Toshiaki Kameya

Production of somatic hybrid plants, ‘Brassicomoricandia’, through protoplast fusion between Moricandia arvensis and Brassica oleracea

Intergeneric hybrid plants were obtained through protoplast fusion between Brassica oleracea L. and Moricandia arvensis (L.)DC. (Toriyama et al 1987). M. arvensis is classified as a member of the subtribe Moricandiinae of the family Brassicaceae, and is known to be a C 3 -C 4 intermediate species with respect to low CO 2 compensation point (Holaday et al 1981). We have been studying (i) the production of somatic hybrids between distantly related species, and (ii) the genetic regulation of photosynthetic systems in the hybrids.

Embryoids derived from isolated protoplasts of carrot.

SummaryProtoplasts isolated enzymatically from carrot root tissues developed into cell clusters in a liquid medium containing coconut milk and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Cells of the resulting calluses differentiated into embryoids on an agar medium containing coconut milk or kinetin.

The modified ABC model explains the development of the petaloid perianth of Agapanthus praecox ssp. orientalis (Agapanthaceae) flowers.

The class B genes, which belong to the MADS-box gene family, play important roles in regulating the development of petals and stamens in flowering plants. To understand the molecular mechanisms of floral development in Agapanthuspraecox ssp. orientalis (Agapanthaceae), we isolated and characterized the homologs of the Antirrhinum majus genes GLOBOSA and DEFICIENS in this plant. These were designated as ApGLO and ApDEF, respectively. ApGLO and ApDEF contain open reading frames that encode deduced protein with 210 and 214 amino acid residues, respectively. Phylogenetic analysis indicated that ApGLO and ApDEF belong to the monocot class B gene family. In situ hybridization experiments revealed that hybridization signals of ApGLO and ApDEF were observed in whorl 1 as well as in whorls 2 and 3. Moreover, the flowers of transgenic Arabidopsis plants that ectopically expressed ApGLO formed petal-like organs in whorl 1. These observations indicate that the flower developmental mechanism of Agapanthus follows the modified ABC model.

Selection of a universal hybridizer in Sinapis turgida Del. and regeneration of plantlets from somatic hybrids with Brassica species.

A double-mutant cell line, which was unable to grow in a medium with NO3-as the sole nitrogen source and was resistant to 5-methyl-tryptophan (5MT), was selected from cell suspensions of Sinapis turgida Del. (Brassicaceae) by culturing the cells in AA medium (Toriyama and Hinata, 1985, Plant Sci. 41, 179–183) supplemented with 50 mM chlorate and 229 μM 5MT. Protoplasts of this cell line were fused with mesophyll protoplasts of Brassica oleracea L. with dextran, and six somatic hybrids were selected initially by culture in the NO3-medium and then by transfer to the NO3-medium supplemented with 229 μM 5MT. The somatic hybrids produced embryoids, leaves and plantlets on a regeneration medium. The hybrid characters were confirmed by investigations of acid phosphatase (EC 3.1.3.2) and peroxidase (EC 1.11.1.7) isoenzymes, chromosome number, growth on NO3-medium, 5MT resistance, and capacity to regenerate plants. Somatic hybrids between S. turgida Del. and B. nigra (L.) Koch were also obtained using this method. These results indicate that the double-mutant cell line established here will be able to serve as a universal hybridizer to select somatic hybrids after protoplast fusion with any other wild-type partner.

Intergeneric somatic hybridization of rice (Oryza sativa L.) and barley (Hordeum vulgare L.) by protoplast fusion

Abstract An intergeneric somatic hybrid was obtained upon fusion of protoplasts of rice and barley. Protoplasts isolated from suspension cultures of rice cells were fused by electrofusion with protoplasts that had been isolated from young barley leaves. Some of the resultant calli formed green spots and shoots. Only one shoot formed roots, and it was subsequently successfully transferred to soil in a greenhouse. Its morphology closely resembled that of the parental rice plant. Cytological analysis indicated that the plant had both small chromosomes from rice and large chromosomes from barley. Southern hybridization analysis with a fragment of the tryptophan B (trpB) gene revealed both a rice-specific band and a barley-specific band. Mitochondrial (mt) and chloroplast (cp) DNAs were also analyzed using the same method. The plant was shown to contain novel mitochondrial and chloroplast sequence rearrangements that were not detected in either of the parents.

Transformation of radish (Raphanus sativus L.) via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group 3 LEA gene from B. napus

A protocol for producing transgenic radish (Raphanus sativus) was obtained by using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation. The Agrobacterium strain LBA4404 contained the binary vector pBI121-LEA (late embyogenesis abundant), which carried a Group 3 LEA gene, from Brassica napus. Among six combinations, Agrobacterium-mediated transformation assisted by a combination of 5-min sonication with 5-min vacuum infiltration resulted in the highest transformation frequency. The existence, integration and expression of transferred LEA gene in transgenic T1 plants were confirmed by PCR, genomic Southern and Western blot analysis. Transgenic radish demonstrated better growth performance than non-transformed control plants under osmotic and salt stress conditions. Accumulation of Group 3 LEA protein in the vegetative tissue of transgenic radish conferred increased tolerance to water deficit and salt stress.

Cool-Temperature-Induced Chlorosis in Rice Plants (I. Relationship between the Induction and a Disturbance of Etioplast Development)

We have established an experimental system for mimicking the phenomenon of cool-temperature-induced chlorosis (CTIC) in rice plants (Oryza sativa L.). Rice seedlings were initially grown in darkness under cool-temperature conditions and then exposed to light and warm conditions to follow the expression of CTIC. Induction of CTIC in the sensitive cultivar (cv Surjamukhi) was bimodally dependent on the temperatures experienced during the initial growth in darkness. CTIC was maximally induced between 15 and 17[deg]C. A positive correlation was demonstrated between induction of CTIC and the growth activity of shoots during growth in darkness. Electrophoretic and immunoblot analysis revealed that accumulation of NADPH-protochlorophyllide oxidoreductase in plastids was also bimodally dependent on the temperatures during the growth in darkness with minimum accumulation between 15 and 17[deg]C, suggesting that the reduction of NADPH-protochlorophyllide oxidoreductase accumulation in plastids might be closely linked to a disturbance in transformations of plastids to etioplasts during the dark growth under the critical temperatures and thereby to the CTIC phenomenon. This was corroborated by electron microscopic observations. These results suggest that growth is one of the determining factors for the expression of CTIC phenotype in rice under cool temperature.

Spatiotemporal expression of duplicate AGAMOUS orthologues during floral development in Phalaenopsis

The AGAMOUS (AG) family of MADS-box genes plays important roles in controlling the development of the reproductive organs of flowering plants. To understand the molecular mechanisms behind the floral development in the orchid, we isolated and characterized two AG-like genes from Phalaenopsis that we denoted PhalAG1 and PhalAG2. Phylogenetic analysis indicated that PhalAG1 and PhalAG2 fall into different phylogenetic positions in the AG gene family as they belong to the C- and D-lineages, respectively. Reverse transcription-polymerase chair reaction (RT-PCR) analyses showed that PhalAG1 and PhalAG2 transcripts were detected in flower buds but not in vegetative organs. Moreover, in situ hybridization experiments revealed that PhalAG1 and PhalAG2 hybridization signals were observed in the lip, column, and ovule during the floral development of Phalaenopsis, with little difference between the expression patterns of the two genes. These results suggest that both AG-like genes in Phalaenopsis act redundantly with each other in floral development.

Production and analysis of asymmetric hybrid plants between monocotyledon (Oryza sativa L.) and dicotyledon (Daucus carota L.).

Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20–22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.

Production and analysis of plants that are somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.)

Abstract In order to obtain plants that were somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.), we fused protoplasts that had been isolated from 6-month-old suspension cultures of carrot cells with protoplasts isolated from barley mesophyll by electrofusion. After culture for 1 month at 25°C , the cells were cultured for 5 weeks at 4°C , and were then returned to 25°C for culture on a shoot-inducing medium. Three plants (nos. 1, 2 and 3) were regenerated from the cells. The morphology of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells had about 24 chromosomes, fewer than the sum of the numbers of parent chromosomes which was 32. Southern hybridization analysis with fragments of the rgp1 gene used as probe showed that the regenerated plants contained both barley and carrot genomic DNA. Chloroplast (ct) and mitochondrial (mt) DNAs were also analyzed with several probes. The ctDNA of the regenerated plants yielded hybridization bands specific for both barley and carrot when one fragment of rice ctDNA was used as probe. Furthermore, the regenerated plants yielded a barley specific band and a novel band with another fragment of rice ct DNA as a probe. One of the regenerated plants (no. 1) yielded a novel pattern of hybridized bands of mt DNA (with an atp6 probe) that was not detected with either of the parents. These results indicated that the regenerated plants were somatic hybrids of barley and carrot and that recombination of both the chloroplast genomes and the mitochondrial genomes might have occurred.