Impact of Environmental Factors on Seed Germination and Seedling Emergence of White Clover (Trifolium repens L.)

White clover (Trifolium repens L.) is cultivated as a forage crop and planted in various landscapes for soil conservation. There are numerous reports of failed white clover stands each year. A good understanding of the seed germination biology of white clover in relation to environmental factors is essential to achieve successful stand establishment. A series of experiments were conducted to investigate the impacts of light, temperature, planting depth, drought, and salt stress on seed germination and the emergence of white clover. White clover is negatively photoblastic, and seed germination averaged 63 and 66% under light and complete dark conditions 4 weeks after planting (WAP), respectively. Temperature affected the seed germination speed and rate. At 1 WAP, seeds incubated at 15 to 25 °C demonstrated a significantly higher germination rate than the low temperatures at 5 and 10 °C; however, the germination rate did not differ among the temperature treatments at 4 WAP. The results suggest that white clover germination decreases with increasing sowing depths, and the seeds should be sown on the soil surface or shallowly buried at a depth ≤1 cm to achieve an optimal emergence. White clover seeds exhibited high sensitivity to drought and salinity stress. The osmotic potential and NaCl concentration required to inhibit 50% seed germination were −0.19 MPa and 62.4 mM, respectively. Overall, these findings provide quantifiable explanations for inconsistent establishment observed in field conditions. The results obtained in this research can be used to develop effective planting strategies and support the successful establishment of white clover stands.

Two Agrobacterium-mediated Transformation Protocols of White Clover (Trifolium Repens) Through the Callus System

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.

Condensed Tannins in White Clover (Trifolium repens) Foliar Tissues Expressing the Transcription Factor TaMYB14-1 Bind to Forage Protein and Reduce Ammonia and Methane Emissions in vitro

Grazing ruminants contribute to global climate change through enteric methane and nitrous oxide emissions. However, animal consumption of the plant polyphenolics, proanthocyanidins, or condensed tannins (CTs) can decrease both methane emissions and urine nitrogen levels, leading to reduced nitrous oxide emissions, and concomitantly increase animal health and production. CTs are largely absent in the foliage of important temperate pasture legumes, such as white clover (Trifolium repens), but found in flowers and seed coats. Attempts at enhancing levels of CT expression in white clover leaves by mutagenesis and breeding have not been successful. However, the transformation of white clover with the TaMYB14-1 transcription factor from Trifolium arvense has resulted in the production of CTs in leaves up to 1.2% of dry matter (DM). In this study, two generations of breeding elevated foliar CTs to >2% of DM. The CTs consisted predominantly of prodelphinidins (PD, 75–93%) and procyanidins (PC, 17–25%) and had a mean degree of polymerization (mDP) of approximately 10 flavan-3-ol subunits. In vitro studies showed that foliar CTs were bound to bovine serum albumin and white clover proteins at pH 6.5 and were released at pH 2.-2.5. Using rumen in vitro assays, white clover leaves containing soluble CTs of 1.6–2.4% of DM significantly reduced methane production by 19% (p ≤0.01) and ammonia production by 60% (p ≤ 0.01) relative to non-transformed wild type (WT) controls after 6 h of incubation. These results provide valuable information for further studies using CT expressing white clover leaves for bloat prevention and reduced greenhouse gas emissions in vivo.

Dynamics of the Species Composition of Phytocenoses of Floodplain Mountain Meadows of the Carpathians Subject to Superficial Improvement

The conducted research of various authors has developed a number of measures for surface improvement of meadowland in various soil and climatic conditions. However, studies on the influence of such surface improvement measures as the use of organic and mineral fertilizers and sowing of perennial grasses and legumes in the sod on the dynamics of the species composition of Floodplain Mountain meadows of the Carpathians have not yet been conducted, which confirms the relevance of this study. The article presents the results of three-year studies conducted during 2017-2019 in the peasant farm of V.F. Martyshchuk (the Verkhovinsky District of the Ivano-Frankivsk region) to study the features of transformation of the species composition of natural phytocenoses of floodplain Meadows of the Black Cheremosh river of the mountain and forest belt of the Carpathians under the influence of their surface improvement with the use of organic and mineral fertilizers and sowing of a cereal mixture for haymaking use and Tribolium repens L. for multipurpose use. It was found that floodplain Meadow phytocenoses were mainly cereals and mixed grasses with a share of wild cereals of 57-58% (up to 15 of them % Festuca rubra L. s. str. and 5-6% of low-value ones in feed terms Calamagrostis arundinaceae (L.) Roth and Deschampsia caespitosa (L.) Beauv.), mixed grasses – 35-38% and unseeded Fabaceae 5-7%. When applying a set of measures for surface improvement of cereals and mixed grasses, low productive (within 1.95-2.15 t/ha of dry weight) floodplain mountain meadowlands, their species composition improved. For making P30K60 the content of wild plants increased by 2-5% Fabaceae, and N60P30K60 – by 9-10% unseeded Poaceae. For haymaking use, Poaceae with Phleum pratense L. and Festuca pratensis Huds. against the background of making N60P30K60 for sowing a mixture in the sod in the spring, their share increased to 74%, and for multi-year use and sowing Trifolium repens L. against the background of making P30K60 the quantity of Fabaceae increased to 44% or by 32%. The highest floral saturation (43 species from 19 families and 93-98% from perennials) was observed in variants without fertilization and with the introduction of P30K60 for multi-year use, which is 4-14 species more compared to haymaking use, or with the introduction of N60P30K60 and or with sowing a mixture of cereals on the background of N60P30K60 for haymaking use, or sowing Trifolium repens L. against the background of P30K60 for multipurpose use

Primera aproximación de carga animal óptima en cuyes al pastoreo durante la época lluviosa en la sierra peruana

Se realizó una primera aproximación de la carga animal óptima en cuyes al pastoreo durante la época lluviosa en la sierra del Perú, utilizando una pastura perenne asociada conformada por Lolium multiflorum, Lolium perenne, Dactylis glomerata, Trifolium pratense, Trifolium repens y Medicago sativa en función a la ganancia de peso vivo individual y por unidad de área, consumo de alimento, conversión alimenticia, rendimiento de pastura, costos de producción y ratio beneficio costo. Se emplearon 132 cuyes machos G de 15 días de edad en un diseño de bloques completos al azar con tres ciclos de engorde de 60 días como bloque y cuatro tratamientos correspondientes a cargas de 400 (T1), 500 (T2), 600 (T3) y 700 (T4) cuyes/ha/año. Se trabajó en áreas de 200 m2 de pastura donde pastorearon grupos de 8, 10, 12 y 14 cuyes en T1, T2, T3 y T4, respectivamente. Los resultados demostraron que no hubo efecto de la carga animal sobre la ganancia de peso individual, consumo de alimento, conversión alimenticia y rendimiento de pastura (p>0.05). Los efectos sobre la ganancia por unidad de área y ratio beneficio costo (p<0.05) fueron consecuencia directa y esperada del diferente número de animales entre cargas animales. El rango de carga animal fue insuficiente para determinar una carga óptima, de manera que tentativamente se sugiere utilizar la carga de 700 cuyes/ha/año, por ser la carga que favorece lograr más cuyes con parámetros productivos similares a cargas menores y mejores parámetros económicos.

Time dynamics of stress legacy in clonal transgenerational effects: a case study on Trifolium repens

Stress can be remembered by plants in a form of stress legacy that can alter future phenotypes of previously stressed plants and even phenotypes of their offspring. DNA methylation belongs among the mechanisms mediating the stress legacy. It is however not known for how long the stress legacy is carried by plants. If the legacy is long lasting, it can become maladaptive in situations when parental-offspring environments do not match. We investigated for how long after the last exposure of a parental plant to drought can the phenotype of its clonal offspring be altered. We grew parental plants of three genotypes of Trifolium repens for five months either in control conditions or in control conditions that were interrupted with intense drought periods applied for two months in four different time-slots. We also treated half of the parental plants with a demethylating agent (5-azaC) to test for the potential role of DNA methylation in the stress legacy. Then, we transplanted parental cuttings (ramets) individually to control environment and allowed them to produce offspring ramets for two months. The drought stress experienced by parents affected phenotypes of offspring ramets. The stress legacy resulted in enhanced number of offspring ramets originating from parents that experienced drought stress even 8 weeks before their transplantation to the control environment. 5-azaC altered transgenerational effects on offspring ramets. We confirmed that drought stress can trigger transgenerational effect in T. repens that is very likely mediated by DNA methylation. Most importantly, the stress legacy in parental plants persisted for at least 8 weeks suggesting that the stress legacy can persist in a clonal plant Trifolium repens for relatively long period. We suggest that the stress legacy should be considered in future ecological studies on clonal plants.

CREEPING CLOVER (Trifolium repens L.): BIOLOGICAL FEATURES, ECONOMIC SIGNIFICANCE, VARIETIES AND METHODS OF SEED PRODUCTION WHEN GROWN IN GRASS MIXTURES WITH A CEREAL COMPONENT

Creeping clover (Trifolium repens L.) is one of the best crops for creating meadow and pasture agrophytocenoses of long-term use. The biological features of this crop, such as durability, good yield, resistance to grazing, high leafiness, along with good feed quality indicators, its high protein nutritional value, make it indispensable when creating pastures not only in Russia, but also abroad. Creeping clover is one of the main components of grass mixtures for improving pastures and developing fallow lands; it is also an excellent phyto-meliorative crop that restores soil fertility. The most important problem of creeping clover seed production is harvesting due to the short stature of pasture varieties. Therefore, it is recommended to grow creeping clover for seeds in a mixture of grass together with cereal components such as perennial ryegrass, reed fescue, meadow fescue, etc. Due to the use of biological nitrogen of the legume component, energy consumption for the production of 1 kg of cereal grass seeds is reduced by 20–30% in comparison with uncoverless single-species crops. In addition to clover seeds, growing in grass mixtures allows you to get high yields of grass seeds in the second year of use without the use of nitrogen fertilizers.

Reducing Phenanthrene Contamination in Trifolium repens L. With Root-Associated Phenanthrene-Degrading Bacterium Diaphorobacter sp. Phe15

Some root-associated bacteria could degrade polycyclic aromatic hydrocarbons (PAHs) in contaminated soil; however, their dynamic distribution and performance on root surface and in inner plant tissues are still unclear. In this study, greenhouse container experiments were conducted by inoculating the phenanthrene-degrading bacterium Diaphorobacter sp. Phe15, which was isolated from root surfaces of healthy plants contaminated with PAHs, with the white clover (Trifolium repens L.) via root irrigation or seed soaking. The dynamic colonization, distribution, and performance of Phe15 in white clover were investigated. Strain Phe15 could efficiently degrade phenanthrene in shaking flasks and produce IAA and siderophore. After cultivation for 30, 40, and 50 days, it could colonize the root surface of white clover by forming aggregates and enter its inner tissues via root irrigation or seed soaking. The number of strain Phe15 colonized on the white clover root surfaces was the highest, reaching 6.03 Log CFU⋅g–1 FW, followed by that in the roots and the least in the shoots. Colonization of Phe15 significantly reduced the contents of phenanthrene in white clover; the contents of phenanthrene in Phe15-inoculated plants roots and shoots were reduced by 29.92–43.16 and 41.36–51.29%, respectively, compared with the Phe15-free treatment. The Phe15 colonization also significantly enhanced the phenanthrene removal from rhizosphere soil. The colonization and performance of strain Phe15 in white clove inoculated via root inoculation were better than seed soaking. This study provides the technical support and the resource of strains for reducing the plant PAH pollution in PAH-contaminated areas.