Methods and Compositions for Increasing Storage-Life of Fruit

Abstract

The invention provides methods and compositions for producing plants with fruit having increased post-harvest storage life, the method comprising reducing the expression or activity in the plant, of a polypeptide with the amino acid sequence of SEQ ID NO: 1, or a variant of the polypeptide. The invention provides host cells, plant cells and plants transformed with the polynucleotides of the invention. The invention also provides methods for selecting plants with fruit having increased post-harvest storage life. The invention also provides plants produced and selected by the methods of the invention.

Claims

1. A method for producing fruit having, or a plant with fruit having increased firmness during or after post-harvest storage the method comprising reducing the expression or activity, in the fruit or the plant, of a polypeptide with the amino acid sequence of SEQ ID NO: 1, or a variant of the polypeptide with at least 90% identity to the amino acid sequence of SEQ ID NO: 1, wherein the variant has polygalacturonase activity, wherein the method comprises the step of introducing a polynucleotide into a plant cell, the fruit or the plant to reduce the expression of the polypeptide or variant, wherein the increased firmness is relative to that of a control fruit under the same conditions, and wherein the fruit or plant is from a Malus species.

2. The method of claim 1, wherein the fruit produced additionally has at least one of: a) reduced water loss, b) reduced cell separation, c) increased juiciness, d) increased crispiness; e) increased waxiness, and f) reduced susceptibility to necrophytic pathogens, during, or after, post-harvest storage, relative to that of the control fruit under the same conditions.

3. The method of claim 1, wherein the polynucleotide comprises 21 contiguous nucleotides complementary to part of an endogenous gene, or nucleic acid, that encodes the polypeptide or variant thereof.

4. The method of claim 3, wherein the endogenous gene comprises at least one of: a) a sequence with at least 90% identity to the sequence of SEQ ID NO: 4, b) the sequence of SEQ ID NO: 4, c) a sequence with at least 90% identity to the sequence of SEQ ID NO: 5, and d) the sequence of SEQ ID NO: 5.

5. The method of claim 1, wherein the polynucleotide is introduced into the plant as part of a genetic construct.

6. The method of claim 5, wherein the genetic construct is an expression construct comprising a promoter operably linked to the polynucleotide.

7. The method of claim 6, wherein the polynucleotide is in an antisense orientation relative to the promoter.

8. The method of claim 1, wherein the polynucleotide comprises at least one of: a) at least 21 contiguous nucleotides of a sequence with at least 90% identity to the sequence of SEQ ID NO: 4, b) at least 21 contiguous nucleotides of the sequence of SEQ ID NO: 4, c) at least 21 contiguous nucleotides of a sequence with at least 90% identity to the sequence of SEQ ID NO: 5, and d) at least 21 contiguous nucleotides of the sequence of SEQ ID NO: 5.

9. The method of claim 1, wherein the plant with reduced expression of the polypeptide is regenerated from the plant cell.

10. A fruit or plant produced by the method of claim 1, wherein the fruit or plant is genetically modified to contain the polynucleotide.

11. A fruit comprising, or a plant producing fruit comprising an expression construct comprising a promoter operably linked to a polynucleotide comprising at least one of: i) a fragment of at least 21 contiguous nucleotides of a sequence with at least 90% identity to any one of SEQ ID NO: 4, 5, 6 and 7, wherein the sequence with 90% identity to any one of SEQ ID NO: 4, 5, 6 and 7, encodes a polypeptide with polygalacturonase activity, and wherein the polynucleotide is in an antisense orientation relative to the promoter, and ii) a fragment of at least 21 contiguous nucleotides from any one of SEQ ID NO: 4, 5, 6 and 7, wherein the promoter is heterologous to the polynucleotide, wherein the fruit has increased firmness during or after post-harvest storage relative to that of a control fruit under the same conditions and wherein the fruit or plant is from a Malus species.

12. The fruit or plant of claim 11, wherein the expression construct is an RNAi construct.

13. The fruit or plant of claim 11 that has reduced expression of an endogenous nucleic acid corresponding to the polynucleotide.

14. The fruit or plant of claim 13, wherein the endogenous nucleic acid encodes a polypeptide with polygalacturonase activity.

15. The fruit or plant of claim 11, wherein the fruit additionally has at least one of: a) reduced water loss, b) reduced cell separation, c) increased juiciness, d) increased crispiness, e) increased waxiness, and f) reduced susceptibility to necrophytic pathogens, during, or after, post-harvest storage, relative to that of the control fruit under the same conditions.

16. A plant part, seed, fruit, propagule or progeny of the plant of claim 10 that is genetically modified to contain the polynucleotide.

17. A plant part, seed, fruit, propagule or progeny of a plant of claim 11 that is genetically modified to contain the construct.

18. The method of claim 1, wherein the level of the polypeptide or variant in the fruit is less than 10% of that in the control fruit after 16 weeks post-harvest storage at 5 C.

19. The method of claim 18, wherein the fruit have an increase in firmness of at least 40% versus control fruit after 16 weeks post-harvest storage at 5 C.

20. The method of claim 1, wherein the fruit have an increase in firmness of at least 40% versus control fruit after 16 weeks post-harvest storage at 5 C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0251] The present invention will be better understood with reference to the accompanying drawings in which are described as follows:

[0252] FIG. 1: Selection of MdPG1 as a key gene in ripening apple fruit

[0253] A) The Venn diagram shows ESTs that significantly changed in expression using microarray analysis of RNA derived from three different treatments as described below:

[0254] Fruit development: 8 time points from anthesis to ripe fruit, 0, 14, 25, 35, 60, 87, 132 and 146 days after full bloom. Ethylene induction: Ethylene knockout mutant apples induced to ripen with ethylene, and harvested 0, 4, 18, 96 and 192 hours after ethylene induction. A 192 hour control (C) was also measured. Storage induction: Three different cultivars of apples were compared to each other before storage (Cultivar comparisons) and after storage at 0 C. for 4 weeks.

[0255] The expression profiles of the 143 cell wall related genes were assessed in 3 unrelated microarray experiments that covered periods of physiological fruit softening: Microarray experiments were performed and analysed as described in Schaffer et al (2007).

[0256] B) Relative expression pattern of MdPG1 measured using qPCR

[0257] RNA samples were extracted from ACC oxidase mutant apples to measure the effects of ethylene induced softening on PG expression with or without a cold storage treatment. Timepoints analysed by qPCR are shown in the schematic below

##STR00001##

[0258] cDNA was synthesized from 2 ug of total RNA in a total volume of 50 mL with Superscript III reverse transcriptase according to the manufacturer's instructions (Invitrogen). Controls with no Superscript III reverse transcriptase were used to assess for potential genomic DNA contamination. cDNA used for real-time RT-PCR was synthesized in triplicate and optical density was measured for each sample. qPCR reactions and normalization was performed as described in Schaffer et al (1998).

[0259] Results show the strong induction of MdPG1 expression by ethylene treatment after 96 h and 192 h and by cold+ethylene treatment at 96 h and 192 h. MdPG1 expression is induced by cold treatment alone but more slowly (after 192 h).

[0260] FIG. 2: Firmness in transgenic PG knockout plants vs Royal Gala controls

[0261] Fruit firmness was measured destructively on individual fruit (n=6-10 fruit per timepoint) at harvest, after 2 weeks storage at room temperature (20 C.) and after 16 weeks storage at 5 C.

[0262] Firmness was measured using a puncture test according to standard industry practise (Blanpied et al., 1978). This involved the localised removal of skin from two opposing locations on the fruit equator, and recording the maximum force while driving a 7.9 mm cylindrical probe into the outer cortex to a constant depth (8 mm) at a fixed speed (4 mm/s). The puncture test and data capture was performed using a Stable Micro Systems TA-XT plus Texture Analyser (Hertog et al 2001;). [0263] A) Fruit firmness in 10 knockout lines vs RG (Royal Gala) control. [0264] B) Fruit firmness measurement expressed as a % of firmness at harvest.

[0265] SPI=starch pattern index measurements were made against commercial standards.

[0266] FIG. 3: Western analysis of PG protein levels in transgenic Royal Gala apple fruit at harvest

[0267] Fruit tissues were ground to a powder with mortar and pestle under liquid nitrogen. Protein was extracted as described in Langenkapmper et al (1998). Proteins were separated on 12% (w/v) SDS-Tris-Tricine gels using a Mini-PROTEAN3 electrophoresis system (Bio-Rad, Hercules, Calif., USA). Protein concentrations in each sample were measured using the QuBit Quantitation System (Invitrogen) and verified on gels by Coomassie staining. A polyclonal antibody raised to apple polygalacturonase was used to immunolocalise PG protein in transgenic and control Royal Gala apple plants.

[0268] Ladder=Precision Plus Protein Dual Colour Standards (Bio-Rad), sizing is in kDa.

[0269] FIG. 4: Western analysis of PG protein levels in transgenic Royal Gala apple fruit after 2 weeks storage at room temperature (20 C.)

[0270] Protein samples were extracted and analysed as described in FIG. 3.

[0271] FIG. 5: Western analysis of PG protein levels in transgenic Royal Gala apple fruit after 4 weeks storage at 5 C.

[0272] Protein samples were extracted and analysed as described in FIG. 3.

[0273] FIG. 6: Western analysis of PG protein levels in transgenic Royal Gala apple fruit after 16 weeks storage at 5 C.

[0274] Protein samples were extracted and analysed as described in FIG. 3.

[0275] FIG. 7: Western analysis of PG protein levels in six selected transgenic Royal Gala apple fruit

[0276] Protein samples were extracted and analysed as described in FIG. 3.

[0277] H=fruit at harvest

[0278] 2w=fruit after 2 weeks storage at 5 C.

[0279] 16w=fruit after 16 weeks storage at 5 C.

[0280] L=ladder, Precision Plus Protein Dual Colour Standards (Bio-Rad)

[0281] FIG. 8: Rate of water loss in transgenic vs control fruit

[0282] ARate of water loss from PG knockout and Royal Gala fruit after 16 weeks storage at 5 C. followed by 5 weeks storage at room temperature. Water loss is expressed as grams of weight lost per day per g fresh weight of the fruit. Each bar represents an individual fruit. Conclusion: Compared to control RG (Royal Gala) fruit, the two lines in which PG has been down-regulated most strongly (lines PG41 and PG275) show the lowest rate of water loss.

[0283] BComparison of water loss from Royal Gala control fruit and fruit in which the apple ACC oxidase gene has been knocked out (see Schaffer et al 1998 for description of this line). Nb this fruit was not cold stored suggesting that cold storing the apples may further accelerate water loss.

[0284] FIG. 9: Reduced wrinkling in transgenic Royal Gala fruit down-regulated for PG compared to Royal Gala control fruit

[0285] Fruit were stored for 16 weeks at 5 C. then transferred to room temperature for 5 weeks at room temperature. After this period transgenic fruit were substantially less wrinkled compared to controls which correlates with the reduced loss of water from these fruit.

[0286] FIG. 10: Toluidine blue stained sections of apple from Royal Gala control and PG41 PG knockout line

[0287] Apple fruit cortex sections were fixed in glutaraldehyde and embedded in LR-white resin. Thin sections of 1 uM were stained with toluidine blue (0.1%). Sections were prepared from control Royal Gala fruit and the PG41 PG knockout line stored for 16 weeks at 5 C. Arrows indicate points of pectin adhesion.

[0288] NB in control fruit the adhesion is reduced whilst in the PG41 lines the adhesion is maintained. This difference will have an effect on fruit softening and texture.

[0289] FIG. 11: Immunolocalisation of non-esterified pectin from Royal Gala control and PG41 PG knockout line

[0290] Fixation of apple fruit tissues and immunolocalisation using JIM5 antibodies was performed as described in Atkinson et al. (2002). Sections were prepared from control Royal Gala fruit and the PG41 PG knockout line stored for 16 weeks at 5 C. Magnification=40. Arrows indicate points of JIM5 fluorescence.

[0291] NB in control fruit the fluorescence is reduced whilst in the PG41 lines the fluorescence is stronger. This result suggests that more demethylated homogalacturonan is present at the junction points between cells in PG41 fruit vs control fruit. This difference will have an effect on fruit softening and texture.

[0292] FIG. 12: Tensile [pull apart] strength (upper panel) and flesh firmness measured with an 8 mm probe (lower panel) for fruit of PG41 lines versus control (Royal Gala) lines at harvest (yellow bars) and after 10 weeks storage at 0.5 C. (magenta bars).

[0293] FIG. 13: Amino acid sequence of MDPG1 highlighting conserved polygalacturonase domains

[0294] FIG. 13 shows the position of four conserved domains (I to IV) that are present in the plant and fungal sequences (Torki et al. 2000 (incorporated herein by reference)). The carboxylate group in the three aspartic acids in NTD and DD structures (domains I and II, respectively) may be a component of the catalytic site and the histidine residue in domain III is thought to participate to the catalytic reaction. The well-conserved positively charged domain IV (RIK) constitutes a likely candidate for ionic interactions with carboxylate groups present in the substrate.

EXAMPLES

[0295] The invention will now be illustrated with reference to the following non-limiting examples.

Example 1: Selection of MdPG1 as a Candidate Gene for Altering Post-Harvest Storage Life in Apple Fruit

[0296] Three microarray experiments that measured apples that underwent ripening were analysed. These included an ethylene induced ripening series (Schaffer et al 2007), a fruit development series (Janssen et al 2008) and a cold storage treatment (manuscript in preparation). 290 cell wall related genes were identified by homology screening in the HortResearch Apple EST collection. Of these, 10 increased in expression late in fruit development, 9 increased in expression upon the addition of exogenous ethylene, and 10 increased in expression during 2 and a half months of cold storage. Of these genes, three were found to be in common to all treatments. Of the three, MdPG1 showed the greatest change in expression. Further analysis of this gene in transgenic apple lines down-regulated for the MdACO gene showed that MdPG1 is up-regulated in an ethylene dependent and cold dependent ripening manner (FIG. 1).

[0297] Although reduction of expression of polygalacturonases (PGs) has been proposed as an approach to improving storage characteristics in fruit, success has been limited. This may be partly due to the large number of PGs that appear to be present in plants. For example Arabidopsis alone has been reported to contain approximately 52 different PG genes.

[0298] Transgenic plants have been used to study the role of endo-PGs in vivo. In tomato (Lycopersicon esculentum), down-regulation of the fruit-specific PG gene pTOM6 under the control of the constitutive cauliflower mosaic virus 35S promoter showed reduced depolymerization of pectin polymers in fruit (Smith et al., 1990). Overexpression of PG in the ripening inhibited mutant rin background restored PG activity and pectin degradation in fruit (Giovannoni et al., 1989). In both cases, only the fruit was affected by the transgene expression; therefore, the gene product isolated from tomato fruit appeared to have fruit specific PG activity. Further experiments where the pTOM6 gene was overexpressed in tobacco (Nicotiana tabacum; Osteryoung et al., 1990) showed that the tomato protein was properly processed and localized in the cell walls of leaves in tobacco. The enzyme showed activity when extracted from transgenic tobacco leaves and tested against tobacco cell wall extracts in vitro. However, no changes in leaf phenotype were observed, nor were there any alterations to the pectins in the tobacco cell walls in vivo. Expressing and given PG gene in plants therefore may give unpredictable results.

[0299] Apple (Malus domestica Borkh. cv Royal Gala) ripens very differently than tomato and many other fruits (Redgwell et al. 2008a; 2008b), because cell wall swelling is not one of the cell wall modifications occurring during apple ripening (Redgwell et al., 1997). There is minimal change in viscosity of cell walls, and minimal pectin solubilization or degradation during fruit ripening. This implies that any endo-PG isolated from ripening fruit of apple may have different characteristics to endo-PGs isolated from ripening tomato fruit. In a range of apple cultivars there is a suggestion that levels of polygalacturonase correlate with fruit firmness irrespective of ethylene production rate (Wakasa 2006).

Example 2: Production of Plants with Reduced Expression of MdPG1

[0300] Ten transgenic Royal Gala lines were created containing MdPG1 expressed in an antisense orientation driven by a strong constitutive promoter (35S promoter). The fruit-specific polygalacturonase cDNA clone MdPG1 (formerly GDPG1, Atkinson 1994), was cloned into pART7 as described previously (Atkinson et al. 2002). A clone with the PG gene in the antisense orientation was digested with NotI and cloned into the binary vector pART27. The binary was electroporated into Agrobacterium tumefaciens strain LBA4404. Transgenic apple Royal Gala shoots were produced using the method of Yao et al. (1995) and maintained in a containment greenhouse under identical conditions (ambient light and temperature) to wild-type plants. Plants were transferred to chillers for 8-10 weeks each year to meet winter chilling requirements. Flowers were hand-pollinated each spring and fruit harvested in autumn when aroma volatiles could be detected.

Example 3: Fruit of Plants Produced by the Methods of the Invention Show Reduced Softening During Post-Harvest Storage

[0301] Five lines, of the 10 described in Example 2, showed less softening than the wild type control after two weeks at room temperature (FIG. 2), and 2 lines (PG275 and PG41) showed significantly less softening after 16 weeks at 5 C. storage. This correlated with previous experiments where apples from the line PG41 showed much reduced softening compared to the control apples. The decreased softening in this line has been shown for fruit collected over 3 growing seasons (Table 1).

[0302] Firmness was measured using a puncture test according to standard industry practise (Blanpied et al., 1978). This involved the localised removal of skin from two opposing locations on the fruit equator, and recording the maximum force while driving a 7.9 mm cylindrical probe into the outer cortex to a constant depth (8 mm) at a fixed speed (4 mm/s). The puncture test and data capture was performed using a Stable Micro Systems TA-XT plus Texture Analyser (Hertog et al 2001).

TABLE-US-00004 TABLE 1 Firmness in PG41 vs control apples over 3 years Storage Storage Storage 2005 30 wks, 2007 32 wks, 2008 16 wks, Harvest 5 C. Harvest 5 C. Harvest 5 C. Probe 11 11 11 11 8.5 8.5 (mm) firmness firmness firmness firmness firmness firmness PGA41 no data 6.18 9.25 7.0 5.06 3.50 control no data 2.83 4.7 4.0 4.64 2.49

[0303] Firmer fruit is a desirable characteristic as sensory/consumer trials show that consumers prefer firmer fruit.

Example 4: Levels of MdPG1 Protein Correlate with Rate of Softening

[0304] The mature ORF of MdPG1 was amplified by PCR using primers RA136 5-ACGGGATCCG CTCCGGCCAA AACCATTAGC-3 and RA137 5-ATAGTTTAGC GGCCGCTTAA CATCTAGGGG AGACAAC-3. The insert was excised with BamHI and Nod (underlined in the primers) and ligated into corresponding sites of the pET-30a(+) vector (Novagen, Madison, Wis., USA). pETMdPG1 was transformed into BL21 cells containing the pLysS plasmid and recombinant His-tagged protein purified by Ni-affinity chromatography under denaturing conditions (Schroder et al. 1998). Purified recombinant forms of MdPG1 protein cut from a polyacrylamide gel was used to raise a polyclonal antibody in rabbits.

[0305] Levels of MdPG1 protein were measured on western blots using polyclonal antibodies raised to the mature MdPG1 protein. For each transgenic line, described in Example 3 above, protein was extracted from apples at harvest, after two weeks room temperature storage and after 16 weeks cold storage. At harvest no MdPG1 was detected in any of the apples except line PG290 (FIG. 3), After 2 weeks storage at room temperature (RT) it was found that both the Royal Gala lines and PG290 showed a significant level of PG. Lines PG7, PG8, PG17, and PG164 had a detectable level of PG (FIG. 4). After 4 weeks of cold storage lines PG7, PG8, PG30 and PG164 had a detectable level of PG (lines 213B, PG275 and PG290 were not assayed) (FIG. 5). At 16 weeks storage lines except PG41 and PG275 showed significant levels of PG (FIG. 6). Comparison across time points there was a strong correlation of levels of PG and rate of softening. Lines PG30 and PG40 showed little softening at 2 weeks RT and showed very low levels of PG at this time point. PG41 showed no detectable PG and PG275 showed very low levels of PG both of which were the firmest apples after 16 weeks storage (FIG. 7).

Example 5: Fruit of Plants Produced by the Methods of the Invention Show Reduced Water Loss During Post-Harvest Storage

[0306] Apples from 8 independent transformant lines, along with the control Royal Gala apples were left at room temperature for 1 month following a 16 week storage period at 4 degrees, and weighed every two weeks. It was found that the lines PG41 and PG275 showed a lower rate of water loss (0.00273 and 0.00237 g/day/g FW respectively) compared to the untransformed control (0.0046 g/day/g FW). The other transgenic lines showed a range between 0.00299 and 0.00317 g/day/g FW) (FIG. 8). These numbers were much larger than those found in a separate water loss experiment with a non-ripening mutant ACO antisense apples and Royal Gala lines that had not been cold stored (FIG. 8), suggesting that cold storing the apples may further accelerate water loss. Apples from the PG41 and PG275 lines also showed less shrivelling compared at 5 weeks RT after transfer from 16 weeks cold storage (FIG. 9).

Example 6: Fruit of Plants Produced by the Methods of the Invention Show Increased Juiciness During Post-Harvest Storage

[0307] Microscopic analysis of PG41 lines and untransformed controls Sectioning cells from Royal Gala apples following a 16 week cold storage period revealed that the cell-to-cell adhesion was significantly weakened (with presumably pectin junctions between cells showing clear regions that have pulled apart FIG. 10A). Sections of cells in the PG41 lines show no pulling apart (FIG. 10B). Additionally antibody staining of the PG41 lines showed a maintainance of the demethylated homogalacturans in cell corners (FIG. 11) compared to the Royal Gala control, that are targeted by PG (identified using a JIM5 antibody). This suggests that decreasing the level of PG reduces cells breaking between the cell boundaries rather than across the cells. It has been proposed that that the difference between juicy apples and mealy apples is due to the way that the cells are disrupted during a bite action. Hallett et al have shown that juicyness is not a measure of water content rather as mealy and juicy apples contain the same amount of water. It has been suggested that juicy apples break across cells releasing the juice while mealy apples break between cells giving a much dryer mouth feel. The loss of cell to cell adhesion in the control lines suggest that the apples would have a more mealy texture, and the PG41 apples would be more juicy (this cannot be confirmed due to restrictions on eating transgenic apples in this country). From these results it is anticipated that the PG knockout apples would also be crisper and crunchier than the Royal gala controls. When cutting the apples after storage they appeared to maintain their crispness.

Example 7: Fruit of Plants Produced by the Methods of the Invention Show Altered Wax Composition

[0308] PG antisense lines PG17 and PG275 both had a waxy feel compared to the Royal Gala control providing evidence that altered expression in the method of the invention can result in altered wax production.

Example 8: Fruit of Plants Produced by the Methods of the Invention Show Reduced Post-Harvest Storage Rots/Infections

[0309] Control fruit were subject to infection by postharvest pathogens after long term storage at 5 C. In contrast PG41 lines rarely showed infection. This effect may be due to a reduction in microcracks on the surface of the fruit which provide an entry point for pathogen invasion.

Fruit Storage at 5 C.

[0310] Commercial fruit storage is carried out at 1 C. in controlled/modified atmosphere conditions. PG41 apples in this study were stored at less than optimal conditions and still maintained fruit quality. This may allow fruit to stored at slightly higher temperatures thereby reducing costs.

Example 9: Fruit of Plants Produced by the Methods of the Invention Show Mostly Normal Ripening Attributes

PG41 Apples Show Normal Ripening Attributes

[0311] To assess whether any other ripening attribute was altered in the PG41 mutant that may contribute to the phenotype, internal ethylenes, starch pattern index (SPI) and soluble solids content (SSC) were measured at harvest, and ethylene was measured 16 weeks after cold storage. From the SPI the Royal Gala apples appeared to be slightly more mature than the PG knock out lines at harvest, but after 16 weeks cold storage the PG41 lines and Royal Gala controls were producing similar amounts of ethylene (FIG. 12), suggesting that the reduced softening is not due to decreased levels of ethylene.

Example 10: Identification of Variants of the MdPG1

[0312] The MdPG1 sequence was used to identify orthologous PG genes from HortResearch proprietary sequence databases.

[0313] Two variant sequences were identified as summarised in the table below.

TABLE-US-00005 Malus Polynucleotide Polypeptide Polygalacturonase species SEQ ID NO: SEQ ID NO: MsPG1 sieboldii 6 2 MsPG2 sieboldii 7 3

[0314] The table below shows the % identity between the MdPG1 and variant polypeptide sequences.

TABLE-US-00006 MdPG1 MsPG1 MsPG2 MdPG1 100% 92.3 95.2 MsPG1 100% No overlap MsPG2 100%

[0315] The function of these variants can be confirmed using the methods described in the examples above.

Example 11: Fruit of Plants Produced by the Methods of the Invention Show Increased Tensile Strength and Firmness in Commercial Storage Conditions

[0316] 30 fruit from the PG41 lines which had no detectable fruit ripening endopolygalacturonasel were harvested along with 30 untransformed Royal Gala (RG) controls. 15 fruit had 1 mm skin removed in 4 quadrants in the equatorial region of the apple and were measured at harvest from each line for puncture firmness was measured (with 4 different probe sizes) (Table 1a) using a TA.XT texture analyzer (Stable Micro Systems, Ltd, UK) as described in Johnston et al (2009). Cores from the cortex tissue were taken and tensile strength of these were measured using the TA.XT texture analyzer

[0317] 15 apples from each line were stored at 0.5 C. for 10 weeks under commercial storage conditions. Following this time apples were tested again for tensile strength and flesh puncture firmness. Additionally these samples were also assessed for amount of juice released by a commercial juicer to assess levels of juiciness.

TABLE-US-00007 TABLE 1 Results of tensile strength and firmness % change during Storage Absolute values storage Data time (wks) Control PG41 Control PG41 Average of Tens Max Force 0 13.1 1.0 12.5 0.6 51.40 34.26 (N) 10 6.3 0.7 8.2 1.0 Average of Force 11 mm 0 84.4 2.2 78.6 3.3 30.94 16.91 Probe (N) 10 58.3 1.4 65.3 2.4 Average of Force 8 mm 0 45.8 1.5 44.3 2.0 33.08 23.70 Probe (N) 10 30.6 0.6 33.8 1.0 Average of Force 5 mm 0 18.7 0.6 18.9 0.8 28.37 22.73 Probe (N) 10 13.4 0.3 14.6 0.5 Average of Force 2 mm 0 4.1 0.2 4.2 0.2 34.97 28.20 Probe (N) 10 2.7 0 3.0 0

[0318] There were no clear differences between the control RG lines and the PG 41 lines at harvest. But after 10 weeks storage there was a significant increase in both tensile strength and firmness. The 7 N increase (65.3 N-58.3) in firmness of PG41 apples relative to RG control apples measured with the 11 mm probe following storage is larger than the minimum 6 N difference that a trained sensory panel can detect (Harker et al 2002) strongly indicating that in a sensory trial the PG41 fruit would be scored as better textured than the RG control after storage (FIG. 12). When the original firmness of the PG41 fruit is taken into account then there is only a 16% loss of firmness compared to a 31% loss of firmness in the control fruit.

REFERENCES

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TABLE-US-00008 SUMMARY OF SEQUENCES SEQ ID NO: Type Species Reference 1 polypeptide Malus domestica MdPG1, polygalacturonase, Genbank accession number L27743 2 polypeptide Malus sieboldii MsPG1, polygalacturonase 3 polypeptide Malus sieboldii MsPG2, polygalacturonase 4 polynucleotide Malus domestica, MdPG1, polygalacturonase promoter sequence and part of first exon. Genbank accession number AF031233) 5 polynucleotide Malus domestica, MdPG1, polygalacturonase, cDNA 6 polynucleotide Malus sieboldii MsPG1, polygalacturonase, cDNA 7 polynucleotide Malus sieboldii MsPG2, polygalacturonase, cDNA