Establishment of regenerative callus, cell suspension system, and molecular characterization of Taxus wallichiana Zucc. for the in vitro production of Taxol

Abstract

Taxus wallichiana, an endangered Taxus species native to Nepal, is a source of Taxol (paclitaxel), an anticancer drug, which if produced via plant cell culture in vitro not only reduces the pressure on natural habitat but also allows continuous production throughout the seasons. Gamborg's B5 medium was the culture medium to develop callus and suspension culture. Quantification of Taxol was carried out via high performance liquid chromatography (HPLC) using C18 column, water-acetonitrile mobile phase, and 1 ml/minute flowrate at 227 nm wavelength. Genetic variation among population was determined using the POPGEN32, version 1.31. Best callus response was in medium with 1 mg/l 2,4-D for both stem and needle explants. For cell suspension culture, callus from needle explants showed better response compared to stem. Fructose and sucrose proved to be the best carbon sources. Cell count increased as carbon concentration increased. 2,4-D at low concentration (<2 mg/l) and naphthaleneacetic acid at high concentration (>5 mg/l) showed higher cell growth rate. Inoculum volume was directly proportional to cell count. Extra addition of macronutrients had inhibitory effect on cell count, but the effect was reversed for MgSO 4 . Using HPLC, for bark, the highest Taxol yield was 0.057 mg/g distilled water (DW), while for needle was 0.056 mg/g DW. For suspension cultured HPLC samples, Taxol yield ranged at 0.004%. Size range of amplified Deoxyribonucleic acids was between 200 and 2,000 bp. Thus, this investigation generated a concrete data for Taxus population of Nepal providing efficient protocols for establishment of callus and cell suspension culture with the aim of producing Taxol in in vitro conditions.

Engineering Considerations to Produce Bioactive Compounds from Plant Cell Suspension Culture in Bioreactors

“…Even though there are several studies where the production of PDSM from callus cultures and differentiated cells/tissues are used, the cell suspension culture from dedifferentiated cells is mostly preferred [51,52]. Cell suspension cell culture (CSC) is considered as a simple and cost-effective method, allowing suitable conditions for cells to produce compounds identical to those from parental cells to be achieved, offering advantages such as setting stable systems for continuous PDSM production with homogeneity in yields and quality, as well as offering the possibility of synthesizing new compounds and greater potential for PDSM commercial application [51,53]. Therefore, CSC has been demonstrated to be the selected biotechnological tool for obtaining high-value PDSM, such as taxol [53,54], resveratrol [55,56], and ginsenosides [57], among others.…”

“…Cell suspension cell culture (CSC) is considered as a simple and cost-effective method, allowing suitable conditions for cells to produce compounds identical to those from parental cells to be achieved, offering advantages such as setting stable systems for continuous PDSM production with homogeneity in yields and quality, as well as offering the possibility of synthesizing new compounds and greater potential for PDSM commercial application [51,53]. Therefore, CSC has been demonstrated to be the selected biotechnological tool for obtaining high-value PDSM, such as taxol [53,54], resveratrol [55,56], and ginsenosides [57], among others. To this end, further discussions will be centered on CSC for producing PDSM at laboratory and larger scales using different bioreactor configurations.…”

Effects of Low-Dose Kinetin, 2,4-D and Monochromatic Light Conditions on Flavonoid Content in Callus Culture of Dioscorea esculenta

“…Various studies have shown the combinations and concentrations effect of kinetin and 2,4-D in callus culture; however, the results vary depending on the plant species and organ or tissue origin [14,15]. In Taxus brevifolia Nutt., a combination of kinetin and 2,4-D showed a linear stimulating effect on callus growth [16]. Furthermore, applying exogenous kinetin increases the content of crucial phytohormones in tomatoes grown under salinity stress.…”