紫花苜蓿（Medicago sativa L.）作为多年生异源四倍体（2n=4x=32）豆科牧草，因其突出的农艺性状和生态价值而成为全球范围内广泛栽培的重要饲草作物。在植物细胞Ca²⁺信号转导系统中，类钙调素蛋白（Calmodulin-like protein，CMLs）作为钙结合蛋白，通过其EF-hand结构域特异性识别Ca²⁺信号，诱导构象变化，进而作用于靶蛋白调控具体的生理生化功能。钙依赖蛋白激酶（Ca²⁺-dependent protein kinase，CDPKs）则通过Ca²⁺依赖的激酶活性，选择性磷酸化下游靶蛋白，从而参与调控植物生长发育及逆境响应等生理过程。本研究基于紫花苜蓿CDPKs家族分析，筛选鉴定出一个基因——MsCDPK33，系统解析了其在紫花苜蓿耐寒耐旱中的功能，为抗逆育种提供了重要基因资源。并针对该基因在‘中苜1号’紫花苜蓿遗传转化中出现的愈伤组织诱导率低、再生周期长等问题，我们进一步优化培养基配方，成功建立了高效稳定的遗传转化体系。并进一步将该体系拓展应用于黄花苜蓿耐寒基因资源开发，采用MfCML50/MfAOC2过表达载体与改造的1300-FMV-CAL-35S-G10evo载体共转化策略，成功创制出兼具草甘膦抗性和显著增强耐寒性的紫花苜蓿新种质，为苜蓿抗逆育种提供了新材料基础。研究结果如下：

1. 紫花苜蓿MsCDPK33耐寒耐旱功能分析

基于前期研究基础，本研究选择MsCDPK33进行功能分析。组织表达模式分析显示，MsCDPK33在根、茎、叶和花中都有表达，尤其在根中表达量最高。对MsCDPK33的蛋白序列分析表明，其编码的蛋白具有CDPKs家族蛋白特有的Ser/Thr激酶结构域及4个EF-Hand结构域。烟草亚细胞定位分析显示该蛋白定位于细胞质膜，且当棕榈酰化位点与豆蔻酰化位点发生突变时，其定位模式由单一的细胞质膜分布转变为细胞核与细胞质膜共定位，且具有激酶活性。通过低温半致死和存活率实验对MsCDPK33的过表达和RNAi转基因株系进行耐寒性表型分析，发现MsCDPK33负调控紫花苜蓿的耐寒性。进一步研究发现低温下MsCBF1、MsLEA3和MsTL16B等COR基因的表达在MsCDPK33过表达或RNAi株系中表现出显著差异，说明MsCDPK33参与调控COR基因的表达。此外，MsCDPK33可能作为负调控因子通过抑制MsP5CS1的表达来限制脯氨酸的积累，进而影响紫花苜蓿的低温胁迫响应。对紫花苜蓿进行干旱胁迫处理后，通过相对电导率测定发现，MsCDPK33负调控植株的耐旱性。进一步研究发现，干旱胁迫显著诱导了干旱响应基因MsDREB2A、MsP5CS1和MsLEA3的表达，且其在干扰株系中的表达量较野生型显著升高，而MsP5CS1和MsLEA3在过表达株系的表达则显著降低，而MsDREB2A的表达量在过表达株系中与野生型无显著差异。

MsCDPK33干扰株系在生长初期，生长速度较野生型明显加快。为了探究MsCDPK33对紫花苜蓿生长发育的影响，对扦插同一环境生长60 d紫花苜蓿生长形态进行比较分析。研究发现，过表达MsCDPK33转基因植株较野生型显著增加了叶片尖端角度，但主茎分枝数、节间长度、茎节数、根长、株高和叶面积则与野生型无显著性差异。而MsCDPK33干扰株系的根长、节间长度、茎节数、主茎分枝数和株高较野生型显著增加、叶片尖端角度和叶面积较野生型显著减小。为深入解析MsCDPK33的分子调控机制，我们采用核体系酵母双杂交技术对紫花苜蓿酵母cDNA文库进行筛选，获得候选互作蛋白。并进一步通过LCI、Y₂H和体外磷酸化实验，验证了MsCDPK33与MsHIPP26之间存在相互作用关系。

2. 过表达MfCML50/MfAOC2转基因紫花苜蓿分析

基于前期研究结果，我们发现MfCML50、MfAOC2、MtAOC2和MtCML50四个关键基因，通过JA信号通路、CBF途径、抗氧化防御系统以及糖代谢途径的协同作用，共同增强了苜蓿的耐寒性。本研究以 “中苜1号”叶片为外植体，通过培养基配方优化，采用SH+2,4-D（4 mg/L）+6-BA（0.5 mg/L）+蔗糖（30 g/L）+肌醇（0.1 g/L）+植物凝胶（4 g/L）为愈伤诱导培养基，MS（4.41 g/L）+6-BA（0.5 mg/L）+ KT（1 mg/L）+蔗糖（20 g/L）+琼脂（8.5 g/L）为分化培养基，MS（2.205 g/L）+蔗糖（15 g/L）+NAA（0.5 mg/L）+琼脂（8.5 g/L）为生根培养基的三阶段培养方案，显著提高了再生效率。同时，建立了基于EPSPS筛选剂的梯度筛选体系：愈伤诱导阶段（6 mg/L）、胚性愈伤形成阶段（2-4 mg/L）和植株再生阶段（1-2 mg/L）。

本研究构建了pCAMBIA3301-MfCML50/MfAOC2基因与G10evo基因共转化载体，通过分子鉴定证实了过表达MfCML50/MfAOC2基因与G10evo基因稳定整合与表达。并通过对其耐寒性、生物量和茎生长形态特征进行测定，发现MfCML50过表达株系在显著增强抗寒性和草甘膦抗性的同时，保持了其他农艺性状的稳定。MfAOC2过表达的高表达株系虽提高了抗逆性，但表现出典型的生长抑制表型。后期还需通过系统的田间试验筛选在保持优良农艺性状的同时兼具双抗特性的优势株系，并深入分析T-DNA拷贝数和插入位点特异性对转基因表达及功能的影响机制，以期获得双抗（抗寒和抗草甘膦）但其它农艺性状不变的紫花苜蓿种质资源。

Alfalfa (Medicago sativa L.), a perennial allopolyploid (2n=4x=32) leguminous forage crop, is widely cultivated worldwide due to its outstanding agronomic traits and ecological value. In the plant calcium signaling system, calmodulin-like proteins (CMLs) function as Ca²⁺-binding proteins that specifically recognize Ca²⁺ signals through their EF-hand domains, inducing conformational changes to directly interact with target proteins and regulate their activity. Meanwhile, calcium-dependent protein kinases (CDPKs) selectively phosphorylate downstream target proteins via their Ca²⁺-dependent kinase activity, thereby participating in the regulation of plant growth, development, and stress responses (Zou et al., 2010). Based on the analysis of the CDPKs gene family in Medicago sativa L. , this study identified a calcium-dependent protein kinase gene, MsCDPK33, which is significantly induced by low temperature and drought stress. We systematically elucidated its functional role in drought and cold resistance in alfalfa, providing a valuable genetic resource for stress-resistant breeding.To address the challenges of low callus induction rate and prolonged regeneration period during the genetic transformation of MsCDPK33 in the alfalfa cultivar 'Zhongmu No. 1', we optimized the culture medium formulation and successfully established a highly efficient and stable genetic transformation system.Furthermore, we extended this system to explore cold-tolerance gene resources in yellow-flowered alfalfa (Medicago falcata). By employing a co-transformation strategy with the MfCML50/MfAOC2 overexpression vector and a modified 1300-FMV-CAL-35S-G10evo vector, we successfully developed novel alfalfa germplasm exhibiting both glyphosate resistance and significantly enhanced cold tolerance. This achievement provides innovative technical approaches and foundational breeding materials for stress-resistant alfalfa improvement. The key results are as follows:

1. Functional analysis of MsCDPK33 in cold and drought tolerance

Based on previous research, this study selected MsCDPK33 for functional analysis. Tissue expression pattern analysis revealed that MsCDPK33 is expressed in roots, stems, leaves, and flowers, with the highest expression level observed in roots. Protein sequence analysis of MsCDPK33 indicated that the encoded protein contains the characteristic Ser/Thr kinase domain and four EF-hand domains typical of the CDPKs family. Subcellular localization analysis in tobacco showed that this protein is localized to the plasma membrane. However, when the palmitoylation and myristoylation sites were mutated, its localization pattern shifted from exclusive plasma membrane distribution to co-localization in both the nucleus and plasma membrane. Further studies revealed that in the RNAi lines, the expression of cold-responsive genes MsLEA3, MsCBF1, and MsLT165 was significantly induced under low-temperature stress, with their transcript levels markedly higher than in the wild-type (WT), whereas the overexpression lines showed the opposite trend. Additionally, MsCDPK33 likely functions as a negative regulator by suppressing MsP5CS1 expression, thereby limiting proline accumulation and subsequently affecting alfalfa's response to cold stress.Relative electrolyte leakage assays demonstrated that MsCDPK33 negatively regulates drought tolerance in alfalfa. Further analysis showed that drought-responsive genes MsP5CS1 and MsLEA3 were significantly induced under drought conditions, with their expression levels substantially elevated compared to the WT. Notably, the core drought-responsive transcription factor gene MsDREB2A exhibited significantly higher expression in the RNAi lines, while no difference was observed between the overexpression lines and the WT.

Phenotypic analysis revealed that MsCDPK33 plays a critical role in regulating alfalfa growth and development. MsCDPK33-knockdown lines exhibited significantly accelerated early-stage growth compared to wild-type (WT) plants. To further investigate the effects of MsCDPK33 on morphological traits, we measured plant height and root length in uniformly propagated seedlings grown under controlled conditions for 40 days.Overexpression of MsCDPK33 resulted in a significant increase in leaf tip angle, while other traits—including branch number, internode length, node number, root length, plant height, and leaf area—remained unchanged relative to WT. In contrast, MsCDPK33-knockdown lines displayed increased root length, internode length, node number, branch number, and plant height, along with significantly reduced leaf tip angle and leaf area compared to WT.To elucidate the molecular mechanism underlying MsCDPK33-mediated growth regulation, we performed a nuclear yeast two-hybrid (Y₂H) screen against an alfalfa cDNA library, identifying potential interacting partners. Subsequent validation via luciferase complementation imaging (LCI), Y₂H, and in vitro phosphorylation assays confirmed a direct interaction between MsCDPK33 and the metallochaperone MsHIPP26.

2. Analysis of MfCML50/MfAOC2 Overexpression Transgenic Alfalfa

Previous studies indicated that MfCML50, MfAOC2, MtAOC2, and MtCML50 form a complex regulatory network enhancing cold tolerance via jasmonic acid (JA) signaling, the CBF pathway, antioxidant defense, and sugar metabolism.This study optimized a three-stage tissue culture system consisting of: (1) Callus induction: SH + 2,4-D (4 mg/L) + 6-BA (0.5 mg/L) + Sucrose (30 g/L) + myo-inositol (0.1 g/L) + phytagel (4 g/L) (2) Differentiation: MS (4.41 g/L) + 6-BA (0.5 mg/L) + KT (1 mg/L) + Sucrose (20 g/L) + Agar (8.5 g/L) (3) Rooting: MS (2.205 g/L) + Sucrose (15 g/L) + NAA (0.5 mg/L) + Agar (8.5 g/L) .A gradient glyphosate selection system was established: (1) Callus induction: 6 mg/L (2) Embryogenic callus formation: 2–4 mg/L (3 )Plant regeneration:1–2 mg/L.

In this study, we developed a binary co-expression vector, pCAMBIA3301-MfCML50/MfAOC2::G10evo, through molecular cloning, and molecular characterization confirmed the stable integration and expression of both MfCML50/MfAOC2 and G10evo genes. Through comprehensive evaluation of cold tolerance, biomass, and stem growth morphology, we found that MfCML50-overexpressing lines exhibited significantly enhanced cold tolerance and glyphosate resistance while maintaining stable performance in other agronomic traits. In contrast, high-expression lines of MfAOC2 showed improved stress resistance but displayed typical growth inhibition phenotypes.Further systematic field trials are required to select elite lines that combine dual stress resistance (cold and glyphosate tolerance) with optimal agronomic performance. Additionally, in-depth analysis of T-DNA copy number and insertion site specificity is needed to elucidate their effects on transgene expression and functionality. These efforts aim to develop novel alfalfa germplasm with dual resistance while preserving other desirable agronomic characteristics.

[1] Ali Z, Merrium S, Habib-ur-Rahman M, et al. Wetting mechanism and morphological adaptation; leaf rolling enhancing atmospheric water acquisition in wheat crop-a review [J]. Environmental Science and Pollution Research. 2022, 29(21): 30967-30985.

[2] An J-P, Wang X-F, Zhang X-W, et al. Apple B-box protein BBX37 regulates jasmonic acid mediated cold tolerance through the JAZ-BBX37-ICE1-CBF pathway and undergoes MIEL1-mediated ubiquitination and degradation [J]. New Phytologist. 2021, 229(5): 2707-2729.

[3] Asano T, Hayashi N, Kobayashi M, et al. A rice calcium-dependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resistance [J]. Plant Journal. 2012, 69(1): 26-36.

[4] Barth O, Vogt S, Uhlemann R, et al. Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29 [J]. Plant Molecular Biology. 2009, 69(1-2): 213-226.

[5] Bian C, Wang H, Li W, et al. Expression Profiling of the 56 R2R3-MYB Family Genes in Response to Cold, Drought, and Salt Stress in Blue Honeysuckle (Lonicera caerulea L.) [J]. Plant Molecular Biology Reporter. 2024, 43(1): 103-120.

[6] Boudsocq M, Sheen J CDPKs in immune and stress signaling [J]. Trends in Plant Science. 2013, 18(1): 30-40.

[7] Cai G, Wang G, Wang L, et al. ZmMKK1, a novel group A mitogen-activated protein kinase kinase gene in maize, conferred chilling stress tolerance and was involved in pathogen defense in transgenic tobacco [J]. Plant Science. 2014, 214: 57-73.

[8] Chaudhuri S, Seal A, DasGupta M Autophosphorylation-dependent activation of a calcium-dependent protein kinase from groundnut [J]. Plant Physiology. 1999, 120(3): 859-866.

[9] Chen J H, Xue B, Xia X L, et al. A novel calcium-dependent protein kinase gene from Populus euphratica, confers both drought and cold stress tolerance [J]. Biochemical and Biophysical Research Communications. 2013, 441(3): 630-636.

[10] Collin A, Daszkowska-Golec A, Szarejko I Updates on the Role of Abscisic Acid Insensitive 5(ABI5) and Abscisic Acid-Responsive Element Binding Factors (ABFs) in ABA Signaling in Different Developmental Stages in Plants [J]. Cells. 2021, 10(8): 1996-2015.

[11] Cosson V, Eschstruth A, Ratet P. Medicago truncatula Transformation Using Leaf Explants [C] //K. WANG. Agrobacterium Protocols, Vol 1, 3rd Edition. 2015: 43-56.

[12] Cushman J C Crassulacean acid metabolism. A plastic photosynthetic adaptation to arid environments [J]. Plant Physiology. 2001, 127(4): 1439-1448.

[13] De Zelicourt A, Colcombet J, Hirt H The Role of MAPK Modules and ABA during Abiotic Stress Signaling [J]. Trends in Plant Science. 2016, 21(8): 677-685.

[14] DeFalco Thomas A, Bender Kyle W, Snedden Wayne A Breaking the code: Ca2+ sensors in plant signalling [J]. Biochemical Journal. 2009, 425(1): 27-40.

[15] Dhalluin K, Botterman J, Degreef W Engineering of herbicide-resistant alfalfa and evaluation under field conditions [J]. Crop Science. 1990, 30(4): 866-871.

[16] Ding F, Wang C, Xu N, et al. Jasmonic acid-regulated putrescine biosynthesis attenuates cold-induced oxidative stress in tomato plants [J]. Scientia Horticulturae. 2021, 288.

[17] Ding S-j, Zhong Q-p, Yuan T-t, et al. Effects of Endogenous Hormones on Camellia oleifera Leaves and Fruit Growth under Drought Stress [J]. Forest Research. 2016, 29(6): 933-939.

[18] Ding Y L, Yang S H Surviving and thriving: How plants perceive and respond to temperature stress [J]. Developmental Cell. 2022, 57(8): 947-958.

[19] Dong H, Wu C, Luo C G, et al. Overexpression of MdCPK1a gene, a calcium dependent protein kinase in apple, increase tobacco cold tolerance via scavenging ROS accumulation [J]. Plos One. 2020, 15(11): 1-19.

[20] Du W X, Yang J F, Li Q, et al. Genome-Wide Identification and Characterization of Growth Regulatory Factor Family Genes in Medicago [J]. International Journal of Molecular Sciences. 2022, 23(13).

[21] Duke S O, Powles S B Glyphosate: a once-in-a-century herbicide [J]. Pest Management Science. 2008, 64(4): 319-325.

[22] Dykema P E, Sipes P R, Marie A, et al. A new class of proteins capable of binding transition metals [J]. Plant Molecular Biology. 1999, 41(1): 139-150.

[23] Echeverria A, Larrainzar E, Li W, et al. Medicago sativa and Medicago truncatula Show Contrasting Root Metabolic Responses to Drought [J]. Frontiers in Plant Science. 2021, 12.

[24] Edwards R, Hannah M Focus on Weed Control [J]. Plant Physiology. 2014, 166(3): 1087-1089.

[25] Fan W, Zhang S, Du H, et al. Genome-Wide Identification of Different Dormant Medicago sativa L. MicroRNAs in Response to Fall Dormancy [J]. Plos One. 2014, 9(12).

[26] Fang L, Liu T, Li M, et al. MODMS: a multi-omics database for facilitating biological studies on alfalfa (Medicago sativa L.) [J]. Horticulture Research. 2024, 11(1): uhad245.

[27] Feng Y, Wang J, Luo S, et al. Costs of Jasmonic Acid Induced Defense in Aboveground and Belowground Parts of Corn (Zea mays L.) [J]. Journal of Chemical Ecology. 2012, 38(8): 984-991.

[28] Franz S, Ehlert B, Liese A, et al. Calcium-Dependent Protein Kinase CPK21 Functions in Abiotic Stress Response in Arabidopsis thaliana [J]. Molecular Plant. 2011, 4(1): 83-96.

[29] Frattini M, Morello L, Breviario D Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development [J]. Plant Molecular Biology. 1999, 41(6): 753-764.

[30] Fu C, Hernandez T, Zhou C, et al. Alfalfa (Medicago sativa L.) [C] //K. WANG. Agrobacterium Protocols. 2015: 213-221.

[31] Gao X, Chen X, Lin W, et al. Bifurcation of Arabidopsis NLR Immune Signaling via Ca2+-Dependent Protein Kinases [J]. Plos Pathogens. 2013, 9(1): e1003127.

[32] Garcia B J, Labbe J L, Jones P, et al. Phytobiome and Transcriptional Adaptation of Populus deltoides to Acute Progressive Drought and Cyclic Drought [J]. Phytobiomes Journal. 2018, 2(4): 249-260.

[33] Geiger D, Scherzer S, Mumm P, et al. Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities [J]. Proceedings of the National Academy of Sciences. 2010, 107(17): 8023-8028.

[34] Gong X, Zhang J, Hu J, et al. FcWRKY70, a WRKY protein of ortunella crassifolia, functions in drought tolerance and modulates putrescine synthesis by regulating arginine decarboxylase gene [J]. Plant Cell and Environment. 2015, 38(11): 2248-2262.

[35] Han B, Dong X, Shi C, et al. Genome-wide identification and characterization of Calcium-Dependent Protein Kinase (CDPK) gene family in autotetraploid cultivated alfalfa (Medicago sativa subsp. sativa) and expression analysis under abiotic stresses [J]. BMC Plant Biol. 2024, 24(1): 1241.

[36] Harmon A C, Putnamevans C, Cormier M J A calcium -dependent but calmodulin -independent protein-kinase from soybean [J]. Plant Physiology. 1987, 83(4): 830-837.

[37] Heuzé V, Tran G, Boval M, et al. Alfalfa (Medicago sativa). Feedipedia, a programme by INRAE, CIRAD, AFZ and FAO [J]. 2016.

[38] Hill A E, Shachar-Hill Y Are Aquaporins the Missing Transmembrane Osmosensors? [J]. Journal of Membrane Biology. 2015, 248(4): 753-765.

[39] Hu X, Liu R, Li Y, et al. Heat shock protein 70 regulates the abscisic acid-induced antioxidant response of maize to combined drought and heat stress [J]. Plant Growth Regulation. 2010, 60(3): 225-235.

[40] Hu Y, Jiang Y, Han X, et al. Jasmonate regulates leaf senescence and tolerance to cold stress: crosstalk with other phytohormones [J]. Journal of Experimental Botany. 2017, 68(6): 1361-1369.

[41] Huang Y, Jiao Y, Xie N, et al. OsNCED5, a 9-cis-epoxycarotenoid dioxygenase gene, regulates salt and water stress tolerance and leaf senescence in rice [J]. Plant Science. 2019, 287: 109455.

[42] Ivashuta S, Liu J Y, Liu J Q, et al. RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development [J]. Plant Cell. 2005, 17(11): 2911-2921.

[43] Jain M, Pathak B P, Harmon A C, et al. Calcium dependent protein kinase (CDPK) expression during fruit development in cultivated peanut (Arachis hypogaea) under Ca2+-sufficient and -deficient growth regimens [J]. Journal of Plant Physiology. 2011, 168(18): 2272-2277.

[44] Jan S, Abbas N, Ashraf M, et al. Roles of potential plant hormones and transcription factors in controlling leaf senescence and drought tolerance [J]. Protoplasma. 2019, 256(2): 313-329.

[45] Jensen N B, Ottosen C-O, Fomsgaard I S, et al. Elevated CO2 induce alterations in the hormonal regulation of stomata in drought stressed tomato seedlings [J]. Plant Physiology and Biochemistry. 2024, 212:108762-108774.

[46] Jiang C J, Liu X L, Liu X Q, et al. Stunted Growth Caused by Blast Disease in Rice Seedlings Is Associated with Changes in Phytohormone Signaling Pathways [J]. Frontiers in Plant Science. 2017, 8.

[47] Jiang F, Chang G, Li Z, et al. The HSP/co-chaperone network in environmental cold adaptation of Chilo suppressalis [J]. International Journal of Biological Macromolecules. 2021, 187:780-788.

[48] Jiang S, Zhang D, Wang L, et al. A maize calcium-dependent protein kinase gene, ZmCPK4, positively regulated abscisic acid signaling and enhanced drought stress tolerance in transgenic Arabidopsis [J]. Plant Physiology and Biochemistry. 2013, 71: 112-120.

[49] Kahrizi D, Salmanian A H, Afshari A, et al. Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of E-coli (k12) and transformation of rapeseed (Brassica napus L.) in order to make tolerance to glyphosate [J]. Plant Cell Reports. 2007, 26(1): 95-104.

[50] Kerbler S M, Taylor N L, Millar A H Cold sensitivity of mitochondrial ATP synthase restricts oxidative phosphorylation in Arabidopsis thaliana [J]. New Phytologist. 2019, 221(4): 1776-1788.

[51] Kim C-G, Koh M-H, Seo J-H, et al. Effects of drought stress at different growth stages on the growth and yield of peanut (Arachis hypogaea L.) [J]. RDA Journal of Agricultural Science Upland and Industrial Crops. 1996, 38(2): 143-148.

[52] Kim S H, Kim H S, Bahk S, et al. Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in Arabidopsis [J]. Nucleic Acids Research. 2017, 45(11): 6613-6627.

[53] Kim T-W, Guan S, Sun Y, et al. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors [J]. Nature Cell Biology. 2009, 11(10): 1254-1260.

[54] Kobayashi M, Ohura I, Kawakita K, et al. Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase [J]. Plant Cell. 2007, 19(3): 1065-1080.

[55] Kong X, Lv W, Jiang S, et al. Genome-wide identification and expression analysis of calcium-dependent protein kinase in maize [J]. Bmc Genomics. 2013, 14(1): 433.

[56] Kurepin L V, Dahal K P, Savitch L V, et al. Role of CBFs as Integrators of Chloroplast Redox, Phytochrome and Plant Hormone Signaling during Cold Acclimation [J]. International Journal of Molecular Sciences. 2013, 14(6): 12729-12763.

[57] Lee S, Park C-M Regulation of reactive oxygen species generation under drought conditions in arabidopsis [J]. Plant Signaling & Behavior. 2012, 7(6): 599-601.

[58] Lee S B, Kim H U, Suh M C MYB94 and MYB96 Additively Activate Cuticular Wax Biosynthesis in Arabidopsis [J]. Plant and Cell Physiology. 2016, 57(11): 2300-2311.

[59] Lee S S, Cho H S, Yoon G M, et al. Interaction of NtCDPK1 calcium-dependent protein kinase with NtRpn3 regulatory subunit of the 26S proteasome in Nicotiana tabacum [J]. Plant Journal. 2003, 33(5): 825-840.

[60] Li Q, Jiang W B, Jiang Z H, et al. Transcriptome and functional analyses reveal ERF053 from Medicago falcata as key regulator in drought resistances [J]. Frontiers in Plant Science. 2022, 13.

[61] Li X, Liu C, Zhao Z, et al. COR27 and COR28 Are Novel Regulators of the COP1–HY5 Regulatory Hub and Photomorphogenesis in Arabidopsis [J]. Plant Cell. 2020, 32(10): 3139-3154.

[62] Liang C, Sun B, Meng Z, et al. Co-expression of GR79 EPSPS and GAT yields herbicide-resistant cotton with low glyphosate residues [J]. Plant Biotechnology Journal. 2017, 15(12): 1622-1629.

[63] Liese A, Romeis T Biochemical regulation of in vivo function of plant calcium-dependent protein kinases (CDPK) [J]. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2013, 1833(7): 1582-1589.

[64] Lin L, Wu J, Jiang M, et al. Plant Mitogen-Activated Protein Kinase Cascades in Environmental Stresses [J]. Int J Mol Sci. 2021, 22(4).

[65] Lin Z F, Peng C L, Lin G Z Photooxidation in leaves of facultative CAM plant Sedum spectabile at C3 and CAM mode [J]. Acta Botanica Sinica. 2003, 45(3): 301-306.

[66] Linghu B, Xu Z L, Chu Y Q, et al. Genome-wide analysis of calcium-dependent protein kinase (CDPK) family and functional characterization of TaCDPK25-U in response to drought stress in wheat [J]. Environmental and Experimental Botany. 2023, 209:105277.

[67] Liou Y C, Daley M E, Graham L A, et al. Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria [J]. Protein Expr Purif. 2000, 19(1): 148-157.

[68] Liu J, Li F, Liang L, et al. Fibroin Delays Chilling Injury of Postharvest Banana Fruit via Enhanced Antioxidant Capability during Cold Storage [J]. Metabolites. 2019, 9(7): 52-69.

[69] Liu N, Shen Y, Huang B Osmoregulants Involved in Osmotic Adjustment for Differential Drought Tolerance in Different Bentgrass Genotypes [J]. Journal of the American Society for Horticultural Science. 2015, 140(6): 605-613.

[70] Lu S X, Hrabak E M The myristoylated amino-terminus of an Arabidopsis calcium-dependent protein kinase mediates plasma membrane localization [J]. Plant Molecular Biology. 2013, 82(3): 267-278.

[71] Lv X, Li Y, Chen R, et al. Stomatal Responses of Two Drought-Tolerant Barley Varieties with Different ROS Regulation Strategies under Drought Conditions [J]. Antioxidants. 2023, 12(4).

[72] Ma F-j, Li D-d, Cai J, et al. Responses of wheat seedlings root growth and leaf photosynthesis to drought stress [J]. Yingyong Shengtai Xuebao. 2012, 23(3): 724-730.

[73] Ma S-Y, Wu W-H AtCPK23 functions in Arabidopsis responses to drought and salt stresses [J]. Plant Molecular Biology. 2007, 65(4): 511-518.

[74] Maidment J H R, Franceschetti M, Maqbool A, et al. Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defense [J]. Journal of Biological Chemistry. 2021.

[75] Manimaran P, Mangrauthia S K, Sundaram R M, et al. Constitutive expression and silencing of a novel seed specific calcium dependent protein kinase gene in rice reveals its role in grain filling [J]. Journal of Plant Physiology. 2015, 174:41-48.

[76] Martens J R T, Entz M H, Hoeppner J W Legume cover crops with winter cereals in southern Manitoba: Fertilizer replacement values for oat [J]. Canadian Journal of Plant Science. 2005, 85(3): 645-648.

[77] Matthews C, Arshad M, Hannoufa A Alfalfa response to heat stress is modulated by microRNA156 [J]. Physiologia Plantarum. 2019, 165(4): 830-842.

[78] Medina C A, Hansen J, Crawford J, et al. Genome-Wide Association and Genomic Prediction of Alfalfa (Medicago sativa L.) Biomass Yield Under Drought Stress [J]. International Journal of Molecular Sciences. 2025, 26(2).

[79] Meng Y, Zhang W, Wang Z, et al. Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues [J]. aBIOTECH. 2023, 4(4): 352-358.

[80] Miller G, Suzuki N, Ciftci-Yilmaz S, et al. Reactive oxygen species homeostasis and signalling during drought and salinity stresses [J]. Plant Cell and Environment. 2010, 33(4): 453-467.

[81] Molla M R, Ali M R, Hasanuzzaman M, et al. Exogenous Proline and Betaine-induced Upregulation of Glutathione Transferase and Glyoxalase I in Lentil (Lens culinaris) under Drought Stress [J]. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2014, 42(1): 73-80.

[82] Nadeem M, Thomas R, Adigun O, et al. Root membrane lipids as potential biomarkers to discriminate silage-corn genotypes cultivated on podzolic soils in boreal climate [J]. Physiologia Plantarum. 2020, 170(3): 440-450.

[83] Naghizadeh M, Reiter R J, Kabiri R, et al. Melatonin improves antioxidant defense mechanism of basil under drought stress [J]. Horticulture Environment and Biotechnology. 2024, 65(1): 83-94.

[84] Oikawa K, Fujisaki K, Shimizu M, et al. The blast pathogen effector AVR-Pik binds and stabilizes rice heavy metal-associated (HMA) proteins to co-opt their function in immunity [J]. Plos Pathogens. 2024, 20(11).

[85] Olvera C Y, Campos F, Reyes J L, et al. Functional Analysis of the Group 4 Late Embryogenesis Abundant Proteins Reveals Their Relevance in the Adaptive Response during Water Deficit in Arabidopsis [J]. Plant Physiology. 2010, 154(1): 373-390.

[86] Oukarroum A, El Madidi S, Strasser R J Exogenous glycine betaine and proline play a protective role in heat-stressed barley leaves (Hordeum vulgare L.): A chlorophyll a fluorescence study [J]. Plant Biosystems. 2012, 146(4): 1037-1043.

[87] Park B J, Liu Z C, Kanno A, et al. Genetic improvement of Chinese cabbage for salt and drought tolerance by constitutive expression of a B. napus LEA gene [J]. Plant Science. 2005, 169(3): 553-558.

[88] Peng C, Mei Y, Ding L, et al. Using Combined Methods of Genetic Mapping and Nanopore-Based Sequencing Technology to Analyze the Insertion Positions of G10evo-EPSPS and Cry1Ab Cry2Aj Transgenes in Maize [J]. Frontiers in Plant Science. 2021, 12.

[89] Peng X, Teng L, Yan X, et al. The cold responsive mechanism of the paper mulberry: decreased photosynthesis capacity and increased starch accumulation [J]. Bmc Genomics. 2015, 16.

[90] Pennycooke J C, Cheng H, Stockinger E J Comparative genomic sequence and expression analyses of Medicago truncatula and alfalfa subspecies falcata Cold-Acclimation-Specific genes [J]. Plant Physiology. 2008, 146(3): 1242-1254.

[91] Pereira L F, Erickson L Stable Transformation of Alfalfa(Medicago Sative L.) by Particle Bombardment [J]. Plant Cell Reports. 1995, 14(5): 290-293.

[92] Powles S B Evolved glyphosate-resistant weeds around the world: lessons to be learnt [J]. Pest Management Science. 2008, 64(4): 360-365.

[93] Ren Z-j, Cao G-y, Zhang Y-w, et al. Overexpression of a modified AM79 aroA gene in transgenic maize confers high tolerance to glyphosate [J]. Journal of Integrative Agriculture. 2015, 14(3): 414-422.

[94] Romeis T, Ludwig A A, Martin R, et al. Calcium‐dependent protein kinases play an essential role in a plant defence response [J]. The EMBO Journal. 2001, 20(20): 5556-5567.

[95] Romeis T, Piedras P, Jones J D G Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response [J]. Plant Cell. 2000, 12(5): 803-815.

[96] Rushton P J, Somssich I E, Ringler P, et al. WRKY transcription factors [J]. Trends in Plant Science. 2010, 15(5): 247-258.

[97] Rutschmann F, Stalder U, Piotrowski M, et al. LeCPK1, a calcium-dependent protein kinase from tomato.: Plasma membrane targeting and biochemical characterization [J]. Plant Physiology. 2002, 129(1): 156-168.

[98] Saha G, Park J-I, Ahmed N U, et al. Characterization and expression profiling of MYB transcription factors against stresses and during male organ development in Chinese cabbage (Brassica rapa ssp pekinensis) [J]. Plant Physiology and Biochemistry. 2016, 104:200-215.

[99] Saijo Y, Hata S, Kyozuka J, et al. Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants [J]. Plant Journal. 2000, 23(3): 319-327.

[100] Saito S, Hamamoto S, Moriya K, et al. N-myristoylation and S-acylation are common modifications of Ca2+-regulated Arabidopsis kinases and are required for activation of the SLAC1 anion channel [J]. New Phytologist. 2018, 218(4): 1504-1521.

[101] Sakamoto T, Kimura S Plant Temperature Sensors [J]. Sensors. 2018, 18(12): 4365-4376.

[102] Santin F, Bhogale S, Fantino E, et al. Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development [J]. Physiologia Plantarum. 2017, 159(2): 244-261.

[103] Schulz P, Herde M, Romeis T Calcium-Dependent Protein Kinases: Hubs in Plant Stress Signaling and Development [J]. Plant Physiology. 2013, 163(2): 523-530.

[104] Shao W, Zhang X, Zhou Z, et al. Genome- and transcriptome-wide identification of trehalose-6-phosphate phosphatases (TPP) gene family and their expression patterns under abiotic stress and exogenous trehalose in soybean [J]. BMC Plant Biol. 2023, 23(1): 641-659.

[105] Shen C, Du H, Chen Z, et al. The Chromosome-Level Genome Sequence of the Autotetraploid Alfalfa and Resequencing of Core Germplasms Provide Genomic Resources for Alfalfa Research [J]. Molecular Plant. 2020, 13(9): 1250-1261.

[106] Shen Y, Li L, Du P, et al. Appropriate Drought Training Induces Optimal Drought Tolerance by Inducing Stepwise H2O2Homeostasis in Soybean [J]. Plants-Basel. 2024, 13(9).

[107] Shi Y H, Huang J Y, Sun T S, et al. The precise regulation of different COR genes by individual CBF transcription factors in Arabidopsis thaliana [J]. Journal of Integrative Plant Biology. 2017, 59(2): 118-133.

[108] Siddiqui M H, Khan M N, Mukherjee S, et al. Hydrogen sulfide (H2S) and potassium (K+) synergistically induce drought stress tolerance through regulation of H+-ATPase activity, sugar metabolism, and antioxidative defense in tomato seedlings [J]. Plant Cell Reports. 2021, 40(8): 1543-1564.

[109] Solomon W, Mutum L, Janda T, et al. Potential benefit of microalgae and their interaction with bacteria to sustainable crop production [J]. Plant Growth Regulation. 2023, 101(1): 53-65.

[110] Stout D G, Hall J W Fall Growth and Winter Survival of Alfalfa in Interior British-Columbia [J]. Canadian Journal of Plant Science. 1989, 69(2): 491-499.

[111] Taïbi K, Del Campo A D, Vilagrosa A, et al. Distinctive physiological and molecular responses to cold stress among cold-tolerant and cold-sensitive Pinus halepensis seed sources [J]. BMC Plant Biology. 2018, 18(1): 236-147.

[112] Tang M, Liu L, Hu X, et al. Genome-wide characterization of R2R3-MYB gene family in Santalum album and their expression analysis under cold stress [J]. Frontiers in Plant Science. 2023, 14:1142562.

[113] Uno Y, Furihata T, Abe H, et al. Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions [J]. Proceedings of the National Academy of Sciences of the United States of America. 2000, 97(21): 11632-11637.

[114] Verma D P S, Hu C A A, Delauney A J. Genetic manipulation for proline overproduction and the control of osmoregulation in plants [C] //International Symposium on Adaptation of Food Crops to Temperature and Water Stress. Taipei, Taiwan, 1992:47-58.

[115] Wang X, Li Q, Xie J J, et al. Abscisic acid and jasmonic acid are involved in drought priming-induced tolerance to drought in wheat [J]. Crop Journal. 2021, 9(1): 120-132.

[116] Wang Y, Dong B, Wang N, et al. A WRKY Transcription Factor PmWRKY57 from Prunus mume Improves Cold Tolerance in Arabidopsis thaliana [J]. Molecular Biotechnology. 2023, 65(8): 1359-1368.

[117] Wang Y J, Zhang Z G, He X J, et al. A rice transcription factor OsbHLH1 is involved in cold stress response [J]. Theoretical and Applied Genetics. 2003, 107(8): 1402-1409.

[118] Wang Z, Zhu Y, Wang L, et al. A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter [J]. Planta. 2009, 230(6): 1155-1166.

[119] Wei W, Liang D-W, Bian X-H, et al. GmWRKY54 improves drought tolerance through activating genes in abscisic acid and Ca2+ signaling pathways in transgenic soybean [J]. The Plant Journal. 2019, 100(2): 384-398.

[120] Wei Z, Fang L, Li X, et al. Effects of elevated atmospheric CO2 on leaf gas exchange response to progressive drought in barley and tomato plants with different endogenous ABA levels [J]. Plant and Soil. 2020, 447(1): 431-446.

[121] Wen W, Wang R, Su L, et al. MsWRKY11, activated by MsWRKY22, functions in drought tolerance and modulates lignin biosynthesis in alfalfa (Medicago sativa L.) [J]. Environmental and Experimental Botany. 2021, 184:104373.

[122] Witte C-P, Keinath N, Dubiella U, et al. Tobacco Calcium-dependent Protein Kinases Are Differentially Phosphorylated in Vivo as Part of a Kinase Cascade That Regulates Stress Response [J]. Journal of Biological Chemistry. 2010, 285(13): 9740-9748.

[123] Wu J, Nadeem M, Galagedara L, et al. Recent insights into cell responses to cold stress in plants: Signaling, defence, and potential functions of phosphatidic acid [J]. Environmental and Experimental Botany. 2022, 203:105068.

[124] Xiang J, Chen X, Hu W, et al. Overexpressing heat-shock protein OsHSP50.2 improves drought tolerance in rice [J]. Plant Cell Reports. 2018, 37(11): 1585-1595.

[125] Xiao P-y, Liu Y, Cao Y-p Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance [J]. Journal of Integrative Agriculture. 2019, 18(8): 1851-1858.

[126] Xu M Z, Xu Z P, Liu Y R, et al. Transcriptome Analysis Reveals the Vital Role of ABA Plays in Drought Tolerance of the ABA-Insensitive Alfalfa (Medicago sativa L.) [J]. Agronomy-Basel. 2024, 14(3): 406-420.

[127] Xu Z, Ma J, Lei P, et al. Poly-γ-glutamic acid induces system tolerance to drought stress by promoting abscisic acid accumulation in Brassica napus L [J]. Scientific Reports. 2020, 10(1): 252.

[128] Yamori W Photosynthetic response to fluctuating environments and photoprotective strategies under abiotic stress [J]. Journal of Plant Research. 2016, 129(3): 379-395.

[129] Yang G X, Shen S H, Yang S H, et al. OsCDPK13, a calcium-dependent protein kinase gene from rice, is induced in response to cold and gibberellin [J]. Plant Physiology and Biochemistry. 2003, 41(4): 369-374.

[130] Yang L, Sun Q, Geng B, et al. Jasmonate biosynthesis enzyme allene oxide cyclase 2 mediates cold tolerance and pathogen resistance [J]. Plant Physiol. 2023a, 193(2): 1621-1634.

[131] Yang L, Sun Q G, Geng B H, et al. Jasmonate biosynthesis enzyme allene oxide cyclase 2 mediates cold tolerance and pathogen resistance [J]. Plant Physiology. 2023b, 193(2): 1621-1634.

[132] Yang Q, Chen Z Z, Zhou X F, et al. Overexpression of SOS (Salt Overly Sensitive) Genes Increases Salt Tolerance in Transgenic Arabidopsis [J]. Molecular Plant. 2009, 2(1): 22-31.

[133] Yang T, Shad Ali G, Yang L, et al. Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants [J]. Plant Signal Behav. 2010, 5(8): 991-994.

[134] Yao C, Li W, Liang X, et al. Molecular Cloning and Characterization of MbMYB108, a Malus baccata MYB Transcription Factor Gene, with Functions in Tolerance to Cold and Drought Stress in Transgenic Arabidopsis thaliana [J]. International Journal of Molecular Sciences. 2022a, 23(9): 4846.

[135] Yao C, Li X, Li Y, et al. Overexpression of a Malus baccata MYB Transcription Factor Gene MbMYB4 Increases Cold and Drought Tolerance in Arabidopsis thaliana [J]. International Journal of Molecular Sciences. 2022b, 23(3): 1794.

[136] Ye Q, Meng X, Chen H, et al. Construction of genic male sterility system by CRISPR/Cas9 editing from model legume to alfalfa [J]. Plant Biotechnology Journal. 2022, 20(4): 613-615.

[137] Yi D, Ma L, Lin M, et al. Development of glyphosate-resistant alfalfa (Medicago sativa L.) upon transformation with the GR79Ms gene encoding 5-enolpyruvylshikimate-3-phosphate synthase [J]. Planta. 2018, 248(1): 211-219.

[138] Yin H, Wang Z Y, Li H, et al. MsTHI1 overexpression improves drought tolerance in transgenic alfalfa (Medicago sativa L.) [J]. Frontiers in Plant Science. 2022, 13.

[139] Yu R, Hou Q, Deng H, et al. Molecular identification and expression patterns of sweet cherry HIPPs and functional analysis of PavHIPP16 in cold stress [J]. Planta. 2024, 260(6): 134.

[140] Yu S H, Wu J X, Sun Y M, et al. A calmodulin-like protein (CML10) interacts with cytosolic enzymes GSTU8 and FBA6 to regulate cold tolerance [J]. Plant Physiology. 2022, 190(2): 1321-1333.

[141] Yuan F, Yang H, Xue Y, et al. OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis [J]. Nature. 2014, 514(7522): 367-371.

[142] Zargar S M, Nagar P, Deshmukh R, et al. Aquaporins as potential drought tolerance inducing proteins: Towards instigating stress tolerance [J]. Journal of Proteomics. 2017, 169:233-238.

[143] Zhang C M, Shi S L, Liu Z, et al. Drought tolerance in alfalfa (Medicago sativa L.) varieties is associated with enhanced antioxidative protection and declined lipid peroxidation [J]. Journal of Plant Physiology. 2019a, 232:226-240.

[144] Zhang H, Liu D, Yang B, et al. Arabidopsis CPK6 positively regulates ABA signaling and drought tolerance through phosphorylating ABA-responsive element-binding factors [J]. Journal of Experimental Botany. 2019b, 71(1): 188-203.

[145] Zhang H, Zhang Y, Deng C, et al. The Arabidopsis Ca2+-Dependent Protein Kinase CPK12 Is Involved in Plant Response to Salt Stress [J]. International Journal of Molecular Sciences. 2018, 19(12): 4062-4079.

[146] Zhang J Y, Broeckling C D, Blancaflor E B, et al. Overexpression of WXP1, a putative Medicago truncatula AP2 domain‐containing transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa (Medicago sativa L.) [J]. The Plant Journal. 2005, 42(5): 689-707.

[147] Zhang L, Song J, Lin R, et al. Tomato SlMYB15 transcription factor targeted by sly-miR156e-3p positively regulates ABA-mediated cold tolerance [J]. Journal of Experimental Botany. 2022, 73(22): 7538-7551.

[148] Zhang L, Zhao L, Wang L, et al. TabZIP60 is involved in the regulation of ABA synthesis-mediated salt tolerance through interacting with TaCDPK30 in wheat (Triticum aestivum L.) [J]. Planta. 2023, 257(6): 107-120.

[149] Zhang W J, Wang T Enhanced salt tolerance of alfalfa (Medicago sativa L.) by rstB gene transformation [J]. Plant Science. 2015a, 234:110-118.

[150] Zhang X, Feng H, Feng C, et al. Isolation and characterisation of cDNA encoding a wheat heavy metal-associated isoprenylated protein involved in stress responses [J]. Plant Biology. 2015b, 17(6): 1176-1186.

[151] Zhang Y M, Wang H Q, Liu D M, et al. Three tandemly aligned LEA genes from Medicago truncatula confer differential protection to Escherichia coli against abiotic stresses [J]. Biologia Plantarum. 2020, 64:95-103.

[152] Zhang Z, Li J, Li F, et al. OsMAPK3 Phosphorylates OsbHLH002/OsICE1 and Inhibits Its Ubiquitination to Activate OsTPP1 and Enhances Rice Chilling Tolerance [J]. Developmental Cell. 2017, 43(6): 731-743.

[153] Zhang Z H, Cao B, Gao S, et al. Grafting improves tomato drought tolerance through enhancing photosynthetic capacity and reducing ROS accumulation [J]. Protoplasma. 2019c, 256(4): 1013-1024.

[154] Zhao C, Zhang Z, Xie S, et al. Mutational Evidence for the Critical Role of CBF Transcription Factors in Cold Acclimation in Arabidopsis [J]. Plant Physiology. 2016, 171(4): 2744-2759.

[155] Zhao J, Zhang W, Zhao Y, et al. SAD2, an importin β-like protein, is required for UV-B response in Arabidopsis by mediating MYB4 nuclear trafficking [J]. Plant Cell. 2007, 19(11): 3805-3818.

[156] Zhao P C, Liu Y J, Kong W Y, et al. Genome-Wide Identification and Characterization of Calcium-Dependent Protein Kinase (CDPK) and CDPK-Related Kinase (CRK) Gene Families in Medicago truncatula [J]. International Journal of Molecular Sciences. 2021, 22(3): 1044-1060.

[157] Zhao T, Lin C-y, Shen Z-c Development of Transgenic Glyphosate-Resistant Rice with G6 Gene Encoding 5-Enolpyruvylshikimate-3-Phosphate Synthase [J]. Agricultural Sciences in China. 2011, 10(9): 1307-1312.

[158] Zhu Q, Maher E A, Masoud S, et al. Enhanced protection against fungal attack by constitutive coexpression of chitinase and glucanase genes in transgenic tobacco [J]. Bio-Technology. 1994, 12(8): 807-812.

[159] Zinn K E, Tunc-Ozdemir M, Harper J F Temperature stress and plant sexual reproduction: uncovering the weakest links [J]. Journal of Experimental Botany. 2010, 61(7): 1959-1968.

[160] Zou J J, Wei F J, Wang C, et al. Arabidopsis Calcium-Dependent Protein Kinase CPK10 Functions in Abscisic Acid- and Ca2+-Mediated Stomatal Regulation in Response to Drought Stress [J]. Plant Physiology. 2010, 154(3): 1232-1243.

[161] Zschiesche W, Barth O, Daniel K, et al. The zinc-binding nuclear protein HIPP3 acts as an upstream regulator of the salicylate-dependent plant immunity pathway and of flowering time in Arabidopsis thaliana [J]. New Phytologist. 2015, 207(4): 1084-1096.

[162] Zuo Z-F, Sun H-J, Lee H-Y, et al. Identification of bHLH genes through genome-wide association study and antisense expression of ZjbHLH076/ZjICE1 influence tolerance to low temperature and salinity in Zoysia japonica [J]. Plant Science. 2021, 313:111088.

[163] 崔国文, 马春平 紫花苜蓿叶片形态结构及其与抗寒性的关系 [J]. 草地学报. 2007, 15(01): 70-75.

[164] 范云六, 张春义 21世纪农作物生物工程的发展与展望 [J]. 中国工程科学. 2000, 2(01): 30-35.

[165] 梁慧敏, 黄剑, 夏阳, 等. 苜蓿外植体再生系统的建立研究 [J]. 中国草地. 2003, 25(04): 9-15.

[166] 亓春宇, 刘凤歧, 刘杰淋, 等. 低温胁迫下紫花苜蓿杂交代抗氧化酶及可溶性蛋白的动态聚类分析 [J]. 中国草地学报. 2017, 39(02): 53-70.

[167] 王强龙, 王锁民, 张金林, 等. 紫花苜蓿体细胞胚高频再生体系的建立 [J]. 草业科学. 2006, 23(11): 21-27.

[168] 张立全, 牛一丁, 郝金凤, 等. 通过花粉管通道法导入红树总DNA获得耐盐紫花苜蓿T0代植株及其RAPD验证 [J]. 草业学报. 2011, 20(03): 292-297.