Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue

A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation using embryogenic suspension cultures in sweetpotato, <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.

Efficient Agrobacterium tumefaciens-mediated transformation was achieved using embryogenic suspension cultures of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lizixiang. Cell aggregates from embryogenic suspension cultures were cocultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hygromycin phosphotransferase II gene (hpt II) genes. Selection culture was conducted using 25 mg l⁻¹ hygromycin. A total of 2,218 plants were regenerated from the inoculated 1,776 cell aggregates via somatic embryogenesis. β-glucuronidase (GUS) assay and PCR, dot blot and Southern blot analyses of the regenerated plants randomly sampled showed that 90.37% of the regenerated plants were transgenic plants. The number of integrated T-DNA copies varied from 1 to 4. Transgenic plants, when transferred to soil in a greenhouse and a field, showed 100% survival. No morphological variations were observed in the ex vitro transgenic plants. These results exceed all transformation experiments reported so far in the literature in quantity of independent events per transformation experiment in sweetpotato.     

Regeneration of transgenic Picea glauca, P. Mariana, and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens

Summary Transgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58/pMP90/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (β-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation procedure. Of the three species tested, P. mariana was transformed at the highest frequency, followed by P. glauca and P. abies. The transgenic state of the embryogenic tissue was initially, confirmed by histochemical β-glucuronidase (GUS) assay followed by Southern hybridization. One to over five copies of T-DNA were detected in various transgenic lines analyzed. Transgenic plants were regenerated for all species using modified protocols for maturation and germination of somatic embryos.     

Assessment of simple marker-free genetic transformation techniques in alfalfa

Methods to avoid the presence of selectable marker genes (SMG) in transgenic plants are available but not implemented in many crop species. We assessed the efficiency of simple marker-free Agrobacterium-mediated transformation techniques in alfalfa: regeneration without selection, or marker-less, and co-transformation with two vectors, one containing the SMG and one containing a non-selected gene. To easily estimate the efficiency of marker-less transformation, the nptII and the GUS markers were used as non-selected genes. After Agrobacterium treatment, somatic embryos were regenerated without selection. The percentage of transgenic embryos was determined by a second cycle of regeneration using the embryos as starting material, in the presence of kanamycin, by PCR screening of T1 progenies, and by the GUS test. In two experiments, from 0 to 1.7% of the somatic embryos were transgenic. Co-transformation was performed with two vectors, one with the hemL SMG and one with the unselected nptII gene, each carried by a different culture of Agrobacterium. Only 15 putative co-transformed plants were regenerated from two experiments, with an average co-transformation percentage of 3.7. Southern blot hybridizations and/or T(1) progeny segregation were used to confirm transgene integration, and qPCR was also used to estimate the T-DNA copy number. In the T(1) progenies obtained by crossing with a non-transgenic pollinator, marker-free segregants were obtained. Both marker-free approaches showed very low efficiency.     

An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens.

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca [Moench] Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the beta-glucuronidase (uidA) reporter gene, was used as binary vector. The highest frequency of transformation (15 transformed tissues g(-1) FW of treated embryogenic tissue) was obtained with 5-d-old tissues grown in liquid medium and co-cultivated with Agrobacterium for 2 d in the same medium but containing 50 microM acetosyringone. Recovery of kanamycin-resistant tissues was improved when tissues were first grown for 10 d on a timentin-containing medium (400 mg l(-1)), to prevent bacterial overgrowth, before application of the selection pressure. After 6 weeks on kanamycin-selection medium, resistant tissues were obtained and showed stable uidA expression. The presence of the transgenes was demonstrated by PCR analysis and their integration into the genome was confirmed by Southern hybridization. Transgenic plants were regenerated from transformed tissues within 4 months after co-culture.     

Production of transgenic <Emphasis Type="Italic">Podophyllum peltatum via Agrobacterium tumefaciens</Emphasis>-mediated transformation

Transgenic Podophyllum peltatum plants were successfully produced by Agrobacterium tumefaciens-mediated transformation. Embryogenic callus was co-cultivated with Agrobacterium tumefaciens harboring a binary vector pBI 121 carrying β-glucuronidase (GUS) and neomycinphosphotransferase (NPT II) gene. GUS-histochemical analysis revealed that, 50 μM acetosyringone treatments during Agrobacterium infection and 3 d co-cultivation with Agrobacterium showed enhanced transformation efficiency. Percentage of GUS positive callus increased rapidly as the subculture time proceeded on selection medium containing 100 mg dm⁻³ kanamycin. Kanamycin resistant somatic embryos were formed from embryogenic callus after cultivation with 11.35 μM abscisic acid (ABA) for 3 weeks and then on hormone-free selection medium. Somatic embryos were germinated and converted into plantlets on medium containing 2.89 μM gibberellic acid (GA₃). The integration of GUS and NPT II gene into transgenic plants was confirmed by polymerase chain reaction and Southern analysis.     

Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens

A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein (GFP) gene were introduced and -glucuronidase (GUS) activity was followed over time. The expression of the uidA gene was lowest with a 35S-gus-intron construct and was 20-fold higher with a 35S-35S-AMVgus::nptII construct. The addition of scaffold attachment region (SAR) sequences surrounding the gus::nptII fusion did not significantly enhance the GUS activity. Transformed mature somatic embryos have been germinated and plantlets are presently being acclimatized.     

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm2 embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).     

Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.)

A system for the genetic transformation of rye by co-cultivation with Agrobacterium tumefaciens is described. A total of 45 independent transgenic plants were regenerated with a transformation efficiency of 1 to % of the inoculated explants. The co-cultivation of Agrobacterium-strain AGL0, harboring plasmid pJFnptII and rye im-mature embryos in liquid medium allowed a high throughput and facilitated washing of the cultures to avoid Agrobacterium overgrowth. Transgenic plants were phenotypically normal and fully fertile, which might be aconsequence of the short time in tissue culture. The selection with paromomycin exclusively during the regen-eration allowed the efficient recovery of transgenic events without interfering with somatic embryogenesis. Southern blot analysis confirmed the independent nature of the analyzed plants and indicated single copy inserts in more than 50% of them. Segregation analysis confirmed single locus integration and stable transgene expression in most of the lines, while one line with multiple locus integration was also observed. The analysis of T-DNA:: plant DNA boundary sequences revealed examples of exclusion of vector sequences, deletion of a few bases of the T-DNA or insertion of up to 29 bases of the vector backbone. This stresses the importance of detailed analysis of the inserted transgenes in order to identify events with the desired integration profile.     

Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare)

Barley (Hordeum vulgare L. cvs. Igri and Dissa) cell suspension cultures, which had been initiated from immature embryo-derived (IED) and microspore-derived (MSD) callus, were co-cultivated with various Agrobacterium tumefaciens strains. The T-DNA vectors contained visually-detectable marker genes (C1/Lc orgusA-intron), as reporters of transient T-DNA transfer, and also drug resistance genes (hph or bar) to facilitate selection of stably transformed cell lines. A set of normal binary vectors in a super-virulent Agrobacterium strain [EHA101(pBECKS)] and also a super-binary vector [LBA4404(pTOK233)] were used in this study. Cells of the suspension cultures which received T-DNA were able to proliferate under selection regimes and a number of hygromycin- or phosphinothricin-resistant barley callus lines were isolated which expressed a co-transferred gusA gene. To ensure homogeneity of the cell lines, prolonged tissue culture regimes were used but these resulted in a loss of the capacity to regenerate plants from the transgenic callus lines. The frequency of recovery of transformed callus lines ranged from 0.3% to 2.9%. Southern blot analyses of the transformed callus lines confirmed the presence of the marker genes and demonstrated them to be associated with DNA which was distinct from that of the original Agrobacterium plasmid. Furthermore, independent transgenic lines showed diverse patterns of hybridising bands. These data suggest that the T-DNA fragment was stably maintained through integration into the genomes of the barley cell lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

用基因枪法介导OSISAP1基因遗传转化洋葱

以洋葱栽培品种‘HG400B’的鳞茎盘胚性愈伤组织为受体,利用基因枪介导法将水稻锌指蛋白基因OSISAP1导入洋葱中。组织化学染色检测到GUS基因在胚性愈伤组织中的瞬间表达活性,PCR、Southern杂交和RT-PCR分析,证实OSISAP1基因已整合到洋葱基因组中并实现高水平表达,转化率约为10%。对获得的转基因植株进行NaC1和NaHCO_3胁迫处理,当总浓度为200 mmol/L、处理1周后,未转基因植株会黄化、枯萎、死亡,而转基因植株却有很强的抗性,能耐受400mmol/L浓度的胁迫,表明OSISAP1基因的导入提高了转基因植株的耐盐碱性。     

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.     

Gene Manipulation of a Heavy Metal Hyperaccumulator Species Thlaspi caerulescens L. via Agrobacterium-mediated Transformation

Thlaspi caerulescens L. is well known as a Zn/Cd hyperaccumulator. The genetic manipulation of T. caerulescens through transgenic technology can modify plant features for use in phytoremediation. Here, we describe the efficient transformation of T. caerulescens using Agrobacterium tumefaciens strain EHA105 harboring a binary vector pBI121 with the nptII gene as a selectable marker, the gus gene as a reporter and a foreign catalase gene. Based on the optimal concentration of growth regulators, the shoot cluster regeneration system via callus phase provided the basis of the genetic transformation in T. caerulescens. The key variables in transformation were examined, such as co-cultivation period and bacterial suspension density. Optimizing factors for T-DNA delivery resulted in kanamycin-resistant transgenic shoots with transformation efficiency more than 20%, proven by histochemical GUS assay and PCR analysis. Southern analysis of nptII and RT-PCR of catalase gene demonstrated that the foreign genes were integrated in the genome of transformed plantlets. Moreover, the activity of catalase enzyme in transgenic plants was obviously higher than in wild-type plants. This method offers new prospects for the genetic engineering of this important hyperaccumulator species.     

Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method

A system for the production of transgenic papaya (Carica papaya L.) plants using zygotic embryos and embryogenic callus as target cells for particle bombardment is described. Phosphinothricin (bar ) and kanamycin (npt II) resistance genes were used as selectable markers, and the gus gene (uidA) as a reporter gene. Selection with 100 mg/l kanamycin and 4 mg/l phosphinothricin (PPT) yielded a total of over 90 resistant embryogenic colonies from three independent experiments using embryogenic callus as a target tissue. This represents an efficiency of 60 transgenic clones per gram of fresh weight callus bombarded. The efficiency of genetic transformation using zygotic embryos was lower, as only 8 independent resistant clones were recovered out of 645 bombarded zygotic embryos, giving a efficiency of 1.24%. Subsequent subculture of transgenic somatic embryos both from zygotic embryos and embryogenic callus led to the development of plants with apparently normal morphology. Histological, fluorimetric assay for GUS, NPT II assay and DNA analysis (Southern hybridization) showed that kanamycin /PPT resistant plants carried and expressed the transgenes.Abbreviations Gus -glucuronidase - NPTII neomycin phophotransferase II - bar phophinothricin acetyl transferase gene - Pat phosphinothricin acetyl transferase - PPT phosphinothricin - Km kanamycin - 2,4-D 2,4-dichlorophenoxyacetic acid - K kinetin - BAP benzylaminopurine - IBA indolbutyric acid     

Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants

Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T₁ progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T₂ progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations.     

Agrobacterium-mediated transformation of sea urchin embryos

Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. In the present work, we studied whether Agrobacterium can transfer genetic information to animal (sea urchin) embryos. Sea urchin embryos were co-cultivated with A. tumefaciens strains carrying binary vectors containing the nptII marker gene and agrobacterial rolC and rolB oncogenes. Bacterial plasmid T-DNA-sea urchin DNA junction sites were identified in the genome of these embryos, thus indicating successful transformation. The nptII and both rol genes were expressed in the transformed embryos. The processes of transgene integration and transgene expression were suppressed when Agrobacteria contained mutated virA, virB or virG genes, suggesting that Agrobacterium transforms sea urchin cells by a mechanism similar to that which mediates T-DNA transfer to plants. Some of the embryos co-cultivated with Agrobacterium developed teratoma-like structures. The ability of Agrobacterium strains to trigger formation of teratoma-like structures was diminished when they contained the mutated vir genes. In summary, our results demonstrate that Agrobacterium is able to transform animal (sea urchin) embryonic cells, thus indicating a potential of this natural system for gene delivery to animal hosts. We also discuss the possibility of horizontal gene transfer from Agrobacterium to marine invertebrates.     

Production of<Emphasis Type="Italic"> Hevea brasiliensis</Emphasis> transgenic embryogenic callus lines by<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>: roles of calcium

A procedure has been established for Agrobacterium tumefaciens-mediated genetic transformation of Hevea brasiliensis embryogenic friable calli. Precultivation of tissues on a CaCl(2)-free maintenance medium dramatically enhanced the transient activity of the reporter gene, gusA encoding beta-glucuronidase (GUS). The increase was first noticed in highly active cells (undifferentiated or/and embryogenic), in tissues precultured for 2-8 weeks. Beyond 8 weeks of preculture, GUS activity increased again, but this time in tissues consisting of differentiated cells accumulating polyphenols. Out of five Agrobacterium strains cocultivated with CaCl(2)-free precultured tissues, only inoculation with EHA105pC2301 led to high transient GUS activity. Paromomycin proved more effective than kanamycin for the selection of transformed cells, as it inhibits the growth of non-transformed cells more radically. Five paromomycin-resistant callus lines were established. The presence of gusA and neomycin phosphotransferase ( nptII) genes in the plant genome was confirmed by DNA amplification, and by Southern hybridization. These results confirmed that A. tumefaciens is an effective system for mediating stable transformation of rubber tree calli with a low copy number of transgenes. Transgenic callus lines constitute a useful tool for studying genes of interest on a cellular level and for regenerating transgenic rubber trees.     

Production of herbicide-resistant transgenic <Emphasis Type="Italic">Panax ginseng</Emphasis> through the introduction of the phosphinothricin acetyl transferase gene and successful soil transfer

Herbicide-resistant transgenic Panax ginseng plants were produced by introducing the phosphinothricin acetyl transferase (PAT) gene that confers resistance to the herbicide Basta (bialaphos) through Agrobacterium tumefaciens co-cultivation. Embryogenic callus gathered from cotyledon explants of P. ginseng were pre-treated with 0.5 M sucrose or 0.05 M MgSO(4 )before Agrobacterium infection. This pre-treatment process markedly enhanced the transient expression of the beta-glucuronidase (GUS) gene. Embryogenic callus was initially cultured on MS medium supplemented with 400 mg/l cefotaxime for 3 weeks and subsequently subcultured five times to a medium containing 25 mg/l kanamycin and 300 mg/l cefotaxime. Somatic embryos formed on the surfaces of kanamycin-resistant callus. Upon development into the cotyledonary stage, these somatic embryos were transferred to a medium containing 50 mg/l kanamycin and 5 mg/l gibberellic acid to induce germination and strong selection. Integration of the transgene into the plants was confirmed by polymerase chain reaction and Southern analyses. Transfer of the transgenic ginseng plantlets to soil was successfully accomplished via acclimatization in autoclaved perlite. Not all of the plantlets survived in soil that had not been autoclaved because of fungal infection, particularly in the region between the roots and leaves. Transgenic plants growing in soil were observed to be strongly resistant to Basta application.     

Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue.

A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inoculation/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.