Tomatoes That Soften More Slowly Post-Harvest Due To Non-Transgenic Alterations In An Expansin Gene
A series of independent human-induced non-transgenic mutations found in an expansin gene (LeExp1) of tomato; tomato plants having these mutations in their LeExp1 genes; and a method of creating and identifying similar and/or additional mutations in the LeExp1 gene by screening pooled and/or individual tomato plants. The tomato plants of the present invention exhibit fruit that soften more slowly post-harvest without having the inclusion of foreign nucleic acids in their genomes.
1 . A method of producing a tomato plant with a human-induced non-transgenic mutation in an expansin gene having substantial homology to SEQ ID NO: 6 comprising the steps of:
a. obtaining plant material from a parent tomato plant;
b. treating said plant material with a mutagen to create mutagenized plant material;
c. analyzing said mutagenized plant material to identify a plant having at least one mutation in at least one expansin gene having substantial homology to SEQ ID NO: 6.
2 . The method of claim 1 wherein the mutation is selected from the group consisting of G220T, G274A, C305T, G403A, C460T, G937A, G940T, C986T, A991G, and G1001A
3 . The method of claim 1 wherein the plant material is selected from the group consisting of seeds, pollen, plant cells, or plant tissue.
4 . The method of claim 1 wherein the mutagen is ethyl methanesulfonate.
5 . The method of claim 4 wherein the concentration of ethyl methanesulfonate used is from about 0.4 to about 1.2%.
6 . The method of claim 1 further comprising the steps of:
a. isolating genomic DNA from the mutagenized plant material or a progeny tomato plant; and
b. amplifying the isolated genomic DNA using at least one primer specific to an expansin gene having substantial homology to SEQ ID NO: 6.
7 . The method of claim 1 further comprising the steps of:
a. isolating genomic DNA from the mutagenized plant material or a progeny tomato plant; and
b. amplifying the isolated genomic DNA using at least one primer specific to DNA sequences adjacent to an expansin gene having substantial homology to SEQ ID NO: 6.
8 . Tomato fruit, seeds, pollen, plant parts or progeny of the tomato plant of claim 1 .
9 . The tomato fruit of claim 8 , wherein the tomato fruit have a decreased rate of post-harvest softening caused by a human-induced non-transgenic mutation in at least one LeExp1 gene.
10 . Food and food products incorporating the fruit of claim 9 .
11 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation G65* in SEQ ID NO: 11.
12 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation G83R in SEQ ID NO: 11.
13 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation T93I in SEQ ID NO: 11.
14 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation G126S in SEQ ID NO: 11.
15 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation L145F in SEQ ID NO: 11.
16 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation G190R in SEQ ID NO: 11.
17 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation D191Y in SEQ ID NO: 11.
18 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation P206L in SEQ ID NO: 11.
19 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation 5208G in SEQ ID NO: 11.
20 . The method of claim 1 wherein the at least one mutation in at least one expansin gene codes for a protein comprising the mutation W211* in SEQ ID NO: 11.
21 . An endogenous expansin gene having substantial homology to SEQ. ID NO: 6 and having at least one human-induced non-transgenic mutation within said endogenous expansin gene, wherein said at least one human-induced non-transgenic mutation is selected from the group consisting of G220T, G274A, C305T, G403A, C460T, G937A, G940T, C986T, A991G, and G1001A.
22 . A tomato plant containing the endogenous expansin gene of claim 21 .
23 . Tomato fruit, seeds, pollen, plant parts, and progeny of the tomato plant of claim 22 .
24 . The tomato fruit of claim 23 , wherein the tomato fruit have a decreased rate of post-harvest softening caused by a human induced non-transgenic mutation in at least one LeExp1 gene.
25 . Food and food products incorporating the fruit of the tomato plant of claim 24 .
26 . The endogenous expansin gene of claim 21 wherein said human-induced non-transgenic mutation creates an amino acid change in the expansin protein expressed from the expansin gene, wherein said amino acid change is selected from G65*, G83R, T93I, G126S, L145F, G190R, D191Y, P206L. S208G, W211*.
27 . An expansin protein expressed from the endogenous expansin gene of claim 26 .