Growth and functional responses of different cultivars of Lotus corniculatus to aluminum and low pH stress

Aluminum toxicity is an important stress factor in acid soils. Growth, respiration and permeability properties of root cells were studied in five cultivars of Lotus corniculatus subjected to aluminum (Al) or low pH stress. The cultivars showed significant differences in root elongation under stress conditions, which correlated with changes in membrane potential (E_M) of root cortical cells. A pH drop from 5.5 to 4.0 resulted in significant membrane depolarization and root growth inhibition. The strongest inhibition was observed in cv. São Gabriel (33.6%) and least in cv. UFRGS (25.8%). Application of an extremely high Al concentration (2 mM) stopped the root growth in cv. INIA Draco, while inhibition in cv. UFRGS reached only 75%.The E_M values of cortical cells of Lotus roots varied between −115 and −144 mV. Treatment with 250 μM of AlCl₃ (pH 4) resulted in rapid membrane depolarization. The extent of the membrane depolarization ranged between 51 mV (cv. UFGRS) and 16 mV (cv. INIA Draco). The membrane depolarization was followed by a loss of K⁺ from Al-treated roots (2 mM Al) and resulted in a decrease of the diffusion potential (E_D). The total amount of K⁺ in Al-treated roots dropped from 31.4 to 16.8 μmol g⁻¹ FW in sensitive cv. INIA Draco, or from 26.1 to 22.7 μmol g⁻¹ FW in tolerant cv. UFGRS. The rate of root respiration under control conditions as well as under Al treatment was higher in cv. INIA Draco than in cv. UFRGS. Al-induced inhibition of root respiration was 21–34% of the control.     

Ginsenoside production in different phenotypes of Panax ginseng transformed roots

Transformed roots were obtained after the inoculation of sterile root discs of Panax ginseng C.A. Meyer with Agrobacterium rhizogenes A4. The established hairy root lines displayed three morphological phenotypes when cultured on hormone-free liquid Schenk and Hildebrandt medium. Most of the cultures showed the characteristic traits of hairy roots (HR-M), while others were either callus-like (C-M) or thin (T-M) without branching. The growth rate of the transformed root lines was always higher than that of untransformed roots, showing that the genetic changes caused by the A. rhizogenes transformation conditioned a higher biomass formation. When considering the different transformed root phenotypes, we can observe that the highest ginsenoside production was achieved by HR-M root lines, closely followed by C-M ones, whereas the lowest yield was reached by T-M root phenotype. The study of the integration of the TL-DNA and TR-DNA fragments of the pRiA4 in the root genome showed that the aux1 gene was always detected in HR-M and C-M root phenotypes which presented the highest biomass and ginsenoside productions. This fact suggests a significant role of aux genes in the morphology of Panax ginseng transformed roots. The ginsenoside pattern of transformed roots varied according to their morphology, although the ginsenoside contents of the Rg group was always higher than that of the Rb group. From our results, we can infer the potential of some root phenotypes of Panax ginseng hairy root cultures for an improved ginsenoside production.     

Trypanosoma theileri: In vitro cultivation in tsetse fly and vertebrate cell culture systems

Epimastigote forms of Trypanosoma theileri were grown at 25°C in insect cell culture media and in Glossina tissue cultures for more than 6 months. Doubling times of 10–14 h during exponential growth were observed. In cell cultures which had been derived from pupal tsetse flies growth rates were higher than in cell free media; in a larval cell line, however, growth of T. theileri was inhibited. Ecdysteroids and juvenile hormone I reduced multiplication of T. theileri in cell free media. When T. theileri was incubated in different sera only fetal calf serum (FCS) supported growth. Epimastigote forms transformed into trypomastigote bloodforms when cultured at 37°C in FCS, vertebrate cell cultures, and Eagle's medium, but not in insect media or Glossina cell cultures. Oxygen uptake of epimastigotes could be inhibited by rotenone antimycin A and cyanide; trypomastigotes were not affected by these inhibitors.     

刺槐核糖体蛋白同源基因RpRPL22在共生结瘤过程中功能研究

目的: 核糖体蛋白(RPs)属于多功能蛋白,能够参与调控细胞生长和响应胁迫条件。RpRPL22是一个从豆科植物刺槐中分离得到的结瘤相关基因,通过序列比对发现其与核糖体大亚基蛋白RPL22高度同源。对其如何通过调控根瘤菌侵染而在共生结瘤过程中发挥重要作用进行了较为深入的探索。方法: 利用实时荧光定量PCR技术(qRT-PCR)分析RpRPL22在接菌后不同时间及不同植物组织的表达变化。利用cDNA末端快速扩增技术(RACE)获得目的基因cDNA全长。通过GFP报告基因进行RpRPL22亚细胞定位分析。通过Gateway BP重组技术构建RNA干扰(RNAi)重组载体,借助电转化法将重组载体转至农杆菌K599,利用农杆菌介导植物根部,接菌后观察和测量植株表型。首先从宏观水平统计观察目的基因是否对结瘤过程有影响,其次从分子水平揭示目的基因在共生结瘤过程的重要功能。结果: 不同接菌时期、不同植物组织目的基因qRT-PCR相对表达量结果显示,几乎在所有取样的接菌时间,目的基因RpRPL22在接菌根中的相对表达量都低于未接菌对照根,只有接菌后第25天除外。在成熟的根瘤中,接菌后第25天该基因的表达量也最高。洋葱表皮和毛状根亚细胞定位结果均显示在椰菜花叶病毒(CaMV)的35S启动子控制下,RpRPL22融合绿色荧光蛋白GFP的荧光信号在细胞核和细胞质有明显的表达。RNAi转化植株的表型统计观察结果,比如植株鲜重、植株的有效结瘤数目较对照组均有明显的降低;同时RNAi转化植株在根瘤菌侵染过程形成的侵染线数目和根瘤原基数目较对照均显著降低。根瘤切片实验用于观察根瘤显微超微结构,结果显示RNAi植株根瘤中固氮区的受菌侵染细胞数目与对照相比明显减少。电镜观察根瘤单个受菌侵染细胞中类菌体形态显示,RNAi根瘤中类菌体侵染细胞胞体多呈不规则形状,皱缩变形严重,环类菌体周间隙空间增大,多共生体融合,表现出细胞凋亡的迹象。对照根瘤中的受菌侵染细胞胞体多呈圆形椭圆形,胞质饱满丰富且分布均匀,细胞发育正常,表明RNAi植株根瘤发育过程明显受阻。结论: 核糖体蛋白(RP)能够参与调控豆科植物共生结瘤过程,相关同源基因RpRPL22可能在起始根瘤菌侵染植物和阻止类菌体降解过程中起重要作用。     

Transformation of Saussurea involucrata by Agrobacterium rhizogenes: Hairy root induction and syringin production

Agrobacterium rhizogenes-mediated genetic transformation of Saussurea involucrata was investigated. Four bacterial strains, A4, LBA 9402, R1000 and R1601 and three explant types, leaf blade, petiole and root, were examined. Over 100 hairy root lines were successfully established with strains R1601, R1000 and LBA9402, but none with A4. The highest transformation efficiency of 67% was achieved by using strain R1601 with root explants. One hairy root line isolated from this combination, HR1601-1, produced up to 43.5 ± 1.13 mg syringin g⁻¹ dw, which is about 50-fold higher than that in the wild type plants.Two other lines, HR1000-1 and HRLBA9402-1, isolated from R1000- and LBA9402-transformed roots, respectively, also displayed high capacity of syringin production, being 32.5 ± 3.08 and 39.7 ± 1.37 mg syringin g⁻¹ dw. These three lines were characterized in detail. Polymerase chain reaction analyses confirmed these root lines were of A. rhizogenes origin.     

Biotransformation of paeonol by Panax ginseng root and cell cultures

Panax ginseng root and cell cultures were shown to biotransform paeonol (1) into its 2-O-β-d-glucopyranoside (2). P. ginseng root cultures were also able to biotransform paeonol (1) into its 2-O-β-d-xylopyranoside (3), 2-O-β-d-glucopyranosyl(1 → 6)-β-d-glucopyranoside (4) and 2-O-β-d-xylopyranosyl(1 → 6)-β-d-glucopyranoside (5), and its demethylated derivate, 2′,4′-dihydroxyacetophenone (6). Compounds 3 and 4 are new glycosides. It is the first example that the administrated compound was converted into its xylopyranoside by plant biotransformation.     

茄病镰刀菌单克隆抗体的制备及胶体金免疫层析试纸条的研发*

Objective: The cell lines secreting specific monoclonal antibodies (McAbs) were prepared by using Fusarium solani, one of the pathogenic fungi causing root rot of Fritillaria thunbergii, and the colloidal gold immunochromatographic test strip based on McAbs was developed to provide scientific basis for detecting root rot of F. thunbergii. Methods: Hybridoma technology was used to obtain cell lines that could secrete specific McAbs against F. solani using the whole protein extract of F. solani as the antigen. The specificity, titer, sensitivity and binding protein of McAbs were detected by indirect ELISA and Western blot. Colloidal gold particles were prepared by trisodium citrate reduction method and McAbs were labeled to prepare colloidal gold immunochromatographic strip. Results: Three cell lines secreting specific McAbs against F. solani were obtained, which were named as FsA3, FsG6 and FsD4. The detection sensitivity of FsA3 was 24.41 ng / mL, and that of both FsG6 and FsD4 was 12.21 ng / mL. FsA3, FsG6 and FsD4 had strong reactions to F. solani, and had no cross-reaction to Alternaria tenuissima, A. alternata, Botrytis cinerea, F. equiseti, F. incarnatum, F. oxysporum, Phoma sp., and Phomopsis oblonga. The colloidal gold immunochromatographic strip based on FsG6 showed only a quality control line when detecting the tissue culture seedlings of F. thunbergii. When 100 ng F. solani antigen or the samples of F. thunbergii infested with root rot disease were detected, there were visible quality control lines and test lines. Conclusion: The specificity and sensitivity of the McAbs and test strip are sufficient to detect F. solani isolated from diseased strains of F. thunbergii, which provides the technical support for the rapid detection of root rot of F. thunbergii in the field.     

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin     

锁阳及其寄主白刺内生真菌的生态分布及遗传关系

首次研究了寄生植物锁阳及其寄主白刺内生真菌的分布特征及遗传关系.采用组织块法分离天然白刺、寄生体中锁阳和白刺的内生真菌,利用ITS-rDNA分子序列并结合形态学方法鉴定菌种,研究内生真菌的分离率、定殖率、分离频率、多样性指数、均匀度指数及相似性系数等的差异,以及寄生关系中内生真菌的多样性、遗传关系及分布特征等.结果表明: 本次获得的49株内生真菌隶属于18个分类单元,95.9%为子囊菌,4.1%为担子菌;内生真菌总分离率为15.3%,总定殖率为25.0%;天然白刺中内生真菌Shannon多样性指数最大,为2.13;锁阳花序与锁阳茎的内生真菌相似性系数最大,为0.50;镰孢菌属为白刺的优势菌属,青霉属为锁阳的优势菌属.锁阳与白刺寄生体中真菌类群的差异性分布表明寄生关系对内生真菌群落存在一定影响.     

The influence of plant growth regulators on callus induction in pumpkin (Cucurbita pepo L.) hairy roots

Following in vitro infection with Agrobacterium rhizogenes wild strain (mannopine, 8196) and two A. tumefaciens transconjugant strains (C58C1 pArA4abc and C58C1 pArA4b) transformed (hairy) roots were induced in pumpkin (C. pepo L.) cotyledons. The presence of pRi T-DNA in pumpkin long-term hairy root cultures was determined by Southern hybridization. The influence of plant growth regulators on callus induction in root explants from hairy root lines, which differed mutually in morphology and growth rate, was tested by the addition of growth regulators to basal nutrient medium; while 2.4-D inhibited root proliferation in all hairy root lines tested, callus induction depended both on plant growth regulators and the root line.     

Grafting tomato (Solanum lycopersicum) onto the rootstock of a high-altitude accession of Solanum habrochaites improves suboptimal-temperature tolerance

Grafting is regarded as a promising tool to broaden the temperature optimum of elite tomato cultivars. However, suitable low-temperature tolerant tomato rootstocks are not yet available and its breeding is hampered by a lack of variation in low-temperature tolerance within the cultivated tomato. In this study, therefore, the impact of grafting tomato (Solanum lycopersicum Mill. cv. Moneymaker, Sl) onto the rootstock of a cold-tolerant high-altitude accession of a related wild species (Solanum habrochaites LA 1777 Humb. & Bonpl., Sh) was examined at different combinations of optimal (25 °C) and/or suboptimal (15 °C) air/root-zone temperatures (RZT), i.e. 25/25, 25/15, 15/25 and 15/15 °C. Self-grafted tomato plants were used as controls. Both scion/rootstock combinations, Sl/Sl and Sl/Sh, were grown hydroponically and compared for biomass production and partitioning, plant morphology, carbohydrate partitioning and leaf C and N status. Grafting tomato onto Sh increased the relative growth rate of shoots with 26 and 11% at 25/15 and 15/15 °C, respectively. This increase could be attributed to stimulation of the leaf expansion rate. Graft combinations with Sh rootstocks were characterized by higher root mass ratios, particularly at 15 °C RZT. Suboptimal RZT strongly reduced the relative growth rate of Sl roots but not of Sh. This was correlated to differences in inhibition of root elongation. In contrast to tomato grafted onto Sh, leaf total C and total N concentrations increased in self-grafted tomato plants in response to 15 °C RZT. The increase in leaf total C concentration of Sl/Sl graft combinations at 15 °C RZT could be ascribed largely to starch accumulation. This study illustrates that growth of vegetative tomato plants at suboptimal temperature is for a significant part inhibited by its poor root development. Grafting tomato onto a low-temperature rootstock provides an alternative tool to reduce, in part, the grow-limiting effects of suboptimal RZ temperature on the shoot. To improve the low-temperature tolerance of existing commercial tomato rootstocks, S. habrochaites LA 1777 appeared to be a valuable germplasm pool.     

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l⁻¹ with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg l ⁻¹ with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.     

Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: a valuable tool for functional genomics

Background  

Transgenic approaches provide a powerful tool for gene function investigations in plants. However, some legumes are still recalcitrant to current transformation technologies, limiting the extent to which functional genomic studies can be performed on. Superroot of Lotus corniculatus is a continuous root cloning system allowing direct somatic embryogenesis and mass regeneration of plants. Recently, a technique to obtain transgenic L. corniculatus plants from Superroot-derived leaves through A. tumefaciens-mediated transformation was described. However, transformation efficiency was low and it took about six months from gene transfer to PCR identification.     

Transformation of callus cultures of nine plant species mediated by agrobacterium

A simple method for the Agrobacterium-mediated transformation of callus cultures of nine plant species, Lycopersicum esculentum Mill, Petunia hybrida Vilm, Pimpinella anisum L., Solanum melongena L., S. tuberosum L., Nicotiana glauca Graham, N. glutinosa L., N. plumbaginifolia Viviani and N. tabacum L., is described. Plant calli were resuspended in liquid media, co-cultivated with A. tumefaciens, and plated on restrictive media. The combination of a gene for kanamycin resistance and a gene for firefly luciferase was convenient in the selection and confirmation of hundreds of transformants. Four strains of A. tumefaciens, A208, A348, A281, and PC2760, were employed. All of the callus cultures were successfully transformed with at least one strain of A. tumefaciens, and A281 was the most effective of the four strains. N. glutinosa, N. plumbaginifolia, N. tabacum, P. hybrida and L. esculentum were transformed more efficiently than the other species tested.     

Selection and characterization of somatic hybrids between Lycopersicon esculentum and Lycopersicon peruvianum

Somatic hybrids of the cultivated tomato, Lycopersicon esculentum, and a wild species, L. peruvianum, were obtained by fusion of leaf protoplasts from both species in the presence of poly-ethylene-glycol (PEG) or in an electric field. The somatic hybrids were selected on the basis of kanamycin resistance of L. esculentum and the plant regeneration capacity of L. peruvainum. Chromosome counts in root tips and the determination of the number of chloroplasts in guard cell pairs revealed that the majority of these hybrids was tetraploid (2n = 4x = 48). The remaining hybrids were at the hexaploid level with chromosome numbers between 64 and 72. The hybrid nature of the regenerated plants was confirmed by analysis of isozyme markers and by their morphology. Most hybrids did flower and set fruits and seeds after selfing. According to RFLP analysis 6 out of the 10 hexaploid hybrids contained two genomes of L. esculentum and four genomes of L. peruvianum. One of these hexaploids had genomes of two different L. peruvianum genotypes and was therefore considered to be derived from a triple protoplast fusion. The hexaploid plants were less fertile than the tetraploids and more resembled L. peruvianum.     

尖孢镰刀菌亚麻专化型Folprp4基因参与调控菌丝生长和分生孢子发生

目的: 通过对尖孢镰刀菌中Folprp4基因的鉴定,揭示其在尖孢镰刀菌中的功能及致病相关性。方法: 基于同源重组原理,根据测定出的Folprp4基因序列,应用Split-Marker重组技术构建含有潮霉素抗性基因(hph)的基因缺失盒。将基因缺失盒经PEG介导转化到野生型原生质体中,在含有潮霉素B的TCC培养基上筛选转化子,通过PCR正负筛查获得Folprp4基因缺失突变株(ΔFolprp4)。构建含有Folprp4基因的载体pZDH1,并将其转化到敲除突变体中进行互补测验。结果: 与野生型(hm)和异位插入突变体(ecFolprp4)相比,敲除突变体菌丝生长受到严重阻碍,当野生型和异位插入突变体长满整个平板时,敲除突变体菌落呈小点状。敲除突变体的另一个显著变化是ΔFolprp4的分生孢子产量显著下降。侵染实验表明,ΔFolprp4对亚麻幼苗的毒力显著降低。互补实验表明,该互补载体的回复子(Folprp4-C)在菌落形态、生长速率、分生孢子产量和毒力方面均恢复到了野生型菌株。结论: Folprp4基因与尖孢镰刀菌的菌丝生长、分生孢子发生和致病性有关。     

Occurrence of azoxyglycosides in Macrozamia riedlei and their effects on the free-living isolated Nostoc PCC 73102

The presence of azoxyglycosides in the Australian cycad Macrozamia reidlei was examined using high performance liquid chromatography. Cycasin and macrozamia were present in all tissues examined, cycasin being three to 17 times more abundant than macrozamin. The symbiotic organ, the coralloid root, contained 0.16% [g/g (fresh weight)] cycasin and 0.01 % (same unit) macrozamin. Addition of these azoxyglycoside concentrations to nitrogen-fixing Nostoc PCC 73102 cultures, a filamentous heterocystous cyanobacterium originally isolated from Macrozia, inhibited light and dark nitrogenase (acetylene reduction) activity. No effects were observed on in vitro glutamine synthetase activity or net in vivo CO₂ fixation. Cycasin (1.6%) and macrozamin (0.1%), i.e. 10 times the concentrations observed in the coralloid root, decreased phycobiliprotein content by 25 and 45%, 1 and 4 hr after the addition, respectively. The relative distribution of individual phycobiliproteins was not affected.     

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Stable expression of insect GABA receptors in insect cell lines promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines

We are interested in establishing stably transformed insect cell lines efficiently expressing the insect γ-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selectable marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chloride ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the genetic transformation of non-Drosophilid insects.