Background : In this study, we investigated the formation of Abeliophyllum distichum callus which has not been reported until now and the culture optimization. The potential of callus cultures was examined by comparing the components and antioxidant activities of callus and wild planting. Furthermore, it was intended to provide data on the possibility of substitution for the active ingredient production of Abeliophyllum distichum callus extraction. Methods and Results : We tried to induce callus by introducing it in vitro culture by using leaves and stem of Abeliophyllum distichum grown in Goesan Chungbuk. In order to investigate the effect of growth regulators on the callus, TDZ, NAA, 2,4-D, IBA and BAP were treated with 0.25 to 5 ㎎/ℓ of single hormone, and the combination hormones were treated with two concentrations of BAP (0.1, 1.0 ㎎/ℓ) and 2,4-D, NAA, IBA at 0.1 ㎎/ℓ and 1.0 ㎎/ℓ. And investigate the effect of inorganic salt concentration on somatic embryogenesis, the incidence of callus was examined by culturing 1X, 1/2X MS medium for 4 weeks. As a result, the effect of growth regulator and treatment concentration on the callus formation of Abeliophyllum distichum, callus induction was the fastest in the 2,4-D condition, but the callus indection was slow. The highest amount was produced ine the NAA condition. The axillary bud was best grown in TDZ condition, but no root was formed. LC chromatogram was able to compare the contents of leaf, stem extracts and callus extracts. Antioxidant ativity showed excellent antioxidative activity in the extracts of the wild planted stem. Conclusion : The optimum culture conditions for callus were 1/2X MS, 1 ㎎/ℓ BAP and 0.1 ㎎/ℓ of 2,4-D. As a result of LC chromatogram, it was confirmed that callus extract contained a large amount of the same components as the stem and leaf extracts grown on the wild planting. Currently, Studies on mass culture using bioreactor to utilize this are underway, We could confirm the potential possibility of Abeliophyllum distichum cultured products.

Background : Rhododendron brachycarpum belong to Ericaceae family is northern herbaceous plant and grows high in mountains of Korea. Traditionally, this plants have been used to treat arthralgia, neuralgia, hypertension, roborant and diuretic. In spite of medicinal values, natural populations are decresing due to climate change and grows slowly. Therefore it need to secure plant materials and in vitro culture is able to the alternative methods. In this study, we examined the effect of PGRs treatment using leaf and petiole explants. Methods and Results : The effects of the different surface sterilization agents (NaOCl and HgCl2) and time were tested. Best results with lower contamination and higher explants survival were recorded with 2 - 4% NaOCl for 1 minutes and 0.5 - 1.0% AgNO3. Callus was obtained when cultured onto MS medium using different concentration and combination of 2,4-D, BA and NAA. Maxium induction of callus was obtained from combinations of 2.0 ㎎ l-1 2,4-D and 1.0 - 2.0 ㎎ l-1 BA from leaf explants. Petiole explants were more effective to induce callus than leaf explants from combination of 1.0 ㎎ l-1 2,4-D and 0.1 - 1.0 ㎎ l-1 BA. Conclusion : The resultes provide that different explants and PGRs combinations were good source of callus induction.

Background : Korean ginseng require 3 - 4 years to produce mature seeds from their mother plants. Therfore, it takes over 20 years to genetic fixation by artificial crossing of 8 generation. Anther culture is a useful method for obtaining homozygotes in only one generation. However, there is not much research on ginseng yet. In this study, we investigated the callus induction of anther depending on the type and concentration of the plant growth regulators in Panax ginseng C. A. Meyer. Methods and Results : Flower buds of P. ginseng were cold pretreated at 2 days before the anthers were plated on the induction medium. The flower buds were immersed in 70% ethanol for 30 sec min, washed two times with sterile distilled water, surface-sterilized in 2% sodium hypochlorite solution for 20 min, then rinsed five times with sterile distilled water. The anthers were placed on Petri dishes containting fifteen different concentrations and combinations of 2,4-D, NAA, BAP and KT. Callus induction was significantly influenced by the type and combination of plant growth regulators. The highest callus induction rate was observed in GR5 medium at 79.2%. The 2,4-D mediums had significantly higher callus induction than the NAA medium, and 2,4-D 1 ㎎/ℓ have a higher callus induction rate than the other concentrations. The increase of callus induction rate was not observed by the addition of cytokinin, but the callus induction rate was gradually decreased as the BAP concentration was increased. There was no difference in callus induction rate between BAP and KT. Conclusion : The important factor for inducing callus of ginseng anther was the addition of 2,4-D, and no effect of cytokinin addition could be found.

본 연구에서는 천연 화장품 소재 개발을 위하여 접시꽃 캘러스 생리활성 효능에 대해서 평가하였다. 접시 꽃캘러스 추출물의 항산화 효능은 DPPH, ABTS, FRAP assay를 통해서 확인하였다. 그 결과 접시꽃캘러스 추출물은 농도 의존적으로 강력한 항산화 능력을 확인되었다. 더불어, 접시꽃 캘러스 추출물은 10 mg/mL 의 농도에서 세포 내 ROS를 효과적으로 감소시키는 것으로 확인되었다. 접시꽃 캘러스 추출물의 미백효능을 평가 하기 위해 tyrosinase 저해 효과를 확인한 결과, 접시꽃 캘러스 추출물은 10 mg/mL 의 농도에서 tyrosinase 활성을 51% 감소시키는 것으로 확인되었다. 이상의 결과를 바탕으로, 접시꽃 캘러스 추출물은 항산화 및 미백 효능을 지닌 화장품 기능성 소재로서 적용 가치가 높은 천연소재로 사료된다.

Plant molecular farming has attracted a lot of attention lately in the field of mass production of industrially valuable materials by extending application of the plant as a kind of factory concept. Among them, protein expression system using rice(Oryza sativa L.) callus is a technology capable of mass culture and industrialization because of a high expression rate of a target protein. This study was carried out to develop an Agrobacterium-mediated transformation system to increase the utilization of rice callus. The transformation efficiency was improved by using the hand when seeds were de-husked for callus induction. Furthermore, we were possible induction of callus from 6 years old seed smoothly. Selection of the callus contained the target gene was required a cultivation period of at least 3 weeks, and the most efficient selection period was after 6 weeks of culture including one passage. This selection was confirmed that the gene was stably inserted into the genomic DNA of the plant cell by the southern blot analysis and progeny test. Such an efficient selection system of rice callus that can be cultured in the long term will be contribute to the industrialization of useful recombinant proteins using rice.

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.

억새(Miscanthus spp.)는 화본과에 속하는 다년생 C4 식물로 국내에 자생하는 대표적인 바이오에너지 원료작물이 다. 농촌진흥청에서 개발한 ‘거대 1호’는 4배체 물억새로 초장 및 경태 등이 일반 억새의 두 배 크기로 탁월한 건물 생산성을 보여 유망한 바이오매스 자원으로 여겨지고 있다. 본 연구에서는 ‘거대 1호’의 미성숙 화기를 이용한 안 정적인 캘러스 유도 및 식물체 재분화 조건을 확립하여 대량증식 및 분자육종을 위한 기초자료를 확보하고자 하였 다. 재료는 억새의 2mm 이하인 미성숙화기를 사용하였으며, 수집한 재료는 70% EtOH로 2분, 0.45% NaOCl으로 20 분간 표면살균 후 미성숙화기의 정단부위를 실체현미경 하에서 적출 후 사용하였다. MS배지에 2,4-D(Auxin)와 BA(Cytokinin)를 각 농도 별로 첨가하여 캘러스 유도율을 조사한 결과, 2,4-D 5 mg/L와 BA 0.1 mg/L를 혼합 처리한 배지에서 가장 높은 캘러스 유도율을 나타내었다. 유도된 캘러스로부터 신초 재분화를 위해 MS 배지에 NAA와 BA, 2,4-D와 BA 등을 농도별로 첨가하여 배양한 결과, NAA 1mg/L와 BA 1mg/L이 첨가된 MS 배지와 5 mg/L BA와 0.1 mg/L NAA가 첨가된 배지에서 각각 15.4, 15.2 %의 신초 재생율을 보였다.

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.

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.

Protoplasts of Panax ginseng C. A. Meyer and Aralia continentalis K. (Araliaceae) were isolated from callus cells and mesophyll cells, respectively. The maximum yield of protoplasts isolated from callus cells of P. ginseng were obtained by incubation for 3 hrs in the enzyme mixture of 0.5% macerozyme, 1.5% cellulase, and 0.5 M mannitol as an osmoticum. In the case of mesophyll cells of A. continentalis, the highest yield of protoplasts were obtained by incubation for 5 hrs in the enzyme mixture of 1% macerozyme, 2% cellulase, and 0.6 M mannitol. A polyethylene glycol (PEG) treatment induced an intergeneric fusion of the protoplasts. The fusion products, that is, heterokaryocytes were obtained by treatment of 50% PEG containing 0.05 M Ca salts.

배발생 캘러스 유도 실험을 통해 음나무의 대량 증식의 가능성을 제시하였다. 절편체의 부위 및 호르몬 농도에 따라 약간의 차이는 보였으나 대부분의 조건에서 배발생 캘러스가 유기되었다. 특히 엽병 절편체에 비하여 잎 절편체에서 배발생 캘러스 유기가 더 활발히 이루어졌으며, 2.0mg/l의 2,4-D와 0.1mg/l의 BA를 혼합 처리한 조건에서 가장 많이 유기됨을 확인할 수 있었다. 배발생 캘러스의 대량 증식을 위하여 현탁배양을 실시한 결과 1.0mg/l의 2,4-D를 첨가한 배양액이 증식에 가장 좋은 것으로 나타났으며, 무기염류를 1/2로 반감한 B5 배지와 White 배지가 적당한 것으로 나타났다. 또한 탄소원은 3%의 농도를 사용하였을 때 캘러스 증식 뿐만 아니라 체세포 배의 발아에도 적당한 것으로 나타났다. 따라서 본 실험 결과는 생물반응기를 이용한 음나무의 대량 생산을 가능하게 하는 기초 자료로 이용될 수 있을 것으로 생각된다.