Ectopic Expression of an Arabidopsis H+/Ca2+ Antiporter Gene in Bottle Gourd (Lagenaria siceraria)

한증술,김창길,우종규,Dae-Geun Oh,Jin-Hwa Kim 한국원예학회 2005 Horticulture, Environment, and Biotechnology Vol.46 No.4

An Arabidopsis thaliana H+/cation antiporter gene (CAX2-B) was introduced into bottle gourd (Lagenaria siceraria) by Agrobacterium-meduated transformation. Transgenic plants were obtained at frequencies of 0.8% under the selection pressure of 100 mgL-1 kanamycin monosulfate, in which integration and transcription of the transgenes were confirmed by polymerase chain reaction (PCR), Southern blot, and reverse transcriptional PCR analyses. The roots of T1 plants expressing CAX2-B grown hydroponically contained 8.7% more Ca than control plants. The Mn contents were not significantly different. In addition to CAX2-B expression, nptII evenly expressed kanamycin resistance in the T1 plants. To test Ca-related tolerance to salinity, we are currently investigating the phenotypes of T3 bottle gourd lines and grafted-watermelon onto the lines that suffered from extreme salinity. To our knowledge, this is the first study on the transgenic plant containing one of the Arabidopsis-CAX2 mutant genes.

갯봄맞이(Glaux maritima L.)의 캘루스 배양을 통한 식물체 재분화

한증술 한국식물생명공학회 2017 JOURNAL OF PLANT BIOTECHNOLOGY Vol.44 No.2

A callus-mediated regeneration protocol for sea-milkwort, an endangered coastal plant species in South Korea, is reported here. The explants of in vitro-plantlets generated from a node culture revealed distinguishable responses in callus induction depending on genotype, explant source, light condition, and 2,4-D concentration. Especially, continuous darkness exclusively facilitated callus induction from explants prior to other treatments. The calli initiated on the media with 2,4-D ranging from 0.1 mg/L to 3.0 mg/L in the dark vigorously proliferated when subcultured on the same media in continuous darkness. Given 1.0 mg/L zeatin in addition to darkness to the calli of the ‘Pistachio’ genotype, normal adventitious shoots were only regenerated from nodular structures that formed earlier from the calli at the frequency of 24.4 percent. Regenerated shoots easily grew into plantlets with roots and green color on a phytohormone-free MS medium under lighted condition, that were used for node culture as plant materials. Node culture effectively multiplied plantlets in accordance with protocol by Bae et al. (2016). Acclimatized plantlet clusters developed mature plant clusters under inland environment, followed by flowering the following April. Results were merged with node culture protocol suggested by Bae et al. (2016), which, as an in vitro propagation system for sea-milkwort, may contribute to natural habitat restoration.

갯봄맞이(Glaux maritima L.)의 캘루스 배양을 통한 식물체 재분화

한증술,Han, Jeung-Sul 한국식물생명공학회 2017 식물생명공학회지 Vol.44 No.2

우리나라에서 멸종위기 해안식물로 지정된 갯봄맞이의 캘루스배양을 통한 재분화 프로토콜을 보고한다. 마디배양을 통해 육성한 기내 유식물체의 절편은 유전자형, 절편의 종류, 광조건 및 2,4-D 첨가농도에 따라 캘루스 형성에 상이한 반응을 보였다. 특히, 다른 처리와는 무관하게 연속 암배양이 절편으로부터 캘루스 유도에 결정적인 영향을 미쳤다. 2,4-D 0.1-3.0mg/L 농도 범위의 배지에서 유도된 캘루스를 동일한 조성의 배지로 이식하여 암배양했을 때왕성하게 캘루스가 증식하였다. 연속 암조건에서 zeatin 1.0mg/L 첨가배지에 'Pistachio' 유전자형의 캘루스를 배양했을 때만 유일하게 정상적인 신초가 재분화 되었고, 이 때 신초는 캘루스로부터 형성된 마디구조에서 발달하였으며 신초의 재분화율은 24.4%였다. 재분화 신초를 식물호르몬 무첨가 배지로 이식하여 명배양 했을 때 지상부 녹화와 더불어 뿌리가 발생하여 유식물체로 발달하였다. 기내 유식물체를 기 보고된 마디배양 프로토콜(Bae et al. 2016)에 따라 배양했을 때 효과적으로 유식물체를 증식시킬 수 있었다. 기외 순화시킨 유식물체는 내륙 자연 환경하에서 성숙식물체로 생육하였으며 이듬 해 4월에 개화하였다. 본 연구의 결과는 Bae et al. (2016)이 제안한 마디배양 프로토콜과 통합하여 운용될 수 있고, 통합된 프로토콜은 갯봄맞이의 기내 번식 시스템으로서 갯봄맞이 자생지 복원에 기여할 수 있을 것이다. A callus-mediated regeneration protocol for sea-milkwort, an endangered coastal plant species in South Korea, is reported here. The explants of in vitro-plantlets generated from a node culture revealed distinguishable responses in callus induction depending on genotype, explant source, light condition, and 2,4-D concentration. Especially, continuous darkness exclusively facilitated callus induction from explants prior to other treatments. The calli initiated on the media with 2,4-D ranging from 0.1 mg/L to 3.0 mg/L in the dark vigorously proliferated when subcultured on the same media in continuous darkness. Given 1.0 mg/L zeatin in addition to darkness to the calli of the 'Pistachio' genotype, normal adventitious shoots were only regenerated from nodular structures that formed earlier from the calli at the frequency of 24.4 percent. Regenerated shoots easily grew into plantlets with roots and green color on a phytohormone-free MS medium under lighted condition, that were used for node culture as plant materials. Node culture effectively multiplied plantlets in accordance with protocol by Bae et al. (2016). Acclimatized plantlet clusters developed mature plant clusters under inland environment, followed by flowering the following April. Results were merged with node culture protocol suggested by Bae et al. (2016), which, as an in vitro propagation system for sea-milkwort, may contribute to natural habitat restoration.

토마토에서 칼슘수송체와 칼슘결합단백질 공동발현에 의한 칼슘결핍유사증상의 완화

한증술,강호주,김창길 한국식물생명공학회 2010 식물생명공학회지 Vol.37 No.4

본 연구는 칼슘 수송체의 하나인 sCAX1과 칼슘결합단백질의 하나인 CRT가 토마토에서 발현했을 때 tip-burn과배꼽썩음병 (BER)이 토마토의 전 생육기간에 걸쳐 어떠한 양상으로 나타나는지를 관찰하는 것에 초점을 맞추어수행하였고, 최종 연구 결과는 CRT 공동발현이 sCAX1 발현에 의해 야기된 병징을 억제시킬 수 있음을 확인시켰다. 양분공급이 불량한 환경하에 놓인 sCAX1 단독 발현토마토 식물체는 그 근단 조직, 정단 조직 및 과실의 암술부착부위 조직에 토마토 재배 농가포장에서 빈번하게관찰되는 칼슘결핍 증상과 일치하는 괴사현상을 나타내었다. sCAX1 형질전환체와 비형질전화체를 각각 대목과접수로 달리 사용한 교호접목 실험을 통해 sCAX1 발현에따른 tip-burn 병징이 토양으로부터의 칼슘이온 흡수에 장애를 받아 생긴 결과가 아님을 확인하였다. 형질전환을통해 CRT 발현 토마토를 획득한 후 CRT가 sCAX1에 의한불량한 특성 발현을 제어할 수 있는지 확인하기 위하여양자간 교배조합을 작성하였다. sCAX1과 CRT를 토마토에서 공동발현 시켰을 때 완벽하지는 않지만 양극 조직의 괴사와 BER이 뚜렷하게 감소되었다. 본 연구의 결과를 바탕으로 sCAX1 발현에 의해 방해 받은 세포 내 칼슘이온 관련 어떤 기구는 CRT 공동발현을 통해 복구될 수있다는 하나의 모델을 제시할 수 있을 것이다. Here we focused on tip-burn and blossom-end rot (BER) symptoms in the tomato plants expressing the constitutively active form of Ca2+/H+ antiporter (sCAX1) and/or a Ca-binding protein (calreticulin, CRT) genes during their whole growth period. Conclusively we demonstrated that CRT is able to suppress the tip-burn and BER symptoms that were induced by sCAX1. Under poor nutrition condition, tomato plants overexpressing sCAX1 showed severe necrotic collapses in both roots and shoot polar tissues, which are in accordance with Ca2+ deficient symptoms frequently observed in an agricultural cultivation of tomato. Reciprocal grafting trials using sCAX1 and wild type plants revealed that the tip-burn symptom by sCAX1 overexpression is not caused by hindrance of Ca2+ uptake from soil. We constructed CRT overexpressing transgenic tomatoes, and crossed them with sCAX1 transgenic plants to investigate the effects of CRT on the symptoms of sCAX1 transgenic plants. Coexpression of sCAX1 and CRT significantly suppressed the Ca2+ deficient symptoms of sCAX1 transgenic plants. Those results suggest the model that Ca2+ homeostasis disturbed by the overexpression of sCAX1 may be suppressed by the co-expression of CRT.

대목용 박에서 애기장대 유래 H⁺/Ca²⁺ 수송체 유전자(CAX2-B)의 발현

한증술,오대근,우종규,김진화,김창길 한국원예학회 2006 한국원예학회 학술발표요지 Vol.2006 No.5

옥수수 calreticulin 과발현 토마토에서 tobamovirus의 상엽 이동 억제

한증술 한국식물생명공학회 2010 식물생명공학회지 Vol.37 No.4

To ascertain the effect of over-expressed maize calreticulin in tomato plant on tobamovirus movement in addition to validating potentiality of the gene (ZmCRT) as a means for the virus-resistance resource, four ZmCRT-expressing homozygous lines were generated from the T0 plants as using an Agrobacterium-mediated transformation, nucleic acid analyses, and a conventional breeding method. Of them, a line was subjected to the bioassay for tolerances to tobacco mosaic virus-U1 (TMV-U1) and tomato mosaic virus (ToMV) followed by RT-PCR and a chlorophyll fluorescence quenching analyses. Both transgenic plants transcribing ZmCRT and wild-type plants showed no symptom by 20 days after viruses inoculation, however the photosystem II quantum yield parameter measured from the upper leaves of ToMV-inoculated plants revealed that ZmCRT transgenic plants have higher photosynthetic ability than wild-type ones at that time, which indirectly implies that over-expressed ZmCRT product acts as a barrier to the cell-to-cell and/or systemic movement of ToMV. Moreover, ZmCRT transgenic plants showed remarkably longer shoot length than wild-type ones in 40 days after TMV-U1 or ToMV inoculation each, which might be resulted from higher photosynthetic ability during the phase not yet showing any external symptoms. Collectively, over-expressed ZmCRT protein in tomato plants is able to interrupt the systemic movement of infected TMV-U1 and ToMV even though not perfect.

배추 원형질체 배양에 미치는 다양한 처리의 효과와 원형질체 유래 캘러스로부터 신초 재분화

한증술,윤무경,정미혜 한국식물생명공학회 2008 식물생명공학회지 Vol.35 No.3

Here we describe a procedure for Chinese cabbage protoplast culture and effects of various treatments. Chinese cabbage protoplasts were isolated from different parts of young seedlings as using an enzyme mixture, of which yield was maximized in seven hours around after digestion. The highest rate of initial cell division followed by micro-callus formation was obtained in the medium with 1.0 mg/L 2,4-D, 0.5 mg/L NAA, and 1.0 mg/L BA when the cotyledon-derived protoplasts were cultured. Initiation of cell division and micro-callus proliferation significantly depended upon Chinese cabbage genotype under a same culture circumstances. The micro-calli developed from cotyledon tissue of Norang-Bom cultivar successfully grew toward callus colonies on the solidified medium with 0.2 mg/L zeatin and 0.1 mM spermidine. The callus colonies generated de novo shoots at the maximum frequency of 4.3% on the medium with 5.0 mg/L BA and 1.0 mg/L NAA. Our results might be helpful for further studies to enhance the regeneration efficiency in Chinese cabbage protoplast culture.

에틸렌 억제제가 박의 재분화와 신초의 배수성에 미치는 영향

한증술(Jeung Sul Han),오대근(Dae Geun Oh),목일진(Il Gin Mok) 한국원예학회 2001 한국원예학회 학술발표요지 Vol.2001 No.10

고온 스트레스에 대한 국화의 생리적 반응과 산화환원 관련 유전자의 형질전환

한증술(Jeung-Sul Han),김창길(Chang Kil Kim),배수지(Su-ji Bae),강범창(Beum-Chang Kang) 한국원예학회 2013 한국원예학회 학술발표요지 Vol.2013 No.10

박의 Agrobacterium 이용 유전자전환에 있어 aminoethoxy -vinylglycine(AVG)에 의한 접종 효율 향상

한증술(Jeung-Sul Han),오대근(Dae-Geun Oh),박효근(Hyo-Geun Park) 한국원예학회 2002 한국원예학회 학술발표요지 Vol.2002 No.3