Simultaneous oxygen production and nitrogenase activity of an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis in a co-culture with wheat

Summary A mutant strain of Anabaena variabilis, strain SA-1 that supported growth of wheat plants in a hydroponic co-culture in nitrogen (N) free medium also produced enough oxygen (O₂) to support root respiration. The steady-state concentration of net O₂ in the co-culture was dependent on incident light intensity. At an incident photosynthetic photoflux (PPF) of 1000 mol photons·m^–2·s^–1, net O₂ evolution by the co-culture in the root zone reached a maximum value of about 220 mol O₂ evolved·h^–1·mg chlorophyll (Chl)^–1. The O₂ concentration in the rhizosphere of the co-culture stayed above the ambient air level. O₂ uptake in the dark by strain SA-1-supplemented wheat roots washed free of cyanobacterium was higher than the root respiration of nitrate-grown plants. Nitrate-grown plants required aeration for maximum growth while the wheat-cyanobacterial co-culture can be cultured without aeration. These results show that O₂ produced by strain SA-1 can be used to supply the O₂ needs for root respiration of wheat. Respiration reduced net O₂ evolution by the mutant SA-1, decreasing the partial pressure of O₂ at the sites of cyanobacterial attachment to the roots. This led to an increase in the specific activity of nitrogenase of the co-culture at the high light intensities used to support wheat growth. This activity of about 30 mol ethylene produced·mg Chl^–1·h^–1 was three-fold higher than the activities obtained with the free-living strain SA-1 assayed at the same light intensity. In the co-culture, ammonia produced by the mutant strain SA-1 was not detectable. The NH _inf4 ^sup+ produced by strain SA-1 was used by the wheat plants and, under these conditions, the total N content of the plants reached as high as 85% of the total N content of nitrate-grown plants. In the co-culture system the metabolism of wheat and the cyanobacterium complemented each other, leading to higher plant growth in N-free medium. Offprint requests to: M. Gunasekaran     

Requirement for direct association of ammonia-excreting Anabaena variabilis mutant (SA-1) with roots for maximal growth and yield of wheat

Direct association between wheat roots and an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis, strain SA-1, was required for maximal enhancement of growth of wheat plants in nitrogen (N)-free, hydroponic medium. Over 85% of the cyanobacterial mutant SA-1 inoculated to the roots were adsorbed under non-saturating conditions. The adsorption process of SA-1 to wheat roots was biphasic: an initial rapid adsorption was followed by a slow phase with about 10% of the initial adsorption rate. The maximal adsorption rate of filaments observed was 1.6 mg dry wt. SA-1 adsorbed·plant^–1·h^–1. Bypassing CO₂ fixation and sugar formation, the ¹⁴C label from [¹⁴C]sucrose was directly applied to leaf blades to study sugar translocation. The ¹⁴C label from this treatment appeared in the wheat culture medium within an hour. Nitrate-grown plants excreted about 30% of the ¹⁴C label into the medium, compared to only 10% excreted by wheat/Anabaena co-cultures. SA-1 assimilated 27% of all ¹⁴C translocated from [U-¹⁴C]sucrose applied to wheat leaves, and ¹⁴C label from this treatment was recovered from strain SA-1 after 30 min. Roots and cyanobacteria accounted for 51% of all radioactive label recovered in the plants co-cultured with SA-1 vs 20% for nitrate-grown plants. We studied the activity of -fructosidase (invertase) in wheat of variety Yecora rojo. Roots from nitrate-grown wheat plants produced high levels of invertase activity, which converted over 85% of 3 mm sucrose into glucose and fructose in 24 h. The rate of sucrose disappearance in the medium of co-cultures using A. variabilis SA-1, was 70% of that of nitrate-grown plants, but the levels of glucose and fructose in these cultures were always very low during sucrose conversion, suggesting hexose assimilation. To study the role of diffusible metabolites, a dialysis membrane was employed to separate the ammonia-excreting SA-1 from the wheat roots. Containing SA-1 in a dialysis bag away from direct root contact, severely limited leaf growth to less than one-third of the growth rate of nitrate control cultures. Ammonia produced by mutant SA-1 in dialysis bag cultures was excreted into the medium at 0.4 mm vs 1.2 mm in free-living cultures, but ammonia was not detectable in co-cultures with or without the dialysis bag containing the mutant. The nitrogenase activity derepressed in the mutant and responsible for ammonia excretion was always higher in the association co-cultures than in either free cells or in dialysis-bag cultures. The nitrogenase activity of strain SA-1 was highest (200 mol ethylene formed·mg^–1 Chl·h^–1) when the cyanobacterium was associated with the root tips. Dialysis membrane separation of plant and cyanobacterium severely inhibited growth of wheat during a complete growth cycle of 2 months. Total biomass and grain yield were very similar for control cultures without inorganic N or SA-1, and for diffusion cultures containing SA-1, kept in a dialysis bag around the roots. Total biomass of the association co-culture attained 75% of the biomass of the nitrate-grown control. It is proposed that wheat roots supplied fructose derived from sucrose for growth and nitrogen fixation of SA-1 in the light, and that ammonia excreted by SA-1 was utilized by the wheat plant for its own growth. Correspondence to: H. Spiller     

Ammonia-excreting mutant strain of the cyanobacterium Anabaena variabilis supports growth of wheat

Summary Growth of wheat in a nitrogen-free hydroponic co-culture with a mutant strain of the cyanobacterium Anabaena variabilitis (strain SA-1) was enhanced over plants grown with the parent strain SA-0. This increase was achieved in the dry weight, grain yield, and total nitrogen content of the plants. Nitrogenase activity of the mutant strain SA-1 was increased in a co-culture of the cyanobacterial mutant with wheat plants compared to the activity of the wild-type strain in association with wheat. Offprint requests to: M. Gunasekaran     

Effect of calcium and sodium salts on ethanol production in high sugar fermentation by free cells ofZymomonas mobilis

Summary Addition of calcium carbonate enhanced ethanol production byZymomonas mobilis ZM4 and a mutant (ZMI₂), especially at higher concentrations (200–400 g/L) of glucose and sucrose, as well as at higher temperature (42°C) by the mutant. Calcium and sodium carbonates neutralized the acid produced in the medium and enhanced the ethanol production. The Na salts were less effective in the parent strain and were not favourable for the mutant. Ca²⁺ ions played a direct role in augmenting ethanol production as evidenced by the effect of calcium chloride at controlled pH (5.5).     

Plant regeneration from protoplast-derived callus of rice (Oryza sativa L.)

Protoplasts isolated from cultured rice cells of an A-58 cytoplasmic male sterile line (A-58 MS)(Oryza sativa L.) were used to investigate the regeneration of rice plants. A cultured cell line (T₃) of A-58 MS with a high growth rate and dense cytoplasm was selected. About 10% of the protoplasts prepared from this established cell line plated in RY-2 (a new medium) formed colonies. The calli formed shoots and roots in the regeneration medium and developed into whole plants.Protoplasts also were prepared from suspension cultures of 25 other varieties of rice using the same methods. The protoplasts isolated from two of the 25 varieties, Fujiminori and Toyotama, had high rates of cell division in RY-2 medium. Only protoplastderived calli from Fujiminori, produced whole plants in the regeneration medium.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MES 2-(N-Morpholino)ethanesulfonic acid, monohydrate     

Growth,nitrogen fixation,and mass culture of isolatedAnabaena azollae

Summary An independent strain ofAnabaena azollae was evaluated for its potential as a biofertilizer in wetland rice fields. Sustained rapid growth (doubling time=10.5 h) and nitrogenase activity (32 nmol C₂H₄ h^–1 g^–1 chl) was recorded. Mass cultivation (up to 300 litres) for the first time with this species was also achieved.     

Lycopersicon esculentum: Trifoliate plants recovered from anther cultures of heterozygous tftf plants

The effects of the genotype and growth medium composition on callus induction and shoot regeneration from tomato (Lycopersicon esculentum Mill) anthers were studied. Five male sterile varieties, homozygous for the recessive gene ms 10³⁵, their isogenic fertile counterparts, and nineteen sterile mutants from an F₂ population segregating for ms 10³⁵, were tested. Callus induction and shoot formation were found to be affected by the genotype. The presence of the mutant gene ms 10³⁵ greatly increased callus induction. A significant interaction concerning callus induction was found between the ms 10³⁵ gene and the general genetic background. In most of the plants shoot regeneration from the anthers was associated with various degrees of callus production. However, there was no correlation between callus production and the ability to regenerate plants from that callus. Anthers isolated from plants which were heterozygous for the recessive leaf marker trifoliate, regenerated diploid plants with trifoliate leaves. The plants retained the trifoliate phenotype for over six months in culture under non-aseptic condition. Since the trifoliate phenotype appears only in the homozygous recessive state, the evidence that these trifoliate plants are doubled haploids of sporogenic origin is discussed.     

Use of wild type and nickel resistantNeurospora crassa for removal of Ni2+ from aqueous medium

Summary A novel nickel resistant, hyperaccumulatingN.crassa nir-2 mutant, isolated by us, sequestered 90% of Ni²⁺ from medium with 120 mg/l Ni²⁺. The parent wild strain showed comparable efficiency only at much lower concentrations (<10mg/l). The initial rapid rate and efficiency of Ni²⁺ removal could be maintained beyond 2 h by fresh addition of mycelial biomass. The results have been discussed from the stand point of the utility of metal resistant fungi in the control of environmental pollution.     

Establishment of new axenic hairy root lines by inoculation with Agrobacterium rhizogenes

Cultured hairy root lines resulting from infection by Agrobacterium rhizogenes are known for approximately thirty plant species. We extend this range by establishing forty original dicotyledonous hairy root lines with A. rhizogenes strain A₄. Hairy roots have been cultured for at least 2–6 years on Murashige & Skoog medium. Some hairy root cultures such as Anagallis arvensis and Antirrhinum majus spontaneously regenerated whole plants.     

Production of glucoamylase by 2-deoxy-D-glucose resistant mutant ofAspergillus terreus 4

Summary A catabolite derepressed mutant of a glucoamylolytic strain ofAspergillus terreus 4 produced 1.8 times more glucoamylase than its parent; the crude enzyme preparation appreciably degraded raw starchy materials like wheat, rice, potato and barley.     

Plasmid stability during continuous culture in aSaccharomyces cerevisiae double mutant transformed by a plasmid carrying a eukaryotic gene

Summary An investigation of plasmid stability in aSaccharomyces cerevisiae double mutant has been performed. The host was a double recombinantura3 furl mutant containing a plasmid bearing the yeast URA3⁺ allele and an expression cassette for human ₁-antitrypsin. The mutant was grown in continuous culture employing a semi-defined medium containing added uracil to provide non selective growth conditions. After 150 generations of continuous growth, no cured cells had been detected: the specific expression level of ₁-antitrypsin remained constant throughout the experiment.     

Partial characterization of anEscherichia coll strain harboring a xylanase encoding plasmid

Summary The plasmid pRH271, harboring a xylanase gene cioned fromBacilius subtilis, has been transferred into a mutant ofE. coli SK2284 which allowed the release of part of the xylanase in the culture supernatant. Kinetic parameters of this recombinantE. coll strain were determined in microscale batch culture with and without the selective pressure of antibiotics. No significant difference in µ_max was observed for the nontransformedE. coli strain when compared to the recombinant strain. However, K₅ values for glucose were two times higher in the case of the recombinant strain. Preliminary study of xylanase production in a large batch farmenter was also described.     

Effect of cinnamoyl putrescines on in vitro cell multiplication and differentiation of tobacco explants

Hydroxycinnamoyl putrescines promote the cell multiplication of leaf discs of a tobacco mutant, RMB₇, cultivatedin vitro on the Murashige and Skoog medium. This mutant never accumulates these molecules during its development and does not enter in floweirng. Maximal effect is obtained at 2.5·10^–4M. The same molecules inhibit bud formation ofNicotiana tabacum var. Xanthi nc, at 5·10^–5 M but promote callus formation. From 10^–4 M to 5·10^–3 M they strongly inhibit cell multiplication and bud formation without toxic effect. Their possible role in plant metabolism is discussed.Abbreviations IAA indole 3-acetic-acid - BA benzyladenine     

Characterization of a superior thermotolerant mutant ofZymomonas mobilis ZM4 for ethanol production in glucose medium

Summary Nitrosoguanidine-induced, stable theromotolerant mutant (ZMI₂) ofZymomonas mobilis ZM4 was found to possess almost normal cell morphology, and a better ethanol tolerance at 42°C than the parent strain (ZM4). Its kinetic parameters, in converting different concentrations of glucose to ethanol, were comparable to ZM4 at 30°C, and significantly superior at 42°C. In a 200 g/L glucose medium in a pH-stat (5.0) at 42°C, the mutant yielded more ethanol (71.0 g/L) (improved to 73.7 g/L at pH 5.5) and alcohol dehydrogenase (ADH) than the parent strain. The ADH levels in both the strains were repressed, depending upon the increased level of sugar and degree of temperature.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

Selective production of phenylalanine from phenylpyruvate using growing cells ofCorynebacterium glutamicum

Summary Phenylalanine was produced from phenylpyruvate by growing cells of a mutant strain ofCorynebacterium glutamicum over-producing valine. A defined medium was used to minimize the accumulation of valine. The maximum phenylalanine concentration achieved was 44.5 mM (7.5 g/l) with a phenylpyruvate molar conversion of 75%.     

A new <Emphasis Type="Italic">Rhizobium</Emphasis> species isolated from the water of a crater lake,description of <Emphasis Type="Italic">Rhizobium aquaticum</Emphasis> sp. nov.

A novel isolate, strain SA-276^T, was isolated from the water of Lake St. Ana, a crater lake which is located in Romania. Phylogenetic analysis based on the 16S rRNA gene revealed that the new strain is a member of the family Rhizobiaceae, showing a high pairwise similarity value (97.65%) to Rhizobium tubonense CCBAU 85046^T (=?DSM 25379^T), Rhizobium leguminosarum USDA 2370^T (=?LMG 14904^T), Rhizobium anhuiense CCBAU 23252^T and Rhizobium laguerreae FB206^T. Cells of strain SA-276^T were rod-shaped, motile, oxidase negative and weakly catalase positive. The predominant fatty acids were C_18:1ω7c and cyclo C_19:0ω8c, the major respiratory quinones were Q-10 and Q-9, and the main polar lipids were phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine. The G?+?C content of the genomic DNA of strain SA-276^T was 60.8 mol%. The novel isolate can be distinguished from the closest related type strain R. tubonense DSM 25379^T based on its broader substrate specificity and positive trypsin enzyme activity. On the basis of the phenotypic, chemotaxonomic and molecular data, strain SA-276^T is considered to represent a new species, for which the name Rhizobium aquaticum sp. nov. is proposed. The type strain is SA-276^T (=?DSM 29780^T?=?JCM 31760^T).     

Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens

A chimaeric neomycin phosphotransferase II (NPT II) gene was introduced in Brassica oleracea using an oncogenic strain of Agrobacterium tumefaciens harbouring Ti plasmid which contains Nos/NPTII in its T-DNA. The transformation of B. oleracea with the oncogenic Ti plasmid, resulted in regeneration of shoots and roots without any exogenous requirement of phytohormones. The presence of NPT II gene was determined by hybridization of Tn5 encoded NPT II gene with DNA of kanamycin resistant regenerated plants. The expression of NPT II was demonstrated by kanamycin phosphorylation assay. Several regenerated plants were obtained, a few of them were found to be morphological variants and a chlorophyll deficient mutant plant was also obtained.     

Semi-pilot scale studies on citric acid fermentation by a gamma-ray induced mutant of Aspergillus niger

Summary Semi-pilot scale production of citric acid was investigated with a gamma-ray induced mutant (HB3) of Aspergillus niger using 500, 1000 and 1500 ml medium in 51 fermentation jars. Yield of citric acid was found to be seven-fold higher compared to the parent in 1000 ml medium and the corresponding increase was two-fold in the 500 ml medium. With 1500 ml/fermentation jar the yield was low with both the parent and the mutant strain though the mutant gave higher yield compared to the parent.