Alfalfa, transgenic

Wigdorovitz, A., Carrillo, C., Dus Santos, M.J., Trono, K., Peralta, A., Gomez, M.C., Rios, R.D., Franzone, P.M., Sadir, A.M., Escribano, J.M., and Borca, M.V. (1999) Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the structural protein VPl. Virology 255 347-353. 

Wigdorovitz A, Carrillo C, Dus Santos M), et al. Induction of a protective antibody response to foot and mouth disease vims in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral stmctural protein VPl Virology,... 

Marita, J. M. Ralph, J. Hatheld, R. D. Guo, D. Chen, F. Dixon, R. A. Structural and compositional modihcations in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase. 

The first hurdle encountered during the development of alfalfa as a recombinant protein production system was the relative inefficiency of the available expression cassettes. A study in which a tomato proteinase inhibitor I transgene was expressed in tobacco and alfalfa under the control of the cauliflower mosaic virus (CaMV) 35S promoter showed that 3-4 times more protein accumulated in tobacco leaves compared to alfalfa leaves [5]. Despite the low efficiency of the CaMV 35S promoter in alfalfa, bio-pharmaceutical production using this system has been reported in the scientific literature. Such reports include expression of the foot and mouth disease virus antigen [6], an enzyme to improve phosphorus utilization [7] and the anti-human IgG C5-1 [8]. In this last work, the C5-1 antibody accumulated to 1% total soluble protein [8]. 

Fig. 1.1 Promoter activity in alfalfa leaves. Accumulation ofp-glucuroni-dase achieved in transgenic alfalfa leaves expressing the gusA gene under the control of CaMV 35S and alfalfa promoters. %TSP, percentage of total soluble proteins.

In order to reduce the time required to confirm the accumulation of a given recombinant protein, we have developed a cell culture system in which transgenic alfalfa callus material produced at the proliferation step of Agrobacterium-based transformation is used to initiate cell cultures. These cell suspensions can be subcultured to sustain batch production of modest protein amounts. The protein blot shown in Fig. 1.2 demonstrates our ability to detect a recombinant protein in total... 

Fig. 1.3 Prediction of the most appropriate subcellular targeting strategies by agroinfiltration. The levels of an industrial enzyme (IE) are shown in agroinfiltrated and transgenic alfalfa leaves using different subcellular targeting peptides. Equal amounts of total soluble leaf proteins were separated by SDS-PAGE and blotted onto a PVDF membrane. Polyclonal anti-IE IgGs were used for detection.

The most widely studied therapeutic proteins produced in plants include monoclonal antibodies for passive immunotherapy and antigens for use as oral vaccines [40]. Antibodies against dental caries, rheumatoid arthritis, cholera, E. coli diarrhea, malaria, certain cancers, Norwalk virus, HIV, rhinovirus, influenza, hepatitis B virus and herpes simplex virus have been produced in transgenic plants. However, the anti-Streptococcus mutans secretory antibody for the prevention of dental caries is the only plant-derived antibody currently in Phase II clinical trials [40]. Until recently, most antibodies were expressed in tobacco, potato, alfalfa, soybean, rice and wheat [9], It has been estimated that for every 170 tons of harvested tobacco, 100 tons represents harvested leaves. A single hectare could thus yield 50 kg of secretory IgA [3, 41]. Furthermore, it has been estimated that the cost of antibody production in plants is half that in transgenic animals and 20 times lower than in mammalian cell cul-... 

Fig. 15.4 Structure of glycans N-linked to IgG molecules expressed in hybridomas and transgenic plants. Glycans N-linked to plant-derived antibodies are structurally different from their mammalian counterparts. In contrast with antibodies produced in alfalfa, antibodies produced in tobacco plants present a very high glycan heterogeneity.

The first system used to produce vaccines was potato plants and transgenic tubers have being employed in some clinical trials. Expression of vaccines is also reported in other hosts such as tomato, banana, carrot, lettuce, maize, alfalfa, white clover and Arabidospis. Hepatitis B surface antigen was successfully expressed in cherry tomatillo and potato. Expression of human cholera toxin p subunit was achieved in tomato and tobacco plants. ... 

Cooper, J.D., Qiu, F., and Paiva, N.L., Biotransformation of an exogenously supplied isoflavo-noid by transgenic tobacco cells expressing alfalfa isoflavone reductase. Plant Cell Rep., 20, 876, 2002. 

Guo D, Chen F, Wheeler J, Winder J, Selman S, Peterson M, Dixon RA. 2001. Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases. Transgenic Res 10 457-464. 

Hipskind JD, Paiva NL. 2000. Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis. Molec Plant Microbe Interact 13 551-562. 

Oommen A, Dixon RA, Paiva NL. 1994. The elicitor-inducible alfalfa isoflavone reductase promoter confers different patterns of developmental expression in homologous and heterologous transgenic plants. Plant Cell 6 1789-1803. 

Fig. 2. UDP-Glc dehydrogenase activity in control and transgenic alfalfa plants. Stem internodes of uniform age from greenhouse-grown plants were assayed for production of UDP-GlcA. Activity is expressed as percentage of the untransformed control Regen-SY. Plant lines in bold were selected for field testing.

Stem Cell-Wall Concentration and Composition of Control and Transgenic Alfalfa Plants Averaged Across Two Harvests from 1999"... 

No obvious differences in tissue distribution, shape, or staining for the presence of pectin were observed among the alfalfa lines. The transgenic lines appeared normal in tissue structure. 

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