Toward routine basil (Ocimum basilicum L.) callus culture analysis using non-destructive Raman spectroscopy

Area of Science:

• Plant biotechnology
• Spectroscopy
• Biochemistry

Background:

• Plant tissue culture techniques are crucial for secondary metabolite production.
• Understanding biochemical changes in callus culture (CC) under different conditions is vital.
• Traditional analytical methods for CC composition often require sample destruction.

Purpose of the Study:

• To investigate the utility of FT-Raman spectroscopy for non-destructive detection of biochemical alterations in Ocimum basilicum L. var. minimum Alef. callus culture.
• To assess the influence of different culture media and spectral light treatments on CC biochemical composition.
• To compare FT-Raman spectroscopy with spectrophotometric analysis for evaluating CC changes.

Main Methods:

• Callus cultures were established on two different media formulations (2,4-D + BAP and NAA + KIN).
• Cultures were subjected to various spectral light treatments and compared to dark-grown controls over four weeks.
• Biochemical composition was analyzed using FT-Raman spectroscopy (1064 nm laser) and spectrophotometry.
• Principal Component Analysis (PCA) was employed to analyze spectral data.

Main Results:

• Spectral composition of light significantly influenced CC chemical composition, particularly on NAA + KIN medium.
• FT-Raman spectroscopy revealed significant differences in vibrational bands associated with proteins and carbohydrates.
• Specific peaks were identified and assigned to carotenoids (1525 cm⁻¹), phenolics (1606 cm⁻¹), and major Raman peaks at 1606, 1629, and 1633 cm⁻¹.
• PCA indicated higher carotenoid and ester group content under blue-red light ± UVa (on 2,4-D + BAP medium), and higher chlorophyll a and phenolics under blue-red light ± far-red (on NAA + KIN medium).

Conclusions:

• FT-Raman spectroscopy is a valuable non-destructive tool for identifying major biochemical changes in basil CC.
• The technique allows for analysis without sample preparation, extraction, or destruction.
• FT-Raman spectroscopy provides insights into biochemical profiles influenced by culture conditions and light treatments, complementing traditional methods.