Plant Cell Rep (2003) 22:122–128 DOI 10.1007/s00299-003-0654-1
GENETIC TRANSFORMATION AND HYBRIDIZATION
R. L. Boscariol · W. A. B. Almeida · M. T. V. C. Derbyshire · F. A. A. Mour¼o Filho · B. M. J. Mendes
The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck) Received: 29 November 2003 / Revised: 2 May 2003 / Accepted: 5 May 2003 / Published online: 19 July 2003 Springer-Verlag 2003
Abstract A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides. Keywords Agrobacterium tumefaciens · Citrus transformation · Sweet orange · Mannose selection marker Communicated by L. Pea R. L. Boscariol · B. M. J. Mendes ()) Laboratrio de Biotecnologia Vegetal, Centro de Energia Nuclear na Agricultura, Universidade de S¼o Paulo, Piracicaba 13 400-970 S¼o Paulo, Brazil e-mail:
[email protected] Tel.: + 55-19-34294686 Fax: + 55-19-34294610 M. T. V. C. Derbyshire Laboratrio de Melhoramento de Plantas, Centro de Energia Nuclear na Agricultura, Universidade de S¼o Paulo, Piracicaba 13 400-970 S¼o Paulo, Brazil W. A. B. Almeida · F. A. A. Mour¼o Filho Departamento de Produ¼o Vegetal, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de S¼o Paulo, Piracicaba 13 418-900 S¼o Paulo, Brazil
Abbreviations BAP: Benzylaminopurine · CPR: Chlorophenol red · EGTA: Ethylene glycol-0-00 - bis (2, aminoethyl) N0 , N0 , N0 , N0 tetraacetic acid · MTT: [3-(4,5-Dimethyl thiazol-2-YL)-2,5-diphenyl] tetrazolium bromide · PMI: Phosphomannose isomerase (EC 5.3.1.8) · PMS: Phenazine methosulphate
Introduction Conventional breeding methods have demonstrated limitations with respect to citrus improvement due to some of the biological characteristics of woody plants such as nucellar polyembryony, high heterozygosity, a long juvenile period and auto-incompatibility (Ghorbel et al. 1999). The development of biotechnological tools have made it possible to overcome some of these problems. In the specific case of citrus breeding programs, somatic hybridization (Grosser and Gmitter 1990; Mendes et al. 2001) and genetic transformation (Pea and Navarro 1999; Costa et al. 2002) have been applied in many countries (Gutirrez-E. et al. 1997; Cervera et al. 2000a; Mendes et al. 2002). In vitro regeneration and genetic transformation protocols have been developed for several citrus cultivars and explant sources (Gmitter et al. 1992). Most of these use epicotyl or internodal segments excised from seedlings germinated either in vitro or in the greenhouses and transformed via Agrobacterium tumefaciens with the nptII (neomycin phosphotransferase II) selection gene in combination with the antibiotic kanamycin as a selection agent. Important genes such as the coat protein (Gutirrez-E. et al. 1997; Domnguez et al. 2000, 2002a; Yang et al. 2000; Febres et al. 2003) and the p23 (Ghorbel et al. 2001) from the citrus tristeza virus, the HAL2 (Cervera et al. 2000b), the LEAFY and APETALA1 (Pea et al. 2001) and the PR-5 pathogenesis-related protein gene (Fagoaga et al. 2001) have been introduced into citrus genomes. Although several promising results have been obtained, citrus genetic transformation protocols can still be improved. Among the problems reported so far are those
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related to the regeneration of a high number of escapes and the low rooting efficiency of the transgenic plants selected on antibiotic-supplemented medium. Because of these limitations and of public concern about the use of antibiotics or herbicides as selection agents, it is important to evaluate the efficiency of alternative selection systems, such as systems based on non-metabolizable agents (Haldrup et al. 1998; Joersbo and Okkels 1996; Joersbo et al. 1998), in citrus genetic transformation. One of these alternative systems, the manA gene, is very promising. This gene is derived from Escherichia coli (Miles and Guest 1984) and encodes for phosphomannose-isomerase (PMI), which catalyzes the reversible interconversion from mannose-6-phosphate to fructose-6phosphate (Privalle et al. 1999). ManA has been tested as a selection gene in a variety of crops such as sugar beet (Joersbo et al. 1998), maize (Negrotto et al. 2000; Wang et al. 2000; Wright et al. 2001), wheat (Wright et al. 2001) and rice (Lucca et al. 2001). The supporting principle in this approach is the inability of some plants to use mannose as a carbon source. The major difference from selection based on antibiotics or herbicides, which kill non-transformed cells, is that the non-transformed cells in the mannose system have their growth and development arrested by carbohydrate starvation (Wang et al. 2000) but still survive (Haldrup et al. 1998). Owing to the growth advantage of transformed cells, this strategy is called “positive selection” (Joersbo and Okkels 1996). In order to avoid the use of antibiotics or herbicides in genetic transformation, both Domnguez et al. (2002b) and Ghorbel et al. (1999) showed that it is possible to regenerate transgenic citrus plants without the use of selection genes or by using the reporter gene gfp (green fluorescent protein), respectively. Although the efficiency of these systems was low, the possibility of generating transgenic citrus plants without using kanamycin as a selectable marker was demonstrated. We report here the use of mannose as an alternative selection agent in sweet orange genetic transformation. This is the first report of this system being used in the genetic transformation of woody plants.
Materials and methods Plant material Seeds were extracted from mature fruits of Valencia, Natal, Hamlin and Pera sweet oranges (Citrus sinensis L. Osbeck) and dried at room temperature for 24 h. The seed coat was then removed, and the seeds were treated with a sodium hypochlorite solution (0.5%) for 15 min. They were then cultured in test tubes (25150 mm) containing 10 ml of MS solid medium (Murashige and Skoog 1962) and incubated in the dark at 27C for 2–3 weeks. Seedlings 12– 15 cm in height were transferred to a 16/8-h (day/night) photoperiod for 10 days. All media were solidified with 0.8% agar (Sigma, St. Louis, Mo.), and the pH was adjusted to 5.8 before autoclaving (121C, 20 min).
Fig. 1 Structure of the pNOV2116 plasmid. Act2int Promoter, NOST nopaline synthase terminator, RB right border, LB left border, Spec gene encoding for the resistance to spectinomycin, PMI phosphomannose isomerase Explant sensitivity to the selection agent Epicotyl segments (0.8–1.0 cm-long) from in vitro-germinated Pera and Natal sweet orange seedlings were plated on EME medium (Grosser and Gmitter 1990) supplemented with mannose, fructose or sucrose (73 mM) to determine whether citrus explants could metabolize these sugars as a carbon source during organogenesis. The experimental design was completely randomized with five replications, each consisting of one petri dish (10015 mm) with 25 explants, with a total of 125 explants per treatment. After 4 weeks, the explants were scored with the aid of a stereomicroscope for the number of explants forming adventitious buds. Agrobacterium strain and vector Plasmid pNOV2116 provided by Syngenta was introduced into A. tumefaciens strain EHA101 using the freeze-thaw method (Lacorte and Romano 1998). This plasmid contained the E. coli-derived manA gene (Miles and Guest 1984) driven by the Act2 promoter (An et al. 1996) that consisted of the 50 flanking sequences of the Act2 gene (Fig. 1). A. tumefaciens was streaked onto YEP medium (5 g/l yeast extract, 10 g/l peptone, 5 g/l NaCl and 15 g/l agar). After 2–3 days of growth, one single colony was inoculated in YEP liquid medium supplemented with antibiotics for bacterial selection and cultured at 28C, 180 rpm for 12–16 h until it reached a density of 0.5–1.0 (OD600). The bacterial suspension was centrifuged (5,000 rpm/10 min) and re-suspended in liquid MS medium to achieve a final concentration of 5.0108 cfu/ml. Determination of mannose concentration for efficient selection Epicotyl segments of Valencia and Natal sweet oranges were inoculated and co-cultured with Agrobacterium EHA101. Following the co-culture period, the explants were transferred to EME selection medium supplemented with various combinations of mannose:sucrose—73:0, 54:18, 36:36, 18:54 and 0:73 (in millimoles)—to determine the best ratio for citrus transgenic plants selection. The number of explants with buds and the number of
124 PCR-positive buds for the manA gene were determined after 4 weeks of incubation. Plant transformation Epicotyl segments of Valencia, Natal, Hamlin and Pera sweet oranges were inoculated with A. tumefaciens EHA101-pNOV2116 for 20 min, blotted dry and transferred to regeneration medium (EME supplemented with 1.0 mg/l BAP) for a 3-day co-cultivation period in the dark at 24C. After co-cultivation, explants were transferred to selection medium supplemented with mannose and cefotaxime (500 mg/l). The mannose concentration during selection ranged from 73 mM to 112.3 mM. Cultures were maintained either under a 16/8-h (day/night) photoperiod (40 mmol m-2 s-1) or in the dark during the first 5 weeks, after which a 16/8-h photoperiod was used. Well-developed shoots were separated from the explants and micrografted onto Carrizo citrange seedlings (Pea et al. 1995b) for plant development and further analysis. Analysis of putative transformants Polymerase chain reaction assay (PCR) For the detection of the manA gene in the regenerated plants, DNA was extracted from leaves of in vitro plantlets (Doyle and Doyle 1990). PCR amplification was performed using 20–80 ng DNA, 200 mM of all dNTPs, 2.0 mM MgCl2, 0.25 mM of each of the primers and 2 U Taq polymerase (Promega, Madison, Wis.). A left primer 50 -CACTGCGTGATGTGATTGAGAGTG-30 and a right primer 50 -ACTAAGGTCATGCAGCGAGAAGGC-30 (Wang et al. 2000) were used to amplify a 820-bp fragment corresponding to the manA gene. The PCR reactions were performed using an MJ Research (Waltham, Mass.) thermal cycler with settings of 30 cycles of 30 s at 94C, 30 s at 65C and 45 s at 72C. PCR-positive plants were acclimatized in the greenhouse. Primers 50 -CTGGCGGCAAAGTCTGAT-30 and 50 -TGTCGTAAACCTCCTCGT-30 (Negrotto et al. 2000) were used to amplify a 450-bp specific fragment of the virG gene and to detect possible contamination of the plant tissue with Agrobacterium. PCR reactions were performed at a setting of 2 min at 96C, followed by 25 cycles of 15 s at 94C, 30 s at 53C and 30 s at 72C with a terminal elongation step of 5 min at 72C. The transformation efficiency rate was calculated as the percentage of PCR-positive plants in relation to the total number of explants inoculated. Southern blot analysis DNA was extracted from leaves of acclimatized transgenic plants, using the protocol of Doyle and Doyle (1990). A 20-mg aliquot of DNA was digested either with KpnI or HindIII restriction enzymes, which cut the T-DNA once outside the gene area. The DNA was then separated on a 1% agarose gel by electrophoresis and blotted onto nylon membranes (Hybond-N+, Amersham-Pharmacia Biotech, Piscataway, N.J.). A PCR-amplified fluorescein-labelled (Amersham-Pharmacia Biotech) fragment from the coding region of the PMI DNA (550 bp) was used as a probe. The hybridization, washing and detection were performed following the supplier’s instructions. Gene expression assays A chlorophenol red assay was conducted to verify PMI activity (Wright et al. 2001). Leaf segments of regenerated plants were incubated for 4–5 days in the dark at 27C in MS liquid medium supplemented with mannose or sucrose (control), pH 6.0, and with chlorophenol red (50 mg/l). Evaluation was based on the color reaction. Red or purple indicated no enzyme activity and yellow or orange indicated PMI activity.
Cold pieces of fresh leaves (40–50 mg) from transgenic plants were used to assay for PMI activity. Non-dissociated protein was extracted in Nankoong medium (Alfenas et al. 1991) for analysis on a 13% maize starch gel. Electrophoresis was performed using a lithium-borate/Tris-citrate buffer, pH 8.3 (Ashton and Braden 1961), and carried out at 4C and 300 V for 24 h. Isoenzymes were detected by incubating the gels in the presence of the following specific substrate-stain solution: 10 ml 0.2 M Tris-HCl, pH 8.0 plus 2 g EGTA; 20 mg d-mannose-6-P; 20 mg pyruvic acid; 1 ml 1% bnicotinamide adenine dinucleotide phosphate; 0.5 ml 1% nicotinamide adenine dinucleotide phosphate; 6 ml phosphoglucose isomerase (10 U); 6 ml glucose-6-P dehydrogenase (17 U); 1 ml 1% MTT; 0.25 ml 1% PMS; 10 ml 1.5% agarose. This solution was added over the gel and incubated at 37C for 20–30 min. In order to quantify PMI activity, leaves of transgenic plants (200 mg) were ground in 600 l Nankoong buffer (Alfenas et al. 1991) and centrifuged for 20 min at 14,000 rpm. The supernatant was transferred to a new tube and centrifuged. The new supernatant (500 l) was mixed with substrate (300 l) and measured at 340 nm in a spectrophotometer. This substrate consisted of a solution containing 100 l nicotinamide adenine dinucleotide phosphate (10 mM), 100 l phosphoglucose isomerase (10 U/ml, Sigma), 50 l glucose-6-P dehydrogenase (10 U/ml, Sigma), 20 l d-mannose-6-P (50 mM) and 130 l Tris-HCl (50 mM, pH 7.5).
Results and discussion Effect of carbohydrates on organogenesis and genetic transformation The use of the PMI/mannose selection system in genetic transformation is based on its ability to inhibit in vitro morphogenesis when non-transformed explants are cultured in medium using mannose as a carbon source. In vitro citrus organogenesis occurs when non-meristematic explants such as epicotyl or internodal segments are cultured in media containing salts, vitamins, growth regulators and sucrose as a carbon source (Perz-MolpheBalch and Ochoa-Alejo 1997; Moreira-Dias et al. 2000). The inhibition of in vitro organogenesis in citrus epicotyl segments cultured in medium supplemented with only mannose (73 mM) indicates that the explants are not able to utilize this sugar as a carbon source (Table 1). This is probably due to a deficiency in endogenous PMI activity in this species. The concentration of mannose selected— 73 mM—was based on previous experiments using sucrose as the carbohydrate source for citrus epicotyl
Table 1 Adventitious bud formation from citrus epicotyl segments of Pera and Natal sweet oranges cultured in EME culture medium supplemented with different carbon sources (73 mM) Carbohydrate
Sucrose Fructose Mannose a Explants b
Responsivea explants (%) Perab
Natalb
96.8a 93.6a 7.2b
98.3a 90.0a 3.3b
with at least one adventitious bud Each value represents the mean (%) of five replications for a total of 125 and 150 explants per treatment for Pera and Natal, respectively. Means followed by the same letter are not significantly different at the 0.01 level by Tukey’s multiple range test
125 Table 2 Adventitious bud formation and recovery of genetically transformed plants from Valencia and Natal sweet oranges under different concentrations of mannose and sucrose during selection Mannose:sucrose (mM)
Valencia Responsive /total explants (%)
PCR+ plants/analyzed plants (%)
Responsivea/total explants (%)
PCR+ plants/analyzed plants (%)
73:0 54:18 36:36 18:54 0:73
30/104 (28.8) 64/75 (85.3) 107/126 (84.9) 104/114 (91.2) 150/150 (100.0)
5/24 (20.8) 1/11 (9.09) 5/55 (9.09) 1/47 (2.12) 0 (0.0)
15/139 15/138 70/135 118/144 142/150
2/6 (33.3) 2/5 (40.0) 4/15 (26.6) 0/26 (0.0) 0 (0.0)
a
Natal a
(10.8) (10.9) (51.8) (81.9) (94.6)
Explants with at least one adventitious bud
Table 3 Plant recovery and transformation efficiency from epicotyl segments of sweet orange using the PMI/mannose selection system Cultivara
Mannose (mM)
Number of plants recovered
Number of PCR+ plants (%)
Transformation efficiencyb (%)
Pera Valencia Natal Hamlin
73 73 84.2 112.3
25 120 38 4
16 50 12 3
7.6 23.8 12.0 3.0
(64.0) (41.6) (31.6) (75.0)
a A b
total of 210 explants were used for Pera and Valencia and 100 for Natal and Hamlin PCR+ plants/total explants inoculated
segments in vitro organogenesis (Mendes et al. 2002). Through PMI enzyme action, mannose-6-phosphate is converted into fructose-6-phosphate, making the incorporation into glycolysis possible. The efficient development of shoots in culture media supplemented with either sucrose or fructose (Table 1) indicates that fructose also functions as a carbon source for citrus epicotyl segments, thereby allowing the use of the PMI/mannose system for citrus transgenic plants selection. Genetic transformation using the PMI/mannose system was carried out using a selection medium supplemented with either mannose or a combination of mannose and sucrose as the carbon source (Negrotto et al. 2000; Reed et al. 2001). The carbohydrate concentration varied among species, ranging from 0.2% for sugarbeet (Joersbo et al. 1998) to 1–5% for rice (Lucca et al. 2001), maize and wheat (Negrotto et al. 2000; Wright et al. 2001). Table 2 shows the number of transgenic plants obtained using different concentrations of mannose combined with sucrose. An increase in sucrose concentration resulted in a higher number of explants with shoots but a decrease in the number of PCR-positive plants in both of the varieties tested. The increase in the number of escapes when the mannose concentration was lowered or combined with sucrose in the selection medium has also been reported for maize (Negrotto et al. 2000; Wright et al. 2001). In this species, a combination of mannose and sucrose was used only after selection, at the regeneration step. Escapes could also be controlled either by continued selection at the rooting stage (Wright et al. 2001) or by a stepwise increase in the mannose concentration during selection (Joersbo et al. 1998; Lucca et al. 2001). Genetic transformation experiments were then carried out with four sweet orange cultivars using at least 73 mM
mannose in the selection medium (Table 3). Based on PCR analysis, it was possible to recover transgenic plants at a 3–23% efficiency rate, depending on the citrus cultivar and mannose concentration. Figure 2A shows the results of PCR analysis of some putative transgenic shoots. Figures 2B–C shows the Southern blot analysis, confirming the integration of the manA gene in sweet orange plants. The pattern of hybridizing bands indicated integration of one copy of the gene in most plants, except for one plant in HindIII digestion, where two copies were found (Fig. 2C). The negative results for virG gene amplification in all of the plants tested confirmed that no contamination with residual Agrobacterium had occurred in the tissue (data not shown). Our results on the transformation efficiency rate using the PMI/mannose selection system are similar to those reported for other crops, such as sugarbeet (0.94%) (Joersbo et al. 1998), maize (45%) (Wright et al. 2001) and rice (44%) (Lucca et al. 2001). The transformation efficiency rate that we obtained using the PMI/mannose selection system in citrus genetic transformation is also similar to that obtained by traditional selection systems. Citrus genetic transformation efficiency rate using the nptII selection gene that confers resistance to kanamycin varies among cultivars, with values ranging from 0.52% to 55.2% for Carrizo citrange and related hybrids (Moore et al. 1992; Kaneyoshi et al. 1994; Pea et al. 1995a) and from 10% to 20% for Key or Mexican lime (Gutirrez-E. et al. 1997; Pea et al. 1997; Domnguez et al. 2000) and 3.6% for grapefruit (Yang et al. 2000). For sweet orange cultivars (C. sinensis), reported values are 7.6% for pineapple (Pea et al. 1995a) and 15% for Hamlin (Mendes et al. 2002). In most of the citrus genetic transformation experiments using kanamycin as a selection agent, explants
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Fig. 2A–E Analysis of putative transformants and gene expression. A PCR analysis of DNA from Valencia (lanes 4–7) and Natal (lanes 8–12) plantlets. Lanes: 1, 14 100-bp ladder (AmershamPharmacia Biotech), 2, 3 negative control (DNA of non-transformed plants), 4–12 DNA of regenerated shoots, 13 positive control (plasmid pNOV2116). B, C Detection of manA transgene integrated in Valencia (lanes 2, 3, 5) and Natal (lanes 4, 6) sweet orange plants by Southern blot analysis of genomic DNA. The probe used was a PCR-amplified fragment corresponding to 550 bp from the manA gene. Lanes: 1 pNOV2116 plasmid digested either with KpnI (B) or HindIII (C), used as positive control, 2–6 DNA
extracted from manA-transgenic sweet orange plants, 7 DNA extracted from non-transformed plants, used as negative control. Lambda DNA cut with HindIII was used as a size marker. D Chlorophenol red assay with citrus leaf pieces. Lanes: 1–3 Leaves of putatively transformed citrus plants, 4 control leaves (nontransformed plant); red indicates negative enzyme activity, yellow indicates phosphomannose-isomerase activity. E Starch gel in LB buffer, pH 8.3. Lanes: 1 Positive control (phosphomannoseisomerase, Sigma), 2 negative control (non-transformed plant), 3– 8 transgenic plants from Valencia (lanes 3–5) and Natal (lanes 6–8) sweet oranges. Rm PMI relative mobility
were cultured in the dark following the co-culture period and up to the formation of callus and shoot development initiation (Cervera et al. 1998; Pea et al. 1995a). Using the PMI/mannose selection system, we recovered shoots only after the co-culture period when the explants were cultured under a 16/8-h (day/night) photoperiod (data not shown). This procedure favored direct organogenesis without callus formation.
Wang et al. 2000). Enzyme activity ranged from 0.176 to 0.778 (OD at 340 nm) in transformed plants, while the control plant (non-transformed) did not show enzyme activity and retained the value zero during the assay. These values are in agreement with those of Wang et al. (2000), who described OD values of 0.17 to 1.32 in transformed maize plants and no PMI activity in nontransformed plants. As discussed by these authors, the difference in the PMI expression level may have been caused by differences either in the position of the insertion site or in gene copy number. However, PMI activity quantification was unstable in citrus plants. When citrus leaves were ground in Tris-HCl buffer, as done by Wang et al (2000) in maize, no activity was found. Enzyme activity was only detected, with some variations, when the leaves were ground in the Nankoong buffer. For this reason, we think that there may be some component in citrus leaves that may either cause this variation or inhibit the enzyme activity. Consequently, confirmation of PMI activity was accomplished using a starch gel and a
Gene expression assays Plants tested for PMI activity in the CPR assay showed variations in shades ranging from yellow to orange (Fig. 2D), which indicates acidification of the medium caused by metabolic activity of the cells. Non-transformed plant tissues were not able to metabolize the carbohydrate, thereby the initial color (purple) was preserved. PMI activity has also been measured in a substrate using a spectrophotometer (Joersbo et al. 1998;
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specific substrate with all six plants analyzed. It was possible to detect a bluish shade, representing the reaction of the PMI with the substrate (Fig. 2E). Different shades of blue were observed, indicating that there were different levels of PMI activity in these plants. Detection using this method showed better results in citrus than did spectrophotometric measurements, and this suggests that it could be also used for detection of PMI activity in other plant species. Concluding remarks The PMI-positive selection system using mannose as a selection agent is not only efficient in citrus genetic transformation but also avoids the undesirable antibiotic selection systems. In our study, gene expression was better observed by enzymatic analysis. ManA can be regarded as an alternative selection gene and can also be used as a reporter gene for citrus as well as for other woody plants in genetic transformation processes. Acknowledgements The authors acknowledge receiving financial support for this research from FAPESP (99/04073-3) and FUNDECITRUS. RLB and WABA both acknowledge FAPESP and CAPES/PICDT for receipt of a scholarship. BMJM and FAAMF acknowledge CNPq for research fellowships. The authors wish to thank Adriana P.M. Rodriguez for critical comments, Myriam R. Orsi for technical assistance and Syngenta for supplying pNOV2116.
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