The transgenic petunia line 17-R contains one copy of the maize A1 gene which mediates brick-red pelargonidin pigmentation of the flower. A white derivative, 17-W, was isolated from homozygous progeny of this line in which no pelargonidin pigmentation was observed. In 17-W the 35S promoter driving the A1 gene was hypermethylated, in contrast to its hypomethylated state in 17-R. Progeny plants carrying both the 17-R and 17-W allele did not show the expected A1 phenotype. Predominantly white progeny and variable plants were observed which showed a continuous change in pattern and intensity of pelargonidin pigmentation. This reduction of A1 activity argues for a semidominant effect of the 17-W allele which inhibits the activity of its homologue, 17-R. This system shows striking similarities to some paramutation phenomena in plants which represent a heritable change in gene function of a paramutable allele directed by a paramutagenic homologue. The analysis of the methylation patterns of the A1 alleles suggests that interactions between differentially methylated alleles are responsible for the paramutation-like effect which is mediated by somatic pairing. The analogy of this system to other phenomena based on homology-dependent interlocus trans-inactivation supports the assumption that those may be based on a related mechanism which includes an interaction between ectopic homologues.

30,000 transgenic petunia plants carrying a single copy of the maize A1 gene, encoding a dihydroflavonol reductase, which confers a salmon red flower colour phenotype on the petunia plant, were grown in a field test. During the growing season plants with flowers deviating from this salmon red colour, such as those showing white or variegated phenotypes and plants with flowers exhibiting only weak pigmentation were observed with varying frequencies. While four white flowering plants were shown at the molecular level to be mutants in which part of the A1 gene had been deleted, other white flowering plants, as well as 13 representative plants tested out of a total of 57 variegated individuals were not mutants but rather showed hypermethylation of the 35S promoter directing A1 gene expression. This was in contrast to the homogeneous fully red flowering plants in which no methylation of the 35S promoter was observed. While blossoms on plants flowering early in the season were predominantly red, later flowers on the same plants showed weaker coloration. Once again the reduction of the A1-specific phenotype correlated with the methylation of the 35S promoter. This variation in coloration seems to be dependent not only on exogenous but also on endogenous factors such as the age of the parental plant from which the seed was derived or the time at which crosses were made.

A considerable proportion of cytosine residues in plants are methylated at carbon 5. According to a well‐accepted rule, cytosine methylation is confined to symmetrical sequences such as CpG and CpNpG, which provide the signal for faithful transmission of symmetrical methylation patterns by maintenance methylase. Using a genomic sequencing technique, we have analysed cytosine methylation patterns within a hypermethylated and a hypomethylated state of a transgene in Petunia hybrida. Examination of a part of the transgene promoter revealed that in both states m5C residues located within non‐symmetrical sequences could be detected. Non‐symmetrical C residues in the two states were methylated at frequencies of 5.9 and 31.9%, respectively. Methylation appeared to be distributed heterogeneously, but some DNA regions were more intensively methylated than others. Our results show that at least in a transgene, a heterogeneous methylation pattern, which does not depend on symmetry of target sequences, can be established and conserved.

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A 2-kilobase (kb) genomic fragment was selected from Petunia hybida that increased transformation efficiencies by at least a factor of 20 after direct DNA transfer to petunia and tobacco protoplasts when supercoiled plasmid DNA was used. Because of this effect this fragment was named transformation booster sequence (TBS). Increased transformation frequencies were observed for plasmids that contained either the 2-kb fragment in dimeric or monomeric form or an internal 1.1-kb fragment of TBS. Analysis of ranformants revealed that preferentially one copy of foreign DNA is integrated. Thus, TBS improves the poor transformation frequencies of direct gene transfer using circular plasmids, while it conserves the simple integration pattern that is important for practical applications. Possible mechanisms of TBS action are discussed.sequences has been reported for other transformations that also used circular plasmid DNA (10).Since synchronization procedures are not tolerated by all species and the technique is time consuming, alternatives for M-phase transformation were examined. Therefore, genomic DNA fragments were analyzed for a positive effect on transformation frequencies to select for autonomously replicating sequences, as has been performed in yeast (11) or for a transformation-enhancing fragment like the one described for Aspergillus nidulans (12).Here,we report on the isolation of a particular DNA fragment from Petunia hybrida, which was named transformation booster sequence (TBS), as it increased transformation frequencies without altering the simple integration pattern.Although DNA transfer systems mediated by Agrobacterium tumefaciens are very efficient (for review, see ref. 1), direct DNA transfer into plant cells has gained some importance since its first application (2, 3). Several strategies to increase the relatively low transformation frequencies of direct DNA transfer have been tested. Considerable improvement was gained through transformation by electroporation (4) or using optimized Mg2e and PEG concentrations (5). However, both these methods include the use of linearized plasmid DNA or of an excess of carrier DNA. Although transformation frequencies are remarkably high, the DNA integration pattern is complex (4) and it remains to be shown whether, by using this technique, a sufficient number oftransformants can be generated that contain and express other genes of the introduced plasmid apart from the selectable marker. The present advantages ofA. tumefaciens-mediated transformation are not only the high transformation frequencies obtained but, importantly, the simple integration pattern of the transferred DNA that guarantees practicability. Therefore, any techniques for direct DNA transfer should improve the quality of the transformation products as well as the frequency of transformation. One way to achieve a more simple integration pattern is the use of supercoiled plasmid DNA without the addition of carrier DNA, since it has been shown that in animal systems this leads to a greater propo...