Shoot apical meristem injection: A novel and efficient

South African Journal of Botany 103 (2016) 210–215

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Shoot apical meristem injection: A novel and efficient method to obtain transformed cucumber plants P. Baskaran a, V. Soós b, E. Balázs a,b, J. Van Staden a,⁎ a b

Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal, Pietermaritzburg, Scottsville 3209, South Africa Department of Applied Genomics, Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, H-2462 Martonvásár, Brunszvik u 2, Hungary

a r t i c l e

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Article history: Received 26 January 2015 Received in revised form 9 July 2015 Accepted 10 September 2015 Available online xxxx Edited by PN Hills Keywords: Agrobacterium shooter strain Cucumber Gene transfer Microinjection Shoot apical meristem

a b s t r a c t The objective of this study was to develop a simple and efficient system for the genetic transformation of cucumber (Cucumis sativus L.). This study investigated the factors influencing the efficiency of gene transfer by microinjection of the shoot apical meristem (SAM) with Agrobacterium shooter strain. The SAM microinjected with Agrobacterium cells containing binary plasmid was incubated in co-cultivation medium without plant growth regulators (PGRs) for 2 days in the dark and subsequently transferred to solid bacterial elimination shoot regeneration medium containing MS salts plus 20 g l−1 glucose, 8 g l−1 agar and 300 mg l−1 cefotaxime. Developed transgenic shoots were screened in selection medium containing the antibiotic kanamycin (Km). The efficiency of gene transfer using various germination media and infection conditions was analyzed by root growth in the presence of Km. The present investigations show that the maximum efficiency of gene transfer was sixty-five percent by Km-resistant plants. In the putative transgenic plants the presence of the transgene was confirmed by polymerase chain reaction (PCR). This transformation system was reproducible, suggesting that it could be adopted for other plant species. © 2015 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Cucumber (Cucumis sativus L.) is one of the world's most important vegetable crops and has received much attention as a model plant for research on Cucurbitaceae. Phytopathogenic fungi, bacteria and viruses severely affect and reduce the yield of cucumber worldwide. Breeding systems for disease resistance is one of the most crucial objectives in cucumber cultivation. Conventional breeding of cucumber to improve disease resistance and other horticultural traits is limited by its narrow genetic base and severe incompatibility barriers to related species (Kho et al., 1980). Recombinant DNA technology provides a novel and powerful way to minimize the loss by improving yield and supplementing traditional plant breeding. The transgenic approach provides a plethora of opportunities to genetically manipulate plants across the species barrier thus making it possible to transfer any gene, be it of bacterial, animal or plant origin to the desired crop (Birch, 1997). These efficient genetic engineering tools could be applied to improve the cucumber crop. Agrobacterium tumefaciens-mediated gene transfer is far from routine in many recalcitrant plants, including C. sativus (Rajagopalan Abbreviations: AS, acetosyringone; GM, germination medium; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; IFM1, infection medium 1; IFM2, infection medium 2; Km, kanamycin; LM, liquid medium; MS, Murashige and Skoog medium; NAA, αnaphthaleneacetic acid; PCR, polymerase chain reaction; PGR, plant growth regulator; PPF, photosynthetic photon flux; SAM, shoot apical meristem; SM, solid medium. ⁎ Corresponding author. E-mail address: [email protected] (J. Van Staden).

http://dx.doi.org/10.1016/j.sajb.2015.09.006 0254-6299/© 2015 SAAB. Published by Elsevier B.V. All rights reserved.

and Perl-Treves, 2005; Nanasato et al., 2012). Utilization of this method for gene transfer requires both a susceptibility to infection by A. tumefaciens and an ability to regenerate plants from individual transformed cells via tissue culture (Fang and Grumet, 1990). To establish a successful strategy for practical plant genetic engineering, it is important to develop systems for recovering large numbers of whole plants from primary explants (Vasudevan et al., 2007). The lack of an efficient transformation and regeneration system often limits the use of gene transfer technologies in vegetable crops (Wang et al., 2012). Therefore, the development of a simple and effective approach for gene transfer to tissues amenable for regeneration is of major interest. A shoot meristem-based transformation system uses a strategy to multiply transgenic shoot apical meristem cells and/or germline cells in vitro, which can be reprogrammed in the developmental direction. Transient and/or stable gene expression in cereals has been reported after delivery of DNA into cells via Agrobacterium-mediated transformation (Sticklen and Oraby, 2005; Baskaran and Dasgupta, 2012). The key steps of shoot apical meristems (SAM) tissue culture involve separation of the tissues surrounding the meristem followed by dissection to expose the meristem for gene transfer (Walden et al., 1989). Overall, the SAM is an optimal explant for genetic manipulation of crops because it is easily cultured in vitro, quickly regenerable, competent to genetic transformation, produces plants genetically identical to the parent, and can be sustained in vitro for long periods of time. Recently, SAM has been used for quick regeneration, genetic transformation, clonal propagation and in vitro sustainability in cereals and

P. Baskaran et al. / South African Journal of Botany 103 (2016) 210–215

In vitro germination

-1

211

-1

MS + 30 g l sucrose + 8 g l agar + 100 µM AS

3 day Seedlings

Shoot apical meristem of shoot-tip

Microinjection

Needle + Bacteria + IFM2 containing 100 µM AS and agitation (3 min)

Co-cultivation

MS + 20 g l glucose + 8 g l agar and 100 µM AS

-1

-1

2 day Bacterial elimination

-1

-1

Liquid MS + 20 g l glucose + 300 mg l cefotaxime

10 min Bacterial elimination SM and Shoot regeneration

-1

-1

-1

MS + 20 g l glucose + 8 g l agar + 300 mg l cefotaxime

6 weeks Selection medium

-1

-1

-1

MS + 20 g l glucose, 8 g l agar, 5.0 µM IBA + 25 mg l kanamycin

6 weeks Transgenic plantlets and ex vitro Fig. 1. Steps for transformation by microinjection protocols in C. sativus L. cv. Ashley.

Fig. 2. Gene delivery and plant regeneration using microinjection and Agrobacterium-mediated transformation of C. sativus L. cv. Ashley: A Microinjection on shoot apical meristem (SAM) explant. B Shoot development after elimination of excess Agrobacterium growth during gene transfer by microinjection. C Necrosis of the shoots in 5.0 μM IBA and 25 mg l−1 kanamycin. D Single shoot induction from SAM after microinjection without bacteria. E Killing of the non-Km resistant shoots. F Development of shoots from SAM after bacterial microinjection. G Rooting of kanamycin-resistant shoots from SAM. H Cotyledon explants after bacterial infection. I Shoot regeneration from cotyledon explants after bacterial infection. J Rooting of kanamycin-resistant shoots from cotyledon. K The transgenic plants growing in a growth room and L in the greenhouse. Bar (A–J) = 5 mm.

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Table 1 Standardization of cefotaxime concentration and exposure period for elimination of excess bacterial growth on microinjected shoot apical meristem of Cucumis sativus L. Bacterial elimination LM + Cefotaxime (mg l−1)

Elimination period (min)

SM for bacterial elimination + cefotaxime (mg l−1)

Bacterial growth (%)

Shoots per explant (#)

Phenotype

LM + 100 LM + 200 LM + 300 LM + 400 LM + 300 LM + 300 LM + 300 LM + 300

0 0 0 0 1 10 30 60

SM +100 SM + 200 SM + 300 SM + 400 SM + 300 SM + 300 SM + 300 SM + 300

100 40.3 13.3 6.7 10.0 0 0 0

0 3.2 ± 0.24 a 3.4 ± 0.24 a 3.8 ± 0.27 a 3.2 ± 0.51 a 3.6 ± 0.47 a 3.8 ± 0.51 a 3.8 ± 0.51 a

Normal shoots Normal shoots Normal shoots Normal shoots Normal shoots Normal shoots Hyperhydric shoots Hyperhydric shoots + WCBC

Liquid medium (LM). Solid medium (SM). WCBC = White compact basal callus. Data recorded after 4 weeks of culture. Results are means ± SEM of shoots from 10 replicates per treatment. Means followed by same letters in each column are not significantly different (P = 0.05) using the Duncan's multiple range test.

crops (May et al., 1995; Katageri et al., 2007; Baskaran and Dasgupta, 2012; Sivakumar et al., 2014). For transformation of cucumber, several approaches have been used to improve Agrobacterium-mediated transformation in order to produce a high number of transformants for gene targeting, agronomically important traits and reverse-genetic studies (Rajagopalan and Perl-Treves, 2005; Nanasato et al., 2012; Wang et al., 2012). Nevertheless, difficulties in the transformation of cucumber remain. Accordingly, we aimed to develop efficient rapid cucumber transformation by establishing optimal conditions for Agrobacteriummediated gene transfer through microinjection using a fine needle on SAM of the commercially grown cucumber cultivar ‘Ashley’. 2. Materials and methods 2.1. Plant material, Agrobacterium strain and binary vector Seeds of C. sativus L. (cv. Ashley, lot No: YS 034AA) were purchased from McDonalds Nursery, Pietermaritzburg, South Africa. Seeds were soaked in water for 30 min and then manually dehusked. Seeds were decontaminated with 3.5% sodium hypochlorite solution with one drop of Tween 20 for 15 min, and subsequently rinsed five times with sterile distilled water. Disinfected seeds were inoculated onto germination medium (GM) consisting of MS (Murashige and Skoog, 1962) plus 30 g l−1 sucrose and 8 g l−1 agar. All media were adjusted to pH 5.8 with 0.1 N NaOH before gelling with 8 g l−1 agar and autoclaved at 121 °C for 20 min. The seeds were germinated at 25 ± 2 °C in the dark. Three-dayold shoot apical meristem (SAM) shoot-tip explants were used for optimization of culture media and conditions for gene transfer. The ‘shooter’ Agrobacterium strain (‘ShooterG’) (Mihálka et al., 2003) is a mutant strain inducing extensive shoot formation in tobacco leaf disk transformation. The pRGGneoNsCP binary vector was introduced into ShooterG by triparental mating (Ditta et al., 1980) using the pRK2013 helper vector (Figurski and Helsinki, 1979). Details of binary vector has been described previously (Balázs et al., 2008) for pepper transformation. The resulting shooter strain, ShooterG-NsCP was cultured in 20 ml of YEB medium (Vervliet et al., 1975) containing 100 mg l− 1 kanamycin (Km), 100 mg l−1 rifampicin, and 100 μM of

acetosyringone (AS) shaken (100 rpm) in the dark at 25 ± 2 °C until an optical density at 600 nm (OD600) of 0.5–0.6 was achieved. The bacteria were centrifuged (5000 rpm for 10 min) and resuspended in liquid microinjection medium (MS and 20 g l− 1 glucose) and then shaken (30 rpm) at 25 ± 2 °C for 5 min. This bacterial culture was used for microinjection of SAM and infection of cotyledon explants.

2.2. Optimization of culture media and conditions for gene transfer The bacteria was microinjected (0.5–1.0 mm depth) three times (1– 2 μl) into SAM region of shoot-tip explants from three-day-old in vitro germinated seedlings using a needle (about 0.1 mm) followed by blotting the shoot-tip on filter paper (Whatman No. 1). The microinjected SAM of the shoot-tip was co-cultivated on MS medium containing 20 g l− 1 glucose, 8 g l− 1 agar and 100 μM acetosyringone (AS) for 2 days at 25 ± 2 °C in the dark. The explants were transferred to different bacterial elimination media [LM (liquid medium: MS + 20 g l−1 glucose + 100–400 mg l− 1 cefotaxime) for different elimination periods (0–60 min) followed by transfer to bacterial elimination SM (solid medium: MS + 20 g l− 1 glucose + 8 g l− 1 agar + 100– 400 mg l−1 cefotaxime)] to standardize the cefotaxime concentration and period needed for elimination of excess bacterial growth. The shoots (2.0–2.5 cm) of one-week-old in vitro seedlings were collected and transferred to MS medium containing 20 g l− 1 glucose, 8 g l− 1 agar, 5.0 μM indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA) and 10–50 mg l−1 Km to standardize the auxin and Km concentration for rooting and selection of putative transformed shoots. The chemicals used were of analytical grade (Biolab, South Africa; Oxoid, England and Sigma, USA). The pH of the co-cultivation, bacterial elimination and selection media were 5.8. Different infection conditions along with GM plus AS were tested for optimal infection conditions and germination medium for efficient gene transfer through microinjection. The precise infection conditions and germination media are outlined in Table 3. No growth regulators were applied during the entire tissue culture procedure, except during the rooting of the regenerated shoots of cucumber.

Table 2 Optimization of auxin and kanamycin concentrations for selection medium for gene transfer of Cucumis sativus L. Auxin (μM)

Kanamycin (mg l−1)

Root growth (%)

Number of roots (length in cm and phenotype)

Hardening (%)

5.0 IAA 5.0 IBA 5.0 NAA 5.0 IBA 5.0 IBA 5.0 IBA 5.0 IBA 5.0 IBA

0 0 0 10 20 25 30 50

100 100 100 40 6.7 0 0 0

13.2 ± 0.92 a (N7 white thin root) 9.6 ± 0.51 b (N5 white thick root) 4.8 ± 0.58 d (N10 white thin root) 7.2 ± 0.58 c (N4 white thick root) 2.0 ± 0.45 e (N2 white thick root) 0 0 0

41.3 62.6 28.0 9.3 0 0 0 0

Data recorded after 6 weeks of culture. Results are means ± SEM from 10 replicate per treatment. Proportion of hardening (%) was determined with three replicates of 25 microplants. Means followed by same letters in each column are not significantly different (P = 0.05) using the Duncan's multiple range test.

P. Baskaran et al. / South African Journal of Botany 103 (2016) 210–215 Table 3 Optimization of germination medium and infection conditions for efficiency of gene transfer with kanamycin (25 mg l−1) resistant Cucumis sativus L. Germination medium

Infection condition

Km-resistant rooted

+ AS (μM)

+ AS (μM)

plants

GM GM GM GM GM GM GM + 100 AS GM + 100 AS GM + 100 AS GM + 100 AS GM + 100 AS GM + 100 AS

IFM1 IFM1 + 100 AS IFM1 + 100 AS + agitation IFM2 IFM2 + 100 AS IFM2 + 100 AS + agitation IFM1 IFM1 + 100 AS IFM1 + 100 AS + agitation IFM2 IFM2 + 100 AS IFM2 + 100 AS + agitation

20/105 (19.04%) 32/105 (32.0%) 43/105 (40.95%) 31/105 (29.52%) 45/105 (42.86%) 51/105 (48.50%) 28/105 (26.66%) 41/105 (39.05%) 49/105 (46.66%) 47/105 (44.76%) 59/105 (56.19%) 68/105 (64.76%)

Germination medium (GM). Infection medium 1 (IFM1) = MS with 20 g l−1 glucose. Infection medium 2 (IFM2) = YEB. Infection period = 3 min. Data recorded after 6 weeks of culture. Results are percentage (% = number of Km-resistant shoots rooted/number of shoots inoculated × 100) of Km-resistant rooted plants.

2.3. Gene transfer through microinjection and direct regeneration Microinjection on three-day-old SAM were conducted from optimized culture media and conditions [germination medium (GM + 100 μM AS), infection medium 2 (IFM2: YEB + 100 μM AS + agitation), co-cultivation medium (MS medium + 20 g l − 1 glucose + 8 g l − 1 agar and 100 μM AS), bacterial elimination LM containing 300 mg l− 1 cefotaxime for 10 min and bacterial elimination SM containing 300 mg l − 1 cefotaxime]. The culture was refreshed every 3 weeks for 6 weeks. No exogenous growth regulators were applied in the experiments. The shoots were transferred to optimized selection medium (MS medium plus 20 g l−1 glucose, 8 g l− 1 agar, 5.0 μM IBA and 25 mg l−1 Km). The cultures were maintained at 25 ± 2 °C under a 16 h photoperiod and photosynthetic photon flux (PPF) of 40 μmol m− 2 s−1 provided by cool white fluorescent tubes (OSRAM L 58 W/740, Germany). Non-microinjected and microinjected without bacteria SAM shoot-tips were used as controls. After 6 weeks, Km-resistant rooted plants were separated and washed in sterile distilled water and then blotted on filter paper. The plants were transferred to Magenta® GA-7 Plant Culture Boxes containing 1:1 (v/v) vermiculite:sand mixture and moistened with sterile water. These plantlets were maintained in the growth room (25 ± 2 °C and 16hour light intensity with 40 μmol m− 2 s−1) for 3 weeks followed by transfer to a greenhouse (25 ± 2 °C) under natural photoperiod conditions and a midday PPF of 950 ± 50 μmol m−2 s−1 for acclimatization ex vitro (Fig. 1). Three-day-old cotyledon explants were removed from germination medium (GM + 100 μM AS) and were immersed in infection medium 1 (IFM1: MS medium with 20 g l−1 glucose + 100 μM AS) containing bacterial suspension (OD600 of 0.5–0.6) for 3 min on a shaker (80 rpm). Excess bacteria were blotted off by filter paper and explants

213

then transferred onto co-cultivation medium (MS medium containing 20 g l−1 glucose, 8 g l−1 agar and 100 μM AS) for 2 days at 25 ± 2 °C in the dark. All further experiments were conducted as outlined in the previous protocol. Non-infected cotyledon explants were used as controls. 2.4. Statistical analysis All experiments were repeated three times with 50 explants per treatment. The data were subjected to one-way analysis of variance (ANOVA) using SPSS version 19.0 for Windows (Chicago, USA). Significantly different means were separated using the Duncan's multiple range test (P = 0.05). 2.5. Polymerase chain reaction analysis Genomic DNA extraction [young leaves and shoot-tips of in vitro, hardened off and control plants (control 1: non-microinjection, control 2: microinjection without bacteria and control 3: leaf of one-week-old in vitro seedlings)] of cucumber and PCR analysis of transgenic plants were performed as described previously (Balázs et al., 2008). The polymerase chain reaction was carried out under standard conditions with 30 s denaturation, 30 s annealing and 45 s extension at 94 °C, 60 °C and 72 °C, respectively. The specific primers (NsCP-for: 5′ CATGGACA AATCTGAATCAACCAGTGC 3′ and NsCP-rev: 5′ CGGAATCAGACTGGGA GCACTCC 3′) amplified a 660 bp sequence of the introduced coat protein gene, Ns-CMV RNA 3 clone (Balázs et al., 2008). Three PCR replicates were carried out for each Km-resistant plant per explant to confirm putative transgenic plants (PCR-positive plants). The gene transfer efficiency was calculated as the number of PCR-positive plants/total number of Km-resistant plants × 100. 3. Results and discussion 3.1. Optimal culture conditions and microinjection The SAM explants were used under different culture conditions to determine the optimum conditions for efficient gene delivery by microinjection (Fig. 2A). Among the different concentrations of cefotaxime and elimination periods tested, 300 mg l−1 and 10 min were found to be a suitable concentration and period for elimination of excess bacterial growth after gene transfer by microinjection in cucumber shoot development (Table 1; Fig. 2B). This was concluded to be the minimum concentration and period for bacterial elimination and was selected for transformation studies. Controlling bacterial growth differs with different antibiotics, concentration, cultivars and explant type (Tabei et al., 1998; Nanasato et al., 2012; Wang et al., 2012). Increased elimination periods (N 30 min) caused the tissue to produce hyperhydric shoots (Table 1). The rooting of shoots and hardening varied with auxin and Km concentrations after 6 weeks of culture (Table 2). Km (25 mg l−1) inhibited rooting and with time shoots deteriorated drastically (Table 2; Fig. 2C), indicating minimal inhibitory concentration for

Table 4 Efficiency of gene transfer by microinjection and infection with Agrobacteria of Cucumis sativus L. shoot apical meristem and cotyledon explants. Microinjection and infection condition

Shoots/explant [#]

Km-resistant plant [number of Km-resistant shoots rooted / number of shoots inoculated (%)]

No. of PCR-positive plants

Efficiency (%)

Control: non-microinjected Control: microinjected without bacteria Microinjected with bacteria Control: non-infected cotyledon Bacterial infected with cotyledon

0 0 4.0 ± 0.45 a 0 3.2 ± 0.45 ab

0 0 78/120 (65%) 0 42/120 (35%)

0 0 50.7 ± 0.98 a 0 18.4 ± 1.27 b

0 0 65.0 0 43.8

Data recorded after 6 weeks of culture. Results are means ± SEM from 10 replicate per treatment. Results are percentage (% = number of Km-resistant shoots rooted / number of shoots inoculated × 100) of Km-resistant rooted plants from 10 replicates per treatment. Efficiency = (number of PCR-positive plants / total number of Km-resistant plants) × 100. Means followed by same letters in each column are not significantly different (P = 0.05) using the Duncan's multiple range test.

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MW

1

2

3

4

5

6

MW

7

8

660 bp

MW

660 bp

Fig. 3. PCR analysis of putative transgenic plants. Arrows indicate the amplification of the nptII gene (660 bp). Lane MW molecular weight marker (100 bp ladder), Lane 1 and 2 DNA from shoot-tip of non-transformed shoots (negative control 1 and 2), Lane 3 and 4 DNA from leaf and shoot-tips of transformed plants by Agrobacterium-mediated transformation of cotyledon explants, Lane 5 and 6 DNA from shoot-tip and leaves of a transformed plant by microinjection of SAM explants, Lane 7 DNA from a leaf of a transformed plant after microinjection isolated from adult greenhouse plants, Lane 8 DNA from leaf of one-week old cucumber seedlings (negative control 3).

selection. This concentration of Km varied between and within cucumber genotype and explant type (Tabei et al., 1998; Rajagopalan and Perl-Treves, 2005; Nanasato et al., 2012; Wang et al., 2012). Different germination media (GM) and infection conditions were tested in this study. The GM plus 100 μM AS, IFM2 plus 100 μM AS and agitation was found to be an optimal germination medium and infection condition for gene transfer (Table 3), which favored the penetration of bacteria into cucumber shoot meristems or access to susceptible cells, including the germline (the L1, L2 and L3 layer in shoot meristem). The microinjection treatment with SAM allowed successful transfer and establishment of T-DNA to the cell possibly by inducing production of vir gene-inducing compounds. A similar phenomenon was also reported (Baskaran and Dasgupta, 2012). Optimized germination medium and infection condition were selected for transformation studies.

coat protein gene of cucumber mosaic virus besides kanamycin resistance. Future work will focus on the responses of transgenic plants under cucumber mosaic virus tolerance. In conclusion, the present study succeeded in establishing high efficiency cucumber gene transfer by SAM microinjection using an Agrobacterium ‘shooter strain’. This protocol minimizes the recovery of false positive and chimeric plants by careful explant selection, infection conditions and suitable kanamycin concentration. The presence of the foreign gene in the transformed cucumber was confirmed by PCR. Further study is needed to clarify whether the expression of the gene are transient or stably integrated into the plant chromosome. The presented transformation techniques will contribute not only to the basic study of cucumber but also to the molecular breeding of cucumber and other crop plants using various genetic traits.

3.2. Production of transgenic cucumber

Conflict of interest

The transgenic cucumber from optimized culture media and conditions were produced from microinjected SAM and cotyledon explants (Table 4). Shoots were produced from transformed SAM and cotyledon explants except the controls after 6 weeks (Table 4; Fig. 2D). The shoots susceptible to Km died after 6 weeks of culture (Fig. 2E). However, microinjected SAM produced 4.0 ± 0.45 shoots per explant with 65% being Km-resistant plants (Table 4; Fig. 2F, G), while cotyledons produced 3.2 ± 0.45 shoots per explant with 35% being Km-resistant plants (Table 4; Fig. 2H, I, J). The results indicate that microinjection provides efficient penetration of bacteria into the young actively dividing cells of SAM to increase the efficiency of gene transfer. Accordingly, the choice of explants is essential for cucumber and it plays a key role in transformation success. A similar phenomenon was also reported in cucumber (Nanasato et al., 2012; Wang et al., 2012). In this study, manipulation of meristem by treatment, following gene transfer, induced multiple shoot regeneration from the meristem, which has potential to generate stable transformants. The plantlets were transferred to a vermiculite:sand (1:1 v/v) mixture and subsequently maintained in the growth room and greenhouse (Fig. 2K and L). The transformation was confirmed by PCR for in vitro Km-resistant and Km-resistant adult putative transgenic plants from the greenhouse and control plants. The transgene was successfully amplified from putative transgenic plants (Fig. 3). The efficiency of gene transfer was higher in microinjected SAM than cotyledon explants (Table 4). The results showed that the Km-resistant plants contained the transgene, indicating high stringency of selection. These transformed plants are expected to be resistant against cucumber mosaic virus due to the successful previous work (Balázs et al., 2008) with this shooter strain which bears the

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