Takashi Handa

Transformation of sweet potato (Ipomoea batatas (L.) Lam.) plants by Agrobacterium rhizogenes

Abstract Transgenic sweet potato plants were obtained after Agrobacterium rhizogenes -mediated transformation. Leaf disks of in vitro plants were inoculated with different Agrobacterium rhizogenes strains. Numerous hairy roots were induced on leaf disks by both agropine-type and mikimopine-type strains. Whole plants transformed with Ri-T-DNA were regenerated from the hairy roots in five cultivars. These plants had wrinkled leaves, altered shape of flowers, reduced apical dominance, shortened internodes, small storage roots and abundant, frequently branching roots that showed reduced geotropism. Transgenic sweet potato plants possessing both NPT II gene and GUS gene were also obtained from the hairy roots by infection with Agrobacterium rhizogenes containing the binary vector pBI121 in addition to the wild-type Ri-plasmid.

Genetic transformation of Antirrhinum majus L. and inheritance of altered phenotype induced by Ri T-DNA

Abstract Hairy roots were obtained from snapdragon (Antirrhinum majus L.) plants infected with Agrobacterium rhizogenes harbouring the agropine type Ri plasmid (pRiA4). Plant regeneration occurred spontaneously on Linsmaier-Skoog (LS) medium lacking phytohormones under a 16-h/day photoperiod. Phenotypic alterations such as dwarfness, decrease in apical dominance, increase in number of flowers and stimulation of root production were observed in transformed plants. Opine analysis suggested the integration of TR-DNA and Southern analysis confirmed multiple integrations of TL-DNA. One of the transformed plants was fertile, and the progeny also exhibited the dwarf phenotype and had small flowers and elliptical leaves. Progeny expressing phenotypic alterations retained core T-DNA including rol genes.

Efficient shoot regeneration from hairy roots of Antirrhinum majus L. transformed by the rol type MAT vector system

Abstract Eleven independent GUS-positive hairy roots were induced by co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing the rol type MAT vector pNPI702. The MAT vector pNPI702 possesses a GUS gene under the 35 S promoter and a removal element in which the 7.6-kb DNA fragments containing the rolA, B, C and D genes and recombinase gene with a 35 S promoter are located between two directly oriented recombination site sequences. A total of 326 adventitious shoots regenerated from 11 independent hairy root lines cultured on 1/2MS medium without plant growth regulators at 25  °C under a 16/8 h (day/night) photoperiod after 8 weeks of stock-culture of hairy roots and 4 weeks of culture of the green segments of hairy roots. Regenerated plants showed either a normal or dwarf morphology. GUS activity was observed in the hairy roots and regenerated shoots. The presence of the GUS gene in the regenerated, morphologically normal plants was confirmed by PCR analysis.

Sectional relationships in the genus Rhododendron (Ericaceae): evidence from matK and trnK intron sequences

Abstract. Phylogenetic relationships among all eight subgenera and 12 sections in Rhododendron as well as its related genera were inferred from matK and trnK intron sequences. The results of this study provided the following insights: (1) Rhododendron is paraphyletic because Menziesia is nested within Rhododendron. (2) Subgenus Therorhodion forms a basal lineage of tribe Rhodoreae. (3) Subgenera Hymenanthes and Tsutsusi are monophyletic. (4) Subgenera Azaleastrum and Pentanthera are polyphyletic. (5) Subgenus Rhododendron is monophyletic, if section Rhododendron subsection Ledum is excluded.

Transformation of Antirrhinum majus L. by a rol-type multi-auto-transformation (MAT) vector system

A total of 11 independent beta-glucuronidase (GUS) positive hairy roots were induced following co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing rol-type multi-auto-transformation (MAT) vector pNPI702. A total of 326 adventitious shoots were regenerated from the hairy root lines on 1/2 MS medium without plant growth regulators at 25 degrees C under a 16 h/day photoperiod condition 4 months after infection of the A. tumefaciens GV2260. The absence of the rol genes in five plants was verified by polymerase chain reaction (PCR) and Southern blot analysis. Acclimatized transformants exhibited normal phenotypes in height and in the morphology of leaves and flowers. Furthermore, the GUS gene was strongly expressed in the leaves, inflorescence of the transformed plant, and the progeny. This result demonstrates that the rol-type MAT vector can be used to study gene functions controlling the morphogenesis of Antirrhinum majus plants.

EVIDENCE FOR INTROGRESSIVE HYBRIDIZATION BASED ON CHLOROPLAST DNA POLYMORPHISMS AND MORPHOLOGICAL VARIATION IN WILD EVERGREEN AZALEA POPULATIONS OF THE KIRISHIMA MOUNTAINS, JAPAN

For the purpose of determining the origin of horticultural evergreen azalea cultivars, this study was focused on the natural populations of azalea in Kyushu (south main island of Japan). The Kirishima mountains, the volcanic mountain mass in Kyushu, are an important centre of diversity for the Japanese evergreen azaleas. Rhododendron kiusianum Makino grows above 1000m alt., whereas R. kaempferi Planch. is distributed below 600m alt. Putative natural hybrid populations of these two species are found in the intermediate region (1000–600m alt.). These two species have been clearly distinguished by their respective morphological features. Rhododendron kiusianum has small pink-purple flowers and small elliptical leaves, whereas R. kaempferi has larger red-orange flowers with dark blotches and large oblong leaves. Interspecific hybrids show phenotypes within the range of the two species, especially with regard to flower colour and leaf shape. A morphological cline of these characteristics corresponding to altitude has been observed between these two species. PCR-RFLP analysis of chloroplast DNA detected specific bands for the two species in the 16S rDNA region when digested with Hha I restriction enzyme. Populations of interspecific hybrids were composed of individuals that had a banding pattern of either R. kiusianum or R. kaempferi . This indicates that R. kiusianum and R. kaempferi are clearly distinct species. Furthermore, natural hybrid populations consist of individuals that have one of two cpDNA. Some individuals in the populations of R. kiusianum (T-1430 and T-1030) possess the cpDNA pattern of R. kaempferi , which suggests that cytoplasmic introgression has occurred in the populations of R. kiusianum from R. kaempferi .

Organ-specific mRNA in transgenic tobacco plants possessing T-DNA of Ri plasmids

Abstract The structure and mRNA accumulation of T-DNA of Ri (A4) plasmids integrated into the genome of Nicotiana tabacum var. Samsun were investigated. Two inserts of TL-DNA were found in the transformed plant (R1) and one (R2) of the progenies obtained by self-crossing of the R1 plant. The expression level of T-DNA-derived transgenic plants was high in roots and stems, but very low in leaves. The gene encoding the major transcript of integrated Ri TL-DNA coincided with the ORF12 (Slightom et al., J. Biol. Chem., 261 (1986) 108).

An improved protocol for Agrobacterium-mediated transformation of Antirrhinum majus L.

Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.

A contribution to the classification of evergreen azalea cultivars located in the Lake Maggiore area (Italy) by means of AFLP markers

The amplified fragment length polymorphism (AFLP) technique was employed to assess the genetic conformity within a gene pool of evergreen azaleas. Ninety-three genotypes grown in historical gardens and nurseries of the Lake Maggiore area (Northern Italy), locally classified in the groups Indica, Japonica and Amoena, 44 reference cultivars of the four more or less universally recognized groups Belgian, Hirado, Kurume and Satsuki, and 40 species and species accessions that most concurred in the origin of the reference groups were included. Aims of the study were to define the genetic profiles of the Italian accessions and to provide useful information for their classification and pedigree reconstruction. Ordination analyses were used to evaluate if the AFLP technique can be applied to establish phylogenetic relationships and to investigate the DNA-based relatedness within the Italian gene pool and between the latter and the reference one. The results regarding the Italian accessions were compared with morphological data by means of Mantel’s test. Assignment tests both on the level of the groups and on individual plant level for the reference and Italian genotypes were applied to further evaluate the relatedness of the groups or individual accessions. Results demonstrated that the AFLP technique together with morphological characterization can contribute to depict the phylogeny of the evergreen azalea. Comparing the reference and Italian genotypes, conclusions about the classification of the evergreen azalea cultivars located in the Lake Maggiore area were drawn.

Molecular phylogenetics and character evolution of morphologically diverse groups, Dendrobium section Dendrobium and allies

The genus Dendrobium, one of the largest genera in Orchidaceae, exhibits enormous vegetative diversification. Such a situation has hindered the establishment of consistent classification systems. To clarify phylogenetic relationships in Dendrobium section Dendrobium and allied groups, we performed molecular phylogenetic analyses of 210 taxa. The results showed that many sections are not monophyletic. Most of the morphological characters that have been believed to reflect phylogenetic relationships are, in fact, the results of convergence. Consequently, recircumscription of the infrageneric classification is required.