High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

Control of <Emphasis Type="Italic">Bemisia tabaci</Emphasis> and <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> in cucumber by <Emphasis Type="Italic">Amblyseius swirskii</Emphasis>

Bemisia tabaci Genn. (Hemiptera: Aleyrodidae) and Frankliniella occidentalis (Thysanoptera: Thripidae) are major pests in greenhouse grown cucumber crops. Recently, Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) was shown an effective biological control agent of both pests. Hence, perhaps both pests can be controlled simultaneously by this predator. However, with simultaneous infestation of both pests, synergistic effects, or interference could affect biological control and perhaps require changes in release rates of the predator. Thus, the aim of the present study was to evaluate different release rates of A. swirskii to control both pests under a worst case scenario of rapid immigration into a cucumber greenhouse. Two experiments were conducted, one simulating the influx of whiteflies alone (whitefly experiment) and the other immigration of whiteflies and thrips together (whitefly plus thrips experiment). Three treatments were compared in the whitefly experiment: (1) B. tabaci alone, (2) B. tabaci + 25 A. swirskii m⁻² and (3) B. tabaci + 75 A. swirskii m⁻². The high release rate was more effective than the low rate in controlling B. tabaci alone. The high rate was subsequently tested against B. tabaci and F. occidentalis for the whitefly and thrips experiment in which five treatments were compared: (1) B. tabaci alone, (2) F. occidentalis alone, (3) B. tabaci + 75 A. swirskii m⁻², (4) F. occidentalis + 75 A. swirskii m⁻² and (5) B. tabaci + F. occidentalis + 75 A. swirskii m⁻². This rate of A. swirskii controlled whiteflies and thrips either alone or together. Therefore, 75 A. swirskii m⁻² should be an adequate rate for controlling both pests either alone or simultaneously in cucumber greenhouses.     

Genetic analysis of loci associated with partial resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Sclerotinia stem rot is the most devastating disease of rapeseed (Brassica napus L.) in China. Quantitative trait loci (QTLs) involved in resistance to Sclerotinia sclerotiorum were detected in a rapeseed population of 128-F(2:3) families derived from a cross between the male sterility restorer line H5200 and a partial resistant line Ning RS-1. A total of 107 molecular markers including 72 RFLPs, 30 AFLPs, 3 SSRs and 2 RAPDs were employed to construct a genetic linkage map with 23 linkage groups covering 1,625.7 cM with an average space of 15.2 cM. Resistance was assessed empirically at two developmental stages: with a detached leaf inoculation at the seedling stage and in vivo stem inoculation at the mature plant stage. The observed resistance was scored for each plant as leaf resistance at the seedling stage (LRS) and stem resistance at the mature plant stage (SRM). A total of 13 loci were identified by one-way ANOVA and six QTLs were detected with MapMaker-QTL. We found that three of the six QTLs were associated with leaf resistance at the seedling stage and collectively accounted for 40.7% of the total phenotypic variation, each accounting for 23.2%, 16.6% and 13.6% respectively. Three QTLs were found corresponding to the disease resistance at the mature plant stage, explaining 49.0% of the phenotypic variation. Epistasis was observed for the resistance and the additive by additive interactions were the predominant type of epistasis. It was concluded that both single-locus QTLs and epistatic interactions played important roles in Sclerotinia resistance in rapeseed.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Compatibility of <Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> as dual inoculants in black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

The compatibility of two biological inoculants, Trichoderma harzianum, a mycoparasitic biological control fungus and Piriformospora indica, a root colonizing plant-growth promoting endophytic fungus was evaluated using tissue cultured black pepper plantlets. We report, for the first time, the ability of P. indica to colonize black pepper, a perennial climber. T. harzianum inhibited the growth of P. indica in an in vitro dual culture plate assay. Simultaneous inoculation with both biological inoculants of tissue cultured black pepper plantlets negatively influenced root colonization by P. indica. However, when P. indica was applied initially followed 30 days later by T. harzianum, there was increased root colonization by the root endophyte P. indica and beneficial effects were found on the growth of the black pepper plants. The present study also showed that the efficacy of inoculation of the two fungal biological agents can be increased by sequential application of P. indica at the hardening stage followed by T. harzianum during transplanting into a soil-sand mixture.     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) with resistance to the mustard aphid (<Emphasis Type="Italic">Lipaphis erysimi</Emphasis> Kalt.)

. Wheat germ agglutinin (WGA), the chitin-binding lectin from wheat germ, has been shown to be antimetabolic, antifeedant and insecticidal to the mustard aphid (Lipaphis erysimi. Kalt). A cDNA encoding WGA was transferred to Indian mustard (Brassica juncea cv. RLM-198) through Agrobacterium-mediated transformation. Southern analysis of the transgenics showed the integration of the transgene, while Northern and Western analyses demonstrated that the transgene was expressed in the transgenics. Bioassays using leaf discs showed that feeding on transgenics induced high mortality and significantly reduced fecundity of aphids.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of gypsophila (<Emphasis Type="Italic">Gypsophila paniculata</Emphasis> L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA₃-pretreated mother plants and a two-step selection scheme. The GA₃ treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.     

Exploring the quantitative resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">phaseolicola</Emphasis> in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Pseudomonas syringae pv. phaseolicola is an important disease that causes halo blight in common bean. The genetic mechanisms underlying quantitative halo blight resistance are poorly understood in this species, as most disease studies have focused on qualitative resistance. The present work examines the genetic basis of quantitative resistance to the nine halo blight races in different organs (primary and trifoliate leaf, stem and pod) of an Andean recombinant inbred line (RIL) progeny. Using a multi-environment quantitative trait locus (QTL) mapping approach, 76 and 101 main-effect and epistatic QTLs were identified, respectively. Most of the epistatic interactions detected were due to loci without detectable QTL additive main effects. Main and epistatic QTLs detected were mainly consistent across the environment conditions. The homologous genomic regions corresponding to 26 of the 76 main-effect detected QTLs were positive for the presence of resistance-associated gene cluster encoding nucleotide-binding and leucine-rich repeat (NL) proteins and known defence genes. Main-effect QTLs for resistance to races 3, 4 and 5 in leaf, stem and pod were located on chromosome 2 within a 3.01-Mb region, where a cluster of nine NL genes was detected. The NL gene Phvul.002G323300 is located in this region, which can be considered an important putative candidate gene for the non-organ-specific QTL identified here. The present research provides essential information not only for the better understanding of the plant-pathogen interaction but also for the application of genomic assisted breeding for halo blight resistance in common bean.     

Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">S</Emphasis>-Allele characterization in self-incompatible pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.)

Gametophytic self-incompatibility, a natural mechanism occurring in pear and other fruit-tree species, is usually controlled by the S-locus with allelic variants ( S1, S2, Sn). Recently, biochemical and molecular tools have determined the S-genotype of cultivars in various species. The present study determined the S-locus composition of ten European pear cultivars via S-PCR molecular assay, thereby obviating time-consuming fieldwork whose results are often ambiguous because of environmental effects. To verify the S-PCR assay, two putative S-allele DNA fragments of Japanese pear were isolated; their sequences proved to be identical to those reported in the databank. Six S-allele fragments of European pear were then sequenced. While field data confirmed the molecular results, fully and half-compatible field crosses were not distinguishable.     

Evidence of intralocus recombination at the <Emphasis Type="Italic">Glu-3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

Recombination at the Glu-3 loci was identified, and strong genetic linkage was observed only between the amplicons representing i-type and s-type genes located, respectively, at the Glu-A3 and Glu-B3 loci.

Abstract

The low-molecular weight glutenin subunits (LMW-GSs) are one of the major components of wheat seed storage proteins and play a critical role in the determination of wheat end-use quality. The genes encoding this class of proteins are located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3). Due to the complexity of these chromosomal regions and the high sequence similarity between different LMW-GS genes, their organization and recombination characteristics are still incompletely understood. This study examined intralocus recombination at the Glu-3 loci in two recombinant inbred line (RIL) and one doubled haploid (DH) population, all segregating for the Glu-A3, Glu-B3, and Glu-D3 loci. The analysis was conducted using a gene marker system that consists of the amplification of the complete set of the LMW-GS genes and their visualization by capillary electrophoresis. Recombinant marker haplotypes were detected in all three populations with different recombination rates depending on the locus and the population. No recombination was observed between the amplicons representing i-type and s-type LMW-GS genes located, respectively, at the Glu-A3 and Glu-B3 loci, indicating tight linkage between these genes. Results of this study contribute to better understanding the genetic linkage and recombination between different LMW-GS genes, the structure of the Glu-3 loci, and the development of more specific molecular markers that better represent the genetic diversity of these loci. In this way, a more precise analysis of the contribution of various LMW-GSs to end-use quality of wheat may be achieved.

     

Genetic mapping of stem rust resistance gene <Emphasis Type="Italic">Sr13</Emphasis> in tetraploid wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis> L.)

Wheat stem rust caused by Puccinia graminis f. sp. tritici, can cause significant yield losses. To combat the disease, breeders have deployed resistance genes both individually and in combinations to increase resistance durability. A new race, TTKSK (Ug99), identified in Uganda in 1999 is virulent on most of the resistance genes currently deployed, and is rapidly spreading to other regions of the world. It is therefore important to identify, map, and deploy resistance genes that are still effective against TTKSK. One of these resistance genes, Sr13, was previously assigned to the long arm of chromosome 6A, but its precise map location was not known. In this study, the genome location of Sr13 was determined in four tetraploid wheat (T. turgidum ssp. durum) mapping populations involving the TTKSK resistant varieties Kronos, Kofa, Medora and Sceptre. Our results showed that resistance was linked to common molecular markers in all four populations, suggesting that these durum lines carry the same resistance gene. Based on its chromosome location and infection types against different races of stem rust, this gene is postulated to be Sr13. Sr13 was mapped within a 1.2–2.8 cM interval (depending on the mapping population) between EST markers CD926040 and BE471213, which corresponds to a 285-kb region in rice chromosome 2, and a 3.1-Mb region in Brachypodium chromosome 3. These maps will be the foundation for developing high-density maps, identifying diagnostic markers, and positional cloning of Sr13.     

Genetic mapping of <Emphasis Type="Italic">psl</Emphasis> locus and quantitative trait loci for angular leaf spot resistance in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

One of the most important cucumber diseases is bacterial angular leaf spot (ALS), whose increased occurrence in open-field production has been observed over the last years. To map ALS resistance genes, a recombinant inbred line (RIL) mapping population was developed from a narrow cross of cucumber line Gy14 carrying psl resistance gene and susceptible B10 line. Parental lines and RILs were tested under growth chamber conditions as well as in the field for angular leaf spot symptoms. Based on simple sequence repeat and DArTseq, genotyping a genetic map was constructed, which contained 717 loci in seven linkage groups, spanning 599.7 cM with 0.84 cM on average between markers. Monogenic inheritance of the lack of chlorotic halo around the lesions, which is typical for ALS resistance and related with the presence of recessive psl resistance gene, was confirmed. The psl locus was mapped on cucumber chromosome 5. Two major quantitative trait loci (QTL) psl5.1 and psl5.2 related to disease severity were found and located next to each other on chromosome 5; moreover, psl5.1 was co-located with psl locus. Identified QTL were validated in the field experiment. Constructed genetic map and markers linked to ALS resistance loci are novel resources that can contribute to cucumber breeding programs.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)₃AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l^–1 thidiazuron, 0.5 mg l^–1 -naphthaleneacetic, 10 mg l^–1 kanamycin (Km), and 250 mg l^–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m^–2 s^–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.