Transgenic White Clover Plants Research Articles

Assessment of nutritional characteristics of virus-resistant transgenic white clover (Trifolium repens L.) grown under field and glasshouse conditions

White clover (Trifolium repens L.) is an important pasture legume in temperate areas throughout the world, providing fodder for grazing animals and improving soil fertility via symbiotic nitrogen fixation. However, the persistence and stress tolerance of white clover are affected by a number of viruses including alfalfa mosaic virus. Transgenic white clover plants with ectopic expression of the alfalfa mosaic virus coat protein were resistant to the virus under field and greenhouse conditions. With all genetic modifications of major consequence, there is the possibility of unintended effects on forage quality and natural toxicant levels. In this paper, we describe the evaluation of a range of parameters related to the nutritive value of white clover herbage to grazing animals and a suite of naturally occurring secondary metabolites that have the potential to be natural toxicants in transgenic white clover plants and wild-type control plants with a similar genetic background. Samples were collected from plants grown under both field and glasshouse conditions. Several commercial cultivars were included for comparison. Although there was plant-to-plant variation, as expected from an obligate outcrossing species, there were no significant differences in the range of this variation between transgenic and wild-type plants. Furthermore, no consistent significant differences were found between groups of transgenic and wild-type plants from the same generation, when mean nutritional parameters (crude protein, in vitro dry matter digestibility, neutral detergent fibre and water-soluble carbohydrates) and natural toxicants (cyanogenic glucosides, phytoestrogens and saponins) were compared.

Biosynthesis of proanthocyanidins in white clover flowers: cross talk within the flavonoid pathway.

Proanthocyanidins and anthocyanins are produced by closely related branches of the flavonoid pathway and utilize the same metabolic intermediates. Previous studies have shown a flexible mechanism of flux diversion at the branch-point between the anthocyanin and proanthocyanidin pathways, but the molecular basis for this mechanism is poorly understood. Floral tissues in white clover plants (Trifolium repens) produce both proanthocyanidins and anthocyanins. This makes white clover amenable to studies of proanthocyanidin and anthocyanin biosynthesis and possible interactions within the flavonoid pathway. Results of this study show that the anthocyanin and proanthocyanidin pathways are spatially colocalized within epidermal cells of petals and temporally overlap in partially open flowers. A correlation between spatiotemporal patterns of anthocyanin and proanthocyanidin biosynthesis with expression profiles of putative flavonoid-related genes indicates that these pathways may recruit different isoforms of flavonoid biosynthetic enzymes. Furthermore, in transgenic white clover plants with down-regulated expression of the anthocyanidin reductase gene, levels of flavan 3-ols, anthocyanins, and flavonol glycosides and the expression levels of a range of genes encoding putative flavonoid biosynthetic enzymes and transcription factors were altered. This is consistent with the hypothesis that flux through the flavonoid pathway may be at least partially regulated by the availability of intermediates.

Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T(0) and T(1) generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T(4) generation in an agronomically elite 'Grasslands Sustain' genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T(4) generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.

Improvement of drought tolerance in white clover (Trifolium repens) by transgenic expression of a transcription factor gene WXP1

White clover (Trifolium repens L.) is an important pasture legume in many regions of the world. A commercial cultivar of white clover (cv. Patriot) was transformed with a Medicago truncatula L. transcription factor gene, WXP1, and a reporter gene, β-glucuronidase (GUS). The WXP1 gene and the GUS gene were placed under control of the Arabidopsis CER6 promoter. GUS staining and cross-section analysis revealed the CER6 promoter directed constitutive expression in leaves and epidermis preferential expression in petioles of white clover. Independent transgenic WXP1 lines, empty vector and wild-type controls were subjected to drought stress treatment. The plants were characterised by measuring several physiological parameters including gas exchange, chlorophyll fluorescence, relative water content and leaf water potential. The WXP1 transgenic lines had higher net photosynthetic rates, higher efficiency of PSII, higher relative water content and leaf water potential under drought-stressed conditions. Consistent with the results from physiological analyses, the transgenic white clover plants carrying WXP1 showed improved tolerance to drought stress.

Post-transcriptional gene silencing as an efficient tool for engineering resistance to white clover mosaic virus in white clover ( Trifolium repens)

The lack of naturally occurring resistance to white clover mosaic virus (WCMV) has demanded exploration of a transgenic approach for the development of WCMV-resistant white clover plants. Transgenic white clover plants producing sense (co-suppression), antisense and hairpin RNA (hpRNA) transcripts corresponding to the WCMV replicase gene were produced and analysed at the molecular and phenotypic levels. Expression of hpRNA and antisense transgenes provided a high level resistance to WCMV, while the sense transgene provided partial resistance. The presence of small interfering RNA molecules (siRNAs) in the transgenic white clover plants prior to virus challenge indicated that WCMV resistance was due to pre-activated RNA silencing, and the presence of siRNAs acted as reliable biomarkers for prediction of the degree of virus resistance in these plants.

Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants

A novel, constitutively expressed gene, designated MtHP, was isolated from the model legume species Medicago truncatula. Sequence analysis indicates that MtHP most likely belongs to the PR10 multi-gene family. The MtHP promoter was fused to a β-glucuronidase gene to characterize its expression in different plant species. Transient assay by microprojectile bombardment and hairy root transformation by Agrobacterium rhizogenes revealed GUS expression in leaf, stem, radicle and root in M. truncatula. Detailed analysis in transgenic Arabidopsis plants demonstrated that the promoter could direct transgene expression in different tissues and organs at various developmental stages; its expression pattern was similar to that of CaMV35S promoter, and the level of expression was higher than the reporter gene driven by CaMV35S promoter. Deletion analysis revealed that even a 107 bp fragment of the promoter could still lead to a moderate level of expression. The promoter was further characterized in white clover (Trifolium repens), a widely grown forage legume species. Strong constitutive expression was observed in transgenic white clover plants. Compared with CaMV35S promoter, the level of GUS activity in transgenic white clover was higher when the transgene was driven by MtHP promoter. Thus, the promoter provides a useful alternative to the CaMV35S promoter in plant transformation for high levels of constitutive expression.

Organ-specific, developmentally-regulated and abiotic stress-induced activities of four Arabidopsis thaliana promoters in transgenic white clover ( Trifolium repens L.)

Expression patterns of chimeric gusA reporter genes encoding bacterial β-glucuronidase (GUS) with four differentially regulated promoters from Arabidopsis thaliana were assessed in transgenic white clover ( Trifolium repens) plants generated by Agrobacterium-mediated transformation. Molecular analysis of independent white clover transformants confirmed the stable integration of T-DNAs containing the various promoter- gusA reporter genes. Histochemical staining of plant tissues and organs revealed that the atmyb32 promoter directed gusA expression in leaf and root vascular tissue including lateral roots and nodules with low levels of expression in reproductive organs. Wound-response of the atmyb32 promoter in white clover leaves and stolons was also shown. The adh promoter showed anaerobic stress and dehydration stress response. The xero2 promoter directed strong expression in roots, leaf vascular tissue, inflorescences, anther filaments and pollen grains, while the A. thaliana SAG12 promoter resulted in senescence-associated gusA expression in white clover leaves.

Fructan formation in transgenic white clover expressing a fructosyltransferase from Streptococcus salivarius.

White clover (Trifolium repens L.) is an important pasture legume that does not normally accumulate fructan as a storage carbohydrate. We have generated transgenic white clover plants that accumulate fructan, by expressing the fructosyltransferase (Ftf) enzyme from the bacterium Streptococcus salivarius under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Fructan accumulated in leaves, petioles, stolons, flowers, and roots of transgenic plants. Levels of fructan up to approximately 2% dry weight were measured in leaves. The fructan was of high molecular mass ( > 5000 kDa), typical of bacterial fructans. Ftf enzyme activity up to 120 nmol min-1 g-1 fresh weight was determined in leaf extracts of the transformed plants, and appeared to be stable throughout leaf development. Most transformed lines appeared normal, flowered and produced seed, but the growth rate of some transformed lines decreased. Photosynthetic carbon assimilation and levels of endogenous carbohydrates (hexoses, sucrose and starch) were not substantially changed in a clonal line with relatively low fructan. However, in a clonal line with relatively high fructan accumulation, plant growth was reduced, leaf photosynthesis was decreased by 60%, and carbohydrate contents were reduced. The results are discussed in the context of manipulating soluble carbohydrate composition in pasture species to improve nutritive quality for grazing animals.

Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides

The expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot-inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non-host rhizobia, lipo-chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40-1 peptide or different flavonoids. The results show that clover-nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot-inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O-acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot-inoculation, but only after delivery by microtargeting. Non-O-acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40-1 peptide failed to induce any detectable changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.

Inheritance and expression of transgenes in white clover

White clover plants from a range of cultivars can now be routinely transformed with cloned foreign genes. However, before transgenic white clover cultivars can be developed, these inserted genes must be stably inherited and expressed at appropriate levels in progeny. Primary transgenic white clover plants containing a uidA (GUS) reporter transgene were outcrossed and the inheritance and expression of the uidA gene was examined over two generations, in several different cultivar backgrounds. Both Mendelian inheritance and consistent expression in different genetic backgrounds were obtained from strongly expressing primary transgenic plants. However, primary transgenic plants with weak or variable expression gave non-Mendelian inheritance and inconsistent expression of the transgene in progeny plants. Transgenic BC1 plants were also intercrossed to produce a segregating F2 population containing individuals heterozygous or homozygous for the transgene. In these populations heterozygous and homozygous plants had similar levels of uidA gene expression. These results indicate that F2 plants, homozygous for a transgene, might be used to develop a transgenic cultivar. However, both selection of a primary transgenic plant with stable, high level expression of the introduced gene and progeny testing, to determine the influence of genetic background, are prerequisites to such a development. Keywords: gene expression, inheritance, transgene, uidA reporter, white clover

Expression of the pea albumin 1 gene in transgenic white clover and tobacco.

In order to improve the quality of pasture protein for ruminant animal nutrition, we are introducing genes encoding rumen-protected proteins, rich in essential amino acids, into white clover (Trifolium repens L.). We have introduced a chimaeric gene transcribed from the 35S CaMV promoter, and encoding the pea albumin 1 (PA1) protein, rich in sulphur amino acids, into the white clover genotype WR8 by Agrobacterium-mediated transformation. A transgenic plant with high levels of PA1 mRNA was crossed with a commercial genotype from cv. Regal Ladino and both the parent and progeny plants were analyzed for expression and accumulation of PA1 gene products. Steady-state mRNA levels and transcript sizes in transgenic parent and progeny were comparable. The abundance and stability of the PA1 protein in transgenic white clover plants was examined by immunoselection of in vivo [35S]Na2SO4-labelled plant proteins. Evidence is presented here, that the 11 kDa PA1 proprotein precursor is processed correctly in petiole tissues of newly regenerated white clover plantlets but only the 6 kDa PA1a subunit accumulates in leaflets of tissue-culture-grown and older glasshouse-grown clover plants. Attempts to enhance PA1 abundance by altering its subcellular target in transgenic tobacco plants suggest that the endomembrane system is a relatively stable environment compared with the cytoplasm or chloroplast, for the accumulation of PA1, despite its low abundance there (< 0.001% total cell protein).

Release of transgenic white clover plants expressingBacillus thuringiensisgenes: An ecological perspective

The destructive effects of indigenous insects on pasture plants are a significant cause of diminished pastoral productivity in New Zealand. Identification of white clover genotypes with natural immunity against native insects has not been successful, so farmers use chemical sprays to control pest populations. An alternative strategy to reduce insect damage to white clover is through development of a transformation system using Agrobacterium‐mediated transfer DNA. We have developed a rapid procedure, where transgenic white clover plants regenerate directly from the cotyledonary axil of germinated seedlings and can be transferred into the glasshouse within 42 days of culture initiation. This technology offers the opportunity to improve the sustainability of white clover in New Zealand pastures by introducing pest resistance genes directly into these plants. Insect feeding bioassays have identified several serotypes of Bacillus thuringiensis (Bt) with δ‐endotoxins active against larvae of the lepidopteran pe...