龙葵愈伤组织生长特性及次生代谢产物积累研究

Growth characteristics and secondary metabolite accumulation of Solanum nigrum

目的建立龙葵Solanum nigrum愈伤组织最优培养体系,研究愈伤组织在不同培养时间下各生理指标变化和澳洲茄碱、澳洲茄边碱的积累,为工业化制备澳洲茄碱和澳洲茄边碱奠定基础。方法研究不同外植体及培养基对龙葵愈伤组织诱导的影响,采用响应面试验设计方法优化愈伤组织诱导的激素配比,运用考马斯亮蓝G-250法测定可溶性蛋白含量,TTC还原法测定细胞活力,邻苯二酚法测定多酚氧化酶(polyphenol oxidase,PPO)活性,氮蓝四唑光化还原法测定超氧化物歧化酶(superoxide dismutase,SOD)活性,愈创木酚比色法测定过氧化物酶(peroxidase,POD)活性,硫代巴比妥酸显色法测定丙二醛(malondialdehyde,MDA)含量,高效液相色谱法测定澳洲茄碱及澳洲茄边碱含量。结果愈伤组织诱导最佳外植体为龙葵茎段,最适培养基为MS+1.43 mg/L NAA+2.15 mg/L 6-BA+2.48 mg/L KT,愈伤组织增殖最适培养基为MS+1.0 mg/L 6-BA+0.2 mg/L NAA。愈伤组织鲜质量增长量呈“S”型趋势,20 d时细胞活力达到最高峰值,可溶性蛋白含量与POD活性均呈先升高后降低的趋势,SOD活性在20 d与30 d出现2个峰值,MDA含量呈先降低后升高的变化趋势,在培养后期PPO活性下降,可能与愈伤组织褐化有关,澳洲茄碱与澳洲茄边碱含量变化趋势一致,在第30天时达到峰值。结论龙葵愈伤组织生长期为25 d,同时也为最适的愈伤组织鲜质量收获期,25 d后会出现不同程度褐化。其愈伤组织中的次生代谢产物积累和细胞生长过程通常是负相关的,次生代谢产物的合成大多在愈伤组织的生长后期。

Objective To establish an optimal culture system for Solanum nigrum callus,study the changes of physiological indicators and the accumulation of solasonine and solamargine in callus at different culture time,and lay the foundation for the industrial preparation of solasonine and solamargine.Methods The effects of different explants and culture media on callus induction of S.nigrum were investigated,response surface test design method was used to optimize the hormone ratio of callus induction,Coomassie brilliant blue G-250 method was used to determine soluble protein content,TTC reduction method was used to determine cell viability,catechol method was used to determine polyphenol oxidase(PPO)enzyme activity,nitrogen blue tetrazolium photochemical reduction method was used to determine superoxide dismutase(SOD)enzyme activity,guaiacol colorimetry was used for the determination of POD enzyme activity,thiobarbituric acid color method was used to determine the content of MDA,and the content of solasonine and solamargine was determined by HPLC.Results The best explant for callus induction was the stems of S.nigrum,and the best medium was MS+NAA1.43 mg/L+6-BA 2.15 mg/L+KT 2.48 mg/L,and the most suitable medium for callus proliferation was MS+6-BA 1.0 mg/L+NAA 0.2 mg/L.The fresh weight growth of callus showed an"S"-shaped trend,and the cell viability reached the highest peak at 20 d,the soluble protein content and POD activity both increased first and then decreased.SOD activity showed two peaks at 20 d and 30 d,and the MDA content decreased first and then increased.In the later period of culture,the decrease of PPO activity may be related to callus browning.Solasonine and solamargine content changed in the same trend,reaching a peak on the 30th day.Conclusion The callus growth period of S.nigrum is 25 d,and it is also the most suitable harvest period for callus fresh weight.After 25 d,browning will appear in varying degrees.The accumulation of secondary metabolites in the callus and the cell growth process are usually negatively correlated,and the synthesis of secondary metabolites is mostly in the late growth period of the callus.