New plant binary vectors with selectable markers located proximal to the left T-DNA border

Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and -glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.     

A plant transformation vector with a minimal T-DNA

Plant transformation, viaAgrobacterium tumefaciens, is usually performed with binary vectors. Most of the available binary vectors contain within the T-DNA (which is transferred to the plant genome) components not required for the intended modification. These additional sequences may cause potential risks during field testing of the transgenic plants or even more in the case of commercialization. The aim of this study was to produce a plant transformation vector which only contains a selectable and screenable marker gene and a multiple cloning site for insertion of promoter::foreign gene::terminator cassettes from other plasmids.     

Bleomycin resistance: a new dominant selectable marker for plant cell transformation

Summary Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. The expression of this determinant in plant cells confers resistance to bleomycin and allows selection of transformed plant cells.     

Genetic transformation in two potato cultivars with T-DNA from disarmed Agrobacterium

Summary Derivatives of potato (Solanum tuberosum cv.'s Maris Bard and Desiree) transformed with disarmed T-DNA from genetically engineered Agrobacterium tumefaciens strains were isolated. The transformed plants were recovered from shoot-forming tumours induced by infection of wounds with mixedcultures of shoot-inducing A. tumefaciens strains T37 and either Agrobacterium strain LBA1834(pRAL1834), (Hille et al. 1983) or LBA4404(pBIN6; pRAL4404), (Bevan 1984). Two small-scale feasibility experiments gave at least four Maris Bard plants transformed with pRAL1834 T-DNA and two Desiree plants with pBIN6 T-DNA. The transformed Maris Bard plants were morphologically abnormal and highly aneuploid. This was probably an unfortunate side-effect of a tissue culture-step introduced to promote the efficiency of shoot regeneration. The transformed Desiree plants, in contrast, were isolated without promoting additional shoot-growth. They were morphologically normal, contained 47 and the euploid 48 chromosomes per cell respectively and had improved growth on media containing kanamycin.     

Phleomycin resistance as a dominant selectable marker for plant cell transformation

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance (Ble) genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however the highest frequency of rooted plants was obtained when transformation was carried out with the Sh Ble gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.     

Simple binary vectors for DNA transfer to plant cells

Summary Cosmid binary vectors for the introduction of DNA into plant cells have been constructed. These vectors are derived from the replicon of the broad host range plasmid pRK2 and contain the T-DNA border regions between which have been placed a chimaeric gene conferring resistance to kanamycin in plant cells. Appropriate restriction endonuclease targets have also been placed between the border regions. These binary vectors, in conjunction with appropriate Agrobacterium strains, are capable of delivering DNA to plant cells in cocultivation experiments with very high efficiency. The transformation frequency is shown to be somewhat dependent on the replicon used. re]19850121 rv]19850506 ac]19850513     

pBECKS

A series of binary T-DNA vectors (pBECKS) has been created for use in theAgrobacterium-mediated genetic transformation of plants. The pBECKS series has corrected the undesirable features of the popular pBIN19 vector; the deleterious mutation within the coding sequence ofnptII has been amended and the cloning sites are now adjacent to the right border repeat in order to reduce the possibility of producing truncated sequences of novel genes within transformants. One set of vectors incorporates various combiantions of the marker genesgusA,C1/Lc,nptII,hph, andbar, for pursuit of early and stable transformation events. A set of constructs which contain deleted T-DNA borders in various combinations and display predictably altered efficacies for gene transfer has also been created. A modular set of vectors has been designed to facilitate the insertion and transfer of novel gene sequences by providing anptII-linked plant expression cassette orlacZ-multiple cloning site. A range of antibiotic resistance genes has been incorporated into the non-T-DNA part of the vectors in order to facilitate their selection across the range ofAgrobacterium virulence strains.     

T-DNA tagging in Brassica napus as an efficient tool for the isolation of new promoters for selectable marker genes

A simple strategy to identify and isolate new promoters suitable for driving the expression of selectable marker genes is described. By employing a Brassica napus hypocotyl transformation protocol and a promoterless gus::nptII tagging construct, a series of 20 kanamycin-resistant tagged lines was produced. Most of the regenerated plants showed hardly any GUS activity in leaf, stem and root tissues. However, expression was readily restored in callus tissue induced on in vitro leaf segments. Genomic sequences upstream of the gus::nptII insertions were isolated via plasmid rescue. Three clones originating from single copy T-DNA lines were selected for further evaluation. The rescued plasmids were cloned as linear fragments in binary vectors and re-transformed to Brassica napus hypocotyl and Solanum tuberosum stem segments. The new sequences maintained their promoter activity, demonstrated by transient and stable GUS activity after transformation. Furthermore, the promoters provided sufficient expression of the nptII gene to yield transgenic plants when using kanamycin as selective agent. Database searching (BLASTN) revealed that the promoters have significant homology with three Arabidopsis BAC clones, one Arabidopsis cDNA and one Brassica napus cDNA. The results presented in this paper illustrate the strength of combined methods for identification, isolation and testing of new plant promoters.     

Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens

Summary Leaves of the in vitro grown potato cultivars Bintje, Berolina, Desiree, and Russet Burbank were wounded and co-cultivated with Agrobacterium strains having chimeric bar and nptII genes on a disarmed T-DNA. Each leaf from these cultivars formed numerous calli on kanamycin-containing medium, and almost all calli regenerated shoots. For Russet Burbank, it was necessary to include AgNO₃ in the medium to obtain efficient shoot regeneration. The transformed plants have one to a few copies of the T-DNA, show NPT-II and PAT activities, and are resistant to high doses of the commercial preparation of phospinotricin (glufosinate). Almost no somaclonal variation was detected in trans-genic plants.     

New plant promoter and enhancer testing vectors

We describe here a set of binary vectors suitable forAgrobacterium-mediated gene transfer and specially designed for studying plant promoters. These vectors are based on the use of thegus reporter gene, contain multiple unique restriction sites upstream of thegus gene, and minimal promoters for testing the effect of enhancers or activator elements. In addition, an intron-containinggus (uidA) gene was introduced into one of these vectors in order to examine reporter gene activity in tissues whereAgrobacterium contamination may be a problem or in transient expression assays.     

Termini and telomeres in T-DNA transformation

A T-DNA vector for plant transformation has been constructed in which the cloning site is located 9 bp from the right-border (RB) end and 27 bp from the left-border (LB) end. In this vector cloned DNA homologous to plant chromosomal sequences is located at the T-DNA termini, and will thus be exposed by even limited exonucleolysis in planta. The arabidopsis ADH (alcohol dehydrogenase) locus was mobilized from Agrobacterium, and integration into the recipient genome was studied. Despite the terminal location of ADH homology in this vector, the T-DNA integrated essentially at random in the Arabidopsis genome rather than at the endogenous ADH locus. T-DNA integration was blocked, however, when Arabidopsis telomeric sequences were added to the construct at each end of the ADH homology. Thus the predominant mode by which incoming T-DNA is integrated into the continuity of chromosomal DNA involves free DNA ends, but, in contrast to modes of recombination such as gap repair, does not involve extensive terminal DNA sequence homology.     

Isolation and characterization of potato-tomato somatic hybrids using an amylose-free potato mutant as parental genotype

Summary Using different genotypes of tomato and diploid potato, possessing alien selectable markers as well as endogenous markers, very high frequencies of protoplast fusion hybrids were obtained. One endogenous genetic marker, the amylose-free (amf) mutant of potato, was helpful not only for the confirmation of fusion products but also for the study of genetic complementation and the segregation of amylose-free starch in microspores. Cytological analysis of the fusion hybrids indicated that except for one which was hexaploid, all of them had a perfectly balanced chromosome number of allotetraploid constitution (2n = 4x = 48). Despite normal chromosome pairing and a diploid behaviour, the microspores in some of the fusion hybrids segregated for the recessive amf-locus. This anomalous segregation of a recessive character in these hybrids was shown not to be due to chromosome elimination or to the absence of the wild-type tomato Amf gene. Although all fusion hybrids were totally sterile, the hexaploid produced stainable pollen and berries with badly developed seeds. Embryo rescue has so far failed to produce backcross progeny.     

Pollen markers for gene-centromere mapping in diploid potato

The utility of two pollen genetic markers for estimating the extent of meiotic recombination between the centromere and a marker gene was tested in 2n pollen of diploid potato clones. One of these markers was the distal locus amylose-free (amf) on chromosome 8 and the other was the isozyme locus alcohol dehydrogenase (Adh-1) on chromosome 4. In the case of the amf locus, the gene-centromere distance was estimated in a normal synaptic and a desynaptic genotype. In both cases the genetic analysis was confined to: (1) a direct estimation of the phenotypic (blue vs red) segregation ratios in FDR (first-division restitution) 2n pollen and (2) a classification of the 4 x progeny from 4x (nulliplex amf) x 2x (Amf/amf) crosses into duplex, simplex and nulliplex classes. The recombination frequency between the centromere and the amf locus in the normal synaptic genotype B92-7015-4 corresponded to a gene-centromere distance of 48.8 cM, whereas this distance amounted to 13.3 cM in the desynaptic genotype RS93-8025-1. Hence desynapsis reduced crossing-over by 73%. The observed genetic distance of 48.8 cM in the normal synaptic clone, B92-7015-4, is the highest gene-centromere distance reported so far in potato and this could be explained on the assumption of absolute chiasma interference. For the Adh-1 locus, it was found that heterozygous 2n pollen grains could be detected in pollen samples of the diploid clones, because of the occurrence of a heterodimeric band of the isozyme. Unlike the amf locus, the genecentromere distance for the Adh-1 locus was estimated only on the basis of the duplex, simplex and nulliplex classes in the progenies from 4x (nulliplex Adh-1 ² )x B92-7015-4 (Adh-1 ¹ /Adh-1 ² )crosses and was found to be 19.4 cM. Because the accurate positions of centromeres in relation to other loci are not available in the existing genetic maps of potato, which are saturated with molecular markers, halftetrad analysis is a promising additional approach to the basic genetics of this crop.     

Transformation of potato using mannopine and cucumopine strains of Agrobacterium rhizogenes

Mannopine and cucumopine strains of Agrobacterium rhizogenes were used for genetic transformation in two cultivars of potato (Solanum tuberosum L.). An overnight pretreatment of stem fragments with NAA prior to bacterial infection was necessary to induce root formation, otherwise very few roots were produced. Whatever the potato cultivar used, rhizogenesis induced by NAA pretreament depended on the bacterial strain. In fact, when explants from both potato cultivars were pretreated with 26.5 M NAA, on average 84.4% and 71.9% produced roots after inoculation with the strains 2659 and 2659 GUS respectively. On the contrary, few rhizogenic responses (2.0–17.0%) or no response at all (0.0%) were obtained with the strains 15834 and 8196 GUS whatever NAA concentration used. Tests for confirming stable transformation of plant explants by examining both -glucuronidase activity and the presence of opines showed that 85% of the selected roots were cotransformed. Most of the transformed roots were highly branched and grew rapidly, compared to non-transformed roots with no branching and poor growth. Transgenic plants were readily regenerated with a frequency reaching 80% of total explants tested for both potato cultivars.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - df degree of freedom - F F distribution - GA₃ gibberellic acid - MS Murashige and Skoog basal medium - NAA -naphthaleneacetic acid - P probability - T-DNA transferred DNA     

The potato in Spain during the late 16th century

A study is presented of the Hospital de la Sangre account books in Seville at the Archivo Hispalense for the period 1546 to 1601, to verify purchases of potatoes (Solanum tuberosum) during that period. Potatoes were bought regularly in the Seville market from 1580 onwards, with the first record appearing in 1573 thus agreeing with Salaman’s conclusion that potatoes became established in Spain by about 1570. Purchases were confined almost entirely to December and January each year, lending weight to the hypothesis that these were short day adaptedS. tuberosum ssp.andigena, actually grown in Spain and forming tubers in the short days at the end of the year. If potatoes had been imported for direct consumption from South America shipping records indicate that they could have arrived in Seville at all times of the year. A listing of other fruits and vegetables bought in the Seville market shows a wide range of mostly mediterranean crops with some of Near Eastern origin and certain spices imported through Lisbon from the Ear East. Surprisingly, very few New World crops are mentioned.     

Genetic localisation of transformation competence in diploid potato

In the course of improving diploid potato genotypes for transformation ability, selection for specific components affecting regeneration and transformation was carried out. From a segregating population between two good regenerating clones a selection was made to yield an optimal well-transforming and fertile genotype J92-6400-A16. This plant yielded predominantly diploid transformants and was heterozygous for the gene R1, conferring resistance to Phytophthora infestans. The speed of, and competence for, regeneration and transformation on both sides of the stem explant were improved. A competence factor for tranformation was found to be linked with the R1 locus and a molecular marker on chromosome 5. The male fertility of transformants was frequently decreased to a great extent, whereas female fertility was not so markedly affected.     

The small,versatilepPZP family ofAgrobacterium binary vectors for plant transformation

The newpPZP Agrobacterium binary vectors are versatile, relatively small, stable and are fully sequenced. The vectors utilize the pTiT37 T-DNA border regions, the pBR322bom site for mobilization fromEscherichia coli toAgrobacterium, and the ColE1 and pVS1 plasmid origins for replication inE. coli and inAgrobacterium, respectively. Bacterial marker genes in the vectors confer resistance to chloramphenicol (pPZP100 series) or spectinomycin (pPZP200 series), allowing their use inAgrobacterium strains with different drug resistance markers. Plant marker genes in the binary vectors confer resistance to kanamycin or to gentamycin, and are adjacent to the left border (LB) of the transferred region. A lacZ -peptide, with the pUC18 multiple cloning site (MCS), lies between the plant marker gene and the right border (RB). Since the RB is transferred first, drug resistance is obtained only if the passenger gene is present in the transgenic plants.