Studies on the growth of callus and suspension cell culltures of citrus suhuiensis using statistical optimization and metabolic flux analysis /
The manipulation of culture medium on callus and suspension culture is one of the main factors to determine the best condition of cell growth. Until now, studies on in vitro of C. suhuiensis growth are still lacking and the production of secondary metabolites from citrus species are low due to crop...
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| Format: | Thesis |
| Language: | English |
| Published: |
Kuala Lumpur :
Kulliyyah of Engineering, International Islamic University Malaysia,
2019
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| Subjects: | |
| Online Access: | Click here to view 1st 24 pages of the thesis. Members can view fulltext at the specified PCs in the library. |
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| Summary: | The manipulation of culture medium on callus and suspension culture is one of the main factors to determine the best condition of cell growth. Until now, studies on in vitro of C. suhuiensis growth are still lacking and the production of secondary metabolites from citrus species are low due to crop plantation problems. Nevertheless, the optimum growth must be examined first before identifying or producing secondary metabolites. Therefore, in this study, several factors were studied to determine the optimum condition for C. suhuiensis growth. Carbon sources (sucrose, glucose, fructose and glycerol), PGRs (2,4-D, BAP, NAA and kinetin) and inoculum size (for suspension only) were the selected factors in optimizing the culture conditions for maximum growth of C. suhuiensis callus and suspension cultures. One-factor-at-one-time (OFAT) method was firstly done before optimization using Face Centred Central Composite Design (FCCCD) in Design Expert 7.0 software on both types of cultures. Carbon sources at 30 g/L and various concentrations of PGRs (0.5, 1.0, 2.0, 3.0 and 4.0 mg/L) were tested to induce callus from the seeds of C. suhuiensis under the continuous dark condition. The fresh weight and callus growth percentage were measured after four weeks period of time. OFAT results indicated that glucose and 1.0 mg/L 2,4-D produced the highest callus weight, 0.216 g and 0.175 g, respectively. Then, the optimization of callus induction was done to investigate the effect of PGRs combination between 2,4-D with BAP and 2,4-D with kinetin. The maximum callus weight (0.372 g) was achieved in the medium of 1.0 mg/L 2,4-D with 1.5 mg/L BAP. For the establishment of cell suspension cultures, friable callus was inoculated into liquid medium. Different concentrations of carbon source (30 and 50 g/L), PGRs (0.5, 1.0, 2.0, 3.0 and 4.0 mg/L) and inoculum size (10, 20 and 30 %) were studied to produce maximum CDW of C. suhuiensis suspension cultures. The samples were collected for CDW measurement for every three days throughout 24 days of incubation. Based on the OFAT experiments, 50 g/L glucose, 0.5 mg/L 2,4-D and 10% (v/v) of inoculum size attained the maximum CDW. These results were then subjected into FCCCD with one response (maximum CDW). Out of 17 runs, cells in Run 8 showed the highest CDW (21.42 g/L) in medium with glucose, 2,4-D and inoculum size at 50 g/L, 0.5 mg/L and 15% of inoculum size, respectively. Three experiments from model validation given by FCCCD were carried out and lower percentage differences (less than 10%) were obtained in all runs indicating the accuracy and applicability of Response Surface Methodology (RSM). Finally, Flux balance analysis (FBA) was applied by using General Algebraic Modeling System (GAMS) 25.1.2 software to study the flux distribution and metabolites that affected the cell growth. Primary metabolic network of Arabidopsis thaliana (A. thaliana) was used as the metabolic model. The specific uptake rates of glucose measured from the validation experiments were then used as one of the constraints in FBA and maximization of growth rate was set as the objective function. The in silico results indicated that high percentage of carbon was directed into CO2 production (67%), TCA cycle (53%) and biomass (45%) whereas flux through PPP was insignificant for biomass production where only 1.90% of carbon was diverted into the pathway. The sensitivity analysis revealed that the growth rate of C. suhuiensis cultures was affected the most by the changes in fatty acids, protein, DNA, RNA and carbohydrate. The optimization on C. suhuiensis culture medium had improved the cell growth on callus and suspension cultures whereas the application of FBA helps in understanding the distribution of fluxes in central metabolic pathway during the growth of C. suhuiensis cultures. |
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| Physical Description: | xix, 164 leaves : colour illustrations ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 126-137). |