This paper reviews the current status, methodology, achievements, and prospects of alfalfa (Medicago sativa L.) breeding in China. There are 77 cultivars that have been registered in the country, these include 36 cultivars bred through breeding programs, 17 introduced from overseas, 5 domesticated from wild ecotypes, and 19 through regional collection/breeding programs. Cultivars have been selected for cold resistance, disease resistance, salt tolerance, grazing tolerance, high yield, and early maturity. Most of these cultivars have been developed through conventional breeding techniques, such as selective and cross breeding, and some are now being evaluated that have been developed through the application of transgenic technology. The major problems for alfalfa breeding in China include low success rate, slow progress to breed resistant varieties, lack of breeding materials and their systematic collection, storage and evaluation, lack of advanced breeding techniques, and low adoption rate of new cultivars. There are gaps in alfalfa breeding between China and the developed world. Improvement of alfalfa breeding techniques, development of cultivars with adaptations to different regions within China, and the protection and utilization of alfalfa germplasm resources have been identified as major strategies to improve the efficiency of alfalfa breeding in China.

Drought severely limits global plant distribution and agricultural production. Elucidating the physiological and molecular mechanisms governing alfalfa stress responses will contribute to the improvement of drought tolerance in leguminous crops. In this study, the physiological and proteomic responses of two alfalfa (Medicago sativa L.) varieties contrasting in drought tolerance, Longzhong (drought-tolerant) and Gannong No. 3 (drought-sensitive), were comparatively assayed when seedlings were exposed to -1.2 MPa polyethylene glycol (PEG-6000) treatments for 15 days. The results showed that the levels of proline, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), hydroxyl free radical (OH^•) and superoxide anion free radical (O₂^•-) in both varieties were significantly increased, while the root activity, the superoxide dismutase (SOD) and glutathione reductase (GR) activities, and the ratios of reduced/oxidized ascorbate (AsA/DHA) and reduced/oxidized glutathione (GSH/GSSG) were significantly decreased. The soluble protein and soluble sugar contents, the total antioxidant capability (T-AOC) and the activities of peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) first increased and then decreased with the increase in treatment days. Under osmotic stress, Longzhong exhibited lower levels of MDA, H₂O₂, OH^• and O₂^•- but higher levels of SOD, CAT, APX, T-AOC and ratios of AsA/DHA and GSH/GSSG compared with Gannong No.3. Using isobaric tags for relative and absolute quantification (iTRAQ), 142 differentially accumulated proteins (DAPs) were identified from two alfalfa varieties, including 52 proteins (34 up-regulated and 18 down-regulated) in Longzhong, 71 proteins (28 up-regulated and 43 down-regulated) in Gannong No. 3, and 19 proteins (13 up-regulated and 6 down-regulated) shared by both varieties. Most of these DAPs were involved in stress and defense, protein metabolism, transmembrane transport, signal transduction, as well as cell wall and cytoskeleton metabolism. In conclusion, the stronger drought-tolerance of Longzhong was attributed to its higher osmotic adjustment capacity, greater ability to orchestrate its enzymatic and non-enzymatic antioxidant systems and thus avoid great oxidative damage in comparison to Gannong No. 3. Moreover, the involvement of other pathways, including carbohydrate metabolism, ROS detoxification, secondary metabolism, protein processing, ion and water transport, signal transduction, and cell wall adjustment, are important mechanisms for conferring drought tolerance in alfalfa.

Grazing exclusion is an effective management strategy for restoring degraded grasslands worldwide, but the effects of different exclusion durations on vegetation structure and soil properties remain unclear. Therefore, we evaluated vegetation characteristics and soil properties in an alpine meadow-steppe under grazing exclusion of different lengths (with grazing and with 3-year, 6-year, 9-year and 11-year grazing exclusions) on the Qinghai-Tibetan Plateau (QTP). We also explored the relationships among above-ground biomass, biodiversity and soil properties to ascertain the mechanism underlying the impact of grazing exclusion on these factors. The results showed that the above- and below-ground biomass, total number of plant species, community density, Shannon–Wiener diversity index, evenness index, richness index, soil and vegetation carbon (C) and nitrogen (N) storage and ecosystem C and N storage exhibited a hump-shaped pattern in response to the length of grazing exclusion with a 6-year threshold. In addition, structural equation modelling showed that the bulk density, soil moisture content, micro sand content and clay and silt contents were the most important determining factors leading to an increase in above-ground biomass in the alpine meadow-steppe after grazing exclusion, whereas the soil total N, available N, available phosphate and soil organic C content were the most important determining factors leading to a decrease in biodiversity. Considering the stability of the plant community and the C and N pools, long-term grazing exclusion (>9 years) is unnecessary, and the optimum exclosure duration of the moderately degraded Elymus nutans - Kobresia humilis type alpine meadow-steppe is six years on the north-eastern QTP.

Freezing stress is a major limiting environmental factor that affects the productivity and distribution of alfalfa (Medicago sativa L.). There is growing evidence that enhancing freezing tolerance through resistance-related genes is one of the most efficient methods for solving this problem, whereas little is known about the complex regulatory mechanism of freezing stress. Herein, we performed transcriptome profiling of the leaves from two genotypes of alfalfa, freezing tolerance “Gannong NO.3” and freezing-sensitive “WL326GZ” exposure to −10°C to investigate which resistance-related genes could improve the freezing tolerance. Our results showed that a total of 121,366 genes were identified, and there were 7,245 differentially expressed genes (DEGs) between the control and treated leaves. In particular, the DEGs in “Gannong NO.3” were mainly enriched in the metabolic pathways and biosynthesis of secondary metabolites, and most of the DEGs in “WL326GZ” were enriched in the metabolic pathways, the biosynthesis of secondary metabolites, and plant-pathogen interactions. Moreover, the weighted gene co-expression network analysis (WGCNA) showed that ATP-binding cassette (ABC) C subfamily genes were strongly impacted by freezing stress, indicating that ABCC8 and ABCC3 are critical to develop the freezing tolerance. Moreover, our data revealed that numerous Ca²⁺ signal transduction and CBF/DREB1 pathway-related genes were severely impacted by the freezing resistance, which is believed to alleviate the damage caused by freezing stress. Altogether, these findings contribute the comprehensive information to understand the molecular mechanism of alfalfa adaptation to freezing stress and further provide functional candidate genes that can adapt to abiotic stress.