WO2013033094A2

WO2013033094A2 - Elimination of seed formation by ablating floral organs

Info

Publication number: WO2013033094A2
Application number: PCT/US2012/052662
Authority: WO
Inventors: Chunsheng Zhang; Kimberly Ann Winkeler; Kim Norris-Caneda
Original assignee: Arborgen Inc
Current assignee: Arborgen Inc
Priority date: 2011-08-31
Filing date: 2012-08-28
Publication date: 2013-03-07
Anticipated expiration: 2014-02-28
Also published as: WO2013033094A3

Abstract

The present invention relates to methods to limit or eliminate migration of pollen and seeds from transgenic plants into wild plant populations by producing sterile transgenic plants capable of achieving full vegetative growth. Specifically, the present invention provides targeted ablation of reproductive structures in plants by devising a promoter specific for carpel tissue and transforming plants with DNA constructs in which the promoter mediates expression of cytotoxic genes.

Description

ELIMINATION OF SEED FORMATION BY ABLATING FLORAL

ORGANS

RELATED APPLICATION

This application claims priority from U.S. Provisional Application Serial No.

61/529,595, filed August 31, 2011, which is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates to the regulation of reproductive development. In particular, this invention relates to the genetic ablation of reproductive tissues in angiosperm and gymnosperm species. Reproductive-preferred promoters, regulatory elements, and cytotoxic nucleotide sequences are provided. Constructs and methods for genetic ablation are also included in the invention.

BACKGROUND OF THE INVENTION

With the advent of plant genetic engineering technology and agro-biotechnology, the ecological implications of genetically modified crops and foods raised by growers and consumers, including the potential spread of pollen from transgenic crops to wild plants, must be considered. In some cases, the introgression of transgenic crop genes into the genome of related wild species could pose undesired ecological and societal concerns Liu et al. Plant Ce// i?e/? 27: 995-1004 (2008); Bergelson et a/. Nature 395: 25 (1998). Limiting transgene flow is therefore a useful tool for the successful development of agro-biotechnology and the preservation of the natural ecosystem. While the production of seeds and fruits is critical in crop plants, most ornamental plants and trees grown for forestry are valued for their vegetative growth and their seeds and fruits have no economical value. Accordingly, one possible strategy for preventing transgene flow is the development of male and female sterility in a desired plant by ablation of its reproductive structures. Although tissue-specific ablation has been achieved with the use of cytotoxic genes in combination with male or female tissue-specific promoters for engineering male and female sterility (Mariani et al. 1990 Nature 347: 737-41; Mariani et al. 1992 Nature 357: 384-87; Beals and Goldberg 1997 Plant Cell 9: 1527-45; Block et al. 1997 Theor. Appl. Gent. 95: 125-31; Gleba et al. 2004 Biotechnol. Genet. Eng. Rev. 21 : 325-67; Skinner et al. 2003 Mol. Breed. 12: 119-32; Wei et al. 2007 Mol. Breed. 19: 69-85), the practical application for transgene flow containment has been hampered by possible negative effects on vegetative growth in the resulting transgenic plants. These detrimental effects on vegetative growth are due to the lack of strongly-regulated floral organ- or gamete-specific promoters that target the reproductive organs of the plants without compromising their vegetative growth (Lannepaa et al. 2005 Plant Cell Rep. 24:69-78). Accordingly, there is a need in the art for floral-specific gene promoters that drive cytotoxic genes to specifically ablate the reproductive system in ornamental plants and forest trees without impairing vegetative growth. The present invention satisfies this need.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide solutions to the aforementioned deficiencies in the art.

To this end, the invention provides for methods to limit or eliminate migration of pollen and seeds from transgenic plants into wild plant populations by producing sterile transgenic plants capable of achieving full vegetative growth. Specifically, the present invention provides targeted ablation of reproductive structures in plants by devising a promoter specific for carpel tissue and transforming plants with DNA constructs in which the promoter mediates expression of cytotoxic genes.

Thus, in one embodiment, the present invention provides an isolated polynucleotide comprising a Eucalyptus AGAMOUS promoter. In one aspect of the invention, the isolated polynucleotide is a polynucleotide that comprises the Eucalyptus AGAMOUS promoter of SEQ ID NO: 1, or a polynucleotide that comprises a sequence that is complementary to SEQ ID NO: 1, or a reverse complement of SEQ ID NO: 1. In a different aspect of the invention, the isolated polynucleotide is a polynucleotide that comprises the Eucalyptus AGAMOUS promoter of SEQ ID NO: 13 or a functional fragment thereof, or a polynucleotide that comprises a sequence that is complementary to SEQ ID NO: 13or is a reverse complement of SEQ ID NO: 13.

In another embodiment, the invention provides an ablation cassette comprising a polynucleotide comprising the Eucalyptus AGAMOUS promoter of SEQ ID NO: 1 or SEQ ID NO: 13, or a sequence that is complementary to SEQ ID NO: 1 or SEQ ID NO: 13, or a reverse complement of SEQ ID NO: 1 or SEQ ID NO: 13, or a functional fragment thereof, operably linked to a desired nucleic acid sequence that encodes a protein that alters the development of a reproductive structure of a plant. The Eucalyptus AGAMOUS promoter may upregulate or downregulate expression of the nucleic acid sequence in a plant. In a preferred embodiment, the plant is not Arabydopsis. In a preferred aspect of the invention, the Eucalyptus AGAMOUS promoter upregulates the expression of the desired nucleic acid sequence, and the desired nucleic acid sequence encodes a cytotoxic protein that destroys a reproductive structure in the plant. In a preferred embodiment, the reproductive structure is a carpel.

In a preferred aspect of the invention, the desired nucleic acid sequence in the ablation cassette encodes a barnase mutant. In one aspect of the invention, the barnase mutant is a barnaseE73G mutant comprising SEQ ID NO: 2 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 3. In a different aspect of the invention, the barnase mutant is a barnaseK27A mutant comprising SEQ ID NO: 4 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 5. Preferably, the polynucleotide comprises a floral enhancer that controls the floral specificity and activity of the Eucalyptus AGAMOUS promoter. In a preferred aspect of the invention, the floral enhancer is the second intron sequence of the AGAMOUS gene and is located at the 5 ' end of the Eucalyptus AGAMOUS promoter. In an even more preferred aspect of the invention, the floral enhancer comprises SEQ ID NO: 6. In another preferred aspect of the invention, the floral enhancer comprises SEQ ID NO: 15. In another embodiment, the invention provides a DNA construct that comprises an isolated polynucleotide comprising the Eucalyptus AGAMOUS promoter of SEQ ID NO: 1 or SEQ ID NO: 13, or a sequence that is complementary to SEQ ID NO: 1 or SEQ ID NO: 13, or a reverse complement of SEQ ID NO: 1 or SEQ ID NO: 13, or a functional fragment thereof, operably linked to a desired nucleic acid sequence, wherein the promoter regulates the expression of the desired nucleic acid sequence. In a preferred aspect of the invention, the plant is not Arabidopsis. In one embodiment, the promoter upregulates or downregulates the expression of the desired nucleic acid sequence. In a preferred aspect of the invention, the promoter upregulates the expression of a nucleic acid sequence that encodes a protein that alters the development of a reproductive structure of a plant. In an even more preferred aspect of the invention, the desired nucleic acid sequence encodes a cytotoxic protein that destroys a reproductive structure in a plant.

In a preferred aspect of the invention, the desired nucleic acid sequence in the ablation cassette encodes a barnase mutant. In one aspect of the invention, the barnase mutant is a barnaseE73G mutant comprising SEQ ID NO: 2 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 3. In a different aspect of the invention, the barnase mutant is a barnaseK27A mutant comprising SEQ ID NO: 4 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 5. Preferably, the polynucleotide comprises a floral enhancer that controls the floral specificity and activity of the Eucalyptus AGAMOUS promoter. In a preferred aspect of the invention, the floral enhancer is the second intron sequence of the AGAMOUS gene and is located at the 5 ' end of the Eucalyptus AGAMOUS promoter. In an even more preferred aspect of the invention, the floral enhancer comprises SEQ ID NO: 6. In another preferred aspect of the invention, the floral enhancer comprises SEQ ID NO: 15.

In another embodiment, the invention provides a plant cell transformed with any of the DNA constructs described above or herein. Also provided by the invention is a transgenic plant comprising one or more plant cells transformed with any of the DNA constructs described above or herein. In one aspect of the invention, the transgenic plant exhibits a phenotype that is different from the phenotype of a non-transgenic plant of the same species. In a preferred aspect of the invention, the different phenotype is a stunted carpel. In another preferred aspect of the invention, the different phenotype is characterized by reduced or no seed formation. In yet another preferred aspect of the invention, the different phenotype is characterized by a stunted carpel and reduced or no seed formation and the transgenic plant shows no difference in vegetative growth rate, height and leaf color when compared to a non-transgenic plant of the same species. In one aspect of the invention, the transgenic plant is a monocotyledonous plant. In a different aspect of the invention, the transgenic plant is a dicotyledonous plant. In one embodiment, the transgenic plant is a gymnosperm. In a different embodiment, the transgenic plant is an angiosperm. In a preferred embodiment, the transgenic plant is a transgenic tree. In another preferred embodiment, the transgenic plant is a transgenic ornamental plant.

In a further embodiment, the invention provides a method for producing a transgenic plant with an ablated reproductive organ, comprising: (a) transforming a plant cell with a DNA construct that comprises an isolated polynucleotide comprising the Eucalyptus

AGAMOUS promoter of SEQ ID NO: 1 or SEQ ID NO: 13, or a sequence that is

complementary to SEQ ID NO: 1 or SEQ ID NO: 13, or a reverse complement of SEQ ID NO: 1 or SEQ ID NO: 13, or a functional fragment thereof, operably linked to a desired nucleic acid sequence, wherein the promoter regulates the expression of the desired nucleic acid sequence; (b) culturing the transformed plant cell under conditions that promote growth of a transgenic plant; and (c) selecting a transgenic plant with a stunted carpel and reduced seed formation phenotype, wherein the transgenic plant shows no difference in vegetative growth rate, height and leaf color when compared to a non-transgenic plant of the same species. In a preferred aspect of the invention, the plant is not Arabidopsis. In one embodiment, the promoter upregulates or downregulates the expression of the desired nucleic acid sequence. In a preferred aspect of the invention, the promoter upregulates the expression of a nucleic acid sequence that encodes a protein that alters the development of a

reproductive structure of a plant. In an even more preferred aspect of the invention, the desired nucleic acid sequence encodes a cytotoxic protein that destroys a reproductive structure in a plant. In a preferred aspect of the invention, the desired nucleic acid sequence in the ablation cassette encodes a barnase mutant. In one aspect of the invention, the barnase mutant is a barnaseE73G mutant comprising SEQ ID NO: 2 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 3. In a different aspect of the invention, the barnase mutant is a barnaseK27A mutant comprising SEQ ID NO: 4 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 5. Preferably, the polynucleotide comprises a floral enhancer that controls the floral specificity and activity of the Eucalyptus AGAMOUS promoter. In a preferred aspect of the invention, the floral enhancer is the second intron sequence of the AGAMOUS gene and is located at the 5 ' end of the Eucalyptus AGAMOUS promoter. In an even more preferred aspect of the invention, the floral enhancer comprises SEQ ID NO: 6. In another preferred aspect of the invention, the floral enhancer comprises SEQ ID NO: 15.

In a further embodiment, the invention provides a method for preventing or reducing seed formation and natural pollination in a plant without impairing the vegetative growth of the plant, comprising: (a) transforming a plant cell with a DNA construct that comprises an ablation cassette comprising an isolated polynucleotide comprising the Eucalyptus

AGAMOUS promoter of SEQ ID NO: 1 or SEQ ID NO: 13, or a sequence that is complementary to SEQ ID NO: 1 or SEQ ID NO: 13, or a reverse complement of SEQ ID NO: 1 or SEQ ID NO: 13, or a functional fragment thereof, operably linked to a desired nucleic acid sequence that encodes a cytotoxic protein that destroys a reproductive structure of a plant; and (b) culturing the transformed plant cell under conditions that promote growth of a plant, wherein expression of the ablation cassette in the plant results in a reduction in seed formation and natural pollination in the plant and has no deleterious effect in the plant. In a preferred aspect of the invention, the desired nucleic acid sequence in the ablation cassette encodes a barnase mutant. In one aspect of the invention, the barnase mutant is a barnaseE73G mutant comprising SEQ ID NO: 2 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 3. In a different aspect of the invention, the barnase mutant is a barnaseK27A mutant comprising SEQ ID NO: 4 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 5. Preferably, the polynucleotide comprises a floral enhancer that controls the floral specificity and activity of the Eucalyptus AGAMOUS promoter. In a preferred aspect of the invention, the floral enhancer is the second intron sequence of the AGAMOUS gene located at the 5 ' end of the Eucalyptus AGAMOUS promoter. In yet another embodiment, the invention provides a method for conferring complete reproductive sterility to a plant without impairing the vegetative growth of the plant comprising (a) transforming a plant cell with a DNA construct comprising (i) a first cassette comprising a male or female reproductive structure -preferred promoter operably linked to a nucleic acid sequence that encodes a protein that ablates reproductive development in the plant, wherein the reproductive structure -preferred promoter regulates the expression of the nucleic acid sequence; and (ii) a second cassette comprising a Eucalyptus AGAMOUS promoter operably linked to a desired nucleic acid sequence that encodes a cytotoxic protein that destroys a reproductive structure of a plant; (b) culturing the transformed plant cell under conditions that promote growth of a plant; and (c) selecting a plant that expresses both the first and the second cassettes of the DNA construct, wherein expression of both the first and the second cassettes in the plant results in complete sterility of the plant and has no deleterious effect on the vegetative growth of the plant. In a preferred aspect of the invention, the first cassette comprises a Pinus radiata male cone (PrMC) promoter comprising SEQ ID NO: 7 or SEQ ID NO: 8 operably linked to a nucleic acid sequence encoding a barnaseH102E mutant comprising SEQ ID NO: 9 or a nucleic acid sequence encoding a barnaseH102E mutant comprising SEQ ID NO: 10, and the second cassette comprises the Eucalyptus AGAMOUS promoter of SEQ ID NO: 13 or a fragment thereof comprising SEQ ID NO: 16 operably linked to a nucleic acid sequence encoding a barnaseK27A mutant. Preferably, the nucleic acid sequence encoding the barnaseK27A mutant comprises SEQ ID NO: 4 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 5.

In yet a further embodiment, the invention provides a completely sterile transgenic plant obtained by a method comprising (a) transforming a plant cell with a DNA construct comprising (i) a first cassette comprising a male or female reproductive structure-preferred promoter operably linked to a nucleic acid sequence that encodes a protein that ablates reproductive development in the plant, wherein the reproductive structure -preferred promoter regulates the expression of the nucleic acid sequence; and (ii) a second cassette comprising a Eucalyptus AGAMOUS promoter operably linked to a desired nucleic acid sequence that encodes a cytotoxic protein that destroys a reproductive structure of a plant; (b) culturing the transformed plant cell under conditions that promote growth of a plant; and (c) selecting a plant that expresses both the first and the second cassettes of the DNA construct. In the most preferred embodiment, the transgenic plant shows no difference in vegetative growth rate, height and leaf color when compared to a non-transgenic plant of the same species.

Preferably, the transgenic plant is a transgenic tree or a transgenic ornamental plant.

In yet another embodiment, the invention provides wood and wood products obtained from the transgenic plants according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the sequence alignment of the AG protein of Eucalyptus occidentalis and Eucalyptus grandis.

Figure 2 depicts the alignments of AG cDNA sequences obtained from Eucalyptus occidentalis and Eucalyptus grandis (EucSppO 13502). The top line in the alignment is the cDNA sequence for the Eucalyptus occidentalis AG cDNA. The bottom line is the sequence from the Eucalyptus grandis ArborGen 4000 AG cDNA. The intervening lines are sequencing data of the Eucalyptus occidentalis 3 'RACE clones.

Figure 3 shows the results obtained for Eucalyptus occidentalis AG expression by semi-quantitative PCR analysis. AG expression was determined in floral buds, leaves and shoot tips. Appearance of a 350 bp band shows AG high expression in floral bud tissue, no expression in leaf tissue and weak expression shoot tips.

Figure 4 shows the genomic DNA partial alignment between three clones having identical 1.4 kb long sequences which share a sequence identical to a region of the

Eucalyptus occidentalis AG gene and the known sequence of the Eucalyptus grandis AG gene. The shared sequence of the three clones was identified as the Eucalyptus occidentalis AG gene promoter. The sections highlighted in yellow represent the overlap between the three clones and the known Eucalyptus occidentalis AG gene sequence; the translational start is highlighted in green.

Figure 5 depicts the PCR primer locations for the cloning of a missed 300 bp genomic DNA fragment. Figure 6 shows the genomic DNA sequence alignment of the Eucalyptus occidentalis AG gene with the location of the missed 300 bp sequence.

Figure 7 depicts the maps of the plasmids pAGW52, pAGW53 and pAGW55.

Figure 8A depicts the flowers of transgenic plants transformed with a floral ablation construct (pAGW53) as compared to the flowers of control plants transformed with a GUS control construct (pAGW55).

Figure 8B compares a GUS (control) tobacco plant carrying naturally-pollinated fruits to a transgenic tobacco plant transformed with the carpel ablation construct pAGW53 carrying degenerated flowers.

Figure 9 compares the seeds of transgenic tobacco plant transformed with the carpel ablation construct pAGW53 to the seeds of control tobacco plants transformed with the GUS control construct pAGW55. Magnification = 35X.

Figure 10 shows the seeds collected from hand-pollinated small fruits of transgenic tobacco plant transformed with the carpel ablation construct pAGW53 germinated on agar Petri dish in the presence of 1% of sucrose.

Figure 11 depicts the plasmid maps of the constructs pAGK Ol (Figure 11A), pAGK 02 (Figure 1 IB), pAGK 03 (Figure 11C), and pAGK 04 (Figure 1 ID).

Figure 12 shows the morphology of stunted carpels in transgenic tobacco plants transformed with the construct pAGK 03.

Figure 13 depicts the plasmid map of the construct pAGK 05.

Figure 14 shows the alignment of the nucleic acid sequence of the second intron of the Eucalyptus occidentalis AGAMOUS gene (3981 bp) and the nucleic acid sequence of the second intron of the Eucalyptus occidentalis AGAMOUS gene (3972 bp).

Figure 15 shows the amplification plot of the Eucalyptus occidentalis TE0558868 transgenic line transformed with the pAGW52 plasmid comprising the AGAMOUS gene promoter (EoAG) operably linked to the barnaseK27A, as compared to a Eucalyptus occidentalis non-transgenic line.

Figure 16 provides a phenotypic comparison of the Eucalyptus occidentalis

TE0558868 transgenic line transformed with the pAGW52 plasmid comprising the

AGAMOUS gene promoter (EoAG) operably linked to the barnaseK27A, and the Eucalyptus occidentalis non-transgenic line.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

AGAMOUS genes encode regulatory proteins which contain a conserved MADS-box region. These regulatory proteins are involved in the formation of reproductive structures in plants, in particular in the formation of stamen and carpel for flowering.

In the past, tissue-specific ablation has been obtained in the laboratory and under greenhouse conditions using the cytotoxic barnase gene under the control of male or female tissue-specific promoters. This approach, however, has led to severe damage to vegetative growth in the transgenic plants because of promoter activity leakage to non-targeted tissues. Use of the Barstar gene system to repress the damaging activity of barnase has not prevented the negative effects on vegetative growth in the transgenic plants.

The aim of the present invention is to provide effective methods for tissue-specific ablation of male and female reproductive organs in gymnosperms and angiosperms by driving the expression of proteins encoded by cytotoxic genes under the control of novel promoters. The inventors of the present application have made the surprising discovery that the transgenic plants thus obtained are fully sterile and have increased vegetative growth and biomass.

The current invention utilizes a Eucalyptus occidentalis AGAMOUS floral gene promoter driving the barnase mutant gene to specifically ablate the carpel of flowers. In one aspect of the invention, the Eucalyptus occidentalis AGAMOUS floral gene promoter comprises SEQ ID NO: 1. In a preferred aspect of the invention, the Eucalyptus occidentalis AGAMOUS floral gene promoter comprises SEQ ID NO: 13 or a functional fragment thereof. In the Eucalyptus occidentalis AGAMOUS floral gene promoter of SEQ ID NO: 13, the second intron is located at the 5' end of the promoter, and the promoter does not comprise a 180 bp first exon, which is the MADS box domain and is present in SEQ ID NO: 1. Since the Eucalyptus occidentalis AGAMOUS floral gene promoter of SEQ ID NO: 13 and functional fragments thereof lack the first exon, these sequences drive the expression of a barnase protein which is free of extra amino acids at its N terminus. To date, this is the first time that a eucalyptus promoter has been successfully used to eliminate the formation of seeds in transgenic tobacco without any side effects. The DNA construct of the invention may also be successfully used to eliminate seed formation in forest trees, such as eucalyptus, by ablating carpel, resulting in an increase in vegetative growth and biomass of trees.

Definitions

The present invention uses terms and phrases that are well known to those practicing the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, microbial culture, cell culture, tissue culture, transformation, transfection, transduction, analytical chemistry, organic synthetic chemistry, chemical syntheses, chemical analysis, and pharmaceutical formulation and delivery. Generally, enzymatic reactions and purification and/or isolation steps are performed according to the manufacturers' specifications. The techniques and procedures are generally performed according to conventional methodology. See, e.g., Sambrook & Russel, MOLECULAR CLONING: A LABORATORY MANUAL, 3.sup.rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.

Agrobacterium: Agrobacteria that are used for transforming plant cells are disarmed and virulent derivatives of, usually, Agrobacterium tumefaciens or Agrobacterium rhizogenes that contain a vector. The vector typically contains a desired polynucleotide that is located between the borders of a T-DNA. Angiosperms: Angiosperms are vascular plants having seeds enclosed in an ovary. Angiosperms are seed plants that produce flowers that bear fruits. Angiosperms are divided into dicotyledonous and monocotyledonous plants.

Angiosperm Reproductive Structure: includes the male and female tissues that comprise a flower. Typically, angiosperm flowers have four different floral organs: sepals (calyx), petals (corolla), stamens (androcecium), and pistils (gynoecium).

Angiosperm reproductive structure also embraces pre-male and pre-female reproductive structures. Pre-male and pre-female reproductive structures embrace cells and tissues that form before development and differentiation of male and female tissues.

Complement: a second nucleic acid that is capable of hybridizing with a first nucleic acid to produce a double-stranded nucleic acid, as depicted in the following example.

First nucleic acid: 5 '-AACCGTTGTT-3 '

Second nucleic acid: 3 ' -TTGGC AACAA-5 ' , which is equivalent to

5 '-AACAACGGTT-3 ' .

Nucleic acid sequences that are capable of hybridizing to form double-stranded structures are said to be complementary. The double-stranded nucleic acid that results from the hybridization of two complementary nucleic acid sequences may be DNA, RNA or a hybrid of DNA and RNA. In the case of RNA, the T's in the above example would be replaced with U's. Historically, the complement as described here has been referred to formally as the reverse complement. In this document, "complement" is considered to be a shortened form of "reverse complement", and the two terms are synonymous.

Desired Polynucleotide: a desired polynucleotide of the present invention is a genetic element, such as a promoter, enhancer, or terminator, or gene or polynucleotide that is to be transcribed and/or translated in a transformed cell that comprises the desired polynucleotide in its genome. If the desired polynucleotide comprises a sequence encoding a protein product, the coding region may be operably linked to regulatory elements, such as to a promoter and a terminator, that bring about expression of an associated messenger RNA transcript and/or a protein product encoded by the desired polynucleotide. Thus, a "desired polynucleotide" may comprise a gene that is operably linked in the 5'- to 3 '-orientation, a promoter, a gene that encodes a protein, and a terminator. Alternatively, the desired polynucleotide may comprise a gene or fragment thereof in an "antisense" orientation, the transcription of which produces nucleic acids that may form secondary structures that affect expression of an endogenous gene in the plant cell. A desired polynucleotide may also yield a double-stranded RNA product upon transcription that initiates RNA interference of a gene to which the desired polynucleotide is associated. A desired polynucleotide of the present invention may be positioned within a T-DNA, such that the left and right T-DNA border sequences flank or are on either side of the desired polynucleotide. The present invention envisions the stable integration of one or more desired polynucleotides into the genome of at least one plant cell. A desired polynucleotide may be mutated or may be a variant of its wild-type sequence. It is understood that all or part of the desired polynucleotide can be integrated into the genome of a plant. It also is understood that the term "desired polynucleotide" encompasses one or more of such polynucleotides. Thus, a T-DNA of the present invention may comprise one, two, three, four, five, six, seven, eight, nine, ten, or more desired polynucleotides.

Dicotyledonous plant (dicot): a flowering plant whose embryos have two seed halves or cotyledons, branching leaf veins, and flower parts in multiples of four or five. Examples of dicots include but are not limited to, Eucalyptus spp, Populus spp., Liquidambar spp., Salix spp., Acacia spp., Tectona spp., Swietenia spp., Quercus spp., Acer spp., Juglans spp., Persea americana, Gossypium spp., Nicotiana spp., Arabidopsis, Solarium spp., Beta spp., Brassica spp., Manihot esculenta, Ipomoea batatas, Euphorbia spp. Glycine spp., Phaseolus spp. Medicago spp., Daucus spp., Fragaria spp. , Lactuca spp., , Rosa spp., , Mentha spp, Cucurbita spp., Chrysanthemum spp. , Pelargonium spp., , Opuntia spp., , Linum spp., Heliothus spp., Arachis spp., Jatropha curcas and Dichondra spp..

Endogenous: refers to a gene that is native to a plant genome.

Female reproductive tissues: include, for example, stigma, style, ovary, megaspore, female cones (ovuliferous cones), female gamete, female zygote, megasporocyte, and pre- female reproductive structures. Female-Sterility Gene: refers to a nucleic acid molecule encoding an RNA, protein, or polypeptide that disrupts growth and development of a female gametophyte, female gamete, female zygote, seed, ovuliferous cone, or pre-female reproductive structure. A plant expressing a female-sterility gene produces no viable seed. There are many different mutations that can lead to female-sterility, involving all stages of development of a specific tissue of the female reproductive organ or pre-female reproductive structure. Examples of female-sterility genes include, but in no way limiting, genes that encode enzymes which catalyze the synthesis of phytohormones, such as: isopentenyl transferase, which is an enzyme that catalyzes the first step in cytokinin biosynthesis and is encoded by gene 4 of Agrobacterium T-DNA; or one or both of the enzymes involved in the synthesis of auxin and encoded by gene 1 and gene 2 of Agrobacterium T-DNA. Yet other examples of female- sterility genes encode: glucanases; lipases such as phospholipase A₂ (Verheij et al. Rev.

Blochem. Pharmacol. 91:92-203 (1981)); lipid peroxidases; or plant cell wall inhibitors. Still other examples of female-sterility genes encode proteins toxic to plants cells, such as a bacterial toxin (e.g., the A-fragment of diphtheria toxin or botulin). Still another example of a female-sterility gene is an antisense nucleic acid, or RNA involved in RNA interference (RNAi) such as small interfering RNA (siRNA), which can be useful for inhibiting or completely blocking the expression of a targeted gene. For example, an antisense or RNAi molecule of the invention encodes a nucleic acid strand complementary to a strand that is naturally transcribed in a plant's reproductive cells under the control of an endogenous promoter as described, for example, in European Patent Publication 0,223,399. Such an antisense nucleic acid or RNAi molecule may be capable of binding to the coding and/or non- coding portion of an RNA, naturally produced in the reproductive cell, so as to inhibit the translation of the naturally produced RNA. The use of RNAi inhibition of gene expression is described generally in Paddison et al., Genes & Dev. 16: 948-958 (2002), and the use of RNAi to inhibit gene expression in plants is specifically described in WO 99/61631, both of which are herein incorporated by reference. A further example of a female-sterility gene encodes a specific RNA enzyme (i.e., a "ribozyme"), capable of highly specific cleavage against a given target sequence as described by Haseloff and Gerlach et al. Nature 334: 585- 591 (1998). Fiber Quality: as used herein, fiber quality refers to a trait that can be modified to change the structure, appearance, or use of fiber. Traits that determine fiber quality include but are not limited to chemical composition, fiber length, coarseness, strength, color, cross- sectional, width, and fiber density. For example, it is known that fiber length imparts strength, whereas fiber coarseness determines texture and flexibility.

Floral Meristems: in angiosperms floral meristems initiate a floral structure having four different types of floral organs: sepals (calyx), petals (corolla), stamens (androecium), and pistils (gynoecium). Each floral organ is initated as a whorl, comprising concentric rings around the flanks of a floral meristem. The floral structure is supported by a pedicel or peduncle.

Flowering plants produce meiospores that are either microspores (male) or megaspores (female).

Foreign: "foreign," with respect to a nucleic acid, means that that nucleic acid is derived from non-plant organisms, or derived from a plant that is not the same species as the plant to be transformed or is not derived from a plant that is not interfertile with the plant to be transformed, or does not belong to the species of the target plant. According to the present invention, foreign DNA or R A may include nucleic acids that are naturally occurring in the genetic makeup of fungi, bacteria, viruses, mammals, fish or birds, but are not naturally occurring in the plant that is to be transformed. Thus, a foreign nucleic acid is one that encodes, for instance, a polypeptide that is not naturally produced by the transformed plant. A foreign nucleic acid does not have to encode a protein product.

Gene: A gene is a segment of a DNA molecule that contains all the information required for synthesis of a product, polypeptide chain or RNA molecule, and includes both coding and non-coding sequences.

Genetic element: a "genetic element" is any discreet nucleotide sequence including, but not limited to, a promoter, a gene, a terminator, an intron, an enhancer, a spacer, a 5'- untranslated region, a 3 '-untranslated region, or a recombinase recognition site. Genetic modification: stable introduction of DNA into the genome of certain organisms by applying methods in molecular and cell biology.

Gymnosperm: as used herein, refers to a seed plant that bears seed without ovaries. Examples of gymnosperms include conifers, cycads, ginkgos, and ephedras. In

gymnosperms, reproductive shoot primordia develop into either male cones (staminate cones) or female cones (ovulate cones).

Gymnosperm Reproductive Structure: includes the male tissues that comprise male pollen cones (staminate cones) and female tissues that comprise female cones (ovulate cones). Gymnosperm reproductive structure also embraces pre-male and pre-female reproductive structures. Pre-male and pre-female reproductive structures embrace cells and tissues that form before development and differentiation of male and female tissues.

Introduction: as used herein, refers to the insertion of a nucleic acid sequence into a cell, by methods including infection, transfection, transformation or transduction.

Lignin: as used herein, refers to a polymeric composition composed of

phenylpropanoid units, including polymerized derivatives of monolignols coniferyl, coumaryl, and sinapyl alcohol. Lignin quality refers to the ability of a lignin composition to impart strength to cell wall matrices, assist in the transport of water, and/or impede degradation of cell wall polysaccharides. Lignin composition or lignin structure may be changed by altering the relative amounts of each of monolignols or by altering the type of lignin. For example, guaiacyl lignins (derived from ferulic acid) are prominent in softwood or coniferous species, whereas guaiacyl-syringyl lignins (derived from ferulic acid and sinapic acid) are characteristic of hardwood or angiosperm species. The degradation of lignin from softwoods, such as pine, requires substantially more alkali and longer incubations, compared with the removal of lignin from hardwoods. Lignin composition may be regulated by either up-regulation or down-regulation of enzymes involved lignin biosynthesis. For example, key lignin biosynthsesis enzymes include, but are not limited to, 4-coumaric acid: coenzyme A ligase (4CL), Cinnamyl Alcohol dehydrogenase (CAD), and Sinapyl Alcohol Dehydrogenase (SAD). Male gametophytes or pollen grains: in angiosperms they develop in anthers, and the anthers are borne on stamens. Anther development occurs in two stages that correlate with pollen development. During phase I, sporogenic cells in the anther undergo

microsporogenesis; nonsporogenic cells form the epidermis and tapetum. The tapetum is a tissue that surrounds sporogenic cells and provides nutritional materials for developing pollen. Additionally, the tapetum secretes the enzyme callase. During phase II, the anther enlarges and the filament elongates. At this time, pollen grains form, dehiscence occurs, and pollen grains are released. In gymnosperms, such as conifers, a male pollen cone consists of an axis bearing a series of scales and two pollen sacs on the undersurface of each scale. Male cones consist of numerous microsporophylls that are tightly clustered in a spiral arrangement on a fertile shoot axis. Each microsporophyll bears two microsporangia, also called pollen sacs, on its lower, abaxial side. Within each microsporangium, sporangenous tissue lies. The sporangenous tissue consists of numerous diploid cells, called microsporocytes, which undergo meiosis. Around the periphery of each microsporangium lies the tapetum. Within the microsporangia, the microspores undergo mitosis and following two mitotic divisions, a four- celled male gametophyte is produced. The pollen grain comprises the microspore wall and the contained male gametophyte. In gymnosperms, a female cone is formed by the fusion of numerous highly modified fertile shoots. In pines, for example, the female cone is comprised of individual units attached to a single, central axis. The individual units are made of an ovuliferous scale (ovule-bearing) and a subtending bract that is almost completely fused to the ovuliferous scale above it. Each ovuliferous scale is formed by the fusion of

megasporophylls and other fertile shoot components. On the upper, adaxial surface of each ovuliferous scale are two ovules. The ovules are oriented with their micropyles toward the central cone axis and are partially imbedded in the tissues of the ovuliferous scale. Each ovule has an integument (one multicellular layer) that, except for the micropyles, completely surrounds the megasporangium. The integument or nucellus functions as the nutritive tissue and each nucellus has a single megasporocyte. The megasporocyte is the diploid cell that undergoes meiosis. The micropylar chamber is located within each ovule between the nucellus and the micropyle. Male reproductive tissues: include, for example, pollen grains, tapetum, anther, filament, pollen mother cells, microspores, microsporocyte, male pollen cones (staminate cones), pollen sacs, and pre-male reproductive structures.

Male- Sterility Gene: refers to a nucleic acid molecule encoding an RNA, protein, or polypeptide that disturbs the proper metabolism, functioning and/or development of any reproductive cell in which the male-sterility gene is expressed, thereby leading to the death and/or destruction of any such reproductive cell. There are many different mutations that can lead to male-sterility, involving all stages of development of a specific tissue of the male reproductive organ or pre-male reproductive structure. The expression of a male-sterility gene, for example, renders a plant incapable of producing fertile pollen. The expression of a male-sterility gene in a transformed plant may result in a plant producing pollen, though the pollen may be aberrant and non-functional for fertilization. For example, a non-functional pollen may fail to germinate a pollen tube. While by no means limiting, examples of male- sterility genes encode: RNases such as R ase Tl (which degrades RNA molecules by hydrolyzing the bond after any guanine residue) and barnase; DNases such as an

endonuclease (e.g., EcoRI); or proteases such as a papain (e.g., papain zymogen and papain active protein). Other male-sterility genes encode enzymes which catalyze the synthesis of phytohormones. For example, isopentenyl transferase, an enzyme that catalyzes the first step in cytokinin biosynthesis, and enzymes involved in the synthesis of auxin may be used for inducing male-sterility. Other male-sterility genes encode glucanases; lipases such as phospho lipase A₂ (Verheij et al. Rev. Biochem. Pharmacol. 91: 92-203 (1981)); lipid peroxidases; or plant cell wall inhibitors. Still other examples of male-sterility genes encode proteins toxic to a plants cell, such as a bacterial toxin (e.g., the B-fragment of diphtheria toxin or botulin). Still another example of a male-sterility gene is an antisense nucleic acid, or RNA involved in RNA interference (RNAi) such as small interfering RNA (siRNA), which can be useful for inhibiting or completely blocking the expression of a targeted gene. For example, an antisense or RNAi molecule of the invention encodes a nucleic acid strand complementary to a strand that is naturally transcribed in a plant's reproductive cells under the control of an endogenous promoter as described, for example, in European Patent Publication 0,223,399. Such an antisense nucleic acid or RNAi molecule may be capable of binding to the coding and/or non-coding portion of an RNA, naturally produced in the reproductive cell, so as to inhibit the translation of the naturally produced RNA. In one embodiment, an antisense nucleic acid and RNAi molecule of the invention can be expressed in pollen grains, tapetum, anther, filament, pollen mother cells, microspores, microsporocyte, male pollen cones (staminate cones), pollen sacs, and pre-male reproductive structures.

Microsporogenesis is the process by which a diploid cell, the microsporocyte, undergoes meiotic division to produce four, haploid microspores (microspore tetrad). The microspore tetrad is encased in a callose cell wall. In angiosperms, microsporogenesis occurs in the stamens, the male reproductive tissues of a flower. Each stamen has a filament and an anther. Each anther has one to four chambers, called pollen sacs or anther sacs. Each anther sac produces numerous microsporocytes, also called pollen mother cells.

In gymnosperms, microsporogenesis occurs in the microsporangia or pollen sacs of the microsporophyll. Within the microsporangia, the microspores undergo mitosis and produce a four-celled male gametophyte. A gymnosperm pollen grain comprises the microspore wall and the contained male gametophyte.

Monocotyledonous plant (monocot): a flowering plant having embryos with one cotyledon or seed leaf, parallel leaf veins, and flower parts in multiples of three. Examples of monocots include, but are not limited to, maize, rice, oat, wheat, barley, sorghum, orchid, iris, lily, onion, palm, turf grasses, and bioenergy grasses. Examples of turf grasses include, but are not limited to, Agrostis spp. (bentgrass species including colonial bentgrass and creeping bentgrasses), Poa pratensis (kentucky bluegrass), Lolium spp. (ryegrass species including annual ryegrass and perennial ryegrass), Festuca arundinacea (tall fescue), Festuca rubra commutata (fine fescue), Cynodon dactylon (common bermudagrass varieties including Tifgreen, Tifway II, and Santa Ana, as well as hybrids thereof); Pennisetum clandestinum (kikuyugrass), Stenotaphrum secundatum (St. Augustine grass), and Zoysia japonica

(zoysiagrass). Examples of bioenergy grasses include Saccharum spp., including S.

qfficinarum (sugar cane), Miscanthus spp., and Panicum virgatum (switchgrass).

Operably linked: combining two or more molecules in such a fashion that in combination they function properly in a plant cell. For instance, a promoter is operably linked to a structural gene when the promoter controls transcription of the structural gene. Phenotype: phenotype is a distinguishing feature or characteristic of a plant, which may be altered according to the present invention by integrating one or more "desired polynucleotides" and/or screenable/selectable markers into the genome of at least one plant cell of a transformed plant. The "desired polynucleotide(s)" and/or markers may confer a change in the phenotype of a tranformed plant by modifying any one of a number of genetic, molecular, biochemical, physiological, morphological, or agronomic characteristics or properties of the transformed plant cell or plant as a whole. Thus, expression of one or more, stably integrated desired polynucleotide(s) in a plant genome may yield a phenotype selected from the group consisting of, for example, increased drought tolerance, enhanced cold and frost tolerance, improved vigor, enhanced color, enhanced health and nutritional

characteristics, improved storage, enhanced yield, enhanced salt tolerance, enhanced heavy metal tolerance, increased disease tolerance, increased insect tolerance, increased water-stress tolerance, enhanced sweetness, improved vigor, improved taste, improved texture, decreased phosphate content, increased germination, increased micronutrient uptake, improved starch composition, and improved flower longevity.

Plant tissue: a "plant" is any of various photosynthetic, eukaryotic, multicellular organisms of the kingdom Plantae characteristically producing embryos, containing chloroplasts, and having cellulose cell walls. A part of a plant, i.e., a "plant tissue" may be transformed according to the methods of the present invention to produce a transgenic plant. Many suitable plant tissues can be transformed according to the present invention and include, but are not limited to, somatic embryos, pollen, leaves, stems, calli, stolons, microtubers, and shoots. Thus, the present invention envisions the transformation of angiosperm and gymnosperm plants such as turfgrass, wheat, maize, rice, barley, oat, sugar beet, potato, tomato, tobacco, alfalfa, lettuce, carrot, strawberry, cassaya, sweet potato, geranium, soybean, oak, apple, grape, pine, fir, acacia, eucalyptus, walnut, and palm.

According to the present invention "plant tissue" also encompasses plant cells. Plant cells include suspension cultures, callus, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, seeds and microspores. Plant tissues may be at various stages of maturity and may be grown in liquid or solid culture, or in soil or suitable media in pots, greenhouses or fields. A plant tissue also refers to any clone of such a plant, seed, progeny, propagule whether generated sexually or asexually, and descendents of any of these, such as cuttings or seed. Of particular interest are conifers such as pine, fir, and spruce, monocots such as Kentucky bluegrass, creeping bentgrass, maize, and wheat, and dicots such as cotton, tomato, lettuce, Arabidopsis, tobacco, apple and geranium.

Plant transformation and cell culture: broadly refers to the process by which plant cells are genetically modified and transferred to an appropriate plant culture medium for maintenance, further growth, and/or further development. Such methods are well known to the skilled artisan.

Pollen refers to the microspores of seeds plants and the mass of microspores shed from anthers and staminate pollen cones.

Pre-female reproductive structures: refers to cells and tissues that form before development and differentiation of female tissues in angiosperm and gymnosperm species.

Pre-male reproductive structures: refers to cells and tissues that form before development and differentiation of male tissues in angiosperm and gymnosperm species.

Progeny: a "progeny" of the present invention, such as the progeny of a transgenic plant, is one that is born of, begotten by, or derived from a plant or the transgenic plant. Thus, a "progeny" plant, i.e., an "Fl " generation plant is an offspring or a descendant of the transgenic plant produced by the inventive methods. A progeny of a transgenic plant may contain in at least one, some, or all of its cell genomes, the desired polynucleotide that was integrated into a cell of the parent transgenic plant by the methods described herein. Thus, the desired polynucleotide is "transmitted" or "inherited" by the progeny plant. The desired polynucleotide that is so inherited in the progeny plant may reside within a T-DNA construct, which also is inherited by the progeny plant from its parent. The term "progeny" as used herein also may be considered to be the offspring or descendants of a group of plants.

Promoter: is intended to mean a nucleic acid, preferably DNA, that binds RNA polymerase and/or other transcription regulatory elements. As with any promoter, the promoter sequences of the current present invention will facilitate or control the transcription of DNA or RNA to generate an mRNA molecule from a nucleic acid molecule that is operably linked to the promoter. The RNA generated may code for a protein or polypeptide or may code for an RNA interfering, or antisense molecule. A promoter, as used herein, may also include regulatory elements. Conversely, a regulatory element may also be separate from a promoter. Regulatory elements confer a number of important characteristics upon a promoter region. Some elements bind transcription factors that enhance the rate of transcription of the operably linked nucleic acid. Other elements bind repressors that inhibit transcription activity. The effect of transcription factors on promoter activity may determine whether the promoter activity is high or low, i.e. whether the promoter is "strong" or "weak."

Plant promoter is a promoter capable of initiating transcription in plant cells, whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria such as

Agrobacterium or Rhizobium which comprise genes expressed in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as tapetum, xylem, leaves, roots, or seeds. Such promoters are referred to as tissue preferred promoters. Promoters which initiate transcription only in certain tissues are referred to as tissue specific promoters. A cell type specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An inducible or repressible promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, heat, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of non-constitutive promoters. A constitutive promoter is a promoter which is active under most environmental conditions, and in most plant parts.

Polynucleotide is a nucleotide sequence comprising a gene coding sequence or a fragment thereof (comprising at least 15 consecutive nucleotides, at least 30 consecutive nucleotides, or at least 50 consecutive nucleotides), a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like. The polynucleotide may comprise single stranded or double stranded DNA or RNA. The polynucleotide may comprise modified bases or a modified backbone. The polynucleotide may be genomic, an RNA transcript (such as an mRNA) or a processed nucleotide sequence (such as a cDNA). The polynucleotide may comprise a sequence in either sense or antisense orientations.

An isolated polynucleotide is a polynucleotide sequence that is not in its native state, e.g., the polynucleotide is comprised of a nucleotide sequence not found in nature, or the polynucleotide is separated from nucleotide sequences to which it typically is in proximity, or is in proximity to nucleotide sequences with which it typically is not in proximity.

Regenerability: as used herein, refers to the ability of a plant to redifferentiate from a de-differentiated tissue.

Reproductive-preferred promoter refers to a promoter preferentially expressed in a plant's reproductive tissue. Reproductive plant tissue includes both male and female portions of the reproductive structure, as well as pre -male and pre-female reproductive structures. Male reproductive tissues include, for example, pollen grains, tapetum, anther, filament, pollen mother cells, microspores, male pollen cones (staminate cones), and pre-male reproductive structures. Female reproductive tissues include, for example, stigma, style, ovary, megaspores, ovuliferous scale, bract, female pollen cones (ovuliferous cones), and pre- female reproductive structures. Accordingly, a reproductive-preferred promoter may be preferentially expressed in any angiosperm reproductive structure or gymnosperm

reproductive structure.

Seed: is a ripened plant ovule containing an embryo, and a propagative part of a plant, as a tuber or spore. Seeds may be incubated prior to Agrobacterium -mediated transformation, in the dark, for instance, to facilitate germination. Seeds also may be sterilized prior to incubation, such as by brief treatment with bleach. The resultant seedling can then be exposed to a desired strain of Agrobacterium.

Selectable/screenable marker: a gene that, if expressed in plants or plant tissues, makes it possible to distinguish them from other plants or plant tissues that do not express that gene. Screening procedures may require assays for expression of proteins encoded by the screenable marker gene. Examples of such markers include the beta glucuronidase (GUS) gene and the luciferase (LUX) gene. Examples of selectable markers include the neomycin phosphotransferase (NPTII) gene encoding kanamycin and geneticin resistance, the hygromycin phosphotransferase (HPT or APHIV) gene encoding resistance to hygromycin, acetolactate synthase (als) genes encoding resistance to sulfonylurea-type herbicides, genes (BAR and/or PAT) coding for resistance to herbicides which act to inhibit the action of glutamine synthase such as phosphinothricin (Liberty or Basta), or other similar genes known in the art.

Sequence identity: as used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified region.

As used herein, percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

Stamen: refers to the organ of the flower that produces the male gamete and includes an anther and filament.

Tapetum: refers to a layer of cells surrounding microsporogenous cells in the anther of an angiosperm or the microsporangeous cells within a staminate cone of a gymnosperm. Given its close proximity to the developing microspores, the tapetum likely provides nutrients, such as reducing sugars, amino acids and lipids to the developing microspores. Reznickova, C. R., Acad. Bulg. Sci. 57:1067 (1978). Nave et al, J. Plant Physiol. 725:451 (1986). Sawhney et al, J. Plant Physiol 125:467 (1986). Tapetal cells also produce beta(l,3) glucanase (callase) which promotes microspore release by digesting the callose cell wall. Therefore, a fragile relationship exists between the tapetum and the microsporogenous cells, and any disruption of tapetal function is likely to result in non-functional pollen grains. It has been shown, for example, lesions in tapetal biogenesis result in male sterility mutants (Kaul, "Male Sterility in Higher Plants" in Monographs on Theoretical and Applied Genetics;

Frankel et al. eds.; Springer Verlag; Vol. 10; pp. 15-95; (1988)). Therefore, a gene encoding callase can be used for disrupting male reproductive development. Thus, a failure of the microspores to develop into mature pollen grains can be induced using, for example, a recombinant DNA molecule that comprises a gene capable of disrupting tapetal function under the control of tapetum-specific regulatory sequences.

Transcription factor: refers to a polypeptide sequence that regulates the expression of a gene or genes by either directly binding to one or more nucleotide sequences associated with a gene coding sequence or indirectly affecting the activity of another polypeptide(s) that bind directly to one or more nucleotide sequences associated with a gene coding sequence. A transcription factor may activate (up-regulate) or repress (down-regulate) expression of a gene or genes. A transcription factor may contain a DNA binding domain, an activation domain, or a domain for protein-protein interactions. In the present invention, a transcription factor is capable of at least one of (1) binding to a nucleic acid sequence or (2) regulating expression of a gene in a plant.

Transcription and translation terminators: The expression DNA constructs of the present invention typically have a transcriptional termination region at the opposite end from the transcription initiation regulatory element. The transcriptional termination region may be selected, for stability of the mRNA to enhance expression and/or for the addition of polyadenylation tails added to the gene transcription product.

Transfer DNA (T-DNA): an Agrobacterium T-DNA is a genetic element that is well- known as an element capable of integrating a nucleotide sequence contained within its borders into another genome. In this respect, a T-DNA is flanked, typically, by two "border" sequences. A desired polynucleotide of the present invention and a selectable marker may be positioned between the left border-like sequence and the right border-like sequence of a T- DNA. The desired polynucleotide and selectable marker contained within the T-DNA may be operably linked to a variety of different, plant-specific (i.e., native), or foreign nucleic acids, like promoter and terminator regulatory elements that facilitate its expression, i.e., transcription and/or translation of the DNA sequence encoded by the desired polynucleotide or selectable marker.

Transformation of plant cells: A process by which a nucleic acid is stably inserted into the genome of a plant cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of nucleic acid sequences into a prokaryotic or eukaryotic host cell, including Agrobacterium-rnQdiated transformation protocols, viral infection, whiskers, electroporation, microinjection, polyethylene glycol-treatment, heat shock, lipofection and particle bombardment.

Transgenic plant: a transgenic plant of the present invention is one that comprises at least one cell genome in which an exogenous nucleic acid has been stably integrated.

According to the present invention, a transgenic plant is a plant that may comprise only one genetically modified cell and cell genome, or it may comprise several or many genetically modified cells, or all of the cells may be genetically modified. A transgenic plant of the present invention may be one in which expression of the desired polynucleotide, i.e., the exogenous nucleic acid, occurs in only certain parts of the plant. Thus, a transgenic plant may contain only genetically modified cells in certain parts of its structure.

Variant: as used herein, is understood to mean a nucleotide sequence that deviates from the reference (i.e., native, standard, or given) nucleotide sequence of a particular gene. The terms, "isoform," "isotype," and "analog" also refer to "variant" forms of a nucleotide sequence. Variant may also refer to a "shuffled gene" such as those described in Maxygen- assigned patents. For instance, a variant of the present invention may include variants of sequences and desired polynucleotides that are modified according to the methods and rationale disclosed in U.S. Pat. No. 6,132,970, which is incorporated herein by reference.

Vegetative growth: refers to the general, overall development of a plant. After reproduction, meristem cells differentiate into apical-, lateral meristems that ultimately develop into roots and shoots and, later, into leaves and flowers, for instance. Shoot and root architecture, branching patterns, development of stems, axillary buds, and primordial cells into leaves, petals, flowers, and fruit etc. are all considered "vegetative" and part of the "vegetative growth" cycle of a plant. The rate of development of such features depends on a variety of factors, such as the species of the plant, photosynthesis, availability of nutrients, and the general environment in which the plant is growing. Genetics also plays an important literal and figurative role in shaping a plant's development. For instance, the "simple" or "compound" shape of a leaf, i.e., whether it is characterized by smooth-edges, deep lobes, individual leaflets, or tendrils can be dictated by gene expression. The "LEAFY" gene, for example, plays a role in compound leaf development and is essential for the transition from vegetative to reproductive development. LEAFY was identified in Arabidopsis and snapdragon, and has homologues in other angiosperms. The pea homologue, Unifoliata, has a mutant phenotype in which compound leaves are reduced to simple leaves, which may indicate a regulatory relationship between shoots and compound leaves. Similarly, the acacia mutant, "tl," converts tendrils to leaflets, whilst the mutation, afilia, "af," converts leaflet to tendrils. The "af tl" double mutant has a complex architecture, resembling a parsley leaf. Likewise, other genes, which are expressed throughout such "vegetative" plant cells and tissues, coordinate and connote developmental, physiological, and structural characteristics to other discreet parts of the plant. Thus, there are many "vegetative-specific" genes that are expressed, either specifically or predominantly, in all vegetative tissues, such as roots, shoots, stems, and leaves, or which are vegetative-tissue specific. The promoters of such genes are, therefore, useful in directing the expression of a desired gene, endogenous or foreign, to a particular vegetative tissue. Thus, it is possible to preferentially express a gene product in one or more vegetative tissues, whilst avoiding expression of that same product in non- vegetative tissues, such as in reproductive tissue cells.

Wood Quality refers to a trait that can be modified to change the chemical makeup, structure, appearance, or use of wood. While not limiting, traits that determine wood quality include cell wall thickness, cell length, cell size, lumen size, cell density, microfibril angle, tensile strength, tear strength, wood color, cell wall chemistry/lignin modification, and length and frequency of cell division. Wood pulp: refers to fiber generated from wood having varying degrees of purification. Wood pulp can be used for producing paper, paper board, and chemical products.

It is understood that the present invention is not limited to the particular methodology, protocols, vectors, and reagents, etc., described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art and so forth. Indeed, one skilled in the art can use the methods described herein to express any native gene (known presently or subsequently) in plant host systems.

Nucleic Acids

By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules, according to the present invention, further include such molecules produced synthetically. Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 3700 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 95% identical, more typically at least about 96% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence may be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each "nucleotide sequence" set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G, C and T). However, by "nucleotide sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U) where each thymidine deoxynucleotide (T) in the specified deoxynucleotide sequence in is replaced by the ribonucleotide uridine (U). For instance, reference to an RNA molecule having the sequence of SEQ ID NO: 1 set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxynucleotide A, G or C of SEQ ID NO: 1 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxynucleotide T has been replaced by a ribonucleotide U.

The present invention is also directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequences disclosed herein is intended DNA fragments at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides in length, which are useful as diagnostic probes and primers is discussed in more detail below. Of course larger nucleic acid fragments of up to the entire length of the nucleic acid molecules of the present invention are also useful diagnostically as probes, according to conventional hybridization techniques, or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A Laboratory Manual, 3rd. edition, edited by Sambrook, J and Russel, D. W., (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., the entire disclosure of which is hereby incorporated herein by reference.

By a fragment at least 20 nucleotides in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the as disclosed herein, i.e., SEQ ID NOs. 1-14. Nucleic acids comprising the nucleotide sequences disclosed herein can be generated using conventional methods of DNA synthesis which will be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes. Alternatively, the DNA fragments of the present invention could be generated synthetically according to known techniques.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above. By a

polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a

polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, and more than 30 nucleotides of the reference polynucleotide. These fragments that hybridize to the reference fragments are useful as diagnostic probes and primers. A probe, as used herein is defined as at least about 50 contiguous bases of one of the nucleic acids disclosed herein. For the purpose of the invention, two sequences hybridize when they form a double-stranded complex in a hybridization solution of 6XSSC, 0.5% SDS, 5XDenhardt's solution and 100μg of nonspecific carrier DNA. See Ausubel et al, section 2.9, supplement 27 (1994). Sequences may hybridize at "moderate stringency," which is defined as a temperature of 60°C. in a hybridization solution of 6XSSC, 0.5% SDS, 5XDenhardt's solution and 100 g of nonspecific carrier DNA. For "high stringency" hybridization, the temperature is increased to 68°C. Following the moderate stringency hybridization reaction, the nucleotides are washed in a solution of 2XSSC plus 0.05%> SDS for five times at room temperature, with subsequent washes with 0.1XSSC plus 0.1% SDS at 60°C. for 1 h. For high stringency, the wash temperature is increased to 68°C. For the purpose of the invention, hybridized nucleotides are those that are detected using 1 ng of a radiolabeled probe having a specific radioactivity of 10,000 cpm/ng, where the hybridized nucleotides are clearly visible following exposure to X- ray film at -70°C. for no more than 72 hours.

The present application is directed to such nucleic acid molecules which are at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence described above. One embodiment encompasses nucleic acid molecules which are at least 95%, 96%, 97%), 98%o, 99%) or 100% identical to the nucleic acid sequences of the invention. By a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence, is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5 ' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule is at least 95%, 96%), 97%), 98%) or 99% identical to a reference nucleotide sequence refers to a comparison made between two molecules using standard algorithms well known in the art. Although any sequence algorithm can be used to define sequence identity, for clarity, the present invention defines identity with reference to the Basis Local Alignment Search Tool (BLAST) algorithm (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), where a promoter sequence set forth in the disclosure is used as the reference sequence to define the percentage identity of

polynucleotide homologs over its length. The choice of parameter values for matches, mismatches, and inserts or deletions is arbitrary, although some parameter values have been found to yield more biologically realistic results than others.

When using BLAST or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed. Relatedness between two polynucleotides also may be described by reference to their ability to hybridize to form double-stranded complexes by the formation of

complementary base pairs. Hybridization conditions have been described previously herein. An increase in temperature can be used to break apart these complexes. The more structurally identical two sequences are, the higher the temperature required to break them apart or "melt" them. The temperature required to melt a double-stranded complex is called the "Tm." The relationship between the Tm and other hybridization parameters is given by:

Tm°C.)=81.5+16.6(logio[Na.+])+0.41(fraction G+C)-0.63(% formamide)-(600/l), where Tm is the melting temperature of a DNA duplex consisting of the probe and its target; and l=the length of the hybrid in base pairs, provided 1>100 base pairs. Bolton et al., Proc. Natl. Acad. Sci. 45: 1390 (1962). Generally, a change of 1°C. in the melting point represents from 0.7% to 3.2% difference in DNA sequence similarity. Bonner et al., Journal of

Molecular Biology 57:123-35 (1973); McCarthy et al, In EVOLUTION OF GENETIC SYSTEMS, H. H. Smith (ed.), Brookhaven Symposium in Biology No. 23, Gordon and Breach, New York, pp. 1-43 (1972). The formation of a stable DNA duplex at 60° C.

typically requires at least an 80%> sequence identity between sequences. Sibley et al, ACTA 1: 83-121 (Proceedings of the 18th International Ornithological Congress, Moscow, Aug. 16- 24, 1982, Academy of Sciences of the USSR).

In one embodiment, the nucleic acids of the present invention confer preferential expression of polypeptides or proteins in the reproductive tissues of angiosperm and gymnosperm plants. The nucleic acids of the present invention can also preferentially direct the expression of antisense RNA, or RNA involved in RNA interference (RNAi) such as small interfering RNA (siRNA), in the reproductive tissue of plants, which can be useful for inhibiting or completely blocking the expression of targeted genes.

Reproductive plant tissue includes both male and female portions of reproductive organs. Male tissues include, for example, pollen, tapetum, anther, filament, pollen mother cells, microspores, male pollen cones (staminate cones), and pre -male reproductive structures. Female reproductive tissues include, for example, stigma, style, ovary, megaspores, female cones (ovuliferous cones), and pre-female reproductive structures.

Reproductive-preferred promoter refers to a promoter preferentially expressed in a plant's reproductive tissue. Reproductive plant tissue includes both male and female portions of the reproductive structure, as well promoters expressed in pre -male and pre-female reproductive structures. Male reproductive tissues include, for example, pollen grains, tapetum, anther, filament, pollen mother cells, microspores, and pollen cones. Female reproductive tissues include, for example, stigma, style, ovary, megaspores, and ovuliferous cones. Accordingly, a reproductive-preferred promoter may be preferentially expressed in any reproductive structure of any angiosperm or gymnosperm species, in addition to expression in any pre-male or pre-female tissue of gymnosperm and angiosperm species.

In one embodiment, a reproductive -preferred promoter confers expression of a gene in a male-reproductive tissue. In one embodiment, a reproductive -preferred promoter confers gene expression in the anther, pollen or filament cells of an angiosperm species. In a further embodiment, the reproductive-preferred promoter confers gene expression in the tapetum or anther epidermal cells. In another embodiment, a reproductive-preferred promoter confers gene expression in a male pollen cone, tapetum, microsporophyll, or any other male reproductive tissue present in a gymnosperm. For both angiosperm and gymnosperm species, a reproductive-preferred promoter confers gene expression in a pre-male or pre-female reproductive structure.

A reproductive-preferred promoter can be used for example, to render a plant male- sterile. For example, a reproductive-preferred promoter can be operably linked to a cytotoxic gene, such that expression of the cytotoxic gene in a male reproductive tissue renders the plant incapable of producing fertile male gametes. In another embodiment, a reproductive- preferred promoter may be selected and isolated such that the promoter does not express an operably-linked gene in a non-reproductive tissue, such as a vegetative tissue.

In one embodiment, a reproductive -preferred promoter confers expression of a gene in a female-reproductive tissue. In one embodiment, a reproductive -preferred promoter confers gene expression in the stigma, style, or ovary of an angiosperm species. In another embodiment, a reproductive-preferred promoter confers gene expression in a female cone (ovuliferous cone), megasporophyll, or any other female reproductive tissue present in a gymnosperm species. For both angiosperm and gymnosperm species, a reproductive- preferred promoter confers gene expression in a pre-male or pre-female reproductive structure.

A reproductive-preferred promoter can be used for example, to render a plant female- sterile. In one embodiment, a reproductive-preferred promoter can be operably linked to a cytotoxic gene, such that expression of the cytotoxic gene in a female reproductive tissue renders the plant incapable of producing fertile female gametes, female zygote, and/or seed. In another embodiment, a reproductive-preferred promoter may be selected and isolated such that the promoter does not express an operably-linked gene in a non-reproductive tissue, such as a vegetative tissue. For example, a reproductive -preferred promoter may be identified by searching for an mR A which is only present during reproductive development.

Additionally, a reproductive-preferred promoter may be present in pre-male and pre-female reproductive tissues. In one embodiment, a reproductive -preferred promoter is identified from mRNA present during development of a plant's male reproductive tissues, including, for example, anthers, pollen, filament, male staminate cones, and pre-male reproductive tissues. In one embodiment, a reproductive -preferred promoter is identified from mRNA present during development of a plant's female reproductive tissues, including, for example, stigma, style, ovary, ovuliferous cones, and pre-female reproductive tissues. Following identification and isolation of a reproductive-preferred mRNA, cDNA is prepared from this reproductive- preferred mRNA. The resultant cDNA may be used as a probe to identify the regions in a plant genome containing DNA coding for a reproductive-preferred mRNA. Once a DNA has been identified, the sequence upstream (i.e., 5') from the DNA coding for a reproductive- preferred promoter may be isolated. As used herein, promoter is intended to mean a nucleic acid, preferably DNA, that binds RNA polymerase and/or other transcription regulatory elements. As with any promoter, the promoters of the current invention will facilitate or control the transcription of DNA or RNA to generate an mRNA molecule from a nucleic acid molecule that is operably linked to the promoter. As stated earlier, the RNA generated may code for a protein or polypeptide or may code for an RNA interfering, or antisense molecule. As used herein, "operably linked" refers to the chemical fusion, ligation, or synthesis of DNA such that a promoter-nucleic acid sequence combination is formed in a proper orientation for the nucleic acid sequence to be transcribed into an RNA segment. The promoters of the current invention may also contain some or all of the 5' untranslated region (5' UTR) of the resulting mRNA transcript. On the other hand, the promoters of the current invention do not necessarily need to possess any of the 5^* UTR.

A promoter, as used herein, may also include regulatory elements. Conversely, a regulatory element may also be separate from a promoter. Regulatory elements confer a number of important characteristics upon a promoter region. Some elements bind

transcription factors that enhance the rate of transcription of the operably linked nucleic acid. Other elements bind repressors that inhibit transcription activity. The integrated effect of transcription factors on promoter activity may determine whether the promoter activity is high or low, i.e. whether the promoter is "strong" or "weak." Transcription factors that bind regulatory elements may themselves be regulated by the interaction with other bound proteins or by covalent modification, e.g. phosphorylation, in response to extracellular stimuli. The activity of some transcription factors is modulated by signaling molecules, such as intracellular metabolites or chemicals exogenous to the organism that communicate with the cellular nucleus. Promoters that are unaffected by changes in the cellular environment are referred to as constitutive promoters.

In another embodiment, the nucleic acids of the invention encode expression products that disrupt the metabolism, function, and/or development of the cell in which the nucleic acid is expressed. In one embodiment, the nucleic acids of the invention encode a cytotoxic expression product. In one embodiment, the nucleic acids of the invention embrace barnase. In a further embodiment, the barnase may be mutated by methods known in the art for increasing and/or decreasing barnase activity. In one embodiment, a mutated barnase may have attenuated cytotoxic activity.

The present invention also provides vectors comprising the isolated nucleic acid molecules and polypeptides of the invention. In one embodiment, the vectors of the present invention are Ti-plasmids derived from the A. tumefaciens.

In developing the constructs of this invention, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector, e.g., a plasmid that is capable of replication in a bacterial host, e.g., E. coli. Numerous vectors exist that have been described in the literature, many of which are commercially available. After each cloning, the cloning vector with the desired insert may be isolated and subjected to further manipulation, such as restriction digestion, insertion of new fragments or nucleotides, ligation, deletion, mutation, resection, etc. to tailor the components of the desired sequence. Once the construct has been completed, it may then be transferred to an appropriate vector for further manipulation in accordance with the manner of transformation of the host cell.

A recombinant DNA molecule of the invention typically includes a selectable marker so that transformed cells can be easily identified and selected from non-transformed cells. Examples of such markers include, but are not limited to, a neomycin phosphotransferase (nptll) gene, which confers kanamycin resistance. Potrykus et al, Mol. Gen. Genet. 7 : 183- 188 (1985). Cells expressing the nptll gene can be selected using an appropriate antibiotic such as kanamycin or G418. Other commonly used selectable markers include the bar gene, which confers bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al,

Bio/Technology 6: 915-922 (1988)), which confers glyphosate resistance; a nitrilase gene, which confers resistance to bromoxynil (Stalker et al. J. Biol. Chem. 2(55:6310-6314 (1988)); a mutant acetolactate synthase gene (ALS), which confers imidazolinone or sulphonylurea resistance (European Patent Application 154,204, 1985); and a methotrexate resistant DHFR gene (Thillet et al. J. Biol. Chem. 265: 12500-12508 (1988)). Additionally, vectors may include an origin of replication (replicons) for a particular host cell. Various prokaryotic replicons are known to those skilled in the art, and function to direct autonomous replication and maintenance of a recombinant molecule in a prokaryotic host cell. The vectors may include selectable markers. Numerous selectable markers may be used in selecting transformed plant cells including, but not limited to, kanamycin, phosphinothricin, and glyphosate resistance genes, and the vectors may include tetracycline or ampicillin resistance for culturing in E. coli, A. tumefaciens and other bacteria.

A plasmid vector suitable for the introduction of nucleic acid of the current invention into monocots using microprojectile bombardment is composed of the following: the promoter of choice; an intron that provides a splice site to facilitate expression of the gene, such as the Hsp70 intron (PCT Publication WO 93/19189); and a 3' polyadenylation sequence such as the nopaline synthase 3' sequence (NOS 3'). Fraley et al. Proc Natl Acad Sci USA 80: 4803-4807 (1983). This expression cassette may be assembled on high copy replicons suitable for the production of large quantities of DNA.

A particularly useful Agrobacterium-based plant transformation vector for use in transformation of dicotyledonous plants is plasmid vector pMON530 (Rogers et al. (1987) Improved vectors for plant transformation: expression cassette vectors and new selectable markers. In Methods in Enzymology. Edited by R. Wu and L. Grossman, p 253-277. San Diego: Academic Press). Plasmid pMON530 is a derivative of pMON505 prepared by transferring the 2.3 kb Stul-Hindlll fragment of pMON316 (Rogers et al. (1987) Improved vectors for plant transformation: expression cassette vectors and new selectable markers. In Methods in Enzymology. Edited by R. Wu and L. Grossman, p 253-277. San Diego:

Academic Press) into pMON526. Plasmid pMON526 is a simple derivative of pMON505 in which the Smal site is removed by digestion with Xmal, treatment with Klenow polymerase and ligation. Plasmid pMON530 retains all the properties of pMON505 and the CaMV35S- NOS expression cassette and now contains a unique cleavage site for Smal between the promoter and polyadenylation signal. Binary vector pMON505 is a derivative of pMON200 (Rogers et al., 1987) in which the Ti plasmid homology region, LIH, has been replaced with a 3.8 kb Hindlll to Smal segment of the mini RK2 plasmid, pTJS75 (Schmidhauser and Helinski. J. Bacteriol. 164-155 (1985). This segment contains the RK2 origin of replication, oriV, and the origin of transfer, oriT, for conjugation into Agrobacterium using the tri- parental mating procedure (Horsch and Klee Proc. Natl. Acad. Sci. USA 83 :4428-4432 (1986). Plasmid pMON505 retains all the important features of pMON200 including the synthetic multi-linker for insertion of desired DNA fragments, the chimeric

NOS/NPTII'/NOS gene for kanamycin resistance in plant cells, the

spectinomycin/streptomycin resistance determinant for selection in E. coli and A.

tumefaciens, an intact nopaline synthase gene for facile scoring of transformants and inheritance in progeny, and a pBR322 origin of replication for ease in making large amounts of the vector in E. coli. Plasmid pMON505 contains a single T-DNA border derived from the right end of the pTiT37 nopaline-type T-DNA. Southern blot analyses have shown that plasmid pMON505 and any DNA that it carries are integrated into the plant genome, that is, the entire plasmid is the T-DNA that is inserted into the plant genome. One end of the integrated DNA is located between the right border sequence and the nopaline synthase gene and the other end is between the border sequence and the pBR322 sequences.

Another particularly useful Ti plasmid cassette vector is pMON 17227. This vector is described in PCT Publication WO 92/04449 and contains a gene encoding an enzyme conferring glyphosate resistance (denominated CP4), which is an excellent selection marker gene for many plants, including potato and tomato. The gene is fused to the Arabidopsis EPSPS chloroplast transit peptide (CTP2), and expression is driven by the promoter of choice.

In one embodiment, the vectors of the current invention are designed in a manner such that the nucleic acids described herein are tissue-specific promoters which are operably linked to DNA encoding a polypeptide of interest. In another embodiment, the polypeptide of interest is a protein involved in an aspect of reproductive development or regulating reproductive development. Polynucleotides encoding many of the proteins involved in reproductive development include, but are not limited to, AGAMOUS (AG), APETALA1 (API), APETALA3 (AP3), PISTILLATA (Pt), LEAFY (LFY), and LEUNIG (LUG). In another embodiment, the coding sequence operably linked to a promoter may code for a gene product that inhibits the expression or activity of proteins involved in reproductive development. For example, a gene encoding the enzyme callase, which digests the callose cell wall surrounding the developing pollen grains, could be operably linked to a tapetum- preferred promoter and expressed before pollen maturation, thereby disrupting pollen development. In another embodiment, the coding sequence operably linked to a promoter may encode a cytotoxic gene product. For instance, a gene encoding barnase may be operably linked to a reproductive-preferred promoter and expressed in a reproductive tissue. In a further embodiment, standard molecular biology methods may be used for mutating barnase activity. In one embodiment, a mutated barnase has reduced RNase activity compared with a wild type barnase protein. In a further embodiment, a mutated barnase having reduced RNase activity is operably linked to a reproductive-preferred promoter and expressed in a reproductive tissue. In a further embodiment, the expression of a mutated barnase having reduced RNase activity in a reproductive tissue does not compromise vegetative growth and development.

In a further embodiment, the vectors of the current invention are designed such that the nucleic acids of the current invention are operably linked to a nucleic acid encoding an antisense RNA or interfering RNA, which corresponds to a gene that code for a polypeptide of interest, resulting in a decreased expression of a targeted gene product. In one

embodiment, the gene products targeted for suppression are proteins involved in reproductive development. The use of RNAi inhibition of gene expression is described generally in Paddison et al, Genes & Dev. 16: 948-958 (2002), and the use of RNAi to inhibit gene expression in plants is specifically described in WO 99/61631, both of which are herein incorporated by reference.

In one embodiment of the method of making a plant of the invention, an exogenous DNA capable of being transcribed inside a plant to yield an antisense RNA transcript is introduced into the plant, e.g., into a plant cell. The exogenous DNA can be prepared, for example, by reversing the orientation of a gene sequence with respect to its promoter.

Transcription of the exogenous DNA in the plant cell generates an intracellular RNA transcript that is "antisense" with respect to that gene.

The invention also provides host cells which comprise the vectors of the current invention. As used herein, a host cell refers to the cell in which the coding product is ultimately expressed. Accordingly, a host cell can be an individual cell, a cell culture or cells as part of an organism. The host cell can also be a portion of an embryo, endosperm, sperm or egg cell, or a fertilized egg. The vectors of the current invention are introduced into the host cells by standard procedures known in the art for introducing recombinant vector DNA into the target host cell. Such procedures include, but are not limited to, transfection, infection, transformation, natural uptake, electroporation, biolistics and Agrobacterium. Methods for introducing foreign genes into plants are known in the art and can be used to insert a gene construct of the invention into a plant host, including, biological and physical plant transformation protocols. See, for example, Miki et al., 1993, "Procedure for Introducing Foreign DNA Into Plants", In: Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88. The methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium (Horsch et al., Science 227: 1229-31, (1985)), electroporation, micro-injection, and biolistic bombardment.

Accordingly, the present invention also provides plants or plant cells, comprising the vectors of the current invention. In one embodiment, the plants are angiosperms or gymnosperms. In another embodiment, the plants are trees. In another embodiment, the plants are ornamentals . In another embodiment the plants are trees grown for industrial or commercial applications. In another embodiment the plants are grown to produce feedstocks for bioenergy. Alternatively, the plant may be selected from pine species and their hybrids (e.g., Pinus taeda, Pinus taeda x P. rigida, Pinus radiata, Pinus banksiana, Pinus brutia, Pinus caribaea, Pinus clasusa, Pinus contorta, Pinus coulteri, Pinus echinata, Pinus eldarica, Pinus ellioti, Pinus jeffreyi, Pinus lambertiana, Pinus massoniana, Pinus monticola, Pinus nigra, Pinus palustrus, Pinus pinaster, Pinus ponderosa, , Pinus resinosa, Pinus rigida, Pinus serotina, Pinus strobus, Pinus sylvestris, and Pinus virginiana), fir species and their hybrids (e.g., Abies amabilis, Abies balsamea, Abies concolor, Abies grandis, Abies lasiocarpa, Abies magnifica, and Abies procera), Chamaecyparis species, Juniperus virginiana, larch species and their hybrids (e.g., Larix decidua, Larix laricina, Larix leptolepis, Larix occidentalis, and Larix siberica), Libocedrus decurrens, spruce species and their hybrids (e.g., Picea abies, Picea engelmanni, Picea glauca, Picea mariana, Picea pungens, Picea rubens, and Picea sitchensis), Pseudotsuga menziesii, Sequoia gigantea, Sequoia sempervirens, Taxodium distichum, hemlock species and their hybrids (e.g.,Tsuga canadensis, Tsuga heterophylla, and Tsuga mertensiana) Thuja occidentalis, and Thuja plicata, Acacia species and their hybrids (e.g. Acacia mangium), Eucalyptus species and their hybrids, (e.g., Eucalyptus grandis, Eucalyptus urophylla, Eucalyptus urophylla x E. grandis, Eucalyptus alba, Eucalyptus bancroftii, Eucalyptus benthamii, Eucalyptus botryoides, Eucalyptus bridgesiana, Eucalyptus calophylla, Eucalyptus camaldulensis, Eucalyptus citriodora, Eucalyptus cladocalyx, Eucalyptus coccifera, Eucalyptus curtisii, Eucalyptus dalrympleana, Eucalyptus deglupta, Eucalyptus delagatensis, Eucalyptus diversicolor, Eucalyptus dunnii, Eucalyptus ficifolia, Eucalyptus globulus, Eucalyptus gomphocephala, Eucalyptus gunnii, Eucalyptus henryi, Eucalyptus laevopinea, Eucalyptus macarthurii, Eucalyptus macrorhyncha, Eucalyptus maculata, Eucalyptus marginata, Eucalyptus megacarpa, Eucalyptus melliodora, Eucalyptus nicholii, Eucalyptus nitens, Eucalyptus nova- angelica, Eucalyptus obliqua, Eucalyptus occidentalis, Eucalyptus obtusiflora, Eucalyptus oreades, Eucalyptus pauciflora, Eucalyptus polybractea, Eucalyptus regnans, Eucalyptus resinifera, Eucalyptus robusta, Eucalyptus rudis, Eucalyptus saligna, Eucalyptus

sideroxylon, Eucalyptus stuartiana, Eucalyptus tereticornis, Eucalyptus torelliana,

Eucalyptus urnigera, Eucalyptus viminalis, Eucalyptus viridis, Eucalyptus wandoo, and Eucalyptus youmanni), Hevea species and hybrids (e.g., Hevea brasiliensis), sweetgum species and their hybrids (e.g., Liquidambar styraciflua, Liquidambar formosana, and Liquidambar orientalis ), poplar/ aspen/ cottonwood species and their hybrids (e.g., Populus alba, Populus balsamifera, Populus x canescens, Populus x canadensis, Populus deltoides, Populus grandidentata, Populus maximowiczii, Populus nigra, Populus tremula, Populus tremula x Populus alba, Populus tremuloides, and Populus trichocarpa), Tectona grandis, and willow species and their hybrids (e.g., Salix alba, Salix nigra, and Salix viminalis). In particular, the transgenic plant may be of the species Eucalyptus grandis, Pinus radiata, Pinus taeda L (loblolly pine), Populus nigra, Populus deltoides, and hybrid Populus, Hevea species and hybrids (e.g. Hevea braziliensis), Acacia species and hybrids, Tectona grandis, Liquidambar species and their hybrids, Salix species and their hybrids, or Acacia mangium.

Beyond the ordinary meaning of plant, the term "plants" is also intended to mean the fruit, seeds, flower, strobilus etc. of the plant. The plant of the current invention may be a direct transfectant, meaning that the vector was introduced directly into the plant, such as through Agrobacterium, or the plant may be the progeny of a transfected plant. The second or subsequent generation plant may or may not be produced by sexual reproduction, i.e., fertilization. Furthermore, the plant can be a gametophyte (haploid stage) or a sporophyte (diploid stage).

The present invention also provides a method for controlling reproductive

development in a plant comprising cultivating a plant or seed comprising the vectors of the current invention. Proper cultivation to induce or sustain the growth or germination of the plants or seeds of the current invention is species-specific, and within the level of ordinary skill in the art. The setting for cultivation may be anywhere which fosters the growth or germination of the plant or seed. Furthermore, cultivation can also include steps such as, but not limited to, providing a stress treatment, (e.g., nitrogen deprivation, heat shock, low temperatures, sucrose deprivation) which can induce embyro genesis.

The invention further provides isolated regulatory elements that bind transcription factors and are capable of regulating tissue-preferred or tissue-specific expression. The degree of regulation conferred by the regulatory elements may be complete, meaning that transcription is not detectable without the transcription factors, or partial, meaning that transcription is enhanced in the presence of the transcription factors. In one embodiment, at least one regulatory element is operably linked to a heterologous promoter to provide a composite promoter. The composite promoter is expressed preferentially or specifically in reproductive tissue. As used herein, heterologous promoters is a phrase whose meaning term that is relative to the regulatory elements. If a regulatory element and a promoter do not associate with one another in a natural setting, the promoter would be considered

heterologous to the regulatory element. Typically, the precise orientation of a regulatory element within a promoter region will not affect its activity. Furthermore, regulatory elements can function normally when inserted into heterologous promoter regions. Thus, for example, reproductive -preferred regulatory elements can be removed from their endogenous promoter and can be inserted into heterologous promoter regions to confer reproductive-specificity or preference. The heterologous promoter may be, for example, a minimal CaMV 35S promoter. Promoters that direct expression in plant cells which are suitable for modification to minimal promoters include the cauliflower virus (CaMV) 35 S promoter (Jefferson et ah, EMBO J., 6: 3901-07 (1987)), the rice actin promoter (McElroy et al, Plant Cell, 2: 163-71 (1990)), the maize ubiquitin-1 promoter (Christensen et al., Transgenic Research, 5: 213-18 (1996)), and the nopaline synthase promoter (Kononowics et al, Plant Cell 4: 17-27 (1992)).

The nucleic acids of the invention may be prepared by constructing genomic libraries using a variety of restriction endonucleases to digest the genome into discrete fragments. Genomic libraries can be constructed from any plant species from which it is desirable to obtain tissue-selective promoters. An adaptor is ligated to each of these genomic sequences, according to the procedure provided by Clontech for use of its GenomeWalker.TM. Systems (Clontech, Palo Alto, Calif). Promoter sequences then are PCR-amplified using adaptor- specific primers and "gene-specific primers." Alternatively, this PCR amplification step optionally may be conducted by the methodology described in U.S. Pat. No. 5,565,340 and No. 5,759,822, herein incorporated by reference, to yield reaction products of long length and minimal background. Using this general PCR amplification methodology, the identification of the promoter of the invention and its identification as a tissue-selective promoter, is governed by the choice of the "gene-specific primer."

In the present invention, a gene-specific primer is a fragment of, or is complementary to, an mRNA that is expressed at high levels in reproductive tissue. In one embodiment, the gene-specific primer is selected by its homology to genes that are known to be expressed specifically in a particular reproductive tissue type. Genes of particular interest are those that are expressed in a particular reproductive tissue at high levels, which typically is an indicator of reproductive -preferred activity of the corresponding promoter.

Expressed sequence tags (ESTs) provide another source of gene-specific primers. An EST is a cDNA fragment of a corresponding mRNA that is present in a given library. Any plant EST database may be searched electronically to find ESTs that share identity to segments of genes that are known to be expressed specifically in a desired tissue type ("in silico screening"). These ESTs thus will provide gene-specific primers for the amplification of the promoter of the corresponding gene in a given genomic library. The amplified gene promoter need not be from the same species from which the EST database was obtained. All that is required is that the EST bears sufficient sequence similarity to the gene promoter of interest to act as a primer for PCR amplification of the target segment of the gene. An alternative methodology to identify tissue-specific promoters rests on detection of mRNAs that are expressed in one tissue type, but not in another, implying that they are transcribed from a tissue-specific promoter. Populations of mRNAs can be distinguished on this basis by subtractive hybridization, for example. One such suitable subtractive

hybridization technique is the PCR-Select.TM. described by Clontech.

Alternatively, a tissue-specific mRNA distribution can be determined by in situ hybridization of thin slices of plant tissue with radiolabeled probes. Probes that radioactively stain a particular tissue type are then used to detect the promoter associated with the mRNA by Southern analysis of genomic libraries, using the methodologies described below. All of the aforementioned techniques require the preparation of mRNA libraries from the tissue of interest, in this case, reproductive tissue. cDNA libraries may be made from reproductive tissues isolated from woody plant species. Briefly, total RNA is isolated using standard techniques, and poly(A) RNA then is isolated and reverse transcribed to construct a reproductive-preferred tissue cDNA library. The cDNA library may be constructed in the .lamda.ZAP-XR vector, employing Strategene cDNA synthesis and Gigapakll Gold.TM. packaging kits. Reproductive-specific promoters can, in turn, be isolated from such cDNA libraries by PCR using a gene-specific probe and a primer that recognizes a sequence at the 5' end of the promoter. A gene-specific probe can be obtained by the in silico approach described above, or by designing a specific probe based on the sequence of the mRNA, if known. Furthermore, a primer can be synthesized which is complementary to the 5' UTR of the desired target gene. Alternatively, the primer can be designed from a partial amino acid sequence of the encoded protein, as a so-called degenerate primer.

Following isolation of the promoter of interest, various methods can be used to characterize its tissue-specific expression pattern and promoter strength. One commonly employed method is to operably link the promoter to a readily assayed reporter gene. For example, a reproductive -preferred promoter is operably linked to the gene encoding β - glucuronidase (GUS). Lacombe et al, Plant J. 23: 663-76 (2000). Suitable expression constructs can be made using well-known methodologies. Transformation of plants can be accomplished by any one of many suitable techniques, including Agrobacterium-mediatGd transformation, as described in U.S. Pat. No. 6,051,757. Other methods for transforming trees are known in the art, as exemplified by U.S. Pat. No. 5,681,730, which discloses an accelerated particle transformation method of gymnosperm somatic embryos. Other transformation methods include micro-projectile bombardment (Klein et al., Biotechnology 6: 559-63 (1988)), electroporation (Dhalluin et al., Plant Cell 4: 1495-1505 (1992)), and polyethylene glycol treatment (Golovkin et al., Plant Sci. 90: 41-52 (1993)). Further, U.S. Pat. No. 6,187,994 discloses a recombinase-assisted insertion of the expression construct into a specific, selected site within a plant genome. All of the aforementioned patents and publications are herein incorporated by reference.

When adequate numbers of cells (or protoplasts) containing the nucleic acid of interest are obtained, the cells (or protoplasts) are regenerated into whole plants. Choice of methodology for the regeneration step is not critical, with suitable protocols being available for hosts from Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, canola/rapeseed, etc.), Cucurbitaceae (melons and cucumber), Gramineae (wheat, barley, rice, maize, etc.), Solanaceae (potato, tobacco, tomato, peppers), various reproductive crops, such as sunflower, and nut-bearing trees, such as almonds, cashews, walnuts, and pecans. See, e.g., Ammirato et al. (1984) Handbook of Plant Cell Culture-Crop Species. Macmillan Publ. Co.; Fromm, M., (1990) UCLA Symposium on Molecular Strategies for Crop Improvement, Apr. 16-22, 1990. Keystone, Colo.; Vasil et al. Bio/Technology 5:429-434 (1990); Vasil et al. Bio/Technology 10:661-61 A (1992);

Hayashimoto et al. Plant Physiol. J:857-863 (1990); and Datta et al. (1990).

The vector comprising the promoter and reporter gene includes a mechanism to select those plant cells successfully transformed with the vector, which may be, for example, kanamycin resistant. The presence of the GUS gene in transformants may be confirmed by a PCR approach, using GUS-specific PCR primers (Clontech, Palo Alto). Segregation of kanamycin resistance in the progeny of the transformed plant cells can be used in conjunction with Southern analysis to determine the number of loci harboring the stably inserted vector. The temporal and spatial pattern of promoter expression is then inferred from a quantification of the reporter gene expression, as described in Jefferson et al, EMBO J. 6: 3901-07 (1987). Generally, GUS expression is determined histochemically in thin slices of plant tissues that are fixed first in 90% acetone and then in a buffered solution containing a GUS substrate, 5- bromo-4-chloro-3-indoyl-.beta.-D-glucuronic acid (X-Gluc). The presence of the GUS expression product is indicated by a colorimetric reaction with the X-Gluc.

Reproductive-specific expression, for example, can be conferred by the presence of regulatory elements that specifically bind transcription factors in reproductive tissue. The interaction between reproductive-specific regulatory elements and reproductive-preferred transcription factors depends on the alignment between a subset of base pairs of the regulatory element with amino acid residues of the transcription factor. Likewise, tapetum- specific expression, for example, can be conferred by the presence of regulatory elements that specifically bind transcription factors in tapetal tissue. Base pairs that do not interact with the bound transcription factor may be substituted with other base pairs, while maintaining the overall ability of the regulatory element to bind specifically the tissue-specific transcription factor.

Various methodologies can be used to identify and characterize regulatory elements that affect tissue-preferred or tissue-specific promoter activity, once a promoter is identified as tissue-preferred or specific. In one methodology, the promoter region is sequentially truncated at the 5' end and the series of truncated promoters are each operably linked to a reporter gene. When a regulatory element is deleted, the effect on the promoter activity is inferred by the loss of tissue-specific expression of the reporter gene. Alternatively, a putative regulatory element can be inserted into an expression construct containing a minimal promoter, such as the CaMV 35S minimal promoter (Keller et al., Plant Mol. Biol. 26: 747- 56) to ascertain if the putative regulatory element confers tissue-specific expression. A minimal promoter contains only those elements absolutely required for promoter activity, such as a RNA polymerase binding site.

Functional Variants or Fragments of the Promoters of the Invention

Additional variants or fragments of the promoters of the invention are those with modifications interspersed throughout the sequence. Functional variants or fragments, as used herein, are nucleic acids that have a nucleic acid sequence at least about 70% identical to the reference nucleic acid, but still confer tissue-specific expression of coding products. The tissue-specificity or preference of the functional variant must be towards the same tissue as the reference nucleic acid. However, even if the functional variant is not as preferential or as specific as the reference nucleic acid, the variant is still considered a functional variant as used herein. In one embodiment, the sequence of the functional variant or fragment is at least about 75% identical to the reference nucleic acid. In other embodiments, the sequence of the functional variant or fragment is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

Modifications that can produce functional variants may be made by sequential deletion of residues from the 5' end or the deletion of 5' UTR sequences from the 3' end. Alternatively, internal residues may be modified. Modifications that do not affect the function of the promoter regions most likely will be those that do not affect the binding of

transcription factors. The modifications encompassed by the invention also include those that occur naturally in the form of allelic variants of the promoters of the invention.

Methods of Making the Nucleic Acids of the Present Invention

The nucleic acids of the invention can be obtained by using well-known synthetic techniques, standard recombinant methods, purification techniques, or combinations thereof. For example, the isolated polynucleotides of the present invention can be prepared by direct chemical synthesis using the solid phase phosphoramidite triester method (Beaucage et al., Tetra. Letts. 22: 1859-1862 (1981)), an automated synthesizer (Van Devanter et al., Nucleic Acids Res. 12: 6159-6168 (1984)), or the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide, which can be converted into double stranded oligonucleotides by hybridization with a complementary sequence, or by polymerization, using the single strand as a template. Also, longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, the nucleic acids of the present invention can be obtained by

recombinant methods using mutually priming oligonucleotides. See e.g. Ausubel et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1990). Also, see Wosnick et al, Gene 60: 115 (1987); and Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3^rd ed., (John Wiley & Sons, Inc. 1995).

Established techniques using the polymerase chain reaction provide the ability to synthesize polynucleotides at least 2 kilobases in length. Adang et al., Plant Mol. Biol. 21 : 1131 (1993); Bambot et al., PCR Methods and Applications 2: 266 (1993); Dillon et al., "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in METHODS IN MOLECULAR BIOLOGY, Vol. 15: PCR PROTOCOLS: CURRENT METHODS AND APPLICATIONS, White (ed.), pages 263-268, (Humana Press, Inc. 1993); Holowachuk et al, PCR Methods Appl. 4: 299 (1995).

Methods of Using the Nucleic Acids of the Invention

The nucleic acids of the current invention are useful for altering characteristics of a plant. The nucleic acids may be operably linked to a gene of interest to increase the levels of a molecule found in the reproductive tissue. Alternatively, the gene of interest may inhibit reproductive development, thereby conferring sterility to a plant. One of the primary targets of such manipulated expression is reproductive development. For the reasons set forth above, there is considerable interest in regulating reproductive development, accomplished through genetic ablation. For example, a cytotoxic barnase molecule under the control of a tapetum- preferred promoter has been used for regulating reproductive development. European Patent Publication 344, 029.

For example, a mutant barnase gene having reduced RNase activity may be used for regulating reproductive development. In one embodiment, the mutant barnase gene may be operably linked to a promoter such that expression of the barnase gene could impose little or no damage to vegetative tissues, yet the mutant barnase may provide adequate RNase activity for reproductive ablation.

The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention. EXAMPLES

Example 1: Cloning of 3' cDNA of AGAMOUS gene from Eucalyptus occidentalis

The AGAMOUS (AG) cDNA from Eucalyptus occidentalis was cloned by PCR amplification of floral cDNA using primers designed for the AG gene. Agarose gel analysis of the PCR product revealed a very strong band of the predicted size of ~1 kb. The PCR product was excised from the gel, purified and then inserted into a T/A cloning vector. The resulting clones were tested by PCR to verify that they contained inserts of the correct size and that the inserts were PCR-amplifiable with the AG-specific primers. The clones were analyzed for sequence analysis and the sequencing data were then compared to the sequence of ArborGen Euc 4000 clone Eucgra2L1823 1 from Eucalyptus grandis (Figure 1). The high degree of homology between the amino acid sequence obtained from the Eucalyptus occidentalis AGAMOUS gene and the amino acid sequence obtained from the Eucalyptus grandis AGAMOUS gene clearly demonstrated that the Eucalyptus occidentalis AGAMOUS gene was successfully cloned. Accordingly, the DNA sequence for the Eucalyptus occidentalis gene was used to design primers for 3' RACE and for QPCR analyses.

Example 2: Designing of Primers

The sequence of the cloned Eucalyptus occidentalis AG gene was used to design primers for Rapid Amplification of cDNA 3' Ends (3 - RACE) of the AGAMOUS gene. The 1^st strand cDNA was used as template in a PCR reaction with a gene-specific forward primer and the oligo-dT adapter primer (aka 3 'RACE adapter). Touch-down PCR cycling conditions and a proofreading polymerase mix were used with an AG forward primer and 3 'RACE adapter reverse primer. Agarose gel analysis of the PCR reaction revealed two distinct bands— one of ~250bp and one of ~1.3kb.

Since the poly A site in the sequence of a ArborGen 4000 Eucalyptus grandis clone AG cDNA, which contained the 3' untranslated region (3'UTR), was ~250bp from the stop codon, it was concluded that the 250bp PCR product was the 3'UTR of Eucalyptus occidentalis AG. This band was excised from the gel, cloned into a T/A vector, and the colonies were then screened by colony PCR. Three of the positive clones were mini-prepped and analyzed for sequencing. The DNA alignment (see Figure 2) clearly showed that the 3'UTR of Eucalyptus occidentalis AG was successfully cloned. The 3'UTR sequence was used to design primers for reverse transcriptase-PCR (RT-PCR) analysis of AG expression in different Eucalyptus occidentalis tissues (floral, leaf, shoot tip).

Example 3: Analysis of Eucalyptus occidentalis A G Expression

In order to determine whether the AG gene promoters of Eucalyptus occidentalis thus obtained are suitable for use in flowering control constructs, AG expression was determined in floral and non-floral tissues. Specifically, AG expression levels was determined in floral buds, leaves, and shoot tips of Eucalyptus occidentalis by semi-quantitative RT-PCR using the Eucalyptus occidentalis 3'UTR sequence to design primers. RNA was isolated and used as template for 1^st strand cDNA synthesis with oligo-dT. The cDNA was then used as template for PCR with the A G- specific primers. AG was expressed very strongly in floral bud tissue {see 350 bp band in Figure 3), was not expressed in leaf tissue, and had low expression in one of the shoot tip preps. The weak expression in the shoot tip prep could be the result from newly developing floral tissue, not yet visible to the eye. AG strong expression in the floral buds and the lack of or weak expression in the non-floral tissues provided strong evidence that the Eucalyptus occidentalis AG promoter could be used as a floral-specific promoter for future flowering control experiments.

Example 4: Isolation of the Eucalyptus occidentalis AG gene promoter

A genome walking technique was employed to clone a portion of the 5'UTR of Eucalyptus occidentalis AGAMOUS gene that would potentially contain the promoter for this gene. This technique involves performing one round of PCR using one of four semi- degenerate primers in combination with a biotinylated gene-specific primer. The PCR products containing the biotinylated primer are then affinity-purified using streptavidin- coated paramagnetic beads. The purified products are then used as template for a secondary, nested PCR reaction. A portion of each secondary reaction was analyzed by agarose gel electrophoresis. The PCR products were cloned in T/A vector for sequencing. The sequences of clones with insert sizes above 500 bp were analyzed. Three of the clones had identical sequences, were 1.4 kb in length and shared a sequence identical to a region of the Eucalyptus occidentalis AG gene. This confirmed that the cloned fragments are from the 5'UTR of Eucalyptus occidentalis AG gene. Partial alignment between the three clones and the known sequence of the Eucalyptus grandis AG gene is shown in Figure 4. The sections highlighted in yellow represent the overlap between the three clones and the known sequence; the translational start is highlighted in green.

Example 5: Isolation of the second intron sequence of the Eucalyptus occidentalis AGAMOUS gene and construction of floral ablation vectors

A 4 kb second intron of the Eucalyptus occidentalis AG gene was cloned and used for the construction of floral ablation vectors. Floral ablation constructs containing the 1.4 kb sequence at 5 ' of the translation start codon ATG, the first exon sequence and the second intron sequence of the Eucalyptus occidentalis AG gene operably linked to the wild-type barnase coding region were used to transform tobacco plants. These constructs did not show any activities in transgenic tobacco. To determine whether the constructs were missing sequences involved in regulation of the promoter, PCR analysis of the Eucalyptus occidentalis genomic DNA was performed using a forward primer within the promoter fragment at the 5 ' of the translation start codon ATG and a reverse primer within the 2^nd intron {see Figure 5). It was expected that this primer pair would produce a product of ~750bp. The plasmid pAGW47, which was used as a PCR positive control, generated a product of the expected size. In contrast, the Eucalyptus occidentalis genomic DNA produced a ~1 kb fragment . To determine whether the 1 kb fragment is part of the

Eucalyptus occidentalis AG gene, the 1 kb band was cut out of the gel, cloned and sequenced. The sequence data showed an insert of -300 bp immediately upstream of the translation start site {see Figure 6). Since the 300 bp sequence was not in the original Eucalyptus occidentalis cDNA clone and the fragment contained the classic intron conserved sequences of "GT" at the 5' end of the fragment and "AG" at the 3' end, it was determined that the insert is the first intron located in the 5' UTR of the Eucalyptus occidentalis gene. The promoter was cloned via genome walking using the primer GW2 {see above).

Surprisingly the 'insertion point' for the intron was in the middle of the primer GW2. This suggested that the 11 bases at the 3 ' end of the primer hybridized strongly enough to the genomic DNA as to be sufficient for priming of the PCR reaction. Accordingly, the intron was not included as part of the original promoter clone. To determine whether the intron plays a role in promoter functions, new floral ablation constructs were made, in which the intron was added to the promoter sequence. Two new floral ablation constructs of pAGW52 and pAGW53 and the control construct of pAGW55 (GUS) were synthesized (see Figure 7), and introduced into tobacco.

Example 6: Evaluation of floral ablation constructs containing the Eucalyptus occidentalis AG promoter operably linked to a gene encoding a cytotoxic protein

Transgenic tobacco flowers transformed with either the pAGW52 or pAGW53 plasmid had a phenotype characterized by a stunted carpel (see Figure 8 A). 19 transgenic lines of pAGW53 carrying the Eucalyptus occidentalis _^4G-barnaseE73G cassette were investigated. The ratio of carpel length to petal length was used as an indicator of the degree of carpel ablation. Untransformed tobacco plants have a ratio of 1, since the carpel has a length similar to the length of the petal. Values of the ratio in Table 1 were the average of the measurements of 5 individual flowers. pAGW53 lines with a ratio equal to or above 0.9 were selected for PCR analysis to confirm the presence of the Eucalyptus occidentalis AG promoter and barnase coding region. Tobacco plants are highly self-pollination compatible and all 15 lines of tobacco plants transformed with the pAGW55 construct carrying the Eucalyptus occidentalis AG-GXJS control cassette had self-pollinated or naturally-pollinated fruits (see Table 1 and Figure 8B). In contrast, 10 of 19 pAGW53 lines (53%) were not able to perform natural pollination (see Table 1 and Figure 8B). To determine seed formation, the stigma of pAGW53 transgenic tobacco plants and GUS control tobacco plants were hand- pollinated with the pollen collected from GUS tobacco control plants and five individual flowers of each of the 19 pAGW53 lines and five GUS lines were hand-pollinated. The results showed that 7 of 19 pAGW53 lines (37%) were not able to bear fruits after hand pollination, such that no seeds were formed in these plants (see Table 1). All five GUS lines were successfully hand-pollinated (Table 1). Five pAGW53 lines (E2, Y2, Ql, P and N2 in Table 1) showed heavy reduction of seed formation as less than 100 seeds were collected from 4 or 5 fruits, compared to thousands of seeds collected from 4 or 5 GUS fruits. The fruits of the five pAGW53 lines were very small. Seeds collected from the pAGW53 small fruits were compared to the GUS seeds under a dissecting microscope. No obvious differences were observed in seed morphology, except that the size of pAGW53 seeds was larger than that of GUS seeds (see Figure 9). Seed germination tests using 0.8% agar and 1%

sucrose showed that the seeds collected from the small fruits of the five pAGW53 lines were able to germinate (see Table 1 and Figure 10). No differences in time required for

germination and seedling morphology were detected.

5 Table 1 : Phenotypic survey, hand pollination, seed formation, and seed germination of

tobacco carrying £O_^4G-barnaseE73G (pAGW53)

Natural Hand Number of seed collected Seeds capable

Transgenic Ratio of carpel pollination pollination from 4 or 5 hand-pollinated of germinating tobacco line Carpel length to petal occurred? performed? or naturally-pollinated on agar Petri ID phenotypes length (Y/N) (Y/N) fruits dishes? (Y/N)* pAGW53-H stunted 0.48 no yes no seeds na pAGW53-L2 stunted 0.60 no yes no seeds na pAGW53-Gl stunted 0.64 no yes no seeds na pAGW53-Bl stunted 0.66 no yes no seeds na pAGW53-M stunted 0.67 no yes no seeds na pAGW53-V2 stunted 0.70 no yes no seeds na pAGW53-U stunted 0.74 no yes no seeds na pAGW53-E2 stunted 0.55 no yes less than 100 seeds yes pAGW53-Y2 stunted 0.66 no yes less than 100 seeds yes pAGW53-Ql stunted 0.80 no yes less than 100 seeds yes pAGW53-P stunted 0.80 yes yes less than 100 seeds yes pAGW53-N2 stunted 0.83 yes yes less than 100 seeds yes pAGW53-Sl stunted 0.80 yes yes thousands of seeds yes pAGW53-Wl stunted 0.83 yes yes thousands of seeds yes pAGW53-I stunted 0.86 yes yes thousands of seeds yes pAGW53-02 stunted 0.89 yes yes thousands of seeds yes pAGW53-Dl normal 0.91 yes yes thousands of seeds yes pAGW53- l normal 0.95 yes yes thousands of seeds yes pAGW53-C normal 0.98 yes yes thousands of seeds yes pAGW55-N normal 1.00 yes yes thousands of seeds yes pAGW55-0 normal 1.00 yes yes thousands of seeds yes pAGW55-D normal 0.98 yes yes thousands of seeds na pAGW55-B normal 1.00 yes yes thousands of seeds na pAGW55-C normal 1.00 yes yes thousands of seeds na pAGW55-A normal 0.99 yes no thousands of seeds yes pAGW55-E nonnal 1.00 yes no thousands of seeds yes pAGW55-G nonnal 1.00 yes no thousands of seeds yes pAGW55-M nonnal 0.96 yes no thousands of seeds na pAGW55-F nonnal 0.98 yes no thousands of seeds na pAGW55-H nonnal 0.98 yes no thousands of seeds na pAGW55-I nonnal 0.98 yes no thousands of seeds na pAGW55- nonnal 0.98 yes no thousands of seeds na pAGW55-J nonnal 1.00 yes no thousands of seeds na pAGW55-L nonnal 1.00 yes no thousands of seeds na

* na, not performed

In a second experiment, 13 transgenic tobacco lines transformed with the pAGW52

construct carrying the Eucalyptus occidentalis A G-barnaseK27A cassette and 13 control

tobacco plants transformed with the GUS control construct pAGW55 were transplanted into the soil about three weeks later than the pAGW53 and pAGW55 tobacco plants in Table 1. The experimental procedures and data collection of pAGW52 tobacco were identical to those of pAGW53 tobacco. The results are reported in Table 2. The control tobacco plants

transformed with the GUS control construct pAGW55 used for the experiments reported in Table 2 are not the same individual plants used for the experiments reported in Table 1 ,

although some plants were the ramets of the same transgenic lines reported in Table 1. The results showed that only one of the 13 pAGW52 lines (8%) was able to perform natural

pollination (self pollination). In contrast, 13 of the 13 pAGW55 lines (100%) performed

natural pollination (Table 2). After hand pollination, 9 of the 13 pAGW52 lines (69%) did not produce any seeds and in three of the 13 (23%) pAGW52 lines seed formation was highly reduced (Table 2). Based on the germination results obtained from pAGW53 seeds, it is very likely that the pAGW52 seeds collected from the small fruits of the 3 lines will germinate on agar medium.

Table 2: Phenotypic survey, hand pollination, seed formation and seed germination of

transgenic tobacco lines transformed with the pAGW52 construct carrying the Eucalyptus

20 occidentalis A G-barnaseK27A cassette

Number of seed colleted Seeds capable of

Transgenic Ratio of carpel Natural Hand from 4 or 5 hand-pollinated germinating on tobacco line Carpel length to petal pollination pollination or naturally-pollinated agar Petri ID phenotypes length occurred? performed? fruits dishes ? pAGW52-N stunted 0.47 no yes no seeds na pAGW52-B2 stunted 0.56 no yes no seeds na pAGW52-H2 stunted 0.56 no yes no seeds na pAGW52-Al stunted 0.57 no yes no seeds na pAGW52-L stunted 0.57 no yes no seeds na pAGW52-G2 stunted 0.59 no yes less than 100 seeds na pAGW52-S stunted 0.60 no yes no seeds na pAGW52-Cl stunted 0.62 no yes no seeds na pAGW52-Kl stunted 0.65 no yes less than 100 seeds na pAGW52-Il stunted 0.65 no yes no seeds na pAGW52-J2 stunted 0.66 no yes no seeds na pAGW52-M stunted 0.76 no yes less than 100 seeds na pAGW52- l normal 1.00 yes yes thousands of seeds na pAGW55-V normal 0.97 yes no thousands of seeds na pAGW55-W normal 0.97 yes no thousands of seeds na pAGW55-A normal 0.98 yes no thousands of seeds na pAGW55-R normal 0.98 yes no thousands of seeds na pAGW55-S normal 0.98 yes no thousands of seeds na pAGW55-U normal 0.98 yes no thousands of seeds na pAGW55-Q normal 0.99 yes no thousands of seeds na pAGW55-X normal 0.99 yes no thousands of seeds na pAGW55-G normal 1.00 yes no thousands of seeds na pAGW55-T normal 1.00 yes no thousands of seeds na pAGW55-B normal 1.01 yes no thousands of seeds na pAGW55-N normal 1.02 yes no thousands of seeds na pAGW55-P normal 1.05 yes no thousands of seeds na

Table 3 summarizes and compares the results obtained from carpel ablation studies made in tobacco plants transformed with constructs containing two genes encoding different cytotoxic barnase enzymes under the control of the Eucalyptus occidentalis AG promoter.

5 The results clearly show that the Eucalyptus occidentalis AG promoter is specifically active in tobacco carpels. In fact, while no differences in growth rate, plant height, size and color of leaves were detected between transgenic tobacco plants transformed with the pWAG52 or the pWAG53 ablation constructs and the control tobacco (GUS) plants in the greenhouse, 69% of the transgenic tobacco lines transformed with an ablation construct carrying the Eucalyptus occidentalis A G-barnaseK27A cassette were not able to produce seeds after hand pollination. The Eucalyptus occidentalis _^4G-barnaseK27A ablation cassette provided stronger prevention of seed formation than the Eucalyptus occidentalis _^4G-barnaseE73G The difference is likely due to the stronger activity of R ase in barnaseK27A.

Table 3: Carpel ablation in transgenic tobacco plants transformed with ablation constructs containing genes encoding barnase enzymes under the control of the Eucalyptus occidentalis A G promoter as compared to control tobacco (GUS) plants.

Example 7: Analysis of the second intron of the Eucalyptus occidentalis AG gene

To investigate whether the second intron of the Eucalyptus occidentalis AG promoter in the pAGW52 and pAGW53 ablation constructs affects floral specificity, four constructs, pAGKNOl, pAGKN02, pAGKN03 and pAGKN04, were synthesized and transformed into tobacco {see Figure 11 A-D). The four constructs were re-engineered versions of the

Eucalyptus occidentalis AG promoter: :barnase mutant floral ablation cassette combinations from pAGW52 (EoAG::barnaseK27A) and pAGW53 (EoAG::barnaseE73G). The

Eucalyptus occidentalis AG 2^nd intron, which carries an 'enhancer' motif, was removed completely to generate the pAGKNOl and pAGKN02 constructs and moved to the 5' of the translation initiation ATG codon to create the pAGKN03 and pAGKN04 constructs. In contrast, in the pAGW52 and pAGW53 constructs, the 2^nd intron is located 3' of the translation initiation ATG codon. All four constructs were additionally re-engineered to remove a 180 bp exon sequence from between the ATG site and the beginning of the barnase coding region to prevent addition of any additional amino acid to the N terminus of the barnase protein.

Tobacco plants were transformed with these constructs. 11 tobacco lines transformed with pAGKNOl, 4 lines transformed with pAGKN02, 20 lines transformed with pAGKN03, and 13 lines transformed with pAGKN04 were obtained. The transgenic tobacco plants showed the following phenotypes: only 1 of the 11 lines transformed with pAGKNOl and only 1 of the 4 lines transformed with pAGKN02 showed the stunted carpel phenotype; in contrast, 17 of the 20 lines transformed with pAGKN03 and 10 of the 13 lines transformed with pAGK 04 showed the stunted carpel phenotype (see Table 4 and Figure 12). The results from these experiments showed that the second intron of the Eucalyptus occidentalis AG gene functions as a floral enhancer and controls the floral specificity and activity of Eucalyptus occidentalis AG promoter. All transgenic flowers with normal carpel morphology (the ratio of carpel length to petal length is about 1.0) could be pollinated by natural pollination while the majority of the stunted carpels could not be pollinated by either hand- or natural-pollination (Table 4). A few transgenic tobacco lines transformed with pAGK 03 or pAGKN04, which showed the stunted carpel phenotype did produce seeds but only via hand pollination; however, the number of the seeds was greatly reduced (Table 4). Similar to the results obtained from the experiments with pAGW52 and pAGW 53 plasmids carrying the Eucalyptus occidentalis AG::barnase mutant cassettes, transformation with these constructs affected only the carpel, whereas petals, sepals and anthers appeared normal. The overall length of the carpel as well as the morphology of the stigma were altered (Figure 12).

Table 4: Floral phenotypic survey and seed formation of transgenic tobacco lines

transformed with the constructs pAGKNOl, pAGKN02, pAGKN03, or pAGKN04

Transgenic tobacco Ratio of carpel Number of seed collected ID length to petal Hand from 5 hand-pollinated or

Carpel phenotype length pollinated naturally-pollinated fruits pAGKNOl .7 stunted 0.29 yes no seeds pAGKNOl .9 normal 1.00 - hundreds of seeds pAGKNOl .8 normal 1.05 - thousands of seeds pAGKNOl .5 normal 1.08 - hundreds of seeds pAGKNOl .4 normal 1.02 - hundreds of seeds pAGKNOl .3 normal 1.03 - thousands of seeds pAGKNOl .14 normal 1.02 - thousands of seeds pAGKNOl .13 normal 1.08 - thousands of seeds pAGKNOl .12 normal 1.05 - thousands of seeds pAGKNOl .10 normal 1.00 - thousands of seeds pAGKNOl . l normal 1.00 - thousands of seeds pAGKN02.1 stunted 0.39 yes no seeds pAGKN02.3 normal 1.12 - thousands of seeds pAGKN02.9 normal 1.01 - thousands of seeds pAGKN02.11 normal 1.02 - hundreds of seeds pAGKN03.11 stunted 0.82 yes < 100 seeds pAGKN03.14 stunted 0.77 yes no seeds pAGKN03.21 stunted 0.73 yes < 100 seeds pAGKN03.5 stunted 0.65 yes no seeds pAGKN03.2 stunted 0.63 yes no seeds pAGKN03.8 stunted 0.61 yes no seeds pAGKN03.17 stunted 0.59 yes no seeds pAGKN03.3 stunted 0.58 yes no seeds pAGKN03.6 stunted 0.58 yes no seeds pAGKN03.1 stunted 0.57 yes no seeds pAGKN03.20 stunted 0.57 yes no seeds pAGKN03.4 stunted 0.52 yes < 100 seeds pAGKN03.13 stunted 0.49 yes no seeds pAGKN03.19 stunted 0.49 yes no seeds pAGKN03.7 stunted 0.49 yes no seeds pAGKN03.10 stunted 0.39 yes no seeds pAGKN03.18 stunted 0.35 yes no seeds pAGKN03.16 normal 1.00 - thousands of seeds pAGKN04.1 stunted 0.76 yes < 100 seeds pAGKN04.20 stunted 0.75 yes no seeds pAGKN04.12 stunted 0.74 yes < 100 seeds pAGKN04.4 stunted 0.73 yes < 10 seeds pAGKN04.19 stunted 0.69 yes no seeds pAGKN04.18 stunted 0.63 yes no seeds pAGKN04.8 stunted 0.63 yes no seeds pAGKN04.16 stunted 0.56 yes no seeds pAGKN04.6 stunted 0.47 yes no seeds pAGKN04.3 stunted 0.39 yes no seeds wt Tobacco line 1 normal 1.09 - thousands of seeds wt Tobacco line 2 normal 1.05 - thousands of seeds wt Tobacco line 3 normal 1.09 - thousands of seeds Genomic DNA was isolated from the ten transgenic tobacco lines transformed with pAGKNOl and one transgenic tobacco line transformed with pAGKN03 which showed normal carpel phenotype. PCR analysis was performed to check for the presence of the barnase and NPT II genes. The results showed that the pAGKN03 transgenic line and nine of the ten pAGKNOl transgenic lines did not contain the barnase mutant gene. Only one pAGKNOl transgenic line (pAGKNOl .4) contained the barnase sequence, although it expressed a normal phenotype, probably because of a sequence change in the cassette that affected gene expression (see Table 5). All transgenic tobacco lines contained the nptll gene. The lower transformation efficiency of the two constructs without the 2^nd intron, pAGKNOl and pAGKN02, with respect to the presence of barnase mutant genes, indicated that the expression of the

Eucalyptus occidentalis AG promoter without the second intron driving the barnase mutant is not specific and results in vegetative toxicity and loss of plants carrying the barnase cassette. PCR control analysis was performed using genomic DNA isolated from transgenic lines expressing the ablated carpel phenotype as well as wild type control plants. These results, in combination with the carpel phenotype observations and the fact that a change in location of the second intron does not affect its function, provided evidence that the second intron of the Eucalyptus occidentalis AGAMOUS gene functions as a floral enhancer which controls the floral specificity and activity of the Eucalyptus occidentalis AG promoter. In addition, these studies showed once again that the barnaseK27A is more effective at carpel ablation than the barnaseE73G when driven by the Eucalyptus occidentalis AG promoter (see Table 6).

Table 5: PCR analysis of barnase and nptll in selected transgenic Tobacco lines transformed with the constructs pAGKNOl or pAGKN03

Carpel Barnase NPT II TUA*

Tobacco Lines

Phenotypes PCR PCR PCR

pAGKNOl .1 normal neg + +

pAGKNOl .3 normal neg + +

pAGKNOl .4 normal + + +

pAGKNOl .5 normal neg + +

pAGKNOl .8 normal neg + +

pAGKNOl .9 normal neg + +

pAGKNOl .10 normal neg + +

pAGKNOl .12 normal neg + (faint) +

pAGKNOl .13 normal neg + + pAGK 01.14 normal neg + +

pAGKN03.16 normal neg + +

τ ie transformation study results are summar ized in the Table 6

Table 6: Summary of the transformation study results obtained from transgenic tobacco lines transformed with the barnaseK27A or barnaseE73G

The results of the tobacco transformation studies provided clear evidence that transformation with the Eucalyptus occidentalis AGAMOUS promoter driving a barnase mutant affects carpel development and seed formation. These results were consistent with the results obtained from the experiments performed with the pAGW52 and pAGW53 constructs. Accordingly, a plasmid carrying both cassettes for pollen control and carpel ablation was generated {see Figure 13). This plasmid, pAGKN05 contained a pollen control cassette, PrMC2.400-3::barnaseH102E::RNS2ter (SEQ ID NO: 11) and the

EoAGpromoter::barnaseK27A:: E9TER cassette (SEQ ID NO: 14). Tobacco plants transformed with this plasmid were grown in the greenhouse and displayed complete sterility. The phenotypic carpel data collected from these transgenic plants to date are summarized in Table 7.

Table 7: Floral phenotypic survey of transgenic tobacco lines transformed with the pAGKN05 construct. ratio of

transgenic carpel

tobacco lines ID carpel phenotype to petal

KN05.22 stunted 0.73

KN05.26 stunted 0.68

KN05.8 stunted 0.67

KN05.4 stunted 0.65

KN05.12 stunted 0.65 KN05.18 stunted 0.65

KN05.6 stunted 0.60

KN05.2 stunted 0.59

KN05.14 stunted 0.53

KN05.11 stunted 0.52

KN05.16 stunted 0.51

KN05.1 stunted 0.50

KN05.13 stunted 0.50

KN05.25 stunted 0.45

KN05.9 stunted 0.34

KN05.15 stunted 0.21

Example 8: Analysis of fragments of the second intron of the Eucalyptus occidentalis AG gene

The results reported in Example 7 clearly showed that the second intron of the

Eucalyptus occidentalis AG gene functions as a floral enhancer and controls the floral specificity and activity of the Eucalyptus occidentalis AG promoter. The sequence of the second intron of the Eucalyptus occidentalis AG gene is 3981 bp long (SEQ ID NO: 6). In order to determine whether the enhancer activity of the second intron is linked to the entire sequence length of the second intron, or whether fragments of the second intron containing regulatory motifs would also have enhancer activity, the second intron of SEQ ID NO: 6 in the Eucalyptus occidentalis AG promoter: :barnase mutant floral ablation cassette combinations in the pAGKN03 and pAGKN04 constructs was replaced with different length fragments of the second intron. The constructs were additionally re-engineered to remove a 180 bp exon sequence from between the ATG site and the beginning of the barnase coding region to prevent addition of any additional amino acid to the N terminus of the barnase protein.

Tobacco plants were transformed with these constructs. It was found that 80% of the transgenic tobacco plants transformed with fragments of the second intron containing at least two putative LEAFY binding sites and at least two CCAATCA boxes showed a stunted carpel phenotype (see SEQ ID NO: 15, which is a 2279 bp fragment of the second intron comprising regulatory motifs, and SEQ ID NO: 16, which is 4019 bp long and comprises the nucleic acid sequence of SEQ ID NO: 15 comprising a fragment of the second intron comprising regulatory motifs fused to the 5' end of the Eucalyptus occidentalis AGAMOUS promoter). The results from these experiments clearly showed that fragments of the second intron sequence of the Eucalyptus occidentalis AG gene containing regulatory motifs function as a floral enhancer and control the floral specificity and activity of Eucalyptus occidentalis AG promoter.

Example 9: Analysis and Comparison of the second intron of the Eucalyptus occidentalis AG gene and the Eucalyptus grandisAG gene

The examples above clearly demonstrated that the second intron of the Eucalyptus occidentalis AG gene function as a floral enhancer and control the floral specificity and activity of Eucalyptus occidentalis AG promoter. In order to determine whether the second intron of the AGAMOUS gene in any Eucalyptus species functions as an enhancer and regulates expression of the AGAMOUS gene, the sequence of the second intron of the Eucalyptus grandis AG gene (SEQ ID NO: 17) was analyzed and compared to the sequence of the second intron of the Eucalyptus occidentalis AG gene. Figure 14 shows the alignment of the two sequences.

The comparison of the second intron of the Eucalyptus occidentalis AG gene and the Eucalyptus grandis AG gene showed 95% identity in nucleotide sequences. The EgAG second intron contains two putative LEAFY binding sites (Deyholos and Sieburth, 2000 Plant Cell 12(10): 1799-1810) and the EoAG second intron contains three putative LEAFY binding sites. The two LEAFY binding sites of EgAG are in the same locations as in the sequence of EoAG second intron (Figure 14, green-highlighted sequences). The two CCAATCA boxes, which are conserved in the second intron of AGAMOUS genes in all plant species, are also in the same locations of the second introns of both eucalyptus A G genes (Figure 14, red-colored sequences). This structural similarity of the two eucalyptus A G second introns strongly suggested that they could function in a similar way as an enhancer for regulating the eucalyptus A G gene expression.

Example 10: Analysis of the Second Intron of the Eucalyptus grandis

AGAMOUS Gene

In order to determine whether the second intron of the Eucalyptus grandis

AGAMOUS gene functions as a floral enhancer for carpel ablation, the second intron sequence of the Eucalyptus grandis AGAMOUS gene is fused to the 5 'end of the CaMV 35S minimal promoter (-60). The CaMV 35S minimal promoter has vary low level activities in all plant organs including leaves, vegetative shoots, stems, flowers and roots. A carpel ablation construct carrying the fused promoter driving barnaseK27A and a control construct carrying the CaMV 35 S minimal promoter driving barnaseK27A are synthesized and introduced into Arabidopsis and tobacco plants via Agrobacterium transformation.

Transgenic plants expressing the floral ablation construct comprising the CaMV 35S minimal promoter fused to the second intron sequence of the Eucalyptus grandis AGAMOUS gene show a stunted carpel phenotype and are not able to produce seeds. In contrast, plants transformed with the control construct show a normal carpel phenotype and are not different from untransformed plants.

These results show that the EgAG second intron functions as a floral enhancer and controls the floral specificity and activity of the CaMV 35S minimal promoter.

Example 11: Crossing of Tobacco Lines with a Stunted Carpel with Tobacco Lines with Pollen Ablation

Transgenic tobacco lines each carrying a different trait were crossed to generate offspring lines carrying both traits. The parental transgenic tobacco lines carrying

reproductive ablation cassettes were: (1) pAGKN03 lines carry the

EoAGAMOUS::barnaseK27A::noster cassette and expressing a phenotype in which the female reproductive structure, the carpel, is stunted in length and the normal stigma morphology is altered resulting in reduced fertility; (2)pAGF243 lines carrying the

PrMC2.400-3::barnaseH102E::R S2ter cassette and expressing a phenotype in which the male reproductive structure, the anther, does not produce pollen. Six hand pollinated control crosses were set up between selected transgenic lines of pAGKN03 (carpel ablation) and pAGF243 (pollen ablation), as illustrated in Table 8 below.

Table 8: Summary of Crosses cross

pAGF243-2 x pAGKN03-2

pAGF243-25 x pAGKN03-23

pAGF243-20 x pAGKN03-22

pAGF243-18 x pAGKN03-14

pAGF243-4 x pAGKN03-12

pAGF243-08 x pAGKN03-14

Tobacco flowers with freshly dehisced anthers with visible pollen were removed from a selected pAGKN03 line (pollen donor) and used to pollinate the stigma on the flowers of a selected pAGF243 line (carpel donor). A total of five flowers on the carpel donor line (pAGF243) were pollinated with pollen from flowers from the pollen donor line

(pAGKN03). These hand pollinated flowers were tagged and monitored for the presence of fruit capsule formation, indicating successful fertilization had occurred. Mature fruit capsules were removed and seeds collected. Seeds were germinated directly in soil and grown under mist in the greenhouse. Within 7-10 days seedlings were observed. Leaf tissue was collected on each seedling and the DNA was isolated and PCR tested for the presence of the reproductive ablation cassettes.

Ninety-three seedlings produced from the six hand pollinated control crosses set up between selected transgenic lines of pAGKN03 (carpel ablation) and pAGF243 (pollen ablation) were PCR screened for the presence of both ablation cassettes:

EoAGAMOUS::barnaseK27A::noster and PrMC2.400-3::barnaseH102E::RNS2ter. All plants that contained both ablation cassettes (PCR positive) were transplanted to the greenhouse and allowed to flower in order to collect phenotypic data. Additionally, two plants that were PCR positive for male cassette only, two plants that were PCR positive for the female cassette only, and five plants that were negative for both cassettes were selected for transplanting to the greenhouse to gather phenotypic data at flowering. A summary of the PCR results with phenotypic data is shown in Table 9 below. PCR controls included testing for an endogenous gene, alpha-tubulin, and results were positive for all lines (data not shown). The phenotypes of the two plants that were positive for the female cassette only were unavailable due to a greenhouse error. Table 9. Tobacco pAGF243 x pAGKN03 Control Cross Floral Phenotypic Data

PCR

PCR results results for

for female male Flower phenotypic tag label Parents cassette cassette observation cross - 001 pAGF243-2 x pAGKN03-2 + + stunted carpel ; no pollen cross - 002 pAGF243-2 x pAGKN03-2 + + stunted carpel ; no pollen cross - 003 pAGF243-2 x pAGKN03-2 + + stunted carpel ; no pollen cross - 004 pAGF243-2 x pAGKN03-2 + + stunted carpel ; no pollen cross - 008 pAGF243-2 x pAGKN03-2 + + stunted carpel ; no pollen cross - 013 pAGF243-2 x pAGKN03-2 + + stunted carpel ; no pollen cross - 019 pAGF243-2 x pAGKN03-2 + + stunted carpel ; no pollen cross - 020 pAGF243-25 x pAGKN03-23 + + stunted carpel ; no pollen cross - 021 pAGF243-25 x pAGKN03-23 + + stunted carpel ; no pollen cross - 025 pAGF243-25 x pAGKN03-23 + + stunted carpel ; no pollen cross - 033 pAGF243-08 x pAGKN03-14 + + stunted carpel ; no pollen cross - 039 pAGF243-25 x pAGKN03-23 + + stunted carpel ; no pollen cross - 041 pAGF243-25 x pAGKN03-23 + + stunted carpel ; no pollen cross - 042 pAGF243-25 x pAGKN03-23 + + stunted carpel ; no pollen cross - 052 pAGF243-20 x pAGKN03-22 + + stunted carpel ; no pollen cross - 066 pAGF243-18 x pAGKN03-14 + + stunted carpel ; no pollen cross - 078 pAGF243-4 x pAGKN03-12 + + stunted carpel ; no pollen cross - 090 pAGF243-4 x pAGKN03-12 + + stunted carpel ; no pollen cross - 075 pAGF243-4 x pAGKN03-12 - - normal carpel ; pollen present cross-055 pAGF243-20 x pAGKN03-22 - - normal carpel ; pollen present cross-064 pAGF243-18 x pAGKN03-14 - - normal carpel ; pollen present cross-067 pAGF243-18 x pAGKN03-14 - - normal carpel ; pollen present cross-072 pAGF243-18 x pAGKN03-14 - - normal carpel ; pollen present cross - 006 pAGF243-2 x pAGKN03-2 + - Phenotypic data unavailable cross - 076 pAGF243-4 x pAGKN03-12 + - Phenotypic data unavailable cross - 005 pAGF243-2 x pAGKN03-2 - + normal carpel ; no pollen cross - 071 pAGF243-18 pAGKN03-14 - + normal carpel ; no pollen

The results show that both floral ablation cassettes were inherited from the parent genotypes. Thus, seeds carrying both ablation (barnase mutants) cassettes can successfully survive and develop into mature plants that express floral ablated phenotypes. The plants generated by the control crosses carrying both reproductive ablation cassettes did not produce fruit capsules. These results indicated lack of fertilization which resulted in seed elimination.

Example 12: Floral Ablation Using EoAG-barnaseK27A in Eucalyptus occidentalis

A Eucalyptus occidentalis TE0558868 transgenic line transformed with the pAGW52 construct comprising the Eucalyptus occidentalis AGAMOUS gene promoter (EoAG) operably linked to the barnaseK27A was identified following quantitative PCR analysis of about 100 calluses or leaf samples of putative transgenic lines. Figure 15 shows the amplification plot of TE0558868, as compared to the spiked control, which was prepared by adding a small quantity of pAGW52 plasmid DNA into the genomic DNA extracted from an untransformed Eucalyptus occidentalis tree. A Eucalyptus occidentalis TE0558869 transgenic line transformed with the pAGW55 construct containing the GUS gene (EoAG- GUS) was also obtained and used for comparison in the amplification plot.

Eight ramets of the barnaseK27A or GUS transgenic line identified by the quantitative PCR were planted in soil in 1 -gallon pots on April 1, 2011. Eight untransformed Eucalyptus occidentalis trees were also concomitantly planted in the pots. When the survival rate was checked several months later, it was found that five barnaseK27A Eucalyptus occidentalis trees, six GUS Eucalyptus occidentalis trees and six untransformed Eucalyptus occidentalis trees had survived.

In early October 2011, young floral buds were observed on some of the GUS and untransformed Eucalyptus occidentalis trees, but no floral buds were found on the five barnaseK27A Eucalyptus occidentalis transgenic lines. Visual observation showed similar morphologies between the barnaseK27A Eucalyptus occidentalis transgenic lines and the untransformed trees, with just a minimal reduction in height and stem diameter of the barnaseK27A Eucalyptus occidentalis transgenic lines as compared to the untransformed or GUS tree lines.

In the second half of November 2011, all six GUS trees and five of the six

untransformed trees were carrying floral buds while one untransformed tree did not have any floral buds. By contrast, none of the five barnaseK27A Eucalyptus occidentalis trees had floral buds. The phenotypic comparison of the barnaseK27A Eucalyptus occidentalis trees and the untransformed trees is presented in Figure 16. The picture in Figure 16 was taken on November 29, 2011.

In the control untransformed tree, the apical vegetative shoot primordia developed into floral primordia and floral buds were initiated on the tree apex. In these trees, the floral buds were also initiated in all axillary locations on the shoot and leaf buds were initiated in the same axillary locations. In the barnaseK27A Eucalyptus occidentalis trees, initiation of floral buds was suppressed in most axillary locations and no floral buds were observed. In a few axillary locations of these transgenic lines, initiation of floral buds was observed, but all initiated floral buds soon developed into "vegetative shoot buds" and died later on. The development of floral buds into vegetative buds seen in the barnaseK27A Eucalyptus occidentalis trees has also been reported in other species, including Arabidopsis and tobacco. Upon observation it was found that some vegetative shoot primordia and leaf primordia immediately adjacent to the floral primordia were also ablated in the barnaseK27A

Eucalyptus occidentalis trees after the trees entered the development stage of reproductive growth (flowering).

In April 2012, one year after soil transplanting, the remaining one untransformed tree finally flowered, whereas all five K27A barnase Eucalyptus occidentalis trees showed no floral buds.

These results clearly show that transformation with the EoAG-barnaseK27A construct (pAGW52) successfully prevents flower formation in Eucalyptus with minimal effects on vegetative growth. Missing Pages 69

Claims

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising an Eucalyptus AGAMOUS promoter selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 13, a sequence that is complementary to SEQ ID NO: 1 or SEQ ID NO: 13, a reverse complement of SEQ ID NO: 1 or SEQ ID NO: 13, and a functional fragment of SEQ ID NO: 1 or SEQ ID NO: 13.

2. An ablation cassette comprising the isolated polynucleotide of claim 1 operably linked to a desired nucleic acid sequence that encodes a protein that alters the development of a reproductive structure of a plant, wherein the promoter upregulates or downregulates the expression of the nucleic acid sequence, and wherein the plant is not Arabidopsis.

3. The ablation cassette of claim 2, wherein the promoter upregulates the expression of the desired nucleic acid sequence, and wherein the desired nucleic acid sequence encodes a cytotoxic protein that destroys a reproductive structure in a plant.

4. The ablation cassette of claim 3, wherein the reproductive structure is a carpel.

5. The ablation cassette of claim 3, wherein the desired nucleic acid sequence is selected from the group consisting of a nucleic acid sequence that encodes an barnaseE73G mutant comprising SEQ ID NO: 2, a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 3, a nucleic acid sequence that encodes a barnaseK27A mutant comprising SEQ ID NO: 4,and a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 5, and wherein the isolated polynucleotide comprises a floral enhancer that controls the floral specificity and activity of the Eucalyptus AGAMOUS promoter.

6. The ablation cassette of claim 5, wherein the floral enhancer is the second intron sequence of the Eucalyptus AGAMOUS gene and wherein the floral enhancer is located at the 5 ' end of the Eucalyptus AGAMOUS promoter.

7. The ablation cassette of claim 6, wherein the floral enhancer comprises SEQ ID NO: 6 or a fragment thereof, or SEQ ID NO: 15 or a fragment thereof.

8. A DNA construct comprising the ablation cassette of claim 5.

9. A plant cell transformed with the DNA construct of claim 8.

10. A transgenic plant comprising the plant cell of claim 9 and exhibiting a phenotype that is different from the phenotype of a non-transgenic plant of the same species, wherein the phenotype is a stunted carpel and reduced seed formation, and the transgenic plant shows no difference in vegetative growth rate, height and leaf color when compared to a non-transgenic plant of the same species.

11. A transgenic plant comprising the plant cell of claim 11 and exhibiting a phenotype that is different from the phenotype of a non-transgenic plant of the same species, wherein the phenotype is a stunted carpel and reduced seed formation, and the transgenic plant shows no remarkable difference in vegetative growth rate, height and leaf color when compared to a non-transgenic plant of the same species.

12. The transgenic plant of claim 11?, wherein the transgenic plant is a transgenic ornamental plant or a transgenic tree.

13. A method for producing a transgenic plant with an ablated reproductive organ without impairing the vegetative growth of the plant, comprising: (a) transforming a plant cell with the DNA construct of claim 8; (b) culturing the transformed plant cell under conditions that promote growth of a transgenic plant; and (c) selecting a transgenic plant with a stunted carpel and reduced seed formation phenotype, wherein the transgenic plant shows no remarkable difference in vegetative growth rate, height and leaf color when compared to a non-transgenic plant of the same species..

14. A method for preventing or reducing seed formation and natural pollination in a plant without significantly impairing the vegetative growth of the plant, comprising: (a) transforming a plant cell with a DNA construct comprising an ablation cassette comprising the isolated polynucleotide of claim 1 operably linked to a desired nucleic acid sequence that encodes a cytotoxic protein that destroys a reproductive structure of a plant; and (b) culturing the transformed plant cell under conditions that promote growth of a plant; wherein expression of the ablation cassette in the plant results in a reduction in seed formation and natural pollination in the plant and has no deleterious effect on the vegetative growth of the plant.

15. The method of claim 14?, wherein the desired nucleic acid sequence is selected from the group consisting of a nucleic acid sequence that encodes an barnaseE73G mutant comprising SEQ ID NO: 2, a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 3, a nucleic acid sequence that encodes a barnaseK27A mutant comprising SEQ ID NO: 4,and a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 5, and wherein the isolated polynucleotide comprises the second intron sequence of the Eucalyptus AGAMOUS gene.

16. A method for conferring complete reproductive sterility to a plant without impairing the vegetative growth of the plant comprising (a) transforming a plant cell with a DNA construct comprising (i) a first cassette comprising a male or female reproductive structure -preferred promoter operably linked to a nucleic acid sequence that encodes a protein that ablates reproductive development in the plant, wherein the reproductive structure- preferred promoter regulates the expression of the nucleic acid sequence; and (ii) a second cassette comprising a Eucalyptus AGAMOUS promoter operably linked to a desired nucleic acid sequence that encodes a cytotoxic protein that destroys a reproductive structure of a plant; (b) culturing the transformed plant cell under conditions that promote growth of a plant; and (c) selecting a plant that expresses both the first and the second cassettes of the DNA construct, wherein expression of both the first and the second cassettes in the plant results in complete sterility of the plant and has no deleterious effect on the vegetative growth of the plant.

17. The method of claim 16?, wherein the first cassette comprises a Pinus radiata male cone (PrMC) promoter comprising SEQ ID NO: 7 or SEQ ID NO: 8 operably linked to a nucleic acid sequence encoding a barnaseH102E mutant comprising SEQ ID NO: 9 or a nucleic acid sequence encoding a barnaseH102E mutant comprising SEQ ID NO: 10; and the second cassette comprises the Eucalyptus AGAMOUS promoter of SEQ ID NO: 13 or a fragment thereof comprising SEQ ID NO: 16 operably linked to a nucleic acid sequence encoding a barnaseK27A mutant.

18. The method of claim 17?, wherein the nucleic acid sequence encoding the barnaseK27A mutant comprises SEQ ID NO: 4 or a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 5.

19. A completely sterile transgenic plant obtained by the method of claim 18, wherein the transgenic plant shows no remarkable difference in vegetative growth rate, height and leaf color when compared to a non-transgenic plant of the same species, wherein the transgenic plant is a transgenic tree or a transgenic ornamental plant.

20. Wood and wood products obtained from the transgenic plant of claim 19.

21. An Fl plant having a phenotype characterized by a stunted carpel and lack of pollen.

22. An Fl plant having a phenotype characterized by lack of seed production.

23. A method of producing an Fl plant having a phenotype characterized by lack of seed production comprising (a) hand-pollinating the flowers of a plant having a phenotype characterized by lack of pollen production with pollen produced by a plant having a phenotype characterized by a stunted carpel; (b) collecting the seeds from mature fruit capsules formed upon fertilization; (c) germinating the seeds to obtain seedlings; (d) screening the seedlings for the presence of at least one ablation cassette by PCR; and (e) growing PCR-positive seedlings under conditions that promote plant growth to obtain an Fl plant having a phenotype characterized by lack of seed production.

24. The method of claim 23, wherein the plant having a phenotype characterized by lack of pollen production expresses a cassette comprising a Eucalyptus AGAMOUS promoter operably linked to a desired nucleic acid sequence that encodes a cytotoxic protein that destroys a reproductive structure of a plant, and the plant having a phenotype

characterized by a stunted carpel expresses a cassette comprising a male or female reproductive structure -preferred promoter operably linked to a nucleic acid sequence that encodes a protein that ablates reproductive development in the plant.

25. The method of claim 24, wherein the Eucalyptus AGAMOUS promoter comprises SEQ ID NO: 13 or a fragment thereof comprising SEQ ID NO: 16 and is operably linked to a nucleic acid sequence encoding a barnaseK27A mutant; and wherein the male or female reproductive structure -preferred promoter is a Pinus radiata male cone (PrMC) promoter comprising SEQ ID NO: 7 or SEQ ID NO: 8 and is operably linked to a nucleic acid sequence encoding a barnaseH102E mutant comprising SEQ ID NO: 9 or a nucleic acid sequence encoding a barnaseH102E mutant comprising SEQ ID NO: 10.

26. A method of controlling pollination in an Fl plant comprising crossing a transgenic plant with a phenotype characterized by a stunted carpel and that expresses a reproductive ablation cassette comprising a Eucalyptus AGAMOUS promoter operably linked to a desired nucleic acid sequence that encodes a cytotoxic protein that alters the stigma morphology in the plant, with a transgenic plant in which the anthers do not produce pollen and that expresses a cassette comprising a a Pinus radiata male cone (PrMC) promoter operably linked to a nucleic acid sequence that encodes a protein that ablates pollen production; and generating an Fl plant that expresses both ablation cassettes and does not produce fruit capsules.

27. The method of claim 26, wherein the EoAGAMOUS promoter comprises SEQ ID NO: 13 or a fragment thereof comprising SEQ ID NO: 16, and is operably linked to a barnaseK27A mutant; and the Pinus radiata male cone (PrMC) promoter comprises SEQ ID NO: 7 or SEQ ID NO: 8 and is operably linked to a nucleic acid sequence encoding a barnaseH102E mutant comprising SEQ ID NO: 9 or a nucleic acid sequence encoding a barnaseH102E mutant comprising SEQ ID NO: 10.