Transgenic White Clover Research Articles

Optimization of Protocols for the Induction of Callus and Plant Regeneration in White Clover (Trifolium repens L.).

White clover is a widely grown temperate legume forage with high nutritional value. Research on the functional genomics of white clover requires a stable and efficient transformation system. In this study, we successfully induced calluses from the cotyledons and leaves of 10 different white clover varieties. The results showed that the callus formation rate in the cotyledons did not vary significantly among the varieties, but the highest callus formation rate was observed in 'Koala' leaves. Subsequently, different concentrations of antioxidants and hormones were tested on the browning rate and differentiation ability of the calluses, respectively. The results showed that the browning rate was the lowest on MS supplemented with 20 mg L-1 AgNO3 and 25 mg L-1 VC, respectively, and the differentiation rate was highest on MS supplemented with 1 mg L-1 6-BA, 1 mg L-1 KT and 0.5 mg L-1 NAA. In addition, the transformation system for Agrobacterium tumefaciens-mediated transformation of 4-day-old leaves was optimized to some extent and obtained a positive callus rate of 8.9% using green fluorescent protein (GFP) as a marker gene. According to our data, by following this optimized protocol, the transformation efficiency could reach 2.38%. The results of this study will provide the foundation for regenerating multiple transgenic white clover from a single genetic background.

Correction to: Application of linked and unlinked co‑transformation to generate triple stack, marker‑free, transgenic white clover (Trifolium repens L.)

In the original article, the order of transgene names in the column headings of Table 3 was incorrect. The correct Table 3 is printed below. The error does not change the outcome of the study. We apologize for any inconvenience.

Application of linked and unlinked co-transformation to generate triple stack, marker-free, transgenic white clover (Trifolium repens L.)

Application of linked and unlinked co-transformation to generate triple stack, marker-free, transgenic white clover (Trifolium repens L.)

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.

Development and validation of protocols for product stewardship in transgenic white clover (Trifolium repens L.): detection of the AMV CP and npt2 transgenes in seeds, herbage and hay

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 is affected by several viruses, chiefly Alfalfa mosaic virus (AMV), Clover yellow vein virus (ClYVV) and White clover mosaic virus (WClMV). Efforts to introgress natural forms of virus resistance from other Trifolium spp. into white clover and lucerne (alfalfa) have had only limited success. This has been addressed by developing white clover germplasm exhibiting viral-coat-protein-mediated resistance to AMV and non-transgenic resistance to ClYVV. This report describes PCR-based assays for detecting the transgenes associated with the H6 transformation event in seeds, fresh leaves, air-dried leaves and mixtures of air-dried herbage of white clover and perennial ryegrass (hay). Although further development is required to convert these assays for use in the field, this paper demonstrates the ability to detect these transgenes in a range of agricultural products associated with the commercial use of white clover.

Development and validation of protocols for product stewardship in transgenic white clover (Trifolium repens L.): detection of the AMV CP and npt2 transgenes in pollen, honey and honey bees

There are no current commercial releases of genetically modified white clover, but several research groups are working on traits such as virus resistance, stress tolerance and bloat safety that are likely to provide large economic benefits for livestock farmers. However, white clover pollen is a common constituent of honey produced by bees foraging white clover flowers. Therefore, there is a need to develop tools to detect the presence of genetically modified pollen in white clover honey. The results presented in this paper describe the development and application of PCR-based techniques to detect the Alfalfa mosaic virus coat protein gene (AMV CP) and the neomycin phosphotransferase 2 selectable marker gene (npt2) in genetically modified white clover pollen, whether this pollen is collected fresh, from honey bees that have been foraging white clover, or from honey. Further research and development will be required to develop ‘field-ready’ tools for the detection and quantification of these transgenes in pollen and honey products. However, this paper demonstrates prospects and principles in pollen and honey from honeybees foraging transgenic white clover.

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.

Improving organic phosphate utilization in transgenic white clover by overexpression of Aspergillus niger PhyA gene

Using the cotyledon of white clover as explants, the transgenic white clover lines ectopic expression of the PhyA gene were established based on Agrobacterium tumefaciens-mediated transformation method. It was found that the tested transgenic lines were all Polymerase Chain Reaction (PCR) positive. The transgenic lines 1 to 4 were used for further Southern blot and Northern blot analysis. The lines 1 and 3 with higher level of PhyA expression were used to assay the phytase activities in root and its intercellular space. When the phytate was the sole phosphorus source, the phytase activities in root in lines 1 and 3 were 31.43% and 44.76% higher than those in control (CK), respectively. Meanwhile, the phytase activities in the root intercellular space in lines 1 and 3 were 3.3-fold and 5.12-fold higher than those in CK, respectively. The phosphorus concentration of plants, the accumulative P amount per plant, plant fresh weight, and plant dry weight were all much higher in lines 1 and 3 than in CK. Thus, it is clearly shown that ectopic expression of Aspergillus niger PhyA gene could significantly increase the ability for white clover to utilize organic phosphate under inorganic phosphate (Pi)-deficient condition.

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.

Edible vaccine development: stability of Mannheimia haemolytica A1 leukotoxin 50 during post-harvest processing and storage of field-grown transgenic white clover

Mannheimia haemolytica is the principal microorganism responsible for bovine pneumonic pasteurellosis, or shipping fever. We have previously expressed a fragment of leukotoxin, a major virulent factor of M. haemolytica A1, as a fusion protein with green fluorescent protein (GFP) in transgenic white clover and demonstrated that this antigen was immunogenic and elicited toxin neutralizing antibodies in rabbits. These previous results showed that using plants to produce M. haemolytica antigen for use as a vaccine against this disease is a viable strategy. In this present study, we examined the stability of the truncated leukotoxin GFP-fusion protein (Lkt50-GFP) in field-grown transgenic white clover. Transgenic clover expressing Lkt50-GFP was clonally propagated and a confined field trial was established. Western immunoblotting showed that the level of Lkt50-GFP expression in field plants was the same as in transgenic plants maintained under optimal conditions in the greenhouse. We also observed that after harvesting and oven drying at 50 °C, the antigen was still present in the dried clover after 1 year of storage at ambient temperature. As special post-harvest conditions (e.g., refrigeration) are not required, the use of transgenic plants to deliver an oral vaccine against shipping fever appears to be economically feasible.

The White Clover enod40 Gene Family. Expression Patterns of Two Types of Genes Indicate a Role in Vascular Function

Enod40 is one of the genes associated with legume nodule development and has a putative role in general plant organogenesis. We have isolated a small enod40 gene family from white clover (Trifolium repens), with three genes designated Trenod40-1, Trenod40-2, and Trenod40-3, all containing the conserved enod40 regions I and II. Trenod40-1 and Trenod40-2 share over 90% homology in the transcribed regions and high levels of similarity in their upstream regulatory sequences. Trenod40-1 and Trenod40-2 are similar to the enod40 genes of legumes forming indeterminate nodules (group II) and are predominantly expressed in nodules. Trenod40-3 shares only 32.8% identity with Trenod40-1 and Trenod40-2 within the transcribed region. Trenod40-3 is similar to the enod40 genes of legumes with determinate nodules (group I) and is not predominantly expressed in nodules. To our knowledge, this is the first report of both group I- and group II-type enod40 genes being expressed in a single legume species. In situ hybridization studies revealed that Trenod40 genes were highly expressed in non-symbiotic tissues, particularly in stolon nodes during nodal root and lateral shoot development. High levels of Trenod40 transcripts were also present in the vascular bundles of mature plant organs, mainly at sites of intensive lateral transport, suggesting a role in vascular tissue function. The expression pattern of Trenod40 genes was analyzed further using Trenod40 promoter-gus fusions in transgenic white clover and tobacco (Nicotiana tabacum), indicating that white clover and tobacco share the regulatory mechanisms for Trenod40-1/2 promoters and some aspects of Trenod40-3 regulation.

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.

Heterologous rhizobial lipochitin oligosaccharides and chitin oligomers induce cortical cell divisions in red clover roots, transformed with the pea lectin gene.

Division of cortical cells in roots of leguminous plants is triggered by lipochitin oligosaccharides (LCOs) secreted by the rhizobial microsymbiont. Previously, we have shown that presence of pea lectin in transgenic white clover hairy roots renders these roots susceptible to induction of root nodule formation by pea-specific rhizobia (C. L. Díaz, L. S. Melchers, P. J. J. Hooykaas, B. J. J. Lugtenberg, and J. W. Kijne, Nature 338:579-581, 1989). Here, we report that pea lectin-transformed red clover hairy roots form nodule primordium-like structures after inoculation with pea-, alfalfa-, and Lotus-specific rhizobia, which normally do not nodulate red clover. External application of a broad range of purified LCOs showed all of them to be active in induction of cortical cell divisions and cell expansion in a radial direction, resulting in formation of structures that resemble nodule primordia induced by clover-specific rhizobia. This activity was obvious in about 50% of the red clover plants carrying hairy roots transformed with the pea lectin gene. Also, chitopentaose, chitotetraose, chitotriose, and chitobiose were able to induce cortical cell divisions and cell expansion in a radial direction in transgenic roots, but not in control roots. Sugar-binding activity of pea lectin was essential for its effect. These results show that transformation of red clover roots with pea lectin results in a broadened response of legume root cortical cells to externally applied potentially mitogenic oligochitin signals.

Anthocyanin regulatory gene expression in transgenic white clover can result in an altered pattern of pigmentation

This study presents the first evidence of heterologous anthocyanin regulatory genes altering anthocyanin expression in stably transformed leguminous plants. Two families of anthocyanin regulatory genes, myc (delila, B-Peru) and myb (myb.Ph2, C1), are involved in the activation of the phenylpropanoid pathway. White clover (Trifolium repens cv. Haifa) plants were transformed with dicotyledonous and monocotyledonous myb or myc genes. Some of these transformed plants exhibited enhanced anthocyanin accumulation in a range of tissues. One plant, transformed with the B-Peru gene driven by the Cauliflower Mosaic Virus 35S promoter, displayed a unique pattern of anthocyanin accumulation in the leaf. The accumulation of anthocyanin in this plant was closely associated with the crescent of leaves, which is normally white. The red pigmentation declined in intensity in the oldest leaf stage. The B-Peru message was detected in all leaf stages of this white clover plant. This anthocyanin pattern was shown to be heritable.

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.

The Activity of a Tobacco Basic Chitinase Promotor in Transgenic White Clover Provides Insights into Plant Development and Symbiosis

White clover was transformed with a tobacco basic chitinase promoter:GUS fusion. Basic chitinase promoter activity was detected by histochemical staining. Comparison of the spatial and temporal expression of the chitinase promoter-driven GUS gene in tobacco to that in white clover indicates that transcription from the promoter is induced by similar developmental and environmental response programs in each species. Wound-responsiveness of the white clover transgene was rapid and localised following mechanical and aphid (Family Aphididae) wounding. Developmental expression of the transgene during root morphogenesis reveals strong expression in tap and lateral root meristems but expression in lateral root meristems was observed only after the emergence through the tap root epidermis. No expression of the transgene was detected in the pericycle or the dividing cells of the developing lateral root. The expression of the tobacco basic chitinase promoter:GUS transgene in white clover was then used as a marker to examine the differences between the early developmental pathways leading to lateral root formation and those involoved in nodule formation in response to Rhizobium inoculation. Inoculation of the zone of emerging root hairs with a nodulation-competent Rhizobium strain ANU845(pRI4003), triggered transient transgene expression 2 to 4 h post-inoculation. No transgene expression was detectable after inoculation with purified Nod factor from strain ANU843. Our results suggest that lateral roots and nodules differ both in some of the mechanisms required to initiate cell division, and in their ongoing development after the emergence from the root epidermis.