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.

First Report of Fusarium meridionale Causing Stalk Rot of Ryegrass in China

Members of the Fusarium graminearum species complex (FGSC) are the main causing agents of head blight, seedling blight, or stalk rot in wheat and other cereals worldwide. Surveys on species composition and mycotoxin production of FGSC populations have mainly focused on food crops such as wheat, maize, and barley, but little is known about the identity of FGSC pathogens present in pasture grass. In April 2021, a survey of grass diseases in the Hongya County (29.90661 N; 103.37313 E) in Sichuan Province was conducted to understand the etiology of stalk rot in perennial ryegrass (Lolium perenne). It was observed in several pastures that about 10% of yield loss in perennial ryegrass was caused by stalk rot. Affected plant stalks were brown to dark brown in colour and appeared soggy. As infections continued or under conditions of high humidity, some plant stalks also became flattened. Perennial ryegrass samples with symptoms of stalk rot or browning of the stem were collected. Symptomatic tissues were cut into short segments (approximately 5 mm), surface-sterilized in 3% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, air dried, plated onto potato dextrose agar (PDA), and then incubated in the dark at 28 °C. After 3 to 5 days, Fusarium-like fungal colonies with reddish-orange mycelium were collected and transferred to new PDA plates for further purification, and the purified cultures were obtained by single spore isolation. Four uniform isolates were obtained and their colonies on PDA resembled typical FGSC colonies (Leslie and Summerell 2006; O’Donnell et al. 2004). Colonies had an average radial growth rate of 8.5 to 11.0 mm/day at 28 °C in the dark on PDA. Conidial characteristics were studied on Spezieller Nährstoffarmer agar (SNA) as described by Wang et al. (2014). Macroconidia were falcate to almost straight, usually with parallel dorsal and ventral lines, 3- to 5-septate, 20.65 to 55.22 μm in length (average 39.16 μm), and 2.38 to 6.93 μm in width (average 4.42 μm) (n = 200). No microconidia were observed. The pathogenicity of the isolated Fusarium strains was then tested on healthy perennial ryegrass (variety Changjiang 1). Ryegrass plants grown for 2 months were inoculated by punching a hole in the stem using a sterile toothpick, followed by an injection of 20 μL macroconidia suspension at a concentration of 105 spores/mL. Ryegrass stems treated with water served as the control. Twenty plants were included in each treatment. After inoculation, the plants were grown in a growth chamber at 25 °C and 90% humidity for 24 h. Stalk tissues at the wound site turned brown after 3 days and the brown area then extended to regions above and below. No symptoms were observed in the water-treated controls. As well, the same pathogen was reisolated from the infected grass stems, but not from the controls. Thus, the isolated Fusarium spp. are a cause of stalk rot in perennial ryegrass based on the fulfillment of Koch’s postulates. To identify the Fusarium spp. to species level, portions of the translation elongation factor 1-α (TEF) gene sequences from all four strains were amplified and sequenced as described by Wang et al. (2015). The obtained sequences were identical, and a sequence of isolate SC1 was submitted to GenBank (accession no. MZ964308). BLASTn searches were conducted on the TEF sequence (607 bp) in two databases, revealing it had 100% similarity to the sequence of Fusarium meridionale strain DS27 (accession no. MN629330) in NCBI and strain NRRL28723 from FUSARIUM-ID (http://isolate.fusariumdb.org/). A concatenated four-gene phylogeny (supplementary figure) resolved SC1 and the type specimens of F. meridionale (NRRL28723, 29010, and 28436) in a monophyletic clade with 100% bootstrap support, confirming that the strain SC1 belongs to F. meridionale. Finally, trichothecene productions of F. meridionale strains were evaluated using rice cultures kept at 28 °C in the dark for two weeks, as described by Desjardins and Proctor (2011). LC-MS/MS analysis indicated that the fungus could produce NIV and 4ANIV in rice cultures with average concentrations of 1400.44 and 3144.10 μg/kg, respectively. To the best of our knowledge, this is the first report of F. meridionale causing disease in perennial ryegrass in China. Further research will be necessary to determine its distribution, aggressiveness, and trichothecene production.

Persistence of perennial ryegrass, tall fescue and cocksfoot following annual sowings: influence of grass species, ryegrass cultivar and pasture age on yield, composition and density

Persistence is an important component of perennial pasture-grass productivity. Defining traits that affect persistence is essential for improving pasture longevity through plant breeding and for identifying persistence traits that should be included in cultivar ranking indices. Compared with conventional longitudinal studies, where a single sowing is monitored over time, repeated annual sowings allow the effects on persistence of sowing year and the ensuing interactions between environment and age of pasture to be identified. An experiment was commenced in 2015 under sheep grazing in Canterbury and in 2016 under cattle grazing in Waikato, where eight cultivars of perennial ryegrass representing different ploidy, flowering date, and cultivar age (release date), and one cultivar each of tall fescue and cocksfoot were sown in four randomised complete blocks in autumn each year. This paper reports interim data on spring and autumn pasture yield, composition, and density of 3-year-old, 2-year-old and 1-year-old pastures exposed to the same environmental conditions within the same, single year. There were significant effects on yield, botanical composition, basal cover and tiller density due to cultivar, pasture age, and their interaction. When the confounding effect of year-to-year variation was removed by comparing each age cohort in the same year, the underlying differences among grass species and cultivars, and ages of pasture, is starting to reveal the nature of this influence on pasture persistence.

Effect of different pasture legumes on growth profile and forage production of the selected native pasture grasses mix at different growth stages

The present experiment aimed to investigate the effect of introducing different pasture legumes on the growth profile and forage production of the selected native pasture grass species at different stages of growth. In a completely randomized design with 5 treatments and 5 replications, the mixture of Sorghum plumosum (SP) and Bothriochloa pertusa (BP) was introduced respectively with one of the forage legumes ie. Alysicarpus vaginalis (AV), Pueraria phasoloides (PP), Desmodium incanum (DI), and Clitoria ternatea (CT). Growth profile and forage production were measured at 40, 60, and 80 days after planting. Results showed that CT and PP significantly improved the growth and DM production of SP and suppressed (P<0.05) the growth of BP during the early vegetative stage but did not during the late vegetative stage. Introduction of legumes reduced (P<0.05) DM production of SP and the total forage production but improved (P<0.001) the DM production of B. pertusa as well as a leaf:stem ratio of both types of grass at the generative stage. PP had the highest (P<0.05) contribution of legumes to the total DM forage production during early and vegetative stages, meanwhile AV and DI during the generative stage. In conclusion, the introduction of forage legumes did not improve the DM production of both grass species but modify their growth profile toward a better quality as shown by increased leaf:stem ratio. P. phasoloides provide the highest foliage during the vegetative stage and A. vaginalis and D. incanum during the generative stage.

267 Effect of Hay Type on the Winter Growth of Bos Grunniens Raised in Eastern Kentucky

Bos grunniens (yak) have been utilized as a source of fiber, milk, dung, meat and transportation in the high-altitude regions of Asia for centuries. Limited research has been conducted on the species in North America. The objective of this on-farm research was to evaluate the effect of hay type on growth rate of growing yaks. Forages evaluated included alfalfa (A), orchardgrass-red clover (O) and a cool-season pasture grass species mixture (P). The experimental design was a partially balanced 3x3 Latin square with an extra pen. Yaks were stratified to pens by age and sex resulting in a pen of mixed sex (3 males/2 females) recently weaned calves, a pen of five yearling females, and two pens of five yearling males. Each period lasted for 45 days and animal weights were collected at the beginning and end to assess weight change and daily gain. Hay offered was weighed and feeding losses were visually estimated to determine hay DM disappearance. Forage samples were collected and analyzed for nutrient content. Data were analyzed using the PROC GLM procedure of SAS. No carryover effects were noted and means were separated using Tukey’s multiple comparison test where α=0.10. Forage type had a significant impact on period weight change of growing yaks during the winter with O having greater weight gain (9.5 kg) than P (-4.7 kg) with A being intermediate (8.5 kg). Forage DM disappearance when expressed as percent of body weight was greater for A (2.70) compared to P (2.25) while O (2.28) was not different from either. Forage type and quality can impact weight gain and forage disappearance during the winter months for growing yaks with higher quality forage providing greater weight gain.

Spatial structure and motor activity of horses of Zabaikalsky breed

The total influence of external environmental factors by seasons on the spatial structure and motor activity of herd horses of Zabaikalsky breed with year-round grazing was studied. The scientific and economic research was carried out in Trans-Baikal Territory. Grazing of 10 herds of horses (20-27 heads) was carried out on the area of 301.5 km2 at different distances from each other. The distance from the horse-breeding camp was 2300-7900 m. According to the motor activity assessment of horses in the spring and summer seasons during 14 hours of grazing, the average speed was 1.4 and 1.1 km/h, the distance traveled was 16828 and 14827 m. The average air temperature during these periods was 10.4 and 12.2 °С, average air velocity was 4.5 and 2.1 m/s, average relative air humidity was 32.2 and 53.3%. In the autumn period, during 10 hours of grazing, the distance was 13879 ± 195.7 m at a horse speed of 1.2 ± 0.07 km/h, average air temperature of minus 1.1 °C, air speed of 2.3 m/s, and air humidity of 54.4%. In winter, the grazing time was 8 hours, the horses in the experiment covered the distance of 12190 ± 142.3 m at a speed of 1.8 ± 0.03 km/h with an average temperature of minus 24.7 °C, wind speed of 1.5 m/s and relative humidity of 64.7%. In the spring, the horses covered the distance greater than in summer, by 13.5% (p < 0.01), in autumn - by 21.2% (p < 0.001) and in winter - by 38.0% (p < 0.001). The highest nutritional value of pasture herbage was noted in the summer (0.60 feed units/kg of dry weight, 102.3 g of digestible protein/ feed unit). In the spring, the nutritional value of pasture grass was 0.35 feed units/kg of dry weight with 31.5 g of digestible protein.

Production of young horse meat when pasture and winter grazing housing under the conditions of forest-steppe area of Transbaikal

The results of research on the meat productivity of horses, the morphological composition of carcasses, the chemical composition of horse meat, its nutritional value and environmental safety have been presented in the article. The content of vitamins and minerals in young horse meat at the age of 6 and 18 months has been established. The chemical composition of pasture grass has been studied. According to the content of vitamins and minerals the environmental safety of young horse meat corresponds to SanPiN 2,3.2. – 1078–01. The carcass weight in foals aged 6 months was 117,3 kg, the slaughter yield was 54,1 %, and in 18 months the carcass weight was 171,4 kg, the slaughter yield was 53,6 %. The yield of by-products of the 1st category was 3,94 and 4,19 kg, respectively, by age. In foals at the age of 6 months, the weight of muscle tissue was 83,6 kg, fat – 5,3 kg, bone – 22,5 kg, connective tissue – 5,9 kg, and at the age of 18 months, the weight of muscle tissue was 121,4 kg, fat – 10,1 kg, bone – 32,2 kg, connective tissue – 7,7 kg. The results of the average meat sample have shown that the meat of foals at the age of 6 months contains 71,7 % water, 21,0 % protein, 6,2 % fat, 1,1 % ash, the caloric content was 1536,4 kcal, and at the age of 18 months, respectively, 70,4, 21,0, 6,0, 6,8, 1,2 % and 1619,2 kcal. The caloric content of meat was higher than at 6 months of age by 82,8 kcal. The benefits of young horse meat are that the amino acids, vitamins and minerals contained in it contribute to the normalization of metabolism in the human body, and the meat itself is a supplier of high-quality animal protein. It has been established that according to the requirements of environmental safety, horse meat meets the maximum permissible concentration of the studied substances according to regulatory documents.

Environmental Impacts of Renewable Insulation Materials

According to the IEA Global Status Report for Buildings and Construction 2019, one of the main industry sectors causing environmental impacts is the construction sector. Hence, construction materials from renewable resources are expected to have a large potential to decrease these impacts. In this study, a Life Cycle Assessment (LCA) was conducted for four different insulation materials from renewable feedstock: insulation made from pasture grass, seaweed, reed, and recycled jute fibres. Additionally, the effects on land use change were evaluated for pasture grass insulation using the LANCA® methodology. To put the LCA results in relation to those of non-renewable resources, a comparison of standardized LCA values for conventional insulation materials is presented. In general, the renewable insulation materials show fewer environmental impacts than their conventional counterparts. In particular, these materials have advantages regarding greenhouse gas emissions and their impact on climate change. Of the analyzed materials, seaweed showed the overall lowest emissions. It can be concluded that insulation materials from non-mineral, non-fossil, and non-wooden resources are still fairly niche in terms of market share, but they have extraordinary potential in decreasing the environmental impacts of construction ventures.

EFFECT OF TECHNOLOGICAL PASTURE SYSTEMS ON THE BOTANICAL COMPOSITION OF LONG-TERM GRASS STANDS AND FEED QUALITY

The results of long-term studies on the influence of multi-variant pasture management systems (techno-genic, integrated, technogenic-mineral, technogenic-organic) on the botanical composition and quality of feed of long-term phytocenoses are presented. The possibility of preserving the valuable botanical com-position and high quality of feed for a 75-year period under the condition of a rational mode of use and an optimal level of fertilizer is justified. All the studied systems of pasture management belong to the category of energy-saving, since with the 75-year use of grass stands, the capital costs planned for periodic re-servicing are reduced to 10 or more times. Experimental data on the botanical composition of pasture grass stands for 2005 and 2020 and the quality of green feed in terms of protein nutrition and mineral content for 1976–2020 are presented in accordance with the requirements of the technical specifications of GOST R 57482-2017 "Pasture feed". The content of seeded grasses, meadow foxtail and meadow bluegrass, in the herbage of 75 years of life against the background of N180P45K120 was 43%. The content of crude protein (at a rate of at least 14%) and crude fat (at a rate of at least 3.2%) in all studied systems exceeded the standard values.