Dec 10, 2012 (5 years and 7 months ago)



J. Faragó
, D. Mihálik
, N. Faragová
, M. Fári
, J. Kraic

Slovak Agricultural Research Centre, Research Institute of Plant Production, Dept. of Agricultural
Biotechnology, Bratislavská cesta 122, SK-921 68 Piešťany, Slovak Republic

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.