Prunus Virginiana L

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HORTSCIENCE 42(1):140–142. 2007. or leaves from a juvenile plant and very few studies recovered whole transgenic plants. To our knowledge, only Dolgov et al. (1999) Agrobacterium-Mediated and Song and Sink (2006) reported that transgenic plants were regenerated from leaf tis- Transformation of Chokecherry sues of mature cherry species and no research has been reported on chokecherry transformation. (Prunus virginiana L.) This study was carried out to develop a gene transformation protocol for future gene 1,4 2 3 Wenhao Dai, Victoria Magnusson, and Chris Johnson transfer of chokecherry. The method of ge- Department of Plant Sciences, North Dakota State University, Fargo, ND netic transformation of chokecherry might be 58105 also useful for genetic engineering of other Prunus species. Additional index words. organogenesis, mature woody plant, leaf explants Abstract. Chokecherry (Prunus virginiana L.) was transformed using Agrobacterium Materials and Methods tumefaciens strain EHA105 harboring binary vector pBI121 carrying the neomycin Plant materials. In vitro cultures of phosphotransferase gene (nptII) and b-glucuronidase (GUS) gene (uidA). Plants were chokecherry clone NN were initiated by regenerated from the Agrobacterium-infected leaf tissues through organogenesis on Zhang et al. (2000) using shoot tips from woody plant medium (WPM) supplemented with MS (Murashige and Skoog) vitamins, a mature seed-propagated chokecherry plant 10 mM 6-benzyladenine (BA), and 250 mgÁL–1 cefotaxime plus 500 mgÁL–1 carbenicillin –1 grown at the USDA Plant Materials Center in plus 15 mgÁL kanamycin (CCK15). Transformation was verified with polymerase chain reaction (PCR) and Southern blot analysis. Four of 150 (2.67%) initial explants produced Bismarck, N.D. In vitro shoots were main- GUS- and PCR-positive shoots. Southern blot analysis confirmed that the transgenes tained in Murashige and Skoog (1962) were integrated into the chokecherry genome. Transgenic in vitro shoots were rooted in medium (MS) supplemented with 2.5 mM 6- benzyladenine (BA), 3% sucrose, and solid- half-strength MS medium containing 10 mM naphthalene acetic acid. Rooted plants were transferred to potting mix and grown in the greenhouse. This research shows a potential ified with 0.7% agar (Difco Co., Detroit, for future improvement of chokecherry and other Prunus species. Chemical names used: Mich., #0140–01–0). The pH was adjusted 6-benzyladenine (BA), naphthalene acetic acid (NAA), acetosyringone (AS), 5-Bromo-4- to 5.7–5.8 before autoclaving. In vitro shoots chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (X-Glu), cefotaxime, carbeni- were subcultured every 4 weeks to fresh cillin, kanamycin. media in 100-mL baby food jars containing 25 mL medium each and cultured at 25 C under cool-white light at 36 mmolÁm–2Ás–1 Chokecherry (Prunus virginiana L.) is a These diseases and infected trees can only be with a 16- to 8-h photoperiod. All other exper- small tree or large shrub widely distributed removed. Therefore, utilization of disease- iments were performed under these condi- across the northern Great Plains in the United resistant plants is the best method to manage tions unless otherwise noted. States and Canada. Native to North America, these diseases. Conventional approaches for Plant transformation. Agrobacterium chokecherry is well adapted to a variety of chokecherry breeding is generally difficult strain EHA105 (Hood et al., 1993), carrying severe conditions such as alkaline soils and and time-consuming because of its high pBI121 (Clontech, Palo Alto, Calif.) contain- harsh Winters and is a valuable food resource heterozygosity, polyploidy, and long juve- ing the nptII gene encoding for neomycin and shelter for wildlife. Chokecherry is one nile period. Thus, genetic engineering offers phosphortransferase and the uidA coding for of the native species (pincherry, cranberry, a useful tool to complement the conven- b-glucuronidase (GUS) (Fig. 1), was grown blueberry, and so on) used in small fruit pro- tional breeding method for chokecherry overnight in LB (Luria-Bertani) medium duction for beverages, fresh fruit, dried fruit improvement. with 100 mgÁL–1 kanamycin at 28 Cina products, and wine. It is also used as an orna- Transgenic plants have been obtained in shaker at 150 rpm. Cells were collected by mental plant because of the beautiful white many woody species (Poupin and Arce- centrifugation at 6000 rpm for 15 min, flowers in Spring and colorful leaves and Johnson, 2005). In the genus Prunus, trans- resuspended to 1.0 O.D. at Abs600 in fresh fruits in Fall. formation of several species have been LB medium supplemented with 20 mM ace- The development of the native fruit in- documented, including peach (Hammerschlag tosyringone (AS) without kanamycin, and dustry in the northern Great Plains is largely and Smigocki, 1998; Pe´rez-Clemente et al., incubated at 28 C in a shaker at 150 rpm impeded by lack of high-quality and high- 2004; Scorza et al., 1995a), plum (Scorza for 2 h. One-month-old in vitro leaves were yield cultivars. Chokecherry suffers several et al., 1995a, 1995b; Yancheva et al., 2002), cut through the main vein once and sub- diseases, including black knot and X-disease almond (Ainsley et al., 2002; Miguel and merged in a bacterial culture solution for 30 (incited by a cell-wall-less prokaryotic phy- Oliveira, 1999), apricot (Petri et al., 2004), min at 28 C. Leaf explants (0.5 · 0.5 cm) toplasma) (Guo et al., 1996). The damage by and cherry (Dolgov et al., 1999; Song and were then removed from the culture, blotted these diseases is severe. No effective meth- Sink, 2006). However, most of these studies on sterilized paper towels, and transferred to ods are available to control these diseases. used immature tissues (immature embryos) a woody plant medium (Lloyd and McCown,

Received for publication 30 June 2006. Accepted for publication 17 Aug. 2006. This research was supported in part by McIntire- Stennis Project ND06212 and USDA-CSREES- 2005-35300-15457. We thank Drs. E. Deckard, C.W. Lee, and H. Hatterman-Valenti for their valuable sugges- tions and comments when we were preparing the manuscript. 1Assistant Professor. Fig. 1. Schematic representation of the T-DNA portion of pBI121 plasmid (Clontech, Palo Alto, Calif.). 2Research specialist. The vector was introduced into the disarmed Agrobacterium tumefaciens EHA 105. RB and LB: 3Graduate student. T-DNA right and left borders; Nos-pro: nopaline synthase promoter; Nos-ter: nopaline synthase 4To whom reprint requests should be addressed; terminator; nptII: neomycin phosphotransferase gene; uidA: b-glucuronidase gene; 35S-Pro: CaMV e-mail [email protected]. 35S promoter from cauliﬂower mosaic virus.

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1980) supplemented with MS vitamins, 10 mM BA, 0.7% agar, and 200 mM acetosyringone in Petri dishes (100 mm · 15 mm, 25 mL medium) for cocultivation in the dark for 72 h at room temperature. Each Petri dish con- tained 25 leaf explants and replicated three times (Petri dishes). The experiment was cocurrently repeated twice. After cocultivation, a total of 150 leaf explants were washed three times with sterile deionized and dis- Fig. 2. A transformed plant was recovered (A); b-glucuronidase staining of a young leaf from tilled water (ddH2O) and once with sterile –1 a nontransformed (B) and a transformed (C) chokecherry. The blue (see the online version in color ddH2O plus 250 mgÁL cefotaxime and 500 mgÁL–1 carbenicillin, and then 10 explants per at www.ashs.org) color in C is the result of active beta-glucuronidase activity. Petri dish (100 mm · 15 mm) containing 25 CAACCCGTG-3#, which produced 410 bp indicating that all bacteria had been killed by mL regeneration medium [WPM supple- and 365 bp products, respectively. antibiotics (cefotaxime and carbenicillin) mented with MS vitamins, 10 mM BA, 0.7% Southern blot analysis. Approximately 25 during subculturing. GUS staining identified agar, and 250 mg L–1 cefotaxime plus 500 Á to 35 mg of genomic DNA was digested in four regenerated lines that stained GUS- mgÁL–1 carbenicillin plus 15 mgÁL–1 kanamy- a 50-mL reaction with 1 mL EcoRI+1mL positive in leaves (Fig. 2), indicating that cin (CCK )] (antibiotics were added into the 15 HindIII restriction enzymes at 37 C for 2.5 h, the uidA gene was being expressed in the leaf autoclaved medium when the medium tem- electrophoresed on a 0.8% TAE (Tris-acetate tissue. perature cooled down to 50 C) for re- EDTA) agarose gel, and blotted to a posi- Polymerase chain reactions. Four GUS- generation under shoot culture conditions tively charged Hybond-N+ nylon mem- positive regenerated lines were proliferated previously mentioned. Calluses developed brane (Amersham Pharmacia Biotech, Little from leaf tissues after the first month of to have enough leaf tissue for genomic DNA Chalfont Buckinghamshire, U.K.). Similarly, culture in CCK medium. Calluses were extraction and subjected to PCR verification. 15 restricted DNA from untransformed clone then detached from original leaf explants The expected fragments of uidA (356 bp) and NN was used as a negative control, whereas and transferred to fresh regeneration medium nptII (410 bp) genes were successfully am- 25 to 40 ng of pBI121 plasmid DNA was used for shoot regeneration. Shoots regenerated plified from all transformants using the spe- from CCK -containing medium were pro- as a positive control. The blot was probed cific primers (Fig. 3). 15 with randomly primed 32P-labeled uidA PCR liferated in MS supplemented with 2.5 mM Southern blot analysis. Genomic DNA product (4 mLdH2O, 1 mL 6-mer oligo was digested with HindIII and EcoRI. Gel BA and CCK15. Proliferated shoots were rooted based on the method of Dai et al. primers, 1.5 mL5mM dNTPs, 1.5 mL10· electrophoresis showed that DNA was well (2004). Rooted plants were transferred to Klenow buffer, 1 mL Klenow polymerase, digested and separated. The double-digested 32 32 Sunshine Mix #1 (Fisons Western Corp., and 5 mL dCTP P). Blot was prehybridized DNA samples were hybridized with the P- Vancouver, Canada) and grown in the green- at 65 C for 6 h in the hybridization solution labeled fragment of the 356-bp probe pre- house. [1% bovine serum albumin (BSA) Fraction V pared from the PCR product of the uidA gene. Histochemical b-glucuronidase assay. (Sigma), 0.5 M NaH2PO4 (pH 7.0), 7% The result showed that all four regenerated Leaves from these regenerated shoots were sodium dodecyl sulfate (SDS), 1 mM ethyl- lines exhibited one distinctive restriction subjected to GUS screening as described by enediamin tetraacetic acid (EDTA)]. DNA fragment (Fig. 4), confirming that the uidA Jefferson (1987). In brief, in vitro young probe was added directly to the blot in the gene was integrated into chokecherry leaves were submerged in a GUS staining prehybridization mixture, hybridized at 65 C genome. for 16 h, and then washed with 2 · SSC (200 solution containing 200 mLdH2O and 200 mL In this study, transformation of four re- X-Glu solution (2 mgÁmL–1; Gold Biotech- mM sodium chloride and 200 mM sodium generated lines (T1–2, T1–4, T1–5, and T1– nology, Inc., St. Louis, Mo.). Submerged citrate) for 35 min followed by 0.5 · SSC + 6) of 150 initial leaf explants has been leaves were incubated at 37 C overnight 0.1% SDS, and 0.1 · SSC + 0.1% SDS for 10 confirmed. The transformation frequency of and then bleached with 70% to 100% ethanol min each at 65 C on a shaker. The blot was gradually. GUS staining was observed under exposed to x-ray film (Kodak, N.Y.) at the microscope and photographed. –80 C for 72 h and developed per the Polymerase chain reactions. Genomic manufacturer’s instructions. DNA was extracted from young leaves of in vitro transformed and nontransformed choke- Results and Discussion cherry plants based on the method of Lodhi Fig. 3. Polymerase chain reaction amplification of et al. (1994). Polymerase chain reactions Transformation of chokecherry. After 4 to uidA and nptII genes in transformed plants. (PCRs) were carried out in 25 mL volume 6 weeks of culture, leaf tissue cocultivated Lanes 1–4 and 8–11 are transformed lines T1– containing 200 mM dNTPs, 1 mM each of with Agrobacterium EHA105 carrying 6, T1–5, T1–4, and T1–2 amplified with pri- oligonucleotide primer, 2.5 units DNA Taq pBI121 produced callus on CCK15 media, mers specific for nptII and uidA, respectively. Polymerase (Promega, Madison, Wis.), and whereas no callus developed without cocul- Lanes 6 and 13 are negative controls of un- 25 ng DNA. The reaction conditions were: tivation. Callus was detached from original transformed clone NN. Lanes 7 and 14 are 1-kb leaf explants and transferred to the same DNA ladders. Lanes 5 and 12 are positive one cycle at 94 C for 5 min, 40 cycles of controls of pBI121. 94 C for 1 min, 60 C for 1 min, and 72 C regeneration medium containing CCK15. for 30 s and then one cycle at 72 o C for 7 min. New shoots were regenerated from callus Amplified DNA fragments (10 mL of reac- tissue after 4-week culture. From 150 initial tion) were electrophoresed on a 1% agarose explants, nine callus lines produced shoots. gel and visualized by staining with ethidium b-glucuronidase staining. To prevent bromide. The primers used for screening false-positive GUS staining and PCR from transgenes were: nptII reverse: 5#-GCAGG contamination of remaining Agrobacterium, Fig. 4. Confirmation of transgene (uidA) in transformed chokecherry. Lane L: 1-kb DNA lad- CATCGCCATGGGTCACGACGA-3# and in vitro shoots from nine regenerated lines # der; Lanes NT, P, and 1 to 4 are double- nptII forward: 5 -GCCCTGAATGAACTG were grown in antibiotic-free MS media to digested DNA with EcoRI and HindIII from CAGGACGAGGC-3# and uidA reverse: 5#- detect remaining Agrobacterium. All cultures nontransgenic clone NN, plasmid pBI121, and CCCGGCAATAACATACGGCGTG-3# and remained free of bacteria after being subcul- transgenic lines: T1–6, T1–5, T1–4, and T1–2, uidAforward:5#-CCTGTAGAAACCC tured in CCK15 medium two to three times, showing a predicted band of 3.0 kb.

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chokecherry in this study was 2.7% (four of fection and acclimate to new culture condi- Lodhi, M.A., G.N. Ye, N.F. Weeden, and B.I. 150), which is higher than most transforma- tions, allowing the plant cells to initiate Reisch. 1994. A simple and efficient method tions for Prunus species using mature mate- regeneration more easily. for DNA extraction from grapevine cultivars, rials (Petri and Burgos, 2005). Only one To date, most transgenic plants of Prunus Vitis species and Ampelopsis. Plant Mol. Biol. recent study reported a slightly higher trans- Rep. 12:6–13. species have been initiated with embryogenic Miguel, C.M. and M.M. Oliveira. 1999. Transgenic formation frequency obtained from cherry tissues (immature embryos) or leaves from almond (Prunus dulcis Mill.) plants obtained transformation (Song and Sink, 2006). Many a juvenile plant. This limits application of by Agrobacterium-mediated transformation of factors such as genotype, Agrobacterium gene transformation for improving vegeta- leaf explants. Plant Cell Rept. 18:387–393. strain, conditions of infection, and cocultiva- tively propagated species, especially for elite Murashige, T. and F. Skoog. 1962. A revised tion affect transformation frequency. The cultivar improvement because of the segre- medium for rapid growth and bioassays with chokecherry clone used in this study is very gation of seed-propagated plants. In this tobacco tissue culture. Physiol. Plant. 15:473– 497. amenable to regeneration from leaf tissue study, chokecherry transformation was (Dai et al., 2004). Thus, higher transforma- Pe´rez-Clemente, R.M., A. Pe´rez-Sanjuań, L. achieved using leaf tissue from a mature tree. Garcıá-Fe´rriz, J.P. Beltrań, and L.A. Canãs. tion efficiency may be achieved if some Therefore, this method can be used to improve improvements can be made during the Agro- 2004. Transgenic peach plants (Prunus persica single or several traits without changing any L.) produced by genetic transformation of bacterium infection and transformant selec- other genetic makeup, providing a useful tool embryo sections using green fluorescent pro- tion. For example, preconditioning explants, in development of new cultivars of this and tein (GFP) as an in vivo marker. Mol. Breed. application of a vacuum technique during the other species. 14:419–427. infection process, and using different Agro- Petri, C. and L. Burgos. 2005. Transformation of bacterium strains will increase the transfer Literature Cited fruit trees. Useful breeding tool or continued DNA delivery efficiency. In this transforma- future prospect? Transgenic Res. 14:15–26. tion system, kanamycin was used as a selec- Ainsley, P.J., G.G. Collins, and M. Sedgley. 2002. Petri, C., N. Alburquerque, S. Garcia-Castillo, J. tion agent to identify transgenic cell Factors affecting Agrobacterium-mediated Egea, and L. Burgos. 2004. Factors affecting lines. Preliminary experiments showed that gene transfer and selection of transgenic calli gene transfer efficiency to apricot leaves during the tolerance of chokecherry leaf tissue to in paper shell almond (Prunus dulcis Mill.). early Agrobacterium-mediated transformation kanamycin appeared to vary greatly with the J. Hortic. Sci. Biotechnol. 76:522–528. steps. J. Hort. Sci. Biotechnol. 79:704–712. Dai, W., V. Jacques, J.A. Walla, and Z.M. Cheng. Poupin, M.J. and P. Arce-Johnson. 2005. Trans- developmental stage of in vitro plants (data 2004. Plant regeneration of chokecherry (Pru- genic trees for a new era. In Vitro Cell. Dev. not shown). Regeneration of chokecherry nus virginiana L.) from in vitro leaf tissues. Biol. Plant 4:91–101. from leaf tissue was sensitive to kanamycin J. Environ. Hort. 22:225–228. Scorza, R., F.A. Hammerschlag, T.W. Zimmerman, concentration. High concentration of kana- Dolgov, S.V., A.P. Firsov, B.M.M. Shemyakin, and J.M. Cordts. 1995a. Genetic transformation mycin(>20mgÁL–1) completely inhibited and A. Ovchinnikov. 1999. Regeneration and in Prunus persica (peach) and Prunus domes- regeneration from leaf tissue. However, the Agrobacterium transformation of sour cherry tica (plum), p. 255–268. In: Y.P.S. Bajaj (ed.). tolerance to kanamycin increased dramati- leaf discs. Acta Hort. 484:577–579. Plant Protoplast and Genetic Engineering VI. cally after in vitro shoots formed. In vitro Guo, Y.H., J.A. Walla, Z.M. Cheng, and I.M. Lee. Springer-Verlag, Berlin, Heidelberg. shoots did not exhibit any damage after 8 1996. X-disease confirmation and distribution Scorza, R., L. Levy, V.D. Damsteegt, L.M. Yepes, and J.M. Cordts. 1995b. Transformation of to12 weeks at 45 to 80 mg L–1 kanamycin. To in chokecherry in North Dakota. Plant Dis. Á 80:95–102. plum with the papaya ringspot virus coat pro- increase efficiency of selection, we exposed Hammerschlag, F.A. and A.C. Smigocki. 1998. tein gene and reaction of transgenic plants to leaf explants to selective medium (containing Growth and in vitro propagation of peach plum pox virus. J. Amer. Soc. Hort. Sci. –1 20 mgÁL kanamycin) immediately after co- plants transformed with shooty mutant strain 120:943–952. cultivation with Agrobacterium. No shoots of Agrobacterium tumefaciens.HortScience Song, G.Q. and K.C. Sink. 2006. Transformation of were regenerated and explants were dying in 33:897–899. Montmorency sour cherry (Prunus cerasus L.) the first 4 weeks, whereas control leaf ex- Hood, E.E., S.B. Gelvin, L.S. Melchers, and A. and Gisela 6 (P. cerasus · P. canescens) cherry plants (in the medium without kanamycin) Hoekema. 1993. New Agrobacterium helper rootstock mediated by Agrobacterium tumefa- produced many shoots within 4 weeks. De- plasmids for gene transfer to plants. Transgenic ciens. Plant Cell Rep. 25:117–123. creasing kanamycin concentration only sev- Res. 2:208–218. Yancheva, S.D., P. Druart, and B. Watillon. 2002. eral units resulted in escapes forming many Jefferson, R.A. 1987. Assaying chimeric genes in Agrobacterium-mediated transformation of plant—The GUS gene fusion system. Plant plum (Prunus domestica L.). Acta Hort. nontransformed shoots. It may be helpful to Mol. Biol. Rep. 5:387–405. 577:215–217. culture infected explants in nonselective (no Lloyd, G. and B. McCown. 1980. Commercially- Zhang, Z., W. Dai, Z.M. Cheng, and J.A. Walla. kanamycin) medium before transfer to selec- feasible micropropagation of mountain laurel, 2000. A shoot-tip culture micropropagation tive (with kanamycin) medium. This may Kalmia latifolia, by use of shoot-tip culture. system for chokecherry. J. Environ. Hort. allow infected explants to recover from in- Proc. Intl. Plant Prop. Soc. 30:421–427. 18:234–237.

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