Species specific shoot regeneration response of cotyledonary explants of Brassicas

A study of shoot regeneration from cotyledons of three basic diploid species of Brassica, B. campestris (AA), B. nigra (BB), B. oleracea (CC) and their amphidiploids B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC) showed species-specific responses for in vitro shoot regeneration. Analysis of the species mean shoot regeneration response over a range of growth regulator combinations revealed that i) B. campestris is the lowest regenerating species, ii) B. nigra and B. oleracea regenerate with high frequencies, iii) In amphidiploids, the presence of B. campestris component brings down shoot regeneration frequency below the value of B. oleracea in B. napus combination and is additive of the combining genomes in B. juncea combination. In B. carinata regeneration frequencies are less than the parental diploid species, iv) Significant intraspecific genotypic differences were observed for B. nigra and B. oleracea among diploids and B. juncea and B. carinata among amphidiploids, when cotyledons of eighteen genotypes were tested in one growth regulator combination.Abbreviations MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - IAA Indole 3-acetic acid - BA 6-Benzyl aminopurine     

Improved L-lysine production by the amplification of theCorynebacterium glutamicum dapA gene encoding dihydrodipicolinate synthetase inE. coli

Summary ThedapA gene (L-2,3-dihydrodipicolinate synthetase: DHDP synthetase) ofCorynebacterium glutamicum JS231, a lysine overproducer, cloned and subcloned inE. coli/C. glutamicum shuttle vector pECCG117 was used to transformE. coli threonine producer and threonine and lysine coproducer. The plasmid pDHDP5812 carryingdapA gene ofC. glutamicum led to increase in lysine production in theseE. coli strains. Threonine and lysine co-producerE. coli TF1 with pDHDP5812 produced lysine with small amount of threonine. The DHDP synthetase activity ofE. coli TF1 carrying pDHDP5812 showed high resistance toward inhibition by lysine.     

Plasmid-encoded ropiness production inLactobacillus casei SSP.casei

Summary Genetic determinants of the Muc⁺ character were investigated in two ropy strains,Lactobacillus delbrueckii ssp.bulgaricus 201 andL. casei ssp.casei NCIB 4114, which secrete a large amount of slime in culture media. Plasmid DNA analysis revealed the presence of two plasmids (4.5 and 2.3 Mdal) inL. casei ssp.casei, whileL. delbrueckii ssp.bulgaricus was plasmid free, suggesting a chromosomal location of Muc⁺ character in this strain. Curing experiments carried out onL. casei ssp.casei NCIB 4114 indicated a correlation between the Muc⁺ phenotype and the 4.5 Mdal plasmid.     

Instability of lactose and citrate metabolism of Leuconostoc strains

Summary The instability of Lac⁺ and Cit⁺ phenotypes was investigated inLeuconostoc mesenteroides subsp.cremoris ATCC 19245 and in four strains ofLeuconostoc mesenteroides subsp.dextranicum. The two phenotypes were linked respectively to a 14 Mdal and a 34 Mdal plasmid in Leuconostoc mesenteroides subsp.cremoris ATCC 19245. InLeuconostoc mesenteroides subsp.dextranicum the character Lac⁺ was linked to a 28 Mdal plasmid, while the Cit⁺ phenotype was stable.     

A high molecular weight plasmid ofZymomonas mobilis harbours genes for HgCl2 resistance

Summary Plasmids fromZ. mobilis could be stably maintained inE. coli HB101 in which the expression of various drug resistance markers could be monitored. A large molecular weight plasmid (5.2 kbp) ofZ. mobilis was found to harbour the genes for mercuric chloride degradation and to confer uponE. coli, resistance to a higher mercuric chloride concentration as compared toZ. mobilis. The introduction of this plamsid madeE. coli sensitive to concentrations of cadmium acetate which were originally non-inhibitory to it.     

Non-polluting wood and pulp delignification: Biomimetic ligninase system

Summary We report the delignification ofPinus radiata D Don,Eucalyptus globulus andEucalyptus grandis woods (formic acid treated and untreated) by 2 h treatment with a hemin/hydrogen peroxide system. The untreated chips and sawdust ofE. globulus were 30% and 50% delignified respectively. No significant effects were found forP. radiata sawdust;P. radiata treated chips (organosolv pulp) did not show any further delignification upon hemin/peroxide action, 25% delignification was achieved in untreated chips. In the case ofE. grandis untreated wood the delignification was better in sawdust than in chips, but in smaller percentage than in the otherEucalyptus species. This relation is maintained in substrates, treated with formic acid or untreated. The delignification of chips in both species ofEucalyptus was improved when they were pre-treated with formic acid. The loss of lignin in theE. grandis andE. globulus sawdust (pre-treated with formic acid) was 79% and 75% respectively.     

Assimilation of cholesterol by some cultures of lactic acid bacteria and bifidobacteria

Summary Cultures ofStr. thermophilus assimilated less cholesterol than those ofLactobacillus delbrueckii subsp.bulgaricus. A significant difference was found between strains ofL. delbrueckii subsp.bulgaricus — LB1 and LB2 and LB3 (p<0.001).Bif. bifidum actively assimilated cholesterol, but no significant difference was observed between their two strains (p>0.05). Cultures ofL. asidophilus assimilated significantly more cholesterol than those ofStr. thermophilus and a commercial yoghurt culture.     

Construction of multiple herbicide resistant ammonia excreting strains of cyanobacteriumNostoc muscorum

Summary Machete resistant (Mat ^r), basalin resistant (Bas ^r), 3(3,4 dichlorophenyl)-1,1-dimethyl urea resistant (DCMU ^r), atrazine resistant (Atr ^r) and propanil resistant (Prp ^r) phenotypes ofGloeocapsa sp. were cotransformed toNostoc muscorum at high frequency. Spontaneously occurring mutants of the multiple herbicide resistant transformant containing L-methionine-DL-sulfoximine resistant (Msx ^r), ethylene diamine resistant (Eda ^r), or phosphinothricin resistant (Ppt ^r) glutamine synthetase (GS) showed extracellular liberation of ammonia resulting from fixation of N₂ under photosynthetic conditions. Results suggest a definite role of GS activity in regulation of extracellular ammonia.     

Thermostable tryptophan synthase ofBacillus stearothermophilus expressed inEscherichia coli

Summary The tryptophan synthase genes,trpA andtrpB, from a moderate thermophile,Bacillus stearothermophilus IFO13737, were expressed efficiently inEscherichia coli. The recombinant tryptophan synthase amounted to 22% of the soluble cellular protein, and was purified to homogeneity by three steps. The enzyme is more thermostable thanE.coli tryptophan synthase, especially the subunit. The enzyme is also more resistant to sodium dodecylsulfate and methanol thanE.coli enzyme.     

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.     

Transformation of the coryneform bacterium cellulomonas flavigena with plasmid DNA via electroporation

Summary Using electroporation we have transformed Cellulomonas flavigena with a shuttle vector (pJA85) derived from the E. coli plasmid pUC8 and the Brevibacterium lactofermentum plasmid pULRS8. Upon transformation this plasmid was found to be stable, not to undergo detectable deletion, and to express antibiotic resistance markers originating in Brevibacterium.     

Microbial transformation of sterols

Summary Arthrobacter simplex, Serratia marcescens, Fusarium and Mycobacterium were tested for their ability to transform phytosterol to Androsta 1, 4 diene 3, 17 dione (ADD). Arthrobacter simplex ATCC 6946 was found to be more efficient than the other species tested.     

Phenylalanine ammonia-lyase activity in suspension cultures of Ulmus pumila and U. campestris treated with spores of Ceratocystis ulmi

Summary Cell suspension cultures of a Ceratocystis ulmi-resistant (Ulmus pumila) and a -susceptible elm (U.campestris) were established from leaf callus tissue. Treatment of cultures with spores of C.ulmi induced a large increase in the activity of phenylalanine ammonialyase, only in the cells of the resistant species U.pumila with a maximum after 24 h. Inoculated U.pumila cells also excreted a red unidentified chemical into the culture medium. Neither responses were induced in inoculated U.campestris cultures. The results are discussed in relation to the development of the elm cell culture system as a model for studying the differential biochemical mechanisms of disease resistance in elms.     

Influence of the inoculum size ofLactobacillus plantarum on the competition withEnterobacter cloacae

Summary The size of the inoculum ofLactobacillus plantarum or its natural density, appears to be of predominant importance in the exclusion ofEnterobacter cloacae in mixed fermentations, such as ensilage. In a liquid medium, simulating adverse silage conditions, an initial density ofL. plantarum at least twice that ofE. cloacae was found necessary in order to obtain a succesful silage.     

Construction of aClostridium acetobutylicum-Escherichia coli shuttle vector

Summary A 4.1-kb cryptic plasmid, designated pCA134, has been isolated fromClostridium species. In order to develop a vector suitable for transforming saccharolytic clostridia three hybrid plasmids were constructed by inserting pCA134 into pHV32 withEcoRI, orBglII andBamHI. The newly constructed plasmids were propagated inEscherichia coli and were used to transformBacillus subtilis andClostridium acetobutylicum. One of them, pCAB32 (10.1 kb), which contains chloramphenicol acetyltransferase gene and an origin of replication derived from pCA134 was introduced intoB.subtilis andC.acetobutylicum as well asE.coli.     

Lactic acid bacteria active during the fermentation of wheat silage in small scale silos

Summary Wheat was ensiled and periodically analyzed for lactic acid bacteria present. Initially Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus cellobiosus and Streptococcus lactis predominated. After two to four days enterococci including S. faecium and S. bovis were present in high populations as well as Lactobacillus plantarum. It was concluded that mixed populations of enterococci and L. plantarum are active in the successful fermentation of wheat silage.     

Studies of dotted,a regulatory element in maize

Summary Dotted (Dt) is the regulatory element of a two-unit controlling system in maize. Dt causes the inherited change from the recessive a (colorless) to its dominant allele, A (anthocyanin production), during the development of the stalk, leaves, and endosperm. The mutation events are observed as sectors of color in an anthocyaninless background.Since its discovery over 40 years ago, Dt has always been found in the terminal knob of the short arm of chromosome 9. This is puzzling because controlling and regulatory elements in general are not located permanently, but change positions (transpose) within the chromosomal complement. To resolve this seeming discrepancy, transpositions were looked for in a homozygous a Dt stock. Because the frequency of aleurone mutations is exponentially related to Dt dosage, a Dt transposition would result in a greatly increased number of dots if the egg or sperm nucleus contained both the transposed Dt and the Dt remaining on chromosome 9. A total of 6 transposed Dt's (Dt-T) were recovered in this manner. Dt-TA was found linked to the gene Y (yellow endosperm) of chromosome 6. Dt-TB no longer showed linkage with yg2 of chromosome 9, but remains unlocated (the original Dt in this stock is separated from yg2 by 6 or 7 cross-over units.). The remaining transpositions (C-F) assorted independently of Dt on chromosome 9.The transposed Dt's had the same effect as Dt on the frequency and timing of aleurone mutations. An increase in transposition frequency and losses of Dt-T's was characteristic of several of the transposed Dt's. Dt-T's B-F transposed so frequently that testcross ratios of 71 (three Dt' s) and 15 1 (four Dt' s) were observed. No secondary transpositions or losses of Dt-TA were detected. Thus, Dt-TA resembles the original Dt with regard to its transposition frequency and stability.Journal Paper No. J-8333 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1880.     

Production of citric acid from sugars present in wood hemicellulose usingAspergillus niger andSaccharomycopsis lipolytica

Summary One strain each of the fungus,Aspergillus niger, and the yeast,Saccharomycopsis lipolytica, were investigated for their ability to produce citric acid from the sugars present in hemicellulose hydrolysates.S. lipolytica produced citric acid as efficiently from mannose as from glucose, but failed to assimilate xylose, arabinose or galactose.A. niger readily assimilated mannose, xylose and arabinose, and produced citric acid from these sugars although the yields were lower than from glucose. A possible inhibitory effect of arabinose on citric acid production from other sugars was observed usingA. niger.     

Molecular cloning of theEscherichia coli gene encoding xylose isomerase

Summary A 2.4 Kb DNA fragment restricted from a Clarke-Carbon ColEl plasmid, pLC32-9, containing the xylose isomerase gene has been inserted into the PstI site of pDB248, a shuttle plasmid between the bacteriumE. coli and the fission yeast,Schizosaccharomyces pombe. This recombinant plasmid, pDB248-XI, can genetically complement xylose isomerase deficientE. coli strains and xylose isomerase gene can be expressed inSchizosaccharomyces pombe.     

Characterization of a cytochrome oxidase-deficient yeast mutant which accumulates porphyrins

A cytochrome oxidase-deficient mutant (pop4) of Saccharomycescerevisiae which accumulates porphyrins has been characterized. The pop4 mutation is recessive and affects a single nuclear gene. The bulk of the accumulated porphyrins consists of uroporphyrin and its partial decar?ylation products, suggesting that the mutation causes a block at the level of uroporphyrinogen decar?ylase. However, pop4 is not allelic to hem6 or pop3, putative decar?ylase structural-gene mutants. These results suggest that there may be a uroporphyrinogen decar?ylase isoenzyme specifically involved in heme a production.