Cell Suspension Culture Of D. Carota Lab Report

Materials and methods
Chemicals
Triton X-100, abscisic acid, sodium nitroprusside, methyl jasmonate (95%) and phenylalanine were purchased from Sigma Aldrich (Mumbai, India), sodium hypochlorite and salicylic acid from Hi-media (Mumbai, India). The other chemicals were of analytical grade and obtained commercially.
Plant material and callus induction
Carrot seeds (var. Atomic red) were collected from Denmark (plantfro). Seeds were surface- sterilized with 75% ethanol for 1 min, and then soaked in sodium hypochlorite (4% w/v Hi-media) for 10 min and rinsed seven times in deionized distilled water. The clean and sterilized seeds were inoculated on solid Murashige and Skoog (MS) medium in the dark. Seedling obtained was used as explant for callus
carota. ABA was dissolved in methanol, SNP, SA, MeJA, and PHE were dissolved in water. The stock solutions of each substance were sterilized by passing them through a 0.22 μm (Nupore Filtration Systems, UP, India) filter. The actively growing cell suspension was treated with elicitors and precursor indiviually on the day of inoculation. The effect of elicitors (ABA, SNP, SA, and MeJA) and precursor (Phenylalnine) on stimulation of anthocyanin in the cell suspension cultures of D. carota was studied at various concentrations viz. 10, 50, 100, 200 μM and phenylalanine viz. 0.5, 1 and 5 mM.The samples were collected at 0, 3, 6, 9, 12, 15 and 18 day, to determine the optimum concentration required for the elicitor to evoke the maximum production.These cultures were incubated at 24 ±2oC in a shaker at 90 rpm. Cultures without elicitors were also included as control group. All experiments were performed in triplicate and data are expressed as the mean of three samples with standard deviation.
Determination of biomass
The plant cell biomass is expressed as the gram fresh cell weight (FCW) per liter. Cell suspension culture was filtered through a no. 1 filter paper (Whatman International Ltd., UK) and washed with distilled
(2010). The absorbance was measured at 520 nm using a UV–visible spectrophotometer.
Yield (mg/L) = Fresh weight (g L-1) * anthocyanin content (mg g-1 FW)
Light microscopy
Microscopic image of D. carota suspension cells was visualized with an Olympus BX 51 light microscope (Olympus, Japan) and image were captured with an imaging software Progres C5 (Germany). To examine the anthocyanin content in the suspension culture from control and treated cells (with ABA, 50 µM) were collected on day 9 and directly observed under the microscope.
Statistical analysis
The data presented in the results are the means of three replicates with the standard deviation. Every experiment was performed thrice, and the values are represented as mean ± SD. All average observations were computed via Two-way analysis of variance (ANOVA) using SPSS Statistics software 23 (IBM SPSS Statistics, Armonk).The Tukey posthoc test was done on ANOVA to give multiple comparison tables with significance level set to