Effects of Macronutrients on Anthocyanin Production in Roselle (Hibiscus sabdariffa L.) Callus Cultures.

Abstract

The effects of various macronutrients on growth and anthocyanin formation in callus cultures of roselle (Hibiscus sabdaritfa L.) were investigated. Of the nutritional factors examined the type and concentration of carbon and nitrogen sources and phosphate concentration showed marked effects on the growth and anthocyanin production. Utilization of an optimized medium based on the results obtained in the present investigation resulted in a 2.5 fold increase in the anthocyanin content Potential exists for application of a two-stage culture method for the production of anthocyanin pigment Introduction Calyces of roselle (Hibiscus sabdariffa L.) contain cyanidin and delphinidin glycosides1) and have been used for making jelly, jams, beverages and food colorants2). Cultured roselle cells might potentially be a suitable source for large scale production of anthocyanin pigments. In a previous paper3) we reported that callus tissues derived from seedlings of roselle could accumulate anthocyanin pigments tentatively identified as cyanidin-3-monoglucoside (major pigment) and cyanidin-3-xylosylglucoside. Anthocyanin formation in the callus markedly enhanced by 2, 4-D and inhibited by gibberelic acid.The present paper describes the effects of macronutrients on cell growth and anthocyanin production of roselle callus as well as an optimized growth and production medium based on the results obtained in this study. Materials and MethodsPlant material and culture method. Callus tissues derived from seedlings of roselle were subcultured at 1-month intervals on Linsmaier and Skoog (LS) basal agar medium4) supplemented with l p M 2, 4-D and l p M kinetin at 25 under 3, 000 lux illumination (16 hr/day). For investigating the influence of various macronutrients, callus tissues (ca 0.2 g) were transferred onto 20 ml of test medium in 50 ml Erlenmeyer flasks and incubated at 25 under illumination for four weeks before harvest. All the test media contained both 2, 4-D and kinetin at 1 uM level.Extraction and quantitative analysis of anthocyanin. Fresh callus tissues were homogenized with 1% methanolic HCl in a mixer. The homogenate was allowed to stand overnight at 4 and then filtered. Absorbance of the filtrate was measured at 530 nm and the anthocyanin content was calculated as a percentage of fresh weight of callus using the molecular extinction coefficient (log E 4.47) for cyanidin-3-monoglucoside5). Results and DiscussionEffects of carbon sourceAlthough sucrose as a carbon source can support both growth and secondary metabolite production in callus and suspension cultures of plant cells, other carbon sources can also be effective. The effects of different carbon sources at 3% concentration on the growth and anthocyanin production of roselle callus are shown in Fig, 1. As regards anthocyanin formation glucose was as effective as sucrose, whereas fructose and maltose could support anthocyanin formation to a limited extent. In contrast, sucrose was most effective for cell growth; glucose, fructose and maltose were much inferior to suc...

Induction of anthocyanin pigments in callus cultures of Rosa hybrida L. in response to sucrose and ammonical nitrogen levels

“…Increase in sucrose concentration in basal media above normally used concentrations (2-3%) has been reported to enhance production of various phenolic compounds including anthocyanin (Mizukami et al 1991) in Hibiscus sabdariffa. Those researchers clearly demonstrated that, among the different carbon sources, only sucrose and glucose were equally effective in inducing anthocyanin production, whereas fructose and maltose improved anthocyanin formation only marginally.…”

Not all anthocyanins are born equal: distinct patterns induced by stress in Arabidopsis

“…Abiotic stresses that induce anthocyanin synthesis include drought in rice and Arabidopsis (Basu et al 2010 ; Sperdouli and Moustakas 2012 ), cold in maize, Arabidopsis , and citrus (Christie et al 1994 ; Crifò et al 2011 ), high salt in tomato and red cabbage (Eryılmaz 2006 ), nutrient deficiency in Arabidopsis , hibiscus, and carrot (Mizukami et al 1991 ; Rajendran et al 1992 ; Jiang et al 2007 ), osmotic stress in carrot callus and grapevine cell cultures (Rajendran et al 1992 ; Suzuki 1995 ), and exposure to low pH of the medium in strawberry suspension cell cultures (Zhang and Furusaki 1997 ; reviewed by Chalker-Scott 1999 ; Winkel-Shirley 2002 ). The presence of sucrose in the culture medium also induces anthocyanin synthesis by a mechanism dependent on the MYB transcription factor, PAP1 (Teng et al 2005 ; Solfanelli et al 2006 ).…”

Bitkilerdeki Antosiyaninlerin Etkinliği, Stabilitesi ve İn Vitro Üretimi

“…Bazı çalışmalara göre diğer şekerlerle karşılaştırıldığında fruktoz içeren ortam, en etkili olan ortam olmuştur. Aralia cordata L. (Sakamoto, 1993), Hibiscus sabdariffa L. (Mizukami, 1991) ve Fragaria vesca L. (çilek) kallus kültürlerinde antosiyanin birikimine neden olmuştur (Mori ve Sakurai, 1994). Laktoz ise mutant bir havuç hücre hattında antosiyanin üretimini artırmıştır (Nagarajan, 1989), Daucus carota L.' nın kallus kültürlerinde sükroza mannitol takviye edildiğinde antosiyanin miktarında önemli artış olmuştur (Rajendran ve ark., 1992).…”