Scientia Horticulturae 123 (2010) 454–458

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Scientia Horticulturae

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Agrobacterium tumefaciens-mediated genetic transformation and plant from a complex tetraploid citrus rootstock

M. Dutt, J. Madhavaraj, J.W. Grosser *

Horticultural Sciences Department, University of Florida-IFAS, Citrus Research and Education Center (CREC), 700 Experiment Station Road, Lake Alfred, FL 33850, USA

ARTICLE INFO ABSTRACT

Article history: Agrobacterium-mediated genetic transformation of a tetraploid ‘‘tetrazyg’’ citrus rootstock selection Received 19 August 2009 ‘Orange #16’ [Nova mandarin (Citrus reticulata Blanco) + Hirado Buntan pummelo (Citrus grandis L. Received in revised form 14 October 2009 Osbeck)] [Cleopatra mandarin (C. reticulata Blanco) + Argentine trifoliate orange (Poncirus trifoliata (L.) Accepted 16 October 2009 Raf.)] was performed. Juvenile epicotyl segments were transformed with a construct containing a bifunctional egfp–nptII fusion under the control of an enhanced double CaMV 35S promoter. Our Keywords: protocol resulted in a reasonable transformation efficiency of 18%. Stable integration of the transgene Agrobacterium tumefaciens was confirmed by visual observation of EGFP expression, PCR and Southern blot hybridization. The Bifunctional gene purpose of this work was to investigate the amenability of novel citrus rootstock germplasm being Citrus EGFP developed for improved tree size control, soil adaptation, and disease resistance, to existing Genetic transformation transformation technologies. Seed trees of such transgenic tetraploids also have potential as trap Tetrazyg plants containing potent insecticidal transgenes, due to their inedible fruit and inherent crossing barriers Tetraploid with conventional commercial diploid scion cultivars, and could be planted around producing citrus groves. ß 2009 Elsevier B.V. All rights reserved.

1. Introduction production systems that utilize high planting densities (Grosser et al., 1996). The model for somatic fusion in citrus is to fuse Tetraploid citrus plants are playing an increasingly important from an embryogenic culture of the first parent with role in citrus cultivar improvement due to their value as potential leaf-derived protoplasts of the second parent (Guo and Grosser, breeding parents for both scion and rootstock improvement 2005). Successful embryogenic cultures in citrus have been limited (Grosser and Gmitter, 2005, 2009; Grosser et al., 2003). The to polyembyonic cultivars that produce seed containing nucellar combination of complementary diploid parents to produce an embryos (Grosser and Gmitter, 1990). The second parent can be allotetraploid plant offers a powerful tool to maximize genetic polyembryonic, or monoembryonic that produce seed with zygotic diversity and thereby incorporate desirable traits into progeny embryos. Somatic hybrids that combine a polyembryonic parent (Grosser et al., 2000). Numerous desirable tetraploid combinations with a monoembryonic parent often produce seed with zygotic have been produced in recent years and targeted somatic embryos, and thus can be used subsequently as females in hybridizations are now routine (Grosser and Gmitter, 1990; conventional breeding at the tetraploid level. This approach has Grosser and Chandler, 2000; Louzada and Grosser, 1994; Guo tremendous potential to maximize genetic diversity in tetraploid et al., 2004; Ananthakrishnan et al., 2006). progeny that can be screened for various traits, and we have been Citrus tetraploids have obvious value in scion improvement, as conducting rootstock breeding and selection at the tetraploid level allotetraploids produced via fusion and autotetraploids for the past few years (Grosser et al., 2003, 2007; Grosser and are routinely used in citrus breeding programs around the world in Gmitter, 2009). We have coined the term ‘tetrazyg’ to identify interploid crosses to produce new commercially important allotetraploid hybrids produced from such crosses of selected seedless triploid hybrids (Grosser and Gmitter, 2005, 2009). For somatic hybrids (Grosser et al., 2003). These unique ‘tetrazyg’ rootstock improvement, tetraploids have been shown to have good plants produce genetic combinations from the of potential for improving tree size control, a desirable trait for new multiple citrus cultivars and this broad genetic base provides them with potential to alleviate many of the common problems facing the citrus industry. The screening of ‘tetrazyg’ hybrids from * Corresponding author. Tel.: +1 863 956 1151; fax: +1 863 956 4631. several crosses has resulted in the identification of promising E-mail address: jgrosser@ufl.edu (J.W. Grosser). rootstock candidates that are showing good tolerance to numerous

0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.10.010 M. Dutt et al. / Scientia Horticulturae 123 (2010) 454–458 455 abiotic conditions as well as pest and diseases (Grosser et al., 2007; 2.3. Agrobacterium-mediated transformation Grosser et al., unpublished). Agrobacterium-mediated transformation of elite citrus culti- Agrobacterium-mediated transformation was carried out as vars has opened up the possibility to produce potential disease or described by Dutt and Grosser (2009). Light green epicotyl pest resistant citrus plants by incorporation of resistance gene(s) segments were cut obliquely into 2.0–2.5-cm-long segments to from sexually compatible or incompatible plant species or other expose the cambial ring. These segments were incubated in organisms (e.g. Dutt et al., 2008; Febres et al., 2008; Barbosa- liquid co-cultivation (CM) medium for 3 h before incubation Mendes et al., 2009). This method of improvement of citrus via with Agrobacterium. A. tumefaciens strain EHA105 containing the genetic transformation is especially useful in cases where it is not binary vector pD35s (Li et al., 2001) was used for transforma- possible to engineer a particular trait of interest to an otherwise tion. This vector contains a bifunctional egfp -nptII fusion gene elite cultivar using conventional breeding. This method also under the control of an enhanced double CaMV 35S promoter. A allows the incorporation of a particular gene of interest without single Agrobacterium colony was cultured in liquid YEP medium otherwise changing the genotypic and phenotypic integrity of a containing 100 mg L1 kanamycin and 20 mg L1 rifampicin on a cultivar. shaker (185 rpm) at 26 8Cfor2days(Dutt et al., 2007). Two Cervera et al. (2000) reported the presence of off type milliliters of a vigorously growing culture was seeded into transgenic autotetraploid plants in a population of diploid Carrizo 48 mL YEP medium containing appropriate antibiotics for 3 h transgenics. Such tetraploids were speculated to have originated before being collected by centrifugation at 5000 g for 6 min at either by the natural occurrence of tetraploid seedlings or by a 25 8C and resuspended in liquid co-cultivation (CM) medium. process of polyploidization during in vitro culture. To date, The OD600 was adjusted to 0.3 or 0.6 before incubation to however, no report of deliberate transformation and regeneration observe the effect of bacterial concentration on transformation. of transgenic shoots from any tetraploid citrus cultivar, especially Explants were incubated in the Agrobacterium suspension for from a promising complex allotetraploid has been reported. 5 min. Subsequently, explants were blotted dry on sterile paper Therefore, there is still a need for development of transformation towels, placed on solid CM medium supplemented with 100 mM protocols for this important group of citrus. In this paper we acetosyringone and incubated in the dark at 25 8Cfor2or3days describe a successful protocol for the Agrobacterium tumefaciens before transfer to regeneration medium. Explants were cultured mediated genetic transformation of a promising complex tetrazyg indarkfor2weeksat268C, followed by transfer to light. The from our tetraploid rootstock breeding program, [Nova mandarin transformation efficiency of putatively transgenic shoots was (Citrus reticulata Blanco) + Hirado Buntan pummelo (Citrus grandis evaluated as the number of EGFP-positive plants per total (L.) Osbeck)] [Cleopatra mandarin (C. reticulata Blanco) + Argen- number of inoculated explants as outlined by Cervera et al. tine trifoliate orange (Poncirus trifoliata (L.) Raf.)]. This selection, (1998). named ‘Orange #16’ has shown wide soil adaptation, good phytophthora resistance, and its seed are highly nucellar averaging 2.4. Selection and regeneration of transformants 28 seeds per fruit (J.W. Grosser, unpublished data). We report herein the effect of medium requirements, optical density of the EGFP-specific fluorescence was evaluated using a Zeiss SV11 Agrobacterium suspension and co-cultivation duration required for epi-fluorescence stereomicroscope equipped with a light source efficient transformation of this tetraploid selection. Polymerase consisting of a 100 W mercury bulb and a FITC/GFP filter set with a chain reaction (PCR) and Southern blot hybridization were also 480 nm excitation filter and a 515 nm longpass emission filter utilized to confirm transgene insertion and copy number of producing a blue light (Chroma Technology Corp., VT, USA). After 2 selected clones. biweekly transfer cycles into fresh regeneration medium contain- ing antibiotics, transgenic shoots that expressed stable, non- 2. Materials and methods chimeric EGFP-specific fluorescence were transferred onto RMG medium. After a month of culture in vitro in this medium, EGFP 2.1. Plant materials expressing shoots were transferred into RMM medium for rooting (Dutt and Grosser, 2009). Rooted plants were hardened off and Fruits harvested from a single tree located in the Citrus their levels confirmed via flow cytometry using a table top Research and Education Center (CREC) grove were used for all Ploidy Analyser (Partec GmbH, Germany) as described by Khan and experiments. The seeds were extracted from fruit and the outer Grosser (2004). seed coat removed before being surface-sterilized for 8 min in a 0.6% (v/v) sodium hypochlorite solution containing a drop of 2.5. Statistical analysis Tween 20 and rinsed with sterile deionized water. Seeds were germinated in 15-cm-long glass culture tubes containing 15 mL of Data were analyzed with SAS version 8.02 (SAS Institute, Cary, solid MS medium [consisting of MS salts and vitamins (Murashige NC, USA) using analysis of variance and Tukey’s test to determine and Skoog, 1962) supplemented with 30 g L1 sucrose and 7 g L1 mean separation between treatments. agar, pH 5.8]. The culture tubes were kept in the dark for 3 week at 25 8C, and subsequently transferred to the light (16 h light/8 h dark 2.6. Analysis of transgene integration cycle using cool white fluorescent light (75 mmol s1 m2)) for an additional week, allowing the etiolated explants to turn green. To confirm presence of the transgene in citrus plants, polymerase chain reaction (PCR) was carried out in a thermal 2.2. Determination of regeneration potential cycler (MJ Research, MA, USA) using GoTaq1 Green Master PCR Mix (Promega Corp, WI, USA) and egfp–nptII fusion gene-specific Regeneration potential was evaluated in three different media. oligonucleotide primers: EN51 (50-GGATCCATGGTGAGCAAG- The media used in the study i.e. RM, RM1 and DBA3 have been GGCGA-30) and EN32 (50-ACTAGTTATCAGAAGAACTCGTCAAG-30). described earlier (Deng et al., 1992; Dutt and Grosser, 2009). Each 50 ng of genomic DNA was used as a template. Amplified DNA medium was supplemented with antibiotics for selection of fragments were electrophoresed on a 1% agarose gel containing transgenic shoots (kanamycin (70 mg L1)) and to get rid of GelRedTM Gel Stain (Biotium Inc., CA, USA) and Agrobacterium (timentin (400 mg L1)). visualized under UV light. 456 M. Dutt et al. / Scientia Horticulturae 123 (2010) 454–458

Table 1 the DIG-labeled egfp probe, chemiluminescence substrate CDP- Regeneration efficiency, number of EGFP-positive shoots and transformation Star was used for immunological detection of hybridization signals efficiency of ‘Orange #16’ following different treatments. using X-ray film autography. Genomic DNA extracted from a non- Treatments No. of inoculated Regeneration No. of EGFP-positive transformed control plant served as negative control. explantsa efficiency (%)b shoots (transformation efficiency (%)c) 3. Results and discussion Medium RM 250 108.3ad 44 (17.6)a 3.1. Genetic transformation of epicotyl explants RM1 265 64.1b 21 (7.9)b DBA3 260 99a 40 (15.4)a Agrobacterium-mediated transformation of juvenile explants of OD600 0.3 200 115a 32 (16)a the tetrazyg selection ‘Orange #16’ was carried out using a binary 0.6 200 101a 36 (18)a vector containing the bifunctional egfp–nptII fusion gene (Li et al., 2001; Dutt et al., 2007) and several transgenic plants regenerated Co-cultivation duration 2 days 248 131.3b 31 (12.5)b that constitutively expressed the EGFP . Production of 3 days 231 90.1a 38 (16.5)a tetrazyg plants offers a novel approach to maximize genetic diversity present in tetraploid progeny (Grosser et al., 2003). a Data from three independent experiments. b Regeneration efficiency was calculated as the total number of shoots produced/ Addition of another trait using allows the total number of explants evaluated 100. incorporation of gene(s) not present in citrus and therefore c Transformation efficiency was calculated as the total number of EGFP-positive impossible to incorporate using conventional methods. Since this shoots/total number of explants evaluated 100. is the first report of Agrobacterium-mediated transformation and d Means within treatments followed by the same letter were not different at a = 0.05 using Tukey’s test. regeneration of tetraploid citrus, we evaluated several media to optimize the regeneration potential. It was observed that there was no difference in regeneration on either RM or DBA3 medium, Southern blot analysis was performed to confirm stable whereas both were superior to RM1 medium (Table 1). Both RM integration of the transgene and determine copy number in four and DBA3 media, although different in their chemical composition randomly selected transgenic plants. Ten micrograms of genomic are similar in their BAP levels. Each medium contains 13.2 mM BAP DNA were digested with HindIII, eletrophoresed on a 0.8% agarose as compared to RM1 which has 4.4 mM BAP. In addition, RM also gel in 1 TAE buffer and transferred onto a positively charged contains 2.7 mM NAA while DBA3 contains 0.05 mM 2,4-D. Our nylon membrane by capillary transfer. Following hybridization to tetrazyg selection behaved similarly as the trifoliate Carrizo

Fig. 1. Transgenic ‘Orange #16’ plants. (A) A young transgenic plant 4 months after soil acclimatization. (B) EGFP-positive shoots visualized under an epi-fluorescent microscope. (C) Closeup of young transgenic leaf as visualized under an epi-fluorescent microscope. Non-transgenic leaf (inset) appears red under blue light due to auto- fluorescence of chlorophyll. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) M. Dutt et al. / Scientia Horticulturae 123 (2010) 454–458 457

significant role in the transformation of ‘Orange #16’. In the levels studied, there was no significant difference in either the regeneration efficiency or transformation efficiency (Table 1).

We have observed earlier that the optimum OD600 was dependent on the cultivar (Dutt and Grosser, 2009). Decreasing OD600 values were also co-related with improved regeneration and transforma- tion frequency in citrange (Yu et al., 2002), but for the range studied here we did not observe any significant difference. We speculate that the ability of explants to tolerate higher levels of Agrobacterium during incubation and subsequent co-cultivation is dependent on the overall morphology and vigor of the epicotyl segments. Thinner epicotyl segments are usually more susceptible to Agrobacterium overgrowth at higher inoculums levels (e.g. Mexican lime or Volkamer lemon) than cultivars with a thicker epicotyl (e.g. Carrizo or Duncan). ‘Orange #16’ is a very vigorous Fig. 2. Flow cytometry analysis of citrus cells. Histogram of the diploid non- cultivar and produces thick epicotyls. Therefore, within the range transgenic Poncirus trifoliata plant (peak 1) and transgenic ‘Orange #16’ tetraploid studied, the higher OD value of 0.6 did not have any detrimental plant (peak 2). 600 impact on the transformation and subsequent regeneration of transgenic plants. citrange rootstock in terms of requirements for a higher level of BAP in the medium (Dutt and Grosser, 2009). It has been reported 3.3. Effect of co-cultivation time that cytokinin levels are critical for callus formation from the cut end of epicotyl explants and subsequent initiation of bud from Our results demonstrated that higher transformation efficiency those calli (Moreira-Dias et al., 2000). Higher transformation was obtained after 3 days of co-cultivation. This was significantly efficiency was also observed from explants placed on RM and higher than results obtained with explants that had been co- DBA3. Similar requirements have also been reported for P. trifoliata cultivated for 2 days. Co-cultivation duration is important as in this transformation by Zou et al. (2008) or during precocious P. period of time the bacterium is able to incorporate its T-DNA into trifoliata transformation by Tan et al. (2009). Culture of explants in the plant’s . In citrus transformation, most cultivars have the dark for a period of 2 weeks also enhanced the callus formation been reported to perform well with co-cultivation duration of 2 or in the cambial cells of cut explants of ‘Orange #16’ and most 3 days. It has been observed that a longer period of co-cultivation transgenic shoots were observed to originate from such cells. (e.g. 5 days) is detrimental for the production of transgenic shoots Callus formation and subsequent indirect organogenesis are due to overgrowth of the bacterium (Cervera et al., 1998). We did essential for high transformation efficiency as demonstrated by not observe any additional improvement in the transformation Pena et al. (2004). Tan et al. (2009) reported similar results during efficiency when explants were co-cultivated for 4 or 5 days. Longer precocious P. trifoliata transformation where they observed the co-cultivation duration also resulted in Agrobacterium overgrowth cambial cells to be receptive to Agrobacterium infection and (data not shown). subsequent callus formation from such cells resulted in high transformation efficiency. Subsequent experiments were therefore 3.4. In vitro regeneration of transgenic plants carried out using RM medium. Rooting of transgenic plants is usually problematic and plants

3.2. Effect of OD600 have to be micrografted into vigorous rootstocks to alleviate this problem. Transgenic ‘Orange #16’ plants constitutively expressing Optical density is considered an important factor in Agrobacter- the EGFP protein (Fig. 1) were however easy to root in culture. We ium-mediated transformation of citrus but it did not play a obtained over 90% rooting efficiency when transgenic explants

Fig. 3. Molecular analyses of transgenic ‘Orange #16’ plants. (A) Amplification products obtained from PCR of genomic DNA of transgenic citrus plants with egfp–nptII gene- specific primers which successfully amplified the expected 1.5 kb gene fragment (arrow). M, 1 kb ladder; 1–8 are eight individual transgenic lines. (B) Southern hybridization analysis of total DNA from leaf tissue of four randomly selected transgenic plants (lanes 1–4) and a non-transgenic plant (CON). Genomic DNA was digested with HindIII, and probed with a DIG-labeled egfp gene fragment. 458 M. Dutt et al. / Scientia Horticulturae 123 (2010) 454–458 were placed in RMM rooting medium (Dutt and Grosser, 2009). Cervera, M., Juarez, J., Navarro, A., Pina, J.A., Duran-Vila, N., Navarro, L., Pena, L., 1998. Genetic transformation and regeneration of mature tissue of woody fruit Numerous roots developed within 2 weeks of culture and plants plants bypassing the juvenile stage. 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