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Agrobacterium-mediated Transformation of Mature Ginseng Embryos

农杆菌介导的成熟人参胚胎的遗传转化

Ok Ran Lee Ok Ran Lee
JH Jin Haeng Han
YK Yoon-Ri Kim

发布: 2014年12月20日第4卷第24期 DOI: 10.21769/BioProtoc.1362 浏览次数: 12537

评审: Tie Liu

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

May 2014

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Abstract

Ginseng refers to species within the genus Panax and is a slow-growing perennial herb from the Araliaceae family. The most widely used Panax species is Panax ginseng Meyer (Korean ginseng). Panax japonicus (Japanese ginseng), Panax notoginseng (Chinese ginseng), and Panax quinquefolium (American ginseng). Due to the various pharmaceutical importance of ginsenosides, ginseng plant has been cultivated for its highly valued root over 2,000 years as a medicinal plant in East Asian countries particularly in China, Korea, and Japan and North America. Korean ginseng (Panax ginseng C. A. Meyer) consists of nine cultivars from three Jakyung, Chungkyung, and Hwangsook lines. Cultivar “Yunpoong” has characteristics to have more axillary shoots and lateral roots compared to other cultivars. Thus “Yunpoong”: Ginseng seeds are relatively more feasible for regeneration of adventitious roots during gene transformation. Here, we describe how to prepare and treat ginseng seeds after harvest till the ginseng immature embryos are ready for the germination, and to be used in Agrobacterium tumefaciens-mediated gene transformation.

Keywords: Panax ginseng (人参) search Adventitious root (不定根) search Agrobacterium tumefaciens (农杆菌介导法) search Binary vector (二进制向量) search Regeneration (再生) search

Materials and Reagents

  1. Ginseng seeds, in which zygotic embryos were in a mature state (4 mm in length)
  2. Agrobacterium tumefaciens C58C1 (pMP90) strain
  3. Binary (pCAMBIA1390) vector
  4. MS powder (Duchefa Biochemie, catalog number: M0222 )
  5. Sucrose (Duchefa Biochemie, catalog number: S0809 )
  6. 2-(N-morpholino) ethanesulfonic acid (MES) monohydrate (MB cell, catalog number: MB-M4837 )
  7. Phytoagar (Duchefa Biochemie, catalog number: P1003) or Gelrite (Duchefa Biochemie, catalog number: G1101)
  8. Yeast extract (Duchefa Biochemie, catalog number: Y1333 )
  9. Peptone (Duchefa Biochemie, catalog number: P1328 )
  10. NaCl (Affymetrix, catalog number: 4171746 )
  11. Bactoagar (BD, catalog number: 214010 )
  12. Hygromycin (Duchefa Biochemie, catalog number: H0192 )
  13. Kanamycin (Duchefa Biochemie, catalog number: K0126 )
  14. Cefotaxime (Duchefa Biochemie, catalog number: C0111 )
  15. 6-benzylaminopurine (BAP) (Duchefa Biochemie, catalog number: B0904 )
  16. 2,4-dichlorophenoxyacetic acid (2,4-D) (Duchefa Biochemie, catalog number: D0911 )
  17. Indole-3-butyric acid (IBA) (Duchefa Biochemie, catalog number: I0902 )
  18. Sucrose
  19. Phyto agar
  20. 70% ethyl alcohol (EtOH)
  21. 2% (v/v) sodium hypochlorite (NaOCl) (Sigma-Aldrich, catalog number: 425044-1L )
  22. B5 medium (Duchefa Biochemie, catalog number: G0209 )
  23. MS (Murashige & Skoog) medium (see Recipes)
  24. YEP medium (see Recipes)
  25. Antibiotics stock solution (in ddH2O) (see Recipes)
  26. Plant growth regulators stock solution (see Recipes)

Equipment

  1. Laminar flow cabinet (HEPA filter 99.99% efficient on particle of 0.3 μ and vertical type)
  2. Shaking incubator (100~200 rpm on a gyratory shaker)
  3. Centrifuge (3,000 x g) (Labnet International, catalog number: C2500-R-230V )
  4. Micropipette
  5. Forceps (Phoenix Technologies, catalog number: ESD-16 )
  6. Erlenmeyer flask (50/100 ml) or autoclavable plastic tube for seed sterilization
  7. Autoclave machine
  8. Growth chamber (23 °C ± 2 °C)
  9. Sterile filter paper
  10. Petri dish

Procedure

English
中文翻译

文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Lee, O. R., Han, J. H. and Kim, Y. (2014). Agrobacterium-mediated Transformation of Mature Ginseng Embryos. Bio-protocol 4(24): e1362. DOI: 10.21769/BioProtoc.1362.
  2. Kim, Y. J., Lee, O. R., Oh, J. Y., Jang, M. G. and Yang, D. C. (2014). Functional analysis of 3-hydroxy-3-methylglutaryl coenzyme a reductase encoding genes in triterpene saponin-producing ginseng. Plant Physiol 165(1): 373-387.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物转化 > 农杆菌介导的转化方法

植物科学 > 植物生理学 > 植物生长

分子生物学 > DNA > 转化

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