​Establishment of a Regeneration System for Wild Alfalfa in Alxa, China

DOI: 10.18805/LRF-679    | Article Id: LRF-679 | Page : 839-843
Citation :- ​Establishment of a Regeneration System for Wild Alfalfa in Alxa, China.Legume Research.2022.(45):839-843
Rui Gu, Zhenyu Jia, Huailin Ma, Yaling Liu, Yuehua Zhang, Bingwen Chang, Fengling Shi nmczysfl@126.com
Address : Key Laboratory of Grassland Resources, College of Grassland, Resources and Environment, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.
Submitted Date : 21-01-2022
Accepted Date : 13-04-2022


Background: The Alxa Region in Inner Mongolia, northwest China is a desert grassland area. Wild alfalfa growing in this area is a precious wild plant resource that can provide resources for improving the stress resistance of alfalfa varieties. Alfalfa is a cross-pollinated plant and its genes are commonly heterozygous. Therefore, establishing a complete tissue culture regeneration system for a single plant is an effective method of protecting wild plant resources. This study used the bud and leaf of wild alfalfa from the Alxa Region as explants to establish an efficient and stable tissue culture regeneration system.
Methods: Two kinds of wild alfalfa tissues were selected as explants. Different concentrations and types of plant growth hormones and regulators were tested, to determine the optimal medium for callus induction, differentiation and rooting culture, including 6-Benzylaminopurine (6-BA), Kinetin (KT), 2, 4-Dichlorophenoxyacetic acid (2, 4-D), Naphylacetic acid (NAA), Indole-3-butyric acid (IBA) and activated carbon (AC). 
Result: The callus induction rate was 93.3% when the bud of wild alfalfa was used as an explant for propagation. With a basic medium (Murashige and Skoog medium with Ca2+, Mg2+ and Mn2+ halved) and hormone regulator (6-BA, KT, NAA, AC), the differentiation rate and rooting rate reached as high as 66.67% and 93.33%, respectively. When leaves were used as explants, the callus induction rate and differentiation rate were 100% and 73.33%, respectively. In the rooting culture process, adding IBA (0.2 mg/L) and AC (0.5 mg/L) to the medium improved the rooting rate of wild alfalfa. Both buds and leaves can be used as explants for plant tissue regeneration and culture, which can help expand and preserve wild alfalfa resources.


​​Callus Differentiation Explant Root Wild alfalfa


  1. Bai, Y., Qu, R. (2010). Factors influencing tissue culture responses of mature seeds and immature embryos in turf-type tall fescue. Plant Breeding. 120(3): 239-242.
  2. Baoler (2021). Process and causes of a regional gale and sand storm in Alxa League. Resources and environment in Arid Areas. 35(4): 112-119
  3. Barbulova, A., Iantcheva, A., Zhiponova, M., Vlahova, M., Atanassov, A. (2002). Agrobacterium-mediated transformation for engineering of herbicide-resistance in alfalfa (Medicago sativa L.). Biotechnology and Biotechnological Equipment. 16(2): 21-27.
  4. Bian, X.Y., Shi, F.L., Wang, D.J., Hao, H.D., Chen, L. (2009). Effects of different hormone proportions on callus induction and differentiation of [Melilotoides ruthenica (L.) Sojak] cv. Zhilixing. Journal of Inner Mongolia University. 40(3): 297-302. 
  5. Gallego, P., Hita, O., Villalobos, N., Dorado, A., Guerra, M.H. (2001). Somatic embryogenesis and plant regeneration in Medicago arborea L. Vitro Cellular and Developmental Biology Plant. 37(2): 199-203.
  6. Geng, H.Z. (1995). Alfalfa in China. China Agricultural Publishing. Beijing. China. 1-9.
  7. Li, F., Zhao, J.M., Shi, F.L., Wang, D.J., Hao, H.D., Guo, Q.H., Bi, J. (2009). Analysis of main factors affecting callus induction of Medicago sativa L. ‘Zhongmu No.2’. Chinese Journal of Grassland. 31(04): 47-52+56.
  8. Liu, S.J., Wang, K.P., Yan, R.Q. (1990). Tissue culture of Medicago ruthenica. Journal of Industrial Technological Economics. 2: 28-29.
  9. Ma, L.L., Liu, X.N., Liu, X.J., Yuan, L.H. (2008). Establishment of tissue culture regeneration system of Medicago sativa L. ‘gannong 4’. Pratacultural Science. 25(12): 67-70.
  10. Markovi, J., Petrovi, M., Terzi, D., Vasi, T., Grubi, G. (2019). Protein fractions as influenced by cultivars, stage of maturity and cutting dates in alfalfa (Medicago sativa L.). Legume Research. 45(1): 46-51.
  11. Shu, Y.J., Li, Wei, Zhao, J.Y., Liu, Y., Guo, C.H. (2018). Transcriptome sequencing and expression profiling of genes involved in the response to abiotic stress in Medicago ruthenica. Genetics and Molecular Biology. 41(3): 638-648.
  12. Wang, J., Li, Y.Z., Shi, S.L. (2010). Isolation and culture of protoplasts from alfalfa callus. Journal of Grassland Science. 18(02): 258-262.
  13. Weeks, J.T., Ye, J., Rommens, C.M. (2008). Development of an in planta method for transformation of Alfalfa (Medicago sativa). Transgenic Research. 17(4): 587. 
  14. Xiao, H.X., Wang, Y. (2007). Study on callus induction and differentiation of Alfalfa Sanditi. Hebei Agricultural Science. (01): 77-81.
  15. Xu, B., Ren, W., Wang, Y.Z., Sun, Q.Z., Guo, W., Lou, Y.J. (2015). Establishment and optimization of high frequency in vitro regeneration system of Alfalfa cotyledons. Hubei Agricultural Sciences. 54(20): 5159-5161.
  16. Xu, B., Wu, R.N., Gao, C.P., Gao, X., Shi, F.L. (2022). Establishment of Tissue Culture Regeneration System for Medicago ruthenica L. cv. ‘Zhilixing’. Legume Research. 45(2): 162-167.
  17. Xu, B., Wu, R.N., Tang, F., Gao, C.P., Gao, X., Shi, F.L. (2021). Haploid culture and double haploid induction in Medicago sativa L. cv. Xin Jiang Da Ye. Legume Research. 44(3): 275-280.
  18. Yang, Q.J., Zhou, H., Sun, Y., Lu, P. (2004). Study on induction callus of Alfalfa and its tissue culture. Journal of Beijing Agricultural College. 19(2): 28-30.
  19. Zare, N., Valizadeh, M., Tohidfar, M., Mohammadi, S.A., Ali, M. (2009). Selection of regenerative genotypes from Iranian alfalfa cultivars. Food Agric. Environ. 7(3-4): 567-572.
  20. Zhang, H., H, Q.M. (2008). Comparative study on callus induction, differentiation and regeneration of different alfalfa varieties. Science and Technology Guide. 02: 38-41.
  21. Zhang, L.Y., Shi, S.L., Yin, C.X., Cui, G.Z. (2013). Establishment of regeneration system of Russian clover. Pratacultural Science. 30(12): 1995-2000.
  22. Zhang, Y.Y., Wang, Z.L., Liu, G.D., Hu, H.F., Du, J.C., Zhao, L.L. (2009). Study on the conditions for callus induction and callus differentiation of Melilotoides ruthenica. Chinese Journal of Grassland. 31(4): 74-79.
  23. Zhao, J.Y., Li, X.Y., Zhu, H.S., Zhang, S.M., Zhang, Y. (2020). Seed Germination and Plant Regeneration System Optimization of Medicago sativa L. ‘Pianguan’. Journal of Grassland Science. 28(04): 947-955.
  24. Zhao, L., Gan, X.Y., Wang, J.D., Chen, Y.C., Shi L, Song, Y.X. (2013). Efficient regeneration system of six cultivars in Medicago sativa. Acta Agriculturaea Sinica. 22(08): 72- 77.

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