3种石斛属植物和细叶石仙桃的红外光谱分析和鉴定

利用傅里叶红外光谱仪和OMINI采样器直接迅速地测定3种石斛属常用原料药植物和常用伪品细叶石仙桃的红外光谱。结果表明,细叶石仙桃与石斛属组3种植物茎叶的红外光谱图存在较大差异,亲缘关系较远,而石斛属组内种间红外光谱图的差异较小。其中,铁皮石斛茎的特征峰为1 254、1 416、1 5453、365 cm-1等,铁皮石斛叶的特征峰为914、1 061 cm-1等。研究表明,铜皮石斛与铁皮石斛具有相似的红外光谱图,它可作为药用石斛品种代用品,而细叶石仙桃则不能作为药用石斛的代用品。     

应用RAPD分子标记技术探讨3种石斛属植物的种间关系

采用RAPD分子标记技术,分析了金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的种间关系。10个引物产生的113条DNA扩增片段中,106条（93.81%）具有多态性,利用113个RAPD标记,计算遗传距离,利用非加权组平均法建立聚类图。结果表明,RAPD标记技术较好地从分子水平揭示金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的遗传背景、亲缘关系,并为后期在DNA水平上对药用石斛的开发利用提供资料。     

金钗石斛生物碱的组织化学定位研究

利用组织化学定位方法对金钗石斛不同生长年限和不同营养器官的生物碱分布和积累进行比较分析。结果表明,金钗石斛茎生物碱主要分布于基本组织,三年生金钗石斛的生物碱含量高于一年生和二年生金钗石斛。金钗石斛根部生物碱含量同样较多,主要分布于皮层薄壁细胞中;金钗石斛叶片生物碱含量较低,存在于上下表皮细胞中,与铁皮石斛和铜皮石斛完全不同。说明三年生金钗石斛作为最适宜采收期,而且金钗石斛根部具有很好的开发利用价值。金钗石斛叶片表皮细胞中呈现生物碱的原因尚未清楚,需要进一步研究。     

水分胁迫和胁迫后复水对玉米叶片生长速率的影响

玉米叶片延伸速率(LER)对水分状况的变化很敏感。快速干旱处理,水分消耗迅速,LER从最大到零需5h,叶水势改变0.5～0.6 MPa,缓慢干旱处理,水分消耗较慢,LER从最大到零需20h,叶水势改变1 MPa。缓慢干旱植株叶片成熟部位的渗透势,在任何LER下,均比快速干旱叶片更负。LER为零时,快速干旱叶渗透势为-1.3 MPa,缓慢干旱为一1.6MPa左右。短时间水分胁迫后复水,LER迅速增加,有部分补偿前期胁迫减少的生长量能力;长时间水分胁迫后复水,LER在6 h内不可能恢复到正常水平。     

石斛属植物茎部总RNA提取方法的研究

获得快速提取高质量石斛属植物茎总RNA的方法,为深入开展分子生物学研究奠定基础。采用7种方法提取铁皮石斛茎总RNA,凝胶电泳和紫外分光光度法检测质量,筛选最佳方法,并对方法进行验证。结果显示,经过比较,用改良华越洋多糖多酚试剂盒法(M7)提取的铁皮石斛茎总RNA的完整性、浓度和纯度均最好。用M7提取铁皮石斛、金钗石斛、鼓槌石斛、球花石斛和重唇石斛的茎总RNA,以及提取不同生长条件(温室和大棚种植)和不同生长时间(大棚生长3个月、1年和2年)铁皮石斛的茎总RNA,所获样品的完整性、浓度和纯度均符合质量要求。M7操作简便,结果重复性好,适于石斛属植物茎总RNA的提取。     

树锦鸡儿、柠条锦鸡儿、小叶锦鸡儿和鹰嘴豆干旱适应能力比较

对1年生豆科作物鹰嘴豆Rupali品种和Almaz品种以及多年生植物树锦鸡儿、柠条锦鸡儿和小叶锦鸡儿叶相对含水量和叶水势对逐渐干旱胁迫的响应进行了分析,比较了两类植物的干旱适应能力。结果表明鹰嘴豆叶相对含水量随叶水势的下降线性下降,树锦鸡儿、柠条锦鸡儿和小叶锦鸡儿叶相对含水量在叶水势分别下降到-2.4MPa、-2.5MPa和-1.5 MPa之前没有下降,之后随水势的下降线性下降。树锦鸡儿、柠条锦鸡儿和小叶锦鸡儿这种叶相对含水量下降的滞后性表明该类植物叶具有较硬而弹性较差的细胞壁,使得植物在干旱胁迫下叶具有良好的保水能力。鹰嘴豆叶具有的最低水势为-4.5MPa,对应的土壤相对含水量为14％,锦鸡儿植物叶具有的最低水势可达-6.7MPa,对应的土壤相对含水量为6％,说明锦鸡儿植物比鹰嘴豆具有更好的干旱适应能力,这种能力可能取决于锦鸡儿植物体内大量渗透调节物质的累积。     

4种石斛属植物花朵挥发性成分分析

为了解石斛属植物花朵中的挥发性成分, 利用固相微萃取(SPME)方法结合GC-MS 技术测定了鼓槌石斛、罗河石斛、细叶石斛和密花石斛盛花期的花朵挥发性成分及其相对含量。结果表明, 从4 种石斛属植物花朵中共鉴定出挥发性成分57 种, 包括酯类、萜烯类、醇类、烷类、醛类、酮类、醌类、芳香族和含氮化合物。4 种石斛花朵挥发性成分的组成和含量差异明显。鼓槌石斛和细叶石斛的主要香气成分是3-蒈烯, 相对含量分别为84.606% 和71.251%。罗河石斛挥发性成分中水杨酸甲酯相对含量最高(57.449%), 其次为D-柠檬烯(22.416%)。密花石斛花朵主要挥发性成分是2-亚甲基-4,8,8-三甲基-4-乙烯基-双环[5.2.0]壬烷(82.013%), 其次为α-法尼烯(4.699%)。这些对于香型石斛兰品种的培育和兰花精油产品开发提供了参考。     

兰科菌根真菌对干旱胁迫下铁皮石斛生长和抗氧化能力及相关基因表达的影响

利用盆栽的方式研究了干旱胁迫下接种兰科菌根真菌(OM)对铁皮石斛生长的影响,并分析了铁皮石斛叶片相对含水量、游离脯氨酸含量、电解质渗透率、丙二醛(MDA)含量、活性氧成分、抗氧化酶活性变化,用定量PCR技术分析了相关抗氧化酶基因的表达特性,以探讨菌根真菌对铁皮石斛干旱胁迫的缓解作用及其机制。结果表明：(1)与正常水分条件相比,干旱胁迫显著降低了铁皮石斛幼苗的生物量和叶片相对含水量,提高了叶片电解质渗透率、脯氨酸含量、MDA含量、O-·2产生速率和H2O2水平。(2)菌根真菌能显著提高干旱胁迫下铁皮石斛叶片相对含水量,降低叶片电解质渗透率、脯氨酸含量、MDA含量、O-·2产生速率和H2O2水平;在不同水分条件下,菌根真菌均能有效促进铁皮石斛幼苗生长,其株高、根重、茎叶重和总生物量均大于未接种组。(3)菌根真菌可诱导干旱胁迫下铁皮石斛超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)基因的表达,提高SOD、POD和CAT的活性,有效缓解干旱胁迫对质膜的过氧化伤害。研究认为,菌根真菌能提高干旱胁迫下铁皮石斛的抗氧化酶活性及其相关基因表达水平,增强铁皮石斛抗氧化防御能力,有效缓解干旱胁迫对铁皮石斛幼苗生长的抑制。     

3种石斛光合作用和叶绿素荧光特性的比较研究

以美花石斛、春石斛和铁皮石斛为材料,对它们的叶片进行光合作用、叶绿素含量和叶绿素荧光等参数的测定.结果表明:3种石斛的光饱和点都低于300 μmol·m-2·s-1,且美花石斛具有较高的净光合速率和较低的光饱和点,容易引起光抑制;美花石斛的羧化效率高于春石斛和铁皮石斛,其净光合速率随着CO2浓度的升高而迅速升高,并在CO2浓度达到500 μmol·m-2·s-1时达到最大值;美花石斛的非循环电子传递速率(ETR)、实际光化学反应量子效率(Yield)和光化学猝灭系数(qP)都明显高于铁皮石斛和春石斛,而春石斛具有较高的非光化学猝灭系数(qN),其光合机构的自我保护能力较强;美花石斛的叶绿素a、叶绿素b以及总叶绿素含量都较低.研究发现,3种石斛的光饱和点均比较低,具有石斛喜阴的生活习性,都表现出对密林和悬崖等独特生长环境的适应,并以美花石斛的这种特性更为突出.     

群体表型性状研究揭示环境与遗传因素对霍山石斛表型及物种分类的影响

石斛属(Dendrobium)种类繁多, 属内物种具有丰富的表型多样性。霍山石斛(D. huoshanense)为我国特有物种, 其与河南石斛(D. henanense)和细茎石斛(D. moniliforme)以及铁皮石斛(D. catenatum)等近缘种表型相似, 在分类处理中存在争议。这种争议很大程度上与植物普遍存在的表型可塑性和代际共存有关。为探究环境和代际间遗传因素对霍山石斛表型性状的影响以及霍山石斛与近缘种的物种边界问题, 本研究观测了安徽省霍山县霍山石斛(野生、林间和温室F₁代、林间和温室F₂代)、野生河南石斛、细茎石斛和铁皮石斛, 共计16个群体2,279株植株的假鳞茎茎长等12个表型性状; 在种内层面, 首次借鉴生态学同质园实验和遗传学代际间性状比较的方法, 对霍山石斛群体表型性状进行差异显著性检验和95%置信区间比较以及主成分和变异系数等统计学分析。在种间层面, 对霍山石斛与河南石斛和铁皮石斛等近缘种群体表型性状进行比较和分析。结果表明, 环境因素对霍山石斛假鳞茎茎长和假鳞茎直径等具有显著的影响, 代际间遗传因素对霍山石斛假鳞茎直径具有显著的影响。霍山石斛与铁皮石斛和细茎石斛等近缘种群体在假鳞茎茎长、假鳞茎直径、花瓣长和花瓣宽等表型性状方面均存在显著性差异和间隔, 但与河南石斛仅在假鳞茎表型性状方面有显著性差异。我们的研究明确了环境和代际间遗传因素对霍山石斛表型性状的影响程度, 为霍山石斛与近缘种等争议物种的分类和鉴定提供了表型证据。     

Leaf water characteristics and drought acclimation in sunflower genotypes

The responses of leaf water parameters to drought were examined using three sunflower (Helianthus annuus L.) genotypes. Osmotic potential at full water saturation (π¹⁰⁰), apoplastic water fraction (AWF) and bulk elastic modulus (BEM) were determined by pressure-volume curve analysis on well watered or on water-stressed plants (−1.0 MPa Ψ₁ < −1.5 MPa) previously drought-pretreated or not. The drought-pretreated plants were subjected to a 7-day drought period (predawn leaf water potential reached −0.9 MPa) followed by 8 days of rewatering. In well watered plants, all genotypes in response to drought acclimation displayed a significantly decreased π¹⁰⁰ associated with a decrease in the leaf water potential at the turgor-loss point (decrease in Ψ_tlp was between 0.15 and 0.21 MPa, depending on the genotype). In two genotypes, drought acclimation affected the partitioning of water between the apoplastic and symplastic fractions without any effect on the total amount of water in the leaves. As a third genotype displayed no modification of AWF and BEM after drought acclimation, the decreased π¹⁰⁰ was only due to the net accumulation of solutes and was consistent with the adjustment of the photochemical efficiency observed previously in this genotype in response to drought acclimation. In water-stressed plants, the osmotic adjustment (OA) can increase further beyond that observed in response to the drought pretreatment. However, the maintenance of photosynthetic rate and stomatal conductance at low leaf water potentials not only depends on the extent of osmotic adjustment, but also on the interaction between OA and AWF or BEM. Adaptative responses of leaf water parameters to drought are thus quite contrasted in sunflower genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Asymbiotic germination of immature seeds,plantlet development and <Emphasis Type="Italic">ex vitro</Emphasis> establishment of plants of <Emphasis Type="Italic">Dendrobium tosaense</Emphasis> makino—A medicinally improrant orchid

Summary Shi-hu (Dendrobium spp. or Dendrobii Herba) is one of the important traditional Chinese medicines. The commercially available crude drug in the traditional medicine market is composed mainly of three species: Dendrobium tosaense, D. nobile, and D. moniliforme. An efficient method of propagation has been developed via asymbiotic germination of seeds in vitro for the medicinally important D. tosaense. Seeds from capsules of D. tosaense collected 8–14 wk after artificial pollination germinated after being cultured on full-strength or half-strength Murashige and Skoog (MS) medium devoid of plant growth regulators and with 3% sucrose. Germination of seeds varied with the medium type and seed maturity. Germinated seedlings after transfer to MS medium with 1.5% sucrose and 8% banana homogenate or potato juice or coconut water and 20 wk of incubation developed into healthy plantlets. Well-developed plantlets were transplanted to moss or moss and tree fern or tree fern as substrates in plastic trays and transferred to a greenhouse for hardening. All plants survived, attained maturity, and developed normal flower and capsule after one and a half years. This protocol of successful plant regeneration by asymbiotic seed germination should permit rapid propagation and conservation of this medicinally important Dendrobium species.     

铁皮石斛SPL膜结合(STM)转录因子的全基因组鉴定及表达分析

SPL转录因子广泛参与植物生长发育、胁迫响应等过程。目前,没有关于铁皮石斛SPL膜结合(SPL with transmembrane motif)转录因子即STM转录因子的研究。为了探究STM转录因子在铁皮石斛生长发育及胁迫响应等方面的作用,该文在铁皮石斛全基因组水平鉴定出4个STM转录因子,并对DoSTM基因家族成员进行生物信息学分析,又利用逆转录PCR研究了DoSTM在不同组织部位及不同逆境处理下的表达情况。结果表明:(1)DoSTM1-4为亲水蛋白,均具有SBP保守结构域和一些激素响应位点。(2)4个DoSTM在根茎叶中均有表达,DoSTM2在叶中的相对表达量最低; DoSTM1/3/4的相对表达水平均无明显差异。(3)DoSTM1-4在低温、高温、干旱胁迫下的相对表达水平都有显著变化,DoSTM1/3/4的表达量降低最为明显,故推测DoSTM与植物体内激素响应、温度变化响应及抗旱性有关。这些结论为后续进一步开展铁皮石斛STM转录因子的研究提供了参考。     

Photosynthesis of <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis> subjected to drought stress is enhanced by choline chloride through alleviating lipid peroxidation and increasing proline accumulation

Rehmannia glutinosa seedlings were pretreated with choline chloride (CC) in concentrations of 0, 0.7, 2.1 and 3.5 mM, and then subjected to drought and rewatering treatment to study the effects of CC on the generation of reactive oxygen species (O₂⁻, H₂O₂), lipid peroxidation, proline accumulation, water status and photosynthesis. The results showed that pretreatment with CC alleviated the inhibition of SOD and APX activity caused by drought stress, and therefore, the rate of O₂⁻ production and H₂O₂ concentration were reduced and lipid peroxidation decreased in pretreated plants. CC pretreatment also accelerated accumulation of proline, maintained higher Ψw and RWC, deferred leaf water loss during drought stress and retarded the drop in proline concentration after rewatering. Consequently, drought-induced decreases in F_m/F₀, F_v/F_m, Φ_PS2, q_P, and A and increase in q_NP were inhibited and the recovery of photosynthesis after rewatering was quicker in pretreated plants. Although differences in F_v/F_m, Φ_PS2 and q_P between treatments were not significant, there was a general trend that the effects of CC increased with the rise of its concentrations. The data suggested that 2.1 mM of CC be suitable for alleviating lipid peroxidation, promoting proline accumulation, retarding leaf water loss and improving photosynthesis of R. glutinosa seedlings under drought stress.     

Maize Leaf Rolling Initiation

Maize plants of CPB2 and CPB8 hybrids were kept under water deficit for 22 d. In the CPB8 hybrid, leaf rolling initiated at the 9^th d of water deficit period, while in CPB2 hybrid it was at the 15^th d. Both hybrids showed leaf rolling initiation at the same leaf water potential, Ψ_W of -0.480±0.095 MPa. At leaf rolling initiation, the leaf osmotic potential, Ψ_S was -0.730±0.085 MPa in CPB8 and 0.630±0.110 MPa in CPB2. The leaf temperature and stomatal conductance were higher in CPB8 than in CPB2. Values of leaf Ψ_W, ribulose-1,5-bisphosphate carboxylase activity, chlorophyll content, and specific leaf area were similar in both hybrids. Phosphoenolpyruvate carboxylase activity and protein content were lower in the CPB2 hybrid than in CPB8. In both hybrids leaf rolling initiation was associated with: (1) higher leaf temperature, with leaf rolling effect related to leaf temperature reduction, and (2) lower leaf Ψ_S, related to osmotic adjustment as an additional component of drought-tolerance strategy. This revised version was published online in June 2006 with corrections to the Cover Date.     

根茎萌发与幼苗期的虉草水分生态幅

为揭示虉草根茎萌发和幼苗期的水分生态幅,该研究通过盆栽控制试验,设置8个水分梯度：(2±0.5)％、(10±0.5)％、(15±0.5)％、(20±0.5)％、(25±0.5)％、(30±0.5)％、(40±0.5)％(水分饱和)和淹水(2 cm),通过萌发率、各形态指标和生理指标对虉草水分生态幅进行了探讨。结果表明：(1)土壤水分含量过低和过高都会降低虉草根茎萌发率。(2)随土壤水分含量的增加,虉草幼苗株高和地上生物量都呈现先上升后下降的趋势,两者并呈指数函数关系y ＝0.2038e0.0486x(R2＝0.803)。(3)虉草通过调整自身结构特点,降低个体大小、减少叶片数量和叶面积来适应胁迫环境,土壤水分含量为10％~30％时虉草含水率能保持相对稳定的水平。虉草的光和色素( Chl a、Chl b、Car)和相对叶绿素( SPAD)含量均随土壤水分含量的增加呈现出先上升后下降的趋势,Chl a/b则相反,呈先下降后上升趋势。(4) Chl/Car各组之间差异不显著。综合上述研究,利用高斯模型分析得出虉草的水分生态幅为7.48％~52.20％,最适水分生态幅为18.99％~40.60％。     

Photosynthetic parameters and leaf water potential of five common bean genotypes under mild water deficit

The leaf water potential, gas exchange and chlorophyll fluorescence were evaluated in five common bean (Phaseolus vulgaris) genotypes A222, A320, BAT477, Carioca and Ouro Negro subjected to moderate water deficit. At the maximum water deficit (10 d of water withholding), the leaf water potential of genotypes A320 and A222 was higher (−0.35 and −0.50 MPa) when compared to the other genotypes (−0.67 to −0.77 MPa). The stomatal conductance and net photosynthetic rate were significantly reduced in all genotypes due to the water deficit. The greater reduction in stomatal conductance of A320 under drought resulted in high intrinsic water use efficiency. Mild water deficit affected the photochemical apparatus in bean genotypes probably by down-regulation since plants did not show photoinhibition. The photochemical apparatus of A222 and A320 genotypes was more sensitive to drought stress, showing reduced apparent electron transport even after the recovery of plant water status. On the other hand, even after 10 d of water withholding, the maximum efficiency of photosystem 2 was not affected, what suggest efficiency of the photoprotection mechanisms.     

Phylogenetic relationship of China Dendrobium species based on the sequence of the internal transcribed spacer of ribosomal DNA

The genetic relationship of 36 Dendrobium species in China was determined based on sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA. Aligned sequences of the complete ITS region obtained from the 36 Dendrobium species and 2 outgroup species (Epigeneium amplum and Epigeneium nakaharaei) by using PCR amplification and direct DNA sequencing. The nrDNA ITS1 of Dendrobium was 225–234 bp and ITS2 was 239–248 bp. Phylogenetic tree was constructed, and seven main clusters were generated among the 36 Dendrobium species. From the results, D. moulmeinense was not grouped in the classification of Dendrobium and E. amplum and E. nakaharaei were shown to be divergent from Dendrobium species. The phylogenetic relationships revealed by ITS DNA analysis partially supported previously published morphological data.     

Isolation and identification of endophytic and mycorrhizal fungi from seeds and roots of <Emphasis Type="Italic">Dendrobium</Emphasis> (Orchidaceae)

The seed germination of orchids under natural conditions requires association with mycorrhizal fungi. Dendrobium nobile and Dendrobium chrysanthum are threatened orchid species in China where they are considered medicinal plants. For conservation and application of Dendrobium using symbiosis technology, we isolated culturable endophytic and mycorrhizal fungi colonized in the protocorms and adult roots of two species plants and identified them by morphological and molecular analyses (5.8S and nrLSU). Of the 127 endophytic fungi isolated, 11 Rhizoctonia-like strains were identified as Tulasnellales (three strains from protocorms of D. nobile), Sebacinales (three strains from roots of D. nobile and two strains from protocorms of D. chrysanthum) and Cantharellales (three strains from roots of D. nobile), respectively. In addition, species of Xylaria, Fusarium, Trichoderma, Colletotrichum, Pestalotiopsis, and Phomopsis were the predominant non-mycorrhizal fungi isolated, and their probable ecological roles in the Dendrobium plants are discussed. These fungal resources will be of great importance for the large-scale cultivation of Dendrobium plants using symbiotic germination technology and for the screening of bioactive metabolites from them in the future.     

In situ seed baiting techniques in <Emphasis Type="Italic">Dendrobium officinale</Emphasis> Kimuraet Migo and <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl. : the endangered Chinese endemic <Emphasis Type="Italic">Dendrobium</Emphasis> (Orchidaceae)

The new in situ seed baiting method using seed packets to assess germination of orchid species within soil provides a means of locating, collecting, and identifying specific fungi that are involved in the lifecycle of orchids in the wild and investigating their relationship with the orchids under natural field conditions. Two isolates (SHH44, SHH53) originating from the seedlings found in the seeds packets in situ were demonstrated to support seed germination and seedling development (in vitro) of two endangered Chinese endemic herbs, Dendrobium officinale and Dendrobium nobile (Orchidaceae). Advanced protocorm development (Stage 3 and greater) in this study of these species of Dendrobium only occurred under the 12/12 h L/D photo-period indicated that illumination may play an important role in seedling recruitment of terrestrial orchid species in their natural environment. The information provided in this study may prove invaluable in the conservation of D. officinale and D. nobile and congener species in China, especially given their rare status.