Shaun L. A. Hobbs

Transgene copy number can be positively or negatively associated with transgene expression.

Two different types of T-DNA insert were found in tobacco plants transformed with Agrobacterium tumefaciens. High-expressing (H) types had one copy of the T-DNA at a locus and produced high expression of the transgene uidA, as measured by uidA RNA levels and β-glucuronidase activity; low-expressing (L) types had inverted repeats of the T-DNA at a locus and produced low uidA expression. H-types from different transformants acted additively, and cross-fertilization between two different homozygous transformants with H-type inserts produced F1 plants with GUS activity that equalled the parents and individual F2 plants with 50%, 100%, 150% and 200% of parental values. However, the L-type inserts worked in trans to suppress uidA expression from H-type inserts when both were present in the same genome. Hence when a transformant homozygous for the L-type insert was crossed to one homozygous for the H-type, all plants in the F1 and F2 generations with both types of insert had low GUS activity while F2 segregants that only had the H-type inserts had high GUS activity again. Suppression of the H-type gene was associated with increased methylation of the insert. Particle acceleration was used to introduce further copies of uidA into tissues of the transformants. Regardless of the promoter used, those plants with endogenous L-type inserts showed none of the distinct loci of GUS activity readily visible in material with no inserts, showing that L-type inserts could suppress not only the uidA expression of genomic homologues, but also of copies added in vitro.

The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants

Inter-transformant variability in the expression of introduced genes was studied in the R1 and R2 generations of 10 tobacco transformants, produced by Agrobacterium-mediated transformation. In replicated and physiologically equivalent material, tranformants showed considerable variability in the expression of the reporter gene uidA as shown by transcript levels and β-glucuronidase (GUS) activity. However, homozygous R2 material could be investigated for seven of the transformants and among these, and in one line in which two inserts could segregate independently, this inter-transformant variability was reduced to simple bimodal expression. The two levels of expression for GUS activity in leaves were high or low (approximately 2.5 or 0.3 nmol cm-2 min-1 respectively), with no continuous variation. Transformants in the high group had single T-DNA insertions, while those in the low group had multiple T-DNA insertions, at the same or different loci. Within each group, although T-DNA was apparently integrated at different sites in the plant genome, there was no evidence of position effects. GUS activity levels of the transformants were very similar in the field and in environmentally controlled conditions under high or low light. Plants with multiple insertions and low expression also tended to have increased methylation of the integrated T-DNA.

Specificity of strain and genotype in the susceptibility of pea to Agrobacterium tumefaciens.

SummaryTo determine the best combination for potential use in transformation of Pisum sativum L., 13 genotypes were inoculated with wild-type Agrobacterium tumefaciens strains A281, C58 and Ach5. A281 appeared to be the most virulent strain, as determined by size and number of tumours, followed by C58 and Ach5. Genotypes differed considerably in their response to inoculation and genotype x strain interaction was evident. Genotypes also responded differently to in vivo or in vitro inoculation. Axenic calli from tumours could be grown on hormone-free medium and the presence of the specific opines for each strain in the callus indicated successful transfer and expression of T-DNA. Southern blot analysis of DNA from callus of A281-inoculated material showed that both TR and TL T-DNA had been incorporated into the pea genome.

Rapid multiple shoot production from cotyledonary node explants of pea (Pisum sativum L.).

SummaryMultiple shoots were produced, from cotyledonary node explants of pea (Pisum sativum L.) cultured on MS medium containing 1 mg 1−1 BAP. The number of buds formed could be increased by scraping the nodes before culture or by increasing the cytokinin concentation. However, cytokinin levels over 5 mg 1−1 increasingly produced shoots that were vitrified and dificult to root. With all the genotypes tested, developing buds were visibile as soon as 5 days after culture and elongated shoots could be removed after 21 days., Histological studies indicated, that the buds and shoots were formed from superficial layers of tissue. The efficiency of this regeneration system compared favorably with previosly published methods. The rapid, genotype-independent, high frequency system described here may be of use in the production of transgenic pea plants.

Effect of promoter-leader sequences on transient reporter gene expression in particle bombarded pea (Pisum sativum L.) tissues

Abstract Particle bombardment was used to deliver plasmids containing promoter-β-glucuronidase (GUS) gene constructs into intact pea embryo axes, leaves and roots. Transient GUS enzyme activity was influenced by the promoter-leader sequence driving the GUS gene, as measured by fluorometric and histochemical assays. In most cases, the untranslated leader sequence from RNA 4 of alfalfa mosaic virus (referred to here as ‘AMV’) significantly increased GUS activity when included between the promoter (nopaline synthase (NOS), cauliflower mosaic virus 35S (35S), or the tandem 35S promoter (35S-35S)) and the GUS coding sequence. The 35S-35S-AMV promoter-leader sequence produced 4- to 10-fold greater levels of transient GUS activity in these pea tissues than the 35S promoter alone. Particle bombardment is a simple and rapid method for the assessment of vector constructs in pea tissues.

Isolation of a pea (Pisum sativum) seed lipoxygenase promoter by inverse polymerase chain reaction and characterization of its expression in transgenic tobacco

Part of the 5′-flanking sequence of a pea (Pisum sativum) lipoxygenase (LOX) gene was cloned, after amplification from genomic DNA by inverse polymerase chain reaction. Translational and transcriptional fusions of 818 bp of the 5′-flanking region and its deletion derivatives (−513 and −356) were made to a β-glucuronidase (GUS)-coding sequence and introduced into tobacco. Analysis of T1 transformants showed that the 818 bp 5′-flanking sequence drove GUS expression in seeds that was temporally regulated in a fashion similar to the accumulation of LOX mRNA in developing pea seeds. Contrary to expectations, however, expression of the 818 bp promoter-GUS fusion was not seed-specific; GUS activity was highest in leaves and also present in stems and, to a lesser extent, roots. Deletion analysis identified the region between −818 and −513 as essential for high-level, temporally regulated expression in seeds and also indicated that the sequence between −513 and −356 plays a negative role in leaf/stem, but not seed, expression. Comparison of translational and transcriptional fusions indicated that the LOX initiation codon was used more efficiently than the GUS initiation codon by the tobacco leaf translational apparatus.

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

SummaryA rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R1 progeny. NPTII activity was observed in the R2 generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.

Genotype- and promoter-induced variability in transient β-glucuronidase expression in pea protoplasts.

SummaryLeaf mesophyll protoplasts isolated from pea (Pisum sativum L.) genotypes Century and PI244253 showed transient expression of β-glucuronidase (GUS) when electroporated with plasmid DNA containing various promoter-leader sequence constructs driving the GUS gene. The optimum conditions for transient expression were: using protoplasts isolated from leaf material that had been kept in the dark for 90 h; electroporating at 250 V and 960 μF; and using 125 μg of calf thymus carrier DNA and 75 μ of plasmid DNA. PI244253 had 5 to 20 times the GUS activity levels of Century. Similar levels of transient expression were obtained using either the nopaline synthase or cauliflower mosaic virus 35S (35S) promoters. These levels were lower than that obtained using a duplicated 35S promoter derivative. The presence of an untranslated coat protein mRNA leader sequence from alfalfa mosaic virus between each promoter and the GUS gene resulted in increased GUS activity. Leaf mesophyll protoplasts and root protoplasts of PI244253 did not differ in levels of transient expression.

Genetic variability in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase and its small subunit mRNA in pea.

The amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was studied during greening in 12Pisum sativum L. genotypes. The proportion of mRNA coding for the small subunit of Rubisco (SSU mRNA) was also monitored by hybridization with a cDNA (complementary DNA) probe from one of the nuclear genes coding for SSU (rbcS). Both the SSU mRNA and Rubisco (in g·FW)−1) contents rapidly increased in all genotypes on illumination of dark-grown seedlings. Natural genetic variability was found in the amounts of SSU mRNA, Rubisco· (g FW)−1, total RNA·(g FW)−1 and mRNA·(μg total RNA)−1. Differences among genotypes in SSU mRNA were apparently the result of differences in the rate of light-induced accumulation of therbcS gene transcripts. Genotype means for SSU mRNA and Rubisco· (g FW)−1 amounts during greening were significantly correlated (r=0.788;P<0.01). This indicates a relationship between genetic differences in the rate of light-induced accumulation of therbcS gene transcripts and the Rubisco amount, and establishes a link between natural genetic variability at the molecular and the physiological levels. Genotypic variability in SSU mRNA during greening was also positively correlated to the Rubisco content per unit leaf area in fully greened leaves. Although Southern-blot analysis indicated that there was also natural genetic variability in the copy number of therbcS genes, this difference in copy number could not account for the differences in SSU mRNA production.

Genetic Control of Photosynthesis in Relation to Growth of Pea (Pisum sativum L.) Plants

The genetic improvement of photosynthesis has often been proposed as a route to increased crop productivity. However, although there is extensive genotypic variability in photosynthesis per unit of leaf area or leaf weight in many crop species, the exploitation of this variability remains difficult. Not only is photosynthesis very sensitive to environmental factors, but there are reports of considerable genotype by environment and genotype by developmental stage interactions. Under such conditions, selection progress would be difficult and large numbers of measurements would be needed to assess phenotypic expression.