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. / Thesis (M.Sc.)-Univesity of Natal, Pietermaritzburg, 1996.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10316 |
| Date | January 1996 |
| Creators | Makunga, Nokwanda P. |
| Contributors | Van Staden, Johannes., Cress, William A. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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