TITLE: INCREASED PRODUCTION OF REPRODUCTIVE ORGANS AND YIELD COMPONENTS IN PLANTS

TECHNICAL FIELD

The present invention relates to methods for increasing grain yields in seed- producing agronomic crops by modulating therein expression of STM genes. BACKGROUND

Over the past few years Brassica napus androgenesis, i.e. the formation of embryos from microspores, has become a model system to study plant embryogenesis and meristem formation (Yeung, 2002; Boutilier et al, 2005; Joosen et al, 2007; Malik et al, 2007; Stasolla et al, 2008). This system is very suitable for

developmental studies since a large number of synchronized embryos can be produced without an intervening callus phase during a short period of time.

Furthermore, the genetic similarities between Brassica and Arabidopsis can be exploited to isolate and characterize Brassica genes involved in embryogenesis. As in any other in vitro embryogenic system, the establishment of a functional shoot apical meristem (SAM) is a prerogative for the successful regeneration of Brassica microspore-derived embryos (MDEs). Reduced post-embryonic growth is commonly observed in those MDEs which develop abnormalities within the apical pole, including vacuolation and differentiation of meristematic cells. Experimental manipulations of culture conditions have been used to alter the structure of the SAM. Applications of DL-buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis (Griffith et al., 1979, Potent and specific inhibition of glutathione synthesis by gluthionine sulfoximine (s-n-Butyl Homocysteine Sulfoximine), J. Biochem. Chem. 254:7558-7560), induced the formation of functional "zygotic-like" meristems able to reactivate at high frequency at germination (Belmonte et al, 2006, Improved development of microspore derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism, Physiol. Plant. 127:690-700). On the contrary, complete loss of SAM functionality was observed when 2,3,5-triiodobenzoic acid (TIBA), an auxin flow inhibitor, was added to the medium. (Ramesar-Fortner et al., 2006, Physiological influences in the development and function of the shoot apical meristem of microspore-derived embryos of Brassica napus cv. Topas. Can. J. Bot. 84:371-383).

The Arabidopsis shoot apical meristem (SAM) is regulated by a set of key genes, including SHOOTMERISTEMLESS (STM), CLA VATA 1 (CLVl), WUSCHEL (WUS), and ZWILLE (ZLL), which establish and maintain the characteristic layering and zonation patterns of the apical pole. The Arabidopsis STM is a member of the class- 1 KNOX homeodomain containing proteins required for the formation and maintenance of the SAM by retaining a pool of undifferentiated cells at the apical pole. Structural abnormalities, including the terminal differentiation of the apical cells, are observed in the shoot meristems of stm plants which are not able to reactivate and grow post-embryonically. Proper expression of STM within the SAM is ensured by ZLL, a member of the ARGONAUTE (AGO) family, which is involved in the formation of RNA-induced silencing complexes in both animal and plants. Unlike STM which is needed for the development of the embryonic SAM and its post-embryonic activity, ZLL is only implicated in the formation of the SAM during embryogenesis. Genetic studies showed that zll embryos fail to develop proper meristems. However, adventitious SAMs originating between the cotyledons ensure post-embryonic development. Proper maintenance of SAM activity also relies on the feedback mechanism of the WUS-CLV signalling. WUSCHEL is a homeobox gene encoding a transcription factor which regulates the "organizing center" of the SAM by maintaining a reservoir of undetermined cells. The expression of WUS is repressed by the CLV signalling upon the interaction of CLV3 with the receptor kinase complex. A mutation of any CLV members, including CLVl, disrupts the balance between cell division and differentiation within the SAM resulting in enlarged meristems and atypical expansion of the WUS domain. It appears the role of CLVl is to promote the differentiation of the meristematic cells thereby reducing the pool of the

undifferentiated stem cell population.

The molecular regulation of SAM is mediated by the activity of plant growth regulators. Numerous studies show that KNOX proteins, which include STM, affect the metabolism of cytokinins and gibberellins. Increasing levels of cytokinins, which are linked to the maintenance of undifferentiated cells in the central domain of the SAM are attributed to the KNOX- activation of isopentenyltransferse genes. By contrast, the levels of GAs in the central zone of the SAM are maintained low by the suppression exercised by KNOX members on GA20 oxidase. High GA levels are observed in the peripheral region of the SAM demarking the initiation of lateral organs. No information is currently available on the regulation of plant growth regulators by other genes that may participate in SAM formation and maintenance.

SUMMARY

This disclosure provides methods for increasing grain yields in agronomic crop plants. The methods pertain to manipulating selected agronomic crop plants to produce therein over-expression of STM genes. Some exemplary methods pertain to transformation of selected agronomic crop plants by incorporating therein expression vectors comprising STM genes.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described in conjunction with reference to the following drawings, in which: Figure 1 is a chart showing expression levels by 10-day-old Arabidopsis seedlings transformed with the Brassica oleracea STM gene (BoSTM), B. rapa STM gene (BrSTM), B. napus STM gene (BnSTM), B. napus CLV1 gene {BnCLVl, B.

napus ZLL-X gene (BnZLL-1), and B. napus ZLL-2 gene (BnZLL-2). Values + SE are means of three independent analyses. Stars indicate values which are significantly different from the WT value (P < 0.05) set at 1 ;

Figures 2A - 2G are micrographs showing immunolocalization of IAA in 25- day-old Brassica napus microspore-derived embryos. The micrographs show cotyledons (2A, 2C, 2E) and shoot apical meristems (2B, 2D, 2F) of embryos cultured in the presence (2A, 2B) or absence (2C, 2D) of 2mM 2,3,5-triodobenzoic acid (TIB A). Embryos were also growth in the presence of TIB A plus 2μΜ 2-(p- chlorophenoxy)-2-methylpropionic acid (PCIB) (2E, 2F). Specificity of staining was demonstrated by the omission of primary antibodies (2G);

Figure 3 is a chart showing the expression profiles of (A) BnSTM, (B) BnCLVl, (C) BnZLL-1, and (D) BnZLL-2 in B. napus microspore-derived embryos cultured in the presence of 2,3,5-triiodobenzoic acid (TIBA), 2-(p-chlorophenoxy)-2- methylpropionic acid (PCIB), and DL-buthionine sulfoximine (BSO);

Figures 4(1) - 4(20) are micrographs illustrating the expression patterns of BnSTM during development of B. napus microspore-derived embryos. The signal for BnSTM was first observed in a small clusters of sub-apical cells in pre-globular embryos 4(1) and retained in the apical pole of globular 4(2), early cotyledonary 4(3) and cotyledonary 4(4) embryos. In some instances BnSTM expression was delocalized to the side of the apical pole throughout development 4(5-7), or in non- adjacent clusters of cells 4(8-11). In the presence of BSO, BnSTM expression expanded in globular 4(12), heart-shaped 4(13), and cotyledonary 4(14) embryos. The signal of BnSTM was reduced in immature embryos treated with TIBA 4(15, 16) and completely lost in fully developed embryos 4(17). These alterations were recovered in immature 4(18) and cotyledonary 4(19) embryos when PCIB was added along with TIBA. No signal was observed with the sense probe 4(20); Figures 5(1) - 5(8) are micrographs illustrating the localization patterns of

BnCLVl in untreated microspore-derived embryos at the globular 5(1), early cotyledonary 5(2) and cotyledonary 5(3) stage of development, in BSO-treated MDEs 5(4, 5), and in TIBA-treated embryos 5(6-7). A localization signal similar to that observed in untreated embryos was also detected in MDEs cultured with TIBA+PCIB (data not shown). No signal was visible with the sense probe 5(8);

Figures 6(1) - 6(9) are micrographs illustrating the expression of BnZLL-1 and BnZLL-2 in longitudinal sections of globular 6(1), early cotyledonary 6(2), middle cotyledonary 6(3) and cotyledonary 6(4) untreated MDEs, as well as in cross sections of cotyledonary embryos 6(5). BnZLL-1 and BnZLL-2 localization in cotyledonary MDEs treated with BSO 6(6), TIBA 6(7), and TIBA+PCIB 6(8). No signal was observed with sense probes 6(9);

Figures 7(1) - 7(3) are micrographs illustrating phenotypic characterization of Arabidopsis lines ectopically expressing the Brassica STM, CLV1, ZLL-1 and ZLL-2: Fig. 7(1) shows the structure of the shoot apical meristem (SAM) in WT cotyledonary embryos, Fig. 7(2) shows the structure of SAM in embryos expressing the Brassica CLV1, and Fig. 7(3) shows the structure of SAM in embryos expressing the Brassica STM (3). The meristematic cells of 35S: .BnCLVl embryos accumulated storage products (2, arrow);

Figure 8 is a chart showing the number of transformed Arabidopsis seedlings producing leaf primordia 5 days after germination; Figures 9(1) - 9(7) are micrographs showing production of ectopic meristems

9(1, arrow) or lobed leaves 9(2, arrow) from high STM over-expressors. The introduction of the Brassica STM also resulted in the production of abnormal leaves 9(3, with WT leaf on the left side), larger inflorescences 9(4) compared to WT plants 9(5), and curved siliques 9(6). Termination of the SAM with a determined stem-like structure (arrow), was often observed in lines ectopically expressing BnCLVl 9(7);

Figures 10(A) - 10(D) are charts showing expression levels measured by semi-quantitative RT-PCR of the meristem-related genes: 109(A) KNAT6, 10(B) WUS, 10(C) CUC1, and 10(D) CLV3 in the transformed Arabidopsis plants after 1 week of germination. The values of the transformed lines were normalized to the value of WT plants set at 100;

Figures 1 1(A) - 1 1(D) are charts showing the effects of hormone treatments on the expression level of BnSTM as measured by semi-quantitative RT-PCR in shoots from 10-day old Brassica napus plants, harvested at different times after application of the hormone treatments. Fig 1 1(A) shows the effects of benzylamine purine (BAP, 100 μΜ), Fig. 10(B) shows the effects of indole acetic-acid (IAA, 30 μΜ), Fig. 11(C) shows the effects of abscisic acid (ABA, 100 μΜ), and Fig. 1 1(D) shows the effects of gibberellin-3 (GA3, 100 μΜ). Figures 1 1(E) - 1 1(H) are charts showing the effects of hormone treatments on the expression level of BnCLVl, wherein Fig. 1 1(E) shows the effects of BAP (100 μΜ), Fig. 1 1(F) shows the effects of IAA (30 μΜ), Fig. 11(G) shows the effects of ABA (100 μΜ), and Fig. 11(G) shows the effects of GA3 (100 μΜ); and

Figure 12(A) is a chart showing the effects of increasing ABA levels on the percentage of seed germination, Figure 12(B) is a chart showing the effects of increasing 2,4-D levels on root growth. DETAILED DESCRIPTION

The present invention relates to methods for increasing seed yields of agronomic crops wherein the numbers of reproductive organs produced per plant are significantly increased in comparison to parental lines. Unless otherwise defined, 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. In order that the invention herein described may be fully understood, the following terms and definitions are provided herein.

As used herein, the term "synthetic DNA" means DNA sequences that have been prepared entirely or at least partially by chemical means. Synthetic DNA sequences may be used, for example, for modifying native DNA sequences in terms of codon usage and expression efficiency.

The word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers.

As used herein, the word "complexed" means attached together by one or more linkages.

The term "a cell" includes a single cell as well as a plurality or population of cells. The term "about" or "approximately" means within 20%, preferably within

10%, and more preferably within 5% of a given value or range.

The term "nucleic acid" refers to a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single- stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semisynthetic DNA.

The term "gene" refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. The term "recombinant DNA molecule" refers to a DNA molecule that has undergone a molecular biological manipulation.

The term "vector" refers to any means for the transfer of a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. The term "vector" includes plasmids, DNA-protein complexes, and biopolymers. In addition to a nucleic acid, a vector may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).

The term "cloning vector" refers to a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. Cloning vectors may be capable of replication in one cell type, and expression in another ("shuttle vector").

A cell has been "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. A cell has been "transformed" by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change. The transforming DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

The term "nucleic acid molecule" refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester anologs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA— DNA, DNA-RNA and RNA— RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Modification of a genetic and/or chemical nature is understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for various purposes, such as in particular that of enhancing its production levels, that of increasing and/or modifying its activity, or that of conferring new pharmacokinetic and/or biological properties on it. Among the derivatives resulting from an addition, there may be mentioned, for example, the chimeric nucleic acid sequences comprising an additional heterologous part linked to one end, for example of the hybrid construct type consisting of a cDNA with which one or more introns would be associated. Likewise, for the purposes of the invention, the claimed nucleic acids may comprise promoter, activating or regulatory sequences, and the like.

The term "promoter sequence" refers to a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.

A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.

The term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including homologous proteins from different species. Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity. This homology is greater than about 75%, greater than about 80%, greater than about 85%. In some cases the homology will be greater than about 90% to 95% or 98%.

"Amino acid sequence homology" is understood to include both amino acid sequence identity and similarity. Homologous sequences share identical and/or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in an aligned reference sequence. Thus, a candidate polypeptide sequence that shares 70% amino acid homology with a reference sequence is one in which any 70% of the aligned residues are either identical to, or are conservative substitutions of, the corresponding residues in a reference sequence.

The term "polypeptide" refers to a polymeric compound comprised of covalently linked amino acid residues. Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group. A polypeptide of the invention preferably comprises at least about 14 amino acids.

The term "protein" refers to a polypeptide which plays a structural or functional role in a living cell.

The term "corresponding to" is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured. A nucleic acid or amino acid sequence alignment may include spaces. Thus, the term "corresponding to" refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.

The term "derivative" refers to a product comprising, for example, modifications at the level of the primary structure, such as deletions of one or more residues, substitutions of one or more residues, and/or modifications at the level of one or more residues. The number of residues affected by the modifications may be, for example, from 1, 2 or 3 to 10, 20, or 30 residues. The term derivative also comprises the molecules comprising additional internal or terminal parts, of a peptide nature or otherwise. They may be in particular active parts, markers, amino acids, such as methionine at position -1. The term derivative also comprises the molecules comprising modifications at the level of the tertiary structure (N-terminal end, and the like). The term derivative also comprises sequences homologous to the sequence considered, derived from other cellular sources, and in particular from cells of human origin, or from other organisms, and possessing activity of the same type or of substantially similar type. Such homologous sequences may be obtained by hybridization experiments. The hybridizations may be performed based on nucleic acid libraries, using, as probe, the native sequence or a fragment thereof, under conventional stringency conditions or preferably under high stringency conditions.

An exemplary embodiment of the present invention pertains to methods for increasing the numbers of reproductive organs produced per plant of a selected seed- producing crop wherein expression of the SHOOT-MERISTEMLESS (i.e., STM) gene is manipulated in a manner such that the STM gene is over-expressed in comparison to non-manipulated plants.

We have surprisingly found that genetically transformed plants having incorporated therein STM genes under control of a 35S promotor, produce significantly more numbers of reproductive organs per plant than do the parent un- transformed plants. A suitable source for STM genes for transforming seed-producing agronomic crop plants is Brassica napus. The STM gene obtained from B. napus is referred to herein as the BnSTM gene. The sequence of the BnSTM gene is given in SEQ ID NO: 1.

Grain-producing agronomic crop plants transformed with an STM gene produce significantly greater numbers of reproductive organs and yield components exemplified by: flowers per plant, siliques per plant, branches per plant, pods per plant, seeds per pods, seeds per plant.

An exemplary embodiment of the present invention relates to a method for increasing the yields of oil-seed crops by manipulating therein the expression of STM genes so that the STM genes are over-expressed thereby increasing flowering and the production of yield components. An aspect pertains to transforming oil-seed crops by incorporating therein a STM gene exemplified by the BnSTM gene. Suitable oil-seed crops are exemplified by canola, mustard, flax, sunflower, camelina and the like. SEQ ID NO: 1.

1 atggaaagtg gttccaacag cacttcttgt ccaatggctt ttgccgggga taatagtgat 61 ggtccgatgt gttctatgat gatgatgatg atgcccgtca taacatcaca tcaacaacat 121 catggtcatg atcaacaaca tcaacatcaa caacaacatg atggttatgc atatcagtca 181 caccaccaac atagtagcct cctttttctt caatcactaa ctcctccgtc tcaagaagcg

241 aagaacaaag ttagatcttc ttgttctcct tcctctggtg ctcctgctta ttctttcatg

301 gagatcaatc accaaaacga actcctcgca ggaggactca atccctgttc ttcagcctct 361 gtcaaggcca aaatcatggg tcatcctcac taccaccgcc tcttgctcac ctatgtcaat 421 tgccagaagg tgggagctcc accggaagtg caggcgaggc tggaagaaac atgctcgtct 481 gcggctgccg ccgcagcgtc gatgggaccc acaggttctt taggtgaaga tccagggctt

541 gatcagttca tggaagcgta ctgtgaaatg ctcgttaagt acgagcaaga actctctaaa 601 ccttttaaag aagctatggt cttccttcaa cacgtcgagt gtcaattcaa atccctctct 661 ctctcctcgc cgtcctcctt ctctggttat ggagaggcag ctattgagag aaacaacaat 721 gggtcatctg aggaagaagt cgatatgaac aatgaatttg tagatccgca ggcagaagat 781 agggagctta aaggacagct cttgcgcaag tacagtggtt acttaggcag tctgaagcaa

841 gagttcatga agaagaggaa gaaaggagag cttcctaaag aagctcgcca gcaactactt 901 gactggtgga gccgacacta caaatggcct tacccttcgg agcagcaaaa gctagcacta 961 gcggaatcaa ctgggctgga ccagaaacag ataaacaact ggttcataaa ccagaggaaa 1021 aggcattgga aaccgtcgga ggatatgcag tttgtagtaa tggacgcaac acatcctcac 1081 cattacttta tggacaatgt catgggaaat cctttcccca ttgatcacat ctcctcgacc

1141 atgctttga

An exemplary embodiment of the present invention relates to a method for increasing the yields of pulse crops by manipulating therein the expression of STM genes so that the STM genes are over-expressed thereby increasing flowering and the production of yield components. An aspect pertains to transforming pulse crops by incorporating therein a STM gene exemplified by the BnSTM gene. Suitable pulse crops are exemplified by soybeans, field peas, lentils, chickpeas, dry beans, faba beans and the like.

An exemplary embodiment of the present invention relates to a method for increasing the yields of cereal crops by manipulating therein the expression of STM genes so that the STM genes are over-expressed thereby increasing flowering and the production of yield components. An aspect pertains to transforming cereal crops by incorporating therein a STM gene exemplified by the BnSTM gene. Suitable cereal crops are exemplified by wheat, winter wheat, malting barley, feed barley, food barley, triticale, oats, and the like.

An exemplary embodiment of the present invention relates to a method for increasing the yields of maize crops by manipulating therein the expression of STM genes so that the STM genes are over-expressed thereby increasing flowering and the production of yield components. An aspect pertains to transforming maize crops by incorporating therein a STM gene exemplified by the BnSTM gene.

EXAMPLES Example 1 : Expression and localization of the Brassica napus genes during normal and abnormal embryo development

To establish a functional relationship between the four Brassica genes i.e., (i) BnSTM, (ii) BnCLVl, (iii) BnZLL-1, and (iv) BnZLL-2, and their effects on the development of shoot apical meristems, their expression and localization patterns in B. napus MDEs in which the SAM structure was experimentally altered through manipulations of the culture medium as disclosed herein.

Plant material and treatments:

Greenhouse culture conditions for growing and maintaining Brassica napus (Topas DH4079) plants and for the production of microspore-derived embryos (MDEs) from the plants followed the methods disclosed by Belmonte et al. (2006, Improved development of microspore derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism, Physiol. Plant. 127:690-700). Brassica napus cv Topaz seeds were grown at 25° C day/16° C night temperature with a 1 -h photoperiod until the first flower buds appeared (approximately 5 weeks). Plants were then transferred to 12° C day/7° C night temperature cold treatment where they remained for bud collection. Flower buds (2-3 mm long) were collected from cold-treated plants and the microspores were isolated and suspended in NLN medium with 13% sucrose (pH 5.8). The microspores were then subjected to a heat shock treatment of 32° C for 72 h, and then transferred onto a gyratory shaker and allowed to develop in the NLN medium up to 35 days under dark conditions.

For microspore-derived embryogenesis, "days in culture" were counted from the imposition of the heat shock treatment. Alterations in MDE and SAM structures were induced by following the methods taught by: (1) Belmonte et al. for 0.1 tnM DL-buthionine sulfoximine (BSO), and (2) by Ramesar-Fortner et al. (2006, Physiological influences in the development and function of the shoot apical meristem of microspore-derived embryos ofBrassica napus cv. Topas. Can. J. Bot. 84:371-383) for 2μΜ 2,3,5-triiodobenzoic acid (TIBA), and 2μΜ 2-(p-chlorophenoxy)-2- methylpropionic acid (PCIB). B. napus cv Topaz seeds were grown at 25° C day/16 ⁰ C night temperature with a 16-h photoperiod until the first flower buds appeared (approximately 5 weeks). Plants were then transferred to 12° C day/7° C night temperature cold treatment where they remained for bud collection. Flower buds (2-3 mm long) were collected from cold-treated plants and the microspores were isolated and suspended in NLN medium with 13% sucrose (pH 5.8). The microspores were then subjected to a heat shock treatment of 32° C for 72 h, and then transferred onto a gyratory shaker and allowed to develop in the NLN medium up to 35 days under dark conditions. Microspore-derived embryos were harvested at day 5, 14, 21, 28, and 35 for gene expression and transcript localization studies. Transgenic Arabidopsis plants ectopically expressing the Brassica napus

BnSTM (GU480584), B. rapa BrSTM (GU480585), B. oleracea BoSTM (AF193813), BnCLVl (GU480585), as well as BnZLL-1 (EU329719) and BnZLL-2 (GU731230) were generated following the methods disclosed by Elhiti et al. (2010, Modulation of embryo-forming capacity in culture through the expression of Brassica genes involved in the regulation of the shoot apical meristem. J. Exp. Bot. 61 :4069-4085). Full-length cDNAs were inserted in the Gateway entry clone vector pDONR221 (Invitrogen Canada Inc., Burlington, ON, Canada) and then transferred into the pK2GW7 vectors carrying the 35S promoter and the 35S terminator (http://www.psb.ugent.be/gateway/index.php). The constructs were introduced into the Agrobacterium tumefaciens strain GV3101 which was utilized to spray transform wild-type (WT) once the plants started flowering. All plants were grown at 22° C under long day conditions (16 h light and 8 h dark). Seeds of transformed plants were screened on l x MS medium supplemented with 1% sucrose and kanamycin (50 μg ml-1).

The lines used in this study were BnSTM (L5, 8, and 23); BrSTM (L5, 15, 21), BoSTM (L5, 7, 1 1, and 33), BnCLVl (L12, L13, and L19), BnZLL-1 (L2 and 16), and BnZLL-2 (L8 and 16) (Figure 1 ).

The reactivation of the embryonic shoot apical meristem in transformed lines was estimated by germinating the Arabidopsis seeds on ½ MS medium containing 1% sucrose, and counting the number of 5-day-old seedlings which displayed newly formed leaf primordia. Expression studies:

Expression studies in Brassica and Arabidopsis were carried out using semiquantitative reverse transcriptase (RT)-PCR. Primers and number of cycles are listed in Table 1. The transcript levels of BnSTM, BnCLVl, BnZLL-1 and BnZLL-2 were measured during Brassica MDE development. The expression levels of KNAT6, WUS, CUC1, and CLV3 were measured in the aerial part (seedling without roots) of 1-week old Arabidopsis plants germinated on ½ MS medium containing 1% sucrose, actin and ubiquitin were used as internal controls for Brassica and Arabidopsis respectively (Table 1).

Histological studies were performed exactly as described by Yeung (1999). Tissue was fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde buffered with 0.05M phosphate buffer pH6.9, dehydrated with methylcellosolve, followed by two changes of absolute alcohol, and then infiltrated and embedded in Historesin. Sectioning was carried out with glass knives on a Reichert-Jung 2040 Autocut microtome. Serial longitudinal sections were cut at a thickness of 3 μπι. The sections were stained with 0.05% toluidine blue O in benzoate buffer, pH 4.4 and observed under a compound microscope. Table 1 : Primer sets and numbers of cycles used to measure the expression levels of

Brassica and Arabidopsis genes by semi-quantitative PCT.

Brassica genes Forward primers Cycles

STM SEQ ID NO:2 5-TGGGACCCACAGGTTCTTTA-3 27

CLV1 SEQ ID NO:4 5 -TCTCCTTC AACG ACCTCTCG-3 23

ZLL1 SEQ ID NO:6 5-GACGACGACGGCGGCAGCTCAGAGCC-3 24

ZLL2 SEQ ID NO: 8 5-ACTCCGACCAAGCTTCTTCAC-3 26

Actin SEQ ID NO: 10 5 -T AAAGTATCCGATTGAGC ATGGT AT-3 22

Brassica genes Reverse primers

STM SEQ ID NO:3 5 -CC AGTTG ATTCCGCTAGTGC-3

CLV1 SEQ ID NO:5 5 -TGGTG AAG ATAAC AC AGTCCTTTCG-3

ZLL1 SEQ ID NO:7 5 -C AGACTCTTTGTAT AGTCTC ACTA-3

ZLL2 SEQ ID NO:9 5 -C AGACTCTTTGT AT AGTCTC ACT A-3

Actin SEQ ID NO: 11 5-CGTAGGCAAGCTTCTCTTTAATGTC-3

Arabidopsis genes Forward primers

KNAT6 SEQ ID NO: 12 5-AGCGGTTTCATATTATTCTTCTTCTTCA-3 27

CUC1 SEQ ID NO: 14 5 -TTCTTCTTGTGCCGAC AATG-3 25

WUS SEQ ID NO: 16 5-CCAGCAAGTTGTTTTCTTGC-3 27

CLV3 SEQ ID NO: 18 5 -TGG ATTCGAAG AGTTTTCTGC-3 29

UBQ10 SEQ ID NO:20 5 -GATCTTTGCGG AAAAC AATTGG AGG ATGG-3 21

Arabidopsis genes Reverse primers

KNAT6 SEQ ID NO: 13 5 - AAAAAGAGGTT ATTTTTATTC ACCG-3

CUC1 SEQ ID NO: 15 5 -TC AG AG AGTAAACGGCC AC A-3

WUS SEQ ID NO: 17 5 -C ATC AT AGAGAT AAACGGTTGTC A-3

CLV3 SEQ ID NO: 19 5-GAAAATCATGAGATATAATAGTGC-3

UBQ10 SEQ ID NO:21 5 -CGACTTGC ATTAGAAAGAAAG AG ATAAC AGG-3 Hormone treatments and hormone analyses

The effects of hormone applications on the expression of Brassica genes were evaluated exactly as described by Liu et al. (2008). Briefly, ten day-old Brassica napus seedlings grown on vermiculite were collected and their roots were immersed in solutions containing difference levels of plant hormones: IAA (30 μΜ), ABA (100 μΜ), BAP (100 μΜ), and GA3 (100 μΜ). Shoots of the seedlings, without the cotyledons, were harvested for RNA extraction at 0, 1, 3, and 6h after hormone treatment. Ten day-old Brassica napus seedlings grown on vermiculite were collected and their roots were immersed in solutions containing difference levels of plant hormones: IAA (30 μΜ), ABA (100 μΜ), BAP (100 μΜ), and GA3 (100 μΜ). Shoots of the seedlings, without the cotyledons, were harvested for RNA extraction at 0, 1 , 3, and 6h after hormone treatment.

For hormonal analyses, 3 week-old shoots from A abidopsis wild type plants and plants over-expressing BoSTM (line 5) and BnCLVl (line 12) were collected and lyophilized. The plant hormone analysis was performed at the National Research Council of Canada (Plant Biotechnology Institute, Saskatoon, SK, CA) by high performance liquid chromatography electrospray tandem mass spectrometry (HPLC- ES-MS/MS) using deuterated internal standards, as described in Chiwocha et al. (2003) and Kong et al. (2008). Sensitivities to ABA and 2,4-D were assayed by performing germination and root growth tests as outlined in Weigel and Glazebrook (2002). For ABA sensitivity assays, seeds were germinated on ½ strength MS medium supplemented with 1 % sucrose and different levels of ABA. The percentage of germinating seeds was scored at different days in culture using a dissecting microspore. For 2,4-D sensitivity assays, seeds were germinated on ½ strength MS medium supplemented 1% sucrose. Established seedlings were then transferred to a similar medium supplemented with different levels of 2,4D, and root growth was measured over time.

Immunolocalization of IAA

Fully developed MDEs were first pre- fixed in freshly prepared 4% aqueous 1- ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride (EDAC; Sigma- Aldrich Canada Ltd., Oakville, ON, CA) at 4°C for 2h, and then post-fixed in FAA (10% formalin, 5% acetic acid, and 50% ethanol) overnight at 4° C. The fixed tissue was dehydrated in a ethanol series, embedded in paraplast, sectioned (10 μηι), and deparaffinised in xylene. The sections were incubated in blocking solution ([10 mM phosphate buffer pH 7 (PBS), 0.1% Tween 20, 1.5% glycine, and 5% BSA) at room temperature for 1 hour. 150 μΐ of monoclonal primary IAA-antibodies (1 mg/ml, Sigma- Aldrich Canada Ltd., Oakville, ON, CA) diluted 1 :200 in 10 mM PBS containing 0.8% BSA were applied to the sections and incubated in a high humidity chamber for 4 hours at room temperature. The slides were washed first in 10 mM PBS containing 0.88 g/L NaCl, 0.1 % Tween 20, and 0.8 % BSA for 5 minutes, and then in 10 mM PBS with 0.8% BSA for 5 minutes in order to remove any excess of Tween 20. The slides were incubated in 200 μΐ secondary antibodies i.e., 1 mg/ml anti- mouse IgG alkaline phosphates conjugate (Promega Corp., Madison, WI, USA), following the manufacturer's instruction overnight in a high humidity chamber, washed 2 times in 10 mM PBS containing 0.88 g/L NaCl, 0.1 % Tween 20, and 0.8 % BSA for 10 minutes, and then incubated in water for 15 min to remove the excess of secondary antibodies. Samples were stained using 250 μΐ Western blue (Promega Corp., Madison, WI, USA) for 40 minutes.

Pharmacological studies The SAM in MDEs was enhanced by inclusions of DL-buthionine sulfoximine

(BSO), which produced better organized SAMs with a zygotic-like appearance. These meristems regenerated shoots at high frequency denoting an improved functionality Formation of the SAM was compromised in MDEs cultured with 2,3,5-triiodobenzoic acid (TIBA). This compound blocks the basipetal flow of auxin resulting in an accumulation of auxin at the tip of the cotyledons and at the SAM (Figure 2). TIBA- treated embryos developed non-functional SAMs and exhibited a characteristic fusion of the cotyledons at their base. These abnormalities were reverted if 2-(p- chlorophenoxy)-2-methylpropionic acid (PCIB), an auxin antagonist, was added with TIBA. The expression levels of all Brassica genes were repressed in MDEs developed in the presence of TIBA (Figures 3A-3D), whereas BSO increased the transcript levels of BnSTM, BnZLL-l and BnZLL-2. Intermediate levels of expression were detected in control embryos and embryos cultured with TIBA+PCIB. The similar expression profiles observed for BnZLL-1 and 2 suggest that these two genes might have a similar function during microspore-derived embryogenesis (Figures 3A- 3D).

In situ hybridization studies revealed that the four B. napus genes are expressed in the SAM during in vitro embryogenesis. In untreated MDEs, three distinct BnSTM localization patterns could be detected. In some MDEs, BnSTM exhibited a "zygotic-like" expression, which was restricted to the central region of the apical pole (Figures 2(1) - 2(4)). In other embryos, transcripts of this gene could be detected in unusual domains, such as the peripheral region of the apical pole (Figures 4(5) - 4(7)), or in separate pockets of cells (Figures 4(8) - 4(11)). In the improved SAMs of BSO-treated MDEs, the localization domain of BnSTM was always central and encompassed a larger group of cells, especially in heart-shaped embryos (Figures 4(12) - 4(14)). In the presence of TIBA, BnSTM expression was restricted to the apical pole in immature embryos (Figures 4(15) - 4(16)) but was lost in cotyledonary embryos with an abnormal SAM (Figure 4(17)). Both structural and expression abnormalities induced by TIBA were reversed in TIBA+PCIB treated MDEs (Figures 4(18) - 4(19)). No signal was detected with a sense probe (Figure 4(20)).

Expression of BnCLVl was first visible in the sub-apical cells of globular MDEs and then extended to a larger domain in middle and late cotyledonary embryos (Figure 5(1) - 5(3)). In the presence of BSO, a larger group of sub-apical cells expressed BnCLVl in globular embryos (Figure 5(4)). The expression in the sub- apical domain was also retained in fully developed BSO-treated MDEs (Figure 5(5)). TIBA reduced the expression domain of BnCLVl in globular embryos, and to a certain extent in cotyledonary embryos (Figure 5(6) - 5(7)). Embryos treated with TIBA+PCIB exhibited a control-like expression pattern (image not shown) whereas no signal was observed in tissue hybridized with sense probe (Figure 5(8)).

The combined expression of BnZLL-1 and BnZLL-2 (the high degree of similarity in the sequences of the two cDNAs did not allow for the design of specific probes) was first detected in the vascular tissue of developing MDEs, and then extended to the SAM of cotyledonary embryos (Figure 6( 1 ) - 6(5)). Within the SAM the signal was often delocalized to the peripheral region (Figure 6(4)). Inclusions of BSO enlarged the localization domain of BnZLL-1 and BnZLL-2 throughout the SAM (Figure 6(6)), whereas no signal was observed in the SAMs of TIBA-treated embryos (Figure 6(7)). The signal was recovered if PCIB was applied with TIB A (Figure 6(8)). Specificity of the hybridization was demonstrated using a sense riboprobe (Figure 6(9)). Phenotypic characterization of Arabidopsis plants ectopically expressing Brassica genes

Given the very low efficiency of Brassica napus Topas (DH4079 embryogenic line) transformation, the effects of ectopic expression of the four Brassica genes during development were analysed in Arabidopsis. During zygotic embryo development the ectopic expression of the Brassica napus BnCLVl resulted in the formation of small SAMs (which also accumulated storage products), whereas an opposite effect was observed with the introduction of STM. Compared to WT, the SAM of the BnSTM over-expressing embryos was dome-shaped and often composed of a larger cluster of meristematic cells (Figures 7(1) - 7(3)). No effects on meristem size or shape were noticed in plants over-expressing the BnZLL-1 and BnZLL-2, which showed a WT phenotype. These structural differences resulted in alterations in the rate of meristem reactivation. Overall SAM reactivation at germination was delayed by the introduction of BnCLVl and accelerated by the over-expression of BnSTM (Figure 8). Similar phenotypic deviations from WT were observed among all the Brassica

STM over-expressing lines (35 S: . BnSTM, BoSTM, and BrSTM), although the most severe phenotypes were obtained in BoSTM plants. In lines with the highest BoSTM expression, such as line 33 (Figure 1) ectopic shoots (Figure 9(1)) and small lobed leaflets (Figure 9(2)) emerged from the adaxal side of the rosette leaves. These lines were sterile and eliminated form further characterization. The remaining lines with lower levels of STM expression produced small lobed and serrated leaves (Figure 9(3)), larger inflorescences characterized by more flowers (Figures 9(4) - 97(5)) and curved siliques (Figure 9(6)). Increased number of siliques and flowers, as well as reduced plant height were observed in almost all STM over-expressors (Table 2). Table 2: Phenotypic characterization of lines over-expressing the Brassica napus (Bn), B. rapa (Br), and B. oleracea (Bo) STM.

H: plant height

B: branches

S: siliques

CL: cauline leaves

RL: rosette leaves

F: flowers

A reduction in hypocotyl elongation and termination of the SAM (Fig. 9(7)) were observed in lines with higher expression levels of BnCLVl. Overall, the introduction of BnCLVl resulted in a reduction in the number of siliques and cauline leaves (Table 3). No major morphological differences were observed in

Arabidopsis lines ectopically expressing BnZLL-1 and BnZLL-2, the only exception being a reduced number of branches (Table 3).

Phenotypic characterization of lines over-expressing the Brassica napus CLV1, ZLL-1 and ZLL-2 genes, expressed as a percentage of controls.

H: plant height

B: branches

S: siliques

CL: cauline leaves

RL: rosette leaves

F: flowers Expression analysis of SAM-related genes in the transformed lines

The expression level of several SAM-related genes was measured in the shoot of some transformed Arabidopsis seedlings after 1 week of germination. The introduction of Brassica STM activated the expression of KNAT6, WUSCHEL (WUS) and CUPSHAPED COTYLEDON- 1 (CUC 1) (Figures 10A - IOC) whereas an opposite trend was observed in the BnCL VI over-expressing line (Figure 10D). A less pronounced increase in expression of the three genes was also obtained in lines ectopically expressing BnZLL-l and BnZLL-2 (Figures 10A - IOC). The expression of CLA VATA 3 (CLV3) was repressed by the introduction of the Brassica STM, whereas it increased in lines over-expressing BnCLVl and BnZLL-2. Effects of hormone treatments on the expression of the Brassica genes

The endogenous levels of BnSTM, BnCLVl, BnZLL-1 and BnZLL-2 were measured in Brassica seedlings treated with different hormones. Compared to control (untreated tissue), applications of the cytokinin BAP and the auxin IAA increased the level of BnSTM after a few hours (Figures 11 A - 1 IB). An opposite expression profile was measured in the presence of ABA and GA3 (Figures 1 1C - 1 ID).

The expression level of BnCLVl increased in both control seedlings and seedlings treated with BAP and IAA (Figures 1 IE - 1 IF). The transcript levels of this gene were induced by ABA (Figure 1 1G) and repressed by GA3 (Figure 11H) applications.

Hormone treatments did not affect the expression of BnZLL-1 and BnZLL-2.

Endogenous hormone analysis of the transformed Arabidopsis plants

For hormone analysis three Arabidopsis lines were selected: WT, BoSTM (L5), and BnCLVl (LI 2); with the last two exhibiting the most severe phenotypes among all the transformed lines. No ZLL over-expressing lines were selected as they did not show any major phenotypic deviation from WT plants. Compared to WT, the introduction of the Brassica STM increased the endogenous levels of the cytokinins trans-zeatin-O-glucoside (t-ZOG), cis-zeatin-O-glucoside (c-ZOG), trans-zeatin riboside (t-ZR), and isopentenyladenosine (iPA). Undetectable or low levels of cytokinins were measured in the shoots of the 35S: .BnCLVl line (Table 4). Zeatin and dehydrozeatin were below detectable levels in all the lines analysed.

Profile of auxin metabolites showed a significant increased level of indole-3- acetic acid (IAA) in the 35S::BoSTM line. IAA-alanine (IAA- Ala) was not detected in the three lines whereas very low levels of IAA-aspartic acid (IAA- Asp) and IAA- glutamic acid (IAA-Glu) were measured.

Compared to the other lines analysed, the line over-expressing the Brassica STM had lower endogenous levels of abscisic acid (ABA), dehydrophaseic acid (DP A), ABA-glucose ester (ABA-GE), and phaseic acid (PA). The highest concentration of DPA was measured in the 35S::BnCLVl line. 7 '-hydroxy- ABA (7'- OH-ABA) was below detectable levels (Table 4).

Table 4: Hormone profiles of Arabidopsis shoots ectopically expressing Brassica oleracea SHOOTMERISTEMLESS (BoSTM) gene and Brassica napus CLA VATA1 (BnCLVl) gene.7

Cytokinins tZOG cZOG t-ZR c-ZR 2iP iPA

WT 28+1 119+2 9+1 11+0 20+1 23+1

BoSTM 45+4* 274+9* 94+1 * 12+1 20+0 60+1*

BnCLVl ND ND < 5+0* < 10+1 *

Auxins IAA IAA-Ala IAA-Asp lAA-Glu

WT 147+13 ND 11+1 ND

BoSTM 254+22* ND 14+0 14±1 *

BnCLVl 138±17 ND < ND

ABA metabolism ABA DPA ABA-GE PA 7ΌΗ-ΑΒΑ

WT 102+2 545+7 157+12 162+4 ND

BoSTM 55+1 * 342+8* 95±18* 75+5* ND

BnCLVl 109+2 745+62 * 83+2* 152+13 ND tZOG: trans-zeatin-O-glucoside cZOG: cis-zeatin-O-glucoside tZR: trans-zeatin riboside cZR: cis-zeatin riboside

2iP: isopentenyl adenonine iPA: isopentenyl adenosine

IAA: indole 3-acetic acid ABA: abscisic acid

IAA-Ala: indole 3-acetic acid alanineDPA: DPA: dehydrophaseic acid

IAA-Asp: indole 3-acetic acid aspartate ABA-GE:abscisic acid glucose ester

IAA-Glu: indole 3-acetic acid glutamic acid PA: phaseic acid

7'-OH-ABA: 7'-hydroxyl abscisic acid

< : indicates that the signals were below the limit for quanitification

ND : not detected Hormone sensitivity assays

Sensitivity to ABA was assayed using a seed germination test. A similar inhibition profile with increasing concentrations of ABA was observed for WT seeds, as well as seeds obtained from 35S::BnCLVl, BnZLL-1 and -2 lines (Figure 12A). Seeds over-expressing the Brassica STM showed a reduced sensitivity to the inhibitory effect of ABA and a significant percentage of germination was still measured at those ABA levels precluding germination in WT seeds.

Root growth assays were performed to test the tissue sensitivity to 2,4-D. Compared to WT, the rate of root growth was generally higher in the 35S::BnCLVl and BnZLL-2 lines. The introduction of the Brassica STM increased sensitivity to high levels of 2,4-D, as estimated by the reduced root growth (Figure 12B).

The Brassica genes are reliable markers of SAM functionality

Meristem functionality in B. napus MDEs, estimated by the ability of the SAMs to convert, i.e. produce viable shoots at germination, can be easily altered in culture. In our system, about 40% of the MDEs produce functional meristems able to reactivate upon transfer onto germination medium. It appears that BnSTM, BnCLVl, and BnZLL-1 and BnZLL-2 are implicated in meristem function during MDE development. In control (untreated) MDEs, BnSTM signal is first activated in a pocket of sub-apical cells in globular-stage embryos where it demarks the future site of the SAM and later extended to the entire apical pole of the embryos (Figs. 4(1)- 4(4)). The inability of many (about 60%) untreated B. napus MDEs to convert correlates with the early (prior to the formation of a meristem) miss-expression of BnSTM either in the peripheral region (Figs. 4(5)-4(7)), or in two separate domains of the SAM (Figs. 4*8)-4(l 1)). A deviation in expression pattern in control MDEs was also observed for BnZLL-1 and BnZLL-2 which are first expressed in the vascular tissue and later in the SAM (Fig. 6). In many MDEs the expression domain of

BnZLL-1 and BnZLL-2 in the SAM is restricted to the peripheral zone (Figure 6(4)).

The BSO-improvement of meristem functionality correlates to the increased transcript levels and enlarged localization patterns of BnSTM, BnZLL-1 andBnZLL -2 (Figures 4 and 6). Of interest, the enlarged BnSTM signal in the apical pole of MDEs cultured with BSO (Figs. 4(12)-4(13)) became apparent before any visible SAM, thereby suggesting that the BnSTM localization pattern in early embryos can be used to estimate the quality of the meristem. The expression of all Brassica genes is compromised in non-functional

(TIBA-treated) MDEs (Figure 3). The SAMs of these embryos do not express BnSTM, BnZLL-1 and -2, while the signal of BnCLVl is reduced. Poor meristem function and repression of gene expression can be attributed to the TIBA-induced accumulation of IAA within the apical regions of the MDEs, especially in the SAM (Figure 2).

Based on this information, it appears that the Brassica genes might be implicated in meristem functionality and can be utilized as "SAM-markers ^" to estimate the quality of the shoot meristems of cultured embyros.

Phenotypic characterization of Arabidopsis ectopically expressing the Brassica genes Ectopic expression of the Brassica STM and CLV1 influence the rate of SAM reactivation at germination, with the former accelerating meristem conversion and the latter having an opposite effect (Fig. 8). These results can be possibly attributed to the different organization of the embryonic SAMs observed in the transformed line (Fig. 7). The role of these two genes during SAM maintenance is well established but their influence on meristem reactivation has not been documented. If the two Brassica genes fulfill similar roles to those assigned to their Arabidopsis orthologs, with STM increasing the domain of meristematic cells and CLV\ repressing the function of STM, then it follows that the rate of meristem reactivation relies on the pool of undifferentiated cells present within the apical pole of the embryos. The accumulation of storage products, a sign of cellular differentiation, in the meristematic cells of 35S::CLV1 embryos (Fig. 7(2)) and the termination of the SAM a few days after germination (Fig. 9(7)) support this observation.

The STM-acceleration of shoot meristem conversion at germination may be due to the early activation of several genes involved in SAM activity, such as KNAT6, WUS, and CUC1 (Fig. 10). KNAT 6, a KNOTTED-like gene, is a downstream effector of the STM pathway required for the promotion of menstematic proliferation. Inactivation of KNAT6 has been shown by others to abolish the residual meristematic activity of weak stm-2 plants. An additional function of this gene is to regulate lateral organ separation, in an integrated pathway including CUC1, which delimits the boundaries of primordia produced in the peripheral zone of the SAM. Fusions of lateral organs often occur in cucl mutants. A third gene induced by the introduction of the Brassica STM is WUSCHEL (WUS), which encodes a WOX-class homeodomain transcription factor required for maintaining the meristematic cells of the central zone in an "indeterminate" state. Mutations of WUS result in the differentiation and incorporation of the cells occupying the central domain of the SAM into leaf primordia, leading to the abortion of the meristem. These three regulators of meristem activity are repressed in BnCLVl lines (Fig. 10) characterized by a delayed conversion of the SAM at germination. The up-regulation of CLV3 in plants ectopically expressing BnCLVl further suggests that the CLAVATA pathway (requiring both CLV1 and CLV3) is highly operative. This would result in the promotion of cellular differentiation (leading to the termination of the SAM, Figs. 7(2)) and depletion of the stem cells through the repression of WUS (Fig. 10). This is in contrast to the STM over-expressors characterized by lower CLV3 expression (Fig. 10). The up-regulation of several genes involved in SAM activity in Arabidopsis lines ectopically expressing the Brassica STM also correlates with the formation of adventitious shoots, abnormal leaf morphology, and increased production of reproductive organs (Fig. 9 and Table 2). The abnormalities in leaf morphopogy observed in our study (Fig. 9) are consistent with the over-expression of other KNOX- 1 genes observed in other systems and can be attributed to meristematic growth originating from the margins of the leaves.

A novel trait linked to the introduction of STM is the enlarged inflorescence size, resulting in a higher number of flowers and siliques (Fig. 9 and Table 2). This phenotype can also be the result of the increased meristematic activity encouraging floral meristem formation. Interaction between the Brassica genes and plant growth regulators

The studies disclosed herein show a positive correlation between STM expression and cytokinins. While applications of BAP activate BnSTM (Fig. 1 1), over-expression of the Brassica STM induces the accumulation of both zeatin-type cytokinins, i.e. c- and t-ZOG, as well as tZR, and iP-type cytokinins, i.e. iPA (Table 4). The concentrations of free zeatin and dehydrozeatin were below detectable levels, implying a fast turnover to ZR. The STM-mediated increase in cytokinins levels might be exercised through the activation of adenylate isopentenyltransferases, which are the first enzymes involved in the biosynthesis of cytokinins in both the methylerythritol phosphate and mevalonate pathways. The introduction of BoSTM in Arabidopsis tissue induces the transcription of adenylate isopentenyltransferase 7. This intimate interaction between STM and cytokinins, which was not observed for CLV1, might explain some of the phenotypic deviations noticed in the transformed plants, including the formation of larger SAMs (Figure 5A), and the production of ectopic meristems (Fig. 9).

The poor architecture of the 35S::CLV1 SAMs in developing embryos and their termination during post-embryonic growth may be caused by the extremely low endogenous levels of cytokinins (Table 4). The lines over-expressing BnCLVl which, besides having low cytokinin levels, may also have reduced WUS expression (Fig. 10 and Table 4).

The GA3 repression on the transcription of STM and CLV1 suggests an interaction between SAM-related genes and GAs. Several studies have shown that proper SAM functions require low levels of GAs and this condition can be attributed to STM (and other KNOXl genes) suppressing the transcription of GA20 oxidase (Hay et al, 2002). All the GAs measured by HPLC-ES-MS/MS, i.e. GA1, 3, 4, 7, 8, 9, 19, 20, 24, 29, 34, 44, 51 and 53 were below detectable levels among the three lines. However, a localized effect of SAM-related genes on GAs cannot be excluded given the large size of the tissue analysed.

The high levels of free IAA (Table 4) and the increased sensitivity to supplied 2,4-D (Fig. 8) exhibited by lines over-expressing the Brassica STM suggests a possible role of auxin in the STM phenotype. During in vitro morphogenesis, auxin might be required for the acquisition of embryogenic fate through the induction of WUS. It is not clear whether a similar interaction occurs in vivo with auxin conferring stem fate to the cells of the organizing center through the activation of WUS. This intriguing possibility cannot be excluded given the positive correlation between high IAA content and elevated WUS expression observed in this study, and the mechanistic parallels between the organizing center in the SAM and the quiescent center in the root meristem. The root quiescent center is specified by the expression of WUS-\ike genes and this specification requires an auxin maximum ensured by its basipetal transport. The lobed leaves and the formation of ectopic meristems observed in the lines over-expressing the Brassica STM (Fig. 9) can be a direct consequence of high auxin levels which maintain the cells within the blastozone of the leaves in a proliferative state.

Compared to the 35S::CLV1 line, the biosynthesis of ABA, as well as its degradation to PA and DP A via the 8'hydroxylation pathway were lower in plants over-expressing STM (Table 4). These plants also exhibited a lower sensitivity to ABA (Fig. 8). The lack of available information relating STM to ABA does not allow any speculation on these results; however, the low endogenous levels and sensitivity to ABA might be responsible for the quick reactivation of the apical meristems at germination.

Example 2: Over-expression of the BnSTM gene in Brassica napus

Brassica transformation with BnSTM in sense orientation was performed following the method taught by Bhalla and Singh (2008, Agrobacterium mediated transformation of Brassica napus and Brassica oleraceae. Nature Protocols 3:181- 189) with some modifications. Briefly, surface sterilized seeds were germinated for 5 days on ½ MS-B5 medium supplemented with 1% sucrose. Hypocotyls (2-3 mm) were inoculated on co-cultivation medium (MS-B5 medium supplemented with 3% sucrose and 2mg/L BA) for 4 days, and then incubated with the same Agrobacterium strain used for Arabidopsis transformation, harboring the pK2GW7 vector for 2 min. Following a 4 day co-cultivation period, the explants were plated on shoot induction medium (MS-B5 supplemented with 5mg/l AgN03, 2mg/l BA, 3% sucrose, 20 mg/1 timentin and 50 mg/1 kanamycin) for 8 weeks. The emerging shoots were first transferred on elongation medium (MS-B5 supplemented with 3% sucrose, 0.1 mg/1 GA3, and 1 mg/1 BA) for 4 weeks and then placed on rooting medium (MS-B5 supplemented with 1% sucrose, 2 mg/1 NAA) for 4 weeks. Rooted shoots were transferred onto soil to generate fully mature plants (F0). Seeds from F0 plants were germinated on ½ MS basal medium with 1% sucrose and 50 mg/1 kanamicin and the resulting Fl generation was screened for the presence of the transgene by RT-PCR using a forward 35S promoter primer (SEQ ID NO:22; 5'- TGGACCCCCACCCACGAG-3') and a reverse gene specific primer (SEQ ID NO:23; ' -GC ACC AGAGGAAGGAG AAC A-3 ' ) . Microspores harvested from the positive Fl plants were cultured to produce haploid MDEs which were germinated on ½ MS medium supplemented with 1% sucrose and 70 mg/1 kanamycin for 4 weeks. Thehaploid seedling were then treated with 0.2% colchicine for 6 h in order to generate homozygous (double haploid) plants. After checking the ploidy level by chromosome counting and confirming the presence of the transgene by qRT-PCR, selected plants were used as a source of seeds to generate the next (F2) generation. The expression level of BnSTM in these plants was measured by quantitative RT- PCR.

The transformed B. napus plants over-expressing BnSTM produced 20% more siliques than were produced by the wild type control plants. In conclusion, this work shows that the expression levels and localization patterns of the Brassica STM, CLVl, and ZLL correlate with the quality of the SAM established during embryo development, and can be utilized to estimate meristem functionality. The ectopic expression of these genes induces profound phenotypic changes during both embryonic and post-embryonic development. While some of these changes are similar to those produced by the ectopic expression of their respective Arabidopsis orthologs, others are unique and might be the consequence of profound alterations in endogenous hormone levels, especially cytokinins, auxins, and ABA.