biolistic transformation of cucumber using embryogenic

ELSEVIER Plant Science 112 (1995) 197-206

Biolistic transformation of cucumber using embryogenic suspension cultures: long-term expression of reporter genes

J. Schulze”, C. Balkob, B. Zellnera”, T. Kopreka, R. Hinsch”, A. Nerlicha, R.R. Mendel*”

aBotanical Institute. Technical University of Braunschweig, Hwnboldtstrasse I. D-38104 Braunschweig, FRG

bFeakral Centre for Breeding Research on Cultivated Plants, Institute of Stress Physiology and Quality of Raw Materials, Institutsplatz 2, I8190 GroJ-Liisewitz. FRG

Received IO August 1995; revision received 21 September 1995; accepted 25 September 1995

Abstract

The generation of transgenic cucumber (Cucumis sativus L.) plants was achieved by biolistic transformation of a highly embryogenic cell suspension culture using the nptlt and uidA gene. Functional expression of the genes in trans- genie plants was determined by neomycin phosphotransferase and /3-glucuronidase enzyme assays. Southern analysis of DNA isolated from kanamycin-resistant plants confirmed stable integration of the genes as well as multicopy integration and rearrangements. A study of gene expression showed activity of the uidA gene in plants regenerated from kanamycin-resistant calli about one year after bombardment, indicating a high stability of the nonselectable gene.

Keywork Cucumber; Transformation; Particle gun; Transgenic plants; nptll; uidA; Kanamycin; Long term gene expression

1. Intro4luction

Cucumber is one of the most important vegetables in the world although its importance varies strongly from region to region. The most

Abbreviations: 2,4-D, 2,4dichlorophenoxyacetic acid; BAP, benzylaminopurine; nptll, gene for neomycin phosphotransferase; NPTII, neomycin phosphotransferasc; uidA, gene for &glucuronidase, GUS, &glucuronidase.

* Corresponding author, Tel.: +49 531 391 5870; Fax: +49 531 391 8128.

’ Present address: Zoological Institute, Ludwig Maximilian University, Luisenstrasse, 80333 Miinchen, FRG.

limiting factor for the outdoor cultivation of cucumber in Central Europe is its yield instability due to its susceptibility to abiotic stress factors such as low temperatures and drought but also to biotic pathogens such as green mottle mosaic virus, angular leaf spot, fusarial wilt, cucurbit scab and damages caused by root nematodes. Resistances against these stress factors can be found in wild species. However, application of conventional plant breeding techniques is strongly limited because of interspecific incompatibility of Cucumis sativus L. to other species in the genus Cucumis excepting the closely related C. sativus

Ol68-9452/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(95)04261-R

198 J. Schulze et al. /Plant Science 112 (1995) 197-204

var. hardwickii (Royle) Alef. [ 11. Somatic hybridization could be used to circumvent this problem provided that there is an efficient protoplast regeneration system. Another, more favoured method for introducing foreign genes into the cucumber genome, is the use of recombinant DNA technologies. Prerequisites are the identification, isolation and cloning of relevant genes as well as an efficient transformation system.

The Agrobacterium-mediated gene transfer is the most frequently used and most ellicient technique to genetically transform dicot cells. Although pro- cedures for plant regeneration for some species of the Cucurbitaceae have been established [2, for re- view 3 and 41 there are only a few reports on transformation of cucumber [5-71 and muskmelon [g-10] using Agrobacterium tumefaciens or A. rhizogenes.

This paper describes the transformation of cucumber via particle gun using a highly regenerable suspension culture [ 1 I].

2. Material and methods

2. I. Callus induction and culture Seeds of Cucumis sativus I._. cv. ‘Libelle’ were

surface sterilized with 3% sodium hypochlorite for 15 min, rinsed four times with sterile water and placed on MS-medium [12] without hormones. After germination in the dark at 27”C, plants were grown at a light intensity of about 160 PE m-’ s-’ 16 h per day. Leaves of 21 day old plants were used for callus induction on MS-medium supplemented with 6 PM 2,4-D and 2 PM BAP (IC-medium) at 25°C in the dark. Callus was transferred after 3 weeks to new K-medium using the same light regime as for plant growth. Subcultures were carried out every third week by transferring only the friable, yellowish embryogenic callus.

2.2. Suspension culture and plant regeneration About 1.5 g friable, yellowish and embryogenic

callus was inoculated in 25 ml liquid IC-medium using 100 ml Erlenmeyer flasks. Cultures were kept on a rotary shaker at 110 rev./min at 25°C and a light intensity of 45 PE me2 s-’ 16 h per day. After the first week of culture, 10 ml of medium were replaced by fresh medium twice a week.

Fast growing cultures could be divided once a week. Four to six weeks after initiation, suspensions were sieved (1 mm pore size) and plated on solid IC-medium. Two weeks later, growing colonies were transferred to MS with 1 PM BAP (RC- medium) using the same temperature and light conditions as for plant and callus growth. Regenerants were transferred to MS-medium without hormones. Regeneration frequency was determined as number of regenerating colonies after subculture to RC-medium.

2.3. Transformation via particle gun Suspensions were sieved 3 days after subculture

and about 0.5 ml packed cell volume were evenly dispersed onto the surface of a 80 mm diameter lil- ter disc and transferred to IC-medium. One day later the cells were bombarded using the DuPont PDS 1000 He System. DNA was coated onto gold particles (0.4- 1.2 pm diameter, Heraeus) according to Sanford [13]. The distance between flying disk and target tissue was 7.5 cm and the pressure used for shooting was adjusted to 1200 psi. The bacterial reporter genes, neomycin phosphotransferase (nptll) and &glucuronidase (uidA) were used both under control of the 35s promotor (pRT99GUS, kindly provided by R. Tiipfer 1141).

2.4. Selection One and four days after transformation, respec-

tively, the bombarded cell aggregates were transferred to callus maintenance medium supplemented with two different kanamycin concentrations (75 or 100 mg/l) by dividing the filter disks. Two weeks later well-growing colonies were subcultured for regeneration onto RC-medium ac- companied by a simultaneous increase of the kanamycin concentration (150 or 200 mg/l). Em- bryogenic calli were subcultured to new regeneration medium plus the appropriate kanamycin concentration every third week. Developing morphogenic structures were transferred for shoot elongation to MS-medium without growth regulators but with the corresponding antibiotic concentration. Well-growing plantlets rooted spontaneously on medium without hormones and kanamycin and were planted into soil.

J. Schulze et al. / Planr Science 112 (1995) 197-206 199

2.5. Enzyme assays GUS-activity was analyzed from young leaves

fluorometrically with 4-methyl-umbelliferyl-/3-D- ghcuronide (CMUG, Biomol) as substrate as well as histochemically with 5-bromo-4-chloro-3- indolyl-&t@ucuronic acid (X-Glut, Biomol) according to Jefferson et al. [15].

NPTII-activity was detected according to Reiss et al. [16] by loading equal amounts of protein. Protein concentration was determined by the method of Bradford [ 171 using the BioRad protein assay.

2.6. Southern hybridization analysis Total DNA was isolated from leaves of putative

transformed and nontransformed plants by the CTAB procedure [18]. Southern blot hybridization analysis was performed as described [ 19,201. The 10 fig high molecular weight DNA, as well as EcoRV- and Hi&II-digested DNA (Boehringer, Mannheim) were electrophoresed in 1% agarose gels (GibcoBRL) and subsequently transferred to Hybond-N+ membranes (Amersham) according to the supplier’s instructions. DNA fragments (uidA 2.0 kb BamHI/E@nI cut from pRT99GUS and nptll 2.2 kb XhoIIHindIII cut from pRTlOONE0) were labelled with 32P (Amersham) using the Random Primed Labelling Kit (Boehr- inger, Mannheim). A 3 h pre-hybridization follow- ed by overnight hybridization was carried out in a hybridization oven (Biometra) at 6Y’C. Filters were washed at 65°C under the following conditions: 2 x 10 min 2 x SSPE, 0.1% SDS; 2 x 10 min 1 x SSPE, 0.1% SDS and 2 x 10 min 0.1 x SSPE, 0.1% SDS and subjected to autoradiography using Kodak X-OMAT AR films.

3. Results

3.1. Ceil culture and plant regeneration Callus formation from leaves of 3 week old in

vitro-grown plants was visible 10 to 14 days after incubation. Application of the growth regulators 2,4-D and BAP led to the formation of a yellow- white and nodular callus. During the following subcultures on induction medium (in general 8 to 10 weeks after starting the culture) a fast pro-

liferating, friable and embryogenic callus developed suitable for establishment of suspensions. Fast growing suspensions were observed 4 to 6 weeks later (Fig. 1) consisting of single cells, small aggregates of isodiametric cytoplasm-rich cells as well as bigger cell clumps. To ensure a relatively homogenous cell population for transformation, suspensions were always sieved before plating. Plating on the same medium as used for suspension culture resulted in the proliferation of embryogenic callus which could be transferred two weeks later to RC-medium in order to induce regeneration (Fig. 2). Frequency of shoot regeneration was 70% 8 weeks after starting suspension culture, ranging between 27% and 47% after 12 weeks depending on the cell line. Mor- phogenic capacity decreased with age of suspension. Six months after suspension initiation, only occasional regeneration was observed. Plantlets could be rooted on hormone free MS-medium without problems.

3.2. Transformation and selection In preliminary experiments, the effect of

kanamycin on growth of plated nontransformed suspensions was investigated in a range of 25 to 100 mg/l. At 75 mg/l kanamycin, only a few colonies were observed but no growth could be detected at 100 mgil.

Selection was started 1 or 4 days post bombardment using 75 or 100 mg/l kanamycin. After 8 days to 2 weeks on selection medium, several small kanamycin-resistant colonies were visible on the filter. These colonies were picked and continued to proliferate on RC-medium supplemented with higher concentrations of the antibiotic (150 and 200 mg/l). Two to three weeks later, first plantlets could be separated. Over a period of 6 months, 189 structures were formed from which 34 developed into plantlets finally resulting in 28 vigorously developed and rooted plants suitable for transfer into soil (Fig. 3). A total of 65% of all kanamycin resistant plants arose from the selection starting 4 days after bombardment, and there was no signifi- cant difference in the number of selected plants using 150 or 200 mg/l kanamycin.

The plants growing well on selection medium could be rooted without problems. Putative trans-

200 J. Schulze et al. /Plant Science I12 (1995) 197-206

J. Schulze et al.. /Plant Science! Il.2 (1995) 197-206 201

Table I GUS expression in plants regenerated from calli, grown for about 1 year on selection medium

Plant no. Callus age before plant regeneration (days)

Plant development NPT activity GUS activity (nmol 4-MU h-t mg-’ protein)

Control 349 Vigorous plant 0.9 127 277 Vigorous plant +++ 2.1 132 329 Plantlet +++ 70.2 133 349 Vigorous plant +++ 2.1 134 366 Vigorous plant +++ 28.7 135 366 Plantlet +++ 18.7 136 366 Vigorous plant + 16.6 141 376 Vigorous plant ++ 5.7 142 376 Vigorous plant +++ 11.5

-, no activity. +, weak activity. ++, medium activity. +++, high activity.

formed plants were transplanted successfully to soil and cultured in a growth chamber. The trans- genie plants looked normal, one of the plants had shorter than normal internodes. After selling, fruits developed (Fig. 4), but none of the harvested fruits contained seeds.

3.3. Analysis of transformants Enzyme analyses of foreign gene expression was

carried out in all selected plants. &Glucuronidase activity was shown in 67% of the kanamycin- resistant plants using the fluorometric assay. His- tochemical staining revealed GUS-activity in leaves, stems, roots (Fig. 5) and petals. In plants regenerated from kanamycin grown calli about 1 year after bombardment, activity of the nonselectable uidA gene could also be detected at a high percentage (Table 1). All selected and well- developed plants were checked for NPT-activity

and were found to be positive (Fig. 6) thus yielding a transformation efficiency of about four plants per bombardment (0.5 ml packed cell volume). As expected, there were transformants with a strong NPT-signal and only low (plant no.13j) or no (plants no.127 and 21) GUS-activity and on the other hand plants with a weak NFT-signal and strong GUS-activity (no. 16). Some randomly chosen plantlets, which did not reach the stage of a vigorous plant, gave a very strong NPT-signal. The high expression of the foreign gene could be the reason for stationary and/or abnormal growth and finally the death of these plantlets.

Evidence for the presence of the introduced nptZZ and uidA gene in the genome of the kanamycin-resistant plants was given by Southern hybridization (Fig. 7). Genomic DNA isolated from NPT-positive and control plants was digested with the enzymes EcoRV and Hi&III,

Fig. 1. Suspension culture of cucumber 8 weeks after initiation suitable for plating.

Fig. 2. Morphogenic capacity of suspension culture 4 weeks after plating.

Fig. 3. Regenerated cucumber plants from bombarded (right) and nonbombarded (left) suspension-derived calli.

Fig. 4. Transgenic cucumber plants in the growth chamber, developing fruits.

Fig. 5. Histcchemical expression of GUS activity in leaf, stem cuttings and roots of a transgenic cucumber plant.

202 J. Schulze et al. ! Plant Science 112 (1995) 197-206

neg.Contr. PlantNol3j PlantNol6 PlantNo PlantNo PlantNo b PlantNol27 PlantNo

0.9 7.5 42.5 1.2 61 11.6 2.1 16.6

Fig. 6. Enzymatic analyses of foreign gene expression from leaf extracts of kanamycin-resistant plants. Upper row: NPTII activity of a negative control and seven transformed cucumber plants as determined by loading equal amounts of protein, 3 h film exposure. Bottom row: Values of GUS expression (nmol 4-MU h-’ mg-’ protein) of the same plants.

respectively. ZZin~II releases a fragment of 2.6 kb (r&4) and a fragment of variable size (minimum 2.8 kb, nptZZ) reaching into the flanking plant DNA, whereas EcoRV excises two internal (0.35

and 0.8 kb) and one flanking (> 1 kb) uidA- fragments and a 2.7 kb nptZZ-fragment. The uidA and nptZZ probes did not show any hybridization to DNA from the control plants. Undigested DNA

Fig. 7. Integration of the uidA and npfll genes in the genome of three transgenic plants as analyzed by Southern hybridization. A IO cg aliquot of undigested genomic DNA (4 left lanes) and of EcoRV- (middle lanes) as well as HindIIIdigested (right lanes) genomic DNA were probed with a 32P-labelled 2.0 kb GUS-Hi&III fragment of pRT99GUS (Panel A) or a 32P-labelled 2.2 kb XhoIIHindIII cut fragment of pRTlOONE0 (Panel B). Lanes 3-5: genomic DNA of a control plant (left undigested, middle EcoRV and right Hitill restricted).

J. Schulze et al. /Plant Science 112 (1995) 197-206 203

Fig. 8. Southern blot analysis of Hi&JlJ-digested DNA from kanamycin-resistant cucumber plants and a control plant (NC). Plants no. 72d and 72e arose from an individual callus, plant no. 71b and 127 were regenerated from a single callus 113 days and 277 days after bombardment, respectively, and plant no. 136 was recovered from callus 1 year after bombardment. DNA was probed with a 32P-labe&d 2.0 kb GUS-Hin&JJ fragment of pRJ-?BGUS.

of the plants selected hybridized only in the high molecular region indicating integration of the introduced foreign DNA into the plant genome. Hy- bridization of the filters with the radiolabelled uidA and nptll probes indicated the expected patterns. Plant no. 16 shows a multicopy-integration and rearrangements. The hybridization signals of plants no. 21 and 29 differ strongly, thus verifying individual transformation events. DNA from plants which were regenerated from single calli (plant no. 13a, 13j and 13k or 72d and 72e) show the same hybridization pattern (Fig. 8).

Furthermore, it should be emphasized that plants regenerated from an individual callus 3 and 9 months after bombardment (no. 71 b and 127) show the same integration pattern (Fig. 8) in-

dicating a high mitotic stability of the foreign genes at callus level.

4. Discussion

The experiments reported here demonstrate for the first time the microprojectile-mediated transformation of cucumber cells and the regeneration of stably transformed plants employing a highly regenerable suspension.

To evaluate the utility of kanamycin as selective agent, the basal level of tolerance of the embryo- genie cucumber suspension to kanamycin was determined. A growth retardation of plated cell aggregates was observed at 75 mg/l and no growth was visible at 100 mg/l kanamycin. Using the antibiotic in a range of 75 to 100 mg/l, a high number of escapes in Agrobacterium-mediated cucumber transformation [5,7] as well as muskmelon [8-lo] was reported although a complete inhibition of development in control experiments was found. Therefore, we raised the concentration to 150 or 200 mg/l kanamycin, respectively, in the second selection cycle in our system.

Calli growing well at the elevated antibiotic concentration gave rise to numerous morphogenic structures, 15% of which developed into vigorously growing plants. There was no indication for a negative influence of the antibiotic on the regeneration capacity as described in other plant transformation systems [21-231. Plants still could be regenerated from kanamycin-resistant calli 11 to 12 months after bombardment (plant no. 127 to 142) displaying no morphological abnormality. The relatively high amount of structures showing no further development in the next selection cycles could be due to escaping from the lower kanamycin concentration at the beginning.

All selected plants were proved to be NPT- positive and no escapes could be detected. In fact, we selected for highly expressing lines by our selection conditions. Cointegration efficiency for the linked unselectable uidA gene was 67%. A similar value was found in plants regenerated about one year after bombardment from kanamycin-resistant calli indicating a high stability of the nonselectable gene in a few callus lines. Until now, long term GUS-expression was only observed for callus lines

204 J. Schul:e et al. /Plant Science 112 (19951 197-206

of perennial ryegrass [24] and yam [25] arising [32], wheat 1331 and oat [34]. Using protoplasts for from biolistic transformation of suspension DNA uptake and stable transformation, sterility

cultures. was reported in maize [35] and rice [36]. Southern analysis performed on I2 transformed

cucumber plants confirmed the integration of both reporter genes in the cucumber genome revealing complex as well as simple integration patterns. Plants regenerated from different calli also differed in their integration pattern whereas plants developed from individual calli showed the same pattern indicating, that the callus developed from a single transformed cell. The pattern of plant no. 21 showing two fragments larger than 2.6 kb when probed with uidA, suggests that integration into the plant genome occurred by opening the plasmid inside the uidA gene resulting in a nonfunctional gene. In fact, no GUS-activity could be detected in this plant. Concerning NPT-activity, plants no. 21 and 29 gave strong signals arising from the inser- tion of several intact gene copies. In case of plant no. 16, a multicopy-integration was detected, nevertheless, there was only a weak NPT-activity but a very strong GUS-activity. On the contrary, for plants no. 71b and 127 showing a weak or no GUS-expression, a multicopy-integration was visualized. Multiple copy integration and rearrangements leading to absence or weak expression after microprojectile-mediated gene transfer has also been reported for soybean callus [26], trans- genie callus lines of tomato [27], red fescue [28] and barley [29] suggesting that deletions, methylation or co-suppression between multiple transgene copies have occurred [30,31]. With respect to the integration pattern using Agrobacterium-mediated transformation in the Cucurbitaceae, single copy (61 as well as multicopy-integration [8] were found, whereas rearrangements were rarely observed.

In the experiments reported here, the relatively long in vitro phase, s+ ess by shooting and, in par- ticular, using a high election pressure seem to be the main obstacles in getting fertile plants. The loss of fertility appears to be primarily associated with time elapsed for establishing suspensions, selection of transformed calli and induction of regeneration, all together giving possible rise to genetic and cytogenetic variations. Antibiotics used for selection, such as kanamycin, were also thought to cause detrimental effects leading to sterility. For Cucumis sativus, a reduced fertility and no data on progeny were reported by Trulson et al. [5] in-

oculating hypocotyls with Agrobacterium. Chee [6] was successful in showing inheritance of the nptZZ gene to the progeny applying the Agrobacterium- mediated transformation protocol for transfer of cucumber mosaic virus coat protein [37].

Furthermore, Chee and Slightom [38] also used the biolistic approach to transfer DNA into embryogenic callus of cucumber. To ensure that all transformed plants, especially those which did not contain a functional npt gene, were included in the investigations, these tissues were not subjected to selection. From a total of 107 independently regenerated cucumber plants, only four expressed the foreign gene and 13 contained the gene nonfunctional due to multiple copies and rearrangements. In contrast, our analysis succeeded in a higher number of NPT-expressing plants which could be caused by several factors like the highly regenerable cell line we used or technical conditions like particle coating, bombardment and other constructs used.

All transplanted plants showed a rich flower development, possessed vital pollen as proved by flu- orescein diacetate staining and showed normal fruit development. However, the harvested fruits did not contain any seed while plants regenerated from non-bombarded suspensions were fully fertile. Sterility is frequently observed in generating transgenic plants independent of the transformation method especially when combined with a long cell culture phase. With regard to the biolistic approach, sterile plants have been described in maize

Although the only cultivar used in our transformation experiments was ‘Libelle’, morphogenic suspensions were also established for six out of ten genotypes tested (unpublished data). Furthermore, embryogenic suspensions were described by other groups [39-421. In conclusion, our results show that a highly morphogenic cucumber suspension described by Balko [l l] and transformation by particle gun can be used to obtain a high number of transgenic cucumber plants (four per bombardment). Shortening the time necessary to establish

J. Schulze er al. /Plant Science 112 (I 995) 197-206 205

embryogenic suspension cultures, should result in getting fully fertile plants.

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sativus L. VI. Conditions for cell suspension. Genetica Polonia, 27 (1986) 249-254. A. Callebaut, J.M. Motte, M. Hoenig and H. Baeten, Substrate utilization by embryogenic and non- embryogenic cell suspension cultures of Cucumis sativus L. J. Plant Physiol., 127 (1987) 271-280. P.P. Chee and D.M. Tricoli, Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L. Plant Cell Rep., 7 (1988) 274-277. J.W. H. Bergervoet, F. van der Mark and J. B. M. Custers, Organogenesis versus embryogenesis from long- term suspension cultures of cucumber (Cucumis safivus L.). Plant Cell Rep.. 8 (1989) 116-l 19.

biolistic transformation of cucumber using embryogenic

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