Results and discussion Greenhouse spring production
Spring production was evaluated in trials performed in two
different locations: Monsampolo and Pontecagnano. Spring
production was divided into early production, consisting of the
first four harvests, and total production, consisting of sixteen
harvests. Early spring production corresponds to the cultivation
period from March to the first half of May, with temperatures
somewhat low for fruit-set and growth. During this period, the
average minimum and maximum temperatures ranged from 7 to 17C in
southern Italy (Pontecagnano) and from 14 to 17C in central
Italy (Monsampolo). The transgenic parthenocarpic hybrids gave a
significantly higher yield, on the average a six-fold increase
during early production, in comparison to their controls (Table
1). The increment in the number of fruits per plant and the
higher average weight of the fruits were the main causes of the
increased early spring production of the parthenocarpic hybrids.
The suboptimal/adverse environmental conditions did not affect
the growth of GM parthenocarpic fruits and the average fruit
weight was significantly higher in the GM eggplants in
comparison to untransformed controls. The traditional
parthenocarpic cultivar Talina produced fruits with an average
weight similar to those of GM eggplant fruits (Table
1).
However, due to the higher number of fruits per plant, the
productivity of the transgenic parthenocarpic hybrids was also
increased, by an average of five-fold, when compared to cv
Talina (Table
1). During the whole harvesting period, the GM
fruits were always seedless (Fig. 1) and were normal in both
size and shape.
The increased productivity of GM hybrids characterised both the
early spring production (i.e. the first four harvests) and the
whole spring production cycle (i.e. sixteen harvests). During
the whole spring production cycle, the hybrids P1 and P2 gave an
average yield that was 46% higher with respect to the
corresponding control C1 (Table
1). The hybrid P5 gave a 37%
higher yield with respect to its control C2. The average total
number of fruits produced per plant in both locations was
similar in all the hybrids (89 fruits/plant). However, the
higher average weight of the GM fruits led to a higher total
yield of transgenic hybrids with respect to their controls. When
considering the whole spring cultivation cycle, the
parthenocarpic cultivar Talina gave a total production that was
not significantly different from either of the three GM hybrids
(Table
1).
Open field (summer) production
Summer production was evaluated in an open field trial carried
out during the optimal period of eggplant cultivation. Plants
were transplanted on May 20th and the last harvest took place on
September 11th. The early production of the transgenic hybrids,
consisting of the first three harvests, was significantly higher
than that of the untransformed hybrids (Table 2): P1 and P10
hybrids yielded, respectively, 36% and 76% more than their
corresponding controls, C1 and C10. The difference in
productivity between P1 and C1 hybrids, which have long-shaped
fruits, was caused by the higher average weight of GM fruits.
When comparing P10 and C10 hybrids, which have sub-oval fruits,
the higher yield obtained with GM plants during the early
harvesting period was due to the increased number of fruits per
plant.
Consisting of ten harvests, the total production of P1 hybrids
was 37% higher than control C1 eggplants (Table 2). The
difference in total yield between P1 and control C1 hybrids was
statistically significant and due both to the higher number of
fruits/plant and to the increased weight of GM fruits. It is
noteworthy to point out that when considered individually,
neither trait (number of fruits/plant or fruit weight) showed
statistically significant differences between the GM and
untransformed plants (Table 2). Although higher in P10 hybrids
in comparison to its control C10, the total yield (the number
and average weight of the fruits) was not significantly
different between the two. During the whole harvesting period,
fruits from both P1 and P10 parthenocarpic hybrids were always
seedless (Fig. 2), whilst control fruits always had seeds.
Therefore, under open field cultivation, the DefH9-iaaM gene had
a positive influence on fruit quality, as GM DefH9-iaaM fruits
were always seedless. Fruit quality affects the economic value
of eggplant production.
Although the environmental conditions were optimal and
consequently did not affect negatively fruit-set, the DefH9-iaaM
gene caused both faster development and growth of the fruits as
indicated by the increased early-summer production (the first
three harvests). In this regard, it is worthwhile to stress that
expression of the DefH9-iaaM gene takes place in the placenta
and ovules before pollen development. As a consequence, in GM
parthenocarpic plants fruits are seedless and fruit development
initiates well before non-GM controls [11].
In all trials we have never used homozygous lines because
growers mostly employ F1 hybrids. The use of hemizygous primary
transformants as pollinator plants allowed us to obtain in
rather short time, by in vivo selection for kanamycin
resistance, F1 plants transgenic for the DefH9-iaaM gene. Young,
healthy and vigorously growing plants did not produce seeds.
However, it is possible to obtain seeds from aged DefH9-iaaM
transgenic plants both by selfing and crossing. By exploiting
the delayed female fertility we have produced the homozygous
plants needed as male parents for rapid seed multiplication of
F1 eggplant hybrids. Therefore, the female sterility of young
and mature plants is not an insuperable hindrance for mass
propagation and commercial fruit production.
Expression of the DefH9-iaaM gene takes place during both flower
and fruit development in transgenic parthenocarpic eggplants
The DefH9 gene is expressed specifically in the placenta and
ovules during early phases of flower development [6]. To
determine whether expression of the DefH9-iaaM gene also occurs
during later stages of fruit growth and whether it is influenced
by pollen fertilization, mRNA from transgenic flowers
and fruits obtained either from emasculated or hand pollinated
flowers was analyzed by RT-PCR at different stages of
development, until the fruit attained a length of 28 cm. An
amplicon of 161 bp corresponding to the spliced DefH9-iaaM mRNA
was detected in all stages analyzed (Fig. 3, lanes 26).
Thus, the presence of the mRNA of the DefH9-iaaM gene and
consequently its action is most likely not limited to early
stages of flower and fruit development. Pollination did not
affect the steady state level of DefH9-iaaM mRNA (Fig. 3,
compare lane 5 and lane 6).
Treatment with auxin often causes parthenocarpic development in
several plant species [for review, see: [12]]. However, in some
species and/or varieties, to efficiently sustain fruit growth,
the hormonal treatment of the flowers must be repeated [13]. The
finding that DefH9-iaaM mRNA is also present during later stages
of fruit development is consistent with the interpretation that
in DefH9-iaaM parthenocarpic plants, the placenta, the ovules,
and the tissues derived therefrom are a source of auxin
during the whole growth of the fruit. As a consequence, they
efficiently sustain fruit growth.
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