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     

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     

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.     

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.     

Ethanolic fermentation of cane molasses by a highly flocculent yeast

Summary A study of the comparative kinetics of standardS.uvarum ATCC 26602 withS.cerevisiae Y-10 (an isolate) and a highly flocculent strain ofS.uvarum in batch mode has shown that both the isolate and the highly flocculentS. uvarum strain have more desirable characteristics than the standard strains for ethanol production from cane molasses.     

Interspecific somatic hybrids ofRudbeckia hirta andR. Laciniata (Compositae)

Interspecific somatic hybrid plants betweenRudbeckia hirta cv. Marmalade andR.laciniata cv. Irish Eyes were regenerated following the electro-fusion of mesophyll protoplasts ofR.hirta with callus protoplasts ofR.laciniata. A hybrid selection scheme was based on the fact that plant regeneration, from parental protoplasts ofR.hirta, was via shoot regeneration of callus, and only via rhizogenesis forR.laciniata. The other half of the selection strategy was based on the presence of anthocyanin-pigmented roots; a characteristic of theR.hirta parent only. Somatic hybrids were regenerated, via rhizogenesis, alongside normalR.laciniata but were distinguished by the presence of pigmented roots (a feature ofR.hirta). Hybrid plants had a floral morphology that was intermediate as compared to that of the two parents, with an expected somatic chromosome number of 2n=(2x+4x)=74. Pollen viability though was low. Esterase and peroxidase isozyme profiles confirmed the hybrid nature of the regenerated plants with pigmented roots, whilst chloroplast DNA restriction analysis showed that these hybrids had aR.laciniata chloroplast DNA. This demonstration of somatic hybridisation not only opens up the possibility of incorporating novel traits between such ornamentalCompositae species, but provides a selection strategy based on rhizogenesis as the route to plant regeneration coupled with heritable pigmentation production of roots as a confirmatory hybrid marker.ABBREVIATIONS BSA bovine serum albumin - EDTA ethylene diamine tetra acetic acid - FDA fluorescein diacetate - f.wt. fresh weight - IAA indole 3-acetic acid - MS Murashige and Skoog (1962) medium - TEMED N,N,N,N-Tetra methyl ethylene diamine - TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid)     

Production and conversion of chitosan with cultures of Mucor rouxii or Phycomyces blakesleeanus

Summary Highly deacetylated chitosan was accumulated in the mycelia ofMucor rouxii orPhycomyces blakesleeanus. These cultures also effected the deacetylation of the chitin ofAspergillus niger mycelium into chitosan. After 96 hours of incubation with these cultures the degree of acetylation of commercial crab shell chitosan was reduced from 25.0% to values between 4.3 and 8.6%. The potential exists for the production of chitosans with tailored physico-chemical properties from waste chitin.     

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.     

Identification of genes specifying proline biosynthesis in marine bacteriumAlteromonas haloplanktis

Summary A gene bank of total DNA of a marine bacterium,Alteromonas haloplanktis, was constructed on pBR322. Two hybrid plasmids pUS2010 and pUS2011 carrying inserts of 8.2 and 5.7 kb, respectively, were isolated that complemented theproBA deletion inE.coli CSH26. Restriction map of the inserts showed that both plasmids in common carried a 5.7 kb fragment. This restriction fragment thus contains both the genes involved in proline biosynthesis inA.haloplanktis and could be expressed inE.coli.     

Dynamic mechanical analysis as a technique for the study of fungal degradation of wood

Summary Dynamic mechanical analysis has been used to follow changes in the properties of wood (Pinus sylvestris) subject to attack by Coniophora puteana. Shear storage modulus fell steadily over the 42 day observation period and correlated with weight loss. Tan shows relatively little change implying uniform degradation of the structural polymers.     

High level extracellular secretion of thermostable α-amylase ofBacillus stearothermophilus inEscherichia coli

Summary Bacillus stearothermophilus BR135 (ATCC 29609)amy gene was cloned in pBR322 from its plasmid DNA and was subcloned in a vector useful both forB. subtilis andE. coli.E.coli HB101 harboring the plasmid pSS099 when grown in L medium in presence of 5. g/ml chloramphenicol produces 70 units/ml of extracellular -amylase. This is nearly twice that ofE.coli cells harboring pSSO76, a plasmid havingamy ofB.stearothermophilus BR135 atHindIII site of pBR322. Characteristically the protein was a 58 kd protein and cross reacted with antiserum developed against purified -amylase of BR135.     

Identification of a penicillin V acylase processing fungus

In our studies with the penicillin V acylase of Bovista plumbea strains NRRL 3501 and NRRL 3824, we wanted to receive spores of these fungi. Surprisingly the fruiting bodies obtained in our work were not identical with those characteristic for Bovista plumbea. We identified them as Pleurotus ostreatus. For this reason we have to correct the name of the fungi known as Bovista plumbea NRRL 3501 and NRRL 3824.     

Resistance of bacterial film to contamination during use as inoculum in repeated batch fermentation

Summary AnEscherichia coli strain constitutive for -galactosidase was immobilized onto cotton cloth. The resultingE.coli film was used as a resident inoculum in repeated batch fermentations for 30 days in the presence ofBrevibacterium ammoniagenes added as a contaminant. Analysis of -galactosidase production shows that contamination did not decrease the capacity of the film to generateE.coli cells, or decrease theE.coli population on the film.     

Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Acid protease is essential for degradation of proteins during soy sauce fermentation. To breed more suitable koji molds with high activity of acid protease, interspecific genome recombination between A. oryzae and A. niger was performed. Through stabilization with d-camphor and haploidization with benomyl, several stable fusants with higher activity of acid protease were obtained, showing different degrees of improvement in acid protease activity compared with the parental strain A. oryzae. In addition, analyses of mycelial morphology, expression profiles of extracellular proteins, esterase isoenzyme profiles, and random amplified polymorphic DNA (RAPD) were applied to identify the fusants through their phenotypic and genetic relationships. Morphology analysis of the mycelial shape of fusants indicated a phenotype intermediate between A. oryzae and A. niger. The profiles of extracellular proteins and esterase isoenzyme electrophoresis showed the occurrence of genome recombination during or after protoplast fusion. The dendrogram constructed from RAPD data revealed great heterogeneity, and genetic dissimilarity indices showed there were considerable differences between the fusants and their parental strains. This investigation suggests that genome recombination is a powerful tool for improvement of food-grade industrial strains. Furthermore, the presented strain improvement procedure will be applicable for widespread use for other industrial strains.     

Intraspecific fusion ofAspergillus niger strains producing citric acid using heat-inactivated and living protoplasts

Protoplasts from a prototrophicAspergillus niger strain were first inactivated at 55°C for 12 min with a regeneration frequency of 3.5×10⁻⁶, and then fused with living protoplasts from an auxotrophic strain. The fusion frequency was 1.1×10⁻⁵. Some fusants segregated sectors of prototrophic recombinants Citric acid production in submerged cultivation of these recombinants was examined. More recombinants were obtained by further treating the fusants with (+)-camphor, a diploidization inducer.     

Stereochemical Behavior of an NADH Model Compound Carrying l-Prolinamide at 3,5-Positions

Interspecific recombinants have been produced between Streptococcus cremoris H-61 and S. lactis J-1 by polyethylene glycol-induced protoplast fusion. All of the fusants obtained showed mixed physiological properties of the two parents, and possessed plasmids derived from both parents at random. Physiological properties of primary colonies were stably maintained among the progenies after the single-colony isolation procedure. Similarly, in most of the fusants the plasmid profiles of the primary colonies were stably maintained, but one lost 2 out of the 7 plasmid bands. However, there was no indication that plasmids from either one of the parents were preferentially lost. These results showed that interspecific genetic transfer occurred on chromosomal and plasmid DNA on the protoplast fusion and that the fusants obtained were not heterokaryons, but true recombinants.     

Intra- and interspecific gametosomatic hybridisation within the genus Petunia

Following PEG and high pH induced fusion, intraspecific gametosomatic hybrid plants (pollen tetrad protoplasts of a normal purple flowered variety of P. hybrida fused with cell suspension protoplasts of a nuclear albino mutant of the variety Blue Lace) and interspecific gametosomatic hybrid plants (tetrad protoplasts (as above) fused with cell suspension protoplasts of a nuclear albino mutant of P. parviflora) were recovered. Hybrid plants of both combinations possessed an intermediate vegetative and floral morphology with chromosome numbers of 2n=3x=21 and 2n=3x=25 respectively. Hybrid cells were in both systems identified as green colonies against an albino background as a result of complementation to chlorophyll proficiency. Pollen tetrad protoplasts did not divide. The production of such plants at the intra- and interspecific level in Petunia has shown that the concept of gametosomatic hybridisation can be extended to genera other than Nicotiana. An alternative selection strategy is available to that as used earlier for Nicotiana.     

Sub-littoral and supra-littoral amphipods respond differently to acute thermal stress

Thermal tolerance was determined in two closely related amphipod species from contrasting environments (sub-littoral and supra-littoral zones of the sea) using HSP expression and the activity of antioxidant enzymes. The levels of HSP70 and small HSPs present in untreated control animals were higher in the supra-littoral Orchestia gammarellus than in the sub-littoral Gammarus oceanicus. Under the acute thermal stress, HSP levels increased less strongly in O. gammarellus than in G. oceanicus. Activities of antioxidant enzymes peroxidase, catalase and glutathione S-transferase, were more pronounced in the supra-littoral O. gammarellus then in the sub-littoral G. oceanicus. We conclude that the environmental temperature regime modifies key cellular defense mechanisms in amphipods. Higher levels of constitutive HSP synthesis and higher levels of antioxidant enzymes in the supra-littoral species likely reflects adaptation to this highly thermally variable environment.     

Acetone-butanol-isopropanol production byClostridium beijerinckii (synonym,Clostridium butylicum)

Isopropanol is a product of the organism formerly known as Clostridium butylicum, which is now included in the speciesClostridium beijerinckii. We tested 52 strains ofC.beijerinckii for their ability to produce acetone,n-butanol, and isopropanol. The 32 butanol-producing strains may be divided into two groups based on whether isopropanol was produced. Isopropanol-producing cultures accumulated acetone only transiently.     

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.