In Vitro Studies of Secondary Metabolite-Related Responses in Some Species of Genus Grifola (Agaricomycetes) from Argentina.

Grifola gargal Singer and Grifola sordulenta (Mont.) Singer mushrooms are related to Grifola frondosa (Dicks.) Gray, which is well known for its medicinal properties. In vitro studies were performed to find a useful guide for optimizing the environmental parameters through biotransformation of lignocellulosic materials and basidiome development, also considering secondary metabolism-related responses (SMRRs) associated with these processes and the variability among species and strains; this optimization is necessary to make the mushroom's industrial cultivation profitable. Morphological features of mycelial cultures revealed that intraspecific variability was of taxonomic relevance. A low ligninolytic capacity in studied Grifola species was observed when compared with 2 control species: G. frondosa and Ganoderma lucidum. Experiments with nutrient media containing different carbohydrate sources indicated that G. gargal mycelia grew better in xylulose and G. sordulenta, in xylulose or cellulose; in addition, the latter species presented cellobiose dehydrogenase activity. An additional study of SMRRs under different light conditions (aroma, pigmentation, and morphogenic manifestations) showed that white light was better than blue, green, or red-filtered light at inducing advanced SMRRs. The results of SMRR stimulation are proposed as useful guidance for optimizing the environmental parameters for bioprocesses aimed at metabolite production.

Submerged Culture of Grifola gargal and G. sordulenta (Higher Basidiomycetes) from Argentina as a Source of Mycelia with Antioxidant Activity.

Submerged culture is an alternative mycelium source for Grifola gargal and G. sordulenta, two rare edible mushrooms related to Grifola frondosa. This work studies their mycelia as a source of antioxidants. The efficient concentrations of methanolic extracts in both radical scavenging (RS) and reducing power (RP) abilities in G. gargal and in G. sordulenta showed a high antioxidant activity. In the experimental design used, the antioxidant activity mainly depended on the culture conditions rather than on the media composition. Irrespective of the basal culture medium, mycelium methanolic extracts of G. sordulenta obtained from culture in Erlenmeyer flasks showed equivalents to ascorbic acid (EQ(AA)) RS-EQ(AA) and RP-EQ(AA) contents higher than the corresponding values obtained with jar cultures. Under stationary cultivation, G. sordulenta produced approximately 50% higher content in both RS-EQ(AA) and RP-EQ(AA) than the medicinal mushroom G. frondosa. Phenolics correlated with RS-EQ(AA) and RP-EQ(AA) in G. gargal and with RP-EQ(AA) in G. sordulenta; besides, thin-layer chromatography showed these compounds to be at least in part related to the RS capacity. It is concluded that G. gargal and G. sordulenta mycelia are excellent sources of antioxidant metabolites.

Optimization of the Yield of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes), Cultivated on a Sunflower Seed Hull Substrate Produced in Argentina: Effect of Olive Oil and Copper.

Sunflower seed hulls were used as the main component of a solid substrate for the cultivation of the lingzhi or reishi medicinal mushroom Ganoderma lucidum. This study evaluated the effects of supplementing the substrate with olive oil and copper (II) on the mushroom production (MP) parameters and fruiting body total triterpenoid content. The addition of 1.5% olive oil increased total MP by 21.7% (dry basis) in 3 flushes. Copper (60 ppm) increased the daily productivity of the first flush (MP per day) by both reducing the time needed to harvest the crop and increasing the MP. However, the MP at the second and third flushes was reduced. When both supplements were combined, the MP at the first flush was 43% higher than with control treatment. No significant change in mushroom total triterpenoid content was observed by the addition of supplements to the substrate. An increase of 145-155% in the mushroom copper content was obtained by the addition of 60 ppm copper to the substrate. It is thus recommended to use substrate formulations containing both olive oil and copper (II) and harvest just the first flush.

Pyrolysis of sunflower seed hulls for obtaining bio-oils.

Bio-oils from pyrolysis of as received sunflower seed hulls (SSH), hulls previously washed with acid (SSHA) and hulls submitted to a mushroom enzymatic attack (BSSH) were analyzed. The concentration of lignin, hemicellulose and cellulose varied with the pre-treatment. The liquid corresponding to SSH presented a relatively high concentration of acetic acid and a high instability to storage. The bio-oil from SSHA showed a high concentration of furfural and an appreciable amount of levoglucosenone. Lignin was degraded upon enzymatic activity, for this reason BSSH led to the highest yield of bio-oil, with relative high concentration of acetic acid and stability to storage.

Enhancement of wheat grain antioxidant activity by solid state fermentation with Grifola spp.

Grifola frondosa, Grifola gargal, and Grifola sordulenta are edible and medicinal mushrooms with antioxidant properties. To obtain wheat flour (Wf ) with a higher antioxidant activity than the one exhibited by regular Wf, solid state fermentation (SSF) of wheat grains with mycelia of those Grifola spp. was used to obtain biotransformed wheat grain (BWG) flour. The methanolic extract of control Wf and BWG flour of G. gargal, G. sordulenta, and G. frondosa (GfWG, GgWG, and GsWG, respectively) were studied for their radical scavenging (RS) activity against 2,2-diphenyl-1-picrylhydracyl (DPPH) and their Fe(III) reducing power (RP). The values for RS-EC50 decreased in BWG flour, therefore presenting a higher antioxidant activity: GgWG (0.56 mg/mL), GfWG (0.81 mg/mL), and GsWG (5.80 mg/mL) in comparison to Wf (57.60 mg/mL). The antioxidant content for this RS activity in terms of ascorbic acid content (RS-EQAA) was highest in GfWG, followed by GgWG and GsWG (71.73, 14.46, and 3.02 mg/g, respectively) and lowest in Wf (0.25 mg/g). The RP-EC50 values in GgWG, GfWG, and GsWG were low (0.55, 0.64, and 4.20 mg/mL, respectively) with respect to Wf (55.00 mg/mL). Compared with Wf (0.56 mg/g), the RP capacity in terms of ascorbic acid content (RP-EQAA) was very high in GfWG (193.67 mg/g) followed by GgWG and GsWG (31.42 and 8.74 mg/g, respectively). The high content in gallic acid equivalents was consistent with RS-EQ(AA) and RP-EQ(AA) contents. TLC revealed that antioxidant activity in BWG could be related to the presence of phenolic compounds. Thus, a valuable food alternative can easily be obtained with wheat grains, that is, by markedly increasing their antioxidant value through SSF with Grifola spp.

Protective effects of new medicinal mushroom, Grifola gargal singer (higher Basidiomycetes), on induced DNA damage in somatic cells of Drosophila melanogaster.

Grifola gargal is an edible mushroom with attributed antioxidant properties. Different sources of G. gargal materials, i.e., fruit bodies and mycelia grown in liquid or solid media, were used to study its potential protective capacity when somatic mutation and recombination is induced in Drosophila melanogaster using DMBA (7-12-dimethyl-benz(α)anthracene) as promutagen. Heterozygote larvae (white/white+) were grown in media with different concentrations of DMBA. Grifola gargal fruit bodies (GgFB) or mycelia from liquid culture (GgLC) or from solid culture (GgWG), i.e., biotransformed wheat kernel flour, were added to the culture media in combined treatments with DMBA. Water, DMBA solvent, or wheat flour (WF) plus DMBA solvent were used as negative controls. Larval mortality increased from 9% to 11% in negative controls to 31% to 36% in DMBA treatments. The addition of GgFB, GgLC, or GgWG materials produced a protective effect on 25 µmol/vial DMBA-induced mortality. Mutations observed in SMART, as light spots per 100 eyes (LS/100 eyes), increased with increasing doses of DMBA; this was also true when considering the mutation incidence expressed as percentage of eyes exhibiting light spots (% eyes with LS). Interestingly, mycelia from GgFB, GgLC, or GgWG, in the presence of 25 µmol/vial DMBA, showed lower values in SMART of both the total LS/100 eyes and the percentage of eyes with LS. Thus, Grifola gargal materials were not only nontoxic, but in combination with 25 µmol/vial DMBA lowered the mortality induced by the promutagen and showed antimutagenic effects. Protective effects of G. gargal against DMBA are discussed in terms of the onset of desmutagenic and/or bioantimutagenic mechanisms of detoxification in the host organism, probably due to some bioactive compounds known to occur in higher mushrooms.

Copper and zinc bioaccumulation and bioavailability of Ganoderma lucidum.

Ganoderma lucidum is a widely recognized medicinal mushroom. The bioaccumulation and potential bioavailability of copper (Cu) and zinc (Zn), which are essentials for human health, were analyzed in G. lucidum mycelium and fruit bodies grown in the presence of these metals to test their potential utility as a food dietary supplement. Mycelia grown in culture medium with non-mycotoxic doses of Cu or Zn (25 and 50 mg/kg) were selected for evaluation of the bioavailability of these metals in the gastrointestinal tract by using an in vitro simulated digestion system. One gram of dried mycelium grown in the presence of 50 mg/kg Cu or Zn showed a bioavailability of 19% for Cu and 2% for Zn of the recommended daily intake (RDI). When production of fruit bodies was evaluated, the highest biological efficiency (23%) was reached when the substrate was enriched with 100 mg/kg Cu. Cu and Zn contents obtained either before or after digestion of fruit bodies from all metal-enriched treatments were substantially lower than those from metal-enriched mycelia. The metal bioavailability was also low: 1.5% of the Cu RDI and almost negligible for Zn. The results are discussed in relation to the RDI values exhibited by two commercial supplements. The potential incorporation of these mineral-enriched mycelia/fruit bodies in capsules, infusions, and dietary supplements is evaluated.

Bioaccumulation and bioavailability of copper and zinc on mineral-enriched mycelium of Grifola frondosa.

Grifola frondosa is an edible and medicinal mushroom. The bioaccumulation and potential biovailability of Cu and Zn were studied to obtain mycelium with potential properties as a food dietary supplement. Mycelia grown in the presence of nonmycotoxic concentrations of 100 and 200 ppm of Cu or 25 and 50 ppm of Zn accumulated 200-322 ppm and 267-510 ppm of Cu or Zn, respectively. When these enriched metal mycelia were subjected in vitro to a simulated gastrointestinal digestion, the solubility in these digestive fluids was 642-669 ppm and 102-530 ppm, which represent 32-33% and 0.7-3.5% of the recommended daily intake (RDI) for Cu and Zn, respectively, in 1 g of mycelium. These results are discussed in relation to the RDI values exhibited by two commercial supplements, and arguments are given on the potential use of these mineral-enriched mycelia in capsules (in the case of Cu-enriched mycelia), in food preparations, and also as a component of cosmetic mixtures.

Copper- and zinc-enriched mycelium of Agaricus blazei Murrill: bioaccumulation and bioavailability.

Agaricus blazei is an edible mushroom with medicinal properties. To obtain organic combinations of potential utility as a food dietary supplement, the accumulation and potential bioavailability of copper (Cu) and zinc (Zn) on mycelium grown in the presence of these metals were studied. At 400 ppm, the mycelium accumulated 449 and 163 times the basal content of Cu and Zn, respectively. When mycelia cultivated with nonmycotoxic concentrations of Cu or Zn (100 and 200 ppm) were subjected to sequential chemical extraction and simulated gastrointestinal digestion, close to 90% of the metals accumulated in the available nonresidual fraction, which was similar to or better than the values found in two commercial supplements. The solubility in the simulated digestive fluids was 30-34% and 18-33%, i.e., 60-98% and 9-11% of the recommended daily intake for Cu and Zn, respectively, with only 1 g of mycelium.

Towards genetic transformation of local onion varieties

The aim of this work was to explore the possibility of obtaining transgenic plants of onion varieties cultivated in Argentina, starting from calli induced from mature zygotic embryos, using two strains of Agrobacterium tumefaciens as transfection vectors. Mature embryos from three varieties of 'Valenciana' onion, Torrentina, Cobriza INTA and Grano de oro were in vitro cultivated for callus induction. After three to four months an average of 57.4 percent success for the three varieties was reached. Transformation was carried out with Agl1 or LBA 4404 A. tumefaciens strains, both carrying a binary vector containing the marker gene gus a and the selection gene nptII. Selection was done in callus induction medium containing 10 mgL-1 geneticin during three subcultures. At the end of the selection period, 342 portions of calli were recovered and transferred to regeneration medium. Of the selected calli evaluated by the expresion of the beta-glucuronidase enzyme, 42 percent presented extensive blue areas or were completely blue. At the end of the first subculture in the regeneration medium, 54 calli were considered potentially organogenic because of the green areas observed. At the end of the wole regeneration period, just one normal plant was obtained, that was negative to PCR analysis using specific primers for gus a and nptII. All selected calli came from the Torrentina variety and the highest quantity of them were transformed with the strain LBA 4404.

Hydrogen peroxide in micropropagation of Lilium. A comparison with a traditional methodology.

The micropropagation of Lilium longiflorum requires adequate equipment which may not be afforded by small laboratories or producers. In this work we compared traditional methodology with a protocol that included easily available elements to sterilize materials and culture media, together with addition of hydrogen peroxide (H2O2) into the nutrient media as chemical sterilizer. A series of H2O2 concentrations (0.005, 0.010, 0.015 and 0.020% p/v) was used to control contamination during in vitro establishment and subsequent cultivation; the explant organogenic response was also examined and compared to the traditional micropropagation technique. The level of culture contamination was within acceptable limits in all treatments, though it was higher in the H2O2 treatments (40%) compared to the traditional methodology (20%). There were not significant differences in the number of bulblets per explant, and at the end of the multiplication phase, bulblets from 0.02% H2O2 treatment had greater biomass than from other treatments, indicating a beneficial effect. These bulblets also had a higher relative growth ratio with respect to the traditional method when cultivated for an additional period and showed the highest average bulblet fresh weight. It is expected that this higher bulblet mass would result in better performance during ex vitro cultivation.

Agrobacterium rhizogenes vs auxinic induction for in vitro rhizogenesis of Prosopis chilensis and Nothofagus alpina.

The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.

Agrobacterium rhizogenes vs auxinic induction for in vitro rhizogenesis of Prosopis chilensis and Nothofagus alpina.

The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA. Rooting in P. chilensis reached 100.0

when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0

of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.

Calcium and boron for in vitro rooting of Nothofagus nervosa

Several mineral compounds influence adventitious rooting, and those containing boron and calcium, play an important role. They are associated to the enzyme regulation, cell division, cellular metabolism (induction stage) and roots enlargement (expression stage). The aims of this work were to study the variation of the endogenous calcium and boron concentrations along the rooting, and determine their optimal concentrations in the rooting basal medium of Nothofagus nervosa. Significant variation in endogenous calcium concentrations was detected along the rooting phase, strongly correlated with rhizogenesis phenomenons and linked to total peroxidase variation as cited elsewhere. Calcium and boron concentrations in the basal medium significantly affected the rooting. Treatment containing 60 mg.L-1 Ca and 1 mg.L-1 B produced root formation in fewer days than other combinations. The variation of these nutrient concentrations in the medium is a useful tool to improve microcutting rooting. Roots are obtained earlier and in a shorter period of time, finally resulting in more homogeneous microplants with a better developed rooting system.(AU)

Calcium and boron for in vitro rooting of Nothofagus nervosa

Several mineral compounds influence adventitious rooting, and those containing boron and calcium, play an important role. They are associated to the enzyme regulation, cell division, cellular metabolism (induction stage) and roots enlargement (expression stage). The aims of this work were to study the variation of the endogenous calcium and boron concentrations along the rooting, and determine their optimal concentrations in the rooting basal medium of Nothofagus nervosa. Significant variation in endogenous calcium concentrations was detected along the rooting phase, strongly correlated with rhizogenesis phenomenons and linked to total peroxidase variation as cited elsewhere. Calcium and boron concentrations in the basal medium significantly affected the rooting. Treatment containing 60 mg.L-1 Ca and 1 mg.L-1 B produced root formation in fewer days than other combinations. The variation of these nutrient concentrations in the medium is a useful tool to improve microcutting rooting. Roots are obtained earlier and in a shorter period of time, finally resulting in more homogeneous microplants with a better developed rooting system.