본 연구는 국내 유통 방풍(S. divaricata, P. japonicum, G. littoralis) 한약재의 원산지 판별을 위해 수행 되었다. 이를 위 해, 형태적 특징비교 뿐만 아니라 엽록체(nrDNA-ITS2) 및 핵 DNA 바코드 유전자(cpDNA-matK, psbA-trnH, rpoB2와 rpoC1)를 이용한 염기서열 분석을 수행하였다. 방풍류 3종의 형태적 특징을 비교한 결과, 잎 모양과 거치의 형태에서 가장 큰 차이를 보인 반면, 건조약재의 경우 육안으로 구별하기에 어려움이 있었다. DNA 수준에서의 차이를 비교하기 위해 DNA 바코드 후보 유전자들을 이용한 방풍류 3종의 식물자원 에 대한 염기서열 분석을 수행한 후 판별 마커 개발 가능성이 있는 ITS2와 matK, psbA-trnH 세 primer를 선발하였다. 이 를 국내 유통 한약재에 적용한 경우 방풍은 S. divaricata와, 식방풍은 P. japonicum과, 해방풍은 G. littoralis와 동일한 것 으로 확인되었다. 중국산 방풍은 S. divaricata와 동일종으로 바르게 표기되어 유통되나, 국산 방풍은 P. japonicum과 동일 종으로 식방풍(갯기름나물)의 뿌리를 건조시켜 방풍으로 혼용 유통됨을 확인하였다. ITS2 구간의 염기서열 분석 결과, 식방 풍의 경우 두 그룹으로 나눠져 동일 종 내에 유전적 변이가 있음이 확인되었다. 따라서 본 연구결과 방풍류 3종 판별을 위 해 nrDNA-ITS2와 cpDNA-matK, psbA-trnH의 사용이 유용 할 것이며, 차후 방풍류 한약재의 판별을 위한 마커 개발 연 구의 기초 자료로서 활용될 수 있을 것으로 사료된다.

This research was conducted to identify the effect of BAP and NAA on plant regeneration from flower bud of A. macrocephala. Flower buds the plant were used as target explants for tissue culture. Plant tissues were sterilized with each 45ml of 50% ethanol and 0.8% sodium hypochlorite containing 20ul of Tween20 for each 5 minutes, respectively, and then washed 5 times with sterile distilled water. Four pieces of the sterilized explants were cultured in a petri-dish at 25±2℃ under 16hr/day light condition. LS basal medium and BAP (0.2 and 2 mg·L-1) and NAA (0.2 and 2 mg·L-1) were used for the experiment. When the target explants were cultured on the media containing both BAP and NAA, explants of the flower buds were swelled about 15~25mm long and then calli were induced from receptacles at about 20 days after planting. However, there were no significant differences between the concentrations of the phytohormones. In results, normal shoots were successfully regenerated on the media supplemented with 2 mg·L-1 BAP and 0.2 mg·L-1 NAA prior to root induction. However, under the conditions of 0.2 mg·L-1 BAP and 2 mg·L-1 NAA, only roots were induced from the calli instead of shoot regeneration.

Until now many strategies have been used to produce marker-free transgenic plants such as co-transformation with negative selectable markers, site-specific recombination system, transposable elements mediated transformation, and etc. In this research, embryogenic calli induced from japonica rices, Ilmibyeo and Dongjinbyeo, were tranformed with the vector which simultaneously constructed with cre/loxP and argE genes in T-DNA. Transformation efficiencies were comparably lower than those of our previous studies, since the constructed genome size was relatively big (>10Kb). For eliminate the transformed tissues which contained positive selectable marker gene, tunicamycin was treated at regeneration and selection stages, since cre recombinase gene is expressed under the presence of this antibiotics. The plants were selected first under 50 mg․L-1 hygromycin at 28℃ for 2 weeks after the Agrobacterium-infection at 25℃ for 7 days. And then, the regeneration plants were successfully obtained on MS basal regeneration medium containing 0.1 mg․L-1 tunicamycin. The regenerated plants are now acclimatizing in greenhouse and molecular analysis are currently accomplished with these plants.