Plant Cell Reports

, Volume 21, Issue 11, pp 1103–1107 | Cite as

Genetic transformation of Pueraria phaseoloides with Agrobacterium rhizogenes and puerarin production in hairy roots

Genetic Transformation and Hybridization

Abstract

An efficient transformation system for the medicinal plant Pueraria phaseoloides was established by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots could be obtained directly from the cut edges of petioles of leaf explants or via callus 10 days after inoculation with the bacteria. The highest frequency of explant transformation by A. rhizogenes ATCC15834 was about 70% after infection for 30 days. Hairy roots could grow rapidly on solid, growth regulator-free Murashige and Skoog medium and had characteristics of transformed roots such as fast growth and high lateral branching. Paper electrophoresis revealed that bacteria-free hairy roots of P. phaseoloides could synthesize agropine and mannopine. The polymerase chain reaction amplification of rooting locus genes showed that left-hand transferred DNA of the root inducing plasmid of A. rhizogenes was inserted into the genome of transformed P. phaseoloides hairy roots. The content of puerarin in hairy roots reached a level of 1.190 mg/g dry weight and was 1.067 times the content in the roots of untransformed plants.

Keywords

Agrobacterium rhizogenes Hairy roots Pueraria phaseoloides  Puerarin 

Abbreviations

HPLC

High performance liquid chromatography

MS

Murashige and Skoog medium

PCR

Polymerase chain reaction

Ri

Root inducing

rolB

Rooting locus B

rolC

Rooting locus C

YEB

Yeast extract broth

References

  1. Bercetche J, Chriqui D, Adam S, David C (1987) Morphogenetic and cellular reorientation induced by Agrobacterium rhizogenes (strains 1855, 2659 and 8196) on carrot, pea and tobacco. Plant Sci 52:195–210CrossRefGoogle Scholar
  2. Chai XS, Wang ZX, Chen PP, Wang LY, Lü XR, Kang B (1985) Anti-arrhythmic action of puerarin. Acta Pharmacol Sin 6:166–168Google Scholar
  3. Christen P, Roberts MF, Phillipson JD, Evans WC (1989) High-yield production of tropane alkaloids by hairy root cultures of a Datura candida hybrid. Plant Cell Rep 8:75–77Google Scholar
  4. Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349PubMedGoogle Scholar
  5. Furner IJ, Huffman GA, Amasino RM, Garfinkel DJ, Gordon MP, Nester EW (1986) An Agrobacterium transformation in the evolution of the genus Nicotiana. Nature 319:422–427Google Scholar
  6. Hamill JD, Parr AJ, Rhodes MJC, Robins RJ, Walton NJ (1987) New routes to plant secondary products. Biotechnology 5:800–804Google Scholar
  7. Hu ZB, Alfermann AW (1993) Diterpenoid production in hairy root cultures of Salvia miltiorrhiza . Phytochemistry 32(2):699–703CrossRefGoogle Scholar
  8. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479Google Scholar
  9. Ottani MP, Schel JHN, Hänisch ten Cate Ch H(1990) Variation in structure and plant regeneration of Agrobacterium rhizogenes transformed and control roots of the potato cv. Bintje. Plant Cell Tissue Organ Cult 20:25–34Google Scholar
  10. Parr AJ, Peerless AC , Hamill JD, Walton NJ, Robins RJ, Rhodes MJC (1988) Alkaloid production by transformed root cultures of Catharanthus roseus. Plant Cell Rep 7:309–312Google Scholar
  11. Petit A, David C, Dahl GA, Ellis IG, Guyon P, Casse-Delbart F, Tempe J (1983) Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol Gen Genet 190:204–214Google Scholar
  12. Sauerwein M, Yamazaki T, Shimomura K (1991) Hernandulcin in hairy root cultures of Lippia dulcis. Plant Cell Rep 9:579–581Google Scholar
  13. Shi HP (2000) Plantlet regeneration from the petiole and stem of Pueraria phaseoloides (in Chinese with English abstract). Zhong Cao Yao (Chinese traditional and herbal drugs) 31:550–552Google Scholar
  14. Song XP, Chen PP, Chai XS (1988) Effects of puerarin on blood pressure and plasma rennin activity in spontaneously hypertensive rats. Acta Pharmacol Sin 9:55–58Google Scholar
  15. Tada H, Murakami Y, Omoto T, Shimomuro K, Ishimaru K (1996) Rosmarinic acid and related phenolics in hairy root cultures of Ocimum basilicum. Phytochemistry 42:431–434CrossRefGoogle Scholar
  16. Takaya K, Itokawa H (1982) Isoflavonoids and the other constituents in callus tissue of Pueraria lobata. Chem Pharm Bull 30:1496–1499Google Scholar
  17. Tepfer D (1984) Genetic transformation of several species of higher plants by Agrobacterium rhizogenes: phenotypic consequences and sexual transmission of the transformed genotype and phenotype. Cell 37:959–967PubMedGoogle Scholar
  18. Tepfer M, Casse-Delbart F(1987)Agrobacterium rhizogenes as a vector for transforming higher plants. Microbiol Sci 4:24–30PubMedGoogle Scholar
  19. White FF, Nester EW (1980) Hairy root: plasmid encodes virulence traits in Agrobacterium rhizogenes. J Bacteriol 41:1134–1141Google Scholar
  20. Yoshikawa T, Furuya T (1987) Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogenes. Plant Cell Rep 6:449–453Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.College of Life ScienceSouth China Normal UniversityGuangzhouChina
  2. 2.Laboratory of Plant Physiology, Department of Agricultural BiotechnologyAgricultural University of AthensAthensGreece
  3. 3.BERA Biosensors CorporationNew YorkUSA

Personalised recommendations