Accumulation of salicylic acid-elicited alkaloid compounds in in vitro cultured Pinellia ternata microtubers and expression profiling of genes associated with benzoic acid-derived alkaloid biosynthesis

The alkaloid compounds found in Pinellia ternata tubers have major bioactive components, and thus, these plant products are one of the most widely used ingredients in traditional Chinese medicines (TCMs). Under field agricultural growth conditions, however, it usually takes 2 years for tuber formation and growth. In vitro induced microtubers provide an alternative approach for the commercial production of P. ternata tubers for use in the TCM industry. The elicitation effect of supplementation with salicylic acid (SA) on the accumulation of alkaloid compounds in tubers and the related molecular regulation mechanism for biosynthesis are not well understood. In this study, we address this knowledge-gap through the development of an efficient induction system of in vitro cultured microtubers subsequently used to study the mechanism for elicitation of alkaloid compound accumulation by SA. Efficient microtuber induction was achieved by inserting petioles inversely into solid Murashige & Skoog medium (MS) followed by subculturing the morphologically expanded lower portion of the culture petioles in suspension culture, without the additional application of plant growth regulators. The in vitro microtuber induction rate achieved was 100% within 25 days of culture. When treated with 50–150 μM of SA, in vitro cultured microtubers showed higher accumulation of alkaloid compounds over the negative control. The highest accumulation detected showed an increase of 2.5-, 2.1-, 2.8-, and 3.1-fold in the concentration of total alkaloid compounds, guanosine, inosine and ephedrine, respectively, in the presence of 100 μM SA, 15 days after induction. qRT-PCR analysis of candidate genes for key enzymes in alkaloid biosynthesis indicated that CNL, CHY and BALDH are most probably responsible for the accumulation of benzoic acid and other alkaloid derivatives in the in vitro cultured microtubers following SA elicitation. This study developed an efficient in vitro microtuber induction system, and used this to determine that SA-promoted accumulation of alkaloids is associated with genes in the benzoic acid and alkaloid derivative biosynthesis pathway in P. ternata.

     

Use of protocorm-like bodies for studying alkaloid metabolism in <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Highly differentiated tissue masses known as protocorm-like bodies (PLBs) have been commonly used for plant regeneration. In this study the potential use of PLBs for studying alkaloid metabolism in the Chinese medicinal herb Pinellia ternata (Thunb.) Breit. was investigated. Tuber, leaf, and petiole explants of P. ternata were incubated on Murashige and Skoog (MS) (1962) basal medium containing different combinations of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). It was observed that 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid (ABA), ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS (1/2 MS) liquid medium supplemented with 0.6 mg/L ABA. Water extracts of PLBs collected from suspension cultures contained guanosine and inosine, two important alkaloids of P. ternata. Levels of guanosine concentrations were tenfold higher in tuber-derived PLBs compared to those in field-grown tubers; whereas, those of inosine were slightly lower in PLBs compared to those from field-grown tubers.     

Expression and characterization of two domains of Pinellia ternata agglutinin (PTA), a plant agglutinin from Pinellia ternata with antifungal activity

Pinellia ternata agglutinin (PTA) from P. ternata f. angustata is a two-domain GNA-related lectin. The current study indicates that the PTA gene encodes a precursor consisting of two tandemly arrayed domains, N-terminal domain (PTA-DOM1) and C-terminal domain (PTA-DOM2). Both domains and the precursor without signal peptide (PTA-P) present different number of activity mannose-binding sites which play key roles for the lectin function. Analyses of the three fusion proteins, PTA-DOM1, PTA-DOM2 and PTA-P, expressed in Escherichia coli revealed that one mannose-binding site the agglutination activity while the additional sites do not possess such activity. However, the number of carbohydrate-binding sites suggests some significant properties on the antifungal effectiveness. In addition, each of the PTA domains has the same function when compared with the natural PTA (N-PTA). The information on PTA gene obtained in this study will served as baseline information in developing this protein as a form of transgenic plant protection.     

In vitro induction and proliferation of protocorm-like bodies (PLBs) from leaf segments of <Emphasis Type="Italic">Phalaenopsis bellina</Emphasis> (Rchb.f.) Christenson

An in vitro culture procedure was established to induce protocorm-like bodies (PLBs) from leaf segments of the Phalaenopsis bellina (Rchb.f.) Christenson directly from epidermal cells without intervening callus on ½ strength modified Murashige and Skoog (MS) (in Physiol Plant 15:473–497, 1962) medium supplemented with 1-Naphthaleneacetic acid (NAA; 0, 0.1, 1 mg/l) and Thidiazuron (TDZ; 0, 0.1, 1, 3 mg/l). The best response was established at 3 mg/l TDZ which induced 78% of leaf segments to form a mean number of 14 PLBs per explant after 16 weeks of culture. No PLBs were found when leaf segments were cultured on ½ strength modified MS media supplemented with 0.1 and 1 mg/l NAA. The best induction percentage for auxin: cytokinin combination was at the combination of NAA and TDZ at 1.0 and 3.0 mg/l which gave 72% induction with 9 PLBs per explant. Semi-solid ½ strength MS and liquid Vacin and Went (VW) (in Bot Gaz 110:605–613, 1949) medium were used in order to find the highest survival and number of PLBs proliferation after 3 months in culture. Half strength MS showed an average of 9 PLBs in comparison with VW with an average of 5.3 PLBs per explants. Histological observations revealed that the regenerated PLBs were generally formed from the epidermal layers of the posterior regions of the leaf segments. Scanning electron micrograph of PLBs showed the origin of newly formed PLB from the peripheral region of leaf segments.     

Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of <Emphasis Type="Italic">Oncidium flexuosum</Emphasis> Sims (Orchidaceae)

The present study describes the direct regeneration of protocorm-like bodies (PLBs) in leaf explants of the tropical species Oncidium flexuosum. The explants were inoculated in a solid, modified Murashige and Skoog (MS) medium with different concentrations of the growth regulator thidiazuron (TDZ) and with or without 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA), and kept away from light or in a 16-h photoperiod. The presence of auxins, 2,4-D, and NAA inhibited the formation of PLBs. The highest frequency of explants that regenerated PLBs (80%) was obtained when they were maintained in a culture medium containing 1.5 μM TDZ under dark conditions. In the same culture medium but under a 16-h photoperiod, 95% of the leaf explants presented necrosis. Therefore, darkness was crucial for the regeneration of PLBs in O. flexuosum leaf explants, which is in disagreement with the literature. PLBs developed from the division of epidermal and subepidermal cells mainly on the adaxial side of the apex region of the explant. Plants with well-developed leaves and roots grew after the PLBs were transferred to growth regulator-free medium under a 16-h photoperiod.     

Chemical constituents from the tubers of Pinellia ternata (Araceae) and their chemotaxonomic interest

This work describes the isolation and characterization of twenty compounds from the tubers of Pinellia ternata (Thunb.) Breit, including 17 lignanoids (1–17) and three alkaloids (18–20). All structures were established based on analysis of their spectroscopic data. It is the first report of compounds 3–11 and 13–18 from the genus Pinellia, and the first report of compounds 3–4, 6–9, 11 and 13–18 from the family Araceae. In addition, the biosynthetic pathways of the 17 lignanoids are summarized. A liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOFMS/MS) method in both positive and negative ion mode was also used to confirm the identity of these seventeen lignanoids from the tubers of P. ternata. The results from the present work provide further information about the diversity of compounds in the genus Pinellia.     

三叶半夏叶片一步成苗离体培养技术

以药用植物三叶半夏叶片为材料,通过比较直接和间接器官发生两种途径,建立了半夏一步成苗的快速繁殖技术体系。结果表明,经过愈伤组织阶段的一步成苗培养基为MS+0.5mg/L2,4-D+1.0mg/LKT,90d左右方可得到再生植株,植株分化率为74%,每个外植体上分化的块茎数为5.61±1.04。附加NAA与BA两种激素对一步成苗培养基进行优化,筛选出一步成苗最佳培养基MS+0.5mg/LNAA+0.5mg/LBA,60d后就可直接发育成完整植株,植株分化率为76%,每个外植体上分化的块茎数高达9.97±0·81,对这种培养基上的再生小植株进行移栽,1个月后,移栽成活率达100%。     

半夏产生物碱内生菌的分离及其抑菌活性的初步研究

用平板分离法从不同产地的半夏根、茎、叶中共分离到内生菌87株.其中,内生真菌48株、细菌34株、放线菌5株;通过点植法、插片法和印片法,并根据菌落、菌丝体、孢子形态特征,将48株内生真菌分为4科、15属;经HPLC法检测,共筛选到12株产生物碱类物质的内生菌,其中,10株为内生细菌,2株为内生真菌;有3株内生细菌对金黄色葡萄球菌有体外抑菌活性.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for "native" lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for “native” lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

Protocorm-like bodies (PLBs) of Dendrobium Sabin Blue: a novel source for in vitro production of dendrobine and anthocyanin

In Vitro Cellular & Developmental Biology - Plant - Dendrobium hybrids have been cultivated as commercially important ornamental plants in the floriculture industry. However, the potential of...     

Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L⁻¹ gellan gum-solidified NDM containing 10 g L⁻¹ sucrose, 20 mg L⁻¹ hygromycin and 40 mg L⁻¹ meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 μM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.     

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.     

Seed bulb size influences the effects of exogenous brassinolide on yield and quality of Pinellia ternata

• In recent years, natural Pinellia ternata populations of have gradually been exhausted, while the cultivated yield has been limited due to lack of research and uncertain climate condition. Therefore, it is necessary to explore methods of improving yield and quality in P. ternata using brassinolide (BR) treatments and choice of a suitable seed bulb size.
• This article reports the effects of BR and two seed bulb sizes (diameter: 0.5–1.0 cm and 1.0–1.5 cm) on active and nutrient components and antioxidant activity in P. ternata. The experiment included six levels of BR (0, 0.05, 0.10, 0.50, 1.00 and 2.00 mg l^?1).
• The tuber yield of the two seed bulb sizes and bulbil yield of small seed bulbs increased 5.67%, 22.66% and 69.23% by day 105 after 0.50 mg l^?1 BR treatment, compared with the control. On day 105, only 0.05 mg l^?1 BR increased scores in principal components analysis (PCA) in tubers of small seed bulbs by 167.29%, and 0.05 and 0.50 mg l^?1 BR increased PCA score in bulbils of large seed bulbs by 145.66% and 252.97%, respectively, compared with the control. Significant BR × seed bulb size interactions were found on yield and quality of P. ternata.
• The results indicate that BR effects on yield and quality of tubers and bulbils of P. ternata are not only related to BR concentration but also to seed bulb size.

     

半夏的繁殖生物学研究

对“泰半夏”（Ｐｉｎｅｌｌｉａｔｅｒｎａｔａ（Ｔｈｕｎｂ．）Ｂｒｅｉｔ．）的块茎和珠芽栽培观察结果表明：（１）不同繁殖体的叶形和珠芽所发生的变化与实验用的播种材料有关,珠芽发生频率与叶形变化呈正相关;（２）半夏倒苗既是对不利环境条件的一种适应,也是一种有效的无性繁殖方式;（３）半夏有性繁殖是属于同株异花传粉类型,但与无性繁殖相比,有性繁殖在种质繁衍上仅起着次要作用。     

Cryopreservation of protocorm-like bodies (PLBs) of <Emphasis Type="Italic">Phalaenopsis bellina</Emphasis> (Rchb.f.) christenson by encapsulation-dehydration

Protocorm-like bodies (PLBs) of Phalaenopsis bellina were successfully cryopreserved by the encapsulation-dehydration approach. Various stages in obtaining successful cryopreservation using this method were optimized. Encapsulated PLBs precultured in half-strength MS medium supplemented with 0.75 M sucrose for 3 days exhibited the highest viability in terms of 2,3,5-triphenyltetrazoliumchloride (TTC) reduction. The amount of sucrose in the PLBs after incubation in different concentrations of sucrose for different periods of time determined by HPLC. The highest sucrose concentration was 7 mg/g of PLBs for the PLBs treated with 0.75 M sucrose for 3 days as compared to the control which had only 1 mg/g sucrose. After sucrose preculture, the PLBs were subjected to desiccation using one of two methods. Desiccation using silica gel was more efficient in reducing PLBs moisture content. After 6 h of desiccation, PLBs desiccated using laminar air flow had 43.5% moisture content while for those desiccated using silica gel had 32% moisture content. PLBs desiccated to different moisture contents were plunged into LN. After storage in LN the encapsulated PLBs were re-warmed. Two weeks after re-warming PLBs viability was determined by TTC reduction and re-growth assessed. Encapsulated PLBs precultured with 0.75 M sucrose for 3 days followed by desiccated using silica gel for 5 h resulting in a moisture content of 39% lead to the highest post re-warming viability in terms of TTC reduction (46.6% of control PLBs) and 30% re-growth.     

贵州半夏块茎腐烂病病原菌的分离与鉴定

【目的】对贵州省半夏块茎腐烂病病原菌进行分离和鉴定,为开发有效的防治技术体系提供依据。【方法】采用组织分离法进行病原菌分离,根据柯氏法则进行致病性测定,结合形态学、生理生化特性和分子生物学方法进行鉴定。【结果】从贵州3个半夏产地患病样品中分离出12株病原细菌和5株病原真菌。经形态学、生理生化特性和分子生物学方法鉴定,12株病原细菌为胡萝卜软腐果胶杆菌胡萝卜软腐亚种;ZJ、Z3病原真菌为尖孢镰刀菌,D3、D5、H1为茄病镰刀菌。【结论】贵州省半夏块茎腐烂病的病原菌有病原细菌和病原真菌。     

Abnormal male meiosis explains pollen sterility in the polyploid medicinal plant Pinellia ternata (Araceae)

Pinellia ternata is an important traditional Chinese medicinal plant. Its different populations in China have various ploidy levels, based on x = 13, as well as extensive aneuploid series. The microsporogenesis process was observed in specimens from three populations from three regions of Hubei Province; they were characterized by normal and abnormal meiotic divisions in pollen mother cells (PMCs) at all stages simultaneously. Meiotic abnormalities including univalents/multivalents, chromosomal laggards/bridges and micronuclei appeared in about 50% of the PMCs, together with abnormal cytokinesis. Chromatin/chromosome transfer between meiocytes occurred only during the first division, at low frequency; this might contribute to these meiotic abnormalities. Although the remaining 50% of the PMCs presented normal cytological behavior, pollen fertility was only about 2%. These results provide cytological explanations for its low seed-set and the general use of asexual reproduction through tubers and bulbils; it also explains the wide variations in chromosome number.     

细茎石斛拟原球茎生长与有效成分积累的关系

目的 :研究细茎石斛拟原球茎生长规律及其与两种有效成分总生物碱和可溶性多糖积累的关系。方法 :采用液体静止培养方法培养细茎石斛拟原球茎 ,在一个周期 ( 60天 )内 ,每隔 1 0天取样 ,测定拟原球茎的鲜、干重 ,总生物碱含量和可溶性多糖含量。结果 :液体培养的细茎石斛拟原球茎生长曲线大致呈“S”型 ,拟原球茎经过 1 0d左右的延迟期后进入对数生长期 ,第 5 0d左右达到生长高峰 ,此后 ,生长缓慢 ,进入静止期。拟原球茎中总生物碱和可溶性多糖含量随着拟原球茎的生长逐渐积累 ,在培养的第 40d左右达到高峰 ,之后含量有所下降 ,这两种有效成分的积累与生长基本同步 ,且拟原球茎中两种有效成分的含量接近或超过野生植株茎中的含量。结论 :液体培养的细茎石斛拟原球茎生长曲线大致呈“S”型 ,总生物碱和可溶性多糖的积累与生长基本同步 ,且拟原球茎中两种有效成分的含量接近或超过野生植株茎中的含量