Plant Cell Reports

Plant Cell Reports (1987) 6:439-442

© Springer-Verlag1987

Transformation of lettuce (Lactuca sativa) mediated by A grobacterium tumefaciens Richard Michelmore, Ellen Marsh, Susan Seely, and Benoit Landry Department of Vegetable Crops, University of California, Davis, CA 95616, USA Received June 23, 1987 / Revised version received September 3, 1987 - Communicated by L. K. Grill

Abstract

Lactuca sa_Lt!¥acan be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R 2 seedlings on media containing G418. Abbreviations

IAA, [ndole acetic acid; KIN, kinetin; BA, benzyladenine; NOS, nopaline synthase; NPTII, neomycin phosphotransferase II; RMNO, tobacco nutrient medium (Marten and Maliga, 1975); SH, Shank & Hildebrandt nutrient medium (Shenk & Hildebrandt, 1972; Michelmore and Eash, 1985). Introduction

Lettuce (Lactuca sativa L.) is a high value vegetable crop species. Several breeding objectives involve single gene traits (Ryder, 1985; Michelmore and Eash, 1985) and may therefore be amenable to manipulation by molecular techniques. A prerequisite for many molecular approaches was the development of a transformation system. Infection by Aarobacterium spp. provides powerful transformation systems for dicotyledenous species; most reports of transformed plants, however, have so far concerned members of the Solanaceae (reviewed in Fraley et al., 1986), although transformed plants are being obtained for non-solanaceous species with increasing frequency. L. sativa is a member of the Compositeae which, while rarely a natural host for Agrobacterium spp., is susceptible to artificial inoculation by A. tumefaciens, k. sativa is also amenable to manipulation in tissue culture and plants can be regenerated readily from explants (reviewed in Michelmore et el., 1985). Regenerabillty is, however, genotype specific and the culture variables have yet to be optimized (Eash and Michelmore, unpublished). This paper describes the development of a transformation system for lettuce mediated by A. tumefaclens. This is a part of our long-term strategy

to clone and manipulate genes for disease resistance (Michelmore et el., 1987). M a t e r i a l s and Methods Plant Material The majority of the studies were made with cv. Cobham Green (Syn. Dark Green Boston; Ferry Morse Seed Co., Modesto, California), because previous studies had shown adequate rates of regeneration with this cultivar (Eash and Michelmore, unpublished). Seeds were surface sterilized by dipping them in 75% EtOH for 15 sec. followed by 60 min in 10% NaOCI (1% available CI?) with a trace of Tween 20 detergent and then two washeg in sterile distilled water. The seeds were then placed (25 to 30 seeds/plate) onto I% agar containing half strength Hoaglands solution (Hewitt, 1966) and 10 ug/ml GA~. Seeds were incubated in the growthroom at 27°C wi~h a 14 h photoperiod (800 lux). After four days the cotyledons were cut into four pieces and used in the experiments. Tobacco Nurse Cultures Many experiments employed a tobacco nurse culture (Rogers et el., 1986). Three ml of a log phase cell suspension culture of Nicotiana olumbaainifolia was pipetted onto plates containing RMNO medium (IAA, 3 mg/l; KIN 0.06 mg/l) two days prior to experimental use and incubated in the growthroom described above. Filter papers (Whatman #2, 9cm) were soaked in liquid RMNO medium and autoclaved. Just prior to the experiment, a filter paper was laid over the nurse cultures. Lettuce explants were then placed on top. Bacterial Strains and Plasmlds The majority of the studies were made with the plasmids pMON120, pMON200, pMON505 and their derivatives in Aerobacterium tumefaciens strain GV3111 containing pTiB6S3 and its derivatives which were kindly supplied by Monsanto Co. (Fraley et al., 1985; Horsch and Klee, 1986; Rogers et al., 1986). pMON200 and pMON505 contained the chimeric NOS.NPTII.NOS gene which can determine resistance to kanamycin in plants. pMON200 is an integrative vector and pMON500 is a binary vector, pMON120 is identical to pMON200 except pMON120 lacks the chimeric NPTII gene. Cultures were grown overnight in Luria broth containing the appropriate antibiotics. Wild type strains of A. tumefaclens. C58 and ACH5, were obtained from C. Kado (U.C. Davis).

* Present address: Agriculture Canada, P.O. Box 457, St. Jean-sur-Richelieu, Quebec, Canada, J3B 6Z8 Offprint requests to: R. Michelmore

440 Experimental Desian and Evaluatlon

Multifactorial studies were conducted to analyze the importance of several experimental variables: bacterial concentration, timing and intensity of selection for antibiotic resistance, media composition, levels of growth regulators, timing of transfers onto different media, presence or absence of a nurse culture, and genotype. In each experiment, each treatment was replicated twice with nine explants in each replicate. Each explant was scored for the amount of callus, leaves, and necrosis and the numbers of shoots with a normal phenotype. Apparently beneficial treatments were repeated in subsequent experiments. Conclusions were based on at least six replications spread over three experiments. Results and Discussion Induction of Galls

The initial experiments were made to confirm the susceptibility of ~. satiya to 4. tumefaciens. Sterile lettuce seedlings were grown in vermiculite and then wounded by squeezing the apex with fine forceps dipped in a bacterial suspension of either wild type C58, ACH5 or GV3111 with pTiB6S3::pMON200 or in a water control. All three inoculations resulted in galls. Galls were most frequently formed with C58. When the galls were analyzed for opines (Rogers et al., 1986), abundant nopaline production was observed with C58, and abundant octopine production with ACH5. Both opines were produced following inoculation with GV3111. This confirmed that the experimental strains were virulent on cv. Cobham Green and indicated that the nopaline synthase (NOS) promoter which was fused to the chimeric kanamycin resistance gene in pMON2DO, was probably active in lettuce. Sensitivitv to Kanamvcin

Explants were plated on callusing medium (SH; IAA I mg/l; KIN 0.5 mg/l) containing varying levels of kanamycin (0, 10, 25, 50, 100, 250, and 500 mg/l). Prolific callus only formed in the absence of kanamycin. Slow, sparse callus grew in the presence of 10 mg/l kanamycin. All other concentrations completely inhibited callus formation. Explants became chlorotic within two weeks on 100 and 250 mg/l kanamycin. Only 500 mg/l kanamycin induced extensive necrosis. Fifty mg/l kanamycin was therefore chosen to provide rigorous selection for kanamycin resistance. C o c u i t i v a t i o n of A. tumefaclens and Cotyledon ExDlants The f i r s t successful t r a n s f o r m a t i o n experiments employed a nurse c u l t u r e of tobacco c e l l s in a s i m i l a r manner t o t h a t described by Horsch e t a l . (1985) f o r the transformation of Petunia sp. Cotyledon explants were soaked in bacterial suspensions for 10 min, blotted on sterile filter paper to remove excess suspension, placed on the nurse cultures, and incubated in the growth room. After 2 days of cocultivation, the explants were plated on SH medium with and without growth regulators (IAA, Img/l; KIN 0.5 mg/l) and with and without kanamycin (50 mg/l). After cocultivation, carbenicillin (500 mg/l) was included in all treatments to inhibit the growth of 4. tumefaciens. Explants in control treatments which were Incubated with no bacteria, callused only on the medium containing growth regulators and no kanamycln. Explants cocultivated with wild type C58, callused profusely on media with and without growth regulators but only in the absence of kanamycin. Explants cocultivated with GV3111 pTiB6S3 pMON200 callused profusely on all media. As a control, explants were cocultivated with GV3111 pTiB6S3::pMON120 which lacks the chimeric NPTII gene; these explants only callused in the absence of kanamycin. Within all treatments, the response of the explants was uniform; in those treatments where callus

formed, it was prolific and occurred along each cut edge. Shoots were only rarely formed as the Ti plasmids contained the hormone auxotrophy genes. These experiments demonstrated that L. satlva could be induced to form prolific callus in the absence of growth regulators and in the presence of normally inhibitory levels of kanamycin by cocultivation with A. tumefaciens containing the appropriate Ti plasmids. Disarmed ~ plasmids, i.e. those with the hormone auxotrophy genes deleted, pTiB6S3SE and pTiB6S3NE (Fraley et al., 1985), then became available and experiments were conducted to optimize conditions for regenerating transformed plants. Previous experiments had shown that maximal regeneration from cotyledons of cv. Cobham Green could be obtained by culturing first on callusing medium (IAA I mg/l; KIN 0.5 mg/l) for 7 days and then on shooting medium (KIN 0.05 mg/l) (Eash and Michelmore, unpublished). After cocultivation, however, this regime resulted in infrequent callus formation and little regeneration in the presence of kanamycin (50 mg/l). The antibiotic seemed to be slowing the development of even the transformed cells. After numerous experiments, the optimal regime appeared to be: 2 days of cocultivation on the nurse culture; 12 days on callus initiation medium (SH; IAA 0.1 mg/l; KIN 0.5 mg/l); then subculturing every 2 weeks on regeneration medium (SH; KIN 0.05 mg/l; zeatin 0.05 mg/l). After cocultivation, all media contained kanamycin (50 mg/l) and carbenicillin (500 mg/l). This regime resulted in confluent callus formation from all explants coincubated with the appropriate plasmids. Callus growth was approximately half as rapid on kanamycin as in the absence of kanamycin. Approximately 10% of the explants formed shoots from 3 weeks onwards when cocultivated with pTiB6S3SE::pMON200. The negative control, cocultivation with pTiB6S3SE::pMON120, was always included and never resulted in callus formation or regeneration in the presence of kanamycin (Fig. I). Preincubation of the explants on the nurse culture prior to cocultivation and cocultivation times of I or 3 days all resulted in lower rates of callus formation and regeneration. Fig. I. Cotyledon explants of lettuce plated on media with and without kanamycin after cocultlvation with A. tumefaciens containing either pMON120 or pMON200.

The nurse cultures were probably beneficial for two reasons. The incubation of the explants on the surface of the filter paper provided an aerobic environment in the presence of large numbers of bacteria and the presence of tobacco cell exudates probably minimized the stress experienced by the lettuce cells. Also, compounds from the tobacco may

441 have stimulated the~LLr_ genes in 4. tumefaciens (Stachel et al., 1985). After the other cultural parameters had been optimized, the importance of the nurse culture was reinvestigated to determine whether the labor involved in maintaining the tobacco suspension cultures and preparing nurse culture plates was necessary. Several transformation experiments were conducted with and without the tobacco nurse cultures. The results were inconsistent. In some experiments the rate of transformation was similar in both treatments; in duplicate experiments, significantly more frequent callus and shoot formation occurred after cocultivation in the presence of the tobacco cells. It was therefore concluded that while the presence of a nurse culture was not absolutely necessary, a nurse culture was beneficial to ensure predictable high rates of transformation. One of the most critical variables seemed to be bacterial concentration. Explants were cocultivated in a range of bacterial concentrations for I, 2 or 3 days. The highest concentration, 3 x 10 bacterla/ml, at any of the three cocultivation times, resulted in submaximal rates of transformation; presumably the plant cells were stressed by such high numbers ~f bacteria. Low concentrations of bacteria (Ixi0 baeteria/ml) resulted in infrequent callus and rare regeneration; presumably insufficient numbers of cells became transformed. Maximal rates of transformation of cv. Cobham Green were obtained by soaking the cotyledons in approximately 5xi0 bacteria/ml for 10 min and cocultivating on the nurse culture for 2 days. Genotypes of ~. s a t ~ a varied in their sensitivity to A. tumefaciens. Several cultivars produced little or no callus and very few shoots under a regime which resulted in high rates of transformation for cv. Cobham Green. The crlsphead cultivars, such as Salinas, Calmar and Vanguard, which are predominant in California, were particularly recalcitrant, even though some can be readily regenerated in the absence of 4. ~ . These cultivars have long, thin cotyledons ~nd frequently became necrotic in the presence of 5xi0 bacteria/ml. Transformation of Winterhaven has now been achieved by stabbing each cotyledon twice with a needle rather than cutting it into segments and by reducing the bacterial concentration a factor of ten. These measures presumably reduced the stress caused by the bacteria. Transformation rates, however, were still lower than with cv. Cobham Green. All the early experiments used pMON200 which is an integrating vector (Rogers et al., 1986). When the binary vector, pMON505 (Horsch and Klee, 1986), became available, its efficiency in transforming lettuce was compared with pMON200. While moderate callus formation and infrequent shoots were obtained with pMON505, the rate of transformation was never as high as with pMON200. This may in part be due to the transformation regime having been developed for pMON200. The difference, however, parallels observations made with these two vectors and tomato (McCormick et al., 1986). Several other binary vectors, pBIN19 (Bevan, 1984), pPMG85/587 (Fillatti et al., 1987), and pAGS112 (Van der Elzen et al.) in strain LBA4404 (Hoekema et al., 1983) were then tested; these resulted in rates of transformation similar to pMON200. The low transformation rates with pMON505 may have been due to plasmid stability problems. R~jeneratlon and C h a r a c t e r i z a t i o n of Transfor~=d Plant~ When shoots had developed on t h e r e g e n e r a t i o n medium to longer than 5 mm, they were excised from the callus, dipped in IAA (10 mg/l), placed on rooting medium (SH; no growth regulators) and incubated in the growthroom. To maintaln the selection for transformed plants, kanamycin (50 mg/l) was included in the medium.

After 2 to 5 weeks, roots began to develop on approximately 65% of the shoots. The base of some shoots callused but did not root. Other shoots became bleached and did not root or grow. These last shoots may have been untransformed escapes or transformed shoots in which the chimeric NPTII gene had become inactive. After roots had developed, plantlets were transferred to vermiculite and incubated in a growthroom at 15°C to prevent bolting. When the plantlets were 6 cM high, they were transplanted into soil mix and grown to maturity in a greenhouse. When the plantlets were transferred to vermiculite, leaf explants were taken and plated on callusing medium with and without kanamycin (50 mg/ml) to confirm transformation. Opine production was also analyzed in sap from these plantlets and from the resultant calli. The majority of plants transformed with pMON200 or pMON505 expressed kanamycin resistance in the callus assay and produced nopaline. Transformation was confirmed by Southern blot analysis. DNA was extracted from leaves of putative transformed plants as described by Landry et al. (1987). Digested DNA was separated electrophoreticallY32transferred to Zetaprobe (Biorad) and probed with P labelled DNA of pMON200 (Fig. 2). Of the 16 plants analyzed, only 2 appear to have single unrearranged insertions. Other hybridization patterns are consistent with multiple independent insertions, tandem insertions or extensive rearrangements of the T-DNA. We are continuing to characterize the integration events. Fig. 2. Southern blot of3½ettuce DNA digested with BamHl, and hybridized to P labelled pMON200. The first two lanes are reconstructions where 10pg would be equivalent to approximately two copies/genome. Lane F contains DNA from an untransformed control plant. F

"

K

Q

Inheritance Transformed (R I) plants were allowed to self and their R2 progeny analyzed for kanamycin resistance. It was not possible to assay for kanamycin resistance by plating seeds onto media containing kanamycin as some germination and growth of untransformed seedlings occurred even on high concentrations of kanamycln (250 mg/I). Media containing the related antibiotic G418 (10 or 15 mg/l; Gibco, Grand Island, N.Y.) gave a clearer distinction between resistant and susceptible progeny; the optimal concentration, however, varied between progeny populations; this was probably due to position effects of T-DNA insertion. Within a progeny population, a wide variation in growth rate of apparently resistant seedlings was observed (Fig. 3); as slow-growing seedlings were approximately twice as frequent as fast growing seedlings, the former were postulated to be hemizygotes and the latter homozygotes for NPTII. This was confirmed when 11 R^ seedlings were allowed to self. Fast-growing z seedlings resulted in non-segregating, kanamycin resistant, R3 progeny; slower-growing seedlings resulted in segregating progeny. Selection on G418 therefore provides a rapid method for enriching for homozygotes from R^ populations and provides an • Z indication of those plants which are expressing the

442 Fig. 3. Seeds of lettuce plated onto water agar containing G418 (15 mg/l). Seedlings of the untransformed control failed to grow. R2 seedlings from a selfed transformed plant, #63, segregated for resistance to G418.

weeks. The segregation of the ability to callus was always unambiguous, even though only sparse callus was formed in some populations (Fig. 4). Segregation in all progeny was consistent with monogenic segregation (Table I) indicating single or linked insertion sites for active NPTII genes even though the Southern hybridization pattern for some of the R i plants had indicated complex integration events. Although only small population sizes were analyzed, all were significantly different from a digenlc segregation ratio. The R I plants frequently had an altered morphology. The R I plants rarely formed a rossette and bolted rapidly. These abnormal morphologies were not, however, displayed in the progeny. Seed set in R I was frequently low probably reflecting the low vigor of the plants. In 30 R2 progeny, no obvious morphological mutants have been observed.

Conclusions highest levels of resistance. Due to the variability in resistance to G418 between progeny populations, inheritance was subsequently using a callus assay. Sterile R2 seedlings were grown for 4 days in the growthroom. Each cotyledon was then excised, halved and plated on callusing medium containing kanamycin (50 mg/l) (Fig. 4). Explants were scored for callus formation after 4 Fig. 4. Explants from R2 seedlings from transformed plants plated onto callusing media contain ng kanamycin, 50 mg/l. Each plate contains 8 sectors; each sector contains explants from a different seedling. Explants from progeny of plant #41 showed a clear segregation of kanamycln resistance after 4 weeks. Explants from progeny of plant #39 show a less prolific response.

Lettuce can now be routinely transformed. Transformation has been achieved with a variety of integrating and binary Ti vectors. Several hundred transformed plants have been regenerated. Transformed seeds can be obtained in only 2-5 weeks longer than the normal llfecycle (80-180 days depending on the genotype and environmental conditions). Several molecular techniques, particularly transposon mutagenesis using heterologous elements, can now be applied to lettuce.

Acknowledgements We thank S. Rogers and R. Horsch (Monsanto Co.), M. Bevan (Plant Breeding Institute), L. Comai (Calgene Inc.), and J. Bedbrook (Advanced Genetic Sciences) for supplying Ti vectors. The financial assistance of the California Iceberg Lettuce Research Board is gratefully acknowledged.

References

Table I.

Inheritance of kanamycin resistance in R2 progeny from lettuce plants transformed with a Ti plasmld carrying a chimeric NPTII gene.

Trans- # of progeny formant + -

011i2a

Integration Events b

I 33 35 39 41 63 65 91 101

0.95 0.21 0.04 0.24 0.23 0.35 0.04 0.13 1.17

not analyzed multiple single complex multiple not analyzed tandem repeat complex not analyzed

41 62 29 25 29 27 25 34 15

18 18 9 10 8 7 9 10 8

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+ = callused on kanamycin; - = no callus on kanamycln. a: Chi values given are for a 3:1 expected ratio; there were no significant deviations from the expected ratio at P = 0.05. Data are also not significantly different from a 2:1 ratio; P = 0.05. b; as indicated bv Southern analysis.

Schenk RV, Hildebrandt AC (1972) Can. J. Bot. 50:199-204. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Nature 318:624-629. Van der Elzen P, Lee K, Townsend J, Bedbrook J (1986) PI. Mol. Biol. 5:149-154.