Tobacco plant and method for manufacturing same

• US 10,640,780 B2
• Filed: 09/27/2018
• Issued: 05/05/2020
• Est. Priority Date: 03/30/2016
• Status: Active Grant

• Current Assignee: Japan Tobacco Inc.
• Original Assignee: Japan Tobacco Inc.
• Inventors: Suzuki, Shoichi, Hamano, Kaori, Sato, Seiki, Arai, Masao, Negishi, Yuta, Nomura, Ayako, Tsukahara, Mai
• Primary Examiner(s): Kallis, Russell
• Application Number:
  US16/144,479
• Publication Number:
  US 20190218564A1
• Time in Patent Office: 586 Days
• CPC Class Codes: A01H 1/121 Plant growth habits
• CPC Class Codes: A01H 5/12 Leaves
• CPC Class Codes: A01H 6/823 Nicotiana, e.g. tobacco
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8262 involving plant development
• CPC Class Codes: C12N 15/827 Flower development or morphology, e.g. flowering promoting factor [FPF]
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Abstract

The present invention provides a tobacco plant which is suitable for cultivation for harvesting leaf tobaccos. The present invention includes (i) a tobacco plant in which a mutation for suppressing the development of axillary buds is introduced and (ii) a method of producing the tobacco plant.

Claims

1. A tobacco plant comprising a mutation which causes functional suppression of

at least one of a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 1 and a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 3;

wherein the functional suppression of said at least one gene,

a) suppresses development of secondary axillary buds which develop after removal of primary axillary buds, without suppressing development of primary axillary buds,

b) decreases the abundance of said polypeptide in comparison with a wild-type plant, and

c) promotes degradation of mRNA transcribed from said at least one gene;

and wherein the mutation is an insertion, into a region of the plant'"'"'s genome outside of the region of the said at least one gene, of a polynucleotide expressing a factor which promotes the degradation of said mRNA, said factor being an antisense RNA molecule or an RNAi molecule.

3. The tobacco plant as set forth in claim 1, wherein the functional suppression causes the number or weight of the secondary axillary buds to decrease to not more than ½

of that of a wild-type plant.

10. The tobacco plant as set forth in claim 1, wherein the tobacco plant belongs to Nicotiana tabacum or Nicotiana rustica.

19. An offspring or a bred progeny, wherein:

the offspring is of a tobacco plant recited in claim 1;

the bred progeny is obtained by crossing a tobacco plant recited in claim 1; and

the following (A) is satisfied;

(A)

a mutation is introduced into a genome, which mutation causes functional suppression of

at least one of a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 1 and a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 3;

wherein the functional suppression of said at least one gene,

a) suppresses development of secondary axillary buds which develop after removal of primary axillary buds, without suppressing development of primary axillary buds,

b) decreases the abundance of said polypeptide in comparison with a wild-type plant, and

c) promotes degradation of mRNA transcribed from said at least one gene;

and wherein the mutation is an insertion, into a region of the plant'"'"'s genome outside of the region of the said at least one gene, of a polynucleotide expressing a factor which promotes the degradation of said mRNA, and

said factor is an antisense RNA molecule or an RNAi molecule.

23. A leaf tobacco harvested from a tobacco plant recited in claim 1.

27. A leaf tobacco harvested from an offspring or a bred progeny recited in claim 19.

2. A tobacco plant in which a mutation is introduced into a genome, which mutation causes functional suppression of

at least one of a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 1 and a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 3;

wherein the functional suppression of said at least one gene suppresses development of secondary axillary buds, which develop after removal of primary axillary buds, without suppressing development of primary axillary buds, and

the mutation is introduced into the at least one gene.

4. The tobacco plant as set forth in claim 2, wherein the functional suppression causes the number or weight of the secondary axillary buds to decrease to not more than ½

of that of a wild-type plant.

5. The tobacco plant as set forth in claim 2, wherein the functional suppression is a decrease in abundance of the polypeptide in comparison with a wild-type plant.

6. The tobacco plant as set forth in claim 4, wherein the functional suppression is a decrease in an amount of translation of the polypeptide in comparison with a wild-type plant.

7. The tobacco plant as set forth in claim 4, wherein the functional suppression is a decrease in an amount of transcription from the at least one gene to an mRNA in comparison with a wild-type plant.

8. The tobacco plant as set forth in claim 4, wherein the functional suppression is promotion of degradation of an mRNA transcribed from the at least one gene.

9. The tobacco plant as set forth in claim 2, wherein the mutation is introduced by mutagen treatment, genome editing, or gene knockout.

11. The tobacco plant as set forth in claim 2, wherein the tobacco plant belongs to Nicotiana tabacum or Nicotiana rustica.

20. An offspring or a bred progeny, wherein:

the offspring is of a tobacco plant recited in claim 2;

the bred progeny is obtained by crossing a tobacco plant recited in claim 2; and

the following (B) is satisfied;

(B)

a mutation is introduced into a genome, which mutation causes functional suppression of

at least one of a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 1 and a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 3;

wherein the functional suppression of said at least one said gene suppresses development of secondary axillary buds, which develop after removal of primary axillary buds, without suppressing development of primary axillary buds, and

the mutation is introduced into the at least one gene.

24. A leaf tobacco harvested from a tobacco plant recited in claim 2.

28. A leaf tobacco harvested from an offspring or a bred progeny recited in claim 20.

12. A method of producing a tobacco plant, comprising the step of:

(a) introducing a mutation that causes functional suppression of

at least one of a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 1 and a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 3;

wherein the functional suppression of said at least one gene, suppresses development of secondary axillary buds, which develop after removal of primary axillary buds, without suppressing development of primary axillary buds,

and wherein step (a), further including inserting, into a region of the plant'"'"'s genome outside of the region of the said at least one gene, of a polynucleotide expressing a factor which promotes degradation of an mRNA transcribed from the said at least one gene, and

said factor being an antisense RNA molecule or an RNAi molecule.

14. The method as set forth in claim 12, further comprising the step of:

(b) selecting, from individuals produced by the step (a), an individual in which development of, of all of the axillary buds, secondary axillary buds that develop after removal of primary axillary buds is suppressed.

16. The method as set forth in claim 14, wherein in the step (b), an individual, in which the number or weight of the secondary axillary buds is decreased in comparison with that of a wild-type plant, is selected.

21. An offspring or a bred progeny, wherein:

the offspring is of a tobacco plant produced by a method recited in claim 12;

the bred progeny is obtained by crossing a tobacco plant produced by a method recited in claim 12; and

the following (A) is satisfied;

(A)

a mutation is introduced into a genome, which mutation causes functional suppression of

at least one of a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 1 and a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 3;

wherein the functional suppression of said at least one gene,

a) suppresses development of secondary axillary buds, which develop after removal of primary axillary buds, without suppressing development of primary axillary buds,

b) decreases the abundance of said polypeptide in comparison with a wild-type plant, and

c) promotes degradation of mRNA transcribed from said at least one gene;

and wherein the mutation is an insertion, into a region of the plant'"'"'s genome outside of the region of the said at least one gene, of a polynucleotide expressing a factor which promotes the degradation of said mRNA, and

said factor is an antisense RNA molecule or an RNAi molecule.

25. A leaf tobacco harvested from a tobacco plant produced by a method recited in claim 12.

29. A leaf tobacco harvested from an offspring or a bred progeny recited in claim 21.

13. A method of producing a tobacco plant, comprising the step of:

(a) introducing a mutation that causes functional suppression of

at least one of a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 1 and a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 3;

wherein the functional suppression of said at least one said gene suppresses development of secondary axillary buds, which develop after removal of primary axillary buds, without suppressing development of primary axillary buds, and

step (a) further including introducing the mutation into the said at least one gene.

15. The method as set forth in claim 13, further comprising the step of:

(b) selecting, from individuals produced by the step (a), an individual in which development of, of all of the axillary buds, secondary axillary buds that develop after removal of primary axillary buds is suppressed.

17. The method as set forth in claim 15, wherein in the step (b), an individual, in which the number or weight of the secondary axillary buds is decreased in comparison with that of a wild-type plant, is selected.

18. The method as set forth in claim 13, wherein the step (a) is carried out by mutagen treatment, genome editing, or gene knockout.

22. An offspring or a bred progeny, wherein:

the offspring is of a tobacco plant produced by a method recited in claim 13;

the bred progeny is obtained by crossing a tobacco plant produced by a method recited in claim 13; and

the following (B) is satisfied;

(B)

a mutation is introduced into a genome, which mutation causes functional suppression of

at least one of a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 1 and a gene containing, as a coding region, a polynucleotide encoding a polypeptide having a sequence identity of 90% or higher with an amino acid sequence represented by SEQ ID NO. 3;

wherein the functional suppression of said at least one said gene suppresses development of secondary axillary buds, which develop after removal of primary axillary buds, without suppressing development of primary axillary buds, and

the mutation is introduced into the at least one gene.

26. A leaf tobacco harvested from a tobacco plant produced by a method recited in claim 13.

30. A leaf tobacco harvested from an offspring or a bred progeny recited in claim 22.