Scientia Horticulturae 232 (2018) 29–39

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Scientia Horticulturae

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Parthenocarpy in accessions with special focus on relatives of T Kunenbo (C. nobilis Lour. var. kunep Tanaka) ⁎ Xiaohui Zhoua, Akira Wakanab, , Jung-Hee Kimc, Kaori Sakaib, Kohei Kajiwarab, Yuki Mizunoeb a Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan b Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan c Graduate School of Science and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8515, Japan

ARTICLE INFO ABSTRACT

Keywords: Parthenocarpy is one of the most essential factors for production of seedless citrus that is a desirable trait Citrus for consumers and producers. However, little research has been done on the degree and type of parthenocarpy in ‘Kunenbo’ citrus, except a few Citrus . To increase the knowledge about parthenocarpy for cultivation and breeding Mandarin of seedless citrus, 113 Citrus accessions including pummelo, mandarin and their relatives were investigated. In Parthenocarpy self-incompatible accessions flower buds just before anthesis were bagged, while in self-compatible accessions Pummelo they were non-pollinated to examine autonomous parthenocarpy or style-removed three days after to examine stimulative parthenocarpy. set rate in each treatment in the 113 accessions was recorded one month after anthesis and the mature fruits were harvested to examine their size and seedlessness. There was no clear difference between stimulative and autonomous parthenocarpy in Citrus accessions examined, indicating that fruit set and development in the accessions is due to autonomous parthenocarpy. The parthenocarpic fruit set occurred in 22 of 50 accessions for pummelo, 26 of 32 accessions for pummelo relatives, 3 of 9 accessions for yuzu and relatives, 12 of 21 accessions for mandarin and relatives, and 63 of 113 accessions in total. Of the 63 accessions, matured seedless fruits were harvested form 19 accessions, of which five accessions ‘Shishiyuzu’, ‘Kunenbo’, ‘Okitsu-wase’ and ‘Aoshima’ satsuma mandarins and ‘Kiyomi’ tangor showed very high degree of autonomous parthenocarpy, followed by ‘Kunenbo’ accessions such as ‘Iyokan’, ‘Hassaku’, ‘Kabosu’ that showed high to moderate degree of autonomous parthenocarpy. Since ‘Kishu’ mandarin (a seed parent of sat- suma) and yuzu (a seed parent of ‘Kabosu’) showed very little or no parthenocarpic fruit growth, it was suggested that ‘Kunenbo’ carried parthenocarpic genes to satsuma mandarin and the other ‘Kunenbo’ relatives showing medium to high autonomous parthenocarpy. It is suggested that these and the other accessions determined to have high to moderate degree of parthenocarpy are useful for production of seedless fruits and breeding of seedless Citrus cultivars.

1. Introduction it avoids seed removal process, increases fruit quality and contributes to laborsaving cultivation. Seedless phenotype is often affected by en- Citrus is one of economically important fruit crops and widely vironmental conditions and depends on a few biological features in cultivated in temperate, subtropical and tropical areas. It has a long which parthenocarpic ability is important (Asins et al., 2015). Parthe- cultivation history for more than 4000 years as nutritional and energy nocarpy is an exclusive way to set fruit and to get high yield under poor sources for human. Pummelo (C. grandis Osbeck), common mandarin pollination and/or fertilization due to male sterility, female sterility, (C. reticulata Blanco) and citron (C. medica Linn.) are considered to be self-incompatibility, long and heavy rainfall and no in ancestral or the basic biological species, from which many horticultural greenhouses (Denna, 1973; Lietzow et al., 2016; Olimpieri et al., 2007; species and/or many cultivars appeared as inter- and intraspecific hy- Pandolfini, 2009). brids (Roose et al., 1995). When a given citrus is able to pro- In general, parthenocarpy is induced autonomously (genetically) or duce normal fruit with a few or no seed, it is defined to be seedless stimulatingly (artificially). Genetically parthenocarpic plants produce (Vardi et al., 2008; Varoquaux et al., 2000). Seedlessness is a desirable fruits without pollination or subsequent fertilization (Gillaspy et al., trait for producers, consumers and fruit processing industries, because 1993). In sterile Citrus accessions such as satsuma mandarin with high

⁎ Corresponding author. E-mail address: [email protected] (A. Wakana). https://doi.org/10.1016/j.scienta.2017.12.045 Received 10 October 2017; Received in revised form 16 December 2017; Accepted 20 December 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved. X. Zhou et al. Scientia Horticulturae 232 (2018) 29–39 degree of parthenocarpy, pollination is unnecessary for commercial pollen of ‘Foster Pink’ grapefruit and/or open-pollinated under the open fruit production. In fertile and self-incompatible accessions such as field conditions, while self-compatible accessions were self-pollinated clementine cultivars, the degree of parthenocarpy is low (Mesejo et al., and/or open-pollinated similarly. 2013; Talon et al., 1992; Vardi et al., 2008). It has been reported that Several accessions reported to have genetic relation with satsuma parthenocarpic fruit set is mainly affected by hormonal status at an mandarin (Fujii et al., 2016; Shimizu et al., 2016) were emasculated early stage of fruit development (Talon et al., 1990b). Seedless citrus and then bagged just before anthesis. In self-incompatible ‘Kunenbo’ (C. fruits can be produced artificially through application of exogenous nobilis Rour. var. kunep Tanaka) reported to be a pollen parent of sat- hormones such as , or inhibitors for auxin transpor- suma mandarin (Fujii et al., 2016; Shimizu et al., 2016), the emascu- tation to the flowers (Lietzow et al., 2016; Mesejo et al., 2016; lated flower buds were sufficiently pollinated with compatible fresh Olimpieri et al., 2007; Serrani et al., 2010; Talon et al., 1992). It has pollen of ‘Suisho Buntan’ (C. grandis complex) until the stigma turned been reported that gibberellin is a crucial factor regulating partheno- yellow and then bagged. The pollinated style was perfectly removed carpy in Citrus (Talon et al., 1997). Gibberellin is commercially used for from the pistil 3 days after pollination (3DAP) and bagged it again. This production in self-incompatible but low parthenocarpic style removal treatment 3DAP to examine pollen-stimulative parthe- Citrus cultivars such as ‘Hyuganatsu’ in Japan (Nakajima, 1973; nocarpy in the plants was also carried out in eight accessions, i.e., Nakajima et al., 1976). Thus, it may be possible to produce seedless ‘Marsh Seedless’ (C. paradisi Macf.), ‘Foster Pink’ (C. paradisi Macf.), fruits with the aid of exogenous gibberellin treatment even in Citrus ‘Valencia’ (C. sinensis Osbeck), ‘Iyokan’ (C. iyo hort. ex Tanaka), ‘Okitsu- cultivars with low degree of parthenocarpy. wase’ (C. unshiu Marc.), ‘Kiyomi’ (C. unshiu cv. Miyagawa × C. sinensis The degree of parthenocarpy in Citrus was focused on a few com- cv. Trovita), ‘Ozaki-mikan’ (C. kinokuni hort. ex Tanaka) and ‘Mukaku mercially important accessions such as highly parthenocarpic satsuma Kishu’ (C. kinokuni hort. ex Tanaka). Here, grapefruit (C. paradisi Macf.) mandarin and low parthenocarpic clementine and (Talon et al., is estimated to be a pummelo-orange hybrid, ‘Iyokan’ (C. iyo hort. ex 1990a, 1992), parthenocarpic ‘Afourer’ mandarin (Gambetta et al., Tanaka) is estimated to be a complex pummelo-Kunenbo hybrid and 2013) and a few highly parthenocarpic cultivars of pummelo (Iwamasa ‘Kishu' (C. kinokuni hort. ex Tanaka) is determined to be a pistillate and Oba, 1980; Hoang et al., 2014). It is unclear at present why satsuma parent of satsuma mandarin (Shimizu et al., 2016). shows such high parthenocarpy and which degree and what type of The fruit set rates of the treated 113 accessions were recorded one parthenocarpy the other accessions have (Montalt et al., 2016). To in- month after anthesis, before the time when natural fruit drop occurred crease our knowledge about parthenocarpy for seedless fruit production in their setting young fruits. In October, the treated and open-pollinated and seedless cultivar breeding, 113 Citrus accessions including pum- fruits were harvested. Fruit number per treatment and seed number per melo, mandarin, yuzu and their relatives were investigated in this fruit were counted, and their fruit weight was measured to compare the study. difference between the seedless and seeded fruits in the same accession or to estimate the enlargement rate of the seedless fruit. The degree of 2. Materials and methods parthenocarpy in each accession was evaluated as a combination value of the fruit set rate and fruit enlargement rate (parthenocarpic fruit 2.1. Plant materials weight rate), i.e., fruit set rate (%) + fruit enlargement rate (%)/2.

A total of 113 Citrus accessions including one accession for lemon 2.3. Statistical analyses (Citrus limon Burm. f.), 61 for pummelo (C. grandis Osbeck) and pum- melo relatives, eight for sour orange (C. aurantium Linn.) and relatives, ANOVA for the significant difference in fruit weight among treat- 13 for sweet orange (C. sinensis Osbeck) and relatives, nine for yuzu (C. ments of non-pollination, cross-pollination, style removal 3DAP, self- junos Sieb. ex Tanaka) and relatives, 21 for common mandarin (C. re- pollination and open-pollination was carried out with a Turkey’s mul- ticulata Blanco) and relatives, were used. Three years investigation tiple comparison test. about their parthenocarpy was carried out from 2014 to 2017, since these citrus trees often show alternate year bearing, i.e., the flowers do 3. Results not form on a bearing shoots in the following spring (Koshita et al., 1999). All of these accessions were about 20-year-old trees grown under In this study, parthenocarpy of a target accession was examined the same field conditions in an orchard of Kyushu University located in from the two aspects of fruit set rate one month after anthesis and fruit Fukuoka, Japan. Tanaka’s Citrus classification system (Tanaka, 1969) weight of nearly mature fruit harvested in October (about five months that includes horticultural species was used for this study to know the after anthesis). During the three-year examination for parthenocarpy of detailed relationship of accessions examined. the 113 accessions, year-to-year variation of the fruit set rate and fruit size were detected. To avoid the year-to-year variation, data of the 2.2. Pollination, non-pollination and bagging treatments three-year results were pooled and averaged (Table 1). When the ac- cessions showed an off year (little or no flower formation in the trees), About ten flower buds with new spring leaves were randomly they were impossible to examine the parthenocarpy. Thus, the results of chosen from two or three trees for each treatment in each accession just 113 accessions for one to three years were listed in Table 1. before anthesis. In pummelo and relative accessions determined to be self-incompatible (Ngo, 2001), two or three flower buds containing a 3.1. Parthenocarpic fruit set rate perfect pistil were selected in a flower cluster with young leaves and bagged without emasculation, since it was known that self-pollination Parthenocarpic fruit set was detected in 63 of 113 accessions ex- in self-incompatible citrus cultivars without high parthenocarpy does amined 30 days after anthesis (Table 1). Of 50 pummelo accessions not stimulate the degree of parthenocarpy and results in flower drop examined, 22 showed parthenocarpic fruit set 30 days after bagging (Iwamasa and Oba, 1980; Mesejo et al., 2013). In self-compatible ac- treatment. In the 22 accessions, the parthenocarpic fruit set rate ranged cessions, flower buds were emasculated and those with a perfect pistil from 1.8% for ‘Ipoh No. 3′ to 28.0% for ‘Akune No. 6′ (Table 1). In were bagged to avoid outcross. One or two flower buds with newly October, mature fruits were harvested in five accessions. Of 11 pum- expanded leaves on an about 2 cm diameter branch were used for this melo relatives, eight showed parthenocarpic fruit set and the parthe- experiment, and the other buds were perfectly removed from the nocarpic fruit set rate ranged from 20.0% to 100.0% (Table 1). branch. To produce seeded fruit as a control for parthenocarpic seedless ‘Shishiyu’ and ‘Hassaku’ showed very high parthenocarpic fruit set rates fruit, self-incompatible accessions were cross-pollinated with fresh of more than 76.5% in bagging and non-pollination treatments, and

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