Genetic engineering of parthenocarpic fruit development in tomato

Genetic engineering of parthenocarpic
fruit development in tomato
Tomato for the fresh
market
DefH9-iaaM gene is able to sustain
parthenocarpic fruit development
in two different tomato types (either
cherry or round-shaped fruits).
Fruit set percentage and fruit
quality were not negatively affected
in the primary transformants in
conparison to selfed flowers of
untrasformed control plants.

DefH9-iaaM gene is able to sustain parthenocarpic fruit development in two different tomato type
(either cherry or round-shaped fruits). = selfed; EM= emasculated.

DefH9-iaaM is expressed in tomato flower buds

Percentage of fruit set
100
90
80
70
60
50
40
30
20
10
0
CM #11 CM #12 CM #13 CM #14 CM Cont L.276 #1 L.276 #4 L.276 #6 L.276 #7 L.276
Cont
genotypes
Fruit set in emasculated flowers of GM DefH9-iaaM tomato plants
Untransformed controls do not set fruits from emasculated flowers

Average fruit weight
140,0
120,0
100,0
Selfed
Emasc.
80,0
g
60,0
40,0
20,0
0,0
CM
#11
CM
#12
CM
#13
CM
#14
CM
Cont
L.276
#1
L.276
#4
L.276
#6
L.276
#7
L.276
Cont
genotypes
Ficcadenti N., Sestili S., Pandolfini T., Cirillo C., Rotino G.L., and Spena, A. (1999) Genetic
engineering of parthenocarpic fruit development in tomato. Mol. Breed. 5, 463-470.

Greenhouse trial for evaluation of parthenocarpic transgenic hybrids
(Spring Cultivation)
Material
3 transgenic hybrids of round type tomato (one of them was F1 Giasone)
1 transgenic hybrid of cherry type tomato (experimental hybrid)
Untransformed isogenic controls

Number of fruits/plant (early production)
25,0
cont rol
transgenic
20,0
15,0
N°
10,0
5,0
0,0
H F1 Giasone H.F1 95-514 H.F1 95-516
hybrids

N°
40,0
35,0
30,0
25,0
20,0
15,0
10,0
5,0
0,0
Number of fruits/plant
total prod control
total prod transgenic
H F1 Giasone H.F1 95-514 H.F1 95-516
genotypes

Average fruit weight (early prod)
g
160
140
120
100
80
60
40
20
0
H F1 Giasone H.F1 95-514 H.F1 95-516
hybrids
control
transgenic

Early production
3500
control
transgenic
3000
2500
g/plant
2000
1500
1000
500
0
H F1 Giasone H.F1 95-514 H.F1 95-516
Hybrids

Total Production
control
5000
4500
transgenic
4000
3500
3000
2500
2000
1500
1000
500
0
H F1 Giasone H.F1 95-514 H.F1 95-516
Hybrids

Giasone untransformed control
Parthenocarpic DefH9-iaaM Giasone

Cherry type tomato
Transgenic line = L CM iaaM x L4
Control line = L CM x L4

Cherry tomato
Number of fruits
60
transgenic
50
control
40
N°
30
20
10
0
I truss II truss I+II+III trusses total

Cherry tomato
Average fruit weight
30
transgenic
control
25
20
N°
15
10
5
0
I truss II truss I+II+III trusses total

Cherry tomato
Yield
1600
1400
transgenic
control
1200
1000
g/plant
800
600
400
200
0
I truss II truss I+II+III trusses total

Conclusions
The DefH9-iaaM gene is able to sustain parthenocarpic fruit
development in different types of tomato for the fresh market,
Marketable fruits are produced under environmental conditions
adverse for pollination and/or fertilization.
No adverse effects on fruit quality
Acciarri N., Ferrari V., Vitelli G., Ficcadenti, N., Pandolfini, T., Spena, A., and Rotino, G.L. (2000) Effetto della
partenocarpia in ibridi di pomodoro geneticamente modificati. Informatore Agrario 4, 117-121.

Genetic engineering of parthenocarpic
fruit development in tomato
Industrial tomato
(The pickelhauben problem)
In the cv UC82, a cultivar typical
for processing tomatoes, the
original gene DefH9-iaaM causes
the production of parthenocarpic
malformed fruits.
The malformations are similar to
those caused by an excess of
exogenous auxin and/or an
higher sensitivity of flowers to the
hormonal treatment.

A genetic solution of the “pickelhauben” problem in tomato
Control fruit
GM malformed fruit
Development of a genetic tool to
reduce the expression and
consequently the action of the
DefH9-iaaM gene
Solution:
Downregulation of gene expression at the
post-transcriptional level by replacing 53
b upstream the AUG initiation codon of
the iaaM gene with 87 b of the rolA intron
mutated at the splicing sites.

In vitro transcription – translation analysis
a) sequence of the mutated rolA intron and scheme of the DefH9-RI-iaaM gene
b) in vitro transcription-translation. Lane 1 and 2 iaaM transcripts with different ULR.
Lane 4 and 5, in vitro translation of iaaM (lane 5) and RI-iaaM (lane 4).
RI= reduced by intron

= selfed; EM= emasculated
RT – PCR analysis
Estimated steady state level of
mRNA in tomato flower buds:
DefH9-RI-iaaM: 1x10 -8 of total mRNA
DefH9-iaaM: 1x10 -7 of total mRNA

Total IAA content in control and GM flower buds
DefH9-iaaM: 100% (47/47) of the transgenic plants with malformed fruits
DefH9-RI-iaaM: 10% (5/44) of the transgenic plants with malformed fruits
IAA content in flower buds
30,00
25,00
20,00
nmol/g
15,00
10,00
5,00
0,00
ctr DefH9-RI-iaaM DefH9-iaaM
genotypes

Average, maximun and minimum values in independent
transgenic plants for DefH9-Ri-iaam (18 plants), DefH9-
iaaM (4 plants) and controls for percentage of fruit set
from emasculated flowers and average fruit weight
derived from emasculated and selfed flowers.
Fruit
set (%)
Fruit weight
(g)
emasculated selfed
aver min-max aver min-max
RI-iaaM 100-73 46 88-25 58 91-42
iaaM 100-87 77 105-32 67 81-39
control 12-0 10.2 48

Conclusions
The modification of the 5’ULR of the DefH9-iaaM
gene (i.e. DefH9-RI-iaaM gene) allows to produce
high quality parthenocarpic fruits in industrial
tomatoes (e.g. UC82).
This method may solve the problem of a too high
expressivity of a trait(s) when using transgenes
affecting phytohormone synthesis.
Pandolfini T., Rotino G.L., Camerini S., R. Defez, Spena A., 2002. Optimisation of transgene action at
the post-transcriptional level: high quality parthenocarpic fruits in industrial tomatoes. BMC
Biotechnology 2(1) http://www.biomedcentral.com/1472-6750/2/1/.