微量元素对大花金挖耳悬浮细胞生长与黄酮含量的影响及其化感作用

李玉平; 李惟雄

doi:10.19303/j.issn.1008-0384.2019.01.001

微量元素对大花金挖耳悬浮细胞生长与黄酮含量的影响及其化感作用

Effects of Trace Elements in Culture Medium on Growth, Flavonoids Content and Intracellular Metabolite Allelopathy of Carpesium macrocephalum

摘要

摘要:
目的筛选微量元素对大花金挖耳细胞培养胞内活性物质的浓度条件，为大花金挖耳大规模细胞培养活性物质开发利用提供依据。
方法以大花金挖耳悬浮培养细胞为材料，研究Zn²⁺、Mn²⁺、Co²⁺、Cu²⁺、Fe²⁺、I^-、BO₃^3-、MoO₄^2-等8种微量元素对大花金挖耳悬浮培养细胞生长和黄酮类化合物累积的影响；并采用微量活性测定法测定细胞培养物的化感潜力。继代培养基为NT+1.0 mg·L^-1 NAA+0.2 mg·L^-1 BA。
结果随培养基中Zn²⁺、Co²⁺、MoO₄^2-、Cu²⁺、BO₃^3-等各处理微量元素浓度增加，大花金挖耳悬浮培养细胞生长量和黄酮含量呈先增后降趋势。MoO₄^2-、Zn²⁺浓度为3.0 μmol·L^-1、0.06 mmol·L^-1时，黄酮含量分别高达1.53%、1.46%，是对照的1.25、1.20倍；Cu²⁺、BO₃^3-、Co²⁺浓度分别为0.4 μmol·L^-1、0.4 mmol·L^-1、0.2 μmol·L^-1时，黄酮含量均高达1.35%，是对照的1.11倍；Mn²⁺浓度0.1~0.8 mmol·L^-1时，黄酮含量逐渐降低，从1.22%下降到0.43%；I^-浓度在5~20 μmol·L^-1范围内时，黄酮含量变化不大；0.1 mmol·L^-1 Fe²⁺浓度最适于细胞生长和黄酮合成；试验中，添加Zn²⁺、Mn²⁺、Co²⁺、Cu²⁺、Fe²⁺、I^-、BO₃^3-、MoO₄^2-等8种微量元素可分别使细胞总黄酮产量达到382.00、291.28、327.34、337.82、288.89、293.90、333.76、379.00 mg·L^-1，微量元素提高细胞总黄酮产量大小顺序依次是：Zn²⁺ > MoO₄^2- > Cu²⁺ > BO₃^3- > Co²⁺ > I^- > Mn²⁺ > Fe²⁺。大花金挖耳悬浮培养细胞的粗提物对供试的苘麻Abutilon theophrasti、鬼针草Bidens pilosa、黄瓜Cucumis sativus和黄豆Glycine max等4种植物幼苗根生长均具有抑制作用，毒力依次为19.86、14.69、19.03、59.07 mg·L^-1。
结论在培养基中适量添加微量元素有利于大花金挖耳细胞的生长和黄酮的生物合成，但过高浓度的微量元素会导致黄酮生物合成能力下降；大花金挖耳悬浮细胞的胞内代谢产物对4种受体植物具有不同程度的化感作用。

Abstract:
Objective Effects of trace elements in medium on the growth, flavonoids content and allelopathy activity by the intracellular metabolites of Carpesium macrocephalum in a suspension culture were studied to promote the development of a full scale utilization of C.macrocephalum culture.
Method Eight minerals in varied concentrations were incorporated in the culture medium to determine their effects on the growth and flavonoids accumulation in the cells. The allelopathic potential of the metabolite extract from each of the resulting cultures on selected plant seedlings was also determined using a microactivity assay. An NT medium with added 1.0 mg·L^-1 NAA +0.2 mg·L^-1 BA was the basal medium applied for the experiment.
Result The cell growth and flavonoids biosynthesis of C. macrocephalum in the suspension culture increased initially followed with a decline as the concentration of Zn²⁺, Co²⁺, MoO₄^2-, Cu²⁺, or BO₃^3- increased in the medium. When the concentration of MoO₄^2- was 3.0 μmol·L^-1 and Zn²⁺ 0.06 mmol·L^-1, the flavonoids content was 1.53% and 1.46%, respectively, representing 1.25 and 1.20 times, respectively, of those of control. The addition of 0.4 μmol·L^-1 Cu²⁺, 0.4 mmol·L^-1 BO₃^3- or 0.2 μmol·L^-1 Co²⁺ yielded 1.35% flavonoids, which was 1.11 times as much as that in control. In between 0.1 and 0.8 mmol·L^-1 of Mn²⁺, the flavonoids content decreased gradually from 1.22% to 0.43%, while 5-20 μmol·L^-1 of I^- did not significantly affect the content. The presence of 0.1 mmol·L^-1 Fe²⁺ appeared most conducive to the cell growth and flavonoids formation. The yield of flavonoids increased in the order of the addition of Zn²⁺, MoO₄^2-, Cu²⁺, BO₃^3-, Co²⁺, I^-, Mn²⁺, and Fe²⁺. The ethanol extracts of metabolites in the cultures exerted varying allelopathic inhibitory effects on the seedlings of Abutilon theophrasti, Bidens pilosa, Cucumis sativus and Glycine max with EC₅₀ of 19.86, 14.69, 19.03 and 59.07 mg·mL^-1, respectively.
Conclusion Appropriate addition of trace elements in the medium was conducive to the growth of C.macrocephalum cells and the biosynthesis of flavonoids. However, excessive concentrations of trace elements would decreased flavonoid biosynthesis. Intracellular metabolites of C.macrocephalum suspension cells had different degrees of allelopathic effects on 4 receptor plants.

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