Taiji Adachi

Isolation and Characterization of a Gene Expressed during Early Embryo Sac Development in Apomictic Guinea Grass (Panicum maximum)

Summary A novel classification method, based on ovary length, for sampling different developmental stages of embryo sac formation in obligate sexual and facultatively apomictic genotypes of guinea grass (Panicum maximum) has been employed to make a cDNA library and to isolate a stage-specific cDNA clone, probably representing a full-length gene. This A2-134 cDNA, designated ASG-1 (Apomixis Specific Gene), was found to be expressed in flower buds of the apomictic but not of the sexual accession. Furthermore, the gene was also not expressed in buds of apomicts until completion of megasporogenesis but could only be found during a phase characterized by the. appearance of aposporous initial cells (AICs) of the embryo sac. The appearance of these cells is strictly limited to apomictic genotypes. Sequence analysis revealed that the A2-134 cDNA (1,177bp) codes for a protein of 305 amino acids with a molecular mass of 34.2 kDa. The amino acid sequence of A2-134 is related to RD22, a seed-specific and drought-induced gene of Arabidopsis thaliana, to USP, an unknown seed protein precursor of Vicia faba, to a polygalaturonase 1 beta chain precursor (Polygl) of Lycopersicon esculentum and to ADR6, an auxin down-regulated gene of Glycine max. Southern blot analysis showed a different hybridization pattern at the genomic level when A2134 cDNA was hybridized with total DNAs isolated from leaves of sexual as well as apomictic plants, indicating that the gene (or related family members) exists in both types. The cDNA library and the ASG-1 gene should be valuable tools toward understanding the molecular mechanism of apomixis, particularly apospory, and toward the transfer of apomixis to important crops. To our knowledge, A2-134 represents the first case of the isolation and characterization of a cDNA related to apomictic embryo sac development.

Plant regeneration from protoplasts of common buckwheat (fagopyrum esculentum).

Protoplasts were isolated from hypocotyls of etiolated seedlings from a diploid and the corresponding autotetraploid variety of common buckwheat (Fagopyrum esculentum). The isolated protoplasts started to divide after 4 days in culture in a modified MS medium. Maximum plating efficiency was approximately 1%. Regenerated calli derived from the tetraploid genotype developed roots easily but were recalcitrant to form shoots. Eighteen months following the initiation of cultures, tetraploid embryoids and shoots emerged after 3 weeks on an MS medium containing 0.1 mg/l gibberellic acid.

Detection of DOPA 4,5-Dioxygenase (DOD) Activity Using Recombinant Protein Prepared from Escherichia coli Cells Harboring cDNA Encoding DOD from Mirabilis jalapa

Betalains are synthesized in flowers, fruits and other tissues of the plant order Caryophyllales. Betalamic acid is the chromophore of betalain pigments synthesized by a ring-cleaving enzyme reaction on l-dihydroxyphenylalanine (DOPA). Although reverse genetic evidence has proven that DOPA 4,5-dioxygenase (DOD) is a key enzyme of betalain biosynthesis, all attempts to detect recombinant plant DOD activity in vitro have failed. Here, we report on the formation of betalamic acid from DOPA under suitable assay conditions using recombinant MjDOD produced by Escherichia coli. This is the first report showing biochemical evidence for DOD activity in vitro.

Somatic embryogenesis and plant regeneration from cultured immature inflorescences of apomictic dallisgrass (Paspalum dilatatum Poir.)

Abstract Plant regeneration from cultured immature inflorescences of Paspalum dilatatum was obtained by somatic embryogenesis. Embryonic callus was initiated from immature inflorescences (5–10 mm length) on Murashige and Skoog (MS) medium supplemented with 2.0–10.0 mg·l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and solidified with 0.2% (w/v) Gellan Gum. Somatic embryos could be induced at all concentrations of 2,4-D, but the maximal frequency of explants which produced somatic embryos occurred on medium containing 10.0 mg·l−1 2,4-D. Somatic embryos developed and germinated precociously when embryogenic calli were transferred to a medium containing 1.0 mg·l−1 each of kinetin and gibberellic acid (GA3). All regenerants were successfully grown to maturity. The embryogenic calli maintained their embryogenic capacity on Murashige and Skoog (MS) medium with 1.0 mg·l−1 2,4-D for more than 1 year.

Inheritance of self-compatibility and flower morphology in an inter-specific buckwheat hybrid

This study was conducted to determine the inheritance of self-compatibility and homomorphic flower type when the wild species Fagopyrum homotropicum was crossed with common buckwheat (F. esculentum). Unidirectional interspecific hybrids between cultivated F. esculentum Moench. (common buckwheat) and its wild relative F. homotropicum were produced after controlled pollination and embryo rescue culture. Cross-compatibility was found to be better when thrum-type common buckwheat was used as the female parent rather than the pin-type. The resulting F1 plants were partially fertile, late maturing and intermediate between the parents in flower shape and plant height. They segregated into heterostylic (thrum only) and homostylic types in equal numbers, indicating that homostyly is controlled by a single dominant gene. The thrum-type F1 hybrids were backcrossed to common buckwheat and the progenies were raised utilizing embryo rescue culture. The homostylic F1 hybrids were advanced to the F2 and F3 generations th...

Chloroplast DNA analysis in buckwheat species: phylogenetic relationships, origin of the reproductive systems and extended inverted repeats

Abstract We have constructed physical maps for the chloroplast genomes of buckwheat (Fagopyrum esculentum) and related species (F. tataricum and F. cymosum) by using six restriction enzymes. The genome sizes were found to be identical with approximately 155.5 kb. A comparison of physical maps revealed 11 altered restriction sites out of 259 analyzed. Phylogenetically, the chloroplast DNA of F. esculentum has been found to be quite distant from those of F. tataricum and F. cymosum. These results also suggest that the allogamous mode could be the ancestral reproductive system for Fagopyrum, whose present-day species display three different mechanisms of reproduction: allogamous, autogamous and vegetative, because both the distantly related species, F. esculentum and F. cymosum, maintain self-incompatibility due to heterostyly. We also observed that the unique 5.05-kb Sma-8 fragments, which extend the inverted repeats in buckwheat species, exhibit little homology with tobacco chloroplast DNA clones.

Evidence that blue light induces betalain pigmentation in Portulaca callus

The wavelength range that activates betalain pigmentation has been studied following selection of light induelble betalain producing callus lines originating from Portulaca sp. ‘Jewel’ seedlings. Light sources with different wave-lengths were used to irradiate the callus, resulting in blue light being effective in inducing betalain pigmentation. In addition, when UV light was combined with blue light, some calluses from this cell line showed high production of the pigment. This is a first report that betalain pigmentation in callus was induced by blue and blue/UV lights.

Plant regeneration from seed-derived embryogenic callus and cell suspension cultures of bahiagrass (Paspalum notatum)

Abstract We have established a high-frequency plant regeneration system via somatic embryogenesis from seed-derived callus and cell suspension cultures in 6 genotypes of bahiagrass ( Paspalum notatum ). Embryogenic callus was initiated from mature seeds on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3.0% sucrose and 0.3% Gellan Gum in the dark. Culture response was found to be correlated with genotype. ‘Pensacola’ had the best response in embryogenic callus formation, and 74% of the calli regenerated plants. Cell suspension cultures were established from embryogenic callus of ‘Pensacola’ in modified N 6 medium containing 1.0 mg/l 2,4-D. The suspension was composed of compact cell clusters. When smaller clusters (approx. 2.0–3.3 mm in diameter) were transferred to a solid MS medium with 3.0% sucrose but without hormones, plant regeneration was initiated at high frequency (28.6%). Morphological evidence is provided that regeneration of suspension cells occurred via embryogenesis. Regenerated plants were established to soil after culture in water at room temperature for 14 days for acclimatization.

Plant regeneration from protoplasts of Iris germanica L.

SummaryProtoplasts were isolated enzymatically from suspension cultures derived from embryogenic calli induced by leaf base culture of Iris germanica. In protoplast culture, the effects of glucose concentration, different sugars and combinations of 2,4-D and KIN on protoplast division and colony formation were examined. N6 medium supplemented with 0.1–1 mg/l 2,4-D, 1 mg/l KIN, 200mg/l casein hydrolysate, 250 mg/l proline, 0.2 M glucose and 20 g/l agarose was suitable for protoplast division and colony formation. When colonies formed were transferred onto hormone-free MS medium, many plantlets were regenerated through somatic embryogenesis. Thus, we could establish a plant regeneration system from protoplasts of I. germanica.

Plant regeneration from suspension cultured-derived protoplasts of apomictic dallisgrass (Paspalum dilatatum Poir.)

Abstract Protoplasts were isolated from embryogenic suspension cells of apomictic dallisgrass (Paspalum dilatatum Poir.). The respective suspension cultures were initiated from immature inflorescence-derived embryogenic callus. Previous to protoplast isolation, suspension cells were treated with Murashige and Skoog (MS) liquid medium without sucrose and hormones. Due to this pretreatment protoplast yield and viability were dramatically increased. A maximum protoplast yield of 4–6 × 106· −1 fresh weight was obtained. Cell division and colony formation from pretreated protoplasts were found to be best in an agarose solidified KM8p medium at a density of 5–8 × 105· ml−1. The plating efficiency, based on colony formation after 2 weeks of culture, was 0.5–0.8%. Protoplast-derived colonies were transferred to a solidified MS medium containing 1.0 mg · 1−1 2,4-dichlorophenoxyacetic acid (2,4-D) for callus proliferation. The calli formed embryonic structures which gave rise to green plants in 0.2% (w/v) Gellan Gum solidified MS medium with 1.0 mg·l−1 naphthaleneacetic acid (NAA) and 0.2 mg·l−1 benzylaminopurine (BAP). The regenerated plantlets were transferred to 1 2 MS hormone-free medium for further growth and root formation. Rooted plants could be transferred to soil.