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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Ligninolytic properties of different white-rot fungi

Summary Seven white-rot fungi were examined for the production of ligninase, manganese peroxidase and laccase. All these enzymes were found inTrametes gibbosa andTrametes hirsuta. Only manganese peroxidase and laccase were produced byPycnoporus cinnabarinus,Coriolopsis polyzona,Stereum hirsutum,Dichomitus squalens andGanoderma valesiacum. All fungi decolorized Poly B-411 and Indulin AT plates with low-N medium. The differences in enzyme pattern indicate that different species of fungi may employ different modes of lignin metabolism.     

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.     

Expression of the threonine operon fromEscherichia coli inBrevibacterium flavum andCorynebacterium glutamicum

Summary The threonine operon fromEscherichia coli was cloned in plasmid pBR322, subcloned into the shuttle vector pCEM300 and the resulting recombinant plasmid was transferred intoBrevibacterium flavum andCorynebacterium glutamicum. The expression ofE. coli threonine genes in these coryneform bacteria was demonstrated by complementing thethrA andthrB mutations and by assaying homoserine dehydrogenase activity.     

Comparative shoot regeneration responses of diploid brassicas and their synthetic amphidiploid products

Comparative shoot regeneration responses of three diploid Brassica species, B. campestris (AA), B. nigra (BB) and B. oleracea (CC) and their synthetic amphidiploid combinations have been investigated. The study indicates that A genome has an inhibitory effect on regeneration as evident from significantly low responses of B. juncea (AABB) and B. napus (AACC) combinations, in comparison with regeneration response of B. nigra and B. oleracea. This inhibition may arise from genomic interactions or from the B. campestris cytoplasm interacting negatively with the alien genome. Significant cytoplasmic influence on regenerability has been observed in B. carinata (BBCC) synthesised from reciprocal crosses of B. nigra and B. oleracea.Abbreviations MS Murashige and Skoog (1962) - IAA Indole-3-acetic acid - NAA -napthaleneacetic acid - BA 6-Benzylaminopurine     

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.     

Intergeneric protoplast fusion between agrobacterium tumefaciens and Bacillus thuringiensis subsp. kurstaki

Summary Intergeneric protoplast fusion betweenA.tumefaciens andB.thuringiensis was performed. The fusants exhibited some properties of both the parental strains. One of the Gram positive fusants with most of theBacillus properties showed tumor inducing capacity in pigeonpea (Cajanas cajan).     

Regulation of mycotoxin production by Fusarium graminearum: Complementation effects between two mutant types

Summary Starting from anileu auxotroph ofFusarium graminearum producing high levels of the mycotoxin zearalenone, selection after UV irradiation gave low-producing mutants of essentially normal morphology,zea,ileu. Heterokaryons betweenzea,ileu strains and an auxotrophic strainlz,inos derived from the lazy morphological mutant ofFusarium graminearum, which has abnormal morphology and also produces little or n zearalenone, produced significant levels (over 50% of the wild-type level) of mycotoxin. The observation confirms views as to the regulatory nature of thelazy mutation.     

Regeneration of plants from callus tissue of the pasture legume Centrosema brasilianum

Plants were regenerated from leaf explants of Centrosema brasilianum cultured in vitro. Callus and buds were produced on Murashige and Skoog medium (MS), 0.8% agar, 0.1 mg/l NAA and 1 mg/l BAP. Regeneration of multiple shoots was achieved by transferring callus onto fresh medium containing 0.01 and 1 mg/l of NAA and BAP, respectively. Shoots formed roots upon transfer to MS with 0.01 mg/l NAA. Plantlets were succesfully transferred to soil. Leaf-derived calli of Centrosema arenarium, C. macrocarpum, C. pascuorum, C. pubescens, and C. virginianum did not produce shoots when cultured in vitro.     

Differentiation in explants from mature leguminous trees

Stem and petiole explants, obtained from mature trees, ofAlbizzia lebbeck,Cassia fistula andC.siamea callused and differentiated shoot-buds and later shoots on B5 medium supplemented with either 0.5 mg/l IAA + 1 mg/l BAP or BM + 2 mg/l NAA + 0.5 mg/l BAP. The stem explants were more responsive than the petiole explants. InA.lebbeck, the IAA substituted medium favoured differentiation from both types of explants. However, inC.fistula, the type of explants rather than the medium composition had an overriding influence on shoot differentiation since those from petiole hardly responded in either medium. It has been possible to obtain plantlets from bothA.lebbeck andC.fistula under conditions conducive to rooting. Plantlets ofA.lebbeck have also been successfully transferred to the field.