Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-01-21
2004-03-02
Horlick, Kenneth R. (Department: 1637)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S284000, C800S286000, C800S287000, C800S285000, C800S288000
Reexamination Certificate
active
06700039
ABSTRACT:
The present invention relates to a method of controlling sprout formation in plants and parts thereof including vegetative storage organs.
Potato tubers are of major economic importance. They represent a carbohydrate resource for many diets and are used as a basis for a variety of processed products. Besides starch, tubers contain high-quality proteins, substantial amounts of vitamins, minerals and trace elements. Continuous production of potato tubers throughout the year is impossible in most regions where potatoes are grown. As a consequence storage of the harvested tubers is required.
One of the potentially most damaging phenomena during storage is premature sprouting. Long term storage involves cooling, forced ventilation and use of chemical sprouting suppressants. The problems directly linked to long term storage are manifold.
Cooling, usually done in Northern Europe by ventilation with air at ambient temperature is one of the methods to inhibit sprouting. Apart from the associated costs, longer term cooling at 4° C. gives rise to the problems of cold sweetening and melanisation (darkening).
Chemical sprouting suppressants are currently the only possibility for inhibiting sprouting in potatoes destined for processing and fresh consumption, since low temperature storage leads to unacceptable accumulation of reducing sugars. However, in recent years, questions have arisen as to the environmental and nutritional impact of chemical suppressants such as chlorinated hydrocarbons. There is therefore a real need for an alternative method of controlling sprouting in vegetative storage organs such as tubers.
An alternative approach to delay sprouting would be the use of transgenic plants with a prolonged quiescence period. Sprouting of potato tubers involves several independent steps which might be targets for genetic engineering. The first step is the mobilisation of reserves, mainly starch. Starch breakdown occurs in amyloplasts and is mediated by starch phosphorylase and/or amylases. In the next step following starch breakdown, the resultant hexoses and/or hexose-phosphates have to be exported from amyloplasts. After transfer into the cytosol the produced hexoses and hexose-phosphates are distributed between glycolysis and sucrose synthesis. The third step is the formation of sucrose in the cytosol. Sucrose synthesis is energy dependent thus glycolysis and respiration are required. The fourth step is the transport of sucrose to the developing sprout. Finally the imported sucrose is utilised in the sprout to support growth and development.
We have now developed a means of controlling sprouting in vegetative storage organs such that sprouting may be turned off and on without any undesirable side effects such as yield loss. This new method involves the targeted expression of genes resulting in the disruption of sprouting in combination with gene switch technology to restore sprouting when required.
According to a first aspect of the present invention there is provided a method for the selective induction or suppression of sprouting in a plant comprising incorporating, preferably stably incorporating, into the genome of said plant by transformation a DNA construct comprising a first polynucleotide sequence comprising at least one DNA sequence operably linked to a tissue or organ selective promoter region and optionally to a transcription terminator region and a second polynucleotide sequence comprising at least one DNA sequence operably linked to and controlled by a controllable promoter region and optionally to a transcription terminator region whereby the DNA sequence(s) in said first polynucleotide sequence is expressed during dormancy of the vegetative organ derived from said transgenic plant resulting in effective suppression of sprouting and the said suppression is neutralised by inducing expression of the DNA sequence(s) in said second polynucleotide sequence from said controllable promoter region by external application of an inducing substance such that restoration of sprouting of said vegetative storage organ is dependent on the application of the inducer.
As used herein the term “tissue or organ selective promoter region” denotes those promoter regions which yield preferential expression of the DNA sequence(s) of interest in the desired tissue or organs.
The DNA sequences in the DNA construct may be endogenous or heterologous with respect to the transformed host.
Examples of DNA sequences which may be used in the method of the present invention to control sprouting include those DNA sequences coding for proteins involved in the mobilisation of reserves during dormancy such as the breakdown of storage compounds e.g starch breakdown, i.e starch phosphorylase, amylase (e.g. &agr; or &bgr; amylase) and maltase; e.g in glycolysis and subsequent metabolism e.g phosphofructokinase, hexokinase; in sucrose biosynthesis e.g sucrose synthase; in the transport of reserves during dormancy such as in phloem loading e.g ATPase; in long distance phloem transport and in phloem unloading e.g inorganic pyrophosphorylase (iPPase); and in the utilisation of reserves during dormancy such as in assimilate breakdown e.g the breakdown of sucrose in the growing sprout, i.e invertase; and in the utilisation of assimilates e.g utilisation of sucrose-derived metabolites, in the provision of energy required for sprout formation e.g. DNA sequences coding for proteins involved in mitochondrial function such as in respiration, such as mitochondrial enzymes and transport proteins such as translocators e.g. adenine nucleotide translocator (ANT) and malate oxoglutarate translocator (MOT) and inhibitors thereof such as uncoupling proteins. Examples of useful DNA sequences also include any other sequences which are involved in potato sprouting
Examples of preferred DNA sequences which may be used in the method of the present invention to control sprouting include those resulting in the production of sense, anti-sense or partial sense sequence(s) to, and/or coding for, proteins involved in the mobilisation and/or utilisation of sucrose, in potato sprouting and in mitochondrial function such as in respiration.
Examples of particularly preferred DNA sequences include those coding for an invertase derived from plant, bacterial or fungal sources e.g. from yeast, a pyrophosphatase derived from plant, bacterial or fungal sources and proteins involved in mitochondrial function such as MOT and ANT derived from plant, bacterial or fungal sources which are described hereinafter.
Suppression of sprouting may be achieved in a variety of ways. The first DNA sequence(s) may be expressed during dormancy of the vegetative storage organ and then down-regulated when sprouting is desired. When sprouting is desired expression of the second DNA sequence(s) is turned on leading to down regulation of the first DNA sequence and consequently restoration of sprouting.
Down regulation of a desired DNA sequence(s) may be achieved using methods well known in the art such as, for example, by use of repressor proteins, sense, anti-sense, partial-sense, and expression of a complementary protein. Examples of suitable operator/repressor systems include for example the lac, tet or lambda 434 systems and mutants thereof such as the Lac I&Dgr; His mutant (Lehming, N., Sartoris, J., Niemoeller, M., Genenger, G., v. Wilcken-Bergman, B. and Muller-Hill, Benno (1987), EMBO J. 6(10) 3145-3153—where the mutant has a change in amino acid 17 of Lac I altering tyrosine for histidine). Alternatively, an Amplicon™ may be used to down-regulate genes (Angell, S. M., Baulcombe, D. C., (1997) 16, 3675-3684). In this regard, the cDNA of replicating potato virus (PVX) RNA which has a transgene inserted therein is used whereby transiently expressed RNA sharing homology with the transgene is suppressed.
Alternatively, expression of the DNA sequence(s) in the first polynucleotide sequence may result in the production of a sense, anti-sense or partial-sense sequence(s) which acts to suppress a gene involved in sprouting or in the expression of an Amplicon™. In this
Ebneth Marcus
Jepson Ian
Sonnewald Uwe
Horlick Kenneth R.
Strzelecka Teresa
Syngenta Limited
Vrana Bruce
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