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 gene-spliced foods come to the agency for pre-market evalu-ation. 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 discus-sion 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 con-travene the scientific consensus on biotechnology regu-lation constrains FDA officials from pushing the scien-tific 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 gene-spliced 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 Orga-nization (WTO) will, in principle, be required to fol-low them, and because they will provide cover for unfair trade practices. Jean Halloran (Consumers Inter-national, 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 Conven-tion 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.
S
eedless fruits are a desirable commodity for con-sumers, and have been produced using traditional farming and breeding methods for many cen-turies. 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 disadvan-tages. 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 currentindustrial 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 chal-lenge. Fruit development comprises early development and maturation (Fig. 1). In most plants, normal early fruit development involves three phases1: (1) fruit set-ting, (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; dur-ing 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 expan-sion 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,
Less is better: new approaches for seedless
fruit production
Fabrice Varoquaux, Robert Blanvillain, Michel Delseny and Patrick Gallois
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 gib-berellins 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 devel-oping 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 uncom-mon and seedless crop plants have existed for many centuries4.
Classification of seedlessness
A plant is considered to be seedless if it is able to pro-duce a fruit with no seed, traces of aborted seeds or a much-reduced number of seeds. Different kinds of seed-lessness 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. Partheno-carpy 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 faculta-tive 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.
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 devel-opment of events preceding, or linked to, seed develdevel-opment. Arrows between (b)and (c)indicate the points in seed formation that might be deficient in seedless varieties.
trends in Biotechnology Anthesis
Pollination
Fertilization
Seed formation
Embryo development
Parthenocarpy
(a)
Fruit development
Ovary development, fertilization
and fruit set
Cell division, seed formation and embryo development
Cell expansion and embryo maturation
(b)
Seed development
Type of seedlessness
(c)
Example of defect
Globular stage
Embryo mature Embryo maturation Fruit set
Cell division
Cell expansion
Mature green fruit
Ripening
Stenospermocarpy Sterile pollen
(cucumber) Abnormal meiosis in triploid plant (watermelon) Defect in endosperm development (grapes) Ovary mutants
(Corinth grapes, cucumber, tomato) Incompatible pollen (citrus fruits) Phase I
Phase II
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 gametic-chromosome imbalance. This triploid plant must be pollinated by a diploid plant in order to produce a seed-less watermelon6. Seedless watermelons are gaining popularity, and the aborted seeds are very soft and pre-sent little inconvenience for consumers. The shape, flavour and yield are as good as seed-producing culti-vars 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, sat-suma 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-incompatibil-ity. 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 sat-suma, 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 well-developed 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 criti-cal in order to obtain an optimum level of bunch loos-ening and is cultivar dependent10. A second spray can
also be carried out to increase the size of the berries. This method of achieving seedlessness is technically dif-ficult, 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 stami-nate flowers on the same plant), androecious (stamistami-nate flowers only), gynoecious (pistillate flowers only), hermaphroditic (hermaphrodite flowers only), andro-monoecious (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 partheno-carpic 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 harm-ful; 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 attrac-tive to the consumer. An illustration of this is the seed-less pickled gherkin, which is more crunchy, firmer and fleshier than its seeded variety12. It is possible to specu-late 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 solids14than seeded cultivars.
Production
tomato pollination occurs in a very narrow range of temperatures: 158C–218C (night) and 308C–358C (day). Parthenocarpic tomato plants (cultivar severianin) pro-duce 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 charac-ter, the transgene would be unable to be disseminated by seed dispersal (e.g. by consumption of the fruit, dis-persal 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, seedless-ness can be used to protect genetically modified crops: linking a transgene with seedlessness would prevent unfair appropriation of the transgene by simply cross-ing the transgenic plant with another commercial vari-ety. 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 pro-duce, mainly because the seed is important for fruit development. Nevertheless, even though the produc-tion 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
affect hormone production, transport and/or metabo-lism in order to promote ovary growth precociously; pollination and fertilization are therefore no longer needed4.
Several parthenocarpic mutants have been studied, in particular, the patmutant 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 aber-rations. 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. More-over, 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 resto-ration 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 thalianacalled SPINDLY(SPY), whose gene product is anticipated to participate in the regulation of the gib-berellin signal-transduction pathway21,22. The phenotype of this mutant, including parthenocarpic siliques and partial male sterility, can be phenocopied in wild-type A. thalianausing 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 SPYgene 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.
of seeds. The same SCAR marker could be used as a starting point to clone this gene. Although the isola-tion 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 partheno-carpy in tomato ovaries has been studied27. At anthe-sis, 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 trans-lation, followed by two-dimensional polyacrylamide gel electrophoresis, revealed the differential expression for at least six in vitrotranslation 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 exogen-ous 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 partheno-carpic ovaries of the tomato33,34. Thus, it is theoreti-cally 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 patent35proposes to fuse an ovary-specific promoter with the Agrobacterium RolBgene, thus interfering with plant auxin produc-tion. The second patent36presents the fusion between pDef H9, an Antirrhinum majuspromoter that is specific to the ovary, and the RolB gene (to obtain partheno-carpy) 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 Def H9 pro-moter and the IaaMgene (tryptophan monooxygen-ase). When the flowers are emasculated, the transgenic eggplants produce marketable seedless fruits. Moreover, transgenic eggplants perform fruit set and growth dur-ing winter conditions, under which non-transgenic lines cannot set fruit. This shows that it is clearly poss-ible to induce parthenocarpy by expressing a gene encoding a step in the auxin biosynthetic pathway in
the ovary. However, one disadvantage is that the flow-ers 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 seed-production 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 patent39lies 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 over-produce auxin in the seed coat using the IamS gene, the product of which converts endogenous tryptophan to indole acetamine, and the IamHgene, whose prod-uct converts indole acetamine to indole acetic acid (an auxin; Fig. 2a); one source of IamSand IamHis Agrobac-terium 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 trans-gene (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 over-expression 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 sys-tem consists of a recombinase (Cre) and recombination sites (loxP). In the presence of Cre, recombination between lox sites occurs on supercoiled, nicked, circu-lar 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, con-taining 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 Cregene 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.
first must have a cytotoxic gene, such as that encoding barnase (an RNAase), under the control of a seed-coat-specific 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 barnasegene is not activated in the seed coat owing to maternal effects. In the F2 seed coat, Creis expressed and the barnasegene is activated. The expression of the barnasegene leads to seed-coat ab-lation and, potentially, to the abortion of seed devel-opment. Experiments have shown that this strategy could be successful for the following reasons: (1) there are several cytotoxic genes such asdiphtheria 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 acti-vate a uidA (GUS) gene in seeds46. Nevertheless, seed-coat-specific promoters are rare and, at present, there is
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 patent51is slightly different from those described above and has attracted much media attention under the derogatory term ‘terminator tech-nology’. 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 tetra-cycline, the tn10 tetrepressor protein binds to tet oper-ators present in a chosen promoter and prevents the expression of the gene under its control. In the pres-ence of tetracycline, the repressor cannot bind to the
Figure 2
(a)Schematic representation of the principle underpinning the production of a cytotoxic compound following the introduction of two inde-pendently 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.
trends in Biotechnology 100% fertile plants Easy to propagate
100% viable F1 seed Sold to farmers F1 seeds
Maternal tissue
Maternal tissue Pseed coat::IamS (Ho)
Pseed coat::IamS (He) Pseed coat::IamH (He)
Pseed coat::IamS (He) Pseed coat::IamH (He)
Pseed coat::IamS (He) Pseed coat::IamH (He)
Other tissues
Plant 2 (male)
Pseed coat::IamH (Ho) Plant 1 (female)
Pseed coat::IamS (Ho)
F1 plants
F2 seeds
IamH and IamS expression in the seed coat leading to
seed destruction
(a)
(b)
Endogenous
tryptophane Indole acetamine Indole acetic acid
Seedless fruits
Iam synthase Iam hydrolase
operators and therefore the transcription of the gene is possible (Fig. 4a).
The principle of the production of fruit with non-viable seeds is shown in Fig. 4b. The main goal is to express a cytotoxic gene in a mature embryo, for exam-ple, the ribosomal-inhibitor protein (RIP). The pro-moter chosen to drive expression in the mature seed is the late-embryogenesis abundant (LEA) class gene. Two parental lines are transformed using two different con-structs; the first parent contains a fusion between the LEA promoter and the cytotoxic gene. A sequence containing the repressor Tn10tetgene under the con-trol 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
the same LEA promoter modified with the tet opera-tor fused to the Cregene. 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 Crein the absence of tetracycline. However, before the seed is purchased, the F1 seeds can be imbibed in an aqueous solution of tetracycline. Dur-ing imbibition,Creexpression takes place and, as a result of Creactivity, 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
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.
trends in Biotechnology Seedless fruits 100% fertile plants Easy to propagate
100% viable F1 seed Sold to farmers Maternal tissue
Pseed coat::lox-polyA-lox::Barnase (Ho)
Pseed coat::lox-polyA-lox::Barnase (He) Other tissues
Plant 2 (male) Pseed coat::Cre (Ho) Plant 1 (female)
Pseed coat::lox-polyA-lox::Barnase (Ho)
F1 plants
F2 seeds
Maternal tissue
Seed-coat destruction
(a)
+ Cre
Inactivated gene Activated gene
Promoter Barnase Promoter
(b)
lox lox lox
lox
Barnase
Pseed coat::Cre (He)
Pseed coat::lox-polyA-lox::Barnase (He) Pseed coat::Cre (He)
Pseed coat::lox-polyA-lox::Barnase (He) Pseed coat::Cre (He)
F1 seeds
Cre expression
lox excision
Barnase activation
lox excision, the efficiency of the repressor in crop plants and the efficiency of the derepression by imbib-ing in tetracyclin solution. Moreover, the use of large amounts of antibiotics in plant agriculture will certainly not be well accepted in many countries.
Advantages and limitations of technologies using recombinant DNA techniques
Parthenocarpy – the leader of the seedlessness strategy Many patents have been developed to induce seedless-ness, and several experiments indicate that seedlessness
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.
trends in Biotechnology 100% fertile plants Easy to propagate
F1 Seeds
pLEA4::lox::tn10::p35s::lox::RIP (He) p35S(3teto)::Cre (He)
Plant 2
p35S(3teto)::Cre (Ho) Plant 1
pLEA4::lox::tn10::p35s::lox::RIP (Ho)
F1 Plants
pLEA4::RIP (He)
F2 Seeds
(a)
(b)
p35S Tn10 tet p35S (otet) RIP
Without tetracycline Active repressor
Transcription
With tetracycline Inactive repressor
Tetracycline application
p35S Tn10 tet p35S (otet) RIP Transcription
Derepression of Cre expression
Excision of lox insert
RIP gene activated
p35S::Cre (He)
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
100% viable F1seed Sold to farmers
can be achieved by many methods. Inducing partheno-carpic 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 pro-duced. 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 fer-tilization 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 partheno-carpic 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-pro-duction 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 tech-nology, 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 technol-ogy also protects the property from rival seed compa-nies. If there are no viable seeds produced in the prog-eny, 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
plants are protected by patents. At present, nobody can predict the results of patent challenges regarding trans-genic 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, conve-nience 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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