Plant Cell Reports

Plant Cell Reports (1993) 12:129-132

9 Springer-Verlag 1993

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants Bruno Delbreil 1, 2 Phifippe Guerche t, and M. Jullien t 1 Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Route de Saint Cyr, 78026 Versailles Cedex, France 2 Laboratoire "in vitro", Jacques Marionnet GFA, 41230 Soings en Sologne, France Received September 8, 1992/Revised version received October 9, 1992 - Communicated by G. Pelletier

Summary. Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens swain harboring 13-glucuronidase and neomycin phosphotransferase II genes. All the lines showed B-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA. Abbreviations: GUS, 13-glucuronidase; X-Glue, 5bromo-4-chloro-3indolyl B-D-glucuronic acid; NPT n, neomycin phosphotransferase II. Key words: Agrobacterium tumefaciens -Asparagus officinalis L. - Somatic embryogenesis - Transformation Introduction

Transformation is now a routine procedure for introducing foreign genes into many plant species including several important crop plants (reviewed by Potrykus, 1991). Traditionally it has been considered that Agrobacteriummediated transformation was limited to dicotyledonous plant species; nevertheless it is now becoming increasingly clear that Agrobacterium can transfer DNA to cells of monocotyledonous plants (Hernalsteens et al. 1984; Schafer et al. 1987; Raineri et al. 1990; Mooney et al. 1991). The limitation in producing transgenic plants from monocotyledonous species still remains in the difficulty of finding a sensitive tissue and in the regeneration of plants from infected explants. The sensitivity of monocotyledonous zygotic embryo tissues to Agrobacteria (Rained et al. 1990; Mooney et al. 1991) and the fact that somatic embryogenesis seems to be the best pathway for regenerating monocots (Ahloowalia I990) are in favour of using somatic embryos for transformation experiments in monocot species. In Asparagus officinalis L. (Liliaceae), somatic embryos can be induced in vitro from several explant sources: Corresnondence to: B. Delbreil t

hypocotyls (Wilmar and Hellendoorn 1968), stems (Reuther 1977), cladophylls (Harada 1973) and cladophyll cell cultures (/ullien 1974). In some cases, Asparagus tissue cultures can produce long-term embryogenic calluses (JuUien 19"14;Delbreil 1992). These embryogenic calluses can be maintained on a hormone-free medium and produce numerous secondary embryos (Delbreil 1992). Asparagus officinalis L. is sensitive to Agrobacteriura tumefaciens. Hernalsteens eta/. (1984) obtained a tumor tissue that grew on hormone-free medium and produced two opines; nopaline and agrocinopine, after inoculating Asparagus stem pieces with a wild-type Agrobacteriura tumefaciens strain, C58. Bytebier et al. (1987) reported T-DNA integration in this tumor line. These authors obtained another tumor line induced by A. tumefaciens strain C58C1 pGV3850::l103neo(dim) and regenerated plants from it. These plants had integrated the A. tumefaciens T-DNA. These two reports showed that Agrobacterium-mediated transformation was possible in Asparagus. The objective of our study was to determine whether long-term embryogenic calluses could be efficiently used for Agrobacterium-mediated transformation, regeneration and multiplication of transgenic Asparagus. Material and methods Somatic embryo cultures. During some embryogenesis induction experiments from different explants of Asparagus oJ~cinalis L such as cladophylls, cells and apices, cultivated first on an inducing medium and then on hormone-free medium, we isolated long-term embryogenic calluses. These embryogeulc calluses were habituated and continuously produced somatic embryos by secondary embryogenesis from the embryo epidermis,which appeared to be of singlecellorigin(Dclbreil 1992). We named these cultures embryogeulc lines. The embryogenic lines were maintained on B medium (Bourgin et al. 1979) in a growth chamber with 16h/day .fluorescent.. light providing 40 to. 70 ,_uE/m2/sec, a t 25QC and 70% relative humidity. Three embryogemc lines LI, L2 and I..3 isolated frc,n three male genotypes were used. L1 and L3 were isolated respectively from two clones C1 and 02 provided by Jacques Marionnet GFA, and L2 from an F1 hybrid (Andreas) provided by INRA. Plants were recovered after embryo germination on B medium and softtransplantation.

130 Agrobacterium strain and inoculation. An A. tumefaciens C58 suspension (p35SGUSINT) (Vancanneyt et al. 1990) was used. Between the border sequences were the following: a chimeric gene encoding the neomycin phosphotransferase II containing the nopaline synthase promoter and terminator, and a chimeric GUS gene harboring an intron and containing the 35S terminator and promoter from cauliflower mosaic virus. The bacteria were grown overnight at 28~ in LB medium (Sambrook et al. 1989) and then washed with B medium. Prior to cocultivation experiments, bacteria were diluted in B liquid medium to an OD600 of 0.6-0.8. Before inoculation, somatic embryos were sieved on various meshes: 0.2, 0.4, 0.8 and 1.6 ram, to separate globular (0.2-0.4 ram) and cylindrical (0.8-1.6 ram) embryos. Somatic embryos, one gram fresh weight, were immersed in 5 ml of the diluted bacterial suspension for 15 min. Embryos were then blotted dry, plated (0.5 gram per dish) on B liquid or solid medium (coculture medium) and cultured for 48 h in the dark. Embryos were then transferred to a B selection medium containing cefotaxime (400 rag/l) and supplemented with kanamycin (100 rag/l). The resistant lines named Rn were maintained by snbculturing monthly on B medium supplemented with kanamycin (100 raM1) and cefotaxime (200 rag0. Histochemicat GUS assay. Histochemical localisation of the GUS genel expression in kanamycm resistant embryogenic lines or regenerated shoots was performed as described by Jefferson (1987) with some modifications. Samples were incubated overnight at 37~ in a 0.5 g/l! solution of X-Glue in 50 mM sodium phosphate buffer (pH 7). DNA isolation and analysis. Genomic DNA was isolated from embryogenic lines or cladophylls according to Dellaporta et al. (1983).i Ten btg of DNA was digested with the restriction endonuclease Hind: III (Bethesda Research Laboratories, UK), separated b y electrophoresis (0.8% agarose (Sigma) gel), blotted and UV-cross' linked to Hybond-N filters (Amersbam, UK). Prehybridization andl hybridization were performed according to standard procedures at 65~ (Sambrook et al. 1989). The 3-2p-labelled probes were synthesized using random oligonucleotide primers and 32p-dCTP as described by Feinberg and Vogelstein (1983). The NPT II gene was detected by using the 1.2 kb EcoR V fragment of pABD 1 as a probe (Paszkowski et al. 1984) and the GUS gene with the 2 kb Pst I/EeoR I fragment of pBI 121 plasmid (Jefferson 1987). The filters were washed 10 min in 6 x SSC (Sambreok et al. 1989) at room temperature, 10 rain in 2 x SSC, 0.1% sodium dodecyl sulphate at 65~ 5 rain in 0.1 x SSC, 0.1% sodium dodccyl sulphate at 65~ Filters were exposed to Kodak X-ARS film for one week with one Quanta HI intensifying screen at -80~

Results

Selection of kanamycin resistant embryogenic lines Preliminary studies indicated that kanamycin at 25 mg/l killed isolated Asparagus somatic embryos. After coculture experiments, resistant embryogenic lines appeared two or three months after the beginning of the selection period. They were characterized by the emergence of white secondary embryos on necrosed primary embryos (Figure 1). We isolated twenty-three kanamycin resistant lines from three embryogenic lines (Table 1). For the L1 line (24 grams inoculated in four manipulations), we obtained 14 kanamycin resistant lines. Six of these lines regenerated only shoots and three others plants after

embryo germination (Table 1). All the kanamycin resistant embryogenic lines (Figure 2B) and plantlets or shoots (Figures 2C,D) regenerated from them, exhibited a positive blue coloration when they were tested for l~glucuronidase activity. The L2 line (2 grams inoculated) produced 8 kanamycin resistant lines and three of these regenerated shoots (Table 1). The L3 line (1 gram inoculated) produced one kanamycin resistant line which regenerated shoots (Table 1). The L2 line seemed to be more sensitive to the Agrobacteria than the two others (Table 1). Embryos and shoots from uninoculated lines did not express GUS activity (Figures 2A,D).

Effects of differentfactors on transformation efficiency In order to optimize the transformation process, we have tested the coculture medium, the selection timing and the embryo age. It appeared that coculture done in liquid medium was less effective than in solid medium for obtaining kanamycin resistant embryogenie lines (Table 2). An early kanamycin exposure (0-7 days after coculture) enhanced emergence of resistant embryos (Table 2). The coculture of globular embryos with Agrobacterium was ineffective in obtaining resistant embryogenic lines in comparison with the coculture of cylindrical embryos (Table 2).

Molecular characterization of kanamycin resistant embryogenic lines and derived plants Southern hybridizations were performed to confirm the presence of the T-DNA from the C58 p35SGUSINT in kanamycin resistant embryogenic lines showing GUS activity and in a plant regenerated from line R4. Embryos and shoots from an uninoculated embryogenic line did not contain either NPT II or GUS sequences (Figure 3). The enzyme Hind III cut at the end of the nos terminator of the NPT II gene and in the flanking plant DNA (see map at Figure 3). Consequently each variant fragment visualized after the use of an NPT II probe corresponded to a single integration event, i.e., one TDNA copy. In the hybridization pattern (Figure 3A), the lines R1, R2, R3 showed two integration events of the NPT II gene respectively of 13 and 10 kb in length, 5.1 and 3 kb, 4.2 and 3.4 kb. Line R4 showed only one fragment of 12 kb. A plant regenerated from the R4 line presented the same integration profile (Figure 3B). The Asparagus genomic DNA was also tested with a GUS probe. Based on the restriction map of the T-DNA region of the p35GUSINT (see map at Figure 3), it could

Table I. Regeneration capacity of kanamyein resistant lines selected after eocultivation of somatic embryos isolated from three ombryogenic lines (L1. [,2. L3) with Agrobacterium tumefaciens C58 (p35SGUSINT).

Embi3rogenic lines L1 L2 L3

Number of kanamycin resistant lines selected

Number of resistant lines regenerating shoots

Number of resistant lines regenerating plants

Transformation rate a

14 8 1

6 3 1

3 0 0

0.6 4 1

a : The transformation rate is the number of kanamycin resistant embryogenic lines produced per gram (fresh weight) of inoculated somatic embryos

131 Table 2. Influence of the cocuhure medium, the stage of embryo development and the timing

of selection on the transformation rate of embryogenic line L1. Embryo stages

Cylindrical Globular

Coculture medium

Transformation ratea 0

timing of selectionb (days) 7 15

30

Liquid Solid

0 1 (4)

0.5 (I)c 2 (8)

0 0

0 0.5 (1)

Liquid

0 0

0 0

0 0

0 0

SoM

a : Transformation rate is the number of kanamycin resistant embryogenic lines produced per gram

kanamycin resistant embryogenic line (arrow) one m o n t h after inoculation of L1 somatic embryos. Bar I cm. Fig.1.

Isolation

of

bfmShweight) of inoculated somatic embryos. : Timing of selection is the time between the coculture period and the beginning of the selection. c : The number of kanamycin resistant lines isolated is indicated in brackets.

Fig.2. Gus expression in different Asparagus explants (A-D). A, uninoculated somatic embryos from L1 line; B, embryos from a kanamycin resistant line (R1) derived from L1 inoculated with Agrobacterium tumefaciens strahl C58 (p35SGUSINT), bar I ram; C, plantlet derived from a kanamycin resistant line (R4), shoot (s) and root meristem (r), bar I mm; D, stems developed from somatic embryos: uninoculated L1 line, R1 a n d R2 kanamycin resistant lines derived from L1 inoculated with Agrobacterium tumefaciens strain C58 (p35SGUSINT), bar I mm.

Fig.3. Southern blots of kanamycin resistant embryogenic Asparaguslines and derived plant (A-C). A: DNA (10 gg) from kanamycin resistant lines was digested by the restriction enzyme Hind Ill and hybridized with an NPT II probe. Lane l, unlnoculated control line (L1); lanes 2 to 5, different kanamycin resistant embryogenic lines isolated from L1, respectively lines R1, R2, R3, R4; lane 6, digestion from DNA corresponding to one copy of the p35GUSINT plasmid. B: DNA (10 p.g) from plants was digested by the restriction enzyme Hind HI and hybridized with an NPT II probe. Lane 1, an mfinoculated plant (C1) oontrol; lane 2, plant regenerated from kanamycin, re.sistant embryogenic line R4 is.olated f.rffmL1; ~ , . C: DNA (10 I~g) from kanamycin resistant lines was digested by the mstncuon enzyme Hind HI ana nyDnmze.a yam a L,u~ pro~e, kane 1, an uninoculated control line (1.1); lanes 2 to 5, different kanamycin resistant embryogenic lines isolated from L1, respectively, lines R1, R2, R3, R4; lane 6, digestion from DNA corresponding to one copy of p35GUSINT plasmid. LB: left T-DNA border, RB: right T-DNA border. t-35S: 35S terminator from cauliflower mosaic virus; p-35S: 35S promoter from cauliflower mosaic vires; t-nos: terminator from nopaline synthase gene; p-nos: promoter from nopaline synthase gene.

132

be predicted that the digestion by Hind HI should give a 2.8 kb internal fragment of the T-DNA containing the GUS gene sequence with the 35S promoter and terminator. All the lines tested with the GUS probe presented a hybridization signal corresponding to a fragment of 2.8 kb in length (Figure 3C). In conclusion, it appeared that the kanamyein resistant lines R1, R2, R3 and R4 have integrated the whole sequence of the T-DNA (Figure 3A,

Acknowledgements. This work was supported by Jacques Marionnet

r

References

Discussion and conclusion

AhloowaliaBS (1990) Somatic embwos in monocots-Thekgenesis and genetic stability. In. Seeds: Genesis of natural and artificial form. Biotechnology~0:InternationalSymposiain Picardy. Amiens 1: 73-84 tsourgin JP, Chupeau Y, Missonler C (1979) Plant regeneration from me~.~phyllprotoplasts of severalNicotiana species. Physiol.Plant. 45:

Kanamycin (100 ms/l) inhibited but did not completely block new embryo formation when somatic embryos were cultured in mass (0.5 gram per dish). Therefore, identification of transformantsrequired repetitive selection on kanamycin for several months. These results were similar to those of McGranahan et al. (1988), who transformed walnut by coculture of somatic embryos and Agrobacterium. The GUS coloration did not show any evident chimeric pattems (transformedand non-transformed cells) in the kanamycin resistant embryos and derived plants. This could be due to the single cell origin of Asparagus secondary somatic embryos (Delbreil 1992). Timing of selection is known to be crucial for efficient recovery of transformed callus (McGranahan et aL 1990). Early kanamycin exposure enhanced the recovery of Asparagus transgenic embryos (Table 2). The cocultured globular embryos did not regenerate kanamycin resistant callus. This could be due to a deleterious effect of the bacterial lysis during the coculture phase on these very young embryos. The transformation rates obtained were low. This poor effiency did not represent a limiting factor in our transformation process because of the great number of embryos used in cocultivation experiments: the embryogenic lines produced about 300 cylindrical embryos per gram of fresh weight. The unique DNA hybridization pattern for each transgenic line confirmed independent insertion events. The hybridization patterns indicated that one to two copies of T-DNA had been incorporated in the embryos comparable to results obtained in dicotyledonous plants (Weising et al. 1988). The number of insertions will be ~. confirmed by progeny analysis. The complete T-DNA sequence, with the GUS and NPT II genes, was integrated in each embryogenic line tested. Only a few transformed embryos germinated, but low rates of germination have often been reported for somatic embryos of Asparagus (Delbreil 1992), and various other species (Gray and Purohit 1991) and seemed to be not especially related to the transformationprocess. This is the fast report of the Agrobacterium tumefaciensmediated transformation of monocotyledonous somatic embryos. The transformation and regeneration of Asparagus offers the opportunity to transfer foreign genetic material which might prove useful for plant improvement, particularly in programs devoted to obtaining phytopathogen resistance, e.g. to Fusarium which causes dramatic reductions in yield. Application of this method to different genotypes will require the

isolation of embryogenic somatic fines for each of them or the transfer of the foreign genes by sexual crossing.

GFA. The authors thank Agnes Vermeulen and Ian Small for reading

theEnglish.

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