3. Results
All four field trials were harvested approximately 19–24 months
after planting and blackheart was observed in control fruit from
each trial. The control lines exhibited the highest incidence (50%)
and greatest severity (34%) of symptoms (Table 2). Blackheart
expression from the transgenic lines, on the other hand, was
much more variable with some lines free from blackheart on each
occasion or exhibited blackheart in some trials and not others.
Severity of symptoms was also variable. Those transgenic lines
that remained free of blackheart over two or more harvests, while
growing under winter production conditions followed by placement
of fruit in cold storage for three weeks prior to assessment,
were regarded as blackheart resistant. Out of a total of 39 transgenic
lines examined, 15 (38%) exhibited no blackheart on two or
more occasions while five consistently performed as well as or better
than the control plants in terms of fruit characteristics and fruit
quality (Table 3). PPO activity averaged 2.24EAUmin−1 g−1 in the
blackheart resistant lines, whereas control fruit and those transgenic
lines exhibiting blackheart symptoms had levels over ten-fold
higher at 25.8EAUmin−1 g−1.
Irrespective of the transformation system employed or the construct
used, blackheart resistant lines were produced, although
the frequency was higher for Agrobacterium-mediated transformation
than for biolistic transformation. Plants transformed with
Agrobacterium produced blackheart resistant plants on 5 of 9 occasions
(56%) with plants surviving the transformation, selection and
regeneration process; while biolistics yielded blackheart resistant
plants on only 10 of 30 occasions (33%). The choice of constructs
also had a bearing on attainment of blackheart resistant plants with
sense ppo constructs producing resistant lines on 7 of 15 occasions
(47%), opp.ppo inverted repeat constructs on 3 of 6 occasions (50%),
opp.i.ppo intron hairpin constructs on 4 occasions of 13 (31%) and
dual gene intron hairpin opp.cca.i.acc.ppo constructs on 1 occasion
of 5 (20%).
Not only was there variation in blackheart expression between
transgenic lines, there was also variation in fruit size, total soluble
solids and titratable acidity (Table 3). Some transgenic blackheartresistant
lines, such as V59(1) and 158-2, consistently produced
heavier fruit (up to 2.6 times) than the poorer performing lines, such
as 91-11 and 152-19. The latter consistently produced aberrantshaped
and also significantly smaller fruit than control plants
(P < 0.05). Most transgenic lines were as sweet as or sweeter than
the control fruit.
However not all transgenic lines were of normal growth and
appearance as 36% could be classed as off-types (or somaclonal
variants) with some of the transgenic lines characterised by small,
spherical fruit, some had extra knobs around the base of the fruit
or they produced an under-developed top of the fruit