Production of Malaysian kenaf Hibiscus cannabinus callus biomass and exopolysaccharide in a cell suspension culture / Danial ‘Aizat Norhisham

Danial ‘Aizat , Norhisham (2023) Production of Malaysian kenaf Hibiscus cannabinus callus biomass and exopolysaccharide in a cell suspension culture / Danial ‘Aizat Norhisham. Masters thesis, Universiti Malaya.

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Abstract

A local fibrous crop, kenaf (Hibiscus cannabinus) is commonly utilised within traditional fields such as paper and pulp production, textiles and bio-composites. Many often overlook its vast potential due to its well-established fibre usage where its future applications can eventually penetrate diverse industries such as medical, pharmaceutical, cosmeceutical and food, owing to its extracts’ therapeutic activities. However, our heavy reliance on traditional farming to ensure a consistent supply of kenaf raw materials is deemed to be unsustainable. This is because concerns about land availability, crop quality and time efficiency will arise if we were to upscale its extract production for industrial purposes. Thus, plant cell suspension culture is opted to solve the aforementioned issues as this method can produce a large amount of kenaf callus biomass (KCB), the source of the extracts. But an optimization study involving its working parameters; sucrose concentration (g/L), agitation (rpm), 2,4-dichlorophenoxy acetic acid (2,4-D) concentration (mg/L) and naphthalene acetic acid (NAA) concentrations (mg/L) is a must. This is to ensure an efficient suspension culture of kenaf in terms of the cost, resources, time, and potential future upscaling activities using bioreactors. Furthermore, it is to maximise the production of the KCB and a potential therapeutic compound, kenaf exopolysaccharide (EPS) from the suspension culture. So, in this dissertation, a preliminary study of kenaf cell suspension culture was conducted using callus that is formed on the stem of 14-days old kenaf seedlings for the establishment of the kenaf callus growth curve. At the end of the suspension culture, KCB and EPS were harvested as the cultivation’s products. It was found that the most suitable time to harvest the cultivated kenaf callus is on the 22nd day of the culture, with a KCB of 9.09 g/L and 1.10 g/L EPS. Then, the optimization of kenaf callus in suspension culture was conducted using response surface methodology (RSM) to study the interaction and optimize the working parameters (sucrose, agitation, NAA and 2,4-D) for the production of KCB and EPS. A central composite design (CCD) was utilised and the polynomial model was fitted to the experimental data. The models generated were significant (p<0.05) with sucrose, agitation and NAA having a strong effect on KCB and EPS production. The maximum predicted outcome of KCB (13.34 g/L) and EPS (1.62 g/L) can be achieved using the optimized parameters; 50 g/L of sucrose, 147.02 rpm and 2 mg/L of NAA. Then a molecular characterization of EPS was performed using Fourier-transform infrared spectroscopy (FTIR). The absorbance at 3299 cm-1, 2933 cm-1, 1628 cm-1, 1081 cm-1 and 865 cm-1 in the spectra suggested that EPS from kenaf is of α-configuration (α-glucan). To conclude, this research provides a blueprint for kenaf callus cultivation in cell suspension culture for mass-production purposes to produce its extracts’ source, callus. This study also determined the type of the excreted polysaccharide during the suspension culture of kenaf which is found to be α-glucan that has been previously proven to have useful properties such as anti-inflammatory, antibacterial and antioxidant.

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