Relationship between Auxin-Induced Cell Proliferation and Somatic Embryogenesis in Culture of Carrot Cotyledons

Kim, Ee Yup,Lee, Eun Kyong,Cho, Duck-Yee,Soh, Woong-Young 한국식물학회 2000 Journal of Plant Biology Vol.43 No.3

We elucidated the relationship between cell proliferation and somatic embryogenesis in the culture of carrot cotyledons. Fresh weights of the cotyledon explants were determined every five days while being cultured on a medium containing 2,4-D. Callus production increased exponentially from Day 20 to Day 25, showing a two-fold rate of proliferation. To examine the embryogenic potential of the callus, we pre-cultured cotyledon explants on an MS medium with 2,4-D, then transferred them to an MS basal medium at five-day intervals. Somatic embryos formed most frequently when the cotyledons were pre-cultured for 20 days on an MS medium that contained 5μM 2,4-D. The frequency of somatic embryo formation was 81%, while that of normal embryos with two cotyledons was 51% among those formed on a hormone-free medium. We used FACScan analysis to relate the embryogenic potential of the callus to the S phase in the cell cycle of cultured cells. The S phase was high after 25 days of culture on the medium with 5μM 2,4-D. In contrast, the frequency of normal embryogenesis was higher at Day 20 of the pre-culture period. Culturing embryogenic calli on a medium with 5μM 2,4-D was most favorable for producing somatic embryos with two cotyledons. We verified that active somatic embryogenesis was apparently related to cell division activity; somatic embryos induced from actively dividing cells were apt to accompany cotyledonary abnormality.

Mass Propagation by Somatic Embryogenesis through In vitro Suspension Culture of Bleeding Heart (Dicentra spectabilis)

Ock-Kyeong Sim,Ee-Yup Kim,Jong-Seon Eun 한국원예학회 2006 한국원예학회 기타간행물 Vol.- No.-

금낭화(Dicentra spectabilis)의 배우체 형성 및 접합자배의 발달

심옥경(Ock-Kyeong Sim),이강섭(Kang-Seop Lee),김이엽(Ee-Yup Kim),은종선(Jong-Seon Eun) 한국자원식물학회 2005 한국자원식물학회지 Vol.18 No.2

본 연구는 금낭화 배우체의 형성과 배 발달특성에 관한 기초 자료로 활용하기 위해 수행되었다. 시원세포로부터 발달된 소포자 모세포는 화뢰의 길이가 1 mm일 때 감수분열을 하여 4면체형(tetrahedral) 4분자가 형성되었다. 4개의 소포자는 분리되어 각각 웅성배우체로 발달하였다. 대포자 모세포는 화뢰의 길이 $4{\sim}5\;mm$ 수식 이미지에서 관찰되었다. 대포자의 발달유형은 정상형(polygonum)이었으며, 배낭의 형태는 굽어있는 곡생배주(amphitropous)였다. 3개의 뚜렷한 반족세포는 배낭이 성숙한 후에도 퇴화하지 않고 남아있었다. 개화 전 자웅배우체는 충분히 성숙하였다. 개화 시에 수술과 암술의 길이는 거의 비슷하거나 수술이 0.5 mm 짧아서 자화수정에 적당한 구조를 가지는 것으로 나타냈다. 수정 후 배는 구형, 심장형을 거쳐 자엽배까지 발달하였으며, 종자 산포시 초기 자엽배를 가지고 있음을 알 수 있었다. This study was carried out to obtain the basic informations on the characteristics of gametophytes formation and embryo developement in Dicentra spectabilis. Microspore mother cells developed from archesporial cells, start meiosis when flower bud length reaches around 1 mm, formed tetrahedral type tetrad. The 4 microspores were separated. They were developed to male gametophytes, respectively. Megaspore mother cells were observed when flower bud length was 4~5mm. The developemental type of megaspore was polygonum and embryo sac was amphitropous. Three large and distinctive antipodals did not degenerated and remained after embryo sac was developed. When the male and female gametophytes was fully developed, the length of stamen and style was very similar or stamen was shorter about 0.5 mm than that of style. This result indicates that self-fertilization can be occurred in this species. After fertilization, developing zygotic embryos showed various stages of development from globular to cotyledonary embryos, and zygotic embryo in seed scattering time seemed to have an early cotyledonary stage.

Retinoic Acid가 Bromodeoxyuridine 표지 PLC / PRF / 5 간암 세포주의 역동성에 미치는 효과

안득수(Deuk Soo Ahn),김대곤(Dae Ghon Kim),송석현(Suck Hyun Song),김이엽(Ee Yup Kim),장동석(Dong Suck Jang),이수택(Soo Taeck Lee) 대한소화기학회 1993 대한소화기학회지 Vol.25 No.6

N/A Retinoie acid (RA) has been reported to show antiproliferative and differentiation-inducing effects on human tumor cells. In this report, we examined the antiproliferative effect and the influence on the cell cycle in PLC/PRF/5 hepatocelluar carcinoma cells, secreting hepatitis B surface antigen (HBs Ag). MTT assay was conducted to prove the antiproliferative effect of RA, and the univariate and the bivariate distribution of BrdU/DNA were analyzed using FACScan, to evaluate the influence of RA on tumor cell kinetics in the labelled cells with the 5-bromodeoxyuridine (BrdU). Following 6 days of exposure, 1uM and 0 1 uM RA containing nontoxic DMSO level showed antiproliferative effect on the cells. The analysis from the tumor cell kinetics showed that actual doubling time (Td) is 69 hrs, potential doubling time (Tpot) is 42 hrs, the mean DNA synthesis time (T5) is 17.4 hrs, and the labelling index is 35.2%. The changes of the tumor cell kinetics in the cells treated with RA were investigated. Early stage of culture (day 2, 4) showed increase in S phase percentage of cells and decrease in G0/G1 phase. But ]ate stage of culture (day 6) show decrease in S phase percentage of cells to minimum and increase in G0/G1 phase at either 1 uM or 0.1 uM concentration of RA as compared with that of control. The influence on cell cyclc was rnore pronounced at 1 uM than at 0.luM concentration. These results demonstrated that RA inhibits the growth of PLC/PRF/5 cells and its antiproliferative effect is suggested to be driven from arresting cell cycle progression from G0/G1 to S phase in tumor cell kinetics.

현탁배양을 통한 금낭화(Dicentra spectabilis L. Lemaire)의 대량증식

이강섭,심옥경,신정순,최용의,김이엽,Lee, Kang-Seop,Sim, Ock-Kyeong,Shin, Jeong-Sun,Choi, Yong-Eui,Kim, Ee-Yup 한국식물생명공학회 2004 식물생명공학회지 Vol.31 No.2

금낭화 (Dicentra spectabilis L.)는 개발가치가 있는 국내 자생화 중의 하나로서 우수한 품종의 대량생산이 절실히 요구되는 실정이다. 본 연구는 현탁배양을 통한 배발생세포의 대량증식과 체세포배 발생 및 식물체재생의 적정조건을 구명하여 효율적인 기내묘목의 대량생산 체계를 확립하고자 시도되었다. 1.0mg/L 2,4-D SH고체배지에서 유도된 배발생캘러스를 현탁배지에서 증식시킨 결과, 1.0mg/L 2,4-D를 포함한 SH배지에서 MS배지에 비해 증식율이 높게 나타났으며, 체세포배 발생율은 1/2배 SH기본배지에서 가장 높게 나타났다. 250$m\ell$ 삼각플라스크에서 5,000개 이상의 고빈도로 체세포배가 생산되었다. 체세포배로부터 발아유도시에 GA$_3$는 정상적인 식물체로의 재생을 억제하는 효과를 나타내었으며, 정상적인 식물체 재생은 1/2희석 SH배지에서 양호하였다, 신초와 뿌리를 갖는 유식물체를 모래와 원예용상토(5:1, vol)가 혼합된 토양이 함유된 포트에 이식하여 1개월 후 58%의 생존율을 나타내었다. 이상의 결과를 활용하면 체세포배로부터 금낭화의 묘목을 대량생산 할 수 있을 것으로 생각된다. Bleeding heart (Dicentra spectabilis L. Lemaire) is one of the most valuable wild flower in Korea. This work was conducted for the mass production of somatic embryos through suspension culture and more effective plant regeneration system in Dicentra spectabilis. High-frequency embryogenic callus proliferation was achieved in SH liquid medium supplemented with 1 mg/L 2,4-D. Half-strength SH medium was suitable concentration for somatic embryo induction and germination. About 5,000 embryos were produced per 250$m\ell$ flask after 4 weeks of culture. Germination rate of somatic embryos was decreased when GA$_3$ was added in medium. The plantlets showed a 58% survival rate when transferred to pots after 1 month of culture. The results indicate that micropropagation procedure via somatic embryogenesis can be applied for an efficient mass propagation of Dicentra spectabilis.

블랙베리(Rubus fruticosus L.)의 맹아절편체로부터 다경유도를 통한 식물체 재분화

신정순(Jeong-Sun Shin),심옥경(Ock-Kyeong Sim),이종천(Jong-Chon Lee),조한직(Han-Jik Cho),김이엽(Ee-Yup Kim),이강섭(Kang-Seop Lee) 한국자원식물학회 2005 한국자원식물학회지 Vol.18 No.3

블랙베리의 다경유도를 통한 식물체 재생을 위하여, 노지에서 생육되고 있는 맹아를 채취하여 1.2% NaOCl 용액에 침지하여 표면 살균한 후, kinetin과 BA가 각각 포함된 MS 배지에서 4주간 배양하여 절편당 형성된 신초의 수를 조사하여 BA가 효과적임을 밝혔다. 동일방식으로 맹아를 IBA와 BA를 첨가한 MS 고체배지에서 4주간 배양하여 신초를 유도하였는데 1.0 mg/L BA가 첨가된 배지에서 신초유도는 100% 이루어졌다. 기내배양한 신초의 절편체를 1.0 mg/L BA가 첨가된 MS 배지에 치상하여 형성된 신초의 수는 절편체당 5.3개로 최고를 나타내었다. 약 10주 정도 배양을 더 오래하였을 때, 다경 형성율이 더 높아질 뿐 아니라 절편체당 신초 수도 많아졌다. 형성된 신초로부터 뿌리 발생을 위하여 glycine의 농도를 달리한 (0~2.0mg/L) MS 기본배지에서 4주간 배양하였을 때, 0.5 mg/L glycine을 첨가한 배지에서 고빈도 (85%의 뿌리발생을 관찰 할 수 있었다. 재생된 식물체는 모래 : 토양 : 버뮤큘라이트 (1:1:1, vol.) 혼합토양에서 순화시켜, 토양에 이식하였을 때 95%의 식물체 생존율을 나타내었다. This study was carried out to induce plant regeneration via shoot formation from sucker explants of Rubus fruticosus L. To induce adventitious shoots, sucker explants were sterilized in 1.2% NaOCl solution, and cultured on the MS solid medium supplemented with kinetin (0.5, 1.0, 3.0 mg/L) and BA (0.5, 1.0, 3.0 mg/L), respectively. As above, to induce adventitious shoots, sucker explants were cultured on the MS solid medium supplemented with IBA (0, 0.1, 1.0 mg/L) and BA (0, 0.1, 1.0, 2.0 mg/L). After 4 weeks of culture, the highest frquency (100% of shoot formation from sucker explants was obtained from the medium with 1.0 mg/L BA. The highest shoot number per explant from in vitro shoot explants was 5.3. After 10 weeks of culture, the number of shoot per explant was increased. The highest frequency (85% of root formation was obtained at 0.5 mg/L glycine medium, when the explant with shoot were cultured on the MS medium containing glycine at various concentrations from 0 to 2.0 mg/L. The survival rate of the plantlets after transfer to plastic pots containing sand, soil, and vermiculite (1:1:1, vol.) was 95%. The results indicate that micropropagation procedure can be applied for an efficient mass propagation of Rubus fruticosus.

교잡종 블랙베리(Rubus fruticosus L. × R. parvifolius L.)의 근맹아 절편체로부터 다경유도를 통한 식물체 재분화

신정순(Jeong-Sun Shin),이종천(Jong-Chon Lee),심옥경(Ock-Kyeong Sim),윤태영(Tai-Young Yoon),조한직(Han-Jik Cho),김이엽(Ee-Yup Kim) 한국자원식물학회 2008 한국자원식물학회지 Vol.21 No.2

노지에 있는 교잡종 블랙베리의 근맹아를 채취하여 1.2% sodium hypochlorite 용액에 침지하여 표면 살균한 후 BA를 첨가한 변형된 × 1, × 1/2 MS 고체배지에서 다경유도를 시도하였다. 4주간 배양하였을 때 BA $1mg·L^-1이 첨가된 기본 MS배지에서 고빈도(83.3%)의 다경유도가 이루어 졌으며, 절편체당 신초수는 $0.5mg·L^-1 BA를 첨가한 MS 기본배지에서 3.7개로 가장 높게 형성되었다. 또한 12주간 배양하였을 때 4주간 한 경우에 비하여 신초형성율이 4.1배 많은 15.4개가 형성됨을 관찰할 수 있었다. 형성된 신초로부터 뿌리발생은 식물생장조절물질이 첨가되지 않은 MS배지를 1/1, 1/2, 1/4배로 희석하여 신초를 배양하였을 때 특히 1/2 MS 배지에서 95%로 가장 양호하였다. 재생된 식물체는 모래 : 원예용상토 : 버미큘라이트(1:1:1, vol.)혼합토양에서 순화하였을 경우 95%의 생존율을 나타내었다. This study was carried out to induce plant regeneration via multiple shoot formation from sucker explants of Rubus fruticosus L. × R. parvifolius L. To induce adventitious shoots, the sucker explants were sterilized in 1.2% sodium hypochlorite (NaOCl) solution, and then were cultured on the full and 1/2 MS solid medium supplemented with BA (0.1, 0.5, 1.0, $2.0mg·L^-1). After 4 weeks of culture, the highest frequency (83.3%) of shoot formation from sucker explants was obtained from the full MS medium with $1.0mg·L^-1 BA. The highest shoot number (3.7) per explant was obtained from the full MS medium with $0.5mg·L^-1 BA. After 12 weeks of culture, the number of shoots (15.4) per explant was increased. The highest frequency (95%) of root formation was obtained from the 1/2 MS medium, when the explant with shoot were cultured on the full, 1/2 and 1/4 MS medium. The survival rate of the plantlets after transfer to plastic pots containing sand, soil, and vermiculite (1.1:1, vol.) was 95%. The results indicate that multiple shoot procedure can be applied for an efficient mass propagation of Rubus fruticosus L. × R. parvifolius L.

Genetic diversity and variation analysis of mutant lines derived from γ-ray and chemical mutagen treatments in blackberry (<i>Rubus fruticosus</i>)

Ryu, Jaihyunk,Ha, Bo-Keun,Kim, Dong Sub,Kim, Jin-Baek,Kim, Sang Hoon,Ahn, Joon-Woo,Jeong, Il Yun,Jo, Han-Jik,Kim, Ee-Yup,Kang, Si-Yong Cambridge University Press 2014 Plant genetic resources Vol.12 No.1

<P>Blackberry is a fruiting berry species with very high nutrient contents. With the recent increasing consumer demand for blackberries, new sources of germplasm and breeding techniques are required to improve blackberry production. This study was carried out to evaluate the genetic diversity (GD) and relationship among 55 blackberry (<I>Rubus fruticosus</I>) mutants derived from γ-ray treatment (52 lines) and <I>N</I>-methyl-<I>N</I>′-nitrosourea (MNU) treatment (three lines) using an inter-simple sequence repeat marker. A total of 18 bands were amplified with an average of 3.6 bands per primer. Among them, eight bands were identified to be polymorphic with a rate of 44.4%. In addition, the GD information content values were highest in the 60 Gy treatment population and the GD values were higher in the γ-ray treatment populations than in the MNU treatment population. According to a cluster analysis, all the mutant lines can be classified into five categories, and the genetic distance was greatest between the 80 Gy-irradiated population and other populations. These results indicate that mutant lines have high GD and can be effectively utilized for improving blackberry breeding.</P>

Fruit Quality and Chemical Contents of Hybrid Boysenberry (Rubus ursinus) Lines Developed by Hybridization and Gamma Irradiation

( Jaihyunk Ryu ),( Soon-jae Kwon ),( Yeong Deuk Jo ),( Hong-il Choi ),( Kyung-yun Kang ),( Bo Mi Nam ),( Dong-gun Kim ),( Chang-hyun Jin ),( Jin-baek Kim ),( Ee-yup Kim ),( Seung Cheol Oh ),( Bo-keun 한국육종학회 2017 Plant Breeding and Biotechnology Vol.5 No.3

The Rubus fruit is an economically important berry crop that contains various functional compounds. The objective of this study was to analyze fruit qualities (i.e., pH, soluble solids content, titratable acidity, and mineral content) as well as fatty acid and phenolic compounds (i.e., ellagic acid and anthocyanins) among hybrid boysenberry lines developed by hybridization and gamma irradiation. There were no significant differences in the hybrid boysenberry fruit pH and titratable acidity (%) among the tested genotypes. However, the soluble solids content was higher in the BSA119 and BSA144 mutants than in the original genotype (BS_Hybrid). Meanwhile, linoleic acid was the most abundant fatty acid in the analyzed hybrid boysenberry fruits. The fatty acid composition did not differ significantly among the genotypes. The ellagic acid content of all genotypes ranged from 8.72 mg/100 g to 46.10 mg/100 g, with the highest concentration observed for the BSB127 genotype. Additionally, cyanidin-3-O-sophoroside (M-H⁺, 611 m/z) and cyanidin-3-O-glucoside (M-H⁺, 449 m/z) were the two major anthocyanins detected in the boysenberry and mutant genotypes, while cyanidin-3-O-glucoside was the predominant blackberry anthocyanin. The total anthocyanin concentrations of four mutant genotypes (i.e., BSA036, BSA078, BSA101, and BSB127) were significantly higher than that of the original genotype (382.0 mg/100 g). The highest total anthocyanin concentration was observed for the BSA078 genotype (467.9 mg/100 g). These results may be useful for identifying the optimal genotypes for breeding new cultivars with enhanced qualities and potential health benefits.

Genetic Diversity and Relationship Assessment based on AFLP Analysis in Blackberry (Rubus fructicosus L.) Mutant Lines

( Jai Hyunk Ryu ),( Bo Keun Ha ),( Dong Sub Kim ),( Jin Baek Kim ),( Sang Hoon Kim ),( Joon Woo Ahn ),( Il Yun Jeong ),( Han Jik Jo ),( Ee Yup Kim ),( Si Yong Kang ) 한국육종학회 2014 Plant Breeding and Biotechnology Vol.2 No.4

This study was carried out to evaluate the genetic diversity and relationships among fifty-six blackberry (Rubus fructicosus) mutants derived from gamma-ray treatment (fifty-two lines) by analysis of Amplified fragment length polymorphism (AFLP) markers. Both cluster analysis and principal coordinate analysis (PCOORDA) were conducted in order to study the genetic diversity, using both morphological traits and AFLP makers. A total of 589 bands were amplified with an average of 58.9 bands per primer. Among them, 560 were identified to be polymorphic, with a rate of 95.08%. A showed a highly significant (P≤0.01) positive correlation with GD and PIC (r2=0.999). MI also showed a significant (P≤0.05) positive correlation with GD and PIC. According to the clustering analysis, all mutant lines could be classified into five categories, but the three gamma-ray treatment lines and the cross-bred line were not clustered into any groups. For the morphological traits, cluster analysis divided the blackberry germplasm into six clusters and two independent groups. In addition, the morphological dendrogram indicated an unclear pattern of division among the groups based on AFLP analysis. The findings of this study indicate that mutant lines have high genetic diversity, and can be effectively utilized as materials for the improvement of breeding.