Plant Cell Reports
Plant Cell Reports (1987) 6:321-325
© Springer-Verlag 1987
Transformation of Brassica napus with A grobacterium tumefaciens based vectors J o y c e Fry, A r l e n e B a r n a s o n , and R o b e r t B. H o r s c h Biological Sciences, Monsanto Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198, USA Received May 28, 1987 / Revised version received July 6, 1987 - Communicated by L. K. Grill
ABSTRACT A reproducible system to produce transgenic Brassica napus plants has been developed using stem segments. Stem segments from 6-7 week old plants were inoculated with an Agrobacterium t~mefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimeric bacterial gene encoding kanamycin resistance (pMON200). Stem explants were cocultnred for 2 days before transfer to kanamycin selection medium. Shoots regenerated directly from the explant in 3-6 weeks and were excised, dipped in Rootone®, and rooted in soil. Transformation was confirmed by opine production, kanamycin resistance, and DNA blot hybridization in the primary transformants. Final proof of transformation was demonstrated by the co-transfer of opine production and kanamycin resistance to progeny in a Mendelian fashion. Over 200 transgenic Brassica napus plants have been produced using this system. ABBREVIATIONS BA, 6-henzyladenine; NAA, R-naphthalene-acetic acid; T-DNA, transferred DNA into plants; IBA, indole butyric acid; IAA, indole acetic acid; TXD, Tobacco Xanthi diploid suspension cells; INTRODUCTION Transgenic B.
by of stem segments, followed by selective regeneration of transformed cells into fertile plants. B. napus is one of the world's most important sources of vegetable oil and protein meal (U.S. Department of Agriculture, 1987). This transformation technology will allow studies of gene expression in B. napus both at the whole plant level and in the seeds using genes derived from plants, animals and/or microbial systems. Agrobacterium
napus plants have been produced tumefaciens mediated transformation
A. tumefaeiens containing disarmed Ti plasmids have been used to stably introduce foreign genes into cells from several plant species (Horseh et al. 1985, Lloyd et al. 1986, DeBloek et al. 1984, McCormick et al. 1986). B. napus has been demonstrated to be a host for Agrobacterium (Holbrook and Miki 1985, Ooms et al. 1985) and phenotypically abnormal plants have been produced by transformation with Agrobacterium rhizogenes (Ooms et al. 1985, Guerche et al. in press). Here we report a reproducible system to produce transgenic B. napus plants which are phenotypically normal, fertile and in which the T-DNA is stably integrated and transmitted to the progeny in a Mendelian fashion.
Offprints requests to: R. Horsch
Tobacco and Tomato plants have been successfully engineered with resistance to herbicides (Shah et al. 1986) and with cross protection to viral infections (Abel et al. 1986). The production of transgenic plants in 6-8 weeks should make agronomic improvements in B. napus proceed rapidly. METHODS AND MATERIALS Plant Material B. napus L. ssp. oleifera
cv. Westar was used in all experiments. Seeds of Westar were obtained from Dr. Wilf Keller~ Plant Research Centre Agriculture Canada, Ottawa, Canada and Dr. Keith Downey, Research Station, Agriculture Canada, Saskatoon, Canada. Seedlings, sown in Metro Mix 350 (Hummert Seed Co., St. Louis) and established in the greenhouse, were transplanted to 4" pots at two weeks of age and transferred to a growth chamber with a day/night temperature of 15/I0°C, relative humidity of 50%, 14 hour photoperiod and a light intensity of 400uEM-2sec -I (Klimaszewska and Keller 1985). The plants were watered four times a day and fertilized weekly with 15:30:15 Peters Hi-Phos Fertilizer (Fogelsville, PA, USA). Bacterial Strains Inoculations were made with A. tumefaciens strain A208 (Sciaky et al. 1978) carrying the disarmed nopaline Interplasmid pTiT37-SE (Rogers et al. in press). mediate plant transformation vectors pMON200 (integrating) (Fraley et al. 1985) and pMON505 (binary) (Horsch and Klee 1986) or their derivatives were introduced into A. tuI~efaciens. The GV3111 strain carrying the disarmed octopine plasmid pTiB6S3-SE (Fraley et al. 1985) was also tested. Transformation/Selection/Regeneration Four terminal internodes from plants just prior to bolting or in the process of bolting but before flowering were removed and surface sterilized in 70% v/v ethanol for 1 minute, 2% w/v sodium hypochlorite for 20 minutes and rinsed 3 times in sterile distilled water. According to the procedure reported by Stringam (1977), stem segments were cut into 5mm discs in a sterile 15xl00mm petri plate, noting the orientation of the basal end. The discs were inoculated for 5 minutes by pouring 2 to 4 mls of an overnight culture of A. tumefaciens over the discs in the petri plate and then blotted dry by placing sterile filter paper in the petri plate and turning the plate over to absorb any excess bacteria. The stem discs were
322 placed basal side down on feeder plates on medium containing 1/10x standard MS salts (Murashige and Skoog basal salts medium purchased from Gibco), B5 vitamins (Gamhorg et al. 1969) 3% sucrose, 0.8% agar, pH 5.7, img/£ BA and l.Sml TXD feeder cells (Horsch et al. 1985). After a 2 to 3 day coculture period, stem discs were transferred, 5 to a deep dish petri plate (25x100mm) containing the same medium with standard MS salts (Gibco), img/£ BA, 500mg/£ carbenicillin, 0.5mM arginine, and 100mg/£ kanamycin for selection. At three weeks the stem explants were transferred to fresh plates containing the same medium. Culture of the explants was in a growth room under continuous cool white light at 26°C. Shoots that developed in the next 1 to 3 week period were excised from the stem explants, dipped in Rootone® and placed in 2 1/4" pots containing water saturated Metro Mix 350 in closed GA7 containers (Magent$ Corp., Chicago, IL, USA) for I0 days in a chamber with a constant temperature of 21°C and a 16 hour photoperiod. After a gradual hardening off period the plants were transplanted to 4" pots and transferred to a growth chamber with a day/night temperature of 18/16°C and 16 hour photoperiod. Expression Assays The B. napus shoots were assayed for the presence of nopaline and kanamycin resistance immediately after being excised from the stem explant while still sterile. The presence of nopaline was assayed by paper electrophoresis (Otten and Schilperoort 1978). Kanamycin resistance was assayed by placing three small leaf pieces from the shoot on media containing standard MS salts (Gibco), B5 vitamins, 3~ sucrose, 5mg/£ BA, .5mg/£ NAA, 500mg/£ carbenicillin, .5mM arginine and 100mg/~ kanamycin (Klimaszewska and Keller 1985). At three weeks the leaf pieces were scored for the presence or absence of green callus. RESULTS/DISCUSSION Regeneration/Transformation We have successfully adapted the leaf disc transformation system widely used for petunia, tobacco, and tomato (Horsch et al. 1985) to B. napus. This method combines the transformation of cells by A. tumefaciens with the very efficient regeneration of shoots from stem segments as first described by Gary Stringam (1977). However, the stock plants were grown under conditions suggested by Wilf Keller (personal communication) and produced more shoots per explant than reported by Stringam. B. napus stem segments are a good explant source for the transformation of cells. Stems provide an abundant source of healthy cells which regenerate at a high frequency. Host of the regeneration comes from the bottom of the stem segments forming a ring of shoots just outside the cambium layer. This is advantageous because it a l l o w s the majority of the cells undergoing regeneration to be in contact with the selection medium. This system, like the leaf disc system, targets transformation to the same set of cells which will regenerate. Stem explants infected with A. tumefaciens strains containing pTiT37-SE::pMON200 cultured in the presence of 100mg/~ kanamycin produced green shoots 3 to 6 weeks after inoculation while uninoculated stem segments or stem segments inoculated with A. tumefaciens strains containing pTiT37-SE::pMON120 (which lacks the kanamycin resistance gene) did not produce any shoots (Fig. I). Green shoots were produced on I0 out of 47 explants selected on lOOmg/~ kanamycin and 3 out of 15 selected on 200mg/~ kanamycin. The kanamycin selection greatly reduces and slows the regeneration response in the stem segments. Without kanamycin in the media 95-I00~ of the inoculated or uninoculated stem segments produced 10-20 shoots each in 3 to 4
Fig. 1 Stem explants inoculated with A. tumefaciens strains, cultured in the presence of 100mg/~ kanamycin. Discs on the plate on the left side of the photo were inoculated with pTiT37-SE::pMON200 which contains the NPTII gene which confers resistance to kanamycin. The discs on the right were inoculated with pTiT37-SE::pMONI20 which lacks the kanamycin resistance gene; they did not produce any shoots.
weeks but with 100mg/£ kanamycin in the media each explant produced 0-2 shoots per explant in 3 to 6 weeks. To prevent regeneration of more than one plant from the same transformation event, a limit of one shoot was excised per stem segment. The number of shoots produced was low using I00 or 200 mg/~ kanamycin selection; lowering the selection to 50mg/~ increased the number of shoots but also increased the number of "escape" shoots, Table I. In 6 experiments stem segments inoculated with a pMON200 derivative produced 37 shoots from 190 explants (19~) when selected on 50mg/£ kanamycin hut only 46 shoots from 365 explants (13~) when selected on I00 mg/~ kanamycin. However the number of kanamycin resistant shoots identified by the ability of leaf tissue to callus on kanamycin was 14 out of 37 and 34 out of 46 on 50 and 100mg/~ kanamycin respectively. Thus a reduction in the concentration of kanamycin for selection resulted in many more shoots which were not transformed and required a greater amount of time to screen. There was also an increased number of bleached shoots produced on the explants selected on 50mg/£ kanamycin, which would not develop further if excised and were considered "escapes." The best experiment using kanamycin as the selectable marker was inoculated with a pMON200 derivative and produced 16 kanamycin resistant shoots out of a total of 75 explants for an overall transformation frequency of 21%. However, an average of 13 experiments (including those in Table l) inoculated with a pMON200 derivative and selected on lO0mg/~ kanamycin produced an average of 7 transgenic shoots per lO0 explants. The production of abnormal shoots greatly reduced the number of transgenic plants we were able to obtain. The transformation frequency based on the detection of nopaline in green structures per explant was 40-60% (data not shown). However, a significant number of explants produced club-like structures without a meristem. These structures usually would not go on to produce a plant with a normal phenotype.
323 The first transgenic plant to be produced by this procedure was transformed with the pMON200 vector and was both kanamycin resistant and nopaline positive. The nopaline synthase gene did not express after the plant was in soil 6 weeks, but leaf tissue taken back into culture produced a strong nopaline signal. Leaf tissue from this plant produced callus on 100mg/£ kanamycin while wild-type tissue died, Fig. 2. A Southern hybridization analysis of DNA from leaf tissue shoWed homology with a radiolabelled NPT II probe confirming the presence of the T-DNA while DNA from a wild-type plant showed no homology to the probe (date not shown).
Table ] The frequency of kanamycin resistant and sensitive shoots produced in 6 B. napus stem segment transformation experiments using a 50 and lOOmg/~ kanamycin selection. 50mg/£ kanamycin ExperTotal iment Explants 1 2 3 4 5 6 Total
80 25 25 15 5 40 190
I
KmR KmS Shoots Shoots 2 3 2 1 3 3 14
I0 0 5 3 1 4 23
100mg/£ kanamycin
I Total Km R FanS IExplants Shoots Shoots 160 50 30 30 20 75
365
II 0 3 3 1 16 34
5 0 1 0 0 6 12
NOTE: The vector was a pMON200 (integrating) or pMON505 (binary) derivative mated into an A208 Agrobacterium strain containing the disarmed pTiT37 nopaline plasmid.
We have produced transgenic plants with Agrobacterium strains carrying either the nopaline (pTiT37) or the octopine (pTiB6S3) Ti plasmids. Holbrook and Miki (1985) found that B. napus was not susceptible to the octopine strains of A. tumefaciens but they were able to produce tumorous callus on B. napus inoculated with nopaline strains. Even though we obtained transformed plants with both the octopine and nopaline strains of Agrobacterium we saw a reduced transformation efficiency with the octopine strains. We also produced plants with both integrating and binary vectors. Highly variable transformation frequencies were observed with both type of vectors. Both NOS and CaMV35S promoters linked to the NPTII coding sequence have been used to produce kanamycin resistant plants. The preparation of explants, inoculation, and coculture is a simple procedure and 500 to 1000 stem segments can easily be put into culture in one day. The most limiting factor is a constant supply of primary apical shoots from 6-7 week old plants. However we found the secondary apical shoots which grew out after removal of the primary shoot also were suitable for producing transgenic plants. Regeneration of Transformants Stem segments placed into culture swelled 5 to I0 times their original size with greater swelling on the basal side. A thin layer of callus would proliferate at both ends of the segments. Shoot buds could be seen as early as I week after inoculation from the basal side of the segment and appeared to be coming from just outside the cambium layer. The shoots appeared to come directly from the explant and did not go through a callus stage. Bypassing a callus stage
Fig. 2 B. napus leaf tissue on medium containing I00 mg/~ kanamycin. Leaf discs on the left forming callus were taken from a plant (#991) transformed with an Agrobacterium strain containing pTiT37-SE:pMON200 which confers resistance to kanamycin. The bleached leaf discs on the right lacking callus were taken from a wild-type B. napus plant.
coupled with a relatively fast regeneration time, should minimize the number of genetic mutations due to tissue culture alone. If the explants were placed apical side down the regeneration was greatly reduced or eliminated. With 100mg/~ kanamycin selection most shoots were large enough to excise from 3 to 6 weeks after inoculation, and the explants were discarded. The average length of an excised shoot was approximately 3 cm but we were able to root much smaller shoots if they had a normal meristem and a leaf or leaves. If shoots were excised from the bottom of the stem segment and were in contact with the media they were more likely to be transformed than if they were excised from the top of the explant away from the selection media. Transgenic shoots were routinely produced 5 to 6 weeks after inoculation. Rooting Rooting of B. napus regenerated shoots was routine in growth media containing NAA, IBA or IAA but rooting of transformed shoots was difficult using the same growth media used to root non-transformed regenerates. The rooting of transformed regenerates took 2-4 weeks with only about 50% of the shoots capable of root formation on growth media with NAA. We were able to improve the rooting efficiency of the transformants by 30% by dusting the cut end of the shoots in Rootone® and planting in a soilless mix rather than rooting in vitro. In our best experiment 82% of the shoots produced roots. Rooting of transformed shoots in vitro in the presence of lOOmg/~ kanamycin was tried in early experiments but since our in vitro rooting efficiency of putative transformed shoots without selection was only 50~ we did not pursue this approach. Leaf Assays The ability of leaf tissue to callus in the presence of kanamycin was used as a screening procedure to eliminate shoots which came through the selection but no longer expressed or contained the T-DNA. Several small 2mm square pieces of leaf tissue were taken from the sterile shoots and placed on MS medium containing 100mg/£ kanamycin just prior to rooting the
324 shoots in soil. The Brassica leaf tissue sometimes expanded I0 to 20 times its original size when put into culture on this media but only leaf tissue transformed with a vector containing the NPTII gene would form callus. Many of the leaf assays produced a small amount of callus or bleached calli which was reported as negative. A minimum of 3 patches of green callus approximately 3mm square was considered a kanamycin resistant leaf assay response. Often the interior portion of the leaf tissue would bleach or brown even on plants considered kanamycin resistant. The wild-type control tissue would not produce callus and it would bleach or brown more quickly than the transformed tissue, usually within 7 days. The expression of the nopaline synthase gene was not a reliable marker for the expression of the T-DNA in primary transgenic B. napus plants. In one experiment the scorable nopaline synthase gene was expressed in only 5 out of 18 of the B. napus transformants which were resistant to the selectable marker. The failure of nopaline synthase to express could be due to "position effects," the loss of T-DNA during transfer to the plant cell, methylation, etc. The reason is not known b u t it is a common phenomenon observed in petunia, tobacco, tomato and Arabidopsis (Horsch et al. 1985, Kahl and Schell 1982, McCormick et al. 1986, Lloyd et al 1986). Southern Hybridization Analysis Transformed plants were examined for the presence of the T-DNA by Southern blot analysis, Fig. 3. Genomic leaf DNA was isolated and digested with BamHl and EcoRl restriction enzymes, fractionated on a 0.7% agarose gel, transferred to nitrocellulose, and hybridized with nick-translated linearized pMON273, a derivative of pMON200. There was hybridization to all transgenic plants, 1082, 1158, 1241, 1337 and 1355 but not to the nontransformed control. The expected internal BamHl fragments are 3.6, 2.5 and 1.7 kb for plants 1082, 1158, 1241 and 1355. The 3.6 and 2.5 kb fragments can be seen in the 3.5 day exposure on each of these plants. The 1.7 kb fragment has very little homology to the probe and can be detected only in plant 1158 which contains 2 insertions of the T-DNA based on first generation progeny data and at least one insertion is a tandem copy based on this Southern blot analysis. Both the 1158 and 1082 plants contain a 4.8 kb fragment which is diagnostic for a tandem insertion of the T-DNA. Plant 1337 is a derivative of pMON200 whose coding region contains only the 3.6 kb internal Bam fragment. The EcoRI digest does not produce any internal fragments but only junction fragments of the T-DNA with the plant DNA and a band the full size of the plasmid where there is a tandem insertion. There are more bands in the EcoRI digest of plant 1158 than would be predicted from the progeny data, indicating either linkage of several T-DNA's or silent copies that do not produce a phenotype in progeny. The Southern blot does show the presence of the correct and unique bands for each individual construct (plasmids not shown), but a detailed analysis of the T-DNA was not made. Transmission of T-DNA to Progeny Parental transgenic plants that produce a good amount of nopaline were chosen for genetic analysis because we have found a strict correlation between nopaline synthase expression in the parent and the progeny plants. Analysis of the progeny from self-pollinations of the transgenic B. napus plants transformed with the Agrobacterium plant vectors revealed that the nopaline synthase and the NPTII genes are faithfully transmitted to the $I progeny just as they are in the Solanaceous plants (Horsch et al. 1985, DeBlock et al. 1984, McCormick et a2. 1986). Progeny from a self-pollination of the transgenic B. napus plant #991
e0
O
Kb
B
pMON200 derivatives 1082 1158 1241 1337 1355 E B E B E B E B E B E
23.72-9.466.66-4.26-
2.25-1.95-
Fig._33 Southern blot analysis of primary B. napus transformants. Leaf genomic DNA was digested with BamHI and EcoRI designated as B and E respectively above each lane. The control plant is a nontransformed B. napus plant cv. Westar. Plants 1082, 1158, 1241, 1337 and 1355 are transgenic B. napus plants transformed with a pMON200 derivative. The blot was probed with a nick translated pMON273 plasmid. The pMON273 plasmid is a pMON200 derivative containing the NPTII gene driven by the CaMV35S promoter. Filter exposed 84hr. at -80°C. The sizes of the molecular weight markers are indicated.
transformed with pMON200 demonstrated that the nopaline synthase and NPTII gene were transmitted as a single dominant factor and produced the typical 3 to 1 Mendelian segregation ratio. In the selfed progeny, 77 plants were nopaline positive, kanamycin resistant and 16 were nopaline negative and kanamycin sensitive as determined by analysis of leaf tissue. The kanamycin leaf assay and the nopaline synthase assays correlated 100%, Fig. 4. The calculated chi-square value predicts a greater than 95~ probability that the deviation from the 3 to 1 ratio results from chance alone. Progeny from self-pollinations of 13 independent transgenic plants, and backcrosses to wild-type plants of 4 independent transgenic plants were analyzed for inheritance of the T-DNA by nopaline production, Table 2. Inheritance data shows that 7 out of 13 plants contain multiple functional inserts of the T-DNA. Three plants could contain more than 2 inserts, and I plant more than 3 inserts. This is in contrast to the single copy T-DNA insertion reported in most transgenic petunia, tobacco, tomato and lettuce (Xavier Delannay and Richard Michelmore, personal communication) plants. A high number of multiple T-DNA inserts has been seen in Arabidopsis (A. Lloyd, personal communication) which also belongs to the Crucifer family. A I to 1 ratio was observed in S 1 progeny of plant 1355 which could be a gametic lethal insertion mutant.
325 Table 2 Segregation ratios based on nopaline assay results from progeny of independent primary transformants. Primary transformants were produced by A. tumefaciens strains containing pMON200 or a pMON200 derivative.
Plant #
Self-Pollinated NOP+:NOP-
991 1082 1124 1127 1145 1158 1185 1198 1202 1220 1241 1276 1355
77:16 68:19 51:9 38:4 71:1 25:2 63:1 34:0 66:0 73:13 51:5 41:2 72:72
Backcross NOP+:NOP-
This procedure for routine production of transgenic B. napus plants will he a useful tool for both basic research and improvement of the agronomic traits of this important crop species.
No. of Inserts a I 1 I 2 or more 2 or more 2 2 or more l'or more 3 or more 1 2 2 ?
11:5 9:4
12:9 37:0
ACKNOWLEDGMENTS We thank Sally Metz for reviewing the manuscript and subsequent confirmation of the results in more recent experiments and we gratefully acknowledge Catherine Hironaka for analysis of the plant DNA. We also thank Dr. Dilip Shah for helpful suggestions on the manuscript, Bill Schuler and Judy Erbschloe for care of the plants and Barbara Schiermeyer for preparation of the manuscript. We would also like to thank Dr. Keith Downey and Dr. Wilf Keller for providing seed and helpful discussion.
abased on Chi-square analysis
OPINE ASSAY Brassica napus Sl P r o g e n y Plant # 9 9 1 ( p M O N 2 0 0 )
is
R R
RI
R
R
R
S 1 Progeny
a. -o
S 1 Progeny
Plant #991
zO ~
Plant #991
RI
Fig. 4 Nopaline assay on leaf tissue from selfed progeny of transgenic plant #991 transformed with A. tumefaciens carrying the pMON200 intermediate vector which confers resistance to kanamycin and contains the nopaline synthase gene as a scorable marker. Leaf tissue was cultured on medium containing 100 mg/£ kanamycin. On the above media leaf tissue from kanamycin resistant progeny produced callus and were nopaline+; kanamycin sensitive progeny and wild-type progeny did not produce callus and were nopaline-. A 2~g nopaline standard was also assayed. The letters R and S denote kanamycin resistance and sensitivity respectively, in the leaf callus assay.
REFERENCES Abel PP, Nelson RS, Barun D, Hoffmann N, Rogers SG, Fraley RT, and Beachy RN (1986) Science 232: 738-743. DeBlock M, Herrera-Estrella L, Schell J, Zambryski P (1984) The EMBO Journal 3: 1681-1689. Eichholtz D, Rogers S, Horsch R, Klee H, Hayford M, Hoffmann N, Bradford S, Fink C, Flick J, O'Connell K, and Fraley R (1987) Somatic Cell and Molecular Genetics, 13: 67-76. Fraley RT, Rogers SG, Horsch RB, Eichholtz D, Flick J, Fink C, Hoffmann N, Sanders P (1985) Bio/Technology 3: 629-635. Guerche P, Jouanin L, Tepfer D, Pelletier G (1987) Mol. Gen. Genetics (in press). Holbrook LA, Miki BL (1985) Plant Cell Reports 4: 329-332. Horsch RB, Klee HJ (1986) Proc. Natl. Acad. Sci. USA 83: 4428-4432. Horsch RB, Fry JE, Hoffmann NL, Wallroth M, Eichholtz D, Rogers SG, Fraley RT (1985) Science 227: 1229-31. Kahl G & Shell J (1982) Molecular Biology of Plant Tumors, Academic, New York. Klimaszewska K and Keller WA (1985) P/ant Cell Tissue Organ Culture 4: 183-197. Lloyd AM, Barnason AR, Rogers SG, Byrne MC, Fraley RT, Horsch RB (1986) Science 234: 464-466. McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R (]986) Plant Cell Reports 5: 81-84. Ooms G, Bains A, B u r r e l l M," Karp A, Twell D, and Wilcox E (1985) Theor. Appl. Genet. 71: 325-329. Otten LABM, Schilperoot RA (1978) Biochimica et Biophgsica Acta 527: 497-500. Rogers S, Klee H, Byrne M, Horsch R, Fraley R (1987) Methods Enzymol. In press. Sciaky D, Montoya AL, Chilton MD (1978) Plasmid 1:238-253. Shah DM, Horsch RB, Klee HJ, Kishore GM, Winter JA, Tumer NE~ Hironaka CM, Sanders PR, Gasser CS, Aykent S, Siegel NR, Rogers SG, and Fraley RT (1986) Science 233: 478-481. Stringam GR (1977) Plant Science Letters 9: 115-119. United States Department of Agriculture, Foreign Agricultural Service, Circular Series, FOP March 1987. World Oilseed Situation and Market Highlights: 9-13.
Plant Cell Reports (1987) 6:321-325
© Springer-Verlag 1987
Transformation of Brassica napus with A grobacterium tumefaciens based vectors J o y c e Fry, A r l e n e B a r n a s o n , and R o b e r t B. H o r s c h Biological Sciences, Monsanto Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198, USA Received May 28, 1987 / Revised version received July 6, 1987 - Communicated by L. K. Grill
ABSTRACT A reproducible system to produce transgenic Brassica napus plants has been developed using stem segments. Stem segments from 6-7 week old plants were inoculated with an Agrobacterium t~mefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimeric bacterial gene encoding kanamycin resistance (pMON200). Stem explants were cocultnred for 2 days before transfer to kanamycin selection medium. Shoots regenerated directly from the explant in 3-6 weeks and were excised, dipped in Rootone®, and rooted in soil. Transformation was confirmed by opine production, kanamycin resistance, and DNA blot hybridization in the primary transformants. Final proof of transformation was demonstrated by the co-transfer of opine production and kanamycin resistance to progeny in a Mendelian fashion. Over 200 transgenic Brassica napus plants have been produced using this system. ABBREVIATIONS BA, 6-henzyladenine; NAA, R-naphthalene-acetic acid; T-DNA, transferred DNA into plants; IBA, indole butyric acid; IAA, indole acetic acid; TXD, Tobacco Xanthi diploid suspension cells; INTRODUCTION Transgenic B.
by of stem segments, followed by selective regeneration of transformed cells into fertile plants. B. napus is one of the world's most important sources of vegetable oil and protein meal (U.S. Department of Agriculture, 1987). This transformation technology will allow studies of gene expression in B. napus both at the whole plant level and in the seeds using genes derived from plants, animals and/or microbial systems. Agrobacterium
napus plants have been produced tumefaciens mediated transformation
A. tumefaeiens containing disarmed Ti plasmids have been used to stably introduce foreign genes into cells from several plant species (Horseh et al. 1985, Lloyd et al. 1986, DeBloek et al. 1984, McCormick et al. 1986). B. napus has been demonstrated to be a host for Agrobacterium (Holbrook and Miki 1985, Ooms et al. 1985) and phenotypically abnormal plants have been produced by transformation with Agrobacterium rhizogenes (Ooms et al. 1985, Guerche et al. in press). Here we report a reproducible system to produce transgenic B. napus plants which are phenotypically normal, fertile and in which the T-DNA is stably integrated and transmitted to the progeny in a Mendelian fashion.
Offprints requests to: R. Horsch
Tobacco and Tomato plants have been successfully engineered with resistance to herbicides (Shah et al. 1986) and with cross protection to viral infections (Abel et al. 1986). The production of transgenic plants in 6-8 weeks should make agronomic improvements in B. napus proceed rapidly. METHODS AND MATERIALS Plant Material B. napus L. ssp. oleifera
cv. Westar was used in all experiments. Seeds of Westar were obtained from Dr. Wilf Keller~ Plant Research Centre Agriculture Canada, Ottawa, Canada and Dr. Keith Downey, Research Station, Agriculture Canada, Saskatoon, Canada. Seedlings, sown in Metro Mix 350 (Hummert Seed Co., St. Louis) and established in the greenhouse, were transplanted to 4" pots at two weeks of age and transferred to a growth chamber with a day/night temperature of 15/I0°C, relative humidity of 50%, 14 hour photoperiod and a light intensity of 400uEM-2sec -I (Klimaszewska and Keller 1985). The plants were watered four times a day and fertilized weekly with 15:30:15 Peters Hi-Phos Fertilizer (Fogelsville, PA, USA). Bacterial Strains Inoculations were made with A. tumefaciens strain A208 (Sciaky et al. 1978) carrying the disarmed nopaline Interplasmid pTiT37-SE (Rogers et al. in press). mediate plant transformation vectors pMON200 (integrating) (Fraley et al. 1985) and pMON505 (binary) (Horsch and Klee 1986) or their derivatives were introduced into A. tuI~efaciens. The GV3111 strain carrying the disarmed octopine plasmid pTiB6S3-SE (Fraley et al. 1985) was also tested. Transformation/Selection/Regeneration Four terminal internodes from plants just prior to bolting or in the process of bolting but before flowering were removed and surface sterilized in 70% v/v ethanol for 1 minute, 2% w/v sodium hypochlorite for 20 minutes and rinsed 3 times in sterile distilled water. According to the procedure reported by Stringam (1977), stem segments were cut into 5mm discs in a sterile 15xl00mm petri plate, noting the orientation of the basal end. The discs were inoculated for 5 minutes by pouring 2 to 4 mls of an overnight culture of A. tumefaciens over the discs in the petri plate and then blotted dry by placing sterile filter paper in the petri plate and turning the plate over to absorb any excess bacteria. The stem discs were
322 placed basal side down on feeder plates on medium containing 1/10x standard MS salts (Murashige and Skoog basal salts medium purchased from Gibco), B5 vitamins (Gamhorg et al. 1969) 3% sucrose, 0.8% agar, pH 5.7, img/£ BA and l.Sml TXD feeder cells (Horsch et al. 1985). After a 2 to 3 day coculture period, stem discs were transferred, 5 to a deep dish petri plate (25x100mm) containing the same medium with standard MS salts (Gibco), img/£ BA, 500mg/£ carbenicillin, 0.5mM arginine, and 100mg/£ kanamycin for selection. At three weeks the stem explants were transferred to fresh plates containing the same medium. Culture of the explants was in a growth room under continuous cool white light at 26°C. Shoots that developed in the next 1 to 3 week period were excised from the stem explants, dipped in Rootone® and placed in 2 1/4" pots containing water saturated Metro Mix 350 in closed GA7 containers (Magent$ Corp., Chicago, IL, USA) for I0 days in a chamber with a constant temperature of 21°C and a 16 hour photoperiod. After a gradual hardening off period the plants were transplanted to 4" pots and transferred to a growth chamber with a day/night temperature of 18/16°C and 16 hour photoperiod. Expression Assays The B. napus shoots were assayed for the presence of nopaline and kanamycin resistance immediately after being excised from the stem explant while still sterile. The presence of nopaline was assayed by paper electrophoresis (Otten and Schilperoort 1978). Kanamycin resistance was assayed by placing three small leaf pieces from the shoot on media containing standard MS salts (Gibco), B5 vitamins, 3~ sucrose, 5mg/£ BA, .5mg/£ NAA, 500mg/£ carbenicillin, .5mM arginine and 100mg/~ kanamycin (Klimaszewska and Keller 1985). At three weeks the leaf pieces were scored for the presence or absence of green callus. RESULTS/DISCUSSION Regeneration/Transformation We have successfully adapted the leaf disc transformation system widely used for petunia, tobacco, and tomato (Horsch et al. 1985) to B. napus. This method combines the transformation of cells by A. tumefaciens with the very efficient regeneration of shoots from stem segments as first described by Gary Stringam (1977). However, the stock plants were grown under conditions suggested by Wilf Keller (personal communication) and produced more shoots per explant than reported by Stringam. B. napus stem segments are a good explant source for the transformation of cells. Stems provide an abundant source of healthy cells which regenerate at a high frequency. Host of the regeneration comes from the bottom of the stem segments forming a ring of shoots just outside the cambium layer. This is advantageous because it a l l o w s the majority of the cells undergoing regeneration to be in contact with the selection medium. This system, like the leaf disc system, targets transformation to the same set of cells which will regenerate. Stem explants infected with A. tumefaciens strains containing pTiT37-SE::pMON200 cultured in the presence of 100mg/~ kanamycin produced green shoots 3 to 6 weeks after inoculation while uninoculated stem segments or stem segments inoculated with A. tumefaciens strains containing pTiT37-SE::pMON120 (which lacks the kanamycin resistance gene) did not produce any shoots (Fig. I). Green shoots were produced on I0 out of 47 explants selected on lOOmg/~ kanamycin and 3 out of 15 selected on 200mg/~ kanamycin. The kanamycin selection greatly reduces and slows the regeneration response in the stem segments. Without kanamycin in the media 95-I00~ of the inoculated or uninoculated stem segments produced 10-20 shoots each in 3 to 4
Fig. 1 Stem explants inoculated with A. tumefaciens strains, cultured in the presence of 100mg/~ kanamycin. Discs on the plate on the left side of the photo were inoculated with pTiT37-SE::pMON200 which contains the NPTII gene which confers resistance to kanamycin. The discs on the right were inoculated with pTiT37-SE::pMONI20 which lacks the kanamycin resistance gene; they did not produce any shoots.
weeks but with 100mg/£ kanamycin in the media each explant produced 0-2 shoots per explant in 3 to 6 weeks. To prevent regeneration of more than one plant from the same transformation event, a limit of one shoot was excised per stem segment. The number of shoots produced was low using I00 or 200 mg/~ kanamycin selection; lowering the selection to 50mg/~ increased the number of shoots but also increased the number of "escape" shoots, Table I. In 6 experiments stem segments inoculated with a pMON200 derivative produced 37 shoots from 190 explants (19~) when selected on 50mg/£ kanamycin hut only 46 shoots from 365 explants (13~) when selected on I00 mg/~ kanamycin. However the number of kanamycin resistant shoots identified by the ability of leaf tissue to callus on kanamycin was 14 out of 37 and 34 out of 46 on 50 and 100mg/~ kanamycin respectively. Thus a reduction in the concentration of kanamycin for selection resulted in many more shoots which were not transformed and required a greater amount of time to screen. There was also an increased number of bleached shoots produced on the explants selected on 50mg/£ kanamycin, which would not develop further if excised and were considered "escapes." The best experiment using kanamycin as the selectable marker was inoculated with a pMON200 derivative and produced 16 kanamycin resistant shoots out of a total of 75 explants for an overall transformation frequency of 21%. However, an average of 13 experiments (including those in Table l) inoculated with a pMON200 derivative and selected on lO0mg/~ kanamycin produced an average of 7 transgenic shoots per lO0 explants. The production of abnormal shoots greatly reduced the number of transgenic plants we were able to obtain. The transformation frequency based on the detection of nopaline in green structures per explant was 40-60% (data not shown). However, a significant number of explants produced club-like structures without a meristem. These structures usually would not go on to produce a plant with a normal phenotype.
323 The first transgenic plant to be produced by this procedure was transformed with the pMON200 vector and was both kanamycin resistant and nopaline positive. The nopaline synthase gene did not express after the plant was in soil 6 weeks, but leaf tissue taken back into culture produced a strong nopaline signal. Leaf tissue from this plant produced callus on 100mg/£ kanamycin while wild-type tissue died, Fig. 2. A Southern hybridization analysis of DNA from leaf tissue shoWed homology with a radiolabelled NPT II probe confirming the presence of the T-DNA while DNA from a wild-type plant showed no homology to the probe (date not shown).
Table ] The frequency of kanamycin resistant and sensitive shoots produced in 6 B. napus stem segment transformation experiments using a 50 and lOOmg/~ kanamycin selection. 50mg/£ kanamycin ExperTotal iment Explants 1 2 3 4 5 6 Total
80 25 25 15 5 40 190
I
KmR KmS Shoots Shoots 2 3 2 1 3 3 14
I0 0 5 3 1 4 23
100mg/£ kanamycin
I Total Km R FanS IExplants Shoots Shoots 160 50 30 30 20 75
365
II 0 3 3 1 16 34
5 0 1 0 0 6 12
NOTE: The vector was a pMON200 (integrating) or pMON505 (binary) derivative mated into an A208 Agrobacterium strain containing the disarmed pTiT37 nopaline plasmid.
We have produced transgenic plants with Agrobacterium strains carrying either the nopaline (pTiT37) or the octopine (pTiB6S3) Ti plasmids. Holbrook and Miki (1985) found that B. napus was not susceptible to the octopine strains of A. tumefaciens but they were able to produce tumorous callus on B. napus inoculated with nopaline strains. Even though we obtained transformed plants with both the octopine and nopaline strains of Agrobacterium we saw a reduced transformation efficiency with the octopine strains. We also produced plants with both integrating and binary vectors. Highly variable transformation frequencies were observed with both type of vectors. Both NOS and CaMV35S promoters linked to the NPTII coding sequence have been used to produce kanamycin resistant plants. The preparation of explants, inoculation, and coculture is a simple procedure and 500 to 1000 stem segments can easily be put into culture in one day. The most limiting factor is a constant supply of primary apical shoots from 6-7 week old plants. However we found the secondary apical shoots which grew out after removal of the primary shoot also were suitable for producing transgenic plants. Regeneration of Transformants Stem segments placed into culture swelled 5 to I0 times their original size with greater swelling on the basal side. A thin layer of callus would proliferate at both ends of the segments. Shoot buds could be seen as early as I week after inoculation from the basal side of the segment and appeared to be coming from just outside the cambium layer. The shoots appeared to come directly from the explant and did not go through a callus stage. Bypassing a callus stage
Fig. 2 B. napus leaf tissue on medium containing I00 mg/~ kanamycin. Leaf discs on the left forming callus were taken from a plant (#991) transformed with an Agrobacterium strain containing pTiT37-SE:pMON200 which confers resistance to kanamycin. The bleached leaf discs on the right lacking callus were taken from a wild-type B. napus plant.
coupled with a relatively fast regeneration time, should minimize the number of genetic mutations due to tissue culture alone. If the explants were placed apical side down the regeneration was greatly reduced or eliminated. With 100mg/~ kanamycin selection most shoots were large enough to excise from 3 to 6 weeks after inoculation, and the explants were discarded. The average length of an excised shoot was approximately 3 cm but we were able to root much smaller shoots if they had a normal meristem and a leaf or leaves. If shoots were excised from the bottom of the stem segment and were in contact with the media they were more likely to be transformed than if they were excised from the top of the explant away from the selection media. Transgenic shoots were routinely produced 5 to 6 weeks after inoculation. Rooting Rooting of B. napus regenerated shoots was routine in growth media containing NAA, IBA or IAA but rooting of transformed shoots was difficult using the same growth media used to root non-transformed regenerates. The rooting of transformed regenerates took 2-4 weeks with only about 50% of the shoots capable of root formation on growth media with NAA. We were able to improve the rooting efficiency of the transformants by 30% by dusting the cut end of the shoots in Rootone® and planting in a soilless mix rather than rooting in vitro. In our best experiment 82% of the shoots produced roots. Rooting of transformed shoots in vitro in the presence of lOOmg/~ kanamycin was tried in early experiments but since our in vitro rooting efficiency of putative transformed shoots without selection was only 50~ we did not pursue this approach. Leaf Assays The ability of leaf tissue to callus in the presence of kanamycin was used as a screening procedure to eliminate shoots which came through the selection but no longer expressed or contained the T-DNA. Several small 2mm square pieces of leaf tissue were taken from the sterile shoots and placed on MS medium containing 100mg/£ kanamycin just prior to rooting the
324 shoots in soil. The Brassica leaf tissue sometimes expanded I0 to 20 times its original size when put into culture on this media but only leaf tissue transformed with a vector containing the NPTII gene would form callus. Many of the leaf assays produced a small amount of callus or bleached calli which was reported as negative. A minimum of 3 patches of green callus approximately 3mm square was considered a kanamycin resistant leaf assay response. Often the interior portion of the leaf tissue would bleach or brown even on plants considered kanamycin resistant. The wild-type control tissue would not produce callus and it would bleach or brown more quickly than the transformed tissue, usually within 7 days. The expression of the nopaline synthase gene was not a reliable marker for the expression of the T-DNA in primary transgenic B. napus plants. In one experiment the scorable nopaline synthase gene was expressed in only 5 out of 18 of the B. napus transformants which were resistant to the selectable marker. The failure of nopaline synthase to express could be due to "position effects," the loss of T-DNA during transfer to the plant cell, methylation, etc. The reason is not known b u t it is a common phenomenon observed in petunia, tobacco, tomato and Arabidopsis (Horsch et al. 1985, Kahl and Schell 1982, McCormick et al. 1986, Lloyd et al 1986). Southern Hybridization Analysis Transformed plants were examined for the presence of the T-DNA by Southern blot analysis, Fig. 3. Genomic leaf DNA was isolated and digested with BamHl and EcoRl restriction enzymes, fractionated on a 0.7% agarose gel, transferred to nitrocellulose, and hybridized with nick-translated linearized pMON273, a derivative of pMON200. There was hybridization to all transgenic plants, 1082, 1158, 1241, 1337 and 1355 but not to the nontransformed control. The expected internal BamHl fragments are 3.6, 2.5 and 1.7 kb for plants 1082, 1158, 1241 and 1355. The 3.6 and 2.5 kb fragments can be seen in the 3.5 day exposure on each of these plants. The 1.7 kb fragment has very little homology to the probe and can be detected only in plant 1158 which contains 2 insertions of the T-DNA based on first generation progeny data and at least one insertion is a tandem copy based on this Southern blot analysis. Both the 1158 and 1082 plants contain a 4.8 kb fragment which is diagnostic for a tandem insertion of the T-DNA. Plant 1337 is a derivative of pMON200 whose coding region contains only the 3.6 kb internal Bam fragment. The EcoRI digest does not produce any internal fragments but only junction fragments of the T-DNA with the plant DNA and a band the full size of the plasmid where there is a tandem insertion. There are more bands in the EcoRI digest of plant 1158 than would be predicted from the progeny data, indicating either linkage of several T-DNA's or silent copies that do not produce a phenotype in progeny. The Southern blot does show the presence of the correct and unique bands for each individual construct (plasmids not shown), but a detailed analysis of the T-DNA was not made. Transmission of T-DNA to Progeny Parental transgenic plants that produce a good amount of nopaline were chosen for genetic analysis because we have found a strict correlation between nopaline synthase expression in the parent and the progeny plants. Analysis of the progeny from self-pollinations of the transgenic B. napus plants transformed with the Agrobacterium plant vectors revealed that the nopaline synthase and the NPTII genes are faithfully transmitted to the $I progeny just as they are in the Solanaceous plants (Horsch et al. 1985, DeBlock et al. 1984, McCormick et a2. 1986). Progeny from a self-pollination of the transgenic B. napus plant #991
e0
O
Kb
B
pMON200 derivatives 1082 1158 1241 1337 1355 E B E B E B E B E B E
23.72-9.466.66-4.26-
2.25-1.95-
Fig._33 Southern blot analysis of primary B. napus transformants. Leaf genomic DNA was digested with BamHI and EcoRI designated as B and E respectively above each lane. The control plant is a nontransformed B. napus plant cv. Westar. Plants 1082, 1158, 1241, 1337 and 1355 are transgenic B. napus plants transformed with a pMON200 derivative. The blot was probed with a nick translated pMON273 plasmid. The pMON273 plasmid is a pMON200 derivative containing the NPTII gene driven by the CaMV35S promoter. Filter exposed 84hr. at -80°C. The sizes of the molecular weight markers are indicated.
transformed with pMON200 demonstrated that the nopaline synthase and NPTII gene were transmitted as a single dominant factor and produced the typical 3 to 1 Mendelian segregation ratio. In the selfed progeny, 77 plants were nopaline positive, kanamycin resistant and 16 were nopaline negative and kanamycin sensitive as determined by analysis of leaf tissue. The kanamycin leaf assay and the nopaline synthase assays correlated 100%, Fig. 4. The calculated chi-square value predicts a greater than 95~ probability that the deviation from the 3 to 1 ratio results from chance alone. Progeny from self-pollinations of 13 independent transgenic plants, and backcrosses to wild-type plants of 4 independent transgenic plants were analyzed for inheritance of the T-DNA by nopaline production, Table 2. Inheritance data shows that 7 out of 13 plants contain multiple functional inserts of the T-DNA. Three plants could contain more than 2 inserts, and I plant more than 3 inserts. This is in contrast to the single copy T-DNA insertion reported in most transgenic petunia, tobacco, tomato and lettuce (Xavier Delannay and Richard Michelmore, personal communication) plants. A high number of multiple T-DNA inserts has been seen in Arabidopsis (A. Lloyd, personal communication) which also belongs to the Crucifer family. A I to 1 ratio was observed in S 1 progeny of plant 1355 which could be a gametic lethal insertion mutant.
325 Table 2 Segregation ratios based on nopaline assay results from progeny of independent primary transformants. Primary transformants were produced by A. tumefaciens strains containing pMON200 or a pMON200 derivative.
Plant #
Self-Pollinated NOP+:NOP-
991 1082 1124 1127 1145 1158 1185 1198 1202 1220 1241 1276 1355
77:16 68:19 51:9 38:4 71:1 25:2 63:1 34:0 66:0 73:13 51:5 41:2 72:72
Backcross NOP+:NOP-
This procedure for routine production of transgenic B. napus plants will he a useful tool for both basic research and improvement of the agronomic traits of this important crop species.
No. of Inserts a I 1 I 2 or more 2 or more 2 2 or more l'or more 3 or more 1 2 2 ?
11:5 9:4
12:9 37:0
ACKNOWLEDGMENTS We thank Sally Metz for reviewing the manuscript and subsequent confirmation of the results in more recent experiments and we gratefully acknowledge Catherine Hironaka for analysis of the plant DNA. We also thank Dr. Dilip Shah for helpful suggestions on the manuscript, Bill Schuler and Judy Erbschloe for care of the plants and Barbara Schiermeyer for preparation of the manuscript. We would also like to thank Dr. Keith Downey and Dr. Wilf Keller for providing seed and helpful discussion.
abased on Chi-square analysis
OPINE ASSAY Brassica napus Sl P r o g e n y Plant # 9 9 1 ( p M O N 2 0 0 )
is
R R
RI
R
R
R
S 1 Progeny
a. -o
S 1 Progeny
Plant #991
zO ~
Plant #991
RI
Fig. 4 Nopaline assay on leaf tissue from selfed progeny of transgenic plant #991 transformed with A. tumefaciens carrying the pMON200 intermediate vector which confers resistance to kanamycin and contains the nopaline synthase gene as a scorable marker. Leaf tissue was cultured on medium containing 100 mg/£ kanamycin. On the above media leaf tissue from kanamycin resistant progeny produced callus and were nopaline+; kanamycin sensitive progeny and wild-type progeny did not produce callus and were nopaline-. A 2~g nopaline standard was also assayed. The letters R and S denote kanamycin resistance and sensitivity respectively, in the leaf callus assay.
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