Transformation of Ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes

Abstract

Transformed root cultures of ipecac (Cephaelis ipecacuanha A. Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15 834. Southern blot analysis of the established roots revealed that only the T_L-DNA was integrated into the plant genome without incorporation of the T_R-DNA. The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark. The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 °C under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants. Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark. The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.

Abbreviations

B5: Gamborg B5 (1968)

HF:phytohormone-free

IAA:indole-3-acetic acid

IBA:indole-3-butyric acid

MS:Murashige and Skoog (1962)

1/2 MS:a half strength Murashige and Skoog

NAA:α-naphthaleneacetic acid

WP:Woody Plant (1980)

Ipecac - Cephaelis ipecacuanha - Agrobacterium rhizogenes - transformation - isoquinoline alkaloids

References

• 1 Evans W C. Ipecacuanha. In: Pharmacognosy, 13th edn. Baillière Tindal London; 1989: pp. 595-9
• 2 Ikeda K, Teshima D, Aoyama T, Satake M, Shimomura K. Clonal propagation of Cephaelis ipecacuanha .  Plant Cell Rep. 1988;  7 288-91
• 3 Yoshimatsu K, Shimomura K. Cephaelis ipecacuanha A. Richard (Brazilian Ipecac): Micropropagation and the production of emetine and cephaeline. In: Bajaj YPS editor Biotechnology in agriculture and forestry, vol 21, Medicinal and aromatic plants IV. Springer Berlin, Heidelberg, New York; 1993: pp. 87-103
• 4 Yoshimatsu K, Shimomura K. Efficient shoot formation on internodal segments and alkaloid formation in the regenerates of Cephaelis ipecacuanha A. Richard.  Plant Cell Rep. 1991;  9 567-70
• 5 Yoshimatsu K, Shimomura K. Plant regeneration on cultured root segments of Cephaelis ipecacuanha A. Richard.  Plant Cell Rep. 1994;  14 98-101
• 6 Yoshimatsu K, Aoi K, Shimomura K. Clonal propagation of Cephaelis ipecacuanha (II): Characteristics of regenerated plants field-cultivated in two districts.  J Plant Physiol. 1994;  144 22-5
• 7 Teshima D, Ikeda K, Satake M, Aoyama T, Shimomura K. Production of emetic alkaloid by in vitro culture of Cephaelis ipecacuanha A. Richard.  Plant Cell Rep. 1988;  7 278-80
• 8 Yoshimatsu K, Shimomura K. Emetic alkaloid formation in root culture of Cephaelis ipecacuanha .  Phytochemistry. 1991;  30 505-7
• 9 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.  Physiological Plantarum. 1962;  15 473-97
• 10 Draper J, Scott R. The isolation of plant nucleic acids: 4.3.3. The isolation of DNA from fresh tissue using SDS. In: Draper J, Scott R, Armitage P, editors Plant genetic transformation and gene expression. A laboratory manual. Blackwell Scientific Publications London; 1988: pp. 212-4
• 11 Saito K, Yamazaki M, Shimomura K, Yoshimatsu K, Murakoshi I. Genetic transformation of foxglove (Digitalis purpurea) by chimeric foreign genes and production of cardioactive glycosides.  Plant Cell Rep. 1990;  9 121-4
• 12 Jouanin L. Restriction map of an agropine-type Ri plasmid and its homologies with Ti plasmids.  Plasmid. 1984;  12 91-102
• 13 Gamborg O L, Miller R A, Ojima K. Nutrient requirements of suspension culture of soybean root cells.  Exp Cell Res. 1968;  50 151-8
• 14 Lloyd G, McCown B. Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tipp culture.  Int Plant Propag Soc Combd Proc for. 1980;  30 421-7
• 15 Yoshimatsu K, Shimomura K. Improved analysis of emetic alkaloids in tissue cultures and regenerates of Cephaelis ipecacuanha by ion pair high performance liquid chromatography.  Photochemical Analysis. 1993;  4 217-9
• 16 Yoshimatsu K, Jaziri M, Kamada H, Shimomura K. Production of diploid and haploid transgenic Atropa belladonna plants: morphological traits and tropane alkaloid production.  Belg Journ Bot. 1997;  130 38-46
• 17 Sevón N, Oksman-Caldentey K M. Agrobacterium rhizogenes-mediated transformation: Root cultures as a source of alkaloids.  Planta Med. 2002;  68 859-68
• 18 Petit A, David C, Dahl G A, Ellis J G, Guyon P, Casse-Delbart F, Tempé J. Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation.  Mol Gen Gent. 1983;  190 204-14
• 19 Toda Y, Aoki T, Yoshimatsu K, Shimomura K. Photosynthetic activity in green hairy roots: Ultrastructure of plastid and correlation of chlorophyll and secondary metabolites.  Natural Medicines. 2002;  56 173-7
• 20 Fujii T, Ohba M. The ipecac alkaloids and related bases. In: Cordell GA, editor The alkaloids, chemistry and biology, vol 51. Academic Press San Diego; 1998: pp. 271-321
• 21 Celma C R, Palazón J, Cusidó R M, Piñol M T, Keil M. Decreased scopolamine yield in field-grown Duboisia plants regenerated from hairy roots.  Planta Med. 2000;  67 249-53
• 22 Sevón N, Dräger B, Hiltunen R, Oksman-Caldentey K M. Characterization of transgenic plants derived from hairy roots of Hyoscyamus muticus .  Plant Cell Rep. 1997;  16 605-11