Effects of arbuscular mycorrhizal fungi on growth and nitrogen uptake of Chrysanthemum morifolium under salt stress.

Soil salinity is a common and serious environmental problem worldwide. Arbuscular mycorrhizal fungi (AMF) are considered as bio-ameliorators of soil salinity tolerance in plants. However, few studies have addressed the possible benefits of AMF inoculation for medicinal plants under saline conditions. In this study, we examined the effects of colonization with two AMF, Funneliformis mosseae and Diversispora versiformis, alone and in combination, on the growth and nutrient uptake of the medicinal plant Chrysanthemum morifolium (Hangbaiju) in a greenhouse salt stress experiment. After 6 weeks of a non-saline pretreatment, Hangbaiju plants with and without AMF were grown for five months under salinity levels that were achieved using 0, 50 and 200 mM NaCl. Root length, shoot and root dry weight, total dry weight, and root N concentration were higher in the mycorrhizal plants than in the non-mycorrhizal plants under conditions of moderate salinity, especially with D. versiformis colonization. As salinity increased, mycorrhizal colonization and mycorrhizal dependence decreased. The enhancement of root N uptake is probably the main mechanism underlying salt tolerance in mycorrhizal plants. These results suggest that the symbiotic associations between the fungus D. versiformis and C. morifolium plants may be useful in biotechnological practice.

Variation in tissue Na(+) content and the activity of SOS1 genes among two species and two related genera of Chrysanthemum.

BACKGROUND: Chrysanthemum, a leading ornamental species, does not tolerate salinity stress, although some of its related species do. The current level of understanding regarding the mechanisms underlying salinity tolerance in this botanical group is still limited. RESULTS: A comparison of the physiological responses to salinity stress was made between Chrysanthemum morifolium 'Jinba' and its more tolerant relatives Crossostephium chinense, Artemisia japonica and Chrysanthemum crassum. The stress induced a higher accumulation of Na(+) and more reduction of K(+) in C. morifolium than in C. chinense, C. crassum and A. japonica, which also showed higher K(+)/Na(+) ratio. Homologs of an Na(+)/H(+) antiporter (SOS1) were isolated from each species. The gene carried by the tolerant plants were more strongly induced by salt stress than those carried by the non-tolerant ones. When expressed heterologously, they also conferred a greater degree of tolerance to a yeast mutant lacking Na(+)-pumping ATPase and plasma membrane Na(+)/H(+) antiporter activity. The data suggested that the products of AjSOS1, CrcSOS1 and CcSOS1 functioned more effectively as Na (+) excluders than those of CmSOS1. Over expression of four SOS1s improves the salinity tolerance of transgenic plants and the overexpressing plants of SOS1s from salt tolerant plants were more tolerant than that from salt sensitive plants. In addition, the importance of certain AjSOS1 residues for effective ion transport activity and salinity tolerance was established by site-directed mutagenesis and heterologous expression in yeast. CONCLUSIONS: AjSOS1, CrcSOS1 and CcSOS1 have potential as transgenes for enhancing salinity tolerance. Some of the mutations identified here may offer opportunities to better understand the mechanistic basis of salinity tolerance in the chrysanthemum complex.

Salicylic acid-induced changes in physiological parameters and genes of the flavonoid biosynthesis pathway in Artemisia vulgaris and Dendranthema nankingense during aphid feeding.

Phloem-feeding aphids cause serious damage to plants. The mechanisms of plant-aphid interactions are only partially understood and involve multiple pathways, including phytohormones. In order to investigate whether salicylic acid (SA) is involved and how it plays a part in the defense response to the aphid Macrosiphoniella sanbourni, physiological changes and gene expression profiles in response to aphid inoculation with or without SA pretreatment were compared between the aphid-resistant Artemisia vulgaris 'Variegata' and the susceptible chrysanthemum, Dendranthema nankingense. Changes in levels of reactive oxygen species, malondialdehyde (MDA), and flavonoids, and in the expression of genes involved in flavonoid biosynthesis, including PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), CHI (chalcone isomerase), F3H (flavanone 3-hydroxylase), F3'H (flavanone 3'-hydroxylase), and DFR (dihydroflavonol reductase), were investigated. Levels of hydrogen peroxide, superoxide anions, MDA, and flavonoids, and their related gene expression, increased after aphid infestation and SA pretreatment followed by aphid infestation; the aphid-resistant A. vulgaris exhibited a more rapid response than the aphid-susceptible D. nankingense to SA treatment and aphid infestation. Taken together, our results suggest that SA could be used to increase aphid resistance in the chrysanthemum.

[Effects of nitrogen form on growth and quality of Chrysanthemums morifolium].

This paper is aimed to study the effects of nitrogen form on the growth and quality of Chrysanthemums morifolium at the same nitrogen level. In order to provide references for nutrition regulation of Ch. morifolium in field production, pot experiments were carried out in the greenhouse at experimental station of Nanjing Agricultural University. Five proportions of ammonium and nitrate nitrogen were set up and a randomized block design was applied four times repeatedly. The results showed that the growth and quality of Ch. morifolium were significantly influenced by the nitrogen form. The content of chlorophyll and photosynthesis rate were the highest at the NH4(+) -N /NO3(-) -N ratio of 25:75; The activities of NR in different parts of Ch. -morifolium reached the highest at the NH4(+) - N/NO3(-) -N ratio of 0: 100. The contents of nitrate nitrogen in the root and leaves reached the highest at the NH4(+) -N/NO3(-) -N ratio of 50:50. The activities of GS, GOGAT and the content of amylum increased with the ratio of NO3(-) -N decreasing and reached it's maximum at the NH4 + -N/NO3 - -N ratio of 100: 0. The content of ammonium nitrogen were the highest at the NH4 + -N /NO3 --N ratio of 75: 25, while the content of soluble sugar reached the highest at the NH4(+)-N/NO3(-) -N ratio of 25: 75. The content of flavones, chlorogenic acid and 3,5-O-dicoffeoylqunic acid were 57.2 mg x g(-1), 0.673% and 1.838% respectively, reaching the maximum at the NH4(+) -N /NO3(-) -N ratio of 25:75; The content of luteoloside increased with the ratio of NO3(-) -N increasing and reached it's maximum at the NH4(+) -N/NO3(-) -N ratio of 0: 100. The yield of Ch. morifolium reached it's maximum at the NH4(+) -N /NO3(-) -N ratio of 25:75. Nitrogen form has some remarkable influence on the nitrogen metabolism, photosynthesis and growth, Nitrogen form conducive to the growth and quality of Ch. morifolium at the NH4(+) -N /NO3(-) -N ratio of 25: 75.

Isolation and characterisation of Chrysanthemum crassum SOS1, encoding a putative plasma membrane Na(+) /H(+) antiporter.

A full-length cDNA homologue of SOS1 (salt overly sensitive 1) was isolated from the salinity-tolerant species Chrysanthemum crassum and found to encode a Na(+) /H(+) antiporter, using degenerate PCR and RACE-PCR. The 3752-bp sequence comprised a 3438 bp open reading frame, encoding a 127-kDa protein with 12 transmembrane domains within its N terminal portion, and a hydrophilic cytoplasmic tail in its C-terminal portion. CcSOS1 appears to be a plasma membrane protein, and shares ∼62% identity at the peptide level with its Arabidopsis thaliana homologue. Expression of CcSOS1 in the roots of C. crassum was sensitive to salinity stress, while in the leaves CcSOS1 was down-regulated in the presence of abscisic acid. CcSOS1 transcript abundance was reduced in both roots and leaves of plants exposed to low temperature, while it was increased in leaves (but not in roots) after drought stress. CcSOS1 expression was not regulated in the presence of CaCl2 . A heterologous complementation assay in yeast suggested that CcSOS1 directs Na(+) efflux, mimicking the function of the endogenous NHA1 protein. Thus CcSOS1 appears to encode a salinity-inducible plasma membrane Na(+) /H(+) antiporter. This gene may be useful in transgenic approaches to improving the salinity tolerance of related ornamental species.

cDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment.

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C.×morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200 mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3 h, 12 h, 24 h and 48 h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.

[Effect of PEG stress on plantlets of Chrysanthemum morifolium induced by endophytic botrytis sp. (C1) and Chaetomium globosum (C4)].

The effect of the endophytic fungi Botrytis sp. (C1) or Chaetomium globosum (C4) on the drought resistance of Chrysanthemum morifolium was studied. Ch. morifolium plantlets were inoculated with C1, C4 and cultured in the pots for 60 days, then the plantlets were stressed by 0%, 10%, 20%, 30%, 40% PEG6000 respectively in order to simulate different drought conditions. Biomass, the activities of SOD, POD, PAL, the contents of MDA and soluble protein of each group were determined. The results showed that endophytic fungi groups grew better than the control (without inoculation endophytic fungi). With the increasing of the concentration of PEG6000, the biomass of Ch. morifolium of each groups decreased, while the biomass of fungi groups was significantly higher than that of control, moreover C4 group higher than C1 group. With the concentration of PEG increasing, the content of MDA of each group increased too, while POD activity and soluble protein content of all treatments increased at first and then decreased. SOD activity and PAL activity of the control were increased with the increase of PEG concentration, but SOD activity of the two fungi groups were stable. After been stressed by different concentrations of PEG, MDA content of two fungi groups were always lower than the control, while SOD activity, POD activity, PAL activity and soluble protein content were higher. In conclusion, endophytic fungi can increase the drought resistance of Ch. morifolium.

[Effects of soil factors on active component content of Chrysanthemum morifolium].

OBJECTIVE: To study the effects of soil factors on the active component content of Chrysanthemum morifolium and screen out the leading factors. METHOD: The active component of water soluble extracts, flavonoids, phenolic compounds and mineral elements were determined and chemical properties and mineral elements of soil were analyzed for studying the effects on Ch. morifolium through correlation, stepwise regression, path and grey correlation analysis. RESULT: Soil available P and K were the most important factors that affected the active component content of Ch. morifolium, followed by urease, phosphatase and invertase activities and organic matter. The mineral elements in Ch. morifolium and in soil correlated well, P and K were enriched in the plant mostly, followed by Cd, Ca, Zn, Cu. The main leading factors of mineral elements in soil were P and K, followed by Fe, Cu and Zn. CONCLUSION: Soil was one of the important factors which affected the active component content of Ch. morifolium.

Development of alternative plant vitrification solutions in droplet-vitrification procedures.

This study aimed at developing alternative vitrification solutions, modified either from the original PVS2 vitrification solution by increasing glycerol and sucrose and/or decreasing dimethylsulfoxide and ethylene glycol concentration, or from the original PVS3 vitrification solution by decreasing glycerol and sucrose concentration. The application of these vitrification solutions to two model species, i.e. garlic and chrysanthemum in a droplet-vitrification procedure, revealed that PVS3 and variants were superior to PVS2 and variants and that most PVS2 variants were comparable to the original PVS2. Both species were sensitive to chemical toxicity of permeating cryoprotectants and chrysanthemum was also sensitive to osmotic stress. The lower recovery of cryopreserved garlic shoot apices dehydrated with PVS2 and variants compared with those dehydrated with PVS3 and variants seemed attributed to cytotoxicity of the vitrification solutions tested as well as to insufficient protection against freezing injury. Chrysanthemum shoot tips were very sensitive to both chemical toxicity and osmotic stress and therefore, induction of cytotoxity tolerance during preconditioning was required for successful cryopreservation. The present study revealed that some of the PVS2 variants tested which have increased glycerol and sucrose and/or decreased dimethylsulfoxide and ethylene glycol concentration can be applied when explants are of medium size, tolerant to chemical toxicity and moderately sensitive to osmotic stress. PVS3 and variants can be used widely when samples are heterogeneous, of large size and/or very sensitive to chemical toxicity and tolerant to osmotic stress.

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line.

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.

[Selection of sodium chloride tolerant mutants in Chrysanthemum morifolium in Anhui].

OBJECTIVE: To select the sodium chloride tolerant mutants of the Chrysanthemum morifolium callus through tissue culture and EMS (ethylmethane sulfonate) treatment. METHOD: Calli were induced from the leaves of C. morifolium. The calli were treated with 0.2% and 0.5% EMS, respectively. After 15 days' culture, the calli were transplanted to selection media with 0.5%, 1.0% and 1.5% NaCl, and the sodium chloride tolerant mutants were selected out. RESULT AND CONCLUSION: After tested the sodium chloride tolerant stability, the callus selected are found to be the mutants indeed.