Euphytica 79: 293-297,1994 .

293
© 1994 Kluwer Academic Publishers . Printed in the Netherlands .

Commercialization of a tomato with an antisense polygalacturonase gene :

The FLAVR SAVRTM tomato story

Matthew G . Kramer & Keith Redenbaugh

Calgene, Inc., 1920 Fifth Street, Davis, CA 95616, USA

Key words: Fruit ripening, antisense, polygalacturonase

Abstract

The FLAVR SAVR TM tomato was developed through the use of antisense RNA to regulate the expression of the
enzyme polygalacturonase (PG) in ripening tomato fruit . This enzyme is one of the most abundant proteins in ripe
tomato fruit and has long been thought to be responsible for softening in ripe tomatoes . The FLAVR SAVRTM
tomato is the first genetically engineered whole food to be sold in commerce . The history of the development of
this product is discussed beginning with the results of the original anti sense work (including conclusions regarding
the role of PG in ripe tomatoes) and will be followed by a description of the regulatory and food safety assessment .
Finally, the development of an operating business to produce, market and distribute a genetically engineered whole
food product is discussed .

Introduction shelf life yet still survive the traditional distribution

system intact .
The FLAVR SAVRTM tomato is the first genetically
engineered whole food to be sold in commerce follow-
ing FDA approval on May 18, 1994 . FLAVR SAVRTM Development and characterization
tomatoes (Lycopersicon esculentum Mill .) are defined
as tomato cultivars or progeny of tomato lines geneti- Plant material used for both research and product devel-
cally engineered using an antisense polygalacturonase opment has included both processing and fresh mar-
gene isolated from tomato (Sheehy et al ., 1987) . These ket tomato varieties . Initial transgenic lines were gen-
tomato cultivars were developed to improve flavor and erated by Agrobacterium-mediated transformation to
taste in fresh market tomatoes . The polygalacturonase develop a set of commercially viable breeding lines
(PG) gene was isolated from tomato and reintroduced which have been used in an ongoing breeding and
in the antisense o rientation . PG is the major enzyme variety development program . In addition, the devel-
involved in pectin metabolism during fruit ripening opmental material has allowed for what is probably
and has historically been associated with fruit soften- the most extensive safety evaluation of tomato that has
ing (Hobson, 1965 ; Brady et al ., 1985) . The use of an ever been undertaken . This was necessary because not
antisense strategy to reduce the expression of the PG only has the antisense technology never before been
gene in tomatoes causes decreased pectin solubliza- applied to a whole food product, but in addition it has
tion in the ripening fruit which in fresh market toma- been necessary to demonstrate the precision and safety
toes results in ripe fruit that remain intact for extended of techniques which utilize Agrobacterium-mediated
periods of time (Kramer et al ., 1992) . In terms of a transformation as a method of creating new and nov-
commercially viable product, the technology allows el genotypes and genotypic combinations . As a result,
for the production of fresh market tomatoes which can the FLAVR SAVRTM tomato currently available in the
be vine-ripened for enhanced flavor and have a longer market represents a unique combination of tradition-
294

al plant breeding technologies and the techniques of gene has no effect on levels of vitamins and nutri-
molecular biology and genetic engineering . ents, on production of potential toxins (tomatine), on
Initial tomato lines for both phenotypic charac- taste, on non-pectin related processing traits, on hor-
terization and product development were produced ticultural traits (growth form, time to flowering, time
through transformation with a PG antisense construct to fruit set, etc.), fruit pH and acidity, and fruit color
pCGN 1416 (Sheehy et al ., 1988) . Approximately 50 and size (Redenbaugh et al ., 1992) . In fact, in its envi-
independent transformation events were generated for ronmental assessments, the USDA concluded, `The
line selection in each variety transformed . Plants were antisense PG gene does not provide the transformed
then selected for further evaluation based on the levels tomato plants with any measurable selective advan-
of PG activity, whole plant morphology and kan' seg- tage over nontransformed tomato plants in their ability
regation ratios . Plant material selected based on these to be disseminated or to become established in the
criteria served as the initial population on which the environment' (USDA APHIS, 1991) .
bulk of phenotypic characterization was carried out
(Kramer et al ., 1990) . Homozygous progeny of select-
ed transformants were then used to produce seed for Safety assessment
further field testing and development .
More than 10 experimental field trials and over 400 Environmental release
acres of commercial production have been conduct-
ed by Calgene, Inc . with FLAVR SAVR TM tomato Historically, the USDA APHIS BBEP (United States
cultivars in the principal tomato producing regions of Department of Agriculture, Animal and Plant Health
California, Florida and Mexico . The experimental field Inspection Service, Biotechnology, Biologics, and
trials have been conducted both to determine the phe- Environmental Protection) has regulatory oversight of
notypic effect of an antisense PG gene on fresh market release of genetically engineered plants into the envi-
and processing tomatoes as well as to determine the ronment, as per Vol . 7 Code of Federal Regulations,
effect of the transgene and the transformation process Part 340. Using these regulations, the USDA has
on overall horticultural performance . approved over 1100 field trials of genetically engi-
In terms of phenotypic evaluation, it has been neered plants in the United States, without any adverse
demonstrated that the absence of PG in the ripening effects .
fruit imparted improved field holding and firmness as In terms of release of genetically engineered plants
well as improved resistance to certain post harvest fun- into the environment, the greatest concern has always
gal pathogens (Kramer et al ., 1990, 1992) . In addition, been the potential to inadvertently produce a new weed
when processing characteristics were evaluated, it was or somehow increase the competitiveness of existing
discovered that the absence of PG resulted in signifi- weeds (Colwell et al ., 1985) . Plant breeders have a long
cant improvements in both juice consistency and serum history of using a variety of plant breeding techniques
viscosity as measured by Bostwick and Ostwald values to select and produce plant cultivars with improved
respectively (Kramer et al ., 1990) . resistance or tolerance to external factors that inhib-
With respect to horticultural performance, observa- it their inherent productivity and/or competitiveness .
tions conducted during all trials and commercial pro- Examples include such traits as resistance to insect
duction demonstrate that cultivars developed through and disease pests, heat, cold and drought tolerance
transformation with the antisense PG gene exhibit sim- as well as earliness and winter hardiness . In theory,
ilar horticultural traits when compared to the identical such improved cultivars are better adapted to persist in
non-transformed genotypes. No unpredicted changes the presence of disease, insects and a variety of envi-
were observed to have occurred, as documented in the ronmental conditions which would normally decrease
field trial reports submitted to the USDA APHIS, sub- productivity and competitiveness . Although there is
sequent publications (Kramer et al ., 1990, 1992), and no evidence that demonstrates that incorporating these
Calgene's Request for Advisory Opinion filing with types of traits into crops has created cultivars which
the FDA . pose a risk of enhanced weediness (USDA APHIS,
More detailed analysis of the fruit expressing the 1991), each gene/crop combination has been evaluat-
antisense PG gene demonstrated that expression of ed on a case by case basis . Since the techniques of
the antisense PG gene affects only the composition molecular biology and genetic engineering are highly
of pectin in the fruit (Kramer et al ., 1990, 1992) . The specific in terms of what genes are being transferred,
 295

Table 1 . Nutritional components for FLAVR SAVR tomatoes and controls as compared to normal ranges for tomato .

Constituent Normal Range Measured Range for FLAVR SAVR Lines Measured Range for Control Lines Unit

Protein 0.85 ( .015 se)* 0.75-1 .14 0.53-1 .05 g

Vitamin A 192-1667 330-1600 420-2200 IU
Vit . Bt (Thiamin) 16-80 38-72 39-64 leg
Vit . B2 (Riboflavin) 20-78 24-36 24-36 µg
Vitamin B6 50-150 86-150 10-140 leg
Vitamin C 8 .4-59 15 .3-29.2 12 .3-29 .2 mg
Nicotinic acid (Niacin) 0.3-0.85 0.43-0 .70 0.43-0 .76 mg
Calcium 4 .0-21 9-13 10-12 mg
Magnesium 5 .2-20 .4 7-12 9-13 mg
Phosphorus 7 .7-53 25-37 29-38 mg
Sodium 1 .2-32.7 2-5 2-3 mg
Iron 0.2-0 .95 0.2-0.41 0.26-0 .42 mg

* Protein measurement is mean ± standard error (se) from USDA Handbook No . 8 . Range is based on ripe fruit constituents per 100 g
fresh tissue .

products developed using these methods will be more 3 . Genes from regulated articles, introduced into
easily defined in terms of the traits being introduced tomato, do not confer characteristics that would
into the plants (USDA APHIS, 1991). present FLAVR SAVRTM tomato as a plant pest
Because of the history of safe field trials in the U .S . risk (e .g. cause tomato to become a weed pest risk) .
and the development of new cultivars containing trans- 4 . No new compounds have been measured in FLAVR
genes, the USDA implemented a petition process for SAVRTM tomato that pose a hazard or are delete-
removing genetically engineered cultivars from reg- rious to the environment .
ulatory oversight . As an integral step in the process
of commercialization, Calgene, Inc . submitted such a Food safety
petition to the USDA APHIS requesting a determina-
tion that FLAVR SAVR tomatoes do not present a plant The data package developed by Calgene, Inc . to
pest risk and are not otherwise deleterious to the envi- demonstrate the safety of the FLAVR SAVRTM toma-
ronment . These tomato cultivars contain specific gene toes was submitted to FDA as two separate requests
sequences introduced into the plant genome via the for advisory opinions. The first, submitted November
binary vectors (pCGN1547, pCGN1548, pCGN1549, 26, 1990, and entitled "kanr Gene : Safety and Use
pCGN1557, pCGN1558, pCGN1559, or pCGN1578) in the Production of Genetically Engineered Plants"
plus the antisense polygalacturonase gene with its (FDA Docket #90A-0416) and addressed the safety
associated promoter and terminator (Sheehy et al ., of the use of the kan' gene as a selectable marker
1987, 1988) . These sequences, as used in producing in food . The second, submitted August 12, 1991, enti-
FLAVR SAVR tomatoes, do not cause these tomatoes tled `FLAVR SAVRT M Tomato : Status as Food' (FDA
to become a plant pest risk the USDA determined on Docket #91A-0330/API) provided a detailed safety
October 19, 1992 (Federal Register. 57 : 47608-47616) assessment of the FLAVR SAVR tomato .
that the FLAVR SAVR tomatoes are not a plant pest From a food safety perspective, one issue of con-
risk, are not deleterious to the environment, and are cern was the APH(3')l protein produced by the kan'
no longer a regulated article under 7 CFR 340 for the gene . APH(3')H is a protein and proteins are general-
following reasons : ly not known to have toxicity in humans . In fact, the
1 . Tomato is not a regulated article . protein is produced naturally by bacteria found in the
human gut . Calgene, Inc . conducted an acute toxici-
2 . Genetic sequences from regulated articles used to ty study which showed no toxicity, mortality or gross
produce FLAVR SAVRTM tomatoes have been necropsy in rats fed FLAVR SAVRTM tomatoes which
disarmed and do not pose a plant pest risk . contain the kanr gene. Experimental results demon-
2 96

specific evidence that the T -DNA region was stably

Table 2 . Comparison of FLAVR SAVR tomatoes with
nontransformed controls . integrated into the tomato genome . FLAVR SAVR T M
tomatoes, stored to the end of their shelf life, had the
Parameter Changed Unchanged same levels of pro-vitamin A and vitamin C as com-
Tomatine levels + pared to controls or compared to the normal range of
Taste (at same stage) + these vitamins in other tomato varieties . The addition-
Viscosity Increased al time the FLAVR SAVRTM tomato will stay in the
Other processing traits + field to reach the vine-ripened stage will not result in
Horticultural traits + any significant changes in agricultural practices that
Fungal resistance Increased will impact the environment . The harvest practices for
Color (pigmentation) + FLAVR SAVRTM tomatoes will be the same as those
Softening rate Decreased used for non-engineered fresh market vine ripened
tomatoes . The FLAVR SAVRTM tomato is less per-
ishable and more durable than tomatoes that do not
contain the FLAVR SAVRTM gene and will provide
strated that the enzyme was inactivated by pepsin in better quality tomatoes to the consumer . Finally, Cal-
simulated gastric and intestinal fluids, as is the case gene, Inc . has in place a quality assurance program
for any other typical protein . Glycosylation and subse- for future transformation events of FLAVR SAVRTM
quent increase in the antigenic capacity of APH(3')II tomatoes to determine the levels of provitamin A and
also would not occur since APH(3')II does not contain vitamin C and the glycoalkaloid tomatine, and ensure
the necessary sequence information for transport to that these levels are within the ranges measured in non-
the subcellular locations at which glycosylation reac- transformed tomatoes .
tions take place . Finally, APH(3')II was shown not An additional concern is the potential for horizon-
to have significant homology with known toxins and tal gene flow from the engineered plant to soil and/or
allergens . gastrointestinal microorganisms . Arguments have been
To demonstrate qualitative and nutritional equiv- made concerning this potential risk, but no data have
alence, nutrient components of FLAVR SAVR toma- been published to support such a concern . There are no
toes were measured . These included protein, vitamins known mechanisms for transfer of genes from plants
A, Bt, B2, B6, and C, niacin, calcium, magnesium, to microorganisms and no cases of such transfer have
phosphorus, sodium and iron (Table 1) . In all cases, been reported . No mechanism by which plant DNA
the range of variation of these nutrients in FLAVR could be incorporated into the genomes of the microor-
SAVRTM tomatoes was the same as found in control ganisms has been proposed . In addition, Zambryski et
fruit . In addition, the range of tomatine levels (a gly- al . (1982) provided evidence that once inserted DNA
coalkaloid related to solanine) was measured (Table is integrated into the plant host genome, it cannot be
2) . These measurements demonstrated that there were remobilized even if acted on again by vir genes . To
no changes in nutrients or potential toxins as a result date, such horizontal gene flow remains speculative
of the process involved in the production of these new with no actual examples .
cultivars . In conjunction with its review of Calgene's data,
The extensive compositional analyses, which FDA developed a policy regarding the safety of genet-
showed no changes in the components essential for ically engineered foods (Department of Health and
a safety evaluation and the verifying feeding studies, Human Services, Food and Drug Administration,
establish that FLAVR SAVRTM tomatoes are as safe Docket No . 92N-0139, Statement of Policy : Foods
for human consumption as other tomatoes that are cur- Derived from New Plant Varieties) . The new policy
rently part of the human diet . A complete character- applies the same regulatory standards to transgenic
ization was done of all DNA sequences between the crops and foods as for those produced using conven-
border of the T-DNA with regard to potential open tional technology. The policy requires companies to
reading frames and any potential corresponding pro- consult with FDA on any safety issue and to thoroughly
teins . The structure of the inserted T-DNA into FLAVR evaluate food safety with respect to allergenicity, tox-
SAVRTM tomatoes remained intact and the inserted T- ins, nutrition, and any newly produced substances .
DNA locus behaved predictably, based on Mendelian
genetics, over five generations . These data provided
 297

Commercial release References

Sale of FLAVR SAVR TM tomatoes began on May Brady, C ., W. McGlasson, J. Pearson, S . Meldrum & E. Kopeliovitch,
1985 . Interaction between the amount and molecular forms of
21, 1994 . This was a historic event in that this prod-
polygalacturonase, calcium, and firmness in tomato fruit . J . Am.
uct represented the first time a genetically engineered Soc . Hort . Sci. 110: 254-258 .
whole food had been sold in the public marketplace . Colwell, R ., P. Brayton, D. Grimes, D . Roszak, S . Huq & L.
Initially fruit was available in only two stores, one in Palmer, 1985 . Viable but non-culturable Vibrio cholerae and relat-
ed pathogens in the environment : Implications for the release of
the midwest (Chicago area) and the other in Califor- genetically engineered microorganisms . Bio/Technology 3 : 817-
nia (Davis) . Consumer acceptance was instantaneous 820 .
and overwhelmingly positive . The first three days of Hobson, G ., 1965 . The firmness of tomato fruit in relation to poly-
galacturonase activity . Hort . Sci . 40 : 66-72 .
sales saw over 3,000 pounds sold from each store. This
Kramer, M ., R . Sanders, H . Bolkan, C . Waters, R . Sheehy & W.
resulted in a shortage of fruit on the store level which Hiatt, 1992. Post-harvest evaluation of transgenic tomatoes with
actually required rationing of fruit for a time so that all reduced levels of polygalacturonase : processing, firmness and
the consumers who wanted to try a FLAVR SAVR TM disease resistance . Post Harvest Biol . Technol . 1 : 241-255 .
Kramer, M ., R . Sanders, R . Sheehy, M . Melis, M . Kuehn & W.
tomato were able . Store numbers are being increased
Hiatt, 1990 . Field evaluation of tomatoes with reduced polygalac-
at a very slow rate in an effort to insure quality and to turonase by antisense RNA . In : Bennett, A. & S . O'Neill (Ed .),
keep consumers satisfied . The emphasis of the FLVR Horticultural Biotechnology, pp. 347-355 . Wiley-Liss, Inc ., New
SAVRTM tomato will continue to be quality and fla- York.
Redenbaugh, K ., W. Hiatt, B . Martineau, M . Kramer, R . Sheehy,
vor. R . Sanders, C. Houck & D . Emlay, 1992 . Safety Assessment of
Genetically-Engineered Fruits and Vegetables : A Case Study the
FLAVR SAVR TM Tomatoes . CRC Press, Boca Raton, FL .
Concluding remarks Sheehy, R ., M . Kramer & W. Hiatt, 1988 . Reduction of polygalac-
turonase activity in tomato fruit by antisense RNA . Proc . Natl .
Acad . Sci . USA 85: 8805-8809.
The FLAVR SAVR TM tomato was developed through Sheehy, R ., J . Pearson, C . Brady & W. Hiatt, 1987 . Molecular
the use of antisense RNA to regulate the expression of characterization of tomato fruit polygalacturonase . Mol . Gen.
Genet. 208 : 30-36 .
the enzyme polygalacturonase (PG) in ripening toma-
USDA APHIS, 1991 . Environmental Assessment and Finding of
to fruit in order to create a commercial vine ripened No Significant Impact on Tomato Containing an Antisense Poly-
tomato product with superior consumer quality and galacturonase Gene . Permit Number 91-268-01 .
flavor. Extensive field testing as well as environmental USDA APHIS, 1992 (October 19) . Interpretive Ruling on Calgene,
Inc., Petition for Determination of Regulatory Status of FLAVR
and food safety assessments determined that FLAVR
SAV R T M Tomato (Docket No . 92-087-2) . Federal Register. 57 :
SAVRTM tomatoes are unchanged in terms of nutri- 47608-47616 .
tion, potential toxins, and horticultural traits and that Zambryski, P, A . Depicker, K. Kruger, & H. Goodman, 1982 .
no unintended technical effects were observed . Data Tumor induction by Agrobacterium tumefaciens : analysis of the
boundaries of T -DNA . J . Mol . Appl . Genet. 1 : 361-370 .
supporting these conclusions were submitted as two
`Requests for Advisory Opinion' to the FDA and as
a `Petition for Determination' with the USDA . The
USDA issued a determination that FLAVR SAVR TM
tomatoes which had previously been field tested under
USDA regulations `will no longer be considered reg-
ulated articles under APHIS regulations' (7 CFR Part
340) on October 19,1992 . On May 18, 1994, FDA con-
cluded that FLAVR SAVR T M tomatoes were as safe
as any other tomato produced through conventional
means . As a result of these determinations, it has been
concluded that these tomato lines pose no risk to either
the environment or consumers. The sale of FLAVR
SAVRTM tomatoes began May 21, 1994 . Consumer
acceptance has been overwhelming positive .