Alan Blowers

Construction of expression vectors based on the rice actin 1 (Act1) 5' region for use in monocot transformation.

SummaryIt has been previously reported that the 5′ region of the rice actin 1 gene (Act1) promoted high-level expression of a β-glucuronidase reporter gene (Gus) in transformed rice cells. In this paper we describe the construction of Act1-based expression vectors for use in monocot transformation. As part of the development of these vectors, we have evaluated the influence of the Act1 first intron, the Act1-Gus junction-encoded N-terminal amino acids, and the sequence context surrounding the Act1 and Gus translation initiation site on Act1-Gus gene expression in rice and maize cells. We have found that addition of Act1 intron 1 to the transcription unit of a Gus reporter gene under control of the cauli-flower mosaic virus (CaMV) 35S promoter stimulated GUS activity more than 10-fold in transformed rice cells. Optimization of the sequence context around the Gus translation initiation site resulted in a 4-fold stimulation of Gus expression in transformed rice cells. By utilizing both the Act1 intron 1 and optimized Gus translation initiation site, a 40-fold stimulation in Gus expression from the CaMV 35S promoter has been achieved in transformed rice cells; very similar results were obtained in transformed maize cells. Taken together these results suggest that the Act1-based expression vectors described here should promote the expression of foreign genes in most, if not all, transformed monocot cells to levels that have not previoulsy been attainable with alternative expression vectors.

Stable transformation of sorghum cell cultures after bombardment with DNA-coated microprojectiles.

Cells from a suspension culture of Sorghum vulgare (sorghum) have been transformed to either hygromycin or kanamycin resistance following uptake of pBC1 or pNGI plasmids, respectively, introduced on DNA-coated high velocity microprojectiles. Hygromycin- and kanamycin-resistant transformants contained hygromycin B phosphotransferase- and neomycin phosphotransferasehybridizing restriction fragments of the expected size, respectively. A second introduced, but unselected for, reporter uidA gene which encodes ß-glucuronidase activity was also detected by DNA gel blot analysis in these transformants and shown to be expressed at low levels in two of the ten transformants analyzed. Transcripts from the introduced foreign genes accumulated to detectable levels in only these two transformants, both of which had a high copy number of genes integrated into their genome. This report further establishes the biolistic method as a useful route for delivery of DNA into the difficult-to-transform monocotyledonous plant species and represents the first stable transformation of this agronomically-important cereal grain.

Stable transformation of tomato cell cultures after bombardment with plasmid and YAC DNA

SummaryStable transformants were obtained after microprojectile particle bombardment of tomato cell suspensions (Lycopersicon esculentum cv VFNT Cherry and L. pennellii). The suspensions were bombarded with tungsten particles coated with either plasmid (∼6.3 kb) or yeast artificial chromosome (YAC) (80 kb) DNA containing the ß-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. The YAC DNA contained an insert of approximately 50 kb of DNA from VFNT Cherry. L. pennellii suspensions were more amenable to transformation than VFNT Cherry; more kanamycin-resistant calli were recovered from L. pennelli after bombardment with plasmid DNA, and only L. pennellii cells produced transformants after bombardment with YAC DNA. DNA gel blot analysis confirmed the presence of the nptll and GUS genes. This analysis also confirmed the integration of YAC DNA into the genome of the kanamycin-resistant calli and suggested that the level of intactness of the integrated YAC DNA was fairly high in four of the five transformants examined. Microprojectile bombardment of regenerable cultures with YACs may ultimately aid in map-based cloning of agriculturally-important genes.

Effect of the cauliflower mosaic virus 35S, actin, and ubiquitin promoters on uidA expression from a Bar-uidA fusion gene in transgenic Gladiolus plants.

SummaryTissue-specific patterns and levels of gene expression were characterized in transgenic Gladiolus plants that contained the phosphinothricin acetyltransferase (bar)-β-glucuronidase (uidA) fusion gene under transcriptional control of the promoter from either the cauliflower mosaic virus 35S (CaMV 35S), duplicated CaMV 35S (2×CaMV 35S), rice actin (Act1), or Arabidopsis ubiquitin (UBQ3) promoters. The bar gene confers resistance to phosphinothricin (PPT)-containing herbicides and allowed selection of transgenic cells. The β-glucoronidase gene encoded by the uidA locus of E. coli functioned as a reporter gene. Maximum levels of β-glucuronidase (GUS) activity in leaves were 173, 112, 50, and 10 nmoles 4-methylumbelliferone h−1 mg−1 protein for transgenic plants with the bar-uidA fusion gene under the control of the CaMV 35S, 2×CaMV 35S, UBQ3, and Act1 promoters, respectively. There was frequently considerable variability in GUS activity between the leaves of a single plant, and levels of uidA expression varied between independently transformed plants for each promoter. Callus derived from transgenic plants showed much less variation in GUS expression than leaves. The mean level of GUS activity was significantly higher (over 3×) for transgenic lines of callus containing the CaMV 35S as compared to the UBQ3 promoter, and this confirmed the higher (2×) level of GUS activity in levels of plants with the CaMV 35S promoter as compared to the UBQ3 promoter. Tissue-specific patterns of uidA expression were determined by histochemical staining. Leaves 5–6 cm long from plants with any of the four promoters tested exhibited uidA expression primarily in the vasculature. Under all four promoters uidA was expressed more frequently in root tips as compared to leaves.

Stable transformation of Gladiolus by particle gun bombardment of cormels

Transgenic plants of Gladiolus were produced following particle bombardment of cormel slices. Plant cells were cotransformed with the gene for phosphinothricin acetyltransferase under control of the cauliflower mosaic virus 35S promoter and the uidA gene coding for β-glucuronidase (GUS) under control of the actin promoter isolated from rice. The optimum concentration for the first selection of transformed plants was 8 mg/l phosphinothricin which resulted in 14% of the bombarded cormel slices regenerating plants that were transformed as confirmed by polymerase chain reaction amplification. Polymerase chain reaction amplification, Southern hybridization and histochemical staining for GUS gene expression on plants after two selective screenings with phosphinothricin indicated that regenerated plants were transformed. Histochemical staining for GUS gene expression showed that the actin promoter resulted in GUS gene expression primarily in callus cells and root meristems. Leaves were typically chimeric for GUS gene expression.

Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation

To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resistant to kanamycin (300 mg/L) were recovered for each size of YAC tested. Two of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also contained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert region of the 80 kb YAC confirmed that one of the two double-resistant lines had integrated a fully intact single copy of the YAC DNA while the other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorporated relatively small portions of the YAC insert DNA distal to the selectable marker. Our data suggest genomic DNA cloned in artificial chromosomes up to 150 kb in size have a reasonable likelihood of being transferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolation of agronomically useful plant genes using map-based cloning strategies.

Genetic Transformation of Gladiolus

Gladiolus is a monocotyledonous floral bulb crop which ranked fifth in 1993 for the number of stems (79 663) shipped worldwide (USDA 1994). In Florida alone, about 3600 acres are devoted to cut flower production, and about 120 acres to the production of stock corms of Gladiolus (Wilfret 1980). Its production areas are severely plagued by viral, microbial, and fungal pathogens, so that a typical cultivar lasts only a few years before it succumbs to disease and is removed from production. It has not been possible to effectively breed pathogen resistance into Gladiolus using conventional breeding methods so this plant represents a commercially important floral crop that would benefit from disease resistance mediated by genetic engineering.