Stable Transformation of Soybean by Electroporation and Root Formation

Home , Electroporation

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 3962-3966, June 1987 Applied Biology Stable transformation of soybean by electroporation and root formation from transformed callus (Glycine max/direct DNA transfer/protoplasts/organogenesis) PAUL CHRISTOU, JEAN E. MURPHY, AND WILLIAM F. SWAIN Agracetus, 8520 University Green, Middleton, WI 53562 Communicated by Oliver E. Nelson, Jr., February 25, 1987 (receivedfor review December 3, 1986)

ABSTRACT Soybean protoplasts from a number of com- to +1550, respectively (11)], and the aminoglycoside 3'- mercially important cultivars have been genetically engineered phosphotransferase II (APHII; kanamycin kinase, EC by way of electroporation using chimeric genes coding for 2.7.1.95) coding region from TnS [nucleotides 1541-2517 resistance to the aminoglycoside antibiotics kanamycin and (12)]. The coding region deletes an AUG triplet present in the G418. Effective electroporation conditions were determined by TnS sequence upstream and out-of-frame with respect to the monitoring transient expression from aminoglycoside 3'-phos- initiator AUG (13). The junctions between the pBR322, photransferase II (APHII) expression plasmids. Electropora- nopaline synthase, and APHII fragments include portions of tion of protoplasts with a chimeric APHII gene and subsequent synthetic polylinkers. A restriction map of pCMC1021 is selection on media supplemented with kanamycin resulted in shown in Fig. 1. Plasmid DNA was prepared by the method the recovery of calli resistant to the antibiotic. Enzyme assays of Ish-Horowicz and Burke (14), was twice-banded by for APHII activity and Southern blot hybridization confirmed isopycnic centrifugation in CsCl/ethidium bromide gradi- the expression ofthe foreign DNA and its stable integration into ents, and was chromatographed on NACS 52 (Bethesda the soybean genome. Root formation was induced from trans- Research Laboratories). formed calli, and these roots maintained expression of the Protoplast Isolation. Four- to eight-millimeter (10-20 day) APHII gene. zygotic embryos were excised from greenhouse grown plants (cultivars Williams 82, Mandarin Ottawa, and Hardin and Soybean (Glycine max) is one of the world's most important also Glycine canescens, a nondomesticated relative of G. agronomic crops. Accordingly, a great deal of effort has been max) and were cut transversely as described by Lu et al. (15). directed towards its genetic improvement by both conven- The chopped embryos were plasmolyzed for 1 hr in a 13% tional breeding techniques and genetic engineering approach- mannitol/salt solution (16) and then were incubated in the es. Successful application of standard genetic engineering enzyme mixture described by Lu et al. (15) for 5 hr at room procedures to soybean has been limited by the lack of an temperature on a gyratory shaker at 20 rpm. Protoplasts were efficient transformation system and the inability to regener- released by squeezing the embryo slices against the flask wall ate transformed tissues. Oncogenic transformation of soy- with a sterile spatula. The digestion mixture was sieved bean by virulent Agrobacterium strains and the axenic through a 54-,um stainless steel screen, and the screen was culture of excised tumors on hormone-free media have been rinsed with 2 ml of a 9% mannitol/salt solution (16). The reported (1-5), but disarmed Ti vectors have not yet been filtrate was transferred to 15-ml conical tubes and was used successfully. Agrobacterium strains have been isolated centrifuged at 135 x g for 8 min. The supernatant was that show increased virulence toward soybean and other discarded, and the protoplasts were washed twice in the same plants that are not particularly susceptible to crown gall solution by resuspension and centrifugation. The pellet was infection, and "disarmed" hosts for Agrobacterium vector collected and suspended for a final wash in a 0.49 M systems have been developed from these strains (6). How- sucrose/salt solution. Aliquots of the protoplast suspension ever, even with these strains, the transformation frequency is were diluted, stained with fluorescein diacetate, and counted very low relative to that obtained in alternative plant hosts on a hemocytometer grid under UV illumination. Protoplasts such as tobacco and petunia. Electroporation has been shown were then diluted with protoplast medium to the desired to be an effective technique for the transformation of mam- density for electroporation. malian cells (7). Fromm et al. (8) have shown that with Electroporation. Soybean protoplasts were electroporated appropriate modification the technique is also applicable to at densities of2-4 x 106 per ml in Kao protoplast medium (17) plant protoplasts, and they have achieved the stable trans- supplemented with 40 mM NaCl. One-milliliter aliquots ofthe formation of maize tissue cultures by this method (9). These protoplast suspension were pipeted into 1.5-ml disposable results, together with our own experience with electropora- cuvettes and were chilled briefly in an ice water bath. An tion of tobacco, prompted us to investigate this method as a electric pulse was delivered to the suspension by way of means of achieving the transformation of soybean. platinum wire electrodes with 1-cm spacings. The electric pulse was supplied by a 490-AF capacitor (Sprague Power- lytic 36D, Marsh Electronics, Milwaukee, WI) charged to the MATERIALS AND METHODS desired voltage with a voltage-regulated power supply. The Plasmids. pCMC1021 was constructed from a fragment of capacitor circuit was similar to that described by Fromm et pBR322 [nucleotides 2522-4361 (10)] containing the replica- al. (8) with the addition of a switchable bleed-resistor. tion origin and the 8-lactamase gene, a chimeric gene con- Voltages were monitored with a voltmeter, and the capacitor sisting of 5' and 3' regulatory regions from the nopaline discharge was followed on an oscilloscope. Under these synthase gene ofpTiT37 [nucleotides -265 to +36 and + 1298 conditions and with the capacitor charged to an initial voltage of 375 V, the discharge was an exponential decay with a The publication costs of this article were defrayed in part by page charge half-time of approximately 45 msec. payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: APHII, aminoglycoside 3'-phosphotransferase II. 3962 Downloaded by guest on October 2, 2021 Applied Biology: Christou et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3963 activity by the method of Reiss et al. (18). The amount of enzyme present in each sample was estimated by comparison to a known amount of purified enzyme included on the same gel. Southern Blots. DNA was prepared from lyophilized tissue by the method of Taylor and Powell (19). DNA was digested with restriction endonucleases under the conditions recommended by the supplier (New England Biolabs), was resolved by electrophoresis on an 0.8% agarose gel, and was transferred to nylon membranes (Biodyne membranes, Pall, Ir- vine, CA) as described by Southern (20). 32P-labeled RNA hybridization probes were synthesized in vitro using an SP6 transcription system (Promega Biotec, Madison, WI) and [a-32P]GTP (300 Ci/mmol; Amersham; 1 Ci = 37 GBq). The template for probe synthesis produced a runoff transcript corresponding to the minus strand ofthe coding sequence for APHII. Reaction conditions recommended by the supplier, using 100 ,uCi of radiolabeled GTP, were used. Incorporation FIG. 1. Restriction map of plasmid pCMC1021. Components of was typically 40-60%. Hybridization and washing conditions the nopaline synthase/APHII chimeric gene are indicated by the were as described by Church and Gilbert (21). Filters were boxed regions. The hatched region represents the APHII coding analyzed by autoradiography using X-Omat AR5 film (Ko- sequence, and the open-boxed regions of the APHII fragment dak) at -80'C with two intensifying screens (Cronex Light- indicate noncoding portions of TnS included in the fragment. Nos ning Plus, DuPont). Promoter and Nos Poly A designate the 5' and 3' control regions of the nopaline synthase gene, respectively, as described in Materials and Methods. The arrow labeled bla indicates the position and RESULTS AND DISCUSSION polarity of the 3-lactamase gene derived from pBR322. The size of the entire plasmid is approximately 3.4 kilobases. A requisite step in recovering a stably transformed soybean tissue from electroporation experiments was to obtain large numbers of viable protoplasts that could be regenerated to Culture of Protoplasts and Selection of Stable Transform- callus. Tissue source was found to be crucial in protoplast ants. Electroporated protoplasts were incubated at 0C for preparation. Root, leaf, stem, and hypocotyl were tested, but approximately 5 min following application of the electric each ofthese tissues was inferior to zygotic embryos in terms pulse and then diluted into 10 ml of Kao's medium (17) in ofboth yield and viability. We found the protoplast yield also Corning 75-cm2 tissue culture flasks. The flasks were incu- to be dependent on embryo size, the optimum size being 4-8 bated in the dark at room temperature without agitation for mm. Larger embryos resulted in fewer protoplasts, presum- 8 days, at which time 5 ml ofKao cell culture medium (17) was ably due to a loss of viability caused by the high starch added dropwise, and the flasks were exposed to low light content of more mature embryos. A modification of the (2000 lux, room temperature). This feeding process was osmotic conditions described by Kao (17) was found to be repeated every 8-10 days; each time the volume was in- suitable for the isolation and culture of protoplasts. Proto- creased by 50%. On alternative feedings, half the contents of plasts isolated from a wild relative of soybean, G. canescens, each flask was subcultured to a new 75-cm2 tissue culture by this method behaved similarly to those isolated from the flask. We observed that cell wall synthesis was initiated cultivars in assays of transient gene expression. This obser- within 12 hr after electroporation, and microcalli could be vation is significant because regeneration ofthis species from observed after 2-3 weeks. Beginning 14 days following protoplasts has been reported (22). electroporation, aliquots of the culture were plated on low Our initial experiments focused on the demonstration and melting point agarose (electrophoresis grade, Bethesda Re- optimization of transient expression of APHII in protoplast search Laboratories) medium containing various levels of cultures using the expression plasmid pCMC1021. Fig. 2 kanamycin as described under Results and Discussion. illustrates the behavior of the soybean system as the voltage Additional aliquots were transferred to a liquid medium is varied. APHII activity increases steadily with increasing containing 25 jig of kanamycin per ml. The remainder of the voltage up to the limiting voltage of the capacitor. However, culture was maintained without selective pressure, and viability ofthe protoplasts is significantly reduced at voltages aliquots were plated as described above at weekly intervals above 350 V (Fig. 3). Electroporations performed at 375 V until day 40, at which time all remaining cells were plated on appear to achieve a reasonable compromise between maxi- low melting point agarose medium containing 25 ,ug of mum cell viability and transient gene expression. At this kanamycin per ml. Kanamycin-resistant calli appeared 2-3 voltage, protoplasts quickly recovered from the shock of the weeks following plating on solid medium. electroporation, and normal cell wall synthesis and cell APHII Assays. To assay transient gene expression, proto- division were observed within 12 hr after application of the plasts were collected by centrifugation (200 x g for 5 min) electric pulse. Therefore, this voltage was used in subsequent 40-50 hr after electroporation. Protoplast pellets were frozen electroporations. As reported by Fromm et al. (8), we found in liquid N2, then were suspended in 50 Al ofextraction buffer the level of transiently expressed enzyme activity to be consisting of 8 mM Tris HCl, 125 mM NaCl, 33 mM EDTA, proportional to the input DNA concentration and also to cell 83 mM NH4Cl, 1.25 mM leupeptin, 2.5 mM phenylmethyl- number (up to 7 x 106 cells per ml). sulfonyl fluoride, 1 mg of soybean trypsin inhibitor per ml, 5 Using the optimal conditions established for transient gene mg of bovine serum albumin per ml, and 15 mM dithiothreitol expression, a series of experiments directed towards the at pH 7.5, and were disrupted by sonication. Callus samples recovery of stable transformants was conducted. In an initial were suspended in 40 ,ul of extraction buffer per 100 mg of experiment, protoplasts (2 x 106 per ml of medium) were tissue, were subjected to three cycles of freezing and thaw- electroporated at 375 V in a mixture containing 10 ,g of ing, and then were homogenized with a glass rod. Cell-free supercoiled pCMC1021 DNA per ml and were cultured in extracts were obtained by centrifugation at approximately liquid medium as described in Materials and Methods. 11,500 x g for 2 min. Extracts were assayed for APHII Fourteen days following electroporation, aliquots of the Downloaded by guest on October 2, 2021 3964 Applied Biology: Christou et al. Proc. Natl. Acad. Sci. USA 84 (1987)

A B

n 0 0 0 0 ._ x m 0 ._ 0

I-

300 350 400 30 350 Voltage (volts) Voltage (volts) FIG. 2. Transient expression from the pCMC1021 gene in FIG. 4. Selection of calli for resistance to kanamycin. Soybean electroporated soybean protoplasts. Protoplasts (4 x 106) were (G. max cultivar Mandarin) protoplasts were electroporated (375 V) electroporated in 1 ml of Kao protoplast medium (17) supplemented and cultured in Kao medium as described in Materials and Methods. with 40 mM NaCl and 10 gg of pCMC1021. The electrical pulse was Fourteen days after electroporation, aliquots of the cultures were varied by charging the capacitor to the indicated voltages. (A) plated on low melting point agarose medium containing 25 Ag of Following electroporation, protoplasts were diluted into 10 ml of Kao kanamycin per ml. After approximately 3 weeks on selective medi- medium (17) in Coming 75-cm2 tissue culture flasks, were incubated um, samples electroporated with pCMC1021 (Right) yield calli 40-50 hr at 250C in the dark, and then were collected for assays of resistant to the antibiotic, whereas control samples electroporated APHII activity (18). Activity is expressed as ng of enzyme per 20 Ag without added DNA (Left) do not. When they are plated on of protein. Each data point is the average of three separate nonselective medium, both samples produce colonies with a plating electroporations. (B) Protoplast viability was measured at 0, 24, and efficiency of about 1%. 48 hr after electroporation. When 47 kanamycin-resistant calli derived from the initial plating were assayed for APHII enzyme activity (18), the culture were plated on low melting point agarose media results shown in Fig. 5 were obtained. Only 4 of the 47 containing either no kanamycin or kanamycin at 25, 50, or 100 apparent transformants exhibited detectable APHII activity. tug/ml. Plating efficiency at this stage was low for all samples. Upon subsequent transfer of these 47 calli to fresh media Approximately 1% ofthe microcalli plated were recovered on containing kanamycin, all of the colonies that did not show plates without kanamycin selection. As shown in Fig. 4, APHII enzyme activity died or were severely inhibited in control samples (protoplasts electroporated without pCMC- their growth. The colonies with measurable APHII activity 1021) did not form colonies when plated on media containing doubled in size within one week of subculture; this permitted kanamycin at any of the concentrations tested. Protoplasts a clear distinction between the two groups. electroporated with the pCMC1021 gene yielded about half as In contrast to results from the early plating, 100 calli many colonies when plated on media containing kanamycin derived from plating 5 weeks after electroporation all showed at 25 Ag/ml as when plated on media without kanamycin. No detectable levels of the enzyme when assayed for APHII colonies were recovered at the higher kanamycin concentra- activity. The increased frequency of APHII activity among tions from this plating. Equal aliquots withdrawn from the kanamycin-resistant calli from later platings suggests that original cultures 5 weeks after electroporation gave much early kanamycin resistance may be due to persistent, low- higher plating efficiencies and a corresponding 10-fold in- level APHII activity derived from nonintegrated copies ofthe crease in the number of kanamycin-resistant calli. In addi- plasmid. At later platings, the microcalli have undergone at tion, large numbers of resistant colonies were recovered at least several additional cell divisions and are, therefore, that time on media containing kanamycin at 25 and 50 ,g/ml. much less likely to maintain nonintegrated plasmid DNA. Large numbers of transformed calli were also obtained when Consequently, calli that display a kanamycin-resistant phe- selection (kanamycin at 25 ;kg/ml) was applied in liquid notype at later platings are more likely to have stably culture 20 days after electroporation. These results indicate integrated, active copies of the selected gene. If this inter- that both plating efficiency and transformation frequency are pretation is correct, it would indicate that the fraction of dependent on the mode and timing of selection. Increased electroporated protoplasts that take up and transiently ex- recovery of transformants occurs when selection pressure is press the input DNA is at least 0.5 in this case. applied later. The presence of pCMC1021 sequences in DNA isolated

I FIG. 3. Effect of pulse voltage on protoplast viability. Protoplasts from electroporations in Fig. 2 were stained with fluorescein diacetate and were visualized by microscopy under UV illumination. (A) Protoplasts prior to application of the electrical pulse; (B) protoplasts electroporated at 375 V; (C) protoplasts electroporated at 500 V. Viable protoplasts exhibit a green fluorescence. (All approximately 120 x.) Photographs were taken 24 hr after electroporation. Downloaded by guest on October 2, 2021 Applied Biology: Christou et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3965

1 2 3 4 5 6 7 Comparison of the intensities of the bands to those of the copy-number standards indicates that each of the transformants contains from one to several copies of the intact fragment per genome. Similar analysis of callus (transform- 4L--.'T"" 2 "tt. ant) 141 DNA digested with HindIII, which cuts 11- pCMC1021 #t;, .;. t -o once, reveals a than the .vl single hybridizing fragment larger unit length of the plasmid. These results are consistent with a single integration of APHII sequences in this callus. In addition to the intact Ava I/HindIII fragment, callus 1 l 'g exhibits many, and callus 5 a few, fragments ofdifferent sizes that hybridize with the APHII probe. Complex restriction patterns from transfected DNA have been extensively char- acterized in mammalian cells (24) and appear to reflect a variety of both homologous and nonhomologous recombina- tion events. Comparably complicated patterns have more recently been observed in tobacco (25-27) and maize (9) and presumably were derived at least in part from similar processes. Altered restriction digest patterns have also been FIG. 5. reported for Robertson's mutator, a transposable element in APHII activity in putative transformants. Extracts pre- maize, but in this case the altered patterns appear to be due pared from calli grown on kanamycin were assayed for APHII activity according to the method of Reiss et al. (18). Lanes 1-3: to DNA modification, which inhibits cleavage by certain wild-type protoplast-derived callus from cultivars Hardin, Williams, restriction enzymes, rather than rearrangement of the se- and Mandarin Ottawa, respectively. Lanes 4 and 5: protoplast- quence (28). Any one process or a combination of similar derived callus from cultivars Mandarin Ottawa and Hardin, respec- processes could be involved in the generation of the restric- tively, electroporated with plasmid pCMC1021. Lanes 6 and 7: tion fragments observed in calli 1 and 5. In any of the cases wild-type soybean callus derived from Mandarin Ottawa zygotic thus far analyzed, we have not observed plasmid unit-length embryos and hypocotyl tissue, respectively. The arrow indicates the fragments in digests obtained with enzymes that cut the position of the kanamycin-dependent autoradiographic signal result- once. ing from APHII activity. plasmid sequence Thus, we have no evidence indicating tandem head-to-tail duplications of the plasmid sequences, which would be expected from homologous recom- from transformed calli was confirmed by Southern analysis as bination among the introduced plasmids or maintenance of shown in Fig. 6. Each of the calli exhibiting APHII enzyme intact plasmids. activity contains a 1-kilobase Ava I/HindIll DNA fragment In attempts to induce somatic embryogenesis, transformed corresponding to the intact APHII coding region (see Fig. 1). calli have been subjected to numerous culture conditions, particularly to a variety of hormone regimes. The formation 1 2 3 4 5 6 7 8 of roots was reliably induced in each cultivar by high auxin concentrations (5-10 mg of naphthalene acetic acid per liter) Origin - or by prolonged incubation on culture plates (starvation) in the presence of kanamycin. When excised from the cultures and assayed for APHII activity, the roots showed high levels of enzyme activity, indicating that organogenesis is compat- 23.1 ible with maintenance of chimeric gene expression. We have extended the application ofelectroporation meth- 9.4 - ods to soybean and have demonstrated the effectiveness of 6.6- these methods for obtaining stable, DNA-mediated transfor- 4.4- mation in this important crop. The advances of tissue culture technology in the area of soybean protoplast isolation, 2.3- culture, and the recovery of callus of clonal origin from these protoplasts (refs. 15-17 and our own results) leave regener- 2.0 ation from protoplasts the only major barrier for the genetic engineering of soybean. Our results with G. canescens, 40 which has been regenerated from protoplasts, suggest an alternative route for transfer of a transformed phenotype to G. max through traditional breeding methods. 0.6- The authors thank Tammy Ford and Barbara Keller for excellent FIG. 6. Southern blot hybridization of APHII coding sequences technical assistance, Scott Downing and Joseph Burkholder for in stably transformed soybean calli (cultivars Williams and Mandarin enzyme assays and photography, David Gelfand for purified APHII, Ottawa). DNA was digested with restriction endonucleases Ava I and and Ken Barton for a critical review of the manuscript. and the were resolved HindIll, resulting fragments by electropho- 1. Faccioti, D., O'Neal, J. K., Lee, S. & Shewmaker, C. K. resis and transferred to a nylon membrane (20). The membrane was with a (1985) Biotechnology 3, 241-246. hybridized 32P-labeled RNA probe corresponding to the minus 2. Owens, L. D. & Cress, D. E. (1985) Plant Physiol. 77, 87-94. strand of the APHII coding region. Hybridizing fragments were detected by autoradiography for 26 hr. Lane size markers 3. Lianzheng, W., Guangchu, Y., Jiaofen, L., Bojun, L., Jian, 1, (HindIII W., Zhenchun, Y., Xiulan, L., Qiquan, S., Xingcun, J. & Zeqi, digest of X DNA; positions and sizes in kilobases of the fragments given at left); lane 2, 60 pg of pCMC1021 plus 10 Ag of soybean callus Z. (1984) Sci. Sin. B 27, 391-397. DNA; lane 3, 6 pg of pCMC1021 plus 10 Ag of soybean callus DNA; 4. Pedersen, H. C., Christiansen, J. & Wyndaele, R. (1983) Plant lane 4, blank; lane 5, 10 ,g of soybean callus DNA; lanes 6-8, 10 jig Cell Rep. 2, 201-204. of DNA from transformants 1, 5, and 141, respectively. Lanes 2 and 5. Wyndaele, R., Van Onckelen, H., Christiansen, J., Rudel- 3 correspond to approximately 10 and 1 copies of pCMC1021 per sheim, P., Hermans, R. & De Greef, J. (1985) Plant Cell soybean genome, respectively, based on a genome size of 6 x 109 Physiol. 26, 1147-1154. base pairs (23). 6. Hood, E. A., Jen, G., Kayes, L., Fraley, R. T. & Chilton, Downloaded by guest on October 2, 2021 3966 Applied Biology: Christou et al. Proc. Nadl. Acad. Sci. USA 84 (1987)

M.-D. (1984) Biotechnology 7, 702-709. 17. Kao, K. N. (1977) Mol. Gen. Genet. 150, 225-230. 7. Potter, H., Weir, L. & Leder, P. (1984) Proc. Natl. Acad. Sci. 18. Reiss, B., Sprengel, R., Will, H. & Schaller, H. (1984) Gene USA 81, 7161-7165. 30, 211-218. 8. Fromm, M., Taylor, L. P. & Walbot, V. (1985) Proc. Natl. 19. Taylor, B. & Powell, A. (1982) Focus 4, 4-6. Acad. Sci. USA 82, 5824-5828. 20. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. 9. Fromm, M. E., Taylor, L. P. & Walbot, V. (1986) Nature 21. Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. (London) 319, 791-793. USA 81, 1991-1995. 10. Sutcliff, J. G. (1978) Cold Spring Harbor Symp. Quant. Biol. 22. Newell, C. A. & Luu, H. T. (1985) Plant Cell Tissue Organ 43, 77-90. Cult. 4, 145-149. 11. Depicker, A., Stachel, S., Dhaese, P., Zambryski, P. & 23. Bennett, M. D. & Smith, J. B. (1976) Philos. Trans. R. Soc. Goodman, H. M. (1982) J. Mol. Appl. Gen. 1, 561-573. London Ser. B 274, 227-274. 12. Beck, E., Ludwig, G., Awerswald, E. A., Reiss, B. & Schal- & ler, H. (1982) Gene 19, 327-336. 24. Perucho, M., Hanahan, D. Wigler, M. (1980) Cell 22, 13. Rogers, S. G., Fraley, R. T., Horsch, R. B., Levine, A. D., 309-317. Flick, J. S., Brand, L. A., Finks, C. L., Moyer, T., O'Con- 25. Krens, F. A., Mans, R. M. W., van Slogteren, T. M. S., nell, K. & Sanders, R. P. (1985) Plant Mol. Biol. Rep. 3, Hoge, J. H. C., Wullems, G. J. & Schilperoort, R. A. (1985) 111-116. Plant Mol. Biol. 5, 223-224. 14. Ish-Horowicz, D. & Burke, J. (1981) Nucleic Acids Res. 9, 26. Paszkowski, J., Schillito, R. D., Saul, M., Mandak, V., Hohn, 2989-2998. T., Hohn, B. & Potrykus, I. (1984) EMBO J. 3, 2717-2722. 15. Lu, D. Y., Cooper-Bland, S., Pental, D., Cocking, E. C. & 27. Riggs, C. D. & Bates, G. W. (1986) Proc. Natl. Acad. Sci. Davey, M. R. (1983) Z. Pflanzenphysiol. 111, 389-394. USA 83, 5602-5606. 16. Frearson, E. M., Power, J. B. & Cocking, E. C. (1973) Dev. 28. Chandler, V. L. & Walbot, V. (1986) Proc. Natl. Acad. Sci. Biol. 33, 130-137. USA 83, 1767-1771. Downloaded by guest on October 2, 2021