Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

基因枪轰击谷子幼穗获得转基因植株

以ＪＱ－７００型国产基因枪轰击豫谷２号谷子幼穗,在１５０ｍｇ／Ｌ卡那霉素选择培养基上筛选到９０８块抗性愈伤组织,其中,绿芽块愈伤５块,共分化出１６株绿苗,组织化学检测ＧＵＳ表达,获得３株阳性植株,Ｓｏｕｔｈｅｒｎ杂交证明１株为阳性。以轰击总外植体计算的转基因植株频率为０．０５％。     

Production of transgenic gentian plants by particle bombardment of suspension-culture cells

 Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l^–1 hygromycin in liquid MS medium that contained 10 mg l^–1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l^–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l^–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l^–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA. Received: 3 June 1999 / Revision received: 21 September 1999 / Accepted: 20 September 1999     

Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment

The relationship between transgene copy number, rearrangement levels, inheritance patterns, expression levels, transgene stability and plant fertility was analysed in a random population of 95 independently transformed rice plant lines. This analysis has been conducted for both the selectable marker gene ( aphIV) and the unselected reporter gene ( gusA), in the presence or absence of flanking Matrix Attachment Regions (MARs) in order to develop a better understanding of transgene behaviour in a population of transgenic rice plants created by particle bombardment. In the first generation (T(0)), all the independently transformed plant lines contained and expressed the aphIV gene conferring resistance to hygromycin, but only 87% of the lines were co-transformed with the unselected gusA marker gene. Both transgenes seemed to be expressed independently. Most lines exhibited complex transgene rearrangements as well as an intact transgene expression unit for both aphIV and gusA transgenes. Transgene copy number was proportional to the quantity of DNA used during bombardment. In T(0) plants, high gusA copy number significantly decreased GUS expression levels but there was no correlation between expression level and transgene copy number across the entire population of lines. Four main factors impaired transgene expression in primary transgenic plants (T(0)) and their progeny (T(1)): (1) absence of transgene expression in T(0) plants (41% of lines), (2) sterility of T(0) plants (28% of lines), (3) non-transmission of intact transgenes to some or all progenies (at least 14% of lines), and (4) silencing of transgene expression in progeny plants (10% of lines). Transgene stability was significantly related to differences in transgene structure and expression levels. The presence of Rb7 MARs flanking the gusA expression unit had no effect on plant fertility or non-transmission of transgenes, but provided copy number-dependent expression of the transgene and improved expression levels and stability over two generations. Overall, only 7% of the plant lines without MARs and 17% of the lines with MARs initially generated, exhibited stable transgene expression over two generations.     

Stable genetic transformation of garlic plants using particle bombardment

The improvement of garlic plants (Allium sativum L.) via biotechnological approaches is currently limited by the lack of an applicable direct gene transfer system. In this paper, we present the development of a genetic transformation system using particle bombardment for gene delivery and immature clove-derived callus as the gene target. Plasmid DNA (pBI221.23), containing the selectable "hpt" gene for hygromycin resistance and the reporter "gus" gene, was delivered into callus tissue that had been previously treated with aurintricarboxylic acid as an endogenous nuclease inhibitor. The transformed calli were selected using hygromycin B, regenerated, and analysed at the molecular level using DNA hybridization, transgenome rescue and histochemical beta-glucuronidase assay. The results indicated that biolistic transformation can lead to the transfer, expression and stable integration of a DNA fragment into garlic chromosomal DNA. The relative simplicity of this system is a good recommendation for its future use in the production of genetically modified garlic plants.     

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.     

Recovery of transgenic orchid plants with hygromycin selection by particle bombardment to protocorms

Protocorms of orchid (Dendrobium hybrid) were transformed by microprojectile bombardment with a helium-pressured PDS 1000 particle gun. Gold particles coated with plasmid DNA containing ß-glucuronidase (GUS) and hygromycin phosphotransferase (Hpt) marker genes were used. Potentially transformed tissues were identified by active growth on MS medium supplemented with 50mg l^-1 hygromycin. After 4–6 months of continuous selection, 15 hygromycin-resistant lines were recovered. Integration of transgenes into the genome of the transformed protocorms and plantlets were confirmed by GUS histochemical assay and Southern blot hybridization. The transgenic protocorms have gone through propagation for more than 8 months and maintained their transgenic characters. These results indicate that we have established a system for orchid transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants.     

Stable transformation of papaya via microprojectile bombardment

Summary Stable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1^–1 2,4-D, 400 mg 1^–1 glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1^–1 kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS,Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - X-gluc 5-Br-4-Cl-3-indolyl--D-glucuronic acid - CaMV cauliflower mosaic virus - NOS nopaline synthase - NPTII neomycin phosphotransferase II Journal Series no. 3448 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.     

Generation of chlorsulfuron-resistant transgenic garlic plants (Allium sativum L.) by particle bombardment

We established an effective biolistic transformation procedure fortransferring foreign genes into garlic (Allium sativumL.),which we demonstrated by generating transgenic plants resistant tochlorsulfuron, a sulfonylurea herbicide. We subcultured callus tissue from theapical meristem of garlic cloves and repeatedly selected calli with brittle,non-mucilaginous surfaces for over six months, to increase transformationefficiency. We then constructed recombinant DNA that contained the acetolactatesynthase (ALS) gene from a chlorsulfuron-resistantArabidopsis mutant, the cauliflower mosaic virus 35Spromoter, the -glucuronidase (GUS) reporter gene, and the hygromycinphosphotransferase (HPT) selectable marker gene. The garlic calli werebombarded twice with tungsten particles coated with the DNA constructs. Transformed calliwere efficiently selected by embedding them in solid agar medium containing 50mg l^–1 hygromycin B. Selected propagules wereregenerated into 12 independent plants. We confirmed that the transgenes wereintegrated and expressed in the plants using PCR-Southern and Northern blotanalyses and by -glucuronidase expression assay forGUS. The regenerated plants survived in the presence of 3mg l^–1 chlorsulfuron, demonstrating that theirALS was insensitive to this herbicide. These results illustrate the successfultransformation of foreign genes into garlic plants. The set of proceduresdeveloped in this study is applicable to the generation of transgenic garlicplants with other agronomically beneficial traits. These authors contributed equally to this work     

Genetic transformation of Linum by particle bombardment

Summary Linseed flax (Linum usitatissimum L.) was transformed by bombarding hypocotyl tissues with gold particles coated with plasmid DNA carrying the β-glucuronidase (GUS) (uid-A) and neomycin phosphotransferase II (npt-II) genes. Transient expression of the introduced β-glucuronidase gene was used to study factors influencing the DNA delivery, while progeny analyses confirmed stable transformation. The efficiency of DNA delivery, uptake and expression was significantly affected by the duration of hypocotyl preculture, bombardment distances, the level of chamber vacuum, the quantity of DNA, and the size of particles. Nineteen independent GUS-positive shoots were recovered and regenerated into whole plants, from which 10 plants successfully produced viable seeds. Analysis of T₁ and T₂ self pollinated progeny for histochemical and fluorometric GUS assays and polymerase chain reaction (PCR) analyses for uid-A, plus npt-II PCR and germination assays in progeny plants demonstrated that the transgenes were expressed in selected plants and transmitted to progeny, usually via a single Mendelian locus. The results show that particle bombardment can be used to produce transgenic Linum plants. The system is rapid, simple and offers an alternative to Agrobacterium methods.     

Genetic transformation of maize cells by particle bombardment

Intact maize cells were bombarded with microprojectiles bearing plasmid DNA coding for selectable (neomycin phosphotransferase [NPT II]) and screenable (β-glucuronidase [GUS]) marker genes. Kanamycin-resistant calli were selected from bombarded cells, and these calli carried copies of the NPT II and GUS genes as determined by Southern blot analysis. All such calli expressed GUS although the level of expression varied greatly between transformed cell lines. These results show that intact cells of important monocot species can be stably transformed by microprojectiles.     

Genetic transformation of Cymbidium orchid by particle bombardment

 A protocol is presented for genetically engineering Cymbidium orchid using particle bombardment. This protocol enabled the routine transformation of orchid plants that were previously difficult to transform. Liquid culture was used to generate a large number of protocorm-like bodies (PLBs) to be bombarded and to promote continued development of the bombarded meristematic tissue. Plasmid DNA (pKH200) carrying the GUS-INT and NPTII genes flanked by tobacco matrix attachment regions was introduced into the meristematic cells of PLBs by particle acceleration. The transformed PLBs were proliferated and selected for kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was then induced from the kanamycin-resistant PLBs, and transgenic plantlets were produced. Both the kanamycin-resistant PLBs and regenerated shoots expressed the GUS-INT gene. The presence of the introduced gene in the transformed orchid plants was confirmed by PCR analysis, sequencing and Southern blot analysis of the PCR product. The recovered transgenic plants were established in soil and acclimatized in the greenhouse. Received: 20 July 1998 / Revision received: 2 December 1998 / Accepted: 17 December 1998     

Stable transformation of Eustoma grandiflorum by particle bombardment

Explants (7.5±2.5 mm) cut from stems and roots of 3-week-old Eustoma grandiflorum Grise, (lisianthus) cv. Glory White seedlings were bombarded with plasmid pBI221, which harbors the uidA gene encoding β-glucuronidase (GUS) driven by the cauliflower mosaic virus (CaMV) 35S promoter. More than 800 blue spots of GUS-expressing cells were observed per 90 explants. Explants bombarded with pARK22 harboring the bar gene encoding phosphinothricin acetyltransferase driven by the CaMV 35S promoter were selected for bialaphos resistance. Putative transgenic plants were obtained about 3 months after bombardment. Southern blot analysis of putative transgenic plants revealed the presence of the bar gene in their genome. Received: 10 April 1996 / Revision received: 7 November 1997 / Accepted: 22 November 1997     

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     

Development of an efficient transformation system for Catharanthus roseus cell cultures using particle bombardment

We have developed an efficient direct DNA transfer procedure for the facile engineering of Catharanthus roseus cell cultures. Particle bombardment of callus derived from leaf material permitted rapid selection and establishment of transgenic cell lines. Transgenic callus were recovered at a frequency of between 60–80% of total callus bombarded with a single plasmid. Bombardment using two separate plasmids resulted in a 25–60% frequency of transgenic callus recovered, up to 90% containing both input plasmids. Between 10–20 g FW of transgenic material was produced within 3 months of bombardment, providing sufficient material for molecular and biochemical analyses. We developed two complementary systems allowing selection on either hygromycin or kanamycin to permit re-transformation using plasmids carrying additional genes of interest. Use of leaf tissue as explant for transformation avoids time-consuming and labor intensive procedures involving suspension cultures. We provide molecular data on integration and expression of selected and non selected transgenes in a number of transgenic callus lines. Transgene integration events for co-transformed plasmids were relatively simple, occurring at one or two sites in the genome for most of the lines we analysed. Molecular analysis of callus resulting from co-transformation experiments using two different plasmids revealed that in nine of 10 putative transgenic lines we selected for analysis both plasmids had integrated into the genome. RNA gel-blot analysis and histochemical staining showed that an unselected transgene, gusA, was expressed in seven of the ten lines we analysed.     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.