Plant Physiol. (1996) 110: 1097-1107

The Competence of Maize Shoot Meristems for lntegrative Transformation and lnherited Expression of Transgenes'

Heng Zhong, Baolin Sun, Donald Warkentin, Shibo Zhang, Ray Wu, Tiyun Wu, and Mariam B. Sticklen*

202 Pesticide Research Center, Department of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824-1 31 1 (H.Z., B.S., D.W., S.Z., M.B.S.); and Section of Biochemistry, Molecular and Cell

Biology, Cornell University, Ithaca, New York 14853 (R.W., T.W.)

We have developed a nove1 and reproducible system for recovery of fertile transgenic maize (Zea mays L.) plants. The transformation was performed using microprojectile bombardment of cultured shoot apices of maize with a plasmid carrying two linked genes, the Strepfomyces bygroscopicus phosphinothricin acetyltransferase gene (bar) and the potato proteinase inhibitor II gene, either alone or in combination with another plasmid containing the 5' region of the rice actin 1 gene fused to the Escbericbia coli p-glucuronidase gene (gus). Bombarded shoot apices were subsequently multiplied and selected under 3 to 5 mg/L glufosinate ammonium. Co-trans- formation frequency was 100% (146/146) for linked genes and 80% (41/51) for unlinked genes. Co-expression frequency of the bar and gus genes was 57% (29/51). The co-integration, co-inheritance, and co-expression of bar, the potato proteinase inhibitor II gene, and gus in transgenic R,, R,, and R, plants were confirmed. Local- ized expression of the actin 1-CUS protein in the R, and R, plants was extensively analyzed by histochemical and fluorometric assays.

The shoot tip, or shoot apex, consists of the shoot apical meristem, a region in which lateral organ primordia form, a subapical region of cell enlargement, and severa1 leaf primordia (Steeves and Sussex, 1989). The meristem region contains apical initial cells and subepidermal cells from which the gametes are derived (Medford, 1992). Theoreti- cally, there are two possibilities for recovering transgenic plants via transfer of DNA into the shoot apical meristem. One possibility is that transgenic progeny may be directly produced via transformation of the subepidermal germ- line cells followed by the development of a partially trans- genic reproductive organ. In this case, the primary trans- formants will always be chimeric. An alternative possibility is to multiply transgenic apical meristem cells and/or germ-line cells, which can be reprogrammed in the developmental direction under in vitro conditions. Trans- genic plants can be regenerated from these cells with or without selection. Our previous research on maize (Zea

This research was supported by the Office of USAID/CAIRO/ AGA/A, under Cooperative Agreement No. 263-0152-A-00- 3036-00 and Michigan State University Bridging Funds to M.B.S. The construction of plasmids was performed at Cornell University. The development of the transformation system, the transforma- tion, and the analysis of transformants were performed at Michi- gan State University.

* Corresponding author; e-mail sticklelQpilot.msu.edu; fax 1-517-353-1698.

mays L.) morphogenesis demonstrated that the maize mer- istem is morphogenetically plastic and can be manipulated to produce multiple shoots, somatic embryos, tassels, or ears in a relatively genotype-independent manner by sim- ple variation of in vitro culture conditions (Zhong et al., 1992a, 1992b). Based on this concept, we transformed maize meristems via microprojectile bombardment with a series of chimeric genes, including bar, pin2, and gus.

In this paper, we report the efficient recovery of fertile transgenic maize plants via a shoot-multiplication system after microprojectile bombardment of shoot tips. Maize shoot apices were transformed with a plasmid incorporat- ing bar driven by the CaMV 35s promoter and pin2 with the wound-inducible pin2 promoter (Fig. l), either alone or in combination with another plasmid containing gus driven by the 5' region of Actl (Fig. 1). The co-integration and co-inheritance of linked and unlinked genes in transgenic R,, RI, and R, maize plants were confirmed. The functional activity of Actl-gus in transgenic R, and RI plants was extensively analyzed.

MATERIALS A N D METHODS

Plant Materiais

Following our previous work on shoot multiplication of 20 genotypes of maize (Zea mays L.) (Zhong et al., 1992a), another 16 genotypes were tested. A11 of them responded to form multiple-shoot-tip clumps (data not shown). We ran- domly selected 12 genotypes (HNP, IGES, B73, VA22, and FR632 from Illinois Foundation Seeds, Champaign, IL; 509, 5922, and 482 from the Michigan Agricultural Experiment Station, East Lansing, MI; K1 from the Institute of Botany, Academy of Science, Beijing, China; A188 from the Crop Breeding Project, Department of Agronomy, University of Minnesota, St. Paul; and L6 and L9 from the Agricultural

Abbreviations: Actl , rice actin 1 gene; BA, N6-enzyladenine; bar, bacterial phosphinothricin acetyltransferase gene; CaMV, cauli- flower mosaic virus; CSM, MS medium containing 2 mg/L BA; CSMD, CSM containing 0.5 mg/L 2,4-D; CSMDSG, CSMD contain- ing 3 mg/L G; CSMD5G, CSMD containing 5 mg/L G; gus, bac- teria1 GUS gene; HNP, Honey N Pearl; IBA, indole-3-butyric acid; IGES, Illinois Golden Extra Sweet; G, glufosinate ammonium [monoammonium 2-amino-(hydrooxymethyl-phosphiny)butano- ate]; MS, Murashige and Skoog basal medium; nos, nopaline syn- thase gene; PAT, phosphinothricin acetyltransferase; pin2, potato proteinase inhibitor I1 gene.

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1098 Zhong et al. Plant Physiol. Vol. 'I 1 O, 1996

H B B B,H EV*;E B E v v v v v

I O 0.5 0.6 0.9 0.8 0.6 0.3 2 7

X* E' S B B S N'

pAcf/-F A ! FS ] \A ! * I i

0.38 O 51 0.20 0.35 1.87 0.26 2.90

Figure 1. Schematic representation of plasmids. pTW-a contains the pin2 promoter (pin2 5'), the rice Actl 5' intron (As'l), the pin2 coding sequence (pin2), and the pin2 terminator (pin2 3') followed by the CaMV 35s promoter (35S), the barcoding sequence (bar), and the nos terminator (nos) in the vector pUC 19. pActl-F contains the 5' flanking sequence (A 5' FS), the noncoding portions of Actl exon (open box), the Actl intron (A5'I), the 5' coding portions of Actl exon (open box), the gus coding sequence (pus), and the nos termi- nator (nos) in the vector pBluescript KS (pBlu. KS). The restriction enzyme sites are abbreviated as follows: B, BamHI; E, EcoRI; EV, EcoRV; H, Hindlll; N, Nod; S, Sacl; and X, Xhol. *, Unique site in plasmids. Numerals below each region are length of fragments in kb.

Genetic Engineering Research Institute, Giza, Egypt) for this research. Mature kernels of 12 genotypes were surface- sterilized with 70% ethanol and 2.6% sodium hypochlorite and germinated on MS (Murashige and Skoog, 1962) basal medium (Sigma) as described (Zhong et al., 1992a).

A 5-mm-long section of a 7-d-old seedling containing a shoot tip, leaf primordia, and a portion of young leaves and stem proximal to the shoot tip was excised, cultured in darkness for 4 weeks, and then maintained in light (24 h, 60 pmol [quanta] m-* s-') at 4-week intervals on shoot mul- tiplication medium CSMD or CSM (Zhong et al., 1992a). Initiation of shoot-tip clumps from the original shoot tips occurred 2 to 4 weeks after culture. Repeated subculture of divided shoot-tip clumps resulted in a higher frequency of shoot-tip formation and more compact shoot-tip clusters. Morphologically, greater numbers of adventitious shoot tips but less leaf and stem elongation were produced in CSMD than in CSM, although both CSM and CSMD fa- vored the axillary and adventitious bud formation (Zhong et al., 1992a) for all genotypes tested. Shoot tips from the multiplied clumps at the end of each subculture (4 weeks) usually have severa1 germinated leaves. The shoot tips with one to three visible leaf primordia were selected for bombardment under a stereomicroscope (Carl Zeiss).

Because the sweet corn genotypes HNP and IGES were our model materials for morphogenesis studies, shoot mul- tiplication cultures of these two genotypes initiated on July 15, 1990, were also used for this research.

Plasmids

The plasmid pTW-a (7.4 kb) contained the Streptomyces kygroscopicus bar gene (Thompson et al., 1987) and the gene pin2 (Keil et al., 1986) in the pUC19 vector. The bar coding region was driven by the CaMV 355 promoter and was terminated with the nos 3' region (Bevan et al., 1983). The

pin2 coding region was driven by the wound-inducible pin2 promoter and rice actin 1 (Actl) 5' intron and terminated with the pin2 3' region (Fig. 1).

The plasmid pActl-F (6.5 kb; McElroy et al., l990) con- tained the Esckerichia coli u i d A gene (gus) (Jefferson, 1987) driven by a region 1.3 kb upstream of the rice Act l trans- lation codon (McElroy et al., 1990) and followed by the nos 3' terminator in the pBluescript KS vector (Fi,g. 1). The plasmid DNA was suspended in 10 mM Tris-HC1 and 1 mM EDTA buffer (pH 8.0) at a concentration of 1 pg/pL.

Microprojectile Bombardment

Plasmid DNA was precipitated onto gold particles (1.0 and 1.6 pm in diameter; Bio-Rad) or tungsten particles (0.9-1.2 pm in diameter; GTE Sylvania, Towanda, PA) following a modification of the original protocol described by Bio-Rad. Briefly, 30 mg of particles were sterilized in 1 mL of 100% ethanol with vortexing for 30 min. A 50-pL aliquot of the particle-ethanol suspension was pipetted into a microcentrifuge tube while vortexing continuously. After washing twice with sterile-distilled water, the particles were resuspended in 332 pL of sterile-distilled water. A total of 15 pL of plasmid DNA (15 pg of plasmid or a 1:l mixture of two plasmids), 225 pL of CaCI, (2.5 M), and 50 pL of spermidine (0.1 M) were successively added and continuously vortexed for 5 min at room tempel-ature. The mixture was then incubated on ice for 10 min. The DNA- coated particles were pelleted by centrifuging at 13,000 rpm for 1 min. After discarding the supernatant, the par- ticles were washed with 500 pL of absolute ethanol by vortexing for 30 s, centrifuging for 1 min, and removing the supernatant and were finally resuspended in 100 or 200 pL of absolute ethanol. For each bombardment, 10 pL of the particle suspension (150 or 75 pg of particles per shot) was pipetted onto the center of the macrocarriers. The prepared macrocarriers were used as soon as the ethanol evaporated.

Prior to bombardment, shoot tips or shoot-tip clumps from cultures of various stages were physically exposed by remova1 of the coleoptile and leaves if necessary. The shoot tips (placed in a circular area of about 1.5 cm diameter) were positioned on multiplication medium solidified with 0.5% Phytagel (Sigma) in a Petri dish below the micro- projectile stopping screen.

Bombardments were carried out using a Biolistic particle acceleration device (PDS 1000/He, Bio-Rad) under a cham- ber pressure of 26 mm of Hg at distances of 1.5,2.0, and 6.5 cm from the rupture disc to the macrocarriers to the stop- ping screen to the target, respectively, with three helium pressures (1100, 1550, and 1800 p.s.i.) and single or multi- ple shots per plates.

Selection and Regeneration of Transformants

For analysis of the variation in stable expression in shoot tips among different genotypes, bombarded explants were immediately transferred to fresh multiplication medium without selection for 6 weeks with one subculture. The explants used for bombardment were at least 1-month-old

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Maize Transformation via Bombardment of Shoot Tips 1 099

cultured shoot tips due to the intensive labor required to dissect shoot tips from young maize seedlings.

For recovery of the transformants, bombarded shoot tips were cultured on multiplication medium CSMD for 4 weeks. The clumps then were divided and subcultured on CSMD3G for 4 weeks. Subsequent subculture at 4-week intervals was carried out by selecting, dividing, and cul- turing green clumps on CSMD5G.

Regeneration of plants was obtained by transfer of CSMD5G-selected shoot-tip clumps to MS medium con- taining 0.5 mg/L BA, 0.5 mg/L IBA, and 5 to 10 mg/L G. Developed shoots were rooted on MS medium containing 1 mg/L IBA and 10 mg/L G. Plantlets then were trans- planted to a soil mixture composed of 1:l (v/v) peat:perlite and grown to maturity.

Histochemical and Fluorometric Assay of CUS

The modified histochemical assay buffer consisted of 100 mM NaPO, buffer, 100 mM Na,EDTA, 50 mM K,Fe(CN), 3H,O, and 0.1% Triton X-100 (pH 7.0). 5-Bromo- 4-chloro-3-indolyl-/3-~-glucuronic acid (Clontech Laborato- ries, Palo Alto, CA) was dissolved in 50% ethanol, stored at -2O"C, and added to buffer to a final concentration of 0.5 mg/mL prior to assay.

The fluorometric assay buffer consisted of 50 mM NaPO, buffer, 10 mM P-mercaptoethanol, 10 mM Na,EDTA, 0.1% sodium lauryl sarcosine, and 0.10/, Triton X-100. 4-Methy- lumbelliferyl /3-D-glucuronide was added to buffer at a final concentration of 1 mg/mL prior to assay.

Whole plantlets and organs from the transgenic and untransformed control plants were immersed in GUS sub- strate mixture immediately followed by vacuum treatment, then incubated at 37°C. The histochemical and fluorometric localization of GUS activity was carried out using free- hand cross-sections. The stained materials were examined under a Zeiss SV8 stereomicroscope or a Zeiss Axioskop routine microscope.

Based on our studies, transient expression of gus in shoot tips of maize 1 d after bombardment was comparable to that in type-I and type-I1 embryogenic calli and immature embryos of maize. A preliminary study using multiple shoot tips of maize genotype HNP showed that the number of blue sectors in shoot-tip clumps was similar 3 weeks and 3 months after bombardment. Therefore, expression of gus in shoot tips 6 weeks after bombardment was considered to be stable. The expression of gus in bombarded shoot-tip clumps was observed under a stereomicroscope. A single isolated cell or an aggregate of cells exhibiting dark blue color in shoot meristems other than in leaves was consid- ered as one GUS-expressing unit. The efficiency of stable expression in shoot tips was relative to the number of shoot tips bombarded. To localize the position of cells with stable expression in the shoot tips, tissues with blue foci were carefully dissected and examined under the microscope.

Chl was extracted by successive incubation in 70% eth- ano1 for 2 h and 100% ethanol overnight before the samples were photographed.

PAT Activity Assay

The PAT activity was determined indirectly by resistance of plants to herbicide application. Two applications of Ig- nite nonselective herbicide (also known as Basta; Hoechst- Roussel Agri-Vet Company, Somerville, NJ) containing 200 g/L G, the active ingredient, were performed. For R, plants, a 1% solution of herbicide (v/v) was sprayed on whole plants at the three-leaf and six-leaf stage in the greenhouse. For RI and R, plants, the herbicide (1%) was also locally painted on the youngest leaf at the three-leaf stage and sprayed on whole plants at the six-leaf stage.

D N A Ce l Blot Analysis

Genomic DNA was isolated from G-resistant multiple shoot-tip clumps and from leaf tissues of untransformed plants, primary transformants, and their progeny using the hexadecyltrimethylammonium bromide method (Rogers and Bendich, 1985). Ten micrograms of DNA per sample were digested with restriction enzymes, fractionated in a 1% agarose gel, and transferred to Nytran membranes (Schleicher & Schuell). Following the procedure recom- mended by Schleicher & Schuell, after prehybridization, hybridizations were carried out with an [a-32P]dCTP probe labeled with T7 Quick Prime (Pharmacia) as described by the manufacturer. Filters were analyzed by autoradiogra- phy using X-Omat film (Kodak).

Production and Analysis of Progeny

Transgenic plants were self-pollinated. Screening of transgenic progeny containing bar was done either through germination of immature embryos or mature caryopses on MS basal medium with 5 mg/L G to minimize the time required to obtain transgenic R, and R, plants, or through treatment of greenhouse-germinated progeny with the her- bicide Ignite. To detect the transgenic progeny containing gus, roots, immature embryos, young leaf sections, and pollen grains of RI and R, progeny were subjected to histochemical assay. The segregation data were statistically analyzed by 2 test (Strickberger, 1985).

RESULTS

Stable Expression of gus in Shoot Tips after Bombardments

Bombardment of undissected shoot tips using GUS as a visible marker revealed that most expression events oc- curred on the leaves that covered the shoot tip or that developed from leaf primordia. No expression was ob- served in shoot meristems after further development of shoot-tip explants without multiplication (data not shown).

The efficiency of both transient and stable expression in shoot tips was greatly affected by the penetration of parti- cles, which depended on the size, quality, density, and velocity of the particles. The larger gold particles (1.6 pm) incorporated with high density, multiple bombardments with 1800 p.s.i. acceleration pressure showed the lowest transient and stable expression, but reducing the particle density and/or acceleration pressure improved the effi-

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1100 Zhong et al. Plant Physiol. Vol. 110, 1996

ciency of stable expression. Using smaller gold particles,the most efficient level of stable expression was obtainedwith a medium velocity (1550 p.s.i.), multiple shots (up tofour), and a low density of particles (75 /xg/shot). Tungstenparticles and gold particles gave comparable results. Thelow density of particles (75 jag/shot) with multiple shotsgave a higher ratio of stable expression than the highdensity of particles (150 jug/shot) regardless of single ormultiple shots.

The efficiency of stable expression in shoot tips of HNPvaried from 2% (bombarded twice with 150 ,̂g of 1.6-jj.mgold particles at 1800 p.s.i.) to 32% (bombarded four timeswith 75 pig of 1.0-jum tungsten particles at 1550 p.s.i.). Themajority of blue sectors were observed out of the center ofthe target 6 weeks after bombardment. From 530 shoot tipsexamined, the percentage of blue foci below the epidermallayer of the meristem was about 65% with gold and 72%with tungsten with 1550 p.s.i. pressure, and about 22%with gold and 10% with tungsten with 1100 p.s.i.

Shoot tips from 1-month-old cultures showed lower ef-ficiency of stable expression after bombardment than oldercultures, although the level of transient expression wassimilar. However, the efficiency of stable expression wasmuch higher in shoot tips bombarded at 1 or 2 weeks (30%)than in shoot tips bombarded at 4 weeks after subculture(8%). There was no significant difference in transient orstable expression between the two media, CSM and CSMD.

Different genotypes did not show any significant differencein either transient or stable expression of gus.

Thus, the optimal delivery of DNA into subepidermalcells of shoot tips was achieved by the bombardment ofmore than 2-month-old shoot-tip clumps that were subcul-tured every 2 weeks, with a low density (75 fig/shot) of1.0-nm gold or tungsten particles with multiple shots (four)and 1550 p.s.i. acceleration pressure.

Recovery of Stably Transformed Shoot-Tip Clumps andRegeneration of Transformants

HNP and IGES were chosen for recovery of transfor-mants because they were model materials for our morpho-genesis study. Their shoot-tip clumps were regularly initi-ated and continuously subcultured for more than 4 yearswithout loss of regeneration ability or fertility. The shoottips were carefully selected and gathered under a stereomi-croscope as shown in Figure 2A prior to bombardmentunder the optimal conditions described above. As many asfive blue sectors showing stable expression were found ina single shoot tip 1 month after bombardment (Fig. 3A).Further development of the bombarded shoot tips withoutmultiplication gave rise to different patterns of chimericshoots after GUS histochemical assay (Fig. 3B). The bluesectors at the leaf tips or on the leaf blade were mostprobably from the transformed cells of the leaf primodium,

Figure 2. Recovery of transgenic maize plants.A, Shoot tips prior to bombardment (X40). B,Untransformed controls after 1 month of selec-tion (1) and recovery and multiplication of G-resistant multiple shoot-tip clumps bombardedwith pTW-a after 1st month (2), 2nd month (3),and 3rd month (4) of selection (X0.4). Arrowsshow some of the G-resistant clumps. C, A tasselof an herbicide-resistant R0 plant (X0.1). D, Earsof an herbicide-resistant R0 plant (X0.1). E, R,progeny 10 d after foliar spray of 1% Ignite. Allof the nontransformed control plants (1 and 2)are sensitive, whereas transformed plants (3-6)segregated into resistant and sensitive individu-als (marked by arrows) (X0.25).

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Maize Transformation via Bombardment of Shoot Tips 1101

which could not produce multiple shoot tips under our culture conditions, whereas the development of trans- formed meristematic cells could result in the successive formation of blue sectors through the leaf blades and sheaths. The 1-month multiplication of bombarded shoot- tip clumps without selection pressure gave similar devel- opmental patterns.

Production of PAT- and CUS-Positive Shoot-Tip Clumps

The transformation results with plasmid pTW-a (which contains the bar gene) from eight independent experiments with a total of 22 plates are summarized in Table I. The efficiency of recovery of G-resistant shoot-tip clumps after the first round of selection (1 month) with 3 mg/L G varied from O to 6.7% under different culture periods prior to selection.

Six independent co-transformation experiments with two unlinked genes (bar and gus in plasmids pTW-a and pAct2-F, respectively) gave a slightly lower efficiency of recovery of G-resistant shoot-tip clumps after the first round of selection, ranging from O to 3.4% (Table 11), com- pared to the efficiency of two linked genes, bar and pin2, in plasmid pTW-a (Table I).

Our results indicated that 1 month of multiplication of bombarded shoot-tip clumps without selection pressure is necessary to achieve a high efficiency of transformation. More than 1 month of multiplication did not improve the recovery of resistant shoot-tip clumps. During the 1st month of selection on CSMD3G, most of the bombarded and unbombarded (control) shoot-tip clumps turned brown and necrotic within 2 weeks. A small number of G-resistant shoot tips emerged clearly with a healthy green color from these necrotic tissues near the end of the 1st month of selection (Fig. 28). The shoot-tip clumps from control experiments rarely survived in this selection scheme. The size of the G-resistant clumps varied from less than 0.1 to 0.3 cm’. Each clump contained 5 to 20 visible shoot tips. Since the multiple shoot-tip clumps were very compact, intimate signal communication and substance ex- change between cells in the clumps provided a great pos- sibility of cross-protection between transformed cells and nontransformed cells. Chimeric clusters of shoots without selection pressure were produced at such a stage (Fig. 3C). Further selection of transformed shoot-tip clumps devoid of nontransformed cells was carried out by dissecting, di- viding, and culturing of the surviving shoot tips on CSMD5G until a11 divided shoot-tip clumps uniformly sur- vived in CSMD5G. Clumps resistant to 5 mg/L G were also resistant to 10 mg/L G. In both HNP and IGES, stable uniform growth clumps were obtained after three or four rounds of selection (1 month in CSMD3G and 3 months in CSMD5G) (Fig. 28). One independent shoot-tip clump (one transformation event) from the 1st month of selection was multiplied to about 15 shoot-tip clumps at 5 months after bombardment and was capable of regenerating more than 1500 transgenic plantlets.

A11 independent G-resistant clumps were further sub- jected to DNA hybridization analysis for the presence of bar, pin2, and gus (Fig. 4). Hybridization to a11 three trans-

genes in high-molecular-weight DNA in a11 independent G-resistant clumps, but not in controls, confirmed that the transgenes bar, pin2, and/or gus were integrated into the chromosomal DNA of a11 resistant transformants. The transformation efficiency of two linked genes (bar and pin2) was 100% in both transformation (95/95) and co-transfor- mation (51/51) experiments (Tables I and 11). The co-trans- formation frequency of two unlinked genes (bar and gus) was 80% (41/51) (Table 11). Histochemical assays of GUS activity in a11 independent G-resistant transgenic clumps (Fig. 3D) revealed a co-expression frequency of 57% (29/ 51) for two unlinked genes (gus and bar). High co-transfor- mation frequency with lower co-expression frequency of unlinked genes suggested that the expression of gus in some PAT-positive events did not occur or occurred at a histochemically undetectable level.

Regeneration and Analysis of Primary Transformants (R,)

Over 2000 plantlets regenerated from 26 independent transgenic events of both genotypes, HNP and IGES, were transferred to the greenhouse for further development. About 90% of them survived to maturity in the greenhouse.

Five plants from each of 10 independent transformed clumps were analyzed for the presence of bar, pin2, and/or gus using Southern blot hybridization. DNA hybridization analysis of genomic DNA from each plant digested with EcoRV (which has a unique site in pTW-a) and/or EcoRI (which has a unique site in pAct2-F) revealed that the patterns of hybridization bands with probes containing the bar, pin2, or gus coding region were identical in the five plants from the same clump, confirming that they were from the same transformed cell, and varied from one clump to another, suggesting that they were independent transformation events (data not shown). A comparison of the copy number and insertion site of a11 of the transgenes in each different transformation event was also performed (eg. see Fig. 5). Genomic DNA digested with EcoRI gave a 0.9-kb bar-hybridizing band that comigrated with the 0.9-kb fragment, containing the bar and nos 3’ terminator, from the EcoRI digestion of pTW-a (Fig. 5A). Genomic DNA digested with HindIII showed a 3.0-kb pin2-hybrid- izing band that comigrated with the 3.0-kb fragment, con- taining the pin2 promoter, the Actl 5’ intron, the pin2 coding region, and the pin2 terminator, from the HindIII digestion of pTW-a (Fig. 5B). The EcoRV-digested genomic DNA showed different hybridization band patterns, indi- cating a different integration site for both bar and pin2 in different transformation events with various copy numbers (Fig. 5, A and B). Following the digestion of genomic DNA with EcoRI and NotI, a 3.3-kb gus-hybridizing band was seen that comigrated with the 3.3-kb fragment, containing a portion of the rice Actl promoter, the gus coding region, and the nos 3‘ terminator, from the EcoRI-NotI digestion of pAct2-F. Following the digestion of genomic DNA with EcoRI (there is only one site in the plasmid pAct2-F), the pattern of mobility and intensity of the EcoRI hybridization bands in Figure 5C suggested that the intact gus was inte- grated into different sites in different independent trans-

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1102 Zhong et al. Plant Physiol. Vol. 110, 1996

Figure 3. Histochemical and fluorometric assay of Acf/-gus expression in transgenic maize plants. A, SUiblr expression ofGUS in a shoot meristem 6 weeks after bombardment without selection (X300, 30 min of staining); I, leaf; m, meristem. B,Shoots directly developed from control (1) and transformed (2-4) shoot tips without selection 6 weeks after bombardmentwith pActl-f (X0.2, 1 h of staining). Arrows show successive formation of the chimeric blue sectors through the entireleaves. C, Shoot clumps regenerated from control (1) and 1-month G-resistant (2 and 3) shoot-tip clumps (X0.2, 1 h ofstaining). D, Shoot-tip clumps from controls (column 1) and from gus-positive, G-resistant (columns 2-8) independentlytransformed events (X0.35). E, Cross-section of a G-resistant multiple-shoot-tip clump 4 months after bombardment (X25,1 h of staining). F, Plantlets regenerated from untransformed control (1) and G-resistant (2) shoot-tip clumps (X0.4, 3 h ofstaining); sr, secondary lateral root. G, Shoot tips, TA10 (XI 20, 10 min of staining); st, shoot tip. H, Transverse section ofleaves, TA10 (X10, 20 min of staining); Ib, leaf blade; Is, leaf sheath; Iv, lateral vein; iv, intermediate vein. I, Transverse

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Maize Transformation via Bombardment of Shoot Tips 1103

Table 1. Summary o f transformation experiments with pTW-a

No. of No. of Age of Days No. of lndependent bar+/pinPd Plates Shoot Tips Cultures" Postcultureb EventsC Genotype

HNP 4 622 50 3 O 2 181 2.5 3 1 (0.6) 1 /I 2 284 2.5 30 19 (6.7) 1 911 9 3 291 52 30 16 (5.5) 16/16 4 560 23 30 21 (3.8) 21/21

IGES 3 41 2 45 30 15 (3.6) 1 5/15 2 21 9 3 30 9 (4.1) 9/9 2 260 21 30 14 (5.4) 14/14

Subtotal 22 2829 95 (3.4) 95/95

a Months after the initiation of shoot tip multiplication from the 7-d-old seedlings. All of the shoot tips for bombardment were selected from the shoot-multiplication cultures 2 weeks after subculture. The number of independent events was the total number of independent G-resistant clumps obtained at the end of the 1st month of selection. The number in parentheses is the relative efficiency (%) of stable transformation measured as total G-resistant clumps divided by total number of bombarded shoot t i ix, X100%.

Days of multiplication after bombardment before first-round selection.

bar+/~in2+ shows the number of indeDendent c lumm with each gene. -

formants. The estimated copy number varied from approx- imately 4 to more than 10.

To assess the expression of bar, Ignite solution (200 g/L [16.22%] G, the active ingredient) was sprayed on a11 of the putatively transformed plants. A11 of the plants were resis- tant to 1% Ignite, whereas untransformed control plants showed necrosis in 2 d and died in 10 d after the applica- tion of Ignite.

More than 90% of R, transformants produced one nor- mal tassel (Fig. 2C) and one to three fertile ears per plant (Fig. 2D) 2 to 3 months after transfer to the greenhouse. About 50% of them showed reduced stature compared to seed-derived plants. Morphologically abnormal character- istics such as pistillate flowers on tassels and curly leaves were observed in less than 5% of the plants. Less than 1% of the plants that survived failed to develop to maturity. The yield of kernels per ear varied from 6 to 200 after self-pollination, although most of the plants yielded 50 to 80 kernels. Mature kernels were harvested 9 months after bombardment.

Histochemical and Fluorometric Localization of CUS in R,, R,, and R, Plants

The visualization of the Act l -ps activity pattern in trans- genic R, plants is summarized in Figure 3, E to S. High GUS activity was observed in the multiplication areas of shoot-tip clumps after histochemical assay (Fig. 3E). Whole plantlets regenerated from in vitro transgenic and untransformed shoot-tip clumps were immersed in 6 mL of 5-bromo-4- chloro-3-indo~y~-~-~-g~ucuro~c acid assay buffer mixture.

Ten minutes after incubation at 37"C, the root hairs presented the first sign of GUS activity, followed by the emergence of blue coloration in the root tips, secondary roots, and root elongation zones within 30 min. The appearance of the blue color on the younger leaves began from the base of the leaves and gradually extended to the entire leaves and the older leaves. The entire plantlets turned to intense blue after 2 h of incubation at 37°C (Fig. 3F). Shoot tips, including meristems and leaf primordia, showed the strongest GUS activity after 5 min of exposure to the assay buffer (Fig. 3G).

A series of hand-cut cross-sections from control and transgenic plants was subjected to histochemical and flu- orometric staining to localize the cellular pattern of GUS activity. After 10 min of incubation, the sections were observed under the microscope. The younger the leaves, the more intense the blue (Fig. 3H). Stems proximal to shoot tips had more intense blue staining than internodes (Fig. 31). The highest intensity of GUS staining was located in the shoot and root tip areas, axillary buds, and vascular system (Fig. 3J). Fluorometric localization of GUS revealed that the high GUS activity existed in phloem cells of the vascular system of transgenic plants (Fig. 3K), whereas no fluorescence was observed in the sections of the control plants. The pattern of GUS intensity in cells was phloem > epidermis > mesophyll cells in leaves, and phloem > root hair > endodermis > epidermis > cortex cells in roots.

Reproductive meristems showed blue color as intense as that of the vegetative meristems, particularly in a11 spikelet primordia of reproductive organs (Fig. 3, L and M). Silks over 6 cm long from most independent transformants dis-

Figure 3. (Continued from previous page.) section of leaves and a stem proximal to the shoot tip, TA10 (XlO, 20 min of staining); Is, leaf sheath, sm, stem. J, Transverse section of a stem with an axillary bud, TAlO (XlO, 20 min of staining); ab, axillary bud; Ih, leaf hair; ls, leaf sheath; sm, stem. K, Fluorometric assay of GUS in a transverse section of leaf and stem, TAlO (X10, l 0 . m i n of staining); Ih, leaf hair; ls, leaf sheath; pl, phloem; sm, stem; sv, stem vascular bundle. L, An ear primordium, TA10 (X32, 15 min of staining); sp, spikelet-pair primordium. M, lmmature ear, TAlO (X10, 1 h of staining); s, spikelet primordium. N, lmmature ear, TA34 (X0.5, 1 h of staining); SI, silk. O, Mature pollen, TAlO (X20, 30 min of staining). P, lmmature tassel, TA1 O (X 12, 1 h of staining); br, lateral branch; cr, central rachis. Q, lmmature florets, TA1 O (X3.5, 1 h of staining); an, anther; gi, inner glume; go, outer glume. R, lmmature embryos from heterozygote, TA 3-1 1 (X6, 30 min of staining). S, Mature pollen from homozygote, TA 10-27 (X25, 30 min of staining).

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1104 Zhong et al. Plant Physiol. Vol. 110, 1996

Table II. Summary of co-transformation experiments with pTW-a and pActl-F

Genotype

HNP

ICES

Subtotal

No. ofPlates

232532

17

No. ofShoot Tips

312421194607377201

2112

Age ofCultures"

33262.52.5

5329

DaysPostcultureb

33030303030

No. of IndependentEvents'7

013(3.1)

5 (2.6)21 (3.4)

9 (2.4)3(1.5)

51 (2.4)

faar+/pm2+/gus+/GUS+d

13/13/11/105/5/4/2

21/21/16/119/9/7/43/3/3/2

51/51/41/29a Months after the initiation of shoot tip multiplication from the 7-d-old seedlings. All of the shoot tips for bombardment were selected from

the shoot-multiplication cultures 2 weeks after subculture. h Days of multiplication after bombardment before first-round selection. c Thenumber of independent events was the total number of independent G-resistant clumps obtained at the end of the 1st month of selection. Thenumber in parentheses is the relative efficiency (%) of stable transformation measured as total G-resistant clumps divided by total number ofbombarded shoot tips, X100%. d bar+/pin2+/gus+/G\JS+ shows the number of independent clumps with each gene or enzyme activity.

played no detectable GUS activity, but a strong blue col-oration was observed throughout the entire developmentof silks in several independent transformants containingmultiple GUS transgenes (Fig. 3N). Mature pollen grainsshowed relatively high GUS activity, but were variable(Fig. 3O). Florets, glumes, young anthers, and young silksdisplayed intense GUS staining (Fig. 3, P and Q). Matureflowers except for pollen did not exhibit GUS activity evenwith overnight incubation. No reproductive organs fromthe control untransformed plants showed any blue staining.

Histochemical assays of hand-cut cross-sections of veg-etative organs from in vitro plantlets from different trans-

formation events and their R: and R2 progeny (containingsingle or multiple copies of transgenes) revealed that thepattern of GUS activity was similar with the exception ofabout 2 to 4% of plantlets, in which the elongation zones ofroots showed higher intensities of GUS staining than theroot tips (data not shown).

GUS activity in immature embryos of transformants varieddepending on their developmental stage (Fig. 3R). However,all immature embryos exhibited a low intensity of GUS stain-ing in the coleoptile and the center of scutellum tissues.

Mature pollen grains from primary transformants (R0)from most of the independent transformation events had a

Probes

bar

. : «M 1*1 **> «-,R kh - - ' l ' - ' - : - f - f

7.2 —pin2

bar

gus

Figure 4. DNA gel blot analysis of undigested genomic DMA (10 /u-g/lane) from independent G-resistant (T1-T56) andGUS-positive (TA1-TA19) shoot-tip clumps. pTW-a and f>Act1-f were linearized with fcoRV or fcoRI, respectively. 1, 2,and 5 each represents reconstruction with number of copies of the plasmid pTW-a or pAct1-f to correspond to that copynumber of transgenes in transformed plants. CO, Untransformed control. A and B, Transformed with pTW-a; C and D,transformed with pTW-a and pAcfJ-F. Each filter shown was hybridized with the indicated coding region probe. www.plantphysiol.orgon February 1, 2020 - Published by Downloaded from

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Maize Transformation via Bombardment of Shoot Tips 1105

pTW-a

A k h 3 3 5 1 C O TA10 TA23 TA34 TA41 TA50c b a a a b a b c a b c a b c a b c a b c

pT\V-a

B kh 3 3 10 4 CO TA10 TA23 TA34 TA41 TA50c b d d d b d b c d b c d h c r i h t d b c

14.1 —

oAcil-¥C kh 3 3 in 4 CO TA10 TA23 TA34 TA41 TA50

c a e c e a e a c e a c e

Figure 5. Southern blot analysis of five indepen-dent transformed R0 plants. 1, 3, 4, 5, and 10each represents the amount of the plasmidpTW-a or pActl-f that would correspond to thatnumber of the transgenes in transformed plants.Each lane contains 10 ̂ g of genomic DMA froma nontransformed control maize plant (CO) orputatively transformed maize plants (TA10,TA23, TA34, TA41, and TA50). Lanes a, DMAdigested with fcoRI; lanes b, DNA digested withfcoRV; lanes c, undigested DNA; lanes d, DNAdigested with H/ndlll; lanes e, DNA digestedwith fcoRI and Noti. A, Filter was hybridizedwith a-32P-labeled bar coding region probe. B,Filter was hybridized with a-32P-labeled pin2coding region and terminator probe. C, Filterwas hybridized with a-32P-labeled gus codingregion probe.

3.3 —

ratio of GUS positive:negative of approximately 1:1. Ma-ture pollen grains from single anthers of RQ, Rj, and R2transformants displayed different intensities of GUS stain-ing. All mature pollen grains from homozygous Rj and R2progeny showed GUS activity (Fig. 3S).

Genetic and Molecular Analysis of R, and R2 Progeny

The phenotypes of R, and R2 plants were identical tothose of plants derived from native self-pollinated kernelsin the greenhouse. To determine the inheritance of selectedand unselected genes, bar and gus, in self-pollinated Ra andR2 progeny, expression of gus and/or bar in progeny from13 independent primary transformants was analyzed. GUShistochemical assay of roots, immature embryos, youngleaf sections, and pollen grains was used to assess thepresence and expression of gus in progeny. The presence ofbar, corresponding to PAT activity, in progeny was deter-minated by resistance of the plantlets to 5 mg/L G in vitroand resistance of the plants to 1% Ignite in the greenhouse.

Figures 2E and 3R present the segregation of expression ofbar in Rj progeny and gus in immature embryos of Rtprogeny, respectively. The results of segregation of bothbar and gus expression in Ra and R2 progeny are summa-rized in Table III.

The x2 analysis indicates that the inheritance of bar andgus expression followed Mendelian inheritance for a singledominant locus (3:1 segregation ratio) in all of the R2progeny tested but not in all of the Rj progeny tested. Thesegregation ratio for PAT activity in the progeny of trans-formant T93 was significantly different from the expectedratio of 3:1. The segregation ratio of PAT and/or GUSactivity in transformants T77 and TA50 fit a 15:1 model forthe presence of two independent dominant loci. Segrega-tion ratios for plants T8, T20, TA3, TA10, and TA34 fit a 1:1model, consistent with lack of either male or female trans-mission.

Southern blot analysis of selected events revealed thepresence of transgenes in Ra and R2 progeny, concordant www.plantphysiol.orgon February 1, 2020 - Published by Downloaded from

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1106 Zhong et al. Plant Physiol. Vol. 110, 1996

Table III. Segregation of expression of bar and gus in self-pollinated R , and R, progeny

Generation Transformant Total Assayed

RI HNP T8 HNP T16 ICES T20 HNP T44 ICES T63 ICES T77 HNP T93 HNP TA3 HNP TA10 HNP TA23 HNP TA34 ICES TA41 ICES TA50

R 2 HNP T8-4 HNP T8-6 HNP T44-3 IGES T77-9 HNP TA3-11 HNP TA1 0-1 3 HNP TA23-7

43 36 51 16

147 78 74 18 32 69 44 93 22 53 86 41 46 13 50 21

PAT(+/-)"

In vitro In vivo

1511 3 1114 813 1 716

31/20 1313

9 215 5 7414 2 015 4

511 3 1917 016 3011 4 1 511 O

68/25 713 2011 4

2111 4111 2 59/27 31/10 3011 6 1013

1 614 2 515 815 810

XZh

4.1d O 4.76d 0.08

1 5.38d 88.2gd 1 8.96d

3.02 3.67 0.09 3.88d 0.06 1.55 0.01 1.86 0.02 0.96 0.02

11 .43d

3.37d

GUS(+/-)'

In vitro In vivo

1018 1 719 O16 3311 1 1 511 O

614 1911 5 65/28

1814

N De 1218 2011 o

815 810

XZh

2.67 7.04d 0.82 6.82d 1 .O4 0.24

2.67 0.02

a Number of individual progeny resistanvsensitive to 5 mg/L C in vitro or 1% lgnite in the greenhouse (in vivo). x2 values calculated using Number of individual progeny staining positivelnegative by GUS histochemical assay on the roots or immature

Values are Yates' correction factor. embryos of progeny germinated in vitro or on the young leaves and pollen grains of progeny grown in the greenhouse (in vivo). significantly different from the expected ratio of 3:l at P < 0.05 with 1 degree of freedom. e ND, Not determined.

with the corresponding enzyme activity of PAT and GUS (data not shown).

DI SCUSSION

In this paper, we describe a nove1 transformation system that we believe will be a useful tool for both basic and ap- plied research on maize. The competence of in vitro- proliferated maize shoot meristems for integrative trans- formation and inherited expression of transgenes was demonstrated. Explants previously used for maize trans- formation are immature embryos, their derived type-I and type-I1 calli, or suspension-cultured cells (Rhodes et al., 1988; Fromm et al., 1990; Gordon-Kamm et al., 1990; D'Halluin et al., 1992; Golovkin et al., 1993; Koziel et al., 1993; Frame et al., 1994; Laursen et al., 1994; Lowe et al., 1995; Wan et al., 1995). Both dissection of zygotic coleop- tile-stage embryos and maintenance of donor plants in the greenhouse or field are very time consuming and labor intensive. In contrast, the explants (shoot tips) we used were easily and simply obtained from in vitro-regenerated shoot tips derived from germinated seedlings. Uniform explant sources can be obtained at any time of the year. In our system, the cycle from mature kernels of donor plants to the mature transgenic kernels was completed within 10 months.

To date, we have regenerated plantlets of 36 genotypes tested via shoot-tip multiplication, but the frequencies of fertile plant production in a11 genotypes other than HNP and IGES are still unknown. The frequency of plantlet production from multiple shoot tips varied from 24 to 97% in 36 genotypes tested. To increase the transformation fre-

quency and apply the system to a wide range o1 genotypes, further research is required on aspects such as develop- mental failure of shoot tips. Particularly, the failure of elite inbred lines on both multiplication and regeneration has been reported (Lowe et al., 1995). We have transformed two sweet corn genotypes and observed stable expression of gus in shoot meristems in 10 other genotypes tested. Three keys to increase the transformation efficiency and to make the co-transformation possible were the selection of newly developed shoot tips, the multiplication of bom- barded shoot tips for 1 month without selection pressure, and the use of selectable marker genes, such as bar, with the appropriate initial selection pressure. In combination with the previous result that chimeric plants were produced in a11 genotypes tested and stable transformants were recov- ered in two genotypes with shoot multiplicaticln responses at opposite ends of the spectrum (Lowe et a]., 1995), we believe our system could be developed to be truly geno- type independent and applied to other cereals. The system also has the potential to be used for studying the formation and function of shoot meristems.

In our study, both CaMV 35s and the Actl 5' region were used to control the expression of bar and gus. The activity of the CaMV 35s promoter in our transgenic maize was adequate to express bar for herbicide resistance in the veg- etative stage and was stably transmitted to their progeny, as was the case in transgenic rice (Cao et al., 1992). Results from our extensive histochemical and fluorometric assays of GUS activity in vegetative and reproductive organs of primary transformants and their progeny demonstrated activity of the rice Actl 5' region in different cells, tissues, and organs throughout development. Combined with sim-

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Maize Transformation via Bombardment of Shoot Tips 1107

ilar results reported earlier i n other monocots (Zhang et al., 1991; Zhong et al., 1993), our results indicate that the rice Actl 5‘ region could be a good promoter for monocot transformation even without modification of the expres- sion structure with other features.

Received November 27, 1995; accepted January 10, 1996. Copyright Clearance Center: 0032-0889/96/110/lO97/11.

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