CA2262779A1 - Methods for altering the rate of plant development and plants obtained therefrom - Google Patents

Abstract

The present invention is based on the unexpected observation that DNA
methylation, particularly at cytosine residues, regulates the rate of development of a plant. Based on this observation, the present invention provides methods of increasing or decreasing the rate of development of a plant by either increasing or decreasing the amount of methylated DNA found in the plant. The present invention further provides plants that have been altered such that their rate of maturation is either increased or decreased relative to the rate of maturation of a non-altered plant.

Description

wo 98/04725 PCT/US97/13358 METHODS FOR ALTERING THE RATE OF PLANT D~VELOPMENT AND
PLANTS OBTAINED THEREFROM

This application claims the benefit of U.S. Provisional Application No.60/023,314. filed July 31, 1996, the specification of which is hereby incorporated by reference in its entirety.
STATEMENT OF RIGHTS TO INVENTIONS M~DF UNI)FR
5FEDERALLY SPONSORFT) RFSEARCH
This invention was made with governm~nt~l support under National Institutes of Health grant GM38148 and National Science Council grant 81 -0203-B001-14 The Government has certain rights in the invention.
TECHNICAL FIELD
10The present application is in the field of plant developmental biology and relates to methods for altering the rate at which a plant develops using molecular genetic techniques BACKGROUNl~ ART
Plant genomes contain relatively large amounts of the modified nucleotide 155-methylcytosine (SmC) (Y GrePnh~llm, et al., Nature 292: 850 (1981)). Despiteevidence implicating cytosine methylation in plant epigenetic phenomena, such asrepeat-induced gene silencing (TIGS), co~u~ es~ion, and inactivation of transposable elements (F. F. Assaad, et al,, Plant Mol, Biol. 22: 1,057 (1993); C. Napoli, et al., SUE~STITUTE SHEET (RULE 26) CA 02262779 l999-0l-29 W O g8/04725 PCT~US97/13358 Plant Cell 2: 279 (1990); J. Bender et al., Cell 83: 725 (1995); P.S. Chomet, et al., Genetics 138: 213 (1994); R.A. Martienssen, et al., Curr. Opin. Genet. Dev. 5: 234 (1995); M.A. Matzke, et al., Plant Physol., 107: 679-685 (1995)), the role of cytosine methylation in plant developmental processes is not clear.
In Arabidopsis, ddm mutants (decrease in DNA methylation) have been isolated with reduced levels of cytosine methylation in repetitive DNA sequences, although these mutations do not result in any detectable change in DNA
methyltransferase enzymatic activity (A. Vongs, et al., Science, 260: 1,926 (1993), T.
Kakutani, et al., Nucleic Acids Res. 23: 130 (1995)). After several generations of self-pollination, ddm mutants exhibit a slight delay (1.7 days) in flowering, altered leaf shape, and an increase in cauline left number (T. Kakutani, et al. (1995)).The exact mech~nisms that mediate plant development are l~iesel1lly not well understood. Plants that have an increased rate of development would be highly useful in plant breeding programs. Specifically, numerous plants, such as tree species, have extremely long generation times and therefore the number of crosses that can be generated within a given year or plant cycle is limited. In one extreme case, certain species of bamboo flower only once every one hundred years. Methods which could be used to decrease the maturation time would be highly beneficial in breeding programs involving many plants.
A reduction in the rate a plant matures can be used to increase the biomass production of a given plant. For numerous plants, increases in biomass yields would SUBSTITUTE SHEET (RULE 26) W 098/04725 PCTrUS97113358 increase the economic value of the commercial plant. For example, flax, tobacco, alfalfa, spinach, lettuce, etc.
It is therefore the focus of the present invention to provide methods for increasing or decreasing the time required for a plant to mature as well as plants 5 which are produced by these methods.
All references disclosed throughout this application are hereby incorporated by reference in their entirety.
DISCLOSURF OF THF INVENTION
The present invention is based on the unexpected observation that a decrease 10 of about 70% in the amount of methylated DNA present in a plant genome results in a plant that requires more time to mature while an increase in the amount of methylated DNA present in a plant genome results in a plant that requires less time to mature.
Based on these observation the present invention provides methods of altering a plant, plant cells, plant tissues or plant seeds, so as to obtain a plant that has an altered rate 1 S of maturation. In one method, the rate of maturation is increased by altering the plant, plant cells, plant tissues or plant seeds, using molecular techniques, such that the plant has a sufficient increase in methylated DNA so as to yield a plant that matures faster than a non-altered plant. In another embodiment, the rate of maturation is decreased ~y altering a plant, plant cells, plant tissues or plant seeds, using molecular 20 techniques, such that the plant has a sufficient decrease in methylated DNA so as to yield a plant that matures slower than a non-altered plant.

SUBSTITUTE SHEET (RULE 26) .

W O 98/04725 PCTrUS97113358 The present invention further provides plants that have an altered rate of maturation that have been produced using the methods herein described.

BRIEF DESCRIPTION OF THE DRAWrNGS
Figure l. The predicted METI gene product and antisense construct.
S A diagramrnatic representation of the predicted gene product of the METl locus is provided. The METl protein is a 1,534-amino acid protein with a high degree of homology to the mouse MTase, particularly in the catalytic and NH2 -tt~.T min~l foci targeling domains (E. J. Finnegan, et al., N2~cleic Acids Res. 21: 2,383 (1993)). The METI antisense construct is shown in the bottom of the figure. See Example 1.

Fi~ure 2. Southern analysis of ,e~elili~fe and single-copy DNA methylation patterns.
Total genomic DNA (3 ~g/lane) from ~ntisen~e lines, wild-type, and the ddml mutant were digested with Hpa II (left panels) or Msp I (right panels), subjected to electrophoresis in 0.8% agarose, and transferred to Zeta-Probe membranes (Bio-Rad) and hybridized to probes as follows: (Upper panels) filters were probed with a l 80-bp centromere repeat (J. M. Martinez-Zapater, et al., Mol. Gen. Genet, 204: 417 (1985)) and 55 rDNA (B. R. Campbell, e~ al., Gene 112: 225 (1992)); (Lower panels) filters were probed with four single-copy gene probes--PHOSPHORIBOSYLANTHRANILATE TRANSFERASE 1 (PAT1), PROLIFERA
(PRL), CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), and ERECTA

SUBSTITUTE SHEET (RULE 26) (ER). Digestion of wild-type genomic DNA with Eco RII and Bst Nl, isochizomers with differential sensitivity to cytosine methylation in the motif C5mC(A/T)GG, showed only minor differences in wild-type DNA when probed with the centromere repeat and SS rDNA probes suggesting C(A/T)G methylation may not be 5 prevalent-shown by dirr~elllial Hpa II-Msp I digestion of wild-type DNA to contain C5mCGG methylation M. J. Ronemus, et al., data not shown. The panel on the right was hybridized to a control gene, LEAFY (LFY) (D. Weigel, et al., Cell 59: 843 (1992)), shown not to be methylated in wild-type DNA.

Fi~ure 3. Southern and phenotypic arlalyses of Tr246 outcross progeny.
Genomic DNAs (4 ~Lg/lane) from outcross progeny of strong antisense line Tr246 to wild-type Arabidopsis (Columbia strain with no mutations) were digested with Hpa 11 (Fig. 3 upper panels) or Eco RI (Fig. 3 lower panels). Southern analyses were done as described above. Filters were probed with the 180-bp centromere repeat (Martinez-Zapater et al. (1985) (Fig. 3 upper panels) or the CaMV 35S promoter 15 fragment from pMON530 (Fig. 3 lower panels). Symbols indicate the presence (+) or absence (-) of the antisense transgene. Phenotypic data for each individual plant are shown below each lane. Plants were grown under continuous light at 21~C.

SIJ~S 1 l l UTE SHEET (RULE Z6) ., .

W O 98/04725 PCTrUS97/13358 GENERAL DESCRIPTION
BEST MODE FOR CARRYING OUT THE INVENTION
General Description The present invention is based on the unexpected observation that DNA
5 methylation, particularly at cytosine nucleotides, is involved in regulating the rate of plant development and maturation, particularly the rate at which flower producing organs and structures mature. Specifically, the present invention is based on the observation that decreasing the amount of methylated DNA results in plants that mature at a slower rate than plants cont~ining normal amounts of methylated DNA
10 while increasing the amount of methylated DNA leads to plants that mature at a faster rate than plants with normal amounts of methylated DNA.
Based on these observations, the present invention provides methods for altering the rate of maturation of a plant which comprises the step of genetically altering the plant, using molecular techniques, so the plant has an altered degree of 15 DNA methylation when compared to a non-altered plant sufficient to alter the rate at which the plant develops.
As used herein, maturation refers to the process of plant differentiation leading to the production of flowers and other reproductive tissues. The rate of maturation is said to be altered when the rate at which the plant develops flowers is either increased 20 or decreased relative to a non-altered plant. The degree that the rate of maturation is SUBSTITUTE SHEET (RULE 26) altered will vary from plant to plant as well as between plants that have been altered using dir~lent methods.
In a ~.lef~.led embodiment, the method will produce plants with an increased rate of maturation of about 10% to about 25% faster, more preferably about 25% to 5 about 50% faster, than the normal rate of maturation. For exarnple, in Arabidopsis, flowers typically develop after 26 days under long day conditions. In the Exarnples, Arabidopsis plants were obtained which developed flowers after 10 to 12 days.
The result of accelerating plant development is the ability to decrease the generational time in plant breeding prograrns. Plants that have been altered using the 10 methods herein described for decreasing maturation times can be used in any conventional breeding program, producing accelerated results.
In another plefell~d embo~iim~nt the methods of the present invention will produce plants with a decreased rate of maturation that is about 10% to about 25%
slower, more preferably about 25% to about 50% slower, most preferably about 50%
15 to about 100% or more slower than the normal rate of maturation. In the Examples, altered Arabidopsis plants were obtained that matured and produced flowers at 45-47 days, representing an almost 100% increase in the days required for m~tl-r~tion when compared to non-altered plants.
When used to slow the rate of maturation, the present methods produce plants 20 that have increased biomass. As used herein, biomass refers to the total plant weight, SUBSTITUTE SHEET (RULE 26) ... ..

particularly leafy material. The amount of leaf material is increased in plants altered to have a slower rate of maturation.
Another consequence of delaying flower production is to produce plants that have more secondary branches and axial nodes. As a result, the plant, when it 5 matures, produces a larger number of flower organs than does the non-altered plant.
Any plant that can be genetically altered using molecular techniques and any plants propagated cont~inin~ the genetic alteration can be used in the present method.
Methods known in the art for molecularly altering a plant are discussed in detail below. The preferred plants include both dicot and monocotyledonous plants. The 10 most preferred plants are plants with economic value as a food or biomass source, or plants with long maturation times. Such plants include, but are not limited to, leafy plants such as tobacco, spinach, lettuce, and seed bearing plants such as rice, corn, soy bean etc.
The methods of the present invention rely on altering plants using molecular 15 techniques. As used herein, molecular techniques exclude classical genetic techniques such as breeding/selection, identific~ti- n of random mutagenesis and chemical mutagenesis techniques. Molecular techniques refers to procedures in which DNA is manipulated in a test tube during at least one stage of the process, such as the direct manipulation of DNA or the use of shuttle host such as bacteriurn. Such methods are 20 well known in the art and are described in, for example, Sambrook, et al., Molecular Cloning. a ~aboratory Manual, Cold Spring Harbor Press (1g89). Some of the SU~3 111 UTE SHEET (RULE 26) techniques that are used to alter a plant are ~i~c~se(l in more detail below.
Molecular techniques are dirr~ t~cl from classical genetics in which randomly occurring spontaneous mutants or classical mutagenic techniques are applied to agiven plant type.
The requirement of the use of molecular techniques is to avoid the present invention reading on altered plants presently known in the art that have been generated through non-molecular techniques such as random mutation. Such plants may be known to have an altered rate of development but the underlying mech~ni~mwas not known prior to the present invention. Based on the present invention, it is likely that such plants will be found to have alterations in DNA methylation.
Although the present invention is based on the use of molecular techniques, a skilled artisan can now employ a new selection criteria when altering plants using non-molecular methods; namely selecting plants generated tllrough methods such as chemical mutagenesis for increased or decreased DNA methylation. Specifically, plants and plant cells can be subjected to chemical mutagenesis, physical mutagenesis and the like and the resulting plant selected based on alterations in amount of DNA
methylation or in the activity of the DNA methyl transferase. Plants can then befurther propagated from the isolated and idçntifiç~l variants as having altered amounts of DNA that then correlates to an altered rate of maturation.

SUBSTITUTE SHEET (RULE 26) . . .

W 098/0472~ PCT~US97/13358 Methods to Decrease the Rate of Maturation As provided above, the methods of the present invention can be used to alter plants such that the rate of maturation is decreased, thus producing plants that require more time to mature. To obtain a plant that has a reduced rate of maturation when S compared to a non-altered plant, molecular techniques are used to reduce the amount of methylated DNA present in the altered plant.
As used herein, a plant is said to have a reduced or decreased amount of methylated DNA when the plant has less methylated DNA than the non-altered plant.
Of the four nucleotides, cytosine has been seen to be methylated in plants. The 10 present methods are therefore accomplished by targeting the methylation of cytosine.
What is contemplated is a reduction sufficient and effective to alter the rate of maturation in a manner useful for the purposes outlined herein. Thus any alteration in the amount of DNA methylation, so long as it results in an altered rate of development, is contemplated by the present invention.
In the plef~cd embodiment, the method of the present invention will result in plants having about a 10% to about a 25% reduction, more preferable about a 25% to about a 50% reduction, most preferably about a 50% to about a 70% or greater reduction in the amount of methylated DNA present when compared to non-altered plants. In the Examples that follows, plants having a 75% reduction in methylated 20 cytosine were obtained.

SUBSTITUTE SHEET (RULE 26) W O 98/04725 PCT~US97/13358 A variety of targets, strategies and molecular techni~ues can be used by a skilled artisan to reduce the amount of methylated DNA present in a plant. For example, to obtain a reduction in the amount of methylated DNA, molecular techniques can be used to reduce the level of ~ es~ion or inactivate the gene 5 encoding one or more DNA methyl transferases that are normally produced in the plant. The methyl transferase can be altered in a variety of ways by a skilled artisan so as to obtain a reduction in the amount of methylated DNA in the plant.
Ally of a plant's DNA methyl transferases genes can be used as a target in the present method. The most ple~ d targets are the genes encoding a cytosine methyl 10 transferase. Examples of DNA methyl transferase genes known in the art include cytosine methyll~ rel~se, such as the METI Arabidopsis gene herein used, the human and mouse analogs, and microbial methyl transferases such as bacterial GC
methyl transferase (for example, see Finnegan ef al., ( 1995)).
In the exarnples that follow, an ;~ntisen.~e DNA expression element was created 15 in which a fragment of a plant DNA methyl transferase gene was placed into an expression vector and inserted to a plant such that an mRNA was produced that is complimentary to mRNA encoding a plant DNA methyl transferase. A skilled artisan can readily follow this procedure with any DNA methyl transferase gene, or fragment thereof. Further, a skilled artisan can readily obtain a DNA methyl transferase gene 20 from any desired plant for use in the present methods. The ple~ lion of ~nti~n~e e~l.,s~ion vectors is discussed in detail below.

SUBSTITUTE SHEET (RULE 26) W O 98/04725 rCT~US97/13358 An alternative strategy to decrease the amount of methylated DNA present in a plant is to create knoclcout mutants in the plant in which one or more of the plant's DNA methyl transferase genes are inactivated. This can be accomplished through the use of homologous recombination to insert stop codons or large DNA fragments 5 within the coding region of a plant DNA methyl transferase gene. Alternatively, transposon mediated mutagenesis can be used for the same purpose. The p.ep~dlion of recombination vectors is discussed in detail below.
Plants that have been altered to contain a reduced amount of methylated DNA
can be further altered so as to contain an ~ression unit that expresses a DNA methyl 10 transferase that is expressed or is functional under inducible conditions. Such plants are of additional value because of the ability to induce expression of a DNA methyl transferase gene, allowing one to activate or deactivate methyl transferase ~ ession thus creating the ability to control alterations in the rate of maturation. The p~ lion of inducible e~pr~s~ion vectors is discussed in detail below.

15 Methods to Increase the Rate of Maturation As provided above, the methods of the present invention can be used to alter plants such that the rate of maturation is increased, thus producing plants that require less time to mature. To obtain a plant that has an increased rate of maturation when compared to a non-altered plant, molecular techniques are used to increase the amount 20 of methylated DNA present in the altered plant.

SUBSTITUTE SHEET (RULE 26) W O 98/04725 PCTrUS97/13358 As used herein, a plant is said to have a reduced or decreased amount of methylated DNA when the plant has less methylated DNA than the non-altered plant.
Of the four nucleotides, cytosine has been seen to be methylated in plants. The present methods are therefore accomplished by targeting the methylation of cytosine.
In the preferred embodiment, the method of the present invention ~vill result inplants having about a 10% to about a 25% increase, more preferable about a 25% to about a 50% inclease, most preferably about a 50% to about a 70% or greater increase in the amount of methylated DNA present when co~ ~ed to non-altered plants. In the Examples that follows, plants having an increase in methylated cytosine were1 0 obtained.
A variety of targets, strategies and molecular techniques can be used by a skilled artisan to increase the amount of methylated DNA present in the plant. For example, to obtain an increase in the amount of methylated DNA, molecular techniques can be used to increase the level of one or more DNA methyl transferases in the plant cell. The methyl transferase can be altered in a variety of ways by a skilled artisan so as to obtain an increase in the amount of methylated DNA in the plant.
For example, DNA methyl transferase encoding e~ iOn units can be inserted into a plant genome using molecular techniques. Such methyl transferaseexpression units can be controlled either by a constitutive promoter, providing continuous ex~leS~iOn of the DNA methyl transferase gene, or using an inducible SUBSTITUTE SHEET (RULE 26) W O 98/04725 PCTrUS97/13358 promoter, allowing one to turn on or offthe expression of DNA methyl transferase.
By controlling the level at which the DNA methyl transferase is expressed, through the use of specific promoter sequences or by altering a plant to contain one or more additional copies of a DNA methyl transferase gene, the arnount of methylated DNA
5 found in the plant can be dramatically increased.
Any DNA methyl transferase gene, or active fragment thereof, can be used to alter a plant such that the plant will contain an increased amount of methylated DNA, so long as the DNA methyl transferase gene can be modified so that it is expressed in a plant. In the examples that follow, the METl DNA methyl transferase gene from Arabidopsis was ~ltili7.od This gene, or its homologue from another plant, can readily be used by a skilled artisan in any plant system. The construction of e~.ession units encoding a DNA methyl ~ sr~-~se is described in detail below.
Plants that have been altered to contain a increased amount of methylated DNA can be further altered so as to contain an expression unit that expresses a DNA
15 methyl transferase that is expressed or is functional under inducible conditions. Such plants are of additional value because of the ability to induce expression of a DNA
methyl transferase gene, allowing one to activate or deactivate methyl transferase e~5l,r~s~ion thus creating the ability to control alterations in the rate of maturation.
The ~ aldtion of inducible expression vectors is discussed in detail below.

SUBSTITUTE SHEET (RULE 26) . .. . . .

W O 98/~4725 PCTrUS97/13358 Plants with Altered Maturation Rates.
The present invention further provides plants that have been altered using molecular techniques so that they have an altered rate of maturation. As provided above, such plants will mature at a rate that is either slower than, or faster than, 5 non-altered plants, depending on whether the arnount of methylated DNA present is either increased or decreased relative to the amount present in a non-altered plant cell respectively. The plants of the present invention fall within two types. The first type are plants that have an increased rate of maturation while the second type are plants that have a reduced rate of maturation.
The ~Ic;fell~d plants of the present invention that have an increased rate of maturation, will mature at a rate which is about 10% to about 25% faster, more preferably about 25% to about 50% faster, than the non-altered plant. The preferred plants of the present invention that have a slower rate of maturation will mature at a rate which is about 10% to about 25% slower, more preferably about 25% to about 15 50% slower~ most preferably about 50% to about 100% slower than a non-altered plant.
The plants or the present invention include those that have been altered, using molecular techniques, to have an altered amount of methylated DNA sufficient to alter the rate of maturation of the plant. The plants of the present invention therefore 20 include any plant that can be altered using molecular technique so as to alter the amount of methylated DNA present in the plant. The preferred plants are plants for SUBSTITUTE SHEET (RULE 26) .. . .. .. .... . ... .

W 098/04725 PCT~US97/13358 which it is desirable to reduce generation times for breeding or seed production purposes or plants that are used for biomass production. Particularly useful plants include, but are not limited to, corn, rice, soy bean, wheat, spinach, lettuce, alfalfa, etc.

5 Expression Units to Express Exo~enous DNA in a Plant As provided above~ several embodiments of the present invention employ t;x~ ession units (or expression vectors or systems) to express an exogenously supplied gene, such as a DNA methyl transferase, or ~nti~n~e molecule, in a plant.
Methods for generating expression unitslsystems/vectors for use in plants are well 10 known in the art and can readily be adapted for use in altering the amount of methylated DNA present in a plant cell. Typically, such units employ a protein or ~nti~en.~e coding region, such as the METl Arabidopsis gene, or a homologue thereof, and one or more ~ es~ion control elements. The choice of the protein/antisense coding region, as well as the control elements, employed will be based on the effect 15 desired (i. e., reduction or increase in the amount of DNA methylation), the plant that is to be altered, the method chosen for altering the amount of methylated DNA and the transforrnation system used. A skilled artisan can readily use any appl~pl;ate plant/vector/expression system in the present methods following the outline provided herein.

SUBSTITUTE SHEET (RULE 26) The e~yl~ession control elements used to regulate the ~yiession of the protein or ~nti~en~e coding region can either be the e~yression control element that is normally found associated with the coding sequence (homologous expression element) or can be a heterologous e~yl~ession control element. A variety of S homologous and heterologous ~yl~ssion control elements are known in the art and can readily be used to make e~yltssion units for use in the present invention.
Transcription initiation regions, for example, can include any of the various opine initiation regions, such as octopine, mannopine, nopaline and the like that are found in the Ti plasmids of Agrobacterium tumafacians. ~lt~ tively, plant viral promoters ~ 10 can also be used, such as the cauliflower mosaic virus 3 5 S promoter to control gene ~yl~s~ion in a plant. Lastly, plant promoters such as prolifera promoter, fruit-specific promoters, Ap3 promoter, heat shock promoters, seed-specific promoters, etc. can also be used. The most plere~l~d promoters will be active in dividing tissue, particularly meristematic cells.
Either a constitutive promoter (such as the CaMV or Nos promoter illustrated above), an organ-specific promoter (such as the E8 promoter from tomato) or an inducible promoter is typically ligated to the protein or ~nti~n~e encoding region using standard techniques known in the art. The expression unit may be further optimized by employing supplement~l elements such as Ll~lscliplion te-tnin~tors 20 and/or enhancer elements.

SUBSTITUTE SHEET (RULE 26) CA 02262779 l999-0l-29 W O 98/04725 PCT~US97/13358 Thus, for expression in plants, the ~ c3sion units will typically contain, in addition to the protein or antisense coding sequence, a plant promoter region, atranscription initiation site and a transcription t~rrnln~tion sequence. Unique restriction enzyme sites at the S' and 3' ends of the e~s~ion unit are typicallyincluded to allow for easy insertion into a preexisting vector. In the construction of heterologous promoter/structural gene or ~ntic~n.~e combinations, the promoter is preferably positioned about the sarne distance ~rom the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this ~ t~n~e can be accornrnodated without loss of promoter fùnction.
In addition to a promoter sequence, the ~ Le~ion s~ette can also contain a transcription terrnination region downstrearn of the structural gene to provide for efficient terrnination. The tçrmin~tion region may be obtained from the sarne gene as the promoter sequence or may be obtained from dirre,el~t genes. If the rn~NA
encoded by the structural gene is to be efficiently processed, DNA sequences which direct polyadenylation of the RNA are also cornrnonly added to the vector construct.
Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., EMBO J3: 835-846 (1984)) or the nopaline synthase signal (Depicker et al., Mol. and Appl. Genet. 1: 561-573 (1982)).
The resulting expression unit is ligated into or otherwise constructed to be included in a vector which is a~,plo,ul,ate for higher plant transforrnation. The vector SUBSTITUTE ''I._t I (RULE 26) will also typically contain a selectable marker gene by which transformed plant cells can be identified in culture. Usually, the marker gene will encode antibiotic resistance. These markers include re~i~t~nce to G418, hygromycin, bleomycin, kanamycin, and ge,~ icin. After transforming the plant cells, those cells having the 5 vector will be identified by their ability to grow on a mediurn cO.~t~ g the particular antibiotic. Replication sequences, of bacterial or viral origin, are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range prokaryotic origin of replication is included. A selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the 10 desired construct. Suitable prokaryotic selectable markers also include resistance to antibiotics such as kanamycin or tetracycline.
Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art. For instance, in the case of Agrobac~erium transformations, T-DNA sequences will also be included for subsequent transfer to 15 plant chromosomes.

Methods to produce antisense encoding vectors As tlicc~ ed above, plants having an altered arnount of methylated DNA can be produced by using ~nti~n~e sequences for i~lt~"u~ing the expression of one or more DNA methyl transferases in a plant cell. As evidenced by the behavior of 20 ~nti~Pn~e mutants described in the Examples, reduction in DNA methylation results in SUBSTITUTE SHEET (RULE 26) W O 98/04725 PCT~US97/133S8 plants ~vith reduced maturation rates while increases in DNA methylation results in plants with increased maturation rates. Accordingly, antlsense sequences of suitable length can be transfected into plant cells, using the methods described herein, to obtain plants that take longer or shorter to mature than the non- altered plant.
S Methods for inhibiting e~lession in plants using ~nticPn~e constructs, including generation of antisense sequences in situ are described, for exarnple, in U.S. Patents 5,107,065 and 5,254,800.
Other methods that can be used to inhibit expression of an endogenous gene in a plant may also be used in the present methods. For example, forrnation of a triple 10 helix at an ec~Pnti~I region of a duplex gene serves this purpose. The triplex code, permitting design of the proper single stranded participant is also known in the art.
(See H. E. Moser, et al., Science 238: 645-650 (1987) and M. Cooney, et al., Science 241: 456-459 (1988)). Regions in the control sequences cont~ining stretches of purine bases are particularly attractive targets. Triple helix forrnation along with 15 photocrossIinking is described, e.g., in D. Praseuth, et al., Proc. Nat 'I Acad Sci. USA
85: 1,349-1,353 (1988).

Inactivation of Endogenous Methyl transferases or transferases Another approach to inactivate one or more endogenous DNA plant genes that encode a DNA methyl transferase employs homologous recombination to disrupt the 20 gene. The techniques for recombinational inactivation are known in the art and can SU~:i 111 ~JTE SHEET (RULE 26) .. , .. . ~ .

readily be adapted to the present invention, for example see D. K. Asch, et al., Mol.
Gen. Genet. 221: 37-43 (1990); K. K. Asch, etal., Genetics 130: 737-748 (1992).
Transformation of Plant Cells When an apl)r~ ;ate vector is obtained, for exarnple as described above, 5 transgenic plants are plepaled which contain the desired ~ ion unit or into which a recombination inactivation vector has been introduced. In one method of transformation, the vector is microinjected directly into plant cells by use of micru~ clles to mechanically transfer the recom~inant DNA into the plant cell (Crossway, Mol. Gen. Genetics 202: 179-185 (1985)). In another method, the genetic 10 material is transferred into the plant cell using polyethylene glycol (Krens, et al., Nature 296: 72-74 (1982)), or high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, is used (Klein, et al., Nature 327: 70-73 (1987)). In still another method protoplasts are fused with other entities which contain the DNA whose introduction is l S desired. These entities are minicells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley, et al., Proc. Nat'I Acad. Sci USA 79: 1,859-1,863 (1982)).
DNA may also be introduced into the plant cells by electroporation (From et al., Proc. Nat'l Acad. Sci. USA 82: 5,824 (1985)). In this technique, plant protoplasts are electroporated in the presence of plasmids cont~inin~ the expression c~sette.

SUBSTITUTE SHEET (RULE 26) .~.. . . . , . ~

wo 98/04725 PCT/usg7/l33s8 Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
For transformation mediated by bacterial infection, a plant cell is infected with 5 Agrobacterium tumefaciens or A. rhizogenes previously transformed with the DNA to be introduced. Agrobacterium is a representative genus of the grarn-negative family Rhizobiaceae. Its species are responsible for crown gall (A. tumefaciens) and hairy root disease (~. rhizogenes). The plant cells in crown gall tumors and hairy roots are induced to produce arnino acid derivatives known as opines, which are catabolized 10 only by the bacteria. The bacterial genes responsible for ~ ession of opines are a convenient source of control elements for chimeric expression cassettes. In addition, assaying for the presence of opines can be used to identify transformed tissue.
Heterologous genetic sequences can be introduced into applo}~liate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The 15 Ti or Ri plasmid is transmined to plant cells on infection by Agrobacterium and is stably integrated into the plant genome (J. Schell, Science 237: 1,176-1,183 (1987)).
Ti and Ri plasmids contain two regions essential for the production of transformed cells. One of these, narned transferred DNA (T-DNA), is transferred to plant nuclei and induces tumor or root forrnation. The other, terrned the virulence (_) region, is 2~) e~.s~nti~l for the transfer of the T-DNA but is not itself transferred. The T-DNA will be transferred into a plant cell even if the vir region is on a different plasmid SUBSTITUTE SHEET (RULE 26) W O 98/04725 PCTrUS97/13358 (Hoekema, et al., Nature 303: 179-189 (1983)). The transferred DNA region can be increased in size by the insertion-of heterologous DNA without its ability to be transferred being affected. Thus a modified Ti or Ri plasmid, in which the disease-c~ ing ~enes have been deleted, can be used as a vector for the transfer of the S gene constructs of this invention into an al~pr~pl iate plant cell.
Construction of recombinant Ti and RI plasmids in general follows methods typically used with the more cornmon bacterial vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with the nativ plasmids and sometimes constructed from foreign sequences. These may include but 10 are not limited to "shuttle vectors," (Ruvkum and Ausubel, Nature 298: 85-88 (1981)), promoters (Lawton et al., Plant Mol. Biol. 9: 315-324 (1987)) and structural genes for antibiotic resistance as a selection factor (Fraley et al., Proc. Nat 'I Acad Sci. 80: 4,803-4,807 (1983)).
There are two classes of recombinant Ti and Ri plasmid vector systems now in 15 use. In one class, called "co-integrate," the shuttle vector cont~ining the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the pMLJ1 shuttle vector of DeBlock et al., EMBO
J3: 1,681-1,689 (1984) and the non-oncogenic Ti plasmid pGV3850, described by 20 Zambryski et al., EMBO J2: 2,143-2,150 (1983). In the second class or "binary"
system, the gene of interest is inserted into a shuttle vector cont~ining the cis-acting SUBSTITUTE SHEET (RULE 26) , . ~ .

WO 98/0472~ PCTAUS97/13358 elements required for plant transforrnation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid, as exemplified by the pBIN19 shuttle vector described by Bevan, Nucleic Acids Res. 12: 8,711-8,721 (1984) and the non-oncogenic Ti plasmid pAL4404 described by Hoekma, et al., (1983). Some of 5 these vectors are commercially available.
There are two common ways to transform plant cells with Agrobacterium:
co-cultivation of Agrobacterium with cultured isolated protoplasts, or transforrnation of intact cells or tissues with Agrobacterium. The first requires an established culture system that allows for culturing protoplasts and subsequent plant regçrlPr~tion from 10 cultured protoplasts. The second method requires (a) that the intact plant tissues, such as cotyledons, can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
Most dicot species can be transformed by Agrobacterium as all species which are a natural plant host for ~grobacterium are transformable in vitro.
15 Monocotyledonous plants, and in particular, cereals, are not natural hosts to Agrobacterium. Attempts to transform them using Agrobacterium have been unsuccessful until recently (Hooykas-Van Slogteren et al., Nature 311: 763-764 (1984)). However, there is growing evidence now that certain monocots can be transforrned by Agrobacterium. Using novel experimental approaches cereal species SUBSTITUTE SHEET (RULE 26) . .

CA 02262779 l999-0l-29 W O 9810472S PCTrUS97/13358 such as rye (de la Pena et al., Nature 325: 274-276 (1987)), maize (Rhodes et al., Science 240: 204-207 (1988)), and rice (Shim~n-)to et al., Nature 338: 274-276 (1989)) may now be transformed.
Identification of transformed cells or plants is generally accomplished by 5 including a selectable marker in the tr~n.cf )rming vector, or by obtaining evidence of successful bacterial infection.
Regeneration of Transformed Plants Plant cells which have been transformed can also be regenerated using known techniques. For example, plant regeneration from cultured protoplasts is described in 10 Evans et al., Handbook of Plant Cell Cultures, Vol. I: (MacMillan Publishing Co.
New York, 1983); and I. R. Vasil (ed.), Cell Culture and Somatic Cell Genetics of Plants, (Acad. Press, Orlando, Vol. I, 1984, and Vol. II, 1986). It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beet, cotton, fruit trees, and 15 legumes.
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate cont~ining transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, somatic embryo formation can be in~ ced in the 20 callus tissue. These somatic embryos germinate as natural embryos to form plants.
The culture media will generally contain various amino acids and plant hormones, SUBSTITUTE SHEET(RULE 26) .. _ , . .. .

W O 98/04725 PCTrUS97/13358 such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and 5 repeatable.
Silencing of Transgenes It has been observed that many exogenously supplied expression units that are introduced into a plant are not expressed the level that might be expected based on the promoter and control sequences employed (M. Matzke, et al., (1995)). The 10 inactivation of transgenes is known as silencing. Using the plants of the present invention, it has been observed that many introduced exl,lession units become inactivated as a result of DNA methylation. Accordingly, the methods used to alter the amount of DNA methylation present in a plant, and plants generated through such methods, can be further used to control the level of expression of an introduced 15 expression unit.
The following examples are provided to illustrate, but not limit, the present invention. All references herein referred to are hereby incorporated by reference.

SUBSTITUTE SHEET (RULE 26) , . _,_ . .

CA 02262779 l999-0l-29 W O 98/04725 PCT~US97/13358 ~XAMPLE 1 Plants With Reduced Rates of Maturation 1. Construction of antisense ~pl~ssion vectors A 4.3 kb METl cDNA sp~nning the positions indicated in Figure 1 was 5 inserted in the reverse orientation with respect to the CaMV 35S promoter in the pMON530 T-DNA vector. METI cDNA BS-2 was cloned into the Eco ~I site of T-DNA vector pMON530 (Monsanto) in the antisense orientation to the CaMV 35S
promoter through use of Eco RI sites present in cDNA linkers. The pMON530 T-DNA confers rPsi~t~nce to kanamycin (50 ,uglmi) on transgenic plants.

2. Southern analysis of repetitive and sinyle-copy DNA methylation patterns.
Total genomic DNA (3 ~g/lane) from antisense lines, wild type, and the ddml mutant were digested with Hpa Il (Fig. 2 left panels) or Msp I (Fig. 2 right panels), subjected to 15 electrophoresis in 0.8% agarose, and transferred to Zeta-Probe membranes (Bio-Rad) and hybridized as described to various probes (J. Chen, et al., in The Maize Handbook, M. Freeling, et al., (Springer-Verlag, New York, 1994), pp. 525~527 and S.L. Dellaporta, et al., ibid, pp.559-572). The filters were probed with a 1 80-bp centromere repeat (J. M. Martinez-Zapater, et al., (1985)) and 55 rDNA (B. R.
20 Campbell, et al., ( 1992)) (Upper panels), or were probed with four single-copy gene SUBSTITUTE SHEET (RULE 26) W 098/04725 PCT~US97/13358 probes--PHOSPHORIBOSYLANTHRANILATE TRANSFERASE 1 (PAT1), PROLIFERA (PRL), CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), and ERECTA (ER) (Lower panels).

3. Southern and phenotypic analyses of Tr246 outcross progeny S Genomic DNAs (4 ,ug/lane) from outcross progeny of strong :~nti.~n~e line Tr246 to wild-type Arabidopsis (Columbia strain with no mutations) were digestedwith Hpa II (Fig. 3 upper panels) or Eco Rl (Fig. 3 lower panels). Southern analyses were done as described above. Filters were probed with the 1 80-bp cenl,olllere repeat (J. M. Martinez-Zapater, et al., (1985)) (Fig. 3 upper panels) or the CaMV 355 promoter fragment from pMON530 (Fig. 3 lower panels). Symbols indicate the presence (+) or absence (-) of the antisense transgene. Phenotypic data for eachindividual plant are shown below each lane. Plants were grown under continuous light at 21~C.

4. Control of DNA Methylation Through Antisense Expression To address the role of DNA methylation in plant development, an antisense strategy was used to inter~ere with METI a DNA methyltransferase (MTase) gene ofArabidopsis, previously cloned by homology to the mouse gene (Fig. 1) (E. J.
Finnegan, et al., (1993)). The METl (cDNA B5-2) used in these experiments was cloned independently; it lacked 436 base pairs (bp) do~ll~Llealll of the deduced SUBSTITUTE SHEET (RULE 26) transaction start site and differed from the published sequence by a thymine-to-cytosine substitution at position +2,981. The METl gene represents one member of a small gene family in Arabidopsis (E. J. Firmegan, et al., (1993)) that maps to position 68.9 on chromosome 5, nonallelic to the ddml locus (The METI gene was mapped S with an Eco RI polymorphism between Arabidopsis ecotypes WS and Wl 00 with RI
lines (Dupont) and is nonallelic to ddml locus. T. ~kut~ni, et al., (1995)). The MET1 gene is expressed in see(lling, vegetative, and floral tissues; in the inflor~qsc~nce7 t;~ s~ion is seen at highest levels in mçri.ctern~tic cells by in situ RNA
hybridization.
To inhibit ~ ession of the METI gene, an ~nti.cçn~e construct (Fig. 1), consisting of a 4.3-kb M~Tl cDNA in the ~nti~ense orientation under the control of a constitutive viral promoter (CaMV 355), was introduced, into Arabidopsis strain Columbia. ~n one ~ hllent, a total of nine primary transformants (TO generation) were recovered; progeny tests indicated that six lines that were further characterized 15 contained single-locus transferred DNA (T-DNA) insertions. All transformations and analyses used an Arabidopsis strain Columbia line homozygous for a mutation at the g/l locus as a marker (but otherwise wild type), except as noted by Agrobacterium-mediated transformation (N. Bechtold, et al., Acad. Sci. Paris 315: 1,194 (1993)).
Six single-locus lines were identified by kanamycin segregation. Southern blot 20 analyses revealed that all lines contained independent insertions consi~ting of tandem repeats of the T-DNA. The plants used in this study represent the kanamycin-resistant SUBSTITUTE SHEET (RULE 26) CA 02262779 l999-0l-29 W 098/04725 PCT~US97/13358 progeny of TI outcrosses from single-locus lines Tr242, 243, 244, 245, and 248.
Before gerrnination, all seeds were plated on MS media (Gibco) with (all transgenic lines) or without (controls) kanamycin (50 ~glml, Sigma) and incubated for 3 days at 4~C in the dark. Growth conditions were 16 hours light, 8 hours dark at 21~C, except as noted. Seedlings were transplanted into soil 8 days after germination.
Methylation p~ttPrn~ in repetitive DNA sequences were examined by Southern (DNA) hybridization (J. Chen, et al., in T7ze Maize Handbook, M. Freeling, et al., (Springer-Verlag, New York, 1994), pp. 525-527 S.L. Dellaporta, et al., ibid, pp.
559-572). Genomic DNAs were digested with the isochizomers Hpa n or Msp I (Fig.
2, upper panels) and probed with a centromeric repeat or a 55 ribosomal DNA (rDNA) sequence; both repeats are methylated in wild-type genomic DNA (A. Vongs, et al., (1993)). Hpa II digestion is inhibited if either cytosine in the CCGG target site is methylated; Msp I can cleave CsmCGC but not 5mCCGG (M. Nelson~ et al., Nucleic Acids Res. 19: 2,045 (1991)).
With both probes, Hpa II digestion revealed a high extent of demethylation in three of six antisense lines (Tr244, 246, and 248; designated "weak") showed near ~ild-type levels of methylation. Msp I digestion was more complete in strong ~nti~n~e lines contain substantial demethylation of these repeated sequences at C5mCGG and 5mCCGG sites.
Digestion of wild-type genomic DNA with Eco RII and Bst NI, isochizomers with dirr~ ial sensitivity to cytosine methylation in the motif C5mC(A/T)GG~

SUBSTITUTE SHEET (RULE 26) W O 98/04725 PCTrUS97/13358 showed only minor differences in wild-type DNA when probed with the centromere repeat and 55 rDNA probes suggesting C(A/T)G methylation may not be prevalent.
DNA methylation was examined at four single-copy gene sequences (A. B.
Rose, et al., Plant Physiol. 100: 582 (1992); P. S. Springer, et al., Science 268: 877 (1995); X. W. Deng, et al., Cell 71: 191 (1992); K. U. Torii, et al., Plant Cell 8: 735 (1995)) (Fig. 2, lower panels). Substantial demethylation of all four genes was seen only in the strong ~ntice~e lines; the ddml mutant showed little or no demethylation relative to wild-type DNA, consistent with published reports (A. Vongs, et al., (1993)). Total genomic levels of smc were also measured in these lines by high-l.elro,lllance liquid chromatography HPLC conditions were as described (C. W.
Gehrke et al., J. Chromatogr. 301: 199 (1984), with the following exceptions:
genomic DNAs were isolated from plants immediately after the onset of flowering,treated with 20 ~g of ribonuclease A (Sigma) for 30 min. at 37~C, followed by passage through a Sepharose CL-SB (Pharmacia) spin column. Nucleosides were resolved on a Varian Vista 5500 Liquid Chromatograph with a Rainin Dynamax S ~m Spherical Microsorb C18 column (100 A pore size, 4.5 mm inner diameter by 15 cm length) with a 20 min. isocratic gradient of 2.5% methanol, 50 mM KH2PO5 (pH 4.0), followed by a 10 min. linear gradient to 8.0% methanol, 50 ml KH2PO5 (pH 4.0).
(Table 1).
Cytosine methylation levels in wild-type (6.4% of total genomic cytosine) and the decrease in the ddml mutant (75%) agree with previously published estimates SUBSTlTUTE SHEET (RULE 26) W 098/04725 PCTrUS97113358 (A. Vongs, et al., (1993)). Total genomic 5mc content in strong lines Tr246 and Tr248 was reduced 71% relative to wild-type levels; the weak line Tr245 showed a 34% reduction. The decrease in smc levels in the strong lines by a factor of 3.5 is comparable to reductions seen in the ddml mutant and the MTase knockout mouse 5 (R. Li, e~ al., Cell 59: 915 (1992)). Unlike the pattern of demethylation of the ddml mutant, however, METI antisense expression resulted in substantial demethylation of both repetitive DNA and single-copy gene sequences.
Cytosine methylation levels in Arabidopsis wild-type, ddml mutant, and three antisense lines was determined by reversed-phase HPLC using the method of Gehrke 10 e~ al., (1984), with the following exceptions: genomic DNAs were isolated from plants immediately after the onset of flowering, treated with 20 ,Llg of ribonuclease A
(Sigma) for 30 min. at 37~C, followed by passage through a Sepharose CL-SB
(Pharmacia) spin column. Nucleosides were resolved on a Varian Vista 5500 Liquid Chromatograph with a Rainin Dynarnax 5 ,um Spherical Microsorb Cl g column (100 15 ~ pore size, 4.5 mm inner diameter by 15 cm length) with a 20 min. isocratic gradient of 2.5% methanol, 50 mM KH2PO5 (pH 4.0), followed by a 10 min. Iinear gradient to 8.0% methanol, 50 ml KH2PO5 (pH 4.0). All values presented in Table 1 represent the averages of two to four individual replicates and were calculated by integration of peak areas with Dynamax HPLC Method Manager v I .2 (Rainin). Percentages of total 20 5mc content [SmC/(5mC + C)] are norm~li7,o~1 for absorbance dirl~Iences between cytosine and smc.

SUBSTITUTE Sl,-, I (RULE 26) W O 98/04725 PCT~US97/13358 Table ITotal Line smc %WT %
(%) levels Decrease Wild type 6.38 ~ 0.69 100 0 Tr2454.24+0.59 66.533.5 S Tr246 1.84+0.27 28.9 71.1 Tr248 1.83~0.16 28.7 71.3 ddml 1.60+0.04 25.0 75.0 Quantitative aspects of vegetative and inflorescence traits are given for wild-type Columbia strain and six ~nti~on~e lines in Table 2. Flowering time is 10 measured in days after ger~in~tion. Flowering time refers to the number of days elapsed from seed germin~tion until emergence of an inflorescence bolt 0.5 to 1.0 cm in height; leaf number refers to the number of vegetative leaves initiated before emergence of the primary inflorescence; and secondary branches refers to the number of branches initiated on the primary inflorescence axis. Values are calculated from a 15 minimum of four individual plants from each line. Pool sizes for each line were as follows: wild-type; TR242; TR245, n = 5; Tr243 and Tr244, n = 4; Tr245, n = I l; and Tr248, n = 15.

SUBSTITUTE SHEET (RULE Z6) WO 98/04725 PCTrUS97/13358 Table 2 Flowering Leaf Secondary Line time number branches Wild type 26.2~1.7 9.2~1.3 4.3+0.5 Tr242 24.5+2.110.0+1.44.5~1.1 Tr243 27.8~0.411.0+0.7 4.3+0.4 Tr244 47.7+4.034.0+5.7 20.3+4.9 Tr245 27.7~7.511.5~1.5 4.3iO.8 Tr246 45.9~3.632.5~3.2 20.7+1.4 Tr248 46.3+4.732.9+2.7 20.5+2.0 Normal patterns of development were perturbed in strong ~nti~n~e lines.
Under long day conditions, six single-locus lines were identified by kanamycin segregation. Southem blot analyses revealed that all lines contained independent insertions consisting of tandem repeats of the T-DNA. The plants used in this study represent the kanamycin-resistant progeny of Tl outcrosses from single-locus lines l S Tr242, 243, 244, 245, and 248. Before germination, all seeds were plated on MS
media (Gibco) with (all transgenic lines) or without (controls) kanamycin (50,ug/ml, Sigma) and incubated for 3 days at 4~C in the dark. Growth conditions were 16 hours light, 8 hours dark at 21 ~C, except as noted. Seedlings were transplanted into soil 8 days after germination; Tr244, Tr246 and Tr248 plants initiated 30 to 35 vegetative SlJ~ JTE SHEET (RULE 26) W O 98/04725 PCT~US97/13358 nodes with delayed abaxial trichome production and flowered after 45 to 48 days (Table 2); these phenotypes were fully penetrant in all T-DNA-cont~ining progeny of the strong antisense lines.
After a transition from vegetative to reproductive development, wild-type S plants initiated a primary inflorescence axis with two to five secondary inflorescence branches subtended by cauline leaves, followed by an abrupt transition to the production of solitary floral meristems (Table 2) (S. Shannon, et al., Plant Cell 5: ~39 (1993)). In strong ~nti~en~e lines, the primary inflorescellce shoot produced an average of 20 secondary branches (Table 2) before the production of flowers. The 10 basal-most branches in strong ~nti~n.ce lines often assumed characteristics of vegetative rosettes, including enh~nce~l spiral phyllotaxy of 10 to 20 vegetative-like leaves and shortened internodes, followed by the emergence of an inflorescence bolt recapitulating the primary inflorescence. Unlike wild-type plants, occasional secondary branches were produced apically to flowers in the transition zone. Early 15 initiating flowers from strong ~nti~f~n.~e lines were normal in appearance and male-fertile, but were often female-sterile. Apical flowers initiated on secondary and tertiary inflorescences showed gross abnormalities, including a threefold increase in stamen number and sterile, incompletely fused carpels lacking stigmas. Despite the severe disruptions in floral morphology seen in these late-initiating flowers, no SUBSTITUTE SHEET (RULE 26) ~ ... .. " ... ~............. . .... .

W O 98/04725 PCTrUS97/13358 obvious defects in pollen viability or paternal transgene tr~mmi~ion were observed on the basis of segregation ratios of transgenes in outcrosses Kanamycin resistance segregated in a l: l ratio in all outcrosses from strong antisense lines.
One trivial explanation for the METl antisense pleiotropy is that it lcples~
5 an indirect effect on the transgene rather than a direct consequence of genomic demethylation; however, several lines of evidence support a direct role for DNA
methylation in development. Data collected and analyzed in additional experiments revealed an identical pleiotropy in 12 independent transgenic lines. In analyses of outcross progeny from strong ~ntis~nce lines, the severe phenotype co-segregated with 10 the presence of the transgene, and a slightly ~tt~ml~te~ pleiotropy was seen in progeny that had lost the transgene but retained a demethylated genome (Fig. 3). The latter finding is not unexpected, because the rate of genomic remethylation is slow (A.
Vongs, et al., (1993)). Phenotypic revertants seen among outcross progeny had reestablished near wild-type levels of genomic methylation (Fig. 3, lanes 5 and l l ).
15 In surn, it appears that demethylation is sufficient to maintain developmental pleiotrophy in the absence of the transgene, whereas genomic remethylation is required to restore a wild-type phenotype.
The demethylation phenotype produced by antisense inhibition of Arabidopsis MTase differs markedly from the phenotype produced by the tre~tment of Arabidopsis 20 seeds with the nonmethylatable cytosine analog 5-azacytidine (5-azaC), but bears some similarity to the ddml mutant phenotype. 5-AzaC produces early flowering in SUBSTITUTE SHEET(RULE 26) . . .

W O 98/04725 PCTrUS97/13358 some late-flowering strains and mutants of Arabidopsis (J. E. Burn, et al., Proc.
Nat'l ~cad. Sci. U.S.~. 90: 287 (1993)), but this effect has not been correlated with a quantitative decrease in cytosine methylation. More recent evidence suggests that the primary effect of 5-azaC treatment is due to toxicity resulting from covalent trapping 5 of DNA MTase (R. Jiitterman, et al., Proc. Nat 'I Acad. Sci. USA 91: 11,797 (1994)) or 5-azaC incorporation into RNA. The overall level of demethylation in the ddml mutant is equivalent to that seen in the strong METl ~nti~n~e lines, yet the phenotype exhibited by the METl antisense lines is much more severe and pleiotropic. This discrepancy may be due to substantial demethylation observed at single-copy gene 10 se~uences in METI antisense lines not seen in the ddml mutant.
On the basis of these studies, DNA methylation is shown to be an esser ti~l component in the process of phase transitions and meristem (letermin~y.
Methylation may serve as a primary signal to restrict meristem determinacy, or it may represent a secondary process required to m~int~in an epigenetic state once 15 established. Phase transitions involve an interplay of both cell autonomous and diffusible signals (R. S. Poethig, Science 250: 923 (1990)). Evidence that methylation may represent an autonomous component in this process comes from the observation that phase transitions are often irregular in strong ~n~i~Pn~e plan--the location of branches apical to flowers in the inflorescence transition zone may represent an 20 autonomous switching of individual cells in the meristem resulting in developmental mosaicism. Methylation effects have also been shown to be progressive in the plant SU~a~ )TE ''1._~1 (RULE 26) meristem (R. Martienssen, e~ al., Genes Dev. 4: 331(1990)), and it is intriguing to speculate that a methylation gradient might be established during m~.ri~tem growth to serve as an autonomous signal that directs meristem determinacy. In this light, the strong antisense phenotype could be explained by delayed establishmen~ of this 5 hypothetical gradient. Such a model predicts that meristem potential becomes progressively more epigenetically restrictive and that repression c~c~fles will be an underlying theme in plant determinacy, a process implicit in the pathway controlling inflorescence and floral development in Arabidopsis (M. D. Wilkinson, et al., Plant Cell 7: 1,485 (1995)).

EXAMPT F. 2 Plants with increased rates of development To generate plants with increased arnount of methylated DNA, Arabidopsis plants were altered to contain an expression unit that expresses cytosine methyl transferase under the control of the CaMV 355 promoter or another plant promoter.
15 The cDNA encoding the MET1 protein from Arabidopsis described above was used to generate ~xpl~ssion units in which the METI encoding sequence was placed under the regulatory control of the CaMV 355 promoter or the flower promoter AP3.
The expression units were introduced into a plant using Agrobacterium as described above. Transformed tissues were identified and plants were regenerated 20 from the transformed tissue.

SUBSTITUTE SHEET (RULE 26) W O 98/04725 PCT~US97113358 An analysis of the rate of development was performed and it was found that plants having an increased amount of methylated DNA produced flowers at 10 to 12days following gennin~tion, compared to 26 days for non-altered plants.

General methods for decreasing the rate of development in a plant The Description of the Invention above provides a detailed outline for the various methods herein disclosed for ~It~ring the amount of DNA methylation present in a plant. The Fx~mples provide the results of applications of the methods herein described. The following is int~.n~le~ to provide a non-limiting summary of the typical steps employed when altering a plant using the present methods.
1. Select Plant The present methods can be applied to any plant that can be altered using molecular techniques. As discussed above, there are numerous commercially exploited plants whose value would be increased by either shortening the time required for maturation into seed producing plants or where it is desirable to increase biomass or seed yield. The first step in applying the present methods is to select as plant. Although it is preferable to select a plant that has had methods for expression, transformation and regeneration developed, most plants, though applied effort, can be transformed and regenerated using methods known in the art.

SUBST~TUTE SHEET (RULE 26) As part of choosing a plant, one needs to choose whether to increase or decrease the rate of maturation. As provided above, to increase the rate of maturation, the plant is altered so as to contain an increased arnount of methylated DNA while to decrease the rate of maturation, the plant is altered so as to contained a decreased 5 amount of methylated DNA.
2. Chose the alteration method/tar~et that is to be employed The second step may be to decide which method to employ to alter the plant.
As provided above, the amount of DNA methylation can be decreased by inactivating or decreasing the activity of the methylase gene while the amount of DNA
10 methylation can be increased by activating or increasing the methylase activity within the cell.

SUBSTITUTE SHEET (RULE 26) W O 98/04725 PCT~US97/13358 selected based on the level of DNA methylation, the expression of the transgene, physiologic effects and equivalent methods thereto.
It should be understood that the foregoing discussion and examples merely present a detailed description of certain prefe-~cd embodiments. It therefore should be 5 a~)palCI~ to those of ordinary skill in the art that based on the above description of the invention and the specific Examples provided, a skilled artisan can readily adapt the present invention for use with any plant that can be altered by using molecular techniques.. All articles and texts that are i~entifiecl above are incorporated by reference in their entirety.

SUBSTITUTE SHEET (RULE 26)

Claims (38)

1. A method to alter the rate of maturation in a plant comprising the step of genetically altering said plant using molecular techniques so that said plant has an altered amount of methylated DNA when compared to a non-altered plant sufficient to alter the rate of maturation of said plant.

2. The method of claim 1 wherein said alteration leads to a reduction in the amount of methylated DNA and said plant matures at a slower rate than said non-altered plant.

3. The method of claim 2, wherein said alteration results in about a 10%
to about a 25% decrease, or greater, in the amount of methylated DNA present in said plant.

4. The method of claim 2, wherein said alteration results in about a 25%
to about a 50% decrease, or greater, in the amount of methylated DNA present in said plant.

5. The method of claim 2, wherein said alteration results in about a 50%
or about a 70% decrease, or greater, in the amount of methylated DNA present in said plant.

6. The method of claim 2, wherein said alteration leads to a reduction in the amount of methylated cytosine bases.

7. The method of claim 2 wherein said plant has had the activity of one or more DNA methyl transferase encoding genes altered using said molecular techniques.

8. The method of claim 7 wherein said DNA methyl transferase encoding gene is the METI gene of Arabidopsis, or a homologue thereof.

9. The method of claim 2 wherein said plant has been altered using said molecular techniques so as to express an antisence nucleic acid molecule that is complementary to an mRNA that encodes a DNA methyl transferase.

10. The method of claim 9 wherein said mRNA is encoded by the METI
gene of Arabidopsis, or a homologue thereof.

11. The method of claim 1 wherein said alteration leads to an increase in the amount of methylated DNA and said plant matures at a faster rate than said non-altered plant.

12. The method of claim 11, wherein said alteration results in about a 10%
to about a 25% increase, or greater, in the amount of methylated DNA present in said plant.

13. The method of claim 11, wherein said alteration results in about a 25%
to about a 50% increase, or greater, in the amount of methylated DNA present in said plant.

14. The method of claim 11, wherein said alteration results in about a 50%
to about a 70% increase, or greater, in the amount of methylated DNA present in said plant.

15. The method of claim 11, wherein said alteration leads to an increase in the amount of methylated cytosine bases.

16. The method of claim 15 wherein said plant has had one or more DNA
methyl transferase encoding genes introduced into said plant using said molecular techniques.

17. The method of claim 16 wherein said DNA methyl transferase encoding gene is the MET1 gene of Arabidopsis, or a homologue thereof.

18. A plant that has been altered according to the method of claim 1.

19. A plant that has been altered according to the method of claim 2.

20. A plant that has been altered according to the method of claim 11.

21. A method to increase the biomass production of a given plant comprising the step of genetically altering said plant using molecular techniques so that said plant has a reduced amount of methylated DNA when compared to a non-altered plant sufficient for said plant to mature at a slower rate than said non-altered plant.

22. The method of claim 21 wherein said plant has had the activity of one or more DNA methyl transferase encoding genes altered using said molecular techniques.

23. The method of claim 22 wherein said DNA methyl transferase encoding gene is the METI gene of Arabidopsis, or an equivalent gene thereof.

24. The method of claim 21 wherein said plant has been altered using said molecular techniques so as to express an antisence nucleic acid molecule that is complementary to an mRNA that encodes a DNA methyl transferase.

25. The method of claim 21 wherein said mRNA is encoded by the MET1 gene of Arabidopsis, or a homologue thereof.

26. A method to decrease the generation times in a plant breeding program comprising the step of using one or more parent strains that have been genetically altered using molecular techniques so that said parent strains have an increased amount of methylated DNA when compared to a non-altered plant sufficient to cause said parent strain to mature at a faster rate than said non-altered plant.

27. The method of claim 26 wherein said plant has had one or more DNA
methyl transferase encoding genes introduced into said plant using said molecular techniques.

28. The method of claim 27 wherein said DNA methyl transferase encoding gene is the MET1 gene of Arabidopsis, or a homologue thereof.

29. A method to select a plant that has an altered rate of development comprising the step of selecting a plant based on the criteria of having an altered amount of methylated DNA or an altered DNA methylase activity when compared to a non-altered plant sufficient to alter the rate of maturation of said plant.

30. The method of claim 29 wherein said alteration leads to a reduction in the amount of methylated DNA and said plant matures at a slower rate than said non-altered plant.

31. The method of claim 30, wherein said alteration results in about a 10%
to about a 25% decrease, or greater, in the amount of methylated DNA present in said plant.

32. The method of claim 30, wherein said alteration results in about a 25%
to about a 50% decrease, or greater, in the amount of methylated DNA present in said plant.

33. The method of claim 30, wherein said alteration results in about a 50%
or about a 70% decrease, or greater, in the amount of methylated DNA present in said plant.

34. The method of claim 29 wherein said alteration leads to an increase in the amount of methylated DNA and said plant matures at a faster rate than said non-altered plant.

35. The method of claim 34, wherein said alteration results in about a 10%
to about a 25% increase, or greater, in the amount of methylated DNA present in said plant.

36. The method of claim 34, wherein said alteration results in about a 25%
to about a 50% increase, or greater, in the amount of methylated DNA present in said plant.

37. The method of claim 34, wherein said alteration results in about a 50%
to about a 70% increase, or greater, in the amount of methylated DNA present in said plant.

38. A plant that has selected according to the method of claim 29.