• Journal of Plant Biotechnology
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• 제4권1호
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• pp.23-27
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• 2002

To establish an in vitro mass production system of grape anthocyanin pigments through callus and cell suspension culture, the effects of nitrogen source and sucrose on fresh weight and anthocyanin production in cell suspension culture of 'Sheridan' grape level were studied. When the medium was devoid of $NO_3^-$, cell fresh weight was either remained stable (1% sucrose) or slightly decreased with culture time (2,3, and 4% sucrose). When $NH_4^-$ was lacking, 3% sucrose was most favorable for cell growth. When $NH_4^-$ was supplied as N source, the anthocyanin content of 2% sucrose containing medium was maintained 2 times higher than other levels till day 8 in culture, then that of 3 and 4% sucrose which peaked at day 12 thereafter. The anthocyanin content was low than $NO_3^-$-free media. Total anthocyanin content in $NH_4^-$-free medium was just about a half of that of $NH_4^+$ medium. Anthocyanin production of 2% sucrose in $NH_4^+$ medium was maintained about 3-fold till day 8, then decreased thereafter. In $NH_4^+$ medium, pH decreased gradually with final pH of 3.5 to 4.0, while pH in $NH_4^+$-free medium increased with final pH of 6.5 to 7.5.

• Journal of Plant Biotechnology
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• 제1권2호
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• pp.117-121
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• 1999

Effects of jasmonic acid treatment, UV-B and white light treatment on the anthocyanin biosynthesis and cell growth were investigated using the cell suspension culture system of grape (Vitis vinifera L.). Cell growth was not affected by white light irradiation, while it was remarkably suppressed by UV-B irradiation from 8 to 32 h. Anthocyanin accumulation dramatically increased after 16 h from irradiation of UV-B. Simultaneous treatment of jasmonic acid and UV-B increased anthocyanin accumulation by 10-fold. The cell division was restored when anthocyanin was abundantly accumulated after 32 h from UV-B irradiation. Optimum concentration of jasmonic acid was found to be 5 uM for maximum accumulation of anthocyanin. Application of jasmonic acid to grape suspension cells rapidly induced the expression of CHS gene after 2 h from treatment and showed maximum level at 32 h. Simultaneous treatment of jasmonic acid and light also induced CHS gene expression after 2 h, but the maximum level of CHS transcript was observed at 16 h with white light and 8 h with UV-B exposure. The synergistical effects could be explained by the defense mechanism that UV irradiation is mediated in part by alterations in JA and its signaling pathway.

• Journal of Plant Biotechnology
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• 제31권3호
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• pp.239-248
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• 2004

포도 현탁배양계를 이용하여 안토시아닌 생합성 경로에 관여하는 phytochrome 신호 전달에 관하여 연구하였다. 그 결과 세포의 생중량과 세포수의 생장은 배양 시 광의 유무 및 단색광의 종류와 관계없이 배양 4일째부터 8일째까지 급격히 증가하였고 8일 이후부터 정상기에 들어서서 최대 생장을 보였다. 그러나 안토시아닌 색소 축적은 청색광에서 가장 높은 효과를 나타내었으며 적색광에서도 높았으나 원적색광하에서는 암소같이 색소생성이 억제되었다. 배양시기에 따른 색소생성에 미치는 광 조사의 효과는 대수증가기인 배양4일째에 조사한 단색광간에 차이가 인정되지 않았으나, 정상기인 배양 7일째 조사한 단색광의 효과는 그 종류에 따라 색소 축적 패턴에 큰 차이를 나타내는 것으로 보아 배양 7일째가 효과적이었다. Phytochrome의 광가역적 효과를 알아보기 위하여 암배양 7일째세포에 다양한 조건의 단색광 (B/R, B/FR, B/R/FR)을 가역적으로 처리했을 경우, 청색광 단독 처리한 구와 비슷한 양의 색소 축적양상을 보였으며, B/R/R 조건의 단색광이 가역적으로 처리된 세포주에서 가장 많은 색소 축적을 보였다. 그러나 배양 7일째 R/FR 광가역 처리시에는 적색광 단독 처리에 비해 색소 축적이 억제되었으나, R/FR/R 가역처리시에는 적색광의 색소 축적양과 동일한 수준으로 회복되었다. 안토시아닌 생합성의 주요효소인 PAL과 CHS의 발현분석에서 PAL의 발현은 7일째 청색광이 처리된 세포주에서 조금 증가하는 현상을 보였으나, 적색광과 근적외광 처리구에서는 거의 반응이 없었다. CHS의 발현은 단색광 처리에서 높은 변화를 보였으며, 광을 처리한 7일째 높은 발현을 보이다 12일 이후 다시 증가하는 패턴을 보였다. 또한 적색광과 원적색광이 가역적으로 처리된 세포주에서 CHS 발현양도 가역적으로 증가하였다. PHYA와 PHYB는 포도세포에서 단일유전자로 존재하였으며, 단색광 처리에 따라 색소축적이 이루어질 때 이들 이들 두 유전자의 발현도 증가하였다.

• Journal of Photoscience
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• 제7권2호
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• pp.53-58
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• 2000

We performed the cloning of a chalcone synthase (CHS) gene, the key enzyme in the anthocyanin biosynthesis, from the cDNA library constructed with grape suspension cells irradiated UV-B. The PCR fragment was used to cloning the CHS gene. One CHS cDNA clone containing an open reading frame and a partial stilbene synthase (STS)cDNA, the stilbene-type phytoalexin, were isolated. The CHS cDNA clone (VCHS) showed 87% sequence homology with VvCHS (V.vinifea) and 72.3% identity with VSTSY(V.vinifea). its amino acid sequences were longer than any other CHS genes as 454 residues. Two genes were weakly expressed in white light irradiated cells, but highly induced in UV-B irradiated condition during 32 hours. Interestingly, the STS was quickly and abundantly expressed from 2 hours when supplemented with jasmonic acid (JA) and the maximum expression was observed at 4 hours and then gradually decreased. But, the additional UV-B or white light quickly degraded the STS expression than only JA treated grape suspension cells. The CHS also was rapidly induced with JA and the synergistical effect was observed at the addigional light treatment of UV-B or white light. These results are indicated that CHS and STS have different response mechanisms against the environmental stresses.

• Journal of Plant Biotechnology
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• 제4권2호
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• pp.77-82
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• 2002

To elucidate the effect of sucrose on cell growth and anthocyanin production, 1, 3, 5, and 7% sucrose were applied to liquid MS basal medium supplemented with 0.5 mg/L BA + 0.1 and 1 mg/L 2,4-D. Higher sucrose concentration decreased the cell growth regardless of the hormonal composition. Cain in fresh weight was gradual, showing the peak at day 12 in culture, and then decreased. Anthocyanin content increased with sucrose concentration in the medium, and practically there was no difference in anthocyanin content between the two media differing in 2,4-D content. Sucrose concentration for appropriate anthocyanin production was 7%, while 5% was more suitable for increase in total anthocyanin content. At higher sucrose levels, anthocyanin content was high due to the cessation of the cell growth. Medium pH decreased at the early stage and gradually increased thereafter.

• Journal of Plant Biotechnology
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• 제4권2호
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• pp.83-89
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• 2002

To establish in vitro mass production system of grape anthocyanin pigments through callus and cell suspension culture, the effects of nitrogen amount and the ratio of $NO_3^-$/$NH_4^+$ in the medium on cell growth and anthocyanin production were investigated. Total nitrogen amount and the ratio of $NO_3^-$/$NH_4^+$ in the medium strongly affected anthocyanin production and cell growth. When $NH_4^+$ was fixed, the cell growth was promoted by 50 mM total nitrogen (20 mM $NO_3^-$ : 30 mM $NH_4^+$ ) than other nitrogen combinations, and was strongly inhibited when $NO_3^-$ was lacking (0 mM $NO_3^-$ : 60 mM $NH_4^+$ ) while anthocyanin production was increased. When $NO_3^-$ was fixed, the cell growth was promoted by 70 mM total nitrogen (40 mM $NO_3^-$ : 30 mM $NH_4^+$) than other nitrogen combinations, and was strongly inhibited when $NO_3^-$ was lacking (0 mM $NO_3^-$ : 60 mM $NH_4^+$ ) while anthocyanin production was increased. Cell growth was gradually increased by all nitrogen combinations, but anthocyanin production reached its peak on day 4 in culture. Anthocyanin content increased with decreasing cell density. Sucrose was rapidly hydrolyzed to fructose and glucose within 4 days. Glucose and fructose concentrations in the medium increased and peaked at the 4th day. The anthocyanin content of $NH_4^+$-free 2% sucrose media was 2 times (200 $\mu\textrm{g}$/g) higher than that of 1% sucrose. When $NO_3^-$ was lacking, the highest anthocyanin production was observed at 4% sucrose after 12 days of culture, and increased along with the sucrose concentration.

• Journal of Plant Biotechnology
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• 제4권1호
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• pp.17-21
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• 2002

To establish an the mass production system of grape anthocyanin pigments through callus and cell suspension culture, the effects of various combinations of auxins and cytokinins on friable callus production were studied. for friable callus production, 2,4-D was superior to other regulators. IAA at 2 mg/L induced callus from stem and petiole while NAA resulted in rooting. Callus induction rate increased with the 2,4-D level, and stem segments were superior to leaf blade or petiole, showing nearly 100% with 1 and 2 mg/L 2,4-D from petiole and stem. Combined treatments of 2,4-D + kinetin and NAA + BA also yielded friable callus from stem segments. In treatments with 1 mg/L 2,4-D + 1 mg/L kinetin and 1 mg/L NAA + 1 mg/L BA, callus induction rate was nearly 100%. The combination effect of 2,4-D and BA on anthocyanin production was not significant.

• 한국자원식물학회지
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• 제30권5호
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• pp.542-548
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• 2017

8종의 잿빛곰팡이병 균주를 순무잎에 접종하여 병반의 크기를 확인한 결과 가장 강한 감염력을 보인 '포도-01' 균주와 병반의 확산이 가장 적은 '오랜지'를 선발하였다. 순무잎이 저항성을 보인 '오랜지'균주를 처리한 잎이 감수성을 보인 '포도-01'균주를 처리한 잎보다 indole-3-ylmethyl glucosinolate (I3M-GLS) 함량이 무처리 보다 2.5배 이상 높았으나 '포도-01' 균주를 처리한 잎에서는 무처리 보다 낮은 함량을 보였다. 균주의 메탄올 추출액과 물추출물을 식물배양세포에 처리한 결과 '오랜지'균주의 추출물이 '포도-01' 균주의 추출물보다 배양세포의 생장을 더 강하게 억제 한 것으로 나타났는데 '오랜지' 균주의 메타놀 및 물 추출물 처리에서 배양세포의 활력은 각각 22.7% 및 16.5% 감소시키는 것으로 나타났다. 한편 '오랜지'균주 추출물을 처리한 배양세포에서 I3M-GLS의 생합성이 '포도-01' 균주 추출물보다 현저히 높은 것으로 나타났다. 본 결과로 보아 식물체내에 생합성되는 I3M-GLS 함량은 잿빛곰팡이균에 대한 식물세포의 저항성과 밀접한 관계가 있는 것으로 판단된다.

• 한국미생물·생명공학회지
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• 제17권3호
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• pp.257-262
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• 1989

착색용 포도품종(Vitis hybrid)의 조직으로부터 callus를 유도하고 액체 진탕배양을 행하여 세포 생육 및 anthocyanin의 생성에 미치는 각종 인자들에 대하여 조사한 결과 광조사량 및 무기질소원 농도가 가장 큰 영향을 미치는 것으로 나타났다. 즉, 광조사량이 10,000lux 였을 때는 암소에서 배양했을 때보다 anthocyanin 생성량은 약 2배 증가하였다. 또한 기존 MS 배지의 무기질소원 농도를 20배로 감소시켜 배양했을 때 전체 anthocyanin 생성량은 약 5-6배, 건조 중량당 함량은 약 16배 증가함을 알 수 있었다. 또한 무기질소원으로 nitrate만을 첨가했을 때 가장 높은 수율을 나타냈다. 고농도의 Co$^{2+}$, Fe$^{2+}$와 Miller 배지, Gamborg 배지 등이 anthocyanin 생산에 적합한 것으로 나타났으며, MS medium 및 modified MS medium을 이용한 2단계 배양을 시도하여 약 40mg/flask의 높은 anthocyanin 수율을 얻을 수 있었다.

• 대한약침학회지
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• 제13권3호
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• pp.63-71
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• 2010

Objectives: Gallic acid (GA) is the major component of tannin which could be easily founded in various natural materials such as green tea, red tea, grape juice, and Corni Fructus. The purpose of this study is to investigate the effect of Gallic acid (GA) on production of interleukin (IL) in mouse macrophage Raw 264.7 cells stimulated by lipopolysaccharide (LPS). Methods: Productions of interleukins were measured by High-throughput Multiplex Bead based Assay with Bio-plex Suspension Array System based on $xMAP^{(R)}$ (multi-analyte profiling beads) technology. Firstly, cell culture supernatant was obtained after treatment with LPS and GA for 24 hour. Then, it was incubated with the antibody-conjugated beads for 30 minutes. And detection antibody was added and incubated for 30 minutes. And Strepavidin-conjugated Phycoerythrin (SAPE) was added. After incubation for 30 minutes, the level of SAPE fluorescence was analyzed on Bio-plex Suspension Array System and concentration of interleukin was determined. Results: The results of the experiment are as follows. 1. GA significantly inhibited the production of IL-3, IL-10, IL-12p40, and IL-17 in LPS-induced mouse macrophage RAW 264.7 cells at the concentration of 25, 50, 100, 200 uM (p<0.05). 2. GA significantly inhibited the production of IL-6 in LPS-induced mouse macrophage RAW 264.7 cells at the concentration of 50, 100, 200 uM (p<0.05). 3. GA diminished the production of some cytokine such as IL-4, IL-5, and IL-13 in LPS-induced mouse macrophage RAW 264.7 cells. 4. GA did not show the inhibitory effect on the production of IL-$1{\alpha}$ and IL-9 in LPS-induced mouse macrophage RAW 264.7 cells. Conclusions: These results suggest that GA has anti-inflammatory activity related with its inhibitory effects on the production of interleukins such as IL-3, IL-10, IL-12p40, IL-17, and IL-6 in LPS-induced macrophages.