Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

Promoters active in mammalian cells (pSV40) and in plants (pNOS) permit expression in yeast of the Tn5 APH(3') II gene

Abstract Four plasmids were constructed by associating Escherichia coli and yeast selection markers and replication origins to a structural gene coding for aminoglycoside phosphotransferase (APH(3')) controlled by different flanking sequences. We used the two bacterial genes of Tn5 (APH(3')II) and Tn903 (APH(3')I) as such and the chimeric pSVneo (APH(3')II) and pNOSneo (APH(3')II) constructs, functional in mammalian and plant cells, respectively. Yeast clones resistant to G418 were obtained with all plasmids except with that bearing the bacterial APH(3')II gene. The three plasmids harbouring the functional APH genes, however, conferred different levels of G418 resistance to yeast.     

Co-expression and inheritance of foreign genes in transformants obtained by direct DNA transformation of tobacco protoplasts

Summary A plasmid containing two marker genes for expression in plants was constructed. This 16 kb vector, pCT1T3, contains an intact nopaline synthase gene and a chimaeric gene consisting of the promoter and terminator regions from cauliflower mosaic virus gene VI and a structural gene, aminoglycoside phosphotransferase (APH(3′)II), from the bacterial transposon Tn5. After transformation of tobacco mesophyll protoplasts with this plasmid, several kanamycin-resistant transformants were obtained. Intensive studies on the drug tolerance of growth and differentiation of the transformants showed that the chimaeric gene was stably expressed. Of 17 independent transformants, 3 (about 18%) expressed the two marker genes, regardless of the state of differentiation, as did individual plants regenerated from the same callus. Multiple copies of the inserted DNA were found in some transformants. Viable seeds were produced by 12 out of 15 independent transformants. Seeds obtained by self-pollination were germinated on medium containing kanamycin sulphate. With the exception of one clone, resistant seedlings with green leaves and sensitive seedlings with white leaves were found to segregate in a 3:1 ratio. This suggests that the inheritance of the inserted gene is Mendelian. A reciprocal cross between the transformants and wild-type tobacco also showed nuclear transmission of the APH(3′)II gene. This was consistently maintained in a subclone of the same transformant derived from the same callus line. Stable inheritance of the single dominant character was also seen among seeds formed in several different flower pods of the same individual plants. Two clones were also found to synthesize nopaline in addition to expressing APH(3′)II. Analysis of the progeny obtained by self-crosses of such transformants revealed the simultaneous expression of these two enzymes, indicating that the two marker genes are linked on the same chromosome.     

Expression of foreign genes in regenerated plants and in their progeny

Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km^R and Cm^R). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.     

A set of novel Ti plasmid-derived vectors for the production of transgenic plants

Summary We describe in this paper the construction and use of a set of novel Ti plasmid-derived vectors that can be used to produce transgenic plants. These vectors are based on one of two strategies: 1) double recombination into the wild-type Ti plasmid of genetic information flanked by two T-DNA fragments on a wide-host range plasmid; 2) the binary vector strategy. The vector based on the double recombination principle contains a kanamycin resistance gene for use as a plant selectable marker, a polylinker for the insertion of foreign genes, and a nopaline synthase gene. The vector was constructed such that a disarmed T-DNA results from the double recombination event. The binary vector combines several advantageous features including an origin of replication that is stable in Agrobacterium in the absence of selection, six unique sites for insertion of foreign genes, an intact nopaline synthase gene, and a kanamycin resistance marker for selection of transformed plant cells. All of these vectors have been used to produce tobacco plants transformed with a variety of foreign genes.     

Gene transfer to cereal cells mediated by protoplast transformation

Summary Direct gene transfer to cereal cells was achieved by transformation of protoplasts with naked DNA. Protoplasts isolated from cultured cells of Triticum monococcum were incubated in the presence of polyethylene glycol (PEG) with circular and linear plasmid DNA. The pBR322-derived plasmid, pBL1103-4, contained a selectable chimeric gene comprising the protein coding region of the Tn5 aminogly-coside phosphotransferase type II gene (NPT II), the nopaline synthase promoter (pNOS) and the polyadenylation signal of the octopine synthase gene. Transformed cells were selected in medium containing kanamycin and identified by detection of aminoglycoside phosphotransferase II activity.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Genetic analysis of T-DNA insertions into the tobacco genome

A genetic test was performed on seeds from 283 transgenic tobacco plants obtained by T-DNA transformation. Seeds from self-fertilized transgenic plants were germinated on kanamycin-containing medium, and the percentage of seeds which germinated, as well as the ratio of kanamycin-resistant to kanamycin-sensitive seedlings were scored. Nine categories of transformants could be distinguished according to the number of loci into which T-DNA had inserted, and according to the effects of T-DNA integration on seed or seedling development. In most of the plants, T-DNA was inserted into a single site; others contained multiple independent copies of T-DNA. The number of T-DNA integration sites was found to be independent of whether a binary vector system or a cointegrate Ti plasmid had been used to obtain the transgenic plant. Loss of marker genes or marker gene expression from generation to generation appeared to be a quite frequent event. Plants which appeared to be insertional recessive embryo-lethal mutants did not exhibit this trait in the next generation.Abbreviations Kan^R kanamycin resistant - Kan^S kanamycin sensitive - NOP nopaline - NOS nopaline synthase - NPT II neomycin phosphotransferase II     

Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens

Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R₂ seedlings on media containing G418.Abbreviations IAA indole acetic acid - KIN kinetin - BA benzyladenine - NOS nopaline synthase - NPTII neomycin phosphotransferase II - RMNO tobacco nutrient medium (Marton and Maliga, 1975) - SH Shenk & Hildebrandt nutrient medium (Shenk & Hildebrandt, 1972; Michelmore and Eash, 1985) Present address: Agriculture Canada, P.O. Box 457, St. Jean-sur-Richelieu, Quebec, Canada, J3B 6Z8     

Inheritance and structure of foreign DNA in progenies of transgenic tobacco obtained by direct gene transfer

Summary One of the transformed tobacco plants obtained by direct DNA transformation possessed two marker genes, a chimeric aminoglycoside phosphotransferase and nopaline synthase genes. Selfed progenies of this plant (T3-d) showed stable inheritance of these two genes. The minimum size of foreign DNA integrated into tobacco genome was estimated to be 5.4 kbp. A deleted nopaline synthase gene co-existed with an intact gene. The linkage analysis indicated that two transformants, T1-b and T3-c, possessed foreign DNA inserted in different chromosomes or in different sites of the same chromosome that recombine freely.     

Inactivation of the nopaline synthase gene by double transformation: reactivation by segregation of the induced DNA

Tobacco plants were transformed with derivatives of a binary vector pMON505 and two kanamycin resistant lines that were nopaline positive were selected for second transformation. The plasmids used for the second transformation were derivatives of pMON850 which carries the nopaline synthase gene in addition to a gene for gentamicin resistance. Insertion of each transgene was confirmed by Southern hybridization. Surprisingly, we found that more than 50% of the doubly transformed tobacco plants were nopaline negative. Tobacco plants that were transformed only by the second vector exhibited nopaline accumulation. DNA methylation patterns at the HpaII site in the promoter region of the nopaline synthase gene did not correlate with the nopaline phenotype. In some plant lines, seedlings of the R1 generation which segregated out the second T-DNA insertion recovered the nop⁺ phenotype. These results indicate that nopaline accumulation was inhibited by the presence of the second T-DNA.Abbreviations T-DNA transferred DNA - NPTII neomycin phosphotransferase II - uidA -glucuronidase - Km kanamycin - Gm gentamicin - nop⁺ nopaline positive - nop^– nopaline negative - MS medium, Murashige-Skoog medium     

Monohybrid and dihybrid segregations in the progenies of tobacco transformed for kanamycin resistance with a Ti-vector system

A chimeric DNA construction having nopaline synthase promoter, coding sequences of neomycin phosphotransferase gene conferring resistance to antibiotic kanamycin and OCS (octopine synthase) polyadenylation sequences bracketed by T-DNA ends was transferred to tobacco. Leaf discs were infected withA. tumefaciens containing disarmed, cointegrate plasmid pGV3850:: 1103 and allowed to form a callus in the presence of kanamycin. Shoots regenerated from infected leaf discs either through the callus or arising directly were further selected for their ability to root in kanamycin-containing media. Among the nine transgenic plants that were progeny tested, the transferred bacterial gene segregated as monohybrid ratio (3 Kan^R: 1 Kan^s) in seven. Segregation data of two plant progenies indicated the presence of two independent loci of Kan^R DNA insertion (15 Kan^R: 1 Kan ^s ). Back-cross segregation data were consistent with the monohybrid or independent assortment of duplicate factors. Thus in the two cases, a minimum independent integration of two copies of T-DNA each with a Kan^R marker is inferred.     

Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts

Summary Genetic transformation of Nicotiana tabacum protoplasts was achieved by incubation of protoplasts with a plasmid DNA-calcium phosphate coprecipitate, followed by fusion of the protoplasts in the presence of polyvinyl alcohol and subsequent exposure to high pH. A derivative of the plasmid pBR322 containing a chimaeric gene, consisting of the nopaline synthase promoter, the coding region of the aminoglycoside phosphotransferase gene of Tn5 and the polyadenylation signal region of the octopine synthase gene, was used for these transformation experiments. This chimaeric gene confers resistance of transformed plant cells to kanamycin. This novel transformation procedure reproducibly yielded transformants at frequencies of approximately 0.01%. Aminoglycoside phosphotransferase II activity was detected in both transformed calli and in regenerated plants. DNA from some of the transformed clones was analyzed by Southern blot hybridization. The input DNA appears to be integrated into high molecular weight cellular DNA. Genetic analysis of one of the kanamycin resistant plants shows that the chimaeric gene is transmitted to the progeny as a single dominant trait in a Mendelian fashion. As a comparison the input DNA was also introduced into tobacco protoplasts using Agrobacterium tumefaciens and Ti-plasmid derived gene vectors.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Expression of a foreign gene in callus derived from DNA-treated protoplasts of rice (Oryza sativa L.)

Summary Protoplasts isolated from suspension cultures of rice cells were treated with bacterial plasmid DNA carrying a chimaeric gene consisting of the nopaline synthase promoter, the aminoglycoside phosphotransferase II (APH(3)II) structural gene from bacterial transposon Tn5 and the terminator region from cauliflower mosaic virus DNA. Colonies capable of proliferating in medium containing kanamycin (100 g/ml) were selected. A transformation frequency of approximately 2% to 3% was recorded in several experiments. The enzyme (APH(3)II) was also detected in kanamycin-resistant callus, which had survived after repeated selection. There was some variation in the APH(3)II activity in the transformants which paralleled the copy number of the inserted genes.     

Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens

Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens harbouring pGV 38501103 neo (dimer) containing chimaeric kanamycin resistance reporter genes. Transformed protoplasts were regenerated to fertile and phenotypically normal plants. Transformation was confirmed by kanamycin resistance, nopaline production, neomycinphosphotransferase II activity, and Southern blot hybridization. Seed progeny from self-pollinated transformants expressed the introduced kanamycin resistance as a Mendelian trait.Abbreviations BAP 6-benzylaminopurine - Cf Claforan^R - 2.4D 2,4-dichlorophenoxy acetic acid - Km kanamycin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - NPT II neomycinphosphotransferase - npt II neomycinphosphotransferase II gene - NOS nopaline synthase - nos nopaline synthase gene - ocs octopine synthase gene - IAA indole-3-acetic acid     

The opine synthase genes carried by Ti plasmids contain all signals necessary for expression in plants

Signals necessary for in vivo expression of Ti plasmid T-DNA-encoded octopine and nopaline synthase genes were studied in crown gall tumors by constructing mutated genes carrying various lengths of sequences upstream of the 5' initiation site of their mRNAs. Deletions upstream of position -294 did not interfere with expression of the octopine synthase gene while those extending upstream of position -170 greatly reduced the gene expression. The estimated size of the octopine synthase promoter is therefore 295 bp. The maximal length of 5' upstream sequences involved in the in vivo expression of the nopaline synthase gene is 261 bp. Our results also demonstrated that Ti plasmid-derived sequences contain all signals essential for expression of opine synthase genes in plants. Expression of these genes, therefore, is independent of the direct vicinity of the plant DNA sequences and is not activated by formation of plant DNA and T-DNA border junction.     

Intermolecular homologous recombination in plants.

To study DNA topological requirements for homologous recombination in plants, we have constructed pairs of plasmids that contain nonoverlapping deletions in the neomycin phosphotransferase gene [APH(3')II], which, when intact, confers kanamycin resistance to plant cells. Protoplasts isolated from Nicotiana tabacum were cotransformed with complementary pairs of plasmids containing these truncated gene constructs. Homologous recombination or gene conversion within the homologous sequences (6 to 405 base pairs) of the protein-coding region of the truncated genes led to the restoration of the functional APH(3')II gene, rendering these cells resistant to kanamycin. Circular plasmid DNAs recombined very inefficiently, independent of the length of the homologous region. A double-strand break in one molecule only slightly increased the recombination frequency. The most favorable substrates for recombination were linear molecules. In this case, the recombination frequency was positively correlated with the length of the homologous regions. The recombination frequency of plasmids linearized at sites proximal to the deletion-homology junction was significantly higher than when linearization was distal to the homologous region. Vector homology within cotransformed plasmid sequences also increased the recombination frequency.     

Nucleotide sequence of the kanamycin resistance determinant of plasmid RP4: Homology to other aminoglycoside 3′-phosphotransferases

The kanamycin resistance determinant of the broad-host-range plasmid RP4 encodes an aminoglycoside 3'-phosphotransferase of type I. The nucleotide sequence of the kanamycin resistance gene (Kmr) and the right end of the insertion element IS8 of plasmid RP4 has been determined. The gene (816 bp) is located between IS8 and the region (Tra 1) encoding plasmid factors mediating bacterial conjugation. Kmr and Tra 1 are transcribed toward each other. The nucleotide sequence has been compared to five related aphA genes originating from gram-negative and gram-positive organisms and from antibiotic producers. Among these that of Tn903 shares the highest degree of similarity (60%) with the RP4 gene. Significant similarities were also detected between the amino acid sequences of the six enzymes. The C-terminal domains of six different aminoglycoside 3'-phosphotransferases (APH(3'] are highly conserved. They are substantially similar to segments of a variety of enzymes using ATP as cofactor. The role of the C-terminal sequences of APH(3') as potential domains for ATP recognition and binding is discussed.     

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid