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Transgenic technologies for enhanced molecular breeding of white clover (Trifolium repens L.)
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White clover is an important pasture legume of temperate regions, generally through co-cultivation with a pasture grass in a mixed-sward setting. White clover provides herbage with high nutritional quality to grazing animals, along with the environmental benefit of biological nitrogen fixation. Several key agronomic traits are amenable to modification in white clover through use of transgenic technology. Efficient methods for Agrobacterium-mediated transformation of white clover have been developed. The current status of transgenic research is reviewed for the following traits: resistance to viruses and insect pests; aluminium tolerance and phosphorus acquisition efficiency; control of leaf senescence and seed yield; biosynthesis of flavonoids and rumen bypass proteins for bloat safety and enhanced ruminant nutrition; cyanogenesis; and drought tolerance. Future prospects for transgenic technology in molecular breeding in white clover are also discussed.
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... White clover (Trifolium repens L.) is one of the most extensively grown perennial leguminous forage grasses in the world. It is native to Europe and is widely distributed in temperate and subtropical high-altitude regions [1]. It is estimated that 3-4 million hectares of white clover are planted annually worldwide [2]. ...
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.
... However, despite the global importance of white clover, it has remained behind other major pastures primarily Medicago sativa (Jiang et al. 2019; Kumar et al. 2018) in developing genomic tools for its improvement. Most functional genomic researches in white clover typically relied on heterologous expression, depending on the Arabidopsis or Medicago truncatula as the model plant (Forster et al. 2013). Therefore, it is still important to develop a highly reproducible and widely applicable transformation system to bene t the white clover transformation network. ...
White clover ( Trifolium repens ) is one of the most widely cultivated livestock forage legumes co-cultivated worldwide with pasture grass in a mixed-sward setting, however, its persistence and aesthetic quality are severely affected by abiotic stresses. In this study, regeneration of white clover plants was conducted through a callus system for 4-5 months with a regeneration frequency of 36-41%. Inoculating 4-day-old cotyledons into MS media fortified with 0.4 mg·L ⁻¹ 6-BA and 2 mg·L ⁻¹ 2,4-D significantly increased the callus formation rate. Roots and cotyledons were better induced, followed by hypocotyls, leaves, and petioles. The development of differentiated structures performed effectively on MS supplemented with 1 mg·L ⁻¹ 6-BA and 0.1 mg·L ⁻¹ NAA. Further, we determined factors affecting the Agrobacterium tumefaciens -mediated transient transformation for root-derived callus and 4-day-old cotyledons. The parameters that facilitated transient transformation were: Agrobacterium suspension density of 0.5 (OD600), 20 mg·L ⁻¹ AS, and 4-days co-cultivation duration. Subsequently, we developed two transformation protocols: transformation after callus formation in root segments (Protocol A) and transformation before callus initiation in 4-day-old cotyledons (Protocol B). The transformation frequencies varied from 1.92% to 3.17% in Protocol A and from 2.76% to 3.47% in Protocol B. We offer the possibility to regenerate multiple transgenic white clover from a single genetic background. In addition to assistance in identification of functional genes associated with yield, resistance and aesthetic quality, our research will also contribute to successful genetic manipulation and genome editing in white clover.
... Genetic analysis has shown there is useful variation for virus resistance in white clover (Pederson and McLaughlin, 1994) which can be exploited through conventional means. However, a sustained effort to develop field tests and release white clover with engineered resistance to virus (Panter et al., 2012;Forster et al., 2013) has proceeded. ...
The expected move towards more sustainable crop-livestock systems implies wider cultivation of perennial forage legumes. Alfalfa (Medicago sativa subsp. sativa) is the main perennial legume in most temperate regions, especially where farm systems rely largely on forage conservation. White clover (Trifolium repens) and red clover (Trifolium pratense) are dominant in specific regions and farm systems. Although breeding progress for disease and insect resistance has been achieved, these crops have shown lower rates of genetic gain for yield than major grain crops, owing to lower breeding investment, longer selection cycles, impossibility to capitalize on harvest index, outbreeding mating systems associated with severe inbreeding depression, and high interaction of genotypes with cropping conditions and crop utilizations. Increasing yield, persistence, adaptation to stressful conditions (drought; salinity; grazing) and compatibility with companion grasses are major breeding targets. We expect genetic gain for yield and other complex traits to accelerate due to progress in genetic resource utilization, genomics resource development, integration of marker-assisted selection with breeding strategies, and trait engineering. The richness in adaptive genes of landraces and natural populations can be fully exploited through an ecological understanding of plant adaptive responses and improved breeding strategies. Useful genetic variation from secondary and tertiary gene pools of Medicago and Trifolium is being increasingly accessed. Genome sequencing projects in alfalfa and white clover will enrich physical, linkage and trait maps. Genome sequences will underpin fine mapping of useful loci and subsequent allele mining, leveraging the synteny of these crops with M. truncatula. Low-cost genome-wide markers generated through genotyping-by-sequencing will make genomic selection for adaptation and forage yield possible for these crops. Genetic markers will also be used for dissecting quantitative traits and developing toolboxes of functional markers for stress tolerance and other traits. Under current regulatory policies, transgenic approaches are likely to be limited to a few breakthrough traits. The key challenge for future applications of genomics technologies is their seamless integration with breeding system logistics and breeding schemes.
... It is estimated that CTs content of 2-4 % in forage DM can effectively reduce protein degradation (Kingston-Smith et al. 2013). Many forage species naturally contain CTs, i.e. bird-foot trefoil (Lotus corniculatus L.), but most of them do not, i.e. white clover, alfalfa (Forster et al. 2013). Legumes high in CTs content can be mixed with white clover forage low in CTs to reduce protein degradability. ...
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Forages are integral components of grassland and pasture agro-ecosystems. They are the major source of feed and nutrition for livestock. As a primary production, they are converted by livestock into secondary production in the food chain. Forage breeding is a complex process involving plant morpho-physiological aspects (perenniality, mode of reproduction, matting systems) and aberrant plant-environment correlations affecting plant performance under various sward conditions. The ultimate aim of forage breeding is to develop cultivars with high and sustainable herbage yield under various management systems. It also encompasses development of cultivars with beneficial impacts on ecosystem functions, animal growth and health. This chapter addresses challenges for forage producers and breeders due to rapidly diminishing grassland areas and the impact on the bio-diversity of grassland ecosystems and their productivity. Approaches to conserve genetic diversity and utilize forage genetic resources in an efficient way as well as breeding procedures practical use in selected forage crops are discussed.
... Over last year's research in our group was focused on (i) functional analysis of the lignin specific genes in C3 and C4 grasses; (ii) understanding the roles, and substrate promiscuity, of catalytic enzymes of COMT family in shaping the composition of lignin polymers; and (iii) metabolic reprogramming of lignin pathway redirecting metabolic fluxes between S and G-pathways (changing S/G ratio) and between lignin and flavonoid pathways under reverse genetics conditions (Louie et al., 2010;Tu et al., 2010;Forster et al., 2013;Giordano et al., 2014a,b). Downregulation of LpCCR1, the first lignin-specific gene in Lolium perenne resulted in a decreased level of the downstream products in the lignin biosynthesis pathway, such as H, G, and S units, caffeoyl aldehyde, and coniferaldehyde (these compounds are shown in red in Figure 5). ...
From an evolutionary perspective, the emergence of the sophisticated chemical scaffolds of flavonoid molecules represents a key step in the colonization of Earth’s terrestrial environment by vascular plants nearly 500 million years ago. The subsequent evolution of flavonoids through recruitment and modification of ancestors involved in primary metabolism has allowed vascular plants to cope with pathogen invasion and damaging UV light. The functional properties of flavonoids as a unique combination of different classes of compounds vary significantly depending on the demands of their local real estate. Apart from geographical location, the composition of flavonoids is largely dependent on the plant species, their developmental stage, tissue type, subcellular localization, and key ecological influences of both biotic and abiotic origin. Molecular and metabolic cross-talk between flavonoid and other pathways as a result of the re-direction of intermediate molecules have been well investigated. This metabolic plasticity is a key factor in plant adaptive strength and is of paramount importance for early land plants adaptation to their local ecosystems. In human and animal health the biological and pharmacological activities of flavonoids have been investigated in great depth and have shown a wide range of anti-inflammatory, anti-oxidant, anti-microbial, and anti-cancer properties. In this paper we review the application of advanced gene technologies for targeted reprogramming of the flavonoid pathway in plants to understand its molecular functions and explore opportunities for major improvements in forage plants enhancing animal health and production.
... T. repens belongs to Fabaceae family. A few studies concerning enzyme activity and gene expression have been performed with this species which is well-studied in agronomy (Harmoney et al., 2001;Sanderson et al., 2005Sanderson et al., , 2012Deak et al., 2007;Forster et al., 2013). Thus, an expanded bibliography on the Fabaceae family has been made and is presented in this section. ...
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White clover (Trifolium repens) is one of the most widely cultivated livestock forage plants whose persistence is severely affected by abiotic stresses. For the white clover, efficient regeneration systems is still a great necessity. In this study, inoculating 4-day-old cotyledons into MS media fortified with 0.4 mg·L-1 6-BA and 2 mg·L-1 2,4-D significantly increased the callus induction rate. Roots and cotyledons proved to be better explants, followed by hypocotyls, leaves, and petioles for callus induction. The development of differentiated structures occurred effectively on MS supplemented with 1 mg·L-1 6-BA and 0.1 mg·L-1 NAA. To increase transformation, we investigated various factors affecting the Agrobacterium tumefaciens transformation in white clover. The optimal conditions for root-derived callus and 4-day-old cotyledons were as follows: Agrobacterium suspension density with OD600 of 0.5, 20 mg·L-1 AS, and 4 days of co-cultivation duration. Subsequently, we developed two transformation protocols: transformation after callus induction from 4-day-old roots (Protocol A) and transformation before initiation of callus from cotyledons (Protocol B). The transformation frequencies varied from 1.92 to 3.17% in Protocol A and from 2.76 to 3.47% in Protocol B. We report the possibility to regenerate multiple transgenic white clover plants from a single genetic background. Our research may also contribute to successful genetic manipulation and genome editing in white clover.
Supplementary information:
The online version contains supplementary material available at 10.1007/s13205-023-03591-2.
This study focused on expression analysis of antioxidant defense genes in Brassica oleracea and in Trifolium repens. Plants were exposed for 3, 10 and 56 days in microcosms to a field-collected suburban soil spiked by low concentrations of cadmium and/or lead. In both species, metal accumulations and expression levels of genes encoding proteins involved and/or related to antioxidant defense systems (gluthatione transferases, peroxidases, catalases, metallothioneins) were quantified in leaves in order to better understand detoxification processes involved following exposure to metals. It appeared that strongest gene expression variations in T. repens were observed when plants are exposed to Cd (metallothionein and ascorbate peroxidase up-regulations) whereas strongest variations in B. oleracea were observed in case of Cd/Pb co-exposures (metallothionein, gluthatione transferase and peroxidase up-regulations). Results also suggest that there is a benefit to use complementary species in order to better apprehend the biological effects in ecotoxicology.
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.
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.
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, beta-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.
A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.
Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity.
Viruses such as alfalfa mosaic alfamovirus (AMV), white clover mosaic potex virus (WCMV) and clover yellow vein potyvirus (CYVV), which are members of the three largest groups of plant viruses, have been found to have significant adverse effects on forage legumes. Each of these viruses individually infects a large number of plant species, has a worldwide distribution, and causes significant losses, especially in pasture and grain legumes.
Pasture legumes contribute to the input base of the rural industries by providing improved pastures for grazing and nitrogen for cropping. However, a widespread gradual decline in pasture yield and persistence and reduced winterhardiness is harming farm profitability. Virus diseases are major causes of this reduced pasture performance, and it is estimated that controlling AMV, WCMV, and CYVV could increase profitability for Australian rural industries by over AUD 860 million per year.
Most of the classical methods for preventing plant virus infections are laborious and economically unsustainable. There is no effective, readily transferable natural resistance to viruses in forage legumes. This makes the production of virus resistant varieties by means of gene technology an attractive option due to the ability to overcome species-specific barriers, to develop multi-gene resistance and to manipulate levels and sites of expression. Strategies for developing pathogen-derived resistance to virus diseases in forage legumes depend on the availability of sequence information of virus genomes and robust regeneration and transformation protocols for the major pasture legumes. In this paper the current status of molecular breeding of crop plants including forage legumes for virus resistance is reviewed.
Plants produce a wide array of natural products, many of which are likely to be useful bioactive structures. Unfortunately, these complex natural products usually occur at very low abundance and with restricted tissue distribution, thereby hindering their evaluation. Here, we report a novel approach for enhancing the accumulation of natural products based on activation tagging by Agrobacterium-mediated transformation with a T-DNA that carries cauliflower mosaic virus 35S enhancer sequences at its right border. Among ∼5000 Arabidopsis activation-tagged lines, we found a plant that exhibited intense purple pigmentation in many vegetative organs throughout development. This upregulation of pigmentation reflected a dominant mutation that resulted in massive activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and flavonoids, including various anthocyanins that were responsible for the purple color. These phenotypes, caused by insertion of the viral enhancer sequences adjacent to an MYB transcription factor gene, indicate that activation tagging can overcome the stringent genetic controls regulating the accumulation of specific natural products during plant development. Our findings suggest a functional genomics approach to the biotechnological evaluation of phytochemical biodiversity through the generation of massively enriched tissue sources for drug screening and for isolating underlying regulatory and biosynthetic genes.
White clover is one of the most important pasture legumes in global temperate regions. It is an outcrossing, insect-pollinated species with gene flow occurring naturally between plants. A 2-year study was conducted to assess the relationship between gene flow and physical distance in white clover under field conditions in southern Australia. White clover plants exhibiting a red leaf mark phenotypic trait acted as pollen donors to recipient plants lacking leaf markings at distances up to 200 m distant from the donor plants. Progeny were scored for the dominant red-leafed phenotype and gene flow was modelled. Paternity was confirmed using simple sequence repeat markers. A leptokurtic pattern of gene flow was observed under conditions designed to measure maximised gene flow with the majority of pollination occurring in the first 50 m from the donor pollen source. The combined use of simple sequence repeat and visual markers confirmed that there was also a white clover pollen source in addition to the donor plants. This research confirms the difficulty in ensuring absolute containment of gene flow in an outcrossing species grown in an environment when endemic populations are known to exist.
Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil bacteria which transfer DNA (T-DNA) to plant cells. Two Agrobacterium strains, each with a different T-DNA, can infect plants and give rise to transformed tissue which has markers from both T-DNAs. Although marker genes from both T-DNAs are in the tissue, definitive proof that the tissue is a cellular clone and that both T-DNAs are in a single cell is necessary to demonstrate cotransformation. We have transferred two distinguishable T-DNAs, carried on binary vectors in separate Agrobacterium rhizogenes strains, into tomato cells and have recovered hairy roots which received both T-DNAs. Continued expression of marker genes from each T-DNA in hairy roots propagated from individual root tips indicated that both T-DNAs were present in a single meristem. Also, we have transferred the two different T-DNAs, carried on identical binary vector plasmids in separate Agrobacterium tumefaciens strains, into tobacco cells and recovered plants which received both T-DNAs. Transformed plants with marker genes from each T-DNA were outcrossed to wild-type tobacco plants. Distribution of the markers in the F1 generation from three cotransformed plants of independent origin showed that both T-DNAs in the plants must have been present in the same cell and that the T-DNAs were genetically unlinked. Cotransformation of plant cells with T-DNAs from two bacterial strains and subsequent segregation of the transferred genes should be useful for altering the genetic content of higher plants.