Genome protoplast fusion

Two major advances describe the construction of mutants with an apparently significantly higher probability of improved phenotype. The method involves whole-genome shuffling - with protoplast fusion - of a small number of parental strains... 

Similar techniques were used by Shinohara et al (71) to develop hybrids with increased production of fusel alcohols and esters. Protoplast fusion techniques have been used to confer amylolytic activity to brewery yeasts (22) and ethanol tolerance to wine yeasts (70) Farris et al (72) used protoplast fusion to produce hybrids with killer factor that is, the ability to secrete proteinic toxins. Kunkee and coworkers (25) utilized a leucine auxotrophic mutant strain of S. cerevisiae (UCD Montrachet 522) to produce base wine for brandy production the mutant strain produces less isoamyl alcohol, reducing the quantity of fusel alcohols in the subsequent brandy. And Thornton (48) discussed the progress in utilizing plasmid vectors to introduce new genes into wine yeasts he cautioned, however, that until the yeast genome is better understood that direct gene manipulation techniques will be of limited value. 

One of the drawbacks in the current commercial fermentation process is that the predominant form of the product is the deprotonated lactate rather than lactic acid, requiring more expensive and wasteful product purification steps. This is because the Lactobacillus fermentation operates at a minimum pH of 5.0-5.5 which is above the pA a of lactic acid (3.87). To overcome this limitation, a powerful strain improvement method, genome shuffling, was used to improve the acid tolerance of a poorly characterized industrial strain of LactobacillusA population of strains with subtle improvement in pH tolerance was isolated using classical strain improvement methods such as chemostats, and were then shuffled by recursive pool-wise protoplast fusion to create mutant strains that grow at substantially lower pH than does the wild-type strain. 

Mutation/selection, strain recombination (e.g., breeding/protoplast fusion), directed genomic a Ite ration/metabolic engineering/enhance me n t of gene expression rates Amino acid exchange/combinatorial... 

Genetic techniques for obtaining chloroplast-encoded herbicide resistance in crop plants are reviewed. These are based on the use of cell culture methods and Include mutant selection in cultured cells, chloroplast transfer via protoplast fusion, and genetic recombination between chloroplast genomes. The first two methods are described in detail using trlazine resistance as an example. 

Cell culture-based methods suitable for obtaining chloroplast-encoded herbicide resistance in crops will be reviewed. These will Include mutant selection in cultured cells, transfer of chloroplasts via protoplast fusion, and selection of recombinant chloroplast genomes. Examples will be limited to resistance to triazine herbicides. Chloroplast transformation involving introduction of novel DNA into chloroplasts, will not be covered. Preliminary data indicate that chloroplast transformation may be feasible (4) but have not been confirmed. The genetics, physiology... 

Protoplast fnsion is a technique that enables the asexual combination of two complex genomes. However, similarly to conventional breeding, the final result is uncertain because of unpredictable gene transfer with little or no understanding of the specific genetic perturbations (Piddocke Olsson, 2010). As an example of protoplast fusion, the conversion of non-flocculent brewer s yeast to a flocculent one by electrofusion can be cited (Urano, Sahara, Koshino, 1993 Urano, Sato, Sahara, Koshino, 1993). 

The cultivated potato and many of its relatives are amenable to eell and tissue culture procedures, ineluding protoplast isolation and fusion. Somatic cell fusions have frequently been used to eombine the genomes of Solarium speeies that are sexually ineompatible because of pollen-stylar interactions or mismatched EBN numbers. Somatic fusion circumvents sexual reproduetion and results in novel eombinations of not only nuclear genomes, but also cytoplasmic genomes (Trabelsi et al., 2005 Bidani et al., 2007 Lovene et al., 2007). However, recalcitrant genotypes... 

Enhancement of the productivity is achieved at first by rounds of random mutation and selection and, if possible, by breeding, and/or by DNA injection or fusion techniques like protoplasting and at further stages, after a comprehensive characterization of the usually complex biosynthetic pathways, directed genomic alterations and metabolic engineering. 

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