R. I. S. Brettell

Heritable somaclonal variation in wheat

SummaryEfficient tissue culture and regeneration methods were established using immature wheat embryos as expiants. Genotype differences in culturability were evident, and from the ten accessions most amenable to culture, a total of 2,846 plants were regenerated. Extensive somaclonal variation for morphological and biochemical traits was observed among 142 regenerants of a Mexican breeding line, ‘Yaqui 50E’, and their progeny. Variant characters included height, awns, tiller number, grain colour, heading date, waxiness, glume colour, gliadin proteins and α-amylase regulation. The variant characters were heritable through two seed generations and included traits under both simple and quantitative genetic control. Segregation data suggested that mutations both from dominance to recessiveness, and from recessiveness to dominance, had occurred. Most mutations in the primary regenerants were in the heterozygous state but some were true-breeding and presumed to be homozygous. Chromosome loss or addition did not account for the variation and none of the variant phenotypes was observed in over 400 plants from the parental seed source. The distinctive parental gliadin pattern was maintained in the somaclones thus excluding seed contamination or cross-pollination as a source of the variation.

pEmu: an improved promoter for gene expression in cereal cells

SummaryA recombinant promoter, pEmu, has been constructed to give a high level of gene expression in monocots. It is based on a truncated maize Adh1 promoter, with multiple copies of the Anaerobic Responsive Element from the maize Adh1 gene and ocs-elements from the octopine synthase gene of Agrobacterium tumefaciens. The pEmu promoter was one of 12 different promoter constructs that were linked to the β-glucuronidase (GUS) marker gene. Promoter activity was measured 48 h after introduction of the constructs into protoplasts of five different monocot species [wheat, maize, rice, einkorn (Triticum monococcum), and Lolium multiflorum] and one dicot (Nicotiana plumbaginifolia). In suspension cell protoplasts, the most highly expressing construct (pEmuGN) gave 10- to 50-fold higher expression than the CaMV 35S promoter in all the monocot species. The pEmu promoter should be valuable where a high level of gene expression is required in monocots. The pEmu promoter showed instability in several widely used Escherichia coli strains but was stable in a recA, recD strain AC001, which is described. Another construct, p4OCSΔ35SIGN, gave a tenfold increase in expression over the CaMV 35S promoter in dicot (Nicotiana plumbaginifolia) protoplasts.

Foreign gene expression in transgenic cereals

Abstract Recent advances in transformation technology have resulted in the routine production of transgenic plants for an increasing number of cereal species. With a view to improving cereal quality and agronomic performance by genetic engineering, attention is beginning to focus on the characterization of those molecular elements that will be used to regulate foreign gene expression in transgenic cereals.

Molecular analysis of a somaclonal mutant of maize alcohol dehydrogenase

SummaryPlants regenerated from tissue cultures of maize were screened for variants of ADH1 and ADH2. Root extracts of 645 primary regenerant plants were tested, and one stable mutant of Adh1 was detected. The mutant gene (Adh1-Usv) produces a functional enzyme with a slower electrophoretic mobility than that of the progenitor Adh1-S allele, and is stably transmitted to progeny. The mutant was not present among four other plants derived from the same immature embryo, and therefore arose as a consequence of the culture procedure. The gene of Adh1-Usv was cloned and sequenced. A single base change in exon 6 was the only alteration found in the gene sequence. This would translate in the polypeptide sequence to a valine residue substituting for a glutamic acid residue, resulting in the loss of a negative charge and the production of a protein with slower electrophoretic mobility.

Variation at the Nor loci in triticale derived from tissue culture.

SummaryPlants derived from tissue cultures of six triticale genotypes were the subject of an analysis for changes in the rRNA genes located at the site of nucleolar organizer regions (the Nor loci) on chromosomes 1B, 6B and 1R. In addition whole plant phenotypes and the chromosomal constitutions of their progenies were examined for alterations. Following treatment of DNA with the restriction endonuclease Taq1, it was possible to assign electrophoretic bands representing rDNA spacer sequences to each of the chromosomes known to carry a major Nor locus. In general, the rRNA genes were found to be stable except in one family where a marked reduction in the number of rDNA units was observed. This reduction in 1R rDNA spacer sequences was heritable and correlated with reduced C-banding at the position of Nor-R1 on chromosome 1R. The change was clearly a consequence of tissue culture since six other plants regenerated from the same culture, and the original parent, did not carry the alteration.

Embryo yield in wheat anther culture is influenced by the choice of sugar in the culture medium

SummaryThe effect of employing different sugars in wheat anther culture has been investigated using four Spring wheat cultivars. The most responsive cultivar, Orofen, gave a three to four-fold increase in embryo yield when maltose was used in place of sucrose, with 50 embryos being produced for every 100 anthers cultured. Measurement of sugar concentrations in the culture media indicated that sucrose was more rapidly hydrolysed than maltose. However, neither the osmotic potential of the medium nor the concentration of glucose appeared to be critical factors in determining embryo yield.

Reactivation of a silent Ac following tissue culture is associated with heritable alterations in its methylation pattern

SummaryTissue cultures were initiated from embryos with an inactive form of Ac in the wx-m9 Ds-cy allele. Plants regenerated from the cultures showed a high frequency of activation of Ac. That activation was shown to be associated with reduced methylation of cytosine residues at the 5′ end of the transposable element. An examination of Ac activity and methylation status of the Ac element in progenies of the regenerant plants demonstrated transmission of the altered epigenotype through two sexual generations. In these progenies no evidence for trans activation of inactive, partially methylated, Ac elements was obtained. These results confirm that in certain instances altered methylation patterns can be inherited through the germ line.

The role of DNA methylation in the regulation of plant gene expression

The most common modification of DNA in plant cells is methylation of cytosine residues at carbon 5. In contrast to mammalian cells in which 3–8% of cytosine residues are methylated (Shapiro, 1975), in plants up to 30% of cytosine residues are modified (Adams and Burdon, 1985). There is considerable inter-species variation in the level of cytosine methylation, ranging from 4.6% in Arabidopsis thaliana (Leutwiler et al., 1984), which has a small genome with relatively little highly repeated DNA, to 33% in rye, Secale cereale (Thomas and Sherratt, 1956). The difference in the extent of methylation, between plants and animals, is due to two factors. The CG dinucleotide, which is methylated to about the same extent (70–80%) in plants and animals, occurs more frequently in plant DNA. In addition plant DNA is methylated in CNG trinucleotides where N can be any base (Gruenbaum et al., 1981). The CG dinucleotide has symmetrical cytosine residues in the two DNA strands and it has been observed that, when modified, both cytosines are methylated (Bird, 1978; Cedar et al., 1979). It is this symmetry that allows the pattern of methylation to be maintained through DNA replication. Newly replicated DNA is hemimethylated with methylation at specific sites on the parental strand. Methylation of the new strand at the unmethylated cytosine of a hemimethylated CG dinucleotide restores the original pattern of methylation; this is termed maintenance methylation (Holliday and Pugh, 1975; Riggs, 1975; Razin and Riggs, 1980). The CNG motif also has strand symmetry suggesting that methylation of this motif will probably be maintained through replication by the same mechanism.

A tissue culture induced Adh1 null mutant of maize results from a single base change

SummaryWe have found a null Adh1 allele which arose as a somaclonal variant following tissue culture of maize embryos carrying Adh1-1S and Adh1-1F alleles. Cloning and sequencing shows that the mutant allele derives from Adh1-1S and that there has been a single base change in the coding region of the gene which converts and AAG lysine codon to a TAG stop codon. The rate of nucleotide substitution (two per 218 embryos cultured) is much greater than normal mutation rates.

Comparison of three selectable marker genes for transformation of wheat by microprojectile bombardment

Three selectable marker genes were compared for their efficacy in the production of transgenic wheat plants following microprojectile bombardment of cultured immature embryos. While transformed plants were recovered using the bar (phosphinothricin acetyltransferase) gene in combination with bialaphos, and the aphA (neomycin phosphotransferase) gene in combination with geneticin or paromomycin, no transgenic material was obtained with the hpt (hygromycin phosphotransferase) gene and hygromycin B. Southern analysis revealed single copy as well as multiple copy insertions of the bar and aphA transgenes. Inheritance of these selectable marker genes was demonstrated in the T1 generation progenies.