Nobumasa Nito

In vitro flowering of Fortunella hindsii (Champ.)

Branch internodes of mature plants and stem internodes of seedlings of Fortunella hindsii flowered in vitro on half-strength MT (Murashige and Tucker 1969) basal medium supplemented with benzyladenine, adenine, 6-γ-γ-dimethylallylaminopurine and kinetin. The highest percentage of flowering was achieved with explants originating from branch internodes of flowering plants close to the apex on half-strength MT basal medium containing 5% sucrose and 0.01 mg 1−1 BA in light. Exposure to darkness for more than 3 weeks followed by re-exposure to light reduced flowering. Flowering required a 4-day exposure to BA, but shoot formation could be initiated even without exposure to BA. First branch internode segments on MT basal medium containing 5% sucrose were prolific in flower (85%) production. The sucrose treatment affected the flower bud size distribution. There were about 13 flower buds per culture in the largest size category (>5 mm).

Plant regeneration via somatic embryogenesis from protoplasts of six plant species related to Citrus.

Protoplasts isolated from embryogenic callus of Fortunella polyandra (Ridl.), Atalantia bilocularis (Pieree ex Guill.), Hesperethusa crenulata (Roxb.), Glycosmis pentaphylla (Retz.) Corr., Triphasia trifolia (Burm. f.) P. Wils. and Murraya koenigii (L.) Spreng. were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l−1 BA and 0.6 M sorbitol. The highest plating efficiencies for all species were obtained on MT basal medium containing 5% sucrose supplemented with 0.001 mg l−1 BA. F. polyandra produced higher percentages of globular somatic embryo development, while A. bilocularis consistently showed a lower percentage of globular somatic embryo development in all 5 concentrations of BA. MT basal medium containing 5% sucrose and supplemented with 0.001 mg l−1 BA was found to be a suitable medium for development of globular somatic embryos derived from protoplasts to form heart-shaped somatic embryos with cotyledon-like structures. The highest percentages of shoot formation for all 6 species were obtained using 0.1 mg l−1 GA3. A complete protoplast-to-plant system was developed for F. polyandra, A. bilocularis and T. trifolia, which could facilitate the transfer of nuclear and cytoplasmic genes from these species into cultivated Citrus through protoplast fusion.

In vitro plantlet formation from juice vesicle callus of satsuma (Citrus unshiu Marc.)

Callus was induced from juice vesicles of satsuma mandarin on Murashige & Skoog medium supplemented with α-naphthaleneacetic acid (NAA), kinetin (K) and gibberellin (GA). Adventitious embryoids arose from the callus tissue on the medium containing 1 mgl−1 NAA alone. The embryoids grew into embryos which resulted in a plantlet on medium containing 1 mgl−1 GA.

Plant regeneration from protoplasts of calamondin (Citrus madurensis Lour.)

Abstract Diploid callus cells arose from the hypocotyl region of plantlets differentiated by anther culture of Calamondin (Citrus madurensis Lour.). The callus grew vigorously on Murashige and Tuckers medium containing 10 mg l−1 benzyladenine, and differentiated embryoids on a medium with 5% lactose and no plant growth regulators. This embryogenic potential was transferred to protoplasts which regenerated plantlets. Preculture of callus in liquid medium containing 5% lactose, for 4–7 days prior to protoplast isolation, effected a high yield of protoplasts. Plating efficiency was increased in a medium solidified with agarose and/or layered with liquid medium.

Cultivar identification of 'Yuzu' (Citrus junos Sieb. ex Tanaka) and related acid citrus by leaf isozymes

Abstract Leaf extracts of 27 ‘Yuzu’ and related acid citrus cultivars were analyzed using polyacrylamide gel electrophoresis for isozyme variation of glutamate oxaloacetate transaminase (GOT) and shikimate dehydrogenase (SDH). SDH yielded 12 different isozyme phenotypes and six cultivars were discriminated by this enzyme alone. GOT produced 10 different isozyme phenotypes and four cultivars were separated. When both enzyme systems were taken together, 16 cultivars (59%) were uniquely discriminated and the rest could be classified into four groups of 2–4 cultivars each. Mutation originated cultivars could not be discriminated. Differences between cultivars suggested that isozymes may provide useful markers for cultivar identification.

Embryogenic protoplast cultures of orange jessamine (Murraya paniculata) and their regeneration into plants flowering in vitro

Embryogenic callus was induced from the hypocotyl region of seedlings germinated from immature embryos of orange jessamine (Murraya paniculata (L.) Jack) on Murashige & Tucker (1969) medium containing 50 g l-1 sucrose, 5.0 mg l-1 benzyladenine, 2.5 mg l-1 2,4-dichlorophenoxyacetic acid and 600 mg l-1 malt extract. Isolated protoplasts divided to produce callus on Murashige & Tucker (1969) medium containing 50 g l-1 sucrose, 0.01 mg l-1 gibberellin A4+7 and 600 mg l-1 malt extract. Callus developed to plantlets via somatic embryogenesis on Murashige & Tucker (1969) medium with 50 g l-1 lactose but no plant growth regulators. These plantlets flowered in vitro on half strength Murashige & Tucker (1969) medium containing 50 g l-1 sucrose after 2 months culture.

Phylogenetic relationships in the kumquat (Fortunella) as revealed by isozyme analysis

Abstract Nine isozyme systems were investigated by polyacrylamide gel electrophoresis as a means of identifying the six species of Fortunella . By examining the characteristic isoenzymatic patterns, it was found that there was considerable variability between the different species. The relationships among the six species of Fortunella were inferred from the similarity matrix cata. The results indicate that no pairs of species are identical and the degree of relationship between the species was low, suggesting that the six species of Fortunella are independent.

Use of glutamate-oxaloacetate transaminase isozymes for detection of hybrids among genera of the «true citrus fruit trees»

Abstract Intergeneric hybrids between Microcitrus (Microcitrus inodora) × Eremocitrus (Eremocitrus glauca), Microcitrus (Microcitrus warburgiana) × Fortunella (Fortunella margarita), Fortunella (F. margarita) × Microcitrus (M. warburgiana) , and Citrus (Citrus grandis cultivar ‘Tosa-buntan’) × Poncirus (Poncirus trifoliata) were developed by controlled pollination. Hybridization was confirmed by isozyme analysis using polyacrylamide gel electrophoresis. Genetic analysis of glutamate-oxaloacetate transaminase (GOT) clearly indicated that a dimeric nature of GOT isozymes in ‘true citrus fruit trees’ of the subtribe ‘Citrinae’ and their genetic control by two loci with six alleles at Got-1 and four alleles at Got-2 . Four new species-specific alleles were discovered, one for E. glauca (Got-1 ‘E’) and three for M. warburgiana (Got-1 ‘W’, Got-2 ‘W’ and ‘G’). Common alleles were observed among the genera of Microcitrus, Fortunella and Eremocitrus and homologous loci were observed among the studied genera.

Plant regeneration via somatic embryogenesis from protoplasts of Uganda cherry orange (Citropsis schweinfurthii)

Protoplasts isolated from embryogenic callus of Citropsis schweinfurthii (Engl.) Swing. & M. Kell were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l−1 BA, 0, 300, 600 or 900 mg l−1 malt extract and 0.6 M sorbitol. The highest plating efficiency was obtained on MT basal medium containing 5% sucrose supplemented with 0.01 mg l−1 BA and 600 mg l−1 malt extract. MT basal medium containing 5% sucrose and supplemented with 0.01 mg l−1 kinetin was found to be a medium suitable for the development of globular somatic embryos derived from protoplasts into heart-shaped somatic embryos with cotyledon-like structures. The highest percentage of shoot formation was obtained using 0.1 mg l−1 GA3. A complete protoplast to-plant system was developed for C. schweinfurthii, which could facilitate the transfer of nuclear and cytoplasmic genes from this species into cultivated Citrus through protoplast fusion.

Genetic analysis of phosphoglucoisomerase isozymes in ‘true citrus fruit trees’

Abstract Isozyme inheritance and variation of phosphoglucoisomerase (PGI) were investigated for six genera of ‘true citrus fruit trees’ (Rutaceae) and their intergeneric hybrids. The progeny segregated showing Mendelian inheritance at locus Pgi-1 in Fortunella, Eremocitrus and Microcitrus . Some common isozymes were detected among the six genera. These results suggest that a genetic relationship exists among all the genera of the ‘true citrus fruit trees’ group.