NUTRITIONAL ENHANCEMENT OF ALFALFA THROUGH GENETIC ENGINEERING
J. Faragó, D. Mihálik, N. Faragová, M. Fári, J. Kraic
(1)Slovak Agricultural Research Centre, Research Institute of Plant Production, Dept. of Agricultural Biotechnology, Bratislavská cesta 122, SK-921 68 Piešťany, Slovak Republic
(2)University of Debrecen, Faculty of Agricultural Science, Dept. of Horticulture and Plant Biotechnology, Böszörményi út 138, HU-4015 Debrecen, Hungary
Alfalfa (Medicago sativa L.) is a pasture legume crop of primary importance to animal production throughout the world. The nutritional quality of alfalfa, as of other leguminous forage crops, is mainly determined by their content in selected essential amino acids (EAAs), such as methionine (Met) and cysteine (Cys). In alfalfa, however, these S-containing amino acids constitute only about 1% or less of crude proteins (Frame et al., 1998). This is significantly less than the 3.5% Met+Cys content in the recommended FAO reference protein (FAO, 1973). Recent advances in genetic engineering allow to use the transgenic approach to increase the content of specific essential amino acids in target plant species. A number of different molecular approaches have been developed to address this issue, such as over-expression of a heterologous or homologous Met-rich protein, expression of a synthetic protein, modification of protein sequence, and metabolic engineering of the free amino acid pool and protein sink.
To study the possibility of transgenic enhancement of nutritional quality of alfalfa, we used the approach of expression of a heterologous protein rich in Met+Cys in cells of alfalfa. The T-DNA introduced into the genome of alfalfa, using Agrobacterium tumefaciens-mediated genetic transformation, contained the selectable merker gene nptII for kanamycin (Kn) resistance, and a cDNA of Ov gene from Japanese quail (Coturnix coturnix) coding for a high Met+Cys containing ovalbumine (Mucha et al., 1991), both under constitutive promoters. After cocultivation of petiole segment- and leaf blade-explants of two highly embryogenic alfalfa genotypes Rg9/I-14-22 and Rg11/I-10-68 (Faragó et al., 1997) with cells of A. tumefaciens strain AGL1 carrying the nptII and Ov genes, and selection of transgenic cells on Kn containing selective media, more than one hundred putatively transgenic regenerants were obtained through somatic embryogenesis. Biological (Kn rooting assay, paromomycin leaf bleach assay) and molecular (PCR, Western blotting) analyses were performed to confirm the transgenic nature of regenerants. Of the selected lines 96.3% showed the presence of 496 bp fragment of Ov gene. Accumulation of ≈43 kDa Ov protein was detected by Western blot analysis in leaf samples of 8 of 27 analysed transgenic lines. HPLC analysis was performed to analyse the amino acid composition of bulked leaf+stem samples of 32 transgenic and 3 non-transgenic control lines of alfalfa. Of these, three lines, SE/22-14-9-1, SE/22-16-1-3 and SE/22-16-2-2, were found to contain 1.9- to 2,2-fold higher concentration of Me+Cys, in comparison with 0.23 %DW of the non-transformed control.