Seedlessness at the Diploid Level

Male and female sterility

Various levels of male sterility at the diploid level have been reported in citrus (Iwamasa, 1966) (Table 8.1). Chromosome

aberration is one of the most important phe- nomena which cause pollen sterility. Asynapsis in ‘Mukaku yuzu’ is genetically controlled, while that in ‘Eureka’ lemon and ‘Mexican’ lime is induced by low tem- perature (Nakamura, 1943; Iwamasa and Iwasaki, 1962; Iwamasa, 1966). Reciprocal translocation is the main cause of sterility of ‘Valencia’ sweet orange, and this sterility is not found in other sweet oranges (Iwamasa, 1966). Inversion caused partial pollen sterility of ‘Mexican’ lime. Pollen sterility of ‘Marsh’ grapefruit is due to the failure of spindle formation (Raghuvanshi, 1962). Male sterility which is not caused by chromosome aberration is also well known. Anther abortion in satsuma hybrids is taken to be due to strict male sterility in citrus (Iwamasa, 1966). The sterile stamen appears only as a fi lament, and no pollen grains are produced (Fig. 8.1). Early degen- eration of pollen mother cells (PMCs) was found in ‘Washington’ navel, ‘Tahiti’ lime and some other hybrids (Osawa, 1912; Uphof, 1931; Frost, 1948). Pollen sterility of satsuma mandarin is caused by plural sterility such as abnormal behaviour and degeneration of pollen grains (Nakamura, 1943; Yang and Nakagawa, 1970). A close relationship between the free proline con- tent in citrus anther and pollen fertility has been found (Liu et al., 1995), and the culti- vars with normal fertility have a higher pro- line concentration than those with partial or complete sterility. To develop new seedless

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Fig. 8.1. Flowers of male sterile (aborted anthers, MS) and male fertile (normal anthers, MF).

Table 8.1. Diagramatic representation of various kinds of male sterility occurring in citrus, according to the sequential order of development (from Iwamasa, 1966).

Meiosis

Division I Asynapsis (genic) Mukaku yuzu Iwamasa, 1966 Asynapsis Eureka lemon Nakamura, 1943

(by low temperature) Mexican lime Iwamasa et al., 1962 Translocation C. assamensis Naithani et al., 1958

Valencia orange Iwamasa, 1966

Inversion C. assamensis, etc. Raghuvanshi, 1962a Mexican lime Iwamasa, 1966

Division II Failure of spindle Marsh grapefruit Raghuvanshi, 1962b

Liberation from tetrad Mitotic division Mature pollen grain

Degeneration Jaffa orange Oppenheim, 1929

Degeneration Satsuma Nakamura, 1943

cultivars effi ciently, genetic analysis of male sterility has been carried out. Among these studies, genetic analysis of anther abortion has progressed remarkably. This male steril- ity is due to gene–cytoplasmic interaction (satsuma mandarin and ‘Encore’ mandarin possess sterile cytoplasm) and is probably controlled by more than two major genes (Iwamasa, 1966; Yamamoto et al., 1992, 1997; Nakano et al., 2001). New seedless cultivars with aborted anthers have been released in Japan (Nishiura et al., 1983; Matsumoto et al., 1991). Anther male steril- ity and inheritance of pollen fertility/steril- ity were also studied (Ueno, 1986). Some pollen-sterile progeny had arisen from par- ents that were both pollen fertile. Vardi and Spiegel-Roy (1981) postulated that asynap- sis was controlled by a single recessive gene because about a quarter of progeny arising from self-pollination of ‘Wilking’ showed

asynapsis. Nakano et al. (2000) found DNA markers linked to male sterility for juvenile screening of male-sterile plants.

Female sterility is a very important trait closely related to seedlessness and is a heritable characteristic (Yamamoto et al., 1995, 2001). ‘Mukaku kishiu’, a bud varia- tion of the seedy kinokuni mandarin, is complete seedless, and is considered to have the strongest female sterility in citrus. This female sterility causes abortion of the zygote, and is controlled by two genes (Nesumi et al., 2001). A new seedless culti- var and parental line with this sterility was bred in Japan. Navel orange and satsuma mandarin have strong female sterility. Only a few seeds developed when they were hand pollinated (Nishiura and Iwasaki, 1963). Osawa (1912) observed degeneration of the embryo sac in both navel orange and satsuma mandarin. Nesumi et al. (2000)

assumed that the female sterility of satsuma mandarin is controlled by two major genes, and they were genetically mapped (Omura et al., 2000). Seediness of hand-pollinated fruits is low in both ‘Valencia’ orange and ‘Marsh’ grapefruit (Wong, 1939). Chromosome aberration, as mentioned with regard to male sterility, probably occurs in the embryo sac. In non-functional pistils of lemon, possible blocking of further stigma and style development is related to the presence or absence of receptive embryo sacs in the ovule (Wilms et al., 1983).

Mutagenesis is effi cient in developing seedless plants from seeded accessions because sterility is one of the most frequent effects of treatment with a mutagen. Hensz (1971) developed the seedless ‘Star Ruby’ grapefruit through irradiation of seed of the seedy ‘Hudson’ by thermal neutrons. After his success in developing a seedless cultivar using irradiation, this method was applied to several seedy cultivars. Hearn (1984) pro- duced seedless strains of ‘Pineapple’ orange and ‘Duncan’ grapefruit from g-ray irradia- tion of seeds. He also developed seedless strains of ‘Foster’ grapefruit through g-irra- diation of buds. Seedless strains of ‘Eureka’ lemon and ‘Monreal’ clementine were developed through g-irradiation of budsticks (Spiegel-Roy et al., 1985; Starrantino et al., 1988). Chen et al. (1991) produced seedless strains of ‘Jin Cheng’ orange through g-irra- diation of seeds, and chromosome aberra- tions were observed in these strains. South Africa has developed an ambitious pro- gramme of mutagenesis by g-irradiation of budwoods to obtain seedless cultivars (Froneman et al., 1996). About 400 trees with 2400 branches exhibiting seedless fruits have been selected.

Biotechnology procedures such as transformation and genome analysis have been conducted to develop new seedless citrus (Koltunow et al., 1998). Tobacco and Arabidopsis transformants have been regen- erated containing chimeric genes of soy- bean conglycinin and storage protein gene promoters linked to the bacterial RNase gene, barnase. Reduction in seed size was only observed in Arabidopsis seeds (exal-

buminous), and not in tobacco (albu- minous). Some transformants of both species were male sterile and this corre- lated with the gene expression in anthers. Citrus forms exalbuminous seeds. The bar- nase constructions may be useful in elicit- ing a reduction in seed size. Transgenic West Indian lime plants containing genes for decreased seed set have already been produced from seedling hypocotyl and epi- cotyl segments by Agrobacterium -mediated gene transfer (Koltunow et al., 2000). The shorter juvenile period of lime provides the opportunity to test the introduced genes for their ability to induce reduced seed set. Garcia et al. (2000) found a DNA marker for the number of seeds obtained from 69 DNA and isozyme markers, and this marker must be related to female sterility.

Self-incompatibility

Self-incompatibility is a genetically con- trolled phenomenon preventing seed set in self-pollinated plants producing functional gametes. In self-incompatible accessions, in conditions of self-pollination, no pollen tubes were found in the ovaries (Ton and Krezdorn, 1967). Nagai and Tanikawa (1928) found that some self-incompatible accessions produced seedless fruits when they were self-pollinated. Almost all pum- melos, some mandarins and several natural or artifi cial hybrids are self-incompatible (Hearn, 1969). Some of the self-incompati- ble cultivars are seedy because of their female fertility and lack of parthenocarpy requiring cross-pollination to set fruits (Miwa, 1951; Mustard et al., 1956; Krezdorn and Robinson, 1958). Some self- incompatible accessions can produce seed- less fruits in a single planting, and clementine is probably the most famous of these. These fruits are sometimes smaller than seeded ones which tend to have reduced setting of fruit. Mixed cultivation of these accessions with male-fertile plants often yield seedy fruits unless they have female sterility (Soost, 1956; Reece and Register, 1961; Hearn, 1969; Iwamasa and

Oba, 1980; Li, 1980). However, seedless fruits can be produced in self-incompatible accessions without parthenocarpic ability by pollination with tetraploid pollen or application of growth regulators (Soost, 1961; Yamashita, 1976).

The incompatibility system of citrus is the gametophytic type, and Soost (1965, 1968) proposed ‘S’ genotypes in some acces- sions. At least three alleles are present in the pummelos tested. At least two of these differ from the alleles in ‘Sukega’. At least four alleles are present in ‘Dancy’, clementine, ‘Minneola’, ‘Orlando’, ‘Sukega’ and ‘Duncan’ grapefruit. Incompatibility S alleles are dis- tributed widely; not only in self-incompati- ble accessions but also in self-compatible ones such as satsuma mandarin, grapefruit and ‘Dancy’ (Soost, 1965, 1968; Vardi et al., 2000). Thus, a self-incompatible individual can be produced from cross-combinations between both self-compatible parents, e.g. ‘Orlando’ and ‘Minneola’ arose from the combination ‘Duncan’ grapefruit and ‘Dancy’ (Swingle et al., 1931). Wakana et al. (1998) demonstrated that glutamate oxaloac- etate trans-minase (GOT) isozyme loci and the incompatibility locus are linked, which should be useful for early screening for self- incompatibility.