油樟精油对半夏开放式组培愈伤组织诱导及分化的作用

以半夏(Pinellia ternata)叶柄、叶片作为外植体,采用组培方法研究不同浓度油樟精油对半夏愈伤组织诱导及分化的作用。结果表明,在基本培养基MS配方基础上,TDZ 0.3 mg/L和油樟精油体积浓度为0.20 mL/L的配方组合,半夏叶柄及叶片愈伤组织诱导率分别为92.3%、95.7%;TDZ 0.3 mg/L和油樟精油体积浓度为0.10 mL/L的配方组合时,分化率为100.0%,平均丛生苗数达5.25个,分化效果最佳。发现油樟精油体积浓度为0.20 mL/L时的促进效果相对较好,可以有效促进愈伤组织的诱导,提高愈伤诱导率,愈伤组织质地较紧密,呈浅绿色,增殖效果较好,分化率达100%,愈伤组织出现的芽点相对明显,且数量相对较多。综合培养成本来看,油樟精油在半夏开放式组培体系的最适浓度为0.20 mL/L。

Oxalic acid in ginger specifically denatures the acrid raphides in the unprocessed dried tuber of Pinellia ternata

Objective:Pinellia Tuber,the dried tuber of Pinellia ternata,is widely used in Japanese Kampo medicines and traditional Chinese medicines.The unprocessed Pinellia Tuber is known to cause very strong acrid irritation at oral and laryngopharynx mucosa.Recent studies have shown that the sharp needle-like crystals called raphides,that are composed of calcium oxalate and proteins,are the main causative substances of the irritation.Ginger,the rhizome of Zingiber officinale,has been used in the processing to reduce the acridity of Pinellia Tuber since before the sixth century,however,the mechanisms of reducing acridity have not been scientifically proved yet.Methods:We developed the raphides denaturation assay(RDA)to quantify the degree of denaturation in the raphides to cause irritation.By their lipophilic characters,the raphides could be extracted in petroleum ether(PE)layer from powdered Pinellia Tuber suspended in water,and the contents of the raphides in PE layer were measured by the absorbance.By this assay,we conducted the activity-guided fractionation from the boiling water extract of ginger to find the ingredients to denature the raphides.We also conducted the gustatory tests to detect the change of the irritation of the denatured raphides.Results:The treatment of powdered Pinellia Tuber suspension with ginger extract reduced the distribution of raphides in PE layer in RDA in a concentration-dependent manner.The activity-guided fractionation using RDA revealed that oxalic acid was the main active ingredient in ginger extract to denature the raphides of Pinellia Tuber.Oxalic acid reduced the lipophilicity of the raphides in the thermo-,time-,and concentration-dependent manners,and its activity was affected by pH.The treatment of powdered Pinellia Tuber suspension with oxalic acid significantly reduced its acrid irritation in gustatory test in human.Conclusions:We found that oxalic acid is the main active ingredient in ginger to reduce the acrid irritation of Pinellia Tuber.

Research Progress of the Wild Medicinal Plant,Pinellia ternata

Based on literature reviews and analysis of research reports on Pinellia ternata found locally and abroad in recent years,this article summarizes and arranges them.The research on Pinellia ternata mainly focuses on its cultivation,tissue culture,and so on.There are only a few research on its active components and its regulation mechanism.The wild resources of Pinellia ternata are gradually decreasing,hence it is urgent to take effective measures to protect these wild resources as well as to establish germplasm resources bank and nursery.In order to meet the needs of the domestic market,it is necessary to investigate the distribution of wild Pinellia ternata resources,explore the best growing environment and conditions,artificially cultivate Pinellia ternata,as well as implement resource industrialization,sustainable development,and utilization.

内生菌协同发酵对半夏多糖及其生物活性的影响

利用内生菌协同发酵提高半夏[Pinellia ternata(Thunb.)Breit.]多糖含量,并探究对其生物活性的影响。以半夏粉末为底物,采用筛选得到的高产胞外多糖半夏内生菌进行固态协同发酵;以多糖得率为指标,筛选出菌种的最适添加比例;通过单因素试验与正交试验对发酵条件进行优化;并对发酵前、后半夏多糖的电镜结构、抗氧化和降血糖能力进行测定。结果表明,菌种的最适添加比例为土曲霉∶亚黑管菌=2∶1;最佳发酵条件为发酵温度31℃、发酵时间110 h、接种量12%,最终半夏多糖的得率为21.76%,相较于未发酵得到显著提高(P<0.05);发酵后的半夏多糖对DPPH自由基、ABTS自由基、羟基自由基的IC50分别为0.355、0.600、0.585 mg/mL,相比发酵前半夏多糖的IC50分别减少了0.137、0.231、0.218 mg/mL;其中,它对3种自由基的清除能力从大到小依次为DPPH自由基、ABTS自由基、羟基自由基;发酵后半夏多糖抑制α-葡萄糖苷酶的IC50比发酵前减小了0.293 mg/mL。由此可知,内生菌协同发酵能够提高半夏多糖的提取率以及抗氧化与降血糖能力。

荆半夏多倍体新种质诱导研究

设置不同浓度和不同诱导时间的秋水仙素,采用混培法和覆棉法对荆半夏[Pinellia. ternata(Thumb) Berit]进行离体和活体的多倍体诱导,以期建立荆半夏多倍体的化学诱变技术体系。结果表明:采用混培法对荆半夏进行离体多倍体诱导的最佳条件是0.1%秋水仙素溶液处理3d,成活率为48.7%,诱导率28.53%;采用覆棉法进行活体多倍体诱导的最佳条件是0.1%秋水仙素溶液处理6d,诱导率为60.91%,成活率为31.83%;经倍性鉴定,获得了荆半夏多倍体植株(2n=16x=208),其多倍体植株叶片大而肥厚、叶绿素含量增加,植株紧凑、粗壮,且叶片的气孔变大、密度减小。

间作大豆对半夏生长环境及产量与药材质量的影响

为探究间作大豆对半夏[Pinellia ternata(Thunb.)Breit]生长及药材质量的影响,在固定半夏播种密度下,设置35、50、65 cm的大豆株距,分析间作大豆条件下半夏生长环境的改变、生长状况、间作系统的土地当量比和种间竞争力以及半夏产量和药材质量的变化。结果表明,随着大豆种植密度的增加,半夏冠层光照度降低,叶面温度降低,土壤温度降低;间作大豆显著增加了半夏主叶叶面积、出土部分叶柄长及叶片SPAD值;间作系统中土地当量比增加,大豆竞争力强于半夏;间作大豆显著增加了半夏产量以及千粒重,其中以大豆50 cm株距间作下半夏产量最高,较半夏单作增产42.9%;间作大豆下的半夏药材质量和对照无显著差异,均符合药典要求。间作大豆为半夏提供了更良好的生长环境,增强了半夏生长势,提高了产量。

Physiological and transcriptional responses to heat stress in a typical phenotype of Pinellia ternata

Pinellia ternata is an important medicinal plant,and its growth and development are easily threatened by high temperature.In this study,comprehensive research on physiological,cytological and transcriptional responses to different levels of heat stress were conducted on a typical phenotype of P.ternata.First,P.ternata exhibited tolerance to the increased temperature,which was supported by normal growing leaves,as well as decreased and sustained photosynthetic parameters.Severe stress aggravated the damages,and P.ternata displayed an obvious leaf senescence phenotype,with significantly increased SOD and POD activities(46%and 213%).In addition,mesophyll cells were seriously damaged,chloroplast thylakoid was fuzzy,grana lamellae and stroma lamellae were obviously broken,and grana thylakoids were stacked,resulting in a dramatically declined photosynthetic rate(74.6%).Moreover,a total of 16808 genes were significantly differential expressed during this process,most of which were involved in photosynthesis,transmembrane transporter activity and plastid metabolism.The number of differentially expressed transcription factors in MYB and bHLH families was the largest,indicating that these genes might participate in heat stress response in P.ternata.These findings provide insight into the response to high temperature and facilitate the standardized cultivation of P.ternata.

Explanation of Banxia Xiexin decoction based on “Xinkai Kujiang”

Banxia Xiexin decoction(BXD)is a representative formula of Zhang Zhongjing(Eastern Han Dynasty)for the treatment of pi syndrome,which is a combination of cold and heat,and its application in the treatment of digestive system diseases is remarkable.In this paper,we discuss how Chinese medicine recognizes“spleen”and“Pi syndrome”to explain and explore the basis of BXD prescription and analyze both clinical examples and drug toxicity to provide more basis for the clinical application of BXD.

4种不同居群半夏在林下种植的适宜性比较

在林下用沙壤土盆栽种植4种不同居群半夏(Pinellia ternata),测定4种不同居群半夏在林下的发芽指标、农艺性状、抗氧化能力,以进一步比较4种不同居群半夏在林下种植的优劣及改善方式。结果发现A居群半夏的综合发芽能力最佳,A和C居群半夏的农艺性状综合优于B和D,A居群半夏抗氧化能力最强,结果说明4种不同居群半夏中,A(四川南充)和C(河北保定)2个居群半夏在林下种植综合性状更优良,更适宜在林下种植。

半夏悬浮培养细胞分化形成人工种胚的同步化条件

为获得同步化的人工种胚,以半夏疏松愈伤组织为试验材料,采用悬浮培养的方法,探讨温度、咖啡因、蔗糖浓度对半夏悬浮培养细胞分化形成人工种胚的同步化作用,为半夏人工种子制作提供同步化种胚。结果表明:以MS为基本培养基,添加1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L sucrose+300 mg/L CH进行悬浮培养,温度为8℃,处理12h,恢复36h,同步化效果最好,细胞分裂指数为13.86%;咖啡因浓度为2.00 mmol/L,处理2 h,恢复48 h,效果较好,细胞分裂指数为9.42%;蔗糖浓度为40 g/L,处理5 d,恢复4 d,效果一般,细胞分裂指数为5.33%。分别先后继代3次于MS+1.0 mg/L IBA+0.5 mg/L 6-BA+30 g/L sucrose+300 mg/L CH进行膨大培养及MS+0.1 mg/L IBA+0.5 mg/L 6-BA+10 g/L sucrose+300 mg/L CH进行分化培养。温度处理组微细胞团为42.33个,种胚分化率为71.11%;咖啡因处理组微细胞团为34.33个,种胚分化率为55.56%;蔗糖处理组微细胞团为40个,种胚分化率为57.78%。因此,半夏悬浮培养细胞同步化形成人工种胚的适宜条件为:MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L sucrose+300 mg/L CH,8℃处理12 h,恢复36 h,再转接进膨大及分化培养基继续培养。本研究为半夏人工种子生产提供技术参考。

半夏居群遗传多样性及遗传结构分析

为了研究中国野生半夏的遗传多样性及谱系结构,揭示其地理分布格局的形成原因,推测该物种潜在的冰期避难所。对分布于中国16个省份20个居群(205个个体)的半夏核基因ITS序列进行PCR扩增、测序分析。结果发现:在所有的半夏居群中共检测到14个单倍型,其中单倍型H3、H6在居群中分布较广。分子方差分析(AMOVA)结果显示,半夏居群的遗传变异主要发生在居群间(75.83%),居群间遗传分化系数(GST=0.7100.05),具有明显的谱系地理结构且基因流较低(Nm=0.019);中性检验结果Tajima’s D为0.37629(P>0.10),Fu’s Fs值为-1.883,结合失配分析的单峰曲线分布表明半夏居群近期经历过小范围快速扩张。研究推测,半夏在第四纪冰期时可能存在多个避难所。

旱半夏ISSR-PCR反应体系的优化

为了建立和完善旱半夏的ISSR-PCR反应体系,本研究以四川地区野生旱半夏为试验材料。以旱半夏整株的基因组DNA为模板,初筛的UBC818号引物用于ISSR-PCR体系,采用正交实验L_(16)(4^(5))并结合单因素实验方法对体系进行优化。通过正交试验结果直观评分可知各因素对该实验结果的影响强度从高到低依次为:d NTPs,引物,Mg^(2+),Taq DNA聚合酶,DNA模板;单因素实验结果表明,最优的20μL反应体系为:d NTPs 0.2 mmol/L,引物0.8μmol/L,Mg^(2+)2.0 mmol/L,Taq DNA聚合酶0.075 U/μL,DNA模板15 ng,退火温度为56℃。本研究通过优化ISSR-PCR反应体系,此体系将应用于半夏组培苗遗传稳定性的检测。