Fertile transgenic wheat from microprojectile bombardment of scutellar tissue

A reproducible transformation system for hexaploid wheat was developed based on particle bombardment of scutellar tissue of immature embryos. Particle bombardment was carried out using a PDS 1000/He gun. Plant material was bombarded with the plasmid pDB1 containing the β-glucuronidase gene ( uidA ) under the control of the actin-1 promoter of rice, and the selectable marker gene bar (phosphinothricin acetyltransferase) under the control of the CaMV 35S promoter. Selection was carried out using the herbicide Basta (Glufosinate-ammonium). From a total number of 1050 bombarded immature embryos, in seven independent transformation experiments, 59 plants could be regenerated. Putative transformants were screened for enzyme activity by the histochemical GUS assay using cut leaf material and by spraying the whole plants with an aqueous solution of the herbicide Basta. Twelve regenerants survived Basta spraying and showed GUS-activity. Southern-blot analysis indicated the presence of introduced foreign genes in the genomic DNA of the transformants and both marker genes were present in all plants analysed.
To date, four plants have been grown to maturity and set seed. Histochemically stained pollen grains showed a 1:1 segregation of the uidA gene in all plants tested. A 3:1 segregation of the introduced genes was demonstrated by enzyme activity tests and Southern blot analysis of R₁ plants.     

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     

Production of herbicide-resistant sweet potato plants transformed with the <Emphasis Type="Italic">bar</Emphasis> gene

Herbicide-resistant sweet potato plants were produced through biolistics of embryogenic calli derived from shoot apical meristems. Plant materials were bombarded with the vectors containing the β-glucuronidase gene (gusA) and the herbicide-resistant gene (bar). Selection was carried out using phosphinothricin (PPT). Transformants were screened by the histochemical GUS and Chlorophenol Red assays. PCR and Southern-blot analyses indicated the presence of introduced bar gene in the genomic DNA of the transgenic plants. When sprayed with Basta, the transgenic sweet potato plants was tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to sweet potato.     

An efficient particle bombardment system for the genetic transformation of asparagus (Asparagus officinalis L.)

The microprojectile bombardment method was used to transfer DNA into embryogenic callus of asparagus (Asparagus officcinalis L.) and to produce stably transformed asparagus plants. Embryogenic callus, derived from UC 157 and UC72 asparagus cultivars, was bombarded with tungsten particles coated with plasmid DNA that contained genes encoding hygromycin phosphotransferase, phosphinothricin acetyl transferase and -glucuronidase. Putatively transformed calli were identified from the bombarded tissue after 4 months selection on 25 mg/L hygromycin B plus 4 mg/L phosphinothricin (PPT). By selecting embryogenic callus on hygromycin plus PPT the overall transformation and selection efficiencies were substantially improved over selection with hygromycin or PPT alone, where no transgenic clones were recovered. The transgenic nature of the selected material was demonstrated by GUS histochemical assays and Southern blot hybridization analysis. Transgenic asparagus plants were found to withstand the prescribed levels of the PPT-based herbicide BASTATM for weed control.Abbreviations GUS -glucuronidase - HPT hygromycin phosphotransferase - bar phosphinothricin acetyl transferase gene - PPT phosphophinothricin - NAA naphthalene acetic acid - 2iP 2-isopentenyl adenine     

Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells

Summary Suspension cells of Oryza sativa L. (rice) were transformed, by microprojectile bombardment, with plasmids carrying the coding region of the Streptomyces hygroscopicus phosphinothricin acetyl transferase (PAT) gene (bar) under the control of either the 5 region of the rice actin 1 gene (Act1) or the cauliflower mosaic virus (CaMV) 35S promoter. Subsequently regenerated plants display detectable PAT activity and are resistant to BASTA^TM, a phosphinothricin (PPT)-based herbicide. DNA gel blot analyses showed that PPT resistant rice plants contain a bar-hybridizing restriction fragment of the expected size. This report shows that expression of the bar gene in transgenic rice plants confers resistance to PPT-based herbicide by suppressing an increase of ammonia in plants after spraying with the herbicide.     

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbi1-gas plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs.Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T1 generation.     

Establishment of a micro-particle bombardment transformation system for Dunaliella salina

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. salina with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 microg/ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.     

Optimization of particle bombardment parameters for DNA delivery into the male flowers of banana

The possibility of increasing the efficiency of banana transformation was investigated by particle bombardment of the male flowers of banana plants for constitutive expression of gfp gene. The effects of particle bombardment parameters, such as acceleration pressure, bombardment distance, chamber vacuum pressure, gold microcarrier size, gold quantity, DNA quantity, number of bombardments and pre-culture were examined. Single cauliflower-like bodies (CLBs) clusters, induced from meristemic parts of Musa sapientum cv. Nangka (AAB) male flowers, were bombarded by pCambia1304 plasmid carrying gfp gene driven by the CaMV 35S promoter. Optimal transient expression of green-fluorescent protein (GFP) was obtained when the three-day old cultured tissues were bombarded two times at 1100 psi helium pressure. However, the highest GFP expression was observed when 9 cm was applied as bombardment distance with 28 mmHg chamber vacuum pressure. Gold particle with 1 μm diameter at 60 μg/μL concentrations coated with 1.5 μg/μL of DNA have been used as the optimum bombardment parameter since GFP expression was significantly different compared to other conditions. Application of optimized condition proved effective for the generation of stable transgenic banana plants. PCR and southern blot analyses confirmed the presence and integration of gfp gene in genomic DNA of transformed plants. Transformation frequency achieved with the optimized protocol was 7.5% which was significantly higher than the conventional protocol.     

Self-Fertile Transgenic Wheat Plants Expressing Herbicide Bromoxynil Resistance

Genetic manipulation of wheat (Triticum aestivum L. ) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation method. The authors report a reproducible protocol for rapid production of transgenic wheat via microprojectile bombardment. The experiment was carried out by using the immature embryo excised from caryopsis 14 to 18 days postanthesis and the plant expression plasmid carrying a CaMV 35S-controlled bxn gene, for resistance to herbicide bromoxynil and a selectahle marker gene NPT I. After bombarding the precultured immature embryos isolated from 13 wheat varieties with plasmid DNA-coated tungsten particle, these embryos were transferred on MS medium containing 10 mg/L geneticin G418 sulphate to select and regenerate transformants step by step. As a result, 16 transformed plants were obtained from a total of 849 bombarded embryos. The characterization of these plants by inoculation with herbicide bromoxynil and Southern analysis with bxn gene as a probe showed that 4 of the self-fertile transformed plants contained the target gene and presented herbicide resistance. In several independent transformation experiments, the fastest one took only 6 months from embryo excision to characterization of regenerated plants. Therefore, this procedure is a rapid and efficient technique for delivering foreign DNA into wheat.     

Genetic transformation of Leymus chinensis with the PAT gene through microprojectile bombardment to improve resistance to the herbicide Basta

Chinese leymus [Leymus chinensis (Trin.) Tzvel.] is a perennial grass (tribe Gramineae) that is widely distributed throughout northern China and Mongolia where it is produced as a forage product. Severe production losses due to weed growth have serious economic consequences, and as non-selective herbicides not only kill the weeds but are also harmful to this forage grass, the introduction of a foreign gene for resistance to the herbicide Basta is necessary since this species lacks herbicide resistance. We have investigated the transformation of a gene for phosphinothricin acetyltransferase (PAT) through microprojectile bombardment in Chinese leymus. Calli from immature inflorescences cultured on N6 medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/l of glutamine were bombarded. The bombarded calli survived on selection medium with 1.0 mg/l of phosphinothricin (PPT). Twenty-three plantlets regenerated from resistant calli on differentiation medium supplemented with 1.0 mg/l 6-benzylaminopurine, 1.0 mg/l kinetin, and 1.0 mg/l PPT, and five of these regenerated plantlets survived on rooting medium with 1.0 mg/l of PPT. PCR and Southern blotting analyses indicated that the PAT gene had been integrated into the genomes of two Chinese leymus plantlets and that the gene was stably transferred to its clonal offsprings. There were no other phenotypic effects associated with transgene expression during vegetative growth except tolerance to the herbicide Basta.The Biotechnology of Pasture Plant Program is funded by the Key Project of the Chinese Academy of Sciences (KSCX1-08)     

Transformation of potato (Solanum tuberosum) using particle bombardment

Internodes, leaves and tuber slices from potato (Solanum tuberosum), genotype 1024-2, were subjected to particle bombardment. Transient expression was optimized using the uidA and the luc reporter genes that encode #-glucuronidase (GUS) and luciferase, respectively. Stable transformation was achieved using the neomycin phosphotransferase (nptII) gene, which confers resistance to the antibiotic kanamycin. The influence of biological parameters (tissue type, growth period before bombardment, pre- and post-bombardment osmoticum treatment) and physical parameters (helium pressure, tissue distance) that are known to possibly affect stable transformation were investigated. Putative transgenic plants, which rooted in media containing kanamycin, were obtained from all of the tissues tested although there were large differences in the efficiency: internodes (0.77 plants per bombarded explant), microtuber slices (0.10 plants per bombarded explant) and leaves (0.02 plants per bombarded explant). Southern blot analysis of putative transgenic plants confirmed the integration of the transgenes into plant DNA. The results indicate that an efficient particle bombardment protocol is now available for both transient and stable transformation of potato internodal segments, thus contributing to an enhanced flexibility in the delivery of transgenes to this important food crop.     

Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences

Summary Transgenic sorghum plants (Sorghum bicolor L. Moench, cv. SRN39) were obtained by microprojectile-mediated DNA delivery (Bio-Rad PDS 1000/He Biolistic Delivery System) to explants derived from immature inflorescences. Explants were precultured on medium supplemented with 2.5 mg/l (11.31 μM) 2,4-D, 0.5 mg/l (2.32 μM) kinetin, and 60 g/l sucrose for 1 to 2 wk prior to bombardment. Bialaphos selectron pressure was imposed 2 wk after bombardment and maintained throughout all the culture stages leading to plant regeneration. More than 2500 explants from 1.5 to 3.0 cm inflorescences were bombarded and subjected to bialaphos selection. Out of more than 190 regenerated plants, 5 were determined to be Ignite resistant. Southern analyses confirmed the likelihood that the 5 herbicide resistant plants derived from two independent transformation events. The phosphinothricin acetyltransferase gene (bar) was inherited by and functionally expressed in T₁ progeny. However, no β-glucuronidase (GUS) activity could be detected in T₁ plants that contained uidA restriction fragments. Histological analyses indicated that in the absence of bialaphos morphogenesis was primarily via embryogenesis while organogenesis was more predominant in callus maintained with herbicide selection.     

Genetic transformation of Nicotiana africana Merxm. with plasmids containing lox recombination

An efficient genetic transformation method for african tobacco Nicotiana africana Merxm. has been established. African tobacco is a valuable source for cytoplasmic male sterility (CMS) and nuclear encoded resistance to potato virus Y (PVY). N. africana transgenic plants have been obtained using both Agrobacterium-mediated and direct transformation of leaf explants with gold particle bombardment using particle inflow gun. Plasmid vectors containing phosphinothricin resistance gene (bar gene) coding region without promoter and independent 35S promoter between lox sites (lox-bar-35S-lox) and nptII gene were used. Transgenic plants were selected according to growth capacity on the selective medium containing 50 mg/l kanamycin. PCR analyses of kanamycin-resistant plants confirmed the presence of nptII and bar genes in their genome. Agrobacterium-mediated transformation of root explants has proved to be the most efficient transformation method for N. africana.     

Rapid Production of Multiple Independent Lines of Fertile Transgenic Wheat (Triticum aestivum)

Improvement of wheat (Triticum aestivum) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation methodology. In this report, we detail a protocol for transformation of a highly embryogenic wheat cultivar, Bobwhite. Calli derived from immature embryos, 0.5 to 1 mm long, were bombarded with microprojectiles coated with DNA containing as marker genes the bar gene, encoding phosphinothricin-resistance, and the gene encoding [beta]-glucuronidase (GUS), each under control of a maize ubiquitin promoter. The bombardment was performed 5 d after embryo excision, just after initiation of callus proliferation. The ability of plantlets to root in the presence of 1 or 3 mg/L of bialaphos was the most reliable selection criteria used to identify transformed plants. Stable transformation was confirmed by marker gene expression assays and the presence of the bar sequences in high molecular weight chromosomal DNA of the resultant plants. Nine independent lines of fertile transgenic wheat plants have been obtained thus far, at a frequency of 1 to 2 per 1000 embryos bombarded. On average, 168 d elapsed between embryo excision for bombardment and anthesis of the T0 plants. The transmission of both the resistance phenotype and bar DNA to the T1 generation verified that germline transformation had occurred.     

Engineering herbicide resistance in plants by expression of a detoxifying enzyme

Phosphinothricin (PPT) is a potent inhibitor of glutamine synthetase in plants and is used as a non-selective herbicide. The bar gene which confers resistance in Streptomyces hygroscopicus to bialaphos, a tripeptide containing PPT, encodes a phosphinothricin acetyltransferase (PAT) (see accompanying paper). The bar gene was placed under control of the 35S promoter of the cauliflower mosaic virus and transferred to plant cells using Agrobacterium-mediated transformation. PAT was used as a selectable marker in protoplast co-cultivation. The chimeric bar gene was expressed in tobacco, potato and tomato plants. Transgenic plants showed complete resistance towards high doses of the commercial formulations of phosphinothricin and bialaphos. These data present a successful approach to obtain herbicide-resistant plants by detoxification of the herbicide.     

Herbicide-tolerant Transgenic Pineapple (Ananas comosus) Produced by Microprojectile Bombardment

Transformed plants of the commercially important Thai pineapple(Ananas comosus‘Phuket’) were produced followingmicroprojectile-mediated delivery of the plasmid AHC25, carryingthe ß-glucuronidase (gus) reporter gene and the bialaphosresistance (bar) gene for herbicide tolerance, into leaves ofmicropropagated shoots. Transformed plants were regeneratedfrom bombarded leaf bases on Murashige and Skoog-based mediumcontaining 0.5 mg l^-12,4-dichlorophenoxyacetic acid, 2.0 mgl^-16-benzylamino purine and 0.5 mg l^-1phosphinothricin. Integrationand expression of thebar gene in regenerated plants was confirmedby Southern analysis and RT-PCR, respectively. Regenerated plantswere assessed in vitro and under glasshouse conditions for theirtolerance to the commercial herbicide Basta^TM, containing glufosinateammonium as the active component. Plants sprayed with Basta^TMcontainingconcentrations of glufosinate ammonium up to 1400 mg l^-1remainedhealthy and retained their pigmentation. The generation of herbicide-tolerantpineapple will facilitate more efficient weed control in thiswidely cultivated tropical crop. Copyright 2001 Annals of BotanyCompany bar gene, Biolistics, herbicide tolerance, pineapple, phosphinothricin (PPT)     

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     

Transformation of peanut with a soybean vspB promoter‐uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut ( Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi‐quantitation of transient expression. One hundred and sixty hygromycin‐resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R₁ seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.     

Multicellular genesis of leaf primordium was demonstrated via chimaeric transgenic plant of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) regenerated from Type II calli

Type-II embryonic calli were induced from immature embryos of maize (Zea mays L.) genotype YD and bombarded with beta-glucuronidase gene. Bombarded calli were proliferated on normal N6 medium for 2 weeks at 26°C in the dark and selected on N6 medium containing 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 mg/l phosphinothricin (PPT) but without casamino acids and proline under the same conditions for 14 days. Regeneration was carried out on hormone-free MS medium containing 5 mg/l phosphinothricin at 26°C under 3000 lux illumination. Plants over 8 cm were transplanted into soil and sprayed with 250 mg/l phosphinothricin when two new leaves appeared. Except normal transgenic plants, chimaeric transgenics also were regenerated in the present work. The expression pattern of beta-glucuronidase gene in leaves of chimaeric transgenic plant revealed that more than one cell formed leaf primordium at the initial stage, and filial cells stemed from each cell in leaf primordium arranged in a row longitudinally from leaf base to leaf apex. There was a clear boundary as a straight line between the area formed by transformed cells and the area formed by normal cells. A hypothesis was put forward that the primitive cells in leaf primordium divided in a longitudinal style, resulted in leaf elongation, then the filial cells divided transversally and synchronously toward the outside to broaden the leaf.     

Bialaphos selection of stable transformants from maize cell culture

Summary Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for -glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.