Background:Callus cultivation has the advantage of producing a large amount of tissue of a plant in a laboratory regardless of the environment, for extracting an active substance. In the present study, callus formation was induced in the leaves of the succulent plant Adenium obesum (Forssk.) Roem & Schult. After callus cultivation, anti-inflammatory activity tests were conducted, because leaves and stems of A. obesum have been reported to possess biological activity.Methods and Results:In order to induce callus formation, various concentrations of plant growth factors, such as kinetin, naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and indole-3-acetic acid (IAA) were added to MS solid medium. The maximum callus proliferation was induced by mixed medium consisting of NAA (2㎎/ ℓ ) and BA (1㎎/ ℓ ). In addition, an elicitor was added to the medium under optimal conditions for initiating suspension culture. After suspension culturing, the activities of the callus extracts were compared and analyzed. The cytotoxicity and anti-inflammatory activity tests revealed that the anti-inflammatory activity of the callus extract and the content of phenolic compounds were elevated after treatment of the callus culture with the elicitior.Conclusions: A. obesum callus might be considered as potential source of biologically active anti-inflammatory material.

Antigen production in plant is a safe and effective strategy for vaccine development. In this study, rice transformants were developed for oral vaccine against pigs diarrhea disease. DNA cassette composed with the cholera toxin subunit B (CTB) connected to the 987P-fasG, for stimulating a strong oral immune response, was introduced to rice through Agrobacterium mediated genetic transformation. Copy number analysis by TaqMan real-time PCR for transgenes revealed that transgene of 1 to 8 copies have been introduced into T1 and T2 rice seeds. The expression level of mRNA in the transformants T1 and T2 generations were up to 35 times higher than the reference value in the result of analysis by Quantitative real time-PCR. In addition, the callus cultured from rice transformants was confirmed that the introduced gene has been maintained till 9-month subculture duration. The amount of mRNA expression value was also confirmed in callus, which was maintained above 2.6 times compared with that of the standard control for a long time. These results provide that the introduced antigen for plant-based vaccine against bacterial diarrhea disease can be maintained in the callus as well as in the transgenic plant and suggest that the callus culture of plant transformant will be an effective way to obtain a plant-derived edible vaccine.

Background : With the increasing demand of the mistletoe in larger quantities for cancer therapy, it has been depleted from its natural habitat in the Far East countries including Korea because of overharvesting for high-value products (e.g., lectins and viscotoxins). The rapid multiplication of mistletoe by tissue culture can help this problem and provide the benefits in the phamaceutical industry. Methods and Results : Mistletoe plants growing on oak trees were collected and their leaf and stem segments were inoculated on MS basal medium supplemented with various concentrations of growth regulators. Calli were induced only from stem explants on MS medium containing 2,4-D and transferred onto MS medium supplemented with various concentrations of 2,4-D, NAA and BAP, respectively, for their propagation. The best callus multiplication rate of more than 15 folds (759 mg) was obtained in treatment of 2,4-D (4 mg/L) that produced yellowish-green, white and friable callus on this medium. To compare biochemical characterization, lectins were partially purified from natural mistletoe plants (nML) and in vitro cultured mistletoe calli (cML), respectively. The former was purified by lactose-agarose affinity chromatography and the latter was done by ammonium sulfate precipitation followed Sephadex G-25 chromatography. Both nML and cML showed similar molecular weight on SDS-PAGE and Western blot analyses. In addition, they showed similar carbohydrate-binding specificities and hemagglutination activities. Conclusion : From the above results, we may suggest that nML and cML showed the similarity in biochemical characters.

Background : Lectins were individually isolated from natural Korean mistletoe (nML) and its in vitro cultured calli (cML). Both of the lectins showed the difference in bioactivities such as cytotoxicity and cytokine induction. Methods and Results : Target cells (1 x 104 cells/well) were seeded independently into each well of a 96-well culture plate and incubated with different concentrations of each lectin. Survivability of target cells was determined by CCK-8 kit (Sigma, USA) according to manufacturer’s directions. The nML showed 46, 34 and 5.5 times stronger than cML in cytotoxicity (IC50) to human melanoma cell line (SK-MEL-28), human carcinoma cell line (NCI-H1650) and murine macrophage (RAW 264.7), respectively. In addition, respective lectins directly stimulated macrophage RAW 264.7 but they showed the difference in enhanced productivity of some inflammatory cytokines. Compared with cML, the nML induced both TNF-α and IL-1β at its low concentration. Administration of two kinds of lectins (10-1000 μ g/kg body weight) to ICR mouse did not show any significant changes on the level of alanine transaminase (ALT), glutamate-oxaloacetic transaminase (GOT) and blood urea nitrogen (BUN) in sera. Conclusion : From the above results, we may suggest that nML and cML showed differences in cytotoxic effects and cytokine production due to the difference in amounts from sources.

Miraculin can modify a sour taste into a sweet taste and is an alternative to traditional sweeteners. Mirculin gene was introduced into the Miyagawa Wase Satsuma mandarin (Citrus unshiu Marc.) callus by Agrobacterum-mediated transformation to produce citrus transgenic plant. Transgenic plant candidates were selected via hygromycin resistance. Transformation was confirmed by Polymerase Chain Reaction, Southern blotting, and Western blot analysis. Expression of this gene in transgenic citrus resulted in the accumulation of miraculin protein in the leaves. Multiple Shoots of transgenic citrus planets were micrografted onto trifoliate rootstocks in the sterile soil. Plants were established in the greenhouse 2 years after planting.

활용가치가 높은 부존식물자원인 갈대의 기내 번식을 통한 배양체계를 확립하고 재분화 식물체들의 유전적 다양성을 검토한 결과, 성숙종자 유래의 캘러스를 통한 기내 식물체 재분화는 N6배지에서 MS배지보다 양호하였고, 0.25~0.5 mg/L의 BA를 포함한 N6배지에서 가장 높았다. ISSR 마커를 이용하여 재분화 식물체의 유전적 안정성을 분석한 결과, 검출된 총 94 유전좌중 유전적 다형성은 17%였고, 평균 유전자다양도 값(h)은 0.03, BA 5 mg/L를 포함한 N6배지에서 0.008, NAA 0.1 mg/L와 kinetin 2 mg/L를 포함한 MS 배지에서 0.040으로 나타났다. 이것은 재분화된 갈대식물체 개체간에 유전적으로 구조가 매우 단순하고 균일하며, 유전적 다양성 진단에 ISSR 마커가 효과적임을 시사한다.

차세대 바이오매스로 주목받고 있는 섬유질계 에너지작물 중 국내에서 활용 가능성이 높은 갈대(Phragmites communis)를 바이오에너지 작물로 개발하기 위하여 미성숙 화기로부터 callus 유도 및 유도된 callus로부터 식물체 재생에 미치는 배지조성의 영향을 조사하였다. 갈대의 미성숙 화기를 절단한 절편체를 callus 유도 배지에 치상하였으며, 배양 4주 후 callus 형성율과 cluster 당 형성된 callus 수를 조사하였다. Callus 유도를 위해서 MS 배지에 2,4-D 1.0mg/L를 첨가하고 배지고형제로 Phytagel 4.0g/L를 첨가한 배지가 callus 유도율 88.9%와 callus 수 8.7개로 가장 좋았다. 배양 4주 후 callus를 분리하여 동일배지로 옮겨주었으며, 4주 간격으로 계대배양 하였고, 이를 재생배지에 치상하여 식물체 재생을 유도하였다. 미성숙 화기 유래 callus를 N6 배지에 BA 0.25mg/L, NAA 0.1mg/L가 첨가된 배지에서 배양하여 식물체 재생을 유도하였을 때, 재생율은 100%, callus당 재생된 식물체수는 17.4개로 가장 양호하였다. 식물체를 분리하여 MS 배지에서 신장을 유도하였고, 배양 4주 후 8～10cm 길이의 소식물체가 형성되었다. 소식물체를 배양용기에서 꺼내어 육묘판으로 이식하였고, 6주 후 지상부가 20cm 이상 발달하면서 성공적으로 활착되었다. 본 연구를 통하여 갈대의 미성숙화기로부터 callus 유도를 거쳐 식물체를 재생시키는 시스템을 확립하였다.

바이오에너지 작물로 주목받고 있는 억새(Miscanthus sinensis)의 성숙 종자로부터 캘러스를 유도하고 식물체를 재생시키는 방법을 개발하였다. 억새 종자로부터의 캘러스 유도에 적합한 조건을 알아보고자 기본배지와 2,4-D의 농도, proline의 농도, 배지고형제의 종류와 농도 처리를 각각 실시하였다. 기본배지로 MS배지와 N6배지를 두고 2,4-D를 농도별로 처리하였을 때, MS 배지에 2,4-D 4.0 mg/L를 첨가한 처리구에서 캘러스 형성율이 42.2%로 가장 높았다. Proline은 종자로부터 캘러스 형성을 증가시키는 효과가 있었고, 3.0 g/L 이하의 저농도에 비해 12.0 g/L를 첨가하는 것이 캘러스 형성에 좋았다. 배지 고형제는 agar에 비해 Phytagel이 효과적이었으며 Phytagel 2.25 g/L를 첨가한 처리구에서 캘러스 형성율이 86.1%로 가장 높았다. 억새의 종자로부터 유도된 캘러스는 4주 간격으로 계대배양하였고, 캘러스 유도 3개월 후 진한 노란색의 둥근 돔 형태를 갖는 캘러스를 분리하여 신초 재생에 이용하였다. 종자로부터 유도된 캘러스를 호르몬 무첨가 MS 배지 또는 MS 배지에 BA 1.0 mg/L를 첨가한 배지에서 배양하여 신초 재생을 유도할 수 있었다. 재생된 신초는 신장과 발근을 거쳐 소식물체로 발달하였으며, 소식물체는 성공적으로 활착되었고 정상적인 표현형을 나타냈다.

본 연구는 최근 한방자원, 사료자원, 바이오에너지 자원 등 다양하게 이용되는 국내 자생 억새(Miscanthus sinensis)의 대량생산 및 신품종 개발을 위한 조직배양체계 확립을 위해 수행되었다. 이를 위해 억새 완숙종자로부터의 캘러스 유도와 재분화를 위한 식물생장조절제의 적정농도를 규명하였다. 억새의 성숙종자유래 배발생 캘러스 유도를 위해 2,4-D, IBA, NAA를 1~10 mg·L-1의 농도로 단용 처리한 결과, 5 mg·L-1 2,4-D 처리에서 가장 높은 85.3%의 캘러스 유도율과 캘러스의 증식을 보였으며 조직배양 과정 중 갈변화율도 가장 낮았다. 또한, 캘러스의 재분화를 위해 옥신인 NAA와 Kinetin, 2-iP, 또는 BAP 등의 사이토키닌을 혼용 처리한 결과, 각각 19.0%~59.0%, 23.0%~67.3%, 14.7%~83.7%의 재분화율을 보여 NAA와 BAP의 혼용 처리구가 NAA와 Kinetin 또는 2-iP와 혼용 처리구보다 식물체 재분화에 효과적이었다. 특히 3 mg·L-1 NAA와 5 mg·L-1 BAP 혼용 처리된 배지에서의 재분화율이 83.7%로 가장 높게 나타났으며, 캘러스 당 재분화 식물체 개수도 5.5개로 동일농도의 2-iP 또는 Kinetin 혼용 처리 시 2.1 및 2.0개보다 많았다. 본 연구결과 억새 성숙 종자로부터의 배발생 캘러스 유도에는 5 mg·L-1 2,4-D가 그리고 캘러스의 재분화에는 3 mg·L-1 NAA와 5 mg·L-1 BAP 혼용 처리가 가장 효율적이었다. 본 연구를 통해 확립된 조직배양체계는 억새의 대량생산 및 신품종 개발에 유용할 것으로 판단된다.

본 연구에서는 조직배양을 통해 은행나무 잎으로부터 캘러스를 유도하였고, 최대 생장량과 항산화능이 우수한 배양조건을 확립하였다. 즉, 은행잎을 이용하여 조직배양한 결과 암과 광조건에서 모두 NAA 첨가 조건이 2,4-D 첨가 조건에 비교하여 양호한 캘러스 생장을 나타냈다. 가장 우수한 캘러스 생장을 나타낸 배양조건은 광조건의 10 μm NAA와 5 μm kinetin의 조합 처리시였다. 따라서 현탁배양에서 캘러스의 지속적인 유지를 위한 효과적인 생장조절제는 10 μm NAA/0.5 μm BA, 10 μm NAA/0.5 μm kinetin으로 나타났다. 또한 은행잎 유래 캘러스 추출물의 항산화능은 광조건에서 10 μm NAA가 처리된 기본 MS배지에 캘러스를 현탁 배양하였을 때 가장 높게 나타났다. HPLC를 통한 flavonol glycosides를 분석한 결과 10 μm NAA가 처리된 조건에서 현탁 배양된 녹색 캘러스에서 quercetin dehydrate와 keamperol이 각각 0.556, 0.157 μg/20 μl 검출되었다. 잎 추출물에 비해 표적물질을 제외한 불순물이 적어 표적물질의 순수생산이 가능할 것으로 기대되며, 특히 keamperol의 경우 기존의 연구보고에 비해 약 7배 높은 함량이 검출되었다.

Brachypodium distachyon is rapidly emerged in biological study and has been currently used as a model system for genetics and functional studies for crop improvement and biofuel production. Phosphinothricin (PPT) has been widely used as a selectable agent, which raises ammonium content and induces toxicity in non-transformed plant cells. However PPT selection is not much effective on Brachypodium callus consequently reducing transformation efficiency. In order to identify the efficient conditions of PPT selection, calli obtained from mature seeds of Brachypodium (PI 254867) were cultured on the callus inducing medium (CIM) or regeneration medium (ReM) containing serial dilutions of the PPT (0, 2, 5, 10, and 15 mg/l) in dark or light condition. Callus growth and ammonium content of each treatment were measured 2 weeks after the treatment. Although callus growth and ammonium content did not show much difference in CIM, slow callus growth and increased ammonium accumulation were found in ReM. No significant difference of ammonium accumulation in response to PPT was found between dark and light conditions. In order to identify major factors affecting increased ammonium accumulation, callus was cultured on the media in combined with phytohormones (2,4-D or kinetin) and carbon sources (sucrose or maltose) containing with PPT (5 mg/l). The highest ammonium content in callus was found in the kinetin and maltose media.

Glutamine synthetase (GS) plays important roles in plants like assimilation of ammonium and detoxification of the ammonium released from many metabolic processes such as amino acid degradation or photorespiration. Using ATP, ammonia is combined with glutamate to yield glutamine by the action of GS. Phosphinothricin (PPT) is widely used as a herbicide because it competes with glutamate to bind the active site of GS. PPT has been used to produce transgenic Brachypodium distachyon callus and plants as a selectable agent. PPT treatment raises ammonium content and induces toxicity in non-transformed cells. To find out efficient condition for selecting transformed callus, ammonium content were measured in this study. Non-transformed callus were derived from mature seeds of Brachypodium distachyon (Bd21). The callus were cultured on the callus inducing media (CIM) or regeneration media (RM) containing serial dilutions of the PPT (2, 5, 10 and 15 mg/l) with or without light. Ammonium content was measured 2 weeks after PPT application. Ammonium toxicity associated with PPT treatment was dose-dependent on RM whereas PPT treatment was not significantly influenced on CIM. There is no influence on dark or light condition. Additionally, callus were cultured on the media containing phytohormones combined with PPT (5 mg/l) and the most affecting element causing increased ammonium content has been identified. Acknowledgements: This work was supported by a grant (No. 20070301-034-016-007) from BioGreen 21 Program, RDA, Republic of Korea.

Italian ryegrass (Lolium multiflorum Lam.) is one of important forage crop grass widely cultivated in Korea. Progress in breeding using conventional selection procedure is very slow, since Italian ryegrass is highly self-infertile. Biotechnological approaches, therefore, may contribute to the development of improved cultivars for forage crops. In an effort to optimize tissue culture responses of Italian ryegrass (Lolium multiflorum Lam.) for future genetic manipulations to improve forage characteristics, the effects of culture medium supplements on tissue culture responses were investigated with mature seeds of Korean Italian ryegrass (Lolium multiflorum Lam.) seven cultivars as explant tissues.

Somaclones regenerated from seed-derived callus culture of a rice cultivar, 'Ilpum', were evaluated for the agronomic and morphological characteristics. A total of 424 plants regenerated and the seeds collected from 297 lines were used for further analysis on morphological and agronomic traits. The results indicated that 21.5% (64 lines) of progenies showed significant differences from the donor cultivar 'Ilpum'. Somaclonal variations were induced leaf character, plant height, grain characters, heading date, and sterility. Among the variants, 8% (24 lines) of sterility, 1.4% (4 lines) of dwarf/semi dwarf, 1.7% (5 lines) of opaque, and 1.3% (4 lines) of leaf-rolling phenotypes were observed. The highest frequency of somaclonal variation was observed on heading date which occupied 9.1% (27 lines) of the entire lines included 6.4% (19 lines) of early heading and 2.7% (8 lines) of late heading. These results suggest that the somaclonal variation will be one of the promising genetic resources due to its wide variation and comparatively high frequency of mutation.

Camptotheca acuminata, a native of South China is a well known natural source of monoterpene-indole alkaloid camptothecin(CPT), one of the most promising anti-tumoural compounds. This study was conducted to optimize plant growth regulators and culture conditions on plantlets regeneration through organogenesis from callus of Camptotheca acuminta. Callus were induced from various explants of in vitro germinated plantlets of C. acuminta using WPM medium containing 0.2 ㎎/L 2,4-D. Hypocotyl segments were exhibited higher embryogenic callus than the other explants. Shoot buds formation from embryogenic callus was affected by plant growth regulators, pre-treated dark condition and liquid culture. Organogenesis was optimal in WPM liquid medium containing 0.5 ㎎/L BA. The dark pre-treatment for 2 weeks before the solid culture was effective for organogenesis. The regenerated shoots were rooted in WPM medium with 0.2 ㎎/L NAA and successfully acclimated in green-house conditions.

참돌꽃 캘러스로부터 elicitor와 전구체가 salidroside 생산에 미치는 영향을 조사하였다. Elicitor로서 효모추출물, 연자성 세라믹, methyl jasmonate, jasmonic acid, ascorbic acid, 및 중금속 (CuCl2/CdCl2)을 캘러스 배양에 처리하였다. 효모추출물 0.2g/l농도로 처리한 결과 처리하지 않은 대조구에 보다 3.45배 증가시켰다. 사용된 elicitor 중 효모추출물이 가장 높은 salidroside 생산을 보여 가장 적합한 elicitor로 사료된다. 전구체로서 L-phenylalanine과 L-tyrosine을 배지에 첨가하여 4일 간 배양 처리하였다. Salidroside 함량분석 결과 캘러스로부터 전구체들은 유용물질 생합성에 영향을 주지는 않았다. 캘러스 배양에 첨가 처리된 L-tyrosine의 모든 농도의 경우에는 캘러스 생장뿐만 아니라 salidroside 생산을 감소시켰다.