Transgenic plants were screened for presence of the
recombinant protein in leaf tissue and Lf expression
levels were determined by indirect ELISA method. The
amount of plant produced human Lf was calculated by
comparison of the absorbance values obtained for extracts Lf from human milk with a known protein concentration.
Quantitative analysis revealed that human Lf content was
0.0047 % of total soluble protein (TSP) in the clone 53.
Transgenic clone 35, in which human Lf transcripts were
not detected by RT-PCR, expressed the recombinant
protein but in lower level 0.0035 % of TSP. In the other
clones tested, the recombinant protein was not detected.
To confirm expression and the size of the recombinant
human Lf, Western blot analysis was performed. The
protein detected in alfalfa leaf tissue from the clone 53
had a molecular mass of approximately 80 kDa, similar to
that of the full-length native human Lf. A band of the
same size was also detected in leaf extracts from the
clone 35 but the signal was too weak (not shown).
Extracts of non-transformed alfalfa plants did not react
with the anti-human Lf antibody.
In order to evaluate disease resistance due to
expression of human Lf cDNA, two bioassays using
strains of bacteria Pseudomonas syringae pv. syringae,
causative agent of bacterial stem blight, and Clavibacter
michiganensis, causing bacterial wilt, were performed.
For the experiment, 6 alfalfa plants from the transgenic
clone 53, for which expression of human Lf was
confirmed, and 1 plant from the clone 79, for which the
recombinant protein was not detected, were used. Nontransformed
alfalfa plants were used as controls.
After Pseudomonas syringae pv. syringae inoculation
on detached alfalfa leaves, symptom progress was
followed for 5 d. Four plants expressing human Lf cDNA
showed less severe Pseudomonas syringae pv. syringae
infection than the plant not expressing human Lf and the
non-transformed controls (Fig. 2I). Inoculation of fresh
branches of alfalfa with Clavibacter michiganensis
caused different level of symptom development. Four
transgenic plants of the clone 53 showed increased
resistance to the pathogen, one developed no symptoms
(-) and the rest showed reduction of symptoms evaluated
by the scale as (+), compared to the non-transformed
control (++) and the plant from the clone 79 (+++). As a
result from these bioassays, four transgenic plants were
determined as less susceptible to both the pathogens
presumably due to expression of human Lf.