Optimization of Inducing Conditions for Loose Callus of Pinellia ternata

[Objective] The research aimed to establish the cell suspension culture system of Pinellia ternate.[Method] The petiole of Pinellia ternate was as the explant.The orthogonal test was used to study the influences of four plant growth substances（2,4-D,NAA,picloram and KT）and their mixture ratios on the formation of loose callus.[Result] The induction effect of 2,4-D and picloram on the petiole callus of Pinellia ternate was the most significant.Then,the second ones were KT and NAA.The optimal medium which induced Pinellia ternate petiole to form the loose callus was MS＋0.5 mg/L of 2,4-D＋1.0 mg/L of NAA＋1.0 mg/L of picloram＋1.5 mg/L of KT.[Conclusion] The research laid the foundation for extracting the active ingredient from the cell suspension of Pinellia ternate and producing the artificial seed.

三叶半夏(Pinellia ternata)雄配子败育的遗传分析

从减数分裂过程，小池子发育和染色体数目与组成三方面探讨了三叶半夏雄配子败育的原 因。三叶半夏（Ｐｉｎｅｌｌｉａ ｔｅｒｎａｔａ）体细胞染色体数目２ｎ＝９０，其减数分裂异常，多数同源染色体能配对形 成二价体，部分同源染色体呈单价体形式或配对成多价体。减数分裂的各时期都能观察到导致遗传物 质不均衡分离的异常结构，如落后染色体，染色体桥和微核等。根据结果推测：三叶半夏是具有染色体 结构变异的多倍体复合种。染色体结构变异是三叶半夏雄配子败育的主要原因。小孢子母细胞都能完 成减数分裂过程，且单核小孢子能通过两次有丝分裂形成三核花粉粒。成熟花粉粒中可育花粉不足 １％。三核期是雄配子败育的时期。

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.

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.

Characterization and biological activities of lectin isolated from <i>Pinellia ternata</i>

In this paper, Pinellia ternata lectin (PTL) purified from 95% saturated ammonium sulfate precipitation by applying mannose-Sepharose 4B affinity column chromatography was first described. The mannose-Sepharose 4B affinity adsorption gel has already been designed and prepared before the experiments according the combinative characterization between lectin and mannose. Mass spectrometry analysis result shows that PTL is a glycoprotein with the molecular weight of 12.165 kD. It was conclude that PTL had strong agglutination effects on mouse red blood cells, and the minimum reaction concentration was 25 μg/ml according the Hemagglutination. Results of automatic amino acid analysis indicated that PTL mainly contained 15 varieties of amino acids, of which the minimum content was cysteine and aspartate was the maximum. Cell experiment results suggested that PTL of low concentrations (0.004 mg/ml, 0.02 mg/ml and 0.1 mg/ml) promoted HeLa cell proliferation, but the effect weakened with the concentrations and treated-time increasing. However, the HeLa cells pro- liferation was intensively inhibited by higher PTL concentrations (0.5 mg/ml and 1 mg/ml), and the effect increased in a dose and time dependent manner.

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.

Preliminary analysis of the protein contents in Pinellia ternata with different types of leaves

Total proteins extracted from Pinellia ternata with different types of leaves have been assayed by UV absorbance, blue native polyacrylamide gel electrophoresis （BN-PAGE） and sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. We found that the total protein contents of P. ternata with the herbaceous peony and claw types of leaves were much higher than that with other types of leaves. P. ternata collected from Nanchuan County, except the ones with herbaceous peony leaves, displayed a high similarity with each other. Both UV absorbance and cluster analysis indicated that the protein content in P. ternata with the herbaceous peony leaves was less affected by habitats than that with other types of leaves. The results showed that it was necessary to increase the homogeneity of seeds for improving the protein content ofP. ternata.

三叶半夏(Pinellia ternata(Thunb.)Breit)的间歇浸没培养

目的:利用间歇浸没培养反应器对药用植物三叶半夏(Pinellia ternata(Thunb.)Breit)进行组培快繁研究。方法:以增殖倍数、鲜重、平均鲜重、折干率和组培苗状态为指标,一方面对三叶半夏叶片、叶柄及丛生芽体进行培养,考察适合间歇浸没培养的外植体类型;另一方面以叶柄为外植体对三叶半夏进行间歇浸没培养、固体培养和液体摇瓶培养,考察三种培养方式对其生长的影响。结果:不同的外植体在反应器内培养时,从增殖情况来看,丛生芽作为外植体得到的增殖倍数高达39.15,叶柄为27.65,叶片为18.05。鲜重方面与增殖倍数具有相同的规律。从平均鲜重和折干率来看,无显著差异。由组培苗形态来看,以叶柄诱导的组培苗最为健壮,而以丛生芽诱导的组培苗则较为细弱。不同培养方式下的增殖倍数分别为:间歇浸没培养方式最高为24.73,其次固体培养为14.75,液体摇瓶培养为12.26,差异显著。平均鲜重和折干率均为间歇浸没培养最优。由组培苗形态来看固体培养优于间歇浸没培养,反应器得到的组培苗较为细弱。在保卫细胞长宽和叶绿体个数上两者没有显著性差异。而液体摇瓶培养玻璃化导致叶绿体个数减少,不适合培养三叶半夏的。结论:较适合间歇浸没培养的外植体类型为叶柄和丛生芽,以叶柄为优。间歇浸没培养方式相对于传统的固体和液体培养在各项生理指标上表现较好,具有大规模培养三叶半夏的潜力。

Discrimination of toxic ingredient between raw and processed Pinellia ternata by UPLC/Q-TOF-MS/MS with principal component analysis and T-test

Objective: To investigate the toxicity difference between raw and processed Pinelliae Rhizoma(Banxia in Chinese, BX), the rhizoma of Pinellia ternata, from the view of chemical composition.Methods: Sixteen samples of raw and processed BX were prepared and analyzed by UPLC/Q-TOF-MS/MS.The discrimination(chemical marker) between the two group was investigated by principal component analysis(PCA) and T-test analysis. According to the accurate charge-to-mass ratio, MS/MS fragments, and comparison of corresponding data with the reference or database, the chemical markers were identified preliminarily.Results: Liquiritin, liquiritigenin, and lysophosphatidylcholine(LPC) were identified as the characteristic markers. The reducing of LPC in processed BX was one of the main reasons for detoxification because LPC could induce the inflammatory response;Liquiritin and liquiritigenin showed the anti-inflammatory effect and reduced liver injury, therefore the appearance of them in processed BX was an another reason for detoxification.Conclusion: An approach to explain the mechanisms of reducing the toxicity in medicinal plants by processing was proposed. Moreover, the chemical markers of toxicity could be used to differentiate the raw material from processed herbs for the quality control and safety application in clinical practice.

Cation Selectivity of Channels Formed at Planar Lipid Bilayer by Pinellia ternata Lectin

By means of the two-compartment system. PTL-channels, the first lectin channels formed on planar lipid bilayers by Pinellia ternata lectin (PTL) have been studied. The results show (i) PTL-channels are voltage-independent and have apparent subunits; (ii) in 50 mmol/L KC1 and 25 mmol/L BaCl2 solutions, a single channel has unit conductance of 21 pS and 42 pS, respectively; (iii) the channel exhibits a slightly higher permeability to divalent than monovalent cation (PBa/PK=4.1), and (iv) the selectivity among divalent or monovalent cations is poor. The cation selectivity sequence for the channel will follow PBa(7. 0)-Psr (6. 4)~PMg(6. 4)>PK(1. 7)>PN.(1. 0)~PLi(l. 0).Moreover, these data also give explanation to the facilitatory action of PTL on the release of acetyl-choline from motor nerve terminals.

Effect of Pinellia ternata Lectin on Membrane Currents of Mouse Motor Nerve Terminals

Pinellia ternata lectin (PTL) extracted from the fresh juice of rhizome of pinellia ternata used as a traditional Chinese medicine facilitated the quantal release of acetylcholine (ACh) in the mouse motor nerve terminals and formed cation channels in artificial lipid bilayer. Here we report the action of PTL on presynaptic membrane currents of motor nerve terminals.The experiments were performed on the intercostal nerve triangularis sterni muscle preparations. By means of the perineurial recording, the effects of PTL on the sodium current in the preterminal part , three potassium currents and two calcium currents generated from the nerve terminals were investigated. The results show that PTL increases voltage-dependent fast Ca2+ current (ICa,f), Na+ current (INa) and Ca2+-acti-vated K+ current (IK,Ca) without action on either the voltage-dependent fast K+ current (IK,f) or the slow K+ current (IK,S). These effects are irreversible, but can be reversed by mannan, the specific binding sugar for PTL.The total action of PTL can be considered to raise [Ca2+], by promoting Ca2+ influx, and then facilitate the neurotransmitter release. To our knowledge, this is the first report about the influence of lectin on presynaptic membrane currents at mammalian motor nerve terminals.

息半夏总生物碱的提取工艺优化及其生物活性分析

以息半夏的块茎为原料,乙醇为超声波辅助提取溶剂,考察乙醇体积分数、超声时间、料液比、超声温度等单因素对总生物碱提取率的影响,设计正交试验优化提取工艺,并测试总生物碱的体外抗氧化和降血糖活性。结果表明,最佳提取条件为乙醇体积分数65%、提取时间60 min、料液比1∶45、超声温度65℃,总生物碱的提取率达0.502%;总生物碱对DPPH、ABTS自由基的清除率分别达89.90%和43.60%,对α-葡萄糖苷酶的抑制率达86.40%。该工艺简单可行,总生物碱表现出良好的体外抗氧化和降血糖生物活性。

半夏提取物对淡色库蚊幼虫的杀虫活性

通过不同萃取相(石油醚、氯仿、乙酸乙酯、正丁醇和水)提取半夏(Pinellia ternata)的活性物质,采用浸液法测定半夏提取物对淡色库蚊(Culex pipiens pallens)幼虫的杀虫活性。结果表明,半夏甲醇提取物对淡色库蚊幼虫具有显著的毒杀活性;在0.5 mg·mL^(-1)浓度处理下,石油醚、氯仿萃取相对淡色库蚊幼虫具有显著的杀虫活性,致死率在50%以上。对石油醚、氯仿萃取相进行室内毒力测定,LC50分别为0.21和0.31 mg·mL^(-1)。研究结果可为新型植物源农药综合利用提供参考。

‘唐半夏’无菌繁殖体系建立

以‘唐半夏’块茎为试材,采用组织培养的方法,研究了不同种类及浓度的植物生长调节剂对不定芽诱导、增殖、生根的影响以及电流刺激对不定芽增殖的影响,以期为‘唐半夏’无性快速繁殖体系的建立提供参考依据。结果表明:最适不定芽诱导培养基为MS+1.5 mg·^(L-1)6-BA+0.1 mg·L^(-1)NAA,平均每个外植体诱导的不定芽数最多(9.38个)且诱导率为93.75%;采用电流刺激不仅提高不定芽的增殖倍数,且对‘唐半夏’植株的生长产生明显的促进作用。特别是20 mA、5 min处理下,不定芽增殖倍数最大(13.31倍),组培苗生长健壮且出现生根的现象。最适生根诱导培养基为1/2 MS+0.2 mg·L^(-1)NAA,生根率为100%,生根数为23.75条。

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。

5%苯醚·咯菌腈悬浮种衣剂的制备及其在半夏种子处理中的应用

为探明成膜剂对悬浮种衣剂成膜效果的影响,采用湿法研磨法,对成膜剂进行筛选,研究5%苯醚·咯菌腈悬浮种衣剂配方的稳定性和对半夏种子安全性。确定5%苯醚·咯菌腈悬浮种衣剂的配方为:咯菌腈3%、苯醚甲环唑2%、脂肪醇聚氧乙烯醚1%、十二烷基苯磺酸钠1%、环氧大豆油1%、乙二醇1%、黄原胶0.5%、壳聚糖0.5%、碱性玫瑰精0.5%,水补足100%。所制悬浮种衣剂各项指标均符合产品质量标准的要求,成膜效果良好,对半夏种子发芽率无影响。

不同产地半夏农艺性状及光合生理特性研究

为分析不同产地半夏在同一试验地的农艺性状及光合生理特性差异,以2年生20个不同产地的半夏为试验材料,测定其农艺性状、光合生理指标及叶绿素荧光参数。结果表明,不同产地半夏在农艺性状上存在显著差异,其中,陕西商洛产半夏的植株最高、种球最大;贵州贵阳产半夏的叶面积最大;河北保定产半夏的农艺性状整体较低。不同产地半夏的光合参数也存在明显差异,推测其光合特性和光能利用率具有较大差别,四川和贵州产半夏具有较高光合潜力。相关性分析发现,净光合速率(net photosynthetic rate,Pn)与气孔导度(stomatal conductance,Gs)、水分利用率(water use rate,WUE)、最大荧光(maximal fluorescence,Fm)、电子传递速率(electron transport rate,ETR)和叶绿素a(chlorophyll a)含量呈极显著(P<0.01)正相关,与叶绿素b(chlorophyll b)含量呈显著(P<0.05)正相关。主成分分析发现,种球直径、中裂叶长、株高、蒸腾速率(transpiration rate,Tr)、光化学淬灭系数(photochemical quenching,qP)和胞间CO_(2)浓度(intercellular CO_(2) concentration,Ci)是影响半夏生长的主要指标。聚类分析发现,四川、贵州、甘肃、河南、湖南和陕西等产地的半夏农艺性状表现较好,且具有较强的光合潜力,被聚为第Ⅰ类;保定、邢台、张家口和文山等产半夏的各项指标较低,被聚为第Ⅱ类。综上所述,四川、贵州、甘肃、河南、湖南和陕西的半夏种源具有明显优势,有良好的培育前景。以上研究结果为半夏良种选育提供了理论依据。

高效防治半夏软腐病生防菌的筛选及其生物防治效果研究

[目的]本研究旨在从实验室现有的生防细菌菌库中筛选对半夏软腐病具有较高防治效果的生防菌株,为半夏软腐病的绿色防控技术提供新的菌株资源。[方法]通过平板拮抗从实验室保存的300株具有生防潜力的菌株中,筛选对半夏软腐病具有拮抗效果的菌株。进一步测定拮抗菌株的水解酶活性和透明圈半径,通过对抑菌圈和透明圈半径进行统计赋值得到对半夏软腐病较高防治效果的复筛菌株。通过离体及温室防效试验验证复筛菌株对半夏软腐病的防治效果。[结果]初筛得到20株对半夏软腐病具有生防潜力的菌株,测定抑菌圈、透明圈半径大小,按照统计结果对20株菌株进行赋值,结果显示5YN8、XZ231和7Ze3赋值分数最高,分别为11.0、11.0和10.5。经离体防效验证得到20株初筛菌株对半夏软腐病腐烂程度的不同影响,对比赋值大小及半夏块茎的腐烂程度发现建立的赋值体系与菌株的生防效果具有较好的相关性,即赋值分数越高,半夏块茎的生防效果越好。将筛选得到的赋值分数最高的3株生防菌株5YN8、XZ231与7Ze3进行温室防效验证,发现它们对半夏软腐病防治效果分别为85.01%、79.55%和79.62%。通过16S rRNA基因测序比对发现5YN8为贝莱斯芽胞杆菌(Bacillus velezensis),XZ231和7Ze3均为枯草芽胞杆菌(Bacillus subtilis)。[结论]5YN8、XZ231与7Ze3三株生防菌株能够有效防治半夏软腐病,具有潜在的应用价值。

利用组培技术构建半夏杂交体系

本研究通过构建半夏品系的杂交体系并研究杂交种苗的扩繁周期,以解决半夏自然杂交成功率低、杂交种苗繁育效率低的问题。对消毒过的半夏花粉和雌蕊以TTC法测定其活力,并通过对比实验确定雌蕊的最佳培养基,对3种新的半夏品系持续培养出F_(1)代杂交苗,通过杂交苗继代探究转接周期和成苗周期。结果表明,半夏雌蕊和花粉的最佳消毒时间为6 min;雌蕊的最佳培养基配方是半糖无琼脂MS培养基20.00 g/L+蔗糖15.00 g/L+琼脂5.00 g/L+6-BA 1.00 mg/L+NAA 0.01 mg/L,pH=5.8。以此为基础,培育出F_(1)代苗;4种半夏品系的F_(1)代杂交苗与亲本组培苗叶型有明显区别,经过扩繁发现了两个杂交种苗的扩繁周期。本研究建立了基于植物组织培养技术的半夏杂交体系并获得杂交苗,并探明杂交种苗的繁育周期。