Factors Affecting Agrobacterium Tumefaciens-Mediated Gusa Expression and Opine Synthesis in Gladiolus

Plant Cell Reports (1997) 16: 389Ð392 © Springer-Verlag 1997

K. Kamo Factors affecting Agrobacterium tumefaciens-mediated gusA expression and opine synthesis in Gladiolus

Received: 9 April 1996 / Revision received: 8 July 1996 / Accepted: 27 September 1996

Abstract Five tumorigenic strains of Agrobacterium tu- the frequency of transformation for various Gladiolus tis- mefaciens were used to inoculate corms, cormels, plants sues was not reported, it is not known how susceptible grown in vitro, and seed-derived seedlings of several cul- Gladiolus is to transformation by Agrobacterium. tivars of Gladiolus. Tumors formed on 12% of the plant Binary vectors containing a gusA gene that direct low tissues inoculated, and 1% of these tumors synthesized ei- or undetectable levels of β-glucuronidase (GUS) activity ther octopine or nopaline. A. tumefaciens-mediated β-glu- in A. tumefaciens have been employed to elucidate the fac- curonidase (GUS) expression showed 0.5% and 3.5% GUS tors that affect Agrobacterium-mediated GUS expression expression for plants grown in vitro and regenerable cal- in rice and maize seedlings (Li et al. 1992; Liu et al. 1992; lus, respectively. GUS expression ranged from 40% to 61% Ritchie et al. 1994) and floral stalks of Tulipa sp. (Wilmink when A. tumefaciens was incubated with leaves from seed- et al. 1992). lings grown in the dark, whereas leaves from seedlings In the study reported here, five tumorigenic strains of grown under a 16-h light photoperiod showed no GUS, in- A. tumefaciens and A. tumefaciens containing binary vec- dicating the significant effect of etiolation on transient tors encoding the gusA gene were used to determine pa- GUS expression mediated by A. tumefaciens. rameters that resulted in transient GUS expression in Gladiolus tissues. Key words Floral bulb crops á GUS expression á A. tumefaciens

Materials and methods

Introduction Plant materials and cultures Seeds of a commercially available hybrid were obtained from Glad- Agrobacterium tumefaciens has been used successfully for A-Way Gardens (Union City, Calif.), and Gladiolus carneus seeds the transformation of some monocots including rice (Rai- were obtained from Dr. F. Meyer (Escondido, Calif.). Seeds were neri et al. 1990), Asparagus (Bytebier et al. 1987), Dio- surface-disinfected by washing 10 min in 70% ethanol, 30 min in scorea (Schafer et al. 1987), Anthurium (Kuehnle and Su- 50% (v/v) Clorox with 20 drops/l Tween-20, and then three rinses, 5 min each, in sterile deionized water. Seeds were germinated on gii 1991), and Musa (May et al. 1995). Graves and Gold- Murashige and Skoog’s (Murashige and Skoog 1962) (MS) basal man (1987) reported Gladiolus transformation by A. tume- salts medium solidified with 0.2% (w/v) Phytagel (Sigma Chemi- faciens. Slices of Gladiolus corms were inoculated with cal, St. Louis, Mo.). Inoculations using tumorigenic A. tumefaciens A. tumefaciens, and evidence of transformation was based strains, A208, A281, A348, B6, C58, were performed on seedlings 2Ð3 cm long that had germinated in the dark. Seedlings used for co- upon the presence of nopaline and octopine in the result- cultivation with A. tumefaciens containing binary vectors encoding ing tumors cultured on growth regulator-free medium. As the gusA gene were from seeds that had germinated under a 16-h light photoperiod at 25¡C until the seedlings were approximately 5 cm long. For the etiolation experiments the seedlings were placed in the dark for 7Ð14 days. Mature corms of cv ‘Florida Flame’ (from Walter Preston, Man- atee Fruit Company, Palmetto, Fla.) were surface-disinfected in 15% Communicated by R. N. Trigiano (v/v) Clorox with 20 drops/l Tween-20 for 20 min, followed by three K. Kamo rinses, 5 min each, in sterile water. Floral and Nursery Plants Research Unit, Plants of cvs ‘Peter Pears’, ‘Rosa Supreme’, and ‘Jenny Lee’ B-010A Room 238, BARC West, (from Oglesby Laboratories, Altha, Fla.), ‘Daydream’ and ‘Lemon United States Department of Agriculture National Arboretum, Lime’ (from Beth Lamb, Twyford Plants, Sebring, Fla.), and ‘Florida Beltsville, MD 20705-2350, USA Flame’ were propagated vegetatively in vitro according to Logan 390 and Zettler (1985). Cormels were collected from the plants grown um phosphate buffer pH 5.6, and 100 µM acetosyringone. Seedlings in vitro. were cocultivated with A. tumefaciens 24 h at 25¡C under a 16-h light Regenerable callus was induced and cultured for 6 months on MS photoperiod. medium supplemented with 2.2 µM 2,4-D (Kamo et al. 1990) before Each strain of A. tumefaciens, At648, At644, At645, At646, was it was used for cocultivation. cocultivated with approximately 200 callus, 100 seedling-derived leaf, or 60 seedling-derived root pieces. The same number of pieces for each type of tissue were cocultivated without A. tumefaciens to Tumorigenic A. tumefaciens strains A208, A281, A348, B6, C58: check for background staining of plant tissue. culture, inoculation of and cocultivation with plant tissue, and cell selection GUS expression Tumorigenic A. tumefaciens strains A208, A281, A348, C58, and the non-tumorigenic (lacking a Ti-plasmid) strain A136 were from GUS expression was determined 5 days after cocultivation with Dr. L. Owens (USDA, Beltsville, Md.). A. tumefaciens B6 was from A. tumefaciens. The substrate 5-bromo-4-chloro-3-indoly-β-D-glu- Dr. G. Moore (University of Florida, Gainesville, Fla.). Bacteria were curonide was solubilized to a final concentration of 1 mM in a ster- grown on YEP plates (Chilton et al. 1974), and a single colony was ile solution of 100 mM sodium phosphate buffer, 100 mM EDTA, used to inoculate YEP broth containing 100 µM acetosyringone. 0.3% Triton X-100, and added directly to the tissues in a petri dish. Cells were placed on a gyratory shaker at 25¡C and shaken 16 h at The dishes were incubated at 37¡C for 16 h prior to observation (Jef- ferson et al. 1987). A sample was scored as GUS positive if there 150 rpm. The next morning the bacterial culture (OD600 ranged from 1.0 to 1.5) was used to inoculate plant material. was at least one discrete dark-blue region on the tissue. Surface-disinfected corms and cormels grown in vitro were sliced transversely into approximately 2- to 3-mm sections immediately prior to being dipped into the broth containing A. tumefaciens. Plants grown in vitro were inoculated by piercing the basal meristem re- Results and discussion gion of the plants with a 22-gauge needle, and seedlings were inoculated by piercing the mesocotyl region with a 27-gauge needle, both of which had been dipped into Agrobacterium-containing broth. Opine and tumor formation by A. tumefaciens strains, After a 2- to 5-min incubation with Agrobacterium-containing broth, A208, A281, A348, B6, C58 the corms, cormel slices, seedlings, and plants grown in vitro were blotted onto Whatman filter paper and then placed on solidified MS Tumors formed on all cultivars of the plants grown in vi- basal salts medium. After 2Ð4 days they were transferred to solidified MS basal salts medium supplemented with 1000 mg/l carbeni- tro, but there was no specific strain of A. tumefaciens that cillin and 100 mg/l cefotaxime. Seedlings were transferred to solid- resulted in significantly more tumors on a particular Glad- ified MS basal salts medium supplemented with 200 mg/l carbeni- iolus cultivar (data not shown). cillin and 200 mg/L cefotaxime 2Ð4 days later. Graves and Goldman (1987) previously reported opine Any tumorous tissue that proliferated from the wound site was removed from the explant and cultured by monthly transfers to so- synthesis by Gladiolus corms inoculated with A. tumefa- lidified MS basal salts medium without growth regulators. Sufficient ciens B6, C58, and LBA4013. In this study the tumorigenic tissue from tumor culture was available after 1 year for opine anal- strains of A. tumefaciens, A208, A281, A348, B6 and C58, ysis. were used to inoculate 709 Gladiolus corms and cormels. Octopine was detected in 0.1% of the corms inoculated Octopine and nopaline analysis with A. tumefaciens strain B6. A. tumefaciens transformation of bulbs based upon opine synthesis has been demon- Octopine and nopaline were analyzed according to Otten and strated for Lilium longiflorum (Cohen and Meredith 1992), Schilperoort (1978) and Aerts et al. (1979). Tumor tissue was cul- Allium cepa (Dommisse et al. 1990), and Dioscorea tured 14 days on MS basal salts medium either without or supplemented with 5 mM arginine. About 50 mg fresh weight tissue was (Schafer et al. 1987). homogenized with a mortar and pestle then centrifuged in a micro- The five tumorigenic A. tumefaciens strains used to in- centrifuge tube at 10 000 g for 2 min. Supernatant samples (5 µl) oculate the seedlings resulted in tumor formation, and 0.6% were used for paper electrophoresis (Whatman 3MM, 21 53 cm) at of the seedlings inoculated with strain C58 synthesized 25 mamp and 1500 V for about 1 h. The running buffer was formic acid: acetic acid: water (5:15:80 v/v). Following electrophoresis, the nopaline. Seedlings of maize inoculated with strains B6 paper was air-dried and then dipped in a (1:1 v/v) solution of phe- and C58 also reportedly synthesized opines (Graves and nanthrene-quinone (0.02% w/v in 95% ethanol) and 10% NaOH (w/v Goldman 1986). Stem segments of lily inoculated with in 60% ethanol). The dried chromatogram was visualized using UV strain C58 showed GUS expression (Langeveld et al. light (366 nm), and the Rf values of nopaline, arginine, and octopine 1995). standards compared with spots from tissue extracts.

A. tumefaciens strains carrying a binary vector containing Transient GUS expression in various tissues a gusA gene: description, culture, preinduction, and cocultivation of Gladiolus grown in vitro with plant tissue

The following A. tumefaciens strains used: At644, At645, At646, Parts of the in vitro-grown plants were used for inocula- At648 (Li et al. 1992; Ritchie et al. 1994), At658 and At659 (Liu tion with A. tumefaciens At644, At645, At646, which have et al. 1992) were received from Dr. S. Gelvin, Department of Bio- vir genes on either an agropine, nopaline, or octopine-type logical Sciences, Purdue University, West Lafayette, Ind. All A. tu- of Ti-plasmid, respectively. These strains of A. tumefaci- mefaciens strains used for cocultivation were grown according to Li et al. (1992), except for the following changes. The preinduction ens carry the binary vector pCNL1 that contains the gusA medium consisted of AB salts (Chilton et al. 1974), 1% glucose, gene under the mas/CaMV 35S promoter. A. tumefaciens 20 mM 2-[N-morpholino] ethane-sulfonic acid pH 5.6, 2 mM sodi- carrying pCNL1 has been demonstrated to show a low level 391 of GUS expression depending on the culture medium that the bacteria was cultured on (Li et al. 1992; Ritchie et al. 1994). Low levels of GUS expression by A. tumefaciens carrying pCNL1 occurred when the A. tumefaciens was cultured on either N6 or MS medium solidified with Bacto- agar or when the N6 or MS medium was supplemented with either 1 g/l casein hydrolysate or 20 mM proline. GUS expression was not detected when A. tumefaciens strains At644, At645, and At646 were cultured on MS medium solidified with agarose. A. tumefaciens carrying pCNL1 binary vectors grew better on N6 and MS medium when it was supplemented with casein hydrolysate or proline or solidified with Bacto-agar than on MS medium supplemented with agarose (Liu et al. 1992; Ritchie et al. 1994). In the present experiments with Gladiolus, A. tumefaciens strains At644, At645 and At646 were cocultivated on MS basal salts medium solidified with 0.2% Phytagel. On this medium the plant tissues did not exhibit low levels of GUS expression following histochemical staining. A. tumefaciens strains At644, At645, and At646 grew slowly on MS medium solidified with Phytagel during cocultivation with Gladiolus tissues, and overgrowth of the bacteria was rarely observed. When overgrowth of the bacteria did occur, there was no light-blue staining indicative of low levels of GUS expression by A. tumefaciens strains At644, At645 and At646. The maximum frequency of GUS expression was 0.5% in in vitro-grown plants of cvs ‘Peter Pears’ and ‘Jenny Lee’ inoculated with either At644 or At646. Regenerable callus of cv ‘Jenny Lee’ was inoculated with A. tumefaciens, and the maximum frequencies of GUS expression were 3.3Ð3.5% using A. tumefaciens At644 and At646, respectively, as compared to 0.9% for At645. GUS expression occurred on both organized tissue of the callus capable of regenerating plants as well as on unorganized cells (Fig. 1A). In all tissues grown in vitro there was never any GUS expression when the tissue was incubated without A. tumefaciens or with A. tumefaciens At648, which lacks a Ti plasmid. Fig. 1A, B GUS expression in various tissues of Gladiolus. A ‘Jenny Lee’ callus after inoculation with A. tumefaciens, B etiolated leaves from Gladiolus seedlings 5 days after inoculation with A. tumefaciens Transient GUS expression in seedlings

Seedlings of G. carneus, a wild species, were grown under a 16-h light photoperiod and inoculated with A. tume- ing of Gladiolus tissues and showed the necessity of a Ti faciens At644, At645, and At646, and there was no GUS plasmid for gusA-mediated expression by A. tumefaciens. expression from the roots or leaves inoculated. In com- Similar results were obtained using a commercially avail- parison, leaves of G. carneus seedlings that were grown able hybrid of Gladiolus (Table 1). The ability of A. tume- 7Ð14 days in darkness prior to inoculation with A. tumefa- faciens to infect both a wild species and a cultivated hy- ciens showed a maximum 60Ð61% frequency of GUS ex- brid may have been possible because cultivated plants are pression following cocultivation with At645 and At646, derived from hybridization between only five wild species. respectively, indicating the significant effect of etiolation These results show that transient GUS expression of on A. tumefaciens-mediated gusA expression. GUS was Gladiolus mediated by A. tumefaciens was best achieved expressed as a dark-blue color over large areas of the leaf using A. tumefaciens strains containing either an octopine pieces (Fig. 1B). There was no GUS expression by leaves or nopaline Ti-plasmid for inoculating etiolated seedlings. and roots that were not incubated with A. tumefaciens or It appears that in future studies A. tumefaciens-mediated with A. tumefaciens At648 that lacks a Ti plasmid. This transformation of seedlings may possibly be applied to confirmed the absence of background histochemical stain- many cultivars of Gladiolus. 392 Table 1 GUS expression from either etiolated or non-etiolated Cohen A, Meredith CP (1992) Agrobacterium-mediated transforma- leaves and roots of a Gladiolus hybrid cocultivated with various tion of Lilium. Acta Hortic 325:611Ð618 strains of A. tumefaciens Dommisse EM, Leung DWM, Shaw ML, Conner AJ (1990) Onion is a monocotyledonous host for Agrobacterium. Plant Sci 69: A. tumefaciens Number of Plant Etiolation GUS positive 249Ð257 strain samples tissue (+) samples Graves ACF, Goldman SL (1986) The transformation of Zea mays (% total)a seedlings with Agrobacterium tumefaciens. Plant Mol Biol 7: 43Ð50 None 105 Leaves Ð 0 b Graves ACF, Goldman SL (1987) Agrobacterium tumefaciens-me- None 96 Roots Ð 0 b diated transformation of the monocot genus Gladiolus: detection None 226 Leaves + 0 b of expression of T-DNA-encoded genes. J Bacteriol 169:1745Ð None 154 Roots + 0 b 1746 Jefferson RA, Kavanagh RA, Bevan MW (1987) GUS-fusions: At648 215 Leaves Ð 0 b β-glucuronidase as a sensitive and versatile fusion marker in At648 227 Roots Ð 0 b higher plants. EMBO J 6:3901Ð3907 At648 249 Leaves + 0 b Kamo K, Chen J, Lawson R (1990) The establishment of cell sus- At648 106 Roots + 0 b pension cultures of Gladiolus that regenerate plants. In Vitro Cell Dev Biol 26:425Ð430 At644 105 Leaves Ð 0 b Kuehnle AR, Sugii N (1991) Induction of tumors in Anthurium an- At644 108 Roots Ð 0 b draeanum by Agrobacterium tumefaciens. HortScience 26:1325Ð At644 226 Leaves + 4 b 1328 At644 193 Roots + 0.5 b Langeveld SA, Gerrits MM, Derks AFLM, Boonekamp PM, Bol JF (1995) Transformation of lily by Agrobacterium. Euphytica 85: At645 109 Leaves Ð 0 b 97Ð100 At645 107 Roots Ð 0 b Li X-Q, Liu C-N, Ritchie SW, Peng J-Y, Gelvin SB, Hodges TK At645 235 Leaves + 40 a (1992) Factors influencing Agrobacterium-mediated transient At645 152 Roots + 1 b expression of gusA in rice. Plant Mol Biol 20:1037Ð1048 Liu C-N, Li X-Q, Gelvin SB (1992) Multiple copies of virG enhance At646 105 Leaves Ð 0 b the transient transformation of celery, carrot and rice tissues by At646 110 Roots Ð 0 b Agrobacterium tumefaciens. Plant Mol Biol 20:1071Ð1087 At646 503 Leaves + 51 a Logan AE, Zettler FW (1985) Rapid in vitro propagation of virus- At646 421 Roots + 0 b indexed Gladiolus. Acta Hortic 164:169Ð180 May GD, Afza R, Mason HS, Wiecko A, Novak FJ, Arntzen CJ a Letters indicate significant differences at P≤0.01 by Duncan’s New (1995) Generation of transgenic banana (Musa acuminata) plants Multiple Range Test via Agrobacterium-mediated transformation. Bio/Tech 13:486Ð 492 Murashige T, Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: Acknowledgements Dr. S. Gelvin is gratefully thanked for his ad- 473Ð492 vice regarding this study and Anne O’Connor for providing excel- Otten LABM, Schilperoort RA (1978) A rapid microscale method lent technical assistance. for the detection of lysopine and nopaline dehydrogenase activ- ities. Biochim Biophys Acta 527:497Ð500 Raineri DM, Bottino P, Gordon MP, Nester EW (1990) Agrobacte- rium-mediated transformation of rice (Oryza sativa L.) Bio/Tech References 8:33Ð38 Ritchie SW, Liu C-N, Sellmer JC, Kononowicz H, Hodges TK, Gel- Aerts M, Jacobs M, Hernalsteens J-P, van Montagu M (1979) Induc- vin SB (1994) Agrobacterium tumefaciens-mediated expression tion and in vitro culture of Arabidopsis thaliana crown gall tu- of gusA in maize tissues. Transgenic Res 2:252Ð265 mors. Plant Sci Lett 17:43Ð50 Schafer W, Gorz A, Kahl G (1987) T-DNA integration and expres- Bytebier B, Deboeck F, DeGreve H, van Montagu M, Hernalsteens sion in a monocot crop plant after induction of Agrobacterium. J-P (1987) T-DNA organization in tumor cultures and transgen- Nature 327:529-532 ic plants of the monocotyledon Asparagus officinalis. Proc Natl Wilmink A, van de Ven BCD, Dons JJM (1992) Expression of the Acad Sci USA 84:5345Ð5349 GUS-gene in the monocot tulip after introduction of particle bom- Chilton MD, Currier TC, Farrand SK, Bendich AJ, Gordon MP, bardment and Agrobacterium. Plant Cell Rep 11:76Ð80 Nester EW (1974) DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci USA 71:3672Ð3676