Patent Application: US-76638904-A

Abstract:
adding at least one gene involved in plant host cell t - dna integration enhances transformation by agrobacterium . the histone h2a gene encoded by the arabidopsis rat5 gene increases transformation frequencies of plants , most likely by causing overexpression of a product needed for t - dna integration . agrobacterium tumefaciens genetically transforms plant cells by transferring a portion of the bacterial ti - plasmid , designated the t - dna , to the plant , and integrating the t - dna into the plant genome . however , not all plants are transformable by agrobacterium and transformation frequencies may be too low to be useful . little is known about the t - dna integration process , and no plant genes involved in integration have been identified prior to the present invention .

Description:
several t - dna tagged [ plants which genes have randomly been disrupted by integration of a t - dna ] mutants of arabidopsis were identified that are recalcitrant to agrobacterium root transformation . these are called rat mutants ( r esistant to a grobacterium t ransformation ). in most of these mutants agrobacterium transformation is blocked at an early step , either during bacterial attachment to the plant cell or prior to t - dna nuclear import . in some of the mutants , however , the t - dna integration step is most likely blocked . because plant factors involved in illegitimate recombination of t - dna into the plant genome have not previously been identified , the characterization of a t - dna tagged arabidopsis mutant , rat5 , that is deficient in t - dna integration , is an aspect of the present invention . characterization of the rat5 mutant . rat5 , an arabidopsis t - dna tagged mutant , was previously identified as resistant to agrobacterium root transformation . an in vitro root inoculation assay was performed using the wild - type agrobacterium strain a208 ( at10 ). after one month , the percentage of root bundles that formed tumors was calculated . greater than 90 % of the root bundles of the wild - type plants ( ecotype ws ) formed large green teratomas . in contrast , fewer than 10 % of the root bundles from the rat5 plants responded to infection , forming small yellow calli ( fig1 a ). a homozygous rat5 plant ( pollen donor ) was crossed to a wild - type plant ( egg donor ) and the resulting f1 progeny tested for susceptibility to agrobacterium transformation . this analysis indicated that rat5 is a dominant mutation ( 7 ; fig1 a ). further analysis of f2 progeny indicated that kanamycin resistance segregated 3 : 1 , indicating that a single locus had been disrupted by the mutagenizing t - dna . kanamycin resistance co - segregated with the rat5 phenotype , indicating that a gene involved in agrobacterium transformation had most likely been mutated by the t - dna insertion . recovery of a t - dna - plant junction from rat5 . the t - dna integration pattern in the rat5 mutant was determined by dna blot analyses . the results indicated that there are only two copies of the mutagenizing t - dna integrated into the genome of the rat5 mutant . further analysis indicated that these two t - dna copies are present as a direct tandem repeat , as shown in fig1 c . a left border ( lb ) t - dna - plant junction was recovered from rat5 using a plasmid rescue technique ( see materials and methods ) and a restriction endonuclease map of this t - dna - plant junction was constructed . an approximately 1 . 7 kbp ecori fragment that contains both plant and lb dna was subcloned into pbluescript and subsequently sequenced at the purdue university sequencing center . the sequence of this fragment is shown in fig1 b . dna sequence analysis of this junction region indicated that the t - dna had inserted into the 3 ′ untranslated region ( utr ) of a histone h2a gene ( fig1 b ). the histone h2a genes of arabidopsis were further characterized by isolating and sequencing numerous cdna and genomic clones . six different gene variants of histone h2a were identified , indicating that the histone h2a genes of arabidopsis comprise a small multi - gene family . in a lambda genomic dna library a clone was identified containing the wild - type histone h2a gene corresponding to rat5 . dna sequence analysis of this genomic clone indicated that in rat5 the t - dna had inserted upstream of the consensus polyadenylation signal ( aataa ). dna blot analysis of ws and rat5 dna indicated that the t - dna insertion in rat5 did not cause any major rearrangements in the plant dna immediately around the site of insertion . disruption of the 3 ′ utr of the rat5 histone h2a gene is likely the sole cause for the rat phenotype in the rat5 mutant . fig1 shows characterization of the rat5 mutant . ( a ) stable transformation of wild - type arabidopsis ecotype ws , the rat5 mutant , and the f1 progeny . sterile root segments were infected with a . tumefaciens a208 . two days after cocultivation , the roots were transferred to ms medium lacking phytohormones and containing timentin as an antibiotic . tumors were scored after four weeks . ( b ) sequence of the rat5 / t - dna junction region . ( c ) pattern of t - dna integration in rat5 . lb , t - dna left border ; rb , t - dna right border ; pbr322 , pbr322 sequences containing the β - lactamase gene and cole1 origin of replication ; tn903 , kanamycin resistance gene for e . coli selection ; tn5 , kanamycin resistance gene for plant selection . five μg of genomic dna from the rat5 mutant was digested with either ecori or sali and was blotted onto a nylon membrane . an ecori - sali fragment of pbr322 was used as the hybridization probe . restriction fragment sizes shown above the t - dna were detected by ecori digestion and the sizes shown below the t - dna were detected by sali digestion . complementation of the rat5 mutant with a wild - type histone h2a gene ( rat5 ). two different constructions were made to perform a complementation analysis of the rat5 mutant . first , a nopaline synthase terminator ( 3 ′ nos ) was fused to the 3 ′ region of the 1 . 7 kbp junction fragment ( the sequence of this 1 . 7 kbp fragment is shown in fig1 b ). this construction contains the rat5 histone h2a gene with its own promoter and a 3 ′ nos . this fragment ( rat5 plus 3 ′ nos ) was cloned into the binary vector pgtv - hpt of beaker containing a hygromycin resistance gene between the left and the right t - dna borders , resulting in the binary vector pkm4 ( fig2 a ). for the second construction , a 9 . 0 kbp saci genomic fragment of wild - type ws dna containing a histone h2a gene ( rat5 ) plus at least 2 . 0 kbp sequences upstream and downstream of rat5 was cloned into the binary vector pgtv - hpt , resulting in the binary vector pkm5 ( fig2 b ). pkm4 and pkm5 were transferred separately into the non - tumorigenic agrobacterium strain gv3101 , resulting in strains a . tumefaciens at1012 and at1062 , respectively . both strains at1012 and at1062 were separately used to transform rat5 plants using a germ - line transformation method ( bent et al ., 1998 ) and transgenic rat5 plants were selected for resistance to hygromicin ( 20 μg / ml ). several transgenic plants ( t1 ) were obtained . these transgenic plants were allowed to self fertilize and t1 seeds were collected . six transgenic lines obtained by transformation with at1012 ( the wild - type histone h2a with 3 ′ nos ) were randomly selected and their seeds were germinated in the presence of hygromycin . tumorigenesis assays were performed as described in nam et al . ( 1999 ) using a . tumefaciens at10 and a sterile root inoculation protocol , on at least five different plants from each of the six transgenic lines . the results indicated that in five of the six transgenic rat5 lines tested , the tumorigenesis - susceptibility phenotype was recovered ( fig2 c ; table 1 ). teratomas incited on the roots of these plants appeared similar to tumors generated on a wild - type plant . one of the transgenic plants tested did not recover the tumorigenesis - susceptibility phenotype , probably because of an inactive transgene . transgenic t1 plants of rat5 obtained by transformation with at1062 ( containing a genomic encoding rat5 from the wild - type plant ) were also tested for restoration of the tumorigenesis - susceptibility phenotype . some of these plants were also able to recover the tumorigenesis - susceptibility phenotype , indicating complementation of the rat5 mutation . hygromycin - resistant transgenic plants generated by transforming the rat5 mutant with pgptv - hpt alone did not form tumors upon infection with a . tumefaciens a208 . to confirm the genetic basis of the complementation experiment , a co - segregation analysis was performed on one of the rat5 transgenic lines ( rat5 at1012 - 6 ) obtained by transformation of the rat5 mutant with a . tumefaciens at1012 . to examine the co - segregation of the complementing t - dna containing the wild - type rat5 gene with the tumorigenesis - susceptibility phenotype , seeds from a t2 plant homozygous for the rat5 mutation but heterozygous for hygromycin resistance were germinated and grown on b5 medium without selection . roots of these plants were subsequently tested for hygromycin - resistance and susceptibility to crown gall tumorigenesis . all plants that were sensitive to hygromycin were also resistant to tumor formation in a manner similar to that of the rat5 mutant . of the 25 hygromycin - resistant plants , at least 8 were susceptible to tumorigenesis . however , 17 hygromycin - resistant plants remained recalcitrant to agrobacterium - mediated transformation . it is likely that these plants are heterozygous with respect to the complementing rat5 gene and did not express this gene to a level high enough to restore susceptibility to tumorigenesis . this possibility corresponds to the finding that the rat5 mutation is dominant , and that therefore one active copy of rat5 is not sufficient to permit agrobacterium - mediated transformation . taken together , the molecular and genetic data strongly indicate that in the rat5 mutant disruption of a histone h2a gene is responsible for the tumorigenesis - deficiency ( rat ) phenotype . overexpression of a histone h2a ( rat5 ) gene in wild - type plants improves the efficiency of agrobacterium transformation . to determine further whether the rat5 gene plays a direct role in agrobacterium - mediated transformation , a . tumefaciens at1012 was used to generate several transgenic arabidopsis plants ( ecotype ws ) containing additional copies of the rat5 histone h2a gene . these transgenic plants were allowed to self - pollinate , t1 seeds were collected , and t2 plants were germinated in the presence of hygromycin . tumorigenesis assays were performed as described herein at least five plants from each of four different transgenic lines . because ecotype ws normally is highly susceptible to agrobacterium transformation , the tumorigenesis assay was altered to detect any subtle differences between the transformation - susceptible wild - type plant and transgenic wild - type plants overexpressing rat5 . these alterations included inoculation of root segments with a 100 - fold lower concentration ( 2 × 10 7 cfu / ml ) of bacteria than that normally used ( 2 × 10 9 cfu / ml ), and spreading individual root segments rather than bundles of root segments on ms medium to observe tumor production . the results , shown in table 1 and fig2 d , indicate that transgenic plants overexpressing rat5 are approximately twice as susceptible to root transformation as are wild - type ws plants . these data indicate that the rat5 histone h2a gene plays a direct role in t - dna transformation , and that overexpression of rat5 can increase susceptibility to transformation . transient expression of histone h2a is sufficient to permit transformation of rat5 and to increase the transformation efficiency of wild - type ws plants . expression of the rat5 histone h2a gene from the incoming t - dna complement the rat5 mutant . although transformation of this mutant with an agrobacterium strain harboring pgptv - hyg ( lacking a histone h2a gene ) resulted in only a few , slow - growing calli on hygromycin selection medium , agrobacterium strains harboring pkm4 or pkm5 incited rapidly growing hygromycin - resistant calli on 60 ± 21 % and 54 ± 22 % of the rat5 root segment bundles , respectively . in addition , when wild - type plants were infected ( at low bacterial density ) with a tumorigenic agrobacterium strain ( a208 ) harboring pkm4 , 78 ± 8 % of the root segments developed tumors , compared to 36 ± 9 % of the root segments infected with a tumorigenic bacterial strain harboring pgptv - hyg . these transformation experiments indicate that agrobacterium strains containing the binary vectors pkm4 or pkm5 are able to transform rat5 mutant plants at relatively high efficiency , and on wild - type plants are two - fold more tumorigenic , and are better able to incite hygromycin - resistant calli , than are agrobacterium strains containing the “ empty ” binary vector pgptv - hyg . transiently produced histone h2a may improve the stable transformation efficiency of plants by agrobacterium . the rat5 mutant is deficient in t - dna integration . agrobacterium - mediated transformation of the arabidopsis rat5 mutant results in a high efficiency of transient transformation but a low efficiency of stable transformation , as determined by the expression of a gusa gene encoded by the t - dna . this result suggested that rat5 is most likely deficient in t - dna integration . to test this hypothesis directly root segments from ws and rat5 plants were inoculated with a . tumefaciens gv3101 harboring the t - dna binary vector pbisn1 . pbisn1 contains a gusa - intron gene under the control of a “ super - promoter ” ( ni et al ., 1995 ; narasimhulu et al ., 1996 ). two days after cocultivation , the root segments were transferred to callus inducing medium containing timentin ( 100 μg / ml ) to kill the bacteria . three days after infection , a few segments were stained for gus activity using the chromogenic dye x - gluc . both the wild - type and the rat5 mutant showed high levels of gus expression ( approximately 90 % of the root segments stained blue ; fig3 a ). the remaining root segments were allowed to form calli on callus inducing medium containing timentin to kill agrobacterium , but lacking any antibiotic for selection of plant transformation . after four weeks numerous calli derived from at least five different ws and rat5 plants were stained with x - gluc . of the ws calli sampled , 92 ± 12 % showed large blue staining areas , whereas only 26 ± 10 % of the rat5 calli showed gus activity , and most of these blue staining regions were small ( fig3 a ). these data indicate that although the rat5 mutant can transiently express the gusa gene at high levels , it fails to stabilize gusa expression . suspension cell lines were generated from these ws and rat5 calli and after an additional month the amount of t - dna was assayed ( using as a hybridization probe the gusa - intron gene located within the t - dna of pbisn1 ) integrated into high molecular weight plant dna from ws and rat5 calli ( nam et al ., 1997 ; mysore et al ., 1998 ). fig3 b shows that although t - dna integrated into the genome of wild - type ws plants was easily detectable , t - dna integrated into the rat5 genome was not . these data directly demonstrate that rat5 is deficient in t - dna integration . to demonstrate equal loading of plant dna in each of the lanes , the gusa probe was stripped from the blot and rehybridized the blot with an arabidopsis phenylalanine ammonia - lyase ( pal ) gene probe . fig2 shows complementation of the rat5 mutant and overexpression of rat5 in wild - type arabidopsis plants . maps of the binary vectors pkm4 ( a ) and pkm5 ( b ). rb , t - dna right border ; lb , t - dna left border ; panos , nopaline synthase polyadenylation signal sequence ; histone h2a , coding sequence of the rat5 histone h2a gene ; ph2a , promoter sequence of the rat5 histone h2a gene ; pnos , nopaline synthase promoter ; hpt , hygromycin resistance gene ; pag7 , agropine synthase polyadenylation signal sequence ; uida , promoterless gusa gene . arrows above the histone h2a , uida , and hpt genes indicate the direction of transcription . ( c ) complementation of the rat5 mutant . rat5 mutant plants were transformed with an agrobacterium strain containing the binary vector pkm4 ( at1012 ). hygromycin - resistant transgenic plants were obtained and were self - pollinated to obtain t2 plants . sterile root segments of t2 plants expressing rat5 , wild - type ws plants , and rat5 mutant plants were infected with the tumorigenic strain a . tumefaciens a208 . two days after cocultivation , the roots were moved to ms medium lacking phytohormones and containing timentin . tumors were scored after four weeks . ( d ) tumorigenesis assay of ws transgenic plants overexpressing the rat5 histone h2a gene . ws plants were transformed with a . tumefaciens at1012 containing the binary vector pkm4 . hygromycin - resistant transgenic plants were obtained and were self - pollinated to obtain t2 plants . sterile root segments of t2 plants overexpressing rat5 and wild - type ws plants were infected at low bacterial density with a . tumefaciens a208 . after two days cocultivation , the roots were moved to ms medium lacking phytohormones and containing timentin . tumors were scored after four weeks . teratomas incited on the roots of these plants appeared similar to tumors generated on a wild - type plant . one of the transgenic plants tested did not recover the tumorigenesis - susceptibility phenotype , probably because of an inactive transgene . transgenic t1 plants of rat5 obtained by transformation with at1062 ( containing a genomic encoding rat5 from the wild - type plant ) were also tested for restoration of the tumorigenesis - susceptibility phenotype . some of these plants were also able to recover the tumorigenesis - susceptibility phenotype , indicating complementation of the rat5 mutation . hygromycin - resistant transgenic plants generated by transforming the rat5 mutant with pgptv - hpt alone did not form tumors upon infection with a . tumefaciens a208 . fig3 shows t - dna integration assays of rat5 and ws plants ; ( a ) transient and stable gus expression in ws and rat5 ; sterile root segments of ws and rat5 plants were infected with the non - tumorigenic agrobacterium strain gv3101 containing the binary vector pbisn1 . two days after cocultivation , the roots were transferred to callus inducing medium ( cim ) containing timentin . three days after infection , half of the segments were stained with x - gluc to determine the efficiency of transient gus expression . the other group of segments was allowed to form calli on cim . after four weeks these calli were stained with x - gluc to determine the efficiency of stable gus expression . ( b ) t - dna integration in rat5 and ws plants . suspension cells were derived from the calli generated from ws and rat5 root segments infected with the non - tumorigenic agrobacterium strain gv3101 containing the binary vector pbisn1 . the suspension cell lines were grown for three weeks ( without selection for transformation ) in the presence of timentin or cefotaxime to kill agrobacterium . genomic dna was isolated from these cells , subjected to electrophoresis through a 0 . 6 % agarose gel , blotted onto a nylon membrane , and hybridized with a gusa gene probe . after autoradiography , the membrane was stripped and rehybridized with a phenylalanine ammonia - lyase ( pal ) gene probe to determine equal loading of dna in each lane . nucleic acid manipulation . total plant genomic dna was isolated according to the method of dellaporta et al . ( 1983 ). restriction endonuclease digestions , agarose gel electrophoresis , plasmid isolation , and dna blot analysis were conducted as described ( sambrook et al ., 1982 ). plasmid rescue . genomic dna ( 5 μg ) of rat5 was digested to completion with sali . the digested dna was extracted with phenol / chloroform and precipitated with ethanol . the dna was self - ligated in a final volume of 500 μl in 1 × ligation buffer ( promega ) with 3 units of t4 dna ligase at 16 ° c . for 16 hr . the ligation mixture was precipitated with ethanol , transformed into electrocompetent e . coli dh10b cells ( mcrbc -; life technologies , inc ., gaithersburg , md .) by electroporation ( 25 μf , 200 ω , and 2 . 5 kv ) and plated on lb medium containing ampicillin ( 100 μg / ml ). ampicillin - resistant colonies were lifted onto a nylon membrane , the bacteria were lysed , and dna was denatured in situ ( sambrook et al ., 1982 ). a radiolabeled left border ( lb ) sequence ( 3 . 0 kbp ecori fragment of pe1461 ) was used as a hybridization probe to identify a plasmid containing the lb . positive colonies were picked and plasmid dna was isolated . by restriction fragment analysis a plasmid containing both the lb and plant junction dna was identified . the plant junction fragment was confirmed by hybridizing the junction fragment to wild - type plant dna . a restriction map of this plasmid , containing the lb - plant junction dna , was made . a 1 . 7 kbp ecori fragment that contained plant dna plus 75 base pairs of lb sequence was subcloned into pbluescript , resulting - in pe1509 . this fragment was subsequently sequenced at the purdue university sequencing center . growth of agrobacterium and in vitro root inoculation of arabidopsis thaliana these were performed as described previously by nam et al . ( 1997 ). plant growth conditions seeds of various arabidopsis thaliana ecotypes were obtained from s . leisner and e . ashworth ( originally from the arabidopsis stock centre , nottingham , uk , and the arabidopsis biological resource center , ohio state university , columbus , respectively ). seeds were surface sterilized with a solution composed of 50 % commercial bleach and 0 . 1 % sds for 10 min and then rinsed five times with sterile distilled water . the seeds were germinated in petri dishes containing gamborg &# 39 ; s b5 medium ( gibco ) solidified with 0 . 75 % bactoagar ( difco ). the plates were incubated initially at 4 ° c . for 2 days and the fro 7 days under a 16 - hr - lights / 8 - hr - dark photoperiod at 25 ° c . seedlings were individually transferred into baby food jars containing solidified b5 medium and grown for 7 to 10 days for root culture . alternatively , the seedlings were transferred into soil for bolt inoculation . growth of agrobacterium tumerfaciens all agrobacterium strains were grown in yep medium ( lichtenstein and draper , 1986 ) supplemented with the appropriate antibiotics ( rifampicin , 10 μg / ml ; kanamycin , 100 μg / ml ) at 30 ° c . overnight bacterial cultures were washed with 0 . 9 % nacl and resuspended in 0 . 9 % nacl a 2 × 10 9 colony - forming units per ml for in vitro root inoculation or at 2 × 10 11 colony - forming units per ml for bolt inoculation . in vitro root inoculation and transformation assays roots grown on the agar surface were excised , cut into small segments (˜ 0 . 5 cm ) in a small amount of sterile water , and blotted onto sterile filter paper to remove excess water . for some experiments , excised roots were preincubated on callus - inducing medium ( cim ; 4 . 32 g / l murashige and skoog [ ms ] minimal salts [ gibco ], 0 . 5 g / l mes , ph 5 . 7 , 1 ml / l vitamin stock solution [ 0 . 5 mg / ml nicotinic acid , 0 . 5 mg / ml pyridoxine , and 0 . 5 mg / ml thyamine - hcl ], 100 mg / l myoinositol , 20 g / l glucose , 0 . 5 mg / l 2 , 4 - dichlorophenoxyacetic acid , 0 . 3 mg / l kinetin , 5mg / l indoleacetic acid , and 0 . 75 % bactoagar ) for 1 day before cutting them into segments . dried bundles of root segments were transferred to ms basal medium ( 4 . 32 g / l ms minimal salts , 0 . 5 g / l mes , ph 5 . 7 , 1 ml / l vitamin stock solution , 100 mg / l myoinositol , 10 g / l sucrose and 0 . 75 % bactoagar ), and 2 or 3 drops of bacterial suspension were placed on them . after 10 min , most of the bacterial solution was removed , and the bacteria and root segments were cocultivated at 25 ° c . for 2 days . for transient transformation assays , the root bundles were infected with agrobacterium strain gv3101 was used ( koncz and schell , 1986 ) containing the binary vector pbisn1 ( narasimhulu et al ., 1996 ). after various periods of time , the roots were rinsed with water , blotted on filter paper , and stained with x - gluc staining solution ( 50 mm nah 2 hpo 4 , 10 mm na 2 edta , 300 mm mannitol , and 2 mm x - gluc , ph 7 . 0 ) for 1 day at 37 ° c . for quantitative measurements of β - glucuronidase ( gus ) activity , the roots were ground in a microcentrifuge tube containing gus extraction buffer ( 50 mm na 2 hpo 4 , 5 mm dtt , 1 mm na 2 edta , 0 . 1 % sarcosyl , and 0 . 1 % triton x - 100 , ph 7 . 0 ), and gus specific activity was measured according to jefferson et al . ( 1987 ). to quantitate tumorigenesis , root bundles were infected with wild - type agrobacterium strains . after 2 days , the root bundles were rubbed on the agar surface to remove excess bacteria and then washed with sterile water containing timentin ( 100 μg / ml ). individual root segments ( initial assay ) or small root bundles ( 5 to 10 root segments ; modified assay ) were transferred onto ms basal medium lacking hormones but containing timentin ( 100 μg / ml ) and incubated for 4 weeks . for transformation of root segments to kanamycin resistance , root bundles were inoculated with agrobacterium strain gv3101 containing pbisn 1 . after 2 days , small root bundles ( or individual root segments ) were transferred onto cim containing timentin ( 100 μg / ml ) and kanamycin ( 50 μg / ml ). kanamycin - resistant calli were scored after 4 weeks of incubation at 25 ° c . to determine stable gus expression , roots were inoculated as given above and the root segments were transferred after 2 days to cim containing timentin ( 100 μg / ml ) without any selection . after 4 weeks , gus activity was assayed either by staining with x - gluc or by measuring gus specific activity by using a 4 - methylumbelliferyl β - d galactoside ( mug ) fluorometric assay , as described above . to determine the kinetics of gus expression , root bundles were infected , the root segments were transferred after 2 days to cim containing timentin ( 100 μg / ml ), and calli were grown on cim without selection . root bundles were assayed at various times , using a mug fluorometric assay as described above , to measure gus specific activity . construction of the binary vectors pkm4 and pkm5 . the plasmid pe1509 containing the 1 . 7 kbp junction fragment cloned into pbluescript was digested with ecori to release the junction fragment . the 5 ′ overhanging ends were filled in using the klenow fragment of dna polymerase i and deoxynucleotide triphosphates . the t - dna binary vector ( pe1011 ) pgtv - hpt ( becker et al ., 1992 ) was digested with the enzymes saci and smai , releasing the promoterless gusa gene from pgtv - hpt . the 3 ′ overhanging sequence of the larger fragment containing the origin of replication and the hygromycin resistance gene ( hpt ) were removed using the 3 ′- 5 ′ exonuclease activity of klenow dna polymerase , and the resulting 1 . 7 kbp blunt end fragment was ligated to the blunt ends of the binary vector . a binary vector plasmid containing the 1 . 7 kbp fragment in the correct orientation ( panos downstream of the histone h2a gene ) was selected and named pkm4 ( strain e1547 ). an approximately 9 . 0 kbp wild - type genomic saci fragment containing the histone h2a gene ( rat5 ) from a lambda genomic clone was cloned into the saci site of the plasmid pbluescript . this 9 . 0 kbp saci fragment was subsequently released from pbluescript by digestion with saci and was cloned into the saci site of the binary vector pgtv - hpt , resulting in the plasmid pkm5 ( strain e1596 ). both pkm4 and pkm5 were separately transferred by triparental mating ( ditta et al ., 1980 ) into the non - tumorigenic agrobacterium strain gv3101 , resulting in the strains a . tumefaciens at1012 and at1062 , respectively . germ - line transformation of arabidopsis . germ - line transformations were performed as described in ( bent and clough , 1998 ). transgenic plants were selected on b5 medium containing hygromycin ( 20 μg / ml ). ballas , n . & amp ; citovsky , v . nuclear localization signal binding protein from arabidopsis mediates nuclear import of agrobacterium vird2 protein . proc . natl . acad . sci . usa 94 , 10723 - 10728 ( 1997 ). bent , a . f . & amp ; clough , s . j . in plant molecular biology manual , ( eds gelvin , s . b . & amp ; verma , d . p . s .) vol . 3 , pp . b7 / 1 - 14 ( kluwer academic publishers , netherlands , 1998 ). britt , a . b . dna damage and repair in plants . annu . rev . plant physiol . plant mol . biol . 47 , 75 - 100 ( 1996 ). dellaporta , s . l ., wood , j ., & amp ; hicks , j . b . plant mol . biol . rep . 1 , 19 - 22 ( 1983 ). deng w . et al . agrobacterium vird2 protein interacts with plant host cyclophilins . proc . natl . acad . sci . usa 95 , 7040 - 7045 ( 1998 ). ditta , g ., stanfield , s ., corbin , d ., & amp ; helinski , d . r . proc . natl . acad . sci usa 77 , 7347 - 7351 ( 1980 ). gheysen , g ., villarroel , r . & amp ; van montagu , m . illegitimate recombination in plants : a model for t - dna integration . genes dev . 5 , 287 - 297 ( 1991 ). jefferson , r . a . m kavanagh , t . a ., bevan , m . w . gus fusions : beta - glucuronidase as a sensitive and versitile gene fusion marker in higher plants . embo j . 6 , 391 - 3907 ( 1987 ). koncz , c . and schell , j . mol . gen . genet . 204 , 383 - 396 ( 1986 ). lichtenstein , c . and draper , j . genetic engeneering of plants . in glover , d . m . 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