Production of Transgenic Tall Fescue Plants with Enhanced Stress Tolerances by Agrobacterium tumefaciens-Mediated Transformation

In order to improve stress tolerances of turf-type tall fescue （Festuca arundinacea Schreb.）, Agrobacterium tumefaciens strain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidopsis thaliana was used to transform mature seeds-derived embryogenic calli of four cultivars. A total of 112 transgenic plants were regenerated from 32 independent lines and verified by histochemical detection of GUS activity, PCR assay and Southern hybridization analysis. The transformation frequency ranged from 0.92 to 2.87% with apparent differences among the cultivars. Stress tolerances of transgenic plants were enhanced, which was shown by the facts that transgenic plants had distinct growth superiority and significantly higher survival rate than non-transformed ones under high salinity and high osmosis stresses, and that relative electronic conductivity of in vitro leaves treated with low and high temoeratures, dehvdration and high salinity stresses was 25-30% lower in transgenic plants than in control plants.In addition,it was observed that growth of transgenic plants was inhibited due to constitutive overexpression of CBF1 gene under normal environmental conditions.

Effects of Seed Maturity of Turf-Type Tall Fescue on the Seed Vigor and the Optimal Harvesting Time

By assay of accelerated aging germination, germination index, vigor index, seedling length, seedling weight, electric conductivity, dehydrogenase activity, ATP content and acid phosphoesterase activity during seed development of turf-type tall fescue, the seed vigor of tall fescue were studied. Combining with seed moisture content and yield, the optimal harvesting time of tall fescue was determined. The results indicated that the seed vigor increased continuously along with seed maturity, and the higher seed vigor was achieved at 19th day after perk anthesis and maintained continuously until 31st day after peak anthesis. At 25th day after peak anthesis, the highest yield of 3 533 kg ha-1 and the good quality of seeds of tall fescue were harvested with 32.19% moisture content. From 22nd to 31st day after peak anthesis, the seed yield of 3 300 kg ha-1 and the good quality seeds of tall fescue were harvested with 40 - 12. 43% moisture content, and the span was the optimal harvesting time.

Metabolomics Analysis of Tall Fescue Leaves under Drought Stress

Abstract The metabolites of tall fescue leaves under normal and drought conditions were analyzed with LC-ESI-MS （ liquid chromatography tandem mass spec- trometry）. A total of 282 metabolites were stably detected in the two groups using OPI.S-DA （orthogonal partial least squares discriminant analysis） method; 148 of which were down-regulated while 134 were up-regulated. After analyzed with MZ-minc software, 10 major metabolites （4 down, 6 up） were identified through searehing public database. Aromatic compounds, 3-p-eoumaroyll, 5-quinolaetone, aromatic free amino acids, styraxin mad methanofuran, were up-regulated ; grease metabolites, methyl 15-hydroperoxy-9Z, 12Z, 16E-octadecatrienoate, 9,12,13-Tri-HOME （10）, 3-oxo-dodecanoic acid, and 13 （S）-HpOTrE acid ester, were down-regulated; piperanine （alkaloids） was down-regulated while Methyl 7-epi-12-hydroxyjasmonate glucoside （indicant） was up-regulated. In a word, aromatic compounds and liquid metabolites may play vital roles in drought-resistant regulation of tall fescue.

Effect of Low Nitrogen Stress on IAA Metabolome of Tall Fescue

Forages generally suffer more severe nutrition stress than other crops, but the corresponding theoretical studies still lag behind. In this study, metabo- lites in tall fescue leaves under normal and low nitrogen conditions were analyzed with LC-ESI-MS （ liquid chromatography-electrospray ionization-mass spectrome- try）. A total of 1 424 and 1 251 metabolites were detected from two groups of samples respectively using OPLS-DA （orthogonal partial least squares discriminant analysis）. The content of 13 major metabolites changed under low nitrogen stress, including auxin （indale acetic acid, IAA）. Quantitative real-time PCR （qRT- PCR） showed that eight genes related to IAA signaling pathway were up-regulated after nitrogen stress. Enzyme-linked immuno sorbent assay （ELISA） revealed that the IAA concentration was elevated in tall fescue leaves after nitrogen stress. Our research provides valuable information for studying the response mechanisms of tall feseue to low nitrogen. The results suggest that application of IAA could be used to alleviate the harmful effects of nitrogen deficiency.

Construction of RNAi Expression Vector Targeting Vernalizational Gene FaCONSTANS in Tall Fescue

[ Objective ] This study aimed to construct RNAi expression vector targeting vemalizational gene FaCONSTANS （GenBank accession number： GU214996） in tall rescue. [ Method] A 145 bp long Arabidopsis actin gene intron was inserted into the expression vector to construct an intermediate vector pBI121-M-INT. Two pairs of specific primers with restriction sites were designed to amplify a 351 bp long cDNA conserved sequence fragment of vemalizational gene FaCONSTANS for RT-PCR. After restriction enzyme digestion, the amplified fragment was inserted forwardly and reversely at two sides of the intron of intermediate vector to construct an RNAi expression vector with hairpin structure. [ Result ] Double digestion （HindIII ＋ BamHI） showed that the intron was successfully insert- ed into the vector pBI121. PCR amplification and double digestion indicated that target fragment FaCONSTANS was successfully inserted forwardly and reversely in- to the intermediate vector. [ Conclusion] This study laid foundation for breeding novel flowering-inhibited tall rescue cultivars.

Breeding for drought tolerance in perennial ryegrass(Lolium perenne L.)and tall fescue(Lolium arundinaceum[Schreb.]Darbysh.)by exploring genotype by environment by management interactions

Background:Drought‐tolerant turfgrass cultivars are becoming more important each year as landscape irrigation practices are becoming more restrictive.Methods:In this study,we quantified the level of drought tolerance(DT)in perennial ryegrass and tall fescue genotypes across years under different mowing(MH)and fertility(FL)rates.A total of 48 ryegrass and 38 tall fescue genotypes were planted in 2011 at NexGen,Albany,OR.The trial was divided into three subtrials:(i)DT;(ii)MH and DT;and(iii)FL and DT.Results:The results showed that there was significant genetic variance for most of the traits in both species.The genotype×year interaction was predominantly simple,indicating small changes in genotype ranking for both species.Lower MH improved DT in perennial ryegrass,whereas higher MH resulted in greater DT in tall fescue.For both species,higher MH improved the population mean for turf quality.Higher FL inputs led to higher turf quality for both species,but it did not affect the DT response.Conclusions:Using a selection index,six genotypes for perennial ryegrass and four for tall fescue were selected.

Determining the earliest growth stage to detect the presence of endophytes in tall fescue and perennial ryegrass seedlings using molecular markers

Background:Tall fescue(Festuca arundinacea[Schreb.],Lolium arundinaceum[Schreb.]Darbysh)and perennial ryegrass(Lolium perenne)are important cool-season forage and amenity grasses that have a mutualistic association with an endophytic fungus.Endophytes confer insect and drought resistance to plants but can produce mammalian toxins.Novel endophytes that do not produce mammalian toxins have been introduced to elite cultivars for commercial production.Seed companies need to maintain adequate levels of novel endophytes within the elite forage cultivars.Endophyte detection is performed using immunochemical and molecular techniques because of their speed and reliability.Early detection in seedlings is essential to evaluate the viability of the endophyte within seed lots.Methods:This research aimed to identify the earliest growth stage in which immunochemical and molecular methods can detect viable endophyte in seedlings of tall fescue cultivars BarOptima(e34),Texoma MaxQII(584),and Jesup MaxQ(542),as well as the perennial ryegrass cultivar Remington(NEA2).Results:Immunochemical testing detected endophytes in seedlings 14 days after germination(DAG),but the detection rate increased until 42 DAG in some cultivars tested.The molecular marker Tef1exon detected endophytes at a lower rate than the immunochemical method at 28–42 DAG.However,there was insufficient DNA to detect endophytes in 14 DAG seedlings using markers.Conclusions:We conclude that the most accurate detection of viable endophytes in seedlings was 42 DAG,at which sufficient and consistent endophyte colonization occurred.

Tiller demography of tall fescue and tall wheatgrass cut at different times during the reproductive phase

Background:Tall fescue(TF)and tall wheatgrass(TW)are the main temperate perennial grasses used for livestock grazing in the Pampas region of Argentina.In this region,pastures based on these grasses show low tiller density and the presence of bare soil in winter.Methods:An experiment was performed in pots kept outdoors.Plants of TF and TW were cut every 550°C day and 700°C day,respectively.In each species,reproductive tillers were removed at different times:(i)at each cut,(ii)when they reached the flag leaf stage,or(iii)when they reached full bloom.Tiller appearance,tiller death,and type of bud(basal or axillary)of the tillers present were determined.Results:Tiller appearance decreased from July to April.Minimum values were 0.2 and 1.1 tillers 100 tillers−1 day−1 in TF and TW,respectively.In TF,tiller death began in September–October and increased from 0.2 to 0.8 tillers 100 tillers−1 day−1 in March–April.In TW,tiller death began in October–November and increased from 0.6 to 0.8–1.0 tillers 100 tillers−1 day−1 from February to April.Conclusions:Defoliation of reproductive tillers affected the tiller density of TF plants in spring and in the following fall.In TW,the treatments did not modify the tiller density of the plants.

Screening of tall fescue genotypes for relative water content and osmotic potential under drought stress

Background:Tall fescue(Festuca arundinacea Schreb.)is an important coolseason perennial grass.Its persistence and forage yield can be severely affected by drought stresses during the hot,dry summers of the southern USA.Methods:One thousand tall fescue genotypes were evaluated in the greenhouse for high relative water content(RWC)and low cell sap osmotic potential(OP).Fifty contrasting genotypes for the two traits were identified and used in further greenhouse and field studies.These genotypes were also screened with 30%PEG8000.Root and shoot characteristics were studied in 10 genotypes.Results:The genotypes differed for RWC(33.7%–97.3%,mean:79.7%)and had an almost fivefold difference in OP(−0.5 to−2.4 MPa,mean:−1.2 MPa).Significant variation(p<0.001)for the main effects of environment and genotypes was found for RWC and OP.Apart from the greenhouse trial,no correlation was found between RWC and OP,indicating that differences in RWC might have been due to factors other than osmotic adjustment.Genotypes with either long roots or high root weights,and high root/shoot ratios demonstrated high RWC and low OP.Conclusions:Genotypes with consistently high RWC and low OP were identified and used for the development of mapping populations and transcriptome studies.

Approach of water-salt regulation using micro-sprinkler irrigation in two coastal saline soils

This study aimed to investigate whether saline silt and sandy loam coastal soils could be reclaimed by micro-sprinkler irrigation.The experiments were run using moderately salt-tolerant tall fescue grass.Micro-sprinkler irrigation in three stages was used to regulate soil matric potential at a 20-cm soil depth.Continued regulation of soil water and salt through micro-sprinkler irrigation consistently resulted in an increasingly large low-salinity region.The application of the three stages of soil wateresalt regulation resulted in an absence of salt accumulation throughout the soil profile and the conversion of highly saline soils into moderately saline soils.There were increases in the plant height,leaf width,leaf length,and tiller numbers of tall fescue throughout the leaching process.The results showed that micro-sprinkler irrigation in three soil water and salt regulation stages can be used to successfully cultivate tall festuca in highly saline coastal soil.This approach achieved better effects in sandy loam soil than in silt soil.Tall fescue showed greater survival rates in sandy loam soil due to the rapid reclamation process,whereas plant growth was higher in silt soil because of effective water conservation.In sandy loam,soil moisture should be maintained during soil reclamation,and in silt soil,soil root-zone environments optimal for the emergence of plants should be quickly established.Micro-sprinkler irrigation can be successfully applied to the cultivation of tall fescue in coastal heavy saline soils under a three-stage soil wateresalt regulation regime.

Forage and breed effects on behavior and temperament of pregnant beef heifers

Background： Integration of behavioral observations with traditional selection schemes may lead to enhanced animal well-being and more profitable forage-based cattle production systems.Brahman-influenced（BR;n = 64） and Gelbvieh × Angus（GA;n = 64） heifers consumed either toxic endophyte-infected tall fescue（E＋） or one of two nontoxic endophyte-infected tall fescue（NT） cultivars during two yr.Heifers were weighed at midpoint and termination of grazing.Grazing behavior（grazing,resting in the shade,lying,or standing without grazing） was recorded（n = 13 visual observations per yr in June and July） for each pasture.During yr 2,exit velocity（EV） and serum prolactin（PRL） were determined.Results： Grazing behavior was influenced（P 0.05） by an interaction between fescue cultivar and breed type.Gelbvieh × Angus heifers assigned to E＋ pastures had the lowest percentage of animals grazing and the largest percentage of animals resting in the shade.Brahman-influenced heifers had faster EV（P 0.001） than GA heifers（0.52 vs.0.74 ± 0.04 s/m,respectively）.Body weight（BW） was affected（P 0.01） by an interaction of tall fescue cultivar and d,and an interaction of tall fescue cultivar and breed type.Heifers grazing NT pastures were heavier（P 0.01） than heifers grazing E＋ pastures at midpoint and termination.Gelbvieh × Angus heifers grazing NT pastures were heavier（P 0.01） than GA and BR heifers grazing E＋ and BR heifers grazing NT pastures.An interaction of forage cultivar and breed type occurred on serum PRL（P 0.01）.Conclusion： Collectively fescue cultivar,EV,and concentrations of serum PRL were associated with grazing behavior.Heifers grazing NT pastures were observed to be grazing more than heifers assigned to E＋ pastures,regardless of breed type,which may have contributed to changes in BW and average daily gain（ADG） in heifers.Integration of behavioral observations along with traditional selection schemes may lead to enhanced animal well-being and more profitable forage-based cattle production systems.

Do High-Magnesium Cool-Season Grasses Contemplate Grass Tetany Risk?

Economic losses associated with grass tetany either of death or poor growth performance of livestock are a growing concern. Breeding of high magnesium (high-Mg) cool-season forage grass (C3) has been a challenging process to minimize the hazards of grass tetany. For appraising the breeding of high Mg-containing cultivars in Japanese Andisol, agronomic properties and grass tetany risk of high-Mg cultivars were compared with commercial cultivars. The high-Mg cultivars of Italian ryegrass (Lolium multiflorum L.), tall fescue (Festuca arundinacea Schreb.), and orchardgrass (Dactylis glomerata L.) were “Magnet”, “HiMag” and “Mgwell”, respectively. The commercial cultivars were viz., Ace, Tachiwase, and Waseyutaka of Italian ryegrass, Hokuryo, Kentucky-31 (Ky-31), and Fawn of tall fescue, and Akimidori and Okamidori of orchardgrass, respectively. Grasses were grown in temperate Andisol under field conditions with standard management practices and were harvested four times during the year. The average plant heights of the high-Mg containing cultivars were lower than the commercial cultivars with the relative range of −7.4%, −3.7%, and −1.5% for Italian ryegrass, tall fescue, and orchard grass species, respectively. The seasonal yield of high-Mg cultivars was ranked as Magnet > HiMag > Mgwell. The Mgwell orchard grass had lower potassium (K) content compared with their respective commercial cultivars with a relative range of −2.2%. Across four harvests, a significantly higher calcium (Ca) and magnesium (Mg) content, and lower grass tetany potential were recorded in high-Mg cultivars when compared to commercial cultivars, irrespective of species. Across four harvests, the lowest grass tetany index, [K/(Ca + Mg)] of 1.36 was recorded in Mgwell orchard grass. The high-Mg cultivars showed the lowest [K/(Ca + Mg)] across four harvests compared to commercial cultivars, promoting the effectiveness of breeding cool-season grass species to control grass tetany in temperate regions (Andisol) and climatic conditions.

Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses

Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system has been widely used for precise gene editing in plants.However,simultaneous gene editing of multiple homoeoalleles remains challenging,especially in self-incompatible polyploid plants.Here,we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue,using both CRISPR/Cas9 and LbCas12a(LbCpf1)systems.Loss-of-function mutants of FaPDS exhibited albino leaves,while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue.Moreover,these mutations were inheritable.Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.

Synthesis of Ordered Biosilica Materials

Biogenic silica with amazing diversity of nanostructure shells, fibers andgranules in diatoms and sponges is mediated by proteins and polysaccharides and forms at ambientpressure and temperatures. Chemical synthetic methods, in contrast, have to rely on extreme pHand/or surfactants to induce the condensation of silica precursors into specific patterns. One kindof benign synthesis method through plant cell wall template-directed ordered biosilica materialsunder ambient conditions is intriguing in this context. Organized silica materials in intercellularspaces of epidermal cells of tall fescue leaves were synthesized through molecular recognitionbetween Si-OH and polysaccharide-OH or glycoprotein-OH of main components of plant cell walls andcellular processing as well when Si(OEt)_4 was supplied rather than monosilicic acid. Thebiosynthesis of structural silica in tall fescue plant was correlated with the Si species applied,reflecting the slower condensation from tetraethoxysilane (TEOS) and thus providing greateropportunities for structural control by the underlying matrix of cell walls. The composition wasestimated by energy dispersive X-ray (EDX) spectra on a scanning electron microscope. All organizedstructures showed carbon, oxygen and silicon peaks, indicating that their formations differ fromnatural siliceous process.