Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation

Shoot-tips from in vitro cultured Hypericum perforatum L. genotypes were subjected to assessments of developmental competence, genetic stability, and biosynthetic ability to identify critical points during cryopreservation. Survival rate, chromosome number stability, alteration in VNTR sequences and hypericin content were evaluated, in plants after pre-culture, and two subsequent cryogenic steps (cryoprotection and cooling) and those recovered from cryopreserved meristems. Pre-culture and cryoprotection treatments, did not reveal any significant differences, in these studied characteristics. Genetic stability was assessed by chromosome counts and analysis of variability in the VNTR sequences. No changes in chromosome number were detected in comparison with the untreated control but minor alterations were revealed in non-coding sequences. The content of hypericin after the recovery of cryopreserved meristems remained comparable with the unfrozen control. The controlled rate freezing technique used for cryopreservation was relevant for restoration of genetic and biochemical stability in Hypericum perforatum L. shoot-tips.     

Pharmacodynamic study of Hypericum perforatum L.

The effects of Hypericum perforatum L. (Hypericaceae) crude ethanol extract (A), ethyl acetate extract (B), aqueous extract (C) and infusion (I), on pentobarbital induced sleeping time, intestinal motility, and their analgesic activity, have been investigated. Extracts A and B exhibited significant stimulatory and antidepressant effects on the CNS. Both extracts prolonged sleep, increasing time up to more than 25 min. The antidepressive activity of extract A was also achieved by significant reduction of the myorelaxant activity of diazepam. Extract B exhibited strong analgesic activity reducing abdominal stretching induced by acetic acid by nearly 50 %. Extracts A, B and C exhibited spasmolytic activity, significantly reducing intestine motility.     

Root cryopreservation to biobank medicinal plants: a case study for Hypericum perforatum L.

In this study, an effective root-based cryopreservation method was developed for Hypericum perforatum L., an important medicinal species, using in vitro plants. A systematic approach was applied to determine effective combinations of protocol steps such as preculture, osmoprotection, vitrification solution treatment, and unloading, followed by protocol optimization using a single-factor approach. The effects of root section type (root tips, middle sections, or basal sections), duration of root section culture after excision, and donor plant age were also investigated. In a wild genotype, middle and basal root sections excised from 8-wk-old plants and cryopreserved at the age of 10 d after excision showed the highest plant regrowth after cryopreservation. In the optimized protocol, root sections were precultured in 10% (w/v) sucrose for 17 h, osmoprotected with a solution composed of 17.5% (w/v) glycerol and 17.5% (w/v) sucrose for 20 min, followed by a vitrification solution of 40% (w/v) glycerol and 40% (w/v) sucrose for 30 min, and cryopreserved using aluminum foil strips (droplet-vitrification). After rewarming in preheated 25% (w/v) sucrose solution and 30-min unloading, root segments were recovered on medium supplemented with 1.0 mg L⁻¹ gibberellic acid and showed 78% plant regrowth. This cryopreservation method was successfully adapted for five elite lines of H. perforatum with a 45 to 87% regrowth rate after cryopreservation. These results suggest that root cryopreservation may be an effective method for medicinal plant conservation and should be tested with a broader range of species.

     

In vitro pollen germination in Hypericum perforatum L. and Hypericum rumeliacum Boiss

In vitro pollen germination and pollen tube growth investigations are valuable tools used in identification of the effects of environmental factors and genotypic differences on pollen viability, pollen germination and tube elongation. In this study pollen viability, in vitro pollen germination capacity, abnormality ratios and tube length in germinated pollens of Hypericum perforatum L. and H. rumeliacum Boiss. were investigated. Both of these species has spheroid-shaped and tricolporate pollen grains. The diameters of Hypericum perforatum and H. rumeliacum pollens were found as 24 +/- 3 microm and 19 +/- 2 microm, respectively. Pollen viability of H. perforatum and H. rumeliacum was found as 83% and 72%, respectively. The germination percentages were found as 12.85% for H. perforatum and 64.42% for H. rumeliacurm. Tube lengths in germinated pollens of both taxa were measured approximately as 95.25 +/- 38 microm in H. perforatum and 165.92 +/- 53 microm in H. rumeliacium 4 h after inoculation. In germinated pollen grains of H. perlbratum and H. rumeliacumn abnormality percentages were determined as 13.23% and 43.97%, respectively. In germinated pollens of these two species, highly significant (P < 0.00001) differences in in vitro germination percents and abnormality percents were observed. Abnormalities such as swollen tube tip, branched tube, spiralled tube and excessive tube formation were observed in pollen tubes. The results of this study showed that there were obvious differences in pollen germinability between these two species growing under the same environmental conditions.     

贯叶连翘中金丝桃素的合成与积累研究进展

金丝桃素是贯叶连翘的主要药理活性成分。本文概述了金丝桃素的化学与生物合成途径,介绍了金丝桃素在贯叶连翘个体发育过程中的积累以及利用贯叶连翘的细胞和组织培养技术生产与积累金丝桃素的研究进展。最后,指出分子生物学和电子显微镜技术的发展为深入研究金丝桃素的产生和积累提供了有利工具。     

植物应答低温胁迫机制的研究进展

低温是植物生长过程中遇到的主要环境胁迫因子之一,而植物响应低温胁迫是一个多因素协同作用的过程,涉及到复杂的基因表达调控网络。尤其是低温下植物体内生理生化、细胞骨架结构及基因表达调控等方面的改变及相关机制,一直受到研究者的普遍关注。该文主要从细胞学及分子生物学等角度入手,将低温胁迫下植物对低温的响应及可能机制进行综述,着重对植物通过细胞内部细胞器结构与功能的改变来抵御或适应低温,尤其对细胞骨架,以及低温信号转导受体及中间体、下游胁迫相关基因的表达及其在细胞内部的调控及应答机制等方面的作用进行探讨,为耐低温植物新品种的培育及农业生产实践提供理论指导。     

贯叶连翘野生转家化前后生物学性状特征观察

野生贯叶连翘 (HypericumperforatumL .)在江苏经过了 3a的引种栽培 ,其生物学特性检测结果表明 ,栽培贯叶连翘的生物量、开花的一致性和生长的适应性等明显优于野生贯叶连翘 ;良好的繁殖方式使贯叶连翘的收获期提前了 1a ;成熟后 3个月采收的种子萌发率较高 ,不同浓度赤霉素处理能明显提高其种子的萌发率 ;贯叶连翘的黑色腺体即分泌细胞球数量与金丝桃素间也基本存在着正相关关系 ,贵州贵阳居群和陕西丹凤居群黑色腺体明显多于其他居群 ,其金丝桃素含量在所有居群中为最高 ,而甘肃武都居群黑色腺体最少 ,其金丝桃素含量在 6个居群中最低。 6个野生居群栽培后生物量相近 ,在 4个不同土壤pH的基地大田中均能良好生长 ,说明江苏地区环境条件完全适合贯叶连翘引种栽培。     

Oxidative stress and antioxidant responses of mulberry (Morus alba) plants subjected to deficiency and excess of manganese

The aim of the study was to relate the effects of deficiency and excess of Mn with the generation of reactive oxygen species (ROS) and altered cellular redox environment in mulberry (Morus alba L.) cv. Kanva-2 plants. Mn deficiency symptom appeared as mild interveinal chlorosis in middle leaves. Mn-excess did not produce any specific symptom. Leaf water potential (Ψ) was increased in Mn-deficient and Mn-excess mulberry plants. Mn-deficient leaves contained less Mn, less chloroplastic pigments and high tissue Fe, Zn and Cu concentrations. Starch content was increased with increasing Mn supply. While reducing sugar content increased in Mn-deficient and Mn-excess plants as well, non-reducing sugars remained unaffected in Mn-deficient plants and decreased in Mn-excess plants. Moreover, study of antioxidative responses, oxidative stress (H₂O₂ and lipid peroxidation) and cellular redox environment [dehydroascorbate (DHA)/ascorbic acid (AsA) ratio] in Mn-stressed mulberry plants was also undertaken. Both hydrogen peroxide and lipid peroxidation were enhanced in the leaves of Mn-deficient plants. Increased H₂O₂ concentration in Mn-excess leaves did not induce oxidative damage as indicated by no change in lipid peroxidation. The ratio of the redox couple (DHA/AsA) was increased both in Mn-deficient or Mn-excess plants. The activities of superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) increased in Mn-deficient plants. The activity of ascorbate peroxidase (EC 1.11.1.11) increased with increasing Mn supply. The results suggest that deficiency or excess of Mn induces oxidative stress through enhanced ROS generation and disturbed redox couple in mulberry plants.     

Anti-Influenza A Virus Effect of Hypericum perforatum L. Extract

To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus (IAV) (H1N1) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney (MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC50) of 40 μg/mL. The mean 50% cytotoxic concentration (CC50) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 μg/mL; the mean CC50 for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin's minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.     

Anti-influenza A virus effect of Hypericum perforatum L. extract

To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus (IAV) (H₁N₁) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney (MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC₅₀) of 40 μg/mL. The mean 50% cytotoxic concentration (CC₅₀) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC₅₀ values of 5.0 μg/mL; the mean CC₅₀ for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin’s minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD₅₀ dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza. Foundation items: One Hundred Person Project of The Chinese Academy of Sciences (2008-287); The Project of Basic Scientific Research Fund for Central Public-Welfare of Institute of Sciences (BRF070402).     

Changes in antioxidant levels in Oryza sativa L. roots subjected to NaCl-salinity stress

Imposition of NaCl-salinity stress induced oxidative reactions in root tissue of rice seedlings. A uniform accumulation of proline was marked with the increasing NaCl concentrations. Both peroxide content and lipid peroxidation level (MDA) increased with the salt treatment from the control. CAT, GPx and SOD activities decreased with the increasing NaCl concentrations suggesting a possible oxidative damage to root tissue.     

Essential oil composition and variability of Hypericum perforatum L. growing in Lithuania

The composition of essential oils obtained from flowers and leaves in 11 accessions of Hypericum perforatum L. was analysed by GC and GC–MS. All the analysed oils were dominated by their oxygenated sesquiterpene fraction. Differences were attributed to the main components: caryophyllene oxide, spathulenol and viridiflorol. There were only a few monoterpenes in essential oil of all accessions. The data indicated some differences in sesquiterpene and aliphatic hydrocarbons, as well as in oxygenated aliphatics biosynthesis in flowers and leaves. The concentrations of β-caryophyllene and caryophyllene oxide in essential oils from leaves were higher than those from flowers, whereas dodecanol, spathulenol, viridiflorol, carotol and tetradecanol were present in higher quantities in flowers. Cluster analysis of all identified components grouped oils from flowers and from leaves into three clusters that corresponded with their major components: caryophyllene oxide, tetradecanal, tetradecanol and manool. Chemical variability of the analysed accessions seems likely to result from the genetic variability, since the influence of different environmental factors has been eliminated.     

Anti-inflammatory and analgesic activity of Indian Hypericum perforatum L

A standardised 50% aqueous ethanolic extract of the Indian variety of Hypericum perforatum (IHp) was examined for its putative anti-inflammatory and analgesic activity at the doses of 100 and 200 mg/kg, po. The experimental paradigms used were carrageenan induced pedal edema and cotton pellet induced granuloma for anti-inflammatory activity, whereas the tail flick, hot plate and acetic acid induced writhing methods were used to asses analgesic activity. Indomethacin (20 mg/kg, ip) was used as the standard anti-inflammatory drug. Pentazocine (10 mg/kg, ip) and aspirin (25 mg/kg, ip), both clinically used analgesics, were used as standard analgesics for comparison. IHp extract showed significant anti-inflammatory and analgesic activity at both dose levels, in all the paradigms used. Additionally, IHp potentiated the anti-inflammatory activity of indomethacin and analgesic activities of pentazocine and aspirin.     

Identification and genetic analysis of the APOSPORY locus in Hypericum perforatum L.

The introduction of apomixis – seed formation without fertilization – into crop plants is a long‐held goal of breeding research, since it would allow for the ready fixation of heterozygosity. The genetic basis of apomixis, whether of the aposporous or the diplosporous type, is still only poorly understood. Hypericum perforatum (St John’s wort), a plant with a small genome and a short generation time, can be aposporous and/or parthenogenetic, and so represents an interesting model dicot for apomixis research. Here we describe a genetic analysis which first defined and then isolated a locus (designated HAPPY for H ypericum AP OSP ORY ) associated with apospory. Amplified fragment length polymorphism (AFLP) profiling was used to generate a cleaved amplified polymorphic sequence (CAPS) marker for HAPPY which co‐segregated with apospory but not with parthenogenesis, showing that these two components of apomixis are independently controlled. Apospory was inherited as a dominant simplex gene at the tetraploid level. Part of the HAPPY sequence is homologous to the Arabidopsis thaliana gene ARI7 encoding the ring finger protein ARIADNE7. This protein is predicted to be involved in various regulatory processes, including ubiquitin‐mediated protein degradation. While the aposporous and sexual alleles of the HAPPY component HpARI were co‐expressed in many parts of the plant, the gene product of the apomict’s allele is truncated. Cloning HpARI represents the first step towards the full characterization of HAPPY and the elucidation of the molecular mechanisms underlying apomixis in H. perforatum.     

Reproduction pathway analysis of several Hypericum perforatum L. somaclonal families

Flow cytometry seed screen of mature seeds originating from several in vitro regenerated Hypericum perforatum L. somaclones and their seed progenies were used to screen the ways of reproduction of 4 subsequent generations of several somaclonal families and to search for the relation between the ploidy and prevalent mode of reproduction. The prevalent reproduction pathway of diploid plants was sexual reproduction. Seed samples of plants with higher ploidy levels showed an extensive variation in the mode of reproduction: BII and BIII hybrid formation and/or aposporous pseudogamy including parthenogenetic development of a reduced embryo sac.     

Identification and quantification of phenolic compounds in Hypericum perforatum L. transgenic shoots

Regeneration of transgenic shoots was achieved from Hypericum perforatum L. hairy roots on hormone-free MS/B₅ medium for a period of 4 weeks under a photoperiod of 16-h light. A control experiment was set up with root segments obtained from in vitro grown seedlings. Investigations have been made to study the production of phenolic compounds in non transgenic and transgenic shoot cultures. Six groups of phenolic compounds such as phenolic acids, flavonols, flavan-3-ols, naphtodianthrones, phloroglucinols, and xanthones were recorded in the transgenic shoots. Chlorogenic acid was found as the most representative phenolic acid in shoot extracts. With regard to the class of quercetin derivatives in transformed shoots, quercetin 6-C-glucoside usually dominated among the glycosides followed by quercitrin and hyperoside. The analysis of flavan-3-ols in transgenic shoots resulted in the identification of epicatechin and proanthocyanidin dimers. One of the main achievements in this study was considerably enhanced hypericin and pseudohypericin production in transgenic shoots. The concentration of identified naphtodianthrones was about 12-fold higher in transformed shoots compared to control. Chromatographic analysis of phloroglucinols in transgenic shoots resulted in the identification of hyperforin, while its homolog adhyperforin was detected in traces. A twofold higher content of hyperforin was observed in transgenic shoots compared to control. Although mangiferin was found as the main representative xanthone in shoot extracts, several other xanthones identified as γ-mangostin isomers, trihydroxy-1-methoxy-C-prenyl xanthone, garcinone E, and banaxathone E were de novo synthesized in transformed shoots. Therefore, H. perforatum transgenic shoots could be considered as a source for rapid and increased production of naphtodianthrones and other specific phenolic compounds.     

激素对贯叶连翘器官分化的影响

贯叶连翘 (ＨｙｐｅｒｉｃｕｍｐｅｒｆｏｒａｔｕｍＬ .)为多年生草本 ,中国民间主要用于止血、抗炎、妇科病等[1] ,欧洲民间用于治疗创伤也有相当长的历史。近年来 ,欧、美等国家和地区将其应用于抑郁症的治疗 ,取得了很好的疗效。 80年代后期 ,由于发现该植物体内含有显著抗     

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.