Nucleic acid molecules coding for debranching enzymes from potato

Nucleic acid molecules are described, which encode debranching enzymes from potato, as well as transgenic plant cells and plants in which an amylopectin with modified properties is synthesized due to the expression of a debranching enzyme from potato or due to the inhibition of such an endogeneous debranching enzyme activity.

FIELD OF THE INVENTION
 Nucleic acid molecules coding for debranching enzymes from potato The
 present invention relates to nucleic acid molecules encoding proteins from
 potato with the enzymatic activity of a debranching enzyme. The invention
 further relates to transgenic plants and plant cells, in which an
 amylopectin with an altered degree of branching is synthesized due to the
 expression of an additional debranching enzyme activity from potato or due
 to the inhibition of an endogeneous debranching enzyme activity. The
 invention also relates to the starch obtainable from said transgenic plant
 cells and plants.
 Starch plays an important role as storage substance in a multitude of
 plants and also as a regenerative, industrially usable raw material and
 has gained increasing significance. For the industrial use of starch it is
 necessary that it meets the demands of the processing industry with
 respect to its structure, form and/or other physico-chemical parameters.
 In order to enable the use in as many areas as possible it is furthermore
 necessary to achieve a large variety of substances. The polysaccharide
 starch is made up of chemically homogeneous basic components, namely the
 glucose molecules. However, it constitutes a highly complex mixture of
 various types of molecules which differ from each other in their degree of
 polymerization and in the degree of branching. One differentiates between
 amylose-starch, a basically non-branched polymer made up of
 .alpha.-1,4-glycosidically branched glucose molecules, and
 amylopectin-starch, a branched polymer, in which the branching results
 from additional .alpha.-1,6-glycosidic interlinkings.
 In plants used typically for the production of starch, such as maize or
 potato, the synthesized starch consists of approximately 25%
 amylose-starch and of about 75% amylopectin-starch. In the case of maize,
 for example, a further branched polysaccharide, apart from amylopectin,
 occurs, namely the so-called phytoglycogen which differs from amylopectin
 by exhibiting a higher degree of branching and different solubility (see
 e.g. Lee et al., Arch. Biochem. Biophys. 143 (1971), 365-374; Pan and
 Nelson, Plant Physiol. 74 (1984), 324-328). In the scope of the present
 application the term amylopectin is used in such a way as to comprise the
 phytoglycogen.
 With respect to the homogeneity of the basic component starch for its use
 in the industrial area, starch-producing plants are needed which contain,
 for example, only the component amylopectin or only the component amylose.
 For a number of other uses plants are needed that synthesize amylopectin
 types with different degrees of branchings.
 Such plants may for example be obtained by breeding or by means of
 mutagenesis techniques. It is known for various plant species, such as for
 maize, that by means of mutagenesis varieties may be produced in which
 only amylopectin is formed. Also in the case of potato a genotype was
 produced from a haploid line by means of chemical mutagenesis. Said
 genotype does not form amylose (Hovenkamp-Hermelink, Theor. Appl. Genet.
 75 (1987), 217-221).
 Apart from conventional breeding and mutagenesis techniques, recombinant
 DNA techniques are now increasingly used in order to specifically
 interfere with the starch metabolism of starch storing plants. A
 prerequisite for this is that DNA sequences be provided which encode
 enzymes involved in the starch metabolism. In the case of potato, for
 example, DNA sequences have by now been found which encode a granule-bound
 starch synthase or a branching enzyme (Q enzyme), and they have been used
 in order to genetically modify plants.
 For a further targeted modification of the starch in plants, in particular
 of the degree of branching of starch synthesized in plants by means of
 recombinant DNA techniques, it is still necessary to identify DNA
 sequences that encode enzymes participating in the starch metabolism,
 particularly in the branching of starch molecules.
 Apart from the Q enzymes that introduce branchings into starch molecules,
 enzymes occur in plants which are capable of dissolving branchings. These
 enzymes are called debranching enzymes.
 In the case of sugar beet, Li et al. (Plant Physiol. 98 (1992), 1277-1284)
 could only prove the occurrence of one debranching enzyme, apart from five
 endo- and two exoamylases. This enzyme having a size of approximately 100
 kD and an optimum pH value of 5.5 is located within the chloroplasts. A
 debranching enzyme was also described for spinach. The debranching enzyme
 from spinach as well as that from sugar beet exhibit a fivefold lower
 activity in a reaction with amylopectin as substrate when compared to a
 reaction with pullulan as a substrate (Ludwig et al., Plant Physiol. 74
 (1984), 856-861; Li et al., Plant Physiol. 98 (1992), 1277-1284). The
 isolation of a cDNA encoding a debranching enzyme was described for
 spinach (Renz et al., Plant Physiol. 108 (1995), 1342).
 The existence of a debranching enzyme for maize has been described in the
 prior art. The corresponding mutant was designated su (sugary). The gene
 of the sugary locus was cloned recently (see James et al., Plant Cell 7
 (1995), 417-429). In the case of the agriculturally significant
 starch-storing cultured plant potato, the activity of a debranching enzyme
 was examined by Hobson et al. (J. Chem. Soc., (1951), 1451). It was proven
 that the respective enzyme, contrary to the Q enzyme, does not exhibit any
 activities leading to an elongation of the polysaccharide chain, but
 merely hydrolyses .alpha.-1,6-glycosidic bonds. Methods for the
 purification of a debranching enzyme from potato as well as partial
 peptide sequences of the purified protein have already been described (WO
 95/04826).
 So far no indication as to the existence of further debranching enzyme
 types from potato could be found. Should this, however, be the case, all
 debranching enzyme types occurring in potato would have to be identified
 and the corresponding genes or cDNA sequences would have to be isolated in
 order to produce transgenic potato plants that do no longer exhibit any
 debranching enzyme activity for the purpose of achieving a modification of
 the degree of branching of the amylopectin starch.
 Therefore, the technical problem underlying the present invention is to
 identify further debranching enzymes possibly occurring in potato and to
 isolate corresponding nucleic acid molecules encoding these enzymes.
 This problem is solved by the provision of the embodiments as defined in
 the claims.
 SUMMARY OF THE INVENTION
 Thus, the present invention relates to nucleic acid molecules encoding
 proteins with the biological activity of a debranching enzyme from potato.
 Such a nucleic acid molecule preferably encodes a protein with the
 biological activity of a debranching enzyme from potato that exhibits the
 amino acid sequence depicted in SEQ ID No. 2. In a particularly preferred
 embodiment such a nucleic acid molecule comprises the nucleotide sequence
 depicted under SEQ ID No. 1, in particular the coding region.
 The present invention also relates to nucleic acid molecules encoding
 proteins with the biological activity of a debranching enzyme from potato
 and hybridizing to one of the above-described nucleic acid molecules or to
 the complementary strand thereof. Furthermore, the present invention
 relates to nucleic acid molecules the sequence of which differs from the
 sequences of the above-mentioned nucleic acid molecules due to a
 degeneracy of the genetic code, and which encode a protein exhibiting the
 biological activity of a debranching enzyme from potato.
 The term "from potato" means that the debranching enzymes encoded by the
 nucleic acid molecules of the invention are typical for the species
 Solanum tuberosum, i.e. they either occur naturally in such plants, for
 example encoded by genomic or RNA molecules or by molecules derived
 therefrom. Derived molecules may for example be produced by the reverse
 transcription of RNA molecules, amplification, mutation, deletion,
 substitution, insertion etc. I.e. the term also comprises enzymes encoded
 by alleles and derivatives of sequences naturally occurring in potato.
 These may for example be produced by in vitro by means of recombinant DNA
 techniques.
 DETAILED DESCRIPTION OF THE INVENTION
 In the scope of the present invention the term "hybridization" signifies
 hybridization under conventional hybridizing conditions, preferably under
 stringent conditions, as described for example in Sambrook et al.,
 Molecular Cloning, A Laboratory Manual, 2nd Edition (1989) Cold Spring
 Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Nucleic acid molecules
 hybridizing to the nucleic acid molecules of the invention may basically
 be derived from any desired type of potato plant. Nucleic acid molecules
 hybridizing to the molecules of the invention may for example be isolated
 from genomic or cDNA libraries.
 The identification and isolation of such nucleic acid molecules may take
 place by using the molecules of the invention or parts of these molecules
 or, as the case may be, the reverse complements of these molecules, e.g.
 by hybridization according to standard methods (see e.g. Sambrook et al.,
 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring
 Harbor Laboratory Press, Cold Spring Harbor, N.Y.) or by means of
 amplification via PCR.
 As a probe for hybridization e.g. nucleic acid molecules may be used which
 exactly or basically contain the nucleotide sequence indicated under Seq
 ID No. 1 or parts thereof. The fragments used as hybridization probe may
 also be synthetic fragments which were produced by means of the
 conventional synthesizing methods and the sequence of which is basically
 identical to that of a nucleic acid molecule of the invention. After
 identifying and isolating the genes hybridizing to the nucleic acid
 sequences of the invention, the sequence has to be determined and the
 properties of the proteins encoded by this sequence have to be analyzed.
 The molecules hybridizing to the nucleic acid molecules of the invention
 also comprise fragments, derivatives and allelic variants of the
 above-described DNA molecules which encode a protein with the enzymatic
 activity of a debranching enzyme from potato or a biologically, i.e.
 enzymatically active fragment thereof. In this context, fragments are
 defined as parts of the nucleic acid molecules, which are long enough in
 order to encode a polypeptide with the enzymatic activity of a debranching
 enzyme. In this context, the term derivative means that the sequences of
 these molecules differ from the sequences of the above-mentioned nucleic
 acid molecules at one or more positions and that they exhibit a high
 degree of homology to these sequences. In this regard, homology means a
 sequence identity of at least 70%, in particular an identity of at least
 80%, preferably of more than 90% and still more preferably a sequence
 identity of more than 95%. The deviations occurring when compared to the
 above-described nucleic acid molecules might have been caused by deletion,
 addition, substitution, insertion or recombination.
 Moreover, homology means that the respective nucleic acid molecules or the
 proteins they encode are functionally and/or structurally equivalent. The
 nucleic acid molecules which are homologous to the above-described
 molecules and represent derivatives of these molecules, are generally
 variations of these molecules that constitute modifications exerting the
 same biological function. These variations may be naturally occurring
 variations, for example sequences derived from other potato plants or
 varieties, or mutations, wherein these mutations may have occurred
 naturally or they may have been introduced by means of a specific
 mutagenesis. Moreover, the variations may be synthetically produced
 sequences. The allelic variants may be naturally occurring as well as
 synthetically produced variants or variants produced by recombinant DNA
 techniques.
 The proteins encoded by the various variants of the nucleic acid molecules
 according to the invention exhibit certain common characteristics. Enzyme
 activity, molecular weight, immunologic reactivity, conformation etc. may
 belong to these characteristics as well as physical properties such as the
 mobility in gel electrophoresis, chromatographic characteristics,
 sedimentation coefficients, solubility, spectroscopic properties,
 stability; pH-optimum, temperature-optimum etc.
 The enzymatic activity of the debranching enzyme may for example be shown
 by means of a staining test, as described in WO 95/04826. This test is
 based on the fact that a protein with a starch-modifying activity may be
 shown by separating protein extracts, for example from potato tubers, in
 non-denaturing amylopectin- containing polyacrylamide gels (PMG) and the
 gel is subsequently, after incubation with a suitable buffer, subjected to
 iodine staining. While unbranched amylose treated with iodine shows a blue
 staining, amylopectine exhibits a reddish purple staining. In
 amylopectin-containing polyacrylamide gels which turn reddish purple when
 treated with iodine, the color of the gel tends to turn into blue at
 positions where a debranching activity is localized, since the branchings
 of the purple-staining amylopectin are dissolved by the debranching
 enzyme.
 Alternatively, the debranching enzyme activity may be shown by means of the
 DNSS test (see Ludwig et al., Plant Physiol. 74 (1984), 856-861).
 The nucleic acid molecules of the invention may be any desired nucleic acid
 molecules, in particular DNA or RNA molecules, such as cDNA, genomic DNA,
 mRNA etc. They may be naturally occurring molecules or they may be
 produced by means of recombinant DNA or chemical synthesizing techniques.
 The nucleic acid molecules according to the present invention encode a so
 far unknown protein from potato with the enzymatic activity of a
 debranching enzyme. So far only one debranching enzyme had been described
 for potato. There was no indication in the prior art that genes exist in
 potato encoding further debranching enzymes. Now it was surprisingly found
 that, apart from the debranching enzyme from potato known so far, at least
 one further enzyme with debranching activity exists. Thus, the molecules
 of the invention encode a novel type of debranching enzymes from potato.
 By means of these molecules it is now possible to specifically interfere
 with the starch metabolism of potato and other starch-storing plants and,
 thus, to enable the synthesis of a starch modified in its chemical or
 physical properties. This may be carried out by over-expressing the
 nucleic acid molecules of the invention in any desired, preferably
 starch-storing plants or by reducing the debranching enzyme activity in
 potato plants by making use of the nucleic acid sequences of the
 invention, for example by antisense or ribozyme effects.
 Furthermore, the present invention relates to nucleic acid molecules with a
 length of at least 15, preferably of more than 50 and most preferably of
 more than 200 base pairs, which specifically hybridize to the nucleic acid
 molecules of the invention. In this context, specifically hybridizing
 means that these molecules hybridize to nucleic acid molecules encoding
 the novel debranching enzymes from potato, however, not to nucleic acid
 molecules encoding other proteins. In this regard, hybridizing preferably
 means hybridization under stringent conditions (see above). The invention
 particularly relates to such nucleic acid molecules that hybridize to
 transcripts of the nucleic acid molecules of the invention, thereby
 preventing their translation. Such nucleic acid molecules specifically
 hybridizing with the nucleic acid molecules of the invention may for
 example be parts of mRNA constructs or ribozymes or may be used as primers
 for the amplification by means of PCR.
 Furthermore, the invention relates to vectors, especially plasmids,
 cosmids, viruses, bacteriophages and other vectors common in genetic
 engineering, which contain the above-mentioned nucleic acid molecules of
 the invention.
 In a preferred embodiment the nucleic acid molecules contained in the
 vectors are linked to regulatory elements that ensure the transcription
 and translation in prokaryotic or eukaryotic cells.
 In a further embodiment the invention relates to host cells, in particular
 prokaryotic or eukaryotic cells, which have been transformed by an
 above-mentioned nucleic acid molecule or vector, as well as to cells
 derived from such host cells and containing the described nucleic acid
 molecules or vectors. The host cells may be bacterial or fungal cells, as
 well as plant or animal cells.
 The invention also relates to proteins with the biological activity of a
 debranching enzyme from potato which are encoded by the nucleic acid
 molecules of the invention, or to biologically active fragments thereof.
 Furthermore, the present invention relates to methods for the production of
 a protein with the biological activity of a debranching enzyme from potato
 or a biologically active fragment thereof, wherein host cells of the
 invention are cultivated under suitable conditions and wherein the protein
 is isolated from the culture, i.e. from the cells and/or the culture
 medium.
 In a preferred embodiment the host cells of the invention are transgenic
 plant cells which due to the presence and expression of an introduced
 nucleic acid molecule of the invention either exhibit a novel or an
 increased debranching enzyme activity when compared to untransformed
 cells.
 By the provision of the nucleic acid molecules according to the present
 invention it is now possible to modify plant cells by means of recombinant
 DNA techniques in such a way that they exhibit a novel or increased
 debranching enzyme activity when compared to wildtype cells.
 Such transgenic plant cells differ from untransformed cells in that the
 introduced nucleic acid molecule is either heterologous to the transformed
 cell, i.e. derived from a cell with a different genomic background, or in
 that the introduced nucleic acid molecule, if it is homologous to
 transformed plant species, is localized at a position in the genome where
 it does not naturally occur in non-transformed cells. The introduced
 nucleic acid molecule may either be subjected to the control of its
 natural promoter or be linked with regulatory elements of foreign genes.
 Transgenic plants containing the above-described transgenic plant cells are
 also the subject matter of the present invention.
 The plant which is transformed with the nucleic acid molecules of the
 invention and in which a debranching enzyme from potato is synthesized due
 to the introduction of such a molecule may principally be any desired kind
 of plant. It is preferably a monocotyledonous or dicotyledonous useful
 plant, in particular a starch storing plant, such as cereals, Leguminosae,
 potatoes or cassava.
 The cereals are in particular monocotyledonous plants belonging to the
 Poales order, in particular of the family of the Poaceae. Examples thereof
 are plants belonging to the genuses Avena (oats), Triticum (wheat), Secale
 (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria,
 Sorghum (millet), Zea (maize) etc. Starch-storing Leguminosae are e.g.
 some types of the genus Pisum (e.g. Pisum sativum), Vicia (e.g. Vicia
 faba), Cicer (e.g. Cicer arietinum), Lens (e.g. Lens culinaris), Phaseolus
 (e.g. Phaseolus vulgaris and Phaseolus coccineus), etc.
 The present invention also relates to starch obtainable from the transgenic
 plant cells or plants. The expression of a novel or additional debranching
 enzyme activity from potato in the transgenic plant cells and plants of
 the invention influences the degree of branching of the amylopectin
 synthesized in the cells and plants. Therefore, a starch synthesized in
 these plants exhibits modified physical and/or chemical properties when
 compared to starch from wildtype plants.
 Furthermore, the present invention relates to propagation material of the
 transgenic plants of the invention, such as seeds, fruits, cuttings,
 tubers, rootstocks etc., wherein this propagation material contains the
 above-described transgenic plant cells. In the case of potato plants the
 propagation material are preferably tubers.
 Furthermore, the present invention relates to transgenic plant cells from
 potato in which the activity of the debranching enzyme of the invention is
 reduced due to the inhibition of the transcription or translation of
 endogeneous nucleic acid molecules encoding a such a novel debranching
 enzyme. This is preferably achieved by expressing a nucleic acid molecule
 of the invention or a part thereof in the corresponding plant cells in
 antisense orientation and by the fact that due to the antisense effect the
 described debranching enzyme activity is reduced. A further possibility in
 order to reduce the debranching enzyme activity in plant cells is to
 express suitable ribozymes that specifically cleave transcripts of the DNA
 molecules of the invention. The production of such ribozymes by means of
 the DNA molecules of the invention is known to the skilled person. It is
 also possible to express molecules which exert an antisense effect in
 combination with a ribozyme effect. Alternatively, the debranching enzyme
 activity in the plant cells may be reduced by means of a cosuppression
 effect. This method is known to the skilled person and has e.g. been
 described in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel et
 al. (Curr. Top. Microbiol. Immunol. 197 (1995), 91-103), Flavell et al.
 (Curr. Top. Microbiol. Immunol. 197 (1995), 43-46), Palaqui and Vaucheret
 (Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al. (Mol. Gen. Genet.
 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994),
 613-621) and other sources. The expression of ribozymes in order to reduce
 the activity of a particular enzyme in cells is also known to the skilled
 person and has for example been described in EP-B1 0 321 201. The
 expression of ribozymes in plant cells has for example been described by
 Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338). Other possibilities
 in order to reduce the activity of the described novel debranching enzymes
 in plant cells are known to the skilled person, for example mutagenesis of
 genomic sequences encoding such enzymes, e.g. by gene tagging or
 transposon mutagenesis or by expressing antibodies which specifically
 recognize the novel debranching enzymes. The mutagenesis of genomic
 sequences may apply for coding regions of the gene (introns and exons) or
 also to regulatory regions, in particular to those necessary for
 initiating transcription.
 The invention further relates to transgenic potato plants containing the
 above-described transgenic plant cells with reduced debranching enzyme
 activity.
 The modified starch obtainable from the transgenic cells or plants is also
 the subject matter of the present invention. When compared to
 non-transformed plants, the amylopectin starch of the transgenic cells and
 plants exhibits a modified degree of branching due to the reduced
 debranching enzyme activity.
 The invention also relates to propagation material of the above-described
 transgenic plants, in particular to seeds and tubers, wherein said
 material contains the above-mentioned transgenic plant cells.
 Transgenic plant cells forming an amylopectin starch with a modified degree
 of branching in comparison to amylopectin starch synthesized in wildtype
 plants due to the expression of a novel or additional debranching enzyme
 activity, may for example be produced by a method comprising the following
 steps:
 (a) Production of an expression cassette comprising the following DNA
 sequences:
 (i) a promoter ensuring the transcription in plant cells;
 (ii) at least one nucleic acid sequence of the invention which encodes a
 protein with the enzymatic activity of a debranching enzyme or a
 biologically active fragment thereof and which is coupled to the 3'-end of
 the promoter in sense-orientation; and
 (iii) optionally, a termination signal for the termination of transcription
 and the addition of a poly-A-tail to the developing transcript, which is
 coupled to the 3'-end of the coding region; and
 (b) transforming plant cells with the expression cassette produced in step
 (a).
 Transgenic plant cells forming an amylopectin starch with a modified degree
 of branching in comparison to amylopectin starch synthesized in wildtype
 plants due to the reduction of the described debranching enzyme activity,
 may for example be produced by a method comprising the following steps:
 (a) Production of an expression cassette comprising the following DNA
 sequences:
 (i) a promoter ensuring the transcription in plant cells;
 (ii) at least one nucleic acid sequence of the invention which encodes a
 protein with the enzymatic activity of a debranching enzyme or a
 biologically active part thereof and which is coupled to the 3'-end of the
 promoter in antisense-orientation; and
 (iii) optionally, a termination signal for the termination of transcription
 and the addition of a poly-A-tail to the developing transcript, which is
 coupled to the 3'-end of the coding region; and
 (b) transforming plant cells with the expression cassette produced in step
 (a).
 Basically every promoter functional in the plants selected for
 transformation may be used as the promoter mentioned under (i). The
 promoter may be homologous or heterologous with respect to the used plant
 species. Use may, for example, be made of the 35S promoter of the
 cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812) which
 ensures a constitutive expression in all plant tissues and also of the
 promoter construct described in WO/9401571. Another example are the
 promoters of the polyubiquitin genes from maize (Christensen et al., Plant
 Mol. Biol. 18 (1992) 675-689). However, use may also be made of promoters
 which are only activated at a point of time determined by exogeneous
 factors (such . as in WO/9307279). In this regard, promoters of heat-shock
 proteins allowing for simple induction may be of particular interest.
 Furthermore, promoters may be used that lead to the expression of
 downstream sequences in a particular tissue of the plant (see e.g.
 Stockhaus et al., EMBO J. 8 (1989), 2245-2251). Promoters which are active
 in the starch-storing parts of the plant to be transformed are preferably
 used. In the case of maize these parts are the maize kernels, in the case
 of potatoes the tubers. In order to overexpress the nucleic acid molecules
 of the invention in potatoes, the tuber-specific B33-promoter (Rocha-Sosa
 et al., EMBO J. 8 (1989), 23-29) may for example be used.
 Seed-specific promoters have already been described for various plant
 species, such as the USP promoter from Vicia faba which ensures a
 seed-specific expression in V. faba and other plants (Fiedler et al.,
 Plant Mol. Biol. 22 (1993), 669-679; Baumlein et al., Mol. Gen. Genet. 225
 (1991), 459-467). In the case of maize, for example, promoters of the zein
 genes ensure a specific expression within the endosperm of the maize
 kernels (Pedersen et al., Cell 29 (1982), 1015-1026; Quattrocchio et al.,
 Plant Mol. Biol. 15 (1990), 81-93).
 In the case that the nucleic acid sequence mentioned under process step
 (a)(ii), which encodes a protein with the enzymatic activity of a
 debranching enzyme from potato, is linked to the promoter in
 sense-orientation, this nucleic acid sequence may be of native or
 homologous origin as well as of foreign or heterologous origin with
 respect to the plant species to be transformed, i.e. potato plants as well
 as any desired other plants (preferably the above-mentioned,
 starch-storing plants) may be transformed with the described expression
 cassette.
 The synthesized protein may in principle be located in any desired
 compartment within the plant cell. Plant debranching enzymes are generally
 located within the plastids and therefore possess a signal sequence for
 the translocation into these organelles. In order to achieve localization
 within another compartment, the DNA sequence encoding this signal sequence
 must be deleted and the coding region has to be linked to DNA sequences
 which ensure localization in the respective compartment. Such sequences
 are known (see e.g. Braun et al., EMBO J. 11 (1992), 3219-3227; Wolter et
 al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al.,
 Plant J. 1 (1991), 95-106).
 In case that the nucleic acid sequence from potato mentioned under process
 step (a)(ii), which encodes a protein with the enzymatic activity of a
 debranching enzyme, is linked to the promoter in antisense-orientation, it
 is preferably a nucleic acid sequence of homologous origin with respect to
 the plant species to be transformed. However, also nucleic acid sequences
 may be used which exhibit a high degree of homology to endogeneously
 present debranching enzyme genes, in particular homologies of more than
 80%, preferably homologies of between 90% and 100% and most preferably
 homologies of more than 95%.
 Sequences with a minimum length of 15 bp may be used. Even when using
 shorter sequences, an inhibiting effect cannot be excluded. Longer
 sequences ranging between 100 to 500 base pairs are preferably used; for
 an efficient antisense inhibition, sequences with a length of more than
 500 base pairs are used. Usually, use is made of sequences that are
 shorter than 5000 base pairs, preferably sequences that are shorter than
 2500 base pairs.
 Termination signals for the transcription in plant cells are described and
 may be interchanged as desired. For example, use can be made of the
 termination sequence of the octopinsynthase gene from Agrobacterium
 tumefaciens.
 The transfer of the expression cassette constructed according to process
 step (a) is preferably carried out by using plasmids, in particular by
 means of plasmids ensuring a stable integration of the expression cassette
 into the plant genome.
 The above-described method for overexpressing a novel debranching enzyme
 from potato may principally be used for all plant species. In this
 context, monocotyledonous and dicotyledonous plants and in particular the
 above-mentioned starch-storing plants are of interest. The above-described
 method for reducing the debranching enzyme activity is preferably used for
 dicotyledonous plants, in particular for potatoes.
 Due to the introduction of an expression cassette constructed according to
 the above-described methods, an RNA is formed within the transformed plant
 cells. If the nucleic acid sequence encoding a debranching enzyme from
 potato is linked to the promoter in sense-orientation in the expression
 cassette, an mRNA is synthesized which may serve as a matrix for the
 synthesis of an additional or novel debranching enzyme from potato in the
 plant cells. As a consequence thereof, these cells exhibit an activity or,
 as the case may be, increased activity of the debranching enzyme from
 potato, which leads to a modification of the degree of branching of the
 amylopectin formed in the cells. Thereby, a starch is made accessible
 which in comparison to naturally occuring starch is characterized by a
 more clearly ordered structure as well as by an increased homogeneity.
 This may, among other things, favorably influence the film forming
 properties.
 If, however, the nucleic acid sequence encoding a debranching enzyme from
 potato is linked to the promoter in antisense-orientation, an
 antisense-RNA is synthesized within the transgenic plant cells inhibiting
 the expression of endogeneous debranching enzyme genes. As a consequence,
 these cells exhibit a reduced activity of the novel debranching enzyme
 from potato, which leads to the synthesis of a modified starch. By means
 of the antisense technique it is possible to produce plants in which the
 expression of an endogeneous debranching enzyme gene in potato is
 inhibited to different degrees within the range of 0% to 100%. This
 enables in particular the production of potato plants synthesizing
 amylopectin starch with most various variations of the degree of
 branching. This constitutes an advantage with regard to conventional
 breeding and mutagenesis techniques in which a lot of time and costs are
 required in order to provide such a variety. Highly branched amylopectin
 has a particularly large surface and is therefore particularly suitable as
 a copolymer. A high degree of branching furthermore leads to an
 improvement of the amylopectin's solubility in water. This property is
 very advantageous for certain technical applications.
 Potato is particularly suitable for the production of modified amylopectin
 by using the nucleic acid molecules of the invention encoding debranching
 enzymes. The application of the invention is, however, not limited to this
 plant species. Any desired other plant species may be used for
 overexpression.
 The modified starch synthesized in the transgenic plants may be isolated
 from the plants or from the plant cells by means of conventional methods
 and may be used for the production of foodstuffs and industrial products
 after purification.
 The starch according to the invention can be modified by the person skilled
 in the art by known methods and can be used in modified or unmodified form
 for different uses in the food or non-food industry.
 Basically, the uses of starch can be subdivided into two major fields. One
 field comprises the hydrolysis products of starch and the so-called native
 starches. The hydrolysis products essentially comprise glucose and glucans
 components obtained by enzymatic or chemical processes. They can be used
 for further processes, such as fermentation and chemical modifications. In
 this context, it might be of importance that the hydrolysis process can be
 carried out simply and inexpensively. Currently, it is carried out
 substantially enzymatically using amyloglucosidase. It is thinkable that
 costs might be reduced by using lower amounts of enzymes for hydrolysis
 due to changes in the starch structure, e.g. increasing the surface of the
 grain, improved digestibility due to less branching or a steric structure,
 which limits the accessibility for the used enzymes.
 The use of the so-called native starch which is used because of its polymer
 structure can be subdivided into two further areas:
 1. Use in Foodstuffs
 Starch is a classic additive for various foodstuffs, in which it
 essentially serves the purpose of binding aqueous additives and/or causes
 an increased viscosity or an increased gel formation. Important
 characteristic properties are flowing and sorption behavior, swelling and
 pastification temperature, viscosity and thickening performance,
 solubility of the starch, transparency and paste structure, heat, shear
 and acid resistance, tendency to retrogradation, capability of film
 formation, resistance to freezing/thawing, digestibility as well as the
 capability of complex formation with e.g. inorganic or organic ions.
 2. Use in Non-Foodstuffs
 The other major field of application is the use of starch as an adjuvant in
 various production processes or as an additive in technical products. The
 major fields of application for the use of starch as an adjuvant are,
 first of all, the paper and cardboard industry. In this field, the starch
 is mainly used for retention (holding back solids), for sizing filler and
 fine particles, as solidifying substance and for dehydration. In addition,
 the advantageous properties of starch with regard to stiffness, hardness,
 sound, grip, gloss, smoothness, tear strength as well as the surfaces are
 utilized. 2.1 Paper and Cardboard Industry
 Within the paper production process, a differentiation can be made between
 four fields of application, namely surface, coating, mass and spraying.
 The requirements on starch with regard to surface treatment are
 essentially a high degree of brightness, corresponding viscosity, high
 viscosity stability, good film formation as well as low formation of dust.
 When used in coating the solid content, a corresponding viscosity, a high
 capability to bind as well as a high pigment affinity play an important
 role. As an additive to the mass rapid, uniform, loss-free dispersion,
 high mechanical stability and complete retention in the paper pulp are of
 importance. When using the starch in spraying, corresponding content of
 solids, high viscosity as well as high capability to bind are also
 significant.
 2.2 Adhesive Industry
 A major field of application is, for instance, in the adhesive industry,
 where the fields of application are subdivided into four areas: the use as
 pure starch glue, the use in starch glues prepared with special chemicals,
 the use of starch as an additive to synthetic resins and polymer
 dispersions as well as the use of starches as extenders for synthetic
 adhesives. 90% of all starch-based adhesives are used in the production of
 corrugated board, paper sacks and bags, composite materials for paper and
 aluminum, boxes and wetting glue for envelopes, stamps, etc.
 2.3 Textiles and Textile Care Products
 Another possible use as adjuvant and additive is in the production of
 textiles and textile care products. Within the textile industry, a
 differentiation can be made between the following four fields of
 application: the use of starch as a sizing agent, i.e. as an adjuvant for
 smoothing and strengthening the burring behavior for the protection
 against tensile forces active in weaving as well as for the increase of
 wear resistance during weaving, as an agent for textile improvement mainly
 after quality-deteriorating pretreatments, such as bleaching, dying, etc.,
 as thickener in the production of dye pastes for the prevention of dye
 diffusion and as an additive for warping agents for sewing yarns.
 2.4 Building Industry
 Furthermore, starch may be used as an additive in building materials. One
 example is the production of gypsum plaster boards, in which the starch
 mixed in the thin plaster pastifies with the water, diffuses at the
 surface of the gypsum board and thus binds the cardboard to the board.
 Other fields of application are admixing it to plaster and mineral fibers.
 In ready-mixed concrete, starch may be used for the deceleration of the
 sizing process.
 2.5 Ground Stabilization
 Furthermore, the starch is advantageous for the production of means for
 ground stabilization used for the temporary protection of ground particles
 against water in artificial earth shifting. According to state-of-the-art
 knowledge, combination products consisting of starch and polymer emulsions
 can be considered to have the same erosion- and encrustation-reducing
 effect as the products used so far; however, they are considerably less
 expensive.
 2.6 Use in Plant Protectives and Fertilizers
 Another field of application is the use of starch in plant protectives for
 the modification of the specific properties of these preparations. For
 instance, starches are used for improving the wetting of plant protectives
 and fertilizers, for the dosed release of the active ingredients, for the
 conversion of liquid, volatile and/or odorous active ingredients into
 microcristalline, stable, deformable substances, for mixing incompatible
 compositions and for the prolongation of the duration of the effect due to
 a reduced disintegration.
 2.7 Drugs, Medicine and Cosmetics Industry
 Starch may also be used in the fields of drugs, medicine and in the
 cosmetics industry. In the pharmaceutical industry, the starch may be used
 as a binder for tablets or for the dilution of the binder in capsules.
 Furthermore, starch is suitable as disintegrant for tablets since, upon
 swallowing, it absorbs fluid and after a short time it swells so much that
 the active ingredient is released. For qualitative reasons, medicinal
 flowance and dusting powders are further fields of application. In the
 field of cosmetics, the starch may for example be used as a carrier of
 powder additives, such as scents and salicylic acid. A relatively
 extensive field of application for the starch is toothpaste.
 2.8 Starch as an Additive in Coal and Briquettes
 The use of starch as an additive in coal and briquettes is also thinkable.
 By adding starch, coal can be quantitatively agglomerated and/or
 briquetted in high quality, thus preventing premature disintegration of
 the briquettes. Barbecue coal contains between 4 and 6% added starch,
 calorated coal between 0.1 and 0.5%. Furthermore, the starch is suitable
 as a binding agent since adding it to coal and briquette can considerably
 reduce the emission of toxic substances.
 2.9 Processing of Ore and Coal Slurry
 Furthermore, the starch may be used as a flocculant in the processing of
 ore and coal slurry.
 2.10 Additive for Casing Materials
 Another field of application is the use as an additive to process materials
 in casting. For various casting processes cores produced from sands mixed
 with binding agents are needed. Nowadays, the most commonly used binding
 agent is bentonite mixed with modified starches, mostly swelling starches.
 The purpose of adding starch is increased flow resistance as well as
 improved binding strength. Moreover, swelling starches may fulfill more
 prerequisites for the production process, such as dispersability in cold
 water, rehydratisability, good mixability in sand and high capability of
 binding water.
 2.11 Rubber Industry
 In the rubber industry starch may be used for improving the technical and
 optical quality. Reasons for this are improved surface gloss, grip and
 appearance. For this purpose, the starch is dispersed on the sticky
 rubberized surfaces of rubber substances before the cold vulcanization. It
 may also be used for improving the printability of rubber.
 2.12 Production of Leather Substitutes
 Another field of application for the modified starch is the production of
 leather substitutes.
 2.13 Starch in Synthetic Polymers
 In the plastics market the following fields of application are emerging:
 the integration of products derived from starch into the processing
 process (starch is only a filler, there is no direct bond between
 synthetic polymer and starch) or, alternatively, the integration of
 products derived from starch into the production of polymers (starch and
 polymer form a stable bond).
 The use of the starch as a pure filler cannot compete with other substances
 such as talcum. This situation is different when the specific starch
 properties become effective and the property profile of the end products
 is thus clearly changed. One example is the use of starch products in the
 processing of thermoplastic materials, such as polyethylene. Thereby,
 starch and the synthetic polymer are combined in a ratio of 1:1 by means
 of coexpression to form a `master batch`, from which various products are
 produced by means of common techniques using granulated polyethylene. The
 integration of starch in polyethylene films may cause an increased
 substance permeability in hollow bodies, improved water vapor
 permeability, improved antistatic behavior, improved anti-block behavior
 as well as improved printability with aqueous dyes.
 Another possibility is the use of the starch in polyurethane foams. Due to
 the adaptation of starch derivatives as well as due to the optimization of
 processing techniques, it is possible to specifically control the reaction
 between synthetic polymers and the starch's hydroxy groups. The results
 are polyurethane films having the following property profiles due to the
 use of starch: a reduced coefficient of thermal expansion, decreased
 shrinking behavior, improved pressure/tension behavior, increased water
 vapor permeability without a change in water acceptance, reduced
 flammability and cracking density, no drop off of combustible parts, no
 halides and reduced aging. Disadvantages that presently still exist are
 reduced pressure and impact strength.
 Product development of film is not the only option. Also solid plastics
 products, such as pots, plates and bowls can be produced by means of a
 starch content of more than 50%. Furthermore, the starch/polymer mixtures
 offer the advantage that they are much easier biodegradable.
 Furthermore, due to their extreme capability to bind water, starch graft
 polymers have gained utmost importance. These are products having a
 backbone of starch and a side lattice of a synthetic monomer grafted on
 according to the principle of radical chain mechanism. The starch graft
 polymers available nowadays are characterized by an improved binding and
 retaining capability of up to 1000 g water per g starch at a high
 viscosity. These super absorbers are used mainly in the hygiene field,
 e.g. in products such as diapers and sheets, as well as in the
 agricultural sector, e.g. in seed pellets.
 What is decisive for the use of the novel starch modified by recombinant
 DNA techniques are, on the one hand, structure, water content, protein
 content, lipid content, fiber content, ashes/phosphate content,
 amylose/amylopectin ratio, distribution of the relative molar mass, degree
 of branching, granule size and shape as well as crystallization, and on
 the other hand, the properties resulting in the following features: flow
 and sorption behavior, pastification temperature, viscosity, thickening
 performance, solubility, paste structure, transparency, heat, shear and
 acid resistance, tendency to retrogradation, capability of gel formation,
 resistance to freezing/thawing, capability of complex formation, iodine
 binding, film formation, adhesive strength, enzyme stability,
 digestibility and reactivity.
 What is decisive for the use of the novel starch modified by recombinant
 DNA techniques are, on the one hand, structure, water content, protein
 content, lipid content, fiber content, ashes/phosphate content,
 amylose/amylopectin ratio, distribution of the relative molar mass, degree
 of branching, granule size and shape as well as crystallization, and on
 the other hand, the properties resulting in the following features: flow
 and sorption behavior, pastification temperature, viscosity, thickening
 performance, solubility, paste structure, transparency, heat, shear and
 acid resistance, tendency to retrogradation, capability of gel formation,
 resistance to freezing/thawing, capability of complex formation, iodine
 binding, film formation, adhesive strength, enzyme stability,
 digestibility and reactivity.
 The production of modified starch by genetically operating with a
 transgenic plant may modify the properties of the starch obtained from the
 plant in such a way as to render further modifications by means of
 chemical or physical methods superfluous. On the other hand, the starches
 modified by means of recombinant DNA techniques might be subjected to
 further chemical modification, which will result in further improvement of
 the quality for certain of the above-described fields of application.
 These chemical modifications are principally known to the person skilled
 in the art. These are particularly modifications by means of
 heat treatment
 acid treatment
 oxidation and
 esterification
 leading to the formation of phosphate, nitrate, sulfate, xanthate, acetate
 and citrate starches. Other organic acids may also be used for the
 esterification:
 formation of starch ethers
 starch alkyl ether, O-allyl ether, hydroxylalkyl ether, O-carboxylmethyl
 ether, N-containing starch ethers, P-containing starch ethers and
 S-containing starch ethers.
 formation of branched starches
 formation of starch graft polymers.
 Furthermore, the present invention relates to the use of the nucleic acid
 molecules of the invention for producing plants synthesizing an
 amylopectin starch with a modified degree of branching in comparison to
 wildtype plants.
 A further subject matter of the present invention is the use of the nucleic
 acid molecules of the invention or parts thereof or, as the case may be,
 of the reverse complements thereof in order to identify and isolate from
 plants or other organisms homologous molecules encoding proteins with the
 enzymatic activity of a debranching enzyme or fragments of such proteins.
 For the term "homology", please refer to the above definition.
 In principle, the nucleic acid molecules of the invention may also be used
 in order to produce plants in which the activity of the debranching enzyme
 of the invention is increased or reduced and in which at the same time the
 activities of other enzymes involved in the starch biosynthesis are
 modified. In this regard, all kinds of combinations and permutations are
 conceivable. For example, nucleic acid molecules encoding a protein of the
 invention, or corresponding antisense- constructs may be introduced into
 plant cells in which the synthesis of endogeneous debranching enzymes,
 GBSS I-, SSS I-, II- or GBSS Il-proteins is already inhibited due to an
 antisense-effect or a mutation, or in which the synthesis of the branching
 enzyme is inhibited (as described e.g. WO92/14827 or in connection with
 the ae mutant of maize (Shannon and Garwood, 1984, in Whistler, BeMiller
 and Paschall, Starch: Chemistry and Technology, Academic Press, London,
 2.sup.nd edition (1984) 25-86)).
 If the inhibition of the synthesis of several debranching enzymes in
 transformed plants is to be achieved, DNA molecules can be used for
 transformation, which at the same time contain several regions in
 antisense-orientation encoding the respective debranching enzymes and
 which are controlled by a suitable promoter. In such constructs, each
 sequence may alternatively be controlled by its own promoter or else the
 sequences may be transcribed as a fusion from a common promoter. The last
 alternative will generally be preferred as in this case the synthesis of
 the respective proteins should be inhibited to approximately the same
 extent.
 Furthermore, it is possible to construct molecules in which, apart from
 sequences encoding debranching enzymes, other DNA sequences are present
 encoding other proteins involved in the starch synthesis or modification.
 These are linked in antisense orientation to a suitable promoter. Again,
 the sequences may be connected up in series and be transcribed from a
 common promoter or each may be transcribed by a promoter of its own. For
 the length of the coding regions used in such a construct the same applies
 as already set forth above for the antisense constructs. There is no upper
 limit for the number of antisense fragments transcribed from one promoter
 in such a DNA molecule. The resulting transcript, however, should usually
 not be longer than 20 kb, preferably not longer than 5 kb. Coding regions
 which are located in antisense orientation downstream of a suitable
 promoter in such DNA molecules in combination with other coding regions
 may be derived from DNA sequences encoding the following proteins:
 granule-bound starch synthases (GBSS I and II), and soluble starch
 synthases (e.g. SSS I and II), branching enzymes, other debranching
 enzymes, disproportionizing enzymes and starch phosphorylases. This
 enumeration merely serves as an example. The use of other DNA sequences
 within the framework of such a combination is also conceivable.
 By means of such constructs it is possible to inhibit the synthesis of
 several enzymes at the same time within the plant cells transformed with
 these constructs. Furthermore, the constructs may be introduced into
 classical mutants which are defective for one or more genes of the starch
 biosynthesis. These defects may be related to the following proteins:
 granule-bound (GBSS I and II) and soluble starch synthases (e.g. SSS I and
 II), branching enzymes (BE I and II), debranching enzymes,
 disproportionizing enzymes and starch phosphorylases. Again, this
 enumeration merely serves as an example.
 In order to prepare the introduction of foreign genes into higher plants a
 high number of cloning vectors are at disposal, containing a replication
 signal for E.coli and a marker gene for the selection of transformed
 bacterial cells. Examples for such vectors are pBR322, pUC series, M13mp
 series, pACYC184 etc. The desired sequence may be integrated into the
 vector at a suitable restriction site. The obtained plasmid is used for
 the transformation of E.coli cells. Transformed E.coli cells are
 cultivated in a suitable medium and subsequently harvested and lysed. The
 plasmid is recovered. As an analyzing method for the characterization of
 the obtained plasmid DNA use is generally made of restriction analysis,
 gel electrophoresis and other biochemico-molecularbiological methods.
 After each manipulation the plasmid DNA may be cleaved and the obtained
 DNA fragments may be linked to other DNA sequences. Each plasmid DNA may
 be cloned into the same or in other plasmids.
 In order to introduce DNA into a plant host cell a wide range of techniques
 are at disposal. These techniques comprise the transformation of plant
 cells with T-DNA by using Agrobacterium tumefaciens or Agrobacterium
 rhizogenes as transformation medium, the fusion of protoplasts, the
 injection and the electroporation of DNA, the introduction of DNA by means
 of the biolistic method as well as further possibilities. In the case of
 injection and electroporation of DNA into plant cells, there are no
 special demands made to the plasmids used. Simple plasmids such as pUC
 derivatives may be used. However, in case that whole plants are to be
 regenerated from cells transformed in such a way, a selectable marker gene
 should preferably be present.
 Depending on the method of introducing desired genes into the plant cell,
 further DNA sequences may be necessary. If the Ti- or Ri-plasmid is used
 e.g. for the transformation of the plant cell, at least the right border,
 more preferably, however, the right and left border of the Ti- and
 Ri-plasmid T-DNA should be connected to the foreign gene to be introduced
 as a flanking region.
 If Agrobacteria are used for the transformation, the DNA which is to be
 integrated should be cloned into special plasmids, namely either into an
 intermediate vector or into a binary vector. Due to sequences homologous
 to the sequences within the T-DNA, the intermediate vectors may be
 integrated into the Ti- or Ri-plasmid of the Agrobacterium due to
 homologous recombination. This also contains the vir-region necessary for
 the transfer of the T-DNA. Intermediate vectors cannot replicate in
 Agrobacteria. By means of a helper plasmid the intermediate vector may be
 transferred to Agrobacterium tumefaciens (conjugation). Binary vectors may
 replicate in E.coli as well as in Agrobacteria. They contain a selectable
 marker gene as well as a linker or polylinker which is framed by the right
 and the left T-DNA border region. They may be transformed directly into
 the Agrobacteria (Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187).
 The Agrobacterium acting as host cell should contain a plasmid carrying a
 vir-region. The vir-region is necessary for the transfer of the T-DNA into
 the plant cell. Additional T-DNA may be present. The Agrobacterium
 transformed in such a way is used for the transformation of plant cells.
 The use of T-DNA for the transformation of plant cells was investigated
 intensely and described sufficiently in EP 120 516; Hoekema, In: The
 Binary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam
 (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and An
 et al. EMBO J. 4 (1985), 277-287.
 For transferring the DNA into the plant cells, plant explants may suitably
 be co-cultivated with Agrobacterium tumefaciens or Agrobacterium
 rhizogenes. From the infected plant material (e.g. pieces of leaves, stem
 segments, roots, but also protoplasts or suspension-cultivated plant
 cells) whole plants may then be regenerated in a suitable medium which may
 contain antibiotics or biozides for the selection of transformed cells.
 The plants obtained in such a way may then be examined as to whether the
 introduced DNA is present or not. Other possibilities in order to
 introduce foreign DNA by using the biolistic method or by transforming
 protoplasts are known to the skilled person (cf. e.g. Willmitzer, L., 1993
 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive
 Treatise (H. J. Rehm, G. Reed, A. PUhler, P. Stadler, editors), Vol. 2,
 627-659, VCH Weinheim-New York-Basel-Cambridge).
 Whereas the transformation of dicotyledonous plants via Ti-plasmid vector
 systems by means of Agrobacterium tumefaciens is well established, more
 recent studies indicate that also monocotyledonous plants may be suitable
 for the transformation by means of vectors based on Agrobacterium (Chan et
 al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994),
 271-282, Deng et al., Science in China 33 (1990), 28-34; Wilmink et al,
 Plant Cell Reports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995),
 486-492; Conner and Domisse; Int. J. Plant Sci. 153 (1992), 550-555;
 Ritchie et al., Transgenic Res. 2 (1993), 252-265).
 Alternative Systems for the transformation of monocotyledonous plants are
 the transformation by means of a biolistic approach (Wan and Lemaux, Plant
 Physiol. 104 (1994), 37-48; Vasil et al., Bio/Technology 11 (1993),
 1553-1558; Ritala et al., Plant Mol. Biol. 24 (1994), 317-325; Spencer et
 al., Theor. Appl. Gent. 79 (1990), 625-631), protoplast transformation,
 the electroporation of partially permeabilized cells, the introduction of
 DNA by means of glass fibers.
 There are various references in the relevant literature dealing
 specifically with the transformation of maize (cf. e.g. WO95/06128, EP 0
 513 849; EP 0 465 875; Fromm et al., Biotechnology 8 (1990), 833-844;
 Gordon-Kamm et al., Plant Cell 2 (1990), 603-618; Koziel et al.,
 Biotechnology 11 (1993), 194-200). In EP 292 435 a method is described by
 means of which fertile plants may be obtained starting from mucousless,
 friable granulous maize callus. In this context it was furthermore
 observed by Shillito et al. (Bio/Technology 7 (1989), 581) that for
 regenerating fertile plants it is necessary to start from
 callus-suspension cultures from which a culture of dividing protoplasts
 can be produced which is capable to regenerate to plants. After an in
 vitro cultivation period of 7 to 8 months Shillito et al. obtain plants
 with viable descendants which, however, exhibited abnormalities in
 morphology and reproductivity.
 Prioli and Sondahl (Bio/Technology 7 (1989), 589) have described how to
 regenerate and to obtain fertile plants from maize protoplasts of the
 Cateto maize inbreed line Cat 100-1. The authors assume that the
 regeneration of protoplast to fertile plants depends on a number of
 various factors such as the genotype, the physiological state of the
 donor-cell and the cultivation conditions. The successful transformation
 of other cereals has by now also been described, such as for barley (Wan
 and Lemaux, loc. cit.; Ritala et al., loc. cit.) and for wheat (Nehra et
 al., Plant J. 5 (1994), 285-297).
 Once the introduced DNA has been integrated in the genome of the plant
 cell, it usually continues to be stable there and also remains within the
 descendants of the originally transformed cell. It usually contains a
 selectable marker which confers resistance against biozides or against an
 antibiotic such as kanamycin, G 418, bleomycin, hygromycin or
 phosphinotricine etc. to the transformed plant cells. The individually
 selected marker should therefore allow for a selection of transformed
 cells against cells lacking the introduced DNA.
 The transformed cells grow in the usual way within the plant (see also
 McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting
 plants can be cultivated in the usual way and cross-bred with plants
 having the same transformed genetic heritage or another genetic heritage.
 The resulting hybrid individuals have the corresponding phenotypic
 properties. Seeds may be obtained from the plant cells.
 Two or more generations should be grown in order to ensure whether the
 phenotypic feature is kept stably and whether it is transferred.
 Furthermore, seeds should be harvested in order to ensure that the
 corresponding phenotype or other properties will remain.
 The examples illustrate the invention.
 In the examples the following methods were used.
 1. Cloning Methods
 For cloning in E.coli the vector pBluescript II SK (Stratagene) was used.
 2. Bacterial Strains
 For the Bluescript vector and for the pUSP constructs use was made of the
 E.coli strain DH5.alpha. (Bethesda Research Laboratories, Gaithersburgh,
 USA). The E.coli strain XL1 -Blue was used for in vivo excision.
 3. Radioactive Labeling of DNA Fragments
 The radioactive labeling of DNA fragments was carried out by means of a
 DNA-Random Primer Labeling Kits by Boehringer (Germany) according to the
 manufacturer's instructions.

EXAMPLE 1
 Cloning of a cDNA Encoding a Novel Debranching Enzyme from Solanum
 Tuberosum
 In order to isolate cDNA molecules encoding a novel debranching enzyme from
 Solanum tuberosum, a cDNA library was constructed within the vector Lambda
 ZAPII (Stratagene) starting from polyA.sup.+ RNA from tuber material and
 packed into phage heads. E.coli cells of the XL1 Blue strain were
 subsequently infected with the phages containing the cDNA fragments
 (1.times.10.sup.6 pfu) and plated on medium in Petri dishes with a
 densitiy of approximately 30,000 per 75 cm.sup.2. After an 8-hour
 incubation, nitrocellulose membranes were put on the lysed bacteria and
 removed after one minute. The filters were first incubated in 0.5 M NaOH;
 1.5 M NaCl for 2 minutes and then in 0.5 M Tris/HCl pH 7.0 for 2 minutes
 and finally in 2.times.SSC for 2 minutes. After drying and fixing the DNA
 by means of UV crosslinking, the filters were incubated in hybridization
 buffer for 3 hours at 48.degree. C. before a radioactively labeled probe
 was added.
 As a probe, use was made of a cDNA from maize encoding a debranching enzyme
 (see James et al., Plant Cell 7 (1995), 417-429, nucleotide 1150-2128).
 The hybridization was carried out in 2.times.SSC, 10.times.Dehnhardt's
 solution; 50 mM Na.sub.2 HPO.sub.4, pH 7.2; 0.2% SDS; 5 mM EDTA and 250
 pg/ml denatured herring sperm DNA at 48.degree. C.
 Hybridizing phage clones were singled out and further purified by means of
 standard methods. By means of in vivo excision E.coli clones were obtained
 from positive phage clones. The E.coli clones contained a double-stranded
 pBluescript plasmid with the respective cDNA insertions. After examining
 the size and the restriction pattern of the insertion, plasmid DNA was
 isolated from suitable clones. Iso5, a plasmid isolated in such a way,
 contained an insertion of 2295 bp.
 EXAMPLE 2
 Sequence Analysis of the cDNA Insert of the Plasmid Iso5
 In the case of the plasmid Iso5, which was isolated as described in Example
 1, the nucleotide sequence of the cDNA insert was determined in a standard
 routine by means of the didesoxynucleotide-method (Sanger et al., Proc.
 Natl. Acad. Sci. USA 74 (1977), 5463-5467). The insert has a length of
 2295 bp. The nucleotide sequence of 2133 bp of this insert and the derived
 amino acid sequence are indicated under Seq ID No. 1.
 Homology comparisons showed that the encoded protein was a novel
 debranching enzyme from potato.
 The nucleotide sequence depicted under SEQ ID No. 1 represents a partial
 cDNA encoding a so far unknown debranching enzyme from potato. By means of
 this sequence it is possible to isolate a complete cDNA sequence or a
 genomic sequence from suitable cDNA or genomic libraries by means of
 standard techniques.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 2
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 2133
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Solanum tuberosum
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (2)..(1819)
 &lt;223&gt; OTHER INFORMATION: Clone Iso5
 &lt;400&gt; SEQUENCE: 1
 g aat tcg gca cga ggg cca gag gat gat tgt tgg ccc cca atg gca ggc 49
 Asn Ser Ala Arg Gly Pro Glu Asp Asp Cys Trp Pro Pro Met Ala Gly
 1 5 10 15
 atg gta cct tct gct tct gat cag ttt gat tgg gaa gga gat cta tta 97
 Met Val Pro Ser Ala Ser Asp Gln Phe Asp Trp Glu Gly Asp Leu Leu
 20 25 30
 ctg aag ttt cca cag aga gat ctt gta atc tat gaa atg cat gtt cgt 145
 Leu Lys Phe Pro Gln Arg Asp Leu Val Ile Tyr Glu Met His Val Arg
 35 40 45
 gga ttt aca aat cat gag tcg agt gaa aca aaa tat cct ggt act tac 193
 Gly Phe Thr Asn His Glu Ser Ser Glu Thr Lys Tyr Pro Gly Thr Tyr
 50 55 60
 ctt ggt gtt gtg gag aaa ctt gat cac ttg aag gaa ctt ggt gtc aac 241
 Leu Gly Val Val Glu Lys Leu Asp His Leu Lys Glu Leu Gly Val Asn
 65 70 75 80
 tgt ata gag cta atg ccc tgt cac gag ttc aat gag ctg gag tac tat 289
 Cys Ile Glu Leu Met Pro Cys His Glu Phe Asn Glu Leu Glu Tyr Tyr
 85 90 95
 agt tat aac tct gta ttg ggc gac tac aag ttt aac ttt tgg ggc tat 337
 Ser Tyr Asn Ser Val Leu Gly Asp Tyr Lys Phe Asn Phe Trp Gly Tyr
 100 105 110
 tct act gtc aat ttc ttt tct cca atg gga aga tac tcg tct gct ggt 385
 Ser Thr Val Asn Phe Phe Ser Pro Met Gly Arg Tyr Ser Ser Ala Gly
 115 120 125
 cta agt aat tgc ggc ctc ggt gca ata aac gaa ttt aag tat ctt gtc 433
 Leu Ser Asn Cys Gly Leu Gly Ala Ile Asn Glu Phe Lys Tyr Leu Val
 130 135 140
 aag gaa gca cat aaa cgt gga atc gag gtt atc atg gat gtt gtt ttc 481
 Lys Glu Ala His Lys Arg Gly Ile Glu Val Ile Met Asp Val Val Phe
 145 150 155 160
 aat cac act gct gaa gga aat gaa aat ggt ccc ata cta tca ttt aga 529
 Asn His Thr Ala Glu Gly Asn Glu Asn Gly Pro Ile Leu Ser Phe Arg
 165 170 175
 ggc att gac aac agt gtg ttt tat acg cta gct cct aag ggt gaa ttt 577
 Gly Ile Asp Asn Ser Val Phe Tyr Thr Leu Ala Pro Lys Gly Glu Phe
 180 185 190
 tac aac tac tca gga tgt gga aat acc ttc aac tgt aat aat ccc att 625
 Tyr Asn Tyr Ser Gly Cys Gly Asn Thr Phe Asn Cys Asn Asn Pro Ile
 195 200 205
 gta cgt caa ttt ata gtg gat tgc ttg aga tat tgg gtt acc gaa atg 673
 Val Arg Gln Phe Ile Val Asp Cys Leu Arg Tyr Trp Val Thr Glu Met
 210 215 220
 cac gta gat ggc ttc cgc ttt gat ctt gct tct atc ctt aca aga agt 721
 His Val Asp Gly Phe Arg Phe Asp Leu Ala Ser Ile Leu Thr Arg Ser
 225 230 235 240
 agc agc tcg tgg aat gct gta aat gtc tat gga aat tca att gac ggt 769
 Ser Ser Ser Trp Asn Ala Val Asn Val Tyr Gly Asn Ser Ile Asp Gly
 245 250 255
 gac atg atc acc aca ggc act cct ctc aca agc cca cca ttg att gat 817
 Asp Met Ile Thr Thr Gly Thr Pro Leu Thr Ser Pro Pro Leu Ile Asp
 260 265 270
 atg att agc aat gat cca ata ctt agt gga gta aag ctt ata gct gaa 865
 Met Ile Ser Asn Asp Pro Ile Leu Ser Gly Val Lys Leu Ile Ala Glu
 275 280 285
 gca tgg gat tgt gga ggc ctt tac caa gtt ggc atg ttt ccg cac tgg 913
 Ala Trp Asp Cys Gly Gly Leu Tyr Gln Val Gly Met Phe Pro His Trp
 290 295 300
 ggt atc tgg tcg gag tgg aac gga aag tac cgt gac atg gta cgt cag 961
 Gly Ile Trp Ser Glu Trp Asn Gly Lys Tyr Arg Asp Met Val Arg Gln
 305 310 315 320
 ttc atc aaa ggc act gat ggg ttt tct ggg gct ttt gct gaa tgc ctt 1009
 Phe Ile Lys Gly Thr Asp Gly Phe Ser Gly Ala Phe Ala Glu Cys Leu
 325 330 335
 tgt gga agc cca aat cta tac cag aaa gga gga aga aaa cca tgg aac 1057
 Cys Gly Ser Pro Asn Leu Tyr Gln Lys Gly Gly Arg Lys Pro Trp Asn
 340 345 350
 agt ata aat ttc gtg tgt gcc cac gat ggt ttt act ttg gct gat tta 1105
 Ser Ile Asn Phe Val Cys Ala His Asp Gly Phe Thr Leu Ala Asp Leu
 355 360 365
 gtg aca tac aac aat aaa cac aat ttg gca aat gga gag gac aac aaa 1153
 Val Thr Tyr Asn Asn Lys His Asn Leu Ala Asn Gly Glu Asp Asn Lys
 370 375 380
 gat ggg gag aat cac aat aat agt tgg aat tgt ggc gag gaa gga gaa 1201
 Asp Gly Glu Asn His Asn Asn Ser Trp Asn Cys Gly Glu Glu Gly Glu
 385 390 395 400
 ttt gca agt atc ttt gtg aag aaa ttg agg aaa aga caa atg cgg aac 1249
 Phe Ala Ser Ile Phe Val Lys Lys Leu Arg Lys Arg Gln Met Arg Asn
 405 410 415
 ttc ttc ctc tgc ctt atg gtt tcc caa ggt gtt ccc atg ata tat atg 1297
 Phe Phe Leu Cys Leu Met Val Ser Gln Gly Val Pro Met Ile Tyr Met
 420 425 430
 ggt gat gaa tat ggt cac act aag gga gga aac aac aac acg tat tgc 1345
 Gly Asp Glu Tyr Gly His Thr Lys Gly Gly Asn Asn Asn Thr Tyr Cys
 435 440 445
 cat gac aat tat att aat tac ttc cgt tgg gat aag aag gat gaa tct 1393
 His Asp Asn Tyr Ile Asn Tyr Phe Arg Trp Asp Lys Lys Asp Glu Ser
 450 455 460
 tca tct gat ttt ttg aga ttt tgc ggc ctc atg acc aaa ttc cgc cat 1441
 Ser Ser Asp Phe Leu Arg Phe Cys Gly Leu Met Thr Lys Phe Arg His
 465 470 475 480
 gaa tgt gaa tca ctg gga tta gat ggt ttc cct aca gca gaa agg ctg 1489
 Glu Cys Glu Ser Leu Gly Leu Asp Gly Phe Pro Thr Ala Glu Arg Leu
 485 490 495
 caa tgg cat ggt cac act cct aga act cca gat tgg tct gaa aca agt 1537
 Gln Trp His Gly His Thr Pro Arg Thr Pro Asp Trp Ser Glu Thr Ser
 500 505 510
 cga ttc gtt gca ttt aca ctg gtc gac aaa gtg aag gga gaa cta tat 1585
 Arg Phe Val Ala Phe Thr Leu Val Asp Lys Val Lys Gly Glu Leu Tyr
 515 520 525
 att gcc ttt aac gcc agc cat ttg cct gta acg att aca ctt cca gaa 1633
 Ile Ala Phe Asn Ala Ser His Leu Pro Val Thr Ile Thr Leu Pro Glu
 530 535 540
 aag cct ggt tat aga tgg cag ccg ttt gtg gac aca ggc aaa cca gca 1681
 Lys Pro Gly Tyr Arg Trp Gln Pro Phe Val Asp Thr Gly Lys Pro Ala
 545 550 555 560
 cca ttt gac ttc ctg aca gac gat gtt cct gag aga gag aca gca gcc 1729
 Pro Phe Asp Phe Leu Thr Asp Asp Val Pro Glu Arg Glu Thr Ala Ala
 565 570 575
 aaa caa tat tct cat ttt ctg gac gcg aac cag tat ccg atg ctc agt 1777
 Lys Gln Tyr Ser His Phe Leu Asp Ala Asn Gln Tyr Pro Met Leu Ser
 580 585 590
 tat tca tcc att att ctt tta cta tca tct gct gat gat gcg 1819
 Tyr Ser Ser Ile Ile Leu Leu Leu Ser Ser Ala Asp Asp Ala
 595 600 605
 tagtttcatt caacaagcca ggtgaggtaa agcagcttca gattttgtta tatgcagtga 1879
 ggtgttactt tgtaaataaa gtaagaaaca ggacagaaca gaactgcaaa cagatagaac 1939
 tggtgaggaa gaagctgatg atttataaga tacaccttgt attataattg tatttatata 1999
 aaataaaaaa aaaaaactag tgaacttgtc tgtgcgaaat aaaatgtata gttgatttca 2059
 aaaaaaaaaa aaaaaaaaaa aaaaaaactc gagctctctc tctctctctc tctctctctc 2119
 tctctctctc tctc 2133
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 606
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Solanum tuberosum
 &lt;400&gt; SEQUENCE: 2
 Asn Ser Ala Arg Gly Pro Glu Asp Asp Cys Trp Pro Pro Met Ala Gly
 1 5 10 15
 Met Val Pro Ser Ala Ser Asp Gln Phe Asp Trp Glu Gly Asp Leu Leu
 20 25 30
 Leu Lys Phe Pro Gln Arg Asp Leu Val Ile Tyr Glu Met His Val Arg
 35 40 45
 Gly Phe Thr Asn His Glu Ser Ser Glu Thr Lys Tyr Pro Gly Thr Tyr
 50 55 60
 Leu Gly Val Val Glu Lys Leu Asp His Leu Lys Glu Leu Gly Val Asn
 65 70 75 80
 Cys Ile Glu Leu Met Pro Cys His Glu Phe Asn Glu Leu Glu Tyr Tyr
 85 90 95
 Ser Tyr Asn Ser Val Leu Gly Asp Tyr Lys Phe Asn Phe Trp Gly Tyr
 100 105 110
 Ser Thr Val Asn Phe Phe Ser Pro Met Gly Arg Tyr Ser Ser Ala Gly
 115 120 125
 Leu Ser Asn Cys Gly Leu Gly Ala Ile Asn Glu Phe Lys Tyr Leu Val
 130 135 140
 Lys Glu Ala His Lys Arg Gly Ile Glu Val Ile Met Asp Val Val Phe
 145 150 155 160
 Asn His Thr Ala Glu Gly Asn Glu Asn Gly Pro Ile Leu Ser Phe Arg
 165 170 175
 Gly Ile Asp Asn Ser Val Phe Tyr Thr Leu Ala Pro Lys Gly Glu Phe
 180 185 190
 Tyr Asn Tyr Ser Gly Cys Gly Asn Thr Phe Asn Cys Asn Asn Pro Ile
 195 200 205
 Val Arg Gln Phe Ile Val Asp Cys Leu Arg Tyr Trp Val Thr Glu Met
 210 215 220
 His Val Asp Gly Phe Arg Phe Asp Leu Ala Ser Ile Leu Thr Arg Ser
 225 230 235 240
 Ser Ser Ser Trp Asn Ala Val Asn Val Tyr Gly Asn Ser Ile Asp Gly
 245 250 255
 Asp Met Ile Thr Thr Gly Thr Pro Leu Thr Ser Pro Pro Leu Ile Asp
 260 265 270
 Met Ile Ser Asn Asp Pro Ile Leu Ser Gly Val Lys Leu Ile Ala Glu
 275 280 285
 Ala Trp Asp Cys Gly Gly Leu Tyr Gln Val Gly Met Phe Pro His Trp
 290 295 300
 Gly Ile Trp Ser Glu Trp Asn Gly Lys Tyr Arg Asp Met Val Arg Gln
 305 310 315 320
 Phe Ile Lys Gly Thr Asp Gly Phe Ser Gly Ala Phe Ala Glu Cys Leu
 325 330 335
 Cys Gly Ser Pro Asn Leu Tyr Gln Lys Gly Gly Arg Lys Pro Trp Asn
 340 345 350
 Ser Ile Asn Phe Val Cys Ala His Asp Gly Phe Thr Leu Ala Asp Leu
 355 360 365
 Val Thr Tyr Asn Asn Lys His Asn Leu Ala Asn Gly Glu Asp Asn Lys
 370 375 380
 Asp Gly Glu Asn His Asn Asn Ser Trp Asn Cys Gly Glu Glu Gly Glu
 385 390 395 400
 Phe Ala Ser Ile Phe Val Lys Lys Leu Arg Lys Arg Gln Met Arg Asn
 405 410 415
 Phe Phe Leu Cys Leu Met Val Ser Gln Gly Val Pro Met Ile Tyr Met
 420 425 430
 Gly Asp Glu Tyr Gly His Thr Lys Gly Gly Asn Asn Asn Thr Tyr Cys
 435 440 445
 His Asp Asn Tyr Ile Asn Tyr Phe Arg Trp Asp Lys Lys Asp Glu Ser
 450 455 460
 Ser Ser Asp Phe Leu Arg Phe Cys Gly Leu Met Thr Lys Phe Arg His
 465 470 475 480
 Glu Cys Glu Ser Leu Gly Leu Asp Gly Phe Pro Thr Ala Glu Arg Leu
 485 490 495
 Gln Trp His Gly His Thr Pro Arg Thr Pro Asp Trp Ser Glu Thr Ser
 500 505 510
 Arg Phe Val Ala Phe Thr Leu Val Asp Lys Val Lys Gly Glu Leu Tyr
 515 520 525
 Ile Ala Phe Asn Ala Ser His Leu Pro Val Thr Ile Thr Leu Pro Glu
 530 535 540
 Lys Pro Gly Tyr Arg Trp Gln Pro Phe Val Asp Thr Gly Lys Pro Ala
 545 550 555 560
 Pro Phe Asp Phe Leu Thr Asp Asp Val Pro Glu Arg Glu Thr Ala Ala
 565 570 575
 Lys Gln Tyr Ser His Phe Leu Asp Ala Asn Gln Tyr Pro Met Leu Ser
 580 585 590
 Tyr Ser Ser Ile Ile Leu Leu Leu Ser Ser Ala Asp Asp Ala
 595 600 605