LETTUCE WITH INCREASED SHELF LIFE
The present application is directed to lettuce plants comprising a mutation in each of at least two different PPO genes, where said mutations reduce the activity of PPO protein compared to a wild type lettuce plant. The present application is also directed to methods for making such lettuce plants.
1 . A lettuce plant comprising a mutation in each of at least two different PPO genes, wherein said mutations reduce the activity of PPO protein compared to a wild type lettuce plant.
2 . The lettuce plant of claim 1 , wherein at least one of said mutations occurs in both alleles of the PPO gene.
3 . The lettuce plant of claim 1 , wherein at least one of said mutations occurs in only one allele of the PPO genes.
4 . The lettuce plant according any one of the preceding claims , wherein the at least two different PPO genes are selected from the group consisting of PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-O, PPO-P, PPO-R, PPO-N, PPO-M, PPO-J, PPO-Q, and PPO-S.
5 . The lettuce plant according to any one of the preceding claims , wherein the at least two different PPO genes comprise PPO-B and at least one of PPO-S and PPO-G.
6 . The lettuce plant according to claim 5 further comprising:
a mutation in one or more of PPO-A, PPO-C, PPO-O, PPO-P, and PPO-R.
7 . The lettuce plant according to claim 5 or claim 6 , wherein the plant comprises a mutation in PPO-S.
8 . The lettuce plant according to any one of claims 5-7 , wherein the plant comprises a mutation in PPO-G.
9 . The lettuce plant according to any one of claims 5-8 further comprising:
a mutation in one or more of PPO-D and PPO-E.
10 . The lettuce plant of any one of the preceding claims , wherein said lettuce is leaf lettuce, romaine lettuce, Frisée lettuce, butter lettuce, Bibb lettuce, Boston lettuce, iceberg lettuce, kale, spinach, radicchio, or endive.
11 . The lettuce plant of claim 10 , wherein said lettuce is romaine lettuce.
12 . The lettuce plant of any one of the preceding claims , wherein said mutations reduce the amount of active PPO protein in the lettuce plant.
13 . The lettuce plant of any one of the preceding claims , wherein said mutations reduce the activity of PPO protein in the lettuce plant.
14 . The lettuce plant of any one of the preceding claims , wherein the activity of PPO protein is reduced by at least 10%.
15 . The lettuce plant of any one of the preceding claims , wherein the activity of PPO protein is reduced by at least 30%.
16 . The lettuce plant of any one of the preceding claims , wherein the activity of PPO protein is reduced by at least 50%.
17 . The lettuce plant of any one of the preceding claims , wherein the lettuce plant at harvest comprises a characteristic selected from the group consisting of: reduced tip burn compared to a wild type variety under the same conditions; higher levels of polyphenolics compared to a wild type variety under the same conditions; higher levels of vitamin A or beta-carotene compared to a wild type variety under the same conditions; higher levels of vitamin C compared to a wild type variety under the same conditions; and higher levels of vitamin K compared to a wild type variety under the same conditions.
18 . The lettuce plant of any one of the preceding claims , wherein the lettuce plant post-harvest comprises a characteristic selected from the group consisting of: reduced browning; reduced tip burn; increased shelf life; higher levels of vitamin A; higher levels of vitamin C; lower levels of CO 2 production; higher levels of carbohydrates; less yellowing as assessed by midvein scoring; and higher levels of polyphenolics compared to a wild type variety under the same conditions.
19 . The lettuce plant of claim 18 , wherein said post-harvest is 7, 13, 14, 21, 27, or 30 days after harvest.
20 . The lettuce plant of any one of the preceding claims , wherein the mutations encode a knockout mutation.
21 . The lettuce plant of any one of the preceding claims , wherein the mutation is an insertion between nucleotides 1112 and 1113 of SEQ ID NO:9.
22 . The lettuce plant of any one of the preceding claims , wherein the mutation is an insertion between nucleotides 988 and 989 of SEQ ID NO:11, a deletion at nucleotide 987 of SEQ ID NO:11, or a deletion between nucleotides 986 and 987 of SEQ ID NO:11.
23 . The lettuce plant of any one of the preceding claims , wherein the mutation is an insertion between nucleotides 1131 and 1132 of SEQ ID NO:7.
24 . The lettuce plant of any one of the preceding claims , wherein the mutation is an insertion between nucleotides 1044 and 1045 of SEQ ID NO:27, a deletion of three nucleotides between nucleotides 1045-1047 of SEQ ID NO:27, or a deletion of seven nucleotides between nucleotides 1045-1051 of SEQ ID NO:27.
25 . The lettuce plant of any one of the preceding claims , wherein the mutation is an insertion between nucleotides 976 and 977 of SEQ ID NO:1, a deletion of 26 nucleotides between nucleotides 971-996 of SEQ ID NO:1, or a deletion of 27 nucleotides between nucleotides 971-997 of SEQ ID NO:1.
26 . The lettuce plant of any one of the preceding claims , wherein the mutation is an insertion between nucleotides 1007 and 1008 of SEQ ID NO:3 or a deletion of nucleotide 1008 of SEQ ID NO:3.
27 . The lettuce plant of any one of the preceding claims , wherein the mutation is an insertion between nucleotides 1023 and 1024 of SEQ ID NO:25.
28 . The lettuce plant of any one of the preceding claims , wherein the lettuce plant is free of any plant pest sequences.
29 . The lettuce plant of any one of the preceding claims , wherein the lettuce plant is free of any selectable marker.
30 . A method of making a lettuce plant with reduced PPO activity, said method comprising:
introducing a mutation into each of at least two different PPO genes of a lettuce plant, wherein said mutations reduce the amount and/or activity of PPO protein compared to a wild type lettuce plant.
31 . The method of claim 30 , wherein the mutations encode a knockout or a knock-down mutation.
32 . The method of claim 30 or claim 31 , wherein the mutation is an insertion between nucleotides 1112 and 1113 of SEQ ID NO:9.
33 . The method of any one of claims 30-32 , wherein the mutation is an insertion between nucleotides 988 and 989 of SEQ ID NO:11, a deletion at nucleotide 987 of SEQ ID NO:11, or a deletion between nucleotides 986 and 987 of SEQ ID NO:11.
34 . The method of any one of claims 30-33 , wherein the mutation is an insertion between nucleotides 1131 and 1132 of SEQ ID NO: 7.
35 . The method of any one of claims 30-34 , wherein the mutation is an insertion between nucleotides 1044 and 1045 of SEQ ID NO:27, a deletion of three nucleotides between nucleotides 1045-1047 of SEQ ID NO:27, or a deletion of seven nucleotides between nucleotides 1045-1051 of SEQ ID NO:27.
36 . The method of any one of claims 30-35 , wherein the mutation is an insertion between nucleotides 976 and 977 of SEQ ID NO:1, a deletion of 26 nucleotides between nucleotides 971-996 of SEQ ID NO:1, or a deletion of 27 nucleotides between nucleotides 971-997 of SEQ ID NO:1.
37 . The method of any one of claims 30-36 , wherein the mutation is an insertion between nucleotides 1007 and 1008 of SEQ ID NO:3 or a deletion of nucleotide 1008 of SEQ ID NO:3.
38 . The method of any one of claims 30-37 , wherein the mutation is an insertion between nucleotides 1023 and 1024 of SEQ ID NO:25.
39 . The method of any one of claims 30-38 , wherein said lettuce is leaf lettuce, romaine lettuce, Frisée lettuce, butter lettuce, Bibb lettuce, Boston lettuce, iceberg lettuce, kale, spinach, radicchio, or endive.
40 . The method of claim 39 , wherein said lettuce is romaine lettuce.
41 . The method of any one of claims 30-40 , wherein said mutations reduce the amount of active PPO protein in the lettuce plant.
42 . The method of any one of claims 30-41 , wherein said mutations reduce the activity of PPO protein in the lettuce plant.
43 . The method of any one of claims 30-42 , wherein active PPO protein is reduced by at least 10%.
44 . The method of any one of claims 30-43 , wherein active PPO protein is reduced by at least 30%.
45 . The method of any one of claims 30-44 , wherein active PPO protein is reduced by at least 50%.
46 . The lettuce plant of any one of the preceding claims , wherein the lettuce plant at harvest comprises a characteristic selected from the group consisting of: reduced tip burn compared to a wild type variety under the same conditions; higher levels of polyphenolics compared to a wild type variety under the same conditions; higher levels of vitamin A or beta-carotene compared to a wild type variety under the same conditions; higher levels of vitamin C compared to a wild type variety under the same conditions; and higher levels of vitamin K compared to a wild type variety under the same conditions.
47 . The lettuce plant of any one of the preceding claims , wherein the lettuce plant post-harvest comprises a characteristic selected from the group consisting of reduced browning; reduced tip burn; increased shelf life; higher levels of vitamin A; higher levels of vitamin C; lower levels of CO 2 production; less rigorous modified atmosphere packaging; higher levels of carbohydrates; less yellowing as assessed by midvein scoring; and higher levels of polyphenolics compared to a wild type variety under the same conditions.
48 . The lettuce plant of claim 47 , wherein said post-harvest is 7, 13, 14, 21, 27, or 30 days after harvest.
49 . A method of editing PPO genes of lettuce plant, said method comprising:
introducing into a lettuce plant a polynucleotide construct comprising:
a first nucleic acid sequence encoding a gene editing nuclease;
a promoter that is functional in plants operably linked to said first nucleic acid sequence;
a second nucleic acid sequence encoding one or more gRNA in a polycistronic arrangement targeting at least two PPO genes of choice to edit; and
a second promoter that is functional in plants, operably linked to said second nucleic acid sequence.
50 . The method of claim 49 , wherein said gene editing nuclease is a Cas9 nuclease, a MAD7 nuclease, a Cpf1 nuclease, or a chimeric nuclease.
51 . The method of claim 50 , wherein said chimeric nuclease comprises the amino acid sequence of SEQ ID NO:30.
52 . The method of any one of claims 49 to 51 , wherein said first promoter and said second promoter are each selected from AtU6 promoter, 35S promoter, and CsVMV promoter.
53 . The method of any one of claims 49 to 52 further comprising:
a third nucleic acid sequence encoding a selectable marker operably linked to a third promoter that is functional in plants.
54 . The method of claim 53 , wherein said selectable marker is a drug resistance gene, a fluorescent protein, or an enzyme.
55 . The method of claim 54 , wherein said selectable marker is a kanamycin resistance gene or a neomycin resistance gene.
56 . The method of any one of claims 49 to 55 further comprising:
regenerating said protoplast into a plant.
57 . A polynucleotide construct for editing polyphenol oxidase genes of a lettuce plant comprising:
a nucleic acid sequence encoding a plurality of gRNAs targeting at least two PPO genes operably linked to a promoter.
58 . The polynucleotide construct of claim 57 , wherein the PPO genes are selected from the group consisting of PPO-A, PPO-B, PPO-C, PPO-D, PPO-E, PPO-G, PPO-O, PPO-P, PPO-R, PPO-N, PPO-M, PPO-J, PPO-Q, and PPO-S.
59 . The polynucleotide of claim 57 or claim 58 , wherein said polynucleotide comprises a second nucleic acid sequence encoding a gene editing enzyme.
60 . The polynucleotide of claim 58 , wherein said gene editing enzyme is Cas9, MAD7, Cpf1, or SynNuc1.