Alfalfa ( <i> Medicago sativa </i> L. ) is an important leguminous crop worldwide and it has important roles in different aspects of the agriculture system, including livestock feed, crop soil conservation, and improvement of soil nitrogen supply. As a perennial crop, alfalfa breeding for trait improvement takes longer periods of time compared to many other crops, therefore, genetic engineering is a faster route for alfalfa trait modification and improvement. Alfalfa was a first crop in which somatic embryogenesis was developed. Alfalfa was also a pioneering crop in which transgenic technology was developed and applied for trait improvement. Transgenic technology in alfalfa has since advanced steadily in various areas. In this review, we update the recent research progress in alfalfa genetic engineering, focusing on genetic transformation and use of transgenic technology for trait improvement and new trait development.
Robins, J.G., Luth, D., Campbell, I.A., Bauchan, G.R., He, C.L., Viands, D.R., Hansen, J.L. and Brummer, E.C. (2007) Genetic Mapping of Biomass Production in Tetraploid Alfalfa. Crop Science, 47, 1-10. https://doi.org/10.2135/cropsci2005.11.0401
Bouton, J. (2007) The Economic Benefits of Forage Improvement in the United States. Euphytica, 154, 263-270. https://doi.org/10.1007/s10681-006-9220-6
Pacini, F., Molinaro, E., Castagna, M.G., Agate, L., Elisei, R., Ceccarelli, C., Lippi, F., Taddei, D., Grasso, L. and Pinchera, A. (2003) Recombinant Human Thyrotropin-Stimulated Serum Thyroglobulin Combined with Neck Ultrasonography Has the Highest Sensitivity in Monitoring Differentiated Thyroid Carcinoma. JCEM, 88, 3668-3673.
Radovic, J.S.D. and Markovic, J. (2009) Alfalfa-Most Important Perennial Forage Legume in Animal Husbandry. Biotechnology in Animal Husbandry, 25, 465-475. https://doi.org/10.2298/BAH0906465R
Ziauddin, A., Lee, R.W.H., Lo, R., Shewen, P. and Strommer, J. (2004) Transformation of Alfalfa with a Bacterial Fusion Gene, Mannheimiahaemolytica A1 Leukotoxin50-gfp: Response with Agrobacterium tumefaciens Strains LBA4404 and C58. Plant Cell, Tissue and Organ Culture, 79, 271-278. https://doi.org/10.1007/s11240-004-4540-5
Bellucci. M., De Marchis, F. and Arcioni, S. (2007) Zeolin Is a Recombinant Storage Protein That Can Be Used to Produce Value-Added Proteins in Alfalfa (Medicago sativa L.). Plant Cell, Tissue and Organ Culture, 90, 85-91. https://doi.org/10.1007/s11240-007-9258-8
Stefanova, G., Slavov, S., Gecheff, K., Vlahova, M. and Atanassov, A. (2013) Expression of Recombinant Human Lactoferrin in Transgenic Alfalfaplants. Biologia Plantarum, 57, 457-464. https://doi.org/10.1007/s10535-013-0305-5
Sun, X., Chi-Ham, C.L., Cohen-Davidyan, T., DeBen, C., Getachow, G., DePeters, E. and Bennett, A. (2015) Protein Accumulation and Rumen Stability of Wheat γ-Gliadin Fusion Proteins in Tobacco and Alfalfa. Plant Biotechnology Journal, 13, 974-982. https://doi.org/10.1111/pbi.12338
Saunders, J.W. and Bingham, E.T. (1972) Production of Alfalfa Plants from callus Tissue. Plant Science, 12, 804-808.
McKersie, B.D., Senaratna, T., Bowley, S., Brown, D., Krochko, J. and Bewley, J. (1989) Application of Artificial Seed Technology in the Production of Hybrid Alfalfa (Medicago sativa L.). In Vitro Cellular & Developmental Biology-Plant, 25, 1183-1188. https://doi.org/10.1007/BF02621272
McKersie, B.D. (1996) Somatic Embryogenesis and Artificial Seeds in Forage Legumes. Seed Science Research, 6, 109-126. https://doi.org/10.1017/S0960258500003135
Kumar, S. (2011) Biotechnological Advancements in Alfalfa Improvement. Journal of Applied Genetics, 52, 111-124. https://doi.org/10.1007/s13353-011-0028-2
Wang, D., Khurshid, M., Sun, Z.M., Tang, X.Y., Zhou, M.L. and Wu, Y.M. (2016) Genetic Engineering of Alfalfa (Medicago sativa L.). Protein and Peptide Letters, 23, 495-502. https://doi.org/10.2174/0929866523666160314152618
Brown, D.C.W. and Atanassov, A. (1985) Role of Genetic Background in Somatic Embryogenesis in Medicago. Plant Cell, Tissue and Organ Culture, 4, 111-122. https://doi.org/10.1007/BF00042269
Raemakers, C.J.M., Jacobsen, E. and Visser, R.G.F. (1995) Secondary Somatic Embryogenesis and Application in Plant Breeding. Euphytica, 81, 93-107. https://doi.org/10.1007/BF00022463
Raimondi, J.P., Camadro, E. and Babinec, F.J. (2001) Somatic Embryogenesis in Asparagus officinalis L. cv. Argenteuil: Interaction between Genotype, Explant Type and Growth Regulators on Callus Induction, Growth and Embryogenic Differentiation. Biocell, 25, 147-154.
Bhojwani, S.S. and Dantu, P.K. (2013) Somaclonal Variation. In: Plant Tissue Culture: An Introductory Text, Springer, India, 141-154. https://doi.org/10.1007/978-81-322-1026-9_12
Elmabrouk, K. and Elmeer, S. (2013) Factors Regulating Somatic Embryogenesis in Plants. In: Somatic Embryogenesis and Gene Expression, Narosa Publishing House, New Delhi, 56-81.
Du, S.E.L. and Bowley, S. (1994) Effect of Plant Genotype on the Transformation of Cultivated Alfalfa (Medicago sativa) by Agrobacterium tumefaciens. Plant Cell Reports, 13, 330-334. https://doi.org/10.1007/BF00232631
Desgagnes, R., Laberge, S., Allard, G., Khoudi, H., Castonguay, Y., Lapointe, J., Michaud, R. and Vezina, L.P. (1995) Genetic Transformation of Commercial Breeding Lines of Alfalfa (Medicago sativa). Plant Cell, Tissue and Organ Culture, 4, 129-140. https://doi.org/10.1007/BF00034229
Fiore, M.C., Trabace, T. and Sunseri, F. (1997) High Frequency of Plant Regeneration in Sunflower from Cotyledons via Somatic Embryogenesis. Plant Cell Reports, 16, 295-298. https://doi.org/10.1007/BF01088284
Trinh, T.H., Ratet, P., Kondorosi, E., Durand, P., Kamaté, K., Bauer, P. and Kondorosi, A. (1998) Rapid and Efficient Transformation of Diploid Medicago truncatula and Medicago sativa ssp. Falcata Lines Improved in Somatic Embryogenesis. Plant Cell Reports, 17, 345-355. https://doi.org/10.1007/s002990050405
Zhang, H., Huang, Q. and Su, J. (2010) Development of Alfalfa (Medicago sativa L.) Regeneration System and Agrobacterium-Mediated Genetic Transformation. Agricultural Sciences in China, 9, 170-178. https://doi.org/10.1016/S1671-2927(09)60081-X
Liu, W.T., Liang, Z., Shan, C.J., Marsolais, F. and Tian, L. (2013a) Genetic Transformation and Full Recovery of Transgenic Alfalfa Plants via Secondary Somatic Embryogenesis. In Vitro Cellular Developmental Biology-Plant, 49, 17-23.
Liu, W., Liang, Z., Sibbald, S., Hunter, D. and Tian, L. (2013b) Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa. PLoS ONE, 8, e56699. https://doi.org/10.1371/journal.pone.0056699
Aung, B., Gruber, M., Amyot, L., Omari, K., Bertrand, A. and Hannoufa, A. (2015b) MicroRNA156 as a Promising Tool for Alfalfa Improvement. Plant Biotechnology Journal, 13, 779-790. https://doi.org/10.1111/pbi.12308
Ninkovic, S., Miljus-Djukic, J. and Neskovic, M. (1995) Genetic Transformation of Alfalfa Somatic Embryos and Their Clonal Propagation through Repetitive Somatic Embryogenesis. Plant Cell, Tissue and Organ Culture, 4, 255-260. https://doi.org/10.1007/BF00029996
Ninkovic, S., Miljus-Djukic, J., Vinterhalter, B. and Neskovic, M. (2004) Improved Transformation of Alfalfa Somatic Embryos Using a Superbinary Vector. Acta Biologica Cracoviensia Series Botanica, 46, 139-143.
Tian, L., Wang, H., Wu, K., Latoszek-Green, M., Hu, M., Miki, B. and Brown, D. (2002) Efficient Recovery of Transgenic Plants through Organogenesis and Embryogenesis Using A Cryptic Promoter to Drive Marker Gene Expression. Plant Cell Reports, 20, 1181-1187. https://doi.org/10.1007/s00299-002-0459-7
Samac, D.A. and Austin-Phillip, S. (2006) Alfalfa (Medicago sativa L.). Agrobacterium Protocols. In: Wang, K., Ed., Methods in Molecular Biology, Springer, Berlin, 301-311. https://doi.org/10.1385/1-59745-130-4:301
Liu, W.T., Tian, L., Nie, X.N., Liang, Z.S. and He, W.J. (2011) Exogenous Protein Expression in Somatic Embryos in Alfalfa. Journal of Northwest A&F Univesity, 39, 194-200.
Liu, L., Fan, X.D., Wang, F.W., Wang, N., Dong, Y.Y., Liu, X.M., Yang, J., Wang, Y.Y. and Li, H.Y. (2013) Coexpression of ScNHX1 and ScVP in Transgenic Hybrids Improves Salt and Saline-Alkali Tolerance in Alfalfa (Medicago sativa L.). Journal of Plant Growth Regulation, 32, 1-8. https://doi.org/10.1007/s00344-012-9270-z
Tohidfar, M., Zare, N., Jouzani, G.S. and Eftekhari, S.M. (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, 113, 227-235.
Ferradini, N., Giancaspro, A., Nicolia, A., Gadaleta, A., Veronesi, F. and Rosellini, D. (2016) Efficient, Antibiotic Marker-Free Transformation of a Dicot and a Monocot Crop with Glutamate 1-Semialdehyde Aminotransferase Selectable Marker Genes. In: MacDonald, J., Kolotilin, I. and Menassa, R., Eds., Protocol Recombinant Proteins from Plants: Methods and Protocols, Springer, Berlin, 89-98.
Tian, L., Wu, K., Ouellet, T., Foster, E.A., Latoszek-Green, M., Sibbald, S., Brown, D. and Miki, B. (2003) Activity of Elements from the Tobacco Cryptic Promoter, tCUP, in Conifer Tissues. In Vitro Cellular & Developmental Biology—Plant, 39, 193-202. https://doi.org/10.1079/IVP2002365
Shetty, K. and McKersie, B.D. (1993) Proline, Thioproline and Potassium Mediated Stimulation of Somatic Embryogenesis in Alfalfa (Medicago sativa L.). Plant Science, 88, 185-193. https://doi.org/10.1016/0168-9452(93)90090-M
Uzelac, B., Zare, N., Smigocki, A. and Budimir, S. (2007) Origin and Development of Secondary Somatic Embryos in Transformed Embryogenic Cultures of Medicago sativa. Biologia Plantarum, 51, 1-6. https://doi.org/10.1007/s10535-007-0001-4
Weeks, J.T., Ye, J. and Rommers C.M. (2008) Development of an in Planta Method for Transformation of Alfalfa (Medicago sativa). Transgenic Research, 17, 587-597. https://doi.org/10.1007/s11248-007-9132-9
Trieu, A.T., Burleigh, S.H., Kardailsky, I.V., Maldonado-Mendoza, I.E., Versaw, W.K. and Blaylock, L.A. (2000) Transformation of Medicago truncatula via Infiltration of Seedlings or Flowering Plants with Agrobacterium. The Plant Journal, 22, 531-541. https://doi.org/10.1046/j.1365-313x.2000.00757.x
Blaydes, D.F. (1966) Interaction of Kinetin and Various Inhibitors in the Growth of Soybean Tissue. Physiologia Plantarum, 9, 748-753. https://doi.org/10.1111/j.1399-3054.1966.tb07060.x
Schenk, R.U. and Hildebrandt, A.C. (1972) Medium and Techniques for Induction and Growth of Monocotyledonous and Dicotyledonous Plant Cell Cultures. Canadian Journal of Botany, 50, 199-204. https://doi.org/10.1139/b72-026
Amina. M., Delco, A. and Naziabad, H.S. (2016) Improvement of In Vitro Embryo Maturation, Plantlet Regeneration and Transformation Efficiency from Alfalfa (Medicago sativa L.) Somatic Embryos Using Cuscuta campestris Extract. Physiology and Molecular Biology of Plants, 22, 321-330.
Han, S., Kowalczys, K., Latoszek-Green, M. and Brown, D.C.W. (2011) Agrobacterium-Mediated Genetic Transformation of Alfalfa through Somatic Embryogenesis. In: Aslam, J., Srivastava, P.S. and Sharma, M.P., Eds., Somatic Embryogenesis and Genetic Transformation in Plants, Narosa Publishing House, New Delhi, 219-250.
Montague, A., Ziauddin, A., Lee, R., Ainley, W.M. and Strommer, J. (2007) High-Efficiency Phosphinothricin-Based Selection for Alfalfa Transformation. Plant Cell, Tissue and Organ Culture, 91, 29-36. https://doi.org/10.1007/s11240-007-9274-8
Rosellini, D., Capomaccio, S., Ferradini, N., Sardaro, M.L.S., Nicolia, A. and Veronesi, F. (2007) Non-Antibiotic Efficient Selection for Alfalfa Genetic Engineering. Plant Cell Reports, 26, 1035-1044. https://doi.org/10.1007/s00299-007-0321-z
Ferradini, N., Nicolia, A., Capomaccio, S., Veronesi, F. and Rosellini, D. (2011) A Point Mutation in the Medicago sativa GSA Gene Provides a Novel, Efficient, Selectable Marker for Plant Genetic Engineering. Journal of Biotechnology, 156, 147-152. https://doi.org/10.1016/j.jbiotec.2011.08.015
Giancaspro, A., Rosellini, D., Blanco, A. and Gadaleta, A. (2012) Gabaculine Selection Using Bacterial and Plant Marker Genes (GSA-AT) in Durum Wheat Transformation. Plant Cell, Tissue and Organ Culture (PCTOC), 109, 447-455. https://doi.org/10.1007/s11240-011-0109-2
Foster, E., Hattori, J., Labbe, H., Ouellet, T., Fobert, P., James, L. and Miki, B. (1999) A Tobacco Cryptic Constitutive Promoter, tCUP, Revealed by T-DNA Tagging. Plant Molecular Biology, 16, 45-55. https://doi.org/10.1023/A:1006229501860
Bennetzen, J.L., Springer, P.S., Cresse, A.D. and Hendrickx, M. (1993) Specificity and Regulation of the Mutator Transposable Element System in Maize. Critical Reviews in Plant Sciences, 12, 57-95. https://doi.org/10.1080/07352689309382356
Qiao, H., Wang, F., Zhao, L., Zhou, J. and Lai, Z. (2004) The F-Box Protein AhSLF-S2 Controls The Pollen Function of S-RNase-Based Self-Incompatibility. The Plant Cell, 16, 2307-2322. https://doi.org/10.1105/tpc.104.024919
Bavage, A.D., Buck, E., Dale, P., Moyes, C. and Senior, I. (2002) Analysis of a Backcross Population from a Multi-Copy Transgenic Brassica napus Line. Euphytica, 124, 333-340. https://doi.org/10.1023/A:1015799901176
Senaranta, T., McKersie, B.D. and Bowley, S.R. (1989) Desiccation Tolerance of Alfalfa (Medicago sativa L.) Somatic Embroys. Influence of Abscisic Acid, Stress Pretreatments and Drying Rates. Plant Science, 65, 253-259. https://doi.org/10.1016/0168-9452(89)90072-1
Senaranta, T., McKersie, B.D. and Bowley, S.R. (1990) Artificial Seeds of Alfalfa (Medicago sativa L.). Induction of Desiceation Tolerance in Somatic Embryos. In Vitro Cellular and Developmental Biology, 26, 85-90. https://doi.org/10.1007/BF02624160
Anandarajah, K. and McKersie, B.D. (1990) Manipulating the Desiccation Toleranceand Vigor of Dry Somatic Embryos of Medicago sativa L. with Sucrose, Heat Shockand Abscisic Acid. Plant Cell Report, 9, 451-455. https://doi.org/10.1007/BF00232271
Hoshino, T. and Cuello, J.L. (2005) Environmental Design Considerations for Somatic Embryogenesis. In: Mujib, A. and Samaj, J., Eds., Somatic Embryogenesis, Springer, Berlin, 25-34.
Vdovitchenko, M.Y. and Kuzovkina, I.N. (2011) Artificial Seed Preparation as the Efficient Method for Storage and Production of Healthy Cultured Roots of Medicinal Plants. Russian Journal of Plant Physiology, 58, 524. https://doi.org/10.1134/S1021443711030174
Zhang, W.J. and Wang, T. (2014) Enhanced Salt Tolerance of Alfalfa (Medicago sativa) by rstB Gene Transformation. Plant Science, 11, 1-21.
Sim, S., Weinberg, D.E., Fuchs, G., Choi, K., Chung, J. and Wolin, S.L. (2009) The Subcellular Distribution of an RNA Quality Control Protein, the Ro Autoantigen, Is Regulated by Noncoding Y RNA Binding. Molecular Biology of the Cell, 20, 1555-1564. https://doi.org/10.1091/mbc.E08-11-1094
Volenec, J.J., Cunningham, S.M., Haagenson, D.M., Berg, W.K., Joern, B.C. and Wiersma, D.W. (2002) Physiological Genetics of Alfalfa Improvement: Past Failures, Future Prospects. Field Crops Research, 75, 97-110. https://doi.org/10.1016/S0378-4290(02)00020-5
Lamb, J.F.S., Sheaffer, C.C., Rhodes, L.H., Mark Sulc, R., Underander, D.J. and Brummer, E.C. (2006) Five Decades of Alfalfa Cultivar Improvement: Impact on Forage Yield, Persistence, and Nutritive Value. Crop Science, 46, 902-909. https://doi.org/10.2135/cropsci2005.08-0236
Heendeniya, R.G. and Yu, P. (2017) Gene-Transformation-Induced Changes in Chemical Functional Group Features and Molecular Structure Conformation in Alfalfa Plants Co-Expressing Lc-bHLH and C1-MYB Transcriptive Flavanoid Regulatory Genes: Effects of Single-Gene and Two-Gene Insertion. International Journal of Molecular Sciences, 18, 664.
Arcalis, E., Stadlmann, J., Rademacher, T., Marcel, S., Sack, M., Altmann, F. and Stoger, E. (2013) Plant Species and Organ Influence The Structure and Subcellular Localization of Recombinant Glycoproteins. Plant Molecular Biology, 83, 105-117. https://doi.org/10.1007/s11103-013-0049-9
Winkelmann, T., Heintz, D., Van Dorsselaer, A., Serek, M. and Braun, H.P.（2006）Proteomic Analyses of Somatic and Zygotic Embryos of Cyclamen persicum Mill. Reveal New Insights into Seed and Germination Physiology. Planta, 224, 508-519.
Fu, G., Grbic, V., Ma, S. and Tian, L. (2015) Evaluation of Somatic Embryos of Alfalfa for Recombinant Protein Expression. Plant Cell Reports, 34, 211-221. https://doi.org/10.1007/s00299-014-1700-x
Chanvivattana, Y., Bishopp, A., Schubert, D., Stock, C., Moon, Y.H., Sung, Z.R. and Goodrich, J. (2004) Interaction of Polycomb-Group Proteins Controlling Flowering in Arabidopsis. Development, 131, 5263-5276. https://doi.org/10.1242/dev.01400
Schubert, S., Dalhoff, A., Stress, H. and Ullmann, U. (2005) Pharmacodynamics of Moxifloxacin and Levofoxacin Simulating Human Serum and Lung Concentrations. Infection, 33, 15-21. https://doi.org/10.1007/s15010-005-8203-1
Kaldis, A., Ahmad, A., Reid, A., McGarvey, B., Brandle, J., Ma, S., Jevnikar, A., Kohalmi, S.E. and Menassa, R. (2013) High-Level Production of Human Interleukin-10 Fusions in Tobacco Cell Suspension Cultures. Plant Biotechnology Journal, 11, 535-545. https://doi.org/10.1111/pbi.12041
Barnes, D.K., Bingham, E.T., Murphy, R.P., Hunt, O.J., Beard, D.F., Skrdla, W.H. and Teuber, L.R. (1977) Alfalfa Germplasm in the United States: Genetic Vulnerability, Use, Improvement, and Maintenance. USDA Technical Bulletin, No. 1571.
Gallego-Giraldo, L., Bhattarai, K., Pislariu, C.I., Nakashima, J., Jikumaru, Y., Kamiya, Y., Udvardi, M.K., Monteros, M.J. and Dixon, R.A. (2014) Lignin Modification Leads to Increased Nodule Numbers in Alfalfa. Plant Physiology, 164, 1139-1150. https://doi.org/10.1104/pp.113.232421
Aung, B., Gruber, M., Amyot, L., Omari, K., Bertrand, A. and Hannoufa, A. (2015a) Ectopic Expression of LjmiR156 Delays Flowering, Enhances Shoot Branching, and Improves Forage Quality in Alfalfa. Plant Biotechnology Reports, 9, 1-15.
Jia, B., Sun, M., Sun, X., Li, R., Wang, Z., Wu, J. and Zhu, Y. (2016) Overexpression of GsGSTU13 and SCMRP in Medicago sativa Confers Increased Salt-Alkaline Tolerance and Methionine Content. Physiologia Plantarum, 156, 176-189.
Marita, J.M., Rancour, D., Hatfield, R. and Weimer, P. (2016) Impact of Expressing p-Coumaryl Transferase in Medicago sativa L. on Cell Wall Chemistry and Digestibility. American Journal of Plant Sciences, 7, 2553-2569. https://doi.org/10.4236/ajps.2016.717221
Aung, B., Gao, R., Gruber, M.Y., Yuan, Z.C., Sumarah, M. and Hannoufa, A. (2017) MsmiR156 Affects Global Gene Expression and Promotes Root Regenerative Capacity and Nitrogen Fixation Activity in Alfalfa. Transgenic Research, 26, 541-557. https://doi.org/10.1007/s11248-017-0024-3
Bajaj, S. and Mohanty, A. (2005) Recent Advances in Rice Biotechnology—Towards Genetically Superior Transgenic Rice. Plant Biotechnology Journal, 3, 275-307. https://doi.org/10.1111/j.1467-7652.2005.00130.x
Jiao, Y., Wang, Y., Xue, D., Wang, J., Yan, M., Liu, G., Dong, G., Zeng, D., Lu, Z., Zhu, X., Qian, Q. and Li, J. (2010) Regulation of OsSPL14 by OsmiR156 Defines Ideal Plant Architecture in Rice. Nature Genetics, 42, 541-544. https://doi.org/10.1038/ng.591
Wang, S., Wu, K., Yuan, Q., Liu, X., Liu, Z., Lin, X., Zeng, R., Zhu, H., Dong, G., Qian, Q., Zhang, G. and Fu, X. (2012) Control of Grain Size, Shape and Quality by OsSPL16 in Rice. Nature Genetics, 44, 950-954. https://doi.org/10.1038/ng.2327
Yadav, D., Shavrukov, Y., Bazanova, N., Chirkova, L., Borisjuk, N., Kovalchuk, N., Ismagul, A., Parent, B., Langridge, P., Hrmova. M. and Lopato, S. (2015) Constitutive Overexpression of the TaNF-YB4 Gene in Transgenic Wheat Significantly Improves Grain Yield. Journal of Experimental Botany, 6, 6635-6650. https://doi.org/10.1093/jxb/erv370
Hannah, L.C., Futch, B., Bing, J., Shaw, J.R., Boehlein, S., Stewart, J.D., Beiriger, R., Georgelis, N. and Greeneb, T. (2012) A shrunken-2 Transgene Increases Maize Yield by Acting in Maternal Tissues to Increase the Frequency of Seed Development. The Plant Cell, 24, 2352-2363. https://doi.org/10.1105/tpc.112.100602
Schilling, R.K., Marschner, P., Shavrukov, Y., Berger, B., Tester, M., Roy, S.J. and Plett, D.C. (2014) Expression of the Arabidopsis Vacuolar H+-Pyrophosphatase Gene (AVP1) Improves the Shoot Biomass of Transgenic Barley and Increases Grain Yield in a Saline Field. Plant Biotechnology Journal, 12, 378-386. https://doi.org/10.1111/pbi.12145
Fu, C., Sunkar, R., Zhou, C., Shen, H., Zhang, J.Y., Matts, J., Wolf, J., Mann, D.G., Stewart Jr., CN., Tang, Y. and Wang, Z.Y. (2012) Overexpression of miR156 in Switchgrass (Panicum virgatum L.) Results in Various Morphological Alterations and Leads to Improved Biomass Production. Plant Biotechnology Journal, 10, 443-452. https://doi.org/10.1111/j.1467-7652.2011.00677.x
Tabe, L.M., Wardley-Richardson, T., Ceriotti, A., Aryan, A., McNabb, W., Moore, A. and Higgins, T.J. (1995) A Biotechnological Approach to Improving the Nutritive Value of Alfalfa. Journal of Animal Science, 73, 2752-2759. https://doi.org/10.2527/1995.7392752x
Baucher, M., Bernard-Vailhe, M.A., Chabbert, B., Besle, J.M., Opsomer, C., Montagu, M.V. and 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, 39, 437-447. https://doi.org/10.1023/A:1006182925584
Samac, D.A., Bucciarelli, B., Miller, S.S., Yang, S.S., O’Rourke, J.A., Shin, S. and Vance, C.P. (2015) Transgene Silencing of Sucrose Synthase in Alfalfa (Medicago sativa L.) Stem Vascular Tissue Suggests a Role for Invertase in Cell Wall Cellulose Synthesis. BMC Plant Biology, 15, 283. https://doi.org/10.1186/s12870-015-0649-4