with plasmid pCNL68 (acb, nptII, gus under napin, nos and 35S promoters, correspondingly),
Escherichia coli DH5ƒ¿ with pBluescript harboring the gene ugt under pT3 and Escherichia coli K802
with pRT104 (Kozack sequence for improving eucaryotic translation and gus under p35). PCR and
Southern blot hybridization were used for determination of the presence of the genes ugt and acb in
transconjugant and in tomato plants. Activities of GUS and UDPG-transferase were estimated as
described earlier (Rekoslavskaya et al., 1999, 2002).
In the 2001 vegetation period the transgenic plant T0 was obtained via infection of tomato
seedlings with the transconjugant cells. One plant of choice was distinguished by its fast growth and
good productivity during growth in greenhouse earth beds. The harvest of this particular plant was
14,7 kg against 4 kg in nontransformed control plants. From fruits of this plant, 308 seeds were
harvested and the growth and development of T1 seedlings were studied. To study germination, the
seeds were placed in water between two disks of filter paper in Petri dishes. The dynamics of
germination and growth of seedlings were monitored for two weeks. In order to study the expression
of marker gene nptII, six drops of 200 g/l kanamycin per leaf were put on nontransformed and
transgenic plants at the four leaf stage of growth in greenhouse soil boxes.
Results and Discussion
The germination in water of seeds isolated from fruits of transgenic tomato plant (T1
generation) began two days earlier, their growth was more energetic than that of control
nontransgenic ones (Table 1). Transgenic T1 seedlings have had more root formation, longer
hypocotyls, and as a whole larger seedling mass.
Table 1. The dynamics of germination and growth of nontransformed and transgenic seedlings of
T1 generation of tomato cv. Ventura
Length
of
hypoco-
tyl (cm)
Length
of roots
(cm)
Mass
of one
seedling
(g)
Length
of
hypoco-
tyl (cm)
Length
of roots
(cm)
Mass/
seedling
(g)
Variant
7 days old seedlings
15 days old seedlings
Nontrans-
formed
3.21±2.83
£0-2
0.05±0.02
7.44±4.10
6.44±5.66
0.10±0.04
Transgenic 8.57±1.60 8.50±2.59 0.18±0.02 18.60±5.08 15.55±5.13 0.22±0.04
In separate experiments the germination of tomato was conducted in boxes with soil in a
greenhouse and plants were monitored for height and leaf area. When tomato seedlings formed 4
true leaves, the treatment with kanamycin was done placing 6 drops of 200 g/l of kanamycin
solution per leaf. As shown in Table 3, transgenic tomato were more tolerant to such treatment
with kanamycin. The size and leaf formation were better in transgenic T1 generation during the
growth in soil. Nontransformed seedlings exposed to kanamycin solution revealed more sensitivity
to antibiotic in comparison to transgenic plants one week after treatment. After 2-3 weeks
nontransformed plants treated with kanamycin mostly died. The survival of transgenic plants was
75-80% under the same conditions. This means that the expression of the marker enzyme
neomycinphosphotranferase occurs in transgenic tomato T1 plants.
No navigation control above? Click here!
