Plant Cell Rep (2005) 24: 350–356 DOI 10.1007/s00299-005-0946-8
GENETIC TRANSFORMATION AND HYBRIDIZATION
Sang Mi Park · Jung Suk Lee · Sung Jegal · Bo Young Jeon · Min Jung · Yoon Sik Park · Sang Lyul Han · Yoon Sup Shin · Nam Han Her · Jang Ha Lee · Mi Yeon Lee · Ki Hyun Ryu · Seung Gyun Yang · Chee Hark Harn
Transgenic watermelon rootstock resistant to CGMMV (cucumber green mottle mosaic virus) infection Received: 28 August 2004 / Revised: 22 February 2005 / Accepted: 23 February 2005 / Published online: 14 June 2005 C Springer-Verlag 2005
Abstract In watermelon, grafting of seedlings to rootstocks is necessary because watermelon roots are less viable than the rootstock. Moreover, commercially important watermelon varieties require disease-resistant rootstocks to reduce total watermelon yield losses due to infection with viruses such as cucumber green mottle mosaic virus (CGMMV). Therefore, we undertook to develop a CGMMV-resistant watermelon rootstock using a cDNA encoding the CGMMV coat protein gene (CGMMV-CP), and successfully transformed a watermelon rootstock named ‘gongdae’. The transformation rate was as low as 0.1–0.3%, depending on the transformation method used (ordinary co-culture vs injection, respectively). However, watermelon transformation was reproducibly and reliably achieved using these two methods. Southern blot analysis confirmed that the CGMMV-CP gene was inserted into Communicated by I.S. Chung S. M. Park · J. S. Lee · S. Jegal · B. Y. Jeon · M. Jung · Y. S. Park · S. L. Han · Y. S. Shin · N. H. Her · J. H. Lee · S. G. Yang · C. H. Harn () Biotechnology Center, Nong Woo Bio, Jeongdan, Ganam, Yeoju, Gyeonggi 469-884, Korea e-mail:
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[email protected] M. Y. Lee · K. H. Ryu Department of Environmental and Life Science, Seoul Women’s University, Seoul 139-774, Korea e-mail:
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different locations in the genome either singly or multiple copies. Resistance testing against CGMMV showed that 10 plants among 140 T1 plants were resistant to CGMMV infection. This is the first report of the development by genetic engineering of watermelons resistant to CGMMV infection. Keywords Transformation . Cucumber green mottle mosaic virus . Coat protein . Selection . Agrobacterium
Introduction In Korea and Japan, rootstock grafting is popularly used in the cultivation of Cucurbitaceae crops such as watermelon, cucumber, and melons, because of the poor viability of cultivar roots. The watermelon rootstock, Citrullus lanatus (Twinser) cv. gongdae, is one of the rootstocks commonly used for grafting commercially important watermelon varieties. Although this rootstock is hardier in the soil environment, it is equally vulnerable to virus infection by cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus. CGMMV infects many Cucurbitaceae species causing mosaic symptoms, a yellowish leaf, and finally fruit deterioration (Lee et al. 1990; Lee 1996; Choi 2001). In fact, CGMMV outbreaks have caused marked losses in the total yields of Cucurbitaceous crops in Korea over the past several years. Since CGMMV is easily transmitted by soil, the development of a virus-resistant rootstock offers a viable solution. Unfortunately, no genetic source is available for resistance against CGMMV infection, and therefore breeding management offers no access to a solution. Thus, the alternative was to utilize a viral gene, such as a coat protein (CP) gene, and transform it into a watermelon rootstock to induce resistance to CGMMV. Watermelon is recognized as one of the most recalcitrant plants regarding transformation by Agrobacterium. However, relatively few reports have been published (Choi et al. 1994; Chen et al. 1998; Ellul et al. 2003) on the
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topic and no reproducible system has been established. Gongdae is a wild watermelon and is not used for breeding commercial watermelon varieties, other than as a grafting stock. In addition, most watermelon lines are not crossed with gongdae (Y.S. Shin, personal communication). Here, we describe the successful transformation of a watermelon rootstock (gongdae) by Agrobacterium-mediated transformation with the CGMMV-CP gene. Stable transformation was obtained in the frequency range 0.1– 0.3, and some of the transformants were resistant to CGMMV. To the best of our knowledge, this is the first report of a transgenic watermelon with virus resistance conferred by the introduction of a gene encoding a viral CP. Materials and methods Plant materials Seeds of the watermelon rootstock gongdae were surfacedisinfected in 70% ethanol for 30s and 25% bleach (Yuhanrox) for 30 min, and then rinsed three times with sterilized water. The sterilized seeds were placed in one-half strength MS medium (Murashige and Skoog 1962) and allowed to germinate in the dark at 25◦ C. Cotyledons from 3-day-old seedlings were excised to nine segments and used as explants for regeneration and transformation. Shoot formation and regeneration Explants were transferred to a regeneration medium consisting of modified MS medium supplemented with 1.0 mg l−1 6-benzylaminopurine (BA) and 0.1 mg l−1 indole acetic acid (IAA). The hormone compositions and antibiotic concentrations used to select transformed shoots are described in Table 2. Shoot formation rates were measured by dividing the number of shoots transferred to rooting medium by the total number of explants. Explants were incubated on selection medium (1.0 mg l−1 BA + 0.1 mg l−1 IAA) for 4–5 weeks, and on rooting medium (hormone free) for 5–6 weeks. The regenerated plants were acclimated for 2 weeks in zippy pot soil in a growth chamber at 25◦ C under a 16 h light/8 h dark cycle. Agrobacterium-mediated transformation Explants were transferred to a pre-culture medium consisting of MS medium supplemented with BA (1.0 mg l−1 ) and IAA (0.1 mg l−1 ), and then placed in a light room at 25◦ C for 24 h. For ordinary co-culture with Agrobacterium, strains LBA4404 or EHA105 were transformed using a binary vector containing the CGMMV-CP gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and the NPTII gene for kanamycin selec-
tion. Transformed Agrobacterium grown to log phase in YEP liquid medium (OD600 : 0.7–0.9) were centrifuged at 3,500 rpm for 10 min and the pellets so obtained were resuspended in MS containing 1.0 mg l−1 BA. Explants were infected by immersing them in the above Agrobacterium inoculum for 30 min and then co-cultured in MS medium containing 1.0 mg l−1 BA and 0.1 mg l−1 IAA for 3 days. They were then briefly washed with 400 mg l−1 lilacilline and placed on agar (0.8%)-solidified shoot regeneration medium supplemented with 1.0 mg l−1 BA, 0.1 mg l−1 IAA, and 40 mg l−1 kanamycin or 7.5 mg l−1 hygromycin, and incubated for 4–5 weeks at 25◦ C. Green shoots were then transferred to rooting medium (basic medium, 20 mg l−1 kanamycin) for 5–6 weeks, and young plants were transferred to zippy pots in a growth chamber at 25◦ C under a 16 h light/8 h dark cycle. After 1 month in the chamber, plants were grown in a plastic house. In addition to ordinary co-culture and kanamycin selection, we experimented with an injection-based method. Explants were injected by multi-wire points of electric cord in the lower meristem tissue region several times to cause wounding. The medium and pre-culture conditions used were the same as those used for co-culture, the only difference being antibiotic selection, i.e., 7.5 mg l−1 hygromycin rather than 40 mg l−1 kanamycin. PCR analysis To detect the CGMMV-CP gene in transformed gongdae by PCR, total DNA was isolated using a DNA extraction kit (iNtRon Biotechnology, http://www.intronbio.com). The PCR primer sequences used for detecting the CGMMV-CP gene insertion were: 5 - TCCGATCACACCTAGCAAAC-3 (sense: 35S promoter region at 3,185–3,204 bp of accession number X84105) and 5 -GACCAGACTACCGAAAACG-3 (antisense: CGMMV-CP gene at 489–471 bp of accession number AJ243831). PCR analysis was carried out using 0.65 µM of each primer, 299 µM dNTP, 1 U/µM Taq DNA polymerase (BioLabs, http://www.neb.com) in 50 mM KCl, 1.5 mM MgCl2 and 10 mM Tris-HCl pH 8.3. The PCR program consisted of 35 amplification cycles of 94◦ C, 55◦ C, and 72◦ C, each for 1 min. Southern, northern and western blot analysis For genomic Southern blot analysis, 20 µg T0 plant DNA was isolated using the method described by Sambrook et al. (1989), and digested with XbaI, BglII, or EcoRV and fractionated on a 0.8% agarose gel. For northern blot analysis, total RNA was extracted from the leaves of T0 plants using a modification of the method described by Chomczynski and Sacchi (1987), and 30 µg RNA was fractionated on 1% agarose gel. Southern and northern blotting were performed as previously described (Church and Gilbert 1984; Sambrook et al. 1989) using Hybond N membranes (Amersham Biosciences, http://www.amershambioscien
352 Table 1 Regeneration frequencies of watermelon rootstock. The numbers indicate the percentage of explants able to regenerate shoots. For each hormone combination, 50 explants were used. BA 6-Benzylaminopurine, IAA indole acetic acid IAA (mg l−1 )
BA (mg l−1 ) 0.0 0.5
1.0
2.0
4.0
0.0 0.1 0.5
30 46 30
60 80 69
45 66 76
50 44 50
33 42 22
ces.com) and hybridized with a32 P-labeled probe containing the CGMMV-CP gene (524 bp; AJ243831) according to the manufacturer’s instructions (Amersham Biosciences). For western blot analysis, the CP was separated on 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were probed with rabbit antibody (1: 1,500 dilution; immunoglobulin G fraction; 1 mg/ml) raised against CGMMV-CP. To visualize antibody-specific proteins, the method devised by Lee et al. (2003) was used.
CGMMV resistance test of T1 plants A total of 140 T1 gongdae seedlings (two-leaf stage) from a T0 plant, were exposed to CGMMV by carborundum and then re-exposed 2 weeks later. The CGMMV strain was provided by the Plant Virus Gene Bank (http://www.virusbank.org) at Seoul Woman’s University. A leaf disk was taken from each T1 plant and an indirect ELISA was performed as described by Shin et al (2002). Readings were taken at A405 nm using an ELISA Thermo Max Microplate Reader (Molecular Devices, http://www.moleculardevices.com).
Results Regeneration rate To establish a transformation system for gongdae rootstock, regeneration frequencies were investigated by culturing explants on a set of hormone combinations with BA and IAA (Table 1); 50 explants were used per combination. Shoots appeared from explants 3 weeks after being placed on the medium. A combination of 0.1 mg l−1 IAA and 1.0 mg l−1 BA showed the highest regeneration frequency (80%). Generally, medium containing 1.0 mg l−1 BA or higher, with or without IAA, showed regeneration frequencies in the range 60–80%, and lower levels of BA generated shoots at much lower frequencies. Without hormone, the regeneration frequency was only ca. 30%. Transformation rate The protocol used to transform gongdae is detailed in Table 2. Generally, the strain of Agrobacterium used, LBA4404 or EHA105, did not influence transformation efficiency, and either kanamycin or hygromycin was used for selection. For both selection methods, shoots formed from explants 4 weeks after placing on selection medium. Shoots formed directly from the explant cut region, and no callus formed around the cut region; roots regenerated 5–6 weeks after placing shoots in rooting medium. When shoots had grown to about 10 cm in height, seedlings were acclimated gently in zippy pots. There were no differences in phenotype or developmental processes whether kanamycin (Fig. 1) or hygromycin selection (Fig. 2) was used, and both methods gave rise to fruits and seeds as did non-transformed plants.
Table 2 A protocol for transformation of watermelon rootstock. CGMMV Cucumber green mottle mosaic virus, CP coat protein Step Germination
Description
Germination condition under dark did not influence the transformation (1/2 MS + 3% sucrose + 0.8% agar, pH 5.8) Explant Cotyledon Pre-culture Basic medium (MS-B5 + 3.0% sucrose + 0.8% agar, pH 5.8) 1.0 mg l−1 BA + 0.2 mg l−1 IAA cDNA insert CGMMV-CP Agrobacterium strains LBA4404, EHA105 Inoculation Basic medium addition of 200 µM acetosyringone OD600nm : 0.7–0.9 Co-culture Basic medium 1.0 mg l−1 BA + 0.1 mg l−1 IAA + 200 µM acetosyringone washing buffer (0.2 M citric acid + 2.0% sucrose, pH 5.8 + 400 mg l−1 lilacilline) Selection and shooting Basic medium 40 mg l−1 kanamycin + 200 mg l−1 lilacilline with 1.0 mg l−1 BA + 0.1 mg l−1 IAA + 2 mg l−1 AgNO3 (7.5 mg l−1 hygromycin + 500 mg l−1 cefotaxim Rooting Basic medium 20 mg l−1 kanamycin + 200 mg l−1 lilacilline without hormone
Duration 3–5 days
1–3 days
30 min
3 days in dark
Shoot formation: 4–5 weeks
Root formation: 5–6 weeks 10 cm height: 2–3 weeks
353 Table 3 Transformation efficiencies
Seeds
Explants
PCR positive
Acclimated Grown in plastic house
Transformation by co-culture (kanamycin selection) 1,000 9,000 21 12 0.23% (21/9,000) Transformation by injection (hygromycin selection) 320 640 50 10 7.8% (50/640)
A
B
Southern (T0 )
11
9 0.1% (9/9,000)
5
2 0.3% (2/640)
A total of 9,000 explants were prepared by germinating around 1,000 seeds for kanamycin selection (Table 3). To determine the transformation rate, shoots grown with rooting were tested by PCR to confirm the presence of the CGMMV-CP insert 21 shoots of 9,000 explants (0.23%) showed a 550 bp band. Eleven T0 plants were then selfcrossed in a plastic house to produce T1 seeds. For hygromycin selection, 640 explants were transformed using the injection method, and 50 shoots contained the CGMMVCP insert (7.8%) (Table 3). The shoot acclimation process proved difficult and many PCR-positive shoots failed to survive in zippy pots. Only five plants grew in a plastic house and those were self-crossed to obtain T1 seeds. Southern and northern blot analysis
C
D
Fig. 1A–D Developmental stages of C. lanatus (Twinser) cv. gongdae grown under kanamycin selection after co-culture with Agrobacterium tumefaciens. A In vitro culture, B rooting stage, C 1-monthold plant after acclimation, D 2-month-old plant
A
B
Genomic DNA (20 µg) isolated from T0 plants was digested with XbaI, and fractionated on a 0.8% agarose gel. The restriction bands produced confirmed the presence of the CGMMV-CP gene (Fig. 3a). Several distinct T0 origins were found in terms of band number: T0 4, 7, 8 vs 5, 6 vs 12, 26 vs 41, 42. The same copy number or band pattern was assumed to be due to the propagation of multishoots. The genomic DNA of T0 106 was also digested with BglII or EcoRV, which do not cut within the insert and thus generate a single band (Fig. 3b). Two bands were generated when genomic DNA of T0 106 was digested with XbaI. Since XbaI cuts the insert once, T0 106 possessed only one inserted copy of the CGMMV-CP gene. We confirmed by genomic Southern blotting that the T1 plants self-crossed from T0 106 also contained one copy of the CGMMV-CP gene (data not shown). The transcript levels of PCR-positive T0 plants were determined by northern blotting (Fig. 4). T0 2, 4, and 25 contained a 550 bp band, but T0 9, 29 and the non-transformed gongdae (N) did not. Resistance to CGMMV test of T1
C
D
Fig. 2A–D Developmental stages of C. lanatus cv. gongdae grown under hygromycin selection after co-culture with A. tumefaciens. A Injection, B selection for shoot formation, C 1-month-old plant after acclimation, D 2-month-old plant. Arrow Injection site
To determine the resistance of transformed watermelon rootstock against CGMMV infection, T0 106 plants were self-crossed, and the 140 T1 plants obtained were exposed to CGMMV. Leaves from 4-week-old T1 plants were inoculated with CGMMV twice with a 2-week interval. Three weeks after the second inoculation, the plants were inspected visually and ELISA was performed. A mosaic pattern was identified in the leaves of susceptible plants,
354 Fig. 3a,b Southern blot analysis of T0 plants. Genomic DNA was digested with XbaI (a), or XbaI, BglII or EcoRV (b). Membranes were hybridized with a CGMMV-CP probe labeled with32 P. Lanes: N Non-transformed; 4–8, 12, 26, 41, 42, 106 transformed T0 plants. X XbaI, B BglII, E EcoRV
a
bX
Xba I N
4
5
6
7
8
12
26
41
42
106
B E 106 106
12.0 kb
12 kb
5.0 kb
2.0 kb 1.4 kb 0.7 kb
Fig. 4 Northern blot analysis of T0 plants. Upper panel Membranes were hybridized with a CGMMV-CP probe labeled with32 P. Lower panel Ethidium bromide-stained gel to verify equal loading. Lanes: N Non-transformed; 2, 4, 9, 25, 29 transformed T0 plants
2
4
9
25
29
N
550 bp
rRNA
whereas no mosaic spots developed in resistant plants. From the 140 T1 plants, we obtained 10 plants resistant to CGMMV infection (Table 4). Non-transformed control plants were all infected with CGMMV. PCR analysis showed that all of the 10 resistant T1 plants contained the CGMMV-CP insert, and that 89% (116/130) of the susceptible T1 plants also possessed the CGMMV-CP insert; however, 14 T1 plants did not contain the CGMMV-CP insert. The phenotypes of the leaves of susceptible and resistant plants at 7 weeks after CGMMV inoculation were clearly different (Fig. 5). Susceptible leaves showed a severe mosaic pattern and a yellow discoloration. Figure 6 shows northern and western blots from PCRpositive T1 plants. Transcripts from six T1 plants were hybridized with a CGMMV-CP gene probe and only lane 5 showed a transcript. This latter transcript produced a protein that cross-reacted with the CGMMV-CP antibody. Although the six T1 plants showed resistance against CGMMV infection, resistance levels by ELISA testing were Table 4 Resistance test performed by exposing T1 plants to CGMMV twice at 2-week intervals. Resistance was determined by ELISA, observing absorbance values less than the control (nontreated) value
T1
Number of plants tested
Susceptible
140
130 (89% 10 (100% PCR PCR-positive) positive) 40 (0% 0 PCR-positive)
Non-transformed 40
Resistant
Resistant
Susceptible
Fig. 5 Phenotypical differences between resistant and susceptible T1 plants at 7 weeks after inoculation with cucumber green mottle mosaic virus (CGMMV)
not related to levels of protein or transcript. Transgenic plants without the transcript showed resistance, suggesting that the defense mechanism of the CP gene is rather complicated. Discussion Watermelon is one of the most difficult plants to transform with Agrobacterium, and thus few reports are available on
355 Fig. 6 Levels of transcript, protein and resistance. Lanes: 1, 2, 5, 6, 7, 11 T1 plants showing resistance against CGMMV; N non-transformed, P Transformation vector with subcloned CGMMV-CP. Northern blot membranes were hybridized with a 32 P-labeled CGMMV-CP probe, and CGMMV-CP on the western blot membrane was probed with rabbit antibody (1:1,500 dilution) raised against CGMMV-CP. The Western band on the right is a CGMMV-CP band used as a positive control
1
2
5
6
7
11
PCR
N
P
800 bp
Northern 500 bp
Western
Resistance Levels
the topic (Choi et al. 1994; Chen et al. 1998; Ellul et al. 2003). Culture conditions and media composition in the literature are similar except for kanamycin concentrations, which suggests that antibiotic selection shows genotype specificity. Choi et al. (1994) and Ellul et al. (2003) used 100 mg l−1 kanamycin and 125–175 mg l−1 kanamycin, respectively, for different cultivars. However, hygromycin has not previously been used as a selection marker. In the present study, we successfully established a transformation system for the watermelon rootstock gongdae. Two separate transformation methods—an ordinary co-culture method and an injection method—were used to introduce the CGMMV-CP gene into the gongdae genome. The transformation rates obtained were 0.1% and 0.3%, respectively (Table 3). However, although these rates are admittedly low, transformation was successful and reproducible, thus indicating that the protocol described in Table 2 is dependable. Most shoots grew well near or on the wounded surfaces of explants on selection medium, i.e., via direct organogenesis from the explants, and no callus was found around the cut region, indicating that shoot formation had nothing to do with callus-mediated or -induced transformation (Lee et al. 2004). The growth patterns of transformed gongdae selected using kanamycin or hygromycin were nearly identical, but acclimation recovery was more difficult for hygromycin-selected plants, and for some reason the hygromycin-selected seedlings did not recover easily in zippy pots. The idea of injecting Agrobacterium below the meristem region of watermelon by using a multi-wire point electric cord is new, and was carried out to induce a different type of wounding. In the present study, the transformation frequency obtained using the injection method was slightly higher than that of the co-culture method (0.3% vs. 0.1%), but there was no significant difference between the two methods. Although the mechanism of transformation by injection is unclear, multi-wounding may allow more efficient foreign DNA transfer. In fact, Chen et al. (1998) transformed watermelon by injecting squash DNA
17kDa
+ + + +
+
+ +
+
+
into the central part of a watermelon ovary via the pollentube pathway to obtain Fusarium-wilt-resistant watermelon with a 5% transformation rate (i.e., 10 in 200). Southern blot analysis of T0 plants supported gongdae transformation due to the presence of the 550 bp CGMMVCP gene insert (Fig. 3). Some T0 plants possessed several copies of the gene, whereas others contained only one copy. Northern blotting demonstrated that transcription levels differed among T0 plants. T0 9 and 29 did not contain the CGMMV-CP gene transcript (Fig. 4), whereas 2, 4, and 25 did. This raises the question as to whether transcript levels are related to resistance levels. We do not yet have a definitive answer to this question, although preliminary results indicate that the level of resistance is not correlated with the transcript level (Fig. 6). More information is required on the defense mechanism utilized by the CP gene. Nevertheless, several mechanisms can be proposed. First, gene silencing caused by transcription of the inserted gene (RNA-mediated resistance) might not have occurred. Second, the CP, which is presumed to interact with viral RNA to prevent replication (protein-mediated resistance), was not expressed in T1 plants. Third, resistance levels may have been influenced by somaclonal variation instead of by transcript- or translation-mediated resistance mechanisms. We also observed this non-correlation between transcript levels and resistance levels from T1 plants of transgenic pepper transformed with the tobacco mosaic virus coat protein (TMV-CP) gene (data not shown). Further studies on resistance with more T generations may elucidate the mechanism involved. The transformation of gongdae reported here was achieved by direct shoot formation from the explant cut region. Indirect shoot formation from callus induction has been applied in rice (Hiei et al. 1994; Rashid et al. 1996), grass (Kusano et al. 2003) and in pepper (Lee et al. 2004), when plants were not successfully transformed by Agrobacterium. Since the transformation rate of gongdae is low, our lab has been involved in the development of a callus
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induction system using hormones to monitor the transformation efficiency of Agrobacterium using expression of green fluorescent protein. Preliminary data shows that watermelon callus can be successfully transformed by Agrobacterium (data not shown). The use of grafting rootstocks before planting for vegetables has become a very popular planting method in Korea and Japan, and farmers cultivate most vegetables in this manner. Protecting plants from initial contamination by a pathogen such as CGMMV in soil, would help considerably to reduce watermelon yield losses. In addition, the use of a rootstock transformant for grafting offers a means of circumventing controversial genetically modified (GM) crop issues. Therefore, we believe that this work opens a door to the development of GM rootstocks that are resistant to crop infections. Acknowledgements This research was supported by grants to C.H. Harn from the Biogreen 21 research fund of the Rural Development of Administration and from the ARPC research fund of the Ministry of Agriculture and Forestry in Korea. We also thank H. S. Kim (Nong Woo Bio Co.) and Dr. N. I. Hyung (Sangmyung University) for their technical assistance.
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