Shaza Hasan Besher* (1)

(1)- National Commission for Biotechnology, Damascus (*Corrsponding author: Shaza Hasan Besher E-Mail: sh.hasan85@gmail.com).

Received: 30/01/2020 Accepted: 04/04/2019

Abstract

This research was carried out at the lab of National Commission for Biotechnology during 2018-2019, in order to determine the response of golden henbane’s callus (H. aureus), and to study its growth dynamics on growth regulators-free medium, based on the fresh weight of biomass as an indicator of the callus growth strength, also to determine the effect of the nutritive medium components and change in gene expression of Tropinon Reductase I (TR I) and Tropinon Reductase II (TR II) on the alkaloids concentration in the cellular lines of H. aureus, by estimating the mRNA transcriptional levels of these genes using Real-Time PCR technique. The results of study showed that the growth curve of callus cells in all tested medium followed the sigmoid trend, where growth speed of cells reached the highest division rate at the exponential phase (15 to 35 day) with superiority of the control medium 5C01 in biomass weight. Also the results showed that superiority of the control 5C01 was significant in alkaloid content compared with 5C, 6C and 10C cellular lines. Whereas highest concentration of alkaloids was at the exponential phase (30 days) (0.49%), (0.36%), (0.29%) and (0.21%) for hyoscyamine in 5C01, 5C, 6C and 10C lines, respectively, while the concentration of scopolamine was very low. Measurement of gene expression showed that the highest value of TR I gene expression was at the exponential phase (30 days), registered (9.781), (2.144) and (8.168) times compared to the control 5C01 in 5C, 6C and 10C cellular lines respectively. While the highest value of TR II gene expression was at the lag phase (5 days). However, this superiority in gene expression was not associated with an increase in the alkaloids concentration, this is mainly due to the failure of TR I enzyme in Tropine formation, due to its failure in competing with carbon flowing from Calystegine towards Tropine synthesis, whereas TR II enzyme induces Pseudotropine formation resulting in accumulation of Calystegine, not tropane alkaloids.

Keyword: Hyoscyamus aureus, Real Time PCR, TR I, TR II, Hyoscyamine, Scopolamine, Callus culture.

Full paper in Arabic: PDF