Micropropagation ofPanax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets

Somatic embryogenesis was induced in callus tissues derived from young flower buds ofPanax notoginseng via callus within 18 weeks of culture. The mature somatic embryos were germinated on half-strength Murashige and Skoog's (MS) medium supplemented with gibberellic acid A₃(GA) and 6-benzyladenine (BA). The most suitable medium for optimal root formation proved to be MS medium supplemented with 1-naphthaleneacetic acid (NAA). Total DNA was extracted from the leaves of the regenerated plantlets ofP. notoginseng. Analysis of random-amplified polymorphic DNA (RAPD) using 21 arbitrary oligonucleotide 10-mers, showed the genetic homogeneity ofP. notoginseng. The amplification products were monomorphic for all of the plantlets ofP. notoginseng regenerated by embryogenesis, suggesting that somatic embryogenesis can be used for clonal micropropagation of this plant.     

Enhanced somatic single embryo formation by plasmolyzing pretreatment from cultured ginseng cotyledons

Cotyledon explants of Panax ginseng zygotic embryos directly produced somatic embryos on Murashige and Skoog medium without growth regulators. Somatic embryos were formed only near the proximal excised region of cotyledons. Multiple and/or single embryos were formed and the frequency of these formations differed according to the degree of maturity of the zygotic embryos used as the explant source. When cotyledon explants pre-plasmolysed (1.0 M sucrose for 24 h), the frequency of single embryo formation was enhanced regardless of cotyledon maturity. In addition, the distribution pattern of somatic embryos changed markedly because the embryos were formed over the whole surface of the cotyledons. Histological observation revealed that plasmolyzing pretreatment broke the plasmodesmatal connection between cells and when the embryogenic cell divisions commenced, plasmodesmatal strands were hardly observed except for newly formed cell walls. This indicates that the enhanced single embryo formation over the entire surfaces of cotyledon explants might be the result of an interruption of cell–cell interaction by plasmolyzing pretreatment.     

林下人参生理特性和生长与林内生态因子的关系

结合模拟试验,对林下人参的生理活动及生长过程与林内生态因子的关系进行 了观测研究．结果表明,林内光照、水分、温度等因子与林下人参生长的关系极为密切．一 般林内光照在中等条件下,即相对光照在１０～３５％左右．林分郁闭度约０．６～０．８,土壤含 水量在 ３５～４０％时人参生长最好;林下人参的物候进程及生长与温度的关系密切．林内 温度在５℃左右人参芽胞开始萌动,１５℃左右地上茎高生长进入速生期,６月上旬生长停 止．因此,人为调节各种生态因子在适宜的范围内有利于林下人参的生长．     

Intranuclear and Cytoplasmic Inclusions in Embryogenic and Embryoidal Cells of Panax quinque folius

The presence of intranuclear and cytoplasmic inclusions in cells of embryogenic callus and developing embryoids from tissue cultures of Panax quinquefolius L. was described. These cellular structures were not found in non-embryogenic cells. The size of intranuclear and cytoplasmic inclusions seemed to be related to the developmental status of the cell.     

培养条件对三七愈伤组织生长和皂苷积累的影响

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-12,4-D+1.0 mg·L-16-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-1KNO3+7.5 mmol·L-1NH4NO3(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。     

三七、栀子有效组分给药的APP/PS1小鼠血清神经递质与行为学的相关性

目的:探讨三七、栀子有效组分及配伍给药对APP/PS1小鼠血清中乙酰胆碱和五羟色胺含量的影响及其与行为学的相关性分析。方法:APP/PS1小鼠随机分成转基因模型组(Tg)、三七总皂苷组(PNS)、栀子苷组(GP)、配伍组(PNS+GP),同窝非转基因小鼠为野生型组(WT),从4月龄开始,以自主进食的方式,分组给药3个月,7月龄时,进行八臂迷宫测试和血清中乙酰胆碱及五羟色胺含量的检测。结果:(1)乙酰胆碱含量测定结果,与野生型组相比,转基因模型组有显著性降低(P0.05);三七总皂苷组、栀子苷组可以显著升高转基因模型组的乙酰胆碱含量(P0.05)。(2)五羟色胺含量测定结果,与野生型组相比,转基因模型组有降低趋势,但无统计学意义(P0.05);三七总皂苷组、栀子苷组和配伍组对转基因组的五羟色胺含量有升高趋势,但都没有统计学意义(P0.05)。(3)相关性分析结果,八臂迷宫测试中小鼠进入食物臂的时间百分比与血清中乙酰胆碱的含量成正相关(r=0.35,P0.05),与血清中五羟色胺的含量没有相关性(rs=0.16,P=0.39)。结论:三七、栀子有效组分及配伍给药对APP/PS1小鼠血清中乙酰胆碱的含量有调节作用,APP/PS1小鼠血清中乙酰胆碱的含量与八臂迷宫行为学测试体现的空间学习记忆能力成正相关。     

Biotransformation of p-, m-, and o-hydroxybenzoic acids by Panax ginseng hairy root cultures

The regioselective glycosylation of three isomers of hydroxybenzoic acids was observed in Panax ginseng hairy root cultures. p-Hydroxybenzoic acid (1) and m-hydroxybenzoic acid (2) were converted into their corresponding glycosides (1a and 2a) and glycosyl esters (1b and 2b) while no metabolite of o-hydroxybenzoic acid (3) was detected. A new compound, m-hydroxybenzoic acid β-d-xylopyranosyl (1 → 6)-β-d-glycopyranosyl ester (2c) was identified as a biotransformation product of 2. Further time-course studies of the biotransformation reactions showed that the glycosides were major products in the latter stage. The addition of carbohydrates or antioxidants increased glycosyl esters formation.     

Elicitation of different Panax ginseng transformed root phenotypes for an improved ginsenoside production

We tested the effect of the presence in the culture medium of chitosan, vanadyl sulfate or methyl jasmonate on growth and ginsenoside production of three stable hairy root lines of Panax ginseng C.A. Meyer showing different morphological phenotypes C-M, HR-M and T-M. The response depended upon line phenotype, specificity of the elicitor and the stage of growth at which the lines were treated. The highest ginsenoside yield was found when methyl jasmonate was added during the progressive deceleration growth phase (on day 25 of culture). In this case, the ginsenoside content reached at the end of the culture (day 28) by root lines C-M, HR-M and T-M was, respectively, 2, 1.8 and 4 times higher than the highest content achieved, also at the end of the culture, by the corresponding untreated root lines. Under the same conditions, the ginsenoside content in the presence of vanadyl sulfate also increased considerably, while with chitosan it clearly decreased. The ginsenoside pattern in response to the presence of the elicitors is also considered.     

Identification and differentiation of Panax species using ELISA, RAPD and eastern blotting

Immunochemical and genetic methods have been developed in order to distinguish Panax spp. With the aim of establishing immunochemical methods, two hybridomas (3H4 and 5H8), each secreting a monoclonal antibody (MAb) against proteins of Panax ginseng, were prepared by fusing splenocytes immunized with two kinds of ginseng water-soluble fractions and a hypoxanthine-thymidine-aminopterin-sensitive mouse myeloma cell line, P3-X63-Ag8-U1. MAb 3H4 cross-reacted with four Panax spp., whereas the MAb 5H8 cross-reacted with P. ginseng in the enzyme-linked immunosorbent assay (ELISA). ELISA and western blotting methods using a ginseng water-soluble fraction as the solid-phase antigen were developed for the unambiguous authentication of P. ginseng. A combination of random amplified polymorphic DNA (RAPD) and eastern blotting analyses using anti-ginsenoside Rb1 and Rgl monoclonal antibodies was used for the identification of P. notoginseng, P. quinquefolius and P. japonicus. RAPD can be used to differentiate the species of Panax from each other. An important parameter used for differentiating P. notoginseng is the absence of ginsenoside Rc in the extract of P. notoginseng with eastern blotting. The combination of these methods enabled a reliable identification of Panax spp.