Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrohacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7-8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T1 plants and the fact that none of the T2 progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.

The chromosomes of Arabidopsis thaliana and Brassica oleracea have been extensively rearranged since the divergence of these species; however, conserved regions are evident. Eleven regions of conserved organization were detected, ranging from 3.7 to 49.6 cM in A. thaliana, spanning 158.2 cM (24.6%) of the A. thaliana genome, and 245 cM (29.9%) of the B. oleracea genome. At least 17 translocations and 9 inversions distinguish the genomes of A. thaliana and B. oleracea. In one case B. oleracea homoeologs show a common marker order, which is distinguished from the A. thaliana order by a rearrangement, indicating that the lineages of A. thaliana and B. oleracea diverged prior to chromosomal duplication in the Brassica lineage (for at least this chromosome). Some chromosomal segments in B. oleracea appear to be triplicated, indicating the need for reevaluation of a classical model for Brassica chromosome evolution by duplication. The distribution of duplicated loci mapped for about 13% of the DNA probes studied in A. thaliana suggests that ancient duplications may also have occurred in Arabidopsis. The degree of chromosomal divergence between A. thaliana and B. oleracea appears greater than that found in other confamilial species for which comparative maps are available.

Disruption of genes encoding endogenous transport proteins in Saccharomyces cerevisiae has facilitated the recent cloning, by functional expression, of cDNAs encoding K+ channels and amino acid transporters front the plant Arabidopsis thaliana [1-4]. In the present study, we demonstrate in whole-cell patch clamp experiments that the inability of trk1 Delta trk2 Delta mutants of S. cervisiae to grow on submillimolar K+ correlates with the lack of K+ inward currents, which are present in wild-type cells, and that transformation of the trk1 Delta trk2 Delta double-deletion mutant with KAT1 from Arabidopsis thaliana restores this phenotype hy encoding a plasma membrane protein that allows large K+ inward currents. Similar K+ inward currents are induced by transformation of a trk1 mutant with AKT1 from A. thaliana.

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. The reproducibility and the overall efficiency of this simple in planta transformation procedure was investigated. In addition, the T-DNA copy number and inheritance in the transformants was determined and it was examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. The results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7-8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci.

Differentiation of sclereids was induced in the pith of Arabidopsis thaliana by repeated cutting of developing inflorescences for 4 weeks. This is the first report of sclereids in A. thaliana. The treatment also resulted in large rosettes and enhanced cambial activity that formed considerable amounts of secondary xylem.

We report here the isolation and nucleotide sequence of genomic clones encoding the chloroplast enzyme sedoheptulose-1,7-bisphosphatase (SBPase) from Arabidopsis thaliana. The coding region of this gene contains eight exons (72-76 bp) and seven introns (75-91 bp) and encodes a polypeptide of 393 amino acids. Unusually, the 5' non-coding region contains two additional AUG codons upstream of the translation initiation codon. A comparison of the deduced Arabidopsis and wheat SBPase polypeptide sequences reveals 78.6%, identity. Expression studies showed that the level of SBPase mRNA in Arabidopsis and wheat is regulated in a light-dependent manner and is also influenced by the developmental stage of the leaf. Although the Arabidopsis SBPase gene is present in a single copy, two hybridizing transcripts were detected in some tissues, suggesting the presence of alternate transcription start sites in the upstream region.

Degenerate oligonucleotide primers corresponding to two conserved regions in animal cyclins were used to amplify cyclin sequences from Arabidopsis thaliana by polymerase chain reaction (PCR). Screening of a floral meristem cDNA library with two distinct PCR-generated cyclin sequences resulted in the isolation of four different cyclin cDNAs. Southern analysis of genomic DNA and sequence information from the isolated clones suggest that there is a family of cyclins in Arabidopsis.