Dec 11, 1991 - Jack M. Widholm. Department of Agronomy ..... Barker RF, Idler KB, Thompson DV, Kemp JD (1983) Nucleo- tide sequence of the T-DNA region ...
Plant Physiol. (1992) 99, 81-88
Received for publication August 20, 1991 Accepted December 11, 1991
0032-0889/92/99/0081 /08/$01 .00/0
Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts' Sarwan K. Dhir*2, Seema Dhir, Michael A. Savka, Faith Belanger3, Alan L. Kriz, Stephen K. Farrand, and Jack M. Widholm Department of Agronomy (S.K.D., S.D., A.L.K., J.M. W., F.B.) and Plant Pathology (M.A.S., S.K.F.), Plant and Animal Biotechnology Laboratory, University of Illinois, Urbana, Illinois 61801 ABSTRACT
through direct uptake of DNA into protoplasts that were permeabilized by electroporation (5, 6, 20). Soybean protoplasts have also been transformed at low frequency by using Agrobacterium (2). However, no transgenic plants were regenerated from these transformed soybean protoplasts because regeneration procedures were not available. We have established a reproducible method for plant regeneration from immature cotyledon-derived protoplasts of Glycine max cv Clark 63. Field performance of these plants showed no somaclonal variation (1 1). The regeneration protocol developed for Clark 63 is also applicable to several other commercial genotypes (our unpublished data). We have also reported transformation of soybean protoplasts via electroporation at high frequency and have recovered transgenic plants (10, 12). Soybean seedlings have been transformed using Agrobacterium (4, 17, 30), and apical meristems have been transformed with particle bombardment (22). In several cases, the transforming DNA can be stably passed on to the progeny, but the transformation frequency is low, and chimeric plants are usually produced. Co-transformation using the direct gene transfer system has become an established technique of introducing two plasmids containing selectable and nonselectable marker genes or vectors that have two or more genes on the same plasmid. Plant cells or protoplasts of tobacco (27), Arabidopsis (7), soybean (6), rice (24), and maize (1) have been co-transformed using mixtures of two plasmids. The efficiency of co-transformation in these systems is lower than the transformation efficiency obtained with two chimeric linked genes (24). In this study, we compared the efficiency of co-transformation for selectable and nonselectable, linked or unlinked, genes for the development of an efficient system for the incorporation of multigenes into the cultivated soybean and to study the expression of genes in the transformed plants.
Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 x 106 electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 x 10-4. More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southem blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.
Plant genetic engineering techniques have unprecedented potential for crop improvement. To obtain transgenic plants through a protoplast system, one requires an efficient transformation method and effective selection and cell culture conditions. In the light of recent developments in transformation techniques, the basic advantage of the protoplast system may be that a large amount of uniformly transformed material can be generated and screening can be done at very early stages to produce nonchimeric cell clones that exhibit proper integration and expression of the introduced genes. Stable transformation of soybean cells has been achieved
MATERIALS AND METHODS Protoplast Isolation Protoplasts were isolated from immature cotyledons of soybean (Glycine max [L.] Merr. cv Clark 63) and purified as described earlier (1 1). Protoplast viability was determined by staining with fluorescein diacetate (29).
Funds from the Illinois Agricultural Experiment Station, Upjohn Company (Kalamazoo, MI), and the Illinois Soybean Program Operating Board were used to support the work. 2 Present address: Monsanto Agricultural Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198. 3Present address: Department of Crop Science, Rutgers University, New Brunswick, NJ 68903.
Plasmid Constructs The BamHI-KpnI TR fragment from pSaK5 (pSa4 vector containing KpnI fragment 5 from pTi15955; S.K. Farrand, 81
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unpublished data) was cloned between the unique BamHI and KpnI sites of pUCD200 1, resulting in the T-DNA vector pMAS2. The insert includes TR, Tc, and part of the TL region of pTil5955 (3). The TR-region contains ORFeS 24, 25, and 26 corresponding to transcripts 2', 1', and 0', which are necessary for the biosynthesis of the mannityl opines (13). An NPTII gene fused to the 35S promoter of the CaMV (14) was cloned into the XbaI site located in TR ORF 21 of pMAS2 (3). Plasmid pUCD200 1 also contains an XbaI site located in the pTAR par region (15). During the cloning, the 5.9-kb fragment between the XbaI sites in ORF 21 and the par locus of pMAS2 were deleted. In plasmid screenings, a single recombinant clone, pMAS4, was isolated which contains the TR right border, ORFs 24, 25 and 26, and the NPTII cassette. The 3' 456 bp of ORF 21 corresponding to gene 3' remain in pMAS4 (Fig. 1). However, the 5' structural and regulatory
sequence of this gene have been deleted. This plasmid lacks about 200 bp of the pTAR par locus and the 5.7-kb BamHIXbaI fragment of the T region including the TR left border, Tc, and the TL segment of KpnI fragment 5. The deletion has no detectable effect on the stability of MAS4 in Agrobacterium or Escherichia coli hosts (data not shown).
Electroporation Purified protoplasts were resuspended at 1 x 106/mL in electroporation buffer containing 10 mM Hepes (pH 7.2), 150 mM NaCl, 5 mm CaCl2, and 0.2 M mannitol. Supercoiled plasmid DNA (20 ,g/mL) in TE buffer was then added. No carrier DNA was used in these experiments. Electroporation was performed at 500 V/cm, 1000 uF, using a single pulse as described previously (9) with 1 mL volume. Co-transformation Co-transformation experiments were performed essentially as described above using pCaMVNeo (4.4 kb [13]) and
4Abbreviations: ORF, open reading frame; NPTII, neomycin phosphotransferase; kb, kilobase; bp, base pair.
I
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T-DHA Vector pMAS4 approx.
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24 = conjugase 25 = reductase
Plant Physiol. Vol. 99, 1992
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26
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H~ = 0.25 Mannityl Opines biosynthetic region from pTi15955 nucleotides i8989-a4337
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.4 C PBR322 AP pTAR , ori ori CL
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C
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.0
c
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Figure 1. Restriction
maps
.7S
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of chimeric NPTII and mannityl opine synthesis genes from pMAS4.
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TRANSGENIC SOYBEAN REGENERATION
pMAS2 plasmids. DNA concentrations, at ratios of 1:1, 1:5, or 5:1 of pMAS2 and pCaMVNeo, were mixed in 100 ,L TE buffer and added to 1 x 106 protoplasts in 1 mL electroporation buffer. Protoplast Culture and Selection of Kanamycin-Resistant Colonies
Electroporated protoplasts were resuspended in Kp8 medium (18) at densities from 2 to 4 x 105/mL and were allowed to grow for 2 weeks without selection. On day 15, protoplastderived cells, at the two- to four-cell stage, were washed with K8 medium (18) and resuspended in an equal volume of 1.2% agarose medium (5 mL) containing 50 ,g/mL kanamycin sulfate (Sigma). The agarose cultures were cut into slices and allowed to grow for 6 weeks under the antibiotic selection in K8 and MSB medium as described earlier (10). Some of the electroporated protoplast-derived colonies were maintained in medium as described above without exposure to kanamycin to ensure that these samples were capable of colony formation. The absolute transformation frequency was calculated as the number of resistant colonies produced after 8 weeks divided by the initial number of protoplasts plated after electroporation. The relative transformation frequency is described as the ratio between the number of resistant colonies in selected cultures and the developing colonies in unselected cultures. Plant Regeneration
After 8 to 10 weeks of selection, individual kanamycinresistant callus pieces, 1 to 2 mm in size, were transferred to different media as previously described for callus formation and shoot and root induction (11). The regenerated plants were transferred to pots containing vermiculite and soil mixture and later were kept under greenhouse conditions. NPTII Assay
The NPTII enzymatic activity was qualitatively detected in callus and leaves from individual transformed clones by the dot blot procedure of McDonnell et al. (23) with some modifications. Tissue (100 mg fresh weight) extracts were made in 50 ,uL extraction buffer (23). After the cell extract was incubated with reaction buffer, 20 gL reaction mixture was blotted onto Whatman p81 (cellulose phosphate) paper, and the paper was washed, dried, and exposed to X-ray film for 24 h at
-70°C. Opine Assay For the detection of mannityl opines, 30 to 50 mg fresh weight of kanamycin-resistant callus, leaf, or root tissue from individual plants was homogenized in 50 ,L of 70% ethanol containing 5 ML electrophoresis running buffer (formic acid:acetic acid:water, 3:6:91, v/v, pH 1.9). Tissue extract was spotted onto Whatman No. 3 filter paper, and opines were separated and visualized as described by Savka et aL (26).
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Southem Hybridization DNA was extracted from callus or leaf tissue essentially as described by Dellaporta et al. (8). DNA digested (10 Mg) with XbaI was electrophoresed in a 1.0% (w/v) agarose gel. The DNA was blotted onto a nitrocellulose filter (28) and hybridized with the 32P-labeled 1.7-kb XbaI fragment containing the NPTII gene from pMAS4. The filter was washed twice with a solution of 2x SSPE (25) and 0.5% (w/v) SDS for 15 min at room temperature followed by 15 min at 68C. Subsequently, the filter was washed with 0.2x SSPE and 0.2% SDS for 15 min at 68°C with agitation, dried, and exposed to X-ray film using an intensifying screen for 24 to 72 h at -70C.
RESULTS AND DISCUSSION Selection of Transformants and Calculation of Transformation Efficiency
Protoplasts prepared from immature seeds were electroporated with pMAS4 plasmid carrying kanamycin resistance and opine synthesis genes, and the transformants were selected on kanamycin medium. No resistant colonies were recovered if kanamycin selection (at 50 ug/mL) was applied directly at the time of culture or to 1-week-old protoplast-derived colonies. Only a few protoplasts (
