Monochromatic light effect on mass and phytocompound production of Bacopa monnieri (L.) Wettst. in the temporary immersion system

Description

Brahmi (Bacopa monnieri (L.) Wettst.), a member of the Plantaginaceae family, is a medicinal herb widely used for both nutritional and therapeutic purposes, particularly for its neuroprotective properties. Despite its wide distribution in Thailand and other tropical regions, natural production is often inconsistent due to environmental variability and potential contamination from heavy metals and toxins. Plant tissue culture offers a viable alternative for propagation; however, conventional methods are labour-intensive and space demanding. The Temporary Immersion System (TIS) has emerged as an efficient approach for large-scale propagation while minimizing physiological disorders associated with prolonged liquid exposure. In addition, light quality is a critical factor influencing plant growth, development, and secondary metabolite accumulation. Therefore, this research aims to investigate the influence of light quality on the growth and development of B. monnieri in TIS.

Steps to reproduce

Chlorophyll and carotenoid measurement Leaf samples (0.05 g) from the 2nd to 4th nodes were cut from the stem. The leaves were ground using a mortar and pestle until the green pigment was visible on the equipment. Then, mixture of 1 mL of 80% cold acetone: absolute ethanol solution in a 1:1 ratio was added. The mixture was centrifuged at 10,000 rpm for 5 minutes, after which 200 L of the supernatant was transferred to a 96-well plate. Chlorophyll content was measured using a microplate reader (Synergy H1) at OD441, OD645, and OD663 nm. The absorbance values were then used to calculate the chlorophyll concentration according to the following formula (Marco Ramírez-Mosqueda 2016). Where W refers to the weight of the fresh leaves (g). Chlorophyll a = [(12.25  A663) - (2.55  A645)]  [1/(100  W)] Chlorophyll b = [(20.30  A645) - (4.91  A663)]  [1/(100  W)] Total chlorophyll = [(7.34  A663) + (17.76  A645)]  [1/(100  W)] Carotenoid = [(4.46  A441) – Chl a + Chl b]  [1/(100  W)] Bacopa monnieri extraction for HPLC technique A dry shoot (0.1 g) was placed into a 2 mL screw-cap tube and incubated in a 45°C hot oven for 60 minutes. The sample was then ground using a BeadBug™ Microtube homogenizer (Benchmark Scientific, USA) at 300 rpm for 90 seconds. Methanol (1 mL) was added to the sample, which was then vortexed for 1 minute and sonicated at 40°C for 15 minutes using the Ultrasonic Bath Sonicator (Ravi Scientific Industries, India). The mixture was centrifuged at 10,000 rpm for 3 minutes, and the supernatant was transferred to a new Eppendorf tube. The extraction process was repeated once. Subsequently, a total of 2 mL of methanol was placed in a 45°C hot oven for 24 hours. After the volume was adjusted to 1 mL, the mixture was filtered through a 0.45 µm nylon syringe filter and transferred to a 1.5 mL vial. Before quantification, the extracts were stored at −80°C. High-performance liquid chromatography (HPLC) with diode array detection (HPLC-DAD) (Agilent 1100 series, Agilent Technologies, Germany) was used for analysis. A Purospher® STAR RP-18 column (250 x 4.6 mm, 5 µm particle size) was utilized for separation. The mobile phase consisted of acetonitrile (65:35 v/v) and 0.2% aqueous phosphoric acid. A flow rate of 1.0 mL/min was applied for a saturation time of 60 minutes. The bacoside content was determined by comparing the respective retention times with the bacoside standard, detected at 205 nm (Kunakhonnuruk et al. 2023). Statical analysis The experiment was designed using a Completely Randomized Design (CRD). The data were analyzed for variance using ANOVA, and the differences between means were compared using Duncan's New Multiple Range Test (DMRT) at a 95% confidence level (p  0.05) by SPSS software 17.0 version.

Categories

Funders

• Naresuan University

  Phitsanulok

Licence