Gert Ooms

Octopine Ti-plasmid deletion mutants of agrobacterium tumefaciens with emphasis on the right side of the T-region.

Abstract One hundred and twelve Agrobacterium tumefaciens mutants with a deleted octopine Ti plasmid were isolated. They originated from four insertion mutants, each of which carried the transposon Tn904 at a different position in the Ti plasmid. The deletion mutants were selected on the basis of loss of the capacity encoded by the Ti plasmid to degrade octopine. They were tested for the expression of other Ti-plasmid coded functions: tumor induction, presence of lysopine dehydrogenase activity in the tumor, and exclusion of phage Ap-1. For 21 mutants affected in at least one of these functions, the map position of the deletions was determined. It was found that deletions at two separated loci give rise to an Occ− phenotype. Genes for Ap-1 exclusion were mapped on a small region just outside and to the right of the TL + TR region. Most of the TR region, present as TR-DNA in a limited number of crown gall tissues only, was shown to be unnecessary for tumor formation, since it could be deleted without affecting virulence (tested on various plant species) of the mutants. However, if the TR region together with a small part of the adjacent TL region, which is always present as TL-DNA in normal crown gall tissues, was deleted the mutants became weakly virulent on Kalanchoe and Nicotiana rustica and avirulent on tomato. We hypothesize that in this case a region necessary for T-DNA integration has been deleted. The same region was found to be essential for lysopine dehydrogenase activity in the tumors.

Genetic modification of potato development using Ri T-DNA

SummaryForty-two potato plants were regenerated from a hairy-root line obtained after infection of a shoot of Solanum tuberosum cv ‘Desiree’ with Agrobacterium rhizogenes strain LBA 9402 (pRil855). Transformed plants were uniform and had a distinct phenotype and development compared with untransformed controls. Their growth was vigorous, especially early in their development, their roots were abundant and showed reduced geotropism, their leaves were slightly crinkled and glossy and they produced longer tubers with more frequent, prominent eyes. Cytological examination showed that ten of the forty-two transformed plants had either 47 or 49 chromosomes instead of the normal 48. In two of these aneuploids structural changes were observed.

Retention of tumor markers in F1 progeny plants from in vitro induced octopine and nopaline tumor tissues

Tumorous tobacco shoots have been derived from callus tissues produced by Agrobacterium tumefaciens--induced transformation of tobacco protoplasts and by fusion of normal protoplasts with those from crown gall tumors. The continued presence of T-DNA sequences in shoots is directly demonstrated by Southern blotting and is also revealed by the presence of the tumor markers octopine and nopaline. When grafted onto normal tobacco plants, both octopine- and nopaline-type shoots (including those from somatic hybrids) produced flowers and set seed. Germination of these seeds gave F1 progeny that showed retention of morphological markers of their parental shoots, and one seedling retained the ability to synthesize nopaline. The data demonstrate that T-DNA markers can be retained during meiosis and are expressed in F1 plants.

Genetic map of an octopine TI-plasmid

Abstract Several deletion mutants of an octopine TI-plasmid were mapped by digestion with the restriction enzyme Sma I. The T region, as it is defined on the B6-806 plasmid, does not appear to be an essential area for tumour induction on the plasmid of Ach5. The genes for octopine breakdown, plasmid transfer, and the replicator were roughly localized. The possibility of using mutants with large deletions as a cloning vehicle in Agrobacterium tumefaciens is discussed.

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

SummaryDerivatives 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.

Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

SummaryThe response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.

Structural diversity of the patatin gene family in potato cv. Desiree

SummaryWe have used a combined genetical and molecular approach to study the structural diversity of the patatin gene family in tetraploid Solanum tuberosum L. cv. Desiree (2n=4x=48). Nine dihaploid derivatives (2n=2x=24) of cv. Desiree were isolated by gynogenesis through prickle pollination with S. phureja Juz et Buk. Patatin DNA sequences in Desiree and in the dihaploids were examined by probing Southern blots of restriction endonucleases Hin-dIII and XbaI digested DNA with patatin cDNA region-specific gene probes and by more detailed examination (restriction endonuclease mapping and partial DNA sequencing) of 10 patatin genomic clones in bacteriophage λ replacement vector EMBL4. This provided positive identification for most individual patatin gene family members and some estimate of their organisation and diversity. Most of the 64–72 patatin DNA copies showed little allelic variation based on HindIII and XbaI restriction fragment length polymorphism (RFLP) mapping and did not appear to be very tightly clustered. Four of the 6–8 class I patatin genes (without a characteristic 22 bp insert in their untranslated leader DNA), showed apparent allelic homogeneity, whilst the remaining class I genes comigrated with a single class II patatin gene RFLP subclass. Of the isolated clones, 4 contained apparent pseudogenes lacking 5′ control sequences and exon-1 DNA while another clone contained a patatin gene truncated at the 5′ region due to the cloning event. The remaining 5 all contained class II genes (with the 22 bp insert) and these showed varying degrees of sequence homology for 400 bp of conserved 5′ coding and non-coding DNA (from 77%–95%). In one case the extent of homology differed, with complete sequence divergence upstream of position-80 from the start of transcription. The structural diversity of the patatin gene family is discussed in relation to expression of individual patatin genes and the use of cv. Desiree as a host for potato transformation experiments.

Developmental regulation of RI TL-DNA gene expression in roots, shoots and tubers of transformed potato (Solanum tuberosum cv. Desiree)

SummaryExpression of TL-DNA from Agrobacterium rhizogenes plasmid pRi 1855 was examined in a transformed derivative of Solanum tuberosum cv. Desiree, D9X8a. Northern blot analysis identified at least nine TL-DNA coded transcripts in roots, shoots and tubers but their relative abundance differed within and between organs. This revealed a distinctive pattern of organ specified differential expression. Grafting experiments showed that the abnormal shape of tubers of transformed potato was probably determined by TL-DNA products synthesised within the tuber and not by diffusable products synthesised in other parts of the plant. The abundance of at least one transcript, tr5, was probably determined by culture conditions. Implications for functions and control of expression of Ri TL-DNA genes are discussed. It is suggested that Ri TL-DNA provides a convenient and extensive set of model genes to study variation and stability of expression of linked foreign genes introduced into plants.

From tumour to tuber; tumour cell characteristics and chromosome numbers of crown gall-derived tetraploid potato plants (Solanum tuberosum cv. ‘Maris Bard’)

SummaryAgrobacterium tumefaciens strains, known to induce tobacco crown galls that spontaneously develop shoots, were used to induce galls on cultured shoots of a tetraploid potato cultivar (Solanum tuberosum cv. ‘Maris Bard’). Shoots also appeared spontaneously from the induced potato galls, although only after 2–4 months. The shoots were excised and cultured separately. Some of these frequently developed side-shoots from their axillary buds. They did not form roots and they produced opines, a strong indication that they were transformed and carried T-DNA. Grafts of the transformed plants were still able to develop tubers. Most of the tumour-derived shoots, however, formed roots, did not produce opines and were indistinguishable from the parental plants on the basis of morphology and chromosome numbers (48 chromosomes per cell). The results are discussed in relation to the origin of previously described variation among protoplast-derived potato plants and with respect to genetic engineering of tetraploid potato cultivars.

The 5' flanking DNA of a patatin gene directs tuber specific expression of a chimaeric gene in potato.

A member of the patatin multigene family which encodes the major soluble tuber protein was isolated from potato cultivar Désirée. Analysis by strategic nucleotide sequencing demonstrated close homology to analogous regions of previously isolated patatin genomic clones from different cultivars. A 3.8-kb fragment containing the promoter and 5′ flanking DNA of the patatin gene was used to construct a transcriptional fusion gene with the coding DNA of the bacterial chloramphenicol acetyltransferase (CAT) gene and the polyadenylation/termination sequences of the nopaline synthase gene (nos). The chimaeric gene was reintroduced into potato cultivar Désirée by agrobacterial transformation of tissue slices. Regenerated transformed plants showed expression of the chimaeric gene (as determined by CAT activity) in tubers, but not in leaves, stems or roots of in vitro grown plants. Independent transformants did not show substantial variation in the level of induced tuber-specific CAT activity. Thus, information contained within 3.8 kb of the 5′ flanking DNA of the patatin gene analysed is sufficient to direct tuber-specific expression, largely independent of position effects.