Stable Production of Transgenic Pepper Plants Mediated by Agrobacterium tumefaciens

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Moon Kyung Ko Kumho Life & Environmental Science Laboratory (KLESL), Chonnam National University, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea

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Hyunchul Soh Kumho Life & Environmental Science Laboratory (KLESL), Chonnam National University, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea

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Kyung-Moon Kim Kumho Life & Environmental Science Laboratory (KLESL), Chonnam National University, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea; and Agricultural Plant Stress Research Center (APSRC), Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea

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Young Soon Kim Kumho Life & Environmental Science Laboratory (KLESL), Chonnam National University, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea; and Agricultural Plant Stress Research Center (APSRC), Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea

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Kyunghoan Im Department of Biology, University of Incheon, 177 Dohwa-dong, Nam-gu, Incheon 402-749, Republic of Korea

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Abstract

The aim of this study was to establish a stable transformation method for hot pepper using the hygromycin phosphotransferase (hpt)/hygromycin selection strategy. Explants from aseptic pepper seedlings were inoculated with Agrobacterium tumefaciens carrying pCAMBIA1301. A number of calli were developed on the medium containing hygromycin to discriminate the induction of “false-positive buds,” and then shoots were successfully regenerated from the hygromycin-resistant calli. Southern and Northern hybridization analysis indicated that the hpt gene was integrated and expressed in the transgenic pepper plants (T0) and transmitted to the progeny (T1) without genetic modification. Most T1 progenies derived from self-pollination revealed a 3:1 segregation ratio for hygromycin resistance, indicating that one copy of the T-DNA was integrated into the respective transgenic lines. Both uidA and hpt genes were stably expressed in the T1 generation and coinherited in the progenies. Finally, homozygous progenies were identified in the T1 generation of the transgenic peppers, and the homozygous state was maintained in all progenies tested (T2). The results show the reliability and stability of the hpt/hygromycin selection protocol for pepper transformation.

Peppers are important horticultural crops worldwide, and a hot pepper is cultivated extensively in Northeast Asia. Agronomically important traits have been introduced into the pepper by conventional breeding, but the application is currently limited by the lack of genetic resources or by sexual incompatibility between species. Genetic transformation of the plant has become an important alternative for both basic and commercial plant breeding programs. However, genetic engineering of hot pepper has been hindered by the difficulty in transforming pepper plants. Only a few papers on Agrobacterium-mediated transformation systems have been published (Cai et al., 2002; Lee et al., 2004; Shin et al., 2002; Zhu et al., 1996), and there have been few reports on the reliable inheritance of transformed genes. Therefore, development of more efficient procedures for Agrobacterium-meditated transformation could facilitate routine production of transgenic pepper lines.

In relation to tissue culture of pepper, initial efforts were concentrated on androgenesis and adventitious regeneration through organogenesis (Dumas de Vaulx et al., 1981; Ebida and Hu, 1993; Phillips and Hubstenberger, 1985). The somatic embryogenesis system was also used for in vitro regeneration in peppers but failed to develop further in some Capsicum varieties (Binzel et al., 1996; Steintiz et al., 2003). In organogenesis, highly morphogenic tissues from seedlings exhibit a higher bud induction response, but shoot elongation was often problematic in whole-plant regeneration (Gunay and Rao, 1978). Multiple shoots could develop directly from the explant surface or indirectly through callus derived from explant tissues (Agrawal et al., 1989). However, direct shoots were often laterally fused into leaf-like structures rather than organized into a shoot bud so that the structure was unable to elongate into a normal shoot (Liu et al., 1990; Wolf et al., 2001). Lee et al. (2004) reported that none of the regenerants from direct shooting turned out to be true transgenic plants. In indirect regeneration, shoot development from callus tissues has rarely occurred. So, despite numerous reports on tissue culture, plant regeneration from cultured pepper explants has been hampered, especially in the genetic engineering of pepper.

In transforming the pepper plant, the use of a suitable selectable marker is very important for the efficient selection of transformed event. The neomycin phosphotransferase II (nptII) gene has been previously used as a selection marker for pepper transformation (Cai et al., 2003; Zhu et al., 1996). However, pepper explants show some intrinsic resistance to kanamycin, as shown in some other crops (Mihalka et al., 2000). This causes poor selection of transformed cells, which results in extremely low transformation efficiency. Therefore, an alternative strategy for strict selection of transformed pepper cells is needed to avoid the growth of untransformed escapes. The hygromycin phosphotransferase (hpt) gene has been used in crop transformation as a marker to allow stringent selection of the transformed event (Van den Elzen et al., 1985). Thus, in the present study, the reliability of the use of hygromycin as a selection agent was examined and assessed for its efficacy in pepper transformation.

Once transgenic plants have been established, the transgenes should be stably integrated and expressed over generations. However, the expression level and patterns of transgene inheritance vary widely among transformed plants. Factors responsible for transgene instability include the site of integration in the genome, the transgene copy number, transgene rearrangement, transformation system (Agrobacterium-mediated, microprojectile bombardment, or PEG, and so on), the selection strategy, and the plant tissue culture system (Birch, 1997; Walden and Wingender, 1995).

In the present study, we used the hpt gene in pepper transformation as a selectable marker for the first time. Fertile transgenic plants were regenerated from hygromycin-resistant callus transformed by Agrobacterium-mediated method. Gene expression and stable inheritance of hpt and uidA genes were also analyzed in advanced generations of transgenic progenies.

Materials and Methods

Plant material.

Seeds of a hot pepper (Capsicum annuum L. cv. Nockkwang) were obtained from a commercial source (Heungong Seed Co. Ltd., Ansung, Korea). The seeds were surface-sterilized in 70% EtOH (v/v) for 5 min and in 30% commercial bleach [Clorox (Yuhan Clorox, Incheon, Korea)] for 10 min and then rinsed three times with sterile ddH2O. The surface-sterilized seeds were placed in a magenta box containing MS (Murashige and Skoog, 1962) medium for germination. The seeds were allowed to germinate in the dark at 25 °C. The cotyledons and hypocotyls from 7-day-old seedlings were excised and used as explants for transformation.

Plant expression vectors and preparation of Agrobacterium tumefaciens.

The plasmid vector pCAMBIA1301 (CAMBIA, Clayton Australia), which contains β-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes under the control of the CaMV35S promoter, was transferred into A. tumefaciens strain GV3101. Agrobacterium cells harboring pCAMBIA1301 were grown overnight in LB medium supplemented with 50 mg·L−1 of kanamycin monosulfate and 50 mg·L−1 of rifampicin in a shaking incubator at 28 °C. Cultured Agrobacterium cells were harvested and then diluted with MS medium containing 100 μm acetosyringone (AS) to an optical density of 0.3 to 0.5 at 600 nm.

Transformation and regeneration.

The explants were inoculated with Agrobacterium cells in suspension for 10 min and then blotted on sterile filter papers to remove excess bacterial cells. The infected explants were cocultured in semisolid MS medium containing 100 μm AS in the dark for 48 h. After cocultivation, explants were washed two to three times with 500 mg·L−1 cefotaxime and transferred onto MS medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) plant agar, 2 mg·L−1 zeatin (Duchefa, Haarlem, The Netherlands), 0.2 mg·L−1 indolacetic acid (IAA), 20 mg·L−1 hygromycin, and 300 mg·L−1 cefotaxime. Explants were subcultured onto a fresh medium at 2- to 3-week intervals. For shoot induction, the selected green calli were transferred onto regeneration medium, which was supplemented with 0.05 mg·L−1 IAA, 2 mg·L−1 zeatin, 10 mg·L−1 hygromycin, and 300 mg·L−1. Plantlets resistant to hygromycin were transferred onto rooting medium containing MS basal salts and 300 mg·L−1 cefotaxime without the selectable agent. All cultures were incubated at 24 °C under a 16/8 h (light/dark) photoperiod. Plants having well-developed roots were transplanted to a pot in a greenhouse and allowed to grow until they set seeds.

Histochemical and fluorometric β-Glucuronidase (GUS) assay.

β-Glucuronidase (GUS) activity was histochemically and fluorometrically assayed in plant tissues and organs from transgenic pepper plants. Histochemical staining for GUS activity was based on the method of Jefferson (1987) with a 5-bromo-4-chloro-3-indoyl-β-glucuronic acid (X-gluc) solution as a substrate. The samples were soaked overnight in X-gluc solution at 37 °C. In addition, fluorometric analysis of GUS activity was carried out in the flower, shoot apex, leaf, stem, and root from transgenic lines with the modification of Jefferson (1987). A 100 mg of plant tissue was homogenized in 1 mL extraction buffer containing 50 mm NaPO4 (pH 7.0), 10 μm β-mercaptoethanol, 10 mm EDTA, 0.1% sarcosyl, and 0.1% Triton X-100. Five microliters of supernatant was reacted in the extraction buffer containing 1 mm of 4-methyl-β-D-unibelliferyl glucuronide. The enzymatic reaction was measured by a fluorometer TD-700 (Turner Designs, Sunnyvale, CA) with excitation at 365 nm and emission at 455 nm. The concentration of protein was determined by the Bradford method (Bradford, 1976).

Progeny analysis by hygromycin resistance (leaf assay in hygromycin solution).

Leaf assay in hygromycin solution for the expression of the hpt gene was conducted to distinguish transformed plants from nontransformed plants. Leaf samples from young plants were placed in a 24-well culture plate containing 0.5 mL of 100 mg·L−1 hygromycin or distilled water. Three leaf disks from a wild-type pepper plant at a similar growth stage were used as a negative control. The leaf disks were placed under light at 25 °C for 3 to 5 d. The leaf disks resistant to hygromycin remained green, indicating that the hpt gene was successfully integrated and expressed in pepper. In addition, primary transgenic plants (T0) were self-pollinated and their T1 progeny were germinated in MS medium containing 20 mg·L−1 hygromycin. The data were then analyzed by χ2 test to determine the number of functional hpt gene loci on the pepper genome. T2 progeny were also tested for hygromycin resistance to identify homogeneity.

Southern and Northern blot analysis.

Pepper genomic DNA was isolated by using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) as described by the manufacturer. Fifteen micrograms of each DNA sample was digested with HindIII in 50 μL of the manufacturer's (NEB, Beverly, MA) buffer overnight and separated on a 0.8% (w/v) agarose gel. For blot analysis, DNA was transferred to a Hybond+ nylon membrane and DNA was fixed to the membrane by ultraviolet crosslinking. Prehybridization was carried out at 65 °C for 3 h in Rapid-hyb buffer (Amersham Biosciences, Buckinghamshire, UK) and hybridization was carried out for 20 h at 65 °C in Rapid-hyb buffer with α-32P-labeled probe. The probe DNA consisted of part of the hpt coding region amplified from pCAMBIA1301 and labeled with [α-32P]dCTP using the Random Prime labeling system (Amersham Biosciences). After hybridization, membranes were rinsed with 1X SSPE, 0.2% SDS at room temperature, and washed twice for 45 min each at 65 °C in 0.25X SSPE, 0.2% SDS. The filter was exposed at −80 °C on Kodak XAR-5 film (Kodak, Windsor, CO) with an intensifying screen.

Polymerase chain reaction analysis.

Two primer sets were used on each DNA sample for amplification: 1) a 871-base pair (bp) fragment containing the uidA gene (forward: gaa ggt tat ctc tat gaa ctg tgc gtc; reverse: aag acg cgg tga tac ata tcc agc ca) and 2) a 412-bp fragment containing the hpt gene (forward: gaa gaa tct cgt gct ttc ag; reverse: gtg tcg tcc atc aca gtt t). Polymerase chain reaction (PCR) was performed in the DNA Thermal Cycler (Perkin Elmer, Norwalk, CT) programmed for 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s. PCR products were resolved by agarose-gel electrophoresis.

Results and Discussion

Selection of selectable marker gene and plant explant.

To find a suitable selectable marker gene/agent for pepper transformation, the regenerability of cotyledons was compared on medium containing various concentrations of kanamycin and hygromycin (Table 1). The explants were highly resistant to kanamycin; even 150 mg·L−1 did not inhibit callus induction, and, at 100 mg·L−1 kanamycin, over 80% of cotyledonary explants developed shoot buds. In contrast, the explants were highly sensitive to hygromycin with complete inhibition of callus induction and bud regeneration at 10 mg·L−1 and 5 mg·L−1, respectively.

Table 1.

Determination of antibiotic sensitivity on the growth and shoot development of pepper explants on various concentration of antibiotics.

Table 1.

Several crops are known to be resistant to kanamycin so the nptII/kanamycin system is not applicable for the selection of the transformed cells. In pepper, despite the report that the system resulted in an unacceptably high proportion of escapes (Mihalka et al., 2000), the nptII gene has been mainly used as a selection marker to screen transgenic pepper cells (Lee et al., 2004; Li et al., 2003; Ochoa-Alejo and Ramirez-Malagon., 2001). Our results also show that pepper cells exhibit an intrinsic resistance to kanamycin. Thus, application of kanamycin might not be enough to screen pepper cells carrying the nptII gene during the transformation procedure.

The hpt/hygromycin system has allowed stringent selection of transformed events in cassava (Zhang et al., 2000), sweetpotato (Kimura et al., 2001), cotton (Rajasekaran et al., 2000), and cucumber (Nishibayashi et al., 1996). Therefore, the hpt/hygromycin selection strategy was chosen for pepper transformation in a further experiment. On the basis of the present study, 20 mg·L−1 hygromycin was used at the callus induction stage and 10 mg·L−1 at the regeneration stage, respectively, to select transformed callus and regenerate transgenic shoots.

Transformation of pepper explants.

Cotyledonary explants were infected with Agrobacterium cells harboring pCAMBIA1301 (Fig. 1A). After three to four subcultures of explants onto callus induction medium containing hygromycin, the hygromycin-resistant calli were clearly identified (Fig. 1B) and the rate of callus induction was ≈20%. The explant of the wild type was highly sensitive to hygromycin so that it was necrotized and finally died within three continuous subcultures. Only green calli survived on hygromycin were transferred to the regeneration medium containing hygromycin and within three to four continuous subcultures, adventitious buds developed from green sectors of the calli (Fig. 1D). The hygromycin-resistant shoots were transferred to rooting medium; 1 month later in the rooting medium, nine independent plants were produced (Fig. 1E). In the greenhouse, these plants were phenotypically normal (Fig. 1F) and seeds were obtained by self-pollination.

Fig. 1.
Fig. 1.

Steps for the development of transgenic pepper plants. (A) Cotyledonary explants cocultivated with A. tumefaciens containing pCAMBIA 1301; (B) development of calli from the cutting edge of the infected explant on medium containing hygromycin (20 mg l−1); (C) localization of histochemical GUS activity in developing calli and regenerated shoot; (D) shoot regenerated from the callus; (E) rooting of the regenerated pepper shoot; (F) pepper plants acclimated in pots.

Citation: HortScience horts 42, 6; 10.21273/HORTSCI.42.6.1425

The time required from infection to transfer of plants to the green house was 5 to 7 months. With nine transgenic plants from ≈1500 infected explants, the transformation frequency was ≈0.6%. Although the timeframe was similar to that reported by Lee et al. (2004), this transformation frequency with hpt/hygromycin is higher than the 0.03% to 0.19% reported for nptII/kanamycin in that experiment. We found that the higher transformation frequency is attributable to the use of a stringent selection system resulting in the growth of transformed cells from the early stage. Thus, a pepper transformation system using the hpt/hygromycin selection strategy has the potential to allow fewer escapes and require less effort in terms of time, cost, and labor. Further studies are under progress to increase transformation efficiency of pepper in combination with the hpt/hygromycin selection system. Development of an advanced propagation technology such as somatic embryogenesis will assist in more efficient genetic transformation of hot pepper for biotechnological purpose.

GUS expression in the transgenic peppers.

GUS activity was measured histochemically as well as quantitatively in the primary transgenic pepper plants. Histochemical analysis showed that the uidA gene was constitutively expressed in the primary transgenic event (Fig. 1C) and in all organs, including leaves, floral organ, root, and fruit at different levels (Fig. 2A). This was also supported by fluorometric GUS assays that measured quantitatively in flower, shoot apex, leaves, stem, and root (Fig. 2B). The results indicate that the uidA gene was stably expressed in the transgenic pepper.

Fig. 2.
Fig. 2.

GUS activity in the primary transgenic peppers. Quantitative assays for GUS-specific activity conducted (A) in situ or (B) in extracts of various tissues from a transgenic pepper that carry the uidA reporter gene. (a) Flower; (b) plantlet with root; (c) leaves; (d) fruit.

Citation: HortScience horts 42, 6; 10.21273/HORTSCI.42.6.1425

To observe the GUS activity in the progenies, 21 seeds obtained from a T0 transgenic pepper (no. 2) and the wild type were germinated on MS medium containing 20 mg·L−1 hygromycin. After 2 weeks, all seedlings were evaluated and stained in X-gluc solution for GUS activity. In the transgenic line, 15 of 21 seedlings exhibited resistance to the hygromycin as well as GUS activity, whereas six seedlings did not show either hygromycin resistance or GUS activity (Fig. 3). Wild-type seedlings showed severely retarded growth in the selection medium and eventually died. This result represented that both uidA and hpt genes are transmitted to the next generation and expressed in the progeny.

Fig. 3.
Fig. 3.

Segregation of hygromycin resistance and GUS activity in the transgenic progeny. After germination of 21 seeds harvested from T0 plant (no. 2), 15 seedlings were resistant to hygromycin and GUS positive; the remaining six were not hygromycin-resistant or GUS-positive. As a control, seedlings germinated from wild-type (WT) seeds showed stopping growth, no GUS activity, and no hygromycin resistance.

Citation: HortScience horts 42, 6; 10.21273/HORTSCI.42.6.1425

Segregation analysis of transgenic pepper lines.

To determine whether the transgenes were stably inherited in the next generation, self-pollinated seeds harvested from nine primary transgenic pepper lines were evaluated for the resistance to 20 mg·L−1 hygromycin (Table 2). Hygromycin-resistant and susceptible seedlings were clearly identified within 1 week. A segregation ratio of 3:1 was observed in seven of the nine lines, representing a single functional hpt gene locus in the pepper genome. χ2 analysis indicated a 3:1 segregation for the hpt gene, a ratio that suggested Mendelian segregation of a single dominant gene. The results indicate that transgenic pepper plants produced by Agrobacterium-mediated transformation were genetically and phenotypically stable in advanced generations. However, a non-Mendelian inheritance pattern was also observed in lines 6 and 7, of which the segregation ratio between resistant and sensitive was 10:54 and 82:12, respectively. For line 6, seed development might not be uniform or the introduced transgene might not be functionally expressed. Although we did not carry out Southern blot analysis with line 7, the introduced transgene might be integrated at more than two transgenic loci in the pepper genome. However, this phenomenon is commonly observed in plant transformation research (Christou et al., 1989; De Block et al., 1984).

Table 2.

Segregation of hygromycin resistance in the progeny of primary transgenic peppers.

Table 2.

Molecular characterization of transgenic plants.

Stable integration of the transgenes was further investigated in the primary and T1 progeny of transgenic peppers. Southern blot analysis was conducted with the genomic DNA isolated from the primary transgenic lines 2 and 3, their progenies, and the wild type as a negative control. The genomic DNAs were digested with HindIII and hybridized with a probe consisting of the hpt gene. DNA from the wild-type (nontransformed) plant showed no hybridization signal to the probe DNA (Fig. 4A). Each primary transgenic line showed a single band with a different band pattern, indicating that these two lines represented independent events. Their respective T1 progeny showed a single band with the same band mobility as the parent plant, indicating that the introduced transgene had been successfully transmitted to the progeny without modification. Because the T-DNA of pCAMBIA1301 has a unique HindIII site, the result also represents that a single copy of the transgene was integrated into the pepper genome.

Fig. 4.
Fig. 4.

Southern and Northern blot analyses. (A) Southern blot analysis of two T0 transgenic lines and their T1 progenies. DNA samples from two transgenic lines and an untransformed pepper plant (WT) were digested with HindIII, and the resulting fragments were resolved by electrophoresis and transferred to a membrane. The membrane was hybridized with a 32P-labeled probe DNA corresponding with the hpt coding region. (B) Northern blot analysis of two transgenic pepper lines. WT, wild type; lane 2, line 2 plants (T0); lanes 3–6, T1 progenies from line 2; lane 7, line 3 plants (T0); lanes 8–11, T1 progenies from line 3. The membrane was hybridized with a 32P-labeled hpt.

Citation: HortScience horts 42, 6; 10.21273/HORTSCI.42.6.1425

To confirm that the introduced transgene was stably expressed in the transgenic peppers, Northern blot analysis was carried out with the primary transgenic lines 2 and 3, their progenies, and the wild type. The isolated total RNA was hybridized with hpt probe DNA. At the mRNA level, the introduced transgene was transcriptionally expressed in the two primary transgenic lines as well as their T1 progenies, whereas no signal was detected in the wild type (Fig. 4B). This indicates that the introduced transgenes were stably expressed in the progeny. In general, a single copy of T-DNA insertion results in high levels of transgene expression, whereas multiple copies of transgene expressions may lead to suppression of the chimeric gene in some cases (Van der Krol et al., 1990).

Inheritance of transgenes in an advanced generation (T2).

PCR analysis was performed on the two independent T1 plants and their progenies (Fig. 5). Both the uidA and hpt genes introduced to the pepper genome were revealed in the T1 and T2 generations of transgenic lines 2 and 3, indicating that the transgenes were stably transmitted.

Fig. 5.
Fig. 5.

Transmission of uidA (upper) and hpt (lower) genes to T2 progenies determined by polymerase chain reaction analysis in two independent transgenic pepper lines. 2-1, T1 transgenic pepper; 2-1-1, 2, 5, 6, T2 transgenic progenies from transgenic line 2-1; 3-2, T1 transgenic pepper; 3-2-2, 3, 8, 9, T2 transgenic progenies from transgenic line 3-2; WT, nontransgenic pepper as a negative control; P, pCAMBIA1301 as a positive control.

Citation: HortScience horts 42, 6; 10.21273/HORTSCI.42.6.1425

We investigated whether both of the introduced transgenes, hpt and uidA, were expressed in the T2 generation without segregating or silencing (Table 3). Twenty T2 seeds from lines 2 and 3 were germinated on hygromycin-containing medium and the seedlings were transplanted into pots. Leaf discs were incubated in 100 mg·L−1 hygromycin to test for hygromycin resistance and in X-gluc for histochemical assay of uidA gene expression. All T2 progenies were functionally resistant to hygromycin, whereas nontransgenic plants were bleached or necrotized. In the histochemical GUS assay, leaf samples from all T2 progenies, which had hygromycin resistance, showed GUS activity. Thus, the data demonstrate that hpt and uidA genes were transmitted and stably expressed in the T2 progenies of the two independent transgenic pepper lines.

Table 3.

Inheritance and functional activity of transgenes (hpt and uidA) in homozygous transgenic plants.

Table 3.

To examine the reliability of hpt/hygromycin system in pepper transformation, the sensitivity of pepper explants to hygromycin was compared in transgenic and nontransgenic plants by measuring fresh weight of hypocotyl explants (Fig. 6). With an increasing hygromycin concentration, the fresh weight of hypocotyls was dramatically reduced in wild type. Although untransformed pepper cells were highly sensitive to hygromycin, calli with developing buds were induced in transgenic explants. At over 10 mg·L−1 hygromycin, adventitious healthy shoots were produced only in hypocotyl explants from transgenic plants.

Fig. 6.
Fig. 6.

Growth of hypocotyl explants from nontransformed (WT) and transformed (T) peppers carrying the hpt gene on medium containing 0 to 50 mg·L−1 hygromycin. (A) Callus and shoot formation. (B) Average of fresh weight of 60 explants after 3 weeks of incubation. The bars indicate se.

Citation: HortScience horts 42, 6; 10.21273/HORTSCI.42.6.1425

In conclusion, we report the development of a reliable protocol for production of transgenic pepper plants with the hpt/hygromycin selection strategy. The selection strategy is critical for improving transformation efficiency of pepper. Although the nptII gene was successfully implemented in Agrobacterium-mediated transformation, the nptII/kanamycin selection strategy requires an extended culture in vitro and labor/cost to screen the escapes of nontransformed events. The hpt/hygromycin selection system allows stringent selection of transgenic pepper plants to eliminate or reduce the escape of untransformed or silenced transgenic pepper plants. Using hpt/hygromycin selection strategy, we confirmed stable inheritance of the introduced transgenes into subsequent generations and stable expression of the transgenes in the progenies. Therefore, the hpt marker system described in this study demonstrates its use for an effective transformation of pepper.

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  • Rajasekaran, K. , Hudspeth, R.L. , Cary, J.W. , Anderson, D.M. & Cleveland, T.E. 2000 High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures Plant Cell Rep. 19 539 545

    • Search Google Scholar
    • Export Citation
  • Shin, R. , Han, J.-H. , Lee, G.-J. & Peak, K.-H. 2002 The potential use of a viral coat protein gene as a transgene screening marker and multiple virus resistance of pepper plants coexpressing coat proteins of cucumber mosaic virus and tomato mosaic virus Transgenic Res. 11 215 219

    • Search Google Scholar
    • Export Citation
  • Steintiz, B. , Kusek, S. , Tabib, Y. , Paran, I. & Zelcer, A. 2003 Pepper (Capsicum annuum L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot In Vitro Cell. Dev. Biol. Plant 39 296 303

    • Search Google Scholar
    • Export Citation
  • Van den Elzen, P. , Townsend, J. , Lee, K.Y. & Bedbrook, J. 1985 A chimeric hygromycin resistance gene as a selectable marker in plant cells Plant Mol. Biol. 5 299 302

    • Search Google Scholar
    • Export Citation
  • Van der Krol, A.R. , Mur, L.A. , Beld, M. , Mol, J.N.M. & Stuitje, A.R. 1990 Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to a suppression of a gene expression Plant Cell 2 291 299

    • Search Google Scholar
    • Export Citation
  • Walden, R. & Wingender, R. 1995 Gene-transfer and plant-regeneration techniques Trend. Biotechnol. 13 324 331

  • Wolf, D. , Matzevitch, T. , Steinitz, B. & Zelcer, A. 2001 Why is it difficult to obtain transgenic pepper plants? Acta Hort. 560 229 234

  • Zhang, P. , Potrykus, I. & Puonti-Kaerlas, J. 2000 Efficient production of transgenic cassava using negative and positive selection Transgenic Res. 9 405 415

    • Search Google Scholar
    • Export Citation
  • Zhu, Y. , Ou-Yang, W. , Zhang, Y. & Chen, Z. 1996 Transgenic sweet pepper plants from Agrobacterium mediated transformation Plant Cell Rep. 16 71 75

    • Search Google Scholar
    • Export Citation
  • Steps for the development of transgenic pepper plants. (A) Cotyledonary explants cocultivated with A. tumefaciens containing pCAMBIA 1301; (B) development of calli from the cutting edge of the infected explant on medium containing hygromycin (20 mg l−1); (C) localization of histochemical GUS activity in developing calli and regenerated shoot; (D) shoot regenerated from the callus; (E) rooting of the regenerated pepper shoot; (F) pepper plants acclimated in pots.

  • GUS activity in the primary transgenic peppers. Quantitative assays for GUS-specific activity conducted (A) in situ or (B) in extracts of various tissues from a transgenic pepper that carry the uidA reporter gene. (a) Flower; (b) plantlet with root; (c) leaves; (d) fruit.

  • Segregation of hygromycin resistance and GUS activity in the transgenic progeny. After germination of 21 seeds harvested from T0 plant (no. 2), 15 seedlings were resistant to hygromycin and GUS positive; the remaining six were not hygromycin-resistant or GUS-positive. As a control, seedlings germinated from wild-type (WT) seeds showed stopping growth, no GUS activity, and no hygromycin resistance.

  • Southern and Northern blot analyses. (A) Southern blot analysis of two T0 transgenic lines and their T1 progenies. DNA samples from two transgenic lines and an untransformed pepper plant (WT) were digested with HindIII, and the resulting fragments were resolved by electrophoresis and transferred to a membrane. The membrane was hybridized with a 32P-labeled probe DNA corresponding with the hpt coding region. (B) Northern blot analysis of two transgenic pepper lines. WT, wild type; lane 2, line 2 plants (T0); lanes 3–6, T1 progenies from line 2; lane 7, line 3 plants (T0); lanes 8–11, T1 progenies from line 3. The membrane was hybridized with a 32P-labeled hpt.

  • Transmission of uidA (upper) and hpt (lower) genes to T2 progenies determined by polymerase chain reaction analysis in two independent transgenic pepper lines. 2-1, T1 transgenic pepper; 2-1-1, 2, 5, 6, T2 transgenic progenies from transgenic line 2-1; 3-2, T1 transgenic pepper; 3-2-2, 3, 8, 9, T2 transgenic progenies from transgenic line 3-2; WT, nontransgenic pepper as a negative control; P, pCAMBIA1301 as a positive control.

  • Growth of hypocotyl explants from nontransformed (WT) and transformed (T) peppers carrying the hpt gene on medium containing 0 to 50 mg·L−1 hygromycin. (A) Callus and shoot formation. (B) Average of fresh weight of 60 explants after 3 weeks of incubation. The bars indicate se.

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    • Export Citation
  • Ochoa-Alejo, N. & Ramirez-Malagon, R. 2001 In vitro chili pepper biotechnology In Vitro Cell. Dev. Biol. Plant 37 701 729

  • Phillips, G.C. & Hubstenberger, J.F. 1985 Organogenesis in pepper tissue cultures Plant Cell Tiss. Org. Cult. 4 261 269

  • Rajasekaran, K. , Hudspeth, R.L. , Cary, J.W. , Anderson, D.M. & Cleveland, T.E. 2000 High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures Plant Cell Rep. 19 539 545

    • Search Google Scholar
    • Export Citation
  • Shin, R. , Han, J.-H. , Lee, G.-J. & Peak, K.-H. 2002 The potential use of a viral coat protein gene as a transgene screening marker and multiple virus resistance of pepper plants coexpressing coat proteins of cucumber mosaic virus and tomato mosaic virus Transgenic Res. 11 215 219

    • Search Google Scholar
    • Export Citation
  • Steintiz, B. , Kusek, S. , Tabib, Y. , Paran, I. & Zelcer, A. 2003 Pepper (Capsicum annuum L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot In Vitro Cell. Dev. Biol. Plant 39 296 303

    • Search Google Scholar
    • Export Citation
  • Van den Elzen, P. , Townsend, J. , Lee, K.Y. & Bedbrook, J. 1985 A chimeric hygromycin resistance gene as a selectable marker in plant cells Plant Mol. Biol. 5 299 302

    • Search Google Scholar
    • Export Citation
  • Van der Krol, A.R. , Mur, L.A. , Beld, M. , Mol, J.N.M. & Stuitje, A.R. 1990 Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to a suppression of a gene expression Plant Cell 2 291 299

    • Search Google Scholar
    • Export Citation
  • Walden, R. & Wingender, R. 1995 Gene-transfer and plant-regeneration techniques Trend. Biotechnol. 13 324 331

  • Wolf, D. , Matzevitch, T. , Steinitz, B. & Zelcer, A. 2001 Why is it difficult to obtain transgenic pepper plants? Acta Hort. 560 229 234

  • Zhang, P. , Potrykus, I. & Puonti-Kaerlas, J. 2000 Efficient production of transgenic cassava using negative and positive selection Transgenic Res. 9 405 415

    • Search Google Scholar
    • Export Citation
  • Zhu, Y. , Ou-Yang, W. , Zhang, Y. & Chen, Z. 1996 Transgenic sweet pepper plants from Agrobacterium mediated transformation Plant Cell Rep. 16 71 75

    • Search Google Scholar
    • Export Citation
Moon Kyung Ko Kumho Life & Environmental Science Laboratory (KLESL), Chonnam National University, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea

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Hyunchul Soh Kumho Life & Environmental Science Laboratory (KLESL), Chonnam National University, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea

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Kyung-Moon Kim Kumho Life & Environmental Science Laboratory (KLESL), Chonnam National University, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea; and Agricultural Plant Stress Research Center (APSRC), Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea

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Young Soon Kim Kumho Life & Environmental Science Laboratory (KLESL), Chonnam National University, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea; and Agricultural Plant Stress Research Center (APSRC), Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea

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Kyunghoan Im Department of Biology, University of Incheon, 177 Dohwa-dong, Nam-gu, Incheon 402-749, Republic of Korea

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Contributor Notes

Moon Kyung Ko and Kyung-Moon Kim contributed equally to this paper.

This research was supported by a grant (code # M104KG010016-04K0701-01600) from the Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology (MOST) of the Republic of Korea.

To whom reprint requests should be addressed; e-mail yskim2@chonnam.ac.kr.

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  • Steps for the development of transgenic pepper plants. (A) Cotyledonary explants cocultivated with A. tumefaciens containing pCAMBIA 1301; (B) development of calli from the cutting edge of the infected explant on medium containing hygromycin (20 mg l−1); (C) localization of histochemical GUS activity in developing calli and regenerated shoot; (D) shoot regenerated from the callus; (E) rooting of the regenerated pepper shoot; (F) pepper plants acclimated in pots.

  • GUS activity in the primary transgenic peppers. Quantitative assays for GUS-specific activity conducted (A) in situ or (B) in extracts of various tissues from a transgenic pepper that carry the uidA reporter gene. (a) Flower; (b) plantlet with root; (c) leaves; (d) fruit.

  • Segregation of hygromycin resistance and GUS activity in the transgenic progeny. After germination of 21 seeds harvested from T0 plant (no. 2), 15 seedlings were resistant to hygromycin and GUS positive; the remaining six were not hygromycin-resistant or GUS-positive. As a control, seedlings germinated from wild-type (WT) seeds showed stopping growth, no GUS activity, and no hygromycin resistance.

  • Southern and Northern blot analyses. (A) Southern blot analysis of two T0 transgenic lines and their T1 progenies. DNA samples from two transgenic lines and an untransformed pepper plant (WT) were digested with HindIII, and the resulting fragments were resolved by electrophoresis and transferred to a membrane. The membrane was hybridized with a 32P-labeled probe DNA corresponding with the hpt coding region. (B) Northern blot analysis of two transgenic pepper lines. WT, wild type; lane 2, line 2 plants (T0); lanes 3–6, T1 progenies from line 2; lane 7, line 3 plants (T0); lanes 8–11, T1 progenies from line 3. The membrane was hybridized with a 32P-labeled hpt.

  • Transmission of uidA (upper) and hpt (lower) genes to T2 progenies determined by polymerase chain reaction analysis in two independent transgenic pepper lines. 2-1, T1 transgenic pepper; 2-1-1, 2, 5, 6, T2 transgenic progenies from transgenic line 2-1; 3-2, T1 transgenic pepper; 3-2-2, 3, 8, 9, T2 transgenic progenies from transgenic line 3-2; WT, nontransgenic pepper as a negative control; P, pCAMBIA1301 as a positive control.

  • Growth of hypocotyl explants from nontransformed (WT) and transformed (T) peppers carrying the hpt gene on medium containing 0 to 50 mg·L−1 hygromycin. (A) Callus and shoot formation. (B) Average of fresh weight of 60 explants after 3 weeks of incubation. The bars indicate se.

 

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