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US 2011/0191902 A1 Atkinson Et Al US 2011 0191902A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0191902 A1 Atkinson et al. (43) Pub. Date: Aug. 4, 2011 (54) METHODS AND COMPOSITIONS FOR Publication Classification INCREASING STORAGE-LIFE OF FRUIT (51) Int. Cl. AOIH I/00 (2006.01) (76) Inventors: Ross Graham Atkinson, Auckland CI2N 5/10 (2006.01) (NZ); Robert James Schaffer, AOIH 5/00 (2006.01) Auckland (NZ); Kularajathevan CI2N 5/82 (2006.01) Gunaseelan, Auckland (NZ); C7H 2L/00 (2006.01) Roswitha Schröder, Auckland CI2N 9/14 (2006.01) (NZ) (52) U.S. Cl. ...... 800/278; 435/419: 800/298; 435/320.1; 800/301; 536/23.2:435/195 (21) Appl. No.: 13/060,257 (57) ABSTRACT The invention provides methods and compositions for pro (22) PCT Filed: Aug. 28, 2009 ducing plants with fruit having increased post-harvest storage life, the method comprising reducing the expression or activ (86). PCT No.: PCT/NZ09/00182 ity in the plant, of a polypeptide with the amino acid sequence of SEQID NO: 1, or a variant of the polypeptide. The inven S371 (c)(1), tion provides host cells, plant cells and plants transformed (2), (4) Date: Apr. 14, 2011 with the polynucleotides of the invention. The invention also provides methods for selecting plants with fruit having (30) Foreign Application Priority Data increased post-harvest storage life. The invention also pro vides plants produced and selected by the methods of the Aug. 29, 2008 (NZ) ........................................ 570886 invention. Fruit Development A. - 3594 ^. ^ 1413-14 259035 Xyloglucan ^, endotransglucosylas i 31357 Y,'- 25 170802,: Mot selected . | 147844 xyloglucan v. 3: 15 f endotransglucosylas v. 269270 166334 / - - - - - - - - . S.214025 X 149098 - Ethylene Storage Polygalacturonase expression 3. Samtrol Sethylene 2. sld to sold ethylene 2 | S. 5 | . gi ig Time hours Patent Application Publication Aug. 4, 2011 Sheet 1 of 11 US 2011/O191902 A1 Figure 1 Fruit Development - 91 31549 2: 3:3:ii.33& A 141-1 y ' '. : • 147644A sys& \v, 269095 ::: f y 170802 252022 147644't St. Xyloglucan f endotransglucosylase N 69 \ ... it. 16633-1 A . Ethylene is 214025---...---- X ---...---149098 - Storage Polygalacturonase expression 3. Stil Sethylene sold trol sold ethylene 2000 S i 15 E 1000 aska .*.* 4. f R Time ours Patent Application Publication Aug. 4, 2011 Sheet 2 of 11 US 2011/O191902 A1 Figure 2 ?GAZ?d Patent Application Publication Aug. 4, 2011 Sheet 3 of 11 US 2011/O191902 A1 Figure 3 PG at haWest Lane 1 Ladder 1 - 3 S W 8 11 2 PG 3 PG8 49k 4. PG17 38 - 5 PG30 SPG4O PG4 8 PGS4 9 PG213B O PG275 11 PG290 12 Royal Gala Figure 4 PG 2 weeks room temperature Ladder 1 2 3 4 5 S 8 g g 11 12 2PG a19k 38 K. 11 PG290 12 Royal Gala Patent Application Publication Aug. 4, 2011 Sheet 4 of 11 US 2011/O191902 A1 Figure 5 PG 4 Weeks Cold treatment 1 2 3 4 5 6 F 8 3 10 Lane Ladder 2 PG7 3 PG8 49k 4. PG17 38 - 5 PG30 SPG40 PG4 8 PG 164 9 Royal Gala 10 tomato Figure 6 PG 16 Weeks Cold treatment Lane 3 8 9 11 Ladder 2 PG 3 PG8 49k 4. PG17 5 PG30 38 - SPG4O 7 PG4 8 PG 164 9 PG213B OPG275 11 PG290 12 Royal Gala Patent Application Publication Aug. 4, 2011 Sheet 5 of 11 US 2011/O191902 A1 Figure 7 PGSO PGAO Royal Gala L. H. 2w 18w H 2w 16w H 2w 16w 49. ka 38 kDa PGA PG275 Royal Gala L H 2w 16w H 2w 16w H 2w 16w 49 kDa -- 38 kg -b. Patent Application Publication Aug. 4, 2011 Sheet 6 of 11 US 2011/O191902 A1 Figure 8 Water loss from PG knockout and Royal Gala after cold storage OE OOO. - -- P- P-8 P-3 P- P- - P-3E - R TRL Water loss from ACO knockout and Royal Gala off tree E. GOO fruit Ead fruit GOO fruit Ead fruit (Wrinkled) (Wrinkled & rotten) (Wrinkled) Royal Gala Control ACC OxidaSe AS ent Application Publication Patent Application Publication Aug. 4, 2011 Sheet 8 of 11 US 2011/O191902 A1 Figure 10 Royal Gala 16 weeks cold treatment PG41 16 Weeks Cold treatment Patent Application Publication Aug. 4, 2011 Sheet 9 of 11 US 2011/O191902 A1 Figure 11 Royal Gala 16 Weeks Cold X 40 PG41 16 WeekS COC x 40 JIM5 antibodies (de-methylated Homogalacturanan) target for PG Patent Application Publication Aug. 4, 2011 Sheet 10 of 11 US 2011/O191902 A1 Figure 12 8tactiseferees &aa praise isxxxxxxx sixes & Sws:}sage w assassssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssar % change caused by 10 weeks stored at O.5°C Coto RG41 Tensile strength 51.40 34.26 Pucture force 33.08 23.7 Patent Application Publication Aug. 4, 2011 Sheet 11 of 11 US 2011/O191902 A1 Figure 13 MALKTOLLWSFVVVFVVSFSTTSCSGSSFOEVNALHSYWDHVDDKESGYNSRAY-SYTDTIEGLKVM-ELIRPRTOL I SSRKLYTITGGIASSAPAKTISVDDIGAKGNGADDTO AVKAWKAACS 83GAMVLVVPOKNYLVRPIEISGPCK SOLTLOIYGTIEASEDRSIYKDIDHWIFDNVONLLV VG-GTING NGNIWWKNSCKIKPOPPCGTYAPTAVTFNRC DCIlain DOIClair II NNLV VKYLNICDAOOIHWIFONCINVCASCLTWTAPESENTEGEHVTNTONITISSSWCTEEEEISIWSCSORV Domain II Domain IV OAT DITEGEGHSESGSLGEDGSEDHVSG VEVNGAKLSGTSNGLRKTWKGGSGSATNIVE ON WOMNVTNPIID ONYCDHKT4DCKOOKSAVOWKYWLYCNIRGTSASGDAITLNCSOSVPCOGIWLOS VOLONGRAECNY VOPAYKGVV SPRC US 2011/O 191902 A1 Aug. 4, 2011 METHODS AND COMPOSITIONS FOR radation in fruit. However, no significant effect on fruit soft INCREASING STORAGE-LIFE OF FRUIT ening, ethylene evolution, or color development was detected. The authors reported that “polygalacturonase was TECHNICAL FIELD the primary determinant of cell wall polyuronide degradation, but suggested that this degradation was not sufficient for the 0001. The present invention relates methods and compo induction of softening (Giovannoni et al., 1989). sitions for producing plants with fruit with increased storage 0010 Further experiments where the pTOM6 gene was life. overexpressed in tobacco (Nicotiana tabacum; Osteryoung et al., 1990) showed that the tomato protein was properly pro BACKGROUND ART cessed and localized in the cell walls of leaves in tobacco. The 0002 Post-harvest spoilage is a major problem for the enzyme showed activity when extracted from transgenic fruit industry. It has been estimated that 10-20% of post tobacco leaves and tested against tobacco cell wall extracts in harvest fruit is lost through spoilage before reaching consum vitro. However, no changes in leaf phenotype were observed, ers. This spoilage results in increased cost of the non-spoiled nor were there any alterations to the pectins in the tobacco cell fruit to the consumer. In addition fruit is often discarded by walls in vivo. the consumer because of spoilage after purchase but before 0011 Together these results suggested to researchers that the fruit is eaten. PG only had role in pectin depolymerization primarily in fruit 0003. One of the main causes of spoilage is the natural but the enzyme did not have an affect on fruit softening. ripening of fruit. As fruit ripens it tends to become softer and 0012. Thus in spite of such substantial research the prob more Susceptible to mechanical damage, as well as biological lem of post-harvest softening has not been overcome for most damage from necrophytic pathogens Such as storage rots. fruits and particularly in apple. Apple (Malus domestica 0004. In addition to the problems associated with soften Borkh.cv Royal Gala) ripens very differently than tomato and ing, the flesh of fruits such as apples often develop a “mealy' many other fruits because cell wall swelling is not one of the dry texture during post-harvest storage, which is unpopular cell wall modifications occurring during apple ripening with consumers. Mealy texture is believed to be a result of (Redgwell et al., 1997). There is minimal change in viscosity separation of cells in the flesh when the fruit is bitten, without of cell walls, and minimal pectin Solubilization or degrada associated rupture of cells and release of the apple's juice. tion during fruit ripening. It would therefore be of benefit to This creates the impression of a dry, less juicy apple, as well provide improved or alternative methods to addressing post as a less crunchy or crispy apple. harvest Softening in apples and other fruit. 0005 Water loss from fruit during storage is also a prob 0013. It is an object of the invention to provide improved lem and can lead to fruit developing an unattractive shriveled methods and compositions for producing plants with fruit appearance. having increased post-harvest storage life, or at least to pro 0006 Various approaches have been used to attempt to vide the public with a useful choice. address post-harvest deterioration of fruit. For example, genetic approaches have focused on manipulating the expres SUMMARY OF THE INVENTION sion of ethylene biosynthesic genes such as ACC oxidase (e.g. 0014. In a first aspect the invention provides a method for in apple, Schafferetal 2007; tomato, Hamilton etal 1990; and producing a plant with fruit having increased post-harvest melon, Ayub etal 1996), and ACC synthase (Oelleretal 1991) storage life, the method comprising reducing the expression and on genes that influence cell wall physiology Such as or activity in the plant, of a polypeptide with the amino acid pectin lyase (Santiago-Doménech et al 2008), expansin sequence of SEQID NO: 1, or a variant of the polypeptide. (Brummelletal 1999) and B-galactosidase (Carey etal 2001). 0015. In one embodiment the fruit have at least one of: However, to the applicants knowledge no fruits resulting from 0016 a) increased firmness, Such approaches are currently commercially available. (0017 b) reduced water loss, 0007 Transgenic tomatoes in which expression of a 0018 c) reduced cell separation, polygalacturonase (PG) gene was reduced were commercia 0019 d) increased juiciness, lised as the well documented Flavir Savr tomatoes. However 0020 e) increased crispiness, technical problems reportedly made it difficult to ship the 0021 f) increased waxiness, and delicate GE tomatoes without damage.
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