Reticulata Blanco Research Articles

カンキツの組織培養苗の環境保全への適用 ２ 生産費シミュレーションによるスンタラ（Ｃｉｔｒｕｓ ｒｅｔｉｃｕｌａｔａ ＢＬＡＮＣＯ）の試験管内大量迅速育苗法の適性化

Optimization of micropropagation procedure in suntara orange (Citrus reticulata) was examined by a combination of experimentation and cost simulation. Initially, 20 seeds were cultured in modified Murashige and Skoog's medium for 42 days. Excised single shoots from seed cultures were sub-cultured in modified Murashige and Skoog's medium containing 1 μM BAP, 1 μM IBA, 1.5g/l Gellun gum, and 30g/l sucrose every 28 days. Single shoots were then cultured in modified Murashige and Skoog's medium containing 5 μM IBA for 21 days. Regenerated plants were acclimated in a plastic bag containing 125-ml soil for 10 days. Micropropagation of suntara orange was established through plant regeneration without disorganizing the state. Unit production cost of in vitro plant was calculated to be 76.6 yen at 10, 000 level production. This cost is about 46% of the original cost based on the experimentation and about 60% of the first simulation cost. So, the production cost was remarkably reduced by the present procedure.Further improvement on the present production procedure was examined by cost simulation and the results showed that production cost could be reduced to 68 yen.The present result suggests that cost simulation is useful to optimize the production procedure. The usefulness of cost simulation in the improvement of micropropagation procedure and related subjects are also discussed.

カンキツの組織培養苗の環境保全への適用 （第１報） スンタラ（Ｃｉｔｒｕｓ ｒｅｔｉｃｕｌａｔａ ＢＬＡＮＣＯ）の試験管内大量迅速育苗と生産コストシミュレーションによる生産方式の改善方向の推定

The idea, reforestation of Citrus species in mountainous regions is useful both in providing job opportunities to rural people and in environmental conservation. In vitro plants may meet the demand of desired saplings. However micropropagation of Citrus species has not been well established, although plantlet regeneration from disorganized systems has been reported. Micropropagation of suntara orange (Citrus reticulata BLANCO) from Nepal was attempted and improvement of the procedure was estimated by cost simulation.Micropropagation of suntara orange was established by induction of multiple shoots from seeds born from the same tree, successive shoot multiplication, and rooting from excised single shoots. Sixty percent of the seeds germinated in modified Murashige and Skoog's medium containing 1.5g/l Gellun gum, 10μM benzylamino purine and 30g/l sucrose. Multiple shoots were induced from 60% of seedlings and the average number of shoots per seedling was about 3.4. When shoots 10-15 mm in length were cultured in the medium containing 1μMBAP, 1μM IBA, 30 g/l sucrose and 1.5 g/l Gellun gum for 63 days, the average size of the shoots increased 8.9 times. It was calculated that about 1, 800, 000 can be obtained from one single shoot per year. When shoots 10-15mm in length were cultured in the medium containing 5μM IBA for 21 days, 93% of the shoots rooted. The regenerated plants from 5 mm to 20 mm in height were acclimatized for 30 days and 89% of the plants survived on potted soil. It is suggested that instead of meristem culture, virus free plantlets can be propagated by culturing vegetative embryos from citrus seeds, multi-embryonic seeds.Production cost per in vitro plantlet at 10, 000 plantlet production level was calculated to be 165 yen. The improvement of production procedure was attempted by cost simulation and the results showed that production cost would be reduced to 116 yen.The present results indicate that propagation of plant species by plant tissue culture has a great potential in environmental conservation. The usefulness of cost simulation in the improvement of micropropagation procedure and related subjects are also discussed.

Comparison of Essential Oil Components between Leaf and Peel in Citrus Hybrids ('Seto unshiu'*'Morita ponkan').

The leaf oil components in hybrid seedlings of ‘Seto unshiu’ (Citrus unshiu MARC.) crossed with ‘Morita ponkan’ (Citrus reticulata BLANCO) as the pollen parent were examined. The leaf and peel oil components in the hybrid seedlings were also compared with those of their parents. Twenty eight of the 32 peaks found in the hydrocarbon fraction of the leaf oils were identified. The major compounds were γ-terpinene and β-caryophyllene in ‘Seto unshiu’ and sabinene and β-ocimene in ‘Morita ponkan.’ All 40 peaks found in the oxygenated compound fraction of the leaf oils were identified. The major compounds were linalool, (Z)-3-hexenal, and (Z)-2-hexenal. There was a correlation between the leaf oil and peel oil in the percentage of the oxygenated compound fraction in the hybrid seedlings. A similar correlation was found for the parents. When the hybrids were classified according to the major kinds of monoterpenes, such as sabinene and γ-terpinene, and thymol and α-sinensal, the same groupings were obtained with the leaf and peel oils. These results suggested that the flavor of the fruit can be predicted by analyzing the oil in the leaf, which can be sampled much earlier than the fruit.

Hyaluronidase Inhibitor in the Fruit of Citrus reticulata BLANCO.

Fifty percent of ethanolic extracts from the immature fruits of Citrus reticulata BLANCO, C. tamurana hort. ex TANAKA, C. natsudaidai HAYATA and C. unshiu MARKOVICH showed a significant inhibitory effect on the activation of inactive hyaluronidase induced by compound 48/80. This effect decreased with the maturation of the fruit in all cases. Among the citrus fruits tested, the extract from the immature C. reticulata BLANCO showed the strongest inhibitory effect. The data of gel permeation chromatography and gas chromatography-mass spectrometry showed the hyaluronidase inhibitor in the fruit to be a pectin with a molecular weight of 1.1×105. The content of D-galacturonic acid in the pectin was determined to be 58% using the carbazole method. The pectin showed almost the same inhibitory effect (IC50 : 91 μg/ml) as a commercial pectin from oranges or a current anti-allergic agent, disodium cromoglycate. Hyaluronidase has been suggested to be one of the target enzymes controlling the degranulation of mast cells, and the inhibitory effect on the activation of inactive hyaluronidase has been reported by Kakegawa et al. (Chem. Pharm. Bull. 33 [1985]) to be an index of anti-allergic activity. Therefore, it is expected that the pectin in C. reticulata BLANCO might possess anti-allergic potential.

EFFECT of pH and ENZYMATIC TREATMENT ON MICROFILTRATION and ULTRAFILTRATION of TANGERINE JUICE

ABSTRACTTangerine (Citrus reticulata blanco) juice clarification by crossflow microfiltration and ultrafiltration using polysulphone flat sheet membranes with nominal molecular weight cut off of 25,000, 50,000, 100,000 and 0.1 μm, 0.2 μm pore sizes was studied. the juice was pretreated by polygalacturonase and pH adjustment. the treated juice was clarified with a laboratory scale filtration unit with effective filtration area of 14 cm2. Filtration conditions were transmembrane pressure of 93 to 194 kPa, crossflow velocity of 0.96 to 3.5 m/s and 25°C. Membrane performance was evaluated in terms of volume flux and clarity (% transmittance) of the permeate.Pretreatment of the juice by polygalacturonase and adjustment to pH 2 with HCl resulted in a clearer supernatant than enzyme treatment alone. Maximum flux was obtained with the 0.1 μm microfiltration membrane. Flux increased with transmembrane pressure and crossflow velocity. Flux at 194 kPa and 3.5 m/s was 69 L per square meter per hour. Permeate clarity was better at higher transmembrane pressure and lower velocity, due to the effect of the polarized/fouling layer of solute on the membrane surface, which acted as a secondary “dynamic” filter.

Agrobacterium-mediated transformation and regeneration of Citrus spp. from suspension cells.

Agrobaclerium-mediated transformation of suspension cells of Citrus and regeneration of transgenic plants have been achieved using a binary vector and a cointegrative vector systems. Three embryo callus lines of 'Washington navel' orange (Citrus sinensis OSBECK), 'Ohta' ponkan and 'Kara' mandarin (C. reticulata BLANCO), and a pollen embyoid callus of 'Trovita' orange (C. sinelesis) were cocultlvated with two strains of A. tumefaciens, A415 or GV3010, for 3, 5 and 7 days. The former strain harbors a binary vector pTRA415 containing a neomycin phosphotransferase II (npt-II) gene. The later strain harbors a cointegrative vector pGV3850 : pMPK110-1lpt containing a hygromycin phosphotransferase (hpt) gene. Callus colonies were formed in calli of 'Washington navel' orange and 'Trovita' orange on selection media containing kanamycin or hygromycin. A relatively high frequency of transformation was observed with 3-day-cocultivation but a lower frequency was observed with 7-day-cocultivation. Embryoids were formed from kanamycin or hygromycin resistant calli and developed into plantlets. Transformation was confirmed on calli and a plantlet by Southern hybrydization.