The maize MADS box gene ZmMADS3 affects node number and spikelet development and is co-expressed with ZmMADS1 during flower development, in egg cells, and early embryogenesis.

MADS box genes represent a large gene family of transcription factors with essential functions during flower development and organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in the regulation of the fertilization process and early embryo development, we have isolated two novel MADS box cDNAs, ZmMADS1 and ZmMADS3, from cDNA libraries of maize (Zea mays) pollen and egg cells, respectively. The latter gene is allelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 is additionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1 expression is restricted to cells with the capacity to form somatic embryos, and to globular embryos at later stages. ZmMADS3 is detectable only by more sensitive reverse transcriptase-PCR analyses, but is likewise expressed in embryogenic cultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygotic embryos. During flower development, ZmMADS1 and ZmMADS3 are co-expressed in all ear spikelet organ primordia at intermediate stages. Among vegetative tissues, ZmMADS3 is expressed in stem nodes and displays a gradient with highest expression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3 are reduced in height due to a reduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absence of anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed.

Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family.

Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the extracellular domain, a proline-rich region (SPP) just upstream of the transmembrane domain and a C-terminal extension (C) after the kinase domain identify them as members of the SERK (somatic embryogenesis receptor-like kinase) family. ZmSERK1 and ZmSERK2 are single-copy genes and show 79% identity among each other in their nucleotide sequences. They share a conserved intron/exon structure with other members of the SERK family. In the maize genome, ZmSERK1 maps to position 76.9 on chromosome arm 10L and ZmSERK2 to position 143.5 on chromosome arm 5L, in regions generally not involved in duplications. ZmSERK1 is preferentially expressed in male and female reproductive tissues with strongest expression in microspores. In contrast, ZmSERK2 expression is relatively uniform in all tissues investigated. Both genes are expressed in embryogenic and non-embryogenic callus cultures.

Fertile transgenic wheat from microprojectile bombardment of scutellar tissue.

A reproducible transformation system for hexaploid wheat was developed based on particle bombardment of scutellar tissue of immature embryos. Particle bombardment was carried out using a PDS 1000/He gun. Plant material was bombarded with the plasmid pDB1 containing the beta-glucuronidase gene (uidA) under the control of the actin-1 promoter of rice, and the selectable marker gene bar (phosphinothricin acetyltransferase) under the control of the CaMV 35S promoter. Selection was carried out using the herbicide Basta (Glufosinate-ammonium). From a total number of 1050 bombarded immature embryos, in seven independent transformation experiments, 59 plants could be regenerated. Putative transformants were screened for enzyme activity by the histochemical GUS assay using cut leaf material and by spraying the whole plants with an aqueous solution of the herbicide Basta. Twelve regenerants survived Basta spraying and showed GUS-activity. Southern-blot analysis indicated the presence of introduced foreign genes in the genomic DNA of the transformants and both marker genes were present in all plants analysed. To date, four plants have been grown to maturity and set seed. Histochemically stained pollen grains showed a 1:1 segregation of the uidA gene in all plants tested. A 3:1 segregation of the introduced genes was demonstrated by enzyme activity tests and Southern blot analysis of R1 plants.

Regeneration of transgenic, microspore-derived, fertile barley.

We have developed a system for the biolistic transformation of barley using freshly-isolated microspores as the target tissue. Independent transformation events led, on average, to the recovery of one plant per 1×10(7) bombarded microspores. Putative transformants have been regenerated using phosphinothricin as a selective agent. R0 plants have been transferred to soil approximately 2 months after bombardment. Integration of the marker genes bar and uidA has been confirmed by Southern analysis. The marker genes are inherited in all progeny plants confirming the expected homozygous nature of the R0 plants.

Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts.

Protoplasts isolated from a barley cell suspension (cv Dissa) were transformed with plasmid DNA containing the neomycinphosphotransferase II (NPT) and ß-glucuronidase (GUS) genes, using polyethyleneglycol (PEG) to induce DNA uptake. Transformed microcalli were selected in media containing G418 sulphate. NPT activity was detected in all antibiotic-resistant cell lines, but not all NPT-positive cell lines had GUS activity. Southern analysis confirmed the presence of sequences homologous to the APT and GUS genes in DNA of G418-resistant callus.

UV-inducible transient expression in parsley protoplasts identifies regulatory cis-elements of a chimeric Antirrhinum majus chalcone synthase gene.

It was shown previously that parsley protoplasts retain differential responsiveness to external stimuli, e.g. UV light. This opened the way for the development of transient expression assays to identify DNA sequences acting as regulatory cis-elements in the transcriptional activation of UV-inducible plant genes. The chalcone synthase (chs) gene of Antirrhinum majus (Snapdragon) is inducible by visible and by UV light. We demonstrate that the kinetics of light induction of a chimeric A.majus chs-nptII gene observed during transient expression in the parsley protoplasts reflect the kinetics of UV-induced CHS expression in A.majus seedlings in vivo. We define three regulatory sequence regions, each differing qualitatively and quantitatively in their effects on gene expression. Immediately upstream of the TATA box (-34) a sequence, with coordinates -39 to -197, functions as an orientation independent UV-light responsive element. The complete 1.1 kb promoter or three tandem copies of this element are capable of rendering a heterologous minimal promoter UV responsive. The next upstream region (-197 to -357) contains sequences that do not by themselves cause UV-induced expression, but specifically potentiate the level of UV-induced expression when combined with the TATA-box proximal UV-responsive element. A third element (-661 to -564) has the properties of a general enhancer since it increases the level of both uninduced and UV-induced expression.