AU4048097A - Methods for altering the rate of plant development and plants obtained therefrom - Google Patents

Description

METHODS FORALTERING THE RATE OFPLANTDEVELOPMENT AND

PLANTSOBTAINED 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 MADE UNDER FEDERALLY SPONSORED RESEARCH

This invention was made with governmental 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

The 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.

BACKGROUND ART

Plant genomes contain relatively large amounts of the modified nucleotide

5-methylcytosine (5^mC) (Y. Greenbaum, et al, Nature 292: 850 (1981)). Despite

evidence implicating cytosine methylation in plant epigenetic phenomena, such as

repeat-induced gene silencing (TIGS), cosuppression, and inactivation of transposable

elements (F. F. Assaad, et al., Plant Mo Biol. 22: 1,057 (1993); C. Napoli, et al.. 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 mechanisms that mediate plant development are presently 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 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

which are produced by these methods.

All references disclosed throughout this application are hereby incorporated by

reference in their entirety.

DISCLOSURE OF THE INVENTION

The present invention is based on the unexpected observation that a decrease

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 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 by altering a plant, plant cells, plant tissues or plant seeds, using molecular

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. 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 DRAWINGS

Figure 1. The predicted MET1 gene product and antisense construct.

A diagrammatic representation of the predicted gene product of the MET]

locus is provided. The METJ protein is a 1,534-amino acid protein with a high degree

of homology to the mouse MTase, particularly in the catalytic and NH₂ -terminal foci

targeting domains (E. J. Finnegan, et al, Nucleic Acids Res. 21: 2,383 (1993)). The

MET! antisense construct is shown in the bottom of the figure. See Example 1.

Figure 2. Southern analvsis of repetitive and single-copy DNA methylation patterns.

Total genomic DNA (3 μg/lane) from antisense lines, wild-type, and the ddm I

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

180-bp centromere repeat (J. M. Martinez-Zapater, et al., Mol Gen Genet, 204: 417

(1985)) and 55 rDNA (B. R. Campbell, et 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 (ER). Digestion of wild-type genomic DNA with Eco RII and Bst NI, isochizomers

with differential sensitivity to cytosine methylation in the motif C^5mC(A/T)GG,

showed only minor differences in wild-type DNA when probed with the centromere

repeat and 5S rDNA probes suggesting C(A/T)G methylation may not be

prevalent-shown by differential Hpa II-Msp I digestion of wild-type DNA to contain

C^5mCGG 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.

Figure 3. Southern and phenotvpic analyses of Tr246 outcross progeny.

Genomic DNAs (4 μg/lane) from outcross progeny of strong antisense line

Tr246 to wild-type Arabidopsis (Columbia strain with no mutations) were digested

with Hpa II (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

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 2 PC. GENERAL DESCRIPTION BEST MODE FOR CARRYING OUT THE INVENTION

General Description

The present invention is based on the unexpected observation that DNA

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 D A results in plants that

mature at a slower rate than plants containing normal amounts of methylated DNA

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

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 or decreased relative to a non-altered plant. The degree that the rate of maturation is altered will vary from plant to plant as well as between plants that have been altered

using different methods.

In a preferred embodiment, the method will produce plants with an increased

rate of maturation of about 10% to about 25% faster, more preferably about 25% to

about 50% faster, than the normal rate of maturation. For example, in Arabidopsis,

flowers typically develop after 26 days under long day conditions. In the Examples,

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 programs. Plants that have been altered using the

methods herein described for decreasing maturation times can be used in any

conventional breeding program, producing accelerated results.

In another preferred embodiment, 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%

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 maturation when compared to non-altered plants.

When used to slow the rate of maturation, the present methods produce plants that have increased biomass. As used herein, biomass refers to the total plant weight, 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

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 containing 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

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

techniques. As used herein, molecular techniques exclude classical genetic techniques

such as breeding/selection, identification 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 bacterium. Such methods are well known in the art and are described in, for example, Sambrook, et al, Molecular

Cloning: a Laboratory Manual, Cold Spring Harbor Press (1989). Some of the techniques that are used to alter a plant are discussed in more detail below.

Molecular techniques are differentiated from classical genetics in which randomly

occurring spontaneous mutants or classical mutagenic techniques are applied to a

given 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 mechanism

was 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 through 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 be

further propagated from the isolated and identified variants as having altered amounts

of DNA that then correlates to an altered rate of maturation. 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 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

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 preferred 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

cytosine were obtained. A variety of targets, strategies and molecular techniques 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 expression or inactivate the gene

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.

Any of a plant's DNA methyl transferases genes can be used as a target in the

present method. The most preferred targets are the genes encoding a cytosine methyl

transferase. Examples of DNA methyl transferase genes known in the art include

cytosine methyltransferase, such as the MET1 Arabidopsis gene herein used, the

human and mouse analogs, and microbial methyl transferases such as bacterial GC

methyl transferase (for example, see Finnegan et al, (1995)).

In the examples that follow, an antisense DNA expression element was created

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

from any desired plant for use in the present methods. The preparation of antisense expression vectors is discussed in detail below. An alternative strategy to decrease the amount of methylated DNA present in a

plant is to create knockout 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

within the coding region of a plant DNA methyl transferase gene. Alternatively,

transposon mediated mutagenesis can be used for the same purpose. The preparation

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 expression unit that expresses a DNA 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 expression

thus creating the ability to control alterations in the rate of maturation. The

preparation of inducible expression vectors is discussed in detail below.

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

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

present methods are therefore accomplished by targeting the methylation of cytosine.

In the preferred embodiment, the method of the present invention will result in

plants having about a 10% to about a 25% increase, more preferable about a 25% to

about a 50% increase, most preferably about a 50% to about a 70% or greater increase

in the amount of methylated DNA present when compared to non-altered plants. In

the Examples that follows, plants having an increase in methylated cytosine were

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 expression units can be

inserted into a plant genome using molecular techniques. Such methyl transferase

expression units can be controlled either by a constitutive promoter, providing

continuous expression of the DNA methyl transferase gene, or using an inducible promoter, allowing one to turn on or off the 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 amount of methylated DNA

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 METJ DNA methyl transferase gene from Arabidopsis was utilized. This gene, or its homologue from another plant, can readily

be used by a skilled artisan in any plant system. The construction of expression units

encoding a DNA methyl transferase 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

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

expression thus creating the ability to control alterations in the rate of maturation.

The preparation of inducible expression vectors is discussed in detail below. 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,

non-altered plants, depending on whether the amount 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 preferred 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

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

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 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.

Expression Units to Express Exogenous DNA in a Plant

As provided above, several embodiments of the present invention employ

expression units (or expression vectors or systems) to express an exogenously

supplied gene, such as a DNA methyl transferase, or antisense molecule, in a plant.

Methods for generating expression units/systems/vectors for use in plants are well

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

antisense coding region, such as the METl Arabidopsis gene, or a homologue thereof,

and one or more expression control elements. The choice of the protein/antisense

coding region, as well as the control elements, employed will be based on the effect

desired ( . 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

transformation system used. A skilled artisan can readily use any appropriate

plant/vector/expression system in the present methods following the outline provided

herein. The expression control elements used to regulate the expression of the protein

or antisense coding region can either be the expression control element that is

normally found associated with the coding sequence (homologous expression

element) or can be a heterologous expression control element. A variety of

homologous and heterologous expression control elements are known in the art and

can readily be used to make expression 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. Alternatively, plant viral promoters

can also be used, such as the cauliflower mosaic virus 35S promoter to control gene

expression 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 preferred 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 antisense encoding region

using standard techniques known in the art. The expression unit may be further

optimized by employing supplemental elements such as transcription terminators

and/or enhancer elements. Thus, for expression in plants, the expression units will typically contain, in

addition to the protein or antisense coding sequence, a plant promoter region, a

transcription initiation site and a transcription termination sequence. Unique

restriction enzyme sites at the 5' and 3' ends of the expression unit are typically

included to allow for easy insertion into a preexisting vector. In the construction

of heterologous promoter/structural gene or antisense combinations, the promoter is

preferably positioned about the same distance from 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 distance can be accommodated without loss of

promoter function.

In addition to a promoter sequence, the expression cassette can also contain a

transcription termination region downstream of the structural gene to provide for

efficient termination. The termination region may be obtained from the same gene as

the promoter sequence or may be obtained from different genes. If the mRNA

encoded by the structural gene is to be efficiently processed, DNA sequences which

direct polyadenylation of the RNA are also commonly 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 andAppl Genet. 1: 561-573 (1 82)).

The resulting expression unit is ligated into or otherwise constructed to be included in a vector which is appropriate for higher plant transformation. The vector 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 resistance to G418, hygromycin, bleomycin,

kanamycin, and gentamicin. After transforming the plant cells, those cells having the

vector will be identified by their ability to grow on a medium containing 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

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 Agrobacterium

transformations, T-DNA sequences will also be included for subsequent transfer to

plant chromosomes.

Methods to produce antisense encoding vectors

As discussed above, plants having an altered amount of methylated DNA can

be produced by using antisense sequences for interrupting the expression of one or

more DNA methyl transferases in a plant cell. As evidenced by the behavior of antisense mutants described in the Examples, reduction in DNA methylation results in plants with reduced maturation rates while increases in DNA methylation results in

plants with increased maturation rates. Accordingly, antisense 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.

Methods for inhibiting expression in plants using antisense constructs, including

generation of antisense sequences in situ are described, for example, 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, formation of a triple

helix at an essential 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 containing stretches of purine

bases are particularly attractive targets. Triple helix formation along with

photocrosslinking 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

gene. The techniques for recombinational inactivation are known in the art and can 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, et al, Genetics 130: 737-748 (1992).

Transformation of Plant Cells

When an appropriate vector is obtained, for example as described above,

transgenic plants are prepared which contain the desired expression 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

micropipettes to mechanically transfer the recombinant DNA into the plant cell

(Crossway, Mol. Gen. Genetics 202: 179-185 (1985)). In another method, the genetic

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

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 'I Acad. Sci. USA 82: 5,824 (1985)). In this technique, plant protoplasts

are electroporated in the presence of plasmids containing the expression cassette. 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

Agrobacterium tumefaciens or A. rhizogenes previously transformed with the DNA to

be introduced. Agrobacterium is a representative genus of the gram-negative family

Rhizobiaceae . Its species are responsible for crown gall (A. tumefaciens) and hairy

root disease (A. rhizogenes). The plant cells in crown gall tumors and hairy roots are

induced to produce amino acid derivatives known as opines, which are catabolized

only by the bacteria. The bacterial genes responsible for expression 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 appropriate plant cells,

by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The

Ti or Ri plasmid is transmitted 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, named transferred DNA (T-DNA), is transferred to plant nuclei

and induces tumor or root formation. The other, termed the virulence (yir) region, is

essential 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 yji region is on a different plasmid (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-causing genes have been deleted, can be used as a vector for the transfer of the

gene constructs of this invention into an appropriate plant cell.

Construction of recombinant Ti and RI plasmids in general follows methods

typically used with the more common bacterial vectors, such as pBR322. Additional

use can be made of accessory genetic elements sometimes found with the native

plasmids and sometimes constructed from foreign sequences. These may include but

are not limited to "shuttle vectors," (Ruvku 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 7 Acad.

Sci. 80: 4,803-4,807 (1983)).

There are two classes of recombinant Ti and Ri plasmid vector systems now in

use. In one class, called "co-integrate," the shuttle vector containing the gene of

interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that

contains both the cis-acting and tr< s-acting elements required for plant

transformation as, for example, in the pMLJl shuttle vector of DeBlock et al, EMBO

J3: 1,681-1,689 (1984) and the non-oncogenic Ti plasmid pGV3850, described by

Zambryski et al, EMBOJ2: 2,143-2,150 (1983). In the second class or "binary"

system, the gene of interest is inserted into a shuttle vector containing the cz^'-v-acting elements required for plant transformation. 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

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 transformation

of intact cells or tissues with Agrobacterium. The first requires an established culture

system that allows for culturing protoplasts and subsequent plant regeneration from

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 Agrobacterium are transformable in vitro.

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

transformed by Agrobacterium. Using novel experimental approaches cereal species such as rye (de la Pena et al, Nature 325: 274-276 (1987)), maize (Rhodes et al,

Science 240: 204-207 (1988)), and rice (Shimamoto et al, Nature 338: 274-276

(1989)) may now be transformed.

Identification of transformed cells or plants is generally accomplished by

including a selectable marker in the transforming 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

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

legumes.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing 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 induced in the

callus tissue. These somatic embryos germinate as natural embryos to form plants.

The culture media will generally contain various amino acids and plant hormones, 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

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

inactivation of transgenes is known as silencing. Using the plants of the present

invention, it has been observed that many introduced expression 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

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. EXAMPLE 1 Plants With Reduced Rates of Maturation

1. Construction of antisense expression vectors

A 4.3 kb METl cDNA spanning the positions indicated in Figure 1 was

inserted in the reverse orientation with respect to the CaMV 35S promoter in the

pMON530 T-DNA vector. METl cDNA B5-2 was cloned into the Eco RI 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 resistance to kanamycin (50 μg/mi) on transgenic plants.

2. Southern analvsis of repetitive and single-copy DNA methylation

patterns.

Total genomic DNA (3 μg/lane) from antisense lines, wild type, and the ddml

mutant were digested with Hpa II (Fig. 2 left panels) or Msp I (Fig. 2 right panels),

subjected to 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 180-bp centromere repeat (J. M. Martinez-Zapater, et al, (1985)) and 55 rDNA (B. R.

Campbell, et al, (1992)) (Upper panels), or were probed with four single-copy gene probes— PHOSPHORIBOSYLANTHRANILATE TRANSFERASE 1 (PAT1),

PROLIFERA (PRL), CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COPl), and ERECTA (ER) (Lower panels).

3. Southern and phenotypic analyses of Tr246 outcross progeny

Genomic DNAs (4 μg/lane) from outcross progeny of strong antisense line

Tr246 to wild-type Arabidopsis (Columbia strain with no mutations) were digested

with Hpa II (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

(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 each

individual plant are shown below each lane. Plants were grown under continuous

light at 2 rC.

4. Control of DNA Methylation Through Antisense Expression

To address the role of DNA methylation in plant development, an antisense strategy was used to interfere with METl a DNA methyltransferase (MTase) gene of

Arabidopsis, 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) downstream of the deduced 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. Finnegan, et al, (1993)) that maps to position

68.9 on chromosome 5, nonallelic to the ddml locus (The METl gene was mapped

with an Eco RI polymorphism between Arabidopsis ecotypes WS and Wl 00 with RI

lines (Dupont) and is nonallelic to ddml locus. T. Kakutani, et al, (1995)). The

METl gene is expressed in seedling, vegetative, and floral tissues; in the

inflorescence, expression is seen at highest levels in meristematic cells by in situ RNA

hybridization. To inhibit expression of the METl gene, an antisense construct (Fig. 1),

consisting of a 4.3-kb METl cDNA in the antisense orientation under the control of a

constitutive viral promoter (CaMV 355), was introduced, into Arabidopsis strain

Columbia. In one experiment, a total of nine primary transformants (TO generation)

were recovered; progeny tests indicated that six lines that were further characterized

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/1 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 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 TI outcrosses from single-locus lines 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 μg/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.

Methylation patterns in repetitive DNA sequences were examined by Southern

(DNA) hybridization (J. Chen, et al, in The 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 II 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 /^"digestion is inhibited if either cytosine in the CCGG target site is

methylated; Msp I can cleave C^5mCGC but not ^5mCCGG (M. Nelson, et αl, Nucleic

Acids Res. 19: 2,045 (1991)).

With both probes, Hpα II digestion revealed a high extent of demethylation in

three of six antisense lines (Tr244, 246, and 248; designated "weak") showed near

wild-type levels of methylation. Msp I digestion was more complete in strong antisense lines contain substantial demethylation of these repeated sequences at

C^5mCGG and 5^mCCGG sites. Digestion of wild-type genomic DNA with Eco RII and Bst NI, isochizomers

with differential sensitivity to cytosine methylation in the motif C^5mC(A/T)GG, 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 antisense 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 5^mC were also measured in these lines by

high-performance 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 μg of ribonuclease A (Sigma) for 30 min. at 37°C, followed by

passage through a Sepharose CL-5B (Pharmacia) spin column. Nucleosides were

resolved on a Varian Vista 5500 Liquid Chromatograph with a Rainin Dynamax 5 μm

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 KH,PO₅ (pH 4.0),

followed by a 10 min. linear gradient to 8.0% methanol, 50 ml KH₂PO₅ (pH 4.0).

(Table I).

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 (A. Vongs, et al, (1993)). Total genomic 5^mC 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 5^mC 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

(R. Li, et al, Cell 59: 915 (1992)). Unlike the pattern of demethylation of the ddml

mutant, however, METl 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

et al, (1984), with the following exceptions: genomic DNAs were isolated from

plants immediately after the onset of flowering, treated with 20 μg of ribonuclease A

(Sigma) for 30 min. at 37°C, followed by passage through a Sepharose CL-5B

(Pharmacia) spin column. Nucleosides were resolved on a Varian Vista 5500 Liquid

Chromatograph with a Rainin Dynamax 5 μm Spherical Microsorb C18 column (100

Λ 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). 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 vl.2 (Rainin). Percentages of total

5^mC content [5^mC/(5^mC + C)] are normalized for absorbance differences between

cytosine and 5^mC. Table 1 Total

Line 5^mC %WT %

(%) levels Decrease

Wild type 6.38 ± 0.69 100 0

Tr245 4.24±0.59 66.5 33.5

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 antisense lines in Table 2. Flowering time is

measured in days after germination. Flowering time refers to the number of days elapsed from seed germination 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

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 = 1 1 ; and

Tr248, n = 15.

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±2.1 10.0±1.4 4.5±1.1

Tr243 27.8±0.4 11.0±0.7 4.3±0.4

Tr244 47.7±4.0 34.0±5.7 20.3±4.9

Tr245 27.7±7.5 1 1.5±1.5 4.3±0.8

Tr246 45.9±3.6 32.5±3.2 20.7±1.4

Tr248 46.3±4.7 32.9±2.7 20.5±2.0

Normal patterns of development were perturbed in strong antisense lines.

Under long day conditions, six single-locus lines were identified by kanamycin segregation. Southern 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 TI outcrosses from single-locus lines

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 μg/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 nodes with delayed abaxial trichome production and flowered after 45 to 48 days

(Table 2); these phenotypes were fully penetrant in all T-DNA-containing progeny of

the strong antisense lines.

After a transition from vegetative to reproductive development, wild-type

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: 539

(1993)). In strong antisense lines, the primary inflorescence shoot produced an

average of 20 secondary branches (Table 2) before the production of flowers. The

basal-most branches in strong antisense lines often assumed characteristics of

vegetative rosettes, including enhanced 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

initiating flowers from strong antisense 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 obvious defects in pollen viability or paternal transgene transmission were observed

on the basis of segregation ratios of transgenes in outcrosses Kanamycin resistance

segregated in a 1 :1 ratio in all outcrosses from strong antisense lines.

One trivial explanation for the METJ antisense pleiotropy is that it represents 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 antisense lines, the severe phenotype co-segregated with

the presence of the transgene, and a slightly attenuated 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 1 1 ). In sum, 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 treatment of Arabidopsis

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 some late-flowering strains and mutants of Arabidopsis (J. E. Burn, et al, Proc.

Nat 'I Acad. Sci. U.S.A. 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

of DNA MTase (R. Jϋtterman, et al, Proc. Natl 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 antisense 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

sequences in METl antisense lines not seen in the ddml mutant.

On the basis of these studies, DNA methylation is shown to be an essential

component in the process of phase transitions and meristem determinacy.

Methylation may serve as a primary signal to restrict meristem determinacy, or it may

represent a secondary process required to maintain an epigenetic state once

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 antisense plan — the location of

branches apical to flowers in the inflorescence transition zone may represent an

autonomous switching of individual cells in the meristem resulting in developmental mosaicism. Methylation effects have also been shown to be progressive in the plant meristem (R. Martienssen, et al, Genes Dev. 4: 331(1990)), and it is intriguing to

speculate that a methylation gradient might be established during meristem growth to

serve as an autonomous signal that directs meristem determinacy. In this light, the

strong antisense phenotype could be explained by delayed establishment of this

hypothetical gradient. Such a model predicts that meristem potential becomes

progressively more epigenetically restrictive and that repression cascades 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 l: 1,485 (1995)).

EXAMPLE 2

Plants with increased rates of development

To generate plants with increased amount 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.

The cDNA encoding the METl protein from Arabidopsis described above was used to

generate expression units in which the METl 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

from the transformed tissue. 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 12

days following germination, compared to 26 days for non-altered plants.

EXAMPLE 3

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 altering the amount of DNA methylation present

in a plant. The Examples provide the results of applications of the methods herein

described. The following is intended 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. 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 amount of methylated DNA while to

decrease the rate of maturation, the plant is altered so as to contained a decreased

amount of methylated DNA.

2. Chose the alteration method/target 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

methylation can be increased by activating or increasing the methylase activity within

the cell.

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 preferred embodiments. It therefore should be

apparent 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 identified above are incorporated by

reference in their entirety.

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 METl 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 antisense 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 METl

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 METl 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 METl 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 antisense 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 METl

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 METl 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.