Stable transformation and regeneration of transgenic plants of Pinus radiata D. Don

A biolistic particle delivery system was used to genetically transform embryogenic tissue of Pinus radiata. The introduced DNA contained a uidA reporter gene under the control of either the tandem CaMV 35S or the artificial Emu promoter, and the npt II selectable marker controlled by the CaMV 35S promoter. The average number of stable, geneticin-resistant lines recovered was 0.5 per 200 mg fresh weight bombarded tissue. Expression of the uidA reporter gene was detected histochemically and fluorimetrically in transformed embryogenic tissue and in derived mature somatic embryos and regenerated plants. The integration of uidA and npt II genes into the Pinus radiata genome was demonstrated using PCR amplification of the inserts and Southern hybridisation analysis. The expression of both genes in transformed tissue was confirmed by Northern hybridisation analysis. More than 150 transgenic Pinus radiata plants were produced from 20 independent transformation experiments with four different embryogenic clones. Received: 9 May 1997 / Revision received: 18 September 1997 / Accepted: 18 October 1997     

Stable Transformation of Embryogenic Tissues of <Emphasis Type="Italic">Pinus nigra</Emphasis> Arn. using a Biolistic Method

The stable transformation of embryogenic tissues of Pinus nigra Arn., cell line E104, has been achieved using a biolistic approach. The introduced DNA consisted of the uidA reporter gene under the control of the double CaMV 35S promoter and the nptII selection gene controlled by the single CaMV 35S promoter. Three days after bombardment, putative transformed tissues were selected for continued proliferation on a medium containing 20 mg geneticin l⁻¹. Resistant embryogenic tissue recovery required 10–12 weeks. The integration of the nptII and uidA genes was confirmed by both histochemical/fluorimetric GUS assays and PCR amplification of the inserts in the five geneticin resistant sub-lines of line E104. The activity of the uidA reporter gene in transgenic, embryogenic tissue lines was stable and could be detected after one year of culture. Somatic embryo maturation was, however, poor and no plantlet regeneration could be obtained.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Transgene expression in regenerating cotyledons and embryogenic cultures of Scots pine

Regenerating cotyledons and embryogenic cultures of Scots pine (Pinus sylvestris L.) were used as targets for gene delivery via particle bombardment. Both target tissues differed in their response to selective agents, regulative sequences of transferred genes, and conditions optimal for particle bombardment. Of the gene constructs tested, the pB1410 including CaMV 35S promoter, AMV-translation enhancer and gusA:npt reporter and selectable marker gene fusion was the most suitable for cotyledon transformation, resulting on average in 3.7 ( 0.1 SE) -glucuronidase (GUS) expression units per cotyledon. In embryogenic cell masses the pB1221.1 gene construct including the gusA reporter gene driven by the 35S promoter gave the highest transient expression, 63 (plusmn; 15 SE) GUS expression units of g^-1 fresh weight of embryogenic cell mass. Kanamycin and geneticin concentrations suitable for selection of cotyledons were 10-15 mg dm^-3 and 0.5-1.0 mg dm^-3, respectively. Kanamycin (10 mg dm^-3) and phosphinotricin (1 mg dm^-3) as selectors caused a significant decline in growth, but geneticin did not significantly affect the growth of the embryogenic cultures during the 8 week cultivation period. The production of transgenic plantlets seems to be more dependent on the regeneration and multiplication efficiency of organogenesis and somatic embryogenesis of Scots pine than on gene delivery into regenerating tissues.     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

Genetic transformation of sweet potato by particle bombardment

Transient and stable expression of foreign genes has been achieved in sweet potato using the particle bombardment system of gene delivery. Callus and root isolates of two genotypes (Jewel and TIS-70357) with positive signs of transformation have been recovered. Tungsten microcarriers coated with plasmid DNA (pBI 221 containing the gusA gene) were accelerated at high velocity using a biolistic device into sweet potato target tissues. Histochemical examination of bombarded leaf and petiole explants revealed that most had cells expressing the gusA gene. When explants were cultured, calli and roots developed in most bombarded tissues. Similar results but with a lower frequency of transformation were observed when the plasmid pBI 121 (with gusA and antibiotic resistance npt II genes) was employed and bombarded explants cultured on an antibiotic selection medium. Subcultured roots and calli were positive for gusA expression when tested even after one year of in vitro culture, and thus the expression of the foreign gene is fairly stable. The particle bombardment approach of gene delivery appears to have a potential for generating transgenic sweet potatoes with useful agronomic traits.Abbreviations BA 6-benzylaminopurine - CaMV cauliflower mosaic virus - 2,4-D 2, 4-dichlorophenoxyacetic acid - GUS ß glucuronidase - NAA naphthaleneaceticacid - nos nopaline synthase gene - NPT II neomycin phosphotransferase II - MS Murashige and Skoog (1962) - MS-CP MS cell proliferation medium     

Optimised DNA delivery into Coffea arabica suspension culture cells by particle bombardment

An optimised bombardment protocol to introduce DNA into Coffea arabica suspension culture cells was developed. Osmotic preconditioning of cells and physical bombardment parameters including Helium pressure, gap and target distances affecting DNA delivery were evaluated by monitoring transient expression of the uidA gene driven by the CaMV35S promoter. The highest transient GUS expression was obtained when cells were subjected to a 0.5 M mannitol–sorbitol pre-treatment 4 h prior to bombardment and a Helium pressure of 1550 psi, a 9-mm gap distance and 12 cm target distance as physical bombardment parameters. The optimised protocol was tested with two coffee promoters: -tubulin and arabicin, which presented similar activity to the CaMV35S promoter in suspension culture cells by fluorometric GUS assays. GUS expression was reduced in bombarded tissue culture leaves, and only the CaMV35S and arabicin promoters showed histochemical activity in coffee endosperms. This is the first report of optimization of particle bombardment on coffee suspension culture cells, equivalent CaMV35S activity for a coffee promoter and transient -glucoronidase expression in coffee endo-sperms.     

Transforming embryogenic cell lines of Gladiolus with either a bar-uidA fusion gene or cobombardment

Summary Embryogenic cell lines of Gladiolus were bombarded with the bar-uidA fusion gene under the cauliflower mosaic virus (CaMV) 35S promoter (pDM327) or cobombarded with uidA under the CaMV 35S promoter (pBCG) and bar under the CaMV 35S promoter (pDM307). Over 500 cell lines were isolated for either the fusion gene or cobombarded cells following selection on Murashige and Skoog's medium supplemented with 2 mg 1⁻¹ (9 μM) 2,4-dichlorophenoxyacetic acid and 6 mg 1⁻¹ phosphinothricin. The optimum DNA concentration for, isolating stable transformants was one-tenth that for optimal isolation of lines with gus expression, and three times as many cell lines were isolated following cobombardment as compared to bombardment with the bar-uidA fusion gene. Three times as many cell lines (72% of the cell lines) containing the bar-uidA fusion gene expressed gus as compared to cobombarded cell lines (23%) following histological staining. Gus expression ceased after 1 yr in culture for 5% of the cell lines containing the fusion gene and 3% of the cobombarded cell lines. The bifunctionality and utility of the bar-uidA fusion gene were demonstrated, accompanied by enhanced gus expression.     

Development of an efficient transient transformation protocol for avocado (Persea americana Mill.) embryogenic callus

An efficient protocol for transient transformation of avocado embryogenic callus has been established, using the PDS-1000/He system and the reporter gus gene driven by the sunflower polyubiquitin promoter. Best physical parameters for transient transformation were 900 psi helium pressure and 6 cm target distance. The level of transient gus expression was slightly higher when the amount of DNA per shot was increased from 0.6 to 1.8 μg, but it was not significantly modified by the type of microprojectile used (tungsten vs. gold particles). The transient transformation assay developed in this research was used to test the strength of different promoters and the expression of fluorescent reporter genes. Four constitutive promoters, sunflower polyubiquitin, CaMV35S, CaMV35S with enhancer, and rice actin 1, as well as a trichome-specific promoter, ATP, were analyzed. Polyubiquitin and ATP promoters yielded the highest number of gus expressing foci, while no expression was detected with the Act1 promoter from rice. Embryogenic callus was also bombarded with plasmids pXK7S*NF2 and pXK7RNR2, harboring the enhanced green fluorescent gene, EGFP, and the red fluorescent gene DsRed, respectively. Both fluorescent proteins were detected 24 and 72 h after bombardment, but the observed transformation efficiency was slightly higher in GFP bombarded cells. The transient transformation system described here can be used as a fast way to select suitable promoters and/or fluorescent genes needed to undertake stable transformation studies in avocado using currently available protocols.     

Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.     

Transgenic Pinus radiata from Agrobacterium tumefaciens–mediated transformation of cotyledons

A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed. Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere. Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos. The main drawback to the method is the small number of genotypes that are amenable to transformation and regeneration. Since more than 80% of genotypes of radiata pine can be regenerated using cotyledons from mature seed, cotyledon explants were cocultivated with A. tumefaciens strain AGL1 containing a plasmid coding for the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene (uidA). Transformed shoots were selected using either geneticin or kanamycin. Critical factors for successful transformation were survival of the cotyledons after cocultivation and selection parameters. Of the 105 putative transformants that were recovered from selection media, 70% were positive for integration of the nptII gene when analysed by PCR. GUS histochemical assay for uidA expression was unreliable because of reaction inhibition by unidentified compounds in the pine needles. Further, only 4 of the 26 independent transformants characterised by PCR and Southern analysis contained an intact copy of both genes. The remaining 22 transformants appeared to have a truncated or rearranged copy of the T-DNA. It is possible that the truncation/rearrangements are due to the Cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the T-DNA junction sites and sequencing of the introduced DNA will help elucidate the nature of T-DNA insertion so that genetic modification of radiata pine can be targeted effectively.Communicated by P. Debergh     

Agrobacterium-mediated transformation of white clover using direct shoot organogenesis

Summary White clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and -glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MS Murashige and Skoog - GUS -glucuronidase - X-GLUc 5-bromo-4-chloro-3-indolyl--D-glucuronide - MUG methylumbelliferyl--D-glucuronide - CaMV Cauliflower Mosaic Virus - NPTII neomycin phosphotransferase II - OCS octopine synthase - 4-MU 4-methyl umbelliferone     

Comparison of the activities of CaMV 35S and FMV 34S promoter derivatives in Catharanthus roseus cells transiently and stably transformed by particle bombardment

Activities of several CaMV 35S and FMV 34S promoter derivatives fused to the gusA reporter gene were compared in suspension-cultured Catharanthus roseus cells that were transiently and stably transformed using particle bombardment. Our data demonstrate that the 35S and a deletion derivative of the 34S promoter combined with particle bombardment form useful tools for genetic engineering of C. roseus cells. Our results disagree on several points with activities of 35S and 34S promoter derivatives reported for tobacco, indicating that absolute and relative promoter activities can differ between plant species.     

Conifer genetic engineering: transgenic Pinus radiata (D. Don) and Picea abies (Karst) plants are resistant to the herbicide Buster

A biolistic transformation procedure was applied to co-transform embryogenic tissue of Pinus radiata and Picea abies with two plasmid DNAs. The first vector contained the bar gene, specifying resistance to the herbicide glufosinate, under the control of the maize ubiquitin promoter. This plasmid also contained the Pinus radiata germin cDNA sequence, in either sense or antisense orientation, driven by the ubiquitin promoter. The second vector contained both the nptII gene under control of the CaMV 35S promoter for selection of transgenic tissue on geneticin and the uidA reporter gene under control of the double CaMV 35 promoter. Polymerase chain reaction analysis of selected geneticin-resistant tissue showed that the transformation rates for the co-bombarded plasmid were high in both Pinus radiata (75%) and Picea abies (86%). A combination of phenotypic analysis and Northern hybridisation demonstrated that a number of the transgenic lines expressed all four transgenes. Regenerated plantlets from Pinus radiata and Picea abies transgenic lines were spray-tested with commercial rates of Buster (glufosinate at 0.5, 1.0, 2.0 and 4.0 kg active ingredient per hectare). Transgenic plants survived and continued to grow with minor or no damage to their needles, whereas non-transgenic plants regenerated from the same cell lines died within 8 weeks of spraying. To our knowledge, this is the first report on genetically engineered herbicide resistance in conifers, and the results demonstrate that this trait is a feasible option for plantation forestry.     

不同调控序列作用下GUS基因在烟草中瞬时表达活性的研究

以pBI121为出发质粒, 利用烟草泛素启动子Ubi.U4、CaMV35S启动子以及Kozak序列构建4种GUS基因表达载体,通过叶盘转化法转化烟草叶片, 检测瞬时表达活性, 研究不同调控序列对外源基因表达的调控作用。结果表明: CaMV35S启动子附加Kozak序列后使GUS活性比独立使用CaMV35S提高了近2倍; 双CaMV35S启动子附加Kozak序列驱动GUS基因的表达活性与单CaMV35S附加Kozak序列相当; 烟草泛素启动子附加Kozak序列的表达活性为CaMV35S启动子附加Kozak序列的1.5倍; Ubi.U4-CaMV35S复合启动子附加Kozak序列驱动GUS基因表达水平最高, 其表达效率是双CaMV35S启动子附加Kozak序列调控下GUS表达效率的3倍, 为CaMV35S独立作用时的10倍。     

不同调控序列作用下GUS基因在烟草中瞬时表达活性

以pB1121为出发质粒,利用烟草泛素启动子Ubi．U4、CaMV35S启动子以及Kozak序列构建4种GUS基因表达载体,通过叶盘转化法转化烟草叶片,检测瞬时表达活性,研究不同调控序列对外源基因表达的调控作用。结果表明：CaMV35S启动子附加Kozak序列后使GUS活性比独立使用CaMV35S提高了近2倍：双CaMV35S启动子附加Kozak序列驱动GUS基因的表达活性与单CaMV35S附加Kozak序列相当;烟草泛素启动子附加Kozak序列的表达活性为CaMV35S启动子附加Kozak序列的1．5倍;Ubi．U4-CaMV35S复合启动子附加Kozak序列驱动GUS基因表达水平最高,其表达效率是双CaMV35S启动子附加Kozak序列调控下GUS表达效率的3倍,为CaMV35S独立作用时的10倍。     

Transient expression of a reporter gene in bulbscales and immature embryos of three Lilium species is affected by 5'upstream sequences and culture conditions

In Lilium , a transformation system has not yet been developed. For efficient selection of cells expressing transferred genes following particle bombardment, the effects of 5'upstream regions on the transient expression of the β-glucuronidase gene ( gusA ) were estimated in bulbscales and immature embryos of lily. When four plasmids having the gusA gene under the control of the cauliflower mosaic virus (CaMV) 35S, maize alcohol dehydrogenase gene and rice actin gene ( Actl ) promoters, and the castor bean catalase introm were introduced by particle bombardment, the patterns of transient expression in the bulbscales showed differences among three Lilium species, L. x formolongi, L. dauricum and L. japonicum . In immature embryos of L x formolongi , transient expression was significantly influenced by age of embryos after self-pollination, duration of culture before bombardment, and culture conditions. Moreover, the transient gusA expression driven by six different 5'upstream regions, including the maize ubiquitin gene promoter and a modified CaMV 35S promoter were compared in both bulbscales and immature embryos. Use of the Actl and modified CaMV 35S promoters resulted in the greatest number of cells that transiently expressed gusA in both types of tissue of L. x formolongi . These two promoters are efficient for use in lily transformation.     

Optimization of biolistic bombardment parameters for <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17 calluses using GFP and GUS as the reporter system

Genetic transformation system of Dendrobium Sonia 17 was optimized using green fluorescent protein (GFP) and -glucuronidase (GUS) gene as the reporter systems. The 35S-sgfp-TYG-nos (p35S) and pSMDFR, carrying sgfp and gusA gene, respectively, were co-bombarded into the calluses. Parameters optimized were acceleration pressure, target distance, gold particle size, pre-bombardment cultured time, plasmid DNA precipitation, total plasmid DNA and the ratio of the plasmids co-bombarded. Both reporter systems responded similarly to the bombardment parameters investigated. Based on the GUS/GFP spot counts, the GFP expression rate was higher than that for GUS under the control of the same promoter, CaMV 35S. GFP could be used as the reporter system for the co-bombardment as it was rapid and non-destructive system to monitor the transformed tissues. A combination of GFP and antibiotic resistance gene was used to select stable putative transformants.     

Transient gene expression after electroporation of protoplasts derived from embryogenic maize callus

Protoplasts isolated from embryogenic callus cultures derived from immature embryos ofZea mays L. are suitable for analysis of transient gene expression using electroporation-mediated DNA transfer. Expression of introduced genes is comparable to the levels obtained with protoplasts from Black Mexican Sweet suspension cultures. Two different promoters, that directing synthesis of the 35S RNA of cauliflower mosaic virus and the maizeAdh1 promoter were placed in front of the luciferase reporter gene to assess protoplast gene expression and the impact of an intron on expression level.Abbreviations 35S promoter isolated from CaMV - CaMV cauliflower mosaic virus - Adh1 maize gene encoding Alcohol dehydrogenase-1 enzyme - BMS suspension cultures of the Black Mexican Sweet maize variety     

Stable transformation of Pinus radiata embryogenic tissue by Agrobacterium tumefaciens

Embryogenic cultures from immature zygotic embryos of Pinus radiata seeds were established on semisolid proliferation medium with 2,4-D and BAP. Growing embryogenic masses containing embryonal cells and suspensor cells were subcultured on this media every 2 weeks. After 10 weeks, embryogenic masses (1.5 cm diameter) were transferred to a maturation medium containing ABA. Fully developed somatic embryos were obtained in this medium after 12 weeks. Embryogenic masses were genetically transformed using Agrobacterium tumefaciens. The pBI121 vector containing -glucuronidase (uidA) and the neomycin phosphotransferase (nptll) genes was introduced into this tissue. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to a selection media containing geneticin and carbenicillin. After 1 month of selection, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Under light microscope, blue crystals were seen inside the embryogenic and suspensor cells, and also completely blue somatic embryos were obtained. The uidA gene was also detected by PCR analysis in genomic DNA isolated from transformed embryogenic tissues. These results indicate stable transformation of P. radiata somatic embryogenic tissues using Agrobacterium-mediated transformation.