Table 2. The effect of 200 g/l of kanamycin on the growth of T1 tomato seedlings cv. Ventura in soil
Variants before treatment with kanamycin
Height (cm)
Mean leaf area
(cm2)
Nontransformed
4.27±1.76
2.01±0.11
Transgenic
6.50±1.13
5.97±0.29
Variants after one week of kanamycin treatment
Nontransformed
5.51±1.51
3.21±1.89
Transgenic
8.48±1.71
7.84±2.33
The transgenic nature of T1 tomato plants was confirmed by the expression of the marker gene gus
that encodes the enzyme ƒÀ
-glucuronidase (GUS). GUS was foundonly in leaves of transgenic
plants especially in the fractions enriched with chloroplasts:
Supernatant from transgenic tomato plants
- 1.31 imp .s
-1 .
mg-equivalent -1 of GUS ;
Chloroplasts fraction from transgenic plant – 198.18 imp .s
-1 .
mg-equivalent -1 of GUS ;
Supernatant from nontransformed plants – 0.0 imp .s
-1 .
mg-equivalent -1 of GUS ;
Chloroplast faction from nontransformed plants - 0.0 imp .s
-1 .
mg-equivalent -1 of GUS .
According to the data obtained for expression of marker enzymes, it was concluded that
integration of the genes was inherited in T1 generation of tomato seedlings var. Ventura after
infection of T0 tomato seedlings with the transconjugant of triparental mating.
In order to prove the expression of target gene ugt, the activity of UDPG-transferase was
tested both in nontransformed and transgenic plants of T1 generation. As was shown in Table 3, the
conversion activity of IAA to IAA-glucose was about 4 times more active in cytosol of transgenic
plants and 2 times more active in chloroplasts fractions of transgenic plants compared to
nontransformed plants.
Table 3. Activity of UDPG-transferase in nontransformed and transgenic T1 plants of tomato
cv. Ventura
Variant
UDPG-transferase, nmol of IAA-
glucose/mg of protein/h
Nontransformed
cytosol
32.07±15.14
chloroplasts
66.95±25.50
Transgenic
cytosol
132.50±50.48
chloroplasts
114.70±9.10
The PCR analyses had shown the integrity of the genes ugt and acb in genomic DNA of
transgenic tomato (Figure 1, A, B, C and E). The Southern blot revealed more homology to the
probe (made from PCR product with primers to the gene ugt from cloned pBluescript) in DNA from
roots of transgenic tomato (Figure 1, D).
Higher activity of UDPG-transferase in transgenic plants was perhaps the reason for the
increased level of free IAA and alkali labile bound IAA. The free content of IAA was about two times
higher in transgenic plants compared to
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