Update on Transformation Recent Advances in Legume Transformation David A. Somers*2, Deborah A. Samac2, and Paula M. Olhoft Department of Agronomy and Plant Genetics (Da.A.S., P.M.O.) and United States Department of Agriculture-Agricultural Research Service, Plant Science Research Unit and Department of Plant Pathology (De.A.S.), University of Minnesota, St. Paul, Minnesota 55108 WHY TRANSFORM LEGUMES? than others. Legume transformation systems, like transformation in all organisms, require develop- Legumes are a large, diverse family ranging from ment of: (a) a source of totipotent cells or gametes herbaceous annuals to woody perennials that, be- that serve as recipients of delivered DNA, (b) a cause of their capacity to fix nitrogen, are essential means of delivering DNA into the target cells, and (c) components in natural and managed terrestrial eco- a system for selecting or identifying transformed systems. Legumes have been domesticated for the cells. For legumes that have been regarded as recal- production of food, feed, forage, fiber, industrial and citrant to transformation, regeneration in vitro is medicinal compounds, flowers, and other end uses. highly genotype specific and only rarely are culti- Understanding the molecular basis of nitrogen fixa- vated varieties amenable to regeneration. In these tion and the unique metabolic pathways that result in cases, plant regeneration remains an “art” that re- the myriad of end uses of legumes is both a matter of quires considerable training of the practitioner to scientific curiosity and of economic necessity because develop the skills needed to generate sufficient trans- of their importance in the biosphere and to the sus- genic plants for a thesis or publication. In addition, tainability of the human race. In accordance, model regeneration is often slow and the frequency of trans- legumes are being rapidly developed as experimental formation (no. of transformed plants generated from systems to pursue a number of important biological each explant) is often low. In species that are amena- questions unique to these plants using molecular tools ble to in vitro somatic embryogenesis such as alfalfa, including genomics. A key component of most func- (lucerne; Medicago sativa), relatively rapid and effi- tional genomics approaches is a high-throughput cient transformation methods have been developed transformation system useful for developing various based on cocultivation of tissue pieces (explants) gene identification strategies. Transformation also is with Agrobacterium tumefaciens. Because inducing so- emerging as an important crop improvement tool. matic embryogenesis or organogenesis in many le- This is particularly evident in soybean (Glycine max), in which Roundup Ready soybean cultivars have cap- gume species is difficult, a variety of transformation tured a major stake in market share of soybeans methods have been reported that use cultures of planted in the U.S. and Argentina. Transformation meristematic cells as sources of totipotent cells. Most theoretically expands the sources of genes for plant commonly, transformation has been based on infec- improvement to all organisms, far beyond the gene tion by A. tumefaciens, although Agrobacterium rhizo- pool accessible via sexual hybridization. Transforma- genes is used for transformation of some species. tion also offers strategies for overexpressing or sup- Regeneration of shoots from the cotyledonary node pressing endogenous genes. Thus, introducing new or from other meristematic explants after Agrobacte- genes or manipulating endogenous gene expression rium infection is emerging as a rapid and relatively via transformation generates new phenotypic varia- efficient method of transformation in a number of tion useful for investigating gene function and for legume species including soybean (Olhoft and Som- crop improvement. ers, 2002), Lotus japonicus (Oger et al., 1996), barrel medic (Medicago truncatula; Trieu and Harrison, 1996), and Trifolium repens (Larkin et al., 1996). A number of legume species also have been trans- ARE LEGUMES DIFFICULT TO TRANSFORM? formed by direct DNA transfer methods including The answer to this question is, of course, that some microinjection, electroporation, and microprojectile legume species are much more difficult to transform bombardment (for review, see Christou, 1997; Atkins and Smith, 1997; Babaoglu et al., 2000). 1 In some species, the difficulty in regenerating This is a joint contribution of the Minnesota Agricultural transgenic plants has been circumvented by develop- Experiment Station and the U.S. Department of Agriculture- Agricultural Research Service. ment of rapid and efficient transformation protocols 2 These authors contributed equally to the paper. using A. rhizogenes to produce hairy roots on “com- * Corresponding author; [email protected]; fax 612– posite” plants (an untransformed plantlet with hairy 625–1268. roots). These composite plants have been used in www.plantphysiol.org/cgi/doi/10.1104/pp.102.017681. studies focused on root characteristics such as nod- 892 Plant Physiology, March 2003, Vol. 131, pp. 892–899, www.plantphysiol.org © 2003 American Society of Plant Biologists Recent Advances in Legume Transformation ulation and root diseases. Examples have been re- for bees, and can be used to volatilize selenium from ported in L. japonicus (Stiller et al., 1997; Martirani et soil. A. rhizogenes inoculation of seedlings in vitro al., 1999), soybean (Narayanan et al., 1999), and bar- results in formation of hairy root, which spontane- rel medic (Boisson-Dernier et al., 2001). Composite ously produce shoots in culture (Cho et al., 1998). plants do not transmit the transgenic trait to their Similarly, a number of protocols using A. rhizogenes progeny and, thus, are of little use in crop improve- for production of transgenic Lotus corniculatus have ment efforts. been described (Atkins and Smith, 1997). Transfor- Advancement of molecular genetics in legumes, e.g. mation of L. corniculatus via cocultivation of leaf gene overexpression, gene suppression, promoter explants with A. tumefaciens followed by callus for- analysis, T-DNA tagging, and expression of genes mation and shoot organogenesis was reported by for crop improvement, requires efficient transfor- Webb et al. (1996). In contrast, transformation of red mation systems that produce low frequencies of clover is based on regeneration via somatic embry- tissue culture-induced phenotypic abnormalities in ogenesis after cocultivation of petiole explants with the transgenic plants. The development of the in A. tumefaciens using genotypes selected for high fre- planta transformation system for Arabidopsis (Clough quency of this culture response (Quesenberry et al., and Bent, 1998) radically accelerated research in basic 1996). plant molecular biology. By analogy, development of L. japonicus was suggested as a model system for simple, rapid transformation systems in legumes that legume genomics by Handberg and Stougaard require the minimum amount of “art” will have a (1992). In addition to other positive attributes as a similar impact on legume biology. In this Update,we model system, transformation of hypocotyls with A. report recent advances in transformation of forage and tumefaciens is relatively efficient via shoot organogen- pasture, grain and pulse, and tree legumes updating esis. This method was further optimized and the time the excellent summaries of Babaoglu et al. (2000) and to produce whole plants reduced by Stiller et al. Atkins and Smith (1997). This information is summa- (1997). Somaclonal variation and sterility were sig- rized in Table I such that the DNA delivery method, nificantly reduced by use of the bar gene and selec- source of totipotent target cells, and selection system is tion with PPT (Lohar et al., 2001). presented for each species. Aspects of transformation A highly efficient transformation method has en- system components that have resulted in improve- abled initiation of a T-DNA insertional mutagenesis ments in transformation efficiency of legumes will program for barrel medic (Scholte et al., 2002). Each also be discussed. Finally, we speculate on possible explant of line R108-1(C3), a genotype selected for avenues for developing non-tissue culture transforma- superior regeneration, produces large numbers of tion systems for legumes. somatic embryos, and up to 80% of the embryos regenerate into plants 3 to 4 months after culture initiation (Trinh et al., 1998). Methods with the po- tential to reduce tissue culture manipulations for PROGRESS IN LEGUME TRANSFORMATION transformation of barrel medic have been reported. Forage and Pasture Legumes Trieu and Harrison (1996) described a method based on cocultivation of A. tumefaciens with cotyledonary In the past decade, considerable success has been node explants followed by culture to induce multiple achieved in transformation of forage and pasture le- shoots from explants. Transgenic plantlets were pro- gumes. Efficient transformation protocols have been duced in 2.5 months. Two in planta transformation developed for alfalfa and T. repens that have enabled systems were described by Trieu et al. (2000); one research to advance from expression of marker genes method is based on infiltration of flowers with A. to evaluation of genes for crop improvement. Com- tumefaciens, similar to the Arabidopsis flower infiltra- mercialization of the first transgenic forage crop, tion protocol, and the other on infiltration of seed- Roundup