Protoplast Fusion Research Articles

Cybridization of Grapefruit with ‘Dancy’ Mandarin Leads to Improved Fruit Characteristics

In cybridization, new combinations of nuclear and cytoplasmic genes result in a unique genotype that may bring cellular, physical, physiological, and biochemical changes to the plant. This has been demonstrated in the unexpected cybrids generated from the fusion of citrus (Citrus sp.) protoplasts in two independent experiments. The first experiment was conducted to generate potentially seedless triploids by fusing diploid protoplasts of embryogenic ‘Dancy’ mandarin (Citrus reticulata) suspension culture cells with haploid ‘Ruby Red’ grapefruit (C. paradisi) protoplasts derived from tetrad-stage microspores. After multiple attempts, only one triploid was recovered, but several diploid plants with typical grapefruit morphology were also regenerated. In the second experiment, protoplasts derived from embryogenic ‘Dancy’ mandarin suspension culture were fused with nonembryogenic protoplasts from ‘Duncan’ grapefruit leaves in an effort to produce an allotetraploid somatic hybrid. The fruit from the resulting trees resembled grapefruit in morphology and type, and maintained excellent quality throughout the summer, when commercial grapefruit rapidly loses quality. Fruit on these trees remained firm with exceptional sweetness and good flavor into August, and without seed germination. The regenerants obtained in the protoplast fusion experiments were confirmed as cybrids by genetic marker analyses. The test grapefruit were identical to commercial ‘Ruby Red’ grapefruit at six nuclear simple sequence repeat (SSR) marker loci, but identical to ‘Dancy’ with respect to a mitochondrial intron marker. The plastid genomes of individual trees originated from either fusion partner. In the first experiment, haploid ‘Ruby Red’ protoplast preparations must have also contained contaminant diploid protoplasts. Apart from the value of altered fruit quality attributes in the marketplace, these plants provide an opportunity to understand the contributions of cytoplasmic organelle genetics to important citrus fruit-breeding objectives.

In situ high-valued utilization and transformation of sugars from Dioscorea zingiberensis C.H. Wright for clean production of diosgenin

The industrial production of diosgenin in China generates a large amount of high-sugar wastes with low bioavailability, which causes serious pollution to the environment. In this study, a new clean and efficient process for the production of diosgenin was developed using sugars through in situ high-valued transformation. The sugar mixture from Dioscorea zingiberensis C.H. Wright contained abundant beneficial components. Nine typical microorganisms that produced intracellular products were evaluated. Saccharopolyspora spinosa was selected for recursive protoplast fusion to increase the spinosad yield by 46.3% compared with that of the wildtype. Diosgenin and spinosad co-production was conducted in a 100L bioreactor, with pH controlled by adding glucose. The biological oxygen demand of the effluent water decreased from 15,000mg/L to 450mg/L; hence, the proposed process is environment friendly.

Genome shuffling of the nonconventional yeast Pichia anomala for improved sugar alcohol production.

BackgroundSugar alcohols have been widely applied in the fields of food and medicine owing to their unique properties. Compared to chemical production, microbial production of sugar alcohols has become attractive because of its environmentally friendly and sustainable characteristics. Our previous study identified the nonconventional yeast Pichia anomala TIB-x229 as a potential producer of sugar alcohols from glucose. To further improve strain performance, we combined genome shuffling with optimized high throughput screening methods for the directed improvement of nonconventional yeast and complex phenotypes.ResultsTo accelerate strain improvement, a practical genome shuffling procedure was developed and successfully applied in the nonconventional yeast P. anomala to increase sugar alcohol production. Through two rounds of genome shuffling, an improved P. anomala isolate GS2-3 could produce 47.1 g/L total sugar alcohols from 100 g/L glucose, which was 32.3% higher than the original strain. In this process, a simple and accurate colorimetric assay was optimized and used for high throughput screening of sugar alcohol-producing strains. Moreover, a fluorescence-activated cell sorting method was developed to efficiently screen protoplast fusions for genome shuffling of nonconventional yeast.ConclusionAn efficient genome shuffling procedure was developed and applied to enhance the sugar alcohol production of the nonconventional yeast P. anomala. Our results provide a general platform for strain improvement of polyol-producing microorganisms or nonconventional microorganisms in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0303-8) contains supplementary material, which is available to authorized users.

Application of the Protoplast Fusion Technique to Engineer a Recombinant Microorganism to More Efficiently Degrade Chlorophenols

Recombinant strain Xz6-1 was constructed by the protoplast fusion technique with the goal of endowing it with the ability to efficiently degrade pentachlorophenol (PCP). This compound was considered as a representative of possible compounds that can be obtained during the bleaching of pulp for papermaking. The potential of Xz6-1 and Pseudomonas putida to treat PCP synthetic wastewater was explored. The majority of PCP was removed within the first 20 h; two degradation curves were obtained that followed first-order reaction kinetics. The kinetics data revealed that the rate constant for degradation of PCP for Xz6-1 was 0.063 h-1, a value that was over 50% greater than that of Pseudomonas putida (0.040 h-1). Aerobic granular sludge was highly fortified with Xz6-1 and Pseudomonas putida to provide PCP degradability improvements of 180.9% and 98.3%, respectively, relative to the original sludge. All results demonstrate that the protoplast fusion technique is an effective approach to construct a high-activity chlorophenol-degrading strain.

Enhanced biobutanol production using novel clostridial fusants in simultaneous saccharification and fermentation of green renewable agriculture residues

AbstractThermostable Clostridia species were developed in the present study through protoplast fusion between mesophilic and thermophilic clostridial species. Two parental strains of Clostridia species, Clostridium acetobutylicum (ATCC 4259) and Clostridium beijerinckii (ATCC BA101), along with three fused strains, Clostridium acetobutylicum Clostridium thermocellum (CaCt), Clostridium beijerinckii Clostridium thermocellum (CbCt) and Clostridium acetobutylicum Clostridium beijerinckii (CaCb), were examined for biobutanol production using wheat straw (WS) as a feedstock in a batch process of simultaneous saccharification and fermentation (SSF). The objective of the study was to use the thermotolerant fused strains to enhance enzymatic activity during the SSF by raising the incubation temperature and thereby eventually increasing biobutanol production. Results from the current study indicated that the fused strains were able to produce the required enzymes at higher temperature essential for the saccharification of WS (i.e., endoglucanase, exoglucanase, and β‐glucosidase). This will have a major impact on eliminating costs associated with adding enzymes to the saccharification process. Fused strain CbCt achieved the highest biobutanol production of up to 14.13 g/L (i.e., a yield of 0.29) at 45 °C with total sugar consumption of 82%. Enzymatic activity of CbCt was found to be 61.67 FPU (filter paper unit) which was the highest in comparison with other fused strains evaluated in this study. These results indicate a breakthrough for the technological and economical obstacles associated with industrialization of the SSF process. © 2015 Society of Chemical Industry and John Wiley & Sons, Ltd

Water consumption and biomass production of protoplast fusion lines of poplar hybrids under drought stress.

Woody crops such as poplars (Populus) can contribute to meet the increasing energy demand of a growing human population and can therefore enhance the security of energy supply. Using energy from biomass increases ecological sustainability as biomass is considered to play a pivotal role in abating climate change. Because areas for establishing poplar plantations are often confined to marginal sites drought tolerance is one important trait for poplar genotypes cultivated in short rotation coppice. We tested 9-month-old plants of four tetraploid Populus tremula (L.) × P. tremuloides (Michx.) lines that were generated by protoplast fusion and their diploid counterpart for water consumption and drought stress responses in a greenhouse experiment. The fusion lines showed equivalent or decreased height growth, stem biomass and total leaf area compared to the diploid line. The relative height increment of the fusion lines was not reduced compared to the diploid line when the plants were exposed to drought. The fusion lines were distinguished from the diploid counterpart by stomatal characteristics such as increased size and lower density. The changes in the stomatal apparatus did not affect the stomatal conductance. When exposed to drought the carbohydrate concentrations increased more strongly in the fusion lines than in the diploid line. Two fusion lines consumed significantly less water with regard to height growth, producing equivalent or increased relative stem biomass under drought compared to their diploid relative. Therefore, these tetraploid fusion lines are interesting candidates for short rotation biomass plantation on dry sites.

Enhancement of ε-poly-lysine production in ε-poly-lysine-tolerant Streptomyces sp. by genome shuffling.

ε-Poly-L-lysine (ε-PL) has been widely used as food additive. However, the self-inhibition of ε-PL on cell growth limits the accumulation of ε-PL in the wild-type strain. Here, we screened ε-PL-tolerant strain of Streptomyces sp. with higher ε-PL productivity by genome shuffling and studied the mechanism for the improvement. The initial mutant library was constructed by diethyl sulfate mutagenesis. After four rounds of protoplast fusion, a shuffled strain F4-22 with 3.11g/L ε-PL productivity in shake flask, 1.81-fold in comparison with that of parent strain, was obtained. The higher aspartokinase activity was induced in F4-22 whereas no obvious changes have been found in ε-PL synthetic and degrading enzymes which indicated that the upstream reregulation of the precursor lysine synthesis rather than lysine polymerization or ε-PL degradation in shuffled strain accounted for the higher productivity. The activities of key enzymes in the central metabolic pathway were also enhanced in F4-22 which resulted in increased vigor of the strain and in delayed strain lysis during fermentation. These improved properties of shuffled strain led to the success of combining general two-stage fermentation into one-stage one in 5-L bioreactor with 32.7% more ε-PL production than that of parent strain. The strategy used in this study provided a novel strain breeding approach of producers which suffered from ε-PL-like self-inhibition of the metabolites.

Ornamental Plant Breeding

World’s ornamental plant market, including domestic market of several countries and its exports, is currently evaluated in 107 billion dollars yearly. Such estimate highlights the importance of the sector in the economy of the countries, as well as its important social role, as it represents one of the main activities, which contributes to income and employment. Therefore a well-structured plant breeding program, which is connected with consumers’ demands, is required in order to fulfill these market needs globally. Activities related to pre-breeding, conventional breeding, and breeding by biotechnological techniques constitute the basis for the successful development of new ornamental plant cultivars. Techniques that involve tissue culture, protoplast fusion and genetic engineering greatly aid conventional breeding (germplasm introduction, plant selection and hybridization), aiming the obtention of superior genotypes. Therefore it makes evident, in the literature, the successful employment of genetic breeding, since it aims to develop plants with commercial value that are also competitive with the ones available in the market.

Vegetative hyphal fusion and subsequent nuclear behavior in Epichloë grass endophytes.

Epichloë species (including the former genus Neotyphodium) are fungal symbionts of many agronomically important forage grasses, and provide their grass hosts with protection from a wide range of biotic and abiotic stresses. Epichloë species include many interspecific hybrids with allodiploid-like genomes, which may provide the potential for combined traits or recombination to generate new traits. Though circumstantial evidence suggests that such interspecific hybrids might have arisen from nuclear fusion events following vegetative hyphal fusion between different Epichloë strains, this hypothesis has not been addressed empirically. Here, we investigated vegetative hyphal fusion and subsequent nuclear behavior in Epichloë species. A majority of Epichloë strains, especially those having a sexual stage, underwent self vegetative hyphal fusion. Vegetative fusion also occurred between two hyphae from different Epichloë strains. Though Epichloë spp. are uninucleate fungi, hyphal fusion resulted in two nuclei stably sharing the same cytoplasm, which might ultimately lead to nuclear fusion. In addition, protoplast fusion experiments gave rise to uninucleate putative hybrids, which apparently had two markers, one from each parent within the same nucleus. These results are consistent with the notion that interspecific hybrids arise from vegetative hyphal fusion. However, we also discuss additional factors, such as post-hybridization selection, that may be important to explain the recognized prevalence of hybrids in Epichloë species.

English

Microorganisms are the richest source of phytase which catalyze hydrolysis of phytate to myo-inositol and phosphate. 50 Actinomycete isolates were isolated from heated soil, sand, wastewater, animal faces and some plant ecological wastes on Starch nitrate agar containing 15 µg / mL of antibiotics (Tetracycline + Amphotericin B, w/w). Out of 50 Actinomycetes isolates, 20 isolates (40%) produced extracellular phytase enzyme on solid medium containing wheat bran as carbon source. In liquid medium, the phytase activity was measured as U/mL and the most active isolate in phytase production was R10. Using morphological, physiological and biochemical studies, it was identified as Streptomyces sp. Using 16S rDNA analysis, it was identified as S. luteogriseus R10. Growth in medium containing 1% Na-phytate (pH 6.5) at 40°C for 7days increased phytase production. The maximum phytase activity was achieved using wheat bran, straw, rice husk and hay after seven days of incubation at 40°C but low activity was obtained using Sawyer and baggage as a carbon source. The molecular weight of the purified phytase is 65 kDa and it exhibits optimum activity at pH 5 and 45°C. All tested metal ions at 10 mM enhanced phytase activity except Ba 2+ , Co 2+ , Cu 2+ , Ag, Fe 3+ and Hg 2+ . Improvement of phytase production was carried out using protoplast fusion between S. luteogriseus R10 and S. niveus MM1. Fusant F7 was the best phytase producer (3 time higher) compared to its parents. © 2015 Friends Science Publishers

Genome shuffling of Lactobacillus brevis for enhanced production of thymidine phosphorylase

Thymidine phosphorylase (TPase) plays a vital role in the biosynthesis of nucleosides and their analogs which have tremendous potential in antiviral and anticancer therapies. In this study, genome shuffling was applied to develop new strains of Lactobacillus brevis with an enhanced production of TPase. The parent organisms were mutated using ultraviolet (UV) irradiation and were shuffled by recursive pool-wise protoplast fusion. The parent protoplasts of each cycle were inactivated by UV irritation for 50 min or by heating at 60°C for 60 min. A rapid and efficient pre-screening method for determining L. brevis fusants with increased TPase production was established by adding appropriate concentrations of substrate thymidine and potassium phosphate to the culture broth based on significant differences in the absorption spectra of substrate thymidine and its product, thymine, in alkaline solution at 290 nm. Strains F3-19 and F3-36 showed high TPase activity and favorable hereditary stability and were screened out through three rounds of recursive protoplast fusion. The increase in the TPase activity of F3-19 and F3-36 was 252.6 and 260.5%, respectively, in comparison with the wild type.

Intergenus Protoplast Fusion between Pichia manshurica and Rhodosporidium paludigenum to Increase the Production of Inulinase

The purpose s of this study was to identify the optimum concentration of the lytic enzyme Glucanex for protoplast isolation and to conduct fusion for the purpose of increasing inulinase production . The study performs the protoplast fusion technique using Pichia manshurica and Rh odosporidium paludigenum . Protoplast fusion consists of a series of stages: protoplast isolation , protoplast fusion , protoplast regeneration , and analysis of hybrid fusion results . Protoplast isolation and fusion success rate are determined by various factors, including age of the culture , media type, and type of lytic enzymes used . H ybrid results were analyzed using a fungicide as a marker and measuring specific growth rate ( μ) of the hybrid compared with parental growth rates. Results demonstrated that a concentration of 4 mg/ mL of Glucanex produces the greatest number of protoplasts, 7.2 x 10 10 ( c ell/mL) for P. manshurica and 8.8 x 10 10 ( c ell/mL) for Rh . paludigenum . The results of analysis of hybrid fusions indicate that the study has identified a new fusant , called fusant F4 . Fusant F4 is capable of producing the highest inulinase, 0.6892 IU, compared with parentals P. manshurica, 0557 IU, and Rh . paludigenum, 0.3263 IU. Fusant F4 has specific growth rate ( μ) of 0.3360/h and generation time ( g) of 2.0629 h .

Development and Characterization of Somatic Hybrids of Ulva reticulata Forsskål (×) Monostroma oxyspermum (Kutz.)Doty.

Ulvophycean species with diverse trait characteristics provide an opportunity to create novel allelic recombinant variants. The present study reports the development of seaweed variants with improved agronomic traits through protoplast fusion between Monostroma oxyspermum (Kutz.) Doty and Ulva reticulata Forsskål. A total of 12 putative hybrids were screened based on the variations in morphology and total DNA content over the fusion partners. DNA-fingerprinting by inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) analysis confirmed genomic introgression in the hybrids. The DNA fingerprint revealed sharing of parental alleles in regenerated hybrids and a few alleles that were unique to hybrids. The epigenetic variations in hybrids estimated in terms of DNA methylation polymorphism also revealed sharing of methylation loci with both the fusion partners. The functional trait analysis for growth showed a hybrid with heterotic trait (DGR% = 36.7 ± 1.55%) over the fusion partners U. reticulata (33.2 ± 2.6%) and M. oxyspermum (17.8 ± 1.77%), while others were superior to the mid-parental value (25.2 ± 2.2%) (p < 0.05). The fatty acid (FA) analysis of hybrids showed notable variations over fusion partners. Most hybrids showed increased polyunsaturated FAs (PUFAs) compared to saturated FAs (SFAs) and mainly includes the nutritionally important linoleic acid, α-linolenic acid, oleic acid, stearidonic acid, and docosahexaenoic acid. The other differences observed include superior cellulose content and antioxidative potential in hybrids over fusion partners. The hybrid varieties with superior traits developed in this study unequivocally demonstrate the significance of protoplast fusion technique in developing improved varients of macroalgae.

Molecular characterization of intergeneric hybrid between Aspergillus oryzae and Trichoderma harzianum by protoplast fusion.

Protoplast fusion between Aspergillus oryzae and Trichoderma harzianum and application of fusant in degradation of shellfish waste. The filamentous chitinolytic fungal strains A. oryzae NCIM 1272 and T. harzianum NCIM 1185 were selected as parents for protoplast fusion. Viable protoplasts were released from fungal mycelium using enzyme cocktail containing 5 mg ml(-1) lysing enzymes from T. harzianum, 0.06 mg ml(-1) β-glucuronidase from Helix pomatia and 1 mg ml(-1) purified Penicillium ochrochloron chitinase in 0.8 mol l(-1) sorbitol as an osmotic stabilizer. Intergeneric protoplast fusion was carried out using 60% polyethylene glycol as a fusogen. At optimum conditions, the regeneration frequency of the fused protoplasts on colloidal chitin medium and fusion frequency were calculated. Fusant showed higher rate of growth pattern, chitinase activity and protein content than parents. Fusant formation was confirmed by morphological markers, viz. colony morphology and spore size and denaturation gradient gel electrophoresis (DGGE). This study revealed protoplast fusion between A. oryzae and T. harzianum significantly enhanced chitinase activity which ultimately provides potential strain for degradation of shellfish waste. Consistency in the molecular characterization results using DGGE is the major outcome of this study which can be emerged as a fundamental step in fusant identification. Now it is need to provide attention over effective chitin degradation to manage shrimp processing issues. In this aspect, ability of fusant to degrade shellfish waste efficiently in short incubation time revealed discovery of potential strain in the reclamation of seafood processing crustacean bio-waste.

Somatic Embryo Formation from Co-cultivated Protoplasts of Brassica rapa &amp; B. juncea

Aims: The objectives of this experiment was to establish initial steps of somatic embryogenesis from the fu sed protoplasts between two Brassica species: Brassica rapa L. (var. Agrani, AA, 2n=20) and Brassica juncea L. (var. BINA Sharisha - 7 and var. BINA Sharisha - 8, AABB, 2n=36). Study Design: The experiment was carried out in completely randomized design with t hree replications for each hormone treatment of each variety combination. Place and Duration of Study: An experiment was conducted in growth room of tissue culture laboratory at the Department of Genetics and Plant Breeding, Bangladesh Agricultural Univers ity, Mymensingh during the period from April, 2013 to May, 2014. Methodology: Mesophyll protoplasts were isolated from the young leaves of aseptically grown plants. Variety combinations for protoplasts fusion were: Agrani+BINA Sharisha - 7 and Agrani+ BINA S harisha - 8. 100% polyethylene glycol - 400 was used as a fusogen. Pellieter different compositions of liquid media were used for protoplasts cultured, microcalli, calli and somatic embryo formation. Results: Fused products were cultured in a modified Pelliet er B liquid media with different

NEW CYBRIDS RESULTING FROM ASEXUAL PATHWAY: A PROMISE OF CYBRIDIZATION FOR CREATING NEW CITRUS ROOTSTOCKS AND CULTIVARS

Cybridization should be a way to induce phenotypical variations in highly heterozygous secondary citrus species or genotypes, without breaking their very complex multilocus structure by sexual recombination. It should have particular interest to manipulate the nucleo-cytoplasmic male sterility, to transfer tolerance traits with cytoplasmic determinisms and eventually to improve tolerance to oxidative stress. The present work is focused on the creation of new cybrids through three different approaches of protoplast fusion: (i) symmetric hybridization, (ii) donor-recipient protoplast fusion by UV irradiation of donor protoplasts, and (iii) cytoplasts + protoplast fusion. Sixteen combinations of symmetric protoplast fusion have produced cybrids. No cybrid was obtained after UV irradiation of donor protoplasts while three combinations of cytoplasts + protoplast fusion produced cybrids. Therefore, cytoplast fusion appears much more promising than the UV approach for targeted cybridization. The innovative plant material is currently propagated for further cultivar and rootstock selection and for basic researches on the impact of nucleo-cytoplasmic interaction on desirable traits. (Resume d'auteur)

CHROMOSOME INSTABILITY IN 'CARRIZO' CITRANGE + CITRUS MACROPHYLLA SOMATIC HYBRIDS

Somatic hybridization by protoplast fusion is a very useful technique for citrus rootstock breeding. Indeed, it theoretically allows the accumulation of all genes of the parents, irrespective of their heterozygosity level and therefore the addition of complementary dominant traits. In this work, protoplasts were isolated from callus of Citrus macrophylla and from leaves of 'Carrizo' citrange (C. sinensis × P. trifoliata). Chemical, electro-chemical and electric fusions were performed and the ploidy of regenerated plants was evaluated by flow-cytometry. Five tetraploid plants, one pentaploid plant, one mixoploid (3x-6x) plant and one heptaploid plant were recovered. All these plants were analyzed with SSR and SNP markers, distributed in the nine chromosomes of citrus. Cytoplasmic genomes were characterized with chloroplastic and mitochondrial markers. Mitochondrial genome was inherited from C. macrophylla for all plants while segregation was observed for the chloroplastic genome. Nuclear genome analysis revealed the loss of parental alleles in most of the regenerated plants. In tetraploids, it affected mainly C. macrophylla alleles while P. trifoliata alleles were mostly lost in 5x and 7x plants. The results indicate chromosome instability in this complex intergeneric combination with apparent non random loss of some chromosome fragments. Two allotetraploid somatic hybrids have been selected for evaluation as potential rootstocks. (Resume d'auteur)

Improving the phytoextraction capacity of plants to scavenge metal(loid)-contaminated sites

Plants are able to extract metal(loid) contaminants from the soil or water through their roots and translocate them to harvestable aerial shoots. Of late, this plant potential has been used as a phytotechnology, termed as phytoextraction, for cleaning contaminated sites, and this process has successfully removed elements like As, Cd, Cu, Ni, and Pb, among others. Exploring plants with high metal-accumulation capacity, as well as engineering new hyperaccumulators, is a need of the hour. It is assumed that hyperaccumulators have a &gt;1 shoot:root metal-accumulation ratio, which they achieve by way of (i) overexpression of transport systems for improved sequestration, (ii) tissue-specific protein expression, and (iii) high concentration of metal chelators. Unlike nonhyperaccumulators, the hyperaccumulating species normally bind metal ions to weak oxygen ligands and use strong ligands only for transient binding during transport to storage sites. Adequate understanding of genetics, biochemistry, and molecular biology of metal accumulation is a prelude to developing transgenics with improved phytoremediation capacity. Current research in plant breeding, genomics, and proteomics suggest promising leads to the creation of “remediation” cultivars. Several transporter genes associated with metal uptake, transport, and accumulation have been identified. Efforts are underway to enhance the phytoextraction capacity of relevant species, not only by using chelating agents but also by attempting hybridization, protoplast fusion, as well as genetic engineering through novel gene transfer, overexpression of genes, and (or) reverse gene insertion, to enhance (i) transpiration rate; (ii) uptake, translocation, and metabolism of metals; (iii) activity of enzymes related to rate-limiting steps; and (iv) transformation of accumulated metal to volatile forms, and (or) silencing gene(s) that encode proteases. Genome evolution in hyperaccumulators needs to be understood through a systematic study of ecological and molecular genomics. Sequencing of a complete genome of hyperaccumulators can help in identifying the promising functional noncoding regions in the genome, thus making the experimental analysis more accurate. In addition to the constitutive overexpression of a single gene, simultaneous expression of several genes in specific cellular components has to be focused. Other areas that require expert attention include identification of metal-transporter proteins and the introduction of genes encoding the metal transporters, overexpression of metallothioneins and phytochelatin synthase, and overproduction of nicotianamine and histidine in plants. A comprehensive study of transgenic gene frequency, covering several plant generations growing on polluted as well as nonpolluted soils, may assess the possibility of gene escape into the environment and its transfer to the microorganisms present in the surroundings. This review attempts not only to collect and collate information available on mechanisms of metal accumulation and detoxification in plants and on the factors affecting the tolerance and phytoextraction capacity of plants but also the strategies that have been or can be devised for raising novel plant genotypes with elevated capacity of metal accumulation and toxicity tolerance.

Biocatalyst development for lactic acid production at acidic pH using inter-generic protoplast fusion

Acid tolerance ofL. delbrueckiiMut Uc-3 has been improved using an inter-generic protoplast fusion approach. The fusant was further treated with UV mutagenesis which generated a mutant with improved lactic acid production in acidic environment.

Development of mitochondrial SNP markers in different Populus species

Key message We aim to develop, for the first time, poplar species-specific mitochondrial SNP markers which can be employed as diagnostic tool, e.g., to identify mitochondria belonging to different poplar species used in protoplast fusion experiments and for population genetics approaches.