Method for selective increase of the anticarcinogenic glucosinolates in Brassica sp.

• US 6,340,784 B1
• Filed: 04/08/1999
• Issued: 01/22/2002
• Est. Priority Date: 04/09/1998
• Status: Expired due to Term

• Current Assignee: Plant Bioscience Limited (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Plant Bioscience Limited (Alnylam Pharmaceuticals Incorporated)
• Inventors: Williamson, Gary, Faulkner, Kathy, Mithen, Richard
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): Kruse, David H
• Application Number:
  US09/288,026
• Time in Patent Office: 1,020 Days
• Field of Search: 800/260, 800/265, 800/266, 800/267, 800/269, 800/298, 800/306, 800/264, 800/273, 435/410
• US Class Current: 800/306
• CPC Class Codes: A01H 1/00 Processes for modifying genotypes ; Plants characterised by associated natural traits A01H4/00 takes precedence
• CPC Class Codes: A01H 6/203 Brassica oleraceae, e.g. broccoli or kohlrabi

Abstract

The invention relates to a method for producing Brassica oleracea with elevated anticarcinogenic glucosinolate derivatives. The elevated levels are obtained by crossing wild Brassica oleracea species with Brassica oleracea breeding lines, and subsequently selecting hybrids with levels of 4 -methylsulfinylbutyl and/or 3-methylsulfinylpropyl glucosinolates elevated above that initially found in Brassica oleracea breeding lines. The invention also relates to edible Brassica plants, such as broccoli plants, with elevated levels of 4-methylaulfinylbutyl glucosinolate and/or 3 -methylsulfinylpropyl glucosinolates, and to seeds of such plants.

Claims

1. A method for the production of Brassica oleracea with elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both, which comprises:

(a) crossing a wild Brassica species selected from the group consisting of B. villosa and B. drepanensis with a Brassica oleracea breeding line to produce hybrid plants; and

(b) selecting the hybrid plants of step (a) with levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both, elevated above that initially found in the Brassica oleracea breeding line.

7. The method according to claim 1, wherein only 4-methylsulfinylbutyl glucosinolate is elevated.

8. The method according to any one of claim 1, wherein only 3-methylsulfinylpropyl glucosinolate is elevated.

9. An edible Brassica oleracea plant produced according to the method of claim 1.

10. An edible portion of a broccoli plant produced according to the method of claim 1.

11. Seed of a broccoli plant produced according to the method of claim 1.

2. A method for the production of Brassica oleracea with levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both, elevated above that initially found in Brassica oleracea breeding lines, which comprises:

(a) crossing a wild Brassica species selected from the group consisting of B. villosa and B. drepanensis with a broccoli breeding line to produce hybrid plants;

(b) selecting the hybrid plants of step (a) with elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both;

(c) backcrossing to a broccoli breeding line to produce backcrossed plants; and

(d) selecting the backcrossed plants of step (c) with elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both.

3. The method according to claim 2 which additionally comprises:

(e) selecting a broccoli line with elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both, capable of causing a strong induction of phase II enzymes.

4. A method for the production of Brassica oleracea with levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both, elevated above that initially found in Brassica oleracea breeding lines, and comprising SI alleles, the presence of which in Brassica oleracea results in self- incompatibility of the Brassica oleracea, the method comprising:

(a) crossing a wild Brassica species selected from the group consisting of B. villosa and B. drepanensis with a broccoli double haploid breeding line containing the SI alleles to produce hybrid plants;

(b) selecting the hybrid plants of step (a) with elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both;

(c) screening the hybrid plants of step (b) for the SI alleles with RFLP markers to identify the hybrid plants comprising the SI alleles; and

(d) selecting the hybrid plants identified in step (c).

5. The method according to claim 4, additionally comprising:

backcrossing the plant of step 4(b) to the brocoli breeding line and selecting backcrossed plants;

with elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both; and

(e) selecting a broccoli line with elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both, and SI alleles, which is capable of causing a strong induction of phase II enzymes.

6. A method for the production of Brassica oleracea with levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both, elevated above that initially found in Brassica oleracea breeding lines, which comprises:

(a) crossing a wild Brassica species selected from the group consisting of B. villosa and B. drepanensis with a broccoli double haploid breeding line to produce hybrid plants;

(b) using DNA probes to select the hybrid plants of step (a) with a genetic combination encoding expression of elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both;

(c) backcrossing to the broccoli double haploid breeding line to produce backcrossed plants;

(d) selecting the backcrossed plants of step (c) with the genetic combination encoding the expression of elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both; and

(e) selecting a broccoli line with elevated levels of 4-methylsulfinylbutyl glucosinolates, or 3-methylsulfinylpropyl glucosinolates, or both, capable of causing a strong induction of phase II enzymes.

12. The method according to claim 6 where the DNA probes used are selected from the group consisting of:

pW176, pW141, pW207, pW224, pW114, pW145, pW123, pW138, pW197, pW228 and pW106.

13. A broccoli plant having elevated levels of 3-methylsulfinylpropyl glucosinolates, or 4-methylsulfinylbutyl glucosinolates, or both, elevated above that initially found in Brassica oleracea breeding lines, wherein the broccoli plant is a hybrid plant produced by crossing a broccoli breeding line with a wild Brassica species selected from the group consisting of B. drepanensis and B. villosa.

14. The broccoli plant according to claim 13, wherein the concentration of 3-methylsulfinylpropyl glucosinolates, or 4-methylsulfinylbutyl glucosinolates, or both, is between 10 and 100 μ

moles per gram of dry weight.

15. A broccoli inflorescence having elevated levels of 3-methylsulfinylpropyl glucosinolates, or 4-methylsulfinylbutyl glucosinolates, or both, elevated above that initially found in Brassica oleracea breeding lines, wherein the broccoli inflorescence is obtained from a hybrid plant produced by crossing a broccoli breeding line with a wild Brassica species selected from the group consisting of B. drepanensis and B. villosa.

16. The broccoli inflorescence according to claim 15, wherein the concentration of 3-methylsulfinylpropyl glucosinolates, or 4-methylsulfinylbutyl glucosinolates, or both, is between 10 and 100 μ

moles per gram of dry weight.

17. A Brassica plant cell having levels of 3-methylsulfinylpropyl glucosinolates, or 4-methylsulfinylbutyl glucosinolates, or both, elevated above that initially found in Brassica oleracea breeding lines, wherein the cell is a cell of a hybrid plant produced by crossing a broccoli breeding line with a wild Brassica species selected from the group consisting of B. drepanensis and B. villosa.

18. The plant cell according to claim 17, wherein the cell is an inflorescence cell.

19. The plant cell according to claim 17, wherein the concentration of 3-methylsulfinylpropyl glucosinolates, or 4-methylsulfinylbutyl glucosinolates, or both, is, between 10 and 100 μ

moles per gram of dry weight.