PlantCell Reports

Plant Cell Reports (1993) 13:63-68

9 Springer-Verlag1993

Transformation of maize (Zea mays L.) protoplasts and regeneration of haploid transgenic plants K. Sukhapinda 1, M . E . Kozueh 2, B. Rubin-Wilson 1, W . M . Ainley 1, and D.J. Merlo 1 1 DowElanco Biotechnology Laboratory, 9410 Zionsville Rd., P.O. Box 68955, Indianapolis, IN 46268-1053, USA z Present address: Dow Chemical Co. Building 1803, Midland, MI 48674, USA Received 20 January 1993/Revised version received 10 August 1993 - Communicated by J.M. Widholm

A b s t r a c t . Transgenic haploid maize (Zea mays L.) plants were obtained from protoplasts isolated from m i c r o s p o r e - d e r i v e d cell s u s p e n s i o n c u l t u r e s . Protoplasts were electroporated in the presence of plasmid D N A containing the g u s A and npt II genes e n c o d i n g B - g l u c u r o n i d a s e ( G U S ) and n e o m y c i n p h o s p h o t r a n s f e r a s e I I ( N P T II), r e s p e c t i v e l y . T r a n s f o r m e d calli were selected and c o n t i n u o u s l y maintained on kanamycin containing medium. Stable transformation was confirmed by enzyme assays and DNA, a n a l y s i s . S t a b l y t r a n s f o r m e d tissue was transferred to regeneration m e d i u m and several plants were obtained. Most plants showed NPT II activity, and some also showed GUS activity. Chromosome examinations performed on representative plants showed that they were haploid. As expected, these plants were infertile.

Key Words: Z e a m a y s L.- m i c r o s p o r e - d e r i v e d cultures - haploid - regeneration- transformation Introduction Successful maize transformation systems have been reported in the literature (Fromm et al., 1990; GordonK a m m et al., 1990; Gould et al., 1991; Waiters et al., 1992; D'Halluin et at., 1992). Since organized tissues are used as the transformation target, there exist the possibility that the selected tissues are chimeric and consequently give rise to chimeric transformed plants. The use of a maize protoplast system for generating infertile transgenic plants has been reported (Rhodes et at., 1988). The infertility of those transgenic plants was likely a result of cumulative changes in the source suspension cultures during extended culture time. The reports of the regeneration of fertile (non-transformed) plants directly from protoplasts (Prioli and Sondahl 1989; Shillito et al.,1989; Morocz et al., 1990, Petersen et at., 1992) raise the possibility of using a protoplast-based transformation system to efficiently produce large numbers of fertile, transgenic plants. Correspondence to: K. Sukhapinda

W e report here the use of haploid cell cultures as the source of protoplasts into which marker genes were introduced via electroporation. Stably transformed callus tissues gave rise to transgenic haploid plants. The procedure is reproducible and efficient. The system may potentially be used in generating fertile transgenic plants.

Materials and Methods Cell

suspension

cultures

The genotype of tile starting material originated from a three-way cross, (H99 x FR16) x Pa91, and developed through a series of intermating between highly anther culturable lines (Petolino et al., 1988). Cell suspension cultures were initiated from immature microspores and maintained as described by Mitchell and Petolino (1991). These microspore-derivedcultures (MSD) varied in viscosity and sizes of cell dusters. They required subculture every 7 to 10 days. These cultures were haploid and capable of regenerating haploid plants. Six to 26 month old cell suspensioncultures were used as proloplast source. Protoplast

isolation

Five to 7 days after subculture, the cells were separated from the medium by eentrifugation(150 x g, 25~ C, 10 min). Sterile enzyme solution containing 4% cellulase Onozuka RS and 1% macerozyme R10 (both from Karlan Chemical Corporation) in KMC salts /g/L: KC1, 8.65; MgCI2 96H20, 16.5; CaCI2 ' 2H20, 12.5 (Itarms and Potrykus, 1978; Shillito et al., 1989)] was added at tile rate of 3 ml of enzyme solution per ml of packed cells. The enzyme/cellsmixture was dispensed into 100 X 20 mm polystyrene Petri ~lates (15 ml/plate). The plates were gently shaken in the dark at 26 C. After 10 to 24 hours, tile enzyme treated suspension was poured through four layers of sterile cheese clotil into a 50 ml tube, and centrifuged as above. The supernatant was discarded and the pellet was resuspended in 20 ml of 12/13 KMC salts solution. Twentyml of floating solution (0.6 M sucrose, 0.5% MES, pH 5.6; Shillito et al., 1989) was gently pipetted under the protoplast suspension. The suspension was centrifuged for 10 min at 600 x g. The protoplasts were ctllected in a 50 ml tube and washed twice with filter sterilized electroporation solution [20 mg/L KH2PO4, 115 mg/L NaH2PO4, 444 mg/L CaCI2, 7.5 g/L NaC1, 36.4 g/L mannitol,pH 7.2 (Frommet al., 1986)]. Protoplast density was adjusted to 4 x 106 protoplasts/ml of electroporation solution.

64 EeoRI

Electroporation The protoplast suspension was heat shocked at 42 ~ C for 5 minutes and then placed on ice. Forty-six Ixg of plasmid DNA (pDAB 199, in 20-40 I.tl sterile 1.0 mM Tris, pH 8.0, 1.0 mM EDTA) was placed in a one ml polystyrene electroporation cuvette containing a volume of the electroporation solution to make a total volume of 0.5 ml. One-half ml of the protoplast suspension was pipetted into the cuvette immediately before a single electrical pulse (1550 BF, 200400 v/cm, 15 msec) was applied from a Promega 450/2500 eleetroporator unit. The reaction module held platinum electrodes with a one cm gap. The cuvette was immediately placed on ice for 10 minutes, then the protoplasts were transferred to a 35 x 10 mm polystyrene Petri plate and placed at room temperature for 10 minutes.

Protoplast culture, regeneration

selection,

and

ORF 28 Poly A npt

pDAB 199 8285 bp

19

NO8

plant

Poly A (2624)

A volume of 250 ul of the protoplast suspension (ca. 5 x 105 protoplasts) was spread on a filter (47 mm nylon filter; Micron Separations, Inc.) which was placed over the feeder cells {300 mg of Black Mexican Sweet corn (BMS) cells spread over M1 solid medium (Table 1) in a 60 x 15 mm polystyrene Petri plate}. Unless otherwise indicated, in this and subsequent steps, the plates were incubated at 26 ~ C in the dark. After one week, the filter was transferred to a Petri plate containing M2 medium (Table 1) with 100 mg/L of kanamycin sulfate (Sigma Chemical Co.) and BMS feeder cells. Seven days later, the filter was transferred to M3 medium containing kanamycin sulfate, but without the feeder cells. Kanamycin-resistant colonies could be picked off the filter in 6 to 8 weeks. The callus tissue was transferred every 2 weeks to fresh M3 medium (Table 1) until embryo induction occurred (3 to 4 weeks). Selection pressure was maintained throughout the embryo induction process. Mature embryos were transferred to M4 medium (Table 1), and placed at 26 ~ C in the light (16 hr daylength, 2500 lux). After about two weeks, green shoots were transferred to MS (Murashige and Skoog, 1962)-hormone-free medium for root induction. Plandets were maintained on MS-hormone-free medium until they were about 10 cm tall, at which time they were moved to soil.

nut~ats M i c ~ nuateats vaIIIIMI~ ~ A Fe.SO4-TH20 Stur Mna~itol L-Praline Myo-in~itol Case.anbydzo~mr

Htmmmc: 2,4-D

MI

M2

M3

M4

MS a ld S BMSd

MS MS BMSd

MS MS BMsd

10g

-

N6b B5c N6b 41.3 nag 27.8 nag

20g

20g

30g

54.6 g

18,2 g

2.88 g 99 lag 100 mg

2 mg

2 nag

0.1 mg

DieamI~

pn a b c d

BAP Kinetin

2.5 m g

5:8

s~

s~8

-

Sail

Figure 1. Partial restriction enzyme map of transforming plasmid pDAB 199. Numbers in parentheses indicate base positions of the enzyme recognition sites. Derivations of the various segments are as follows: The 35S/gus A/Nos gene was derived from pBI221 (CLONTECH, Palo Alto, CA) by modification of the 5' untranslated leader to conform more closely to a eukaryotic translation start consensus sequence. This version of the 35S promoter is that described by Baulcombe et al. (1986). The npt II coding region was similarly modified, and placed under the transcriptional control of the -343 deletion mutant of the 35S promoter of Odell et al. (1985). ORF 26 refers to transcript 26 of the pTi-15955 sequence of Barker et al. (1983).

Enzyme assays NPT II activity was detected using the procedure of Reiss et al. (1984). GUS activity was detected using both histoehemical and fluorometrie assays as described by Jefferson (1987).

Genomic DNA was isolated from callus by the procedure of Rogers and Bendich (1988), except that the calli were frozen in liquid nitrogen and ground with a mortar and pestle. Restriction enzyme digested DNA fragments were separated by gel electrophoresis, blotted to nylon filters (Gene Screen Plus, Du Pont NEN R Research Products), and Southern blot analysis was performed essentially as described by Meijer et al. (1991). 32p-labeled probe was prepared from the 2.6 kbp Eco RI fragment of pDAB 199 (Figure 1) using a random primer kit (Prime-ItTM,Stratagene).

-

3.5 rag. or 2.15 nag

5.s

Murashige and Slmog, 1962 Chu,1978 Oamborg et al.. 1968 BMS: 130 mg aspamgine, 1.3 nag niacin, 0.25 mg thiamine. 0.25 mg pyndoxine. 0.25 mg calcittm pentolhenate,200 mg inositol

Plasmid

A

Southern blot analysis

(per liter)

-

II

35S Promoter

35S Prom oter gus

TRJbI* I. Idttdim~ r

(1)

vector

Plasmid pDAB 199 (Figure 1) was assembled using standard recombinant DNA procedures, essentially as described in Maniatis et al. (1982). Two variants of the Cauliflower Mosaic Virus (CaMV) Cabb S 35S promoter were used to drive expression of the 9 Escherichia coli 13-glueuronidase (gus A, Jefferson, 1987) and neomycin phosphotransferase II (npt II, Beck et al., 1982) genes.

Results

Protoplast isolation and electroporation The protoplast isolation procedure described above was applied to several independently derived MSD cell lines. Protoplast yields ranged from 2 to 10 x 106 protoplasts per ml packed cell volume. The size of file cytoplasmically dense protoplasts was between 10 to 20 ~m in diameter (Figure 2a). Approximately 60% of the protoplasts survived electroporation (as judged by trypan blue staining; Technical Information, Sigma 1992 Cell Culture Catalog), and up to 0.1% of the protoplasts showed transient GUS activity 48 hr after electroporation.

Stably transformed callus tissue After 10 days to two weeks on the selection medium, several small, kanamycin resistant colonies were visible on the filter. These colonies were separated and maintained on the selection medium for six to eight weeks (Figures 2b and 2c). Selection frequency appeared to be culture dependent (Table 2).

65

Figure 2: Regeneration of haploid transgenic plants, a: protoplasts isolated from a microspore-derived (MSD) cell suspension culture (bar = 10 pm); b: protoplast culture and selection on medium containing 100 mg/L of kanamycin sulfate in 60 x 15 mm Petri plates; c: a kanamycin-resistant callus isolate grown in a 60 x 15 mm Petri plate; d: shoot and root formation in a 20 x 100 mm Petri plate; e: transgenic haploid plants in 12 inch- pots T . l a . ~ biumlb~r of Iomm~y~a nesimmt iwlmm m~ov=ed born p m m p ~ m bolmed I m m feet m i c e 0 ~ m - ~ e d cea trees CeU Lmr

PImmid I~IA

Voltage Stating (1550 ~tP,15 imec)

N o . o f ~ Pro~plag~s

No. of Resimat l~pm~

#5 a # 5a # 5a # 5a

lloDNA I~DABI99 pDABI99 pDABI99

~0V 200 V 300 V 400 V

0.5 x 2.5 9 0.5 x 1.0 x

106 106 106 106

0 121 23 18

# # # #

uo DNA pDABI99 pDAB199 pDAIU99

300 V 200 V 300 V 400 V

1.0 9 1.0 x 2,0 9 1.0 x

106 106 106 106

0 0 0 0

#2 c #2 c #2 c # 2c

~oDNA pDABI99 I~ABI99 pDABI99

200V 200V 300V 400 V

1.0 x ll)6 1.0 x 106 0.5 x 106 0.5 9106

0 22 8 I0

#4N d #~N d

moDNA pDABI99

300V ~0 V

0.5 x 106 0.5 x 106

12 70

13b 13b 13b 13b

a 18 month old r fast ga'vwing, fum ~ b 18 monthold culture, viscous, slow~ growing, medium sized aggmga~s c 18 mouth old cullarc, viscous, fast growing, fiac aggl~gates d 8 month d d r162 viscous, filSt growing, f ~ aggregates

W h e n N P T II e n z y m e assay was performed on tissue samples o f the k a n a m y c i n resistant isolates (Figure 3, top panel), the majority (60 to 100%) o f these isolates showed N P T II activity (Table 3). Some isolates that did not express the N P T I I activity were considered "escapes". G U S h i s t o c h e m i c a l a s s a y was a l s o performed (Figure 4). A somewhat lower percentage (18 to 63%) o f the kanamycin resistant isolates showed GUS activity (Table 3). Between 38 and 60 % o f the N t r r II positive isolates were GUS positive (Table 3).

Figure 3: NPT II assay. Top panel: extracts from selected calli growing on selection medium; isolates #9, #11, #67, #74, #86, #87, #96 were fast growing, whereas isolates #10, #47, #68 were slow growing, suggesting that they were not transformed. The negative control was a non-transformed MSD callus and the positive control was a transformed BMS callus. Bottom panel: extracts from plants regenerated from different independently transformed callus tissues (the first number on each lane represents the number of the callus isolate, the second number represents the number of an individual pianO. Isolate #67 gave rise to an NPT II positive plant (67-8) and an NPT II-negative plant (67-7). Table 3. Nm~m" of I ~ a m y c t h lesistant isola~ that showed NPT n activity rod/or b U S activip/ Exp.#

lqO. of k,~m~t~ycill r~i~lant isolates

No. of NFT If+/ No. ~ t e d

No, of GUS+I No. tested

Nr of GUS§ lind NPT ll+! No. N v r I1+ (,:o-extm~am)

1 2 3

162 110 18

21/35 (60%) 52/56 (93%) 10/10 (109%)

17/93 (18%) 45/71 (63%) fi/12 ( ~ )

8/21 (38%) 32/52 (62%) 6/10 (60%)

Stably transformed plants Plants were regenerated from several stably transformed callus isolates (Figure 2d and 2e, Table 4). W h e n they were approximately 10 cm tall, samples were taken from a leaf of individual plants, and appropriate enzyme assays were performed. The results in Figure 3 (bottom panel) showed that most regenerable callus isolates expressing NPT II activity gave rise to N t ~ II positive plants. In a few cases, however, the N P T I I - p o s i t i v e callus pieces yielded both N P T II-positive plants as well as NPT IInegative plants. G U S h i s t o c h e m i c a l staining showed many different patterns of G U S expression in the leaves of plants derived from different clonal origins (Figure 4d, 4e). The extent o f the blue staining correlated well with the specific activities o f GUS as measured by fluorometric assays (data not shown).

66

Figure 4: GUS histochemical assay, a: a protoplast 2 days after electroporation expressing GUS (bar = 5 ~m); b: a 10 day old transformed cell clump (bar = 20 lain); c: a transformed somatic embryo showing variegated GUS staining, the arrows show dark blue spots that are commonly observed (bar = 500 lam); d: different patterns of GUS expression in the leaf sections of different transgenic plants (the leaf section in the lower right hand corner is from a non-transformed control plant, bar = 1 cm); e: a close up view of the dark blue spots on a leaf section, and the staining of the leaf hair which appears as light blue background (bar = 1 mm)

67 Talbb 4, Namber e~t"!~laatsregenemed f~m waadot.medcalles i m b . ~ tbOt~l~ GUS m~or ~

w~ty Exp.#

1

2 3

NO.d plainsregene~ed (No. t~epeedem tr,ela~) 93 (12)

79(13) 2~2)

No. of NI~ I] pmttive/ No. tested 38/49

~65 ~

H

No. of GUS IX~ith~ No. e~ed 20/'39

n3tss ~2

All transgenic plants derived from the same clonal origin were phenotypically alike, but those that were derived from different clonal origins showed some morphological differences. Nevertheless, all plants appeared abnormal when compared to seed-derived diploid plants. Transgenic plants showed characteristics of haploid plants similar to those described by Sun et al. (1989), i.e., shortened intemodes, fewer nodes, and smaller leaves. Although some plants developed both ears and tassels at the appropriate positions, many plants developed tassel ears. Cytological examinations of a few plants confirmed that they were haploid. Spontaneous chromosome doubling was not observed.

Southern blot analyses Southern blot analyses of genomic DNAs were performed to verify the presence of the npt II gene in the DNAs of the transformed callus isolates and those of the plants derived from the transformed callus tissues. Between one to three copies of the genes were observed in the transformed callus tissues or transgenic plants (Figure 5a, 5b). No hybridizing bands were seen in DNAs obtained from non-transformed control callus tissues (data not shown), or from non-transformed plant (Figure 5b).

Figure 5a: Southern blot analysis: DNA from 7 transformed callus isolates (lane 1-7). The genomic DNA was digested with the restriction enzyme EcoRl and the DNA fragments were separated on a 0.7% agarose gel, blotted onto Gene Screen Plus, and hybridized to a DNA probe containing the coding sequence of the npt II gene (Figure 1). The presence of a 2.6 kb DNA fragment (arrow) indicates that the EcoRI fragment from pDAB 199 is intact in the plant genome. The presence of fragments larger than 2.6 kb indicates that one of the EcoRI sites flanking the npt II gene in pDAB 199 is no longer present. The sizes (kb) and the mobilities of molecular weight markers are shown on the right of the figure.

Figure 5b: Southern blot analysis: DNA from 9 plants regenerated from different transformed callus isolates (lane 3-11) and a nontransformed control (lane 2). The genomie DNAs from plants were analyzed the same way as those from callus tissues (Figure 5a). A 10 copy-reconstruction of pDAB 199 was presented in lane 1. The presence of DNA fragments that hybridized to the npt II probe shows that the regenerated plants contain the introduced pDAB 199 DNA (arrow indicates 2.6 kb fragment representing the intact npt II gene).

Discussion

We have described a protocol that can be used to obtain protoplasts from several haploid cell lines that differ considerably in viscosity, cell density, doubling time, and pigmentation. Predictably, some variability of protoplast yields between lines was observed, but all lines tested (more than 70 lines) yielded adequate numbers of protoplasts. Electroporation is an effective means of introducing plasmid DNA into MSD protoplasts. Subsequent selection on kanamycin-containing medium results in the isolation of many callus lines that express one or both of the marker genes. Kanamycin selection of transformed tissues is relatively efficient, although some "escapes" are seen. All isolates that demonstrate in vitro NPT II enzyme activity also have intact copies of the npt II gene integrated into their genomes. Individual transformed isolates exhibit different levels of expression of the npt II or gus A genes, and no correlation is seen between the levels of expression of these two genes in a single callus. Although the gene constructs on the transforming plasmid pDAB 199 are not optimized for maize expression, (e.g. no 5' leader intron, Callis et al., 1987), expression levels of the genes under the control of the CaMV 35S promoter are sufficient to allow selection and screening of the transgenic lines. DNA blot analysis demonstrates that the transformed tissues examined contain only single, or a very few, copies o f the transgenes.

68 The plants that were regenerated from transformed callus tissue, in most cases, expressed NPT II or GUS in a pattern that mimicked that of the parent culture. However, a single piece of callus could yield some regenerants that express the transgene(s) and some that do not. To date, we have not determined whether these expression differences are due to either the loss of the introduced gene during cell division/regeneration, the loss of the expression due to DNA modifications such as methylation, or calli that are a mixture of transformed and non-transformed tissue. Plants regenerated from transgenic haploid callus were, as expected, infertile as were non-transformed controls. However, fertile non-transformed plants in which spontaneous chromosome doubling has occurred were obtained at a low frequency (J.C, Mitchell, personal communication). The maize plants from which the MSD suspension cultures were established were specifically bred and selected for the ability to regenerate plants from anther-derived tissue culture (Mitchell and Petolino, 1991). Approximately 5% of the plants regenerated from anther-derived suspensions are fertile, indicating a very low spontaneous doubling rate. Treatment of anther-derived haploid cultures with agents such as pronamide or colchicine, which inhibit spindle fiber formation, significantly increases the recovery of doubled haploid, fertile plants (J. Petolino, personal communication, Wan et al., 1989; 1991). Whether or not these treatments will increase the frequency of regeneration of fertile plants from the nontransformed or transformed MSD cultures has yet to be determined.

Acknowledgments We thank J. Mitchell, J. Petolino for supplying cell cultures and suggestions, T. Wright for doing a part of the DNA analysis, W. Kulka for preparing the plasmid DNA, and K. Crawford, S. McDowell, J. Chichester for laboratory and greenhouse assistance.

References Baulcombe DC, Saunders GR, Bevan MW, Mayo MA, Harrison BD (1986), Nature (London) 321:446-449 Barker RF, Idler KB, Thompson DV, Kemp JD (1983), Plant Molec Biol 2:335-350 Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982), Gene 19:327-336 Callis J, Fromm M, Walbot V (1987), Genes Dev. 1:1183-1200 Chu CC (1978), In Proceedings of Symposium on Plant Tissue Culture, Science Press, Peking :43-50. D'Halluin KD, Boune E, Bossut M, De Beuckeleer M, Leemans J (1992), The Plant Cell 4:1495-1505 Fromm ME, Taylor LP, Walbot V (1986), Nature (London) 319:791-793 Fromm ME, Morrish F, Armstrong C Williams R, Thomas J, Klein TM (1990), Bio/technol 8:833-839 Gamborg OL, Miller RA, and Ojimao K (1968), Exp Cell Res 50: 151-158 Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O'Brian JV, Chambers SA, Adams WR, WiUets NG, Rice TB, Mackey CJ, Krueger RW, Kansch AP, Lemaux PG (1990), Plant Cell 2:603-618 Gould J, Devey M, Hasegawa O, Ulian EC, Peterson G, Smith RH (1991), Plant Physio195:426-434 Harms CT and Potrykus I (1978), Theor Appl Genet 53:57-63 Jefferson RA (1987), Plant Molec Biol Rep 5:387-405 Maniatis T, Fritseh EF, Sambrook J (1982), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York.

Meijer EGM, Schilperoort RA, Rueb S, van Os-Ruygrok PE, Hensgens LAM (1991), Plant Mol Biol 16:807-820 Mitchell J and Petolino J (1991), J. Plant Physiol 137:530-536 Morocz S, Doun G, Nemeth J, Dudits D (1990), Theor Appl Genet 80:721-726 Murastfige T, Skoog F (1962), Physiol Plant 15:473-497 Odell JT, Nagy F, Chua N-H (1985), Nature (London) 313:810-812 Petersen WL, Sulc S, Arn~trong CL (1992), Plant Cell Rep 10: 591594 Petolino JF, Jones AM, Thompson SA (1988) Theor. Appl. Genet. 76:157-159 Prioli LM, Sondahl MR (1989), Bio/Technol 7:589-594 Reiss B, Sprengel R, Will H, Schaller H (1984), Gene 30:211-218 Rhodes CA, Pierce DA, Mettler IJ, Mascarenhas D, Detmer JJ (1988), Science 240:204-207 Rogers SO, Bendich AJ (1988), in: Gelvin SB, Schilperoort RA (eds) Plant Molecular Biology Manual. Kluwer Academic Publishers, Dordrecht, Belgium, A6: pp 1-10 Shillito RD, Carswell GK, Johnson CM, Dimaio JJ, Harms CT (1989), Bio/Technol 7:581-587 Sun CS, Prioli LM, Sondahl MR (1989), Plant Cell Rep 8:313-316 Waiters DA, Vetsch CS, Potts DE, and Lundquist RC (1992), Plant Mol Biol 18:189-200 Wan Y, Petolino JF, Widholm JM (1989), Theor Appl Genet 77:889892 Wan Y, Duncan DR, Rayburn AL, Petolino JF, Widholm JM (1991), Theor Appl Genet 81:205-211