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

Abstract

Here we investigated whether FT-Raman spectroscopy could be used to detect biochemical changes in small-leaved basil (Ocimum basilicum L. var. minimum Alef.) callus culture (CC). To address the effect of culture conditions and elicitor treatments, CC established on 1 mg L⁻¹ 2,4-D + 0.5 mg L⁻¹ BAP, or 2.5 mg L⁻¹ NAA + 0.5 mg L⁻¹ KIN was exposed to various spectral light treatments during four weeks and compared to those grown in dark. The composition of CC was analysed both using an FT-Raman spectrometer equipped with laser 1064 nm, and spectrophotometrically. The spectral composition of light had a higher influence on the chemical composition of CC grown on NAA + KIN than on 2,4-D + BAP medium. Spectrophotometrically, no differences in the content of protein or sugar were determined in relation to the plant growth regulators applied. However, significant differences in frequencies and intensities of vibrational bands associated with proteins (S-S disulfide stretching, tyrosine, cystine, and methionine at lower spectral ranges, and amide III stretching in the higher spectral range), and carbohydrates (C–O–C skeletal mode at lower spectral ranges, and C–O–H vibrations at higher spectral ranges) within the Raman spectra were estimated and discussed. The 1525 cm⁻¹ and 1606 cm⁻¹ peaks with high intensities of vibration bands were identified and assigned to carotenoids and phenolics. In all treatments applied the major Raman peaks were detected at 1606, 1629, and 1633 cm⁻¹. PCA analysis showed that CC under blue-red light and blue-red light + UVa (2,4-D + BAP) had higher content of carotenoids and ester groups, while chlorophyll a and phenolics were found in CC grown on NAA + KIN under blue-red light + UVa and blue-red light + far-red. Compared to traditional methods of analysis, which are preceded by the sample destruction before extraction and analysis, it can be concluded that the FT-Raman spectroscopy may serve as a valuable tool for the non-destructive and non-invasive identification of major biochemical changes in basil CC without any sample preparation.