Patent Application: US-26146594-A

Abstract:
genes controlling gibberellic biosynthesis are used in genetic engineering to alter plant development . alterations in the nature or quantity of products of the genes affects plant development . a family of genes in monocots encodes a cyclase involved in the early steps of gibberellic acid biosynthesis . a member of the family , the gene an1 , is identified in maize and cloned and the function of the gene is characterized . using recombinant genetic technology , ga levels are manipulated . changes in ga levels alter monocot plant phenotypes , for example , height and fertility .

Description:
gibberellic acid ( ga ) levels are important factors in plant development . control of ga levels by genetic engineering technology allows alteration of plant phenotypes such as fertility and size . identification and isolation of genes controlling the biosynthesis of ga , are required for this effort . a family of genes have been identified that is capable of encoding a product that is necessary for the conversion of ggpp to ent - kaurene in the biosynthesis of gibberellic acid , the product is consistent in structure with a cyclase . members of this gene family hybridize with the an1 gene under conditions of high stringency . these genes also encode the functional equivalent of the sequence in fig2 a - 2c within the box . steps catalyzed by kaurene synthetase are as follows : two rings are closed in the conversion of ggpp to cpp by kaurene synthetase a . the third ring is closed , the pyrophosphate group is cleaved , and a carbon - carbon bond is broken and reformed at a nearby site as cpp is converted to ent - kaurene by kaurene synthetase b ( fig1 b ). an illustrative embodiment of a cloned member of this family , as noted above , is the an1 gene in maize . also as noted , an1 is one of five identified genes in maize that are involved in ga biosynthesis . the an1 , d1 , d2 , d3 , and d5 mutants of maize compose a class of recessive mutants that are ga deficient and ga responsive . they all appear to be defective in some step of the ga biosynthetic pathway , and they share a number of phenotypes , including reduced stature and the development of anthers on the normally pistillate ear . within this class of mutants there are two distinct groups relative to stature . alleles of d1 , d2 , d3 , and d5 are typically severe dwarfs , exhibiting an 80 % or greater reduction in final plant height . in contrast , alleles of an1 are less severely dwarfed , typically semi - dwarfed , and in some cases there is no reduction in their final height . the severity of reduction in shoot height for both groups is also reflected in the degree of reduction in their leaf lengths . for the entire class the reduction in height is scorable in both light and dark grown seedlings . in six day - old dark grown an1 seedlings , the basis of the reduced height lies in the cells of the mesocotyl . coleoptile cell number is slightly reduced in an1 seedlings , while the average cell length of coleoptile cells is the same as found in wild - type siblings ( table 1 ). this is in contrast to the mesocotyl where cell number is reduced by one - half and average cell length is reduced to one - fourth of that observed in wild - type seedlings . thus , the reduced stature in dark grown seedlings is due primarily to greatly reduced final cell lengths . table 1______________________________________comparison of cell length and cell number in shootsof dark grown maize seedlings . number of average cell length length ( mm ) cells ( mm ) ______________________________________tall siblingcoleoptile 18 228 0 . 08mesocotyl 70 294 0 . 18total 88 522dwarf ( an1 ) coleoptile 14 171 0 . 08mesocotyl 6 130 0 . 05total 20 301______________________________________ seedlings were grown for six days in total darkness . the an1 gene was cloned using transposon tagging . a key advantage for tagging genes with mutator is the 50 - fold or greater increase in mutation frequency compared to spontaneous rates . see walbot , 1992 for a review . transposon tagging involves using any one of a number of naturally occurring plant transposons -- mu , ac , spm and the like -- to create a &# 34 ; molecular tag &# 34 ; to recover the mutated gene . although it has been used before , the transposon - tagging approach to recovering a gene of interest is unpredictable , is plagued by a low mutation frequency , and is very difficult technically . first , the genetic stocks have to be screened phenotypically for mutants of interest . there is no way to direct the transposon to a particular gene or to produce a particular phenotype . after a mutant phenotype of interest is found , moreover , it is necessary to determine whether the mutant is actually caused by the insertion of a transposon , because not all mutations are caused by transposable elements . a gene can be isolated by transposon tagging only if a particular transposon has inserted into the gene . each transposon system has major advantages and disadvantages . ac and spm , for example , occur in lower copy number per genome than mu and therefore , promote a lower frequency of mutations . because both of these elements excise from the germline at a higher frequency than mu , however , it is possible to use the powerful genetic tool of looking for a reversion of the mutant phenotype as a result of excision of the element from the germline . this provides very strong evidence that a particular mutant was caused by the transposon insertion . mu has the advantage of having a high copy number , so the frequency of causing mutations is higher ( up to 10 - 100x higher than the background mutation rate . because the germline excision frequency is very low (˜ 1 in 10 , 000 ), however , standard tests for reversion are not practical . other , labor - intensive means need to be used to prove that the gene is tagged by the transposon . those methods are molecular detection methods which involve isolating dna from the mutant plants of interest , and probing the dna for the presence of a mu element which co - segregates with the mutant phenotype . with mu this is particularly difficult , because there are many copies of mu per genome -- in fact , some genomes have over 200 copies ( walbot and warren , 1988 ). co - segregation of an an1 - 891339 phenotype and mu2 containing restriction fragments was demonstrated by southern blot analysis . dna from individual homozygous f2 dwarfed an1 - 891339 siblings was analyzed to determine linkage between the mutation and a mu element . dna was restricted with ssti , and the blot was probed with an internal mu2 - dna fragment . a mu2 containing restriction fragment of 5 . 7 kb , common to all tested individuals , was identified . this mu2 containing restriction fragment was cloned into a lambda vector . dna gel blot analysis of a restriction digest of the clone was performed . double digests of the cloned fragment was in lane 2 ( ssti and hindiii ) and lane 3 ( ssti and xbai ). flanking sequence dna was identified , and a 2 . 6 kb flanking sequence fragment ( g2 . 6xba ) was subcloned and used as a probe . southern blot analysis of the deletion mutant ( an1 - bz2 - 6923 ) was performed as follows : southern blots of ssti digested genomic dna of the deletion mutant and wild - type sibling dna were analyzed . a blot probed with genomic flanking sequence subclone g2 . 6xba showed deletion mutant plants lack dna homologous to g2 . 6xba . a sequence comparison of maize an1 and arabidopsis ga1 showed the complete predicted amino acid sequences of an1 and ga1 are similar . overall identity is 47 %, similarity 68 % ( gcg package , genetics computer , inc ., university of wisconsin ). a putative polyprenylpyrophosphorylate binding domain is indicated with a box ( fig2 a - 2c ). the homology between predicted amino acid sequences of maize an1 ( seq id no : 1 ) and arabidopsis ga1 ( seq id no : 1 ) points to a common function for these genes . their overall identity of 47 % ( 68 % similarity ) is striking , but is even stronger in an internal 300 amino acid segment that is 68 % identical ( 94 % similar ). as to the putative polyprenyl - pyrophosphate binding domain within this segment , an1 and ga1 share 100 % similarity . other sequenced plant genes that use polyprenylpyrophosphorylated substrates ( geranyl -, farnysyl - and geranylgeranyl - pyrophosphate ) also share significant homology with an1 in this domain ( facchini et al ., 1992 ), but much less overall homology with an1 ( 20 to 25 % identity ). these sequence homologies clearly indicate that an1 encodes a cyclase which functions in the conversion of ggpp to ent - kaurene . while highly homologous to ga1 , it is important to note that an1 is distinct from ga1 in its amino ( only 11 % identical for first 100 amino acids ) and carboxyl terminus ( only 18 % identical for the last 283 amino acids ). also , the amino terminus of an1 has characteristics expected of a chloroplast targeting sequence including a net positive charge ( 12 of 43 amino acids are basic while only two are acidic ). in addition , the an1 amino terminus also has a greater than 50 % similarity to the amino terminus of an aspartate aminotransferase cdna clone from rice ( gene bank source d16340 ). aspartate aminotransferase has many isoforms , at least one of which is located in the chloroplast ( matthews et al ., 1993 ). this suggests that the amino terminus of an1 serves as a chloroplast - targeting sequence . support for a chloroplastic localization of kaurene synthesis comes from the demonstration that cell free assays of purified chloroplasts synthesize kaurene ( simcox et al ., 1975 ). if an1 and ga1 code for the same chloroplast targeted activity their targeting sequences are distinct . the low homology between an1 and ga1 in their carboxyl termini may be functionally important . while a number of plant cyclase activities share a conserved polyprenylpyrophosphate binding domain , they act on distinct substrates and cyclize by distinct mechanisms . the basis for these differences is not obvious from an examination of the primary amino acid sequences . southern blot analysis using high and low stringency was performed . southern blots of an1 - bz2 - 6923 and wild - type sibling dna compared from high ( 65 °) and low ( 25 °) stringency washes were compared . genomic dnas were digested with bamhi . the probe was an1 - cdna . therefore , at high stringency , dna from the deletion mutant hybridizes to wheat , at low stringency , hybridization occurs with wheat and deletion mutant maize . a related sequence is likely in wheat . fig3 a - 3c shows the cdna sequence of a maize an1 gene ( seq id no : 3 ). northern blot analysis shows an1 transcript accumulation . northern blots from total rna preparations were probed with an1 - cdna . tissues analyzed were : the blot revealed an1 transcript accumulation in all tissues and an enhancement of accumulation in light grown shoots . since ga plays important developmental roles , its control is a useful avenue to altering development for specific purposes . the an1 - bz2 - 6923 allele of an1 is consistent with a robust plant which demonstrates little or no reduction in plant height or leaf length compared to wild - type siblings . despite its similarity in growth , the average first day of pollen shed in this mutant is delayed , in the example shown this delay is 5 days ( fig4 ). lowering ga levels reduces time - to - maturity in maize , possibly by shortening the time required between germination and floral initiation . a comparison of days required to maturity for an1 - bz2 - 6923 and its wild - type siblings is shown in fig4 as a plot of the height of wild - type siblings and an1 - bz2 - 6923 mutants versus gdushd ( heat units to pollen shed , 25 units ≈ 1 day ). although no difference in final height exists , there is an average of 200 gdushds delay for the mutant plants . shortened time to maturity is an advantage in some growing zones ( climates ); whereas , increased time to maturity is an advantage in other growth zones . therefore , the ability to manipulate ga levels by recombinant techniques is advantageous for developing commercial monocots . isolation of genes such as an1 provides some of the tools needed for this endeavor . the an1 gene will be useful to probe for homologous genes in other species . the present invention is illustrated in further detail in the following examples . these examples are included for explanatory purposes and should not be considered to limit the invention . reports in the literature suggest that ga levels may be a partial cause of heterosis . to develop transgenic tools for improving yield in crop plants using genes affecting ga synthesis , a goal was to clone genes which encode enzymes of the ga biosynthetic pathway . several ga - deficient mutants of maize had been described ( d1 , d2 , d3 , d5 , an1 ) which were associated with a dwarf stature and andromonoecious flowering ( perfect flowers on the ear ). if these mutations actually occurred in the genes directly coding for ga biosynthetic enzymes , it was difficult to envision how to identify and isolate the genes without having to purify the as yet uncharacterized enzymes in the ga pathway . one possible approach was to use transposon tagging , which had been successfully used in some cases to tag and isolate genes ( walbot , 1992 ). but dwarfs are very rare and , moreover , no known transposon -- induced alleles had previously been reported for any dwarf mutants . an anther ear ( an1 ) mutation segregating in a mu - containing maize line was obtained from patrick schnable ( iowa state university ), and experiments were carried out to determine whether a transposable element could be found associated with the mutant gene . the likelihood of this was questionable , however , because such transposon - tagged dwarf mutants had never been identified before . the employed mutant - detection method involved isolating dna from the mutant plants of interest and then probing the dna for the presence of a mu element which co - segregates with the mutant phenotype . this was particularly difficult because there are many copies of mu per genome ; in fact , some genomes have over 200 copies ( walbot and warren , 1988 ). in order to reduce the extremely large number of mu - hybridizing bands , it was first necessary to make repeated crosses to plants that inactivated and diluted out most of the mu elements . it was also necessary for the an1 mutant gene search to use southern blots to probe genomic dna separately with a dna fragment that is unique to each of nine distinct mu families . even then , the number of copies per mu family is around 25 , making it very difficult to identify one hybridizing band in the blot that co - segregates with the mu element used as probe . in doing such a dna screen for an1 , it was necessary to prepare dna from 50 different individual plants and probe each of those samples in a southern blot with each of the mu - specific probes , mu1 , mu2 and mu3 , that are characteristic of the sub - family . after a mu - tagged , co - segregating restriction fragment was found , the fragment was isolated by cloning and sequenced to identify the location of the mu insertion . the flanking regions were also sequenced , to locate the structural gene of interest . for a gene like an1 , not identified or isolated previously and , hence , of unknown sequence , it can be very difficult to determine the exact limits of the gene and even to prove that the clone contains the mutant gene of interest . as walbot indicates in her 1992 review of strategies for mutagenesis and gene cloning using transposon tagging , identification of a co - segregating band is not straightforward . moreover , identification of such a band is not proof that the band in question defines the gene of interest . a family with a phenotype characteristic of ga deficiency was observed to segregate as a simple recessive trait in an active mu line . the mutation was shown to be allelic with an1 , and was identified as an1 - 891339 . southern analysis of ssti - restricted genomic dna from an1 - 891339 and its wild type siblings identified a mu2 - containing restriction fragment , of approximately 5 . 4 kb , which co - segregated with the mutation . this fragment was eluted from a preparative agarose gel , cloned into a bacteriophage lambda vector and plaque purified using a mu2 internal fragment as a probe . analysis of the cloned fragment , by restriction with xbai or hindiii , identified fragments of flanking sequence dna . a 2 . 6 kb xbai flanking sequence fragment ( g2 . 6xba ) was subcloned into a plasmid and used as a probe for southerns and screening maize cdna libraries . southern analysis of maize genomic dna demonstrated that g2 . 6xba was single copy dna . using g2 . 6xba as a probe , a number of cdna clones were selected from maize cdna libraries , demonstrating that g2 . 6xba lies in a transcribed region of the genome . the frequency of positive clones in each of two amplified libraries was 8 per 360 , 000 plaques . the longest of the cdnas , 2 . 8 kb , was subcloned into a plasmid and sequenced . this cdna appears to represent full length mrna . comparing cdna and an1 genomic dna sequence identifies a number of exons . the comparison also demonstrates that the mu2 element causing the mutation is inserted within or at the border of an intron , 1 . 6 kbp from the carboxyl terminal of the transcript and 900 bp from the amino terminal . it was necessary to take several approaches to confirm the identity of the putative clone of the an1 gene . tight linkage between the clone and the gene needed to be established by testing to show that the clone did not hybridize to dna from a known genetic deletion mutant of an1 . this evidence placed the clone to within a few map units ( 4 centimorgans ) of the genetic locus for an1 , based on the resolution of this mapping experiment . that distance corresponds to ˜ 8 . 4 mb × 10 6 bp , so it is possible the clone could have been located as far away as 8 . 4 mb from the genetic locus for an1 . the next step was to isolate and sequence a cdna clone . to do this , it was necessary to determine where the putative an1 gene was expressed so that a cdna library could be created that was likely to contain the gene . because the size of the mrna was known to be quite large (˜ 3 kb ), recovery of a full - length clone was very difficult . the first clone was only 2 . 5 kb in size , so it was necessary to screen a second library to recover a longer clone of 2 . 8 kb . the sequence of the cdna showed ˜ 40 % similarity in only one region of the clone to an isoprenoid cyclase type of binding region , based on other known cyclase - type genes . the biochemical function of an1 is known to be required for kaurene accumulation and is likely the cyclase which converts ggpp to cpp . this is known to be the first committed step in ga biosynthesis ( kaurene synthetase a ). homology with other cyclases was consistent with one of the possible functions for the an1 gene product . the homology that was seen was very limited and far less than the overall homology typically seen among cyclases , so only tentative conclusions could be drawn as to the identity of the isolated gene . therefore , additional evidence had to be obtained from other technical approaches . peptides were synthesized that corresponded to predicted antigenic domains of the protein which was encoded by the clone . antibodies were raised against several peptides . only 2 of the 4 antibody preparations were usable . some of the antibodies were shown to precipitate the ggpp - to - cpp cyclase activity of cucurbit endosperm extracts , providing additional evidence to support the possibility that the isolated gene was an1 . finally , a comparison of amino acid sequence between our clone and a ga1 clone from arabidopsis revealed significant homology throughout the length of the protein . ga1 has been shown to encode the ggpp - to - cpp cyclase ( tai - ping sun et al ., personal communication ). these data provide a convincing case that an1 was cloned , but clearly , the process was a difficult and uncertain one . although transposon tagging made it possible to clone the an1 gene , success was far from predictable . the efficiency of obtaining an insertional mutant depends on a variety of factors , including the activity phase of the autonomous element ( s ), the number of mobile elements , the location of the elements and the susceptibility of the target locus ( walbot , 1992 ). as walbot states in her review , &# 34 ; although not often reported , some targeted mutagenesis screens fail completely , despite reasonable progeny sizes &# 34 ;. table 2 in her review indicates a number of examples where attempts to target specific genes by transposon insertion have failed . based on the previous failure to identify any dwarf mutants which were transposon - tagged , it was not unreasonable to assume that the target locus for genes in the ga pathway might not be susceptible to tagging . therefore , it was very uncertain that the an1 mutant from the mu genetic stocks was in fact tagged by mu . however , the an1 gene has been cloned , as shown herein . as described previously , an1 is unlike the other ga deficient / responsive mutants of maize in that it is a semi - dwarf . this is true of all four isolates of an1 examined . an1 plants respond to the application of a number of ga biosynthetic intermediates , including ent - kaurene . since ga biosynthesis is initiated by the conversion of ggpp to cpp , followed by the conversion of cpp to ent - kaurene , an1 appears to be deficient in the conversion of ggpp to ent - kaurene . probing an1 - bz2 - 6923 dna on a southern blot with either g2 . 6xba or full length an1 - cdna resulted in no detectable hybridization of probe . similar results were observed on northern blots of deletion mutant rna . this indicates that the transcript of the an1 gene lies entirely within the deletion and is therefore not present in an1 - bz2 - 6923 plants . it would be expected , therefore , that this mutant would be absolutely defective in ent - kaurene synthesis . yet light - grown an1 - bz2 - 6923 seedlings accumulate ent - kaurene in vivo , albeit at a much reduced rate ( 20 %) compared to their wild - type siblings ( table 2 ). this accumulation must result from a non - an1 activity that supplements an1 production of ent - kaurene . the supplementary activity is thought not to be unique to maize . a deletion mutant of arabidopsis , ga1 - 3 , also is expected to be devoid of ent - kaurene , since the ga1 coding region is entirely deleted ( tai - ping sun et al ., 1992 ). however , ga1 - 3 plants convert ggpp to cpp and cpp to ent - kaurene in cell - free extracts of siliques . notably , there are a number of ga1 isolates that demonstrate a uniform but variable reduction in plant height similar to that observed for the an1 isolates in maize . the accumulation of ent - kaurene is not observed in maize d5 mutants , however . the d5 mutant is believed to be defective in kaurene synthetase b as is the ga2 mutant of arabidopsis which has a , but no b activity in cell free extracts from immature siliques . when the stringency of southerns is lowered for blots of restricted an1 - bz2 - 6923 dna , bands sharing homology to an1 can be identified suggesting that homologous sequences provide an1 functional equivalents . thus , the consistent &# 34 ; leaky &# 34 ; or semi - dwarfed phenotype observed for all documented an1 mutants in maize is likely the result of a redundancy for an1 function . this redundancy does not exist , or is of little significance , for the kaurene synthetase b - encoding maize d5 and arabidopsis ga2 genes , since their block in kaurene synthesis seems complete . transcription of the an1 gene in maize occurs in a number of tissues , as demonstrated by northern blots . vegetative parts of the plant , shoots and roots , contain an1 mrna . reproductive tissues including tassels , developing ears , silks and embryos all contain an1 mrna . interestingly , etiolated shoot tissue appears to have very little if any an1 mrna compared to light - grown shoots . the presence of message in the roots decouples this light - induced transcription from dependence on chloroplast development . recombinant genetic methods make use of an isolated dna molecule encoding a gene product which is necessary to convert ggpp to ent - kaurene in the biosynthesis of ga . the isolated dna molecule is incorporated into a plasmid , such as that shown in fig5 and transferred into a host plant . the expression of the dna in the host will generally increase the endogenous levels of ga . the effect will depend on the species and the increment in ga levels . a strong , constitutive promoter is generally preferred to regulate a gene of the present invention in a host cell . examples of suitable promoters are ubiquitin and 35s . decreasing endogenous ga levels is achieved by introducing an antisense molecule to a gene product of the present invention . knowledge of the binding domain sequence ( fig2 a - 2c ) allows such antisense molecules to be specifically constructed . directed mutation is useful to change a phenotypic gene of the present invention so that ga levels are reduced . the effects of reduced ga levels have been described above . table 2______________________________________kaurene accumulation in shoots of light grown maizeseedlings . ent - kaurene content ( pmoles / gfwt ) leaf length ( mm ) plant no treatment 48 h tetcyclacis 2nd leaf 3rd leaf______________________________________an1 - bz2 - 6923tall 120 1330 42 83dwarf 33 209 30 58an1 - 891339tall 61 710dwarf 54 216d5dwarf not detected not detectedb73 94 1093______________________________________ seedlings were grown in continuous light for six days , at which time mm tetcyclacis ( an inhibitor of kaurene metabolism ) was applied directly to the shoots . fortyeight hours later , the shoots of treated and nontreated plants were analyzed for entkaurene content . a mu2 tagged an1 maize family , an1 - 891339 , was selected from lines with active mu elements ( lines originated from pat schnable , iowa state university ). additional an1 alleles used in this study include ; an1bm2 ( 110d , maize genetics cooperation stock center , u . illinois ), idd *- 2286a and an1 - bz2 - 6923 ( both from g . neuffer , u . missouri ). idd *- 2286a is mutated in both the indeterminate locus ( id ) and the an1 locus ( d ) but does not appear to be a deletion mutant , as progeny of selfs from this material segregate for id and an1 . conversely , an1 - bz2 - 6923 appears to be a deletion mutant . the extent of the deletion is not defined although id ( two map units proximal to an1 ) and ad ( two map units distal from bz2 ) are unaffected by the deletion . total dna was extracted from leaf tissue by the urea extraction method ( dellaporta et al ., 1983 ). southern blots were performed as previously described ( johal , 1992 ) using duralose - uv membranes ( stratagene ). mu2 probes were synthesized by random priming ( amersham ) a gel - eluted internal 650 - bp avai - bsteii mu1 fragment isolated from pa / b5 ( chandler , 1986 ). this internal mu1 fragment contains regions of homology to mu2 , thus allowing for hybridization to both mu1 and mu2 sequences . the genomic dna restriction fragment containing the mu2 element judged to cause the an1 - 891339 mutation was electro - eluted following preparative agarose gel electrophoresis of ssti digested total dna , dialyzed , and concentrated by ethanol precipitation . precipitated fragments were pre - annealed to ssti restricted arms of the bacteriophage vector lambda sep6 / lac5 ( meyerowitz , from marteinssen , csh ) and packaged using gigapack gold ( stratagens ). this library was screened for mu2 containing phage , with the ssti insert of a plaque purified mu2 containing clone then transferred to the bacteriophage vector lambda - zapii ( stratagene ). this insert and other clones used for probing or sequencing were all sub - cloned into the plasmid bluescript sk + and maintained in sure cells ( stratagene ). two cdna libraries , which served as sources for an1 cdnas , were prepared from the shoots of 14 day old light grown b73 seedlings , a gift from a . barkan , university of oregon ( barkan , 1991 ) and from whole kernels ( 30 dap ) of w22 , a gift from karen cone , university of missouri . sequence data from a 2 . 5 kb an1 cdna was generated by loftstrand labs limited . total rna was prepared as previously described ( chomczynski et al ., 1987 ). polya + rna was enriched using polya - tract system iii ( promega ) following the manufacturer &# 39 ; s protocol . northerns were run , blotted and probed as previously described ( johal , 1992 ) using 1 . 5 kb and 1 . 1 kb subclones of an1 cdna to generate random primed probes . analysis of the in vivo accumulation of ent - kaurene in light grown maize seedlings was performed as previously described . cell free assays of kaurene synthetase a and b activities were performed as previously described using immature siliques from arabidopsis seedlings . a transgenic plant containing a construct having a gene of the present invention can be regenerated from a culture transformed with that same construct , so long as plant species involved is susceptible to regeneration . &# 34 ; culture &# 34 ; in this context comprehends an aggregate of cells , a callus , or derivatives thereof that are suitable for culture . a plant is regenerated from a transformed cell or culture , or from an explant , by methods disclosed herein that are known to those of skill in the art . methods vary according to the plant species . seed is obtained from the regenerated plant or from a cross between the regenerated plant and a suitable plant of the same species using breeding methods known to those of skill in the art . example of transformation methods in maize ( may be modified for specific promoters and structural genes ) maize tapetum specific promoter : stable transformations experimental protocols repetition 1 , 2 , and 5 ; goal : recover transgenic colonies , plants and progeny of maize resistant to basta / bialophos and expressing gus driven by the tapetum specific sgb6g1 promoter medium : 237 liquid suspension medium for maize 115 , callus maintenance medium for maize 115e , callus 5 mg / l basta selection medium 115b , callus 3 mg / l bialaphos selection medium plate cells , 0 . 5 ml / plate onto glass filters 934 - ah atop a whatman filter moistened with 1 ml 237 + 3 % peg medium transfer samples to 115e ( repetition 1 ) 48 hours post bombardment . transfer samples to 115b ( repetition 2 and 5 ) 7 days post bombardment maintain samples at 28 c in the dark method of corn transformation to recover stable transgenic plants ______________________________________day - 1 cells placed in liquid media and sieved ( 710 um ), 100 - 200 mg of cells collected on 5 . 5 cm glass fiber filter over an area of 3 . 5 cm . cells transferred to media and incubated media over night . day 0 filter and cells removed from media , dried and bom - barded . filter and cells placed back on media . day 5 cells on filter transferred to selection media ( 3 mg bialophos ). day 12 cells on filter transferred to fresh selection media . day 19 cells scraped form filter and dispersed in 5 ml of selection media containing 0 . 6 % low melting point sea plaque agarose . cells and media spread over the surface of two 100 mm × 15 mm plate containing 20 ml of gel - rite solidified media . day 40 putative transformants picked from plate . day 61 plates checked for new colonies . ______________________________________ the references listed below are incorporated herein by reference to the extent that they supplement , explain , provide a background for or teach methodology , techniques or compositions employed herein . barendse , g . w . m ., dijkstra , a . and moore , t . c . 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( 1992 ). strategies for mutagenesis and gene cloning using transposon tagging and t - dna insertional mutagenesis . ann . rev . plant physiol . 43 : 49 - 82 . pct patent application wo / 9316096 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 3 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 823 amino acids ( b ) type : amino acid ( d ) topology : linear ( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 802 amino acids ( b ) type : amino acid ( d ) topology : linear ( xi ) sequence description : seq id no : 2 : 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( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 2784 base pairs ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear ( xi ) sequence description : seq id no : 3 : 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