Iridoid and phenylethanoid glycoside production and phenotypical changes in plants regenerated from hairy roots of Rehmannia glutinosa Libosch.

Abstract

Spontaneous shoot regeneration was obtained from the hairy roots of Rehmannia glutinosa cultured in liquid woody plant medium for 4 weeks in darkness after induction by Agrobacterium rhizogenes strain A4. These shoots were proliferated on solid Murashige and Skoog (MS) medium in 300 mL glass jars and in liquid MS medium in a 5 L nutrient sprinkle bioreactor. These media were supplemented with 0.1 mg L−1 of indole-3-acetic acid (IAA) and 1.0 mg L−1 of 6-benzylaminopurine. All shoots became rooted on MS agar medium containing IAA or indole-3-butyric acid within 4 weeks. The plantlets were then transferred into soil and grew in a greenhouse. The transgenic nature of the plants was confirmed by polymerase chain reaction (PCR) and reverse-transcriptase PCR analyses. The pRi-regenerated transformants demonstrated a few phenotypical alterations. Production of six iridoid (catalpol, aucubin, loganin, catalposide, harpagide, harpagoside) and two phenylethanoid (verbascoside and isoverbascoside) glycosides was determined in shoots cultured in vitro and leaves and roots of 6- and 12-month-old plants by ultra high performance liquid chromatography. The shoot culture was able to synthesize low amounts of catalpol, verbascoside and isoverbascoside. Leaves and roots of pRi-regenerants accumulated similar (harpagoside, isoverbascoside), higher (catalposide, aucubin, harpagide, verbascoside) or lower (catalpol, loganin) levels of the metabolites in comparison with untransformed R. glutinosa plants and their content varied with the age of the plants and the organ analyzed. However, the increase in shoot and root biomass of transformed plants resulted in a significantly greater yield of all analyzed compounds.