Sakae Suzuki

Production of transgenic plants of the Liliaceous ornamental plant Agapanthus praecox ssp. orientalis (Leighton) Leighton via Agrobacterium-mediated transformation of embryogenic calli

Abstract A system for producing transgenic plants was developed for the Liliaceous ornamental Agapanthus praecox ssp. orientalis (Leighton) Leighton via Agrobacterium -mediated genetic transformation. Leaf-derived embryogenic calli were inoculated with A . tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing β-glucuronidase (GUS-intron) genes in the T-DNA region. Following co-cultivation, the calli were transferred to a medium containing 1 mg l −1 picloram (PIC), 50 mg l −1 hygromycin and 500 mg l −1 cefotaxime, on which several hygromycin-resistant (Hyg r ) cell clusters were obtained 5–6 weeks after transfer. Agrobacterium strain, co-cultivation period and acetosyringone (AS) treatment during co-cultivation affected the number of Hyg r callus lines produced: the best result was obtained when embryogenic calli were co-cultivated with LBA4404/pTOK233 for 7 days in the presence of 20 mg l −1 AS. Hyg r calli were transferred to the same medium, but lacking PIC, for inducing somatic embryos. Somatic embryos thus obtained developed into complete plantlets following their transfer to a medium without PIC and antibiotics. All of them were verified to be stable transformants by GUS histochemical assay, PCR and Southern blot analyses.

Flower color alteration in Lotus japonicus by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.

CALLUS FORMATION AND PLANT REGENERATION IN VARIOUS LILIUM SPECIES AND CULTIVARS

SummaryIn researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.

organogenesis and somatic embryogenesis from callus cultures in muscari armeniacum leichtl. ex Bak.

SummaryEstablishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.

Finding the superior allele of japonica- type for increasing stem lodging resistance in indica rice varieties using chromosome segment substitution lines

BackgroundIn cereal crops, stem lodging can be classified into two types: stem-breaking type and stem-bending type. To improve stem-lodging resistance, the strong culm traits of superior lodging-resistant varieties must be characterized. The identification of quantitative trait loci (QTLs) and the corresponding genes associated with the parameters for bending moment at breaking (M) and flexural rigidity (FR) is expected to enable the efficient development of lodging-resistant varieties. A set of Chromosome Segment Substitution Lines (CSSLs) derived from the cross between Takanari and Koshihikari were used in this study to identify QTLs associated with lodging resistance.ResultsThe indica variety Takanari possesses large M due to its large section modulus (SM) despite its small bending stress (BS), whereas Takanari also has large FR due to its large secondary moment of inertia (SMI) and Young’s modulus (YM). The QTLs for BS were assigned to chromosomes 3, 5, 6, 8, 9, 10, 11, and 12. Koshihikari alleles increased BS in these QTLs. The YM was increased by substitution of the Koshihikari chromosomal segments on chromosomes 2, 10, and 11. Other QTLs mapped to chromosomes 7 and 12, such that the Koshihikari alleles contributed to the decrease of YM. QTLs for cellulose density were assigned to chromosomes 1, 3, and 5, which were replaced by substitutions of Koshihikari segments. The QTLs for hemicellulose, cellulose, and holocellulose densities identified on chromosome 5 overlapped with those for BS, indicating the positive effect of the Koshihikari segment on increasing BS.ConclusionsThese results suggested that the QTLs for the densities of cell wall materials in japonica varieties contributed to increased BS and might be utilized for improving lodging resistance in indica varieties of rice.