음나무 배발생 캘러스의 증식 및 체세포배 발달을 위한 액체 현탁 배양조건 확립
Establishment of suspension culture condition for embryogenic callus proliferation and somatic embryo development of Kalopanax septemlobus
1
조회
0
다운로드
3
인용
0
참고문헌
초록
본 실험은 음나무 배발생 세포의 증식 및 체세포배 발달을 위한 액체 현탁 배양조건의 확립을 위해 수행되었다. 배발생 세포의 생 장율은 접종 밀도가 증가할수록 감소하였다. 배발생 세포의 증식 에 가장 효과적인 접종 밀도는 0.1 g/100 ml 로서 이 농도에서 세포 의 생장율이 가장 높았다. 배양기간에 따른 배발생 세포의 생장 패 턴 및 세포 주기 (G1, S, G2/M) 분석 결과, 세포의 생장은 배양 5일 후부터 증가가 시작되어 15일 까지 급격히 생장하였으며 그 이후 에는 점차 감소하였다. 배양 기간 별 세포 주기 (cell cycle) 의 변화 가 명확하게 관찰되어 배양 5일째 S기는 초기의 5.6% 에서 11.7% 로 두 배 정도 증가하였으며, 배양 15일 이후부터는 다시 초기의 세포 주기로 되돌아 가면서 안정화되는 것으로 나타났다. 따라서 음나무 배발생 세포의 현탁배양은 15일의 주기로 배양하는 것이 증식에 가장 효과적인 것으로 생각되었다. 한편 배발생 세포에서 체세포배의 유도는 배양초기의 접종 밀도가 중요한 것으로 나타났 다. 0.5 g/L 의 낮은 밀도로 접종 시에는 65% 이상의 어뢰형 배가 유도된 반면 접종 밀도가 높아질수록 어뢰형으로의 배발달은 급격 히 감소하였다. 초기의 접종 밀도가 증가할수록 특히 어뢰형 배의 발달은 지연되었으나 구형 및 심장형 배의 유도는 초기 접종밀도 에 영향을 받지 않았다. 이상의 실험결과로 음나무 액체 배양 시 초기 접종 밀도를 조절함으로써 배발생 캘러스의 증식 및 체세포 배를 효과적으로 유도할 수 있었으며 이는 체세포배 생산을 위한 배양 기간의 단축이 가능함을 보여주는 결과이다.
Abstract
This study was conducted to establish the optimal suspension culture system for both the propagation of embryogenic cells (ECs) and the induction of somatic embryos (SEs) of Kalopanax septemlobus. The proliferation rate of ECs was reduced as the inoculum density was increased; the highest rate was obtained when 0.1 g/100 ml of cells was initially inoculated. According to the analysis of cell growth pattern and cell growth cycle (G1, S and G2/M), the cell growth started in 5 days culture initiation, grew rapidly until 15 days and then decreased gradually. Distinctive changes of the cell growth cycle by the culture periods was also observed; the growth cycle was doubled from initial 5.6% to 11.7% of S stage in 5 days culture and then reached in stable stages again. Therefore, the results indicated that a 15-day-cycle was the optimal culture period for the propagation of the ECs through the suspension culture. Furthermore, the cell inoculum density was also important for the induction of SE; more than 65% of SEs at the torpedo stage was induced by using the low level of cell inoculum (0.5 g/L), while the higher inoculum densities were rapidly reduced the proportion of SEs at that stage. Although the higher inoculum density delayed the development of SE, it did not affect the proportion of SEs at the globular and heart stage. In conclusion, this study showed that the suspension culture of the Kalopanax septemlobus ECs through the control of inoculum density was an efficient way for both the propagation of ECs and the induction of SEs, suggesting that the development of this system might help to reduce the culture period for the somatic embryo production.
참고문헌 (23)
- 채영복, "한국유용식물자원 연구 편람", pp.122~ (1988), 한국화학연구소
- Akalezi CO, "Combined effects of initial sucrose concentration and inoculum size on cell growth and ginseng saponin production by suspension culture of Panax ginseng", Proc Biochem, 34, pp.639~642 (1999)
- Chakrabarty D, "Detection of DNA methylation changes during somatic embryogenesis of siberian ginseng (Eleuterococcus senticosus)", Plant Sci, 165, pp.61~68 (2003)
- Choi YE, "High frequency of plant production via somatic embryogenesis from callus or cell suspension cultures in Eleutherococcus senticoccus", Annals Bot, 83, pp.309~314 (1999)
- Contin A, "The inoculum size triggers tryptamine or secologanin biosynthesis in a Catharanthus roseus cell culture", Plant Sci, 139, pp.205~211 (1998)
- Hu WW, "Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioteactors", Biotechnol Prog, 17, pp.838~846 (2001)
- Kanokwaree K, "Effect of inoculum size on growth of Atropa belladonna hairy roots in shake flasks", J Ferment Bioeng, 84, pp.378~381 (1997)
- Kim YS, "Production of ginsenosides through bioreactor cultures of adventitious roots in ginseng (Panax ginseng C.A. Meyer)", 박사, pp.2002
- 최홍근, "In vitro Regeneration of Phragmites australis through Embryogenic Cultures", Journal of Plant Biotechnology, 8(1), pp.21~25 (2006), 한국식물생명공학회
- Masako A, "Induction of proembryos in liquid culture increases the efficiency of plant regeneration from Alstroemeria calli", Plant Sci, 163, pp.475~479 (2002)
- 문흥규, "음나무의 액아배양에 의한 기내증식", 한국임학회지, 91(6), pp.775~780 (2002), 한국임학회
- Moon HK, "Micropropagation of Kalopanax pictus tree via somatic embryogenesis", In Vitro Cell Dev Biol-Plant, 41, pp.303~306 (2005)
- Moon HK, "Somatic embryogenesis and plantlet production using rejuvenated tissues from serial grafting of a mature Kalopanax septemlobus tree", In Vitro Cell Dev Biol-Plant, 44, pp.119~127 (2008)
- Murashige T, "A revised medium for rapid growth and bioassays with tobacco tissue cultures", Physiol Plantarum, 15, pp.473~479 (1962)
- Osuga K, "Cell density is an important factor for synchronization of the late stage of somatic embryogenesis at high frequency", Plant Tiss Cult Lett, 10, pp.180~183 (1993)
- Paek KY, "Micropropagation of woody plants using bioreactor. In Micropropagation of woody trees and fruits.", pp.735~756 (2003), Kluwer Academic Publisher
- Shohael AM, "Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides", J Biotechnol, 120, pp.228~236 (2005)
- Thiruvengadam M, "Development of an embryogenic suspension culture of bitter melon (Momordica charantia L.)", Sci Hort, 109, pp.123~129 (2006)
- Winkelmann T, "Flow cytometric analyses in embryogenic and non-embryogenic calus lines of Cyclamen persicum Mill.: relation between ploidy level and competence for somatic embryogenesis", Plant Cell Rep, 17, pp.400~404 (1998)
- Yeung EC, "The use of histology in the study of plant tissue cultures systems-some practical comments", In Vitro Cell Dev Biol-Plant, 35, pp.137~143 (1999)
- Yeoung YR, "Seed germination and softwood cutting technique of Kalopanax pictus Nakai", Kor J Plant Res, 14, pp.53~59 (2001)
- Zhong JJ, "Effects of plant growth regulators on cell growth and ginsenoside saponin production by suspension cultures of Panax quinquefolium", J Biotechnol, 45, pp.227~234 (1996)
- Zhu ML, "Efficient organogenesis and plantlet regeneration in the timber species Cunninghamia lanceolata (Lamb.) Hook", In Vitro Cell Dev Biol-Plant, 43, pp.449~455 (2007)