Micropropagation of Cyclopia genistoides, an Endemic South African Plant of Economic Importance

Abstract

An effi cient micropropagation protocol of Cyclopia genistoides (L.) Vent., an indigenous South African shrub of economic importance, was established. In vitro shoot cultures were obtained from shoot tip fragments of sterile seedlings cultured on solid Schenk and Hildebrandt (SH) medium supplemented with 9.84 μM 6-(γ,γ-dimethylallylamino)purine (2iP) and 1.0 μM thidiazuron (TDZ). Maximum shoot multiplication rate [(8.2  1.3) microshoots/explant)] was observed on this medium composition. Prior to rooting, the multiplied shoots were elongated for 60 days (two 30-days passages) on SH medium with one-half sucrose concentration, supplemented with 4.92 μM indole-3-butyric acid (IBA). The rooting of explants was only possible in the case of the elongated shoots. The highest root induction rate (54.8%) was achieved on solid SH medium with one-half sucrose and one-half potassium nitrate and ammonium nitrate concentration, respectively, supplemented with 28.54 μM indole-3-acetic acid (IAA) and 260.25 μM citric acid. The plantlets were acclimatized for 30 days in the glasshouse, with the use of peat/gravel/perlite substrate (1:1:1). The highest acclimatization rate (80%) was obtained for explants rooted with the use of IAA-supplemented medium. The phytochemical profi le of the regenerated plants was similar to that of the reference intact plant material. HPLC analyses showed that C. genistoides plantlets obtained by the micropropagation procedure kept the ability to produce xanthones (mangiferin and isomangiferin) and the fl avanone hesperidin, characteristic of wild-growing shrubs.

Xanthone, benzophenone and bioflavonoid accumulation in Cyclopia genistoides (L.) Vent. (honeybush) shoot cultures grown on membrane rafts and in a temporary immersion system

“…These cultures are deposited in the Higher Plants Biotechnology Laboratory at the Department of Pharmacognosy, Medical University of Gdansk, Poland, and is available to other researchers. As demonstrated in previous study (Kokotkiewicz et al 2012), C. genistoides microshoots accumulated xanthones (M and IM), flavanone hesperidin (H), as well as three isoflavone glucosides [7-O-b-glucosides of calycosin (CG), formononetin (CG) and pseudobaptigenin (PG)], absent in intact plant material but otherwise characteristic for undifferentiated cultures of Cyclopia (Kokotkiewicz et al 2013a(Kokotkiewicz et al , 2014. However, since the current research focused on establishing an in vitro source of xanthones and benzophenones, the experiments were conducted using SH medium enriched with indole-3-butyric acid (IBA), which was previously shown to enhance the accumulation of phenolics typical for field-grown plants (Kokotkiewicz et al 2012).…”

“…As demonstrated in previous study (Kokotkiewicz et al 2012), C. genistoides microshoots accumulated xanthones (M and IM), flavanone hesperidin (H), as well as three isoflavone glucosides [7-O-b-glucosides of calycosin (CG), formononetin (CG) and pseudobaptigenin (PG)], absent in intact plant material but otherwise characteristic for undifferentiated cultures of Cyclopia (Kokotkiewicz et al 2013a(Kokotkiewicz et al , 2014. However, since the current research focused on establishing an in vitro source of xanthones and benzophenones, the experiments were conducted using SH medium enriched with indole-3-butyric acid (IBA), which was previously shown to enhance the accumulation of phenolics typical for field-grown plants (Kokotkiewicz et al 2012). Unfortunately, attempts to obtain shaker cultures proved unsuccessful, probably due to high sensitivity of the investigated biomass to mechanical stress and/or explant browning, triggered by excessive contact with the growth medium (Kokotkiewicz et al 2012).…”

“…C. genistoides is characterized by exceptionally high content of the xanthone mangiferin (M), present in the amounts exceeding 5 and 10 % dry weight (DW) in the whole herb and leaves, respectively (Kokotkiewicz et al 2012(Kokotkiewicz et al , 2013bJoubert et al 2014). This compound is known to exhibit a number of biological activities including anti-inflammatory, antidiabetic and chemopreventive (Matkowski et al 2013), thus contributing to the health-promoting properties of the honeybush.…”

“…Two other important C. genistoides constituents, isomangiferin (IM) and iriflophenone-3-C-b-glucoside (IG) constituting ca. 2 and 1 % DW, respectively (Kokotkiewicz et al 2012(Kokotkiewicz et al , 2013bJoubert et al 2014), have so far not been extensively studied with respect to their biological effects. However, both compounds were recently shown to be potent antioxidants (Malherbe et al 2014) and also demonstrated strong pro-apoptotic activity on rheumatoid arthritis synoviocytes (Kokotkiewicz et al 2013b).…”

“…However, since the current research focused on establishing an in vitro source of xanthones and benzophenones, the experiments were conducted using SH medium enriched with indole-3-butyric acid (IBA), which was previously shown to enhance the accumulation of phenolics typical for field-grown plants (Kokotkiewicz et al 2012). Unfortunately, attempts to obtain shaker cultures proved unsuccessful, probably due to high sensitivity of the investigated biomass to mechanical stress and/or explant browning, triggered by excessive contact with the growth medium (Kokotkiewicz et al 2012). Therefore, it was decided to grow the shoots using the MR system, which provides the advantages of liquid cultures (enhanced exchange of nutrient and metabolites between plant tissues and the growth medium) without exerting mechanical stress on the explants (Vágner et al 2005).…”