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Efficient Agrobacterium-mediated transformation and recovery of transgenic fig (Ficus carica L.) plants
Plant Science 168 (2005) 1433–1441 www.elsevier.com/locate/plantsci
Efficient Agrobacterium-mediated transformation and recovery of transgenic fig (Ficus carica L.) plants Svetla D. Yancheva b, Sara Golubowicz a, Zeev Yablowicz a, Avi Perl a, Moshe A. Flaishman a,* a
Department of Fruit Tree Sciences, Institute of Horticulture, ARO, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel b Plant Biotechnology Laboratory, Agricultural University, 12 Mendeleev St., 4000 Plovdiv, Bulgaria Received 3 October 2004; received in revised form 28 November 2004; accepted 3 December 2004 Available online 27 December 2004
Abstract An efficient and reproducible system for regeneration and Agrobacterium-mediated transformation of the common fig (Ficus carica L.) cultivars Brown Turkey (fresh consumption) and Smyrna (dry consumption) was developed. Optimal shoot regeneration (up to 100%) was obtained on MS basal salt mixture supplemented with 100 mg l1 myo-inositol, 1 mg l1 thiamine HCl and addition of 2.0 mg l1 thidiazuron (TDZ), 2 mg l1 indole-3-butyric acid (IBA), 4% sucrose and 0.8% agar. Regeneration was highly dependent on the dorsoventral orientation of the explants. When explants were cultured with the adaxial surface up, 100% regeneration was achieved with more than five shoots per regenerating explant in both studied cultivars. In contrast, if leaves were placed with their abaxial side up, shoot regeneration took place, but still mostly from the adaxial surface. Leaf explants of in vitro propagated plants were co-cultivated with the disarmed Agrobacterium strain EHA105 harboring the plasmid pME504 that carried the uidA-intron and nptII genes. Transformation efficiencies were in a range of 1.7– 10.0% for cv. Brown Turkey and 2.8–7.8% for Smyrna. The transgenic nature of the regenerated plants was confirmed by molecular analyses (PCR and Southern blot) as well as by GUS staining. Similar to regeneration, the orientation of the leaf surface during organogenesis was a key factor for successful transformation. Successful transformation of commercial fig cultivars provides a new promising tool for the introduction of foreign genes into transgenic fig cultivars. The regeneration and transformation methodologies described here may pave the way for transgenic varieties with improved agronomic characteristics, such as storability and disease resistance, and will provide a means for the production of foreign proteins in the edible parts of fig, leading to improved nutritional and/or pharmaceutical composition. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Fig (Ficus carica); Transformation; Regeneration; GUS; nptII; Agrobacterium tumefaciens
1. Introduction Fig trees are one of the earliest fruit-bearing trees cultivated. Ficus carica (Moraceae), the well-known fig of commerce, is indigenous to wide areas ranging from Asiatic Turkey to North India, and natural varieties are cultivated in Abbreviations: BA, 6-benzylaminopurine; CaMV, cauliflower mosaic virus; GA3, gibberellic acid A3; GUS, b-glucuronidase; IBA, indole-3butyric acid; NAA, a-naphthalene acetic acid; nptII, neomycin phosphotransferase II; PCR, polymerase chain reaction; TDZ, thidiazuron (Nphenyl-N0 -1,2,3-thiadiazol-5-yl urea); uidA, b-glucuronidase gene of Escherichia coli * Corresponding author. Tel.: +972 3 968 3394; fax: +972 3 966 9583. E-mail address: [email protected] (M.A. Flaishman).
most Mediterranean countries [1]. Fig fruit is well known for its nutritive value, and is consumed fresh or dry worldwide. Fig fruits are also known for their mild laxative activity and high alkalinity, and substances derived from them are used in various drug preparations. Other parts of fig trees have also been shown to have a commercial value [2]. Traditional breeding methods of F. carica require a longterm effort for improving traits such as production of parthenocarpic varieties. Among F. carica there are trees that bear only female figs and others trees, named caprifig, bear both male and female flowers. Edible fruits can be produced only on trees bearing female synconia, provided cross-pollination is mediated by a specific wasp [3]. Thus, traits originated via pollen are hard to track.
0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.12.007
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The application of genetic engineering techniques to stably incorporate homologous and/or heterologous genetic material into woody species, including fruit trees, offers the potential of obtaining improved planting stocks for agricultural use in a short period of time compared to traditional breeding techniques. In addition, efficient transformation methods can be used for the production of heterologous polypeptides having nutritional and/or pharmaceutical value. The overall efficiency of techniques for genetically modifying plants depends upon the efficiency of the transformation technique(s) used to stably incorporate the desired genetic material into plant cells or tissues, and the regeneration technique(s) used to produce viable plants from transformed cells. Within Ficus sp. adventitious shoot regeneration has been reported only for F. lyrata from young leaves isolated from plants maintained under greenhouse conditions [4]. In vitro studies using F. carica were restricted to the development of protocols for micropropagation and production of figmosaic-virus-free plants by single shoot tip culture [5–10] as well as to biochemical assessment of active compounds in fig calli grown in vitro [11,12]. Within the genus Ficus, several reports described regeneration and organogenesis from calli and other explants. F. religiosa plants were regenerated from calli of stem segments [13] and from axillary buds of mature trees [14]. Regeneration of F. carica plants from the apical buds of a mature tree was also reported [15]. However, in all mentioned in vitro studies, plant regeneration has been restricted to the use of single shoot tips and apical buds. Recently, Yakushiji et al. [16] reported a method for the induction of organogenesis from leaf explants of F. carica using phloroglucinol (PG). However, by this method the frequency of adventitious bud differentiation from leaf fragments was relatively low, and no adventitious buds were observed without PG. Moreover, to date no method for the introduction of isolated genetic material into Ficus species has been reported. The overall objective of the current research was to develop an optimized protocol for in vitro regeneration of fig that allows for successful application of Agrobacteriummediated transformation in two fig (F. carica) commercial cultivars Brown Turkey (fresh consumption) and Smyrna (dry consumption).
2. Materials and methods 2.1. Fig shoot culture maintenance Fig (F. carica) cultivars Brown Turkey (common for obtaining fresh fruit) and Smyrna (common for dry fruit production) were used. In vitro shoot cultures of both cultivars were established according to Pontikis and Melas [6] and subsequently grown on proliferation medium (PM) consisting of MS [17] basal salt mixture supplemented with 100 mg/l myo-inositol, 0.5 mg/l thiamine–HCl, 3% sucrose
(w/v), 0.8% agar (Sigma), 0.25 mg/l BA, 0.05 mg/l IBA, and 0.05 mg/l GA3. The pH was adjusted to 5.7 prior to autoclaving for 20 min at 120 8C. Two shoots were placed horizontally in Magenta boxes containing 50 ml PM and exposed to cool white fluorescent light (40 mmol/m2 s) in a 16/8 h photoperiod at 25 8C for 4–5 weeks before leaf explants were removed. 2.2. Plant regeneration Regeneration experiments were carried out initially with the cultivar Brown Turkey. To examine the effect of culture media on adventitious bud formation, the basal media MS, AP [18] and NN [19], including various combinations of only the auxins IBA and NAA (0.25–2 mg/l), or NAA in combination with TDZ (0.5–5 mg/l) or BA (2–5 mg/l) were examined. A control treatment with only TDZ (0.5–5 mg/l) was performed. The regeneration media contained MS basal salt mixture, supplemented with 100 mg/l myo-inositol, 1 mg/l thiamine–HCl, 2–4% sucrose (w/v) and 0.8% agar (Sigma) at pH 5.7, with or without 0.25% activated charcoal (AC). The three youngest expanding leaves isolated from 3 to 4 weeks old plants were used as explants. Each leaf was wounded by forming three cuts perpendicular to the central vein and placed on regeneration medium with either the abaxial or the adaxial surface facing the medium. At least 10 Petri dishes, each containing 10 explants were used per treatment. The cultures were kept for 7 days in low light intensity (2.5 mmol/m2 s) followed by exposure to high light intensity (40 mmol/m2 s) at 25 8C, in a 16/8 h photoperiod. Leaf explants were examined after 28 and 35 days and the percentage of explant producing shoots (regeneration capacity) and the mean of adventitious shoots formed per regenerating explant (regeneration efficacy) were recorded. All experiments were repeated at least three times. 2.3. Rooting and acclimatization Rooting and acclimatization experiments were carried out initially with the cultivar Brown Turkey. Two methods for rooting were evaluated. According to one method, each shoot was cultivated individually in a tube with root induction (RI) medium composed of half strength MS medium (1/2MS) supplemented with 100 mg/l myoinositol, 1 mg/l thiamine–HCl, 90 mg/l phloroglucinol, 2% sucrose (w/v), 0.25% activated charcoal (AC) and 0.8% agar (pH 5.7), with or without IBA at different concentrations (0, 1, 2 mg/l). Each treatment included 32 individual plants. According to the second method, well developed shoots (up to 3–4 cm long with 3–4 expanded leaves) were directly transplanted from the proliferation medium to rooting cylinders (3.5 cm high 3.0 cm in diameter) with soil mixture comprising 55% granular polypropylene foam, 30% peat and 15% perlite (Tivonchem Ltd., Israel). Soil mixture was moistened by soaking the rooting cylinders in
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liquid RI medium supplemented with either 0, 1 or 2 mg/l IBA. Each treatment consisted of 32 shoots and results were scored after 4 weeks. Each experiment was repeated three times. 2.4. Histology The regeneration process was examined by light microscopy. Samples were fixed in a solution of freshly prepared FAA (5% formalin, 25% acetic acid, 70% alcohol), dehydrated in a graded ethanol series and embedded in Paraplast. Sections were cut at 10 mm thickness, stained in Safranin and Fast-Green, mounted in Permount (Fisher) and examined, using Leica DMLB light microscope. 2.5. Agrobacterium tumefaciens strain and plasmid Super-virulent A. tumefaciens strain EHA 105 [20] harboring the vector pME 504 carrying the nptII and the uidA-intron genes [21] was used. Agrobacterium culture was grown overnight in LB medium (Duchefa L-1704) with appropriate antibiotics. Bacteria were spun down by centrifugation (4000 rpm for 15 min), resuspended in liquid SIM medium [22] supplemented with 100 mM acetosyringone (AS) to obtain a final OD600 of 0.7, and incubated in an orbital shaker at 28 8C and 250 rpm for 4 h. 2.6. Transformation Leaves of 3–4 weeks old micropropagated shoots were wounded across the midrib with a scalpel and immersed in the bacterial suspension for 20 min, dry-blotted on filter paper and cultured on regeneration medium based on MS medium supplemented with 2.0 mg/l TDZ and 2 mg/l IBA, 4% sucrose and 0.8% agar at pH 5.7. Co-cultivation medium was supplemented with 100 mM AS. After co-cultivation for 72 h in the dark at 25 1 8C, the explants were washed in liquid MS medium supplemented with 300 mg/l ticarcillin, dry-blotted and transferred to the regeneration medium with addition of ticarcillin (150 mg/l) and either kanamycin (50, 75 and 100 mg/l) or paromomyci (25 and 50 mg/l). All cultures were kept during the first week at low light intensity (2.5 mmol/m2 s) and subsequently transferred for the next 3 weeks to fluorescent light (40 mmol/m2 s) in 16 h light/8 h dark photoperiod at 25 1 8C. Regenerated shoots were developed after two subcultures in PM supplemented with 100 mg/l kanamycin and 150 mg/l ticarcillin. Transformation rate was calculated as the number of independent transformation events (kanamycin resistant/GUS expressing shoots) obtained out of the total number of explants. Multiplication and maintenance of the selected plants were performed by culturing the shoots horizontally on PM medium supplemented with kanamycin (50 or 100 mg/l) and ticarcillin (150 mg/l). Rooting procedure was carried out in rooting cylinders soaked in the liquid medium described above, with the addition of 100 mg/l kanamycin.
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2.7. Molecular confirmation of transformation To verify the presence and integration of the nptII and GUS genes, all selected clones were subjected to molecular analyses by PCR and Southern blotting. Plant genomic DNA was isolated from the youngest three leaves excised from kanamycin-resistant shoots according to Murray and Tommpson [23]. The oligonucleotide primers used for the PCR amplification of a 645-bp fragment of the nptII gene were: direct primer 50 -GCCGCTTGGGTGGAGAGGCTAT-30 (63.6 8C); reverse primer 50 -GAGGAAGCGGTCAGCCCATTC-30 (60 8C). The primers for a 676-bp fragment of the GUS gene were: GUSup 50 -CGAGCGATTTGGTCATGTGAAG30 (57.5 8C); GUSlow primer 50 -CATTGTTTGCCT CCCTGCTGCGGTT-30 (55.9 8C) (Sigma). Amplification was performed in aliquots of 25 ml using a thermal cycler (Biometra). The PCR conditions for amplification of the nptII gene fragment were 95 8C for 5 min, followed by 35 cycles at 94 8C for 1 min, 62 8C for 1 min, 72 8C for 1 min and a final extension at 72 8C for 10 min. Amplification of the uidA-intron fragment was performed according to the following program: 95 8C for 5 min followed by 35 cycles at 94 8C for 45 s, 55 8C for 45 s, 72 8C for 45 s and a final extension at 72 8C for 10 min. For Southern blot analysis, 10 mg of genomic DNA was digested with HindIII, subjected to electrophoresis on 0.8% agarose gel and transferred to nylon membranes (GeneScreen Plus, NEN, Boston, MA). Southern hybridization was performed using a [32P]-radioactively labeled 524-bp nptII gene fragment as a probe, as described by Wabiko and Minemura [24]. Hybridizations were performed for 20 h at 65 8C. Filters were washed twice, at high-stringency conditions, with 0.1% SDS 2 SSC for 30 min at 65 8C and exposed to autoradiography film with intensifying screens at 80 8C. 2.8. Histochemical GUS assay Histochemical analysis was performed following the procedure of Jefferson et al. [25]. Transient GUS expression was assayed 4 and 7 days after infection. During regeneration, leaf sections were excised from regenerating shoots and cultivated for 4 weeks under selective conditions. GUS expression was also further examined in mature putatively transformed shoots and in rooted plantlets.
3. Results 3.1. Plant regeneration 3.1.1. Effect of growth regulators In preliminary experiments, about 30% of the explants exhibited shoot regeneration when cultured on MS basal medium supplemented with IBA and TDZ while only 15%
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medium supplemented with 2 mg/l IBA and 2 mg/l TDZ, under conditions of 1 week low intensity light followed by transfer to normal light, comprised the optimal conditions for obtaining high regeneration (50%) rate and increased rate of shoot differentiation. Histological observations (Fig. 2A) showed that the regeneration process followed the developmental pattern of direct organogenesis. Adventitious shoots developed from meristematic centers, appearing in both epidermal and mesophyll tissues. Within 25 days, these centers (foci) became visible on the wounded surface of the explant. A prolonged culture of up to 42 days resulted in explant expansion, but the developing shoots were surrounded and covered by significant amount of callus, which inhibited their further growth.
Fig. 1. Effect of growth regulator concentration and light conditions on shoot regeneration of cv. Brown Turkey. Leaf explants were cultured with the abaxial side up followed by (A) 1 week low intensity light or (B) 2 weeks in the dark and 1 week low intensity light followed by transfer to normal light. Vertical bars indicate standard error (S.E.).
shoot regeneration was observed when MS medium was supplemented with NAA and TDZ. On the basis of these observations, we further studied the effects on shoot regeneration of MS medium supplemented with different concentrations of IBA and TDZ. Fig. 1A and B shows the effect of growth regulators concentration and two different light conditions on the regeneration of leaf explants obtained from shoots grown in PM medium. Higher regeneration rate was obtained when leaf explants were cultured for 1 week at low light intensity (2.5 mmol/m2 s) and subsequently transferred to normal light intensity (40 mmol/m2 s). A combination of 2 mg/l IBA and 2 mg/l TDZ yielded the best regeneration rate of 50.2%, with an average of 3.2 0.6 shoots per regenerating explant (Fig. 1A). Similar regeneration efficiencies (42.6–46.6%) were obtained when leaf explants were grown in a medium containing 1 mg/l IBA and 2 mg/l TDZ; however, most of the explants turned brown and formed excessive calli. Culturing of control explants only with TDZ induced significant explant expansion and compact calli formation, however with a very low shoot regeneration rate of ca. 2%. The addition of AC did not positively influence the rate of regeneration (data not shown). Two-week long dark treatment before the exposure of plants to low light intensity was also not advantageous (Fig. 1B). Hence, cultivating leaf explants on MS basal
3.1.2. Effect of sucrose concentration and leaf surface position on the solid medium Our studies demonstrated that the position of the leaf surface placed on the medium, in combination with sucrose concentration, had a significant effect on shoot regeneration from leaf explants. Data presented in Fig. 3 indicate that cultivating the explants with their adaxial surface up led to a considerable increase in their regeneration rate. In media supplemented with 4% sucrose, leaf explants cultured with their adaxial side up showed 100% of adventitious shoot formation with more than five shoots per regenerating explant. Most of the adventitious shoots developed directly, mainly from the wounding sited at the central and distal part of the leaf petiole (Fig. 2B(1)). When the explants were cultured with their abaxial side up, shoot formation still occurred only on the adaxial side of the leaf, in parallel to explant necrogenesis and calli (Fig. 2B(2)). 3.2. Rooting Two methods were examined to obtain root induction (Table 1). According to the first approach, each regenerated shoot was placed in a tube containing RI medium containing 2 mg/l IBA. Using this method, ca. 80% of the shoots formed roots. However, only low percentage of these plants successfully acclimatized under greenhouse conditions. According to the second approach, regenerated shoots were cultured directly in rooting cylinders, with or without 1 mg/l IBA. After 4 weeks, 100% root formation was achieved, independent of the concentration of auxin used (Fig. 2C). These plants had better hardening and underwent easier acclimatization in greenhouse conditions. 3.3. Transformation of F. carica 3.3.1. Choice of selective antibiotics In preliminary studies, we calibrated the killing effect of kanamycin on shoot regeneration from leaf explants derived from both cultivars tested. Wounded leaves were placed on regeneration medium supplemented with kanamycin at
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Fig. 2. Regeneration, rooting and acclimatization of plants from cv. Brown Turkey. (A) Light microscopy observations of shoot organogenesis in fig. Leaf explants of cv. Brown Turkey were cultivated on medium with IBA (2 mg l1) and TDZ (2 mg l1). (1) After 15 days, the first meristematic domes appeared on the adaxial surface of the explant. Bar = 100 mm. (2) After 21 days, appearance of adventitious buds and leaf primordia on the adaxial epidermis. Bar = 200 mm. (3) After 28 days, appearance of adventitious shoots with differentiated apical and axillary meristems and developing leaves. Bar = 100 mm; C – callus, CD – cell division, DL – developing leaves, E – epidermis, M – meristem, VS – vascular system. (B) Adventitious shoot formation in fig cv. Brown Turkey. Stereomicroscopic observation of leaf explants cultured 4 weeks on regeneration medium (1) with the adaxial side up (0.63 0.8) or (2) with abaxial side up (0.63 1.25). (C) Rooting and acclimatization of plants from cv. Brown Turkey. (1) In vitro rooted plant (bar, 1 cm). (2) Plants in rooting cylinders. (3) Potted plants in the greenhouse 1 month after acclimatization.
various concentrations (0, 10, 25, 50, 75 and 100 mg/l). The minimal concentration for inhibition of adventitious shoot induction in cv. Brown Turkey was 25 mg/l. However, with cv. Smyrna a few single buds were formed at 25 mg/l kanamycin, but they remained white and did not develop further (Fig. 4). Therefore, the concentration of 50 mg/l
kanamycin was initially chosen for selection in subsequent transformation experiments. 3.3.2. Regeneration of transgenic shoots Following co-cultivation with A. tumefaciens strain EHA105 harboring the plasmid pME504, explants were initially placed with their adaxial side up on regeneration medium supplemented with 50 mg/l kanamycin and 150 mg/l ticarcillin. Explants were exposed for 7 days to low intensity light and then transferred to normal light. Under selective conditions, up to 30% of the explants of Brown Turkey and up to 50% of Smyrna formed green Table 1 Effect of two rooting methods and IBA concentrations on root formation of cv. Brown Turkey IBA (mg l1)
Fig. 3. Influence of sucrose concentration and orientation of the leaf surface on adventitious shoot regeneration. Leaf explants of cv. Brown Turkey were cultured on medium with TDZ (2 mg l1) and IBA (2 mg l1). Vertical bars indicate standard error (S.E.).
0 1 2
Root formation S.E. (%) In vitro rooting
Rooting cylinders
8.3 1.59 47 5.43 81.3 7.61
100 100 83 6.27
Two methods were examined: in vitro rooting and then after acclimatizing under greenhouse conditions and culturing directly in rooting cylinders.
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Fig. 4. Kanamycin sensitivity of leaf explants of the fig cv. Brown Turkey and Smyrna.
shoots after 4 weeks in culture. The regenerating shoots were transferred to PR medium supplemented with 100 mg/l kanamycin and 150 mg/l ticarcillin for further selection. Cultivation of the new emerging transgenic shoots for more than three subcultures under selective conditions using 100 mg/l kanamycin on proliferation medium resulted in inhibition of their growth and development. Using this selection screen, transformation frequency established for cv. Brown Turkey varied in the different experiments from 1.7 to 10% and for cv. Smyrna from 2.1 to 7.8%. 3.3.3. Histochemical GUS assay High level of explants transiently expressing GUS was observed for leaves that were placed during co-cultivation with their adaxial side up. Histochemical GUS analysis of such leaves carried out 3 and 7 days after infection showed that 95–100% of the explants of both cultivars had large blue sectors (Fig. 5A). However, when cultured with their abaxial side up no transient GUS expression was observed in either of the two fig varieties (data not shown). After 4 weeks under selective conditions some of the regenerating explants (20 leaves) stained positively for GUS. Stereo-microscopic analyses (Fig. 5B) of leaves of cv. Brown Turkey showed that 7.46% (10 out of 134) of the shoots were GUS positive. An in situ b-glucuronidase assay was carried out to further confirm the integration and expression pattern of the uidA gene. Intact plants of all selected shoots propagated on kanamycin-containing medium as well as control (wild type) plants were tested. Strong GUS expression was evident in the leaves and stems of the transgenic plants (Fig. 5C). 3.3.4. Molecular confirmation of transformation PCR analysis of the putative transgenic shoots confirmed the stable incorporation of the transgenes into the F. carica genome. All clones selected after transformation with pME504 showed the predicted bands: the 645 bp for the nptII gene and the 676 bp for the uidA-intron (GUS). No fragment was amplified in the control, untransformed plant (Fig. 6A and B).
Fig. 5. Histochemical GUS analysis: stereomicroscopic observations. (A) Transient GUS expression 3 days after inoculation of leaf explants of cv. Brown Turkey (0.63 1.0). (B) GUS staining of regenerating leaf explants after 4 weeks culture in the presence of 50 mg l1 kanamycin and 150 mg l1 ticarcillin (0.63 2.5). (C) GUS expression detected in an isolated putatively transformed shoot cultured on PM with 100 mg l1 kanamycin and. 150 mg l1 ticarcillin (0.63 2.0). (D) GUS assay of transgenic clone BT 001.
Southern blot analysis of HindIII digested genomic DNA from three randomly selected putative transgenic plants of Brown Turkey and three of Smyrna provided additional molecular evidence for the incorporation of the foreign DNA (Fig. 7A and B). The Southern blot data were consistent with the plasmid pME504, that has one HindIII restriction site, located 2.8 kb from the plasmid right border after the nptII gene. No hybridization bands were present in the control, untransformed plants. Southern blot analysis of HindIII digested DNA from the six putative transgenic plants provided additional evidence for the incorporation of the foreign DNA. The nptII bands were larger than 2.8 kb, varying in size between the six clones. The hybridization pattern in clone 1 of Smyrna cv. indicated insertion of the TDNA into multiple different loci (Fig. 7B).
4. Discussion In the last two decades, transformation technology has played an increasingly important role in the genetic manipulation of crop plants. This approach has led to crop improvement as well as to better understanding of the molecular mechanisms underlying plant gene expression and regulation. However, due to a lack of transformation and regeneration procedures, the application of such biotechnological approaches has not been possible for species in the genus Ficus. The present study provides a highly efficient system for in vitro regeneration and subsequent transformation of fig trees (F. carica). A key factor in the high efficacy
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of both transformation and shoot regeneration is the dorsoventral orientation of the explants. 4.1. The regeneration process in fig
Fig. 6. PCR analysis of the putative transgenic shoots showed the predicted bands for the nptII gene and uidA-intron (GUS) gene in the cvs. Brown Turkey (A) and Smyrna (B). For Brown Turkey (A): M – lambda DNA/ EcoRI + HindIII marker; lanes 1–4, transgenic clones; C, untransformed plants. For Smyrna (B): M – lambda DNA/EcoRI + HindIII marker; lanes 1–3, transgenic clones; C, untransformed plants.
The success of gene transfer techniques is largely dependent on an efficient regeneration system. Only Yakushiji et al. [16] have reported successful regeneration of F. carica, though with a low regeneration frequency of only 22%. The present study is the first report of a highly efficient system for in vitro regeneration in figs using cv. Brown Turkey and Smyrna. Here, adventitious shoots developed directly from the explant when cultured on regeneration medium supplemented with 2 mg l1 IBA, 2 mg l1 TDZ and 4% sucrose. The key factor for shoot regeneration was the dorsoventral orientation of the leaf. Only when leaves were placed with the right (adaxial) side up (Fig. 2B(1)) could we get high regeneration efficiency of up to 100%, with more than five shoots per regenerating explant in both studied cultivars. In contrast, if leaves were placed with their abaxial side up, shoot regeneration took place, but still mostly from the adaxial surface. A similar phenomenon has been observed for African violet [26–28]. Lo et al. [26,27] hypothesized that the difference in regeneration response may be due to morphological differences between the adaxial and abaxial epidermal layers, requiring different threshold levels of hormones for regeneration. The adventitious shoots of Brown Turkey and Smyrna emerged preferably at the central and distal parts of the leaf. We cannot rule out the presence of unidirectional polarity in fig leaves, but further studies are needed to address this question. In contrast, apple explants taken from segments closest to the proximal end of the cotyledons [29] and leaf petiole [30] were more regenerable than distal segments. This polarity could result from topological, developmental, morphological, or chemical influence, and/or a combination of some or all of these factors [29]. TDZ has been reported to be very efficient in stimulating adventitious shoot production in several recalcitrant woody plants [31–36]. Additional evidence for the influence of TDZ in combination with different auxins on the morphogenic potential of apples was recently provided [37]. Here, we confirmed the stimulatory effect of TDZ in combination with IBA on fig adventitious shoot regeneration. Fig plantlets could be transferred directly into rooting cylinders without addition of any auxin. The high efficiency (100%) of root formation was followed by improved hardening and subsequent successful acclimatization of the plants in the greenhouse conditions. 4.2. Transformation
Fig. 7. Demonstration of the T-DNA integration in the cvs. Brown Turkey (A) and Smyrna (B) genome by Southern blot analysis. Total DNA was digested with HindIII and hybridized with the nptII probe. Lanes 1–3, transgenic clones; C, untransformed plants.
The present study describes for the first time the successful transformation of the commercially important fig cultivars Brown Turkey and Smyrna using the uidA-intron and nptII genes. We found that under selective conditions, transforma-
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tion frequencies varied in Brown Turkey from 1.7 to 10.0% and in Smyrna from 2.1 to 7.8%. We also observed that culturing with more than three subcultures in selective conditions of 100 mg l1 kanamycin resulted in inhibited development of the new emerging transgenic shoots. In light of these observations, we suggest the following design of regeneration/selection process: one subculture (4 weeks) on regeneration medium with 50 mg l1 kanamycin and 150 mg l1 ticarcillin, followed by two subcultures (6 weeks) of the isolated shoots on PM with 100 mg l1 kanamycin and 150 mg l1 ticarcillin, and finally, one subculture only with ticarcillin (150 mg l1) before leaves from the putative transgenic plants are taken for histochemical GUS staining. The selection scheme developed in this study enabled early isolation of shoots from the original explant. Transgenic status of the selected clones has been confirmed by GUS histochemical assay and molecular analysis. Positive PCR was shown in all tested clones (Fig. 6A and B). Southern blot provided additional confirmation for the integration of the nptII gene (Fig. 7A and B).
5. Conclusion Successful transformation and subsequent regeneration of the two fig commercial cultivars Brown Turkey (fresh consumption) and Smyrna (dry consumption), using the procedures described here, provide a new means for the introduction of foreign genes into fig species, specifically into commercially valuable fig tree (F. carica). This technology should pave the way for the development of transgenic Ficus varieties with improved agronomic performance characteristics. It also provides a new experimental system for studying gene expression and function in these species, and a means for the production of foreign proteins in edible parts of Ficus species, such as the production of edible vaccines. The use of improved F. carica varieties via the utilization of the transformation and regeneration technology also facilitates the implementation of sustainable agricultural practices in fig tree cultivation.
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[7] A. Demiralay, Y. Yalcin-Mendi, Y. Aka-Kacar, S. Cetiner, In vitro propagation of Ficus carica L. var. Bursa siyahi through meristem culture, Acta Hort. 480 (1998) 165–167. [8] G. Gu˝ nver, E. Ertan, A study on the propagation of figs by the tissue culture techniques, Acta Hort. 480 (1998) 169–172. [9] R. Gella, J.A. Marin, M.L. Corrales, F. Toribio, Elimination of fig mosaic from fig shoot tip cultures by thermotherapy, Acta Hort. 480 (1998) 173–177. [10] J. Grbelja, Z. Eric, Isolation of a potyvirus from Ficus carica L., Acta Bot. Croatia. 4 (1983) 11–14. [11] A.H. Nassar, H.J. Newbury, Ficin production by callus cultures of Ficus carica, J. Plant Physiol. 131 (1987) 171–179. [12] F. Cormier, C. Charest, C. Dufresne, Partial purification and properties of proteases from fig (Ficus carica) callus cultures, Biotechnol. Lett. 11 (1989) 797–802. [13] V.S. Jaiswal, P. Narayan, Regeneration of plantlets from the callus of stem segment of adult plants of Ficus religiosa L., Plant Cell Rep. 4 (1985) 256–258. [14] S.R. Deshpande, P.C. Josekutty, G. Prathapasenan, Plant regeneration from axillary buds of a mature tree of Ficus religiosa, Plant Cell Rep. 17 (1998) 571–573. [15] V. Kumar, A. Radha, S. Kumar Chitta, In vitro plant regeneration of fig (Ficus carica L. cv. gular) using apical buds from mature trees, Plant Cell Rep. 17 (1998) 717–720. [16] H. Yakushiji, N. Mase, Y. Sato, Adventitious bud formation and plantlet regeneration from leaves of fig (Ficus carica L.), J. Hort. Sci. Biotechnol. 78 (2003) 874–878. [17] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473– 497. [18] A. Almehdi, D.E. Parffit, In vitro propagation of peach. I. Propagation of ‘‘Lovell’’ and ‘‘Nemaguard’’ peach rootstocks, Fruit Varieties J. 40 (1986) 12–17. [19] J.P. Nitsch, C. Nitsch, Haploid plants from pollen grains, Science 163 (1969) 85–87. [20] E.E. Hood, S.B. Gevin, L.S. Melchers, A. Hoekema, New Agrobacterium vectors for plant transformation, Transgen. Res. 2 (1993) 208– 218. [21] G. Vancanneyt, R. Schimidt, A. O’Connor-Sanchez, L. Willmitzer, M. Rocha-Sosa, Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation, Mol. Gen. Genet. 220 (1990) 245–250. [22] J. Alt-Mo¨ rbe, H. Ku˝ hlman, J. Schro¨ der, Differences in induction of Ti plasmid virulence genes virG and virD, and continued control of virD expression by four external factors, MPMI 2 (1989) 301– 308. [23] M.G. Murray, W.F. Tommpson, Rapid isolation of high molecular plant DNA, Nucl. Acids Res. 8 (1980) 4321–4325. [24] H. Wabiko, M. Minemura, Exogenous phytohormone-independent growth and regeneration of tobacco plants transgenic for the 6b gene of Agrobacterium tumefaciens AKE10, Plant Physiol. 112 (1996) 939– 951. [25] R.A. Jefferson, T.A. Kavanagh, M.W. Bevan, GUS fusions: b-glucuronidase as a sensitive versatile gene fusion marker in higher plants, EMBO J. 6 (1987) 3901–3907. [26] K.H. Lo, K.L. Giles, V.K. Sawhney, Acquisition of competence for shoot regeneration in leaf discs of Saintpaulia ionantha confusa hybrids (African violet) cultured in vitro, Plant Cell Rep. 16 (1997) 416–420. [27] K.H. Lo, K.L. Giles, V.K. Sawhney, Histological changes associated with acquisition of competence for shoot regeneration in leaf discs of Saintpaulia ionantha confusa hybrid (African violet) cultured in vitro, Plant Cell Rep. 16 (1997) 421–425. [28] S. Ohki, Scanning electron microscopy of shoot differentiation in vitro from leaf explants of the African violet, Plant Cell Tissue Org. Cult. 36 (1994) 157–162.
S.D. Yancheva et al. / Plant Science 168 (2005) 1433–1441 [29] M. Kouider, S.S. Korban, R.M. Skirvin, M.C. Chu, Influence of embryonic dominance and polarity on adventitious shoot formation from apple cotyledons in vitro, J. Am. Soc. Hort. Sci. 109 (1984) 381– 385. [30] M. Welander, G. Maheswaran, Shoot regeneration from leaf explants of dwarfing apple rootstocks, J. Plant Physiol. 140 (1992) 223–228. [31] C. Leblay, E. Chevreau, L.M. Raboin, Adventitious shoot regeneration from in vitro leaves of several pear cultivars (Pyrus communis L.), Plant Cell Tissue Org. Cult. 25 (1991) 99–105. [32] S.D. Yancheva, Preliminary studies on regeneration and transformation of plum (Prunus domestica L.), Acta Hort. 359 (1994) 159–163. [33] O. Perez-Tornero, J. Egea, A. Vanoostende, L. Burgos, Assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot, Plant Sci. 158 (2000) 61–70.
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[34] S.S. Korban, P.A. O’Connor, A. Elobeidy, Effect of thidiazuron, naphthalene acetic acid, dark incubation and genotype on shoot organogenesis from Malus leaves, J. Hort. Sci. 67 (1992) 341–349. [35] M. Sarwar, R.M. Skirvin, Effect of thidiazuron and 6-benzylaminopurine on adventitious shoot regeneration from leaves of three strains of ‘McIntosh’ apple (Malus domestica Borkh.) in vitro, Sci. Hort. 68 (1997) 95–100. [36] N. Gamage, T. Nakanishi, In vitro shoot regeneration from leaf tissue of apple (Cultivar ‘‘Orine’’): high shoot proliferation using carry over effect of TDZ, Acta Hort. 520 (2000) 291–298. [37] S.D. Yancheva, S. Golubowicz, E. Fisher, S. Lev-Yadun, M.A. Flaishman, Auxin type and timing of application determine the activation of the developmental program during in vitro organogenesis in apple, Plant Sci. 165 (2003) 299–309.
Efficient Agrobacterium-mediated transformation and recovery of transgenic fig (Ficus carica L.) plants Svetla D. Yancheva b, Sara Golubowicz a, Zeev Yablowicz a, Avi Perl a, Moshe A. Flaishman a,* a
Department of Fruit Tree Sciences, Institute of Horticulture, ARO, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel b Plant Biotechnology Laboratory, Agricultural University, 12 Mendeleev St., 4000 Plovdiv, Bulgaria Received 3 October 2004; received in revised form 28 November 2004; accepted 3 December 2004 Available online 27 December 2004
Abstract An efficient and reproducible system for regeneration and Agrobacterium-mediated transformation of the common fig (Ficus carica L.) cultivars Brown Turkey (fresh consumption) and Smyrna (dry consumption) was developed. Optimal shoot regeneration (up to 100%) was obtained on MS basal salt mixture supplemented with 100 mg l1 myo-inositol, 1 mg l1 thiamine HCl and addition of 2.0 mg l1 thidiazuron (TDZ), 2 mg l1 indole-3-butyric acid (IBA), 4% sucrose and 0.8% agar. Regeneration was highly dependent on the dorsoventral orientation of the explants. When explants were cultured with the adaxial surface up, 100% regeneration was achieved with more than five shoots per regenerating explant in both studied cultivars. In contrast, if leaves were placed with their abaxial side up, shoot regeneration took place, but still mostly from the adaxial surface. Leaf explants of in vitro propagated plants were co-cultivated with the disarmed Agrobacterium strain EHA105 harboring the plasmid pME504 that carried the uidA-intron and nptII genes. Transformation efficiencies were in a range of 1.7– 10.0% for cv. Brown Turkey and 2.8–7.8% for Smyrna. The transgenic nature of the regenerated plants was confirmed by molecular analyses (PCR and Southern blot) as well as by GUS staining. Similar to regeneration, the orientation of the leaf surface during organogenesis was a key factor for successful transformation. Successful transformation of commercial fig cultivars provides a new promising tool for the introduction of foreign genes into transgenic fig cultivars. The regeneration and transformation methodologies described here may pave the way for transgenic varieties with improved agronomic characteristics, such as storability and disease resistance, and will provide a means for the production of foreign proteins in the edible parts of fig, leading to improved nutritional and/or pharmaceutical composition. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Fig (Ficus carica); Transformation; Regeneration; GUS; nptII; Agrobacterium tumefaciens
1. Introduction Fig trees are one of the earliest fruit-bearing trees cultivated. Ficus carica (Moraceae), the well-known fig of commerce, is indigenous to wide areas ranging from Asiatic Turkey to North India, and natural varieties are cultivated in Abbreviations: BA, 6-benzylaminopurine; CaMV, cauliflower mosaic virus; GA3, gibberellic acid A3; GUS, b-glucuronidase; IBA, indole-3butyric acid; NAA, a-naphthalene acetic acid; nptII, neomycin phosphotransferase II; PCR, polymerase chain reaction; TDZ, thidiazuron (Nphenyl-N0 -1,2,3-thiadiazol-5-yl urea); uidA, b-glucuronidase gene of Escherichia coli * Corresponding author. Tel.: +972 3 968 3394; fax: +972 3 966 9583. E-mail address: [email protected] (M.A. Flaishman).
most Mediterranean countries [1]. Fig fruit is well known for its nutritive value, and is consumed fresh or dry worldwide. Fig fruits are also known for their mild laxative activity and high alkalinity, and substances derived from them are used in various drug preparations. Other parts of fig trees have also been shown to have a commercial value [2]. Traditional breeding methods of F. carica require a longterm effort for improving traits such as production of parthenocarpic varieties. Among F. carica there are trees that bear only female figs and others trees, named caprifig, bear both male and female flowers. Edible fruits can be produced only on trees bearing female synconia, provided cross-pollination is mediated by a specific wasp [3]. Thus, traits originated via pollen are hard to track.
0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.12.007
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The application of genetic engineering techniques to stably incorporate homologous and/or heterologous genetic material into woody species, including fruit trees, offers the potential of obtaining improved planting stocks for agricultural use in a short period of time compared to traditional breeding techniques. In addition, efficient transformation methods can be used for the production of heterologous polypeptides having nutritional and/or pharmaceutical value. The overall efficiency of techniques for genetically modifying plants depends upon the efficiency of the transformation technique(s) used to stably incorporate the desired genetic material into plant cells or tissues, and the regeneration technique(s) used to produce viable plants from transformed cells. Within Ficus sp. adventitious shoot regeneration has been reported only for F. lyrata from young leaves isolated from plants maintained under greenhouse conditions [4]. In vitro studies using F. carica were restricted to the development of protocols for micropropagation and production of figmosaic-virus-free plants by single shoot tip culture [5–10] as well as to biochemical assessment of active compounds in fig calli grown in vitro [11,12]. Within the genus Ficus, several reports described regeneration and organogenesis from calli and other explants. F. religiosa plants were regenerated from calli of stem segments [13] and from axillary buds of mature trees [14]. Regeneration of F. carica plants from the apical buds of a mature tree was also reported [15]. However, in all mentioned in vitro studies, plant regeneration has been restricted to the use of single shoot tips and apical buds. Recently, Yakushiji et al. [16] reported a method for the induction of organogenesis from leaf explants of F. carica using phloroglucinol (PG). However, by this method the frequency of adventitious bud differentiation from leaf fragments was relatively low, and no adventitious buds were observed without PG. Moreover, to date no method for the introduction of isolated genetic material into Ficus species has been reported. The overall objective of the current research was to develop an optimized protocol for in vitro regeneration of fig that allows for successful application of Agrobacteriummediated transformation in two fig (F. carica) commercial cultivars Brown Turkey (fresh consumption) and Smyrna (dry consumption).
2. Materials and methods 2.1. Fig shoot culture maintenance Fig (F. carica) cultivars Brown Turkey (common for obtaining fresh fruit) and Smyrna (common for dry fruit production) were used. In vitro shoot cultures of both cultivars were established according to Pontikis and Melas [6] and subsequently grown on proliferation medium (PM) consisting of MS [17] basal salt mixture supplemented with 100 mg/l myo-inositol, 0.5 mg/l thiamine–HCl, 3% sucrose
(w/v), 0.8% agar (Sigma), 0.25 mg/l BA, 0.05 mg/l IBA, and 0.05 mg/l GA3. The pH was adjusted to 5.7 prior to autoclaving for 20 min at 120 8C. Two shoots were placed horizontally in Magenta boxes containing 50 ml PM and exposed to cool white fluorescent light (40 mmol/m2 s) in a 16/8 h photoperiod at 25 8C for 4–5 weeks before leaf explants were removed. 2.2. Plant regeneration Regeneration experiments were carried out initially with the cultivar Brown Turkey. To examine the effect of culture media on adventitious bud formation, the basal media MS, AP [18] and NN [19], including various combinations of only the auxins IBA and NAA (0.25–2 mg/l), or NAA in combination with TDZ (0.5–5 mg/l) or BA (2–5 mg/l) were examined. A control treatment with only TDZ (0.5–5 mg/l) was performed. The regeneration media contained MS basal salt mixture, supplemented with 100 mg/l myo-inositol, 1 mg/l thiamine–HCl, 2–4% sucrose (w/v) and 0.8% agar (Sigma) at pH 5.7, with or without 0.25% activated charcoal (AC). The three youngest expanding leaves isolated from 3 to 4 weeks old plants were used as explants. Each leaf was wounded by forming three cuts perpendicular to the central vein and placed on regeneration medium with either the abaxial or the adaxial surface facing the medium. At least 10 Petri dishes, each containing 10 explants were used per treatment. The cultures were kept for 7 days in low light intensity (2.5 mmol/m2 s) followed by exposure to high light intensity (40 mmol/m2 s) at 25 8C, in a 16/8 h photoperiod. Leaf explants were examined after 28 and 35 days and the percentage of explant producing shoots (regeneration capacity) and the mean of adventitious shoots formed per regenerating explant (regeneration efficacy) were recorded. All experiments were repeated at least three times. 2.3. Rooting and acclimatization Rooting and acclimatization experiments were carried out initially with the cultivar Brown Turkey. Two methods for rooting were evaluated. According to one method, each shoot was cultivated individually in a tube with root induction (RI) medium composed of half strength MS medium (1/2MS) supplemented with 100 mg/l myoinositol, 1 mg/l thiamine–HCl, 90 mg/l phloroglucinol, 2% sucrose (w/v), 0.25% activated charcoal (AC) and 0.8% agar (pH 5.7), with or without IBA at different concentrations (0, 1, 2 mg/l). Each treatment included 32 individual plants. According to the second method, well developed shoots (up to 3–4 cm long with 3–4 expanded leaves) were directly transplanted from the proliferation medium to rooting cylinders (3.5 cm high 3.0 cm in diameter) with soil mixture comprising 55% granular polypropylene foam, 30% peat and 15% perlite (Tivonchem Ltd., Israel). Soil mixture was moistened by soaking the rooting cylinders in
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liquid RI medium supplemented with either 0, 1 or 2 mg/l IBA. Each treatment consisted of 32 shoots and results were scored after 4 weeks. Each experiment was repeated three times. 2.4. Histology The regeneration process was examined by light microscopy. Samples were fixed in a solution of freshly prepared FAA (5% formalin, 25% acetic acid, 70% alcohol), dehydrated in a graded ethanol series and embedded in Paraplast. Sections were cut at 10 mm thickness, stained in Safranin and Fast-Green, mounted in Permount (Fisher) and examined, using Leica DMLB light microscope. 2.5. Agrobacterium tumefaciens strain and plasmid Super-virulent A. tumefaciens strain EHA 105 [20] harboring the vector pME 504 carrying the nptII and the uidA-intron genes [21] was used. Agrobacterium culture was grown overnight in LB medium (Duchefa L-1704) with appropriate antibiotics. Bacteria were spun down by centrifugation (4000 rpm for 15 min), resuspended in liquid SIM medium [22] supplemented with 100 mM acetosyringone (AS) to obtain a final OD600 of 0.7, and incubated in an orbital shaker at 28 8C and 250 rpm for 4 h. 2.6. Transformation Leaves of 3–4 weeks old micropropagated shoots were wounded across the midrib with a scalpel and immersed in the bacterial suspension for 20 min, dry-blotted on filter paper and cultured on regeneration medium based on MS medium supplemented with 2.0 mg/l TDZ and 2 mg/l IBA, 4% sucrose and 0.8% agar at pH 5.7. Co-cultivation medium was supplemented with 100 mM AS. After co-cultivation for 72 h in the dark at 25 1 8C, the explants were washed in liquid MS medium supplemented with 300 mg/l ticarcillin, dry-blotted and transferred to the regeneration medium with addition of ticarcillin (150 mg/l) and either kanamycin (50, 75 and 100 mg/l) or paromomyci (25 and 50 mg/l). All cultures were kept during the first week at low light intensity (2.5 mmol/m2 s) and subsequently transferred for the next 3 weeks to fluorescent light (40 mmol/m2 s) in 16 h light/8 h dark photoperiod at 25 1 8C. Regenerated shoots were developed after two subcultures in PM supplemented with 100 mg/l kanamycin and 150 mg/l ticarcillin. Transformation rate was calculated as the number of independent transformation events (kanamycin resistant/GUS expressing shoots) obtained out of the total number of explants. Multiplication and maintenance of the selected plants were performed by culturing the shoots horizontally on PM medium supplemented with kanamycin (50 or 100 mg/l) and ticarcillin (150 mg/l). Rooting procedure was carried out in rooting cylinders soaked in the liquid medium described above, with the addition of 100 mg/l kanamycin.
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2.7. Molecular confirmation of transformation To verify the presence and integration of the nptII and GUS genes, all selected clones were subjected to molecular analyses by PCR and Southern blotting. Plant genomic DNA was isolated from the youngest three leaves excised from kanamycin-resistant shoots according to Murray and Tommpson [23]. The oligonucleotide primers used for the PCR amplification of a 645-bp fragment of the nptII gene were: direct primer 50 -GCCGCTTGGGTGGAGAGGCTAT-30 (63.6 8C); reverse primer 50 -GAGGAAGCGGTCAGCCCATTC-30 (60 8C). The primers for a 676-bp fragment of the GUS gene were: GUSup 50 -CGAGCGATTTGGTCATGTGAAG30 (57.5 8C); GUSlow primer 50 -CATTGTTTGCCT CCCTGCTGCGGTT-30 (55.9 8C) (Sigma). Amplification was performed in aliquots of 25 ml using a thermal cycler (Biometra). The PCR conditions for amplification of the nptII gene fragment were 95 8C for 5 min, followed by 35 cycles at 94 8C for 1 min, 62 8C for 1 min, 72 8C for 1 min and a final extension at 72 8C for 10 min. Amplification of the uidA-intron fragment was performed according to the following program: 95 8C for 5 min followed by 35 cycles at 94 8C for 45 s, 55 8C for 45 s, 72 8C for 45 s and a final extension at 72 8C for 10 min. For Southern blot analysis, 10 mg of genomic DNA was digested with HindIII, subjected to electrophoresis on 0.8% agarose gel and transferred to nylon membranes (GeneScreen Plus, NEN, Boston, MA). Southern hybridization was performed using a [32P]-radioactively labeled 524-bp nptII gene fragment as a probe, as described by Wabiko and Minemura [24]. Hybridizations were performed for 20 h at 65 8C. Filters were washed twice, at high-stringency conditions, with 0.1% SDS 2 SSC for 30 min at 65 8C and exposed to autoradiography film with intensifying screens at 80 8C. 2.8. Histochemical GUS assay Histochemical analysis was performed following the procedure of Jefferson et al. [25]. Transient GUS expression was assayed 4 and 7 days after infection. During regeneration, leaf sections were excised from regenerating shoots and cultivated for 4 weeks under selective conditions. GUS expression was also further examined in mature putatively transformed shoots and in rooted plantlets.
3. Results 3.1. Plant regeneration 3.1.1. Effect of growth regulators In preliminary experiments, about 30% of the explants exhibited shoot regeneration when cultured on MS basal medium supplemented with IBA and TDZ while only 15%
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medium supplemented with 2 mg/l IBA and 2 mg/l TDZ, under conditions of 1 week low intensity light followed by transfer to normal light, comprised the optimal conditions for obtaining high regeneration (50%) rate and increased rate of shoot differentiation. Histological observations (Fig. 2A) showed that the regeneration process followed the developmental pattern of direct organogenesis. Adventitious shoots developed from meristematic centers, appearing in both epidermal and mesophyll tissues. Within 25 days, these centers (foci) became visible on the wounded surface of the explant. A prolonged culture of up to 42 days resulted in explant expansion, but the developing shoots were surrounded and covered by significant amount of callus, which inhibited their further growth.
Fig. 1. Effect of growth regulator concentration and light conditions on shoot regeneration of cv. Brown Turkey. Leaf explants were cultured with the abaxial side up followed by (A) 1 week low intensity light or (B) 2 weeks in the dark and 1 week low intensity light followed by transfer to normal light. Vertical bars indicate standard error (S.E.).
shoot regeneration was observed when MS medium was supplemented with NAA and TDZ. On the basis of these observations, we further studied the effects on shoot regeneration of MS medium supplemented with different concentrations of IBA and TDZ. Fig. 1A and B shows the effect of growth regulators concentration and two different light conditions on the regeneration of leaf explants obtained from shoots grown in PM medium. Higher regeneration rate was obtained when leaf explants were cultured for 1 week at low light intensity (2.5 mmol/m2 s) and subsequently transferred to normal light intensity (40 mmol/m2 s). A combination of 2 mg/l IBA and 2 mg/l TDZ yielded the best regeneration rate of 50.2%, with an average of 3.2 0.6 shoots per regenerating explant (Fig. 1A). Similar regeneration efficiencies (42.6–46.6%) were obtained when leaf explants were grown in a medium containing 1 mg/l IBA and 2 mg/l TDZ; however, most of the explants turned brown and formed excessive calli. Culturing of control explants only with TDZ induced significant explant expansion and compact calli formation, however with a very low shoot regeneration rate of ca. 2%. The addition of AC did not positively influence the rate of regeneration (data not shown). Two-week long dark treatment before the exposure of plants to low light intensity was also not advantageous (Fig. 1B). Hence, cultivating leaf explants on MS basal
3.1.2. Effect of sucrose concentration and leaf surface position on the solid medium Our studies demonstrated that the position of the leaf surface placed on the medium, in combination with sucrose concentration, had a significant effect on shoot regeneration from leaf explants. Data presented in Fig. 3 indicate that cultivating the explants with their adaxial surface up led to a considerable increase in their regeneration rate. In media supplemented with 4% sucrose, leaf explants cultured with their adaxial side up showed 100% of adventitious shoot formation with more than five shoots per regenerating explant. Most of the adventitious shoots developed directly, mainly from the wounding sited at the central and distal part of the leaf petiole (Fig. 2B(1)). When the explants were cultured with their abaxial side up, shoot formation still occurred only on the adaxial side of the leaf, in parallel to explant necrogenesis and calli (Fig. 2B(2)). 3.2. Rooting Two methods were examined to obtain root induction (Table 1). According to the first approach, each regenerated shoot was placed in a tube containing RI medium containing 2 mg/l IBA. Using this method, ca. 80% of the shoots formed roots. However, only low percentage of these plants successfully acclimatized under greenhouse conditions. According to the second approach, regenerated shoots were cultured directly in rooting cylinders, with or without 1 mg/l IBA. After 4 weeks, 100% root formation was achieved, independent of the concentration of auxin used (Fig. 2C). These plants had better hardening and underwent easier acclimatization in greenhouse conditions. 3.3. Transformation of F. carica 3.3.1. Choice of selective antibiotics In preliminary studies, we calibrated the killing effect of kanamycin on shoot regeneration from leaf explants derived from both cultivars tested. Wounded leaves were placed on regeneration medium supplemented with kanamycin at
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Fig. 2. Regeneration, rooting and acclimatization of plants from cv. Brown Turkey. (A) Light microscopy observations of shoot organogenesis in fig. Leaf explants of cv. Brown Turkey were cultivated on medium with IBA (2 mg l1) and TDZ (2 mg l1). (1) After 15 days, the first meristematic domes appeared on the adaxial surface of the explant. Bar = 100 mm. (2) After 21 days, appearance of adventitious buds and leaf primordia on the adaxial epidermis. Bar = 200 mm. (3) After 28 days, appearance of adventitious shoots with differentiated apical and axillary meristems and developing leaves. Bar = 100 mm; C – callus, CD – cell division, DL – developing leaves, E – epidermis, M – meristem, VS – vascular system. (B) Adventitious shoot formation in fig cv. Brown Turkey. Stereomicroscopic observation of leaf explants cultured 4 weeks on regeneration medium (1) with the adaxial side up (0.63 0.8) or (2) with abaxial side up (0.63 1.25). (C) Rooting and acclimatization of plants from cv. Brown Turkey. (1) In vitro rooted plant (bar, 1 cm). (2) Plants in rooting cylinders. (3) Potted plants in the greenhouse 1 month after acclimatization.
various concentrations (0, 10, 25, 50, 75 and 100 mg/l). The minimal concentration for inhibition of adventitious shoot induction in cv. Brown Turkey was 25 mg/l. However, with cv. Smyrna a few single buds were formed at 25 mg/l kanamycin, but they remained white and did not develop further (Fig. 4). Therefore, the concentration of 50 mg/l
kanamycin was initially chosen for selection in subsequent transformation experiments. 3.3.2. Regeneration of transgenic shoots Following co-cultivation with A. tumefaciens strain EHA105 harboring the plasmid pME504, explants were initially placed with their adaxial side up on regeneration medium supplemented with 50 mg/l kanamycin and 150 mg/l ticarcillin. Explants were exposed for 7 days to low intensity light and then transferred to normal light. Under selective conditions, up to 30% of the explants of Brown Turkey and up to 50% of Smyrna formed green Table 1 Effect of two rooting methods and IBA concentrations on root formation of cv. Brown Turkey IBA (mg l1)
Fig. 3. Influence of sucrose concentration and orientation of the leaf surface on adventitious shoot regeneration. Leaf explants of cv. Brown Turkey were cultured on medium with TDZ (2 mg l1) and IBA (2 mg l1). Vertical bars indicate standard error (S.E.).
0 1 2
Root formation S.E. (%) In vitro rooting
Rooting cylinders
8.3 1.59 47 5.43 81.3 7.61
100 100 83 6.27
Two methods were examined: in vitro rooting and then after acclimatizing under greenhouse conditions and culturing directly in rooting cylinders.
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Fig. 4. Kanamycin sensitivity of leaf explants of the fig cv. Brown Turkey and Smyrna.
shoots after 4 weeks in culture. The regenerating shoots were transferred to PR medium supplemented with 100 mg/l kanamycin and 150 mg/l ticarcillin for further selection. Cultivation of the new emerging transgenic shoots for more than three subcultures under selective conditions using 100 mg/l kanamycin on proliferation medium resulted in inhibition of their growth and development. Using this selection screen, transformation frequency established for cv. Brown Turkey varied in the different experiments from 1.7 to 10% and for cv. Smyrna from 2.1 to 7.8%. 3.3.3. Histochemical GUS assay High level of explants transiently expressing GUS was observed for leaves that were placed during co-cultivation with their adaxial side up. Histochemical GUS analysis of such leaves carried out 3 and 7 days after infection showed that 95–100% of the explants of both cultivars had large blue sectors (Fig. 5A). However, when cultured with their abaxial side up no transient GUS expression was observed in either of the two fig varieties (data not shown). After 4 weeks under selective conditions some of the regenerating explants (20 leaves) stained positively for GUS. Stereo-microscopic analyses (Fig. 5B) of leaves of cv. Brown Turkey showed that 7.46% (10 out of 134) of the shoots were GUS positive. An in situ b-glucuronidase assay was carried out to further confirm the integration and expression pattern of the uidA gene. Intact plants of all selected shoots propagated on kanamycin-containing medium as well as control (wild type) plants were tested. Strong GUS expression was evident in the leaves and stems of the transgenic plants (Fig. 5C). 3.3.4. Molecular confirmation of transformation PCR analysis of the putative transgenic shoots confirmed the stable incorporation of the transgenes into the F. carica genome. All clones selected after transformation with pME504 showed the predicted bands: the 645 bp for the nptII gene and the 676 bp for the uidA-intron (GUS). No fragment was amplified in the control, untransformed plant (Fig. 6A and B).
Fig. 5. Histochemical GUS analysis: stereomicroscopic observations. (A) Transient GUS expression 3 days after inoculation of leaf explants of cv. Brown Turkey (0.63 1.0). (B) GUS staining of regenerating leaf explants after 4 weeks culture in the presence of 50 mg l1 kanamycin and 150 mg l1 ticarcillin (0.63 2.5). (C) GUS expression detected in an isolated putatively transformed shoot cultured on PM with 100 mg l1 kanamycin and. 150 mg l1 ticarcillin (0.63 2.0). (D) GUS assay of transgenic clone BT 001.
Southern blot analysis of HindIII digested genomic DNA from three randomly selected putative transgenic plants of Brown Turkey and three of Smyrna provided additional molecular evidence for the incorporation of the foreign DNA (Fig. 7A and B). The Southern blot data were consistent with the plasmid pME504, that has one HindIII restriction site, located 2.8 kb from the plasmid right border after the nptII gene. No hybridization bands were present in the control, untransformed plants. Southern blot analysis of HindIII digested DNA from the six putative transgenic plants provided additional evidence for the incorporation of the foreign DNA. The nptII bands were larger than 2.8 kb, varying in size between the six clones. The hybridization pattern in clone 1 of Smyrna cv. indicated insertion of the TDNA into multiple different loci (Fig. 7B).
4. Discussion In the last two decades, transformation technology has played an increasingly important role in the genetic manipulation of crop plants. This approach has led to crop improvement as well as to better understanding of the molecular mechanisms underlying plant gene expression and regulation. However, due to a lack of transformation and regeneration procedures, the application of such biotechnological approaches has not been possible for species in the genus Ficus. The present study provides a highly efficient system for in vitro regeneration and subsequent transformation of fig trees (F. carica). A key factor in the high efficacy
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of both transformation and shoot regeneration is the dorsoventral orientation of the explants. 4.1. The regeneration process in fig
Fig. 6. PCR analysis of the putative transgenic shoots showed the predicted bands for the nptII gene and uidA-intron (GUS) gene in the cvs. Brown Turkey (A) and Smyrna (B). For Brown Turkey (A): M – lambda DNA/ EcoRI + HindIII marker; lanes 1–4, transgenic clones; C, untransformed plants. For Smyrna (B): M – lambda DNA/EcoRI + HindIII marker; lanes 1–3, transgenic clones; C, untransformed plants.
The success of gene transfer techniques is largely dependent on an efficient regeneration system. Only Yakushiji et al. [16] have reported successful regeneration of F. carica, though with a low regeneration frequency of only 22%. The present study is the first report of a highly efficient system for in vitro regeneration in figs using cv. Brown Turkey and Smyrna. Here, adventitious shoots developed directly from the explant when cultured on regeneration medium supplemented with 2 mg l1 IBA, 2 mg l1 TDZ and 4% sucrose. The key factor for shoot regeneration was the dorsoventral orientation of the leaf. Only when leaves were placed with the right (adaxial) side up (Fig. 2B(1)) could we get high regeneration efficiency of up to 100%, with more than five shoots per regenerating explant in both studied cultivars. In contrast, if leaves were placed with their abaxial side up, shoot regeneration took place, but still mostly from the adaxial surface. A similar phenomenon has been observed for African violet [26–28]. Lo et al. [26,27] hypothesized that the difference in regeneration response may be due to morphological differences between the adaxial and abaxial epidermal layers, requiring different threshold levels of hormones for regeneration. The adventitious shoots of Brown Turkey and Smyrna emerged preferably at the central and distal parts of the leaf. We cannot rule out the presence of unidirectional polarity in fig leaves, but further studies are needed to address this question. In contrast, apple explants taken from segments closest to the proximal end of the cotyledons [29] and leaf petiole [30] were more regenerable than distal segments. This polarity could result from topological, developmental, morphological, or chemical influence, and/or a combination of some or all of these factors [29]. TDZ has been reported to be very efficient in stimulating adventitious shoot production in several recalcitrant woody plants [31–36]. Additional evidence for the influence of TDZ in combination with different auxins on the morphogenic potential of apples was recently provided [37]. Here, we confirmed the stimulatory effect of TDZ in combination with IBA on fig adventitious shoot regeneration. Fig plantlets could be transferred directly into rooting cylinders without addition of any auxin. The high efficiency (100%) of root formation was followed by improved hardening and subsequent successful acclimatization of the plants in the greenhouse conditions. 4.2. Transformation
Fig. 7. Demonstration of the T-DNA integration in the cvs. Brown Turkey (A) and Smyrna (B) genome by Southern blot analysis. Total DNA was digested with HindIII and hybridized with the nptII probe. Lanes 1–3, transgenic clones; C, untransformed plants.
The present study describes for the first time the successful transformation of the commercially important fig cultivars Brown Turkey and Smyrna using the uidA-intron and nptII genes. We found that under selective conditions, transforma-
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tion frequencies varied in Brown Turkey from 1.7 to 10.0% and in Smyrna from 2.1 to 7.8%. We also observed that culturing with more than three subcultures in selective conditions of 100 mg l1 kanamycin resulted in inhibited development of the new emerging transgenic shoots. In light of these observations, we suggest the following design of regeneration/selection process: one subculture (4 weeks) on regeneration medium with 50 mg l1 kanamycin and 150 mg l1 ticarcillin, followed by two subcultures (6 weeks) of the isolated shoots on PM with 100 mg l1 kanamycin and 150 mg l1 ticarcillin, and finally, one subculture only with ticarcillin (150 mg l1) before leaves from the putative transgenic plants are taken for histochemical GUS staining. The selection scheme developed in this study enabled early isolation of shoots from the original explant. Transgenic status of the selected clones has been confirmed by GUS histochemical assay and molecular analysis. Positive PCR was shown in all tested clones (Fig. 6A and B). Southern blot provided additional confirmation for the integration of the nptII gene (Fig. 7A and B).
5. Conclusion Successful transformation and subsequent regeneration of the two fig commercial cultivars Brown Turkey (fresh consumption) and Smyrna (dry consumption), using the procedures described here, provide a new means for the introduction of foreign genes into fig species, specifically into commercially valuable fig tree (F. carica). This technology should pave the way for the development of transgenic Ficus varieties with improved agronomic performance characteristics. It also provides a new experimental system for studying gene expression and function in these species, and a means for the production of foreign proteins in edible parts of Ficus species, such as the production of edible vaccines. The use of improved F. carica varieties via the utilization of the transformation and regeneration technology also facilitates the implementation of sustainable agricultural practices in fig tree cultivation.
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