Methods and Materials for Producing Coreless Fruit

Abstract

The invention provides materials and methods for producing coreless fruit, or plants that produce coreless fruit. The invention involves combining reduced expression of AGAMOUS (AG) with parthenocarpy. Parthenocarpy can be induced by hormone treatment, or can be provided by reduced or eliminated expression of PISTILATA (PI) or APETALA3 (AP3). The invention provides methods and materials for producing the plants and coreless fruit by genetic modification (GM) and non-GM means. The invention also provides the plants and coreless fruit.

Claims

1-42. (canceled)

43. A method for producing a coreless fruit, or a plant that produces at least one coreless fruit, the method comprising reducing, or eliminating, expression of at least one AGAMOUS (AG) protein in a plant from a species that produces accessory fruit.

44. The method of claim 43 that includes the additional step of inducing parthenocarpy in the plant.

45. The method of claim 43 wherein the plant in which expression of at least one AGAMOUS (AG) protein is reduced or eliminated, is a parthenocarpic plant.

46. The method of claim 44 in which parthenocarpy is induced by application of plant hormones to flowers of the plant.

47. The method of claim 44 in which parthenocarpy is induced by manipulating expression of genes controlling fruit set.

48. The method of claim 47 in which parthenocarpy is induced by reducing, or eliminating expression, of at least one PISTILLATA (PI) gene or protein.

49. The method of claim 47 in which parthenocarpy is induced by reducing, or eliminating expression, of at least one APETALA3 (AP3) gene or protein.

50. The method of claim 45 in which the parthenocarpic plant is a mutant plant with reduced, or eliminated expression, of at least one PISTILLATA (PI) gene or protein.

51. The method of claim 45 in which the parthenocarpic plant is a mutant plant with reduced, or eliminated, expression of at least one APETALA3 (AP3) gene or protein.

52. A method for producing a coreless fruit, or a plant that produces at least one coreless fruit, the method comprising reducing, or eliminating, in a plant from a species that produces accessory fruit, expression of: a) at least one AGAMOUS (AG) protein, and b) at least one of: i) at least one PISTILLATA (PI) protein, and ii) at least one APETALA3 (AP3) protein

53. the method of claim 52 in which reducing, or eliminating, expression of the PISTILLATA (PI) protein or APETALA3 (AP3) protein induces parthenocarpy.

54. A method of detecting, in a plant from a species that produces accessory fruit, at least one of: a) reduced, or eliminated, expression of at least one AGAMOUS (AG) protein, b) reduced, or eliminated, expression of at least one polynucleotide encoding an AGAMOUS (AG) protein, c) presence of a marker associated with reduced expression of at least one AGAMOUS (AG) protein, and d) presence of a marker associated with reduced expression of at least one polynucleotide encoding an AGAMOUS (AG) protein.

55. The method of claim 54 wherein detecting any of a) to d) indicates that the plant will produce, or be useful for producing, at least one coreless fruit.

56. The method of claim 54, wherein plant identified is a mutant plant with reduced or eliminated expression of an AGAMOUS (AG) gene or protein.

57. The method of claim 54, further comprising detecting in the plant, at least one of: e) reduced, or eliminated, expression of at least one PISTILLATA (PI) or APETALA3 (AP3) protein, f) reduced, or eliminated, expression of at least one polynucleotide encoding a PISTILLATA (PI) or APETALA3 (AP3) protein, g) presence of a marker associated with reduced expression of at least one PISTILLATA (PI) or APETALA3 (AP3) protein, and h) presence of a marker associated with reduced expression of at least one polynucleotide encoding a PISTILATA (PI) or APETALA3 (AP3) protein.

58. The method of claim 57 wherein detecting any of e) to h) indicates that the plant will produce, or be useful for producing, at least one coreless fruit.

59. The method of claim 57, wherein plant identified is a mutant plant with reduced or eliminated expression of a PISTILLATA (PI) or APETALA3 (AP3) gene or protein.

60. A method for producing a plant that produces at least one coreless fruit, the method comprising crossing at least one of: a) a plant with reduced, or eliminated, expression of at least one AGAMOUS (AG) protein, and b) a mutant plant with reduced, or eliminated, expression of at least on one of AGAMOUS (AG) protein, with another plant, wherein the off-spring produced by the crossing is a plant that produces at least one coreless fruit, and wherein the plant in a), the plant in b) and the another plant are from a species that produces accessory fruit.

61. The method of claim 60 in which at least one of the plant of a), the plant of b, and the another plant, is at least one of: i) a parthenogenic plant, ii) a plant with reduced or eliminated expression of at least one PISTILLATA (PI) protein, iii) a plant with reduced or eliminated expression of at least one APETALA3 (AP3) protein.

62. A method for producing a coreless fruit, the method comprising cultivating a plant in which at least one of a) to d) is detected in the method of claim 54.

63. The method of claim 62 which includes the additional step of inducing parthenocarpy in the plant.

64. The method of claim 62 in which the plant produces coreless fruit as a result of having reduced, or eliminated expression, of at least one AGAMOUS (AG) protein, and having reduced, or eliminated expression, of one of an PISTILLATA (PI) protein and an APETALA3 (AP3) protein.

65. A method of producing a coreless fruit the method comprising cultivating a plant, from a species that produces accessory fruit, with reduced, or eliminated, expression of at least one AGAMOUS (AG) protein.

66. The method of claim 65 wherein the plant also has reduced, or eliminated, expression of at least one of: i) at least one PISTILLATA (PI) protein, and ii) at least one APETALA3 (AP3) protein.

67. A coreless fruit produced by a method of claim 65.

68. A coreless fruit, or plant that produces at least one coreless fruit, with reduced or eliminated expression of at least one AGAMOUS (AG) protein, wherein the coreless fruit or plant is from a species that produces accessory fruit.

69. The coreless fruit, or plant of claim 68 that also has reduced or eliminated expression of at least one of: i) at least one PISTILLATA (PI) protein, and ii) at least one APETALA3 (AP3) protein.

70. A coreless fruit or a plant of claim 68, wherein the coreless fruit or plant comprises a construct for reducing, or eliminating, the expression, in a plant, of at least one of: a) an AGAMOUS (AG) protein, b) a PISTILLATA (PI) protein, and c) an APETALA3 (AP3) protein.

71. The coreless fruit or plant of claim 70 wherein the construct comprises part of a gene or polynucleotide that encodes the protein.

72. The coreless fruit or plant of claim 70 wherein the construct is designed to reduce, or eliminate, expression of at least one of: a) an AGAMOUS (AG) protein and a PISTILLATA (PI) protein, and b) an AGAMOUS (AG) protein and an APETALA3 (AP3) protein.

73. Use of a plant part, progeny, or propagule of a plant of claim 66, that has reduced, or eliminated expression of at least on AGAMOUS (AG) protein to produce a plant that produces at least one coreless fruit.

74. The use of claim 73 in which plant part, progeny, or propagule also has reduced, or eliminated expression of at least one of: a) a PISTILLATA (PI) protein, and b) an APETALA3 (AP3) protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0426] The present invention will be better understood with reference to the accompanying drawings in which:

[0427] FIG. 1 shows the Cluster of AGAMOUS like MADS box genes in Arabidopsis and Apple

[0428] FIG. 2 shows an alignment of MdAG (SEQ ID NO:1) with AtAG (SEQ ID NO:X)

[0429] FIG. 3 shows an alignment of MdAG (SEQ ID NO:1) with published MdMADS15 (SEQ ID NO:30).

[0430] FIG. 4 shows expression analysis of AG-like genes in untransformed (WT) apple and 2 independent ag RNAi transgenic lines showing ag phenotype (AS2905 and AS2921)

[0431] FIG. 5 shows the floral phenotype of suppression of AG in apples. ag(AS2921) mutants show whorls of petals and sepals.

[0432] FIG. 6 shows generation of apple through the treatment of ag (AS205) flowers with GA/IAA. These apples have reduced core tissue pushed towards the calex

[0433] FIG. 7 shows generation of apple through the treatment of ag (AS2921) flowers with GA/IAA/cytokinin. This apple has no apparent core tissue.

[0434] FIG. 8 shows a map of pTKO2S_262928, the MdAG sequences are shown as green arrows (KO seq)

[0435] FIG. 9 shows the conserved MADS domain and K domain of proteins MdAG (SEQ ID NO:1), MdPI (SEQ ID NO: 7), MdTM6 (SEQ ID NO: 13) and MdMADS13 (SEQ ID NO: 14).

EXAMPLES

[0436] The invention will now be illustrated with reference to the following non-limiting example.

[0437] It is not the intention to limit the scope of the invention to the abovementioned example only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention.

Example 1

Production of Coreless Fruit by Reducing Expression of the Agamous (AG) Gene and Hormone Application

[0438] Gene Identification

[0439] The AG cluster in Arabidopsis consists of 4 genes which are AG, SEEDSTICK (STK) and SHATTERPROOF (SHP) 1 and 2. The ancient genome duplication in apples means that for each of these Arabidopsis genes there are two similar apple genes

[0440] Using the apple genome (Velasco et al., 2010), apples MDP0000324166 and a homeologous gene MDP0000250080) are the most similar to Arabidopsis AG (atAG), see FIG. 1.

[0441] The first MDP0000324166, has been published as MADS15 (SEQ ID NO:30, van der Linden et al., 2002) The DNA sequence encoding MADS15 is shown in SEQ ID NO:31).

[0442] The applicants identified the equivalent gene for the apple cultivar Royal Gala, and designated this gene MdAG. The sequence of the MdAG protein and the polynucleotide encoding the protein are shown in SEQ ID NO: 1 and 4 respectively

[0443] An alignment of MdAG and AtAG proteins is shown in FIG. 2.

[0444] Expression Analysis

[0445] Expression analysis of the gene as per mRNA seq of developing (balloon stage) flowers and open flowers show that MDP0000324166/MADS15/MdAG is higher expressed in apple flowers compared to MDP0000250080 MADS115.

[0446] Suppression by RNAi of the MDP0000324166/MADS15/MdAG resulted in less transcript abundance (FIG. 3) and also down regulation of STK-like and SHP-like genes. (which maybe expected as these are both downstream of AG in Arabidopsis defining different carpel structures.) The next most similar genes outside this clade (SOC like) were unaffected (FIG. 3).

[0447] Creation of Plants Suppressed for MdAG

[0448] A hairpin construct, containing the first 403 bp (ATGGCCTATGAAAGCAAATCCTTGTCCTTGGACTCTCCCCAGAGAAAATTGGGTAGGGG AAAGATCGAGATTAAGCGGATCGAAAACACAACGAATCGTCAAGTGACCTTCTGCAAGA GGCGCAATGGGTTGCTCAAGAAGGCCTATGAACTCTCTGTGCTCTGTGATGCAGAGGTT GCTCTCATAGTCTTCTCTAACCGTGGCCGCCTCTATGAGTATGCCAACAATAGTGTTAAA GGAACAATTGAGAGGTACAAGAAGGCAAGTGCAGATTCTTCAAATACTGGATCAGTTTCT GAAGCTAGTACTCAGTACTACCAGCAAGAAGCTGCGAAATTGCGTGCGCAGATAGTGAA ATTGCAGAATGACAACCGGAATATGATGGGTGATGCATTGAGTAGTATGTCTGTCAAGG ACCTGAAGAGCCTGG—SEQ ID NO:25) of the MdAG gene of SEQ ID NO:4 was cloned into the a pDONOR (Invitrogen) and inserted into the gateway compatible pTKO2 vector (Snowden et al., 2005) in an inverted repeat (green KO seq—FIG. 8). These were transformed into ‘Royal Gala’ apples as described by (Yao et al., 1995).

[0449] Construction of hairpin knockout vector pTKO2S_262928 (EST 262928)

[0450] The hairpin knockout vector pTKO2S_262928 (EST 262928) was constructed with pTKO2 (Snowden et al 2005) using Gateway Technology (Invitrogen).

[0451] PCR was carried out on pBluescript (SK-) EST_262928 with the primers 262928_F (Gateway attB1—atggcctatgaaagcaaatcc—SEQ ID NO:26) and 262928_R (Gateway attB2—CCAGGCTCTTCAGGTCCTTG—SEQ ID NO:27) to give a PCR product of 430 bp.

[0452] Amplfication was carried out on 10 ng of template DNA with 0.5 mM of each primer, 0.8 mM dNTPs, 1× Taq DNA polymerase buffer, 0.5 U Expand High Fidelity Taq DNA polymerase (Boehringer Mannheim) in a Techne Progene cycler: 94° C. (3 min), followed by 30 cycles of 94° C. (30 s), 60° C. (45 s), 68° C. (1 min).

[0453] The Gateway BP reaction with PCR product and pDONR was carried out as recommended by the manufacturer (Invitrogen). Plasmid DNA of resulting transformants was isolated using Wizard Plus Miniprep DNA Purification System (Promega) and the correct constructs were verified by restriction enzyme analysis for the pENTRY_262928 (430 bp insert).

[0454] Gateway LR reactions with the resulting pENTRY_262928 vector and destination vector pTKO2 was carried out as recommended by the manufacturer. (Invitrogen).

[0455] The final construct was verified by restriction enzyme analysis.

[0456] A map of pTKO2S_262928 is shown in FIG. 8.

[0457] Phenotype of the RNAi Suppressed Lines

[0458] Apples with suppressed AG have floral conversion to whorls of sepals and petals these can be seen in FIG. 4. This is consistent with the literature when you knock out AG in other species such as Arabidopsis (Yanofsky et al., 1990). Microscopy of one of the suppressed lines (AS2905) revealed that there are apparent remnant ovules and possible pollen like formations in the more acropetal whorls of organs (FIG. 5)

[0459] Induction of Parthenocarpy

[0460] Pathenocarpy (production of fruit with no pollination) can be induced with hormone treatment or genetically with the modulation of certain genes detailed in (Sotelo-Silveira et al., 2014).

[0461] In apples extensive work was done to induce parthenocarpy, only the triple combination of GA3, SD8339, and 2-NAA, rather than single or paired application, resulted in parthenocarpy in Cox's Orange Pippin (Kotob and Schwabe, 1971) and GA4+7 alone induced parthenocarpy in frost-damaged Bramley's Seedling and cytokinin SD8339 had no additional benefits; GA3 was not effective. This said, Bramley's Seedling is triploid and partially self-fertile so may be an unusual case (Modlibowska, 1972).

[0462] To induce parthenocarpy in the ag apples, treatments with different concentrations and combinations of Gibberellins (GA), Auxin (IAA) and Cytokinins (BAP) were applied to the flowers. Hormone concentrations: 300 ppm GA4 & 1 ppm IAA, and 300 ppm GA4, 100 ppm 6-BA, & 1 ppm IAA.

[0463] All treatments started at a stage around −7DAFB. All flowers treated −7, −4 and +1 DAFB, three treatments in total for most flowers, 4 treatments for a few.

TABLE-US-00008 final fruit Genotype treatment infor. flowers numbers wild-type GA/IAA 22 110 5 4.5% GA/IAA/BA 23 115 7 6.1% AS2905 GA/IAA 22 110 1 0.9% AS2921 GA/IAA/BA 8 40 1 2.5%

[0464] These apples were allowed to grow to maturity, then they were harvested and assessed for presence of core. Apples from transgenic lines containing partial ovules were able to be induced with GA and IAA alone. Transgenic lines with more severe phenotype (no ovule tissue) needed cytokinins (FIG. 5).

[0465] Properties of Reduced Core and Coreless Apples

[0466] Reduced core and coreless apples are shown in FIGS. 6 and 7 respectively. With reduced cored apples (FIG. 6) having less locule tissue and an increase in relative amounts of flesh tissue compared to untransformed controls. With the complete absence of ovule (Core) tissue (FIG. 7), no locules or seed bearing tissue is present and the flesh tissue is distributed throughout the apple.

Example 2

Production of Coreless Fruit by Reducing Expression of AG and AP3-Like Genes

[0467] It will be understood by those skilled in the art that apple plants that do no express AP3-like genes are parthenocarpic (Yao et al. 2001). Therefore in accordance with the present invention, suppression of both AG and AP3-like genes (to induce parthenocarpy) results in plants than produce coreless fruit.

[0468] Hairpin Construct for Suppressing AP3-Like Genes

[0469] To suppress the two apple AP3-like genes, MdMADS13 and MdTm6, a hairpin construct containing the first 414 bp (ATATATCAAGTAAAACAAGATCAGAAAATTGCTAGGAAAAGGTAAGAAATTTGAGAGAG AGAGAGAAATTATGGGTCGTGGGAAGATTGAAATCAAGCTGATCGAAAACCAGACCAAC AGGCAGGTGACCTACTCCAAGAGAAGAAATGGGATCTTCAAGAAGGCTCAGGAGCTCAC CGTTCTCTGTGATGCCAAGGTCTCCCTCATTATGCTCTCCAACACTAATAAAATGCACGA GTATATCAGCCCTACCACTACGACCAAGAGTATGTATGATGACTATCAGAAAACTATGGG GATCGATCTGTGGAGGACACACGAGGAGTCGATGAAAGACACCTTGTGGAAGTTGAAAG AGATCAACAATAAGCTGAGGAGAGAGATCAGGCAGAGGTTGGGCCATGATCTAAATGG—SEQ ID NO:28) of MdMADS13 (SEQ ID NO:20) can be cloned into the a pDONOR (Invitrogen) and inserted into the gateway compatible pTKO2 vector (Snowden et al., 2005) as an inverted repeat. This construct will suppress both MdTM6 and MdMADS13 because the DNA sequences in this region are highly conserved between the two genes.

[0470] Transformation

[0471] To suppress both AG and the AP3-like genes, this construct and the MdAG suppressing construct (described in Example 1) can both be transformed into ‘Royal Gala’ apples as described in Example 1 and Yao et al., 1995.

[0472] Transgenic plants containing both gene constructs can be identified using PCR analysis and grown in a glasshouse for fruit production and phenotype analysis.

[0473] This will result in an apple plant with reduced, or eliminated, expression of both MdAG and the AP3-like genes, which will produce coreless fruit.

Example 3

Production of Coreless Fruit by Reducing Expression of AG and PI Genes

[0474] It will be understood by those skilled in the art that apple plants that do no express PI genes are parthenocarpic (Yao et al. 2001). Therefore according to the invention, suppression of both AG and PI genes results in plants than produce coreless fruit.

[0475] Hairpin Construct for Suppressing PI Genes

[0476] To suppress the two apple MdPI, a hairpin construct containing the first 414 bp (ATGGGACGTGGGAAGGTTGAGATCAAGAGGATTGAGAACTCAAGTAACAGGCAGGTGA CCTACTCCAAGAGGAGGAATGGGATTATCAAGAAGGCAAAGGAGATCACTGTTCTATGT GATGCTAAAGTATCTCTTATCATTTATTCTAGCTCTGGGAAGATGGTTGAATACTGCAGC CCTTCAACTACGCTGACAGAAATCTTGGACAAATACCATGGACAATCTGGGAAGAAGTTG TGGGATGCTAAGCATGAGAACCTCAGCAATGAAGTGGATAGAGTCAAGAAAGACAATGA CAGCATGCAAGTAGAGCTCAGGCATCTGAAGGGAGAGGATATCACATCATTGAACCATG TAGAGCTGATGGCCTTAGAGGAAGCACTTGAAAATGGCCTTACAAGTATCCGGGACAAG—SEQ ID NO:29) of MdPI (SEQ ID NO:20) can be cloned into pDONOR (Invitrogen) and inserted into the gateway compatible pTKO2 vector (Snowden et al., 2005) as an inverted repeat.

[0477] Transformation

[0478] To suppress both MdAG and the MDPI genes, this construct and the MdAG suppressing construct (described in Example 1) can both be transformed into ‘Royal Gala’ apples as described in Example 1 and Yao et al., 1995.

[0479] Transgenic plants containing both gene constructs can be identified using PCR analysis and grown in a glasshouse for fruit production and phenotype analysis.

[0480] This will result in an apple plant with reduced, or eliminated, expression of both MdAG and the MdPI genes, which will produce coreless fruit.

Example 4

Production of Coreless Fruit by Reducing Expression of AG in a Pistilata (PI) Mutant

[0481] It will be understood by those skilled in the art that apple plants that do no express MdPI genes are parthenocarpic (Yao et al. 2001). Therefore in accordance with the present invention, suppression of AG in a plant that does not express a PI gene results in plants than produce coreless fruit.

[0482] The hairpin construct designed to suppress MdAG (described in Example 1, and shown in FIG. 8) can be transferred into the ‘Rae Ime’ apple mutant (for example) that does not express the apple MdPI gene (Yao et al. 2001) using the method as described in Example 1.

[0483] This will result in an apple plant with reduced, or eliminated, expression of both MdAG and MdPI which will produce coreless fruit.

Example 5

Production Plants Producing Coreless Fruit by Non-Transgenic Means

[0484] In accordance with the invention apple plants with suppressed or eliminated expression if AG and PI, or AG and AP3-like genes will produce coreless fruit.

[0485] Apple plants with reduced, or eliminated, expression of both AG and PI can be produced by combining natural apple mutants using sexual crossing. First, natural mutants of apple AG gene are identified. The AG suppressed apples have increased whorls of petals and can therefore be selected amongst existing cultivars.

[0486] The applicants have identified, within their germplasm collections, natural mutants of AG with apple varieties, such as Malus ioensis ‘Plena’, which show a similar phenotype to the AG suppression transgenic plants, described in Example 1.

[0487] Plants with such a phenotype can optionally be selected for whole genome sequencing to identify mutations in the AG genes, and for q-RT-PCR analysis to confirm the reduced or eliminated expression of the AG gene. Alternatively plants can be screened for reduced expression of the AG gene first.

[0488] The AG mutant plant can be crossed with parthenocarpic plants, such as the PI mutants described herein, by methods well known to those skilled in the art.

[0489] For example the AG and PI mutants can for example be combined with high fruit quality by rapid introgression breeding using a fast flowering ‘Royal Gala’ apple line. A ‘Royal Gala’ apple transgenic line has been established by over-expression of a flowering promotion gene. This line flowered a few weeks after transplanted into greenhouse from tissue culture. Seedlings of this line would be expected to flower within one year, i.e. one year per generation compared to 6-8 years per generation for normal apple plants.

[0490] Resulting plants with reduced, or eliminated, expression of both MdAG and MdPI which will produce coreless fruit.

[0491] If the varieties containing AG and PI mutaions are poor in fruit quality, multiple of back-crosses to premium apple cultivars can be performed, by methods well known to those skilled in the art, in order to maintain the high fruit quality of the future coreless apple cultivars.

REFERENCES

[0492] Kotob M, Schwabe W. 1971. Induction of parthenocarpic fruit in Cox's Orange Pippin apples. J Hort Sci.

[0493] Modlibowska I. 1972. effect of gibberellins and cytokinins on fruit development of Bramley's Seedling apple. J Hort Sci.

[0494] Snowden K C, Simkin A J, Janssen B J, Templeton K R, Loucas H M, Simons J L, Karunairetnam S, Gleave A P, Clark D G, Klee H J. 2005. The Decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 Gene Affects Branch Production and Plays a Role in Leaf Senescence, Root Growth, and Flower Development. The Plant Cell Online 17, 746-759.

[0495] Sotelo-Silveira M, Marsch-Martinez N, de Folter S. 2014. Unraveling the signal scenario of fruit set. Planta 239, 1147-1158.

[0496] van der Linden C G, Vosman B, Smulders M J M. 2002. Cloning and characterization of four apple MADS box genes isolated from vegetative tissue. Journal of Experimental Botany 53, 1025-1036.

[0497] Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar S K, Troggio M, Pruss D. 2010. The genome of the domesticated apple (Malus [times] domestica Borkh.). Nature genetics 42, 833-839.

[0498] Yanofsky M F, Ma H, Bowman J L, Drews G N, Feldmann K A, Meyerowitz E M. 1990. The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346, 35-39.

[0499] Yao J-L, Cohen D, Atkinson R, Richardson K, Morris B. 1995. Regeneration of transgenic plants from the commercial apple cultivar Royal Gala. Plant Cell Reports 14, 407-412.

[0500] Yao, J.-L., Dong, Y.-H. & Morris, B. A. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor. Proceedings of the National Academy of Sciences 98, 1306-1311 (2001).