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From over forty independently isolated potato lines transformed with wild-type and promoter-mutated T-cyt genes, a number of lines were selected for examination of phenotypic changes in growth and development for plants grown in soil in a controlled environment. The three lines chosen for most detailed examination showed a wide spectrum of phenotypic changes. In comparisons with control potato cv. Désirée, the plants of one line had a two- to three-fold increase in biomass production during early vegetative growth, advanced senescence and a shortened plant life-span. Another line showed abnormal cellulytic senescence. In two lines there were increases in tuber numbers and more skewed tuber size distributions which correlated with reduced shoot apical dominance and shortened dormancy of the stored tubers. None of the lines showed altered timing of onset of tuberization or flowering, although tuberization was consistently delayed when expressed as a function of increasing total plant weight. A hypothesis is proposed to explain the diverse phenotypes which postulates that (1) T-cyt transformation causes enhanced sensitivity to cytokinins in specific types of shoot cells which are already targets for regulation by normal root-derived cytokinins; (2) two distinct types of shoot target cells are present, one in shoot meristems and one in leaves; (3) the two types can acquire enhanced sensitivity, either separately or in combination depending on the particular T-cyt transformation event. The scope for using the transformed plants in subsequent physiological, biochemical and molecular studies, aimed at examining the molecular basis of the model or selected consequences of T-cyt transformation in altering regulation of potato plant growth and development, is discussed. The attention is drawn to the possible involvement at the subcellular level of sucrose phosphate synthase in mediating the phenotypic effects caused by T-cyt transformation.

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for beta-glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3' flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11,000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10-15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5' expression control sequences.

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.

Two dimensional gel electrophoresis was used to examine differences in steady state total poly(A) RNA from untransformed potato (Solanum tuberosum cv. Maris Bard) and potato transformed with shoot-inducing TL-DNA from A. tumefaciens. RNA was compared from phenotypically very distinct in vitro cultured shoots, more similar grafted plants and tubers. In each case between 200-400 translation products were identified representing the more abundant poly(A) mRNA's. In general, poly(A) RNA from the transformed tissues gave more high molecular weight products. This increase was most evident in poly(A) RNA from shoot cultures. Depending on the tissue examined, 1-5% of the translation products with a molecular weight <43 KD were observed to increase or decrease in abundance. The influence of T-DNA on cellular gene expression in the different transformed potato tissues is discussed in relation to previously determined changes in T-DNA gene expression (particularly of the T-DNA cytokinin gene) and the corresponding changes in endogenous hormone concentrations. It is concluded that some of the specific changes in low molecular weight products are either directly caused by the increased cytokinin levels or are indirectly involved in maintaining the transformed phenotype. re]19850530 rv]19851206 ac]19851210.

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.

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.

Large amounts of pathogenesis-related (PR) proteins were found inNicotiana tabacum crown gall tissue, following transformation of normal tobacco cells withAgrobacterium tumefaciens. In contrast, PR proteins were not detected in leaves of grafted plants that had been recovered from crown gall tissue even though these plants were still transformed as shown by their inability to form roots and ability to produce octopine. No difference was observed in susceptibility to virus infection between untransformed and transformed plants grafted onto identical rootstocks. The results are discussed in relation to physiological factors controlling PR protein induction and virus resistance.

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.

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.

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.

In potato line Mb1501B one or possibly two normal size Ti TL-DNA copies per tetraploid genome were detected by Southern blot analysis, but no TR-DNA. The TL-DNA probably contained the entire transposon Tn1831 inserted at the T-DNA auxin gene for transcript 2. Northern blot analyses of the steady-state RNA in different Mb1501B tissues isolated from (i) shoots cultured in vitro (ii) grafted plants and (iii) tubers, showed that that TL-DNA transcripts 3, 4, 6a and 7 were expressed most abundantly in the cultured shoots. They formed approximately 0.0023 to 0.0007% of the total poly(A) RNA. Transcripts 1, 5 and 6b were not detected in any of the tissues analysed. This indicated even lower levels of expression (below approximately 0.0001% of the total poly(A) RNA or, making certain assumptions, an abundance of less than one T-DNA derived RNA molecule per cell). As expected, transcript 2 was not detected in any of the Mb1501B tissues. The abundance of the transcripts was reduced in grafted plants and tubers compared with cultured shoots with the greatest decrease (5×) for transcripts 4, 6a and 7. Transcript 4, the one most responsible for the changed growth and development of Mb 1501 B, formed approximately 0.0003% of the poly(A) RNA from both grafted plants and tubers.