The differential effect of N6-benzyl-adenine and N6-(?2-isopentenyl)-adenine on in vitro propagation of Paeonia suffruticosa Andr. is correlated with different hormone contents

Abstract

N(6)-benzyl-adenine (BA) enhanced phyllogenesis and axillary bud development of Paeonia suffruticosa during in vitro culture allowing good propagation while N(6)-(Δ(2)isopentenyl)adenine (iP) did not. During the first five days of culture, the mitotic activity of BA-treated explants was higher than in the iP-treated ones. High BA levels were detected in the BA-treated explants, and this was correlated with the absence of or the low indole-3-acetic acid (IAA) content. The low iP levels measured in iP-treated explants were correlated with high endogenous IAA content; the new cytokinin / auxin ratio could explain the lack of axillary buds and the development of only one leaf. Abscisic acid (ABA) was detected neither in the controls nor in the cytokinin-treated explants during the first week. However, intensive restoration of ABA accumulation was observed in controls from the third week onwards. Both BA and iP-treated explants accumulated less ABA than the controls but this hormone appeared later in the BA-treated explants than in the iP-treated ones.

Relations between auxin and cytokinin contents and in vitro rooting of tree Peony (Paeonia suffruticosa Andr.)

“…After 65 h shaking at +4°C, samples were filtered successively through a nitrocellulose prefilter (Millipore, USA), a 0 .2 pm polypropylene filter (Dynagard, FRG) and a Sep-pak-C18 column (Waters, USA), and successively fractionated in 50 fractions of 0 .8 ml (1 min each) on a high performance liquid chromatography (HPLC) column (4 .6 x 250 mm Superspher C18, Merck, FRG) run by a methanol/formic acid gradient . Four aliquots of each HPLC fraction were taken ; one of them was submitted to scintillation counting for recovery measurements and the three others to an enzyme linked immunosorbent assay (ELISA), using three different polyclonal rabbit antibodies : i) anti9RiP antibodies recognised BA, allowing to measure BA levels in microcuttings coming from a medium containing this cytokinin [8,15] ; anti9RiP antibodies were also used to measure N6 -(0 2 -isopentenyl)adenine (iP) and 9-,3-D-ribofuranosyl-iP (9RiP) levels [15,20] ; ii) anti9RZ antibodies were used to measure zeatin (Z) and 9-,3-D-ribofuranosyl-zeatin (9RZ) levels [3] ; iii) anti-IAA antibodies were used to measure IAA [3,8] . The methanol/formic acid gradient used for HPLC allowed separation of molecules recognized by the same antibody [8] .…”

“…Four aliquots of each HPLC fraction were taken ; one of them was submitted to scintillation counting for recovery measurements and the three others to an enzyme linked immunosorbent assay (ELISA), using three different polyclonal rabbit antibodies : i) anti9RiP antibodies recognised BA, allowing to measure BA levels in microcuttings coming from a medium containing this cytokinin [8,15] ; anti9RiP antibodies were also used to measure N6 -(0 2 -isopentenyl)adenine (iP) and 9-,3-D-ribofuranosyl-iP (9RiP) levels [15,20] ; ii) anti9RZ antibodies were used to measure zeatin (Z) and 9-,3-D-ribofuranosyl-zeatin (9RZ) levels [3] ; iii) anti-IAA antibodies were used to measure IAA [3,8] . The methanol/formic acid gradient used for HPLC allowed separation of molecules recognized by the same antibody [8] . Identification of these molecules was based on their retention times in HPLC compared to that of the tritiated IAA standard and cold standards of all types of hormones measured, which were injected separately into the same HPLC column .…”

Medicinal and Aromatic Plants IX

“…For the in vitro propagation of P. suffruticosa, BA and N6-(~2-isopentenyl) adenine (ip) were added separately in the culture medium, and this resulted in two types of development: explants cultured on a BA medium developed new leaves and axillary buds assuring consequently a good vegetative multiplication, while explants cultured on an ip medium developed only one leaf and no axillary buds (Bouza et al 1993). Effects of exogenous GA 3 , ABA, and BA on epicotyl dormancy of cultured herbaceous paeony embryos were investigated.…”