Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soyb
GENETIC TRANSFORMATION AND HYBRIDIZATION
Tissue culture speci?city of the tobacco ASA2promoter driving hpt as a selectable marker for soybean transformation selection
Olga Zernova ?Wei Zhong ?Xing-Hai Zhang ?Jack Widholm
Received:1May 2008/Revised:14July 2008/Accepted:25July 2008/Published online:5August 2008óSpringer-Verlag 2008
Abstract This study was carried out to determine if the tobacco anthranilate synthase ASA2 2.3kb promoter drives tissue culture speci?c expression and if it is strong enough to drive hpt (hygromycin phosphotransferase)gene expression at a level suf?cient to allow selection of trans-formed soybean embryogenic culture lines.A number of transformed cell lines were selected showing that the pro-moter was strong enough.Northern blot analysis of plant tissues did not detect hpt mRNA in the untransformed control or in the ASA2-hpt plants except in developing seeds while hpt mRNA was detected in all tissues of the CaMV35S-hpt positive control line plants.However,when the more sensitive RT-PCR assay was used all tissues of the ASA2-hpt plants except roots and mature seeds were found to contain detectable hpt mRNA.Embryogenic tis-sue cultures initiated from the ASA2-hpt plants contained hpt mRNA detectable by both northern and RT-PCR analysis and the cultures were hygromycin resistant.Fria-ble callus initiated from leaves of ASA2-hpt plants did in some cases contain hpt mRNA that was only barely detectable by northern hybridization even though the callus was very hygromycin resistant.Thus the ASA2promoter is strong enough to drive suf?cient hpt expression in soybean embryogenic cultures for hygromycin selection and only
very low levels of expression were found in most plant tissues with none in mature seeds.
Keywords ASA2promoter á
Soybean embryogenic cultures áSelectable marker áTissue culture speci?c expression
Abbreviations
ASA2-hpt ASA2promoter driving hpt transformant 35S-hpt CaMV35S promoter driving hpt transformant GUS b -Glucuronidase MUG 4-Methylumbelliferyl-glucuronide RT-PCR Reverse transcriptase polymerase chain
reaction
Introduction
Since most methods used for inserting genes into plant systems are very inef?cient,selectable marker genes are used so that the transformed cells or seedlings can be selected from the untransformed cells or seedlings.There are many possible selectable marker genes,but the most commonly used impart either antibiotic or herbicide https://www.360docs.net/doc/2c18945516.html,ually the selectable marker genes are driven by constitutive promoters that cause high levels of expression during the selection and in most plant tissues throughout the life cycle.There could be reasons to not have the selectable marker gene be expressed in the regenerated plant as in the case of herbicide resistance that might be spread to weeds via pollen or if the expression is detrimental to plant growth,development or fertility.This would include the possibility that production of unneeded proteins could use energy and affect plant performance as a
Communicated by D.Somers.
O.Zernova áW.Zhong áJ.Widholm (&)
Edward R.Madigan Laboratory,Department of Crop Sciences,University of Illinois,1201W.Gregory,Urbana,IL 61801,USA e-mail:widholm@https://www.360docs.net/doc/2c18945516.html,
X.-H.Zhang
Department of Biological Sciences,Florida Atlantic University,777Glades Road,Boca Raton,FL 33431,USA
Plant Cell Rep (2008)27:1705–1711DOI 10.1007/s00299-008-0589-7
consequence of‘‘metabolic drain’’(Purrington and Bergelson1997).However,little evidence to support this hypothesis has appeared but Purrington and Bergelson (1997)showed that Arabidopsis thaliana plants trans-formed with a mutant acetolactate synthase gene that conferred resistance to the herbicide Chlorsulfuron had a 26%reduction in seed yield while plants transformed with a gene conferring kanamycin resistance showed no seed yield decrease.The presence of the selectable marker gene protein in seed or other harvested tissues could also be objectionable to the public.Thus the use of a promoter that is tissue culture speci?c might be of value in some cases.
The feedback insensitive anthranilate synthase gene, ASA2,is expressed in5-methyltryptophan resistant tobacco cell cultures and not in regenerated plants as shown by measurement of the enzyme activity(Brotherton et al.1986)and mRNA and transient expression(Song et al.1998).We have recently transformed soybean with the2.3kb ASA2promoter fragment driving the uidA gene (b-glucuronidase)(Inaba et al.2007b)to determine the ASA2promoter expression in another species.The plants express observable GUS histochemical activity only in pollen and seed while embryogenic cultures and friable callus cultures also show blue staining.However,mea-surement of GUS activity using the quantitative and sensitive4-methylumbelliferyl-glucuronide(MUG)bio-chemical assay showed that the ASA2promoter drove extremely low but measurable levels of expression in leaf and stem tissues as well.
In the study described here we bombarded embryogenic suspension cultures of soybean with a construct containing the ASA2promoter driving the hpt(hygromycin phos-photransferase)gene that imparts hygromycin resistance. Hygromycin selection was used to obtain transformed lines that were regenerated into plants where hpt gene expres-sion was studied.Both embryogenic and friable tissue cultures were also initiated from the plants for the study of hpt expression and hygromycin resistance.
Materials and methods
Tissue culture,transformation and plant regeneration The methods described in Zhang et al.(2005)were used to initiate embryogenic suspension cultures of soybean(Gly-cine max(L.)Merrill cv.Jack).The cultures were bombarded with0.6l m diameter gold particles(Bio-Rad, https://www.360docs.net/doc/2c18945516.html,)coated with0.1l g plasmid DNA per shot using a PDS1,000/He Particle gun(Bio-Rad) using1,100psi rupture disks under27–28in.of mercury vacuum.After5days the cultures were grown in the presence of25mg/l hygromycin with transfer to fresh medium weekly for8weeks.The surviving green clumps were cultured for about4weeks in liquid medium without selection and then plants were regenerated as described by Bailey et al.(1993).Leaf pieces were tested for callus formation on a medium with50mg/l hygromycin(Zhang et al.2005).
Plasmid construction
The hpt coding region was obtained by a Xho I digestion of pCAMBIA1380and was cloned into the Sal I-cut pBlue-script KSII(Stratagene,https://www.360docs.net/doc/2c18945516.html,).Then the Eco RV–Xho I fragment of the hpt coding sequence was ligated with the Sma I–Xho I fragment of pHAG(Inaba et al. 2007a,b)to obtain the intermediate plasmid pHyII,so that the hpt coding region was?anked by the2.3kb ASA2 promoter at the50-end and the Arabidopsis actin2termi-nator at the30-end.Finally,the Sac I–Sac II fragment (3.9kb)of pHyII was ligated into the Sac I–Sac II-cut pBluescript KSII(2.9kb)to complete the transformation vector pXZIII-8(Fig.1a).As a control a soybean line transformed with the plasmid containing CaMV35S-hpt-Nos-T(PGL1H-2,Fig.1b)constructed by Sermsiri Chanprame was also used.
PCR analysis
Genomic DNA was extracted from freeze dried leaves of plants following the method of Dellaporta(1993).The PCR was done using primers to amplify410bp of the hpt gene: HPT-FOR:50-CAT GGC GTG ATT TCA TAT GCG CGA and HPT-REV:50-CGA AAT TGC CGT CAA CCA AGC TCT or primers for the ASA2promoter and hpt gene: 50ASA2:TGT ATT GCC CAT TTC ATT AGA AAG GAT and HPT-REV to amplify1,447bp.The PCR reaction was
carried out using Taq DNA polymerase(New England Biolabs,https://www.360docs.net/doc/2c18945516.html,)with denaturation at95°for 4min,then30cycles at95°for45s,annealing at58°for 45s and extension at72°for40s,and the?nal step at72°for5min.
Southern blot hybridization
Genomic DNA was extracted from leaves using a‘‘Nucleon Phyto Pure,Plant and Fungal DNA Extraction Kit’’(Amersham Life Sci.,https://www.360docs.net/doc/2c18945516.html,).A mea-sure of15l g of extracted genomic DNA was digested with Hin dIII and was run in a0.8%gel,and the gel was blotted onto Hybond XL membrane(Amersham)in209SSC buffer for24h.The hpt gene was obtained by digestion with Hin dIII and Xho I from the plasmid pXZIII-8,run in the gel, puri?ed from the gel with‘‘QIAEX II Gel Extraction Kit’’(Qiagen,https://www.360docs.net/doc/2c18945516.html,),labeled with P32-dCTP with a‘‘Megaprime DNA labeling System’’(Amersham) and used as a probe.The membrane was prehybridized for 1h at65°C in the preheated hybridization buffer that con-sists of59SSC,59Denhardt’s solution,0.5%SDS,ssDNA and then hybridized in the same buffer with radioactive probe at65°C for24h.Washing was done sequentially in 29SSC,0.1%SDS solution for5min twice;in19SSC, 0.1%SDS for10min twice and in0.19SSC,0.1%SDS for 5min four times at room temperature.The membrane was exposed to X-ray?lm for3days at-80°C for radioactive signal development.
Northern blot hybridization
Total RNA was extracted from different organs of plants and cultures with‘‘RNasy Plant Mini Kit’’(Qiagen).20l g of total RNA was run in a MOPS/formaldehyde gel and then gel blotted on Hybond XL membrane in109SSC buffer for24h.Prehybridization was done for1h at63°C with hybridization at the same temperature for24h in the same hybridization buffer as used for the Southern blot. The membrane was washed in29SSC,0.1%SDS solution for5min two times;in19SSC,0.1%SDS solution for 5min two times;in0.19SSC,0.1%SDS solution for 5min four times at room temperature.The membrane was exposed to X-Ray?lm at-80°C for1week.
RT-PCR analysis
Total RNA,extracted as described above,was used after DNA digestion in the samples with rDNase I‘‘One step RT-PCR’’kit using RNase-Free enzyme for the reverse transcriptase reaction(USB Corporation,http://www. https://www.360docs.net/doc/2c18945516.html,)according to the instructions.A total of 410bp of hpt mRNA was ampli?ed with the primers that were used for PCR screening of regenerated plants.The positive control for RT-PCR with RNA extracted from roots,stems,leaves,and?owers was prepared with primers for800bp of IFS cDNA(iso?avone synthase,accession# AF135484):50IFS-76-TTT CTG CAC TTG CGT CCC ACA CCC ACT GCA AAA and30IFS-736-TCG ATC CTC TTC TCA TAC TTT CCA ACC TTG AGA.The positive control for the RT-PCR for pods,seeds,embryo-genic cultures and leaf callus mRNA was done with primers for600bp of the seed lectin gene:Lec50-GTG GTT GTA TCT CTC TCC and Lec30-CAT CGG AGA GGA TAT TGC.A negative control was included for each sample consisting of the same RNA after rDNase treatment used in RT-PCR in the same conditions but without the reverse transcriptase enzyme in order to monitor DNA contamination of the RNA samples.
Data analysis
One-way ANOVA using the algorithm in Microsoft Excel 2002was used to determine the statistical difference between callus weights with a signi?cance of P\0.05. Results and discussion
Production of soybean plants with ASA2-hpt
Embryogenic soybean suspension cultures were bombarded with the pXZIII-8plasmid that contains the ASA2promoter driving hpt(Fig.1)and selection for transformants was done using25mg/l hygromycin.In the?rst set of bom-bardments,seven hygromycin resistant putatively transformed lines were selected after57bombardments. Four of these lines were regenerated into plants that had leaves that formed callus in the hygromycin leaf test and the three that were tested were Southern positive for the hpt gene(data not shown).Plants of two of these lines set seed and are denoted1H10and3H6.In the second set of bom-bardments,23hygromycin resistant lines were selected following18bombardments.Plants were regenerated from the23selected lines and16were PCR and Southern positive for the hpt gene(examples shown in Figs.2,3).Progeny of the two lines that were selected in the?rst experiment that set seed were also PCR as well as Southern positive for the hpt gene(lines1H10and3H6,Figs.2,3).Most of the lines contained more than one copy of the hpt gene(Fig.3).
Measurement of hpt gene expression by northern hybridization and RT-PCR
Systematic hpt expression studies were carried out with the two lines selected in the?rst set of bombardments since
they were in the T4generation and line 3H6was homo-zygous for the transgenes.Since line 1H10was still segregating for the transgene only plants that were hygro-mycin leaf test positive were used.A line denoted 35S-hpt was used as the positive control since it contained hpt driven by the CaMV35S constitutive promoter (Fig.1).Untransformed Jack was used as the negative control.Northern blot hybridizations using mRNA isolated from plants of the two ASA2-hpt lines and Jack showed that no hpt mRNA was detectable in leaf,stem,pod,mature seed,root and ?ower tissues (Fig.4).In contrast the 35S-hpt plants showed detectable hpt mRNA in all these tissues.The levels in ?owers were very low however.The ASA2-hpt lines and the 35S-hpt line did show northern detectable levels of hpt mRNA in developing seeds and embryogenic cultures initiated from developing seeds while Jack did not.Surprisingly the friable callus formed from leaves of the ASA2-hpt lines sometimes contained only barely detect-able levels of hpt mRNA by northern hybridization even
though the callus formed and proliferated on medium with 50mg/l hygromycin that prevented callus formation from leaf segments of Jack (Fig.5a).The 35S-hpt line formed callus on the hygromycin medium and did show hpt mRNA by northern hybridization analysis (Fig.4).
When the mRNA from all the tissues was analyzed by RT-PCR some hpt mRNA was detectable in all of the 35S-hpt tissues (Fig.4).All ASA2-hpt tissues except roots and mature seeds also contained hpt mRNA.Jack tissues did not contain any detectable hpt mRNA as expected.The RT-PCR samples all were done with either a lectin or iso?avone synthase internal positive control depending upon the tissue.
To determine the level of resistance to hygromycin of the friable callus formed from leaf segments (Fig.5)the callus was incubated for 14days on several hygromycin concentrations (25,50and 100mg/l)and then weighed.As shown in Table 1the Jack callus that was initiated on medium without hygromycin is completely inhibited by the hygromycin concentrations used and the 35S-hpt callus grows some on 25mg/l but not 50and 100.However,the ASA2-hpt lines callus grows on all the hygromycin con-centrations.When the callus samples grown on the different hygromycin concentrations were also used for northern blot analysis to determine if growth on hygro-mycin containing medium affects the hpt mRNA level,the ASA2-hpt lines 1H10and 3H6sometimes showed detect-able hpt mRNA levels indicating that hygromycin might have some effect (data not shown).
Embryogenic cultures initiated from immature cotyle-dons of a total of ?ve ASA2-hpt lines and 35S-hpt were able to proliferate in the presence of 18mg/l hygromycin,a concentration that completely inhibits the growth of Jack cultures (data not shown).The hygromycin resistance seen here with the embryogenic cultures of the ASA2-hpt lines might be expected since hpt mRNA was detectable by northern blot hybridization and RT-PCR (Fig.4).
We have previously tested the tobacco ASA2promoter expression in soybean by using it to drive uidA gene expression both using the GUS histochemical and quanti-tative MUG ?uorimetric assays (Inaba et al.2007b ).The GUS histochemical assays showed detectable expression only in embryogenic cultures,developing and mature seed and also in pollen of some lines.Thus our results reported here using northern blot analysis are similar since we see hpt mRNA in embryogenic cultures and developing seeds.We see no expression in mature seeds unlike the GUS results,but it is known that the GUS protein is relatively stable (Jefferson et al.1987)and one could speculate that the enzyme produced during seed development could still be present in mature seeds even though the gene driven by the ASA2promoter may not be expressed there.The northern blot analysis may not be sensitive enough to
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Fig.2PCR analysis of genomic DNA isolated from leaves of independent soybean transformed plants after bombardment with pXZIII-8.Jack is the untransformed control,35S is 35S-hpt and +is the plasmid.The 50ASA2promoter and 30hpt gene primers were used in a while the 50hpt and 30hpt gene primers were used in
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Fig.3Southern blot analysis of genomic DNA isolated from leaves of several ASA-hpt transformed plants following Hin dIII digestion using the hpt gene as probe.Jack is the untransformed control,35S is 35S-hpt and +is the plasmid
measure hpt mRNA in pollen since whole ?owers were analyzed due to their small size.
The MUG assay showed that leaves,stems,?owers,immature and mature seeds and embryogenic cultures of the ASA2-GUS plants contained measurable GUS activity (Inaba et al.2007b ).In all cases the activity levels were much lower than that seen with the CaMV35S-GUS posi-tive control with the highest expression being about 2%of this control in seed coats to much lower levels in all other tissues tested.These results ?t with our RT-PCR results that show the presence of some hpt mRNA in all these tissues except mature seeds.Again the lack of correlation between hpt mRNA and GUS enzyme activity in mature seeds could be due to the stability of the enzyme (Jefferson et al.1987)since immature seeds do contain hpt mRNA (Fig.4).
These results show that the ASA2promoter is strong enough to drive expression of the hpt gene at a level suf-?cient to allow selection of transformed cell lines with the embryogenic soybean system.While we did not do com-parative studies with other promoters driving hpt for selection at the same time as ASA2-hpt,we normally obtain about one transformant per bombardment using constitutive promoters such as CaMV35S or potato ubiq-uitin-3driving hpt .
The rice b -glucanase 9(Gns9)promoter was shown by Huang et al.(2001)to drive GUS expression that was detectable in rice callus but not in plant roots,leaves,mature seeds or germinated seeds using the histochemical GUS assay.They then were able to select transformed rice callus using hygromycin following bombardment with a construct containing the Gns9promoter driving hpt.The regenerated plants did not express hpt .Roberts et al.(2005)demonstrated that the ethanol inducible alc two component system from Aspergillus nidulans drives signi?cant levels of GUS expression in callus and suspension cultures ini-tiated from the transformed tomato plants.The authors suggested that the inducers ethanol and/or acetaldehyde
J a c k
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Fig.4Northern blot and RT-PCR analysis of RNA isolated from different plant tissues of the ASA2-hpt lines 1H10and 3H6,the 35S-hpt line and the untransformed control Jack.Northerns are on the left and RT-PCR on the right for each tissue shown,RNA loading is shown below each northern and a minus reverse transcriptase and lectin or iso?avone synthase (IFS)positive control shown with the hpt RT-PCR.The hpt gene probe was used for the northern and the 50hpt and 30hpt primers used for the RT-PCR
may be produced in response to decreased oxygen avail-ability in the cultured cells.Tomato plants do not show expression unless induced with ethanol or acetaldehyde.Since the ASA2gene itself encodes a naturally occur-ring feedback insensitive form of the tryptophan biosynthetic control enzyme anthranilate synthase (Song et al.1998),expression of the gene in transgenic Astrag-alus sinicus and soybean hairy roots (Cho et al.2000,2004),tobacco (Zhang et al.2001;Tsai et al.2005)and soybean plants (Inaba et al.2007a )leads to the accumu-lation of free tryptophan.This feedback inhibition resistance also means that the ASA2gene can be used as a selectable marker gene using toxic tryptophan analogs for selection of A.sinicus hairy roots (Cho et al.2004)and tobacco shoots (Barone and Widholm 2008).
Most (14of 20)of the PCR positive transformed lines that were selected in our experiments did form friable callus when leaf segments were plated on medium con-taining 50mg/l hygromycin,a concentration that bleaches and kills the untransformed control Jack leaf segments.This shows that the tobacco ASA2promoter in most cases also does operate in a tissue culture speci?c manner to cause hpt expression in soybean.It is worth noting that this tissue culture speci?c pattern of ASA2promoter function was initially observed in its host plant tobacco (Song et al.1998).We have subsequently demonstrated that this pat-tern remains in a different host plant soybean (Inaba et al.2007a ,b and this report),suggesting that the ASA2pro-moter may function in the tissue speci?c fashion in other plants as well.Thus this promoter might be useful for driving selectable markers in tissue cultures that do not express in regenerated plants of many plant species.
Acknowledgments Funds for this research were provided by the United Soybean Board,the Illinois Soybean Association Checkoff,the Illinois Agricultural Experiment Station and the Consortium for Plant Biotechnology Research,Inc.by DOE Agreement No.DE-FG36-02G012026.This support does not constitute an endorsement by DOE or by the Consortium for Plant Biotechnology Research,Inc.of the views expressed in this publication.
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Table 1Effect of hygromycin on the fresh weight of six friable callus pieces of each line after 14days growth Line
Hygromycin (mg/l)
02550
100g fresh weight (±SD)per callus piece
Jack 0.61±0.320.26±0.060.26±0.060.28±0.0435S-hpt 0.95±0.430.42±0.09*0.29±0.050.27±0.061H100.77±0.290.64±0.32*0.49±0.240.52±0.273H6
0.76±0.22
0.78±0.14
*
0.88±0.25
*
0.81±0.28*
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