培养条件对三七愈伤组织生长和皂苷积累的影响

Effects of culture conditions on the growth and accumulation of saponin in Panax notoginseng callus

关键词:  激素配比, 三七, 愈伤组织, 皂苷, 组织培养

Key words:  ratio of phytohormones, Panax notoginseng, callus, saponins, tissue culture

摘要:

以MS为基础培养基,改变激素配比、氮源和光照等因素,以分光光度法和HPLC法分析三七愈伤组织培养过程中皂苷含量的变化。结果表明:培养条件对三七愈伤组织中皂苷积累有一定影响,激素配比对愈伤组织中皂苷含量的影响最大,在0.5 mg·L^-1 2,4-D+1.0 mg·L^-1 6-BA 组合下,培养物中总皂苷含量最多,达到4.72%±0.29%; 在总氮量为60 mmol·L^-1条件下,45 mmol·L^-1 KNO₃+7.5 mmol·L^-1 NH₄NO₃(NO₃^-/NH₄⁺=7:1)时,愈伤组织皂苷含量最多,达到4.71% ± 0.17%; 分别在1 000 lx和500 lx光强下每天光照12 h的愈伤组织,皂苷含量均低于黑暗培养的愈伤组织,三者皂苷含量分别为1.94% ± 0.31%、2.38% ± 0.12%和3.57% ± 0.27%,光照引起愈伤组织表面变绿及细胞分化,可能是抑制愈伤组织中皂苷合成与积累的主要原因; HPLC检测发现,三七愈伤组织和根中均含有Rg₁、Re、Rb₁及Rd四种皂苷,但栽培三七根含有R₁皂苷,而三七愈伤组织中未检测到R₁,其原因需要进一步研究。该研究结果为未来愈伤组织培养成为部分代替人工栽培生产三七天然产物的潜在途径提供了研究基础。

Abstract:

It is well known that the growth and secondary metabolites accumulation of plant cells cultured in vitro are determined by many factors, such as culture conditions, nutrients supply, phytohormone application, etc. This study aims to explore the affecting factors during Panax notoginseng callus culture. On the basis of MS, modulations of phytohormone combination, nitrogen resource and illumination strength were carried out, and then the changes in saponins content during culture of P. notoginseng callus were measured and analyzed with spectrophotometry and HPLC. The results showed that the culture conditions had an considerable effect on the saponin accumulation in Panax notoginseng callus, in detail, combination of phytohormone had significant effects on saponin content with a peak of 4.72% ± 0.29% from the callus cultured on MS supplemented with 0.5 mg·L^-1 2,4-D+1.0 mg·L^-1 6-BA; The total N source concentration was maintained at 60 mmol·L^-1, the saponin accumulated at the highest level of 4.71% ± 0.17% in the callus under the ratio of NO₃^-/NH₄⁺=7:1(45 mmol·L^-1 KNO₃+7.5 mmol·L^-1 NH₄NO₃); When cultures grown in the light(1 000 lx and 500 lx for 12 h·d^-1, respectively)versus dark, their saponin content were calculated as 1.94% ± 0.31%, 2.38% ± 0.12% and 3.57% ± 0.27%, respectively. In addition, greening and cell differentiation were observed on the surface of callus under light, might be the main cause resulting in the inhibition of saponins synthesis and accumulation in callus. Saponins of P. notoginseng have been acknowledged to be the major compounds functioning in medical treatment of diseases, herein they are employed as the indicator of callus evaluation. Four kinds of saponins including Rg₁, Re, Rb₁ and Rd were identified from both the P. notoginseng callus and root by HPLC, and R₁ could not be detected in the former, however, presence in the latter, the reason for which needs to be further investigated. The above results are likely to provide technical support for the production of natural compounds through callus culture in large scale instead of field cultivation partly in the future, furthermore, which would also be a potential alternative to wild medicinal plant resources for its merits of sustainable and rational exploitation.