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BIOTOPICS

and its 20-year-old commitment not to discriminate
against gene-spliced products in general. Within a few
months, according to senior FDA officials, the agency
expects to announce a new requirement that all genespliced foods come to the agency for pre-market evaluation. FDA officials orchestrated a phony ‘demand’ for
such a change by holding public meetings at the end
of 1999 that offered activists an opportunity to stuff the
ballot box, and at which the FDA packed the discussion panels with radical opponents of biotechnology.
This impending change for the worse in domestic
regulatory policy tied the USA delegation’s hands at the
Codex task force, and will do so in other international
forums. Knowing that their own policy will soon contravene the scientific consensus on biotechnology regulation constrains FDA officials from pushing the scientific line. As a result, the Codex task force is en route
to describing and codifying various procedures and
requirements that are more appropriate to potentially
dangerous prescription drugs or pesticides than to
gene-spliced tomatoes, potatoes and strawberries. They
will likely include long-term monitoring for adverse
health effects and a battery of tests for genetic stability,
toxins, allergenicity, and so on. The most egregious is
something called ‘traceability,’ an array of technical,

labeling and record-keeping mechanisms to keep track
of a plant ‘from dirt to dinner plate’, so that consumers
will know whom to sue if they get diarrhea from genespliced prunes, and providing, in the words of the

European Commission delegate, ‘a tool governments
can use to remove products from the market’.
The prospect of unscientific, overly burdensome
Codex standards focused on gene-spliced foods is
ominous, because members of the World Trade Organization (WTO) will, in principle, be required to follow them, and because they will provide cover for
unfair trade practices. Jean Halloran (Consumers International, London, UK) considered Codex standards to
be a tool to stop the import of biotechnology foods.
‘The Codex is important because of the WTO. If there
is a Codex standard, one country cannot file a challenge
(for unfair trade practices) against another country that
is following the Codex standard. But when there is no
Codex standard, countries can challenge each other on
anything.’ Thus, standards such as those promulgated by
Codex, and regulations such as the recent, execrable
biosafety protocol negotiated under the UN’s Convention on Biological Diversity (the Cartegena Protocol
on Biosafety) are intended by their proponents not to

protect human health or the environment, but to stifle
trade in the products of the new biotechnology.
Food production has low profit-margins and cannot
easily absorb the costs of gratuitous regulation. The
over-regulation of gene-splicing prevents its wide
application to food production, deprives farmers of
important tools for raising productivity, and denies to
food manufacturers and consumers greater choice
among improved, innovative products.

Less is better: new approaches for seedless
fruit production
Fabrice Varoquaux, Robert Blanvillain, Michel Delseny and Patrick Gallois

S

eedless fruits are a desirable commodity for consumers, and have been produced using traditional
farming and breeding methods for many centuries. Evidence that seedless forms of Vitis vinifera
grapes have been prized for many centuries as
dried fruit is provided by Greek philosophers such as

Hippocrate, Platon and in the writings of ancient Egypt
of 3000 BC. However, the use of current agricultural
practices to achieve seedlessness has in-built disadvantages. Here we discuss novel approaches that have
emerged over the past few years that open up new
possibilities for breeding seedless plants. These include
quantitative trait loci, manipulating genes that promote
parthenocarpy and interfering with seed development
using ‘terminator’ technology. Several patents based on
recombinant DNA techniques illustrate the current

F. Varoquaux, R. Blanvillain, M. Delseny and P. Gallois (gallois@
univ-perp.fr) are at the Laboratoire Génome et Developpement des
Plantes, CNRS UMR 5096, Université de Perpignan, 52 avenue de
Villeneuve, 66860 Perpignan Cedex, France.
TIBTECH JUNE 2000 (Vol. 18)

industrial interest in this field, and we discuss the likely
positive and negative impacts of these novel strategies
on food production.
To obtain fruits without seeds is a physiological challenge. Fruit development comprises early development

and maturation (Fig. 1). In most plants, normal early
fruit development involves three phases1: (1) fruit setting, (2) cell division, and (3) cell expansion. During the
first phase, the ovary takes the decision to either abort
or to go further in fruit development. The next phase
is the growth of the fruit as a result of cell division; during this phase, the increase in fruit size is low because
the dividing cells are small and tightly compressed. The
final size of a fruit will be highly dependent on the
number of cells. The third and last phase begins after
cell division ceases, the fruit grows by the increase in
cell volume, until it reaches its final size. Cell expansion commonly increases fruit size by 100-fold, and
this makes the greatest contribution to the final size
of the fruit. At the end of early development, a green
fruit is obtained, which has the size of a mature fruit,

0167-7799/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01448-7

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BIOTOPICS

(a)
(b)
Fruit
Seed
development development

Phase I

Ovary development,
fertilization
and fruit set

Ovary mutants
(Corinth grapes,
cucumber, tomato)

Anthesis
Pollination

Type of

seedlessness

Parthenocarpy

Incompatible pollen
(citrus fruits)

Fertilization

Fruit set

Sterile pollen
(cucumber)

Cell division, seed
formation and
embryo development
Phase II

(c)

Example
of defect

Cell division

Abnormal meiosis
in triploid plant
(watermelon)

Seed
formation

Defect in endosperm
development (grapes)

Embryo
development
Cell expansion and
embryo maturation

Stenospermocarpy

Globular stage

Cell expansion
Phase III

Embryo maturation
Mature
green fruit Embryo mature

Ripening

trends in Biotechnology

Figure 1
Schematic representation of the links existing between fruit and seed development: (a) different steps in fruit development, (b) different
steps in seed development, and (c) examples of seedless fruit. Arrows drawn between (a) and (b) indicate the positive effect on fruit development of events preceding, or linked to, seed development. Arrows between (b) and (c) indicate the points in seed formation that might
be deficient in seedless varieties.

and the maturation phase occurs from this point
onwards.
During early fruit development, many pathways
of communication between the sporophyte and the
gametophyte are established. It is generally considered,
for instance, that the decision of whether or not to set
fruit is dependent on the successful completion of
pollination and fertilization. The pollen produces gibberellins and it is well known that the application of
exogenous gibberellins can induce an increase in the
content of auxin in the ovary of an unpollinated flower
of the tomato plant2, and therefore trigger fruit setting
in the absence of fertilization. Additionally, the developing embryo controls the rate of cell division in the
surrounding fruit tissue1, and there is evidence that the
number of developing seeds influences the final size
and weight of a fruit3. It is generally considered that
developing seeds promote cell expansion within the
fruit by the production of auxin and other unknown
molecules1. Therefore, a developing seed has a very
important role to play in the early development of
fruit; producing seedless fruits might therefore be

complicated. Nevertheless, seedlessness is not uncommon and seedless crop plants have existed for many
centuries4.

234

Classification of seedlessness
A plant is considered to be seedless if it is able to produce a fruit with no seed, traces of aborted seeds or a
much-reduced number of seeds. Different kinds of seedlessness can be distinguished depending on the time at
which the development of the seed is disrupted (Fig. 1).
Parthenocarpic fruits are seedless because the ovary is
able to develop without ovule fertilization. Parthenocarpy can also be the only way to produce fruits, or it
can be facultative, depending on the fertility of the plant.
If the plant is sterile, parthenocarpy arises without any
external stimulation and requires a vegetative method of
propagation (e.g. bananas and pineapples). Many tomato
parthenocarpic mutants are good examples of facultative parthenocarpy because they only produce seedless
fruit if fertilization does not occur. Therefore, in many
cases, parthenocarpy can be induced by factors that
inhibit fertilization. The most classical of these factors
include certain environmental conditions such as low

temperature, light, and physical or chemical treatments
of the female flower or of the pollen. Stenospermocarpy
enables another form of seedlessness in which the fruit
contains partially formed seeds that have aborted after
fertilization5. Further, fruits with nonviable seeds must
also be considered to be functionally seedless.
TIBTECH JUNE 2000 (Vol. 18)

BIOTOPICS

Traditional seedless-fruit technologies
Watermelon
The seedless watermelon contains partially developed
seeds, and is a classic example of stenospermocarpy. To
obtain such a plant, a cross is made between a tetraploid
maternal parent and a diploid pollinator, resulting in a
triploid plant that is self-infertile because of a gameticchromosome imbalance. This triploid plant must be
pollinated by a diploid plant in order to produce a seedless watermelon6. Seedless watermelons are gaining
popularity, and the aborted seeds are very soft and present little inconvenience for consumers. The shape,
flavour and yield are as good as seed-producing cultivars and have a longer shelf life; nevertheless, some
problems still exist:
• producing the tetraploid parental line (by treating
seedlings with colchicine);
• finding compatibility between the diploid pollinator
and the tetraploid mother plant7; and
• the triploid seeds have a thicker seed coat, which
decreases their vigour and germability.
Consequently, these difficulties result in a higher cost
of seed production; it should be noted that tetraploid
plants are not viable in most other species.
Citrus fruits
Citrus fruits that have less than five seeds are said to
be seedless. Many mutants exist, and they express
parthenocarpy at different levels. In some cultivars,
where the level of parthenocarpy is high, pollination is
not required for fruit formation (e.g. tahiti lime, satsuma mandarin). In other cases, with a low level of
parthenocarpy, pollination is required to set fruit (e.g.
star-ruby grapefruit); to obtain seedlessness, it is then
necessary to pollinate with dead pollen or, preferably,
to combine genetic seedlessness with self-incompatibility. Therefore, to produce and to select new cultivars
of seedless citrus fruits is not an easy task. In addition,
one of the best sources of seedlessness, the cultivar satsuma, is not a suitable parent for breeding because it
causes embryo-sac degeneration8, sterile pollen and the
few seeds present per fruit are often polyembryonic.
Achieving seedlessness using triploidy has also been
tested; unfortunately, in Citrus, there are very few
tetraploid parent lines to cross with diploids, and
the resulting triploid lines are often fruitless or have
undersized fruits9.
Grapes
The case of the grapevine is interesting because two
kinds of seedlessness exist. The first is observed in the
Corinth cultivars, and is caused by parthenocarpy; the
berries of these plants are very small and spherical, and
their only use is to make dried fruits. The second kind
of seedlessness is caused by stenospermocarpy, for
example, Thompson cultivars. Here, traces of seed are
present but they are not woody. However, to obtain a
bunch of grapes with a substantial number of welldeveloped berries, it is necessary to apply particular,
complex hormone treatments. Gibberellic acid is used
to thin berries from the cluster, elongate the cluster,
increase berry size and reduce the trace of seeds. The
concentration of the first gibberellic-acid spray is critical in order to obtain an optimum level of bunch loosening and is cultivar dependent10. A second spray can
TIBTECH JUNE 2000 (Vol. 18)

also be carried out to increase the size of the berries.
This method of achieving seedlessness is technically difficult, weather dependent, labour intensive, expensive
and has the drawback of potentially introducing synthetic
chemicals into the human diet.
Cucumber
Sex inherence plays an important role in cucumber
breeding. In the cucumber, several primary sex types
can occur: monoecious (separate pistillate and staminate flowers on the same plant), androecious (staminate
flowers only), gynoecious (pistillate flowers only),
hermaphroditic (hermaphrodite flowers only), andromonoecious (staminate and hermaphroditic flowers on
the same plant) and dioecious (plants bearing either
all-male or all-female flowers). The cucumber produced
by hermaphrodite flowers is unmarketable (as a result
of its large seed cavities). It is therefore necessary to
cultivate only monoecious or gynoecious plants, the
latter having the greatest yield potential. The problem
is to sow a suitable ratio of the seeds of gynoecious
plants and the seeds of pollinators, and this can be
overcome by genetically introducing genes for fruit
parthenocarpy into gynoecious plants. Some parthenocarpic cultivars exist and they are intensively cultivated;
in addition, they have the advantage that fruit setting is
not dependent on weather conditions, or on manual or
insect pollination. Stenospermocarpy can also be induced
by the pollination of non-parthenocarpic cultivars using
irradiated pollen.
Advantages of seedlessness
Fruit quality
Seedless fruits have many gustatory advantages. Seeds
are often hard, can have a bad taste and can be harmful; for example, grapeseeds can bring about digestive
problems11. In addition, if the seeds and their cavities
are replaced with edible fruit tissue, this is more attractive to the consumer. An illustration of this is the seedless pickled gherkin, which is more crunchy, firmer and
fleshier than its seeded variety12. It is possible to speculate that this advantage might be even greater for species
with a large seed, such as peaches and mangos, or for
those with a large cavity that is filled with numerous
seeds, such as melons and papayas.
The shelf life of seedless fruit is expected to be longer
than seeded fruit because seeds produce hormones that
trigger senescence. This effect has been observed in
watermelons, in which seeds are the origin of fruit
deterioration. Seedless watermelons develop a mealy
texture and become overripe significantly later than
seeded varieties. Studies have shown that seedless
tomato fruits are tastier than the seeded variety. Indeed,
seedless tomato fruits exceed seeded fruits in dry-matter
content by up to 1% (Ref. 13), contain more sugars,
less acidity13, less cellulose13 and have considerably
more soluble solids14 than seeded cultivars.
Production
One of the most critical steps in fruit formation and
the maintenance of yield is pollination. Parthenocarpy
can be a good way of eliminating problems owing to
poor pollination. Many species, including tomato, eggplant and peppers, will only produce sufficiently fertile
pollen grains in specific climate conditions. For example,

235

BIOTOPICS

tomato pollination occurs in a very narrow range of
temperatures: 158C–218C (night) and 308C–358C (day).
Parthenocarpic tomato plants (cultivar severianin) produce a higher yield and fruit set in colder temperatures
(night temperature ,128C) than seeded cultivars15.
Thus, parthenocarpy is potentially useful for producing
vegetables in winter months16 or, more generally, to
ensure yield stability in case of unfavourable pollination
conditions. Moreover, it has been shown that seed
development in fruits restricts the yield in cucumber17,18
and tomato19.
Technology protection
A new potential use of seedlessness arose with the
development of plant genetic engineering. In the case
of an association of a transgene with a seedless character, the transgene would be unable to be disseminated
by seed dispersal (e.g. by consumption of the fruit, dispersal on the ground, or by birds). In this case, the only
possible dissemination of the transgene would be by
pollen. This might be a problem, depending on the
ability of the species concerned to cross-hybridize with
the surrounding plants. The resulting hybrid plants
would be seedless only if the transgene is genetically
linked to the seedless character. Additionally, seedlessness can be used to protect genetically modified crops:
linking a transgene with seedlessness would prevent
unfair appropriation of the transgene by simply crossing the transgenic plant with another commercial variety. Finally, if the fruits are seedless, new seeds will have
to be bought at the beginning of each planting season,
thus providing secure commercial protection to the
investment in plant breeding. However, the latter point
is highly controversial because rather than saving a part
of their harvested seeds for the next planting season,
the farmers would have to buy their seeds each year.
Few seedless plants exist on the market
Seedless mutants exist in many species but they are
not currently produced as seedless varieties because
mutations for parthenocarpic fruits are often pleiotropic
and associated with unfavourable characteristics for
breeding programmes, such as male or female sterility.
Another problem that is frequently encountered in
parthenocarpic plants is their undersized and misshapen
fruits. Moreover, parthenocarpy is often controlled by
complex multigenic systems, which makes breeding
difficult. Thus, seedless plants are very difficult to produce, mainly because the seed is important for fruit
development. Nevertheless, even though the production of seedless fruit is expensive, interest in seedlessness
is currently increasing because of its advantages; ample
proof of this is reflected by the increase in the number
of patents and articles concerning seedlessness.
Towards the understanding, discovery and use
of genes for seedlessness
What can be a gene for parthenocarpy?
Fruit setting and development is triggered by growth
hormones that are produced and regulated by pollen or
developing seeds. In parthenocarpic plants, the ovary
develops as a result of exogenous hormone treatments
or genetic stimuli. The ovaries of parthenocarpic plants
contain high levels of auxins and gibberellins, and it has
been proposed that genes for parthenocarpy might

236

affect hormone production, transport and/or metabolism in order to promote ovary growth precociously;
pollination and fertilization are therefore no longer
needed4.
Several parthenocarpic mutants have been studied, in
particular, the pat mutant of the tomato. These mutants
produce parthenocarpic fruits, but the genes involved
show some pleiotropic effects, such as male and female
sterility, as a result of some floral developmental aberrations. In the pat mutant, ovary growth begins before
anthesis. This timing and the various aberrations in
flower development suggest that, in this mutant,
parthenocarpy is a secondary effect of the activity of a
gene that controls organ identity at the early stages of
floral development. It is possible that genes affecting
organ identity and development can have a delayed
effect on processes such as ovary development20. Moreover, some deficiencies in cell elongation in different
organs (short anthers, smaller-sized seeds and fruits,
undersized integuments) suggest that the pat gene
product interacts with gibberellin metabolism. The
application of gibberellin to flowers causes the restoration of a wild-type anther phenotype, but does not
restore female fertility.
Another indication of the link between genes
involved in parthenocarpy and gibberellin metabolism
is found in the parthenocarpic mutant of Arabidopsis
thaliana called SPINDLY (SPY), whose gene product
is anticipated to participate in the regulation of the gibberellin signal-transduction pathway21,22. The phenotype
of this mutant, including parthenocarpic siliques and
partial male sterility, can be phenocopied in wild-type
A. thaliana using repeated gibberellin treatments. SPY
is epistatic to mutants affected in gibberellic-acid
biosynthesis23, and also to gai (gibberellin insensitive)24,
an A. thaliana dwarf mutant with reduced gibberellin
perception. Double-mutant analysis suggests that the
SPY gene encodes a negative regulator in part of the
gibberellic-acid signal-transduction pathway. The SPY
gene encodes a protein containing a tetratricopeptide
repeat domain; few genes with tetratricopeptide repeats
have been isolated in plants, but in other organisms,
these repeats are associated with transcriptional repression
or cell-cycle regulation23.
Development of new tools and molecular markers
The genetics of stenospermocarpy have been well
studied in grapevines. It was proposed that seedlessness
might be controlled by three complementary recessive
genes: a1, a2 and a3, independently inherited and
regulated by a dominant gene I 25. There is an urgent
requirement to find molecular markers of seedlessness
because grapevines do not produce bunches of grapes
before the age of three to four years, thus slowing down
selection schemes. Recently, rapid amplified polymorphic DNA (RAPD) markers have been discovered
using bulk segregant analysis that appeared to be tightly
linked to gene I (at 0.7 and 3.5 centimorgans)26. The
importance of the contribution of the gene I to grape
stenospermocarpy was confirmed by statistical analysis
using the closest RAPD marker converted to a codominant sequence-characterized amplified region (SCAR).
It was revealed that this marker accounted for at least
78.7% of dry-matter variation in the seed; dry matter
being a criterion used to evaluate the size and hardness
TIBTECH JUNE 2000 (Vol. 18)

BIOTOPICS

of seeds. The same SCAR marker could be used as a
starting point to clone this gene. Although the isolation of such a gene would be interesting for basic
science, it is not required for a successful breeding
programme.
Identification of proteins associated with parthenocarpic
fruits
Gene expression associated with natural parthenocarpy in tomato ovaries has been studied27. At anthesis, the ovaries of a non-parthenocarpic line and of a
near-isogenic parthenocarpic line (pat-2) of tomato
were isolated and RNA was prepared. An in vitro translation, followed by two-dimensional polyacrylamide
gel electrophoresis, revealed the differential expression
for at least six in vitro translation products27. One of
these, a 30 kD protein, was previously described in the
flowers of other parthenocarpic mutants28 (pat-3 and
pat-4). In the future, cloning of the corresponding
genes might open up new avenues for manipulating
parthenocarpy. In addition, the study of parthenocarpic
mutants in A. thaliana is expected to contribute
additional genes.
Patents for seedlessness in plants
Inducing parthenocarpic fruits in F1 plants
The different patents on methods using recombinant
DNA to induce parthenocarpic fruits are based on the
observation that parthenocarpy is positively correlated
with the level of auxin in the ovary, and that the exogenous application of auxin29, gibberellins30, cytokinins30
and auxin-transport inhibitors31 to cucumber flowers
induces parthenocarpy. It has also been shown that the
application of these hormones causes an increase in the
auxin content of the cucumber ovary32. Moreover, a
high content of auxin has been discovered in parthenocarpic ovaries of the tomato33,34. Thus, it is theoretically possible to induce parthenocarpy in transgenic
plants expressing an auxin or cytokinin biosynthetic
gene in the ovary, between anthesis and early fruit
development. Consequently, the first patent35 proposes
to fuse an ovary-specific promoter with the Agrobacterium
RolB gene, thus interfering with plant auxin production. The second patent36 presents the fusion between
pDef H9, an Antirrhinum majus promoter that is specific
to the ovary, and the RolB gene (to obtain parthenocarpy) or a cytotoxic gene (to obtain female sterility).
In a third patent37, the promoters specific to the ovary
or developing fruit are pGH3, pAGL and pPLE36.
The genes to be expressed encode an isopentenyl
transferase (cytokinin biosynthesis) or a tryptophan
oxygenase (auxin precursor biosynthesis).
All the patent authors claim to have obtained
parthenocarpic fruits. Nevertheless, the only result
published to date is the obtainment of parthenocarpic
eggplant and tobacco38. Eggplant and tobacco have
been transformed by a fusion between the DefH9 promoter and the IaaM gene (tryptophan monooxygenase). When the flowers are emasculated, the transgenic
eggplants produce marketable seedless fruits. Moreover,
transgenic eggplants perform fruit set and growth during winter conditions, under which non-transgenic
lines cannot set fruit. This shows that it is clearly possible to induce parthenocarpy by expressing a gene
encoding a step in the auxin biosynthetic pathway in
TIBTECH JUNE 2000 (Vol. 18)

the ovary. However, one disadvantage is that the flowers must be emasculated in order to produce seedless
fruits. For ease of use, this system must be coupled with
male sterility. Another problem is that, to be useful, this
system must be introduced into an F1 hybrid seedproduction strategy in order to obtain and sell viable
hybrid F1 seeds that will germinate and give rise to
seedless plants. This aspect is not addressed in the patents
described previously.
Preventing the development of F2 seeds
Combining two independently harmless genes to produce a
cytotoxic effect
The principle of this patent39 lies in the use of two
individually harmless genes that are cytotoxic in tissues
when both expressions are combined. The product of
the first gene is capable of converting an endogenous
molecule into a non-toxic molecule, which in turn can
be converted into a cytotoxic molecule by the product
of the second gene. The system chosen was to overproduce auxin in the seed coat using the IamS gene,
the product of which converts endogenous tryptophan
to indole acetamine, and the IamH gene, whose product converts indole acetamine to indole acetic acid (an
auxin; Fig. 2a); one source of IamS and IamH is Agrobacterium tumefaciens40. To introduce this system into a
classical F1 seed-production strategy (Fig. 2b), each
parent has to be transformed with either the first or the
second transgene. When the two parents are crossed,
the F1 seeds obtained are viable because only one transgene (that of the mother plant) is present in the seed
coat. The F1 plants grow normally and carry both
transgenes. When the F1 plants self- or cross-pollinate,
the two transgenes are both expressed in the seed coat.
It is assumed that the seed coat is destroyed by the overexpression of auxin, which leads to seed abortion. This
is based on the fact that auxin overproduction in pollen
has been used previously to produce male sterile plants41.
Site-specific recombination of DNA in a plant cell
This patent42 describes the use of a site-specific
recombination system from bacteriophage P1. The system consists of a recombinase (Cre) and recombination
sites (loxP). In the presence of Cre, recombination
between lox sites occurs on supercoiled, nicked, circular or linear DNA43. The Cre-lox system is efficient in
many organisms and, in particular, in plants wherein
numerous applications have been found. For example,
Cre-lox systems can be used to make a site-specific
insertion44, as a meganuclease45, to induce translocation
or to activate genes46. The principle of gene activation
is described in Fig. 3a. A sequence, for example, containing a polyA signal flanked with two lox sites, is
inserted between a transgene and its promoter. This
insertion renders transcription impossible, and the gene
is inactivated. If the Cre gene is present and expressed,
recombination occurs between the two lox sites, thus
eliminating the polyA sequence and one lox site. Only
one lox site remains between the promoter and the
gene. However, the lox site, being composed of only
23 bp, does not prevent transcription, and therefore the
gene is activated.
The activation of a cytotoxic gene using this system
can be an elegant way of producing seedless fruit
(Fig. 3b). Two transgenic plants have to be made; the

237

BIOTOPICS

(a)

Iam hydrolase

Iam synthase
Endogenous
tryptophane

Indole acetamine

Indole acetic acid
(Cytotoxic)

(b)

Plant 1 (female)
Pseed coat::IamS (Ho)

Plant 2 (male)
Pseed coat::IamH (Ho)

100% fertile plants
Easy to propagate

F1 seeds
Maternal tissue
Pseed coat::IamS (Ho)
Other tissues
Pseed coat::IamS (He)
Pseed coat::IamH (He)

100% viable F1 seed
Sold to farmers

F1 plants
Pseed coat::IamS (He)
Pseed coat::IamH (He)

F2 seeds
Maternal tissue
Pseed coat::IamS (He)
Pseed coat::IamH (He)
Seedless fruits

IamH and IamS expression in
the seed coat leading to
seed destruction
trends in Biotechnology

Figure 2
(a) Schematic representation of the principle underpinning the production of a cytotoxic compound following the introduction of two independently innocuous genes encoding Iam synthase and Iam hydrolase into plants. (b) Schematic representation of the production of seedless
fruits in the F2 generation using the Iam synthase and Iam hydrolase system. The seed coat is a maternal tissue; the genotype of maternal
tissue is thus different from the other parts of the seed. Abbreviations: Ho, homozygous; He, heterozygous; Pseed coat, seed-coat-specific
promoter.

first must have a cytotoxic gene, such as that encoding
barnase (an RNAase), under the control of a seed-coatspecific promoter. A lox-polyA-lox sequence is inserted
between the promoter and the gene. The second plant
contains a seed-coat-specific promoter linked to the Cre
gene. These two transgenic plants are viable, fertile and
easy to propagate, and are crossed to produce F1 seeds.
The F1 seeds and the plants that develop from them are
viable because the barnase gene is not activated in the
seed coat owing to maternal effects. In the F2 seed coat,
Cre is expressed and the barnase gene is activated. The
expression of the barnase gene leads to seed-coat ablation and, potentially, to the abortion of seed development. Experiments have shown that this strategy
could be successful for the following reasons: (1) there
are several cytotoxic genes such as diphtheria toxin A47,
EcoRI48, barnase49 and streptavidin50, which have been
used successfully in cell-ablation experiments; and (2)
the Cre-lox system has been used successfully to activate a uidA (GUS) gene in seeds46. Nevertheless, seedcoat-specific promoters are rare and, at present, there is

238

no experimental evidence that the destruction of the seed
coat leads to the abortion of seed development. Another
possible limitation of the system is the level of excision
of lox elements, which has to occur independently in
most, if not all, cells of the seed coat.
Producing nonviable F2 seeds
The principle of this patent51 is slightly different from
those described above and has attracted much media
attention under the derogatory term ‘terminator technology’. Rather than producing seedless fruits, its main
goal is to produce fruits with seeds that are unable to
germinate. This patent resembles the previous one42
because it also includes the use of Cre-lox-specific
recombination. However, in this patent, the Cre-lox
system is coupled with a repressor (tn10 tet repressor
gene)–operator (tet) system52,53. In the absence of tetracycline, the tn10 tet repressor protein binds to tet operators present in a chosen promoter and prevents the
expression of the gene under its control. In the presence of tetracycline, the repressor cannot bind to the
TIBTECH JUNE 2000 (Vol. 18)

BIOTOPICS

(a)

Inactivated gene
Promoter lox

lox

Activated gene

Barnase

Cre

Promoter lox

Barnase

+
lox

(b)
Plant 1 (female)
Pseed coat::lox-polyA-lox::Barnase (Ho)

Plant 2 (male)
Pseed coat::Cre (Ho)

100% fertile plants
Easy to propagate

F1 seeds
Maternal tissue
Pseed coat::lox-polyA-lox::Barnase (Ho)
Other tissues
Pseed coat::lox-polyA-lox::Barnase (He)

100% viable F1 seed
Sold to farmers

Pseed coat::Cre (He)
F1 plants
Pseed coat::lox-polyA-lox::Barnase (He)
Pseed coat::Cre (He)

F2 seeds
Maternal tissue
Pseed coat::lox-polyA-lox::Barnase (He)
Pseed coat::Cre (He)
Seedless fruits

Cre expression
lox excision

Barnase activation
Seed-coat destruction
Seed abortion
trends in Biotechnology

Figure 3
(a) The principle of gene activation using the Cre-lox system. The lox-lox DNA fragment prevents barnase expression. To activate the barnase
gene, the Cre recombinase excises the lox-lox DNA fragment. (b) Schematic representation of how the activation of a cytotoxic gene, such
as barnase, can induce seedlessness in the F2 generation by specific expression in the seed coat (maternal tissue). Abbreviations: Ho,
homozygous; He, heterozygous; Pseed coat, seed-coat-specific promoter.

operators and therefore the transcription of the gene is
possible (Fig. 4a).
The principle of the production of fruit with nonviable seeds is shown in Fig. 4b. The main goal is to
express a cytotoxic gene in a mature embryo, for example, the ribosomal-inhibitor protein (RIP). The promoter chosen to drive expression in the mature seed is
the late-embryogenesis abundant (LEA) class gene. Two
parental lines are transformed using two different constructs; the first parent contains a fusion between the
LEA promoter and the cytotoxic gene. A sequence
containing the repressor Tn10 tet gene under the control of the 35S promoter and flanked with lox sites must
be inserted between the promoter and the cytotoxic
gene. The other parent contains a construct containing
TIBTECH JUNE 2000 (Vol. 18)

the same LEA promoter modified with the tet operator fused to the Cre gene. These two plants are perfectly
viable and fertile, and can be crossed to produce F1
seeds. The F1 seeds can develop because the repressor
blocks expression of Cre in the absence of tetracycline.
However, before the seed is purchased, the F1 seeds can
be imbibed in an aqueous solution of tetracycline. During imbibition, Cre expression takes place and, as a result
of Cre activity, the excision of the lox insert is induced.
Because the LEA promoter is specific to late-seed
formation, the cytotoxic effect occurs only in the F2
seeds.
Despite considerable media attention, this patent
seems to be technically difficult to carry out because of
several potential problems. These include the level of

239

BIOTOPICS

Without tetracycline
Active repressor

(a)
p35S

Tn10 tet

p35S (otet)

RIP

Transcription

RIP

Transcription

With tetracycline
Inactive repressor

p35S

Tn10 tet

p35S (otet)

(b)
Plant 1
pLEA4::lox::tn10::p35s::lox::RIP (Ho)

F1 Seeds
pLEA4::lox::tn10::p35s::lox::RIP (He)
p35S(3teto)::Cre (He)

Plant 2
p35S(3teto)::Cre (Ho)

100% fertile plants
Easy to propagate

100% viable F1seed
Sold to farmers

Tetracycline application

Derepression of Cre expression
Excision of lox insert

RIP gene activated

F1 Plants
pLEA4::RIP (He)
p35S::Cre (He)

F2 Seeds
1/4pLEA4::RIP (Ho)
1/2 pLEA4::RIP (He)
1/4 without RIP (viable seeds)

RIP expression in mature seed
Fruits with only 25% of viable seed

The fruits contain
75% of nonviable seeds

trends in Biotechnology

Figure 4
(a) Schematic diagram representing the mode of action of the Tn10 tet repressor in the presence or absence of tetracycline. (b) Schematic
representation of the production of fruits containing predominantly nonviable seeds in the F2 generation using a Cre-lox and a Tn10 tet
repressor system. Abbreviations: Ho, homozygous; He, heterozygous; p35S, 35S promoter; Tn10 tet, gene encoding the repressor;
teto and otet, repressor DNA binding site; RIP, ribosomal-inhibitor protein; pLEA4, promoter of a late-embryogenesis abundant gene; nos39,
transcription terminator.

lox excision, the efficiency of the repressor in crop
plants and the efficiency of the derepression by imbibing in tetracyclin solution. Moreover, the use of large
amounts of antibiotics in plant agriculture will certainly
not be well accepted in many countries.

240

Advantages and limitations of technologies
using recombinant DNA techniques
Parthenocarpy – the leader of the seedlessness strategy
Many patents have been developed to induce seedlessness, and several experiments indicate that seedlessness
TIBTECH JUNE 2000 (Vol. 18)

BIOTOPICS

can be achieved by many methods. Inducing parthenocarpic fruits by transforming plants with a transgene
composed of a fusion between an ovary-specific promoter
and an auxin precursor biosynthesis gene has produced
impressive results. Parthenocarpic eggplants with no
deleterious pleiotropic effects have been effectively produced. This strategy seems to be able to induce
parthenocarpy in a wide variety of species, including
tomatoes and watermelons. This is surprising because,
theoretically, parthenocarpy was the basis of the type of
seedlessness that was most difficult to achieve mainly
because fruit development must take place without fertilization and sometimes even with no stimulation via
pollen deposition.
One question, which has not yet been fully answered,
is how the induction of auxin in the ovary is able to
substitute for seeds in the developing fruit. In the
tomato, several natural parthenocarpic cultivars exist,
but all of them have disadvantages that are caused by
the pleiotropy of the genes involved. In the future,
because of the availability of transgene technology, it
thus seems pointless to try to find and use new parthenocarpic mutants in crops. Additionally, parthenocarpy
is the most interesting form of seedlessness because it
is the only one that encompasses all the advantages
brought by this condition. The Cre-lox system could
be a useful tool for enabling the introduction of a
parthenocarpic inducer transgene in an F1 seed-production strategy. The next step towards the realization
of parthenocarpic varieties on a commercial scale is to
develop a combination of the parthenocarpic transgene
with male sterility or self-incompatibility.
What strategies will consumers and farmers accept?
Among all the strategies available to produce seedless
fruits, terminator technology is the only one to have
unleashed the wrath of the media. One potential use
of terminator technology, similar to other seedless
strategies, is to prevent genetically modified plants
being used without payment to the seed companies. If
the seeds produced are nonviable, farmers must buy
new seeds at every planting season. The problem is that
in many parts of the world, an age-old practice of
farmers consists of saving some seeds from the previous
harvest to sow the following year, and this practice is
fiercely protected. In addition, the farmers fear that this
technology might limit the choice of varieties from
which seed saving is possible. Moreover, the fact that
they have to buy seeds at each planting season results
in an increase in the cost of crop production. However,
seed protection by the seed companies is not a novel
concept; the development of the F1 hybrid seed is a
perfect example. F1 seeds need to be bought each
planting season but, in contrast to terminator technology, this strategy is now well accepted by farmers
because F1 hybrids bring clear advantages, such as
productivity gain.
In addition to protecting new transgenic varieties
from the practice of seed saving, terminator technology also protects the property from rival seed companies. If there are no viable seeds produced in the progeny, it is difficult for unscrupulous companies to recover
the transgene by a simple cross with one of its favourite
varieties. In practice, classic varieties are well protected
by plant-variety rights and, by contrast, transgenic
TIBTECH JUNE 2000 (Vol. 18)

plants are protected by patents. At present, nobody can
predict the results of patent challenges regarding transgenic plants because patenting living organisms is still
controversial. For this reason, terminator technology
could be a strategy to limit the occurrence of legal
action, but this argument is unlikely to be of interest to
farmers and consumers.
Genetic engineering of stenospermocarpy or
parthenocarpy in a wider range of species can bring the
same advantages as terminator technology can for the
plant biotechnology industry. However, seedless fruits
are far more attractive for farmers and consumers than
sterile seeds. Seedless watermelon, grapes and citrus
fruits are highly appreciated by consumers. For example,
greater than 80% of the grapes consumed in the world
are now seedless. These improvements in taste, convenience and the ability to eat fruits in all seasons are easily
understandable by consumers and are well accepted.
Genetic engineering of seedlessness could be a base for
the public acceptance of transgenic plants.
In conclusion, the interest of consumers, farmers
and seed companies in seedless fruit and the technical
progress of genetic engineering of seedlessness lead us
to believe that, in the near future, seedlessness could be
an improvement introduced into a wider range of fruits
and vegetables.
Acknowledgments
We are very grateful to P. This for helpful discussion
on seedless fruits. We thank G. Hull and J. Timmis for
critically reading and improving the manuscript before
submission. R. Blanvillain is funded by an EC grant
(EPEN BIO4-CT96-0689).
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