007 (see Table 2); () partial pollination in 2008 (see Table 2); () partial pollination in 2009 (see Table 2); () partial pollination in 2010 (see Table 2); () normal pollination in 2007 (see Table 2); () normal pollination in 2008 (see Table 2); () normal pollination in 2009 (see Table 2); (♦) normal pollination in 2010 (see Table 2).
We investigated the number of ‘Fuji’ fruit seeds shown in Table 2. The number of seeds in ‘Fuji’ apple obtained by partial artificial pollination (using one out of five pistils) was significantly reduced from that of 8.6 ± 0.4 (n = 41) of normal pollination (using five pistils) to 5.1 ± 0.3 (n = 44) (Fig. 1A). We also investigated the difference in the rates of lopsidedness between partial and normal pollinations, and demonstrated that the rates had significantly increased from 8.6 ± 0.6 (n = 41) of normal pollination to 15.1 ± 1.1 (n = 44) of partial pollination (Fig. 1B). The negative correlation between seed number in a fruit and their lopsidedness was obtained using regression analysis (Fig. 2). It was calculated from the formula y = −1.126x + 19.617, which was obtained as a function of a regression line, that <4.10 seeds in a fruit became lopsided (≥15% lopsidedness). Although fruits having less than two seeds exhibited lopsided fruits, those with more than three showed both
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Table 2 Rate of lopsided ‘Fuji’ (S1 S9 ) fruits by artificial pollination with ‘Maypole’ (S10 S16 ) pollen. No. of stigmas pollinated
Year
No. of lopsided fruits
No. of normal fruits
Rate of lopsided fruits (mean ± S.E.%)
1 1 1 1 1 3 5 5 5 5 5
2007 2008 2009 2010 2007–2010 2007 2007 2008 2009 2010 2007–2010
6 6 3 6
10 4 3 6
2 3 0 0 0
16 15 10 3 10
37.5 60.0 50.0 50.0 49.4 ± 4.6 a 11.1 16.7 0.0 0.0 0.0 4.2 ± 4.2 b
Values followed by a different letter within the same column differ significantly at the 0.1% level by Chi-square test.
Table 3 S-RNase allele analyses of individual seeds in a ‘Fuji’ (S1 S9 ) fruit obtained by repeated pollination at 4- or 24-h intervals (order of pollen used: 1st ‘Maypole’ (S10 S16 ), 2nd ‘Orin’ (S2 S7 ), 3rd ‘Chouka 19’ (S3 S5 ). Year
No. of lopsided fruits
Rate of No. of normal fruits lopsided fruits
No. of total seeds
No. of seeds/fruit
Pollination
Intervals (h)
No. of pollinated stigma No. of repeated times
2008 2008 2008 2008 2009 2009 2009 2009 2010
2 0 2 0 1 0 0 0 0
4 5 2 6 4 3 4 6 3
33.3 0.0 50.0 0.0 25.0 0.0 0.0 0.0 0.0
32 61 21 50 34 24 33 56 31
5.3 12.2 5.3 8.3 6.8 8.0 8.3 9.3 10.2
1 5 1 5 1 5 1 5 1
2 2 3 3 2 2 3 3 3
No. of seeds S-RNase alleles detected in seeds
Maypole Orin
24 24 24 24 4 4 4 4 4
Chouka 19
S10 S16 S2
S7
S3
S5
14 20 9 22 6 9 4 7 6
0 0 0 1 11 7 0 13 5
– – 0 0 – – 5 7 11
– – 0 0 – – 15 12 4
18 41 12 27 13 1 9 7 0
0 0 0 0 4 7 0 10 5
Fig. 3. Correlation between the rates of lopsided ‘Fuji’ fruits and seed distribution in the fruit. Sectors observed with one to four seeds were shaded. The 44 fruits investigated were obtained by partial pollination (one out of five pistils used for pollination) (see Table 2). Rate of lopsided fruits (number of lopsided fruits/total fruits), seed number with a standard error (mean ± S.E.) were indicated below each figure in the upper row, and those from fruits containing more than five seeds were indicated in the lower row.
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reduced compared with that (299.1 g ± 13.9) of normal artificial pollination (7th row in Table 2). The five stigmas in apple fused at the middle of styles different from those in Japanese pear, and the hormones released from the pollen tube soon after the germination might be less important for apple lopsided fruit generation. 3.3. Effect of repeated pollination for production of normal-shaped fruits
Fig. 4. Average apple fruit weights (g) with standard error (mean ± S.E.) of lopsided (2007, n = 11; 2009, n = 12) and normal (2007, n = 41; 2009, n = 19) obtained by partial (artificial; 2007) and natural (2009) pollinations. The symbols a and a above the columns denote no significant difference at a 5% risk.
normal and lopsided fruits (Fig. 2). We investigated seed distributions in 44 fruits obtained by partial pollination (Fig. 3). The rate of lopsided fruits increased with the degree of biased seed distribution in a fruit. From 77.8 to 100% of fruits having seeds in only one or two out of five sectors became lopsided, and the rate decreased upon reducing the degree of biased seed distribution, i.e., from 58.3% (having seeds in three sectors), 22.2% (having seeds in four sectors) to 10.0% (having seeds in all five sectors) (Fig. 3). We chose fruits containing more than five seeds, and confirmed the tendency of biased seed distribution to result in the generation of lopsided fruits. This suggested that biased seed distribution in addition to low seed number in a fruit might be related to the formation of lopsided fruits. The lopsided shapes might be formed by the hormone (e.g., indoleacetic acid and gibberellin) derived from seeds in fruits with the uneven distribution of nutrients and water within the pulps. In our experiment, the ‘Fuji’ fruits containing more than five seeds were obtained despite being pollinated at one out of five pistils (Fig. 3). It was suggested that the gynoecium of ‘Fuji’ is perfectly syncarpic, as was indicated in the cultivar Summerland McIntosh (Sheffield et al., 2005). We compared the weights of 41 and 19 normal fruits with those of the 11 and 12 lopsided fruits obtained in 2007 (partial pollination, 1st, 6th and 7th row in Table 2) and 2009 (natural pollination, S-9 in Table 1), respectively. As shown in Fig. 4, the average weight of lopsided fruits (261.9 g ± 13.1, 326.8 g ± 14.8) was slightly reduced compared with those of normal fruits (304.5 g ± 10.5, 368.3 g ± 13.4), but the difference was dismissed at the 5% risk by Student’s t-test. Although Keulemans et al. (1996) found a positive correlation between fruit weights and the number of welldeveloped seeds, we could find no such correlation (Fig. 4). The weight of fruits produced by partial pollination was the same as those produced by normal pollination despite the decreasing number of seeds. As Keulemans et al. (1996) pointed out, this might be a characteristic of ‘Fuji’. The average fruit weight (281.3 g ± 14.7) of partial artificial pollination (1st row in Table 2) was not significantly
As lopsided fruits were not shipped as fresh use material, but instead were shipped as processing material low in price, apple growers prefer to suppress its generation at their orchards to avoid an economic loss. Since lopsided fruits seemed to be produced by inadequate partial pollination, we carried out repeated pollination to determine the proper timing of pollination for suppressing the generation of lopsided fruits. To reveal the accurate contribution of repeated pollination for increasing seed number in a ‘Fuji’ fruit, we used ‘Maypole’ (S10 S16 ), ‘Orin’ (S2 S7 ) and ‘Chouka 19’ (S3 S5 ) pollen, with different S-genotypes. The rate of fruit set of cross pollination of fully compatible combination was identical with that of a semi-compatible combination, however, the seed number per fruit were reduced by the semi-compatible combination (Matsumoto et al., 2006). All three cultivars show full compatibility with ‘Fuji’ (S1 S9 ), and the order of the pollen used, that is, 1st ‘Maypole’, 2nd ‘Orin’ and 3rd ‘Chouka 19’, in the orchard flowering order. The rate of pollen germination was investigated using more than 100 randomly selected pollens spread on a 1% agar dish containing 17% sucrose after 24 h incubation at 23 ◦ C. The rate of pollen germination of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ exhibited 61.8 ± 12.1%, 78.6 ± 6.7% and 76.7 ± 13.0% in 2008, and 60.5 ± 6.7%, 53.0 ± 7.9% and 71.4 ± 13.5% in 2009, respectively, from the thrice-repeated experiments. The average length of randomly selected 12–22 germinated pollens of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ was measured after a 2-h incubation. The length of germination pollen of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ was 152.8 ± 22.8 m, 155.9 ± 14.4 m and 190.4 ± 18.8 m in 2008, and 268.8 ± 15.3 m, 207.8 ± 21.9 m and 244.1 ± 14.8 m in 2009, respectively. The difference between the rate of pollen germination and the average length of germinated pollen among ‘Maypole’, ‘Orin’ and ‘Chouka 19’ was dismissed at the 5% risk by one-factor ANOVA. Repeated pollination using one out of five or five stigma(s) at 24-h intervals was carried out in 2008 because the fine weather continued. As we used undiluted pollen covering each stigma completely throughout artificial pollination experiments, the pollen competition seemed to be strong as had been pointed out by Zhang et al. (2010). In apples, since no distortion for segregation at the Slocus was observed at fully compatible cross-pollination, the pollen competition among pollen containing different S-alleles seemed to be identical with that among pollen containing the same S-allele. In the case of partial pollination using one of five pistils, all the seeds in fruits obtained by repeated pollination were produced by the ‘Maypole’, not ‘Orin’ or ‘Chouka 19’ pollen (Table 3, Fig. 5). The same results (except for 1 out of 50 seeds produced by ‘Orin’ pollen) were obtained by normal pollination using five pistils (Table 3, Fig. 5). Since two and three times repeated pollination at 24-h intervals provided almost no contribution to seed production, it
Table 4 Rate of ‘Fuji’ (S1 S9 ) fruit set fertilized by ‘Maypole’ (S10 S16 ) pollen. Days after flowering
No. of flowers
No. of fruits
Fruit set (%)
No. of seeds/fruits
0 1 2 3 4 5
4 5 5 4 6 7
4 5 5 4 4 0
100 100 100 100 67 0
8.5 9.2 8.6 8.8 7.5 –
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Table 5 Rate of ‘Fuji’ (S1 S9 ) fruit set fertilized by 1st ‘Fuji’ and 2nd ‘Maypole’ (S10 S16 ) or ‘Fuji’ pollen. Pollen cultivar Days of flowering
1 day after flowering
3 days after flowering
Fuji Fuji Fuji
Maypole –
– Maypole Fuji
No. of flowers
No. of fruits
Fruit set (%)
No. of seeds/fruits
5 6 5
5 6 0
100 100 0
8.4 8.7 –
pollination at 4-h intervals contributed to increasing seed number in a fruit and decreasing lopsided fruits, suggesting that artificial pollination within one day was effective in reducing lopsided fruits. As repeated pollination at 24-h intervals contributed nothing to seed production in a fruit, pollinizers not overlapping their flowering periods might be unnecessary in an apple orchard consisting of single cultivar. Fig. 5. Examples of S-RNase allele-specific PCR amplifications. Some seeds in Table 3 possessing either of S10 , S16 , S2 , S7 , S3 and S5 were used. Lanes 1–6 represent the seed possessing S10 (282 bp), S16 (278 bp), S2 (449 bp), S7 (440 bp), S3 (375 bp), and S5 (363 bp).
was suggested that once pollination or fertilization occurred, the gynoecium lost its ability to accept new pollen within 24 h, even though the empty ovules were intact. We investigated the period during which the ‘Fuji’ gynoecium was able to be fertilized. ‘Fuji’ flowers at the popcorn stage were bagged to avoid natural pollination, then artificially pollinated by ‘Maypole’ pollen from the date of flowering to five days later (e.g., five days; bags were removed from the flowers five days after flowering, then pollinated). As shown in Table 4, ‘Fuji’ genoecium kept its ability for fertilization from the day of flower opening to 4 days after flowering. The gynoecium seemed to lose its fertilization ability within one day by pollination or fertilization, in spite of maintaining its ability for fertilization for 4 days. We speculated that this was caused by fertilization rather than by pollination, since the 2nd pollen ‘Maypole’ was accepted two days after the pollination of incompatible pollen ‘Fuji’ (Table 5). In the field, 24 h after hand pollination of P. pyrifolia, pollen tubes in the pistil for the undiluted pollen treatment grew faster than pollen tubes in the diluted pollen treatment, and the pollen tubes had almost reached the base of the style (Zhang et al., 2010). Those results suggested that the 2nd pollination one day after the 1st contributed nothing to increase the seed number in the fruit, which was connected to the diminished generation of lopsided fruits. In 2009, we then carried out repeated pollination at 4-h intervals. In contrast to the 24-h interval results, we detected 15 out of 34, and 14 out of 24 seeds produced in the 2nd pollen ‘Orin’ by partial and normal pollinations, respectively (Table 4). We also detected 20 out of 33, and 19 out of 56 seeds produced by the 3rd pollen ‘Chouka 19’ by partial and normal pollinations, respectively (Table 4). As in the case of a thrice-repeated partial pollination, seeds produced by the 2nd pollination were not detected. For reasons unknown, we detected seeds produced by the 2nd pollen ‘Orin’ as well as the 3rd pollen ‘Chouka 19’ at the rates of 10 out or 31 and 15 out of 31, respectively, in 2010 (Table 4). The seeds in fruits obtained by two- and three-time repeated partial pollination at 4-h intervals increased compared with those at 24-h intervals, and the rate of lopsided fruits decreased compared with those at 24-h intervals. 4. Conclusions Our experiments in inadequate partial pollination using one out of five pistils for pollination contributed to increasing generation of lopsided ‘Fuji’ apple fruits and to decreasing in the seed number in a fruit. Biased seed distribution also seemed related to the generation of lopsided ‘Fuji’ fruits. Two- or three-time repeated
Acknowledgements We are indebted to Ms. A. Kojima and Ms. J. Morita for their valuable technical assistance. This research was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Nos. 17580024 and 23658026), and the Research Project for Utilizing Advanced Technologies in Agriculture, Forestry and Fisheries. References Brault, A., de Oliveira, D., 1995. Seed number and an asymmetry index of ‘McIntosh’ apples. HortScience 30, 44–46. Broothaerts, W., Janssens, G.A., Proost, P., Broekaert, W.F., 1995. cDNA cloning and molecular analysis of two self-incompatibility alleles from apple. Plant Mol. Biol. 27, 499–511. De Nettancourt, D., 1977. Incompatibility in angiosperms. In: Frankel, R., Gal, G.A.E., Linskens, H.F. (Eds.), Monographs on Theoretical and Applied Genetics. Springer, Heide, Germany, pp. 28–57. Drazeta, L., Lang, A., Hall, A.J., Volz, R.K., Jameson, P.E., 2004. Modeling the influence of seed set on fruit shape in apple. J. Hortic. Sci. Biotechnol. 79, 241–245. Janse, J., Verhaegh, J.J., 1993. Effects of varying pollen load on fruit set, seed set and seedling performance in apple and pear. Sex. Plant Reprod. 6, 122–126. Janssens, G.A., Goderis, I.J., Broekaert, W.F., Broothaerts, W., 1995. A molecular method for S-allele identification in apple based on allele-specific PCR. Theor. Appl. Genet. 91, 691–698. Keulemans, J., Brusselle, A., Eyssen, R., Vercammen, J., van Daele, G., 1996. Fruit weight in apple as influenced by seed number and pollinizer. Acta Hort. 423, 201–210. Kitahara, K., Matsumoto, S., 2002. Sequence of the S10 cDNA from ‘McIntosh’ apple and a PCR-digestion identification method. HortScience 37, 187–190. Kobel, F., Steinegger, P., Anliker, J., 1939. Weitere Untersuchungen über die Befruchtungsverhältnisse der Apfel-und Birnsorten. Landvirtschaftliches Jahrbuch der Schweiz 53, 160–191. Matsumoto, S., Komori, S., Kitahara, K., Imazu, S., Soejima, J., 1999. S-genotypes of 15 apple cultivars and self-compatibility of ‘Megumi’. J. Jpn. Soc. Hort. Sci. 68, 236–241. Matsumoto, S., Furusawa, Y., Komatsu, H., Soejima, J., 2003. S-allele genotypes of apple pollenizers, cultivars and lineages including those resistant to scab. J. Hortic. Sci. Biotechnol. 78, 634–637. Matsumoto, S., Kitahara, K., Komatsu, H., Abe, K., 2006. Cross-compatibility of apple cultivars possessing S-RNase alleles of similar sequence. J. Hortic. Sci. Biotechnol. 81, 934–936. Matsumoto, S., Eguchi, T., Bessho, H., Abe, K., 2007. Determination and confirmation of S-RNase genotypes of apple pollenators and cultivars. J. Hortic. Sci. Biotechnol. 82, 323–329. Matsumoto, S., Eguchi, T., Maejima, T., Komatsu, H., 2008. Effect of distance from early flowering pollinizers ‘Maypole’ and ‘Dolgo’ on ‘Fuji’ fruit set. Sci. Hortic. 117, 151–159. Matsumoto, S., Morita, J., Abe, K., Bessho, H., Yamada, K., Shiratake, K., Fukui, H., 2009a. S-RNase genotypes of wild apples necessary for utilization as pollinizers. Hort. Environ. Biotechnol. 50, 213–216. Matsumoto, S., Abe, A., Maejima, T., 2009b. Foraging behavior of Osmia cornifrons in an apple orchard. Sci. Hortic. 121, 73–79. Matsumoto, S., Maejima, T., 2010. Several new aspects of the foraging behaviour of Osmia cornifrons in an apple orchard. Psyche 2010, 6, doi:10.1155/2010/384371. Matsumoto, S., 2010. Development of safe new cultivation system for apples based on S-RNase and SSR information. In: Sampson, A.N. (Ed.), Horticulture in the 21st century. Nova Science Publishers, Inc., New York, pp. 203–211. Noé, N., Eccher, T., 1996. ‘Golden Delicious’ apple fruit shape and russeting are affected by light conditions. Sci. Hort. 65, 209–213.
S. Matsumoto et al. / Scientia Horticulturae 137 (2012) 131–137 Sheffield, C.S., Smith, R.F., Kevan, P.G., 2005. Perfect syncarpy in apple (Malus × domestica ‘Summerland McIntosh’) and its implications for pollination, seed distribution and fruit production (Rosaceae: Maloideae). Ann. Bot. 95, 583–591. Thomas, M., Matsumoto, S., Cain, P., Scott, N.S., 1993. Repetitive DNA of grapevine: class present and sequences suitable for cultivar identification. Theor. Appl. Genet. 86, 173–180.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Influence of repeated pollination on seed number and fruit shape of ‘Fuji’ apples Shogo Matsumoto a,∗ , Junichi Soejima b , Tsutomu Maejima c a b c
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan Apple Research Station, NARO Institute of Fruit Tree Science (NIFTS), Shimokuriyagawa, Morioka 020-0123, Japan Nagano Fruit Tree Experiment Station, Nagano 382-0072, Japan
a r t i c l e
i n f o
Article history: Received 18 September 2011 Received in revised form 20 January 2012 Accepted 25 January 2012 Keywords: Malus × domestica Lopsided fruits S-RNase Pollinizer
a b s t r a c t Lopsided apple fruits were found in an orchard consisting of a single commercial cultivar Fuji and pollinizers of crab-apples under conditions of natural pollination. Speculating that it was caused by inadequate partial pollination, we devised an appropriate model. We artificially pollinated one out of five pistils of apple gynoecium, and then investigated the correlations among inadequate partial pollination, seed number, distribution, and fruit shape. It was speculated that a decrease in seed number and an increase in the rate of lopsidedness were caused by inadequate partial pollination. A deviation in seed distribution was also a factor in the occurrence of lopsided fruits. Since many of the main pollinator Osmia cornifrons visited a flower repeatedly at an orchard consisting of a single cultivar and pollinizers, we analyzed the effect of repeated pollination on increasing and improving seed number in fruits and fruit shape, respectively, using artificial pollination of a pollinizer and cultivars with various S-genotypes. Repeated pollination at 24-h intervals did not contribute to fertilization, but those at 4-h intervals contributed significantly as pollen parents of seeds in fruits. The ability of pistil fertilization was maintained until four days after flowering regardless of pollination, but once fertilization occurred, this pistil was not considered to contribute to seed production 24 h after initial pollination. Those results suggested that pollinizers in full bloom be shifted more than a day seemed unnecessary at an orchard with single commercial cultivar, and repeated artificial pollination must be done within one day. © 2012 Elsevier B.V. All rights reserved.
1. Introduction A new apple (Malus × domestica Borkh.) cultivation system consisting of single commercial cultivars with pollinizers, such as crab-apples, is increasing in Japan because of the sensitive issue of agrochemical sprays for late- or mid-season cultivars drifting to early-season cultivars at harvest time (Matsumoto, 2010). The system contributes to reducing apple production costs through simplification of cultivation management, and optimization of pest control. As apples have gametophytic self-incompatibility controlled by the multi-allelic S-locus (De Nettancourt, 1977; Kobel et al., 1939), we selected pollinizers suitable for commercial cultivars according to their S-genotypes, flowering period synchronization, and so on (Matsumoto et al., 2003, 2007, 2009a). We also selected Osmia cornifrons as a pollinator, and devised a suitable arrangement, such as the proper distance between pollinizers (crab apples) and commercial cultivars, in an orchard for stable commercial apple production (Matsumoto et al., 2008, 2009b).
∗ Corresponding author. Tel.: +81 52 789 4025; fax: +81 52 789 4025. E-mail address: [email protected] (S. Matsumoto). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2012.01.033
We applied the new apple cultivation system to some private apple orchards consisting of ‘Fuji’ and pollinizers. Although it proved the usefulness of this system for reducing pesticide and labor, one issue has remained, i.e., the occurrence of lopsided (asymmetriae) fruits. Many factors influence lopsided ‘Fuji’ fruits, such as the state of fruits borne, the number of keeping inflorescence, the nutrients of tree body storing, and light conditions (Noé and Eccher, 1996). In the new cultivation system, as the rate of ‘Fuji’ fruit produced by the pollinizers decreased to 47% and 39%, respectively, when ‘Fuji’ was 10 m from ‘Maypole’ and ‘Dolgo’ (Matsumoto et al., 2008) and the main pollinator, O. cornifrons, gathered pollen mainly from the area close to the nestiny side, the fruit set was actually significantly reduced at ‘Seirin Spur’ (a ‘Fuji’ sport) Trees 33 m away from the nestiny side and pollinizers ‘Maypole’ (Matsumoto et al., 2009b; Matsumoto and Maejima, 2010; unpublished results). It was suggested that lopsided fruits observed on the trees of low fruit sets might be due to inadequate pollination. Higher levels of pollen density improved the fruit set and size in Japanese pear (Pyrus pyrifolia), and endogenous gibberellin (GA3 and GA4 ) concentrations increased in pollen tubes soon after germination were positively correlated to final fruit size (Zhang et al., 2010). However, the negative effect of reducing pollen load on the apple fruit and seed set was ambiguous (Janse and Verhaegh, 1993).
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On the other hand, the number and distribution of seeds within a developing apple affect its shape and weight (Brault and de Oliveira, 1995; Keulemans et al., 1996). The index of asymmetry increased curvilinearly with decreases in the number of well-formed seeds and of fertilized ovules in ‘McIntosh’ apples (Brault and de Oliveira, 1995). The same influence of seed sets on fruit shape, in which seed is asymmetrically distributed resulted in asymmetrical fruit, and was observed in lopsided ‘Granny Smith’ apples (Drazeta et al., 2004). From the results, it was speculated that the hormone released from seeds in fruits rather than that from pollen tube might be critical for lopsided fruit generation in apple. The five stigmas of ‘Fuji’ flowers are moving away from each other, and inadequate pollination by O. cornifrons, such as pollination of a part of the stigmas might occur since O. cornifrons are passing flowers one after another. To determine whether or not the lopsided apple fruits were caused by such inadequate partial pollination, we planned the artificial pollination experiments, such as pollination of one out of five stigmas of ‘Fuji’ with ‘Maypole’ pollen. Although O. cornifrons showed strong flower constancy during one pollen-nectar foraging trip, the bees seemed to forage for different types of flowers during their 16–22 pollen-nectar foraging trips based on the S-RNase allele and simple sequence repeat analyses of pollen loaves made during those trips (Matsumoto et al., 2009b). As pollination of ‘Fuji’ flowers with different pollinizers by O. cornifrons seemed to occur, we also planned repeated pollination experiments to assess its effect for reducing lopsided fruits, and to apply the information to reduce the lopsided fruit generation. ‘Maypole’ crab apples flowered earlier than did ‘Orin’, and ‘Orin’ flowered earlier than other commercial cultivars, so we used pollen of ‘Orin’ and ‘Chouka 19’ as the 2nd and 3rd pollen sources (1st is ‘Maypole’). In this paper, we investigated the correlation between inadequate partial pollination and the occurrence of lopsided ‘Fuji’ fruits. Increasing the number of seeds in a fruit leading to a decrease in lopsided fruits was observed by repeated pollination experiments. Until now, an effective way to reduce lopsided fruits remains
unknown, despite its attendant economic loss to apple growers. We show the way to produce normal apple fruits efficiently by the careful selection of pollinizers and the timing of repeated pollination. 2. Materials and methods 2.1. Experimental area Our research was carried out from 2007 to 2010 in a 9.0-ha apple orchard of the Nagano Fruit Tree Experiment Station, in Nagano, Japan (Matsumoto et al., 2008). We mainly used two ‘Fuji’ trees grafted onto a JM7 rootstock in 1988, and planted block no. 17 (2000 m2 ) in 1989. 2.2. Pollination Anthers of flowers at the popcorn stage of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ were collected on paper for wrapping powdered medicine and allowed to dehisce for 24–48 h at 25 ◦ C. After dehiscence, the anthers in the paper were placed in a glass bottle with dry silica gel and kept in a cool, dry place. Two ‘Fuji’ trees, now 24 years old, were used as female parents. Flowers randomly chosen at the “popcorn” stage were emasculated and cross-pollinated. We only used terminal, or ‘king’ flower, which is the first to open; flower clusters in lateral buds that open later than those in terminal buds were removed. As for inadequate partial pollination, we cut two or four out of five stigmas with scissors at approx. 3–5 mm below the stigmatic surface and cauterized them with a heated forceps. The remaining stigmas were cross-pollinated with undiluted pollen, then wrapped with paper bags to avoid natural pollination. After 4 or 24 h, repeated pollination was carried out in the order of 1st ‘Maypole’, 2nd ‘Orin’, and 3rd ‘Chouka 19’ pollen.
Fig. 1. Mean seed number in a ‘Fuji’ apple (A), and mean rate of lopsidedness in ‘Fuji’ fruits (B) for each pollination treatment. One out of five or five stigmas were pollinated artificially by ‘Maypole’ pollen. The symbols a and b above the columns of (A) and (B) denote a significant difference at a 5% risk.
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Table 1 Rate of lopsided fruits in a commercial apple orchard consisting of ‘Fuji’ (S1 S9 ) with pollinizer ‘Neville Corpman’ (S24 S26 ). Tree number
Year
Fruit set (%)
No of. lopsided fruits
No. of normal fruits
Rate of lopsided fruits (%)
B-7 S-9 S-2 B-5
2008 2009 2008 2009
37 37 72 99
19 12 6 3
31 19 44 30
38.0 a 38.7 a 12.0 b 9.1 b
Values followed by a different letter within the same column differ significantly at the 1% level by Chi-square test.
2.3. Fruit set, measurement and statistical analyses We judged that the flowers with standing sepals and a tendency toward swollen ovaries and surrounding receptacle tissues had succeeded in fertilizing and setting fruit. Hand-fruit-thinning was performed to achieve a possible rate of one fruit per 50–60 mature leaves (approximately one fruit per five terminal buds) at the end of June. In mid-October, we measured the fruit weight and length of longitudinal diameters using electric vernier calipers. The diameters of seven samples from 2008 and nine samples from 2009 were measured at the end of July. Rates of lopsidedness (%) were obtained by the formula: (maximum longitudinal diameter/minimum longitudinal diameter − 1) × 100. The rate of lopsided fruits (%) denotes the rate of those fruits showing ≥15% lopsidedness. Chi-square test was applied to determine the difference between the rate of lopsided fruits on low and high fruit set trees, and between partial (used one out of five pistils for pollination) and normal (five pistils for pollination) pollinations (Tables 1 and 2). Student’s t-test was applied to test for the difference of seed number in fruits between partial and normal pollinations (Fig. 1A), and in fruit weights between lopsided and normal fruits (Fig. 4). We used Mann–Whitney’s U test to determine the difference in the rate of lopsidedness between partial and normal pollinations (Fig. 1B), since the normality of partial pollination data showed a deviation. The scatter diagram shown in Fig. 2 was obtained using simple regression analysis. The rates of pollen germination among the pollen of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ were compared using a Bartlett test and one-factor ANOVA. 2.4. S-allele-amplification and allele-specific PCR digestion Total DNA was isolated from the leaves of individual plants as described by Thomas et al. (1993). The primers and conditions used for S-allele-specific PCR-RFLP analyses were those described by Broothaerts et al. (1995; for S2 and S3 ), Janssens et al. (1995; for S7 ),
Kitahara and Matsumoto (2002; for S10 ), Matsumoto et al. (1999; for S5 and S7 ), and Verdoodt et al. (1998; for S16(=27) )). 3. Results and discussion 3.1. ‘Fuji’ fruits of oblique shape in an apple orchard, and influence of inadequate pollination on fruit shape O. cornifrons have been used as pollinators for apple cultivar Fuji production at a 9.0-ha apple orchard of the Nagano fruit tree station since its foraging behavior is superior to that of the honey bee (Matsumoto et al., 2009b). The orchard was partitioned into 49 blocks, and no severely lopsided fruits were observed at the block consisting of a single cultivar Fuji and ‘Maypole’ or ‘Dolgo’ pollinizers. Though that was probably due to the pollen flow from commercial cultivars planted outside the block, lopsided fruits were observed in an orchard consisting of commercial cultivar Fuji and pollinizer ‘Neville corpman’ at Shiwa-cho, Iwate Prefecture, Japan. As shown in Table 1, the rate of lopsided fruits on low pollination trees was significantly higher than that on high pollination trees. As ‘Fuji’ is a solely commercial cultivar at that location, we speculated that the high rate of lopsided fruits must have been produced by inadequate pollination. To confirm that such a high rate of lopsided fruits observed at the orchard consisting of ‘Fuji’ and ‘Neville Corpman’ was due to inadequate pollination, we carried out an artificial partial pollination, i.e., we pollinated one out of five stigmas of ‘Fuji’ with ‘Maypole’ pollen, and then investigated the rate of lopsidedness. That rate was significantly higher than that of normal pollination and three out of five stigmas used for pollination in 2007 (Table 2). As the rates of lopsided fruits between the normal and partial pollinations of three out of five pistils used for pollination were not significantly different, we continued the experiments (except for the partial pollination of three out of five stigmas) for more than two years, and confirmed the results (Table 2). Those results suggested that the high rates of lopsided fruits were caused by inadequate partial pollination. 3.2. Correlation between seed number with distribution in fruit and fruit shape, and influence of inadequate partial pollination on fruit weights
Fig. 2. Correlation between seed number and lopsidedness of ‘Fuji’ apple fruits. (䊉) Partial pollination in 2007 (see Table 2); () partial pollination in 2008 (see Table 2); () partial pollination in 2009 (see Table 2); () partial pollination in 2010 (see Table 2); () normal pollination in 2007 (see Table 2); () normal pollination in 2008 (see Table 2); () normal pollination in 2009 (see Table 2); (♦) normal pollination in 2010 (see Table 2).
We investigated the number of ‘Fuji’ fruit seeds shown in Table 2. The number of seeds in ‘Fuji’ apple obtained by partial artificial pollination (using one out of five pistils) was significantly reduced from that of 8.6 ± 0.4 (n = 41) of normal pollination (using five pistils) to 5.1 ± 0.3 (n = 44) (Fig. 1A). We also investigated the difference in the rates of lopsidedness between partial and normal pollinations, and demonstrated that the rates had significantly increased from 8.6 ± 0.6 (n = 41) of normal pollination to 15.1 ± 1.1 (n = 44) of partial pollination (Fig. 1B). The negative correlation between seed number in a fruit and their lopsidedness was obtained using regression analysis (Fig. 2). It was calculated from the formula y = −1.126x + 19.617, which was obtained as a function of a regression line, that <4.10 seeds in a fruit became lopsided (≥15% lopsidedness). Although fruits having less than two seeds exhibited lopsided fruits, those with more than three showed both
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Table 2 Rate of lopsided ‘Fuji’ (S1 S9 ) fruits by artificial pollination with ‘Maypole’ (S10 S16 ) pollen. No. of stigmas pollinated
Year
No. of lopsided fruits
No. of normal fruits
Rate of lopsided fruits (mean ± S.E.%)
1 1 1 1 1 3 5 5 5 5 5
2007 2008 2009 2010 2007–2010 2007 2007 2008 2009 2010 2007–2010
6 6 3 6
10 4 3 6
2 3 0 0 0
16 15 10 3 10
37.5 60.0 50.0 50.0 49.4 ± 4.6 a 11.1 16.7 0.0 0.0 0.0 4.2 ± 4.2 b
Values followed by a different letter within the same column differ significantly at the 0.1% level by Chi-square test.
Table 3 S-RNase allele analyses of individual seeds in a ‘Fuji’ (S1 S9 ) fruit obtained by repeated pollination at 4- or 24-h intervals (order of pollen used: 1st ‘Maypole’ (S10 S16 ), 2nd ‘Orin’ (S2 S7 ), 3rd ‘Chouka 19’ (S3 S5 ). Year
No. of lopsided fruits
Rate of No. of normal fruits lopsided fruits
No. of total seeds
No. of seeds/fruit
Pollination
Intervals (h)
No. of pollinated stigma No. of repeated times
2008 2008 2008 2008 2009 2009 2009 2009 2010
2 0 2 0 1 0 0 0 0
4 5 2 6 4 3 4 6 3
33.3 0.0 50.0 0.0 25.0 0.0 0.0 0.0 0.0
32 61 21 50 34 24 33 56 31
5.3 12.2 5.3 8.3 6.8 8.0 8.3 9.3 10.2
1 5 1 5 1 5 1 5 1
2 2 3 3 2 2 3 3 3
No. of seeds S-RNase alleles detected in seeds
Maypole Orin
24 24 24 24 4 4 4 4 4
Chouka 19
S10 S16 S2
S7
S3
S5
14 20 9 22 6 9 4 7 6
0 0 0 1 11 7 0 13 5
– – 0 0 – – 5 7 11
– – 0 0 – – 15 12 4
18 41 12 27 13 1 9 7 0
0 0 0 0 4 7 0 10 5
Fig. 3. Correlation between the rates of lopsided ‘Fuji’ fruits and seed distribution in the fruit. Sectors observed with one to four seeds were shaded. The 44 fruits investigated were obtained by partial pollination (one out of five pistils used for pollination) (see Table 2). Rate of lopsided fruits (number of lopsided fruits/total fruits), seed number with a standard error (mean ± S.E.) were indicated below each figure in the upper row, and those from fruits containing more than five seeds were indicated in the lower row.
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reduced compared with that (299.1 g ± 13.9) of normal artificial pollination (7th row in Table 2). The five stigmas in apple fused at the middle of styles different from those in Japanese pear, and the hormones released from the pollen tube soon after the germination might be less important for apple lopsided fruit generation. 3.3. Effect of repeated pollination for production of normal-shaped fruits
Fig. 4. Average apple fruit weights (g) with standard error (mean ± S.E.) of lopsided (2007, n = 11; 2009, n = 12) and normal (2007, n = 41; 2009, n = 19) obtained by partial (artificial; 2007) and natural (2009) pollinations. The symbols a and a above the columns denote no significant difference at a 5% risk.
normal and lopsided fruits (Fig. 2). We investigated seed distributions in 44 fruits obtained by partial pollination (Fig. 3). The rate of lopsided fruits increased with the degree of biased seed distribution in a fruit. From 77.8 to 100% of fruits having seeds in only one or two out of five sectors became lopsided, and the rate decreased upon reducing the degree of biased seed distribution, i.e., from 58.3% (having seeds in three sectors), 22.2% (having seeds in four sectors) to 10.0% (having seeds in all five sectors) (Fig. 3). We chose fruits containing more than five seeds, and confirmed the tendency of biased seed distribution to result in the generation of lopsided fruits. This suggested that biased seed distribution in addition to low seed number in a fruit might be related to the formation of lopsided fruits. The lopsided shapes might be formed by the hormone (e.g., indoleacetic acid and gibberellin) derived from seeds in fruits with the uneven distribution of nutrients and water within the pulps. In our experiment, the ‘Fuji’ fruits containing more than five seeds were obtained despite being pollinated at one out of five pistils (Fig. 3). It was suggested that the gynoecium of ‘Fuji’ is perfectly syncarpic, as was indicated in the cultivar Summerland McIntosh (Sheffield et al., 2005). We compared the weights of 41 and 19 normal fruits with those of the 11 and 12 lopsided fruits obtained in 2007 (partial pollination, 1st, 6th and 7th row in Table 2) and 2009 (natural pollination, S-9 in Table 1), respectively. As shown in Fig. 4, the average weight of lopsided fruits (261.9 g ± 13.1, 326.8 g ± 14.8) was slightly reduced compared with those of normal fruits (304.5 g ± 10.5, 368.3 g ± 13.4), but the difference was dismissed at the 5% risk by Student’s t-test. Although Keulemans et al. (1996) found a positive correlation between fruit weights and the number of welldeveloped seeds, we could find no such correlation (Fig. 4). The weight of fruits produced by partial pollination was the same as those produced by normal pollination despite the decreasing number of seeds. As Keulemans et al. (1996) pointed out, this might be a characteristic of ‘Fuji’. The average fruit weight (281.3 g ± 14.7) of partial artificial pollination (1st row in Table 2) was not significantly
As lopsided fruits were not shipped as fresh use material, but instead were shipped as processing material low in price, apple growers prefer to suppress its generation at their orchards to avoid an economic loss. Since lopsided fruits seemed to be produced by inadequate partial pollination, we carried out repeated pollination to determine the proper timing of pollination for suppressing the generation of lopsided fruits. To reveal the accurate contribution of repeated pollination for increasing seed number in a ‘Fuji’ fruit, we used ‘Maypole’ (S10 S16 ), ‘Orin’ (S2 S7 ) and ‘Chouka 19’ (S3 S5 ) pollen, with different S-genotypes. The rate of fruit set of cross pollination of fully compatible combination was identical with that of a semi-compatible combination, however, the seed number per fruit were reduced by the semi-compatible combination (Matsumoto et al., 2006). All three cultivars show full compatibility with ‘Fuji’ (S1 S9 ), and the order of the pollen used, that is, 1st ‘Maypole’, 2nd ‘Orin’ and 3rd ‘Chouka 19’, in the orchard flowering order. The rate of pollen germination was investigated using more than 100 randomly selected pollens spread on a 1% agar dish containing 17% sucrose after 24 h incubation at 23 ◦ C. The rate of pollen germination of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ exhibited 61.8 ± 12.1%, 78.6 ± 6.7% and 76.7 ± 13.0% in 2008, and 60.5 ± 6.7%, 53.0 ± 7.9% and 71.4 ± 13.5% in 2009, respectively, from the thrice-repeated experiments. The average length of randomly selected 12–22 germinated pollens of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ was measured after a 2-h incubation. The length of germination pollen of ‘Maypole’, ‘Orin’ and ‘Chouka 19’ was 152.8 ± 22.8 m, 155.9 ± 14.4 m and 190.4 ± 18.8 m in 2008, and 268.8 ± 15.3 m, 207.8 ± 21.9 m and 244.1 ± 14.8 m in 2009, respectively. The difference between the rate of pollen germination and the average length of germinated pollen among ‘Maypole’, ‘Orin’ and ‘Chouka 19’ was dismissed at the 5% risk by one-factor ANOVA. Repeated pollination using one out of five or five stigma(s) at 24-h intervals was carried out in 2008 because the fine weather continued. As we used undiluted pollen covering each stigma completely throughout artificial pollination experiments, the pollen competition seemed to be strong as had been pointed out by Zhang et al. (2010). In apples, since no distortion for segregation at the Slocus was observed at fully compatible cross-pollination, the pollen competition among pollen containing different S-alleles seemed to be identical with that among pollen containing the same S-allele. In the case of partial pollination using one of five pistils, all the seeds in fruits obtained by repeated pollination were produced by the ‘Maypole’, not ‘Orin’ or ‘Chouka 19’ pollen (Table 3, Fig. 5). The same results (except for 1 out of 50 seeds produced by ‘Orin’ pollen) were obtained by normal pollination using five pistils (Table 3, Fig. 5). Since two and three times repeated pollination at 24-h intervals provided almost no contribution to seed production, it
Table 4 Rate of ‘Fuji’ (S1 S9 ) fruit set fertilized by ‘Maypole’ (S10 S16 ) pollen. Days after flowering
No. of flowers
No. of fruits
Fruit set (%)
No. of seeds/fruits
0 1 2 3 4 5
4 5 5 4 6 7
4 5 5 4 4 0
100 100 100 100 67 0
8.5 9.2 8.6 8.8 7.5 –
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Table 5 Rate of ‘Fuji’ (S1 S9 ) fruit set fertilized by 1st ‘Fuji’ and 2nd ‘Maypole’ (S10 S16 ) or ‘Fuji’ pollen. Pollen cultivar Days of flowering
1 day after flowering
3 days after flowering
Fuji Fuji Fuji
Maypole –
– Maypole Fuji
No. of flowers
No. of fruits
Fruit set (%)
No. of seeds/fruits
5 6 5
5 6 0
100 100 0
8.4 8.7 –
pollination at 4-h intervals contributed to increasing seed number in a fruit and decreasing lopsided fruits, suggesting that artificial pollination within one day was effective in reducing lopsided fruits. As repeated pollination at 24-h intervals contributed nothing to seed production in a fruit, pollinizers not overlapping their flowering periods might be unnecessary in an apple orchard consisting of single cultivar. Fig. 5. Examples of S-RNase allele-specific PCR amplifications. Some seeds in Table 3 possessing either of S10 , S16 , S2 , S7 , S3 and S5 were used. Lanes 1–6 represent the seed possessing S10 (282 bp), S16 (278 bp), S2 (449 bp), S7 (440 bp), S3 (375 bp), and S5 (363 bp).
was suggested that once pollination or fertilization occurred, the gynoecium lost its ability to accept new pollen within 24 h, even though the empty ovules were intact. We investigated the period during which the ‘Fuji’ gynoecium was able to be fertilized. ‘Fuji’ flowers at the popcorn stage were bagged to avoid natural pollination, then artificially pollinated by ‘Maypole’ pollen from the date of flowering to five days later (e.g., five days; bags were removed from the flowers five days after flowering, then pollinated). As shown in Table 4, ‘Fuji’ genoecium kept its ability for fertilization from the day of flower opening to 4 days after flowering. The gynoecium seemed to lose its fertilization ability within one day by pollination or fertilization, in spite of maintaining its ability for fertilization for 4 days. We speculated that this was caused by fertilization rather than by pollination, since the 2nd pollen ‘Maypole’ was accepted two days after the pollination of incompatible pollen ‘Fuji’ (Table 5). In the field, 24 h after hand pollination of P. pyrifolia, pollen tubes in the pistil for the undiluted pollen treatment grew faster than pollen tubes in the diluted pollen treatment, and the pollen tubes had almost reached the base of the style (Zhang et al., 2010). Those results suggested that the 2nd pollination one day after the 1st contributed nothing to increase the seed number in the fruit, which was connected to the diminished generation of lopsided fruits. In 2009, we then carried out repeated pollination at 4-h intervals. In contrast to the 24-h interval results, we detected 15 out of 34, and 14 out of 24 seeds produced in the 2nd pollen ‘Orin’ by partial and normal pollinations, respectively (Table 4). We also detected 20 out of 33, and 19 out of 56 seeds produced by the 3rd pollen ‘Chouka 19’ by partial and normal pollinations, respectively (Table 4). As in the case of a thrice-repeated partial pollination, seeds produced by the 2nd pollination were not detected. For reasons unknown, we detected seeds produced by the 2nd pollen ‘Orin’ as well as the 3rd pollen ‘Chouka 19’ at the rates of 10 out or 31 and 15 out of 31, respectively, in 2010 (Table 4). The seeds in fruits obtained by two- and three-time repeated partial pollination at 4-h intervals increased compared with those at 24-h intervals, and the rate of lopsided fruits decreased compared with those at 24-h intervals. 4. Conclusions Our experiments in inadequate partial pollination using one out of five pistils for pollination contributed to increasing generation of lopsided ‘Fuji’ apple fruits and to decreasing in the seed number in a fruit. Biased seed distribution also seemed related to the generation of lopsided ‘Fuji’ fruits. Two- or three-time repeated
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