Scientia Horticulturae 112 (2007) 172175 www.elsevier.

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Improved protocol for Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene CsZCD
Dongliang Qiu a,b,*, Gianfranco Diretto b, Raffaela Tavarza b, Giovanni Giuliano b
a

College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China b ENEA, Casaccia Research Centre, P.O. Box 2400, Rome 00100AD, Italy

Received 7 October 2005; received in revised form 2 September 2006; accepted 5 December 2006

Abstract Improved protocol for Agrobacterium mediated transformation of tomato (Lycopersicon esculentum) Micro-Tom was developed to use in corporation of the carotenoid biosynthetic genes CsZCD (Crocus zeaxanthin 7,8-cleavage dioxygenase). From these experiment, a transformation methodology using explants from cotyledons cultured for 1 day on the medium with zeatin 2 mg/L, IAA 0.1 mg/L, carefully submerged in the Agrobacterium inoculum for 20 min, then concultured with the agrobacterium for 3 days on the same medium, followed by a transfer to the same medium with 500 mg/L cefotaxin for 3 days and then by a transfer to the same medium with 100 mg/L kanamycin and 500 mg/L carabenillin for 68 weeks and resulted in a greater than 20% transformation efciency in the concentration of Agrobacterium OD600 = 0.2 tested. In this transformation method, no feeder layers were used and the subculture media was minimal. Among the Agrobacterium concentrations of OD600 = 0.2, 0.5 and 1.0, the best transformation efciency, 20.87%, was obtained by using OD600 = 0.2, which was signicantly higher than that of OD600 = 1.0. The presence of the inserted target genes was checked using a rapid and efcient PCR test. The protocol was successfully employed in the production of transgenic Micro-Tom tomato containing the carotenoid biosynthesis CsZCD under constructive promoter. This procedure represents a simple, efcient and general means of transforming tomato. # 2006 Elsevier B.V. All rights reserved.
Keywords: Agrobacterium tumefaciens; Improved protocol; Tomato transformation; Carotenoid; CsZCD

1. Introduction Tomato (Lycopersicon esculentum) is one of the most important vegetable crops and a genetic model for improving other dicotyledonous crop plants (McCormick et al., 1986; Ling et al., 1998). In basic and practical studies for tomato improvement, successful transformation is essential. However, tomato transformation is still time consuming (Van Roekel et al., 1993; Frary and Earle, 1996; Ling et al., 1998). Therefore, the development of an efcient tomato transformation method is crucial. The rst report of tomato transformation was by McCormick et al. (1986). Since then there have been
* Corresponding author at: College of Horticulture, Fujian Agriculture and Forestry University, Jianxin Town, Jinshan Street, Fuzhou 350002, China. Tel.: +86 591 83789284; fax: +86 591 83768251. E-mail addresses: qiudl1970@yahoo.com.cn, qiudl1970@hotmail.com (D. Qiu). 0304-4238/$ see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.12.015

numerous publications of transformation in various cultivars of tomato (Chyi and Phillips, 1987; Fillatti et al., 1987; Fischhoff et al., 1987; Delannay et al., 1989; Van Roekel et al., 1993; Agharbaoui et al., 1995; Hamza and Chupeau,1993; Frary and Earle, 1996; Ling et al., 1998; Tabaeizadeh et al., 1999; Vidya et al., 2000; Costa et al., 2000; Hu and Phillips, 2001; Park et al., 2003; Raj et al., 2005; Sun et al., 2006). For example, genotypes vary in their response to specic treatments, and standardization of the various procedures is incomplete. Transformation efciencies have ranged from 6% (Vidya et al., 2000), more than 20% (Park et al., 2003), to 40% (Sun et al., 2006). In spite of these success of tomato transformations, most of the procedures relied on cumbersome either feeder layers (petunia, tomato, or tobacco), time consuming media formulations, or successive subcultures. No simple general procedure for tomato transformation exists. The miniature cultivar Micro-Tom is well suited for largescale mutagenesis owing to its small size, rapid life cycle, and

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easy transformability. The aim of the study was to simplify tomato transformation by avoiding the feeder layers, simplifying media conditions, eliminating frequent media changes in order to simplify the protocol of Agrobacterium mediated transformation of tomato and production of transgenic plants containing carotenoid biosynthetic gene. Tomato is of great interest because variation in the carotenoid biosynthetic pathway can be readily observed by the accumulation of various carotenoid biosynthetic intermediates. CsZCD codes for a chromoplast enzyme that initiates the biogenesis of three major carotenoid derivativescrocetin glycosides, picrocrocin, and safranal. Therefore, this will make Micro-Tom a good tomato type to be used in rst studies in the production of transgenic plants containing CsZCD. 2. Material and methods 2.1. Equipment and consumables Autoclavable ask, Whatman paper (#50, 9 cm) and lter paper wrapped in aluminium foil, deionised H2O, single-use scalpel blades, and other tissue culture equipment were sterilised by autoclaving for 20 min at 121 8C. A high speed refrigerated centrifuges and 50 mL Falcon tube, Falcon dishes (90 mm) as well as sterilised Eppendorf tube were used for transformation. An environmentally controlled Shaker (28 8C) and growth room (25 8C) were essential for Agrobacterium culture and seed germination and explant growth, respectively. 2.2. Plant material Seeds of tomato, L. esculentum cv Micro-Tom were surface sterilised for 30 s in a 70% alcohol and washed with sterilised water for 10 s and then sterilised for 10 min in a 1% hypochlorite solution and washed two times with sterilised water for 30 min before sowing on Medium A (the composition of various media is described in Table 1). Seeds were sown in a Magenta box and germinated at 24 8C during a 16 h light period and 8 h dark period. Cotyledons of half upright seedling were used after 45 days of germination.
Table 1 Composition of the various media MSB5 salts Medium A, 0.5 Sucrose (%) Glucose Agar (Daichin) (%) pH IAA 0.1 mg/L ZR 2 mg/L IBA 0.1 mg/L Cefotaxime 500 mg/L Carbenicillin 500 mg/L Kanamycin (mg/L) 1 N 0.60 5.8 B, 1 3 N 0.60 5.8 + + B1, 1 3 N N 5.8 C, 1 3 N 0.60 5.8 + + + D, 1 N 1% 0.60 5.8 + + + + 100 E, 1 1 N 0.60 5.8 + + 30

2.3. Media recipes, antibiotics and hormones The media MSB5 (M0404 lot 102K23323) were obtained as powders from Sigma Chemical Co., and stored at 28 8C. Sucrose and glucose were stored at room temperature. 2.4. Antibiotic and hormones (Table 1) Kanamycin, carbenicillin, cefotaxime, rifamicin, ZR, IAA, IBA were prepared as stocks in ddH2O and stored at 20 8C. Antibiotics and hormones were ltered serially before storing at 20 8C. The concentration of the stocks were: kanamycin 10 mg/L, carbenicillin 250 mg/L, cefotaxime 50 mg/L, IAA 0.1 mg/mL, ZR 2 mg/mL, and IBA 10 mg/mL. All media component are for 1 L. Media were autoclaved for 20 min at 121 8C. Then stored at room temperature. Prior to use, solid media were melted in a microwave oven and cooled. Antibiotic and hormones were then added and the media used immediately. 2.5. Bacterial strains and plasmids For transformation experiments, one binary vector containing CsZCD, one binary vector containing b-LCY and one Agrobacterium helper EHA105 were used. The binary vectors used in this study contained the nptII gene as selection maker; plasmid was maintained in Escherichia coli and Agrobacterium tumefaciens under kanamycin selection. The Agrobacterium strains used in this study harboured a rifampicin selection maker. Bacteria were grown overnight in LB medium with antibiotic (rifamicin 30 mg/L, kanamycin 100 mg/L), diluted to OD600 = 0.2 and grown to expected OD600 in LB without antibiotics. Bacterial suspensions were centrifuged at 4000 rpm for 15 min in a 50 mL Falcon tube. Bacteria were resuspended in B1 medium, and used for cocultivation experiments. 2.6. Transformation protocol The cotyledons were prepared as follows. The excision of the cotyledons from the seedling was done extremely carefully to prevent the issue from bruising. Isolated cotyledons were cut on the basal and the lateral side only and placed upside up onto Medium B. Approximately 50 explants were placed on a single Petri dish (9 cm) and incubated overnight under the same conditions as described in Section 2.2. The next day explants were carefully submerged in the Agrobacterium inoculum in a Petri dish (9 cm) for 20 min (swing the plate gently). They were blotted dry on sterile paper and transferred to the new Medium B. After 72 h, explants were transferred to plates containing Medium C. After incubation for another 72 h, the explants were transferred to selection Medium D. Every 3 weeks the explants were subcultured to the same medium. After approximately 68 weeks, shoots were excised and transferred to Medium E. Transformation frequency is expressed as the percentage of the number of cotyledons from which shoots

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D. Qiu et al. / Scientia Horticulturae 112 (2007) 172175 Table 2 Effect of constructs on the transformation efciency of tomato Constructs b-LCY CsZCD Number of explants 343 269 Callus (%) 100 100 Number of regenerant 131 84 Transformation efciency 37.33 3.91aA 29.63 3.76bB

were recovered, with regard to the total number of explants inocubated. A transformant is dened as a rooting shoots on medium containing 30 mg/L kanamycin. 2.7. Molecular verication 2.7.1. Rapid DNA extraction Small leaf samples were collected from the regenerants in a 2 mL Eppendorf tube. A single small leaf (about 520 mg) should be sufcient. Non-transformed samples and wild type were collected as control. One ball was added in each tube. The samples were marked and placed under 80 8C for more than 12 h, and homogenized in 800 mL extraction buffer containing TrisHCl 200 mM pH 7.8, NaCl 250 mM, EDTA 25 mM, SDS 0.5%, 2-mercaptoetanol 0.1% and 200 mL phenol:chloroform. Then, the mixture was centrifuged immediately at 15,000 g for 10 min and 600 mL of the superant was transferred to a fresh tube, and 500 mL isopropyl alcohol was added. After 20 min of centrifugation at 20,000 g, the superant was carefully discarded and 400 mL 80% ethanol was added. After gently mixed and centrifuged for 10 min at 20,000 g, the superant was carefully discarded and 50 mL TE containing RNAsi A 100 ng/mL was added, and incubated at 37 8C for 30 min. Finally, 2 mL sample for each 20 mL volume PCR was used and the rest was stored at 20 8C. 2.7.2. PCR reaction The PCR reaction conditions are selected according to primer design. For the CsZCD gene amplication, the primers 50 -GTCGAGTTTCGTGATG-30 and 50 -CCAGTGAATTCCCGATCTAGTAAC-30 was used. The predicted sizes of amplied DNA fragments were 200 bp. Extractions from control plants, as well as from non-transformed bacteria and a sample from the puried plasmid served as positive controls. PCR was carried out in 20 mL volumes containing 20 mM of each dNTP, 50 ng of each oligonucleotide, 0.5 U Taq polymerase, and 2 mL of sample DNA. The reaction mixture was subjected to the following reaction conditions: a 5 min denaturation step at 94 8C, 45 s at 94 8C, 1 min at 48 8C, 45 s at 72 8C, 34 cycles of 45 s at 94 8C, 1 min at 48 8C, 45 s at 72 8C. The amplied fragments were electrophoresed on a 2.0% (w/v) agarose gel, using Tris-borate buffer (containing 1.3 M Tris, 0.7 M boric acid and 24.5 mM EDTA, pH 8.4). The gel was stained with ethidium bromide, photographed under ultraviolet light and the bands analysed.

Small and capital letters indicated 5 and 1% signicant levels at with Duncans multiple range test (DMRT), respectively. Table 3 Effect of concultivation on the transformation efciency of tomato Concultivation Feeder layer No feeder layer Number of explants 346 313 Callus (%) 100 80.7 Number of regenerant 107 63 Transformation efciency 30.73 3.95aA 20.87 3.26bB

Small and capital letters indicated 5 and 1% signicant levels at with Duncans multiple range test (DMRT), respectively.

3. Result and discussion Six to eight weeks after inoculation on the medium with kanamycin and carabenicillin, selected shoots regenerated from the cut surface of the explants, and these were mainly transformed regenerants. CsZCD has lower transformation efciency in Micro-Tom tomato than b-LCY. The transformation efciency of CsZCD is 29.63%, signicantly lower than that of b-LCY, 37.33% (Table 2). Therefore, CsZCD was used to examine the cocultivation condition with and without feeder layer. The transformation efciency with feeder layer was 30.73%, signicantly higher that that without feeder layer, 20.87%. The results reconrm that feeder cells could simulated tomato cell transformation. But the transformation efciency without feeder layer was still over 20%, which could simply be due to that the Micro-Tom is easily transformable cultivar (Table 3), and this method can not only avoid the feeder layers (petunia, tomato or tobacco), but also simplify media condition as compared with the ones of Van Roekel et al. (1993) and Park et al., 2003, eliminate frequent media changes. The number of Agrobacterium cells in the inoculum is considered to be a factor in the efciency of transformation. Among the Agrobacterium concentrations of OD600 = 0.2, 0.5 and 1.0, the best transformation efciency 20.87% without feeder layer was obtained by using OD600 = 0.2 (Table 4). Shoots were separated from the mother explants and induced to root for about 23 weeks which were the main transformants.

Fig. 1. Analysis of the CsZCD gene in regenerated transgenic plants. Agarose gel of PCR-amplied 200 bp CsZCD fragment M: 1 kb marker; 1, 2: untransformed control; 312, 14: independent transgenic plants; 13: empty; 15: wild type; 16: negative control; 17: positive control; NS: no use.

D. Qiu et al. / Scientia Horticulturae 112 (2007) 172175 Table 4 Effect of Agrobacterium concentration on the transformation efciency of tomato Concentration 0.2 0.5 1.0 Number of explants 313 256 283 Callus (%) 80.7 69.2 48.7 Number of regenerant 63 46 46 Transformation efciency 20.87 3.26aA 16.42 3.69abA 14.67 4.25bA

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Small and capital letters indicated 5 and 1% signicant levels at with Duncans multiple range test (DMRT), respectively.

These transformants were conrmed by PCR analysis. Genomic DNA from 13 randomly selected tomato plants was performed by PCR for the presence of the introduced gene. All of these transformants showed the predicted band for CsZCD gene. Even though the bands in 3 lane and 12 lane were very weak (Fig. 1) which might be resulted from the fewer collection of small leaf sample. 4. Conclusion A transforming sequence using explants cultured for 1 day on the medium with zeatin 2 mg/L, IAA 0.1 mg/L, carefully submerged in the Agrobacterium inoculum with OD600 = 0.2 for 20 min, then concultured with the agrobacterium for 3 days on the same medium, followed by a transfer to the same medium with 500 mg/L cefotaxin for 3 days and then by a transfer to the same medium with 100 mg/ L kanamycin and 500 mg/L carabenillin for 68 weeks and resulted in a greater than 20% transformation efciency. This transformation method is simple, repeatable, does not require feeder layers and may become general means of transforming tomato. Acknowledgement This work was supported by China Scholarship Council. References
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