AU2007237226A1 - Plants with improved morphogenesis and method of constructing the same - Google Patents

Description

P/o00/0o Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Plants with improved morphogenesis and method of constructing the same The following statement is a full description of this invention, including the best method of performing it known to us:

SPECIFICATION

PLANTS WITH IMPROVED MORPHOGENESIS AND ^Z METHOD OF CONSTRUCTING THE SAME 00

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TECHNICAL FIELD The present invention relates to plants with various

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types of improved morphogenesis, in particular to plants with c N morphogenetically improved stems, leaves, flowers, ovaries (pods, fruit) and seeds. The present invention further

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relates a method of producing such plants.

BACKGROUND ART Plants adapt to various types of environmental factors of their roots. For example, plants have a capacity to change shapes corresponding to the environment of growth and development. Once a plant has sent down roots in a given place, if the environment of that place somehow becomes unsuitable to that plant, the plant cannot move, but adapts and continues to grow and develop by changing its own shape.

The ability to change shapes is an essential condition of survival for a plant.

Cross-breeding, breeding using recent genetic engineering techniques, and methods utilizing the action of plant hormones and plant regulators have been implemented in order to improve various types of morphological modification.

Numerous studies have been conducted using Arabidopsis thaliana in order to understand the control mechanisms of the morphogenesis and morphological modification of plants.

There have been reports of LEAFY (LFY), APETALA1 (API), AGAMOUS SUPERMAN (SUP) and FIL (FIL) as genes that 2 participate in flower formation. There have been reports of TCH4 and dbp, etc. as genes that participate in stem 0 Z formation. There have been reports of LAN1 and MAC as genes 00 0, that participate in leaf formation. There have been reports of ttg, gl2, CPC and IRE as genes that participate in root

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eC- formation.

c- Plants with improved morphological modification and (cN morphogenesis have already been created using genetic Sengineering techniques. There have been reports of morphological changes of the flowers of petunia by introducing a transcription factor (PetSPL3) having a zinc finger motif (Japanese Enexamined Patent Publication No.

262390/1999). There have been reports of morphological changes of the roots of Arabidopsis thaliana by introducing a CPC gene or an IRE gene (Japanese Enexamined Patent Publication No. 4978/1998, Japanese Enexamined Patent Publication No. 270873/2000). However, most plants in which these genes have been transgenetically modified have not actually obtained sufficient effect to have industrial applicability, and currently a practical level has not yet been reached.

Polyamines, the general term for aliphatic hydrocarbons with 2 or more primary amino groups, are ubiquitous natural substances in organisms, with more than 20 types discovered so far. Typical polyamines include putrescine, spermidine, and spermine. The known primary physiological action of polyamines includes nucleic acid stabilization and structural modification through interaction with nucleic acids; promotion of various nucleic acid synthesis systems; activation of protein synthesis systems; and (4) stabilization of cell membranes and enhancement of membrane

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Z permeability of substances.

00 00 Reports on the role of polyamines in plants include cell protection and promotion of nucleic acid or protein

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C-q biosynthesis during cellular growth or division. The

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involvement of polyamines in various types of environmental C-i stress has recently attracted attention.

0 There have been several reports on morphological modification and polyamine relating to somatic embryogenesis and adventitious root formation. For example, it has been demonstrated that polyamine promotes somatic embryogenesis in carrots (Planta, 162, 532, 1984, Science, 223, 1433, 1984), oats (Plant Growth Reg., 3, 329, 1985), Chinese cabbage (Plant Sci., 56, 167, 1988), and petunia (Plant Sci., 62, 123, 1989), and promotes adventitious root formation in mung beans (Physiol. Plant., 64, 53, 1985, Plant Cell Physiol., 24, 677, 1983). Thus far, there have been hardly any reports on the participation of polyamine in the morphogenesis of flowers, stems, leaves, and ovaries.

Known polyamine metabolism-related enzymes involved in the biosynthesis of plant polyamines include arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), spermidine synthase (SPDS), and spermine synthase (SPMS). Several polyamine metabolism-related enzyme genes coding for such polyamine metabolism-related enzymes have already been isolated from plants. The ADC gene has been isolated from oats (Mol. Gen. Genet., 224, 431-436 (1990)), tomatoes (Plant 004823679 4 Cq Physiol., 103, 829-834 (1993)), Arabidopsis thaliana (Plant SPhysiol., 111, 1077-1083 (1996)), and peas (Plant Mol. Bioil., Z 28, 997-1009 (1995)); the ODC gene has been isolated from 00 0, datura (Biochem. 314, 241-248 (1996)); the SAMDC gene has been isolated from potatoes (Plant Mol. Biol., 26, 327-338 O (1994)), spinach (Plant Physiol., 107, 1461-1462 (1995)), and C tobacco; and the SPDS gene has been isolated from Arabidopsis thaliana (Plant Cell Physiol., 39(1), 73-79 (1998)).

0 The present invention relates to recombinant plants C 10 with various types of improved morphogenesis by artificially controlling the expression of polyamine metabolism-related enzyme genes, and by varying the polyamine levels.

Brief Description of the Invention Fig. 1 indicates the structure of an expression construct containing a polyamine metabolism-related enzyme gene; Fig. 2 indicates the results of expression of the Cucurbita ficifolia Bouche SPDS gene in transformants. In figure 2, 1. wild type 2. transformant (TSP-14); 3.

transformant (TSP-15); 4. transformant (TSP-16); transformant (TSP-17); 6. transformant (TSP-19).

Fig. 3 indicates a comparison of the number of stems of a wild type plant and of a plant in which a polyamine metabolism-related enzyme gene has been introduced; Fig. 4 is a photograph indicating a comparison of the number of stems, leaves, flowers, ovaries and seeds of a wild type plant and of a plant in which a polyamine metabolismrelated enzyme gene has been introduced; Fig. 5 is a diagram indicating a comparison of the number of main stems of a wild type plant and of a plant in 004823679 c( which a polyamine metabolism-related enzyme gene has been introduced; Fig. 6 is a diagram indicating a comparison of the 00 0C number of side stems of a wild type plant and of a plant in which a polyamine metabolism-related enzyme gene has been

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eCq introduced; Fig. 7 is a diagram indicating a comparison of the day of blooming of a wild type plant and of a plant in which a polyamine metabolism-related enzyme gene has been introduced; Fig. 8 indicates the results of measuring the changes over time in the number of flowers per individual for an individual plant expressing a foreign gene; and Fig. 9 indicates the results of measuring the changes over time in the number of flowers per individual for individual expressing a foreign gene.

DISCLOSURE OF THE INVENTION As a result of extensive research, the inventors discovered that various parameters of morphogenesis, such as stems, leaves, flowers, ovaries (pods, fruit) and seeds (ovule) are improved when polyamine metabolism-related enzyme genes involved in polyamine biosynthesis are isolated and are introduced and overexpressed in plants so as to bring about changes in the polyamine level through the manipulation of polyamine metabolism.

Polyamines are basic substances containing an abundance of amines per molecule, typical examples of which include the diamine putrescine, the triamine spermidine, and the tetraamine spermine. Examples of polyamine metabolismrelated enzymes involved in the biosynthesis of such 004823679 6 c-ipolyamines include ADC and ODC for putrescine, SANDO and SPDS for spermidine, and SAMDC and SPMS for spermine. Polyamine 00

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005059126 7 S metabolism-related enzyme genes coding for such polyamine Cl metabolism-related enzymes have already been isolated in several O plants. Further, some polyamine metabolism-related enzyme genes Z are incorporated in plants. However, there have been no reports 00 C- 5 on improved morphogenesis of stems, leaves, flowers, ovaries and seeds of the resulting transgenic plants.

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CR1 As a result of extensive research in view of the foregoing to improve the morphogenesis of plants, the inventors discovered that S the content of the polyamines spermidine and spermine, in particular, is important for morphogenesis in plant. The inventors actually isolated and identified polyamine metabolismrelated enzyme genes (SPDS, SAMDC, ADC) involved in spermidine and spermine biosynthesis from plant tissue. The inventors perfected the present invention upon discovering that the introduction and over-expression of these genes in plants brought about changes in the polyamine level through the manipulation of polyamine metabolism, leading to improvement of various morphogenesis parameters with respect to stems, leaves, flowers, ovaries (pods, fruit) and seeds (ovules).

The present invention relates to a method for producing a plant that stably retains an exogenous S-adenosylmethionine decarboxylase (SAMDC) gene under the control of one or more promoters that can function within the plant, and that has improved morphogenesis compared to the plant lacking said exogenous SPDS gene, comprising the steps of: transforming cells of a plant lacking said exogenous SAMDC gene with said SAMDC gene, regenerating plants from the transformed cells, and cultivating the plant bodies under normal conditions; 005059126 7A S wherein the improved morphogenesis is selected from the group Cq consisting of:

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Z increased number of stems, main stems, side stems, flower 00 stalks, trunks or branches;

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(iv) extended flower blooming period;

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increased number of ovaries, fruit, cotton balls, pods, ears and capsules; (vi) increased development period from bearing to maturity of ovaries, fruit, pods and capsules; (vii) increased number of seeds, grains or ovules; and (viii) increased number of roots or tuberous roots.

The present invention further relates to a method for producing plants with improved morphogenesis compared to plants lacking an exogenous S-adenosylmethionine decarboxylase (SAMDC) gene, comprising the steps of: transforming cells of a plant lacking said exogenous SAMDC gene with at least one expression vector containing at least one exogenous SAMDC gene under the control of one or more promoters capable of functioning in plants, regenerating plants from the transformed cells, and cultivating the plant bodies under normal conditions; 005059126 7B 0 wherein the improved morphogenesis is selected from the group C-i consisting of:

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Z increased number of stems, main stems, side stems, flower 00 stalks, trunks or branches; ,O 5 (ii) increased number of leaves;

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c- S (iii) increased number of flowers;

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(iv) extended flower blooming period;

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increased number of ovaries, fruit, cotton balls, pods, ears and capsules; (vi) increased development period from bearing to maturity of ovaries, fruit, pods and capsules; (vii) increased number of seeds, grains or ovules; and (viii) increased number of roots or tuberous roots.

The present invention further relate to a method for producing a plant with fixed traits, which is a homozygote with respect to an exogenous S-adenosylmethionine decarboxylase (SAMDC) gene and which has improved morphogenesis compared to plants lacking said nucleic acid sequence, comprising the steps of: transforming cells of a plant lacking said exogenous SAMDC gene with at least one expression vector containing at least one said exogenous SAMDC gene under the control of one or more promoters capable of functioning in plants; regenerating plants with improved morphogenesis compared to plants lacking said exogenous SAMDC gene from the 005059126 0

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transformed cells; cultivating the plant bodies under normal conditions; harvesting seeds by pollination from the plant bodies; and assaying the nucleic acid sequence in the seeds obtained by pollination from the plant bodies, which have been obtained by cultivation of the seeds so as to select a homozygote with respect to the SAMDC gene; wherein the improved morphogenesis is at least one selected from the group consisting of: increased number of stems, main stems, side stems, flower stalks, trunks or branches; (ii) increased number of leaves; (iii) increased number of flowers; (iv) extended flower blooming period; increased number of ovaries, fruit, cotton balls, pods, ears and capsules; (vi) increased development period from bearing to maturity of ovaries, fruit, pods and capsules; (vii) increased number of seeds, grains or ovules; and (viii) increased number of roots or tuberous roots.

005059126 7D The present invention further relates to plants and their progeny, S which stably retain at least one nucleic acid sequence modulating 0 an amount of one or more polyamines under the control of one or 0C more promoters capable of functioning in plants, and the plants and their progeny have at least one kind of improved morphogenesis compared to the plant lacking said nucleic acid sequence.

005059126 8 The present invention further relates to a method for C producing plants that stably retains at least one nucleic acid sequence modulating an amount of one or more polyamines under Z the control of one or more promoters that can function within 00 C- 5 the plant, and that has improved morphogenesis compared to the plant lacking said nucleic acid sequence, comprising the step of IND transforming the cells of a plant lacking said nucleic acid sequence.

c, The present invention further relates to a method for producing plants with improved morphogenesis compared to plants lacking a nucleic acid sequence modulating an amount of one or more polyamines, comprising the step of transforming cells of a plant lacking said nucleic acid sequence with at least one expression vector containing at least one said nucleic acid sequence under the control of one or more promoters capable of functioning in plants.

The present invention further relates to a method for producing a plant with fixed traits, which is a homozygote with respect to a nucleic acid sequence modulating an amount of one or more polyamines and which has improved morphogenesis compared to plants lacking said nucleic acid sequence, comprising the steps of: transforming cells of a plant lacking said nucleic acid sequence with at least one expression vector containing at least one said nucleic acid sequence under the control of one or more promoters capable of functioning in plants; regenerating plants with improved morphogenesis compared to plants lacking said nucleic acid sequence from the transformed cells; 005059126 9 harvesting seeds by pollination from the plant bodies; CI and Z assaying the nucleic acid sequence in the seeds 00 00 obtained by pollination from the plant bodies, which have been obtained by cultivation of the seeds so as to select a ND homozygote with respect to the nucleic acid sequence.

M The present invention further relates to a method for S producing calli with fixed traits, which is a homozygote with respect to a nucleic acid sequence modulating an amount of one [0 or more polyamines and which has improved morphogenesis compared to plants lacking said nucleic acid sequence, comprising the steps of: transforming cells of a plant lacking said nucleic acid sequence with at least one expression vector containing at least one inhibitor of the nucleic acid sequence under the control of one or more promoters capable of functioning in plants; and inducing calli from the transformed cells.

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

005059126 9A According to the invention, "organ" means all organs Cl (tissues) of the plant, and indicates, for example, stems, O tubers, leaves, roots, tuberous roots, buds, flowers, petals, Z ovaries, fruit, pods, capsules, seeds, fibers and ovules, etc.

00 C- 5 The traits relating the form of the organ (tissue) may be of the quantity, the growth and development period, shape, colour, ND properties, and characteristics, etc. Because the amount of S expression of polyamine closely relates to the quantity of c organs, increase of polyamines advances the beginning and delays the end of growth and development with respect to leaves, S flowers, fruit, roots, tubers, tuberous roots, pods and capsules, these can be appreciated and enjoyed for an extended period.

The amount of expression of polyamine in a plant 0 obtained by the present invention may be increased without c- >being influenced by the growing environment (for example 0 Z environmental stress), and can improve morphogenesis.

00 C' 5 Exogenous polyamine metabolism-related enzyme genes or inhibitors of endogenous polyamine metabolism-related enzyme

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(C genes may be cited as nucleic acid sequences that modulate the amount of polyamines. Exogenous polyamine metabolismrelated enzyme genes increase the amount of polyamine in the O 10 plant, and inhibitors of endogenous polyamine metabolismrelated enzyme genes reduce the amount of polyamine in the plant.

With respect to the present invention, "plants that do not have the nucleic acid sequence to modulate the amount of polyamine" means all plants that do not have in the genome the nucleic acid sequence (for example, exogenous polyamine metabolism-related enzyme genes or inhibitors of endogenous polyamine metabolism-related enzyme genes). Consequently, in addition to wild-type varieties, this encompasses cultivars established by ordinary cross-breeding, natural or artificial mutants, and transgenic plants in which exogenous genes other than polyamine metabolism-related enzyme genes have been introduced.

The "polyamines" referred to in the present invention are common natural substances ubiquitous in organisms, and are aliphatic hydrocarbon compounds with two or more primary amine groups. Examples include 1,3-diaminopropane, putrescine, cadaverine, norspermidine, spermidine, homospermidine, aminopropylcadaverine, thermine (norspermine), spermine, 0 thermospermine, canavalmine, aminopentylnorspermidine, N,Nbis(aminopropyl)cadaverine, homospermine, caldopentamine, 0 Z homocaldopentamine, caldohexamine, and homocaldohexamine.

00 0- Polyamine Metabolism-Related Enzyme Genes As used in the present invention, "polyamine

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C metabolism-related enzyme genes" are genes coding for amino acids of enzymes involved in polyamine biosynthesis in plants.

Examples which are believed to be involved, and to be rate 0 limiting, include arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) genes for the typical polyamine putrescine, S-adenosylmethionine decarboxylase (SAMDC) and spermidine synthase (SPDS) genes for spermidine, and S-adenosylmethionine decarboxylase (SAMDC) and spermine synthase (SPMS) genes for spermine.

Arginine decarboxylase (ADC: EC4.1.1.19.) is an enzyme catalyzing the reaction producing agmatine and carbon dioxide from L-arginine. Ornithine decarboxylase (ODC: EC4.1.1.17.) is an enzyme catalyzing the reaction producing putrescine and carbon dioxide from L-ornithine. S-adenosylmethionine decarboxylase (SAMDC: EC4.1.1.50.) is an enzyme catalyzing the reaction producing adenosylmethylthiopropylamine and carbon dioxide from S-adenosylmethionine. Spermidine synthase (SPDS: EC2.5.1.16.) is an enzyme catalyzing the reaction producing spermidine and methylthioadenosine from putrescine and adenosylmethylthiopropylamine.

These genes, any of which may be derived, can be isolated from various plants. Specific examples include dicotyledons such as Cucurbitaceae; Solanaceae; Brassicaceae such as Arabidopsis thaliana; Papilionaceae such as alfalfa O and Vigna unguiculata; Malvaceae; Asteraceae; Chenopodiaceae; and Convolvulaceae; or monocotyledons such as Gramineae, 0 Z including rice, wheat, barley, and corn. Drought-resistant 00 -0 cactus or Mesembryanthemum crystallinum are also included.

Cucurbitaceae are preferred, and Cucurbita ficifolia Bouche

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Ci is especially preferred.

CPlant tissue in which the plant-derived polyamine metabolism-related enzyme genes of the invention are isolated 0 may be in the form of seeds or in the process of growing. The genes may be isolated from part or all of the tissue of growing plants. Any part can be used to isolate genes, but whole plants, buds, flowers, ovaries, fruit, leaves, stems, roots, and the like are preferred.

Preferred examples of polyamine metabolism-related enzyme genes used in the present invention include the spermidine synthase gene, S-adenosylmethionine decarboxylase, and arginine decarboxylase gene. Specific examples include: DNA having the base sequence represented by base numbers 77 through 1060 in the base sequence given in SEQ ID NO. 1; DNA having the base sequence represented by base numbers 456 through 1547 in the base sequence given in SEQ ID NO. 3; and DNA having the base sequence represented by base numbers 541 through 2661 in the base sequence given in SEQ ID NO. Further examples include: DNA having a base sequence capable of hybridizing under stringent conditions with any of the above sequences, and coding for a polypeptide with polyamine metabolism-related enzyme activity equivalent to those sequences.

Still further examples include: DNA comprising any of the above base sequences with 1 or 0 Z more bases deleted, substituted, inserted, or added, and 00 0-0 coding for a polypeptide with polyamine metabolism-related enzyme activity equivalent to those sequences.

(C The "stringent conditions" referred to here mean c conditions under which only base sequences coding for a polypeptide with polyamine metabolism-related enzyme activity Sequivalent to the polyamine metabolism-related enzyme encoded by a specific polyamine metabolism-related enzyme gene sequence form hybrids with the specific sequence (referred to as specific hybrids), and base sequences coding for polypeptides with no such equivalent activity do not form hybrids with the specific sequence (referred to as nonspecific hybrids). One with ordinary skill in the art can readily select such conditions by varying the temperature during the hybridization reaction and washing process, the salt concentration during the hybridization reaction and washing process, and so forth. Specific examples include, but are not limited to, conditions under which hybridization is brought about at 42 0 C in 6 x SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 x SSPE (3M NaCl, 0.2 M NaH 2

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4 20 mM EDTA-2Na, pH and the product is washed with 0.5 x SSC at 420C.

In general, it is well known by those skilled in the art that substitution, deletion, insertion or addition of one or more amino acids in amino acid sequence of biologically active proteins may maintain those physiological activities.

The "base sequences with 1 or more bases deleted, substituted, inserted, or added" referred to here include genes that have such modifications and that code for a polyamine metabolism- 0 Z related enzyme, such genes are included within the scope of O0 the present invention. For example, the poly A tail or 5',3' end nontranslation regions may be "deleted," and bases may be

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(C "(deleted" to the extent that one or more amino acids are c deleted. Bases may also be "substituted," as long as no frame shift results. Bases may also be "added" to the extent Sthat amino acids are added. However, it is essential that such modifications do not result in the loss of polyamine metabolism-related enzyme activity. "Genes with one or several bases deleted, substituted, or added" are preferred.

Such modified DNA can be obtained by modifying the DNA base sequences of the invention so that amino acids at specific sites are substituted, deleted, inserted, or added by site-specific mutagenesis, for example (Nucleic Acid Research, Vol. 10, No. 20, 6487-6500 (1982)).

In the present invention, "antisense genes" refer to genes with a sequence complementary to the base sequence of a polyamine metabolism-related enzyme gene. Antisense DNA is complementary the base sequence of SEQ ID NOS. 1, 3, or for example. Antisense RNA is produced from that.

Inhibitors of endogenous polyamine metabolism-related enzyme genes The inhibitors suppress the expression of the genes, and are not particularly limited, and, for example, include antisense DNA, or double stranded RNA (dsRNA) or RNAi of the gene and sequences selected from upstream or downstream.

O The antisense DNA may be complementary to either a regulatory region on the 5' upstream side that includes an Z intron or exon of the gene, or a promoter of the gene, or a 00 region on the downstream side of the termination codon, which affects gene expression. The length of the antisense DNA is CI at least 20 bases, preferably at least 100 bases, more preferably at least 300 bases, most particularly at least 500 bases. The transcription product of the antisense DNA is Shybridized with mRNA of endogenous polyamine metabolismrelated enzyme genes, or is hybridized to a non-coding region (for example promoter, intron, transcription termination factor) of endogenous polyamine metabolism-related enzyme genes or an upstream or downstream sequence thereof.

RNAi comprises RNA having a sense sequence in relation to a target gene and an antisense sequence in relation to a target gene, and the sense sequence and antisense sequence may be included on 2 strands of RNA respectively and function as double stranded RNA, or may be included on 1 RNA strand and function as a single RNA molecule having a double strand portion and a loop sequence whick links sense and antisense sequences.

Plant and their progeny with improved morphogenesis "Organ" in the present invention, as described above, means all organs (tissues) of the plant, including, for example, stems, tubers, leaves, roots, tuberous roots, buds, flowers, petals, ovaries, fruit, pods, capsules, seeds, fibers and ovules, etc. The traits relating to the form of the organ (tissue) may be of the quantity, the growth and development period, shape, color, properties, and characteristics, etc.

0 Z A "plant in which morphogenesis has been improved" and a

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0- "plant having improved morphogenesis" of the present invention refers to a plant wherein an exogenous polyamine

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N1 metabolism-related enzyme gene or an inhibitor of an endogenous polyamine metabolism-related enzyme gene is introduced to make a trait relating to morphogenesis that, compared to prior to introduction, enhance, improves or suppresses quantity, the growth and development period, shape, color, properties, or characteristics. For example, by introducing into a plant a polyamine metabolism-related enzyme gene or the inhibitor, the traits related to the formation of the stems, leaves, flowers, or seeds are improved compared to a plant not having the exogenous polyamine metabolism-related enzyme gene or the inhibitor.

However, the invention is not limited to these.

Concretely, a "plant with the number of stems improved" by a genetic sequence that modulates the amount of polyamine is a plant with an increased number of plant stems (including main stems, side stems, flower stalks, trunks, branches, etc.) and a plant with a decreased or reduced number of plant stems (including main stems, side stems, flower stalks, trunks, branches, etc.) based on the inhibitor.

A "plant having an improved number of leaves" is a plant with an increased or a decreased number of or reduced plant leaves.

A "plant having an improved number of flowers or flower blooming period" is a plant with an increased or a decreased O number of or reduced plant flowers, or a plant with an early plant flower blooming period or an extended blooming period.

0 Z A "plant having an improved number of ovaries or ovary

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development period" is a plant with an increased or a

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decreased number of or reduced plant ovaries, fruit, pods and

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C-i capsules, or a plant with an increased or a decreased or a "reduced growth or development period from bearing to maturity of ovaries, fruit, pods and capsules.

SA "plant having an improved number of seeds" is a plant with an increased or a decreased number of, or reduced plant seeds (including ovules).

In this way, improvement of product quality, productivity and yield of the plant (organs and tissues, etc.), as well as improved product characteristics can be expected. Moreover, in edible plants such as vegetables and fruit, it is possible that reducing the amount of polyamine of the edible part (leaves, fruit, tuber, tuberous root, etc.) will have a beneficial result on the characteristics, such as the product quality. Consequently, it is possible to attempt to improve the yield while improving the quality by expressing the inhibitor in these edible parts, and by expressing polyamine metabolism-related enzyme genes in the other parts.

Further, improvement of the adaptability (resistance) to various kinds of environmental stress may be expected by improving morphogenesis and varying the shape and formation of the plant. Specifically, it appears that the number of leaves and the green color of leaves based on chlorophyll may be increased; roots (including tuberous roots and tubers) may be elongated; the number and thickness of stems may be increased; consequently, the environmental adaptability of 0 Z the plant may be increased.

00 C- In one of the preferable embodiments of the present invention, the difference in external appearance of the plant

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N during the vegetative growth period is small (the leaves are "darker green), but a notable improvement in such organs as c-i the leaves, flowers and stems is more notable during the Sreproductive growth period, and this reveals that the latent c-i capacity (potential) of the plant was improved.

The plants of the invention include not only the plant in its entirety (whole plant), but also calli, seeds, all plant tissues, leaves, stems, tubers, roots, tuberous roots, buds, flowers, petals, ovaries, fruit, pods, ovules and fibers. Their progeny are also included in the plants of the invention.

cUseful substances obtained from plants and their progeny" in the present invention indicate useful substances produced by plants and their progeny in which the introduction of an exogenous polyamine metabolism-related enzyme gene provides or improves morphogenesis compared to before the gene was introduced. Examples of useful substances include amino acids, oils and lipids, starches, proteins, phenols, hydrocarbons, cellulose, natural rubber, pigment, enzymes, antibodies, vaccines, medicinal products, and biodegradable plastics.

The plants of the present invention are plants with no exogenous polyamine metabolism-related enzyme gene, to which such an exogenous polyamine metabolism-related enzyme gene is introduced by genetic engineering and is retained in a stable

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manner. As used herein, "retained in a stable manner" means 0 Z that the polyamine metabolism-related enzyme gene is 00 0. expressed in the plant at least in which the polyamine metabolism-related enzyme gene has been introduced, and is C-I retained in the plant cells long enough to result in the i improvement of morphogenesis. The polyamine metabolismrelated enzyme gene is, therefore, preferably incorporated on Sthe chromosomes of the host plant. The polyamine metabolismrelated enzyme gene or genes should even more preferably be retained in subsequent generations.

As used herein, "exogenous" means not endogenous to the plant, but externally introduced. Accordingly, an "exogenous polyamine metabolism-related enzyme gene" may be a polyamine metabolism-related enzyme gene homologous to the host plant (that is, derived from the host plant), which is externally introduced by genetic manipulation. The use of a host-derived polyamine metabolism-related enzyme gene is preferred in consideration of the identity of the codon usage.

The exogenous polyamine metabolism-related enzyme gene may be introduced into plants by any method of genetic engineering. Examples include protoplast fusion with heterologous plant cells having the polyamine metabolismrelated enzyme gene, infection with a plant virus having a viral genome genetically manipulated to express the polyamine metabolism-related enzyme gene, or transformation of host plant cells using an expression vector containing the polyamine metabolism-related enzyme gene.

O The plants of the invention are preferably transgenic plants which are obtained by the transformation of cells of 0 Z plants lacking the exogenous polyamine metabolism-related 00 00 enzyme gene in an expression vector containing the exogenous polyamine metabolism-related enzyme gene under the control of Cq a promoter capable of functioning in plants.

Examples of promoters capable of functioning in plants include the 35S promoter of the cauliflower mosaic virus 0 (CaMV) which is constitutively expressed in plant cells, the nopaline synthase gene (NOS) promoter, octopine synthase gene (OCS) promoter, phenylalanine ammonia lyase (PAL) gene promoter, and chalcone synthase (CHS) gene promoter. Other well-known plant promoters not limited to these are also available.

Not only promoters constitutively expressed in the entire organ such as the 35S promoter, but also promoters specific to organs or tissues can be used to express the target gene in only target organs or tissues so as to improve morphogenesis in specific organs or tissues. For example, a polyamine metabolism-related enzyme gene and a promoter capable of function specific to flower organ (such as WO99/43818,Japanese Unexamined patent publication 178572/1999,Japanese Unexamined patent publication 316582/2000) can be used to improve the number of flowers and a flowering period. In addition, growth and duration of development of ovary and fruit can be improved by using promoters specific to ovary and fruit (Plant Mol. Biol., 11, 651-662, 1988).

If a time specific promoter is used, the target gene can

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be expressed only at a specific time, and the morphogenesis 0 Z can be improved at only the specified time period. For 00 0. example, morphogenesis can be improved only during the vegetative growth period by using a polyamine metabolism-

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(CN related enzyme gene, and a promoter that works in the vegetative growth period.

Cc| Promoters regulated by low temperature, elevated Stemperature, stress, drought, light, heat, hormones, damage or the like can be used to express the target gene according to the living environment. For example, a polyamine metabolism-related enzyme gene and a promoter capable of transcription only when the plant is exposed to low temperatures (such as the BN115 promoter: Plant Physiol., 106, 917-928 (1999)) can be used to control the polyamine metabolism of the plant only at low temperatures and to improve morphogenesis. A polyamine metabolism-related enzyme gene and a promoter capable of transcription only when the plant is exposed to drought (such as the Atmyb2 promoter: The Plant Cell, 5, 1529-1539, 1993) can also be used to control the polyamine metabolism of the plant during drought and to improve morphogenesis.

The exogenous polyamine metabolism-related enzyme gene in the expression vector of the present invention is located downstream of the promoter so that transcription is controlled by the promoter capable of functioning in plants.

A transcription termination signal (terminator region) capable of functioning in plants should also be added Sdownstream of the polyamine metabolism-related enzyme gene.

An example is the terminator NOS (nopaline synthase) gene.

0 Z The expression vector of the present invention may also 00 00 contain a cis-regulatory element such as an enhancer sequence.

The expression vector may also contain a marker gene for

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C- selecting transformants such as a drug-resistance gene marker, examples of which include the neomycin phosphotransferase

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(NPTII) gene, the phosphinothricin acetyl transferase (PAT) 0 gene, and the glyophosate resistance gene. Because the incorporated gene is sometimes dropped in the absence of selection pressure, it is advantageous to ensure that a herbicide resistance gene is also present on the vector so that the use of a herbicide during cultivation will always result in conditions involving selection pressure.

To facilitate mass production and purification, the expression vector should also contain a selection marker gene (such as ampicillin resistance gene or tetracycline resistance gene) in E. coli and a replication origin capable of autonomous replication in E. coli. The expression vector of the present invention can be constructed in a simple manner by inserting the selection marker gene as needed and an expression cassette of the polyamine metabolism-related enzyme gene at the cloning site of an E. coli vector (pUC or pBR series).

When the exogenous polyamine metabolism-related enzyme gene is introduced by infection with Agrobacterium tumefaciens or Agrobacterium rhizogenes, the polyamine metabolism-related enzyme gene expression cassette can be inserted in the T-DNA region (region transferred to plant 0 chromosome) on a Ti or Ri plasmid of the cells. At present, binary vector systems are used in standard methods of 0 Z transformation with Agrobacterium. The necessary functions 00 0- for T-DNA transfer are independently provided by both the T- DNA itself and the Ti (or Ri) plasmid, these structural

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C-q elements being divided on separate vectors. The binary plasmid has 25 bp border sequences at both ends necessary for p- cleaving and combining the T-DNA, and the plant hormone gene 0 inducing crown gall (or hairy root) is removed, simultaneously providing room for inserting the exogenous gene. Examples of commercially available binary vectors include pBI101 and pBIl21 (both by Clontech). The Vir region involved in the incorporation of the T-DNA has trans action on the separate Ti (or Ri) plasmid referred to as the helper plasmid.

Various conventionally known methods can be used for the transformation of the plants. Examples include the PEG method in which protoplasts are isolated from plant cells by treatment with a cell wall-degrading enzyme such as cellulase or hemicellulase, and polyethylene glycol is added to a suspension of the protoplasts and an expression vector containing the aforementioned polyamine metabolism-related enzyme gene expression cassette to incorporate the expression vector into the protoplasts by a process such as endocytosis; the liposome method in which an expression vector is introduced by ultrasonic treatment or the like into lipid membrane vesicles such as phosphatidylcholine, and the vesicles are fused with protoplasts in the presence of PEG; methods of fusion in a similar process using micelles; and 0 electroporation in which electrical pulses are applied to a suspension of protoplasts and an expression vector to

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Z incorporate the vectors in the external solution into the 00 protoplasts. However, these methods are complicated in that they require a culturing technique for the redifferentiation

NO

C-i of the protoplasts into plants. Processes for introducing the gene into intact cells with cell walls include direct injection such as microinjection in which a micropipette is Sinserted into cells to inject the vector DNA in the pipettes under hydraulic or gas pressure into the cells, or the particle gun method in which metal microparticles coated with DNA are accelerated through the detonation of an explosive or gas pressure and thus introduced into the cells, and methods involving the use of infection with Agrobacterium. Drawbacks of microinjection are the need for considerable training and the small number of cells that are handled. It is therefore more desirable to transform plants with more convenient methods such as the Agrobacterium method and the particle gun method. The particle gun method is useful in that genes can be directly introduced into the apical meristem of plants while cultivated. In the Agrobacterium method, the genomic DNA of a plant virus such as the tomato golden mosaic virus (TGMV) or another gemini virus is simultaneously inserted between the border sequences into the binary vector, so that the viral infection can spread throughout the entire plant and the target gene can be simultaneously introduced into the entire plant simply by inoculating cells at any location of the cultivated plant with the viral cell suspension.

0 Specific examples of methods for obtaining polyamine metabolism-related enzyme genes as well as methods for

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Z introducing the target gene using Agrobacterium to produce 00 0- transformants are given below. With respect to inhibitors of endogenous polyamine metabolism-related enzyme genes, those

NO

Cq skilled in the art can produce a transgenic plant with reference to the following description.

1. Obtaining polyamine metabolism-related enzyme genes Preparation of cDNA library for PCR Poly(A) RNA is extracted in the usual manner from root tissue of Cucurbita ficifolia Bouche which has undergone 3 days of low temperature treatment at 180C daytime/14 0 C night time. A cDNA library can be prepared for use in PCR from the isolated poly(A) +RNA using a commercially available marathon cDNA Amplification Kit (by Clontech) or the like. The isolated poly(A) +RNA is used as template, and reverse transcriptase and modified lock-docking oligo(dT) primer with two degenerate nucleotide positions at the 3' end are used to synthesize the first-strand cDNA. Double-stranded cDNA is obtained by polymerase reaction. The double-stranded cDNA is blunted with T4 DNA polymerase, and a Marathon cDNA adapter is ligated, giving a library of double-stranded cDNA with the adapter added.

Design of PCR Primer The SPDS gene, SAMDC gene, ADC gene, and ODC gene can be isolated as the polyamine metabolism-related enzyme gene.

The SPDS gene can be isolated from Cucurbita ficifolia Bouche or Hyoscyamus niger, the SAMDC gene can be isolated from potatoes, spinach, or tobacco, the ADC gene can be isolated from soybean, peas, or tomatoes, and the ODC gene can be isolated from Datura. The base sequences have already been Z determined. Extremely well conserved regions can therefore be 00 selected by comparing known base sequences, and DNA oligomers can be synthesized to design primers for PCR.

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C-i Obtaining SPDS gene, SAMDC gene, and ADC gene fragments by PCR C<1 The cDNA library for PCR prepared in above is used 0 as template, and the primers designed in above are used to carry out PCR. The PCR products are isolated by gel electrophoresis and are purified with glass milk or the like.

The purified PCR products are ligated to a cloning vector such as the TA vector.

The base sequences of the cloned cDNA are determined by the method of Maxam-Gilbert, the dideoxy method, or the like.

Either method can be carried out using commercially available kits, and an auto sequencer can be used for automatic sequencing.

Isolation of full-length gene The full-length gene can be obtained in the usual manner by plaque hybridization, RACE (rapid amplification of cDNA ends), Marathon RACE, or the like.

The genes thus obtained are genes that participate in polyamine biosynthesis, and using these genes it is possible to control polyamine levels by controlling the expression of genes on a molecular biological level, and to produce plants with various types of improved morphogenesis.

2. Introduction of target gene with Agrobacterium into Arabidopsis thaliana, and preparation of transgenic plant O The genes obtained in 1. above can be introduced into a plant host to produce transgenic plants with improvement of

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Z various types of morphogenesis such as morphogenesis of stems, 0 leaves, flowers, ovaries, fruit, pods and seeds in particular.

Preparation of expression construct and

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IN transformation of Agrobacterium

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Expression constructs can be prepared by cleaving the (ci polyamine metabolism-related enzyme gene obtained in 1. above Swith suitable restriction enzymes so as to include all of the open reading frame, then ligating suitable linkers as needed, and inserting the gene into a plant transformation vector.

Examples of plant transformation vectors which can be used include pBIl01 and pBIl21.

The resulting expression construct is amplified in E.

coli, and the expression construct is then transformed by tripartite conjugation (Nucleic Acid Research, 12, 8711 (1984)), freeze thawing, electroporation, or the like with Agrobacterium tumefaciens C58, LBA4404, EHA101, or the like.

Tripartite conjugation involves, for example, the culture of E. coli having an expression construct containing the target gene, E. coli having a helper plasmid (such as pRK2013), and Agrobacterium on medium containing an antibiotic (such as rifampicillin, kanamycin, or hygromycin) so as to obtain transformed Agrobacterium.

Production of Transgenic Plant Parts of plants to which genes can be introduced in the present invention include the entire plant, plant organs (such as leaves, stems, roots, flower organs, vegetative points, and seeds), plant tissue (such as epidermis, phloem, 0 parenchyma, xylem, and vascular bundle), and plant cultured cells.

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Z The target gene can be introduced upon infecting plants

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0- with the transformed Agrobacterium prepared in by callus regeneration (Plant Cell Reports, 12, 7-11 (1992)), for CN example. That is, MSO plates (4.6 g Murashige-Skoog mineral salts, 10 g sucrose, 1 mL/L of 1000 x vitamin stock, pH 6.2) can be inoculated with seeds of Arabidopsis thaliana in the Susual manner for aseptic culture. After taking root, slices of root can be used in the culture of callus on CIM plates (MSO plates supplemented with 2 ,4-dichlorophenoxyacetic acid to a final concentration of 0.5 pg/mL and kinetin to a final concentration of 0.05 pg/mL). Agrobacterium transformed with plasmid containing the target gene joined to the promoter, kanamycin and hygromycin resistance genes is cultured, diluted samples are aliquoted into tubes, and slices of the roots on which callus is forming are soaked for several days of co-cultivation on CIM plates. When the strains have grown enough to become visible to the naked eye, they are disinfected for several days of culture on SIMC plates (MSO plates supplemented with N 6 -[2-isopentenyl]adenine to a final concentration of 5 pg/mL, indoleacetic acid (IAA) to a final concentration of 0.15 tg/mL, and claforan to a final concentration of 500 pg/mL). The slices are finally cultured on SIMCS plates (plates containing kanamycin and hygromycin) and repeatedly transplanted to fresh plates every week. The transformed slices continue to be grown, resulting in callus.

Slices that have not been transformed will turn brown, as the selection is based on antibiotics. The transformants are Sabout 5 mm, and are cultured until rosette leaves are formed.

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When the complete rosette form is evident, the roots of the

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Z transformants are cut with a scalpel to leave out the callus

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00 and are transplanted to RIM plates (MSO plates supplemented with IAA to a final concentration of 0.5 pg/mL). When large

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C-i calli are attached, the roots will show through the callus 0 even if they have taken root, and vascular bundles will not

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often become joined to the rosettes. After about 8 to 10 days, 0 they become planted on rock wool soaked with mineral salts mM KN0 3 2.5 mM K-phosphate buffer (pH 2 mM MgS0 4 2 mM Ca(N0 3 2 50 pM Fe-EDTA, 1000 x microelements (70 mM H 3 BO3, 14 mM MnCl 2 0.5 mM CuSO 4 1 mM ZnSO 4 0.2 mM NaMoO 4 10 mM NaC1, 0.01 mM CoC12) 1 mL/L). Plants that have flowered and formed pods are transplanted to soil soaked with mineral salt media, allowing the seeds to be obtained. The seeds are disinfected and are allowed to germinate upon the inoculation of MSH (MSO plates supplemented with hygromycin to a final concentration of 5 U/mL), thereby allowing transformants to be obtained.

Plants can be infected with the transformed Agrobacterium prepared in by infiltration at reduced pressure (The Plant Journal, 19(3), 249-257 (1999)) to introduce the target gene. That is, potting commcercial nursery soil (such as Metromix) is inoculated with seeds of Arabidopsis thaliana, which are cultivated under conditions involving long days (such as 16 hour days and 8 hour nights) at 22 0 C. After about 3 to 4 weeks, the extended main axis (flower stalk) is cut to begin induction of lateral shoots.

After about 1 week of top pruning, the Arabidopsis thaliana is dipped in a suspension of cultured Agrobacterium 0 transformants, is placed in a dessicator, which is suctioned with a vacuum pump to about -0.053 MPa (400 mmHg), and is

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Z then allowed to stand at ambient temperature for 10 minutes.

00 0- The infected pot is transferred to a deep-bottomed tray and tilted on its side to allow a small amount of water to drip

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Ci into the bottom of the tray, a transparent covering is placed on it, and it is then allowed to stand for about 1 day under humid conditions. The infected pot is then raised, and Scultivation is started under conditions involving long days at 22 0 C to harvest the seeds.

The seeds are harvested for about 2 to 4 weeks, and the harvested seeds are strained through a tea strainer or the like to remove debris and husks, and are dried and stored in a dessicator.

The selection of transgenic plants involves sterilizing the harvested seeds in the usual manner and suspending them in about 9 mL of 0.1% agar aqueous solution, then spreading the suspension on selection medium (such as 1 x MS salt, 1 x Gamborg B5 vitamin, 1% sucrose, 0.5 g/L MES, 0.8% agar, 100 mg/L carbenicillin, 50 mg/L kanamycin, 40 mg/L hygromycin) for aseptic culture at 22 0 C. Transgenic plants showing resistance to the antibiotics will grow well and can be identified in about 1 to 2 weeks. Transgenic plants with about 4 to 6 true leaves are transplanted to pots containing potting commcercial nursery soil to begin cultivation during long days at 220C.

DNA is extracted in the usual manner from the resulting transgenic plants, the DNA is cleaved with suitable restriction enzymes, and the polyamine metabolism-related O enzyme gene can be used as probe in Southern hybridization to determine whether or not the gene has been introduced.

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Z RNA can be extracted in the usual manner from 00 C-i transgenic or non-transgenic plants to prepare probes with the polyamine metabolism-related enzyme gene sense sequence C1 or antisense sequence, and the probes can be used in Northern hybridization to study the expression of the target gene.

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0 Callus can be induced from the resulting transgenic O plants to produce callus.

The proportion of transformed progeny T2, created by self-pollination, of transgenic plants (TI) obtained by reduced pressure infiltration will ordinarily follow Mendel's laws. For example, when the polyamine metabolism-related enzyme gene is heterozygously incorporated in a gene locus, the proportion of transformants in T2 progeny will be 3:1. In terms of T3 progeny produced by self-pollination upon cultivation of T2 progeny, when transformants appear in all progeny, the T2 transgenic plants will be homozygotes, and when transformants are isolated in a proportion of 3:1, the T2 transgenic plants will be heterozygotes with respect to the introduced polyamine metabolism-related enzyme gene.

The plants thus selected, which are homozygote with respect to the introduced polyamine metabolism-related enzyme gene, are very useful in the field of seed industry as independent line in which improved morphogenesis is fixed.

The transgenic plant whose gene expression analysis of polyamine metabolism-related enzyme gene is conducted by Southern analysis or Northern analysis can be subjected to evaluation of polyamine content and various types of morphogenesis.

O

Z In the case of polyamine assay, for example, 00 0. perchloric acid aqueous solution is added to 0.05 to 1 g sample to extract the polyamine. Assay of the extracted Cq polyamines involves fluorescent labeling by benzoylation, dansylation, or the like, followed by analysis with an internal standard using high performance liquid Schromatography (HPLC) with a UV detector or fluorescence detector.

For example, there are methods to evaluate the various types of morphogenesis. Stem formation can be evaluated by sowing in a suitable culture medium or potting soil T2 or T3 seeds obtained by self-pollination of a transgenic plant, growing under long day conditions (day length of daylight/dark: 16 hours/8 hours) at 20 to 250 C, and investigating the number and shape of stems (for example, main flower stalks, side flower stalks, branches, etc.).

Leaf formation can be evaluated by sowing in a suitable culture medium or potting soil T2 or T3 seeds obtained by self-pollination of a transgenic plant, growing under long day conditions (day length of daylight/dark: 16 hours/8 hours) at 20 to 250 C, and investigating the number and shape of leaves (for example, rosette leaves, true leaves, etc.).

Flower formation can be evaluated by germinating in a suitable culture medium or potting soil T2 or T3 seeds obtained by self-pollination of a transgenic plant, growing under long day conditions (day length of daylight/dark: 16 hours/8 hours) at 20 to 250 C, and investigating the number, 0 blooming time, blooming period, shape, and color of flowers, etc. Ovary formation can be evaluated by germinating in a 0 Z suitable culture medium or potting soil T2 or T3 seeds 00 0- obtained by self-pollination of a transgenic plant, growing under long day conditions (day length of daylight/dark: 16 CN hours/8 hours) at 20 to 250 C, and investigating the number, shape, color, and development period from setting the fruit 0 to maturation of the ovaries (for example, pods and fruit, Setc.). Seed formation can be evaluated by germinating in a suitable culture medium or potting soil T2 or T3 seeds obtained by self-pollination of a transgenic plant, growing under long day conditions (day length of daylight/dark: 16 hours/8 hours) at 20 to 250 C, and investigating the number and shape of seeds, as well as germination rate after harvesting, etc.

Examples of plants which may be transformed in the invention include, but are not limited to, dicotyledons, monocotyledons, herbaceous plants, and shrubs. Examples include sweet potatoes, tomatoes, cucumbers, squash, melons, watermelon, tobacco, Arabidopsis thaliana, bell peppers, eggplant, beans, taro, spinach, carrots, strawberries, white potatoes, rice, corn, alfalfa, wheat, barley, soybeans, rapeseed or canola, sorghum, Eucalyptus, poplar, kenaf, Eucommia ulmoides, sugarcane or sugarbeet, Chenopodium album, lilies, orchids, carnations, roses, petunia, Torenia fournieri, Antirrhinum majus, Cyclamen persicum, Gypsophila elegans, Pelargonium graveolens, sunflowers, Zoisia japonica, cotton, matsutake mushrooms, shiitake mushrooms, mushrooms, ginseng, citrus fruit, bananas, and kiwi fruit, Manihot utilissima, Metroxylon sagu Roxb. Sweet potatoes, white potatoes, tomatoes, cucumbers, rice, corn, soybeans, wheat, 0 Z petunia, Torenia fournieri, Eucalyptus and cotton are

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preferred.

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According to the present invention, improvement of

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C-i various types of morphogenesis in plants is possible, and it Ccmay be expected that the quality of plant organs and tissue, value, productivity, and yield may be improved, or that resistance to collapsing may be improved by making dwarves, etc. Specifically, it may be expected that when producing the organs as an agricultural product by increasing the number of stems, leaves, flowers, ovaries, fruit, pods, and seeds, increased quality, productivity and yield may be expected. For example, it may be expected that increasing the number of seeds per rice plant will augment the yield of rice that can be harvested. It may be expected that increasing the number of female ears per corn plant will augment the yield of kernels. It may be expected that increasing the number of pods per soybean plant will augment the yield of beans. It may be expected that increasing the number of cotton bolls per plant will augment the yield of fiber harvested. It may be expected that increasing the number of tubers and tuberous roots of white potatoes and sweet potatoes will augment the yield of potatoes. It may be expected of fruit trees that increasing the number of fruit (ovaries) set and lengthening the development period will improve producibility. It may be expected of ornamental plants that increasing the number of stems, leaves, and especially, flowers, as well as delaying the blooming period 0 and making better visual appeal by miniaturization, will increase the commercial value. Further, it may be expected 0 Z that by changing the shape and formation of the plant, 00 0- various environmental adaptability may be improved, stabilization of cultivation and producibility may be C<N improved, and the land under cultivation may be expanded.

BEST MODE FOR CARRYING OUT THE INVENTION The invention is illustrated in further detail by the 0 following examples, but they are provided only as examples and do not in any way limit the scope of the invention.

Example 1: Cloning of Plant-Induced Polyamine Metabolism- Related Enzyme Gene Preparation of Poly(A)+RNA Vermiculite was inoculated with Cucurbita ficifolia Bouche, and the plants were transplanted to pots filled with commercially available fine soil (Sansan soil, by Takii Co., Ltd.) at the cotyledon development stage. The potted Cucurbita ficifolia Bouche was placed in incubators (air temperature: day 26 0 C/night 22 0 C, 13 hour long days) for plant cultivation. At the two leaf stage, the incubator temperature was lowered to day 18 0 C/night 14 0 C to begin low temperature treatment. After 3 days of low temperature treatment, the plants were divided into roots, stems, and leaves for sampling. The samples were stored in an -80 0

C

freezer until RNA extraction.

About 4 g of Cucurbita ficifolia Bouche root tissue was immediately frozen in liquid nitrogen and finely milled in a mortar and pestle in the presence of liquid nitrogen. 10 mL of 0.2 M Tris acetate buffer (5 M guanidine thiocyanate, 0.7% 0 P-mercaptoethanol, 1% polyvinyl pyrrolidone 360,000), 0.62% N-lauroylsarcosine sodium salt, pH 8.5) for extraction 0 Z was then added, and the tissue was milled for 2 minutes while 00 0- cooled on ice using a Polytron homogenizer (by Kinematica).

The mercaptoethanol and polyvinyl pyrrolidone were added just

IO

C-i before use. The milled product was then centrifuged for c-i minutes at 17,000 x g, and the supernatant was recovered.

c-i The supernatant was filtered through mira cloth, the Sfiltrate was gently layered in 1.5 mL of 5.7 M cesium chloride solution placed in an ultracentrifugation tube, the contents were centrifuged for 20 hours at 155,000 x g, the supernatant was then discarded, and the RNA precipitate was recovered. The precipitate was dissolved in 3 mL of 10 mM Tris-HCl, 1 mM EDTA-2Na, pH 8.0 (referred to as TE buffer), an equivalent amount of phenol:chloroform:isoamyl alcohol (volumetric ratio of 25:24:1) was furthermore added, the ingredients were mixed and then centrifuged, and the upper aqueous layer was recovered. 1/10-fold 3 M sodium acetate (adjusted to pH 6.2 with glacial acetic acid) and ethanol were added to the aqueous layer, the ingredients were mixed, and the mixture was allowed to stand over night at 0 C. The mixture was then centrifuged for 20 minutes at 17,000 x g, and the resulting precipitate was washed with ethanol and dried at reduced pressure.

The dried preparation was dissolved in 500 |iL of the aforementioned TE buffer, giving total RNA solution. The RNA solution was incubated for 5 minutes at 650C and then quenched on ice. An equivalent amount of 2 x binding buffer mM Tris-HCl, 5 mM EDTA-2Na, 1 M NaC1, 0.5% SDS, pH 0 was added to the RNA solution, and the solution was layered

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on an oligo dT cellulose column (by Clontech) equilibrated 0 Z with equilibration buffer (10 mM Tris-HCl, 5 mM EDTA-2Na, 00 M NaC1, 0.5% SDS, pH The column was then flushed with about 10-fold equilibration buffer described above, and the C-i poly +RNA was eluted with elution buffer (10 mM Tris-HCl, mM EDTA-2Na, pH 1/10-fold 3 M sodium acetate aqueous solution described above and 2.5-fold ethanol were added to the resulting eluate, the ingredients were mixed, and the mixture was allowed to stand at -70 0 C. The mixture was then centrifuged for minutes at 10,000 x g, and the resulting precipitate was washed with 70% ethanol and dried at reduced pressure. The dried preparation was again dissolved in 500 pL TE buffer and repeatedly purified on an oligo dT cellulose column. The resulting poly *RNA from the roots of the low temperature treated Cucurbita ficifolia Bouche was used to prepare a cDNA library for PCR and a cDNA library for isolating the fulllength gene.

Preparation of cDNA library for PCR The cDNA library was prepared using a Marathon cDNA Amplification Kit (by Clontech). The poly +RNA from the roots of the Cucurbita ficifolia Bouche obtained in was used as template, and reverse transcriptase and modified lock-docking oligo(dT) primer with two degenerate nucleotide positions at the 3' end were used to synthesize doublestranded cDNA according to the method of Gubler, Hoffman, et al (Gene, 25, 263-269 (1983)).

SA Marathon cDNA adapter (the 5' end phosphorylated to facilitate binding to both ends of the ds cDNA with T4 DNA

O

Z ligase) was ligated to both ends of the resulting cDNA. The 00 0- resulting adapter-linked cDNA was used as a Cucurbita ficifolia Bouche root-derived cDNA library for PCR.

Ci Design of PCR primers The base sequences of arginine decarboxylase, S-adenosylmethionine decarboxylase, and spermidine synthase 0 genes already isolated from plants or mammals were compared.

Regions with extremely highly conserved homology were selected to synthesize DNA oligomers (sequence primers I through VI).

SPDS primer I (SEQ ID NO. 5'-GTTTTGGATGGAGTGATTCA-3' SPDS primer II (SEQ ID NO. 5'-GTGAATCTCAGCGTTGTA-3' SAMDC primer III (SEQ ID NO. 5'-TATGTGCTGTCTGAGTCGAGC-3' SAMDC primer IV (SEQ ID NO. 10): 5'-GCTAAACCCATCTTCAGGGGT-3' ADC primer V (SEQ ID NO. 11): 5'-GGGCT(T/G)GGA(G/A)T(G/C)GACTA(C/T)-3' ADC primer VI (SEQ ID NO. 12): 5'-(T/C)CC(A/G)TC(A/G)CTGTC(G/A)CA(G/C)GT-3' Amplification by PCR The cDNA library for PCR obtained in was used as template, and the sequence primers designed in were used for PCR. The PCR steps involved 5 cycles of 30 seconds at 94°C, 1 minute at 450C, and 2 minutes at 720C, followed by cycles of 30 seconds at 940C, 1 minute at 550C, and 2 minutes at 720C.

Agarose Gel Electrophoresis 0 Electrophoresis of the PCR amplified products on

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agarose was followed by ethidium bromide staining of the 0 Z electrophoresed gel and detection of amplified bands on a UV

OC

0- transilluminator.

Verification and Recovery of PCR Amplified Products Ci The detected amplified bands were verified and were cut out of the agarose gel with a razor. The pieces of gel were transferred to 1.5 mL microtubes, and the DNA fragments were 0 isolated and purified from the gel using a QIAEX II Gel Extraction Kit (by QIAGEN). The recovered DNA fragments were subcloned to the pGEMT cloning vector (by Promega), transformed with E. coli, and then used to prepare plasmid DNA in the usual manner.

Sequencing The sequencing of the sequences inserted into the plasmids were determined by the dideoxy method (Messing, Methods in Enzymol., 101, 20-78 (1983)). Three types of SPDS gene, one type of SAMDC gene, and two types of ADC gene were isolated.

Detection of Homology A homology search of the base sequences of these genes against a database of known gene base sequences revealed that the SPDS genes had 70% homology with known plant-derived SPDS genes, that the SAMDC gene had at least 70% homology with known plant-derived SAMDC genes, and that the ADC genes had at least 67% homology with known plant-derived ADC genes.

Obtaining Full-Length Genes Full-length genes were obtained by plaque hybridization.

cDNA libraries were prepared using the ZAP-cDNA Synthesis Kit (Stratagene). The poly 'RNA from the roots of Cucurbita ficifolia Bouche obtained in was used as template, and

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Z oligo(dT) primers were used to synthesize double-stranded

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,q cDNA according to the method of Gubler, Hoffman, et al (Gene, 25, 263-269 (1983)).

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EcoRI adapters (with internal XhoI and Spel sites) were c- 0 ligated to both ends of the resulting cDNA, which was digested with XhoI, the fragments were ligated to the EcoRI 0 and XhoI sites in the arm of a phage vector (X ZAPII) and were then packaged using an in vitro packaging kit (Gigapack Gold, by Stratagene), and the E. coli SURE strain (OD 660=0.5) was infected, giving numerous recombinant X phages.

These were used as the Cucurbita ficifolia Bouche rootderived cDNA library. The library size was 8.0 x 106.

To prepare probe, the insert cDNA was isolated and purified from the plasmid DNA of the SPDS, SAMDC, and ADC genes prepared in the resulting cDNA was used as template, and a Random Primed DNA Labeling Kit (USB) was used to prepare 32 labeled probe. The resulting 32P labeled cDNA was used as probe.

The phages used to construct the Cucurbita ficifolia Bouche root-derived cDNA library were used to infect E. coli for amplification on LB agar medium, and about 50,000 copies of phage DNA were transferred to nylon membranes (HyBond-N, by Pharmacia).

The nylon membranes on which the phage DNA was transferred were transferred onto filter paper containing alkali denaturation solution (0.5 M NaOH, 1.5 M NaC1), the membranes were allowed to stand for 4 minutes, and they were then transferred onto filter paper containing neutralization

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solution (0.5 M Tris-HCl, 1.5 M NaC1, pH 8.0) and allowed to

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Z stand for 5 minutes. The membranes were washed with 2 x SSC 00 0- (0.3 M NaCI, 0.03 M trisodium citrate), and the DNA was then fixed on the membranes using Stratalinker (by Stratagene).

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Cq The nylon membranes on which the DNA had been fixed were cplaced in hybridization solution (50% formamide, 0.5% SDS, 6 x SSPE (3 M NaC1, 0.2 M NaH 2

PO

4 20 mM EDTA-2Na, pH 5 x 0 Denhardt's solution Ficoll, 0.1% polyvinyl pyrrolidone, 0.1% bovine serum albumin), 50 pg/mL denatured salmon sperm DNA) to bring about pre-hybridization for 3 hours at 420C, the cDNA probe that had been prepared was added, and hybridization was brought about for 18 hours at 420C. The membranes were then taken out and washed for 1 to 2 hours at 420C with solution containing 2 x SSC, 1 x SSC, 0.5 x SSC, and 0.1 x SSC. The membranes were dried and were then placed on X-ray film and exposed over night.

Positive clones hybridized with probe obtained from the SPDS, SAMDS, and ADC gene fragments could thus be selected.

Plasmid clones with cDNA inserts were prepared by in vivo excision from the phage DNA of the positive clones. The in vivo excision followed the method in the ZAP-cDNA Synthesis Kit (Stratagene).

A 200 [iL amount of each phage solution containing the SPDS, SAMDC, and ADC genes respectively, 200 pL E. coli XL1- Blue suspension, and 1 pL of helper phage R408 suspension were mixed, the mixtures were incubated for 15 minutes at 37°C, 3 mL of 2 x YT medium was added for 2 hours of shaking culture at 370C, the cultures were treated for 20 minutes at O 70°C and centrifuged (10 minutes at 4,000 x and the >supernatant was recovered. 30 [iL of the resulting

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Z supernatant and 30 [L E. coli SURE suspension were mixed, the 00 Cq mixture was incubated for 15 minutes at 37 0 C, and several jiL was used to inoculate LB agar medium containing 50 ppm

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C- ampicillin for culture over night at 37 0 C. The E. coli forming colonies contained plasmids with cDNA inserts. The

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base sequences of the inserted sequences in the plasmids were Ssequenced by the dideoxy method (Messing, Methods in Enzymol., 101, 20-78 (1983)). The results showed that the plasmids contained start codons.

The resulting full-length spermidine synthase genes from Cucurbita ficifolia Bouche were designated FSPD1 (SEQ ID NOS. 1 and the S-adenosylmethionine decarboxylase gene was designated FSAM24 (SEQ ID NOS. 3 and and the arginine decarboxylase gene was designated FADC76 (SEQ ID NOS. 5 and 6).

The amino acids of the resulting FSPD1 were compared with those of known plant-derived spermidine synthase genes (Table The results of Table 1 show that FSPD1 from Cucurbita ficifolia Bouche roots had high homology at the amino acid level with other plant-derived SPDS genes.

Table 1 Comparison of Cucurbita ficifolia Bouche FSPD1 gene and other SPDS genes Plant Origin Amino acid homology Arabidopsis thaliana 83.8 Nicotiana sylvestris Leaves 82.1 Hyoscyamus niger roots 86.5 The amino acids of the resulting FSAM24 were compared with those of known plant-derived S-adenosylmethionine decarboxylase genes (Table The results of Table 2 show that FSAM24 from Cucurbita ficifolia Bouche roots had high homology at the amino acid level with other plant-derived SAMDC genes.

Table 2 Comparison of Cucurbita ficifolia Bouche FSAM24 gene and other SAMDC genes Plant Origin Amino acid homology Arabidopsis thaliana 66.3 Spinacia oleracea seedlings 63.3 Solanum tuberosum 68.2 Pisum sativum undifferentiated- 65.2 calli The amino acids of the resulting FADC76 were compared with those of known plant-derived arginine decarboxylase genes (Table The results of Table 3 show that FADC76 from Cucurbita ficifolia Bouche roots had high homology at the amino acid level with other plant-derived ADC genes.

Table 3 Comparison of Cucurbita ficifolia Bouche FADC76 gene and other ADC genes Plant Origin Amino acid homology Lycopersicon esculentum fruit 77.1 Nicotiana sylvestris 75.4 Arabidopsis thaliana 70.7 Pisum sativum fruit 70.6 Example 2: Preparation of transgenic Arabidopsis thaliana Preparation of Expression Construct The FSPD1 polyamine metabolism-related enzyme gene given in SEQ ID NO. 1 was cleaved with XhoI in such a way that the entire reading frame of the base sequence was included, and the fragment was purified by the glass milk method. pGEM-7Zf (Promega) was then cleaved with XhoI, and the FSPD1 fragments were subcloned in the sense and antisense directions. The FSPD1 fragments were again cleaved with the XbaI and KpnI restriction enzymes at the multicloning site of pGEM-7Zf, and were subcloned in the sense and antisense direction to the binary vector pBI101-Hm2 to which the promoter had been ligated. The resulting plasmid was designated pBI35S-FSPD1. The structure of this expression construct is given in Fig. 1. Transformed E. coli JM109 was designated Escherichia coli JM109/pBI35S-FSPD1 The polyamine metabolism-related enzyme gene FSAM24 given in SEQ ID NO. 3 was cleaved with NotI in such a way that the entire reading frame of the base sequence was included, and the ends were blunted. The fragments were 0 subcloned to the binary vector pBI101-Hm2 to which the (blunted) 35S promoter had been ligated. The resulting

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Z plasmid was designated pBI35S-FSAM24 Transformed E.

00 coli JM109 was designated Escherichia coli JM109/pBI35S- FSAM24 C' The polyamine metabolism-related enzyme gene FADC76 Sgiven in SEQ ID NO. 5 was cleaved with NotI in such a way that the entire reading frame of the base sequence was Sincluded, and the ends were blunted. The fragments were subcloned to the binary vector pBI101-Hm2 to which the (blunted) 35S promoter had been ligated. The resulting plasmid was designated pBI35S-FADC76 Transformed E.

coli JM109 was designated Escherichia coli JM109/pBI35S- FADC76 Introduction of plasmids to Agrobacterium The E. coli pBI35S-FSPD1 E. coli pBI35S-FSAM24 or E. coli pBI35S-FADC76 obtained in and the E. coli HB101 strain with the helper plasmid pRK2013 were cultured for 1 night at 37 0 C on LB medium containing 50 mg/L kanamycin, and the Agrobacterium C58 strain was cultured for 2 nights at 37 0 C on LB medium containing 50 mg/L kanamycin. Cells were harvested from 1.5 mL of each culture in Eppendorf tubes and then washed with LB medium. The cells were suspended in 1 mL of LB medium, 100 [L each of the three types of cells were mixed to inoculate LB agar medium and cultured at 28 0 C to allow the plasmids to be conjugated with the Agrobacterium (tripartite conjugation). After 1 or 2 days, portions were scraped with a platinum loop and smeared on LB agar medium containing 50 mg/L kanamycin, 20 mg/L hygromycin and 25 mg/L O chloramphenicol. After 2 days of culture at 28 0 C, single colonies were selected. The resulting transformants were 0 ^Z designated C58/pBI35S-FSPD1 C58/pBI35S-FSAM24 and 00 0- C58/pBI35S-FADC76 Transgenic Arabidopsis thaliana was prepared by reduced pressure infiltration through (6)

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C< below) or callus regeneration through (12) below).

Cultivation of Arabidopsis thaliana 0- Potting commcercial nursery soil Metromix (Hyponex 0 Japan) was placed in plastic pots, the surfaces were covered with netting mesh, and 2 to 5 seeds (donated by Professor Takayuki Kohchi of Nara Institute of Science and Technology Graduate University) of Arabidopsis thaliana (referred to below as the "Columbia strain" or "wild type") were inoculated through the interstices of the mesh. The pots were placed for 2 days at 4 0 C in a low temperature chamber to germinate, and were then transferred for cultivation under 22 0 C long-day conditions (16 hour long day/8 hour night).

After about 4 to 6 weeks, lateral shoots were induced by top pruning plants in which the main axis flower stalk was extended to between 5 and 10 cm. After about 1 to 2 weeks of top pruning, the plants were infected with Agrobacterium.

Preparation of Agrobacterium suspension 2 days before infection, the Agrobacterium prepared in above was used to inoculate 10 mL LB medium containing antibiotics (50 tg/mL kanamycin, 20 [ig/mL hygromycin) for 24 hours of shaking culture at 28 0 C. Portions of the culture were transferred to 1000 mL LB medium containing antibiotics jg/mL kanamycin, 20 ig/mL hygromycin) for about another 24 hours of shaking culture at 28 0 C (to an OD 600 of between 0 1.2 and Cells were harvested from the culture at ambient temperature and were resuspended in suspension medium Z for infiltration (0.5 x MS salt, 0.5 x Gamborg B5 vitamin, 1% 00 Ci sucrose, 0.5 g/L MES, 0.44 pM benzylaminopurine, 0.02% Silwet-77) to an OD 600 of between 0.8 and 1.

CA Agrobacterium Infection The potting soil in the pots of Arabidopsis thaliana

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r- prepared in above was watered to prevent the potting soil C from absorbing the Agrobacterium suspension prepared in (4) above. Approximately 200 to 300 mL of the Agrobacterium suspension was placed in 1000 mL beakers, and the potted Arabidopsis thaliana was turned upside down to dip the plants in the suspension. The beakers in which the pots had been placed were put into a dessicator, which was suctioned with a vacuum pump to about -0.053 MPa (400 mmHg), and the plants were then allowed to stand for about 10 minutes. The negative pressure was gradually released, the plants were then taken out of the Agrobacterium suspension, the excess Agrobacterium suspension was wiped off with a Kimtowel, and the pots were placed on their sides in deep-bottomed trays.

A small amount of water was introduced, and the plants were covered with saran wrap. The plants were allowed to stand in this manner for about 1 day. The saran wrap was then removed, and the pots were placed upright and irrigation was stopped for about 1 week. The potting commcercial nursery soil was then gradually watered, and seeds were harvested from matured pods for about 3 to 5 weeks. The harvested seeds were strained through a tea strainer to eliminate debris and husks, O and the seeds were placed in a dessicator and thoroughly dried.

0 Z Obtaining Transformed Plants 00 C 100 pL (about 2000) seeds obtained in above were transferred to 1.5 mL Eppendorf tubes and soaked for 2

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C-I minutes in 70% ethanol and 15 minutes in 5% sodium hypochlorite solution, and the seeds were finally washed five times with sterile water to disinfect the seeds. The 0 disinfected seeds were transferred to 15 mL falcon tubes, about 9 mL of 0.1% aseptic agar solution was added, and the contents were vigorously mixed. A 0.1% agar mixture of seeds was evenly spread on selection medium (1 x MS salt, 1 x Gamborg B5 vitamin, 1% sucrose, 0.5 g/L MES, 0.8% agar, 100 mg/L carbenicillin, 50 mg/L kanamycin, 40 mg/L hygromycin, 8 g/L Phytagar, pH 5.7) like plating the phages. The plates were dried for about 30 minutes in a clean bench, a 4 0 C low temperature treatment was performed for 2 days, the plates were transferred to a 220C growth chamber, and transformants with antibiotic resistance were selected. Plants with about 3 to 5 true leaves were again transferred to fresh selection medium and cultivated until 4 to 6 true leaves had grown.

Transformants with antibiotic resistance (Tl) were planted in pots filled with commcercial nursery soil and acclimated under humid conditions for about 5 to 7 days. After acclimation, the plants were cultivated at 23°C under long day conditions (16 hour long days/8 hour nights). The resulting transgenic plants (Tl) and plants T2 grown from seeds (T2) obtained from the transgenic plants were analyzed for genes introduced by PCR or Southern hybridization and O their levels of expression by Northern hybridization were >analyzed, so as to confirm that the target polyamine Z metabolism-related enzyme genes had been incorporated in a 00 00 consistent manner and that transformants had been expressed.

Seeds T3 were also harvested from the plants T2, and CI antibiotic resistance tests (segregation analysis) were conducted to obtain homozygotes (T2) based on the proportion Sin which transformants appeared. Seeds T2 and seeds T3 0 obtained from the homozygotes (T3 homozygous cell line) were used in the following tests.

Aseptic Arabidopsis thaliana cultivation seeds (donated by Professor Atsuhiko Shinmyo of Nara Institute of Science and Technology Graduate University) of the Arabidopsis thaliana Wassilewskija strain (referred to below as the WS strain) were introduced into 1.5 mL tubes, 1 mL of 70% ethanol was added, and the seeds were allowed to stand for 3 minutes. The seeds were then dipped for 3 minutes in disinfecting solution sodium hypochlorite, 0.02% Triton X-100), washed 5 times with sterilized water, and then planted in MSO plates (4.6 g Murashige-Skoog mineral salts, 10 g sucrose, 1 mL/L 1000 x vitamin stock, pH 6.2).

The plates were allowed to stand for 2 days at 4 0 C to carry out low temperature treatment, and they were then cultured for 21 days in plant incubators (MLR-350HT, by Sanyo) under conditions involving long days (16 hour long days, 8 hour nights) at 220C and a light intensity of 6000 lux. To improve the infection efficiency, the plants were again aseptically plucked out, the roots were spread out on the surface of 0 fresh MSO plates, and the culture was continued for another 2 cdays.

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Z Agrobacterium Infection 00 0- Several roots of the WS strain cultured for 21 days above were arranged and cut with a scalpel to between about

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Cl 1.5 and 2.0 cm, and they were placed alongside each other on CIM plates (MSO plates supplemented with 2,4dichlorophenoxyacetic acid to a final concentration of t pg/mL and kinetin to a final concentration of 0.05 pg/mL).

Samples which had been cultured for 2 days at a light intensity of 3000 lux in 16 hours of light/8 hours of darkness and diluted 3-fold with MS dilution solution (6.4 g/L Murashige-Skoog mineral salts, pH 6.3) were aliquoted in 1 mL portions to tubes, and slices of the roots on which callus was forming were dipped for 10 minutes in the tubes.

The slices were placed on doubled disinfected filter paper, the excess moisture was removed, and the slices were arranged on fresh CIM plates for two days of co-cultivation under the same conditions.

Disinfection Slices on which the strain had grown enough to become visible to the naked eye were transferred to a disinfection solution (MS diluting solution supplemented with claforan to a final concentration of 200 pg/mL) and gently shaken to wash them for 60 minutes. These operations were repeated 5 times, the moisture was removed on sterilized filter paper, and the slices were placed on SIMC plates (MSO plates supplemented with 2-ip to a final concentration of 5 pg/mL, IAA to a final concentration of 0.15 pg/mL, and claforan to a final Sconcentration of 500 ug/mL) for 2 days of culture at a light intensity of 6000 lux in 16 hours of light/8 hours of

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Z darkness.

00 0q (10) Selection of Transformants The slices cultured for 2 days above were transferred C-q to SIMCS plates (SIMC plates supplemented with hygromycin B to a final concentration of 4.6 U/mL) for culture at a light intensity of 6000 lux in 16 hours of light/8 hours of 0 darkness. The slices were subsequently transferred to fresh SIMCS plates every week. The transformed slices continued to be grown, forming dome-shaped callus, but slices that had not been transformed turned brown. After about 2 weeks, the callus of the transformants turned green. After about 1 month, leaves formed, and after that rosettes formed.

(11) Regeneration of Transformants The roots of plants with rosette leaves were cut with a knife or scalpel to leave out the callus and were inserted so as to ride gently on RIM plates. After 8 to 10 days, those on which several roots of about 1 to 2 cm had formed were planted using tweezers and cultivated in rock wool minipots (by Nitto Boseki) soaked with mineral salt medium (5 mM KNO 3 mM K-phosphate buffer (pH 2 mM MgSO 4 2 mM Ca(NO 3 2 jiM Fe-EDTA, 1000 x microelements (70 mM H 3

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3 14 mM MnC1 2 mM CuSO 4 1 mM ZnSO 4 0.2 mM NaMo0 4 10 mM NaC1, 0.01 mM CoC12) 1 mL/L). After flowering and the formation of pods, the plants were transplanted to soil prepared by mixing pearlite and vermiculite (by TES) in a 1:1 ratio, and then soaked with mineral salt media. After about 1 month, several 0 hundred seeds of each strain were obtained. These were subsequently called T2 seeds.

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Z (12) Obtaining Antibiotic Resistant Strains 00 About 100 T2 seeds were sterilized in the same manner as in and used to inoculate MSH plates. Hygromycin B-

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CI resistant strains germinated in a proportion of about 3:1.

(13) DNA Extraction and Southern Hybridization The germinated T2 seeds above were transplanted using 0 tweezers to rock wool minipots soaked with mineral salts and cultured at a light intensity of 6000 lux and a temperature of 22 0 C in 16 hours of light and 8 hours of darkness. After 2 weeks, the top soil was cut away with a scalpel to allow the surface of the rock wool to be stroked with a knife, and samples were immediately frozen with liquid nitrogen. The samples were finely milled in a mortar and pestle in the presence of liquid nitrogen, 3 mL of DNA extraction buffer (200 mM Tris-HCl (pH 100 mM EDTA-2Na, 1% Nlauroylsarcosine sodium, 100 Jg/mL proteinase K) was added per gram, and the ingredients were thoroughly mixed. The mixture was incubated for 1 hour at 60 0 C and then centrifuged minutes at 10,000 x and the supernatant was filtered through mira cloth and transferred to a fresh tube. It was extracted three times with phenol:chloroform:isoamyl alcohol (25:24:1) and then precipitated in ethanol. The precipitate was dissolved in TE buffer. 20 pg each of genomic DNA was obtained from about 2.0 g of each of the plants. 1 ng of DNA was cleaved with the EcoRI and HindIII restriction enzymes for 1% agarose electrophoresis and Southern hybridization.

0 Seeds of untransformed WS strain were germinated and

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allowed to grow, DNA was similarly extracted from the plants 0 Z and digested with the EcoRI and HindIII restriction enzymes 00 0- for 1% agarose electrophoresis and Southern hybridization.

FSPD1, FSAM24, and FADC76 gene fragments were used as C- hybridization probes.

Southern hybridization was performed according to the method in Molecular Cloning, a Laboratory Manual (Chapter 9, 0 pp. 31-58 (Cold Spring Harbor Specifically, electrophoresis of the DNA material on 1% agarose gel was followed by alkali denaturation and Southern blotting overnight on nylon membranes (HyBond-N, by Amersham). The DNA was fixed by 3 minutes of irradiation with a UV transilluminator (254 nm). The membranes were pre-hybridized for 2 hours at 50 0 C in 5 mL of pre-hybridization buffer (5 x Denhardt's, 6 x SSC, 0.1% SDS, 10 [ig/mL salmon sperm DNA) Probes were added for hybridization over night at 50 0

C.

After the hybridization, the membranes were washed twice for minutes with washing solution containing 2 x SSC and 0.1% SDS, and were then washed twice for 30 minutes at 50 0 C with the same solution. The membranes were dried and exposed over night at -80 0 C in cassettes filled with X-ray film (by Kodak) to take autoradiographs. The patterns of the signals detected by Southern hybridization were compared for untransformed strains transformants containing FSPD1, FSAM24, and FADC76 and transformants containing only the vector In addition to the endogenous signal shared in common by and specific signals were observed in EcoRI O digests and HindIII digests of confirming that the target gene had been incorporated in Oz 00 C< Example 3: Northern Blotting Analysis In order to ascertain whether the target gene was

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C0 actually expressed in the T2 transformants obtained in pq Example 2, Northern blotting was performed in the following

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manner.

STotal RNA was extracted from untransformed wild type (WT) and T2 transformant (cell lines: TSP-14, 15, 16, 17, 19) rosette leaves. The RNA was extracted in the same manner as in Example 2. 10 pg of the resulting total RNA was electrophoresed on 1.5% formaldehyde agarose gel and blotted over night on HyBond N nylon membranes. The RNA was fixed with a UV crosslinker and then pre-hybridized for 2 hours at 42 0 C in pre-hybridization buffer (50% formamide, 5 x SSPE, x Denhardt's, 0.1% SDS, 80 pg/mL salmon sperm DNA, pH Probes were prepared with the use of 32 P-dCTP and a random label kit (by Amersham) from the cDNA of the transformed Cucurbita ficifolia Bouche SPDS gene fragment. The probe was added to the pre-hybridization mixture for hybridization over night at 42 0 C. After the hybridization, the membranes were washed twice for 30 minutes at 55 0 C, beginning with a washing solution containing 2 x SSC and 0.1% SDS, and ending with a washing solution containing 0.1 x SSC and 0.1% SDS.

Autoradiographs of the membranes were taken using X-ray film (Kodak).

The results of Northern blotting are given in Fig. 9.

The results in Fig. 9 show that no expression of the 0 exogenous Cucurbita ficifolia Bouche SPDS gene was detected in the wild type but that the Cucurbita ficifolia

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Z Bouche SPDS gene (FSPD1) was expressed in extremely high 00 0- levels in all the T2 transformants.

Example 4: Evaluation of Polyamine Content Ci Selection of cell lines containing target gene Cell lines were selected upon confirmation that the target gene had been introduced by PCR (or Southern analysis) 0 and Northern analysis of the transformants prepared in Example 3, resulting in the selection of cell lines TSP-114, 115, 116, 117, 119, 131, 132, 151, 152, 221 and 222.

Polyamine content was determined with respect to TSP-114 to 115 in which FSPD1 was introduced, TSP-131 and 132 in which FSAM24 was introduced, TSP-151 and 152 in which FADC76 was introduced, TSP-221 and 222 in which FSPD1 was introduced in antisense direction.

Analysis of Polyamine Content Approximately 0.1 to 0.3 g of rosette leaves (or true leaves) from the transgenic plants (TSP) and the wild type plants which were cultivated at the same time, were sampled, frozen and stored in plastic vials which could be tightly sealed. Dilution internal standard solution (1,6hexanediamine, internal standard content 7.5 nmol) and perchloric acid aqueous solution (5 to 10 mL per 1.0 g specimen fresh weight) were added to the sampled specimen, and was thoroughly ground down and extracted using an omnimixer at room temperature. The ground solution was centrifuged at 40 C, 35,000 x g for 20 minutes, and the supernatant was collected and was taken as the free polyamine 0 solution. Four hundred microliters of free polyamine solution, 200 pL of saturated sodium carbonate aqueous 00 Z solution, and 200 pL of dansyl chloride/acetone solution mg/mL) were added into a microtube with a screw cap, and lightly mixed. After firmly closing with a tube stopper and C covering with aluminum foil, dansylation was conducted by pg heating for 1 hour in a 60 0 C water bath. After allowing the

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r tube to cool, 200 iLL of proline aqueous solution (100 mg/mL) Swas added and mixed. The tube was covered with aluminum foil and heated again for 30 minutes in a water bath. After standing to cool, the acetone was removed by spraying nitrogen gas, and then 600 IL of toluene was added and vigorously mixed. After allowing the tube to stand quietly and separate into 2 phases, 300 IL of toluene in the upper layer was separated into a microtube. The toluene was completely removed by spraying nitrogen gas. 100 to 200 pL of methanol was added to the tube and the dansylated free polyamine was dissolved. The free polyamine content of putrescine, spermidine and spermine was assayed by the internal standard method using high performance liquid chromatography connected to a fluorescence detector (exitation wavelength is 365 nm, emission wavelength is 510 nm). A pBondapak C18 (manufactured by Waters, Co.: 027324, 3.9x300 mm, particle size 10 pm) was used for the HPLC column.

The polyamine content in the specimens was calculated by deriving the peak areas of the polyamine and internal standard from the HPLC charts of the standard solution and specimens. The results are indicated in Table 4.

Table 4 Cell line Free polyamine content (nmolg-fw) Putrescine Spermidine Spermine Total S___polyamines Wild type:WT 5.41±3.74 108.99±12.63 11.95±2.92 126.35±16.04 TSP-114 6.06±3.04 149.96±11.64 23.68±2.06 179.70±2'0.82 TSP-115 8.33±2.05 175.76±16.30 21.53±1.29 205.62±20.82 TSP-116 10.66±3.98 182.94±23.73 21.36±6.48 214.96±29.41 TSP-117 12.40±3.89 177.45±12.70 13.33±1.07 203.18±16.77 TSP-119 7.82±3.55 169.13±36.97 21.59±3.10 198.54±41.49 TSP-131 7.02±3.10 165.55±11.54 19.11±2.00 191.68±14.66 TSP-132 8.34±3.65 162.34±24.01 19.04±7.01 189.72±29.89 TSP-151 10.99±3.82 159.39±12.45 18.98±1.51 189.36±15.89 TSP-152 11.98±3.72 158.99±12.93 18.54±2.90 189.51±16.54 TSP-221 4.89±1.69 74.11±8.57 7.37±0.16 86.36±8.08 TSP-222 4.99±1.00 71.31±8.77 7.11±1.03 83.41±9.13 Table 4 shows that cell lines TSP-114, 115, 116, 117, 119, 131, 132, 151 and 152 in which the polyamine metabolismrelated enzyme gene was introduced in sense direction had significantly higher levels of putrescine, spermidine and spermine contents than the wild type and that the total polyamine content was also significantly higher than in the wild type In particular, spermidine and spermine contents were increased remarkably. In addition, TSP-221 and 222 in which the polyamine metabolism-related enzyme gene was introduced in antisense direction had significantly lower levels of, in particular, spermidine and spermine contents than the wild type and that the total polyamine content was also significantly lower than in the wild type (WT).

The results showed that the introduction of the polyamine metabolism-related enzyme gene into plants in sense or actisence direction allowed the polyamine content to be increased or decreased. The results showed that the introduction of the polyamine metabolism-related enzyme gene into plants allowed the polyamine content to be controlled through the manipulation of polyamine metabolism.

0 Z Example 5: Evaluation of various types of morphogenesis 00 ¢c Evaluation of the number of stems, leaves, flowers and pods

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CI Seeds of T3 transgenic plants obtained in example 2 c-i (cell lines: TSP-16-1, 16-2, in which FSPD1 was introduced in the sense direction) and of a wild type plant (WT: Columbia Sstrain) were planted in plastic pots containing Metromix potting soil (manufactured by Hyponex Japan, After planting and treating at a low temperature of 4° C for approximately 2 days, the pots were placed in plastic vats, and cultivation under long day conditions (day length of daylight/dark: 16 hours/8 hours) at 230 C was started. The plants were grown for 15 weeks after planting. After growing, the number of stems (flower stalks) per individual plants in each line was investigated. Thirty individual plants of each line were studied. The results are indicated in Fig. 3.

The results of Fig. 3 demonstrate that compared to the wild type plant, the T3 transgenic plants (TSP-16-1, 16-2) had a significant increase in the number of stems by approximately 7 to 8 stems per plant. Further, it was demonstrated that by increasing the number of stems per plant, the number of leaves (cauline leaves), flowers and pods (ovaries) also increased notably. The plant habit of the wild type plant and TSP-16-1 are indicated in Fig. 4.

Evaluation of the number of main stems and side stems From the results of it was confirmed that the number of stems significantly increased in the T3 transgenic plants Z compared to the wild type plant. Then, because the stems of 00 Arabidopsis thaliana may be divided into main stems (main flower stalks), and side stems (side flower stalks), the CI decision was made to study the respective numbers of main and side stems. Seeds of T3 transgenic plants (cell lines: TSP-

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161B, 162B, in which FSPD1 was introduced in the sense Sdirection) and of a wild type plant (WT: Columbia strain) were planted in plastic pots containing Metromix potting soil (manufactured by Hyponex Japan, After planting and treating at a low temperature of 40 C for approximately 2 days, the pots were placed in plastic vats, and cultivation under long day conditions (day length of daylight/dark: 16 hours/8 hours) at 230 C was started. The plants were grown for 7 weeks after planting, and from week 7 (day 49) until week 13 (day 91) the respective numbers of main stems (main flower stalks) and side stems (side flower stalks) per individual plant in each line were investigated once per week.

Thirty to 32 individual plants of each line were studied.

The results are indicated in Figs. 5 and 6.

From the results of Fig. 5, the number of main flower stalks of the T3 transgenic plants increased significantly from day 49 compared to those of the wild type plant, and on day 70 there was an increase of approximately 1 stem per plant in the T3 transgenic plants compared to the wild type plant. The extent of increase in main flower stalks of the T3 transgenic plants and the wild type plant continued about the same until day 91, and the final number of main flower stalks was higher in the T3 transgenic plants than in the

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wild type plant.

0 Z From the results of Fig. 6, the number of side flower 00 0- stalks of the T3 transgenic plants increased significantly from day 49 compared to those of the wild type plant, and on (C1 day 70 there was an increase of approximately 4 stems per c plant in the T3 transgenic plants compared to the wild type plant. On day 91, there was an increase of approximately Sto 6 stems per plant in the T3 transgenic plants compared to the wild type plant, and the final number of side flower stalks was notably higher in the T3 transgenic plants than in the wild type plant.

Evaluation of the number of leaves, flowers and pods From the results of it was confirmed that the number of leaves, flowers and pods (ovaries) significantly increased in the T3 transgenic plants compared to the wild type plant. Then, a detailed study of the number of various organs was conducted. Seeds of a T3 transgenic plant (cell lines: TSP-15-3, 16-1, in which FSPD1 was introduced in the sense direction) and of a wild type plant (WT: Columbia strain) were planted in plastic pots containing Metromix potting soil (manufactured by Hyponex Japan, After planting and treating at a low temperature of 4° C for approximately 2 days, the pots were placed in plastic vats, and cultivation under long day conditions (day length of daylight/dark: 16 hours/8 hours) at 230 C was started. The plants were grown for 67 days after planting, and the number of leaves (cauline leaves), flowers with white petals, and pods (pod length 5 mm or more) per individual (plant) in each O line was investigated on day 67. Thirty individual plants of each line were studied. The results are indicated in Table ^Z Table 00 (Number per plant) Cell line True leaves Flowers Pods \s Wild type plant (WT) 10.73 9.60 6.67 ri TSP-15-3 19.27 13.93 10.00 TSP-16-1 18.33 14.27 12.67 mc The results of Table 5 demonstrate that T3 transgenic ci plants have a significantly increased number of true leaves, flowers and pods per plant compared to the wild type plant.

After this investigation, the pods of all lines were allowed to mature and dehisce, and the mature seeds were harvested.

No significant difference between the T3 transgenic plants and the wild type plant was revealed in the number of seeds per pod, but it was demonstrated that the total number of seeds harvested per plant increased significantly in the T3 transgenic plants as a result of the number of pods increasing about 1.6 to 2.0 times per plant. When conducting a comparative test of the germination rate using seeds from the wild type plant and T4 seeds harvested from the T3 transgenic plants, a high germination rate of 95 to 100% was obtained for both the T4 seeds and the wild type seeds.

Evaluation of flower blooming period, and ovary (pod) development period Seeds of the T3 transgenic plant obtained in example 2 (cell lines: TSP-15-3, 16-1, in which FSPD1 was introduced in the sense direction) and of a wild type plant (WT: Columbia strain) were planted in plastic pots containing Metromix r- 0 potting soil (manufactured by Hyponex Japan, After planting and treating at a low temperature of 40 C for Z approximately 2 days, the pots were placed in plastic vats, 00 0-0 and cultivation under long day conditions (day length of daylight/dark: 16 hours/8 hours) at 230 C was begun. The

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days to initial bolting and the days to blooming (first f flower) were studied in all plants of the various lines, and the plants were studied from the day that the first flower bloomed until the day that the pod (ovary) matured (day to beginning of dehiscence). Thirty individual plants of each line were studied. The results of the days to blooming are indicated in Fig. 7, and the results of the pod (ovary) development period are indicated in Table 6.

The results of Fig. 7 demonstrate that the days to blooming of the T3 transgenic plant is earlier than that of the wild type plant. It was confirmed that, on average, T3 transgenic plants are about 6 to 7 days earlier than the wild type plant.

Table 6 (Number of days after planting) Line Days to Days to pod Development blooming maturation period Wild type 59 78 19 plant (WT) TSP-15-3 52 76 24 TSP-16-1 53 76 23 The results of Table 6 demonstrate that the pod (ovary) development period of the T3 transgenic plants is significantly increased compared to that of the wild type plant.

0 Example 6: Evaluation of transgenic petunia Production of transgenic petunia 0 Z pBI101-35S-FSPD1+ and pBI101-35S-FSAM24+ were introduced 00 0- into Agrobacterium tumefacience AglO strain (Lazo et al.

BioTechnology 9:963-967 (1991)) by a method using calcium

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C1 chloride (for example, Handbook of New Biochemistry Experiments 3, Kagaku Dojin page 121-122, 1996). The transformed Agrobacterium thus obtained was transmitted Srespectively onto leaf disks derived from the petunia (Petunia hybrida) variety Safinia Purple Mini (Suntory Flowers Co., Ltd.), and transgenic petunia were obtained by selecting plant cells indicating resistance to hygromycin, and by re-differentiation. The transformation of petunia was conducted using well-known methods (Horsch et al. Science 227: 1229-1231 (1985)). The various petunia transformed by pBI101-35S-FSPD1+ and pBI101-35S-FSAM24+ were taken to be Experiment PT144 and Experiment PT146. Twenty-three independent transgenic plants were obtained in PT144, and 43 plants in PT 146.

Analysis of the expression of introduced genes The chromosomal DNA and RNA from these plants were prepared using the DNeasy Plant Mini Kit and RNeasy Plant Mini Kit (Qiagen Co., Ltd., Tokyo) respectively. It was found that PT144 and PT146 petunia are transgenic plants that retain SPDS (FSPD1) and SAMDC (FSAM24) genes respectively based on PCR reactions conducted using chromosomal DNA as a template (for PT144, the PCR reaction was conducted using primer FSPD1F (5'-TAGTAGAGGGATTATTATGTCTGCGGA-3') and FSPD1R (5'-ATGACGCTGATCACAATATAAAGCGAC-3'); and for PT146, primer 0 FSAM24F (5'-TGAAGGCTATGAAAAGAGGCTTGAAGTA-3') and FSAM24R TCATGAGGATTGACCTTGGGATGACG-3'). After maintaining at 95 0

C

Z for 1 minute, the reaction was conducted in 30 cycles of a 00 eCq cycle comprising 95°C 1 minute, 52 0 C 2 minutes, 720 C 3 minutes, and then maintaining at 72 0 C for 1 minute more.) C Moreover, using whole RNAs extracted from these transgenic petunia leaves, the genetic expression of the various individuals were analyzed based on RT-PCR, wherein a reverse Stranscription reaction was conducted using a superscript fast strand synthesis system for RT-PCR (Invitrogen Co., Ltd., Tokyo) and using Oligo(dT)12-18 primer, and wherein PCR was conducted by the same primers and cycles concerning chromosomal DNA using reverse transcription product as a template. The expression of foreign genes (SPDS in PT144; SAMDC in PT146) was confirmed in 9 individuals in PT144 plants, and 27 individuals in PT146 plants.

Evaluation of polyamine content The free polyamine content was analyzed in the individuals of the transgenic plants (cell lines) that had the most expression. Approximately 0.3 to 0.6 g of leaves from the transgenic plants (PT144, PT146) and the wild type plants (original strains: PT144-C, PT146-C), which were cultivated at the same time, were sampled, frozen and stored.

Dilution internal standard solution (1,6-hexanediamine, internal standard content 7.5 nmol) and 5% perchloric acid aqueous solution (5 to 10 mL per 1.0 g specimen fresh weight) were added to the sampled specimen, and was thoroughly ground down and extracted using an omnimixer at room temperature.

The ground solution was centrifuged at 40 C, 35,000 x g for minutes, and the supernatant was collected and was taken as the free polyamine solution. Four hundred microliters of 0 Z free polyamine solution, 200 |tL of saturated sodium carbonate 00 0- aqueous solution, and 200 pL of dansyl chloride/acetone solution (10 mg/mL) were added into a microtube with a screw

\O

Cq cap, and lightly mixed. After firmly closing with a tube stopper and covering with aluminum foil, dansylation was conducted by heating for 1 hour in a 600 C water bath. After 0 allowing the tube to cool, 200 pL of proline aqueous solution (100 mg/mL) was added and mixed. The tube was covered with aluminum foil and heated again for 30 minutes in a water bath.

After standing to cool, the acetone was removed by spraying nitrogen gas, and then 600 pL of toluene was added and vigorously mixed. After allowing the tube to stand quietly and separate into 2 phases, toluene in the upper layer was separated into a 300-pL microtube. The toluene was completely removed by spraying nitrogen gas. 100 to 200 pL of methanol was added to the tube and the dansylated free polyamine was dissolved. The free polyamine content of putrescine, spermidine and spermine was assayed by the internal standard method using high performance liquid chromatography connented to a fluorescence detector. A gBondapak C18 (manufactured by Waters, Co.: 027324, 3.9x300 mm, particle size 10 pm) was used for the HPLC column. The polyamine content in the specimens was calculated by deriving the peak areas of the polyamine and internal standard from the HPLC charts of the standard solution and specimens. The results are indicated in Table 7. The mean value of free putrescine content in wild type plant (144-C, 146-C) was 0 29.46 nmol/gFW (FW: Fresh Weight), while the mean value in transgenic plant (144, 146) was 38.67 nmol/gFW. The mean 00 Z value of free spermidine content in wild type plant was 37.03 C- nmol/gFW (FW: Fresh Weight), while the mean value in transgenic plant was 50.40 nmol/gFW. The amounts of free C< putrescine and free spermidine had both increased. The results of a t-test between the wild type plant and the transgenic plants using a 5% significance level revealed a 0 difference in free spermidine, and demonstrated that the amount of spermidine in particular had increased in the transgenic plant. It was further confirmed that the free spermine content in the PT146 transgenic plant had increased significantly compared to the wild type plant (146-C).

Table 7 Individual Free putrescine Free spermidine No. (nmol/gFW) (nmol/gFW) 144-C-1 24.44 46.2 144-C-2 15.05 31.38 144-6 30.12 65.7 144-7 46.92 55.54 144-8 23.74 59.06 144-9 29.14 71.22 144-12 26.28 80.38 144-13 22.16 43.05 144-15 24.13 48.24 144-17 20.55 33.25 144-18 32.26 36.95 146-C-1 38.56 36.45 146-C-2 39.8 34.08 146-2 58.65 39.87 146-5 47.42 32.66 146-10 36.76 29.12 146-11 29.99 66.14 146-12 39.42 32 146-15 30.26 31.89 146-16 55.48 33.79 146-18 64.25 51.85 146-19 49.87 50.96 146-20 52.55 50.23 146-21 52.55 68.67 146-25 45.15 44.6 146-27 24.02 61.25 146-34 27.01 62.67 146-37 37.69 40.55 146-39 38.9 43.16 146-41 60.02 77.8 Evaluation of morphological modification of transgenic petunia Petunias were cultured in a closed system greenhouse with an air temperature of approximately 250 C and artificial light supplementing natural light to make a 16-hour light, 8hour dark cycle. The changes in the number of flowers per individual plant were measured over time from immediately after pruning to day 60 for individual plants that expressed O foreign genes. The results are indicated in Table 8, Figs. 8, and 9. The results of Table 8, Figs. 8 and 9 clearly reveal 4 that there were increased numbers of branches and flowers in 00 C( the transgenic petunia compared to the original stock (wild type plant). During the measurement period, the number of

NO

(C1 flowers of the original stock Safinia Purple Mini did not r exceed 10, but in 6 of the PT144 plants and in 18 of the PT146 plants the number of flowers exceeded SMoreover, in many of the plants of the original stock, the number of flowers per branch was 0 to 3, and hardly any branches with more than 5 flowers were observed. However, a high percentage of the transgenic plants had branches with more than 5 flowers, including 3 of the PT144 plants (33%) and 5 of the PT146 plants Table 8 Plant Highest number of Number of Number of No. flowers on 1 branch branches flowers 144-6 3 5 9 144-7 2 3 3 144-8 6 9 14 144-9 5 10 14 144-12 2 5 9 144-13 6 8 13 144-15 3 6 144-18 3 4 7 144-C1 2 1 2 144-C2 4 5 8 146-2 3 8 12 146-3 5 7 17 146-4 4 6 11 146-5 4 5 11 146-10 3 6 13 146-11 2 6 8 146-12 2 4 7 146-15 5 5 146-16 4 3 8 146-18 2 2 3 146-19 4 4 11 146-20 3 6 9 146-21 2 6 12 146-22 4 3 6 146-25 3 6 146-26 2 7 12 146-27 5 5 13 146-31 2 6 9 146-32 4 4 8 146-34 1 3 2 146-35 4 6 17 146-36 2 5 4 146-37 5 5 9 146-39 7 3 12 146-41 2 3 6 146-42 2 3 146-43 3 5 9 146-C1 1 1 1 146-C2 2 3 6 The correlation between the increase in the amount of polyamine and the increase in the number of flowers and the 004991823 number of branches in petunia is found, and it has been C demonstrated that introduced genes increase the amount of polyamine and bring about an increase in the number of flowers Z and the number of branches. In order to investigate whether the 00 C- 5 increase in the number of flowers observed here is caused by lengthening the blooming period of the individual flowers, or is ND caused by increasing the number of flower buds simultaneously C(l blooming, the length of the blooming period was investigated in the individual plants that had the most flowers per branch (PT144 5 plants, PT146 9 plants), wherein the period from immediately after the flower blooms until the petals wither was taken as the blooming period; five flowers per individual plant were measured; and the mean values were compared. The mean number of blooming days of the original stock was 8.34 days, and mean number of blooming days of the transgenic plants was 9.35 days, and thus the blooming period tended to increase in the transgenic plants.

Claims (12)

1. A method for producing a plant that stably retains an O Z exogenous S-adenosylmethionine decarboxylase (SAMDC) gene under 00 the control of one or more promoters that can function within the plant, and that has improved morphogenesis compared to the DO plant lacking said exogenous SAMDC gene, comprising the steps (C of: (Nc S transforming cells of a plant lacking said exogenous SAMDC Sgene with said SAMDC gene, [0 regenerating plants from the transformed cells, and cultivating the plant bodies under normal conditions; wherein the improved morphogenesis is selected from the group consisting of: increased number of stems, main stems, side stems, flower stalks, trunks or branches; (ii) increased number of leaves; (iii) increased number of flowers; (iv) extended flower blooming period; increased number of ovaries, fruit, cotton balls, pods, ears and capsules; (vi) increased development period from bearing to maturity of ovaries, fruit, pods and capsules; (vii) increased number of seeds, grains or ovules; and 005059126 72 S (viii) increased number of roots or tuberous roots. (N

2. A method for producing plants with improved morphogenesis O Z compared to plants lacking an exogenous S-adenosylmethionine S decarboxylase (SAMDC) gene, comprising the steps of: (N ,D 5 transforming cells of a plant lacking said exogenous SAMDC Ci gene with at least one expression vector containing at least 1- C one exogenous SAMDC gene under the control of one or more (N promoters capable of functioning in plants, regenerating plants from the transformed cells, and cultivating the plant bodies under normal conditions; wherein the improved morphogenesis is selected from the group consisting of: increased number of stems, main stems, side stems, flower stalks, trunks or branches; (ii) increased number of leaves; (iii) increased number of flowers; (iv) extended flower blooming period; increased number of ovaries, fruit, cotton balls, pods, ears and capsules; (vi) increased development period from bearing to maturity of ovaries, fruit, pods and capsules; (vii) increased number of seeds, grains or ovules; and 005059126 73 S (viii) increased number of roots or tuberous roots. CN

3. A method according to Claim 1 or 2, wherein said O Z S-adenosylmethionine decarboxylase gene comprises a base 0 0 sequence of or below: 5 the base sequence represented by base numbers 456 through C-i 1547 in the base sequence given in SEQ ID NO. 3; 1c a base sequence coding for a protein with S-adenosylmethionine decarboxylase activity and hybridizing under stringent conditions with the base sequence of (a) above; and a base sequence coding for a protein with S-adenosylmethionine decarboxylase activity, comprising the base sequence of or with 1 or more bases deleted, substituted, inserted or added.

4. A method according to any one of Claims 1 to 3, wherein morphogenesis is improved in reproductive stages more than in vegetative stages.

A method according to any one of Claims 1 to 4, wherein the plant with improved morphogenesis is a plant with improved number of stems.

6. A method according to any one of Claims 1 to 4, wherein the plant with improved morphogenesis is a plant with improved number of leaves.

7. A method according to any one of Claims 1 to 4, wherein the plant with improved morphogenesis is a plant having an improved number of flowers and/or flower blooming period (time of initial blooming or period in bloom). 005059126 74 0

8. A method according to any one of Claims 1 to 4 wherein the S plant with improved morphogenesis is a plant having an improved number of ovaries and/or ovary development period. z 00

9. A method according to any one of Claims 1 to 4, wherein the plant with improved morphogenesis is a plant with improved 1D number of seeds. c

10. A method according to any one of Claims 1 to 9, wherein the (N plant with improved morphogenesis is dicotyledons. (<i

11. A method for producing a plant with fixed traits, which is a homozygote with respect to an exogenous S-adenosylmethionine decarboxylase (SAMDC) gene and which has improved morphogenesis compared to plants lacking said nucleic acid sequence, comprising the steps of: transforming cells of a plant lacking said exogenous SAMDC gene with at least one expression vector containing at least one said exogenous SAMDC gene under the control of one or more promoters capable of functioning in plants; regenerating plants with improved morphogenesis compared to plants lacking said exogenous SAMDC gene from the transformed cells; cultivating the plant bodies under normal conditions; harvesting seeds by pollination from the plant bodies; and assaying the nucleic acid sequence in the seeds obtained by pollination from the plant bodies, which have been obtained by cultivation of the seeds so as to select a homozygote with respect to the SAMDC gene; 005059126 0 wherein the improved morphogenesis is at least one selected from 0 N' the group consisting of: Z increased number of stems, main stems, side stems, flower 00 stalks, trunks or branches; 5 (ii) increased number of leaves; S(iii) increased number of flowers; (N (iv) extended flower blooming period; (N increased number of ovaries, fruit, cotton balls, pods, ears and capsules; [0 (vi) increased development period from bearing to maturity of ovaries, fruit, pods and capsules; (vii) increased number of seeds, grains or ovules; and (viii) increased number of roots or tuberous roots.

12. A method according to any one of Claims 1, 2 or 11, substantially as hereinbefore described with reference to the examples.