Genetic Diversity and Breeding of Persimmon Genetic Diversity and Breeding of Persimmon

The genus Diospyros, which is distributed in tropical and subtropical regions of the world, contains hundreds of plant species. However, four species of them have commercial importance. D. kaki Thunb. is the most widely cultivated species of the Diospyros genus. Persimmon ( D. kaki Thunb.) is grown in many parts of the world that display subtropical climate conditions. In recent years, the cultivation of persimmon has found renewed inter- est in various countries of the Mediterranean basin. In China (which is the origin of persimmon) and in Japan and Korea (where it is grown widely), persimmons were selected from some well-known old varieties. Recently in countries such as Italy, Spain, USA, Brazil, Turkey and Israel, persimmons were selected from new cultivars. Currently China, Japan and Korea have the big persimmon germplasm collections with a large number of varieties and other Diospyros species. Also, Italy, Spain, USA, Brazil, Turkey, Israel, Azerbaijan, Uzbekistan and Pakistan have constituted the germplasms by high commercial value cultivars and/or local varieties. In this chapter, we tried to provide an overview of the genetic diversity and breeding of persimmon by combining literature with our studies.


Introduction
Persimmon is fleshy fibrous subtropical and tropical, deciduous fruit belonging to Ebenaceae family. The Oriental persimmon (Diospyros kaki Thunb.) is an exotic fruit rich in vitamins, nutrients and antioxidants vital for optimum health with various medicinal and chemical uses of fruits and leaves. Its fruit is usually consumed as a fresh or dried fruit. It is believed to have originated in the mountain area of southern China and has been cultivated as an important fruit crop in China, Korea as well as in Japan for centuries [1]. It is commonly  (mid-April) to avoid spring frosts. On the other hand, persimmon has been damaged by earlyautumn frosts. Early-autumn frosts can lead to skin blemishes on fruit and early defoliation. Persimmon does not tolerate wind. It does not provide a good fruit yield and quality, if strong winds occur during the growing season. Fruit is also prone to wind rub from leaves and branches causing skin blemish on fruit. Windbreakers can be used to reduce the wind speed.
Full sun with some air movement is recommended for persimmon trees in inland areas, although they will tolerate some partial shade. But trees do not produce well in the high summer heat of desert regions, which sunburn the bark.
Kaki persimmons are drought tolerant. Persimmon trees can withstand drought, but fruit yield and quality (especially size) are reduced. Also, adequate moisture in the soil is required to produce sufficient shoot growth and formation of flower buds for next year's crop. The trees should be irrigated during dry periods.
The persimmons trees can grow well on a wide range of soil types but do best in deep, welldrained loam soils with a good supply of organic matter. Heavy clay loam soils that are prone FAO stat [2]. http://www.faostat.com. * TUİK [3]. Turkish Statistic Council records. to water-logging should be avoided. The preferred soil pH for optimum tree growth is in the range of 6.0-7.5. However, persimmon trees can tolerate a wider variety of conditions than most fruit trees.
Pest and disease problems: protection of fruits from bats and birds are required. Fruit flies are the potential problem as are aphids and mealybugs. Persimmon trees are also susceptible to collar rot, thus keeping mulch clear of the trunk is required.

Origin and history
Zeven and Zhukovsky [5] suggested that persimmon (D. kaki) has a primary center of genetic origin in the mountains of central China and a secondary center in Japan. Persimmon cultivation in China began more than 2000 years ago, and it is also scientifically known as D. chinensis. In China, it is found wild at altitudes up to 6000-8000 ft. [6]. It spread from China to Korea and to Japan many years ago. Since from prehistoric times, permission is consumed as food source in these countries. There are some trees that are 400-500 years old. It was imported in Europe (South France) for the first time in 1760. Thereafter it spread to the Mediterranean coast (Italy, Spain, Greece, Turkey and Algeria). The persimmon plant was introduced in North America (California, Florida), South America (Brazil) and Australia in the mid-1800s. Early in the fourteenth century, the explorer Marco Polo recorded the Chinese trade in persimmons [7]. Its cultivation has recent traditions in western countries where it is present only since the second half of the nineteenth century. Currently, persimmon is one of the most important fruit crops in Asian countries and, there is also steady increase in its production in some European countries.

Botany of persimmon
The genus Diospyros contains hundreds of plant species and are distributed in the tropical and subtropical regions of the world. Four species of them have commercial importance. D. kaki L. is the most widely cultivated species of the Diospyros genus. D. kaki is also known as the persimmon, Japanese persimmon, Oriental persimmon, Japanese persimmon, Kaki, Asian persimmon. It has been reported that wild type D. kaki exists in the forests of China [8,9]. The other species are D. lotus L. (the date plum), D. viriginiaina L. (native American persimmon) and D. oleifera Cheng [10].
The origin of D. kaki and its relationship to other Diospyros species is not well understood. The persimmon culture was known to occur in the fifth or sixth century in China [9]. In addition to D. kaki, D. lotus and D. oleifera also have been cultivated as fruit crop. D. lotus has been consumed as a fresh as well as dried fruit, and it is a source for tannin [9,10]. Another important species known as a fruit crop is D. virginiana, of the eastern United States origin. This species which is consumed as fresh and processed is grown on a much smaller scale and is not yet considered a commercial crop [11]. These species are quite important as horticultural crops among the Diospyros species of temperate origin. On the other hands, D. rhombifolia originated from China is an ornamental plant which bears tiny attractive-colored fruit on a dwarf tree [9].
There are other species such as D. digyna (black sapote), D. discolor and D. decandra that have originated in the tropics and subtropics and produce edible fruits.

Pomological classification
Persimmon fruit is highly astringent due to soluble tannins in the vacuoles of the fruit flesh. However, some cultivars lose astringency naturally on the tree as fruit ripens, while others retain astringency until maturity. Therefore, persimmons are classified into two major groups (based on the presence or absence of astringency in the fruit at maturity) as astringent (A) and non-astringent or sweet (NA) cultivars. Water-soluble tannins which cause astringency in the flesh of astringent types decrease as the fruit softens and becomes edible. However, astringency can be removed by various chemical treatments. Carbon dioxide gas or alcohol can be used to remove astringency, while the fruit remains firm. If ethylene is used for removing astringency, the fruit softens very quickly. Fruit of the non-astringent types naturally loses astringency, while the fruit is still firm. Thus, the fruit of non-astringent types is edible either the flesh is firm or soft [10,12,18].
Each group can be further subdivided, based on their response to pollination [18]. The amount of dark flesh coloration around the seeds varies in cultivars and changes in flesh color are related to seed formation, not pollination. In pollination variant types (PV), the flesh is dark and streaked around the seeds, but clear orange when seedless. When pollination is poor and only one or several seeds are formed, a dark area develops around the seeds, but the remaining flesh is light colored. The pollination variant types include cultivars that are astringent when they have several seeds or seedless (PVA), as well as partially or totally non-astringent when they have only one or a few seeds (PVNA). Also, in astringent cultivars of the pollination variant type, fruit which has a great degree of the dark flesh is non-astringent even when the fruit flesh is firm [10,18].
Pollination constant (PC) types lack the dark streaking regardless of seed formation. The flesh color of pollination constant astringent (PCA) cultivars is not influenced by pollination and it does not develop dark flesh around the seeds. Pollination constant non-astringent (PCNA) persimmons are always edible when still firm, regardless of whether or not pollination has occurred.
PVA types can vary to either PCA or PVNA depending on several situations. If PVA type does not have any seed for some reason or when PVA persimmon varieties are cultivated without pollinators, the fruit has clear orange flesh and remains astringent (PCA) such as the Spainish variety 'Rojo Brillante' and Japanese variety 'Hiratanenashi'. Similarly, when PVA type has enough seeds (usually four or five) after pollination, the fruit has a great degree of dark flesh and loses astringency (PVNA) such as 'Nishimura Wase'.

Commercial and recently improved persimmon varieties
The fruits of two species (D. kaki L. and D. virginiana) in the genus Diospyros have commercial importance. In China, using native persimmon germplasm, several common persimmon varieties were developed. However, they all are PCA with the exception of 'Luo Tian Shi' [19,20]. Persimmon is the main species cultured for edible fruit production in northern China. Recently, 'Jirou', 'Youhou', 'Taishuu' and 'Fuyu' among PCNA cultivars are gaining popularity. Persimmon growing regions are also spreading widely in Japan and Korea, thus some old well-known Persimmon varieties which were still being produced were selected from these countries. Persimmon has been a major fruit crop in Japan for many years [21] and for Japanese persimmon commercial production, 'Fuyu' (PCNA), 'Hiratanenashi' (PVA) and 'Tonewase' have been the three important cultivars. About 57% of the total area is devoted to these varieties [22]. Other varieties growing in Japan are 'Kosyu Hyakume', 'Matsumotowase Fuyu', 'Early ripening Jiro', 'Ichidagaki', 'Jiro Dojohachiya' and 'Taishu'. However, newly released cultivars such as 'Reigyoku' and 'Taiho' are also available. In Korea, non-astringent varieties have increased, while astringent varieties have decreased. In the recent years, amount of production of non-astringent varieties are higher than those of astringent varieties. Major cultivar of non-astringent type is 'Fuyu', which accounts for almost 82% in total production of persimmon, and 'Jiro' with 9.8% [23].
In Taiwan, 'Suzchou', 'Niouhsin' and 'Shihshih' local PCA are the major commercial varieties used. 'Fuyu' and' Jiro' are the main PCNA varieties [24]. Countries such as Azerbaijan and Uzbekistan focused on local astringent cultivars. In Brazil, the most cultivated persimmon cultivars include 'Rama Forte' and 'Giombo', which belong to the PVA group, 'Taubate' which is continually astringent with yellow flesh either with or without seeds (PCA), and 'Fuyu', which belongs to the PCNA group [25,26].
'Fuyu', 'Hana Fuyu' and 'Ichikikei Jiro' cultivars which are PCNA and 'Hachiya' (PCA) are commonly produced in the USA [10,18]. In new persimmon growing countries such as New Zealand and Australia, most of the cultivation area is devoted to 'Fuyu' [27]. In Spain, the most produced cultivars are 'Rojo Brillante' (Figure 1) and 'Triumph' which can be stored for a long time [28]. In Italy, almost 90% of the persimmon production is Kaki Tipo (PVNA), the rest of production is other PVNA varieties (Vainiglia, Mercatelli and Moro) and PCNA cultivars such as Hana Fuyu, Jiro and Gosho [29].
Israel has its own cultivar, Triumph (Figure 2) which is sold under the name of Sharon fruit, and it is planted on 95% of the total area devoted to persimmon [27]. Also, persimmon production in South Africa is based on Triumph [30]. In Turkey, a great amount of production is PCA and PVNA varieties, which are selected from Turkey. However, recently introduced PCNA cultivars such as Fuyu, Hana Fuyu, Jiro and Izu have become popular with the growers. The new orchards with 'Fuyu' and 'Hachiya' cultivars have been established.

Persimmon germplasm resources
Persimmon originated from China, but it has been cultivated and produced mostly in Japan [31]. Persimmon has limited amount of production in the rest of the World. However, Spain, Italy, Israel and Brazil are now producing important amounts and these countries have developed their own cultivars such as 'Rojo Brillante' in Spain, 'Kaki Tipo' in Italy, 'Triumph' in Israel and 'Lama Forte' in Brazil. Recently, Australia and New Zealand have started to produce persimmon mainly for export, and the USA is also producing persimmon on a small scale.
Greene and Morris [32] indicated that germplasm collections are a source of genetic diversity to support crop improvement and botanical research as well as to support conservation efforts [33]. For the specific breeding objectives, these variations can either be created spontaneously or artificially by budwood mutations or cross breeding. The importance of germplasm can be explained by the variation of plant material. Therefore, recording and registration of genetic resources is critical for breeders in terms of improving new varieties. Hance. have been cultivated as fruit crops [35]. There are 550 accessions including most cultivars native to China and some native to Japan and Korea.
Aside from this exceptional existence of a PCNA type cultivar in China, almost all non-astringent type cultivars were developed in Japan. Historical records show that 'Zenjimaru', known to be the oldest PVNA type cultivar, was found in the beginning of the twentieth century, and that 'Gosho', was the first PCNA type cultivar, which was recorded in the seventeenth century [36]. In the beginning of the nineteenth century, 'Fuyu' and 'Jiro' were recorded as the most popular PCNA type cultivars. According to a nationwide survey on persimmon cultivars in Japan (Agricultural Research Station 1912), there were only 6 PCNA type cultivars in contrast to 401 PVNA type cultivars among more than 1000 cultivars collected from all over Japan. This means that in addition to its more recent appearance, the PCNA type probably has very narrow genetic variability. A total of 40 PCNA cultivars, including bud sports, which may cover almost all PCNA type cultivars currently existing in Japan, are now preserved at the National Institute of the Fruit Tree Science (NIFTS) in Akitsu, Hiroshima [9]. There are many astringent and PVNA local cultivars throughout Japan. The current conservation in Japan consists of approx. 600 genotypes [37].
In Korea, 233 local cultivars were collected at the branch of Experimental Station at Kim-hae during 1959-1969, and 74 superior cultivars were selected for persimmon cultivation after identifying the name of 188 cultivars among these local cultivars. In Korea, interest in persimmon cultivation is increasing and two experimental stations for persimmon have been established, the one for non-astringent persimmon was established in 1994 and the other for astringent persimmon established in 1995. In addition, a breeding program for obtaining new PCNA cultivars was started in 1995 by crosses among PCNA cultivars that were introduced from Japan. The breeding objectives in Korea are focused on obtaining superior PCNA cultivars with good eating qualities, large fruit and early ripening characteristics [9].
In Europe, persimmon is considered a secondary fruit tree species; only few countries, located in the Mediterranean area, are interested in a large-scale production.
Persimmon was introduced in Italy at the end of the ninteenth century. Later in Tuscany, the interest for this new species was increased and the genotypes were collected together with exotic and local varieties of fruit tree species (citrus, peaches and plums among others). As early as 1940, the University of Florence collected 11 accessions from the USA and France, or as local varieties and characterized them. The persimmon collection of Florence consisted of 52 cultivars and was totally destroyed by winter frost in 1985. Then a new germplasm orchard was established by introducing new accessions from Japan. A French germplasm was recorded at the beginning of the twentieth century. The Spanish collection was created in 1993 with material from Italy (54.2% of accessions) and from Spanish institutions and nurseries (45.8%) [38].
Persimmon was introduced into Brazil's São Paulo state in 1890. However, its cultivation expanded around 1920 with Japanese immigration. São Paulo is the main persimmon producing state. Rio Grande do Sul state has the second largest persimmon production of Brazil. In recent years, the persimmon acreage has increased and the trend is to continue crop expansion. 'Fuyu', 'Rama Forte', 'Giombo' and 'Taubaté' are the cultivars grown in Brazil.
In Azerbaijan, persimmon production is widely spread since 1998, although its history has deep roots [2]. In terms of persimmon genetic resources in Israel, only high commercial value cultivars are collected.
Although the exact date of the introduction of persimmon to Anatolia is unknown, it is clear that it dates back to rather old times [39,40]. Persimmon was introduced to Turkey from Russia via the Black Sea region. Turkey has main persimmon species (D. kaki, D. lotus and D. oleifera). D. oleifera can be seen only in the Mediterranean region of Turkey, while D. lotus grows as wild in Northern Anatolia and is used as dried fruits in this region. D. kaki and D. oleifera have been introduced from other countries at least 200 years ago. During this time, continuous propagation of persimmon by its seeds resulted in genetic diversity in D. kaki trees due to the high heterozygosity. Therefore, in the northeastern part of Turkey, persimmon trees differ from another in terms of fruit productivity, yield, shape, size, astringency and plant growth. This diversity in persimmon population in Turkey provided a great opportunity to the breeders for selection programs. As a result, the breeders were able to identify many promising clones in different parts of Turkey. A germplasm collection in the Black Sea region in Turkey with selected promising genotypes has been established.
First studies on persimmon in Turkey were started to introduce the foreign cultivars by the Ministry of Agriculture in 1967. Then, some selection studies were done in different parts of Turkey. After 1989, the total number of the known cultivars and types reached up to 74. Most of these varieties were introduced from Italy and some of them were from Israel, Japan, France and Pakistan especially after the attempts made by the Cukurova University, in Eastern Mediterranean, Turkey. Most even totality of the selections is astringent type. It seems to be rather difficult to find non-astringent types in Turkey. Yilmaz et al. [41] established a characterization study on persimmon genetic resources collected from Turkey. These germplasms were preserved with commercial cultivars in an ex situ germplasm preservation orchard located at the Cukurova University, Turkey. Persimmon genotypes were characterized based on their morphological traits. The collection comprising traditional genotypes, local accessions and also global varieties were collected from five different provinces of the Mediterranean region of Turkey where persimmon is widely produced. A total of 48 persimmon genotypes and cultivars were morphologically characterized, using 59 morphological and agronomic traits ( Table 5).

No Cultivar and selections
Scientific Name Origin Type of astringency  From them, 9 traits were related with plant growth, 5 with leaves, 7 with flowers, 32 with fruits and 6 with seeds. As a result of the morphological characterization, persimmon varieties and types were classified by PCA, PVA, PCNA and PVNA. Besides, data obtained by characterization were subjected to similarity coefficient, principal components and cluster analyses to study phenotypic relationships among these genotypes. As a result of their study, the 12 factor scores represented 74.75% of the total multivariate variation, and cluster analysis indicated that the similarity index of the population consisting of the investigated genotypes ranged between 81.09 and 17.32% (Figure 3).

Persimmon breeding and genetic improvement
Breeding aims for persimmon emphasized on enhanced fruit quality such as fruit weight, shape, color, soluble solids content, fruit cracking, fruit ripening time, high productivity, long shelf life, parthenocarpy and sex expression. Selection breeding is the most common breeding technique in persimmon due to the fact that persimmon breeding is mainly hindered by its high ploidy level and by its complex sex expression [42]. Because somatic and bud sport mutations affect the fruit traits, new lines of persimmon are frequently improved by bud mutations [43,44]. Also, there are many seed propagated populations in persimmon growing countries of the world especially in native Asian countries. Many established cultivars are chance seedlings selected by growers or researchers in Japan, China and Korea. There are also many selections from introduced genotypes or seedling populations in the USA, Israel, India, Australia, New Zealand, Taiwan [42]. 'Nantongxiaofangshi' variety having dwarfness character is a persimmon that has been found in Nantong. 'Nantong small persimmon' (D. kaki Linn. cv. Nantongxiaofangshi) is a rare and dwarf variety of persimmon found in 1982 in Jiangsu Province Nantong City Fruit resource survey.
The height of the adult tree is only about 2 m, which is approximately equal to 60% that of the standard type growing under the same conditions [46]. 'Hasshu' persimmon (D. kaki Thunb.) is a dwarf cultivar originated by a bud sport from the leading persimmon cultivar 'Hiratanenashi' in Hiroshima prefecture, Japan in 2005. Its somatic polyploidy (2n = 120 = 8x) was confirmed by flow cytometric analysis and chromosome observation. Although nonaploid 'Hiratanenashi' and some of its bud sports are known to be seedless, 'Hasshu' produces regular seeds with the ability to germinate [16].
Main objective of persimmon breeding has been to produce commercially attractive cultivars of the PCNA type which can be eaten without any postharvest treatment [46]. Therefore, PCNA fruit are the most desirable for fresh consumption because it is not necessary to apply any postharvest treatment in order to remove the astringency. Hence, the breeding of new PCNA cultivars is the most popular objective in the entire persimmon growing countries.
Although persimmon is produced in Brazil, Israel, Italy, Spain, Azerbaijan, Uzbekistan, New Zealand and Australia, new persimmon cultivars developed by cross breeding have been released only in Japan and Korea [21] and also in these countries persimmon cultivars have been selected over time for commercial production. Hybridization method can also be used in persimmon breeding. In Japan, hybridization method has been used for fruit ripening time, crack-resistance and large fruit size in persimmon.
Persimmon breeding is complex, and results are not always as expected, especially when working on the PCNA group [42]. Researchers found that fruit ripening time is under additive and quantitative control. The tendencies of persimmon fruit are quantitatively inherited traits, the non-cracking cultivars are homozygous, whereas cultivars with cracking are heterozygous. Also, it has been claimed that fruit weight is a quantitative characteristic with high broad-sense heritability [21]. Many crosses performed using large and small fruit size parents indicated that small fruit alleles were dominant to large size alleles [48]. Complete loss of astringency is important for commercial persimmon production. Generally, a little astringency remains in PCNA fruit at maturity in cooler regions, so that they are commercially produced in warm regions. Incomplete loss of astringency results from not only environmental factors but also genetic factors [21]. According to the criteria established for persimmon cultivars, persimmon can be categorized into two major groups, PCNA type consisting of two subcategories, Chinese PCNA (CPCNA) and Japanese PCNA (J-PCNA). The second group is non-PCNA type consisting of three subcategories: PCA, PVNA and PVA [49]. Japanese PCNA cultivars are based on a recessive character and their genetic resources are very few. Repeated crossings within the narrow gene pool cause inbreeding depression, which hinders tree vigor, fruit yield and size. Therefore, studies have been ongoing to obtain new cultivars through the backcross (PCNA × non-PCNA) × PCNA since 1990. In 2007, 'Taiten' and 'Taigetsu', which are PVA cultivars, were derived from the cross of 'Kurokuma' (a local PVNA cultivar in Japan) × PCNA cultivar 'Taishu'. Parthenocarpy in 'Taigetsu' is high [19]. The trait of natural astringency loss is dominant and controlled by the single locus CPCNA in Chinese PCNA persimmon [50]. At the end of the hybridization studies in Japan, PCNA cultivars 'Shinshuu', 'Soshu', 'Kanshu' and 'Kishu' were released as early ripening cultivars while 'Suruga', 'Youhou', 'Taishuu' and 'Yubeni' were released as medium to late ripening cultivars [21]. Other persimmon hybrids are 'Fuyuhana' and 'Ito'. 'Fuyuhana' were developed from a 'Fuyu' × 'Hanogosho' cross as an alternative to 'Fuyu' and 'Jiro'. Ito is another hybrid obtained by crossing 'Fuyu' × 'Oku-Ogosho' [51].
In persimmon, different genus can pollinate each other. Native American persimmon (D. virgininiana) and Japanese persimmon (D. kaki) hybridization would set a goal of stabilizing and improving the variable flavors and cold hardiness of the native American persimmon. D. kaki and D. virginiana are apparently cross-incompatible; however a hybrid 'Rossiyanka' has been developed through embryo culture technique [51]. Rossiyanka is cold hardy, nearly seedless and it is smooth textured with Asian persimmon flavor [52]. Nikita's gift hybrid persimmon is unique hybrid of Asian and American persimmon; the fruit is sweet and flavorful [53].
In persimmon breeding programs, mutation breeding technique has also used. The main objectives of persimmon mutation breeding were focused on obtaining new cultivars with the positive agronomic features but with more diversity in ripening date, astringency and fruit characteristics from the PCNA types. However, obtaining PCNA type varieties is difficult due to the dominant inheritance of astringency, the limited number of cultivars which bear male flowers and the hexaploid inheritance of basic persimmon cultivars. Therefore, the PCNA type cultivars have low genetic diversity and crossing among these generally result in negative effects of inbreeding. Mutation breeding has been used as an alternative method for generating diversity in persimmon [54]. Some researchers studied to determine which gamma ray doses can be used in persimmon. Ray [51] claimed that 5-10 kR gamma doses obtained widest range of viability on cuttings, seeds and pollen of persimmon. In Spain, shoot buds of the persimmon 'Rojo Brillante' were subjected to various doses of gamma rays, 15 and 20 gray from a 60 Co source. In this study, Naval et al. [55] found that the most favorable gamma irradiation dose combining survival and mutation induction was 20 gray. Two new varieties with similar fruit quality to 'Rojo Brillante', that allow to enlarge the persimmon harvest season in Spain, were selected [56].

Biotechnology and genomics
Biotechnology refers to the use of living organisms or their components to provide useful products in its broadest sense. Using biotechnology in plant breeding has become the most attractive method due to increasing knowledge in plant biotechnology and genomics. Improvements in the field of genomics have resulted in the development of huge quantities of useful new knowledge that greatly assists scientific plant breeding. Also, improvements in biotechnological techniques like plant tissue culture provided new methods for rapid production of high-quality, disease-free and true to type planting material.
In persimmon, biotechnological advances and molecular biology have been used for the classification of Diospyros species, in vitro propagation, regeneration from callus, root, protoplast and endosperm, ploidy manipulations, agrobacterium-mediated genetic transformation and markerassisted selection. Molecular markers have been widely used for investigating the genetic relationships among persimmon genotypes. Akbulut et al. [57] compared persimmon genotypes by using random amplified polymorphic DNA (RAPD) and fatty acid methyl esters (FAME) data. The results showed that RAPD analyses could differentiate the relationship of persimmon (D. kaki Thunb.) genotypes used in their study. The authors suggested that more cultivars were needed as plant materials in terms of determining the degree of relationships of RAPD and FAME data which could help delimiting taxonomic classes within persimmon. Raddová et al. [58] indicated that RAPD and inter-primer binding site (i-PBS) were reliable enough to detect differences between the genetically close cultivars of persimmon. In addition, Badenes et al. [45] studied the genetic diversity of introduced and local Spanish persimmon cultivars as revealed by RAPD markers. The authors suggested that a correct identification of germplasm material from persimmon collections should be the first step in projects related to breeding or management of cultivar aimed at improving the crop. They also indicated that RAPD technology is adequate for fingerprinting persimmon. Yonemori et al. [59] studied the relationship between the European persimmon (D. kaki Thunb.) cultivars and Asian cultivars using AFLPs. The authors indicated that the placement of several Japanese cultivars within the European cultivar group suggests that European cultivars were developed from Japanese germplasm relatively recent and differences among cultivars are much greater than differences among cultivar groups regarding AFLP markers. In addition, Guo and Luo [60] indicated that SSR markers are a valuable tool for the estimation of genetic diversity and divergence in Diospyros.
The main in vitro tissue culture techniques developed for persimmon deal with direct regeneration (from dormant buds and root tips) and indirect regeneration through callus from dormant buds, apexes and leaves. Kochanová et al. [61] indicated that in the genus Diospyros L., biotechnological researches focused on quality improvement and preservation of the cultivars that has been economically cultivated. The authors also remarked that the genetic variability had been lost as only those limited cultivars that are popular among growers are grown. In recent years, studies were conducted on in vitro micro-propagation of persimmon [62], especially on Jiro [63] and Rojo Brillante [64]. Choi et al. [65] recorded an efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (D. kaki Thunb.). The authors indicated that the frequencies of adventitious shoot regeneration by 'Nishimurawase' and 'Fuyu' reached up to 100% and the regenerated shoots rooted successfully with over 80% efficiency. Yokoyama et al. [66] suggested that the meristematic nodule is a promising material for propagation and long-term conservation of 'Fuyu' variety. Naval et al. [67] recorded a protocol for plant regeneration of D. kaki Thunb. cv. 'Rojo Brillante' via organogenesis from leaf explants by using combined phytohormones and dosages. In addition, Naval et al. [67] studied somaclonal variation of 'Rojo Brillante' as a breeding tool by using various combinations of cytokinin (Z or BA) with different auxins (IAA or NAA). Furthermore, Palla et al. [68] studied in vitro culture and rooting of D. virginiana L. from nodal root explants by using several phytohormones and culture media. The authors indicated that the presence of auxins was not essential but slightly accelerated the organogenic callus formation and organogenesis. Cryopreservation is recognized as having the distinctive advantage of allowing long-term conservation with minimum space and maintenance [69]. Matsumoto et al. [70] studied cryopreservation of persimmon (D. kaki Thunb.) by vitrification of dormant shoot tips. The authors recorded that using dormant shoot tips was promising as a routine method for the cryopreservation of Diospyros germplasm.
After the great progress in in vitro regeneration of plants from protoplasts, several researches focused on plant somatic hybridization which allows combining protoplasts from different cultivars, species or genera for variety improvement [71]. Tao et al. [72] reported plant regeneration from callus protoplast of D. kaki. They used callus as the protoplast source derived from leaf primordia excised from dormant winter buds of adult Japanese persimmon (D. kaki L. cv. Jiro) for plant regeneration. Tamura et al. [73] studied protoplast culture and plant regeneration of D. kaki L. and reported that plantlets could be obtained from the protoplast-derived calli. Tamura et al. [74] indicated that somatic hybrids of Japanese persimmon (D. kaki L.) were obtained by electrofusion of protoplasts. Callus protoplasts of Jiro and Suruga were fused electrically and cultured in modified KM8p medium using agarose-bead culture. The authors recorded that the fused products had the dodecaploid chromosome number of around 2n = 180, which is twice the number of parental plants (2n = 90x = 15). In addition, Tamura et al. [75] recorded interspecific somatic hybrids between D. glandulosa (2n = 2x = 30) and D. kaki cv. Jiro (2n = 6x = 90) produced by electrofusion of protoplasts. Colchicine treatment of actively dividing cells can induce chromosome doubling and has been used to make plants with doubled chromosome number. Colchicine treatment to a protoplast at the very beginning of its division could be one method to overcome the problem because plants can be regenerated from a single cell with doubled chromosome number. Tamura et al. [74] reported production of dodecaploid plants of Japanese persimmon by colchicine treatment of protoplasts.
Improved genomic research and resources, in recent years, have resulted in the development of screening tools via marker-assisted selection (MAS). Using MAS has led to more efficient selections and has increased the efficiency in persimmon breeding programs hastening the release of new cultivar. In order to obtain PCNA offspring in breeding programs, the parental materials considered for choosing the cross combinations have to be PCNA type regarding the inheritance of astringency. However, repeated crosses among PCNA cultivars/selections has led to inbreeding depression for tree vigor, productivity and fruit weight [36]. In these situations, marker-assisted selection should be developed for selecting PCNA offspring efficiently. Recently, Kanzaki et al. [47] have developed molecular markers associated with the trait of natural astringency loss in persimmon fruit and the markers are practically useful in persimmon breeding programs. In addition, Mitani et al. [76] studied if the SCAR markers could reliably distinguish PCNA and non-PCNA genotypes in a large number of offspring derived from backcross between 'Taigetsu' and PCNA 'Kanshu'. The authors indicated that PCNA offspring can be selected by two PCR primers in the progeny derived from 'Taigetsu' × 'Kanshu'. Yonemori et al. [77] reported molecular marker for selecting PCNA type persimmon progenies at the juvenile stage. Yonemori et al. [77] constructed a reliable PCR marker for selecting PCNA type offspring among breeding population of persimmon. In addition Kanzaki et al. [47] and Mitani et al. [76] reported that SCAR markers can practically be used in application of marker-assisted selection in persimmon breeding.
Genetic transformation is also an alternative technique for persimmon genetic improvement. Transgenic persimmon cultivars thus produced have potential for commercial success and grower acceptance because the unique genetic constitution of the cultivars has not been disturbed. Tao et al. [78] reported genetic transformation of persimmon by Aagrobacterium rhizogenes. Phenotypic alterations such as dwarfness and decrease in rooting ability were observed in the transformants. In addition, Gao et al. [79] transformed 'Jiro' persimmon with Arabidopsis FT gene (AtFT) and PmTFL1 gene, a Prunus mume ortholog of Arabidopsis TFL1 gene. The authors indicated that the PmTFL1 transgenic in vitro shoots did not show a different appearance compared with non-transformed 'Jiro' shoots, however, the AtFT transgenic shoots indicated a 'bushy' phenotype having the short internodes.

Conclusions
Persimmon can adapt to a wide range of climatic conditions. Production in many countries having subtropical and tropical climates satisfies domestic demand and creates new export opportunities. Increasing the world persimmon production has been very successful since 1995. Recently, the applications of the technique for removing astringency without losing fruit firmness have been significantly promoted to increase the production. It is expected that the production will significantly increase over the next few decades.
Selection breeding is the most common breeding technique in persimmon because persimmon breeding is mainly hindered by its high ploidy and by its complex sex expression. Fuyu, Hachiya, Hiratatenashi, Izu, Jiro and Saijo cultivars which are extensively growing all over the world were selected from shoots of bud sports in Japan. We should continue the screening of plants coming from spontaneous mutations. Hybridization method can also be used in persimmon breeding. Hybridization studies among D. kaki in Japan have led to the release of a lot of PCNA cultivars which ripen at different times. There is also a unique hybrid of Asian and American persimmon. In Spain, studies on induced mutation have also led to the development of new cultivars.
There are a number of collections including many accessions in institutions of the various persimmon growing countries. The morphological and molecular characterization of all the persimmon accessions needs to be achieved. The information developed from this will be highly beneficial for screening against biotic and abiotic stress factors. Genomics and transcriptomic resources need to be developed for persimmon. It will also lead to the development of new and improved cultivars of persimmon.