朱砂根愈伤组织培养及悬浮细胞系建立

以朱砂根(Ardisia crenata Sims.)无菌苗的茎段、叶片、胚轴和胚根为外植体进行愈伤组织诱导研究。结果表明:胚根在含有2,4-D的培养基中的诱导率最高,在添加5 mg L-1 AgNO3的MS+2,4-D 0.5 mg L-1+KT 0.01 mg L-1培养基中继代培养的增殖系数高达8倍以上。培养中获得了5种类型的愈伤组织(I-白色湿软状、Ⅱ-白色冰砂状、Ⅲ-淡黄色颗粒状、Ⅳ-黄绿色块状和V-绿色块状),其中Ⅱ和Ⅲ型愈伤组织可以成功建立悬浮细胞系,用M S+2,4-D 0.5 mg L-1+KT 0.01 mg L-1培养基进行固-液轮回培养,可以较好地保持悬浮细胞系。     

Purification and properties of glucoamylase from sugar beet cells in suspension culture

Glucoamylase and α-amylase are present in callus and suspension cultures of sugar beets (Beta vulgaris L.) as well as in mature roots. The subcellular localization of glucoamylase differed in callus and suspension-cultured cells: in callus, glucoamylase was present together with α-amylase in the soluble fraction of cells, but in suspension cultures, it was present predominantly in the extracellular fraction while most of the α-amylase activity remained in cells. Glucoamylase activity was considerably lower in callus protoplasts relative to the activities of α-mannosidase and α-galactosidase and the suspension of callus in Murashige-Skoog liquid medium or in mannitol by brief agitation resulted in the release of glucoamylase to the medium. These findings suggest that glucoamylase in callus may be present in a soluble form in the free space in the cell wall. Both mature roots and callus contained α-amylase and glucoamylase in the soluble fraction. Glucoamylases in the soluble fraction of callus and in the medium of suspension cultures were purified separately to homogeneity by the same four-step purification procedure, which included fractionation with ammonium sulfate, column chromatography on carboxymethyl cellulose, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The identity of the glucoamylases from the two sources was confirmed by a comparison of chromatographic behavior during purification, mobility during gel electrophoresis, M_r (83,000 D by SDS PAGE), and enzymic and kinetic properties of the catalytic reaction, such as optimal pH and temperature, heat stability, and K_m value for soluble starch. Glucoamylase from suspension cultures was one of the major proteins that were secreted into the medium. Dedifferentiation of leaves of young plants to callus was accompanied by induction of glucoamylase and repression of some α-amylases and the debranching enzyme.     

Monoterpene synthesis in shoots regenerated from callus cultures

-Shoots regenerated from two-year old callus cultures of Lavandula angustifolia and Rosmarinus officinalis accumulated monoterpenes characteristic of the parent tissue. No such compounds could be detected in undifferentiated callus maintained under a variety of conditions.     

Age and orientation of the cotyledonary leaf explants determine the efficiency of de novo plant regeneration and Agrobacterium tumefaciens-mediated transformation in Jatropha curcas L.

Effects of age and orientation of the explant on callus induction and de novo shoot regeneration from cotyledonary leaf segments of Jatropha curcas were studied. The callus induction and shoot regeneration capacity of cotyledonary leaf segments were found significantly related to the age of the explants and their orientation in culture medium. The youngest explant, derived from the cotyledonary leaf of germinated seed induced the highest regeneration response as compared to one- and two-week-old explants. A gradient response with age of the explant was observed in percentage of callus induction, shoot regeneration from callus and the number of shoots per regenerating callus. The explants cultured with their abaxial side in medium showed significantly higher regeneration response. The youngest explant was found to be most amenable to Agrobacterium-mediated transformation as compared to older explants. The fact that callus induced from the edges of the explant followed by de novo shoot induction, and strong transient gus expression observed in the edges of the explant are significant for routine Agrobacterium-mediated transformation and generation of stable transgenic plants in J. curcas.     

A simple and efficient method for obtaining transgenic soybean callus tissues

In the present study, a simple and efficient method for obtaining transgenic callus tissues of soybean [Glycine max (L.) Merr.] was developed based on Agrobacterium-mediated transformation. Hypocotyl segments of soybean were used as the starting material. Several factors such as soybean genotype, Agrobacterium concentration, inoculation time, co-cultivation period and addition of antioxidants in co-cultivation medium affecting the transformation efficiency were examined. The explants were cultured on callus induction medium containing 0.5 mg L^?1 6-benzylaminopurine and 2.0 mg L^?1, 2,4-Dichlorophenoxyacetic acid for callus induction. Callus tissues were induced at both the acropetal and basipetal ends. CaMV35S::GUS and CaMV35S::GFP transgenic callus tissues were obtained using the optimized protocol. The average transformation efficiency reached up to 87.7 % based on GUS detection. From inoculation with Agrobacterium to obtaining transgenic soybean callus will take about 3 weeks. In order to validate this method for gene function investigation, GVG::GmSARK transgenic soybean callus tissues were obtained and their senescence-associated phenotypes were assessed. To our knowledge, this is the first report using hypocotyl segments as starting materials to obtain transgenic callus, and this system provides a method for high-throughput screening of functional genes of interest in transformed soybean callus.     

Establishment and characterization of a maize Hi-II endosperm culture

An in vitro continuous endosperm callus culture derived from developing endosperm of transformation-amenable maize Hi-II genotype was obtained. The endosperm callus was composed of cells that differentiated into aleurone-like and starchy endosperm-like cell types. This callus has been maintained for 4?yr. Endosperm callus cells transcribe and produce zein proteins at a level similar to developing endosperm tissue. Starchy endosperm cells of the endosperm callus displayed active starch biosynthetic activity. The dual cell physiology of this culture limited the utility of the cell line for promoter analysis and transient assays of gene expression in the current culture conditions. However, because such cell line can be readily initiated and easily maintained for a long period of time, it provides an alternative tool for analysis of transgene expression in endosperm callus derived from transgenic maize lines in Hi-II background.     

Limitations of feeding experiments in studying alkaloid biosynthesis in Peganum harmala callus cultures

Feeding and trapping experiments to Peganum harmala callus cultures were limited by compartmentation; exogenous substrates were detoxified by precipitation, presumably as polymers or conjugates, or by conversion to water-soluble products, such as phenols and glucosides, easily stored in vacuoles. Alkaloid-producing and non-alkaloid-producing callus cultures were readily able to convert tryptamine to 5-hydroxytryptamine and harmaline to dihydroruine (8-hydroxyglucosylharmaline). Phenolic substrates, including 5- and 6-hydroxy-tryptophan, 5- and 6-hydroxytryptamine and harmalol, were not metabolized. In alkaloid-producing callus cultures, radioactivity from [methylene-¹⁴C]-l-tryptophan and [methyl-¹⁴C-]-harmaline was incorporated into harmine. The dilution of radioactivity was 30000- and 2-fold respectively.     

Biological effects of water-soluble soil phenol and soil humic extracts on plant systems

The aim of this research was to extract, with a sequential method, HS free from water-soluble phenols (HS-WP) and water-soluble phenols free from humic substances (WP-HS), and to evaluate and to compare the biological effects of these two extracts to humic substances (HS) and water-soluble phenols (WP) traditionally extracted. In each extract we determined the concentration of low molecular weight organic acids, soluble carbohydrates, fatty acids, phenolic acids and total proteins. We tested the biological activity of each soil extract and of the single identified compounds on different plant organs (Pinus laricio callus, Daucus carota cells, and Pinus laricio, Pinus halepensis, Lens culinaris and Cichorium intybus seeds). The results showed that eliminating from HS the WP fraction, and from WP the HS fraction we obtained extracts chemically different from HS as such, and WP as such. HS and HS-WP increased callus and cell growth and also root elongation of the different species used; in contrast, WP and WP-HS had negative effects inhibiting callus and cell growth and seed germination percentage of coniferous, herbaceous and leguminous species. The negative effects can be ascribed to the presence of phenolic acids in the extracts while the positive biological activity can be attributed to the presence of tartaric acid, and fatty acids. In conclusion, this study helps to discriminate the effects of humic substances against phenolic compounds extracted from soils explaining the different and in some way contradictory biological behaviour of these two main fractions of SOM.     

Reaction of soybean callus to culture filtrates of Phialophora gregata

Soybean [Glycine max (L.) Merr.] calli derived from susceptible and resistant soybean genotypes were exposed to the culture filtrates of pathogenic and non-pathogenic isolates of Phialophora gregata (Allington and Chamberlain) W. Gams. The rate of browning, growth and viability (measured by 2,3,5-triphenyltetrazolium chloride reduction) of the callus were determined after various exposure times to the fungus culture filtrates. Callus from susceptible Century, Cumberland, Corsoy 79, Harosoy and Clark 63 were sensitive to the culture filtrates of pathogenic isolates of P. gregata. Callus from Plant Introductions 437833 and 84946-2, when treated with fungal culture filtrates, did not develop browning and callus growth and cell viability were not decreased compared to untreated controls. Culture filtrates from non-pathogenic isolates of the fungus did not affect the growth of susceptible and resistant callus. Tobacco (Nicotiana tabacum L.) callus was not sensitive to the culture filtrate of a P. gregata isolate pathogenic to soybean. The fungal culture filtrate, based on limited evaluation, appears to be selective towards soybean callus. Based on this initial work, it appears that soybean callus bioassays have utility for evaluating soybean for resistance to P. gregata as well as assessing pathogenicity of fungus isolates.     

The influence of some elicitors on growth and morphogenesis of Hypericum perforatum L. callus cultures

We studied the effects of elicitors, such as mannan, gβ-1,3-glucan, ancymidol, and cork crumbs, on morphogenetic and biosynthetic potencies of shoot cultures of Hypericum perforatum L. In the presence of these elicitors, different morphogenetic structures of H. perforatum callus cultures were formed. A correlation was found between the morphogenetic processes and induction of hypericin and pseudohypericin biosynthesis in the callus cultures.     

Genotypic variation in cytokinin oxidase from phaseolus callus cultures

Genotypic variation in cytokinin oxidase has been detected in enzyme preparations from Phaseolus vulgaris L. cv Great Northern and Phaseolus lunatus L. cv Kingston callus cultures. Although cytokinin oxidase preparations from Great Northern and Kingston callus tissues appear to have very similar substrate specificities, the cytokinin oxidase activities from the two callus tissues were found to differ in a number of other properties. The cytokinin oxidase from P. vulgaris cv Great Northern callus tissue exhibited a pH optimum of 6.5 (bisTris) and had a strong affinity for the lectin concanavalin A. The cytokinin oxidase from P. lunatus cv Kingston callus tissue exhibited a pH optimum of 8.4 (Taps) and did not bind to concanavalin A. The two enzymes also differed in position of elution when chromatographed on DEAE-cellulose. Both cytokinin oxidase activities exhibited enhanced activity and lower pH optima in the presence of copper-imidazole complexes, but the optimum copper-imidazole ratio and the magnitude of enhancement differed for the two activities. In both callus tissues, transient increases in the supply of exogenous cytokinins induced increases in cytokinin oxidase activity. The differences in pH optima and in glycosylation (as evidenced by the observed difference in lectin affinity) of the cytokinin oxidases from Great Northern and Kingston callus tissues suggest that the compartmentation of cytokinin oxidase may differ in the two callus tissues. The possibility that enzyme compartmentation and isozyme variation in cytokinin oxidase may play a role in the regulation of cytokinin degradation in plant tissues is discussed in relation to known differences in the rates of cytokinin degradation in Great Northern and Kingston callus tissues.     

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.     

Callus induction and plant regeneration from hypocotyl explants of the forage legume Astragalus adsurgens

An efficient procedure was developed for inducing callus and plant regeneration using hypocotyl segments of Astragalus adsurgens. The combinations and concentrations of different growth regulators were shown to be critical factors for both the frequency and the type of callus formation as well as for the potential of callus differentiation. Of the four morphologically distinct types of calli that were induced, a friable, yellow callus, i.e. type I, induced on MS medium supplemented with 9.0 μM 2,4-dichlorophenoxyacetic acid and 2.2 μM N⁶-benzylaminopurine (BA), and then transferred to MS medium containing 0.5 μM α-naphthaleneacetic acid and 8.9 μM BA, exhibited the maximum frequency of shoot regeneration (75%). After regenerated shoots were transferred onto half-strength MS medium without growth regulators, they rooted and complete plants were obtained. Plantlet regeneration from callus cultures required 7–8 weeks. Received: 26 February 1997 / Revision received: 28 August 1997 / Accepted: 13 September 1997     

Evidence for the Presence of Bacteria-specific Proteins in Sterile Crown Gall Tumor Tissue

Cross-reacting antigens were found in bacteria-free crown gall tumor tissue tested with serum prepared against Agrobacterium tumefaciens (Smith and Towns.) Conn., but no such antigens were detected in callus tissue. Soluble proteins from tumor tissue, callus tissue, and the crown gall bacteria were fractionated on a DEAE-Sephadex (A-50) column. The diethylaminoethyl-Sephadex elution profile for tumor tissue showed three protein fractions that were not detected in the callus tissue. Two of these protein fractions were shown to be exclusively bacteria specific. Besides these qualitative differences between the two tissues, significant quantitative differences in the amount of protein fractions were also observed. The diethylaminoethyl-Sephadex column fractions from tumorigenic strain of A. tumefaciens corresponding in position to the three additional peaks in the tumor tissue also showed cross-reacting antigens when tested with serum prepared against sterile tumor tissue. It is suggested that tumor formation by A. tumefaciens involves integration of the bacterial genome into the host-cell genome.     

In vitro callus induction from adult tissues of peach (Prunus persica L. Batsch)

We describe an efficient protocol for callus induction from adult tissues of Prunus persica (L.) Batsch. Three different commercial peach genotypes, Early May®, Zise May®, and UFO-3®, plus three other genotypes from hybrid crosses performed in February 2006, PS108, PS208, and PS708, were used in the study. Thirteen explant treatments were tested using nine different plant parts. Murashige and Skoog and woody plant medium salts were assayed with several concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KN), and thidiazuron, and two different photoperiods were tested, a 16-h photoperiod or continuous darkness. In terms of the quantitative response, two parameters were assessed: the number of d to callus induction and relative callus growth recorded after 30 d. Woody plant medium supplemented with 2,4-D and KN significantly increased the rates of callus induction in the majority of treatments. And no significant differences among the P. persica genotypes were found. The explants derived from stem and calyx produced up to 85 and 96% callus induction, respectively. The protocol described here could be used for efficient callus induction in a range of Prunus spp.     

Morphological Variation in Pratylenchus penetrans

Variability of morphological characters used to separate Pratylenchus penetrans from other species of the genus was studied in a population originating from a single gravid female. Pronounced heteromorphism was observed and characterized. About 30% of females had a crenate-tail terminus. Several shapes of stylet knobs were characterized; 50% of them were anteriorly flattened to indented. The outer margin of the cephalic framework extended into the body from one-half to two annules. The shape of the spermatheca varied from round to oval. A fifth lateral line was observed in many specimens. Environmental factors, and particularly the host plant, influenced such morphometric characters as body length, width, esophagus length, stylet length, V value, a and b'' ratios, as well as qualitative characters such as tail terminus, growth of ovary, and shape of median bulb. Nematodes reared on pea and cabbage had a higher percentage of females with a crenate-tail terminus than those from tomato, rye, beet, and alfalfa callus culture. Nematodes from peas were longer and wider; they often had gonads that extended to esophagi, but they had shorter esophagi amt stylets than those from callus culture. Populations from different geographical locations also exhibited variahility in morphological characters, as did the Cornell population. The validity of many characters used in species identification is discussed, and the possibility that other related Pratylenchus species are conspecific with P. penetrans is suggested.     

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).     

Type I callus as a bombardment target for generating fertile transgenic maize (Zea mays L.)

To develop a less genotype-dependent maize-transformation procedure, we used 10-month-old Type I callus as target tissue for microprojectile bombardment. Twelve transgenic callus lines were obtained from two of the three anther-culture-derived callus cultures representing different gentic backgrounds. Multiple fertile transgenic plants (T₀) were regenerated from each transgenic callus line. Transgenic leaves treated with the herbicide Basta showed no symptoms, indicating that one of the two introduced genes, bar, was functionally expressing. Data from DNA hybridization analysis confirmed that the introduced genes (bar and uidA) were integrated into the plant genome and that all lines derived from independent transformation events. Transmission of the introduced genes and the functional expression of bar in T₁ progeny was also confirmed. Germination of T₁ immature embryos in the presence of bialaphos was used as a screen for functional expression of bar; however, leaf painting of T₁ plants proved a more accurate predictor of bar expression in plants. This study suggests that maize Type I callus can be transformed efficiently through microprojectile bombardment and that fertile transgenic plants can be recovered. This system should facilitate the direct introduction of agronomically important genes in to commercial genotypes.