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Chano H. Kim, Jonq H. Park, In S. Chung, Sung R. Kim and Seung W. Lee

Secondary metabolite production by plant cell culture has been become of interest because of its commercial value in use. However, cultured plant cells usually yield lower levels of secondary metabolites than those of intact plants. In order to improve the anthocyanin productivity in hairy root culture of Daucus carota, fungal elicitors from 8 species of Fungi were examined. Through the studies of fungal elicitors in this work, it was turned out that fungal elicitors were very effective to improve the yield of anthocyanin. Despite of its low yield of anthocyanin, high density culture of hairy roots is achieved in fluidized-bed bioreactor, Anthocyanin production in fluidized-bed bioreactor with fungal elicitor treatment was increased greatly. We are currently researching more detailed aeration effects and scale-up in air-lift bioreactors. And these studies could provide important data to establish mass production system for secondary metabolites.

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Dong-Lim Yoo*, Chun-Woo Nam, Jong-Teak Suh, Seung-Yeol Ryu and Seung-Woo Lee

Hanabusaya asiatica has beautiful flowers as ornamental pot plant. It's a famous Korean endemic perennial plant in Korea. Recently many research items has been studying for developing cultivation technology of H. asiatica as a new commercial pot plant. Many endemic plants have much problems associated with maintaining quality for commercial plant. In H. asiatica, as a result of accumulation of anthocyanin in the leaves at reproductive stage, the leaf veins turn to brownish black and whole leaves become to necrosis and dry after all. This study was carried out to find out the suitable method for preventing the accumulation of anthocyanin in leaves by light quality. H. asiatica was treated three light quality, blue, far-red + blue, far-red and control on the middle stage of vegetative growth. Light quality sources were made by diodes. Light quality treatments were done in growth chamber. The photoperiod was 16 hours. Light quality treatments were done for 4 hours as daylight extension after 12 hours lighting by fluorescent lamps. Far-red lighting treatment was very effective to prohibit the formation of anthocyanin in the leaves. Blue lighting treatment was increased the anthocyanin accumulation but blue lighting treatment with far-red showed preventing the formation of anthocyanin. In these results, far-red lighting was very effective for preventing the action of cryptochrome by blue lighting on the anthocyanin formation in the leaves of Hanabusaya asiatica.

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W.S. Lee, J.C. Lee and Y.S. Hwang

The coloration of grape berries depends on the anthocyanin synthesis during maturation. The quality of berries is often decreased due to the poor color development when berries are grown under unfavorable environments and/or inadequate internal factors are involved. It has been well-known that the level of ABA at ripening is closely associated with anthocyanin synthesis; thus, the external application of ABA results in the increase of anthocyanin content even in berries grown under favorable conditions. However, the agricultural use of natural ABA is not possible because of high prices. This experiment was conducted to study the potential of STC-4771 as a substitute for ABA. The effect of STC-4771 was studied in `Kyoho', `Pione', and `Delaware' grapes. Chemicals were applied when ≈10% of berries in a cluster were colored. In `Kyoho', anthocyanin synthesis was enhanced at a concentration of 100 mg/L and there was a trend in color enhancement in `Pione', regardless of treatment concentration, between 10 to 40 mg/L. However, no clear effect was found in `Delaware' at 50 to 100 mg/L. In an in vitro experiment, anthocyanin was only increased when an adequate amount of sucrose (0.6 m) was added in the incubation medium under light. Natural ABA effectively increased the anthocyanin content of berry segments even under shading condition through four bagging materials, but no effect was confirmed in STC treatment.

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A. Howell, W. Kalt, J.C. Duy, C.F. Forney and J.E. McDonald

It is now widely held that the antioxidants contained in fruit and vegetables can provide protection against certain human degenerative conditions that are associated with oxygen free radical damage. This view is supported by epidemiological, in vitro, and more recently, in vivo evidence. Phenolics (polyphenolics) contribute substantially to the antioxidant complement of many small fruit species whose ripe fruit are red, purple or blue in color. Fruit containing high levels of phenolic antioxidants would be attractive to health conscious consumers, therefore optimization of production and processing factors affecting small fruit antioxidant capacity is desirable. In many small fruit crops, antioxidant activity [measured as oxygen radical absorbing capacity (ORAC)] is positively correlated with their content of anthocyanins and total phenolics. Genera, species, and genotypes vary with respect to phenolic content. Both annual and geographical factors appear to influence ORAC, although many years of study are needed to distinguish these effects from other biotic and abiotic factors that influence fruit phenolic content. Antioxidant capacity due to phenolics is decreased by food processing practices, such as heat or aeration.

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George L. Hosfield and Clifford W. Beninger

Seed coat color in dry bean (Phaseolus vulgaris L.) is determined by the presence or absence of tannins, flavonoids, and anthocyanins. Black beans contain three main anthocyanins that are responsible for their black seed coat color: delphinidin 3-O-glucoside, petunidin 3-O-glucoside, and malvidin 3-O-glucoside. Leaching of anthocyanins occurs in many black bean genotypes during thermal processing (i.e., blanching and cooking). Black beans that lose their dark color after processing are unacceptable to the industry. Since the marketability of black beans can be adversely affected by thermal processing, an experiment was conducted to ascertain whether pigment leaching was due to qualitative or quantitative changes in anthocyanins during processing. Four black bean genotypes that showed differential leaching of color were investigated. `Harblack' retains most of its black color after processing while `Raven' loses most of its color. `Black Magic' and `Black Jack' are intermediate between `Harblack' and `Raven' in processed color. Bean samples (119 ± 1.5 g) of the four genotypes were thermally processed in 100 x 75-mm tin cans in a pilot laboratory. Seed coats were removed from the cooked beans, freeze-dried, and placed in solutions of formic 10 acid: 65 water: 25 methanol to extract anthocyanins. The extracts were analyzed by HPLC. Although all genotypes retained some color, there were no detectable anthocyanins in seed coats of the cooked beans. In a second experiment, raw beans of each genotype were boiled in distilled water for 15 minutes. All four genotypes lost color during boiling, but `Harblack' retained most of its color and had a five-fold higher concentration of the three anthocyanins than did the other genotypes. `Harblack' may retain color better than other black beans because of physical characteristics of the seed coat.

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Maria Claudia Dussi, David Sugar and Ronald E. Wrolstad

The anthocyanin in `Sensation Red Bartlett' pear skin was characterized and quantified, and the effect of light quality on fruit color development was evaluated. Anthocyanin concentration was related to fruit chromaticity values. Pigments were analyzed using high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). One of two spots detected in the TLC chromatogram did not change color with molybdate sprays, indicating the possible presence of peonidin. HPLC analysis confirmed the presence of a major and a minor pigment, which co-eluted with cyanidin 3-galactoside and peonidin 3-galactoside. Monomeric anthocyanins in the pear skin extract were 6.83 mg/100 g of fruit peel. To study light quality, gelatin filters allowing passage of different wavelengths of-light were attached over the exposed side of `Sensation Red Bartlett' pears 1 month before harvest. Chromaticity was recorded before the filters were attached and after their removal at harvest using the Commission Internationale del'Eclairage (L*, a*, and b*) color space coordinates. Following color measurements, anthocyanin was extracted from individual skin disks. Skin beneath all filters yielded less hue than the control. Wavelengths that transmit above 600 nm had the largest effect on chroma, a*, and b* values. Fruit wrapped in aluminum foil to obscure all light had the highest luminosity. Wavelengths from 400 to 500 nm gave darker, less chromatic, and redder pear fruit. All treatments yielded higher anthocyanin content than the control. There was a tendency toward increased anthocyanin content with longer wavelengths. The simple linear regression of the log anthocyanin content on L* value and (a*/b*) provided an R 2 = 0.41.

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Shawn A. Mehlenbacher and Maxine M. Thompson

The style color of standard hazelnut (Corylus avellana L.) cultivars ranges from pink to dark purple. Styles with an unusual yellow color were first noted in seedlings of the progeny `Goodpasture' × `Compton', and the ratio was ≈3 red: 1 yellow. Controlled crosses were made to investigate the genetic control of style color. The same 3:1 ratio was observed in four additional crosses in which both parents had red styles. Two crosses of a red and a yellow parent gave ≈50% yellow styles, while a cross of two selections with yellow styles gave only seedlings with yellow styles. These segregation ratios indicate control by a single locus, with yellow style color recessive to red. Seedlings with yellow styles have green buds and catkins and a more upright growth habit than their siblings with red styles. Inspection of the pedigrees of these progenies shows that `Daviana', `Willamette', `Butler', `Compton', `Goodpasture', and `Lansing #1' are heterozygous. `Daviana' appears to be the original source of the allele for yellow styles, as it is a known or suspected parent or ancestor of the others. Ratios in a progeny segregating simultaneously for growth habit (normal vs. contorted) and style color indicated independence of the traits. However, in a progeny segregating simultaneously for leaf color (red vs. green) and style color, no redleaf seedlings had yellow styles. The S-alleles of eight genotypes with yellow styles were determined, and indicate a possible linkage between the yellow style locus and the S locus that controls pollen-stigma incompatibility. One explanation is that the yellow style trait is conferred by an allele (a ys) at the anthocyanin (A) locus that controls leaf color. A second explanation is that there is a yellow style locus closely linked to the A locus. The A locus is known to be loosely linked to the S locus.

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Tomomi Tsuda, Masami Yamaguchi, Chikako Honda and Takaya Moriguchi

We used RNA blot analysis to examine the expression of six genes of the anthocyanin biosynthesis pathway in the flowers and fruit skins at three developmental stages of white and red peaches and a deep-red nectarine [Prunus persica (L.) Batch]. In the red peach `Akatsuki' and the deep-red nectarine `Flavortop', expression levels of anthocyanin biosynthesis genes were related to anthocyanin accumulation in the fruit skin; expression of all six genes dramatically increased at Stage III of fruit development, and anthocyanin concentration also increased at this stage. In the white peach `Mochizuki', however, expression of the chalcone synthase gene (CHS) and the dihydroflavonol 4-reductase gene (DFR) was undetectable in Stage III, although the chalcone isomerase gene (CHI), the flavanone 3-hydroxylase gene (F3H), the anthocyanidin synthase gene (ANS), and the UDP-glucose-flavonoid 3-O-glucosyltransferase gene (UFGT) were expressed. We occasionally found red pigment in the skin of `Mochizuki' peach. In these red skin areas, both CHS and DFR were clearly expressed in Stage III. These results suggest that CHS and DFR are the key regulatory genes in the process of anthocyanin biosynthesis in mature red peach and nectarine.

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Nicholi Vorsa, James Polashock, David Cunningham and Robin Roderick

A diversity of anthocyanins exists among angiosperm species. Studies indicate that various anthocyanins differ in antioxidant potential, their bioavailability, and stability during processing. The fruit of the American cranberry, Vaccinium macrocarpon Ait., is recognized as having six anthocyanins, composed largely of 3-O-galactosides and 3-O-arabinosides, and to lesser amount (≈6%), 3-O-glucosides of the aglycones cyanidin and peonidin. This study analyzed proportions of these six anthocyanins from >250 accessions of a germplasm collection over harvest dates. Fruit samples from 78 selected accessions, based on the first year analysis, were also analyzed a second year. Principal component analysis identified general negative relationships between the proportions of cyanidin versus peonidin, arabinosides versus glucosides, and galactosides versus arabinosides and glucosides. These relationships were consistent across the 2 years. Most variation in germplasm anthocyanin profiles reflected variation of cyanidin versus peonidin proportions, with cyanidin to peonidin ratios ranging from 3.6:1 to 0.5:1. Variation for glycosylation profiles was also evident, with galactoside proportions ranging from 64% to 75%, arabinoside proportions ranging from 20% to 33%, and glucoside proportions ranging from 3% to 9%. Evidence for both significant qualitative and quantitative genetic variation exists for the methoxylation of cyanidin to peonidin. Significant quantitative genetic variation is also apparent for glycosylation.

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Karim M. Farag, Jiwan P. Palta and Elden J. Stang

The application of ethanol for enhancing effectiveness of ethephon under field conditions on cranberry (Vaccinium macrocarpon Ait.) fruit was tested during three seasons (1986 to 1988). The formulation containing ethephon plus the surfactant Tergitol (0.3% or 0.5%, v/v) and ethanol (2.5%, 5%, or 10%) consistently increased anthocyanin content in the fruit by 28% to 54% over the control. In general, fruit size was not affected by the ethephon treatment containing ethanol and Tergitol. The application of ethephon plus surfactant did not increase the anthocyanin content in the fruit. The presence of ethanol in the ethephon and surfactant mixture, however, consistently enhanced the fruit anthocyanin content by 21% to 40% as compared to ethephon plus surfactant. No adverse effect of various treatments on vine growth or appearance was noticed over the three seasons. Chemical name used: (2-chloroethyl) phosphonic acid (ethephon).