Study of Parthenocarpy in Three Kiwifruit Commercial Cultivars

ABSTRACT

To examine parthenocarpy in commercial kiwifruit cultivars in Japan, ‘Rainbow Red’, ‘Kaimitsu’, and ‘Hayward’ cultivars were subjected to four treatments: 1) pistil removal just before flowering; 2) pistil removal just before flowering and soaking with 10 ppm N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU); 3) soaking with 10 ppm CPPU and natural pollination; and 4) artificial dust pollination. The percentage of fruit borne in the pistil removal treatment groups was 0% in ‘Rainbow Red’ and ‘Hayward’, and was 56% in ‘Kaimitsu’. Although 72% had fallen, the fruit borne in ‘Kaimitsu’ was harvestable. CPPU treatment induced parthenocarpy in the three kiwifruit cultivars. There were no viable seeds in the groups with pistil removal, regardless of treatment with or without CPPU. CPPU treatment also promoted fruit maturity at harvesting time in ‘Rainbow Red’ and ‘Kaimitsu’. In ‘Kaimitsu’, parthenocarpic fruits without CPPU treatment were nearly as ripe as pollinated fruits without CPPU treatment at harvest time. After ethylene treatment, fruits ripened, and ethylene was detected in all treatments and cultivars. We confirmed that ‘Kaimitsu’ exhibited fruit development without artificial pollination or CPPU treatment in another season. Furthermore, in ‘Kaimitsu’, parthenocarpic fruits without CPPU treatment were smaller than artificial pollinated fruits, and natural fruit drop was extensive. Thus, parthenocarpy of ‘Kaimitsu’ was thought to be unstable. These results showed that excessive parthenocarpy may be successful in kiwifruit depending on specific cultivars.

KEYWORDS:

• Ethylene
• natural fruit drop
• N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU)
• ripening
• seed

Introduction

Kiwifruit is one of the most important commercial fruit crops in Japan. It belongs to the genus Actinidia, which contains more than 70 species (Ferguson and Huang, 2007). Kiwifruit appears to be a functionally dioecious vine. Female vines have flowers with stamens and produce pollen, but mature pollen is usually empty and therefore sterile (Watanabe et al. 1992). Male vines have ovules and no or greatly reduced styles (Hopping, 1990). Generally, male plants pollinate female plants, which in turn bear fruits. As such, artificial pollination or male to female arrangements are important for reproductive success and fruit set with good yield.

Parthenocarpy produces seedless fruits without fertilization. This phenomenon is vital in fruit cultivation because it obviates the need for artificial pollination or pollinating insects and pollinizers. In kiwifruit, parthenocarpic fruit development does not occur naturally (Hopping, 1990). However, treatment of unpollinated flowers on or before anthesis with N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) induces parthenocarpic fruit development in the kiwifruit cultivar ‘Hayward’ (Iwahori et al., 1988).

The green flesh kiwifruit cultivar ‘Hayward’ is mainly cultivated in Japan and is classified as a hexaploid (2 n = 6x = 192) belonging to Actinidia chinensis var. deliciosa. Yellow flesh kiwifruit cultivar ‘Kaimitsu’ is classified as tetraploid (2 n = 4x = 128), whereas yellow-red flesh cultivar ‘Rainbow Red’ is classified as diploid (2 n = 2x = 64). ‘Kaimitsu’ and ‘Rainbow Red’ cultivars belong to A. chinensis var. chinensis. These three cultivars (‘Hayward’, ‘Kaimitsu’, and ‘Rainbow Red’) are commercially cultivated and have had a good reputation in recent years. We examined parthenocarpic fruit development by CPPU treatment with the omission of artificial pollination in these commercial cultivars in Japan. CPPU treatment induced parthenocarpic fruit development in all three kiwifruit cultivars. Furthermore, fruit development was stimulated in ‘Kaimitsu’ without CPPU treatment and pollination. This article presents the details of these experiments.

Materials and Methods

Inducing Parthenocarpy by CPPU Treatment in Three Kiwifruit Cultivars

Three kiwifruit cultivars, ‘Rainbow Red’ (A. chinensis var. chinensis), ‘Kaimitsu’ (A. chinensis var. chinensis), and ‘Hayward’ (A. chinensis var. deliciosa) were cultivated in an orchard in Shizuoka, Japan. Thirteen-year-old trees of ‘Rainbow Red’, 16-year-old ‘Kaimitsu’, and 33-year-old ‘Hayward’ were used in the present study. Two trees were used per variety. ‘Rainbow Red’ was estimated to be diploid (Yamamoto et al., 2009), ‘Kaimitsu’ is tetraploid (Yamamoto et al., 2009), whereas ‘Hayward’ is hexaploid (Watanabe et al., 1990).

Experiments were performed in the 2015 season. Four treatments of four plot replications and randomly distributed were applied to the orchard plants as follows: 1) pistil removal just before flowering using small scissors; 2) pistil removal just before flowering and soaking with 10 ppm CPPU; 3) soaking in 10 ppm CPPU and natural pollination; and 4) artificial dust pollination (Figure 1). Soaking in 10 ppm CPPU was applied at flowering. The quantity of ‘Tomuri’ (A. chinensis var. deliciosa) pollen dust for artificial pollination was approximately 100 g/10 a and was applied using a rechargeable pollen dust sprayer (New Pollen Duster; Agri. Co., Ltd., Saga, Japan). Treated flowers were picked from five primary scaffold branches of each tree. Flower numbers of each experimental plot ranged from 90 to 150. The flower or flower bud treatment dates for ‘Rainbow Red’ were April 29, 30, and May 1. Flower treatment date for ‘Kaimitsu’ was May 6. The flower treatment dates for ‘Hayward’ were May 17 and 20. Ripe fruits from each treatment were harvested in a single day. The ‘Rainbow Red’ fruit harvest date was September 4, whereas that of Kaimitsu was October 14 and that of ‘Hayward’ was November 17. Harvested fruits were treated with 100 nL g^–1 h^–1 ethylene in a desiccator for 24 h at 20°C. After ethylene treatment, the fruits were ripened in an ethylene-free environment at 20°C for seven days.

Figure 2. Fruit appearance and sections of fruits developed through artificial pollination, pistil removal with CPPU treatment, pistil removal, and natural pollination with CPPU treatment in three kiwifruit cultivars. A, ‘Rainbow Red’, B, ‘Kaimitsu’, and C, ‘Hayward’

Figure 1. Outline of the experimental design in this study

The harvested fruits were examined for fruit weight, fruit quality, and number of viable seeds. Fruit quality was examined based on flesh firmness, soluble solid content (SSC), and titratable acidity (TA) at harvest and after ripening. Ethylene production was determined by incubation of individual fruits in 250 mL plastic containers for 1 h at 25°C, after which 1 mL of headspace gas was withdrawn and injected into a gas chromatograph (GC-2014; Shimadzu Co., Ltd., Kyoto, Japan). Flesh firmness was determined by measuring compression using a fruit hardness tester (Harvesting, KM-5; After ripening, KM-1; Fujiwara Scientific Company Co., Ltd., Tokyo, Japan) with a cone-shaped plunger. SSC of the fruit juice was measured using a digital refractometer (DBX-55A; Atago Co., Ltd., Tokyo, Japan). TA of the fruit juice was determined by titration using 0.1 N NaOH and expressed as the percentage of citric acid equivalents.

The results were analyzed with BellCurve for Excel software (Social Survey Research Information Co., Ltd., Tokyo, Japan) using Tukey’s test (P <0 .05).

Reproducing Parthenocarpy in ‘Kaimitsu’

We confirmed that ‘Kaimitsu’ could develop fruit without artificial pollination or CPPU treatment in the 2015 season. From these results, we studied the reproducibility of parthenocarpy in ‘Kaimitsu’ in the 2017 season. ‘Kaimitsu’ was cultivated in orchards in Hamamatsu and Shizuoka. Eighteen-year-old and five-year-old tress were used in experiments in Hamamatsu and Shizuoka, respectively. Two trees were used at each location. Two treatments, 1) pistil removal just before flowering, and 2) artificial pollination, were performed as per the 2015 study season. Flower numbers in each experimental plot ranged from 54 to 103. The flower treatment date for ‘Kaimitsu’ was May 9 in Hamamatsu and May 15 in Shizuoka. The fruit harvest date for ‘Kaimitsu’ was October 18 in Shizuoka. In Hamamatsu, no harvesting was performed as the borne fruit had dropped.

Results

Induced Parthenocarpy by CPPU Treatment in Three Kiwifruit Cultivars

In the pistil removal treatment groups, ‘Rainbow Red’ and ‘Hayward’ exhibited fruit bearing rates of 0%, compared to 56% in ‘Kaimitsu’ (Table 1). A total of 76–100% of individual trees in the group with pistil removal plus CPPU treatment exhibited fruit bearing in all cultivars. Natural pollination with CPPU treatment showed fruit bearing rates of 79–100% in each cultivar. Artificial pollination resulted in fruit bearing rates of 91–100% in each cultivar.

Table 1. Kiwifruit flowers, fruit bearing rate, and fruit dropping rate in each treatment in 2015

Variety	Treatment	Treated flowers	Bearing fruits	Bearing rate (%) ^a	Harvested fruits	Dropping rate (%) ^b
‘Rainbow Red’	Pistil removal	90	0	0	0	－
	Pistil removal + CPPU	90	90	100	45	50
	CPPU + Natural pollination	91	75	82	72	4
	Artificial pollination	150	136	91	131	3
‘Kaimitsu’	Pistil removal	109	61	56	17	72
	Pistil removal + CPPU	108	82	76	13	84
	CPPU + Natural pollination	106	106	100	55	48
	Artificial pollination	118	118	100	105	11
‘Hayward’	Pistil removal	101	0	0	0	－
	Pistil removal + CPPU	117	103	86	100	3
	CPPU + Natural pollination	104	82	79	80	2
	Artificial pollination	98	89	91	89	0

^aBearing rate (%) = Bearing fruits at day 30 after blooming /Treated flowers x 100.

^bDropping rate (%) = 100 - (Harvested fruits /Bearing fruits at day 30 after blooming x 100.

The percentage of fruit dropping was 72% for ‘Kaimitsu’ in the pistil removal treatment group. In ‘Rainbow Red’ and ‘Kaimitsu’, pistil removal with or without CPPU treatment caused fruit dropping rates of 48–84%. Artificial pollination showed low fruit dropping rates of 0–11% in each cultivar. Fruit dropping was higher since the end of August.

Parthenocarpic fruit characteristics are shown in Figure 2 and Table 2. Fruit weight decreased significantly in ‘Rainbow Red’ and ‘Kaimitsu’ in the pistil removal treatment groups with or without CPPU treatment. Fruit weight with artificial pollination was greater than that with natural pollination with CPPU treatment. CPPU treatment with or without pollination showed increased SSC and decreased flesh firmness at harvest time in ‘Rainbow Red’ and ‘Kaimitsu’. This pattern was not observed in ‘Hayward’. Ethylene was not detected in any treatment or cultivar at the time of harvest (Table 2). SSC increased, whereas flesh firmness and TA decreased in all treatment groups and cultivars to varying degrees after ethylene treatment. Ethylene was detected in all treatments and cultivars. There were no viable seeds in the groups treated with pistil removal, with or without CPPU (Figure 2 and Table 2). Empty seeds were found in parthenocarpic fruits. Artificially pollinated fruits had greater number of viable seeds than naturally pollinated fruits in each cultivar.

Table 2. Fruit weight, seed number, fruit quality, and ethylene production in each treatment in 2015

Variety	Treatment	Fruit weight (g)^a	Seed number per fruit^a	Fruit quality at harvesting time					Fruit quaity after ripening
				Number of fruits sampled	Soluble solid content (Brix)	Flesh firmness (N)	Titratable acidity (%)	Ethylene production (nL･g^−1･h^−1)	Number of fruits sampled	Soluble solid content (Brix)	Flesh firmness (N)	Titratable acidity (%)	Ethylene production (nL･g^−1･h^−1)
‘Rainbow Red’	Pistil removal + CPPU	29 ± 1 c^b	0 ± 0 c	9	14.4 ± 0.6a	20.5 ± 0.8b	1.71 ± 0.07b	n.d.^c	6	21.9 ± 0.3a	2.3 ± 5.2b	1.03 ± 0.11a	6.6 ± 0.4a
	CPPU + Natural pollination	66 ± 3b	209 ± 58b	10	7.4 ± 0.6b	26.9 ± 0.7a	1.94 ± 0.06a	n.d.	6	20.1 ± 0.7ab	4.1 ± 3.9a	0.76 ± 0.04a	2.9 ± 1.4ab
	Artificial pollination	79 ± 3a	680 ± 29a	10	6.2 ± 0.1b	28.7 ± 0.2a	1.86 ± 0.03ab	n.d.	6	19.6 ± 0.4b	3.9 ± 3.9ab	0.76 ± 0.06a	2.4 ± 1.2b
‘Kaimitsu’	Pistil removal	26 ± 4 c	0 ± 0 c	5	8.7 ± 0.6bc	25.0 ± 0.5a	2.47 ± 0.03a	n.d.	6	17.7 ± 0.4bc	4.3 ± 3.0a	1.92 ± 0.09b	4.1 ± 3.8a
	Pistil removal + CPPU	41 ± 2b	0 ± 0 c	5	20.0 ± 1.9a	13.2 ± 2.6b	2.48 ± 0.16a	n.d.	5	18.4 ± 0.6b	1.8 ± 4.8b	2.02 ± 0.10a	0.4 ± 0.4a
	CPPU + Natural pollination	74 ± 4b	46 ± 19b	10	11.1 ± 1.1b	21.6 ± 2.1a	2.32 ± 0.05a	n.d.	10	20.7 ± 1.2a	2.5 ± 7.0b	1.32 ± 0.19bc	0.5 ± 0.2a
	Artificial pollination	126 ± 1a	900 ± 29a	10	6.8 ± 0.1 c	26.8 ± 0.3a	2.32 ± 0.05a	n.d.	10	16.0 ± 0.1c	4.7 ± 1.7a	1.10 ± 0.05 c	5.8 ± 1.4a
‘Hayward’	Pistil removal + CPPU	80 ± 2a	0 ± 0 c	10	7.1 ± 0.1b	21.8 ± 0.4b	1.83 ± 0.02a	n.d.	10	16.3 ± 0.3a	4.2 ± 1.6b	1.13 ± 0.05a	9.2 ± 1.7a
	CPPU + Natural pollination	67 ± 3b	31 ± 10b	10	7.3 ± 0.4b	22.5 ± 0.3b	1.61 ± 0.09b	n.d.	10	16.0 ± 0.1a	5.1 ± 1.4a	1.15 ± 0.07a	2.2 ± 0.9b
	Artificial pollination	79 ± 3a	1114 ± 50a	10	9.4 ± 0.7a	23.9 ± 0.2a	1.95 ± 0.04a	n.d.	10	16.8 ± 0.4a	5.3 ± 1.0a	1.26 ± 0.06a	2.2 ± 0.7b

^aFruit weight was examined for all harvested fruits, whereas seed number per fruit was examined for 10 fruits.

^bMean ± S.E. Values within a column followed by the same latter in each variety are not significantly different at P < 0.05, according to Tukey’s multiple comparison test.

^cNot detected.

Reproducibility of Parthenocarpy in ‘Kaimitsu’

The pistil removal treatment groups displayed fruit bearing rate of 1% in Hamamatsu and 15% in Shizuoka (Table 3). The percentage of fruit dropping was 100% in Hamamatsu and 89% in Shizuoka. Overall, fruit dropping in the 2017 season was much more frequent than in the 2015 season. However, we harvested one fruit in the pistil removal treatment without artificial pollination in Shizuoka in the 2017 season. The harvested fruit had no viable seeds.

Table 3. Kiwifruit flowers, fruit bearing rate, and fruit dropping rate in ‘Kaimitsu’ in a repeated experiment in 2017

Place	Treatment	Treated flowers	Bearing fruits	Bearing rate (%) ^a	Harvested fruits	Dropping rate (%) ^b
Hamamatsu	Pistil removal	100	1	1	0	100
	Artificial pollination	103	89	86	86	3
Shizuoka	Pistil removal	60	9	15	1	89
	Artificial pollination	54	54	100	54	0

^aBearing rate (%) = Number of fruits at day 30 after blooming /Treated flowers x 100.

^bDropping rate (%) = 100 - (Harvested fruits /Number of fruits at day 30 after blooming x 100.

Discussion

Mostly, parthenocarpic fruit development does not occur naturally in kiwifruit (Hopping, 1990). It is reported that CPPU treatment induces parthenocarpic fruit development in kiwifruit (Iwahori et al., 1988). However, Mizugami et al. (2007) reported that the Actinidia arguta cultivar ‘Issai’ showed parthenocarpy without CPPU treatment. Furthermore, kiwifruit cultivar named ‘Xiangi’ (A. chinensis var. deliciosa) possesses the ability to produce parthenocarpic fruits without CPPU treatment (Pei et al., 2011). In the present study, ‘Kaimitsu’ showed fruit development without artificial pollination or CPPU treatment. These results indicate that parthenocarpy occurs in the genus Actinidia. Therefore, the development of parthenocarpic kiwifruit cultivars might be possible in future. However, the parthenocarpic fruits were very small compared to fruits borne through artificial pollination (Table 2). Furthermore

, most parthenocarpic fruits dropped naturally (Tables 1 and 3). Thus, parthenocarpy of ‘Kaimitsu’ was thought to be unstable. Breeding cultivars with parthenocarpy requires the trait to be stable.

Ethylene production following ethylene treatment has been shown to be reduced and delayed in parthenocarpic kiwifruit (Ikoma et al., 1998). In the present study, maturity of the fruit with pistil removal and CPPU treatment was clearly advanced in ‘Rainbow Red’ and ‘Kaimitsu’ (Table 2). These fruits also ripened rapidly after ethylene treatment (Table 3). In ‘Kaimitsu’, parthenocarpic fruits with CPPU treatment clearly ripened by harvest time, whereas parthenocarpic fruits without CPPU treatment were nearly as ripe as pollinated fruits without CPPU treatment. Murakami (2014) reported marked intervarietal differences in the effects of CPPU treatment on kiwifruit. In particular, CPPU treatment promoted fruit maturity at harvest time in ‘Rainbow Red’ and ‘Kaimitsu’. CPPU treatment was thought to have a greater effect on the difference in fruit ripeness at harvest time and after ethylene treatment compared with the number of seeds.

In this study, ‘Kaimitsu’ exhibited fruit development without artificial pollination or CPPU treatment (Table 1). However, ‘Kaimitsu’ did not virtually exhibit parthenocarpy in either Hamamatsu or Shizuoka in the repeat season 2017 (Table 3). Most fruits spontaneously dropped from the end of August to the harvest season. During this period, in Japan, kiwifruit trees were subjected to water stress. Natural fruit drop commonly occurs in several fruits including kiwifruit. This phenomenon has been studied from various perspectives, such as genetic, physiological, and environmental perspectives. In these studies, the relationship between natural fruit drop and water stress or fruit seed number has been investigated. Badal et al. (2013) reported that moderate water stress reduces fruit drop of ‘RojoBrillante’ persimmon. In peach, premature fruit drop after the June-drop was reduced under water stress (Li et al., 1989). On the other hand, Kajiura (1941) reported that natural fruit drop of pollinated fruits is less than that of non-pollinated fruits in persimmon. If the fruit has more than three seeds, natural fruit drop does not occur in ‘Fuyu’ (Yakushiji and Hase, 1991). Previous studies also suggest that natural fruit drop might easily occur in parthenocarpic kiwifruit under water stress conditions. The link between seed number and natural fruit drop requires further research.

‘Kaimitsu’ fruits have comparatively superior characteristics such as fruit bearing without artificial pollination and high–yielding under field conditions. In this study, we confirmed that ‘Kaimitsu’ has weak parthenocarpy. This characteristic may relate to fruit bearing or high yielding. Further studies on parthenocarpy can lead to the development of high yielding cultivars.

Seed number is positively correlated with fruit growth in kiwifruit (Hopping and Hacking, 1983), whereas CPPU treatment promotes fruit enlargement (Kurosaki and Mochizuki, 1990). Seed number and CPPU treatment may be linked to markedly smaller fruit size in parthenocarpic ‘Kaimitsu’.

In the present study, CPPU treatment induced parthenocarpy in three kiwifruit cultivars. CPPU treatment can induce parthenocarpy regardless of cultivar and can be expected to contribute to the omission of artificial pollination. However, CPPU treatment showed marked varietal differences in kiwifruit (Murakami, 2014). Therefore, further studies regarding the use of CPPU for inducing parthenocarpy are required.

In conclusion, the present study confirmed that CPPU treatment induces parthenocarpy in three kiwifruit cultivars. Furthermore, we found that ‘Kaimitsu’ displayed fruit development without CPPU treatment or pollination. The role of seeds during fruit setting, maturation, and the process of ripening are not well understood in kiwifruit. Parthenocarpic fruits without CPPU treatment in ‘Kaimitsu’ are expected to be useful in future studies of seed functions in kiwifruit.