Effects of Drought Stress on Germination of Tall Fescue

[Objective] The aim was to explore the effects of drought stress on germination of tall fescue.[Method] Simulation drought experiment on 14 varieties of tall fescue seeds by PEG-6000 researched its germination effect under drought resistance.[Result] The relative germination rate of all tall fescue varieties declined with intensifying drought stress level with the water potential of-0.6 Mpa as the turning point;the relative germination rate of tall fescue existed linear relationship with drought stress：Y = 70.412X ＋ 103.11,r=0.988 8.The sensible water potential range was ≥-0.40 Mpa for tall fescue under drought stress.LC50 of water potential was-0.76 Mpa,the lethal water potential was-1.33 Mpa.Arid Ⅲ,Crossfire Ⅱ and Traple A had stronger drought resistant ability,but Pixie,Infero and Poppoint had weaker drought resistant ability.[Conclusion] This study had provided reference for cultivated pasture and drought resistant breeding.

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.

Cloning and Differential Expression Analysis of FaGF14-B and FaGF14-C Genes in Tall Fescue

[Objective] The differential expression analysis was performed for FaGF14- B and FaGF14-C genes in tall fescue so as to provide certain basis for follow-up functional analysis of genes. [Method] The sequence fragments of FaGF14-B and FaGF14-C obtained from reverse transcription were used as templates, and the full- length cDNA sequences of FaGF14-B and FaGF14-C were amplified using the 5＇ RACE use 3＇RACE techniques. They were named as FaGF14-B and FaGF14-C, and used for nucleic acid sequence analysis, encoded protein analysis, protein con- served domain analysis, phylogenetic analysis and differential expression analysis. [Result] The FaGF14-B gene has a full length of 1 548 bp. It has a complete open reading frame （ORF, 449-1 228 bp）, and encodes a protein composed of 261 amino acids. The FaGF14-C gene has a full length of 1 250 bp. It also has a complete open reading frame （ORF, 66-848 bp）, and encodes a protein composed of 261 amino acids. The GF14-B and GF14-C proteins all have a typical domain 14-3-3, and their secondary structures all contain 9 conserved co-helical structures and non-conserved N- and C- terminals. The phylogenetic analysis showed that the FaGF14-B and FaGF14-C from tall rescue have high similarities with GF14 protein from gramineous plants, and they are divided into the same clade with closer ge- netic relationship. The real-time fluorescence quantitative PCR analysis showed that the expression of FaGF14-B and FaGF14-C genes is all sensitive to nitrogen stress. [Conclusion] This study will lay a theoretical basis for further screening of low nitrogen-tolerant genes and breeding of low nitrogen-tolerant grass germplasms.

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.

Expression Analysis of 14-3-3 Gene in Tall Fescue under Several Abiotic Stresses

To study the functions of 14-3-3 gene family in tall fescue, the potential functions of 13 14-3-3 proteins in Arabidopsis were investigated by bioinformatic analysis. Based on the sequences of 14-3-3 genes in tall fescue by transcriptome and proteomic sequencing, the full-length cDNA sequences of 4 14-3-3 genes in tall fescue were obtained. Their sequences were aligned by Clustal W2. The results showed that the genetic relationships between 14-3-3A and 14-3-3D, 14-3-3B and 14-3-3C are closer, and their main structures are very conservative. The changes in expression levels of 14-3-3 genes under low nitrogen, drought, high temperature and high salt stresses were investigated by fluorescence quantitative PCR. The expres- sion level of 14-3-3A makes responses to low nitrogen, drought, high temperature and high salt stresses; the expression levels of other genes also make responses to abiotic stresses in varying degrees, but the relevant response mechanisms are not exactly the same. Therefore, it is speculated that the 14-3-3 gene family regu- lates stress resistance of plants through different pathways, and functional differenti- ation occurs during its evolution.

Construction of RNAi Expression Vector Targeting FaVRN1 Gene in Tall Fescue

[Objective] This study was to construct RNAi expression vector targeting FaVRN1 gene. [Method] A 145 bp Arabidopsis actin intron was inserted into the expression vector to generate an intermediate vector pBI121-M-INT. And then two pairs of specific primers with enzyme restriction sites spanning a 351 bp cDNA conserved sequence fragment of FaVRN1 gene were designed for RT-PCR to construct RNAi expression cassette. The amplified fragment was inserted forwardly and reversely at two sides of the intron to construct an RNAi expression vector with hairpin structure. [Result] Double enzyme digestion（HindIII＋BamHI） showed the intron had been successfully into the vector pBI121. PCR amplification and double enzyme digestion indicated the success of forward and reverse ligation of target FaVRN1 fragment into the intermediate vector. [Conclusion] This study laid foundation for breeding novel flowering-inhibited tall fescue varieties.

Cloning and Characterization of Phytochrome A Gene Fa PHYA from Tall Fescue

The sequence fragment of PHYA, obtained from transcriptome sequencing,was used as the template, and the full-length c DNA sequence of PHYA gene in tall fescue was amplified using 3＇RACE and 5＇RACE techniques. The c DNA sequence of PHYA gene has a complete open reading frame（ORF, 293-6 682 bp）, and it encodes a protein composed of 1 129 amino acids. The N-terminal of Fa PHYA is composed of GAF and Phytochrome domains, and its C-terminal contains two repeated PAS domains, one histidine kinase A domain and one histidine kinase-like ATPase domain. The homology analysis showed that the amino acid sequences of Fa PHYA of tall fescue and PHYAs of gramineous plants have higher homologies（85%）, indicating close genetic relationships. However, the homologies between FaPHYA of tall fescue and PHYAs of monocotyledons are lower, indicating far genetic relationships.

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.

Research Progress on Genetic Breeding of Tall Fescue(Festuca arundinacea Schreb.)

Tal fescue （Festuca arundinacea Schreb.） is an important cool-season forage grasses and turfgrasses at home and abroad. In order to provide a reference for tal fescue breeding, we reviewed the genetic constitution and heritability, endo-phytic fungi and new tal fescue variety breeding, application of modern biological technology （expressed sequence tag, genetic linkage map construction and trans-genic breeding） in breeding new varieties of tal fescue and the breeding status of tal fescue in Guizhou region.

Research on Physiological Characteristics of Tall Fescue under Nitrogen Stress

[Objective] This study aimed to investigate the physiological characteristics of tall fescue under nitrogen stress. [Method] A series of physiological changes in protein content, chlorophyll content, enzyme activities and amino acids contents in tall fescue under nitrogen stress were analyzed. [Result] Under nitrogen stress, the leaf chlorophyll content in tall fescue was reduced, and superoxide dismutase（SOD）,peroxidase（POD）, glutamine synthetase（GS） activities were increased. Among the 18 kinds of amino acids analyzed, the contents of asparagine, serine, glycine, glutamine, glutamic acid, aspartic acid, threonine, alanine, arginine, methionine, valine,isoleucine and leucine were significantly reduced in tall fescue under nitrogen stress.[Conclusion] This study will provide certain reference for rational application of nitrogen and exploration of plant ecological potential in the future.

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.

Effects of Soil Nitrate Nitrogen Residues and Leaching for Different Kinds of Slow-release Nitrogen Fertilizers in Tall-fescue Soil

ObjectiveThe aim was to explore the movement of nitrate nitrogen in tall-fescue soils by different kinds of slow release nitrogen fertilizers. MethodBased on infiltration-tanks and test plots, a new and environment friendly fertilizer was explored. ResultThe results show that compared with urea treatment, slow-release nitrogen fertilizer treatments could reduce nitrate nitrogen content and leaching amount in soils. Compared with PCU30 and IU treatments, the PCU60 treatment became more efficient in reducing nitrate content and leaching amount in 0-90 cm soil layer. ConclusionIn summary, slow-release nitrogen fertilizer, which can reduce soil nitrate content and leaching losses, is a kind of novel fertilizer with high environmental benefit and promising application.

Establishment of genetic transformation system via Agrobacterium in tall fescue cultivar

Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the grass using Agrobacterium （EHA105）, where β-glucuronidase gene （uidA） was used as a reporter and hygromycin phosphotransferase gene （hyg） as a selectable marker. An effective expression of transgene was observed in transforming 2-month-old calli derived from mature seeds （cv. Bingo） cultured on MS medium supplemented with 9 mg·L^-1 2, 4-D. A two-step solid medium selection with increasing hygromycin concentration （from 30 to 50 mg· L^-1） was used to obtain resistant calli. Transgenic plants have been produced from many independent transformed calli. The presence of functional β-glucuronidase gene （uidA） was detected in hygromycin-resistant calli. Transgenic plants were regenerated and PCR and Southern blot confirmed transgene integration in the tall fescue genome.

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.

Screening of Highly Effective Sinorhizobium meliloti Strains for 'Vector' Alfalfa and Testing of Its Competitive Nodulation Ability in the Field

Seventeen Sinorhizobium meliloti strains from seven provinces in China were used to screen highly effective strains for alfalfa cultivar in a greenhouse study and their symbiotic relationship and competitive ability were studied in the field. CCBAU30138 was the most effective strain, as evidenced by increase in dry weights. A field experiment showed that the inoculation of alfalfa with CCBAU30138 resulted in increases of 11.9% and 19.6% of dry matter production and crude protein production, respectively, in forage of monocultured plants. The total dry matter yields of alfalfa and tall fescue in binary culture were increased by 16.3% by inoculation of alfalfa with this strain. These results showed that S. rneliloti strain CCBAU30138 was an effective inoculant both in the greenhouse and in the field. The analysis of randomly amplified polymorphic DNA （RAPD） by polymerase chain reaction （PCR） from nodule extracts showed that the strain CCBAU30138 had high competitiveness in the field. It occupied 47.5% of nodules in alfalfa monoculture and 44.4% of nodules in alfalfa-tall fescue binary culture after 20 weeks of growth. In conclusion, a simple system to select highly effective and competitive symbiotic strains specific to alfalfa was established. Using this system, a s.train suitable for the alfalfa cultivar ‘Vector＇ grown in Wuqiao County of Hebei Province was obtained.