US005569829A United States Patent (19) 11 Patent Number: 5,569,829 Bird et al. 45 Date of Patent: Oct. 29, 1996

54) TRANSFORMED TOMATO PLANTS OTHER PUBLICATIONS (75) Inventors: Colin R. Bird, ; Jeremy M. Maunders et al., "Ethylene stimulates the accumulation for Boniwell, Marston; Donald Grierson, ripening-related mRNAs intomatoes", Plant, Cell and Envi Shepshed; John A. Ray, Wooden Hill; ronment (1987) 10, 177-184. Wolfgang W. Schuch, , all Slater et al, "Isolation and characterisation of cDNA clones of for tomato polygalacturonase and other ripening-related proteins", Plant Molecular Biology (1985) 5, 137-147. 73) Assignee: Imperial Chemical Industries, Smith et al, "Antisense RNA inhibition of polygalactyuro London, nase gene expression in transgenic tomatoes', Nature (1988) 334, 724-726. 21 Appl. No.: 73,425 Grierson et al, Phil. Trans., R. Soc. Lond. B314, 399-410 22 Filed: Jun. 9, 1993 (1986). Schuch et al, Plant Molecular Biology (1989) 13, 303-311. Related U.S. Application Data Tanksley, et al (1988) Theor Appl Genet. 75:811-823. Tieman et al (Jun. 1992) The Plant Cell 4:667-679. I63) Continuation-in-part of Ser. No. 598,873, filed as PCT/ Boswell, et al in Computational Molecular Biology (Tesk, GB93/00021 Jan. 8, 1993, Pat. No. 5,254,800. ed) Oxford University Press, Oxford, 1988, pp. 170-171. 30 Foreign Application Priority Data

Oct. 20, 1989 (GB) United Kingdom ...... 8923716 Primary Examiner-Che S. Chereskin Jan. 10, 1992 GB) United Kingdom ...... 9200520 Attorney, Agent, or Firm-Cushman Darby & Cushman (51) Int. Cl...... A01H 4/00 57 ABSTRACT 52 U.S. Cl...... 800/205; 435/172.3; 800/DIG. 44 A method is provided for making fruit (particularly toma 58) Field of Search ...... 435/1723, 240.4, toes) having increased solids content which comprises cul 435/320.1; 800/200, 205, DIG. 44 tivating fruit-bearing plants in which expression of genes homologous to pTOM36 is at least partially inhibited. For 56) References Cited this purpose the fruit may be transformed with DNA con U.S. PATENT DOCUMENTS structs comprising a DNA sequence homologous to some or all of the gene encoded by the clone pTOM36. The clone is 4,801,540 l/1989 Hiatt et al...... 435/240.4 adapted to generate sense or antisense RNA under control of 5,107,065 4/1992 Shewmaker et al...... 800/205 a plant promoter. FOREIGN PATENT DOCUMENTS WO9105865 5/1991 WIPO. 2 Claims, 5 Drawing Sheets

U.S. Patent 5,569,829

5,569,829 1 2 TRANSFORMED TOMATO PLANTS thereof. Fruit according to this aspect of the invention are particularly useful for making processed food products, for This is a continuation-in-part of Ser. No. 07/598,873, example tomato paste or tomato soup. filed Oct. 19, 1990, now U.S. Pat. No. 5,254,800, and Increased fruit solids has been a major target of breeding International Application No. GB93/00021, filed Jan. 8, for processing cultivars of several crops for many years. 1993 designating the U.S. and claiming priority from U.K. Improved flavour is also a breeding target for all fruit crops, Application No. 9200520.6, filed Jan. 10, 1992. especially in cultivars for the fresh market. This application relates to novel DNA constructs, plant The quality of paste produced from processed tomatoes cells containing them and plants derived therefrom. It is in part related to the viscosity of the product which is usually determined by the Bostwick flow rate, reduced flow involves the use of sense or antisease RNA technology to 10 rate being desirable. The factors that interact to give a control gene expression in plants. thicker product with reduced flow rate are complex, involv As is well known, a cell manufactures protein by tran ing interactions between insoluble and soluble components. scribing the DNA of the gene for that protein to produce The characteristics of components in whole fruit will change messenger RNA (mRNA), which is then processed (eg by during processing because of enzyme action and chemical the removal of introns) and finally translated by ribosomes 15 changes brought about by heating which is involved in into protein. This process may be inhibited by the presence tomato processing by the so-called "hot-break' method. in the cell of "antisense RNA'. By this term is meant an The consistency of hot break paste is improved by RNA sequence which is complementary to a sequence of increasing the level of solids in the whole fruit used in bases in the mRNA in question: complementary in the sense processing. Increased levels of soluble and insoluble solids that each base (or the majority of bases) in the antisense 20 in processing tomatoes has been an object of plant breeders sequence (read in the 3' to 5' sense) is capable of pairing with for many years. the corresponding base (G with C, A with U) in the mRNA Soluble solids are the solutes in the tomato serum and sequence read in the 5' to 3' sense. It is believed that this consists primarily of carbohydrates. In ripe fruit, hexoses are inhibition takes place by formation of a complex between the primary component of the soluble solids and account for the two complementary strands of RNA, preventing the 25 about 50% of the fruit dry weight. The free sugars are mainly formation of protein. How this works is uncertain: the glucose and fructose; sucrose is present but rarely exceeds complex may interfere with further transcription, process 1% of the dry weight. Paste is normally sold on the basis of ing, transport or translation, or degrade the mRNA, or have its natural tomato soluble solids (NTSS) content. Because more than one of these effects. Such antisense RNA may be the sugars are the major contributors to NTSS, a higher produced in the cell by transformation with an appropriate 30 sugar content contributes to a higher yield of paste per tonne DNA construct arranged to transcribe backwards part of the of tomatoes. The correlation between NTSS and total solids coding strand (as opposed to the template strand) of the (TS) is very high, although the relationship varies amongst relevant gene (or of a DNA sequence showing substantial tomato cultivars. Sugar content is also a critical component homology therewith). of the flavour of tomatoes. The use of this technology to downregulate the expres 35 Insoluble solids (IS) consist mainly of the polysaccha sion of specific plant genes has been described, for example rides in the cell wall. Residual starch will also contribute to in European Patent publication no 271988 to ICI (corre the IS although, in normal ripening, this forms a small sponding to U.S. Ser. No. 119614). Reduction of gene component. The ISTS ratio partially determines the consis expression has led to a change in the phenotype of the plant: tency of tomato products. Where high consistency is either at the level of gross visible phenotypic difference e.g. required, a greater quantity of IS improves the product lack of anthocyanin production in flower petals of petunia quality. IS are measured as both water-insoluble solids leading to colourless instead of coloured petals (van der Krol (WIS) and alcohol-insoluble solids (AIS). The AIS quanti et al, Nature, 333, 866-869, 1988); or at a more subtle ties are greater than those for WIS because smaller polysac biochemical level e.g. change in the amount of polygalac charides are less soluble in 80% ethanol than in water. turonase and reduction in depolymerisation of pectins during 45 Thus, increasing the solids content is advantageous as it tomato fruit ripening (Smith et al, Nature, 334, 724-726, may improve the processing properties and/or texture and/or 1988; Smith et al., manuscript submitted for publication). sweetness and/or taste of the fruit. Such an increase may be Thus antisense RNA has been proven to be useful in brought about by expression of a pTOM36 sense or anti achieving downregulation of gene expression in plants. sense construct in fruits such as tomato, melon, peach, pear, In work leading to the present invention we have iden 50 etc. tified a gene which expresses an enzyme involved in the DNA constructs useful in the invention comprise at least ripening of tomatoes. This gene has been cloned and char part of a DNA sequence homologous to pTOM36 adapted acterised. We propose that inhibition of this gene be used to for expression under the control of a promoter functional in produce tomatoes and other fruit having an increased solids plants. The DNA sequence homologous to pTOM36 may be content. The gene in question is encoded (almost com 55 arranged to express mRNA that is homologous with or pletely) in the clone pTOM36. complementary to (sense or antisense) natural pTOM36 In particular, we have identified in tomato fruit down mRNA. It preferably comprises a homologous base regulated for production of the gene pTOM36 the properties sequence at least 50 bases in length. There is no theoretical of increased solids content; and of higher content of reduc upper limit to the base sequence-it may be as long as the ing sugars, for example glucose and fructose. 60 relevant mRNA produced by the cell-but for convenience Accordingly, according to the present invention we pro it will generally be found suitable to use sequences between vide a method of producing fruit, especially tomato fruit, 100 and 1000 bases in length. Moreover, if the DNA is having an increased solids content, which comprises culti arranged to express sense RNA, the sequence is preferably vating plants in which expression of genes homologous to shorter than full-length (i.e., long enough to code for a pTOM36 is at least partially inhibited. Such inhibition may 65 functional protein). Full-length sense sequences may (but do conveniently be achieved by transforming plants with suit not necessarily) result in over-expression rather than inhi able constructs containing the gene pTOM36, or part bition. 5,569,829 3 4 The preferred source of DNA for use in the present while by using a tissue specific promoter, functions may be invention is DNA derived from the clone pTOM36. The controlled more selectively. Thus in applying the invention, required DNA can be obtained in several ways: by cutting e.g. to tomatoes, it may be found convenient to use the with restriction enzymes an appropriate sequence of such promoter of the PG gene (Bird et al., 1988, cited above). Use DNA; by synthesising a DNA fragment using synthetic of this promoter, at least in tomatoes, has the advantage that oligonucleotides which are annealed and then ligated the production of RNA is under the control of a ripening together in such a way as to give suitable restriction sites at specific promoter. Thus the RNA is only produced in the each end; by using synthetic oligonucleotides in a poly organ in which its action is required. Other ripening-specific merase chain reaction (PCR) to generate the required frag promoters that could be used include the E8 promoter ment with suitable restriction sites at each end. The DNA is O then cloned into a vector containing upstream promoter and (Diekman & Fischer, EMBO Journal 7, 3315–3320, 1988) downstream terminator sequences. If antisense vectors are and the promoters from the ptOM36 genes. required, the cloning is carried out so that the DNA sequence Vectors according to the invention may be used to is inverted with respect to its orientation in the strand from transform plants as desired, to make plants according to the which it was cut. In the new vector, the strand that was 15 invention. Dicotyledonous plants, such as tomato, may be formerly the template strand then becomes the coding transformed by Agrobacterium Ti plasmid technology, for strand, and vice versa. The new vector will thus encode RNA example as described by Bevan (1984) Nucleic Acid in a base sequence which is complementary to the sequence Research, 12, 8711-8721. Such transformed plants may be of pTOM36 mRNA. Thus the two RNA strands are comple reproduced sexually, or by cell or tissue culture. mentary not only in their base sequence but also in their 20 The degree of production of RNA in the plant cells can orientations (5' to 3"). be controlled by suitable choice of promoter sequences, or As source of the DNA base sequence for transcription, it by selecting the number of copies, or the site of integration, is convenient to use a cDNA clone such as pTOM36. The of the DNA sequences according to the invention that are base sequence of pTOM36 is set out in FIGS. 1A-1C. introduced into the plant genome. In this way it may be Searches in DNA and protein data bases have not revealed 25 possible to modify solids content to a greater or lesser any homology to known genes or proteins. This clone has eXtent. been deposited at the National Collections of Industrial and The constructs of our invention may be used to transform Marine Bacteria, PO Box 31, of 23 St. Machar Drive cells of both monocotyledonous and dicotyledonous plants (formerly of 135 Abbey Road), Aberdeen AB2 1RY, Scot in various ways known to the art. In many cases such plant land, as a plasmid in E.coli, under the reference NCIMB 30 cells (particularly when they are cells of dicotyledonous 40192, on 1 Sep. 1989. Alternatively, a cDNA clone similar plants) may be cultured to regenerate whole plants which to pTOM36 may be obtained from the mRNA of ripening Subsequently reproduce to give successive generations of tomatoes by the method described by Slater et al, Plant genetically modified plants. Examples of genetically modi Molecular Biology 5, 137–147, 1985. In this way may be fied plants according to the present invention include, as obtained sequences coding for the whole, or substantially 35 well as tomatoes, fruits of such as mangoes, peaches, apples, the whole, of the mRNA produced by pTOM36. Suitable pears, strawberries, bananas and melons. lengths of the cDNA so obtained may be cut out for use by As previously stated, the preferred source of antisense means of restriction enzymes. RNA for use in the present invention is DNA showing An alternative source of DNA for the base sequence for homology to the gene encoded by the clone pTOM36. transcription is a suitable gene encoding a protein involved 40 pTOM36 was derived from a cDNA library isolated from in fruit ripening. Such a gene may differ from the cDNA of ripe tomato RNA (Slater et al Plant Molecular Biology 5, pTOM36 in that introns may be present. The introns are not 137-147, 1985). Four other clones (pTOM22, pTOM76, transcribed into mRNA (or, if so transcribed, are subse pTOM77, pTOM89) from the same library cross-hybridise quently cut out). When using such a gene as the source of the to pTOM36 and probably contain related sequences. base sequence for transcription it is possible to use either 45 pTOM36 has been characterised by hybrid select translation, intron or exon regions. but there is some ambiguity about the results of these A further way of obtaining a suitable DNA base sequence experiments. Slater et al (Plant Molecular Biology 5, for transcription is to synthesis it ab initio from the appro 137-147, 1985) reported a product of 44 kD, whereas priate bases, for example using FIG. 1 as a guide. (Maunders et al Plant, Cell and Environment 10, 177-184, Recombinant DNA and vectors according to the present 50 1987) found that it encodes a protein of approximately invention may be made as follows. A suitable vector con 52,000 daltons. DNA sequence analysis has demonstrated taining the desired base sequence for transcription (for that the clone is 1069 bases long with an open reading frame example pTOM36) is treated with restriction enzymes to cut of 271 codons. It is believed to encode a cytoplasmic the sequence out. The DNA strand so obtained is cloned (if protein, as no apparent leader sequence was detected using desired in reverse orientation) into a second vector contain 55 computer analysis of the amino acid sequence derived from ing the desired promoter sequence (for example cauliflower the DNA sequence. mosaic virus 35S RNA promoter or the tomato polygalac We have shown that the mRNA for which pTOM36 turonase gene promoter sequence-Bird et al., Plant codes is expressed in ripening tomato fruit. No expression Molecular Biology, 11, 651–662, 1988) and the desired could be detected in green fruit.pTOM36 is expressed most terminator sequence (for example the 3' of the Agrobacte 60 strongly at the full orange stage of ripening. The level of rium tumefaciens nopaline synthase gene, the nos 3' end). mRNA then declines in line with the general decline in According to the invention we propose to use both synthetic capacity of the ripening fruit. Expression of constitutive promoters (such as cauliflowermosaic virus 35S pTOM36 mRNA could also be induced by exposing mature RNA) and inducible or developmentally regulated promot green fruit to exogenous ethylene. The expression of ers (such as the ripe-fruit-specific polygalacturonase pro 65 pTOM36 is reduced in the ripening inhibitor (rin) tomato moter) as circumstances require. Use of a constitutive pro fruit ripening mutant which mature very slowly. pTOM36 moter will tend to affect functions in all parts of the plant: related sequences are also expressed in senescing leaves. 5,569,829 5 6 The genomic locations of sequences homologous to EXAMPLE 3A pTOM36 have been identified using RFLP mapping: three loci in the tomato genome carry sequences homologous to Construction of pTOM36 antisense RNA vector with the pTOM36. It has also been shown by Southern blotting that polygalacturonase promoter. the gene may be present as a small multigene family. The The fragment produced in Example 2A by cleavage with individual members of the multigene family may be BamHI and KpnI was also cloned into the vector pR2 to expressed differentially in ripening fruit and during senes give the clone pjR236B. p)R2 is a Bin 19 based vector, CCCC. which permits the expression of the antisense RNA under The invention will now be described further with refer the control of the tomato polygalacturonase promoter. This ence to the accompanying drawings, in which: 10 vector includes a nopaline synthase (nos) 3' end termination FIGS. 1A-1C show the base sequence of the clone sequence. This vector does not contain a Kpnl or a BamHI pTOM36 (SEQ ID No:1); site between the promoter and terminator sequences. Con FIG. 2 show the regions of the pTOM36 sequence which sequently, the PCR synthesised fragment was digested with may be synthesised by polymerase chain reaction (PCR) and Kpnl and BamHI, the cut ends were made flush with T4 used in the construction of antisense RNA vectors according 5 polymerase and then cloned into the HincII site of p)R2. to the invention. After synthesis, the vector with the correct inverted orien FIG. 3 show the base sequence of the oligonucleotides tation of pTOM36 sequence was identified by DNA used as primers (SEQ ID No:2 to SEQ ID No:5) for the sequence analysis. polymerase chain reactions to synthesise the fragments illustrated in FIG. 2. 20 EXAMPLE 3B The following Examples illustrate aspects of the inven Clones similar to pR236B were made from the fragments tion. of Example 2B. These are: 1. Bases 1 to 132-pjR236A EXAMPLE 1. 25 2. Bases 1 to 1069-pjR236C Identification of base sequence of pTOM36 The base sequence of pTOM36 was determined by stan EXAMPLE 4 dard DNA sequencing procedures and is shown in FIGS. Construction of pTOM36 sense RNA vectors with the 1A-1C. Knowledge of this sequence is essential for deter 30 CaMV 35 promoter. mining the orientation of the open reading frame and for the The fragments of pTOM36 cDNA described in Example subsequent construction of RNA antisense vectors. 2 were also cloned into the vector pr1 in the sense orientation to give the following clones: EXAMPLE 2A 1. Bases 1 to 132-pjR136AS 35 2. Bases 1 to 538-pjR136BS Construction of pTOM36 antisense RNA vectors with the 3. Bases 1 to 1069-pjR136CS CaMV 35S promoter The PCR generated fragments were digested with KpnI A vector pR136B was constructed using the sequence and BamHI, the cut ends made flush with T4 polymerase and corresponding to Fragment B (bases 1-538) of the pTOM36 then cloned into the HincII site of p)R1. After synthesis, the cDNA as shown in FIG. 2. 40 vectors with the sense orientation of pTOM36 sequence This fragment was synthesised in vitro using polymerase were identified by DNA sequence analysis. chain reactions with the synthetic oligonucleotides 1 and 3 as shown in FIG. 2 as primers and pTOM36 cDNA as EXAMPLE5 template. The synthetic oligonucleotide primers were designed such that a BamHI restriction site was incorporated 45 Experiments with pTOM36 antisense transformed toma at the 5' end of the fragment and a Kpnl site was incorpo toes are described below. rated at the 3' end of the fragment: base sequences are shown Generation of transformed plants in FIG. 3. After cleavage of the fragment with BamHI and Tomato plants were transformed with vector pR136B KpnI, it was cloned into the vector plR1 which had previ (the pTOM36 antisense RNA vector described in Example 2 ously been cut with Kpnl and BamHI, to give a vector which 50 above). was named p)R136B. p.JR1 (Smith et al Nature 334, Vectors were transferred to Agrobacterium tumefaciens 724-726, 1988) is a Bin 19 (Bevan, Nucleic Acids Research, LBA4404 (a micro-organism widely available to plant bio 12, 8711-8721, 1984) based vector, which permits the technologists) and were used to transform tomato plants expression of the antisense RNA under the control of the (Lycopersicon esculentum, vat. Ailsa Craig). Transformation CaMV 35S promoter. This vector includes a nopaline syn 55 of tomato stem segments followed standard protocols (e.g. thase (nos) 3' end termination sequence. Bird et al Plant Molecular Biology 11, 651-662, 1988. Thirty-six plants were selected as transformed by their After synthesis of the vector plR136B, the structure and ability to produce roots on media containing kanamycin. orientation of the pTOM36 sequence it contained were These plants were grown to maturity in the glasshouse. confirmed by DNA sequence analysis. 60 Analysis of transformed plants Visual appearance. EXAMPLE 2B The majority of plants grew normally and produced fruit which appeared to ripen normally. Previous experience with Vectorsp)R136A and pR136C were prepared in the same populations of primary transformants indicates that a few way as pR136B in Example 2A. They contain respectively 65 plants have abnormal growth habit and/or do not set fruit. bases 1 to 132 and bases 1 to 1069 (the complete cDNA) of This was also observed with the pTOM36 antisense plants. pTOM36. pTOM36 related mRNA levels in the fruit. 5,569,829 7 8 In order to determine whether the transformed plants had 1991 Glasshouse trials of modified lines. reduced expression of pTOM36 related genes, total RNA In late Summer 1991 lines L1AC36A (homozygous), was extracted from ripe fruit from 11 plants. RNA (North L2AC36A (azygous) and unmodified Ailsa Craig were ern) blots of this extracted RNA were probed with radiola grown in large scale glasshouse trials in the UK. belled probe for the pTOM36 sense strand. This probe 5 Plants were grown in a fully replicated block trial. Fruit hybridises to RNA molecules of three sizes (1.45, 20, 2.8 were harvested at 5 ripening stages (3, 5, 7, 10 and 14 days kb) from unmodified tomato fruit. These RNA species were post breaker-dpb') and analysed for colour, firmness, detected in RNA extracted from all of the modified plants. survivability, dry weight/fresh weight ratio and sugar con However, major reductions in the abundance of all 3 RNA tent. The experiment was replicated 3 times during the species was observed in RNA from five of the modified 10 season. Analysis of variance was used to identify significant plants. differences between the lines. Selection of plants with single sites of insertion of the Survivability and firmness of the homozygous fruit were antisense gene. not significantly different from the azygous and unmodified In order to select plants with single sites of insertion that fruit. The colour index was slightly enhanced compared to would give simple segregation patterns in future genera- 15 both control lines. tions, genomic DNA was extracted from leaves of the plants Over the 5 ripening stages the mean dry weight as a with reduced levels of pTOM36 related RNA in the fruit. percentage of fresh weight of L1AC36A fruit was signifi DNA (Southern) blots were prepared with this genomic cantly (p<0.001) enhanced (by more than 5%) compared to DNA after digestion with HindIII. The blots were probed both the azygous and the unmodified control lines. The with radiolabelled insert from the pTOM36 cDNA. This 20 greatest increase was observed in later stages of ripening probe hybridised to a DNA fragment of 4.4 kb from unmodi- (13.5% increase at 14 dpb). fied plants. Additional hybridising fragments in the digested The reducing sugar, acids (malic and citric) and aqueous DNA from the modified plants were interpreted to represent alcohol insoluble solids contents of the dried pericarp individual sites of insertion of the pTOM36 antisense gene. samples were determined. There were no significant differ One plant, E56C37, was identified as having a single site of 25 ences in the acids and aqueous alcohol insoluble solids insertion and was selected for analysis of progeny. contents between the homozygous fruit and fruit from the Analysis of dry weights and sugar content of fruit from two control lines. primary transformants. The sugars (which comprise 50% of the solids content of The dry weights and reducing sugar content of pericarps tomato) were significantly greater in the homozygous than in from ripe fruit were analysed. Fruit from some individual 30 the azygous fruit. This increase in sugars content was plants from the population of primary transformants had sufficient to account for the greater solids content of the significantly greater dry weight/fresh weight ratio and Sugar homozygous fruit. content than unmodified fruit. Fruit from E56C37 and a However, there was no significant difference between the second transformant, E56C1, had approximately 5 and 9% sugars content of the homozygous and the unmodified Ailsa increases in dry weight/fresh weight ratio respectively. 35 Craig fruit, despite the greater dry weight content of the Analysis of progeny homozygous fruit. Thus the compositions of the two control Identification of homozygous and azygous selfed progeny. fruit were apparently different. Plants homozygous or azygous (null) for the pTOM36 These experiments confirm that the increased percentage antisense gene were identified in populations of self-fertil- dry weight is associated with the presence of the pTOM36 ised progeny from E56C37. In addition, the pTOM36 anti- 40 antisense fruit. In addition, the difference in sugar contents sense gene segregated as a single site of insertion in selfed of the homozygous and azygous lines indicates that progeny of E56C1. These plants were chosen as the parents increased sugars has segregated with the presence of the of modified or unmodified lines for extensive analysis: pTOM36 antisense gene. The reason for the differences between the sugar contents of the two control lines is not yet Parent Line Genotype 45 clear. Further trials with Lines L1AC36A, L2AC36A, E56C37 LAC36A Homozygous L3AC36A and L4AC36A were carried out in 1992. These L2AC36A Azygous trials did not however show significant increases in solids E56C1 E. itsu content for homozygous as compared with azygous lines. 50 The reason for this is not clear, but may be attributable to environmental factors such as altered growing conditions.

SEQUENCE LISTING

( 1) GENERAL INFORMATION: ( i i i ) NUMBER OF SEQUENCES: 5

( 2) INFORMATION FOR SEQID NO:1: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1080 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear 5,569,829

-continued ( i i ) MOLECULETYPE: cDNA to mRNA ( i i i ) HYPOTHETICAL: NO ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Lycopersicon esculentum (B) STRAIN: Ailsa Craig (D) DEVELOPMENTAL STAGE: Ripening ( xi ) SEQUENCE DESCRIPTION: SEQID NO:1:

A T G G T AAA T G CAA T G G T G A A G GA GT CTT G T T TAT CGAAG G T G A T G CTAA TAT AGAG. CTT 60

GAAAAAT TAG G T GAAT CTAT TAAG CCA C C A T G T CAT ACT T G GATTT ACTA CTT CATAATG 1 20

TT CAT GGTT C T G A T G GAATT ATTGGTTC T C C T CTTTT GTT AATT CAG G T G A CTC GTTTTA 18 O

CTT G T G G T G G AT TT G CT GT T G GATTT AGAT TT AAT CACAC AATGA T G GAT G CT TAT GGCT 2 4 0

T CAAAA T G TT T. CT AAA T G CG T TAA GT GAAT TAATT CAAG G A G CTT CAA CA CCTT CTATAT 3 00

TG CCT GTA T G G GAA AGA CAT C T C CTA AG T G CT AGAT CAT C A C CAA GTATT ACAT GTATTC 360

AT CAT GA GT T TGA T G A G GAA ATTGAAT CAA AAA TT G C G T G G GAA T C T A T G GAA GATA AGT 4 20

T GAT ACA ACA AT CATTTTTC TTT GG AAA T G A G GAGA T G GA A GT CAT TAAA AAT CAA GTTC 48 0

CTCCAAATTA T GAA T G T ACA AAATTC GA GT TAT TAATG GC ATTTTTATGG AAA T G T C GTA 5 40

C CATT GCT. CT TAA TTT GCAC T CT GAT GAAA TT G T T C GTTT GA CATAC GT T A TT AAT ATAC 60 0

G T G GAAAAAA GT CACT CAA C AT T GAATTAC CAATT GGTTA TTA T G G GAA T G CGTTTATTA 6 60

CTCCA GT T G T T G TAT CAAAA G CAGGTTT G T TA T G T T CAAA T C CA GT GACA. TAT G CA GT TG 7 20

AATTGAT CAA GAAA GT TAAA GAT CATA TAA AT GAA GAA TA CAT CAAATCA TT GAT AGATT 80

TAATGGTT AC TAAAG G GAGA C C A GA GT TAA CAAAT CTTGG AATTTTTT G G T C T C A GATAA 8 40

TAGA TATAT T G GATTT GA T G AATTT GATT T. T G GATGG G GA AA C C C CAT TT TT G GA. GGG AT 900

CTTAAAG G CT A TAT CTTT CA CTA GT TTT G G T G T TT C T G T T AAAAATGA CA AAG GAGAAAA 9 60

A G G T G T TTT G AT AG CTATA A GT TAC CT C C AT T G GCC AT G AAAAAA CTTC AA GATA TCTA 1 O 20

CAA CAT GACT TT CAGA GT CA TAATTT CAAA TATAT AG GCT TTT CTATTGA AAAAAAAAAA 1 0 80

( 2) INFORMATION FOR SEQID NO:2: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH:37 basc pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: lincar ( xi ) SEQUENCE DESCRIPTION: SEQID NO:2: GGGGGGGA T C CTA AA TT GCA A T G G T GAAG G A GT CTTG 37

(2) INFORMATION FOR SEQID NO:3: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:3: GGTAC CAATA GAAAA G CCTA TATATTT GAA ATTA T G ACT C T GAA AG 46

(2) INFORMATION FOR SEQID NO:4: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 52 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single 5,569,829

-continued (D) TOPOLOGY: lincar ( xi ) SEQUENCE DESCRIPTION: SEQID NO:4: G G T A C CGA CA TTT C CATAAA AA T G CCA TTA ATA A C T C GAA TTTT G T A CAT TC 5 2.

( 2) INFORMATION FOR SEQID NO:5: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:5: C CAATAATTC CAT CG G T ACC AT GAA CAT TA T GAA GTA GTA AAT C CAAG 48

We claim: wherein the DNA construct comprises a DNA sequence 1. A method of producing fruit having increased solids 20 encoded by the clone pTOM36 under control of a content which comprises transforming fruit-bearing plants promoter functional in plants so that the DNA sequence with a DNA construct adapted to inhibit expression of the pTOM36 gene during ripening, selecting transgenic plants generates RNA during ripening, said RNA being sense in which expression of the pTOM36 gene is at least partially or antisense RNA and inhibited when compared to expression in non-transformed 25 wherein the fruit is tomato. plants, cultivating said transgenic plants or progeny thereof 2. Tomatoes produced by the method of claim 1. and harvesting the fruit which shows an increase in dry weight to fresh weight ratio of about 5-14%, ck k >k cK ck