T. Ohgawara

Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata

SummarySomatic hybrid plants of Rutaceae were obtained by protoplast fusion between Citrus sinensis Osb. (‘Trovita’ orange) and Poncirus trifoliata. Protoplasts isolated from embryogenic cells of C. sinensis and from leaves of P. trifoliata, and the culture of fusion products in the presence of high concentrations of sucrose were essential requirements for the selection of hybrids. Green globular embryoids derived from protoplasts resulted in the regeneration of trifoliate plants. Other morphological characters of these plants were intermediate between both parents. The chromosome number in one of the hybrid plants was 36, which was the sum of C. sinensis (2n=18) and P. trifoliata (2n=18). EcoRI restriction analysis of rDNA confirmed the presence of parental nuclear DNAs in the hybrid.

A somatic hybrid plant obtained by protoplast fusion between navel orange (Citrus sinensis) and satsuma mandarin (C. unshiu)

A somatic hybrid plant was obtained by protoplast fusion between navel orange and satsuma mandarin. Protoplasts isolated from nucellar calli of navel orange and from leaves of satsuma mandarin were fused by the PEG method. The fusion products were cultured in a Murashige & Tucker medium containing 0.6 M sucrose. In this medium, some colonies developed into whole plants through embryogenesis. One of the regenerated plants was shown to be a hybrid, which was proven by restriction endonuclease analysis of nuclear ribosomal DNA. The chromosome number of the hybrid was 36. Both parents have a chromosome number 2n=18.

Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.)

SummaryProtoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somatic embryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding.

Citrus cybrid regeneration following cell fusion between nucellar cells and mesophyll cells

Abstract Through the cell fusion between nucellar callus cells and mesophyll cells in two Citrus combinations, we obtained the regenerated plants resembling mesophyll parent besides the somatic hybrids. In the fusion experiment between sudachi (Citrus sudachi Hort.) and lime (C. aurantifolia Swingle), 4 out of 12 regenerated clones were similar to mesophyll parent (lime) as we already reported. In another experiment of sudachi and lemon (C. limon Burn) 4 out of 6 clones were similar to mesophyll parent (lemon). These regenerated plants had 18 chromosomes, which were equal to diploid parents and showed the same nuclear rDNA fragment patterns as those of mesophyll parents. The composition of the mitochondrial genomes of these regenerated plants was investigated. The mtDNA analysis revealed that all of the clones that resembled mesophyll parents had identical mtDNA fragment patterns to those of the nucellar callus parent, indicating that these regenerated clones resembling mesophyll parents were cybrids. These results suggest that the mitochondria of nucellar cells may play a significant role in Citrus embryogenesis.

Fertile fruit trees obtained by somatic hybridization: navel orange (Citrus sinensis) and Troyer citrange (C. sinensis x Poncirus trifoliata)

SummaryNucellar cell suspension protoplasts of navel orange (Citrus sinsensis Osb.) were chemically fused with mesophyll protoplasts of Troyer citrange (C. sinensis x Poncirus trifoliata) and cultured in hormone-free Murashige and Tucker medium containing 0.6 M sucrose. Two types of plant were regenerated through embryogenesis. One type showed intermediate mono-and difoliate leaves and the other types was identical to Troyer citrange. The regenerated plants with intermediate morphology were demonstrated by chromosome counts and rDNA analysis to be amphidiploid somatic hybrids. Five clones of these somatic hybrids were grafted in the field. After 4 years, they set flowers having a morphology intermediate between those of the two parents. The pollen grains showed high stainability and sufficient germinability, and were larger than those of Troyer citrange. The fruits of the somatic hybrids were large and spherical with thick rinds. Most of them contained seeds with normal germinability. These results indicate that somatic hybridization is a useful tool for Citrus breeding.

Analysis of cytoplasmic genomes in somatic hybrids between navel orange (Citrus sinensis Osb.) and ‘Murcott’ tangor

SummarySomatic hybrid plants were produced by protoplast fusion of navel orange and ‘Murcott’ tangor. Hybridity of the plants was confirmed by the restriction endonuclease analysis of nuclear ribosomal DNA. All of the plants (16 clones) were normal, uniform, and had the amphidiploid chromosome number of 36 (2n=2x=18 for each parent). The cpDNA analysis showed that each of the 16 somatic hybrids contained either one parental chloroplast genome or the other. In all cases, the mitochondrial genomes of the regenerated somatic hybrids were of the navel orange type.

Somatic hybridization in Citrus using embryogenic cybrid callus

Abstract Embryogenic callus of a lime-type cybrid possessing the nuclear genome of lime ( Citrus aurantifolia Swing.) and the mitochondrial genome of sudachi ( C. sudachi Hort. ex Shirai) was induced from a somatic embryo which had been obtained through protoplasts fusion between nucellar callus of sudachi and mesophyll of lime [1]. Protoplasts derived from the cybrid callus were fused electrically with mesophyll protoplasts of lemon ( C. limon Burm.). After 5 months, plants were regenerated from eigth fusion-derived cell clones through embryogenesis. The plants showed two types of leaf morphology; thick and broad leaf shape, identical to lemon. Chromosome counts and nuclear and mitochondrial DNA analysis confirmed that the former plants were somatic hybrids which were allotetraploid (4x = 36), possessing the nuclear genomes of both parents and the mitochondrial genome of sudachi. The latter lemon-type plants were novel cybrids possessing 18 chromosomes with the nuclear genome of lemon and the mitochondrial genome of sudachi. Lime-type cybrid plants, identical with one of the parents, were not found in this study. These results indicate that embryogenic cybrid callus lines can be used as fusion material, resulting in further production of somatic hybrid and cybrid plants.

Acid citrus somatic hybrids between sudachi (Citrus sudachi Hort. ex Shirai) and lime (C. aurantifolia Swing.) produced by electrofusion

Abstract Conditions were established for the production of hybrid plants between sudachi embryogenic nucellar suspension culture and line mesophyll protoplasts by electrofusion. Under these conditions, 12 clones out of a total of 40 embryoids obtained were regenerated to complete plants through embryogenesis. Eight of the regenerated clones were shown to be somatic hybrids on the basis of chromosome number and analysis of nuclear ribosomal DNA. The other 4 were recognized to have lime-type characteristics. This electrofusion method considerably simplified protoplast fusion of citrus plants and allowed successful production of acid citrus somatic hybrids.

Somatic Hybridization Between Citrus sinensis and Poncirus trifoliata

Citrus and Poncirus belong to the family Rutaceae. Citrus includes many commercially important fruit species such as sweet orange (C. sinensis), mandarin (C. reticulata), lemon (C. limon), grapefruit (C. paradisi), and is grown in over 100 countries in tropical and subtropical areas spreading over approximately 40° latitude. On the other hand, Poncirus includes only one species of trifoliate orange. (P. trifoliata), which is a deciduous shrub widely grown in Japan and China. It is an important rootstock which possesses cold hardiness, and is resistant to phytophthora, nematode, and tristeza virus (Saunt 1990). The morphology of Citrus and Poncirus is very different; however, their genomes are similar and the chromosome number is 2n = 18. These two genera are sexually compatible and the most widely grown sexual hybrid is Troyer citrange, which has become a very important rootstock in many countries.