Effects of antioxidant‑rich Lactiplantibacillus plantarum inoculated alfalfa silage on rumen fermentation, antioxidant and immunity status, and mammary gland gene expression in dairy goats

Background Milk synthesis in lactating animals demands high energy metabolism,which results in an increased production of reactive oxygen metabolites(ROM)causing an imbalance between oxidants and antioxidants thereby inducing oxidative stress(OS)on the animals.To mitigate OS and postpartum disorders in dairy goats and gain insight into the impact of dietary choices on redox status during lactation,a feeding trial was conducted using alfalfa silage inoculated with a high-antioxidant strain of Lactiplantibacillus plantarum.Methods Twenty-four Guanzhong dairy goats(38.1±1.20 kg)were randomly assigned to two dietary treatments:one containing silage inoculated with L.plantarum MTD/1(RSMTD-1),and the other containing silage inoculated with high antioxidant activity L.plantarum 24-7(ES24-7).Results ES24-7-inoculated silage exhibited better fermentation quality and antioxidant activity compared to RSMTD-1.The ES24-7 diet elevated the total antioxidant capacity(T-AOC),superoxide dismutase(SOD),glutathione peroxi-dase(GSH-Px),and catalase(CAT)activities in milk,serum,and feces of lactating goats(with the exception of T-AOC in milk).Additionally,the diet containing ES24-7 inoculated silage enhanced casein yield,milk free fatty acid(FFA)content,and vitamin A level in the goats’milk.Furthermore,an increase of immunoglobulin(Ig)A,IgG,IgM,inter-leukin(IL)-4,and IL-10 concentrations were observed,coupled with a reduction in IL-1β,IL-2,IL-6,interferon(IFN)-γ,and tumor necrosis factor(TNF)-αconcentrations in the serum of lactating goats fed ES24-7.Higher concentrations of total volatile fatty acid(VFA),acetate,and propionate were observed in the rumen fluid of dairy goats fed ES24-7 inoculated silage.Moreover,the diet containing ES24-7 inoculated silage significantly upregulated the expression of nuclear factor erythroid 2 like 2(NFE2L2),beta-carotene oxygenase 1(BCO1),SOD1,SOD2,SOD3,GPX2,CAT,glu-tathione-disulfide reductase(GSR),and heme oxygenase 1(HMOX1)genes in the mammary gland,while decreased the levels of NADPH oxidase 4(NOX4),TNF,and interferon gamma(IFNG).Conclusions These findings indicated that feeding L.plantarum 24-7 inoculated alfalfa silage not only improved rumen fermentation and milk quality in lactating dairy goats but also boosted their immunity and antioxidant status by modulating the expression of several genes related to antioxidant and inflammation in the mammary gland.

Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa

Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

Variation Characteristics of Root Traits of Different Alfalfa Cultivars under Saline-Alkaline Stress and their Relationship with Soil Environmental Factors

Soil salinization is the main factor that threatens the growth and development of plants and limits the increase of yield.It is of great significance to study the key soil environmental factors affecting plant root traits to reveal the adaptation strategies of plants to saline-alkaline-stressed soil environments.In this study,the root biomass,root morphological parameters and root mineral nutrient content of two alfalfa cultivars with different sensitivities to alkaline stress were analyzed with black soil as the control group and the mixed saline-alkaline soil with a ratio of 7:3 between black soil and saline-alkaline soil as the saline-alkaline treatment group.At the same time,the correlation analysis of soil salinity indexes,soil nutrient indexes and the activities of key enzymes involved in soil carbon,nitrogen and phosphorus cycles was carried out.The results showed that compared with the control group,the pH,EC,and urease(URE)of the soil surrounding the roots of two alfalfa cultivars were significantly increased,while soil total nitrogen(TN),total phosphorus(TP),organic carbon(SOC),andα-glucosidase activity(AGC)were significantly decreased under saline-alkaline stress.There was no significant difference in root biomass and root morphological parameters of saline-alkaline tolerant cultivar GN under saline-alkaline stress.The number of root tips(RT),root surface area(RS)and root volume(RV)of AG were reduced by 61.16%,44.54%,and 45.31%,respectively,compared with control group.The ratios of K^(+)/Na^(+),Ca^(2+)/Na^(+)and Mg^(2+)/Na^(+)of GN were significantly higher than those of AG(p<0.05).The root fresh weight(RFW)and dry weight(RDW),root length(RL),RV and RT of alfalfa were positively regulated by soil SOC and TN,but negatively regulated by soil pH,EC,and URE(p<0.01).Root Ca^(2+)/Na+ratio was significantly positively correlated with soil TN,TP and SOC(p<0.01).The absorption of Mg and Ca ions in roots is significantly negatively regulated by soilβ-glucosidase activity(BGC)and acid phosphatase activity(APC)(p<0.05).This study improved knowledge of the relationship between root traits and soil environmental factors and offered a theoretical framework for elucidating how plant roots adapt to saline-alkaline stressed soil environments.

不同症状类型苜蓿病毒病AMV病原检测及其寄主范围测定

为明确采自田间不同症状类型苜蓿病毒病病样中苜蓿花叶病毒(alfalfa mosaic virus,AMV)的带毒情况、症状表现与叶绿素含量的相关性及AMV的寄主范围,本试验通过田间调查采样、症状归类、丙酮乙醇混合液法和双抗体夹心酶联免疫吸附测定(DAS-ELISA)法对不同病样的叶绿素和AMV含量进行测定和检测,并对提纯至不同症状类型病样中AMV对9科32种植物的致病性进行测定。结果表明田间苜蓿病毒病病样有轻花叶、重花叶、叶片边缘褪绿黄化型和叶片畸形皱缩花叶矮化型4种症状类型,均带有AMV,且叶绿素含量、AMV含量与症状表现之间具有正相关性,症状表现最严重的叶片畸形皱缩花叶矮化型病样的叶绿素a、b、总叶绿素含量均比对照低58.00%以上,类胡萝卜素含量比对照高134.06%,AMV含量最高,为252.96 pg·mL^(-1);寄主范围测定表明AMV可侵染7科27种植物,对西葫芦致病性最强,症状表现为局部枯斑,30 d时AMV浓度为316.19 pg·mL^(-1)。

Flavonol synthase gene MsFLS13 regulates saline-alkali stress tolerance in alfalfa

Alfalfa(Medicago sativa L.) is one of the most extensively grown leguminous forage worldwide.Environmental saline-alkali stress significantly influences the growth,development,and yield of alfalfa,posing a threat to its agricultural production.However,little is known about the potential mechanisms by which alfalfa responds to saline-alkali stress.Here,we investigated these mechanisms by cloning a saline-alkali-induced flavonol synthase gene(Ms FLS13) from alfalfa,which was previously reported to be significantly upregulated under saline-alkali stress,and examining its function in the saline-alkali response.Overexpression of Ms FLS13 in alfalfa promoted plant tolerance to saline-alkali stress by enhancing flavonol accumulation,antioxidant capacity,osmotic balance,and photosynthetic efficiency.Conversely,Ms FLS13 inhibition using RNA interference reduced flavonol synthase activity and inhibited hairy root growth under saline-alkali stress.Yeast one-hybrid and dual-luciferase reporter assays indicated that the R2R3-MYB Ms MYB12 transcription factor activates Ms FLS13 expression by binding to the MBS motif in the Ms FLS13 promoter.Further analysis revealed that abscisic acid mediates the salinealkali stress response partially by inducing Ms MYB12 and Ms FLS13 expression,which consequently increases flavonol levels and maintains antioxidant homeostasis in alfalfa.Collectively,our findings highlight the crucial role of Ms FLS13 in alfalfa in response to saline-alkali stress and provide a novel genetic resource for creating saline-alkali-resistant alfalfa through genetic engineering.

Overexpression of ZxABCG11 from Zygophyllum xanthoxylum enhances tolerance to drought and heat in alfalfa by increasing cuticular wax deposition

Drought and heat stresses cause yield losses in alfalfa,a forage crop cultivated worldwide.Improving its drought and heat tolerance is desirable for maintaining alfalfa productivity in hot,arid regions.Cuticular wax forms a protective barrier on aerial surfaces of land plants against environmental stresses.ABCG11encodes an ATP binding cassette(ABC) transporter that functions in the cuticular wax transport pathway.In this study,Zx ABCG11 from the xerophyte Zygophyllum xanthoxylum was introduced into alfalfa by Agrobacterium tumefaciens-mediated transformation.Compared to the wild type(WT),transgenic alfalfa displayed faster growth,higher wax crystal density,and thicker cuticle on leaves under normal condition.Under either drought or heat treatment in greenhouse conditions,the plant height and shoot biomass of transgenic lines were significantly higher than those of the WT.Transgenic alfalfa showed excellent growth and 50% greater hay yield than WT under field conditions in a hot,arid region.Overexpression of Zx ABCG11 up-regulated wax-related genes and resulted in more cuticular wax deposition,which contributed to reduction of cuticle permeability and thus increased water retention and photosynthesis capacity of transgenic alfalfa.Thus,overexpression of Zx ABCG11 can simultaneously improve biomass yield,drought and heat tolerance in alfalfa by increasing cuticular wax deposition.Our study provides a promising avenue for developing novel forage cultivars suitable for planting in hot,arid,marginal lands.

Effects of Different Herbicides on Weed Control in Alfalfa Field

[Objectives]The paper was to systematically study the technology of weed control in alfalfa field.[Methods]Reviving alfalfa field and newly sown alfalfa field after emergence were selected,and the effects of different herbicides on weed control and alfalfa yield were discussed.[Results]The optimal herbicides after alfalfa reviving were 5%imazethapyr and 10%imazethapyr,and the optimal dosages were 1.5 and 1.05 L/hm 2,respectively.The optimal herbicides after emergence of newly born alfalfa were 5%imazethapyr and 10%imazethapyr,and the optimal dosages were 1.5 and 0.75 L/hm 2,respectively.[Conclusions]This study will provide a technical support for high quality production of alfalfa.

卫星搭载对紫花苜蓿当代植株生物量的影响(英文)

[Objective] The aim of this study was to investigate the effect of space flight factors on plant biomass in the generation of alfalfa carried by the satellite.[Method]Seeds from three lines of alfalfa were carried by the seed-breeding satellite Shijian-8.After the satellite returned to the ground,stem diameter,primary branch number and current-year individual biomass of alfalfa were studied.[Result]After space flight,primary branch number and current-year individual biomass of alfalfa increased significantly,while the stem diameter had no significant change.Using the value over(the mean value of control + three standard deviation)as a criterion to screen,the variants with enlarged stem diameter,increased primary branch number and individual biomass was two,five and twelve respectively.[Conclusion]The obtained variants can be used in the variety improvement of alfalfa and its new variety breeding,but whether its favorable variation can inherit stably to the progenies needs further study.

国内外苜蓿品种性状分析与评价(英文)

The agronomic and qualitative traits of 17 alfalfa varieties were analyzed in field and lab from 2006 to 2008, and these traits were evaluated by principal component analysis and cluster analysis. A total of 10 main traits were classified as five factors： growth factor, quality factor, stem and leaf factors, plant factor and yield factor. These five factors and their correlation were selected for breeding. These 17 varieties can be divided into five clusters. The domestic varieties have better agronomic traits, while the introduced ones have better qualitative traits.

紫花苜蓿分枝期光合蒸腾特性及其与相关因子的关系

[ Objective] The paper presents the diumal changes of photosynthesis and transpiration of different alfalfa varieties and their relationship with the associated physiological and ecological factors during branching stage, so as to provide a basis for the development, utilization, and breed- ing of alfalfa. [ Method] Under natural conditions, the diurnal changes of net photosynthetic rate （Pn）, transpiration rate （Tr）, the relevant physio- logical factors including leaf temperature （TI）, stomatal conductance （Gs） and intemal COn concentration （Ci）, as well as the relevant physiologi- cal factors including photosynthetic available radiation （PAR）, CO2 concentration in field （Ca） and air temperature （Ta） were measured in four al- falfa varieties （Algonguin, WL323 HQ, WL414, and Millionaire）. The water use efficiency （WUE） and light use efficiency （LUE） were calculated, and the correlation among them was also analyzed. [Result] The Pn, Tr, PAR and Ta of the four varieties appeared to vary in a single-peak curve; the sequence of WUE was WL323 HQ ~ Algonguin ~ WL414 ~ Millionaire; there was no significant difference in LUE of the four alfalfa varieties; coef- ficient analysis showed that Pn was mainly affected by PAR, Gs, and Ci, while Tr by PAR and Ta. [ Conclusion] WL323 HQ is the variety with high Pn, high WUE and low Tr, and it has strong adaptability to drought. In four alfalfa varieties, PAR, Ta, Gs, and TI are the primary determining fac- tors while Ca and Ci the limiting factors of Tr; Gs is the primary determining factor while Ci the limiting factor of Pn.

紫花苜蓿根腐病性病害生防放线菌的分离与筛选(英文)

[Objective] Actinomycetes with high antagonistic effects on alfalfa root rot pathogen was isolated from 10 soil samples in Chifeng Inner Mongolia. [Method] 91 actinomyces were separated from 10 soil samples in Chifeng Inner Mongolia by gradient dilution separation method. In duel culture tests, all isolates were tested for their antagonism by using 3 strains including Fusarium solani, F. oxysoporum, F. avenaceum of alfalfa root rot pathogenic bacteria as indicator strains. [Result] 5 strains with strong antagonistic effect on tested alfalfa root rot pathogen were obtained from No.1, No.4, No.6 and No.7 soil samples which were numbered 1-3-6, 4-4-2, 6-2-27 and 7-2-13 respectively, accounted for 5.50% in separated strains. [Conclusion] This study laid certain foundation for biological control of alfalfa root rot disease.

Alfalfa-containing diets alter luminal microbiota structure and short chain fatty acid sensing in the caecal mucosa of pigs

Background: Pork produced by outdoor-reared pigs raised mostly on alfalfa pastures attracts increasing population of consumer from most of the world. In China, pigs were raised with alfalfa-containing diets to seek for good quality pork.However, the influence of dietary alfalfa involving high level of insoluble dietary fiber(IDF) on pig intestinal luminal microbiota composition remains unclear. The objective of this study was to investigate the effects of alfalfa on luminal microbiota and short chain fatty acids(SCFA) production, and gene expressions involved in SCFA sensing, transporting and absorbing in pig caecal mucosa.Results: Twenty-four growing pigs were randomly allotted to four diets containing 0%, 5%, 10% and 15% alfalfa meal for a 28-d experiment. Ingestion of alfalfa meal-contained diets significantly increased the ratio of body weight gain to feed consumption. Illumina MiS eq sequencing of the V3 region of the 16 S r RNA genes showed that alfalfa-containing diet significantly decreased the relative abundance of genera Turicibacter, Acidiphilium, Paracoccus, Propionibacterium,Corynebacterium, Pseudomonas, Acinetobacter, and Staphylococcus, and increased the relative abundance of genera Lachnospira, Marvinbryantia, and Desulfovibrio in the caecal digesta. Butyrate concentration was significantly increased in the hindgut by the supplementation of alfalfa meal in diets. The m RNA gene expressions of FFAR3, SMCT1, MCT1,PYY, and GCG were significantly increased in the caecal mucosa of pigs fed alfalfa meal.Conclusions: Our results suggested that alfalfa-containing diet has exerted significant impacts on caecal microbiota composition, butyrate concentration and significantly upregulated m RNA expression of host caecal mucosal genes involved in SCFA sensing and absorption as well as regulation of satiety.

Effect of Graded Levels of Fiber from Alfalfa Meal on Nutrient Digestibility and Flow of Fattening Pigs

The fiber level and composition have an important effect on nutrient digestibility of swine diets. Little information is known about the effects of fiber level and composition from alfalfa meal on nutrient digestibility of fattening pigs fed a corn-soybean meal-based diet. The objective of this experiment was to determine the effects of alfalfa fiber on the growth performance, intestinal nutrient flow and apparent total tract digestibility（ATTD） of nutrients in fattening pigs. 24 barrows（Duroc×（Large White×Landrace）, body weight=（60.6±0.7） kg） were randomly allotted to 4 treatments with 6 replicates of 1 pig per replicate. The pigs were provided a control diet or a diet containing 5, 10 or 20% of alfalfa meal during a 14-d experiment period. Average daily gain（ADG） and the ATTD of dry matter（DM）, organic matter（OM）, crude protein（CP）, neutral detergent fiber（NDF）, acid detergent fiber（ADF） and gross energy（GE） reduced linearly as the level of alfalfa meal in the diet increased（P〈0.01）. The total tract flow of DM, OM, CP, NDF, ADF, and GE increased with the increase in dietary alfalfa（linear, P〈0.05）. Growth performance and nutrient digestion were not affected by inclusion of 5% alfalfa meal in the diet（P〉0.05）. A multiple linear regression analysis, taking into account both soluble and insoluble fiber intake, explained approximately 70% of the variation in the ATTD of DM, OM, NDF, and GE（P〈0.01）. In conclusion, alfalfa meal should be limited to less than 5% of the diet in fattening pigs to maximize growth performance and nutrient digestion. Soluble and insoluble fiber from alfalfa meal has the differential roles in nutrient digestion, which may help explain the main variation observed in nutrient digestibility. These findings suggest that knowledge of specific fiber components is necessary to accurately predict the effects of dietary fiber on nutrient digestibility.

Influence of lactic acid bacteria, cellulase, cellulase-producing Bacillus pumilus and their combinations on alfalfa silage quality

This study assessed the effects of lactic acid bacteria(LAB), cellulase, cellulase-producing Bacillus pumilus and their combinations on the fermentation characteristics, chemical composition, bacterial community and in vitro digestibility of alfalfa silage. A completely randomized design involving a 8(silage additives)×3 or 2(silage days) factorial arrangement of treatments was adopted in the present study. The 8 silage additive treatments were: untreated alfalfa(control); two commercial additives(GFJ and Chikuso-1); an originally selected LAB(Lactobacillus plantarum a214) isolated from alfalfa silage; a cellulase-producing Bacillus(CB) isolated from fresh alfalfa; cellulase(C); and the combined additives(a214+C and a214+CB). Silage fermentation characteristics, chemical composition and microorganism populations were analysed after 30, 60 and 65 days(60 days followed by exposure to air for five additional days). In vitro digestibility was analysed for 30 and 60 days. Compared with the other treatments, selected LAB a214, a214 combined with either C or CB, and Chikuso-1 had the decreased(P<0.001) pH and increased(P<0.001) lactic acid concentrations during the ensiling process, and there were no differences(P>0.05) among them. Fiber degradation was not significant(P≥0.054) in any C or CB treatments. The a214 treatment showed the highest(P=0.009) in vitro digestibility of dry matter(595.0 g kg–1DM) after ensiling and the highest abundance of Lactobacillus(69.42 and 79.81%, respectively) on days 60 and 65, compared to all of other treatments. Overall, the silage quality of alfalfa was improved with the addition of a214, which indicates its potential as an alfalfa silage inoculant.

Effects of neutral salt and alkali on ion distributions in the roots,shoots,and leaves of two alfalfa cultivars with differing degrees of salt tolerance

The effects of neutral salt and alkali on the ion distribution were investigated in two alfalfa （Medicago sativa L.） cultivars, including Zhongmu 1, a high salt-tolerant cultivar, and Algonquin, a low salt-tolerant cultivar. The alkali stress expressed more serious growth inhibition than the neutral salt stress at the same Na＋ concentration. Compared with Algonquin, Zhongmu 1 did not exhibit a higher alkali tolerance under the Na2CO3-NaHCO3 treatment with the low Na＋ concentration （50 mmol L-l）. The alkali increased the accumulation of Na＋, Ca2＋, and Mg2＋ in the root and changed the Ca2＋ and Mg2＋ balance in the entire alfalfa plant. The salt and alkali stresses decreased the K＋ and Fe3＋ contents of the roots and leaves, the root Mn2＋ content, and the shoot Zn2＋ content, but they increased the Fe3＋ accumulation of the shoots, the shoot and leaf Cu2＋ contents, and the leaf Zn2＋ content in both alfalfa cultivars. Based on the results obtained under the conditions of this experiment, we found that the salt and alkali stresses reduced the plant growth in both alfalfa cultivars, while the alkali caused a stronger stress than the neutral salt in alfalfa. Thus, we conclude that under hydroponic conditions, the deleterious effects of the alkali on plants are due to the distribution change of some trophic ion balance in the roots, shoots, and leaves of the plants by causing of Na＋, CO3^2-, and/or HCO3- stresses.

Changes in aggregate-associated organic carbon and nitrogen after 27 years of fertilization in a dryland alfalfa grassland on the Loess Plateau of China

Changes in the distribution of soil aggregate sizes and concentrations of aggregate-associated organic carbon （OC） and nitrogen （N） in response to the fertilization of grasslands are not well understood. Understanding these changes is essential to the sustainable development of artificial grasslands. For understanding these changes, we collected soil samples at 0-20 and 20-40 cm depths from a semi-arid artificial alfalfa grassland after 27 years of applications of phosphorus （P） and nitrogen＋phosphorus＋manure （NPM） fertilizers on the Loess Pla- teau of China. The distribution of aggregate sizes and the concentrations and stocks of OC and N in total soils were determined. The results showed that NPM treatment significantly increased the proportions of 〉2.0 mm and 2.0-0.25 mm size fractions, the mean geometric diameter （MGD） and the mean weight diameter （MWD） in the 0-20 cm layer. Phosphorous fertilizer significantly increased the proportion of 〉2.0 mm size fractions, the MGD and the MWD in the 0-20 cm layer. Long-term application of fertilization （P and NPM） resulted in the accumulation of OC and N in soil aggregates. The largest changes in aggregate-associated OC and N in the 0-20 cm layer were found at the NPM treatment, whereas the largest changes in the 20-40 cm layer were found at the P treatment. The results suggest that long-term fertilization in the grassland leads to the accumulation of OC and N in the coarse size fractions and the redistribution of OC and N from fine size fractions to coarse size fractions.

Recent progress in alfalfa(Medicago sativa L.)genomics and genomic selection

Alfalfa(M. sativa L.) is a highly valuable forage crop, providing >58 Mt of hay, silage, and pasture each year in the United States. As alfalfa is an outcrossing autotetraploid crop,however, breeding for enhanced agronomic traits is challenging and progress has historically not been rapid. Methods that make use of genotypic information and statistical models to generate a genomic estimated breeding value(GEBV) for each plant at a young age hold a great deal of promise to accelerate breeding gains. An emerging genomic breeding pipeline employs SNP chips or genotyping-by-sequencing(GBS) to identify SNP markers in a training population, followed by the use of a statistical model to find associations between the discovered SNPs and traits of interest, followed by genomic selection(GS), a breeding program utilizing the trained model to predict breeding values and making selections based on the estimated breeding value(EBV). Much work has been done in recent years in all of these areas, to generate marker sets and discover SNPs associated with desirable traits, and the application of these technologies in alfalfa breeding programs is under way. However, GBS/GWAS/GS is still a new breeding paradigm,and work is ongoing to evaluate different models, software, and methods for use in such programs. In this review, we look at the progress of alfalfa genomics over the past halfdecade, and review work comparing models and methods relevant to this new type of breeding strategy.

Development of Alfalfa (Medicago sativa L.) Regeneration System and Agrobacterium-Mediated Genetic Transformation

The regeneration ability of four alfalfa （Medicago sativa L.） cultivars, Xinjiang Daye, Longdong, Gannong 1 and Gannong 3, was studied, and the effects of various cultivars, explant sources and medium recipes on regeneration were compared. The better callus forming frequency obtained from hypocotyls of Xinjiang Daye is 88.5% and regeneration frequency is 9.8% in our initial experiments. To further optimize regeneration system for genetic transformation, we therefore changed concentrations of plant growth regulators and supplemented with glutamine into callus-induction and shoot-regeneration media. Callus forming frequency and shoot differentiation frequency were increased to 100%. The time taken to generate transgenic plants （16 weeks） was shorter than that for previouse procedure （25 weeks） and regeneration frequency was promoted to 15.1%. The results show that addition of glutamine is particularly important for shortening period of regeneration and promoting regeneration frequency. For study of genetic transformation of alfalfa, Agrobacterium tumefaciens-mediated transformation of Xinjiang Daye was developed based on this optimized regeneration system. The plant expression vector carrying two glutamine synthetases （GS 1 and GS2） and △1-pyrroline-5-carboxylate synthetase （P5CS） gene was used for alfalfa in vitro transformation. Six transgenic alfalfa plantlets with resistance to PPT were obtained. The introduction of foreign genes into plants was assessed in the transformants by PCR analysis and Southern hybridizations.

Nitrogen acquisition,fixation and transfer in maize/alfalfa intercrops are increased through root contact and morphological responses to interspecies competition

Nitrogen(N)fixation by legumes and nitrogen transfer to cereals have been considered as important pathways for overyielding and higher N use efficiency in cereal/legume intercropping systems.However,the extent to which root morphology contributes to N fixation and transfer is unclear.A two-factorial greenhouse experiment was conducted to quantify the N fixation,transfer and root morphology characteristics of the maize/alfalfa intercropping system in two consecutive years using the 15N-urea leaf labeling method,and combining two N levels with three root separation techniques.N application could inhibit N fixation and transfer in a maize/alfalfa intercropping system.Irrespective of the N application level,compared with plastic sheet separation(PSS),no separation(NS)and nylon mesh separation(NNS)significantly increased the total biomass(36%)and total N content(28%),while the N fixation rate also sharply increased by 75 to 134%,and the amount of N transferred with no root barrier was 1.24–1.42 times greater than that with a mesh barrier.Redundancy analysis(RDA)showed that the crown root dry weight(CRDW)of maize and lateral root number(LRN)of alfalfa showed the strongest associations with N fixation and transfer.Our results highlight the importance of root contact for the enhancement of N fixation and transfer via changes in root morphology in maize/alfalfa intercropping systems,and the overyielding system was achieved via increases in maize growth,at the cost of smaller decreases in alfalfa biomass production.

China’s alfalfa market and imports: Development, trends, and potential impacts of the U.S.-China trade dispute and retaliations

This study examines the development and trends of China’s alfalfa market and imports, identifies key factors for the rapid increase in China’s alfalfa imports, and discusses potential impacts of the U.S.-China trade dispute and retaliations on the alfalfa markets and trade in both nations. China’s rapid transition toward larger-scale commercial dairy production, with enhanced feed and cost management as well as quality and safety control, and its limited resources for high-quality alfalfa production are key factors for the dramatic increase in its alfalfa imports, from 19 601 metric tons in 2008 to 1.38 million metric tons(mmt) in 2018. While the United States dominated China’s alfalfa imports with an average share of 97.01% from 2007 to 2017, the share dropped to 83.76% in 2018 and 63.28% in January 2019 due to the trade dispute and retaliations started in 2018. China will likely remain a large importer of alfalfa because of both its growing demand and the comparative advantages of imported alfalfa in quality and price, but the imports from the United States will be highly affected by the ongoing trade dispute and negotiations. China is also expected to make more efforts to reduce its dependence on U.S. alfalfa through increased investment in domestic alfalfa production and identification of alternative sources of alfalfa and other hay imports.