Mihoko Tamura

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

Abstract In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (Sa, Sb, Sc, Sd). We previously reported the cDNA cloning of three of these alleles, namely Sb, Sc and Sd. In this paper we report the cloning and DNA sequence analysis of the Sa allele. The Sa-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (Sb, Sc, and Sd) and this similarity was lower than that observed among the Sb, Sc and Sd-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; Sa (602 bp), Sb (1083 bp), Sc (221 bp) and Sd (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the Sa- and Sb-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the Sa-allele and 556 bp in the Sb-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage.

Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120.

Regeneration of somatic hybrids from electrofused protoplasts of Japanese persimmon (Diospyros kaki L.)

Somatic hybrids of Japanese persimmon (Diospyros kaki L.) were obtained by electrofusion of protoplasts. Callus protoplasts of cvs. Jiro and Suruga were fused electrically and cultured in modified KM8p medium using agarose-bead culture. Relative nuclear DNA contents of calli recovered from the fused protoplasts were determined by flow cytometry. About 20% of the callus lines (22 of 127) regenerated from protoplasts had twice the nuclear DNA content of the parental calli. When the 22 callus lines were transferred to regeneration medium, nine of them formed adventitious buds. RAPD analysis of nine shoot lines showed that they were somatic hybrids between Jiro and Suruga. Neither Jiro + Jiro nor Suruga + Suruga was regenerated. Chromosome number of the somatic hybrids was counted by microscopic observation of root tip cells. They had the dodecaploid chromosome number of around 2n = 180, which is twice the number of parental plants (2n = 90, x = 15).

Production of dodecaploid plants of Japanese persimmon (Diospyros kaki L.) by colchicine treatment of protoplasts

SummaryDodecaploid plants of Japanese persimmon (Diospyros kaki L.) were obtained by colchicine treatment of protoplasts. Callus protoplasts of ‘Jiro’ (2n=90, x=15) were cultured in modified KM8p medium with 0.1% colchicine for 3–9 days. After colchicine treatment, they were cultured using agarose bead culture. Microcalli were recovered from the protoplasts after 3 months. Flow cytometric measurement showed that nine of 31 callus lines obtained from 6 days of colchicine treatment had twice the nuclear DNA content as non-treated controls. Plantlets were regenerated from the calli with twice the nuclear DNA content. Microscopic observation of root tip cells showed that their somatic chromosome number was 2n=180 (x=15). Compared with ‘Jiro’, dodecaploid plants had longer stomatal guard cells and lower stomatal densities, consistent with increased ploidy.

Plant regeneration from callus protoplasts of adult Japanese persimmon (Diospyros kaki L.)

Abstract A large number of viable protoplasts were isolated from calli derived from leaf primordia excised from dormant winter buds of adult Japanese persimmon (Diospyros kaki L. cv. Jiro). The first cell division occurred within 1 week in KM8p agarose medium with an initial plating density at more than 1 × 105 protoplasts/ml. After 2 weeks of culture, the agarose plate was cut into blocks and cultured in bead-type culture. Microcalli, 1–2 mm in diameter, were produced after 12 weeks of culture. When they were subcultured onto KM8 agar medium, half of them developed into callus masses. A high frequency of adventitious bud formation occurred on Murashige and Skoogs (MS) agar medium containing 10 μM zeatin and 0.1 μM 3-indoleacetic acid (IAA). After shoot growth, rooting was induced by 3-indolebutyric acid (IBA) treatment. Plantlets regenerated were then raised in vermiculite.

Regeneration of Plants from Protoplasts of Diospyros kaki L. (Japanese Persimmon)

Japanese persimmon (Diospyros kaki L.) is native to East Asia and has been cultivated for centuries in China, Korea, and Japan (Tao and Sugiura 1992a). Recently, it has been gaining in popularity throughout the world and its culture is spreading to outside the temperate parts of Asia including Italy, Australia, USA, Brazil, Israel, and New Zealand. In spite of recent worldwide interest and requirements of improved fruit quality in this species, conventional crossbreeding is limited because among the hundreds of native cultivars, there are not many strains carrying hermaphrodite and/or male flowers. Furthermore, as is often the case with woody plants, the long juvenile period, large plant size, and high heterozygosity of Japanese persimmon make rapid breeding progress difficult. Therefore the use of somatic hybrids and, in particular, protoclonal variants will have a great impact on Japanese persimmon breeding programs.