Development of an in planta method for transformation of alfalfa (Medicago sativa)
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.
KeywordsMarker-free transformation in planta transformation Intragenic alfalfa Biotechnology
The authors are grateful to Scott Simplot, Bill Whitacre, and Dr. Kathy Swords for fruitful discussion and support. Serena McCoy, Jeff Hein, and Michele Krucker are acknowledged for excellent technical assistance.
- Barton KA, Binns AN, Chilton MM, Matzke AJM (2000) Regeneration of plants containing genetically engineered T-DNA. United States patent 6051757Google Scholar
- Bent AF (2006) Arabidopsis thaliana Floral dip transformation method. In: Wang K (eds) Methods mol biol. Agrobacterium protocols, vol 343, 2nd edn. Humana Press, Totowa, NJ 87–103Google Scholar
- Cheng XY, Gao MW, Liang ZQ, Liu GZ, Hu TC (1992) Somaclonal variation in winter wheat: frequency, occurrence and inheritance. Euphytica 64:1–10Google Scholar
- Edwards GA (1998) Genetic modification of plant material. World Patent application 9856932A1Google Scholar
- Harrison MJ, Trieu AT (2000) Plant transformation process. World Patent application 0037663A2Google Scholar
- Inan G, Zhang Q, Li P, Wang Z, Cao Z, Zhang H, Zhang C, Quist TM, Goodwin SM, Zhu J, Shi H, Damsz B, Charbaji T, Gong Q, Ma S, Fredricksen M, Galbraith DW, Jenks MA, Rhodes D, Hasegawa PM, Bohnert HJ, Joly RJ, Bressan RA, Zhu JK (2004) Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. Plant Physiol 135:1718–1737PubMedCrossRefGoogle Scholar
- Lin J-J, Assad-Garcia N, Kuo J (1994) Effects of Agrobacterium cell concentration on the transformation efficiency of tobacco and Arabidopsis thaliana. Focus 16(3):72–77Google Scholar
- Liu F, Cao MQ, Li Y, Robaglia C, Tourneur C (1998) In planta transformation of pakchoi (Brassica campestris L ssp Chinensis) by infiltration of adult plants with Agrobacterium. Acta Hort 467:187–192Google Scholar
- Opabode JT (2006) Agrobacterium-mediated transformation of plants: emerging factors that influence efficiency. Biotechn Mol Biol Rev 1:12–20Google Scholar
- Rogers SG, Fraley RT (2001) Chimeric genes suitable for expression in plant cells. United States patent 6174724Google Scholar
- Samac DA, Austin-Phillips S (2006) Alfalfa (Medicago sativa L.). In: Wang K (eds) Methods mol biol. Agrobacterium protocols, vol 343, 2nd edn. Humana Press, Totowa, NJ pp 301–311Google Scholar
- Trieu AT, Burleigh SH, Kardailsky IV, Maldonado-Mendoza IE, Versaw WK, Blaylock LA, Shin H, Chiou TJ, Katagi H, Dewbre GR, Weigel D, Harrison MJ (2000) Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J 22:531–541PubMedCrossRefGoogle Scholar