2000-植物转基因 玉米

A.S. Wang 7 R.A. Evans 7 P.R. AltendorfJ.A. Hanten 7 M.C. Doyle 7 J.L. Rosichan

A mannose selection system for production

of fertile transgenic maize plants from protoplasts

Received: 12 July 1999 / Revision received: 11 October 1999 / Accepted: 11 October 1999

AbstractMaize (Zea mays L.) callus cultures cannotuse mannose as a sole carbohydrate source, but canutilize fructose for that purpose. Phosphomannoseisomerase (PMI) can convert mannose to fructose.Transgenic maize plants were obtained by selectingpolyethylene glycol (PEG)-mediated transformedprotoplasts on mannose (20g/l) containing medium.Transgenic calluses and plants carrying the PMI struc-tural gene, manA, were able to convert mannose tofructose. The PEG-mediated protoplast transformationfrequency was 0.06%. Stable transformation wasconfirmed by PCR, PMI activity, germination tests, andby histochemical staining with 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-Gluc). Stable integrationof the transgenes into the maize genome was demon-strated in T1 and T2 plants. Results indicate that themannose selection system can be used for maize PEG-mediated protoplast transformation.

Key wordsMaize 7 Protoplast 7 Transformation 7Mannose-selection marker

Abbreviations2,4-D: 2,4-Dichlorophenoxyacetic acid 7CaMV: Cauliflower mosaic virus 7 GUS:

b-Glucuronidase 7 OD: Optical density 7 PAT:Phosphinothricin-N-acetyl-transferase 7 PCR:Polymerase chain reaction

Introduction

Maize, rice, and wheat are the major cereal crops withgreat potential for biotechnological advances. Biotech-nology has contributed significantly to increasingdisease (Murry et al. 1993) and insect resistance (Kozielet al. 1993; Williams et al. 1997) in maize. For maizetransformation, either antibiotics or herbicides havebeen widely used as selection agents to identify trans-formants. Hygromycin (Armstrong et al. 1990; Ishida etal. 1996), kanamycin (Murry et al. 1993; Omirulleh etal.1993), methotrexate (Golovkin et al. 1993), bialaphos(Gordon-Kamm et al. 1990), chlorsulfuron (Fromm etal. 1990), and phosphinothricin (Koziel et al. 1993)have all been used as selection agents to eliminate thenon-transformed tissues or cells in maize.

Recently, the use of antibiotics and herbicide selec-tion agents has caused widespread public concernbecause of inadequate knowledge of the agents’ impacton the environment and on human health. To avoid theuse of those agents, alternative selection systems havebeen proposed and developed (Joersbo and Okkels1996; Haldrup et al. 1998; Joersbo et al. 1998). Trans-formation has been achieved using the following selec-tion agents: benzyladenine N-3-glucuronide in tobacco,mannose in sugar beet, and xylose in potato, tobaccoand tomato. The selection systems allow for separationof non-transformed from transformed tissue byarresting the growth of the non-transformed cells andtissue through carbohydrate starvation.

Sucrose, a carbon source often used in maize tissueculture media, can be replaced by other sugars such asfructose or glucose, but not by mannose. Maize callusdoes not contain a PMI gene to convert mannose tofructose. The PMI structural gene (manA) of Escheri-chia coli, which is capable of converting mannose tofructose, has been cloned (Miles and Guest 1984). ThemanA gene has been used successfully in maize biolistictransformation as a selectable marker gene (Evans etal. 1996). The manA transgenic maize calluses couldgrow on mannose medium and utilize mannose as a

Communicated by G. Phillips

A.S. Wang (Y) 7 R.A. Evans 7 P.R. Altendorf 7 J.A. HantenM.C. Doyle 7 J.L. Rosichan

Novartis Seeds Inc., Applied Biotechnology,317330th St., Stanton, MN

e-mail: Andy.wang@seeds.novartis.comFax: c507-57519

carbon source. Here, we report a mannose selectionsystem for maize PEG-mediated protoplast transforma-tion.

Materials and methods

Plant materials and culture media

Maize cell lines were established from callus cultures of blackMexican sweet corn (BMS; a gift from C. Donovan, University ofMinnesota), A188/B73 (Hi-II; a gift from Dr. C. Armstrong), andtwo Novartis proprietary lines (7R7B, 7Haf1). The callus cultureswere initiated from 10-days-old immature embryos (1.5mm inlength) on pH (5.7) pre-adjusted MS medium (Gibco BRL)containing MS basic salts (Murashige and Skoog 1962) plus 6mMasparagine, 1mg/1 thiamine-HCl, 1mg/l nicotinic acid, 0.2mg/lpyridoxine, 2mg/l glycine, 1.4g/l proline, 100mg/l casein hydroly-sate, 20g/l sucrose, 1mg/l 2,4-D, and was solidified with 2.3g/lGelrite (Schweizerhall, Inc.). The cultures were maintained onthe same medium with a 14 day subculture interval. For determi-nation of the effect of sugars on callus growth, sucrose in the MSmedium was replaced with fructose, glucose, mannose or xylose(20g/l).

PEG-mediated protoplast transformation

Several suspension cultures were initiated from 7R7B and 7Haf1friable embryogenic callus cultures, maintained in liquid MSmedium supplemented with 30g/l of sucrose (instead of 20g/l) onan orbital shaker (150rpm) in the dark at 267C and subculturedtwice per week. One gram of suspension cells was collected on the3rd day of subculture, pretreated with 10ml of 0.3M sorbitol and0.5M CaCl for 1h in the dark, and then digested with 10ml of2% cellulase Onozuka RS (Yakult Pharmaceutical Ind., Japan)and 0.2% pectolyase Y-23 (Seishin Pharmaceutical Co., Japan)for 2–3h. The enzymes were dissolved in PWS solution (2g/lbovine serum albumin, 0.2M mannitol and 80mM CaCl2,pH7.0). Protoplasts were filtered through a 46 mm screen,collected by centrifuging at 100G for 7min, resuspended in 10mlof PWS, underlaid with 3ml of 20% sucrose, and then centrifugedat 100G for 4min. The white protoplast band was withdrawn,added to 15ml of PWS, and centrifuged. MaCa solution (0.2Mmannitol and 80mm CaClwas 2) was added to the protoplasts. Theprotoplast concentration adjusted to 2!106 protoplasts perml, as determined by a hemacytometer.

A PEG solution (40%) was prepared by dissolving PEG 4000(EM Science) in Krens’ F solution (Krens et al. 1982). The proto-plast solution was gently mixed with the plasmid DNA ofpNKS205 (150mg/l06 protoplasts) and pZO1458 (50mg/106 proto-plasts), then with an equal volume of the 40% PEG solution andgently agitated for 30min. The protoplast/PEG solution wasdiluted with an equal volume of MSMa (liquid MS medium with8% mannitol, pH5.6) five times at 5min intervals, centrifuged,washed with MSMa, and resuspended in MSMa at 1 millionprotoplasts per ml. One milliliter of the protoplast suspension wasplated and cultured on a hydrophobic edge membrane (MilliporeCorp) on top of a layer of 7-day-old BMS feeder cells. The BMSfeeder layer was prepared 7 days earlier by placing 2ml of theBMS suspension (1g/ml) on the solid MS medium plus 4%mannitol. The BMS suspension was derived from a non-regener-able BMS callus culture.

Protoplast colonies were visible in 7 days. The plating effi-ciency of the protoplasts was determined on the 10th day bycounting the total number of protoplast derived colonies dividedby the total number of protoplasts plated on each filter. For selec-tion, the protoplast colonies were transferred to the MS mediumcontaining mannose (20g/l) without sucrose and subcultured onthe same medium at 14 days intervals for 2 months. Recoveredcolonies were analyzed by PCR and histochemically stained with

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X-Gluc. Hormone-free MS medium supplemented with either(20g/l) sucrose or mannose was used for plant regeneration oftransgenic calluses.Plasmids

The pNKS205 and pZO1083 plamids were used for transforma-tion. The pZO1083 was modified from pZO1084 (Murry et al.1993) by replacing the MDMV-B and NPT II coding sequences ofpZO1084 with GUS (1.8kb) and PAT (0.5kb), respectively. ThepNKS205 construct contains an octopine synthase (OCS)promoter driving the manA gene (1.2kb; obtained from DaniscoBiotechnology, Denmark) and a Nopaline synthase (NOS) termi-nator (Fig.1).PMI activity assay

The PMI assays of Feramisco et al. (1973) were modified. Planttissue (250mg) was ground in 250ml of 50mM Tris-HCl andcentrifuged at 14 000rpm for 20min. The supernatant (50ml) wasadded to 100ml of 50mM Tris-HCl (pH7.5), mixed with 100ml ofsubstrate and measured at 340 nm in a spectrophotometer. Thesubstrates consisted of 25ml of nicotinamide adenine dinucleotidephosphate (10mM), 25ml of phosphoglucose isomerase (10U/ml,EC 5.3.1.9, Sigma), 12.5ml of glucose-6-P dehydrogenase (10U/ml, EC 1.1.1.49, Sigma), 5ml of D-mannose-6-P (50mM), and32.5ml of Tris-HCl (50mm, pH7.5).PCR assays

DNA extraction of callus and leaf tissue and PCR assays werecarried out with a procedure similar to Hanten et al. (1996). A leftprimer 5b CACTGCGTGATGTGATTGAGAGTG-3b and aright primer 5b ACTAAGGTCATGCAGCGAGAAGGC-3bwere used to amplify an 820bp fragment of the manA gene. ThePCR reactions were subjected to 30 cycles of: 30s at 947C, 30s at657C, and 45s at 727C.GUS assays

Plant tissues were histochemically stained with X-Gluc accordingto Jefferson (1987) with modifications. The plant materials werestained at 377C overnight in 100mM sodium phosphate buffer(pH7) containing 10mM EDTA, 0.5% (v/v) Triton X-100, andX-Gluc (0.5mg/ml). The leaves were bleached with severalwashes of 70% (v/v) EtOH.

Southern blot analyses of transgenic plants

Genomic DNA of manA-positive Tshoots (derived from 2 maize shoots and non-trans-genic control explants from the same cellline, but not transformed with manA) was prepared according toDellaporta et al. (1983), digested with Pst1, Xho1, EcoR1, andEcoRV and subjected to gel electrophoresis followed by blottingonto MSI nylon transfer membrane (Osmonics Inc. Mass). TheSouthern blot was hybridized with a DNA fragment containingthe PMI gene labeled with 32P-dCTP by random priming(Sambrook et al. 19).

EcoRIBamHI/Pst1EcoRV

SacI/Pst1

HindIIIfff

f

f

OCSpromoter

manAcodingregion

NOSterminator

Fig.1Map of the pNKS205 construct. These plasmids wereprepared using standard procedures and purified by CsClgradient centrifugation (Sambrook et al. 19)

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Results and discussion

Effect of sugars on callus induction and growthThe effects of mannose and sucrose on callus inductionand germination was determined. Immature embryosand mature embryos were isolated from Hi II and7R7B plated on solid MS medium supplemented witheither mannose or sucrose (20g/l). Table1 shows thatnone of the embryos from two cell lines on mannosemedia produced callus or germinated, while 60% (90/150) of the immature embryos produced calluses and97% (107/110) of the mature embryos germinated onsucrose medium. These data suggest that an externalsucrose source is required for callus initiation andembryo germination. Failure of the embryo to germi-nate on mannose medium may be caused by energydepletion via a hexokinase-mediated pathway. Pego etal. (1999) reported that hexokinase was involved in themannose-mediated inhibition of Arabidopsis seedgermination. Phosphorylation of mannose by hexoki-nase triggers a signaling cascade resulting in generepression and energy depletion during seed germina-tion.

Four maize callus lines were grown on solid MSmedium supplemented with either fructose, glucose,mannose, sucrose or xylose to determine the effect ofthese sugars on callus growth and whether mannosecould be used as a carbon source or a selection agent(Table2). After 14 days, the callus weight for these celllines on sucrose medium ranged from 0.87 to 2.04g.BMS growth rate was statistically significantly slowerthan other cell lines at the 1% level because of its largeelongated and vacuolated cells. No increase in callusweight was observed for any cell lines cultured onmannose medium. Slight increases in weight weredetected in most cell lines on xylose medium with anaverage increase in weight of 0.29g after 14 dayscompared with 1.01g on sucrose medium. All of thecell lines grew normally on fructose, glucose andsucrose containing media. The results suggest that themaize callus cultures are not able to utilize mannose asa carbon source.

An additional experiment was conducted to deter-mine if the lack of callus growth on mannose mediumwas due to either carbohydrate starvation or mannosetoxicity. Four non-transformed cell lines were grown on

Table2Effect of various sugars (2%, w/v) on maize callusgrowth. Callus (500mg) was placed in two circles (2cm diameter)on a 100!15mm plate containing 30ml medium. The calluseswere weighed 14 days laterSugars (20g/l)

Cell lines and callus fresh weight (gBSEa)7Haf1

FructoseGlucoseXyloseMannoseSucrose15Sc5Mb10Sc10McabHi-II1.41B0.081.36B0.061.10B0.070.48B0.041.47B0.061.45B0.061.34B0.06

BMS0.82B0.070.79B0.030.56B0.020.41B0.030.87B0.030.90B0.050.75B0.09

7R7B1.93B0.051.84B0.040.79B0.070.51B0.051.80B0.131.94B0.051.67B0.06

2.10B0.121.99B0.070.71B0.080.50B0.141.91B0.142.14B0.222.08B0.20

MeanBSE for five replications15g/l sucrose c5g/l mannosec10g/l sucrose c10g/l mannose

MS medium supplemented either with 20g/l sucrose,15g/l sucrose c5g/l mannose, or 10g/l sucrose c10g/lmannose. As before, the callus growth was determinedafter 14 days (Table2). Addition of mannose to themedia had no effect on callus growth. All of the celllines could grow on sucrose medium supplementedwith up to 10g/l mannose. There was no significantdifference in growth within a cell line. Therefore, theresults demonstrate that mannose is not toxic to maizecallus culture.

PEG-mediated protoplast transformation

Two maize suspension cell lines, 7R7B and 7Haf1, wereused in the protoplast transformation experiments.Transgenic selection was carried out on mannose (20g/l) medium for 8 weeks. A total of 12 transformationexperiments with the manA gene were performed;seven for 7Haf1 and five for 7R7B. The results werepooled together and are presented in Table3. Lessthan one transformed colony per million treated proto-plasts was recovered. When the plating efficiency(0.1%) is taken into account, the average adjustedtransformation frequency for the two cell lines is0.06%. This transformation frequency is lower than thefrequencies obtained from hygromycin-treated BMS(Armstrong et al. 1990) and Kanamycin-treated He(Omirulleh et al. 1993) transformations. Since different

Table1Effect of mannose on callus initiation of immature embryos and germination of mature embryos of maizeCell lines

Callus initiation of immature embryosHi-II

2% Mannose2% Sucrose

aGermination of mature embryos

Total0/16090/150

Hi-II0/5049/50

7R7B0/5058/60

Total0/100107/110

7R7B0/1637/75

0/99a53/75

Number of embryos producing callus (or germinating)/Number of embryos plated, 7 days post isolation

Table3PEG-mediated protoplast transformation frequency inmaize. Plating efficiencyp0.1% based on average of ten plates,five plates/cell lineCell lines

No. of

No. ofAdjusted

protoplastscoloniestransformationplatedrecoveredfrequency (%)7R7B25000000100.047Haf1000000380.07Total ofboth lines

79000000

48

0.06

selection agents and cell lines were used, a directcomparison can not be made. However, recently, wehave made a ten-fold improvement over the currenttransformation frequency by lowering the transforma-tion temperature (87C) and increasing the DNAconcentration (data not shown).

Three months after transformation, portions ofrecovered calluses were analyzed by PCR and the restof the calluses were saved for regeneration. Fig.2shows the results of the PCR assay: 7 of 15 putativetransformed calluses have the expected amplified DNAsize, demonstrating the presence of the manA gene inthe genomic DNA. Using PCR analyses, 50% of thecalluses were found to be negative for the manA gene,and therefore escaped the selection. A longer selectionperiod might be required for reducing the escape rate.In order to compare transformed and non-trans-formed callus growth on mannose medium, 500mg ofthe transformed and non-transformed calluses of celllines 7R4B and 7Haf1were plated on both mannoseand sucrose media. The callus weight was determinedafter 14 days (Table4). The callus weights from the twoindependent transformation events, T07R4B andT07Haf1, were significantly higher than their non-trans-formed counterparts on mannose medium. The resultsindicate that the manA-transformed calluses are able toutilize mannose as a carbon source. No significantdifference in the callus weight of T07Haf1 was observedon either medium (2.14g versus 2.10g). Therefore, theTo 7Haf1 cell line apparently could utilize mannose as

Fig.2Agarose gel electrophoresis of PCR amplified DNA fromputatively transformed calluses. The lanes are as follow: lanes1–10 and 12–16 are15 independent calluses; 11 positive control; 17negative control; 18 plasmid DNA; 19 blank; 20 123bp ladder(Gibco BRL). The band at 820bp represents the amplified DNAfragment of the manA gene

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Table4Effect of mannose on growth of transformed and non-transformed cell lines. These data represent the mean of fivereplicationsBSE in callus fresh weight (g)MediumCell linescontaining:

7R4B

T07R4Ba7Haf1

T07Haf1aMannose (20g/l)0.51B0.051.82B0.070.66B0.262.14B0.07Sucrose (20g/l)1.95B0.042.04B0.092.21B0.122.10B0.18

aTransformed cell lines, others are non-transformed

efficiently as sucrose. On the other hand, T07R4Bweighed less on mannose medium than it did on thesucrose medium (1.82g versus 2.04g), which waspossibly due to lower expression of manA gene in thistransgenic event. There was no significant differencedetected in growth on sucrose medium between trans-formed and non-transformed cell lines of either 7R4Bor 7Haf1 (Table4). Therefore, PEG-mediated transfor-mation had no negative effect on the the callusgrowth.

PMI activity assay

Transgenic calluses and T0 plants were assayed for PMIactivity. Table5 shows that there is PMI activity in thetransgenic cell lines and no activity in the non-trans-formed controls. There also exists a difference in thedegree of PMI activity between two independent trans-genic events, B6–7D and B9–20A. The difference in thePMI expression level may have been caused by differ-ences in chromosomal insertion sites of each transgenicevent or by other factors such as DNA methylation andcopy number.

Transgenic plant analyses

Five months after the transformation, another 24mannose resistant colonies were analyzed by PCR.Among them, 13 manA and GUS, 3 manA, and 8 GUS

Table5PMI activity of the two manA transgenic maize cell lines.PMI activity is measured in terms of OD at 340 nm over 20minCell lines

MannoseSucrosecontainingcontainingmediummedium7Haf1A1.32B0.18a0.40B0.04a7Haf1D

0.17B0.11a0.06bNon-transformed

0.00c0.03B0.02aaThese data represent the mean of at least four replica-tionsBSEbNo replicationcNot detectable

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Fig.3Agrose gel electrophoresis of PCR amplified DNA fromleaf tissue of the transformed T1 maize plants. Lanes 1–16 repre-sent 16 T1 plants; lanes 17, 18, 19, and 20 are negative control,plasmid DNA, positive control and the 1kb ladder, respectively

positive colonies were identified. T0 plants were regen-erated from calluses positive for manA and GUS. T1seeds were harvested 2 months later for furtheranalyses. Two-week old T1 leaf tissues were analyzedby PCR for the manA gene (Fig.3) and histochemicallyassayed for GUS expression by staining with X-Gluc(Fig.4).

Only a few seeds were obtained from each T0 plant.Therefore, T1 plants were self-pollinated and crossed toa non-transformed inbred (7Haf1) in order to deter-mine inheritance and stability of the PMI trait. Integra-tion of a single copy of the manA gene into the maizegenome was detected by Southern analyses (Fig.5).Representative samples of a total of 15 T2 progeny(58–7 and 58–9) and 5 negative controls (57–2) wereanalyzed and these data presented in Figure5. Diges-Fig.4Leaves of stably trans-formed T1 maize plantsshowing GUS expression.Leaves 1–4 from left are fromevent B30A, 5–9 from G14Aand 10–11 are untransformedcontrols

tion with Pst1 cut at both ends of the manA geneyielding the presence of the predicted 1.2kb band inthe T2 progeny and its absence in the negative controls.Digestion of 58-7 and 58-8 by Xho1 produced one bandand demonstrated the presence of a single insert. Thereis apparently some background, nonspecific hybridiza-tion present in the EcoR1 digest of both negativecontrols and transformants since no bands weredetected in any of the other digests of the negativecontrol. Digestion of 58–7 and 58–8 with EcoRV cut anEcoRV site internal to the manA coding region andproduced two bands as predicted for a single insertionevent.

When T2 mature embryos were separated fromendosperm and germinated on mannose regenerationmedium, a 3:1 segregation ratio for the manA gene wasobserved among the self-pollinated T2 progenies of twoT1 plants and a 1:1 ratio detected for their cross-polli-nated T2 progenies (Table6). The segregation data alsosuggest that a single copy of manA gene was stablyintegrated into the maize genome.

The PEG-mediated protoplast transformation hasbeen successfully used to produce fertile transgenicmaize plants (Golovkin et al. 1993; Omirulleh et al.1993). The procedure is effective, inexpensive andsimple (Armstrong et al. 1990). Times required forproducing transgenic plantlets and T1 mature seeds are4 and 7 months, respectively. The results presented inthis paper indicate that the PEG-mediated protoplasttransformation system can be routinely used forproduction of transgenic maize plants.

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Table6Inheritance of manAtransgene expression in maizeT2 progeny by embryo germi-nation assay. Note: only manApositive embryos can germi-nate on mannose regenerationmedium

Transgenic events

Total no. ofT2 progeny8012415659

manApositiveprogeny8229

non-manAprogeny22307430

x2 value forexpectedratio

0.26 for 3:10.04 for 3:10.42 for 1:10.02 for 1:1

Self-pollinated BSelf-pollinated B56B X 7Haf1B56 X 7Haf1

Fig.5Southern blot analysis of T58–7 and 58–8) maize transformants 2 progenies of two PMI-positive(and one non-transformedcontrol (57–2) along with molecular size markers (given in kb).The blot was hybridized with the 1.2kb PMI coding region. Thearrow indicates the predicted 1.2kb Pst1 fragment. PPst1,XXho1, R1EcoR1, RVEcoRV and ll DNA digested with HindIII (Gibco BRL)

In conclusion, the mannose selection system is asubstrate-based selection system that does not causeany risk to animal, human or environmental safety. ThemanA gene can be expressed in maize cells and confersthe ability to utilize mannose as a carbohydrate sourceupon plant culture and tissue. It provides an alternativeselectable marker to antibiotics and herbicides fortransformation of maize and other plant species.

AcknowledgementsWe thank Dr. Ron Meeusen for encourage-ment during the course of this investigation and PMI activityassay, Paul Dietrich for supplying pZO1083, and Michele Kasel,Roger Taylor and Keith Hines for excellent technical support.

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