Browsing by Author "Shaik, Shakira."

Now showing 1 - 4 of 4

In vitro studies and phytocompound analysis in Lessertia frutescens (Fabaceae)
(2011) Shaik, Shakira.; Nicholas, Ashley.; Singh, Nisha.
The cancer bush (Lessertia frutescens L.) is an important leguminous perennial native to southern Africa and has been used for centuries in traditional medicine by the continent’s diverse cultural groups. Like many other legumes, the seeds of this species exhibit dormancy. Moreover, woody plants are typically difficult to propagate in in vitro culture systems. But in vitro shoot cultures are valuable in providing an alternative means of deriving desired secondary metabolites or phytocompounds, under controlled conditions. This study describes novel protocols for breaking seed dormancy, rapid and efficient in vitro propagation, bioreactor culture, and comprehensive phytochemical data following screening and analysis of in vitro and field extracts of L. frutescens. Experiments using physical, mechanical and chemical pre-sowing treatments were conducted to determine the germination response of this species. The results indicated that seeds of L. frutescens exhibited exogenous dormancy due to the inhibitory effect of the hard coat on germination. Seed dormancy was released by mechanical scarification in which 100 % germination was achieved. In vitro propagation studies using single node explants in Murashige and Skoog (MS) medium supplemented with combinations of different concentrations of benzyladenine and naphthaleneacetic acid revealed a maximum number of 10 shoots per explant in solid medium, and 12.9 shoots per explant in liquid medium inside a temporary immersion bioreactor. Indirect shoot organogenesis and plant regeneration using rachis and stem segments was achieved with the highest percentage of explants forming shoots (88.8 %) from rachis explants cultured onto MS medium supplemented with thidiazuron. Direct shoot organogenesis from hypocotyl and cotyledon segments was also achieved in L. frutescens. The highest shoot regeneration using hypocotyls (83 %) was obtained in MS medium supplemented with kinetin whilst the highest shoot regeneration using cotyledons (46 %) was obtained in MS medium supplemented with kinetin in combination with benzyladenine. Successful rooting (up to 80 %) and acclimatization (up to 90 % survival rate) was attained. Spectrophotometric and gravimetric methods indicated that saponins were the most abundant, followed by phenolics, flavonoids and then alkaloids in in vitro leaf extracts then in field leaf extracts and seed extracts, respectively. After qualitative analysis these extracts were also found to contain tannins, phlobatannins and cardiac glycosides of medicinal interest. By using gas and liquid chromatography the presence of the medicinally important L-canavanine, gamma amino-butyric acid and D-pinitol was verified in in vitro leaf, field leaf and seed extracts. In vitro leaves had higher quantities of all compounds, except for D-pinitol. Phytocompound analysis of shoots derived from several of the cytokinin-enhanced media showed that these organs contained higher quantities of L-canavanine compared to the control. This study, therefore, highlights the potential techno-economic production of medicinal phytocompounds from in vitro leaves of L. frutescens following large scale production using the protocols described in this study.
Phytochemical investigation and tissue culture studies on the South African knob trees, Zanthoxylum Capense and Senegalia Nigrescens.
(2017) Sunday, Bodede Olusola.; Moodley, Roshila.; Shaik, Shakira.
Abstract available in PDF file.
Screening, selection and clonal propagation of Amaranthus dubius genotypes with different calcium and iron content.
(2018) Dladla, Phindile.; Shaik, Shakira.; Watt, Maria Paula Mousaco Deoliveira.
Decontamination of Amaranthus dubius field-derived nodal explants was achieved in a 10 min soak with 1% (v/v) NaOCl and 2 drops of Tween 20® followed by three rinses in sterile distilled water, immersion in an antibiotic solution (¼ strength Murashige and Skoog basal salt medium, 50 μg l-1 rifampicin, 100 μg l-1 streptomycin/penicillin), at 1500 rpm for 5 h. Of the tested plant growth regulators, MS media supplemented with 2 mg l-1 benzylaminopurine (BAP) + 0.5 mg l-1 indole-3-acetic acid (IAA), and 0.1 mg l-1 IAA, respectively, gave the best in vitro responses of 4 shoots/nodal explant and 100% rooting. Plantlets were acclimatised over 21 days (d) in soil (S) and 1soil:1vermiculite (v/v) (1S:1V) substrates; a significant increase in the number of leaves occurred up to 21 d (6.8 to 16.2 in S and 6.3 to 13.1 in (v/v) 1S:1V). At 21 d in (v/v) 1S:1V, there were more leaves than in S, in contrast longer plant height and root length were observed in S than in (v/v) 1S:1V. The post-acclimatisation yield was 2 plants/nodal explant. The variation in calcium (Ca) and iron (Fe) content within a population of greenhouse-germinated A. dubius seedlings was then evaluated, and specific genotypes were selected to investigate the effects of micropropagation, acclimatisation in S and (v/v) 1S:1V and physiological age (time) on their growth and Ca and Fe accumulation. After 60 d, using inductively coupled plasma-optical emission spectrometry, the content of leaf Ca ranged from 246.3 to 765.3 mg 100 g-1 dry mass (DM) and the Fe from 5.3 to 26.7 mg 100 g-1 DM. Based on the significant differences of these levels amongst the parent genotypes seven were selected and were ‘ranked’ as G47 > G45 > G11 > G41 = G8 > G39 > G15 for Ca and as G47 = G45 > G39 = G41 > G8 > G15 > G11 for Fe. Nodal explants of the selected parent genotypes were subjected to the established micropropagation protocol (using S and (v/v) 1S:1V during acclimatisation). The post– acclimatisation yield was 2 to 4 plants/nodal explant. Over the 21 d of acclimatisation in the two substrates, there were clear genotypic effects on all the tested growth parameters in S. There were significant increases in S–grown plants in the number of leaves of G39 (7.0 to 10.6) and G47 (7.0 to 13.3), the plant height of G11 (5.6 to 12.3 cm) and the root lengths of G8 (6.6 to 17.3 cm) and G41 (10.0 to 16.6 cm). When grown in (v/v) 1S:1V, the plant height significantly increased from d 0 to 21 for G8 (7.0 to 12.3 cm) and G47 (6.3 to 10.6 cm). With regards to the effect of substrate, only the clones of G8 preferred nutrient-poor soil to produce more leaves (8) than when grown in S at d 21 (5). After transferring the clones to the greenhouse for 90 d, no significant differences in the root:shoot dry masses amongst the clones were observed on each substrate, and the substrate had no effect on the root:shoot dry mass for each genotype. After acclimatisation and transfer of the clones into the greenhouse it was observed that both physiological age (time, i.e. 15, 30, 60, 80 and 90 d) and substrate influenced their accumulation of Ca and Fe. Over time, in both substrates, the Ca content increased while Fe content decreased. Significant interactions were found between the genotype and substrate for both Ca and Fe, and between physiological age (time) and genotype for Fe only. In S, the clones of all the parent genotypes matched the Ca content of their parents at 15 d while for Fe that of five of the seven selected genotype clones were similar in Fe content to their parents at 60 d. Clones of four of the seven selected parent genotypes accumulated higher Ca and Fe levels when grown in (v/v) 1S:1V than in S at certain time intervals. In S, the Ca ‘rankings’ of all the clones did not match their respective parent genotypes at any time interval while in the case of Fe, the clones of two genotypes in S (G47 and G11) and one genotype in (v/v) 1S:1V (G47) matched their respective parents between 60 to 90 d of growth in the greenhouse. In conclusion, nodal explants of the selected A. dubius genotypes with varying Ca and Fe contents, were clonally propagated in vitro using BAP and IAA and the yield after acclimatisation was 2 to 4 plants/nodal explant. The physiological age (time) and substrate affected the number of leaves of the cloned genotypes, whilst in the case of Ca and Fe, these levels were influenced by micropropagation, physiological age and substrate type. Phenotypic plasticity can be further evaluated by exposing the clones of the selected genotypes to varying water, salinity and heat stresses. Additionally, investigations to understand the clones’ ability to accumulate Ca and Fe would be valuable, in this regard, quantifying inhibitory factors and exploring the effects of substrate properties such as pH and porosity are suggested.
Selection and micropropagation of solanum nigrum genotypes with varying calcium and iron content.
(2018) Goordiyal, Kimerra.; Shaik, Shakira.; Watt, Maria Paula Mousaco Deoliveira.
A direct organogenesis protocol was established for Solanum nigrum using leaf explants from seedling plants. The post acclimatisation yield of the seedling-derived leaf explants was 25 plants/explant. It included decontaminating the leaves with 1 % (v/v) sodium hypochlorite and Tween 20® (10 min), shoot multiplication on medium containing 3 mg l-1 benzylaminopurine (BAP) for 4 weeks, elongation on medium containing 0.1 mg l-1 BAP for a week, rooting on hormone-free Murashige and Skoog medium for 3 weeks and acclimatisation in pots (1 soil : 2 vermiculite [1S : 2V]) in a growth room for 2 weeks. A population of fifty 6-week old seedlings were screened using Inductively Coupled Plasma-Optical Emission Spectrometry. They varied in leaf calcium (Ca) (331.05-916.30 mg 100 g-1 dry mass [DM]) and iron (Fe) (0.64-14.95 mg 100 g-1 DM) contents. Based on these results, genotypes for high Ca (G5 and G20), high Fe (G6 and G15), low Ca (G43 and G45) and low Fe (G35 and G50) were selected for further investigation. These were micropropagated using the established protocol to determine whether their clones maintained similar levels of Ca and/or Fe to those of their parents when grown in soil. Micropropagation influenced the Ca and Fe levels of the clones of the selected genotypes, i.e. the 6-week old clones of six (i.e. G5, G20 and G45 for Ca; and G6, G15 and G50 for Fe) out of the eight selected genotypes had either significantly higher (G45 and G50) or lower (G5, G6, G15 and G20) levels of Ca and/or Fe than their 6-week old parents when grown in soil. There were also genotypic differences regarding the in vitro and ex vitro growth responses (i.e. percentage of explants with shoots, number of shoots/explant and post acclimatisation yield) and leaf Ca and Fe levels of the clones of the selected genotypes. The Ca and Fe contents of the clones of most of the selected genotypes were not affected by substrate type, suggesting that both soil and 1S : 2V were adequate to grow the S. nigrum genotypes for the benefit of high Ca and Fe. Four of the selected genotypes (viz. G15, G20, G35 and G43) were then chosen to investigate the effect of physiological age on their leaf Ca and Fe contents when grown in soil and 1S : 2V. Advancing age only affected the Fe levels of the G35 clones in soil and G15 clones in both the tested substrates. The significant differences in the Ca (G20>G43) and Fe (G15>G35) contents of the 6-week old parents were compared with those of their clones at 4, 6 and 8 weeks ex vitro in soil. A similarity (i.e. G20>G43) in the ‘ranking’, was only found at 4 weeks ex vitro for Ca. Initial attempts to establish a minimal growth protocol for storing germplasm of the eight selected genotypes showed that in vitro shoots could be kept on medium containing ¼ MS + 5 g l-1 sucrose for 8 weeks. After this period, the in vitro shoots of the eight selected genotypes that were placed onto shoot multiplication medium produced 7-13 shoots/explant. It can be concluded that micropropagation and genotype influenced both the Ca and Fe levels of the selected genotypes, while physiological age only influenced their Fe content. As the present study was a smaller component of a larger research program, further investigations need to be carried out on the selected S. nigrum genotypes prior to distribution to community gardens. Future work should include evaluating the effects of various environmental conditions (i.e. growth in a greenhouse, shadehouse, glasshouse and/or in the field), different light intensities, watering regimes, fertiliser treatments and soil pH on the growth, and leaf Ca and Fe levels of the clones. Regarding minimal growth storage, future work should include investigating whether the germplasm of the eight selected genotypes can be stored under the same conditions for longer than 8 weeks.