The progress of genetic improvement in alfalfa (Medicago sativa L.) T., Bao A., Bao Z., Wang F., Gao L., Wang S. (2018): The progress of genetic improvement in alfalfa (Medicago sativa L.). Czech J. Genet. Plant Breed., 54: 41-51.
download PDF

Alfalfa (Medicago sativa L.) is a perennial and outcrossing species, widely grown as a forage legume for hay, pasture and silage. The genetic engineering approaches involve the transfer of useful or novel gene(s) into alfalfa to improve desired traits. The recent development of genetic engineering is extensively applied to basic and applied research for alfalfa improvement, including improvement of herbicide resistance, reinforcement of the resistance to biotic and abiotic stresses, production of novel compounds, improved yield for industrial and/or pharmaceutical proteins and renewable energy sources. On the other hand, alfalfa forage needs to possess additional fermentable carbohydrates, proteins with a balanced amino acid profile that are gradually degraded in the rumen of domestic livestock, and zero anti-nutritional factors, which are the major concerns of recent interest in alfalfa. However, an advance of transgenic approach is contentious, requiring vigilant experimental methods and design to contest bio-safety challenges. More importantly, the technology of clustered regularly interspaced short palindromic repeats (CRISPR) is rapidly growing and might be a game player or changer in alfalfa. The present review can enable us to identify the proper direction, get familiar with new research methods and success of genetic engineering technology in alfalfa, to produce maximally improved cultivars.

Aboagye I.A., Lynch J.P., Church J.S., Baah J., Beauchemin K.A. (2015): Digestibility and growth performance of sheep fed alfalfa hay treated with fibrolytic enzymes and a ferulic acid esterase producing bacterial additive. Animal Feed Science and Technology, 203, 53-66
Agrawal R., Singh N. R., Ribeiro F. H., Delgass W. N. (2007): Sustainable fuel for the transportation sector. Proceedings of the National Academy of Sciences, 104, 4828-4833
Austin-Phillips S., Ziegelhoffer T. (2001): The Production of Value-added Proteins in Transgenic Alfalfa. Molecular Breeding of Forage Crops. Dordrecht, Kluwer: 285–301.
Avraham Tal, Badani Hanna, Galili Shmuel, Amir Rachel (2005): Enhanced levels of methionine and cysteine in transgenic alfalfa (Medicago sativa L.) plants over-expressing the Arabidopsis cystathionine γ-synthase gene. Plant Biotechnology Journal, 3, 71-79
Bagga S., Adams H.P., Rodriguez F.D., Kemp J.D., Sengupta-Gopalan C. (2004): Coexpression of the maize delta-zein and beta-zein genes results in stable accumulation of delta-zein in endoplasmic reticulum-derived protein bodies formed by beta-zein. Plant Cell, 99: 1683–1696.
Bao Ai-Ke, Wang Suo-Min, Wu Guo-Qiang, Xi Jie-Jun, Zhang Jin-Lin, Wang Chun-Mei (2009): Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.). Plant Science, 176, 232-240
Bao Ai-Ke, Du Bao-Qiang, Touil Leila, Kang Peng, Wang Qiang-Long, Wang Suo-Min (2016): Co-expression of tonoplast Cation/H + antiporter and H + -pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity, drought and field conditions. Plant Biotechnology Journal, 14, 964-975
Barry T.N., McNabb W.C. (1999): The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants. British Journal of Nutrition, 814: 263–272.
Basak Jolly, Nithin Chandran (2015): Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting. Frontiers in Plant Science, 6, -
Baucher M., Bernard-Vailhe M.A., Chabbert B., Besle J.M., Opsomer C., Van Montagu M., Botterman J. (1999): Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility. Plant Molecular Biology, 393: 437–447.
Belhaj Khaoula, Chaparro-Garcia Angela, Kamoun Sophien, Nekrasov Vladimir (2013): Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods, 9, 39-
Brito A.F., Broderick G.A. (2006): Effect of Varying Dietary Ratios of Alfalfa Silage to Corn Silage on Production and Nitrogen Utilization in Lactating Dairy Cows. Journal of Dairy Science, 89, 3924-3938
Brummer E. Charles (2004): Applying Genomics to Alfalfa Breeding Programs. Crop Science, 44, 1904-
Buxton D.R., Redfearn D.D. (1997): Plant limitations to fiber digestion and utilization. The Journal of Nutrition, 127 (5 Suppl): 814S–818S.
Calderini Ornella, Bovone Tessa, Scotti Carla, Pupilli Fulvio, Piano Efisio, Arcioni Sergio (2007): Delay of leaf senescence in Medicago sativa transformed with the ipt gene controlled by the senescence-specific promoter SAG12. Plant Cell Reports, 26, 611-615
Castroluna A., Ruiz O.M., Quiroga A.M. (2014): Effects of salinity and drought stress on germination, biomass and growth in three varieties of Medicago sativa L. Avances en Investigación Agropecuaria, 18: 39–50.
Chandra Amaresh (2009): Screening global Medicago germplasm for weevil (Hypera postica Gyll.) tolerance and estimation of genetic variability using molecular markers. Euphytica, 169, 363-374
Cheeke P.R. (1996): Biological effects of feed and forage saponins and their impacts on animal production. Advances in Experimental Medical Biology, 405: 377–385.
Chen Fang, Dixon Richard A (2007): Lignin modification improves fermentable sugar yields for biofuel production. Nature Biotechnology, 25, 759-761
Chen Fang, Srinivasa Reddy Marry S., Temple Stephen, Jackson Lisa, Shadle Gail, Dixon Richard A. (2006): Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa ( Medicago sativa L.). The Plant Journal, 48, 113-124
Cole D.J. (1985): Mode of action of glyphosate –a literature analysis. In: Grossbard E., Atkinson A. (eds): The Herbicide Glyphosate. Boston, Butterworth’s & Co: 48–75.
D’Aoust M.A., Lerouge P., Busse U., Bilodeau P. et al. (2004): Efficient and reliable production of pharmaceuticals in alfalfa. In: Fischer R., Schillberg S. (eds): Molecular Farming. Weinheim, Wiley-VCH: 1–12.
Duan Zhen, Zhang Daiyu, Zhang Jianquan, Di Hongyan, Wu Fan, Hu Xiaowen, Meng Xuanchen, Luo Kai, Zhang Jiyu, Wang Yanrong (2015): Co-transforming bar and CsALDH Genes Enhanced Resistance to Herbicide and Drought and Salt Stress in Transgenic Alfalfa (Medicago sativa L.). Frontiers in Plant Science, 6, -
Gallego-Giraldo Lina, Jikumaru Yusuke, Kamiya Yuji, Tang Yuhong, Dixon Richard A. (2011): Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.). New Phytologist, 190, 627-639
Gallego-Giraldo L., Bhattarai K., Pislariu C. I., Nakashima J., Jikumaru Y., Kamiya Y., Udvardi M. K., Monteros M. J., Dixon R. A. (2014): Lignin Modification Leads to Increased Nodule Numbers in Alfalfa. PLANT PHYSIOLOGY, 164, 1139-1150
Guo D. (): Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa: Impacts on Lignin Structure and Implications for the Biosynthesis of G and S Lignin. THE PLANT CELL ONLINE, 13, 73-88
ISAAA (2016): Global Status of Commercialized Biotech/GM Crops: 2016. ISAAA Brief No. 52, Ithaca, ISAAA.
Jiang Qingzhen, Zhang Ji-Yi, Guo Xiulin, Monteros Maria J., Wang Zeng-Yu (2009): Physiological Characterization of Transgenic Alfalfa ( Medicago sativa ) Plants for Improved Drought Tolerance. International Journal of Plant Sciences, 170, 969-978
Jin Taicheng, Chang Qing, Li Wangfeng, Yin Dongxu, Li Zhijian, Wang Deli, Liu Bao, Liu Lixia (2010): Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa. Plant Cell, Tissue and Organ Culture (PCTOC), 100, 219-227
Kang Peng, Bao Ai-Ke, Kumar Tanweer, Pan Ya-Qing, Bao Zhulatai, Wang Fei, Wang Suo-Min (2016): Assessment of Stress Tolerance, Productivity, and Forage Quality in T1 Transgenic Alfalfa Co-overexpressing ZxNHX and ZxVP1-1 from Zygophyllum xanthoxylum. Frontiers in Plant Science, 7, -
Kim W.S., Krishnan H.B. (2003): Allelic variation and differential expression of methionine-rich delta-zeins in maize inbred lines B73 and W23a1. Planta, 2171: 66–74.
Kineman Brian D., Brummer E. Charles, Paiva Nancy L., Birt Diane F. (2010): Resveratrol From Transgenic Alfalfa for Prevention of Aberrant Crypt Foci in Mice. Nutrition and Cancer, 62, 351-361
Kumar Suresh (2011): Biotechnological advancements in alfalfa improvement. Journal of Applied Genetics, 52, 111-124
Kumar S., Chandra A., Pandey K.C. (2008): Bacillus thuringiensis (Bt) transgenic crop: an environment friendly insect-pest management strategy. Journal of Environmental Biology, 295: 641–653.
Kumar Tanweer, Uzma , Khan Muhammad Ramzan, Abbas Zaheer, Ali Ghulam Muhammad (2014): Genetic Improvement of Sugarcane for Drought and Salinity Stress Tolerance Using Arabidopsis Vacuolar Pyrophosphatase (AVP1) Gene. Molecular Biotechnology, 56, 199-209
Laudadio V., Ceci E., Lastella N. M. B., Introna M., Tufarelli V. (2014): Low-fiber alfalfa (Medicago sativa L.) meal in the laying hen diet: Effects on productive traits and egg quality. Poultry Science, 93, 1868-1874
Li J.F., Zhang D., Sheen J. (2014): Cas9-based genome editing in Arabidopsis and tobacco. Methods in Enzymology, 546:459–472
Li Li, Yuan Hui (2013): Chromoplast biogenesis and carotenoid accumulation. Archives of Biochemistry and Biophysics, 539, 102-109
Li X., Weng J.K., Chapple C. (2008): Improvement of biomass through lignin modification. Plant Journal, 544: 569–581.
Liu C.Z., Yan L., Wei L.X., Zhang F., Qian X.J. (2008): Effects of cutting on the population dynamics of main insect pests on alfalfa. Ying Yong Sheng Tai Xue Bao, 193: 691–694.
Mathison G. W., Soofi-Siawash R., Klita P. T., Okine E. K., Sedgwick G. (1999): Degradability of alfalfa saponins in the digestive tract of sheep and their rate of accumulation in rumen fluid. Canadian Journal of Animal Science, 79, 315-319
McCaslin M., Temple S.J., Tofte J.E. (2002): Methods for maximizing expression of transgenic traits in autopolyploid plants. US Patent Appl US-2002-0042928-A1.
McCoy T., Walker K. (1984): Alfalfa. In: Ammirato P.V., Evans D.A., Sharp W.R. Yamada Y. et al. (eds): Handbook of Plant Cell Culture. Vol 3. Crop Species, MacMillan Publishing Company: 171–192.
McKersie B. D., Bowley S. R., Harjanto E., Leprince O. (1996): Water-Deficit Tolerance and Field Performance of Transgenic Alfalfa Overexpressing Superoxide Dismutase. Plant Physiology, 111, 1177-1181
McKersie Bryan D., Murnaghan Julia, Jones Kim S., Bowley Stephen R. (2000): Iron-Superoxide Dismutase Expression in Transgenic Alfalfa Increases Winter Survival without a Detectable Increase in Photosynthetic Oxidative Stress Tolerance. Plant Physiology, 122, 1427-1438
McMahon L. R., McAllister T. A., Berg B. P., Majak W., Acharya S. N., Popp J. D., Coulman B. E., Wang Y., Cheng K.-J. (2000): A review of the effects of forage condensed tannins on ruminal fermentation and bloat in grazing cattle. Canadian Journal of Plant Science, 80, 469-485
MENDIS M. H, POWER J. B., DAVEY M. R. (1991): Somatic Hybrids of the Forage Legumes Medicago sativa L. and M. falcata L.. Journal of Experimental Botany, 42, 1565-1574
Meng Yingying, Hou Yaling, Wang Hui, Ji Ronghuan, Liu Bin, Wen Jiangqi, Niu Lifang, Lin Hao (2017): Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula. Plant Cell Reports, 36, 371-374
Michno J.M., Wang X., Liu J., Curtin S.J., Kono T.J., Stupar R.M. (2015): CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas 9 enzyme. GM Crops & Food, 6: 243–252.
Mizukami Yuko, kato Mitsuru, Takamizo Tadashi, Kanbe Michio, Inami Susumu, Hattori Kazumi (2006): Interspecific hybrids between Medicago sativa L. and annual Medicago containing Alfafa weevil resistance. Plant Cell, Tissue and Organ Culture, 84, 80-89
Nair R. B. (2004): The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 Gene Encodes an Aldehyde Dehydrogenase Involved in Ferulic Acid and Sinapic Acid Biosynthesis. THE PLANT CELL ONLINE, 16, 544-554
Nicolia A., Ferradini N., Molla G., Biagetti E., Pollegioni L., Veronesi F., Rosellini D. (2014): Expression of an evolved engineered variant of a bacterial glycine oxidase leads to glyphosate resistance in alfalfa. Journal of Biotechnology, 184, 201-208
Nutter F. W., Guan J., Gotlieb A. R., Rhodes L. H., Grau C. R., Sulc R. M. (2002): Quantifying Alfalfa Yield Losses Caused by Foliar Diseases in Iowa, Ohio, Wisconsin, and Vermont. Plant Disease, 86, 269-277
Pickering FS, Reis PJ (1993): Effects of abomasal supplements of methionine on wool growth of grazing sheep. Australian Journal of Experimental Agriculture, 33, 7-
Reddy M. S. S., Chen F., Shadle G., Jackson L., Aljoe H., Dixon R. A. (2005): Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.). Proceedings of the National Academy of Sciences, 102, 16573-16578
Ricroch Agnès E., Hénard-Damave Marie-Cécile (2016): Next biotech plants: new traits, crops, developers and technologies for addressing global challenges. Critical Reviews in Biotechnology, , 1-16
Robins Joseph G., Bauchan Gary R., Brummer E. Charles (2007): Genetic Mapping Forage Yield, Plant Height, and Regrowth at Multiple Harvests in Tetraploid Alfalfa ( L.). Crop Science, 47, 11-
Rule D. M., Nolting S. P., Prasifka P. L., Storer N. P., Hopkins B. W., Scherder E. F., Siebert M. W., Hendrix W. H. (2014): Efficacy of Pyramided Bt Proteins Cry1F, Cry1A.105, and Cry2Ab2 Expressed in SmartStax Corn Hybrids Against Lepidopteran Insect Pests in the Northern United States. Journal of Economic Entomology, 107, 403-409
Samac D.A., Jung H-J.G, Lamb J.F.S. (2006): Development of alfalfa (Medicago sativa L.) as a feedstock for production of ethanol and other bio products. In: Minter S.L. (ed): Alcoholic Fuels. CRC Press: 79–98.
Schaart Jan G., van de Wiel Clemens C.M., Lotz Lambertus A.P., Smulders Marinus J.M. (2016): Opportunities for Products of New Plant Breeding Techniques. Trends in Plant Science, 21, 438-449
Schuster Mariana, Schweizer Gabriel, Reissmann Stefanie, Kahmann Regine (2016): Genome editing in Ustilago maydis using the CRISPR–Cas system. Fungal Genetics and Biology, 89, 3-9
Sen Sucharita, Makkar Harinder P. S., Becker Klaus (1998): Alfalfa Saponins and Their Implication in Animal Nutrition. Journal of Agricultural and Food Chemistry, 46, 131-140
Shadle Gail, Chen Fang, Srinivasa Reddy M.S., Jackson Lisa, Nakashima Jin, Dixon Richard A. (2007): Down-regulation of hydroxycinnamoyl CoA: Shikimate hydroxycinnamoyl transferase in transgenic alfalfa affects lignification, development and forage quality. Phytochemistry, 68, 1521-1529
Shahin Elias A., Spielmann Albert, Sukhapinda Kitisri, Simpson Robert B., Yashar Mayer (1986): Transformation of Cultivated Alfalfa Using Disarmed Agrobacterium tumefaciens1. Crop Science, 26, 1235-
Soto-Zarazúa M. Guadalupe, Rodrigues Francisca, Pimentel Filipa B., Bah M. M., Oliveira M. Beatriz P. P. (2016): The isoflavone content of two new alfalfa-derived products for instant beverage preparation. Food & Function, 7, 364-371
Strizhov N., Keller M., Mathur J., Koncz-Kalman Z., Bosch D., Prudovsky E., Schell J., Sneh B., Koncz C., Zilberstein A. (1996): A synthetic cryIC gene, encoding a Bacillus thuringiensis  -endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proceedings of the National Academy of Sciences, 93, 15012-15017
Suárez Ramón, Calderón Cecilia, Iturriaga Gabriel (2009): Enhanced Tolerance to Multiple Abiotic Stresses in Transgenic Alfalfa Accumulating Trehalose. Crop Science, 49, 1791-
Tang Lili, Cai Hua, Ji Wei, Luo Xiao, Wang Zhenyu, Wu Jing, Wang Xuedong, Cui Lin, Wang Yang, Zhu Yanming, Bai Xi (2013): Overexpression of GsZFP1 enhances salt and drought tolerance in transgenic alfalfa (Medicago sativa L.). Plant Physiology and Biochemistry, 71, 22-30
Teotia Sachin, Singh Deepali, Tang Xiaoqing, Tang Guiliang (2016): Essential RNA-Based Technologies and Their Applications in Plant Functional Genomics. Trends in Biotechnology, 34, 106-123
Tesfaye M., Temple S. J., Allan D. L., Vance C. P., Samac D. A. (2001): Overexpression of Malate Dehydrogenase in Transgenic Alfalfa Enhances Organic Acid Synthesis and Confers Tolerance to Aluminum. PLANT PHYSIOLOGY, 127, 1836-1844
Tesfaye Mesfin, Denton Matthew D., Samac Deborah A., Vance Carroll P. (2005): Transgenic alfalfa secretes a fungal endochitinase protein to the rhizosphere. Plant and Soil, 269, 233-243
Tesfaye Mesfin, Silverstein Kevin A. T., Bucciarelli Bruna, Samac Deborah A., Vance Carroll P. (2006): The Affymetrix Medicago GeneChip ® array is applicable for transcript analysis of alfalfa ( Medicago sativa ). Functional Plant Biology, 33, 783-
TIVOLI B., BARANGER A., SIVASITHAMPARAM K., BARBETTI M. J. (2006): Annual Medicago: From a Model Crop Challenged by a Spectrum of Necrotrophic Pathogens to a Model Plant to Explore the Nature of Disease Resistance. Annals of Botany, 98, 1117-1128
Tohidfar Masoud, Zare Naser, Jouzani Gholamreza Salehi, Eftekhari Seide Maryam (2013): Agrobacterium-mediated transformation of alfalfa (Medicago sativa) using a synthetic cry3a gene to enhance resistance against alfalfa weevil. Plant Cell, Tissue and Organ Culture (PCTOC), 113, 227-235
Torregrosa Carine, Cluzet Stéphanie, Fournier Joëlle, Huguet Thierry, Gamas Pascal, Prospéri Jean-Marie, Esquerré-Tugayé Marie-Thérèse, Dumas Bernard, Jacquet Christophe (2004): Cytological, Genetic, and Molecular Analysis to Characterize Compatible and Incompatible Interactions Between Medicago truncatula and Colletotrichum trifolii. Molecular Plant-Microbe Interactions, 17, 909-920
USDA (2005): Determination of Non-regulated Status for Alfalfa Genetically Engineered for Tolerance to the Herbicide Glyphosate. Federal Register, Vol 70, No. 122, June 27, 2005. Available at
Vlahova M., Stefanova G., Petkov P., Barbulova A., Petkova D., Kalushkov P., Atanassov A. (2014): Genetic Modification of Alfalfa (Medicago Sativa L.) for Quality Improvement and Production of Novel Compounds. Biotechnology & Biotechnological Equipment, 19, 56-62
Wang Zhi, Li Hongbing, Ke Qingbo, Jeong Jae Cheol, Lee Haeng-Soon, Xu Bingcheng, Deng Xi-Ping, Lim Yong Pyo, Kwak Sang-Soo (2014): Transgenic alfalfa plants expressing AtNDPK2 exhibit increased growth and tolerance to abiotic stresses. Plant Physiology and Biochemistry, 84, 67-77
Weeks J Troy, Ye Jingsong, Rommens Caius M (2008): Development of an in planta method for transformation of alfalfa (Medicago sativa). Transgenic Research, 17, 587-597
Winicov Ilga (2000): Alfin1 transcription factor overexpression enhances plant root growth under normal and saline conditions and improves salt tolerance in alfalfa. Planta, 210, 416-422
Wu H.S., Shi X., Li J., Wu T.Y., Ren Q.Q., Zhang Z.H., Xiao S.H. (2016): Effects of root exudates of bivalent transgenic cotton (Bt+CpTI) plants on antioxidant proteins and growth of conventional cotton (Xinluhan 33). Journal of Environmental Biology, 37: 13–19.
Yang S., Gao M., Xu C., Gao J., Deshpande S., Lin S., Roe B. A., Zhu H. (2008): Alfalfa benefits from Medicago truncatula: The RCT1 gene from M. truncatula confers broad-spectrum resistance to anthracnose in alfalfa. Proceedings of the National Academy of Sciences, 105, 12164-12169
Zhang H., Gou F., Zhang J., Liu W., Li Q., Mao Y., Botella J.R., Zhu J.K. (2016a): TALEN-mediated targeted mutagenesis produces a large variety of heritable mutations in rice. Plant Biotechnology Journal, 14: 186–194.
Zhang J., Duan Z., Zhang D., Zhang J., Di H., Wu F., Wang Y. (2016b): Co-transforming bar and CsLEA enhanced tolerance to drought and salt stress in transgenic alfalfa (Medicago sativa L.). Biochemical and Biophysical Research Communications, 472: 75–82.
Zhang Wan-Jun, Wang Tao (2015): Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation. Plant Science, 234, 110-118
download PDF

© 2022 Czech Academy of Agricultural Sciences | Prohlášení o přístupnosti