In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

High frequency <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated plant transformation induced by ammonium nitrate

Success in plant genetic transformation depends on the efficiency of explant regeneration and transgene integration. Whereas the former one depends on explant totipotency, the latter depends on the activity of host DNA repair and chromatin organisation factors. We analyzed whether factors that result in an increase in recombination frequency can also increase transformation efficiency. Here, we report that a threefold increase in the concentration of NH₄NO₃ in the growth medium results in more than a threefold increase in the Agrobacterium tumefaciens-mediated transformation frequency of Nicotiana tabacum plants. Regeneration of calli without selection showed that the increase in transformation frequency was primarily due to the increase in transgene integration efficiency rather than in tissue regeneration efficiency. PCR analysis of insertion sites showed a decrease in the frequency of truncations of the T-DNA right border and an increase on the left border. We hypothesize that exposure to ammonium nitrate modifies the activity of host factors leading to higher frequency of transgene integrations and possibly to the shift in the mechanism of transgene integrations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Improving transformation efficiency of<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by modifying the floral dip method

Arabidopsis thaliana transformation with the floral dip method was improved by modifying the cell density and mode of application of theAgrobacterium inoculum. Drops of inoculum were applied 4 times to closed flower buds. The inoculum OD₆₀₀ was increased from 0.8 to more than 2.0. These modifications improved reproducibility and increased transformation efficiencies to 2–3%.     

A stable and reproducible transformation system for the wetland monocot <Emphasis Type="Italic">Juncus accuminatus</Emphasis> (bulrush) mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

A highly reproducible Agrobacterium-mediated transformation system was developed for the wetland monocot Juncus accuminatus. Three Agrobacterium tumefaciens binary plasmid vectors, LBA4404/pTOK233, EHA105/pCAMBIA1201, and EHA105/pCAMBIA1301 were used. All vectors contained the 35SCaMV promoter driven, intron containing, β-glucuronidase (gus), and hygromycin phosphotransferase (hptII) genes within their T-DNA. After 48 h of cocultivation, 21-d-old seedling derived calli were placed on medium containing timentin at 400 mg l⁻¹, to eliminate the bacteria. Calli were selected on MS medium containing 40 or 80 mg l⁻¹ hygromycin, for 3 mo. Resistant calli were regenerated and rooted on MS medium containing hygromycin, 5 mg l⁻¹(22.2 μM) of 6-benzylamino-purine (BA) and 0.1 mg l⁻¹(0.54 μM) of alpha-naphthaleneacetic acid (NAA), respectively. Seventy-one transgenic cell culture lines were obtained and 39 plant lines were established in the greenhouse. All the plants were fertile, phenotypically normal, and set viable seed. Both transient and stable expression of the gus gene were demonstrated by histochemical GUS assays of resistant calli, transgenic leaf, root, inflorescence, seeds, and whole plants. The integration of gus and hptII genes were confirmed by polymerase chain reaction (PCR) and Southern analysis of both F₀ and F₁ progenies. The integrated genes segregated to the subsequent generation in Mendelian pattern. To our knowledge, this is the first report of the generation of transgenic J. accuminatus plants.     

Marker-assisted pyramiding of two cereal cyst nematode resistance genes from <Emphasis Type="Italic">Aegilops variabilis</Emphasis> in wheat

The cereal cyst nematode (CCN) Heterodera avenae, is a significant pathogen of wheat. The wild grass Aegilops variabilis Accession No.1 has been found to be resistant to pathotypes of CCN; at least two genes transferred to wheat, designated as CreX and CreY, are involved in the resistance response. The CreY gene may be the same as Rkn-mn1, which confers resistance to root knot nematode (RKN) Meloidogyne naasi. The objective of this work was to pyramid the two CCN resistance genes in a wheat background through marker-assisted selection. As a first step, molecular markers flanking CreX were identified. The completely linked RAPD marker of Rkn-mn1 (CreY), OpY16-_1065, previously obtained, was converted into a SCAR. All these dominant markers were used to incorporate in the same genotype the two Ae. variabilis chromosome segments carrying the two genes for resistance. CCN bioassays with the Ha12 pathotype showed that the level of resistance of the pyramided line was significantly higher than that of CreX and CreY single introgression lines, but lower than that of Ae. variabilis. This study thus illustrates the utilization of molecular markers in breeding for host resistance.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Genetic transformation and regeneration of transgenic plants from precultured cotyledon and hypocotyl explants of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis> Sm. using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient transformation protocol was developed for Eucalyptus tereticornis Sm. using cotyledon and hypocotyl explants. Precultured cotyledon and hypocotyl explants were cocultured with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the uidA and neomycin phosphotransferase II genes for 2 d and transferred to selective regeneration medium containing 0.5 mg/l 6-benzylaminopurine (BAP), 0.1 mg/l naphthalene acetic acid, 40 mg/l kanamycin, and 300 mg/l cefotaxime. After two passages in the selective regeneration medium, the putatively transformed regenerants were transferred to Murashige and Skoog (MS) liquid medium containing 0.5 mg/l BAP and 40 mg/l kanamycin on paper bridges for further development and elongation. The elongated kanamycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1.0 mg/l indole-3-butyric acid and 40 mg/l kanamycin. A strong β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. This protocol allows effective transformation and direct regeneration of E. tereticornis Sm.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

The transformation of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): applicable methods of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated gene transfer

Three methods of transformation of pea (Pisum sativum ssp. sativum L. var. medullare) were tested. The most efficient Agrobacterium tumefaciens-mediated T-DNA transfer was obtained using embryonic segments from mature pea seeds as initial explants. The transformation procedure was based on the transfer of the T-DNA region with the reporter gene uidA and selection gene bar. The expression of β-glucuronidase (GUS) in the regenerated shoots was tested using the histochemical method and the shoots were selected on a medium containing phosphinothricin (PPT). The shoots of putative transformants were rooted and transferred to non-sterile conditions. Transient expression of the uidA gene in the tissues after co-cultivation and in the course of short-term shoot cultivation (confirmed by histochemical analysis of GUS and by RT-PCR of mRNA) was achieved; however, we have not yet succeeded in proving stable incorporation of the transgene in the analysed plants.     

Efficient<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-mediated transformation of soybeans using an embryonic tip regeneration system

Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans [Glycine max (L.) Merr.]. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l^–1 N⁶-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l^–1 BAP and 0.2 mg l^–1 indolebutyric acid. Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials. Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8%. These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation.Abbreviations GUS -Glucuronidase - T-DNA Transferred DNA - BAP N⁶-Benzylaminopurine - IBA Indolebutyric acid     

Overexpression of <Emphasis Type="Italic">WUSCHEL</Emphasis> in <Emphasis Type="Italic">C. chinense</Emphasis> causes ectopic morphogenesis

Capsicum chinense is a recalcitrant species for in vitro morphogenesis, and up to date there is no efficient system for genetic transformation and regeneration of this species via somatic embryogenesis. Here, we carried out an in vitro transformation of C. chinense via Agrobacterium tumefaciens co-cultivation with a system that expresses the heterologous gene WUSCHEL from Arabidopsis thaliana. WUSCHEL has been shown to promote the transition from vegetative to embryogenic state when overexpressed. We tested if the expression of WUSCHEL in C. chinense would promote an embryogenic response in this species. After 15 days of induction, the segments of transformed stems begun to form globular structures, suggesting that heterologus WUSCHEL was active and involved in the process of morphogenesis.     

Decolourisation and dephenolisation potential of selected <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> strains – <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

Influence of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> strain on the production of transgenic peas (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

We compared the efficiency of two Agrobacterium tumefaciens strains, AGL 1 and KYRT1, for producing transgenic pea plants. KYRT1 is a disarmed strain of Chry5 that has been shown to be highly tumourigenic on soybean. The efficacies of the strains were compared using cotyledon explants from three pea genotypes and two plasmids. The peas were sourced from field-grown plants over three Southern Hemisphere summer seasons. Overall, KYRT1 was found to be on average threefold more efficient than AGL 1 for producing transgenic plants. We suggest that KYRT1 is sensitive to cocultivation temperature as the expected increase in efficiency was not achieved at high laboratory temperatures.Communicated by P. Debergh     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

Influence of the<Emphasis Type="Italic">SMT2</Emphasis> knock-out on hypocotyl elongation in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Inhibitors are very important in the study of hormone function. Brasinazole (Brz) is a specific inhibitor of brassinosteroids (BRs) biosynthesis. To expand our knowledge of the molecular mechanisms of plant steroid signaling, we performed genetic screening using medium containing Brz under dark conditions. Mutants insensitive to Brz developlonger hypocotyls than their wild type counterparts. We isolatedabz453 as a Brz insensitive mutant. TAIL-PCR and the segregation ratio of T2 plants indicated a single T-DNA insertion at the 24-Sterol C-methyltransferase (SMT2) gene in theabz453 mutant. Recapitulation for putative FCP serine phosphatase (FSP), the gene neighboringSMT2, indicated no significant phenotypes, but theSMT2 anti-sense (SMT2-AS) line developed longer hypocotyls than the wild type in medium containing Brz. Additionally, theSMT2-AS line displayed similar phenotypes to theabz453 line in soil including enhanced growth and smaller silique. Theabz453 andSMT2-AS mutants showed phenotypes similar to those of wild type in medium containing benzylaminopurine, pacrobutrazol and ACC (precursor for ethylene) under dark conditions. However, when brassinolide (BL) dose response was observed, theabz453 andSMT2-AS lines showed higher sensitivity than wild type. Theabz453/det2 andabz453/bri1-119 double mutants showed enhanced growth compared to thedet2 andbri1-119 line under both dark and light conditions. Specially, in dark conditions double mutants displayed nearly 2- and 1.5-fold longer hypocotyls thandet2 andbri1-119 plants. Brz insensitivity to theSMT2 knock-out mutant and phenotypes of double mutants indicate that not only do BRI1 and DET2 influence the BRs response, as evidenced by hypocotyl elongation, but another sterol derived signals may also be affected in mutants, suggesting that another pathway is involved in hypocotyl elongation due to SMT2.     

Identification of SNPs and development of functional markers for LMW-GS genes at <Emphasis Type="Italic">Glu-D3</Emphasis> and <Emphasis Type="Italic">Glu-B3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GS) have great effect on wheat processing quality, but were numerous and difficult to dissect by SDS-PAGE. The development of functional markers may be the most effective way for a clear discrimination of different LMW-GS genes. In the present study, three different approaches were used to identify SNPs of different genes at Glu-D3 and Glu-B3 loci in bread wheat for the development of six STS markers (3 for Glu-D3 and 3 for Glu-B3 genes) that were validated with distinguished wheat cultivars. Firstly, seven LMW-GS gene sequences ( AY585350, AY585354, AY585355, AY585356, AY585349, AY585351 and AY585353 ) from Aegilops tauschii, the diploid donor of the D-genome of bread wheat, were chosen to design seven pairs of AS-PCR primers for Glu-D3 genes. By amplifying the corresponding genes from five bread wheat cultivars with different Glu-D3 alleles (a, b, c, d and e) and Ae. tauschii, a primer set, S13F2/S13R1, specific to the gene AY585356, was found to be positive to cultivars with alleles Glu-D3c and d. Nevertheless, the other five pairs of primers designed from AY585350, AY585349, AY585353, AY585354 and AY585355, respectively, did not produce specific PCR products to the cultivars tested. Secondly, all the PCR products from the five primer sets without specific characteristics were sequenced and an SNP from the gene AY585350 was detected in the cultivar Hartog, which resulted in the second STS marker S1F1/S1R3 specific to the allelic variant of AY585350. Thirdly, three Glu-D3 sequences (AB062851, AB062865 and AB062872) and three Glu-B3 sequences (AB062852, AB062853 and AB062860) defined by Ikeda et al. (2002) were chosen to query wheat EST and NR databases, and DNA markers were developed based on the putative SNPs among the sequences. Using this approach, four STS markers were developed and validated with 16-19 bread wheat cultivars. The primer set T1F4/T1R1 was also a Glu-D3 gene-specific marker for AB062872, while T2F2/T2R2, T5F3/T5R1 and T13F4/T13R3 were all Glu-B3 gene specific markers for AB062852, BF293671 and AY831800, respectively. The chromosomal locations of the six markers were verified by amplifying the genomic DNA of Ae. tauschii (DD), T. monococcum (AA) and T. turgidum (AABB) entries, as well as Chinese Spring and its group 1 chromosome nulli-tetrasomic lines. The results are useful to discriminate the corresponding Glu-D3 and Glu-B3 genes in wheat breeding programs.     

A new name in <Emphasis Type="Italic">Pavetta</Emphasis> L. (<Emphasis Type="Italic">Rubiaceae</Emphasis>)

Summary  The name Pavetta modesta (Hiern) S. E. Dawson is a later homonym of P. modesta Bremek. Pavetta crystalensis is proposed as a new name.