Flavones in genetically transformed <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> roots and induction of their synthesis by elicitation with methyl jasmonate

The composition and content of flavones were estimated in pRi T-DNA-transformed skullcap (Scutellaria baicalensis Georgi) roots obtained by the inoculation of axenically grown seedlings with a wild A4 strain of the soil bacterium Agrobacterium rhizogenes. It was elucidated by analytical and preparative HPLC of phenolic compounds in the extracts from the pRi T-DNA-transformed roots and also by ultraviolet spectroscopy and ¹H and ¹³C NMR that cultured skullcap roots contained similar basic flavones as intact roots of this plant species, i.e., baicalein and wogonin and corresponding glucuronides, baicalin and wogonoside. The content of these flavones in cultured roots was threefold lower than in the roots of intact five-year-old plants. When skullcap roots were cultured on B5 or Murashige and Skoog medium, the ratios between major flavones changed but their total content remained unchanged. The treatment of three-week-old cultured roots with methyl ether of jasmonic acid (MeJa) doubled the total concentration of major flavones in roots; the content of aglycons, baicalein and wogonin, increased to a greater degree, e.g., by 2.3 and 3.3 times, respectively. The induction of flavone production by elicitors indicates that flavones behave as phytoanticipins because major flavones of skullcap manifest a distinct antimicrobial activity. The results of the short-term treatment of skullcap roots with MeJa show that stress biotic factors can considerably increase the content of physiologically active flavones.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 90–96.Original Russian Text Copyright © 2005 by Kuzovkina, Guseva, Kovács, Szöke, Vdovitchenko.     

Artificial seed preparation as the efficient method for storage and production of healthy cultured roots of medicinal plants

The technique for the refinement of pRi T-DNA-transformed root cultivation by the root fragment encapsulation in the gel coat, i.e., so-called “artificial seed” (AS) production, was studied. AS were produced from genetically transformed roots of Baikal skullcap (Scutellaria baicalensis Georgi) and common rue (Ruta graveolens L.). The effects of duration of AS storage at 4°C on their subsequent growth activity and a capability for resumption of actively growing root cultures were analyzed. Encapsulation of contaminated Baikal skullcap root culture with the addition of antibiotic and storage during 2–5 weeks at low above-zero temperature resulted in a complete elimination of infection, i.e., obtaining the healthy root culture. Growth activity and total flavone concentration were markedly increased in this culture, so that total productivity of this renewed root culture increased substantially. Using AS produced from the root fragments of common rue, it was shown that, after long-term storage at low above-zero temperature, they are capable of not only root growth resumption but also active shoot formation, which is of interest for plant micropropagation. Long-term retaining growth activity of AS produced from root cultures of valuable medicinal plants permits their usage as a reserve and also, in the case of necessity, for long-distance transport as compact axenic root inocula. The storage of viable root fragments within AS also helps to optimize intervals between numerous subculturings of root cultures required for the maintenance of IPPRAS collection in the active state.     

Flavonoid Production in Transformed Scutellaria baicalensis Roots and Ways of Its Regulation

A root culture of skullcap (Scutellaria baicalensisGeorgi) transformed with pRi T-DNA was initiated by the inoculation of sterile seedlings with Agrobacterium rhizogenes(wild-type strain A-4). The flavonoid concentration in cultured roots comprised 5% of the root dry weight and was maintained essentially constant during a subculture. For four weeks of culturing, the weight of the roots increased by 20–30 times; when the roots were cultured for a longer time and with periodic enrichment of the nutrient medium, their weight increased 50-fold. Skullcap roots were shown to synthesize flavones characteristic of intact roots (wogonin, baicalein, and baicalin). The addition of 0.01–1 mM L-phenylalanine (a precursor of flavonoids) to the nutrient medium affected neither root growth, nor their flavonoid concentration. Root elicitation with 100 M methyl jasmonate for 72 h increased the flavonoid content per flask and per root dry weight by 1.8 and 2.3 times, respectively.     

Effect of auxins on <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis> root cultures

Effects of auxins (IAA, IBA and NAA) on K. humboldtiana root culture cultivated in 16-h photoperiod or in dark have been observed. Light affected positively the production of biomass when cultivated on medium supplemented with NAA in 10 and 25 mol ^–1 concentrations. In the presence of IAA and IBA these values were significantly lower. The growth dynamics of root cultures depended on the auxin used. The best adventitious roots elongation and lateral roots induction on media supplemented with IBA has been ascertained. Morphological and anatomical differences in dependence on auxin used were observed. NAA supported the formation of huge callus-like mass besides mostly very short roots, especially under the light. Similarly IAA induced short roots, and IBA seems to be the most effective substance for the root elongation in this model system. NAA induced roots with larger diameter under the light compared with the other two auxins used. The reason is in the different anatomical structure of roots which was characterized by higher number of cell layers and large intercellulars in the cortex. The shape of cortical cells in the presence of IBA depended on the light conditions. Isodiametric cortical cells were present in roots cultivated in 16-h photoperiod, irregularly-shaped cells in the dark. The effect of light conditions was the smallest in the case of roots grown on IAA enriched media.     

Cadmium stress in wheat seedlings: growth,cadmium accumulation and photosynthesis

Seedlings of wheat (Triticum aestivum L.) cultivars Jing 411, Jinmai 30 and Yangmai 10 were exposed to 0, 10, 20, 30, 40 or 50 μM of CdCl₂ in a solution culture experiment. The effects of cadmium (Cd) stress on wheat growth, leaf photon energy conversion, gas exchange, and Cd accumulation in wheat seedlings were investigated. Gas exchange was monitored at 3, 9, 24 days after treatment (DAT). Growth parameters, chlorophyll content, leaf chlorophyll fluorescence, and Cd concentration in shoot and root were measured at 24 DAT. Seedling growth, gas exchange, chlorophyll content, chlorophyll fluorescence parameters were generally depressed by Cd stress, especially under the high Cd concentrations. Cd concentration and accumulation in both shoots and roots increased with increasing external Cd concentrations. Relationships between corrected parameters of growth, photosynthesis and fluorescence and corrected Cd concentrations in shoots and roots could be explained by the regression model Y = K/(1 + exp(a + bX)). Jing 411 was found to be Cd tolerant considering parameters of chlorophyll content, photosynthesis and chlorophyll fluorescence in which less Cd translocation was from roots into shoots. The high Cd concentrations were in shoots and roots in Yangmai 10 which has been found to be a relative Cd tolerant cultivar in terms of most growth parameters.     

Establishment of <i>Rhodiola quadrifida</i> Hairy Roots and Callus Culture to Produce Bioactive Compounds

Rhodiola quadrifida is a rare mountain medicinal plant whose root extracts are used in traditional Chinese medicine as a hemostatic, antitussive, and tonic in the treatment of gynecological diseases. The aim of the study was to obtain R. quadrifida cultures at different degrees of differentiation in vitro and compare their growth characteristics and the content of salidroside and rosavin. Hairy roots were obtained by incubating cotyledons and hypocotyls in a suspension of Agrobacterium rhizogenes strain A4. The presence of the rolB and rolC genes was proven by polymerase chain reaction. The obtained roots were cultivated in Murashige-Skoog medium (MS). Calluses were obtained from the hairy roots in MS medium with the addition of hormones: 3 mg/L 2,4 D and 0.5 mg/L BAP. The presence of the main secondary metabolites of R. quadrifida, salidroside and rosavin, in calluses and salidroside in hairy roots by HPLC/MS was confirmed. The content of salidroside in callus culture was significantly higher than in hairy roots, 0.158 and 0.047%, respectively. The content of rosavin in callus culture was 0.07%. The content of rosavin and salidroside in callus culture was close to the level of these substances in the rhizomes of R. quadrifida plants growing in vivo, making this culture promising for its possible biotechnological use.     

Some Effects of Light on Excised Wheat Roots with Special Reference to Peroxide Metabolism

When excised wheat roots are exposed to blue light, catalase activity changes in a way to he expected as a result of photodestruction with first order kinetics. Wheat root catalase in vivo is less light sensitive than animal catalase in vitro, possibly due to internal screening. Illumination of the roots with red light causes some increase in catalasc activity. Peroxidase activity is much less affected by light. No relation has been found between catalase destruction and chlorophyll formation. The ability of roots to oxidize pyrogallol to purpurogallin (by other workers interpreted as peroxide production) is decreased by light, especially blue light. On the contrary, one peroxide producing enzyme, glycolic acid oxidase, was detected only in roots grown in blue light. The total flavin content, or the fraction present as FAD, is not affected by light. The ascorbic acid content is low, but slightly increased by blue light.     

不同培育方式对橡胶树实生苗生理指标、养分及长势的影响

橡胶树(Heveabrasiliensis)种子催芽生长一般使用沙床培育,沙子是不可再生资源,为了选择一种适合橡胶树种子培育方式来替代对沙子的依赖,该研究通过水培、悬空培育和传统的沙培比较橡胶树实生苗第1蓬叶稳定时,苗木的生长势、生理指标及养分含量。结果表明,水培实生苗地上部株高、茎粗、叶面积的长势最佳,壮苗指数和生物量的含量最高,但其根太长,根相对较细。水培的叶、茎、根的可溶性糖、丙二醛、游离脯氨酸、超氧化物歧化酶的含量均较低;水培和悬空培育的叶片和茎的叶绿素、类胡萝卜素及根系活力的含量没有显著性差异,均高于沙培。水培的叶、茎、根中的氮和磷含量最低,沙培的最高;而水培实生苗根和茎中钾的含量较高,叶片中含量与悬空培育、沙培均没有显著性差异;悬空培育在叶、茎、根中钾的含量最低。水培促进了苗木的生长,降低干旱胁迫,提高养分利用率,但后续还需调控根系,建设良好根团。悬空培育的苗木长势较弱,还需进一步完善方法。     

Comparative study of mycorrhizal susceptibility and anatomy of four palm species

A morphological and anatomical study of the root systems of the palm species Brahea armata S. Watson, Chamaerops humilis L., Phoenix canariensis Chabaud and Phoenix dactylifera L. has been carried out to determine possible mycorrhizal colonization sites. Furthermore, the arbuscular mycorrhizal (AM) anatomical types formed by the four palm species in association with Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe have been examined. The presence of a continuous sclerenchymatic ring in the outer cortex and aerenchyma in the inner cortex that are anatomical indicators of mycorrhizal nonsusceptibility in all four palm species is observed. The root systems of B. armata and C. humilis present only one group of third-order roots, while the third-order roots of P. canariensis and P. dactylifera may be divided into five different groups: short thick roots, mycorrhizal thickened roots, fine short roots, fine long roots, and pneumatorhizas. Third-order and some second-order roots of B. armata and C. humilis are susceptible to colonization by AM fungi, while only the mycorrhizal thickened roots form mycorrhizas with arbuscules in the Phoenix species. The root system of the Phoenix species also presents AM colonization in fine roots with only intraradical hyphae and spores, but without arbuscules, and pseudomantles of spores anchored in the pneumatorings of the second-order roots, which are described for the first time. The mycorrhizas formed by the four palm species are of an intermediate type, between the Arum and the Paris types, and are characterized by intercalary arbusculate coils and not only by intracellular but also by intercellular fungal growth. Our study suggests that a different degree of adaptation may exist among palm mycorrhizas toward the slow growth of palms and low spore numbers in the soil where they grow.     

黄河三角洲滨海盐碱地11个造林树种细根解剖性状对土壤条件的响应

细根作为植物与土壤连接的重要部位,能够反映植物对生存环境的适应性。以黄河三角洲滨海盐碱地不同立地条件下11个造林树种为对象,基于细根分支等级划分1-4级根序并进行解剖特征测定,分析细根解剖性状对滨海盐碱地不同土壤条件的响应规律。结果表明：（1）不同根序的细根直径存在显著差异,细根直径随根序升高呈增大趋势,而同根序的细根直径在不同树种间表现出显著的种间差异（P < 0.05）。1-2级细根皮层厚度、3-4级细根导管密度在树种间的差异均达显著水平（P < 0.05）。（2）在较为严重盐渍化土壤条件下（立地1）,细根皮层厚度较其他立地显著增大,但细根导管密度较小;在轻度盐碱立地条件下（立地3）,细根导管密度较大;较为严重的盐碱立地具有更为发达的细根直径及维管柱直径。（3）树种1-2级细根解剖结构与土壤环境关系最为密切,其中1级根直径与土壤pH值显著正相关（P < 0.05）,与土壤硝态氮含量呈显著负相关（P < 0.05）。对土壤理化性质与细根解剖性状的冗余分析表明,前两个轴的特征值达0.640和0.196,土壤速效养分含量与轴一（RDA1）呈正相关,低级根解剖性状则与轴二（RDA2）呈显著负相关。低级根解剖结构以及土壤的pH值能解释较多树种的差异性,其中低级根直径与皮层厚度对盐碱环境表现出较强的响应。     

Defining Plant Resistance to Phytophthora cinnamomi: A Standardized Approach to Assessment

Phytophthora cinnamomi is a soil‐borne plant pathogen that causes devastating disease in agricultural and natural systems worldwide. While a small number of species survive infection by the pathogen without producing disease symptoms, the nature of resistance, especially under controlled conditions, remains poorly understood. At present, there are no standardized criteria by which resistance or susceptibility to P. cinnamomi can be assessed, and we have used five parameters consisting of plant fresh weight, root growth, lesion length, relative chlorophyll content of leaves and pathogen colonization of roots to analyse responses to the pathogen. The parameters were tested using two plant species, Zea mays and Lupinus angustifolius, through a time course study of the interactions and resistance and susceptibility defined 7 days after inoculation. A scoring system was devised to enable differentiation of these responses. In the resistant interaction with Z. mays, there was no significant difference in fresh weight, root length and relative chlorophyll content in inoculated compared with control plants. Both lesion size and pathogen colonization of root tissues were limited to the site of inoculation. Following inoculation L. angustifolius showed a significant reduction in plant fresh weight and relative leaf chlorophyll content, cessation of root growth and increased lesion lengths and pathogen colonization. We propose that this technique provides a standardized method for plant–P. cinnamomi interactions that could be widely used to differentiate resistant from susceptible species.     

The Relationship Between Endogenous β‐Glucuronidase Activity and Biologically Active Flavones‐Aglycone Contents in Hairy Roots of Baikal Skullcap

Here, we examine the relationship between contents of principal flavones in hairy roots of Scutellaria baicalensis with the activity of the β‐glucuronidase (sGUS) enzyme during a culturing cycle. Using RP‐HPLC, we show that the highest contents of aglycones, baicalin and wogonin is observed at the growth days 8, 14, and 71 and reach 45, 41, and 62% (based on the total weight of hairy roots of the Baikal skullcap), correspondingly. Their accumulation is accompanied by increase of the sGUS activity, which we determined fluorometrically. Moreover, the enzyme activity is characterized by significant and reasonable correlation only with the wogonin contents. Our results confirm a significant role of sGUS at the final steps of the metabolism in root‐specific flavones of Baikal skullcap and suggest how one can optimize the conditions of culturing the hairy roots for biotechnological production of individual flavonoids. For example, at the culturing day 71 wogonin constituted over 80% of all flavones extracted from cells.     

Optimization and quality assessment of adventitious roots culture in Panax quinquefolium L.

In this study, adventitious roots of Panax quinquefolium L. have been successfully established. The highest induction rate of roots was obtained in MS medium containing 3 mg L^?1 IBA after 4 weeks of culture. The culture conditions of adventitious root were optimized and evaluated with response surface methodology. The best culture conditions for root growth seemed to be 0.75 salt strength MS medium, 4.70 g L^?1 inoculum size and 40 days of culture. The active component contents of adventitious roots were compared with those of native roots. The total saponins content was found to be 16.28 mg g^?1 in native root and 4.64 mg g^?1 in adventitious root. The polysaccharide content of the adventitious root was 1.5 times higher than that in the native P. quinquefolium (30.54 vs. 20.28 mg g^?1).     

Chilling tolerance in February seeded Chaite rices (Oryza sativa) of Nepal

Five Chaite and three main season rices Oryza sativa genotypes were examined to assess the effect of low temperature on the germination and growth of roots and shoots, root regeneration capacity, the extent of chlorophyll synthesis of etiolated seedlings, total chlorophyll content and photo-chemical efficiency (Fv/Fm) of chilled plants. Cultivated differences in the ability to germinate and grow at low temperatures have been found. The poor ability of Chaite rices to tolerate photo-oxidative tissue damage during January and February seemed to be the major factor resulting in poor seedling growth.     

Changes in pathogenesis-related proteins in pepper plants with regard to biological control of phytophthora blight with <Emphasis Type="Italic">Paenibacillus illinoisensis</Emphasis>

To evaluate the biocontrol effectiveness of chitinase-producing bacterium, Paenibacillus illinoisensis strain KJA-424 against pathogenic strain of Phytophthora capsici in pepper plants, growth response and kinetics of pathogen related (PR) proteins were estimated after inoculation with P. capsici (P), and with a combination of P. capsici and strain KJA-424 cell culture (P+A). Fresh weight and chlorophyll content in shoots at P+A-treated plants significantly increased by 23.4 and 34.2%, respectively after 7days of inoculation, compared to P-treated plants. Root mortality in P+A-treated plants was significantly reduced compared to P-treated plants. Seven days after inoculation, the activities of -1,3-glucanase, cellulase and chitinase in P-treated roots had decreased by 54.8, 36.5 and 52.8%, respectively, compared to P+A-treated roots, while those in P-treated leaves increased by 22.8, 36.3 and 23.8%, respectively, compared to those in P+A-treated leaves. The activities of -1,3-glucanase, cellulase and chitinase in roots are negatively correlated with root mortality. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, reduction of PR proteins in roots, and activates of PR proteins in leaves infected by P. capsici.     

Subterranean axes in tribe Diurideae (Orchidaceae): Morphology,anatomy, and SYSTEMATIC SIGNIFICANCE

The morphological and anatomical nature of perennating storage organs of the predominantly Australasian orchid tribe Diurideae (Orchidoideae: Orchidaceae) as well as anatomical concepts in tribes Orchideae and Diseae of Orchidoideae have been problematic for 150 yr, reflected in conflicting or vague terminology and questions about polystely and even monophyly of Orchidoideae. From a representative survey of underground organs of 145 species in 37 ingroup genera (Diurideae) and two outgroup genera (Spiranthes and Disa), the so-called “root-stem tuberoids” are here interpreted as root tubers (except for the stem tubers of Rhizanthella) borne on either droppers or stolonoid roots. All root tubers examined are bounded by a 1–4 layered velamen and exodermis, whereas droppers and stolonoid roots may have velamen-exodermis or a simple epidermis depending on the taxon and often bear multiseriate or uniseriate trichomes associated with mycorrhiza (as do roots of some taxa). The “polystely” reported in tubers of many Orchideae also occurs in tubers of many Diurideae but represents only dissection of the siphonostele into 2–13 traces. Cladistic analyses of data show extraordinarily high levels of homoplasy in characters related to root, dropper/stolonoid root, and tuber, so that these characters alone are of limited usefulness.     

根结线虫对文冠果苗木根系结构与生长及矿质养分分布特性的影响北大核心CSCD

以根结线虫侵染的文冠果一年生苗木为材料,观察分析苗木根系的形态和显微结构,植株生长发育以及主要矿质养分在苗木中的分布特性,并分析不同矿质元素与线虫侵染的关联关系,以揭示根结线虫对文冠果苗木生长发育的影响机制。结果表明:(1)受根结线虫侵染的文冠果苗木根系形成根结,依据根结发生程度分为0(对照,正常植株)、2、3、4级;与对照相比,具有根结的文冠果苗木根系解剖结构特征主要表现为皮层相对较厚,木质部排列扭曲,导管较少,射线数目较少且分布不均匀;韧皮细胞内含物明显较多;多个巨细胞及细胞空腔主要存在于韧皮部。(2)具根结的苗木株高和地径较对照均增加,且株高增幅达显著水平(P<0.05)。(3)具根结苗木根、茎、叶部位的N、P含量均较对照下降,根部降幅达显著水平(P<0.05),其中具2~4级根结的根部N含量分别较对照显著下降7.8%、16.0%和29.5%,P含量分别显著下降15.6%、7.1%和43.3%;根部Fe含量、Zn含量显著上升,2~4级根结根系中Fe含量较对照分别增加1.56倍、0.81倍和3倍,Zn含量分别增加1.11倍、1.56倍和1.78倍;具3和4级根结的苗木根系中K含量较对照显著增加(P<0.05),具2和4级根结的苗木叶片中K含量较对照分别显著下降61.5%和47.0%。(4)苗木根部、茎部N元素含量对于根结线虫侵染的响应最明显,且其含量随侵染程度的增大而降低,叶部Mg元素含量与根结线虫侵染率、K元素含量与整株苗木生物量均呈显著负相关关系(P<0.05)。该研究为根结线虫对文冠果苗木养分吸收利用及运转的影响提供了一定的理论依据。     

Effect of exogenous ammonium gluconate on growth,ion flux and antioxidant enzymes of maize (Zea Mays L.) seedlings under NaCl stress

• Ammonium gluconate (AG) provides both an organic carbon source and a nitrogen source, which can positively improve soil fertility and delay soil degradation.
• We investigated the underlying mechanisms of both NH₄⁺‐ and C₆H₁₁O₇^?‐mediated resistance to high salt concentrations in maize (Zea mays L.), and how they relate to antioxidant cellular machinery, root system architecture, root activity and lignin content in roots.
• Seedlings treated with AG maintained lower Na⁺ content, higher chlorophyll content, higher CAT and POD activity, compared with those without AG and ammonium carbonate (AC). The total size of the root system, primary root length and number of lateral roots detected on the primary root treated with AG decreased compared with those not treated with AG at the same NaCl concentration. However, average root diameter and root activity when treated with AG were significantly higher than roots without AG at the same NaCl concentration. Furthermore, total size of the root system, primary root length and number of lateral roots detected on primary rootsof seedlings treated with AG were higher than those treated with AC at the same NaCl concentration.
• These results suggested that AG may be a good organic fertiliser under salt stress by decreasing Na⁺ content and increasing chlorophyll content, activity of antioxidant enzymes, root diameter and root activity in maize seedlings.

     

闽西南红壤侵蚀区黑莎草功能性状特征及其与土壤因子的关系

为了解黑莎草(Gahnia tristis)在南方红壤侵蚀区的适应状况,测定了长汀县红壤侵蚀区的黑莎草叶片、根系的功能性状及土壤理化性质,并应用数理统计方法分析了黑莎草叶片与根系功能性状之间的相关性,及其对土壤因子的响应。结果表明,黑莎草叶片表型性状在冬夏季间存在显著差异,叶长、叶宽、叶组织密度和叶绿素表现为夏季显著大于冬季,根系表型性状则更具稳定性,冬季的根系养分含量均高于夏季,养分的分配上叶片养分高于根系养分。叶组织密度与叶绿素含量呈显著正相关,与比叶面积呈显著负相关;根组织密度与比根长和比根面积均呈显著负相关,叶片和根系养分间均呈显著正相关,土壤碳、氮、磷含量是影响黑莎草功能性状主要因子。因此,黑莎草可通过调节功能性状以适应环境变化,可作为地带性植物应用于南方红壤侵蚀区的植被恢复和水土流失治理。