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
2000-08-29
2004-01-20
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
C800S295000, C800S265000, C800S263000, C800S260000, C800S270000
Reexamination Certificate
active
06680429
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a
Pisum sativum
variety that contains a recessive gene that produces highly wrinkled seed having a low starch content. Additionally, the present invention relates to nucleotide sequences for said recessive gene and methods for using said nucleotide sequences in site-specific recombination.
BACKGROUND OF THE INVENTION
The garden pea (
Pisum sativum
L.) is a commercially important food crop and the immature seed of the garden pea (“peas”) are widely consumed. There are a large number of genes that affect starch or carbohydrate synthesis in peas. The first reported and best described gene is the r gene (see White, O. E.,
Proceedings of the American Philosophical Society
, 56: 487-588). The r mutant is believed to have occurred spontaneously at the beginning of the seventeenth century (see Lamprecht, H.,
Agri. Hortique Genetica
14: 1-4 (1956)) and its mature, dry seed has a wrinkled appearance (hence “r”, derived from the Latin, rugosus meaning “wrinkled or shriveled”). Wrinkling of mature seed was one of the characteristics used by Mendel in experiments which led him to formulate his laws of inheritance. (see Mendel, G.,
Verhandlungen des Naturforschenden Vereinds in Brünn
4: 3-47 (1865)).
Recessive alleles at the r locus not only have a profound effect on the shape of the seed, but also have numerous effects at all levels of seed development. Wang, T. L., et al.,
Seed Science Research
1:3-14 (1991). Specifically, seeds of the r mutant contain a lower proportion of starch than the wildtype (about 30% dry weight as opposed to about 50%) with the starch composition being altered to contain a higher proportion of amylose and a small proportion of amylopectin (with about 70% of dry weight of the starch of mutant seeds being amylose as opposed to 38% of the wildtype starch). For a long period of time, the molecular basis of the mutation was not clear. The difference in the level and composition of starch in the seed led several investigators to examine the various enzymes of starch biosynthesis. Id. Early studies indicated that the mutation was likely to be in the starch branching enzyme and this was confirmed when it was shown, using a pair of lines near-isogenic except for genes at the r locus that rr embryos lacked one form of the starch-branching enzyme, which is frequently referred to as “SBE1”. Subsequently, it was shown that the mutant was caused by a transposon-like insertion in the gene encoding SBE1. Id.
A second recessive rugosus locus termed “rb” is also known. Mutants homozygous recessive at this locus exhibit a wrinkled seed phenotype similar to that of rr plants. However, the amount of starch and its composition in the rb mutant are different than that of the r mutant. Specifically, seed of the r mutant contains about 30% dry weight of starch, of which about 70% is amylose. Seed of the rb mutant contains about 36% dry weight of starch, of which about 23% is amylose. The rb mutation has been found to result in reduced activity in the enzyme ADP glucose pyrophosphorylase. Purification of the enzyme and western blot experiments have revealed the absence of one of the four polypeptide subunits present in the wildtype enzyme.
WO 98/01574 describes
Pisum sativum
varieties which contain a mutation referred to as “rug3”. Plants containing the rug-3 mutation produce wrinkled seeds having low levels of starch, high levels of sucrose, as well as a high protein and lipid content. The rug-3 mutation has been found to be associated with a reduction in the activity of the enzyme plastid phosphoglucomutase (hereinafter “PGM”). According to WO 98/01574, pea seeds containing the rug-3 mutation have a sucrose content of greater than 6% by weight of the total weight of the seed as harvested at a tenderometer reading exceeding 120 tenderometer units.
The rug-3 mutation described in WO 98/01574 was induced via a mutagenesis program. Twenty thousand phenotypically round genetically wildtype (RR) seeds (referred to as “M1” seed) were treated with ethyl methanesulphonate or N-methyl-N-nitrosourea, which are known mutagens. M1 seed gave rise to M1 plants bearing M2 seed. M2 seed gave rise to M2 plants bearing M3 seed. M3 seeds were analyzed for storage product content. Seeds which exhibited a wrinkled appearance were selected from the M3 generation. These seeds were found to contain a wide range of starch, from 0-60% as a proportion of the dry weight of the mature seed. Within the starch of these seeds, the amylose content ranged from 0-80%. Also, the lipid content of the peas ranged from about 1 to about 8% of the dry weight and protein ranged from about 24 to about 48%. WO 98/01574 does not contain any information characterizing the rug-3 mutant except to describe how this mutation was induced via a mutagen program. Additionally, WO 98/01574 does not contain any information that distinguishes the DNA sequence of the rug-3 mutant from that of the wildtype and contains no information on effects on alcohol insoluble solids content.
WO 98/01574 also describes the isolation and characterization of the nucleotide sequence for wildtype pea plastid phosphoglucomutase. According to WO 98/01574, this nucleotide sequence can be introduced into a plant via recombinant DNA technology to produce transgenic plants in which the plastid PGM gene expression is down regulated or inactivated in developing seeds.
Sweetness in peas, associated with increased sugar content, is generally prized by consumers, who perceive that sweeter peas have a better flavor. Thereupon, because peas are such an important food crop, there is a need in the art for peas having an increased sweetness.
SUMMARY OF THE INVENTION
The present invention relates to a new variety of
Pisum sativum
, which is resistant to Fusarium Wilt Fungus and Powdery Mildew Fungus and which contains within its genome, a homozygous recessive gene, referred to as the bsg gene. It has been determined that the bsg gene contains a mutation in an intron in a 3′ splice site of the gene. More specifically, the bsg gene has the genomic nucleotide sequence shown in Sequence ID NO:1 and contains a mutation at nucleotide 1548 at the 3′ splice site dinucleotide AG, where nucleotide A is replaced with nucleotide T.
A
Pisum sativum
variety that contains the bsg gene within its genome produces peas (known in the art as immature seeds) which exhibit a lower level of starch, an elevated level of sucrose and a decreased level of alcohol insoluble solids when compared with peas produced from a
Pisum sativum
variety that does not contain the bsg gene homozygous within its genome.
Additionally, the present invention contemplates a
Pisum sativum
variety which contains a homozygous bsg gene within its genome. The bsg gene has the nucleotide sequence shown in SEQ ID NO:1 and contains a mutation in an intron at nucleotide 1548 at the 3′ splice site dinucleotide AG, where nucleotide A is replaced with nucleotide T.
The peas of the present invention contain from about 6.0 to about 7.5 percent fresh weight of sucrose when measured at a tenderometer value of from about 90 to about 110 and from about 6.5 to about 8.0 percent by weight of alcohol insoluble solids when measured at a tenderometer value of about 105. Moreover, the peas of the present invention contain from about 5 to about 30 percent fresh weight more sucrose than peas produced from a
Pisum sativum
variety that does not contain the bsg gene homozygous within its genome. Additionally, the peas of the present invention exhibit twenty (20) percent less alcohol insoluble solids when compared with peas from a
Pisum sativum
that does not contain the bsg gene homozygous within its genome.
Additionally, the present invention relates to a process for producing peas of a
Pisum sativum
variety that contain higher levels of sucrose and lower levels of alcohol insoluble solids than peas from a
Pisum sativum
variety that does not contain the bsg gene homozygous within its genome. The said process involves crossing a
Pisum sativum
Burgess Diane
Webster David
Fox David T.
Kallis Russell
Seminis Vegetable Seeds Inc.
Wood Phillips Katz Clark & Mortimer
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