Developmental studies of production methods for seedless pollination-constant non-astringent (PCNA)-type fruits in Japanese persimmon (Diospyros kaki Thunb.)

Japanese persimmon (Diospyros kaki Thunb.) is an important fruit tree in Japan with numerous cultivars, many of which are hexaploid (2n = 6x = 90). One of the major cultivars is 'Fuyu', a pollination-constant non-astringent (PCNA)-type persimmon in Japan. 'Fuyu' does not have parthenocarpic ability; as a consequence, pollination is necessary to prevent June drop, and only seeded fruits are produced. In contrast, several nonaploid cultivars (2n = 9x = 135), such as 'Hiratanenashi' and its bud sports, are increasingly grown in commercial culture for their seedless fruits of good eating quality. Because these nonaploid cultivars are astringent types, astringency must be removed before shipment, which shortens shelf life. The production of non-astringent seedless persimmon fruits is thus of great interest. The purpose of this study is to produce non-astringent-type seedless persimmon fruits both through breeding and cultivation. Chapter 1. Dodecaploid Persimmons Induced by In Vitro Colchicine Treatment and Their Growth Characteristics. Analogous to the production of triploids via pollination of diploids with tetraploids, it should be possible to generate nonaploid persimmons by pollinating hexaploid cultivars with dodecaploids (2n = 12x = 180). Because no naturally occurring dodecaploids exist, however, artificial chromosome doubling must be used. In addition, inter-crossing between PCNA genotypes is necessary for breeding PCNA-type Japanese persimmons, because the PCNA phenotype is recessive to non-PCNA phenotypes. Chromosome doubling induced by colchicine treatment of seedlings derived from a cross between PCNA hexaploid persimmons was carried out to generate dodecaploid persimmons. In vitro colchicine treatment of seedling meristem for 12-24 hours was effective at a concentration of 0.03-0.05%. Induced dodecaploid seedlings generally exhibited weak vigor, but some vigorous seedlings were obtained. Compared with the hexaploid parental cultivars, these dodecaploid seedlings had longer stomatal guard cells and lower stomatal densities. Although the rate of flower bud formation on dodecaploid seedlings was lower than that of the hexaploid seedlings, some of the seedlings bore both pistillate and staminate flowers the pollen germination ability. Dodecaploid seedlings bore PCNA-type fruits; however, they did not form seeds when pollinated with hexaploid pollen. On the other hand, imperfect seeds were formed when pollinated to hexaploid persimmons with dodecaploid pollen. These findings suggest dodecaploid persimmons could be used as a pollen parent for the breeding of nonaploid PCNA persimmons. Chapter 2. Generation of Fruiting Mother Shoots from Water Sprout by Pinching in Japanese Persimmon (Diospyros kaki Thunb.). Two important parental cultivars for persimmon breeding are 'Nishimura-Wase', which is early maturing, and 'Taishuu', which produces large, good-tasting fruits. Persimmon has diclinous flower bud differentiation, 'Taishuu' and 'Nishimura-Wase' produce many more staminate flowers than pistillate flowers with age or weakening tree vigor. Thus, it is very important to have well-nourished shoots bearing numerous pistillate flowers to use these cultivars as seed parents. Because they distort tree shape and block sunlight exposure, water sprouts are usually pruned out during the growing season. As described in this chapter, I tried to form fruiting mother shoots from water shoots by pinching the latter in spring. Summer shoots growing from water shoots pinched from May to June bore more pistillate flowers and fewer staminate flowers than did normal shoots. Pinched water shoots that did not sprout summer shoots produced pistillate and staminate flowers in ratios similar to non-pinched plants. Although no difference was observed in numbers of flowers among pinching dates, pinching in early May is preferable, as it leads to a higher percentage of summer shoots sprouting from water shoots than does pinching after late May. There was no difference in seed numbers per fruit between experimental plots, but total seed numbers from summer shoots produced by water shoot pinching were much higher than those from normal shoots. Based on these findings, pinching water sprouts from May to June is an effective method for generating fruiting mother shoots for the following year. Chapter 3. Generation of Nonaploid Persimmons by Embryo Culture of Imperfect Seeds Derived From a Cross between 'Fuyu' and 'Taishuu'. Because persimmon has the property of long juvenility, fruit production will be extremely delayed when nonaploid seedlings are generated via dodecaploid parents. If nonaploid seedlings are generated directly from inter-crossing between hexaploids, breeding periods can be drastically shortened. In some citrus species, triploid offspring have been derived from imperfect seeds produced by inter-crossing diploids. As detailed in this chapter, I similarly aimed to obtain nonaploid seedlings from imperfect seeds by embryo culture using a cross between the hexaploid PCNA cultivars 'Fuyu' and 'Taishuu'. An additional goal was to clarify the origin of nonaploid embryos from imperfect seeds. Seeds were collected from 300 fruits. Of 1,078 seeds, 68 (6.3%) were imperfect, 10 of which germinated into seedlings. Eight of these seedlings developed into normal-appearing plantlets with normal growth, while the remaining two produced abnormal plantlets without meristems at either shoot apex or root tip. Two seedlings were recovered from the abnormal plantlets via callus, and grew vigorously in the greenhouse. Cytogenetic analysis confirmed that these two seedlings were nonaploids. Parental analysis using four simple sequence repeat (SSR) markers, D.CT-13, 24, 61, and 179, revealed that each nonaploid seedling had alleles originating from the parents, indicating that they were generated by syngamy. The two nonaploid seedlings had 222-bp alleles at D.CT-61, specific to 'Taishuu', the pollen parent, but did not have the 136- or 140-bp alleles at D.CT-179, characteristic of 'Fuyu', the seed parent. These results suggest that the seedlings may have been derived from fertilization of a reduced female gamete with an unreduced male gamete. Chapter 4. Development of Seedless PCNA Persimmon Cultivar 'Fukuoka K1 Gou' and the Effect of Gibberellin Spray and/or Disbudding on Fruit Set. I developed the nonaploid PCNA persimmon cultivar 'Fukuoka K1 Gou' (TM; Akiou) by embryo culture of imperfect seeds derived from a cross between the hexaploid PCNA cultivars 'Fuyu' and 'Taishuu'. 'Fukuoka K1 Gou' was confirmed to bear seedless PCNA-type fruits. 'Fukuoka K1 Gou' fruit ripens from mid-October to early November, nearly the same time period as 'Taishuu'. Fruit-soluble solids content was 19.6 °Brix, which was higher than that of 'Fuyu' and 'Taishuu'. Fruit weight, 365 g, was greater than that of 'Fuyu' and almost identical to that of 'Taishuu'. Fruit skin of 'Fukuoka K1 Gou' at harvest time was less reddish than that of 'Fuyu'. Parthenocarpic and stenospermocarpic abilities of 'Fukuoka K1 Gou' were low, causing unstable fruit set, especially in young trees. Spraying fruit with 200 ppm gibberellin 10 days after full bloom and disbudding both improved fruit set. In addition, a combination of the two treatments was significantly more effective than either treatment alone. Chapter 5. Effect of Pollination with Nonaploid Persimmon Pollen on Fruit Set and Fruit Quality in 'Fuyu', a Hexaploid Persimmon. The nonaploid astringent cultivar 'Hiratanenashi' has both parthenocarpic and stenospermocarpic ability. As a consequence, seeds of 'Hiratanenashi' abort when pollinated with pollen from hexaploid persimmons, preventing June drop. If a hexaploid cultivar was pollinated with pollen from a nonaploid persimmon, stenospermocarpy might be induced. In the experiments described in this chapter, I investigated fruit set and fruit quality of hexaploid 'Fuyu' pollinated with nonaploid persimmon pollen. The goal of this research was to explore the possibility of a novel cultural practice for seedless 'Fuyu' fruit production. Pollen from nonaploids germin ated normally; pollen germination rates were lower than in the hexaploid 'Zenjimaru' but similar to those in 'Akagaki'. Although a much higher rate of fruit set was observed in pollinated than in non-pollinated 'Fuyu', the rate was lower after pollination with pollen from nonaploid plants (nonaploid pollination) than from hexaploid plants (hexaploid pollination). Seeds from nonaploid pollination grew as well as those from hexaploid pollination until 44 days after full bloom. At that point, however, growth of seeds from nonaploid pollination slowed and finally stopped, resulting in a final seed size approximately two-thirds the size of seeds obtained with hexaploid pollination. Fruit weight, total soluble solid content, and skin color of fruit from nonaploid pollination was almost indistinguishable from that of fruits from hexaploid pollination, whereas flesh firmness was lower with nonaploid than hexaploid pollination.