Development of an in Planta Method for Transformation of Alfalfa (Medicago Sativa)

Overview

Journal Transgenic Res

Specialty Molecular Biology

Date 2007 Sep 14

PMID 17851774

Citations 22

Authors

J Troy Weeks

Jingsong Ye

Caius M Rommens

Affiliations

Soon will be listed here.

Abstract

Conventional methods in transforming alfalfa (Medicago sativa) require multiple tissue culture manipulations that are time-consuming and expensive, while applicable only to a few highly regenerable genotypes. Here, we describe a simple in planta method that makes it possible to transform a commercial variety without employing selectable marker genes. Basically, young seedlings are cut at the apical node, cold-treated, and vigorously vortexed in an Agrobacterium suspension also containing sand. About 7% of treated seedlings produced progenies segregating for the T-DNA. The vortex-mediated seedling transformation method was applied to transform alfalfa with an all-native transfer DNA comprising a silencing construct for the caffeic acid o-methyltransferase (Comt) gene. Resulting intragenic plants accumulated reduced levels of the indigestible fiber component lignin that lowers forage quality. The absence of both selectable marker genes and other foreign genetic elements may expedite the governmental approval process for quality-enhanced alfalfa.

Citing Articles

Improved Protocol for Efficient -Mediated Transient Gene Expression in L.

Basak S, Parajulee D, Dhir S, Sangra A, Dhir S Plants (Basel). 2024; 13(21).

PMID: 39519910 PMC: 11547841. DOI: 10.3390/plants13212992.

A comprehensive review of in planta stable transformation strategies.

Gelinas Belanger J, Copley T, Hoyos-Villegas V, Charron J, ODonoughue L Plant Methods. 2024; 20(1):79.

PMID: 38822403 PMC: 11140912. DOI: 10.1186/s13007-024-01200-8.

Cisgenics and intragenics: boon or bane for crop improvement.

Vasudevan S, Pooja S, Raju T, Damini C Front Plant Sci. 2023; 14:1275145.

PMID: 38089798 PMC: 10715053. DOI: 10.3389/fpls.2023.1275145.

Plant Promoters and Terminators for High-Precision Bioengineering.

Brooks E, Elorriaga E, Liu Y, Duduit J, Yuan G, Tsai C Biodes Res. 2023; 5:0013.

PMID: 37849460 PMC: 10328392. DOI: 10.34133/bdr.0013.

Genetic Improvement of Wheat for Drought Tolerance: Progress, Challenges and Opportunities.

Bapela T, Shimelis H, Tsilo T, Mathew I Plants (Basel). 2022; 11(10).

PMID: 35631756 PMC: 9144332. DOI: 10.3390/plants11101331.

References

Bent A . Arabidopsis thaliana floral dip transformation method. Methods Mol Biol. 2006; 343:87-103. DOI: 10.1385/1-59745-130-4:87. View

Guo D, Chen F, Wheeler J, Winder J, Selman S, Peterson M . Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases. Transgenic Res. 2001; 10(5):457-64. DOI: 10.1023/a:1012278106147. View

Konig A . A framework for designing transgenic crops--science, safety and citizen's concerns. Nat Biotechnol. 2003; 21(11):1274-9. DOI: 10.1038/nbt1103-1274. View

Rommens C, van Haaren M, Buchel A, Mol J, van Tunen A, Nijkamp H . Transactivation of Ds by Ac-transposase gene fusions in tobacco. Mol Gen Genet. 1992; 231(3):433-41. DOI: 10.1007/BF00292713. View

Strauss S . Genetic technologies. Genomics, genetic engineering, and domestication of crops. Science. 2003; 300(5616):61-2. DOI: 10.1126/science.1079514. View

Trieu A, Burleigh S, Kardailsky I, Maldonado-Mendoza I, Versaw W, Blaylock L . Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J. 2000; 22(6):531-41. DOI: 10.1046/j.1365-313x.2000.00757.x. View

Rommens C, Humara J, Ye J, Yan H, Richael C, Zhang L . Crop improvement through modification of the plant's own genome. Plant Physiol. 2004; 135(1):421-31. PMC: 429395. DOI: 10.1104/pp.104.040949. View

Miller M, Tagliani L, Wang N, Berka B, Bidney D, Zhao Z . High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Res. 2002; 11(4):381-96. DOI: 10.1023/a:1016390621482. View

Clough S, Bent A . Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1999; 16(6):735-43. DOI: 10.1046/j.1365-313x.1998.00343.x. View

10.

Khoudi H, Vezina L, Mercier J, Castonguay Y, Allard G, Laberge S . An alfalfa rubisco small subunit homologue shares cis-acting elements with the regulatory sequences of the RbcS-3A gene from pea. Gene. 1997; 197(1-2):343-51. DOI: 10.1016/s0378-1119(97)00282-5. View

11.

Rommens C, Bougri O, Yan H, Humara J, Owen J, Swords K . Plant-derived transfer DNAs. Plant Physiol. 2005; 139(3):1338-49. PMC: 1283770. DOI: 10.1104/pp.105.068692. View

12.

Liu Y, Whittier R . Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics. 1995; 25(3):674-81. DOI: 10.1016/0888-7543(95)80010-j. View

13.

Tague B . Germ-line transformation of Arabidopsis lasiocarpa. Transgenic Res. 2001; 10(3):259-67. DOI: 10.1023/a:1016633617908. View

14.

Desfeux C, Clough S, Bent A . Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol. 2000; 123(3):895-904. PMC: 59052. DOI: 10.1104/pp.123.3.895. View

15.

Jefferson R, Kavanagh T, Bevan M . GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987; 6(13):3901-7. PMC: 553867. DOI: 10.1002/j.1460-2075.1987.tb02730.x. View

16.

Gamborg O, Miller R, Ojima K . Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 1968; 50(1):151-8. DOI: 10.1016/0014-4827(68)90403-5. View

17.

Curtis I, Nam H . Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method--plant development and surfactant are important in optimizing transformation efficiency. Transgenic Res. 2001; 10(4):363-71. DOI: 10.1023/a:1016600517293. View

18.

Fox J . US courts thwart GM alfalfa and turf grass. Nat Biotechnol. 2007; 25(4):367-8. DOI: 10.1038/nbt0407-367. View

19.

Zuo J, Niu Q, Moller S, Chua N . Chemical-regulated, site-specific DNA excision in transgenic plants. Nat Biotechnol. 2001; 19(2):157-61. DOI: 10.1038/84428. View

20.

Atlung T, Christensen B, Hansen F . Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12. Plasmid. 1999; 41(2):110-9. DOI: 10.1006/plas.1998.1386. View