Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
1999-07-15
2003-03-25
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C800S278000, C800S286000, C800S295000, C800S298000, C435S069100, C435S320100, C435S468000, C435S419000, C536S023100, C536S023200, C536S023600, C536S024500
Reexamination Certificate
active
06538180
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to plants, and particularly concerns peas (
Pisum sativum
L.), products derived therefrom and methods for genetically altering them, particularly for affecting the sucrose and starch content.
BACKGROUND TO THE INVENTION
Peas are an important crop plant, producing products used for human and animal consumption. The seeds of the pea plant can be harvested either in a dry mature form or in an immature state, with the precise stage of maturity varying according to the end use. Within each of these two categories there are a number of specialized uses and markets. The dry mature seed is used extensively as animal feed, directly as human food and as an ingredient of a variety of prepared foods. Those harvested in an immature form are used directly as a fresh vegetable or are processed by being canned, dehydrated or frozen. Peas harvested by machine at an immature stage for quick freezing are referred to in the art as vining peas.
Conventional cross-breeding methods have been used to develop new varieties and cultivars in order to satisfy different local or national requirements or niche markets. They include varieties with different colour, texture, sugar and starch contents, and size of seed.
The pea is also a useful experimental organism, and the pea is well characterised with many known variants.
Characterised mutants cover the whole spectrum of plant development, morphology and physiology. Some mutants may have had characters which were desirable to man to improve the pea crop and as such have been selected for.
The ‘Rugosus’ Loci
The r and rb Loci
Mendel, in his classic studies of genetics showed that the wrinkled-seeded phenotype of the r (rugosus) mutant is a recessive trait which fitted his newly formulated laws of inheritance. The r mutant became a popular tool for geneticists both classical and modern and is now well characterised. The terms rr, Rr and RR have been used to describe the homozygous recessive, heterozygous and homozygous dominant genotypes, respectively, with the rr genotype leading to the mature seeds being wrinkled in appearance (hence rugosus, which is the Latin word for wrinkled) The presence of the dominant allele (R) causes the mature seeds to be smooth. The original mutation is believed to have arisen spontaneously at the beginning of the seventeenth century. The seeds of the r mutant contain a lower proportion of starch than the wild-type (about 30% dry weight as opposed to about 50%), with the starch composition being altered to contain a higher proportion of amylose and smaller proportion of amylopectin (with about 70% of dry weight of the starch of mutant seeds being amylose as opposed to 38% of the wild-type starch). The effect of the mutation in the r gene has been shown to be caused by reduced activity of one of the branching enzyme isoforms (SBE1). The gene has been cloned and sequenced, and a 0.8 kb transposon-like insertion has been found to be present in the mutant gene.
A second recessive rugosus locus termed rb has also been characterised. Mutants homozygous recessive at this locus have a wrinkled-seeded phenotype similar to that of rr plants, although the amount of starch and its composition differs in that starch comprises about 36% of the dry eight of the seed, about 23% of which 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-blotting experiments have revealed the absence of one of the four polypeptide subunits present in the wild-type enzyme. Manipulation by reduction or suppression of the activity of ADP-glucose pyrophosphorylase (ADPG-PPase) to give an increased level of sucrose in the plant has been described in U.S. Pat. No. 5,498,831 (Burgess et al).
New Rugosus Loci
A mutagenesis programme was carried out by Wang et al, as described in Plant Breeding 105, 311-320 (1990) “An Analysis of Seed Development in
Pisum sativum
. XIII The Chemical Induction of Storage Product Mutants”. The programme employed chemical mutagenesis using ethyl methanesulphonate (EMS) or N-methyl-N-nitrosourea (MNU). Peas have been shown to be susceptible to mutation by chemical agents and these particular mutagens are likely to cause point mutations by alkylation. Twenty thousand phenotypically round genetically wild type (RR) seeds were treated with either of the above chemicals, these being termed M1 (mutagenised) seed. M1 seed gave rise to M1 plants bearing M2 seed. M2 seed gave rise to M2 plants bearing M3 seed. M3 seeds were analysed for storage product content.
Seeds which appeared wrinkled selected from the M3 generation had a wide range of starch content, 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%. The lipid and protein contents of the M3 seeds also appeared to be more varied than had been previously observed in peas, with a lipid content from 1-8% of the dry weight and a protein range of 24-48%, the latter showing a higher maxima than the existing variation of between 24 and 41%. The conclusion from the initial analyses of the M3 seeds was that new rugosus mutants had been induced and that it was likely that some would be mutants affecting starch biosynthesis.
The new mutant lines were each designated by a ‘SIM’ number (SIM=Seed: Induced Mutant). Preliminary allelism tests to the r and rb loci revealed that some of the SIM lines were not allelic to either of these loci and therefore were probably mutants affecting other enzymes in the starch pathway. Other lines were found to be allelic to r or rb and therefore these lines represent new mutant alleles of these loci (see Wang and Hedley, Seed Science Research (1991) 1, 3-14, “Seed Development in peas: knowing your three “r's” (or four of five)”). More detailed complementation analyses involving a complete diallel cross between 24 of the SIM lines and lines with rr and rbrb genotypes placed the mutants into five groups, two of which contained the original rugosus mutants (see Hedley and Wang, Aspects of Applied Biology 27 (1991) Production and protection of legumes, 205-209, “Adding value to the pea crop by genetically manipulating the storage product composition of the seed”). Recently, grouping of the SIM lines has been completed and the three new rugosus loci have been assigned the gene symbols rug3, rug4 and rug5 in accordance with the Pisum Genetics Association (see Wang and Hedley, 1993, Pisum Genetics 25, 64-70, “Seed Mutants in Pisum”). The five complementation groups are shown in Table 1.
TABLE 1
Group
1
2
3
4
5
Gene Symbol
r
rb
rug3
rug4
rug5
SIM Lines
53
14
1
11
51
54
15
32
91
52
55
16
41
201
81
56
101
42
57
102
43
58
103
59
103W
61
71
rug3
Preliminary analysis of the storage product content of the SIM lines showed that those belonging to the rug3 group had a dramatically reduced starch content in the mature seed by comparison to wild-type, round-seeded lines. The mutants in this group appeared to have between 1 and 20% starch as a proportion of the dry weight of the mature seed, compared with about 55% in round seeds (see Wang and Hedley, 1991 referred to above). In addition, these lines seemed to show a complete absence of amylose from the starch that was present. Such a phenotype had never been observed previously in pea.
The SIM lines belonging to the rug3 complementation group have been assingned gene symbols as shown in Table 2 (Wang and Hedley, 1993 referred to above).
TABLE 2
SIM number
Gene Symbol
1
rug3
a
32
rug3
b
41
rug3
c
42
rug3
d
43
rug3
a
Peas of the rug3rug3 genotype (which are referred to herein as rug3 peas for simplicity) are of scientific and potential commercial interest because of the low levels of starches of unusual nature, and also because of their high protein and lipid contents. See, for example, Hedley and Wang, Aspects of Applied Biology 27 (1991) 205-209, Hedley and Wang, Agro-Food Industry Hi-Tech (January/February 1993) 14-17, Farmers Weekly, Apr. 19, 1991, 54-57.
The present inve
Harrison Christopher J.
Hedley Clifford L.
Hughes Stephen G.
Lacey Colin N.
Wang Trevor L.
Ibrahim Medina A.
McElwain Elizabeth F.
Morgan & Lewis & Bockius, LLP
Unilever Patent Holdings B.V.
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