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강호덕,강상구,배한홍,박교수,Kang, Hoduck,Kang, Sang-Gu,Bae, Hanhong,Park, Kyo-Soo,Hall, Richard B. Korean Society of Forest Science 1997 한국산림과학회지 Vol.86 No.3
미류나무의 미성숙 ovule과 줄기로부터 유기된 캘러스에 plasmid pBI221 유전자를 유전자총을 이용하여 인위적으로 삽입하였다. Plasmid pBI221은 CaMV-35S 유전자에 의하여 발현되는 ${\beta}$-glucuronidase(GUS) reporter 유전자를 포함하고 있다. Plasmid pBI221이 물리적으로 삽입된 후 GUS 유전자의 발현정도는 5-bromo-4-chloro-3-indolyl-${\beta}$-gluconide(X-glue)의 반응에 의해 분석되었고, 유전자의 일시적 발현현상은 ovule, 캘러스 시료에 X-glue substrate의 반응에 의하여 나타나는 뚜렷한 점(spot)의 수에 따라서 조사하였다. 본 실험에서 particle bombardment후 GUS유전자의 발현검정에 있어 가장 중요한 요인은 bombardment 횟수와 X-glue substrate에 노출된 시간이었다. X-glue substrate와의 반응결과, 캘러스와 ovule에서 각각 56.8%, 75.9%의 반응 점들을 나타냈다. 여러 처리중 두번의 연속적인 shot와 bombardment 이후, X-glue과 sample을 48시간 반응시킨 후 24시간 동안 alcohol로의 침지가 가장 많은 수의 점을 유기시켰고, 이들 반응으로부터 평균 $25.75{\pm}2.77$(ovule), $11.43{\pm}1.22$(calli)개의 반응 점을 보였다. 유전자총에 의한 외래 유전자 도입에 관한 본 연구는 두종류의 시료로부터 빠른 시간 내에 유전자 발현을 볼 수 있을 뿐만 아니라, 지금까지 Agrobacterium을 이용한 형질전환이 보고되지 않은 미류나무(Populus deltoides)의 형질전환 연구에 대체 방법을 제공하리라 생각된다. Excised immature ovules and calli derived from the stems of cottonwood were bombarded with microprojectiles carrying plasmid DNA containing CaMV-35S promoter and ${\beta}$-glucuronidase(GUS) gene. After bombarded, the expression of GUS gene was detected by the assay of 5-bromo-4-chloro-3-indolyl-${\beta}$-gluconide(X-gluc). Transient gene expression was measured by counting the number of distinct regions of GUS activity per explant. As major parameters, the number of shots and the period of exposure to X-gluc after the bombardment were investigated for detecting GUS gene expression. In this experiment, the percents of GUS gene expression showing spots were 56.8 from immature ovules and 75.9 from micro-calli of cottonwood species. Among the treatments, two consecutive shots and 48 hour exposure produced about $25.75{\pm}2.77$(per ovule), $11.43{\pm}1.22$(per mini petridish) spots, respectively, Microprojectile particle bombardment provides a useful method to assay transient expression in both types of explants. Furthermore, our results represent that the excised ovule and/or the calli might be stably transformed by the biolistics.
Peace 포플러(Populus koreana X P.trichocarpa)의 줄기형성에 미치는 Thidiazuron 효과
강호덕,이민순,문흥규,박인선 한국식물생명공학회 2004 JOURNAL OF PLANT BIOTECHNOLOGY Vol.31 No.1
Shoot formation was investigated from in vitro cultivation of exotic hybrid poplar (Populus koreana × P. trichocarpa) with a specific stomatal character occurring both upper and lower surface of leaves. Two different explants (stem and leaf segment) of Peace poplar were cultured on half strength Murashige and Skoog (MS) basal medium supplemented with the various concentrations of thidiazuron as a plant growth regulator. Most adventitious shoots were produced from excised ends of stem or mid-veins of leaf segments. The highest average numbers of shoots were 7.1 and 5.3 with the treatments of 0.02 mg/L TDZ in both explants of stem and leaf segment. The highest shooting rates were achieved to 83.3% and 47.6% with the concentrations of 0.01 mg/L and 0.02 mg/L TDZ by axillary bud and leaf cultures, respectively. 기공이 잎의 앞뒤 양면에 존재하는 생리·형태적 특징을 가지고 있는 외래 교잡종인 Peace (Populus koreana×P. trichocarpa) 포플러의 효율적인 기내증식법을 개발하고자 액아와 엽육 절편을 이용하여 기내배양을 실시하였다. 몇 종류의 배 지 시험을 통해 1/2 MS 배지를 적정배지로 선정할 수 있었고, 이 배지에 TDZ를 처리하여 multiple shoot 및 adventitious shoot 유도 효과를 조사하였다. 절간액아 및 엽육을 절편으로 TDZ의 농도별 처리로 배양 2주 후부터 multiple shoot 및 adventitious shoot가 유도되었으며 절편 및 TDZ 처리 농도에 따라 차이를 나타냈다. 액아로부터는 TDZ 처리로 절편당 5-7 개의 multiple shoot가 유도된 반면 엽육 절편에서는 2-5개의 adventitious shoot가 유도되었다. 한편 부정아로부터 정상적인 줄기의 성장에 있어서도 절편에 따른 차이를 나타냈다. 이상 의 결과는 저농도의 TDZ의 처리로 peace 포플러의 액아 혹은 엽육 절편의 기내배양을 통해 대량증식이 가능함을 보여주는 결과이다.
형질전환된 포플러의 딱정벌레에 대한 저항성 유전자 ( Proteinase Inhibitor 2 ) 발현
강호덕 ( Ho Duck Kang ) 한국산림과학회 1997 한국산림과학회지 Vol.86 No.4
The resistance of a non-transgenic poplar clone, `Ogy` and three transgenic poplar lines to the cottonwood leaf beetle, Chrysomela scripta F., was evaluated by in vitro feeding. The lines were transformed with neomycin phosphotransferase II (NPT II ) as a selectable marker, proteinase inhibitor II(pin2) as a resistance gene, and CaMV 35S as a promoter. An efficient method of sterilizing the beetle eggs and introducing them into plant tissue cultures was developed. The resistance of the transgenic lines was investigated in terms of effects tin leaf area consumed, insect weight, insect developmental stages, and plantlet root dry weight after feeding. Also, leaf area consumed was examined by leaf age as measured through leaf plastochron index(LPI). The leaf area consumed and insect weight were highly significant between transformants and control, and insect development in vitro was significant among the transgenic lines. Larval infestation was the most severe around LPI 4 to 5 which were young leaves. The system provided a quick, highly controlled method to screen developing transgenic plantlets directly.
포플러의 미성숙 배와 캘러스에서 유전자총에 의한 GUS - gene 의 일시적 발현
강호덕,강상구,배한홍,박교수,( Ho Duck Kang,Sang Gu Kang,Han Hong Bae,Kyo Soo Park,Richard B . Hall ) 한국산림과학회 1997 한국산림과학회지 Vol.86 No.3
Excised immature ovules and calli derived from the stems of cottonwood were bombarded with microprojectiles carrying plasmid DNA containing CaMV-35S promoter and β-glucuronidase(GUS) gene. After bombarded, the expression of GUS gene was detected by the assay of 5-bromo-4-chloro-3-indolyl-β-gluconide(X-gluc). Transient gene expression was measured by counting the number of distinct regions of GUS activity per explant. As major parameters, the number of shots and the period of exposure to X-gluc after the bombardment were investigated for detecting GUS gene expression. In this experiment, the percents of GUS gene expression showing spots were 56.8 from immature ovules and 75.9 from micro-calli of cottonwood species. Among the treatments, two consecutive shots and 48 hour exposure produced abet 25.75±2.77(per ovule), 11.43±1.22(per mini petridish) spots, respectively, Microprojectile particle bombardment provides a useful method to assay transient expression in both types of explants. Furthermore, our results represent that the excised ovule and/or the calli might be stably transformed by the biolistics.
강호덕,Romika Chandra 한국산림과학회 2016 Forest Science And Technology Vol.12 No.2
The present work aimed to study the physiological effects of mixed heavy metals, cadmium (Cd), copper (Cu), chromium (Cr), and zinc (Zn) in four poplar hybrids. Poplar hybrids were treated once with six different concentrations (0, 5, 50, 100, 200, and 500 ppm) of mixed heavy metals for a period of 3 months. The photosynthetic and transpiration rates were measured before and during treatment using an LCiSD portable photosynthesis system whereas the photosynthetic pigments were analytically determined. According to the results obtained, poplar hybrids exhibited a decrease in chlorophyll content and carotenoids except for hybrid 1 (Eco 28) which showed an increase at 200 and 500 ppm heavy metal concentrations. The photosynthetic rate decreased with increasing heavy metal concentrations; however, an increase was observed at the highest concentration of 500 ppm at 14.54 mmol m ¡2 s ¡1. Transpiration rates were significantly affected by increased heavy metals. Hybrid 1 (Eco 28) was deduced as the hybrid that has the ability to tolerate high levels of mixed heavy metals. Among the four poplar hybrids tested, hybrid 1 (Eco 28) was the best candidate for phytoremediative experiments under greenhouse conditions. These results are valuable in understanding how various tree hybrids respond to mixed heavy metal stress, especially when using them as bio indicators for phytoremediation experiments.