Combat of iron-deprivation through a plant growth promoting fluorescent Pseudomonas strain GRP3A in mung bean (Vigna radiata L. Wilzeck)

Microorganisms and plants sustain themselves under iron-deprived conditions by releasing siderophores. Among others, fluorescent pseudomonads are known to exert extensive biocontrol action against soil and root borne phytopathogens through release of antimicrobials and siderophores. In this study, production and regulation of siderophores by fluorescent Pseudomonas strain GRP3A was studied. Among various media tested, standard succinate medium (SSM) promoted maximum siderophore production of 56.59 mg l(-1). There were low levels of siderophore in complex media like King's B medium, trypticase soya medium and nutrient medium (41.27, 29.86 and 27.63 mg l(-1)), respectively. In defferrated SSM, siderophore level was quantified to be 68.74 mg l(-1). Supplementation with iron (FeCl3) resulted in decreased siderophore levels depending on concentration. Siderophore production was promoted by Zn2+ (78.94 mg l(-1)), Cu2+ (68.80 mg l(-1)) whereas Co2+ (57.33 mg l(-1)) and Fe3+ reduced siderophore production (37.44 mg l(-1) as compared to control (55.97 mg l(-1)). Strain GRP3A showed plant growth promotion under iron limited conditions.     

Agrobacterium-mediated transformation of seedling-derived maize callus

Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes including commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l(-1) picloram and 3 mg l(-1) 6-benzylaminopurine, which induced the development of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l(-1) picloram and 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid. For lines L4 and L9 the frequency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induction from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived callus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluorescence protein gene and genes for either neomycin phosphotransferase II or glyphosate selection were used in transformation experiments. Transformation frequency varied from 2 to 11% and about 60% of the T(0) plants had 1-2 copies of transgenes.     

Effects of rare earth elements on the growth of Cistanche deserticola cells and the production of phenylethanoid glycosides

The rare earth elements Nd, La, Ce at proper concentrations had positive effects on the cell growth of Cistanche deserticola and production of phenylethanoid glycosides (PeG). A mixture of rare earth elements (MRE, La(2)O(3):CeO(2):Pr(6)O(11):Sm(2)O(3)=255:175:3:1, mol/mol) showed the most remarkable effects. After 30 day's culture, 0.02 mmoll(-1) MRE gave the highest content (20.8%) and production (1.6 gl(-1)) of PeG, which were 104 and 167% higher than those obtained in control (without rare earth elements).     

Enhanced Ginsenoside Productivity by Combination of Ethephon and Methyl Jasmoante in Ginseng (Panax ginseng C.A. Meyer) Adventitious Root Cultures

Ethephon at 50 μM enhanced both root growth and ginsenoside accumulation in ginseng (Panax ginseng C.A. Meyer) adventitious root cultures, but at 100 μM it inhibited only ginsenoside accumulation. Ginsenoside productivity with 50 μM ethephon was the highest at 1.7 mg l⁻¹ d⁻¹ after 8 days of elicitation. However, elicitation with 50 μM ethephon and 100 μM methyl jasmonate (MJ) improved productivity (6.3 mg l⁻¹ d⁻¹) whereas elicitation with 100 μM MJ alone gave only 2.9 mg l⁻¹ d⁻¹.     

Stimulation of taxol production and excretion in Taxus spp cell cultures by rare earth chemical lanthanum

The trivalent ion of a rare earth element, lanthanum, was tested for elicitor-like effects on taxol production in suspension cultures of four different Taxus spp cells. In T. yunnanensis cell cultures, the lanthanum ion at concentrations from 1.15 to 23.0 microM stimulated taxol production. The lanthanum ion also promoted taxol excretion by the T. yunnanensis cells considerably. The maximum stimulation of taxol production was achieved by the addition of 5.8 microM La3+ to the culture during mid-log growth phase, increasing the volumetric taxol yield by nearly threefold, from 2.61+/-0.37 to 9.89+/-1.92 mg l(-1) over a 28 day culture period. At higher concentrations, i.e. 23.1 and 46.2 microM, however, the lanthanum ion caused significant growth inhibition. For the other three Taxus cell lines, namely an embryo and a leave cell of T. chinensis and a stem cell of T. chinensis marv, the addition of lanthanum ion to the culture only had a significant effect on taxol production by the T. chinensis marv stem cells, increasing the volumetric yield by about threefold to 4.69+/-0.76 mg l(-1). These results suggest that lanthanum has elicitor-like effects on secondary metabolite synthesis of plant cell cultures.     

Establishment of callus and cell suspension cultures of Corydalis saxicola Bunting, a rare medicinal plant

An efficient procedure has been developed for callus induction and cell suspension cultures of C. saxicola for the first time. Explant selection was carried out among leaf, stem and root to select a suitable type of explants capable of higher callus formation. Leaf explants thus selected showed maximum response to callus induction (67.1%). Modified B5 medium supplemented with 0.5 mg l(-1) 2,4-D plus 2 mg l(-1) BA was the most favorable medium for callus formation with the highest induction rate (94.8%) and greatest fresh weight of callus (1.7 g per explant). Cell suspension cultures were established by transferring 2-8 g fresh callus to 80 ml liquid B5 medium. An inoculum size of 8 g produced the greatest biomass accumulation, dehydrocavidine and berberine productions, which was 13.1 g l(-1), 8.0 mg l(-1) and 4.1 mg l(-1), respectively. In response to various sucrose concentrations from 10 g l(-1) to 80 g l(-1), cultures with 60 g sucrose l(-1) not only produced the highest dry biomass (18.5 g l(-1)) but also the highest formation of dehydrocavidine (11.6 mg l(-1)) and berberine (7.6 mg l(-1)). These prepared cell suspension cultures provided a useful material for further regulation of alkaloid biosynthesis and for enhanced production of valuable alkaloids on a large scale.     

Optimization of glycosidases production by Pseudoalteromonas issachenkonii KMM 3549(T)

AIMS: The present work aimed to design an optimized medium to yield a higher production of glycosides by Pseudoalteromonas issachenkonii KMM 3549(T). METHODS AND RESULTS: Higher levels of fucoidan hydrolase, alginase, laminaranase and b-N-acetylglucosaminidase production were obtained with peptone concentrations ranging from 2.5 g l(-1) to 10 g l(-1), while the presence of both yeast extract and glucose did not affect enzyme production. The activity of fucoidan hydrolase and laminaranase increased up to 4.83 microM h(-1) mg(-1) and 19.23 microM h(-1) mg(-1) protein, respectively, in growth media containing xylose (1.0 g l(-1)), laminarin (0.5 g l(-1)) or alginate (0.5 g l(-1)), and production of b-N-acetylglucosaminidase substantially increased in the presence of fucoidan (0.5 g l(-1)) or galactose (1 g l(-1)). All polysaccharides tested in concentrations of 0.5 g l(-1) fucoidan and 0.2 g l(-1) fucose induced production of alginase (up to 5.06 microM h(-1) mg-1 protein). CONCLUSIONS: The production of glycosidases is not only stimulated by the presence of algal polysaccharides, but may also be stimulated by monosaccharides (e.g. xylose). SIGNIFICANCE AND IMPACT OF THE STUDY: The production of glycosidases by Pseudoalteromonas issachenkonii KMM 3549(T) was significantly improved by using a simple nutrient medium containing peptone (2.5 g l(-1)) and xylose (5.0 g l(-1)) in 100% natural seawater.     

Change in colony morphology and kinetics of tylosin production after UV and gamma irradiation mutagenesis of Streptomyces fradiae NRRL-2702

Tylosin is a macrolide antibiotic used as veterinary drug and growth promoter. Attempts were made for hyper production of tylosin by a strain of Streptomyces fradiae NRRL-2702 through irradiation mutagenesis. Ultraviolet (UV) irradiation of wild-type strain caused development of six morphologically altered colony types on agar plates. After screening using Bacillus subtilis bioassay only morphological mutants indicated the production of tylosin. An increase of 2.7±0.22-fold in tylosin production (1500 mg/l) in case of mutant UV-2 in complex medium was achieved as compared to wild-type strain (550 mg/l). Gamma irradiation of mutant UV-2 using ⁶⁰Co gave one morphologically altered colony type γ-1, which gave 2500 mg/l tylosin yield in complex medium. Chemically defined media promoted tylosin production upto 3800 mg/l. Maximum value of q_p (3.34 mg/gh) was observed by mutant γ-1 as compared to wild strain (0.81 mg/gh). Moreover, UV irradiation associated changes were unstable with loss of tylosin activity whereas mutant γ-1 displayed high stability on subsequent culturing.     

Efficient regeneration of fertile barley plants from callus cultures of several Nordic cultivars

The regeneration potential of three major Estonian barley cultivars was tested and compared to that of the Finnish cultivar Kymppi. Two different regeneration systems were used. The first was characterized by the high maltose concentration (60 g l(-1)) and by the use of 2,4D together with two different combinations of amino acids in the callus induction medium followed by the regeneration medium containing BAP (2 mg l(-1)) and 2,4D (0.2 mg l(-1)). The second exploited callus induction medium that contained Dicamba, lower concentrations of maltose (30 g l(-1)) and higher concentrations of myo-inositol and thiamine and different set of amino acids and regeneration medium that contained higher concentrations of Cu2+ and inorganic nitrogen accompanied by lower concentrations of NH4+ and BAP, when compared to the first regeneration system. The second regeneration system used produced significantly higher rates of callus induction, callus growth and regeneration of plantlets. However, it yielded also many albino plants (up to 51%), whereas the first regeneration system used did not produce practically any albino plants. No major genotype-dependent differences were observed in comparison between two regeneration systems - in both systems higher regeneration potential of Anni, Elo and Kymppi contradicted to the low regeneration potential of Teele. It is concluded that the continuous somatic embryogenesis on the regeneration medium allows the regeneration of many plants from the same callus over long periods of time and makes available highly efficient regeneration protocols for Estonian and Finnish barley cultivars.     

An apoplastic mechanism for short-term effects of rare earth elements at lower concentrations

The short-term effects of rare earth elements on pollen germination and tube growth were tested. Concentrations of 2.5 approximately 20 micro m lanthanum(La3+) or cerium (Ce3+)increased pollen germination and pollen tube growth, whereas concentrations higher than 40 micro m La3+ and Ce3+ inhibited this process. The most effective concentration of La3+ needed for promotion shifted from 10 to 40 micro m, depending on the Ca2+ concentration in the medium. Calmodulin (CaM) antagonist W7-agarose and anti-CaM antibody depressed La3+-promoted pollen germination and tube growth in a dose-dependent manner. La3+-CaM complexes (La3+-CaM) increased pollen germination and tube growth more than CaM or La3+ alone. Pertussis toxin (PTX) inhibited La3+-promoted pollen germination and tube growth. Cholera toxin (CTX) partially recovered the inhibition of the above La3+-promoted process by the anti-CaM antibody. Concentrations of 10-7 approximately 10-9 m La3+-CaM increased GTPase activity inside plasma membrane vesicles of the pollen tube, but apo-CaM or La3+ alone had no positive effects. The results suggest that apoplastic CaM may be involved in the promotion effects of lower concentrations of La3+ on pollen germination and tube growth, and the heterotrimeric G-protein on the plasma membrane may transduce La3+-activated CaM signalling. The present studies provide an apoplastic mechanism for short-term effects of rare earth elements at lower concentrations in the pollen system.     

产藏红花素1(crocin)愈伤组织的诱导及其细胞系的筛选

对藏红花(cfocus sativus L)愈伤组织的诱导条件进行了优化.结果表明:Ms是藏红花芽愈伤组织的最佳诱导培养基,而B5是叶子和花愈伤组织的最佳培养基.藏红花芽、叶和花愈伤组织的最佳诱导温度分别是18℃、25℃和21℃.光照是叶子愈伤组织诱导的有利因素,但不利于芽和花愈伤组织的诱导.1.5～2.0 mg·L-1NAA和0.25 mg·L-6-BA是愈伤组织诱导的最佳激素组合.通过目视法和HPLC方法,从229株细胞系中筛选出细胞系Corml,其藏红花素1的含量是1 677 mg·g-,生长较快,且不易褐化.为采用植物细胞工程法解决藏红花素1资源短缺问题打下了基础.     

Effects of stress factors, bioregulators, and synthetic precursors on indole alkaloid production in compact callus clusters cultures of Catharanthus roseus

Compact callus cluster (CCC) cultures established from Catharanthus roseus consist of cohesive callus aggregates displaying certain levels of cellular or tissue differentiation. CCC cultures synthesize about two-fold more indole alkaloids than normal dispersed-cell cultures. Our studies here show that additions of KCl, mannitol, and a variety of synthetic precursors and bioregulators to the CCC cultures markedly improved indole alkaloid production and release of these alkaloids into the medium. Treatment with 250 mM mannitol and 4 g/l KCl yielded 42.3 mg l(-1) and 33.6 mg l(-1)of ajmalicine, respectively; these amounts were about four-fold higher than the control. Succinic acid, tryptamine, and tryptophan feedings also significantly increased ajmalicine (41.5 mg l(-1), 36.9 mg l(-1), and 31.8 mg l(-1), respectively) and catharanthine (21.1 mg l(-1), 17.2 mg l(-1), and 18 mg l(-1), respectively) production by the CCC cultures, while geraniol feeding inhibited biomass and alkaloid accumulation. We also found that tetramethyl ammonium bromide could significantly improve ajmalicine production (49.3 mg l(-1)) and catharanthine production (18.3 mg l(-1)) in C. roseus CCC cultures. The mechanisms responsible for these treatment effects are discussed herein.     

产藏红花素1(crocin)愈伤组织的诱导及其细胞系的筛选

对藏红花(Crocus sativus L.) 愈伤组织的诱导条件进行了优化。结果表明：MS 是藏红花芽愈伤组织的最佳诱导培养基,而B5是叶子和花愈伤组织的最佳培养基。藏红花芽、叶和花愈伤组织的最佳诱导温度分别是18℃、25℃和21℃。光照是叶子愈伤组织诱导的有利因素,但不利于芽和花愈伤组织的诱导。1.5～2.0 mg.L-1 NAA和0.25 mg.L-1 6-BA是愈伤组织诱导的最佳激素组合。通过目视法和HPLC方法, 从229株细胞系中筛选出细胞系Corm1,其藏红花素1 的含量是1 677 mg.g-1,生长较快,且不易褐化。为采用植物细胞工程法解决藏红花素1资源短缺问题打下了基础。     

Effect of calcium, phosphate and nitrogen on cell growth and biosynthesis of cell wall polysaccharides by Silene vulgaris cell culture

Medium nutrients such as calcium, phosphorus, nitrogen and nitrate to ammonium ratio have significant influence on the growth, biosynthetic and biochemical characteristics of polysaccharides produced by Silene vulgaris (M.) G. cell culture. Cell growth and production of polysaccharides was limited by an absence of any of these components in the medium. Optimal growth of the callus and production of arabinogalactan were achieved at 1.5-4.5 microM calcium while the optimal production of pectin named silenan was observed at 3.0-4.5 microM. The phosphate contents in the medium in the range of 0.63-3.75 microM were favorable for callus growth. Production of silenan was maximal at 1.25-3.75 microM phosphate. Optimal growth of the callus was achieved at 30-90 microM nitrogen. Maximal production of silenan was observed at 60 microM nitrogen while the optimal production of arabinogalactan was at 90 microM nitrogen (at ratio of NH(4)(+):NO(3)(-) as 1:2). A presence both of nitrate and ammonium is needed for the silenan biosynthesis (the NH(4)(+):NO(3)(-) ratio as 1:1 and 1:2). Yields and volumetric production of arabinogalactan were maximal at deletion of ammonium from the nutrient medium (ratio 0:1). Absence of calcium or nitrogen in the medium leads to a decrease of the galacturonic acid residues in silenan. The galactose residues contents in arabinogalactan were decreased in the absence of nitrogen and calcium in the medium.     

The influence of nitrogen and biotin interactions on the performance of Saccharomyces in alcoholic fermentations

AIM: To study the impact of assimilable nitrogen, biotin and their interaction on growth, fermentation rate and volatile formation by Saccharomyces. METHODS AND RESULTS: Fermentations of synthetic grape juice media were conducted in a factorial design with yeast assimilable nitrogen (YAN) (60 or 250 mg l(-1)) and biotin (0, 1 or 10 microg l(-1)) as variables. All media contained 240 g l(-1) glucose + fructose (1 : 1) and were fermented using biotin-depleted Saccharomyces cerevisiae strains EC1118 or UCD 522. Both strains exhibited weak growth and sluggish fermentation rates without biotin. Increased nitrogen concentration resulted in higher maximum fermentation rates, while adjusting biotin from 1 to 10 microg l(-1) had no effect. Nitrogen x biotin interactions influenced fermentation time, production of higher alcohols and hydrogen sulfide (H(2)S). Maximum H(2)S production occurred in the medium containing 60 mg l(-1) YAN and 1 microg l(-1) biotin. CONCLUSIONS: Nitrogen x biotin interactions affect fermentation time and volatile production by Saccharomyces depending on strain. Biotin concentrations sufficient to complete fermentation may affect the organoleptic impact of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the necessity to consider nutrient interactions when diagnosing problem fermentations.     

稀土元素对红酵母的生长及类胡萝卜素合成的影响

探索了稀土元素镧Ｌａ^３＋、铈Ｃｅ^３＋和钕Ｎｄ^３＋对红酵母ｐｈａｆｆｉａ　ｒｈｏｄｏｚｙｍａ的生长,类胡萝卜素合成及菌体氨基酸组成的影响。三种稀土元素在所测试的浓度范围内对红酵母细胞生物量仅有轻度的抑制作用,但对类胡萝卜素的合成有促进作用。当培养基中分别加入２０ｍｇ／Ｌ的ＬａＣｌ_３、５ｍｇ／Ｌ的ＣｅＣｌ_３,和５０ｍｇ／ＬＮｄＣｌ_３时,刺激类胡萝卜素合成作用最强,产     

应用稀土调控藏红花胚性愈伤组织的生长与分化

为提高藏红花（Crocus sativus）胚性愈伤组织的繁殖系数与出芽率,以建立藏红花离体快繁体系,解决其资源短缺问题,采用两步法,用稀土调控其胚性愈伤组织的生长与分化。结果表明,在添加了0.25mg·L-1NAA、3mg·L-16-BA和400mg·L-1CH的B5固体培养基中,0.04mmol·L-1La3＋促进胚性愈伤组织生长的效果最佳,繁殖系数为12,是不添加稀土处理组的1.48倍;在添加了0.25mg·L-1NAA、3mg·L-16-BA和400mg·L-1CH的1/2B5固体培养基中,0.06mmol·L-1Ce3＋促进胚性愈伤组织分化出芽的效果最佳,出芽率高达84.5%,是不添加稀土处理组的1.81倍,且高于国外报道的出芽率（40%）。初步解决了藏红花胚性愈伤组织生长慢和出芽率低等问题,为建立高效稳定的藏红花离体快繁体系奠定了基础。     

Plant regeneration from leaf-derived callus cultures of the tropical pasture legume Stylosanthes scabra Vog.

Callus cultures of 5 genotypes of S. scabra Vog. were optimally established from leaf tissue on Murashige and Skoog (MS) basal medium supplemented with 0.5–2.0 mg l^-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 1.0–2.0 mg l^-1 6-benzylaminopurine (BAP). On media containing 2, 4-D only, calli were soft, and rhizogenesis occurred on calli of 4 genotypes. Calli formed on media containing BAP only, but not with kinetin only. In the presence of 2, 4-D, BAP inhibited rhizogenesis and promoted better callus growth than kinetin. High frequency shoot induction was achieved for 3 genotypes on MS +2.0 mg l^-1 BAP. Roots formed on shoots when sub-cultured on half-strenght MS without growth regulators. The form of cytokinin used in the callus induction media appeared to affect subsequent shoot organogenesis. Genotypic differences were observed for shoot organogenesis. There was some morphological variation evident among regenerants.     

Improved cell growth and Taxol production of suspension-cultured Taxus chinensis var. mairei in alternating and direct current magnetic fields

The growth of suspension cultures of Taxus chinensis var. mairei and Taxol production were promoted both by a sinusoidal alternating current magnetic field (50 Hz, 3.5 mT) and by a direct current magnetic field (3.5 mT). Taxol production increased rapidly from the 4th d with the direct current magnetic field but most slowly with the alternating current magnetic field. The maximal yield of Taxol was 490 microg l(-1) with the direct current magnetic field and 425 microg l(-1) with the alternating current magnetic field after 8 d of culture, which were, respectively, 1.4-fold and 1.2-fold of that without exposure to a magnetic field.     

Production of crocin using Crocus sativus callus by two-stage culture system

Saffron callus was grown in a two-stage culture on B5 medium supplemented with casein hydrolysate (300 mg l^–1) at 22 °C in dark with naphthalene acetic acid (2 mg l^–1) and 6-benzyladenine (1 mg l^–1) to give maximum biomass (16 g dry wt l^–1), and with indole 3-acetic acid (2 mg l^–1) and 6-benzyladenine (0.5 mg l^–1) for crocin formation. The maximum crocin production (0.43 g l^–1) was achieved by this two-stage culture method, which was three times that by a one-stage method.