Plant Molecular Biology 18: 301-313, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

301

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens Dennis Bidney, Chris Scelonge, Joanie Martich, Monique Burrus, Lynn Sims and Gary Huffman

Pioneer Hi-Bred International, Inc., Department of Biotechnology Research, 7300 N.W. 62nd Avenue, Johnston, IA 50131, USA Received 31 May 1991; accepted in revised form 16 September 1991

Key words:Agrobacterium tumefaciens, /%glucuronidase, meristem, microprojectile bombardment, neomycin phosphotransferase, sunflower, tobacco, transformation, wounding

Abstract

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/ Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.

Introduction

Disarmed Agrobacterium tumefaciens strains have become widely used to achieve stable transformation of dicotyledonous plant cells [8, 36]. Transformations by engineered Agrobacterium strains can be conveniently performed in some systems using wounded material such as leaf punches or macerated tissue [28]. Phenolic plant metabolites released from damaged cells are required for the activation of the virulence functions

within Agrobacterium [34]. Virulence functions mediate the mobilization of T-DNA from the Ti plasmid into the plant cell and subsequent integration into the plant genome [37]. Presumably, transgenic plants arise from regeneration-competent cells that are transformed following T-DNA transfer in the region of the wound. A recent development in transformation technology has been the advent of DNA delivery into cells by particle bombardment [30, 35]. Transforming DNA is precipitated onto dense micro-

302 carriers made of tungsten or gold and accelerated into cells. D N A introduced in this manner can be stably integrated into the genomes of some of the bombarded cells and transgenic plants recovered [12, 25, 35]. Ideally, particles should enter cells in a manner that will preserve their integrity. In practice, the high-velocity impact of dense particles on plant tissues will generate a large array of macro- and microwounds. Excessive wounding of the target tissue is detrimental to particle/ plasmid methods of stable transformation. However, such wounded tissue may provide numerous sites over the surface area of the target that are able to support Agrobacterium induction and plant cell transformation. This report describes the effect on transformation frequency when two different types of explants, tobacco leaves and sunflower meristems have been wounded by particle bombardment prior to Agrobaeterium treatment. A comparison of the transformation efficiencies of standard microprojectile bombardment methods and Agrobacterium is presented, as well as developmental consequences of transformation of cells within intact meristems.

Materials and methods

Plasmids and Agrobacterium strains Equivalent gene constructs which include the plant-expressible markers neomycin phos-

photransferase II (NPTII) and /%glucuronidase (GUS) [19] were used in plasmids for particle bombardment experiments or in binary T-DNA vectors [ 15] for Agrobacterium-mediated transformation (Table 1). Marker gene expression was driven by a double CaMV 35S promoter [22], the f~' RNA leader sequence [10] and the nopaline synthase 3' region [3]. The binary vectors were constructed by insertion of a linearized marker gene plasmid between the left and right T-DNA borders of pPHI6 such that the genes were positioned near the right T-DNA border sequence. These vectors were directly transformed into Agrobaeterium strain EHA101 using the freezethaw method [16]. Agrobacterium strain A136, which lacks the trans-acting virulence functions of the Ti helper plasmid, was transformed with pPHI236 and used as a control.

Tissue preparation and transformation Nicotiana tabacum The preculture, callus induction and plant regeneration protocols were as described by Tomes et al. [35]. Briefly, sterile seeds, cv. Xanthi, were germinated and grown in vitro under a 16 h day length at 25 °C on medium 272 (MS salts and vitamins, 4~o sucrose, 1.5 ~o Gelrite, pH 5.8). Ten to 14 days after germination the 4-6 m m diameter primary leaves were harvested. They were cultured intact, adaxial side up, for 24 h on filter

Table i. A brief description of the Agrobacterium strains and plasmids used in this study.

Strain/plasmid

Description

Reference/source

Agrobacterium strains EHA 101 A136

Disarmed, hypervirulent strain Strain which lacks a Ti plasmid

[12]

Plasmids pPHI6 pPHI158 pPHI167 pPHI236 pPHI413 pPHI419

Binary vector derivative of pGA473 [ 1] Linearized pPHI419 ligated between the T-DNA borders of pPHI6 Linearized pPHI413 ligated between the T-DNA borders of pPHI6 pPHI6 derivative carrying the BAR and GUS genes Non-binary vector with a plant-expressible GUS gene Non-binary vector with a plant-expressible NPTII gene

This This This This This This

Abbreviations: NPTII, neomycin phosphotransferase II; GUS,/3-glucuronidase; BAR, bialaphos resistance.

[6] study study study study study study

303 paper moistened with medium 530 (MS salts, B5 vitamins, 4 mg/1 p-chlorophenoxyacetic acid, 3 ~o sucrose, 0.25 M sorbitol, pH 5.8). Following culture on medium 530, but prior to Agrobacterium inoculation, leaves were wounded by bombarding with the particle gun or bisecting the lamina longitudinally. Twenty leaves were used per treatment. Control leaves were not treated further but had a cut petiole surface and incidental wounding. Particle gun wounding consisted of arranging four leaves around the perimeter of a 1 cm circle in 60 mm x 20 mm Petri plates and bombarding 2 times in a Biolistics style particle acceleration device with 1.8/~m average diameter tungsten particles (GE, Cleveland). Particles prepared according to Klein et al. [23] were either resuspended in TE buffer and used in bombardments without further treatment or were associated with plasmids pPHI419 or pPHI413 by CaC12 and spermine precipitation methods [24] (10 #g supercoiled DNA per 4.375 mg particles in 35/~1 final volume). In either case the particle concentrations were equivalent. Leaves bombarded with particles were suspended in TE buffer, leaves cut in half and untreated leaves were dipped for 10 minutes in a suspension of EHA101/pPHI158 or EHA101/pPHI167. Bacteria were grown in YEP medium supplemented with 50 #g/ml kanamycin sulfate and 100 #g/ml carbenicillin to an OD6oo of 0.5-1.0. Bacteria were resuspended in a medium composed of 12.5 mM MES pH 5.7, 1 g/1 NH4C1 , 0.3 g/1 MgSO 4 [13] to an OD60o of 0.75. Inoculated leaves were blotted on sterile filter paper and co-cultured for 72 h on filter paper moistened with medium 530 under the conditions described for seedling growth. Helianthus annuus Seeds of Pioneer sunflower line SMF-3 were dehulled and surface-sterilized. The seeds were imbibed in the dark at 26 °C for 18 h on filter paper moistened with water. The cotyledons and root radical were removed, and the meristem explants cultured on 374BGA medium (MS salts, Shepard vitamins [32], 40rag/1 adenine sulfate, 3~o sucrose, 0.8~o Phytagar pH 5.6 plus 0.5 mg/1 BAP, 0.25 rag/1 IAA and 0.1 mg/1 GA). Twenty-four

hours later the primary leaves were removed to expose the apical meristem, and the explants were placed with the apical dome facing upward in a 2 cm circle in the center of a 60 mm x 20 mm Petri plate containing water agar. The explants were bombarded twice with tungsten particles suspended in TE buffer as described above or with particles associated with pPHI413. Some of the TE/particle-bombarded explants were further treated with Agrobacterium tumefaciens strain EHA101/pPHI167 by placing a droplet of bacteria suspended in inoculation medium, OD6o o 2.00, directly onto the meristem. The meristem explants were co-cultured on 374BGA medium in the light at 26 °C for an additional 72 h.

Culture, selection and plant regeneration Nicotiana tabacum 72 h afterAgrobaterium treatment, all material was transferred to medium 526 (MS salts, B5 vitamins, 0.5 rag/1 BAP, 2.0 mg/1 NAA, 3 ~ sucrose, 0.8~o Phytagar pH5.7) supplemented with 100 #g/ml kanamycin sulfate and 250/~g/ml cefotaxime and incubated under a 16 h day length at 26 °C. As cultured leaves of all treatments expanded and curled, they were cut into smaller pieces to ensure as much explant-medium contact as possible. The number of colonies was counted at 2 weeks following transfer to selective medium. Twenty individual colonies from particle-bombarded leaves co-cultured with EHA101/ pPHI158 were excised for continued callus culture and plant regeneration. The calli were transferred at 4 weeks to plant regeneration medium 528 (MS salts, B5 vitamins, 0.5 mg/1 BAP, 3~o sucrose, 0.8~o Phytagar pH 5.7) supplemented with 100 #g/ml kanamycin sulfate and 250 #g/ml cefotaxime. Shoots were excised and transferred to rooting medium 272 supplemented with 50 #g/ml kanamycin sulfate and 250/zg/ml cefotaxime. Callus from 19 of the 20 isolated colonies gave rise to shoots. Leaf tissue from one regenerate of each line was assayed for NPTII activity. NPTII enzyme assays were done according to the procedures of Henderson et al. [ 14].

304 Approximately 60 seeds from selected transgenic plants and from cv. Xanthi untransformed controls were surface-sterilized, then germinated on MS salts plus 4% sucrose and 100 mg/1 kanamycin sulfate. Plants were scored after 14 days for green or white phenotype. Southern hybridization analysis was performed to detect the presence of NPTII sequences in transgenic plants. DNA from leaf tissue of R0 plants 7, 19 and their respective kanamycinresistant progeny was isolated by the CTAB method of Saghai-Maroofet al. [29]. About 10/~g of cut DNA was used per lane and blotted onto a nylon membrane. The NPTII fragment was radiolabeled by the random hexanucleotide method of Feinberg et al. [7]. Attempts were made to isolate protoplasts from control, bombarded or bombarded plus Agrobacterium-treated tobacco leaves. Treated or control leaves were cut into small strips and digested for 12 h at 25 °C in S medium [33] with Macerozyme R10 (0.2~o, w/v), Cellulase R10 (0.2~o) and Driselase (0.2~o). These conditions were selected by examining yield and quality of protoplasts recovered from control leaves when digested by different combinations of the above enzymes (Cellulase YC, Caylase, Caylase T and Caylase M2 were additionally tested) in S, To [6], or NTTo [9] media.

Helianthus annuus Agrobacterium-treated meristems were transferred following the 72 h co-culture period to medium 374 (374BGA with 1~o sucrose and no BAP, IAA or GA3) and supplemented with 250 #g/ml cefotaxime. The plantlets were allowed to develop for an additional 2 weeks under 16 h day and 26 °C incubation conditions. The plantlets were then harvested and histochemically stained by submerging the tissue in X-gluc [19] to detect GUS activity. Meristems bombarded with pPHI413 were sacrificed at 72 h or 2 weeks after bombardment, and explants or resulting plantlets were similarly stained for GUS expression. The plantlets were decolorized with 95 ~o ethanol prior to photography.

Histological methods Meristem explants 48 h old cultured on medium 374BGA or noncultured embryonic axes of seed were fixed in fluoracetic acid and dehydrated in an ethanol series to xylene. Explants were embedded in paraffin, cut into 10 #m ribbons and attached to slides with Haupt's adhesive [2]. Slides were dewaxed in xylene and rehydrated in an ethanol series. Sass's hemalum was used to stain meristematic regions [31]. After staining, slides were dehydrated in ethanol, cleared in xylene, and mounted under cover glass with Permount.

Results

Tobacco leaves The protocols for callus induction and plant regeneration of tobacco cv. Xanthi leaves produced prolific cultures with high regeneration potential. The leaves of in vitro grown plants were able to sustain 2 particle gun bombardments without an excessive amount of damage in most cases. The combination of particle bombardment and Agrobacterium co-cultivation did not prevent callus formation or plant regeneration. Table 1 lists the Agrobacterium strains containing the NPTII and GUS constructs used in the following experiments. Colonies resistant to kanamycin were obtained from the cut surfaces of bisected leaves and the cut petiole region of excised, nonwounded leaves (Fig. la) following exposure to EHA101/ pPHI158 (Table 2). Control treatments included no Agrobacterium inoculation, transformation with the GUS strain EHA101/pPHI167 or immersion of particle-wounded tissue in a solution of the NPTII plasmid pPHI419. None of the control treatments produced colonies on selective callusing medium containing kanamycin. Transformants were recovered at a low frequency from leaves subjected to the standard particle gun transformation protocol using the construct pPHI419. A large increase in transformation fre-

305

Fig. 1. EHA101/pPHI158-treated Xanthi leaves after 2 weeks of culture on medium 526+100#g/ml kanamycin + 250/~g/ml cefotaxime. A. Nonbombarded, prior to Agrobacterium treatment. B. Bombarded twice prior to Agrobacterium treatment.

quency was observed, however, when EHA101/ pPHI158 was utilized as the method of transformation in bombarded leaves (Table 2). Numbers of putative transformants were observed after 3

weeks of culture in the presence of 100/~g/ml kanamycin over the entire area of leaves treated in this manner. A resulting callus mass originated from the growth and merger of a large number of small colonies that arose in the leaf(Fig, lb). Estimation of transformation frequencies was done by counting leaf colonies after two weeks on selective medium or by measuring NPTII activity in Agrobacterium co-cultivated leaves 9 to 12 days after treatment (Table 3). An additional attempt to estimate transformation frequency by culturing protoplasts from bombarded or bombarded and Agrobacterium-treated leaves failed because these treatments prohibited the recovery of viable protoplasts. Attempts were made 2 weeks after EHA101/ pPHI158 treatment to recover individual colonies. Twenty colonies were isolated and plants were regenerated from 19 of the resulting callus lines. One healthy shoot from each callus was excised, rooted in the presence of kanamycin, and assayed for NPTII activity. All of the R 0 plants tested positive for NPTII (Table 4). The R0 plant expressing the highest (No. 7) and the lowest (No. 19) level of enzyme activity was carried forward for recovery of progeny. Kanamycinresistant progeny were obtained from each of these parents. Chi-square analysis was used to test the ratio of resistant to sensitive progeny from both plants. The R 1 closely fit a 3:1 ratio for green:white phenotype, as expected for a singlelocus insertion (X2 = 1.3 and p = 0.25 for plant 7; Z2= 1.1 and p =0.30 for plant 19). Southern hybridization analysis showed that one (No. 19) or two (No. 7) fragments homologous to the NPTII gene were present in the Ro transformants and in five progeny from each. No homology was seen in an untransformed control sample (Fig. 2a). Use of the NPTII gene as a radiolabeled probe and a unique Eco RV site between the T-DNA borders allowed the detection of right-border plant D N A junctions following digestion with Eco RV. The hybridizing bands of tobacco genomic D N A were not equal in size to those of Eco RV-digested pPHI158, thus verifying stable T-DNA integration at the molecular level.

306 Table 2. Colonies arising from cv. Xanthi leaves 2 weeks after treatment on medium 526 + 100 #g/ml kanamycin and 250 #g/ml cefotaxime. Explants were derived from 20 starting leaves. Leaf treatment

Number of explants

Explants w/colonies

Response ~

Colonies explant 1

Colonies/ leaf

Bombarded particles in TE Bisected 2 x bombarded particles in TE + EHA101/pPHI167 2 x bombarded particles in TE + dipped in 0.1/tg/ml PPHI419 Nonwounded lamina + EHA101/pPHI158 2 x bombared with particles associated with pPHI419 Bisected + EHA101/pPHI158 2 x bombarded particles in TE + EHA101/pPHI158

31 40 34

0 0 0

0 0 0

0 0 0

0 0 0

55

0

0

0

0

CMfi ~ cm petiole surface

64 48

4

8.3

0.2±0.1

0.4

37 51

22 44

59.5 86.3

2.5±0.5 14.4±1.9

4.0 36.6

1 Mean number of colonies + standard error.

Table 3. Xanthi leaves were treated, cocultivated with EHA101/pPHI158 for three days, then cultured on selective 526 medium followed by measurement of NPTII activity. Exp. No.

1

2

3

Cocultivation medium

Treatment

Days

NPTII 1 Range

Mean 2

526

Bombarded Bisected Control

9

0.06-1.74 0.09-0.88 0.00-1.05

0.73 + 0.17 0.51 + 0.09 0.36 + 0.19

526

Bombarded Bisected Control

11

0.31-4.27 0.21-1.60 0.12-1.10

1.90 + 0.37 0.61 + 0.18 0.69 + 0.29

530

Bombarded Bisected Control

9

0.20-7.40 0.00-1.60 0.00-0.10

2.59 + 0.83 0.79 + 0.20 0.01 __+0.01

530

Bombarded Bisected Control

12

0.12-12.12 0.19-2.62 0.00-0.37

1.81 + 0.82 0.73 + 0.10 0.18 + 0.04

530

Bombarded Bisected Control

9

0.10-1.43 0.00-0.47 0.00-0.00

0.75 + 0.12 0.19 + 0.05

1 Assay data are presented as nanograms NPTII per microgram totaJ protein. 2 Mean NPTII assay data_+ standard error.

Sunflower meristems The apical meristem of a pretreated, germinating sunflower seed was easily exposed by removal of

the cotyledons and primary leaves. Meristems cultured in the presence of BAP, IAA and G A 3 increased in diameter from 50 ktm to over 200/~m in 2 days, and the structure was such that parti-

307

Table4. NPTII assay results of R0 and R1 Xanthi plants transformedby particle bombardment followedby treatment with EHA101/pPHI158. These data were generatedby samplingone leaf from each plant. Ro

R1

Plant No

NetCPM

NPTII1

7 19

28100 865

160 6

1 2 3 4 5 6 8 9 10 11 12 13 14 15 16 17 18

1190 21140 21580 9800 12700 9800 8450 17400 13300 21000 2600 2580 12700 7900 2680 12600 16500

9 129 120 65 93 82 53 135 92 130 18 20 89 66 16 87 99

115

2

Con~ol

Plant No

Net CPM

NPTII1

7-1 7-2 7-3 7--4 7-5

37200 21400 26300 18450 23500

84 64 63 46 57

19-1 19-2 19-3 19--4 19--4

3500 4500 3400 3000 3700

9 9 9 7 10

1 Assay data are presented as nanograms NPTII per microgramtotal protein.

cles had an unobstructed path into the cells of the meristematic dome (Fig. 3a and 3b). Single plants could be rapidly recovered from the apical domes of control, bombarded, and/or Agrobacteriumtreated meristem explants. Two small axillary shoots would frequently develop at the base and on either side of the large apical shoot. The axillary shoots would develop more rapidly in those explants where the apical meristem was severely damaged by bombardment. Transient GUS expression was high in histochemically stained apical meristems 3 days after bombardment with plasmid pPHI413 (Fig. 3c). As many as 88~o of the explants exhibited GUS activity in the apical dome. Virtually all GUS expression was absent, however, in plantlets developed from bombarded meristems 2 weeks after treatment. Of 2000 apical explants bombarded with pPHI413, only 1 resulting plant had a level of GUS expression indicative of stable transformation (Fig. 3d).

Meristem explants treated with Agrobacterium strain EHA101/pPHI167, the pPHI413 T-DNA analogue, without prior bombardment showed no sectors of GUS expression in the resulting plants 2 weeks after treatment. Meristems bombarded with particles suspended in TE buffer prior to application of EHA101/pPHI167, however, resulted in an average of 14.9~o of the plants exhibiting substantial GUS-stained sectors (Table 5). The sectors appeared in leaves, stems and/ or developing axillary shoots (Fig. 4). Most sectors were confined to lower, single leaves (Fig. 4a), while later developmental events and apparent developmental lineages appeared in others (Fig. 4b--d). Figure 4b is an example of a stem that extended into the leaf, while the plant in Fig. 4c shows a series of 3 leaves on the same side of the plant that had a similar GUS-sectoring pattern. The first 5 leaves of the plant in Fig. 5d were GUS-negative while the 2 youngest leaves

308 merits in which plants from bombarded meristem explants treated with EHA 101/pPHI 158 (pPHI419 T-DNA analogue), were allowed to develop in the presence of 200 mg/1 kanamycin. Green leaves emerging from bleached shoots or green/white sectored leaves (Fig. 4F) were observed in 12.5~o of the explants treated.

Discussion

Fig. 2. Southern hybridization analysis of tobacco transformants. A. Plant genomic DNA and pPHI158 were digested with Eco RV, blotted onto a nylon membrane and probed with the 0.8 kb NPTII fragment shown in Fig. 3b. Lane 1, which is labeled U, is the untransformed plant D N A control. The R 0 transformant, No. 7, and five progeny from this plant follow in lanes 2-7. Lane 8 contains DNA from R 0 plant 19 and lanes 9-13, the progeny of this plant. Eco RV-digested DP158, which was used as a positive control, is shown in the lane designated C. B. Linear map ofpPHI158 showing the unique Eco RV site used to cut between the T-DNA border sequences. The NPTII gene was used as a probe in hybridization analysis. RB and LB are the right and left T-DNA borders. The position of 0.8 kb NPTII gene is shown by the filled box.

to emerge were GUS-positive. Transformation was not restricted to the apical shoot alone, but instances of axillary shoot GUS expression in the absence of apical activity were also observed (Fig. 4e). Examples of the restriction of transformation to epidermal cell layers were also seen, but most sectors of GUS expression included all of the cell layers of the leaf. Plants recovered from meristems bombarded and treated with an Agrobacterium strain (A136) incapable of T-DNA transfer but transformed with a binary vector carrying the GUS gene construct (pPHI236) did not exhibit GUS-stained sectors. A similar frequency of numbers and patterns of sector development were obtained from experi-

Microprojectile bombardment as a means of inducing a wound response in plant tissues is an effective method for improving transformation frequencies by Agrobacterium tumefaciens. The wounds created by the impact of particles on a tobacco leaf produce sites conducive to Agrobacterium infection over much of the surface area. Leaves treated with EHA101/pPHI158 following particle bombardment and cultured under kanamycin selection developed many small colonies that rapidly grew together, forming a large, illdefined callus mass capable of regenerating large numbers of shoots. Attempts were made to estimate the number of transformation events that occurred after various treatments. The data in Table 2 are counts of colonies arising from leaves after 2 weeks of culture on kanamycin-containing medium. Counting colonies probably resulted in an underestimate of the efficiency of the system. Another method attempted was the culture of protoplasts isolated from bombarded or bombarded/Agrobacterium-treated leaves, and quantification of kanamycin-resistant calli. We were unable to isolate protoplasts, however, from this material, even though digestion conditions were used which produced high-quality isolations from untreated leaves. The leaf wounding apparently reduced both the ability of the material to be digested and the viability of the few protoplasts that were recovered. Estimation of relative transformation efficiencies by assaying Agrobacterium-cocultivated leaves for NPTII activity showed that leaves wounded by particle bombardment had higher levels of activity than bisected or unwounded material (Table 3). This result was consistent with

309

Fig. 3. Sunflower meristem explants. A and B are 12 #m longitudinal thin sections. A. Embryonic axis from sunflower dry seed. B. 48 h sunflower shoot apical meristem explant precultured on medium 374BGA. Bar represents 50 #m. C. Sunflower apical dome stained for GUS activity by X-gluc 72 h after bombardment with the GUS construct pPHI413. D. GUS activity in a sunflower plantlet recovered from a meristem explant 2 weeks after bombardment with pPHI413.

the colony development data and suggests that the higher levels of NPTII activity in bombarded leaves correlated to an increased number of transformation events. Regardless, wounding of tobacco leaves by microprojectile bombardment conferred an advantage over wounding by simple bisection of the leaf. Additional wounding of bisected leaves by further sectioning prior to Agrobacterium treatment would generate more cut surfaces. The additional wounded areas would most likely result in the appearance of more transformed colonies on a per leaf basis and the 9-fold transformation advantage Of wounding by particles over manual bisection of the leaf would undoubtedly decline. In our experiments, particle bombardment both increased the percentage of Agrobacterium-

transformed leaf explants (from 60 ~o to 86 ~o) and increased the number of transformed calli per leaf (from 4 to 37) (Table 2). The number of resistant colonies per explant is high as compared to that reported by Burow et al. [4] in a tobacco leaf transformation system. The percentage of explants giving rise to resistant calli exceeds that reported for leaf explants of strawberry [27], tomato [26] and pea [21]. It is difficult to compare the results of different transformation systems because plant species, selectable markers, culture conditions, state of the source tissue, etc., greatly affect the outcome. In spite of these differences, we feel that particle gun wounding could offer comparable improvements in other systems than those we have observed. The data from Table 2 also indicate that transformation in bombarded

310 Table 5. Sectors of G U S expression in meristem-derived sunflower plants 2 weeks after Agrobacterium treatment. Entries under treatment: 2 x bombarded + EHA101/pHI 167 are independent experiments Treatment

Nonbombarded + EHA101/pPHI167 2 x bombarded + A163/pPHI236 2 x bombarded + EHA101/pPHI167

Number of meristem plants

Number of plants with sectors

45

0

0

111

0

0

106 138 80 39 73 70

8 21 14 7 15 8

7 15 17 18 20 11

tobacco leaves by Agrobacterium (EHA101/ pPHI158) was at least 100-fold more efficient in generating kanamycin-resistant colonies than the standard particle/plasmid transformation protocol. Similar trends were observed in plants derived from sunflower meristems. Wounding of the meristem prior to Agrobaeterium application was a prerequisite for the appearance of transformed sectors in the resulting plants (Fig. 4). The standard particle/plasmid protocol of bombardment into meristems produced large, transformed sectors in plant structures at only a 0.05 ~o frequency, i.e. 1 event in 2000 plantlets assayed (Fig. 3b). This is in contrast with the results of McCabe et al. where frequencies of GUS sectors as high as 2~o were observed in plants derived from bombarded soybean embryonic axes [25]. By comparison, large sectors of GUS expression were observed in plants from the particle/ Agrobacterium method at an average frequency of 14.9~o (Table 5). Several interesting patterns of GUS expression, indicative of the complexity of the target

tissue, were observed in plants from bombarded/

Agrobacterium-treated meristem explants (Fig. 4). The GUS expression patterns suggest that the plants appear to be predominantly sectorial or mericlinal chimeras. In our observations of approximately 70 sectored plants, GUS expression was observed as spots on stems or leaves, stem sectors, stem and leaf sectors together or leaf sectors only. These patterns closely resemble those observed by Christou [5] in chimeric soybean transformants. No uniformly transformed sunflower plants were found. Our results are similar to those obtained by Jegla and Sussex [20] who used mutants from irradiated seed to determine the sunflower meristem fate map. The mutant sectors obtained in some of the mature sunflower plants were similar to the GUS sectors we have seen in plantlets derived from bombarded/Agrobacterium-treated meristems. They also found that no mutant sectors were observed throughout the full length of the plant, as was the case with our GUS-sectored plantlets. While our results seem to reflect elements of those from studies designed to determine the fate map of the intact embryonic axis, it must be acknowledged that the plantlets recovered from meristem explants are produced under highly artificial conditions. Hormonal stimulation, bombardment, Agrobacterium treatment, and in vitro development could easily alter normal developmental patterns. The meristem explantderived plants, for example, have 10-15 nodes while those derived from seed have 30-33 nodes. In spite of these complicating factors, it appears that the particle wounding of meristem explants allowed Agrobacterium to access and transform cells that participated in the development of the plants. The work of Hussey et al. has demonstrated, by histological examination, that cells of the apical dome of cultured pea shoots are transformable by Agrobacterium [18]. The assumption in our work is that the transformed cells were

Fig. 4. Sunflower plantlets from meristem explants 2 weeks after particle bombardment and Agrobacterium treatment. A-E. Meristems exposed to EHA101/pPHI167 and stained by X-gluc. F. Meristem exposed to EHA101/pPHI158 and cultured for 2 weeks on 200 #g/ml kanamycin.

311

312 components of the shoot apical meristem as presented in Fig. 4b, however, we have not demonstrated the location of the sites of bacterial attachment or transformation in the explant. The data presented here indicate that the frequency of transformation mediated by Agrobacterium tumefaciens can be greatly enhanced by first generating microwounds in the target tissue through high-velocity microprojectile bombardment. In addition, Agrobacterium is a much more efficient transformation tool in those compatible plant species than the analogous particle gun protocol. We have also found that it is possible to dry Agrobacterium in the presence of gold particle suspensions, bombard tissues directly with particles and associated bacteria, and obtain transformation events (data not shown). Particle/Agrobacterium combinations should be helpful in recovering transgenic plants at a reasonably high frequency in systems where the regenerable cell types are in a region wounded by particle bombardment.

Acknowledgements We would like to thank Kellie Winter for excellent technical assistance with the NPTII assays. In addition, we appreciate the assistance with histological techniques from Marc Albertson and Wendy Nelson.

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