Study on the Tissue Culture of Gentiana rigescens Franch. ex Hemsl. by Fourier Transform Infrared Spectroscopy
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摘要: 野生药用植物资源的不断减少,使得寻找其原植物的合适替代品显得尤为重要。利用组培材料代替野生药用植物作为药源已取得重大进展,但利用傅里叶变换红外光谱(Fourier transform infrared spectroscopy, FTIR)技术筛选合适的组培材料作为野生药用植物替代资源方面的应用鲜有报道。本研究采用FTIR结合偏最小二乘判别分析(partial least squares discriminant analysis, PLS-DA)对滇龙胆组织培养形成的愈伤组织(肉质部、茎、叶)、增殖苗(肉质部、茎、叶)、生根苗(根、茎、叶)进行比较。结果显示:(1)从原始FTIR光谱图上看,滇龙胆肉质部和根部峰形相似,茎和叶峰形相似;(2)二阶导数光谱图扩大了样品间的差异。在龙胆苦苷的主要吸收峰1612 cm-1附近,吸收峰强度依次为:生根苗叶 > 增殖苗叶和生根苗茎 > 增殖苗茎 > 愈伤组织叶,愈伤组织茎及肉质部、增殖苗肉质部和生根苗根部在该处无吸收峰;(3)PLS-DA得分图表明,同一组培阶段相同组织部位样品聚集在一起,而愈伤组织、增殖苗、生根苗及其各组织部位能够较好的分开。其中:肉质部、根部与茎叶之间距离较远,表明其化学成分和含量可能差异较大;肉质部和根部样品间距离较近,茎和叶样品间距离也较近。二阶导数光谱图显示,组培材料有望代替其原植物满足药用需求;若以龙胆苦苷含量为评价对象,生根苗叶则可能具有更大的开发潜能,有望代替野生滇龙胆以缓解其资源稀缺局面。本研究结果表明,采用傅里叶变换红外光谱法可以简便有效地对药用植物不同组培阶段不同组织部位的替代潜力及开发利用进行初步评估。Abstract: It is important to find suitable alternatives for medicinal plants due to the gradual decline of wild resources. Tissue culture exhibits significant advantages in achieving medicinal plant substitutes. However, few studies have reported on the application of Fourier transform infrared (FTIR) spectroscopy to select appropriate material. In this research, FTIR combined with partial least squares discriminant analysis (PLS-DA) was used to compare calli (fleshy part, stem and leaf), proliferation plantlets (fleshy part, stem and leaf) and regenerated plantlets (root, stem and leaf) of Gentiana rigescens Franch. ex Hemsl. formed by tissue culture. Results showed that:(1) FTIR spectra of the fleshy parts and roots of G. rigescens samples were alike, as were the stems and leaves; (2) Second derivative spectra showed clear differences among the samples. Around the main characteristic absorption peak (1612 cm-1) of gentiopicroside, the intensities of absorption peak was, in turn, the regenerated plantlet leaf, proliferation plantlet leaf and regenerated plantlet stem, proliferation plantlet stem. However, the stem and fleshy part of the callus, fleshy part of the proliferation plantlet and the root of the regenerated plantlet had no spectral peaks in this position; (3) Results of PLS-DA demonstrated that samples of the same part and at the same tissue culture stage could be grouped together. The fleshy parts and roots differed from the stems and leaves of the samples. Thus, the chemical constituents and content of the stems and leaves of G. rigescens could be differentiated from the fleshy parts and roots, with the fleshy parts similar to the roots and the stems similar to the leaves. Second derivative spectra showed that material formed by tissue culture could be a viable alternative to the original plants for medical use. In addition, regenerated plantlet leaves exhibited great potential for exploitation based on gentiopicroside, and may replace wild G. rigescens to relieve resource scarcity. Our study showed that FTIR can be used as a simple and effective method for the preliminary assessment of the substitution potential and utilization of different parts of medicinal plants during different stages in tissue culture.
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