Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-02-10
2002-05-21
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S260000, C800S264000, C800S271000, C800S274000, C800S303000
Reexamination Certificate
active
06392127
ABSTRACT:
BACKGROUND OF THE INVENTION
Oilseed from Brassica plants is an increasingly important crop. As a source of vegetable oil, it presently ranks behind only soybeans and palm in commercial importance and it is comparable with sunflowers. The oil is used both as a salad oil and as a cooking oil.
In its original form, Brassica oil, known as rapeseed oil, was harmful to humans due to its relatively high level of erucic acid, Erucic acid is commonly present in native cultivars in concentrations of 30 to 50 percent by weight based upon the total fatty acid content. This problem was overcome when plant scientists identified a germplasm source of low erucic acid rapeseed oil (Stefansson, 1983).
In addition, plant scientists have attempted to improve the fatty acid profile for rapeseed oil (Robbelen, 1984; Ratledge et al., 1984; Robbelen et al., 1975; and Rakow et al., 1973). These references are representative of those attempts.
Particularly attractive to plant scientists were so-called “double-low” varieties: those low in erucic acid in the oil and low in glucosinolates in the solid meal remaining after oil extraction (i.e., an erucic acid content of less than 2 percent by weight based upon the total fatty acid content, and a glucosinolate content of less than 30 &mgr;mol/gram of the oil-free meal). These higher quality forms of rape, first developed in Canada, are known as canola.
More recently, plant scientists have focused their efforts on reducing the glucosinolate content further, to levels of less than 20 &mgr;mol/gram of oil-free meal, as verified by quantifying trimethylsilyl (TMS) derivatives (Sosulski and Dabrowski, 1984) for spring canola, or less than 20 &mgr;mol/gram of whole, ground seed, as determined by high performance liquid chromatography (HPLC) (International Organization for Standardization, reference number ISO 9167-30 1:1992(E)) for winter canola.
Glucosinolates are sulfur-based compounds that remain in the solid component of the seed—the solid meal—after the seed has been ground and its oil has been extracted. Their structure includes glucose in combination with aliphatic hydrocarbons (3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3-butenyl glucosinolate, and 2-hydroxy-4-pentenyl glucosinolate) or aromatic hydrocarbons (3-indoylmethyl glucosinolate, 1-methoxy-3-indoyl methyl glucosinolate). Aliphaitic glucosinolates are also known as alkenyl glucosinolates. Aromatic glucosinolates are also known as indoles.
High levels of glucosinolates are undesirable because they produce toxic by-products when acted upon by the enzyme myrosinase. Myrosinase is a naturally occurring enzyme present in Brassica species. When Brassica seed is crushed, myrosinase is released and catalyzes the breakdown of glucosinolates to produce glucose, thiocyanates, isothiocyanate and nitriles. When separated from glucose, these other products are toxic to certain mammals. Isothiocyanate, for example, inhibits synthesis of thryroxine by the thyroid and has other anti-metabolic effects (Paul et al., 1986). Attempts have been made to inactivate the enzyme myrosinase (using steam, for example). These attempts have not been entirely successful.
Rapeseed possesses high levels of glucosinolates (from 100 &mgr;mol/gram to 200 &mgr;mol/gram of oil-free meal), whereas canola possesses lower levels of glucosinolates (less than 30 &mgr;mol/gram of oil-free meal). The levels of glucosinolates in canola are regulated in many countries. In Europe, for example, winter canola must have a glucosinolate content of less than 25 &mgr;mol/gram of seed at 8.5% moisture, as measured by HPLC. In Canada, spring canola must have a glucosinolate content of less than 30 &mgr;mol/gram of oil-free meal at 0% moisture, as measured by TMS. Many countries are requiring even lower levels of glucosinolates in order to register canola varieties.
In developing improved new Brassica varieties, breeders use self-incompatible (SI), cytoplasmic male sterile (CMS) and nuclear male sterile (NMS) Brassica plants as the female parent. In using these plants, breeders are attempting to improve the efficiency of seed production and the quality of the F
1
hybrids and to reduce the breeding costs. When hybridisation is conducted without using SI, CMS or NMS plants, it is more difficult to obtain and isolate the desired traits in the progeny (F
1
generation) because the parents are capable of undergoing both cross-pollination and self-pollination. If one of the parents is a SI, CMS or NMS plant that is incapable of producing pollen, only cross pollination will occur. By eliminating the pollen of one parental variety in a cross, a plant breeder is assured of obtaining hybrid seed of uniform quality, provided that the parents are of uniform quality and the breeder conducts a single cross.
In one instance, production of F
1
hybrids includes crossing a CMS Brassica female parent, with a pollen producing male Brassica parent. To reproduce effectively, however, the male parent of the F
1
hybrid must have a fertility restorer gene (Rf gene). The presence of a Rf gene means that the F
1
generation will not be completely or partially sterile, so that either self-pollination or cross pollination may occur. Self pollination of the F
1
generation to produce several subsequent generations is important to ensure that a desired trait is heritable and stable and that a new variety has been isolated.
One Brassica plant which is cytoplasmic male sterile and is used in breeding is ogura (OGU) cytoplasmic male sterile (R. Pellan-Delourme et al., 1987). A fertility restorer for ogura cytoplasmic male sterile plants has been transferred from
Raphanus sativus
(radish) to Brassica by Institut National de Recherche Agricole (INRA) in Rennes, France (Pelletier et al., 1987). The restorer gene, Rf1 originating from radish, is described in WO 92/05251 and in Delourme et al., (1991).
However, this restorer is inadequate in that restorer inbreds and hybrids carrying this Rf gene have elevated glucosinolate levels and the restorer is closely related to a decrease in seed set—the number of ovules per silique—(Pellan-Delourme et al., 1988; Delourme et al., 1994). In the case of hybrids, the glucosinolate levels are elevated even when the female parent has reduced glucosinolate content. These levels, typically more than 30 &mgr;mol/gram of oil-free meal, exceed the levels of glucosinolates allowable for seed registration by most regulatory authorities in the world. Thus, the restorer can be used for research purposes, but not to develop directly canola-quality commercial hybrid varieties. To date, there is no other source of a restorer of fertility for ogura cytoplasmic male sterility available.
INRA outlines the difficulties associated with obtaining restorer lines with low glucosinolate levels for ogura cytoplasmic sterility (Delourme, et al., 1994; Delourme, et al., 1995). INRA indicates that these difficulties are due to the linkage between male fertility restoration and glucosinolate content in its breeding material. INRA suggests that more radish genetic information needs to be eliminated in its restorer lines (Delourme, et al., (1995)). Although improvements have been made to restorers during the past few years, isozyme studies performed on the improved restorer lines indicate that radish genetic information still remains around the restorer gene (Delourme et al., 1994).
INRA has attempted to develop a restorer having decreased glucosinolate levels. It reported a heterozygous restorer with about 15 &mgr;mol per gram (Delourme et al., 1995). However, (i) this restorer was heterozygous (Rfrf) not homozygous (RfRf) for the restorer gene, (ii) this restorer was a single hybrid plant rather than an inbred line, (iii) there was only a single data point suggesting that this restorer had a low glucosinolate level rather than multiple data points to support a low glucosinolate level, (iv) there was no data to demonstrate whether the low glucosinolate trait was passed on to the progeny of the restorer, and (v) the restorer was selected and evaluated in a sin
Charne David G.
Grant Ian
Kraling Konrad
Patel Jayantilal D.
Pruvot Jean-Claude M.
Fox David T.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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