Improvement of efficiency of genetic transformation for <Emphasis Type="Italic">Dunaliella salina</Emphasis> by glass beads method

Dunaliella salina has been exploited as a new type of bioreactor due to its unique advantages. However, this bioreactor application was restricted for absence of a high-efficiency and stable transformation method at present. In the present study, the cells of D. salina were transformed by glass beads. The results of histochemical staining revealed that the GUS gene was successfully expressed in the positive transformants, and PCR and PCR-Southern blot analysis further demonstrated that the bar gene was integrated into the D. salina genome. Moreover, the three transformation methods, including glass beads, bombardment particle and electroporation, were compared for screening a high-efficiency transformation method for gene engineering of D. salina. The results showed that transformation efficiency of the glass beads was the highest, approximately 10² transformants/μg DNA. It is concluded that the established glass beads method has been demonstrated to be an optimal transformation way for D. salina.     

Stable transformation of petunia plastids

Plastid transformation results in stably expressed foreign genes, which for most Angiosperms are largely excluded from sperm cells, thereby greatly reducing the risk of foreign gene spread through pollen. Prior to this work, fertile plastid transformants were restricted to tobacco, tomato and Lesquerella . Application of plastid engineering in the important floriculture industry requires the development of stable plastid transformation in a major ornamental plant species such as Petunia hybrida. Here we describe the successful isolation of fertile and stable plastid transformants in a commercial cultivar of P. hybrida (var. Pink Wave). Plastid targeting regions from tobacco were used to integrate aad A and gusA between the acc D and rbc L genes of P. hybrida plastid DNA following particle bombardment of leaves. For three spectinomycin and streptomycin resistant lines, DNA blot analysis confirmed transgene integration into plastid DNA and homoplasmy. Maternal inheritance and homoplasmy resulted in 100 transmission of spectinomycin resistance to progeny after selfing. Plastid transformants expressed the gusA gene uniformly within leaves and to comparable levels in all three lines. Insertion of trait genes in place of gusA coding sequences enables immediate applications of our plastid transformation vector. Establishment of plastid transformation in P. hybrida facilitates a safe and reliable use of this important ornamental plant for research and plant biotechnology.These two authors contributed equally to this work.     

DNA-mediated transformation system in a bipolar basidiomycete, <Emphasis Type="Italic">Pholiota microspora</Emphasis> (<Emphasis Type="Italic">P. nameko</Emphasis>)

We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was about 88.8 transformants/μg pMBsip2 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation was about 122.4 transformants/μg pMBhph1 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.     

High frequency transformation of the industrial erythromycin-producing bacterium Saccharopolyspora erythraea

The DNA transformation in the industrial erythromycin-producing Saccharopolyspora erythraea was investigated as standard protoplast transformation methods are ineffective. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies from 0.05 × 10⁻⁸ to 7.2 × 10⁻⁸ exconjugants generated per recipient. Electroporation-mediated methodologies were also established. More than 10⁵ transformants were acquired per μg DNA. The proposed protocol provides an alternative route for the introduction of DNA into industrial strains.     

<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.     

Development of a system for integrative and stable transformation of the zygomycete<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis> by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated DNA transfer

Two transformation systems, based on the use of CaCl₂/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae. Irrespective of the selection marker used, a pyr4 marker derived from R. niveus or a dominant amdS⁺ marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl₂/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA. Furthermore, these transformants displayed low mitotic stability. In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions. Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R. oryzae at a single locus in independently obtained transformants. In addition, truncation of the transforming DNA was observed, resulting in the integration of the R. niveus pyr4 marker gene, but not the second gene located on the transferred DNA. Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl₂/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R. oryzae. It is likely that the unique mechanism used by A. tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.An erratum to this article can be found at Communicated by C. P. Hollenberg     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Transient expression of the GUS gene in a unicellular marine green alga,<Emphasis Type="Italic">Chlorella</Emphasis> sp.<Emphasis Type="Italic">MACC/C95</Emphasis>, via electroporation

A transient expression system for a unicellular marine green alga,Chlorella sp.MACC/C95, was developed using a reporter GUS gene coded for by plasmid pBI121. The results demonstrated a high transformation efficiency could be achieved by using electroporation to deliver DNA into intact cells and the CaMV35S promoter to drive the foreign gene expression inChlorella sp.MACC/C95. The use of a carrier DNA coupled with osmosis treatment improved the transformation efficiency, while linearization of the plasmid had minor effects. Investigation of the effects of DNA concentration and growth phases ofChlorella sp.MACC/C95 on transformation efficiency indicated that the highest level of transient expression was observed when 6 μg mL⁻¹ of plasmid DNA and cells 2–6 days old were used.     

Transformation of <Emphasis Type="Italic">Lyophyllum decastes</Emphasis> by particle bombardment

The basidiomycete Lyophyllum decastes was transformed by means of particle bombardment. We isolated five transformants under twelve conditions differing in the two parameters of target distance and helium pressure. The transformation frequency was one transformant/μg DNA. In the transformants, plasmid DNAs were integrated into the genomic DNA and stably maintained. This is the first report on transformation of L. decastes by particle bombardment.     

An efficient transformation method for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> DB104

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10³ transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 10⁵ transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.     

Plastid transformation in Arabidopsis thaliana

Plastid transformation is reported in Arabidopsis thaliana following biolistic delivery of transforming DNA into leaf cells. Transforming plasmid pGS31A carries a spectinomycin resistance (aadA) gene flanked by plastid DNA sequences to target its insertion between trnV and the rps12/7 operon. Integration of aadA by two homologous recombination events via the flanking ptDNA sequences and selective amplification of the transplastomes on spectinomycin medium yielded resistant cell lines and regenerated plants in which the plastid genome copies have been uniformly altered. The efficiency of plastid transformation was low: 2 in 201 bombarded leaf samples. None of the 98 plants regenerated from the two lines were fertile. Received: 13 February 1998 / Revision received: 24 April 1998 / Accepted: 5 June 1998     

Establishment of an <Emphasis Type="Italic">Agrobacteriuim</Emphasis>-mediated cotyledon disc transformation method for <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for J. curcas has been established for the first time via Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin. About 55% of the cotyledon explants produced phosphinothricin-resistant calluses on Murashige and Skoog (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA), 0.05 mg l⁻¹ 3–indolebutyric acid (IBA), 1 mg l⁻¹ phosphinothricin and 500 mg l⁻¹ cefotaxime after 4 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, 0.5 mg l⁻¹ gibberellic acid (GA3), 1 mg l⁻¹ phosphinothricin and 250 mg l⁻¹ cefotaxime, and about 33% of the resistant calli differentiated into shoots. Finally, the resistant shoots were rooted on 1/2 MS media supplemented with 0.3 mg l⁻¹ IBA at a rate of 78%. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. 13% of the total inoculated explants produced transgenic plants after approximately 4 months. The procedure described will be useful for both, the introduction of desired genes into J. curcas and the molecular analysis of gene function.     

The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum)

In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L⁻¹ sucrose and 10 g L⁻¹ mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.     

Stable Plastid Transformation in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.     

An improved procedure for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of trifoliate orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.) via indirect organogenesis

Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical assay, PCR, and Southern blot analysis.     

Efficient transformation ofKlebsiella oxytoca by electroporation

A protocol for the transformation ofKlebsiella oxytoca by electroporation was developed. Preparation of competent cells at early exponential phase was most critical to obtain a high transformation efficiency. The highest efficiency of 1.6 × 10⁶ transformants per μg DNA (pBR 322) could be obtained by electroporation ofK. oxytoca cells prepared at the OD₆₀₀ of 0.2 with 1.25 μg DNA at the filed strength of 2.5 kV, the parallel resistance of 200 Ω and capacitance of 25 μF.     

Isolation and characterization of a novel bacterium, <Emphasis Type="Italic">Sphingomonas bisphenolicum</Emphasis> strain AO1, that degrades bisphenol A

Bisphenol A (2,2-bis(4-hydroxyphenyl) propane, BPA), which is used as a synthetic resin material or a plasticizer, is a pollutant that␣possesses endocrine-disrupting activity. Bioremediation of BPA is used to decrease its polluting effects, and here we report a novel bacterial strain AO1, which is able to degrade BPA. This strain was isolated using enrichment cultivation from a soil sample from a vegetable-growing field; the sample was one of 500 soil samples collected across Japan. Strain AO1 degraded 100 mg/l BPA to an undetectable level within 6 h in MYPG medium (containing malt extract, yeast extract, peptone, and glucose) and within 48 h in minimum medium containing 1% glucose at 30°C. Strain AO1 can utilize BPA as a sole source of carbon and as an energy source under aerobic conditions. The estrogenic activity of BPA in MYPG medium was ultimately reduced by strain AO1, although the activity initially increased. Taxonomical analysis showed that strain␣AO1 is closely related to Sphingomonas chlorophenolicum and S. herbicidovorans, neither of which have a capacity for BPA degradation. DNA–DNA hybridization showed that strain AO1 is a novel species of the Sphingomonas genus, and we designated AO1 as S. bisphenolicum.     

Plastid transformants of tomato selected using mutations affecting ribosome structure

Tomato plastid transformants were obtained using two vectors containing cloned plastid DNA of either Nicotiana tabacum or Solanum nigrum and including point mutations conferring resistance to spectinomycin and streptomycin. Transformants were recovered after PEG-mediated direct DNA uptake into protoplasts, followed by selection on spectinomycin-containing medium. Sixteen lines contained the point mutation, as confirmed by mapping restriction enzyme sites. One line obtained with each vector was analysed in more detail, in comparison with a spontaneous spectinomycin-resistant mutant. Integration of the cloned Solanum or Nicotiana plastid DNA, by multiple recombination events, into the tomato plastome was confirmed by sequence analysis of the targeted region of plastid DNA in the inverted repeat region. Maternal inheritance of spectinomycin and streptomycin resistances or sensitivity in seedlings also confirmed the transplastomic status of the two transformants. The results demonstrate the efficacy in tomato of a selection strategy which avoids the integration of a dominant bacterial antibiotic resistance gene.     

Direct transformation of the liverwort <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> L. by particle bombardment using immature thalli developing from spores

The liverwort, Marchantia polymorpha L., belongs to a group of basal land plants and is an emerging model for plant biology. We established a procedure to prepare sporangia of M. polymorpha under laboratory conditions by promoting its transition to reproductive development by far-red light irradiation. Here we report an improved direct transformation system of M. polymorpha using immature thalli developing from spores. Hygromycin-resistant transformants were obtained on selective media by transformation with a plasmid carrying the hygromycin-phosphotransferase gene (hpt) conferring hygromycin resistance in 4 weeks. The aminoglycoside-3'-adenyltransferase gene (aadA) conferring spectinomycin resistance was also successfully used as an additional selectable marker for nuclear transformation of M. polymorpha. The availability of the aadA gene in addition to the hpt gene should make M. polymorpha a versatile host for genetic manipulation. DNA gel-blot analyses indicated that transformed thalli carried a variable number of copies of the transgene integrated into the genome. Although the previous system using thalli grown from gemmae required a two-step selection in liquid and solid media for 8 weeks, the system reported here using thalli developing from spores allows generation of transformants in half the time by direct selection on solid media, facilitating genetic analyses in this model plant.