Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants

Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T₁ progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T₂ progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations.     

Transgene copy number can be positively or negatively associated with transgene expression

Two different types of T-DNA insert were found in tobacco plants transformed with Agrobacterium tumefaciens. High-expressing (H) types had one copy of the T-DNA at a locus and produced high expression of the transgene uidA, as measured by uidA RNA levels and -glucuronidase activity; low-expressing (L) types had inverted repeats of the T-DNA at a locus and produced low uidA expression. H-types from different transformants acted additively, and cross-fertilization between two different homozygous transformants with H-type inserts produced F₁ plants with GUS activity that equalled the parents and individual F₂ plants with 50%, 100%, 150% and 200% of parental values. However, the L-type inserts worked in trans to suppress uidA expression from H-type inserts when both were present in the same genome. Hence when a transformant homozygous for the L-type insert was crossed to one homozygous for the H-type, all plants in the F₁ and F₂ generations with both types of insert had low GUS activity while F₂ segregants that only had the H-type inserts had high GUS activity again. Suppression of the H-type gene was associated with increased methylation of the insert. Particle acceleration was used to introduce further copies of uidA into tissues of the transformants. Regardless of the promoter used, those plants with endogenous L-type inserts showed none of the distinct loci of GUS activity readily visible in material with no inserts, showing that L-type inserts could suppress not only the uidA expression of genomic homologues, but also of copies added in vitro.     

Characteristic analysis of transformants in T-DNA mutation library of <Emphasis Type="Italic">Monascus ruber</Emphasis>

Monascus ruber, a red mold species, has been widely used in the fields of food and medicine. In this research, we transformed Monascus ruber spores using Agrobacterium tumefaciens as a tool for random insertional mutagenesis with the hygromycin phosphotransferase gene as the selected marker. Three types of mutants including citrinin-producing mutants, mutants with abnormal aerial hyphae and pigment change mutants were screened for molecular analysis. Southern blot analysis showed that more than 83.3% of transformants contained single T-DNA insertions. The genomic DNA segments of the transformants flanking the T-DNA could be amplified from their left borders with TAIL-PCR. Homologous comparison using the Blast tool showed that none of the isolated DNA sequences had any similarity to each other, suggesting that the T-DNA was randomly integrated into the fungal genome, which provided the hypothetical reason for the variant phenotypes of the transformants. The successful creation of transformants with a single T-DNA tag insertion may help us to clone functional genes related to the metabolism and differentiation of Monascus spp., which will greatly facilitate the molecular analysis of this important fungus and the improvement of strains at the genetic level.     

Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens

Vectors for transformation of higher plants mediated by Agrobacterium tumefaciens were modified so that one, two or three additional copies of the left border (LB) sequences were inserted close to the original LB of the T-DNA. A gene for -glucuronidase (gusA) was placed outside the T-DNA to monitor the transfer to plants of 'vector backbone' sequences. The expression of GUS in immature embryos of rice that had been co-cultivated with A. tumefaciens carrying these constructs was around one tenth of that with A. tumefaciens carrying an unmodified control vector. Between 88 and 127 of independent transformants were regenerated from rice tissues infected with A. tumefaciens carrying each of these vectors. The GUS expressors among the rice transformed with the modified vectors were much less frequent than ones among the control transformants, and rate of reduction in the ratio of transgenic plants that expressed GUS was higher than 93%. Detection of a fragment across the LB region by the polymerase chain reaction and the gusA gene by Southern hybridization correlated well with GUS expression. These results indicate that transfer of the 'vector backbone' from the control vectors resulted mainly from inefficient termination of formation of the transfer intermediate of the T-DNA and additional LB sequences effectively suppressed such transfer. This approach is simpler than the strategy to place a 'lethal gene' outside the T-DNA and will likely help produce 'clean' transformants efficiently.     

Promoter Trapping in Arabidopsis Using T-DNA Insertional Mutagenesis

A new promoter trap vector was constructed based on the juxtaposition of T-DNA right border to coding sequence of GUS. The new vector pRN-1 carried an intron in the GUS coding region. Promoter trap vectors pGKB5 and pRN-1 vectors were used to transform Arabidopsis ecotype Columbia using the floral dip transformation system. The transformants were selected on appropriate selection media and the primary transformants were confirmed by PCR using gene specific primers. Approximately 50 % of the T₂ lines segregated for a 3:1 ratio indicating presence of T-DNA at single locus. Approximately 15% of the transformed lines showed expression of GUS. Morphological mutants for male sterility and dwarfism were also identified in the T₂ population. A T-DNA tagged line was identified in T₂ with GUS expression specifically in the floral parts. The number of T-DNA loci in this line was confirmed by Southern blot hybridization. T-DNA flanking region isolated from this line suggested insertions into chromosome 2 at two closely linked loci. The results demonstrate that the population generated can be used effectively to identify and characterize gene regulatory elements.     

Transformation of carrot tissues derived from proembryogenic suspension cells: A useful model system for gene expression studies in plants

A method is described for the high frequency transformation of carrot proembryogenic suspension culture cells by a non-oncogenic Ti-plasmid vector (pGV3850::1103) which carried a chimaeric kanamycin resistance gene (nos-NPT-II). Plants were regenerated efficiently from transformed material by somatic embryogenesis in the presence of kanamycin. Transformed tissues expressed readily detectable levels of both NPT-II and nopaline. NPT-II could be detected in total protein extracts by Western blotting. This analysis indicated that NPT-II was produced as a single, full length polypeptide. The T-DNA copy number in individually selected transformants was analysed by Southern blotting and ranged from 1–8 per diploid genome. The copy number and organization of the T-DNA was retained in plants regenerated from these transformants by somatic embryogenesis. These data suggested a clonal origin for the selected kanamycin resistant colonies. NPT-II expression levels appeared to be directly related to gene dosage.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

Composition of endogenous alleles can influence the level of antisense inhibition of granule-bound starch synthase gene expression in tetraploid potato plants

The T-DNA composition was analysed of twelve potato genotypes obtained after transforming a tetraploid cultivar with an antisense granule-bound starch synthase (GBSSI) gene. In five transformants (labelled TB50 nos.) the antisense GBSSI gene was driven by the CaMV 35S promoter, while in the remaining seven (labelled TBK50 nos.) the GBSSI promoter was used. In these twelve transformants the antisense effect on amylose production in potato tuber starch ranged from complete suppression to no discernible inhibition, and the number of T-DNA insertions ranged from one to at least fifteen. The antisense effect of individual T-DNA loci in progeny of these transformants was studied. Progeny containing a single T-DNA showed no inhibition of GBSSI activity. Only multiple, linked T-DNA insertions resulted in substantial antisense inhibition. T-DNA fragments present in duplex in selfed progeny resulted in a larger antisense effect than that in the parent (which contained the T-DNA insertions in simplex). Furthermore, the antisense effects of some T-DNA-containing linkage groups were influenced by the composition of endogenous GBSSI alleles. For practical breeding this implies that (1) the efficiency of obtaining primary potato transformants showing complete inhibition of GBSSI gene expression by antisense RNA is genotype-dependent, and (2) many transformants have to be produced per genotype to be able to select plants with maximum suppression of GBSSI and a minimum number of T-DNA loci.     

The use of the two T-DNA binary system to derive marker-free transgenic soybeans

Summary A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101. Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T₀) representing 10 independent events were characterized. Seven of the 10 independent T₀ events co-expressed GUS. Progeny analysis was conducted by sowing the T₁ seeds and monitoring the expression of the GUS gene after 21 d. Individual T₁ plants were subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mgl⁻¹ solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.     

Evaluation of putative seed-specific promoters for Linum usitatissimum

The GUS reporter gene was used to test four different putativeseed-specific promoters in developing and mature seeds, leaves and roots fromlinseed flax (Linum usitatissimum). The promoters testedincluded the regulatory regions of the -ketoacyl-CoA synthase gene (KCS)and the napin protein gene from Brassica napus, thepromoter regions of the 'unknown seed protein' (USP), and a legumin proteingene(LeB4) from Vicia faba and the CaMV 35S promoter (positivecontrol). The promoter-GUS constructs were inserted into L.usitatissimum via Agrobacterium mediatedtransformation, and GUS activity evaluated using histochemical andfluorimetrical assays. All the promoters showed some activity, but only CaMV35S, LeB4 and USP exhibited an expression level high enough to be useful inlinseed flax. Plants with USP-GUS showed the earliest GUS activity at 5 to 6days after flowering (daf) and persisting until 40 daf. Expression of GUS underthe control of the LeB4 promoter was measurable 11 daf and was still detectableat 40 daf. The KCS-GUS construct showed a low level of GUS activity between 14daf and 40 daf. Plants transformed with USP-GUS or LeB4-GUS exhibited a lowlevel of GUS activity in leaves and roots of some of the transformants,indicating the need for generating large numbers of primary transformants,followed by careful evaluation and selection for ones with not only the desiredlevel of expression, but also the desired spatial and temporal expression.     

The GUS gene fusion system (Escherichia coli beta-D-glucuronidase gene), a useful tool in studies of root colonization by Fusarium oxysporum.

The plant-pathogenic fungus Fusarium oxysporum was successfully transformed with the beta-D-glucuronidase gene from Escherichia coli (gusA) (GUS system) in combination with the gene for nitrate reductase (niaD) as the selectable marker. The frequency of cotransformation, as determined by GUS expression on plates containing medium supplemented with 5-bromo-4-chloro-3-indolyl glucuronide (GUS+), was very high (up to 75%). Southern hybridization analyses of GUS+ transformants revealed that single or multiple copies of the gusA gene were integrated into the genomes. High levels of GUS activity are expressed in some transformants, but activity in F. oxysporum does not appear to be correlated with the copy number of the gusA gene. Since the highest activity was found in a transformant with a single copy, it can be assumed that sequence elements of F. oxysporum integrated upstream of the gene can act as a promoter or enhancer. Expression of the gusA gene was also detected during growth of the fungus in plants, indicating that the GUS system can be used as a sensitive and easy reporter gene assay in F. oxysporum.     

Flavonoid-related regulation of auxin accumulation in<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-induced plant tumors

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.     

High Efficiency Transgene Segregation in Co-Transformed Maize Plants using an Agrobacterium Tumefaciens 2 T-DNA Binary System

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the -glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R₀ plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R₀ events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R₁ generation. Of these R₀ events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R₁ generation. A total of 64.4% of the R₀ 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R₁ progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R₀ plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R₁ phenotypes were highly correlated to genetic segregation of the bar and GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T₀) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T₀ and T₁ generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.     

Transgenic Pinus radiata from Agrobacterium tumefaciens–mediated transformation of cotyledons

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

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.