Patent Application: US-59141805-A

Abstract:
this invention relates to a method for modifying cell proliferation in a plant which comprises the step of transforming a plant , or plant propagating material , with a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within the integuments and / or seed coat and at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulating cell proliferation , whereby on expression of that nucleic acid sequence cell proliferation is modified . the invention also relates to a plant which includes a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within integuments and / or seed coat an at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulated cell proliferation , whereby on expression of that nucleic acid sequence cell proliferation is modified . the invention also relates to reproducible plant material including a nucleic acid molecule comprising at least one regulatory sequence capable of directing expression within integuments and / or seed coat and at least one nucleic acid sequence whose expression or transcription product is capable of directly or indirectly modulated cell proliferation , whereby expression of that nucleic acid sequence cell proliferation is modified . according to another aspect of the invention , there is provided a method for modifying cell proliferation in a plant which comprises the step of modulating the response of the plant to an auxin in which cell proliferation is modified to produce larger or smaller seeds than wild - type .

Description:
methods and products in accordance with the present invention will now be described with reference to the following examples , which should not be construed as in any way limiting the invention . bj36 , bj40 , bj60 ( gift of bart janssen , horticultural & amp ; food research institute of new zealand ) plant transformation protocols are based on clough and bent ( 1998 ) for arabidopsis thaliana and moloney et al . ( 1989 ) for brassica . protein predictions and sequence alignments are carried out with genedoc software version 2 . 6 . 001 ( nicholas and nicholas , 1997 ). we identified the mutant , megaintegumenta - 1 ( mnt - 1 ), in a screen for large seeds yielded by a population of ems ( ethyl methanesulfonate )- mutagenized arabidopsis thaliana in the col - 3 - accession . mature seeds produced by a seed parent homozygous for the mnt - 1 mutation are larger and more pointed than wild - type , with extra cells in the seed coat , and contain larger embryos ( fig1 a ). specifically , fig1 a shows that mnt - 1 mutants produce larger seeds with more cells in the seed coat ( counts are for ii1 , the outer layer of the inner integument ). seeds collected from self - pollinated mnt - 1 mutant plants are up to twice the weight of wild - type col - 3 seeds ( table 1a ). seed weights in μg from mnt - 1 and w . t . col - 3 crosses , self seed , however , mnt - 1 mutant plants are self - sterile until late in development due to floral abnormalities ( see below ), raising the possibility that the mutant produces large seeds because there are few seeds requiring maternal resources . therefore we also conducted controlled pollinations in which only three siliques ( seed pods ) were allowed to set seed per plant , for both mnt - 1 and wild - type ( table 1b ). seed weights in μg from mnt - 1 and w . t . col - 3 crosses , manual ttest [ mnt × mnt ] vs [ w . t . × w . t . ]: p & lt ; 0 . 000 , significant ttest [ mnt × mnt ] vs [ mnt × w . t . ]: p & gt ; 0 . 2 , not significant ttest [ w . t . × w . t .] vs [ w . t . × mnt ]: p & gt ; 0 . 9 , not significant this treatment raised the mean weight of wild - type seeds by 90 % and mnt - 1 seeds by 29 %, indicating that low seed number is a component of large seed size in mnt - 1 mutants but that the mnt - 1 mutation also has a significant effect . on average mnt - 1 seeds weighed 26 % more than wild - type when only three siliques per plant set seed ; the difference in weights was significant at p & lt ; 0 . 000 . we also investigated whether occasional low seed set within individual mnt - 1 siliques might raise seed weight ; however a scatter plot ( fig1 b ) of mean seed weight of each pod vs number of seeds per pod following controlled pollinations in mnt - 1 ( data from table 1b ) shows no correlation . we also compared the weight of seeds produced by mnt heterozygotes with the weight of seeds produced by wild - type plants ( table 1c ). we generated the heterozygotes through crosses in both directions , i . e . [ w . t . x mnt - 1 ] ( designated [ wxm ]) and [ mnt - 1 × w . t .] ( designated [ mxw ]). we found that the weights of seeds from w . t . and mnt - 1 heterozygous plants were significantly different ( t - test , ho w . t .= mnt - 1 heterozygous , p = 0 . 0002 ). seed weights in μg from w . t . col - 3 and mnt - 1 heterozygous plants , we also conducted two further experiments to compare the weights of seeds from ( a ) wild - type plants , ( b ) mnt - 1 homozygotes , and ( c ) mnt - 1 heterozygotes under conditions of restricted pollination . in the first experiment , six siliques on the primary shoot were pollinated and all other siliques on the primary shoot were removed ; but all secondary shoots were allowed to set self - seed ( table 1d ). in the second experiment , only six siliques ; on the primary shoot were pollinated and all other siliques on the plant were removed ( table 1e ). in both experiments we carried out manual pollinations on six siliques per plant to enable young mnt - 1 homozygous mutant plants to set seed . t - test : h 0 w . t . = mnt - 1 homozygous , p & lt ; 0 . 0000 , difference is significant t - test : h 0 w . t . = mnt - 1 heterozygous , p & lt ; 0 . 0000 , difference is significant table 1e seed weights in μg from w . t . col - 3 , mnt - 1 homozygous , and mnt - 1 heterozygous plants , 6 siliques pollinated on primary shoot , secondary shoots removed w . t . mnt homozygous mnt heterozygous plant 1 33 . 0 ( n = 307 ) 38 . 7 ( n = 274 ) 37 . 1 ( n = 53 ) [ w × m ] plant 2 32 . 2 ( 222 ) 37 . 5 ( 221 ) 35 . 1 ( 300 ) plant 3 35 . 1 ( 74 ) 41 . 1 ( 226 ) 37 . 7 ( 347 ) plant 4 35 . 0 ( 252 ) 41 . 0 ( 302 ) 35 . 6 ( 110 ) plant 5 34 . 8 ( 230 ) 38 . 5 ( 205 ) 38 . 1 ( 195 ) plant 6 40 . 9 ( 193 ) [ m × w ] plant 7 37 . 8 ( 245 ) plant 8 36 . 7 ( 280 ) plant 9 38 . 1 ( 210 ) plant 10 39 . 9 ( 222 ) mean seed 34 . 0 ( 1085 ) 39 . 4 ( 1228 ) 37 . 7 ( 2155 ) weight range 23 . 2 to 35 . 1 37 . 5 to 41 . 1 35 . 1 to 40 . 9 standard 0 . 6 0 . 7 1 . 8 error n = number of seeds weighed from each plant t - test : ho w . t . = mnt - 1 homozygous , p = 0 . 0004 , difference is significant t - test : ho w . t . = mnt - 1 heterozygous , p = 0 . 0013 , difference is significant mnt - 1 homozygotes and mnt - 1 heterozygotes produced heavier seeds than wild - type plants in both experiments , and the difference in weight was significant in all cases at p & lt ; 0 . 002 . when secondary shoots were allowed to set seed , seeds from mnt - 1 homozygotes were on average 47 % heavier than seeds from wild - type plants , and seeds from mnt - 1 heterozygotes were on average 18 % heavier than seeds from wild - type plants . when secondary shoots were removed , so that only six siliques set on each plant regardless of genotype , seeds from mnt - 1 homozygotes weighed 16 % more than wild - type , and seeds from mnt - 1 heterozygotes weighed 11 % more than wild - type . the mnt - 1 mutation has a maternal effect on seed size . that is , an mnt - 1 homozygous mutant seed parent yields large seeds regardless of whether it is pollinated by an mnt - 1 or wild - type plant , while a wild - type parent yields normal seeds even if pollinated by an mnt - 1 plant ( fig1 c ). in fig1 c seeds produced by mnt - 1 seed parents are shown on top and seeds from wild - type seed parents are below . h = fertilization products ( embryo and endosperm ) are heterozygous for the mnt - 1 mutation . this shows that seed size in mnt - 1 mutants depends on the genotype of the seed parent , not the fertilization products . this is also shown by the lack of a significant difference between seed weights from [ mnt - 1 × mnt - 1 ] and [ mnt - 1 × w . t ] seeds , and between [ w . t .× w . t .] and [ w . t .× mnt - 1 ] seeds ( table 1b ). the primary difference between mnt - 1 and wild - type seeds is that the mutant seeds contain more cells in the seed coat . comparison of ovule development in mnt - 1 and wild - type plants shows that mnt - 1 ovules are of normal size and morphology until they are near maturity , at which time we observe that both the inner and outer integuments of mnt - 1 ovules are significantly longer than in wild - type , primarily due to a significantly greater number of cells ( fig2 ). in relation to the results depicted in fig2 , in arabidopsis thaliana and other members of the brassicacea most cell division and expansion occurs in the integuments on the abaxial side of the ovule ( marked on wild - type ovule in fig2 a ). similarly , the nucellus in rice is enveloped by the abaxial inner integument ( lopez - dee et al ., 1999 ). in arabidopsis , ii1 , ii1 ′, and ii2 are the three cell layers of the inner integument and oil and oi2 are the two layers of the outer integument . the cells of layer ii1 ′, which does not completely span the embryo sac , significantly expand in width after fertilization as part of seed growth ( beeckman et al ., 2000 ). mnt - 1 ovules have longer integuments with extra cells and in some cases an extra layer ( arrow ), as well as a larger seed cavity ( fig2 a ). c = the ‘ curving zone ’ of the abaxial outer integument ( the region overlying ii1 ′; beeckman et al ., 2000 ), m = the ‘ micropylar zone ’, regions delimited with black bars ( fig2 a ). measurements shown in fig2 b were taken for the abaxial integuments only . layers ii1 ′, ii1 , and the curving zone of oi2 are longer in mnt - 1 mutant ovules , almost exclusively due to greater cell number . mean cell length is greater in the micropylar zone of mnt - 1 ovules but smaller or not significantly different in the oi2 curving zone and the other integument layers measured . there is no difference in mean width between mnt - 1 and wild - type cells of layer ii1 ′. the peripheral endosperm in mnt - 1 mutant seeds also generates more nuclei than in wild - type seeds . the mean number of peripheral endosperm nuclei in mnt - 1 seeds at the heart stage is 1150 , compared with 550 for a wild - type col - 3 seed at a comparable stage ; see scott et al . ( 1998 ) for a description of endosperm morphology and the counting method . however , we consider there are two crucial differences between mnt - 1 mutant seeds and large seeds that show endosperm - led growth . first , the chalazal region of the endosperm , which becomes greatly enlarged in endosperm - led seeds ( e . g . seeds from interploidy crosses generating paternal excess , crosses where the dna of the seed parent is hypomethylated , or fis mutants ), is of roughly normal size in mnt - 1 mutants ( although the pinched shape of the chalazal pole of mnt - 1 seeds results in a longer and narrower chalazal endosperm ) ( fig3 ). we measured the maximum cross - sectional area of the chalazal cyst plus nodules at 6 dap , a stage at which differences are apparent between wild - type and paternalized endosperms ( scott et al ., 1998 ). mean areas were 2690 μm 2 (± s . e . m . 328 ) for wild - type seeds ( n = 4 ) and 2537 μm 2 (± 416 ) for mnt - 1 seeds ( n = 5 ), and there was no significant difference between the mutant and wild - type endosperms ( t - test , ho w . t .= mnt - 1 , p = 0 . 79 ). second , the size difference between mnt - 1 and wild - type seeds follows from differences existing before fertilization i . e . before the endosperm has been created . the overproliferation of peripheral endosperm may follow from the larger seed volume created by enlarged integuments / seed coat . we observe that seeds with enlarged endosperms and seeds with large seed coats have a feature in common : the seed cavity ( i . e . the space within the post - fertilization embryo sac ) is larger than normal , giving the embryo more space to grow ( fig4 a , 4 b ). specifically , endosperm - led seed growth is illustrated by interploidy crosses in the c24 accession of arabidopsis thaliana ( see also scott et al ., 1998 ). as shown in fig4 a extra paternal genomes produce seeds with a large cavity ( top left , 2x × 6x cross ), and ultimately large seeds with large embryos ( 2x × 4x cross , bottom left ). conversely , extra maternal genomes generate seeds with small cavities ( top right , 6x × 2x cross ), and ultimately small seeds with small embryos ( 4x × 2x cross , bottom right ). the control 2x × 2x cross is shown in the middle . in contrast in integument - led seed growth as illustrated in fig4 b the seeds also have a large seed cavity ( top left ) compared with wild - type ( top right ). mature seeds and embryos are compared below . this leads to our ‘ big bag ’ hypothesis , which states that seed and ultimately embryo size is set by the size of the seed cavity , which may be controlled by several factors including extent of endosperm proliferation and extent of integument / seed coat proliferation ( fig4 c ). it is well established in the literature that after fertilization in arabidopsis thaliana there is no further division in the seed coat , and growth occurs only by cell expansion ( leon - kloosterziel et al ., 1994 ; beeckman et al ., 2000 ; windsor et al ., 2000 ). obviously seeds with large endosperms must also have large seed coats ; however , in this case , the seed coat grows by cell expansion after fertilization . in seeds where large seed coat is considered the primary cause of seed enlargement ( integument - led seed growth ), the integument / seed coat contains extra cells , as observed in mnt - 1 mutants . the mnt - 1 mutation affects floral morphology as well as seed size . most flowers fail to open ; this is associated with a deviation from the normal ratio of sepal to petal length , so that the petals are shorter than the sepals . specifically , mnt mutant sepals are about 60 % longer than wild - type . this deviation is mainly due to overgrowth of the sepals caused by extra cell division , although under some conditions the petals also fail to expand normally . this characteristic may be commercially useful in some crop species . a smaller increase in sepal length may be sufficient to prevent flower opening whilst allowing self - fertilization . additionally , pollen is shed from the anthers on to the sides of the carpel rather than the stigma . this is associated with overgrowth of the gynoecium caused by extra cell division , although under some conditions the stamen filaments do not extend normally . the floral phenotypes result in sterility of plants unless manual pollination is carried out ( mnt - 1 homozygotes are female fertile , and the pollen that completes development is also fertile ). however the last few flowers produced by mnt - 1 mutants appear wild - type and these are self - fertile . germination frequency of mnt - 1 seeds is normal , and the seedlings are vigorous . mnt - 1 mutants have thick inflorescence stems compared with wild - type plants ( fig2 ). a comparison of primary inflorescence stems shows that mnt - 1 stems have a 20 % greater diameter than wild - type ( mean diameters mnt - 1 , 1 . 59 mm ± s . e . m . 0 . 04 ; w . t ., 1 . 32 mm :± 0 . 06 ; n = 6 for each ). transverse sections ( fig2 b ) show that cells are of normal size in mnt - 1 mutant stems but many more cells are formed . d . molecular characterization of the wild - type mnt gene and mnt - 1 mutant allele we mapped the mnt locus to a 60 . 9 kb region of chromosome 5 that was annotated by the arabidopsis information resource ( tair ) ( http :// www . arabidopsis . org ) to contain 17 genes . t - dna insertion lines with insertions in these genes generated by the salk institute genome analysis laboratory ( signal ( alonso et al ., 2003 ) ( http :// signal . salk . edu ) were obtained from the nottingham arabidopsis stock centre ( nasc ) ( http :// nasc . nott . ac . uk ). salk line no . 108995 ( nasc stock no . n608995 ), with an insertion in the coding region of the auxin response factor 2 ( arf2 ) gene , included a plant homozygous for the insertion with a similar phenotype to mnt - 1 mutants , including closed flowers and large seeds ( fig5 a - c ). genotypic scoring of segregants from the salk 108995 family , including one heterozygote and the homozygote , is shown in fig5 d . specifically in fig5 d top : scoring for presence of an insertion in the arf2 gene . primers used were 5 ′ tgg ttc acg tag tgg gcc atc g 3 ′, and 5 ′ gag tgg gtg gag tgt gtt tg 3 ′. lanes m and o show presence of the insertion . bottom : scoring for homozygous insertion mutants . primers used were 5 ′ gag tgg gtg gag tgt gtt tg 3 ′ and 5 ′ agt tgg ttt tcg ttt gag cat 3 ′. pcr conditions are set so that the gene will only amplify if there is no insertion : therefore pcr products will be amplified from dna extracted from wild - type plants and also those hemizygous for the insertion , but not homozygous plants . lane m shows no amplification , indicating this plant is homozygous for the insertion . an allelism test was conducted by crossing a seed parent homozygous for the mnt - 1 mutation with the salk 108995 homozygote as pollen parent . f1 progeny were hemizygous for the insertion ( fig5 e ) and had the mnt - 1 mutant phenotype ( fig5 f ), confirming that mnt is the arf2 gene . mnt / arf2 will be referred to as mnt in the remainder of this document . the mnt gene = at5g62000 , accession no . nm — 125593 . the genomic dna for mnt , including the coding region plus 4371 bases of 5 ′ and 525 bases of 3 ′ flanking region , is shown in seq id no . 1 . seq id no . 2 is the complete cdna , and seq id no . 3 , the predicted protein . arfs form part of the system for responding to auxin , a hormone known to be involved in many plant developmental processes including cell division and expansion ( stals and inzé , 2001 ; leyser , 2002 ). arfs are transcription factors that in general are not induced by auxin themselves but which regulate expression of auxin - inducible genes , such as members of the aux / iaa class ( liscum and reed , 2002 ). arfs have been shown to bind to auxin response elements ( ares ) containing the motif tgtctc in the promoters of auxin - inducible genes ( ulmasov et al ., 1999a ). twenty - two arfs predicted to be functional have been annotated in the arabidopsis thaliana genome ( hagen and guilfoyle , 2002 ). arfs contain two conserved domains — an n - terminal dna binding domain and a c - terminal dimerization domain — and a variable middle region . an arf may activate or repress transcription of its targets and this is thought to depend on the sequence of the middle region ( ulmasov et al ., 1999b ). evidence so far suggests that arf2 is likely to be a repressor ( tiwari et al ., 2003 ). we sequenced the coding region from genomic dna of the mnt - 1 allele plus 4371 bases of the 5 ′ and 525 bases of the 3 ′ flanking regions ( this genomic sequence is shown in seq id no . 4 ). a single base change with respect to the wild - type col - 3 sequence , from g to a , was identified at position 665 from translational start , at the end of intron 3 . this was predicted to affect splicing by changing the conserved 3 ′ splice site ( brown and simpson , 1998 ) from the consensus ag sequence to aa . we sequenced the first 837 bases of the mnt - 1 cdna from start of translation and confirmed that four bases are deleted from the beginning of exon 4 . the mnt - 1 cdna from translational start to stop , consisting of the 837 directly sequenced bases plus the remainder of the cdna coding region as predicted from the sequenced mnt - 1 genomic dna , is shown in seq id no . 5 . wild - type mnt and mutant mnt - 1 cdna sequences are aligned in fig6 . the predicted mnt - 1 protein ( seq id no . 6 ) has a frameshift from amino acid position 123 and an early stop codon at position 167 . wild - type mnt and mutant mnt - 1 predicted protein sequences are aligned in fig7 . the frameshift and early stop codon are both within the dna binding domain and therefore the mnt - 1 allele is likely to cause a complete loss of mnt function . value of mnt mutants in understanding and modifying growth of integuments / seed coat the mnt mutant seed phenotype demonstrates that there is a correlation between the size of integuments before fertilization and the size of the mature seed in arabidopsis thaliana ( fig1 , 2 ). due to the similarities in seed structure among even distantly related groups of flowering plants , this leads to the expectation that modification to integument / seed coat size in other species , and certainly in members of the brassicaceae such as brassica napus , will also result in changes to seed size . our knowledge of the mnt mutant phenotype and mnt gene sequence can be exploited in other species through tilling (‘ targeting induced local lesions in genomes ’). in this reverse genetics technique , chemically mutagenized populations are screened for presence of a point mutation in a nucleic acid sequence of interest ; this can be done as a high - throughput procedure and is applicable to many species ( till et al ., 2003 ). for example , tilling could be applied to the brassica napus or rice orthologues of mnt in order to modify seed size in these crop species . our knowledge that mnt - 1 heterozygotes are self - fertile and produce larger seeds than wild - type plants shows that a plant with reduced mnt function ( as in a heterozygote for an mnt mutation or in a plant which has been genetically modified in some way to achieve the same effect as conventional breeding ) will advantageously produce large seeds without a loss of fertility . knowledge of the mnt sequence in arabidopsis thaliana also allows us to search for orthologues in crop species as a necessary first step in targeted modification of the expression of the gene in these species . by way of example , we amplified the putative brassica napus orthologue ( bnarf2 ) of mnt using primers ( seq id no 7 , 8 ) based on the mnt sequence and on publicly available brassica oleracea sequence . the bnarf2 cdna was amplified from total rna isolated from seedlings of brassica napus var . westar . the bnarf2 cdna from translational start to stop is shown in seq id no . 9 and is aligned with arabidopsis thaliana mnt cdna in fig8 . the bnarf2 predicted protein ( seq id no . 10 ) has 85 % identity to arabidopsis thaliana mnt . a family of arfs has also been characterized in rice and one of these , osarf2 ( accession no . ab071293 ), is considered to be the orthologue of arabidopsis thaliana arf2 ( sato et al ., 2001 ). fig9 shows an alignment of the predicted protein sequences of mnt ( arabidopsis thaliana arf2 ), bnarf2 , and osarf2 . orthologues of mmt may be determined for other species using similar techniques . construction , transformation , and analysis of reporter vectors to show where integument / seed coat promoters are expressed in arabidopsis thaliana this is to test which promoters are suitable for driving integument / seed coat - specific or - preferred expression of nucleic acids such as mnt antisense or rnai constructs , or other genes modifying cell proliferation . diagrams of the bj60 , bj40 , pfgc5941 , part7 , and bj36 vectors used in the cloning strategies described in this and following examples are shown in fig1 . a reporter vector based on the promoter of the tt8 gene ( nesi et al ., 2000 ; at4g09820 , accession no . aj277509 ) is constructed as described below . a 1 . 7 kb fragment including the tt8 promoter is amplified by the polymerase chain reaction ( pcr ) from arabidopsis thaliana genomic dna 5 ′ to translational start of the tt8 gene using the primers tt8f and tt8r which introduce an ndei and a psti site at the 5 ′ and 3 ′ ends of the tt8 pcr fragment respectively . the tt8 pcr fragment is a - tailed and ligated into pgemt , then excised with ndei and psti and ligated into the ndei and psti sites of bj60 , 5 ′ to the uida reporter which includes a terminator signal , forming the vector tt8 - bj60 . a reporter vector based on the promoter of the tt12 gene ( debeaujon et al ., 2000 ; at3g59030 , accession no . aj294464 ) is constructed as described below . a 1 . 7 kb fragment including the tt12 promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the tt12 gene using the primers tt12f and tt12r which introduce an ndei and a psti site at the 5 ′ and 3 ′ ends of the tt12 pcr fragment respectively . the tt12 pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of bj60 , 5 ′ to the uida reporter gene forming tt12 - bj60 . 3b construction of binary vectors and transformation into arabidopsis thaliana tt8 - bj60 tt12 - bj60 and ligated into the noti sites of the binary vector bj40 , forming the following vectors for transformation : tt8 - uida - bj40 tt12 - uida - bj40 the binary vectors are transformed into agrobacterium tumefaciens and then into arabidopsis thaliana . the uida gene encodes β - glucuronidase ( gus ), which is assayed using standard protocols ( e . g . jefferson , 1987 ). for fig1 ( below ) the following assay was used . seeds were dissected from siliques into gus staining buffer ( 100 mm tris - hcl ph 7 . 2 , 50 mm nacl , 0 . 1 % triton - x - 100 , 2 mm 5 - bromo - 4 - chloro - 3 - indolyl - beta - d - glucoronic acid ( x - gluc ), 2 mm k 3 fe ( cn 6 ), 2 mm k 4 fe ( cn ) 6 ) and incubated overnight at 37 ° c . fig1 shows a globular stage seed from a plant containing the tt12 :: uida construct assayed for gus expression ; the inner layer of the inner integument is stained ( arrow ), indicating activity of the tt12 promoter fragment in that integument . construction and transformation of an rnai cassette that decreases mnt expression in arabidopsis thaliana , including decreased expression in the integuments / seed coat an rnai vector based on the mnt gene ( see above ) is constructed as described below . a 0 . 57 kb fragment of the mnt cdna (‘ mnti ’) is amplified by pcr from arabidopsis thaliana cdna using the primers farf2 i and rarf2inew which introduce xbai and asci sites at the 5 ′ end of the mnti pcr fragment , and bamhi and swai sites at the 3 ′ end of the pcr fragment . the mnti pcr fragment is a - tailed and ligated into pgemt , and then excised with asci and swai and ligated into the asci and swal sites of the pfgc5941 rnai vector 3 ′ to the 35s promoter and 5 ′ to the chsa intron , which places the fragment in forward orientation . this forms the vector 35s - mnti - pfgc5941 . the mnti pcr fragment is then excised from pgemt with bamhi and xbai and ligated into the bamhi and xbai sites of the 35s - mnti - pfgc5941 vector , 3 ′ to the chsa intron and 5 ′ to the ocs terminator signal , which places the fragment in inverse orientation . this forms the vector 35s - mnti - inv mnti - pfgc5941 . vector 35s - mnti - inv mnti - pfgc5941 is transformed into agrobacterium tumefaciens and then into arabidopsis thaliana . 4c analysis of seed weights in plants transformed with the 35s :: mnt rnai vector wild - type plants transformed with the 35s :: mnt rnai vector described in example 4a , b have the mnt mutant phenotype , including closed flowers for most of the plant &# 39 ; s life cycle ( fig1 b top left ), inflorescence stems with increased diameter ( fig1 b top right ), and large seeds ( fig1 b , bottom ). seeds from four independently transformed lines , along with wild - type plants grown under the same conditions , are shown in fig1 b ( bottom ). the mean weight for these four lines was 35 . 3 μg , compared with 13 . 8 μg for the wild - type control . construction and transformation of an rnai cassette that decreases bnarf2 expression in brassica napus , including decreased expression in the integuments / seed coat an rnai vector based on the b arf2 gene ( example 2 , above ) is constructed as described below . a 0 . 56 kb fragment of the bnarf2 cdna ( bnarf21 ) is amplified by pcr from brassica napus cdna using the primers fbnarf2 i and rbnarf2 i which introduce xbai and asci sites at the 5 ′ end of the bnarf2 i pcr fragment , and bamhi and swai sites at the 3 ′ end of the pcr fragment . the bnarf21 pcr fragment is a - tailed and ligated into pgemt and then excised with asci and swai and ligated into the asci and swai sites of the pfgc5941 rnai vector 3 ′ to the 35s promoter and 5 ′ to the chsa intron using the enzymes ascl and swai , which places the fragment in forward orientation . this forms the vector 35s - bnarf21 - pfgc5941 . the bnarf21 pcr fragment is then excised from pgemt with bamhi and xbai and ligated into the bamhi and xbai sites of the 35s - bnarf21 - pfgc5941 vector 3 ′ to the chsa intron and 5 ′ to the ocs terminator , which places the fragment in inverse orientation . this forms the vector 35s - bnarf21 - inv bnarf21 - pfgc5941 . vector 35s - bnarf21 - inv bnarf21 - pfgc5941 is transformed into agrobacterium tumefaciens and then into brassica napus . construction and transformation of rnai cassettes that decrease mnt expression primarily in the integuments / seed coat of arabidopsis thaliana this is specifically to phenocopy the big seed effect of mnt mutations without other effects on plant growth , development , or fertility . an rnai vector in which the tt8 promoter ( nesi et al ., 2000 ; at4g09820 , accession no . aj277509 ) drives an inverted repeat of an mnt nucleic acid fragment ( see example 4 , above ) is constructed as described below . a 1 . 7 kb fragment including the tt8 promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the tt8 gene using the primers tt8 ecori f and tt8 ncoi r which introduce an ecori and an ncoi site at the 5 ′ and 3 ′ ends of the tt8 pcr fragment respectively . the tt8 pcr fragment is a - tailed and ligated into pgemt , and then excised with ecori and ncoi and exchanged for the 35s promoter in the vector 35s - mnt - inv m1mnti - pfgc5941 ( example 4 , above ), forming the vector tt8 - mnt - inv mnti - pfgc5941 . an rnai vector in which the ino promoter ( villanueva et al ., 1999 ; at1g23420 , accession no . af195047 ) drives an inverted repeat of an mnt nucleic acid fragment ( see example 4 , above ) is constructed as described below . a 1 . 5 kb fragment including the ino promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the ino gene using the primers finoi and rinoi_which introduce an ecori and an ncoi site at the 5 ′ and 3 ′ ends of the ino pcr fragment respectively . the ino pcr fragment is a - tailed and ligated into pgemt , and then excised with ecori and ncoi and exchanged for the 35s promoter in the vector 35s - mnt - inv mnti - pfgc5941 ( example 4 , above ), forming the vector ino - mnt - inv mnti - pfgc5941 . the tt8 - mnt - inv mnti - pfgc5941 and ino - mnt - inv mnti - pfgc5941 vectors are transformed into agrobacterium tumefaciens and then into arabidopsis thaliana . construction and transformation of rnai cassettes that decrease bnarf2 expression primarily in the integuments / seed coat of brassica napus an rnai vector in which the tt8 promoter ( nesi et al ., 2000 ; at4g09820 , accession no . aj277509 ) drives an inverted repeat of a bnarf2 nucleic acid fragment ( see example 5 , above ) is constructed as described below . a 1 . 7 kb fragment including the tt8 promoter with ecori and ncoi linkers is amplified by pcr from arabidopsis thaliana genomic dna as described in example 6a ( i ) above . the tt8 pcr fragment is a - tailed and ligated into pgemt , and then excised with ecori and ncoi and exchanged for the 35s promoter in the vector 35s - bnarf2 - inv bnarf21 - pfgc5941 ( example 5 , above ), forming the vector tt8 - bnarf2 - inv bnarf21 - pfgc5941 . an rnai vector in which the ino promoter ( villanueva et al ., 1999 ; at1g23420 , accession no . af195047 ) drives an inverted repeat of a bnarf2 nucleic acid fragment ( see example 5 , above ) is constructed as described below . a 1 . 5 kb fragment including the ino promoter with ecori and ncoi linkers is amplified by pcr from arabidopsis thaliana genomic dna as described in example 6a ( ii ) above . the ino pcr fragment is a - tailed and ligated into pgemt , and then excised with ecori and ncoi and exchanged for the 35s promoter in the vector 35s - bnarf2 - inv bnarf21 - pfgc5941 ( example 5 , above ), forming the vector ino - bnarf2 - inv bnarf21 - pfgc5941 . the tt8 - bnarf2 - inv bnarf21 - pfgc5941 and ino - bnarf2 - inv bnarf21 - pfgc5941 vectors are transformed into agrobacterium tumefaciens and then into brassica napus . construction and transformation of an expression vector that increases mnt expression in arabidopsis thaliana , including increased expression in the integuments / seed coat construction of an expression vector with the camv 35s promoter driving the mnt gene is described below . the mnt cdna including the translational start and stop is amplified by pcr from arabidopsis thaliana cdna using the primers 35s xho new and 35s bam new which introduce a xhoi and a bamhi site at the 5 ′ and 3 ′ ends of the a4nt pcr fragment respectively . the mnt pcr fragment is a - tailed and ligated into pgemt , and then excised with xhoi and bamhi and ligated into the xhoi and bamhi sites of part7 , 3 ′ to the 35s promoter and 5 ′ to the ocs terminator , forming the vector 35s - mnt - part7 . 8b construction of binary vectors and transformation into arabidopsis thaliana the 35 s :: mnt expression cassette ( including the ocs terminator signal ) is excised from 35s - mnt - part7 with noti and ligated into the noti sites of the binary vector bj40 , forming the vector 35s - mnt - bj40 . the binary vector is transformed into agrobacterium tumefaciens and then into arabidopsis thaliana . 8c analysis of seed weights in plants transformed with the 35s :: mnt cassette wild - type plants transformed with the 35s :: mnt cassette described in example 8a , b have the mnt mutant phenotype , including closed flowers for most of the plant &# 39 ; s life cycle ( fig1 b , top ), and large seeds . seeds from three independently transformed lines , along with wild - type plants grown under the same conditions , are shown in fig1 b , middle . the overall mean weight for these three lines was 25 . 5 μg , compared with 15 . 0 μg for the wild - type control . expression of mnt / arf2 was assayed in transformed and wild - type plants by semiquantitative rt - pcr ( fig1 b , bottom ) using multiplex rt - pcr with primers rtarf2 - f ( 5 ′- gagtgggtggagtgtgtttg - 3 ′) and rtarf2 - r ( 5 ′- agttggttttcgtttgagcat - 3 ′), and control primers to the gapc gene , gapc - f ( 5 ′- cacttgaagggtggtgccaag - 3 ′) and gapc - r ( 5 ′- cctgttgtcgccaacgaagtc - 3 ′). pcr was initiated with rtarf2 primers and run for 4 cycles at an annealing temperature of 55 ° c ., extension time 2 min . gapc primers were added to each reaction mix and the reaction was run for an additional 22 cycles . this showed that plants transformed with the 35s :: mnt cassette did not have lower levels of mnt expression than wild - type plants ; therefore the mutant phenotype was not due to cosuppression . therefore constitutive expression of the mnt gene ( such as achieved under control of the 35s promoter ) provides a further method for producing large seeds . construction and transformation of an expression cassette that increases bnarf2 expression in brassica napus , including increased expression in the integuments / seed coat this is also to produce a plant with altered seed size . construction of an expression vector with the camv 35s promoter driving the bnarf2 gene is described below . the bnarf2 cdna from translational start to stop is amplified by pcr from brassica napus cdna using the primers bnarf2 xhoi f and bnarf2 bamhi r which introduce a xhoi and a bamhi site at the 5 ′ and 3 ′ ends of the bnarf2 pcr fragment respectively . the bnarf2 pcr fragment is a - tailed and ligated into pgemt , and then excised with xhoi and bamhi and ligated into the xhoi and bamhi sites of part7 , 3 ′ to the 35s promoter and 5 ′ to the ocs terminator , forming the vector 35s - bnarf2 - part7 . 9b construction of binary vectors and transformation into brassica napus the 35s :: bnarf2 expression cassette ( including the ocs terminator signal ) is excised from 35s - bnarf2 - part7 with noti and cloned into the noti sites of the binary vector bj40 , forming the vector 35s - bnarf2 - bj40 . the binary vector is transformed into agrobacterium tumefaciens and then into brassica napus . constitutive expression of the bnarf2 gene ( such as achieved under control of the 35s promoter ) provides a further method for producing large seeds . construction and transformation of expression cassettes that increase mnt expression primarily in the integuments / seed coat of arabidopsis thaliana an expression vector based on the tt8 promoter ( nesi et al ., 2000 ; at4g09820 , accession no . aj277509 ) is constructed as described below . a 1 . 7 kb fragment including the tt8 promoter with ndei and psti linkers is amplified by pcr from arabidopsis thaliana genomic dna as described in example 3a ( i ), above . the tt8 pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of bj36 , 5 ′ to the ocs terminator signal , forming the vector tt8 - bj36 . an expression vector based on the promoter of the ino gene ( villanueva et al ., 1999 ; at1g23420 , accession no . af195047 ) is constructed as described below . a 1 . 5 kb fragment including the ino promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the ino gene using the primers inof and inor which introduce an ndei and a psti site at the 5 ′ and 3 ′ ends of the ino pcr fragment respectively . the ino pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of bj36 , 5 ′ to the ocs terminator signal , forming the vector ino - bj36 . the mnt cdna with xhoi and bamhi linkers is amplified by pcr from arabidopsis thaliana cdna and ligated into pgemt as described in example 8a , above . the mnt pcr fragment is excised from pgemt with xhoi and bamhi and ligated into the xhoi and bamhi sites of the tt8 - bj36 vector , 3 ′ to the tt8 promoter , forming the vector tt8 - mnt - bj36 . the mnt pcr fragment is excised from pgemt with xhoi and bamhi and ligated into the xhoi and bamhi sites of the ino - bj36 vector , 3 ′ to the ino promoter , forming the vector ino - mnt - bj36 . the tt8 :: mnt expression cassette ( including the ocs terminator signal ) is excised from tt8 - mnt - bj36 with noti and cloned into the noti sites of the binary vector bj40 , forming the vector tt8 - mnt - bj40 . the ino :: mnt expression cassette ( including the ocs terminator signal ) is excised from ino - mnt - bj36 with noti and cloned into the noti sites of the binary vector bj40 , forming the vector ino - mnt - bj40 . the tt8 - mnt - bj40 and ino - mnt - bj40 binary vectors are transformed into agrobacterium tumefaciens and then into arabidopsis thaliana . construction and transformation of expression vectors that increase bnarf2 expression primarily in the integuments / seed coat of brassica napus an expression vector based on the promoter of the tt8 gene ( nesi et al ., 2000 ; at4g09820 , accession no . aj277509 ) is constructed as described below . a 1 . 7 kb fragment including the tt8 promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the tt8 gene using the primers tt8f and tt8 mlui r which introduce an ndei and an mlui site at the 5 ′ and 3 ′ ends of the tt8 pcr fragment respectively . the tt8 pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and mlui and ligated into the ndei and mlui sites of bj36 , 5 ′ to the ocs terminator signal , forming the vector tt8 ( ndei mlui )- bj36 . an expression vector based on the promoter of the ino gene ( villanueva et al ., 1999 ; at1g23420 , accession no . af195047 ) is constructed as described below . a 1 . 5 kb fragment including the ino promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the ino gene using the primers inof and ino mlui r which introduce an ndei and an mlui site at the 5 ′ and 3 ′ ends of the ino pcr fragment respectively . the ino pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and mlui and ligated into the ndei and mlui sites of bj36 , 5 ′ to the ocs terminator signal , forming the vector ino ( ndei mlui )- bj36 . the bnarf2 cdna with xhoi and bamhi linkers is amplified by pcr from brassica napus cdna and ligated into pgemt as described in example 9a , above . the bnarf2 pcr fragment is excised from pgemt with xhoi and bamhi and ligated into the xhoi and bamhi sites of the tt8 ( ndei mlui )- bj36 vector , 3 ′ to the tt8 promoter , forming the vector tt8 - bnarf2 - bj36 . the bnarf2 pcr fragment is excised from pgemt with xhoi and bamhi and ligated into the xhoi and bamhi sites of the ino ( ndei mlui )- bj36 vector , 3 ′ to the ino promoter , forming the vector ino - bnarf2 - bj36 . the tt8 - bnarf2 expression cassette ( including the ocs terminator signal ) is excised from tt8 - bnarf2 - bj36 with noti and ligated into the noti sites of the binary vector bj40 , forming the vector tt8 - bnarf2 - bj40 . the ino - bnarf2 expression cassette ( including the ocs terminator signal ) is excised from ino - bnarf2 - bj36 with noti and ligated into the noti sites of the binary vector bj40 , forming the vector ino - bnarf2 - bj40 . the binary vectors tt8 - bnarf2 - bj40 and ino - bnarf2 - bj40 are transformed into agrobacterium tumefaciens and then into brassica napus . construction , transformation , and analysis of expression vectors that increase expression of a gene promoting cell division in the integuments / seed coat of arabidopsis thaliana an expression vector based on the tt8 promoter ( nesi et al ., 2000 ; at4g09820 , accession no . aj277509 ) is constructed as described below . a 1 . 7 kb fragment including the tt8 promoter with ndei and psti linkers is amplified by pcr from arabidopsis thaliana genomic dna as described in example 3a ( i ), above . the tt8 pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of bj36 , 5 ′ to the ocs terminator signal , forming the vector tt8 - bj36 . an expression vector based on the tt12 promoter ( debeaujon et al ., 2000 ; at3g59030 , accession no . aj294464 ) is constructed as described below . a 1 . 7 kb fragment including the tt12 promoter with ndei and psti linkers is amplified by pcr from arabidopsis thaliana genomic dna as described in example 3a ( ii ), above . the tt12 pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of bj36 , 5 ′ to the ocs terminator signal , forming the vector tt12 - bj36 . an expression vector based on the ino promoter ( villanueva et al ., 1999 ; at1g23420 , accession no . af195047 ) is constructed as described below . a 1 . 5 kb fragment including the ino promoter with ndei and psti linkers is amplified by pcr from arabidopsis thaliana genomic dna as described in example 10a ( ii ), above . the ino pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of bj36 , 5 ′ to the ocs terminator signal , forming the vector ino - bj36 . an expression vector based on the promoter of the ban gene ( devic et al ., 1999 ; at1g61720 , accession no . af092912 ) is constructed as described below . a 0 . 4 kb fragment including the ban promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the ban gene using the primers banf and banr which introduce an ndei and a psti site at the 5 ′ and 3 ′ ends of the ban pcr fragment respectively . the ban pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of bj36 , 5 ′ to the ocs terminator signal , forming the vector ban - bj36 . 12b construction of expression cassettes containing are integument / seed coat promoter driving a gene promoting cell division construction of expression vectors with an integument seed coat promoter driving the cycd3 ; 1 gene is described below . the cycd3 ; 1 cdna ( formerly cycδ3 ; soni et al ., 1995 ; vandepoele et al ., 2002 ; at4g34160 , accession no . x83371 ) is amplified by pcr from arabidopsis thaliana cdna using the primers cycd3f and cycd3r which introduce a smai and a bamhi site at the 5 ′ and 3 ′ ends of the cycd3 ; 1 pcr fragment respectively . the cycd3 ; 1 pcr fragment is a - tailed and ligated into pgemt , and then excised with smai and bamhi and ligated into the smai and bamhi sites of the following vectors : tt8 - bj36 vector , 3 ′ to the tt8 promoter and 5 ′ to the ocs terminator signal , forming the vector tt8 - cycd3 ; 1 - bj36 tt12 - bj36 vector , 3 ′ to the tt12 promoter and 5 ′ to the ocs terminator signal , forming the vector tt12 - cycd3 ; 1 - bj36 ino - bj36 vector , 3 ′ to the ino promoter and 5 ′ to the ocs terminator signal , forming the vector ino - cycd3 ; 1 - bj36 ban - bj36 vector , 3 ′ to the ban promoter and 5 ′ to the ocs terminator signal , forming the vector ban - cycd3 ; 1 - bj36 construction of expression vectors with an integument / seed coat promoter driving the ipt1 gene is described below . the ipt1 gene ( takei et al ., 2001 ; at1g68460 , accession no . ab062607 ) is amplified by pcr from arabidopsis thaliana genomic dna ( the ipt1 gene contains no introns ) using the primers ipt1f and ipt1r which introduce a smai and a bamhi site at the 5 ′ and 3 ′ ends of the ipt1 pcr fragment respectively . the ipt1pcr fragment is a - tailed and ligated into pgemt , and then excised with smai and bamhi and ligated into the smai and bamhi sites of the following vectors : tt8 - bj36 vector , 3 ′ to the tt8 promoter and 5 ′ to the ocs terminator signal , forming the vector tt8 - ipt1 - bj36 tt12 - bj36 vector , 3 ′ to the tt12 promoter and 5 ′ to the ocs terminator signal , forming the vector tt12 - ipt1 - bj36 ino - bj36 vector , 3 ′ to the ino promoter and 5 ′ to the ocs terminator signal , forming the vector ino - ipt 1 - bj36 ban - bj36 vector , 3 ′ to the ban promoter and 5 ′ to the ocs terminator signal , forming the vector ban - ipt1 - bj36 construction of expression vectors with an integument / seed coat promoter driving the ant gene is described below . the ant gene ( klucher et al ., 1996 ; at4g37750 , accession no . nm — 119937 ) is amplified by pcr from arabidopsis thaliana cdna using the primers antf and antr which introduce a smai and a bamhi site at the 5 ′ and 3 ′ ends of the ant pcr fragment respectively . the ant pcr fragment is a - tailed and ligated into pgemt , and then excised with smai and bamhi and ligated into the smai and bamhi sites of the following vectors : tt8 - bj36 vector , 3 ′ to the tt8 promoter and 5 ′ to the ocs terminator signal , forming the vector tt8 - ant - bj36 tt12 - bj36 vector , 3 ′ to the tt12 promoter and 5 ′ to the ocs terminator signal , forming the vector tt12 - ant - bj36 ino - bj36 vector , 3 ′ to the ino promoter and 5 ′ to the ocs terminator signal , forming the vector ino - ant - bj36 ban - bj36 vector , 3 ′ to the ban promoter and 5 ′ to the ocs terminator signal , forming the vector ban - ant - bj36 construction of expression vectors with an integument / seed coat promoter driving the cycb1 ; 1 gene is described below . the cycb1 ; 1 gene ( formerly cyc1aat ; ferreira et al ., 1994 ; vandepoele et al ., 2002 ; at4g37490 , accession no . nm — 119913 ) is amplified by pcr from arabidopsis thaliana cdna using the primers cycb1f and cycb1r which introduce a smai and a bamhi site at the 5 ′ and 3 ′ ends of the cycb1 ; 1 pcr fragment respectively . the cycb1 ; 1 pcr fragment is a - tailed and ligated into pgemt , and there excised with smai and bamhi and ligated into the smai and bamhi sites of the following vectors : tt8 - bj36 vector , 3 ′ to the tt8 promoter and 5 ′ to the ocs terminator signal , forming the vector tt8 - cycb1 ; 1 - bj36 tt12 - bj36 vector , 3 ′ to the tt12 promoter and 5 ′ to the ocs terminator signal , forming the vector tt12 - cycb1 ; 1 - bj36 ino - bj36 vector , 3 ′ to the ino promoter and 5 ′ to the ocs terminator signal , forming the vector ino - cycb1 ; 1 - bj36 ban - bj36 vector , 3 ′ to the ban promoter and 5 ′ to the ocs terminator signal , forming the vector ban - cycb1 ; 1 - bj36 12c construction of binary vectors and transformation into arabidopsis thaliana expression cassettes ( including the ocs terminator ) are excised with noti from the following vectors and ligated into the noti sites of the binary vector bj40 , forming the following vectors for transformation : the binary vectors are transformed into agrobacterium tumefaciens and then into arabidopsis thaliana . results from some primary transformants using the tt8 promoter are shown in table 2a and fig2 a . the histogram shows that seeds from tt8 :: cycd3 ; 1 and tt8 :: ipt1 plants have a broader distribution and higher peak of weights than the controls . tt8 :: uida lines were used as controls , as expression of the uida gene is not found to affect plant growth and development . individual tt8 :: cycd3 ; 1 plants produced seeds up to 97 % heavier than controls , with a mean increase over 27 lines of 37 %. tt8 :: ipt1 plants produced seeds up to 107 % heavier , with a mean increase over 24 lines of 28 %. the mean weights of tt8 :: cycd3 ; 1 and tt8 :: ipt1 seeds were compared with the controls using t - tests and found to be significantly different from the controls with p & lt ; 0 . 000 . it should be noted that some of the tt8 :: ipt1 lines , including the highest weighing line , also had a vegetative phenotype including dwarfing , serrated leaves , and extremely low fertility , most likely due to the tt8 promoter driving vegetative expression of ipt1 in some lines . however lines with normal vegetative development also produced large seeds . it is likely that vegetative expression of tt8 could be prevented if required by the technique of promoter dissection ( e . g . chandrasekharan et al ., 2003 ). ttest for control vs tt8 :: cycd3 ; 1 and tt8 : ipt1 , p & lt ; 0 . 000 , significant further results from plants transformed with expression vectors to increase seed size are shown in table 2b and fig2 b . for these experiments , we selected kanamycin resistant lines with heavy seeds and confirmed the presence of the expression vector using pcr . for two of the lines below we weighed seeds produced by t3 plants , confirming the heritability of the large seed trait . in table 2b , weights of controls ( in this case the controls were wild - type col - 0 are shown alongside transformants where the controls and transformants were grown together . ban :: cycd3 ; 1 seeds were 35 % heavier than controls grown under the same conditions , and ino :: ant seeds were 53 % heavier . ino :: ant seeds were also misshapen ( fig2 b ), suggesting that the expression cassette indeed affects seed coat development . construction , transformation , and analysis of expression vectors that increase expression of a gene promoting cell division in the integuments / seed coat of brassica napus the binary vectors described in example 12c ( above ) are transformed into brassica napus . construction of an expression vector containing a petal - and stamen - specific promoter driving mnt and transformation into mnt mutants 14a construction of an expression vector based on the ap3 promoter an expression vector based on the promoter of the ap3 gene ( jack et al ., 1992 ; at3g54340 , accession no . ay142590 ) is constructed as described below . a 1 kb fragment including the ap3 promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the ap3 gene using the primers ap3f and ap3r which introduce an ndei and a psti site at the 5 ′ and 3 ′ ends of the ap3 pcr fragment respectively . the ap3 pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of the bj36 vector , 5 ′ to the ocs terminator signal , forming the vector ap3 - bj36 . the mnt cdna with xhoi and bamhi linkers is amplified by pcr from arabidopsis thaliana cdna and ligated into pgemt as described in example 8a , above . the m1t pcr fragment is excised with xhoi and bamhi and ligated into the xhoi and bamhi sites of the ap3 - bj36 vector , 3 ′ to the ap3 promoter and 5 ′ to the ocs terminator , forming the vector ap3 - mnt - bj36 . 14c construction of binary vector and transformation into arabidopsis thaliana the ap3 :: mnt expression cassette ( including the ocs terminator signal ) is excised from ap3 - mnt - bj36 with noti and cloned into the noti sites of the binary vector bj40 , forming the vector ap3 - mnt - bj40 . the binary vector is transformed into agrobacterium tumefaciens and then into arabidopsis thaliana . construction of an expression vector containing a sepal - and petal - specific promoter driving mnt and transformation into mnt mutants 15a construction of an expression vector based on the ap1 promoter an expression vector based on the promoter of the ap1 gene ( mandel et al ., 1992 ; at1g69120 , accession no . nm — 105581 ) is constructed as described below . a 1 . 7 kb fragment including the ap1 promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the ap1 gene using the primers ap1f and ap1r which introduce an ndei and a psti site at the 5 ′ and 3 ′ ends of the ap3 pcr fragment respectively . the ap1 pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of the bj36 vector , 5 ′ to the ocs terminator signal , forming the vector ap1 - bj36 . the mnt cdna with xhoi and bamhi linkers is amplified by pcr from arabidopsis thaliana cdna and ligated into pgemt as described in example 8a , above . the mnt pcr fragment is excised with xhoi and bamhi and ligated into the xhoi and bamhi sites of the ap1 - bj36 vector , 3 ′ to the ap1 promoter and 5 ′ to the ocs terminator , forming the vector ap1 - mnt - bj36 . 15c construction of binary vector and transformation into arabidopsis thaliana the ap1 :: mnt expression cassette ( including the ocs terminator signal ) is excised from ap1 - mnt - bj36 with noti and cloned into the noti sites of the binary vector bj40 , forming the vector ap1 - mnt - bj40 . the binary vector is transformed into agrobacterium tumefaciens and then into arabidopsis thaliana . value of mnt mutants in understanding and modifying growth of the inflorescence stem mnt - 1 mutants have thick inflorescence stems compared with wild - type plants . the increased diameter of mnt - 1 stems is caused by extra cell divisions ( fig2 b ). therefore it is expected that stem thickness may be increased in other species by altering expression of an mnt orthologue and thereby increasing the number of cells in the stem . construction and transformation of an rnai cassette that decreases mnt expression in arabidopsis thaliana , including decreased expression in the stem the cloning and transformation strategy is described in example 4 . the cloning strategy is shown in fig1 a . transformed plants have an increased stem diameter with respect to wild type ( mean inflorescence stem diameter between nodes 2 and 3 : w . t ., 1 . 293 ± s . e . m . 0 . 4 mm , n = 13 ; 35s :: mnt rnai , 1 . 419 ± 0 . 4 , n = 14 ; two - tailed t - test shows that diameters of w . t . and 35s :: mnt rnai stems are significantly different at p & lt ; 0 . 05 ). the stem phenotype of transformed plants compared with wild - type plants is shown in fig1 b . construction of an expression vector containing a flower - preferred promoter driving mnt and transformation into mnt mutants 18a construction of an expression vector based on the lfy promoter an expression vector based on the promoter of the lfy gene ( weigel et al ., 1992 ; at5g61850 , accession no . nm — 125579 ) is constructed as described below . a 2 . 1 kb fragment including the lfy promoter is amplified by pcr from arabidopsis thaliana genomic dna 5 ′ to translational start of the lfy gene using the primers lfyf and lfyr which introduce an ndei and a psti site at the 5 ′ and 3 ′ ends of the ap3 pcr fragment respectively . the lfy pcr fragment is a - tailed and ligated into pgemt , and then excised with ndei and psti and ligated into the ndei and psti sites of the bj36 vector , 5 ′ to the ocs terminator signal , forming the vector lfy - bj36 . the mnt cdna with xhoi and bamhi linkers is amplified by pcr from arabidopsis thaliana cdna and ligated into pgemt as described in example 8a , above . the mnt pcr fragment is excised with xhoi and bamhi and ligated into the xhoi and bamhi sites of the lfy - bj36 vector , 3 ′ to the lfy promoter and 5 ′ to the ocs terminator , forming the vector ap1 - lfy - bj36 . 18c construction of binary vector and transformation into arabidopsis thaliana the lfy :: mnt expression cassette ( including the ocs terminator signal ) is excised from ap1 - lfy - bj36 with noti and cloned into the noti sites of the binary vector bj40 , forming the vector ap1 - lfy - bj40 . the binary vector is transformed into agrobacterium tumefaciens and then into arabidopsis thaliana . 15 f primer for mnt rnai fragment with xbal and asci linkers 16 r primer for mnt rnai fragment with bamhi and sawi linkers 17 f primer for bnarf2 rnai fragment with xbai and asci linkers 18 r primer for bnarf2 rnai fragment with bamhi and swai linkers adams , s ., vinkenoog , r ., spielman , m ., dickinson , h . g ., and scott , r . j . 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