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An experimental and theoretical investigation of the influence of high-frequency acoustic waves on the flow of a liquid through a porous material has been made. Particular attention was paid to the phenomenon of acoustic streaming of the liquid in the porous material due to the damping of the acoustic waves. The experiments were performed on Berea sandstone cores. Two acoustic horns were used with frequencies of 20 and 40 kHz, and with maximum power output of 2 and 0.7 kW, respectively. A high external pressure was applied in order to avoid cavitation. A microphone was used to measure the damping of the waves in the porous material and also temperature and pressure measurements in the flowing liquid inside the cores were carried out. To model the acoustic streaming effect Darcy's law was extended with a source term representing the momentum transfer from the acoustic waves to the liquid. The model predictions for the pressure distribution inside the core under acoustic streaming conditions are in reasonable agreement with the experimental data.

An experimental and theoretical investigation has been made of the influence of high-frequency acoustic waves on the flow of a liquid through a porous material. The experiments have been performed on Berea sandstone cores. Two acoustic horns were used with frequencies of 20 and 40 kHz, and with maximum power output of 2 and 0.7 kW, respectively. Also, a temperature measurement of the flowing liquid inside the core was made. A high external pressure was applied in order to avoid cavitation. The acoustic waves were found to produce a significant effect on the pressure gradient at constant liquid flow rate through the core samples. During the application of acoustic waves the pressure gradient inside the core decreases. This effect turned out to be due to the decrease of the liquid viscosity caused by an increase in liquid temperature as a result of the acoustic energy dissipation inside the porous material. Also, a theoretical model has been developed to calculate the dissipation effect on the viscosity and on the pressure gradient. The model predictions are in reasonable agreement with the experimental data.

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts--250 V/cm; Désirée mesophyll protoplasts--225 V/cm; Désirée suspension culture protoplasts--225 V/cm; and Désirée tuber protoplasts--150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36-48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the beta-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0-10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20-30 pmol/ml) the patatin promoter directed 4-5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.

To obtain transformation of plant cells, we incubated 3-day-old cell wall-regenerating protoplasts from tobacco with Agrobacterium tumefaciens harboring tumor-inducing plasmids. Putative transformed tobacco cells were selected by phytohormone autotrophic growth and were shown to be transformed by the detection of the tumor cell specific enzymes lysopine dehydrogenase or nopaline dehydrogenase. This was substantiated by the detection, in transformed tumor tissues, of DNA sequences homologous to sequences in the tumor-inducing plasmid. Segregation of tumor markers has been observed among the transformants and it is suggested that this happened during the initiation of the transformation. The stable character of the transformed state was shown by the retention of tumor markers in subcloning of primary transformants under nonselective conditions. Suppression of the neoplastic state of transformants could take place, resulting in the development of transformed shoots. Indications were obtained for the inheritance of tumor markers through meiosis from seedlings obtained from seeds of flowering transformed plants that still expressed nopaline synthesis.

The 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.

Ti plasmid mutants derived from Agrobacterium tumefaciens strain Ach5 that induce tumors of abnormal morphology have been analyzed. On tobacco, A. tumefaciens mutant strain LBA4060 induces tumors that specifically give rise to shoots. Shoots continue to grow from in vitro cultured bacteria-free tumor tissue derived from such tumors. The mutant character is shown to be correlated with the insertion of an A. tumefaciens IS element, IS60, into the left arm of the T-region of the octopine Ti plasmid. Evidence is presented showing that IS60 is transferred into the plant cell DNA as part of the T-DNA. A second Ti plasmid insertion mutant A. tumefaciens strain LBA4210, with a Tn904 transposon in the center of the T-region, induces tumors that specifically exhibit a root development on tobacco plants. T-DNA has been detected in sterile amorphous crown-gall tissue derived from these tumors. The transposon Tn904 insertion was shown to result a changed "core" T-DNA. Abnormal tumor morphologies induced by these mutant strains have been observed also on Kalanchoë stems. On tomato plants the mutants induce small unorganized tumors while on Nicotiana rustica unorganized tumors, nearly equal in size to those caused by the wild-type strain have been induced. LBA4060 was shown to be avirulent on Kalanchoë leaves and LBA4210 was weakly virulent. Infection of Kalanchoë leaves or tomato plants with a mixture of separately grown cultures of both mutants resulted in the formation of more or less normal tumors. The exposure of a tomato plant to naphthalene acetic acid (NAA), a synthetic auxin, during development of tumors induced by LBA4060 stimulated tumor formation. Tumor growth induced by LBA4210 was found to be stimulated by kinetin.

Forty-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.

Expression 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.

Agrobacterium 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.

We 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 HindIII 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 lambda 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.

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.

The molecular basis of complementation by a mixture of two different types of octopine T-region mutants (LBA4060 and LBA4210) was studied. Six randomly chosen cellular clones derived from a tumor obtained after mixed infection were analyzed for their T-DNA content via Southern blot hybridization. The clones appeared to contain T-DNA that originated from each of both mutants, indicating that they developed from doubly infected single cells. Genetic complementation, therefore, might explain at least in part the observed complementation phenomenon. However, complementation as a result of cross-feeding between separately transformed cells could not be excluded. Following protoplast isolation, small aggregates might have formed that developed into the clones analyzed.

Potato Line Mb1501B is a derivative of the cultivar Maris Bard (Solanum tuberosum), transformed with T-DNA from A. tumefaciens strain LBA1501. In culture it grew as frequently branching stunted shoots with a basal callus, lacking roots. These shoots did not form tubers. When grafted, Mb1501B shoots gradually became morphologically more normal and aerial tubers formed readily. Cultured Mb1501B shoots contained 100-200-fold higher concentrations of the biologically-active cytokinins zeatin, zeatin riboside and their corresponding side-chain o-glucosides than untransformed Maris Bard shoots. Cultured Mb1501B shoots contained approximately a 3-fold lower concentration of indole acetic acid (IAA). In grafted Mb1501B plants a 3-10-fold higher concentration of the active cytokinins was found compared with untransformed plants and no difference in IAA concentration.

Transfer of octopine Ti plasmids to strains already carrying an octopine Ti plasmid was found to occur at the same (high) frequency as transfer to Ti plasmid lacking recipients, showing that resident Ti plasmids do not exhibit entry exclusion towards incoming Ti plasmids. The resident octopine Ti plasmid was lost by the recipient after the entrance of the incoming Ti plasmid, which is indicative of the incompatibility between the Ti plasmids. Octopine Ti plasmids were found to become established only infrequently in recipients with a nopaline Ti plasmid and, vice versa, nopaline Ti plasmids were only rarely established in recipients with an octopine Ti plasmid. Rare clones in which the incoming octopine (nopaline) Ti plasmid had been established despite the presence of a nopaline (octopine) Ti plasmid appeared to harbor cointegrates consisting of the entire incoming Ti plasmid and the entire resident Ti plasmid. The integration event invariably had occurred in a region of the plasmids that is highly conserved in evolution and that is essential for oncogenicity. These results show that octopine and nopaline Ti plasmids cannot be maintained as separate replicons by one and the same cell. Therefore, be definition, these plasmids belong to the same incompatibility group, which has been names inc Rh-1. Agrobacterial non-Ti octopine and nopaline plasmids were found to belong to another incompatibility group. The tumorigenic properties of strains harboring two different Ti plasmids, in a cointegrate structure, were indicative of the virulence genes of both of them being expressed. The agrobacterial non-Ti octopine and nopaline plasmids did not influence the virulence properties encoded by the Ti plasmid.

Octopine-type tumor tissue was obtained both by infection of plants or isolated protoplasts with Agrobacterium tumefaciens and by somatic hybridization of normal and crown gall tobacco cells. Analysis of T-DNA by Southern blotting of clones and uncloned tissue reveals that, whereas tumors induced on plants are heterogeneous mixtures of cells differing in T-DNA organization, each tissue derived from transformed protoplasts or from somatic hybridization is homogeneous. Detailed analysis of T-DNA organization showed that TL- or "core" T-DNA was always present at one or two copies per diploid genome. However, sometimes it was present in a modified form, either deleted, extended, tandemly duplicated or probably methylated. TR-DNA was not detected. The observed variation in the organization of T-DNA in octopine crown gall tissue did not appear to be a characteristic of the way the tissue was derived.

A transfer-deficient derivative of plasmid RP1-pMG1 was isolated after insertion of Mu cts62. The Tra- R plasmid was used to donate Tn904, encoding streptomycin resistance, to Ti plasmid pAL102 harbored by Agrobacterium tumefaciens Ach5. Under conditions promoting high Ti transfer frequencies, 155 strains were isolated in which the streptomycin marker coupled with Ti plasmid in further transfer experiments. These isolates represent stable insertions of Tn904 into the Ti plasmid. In addition, 19 strains were isolated in which the insertion of Tn904 was apparently unstable. The frequency of stable Tn904 transpositions was estimated to be 3 x 10(4-) per transferred Ti plasmid. Evidence was obtained that Tn904 readily may transpose from the Ti plasmid into the bacterial chromosome. The strains carrying Ti plasmids with stable insertions were characterized with respect to virulence, octopine degradation, octopine synthesis in induced tumors, and Ti plasmid transfer. Thirteen of the strains were found to be affected in tumor-inducing ability.

Stable cointegrates between incRh-1 octopine (Ach5) and nopaline (C58) Ti-plasmids, present in ten independently isolated Agrobacterium tumefaciens strains, showed identical restriction endonuclease patterns. Each cointegration event had taken place in the common sequence between the T-regions of both Ti-plasmids. This illustrates a high preference for this region when used in the formation of cointegrates. Four crown gall tissues, obtained after transformation of Nicotiana tabacum cells by one of the mutants, were analysed by using Southern blot analysis for their T-DNA structure. The borders of T-DNA frequently appeared to differ from T-DNA borders previously detected in tumour tissues that had been induced by Agrobacterium strain C58 or Ach5. Therefore, it was concluded that possibly a less stringent mechanism exists for the integration into plant DNA of T-DNA, derived from a composite (octopine/nopaline) T-region than for integration of T-DNA from a normal (octopine or nopaline) T-region.

Seven Tn904 insertion mutants of pTi Ach5 affecting Agrobacterium tumefaciens virulence were studied. The mutant character was shown to be plasmid borne. Four of these mutants were avirulent and carried an insertion in restriction endonuclease HpaI fragment 12, a 3.3-megadalton fragment, which therefore appears to be a Ti plasmid region essential for virulence. Two mutants were attenuated in virulence. The inserts mapped close to HpaI fragment 12. One mutant giving rise to small tumors with excessive adventitious root formation on Kalanchoe daigremontiana carried an insertion in the right side of the common sequence in the deoxyribonucleic acid of the Ti plasmid detected in crown gall tumors. The insertion behavior of Tn904 was studied by analyzing 11 independently isolated and randomly chosen mutants. The Tn904 inserts did not affect oncogenicity, tumor morphology, bacterial transfer functions, octopine catabolism functions, or vital parts of the Ti plasmid, such as the origin of replication. Most of the Tn904 inserts were concentrated in a small part of the map. The size of additional deoxyribonucleic acid as a result of Tn904 inserts varied between 5 and 15 megadaltons. In two cases a Ti plasmid was found with two Tn904 insertions at different positions.

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.