水蕨

Breakthrough Technologies

Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides1[W][OPEN]

Balasubramaniam Muthukumar,Blake L.Joyce,Mark P.Elless,and C.Neal Stewart Jr.*

University of Tennessee,Knoxville,Tennessee37996(B.M.,B.L.J.,C.N.S.);and Edenspace Systems Corporation,Manhattan,Kansas66502(M.P.E.)

ORCID ID:0000-0002-8220-2823(B.M.).

Ferns(Pteridophyta)are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics,genomics,and molecular biology.We report here,to our knowledge,the?rst instance of stable transformation of fern with recovery of transgenic sporophytes.Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid‘C-fern Express’(Ceratopteris thalictroides)were stably transformed by Agrobacterium tumefaciens with constructs containing the P.vittata actin promoter driving a GUSPlus reporter gene.Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes.Southern-blot analysis con?rmed stable transgene integration in recovered sporophytes and also con?rmed that no plasmid from A.tumefaciens was present in the sporophyte tissues.We recovered seven independent transformants of P.vittata and four independent C.thalictroides transgenics.Inheritance analyses using b-glucuronidase(GUS)histochemical staining revealed that the GUS transgene was stably expressed in second generation C.thalictroides sporophytic tissues.In an independent experiment,the gusA gene that was driven by the23Cauli?ower mosaic virus35S promoter was bombarded into P.vittata spores using biolistics,in which putatively stable transgenic gametophytes were recovered.Transformation procedures required no tissue culture or selectable marker genes.However,we did attempt to use hygromycin selection,which was ineffective for recovering transgenic ferns.This simple stable transformation method should help facilitate functional genomics studies in ferns.

Ferns(Pteridophyta)are non?owering vascular plants comprised of250genera,the second largest group of diversi?ed species in the plant kingdom(Gifford and Foster,1989).Many interesting traits are inherent in various fern species,such as arsenic hyperaccumulation (Pteris vittata;Ma et al.,2001),insecticide production and allelopathy(Pteridium aquilinum;Marrs and Watt, 2006),and antimicrobial compound production(Acros-tichum aureum;Lai et al.,2009).Ferns occupy the evolutionary niche between nonvascular land plants, bryophytes,and higher vascular plants such as gym-nosperms and angiosperms.Therefore,extant fern spe-cies still hold a living record of the initial adaptations required for plants to thrive on land.Of these adapta-tions,the most important is tracheids that comprise xylem tissues for water and mineral transport and structural support.The vascular system allowed pte-ridophytes to grow upright during the sporophyte generation,leading to greater resource acquisition ca-pacity.Ultimately,suf?cient resources allowed for greater spore production and upright growth,which facilitated spore spread.Recent endeavors have inves-tigated lower plant genomics,including the sequencing of the bryophyte Physcomitrella patens(Rensing et al., 2008)and the lycophyte Selaginella moellendorf?i(Banks et al.,2011).Both basic and applied biology of ferns lag far behind that for angiosperms and even bryophytes. Stable genetic transformation has been accomplished in a few species outside angiosperms and gymno-sperms,especially among bryophytes(Schaefer et al., 1991;Chiyoda et al.,2008;Ishizaki et al.,2008),but never in the Pteridophyta.Transient transformation methods have been developed both in Ceratopteris richardii‘C-fern’and P.vittata,which were limited to heterologous expression in gametophytes(Rutherford et al.,2004;Indriolo et al.,2010).While transient ex-pression of transgenic constructs does enable research, there is no substitute for stable transformation in func-tional genomics and plant biology.Researchers have resorted to studying fern gene function using heterol-ogous expression in the angiosperm model plant Arabidopsis(Arabidopsis thaliana;Dhankher et al.,2006; Sundaram et al.,2009;Sundaram and Rathinasabapathi, 2010),which is far from optimal.Overexpression and knockdown analysis of individual genes in a wide va-riety of fern species would undoubtedly accelerate our ability to learn more about their biology and subse-quently develop novel products from ferns.Further-more,a facile transformation system would accelerate functional genomics and systems biology of ferns.For example,knockdown analysis of genes involved in interesting biosynthetic pathways can greatly facilitate gene and biochemical discovery.

1This work was supported by Edenspace Systems Corporation through a National Institute of Health award(R42ES014976),the Uni-versity of Tennessee Ivan Racheff Endowment,and the U.S.Depart-ment of Agriculture-National Institute of Food and Agriculture.

*Address correspondence to nealstewart@https://www.wendangku.net/doc/4817309294.html,.

The author responsible for distribution of materials integral to the ?ndings presented in this article in accordance with the policy described in the Instructions for Authors(https://www.wendangku.net/doc/4817309294.html,) is:C.Neal Stewart,Jr.(nealstewart@https://www.wendangku.net/doc/4817309294.html,).

[W]The online version of this article contains Web-only data.

[OPEN]Articles can be viewed online without a subscription.

https://www.wendangku.net/doc/4817309294.html,/cgi/doi/10.1104/pp.113.224675

P.vittata has the unparalleled ability to accumulate more arsenic per gram biomass than any other plant species and is highly tolerant to arsenic(Gumaelius et al.,2004).It can thrive in soils containing up to 1,500m g mL–1arsenic,whereas most plants cannot survive50m g mL–1arsenic(Ma et al.,2001).Therefore, P.vittata has been the subject of extensive basic and applied research for arsenic hyperaccumulation,trans-location,and resistance(Gumaelius et al.,2004)and has been used for arsenic phytoremediation(Shelmerdine et al.,2009).For example,this fern might be of great utility for the production of a safer rice(Oryza sativa) crop;arsenic can be transported and stored in the grain,resulting in serious human health rami?cations (Srivastava et al.,2012).In a recent greenhouse study, P.vittata has been used to remediate arsenic-contaminated soil.Following remediation,rice plants were subse-quently grown,and it was found that arsenic uptake by rice grains was reduced by52%,resulting in less than1m g mL–1arsenic after two rounds of remediation using P.vittata phytoremediation(Mandal et al.,2012). Furthermore,this treatment also resulted in increased rice grain yield by14%(w/w)compared with control. Ceratopteris is a subtropical-to-tropical fern genus containing four to six species living in aquatic habitats. The C-fern cultivar was developed as a model fern for teaching(https://www.wendangku.net/doc/4817309294.html,)and research owing to its small size,short life cycle(120d),and its ame-nability for in vitro culture(Banks,1999).The C-fern Express cultivar was developed by Leslie G.Hickok by crossing two Japanese varieties of Ceratopteris thalic-troides(L.Hickok,personal communication).cv C-fern Express,a tetraploid,develops spores in60d of cul-ture.Though cv C-fern spores have been shown to be a useful single cell model system and a rapid and ef?-cient system for studying RNA interference in ferns (Stout et al.,2003),no stable transformation studies have been reported.In bryophytes,immature thalli are most often used as explants(Chiyoda et al.,2008; Ishizaki et al.,2008),whereas in fungi,spores are rou-tinely used for stable transformation studies(Michielse et al.,2005;Utermark and Karlovsky,2008).

The objective of our research was to develop,for the ?rst time,a facile stable transformation system for P.vittata and C.thalictroides using spores as the trans-formation targets.This method can be used as an ad-ditional tool to further substantiate and strengthen the molecular mechanism studies in pteridophytes.

RESULTS AND DISCUSSION

Isolation of a Putative Fern Actin Gene and Its Promoter An important missing component of fern molecular biology is effective promoters that can be used for transgene expression.Actin genes are likely candidates to have constitutive promoters.Thus,we identi?ed P.vittata actin genes using degenerate primers designed from algae,ferns,and angiosperms,which resulted in a 1.4-kb PCR fragment.BlastN and tblastX sequence anal-yses con?rmed that the isolated PCR product from P.vittata was an actin gene.A neighbor-joining tree was constructed based on Blast searches that

revealed Figure1.In vitro generation of non-transgenic control and transgenic sporophytes of P.vittata(A–H)and C.thalictroides(I–P).Both A.tumefaciens-mediated transformed spores and non-transgenic control spores were germinated on one-half-strength MS medium. The nontransgenic prothalli(A and I)and transgenic prothalli(B and J)were trans-ferred to the same medium,where they formed gametophytes.The nontransgenic gametophytes(C and K)and transgenic gametophytes(D and L)were transferred to one-half-strength MS medium without Suc and generated nontransgenic sporo-phytes(E and M)and transgenic sporo-phytes(F and N),which were subsequently transferred and established in pots(G and O,nontransgenic;H and P,transgenic). Bar=3mm.

Stable Transformation of Ferns

that the closest neighbors to fern actin were P.patens actin1and Chlamydomonas reinhardtii actin(Supplemental Fig.S1).

Once the actin gene structure was con?rmed,we performed genome walking upstream to isolate the actin promoter.During the?rst genome walk using a Pvu II library,a720-bp PCR product was obtained. Sequencing results con?rmed the isolation of520-bp putative promoter region located59upstream of200-bp actin gene template.A second genome walk was per-formed using the520-bp and a new369-bp upstream region that was obtained from an Eco RV library.An 889-bp putative promoter sequence was sequence con-?rmed and assembled and further used to drive the expression of the GUSPlus gene.Motif analyses using PlantCARE and PLACE regulatory element databases showed that the actin promoter contained several char-acterized higher plant promoter motifs(Supplemental Table S1).

In Vitro Generation of Transgenic P.vittata

and C.thalictroides Sporophytes

In our initial experiments,no antibiotic selection was used throughout the transformation studies for both ferns.In parallel,nontransgenic control spores were also germinated and compared to Agrobacterium tumefaciens-transformed spores(Fig.1).Incubation of suspended spores in carboxy methyl cellulose for15h at room tem-perature before transformation increased the synchroni-zation of growth as well as spore germination by40%in P.vittata and20%in C.thalictroides.GUS staining was used to identify transformants at various life cycle stages. In P.vittata,mature prothalli(more than90%)began to form6weeks after spore transformation in one-half-strength Murashige and Skoog(MS)medium with 20%(w/v)Suc in both transgenic and nontransgenic control samples(Fig.1,A and B).After an additional6weeks, approximately90%of prothalli formed gametophytes in the same medium(Fig.1,C and D).The3-week-old gametophytes that were transferred to the same basal medium without Suc started to produce sporophytes after5weeks from both nontransgenic control and transgenic gametophytes(Fig.1,E and F).Approxi-mately85%of the gametophytes gave rise to sporo-phytes.Ef?ciency calculations were based on visual estimation for three different transformation experiments. The sporophytes were established in potting media after 3weeks(Fig.1,G and H).Approximately60%of the transferred sporophytes were established in soil.The total time to obtain mature sporophytes from spores was23to27weeks.The ef?ciency of sporophyte for-mation from spores was approximately55%to65%. In C.thalictroides,95%of prothalli were formed in 3weeks both in control and transgenic samples(Fig.1, I and J),which gave rise to80%ef?ciency of gameto-phyte production at4weeks posttransformation in both samples(Fig.1,K and L).Seventy percent of gametophytes produced sporophytes3to4weeks after gametophytes were transferred to Suc-free one-half-strength basal medium(Fig.1,M and N).About 60%of the transferred sporophytes survived to grow in potting mix(Fig.1,O and P).Mature C.thalictroides sporophytes were obtained after11to13weeks of cul-ture from spores with50%to60%ef?ciency.All the calculations were based on three independent experi-ments.The established sporophytes started sporulating after8weeks in pots(Fig.1,O and P).

Spores from both species germinated normally in one-half-strength MS medium with20%Suc and con-tinued to grow and produce gametophytes both in nontransgenic control and transgenic samples.Previ-ous studies have suggested that Suc is necessary for germination and early growth development of ferns (Camloh,1993;Shef?eld et al.,2001),yet the in?uence of Suc on the later stages of fern development was unknown.We found that Suc was not necessary for sporophyte development from gametophytes;how-ever,the critical effect of Suc on growth stages was

not

Figure2.GUS histochemical staining of P.vittata T

prothalli and gametophytes.Six-week-old prothalli(B)and gametophytes(D)de-rived from transgenic spores obtained from A.tumefaciens-mediated transformation.A and C are nontransgenic control prothalli and ga-metophytes,respectively.E,Protonemata of P.vittata showing GUS expression1week after bombardment of spores with a2335S cauli-?ower mosaic virus promoter-gusA gene construct.F,GUS expression in gametophytes derived from spores of P.vittata after bombardment. Bar=0.25mm.

Muthukumar et al.

studied.As expected,C.thalictroides was able to gen-erate sporophytes much quicker than the slower growing P.vittata(Gumaelius et al.,2004),but none-theless,generation of sporophytes was possible in both of these hermaphroditic ferns(Schedlbauer,1976; Gumaelius et al.,2004)using same media and similar growth conditions.Samples that were subjected to A.tumefaciens appeared to lag a few days behind the controls,but there was no other phenotypic differences observed between nontransgenic control and trans-genic ferns.

Hygromycin as a Potential Selection Agent for Ferns Hygromycin at3and15mg L–1were the lowest concentrations that severely inhibited growth of P.vittata and C.thalictroides spores,respectively(Supplemental Fig.S2).Thus,these concentrations were used to attempt to select transformants for these species in separate transformation experiments.

After cocultivation with A.tumefaciens for90h, P.vittata spores were immediately transferred to one-half-strength MS medium with and without hygromycin. Apparent normal growth was observed in control plates (0mg L–1hygromycin and400mg L–1timentin)contain-ing transformed and nontransformed spores.Whereas on the hygromycin-containing plates,none of the spores germinated at4weeks.In addition,GUS expression was observed in gametophytes obtained from trans-formed control plates.The same result was obtained for C.thalictroides.

In another set of experiments,both ferns were cultured on hygromycin-containing plates following2weeks of incubation on plates without hygromycin after coculti-vation.The spores germinated and formed prothalli,but then no further growth was observed after transfer to hygromycin-containing plates,wherein the plants sub-sequently died.

Thus,we conclude that more experiments are needed to determine whether hygromycin selection is feasible. Selection regimes can be?ne-tuned and/or a promoter stronger than nopaline synthase(nos)is likely required. Because the nos promoter is not a strong constitutive promoter,even in angiosperms(Christensen and Quail,1996;Wilkinson et al.,1997;Bhattacharyya et al.,2012), the next likely step would be to replace it with a stronger promoter in the selectable marker cassette.We noted that young prothalli are sensitive to hygromycin;lower amounts of hygromycin might be required if antibiotic selection is desired.

Stable GUS Expression in Ferns Transformed by

A.tumefaciens and Particle Bombardment

Because spores were used as the transformation targets,the resulting sporophytes that arise from her-maphrodite gametophytes after fertilization of sperm and eggs are referred to as T1transformants for both species.GUS expression was observed in all life stages of both fern species:prothalli,gametophytes,and sporophytes.In nontransgenic control prothalli,no GUS expression was observed(Fig.2A),but expression was detected as early as6weeks posttransformation in transgenic prothalli of P.vittata(Fig.2B).When trans-genic P.vittata prothalli further developed into gameto-phytes,GUS expression was observed in most of the gametophyte tissues(Fig.2D),which was not observed in nontransgenic control gametophytes(Fig.2C). Particle bombardment of fresh spores as well as5-day-old germinating spores yielded fully transformed young gametophytes with GUS expression in rhizoids and prothalli(Fig.2E).When15-day-old multicellular pro-tonemata were transformed,the result was apparent chimeric expression of GUS in gametophytes(Fig.2F). GUS expression driven by the cauli?ower mosaic virus2335S promoter was observed at an average of 12610out of100,000spores,which corresponds to a mean transformation ef?ciency of0.012%using biolistics.

The choice of targets for transformation plays a crucial role in successful stable transformation by A.tumefaciens(de la Riva et al.,1998;Karami et al., 2009)and by biolistics(Kikkert et al.,2004;Jones and Sparks,2009).When spores were transformed using bombardment,evenly GUS-stained gametophytes were observed,demonstrating that targeting spores for transformation avoids the potential for chimeras.It

also Figure3.GUS histochemical staining of transgenic and control T1P.vittata,T1C.thalictroides gametophytes,and T2spo-

rophytic fronds.Controls are labeled as B,C,and F.Mature sporophytes were generated from transgenic spores of P.vittata(A).

T1gametophytes(D)and T2sporophytes(E)of C.thalictroides were generated from transgenic T1spores obtained from T1 sporophytes.Bar=3mm.

Stable Transformation of Ferns

negates the need for tissue culture.Until this study,only fern gametophytes have been targeted for transient transformation(Rutherford et al.,2004;Indriolo et al., 2010).From our results,it is possible that choosing spores as transformation targets probably played a crucial role in successful transformation of these two fern species, which is the same case in fungi(Michielse et al.,2005; Utermark and Karlovsky,2008),where spores have been used as targets.Moreover,A.tumefaciens growth medium(Utermark and Karlovsky,2008)coupled with the phenolic compound acetosyringone,a known in-ducer of virulence genes(Stachel et al.,1986),has been successfully used in experiments for fungal spore trans-formation as well.

Overall,there was no visible difference in the GUS expression pattern in the?rst generation sporophytes of both ferns.In sporophytes,no endogenous blue his-tochemical GUS staining was observed in nontransgenic control tissues that were treated with or without carbo-rundum,in contrast to the con?rmed transgenic plants (Fig.3B).In P.vittata sporophytes,blue GUS staining was observed only in carborundum-treated mature sporophyte fronds(Fig.3A).Carborundum treatment was not required for effective GUS staining of trans-genic C.thalictroides sporophytes;their fronds GUS stained evenly(Supplemental Fig.S3).One version of the GUSPlus marker gene we used contains a rice signal peptide that facilitates GUS expression in apoplasts.C.thalictroides was transformed with GUSPlus gene with no signal peptide.Nonetheless,a similar pat-tern of GUS expression was observed in fronds of both species.

In P.vittata,GUS expression was visible only after carborundum600-mesh size treatments,possibly be-cause random carborundum perforations helped the GUS staining solution enter into the otherwise impen-etrable frond tissues.Other mesh sizes,i.e.300and 1,200,were not effective in GUS assays.The tape sandwich method(Wu et al.,2009),which was used to peel off epidermal layers in an Arabidopsis protoplast isolation method,also yielded effective GUS staining in P.vittata fronds,but resulted in tearing of fronds, whereas the fronds perforated by carborundum were intact.Although these fronds were intact after carbo-rundum perforation,GUS staining was observed only in those areas where carborundum perforation re-moved the epidermal layers.Carborundum is routinely used to aid screening of plant susceptibility for patho-gens(Thaler et al.,2004);however,to the best of our knowledge,this is the?rst time carborundum was used to aid in GUS assays.

GUS Expression Analysis in T1Gametophytes

and T2Sporophytes of C.thalictroides

The gametophytes along with the T2sporophytes that developed from T1spores from two independent transgenic lines1and7were assayed using histo-chemical GUS staining.Out of15gametophytes screened from each line,13and14of them were positive in each line.GUS expression was not observed in nontransgenic control gametophytes(Fig.3C),whereas in transgenic gametophytes,GUS expression was seen throughout the entire gametophyte tissue(Fig.3D).Seven sporo-phytes from transgenic lines1and7were also screened for GUS expression.In line1,?ve sporophytes were positive for GUS expression,and in line7,four spo-rophytes were positive for GUS expression.GUS ex-pression was seen in almost all parts of the frond tissue

Table I.Transformation ef?ciency of P.vittata and C.thalictroides by A.tumefaciens-mediated transformation using R4pGWB501containing P.vittata actin promoter driving GUSPlus

Rows indicate the results of separate experiments.Transformation ef?ciency was calculated based on gametophytes that were GUS positives based on three independent transformation experiments.Prothalli and gametophytes were used to calculate the ef?ciency because more than90%of spores formed prothalli.Sporophytes were not used to calculate ef?ciency because not all sporophytes were recovered for further growth and analysis.ANOVA was performed to calculate the average and error rate for transformation.

Fern species Spores Transformation Prothalli Analyzed/

GUS Positives

Transformation

Ef?ciency Using Prothalli

in the Numerator

Gametophytes Analyzed/

GUS Positives

Transformation Ef?ciency

Using Gametophytes

in the Numerator

no.%% P.vittata400No,control380Not applicable370Not applicable 300No,control290Not applicable280Not applicable

200No,control185Not applicable180Not applicable 50,000Yes3/10,0000.0317/30,0000.056

40,000Yes9/15,0000.068/17,0000.047

50,000Yes12/17,0000.074/12,0000.0333

Mean,0.05360.021Mean,0.04560.012

C.thalictroides70No,control62Not applicable55Not applicable

90No,control76Not applicable73Not applicable

100No,control93Not applicable85Not applicable 60,000Yes2/10,0000.029/20,0000.046

70,000Yes3/18,0000.1713/40,0000.033

90,000Yes6/25,0000.0247/50,0000.014

Mean,0.0260.004Mean,0.03160.016 Muthukumar et al.

(Fig.3E),while in control tissues,no GUS expression was observed (Fig.3F).In GUS staining,potassium ferrocyanide and potassium ferricyanide aids in rapid oxidation of X-Gluc during hydrolysis (Cervera,2005),which might have prevented GUS gene expression from being observed in all tissues that were analyzed for GUS expression,including the parts of the sporophytes.We expected that all C.thalictroides T1gametophytes and T2sporophytes from otherwise-con ?rmed transgenics would be GUS positive because this is a hermaphrodite species,but this was not the case.It is possible that transfer DNA (T-DNA)integration could have occurred in germinating spores during the 90-h cultivation pe-riod,which could have led to chimeric plants.

Fern Actin Promoter Performance

The 889-bp 59upstream region of the P.vittata actin gene was suf ?cient to highly express the GUSPlus re-porter gene in both ferns in all life stages,including prothalli,the sexual gametophytes,and the asexual diploid sporophytes.Moreover,the actin promoter isolated from P.vittata was able to function ef ?ciently in another Pteridaceae family member,C.thalictroides ,which might indicate regulatory elements in the actin promoter could be expected to function in other fern taxa as well.

Transformation Ef ?ciency

For P.vittata ,approximately 40,000to 50,000spores were subjected to three transformation experiments with no antibiotic selection (Table I).The average transfor-mation ef ?ciency,calculated at the prothalli stage,was 0.053%.If calculated at the gametophyte stage,it was 0.045%.Of the 5,000sporophytes that were still associ-ated with gametophytes that were randomly screened for GUS,only 40GUS-positive sporophytes were transferred

to pots.Of these,29sporophytes survived and were established in pots.This ef ?ciency likely underrepresents the actual transformation ef ?ciency because subsampling for GUS expression was performed at each life stage.Moreover,all sporophytes that were formed from gametophytes were not recovered and analyzed.Hence,for calculating transformation ef ?ciency,sporo-phytes were not considered.All of the 29

established

Figure 5.Southern-blot analysis of T1sporophytes of P.vittata (A,B,and C)and C.thalictroides (D).Genomic DNA from fronds were digested with Cla I and probed with a DIG-labeled hpt gene https://www.wendangku.net/doc/4817309294.html,nes 2to 5,7to 10,and 12to 22:transgenic https://www.wendangku.net/doc/4817309294.html,nes 1,6,and 23:nontransgenic P.vittata .Lanes 11and 24:Cla I-digested R4pGWB 501actin promoter-GUSPlus plasmid https://www.wendangku.net/doc/4817309294.html,nes 26to 35:transgenic C.thalictroides .Lane 34:nontransgenic C.thalictroides .DIG-labeled l DNA was used as a marker in all blots.Each plant sample lane rep-resents individual plants arising each from an independent https://www.wendangku.net/doc/4817309294.html,nes 23and 24were exposed longer (8h)than the rest of the blot (4h)using same

X-ray.

Figure 4.Vector map of R4pGWB501multisite Gateway binary vector containing the P.vittata actin promoter driving the GUSPlus gene with or without an apoplastic signal peptide sequence.There is a unique Bam HI site in the T-DNA that was used to determine insertion (copy)number in transgenic P.vittata and C.thalictroides sporophytes using southern-blot analysis.A 5.7-kb Cla I fragment is noted;it was utilized to diagnose whether A.tumefaciens plasmid-derived contamination was present using southern-blot analysis from putative transgenic frond samples.

Stable Transformation of Ferns

sporophytes were GUS positive and were found to contain T-DNA inserts using southern-blot analysis.For C.thalictroides ,approximately 60,000to 90,000spores were used for three transformation experiments with no antibiotic selection (Table I).If calculated at the prothalli stage,the transformation ef ?ciency was 0.02%.If calculated at the gametophyte stage,it was 0.03%.Of the 6,000sporophytes that were randomly selected for GUS assays,only 28of these were trans-ferred to pots.Of these,10of the 15sporophytes that survived transplanting were GUS stain positive.All of the 10GUS-positive plants were positive for the trans-gene in the southern-blot analysis.

Direct comparison of transformation ef ?ciencies among taxa is dif ?cult because each transformation method is in ?uenced by various factors and ef ?ciency calculations are not standardized.However,our fern spore trans-formation ef ?ciency reported here is nearly identical to that found for fungal (Fusarium oxysporum )spore trans-formation (Mullins et al.,2001),in which there was 0.05%(500transgenic conidia per 106spores)ef ?ciency.Ten-fold higher transformation ef ?ciency (0.8%,800spo-rangia from 105spores)has been reported for liverwort (Marchantia polymorpha )transformation based on initial hygromycin-resistant sporangial development from prothalli (Ishizaki et al.,2008).Similarly,in Arabidopsis plants,0.5%to 3%transformation ef ?ciency has been reported using the ?oral dip transformation method (Bechtold et al.,1993;Clough and Bent,1998).None-theless,?oral dip transformation is highly ef ?cient only in Arabidopsis and,apparently,only in some ecotypes.It is important to demonstrate stable transfor-mation and inheritance,which was shown here in the various life stages of two fern species.

We conservatively chose GUS as the visible marker for these experiments because of its sensitivity;we had

no a priori knowledge about the strength of promoters controlling marker gene expression.In the absence of using antibiotic selection,the experimental ef ?ciency could be increased by using a ?uorescent marker such as an orange ?uorescent protein gene instead of GUS (Mann et al.,2012).If an orange ?uorescence assay could be used at the prothalli stage,for example,subjecting a few thousand spores to transformation would lead to selecting just a few transformed ferns,which would greatly reduce the numbers of ferns handled through-out the life cycle.

Transgene Integration

Even though the ?rst generation of sporophytes be-longs to T1generation,they are largely supported by remaining gametophyte tissues,and hence we thought it might be possible that A.tumefaciens could persist in the sporophyte tissues.Southern-blot analysis was performed to test for stable integration of T-DNA in the genomes of both ferns and to estimate T-DNA copy numbers.

Southern-blot hybridization of Cla I-digested genomic DNA from fronds of sporophytes of both ferns using hygromycin phosphotransferase (hpt )gene probe from pGWB501vector revealed that a 5.7-kb A.tumefaciens plasmid band (Fig.4)was not present in any of the transgenic ferns assayed (Fig.5;Supplemental Fig.S4A).In all 29transgenic P.vittata ,the hpt probe hybridized to an approximately 2.0-to 2.1-kb Cla I fragment (Fig.5,A –C;Supplemental Fig.S4A),whereas in all 10trans-genic C.thalictroides (Fig.5D),strong hybridization was observed at 2.8kb.Additionally,in C.thalictroides ,there was also a weaker hybridization signal observed at 2.6kb.In both ferns,similar hybridization

signals

Figure 6.Southern-blot analysis of T1sporophytes of P.vittata (A and B)and C.thalictroides (C and D)to determine the number of T-DNA inserts.Genomic DNA from fronds were digested with Bam HI and probed with a DIG-labeled GUSPlus gene https://www.wendangku.net/doc/4817309294.html,ne 1:Bam HI-digested R4pGWB 501actin promoter-GUSPlus plasmid https://www.wendangku.net/doc/4817309294.html,nes 2to 7,9to 11,and 13to 22:transgenic P.vittata .Lanes 8and 12:nontransgenic P.vittata .Lanes 25to 28and 31to 36:transgenic C.thalictroides .Lanes 23and 29:Bam H1-digested plasmid positive https://www.wendangku.net/doc/4817309294.html,nes 24and 30:nontransgenic C.thalictroides .DIG-labeled l DNA was used as a marker in all the gels.Each plant sample lane represents in-dividual plants arising each from an independent spore.

Muthukumar et al.

were observed in most of the individual transgenic ferns examined.Moreover,all GUS positive lines were also hpt positive in the southern blots,which implies that all transgenic lines had the full T-DNA insert.

In the pGWB501vector,there is one Bam HI site present within the T-DNA(Fig.4),and Bam HI diges-tion was utilized to estimate number of T-DNA inserts integrated into the P.vittata and C.thalictroides genome among transgenic plants.In P.vittata,most of the trans-genic lines apparently contained more than one insert, and the hybridization patterns were similar among transgenic events.Transgenic lines in lanes2to7and 9and10had at least six inserts of the GUSPlus gene (Fig.6A).Only one line(lane19)had a single insert (Fig.6B).Transgenic lines loaded in lanes14,18,and 22had at least three inserts(Fig.6B).Lines loaded in lanes13,15,16and17,21and22had at least two inserts(Fig.6B).Lines loaded in lanes48to53and 55to57in Supplemental Figure S4had only two in-serts.The band sizes ranged from1.8to9.5kb.Based on the banding pattern and the copy number estima-tion in the southern-blot analysis,there were approx-imately seven independent transgenic P.vitatta events recovered.

C.thalictroides genomic DNA was apparently more dif?cult to digest with Bam HI compared with P.vittata genomic DNA.We used150units of Bam HI to digest 15m g DNA of P.vittata DNA,but C.thalictroides re-quired400units of enzyme to digest most of the genomic DNA.Despite this problem,both Cla I and Bam HI en-zymes effectively digested genomic DNA of both ferns (Supplemental Fig.S5).In C.thalictroides,four lines(lanes 25to28)were found to contain only a single T-DNA insert(Fig.6C).Transgenic lines loaded in lanes31and32 had an estimated four copies,and lines in lanes33and34 had at least six copies of the T-DNA.Lines in lanes 35and36were also found to have at least six copies but in different apparent locations compared with lines loaded in lanes31and32(Fig.6D).The band sizes ranged from1.8to8.5kb.Based on this analysis,we estimate there were up to10independent transgenic events analyzed.

Southern-blot analysis con?rmed that there was no A.tumefaciens-derived plasmid contamination and full-length integration of the T-DNA in transgenic plants. The presence of the2.0-kb hpt gene insert in both ferns and2.8and2.6kb in C.thalictroides depended on the nearest Cla I sites near the left border in the genome where it was integrated.It may be possible that a Cla I site might exist proximal to2kb at the LB-plant ge-nome junction.Multiple inserts that might not be ran-dom have been observed previously in transgenic angiosperms(Gelvin and Kim,2007),fungi(Michielse et al.,2005),and bryophytes(Ishizaki et al.,2008).Similar to our system,in which no selection pressure was used to select transgenic lines,it was found that in Arabidopsis, T-DNA was integrated at multiple locations in the ge-nome without any discrimination of DNA sequence (Kim et al.,2007).Southern blots for both the hpt gene and GUS gene substantiated the integration of the entire T-DNA in the fern genome.However,in eight lines of P.vittata and two lines of C.thalictroides,southern-blot analysis using the GUS probe showed many small-sized bands in both blots,which might be the result of indis-criminate restriction enzyme digestion occurring in both ferns,i.e.Bam HI.Although,in A.tumefaciens-mediated transformation,truncated transgene integration is less likely to occur than in biolistics,it has been observed in higher plants,especially in Pinus strobus and in wheat (Triticum aestivum;Levee et al.,1999;Hensel et al.,2012). In the remaining C.thalictroides lines and in P.vittata,it can be concluded that T-DNAs are mostly integrated intact.Multiple T-DNA insertion events per fern in both species were observed during this study.Because both ferns are homosporous and hermaphroditic in nature, multiple T-DNA insertions might have occurred in the initial haploid stages,as T-DNA insertion can occur multiple times during the transformation process. Multiple T-DNA insertions in a single line have been observed in Arabidopsis(Alonso et al.,2003;O’Malley and Ecker,2010),and multiple T-DNA insertions were also observed even within a single locus in Arabidopsis (Valentine et al.,2012).Based on our results,multiple T-DNA insertions might have occurred within a narrow region of the fern genome.Analyses of more transgenic fern lines will help determine the pattern of T-DNA insertion and will also determine whether more single insertion events can be obtained using this spore trans-formation method.

CONCLUSION

We have demonstrated that two fern species can be stably transformed by targeting spores using either A.tumefaciens or biolistics.We also discovered and validated a fern actin promoter,which is active in two fern species at each life stage.These tools should en-able expanded functional genomics studies in ferns, which will help to provide answers to many questions currently unknown in fern biology,including those regarding sex determination.Furthermore,because cv C-fern Express is emerging as a fern model,the ability to stably transform it will enhance its usefulness as a model.Practical research in P.vittata should also be enabled,especially with regards to arsenic hyper-accumulation(Gumaelius et al.,2004),to further develop its capabilities for phytoremediation and phytosensing applications.We are encouraged that ferns are appar-ently amenable to A.tumefaciens-mediated transforma-tion and that spores could be universal targets for fern transformation.

MATERIALS AND METHODS

Actin Promoter Isolation and Vector Construction Because there were no available fern promoters for transformation studies, we sought likely candidates in Pteris vittata.The P.vittata actin gene was isolated from genomic DNA using plant-conserved degenerate primers:forward primer, 59-ATGGCNGAYGGNGARGA-39and reverse primer(Bhattacharya et al.,

Stable Transformation of Ferns

1993),59-GAAGCAYTTGCGRTGSACRAT-39.The59upstream region of the putative actin gene was isolated by two-step genome-walking procedure us-ing the Genomewalker kit(Clontech).The putative actin promoter was used to regulate the expression of the GUSPlus reporter gene.Two GUSPlus variants, one with and one without a rice(Oryza sativa)signal peptide sequence,were used to determine if one was more effective than the other in fern transfor-mation.pCAMBIA1305.2(GenBank accession no.AF354046)features GUSPlus that contains a castor bean(Ricinus communis)intron and a rice apoplastic signal peptide.This version was used to transform P.vittata.For Ceratopteris thalictroides transformation studies,the same P.vittata actin promoter was used to control GUSPlus gene taken from pCAMBIA1305.1(GenBank accession no. AF354045),which lacks the rice apoplastic signal peptide.The actin pro-moter and GUSPlus genes were subcloned into pDONRP4P1R and PCR8/ GW/TOPO(Life Technologies),respectively,using the manufacturer’s in-structions for primer design and cloning.The promoter and the GUSPlus fragments were cloned into the Gateway binary vector R4pGWB501,which also harbors hpt for plant hygromycin selection(Nakagawa et al.,2008;Fig.4), using a multisite Gateway three fragment vector construction kit(Life Tech-nologies).After sequence con?rmation,the binary vector(12.6kb with T-DNA of6.0kb)was transformed into Agrobacterium tumefaciens strain EHA105(Hood et al.,1993)using a freeze-thaw method(H?fgen and Willmitzer,1988).The P.vittata actin promoter and actin gene sequences were deposited into GenBank (KC463697).

A.tumefaciens-Mediated Transformation of Spores

P.vittata was acquired from Edenspace Systems Corporation,and spores were collected from mature sporophyte fronds.C.thalictroides‘C-fern Express’spores were obtained from Carolina Biological Supply Company.One hun-dred milligrams of spores were suspended in2mL of sterile deionized water, cleaned using a60-m m nylon mesh followed by surface sterilization using 2.5%(v/v)commercial bleach containing a few drops of Tween20for5min, and then washed?ve times in equal volumes of sterile water.After each wash, the spores were pelleted using a tabletop centrifuge at10,000ɡfor3min. Spores were suspended in0.5mL1.5%(w/v)carboxy methyl cellulose(low viscosity,Fisher Scienti?c)as described in Shef?eld et al.(2001).The spore concentration was calculated based on hemocytometer counts.Based on the hemocytometer counts,the spore volume was adjusted to300spores50uL–1. To increase the ef?ciency of spore germination,the suspended spores were incubated at room temperature for15h before transformation or germination.

A2-mL A.tumefaciens inoculant culture was started in induction medium with the required antibiotic(Utermark and Karlovsky,2008)but without acetosyringone.After8h,250m L from the2-mL culture was inoculated into 25mL of the same medium.The culture was grown to0.8optical density for 15h at28°C,and then10mL of the culture was centrifuged at5,000ɡfor 15min and resuspended in20mL of the same A.tumefaciens growth medium. Two hundred micromolar acetosyringone was then added to induce virulence gene expression.The culture was further incubated for24h by shaking the suspension at60rpm at room temperature(25°C)on a rotary shaker.Then, 0.5mL of induced culture was mixed with0.5mL of sterilized spore sus-pension.This mixture was incubated for15min,and the entire suspension was plated as50-m L(300spores per plate)aliquots on top of a hydrophilic polyvinylidene di?uoride membrane(Fisher Scienti?c)in100-mm cocultivation agar plates containing A.tumefaciens growth medium with200m M acetosyr-ingone and2g L–1gellan gum for72h.Membranes were then transferred to one-half-strength MS plus20g Suc plates containing400mg L–1timentin to kill the residual A.tumefaciens.The spores were subcultured every2weeks in the same medium.The transformation experiment was repeated?ve times,and ef?ciency was calculated from three independent experiments.After transformation,we simply allowed the ferns to progress naturally through their life cycle.Spores germinated into gametophytes,which were transferred to one-half-strength MS (0g Suc)plates containing200mg L–1timentin for sporophyte development.All fern cultures were grown at22°C with65m mol m–2s–1light intensity using ?uorescent lights with16-h/8-h light/dark cycle.Mature sporophytes were eventually transferred to4-L pots containing PRO-MIX potting media.Spores were collected and analyzed for transgene inheritance analyses.The mature sporophytes were grown in growth chambers at24°C with105m mol m–2s–1 irradiance from?uorescent lights under a16-h/8-h light/dark cycle.We used nontransgenic ferns from the same accessions as controls,and they were treated identically(except transformation).T1spores were germinated in pots and allowed to grow until they formed gametophytes and sporophytes.Tissues were collected and analyzed for GUS expression.Hygromycin Sensitivity Assay

To determine whether hygromycin could be used as a selection agent,we performed a toxicity analysis by plating spores on one-half-strength MS me-dium containing20g Suc and400mg L–1timentin supplemented with hygro-mycin(0,3,5,10,15,and25mg L–1).After4weeks,hygromycin toxicity was determined based upon live/dead status of each fern.The experiment was repeated twice.

Particle Bombardment of P.vittata Spores

P.vittata spores were surface sterilized using70%(v/v)ethanol and10% (v/v)bleach.Surface sterilized spores were then plated onto polyvinylidene di?uoride membranes on one-half-strength MS with20g Suc medium in1-mL aliquots for biolistic transformation.Fresh spores,5-day-old germinating spores or protonemata,and15-day-old multicellular protenemata were used for transformation.The pMDC32vector containing a dual35S promoter driving the gusA was used for transformation(Curtis and Grossniklaus,2003). Biolistic transformation was carried out using conditions described for Glycine max that used0.6-m m gold particles(Hazel et al.,1998).Nicotiana tabacum ‘Xanthi’leaves grown from in vitro cultures were used as a positive control. P.vittata gametophytes and N.tabacum leaves were GUS stained after3weeks after the gametophytes were fully developed.

Marker Gene Analysis

GUS histochemical staining was performed using a standard protocol (Cervera,2005)with modi?cations to help in?ltrate the P.vittata sporophyte fronds,which were recalcitrant to standard staining.Fronds were perforated with carborundum600(Fisher Scienti?c)using cotton swab by painting on both adaxial and abaxial surfaces,which was subsequently removed by washing three times with0.1M Tris-HCl buffer,pH7.0.Fern tissues were incubated in GUS staining solution,destained using70%(v/v)ethanol to remove chlorophyll,rinsed with50%(v/v)glycerol,and viewed under a light stereoscope.

Southern-Blot Analysis

Genomic DNA from each fern species was isolated using a cetyl-trimethylammonium bromide method(Dong et al.,2005).Between3to5g of young transgenic and nontransgenic control sporophyte fronds were collected and frozen in liquid nitrogen.Tissues were homogenized in500m L extraction buffer containing2%(w/v)cetyltrimethylammonium bromide,100m M Tris-HCl,pH8.0,1.4M NaCl,20m M EDTA,and0.2%(v/v)2-mercaptoetanol and incubated at65°C for20min with50m g mL–1ribonuclease A.Then,an equal volume of chloroform:isoamyl alcohol(24:1,v/v)was added and mixed vigorously.The mixture was centrifuged immediately at6,000ɡfor20min. The aqueous phase was transferred to a fresh tube,and0.7mL–1of isopropyl alcohol was added and incubated at–20°C for at least2h.The DNA was pelleted by centrifuging the mixture at6,000ɡfor20min.The DNA pellet was washed twice with70%(v/v)ethanol and resuspended in400m L sterile distilled water.DNA was quanti?ed using?uorometry as well as visual es-timation using agarose gel electrophoresis.

For each sample,15m g genomic DNA was digested with Bam HI or Cla I restriction endonucleases(Fig.4)and electrophoresed in0.8%(w/v)agarose gels for22h.All gels were loaded with3to4m L of digoxigenin(DIG)-labeled l DNA marker III(Hin dIII-Eco RI digested,Roche Applied Science)as size marker and100picogram mL–1Cla I-or Bam HI-digested R4pGWB501plasmid DNA containing P.vittata actin promoter and GUSPlus,both of which can be detected in x-ray?lm along with experimental samples.DNA was transferred to a nylon membrane(GE Healthcare)by capillary blotting(Sambrook et al., 1989)and cross linked by UV irradiation.

Membranes were hybridized with a570-bp GUSPlus fragment gene or a 580-bp hpt fragment as probes in35mL DIG EasyHyb solution at42°C(for GUS probe,40°C)for2h.The GUSPlus primers were forward,59-CAAG-CACCGAGGGCCTGAGC-39and reverse,59-CTCAGTCGCCGCCTCGTTGG-39. The hpt primers were forward,59-GCGCTTCTGCGGGCGATTTG and re-verse,59-GTCCGAATGGGCCGAACCCG.Each probe was DIG labeled (Roche Applied Science),and hybridizations were conducted at42°C for the hpt gene and40°C for the gus gene in a rotating hybridization oven.The hy-bridization temperature was based on the melting temperature of the probe

Muthukumar et al.

and the formula based on DIG hybridization buffer as speci?ed in the man-ufacturer’s protocol(Roche DIG application manual for?lter hybridization). The membranes were then washed twice in23SSC(Sambrook et al.,1989)and 0.1%(w/v)SDS at room temperature for5min each and twice in0.13SSC and0.1%(w/v)SDS at60°C for15min each(Neuhaus-Url and Neuhaus, 1993;Dietzgen et al.,1999).Detection of the hybridized probe DNA was carried out as described in the manufacturer’s protocol.Ten-fold diluted CDP-Star chemiluminescent reagent(Roche Applied Science)was used(incubation time10min),and signals were visualized on x-ray?lm after6-to18-h exposure. The GUSplus probe was used for Bam HI-digested blots,and the hpt probe was used for Cla I-digested blots.

Sequence data from this article can be found in the GenBank data libraries under accession number KC463697.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1.The phylogenetic relationship of the P.vittata actin gene with selected actin genes from other plant species.

Supplemental Figure S2.Hygromycin kill curve analysis of P.vittata and

C.thalictroides,which were scored based on gametophytes formed after

3to4weeks of culture on one-half-strength MS medium.

Supplemental Figure S3.GUS histochemical staining of transgenic and control T1C.thalictroides sporophytic fronds.

Supplemental Figure S4.Southern-blot analysis of T1sporophytes of P.vittata to screen for A.tumefaciens plasmid contamination and for T-DNA insert number from a subsample of10of29T1P.vittata sporophytes.

Supplemental Figure S5.Agarose gel electrophoresis of Cla I-and Bam HI-digested samples that were used for P.vittata and C.thalictroides southern-blot analysis.

Supplemental Table S1.Putative regulatory motifs of P.vittata actin pro-moter.

ACKNOWLEDGMENTS

We thank Jonathan Willis for helping with DIG southern-blot experiments, Jason Burris for assistance in transformation experiments,Kellie Burris,Jennifer Hinds,Reggie Millwood,Yijia Zhang,and Mike Blaylock for their encour-agement and help,Mitra Mazarei and Reza Hajimorad for their assistance in carborundum perforation experiments,and Les Hickok for sharing in-formation about cv C-fern,which was useful in this study.

Received July10,2013;accepted August6,2013;published August9,2013.

LITERATURE CITED

Alonso JM,Stepanova AN,Leisse TJ,Kim CJ,Chen H,Shinn P,Stevenson DK,Zimmerman J,Barajas P,Cheuk R,et al(2003)Genome-wide inser-tional mutagenesis of Arabidopsis thaliana.Science301:653–657

Banks JA(1999)Gametophyte development in ferns.Annu Rev Plant Physiol Plant Mol Biol50:163–186

Banks JA,Nishiyama T,Hasebe M,Bowman JL,Gribskov M,dePamphilis C, Albert VA,Aono N,Aoyama T,Ambrose BA,et al(2011)The Selaginella genome identi?es genetic changes associated with the evolution of vascular plants.Science332:960–963

Bechtold N,Ellis J,Pelletier G(1993)In planta Agrobacterium mediated gene transfer by in?ltration of adult Arabidopsis thaliana plants.Life Sci 316:1194–1199

Bhattacharya DS,Stickel D,Sogin ML(1993)Isolation and molecular phylo-genetic analysis of actin coding regions from the prymnesiophyte alga, Emilianiahuxleyi,using reverse transcriptase and PCR methods.Mol Biol Evol 10:689–703

Bhattacharyya J,Chowdhury AH,Ray S,Jha JK,Das S,Gayen S,Chakraborty A,Mitra J,Maiti MK,Basu A,et al(2012)Native polyubiquitin promoter of rice provides increased constitutive expression in stable transgenic rice plants.Plant Cell Rep31:271–279Camloh M(1993)Spore germination and early gametophyte development of Platycerium bifurcatum.Am Fern J83:79–85

Cervera M(2005)Histochemical and?uorometric assays for uidA(GUS) gene detection.In L Pena,ed,Transgenic Plants:Methods and Protocols, Vol286.Humana Press,Totowa,New Jersey,pp203–213 Christensen AH,Quail PH(1996)Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants.Transgenic Res5:213–218

Chiyoda S,Ishizaki K,Kataoka H,Yamato KT,Kohchi T(2008)Direct trans-formation of the liverwort Marchantia polymorpha L.by particle bom-bardment using immature thalli developing from spores.Plant Cell Rep 27:1467–1473

Clough SJ,Bent AF(1998)Floral dip:a simpli?ed method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J16:735–743

Curtis MD,Grossniklaus U(2003)A gateway cloning vector set for high-throughput functional analysis of genes in planta.Plant Physiol133: 462–469

de la Riva GA,Gonzalez-Cabrera J,Vázquez-Padrón R,Ayra-Pardo C (1998)Agrobacterium tumefaciens:a natural tool for plant transformation.

Electron J Biotechnol1:18–133

Dhankher OP,Rosen BP,McKinney EC,Meagher RB(2006)Hyper-accumulation of arsenic in the shoots of Arabidopsis silenced for arsenate reductase(ACR2).Proc Natl Acad Sci USA103:5413–5418

Dietzgen RG,Abedina M,Higgins CM,Karunatratne S,Vickers J(1999) Nonradioactive detection of transgenes in plant genomic Southern blots.

Biochemica(Indianap,Ind)1:19–20

Dong R,Formentin E,Losseso C,Carimi F,Benedetti P,Terzi M,Schiavo FL(2005)Molecular cloning and characterization of a phytochelatin synthase gene,PvPCS1,from Pteris vittata L.J Ind Microbiol Biotechnol 32:527–533

Gelvin SB,Kim S-I(2007)Effect of chromatin upon Agrobacterium T-DNA in-tegration and transgene expression.Biochim Biophys Acta1769:410–421 Gifford E,Foster A(1989)Morphology and Evolution of Vascular Plants.

Freeman and Company,New York,pp626

Gumaelius L,Lahner B,Salt DE,Banks JA(2004)Arsenic hyperaccumulation in gametophytes of Pteris vittata.A new model system for analysis of arsenic hyperaccumulation.Plant Physiol136:3198–3208

Hazel CB,Klein TM,Anis M,Wilde HD,Parrott WA(1998)Growth characteristics and transformability of soybean embryogenic cultures.

Plant Cell Rep17:765–772

Hensel G,Oleszczuk S,Daghma DES,Zimny J,Melzer M,Kumlehn J(2012) Analysis of T-DNA integration and generative segregation in transgenic winter triticale(3Triticosecale Wittmack).BMC Plant Biol12:171

H?fgen R,Willmitzer L(1988)Storage of competent cells for Agrobacterium transformation.Nucleic Acids Res16:9877

Hood EE,Gelvin SB,Melchers LS,Hoekema A(1993)New Agrobacterium helper plasmids for gene transfer to plants.Transgenic Res2:208–218 Indriolo E,Na GN,Ellis D,Salt DE,Banks JA(2010)A vacuolar arsenite transporter necessary for arsenic tolerance in the arsenic hyperaccumulating fern Pteris vittata is missing in?owering plants.Plant Cell22:2045–2057 Ishizaki K,Chiyoda S,Yamato KT,Kohchi T(2008)Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L.,an emerging model for plant biology.Plant Cell Physiol49:1084–1091 Jones HD,Sparks CA(2009)Stable transformation of plants.In DJ Somers, P Langridge,JP Gustafson,eds,Methods in Molecular Biology,Vol513.

Humana Press,New York,pp111–130

Karami O,Esna-Ashari M,Kurdistani GK,Aghavaisi B(2009)Agrobacterium-mediated genetic transformation of plants:the role of host.Biol Plant53: 201–212

Kikkert JR,Vidal JR,Reisch BI(2004)Stable transformation of plant cell by particle bombardment/biolistics.In L Pena,ed,Transgenic Plants:Methods and Protocols.Humana Press,Totowa,New Jersey,pp 61–78

Kim S-I,Veena,Gelvin SB(2007)Genome-wide analysis of Agrobacterium T-DNA integration sites in the Arabidopsis genome generated under non-selective conditions.Plant J51:779–791

Lai Hy,Lim YY,Tan SP(2009)Antimicrobial tyrosinase inhibiting activities of leaf extracts from medicinal ferns.Biosci Biotechnol Biochem73:1362–1366 Levee V,Garin E,Klimaszewska K,Seguin A(1999)Stable genetic transfor-mation of white pine(Pinus strobus L.)after cocultivation of embryogenic tissues with Agrobacterium tumefaciens.Mol Breed5:429–440

Ma LQ,Komar KMM,Tu C,Zhang W,Cai Y,Kennelley ED(2001)A fern that hyperaccumulates arsenic.Nature409:579

Stable Transformation of Ferns

Mandal A,Purakayastha TJ,Patra AK,Sanyal SK(2012)Phyto-remediation of arsenic contaminated soil by Pteris vittata L.II.Effect on arsenic uptake and rice yield.Int J Phytoremediation14:621–628 Mann DGJ,Abercrombie LL,Rudis MR,Millwood RJ,Dunlap JR, Stewart CN Jr(2012)Very bright orange?uorescent plants:endoplas-mic reticulum targeting of orange?uorescent proteins as visual re-porters in transgenic plants.BMC Biotechnol12:17

Marrs RH,Watt AS(2006)Biological?ora of the British Isles245:Pteridium aquilinum(L.)Kuhn.J Ecol94:1272–1321

Michielse CB,Hooykaas PJ,van den Hondel CA,Ram AF(2005)Agrobacterium-mediated transformation as a tool for functional genomics in fungi.Curr Genet48:1–17 Mullins ED,Chen X,Romaine P,Raina R,Geiser DM,Kang S(2001) Agrobacterium tumefaciens-mediated transformation of Fusarium oxy-sporum:an ef?cient tool for insertional mutagenesis and gene transfer.

Phytopathology91:173–180

Nakagawa T,Nakamura S,Tanaka K,Kawamukai M,Suzuki T,Nakamura K, Kimura T,Ishiguro S(2008)Development of R4gateway binary vectors (R4pGWB)enabling high-throughput promoter swapping for plant re-search.Biosci Biotechnol Biochem72:624–629

Neuhaus-Url G,Neuhaus G(1993)The use of nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern blot hybridi-zation:A comparison with radioactivity.Transgenic Res2:115–120 O’Malley RC,Ecker JR(2010)Linking genotype to phenotype using the Arabidopsis unimutant collection.Plant J61:928–940

Rensing SA,Lang D,Zimmer AD,Terry A,Salamov A,Shapiro H, Nishiyama T,Perroud P-F,Lindquist EA,Kamisugi Y,et al(2008)The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants.Science319:64–69

Rutherford G,Tanurdzic M,Hasebe M,Banks J(2004)A systemic gene silencing method suitable for high throughput,reverse genetic analyses of gene function in fern gametophytes.BMC Plant Biol4:6.5 Sambrook J,Fritsch EF,Maniatis T(1989)Molecular Cloning:A Laboratory Manual.Cold Spring Harbor Lab Press,Plainview,New York,pp7.6–7.9 Schaefer D,Zryd JP,Knight CD,Cove DJ(1991)Stable transformation of the moss Physcomitrella patens.Mol Gen Genet226:418–424 Schedlbauer MD(1976)Fern gametophyte development:controls of di-morphism in Ceratopteris thalictroides.Am J Bot63:1080–1087Shef?eld E,Douglas GE,Hearne SJ,Huxam S,Wynn JM(2001)En-hancement of fern spore germination and gametophyte growth in arti-?cial media.Am Fern J91:179–186

Shelmerdine PA,Black CR,McGrath SP,Young SD(2009)Modelling phytoremediation by the hyperaccumulating fern,Pteris vittata,of soils historically contaminated with arsenic.Environ Pollut157: 1589–1596

Srivastava S,Suprasanna P,D’Souza SF(2012)Mechanisms of arsenic tolerance and detoxi?cation in plants and their application in transgenic technology:a critical appraisal.Int J Phytoremediation14:506–517 Stachel SE,Nester EW,Zambryski PC(1986)A plant cell factor induces Agrobacterium tumefaciens vir gene expression.Proc Natl Acad Sci USA 83:379–383

Stout SC,Clark GB,Archer-Evans S,Roux SJ(2003)Rapid and ef?cient sup-pression of gene expression in a single-cell model system,Ceratopteris ri-chardii.Plant Physiol131:1165–1168

Sundaram S,Rathinasabapathi B(2010)Transgenic expression of fern Pteris vittata glutaredoxin PvGrx5in Arabidopsis thaliana increases plant tolerance to high temperature stress and reduces oxidative damage to proteins.Planta231:361–369

Sundaram S,Wu S,Ma LQ,Rathinasabapathi B(2009)Expression of a Pteris vittata glutaredoxin PvGRX5in transgenic Arabidopsis thaliana in-creases plant arsenic tolerance and decreases arsenic accumulation in the leaves.Plant Cell Environ32:851–858

Thaler JS,Owen B,Higgins VJ(2004)The role of the jasmonate response in plant susceptibility to diverse pathogens with a range of lifestyles.Plant Physiol135:530–538

Utermark J,Karlovsky P(2008)Genetic transformation of?lamentous fungi by Agrobacterium tumefaciens.Nat Protocols10:

Valentine ME,Wolyniak MJ,Rutter MT(2012)Extensive phenotypic variation among allelic T-DNA inserts in Arabidopsis thaliana.PLoS ONE7:9e44981

Wilkinson JE,Twell D,Lindsey K(1997)Activity of CaMV35S and nos promoters in pollen:implications for?eld release of antisense transgenic plants.J Exp Bot48:265–275

Wu FH,Shen SC,Lee LY,Lee SH,Chan MT,Lin CS(2009)Tape-Arabidopsis Sandwich-a simpler Arabidopsis protoplast isolation method.Plant Methods5:16

Muthukumar et al.