국내육성 오차드그라스 품종들의 캘러스 형성율 및 식물체 재분화 효율

김기용,강경민,배은경,이인애,임용우,최기준,박근제,손대영,조진기 Plant molecular biology and biotechnology research 2003 Plant molecular biology and biotechnology research Vol.2003 No.-

Comparisons of callus formation ratios from seed explants, callus sizes, regeneration ratios from callus and regeneration efficiency for 4 orchardgrass (Dactylis glomerata L.) varieties (Three were developed in Korea and one was imported from foreign nation) are as follows; Jangbeol 102 (67.0%) has the highest callus formation ratio in 4 weeks incubated callus after bedding the seed explants, but Potomac (68.4%) has the highest ratio in 6-seek callus. Potomac (3.93cm) has the highest callus size in 4-week callus, but Jangbeol 101 (4.32cm) has the highest size in 6-week callus. Jangbeol 101 (17.7%) has the highest plant regeneration ratio in 4-week callus, but Potomac (37.4%) has the highest raion in 6-week callus. Jangbeol 102 (11.5%) has the highest plant regeneration efficiency in 4-week callus, but Potomac (25.6%) has the highest efficiency in 6-week callus.

상추 (Lactuca sativa L.) callus로부터 ACAT 억제 활성물질, phytol의 생산

안광희,장태오,백남인,김세영,An, Kwang-Hee,Jang, Tae-O,Baek, Nam-In,Kim, Se-Young 한국식물생명공학회 2002 식물생명공학회지 Vol.29 No.1

상추의 기내 callus 배양으로부터 ACAT (Acyl-CoA: Cholesterol acyltransferase) 억제 활성물질로 알려진 phytol 의 생산가능성이 연구되었다. Callus는 0.5mg.L$^{-1}$ NAA가 첨가된 MS 배지에 4주간 치상된 자엽 절편체로부터 유도되었으며, 다양한 수준의 chlorophyll을 함유하고 있었다. Phytol이 chlorophyll분자를 구성하는 물질로 알려져 있음에 도 불구하고, phytol의 추출량은 녹색의 자엽에서 보다 callus 에서 높게 나타났다. GC 분석결과, phytol의 추출량은 callus의 chlorophyll 함량과 비례하지 않는 것으로 나타났으며, callus의 chlorophyll 함량수준은 callus의 형성능과 관련이 있는 것으로 사료되었다. 본 연구의 결과는 phytol의 기내생산에 있어서 상추의 callus 배양의 잠재성을 제시한다 하겠다. The possibility for mass production of phytol, inhibitory diterpene against ACAT (Acyl-CoA: Cholesterol acyltransferase) was investigated by using callus culture of lettuce. The callus were induced from lettuce cotyledon explants on MS medium containing 0.5 mg.L$^{-1}$ NAA after 4 week's culture. Adventitious roots were formed from the explants on MS medium containing 0.5 mg.L$^{-1}$ IBA or 1.0 mg.L$^{-1}$ NAA. Adventitious shoots and roots were emerged from the callus when the callus was transferred to MS medium containing auxin alone, or with cytokinin. The plant growth regulators and their concentrations for the organogenesis were 1.0 mg.L$^{-1}$ NAA, 0.1 mg.L$^{-1}$ BA, 0.5 mg.L$^{-1}$ NAA with 0.1 mg.L$^{-1}$ kinetin, or 0.5 g.L$^{-1}$ 2.4-D with 1.0 mg.L$^{-1}$ kinetin. Analyses of chlorophyll contents showed that chlorophyll contents were higher in morphogenic calli than in non-morphogenic calli. However, the chemical analyses of gas chromatography indicated that phytol contents were not proportionate to the chlorophyll contents of callus. The content of phytol was higher in callus than in lettuce cotyledon.ledon.

곰취(Ligularia fisheri )를 이용한 callus배양 및 항산화 활성 검정

강현욱,김영윤,김추자,박요셉,서완우,양정호,연지영,이소연,이인순,문혜연 대구대학교 산업기술연구소 2011 産業技術硏究 Vol.22 No.1

We described the effects of antioxidant activity on the extracts of tissue and callus formation by Ligularia fisheri . The callus culture was induced from leaf tissue of Ligularia fisheri in both MS and B5 medium with IAA(0∼1.5 ㎎/ℓ) or/and kinetin (0∼1.0 ㎎/ℓ). As medium for callus culture induced from Ligularia fisheri , B5 and MS medium showed no callus formation. Both media brought to decrease differentiation of callus, on the other hand those induced to accelerate cell development. The hytohormones were required over 0.4 ㎎/ℓ of IAA and kinetin for formation of callus and those conditions were induced the pigmental deposit like dark red color in callus tissue. In order to extract functional material from natural leaf and callus tissue of Ligularia fisheri used distilled water and 75% ethanol as solvent and those extract used to determine antioxidant activity as 1, 1-diphenyl-2-picryl-hydrozyl (DPPH) radicals scavenging effect and TBA(thiobarbituric acid) activity as well as the concentration of total phenol and flavonoid. In result, the effect of DPPH and TBA had high activity more callus extract of red color than callus(green color) and natural leaf tissue and it also had high concentration of flavonoid. That is because the high concentration of phytohormone brought stress tolerance and hypersensitivity in callus tissue of Ligularia fisheri , so this reaction induced to change color of callus tissue like pigment deposit for culture.

홍경천 뿌리 및 캘러스 초음파 추출물의 항암활성 비교

하지혜(Ji Hye Ha),정향숙(Hyang Suk Jeong),정명훈(Myoung Hoon Jeong),김승섭(Seung Seop Kim),김영(Ling Jin),남종현(Jong Hyun Nam),황백(Baik Hwang),마충제(Choong Je Ma),이현용(Hyeon Yong Lee) 한국식품과학회 2009 한국식품과학회지 Vol.41 No.5

홍경천은 높은 약리작용에도 불구하고 특수한 배양 조건 및 낮은 번식율로 인해 홍경천 활성 성분의 대량생산이 불가능한 실정이다. 따라서 본 연구에서는 환경에 구애받지 않고 대량생산 및 품질유지가 가능한 식물조직배양 공정을 통해 홍경천 조직으로부터 유도된 callus를 대상으로 초음파 추출공정을 적용하여 활성성분의 용출을 극대화 및 독성저감을 가능하게 하고 callus에 대한 항암활성을 탐색하여 홍경천 callus의 기능성 식품 소재로서 가치를 발견하고자 하였다. 인간 정상 세포인 HEK293과 HEL299에 대한 세포독성을 측정한 결과, 홍경천과 callus의 독성이 유의적인 차이를 나타내지 않았으며, 홍경천 callus 초음파 추출물이 전체적으로 낮은 독성을 나타내는 것으로 미루어 보아 초음파 공정을 통한 세포독성 저감효과를 확인할 수 있었으며, 조직배양 공정을 통해 홍경천의 독성 저감이 이루어지지 않은 것을 미루어 볼 때 callus가 홍경천과 같거나 비슷한 정도의 활성 성분을 함유하고 있을 것이라 사료된다. 홍경천 및 callus의 소화기, 호흡기 계통의 암세포 생육억제 활성 및 selectivity를 측정한 결과, 특히 간암 세포(HEP3B)에 대해 홍경천 및 callus 추출물의 고농도 첨가 시 암세포 저해 활성이 급격히 증가하는 것을 확인할 수 있었으며, callus 초음파 추출물은 최고농도 1.0 mg/mL에서 73.19%의 암세포 생육저해 활성과 최고 5.94의 selectivity를 나타내었다. 전체적인 항암활성 측정 결과, callus 추출물에서 높은 항암활성을 나타냈으며 특히 위암 및 간암에 대한 탁월한 항암효과가 확인됨에 따라 앞으로 홍경천 및 홍경천 callus의 소화기계 암 또는 특정 암에 대한 항암활성 연구가 필요할 것으로 생각된다. 암의 예방 및 억제에 중요한 기전으로 작용할 수 있는 항산화 활성 평가를 위해 DPPH radical 소거능을 측정한 결과, 초음파 공정을 통한 callus 추출물에서 홍경천의 높은 항산화 작용을 확인할 수 있었다. 또한 인간 면역세포 생존율의 측정을 통해 홍경천 및 callus 추출물에 의해 면역세포가 자극을 받는 것을 확인할 수 있었으며 대식세포의 NO-생성량이 증가하는 것을 확인할 수 있었다. 이러한 결과를 통해 면역계 세포들의 면역체계의 항상성 유지 및 antibody와 cytokine과 같은 면역물질 생성을 통한 암세포 억제 및 제거작용이 가능할 것이라 사료된다. 홍경천 조직으로부터 유도된 callus에 초음파 추출 공정을 적용할 시 세포독성 저감과 활성 성분 용출량의 극대화에 기인한 항암 및 암 발생과 억제 기작에 영향을 주는 항산화 및 면역증강 활성의 증가를 확인함에 따라 홍경천 callus가 기능성 항암 소재로서의 가치가 매우 높다고 사료된다. 하지만 이와 같은 홍경천 callus의 항암 소재로서 활용을 위해서는 활성성분의 용출을 극대화 할 수 있는 추출 공정의 확립과 홍경천 본래의 독성은 줄이고 암세포에 선택적인 활성을 나타낼 수 있는 활성 성분의 규명 및 정상세포 독성을 배제할 수 있는 추출물 농도의 최적화에 대한 연구가 이루어져야 하겠다. In this study, the anticancer activity of the water extract at 100oC was compared to that of the callus extracts via a ultrasonification extraction process. All the extracts were utilized to evaluate cytotoxicity, antioxidant and immune activities. The callus extracted via ultrasonification extraction showed relatively low cytotoxicity on normal human cell lines, HEK293 and HEL299, showing 13.17% and 21.78%, respectively. The callus extract has 59.82% which was similar to 61.70% for water extracts. It was also found that callus extract yielded higher nitric oxide secretion form macrophage than other extracts. The growths of both human stomach adenocarcinoma (AGS) cell and human lung carcinoma (A549) were inhibited up to 70% by adding 1.0 mg/mL of the callus extracts with ultrasonification extraction. This inhibition ratio (70%) was almost close to that of water extract. Human hepatoma carcinoma (HEP3B) cell growth was most significantly inhibited up to 75% by adding 1.0 mg/mL of callus extracts, and its selectivity was highest compared to other extracts. It indicates that the callus extracts could selectively inhibit growth of digestive system-related cancer cells. It can be also concluded from the results of this study that the callus extracts associated with ultrasonification extraction process have the potential for anticancer activity.

고추(Capsicum annuum) callis와 검은 무늬병 유발균의 elicitor를 이용한 항균물질 생산

박지은,구부연,박재영,권혜인,문슬아,조은별,이인순,박흠대 대구대학교 산업기술연구소 2014 産業技術硏究 Vol.25 No.1

We described the effects of antimicrobial activity on a produced material by the co-culture of elicitor inducing black rot's disease and callus of Capsicum annuum that callus was induced in MS medium containing IAA 0.5 mg/ml and kinetin 2.0 mg/ml. the bacterial elicitor used 0.5% Yeast extract and the fungal elicitor made by Alternaria alternata KACC 40019 with PDB medium. In order to produce antimicrobial agent protective balck rot's disease, we co-cultured the fungal elicitor or bacterial elicitor with the red pepper callus tissue for 7days repectively. The result, an antifungal agent was produced extracellular materials into medium during co-culture red pepper callus with the fungal elicitor. The antifungal activity of the extracellular material was showed 23% inhibition rate on Alternaria alternata KACC 40019 on the other hand the intracellular material was showed 2% inhibition rate. A chitinase activity of extracellular material was also showed higher value than a chitinase of the intracellular material. The extracellular material produced by elicitor was had more 15% of andtibacterial activity than the intracellular material. As a result, a biotic stress as a fungal elicitor and a bacterial elicitor brings a defence system in the red pepper callus and induce to synthesis in extracellular material an antimicrobial agent as a chitinase and phytoalexin.

Esterase isozyme patterns in developing plant regeneration from calli of Citrus junos Sieb.

Hyun-Hwa Lee,Sook-Young Lee,Min-Hee Park,Hyun-Kyu Jang,Hong-Sub Kim 한국자원식물학회 1999 Plant Resources Vol.2 No.1

The callus from the hypocotyl region of immature embryo of Citrus junos Sieb. was efficiently induced in the } MS medium containing 454M BA after 8 weeks culture. The callus was developed into the two callus type, embryogenic callus and nonembryogenic callus, which can be distinguished by visual examination depending on color and appearence. In vitro regeneration of callus established efficiently in the hormone-free MS medium from the embryogenic callus. In order to investigate the physiological changes depending on the developmental stage of embryo, the embryo was formed in the MS medium. The embryogenic and nonembryogenic callus, and the various stages of the somatic embryo were examimed the changes of esterase activity, and their isozyme patterns as well. The protein content and esterase activities was gradually increased on the developmental stages of embryo. Total protein pattern were different by the SDS-PAGE and were appeared strong band of 23 KD in the torpedo stage. The pattern of the esterase isozyme was exhibited a difference between embryogenic callus and nonembryogenic callus. It was appered pl 6.0, 8.0, 8.2 in the embryogenic callus. Also the new band of pl 4.75 was appeared in the cotyledon. These results suggest that the changes of esterase activities and isozyme patterns are importent factor in the differentiation and development of citrus.

백합 'Gelia' 캘러스로부터 자구 재분화에 미치는 제요인

박소영,김시동,신세균,이철희,백기엽 한국식물생명공학회 1997 식물생명공학회지 Vol.24 No.3

본 시험은 L. longiflorum 'Gelia'의 캘러스 유지 및 증식, 캘러스 선발, 액체현탁배양 등의 단계로 수행하였다. 재분화를 억제시키면서 캘러스를 유지 증식시키기 위하여 MSH배지에 2.4-D 0.5 mg/L , NAA 1.0 mg/L, BA 0.3 mg/L를 첨가한 배지가 가장 효과적이었으며 재분화를 억제시키기 위해 2,4-D의 첨가는 필수적이었다. 당은 30 g/L 첨가가 캘러스 생육에 가장 적합하였고 50 g/L 이상 고농도는 캘러스 생육에 억제적이었다. 또한 0.42%의 한천을 첨가한 반고형배지에서 캘러스의 생육이 증진되었다. 4~5회 계대배양된 캘러스에서 유사배발생 캘러스(ELC)가 관찰되었다. ELC의 증식과 캘러스의 유연성을 증진시키기 위해 $\textrm{NO}_{3^-}$ 와 $\textrm{NH}_{4^+}$의 비율을 달리하여 배양한 결과, 전반적으로 NO$_3$-의 함량이 높은 배지에서 배양된 캘러스는 생육과 유연성이 양호하였다. 그러나 체세포배발생 가능성이 있는 캘러스의 증식에는 효과적이지 못했다. 액체배양은 MSH배지에 NAA 1.0 mg/L, BA 0.3 mg/L, 16.7% conditioned배지(30 mL당 1 mL), casein hydrolysate 2.0g/L를 첨가한 액체배지에 배지 30 mL당 1.5 g의 캘러스를 배양했을때 가장 캘러스 증식효율이 높았다. 광학현미경으로 조직을 관찰한 결과 1년 이상 장기배양된 캘러스에서 기관분화가 가능했고 배발생 초기단계의 세포도 관찰되어 백합 'Gelia' 캘러스로부터 체세포배 형성이 가능함을 알 수 있었다. Callus from scale segments of Lilium longiflorum 'Gelia' was effectively induced and maintained from unorganized tissue on the semi-solid medium by 0.42% Bacto agar with MS basal salts and vitamins of SH medium supplemented with 0.5 mg/L 2, 4-D, 1.0 mg/L NAA, 0.3 mg/L BA, and 3% sucrose. More than 5% of high sucrose level had inhibiting effect on regeneration capacity of formed callus and decreased callus growth. Various combinations of nitrogen did not effective to proliferate the ELC (Embryogenic-like callus), but friability of callus was increased in the medium containing only nitrate as nitrogen source. 5 mL conditioned medium into 30 mL fresh medium was good for cell growth. However friable cell aggregates during suspension culture had to form hard callus which hindered to establish suspention culture system. Addition of 2 g/L casein hydrolysate increased callus growth and friability of the hard callus. As a result of anatomical observation of callus, organogenesis such as shoots, roots and bulblets was independently induced from callus tissue. Somatic embryogenesis from callus tissue could be observed with low frequency.

형질전환된 토마토 캘러스의 Superoxide Dismutase와 Peroxidase 활성

유정민,정형진,김경민,곽상수 한국식물생명공학회 1998 식물생명공학회지 Vol.25 No.3

This study was carried out to investigate activity difference in the superoxide dismutase (SOD) and peroxidase (POD) of tomato callus transformed with Agrobacterium containing the GUS gene. Than those of other two tomato cultivars, the hypocotyl explant of JA101 was shown to have higher POD and SOD specific activity of 23 unit/mg protein and 2,156 unit/mg protein, respectively. Relatively high frequency of callus formation was obtained from the hypocotyl explant on MS medium containing 1 mg/L 2,4-D for 30 days and its POD(47 unit/mg protein) and SOD (95,786 unit/mg protein) specific activities were higher than other 2,4-D concentration. The hypocotyl explant and callus cocultivated with Agrobacterium for 72 hours were transferred to MS medium supplemented with 1 mg/L 2,4-D, 30 mg/L kanamycin, 30 g/L sucrose and 4 g/L Gelrite. The hypocotyl explants transferred to the medium formed callus with 45.5% effeciency after 8 weeks. The transformation efficiency confirmed by GUS assay was 21.6%. POD specific activity of the transformed callus (54 unit/mg protein) were somewhat lower than the non-transformed callus (64 unit/mgg protein) and SOD specific activity of the transformed callus (30,300 unit/mg protein) were also lower than the non-transformed callus (37,077 unit/mg protein). However there was no significant difference in POD and SOD isozyme patterns between the transformed and the non-transformed calluses. From these results, it revealed that there was no difference of antioxidant enzyme activities between the transformed callus and the non-transformed callus in tomato. 토마토에서 GUS 유전자를 가진 Agrobacterium을 이용하여 형질전환된 캘러스와 형질전환 되지 않은 캘러스의 peroxidase(POD)와 superoxide dismutase(SOD) 활성 차이를 조사하여 형질전환된 세포의 안전성평가의 기초자료로 삼고자 얻어진 결과는 다음과 같다. JA101 종자로부터 형성된 hypocotyl의 POD, SOD 비활성은 각각 23 unit/mg protein와 2,156 unit/mg protein로 공시품종 중 가장 높은 활성을 나타내었다. Hypocotyl 절편체로부터 유도한 캘러스 생장은 1mg/L 2,4-D의 농도에서 가장 좋았으며, 1 mg/L 2,4-D가 함유된 배지에서 유도된 캘러스에서 POD 와 SOD 비활성은 각각 47 unit/mg protein 와 95,786 unit/mg protein로 높게 나타났다. Agrobacterium과 hypocotyl 절편체와의 공동배양으로부터 형성된 캘러스중 21.6%의 형질전환된 캘러스를 얻을수 있었으며, 형질전환된 캘러스의 POD 비활성은 54 unit/mg protein로 형질전환 되지 않은 캘러스의 64 unit/mg protein보다 낮게 나타났고, SOD 비활성은 형질전환된 캘러스가 30,300 unit/mg protein로 형질전환되지 않은 캘런스의 37,077 unit/mg protein 보다 낮게 나타났다. 형질전환된 캘러스와 형질전환되지 않은 캘러스간의 isozyme pattern 차이는 인정할 수 없었다.

성숙종자 유래 캘러스를 이용한 들잔디 (Zoysia japonica Steud.)의 효과적인 식물체 재분화

배창휴,이성춘,임용표,김호일,송필순,이효연 한국식물생명공학회 2001 식물생명공학회지 Vol.28 No.2

들잔디 (Zoysia japonica Steud.)의 성숙종자 유래의 캘러스를 사용하여 캘러스 생장 및 재분화에 적합한 몇 가지 조건과 캘러스 유형에 따른 재분화 효율을 검토하였다. 종자로부터 캘러스 유도는 2 mg/L 2,4-D, 0.2 mg/L BAP, 4 mg/L thiamine-HCl, 100 mg/L $\alpha$-ketoglutaric acid를 포함한 MS 배지에서 가장 좋았다. 캘러스 증식은 0.5 mg/L 2,4-D, 0.05mg/L BAP, 4 mg/L thiamine-HCl, 100 mg/L $\alpha$-ketoglutaric acid를 포함한 MS 배지가 가장 좋았다. 식물체의 재분화는 3% maltose, 1 mg/L BAP 또는 1 mg/L TDZ를 포함한 MS배지에서 좋았다. 캘러스의 Type과 빈도는 2,4-D와 BAP 조합과 농도에 따라 크게 영향을 받았고, 형태적으로 4가지 Type이 뚜렷하게 나타났다. Type I, II, III 캘러스는 계대배양으로 신초가 형성된 반면, 물기가 많은 캘러스인 Type IV에서는 뿌리만이 분화되었다. 이 중 Type I 캘러스는 신초의 재분화율이 82%로 가장 높고 albino체의 출현빈도가 4%로 가장 낮았다. 이와같이 캘러스를 유형별로 구분하여 배양함으로써 잔디 캘러스로부터 식물체의 재분화를 높일 수 있었다. Using mature seed-derived callus, optimal conditions for efficient callus growth and plant regeneration, and regeneration efficiency by callus type were investigated in zoysiagrass (Zoysia japonica steud.). Callus induction was highest when the seeds were cultured on MS medium containing 2 mg/L 2,4-D, 0.2 mg/L BAP, 4 mg/L thiamine-HCl and 100 mg/L $\alpha$-ketoglutaric acid. Callus growth was highest when callus were cultured on MS medium containing 0.5 mg/L 2,4-D, 0.05 mg/L BAP, 4 mg/L thiamine-HCl and 100 mg/L $\alpha$-ketoglutaric acid. Plant regeneration was highest when callus was transferred on MS medium containing 3% maltose and 1 mg/L BAP, or 1 mg/L thidiazuron (TDZ). The combinations and concentrations of 2,4-D and BAP were shown to be critical factors for both the frequency and the type of callus. And four morphologically distinct types of callus were induced from the 2,4-D and BAP treatment. Type I,II and III calli produced shoots upon subculture, while the watery callus, type IV produced roots without shoots. Of four types of callus, type I exhibited the maximum frequency (82%) of shoot regeneration and the minimum frequency (4%) of albinism.

Organogenesis from Callus Derived from In Vitro Root Tissues of Wild Prunus yedoensis Matsumura

Eun Ju Cheong 강원대학교 산림과학연구소 2019 Journal of Forest Science Vol.35 No.1

In vitro organogenesis system of the valuable ornamental species, Prunus yedoensis which is native to Korea, was established through callus culture derived from root tissues. Callus were induced on the medium supplemented with 2,4-D and BA or NAA and kinetin. Organogenesis was differ from the callus type, and NAA and kinetin combination was effective to induce organogenic callus. Growth of callus was influenced by sucrose concentrations. High level of sucrose (over 5%) had adverse effects such as decreased fresh weight and increased mortality of callus. Shoots developed from the callus when GA3 was treated with BA in the medium. Results showed that GA3 is essential for shoot development and elongation from callus in this species.