Lactulose biosynthesis by β-galactosidase from a newly isolated <Emphasis Type="Italic">Arthrobacter</Emphasis> sp.

Lactulose, a ketose disaccharide, is used in both pharmaceutical and food industries. This study was undertaken to screen and isolate potent β-galactosidase-producing bacteria and to evaluate their enzymatic production of lactulose. Soil samples from fruit gardens were collected. One isolate designated LAS was identified whose cell extract could convert lactose and fructose into lactulose. The 16S rDNA gene analysis of LAS revealed its phylogenetic relatedness to Arthrobacter sp. The β-galactosidase produced by LAS was purified 15.7-fold by ammonium sulfate precipitation and subsequent Phenyl-Sepharose hydrophobic chromatography. The optimum pH and temperature for lactulose synthesis by this β-galactosidase were 6.0 and 20°C, respectively. The low optimum temperature of this enzyme compared to the currently used ones for lactulose production has the advantage of reducing the nonenzymatic browning in biotransformations. The results indicated that Arthrobacter could be used as a novel bacterial β-galactosidase source for lactulose production.     

<Emphasis Type="Italic">Sphingomonas rosea</Emphasis> sp. nov. and <Emphasis Type="Italic">Sphingomonas swuensis</Emphasis> sp. nov., rosy colored <Emphasis Type="Italic">β</Emphasis>-glucosidase-producing bacteria isolated from soil

Two strains PB196^T and PB62^T of Gram-negative, non-motile, and non-spore-forming bacteria, were isolated from soil in South Korea and characterized to determine their taxonomic positions. 16S rRNA gene sequence analysis showed that the two strains belonged to the genus Sphingomonas. The highest degree of sequence similarity of strain PB196^T was found with PB62^T (98.9%), Sphingomonas humi PB323^T (98.9%), Sphingomonas kaistensis PB56^T (98.2%), and Sphingomonas astaxanthinifaciens TDMA-17^T (98.0%). The highest degree of sequence similarity of strain PB62^T was found with Sphingomonas humi PB323^T (98.8%), Sphingomonas astaxanthinifaciens TDMA-17^T (98.2%), and Sphingomonas kaistensis PB56^T (98.1%). Chemotaxonomic data revealed that they possessed ubiquinone-10 (Q-10) as common in the genus Sphingomonas, that the predominant fatty acids were summed feature 7 (C_18:1 ω7c/ω9t/ω12t), summed feature 4 (C_16:1 ω7c/C_15:0 iso 2OH), C_16:0, and C_17:1 ω6c, and that they contained sphingoglycolipid, phosphatidylglycerol (PG), and phosphatidyle-thanolamine (PE) in common but they showed difference for diphosphatidylglycerol (DPG). Based on these data, PB196^T (=KCTC 12339^T =JCM 16604^T) and PB62^T (=KCTC 12336^T =JCM 16605^T =KEMB 9004-005^T) should be classified as type strains of two novel species, for which the names Sphingomonas rosea sp. nov. and Sphingomonas swuensis sp. nov. are proposed, respectively.     

High-level productivity of <Emphasis Type="Italic">α,ω</Emphasis>-dodecanedioic acid with a newly isolated <Emphasis Type="Italic">Candida viswanathii</Emphasis> strain

α,ω-Dicarboxylic acids (DC) are versatile chemical intermediates with different chain lengths, which are well-known as polymer building block. In this work, a new strain with high productivity of DC was isolated from oil-contaminated soil. Based on the morphology and phylogenetic analyses of the internal transcribed spacer sequences, it was characterized as Candida viswanathii. It was found that the contribution of carbon flux to the cell growth and DC production from n-dodecane could be regulated by the sucrose and yeast extract concentrations in the medium, and besides the broth pH, a suitable proportioning of sucrose and yeast extract was the key to achieve the optimal transition from cell growth phase to DC production phase. By optimizing culture conditions in a 7.5-L bioreactor, a higher DC productivity of 1.59 g·L^?1 h^?1 with a corresponding concentration of 181.6 g/L was obtained. After the purification of DC from the culture, the results from gas chromatography–mass spectrometry, infrared spectroscopy and ¹H-NMR showed that α,ω-dodecanedioic acid (DC₁₂) was the major product of C. viswanathii ipe-1 using pure n-dodecane as substrate. For the first time, we reported that a high productivity of DC₁₂ could be produced by C. viswanathii.     

β-Fructofuranosidase production by repeated batch fermentation with immobilized <Emphasis Type="Italic">Aspergillus japonicus</Emphasis>

The fungus Aspergillus japonicus ATCC 20236 was immobilized in vegetal fiber and used in repeated batch fermentations of sucrose (200 g/l) for the production of β-fructofuranosidases (FFase). The assays were performed during eight consecutive cycles that were completed in a total period of 216 h. After each 24-h cycle of fermentation (except for the first cycle, which lasted 48 h), the fermented broth was replaced by fresh medium, and the FFase activity was determined in the replaced medium. The average value of FFase activity was a constant 40.6 U/ml at the end of the initial seven cycles, but had decreased by 22% at the end of the eighth cycle. Concurrent with these high and constant FFase values, the hydrolyzing activity of this enzyme increased during the cycles, while the transfructosylating activity decreased. As a consequence, the maximum production of fructooligosaccharides of 134.60 g/l observed in the initial 30 h of fermentation (first cycle) had gradually decreased by the end of the subsequent cycles, reaching approximately 23% of this value during cycles 4–8. Based on these results, we conclude that the present immobilization system has a great potential for application in a semi-continuous process for the production of FFase, but further studies are necessary to maintain the FFase transfructosylation activity at high levels during the overall process.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

Interaction of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>–<Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>–<Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> and feedstock on fuel ethanol fermentation

The alcoholic fermentation for fuel ethanol production in Brazil occurs in the presence of several microorganisms present with the starter strain of Saccharomyces cerevisiae in sugarcane musts. It is expected that a multitude of microbial interactions may exist and impact on the fermentation yield. The yeast Dekkera bruxellensis and the bacterium Lactobacillus fermentum are important and frequent contaminants of industrial processes, although reports on the effects of both microorganisms simultaneously in ethanolic fermentation are scarce. The aim of this work was to determine the effects and interactions of both contaminants on the ethanolic fermentation carried out by the industrial yeast S. cerevisiae PE-2 in two different feedstocks (sugarcane juice and molasses) by running multiple batch fermentations with the starter yeast in pure or co-cultures with D. bruxellensis and/or L. fermentum. The fermentations contaminated with D. bruxellensis or L. fermentum or both together resulted in a lower average yield of ethanol, but it was higher in molasses than that of sugarcane juice. The decrease in the CFU number of S. cerevisiae was verified only in co-cultures with both D. bruxellensis and L. fermentum concomitant with higher residual sucrose concentration, lower glycerol and organic acid production in spite of a high reduction in the medium pH in both feedstocks. The growth of D. bruxellensis was stimulated in the presence of L. fermentum resulting in a more pronounced effect on the fermentation parameters than the effects of contamination by each microorganism individually.     

Ginsenoside Rd production from the major ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermus caldophilus</Emphasis>

Under optimum conditions (pH 5, 75°C, and 0.2 U purified enzyme ml⁻¹), 4 mg ginsenoside Rd was produced from 5 mg reagent-grade ginsenoside Rb₁ in 5 ml after 30 min by β-glucosidase from Thermus caldophilus GK24. Using a ginseng root extract containing 1 mg ginsenoside Rb₁ ml⁻¹ and 3.2 mg additional ginsenosides ml⁻¹, 1.23 mg ginsenoside Rd ml⁻¹ was produced after 18 h; the concentrations of ginsenosides Rb₁, Rb₂, and Rc used for ginsenoside Rd production were 0.77, 0.17, and 0.19 mg ml⁻¹, respectively.     

<Emphasis Type="Italic">Acinetobacter kyonggiensis</Emphasis> sp. nov., a <Emphasis Type="Italic">β</Emphasis>-glucosidase-producing bacterium,isolated from sewage treatment plant

A Gram-negative, non-motile bacterium, designated KSL5401-037^T, was isolated from a sewage treatment plant in Gwangju in the Republic of Korea and was characterized using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that strain KSL5401-037^T belonged to the genus Acinetobacter in the family Moraxellaceae of the Gammaproteobacteria (Brisou and Prevot, 1954). According to a 16S rRNA gene sequence analysis, it was closely related to Acinetobacter johnsonii ATCC 17909^T (97.3%), A. bouvetii 4B02^T (97.2%), and A. beijerinckii 58a^T (96.8%). Chemotaxonomic data revealed that strain KSL5401-037^T possesses an ubiquinone system with Q-8 as the predominant compound and C_16:0 (19.2%), C_18:1 ω9c (19.5%), and summed feature 3 (C_16:1 ω6c / C_16:1 ω7c, 34.1%) as the predominant cellular fatty acids. The major polar lipids detected in strain KSL5401-037^T were diphosphatidylglycerol (DPG) and, phosphatidylethanolamine (PE), followed by phosphatidylglycerol (PG) and moderate amounts of phosphatidylcholine and phosphatidylserine. The G+C content of the genomic DNA was 41.2–42.1 mol%. Strain KSL5401-037^T exhibited relatively low levels of DNA-DNA relatedness with respect to A. johnsonii DSM 6963^T (17.7%) and A. bouvetii 4B02^T (9.3%). The DNA-DNA relatedness values, biochemical, and physiological characteristics of strain KSL5401-037^T strongly support its genotypic and phenotypic differentiation from other recognized type strains of the genus Acinetobacter. Based on these data, strain KSL5401-037^T (JCM 17071^T =KEMC 5401-037^T) should be classified in the genus Acinetobacter as a type strain of novel species, for which the name Acinetobacter kyonggiensis sp. nov. is proposed.     

Bioindicator production with <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis>’ thermal-resistant spores cultivated by solid-state fermentation

Bacillus atrophaeus’ spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost–productivity relation. Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental designs and response surface methodology in which the influence of different variables—particle size, moisture content, incubation time, pH, inoculum size, calcium sources, and medium composition—was studied. The results have suggested that soybean molasses and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time. Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%), initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the control and no significant difference in dry heat resistance was observed.     

Enantioselective biocatalytic hydrolysis of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-mandelonitrile for production of (<Emphasis Type="Italic">R</Emphasis>)-(−)-mandelic acid by a newly isolated mutant strain

(R)-(−)-Mandelic acid (R-MA) is an important intermediate with broad uses. Recently, R-MA production using nitrilase has been gaining more and more attention due to its higher productivity and enantioselectivity. In this work, a new bacterium WT10, which exhibited favorable nitrilase activity and excellent enantioselectivity for production of R-MA by enantioselective biocatalytic hydrolysis of (R,S)-mandelonitrile, was isolated and identified as a strain of Alcaligenes faecalis. In order to improve its nitrilase activity for industrial application, the wild-type strain WT10 was further subjected to mutagenesis using a combined LiCl–ultraviolet irradiation and low energy N⁺ ion beams implantation technique. A valuable mutant strain A. faecalis ZJUTB10 was obtained. The nitrilase specific activity of the mutant strain was greatly improved up to 350.8 U g⁻¹, in comparison with wild-type strain WT10 of 53.09 U g⁻¹. The reaction conditions for R-MA production by mutant strain A. faecalis ZJUTB10 were also optimized. Nitrilase activity in mutant strain showed a broad pH optimum at pH 7.7–8.5. The optimal temperature was 35°C. The highest production rate reached 9.3 mmol h⁻¹ g⁻¹. The results showed that mutant strain A. faecalis ZJUTB10 was a new candidate for efficient R-MA production from (R,S)-mandelonitrile and could potentially be used in industrial production.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.     

Improved cider fermentation performance and quality with newly generated <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> × <Emphasis Type="Italic">Saccharomyces eubayanus</Emphasis> hybrids

Yeast cryotolerance may be advantageous for cider making, where low temperatures are usually employed. Here, we crossed the cryotolerant S. eubayanus with a S. cerevisiae wine strain and assessed the suitability of the hybrids for low-temperature cider fermentation. All strains fermented the juice to 5% ABV, but at different rates; hybrid strains outperformed S. cerevisiae, which was sensitive to low temperatures. The best hybrid fermented similarly to S. eubayanus. S. eubayanus produced sulphurous off flavours which masked a high concentration of fruity ester notes. This phenotype was absent in the hybrid strains, resulting in distinctly fruitier ciders. Aroma was assessed by an independent consumer panel, which rated the hybrid ciders as identical to the wine strain cider. Both were significantly more pleasant than the S. eubayanus cider. Interspecific hybridization can apparently be used effectively to improve low-temperature fermentation performance without compromising product quality.     

Establishment of <Emphasis Type="Italic">Miscanthus sinensis</Emphasis> with decreased lignin biosynthesis by <Emphasis Type="Italic">Agrobacterium</Emphasis>–mediated transformation using antisense <Emphasis Type="Italic">COMT</Emphasis> gene

This study was to determine a transformation system for Miscanthus sinensis, and to optimize factors and conditions required for expression of an antisense caffeic acid O-methyltransferase gene in the M. sinensis (MsCOMT-AS). Transformation of callus derived from seeds and immature inflorescences of M. sinensis was established by using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish the stable transformation system, several transformation factors such as explant type, strain, co-culture periods, acetosyringone concentration, and selective markers were assessed. In this study, seven putative transgenic plants were obtained from callus transformation and plantlet regeneration. Various tests including PCR analysis and RT-PCR were used to detect the transgenic insert. The transgenic plants were also characterized for their agronomic and morphological characteristics, expression of MsCOMT-AS gene, and variation in lignocellulosic content. Biomass related traits such as plant height, number of leaves, length of leaf, stem diameter, fresh weight, dry weight, and cell size of the control plants were superior to transgenic plants. Total lignin content of transgenic plants was lower than that of the control plant due to reduced caffeic acid O-methyltransferase (COMT) gene expression related to lignin production. Cellulose and hemicellulose content in transgenic plants were not increased. Variation in cellulose and hemicellulose content had no correlation with variation in lignin content of transgenic plants. In conclusion, transgenic M. sinensis was obtained with down-regulated COMT gene. Lignin synthesis was decreased what offers possibility of crop modification for facilitated biofuel production.     

AFLP and SSR polymorphism in a <Emphasis Type="Italic">Coffea</Emphasis> interspecific backcross progeny [(<Emphasis Type="Italic">C. heterocalyx</Emphasis> × <Emphasis Type="Italic">C. canephora</Emphasis>) × <Emphasis Type="Italic">C. canephora</Emphasis>]

An interspecific cross (BC 1) involving a species with one of the largest genomes in the Coffea genus [Coffea heterocalyx (HET), qDNA = 1.74 pg] and a species with a medium-sized genome [Coffea canephora (CAN), qDNA = 1.43 pg] was studied using two types of molecular markers, AFLP and SSR. One hundred and eighty eight AFLP bands and 34 SSR primer pairs were suitable for mapping. The total map length was 1,360 cM with 190 loci distributed in 15 linkage groups. The results were compared to those obtained previously on an interspecific BC 1 progeny involving a species with a medium-sized genome (Coffea liberica var dewevrei, DEW) and a species with one of the smallest genomes (Coffea pseudozanguebariae, PSE). They are discussed relative to three main points: (1) the relevance of the different marker types, (2) the genomic distribution of AFLP and SSR markers, and (3) the relation between AFLP polymorphism and genome size.Communicated by H.F. Linskens     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Enhanced hybridization rates in a <Emphasis Type="Italic">Larix gmelinii</Emphasis> var. <Emphasis Type="Italic">japonica</Emphasis> × <Emphasis Type="Italic">L. kaempferi</Emphasis> interspecific seed orchard with a single maternal clone revealed by cytoplasmic DNA markers

We identified diagnostic chloroplast DNA and mitochondrial DNA markers that can (a) discriminate between Larix gmelinii var. japonica and L. kaempferi, and (b) determine the maternal and paternal species of hybrids between them by exploiting the difference in inheritance mode between the two genomes. We also investigated the hybridization rates at a site with two types of interspecific seed orchard—a new type with rows of a single maternal clone of L. gmelinii var japonica among rows of L. kaemferi and a “traditional” type with multiple, intimately mixed clones—in 2 years using chloroplast diagnostic DNA markers. The average hybridization rates in the single maternal clone (SMC) interspecific seed orchards [84.2% (±9.4%) in 2004 and 94.1% (±3.9%) in 2005) were higher than that in the traditional interspecific seed orchards [15.9% (±13.4%) in 2004 and 30.0% (±25.5%) in 2005] because of the self-incompatibility of the L. gmelinii var. japonica clone. We detected significant differences in hybridization rates between the orchard types in both investigated years (P < 0.001, analysis of variance, ANOVA). This finding suggests that SMC interspecific seed orchards can reliably provide seeds with high proportions of hybrids. In the traditional interspecific seed orchard, there were significant differences in the proportions of hybrids among L. gmelinii var. japonica seeds between the two years (P < 0.005, ANOVA), which may have been partly due to differences in the relative amounts of pollen cones produced by L. gmelinii var. japonica and L. kaempferi.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

Statistical optimization of <Emphasis Type="Italic">α</Emphasis>-amylase production by <Emphasis Type="Italic">Bacillus brevis</Emphasis> MTCC 7521 in solid-state fermentation using cassava bagasse

Production of α-amylase under solid-state fermentation by Bacillus brevis MTCC 7521 has been investigated using cassava bagasse as the substrate, one of the major solid wastes released during extraction of starch from cassava (Manihot esculenta). Response surface methodology was used to evaluate the effect of the main variables, i.e. incubation period (36 h), moisture holding capacity (60%), pH (7.0) and temperature (60°C) on enzyme production by applying a full factorial central composite design. The maximum hydrolysis of soluble starch (85%) and cassava starch (75%) was obtained with the application of 4 mL (≈ 14,752 units) of B. brevis crude enzyme after 5 h of incubation.     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.