In vitro anther culture and Agrobacterium-mediated transformation of the AP1 gene from Salix integra Linn.in haploid poplar(Populus simonii×P.nigra)

Jingli Yang?Kun Li?Chunyan Li?Junxiu Li?Bo Zhao?Wei Zheng?Yuchi Gao?Chenghao Li

Introduction

Poplar,as a model forest tree,has been widely used in biotechnology and genetic studies(Taylor 2002;Wullschleger et al.2002).Because poplar is dioecious and undergoes cross-pollination,its genome is characterized by a high level of heterozygosity,which has posed a signi ficant obstacle in poplar breeding and genetic research(Deutscha et al.2004).Haploid techniques are a valuable tool for the rapid production of homozygous transgenic plants,thereby accelerating cultivar improvement and enabling a timely response to changing market requirements.

In the 1950s,haploid culture was used for tree species,but remained at the callus induction stage.Among the various haploid culture methods,anther culture has been the most conducive to recovering homozygous inbred lines and for generating haploid plants of poplar species and other forest trees(Deutscha et al.2004).Although many studies have reported the production of haploid poplar,the results of anther culture of poplars have been disappointing and negligible compared with most of the results for herbaceous crops(Baldursson and Ahuja 1996),and transgenic haploid poplar plants have not yet been generated.

Success of the procedure depends on the culture medium composition(Asaduzzaman et al.2003),culture conditions(Raina and Ifran 1998),pretreatment(Trejo-Tapia et al.2002),microspore developmental stages(Afza et al.2000),callus formation and plantlet regeneration(Orshinsky and Sadasivaiah 1997;Stober and Hess 1997).Low culture ef fi ciency,early spontaneous chromosome doubling and somatic regeneration from the anther wall tissue are the main obstacles to obtaining pollen plants.

For fl owering plants,the transition to fl owering is the most critical event in the life cycle and is tightly controlled by MADS-box genes.The geneAPETALA1(AP1)is a MIKC-type MADS-box factor.It plays a key role in establishing the fl oral meristem identity by regulating genes related to phase transition and fl ower initiation(Kaufmann et al.2010).In addition,theAP1gene is involved in sepal and petal formation(Pabón-Mora et al.2012),and numerous gene homologs ofAP1have been identi fi ed in various plant species(Fernando and Zhang 2005;Mimida et al.2011).Constitutive expression of theAP1gene or homologous genes in herbaceous and woody species accelerates the initiation of fl owering and changes the morphology of transgenic plants.GmAP1 isolated from soybean promotes the fl owering time and determines fl oral organ formation when overexpressed in tobacco(Chi et al.2011).Ectopic expression ofAP1-like genes inArabidopsispromotes early fl owering(Wang et al.2013;Chen et al.2015).Kim et al.(2006)reported that overexpression of theAP1homologMdMADS5/MdAP1in‘Fuji’resulted in a dramatic shortening of the juvenile stage.Overexpression ofAP1also induces early fl owering inBetula platyphylla×B.pendula(Huang et al.2014).These results indicate thatAP1regulates fl ower development,which is necessary and suf fi cient for turning an in fl orescence meristem into a fl oral meristem.Therefore,if the expression ofAP1-like genes that control fl owering time and alter fl oral organ identity is controllable,it may produce valuable phenotypic changes that improve tree species.

The seed hair of female fl oral buds and the pollen of poplar are allergens(An et al.2011;Chen et al.2015),so the ability to control fl owering time and identify speci fi c fl owering-related genes in poplar thus has great practical signi fi cance.However,as Strauss et al.(2004)reported,overexpression ofAP1inPopulusdoes not induce early fl owering,and we do not understand the functions and roles of AP1 inPopulusclearly.

Materials and methods

Plant materials and fl ower induction

Branches of malePopulus simonii×P.nigrawere collected from Zhaoyuan City,Heilongjiang Province in February 2007.The materials were transported to the greenhouse at Northeast Forestry University and stored in a cellar at 0°C for 1 week,then grown hydroponically at room temperature(16–20 °C)to induce fl owering.

Isolation and culture of anthers

Anthers were isolated and developmental stages observed micorscopically from February to April.Mid-and lateuninucleate anthers were agitated in 75%ethanol for 1 min,surface-sterilized using 30%hydrogen peroxide for 10 min,and then rinsed fi ve times with sterile distilled water.Isolated anthers were cultured on MS medium(Murashige and Skoog 1962)containing 30 g L-1sugar supplemented with 2.0 mg L-12,4-dichlorophenoxyacetic acid(2,4-D)and 0.5 mg L-1kinetin(KN)for callus induction.Materials were cultured at 25°C under illumination at 45 μmol m-2s-1with white light,25 °C in the dark,or 21°C under red light with the same wavelength for 20–70 days.

Adventitious bud induction

After 4 weeks of callus induction,the callus was cultured in MS medium supplemented with different concentrations of 6-benzylaminopurine(6-BA),α-naphthaleneacetic acid(NAA)and thidiazuron(TDZ).The cultures were incubated under a 16 h light/8 h dark photoperiod at 25°C under illumination at 45 μmol m-2s-1with cool fl uorescent lights.After 4 weeks,the induction frequency(%)was calculated to determine the optimal culture conditions for adventitious buds induction.

Plant regeneration and transplantation

After culturing in elongation MS medium containing 20 g L-1sucrose supplemented with 0.8 mg L-1BA and 0.01 mg L-1NAA for 1 month,adventitious buds(2–4 cm long)were selected for culture in MS medium containing 20 g L-1sucrose and 0.2 mg L-1indole-3-butytric acid(IBA)for rooting.After 4 weeks on agar medium,regenerated plantlets with a well-developed leaf and root were subcultured at 4-week intervals.

Plantlets that regenerated in vitro with well-developed leaf and root systems were selected and transferred to pots containing autoclaved sand and soil(1:3 mixture).The pots were covered with polyethylene bags to maintain high humidity.The bags were perforated,and the covers were removed after 3 weeks when the plants showed new leaves.The survival rates were calculated after 8 weeks of hardening.

Ploidy level analysis

Ploidy was determined using a PA-I Ploidy Analyzer(Partec,Munster,Germany).Brie fl y,approximately 0.5 cm2of young leaves from 15 lines of regenerated plantlets were soaked in a plastic Petri dish containing 1.5 mL of ice-cold Partec HR-A solution(Lysing solution,Partec,)for 2–5 min and were minced with a sharp razor blade.The sample was fi ltered through a 30-μm CellTrics fi lter(Partec)and incubated in a sample tube for 5 min at room temperature.The fi ltered nuclei were stained with 700 μL of Partec HR-B solution for 5 min three times.The DNA ploidy of nuclei was compared among different plantlets to assess the incidence of polyploidy.The haploid and diploid plantlets that were identi fi ed were used for transformation.

Simple sequence repeat(SSR)analysis

Genomic DNA was isolated from leaf tissue of regenerated haploid and diploid plants that were previously analyzed for ploidy level,and from the donor tree.DNA was extracted using the CTAB protocol and digested with RNase(Takara,Japan)at 37°C for 30 min.The DNA concentration was measured with a photometer(Eppendorf,Hamburg,Germany).Five primers,i.e.,WPMS 9 and WPMS 13(Van der Schoot et al.2000)and primers WPMS 14,WPMS 18,and WPMS 20 designed by Smulders et al.(2001)were referenced for SSR analysis in our study(Table 3).One line of a haploid DNA sample was used for the positive control,and the donor tree was used as the negative control.

The 25-μL PCR reaction mixture,including 1× PCR reaction buffer,1.6 mM MgCl2,0.2 mM of each primer,0.1 mM of each dNTP,0.5 U of Taq DNA polymerase(Eurogentec,Cologne,Germany)and 20 ng of genomic DNA was used for the SSR analysis.An initial denaturation at 94°C for 3 min was followed by 35 cycles of 1 min at 92 °C for denaturation,30 s at 60 °C for annealing and 1 min at 72°C for extension;and a fi nal extension step of 7 min at 72°C.

Cloning and construction of AP1

Female branches ofSalix integraLinn.were collected from Maoer Mountain of Harbin,Heilongjiang Province,then hydroponically cultured(Tomioka et al.2005)for 2 weeks at room temperature.Total RNA was isolated from fl ower shoots using the 2%CTAB method,and the RNA was then subjected to reverse transcription using a Reverse Transcriptase kit(TaKaRa Biotech,Dalian,China).The fulllengthAPETALA1(SpAP1,GenBank accession KF656 720)was ampli fi ed with the gene-speci fi c primers F:5′-TAGGATCCTATGGGAAGAGGTAGGGTT-3′and R:5′-TGAGCTCGATCATGCTCCATAGCCTCCA-3′by PCR and then cloned into the pGEM-T vector(Promega,Madison,WI,USA)for sequencing.

The full length CDS of theSpAP1gene was ampli fi ed by RT-PCR with the gene-speci fi c primer pair mentioned above containing BamHI and SacI restriction sites at the ends of the forward and reverse primers,respectively.The CDS was then cloned into the pROKII vector,which carried the gene encoding hygromycin phosphotransferase(hptII)as a plant selection marker under control of the strong constitutive CaMV35S promoter.The recombinant plasmid pROKII-SpAP1was inserted intoAgrobacterium tumefaciensEHA105 and then used to transformS.purpurea.

Transformation and regeneration of transgenic plants

Both sides of the leaves of haploid and double haploid poplar(diploid)plants were removed,and a slit in the base of the midrib was cut with a knife,and the leaves were then inoculated withAgrobacteriumEHA105 at OD600=0.6 density for 30 min.After 2 days of co-cultivation on induction medium in the dark,the leaves were washed with sterile water containing 250 mg L-1cefotaxime to kill theAgrobacteriumcells.Then the explants were transferred to the same medium supplemented with 50 mg L-1kanamycin(Km)and 250 mg L-1cefotaxime(Cef)for induction and multiplication oftransgenic shoots.After 2 months,multiple shoots were successively transferred to elongation and rooting medium containing 50 mg L-1km and 250 mg L-1Cef.The transgenic plants were successively subcultured at 4-week intervals.

PCR and RT-PCR analysis of putative transformation

Plant genomic DNA was isolated using a DNeasy Plant Minikit(QIAGEN)from the leaves of non-and transgenic lines.The DNA of the plasmid pROKII-SpAP1was used as a positive control,and the DNA from nontransgenic plants was used as a negative control.PCR was performed using a primer pair speci fi c for theSpAP1gene(F:5′-TAGGATCCTATGGGAAGAGGTAGGGTT-3′andR:5′-TGAGCTCGATCATGCTCCATAGCCTCCA-3′). The PCR product was expected to be a 753-bp fragment.The PCR conditions were 94°C for 3 min;followed by 30 cycles of 94 °C for 30 s,58 °C for 30 s and 72 °C for 40 s;and a fi nal 7 min extension at 72°C.

For RT-PCR,total RNA was extracted using a Plant RNA Puri fi cation Reagent(Invitrogen,Carlsbad,CA,USA).First-strand cDNA was synthesized from 0.5 μg of puri fi ed RNA and reverse-transcribed with a Reverse Transcriptase kit(TaKaRa Biotech,Dalian,China).The PCR conditions were identical to those described above.

Culture conditions and statistical comparison

Media used in experiments were adjusted to pH 5.8 then agar(Duchefa,Netherland)was added(0.8%w/w),and then sterilized by autoclaving at 1.1 kg cm-2(121°C)for 15 min.Cultures were performed in 100-mL Erlenmeyer fl asks with 30 mL medium and subcultured at 4-week intervals,and were maintained at 24± 2°C with a 16-h day-1photoperiod using cool white fl uorescent tubes(36 μmol s-1m-2).Means were separated using Duncan’s multiple range test atP=0.05.

Results

Induction of anther callus

To rapidly generate diploid plants,we used anther culture.Mid-and late-uninucleate anthers were cultured in MS medium containing 30 g L-1sugar supplemented with 2.0 mg L-12,4-D and 0.5 mg L-1KN to induce callus.Analysis of variance indicated that the induction frequency was signi fi cantly different after 4 weeks under various conditions(P=0.000)(Table 1).The highest induction frequency occurred in the 25°C dark culture(Fig.1a;Table 1),followed by 25°C white light,which reached 64.1%(Table 1;Fig.1b).The lowest induction frequency was 32%at 21°C white light(Table 1;Fig.1c)and at 21°C red light cultures(Table 1;Fig.1d).As shown in Fig.2a,two types of callus were produced from anthers.Some anthers became swollen after 4-days in culture,and the callus was induced after 2 weeks.Microscopic observation of anther cultures for 4 days showed that most of the anther wall and pollen grains were adherent(Fig.2a).Therefore,the callus induced by anthers is likely to be a source of pollen grains and anther walls.Because the callus from the pollen wall grows differently than that from the pollen grain,many of the adventitious buds are likely not the source of pollen grains.Some anther walls became red after 3-days of culture at 21°C white light,and there was no obvious shape change during the culture process.However,the pollen grain inside gradually expanded until callus formed and then extruded from the anther wall after approximately 7 days of cultivation(Fig.2b).The callus is easily separated from the anther wall when it is yellow,round,smooth,compact and slow growing,which are indicative of haploid cells.Taken together,the culture condition of 21°C white light is more suitable for pollen grain callus induction.

Adventitious bud induction

Different combinations of PEG signi fi cantly affected the induction frequency of adventitious buds(P=0.010).The highest induction frequency(36.1%)was reached when the samples were transferred to MS medium supplemented with 1.0 mg L-16-BA,0.2 mg L-1NAA and 0.1 mg L-1TDZ(Table 2;Fig.3a).

Rooting and transplantation

After culturing in elongation MS medium containing 20 g L-1sugar supplemented with 0.8 mg L-1BA and 0.01 mg L-1NAA for 4 weeks,the stem became longer and more leaves grew(Fig.3b).The elongated shoots were cut to approximately 2–4 cm high for culture in MS medium containing 20 g L-1sugar and 0.2 mg L-1IBA for rooting.After 6 days,the shoots began rooting.A total of 4–7 taproots developed for each shoot,and rooting rate was 100%.At least two types of plantlets were regenerated,a dwarf phenotype with small leaves(Fig.3c)and a phenotype with normal growth(Fig.3d).

A total of 721 plantlets from 16 plant lines with welldeveloped root systems were transplanted into small containers containing autoclaved sand and soil(1:3).During the early transplantation period,a transparent plastic fi lm was used to cover the containers to maintain high humidity.Plantlets were generally grown at 21°C,and the plastic cover was removed 6 weeks later.The survival rate was 95.7%,and development was normal.

Fig.1 Callus formed from isolated immature anther of Populus simonii×P.nigra after 4 weeks of culture.The culture conditions were a 25 °C dark,b 25 °C white light,c 21 °C white light,and d 21 °C red light.Bars 1 cm

Fig.2 Stereomicrographs of anthers after 3 days of culture at 25 °C white light(a)and after 7 days of culture at 21 °C white light(b).Bars a 200 μm,b 800 μm

Ploidy stability analysis

In 87 samples from 16 lines,two plant lines were haploid(Fig.4a),six lineswere diploid (Fig.4b),and the remaining were tetraploid or heterozygote.

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SSR

Among the fi ve primers used,WPM09 produced clear and strong bands,whereas the other four primers were not stable and showed poor repeatability(Fig.3).In Fig.5,one diploid,as measured by fl ow cytometry,revealed the same heterozygous allelic combination as the donor trees,and fi ve diploids had the same homozygous allelic combination as the haploid sample(Table 3).

Table 2 Effect of plant growth regulators on pollenadventitious buds induction of P.simonii×P.nigra

Fig.3 Plant regeneration from pollen grain callus.a Adventitious buds induced from pollen grain callus.b Elongation of adventitious buds.Haploid plant with dwarf phenotype and small leaves(c)and d normal growth.Bars a 1.5 cm,b 4 mm,c 1.5 cm,c 1.5 cm

Evaluation and phenotypic observation of SpAP1-transgenic Populus

To assess whetherSpAP1affects the fl owering time and fl oral development of poplar,we generated transgenic haploid and diploid plants in whichSpAP1was overexpressed under control of the CaMV 35S promoter.The media used in the transformation process are described in Table 4.A total of six transgenic haploid poplar lines that were successively subcultured were con fi rmed by PCR(Fig.6a)and RT-PCR(Fig.6b)analysis.According to PCR,six lines produced a 753-bp bands,whereas the nontransgenic plants yielded no PCR products(Fig.6a).NontransgenicPopulushad multiple shoots(Fig.7a).However,RT-PCR using the same speci fi c primers showed thatSpAP1was expressed in four individuals(Fig.6b)with an early fl owering phenotype(Fig.7b)and was not expressed in two individuals did not fl ower early.There are two types of transgenic poplar phenotypes(Fig.7b),and this type of fl ower cluster is can be cultured in vitro.However,a few phenotypes with fl owers that had obvious petals(Fig.8a)could not be subcultured until after the fl owers faded.Viewed from the side,the transgenic plants had a few white petals(Fig.8b),and in top view,the fl ower had a stamen-like organ in the center(Fig.8c).The relationship betweenSpAP1expression levels and the early fl owering phenotype in transgenic haploid lines indicates that overexpression ofSpAP1is related to early fl owering.This study highlights the need for further research on the mechanism by whichSpAP1triggers early fl owering in haploid but not diploidPopulus.

Fig.4 Determination of ploidy using fl ow cytometry.a Haploid,b diploid.Note Position of the fi rst peak on the left determines the ploidy

Fig.5 SSR analyses.H haploid used as positive control.Ck donor tree used as control plant.Single star haploid;double stars diploid

Table 3 Primers used for SSR analysis

Discussion

In addition to its use in breeding,haploid plants are useful in mutation studies,gene mapping,functional genomics,and as a target for transformation.During transformation,haploid plants can be cocultured withAgrobacteriumto obtain transgenic doubled haploids(DH),resulting in stable transgenes,which has led to a resurgence of interest in haploid research.Haploids are not only used for the application of crop improvement programs but also as an important tool to study gamete and embryo biology and genetics,including gene mapping,gene discovery,and identi fi cation.

For the formation of haploids using anther culture,the pretreatment and cultivation conditions have the greatest impact.In the present study,we regenerated plants from isolated anthers of poplar using direct organogenesis.The effect of low temperature pretreatment,various culture conditions and growth regulators on haploid induction were also investigated.

Table 4 Media used in transformation protocol of P.simonii×P.nigra

Fig.6 Molecular analyses of 35S::SpAP1-transgenic plant lines.a PCR analysis with speci fi c primers.b RT-PCR analysis with speci fi c primers.M DL2000,P positive control,W wild type,1–9 PCR products of SpAP1 gene in transgenic plant

We selected mid-and late-uninucleate pollen grains as the initial material to induce callus because the pollen development stage is a key complex factor that strongly affects the success of anther culture.Sopory and Munshi(1996)reported that the microspore stage affects the ploidy level of the plant produced in the anther culture because in their study,plantlets obtained from pollen at the uninucleate stage were mostly haploids,whereas plants with higher chromosome numbers were produced by anthers at later stages.

Culture temperature and light also affected callus induction in our study.Light is an environmental signal that regulates pollen morphogenesis in vitro.Although darkness or low-intensity light is more suitable than white light,most of the calli that develops from pollen grains are adherent to swollen anther walls under this condition.The optimal condition for pollen grain callus induction is 21°C with white light,and the pollen grain callus induced by these conditions was easily separated from the anther wall callus.

Finally,two lines of haploid and six lines of diploid plants were obtained,of which one line was heterozygote and fi ve diploids were homozygous allelic based on SSR analysis.Although many factors were investigated in herbaceous plants,it was dif fi cult to test these factors in wood plants.Only one report on the embryogenesis and plant regeneration of haploid poplar induction through the anther has been published,Deutscha et al.(2004),and the impact of various treatments,including the storage of donor material,pretreatments,growth regulators and other culture conditions were described.

Fig.7 Plant phenotypes of adventitious buds.a Nontransgenic plant,b 35S::SpAP1 transgenic plant.Bars a 1 mm,b 1 mm

Previous studies showed thatAP1accelerates fl owering in many other plants,such asArabidopsis,citrus and apple tree(Fernando and Zhang 2005;Flachowsky et al.2007).InArabidopsis,AP1belongs to the A class in the ABC model and speci fi es sepal and petal identities.Overexpression ofAtAP1results in early fl owering and converting the in fl orescence into a fl ower-like phenotype(Mandel and Yanofsky 1995).AP1 is positively regulated by the photoperiodic response module,FT/FD(Corbesier et al.2007),and activates B-class fl oral organ identity genesAPETALA3(AP3)andPISTILLATA(PI),which determine the identity of petals and stamens,respectively(Irish 2010).InB.platyphylla,overexpression ofBpAP1induces in fl orescences to emerge within 2 months after transplantation,resulting in a signi fi cantly shortened juvenile period compared with the typical 10–15 years until fl owering.However,As reported by Strauss et al.(2004),35S::AP1is not functional in poplar,as reported by Strauss et al.(2004),likely because the genomic information of poplar is different and complex.But Chen et al.(2015)found ectopic expression of AtAP1M3 in poplar resulted in the expression some key endogenous fl owering-related genes,including fl oral meristem identity geneLEAFY(LFY),B-class fl oral organ identity genesAP3andPI, fl owering pathway integratorFLOWERING LOCUS T1(FT1)and fl ower repressorsTERMINAL FLOWER 1(TFL1)andSHORT VEGETATIVE PHASE(SVP).However,none of the fl owers in AP1-transgenic poplar had anyobviously abnormal sepals,petals and stamens.

Fig.8 Flower of in vitro 35S::SpAP1-transgenic plant lines with petals.a Early fl owering of in vitro.Morphology of transgenic Populus fl ower b side view,c top view of transgenic plant.Bars a 5 mm,b 2 mm,c 2 mm

Interestingly,ectopic expression ofSpAP1signi fi cantly promoted fl owering in haploid poplar in this study.Meanwhile,the transgenicPopulusfl owers had petals instead of in fl orescence.Histological analysis showed that fl ower primordia were present in the shoot apical meristem of the transgenic haploid,whereas the shoot apex of control plants remained in the vegetative stage.Moreover,stamenlike structures appeared in the central of fl ower of transgenicPopulus.These results indicate thatAP1may play an important role in establishing fl oral meristem identity and may be involved in petal formation of the haploid.Therefore,if the expression ofAP1-like genes that control the fl owering time and alter the fl oral organ identity is controllable,valuable phenotypic changes might be generated to improve the tree species.

The ability to control fl owering time and the identi fication of speci fi c fl owering-related genes in poplar are of great practical signi fi cance.This is the fi rst study on AP1 induction of early fl owering inPopulus.Although the precise mechanism by which theAP1gene modi fi es fl owering in haploidPopulusis not clear,these results open new possibilities for genetic improvement and breeding ofPopulustree species.

Conclusions

Thehighestcallusinductionfrequencyoccurredfromanther of haploidP.simonii×P.nigrain the 25 °C dark culture.Two kinds of callus were induced separately from pollen grains or anther walls.Callus that separates from the anther wall is indicative of haploid cells.In the present study,we reported a successful protocol for the culture and plant regeneration of haploid poplar from the isolated anther of poplar.Ectopic expression of anAP1gene isolated fromSalix integraLinn.signi fi cantly promoted fl owering and reduced the vegetative growth period in haploid poplar.

AcknowledgementsThe Fundamental Research Funds for the Central Universities(2572015EA01),the 111 Project(B16010),the Innovation Project of State Key Laboratory of Tree Genetics and Breeding(Northeast Forestry University;Grant Number 2013A04)and Natural Science Fund of Heilongjiang Province (No.QC2015035)supported this study.

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