Production of selectable marker-free transgenic tobacco plants using a non-selection approach: chimerism or escape,transgene inheritance,and efficiency

Public concern and metabolic drain were the main driving forces for the development of a selectable marker-free transformation system. We demonstrated here the production of transgenic tobacco plants using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. A. tumefaciens-infected leaf explants were allowed to produce shoots on a shoot induction medium (SIM) containing no selective compounds. Up to 35.1% of the A. tumefaciens-infected leaf explants produced histochemically GUS⁺ shoots, 3.1% of regenerated shoots were GUS⁺, and 72% of the GUS⁺ shoots were stably transformed by producing GUS⁺ T1 seedlings. When polymerase chain reaction (PCR) was used to screen the regenerated shoots, 4% of the shoots were found to be PCR⁺ for the transgene and 65% of the PCR⁺ shoots were stable transformants. Also, generation of PCR⁺ escapes decreased linearly as the number of subculture increased from one to three on SIM containing the antibiotic that kills the Agrobacterium. Twenty-five to 75% of the transformants were able to transmit transgene activity to the T1 generation in a Mendelian 3:1 ratio, and a transformation efficiency of 2.2–2.8% was achieved for the most effective binary vector. These results indicated that majority of the GUS⁺ or PCR⁺ shoots recovered under no selection were stable transformants, and only one-third of them were chimeric or escapes. Transgenes in these transgenic plants were able to transmit the transgene into progeny in a similar fashion as those recovered under selection.     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

Loss of decreased-rubisco phenotype between generations of wheat transformed with antisense and sense rbcS

The elite UK winter wheat cv. Riband was transformed with constructs containing rbcS in sense and antisense orientations driven by the maize ubiquitin promoter with a transformation efficiency of 1.2%. Of 77 primary transformants 31% of the sense-rbcS transformed lines and 78% of the antisense-rbcS transformed lines had decreased rubisco content compared to wild-type and marker-only controls, with decreases of up to 60%. However, in the T1 progeny which inherited the transgene, only 5% showed significantly decreased rubisco content and these effects were on the margins of significance. Five potential T2 homozygous lines from T1 parents which had transgene segregation consistent with a single locus were identified. There was no significant decrease in rubisco content relative to wild-type in any of these lines (LSD of 8% for P= 0.05). Expression of antisense rbcS transgenes in two of these T2 lines was low but was increased following exposure of the plants to 37°C for 48 h. However this did not induce a significant decrease in rubisco protein content relative to controls. Southern analysis of two antisense lines showed that they had low copy number and 1–2 insertion events. In one of the two lines there was increased methylation of the ubiquitin intron in T2 samples compared to the TO primary transformant. Further work is required to establish whether methylation occurred in all the lines which lost the phenotype, and therefore the likelihood of this being the cause. The disappearance of the decreased rubisco-content phenotype between generations may therefore be attributable to (1) greater activity of the ubiquitin promoter due to greater stress in the T0 generation plants and/or (2) increased methylation of the transgene promoter region between generations.     

Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants

The ease of integrative transformation with foreign genes and the extent of their expression and stability in successive generations determine the applicability of direct gene transfer. InNicotiana plumbaginifolia, one to ten copies of foreign DNA were integrated into the plant genome, resulting in simple to complex patterns of integration. Genetic analysis showed that in more than 50% of the cases, this DNA inserted at two or more loci in the genome. Of the 156 crosses performed between F₁ monogenic transformants, only eight combinations showed linkage of the inserted neomycin phosphotransferase genes (npt). The following instability events were registered: physical loss, alterations in the initial segregation rates in successive meiotic generations observed in either selfing or crossing (reduction or increase in number of segregating loci) and genomic disorders in crosses between transformants. Among them of particular interest were the discordant segregation values observed between corresponding R₁ and F₁ progenies in up to 9% of the evaluated transformants. In addition, 5% of the transformants showed a phenotypic loss of resistance. In the F₃ generation, 5 out of 15 transformants exhibited instability, which was transmitted to the F₄ generation. Further increases in instability rates were observed with higher numbers of insertion loci and in crosses between independent transgenic plants, reaching 100% when a trigenic partner was involved.N. plumbaginifolia exhibited more instability thanN. tabacum under equivalent experimental conditions. The molecular bases of such instability events are discussed in relation to DNA methylation, co-suppression and genomic imbalance.     

Transposon-mediated generation of T-DNA- and marker-free rice plants expressing a Bt endotoxin gene

Transposon-mediated repositioning of transgenes is an attractive strategy to generate plants that are free of selectable markers and T-DNA inserts. By using a minimal number of transformation events a large number of transgene insertions in the genome can be obtained so as to benefit from position effects in the genome that can contribute to higher levels of expression. We constructed a Bacillus thuringiensis synthetic cry1B gene expressed under control of the maize ubiquitin promoter between minimal terminal inverted repeats of the maize Ac-Ds transposon system, which was cloned in the 5' untranslated sequence of a gfp gene used as an excision marker. The T-DNA also harboured the Ac transposase gene driven by the CaMV 35S promoter and the hph gene conferring resistance to the antibiotic hygromycin. Sixty-eight independent rice (Oryza sativa L.) transformants were regenerated and molecularly analysed revealing excision and reinsertion of the Ds-cry1B element in 37% and 25% respectively of the transformation events. Five independent transformants harbouring 2–4 reinserted Ds-Cry1B copies were analysed in the T1 progeny, revealing 0.2 to 1.4 new transpositions per plant. Out segregation of the cry1B gene from the T-DNA insertion site was observed in 17 T1 plants, representing 10 independent repositioning events without selection. Western analysis of leaf protein extracts of these plants revealed detectable Cry1B in all the plants indicating efficient expression of the transgene reinsertions. Stability of position and expression of the cry1B transgene was further confirmed in T2 progeny of T-DNA-free T1 plants. New T-DNA-free repositioning events were also identified in T2 progenies of T1 plants heterozygous for the T-DNA. Furthermore, preliminary whole plant bioassay of T-DNA-free lines challenged with striped stem borer larvae suggested that they are protected against SSB attacks. These results indicate that transposon mediated relocation of the gene of interest is a powerful method for generating T-DNA integration site-free transgenic plants and exploiting favourable position effects in the plant genome.     

Cytosine deaminase as a substrate-dependent negative selectable marker in Brassica napus

The enzyme cytosine deaminase, encoded by the codA gene, catalyzes the deamination of the non- toxic compound 5-fluorocytosine (5-FC) to the highly toxic compound 5-fluorouracil (5-FU). Cytosine deaminase activity is not found in higher plants and Brassica napus seedlings are unaffected by the presence of 5-FC in the growth medium. In codA-transformed B. napus seedlings, expression of cytosine deaminase results in a reduction of root and hypocotyl lengths, and a severe suppression of true leaf development. This phenotype is dependent on the presence of the 5-FC substrate and no effects are seen in plants grown in the absence of the substrate or in sibling plants lacking the transgene. The codA transformants have been assessed over three generations of growth and in each generation the transgene is stably inherited and confers the same 5-FC-sensitive phenotype. Transfer of 5-FC-sensitive seedlings to soil results in the restoration of normal growth in up to 100% of the seedlings. These results indicate that codA is a versatile dominant marker gene that can be used effectively in B. napus for substrate-dependent negative selection. Received: 24 June 1999 / Accepted: 22 July 1999     

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.     

Expression of the cry1EC gene in castor (Ricinus communis L.) confers field resistance to tobacco caterpillar (Spodoptera litura Fabr) and castor semilooper (Achoea janata L.)

Castor (cv. DCS-9) has been transformed through Agrobacterium-mediated and particle gun bombardment methods using appropriate vectors containing the Bt chimeric gene cry1EC driven by enhanced 35S promoter. About 81 and 12 putative transformants were regenerated following selection on hygromycin and kanamycin, respectively. Southern analysis of DNA extracted from T₀ plants confirmed integration of the introduced gene in castor genome. The integration and inheritance of the introduced genes was demonstrated up to T₄ generation by PCR and Southern analysis. Southern analysis of two events having single and two copies showed the same pattern of integration in the subsequent generations. Insect feeding experiments conducted in the laboratory by releasing neonate larvae of castor semilooper and S. litura on leaf tissues excised from transgenic and control plants showed varying degrees of larval mortality and slow growth in larvae fed on transgenic leaf tissue. Field bioassays against Spodoptera litura and castor semilooper conducted for eight events in T₁–T₄ generations under net confinement were more informative and events conferring resistance to the two major defoliators were identified.     

A large-scale<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10^-3 to 10^-5 compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 10³ stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10^-2 using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.Abbreviations AS: Acetosyringone - 5-FU: 5-Fluorouracil - FW: Fresh weight - GT: Gene targeting - HR: Homologous recombination - NHEJ: Non-homologous end joining Communicated by H. Ebinuma     

An improved protocol for<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Antirrhinum majus</Emphasis> L.

Efficient Agrobacterium -mediated transformation of Antirrhinum majus L. was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings. Stable transformants were obtained by inoculating explants with A. tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene (NPT II) as a selectable marker and the gene for the Green Fluorescent Protein (GFP) as a visual marker. Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy. PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants. The transformants had a morphologically normal phenotype. The transgene was shown to be inherited in a Mendelian manner. This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4–5 months of culture, reached 8–9%. This success rate makes the protocol very useful for producing transgenic A. majus plants.Communicated by G. Jürgens     

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency ( 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low ( 7%) from R0 to R3 generations when only one Ac copy was present. The strategy of choosing R0 plants that undergo a low frequency of germinal excision will provide a means to avoid screening non-independent transpositions and increase the efficiency of transposon tagging.     

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop × weed hybrid generations

The level of transgene expression in crop × weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T₁ single-locus insert GFP/Bacillus thuringiensis (Bt) transgenic canola (Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC₁F₁, BC₂F₁) were produced by backcrossing various GFP/Bt transgenic canola (B. napus, cv Westar) and birdseed rape (Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC₂F₂ Bulk) were generated by crossing BC₂F₁ individuals in the presence of a pollinating insect (Musca domestica L.). The ploidy of plants in the BC₂F₂ Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F₁ hybrid generations contained 95–97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15–29% presence in the BC₂F₂ Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC₂F₂ Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F₁, BC₁F₁ and BC₂F₁). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.Communicated by J. Dvorak     

T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method

Designing transformation experiments for either functional genomics or crop improvement requires knowledge of the transgene locus structure, number, transmission and expression resulting from a specific transformation method. We recently reported an improvement to the soybean [Glycine max (L.) Merrill] cotyledonary-node transformation method that resulted in the efficient production of transgenic plants. To characterize the transgene loci resulting from this method, we analysed 270 independent T0 plants and 95 randomly selected T1 progenies for T-DNA locus complexity using Southern analysis. The lines were transformed with Agrobacterium tumefaciens strains LBA4404 or EHA105 carrying the binary plasmids pGPTV, pTOK233, pCAMBIA1303 or pCAMBIA1309, and regenerated in medium supplemented with or without silver nitrate (AgNO3). Analysis in the T0 generation showed that the number of hpt-hybridizing fragments per plant ranged from 1-15, with 31.5% of the lines having a single hpt-hybridizing fragment. Each primary soybean transformant had, on average, 2.0 unlinked transgene loci and that half of the segregating loci in the T1 progenies were single, simple T-DNA insertions. Of the loci containing multiple T-DNA fragments, a low frequency had tandem and inverted repeat T-DNA structures. Integration of binary plasmid backbone sequences occurred in 37% of primary transformants. A. tumefaciens strain, binary plasmid and thiol treatment had no significant effect on transgene locus structure, numbers or expression. Interestingly, exposure of soybean explants to AgNO3 throughout shoot induction and elongation increased T-DNA locus complexity in the primary transformants and decreased silencing of gusA expression in the T1 generation.     

A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants

Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either β-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.     

Elite Indica Transgenic Rice Plants Expressing Modified Cry1Ac Endotoxin of Bacillus Thuringiensis Show Enhanced Resistance to Yellow Stem Borer (Scirpophaga Incertulas)

Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic of Agrobacterium-mediated approaches. A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used. Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system. Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher ( 9%) for Agrobacterium. An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt-transformants and reduced the number of escapes. Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium. In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert. Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India. Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays. However, there was an occasional white head even among such plants when assayed at the reproductive stage. Results are discussed in the light of resistance management strategies for deployment of Bt-rice.     

Isolation of transgenic progeny of maize by embryo rescue under selective conditions

Summary Fertile transgenic maize plants (T₀) and progeny (T₁) were obtained using microprojectile bombardment and callus selection on hygromycin B. To quickly identify progeny expressing the transgene, embryos from T3 generation kernels were excised 20 days after pollination and exposed to different concentrations of hygromycin B. Surviving and non-surviving embryos were assayed for the presence of the hygromycin phosphotransferase (aphIV) gene using polymerase chain reaction. Embryos that germinated and survived on 25, 50, or 100 mg/liter hygromycin possessed theaphIV gene. Embryos that did not germinate lacked the gene. Progeny surviving selection were transferred to the greenhouse and tested for expression of the gene using a leaf disc assay. The results demonstrated that the gene construct was expressed in both embryo and leaf tissue and that selection during germination successfully eliminated progeny lacking the gene of interest. This method is also useful for rapid-cycling of maize generations.     

Generation of fertile transplastomic soybean

We describe here the development of a plastid transformation method for soybean, a leguminous plant of major agronomic interest. Chloroplasts from embryogenic tissue of Glycine max have been successfully transformed by bombardment. The transforming DNA carries a spectinomycin resistance gene (aadA) under the control of tobacco plastid regulatory expression elements, flanked by two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon. All generated spectinomycin resistant plants were transplastomic and no remaining wild type plastome copies were detected. No spontaneous mutants were obtained. The transformation efficiency is similar to that of tobacco plastids. All transplastomic T0 plants were fertile and T1 progeny was uniformly spectinomycin resistant, showing the stability of the plastid transgene. This is the first report on the generation of fertile transplastomic soybean.     

Decreased frequency of the rat growth hormone transgene in mouse populations with or without selection for increased adult body weight

Summary Frequencies of mice with the rat growth hormone (rGH) transgene were examined in lines derived from two genetic bases (P/W and P/C). The genetic bases were developed from males (P) with the rGH transgene, mated with non-transgenic females of different origin: a line previously selected for large body size (W) and a corresponding unselected control line (C). They were maintained for six generations under random mating with or without selection for increased 42-day body weight. The frequencies of P/W and P/C males with the rGH transgene wer 0.075 and 0.300, respectively at generation 0 of the genetic bases. They were significantly (P<0.05) lower than the expected frequency (about 0.5). At generation 6, the frequencies had decreased further both in selected and unselected lines (ranging from 0.025 to 0.125). Decreased frequencies of mice with the transgene were confirmed in a separate experiment testing segregation of the transgene. The reasons for these decreases are not clear. The results suggest that transgenes need to be monitored when transgenic animals are mated with animals of different origin.Animal Research Centre Contribution No. 1697