Shoot and root culture of Hypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS

Abstract

Hairy root cultures of Hypericum perforatum were obtained following inoculation of aseptically germinated seedlings with A. rhizogenes strain A4M70GUS. Effect of sucrose on the growth and biomass production of hairy root cultures was investigated. Hairy root cultures spontaneously regenerated shoots buds from which a number of shoot culture clones was established. Transformed shoot cultures exhibited good shoot multiplication, elongation and rooting on a hormone-free woody plant medium. Plants regenerated from hairy roots were similar in appearance to the normal, nontransformed plants.Additional key words: GUS activity, hairy roots, shoot regeneration, St. John's wort, sucrose. ⎯⎯⎯⎯ Hypericum perforatum L. (St. John's wort) is an important medicinal plant rich in secondary metabolites considered as an interesting drug source for the pharmaceutical industry. Most of the in vitro culture studies on this species are usually dedicated to cell and tissue cultures and their ability to produce various secondary metabolites. The aim of our investigation was to obtain and study hairy root cultures of H. perforatum. For this purpose we selected A. rhizogenes strain A4M70GUS, previously used in our laboratory to obtain hairy root cultures in several species of Gentiana (Momčilović et al. 1997, Vinterhalter et al. 1999), Lotus corniculatus (Nikolić et al. 2003/04), Centaurium erythrea (Subotić et al. 2003/04), Aesculus hippocastanum (Zdravković-Korać et al. 2004) and Blackstonia perfoliata (Bjelović et al. 2004). So far there are no papers published on Agrobacterium mediated transformation of H. perforatum.Hypericum perforatum L. seeds collected on locations around Belgrade were washed with 70 % ethanol for 1 min and than surface sterilized in 15 % commercial bleach (4 -5 % NaOCl) for 20 min. Seeds were rinsed 3 × 5 min in autoclaved water and aseptically germinated. Skoog (1962; MS) microsalts, iron and vitamins. Media were supplemented with 0.62 % agar and 2 % sucrose. Media pH was adjusted to 5.8 prior to autoclaving performed 20 min at 114 °C. Conditions in the growth room were: temperature of 25 ± 2 °C, 16-h photoperiod and irradiance 46.5 μmol m -2 s -1 . Basal media contained woody plant medium (Lloyd and McCown 1981; WPM) macrosalts and Murashige andEpicotyls were excised from seedling and cultured on basal media supplemented with 0.2 -0.5 mg dm -3 kinetin. Shoot cultures derived from epicotyls were subculture in 5 -6 weeks intervals.A. rhizogenes strain A4M70GUS contains a GUS construct integrated into the TL region of the cointegrative plasmid pRiA4 (Tepfer and Casse-Delbart 1987). GUS construct contains uidA sequence under the 70S promoter (enhancer-doubled 35S Ca MV promoter), followed by NOS polyadenylation sequence. Bacterial strains were maintained according to Van Larebake et al. (1977).Shoots comprising 3 -4 internodes were inoculated by wounding with a needle dipped in bacterial suspension. Wounding was done at the first node above ⎯⎯⎯⎯

Hairy roots of Hypericum perforatum L.: a promising system for xanthone production

“…Recent studies on different primary explants infected with A. rhizogenes reported lower HR transformation rates. Efficient transformation with A. rhizogenes A4M70GUS was observed in 21% of infected shoots [10]. Di Guardo et al [9] showed that 25% of leaf explants and only 13% of root segments had been successfully transformed with A. rhizogenes ATCC 15834.…”

“…The first step towards the application of transformation procedures to few Hypericum species has been encountered. Until now, only A. rhizogenes- [9][10][11] and biolistic-mediated [12] transformation methods have been applied. Wild agropine strain A. rhizogenes ATCC 15834 was used in the first successful transformation of H. Perforatum [9].…”

“…Wild agropine strain A. rhizogenes ATCC 15834 was used in the first successful transformation of H. Perforatum [9]. Also, an efficient transformation protocol of this species was reported with A. rhizogenes A4M70GUS [10]. Recently, two other Hypericum species (H. tomentosum and H. tetrapterum) were successfully transformed with A. rhizogenes ATCC 15834 and A4 [11].…”

“…Recently, two other Hypericum species (H. tomentosum and H. tetrapterum) were successfully transformed with A. rhizogenes ATCC 15834 and A4 [11]. HR cultures of H. perforatum exhibited high potential for spontaneous regeneration into whole transgenic plants [9,10]. Selected Hypericum HR regenerated plants have been evaluated for their bioactive secondary metabolites [9,13,14].…”

Increasing gland number and red pigments in St. Johns wort in vitro culture: Influence of mannitol, sucrose and hydrolyzed casein

“…Therefore, many attempts to obtain them both in vivo and in vitro have been undertaken. All this makes topical the production of longterm cell of H. perforatum cultures capable of biosynthesis of secondary metabolites in vitro (Vinterhalter et al, 2006). Many authors focused their attention on the synthesis of bioactive compounds under different conditions of cultivation (dark conditions and growth regulators influenced growth and synthesis of hypericin in H. perforatum) and also, to their distribution in plants in different ontogenetic stages of plants having different activity of metabolism (Kirakosyana et al, 2000Vinterhalter et al, 2006Falk, 1999.…”