Agrobacterium－mediated transformation and assessment of factors influencing transgene expression in loblolly pine（Pinus taeda L.）

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.     

Additional virulence genes and sonication enhance <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated loblolly pine transformation

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.     

DNA delivery into Eucalyptus globulus zygotic embryos through biolistics: optimization of the biological and physical parameters of bombardment for two different particle guns

Summary DNA transfer into zygotic embryos of Eucalyptus globulus by microprojectile bombardment was studied with two devices: a gunpowder apparatus and a compressed-helium system. Using, as a test, the transient expression of a reporter gene, we optimized the physical and biological conditions of bombardment. Six-day-old cultured embryos were found to be the best target material, and osmotic treatment increased the expression rate. Conditions of bombardment (particle acceleration and quality of the particle: DNA mix) were studied. In optimal conditions, we were able to obtain up to 130 GUS expression events per embryo with a good distribution over the tissue.In our transient expression experiments, the gunpowder and helium devices exhibited similar efficiencies, reliabilities and reproducibilities.Abbreviations GUS -glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid     

Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens

Embryos of 24 open-pollinated families of loblolly pine (Pinus teade L.) were used as explants to conduct in vitro regeneration. Then, Agrobacterium tumefaciens strain GV3101 harboring the plasmid pPCV6NFHygGUSINT was used to transform mature zygotic embryos of seven families of loblolly pine. The frequency of transformation varied among families infected with A. tumefaciens. The highest frequency (100%) of transient beta-glucuronidase (GUS)-expressing embryos was obtained from family 11-1029 with over 300 blue spots per embryo. Expression of the GUS reporter gene was observed in cotyledons, hypocotyls, and radicles of co-cultivated mature zygotic embryos, as well as in callus and shoots derived from co-cultivated mature zygotic embryos. Ninety transgenic plants were regenerated from hygromycin-resistant callus derived from families W03. 8-1082 and 11-1029. and 19 transgenic plantlets were established in soil. The presence of the GUS gene in the plant genome was confirmed by polymerase chain reaction. Southern blot, and plant DNA/T-DNA junction analysis. These results suggest that an efficient A. tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for future studies on transferring economically important genes to loblolly pine.     

Analysis of an ABA-responsive rice gene promoter in transgenic tobacco

We have analyzed in transgenic tobacco the expression of a chimeric gene containing 5 sequences of the rice rab-16B gene fused to the -glucuronidase (GUS) reporter gene. This construct, a translational fusion (–482 to +184) including 14 amino acids of the RAB-16B protein, is expressed only in zygotic and pollen-derived embryos. In zygotic embryos, GUS activity begins to accumulate 10 days after flowering (daf), and increases until seed maturation at 25 daf. Immunological measurements of endogenous abscisic acid (ABA) accumulation in these seeds showed a close parallel between hormone levels and GUS activity. However, GUS activity could not be reproducibly induced by treatment of immature embryos with ABA (10 M). Neither GUS activity nor GUS mRNA could be detected in leaves of transgenic tobacco even after ABA treatment. In contrast, GUS activity could be induced to high levels in pollen-derived embryos by treatment with ABA. Our results show that 482 bp of 5 sequences of the rice rab-16B promoter can confer in transgenic tobacco developmentally regulated expression in embryos but not ABA-responsive expression in vegetative tissues.     

Expression of the BBM gene during somatic embryogenesis of Arabidopsis thaliana

The relationship between somatic embryogenesis (SE) and the expression of the BABY BOOM (BBM) gene was studied in cultured immature zygotic embryos (IZEs) using a transgenic line of Arabidopsis thaliana containing a BBMPro::GUS construct. Results showed spatio-temporal differences in BBM expression in explants during culture. BBM promoter activity was observed in freshly isolated IZEs except distal parts of cotyledons. At the beginning of culture, considerable increase of GUS staining intensity was observed in all parts of explants, which maintained at high level over next few days and coincide with cell divisions. Gradual decrease of GUS distribution in explants was observed at about the 5th day of culture. BBM promoter activity became largely restricted to dividing cells, then to developing somatic embryos, shoot-like structures and callus. In parts of explants not involved in morphogenesis BBM promoter activity was absent or hardly seen. Thus the in vitro expression of BBM coincides with cell proliferation and morphogenesis.     

Genetic transformation of Eucalyptus globulus through biolistics: complementary development of procedures for organogenesis from zygotic embryos and stable transformation of corresponding proliferating tissue

Stable transformation of Eucalyptus globulus after biolisticDNA delivery was investigated using zygotic embryos as targetmaterial. Conditions for plant regeneration were first investigated.Six-day-old cultured embryos, which had shown to be a good targetfor DNA transient expression, appeared to be suitable for regeneration.Whole plants were recovered through organogenesis after particlegun bombardment. Transformation experiments were performed witha linear T-DNA fragment harbouring GUS and NPTII genes, usingthe biological and physical conditions defined for optimum transientexpression. After 2 months on a culture medium, neoformed GUS-positivecalli were obtained from the ‘T-DNA’ bombarded embryos.GUS-expressing calli were also recovered after selection withkanamycin following bombardment. The integration of both GUSand NPTII genes into the Eucalyptus genome was confirmed bySouthern blot analysis, demonstrating stable transformationof Eucalyptus globulus cells for the first time. Key words: Eucalyptus, zygotic embryos, biolistics, stable transformation, regeneration     

Dehydrating immature embryo split apices and rehydrating with<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>: A new method for genetically transforming recalcitrant sunflower

Split embryonic axes of 21-day-old immature sunflower (Helianthus annuus L. var. RHA266) embryos were bombarded, vacuum infiltrated, or dehydrated before being infected byAgrobacterium tumefaciens strain EHA 105, bearing a binary vector carrying the selectable markerNPTII and the scorable markeruidA gene. RHA266 was found to be particularly sensitive to in vitro culture and recalcitrant to organogenesis. Of the 3 methods used, only dehydration and rehydration withAgrobacterium gave rise to transient GUS expression in shoot apices and stably transformed plants as confirmed by Southern blotting analysis.     

Agrobacterium tumefaciens– mediated transformation of soybean [Glycine max (L.) Merrill.] using immature zygotic cotyledon explants

Agrobacterium tumefaciens -mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus. Received: 6 December 1999 / Revised: 11 February 2000 / Accepted: 14 March 2000     

Transient expression of β-glucuronidase in different cellular compartments following biolistic delivery of foreign DNA into wheat leaves and calli

Summary Transient expression of -glucuronidase (GUS) in different cellular compartments following biolistic delivery of chloroplast or nuclear expression vectors into wheat leaves or calli, derived from anther culture or immature embryos, is reported here. When pB1121, the nuclear GUS vector, was used to bombard wheat cells, the -glucuronidase product, an insoluble indigo dye, was observed evenly throughout the cytosol. But, when the chloroplast expression vector pHD203-GUS was used for bombardments, the indigo dye (GUS product) was subcellularly localized within the chloroplasts of wheat cells. The observation of GUS expression in albino plastids, when anther culture derived albino leaves were bombarded with the chloroplast expression vector pHD203-GUS, suggests the presence of a functional protein synthetic machinery in these organelles. GUS expression was also observed in regenerable calli derived from wheat immature embryos bombarded with pHD203-GUS. Leaves or calli bombarded with pUC19, as negative controls, did not show any GUS expression. These results constitute the first demonstration of foreign gene expression in chloroplasts of a monocot and that a dicot chloroplast promoter functions in a monocot chloroplast.     

A comparison of methods for direct gene transfer into maize (Zea mays L.)

Summary Techniques for transforming intact tissues of cereals were evaluated for their efficacy in transforming immature embryos and Type II callus of maize (Zea mays L.). The techniques used were particle bombardment, tissue electroporation, tissue electrophoresis, and silicon carbide fibers. Each method was assessed in terms of transient β-glucuronidase (GUS) expression. High levels of GUS expression were observed in A188 Type II callus using both tissue electroporation and particle bombardment, with means of 417.8 and 954.5 blue expression units (beu) per g fresh weight (FW) callus, respectively. Only particle bombardment resulted in high transient gene expression in immature embryos, with a mean transformation frequency of 34.8 b.e.u. per embryo. Very low levels of GUS expression were achieved with silicon carbide-mediated gene transfer, even when employing tissues used in the original publication (Black Mexican Sweet suspension cells). GUS expression was not obtained following tissue electrophoretic gene delivery.     

Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos

We report a new protocol for the stable transformation of Larix gmelinii. Thirty mature zygotic embryos precultured for 3 days on solid medium supplemented with benzyladenine were bombarded with plasmids pUC-GHG (GUS, HPT, and GFP genes) or pBI221-HPT (HPT and GUS genes). After a 2-month culture on selection medium, hygromycin-resistant calli appeared on the surfaces of the necrotic embryos. The frequencies of embryos with resistant calli were 18.4% and 17.4% in the transformations with pUC-GHG and pBI221-HPT DNA, respectively. More than 20 adventitious shoots formed from each of the transgenic calli. Of 17 elongated shoots selected for culturing on a rooting medium, five shoots rooted after 2 months. Expression of the GFP and GUS genes was detected in the resistant tissues by microscopic observations and by a histological GUS activity assay, respectively. PCR and Southern analysis confirmed the stable insertion of the introduced DNA into the genome.     

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     

Transient and stable transformation of wheat with DNA preparations delivered by a biolistic method

An optimized procedure for transformation of wheat with the use of a Biolistic Particle Delivery System PDS 1000/He to deliver foreign DNA is described in detail. The bacterial uidA and bar genes (both driven by plant promoters) were utilized as the reporter and selectable marker genes, respectively. Moderately high gas pressure appeared to be most important to achieve the highest level of transient GUS expression in target tissues. There was, however, no apparent correlation between transient and stable GUS expression. The presence of telomeric DNA sequences in an uidA gene-containing vector did not influence transient GUS expression but, apparently, prevented its stable expression. Mechanical lesions caused by the bombardment (tungsten particles) seemed to be less severe when embryo- derived calli, instead of freshly excised immature embryos, were used as the target tissue. The limited ability of callus cells for regeneration, together with a restricted number of cells that receive the foreign DNA by particle bombardment, result in a low efficiency of wheat stable transformation.     

Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum acstivum L.) mature embryos

We have demonstrated that foreign DNA can be delivered into cells of mature embryos of wheat (Triticum aestivum L.) using silicon carbide fibers (SCF). The highest transient expression of thegusA (GUS) gene was detected when dry embryos were vortexed for 10–30 min in a SCF-DNA solution containing 90–120 g/l of sucrose. Up to 100 (on average 20–40) blue expression units per embryo were observed. Scutellum side and epiblast of the intact wheat embryos are preferentially transformed. When embryos with the coleoptilar tip removed were treated and allowed to germinate, GUS staining was observed in emerging leaf tissues. The potential of this new approach for stable transformation of wheat is under investigation. It has been found that callus tissues induced from the SCF treated embryos contain GUS-expressing sectors one month after treatment.     

Histological analysis of direct somatic embryogenesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.     

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in an important Cavendish banana cv. Robusta (AAA)

Establishment of an efficient protocol for regeneration and genetic transformation is required in banana for the incorporation of useful traits. Therefore an efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of Cavendish banana cultivar Robusta (AAA). Embryogenic cell suspension culture (ECS) was established using immature male flowers. Percentage appearance of embryogenic callus and distinct globular embryos was 10.3 and 11.1, respectively. ECS obtained was cocultivated under different cocultivation conditions with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA 1301 plant expression vector. Up to 30 transgenic plants/50 mg settled cell volume (SCV) was obtained with cocultivation in semisolid medium whereas no transgenics could be obtained with parallel experiments carried out in liquid medium. Histochemical GUS assay in different tissues of putatively transformed plants demonstrated expression of uidA gene. Among the putatively transformed plants obtained, a set of 4 were confirmed by PCR analysis and stable integration of the transgene by Southern analysis. GUS specific activity measured by a MUG (4-methylumbelliferyl-β-d-glucuronide) based flourometric assay revealed increase in transient GUS expression in semisolid as well as liquid cocultivation with centrifugation. This is the first report showing somatic embryogenesis and Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in an important Cavendish banana cultivar Robusta. The present protocol will make possible agronomic improvement of this important commercially grown cultivar by introduction of disease resistance characteristics and antisense-mediated delayed fruit ripening strategies. Further, it will also assist in functional characterization of new gene or promoter elements isolated from this or other cultivars of banana.     

Physical,chemical and physiological parameters for electroporation-mediated gene delivery into rice protoplasts

Transformation of cereal protoplasts has been reported using several methods; however, the efficiencies of transformations are still very low. We have evaluated a number of parameters that influence electroporation-mediated DNA uptake and have also compared the efficiency of transient GUS activity and stable transformation obtained using an optimized electroporation method with that of the PEG method. The electroporation conditions tested were ionic composition of buffer, ionic strength, resistivity of buffer, type of anions, voltage, and capacitance.Protoplasts isolated from suspension cultures derived from immature embryos of rice (cvs Radon and IR-54) were used for this study. Stable transformation or transient GUS expression experiments were carried out using a plasmid construct containing the CaMV 35S promoter driving thebar gene and a rice actin promoter driving thegus A (uid A) gene (pAG35bar). Electroporation under optimized conditions resulted in about 13-fold higher GUS activities compared to the PEG method. Protoplast survival following optimized electroporation conditions was 55–60%, compared to 35–40% with the PEG treatment. Protoplasts isolated from a suspension culture at different ages gave substantially different levels of transient GUS expression following electroporation-mediated DNA uptake. In contrast, the age of the suspension culture did not influence PEG-mediated DNA uptake and transient GUS activities, which remained low throughout the culture period examined (21 months). Putatively transformed calluses were selected after three to four weeks on medium containing phosphinothricin as the selection agent. The transformation frequencies ranged from 6.2×10^–5 to 5.4×10^–4 with the electroporation method compared to 1.3×10^–5 to 5.3×10^–5 with the PEG method. Southern blot analysis of PPT-resistant calluses obtained by the electroporation-mediated transformation showed simple intergration patterns of integrated DNA in most of the transformants.     

The physiological state of suspension cultured cells affects the expression of the β-glucuronidase gene following transformation of tall fescue (Festuca arundinacea) protoplasts

Both the stage of the growth cycle and the age of the cell culture used to isolate protoplasts had a pronounced effect on both transient and stable expression of the GUS gene. A level of GUS gene transient expression of 9000 pmol 4MU/μg protein/h and a frequency of GUS gene stable expression of 5.72% were obtained with protoplasts isolated from suspension cultures 10–20 weeks after initiation and 3–4 days after subculturing when an optimized transformation protocol and a rice actin 1 promoter-uidA gene construct were used. The effect of the cell growth cycle on GUS gene transient expression was closely correlated with the growth rate and the rate of protein synthesis in cell cultures whereas prolonged subculturing of the cells resulted in a gradual decline in both transient and stable expression. The length of time cells were digested in cell wall digestion enzyme and the osmolarity of the transformation medium were found to critically affect both the level of transient and stable GUS gene expression. The composition and osmolarity of the protoplast culture medium was less critical for transient GUS gene expression although the osmolarity of the medium was shown to have a significant effect on stable expression of the GUS gene.