白花蛇舌草悬浮细胞分批培养规律的研究

The rules on suspension cell batch culture of Hedyotis diffusa

目的利用白花蛇舌草茎尖的分生组织建立植物悬浮细胞培养系,确定白花蛇舌草悬浮细胞分批培养时的变化规律。方法通过接种不同量的细胞液确定最适接种量,以细胞干重、蔗糖、铵离子、硝酸盐氮和多糖含量作为检测指标,确定白花蛇舌草悬浮细胞分批培养时的变化规律。结果白花蛇舌草悬浮细胞分批培养的最适接种量为15%,此时细胞干重达到最大值。在此接种量下,当培养时间达到9 d时,pH很快降低到3左右,细胞干重不再增加。培养液中的主要营养成分碳源———蔗糖,在培养开始时,有比较大的降低,在培养后期,细胞干重不再增加时,也不再有大的改变。氮源[NO3-]优于[NH4+]先被利用,而且[NO3-]的利用速率要远高于[NH4+],达到7.14μg/(mL.d);细胞液中多糖的生成和细胞的生长属于非偶联型,在培养后期,逐渐大量生成。结论初步确定白花蛇舌草悬浮细胞分批培养时的变化规律,为以后的培养工艺优化打下了基础。

Purpose Hedyotis diffusa suspension cell was established by the tip of meristem to find out the rules on suspension cell batch culture of Hedyotis diffusa with the time. Methods The optimum in- oculum was determined by different inoculation of cell medium. The rules on suspension cell batch culture of Hedyotis diffusa with the time were found by the detection of the amount of cell dry weight, sucrose, am- monium,nitrate and polysaccharide. Results The suspension cell batch culture of Hedyotis diffusa opti- mum inoculum is 15% ,maximum dry cell weight at the same time. At this inoculation,when the incuba- tion time was of 9 days, and pH quickly reduced to about 3, the cell dry weight did not increase. The main medium nutrients were carbon -sucrose, a relatively large decreased in the beginning of culture, and the cell dry weight no longer had a big change in the late culture stage. Nitrogen [ NO3- ] was used prior to [NH4＋ ] ,and the use rate of [NOa- ] is far higher than [NH4＋ ] ,to 7.14 Ixg/（mL ~ d） ,cell growth and the polysaccharide production were non-coupled and the polysaccharide was generated in the late culture stage. Conclusion It was found that the rules on suspension cell batch culture of Hedyotis diffusa with the time is helpful for later culture technique.