Molecular basis of anthocyanin production in callus and cell cultures of Oxalis reclinata.

Thesis (M.Sc.)-Univesity of Natal, Pietermaritzburg, 1996.

Oxalis reclinata Jacq., is a dicotyledonous plant. O. reclinata belongs to the family Oxalidaceae. This plant produced callus which accumulated red coloured anthocyanin pigments when cultured in vitro. The levels of anthocyanin accumulated by O. reclinata callus were higher than in the intact plant. The major pigment was isolated and identified as cyanjdin-3-glucoside (CROUCH, VAN STADEN, VAN STADEN, DREWES & MEYER, 1993). In nature, anthocyanins are responsible for orange, red, purple and blue colouration of certain tissues of higher plant s. Due to the toxicity of many synthetic red colouring agents, anthocyanins are regarded as potential substitutes for synthetic food colourants. This research was aimed at investigating mechanisms which induce pigment production as well as to optimize anthocyanin yield from callus cultures of O. reclinata, once anthocyanin production was stimulated. Pigmented and non-pigmented callus lines were generated from O. reclinata (CROUCH & VAN STADEN, 1994) and maintained on MURASHIGE & SKOOG (1962) agar medium (O.8% [w/v], pH 5.7) supplemented with 0.5 mgℓ ¯¹ BA, 5 mgℓ ¯¹ NAA, 30 gℓ ¯¹ sucrose and 0.1 gℓ ¯¹ myo-inositol. Plant tissue culture studies were conducted on red and white lines of O. reclinata to optimize callus yield and anthocyanin production in vitro. This involved manipulating contributory factors of the culture environment (carbohydrates, nitrates, phosphates, phytohormones, light and temperature). In vitro studies showed that, light played an inductive role in anthocyanin production in callus cultures of O. reclinata. The auxin, 2,4- dichlorophenoxyacetic acid (2,4-D) reduced pigment production but increased callus biomass. This hormone probably exerted its effect by reducing the pool of anthocyanin precursors, such as phenylalanine, resulting in increased primary metabolic activity. Suspension cultures were shown to be a viable means of propagating pigmented callus cells of O. reclinata. The growth curves for red and white callus cells were determined using the settled cell volume (SCV) method. Pigmented cell cultures grew for longer periods compared to nonpigmented cells of O. reclinata. White callus cells reached the stationary phase after 18 days. Red callus cells continued growing exponentially for an extra three days compared to white callus cells. The vacuole was identified as the organelle where anthocyanins accumulate using the light microscope. The molecular techniques of two-dimensional electrophoresis and in vitro translation were utilized to analyze differences in gene expression between white and red callus cultures of O. reclinata. Thus far, two-dimensional electrophoresis has shown that the red callus of O. reclinata had more polypeptides compared to the white callus. The level of gene expression was higher in the red callus compared to white callus, as revealed by nonradioactive in vitro translation. With optimization of radioactive in vitro translation, identification of specific structural anthocyanin genes which are under regulatory control should be possible. Future research should aim at acquiring a better understanding about the genetic control of anthocyanin biosynthesis in order to manipulate this pathway effectively.