Potato plant producing essentially amylose-free starch

Details Potato plant producing essentially amylose-free starch Potato plant producing essentially amylose-free starch

1994-08-23

2003-07-29

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

C800S278000, C800S284000, C800S286000, C800S287000, C800S288000, C800S294000, C435S101000, C435S468000, C435S469000, C536S023600, C536S024500

Reexamination Certificate

active

06600093

ABSTRACT:

FIELD OF THE INVENTION
The invention is in the field of genetic engineering by recombinant DNA technology, more particularly the genetic engineering of potato plants in order to change the starch composition in the tubers towards essentially amylose-free starch.
BACKGROUND OF THE INVENTION
Starch is the major storage carbohydrate in potato and consists of two components, a linear (1→4)&agr;-D-glucan polymer and a branched (1→4) (1→6)&agr;-D-glucan called amylose and amylopectin, respectively. Amylose has a helical conformation with a molecular weight of 10
4
-10
5
. Amylopectin consists of short chains of &agr;-D-glucopyranose units primarily linked by (1→4)&agr; bonds with (1→6)&agr; branches and with a molecular weight up to 10
7
. In plants starch is found in two types of plastids: chloroplasts and amyloplasts. In both types of organelles the starch occurs as granules. In chloroplasts so-called transitory starch is accumulated for only a short period of time, whereas starch in amyloplasts is accumulated for long term storage and hence is named reserve starch. Generally, amylose makes up 11%-37% of the total reserve starch and variation in the amylose content is not only found among different plant species, but also among different cultivars of the same species. In potato the amylose content in the tuber varies from 18% to 23%. Furthermore, in a number of plant species mutants are known with a starch composition which deviate significantly from the above mentioned percentages.
Transitory and reserve starch are generally considered to be synthesized by the same enzymes. Starch metabolism in leaves follows a diurnal rhythm: synthesis and accumulation occur during the light period while hydrolysis occurs during the night. In storage tissue, starch synthesis occurs during a specific phase of tissue development; the synthesis being the predominant function of amyloplasts. The amount of amylose found in storage tissue of potato is about twice as high as that in leaves.
Sucrose is considered to be the major substrate for starch biosynthesis which involves the following steps: initiation, elongation, branching and granule formation. In the pathway of conversion of sucrose into amylose and amylopectin at least 13 enzymes play a role. Three groups of enzymes are directly involved in the formation of starch. These enzymes are phosphorylase, starch synthases and branching enzymes. Phosphorylase is active in starch breakdown, branching enzyme converts amylose into amylopectin by the breakage of (1→4)&agr;-bonds and the synthesis of (1→6)&agr;-bonds. Starch synthases are responsible for the synthesis of starch by the addition of ADP (UDP) glucose subunits to the non-reducing end of an (1→4)&agr;-D-glucan polymer. Starch synthase has been identified in two forms: one form is soluble while the other is tightly associated with starch granules. The soluble enzyme uses only ADP-glucose as the D-glucosyl donor, whereas the granule bound starch synthase (GBSS) utilizes ADP-glucose and UDP-glucose. Solubilization of the GBSS protein from starch granules of various plants has been reported. Although in maize there are thought to be at least two forms of GBSS, potato seems to have only one form. The presence and activities of the different starch synthases are important to starch biosynthesis not only because they have an effect on the amylose/amylopectin ratio in starch, but also because they can have a large impact on total starch content. In general, it appears that complete suppression of the enzymes producing amylose can be achieved with almost no change in the total amount of starch laid down, whereas suppression of the enzyme system producing amylopectin leads to a marked decrease of the amount of total starch.
Starch as such or in modified form is widely used in the food, paper and textile industries. With the depletion of natural oil resources starch could also become a substitute for oil as a raw material for the chemical industry. Therefore, it could become of major interest to produce starch which meets special requirements for certain applications. Although special forms of starch are already available from mutants of maize and rice and starches from other sources might have certain advantages, genetical engineering could be an option in order to obtain tailor-made starches in plants in which (recessive) mutants are not easily obtained. Selection of mutants is especially difficult in vegetatively propagated crops which are mainly crosspollinators and/or polyploids, such as the potato.
Although recently in a laborious isolation procedure a mutant of potato (amf) was isolated which, in analogy to the wx mutants in maize, lacks GBSS protein, GBSS activity and amylose (Hovenkamp-Hermelink et al. 1987), the breeding of such a mutant into a cultivar will take another number of years. One cause for the long duration of the procedure is the fact that a haploid clone had to be used for the isolation of the recessive mutant. To circumvent problems of isolating recessively inherited mutants in a polyploid crop like potato and to speed up the introduction of such a mutant character in potato varieties, the antisense approach would be a very important alternative, because an antisense gene would act as a dominant suppressor gene. The great advantage is that eventually it will become possible to mimick such a mutant phenotype directly in a tetraploid variety. With the availability of GBSS sequences, both from maize (Shure et al 1983) and potato (Hergersberg 1988; Visser et al 1989d) and an efficient transformation system for potato (Visser et al 1989a, 1989b) this approach could be tested.
It has been shown that antisense RNA transcripts can be used to mimic mutations in pro- and eukaryotes (for review see van der Krol et al. 1989). Antisense RNA was originally found as a naturally occurring mechanism used to control gene expression in bacteria (Tomizawa et al. 1981; Mizuno et al. 1984). Izant and Weintraub (1984, 1985) proposed that antisense RNA could be used to inhibit the expression of eukaryotic genes. By inhibiting the expression of specific target RNAS, this approach has led to the generation of mutant phenotypes in a number of different eukaryotic systems. In plants the use of antisense RNA proved to be successful in effectively inhibiting the activity of nopaline synthase (Rothstein et al. 1987; Sandler et al. 1988), chloramphenicol acetyltransferase (Ecker and Davis 1986; Delauney et al. 1988), chalcone synthase (van der Krol et al. 1988), polygalacturonase (Smith et al. 1988; Sheehy et al. 1988), phosphinotricin acetyl transferase (Cornelissen and Van de Wiele 1989) and &bgr;-glucuronidase (Robert et al. 1989).
Visser (1989) tested whether the antisense approach could be used to inhibit the expression of the gene for granule-bound starch synthase in potato using heterologous antisense constructs, i.e. an antisense gene constructed from a maize genomic GBSS gene.
The antisense gene was fused between the 35S cauliflower mosaic virus promoter and the nopaline synthase terminator in the binary vector pROK-1, which also carries a plant selectable kanamycin resistance gene. Since it was known from the amf-mutant that the mutation is expressed in all tissues in which starch is formed, including columella cells of the root cap, it was expected that also antisense effects would be visible in roots. The presence or absence of amylose could be easily detected because amylose forms a blue staining complex with the iodine present in Lugol's solution (I—KI). Starch without amylose, i.e. only containing amylopectin, forms a reddish-brown staining complex with iodine. In order to efficiently test the introduced antisense gene in potato for a biological effect a transformation system was developed in which the binary antisense vector was incorporated into
Agrobacterium rhizogenes.
The binary vector was present next to the wildtype Ri-plasmid of
A. rhizogenes
which is responsible for the formation of so-called hairy roots on plant explants.
Agrobacterium r

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