Frontiers in Life Science, 2015 Vol. 8, No. 3, 249–255, http://dx.doi.org/10.1080/21553769.2015.1051242 Stable integration of mgfp5 transgenes following Agrobacterium-mediated transformation in Boesenbergia rotunda cell suspension culture a a a , b b Sher Ming Wong , Fatin Iﬀah Rasyiqah Mohamad Zoolkeﬂi , Rezaul Karim , Boon Chin Tan , a , b , c a , b∗ Jennifer Ann Harikrishna and Norzulaani Khalid a b Institute of Biological Sciences, Faculty of Science, University of Malaya, Lembah Pantai, Kuala Lumpur 50603, Malaysia; Center of Biotechnology for Agriculture Research (CEBAR), University of Malaya, Lembah Pantai, Kuala Lumpur 50603, Malaysia; High Impact Research (HIR), University of Malaya, Lembah Pantai, Kuala Lumpur 50603, Malaysia (Received 15 October 2014; accepted 11 May 2015 ) Boesenbergia rotunda, a herb in the ginger family, contains numerous beneﬁcial compounds, such as ﬂavonoids, ﬂavones and cyclohexenyl chalcone derivatives, that have great potential for pharmaceutical applications. However, the low concentration of the bioactive compounds limits their commercial application. In this study, a simple and reliable Agrobac- terium-mediated transformation protocol for B. rotunda cell suspension culture was successfully developed. The minimal −l inhibitory concentration and natural tolerance of the selective agent, hygromycin, against the cells were 20 mg l and 30 −l mg l in liquid media and solid media, respectively. The highest number of transformed regenerants (18 ± 0.00 per ml settled cell volume) was recorded when cells were infected with Agrobacterium tumefaciens harbouring pCAMBIA1304 for 10 min and co-cultivated for 2 days. Prolonged infection time ( > 10 min) and co-cultivation period ( > 2 days), however, did not increase the transformation eﬃciencies. The results clearly show that infection and co-cultivation periods strongly inﬂuenced the transformation eﬃciency in ginger. The transformed cells were recovered and showed green ﬂuorescent sig- nals under ultraviolet excitation. An intense blue colour was observed in the transformed cells after β -glucuronidase (GUS) histochemical staining, further conﬁrming the functionality of the GUS enzymes in the regenerants. Polymerase chain reac- tion analysis of 3-, 6-, 9- and 12-month-old transformed cells conﬁrmed that the protocol enabled stable integration of the mgfp5 gene. Moreover, the comparatively high number of transformed regenerants in this study made it possible to generate a large number of transgenic cells in a short period, which would be useful for high-throughput functional screening of enzymes involved in the biosynthetic pathways of bioactive compounds. Keywords: Agrobacterium transformation; pCAMBIA1304; medicinal ginger; green ﬂuorescent protein Introduction 2007) and the simplicity of enhancing the compound production using chemical elicitors or physical stimulants Plant secondary metabolites are important sources of use- (Zhao et al. 2010; Cai et al. 2011). They also oﬀer a robust ful pharmaceutical drugs. However, the low amounts of platform for the genetic engineering of metabolic pathways these compounds in plants pose a signiﬁcant challenge for owing to their fast growing and homogeneous characteris- drug development to meet industrial needs. The use of tics (Rao & Ravishankar 2002). However, yields of desired plant cell cultures has been widely studied for the pro- compounds may be less than ideal. As a result, there is duction of secondary metabolites (Häkkinen et al. 2013). interest in applying bioengineering approaches to enhance Although chemical synthesis is possible for several sec- the productivity of desired compounds. Metabolic engi- ondary metabolites, the use of toxic chemicals and extreme neering has been used to modulate endogenous metabolic conditions often limits the de novo synthesis of these com- pathways or introduce new metabolic capabilities in order pounds (Wang et al. 2011). To overcome this problem, to increase the production of a desirable compound in an eﬀective strategy to enhance the production of beneﬁ- plants (Farré et al. 2014). Despite the successes achieved cial secondary metabolites is essential. Over the decades, in the past few years, genetic engineering of plants is still many studies have been carried out to improve the pro- in its infancy. This may be due to the limited understand- duction of these high-value compounds through plant cell ing of most secondary metabolite biosynthesis pathways suspension cultures (Aoyagi 2011; Häkkinen et al. 2013). (Weathers et al. 2010), including incomplete pathways Plant cell suspension cultures oﬀer several advantages for and key regulatory mechanisms controlling the pathway the production of secondary metabolites, including the ease ﬂuxes (Oksman-Caldentey & Inzé 2004), as well as the of compound extraction and scaling up (Tepe & Sokmen *Corresponding author. Email: firstname.lastname@example.org © 2015 Taylor & Francis 250 S.M. Wong et al. low success rates in introducing multiple genes into plants concentrations of hygromycin B. Hygromycin B used in (Chen et al. 2015). the experiments was ﬁlter-sterilized using a 0.2 μm syringe An eﬃcient plant transformation can facilitate the ﬁlter and stored at 4°C until use. Gelrite (Duchefa, Haar- metabolic engineering of plants for enhanced production of lem, The Netherlands) at 2% was added only to the semi- a selected secondary metabolite. Agrobacterium-mediated solid culture medium. For the SM medium, the cells were transformation has been the most accessible tool with cultured in 250 ml conical ﬂasks with continuous shaking which to introduce heterologous DNA and manipulate at 80 rpm. All cultures were maintained at 25 ± 2°C under −2 gene expression in plants (Ribas et al. 2011). Therefore, a 16 h photoperiod with a light intensity of 31.4 μmol m −1 a reliable transformation and regeneration system with s provided by cool ﬂuorescent lamps in a growth room. a low degree of chimerism and high regeneration fre- Growth and growth-inhibition of the cell suspension cul- quency is a prerequisite (May et al. 1995; Sági et al. tures were observed by measuring the settled cell volume 1995). There are several factors that aﬀect the transforma- (SCV) with graduated 15 ml centrifuge tubes after 20 days tion eﬃciency, such as Agrobacterium density, infection of culture, whereas cells plated on semi-solid medium were time, co-cultivation duration and conditions (Yong et al. observed after 3 weeks of culture. The number of surviv- 2006; Vrbová et al. 2013), and temperature (Li et al. 2003). ing regenerants on the agar plates was counted under a To date, there is no report of Agrobacterium-mediated or stereomicroscope. direct gene transformation of medicinal ginger, Boesenber- gia rotunda. Boesenbergiarotunda is rich in bioactive com- pounds, such as ﬂavanoids, ﬂavones, cyclohexenyl chal- Plant transformation cone derivatives (Trakoontivakorn et al. 2001; Tan et al. Agrobacterium tumefaciens strain LBA4404 (Hoekema 2012) and essential oils (Jaipetch et al. 1982). This arti- et al. 1983) harbouring a binary vector pCAMBIA1304 cle reports a reliable and eﬃcient Agrobacterium-mediated (CSIRO, Dickson, Australia) was used in transforming the transformation protocol for B. rotunda cell suspension cul- B. rotunda cell suspension. The plasmid includes the nptII ture that may facilitate the development of high numbers of kanamycin selective marker genes for bacteria, an hpt gene multiple independent transformation events in B. rotunda. for plant selection and the reporter genes gus and mgfp5. Before transformation, mid-log phase Agrobacterium cul- tures with an optical density (OD ) of about 1.0 in yeast Materials and methods −1 −1 extract broth (14.0 g l nutrient broth, 1.0 g l yeast −1 Plant material and establishment of cell suspension extract, 10 mM MgSO and 5.0 g l sucrose, pH 7.5) were culture used for all transformation experiments. For infection, bac- teria were collected by centrifugation at 4000 g for 10 min Cell suspension cultures were established according to and resuspended in liquid medium at OD ≈ 1. One vol- Wong et al. (2013). In brief, newly emerged buds 600 ume of the settled cells was submerged in four volumes obtained from the fresh rhizomes of B. rotunda were of bacteria in liquid medium supplemented with 100 μM cultured on callus induction medium consisting of of acetosyringone (SMC) at diﬀerent infection times. Bac- Murashige and Skoog (MS) salts (Murashige & Skoog −1 terial broth was completely removed by careful pipetting 1962), 1.0 mg l α-naphthaleneacetic acid (NAA), 1.0 −1 −1 after the incubation period. The cells were co-cultivated mg l 6-benzyladenine (BA), 1.0 mg l indole-3-acetic with Agrobacteria in 5 volumes of SMC liquid medium acid (IAA), 3% sucrose and 0.2% Gelrite (Sigma, for diﬀerent co-cultivation periods at 28°C in darkness. For St Louis, MO, USA). The induced pro-embryogenic post co-cultivation treatments, the cells were washed in SM masses were then transferred on to propagation medium −1 medium supplemented with an appropriate concentration [MS basal medium supplemented with 3 mg l 2,4- −1 of hygromycin B and 300 mg l cefotaxime and agitated dichlorophenoxyacetic acid (2,4-D), 3% sucrose and 0.2% on a rotary shaker for 1 h at 80 rpm. They were then trans- Gelrite] for 3–4 weeks. To establish cell suspension cul- ferred into 50 ml SM medium and maintained for 20 days tures, embryogenic callus generated was cultured in liquid −1 at 25 ± 2°C under a 16 h photoperiod with a light intensity MS basal medium supplemented with 1 mg l 2,4-D, 680 −2 −1 of 31.4 μmol m s provided by cool ﬂuorescent lamps μM L-glutamine and 2% sucrose. The cell suspension in the growth room. After 20 days of culture, the cells were cultures were agitated at 80 rpm on a rotary shaker and plated on SMA for recovery and regeneration. Transforma- subcultured every 14 days with replacement of fresh liquid tion eﬃciency was expressed as the number of regenerants medium at a ratio of 1:4 (old to fresh media). per millilitre of SCV. Minimal inhibitory concentration of hygromycin B against Boesenbergia rotunda cells Green ﬂuorescent protein visualization and β -glucuronidase histochemical assay The established cells were cultured in MS liquid (SM) or on semi-solid (SMA) selection media supplemented Putative transformed cells were visualized under 460 −1 with 1mgl 2,4-D, 680 μM L-glutamine and diﬀerent nm excitation wavelength with a ﬂuorescent inverted Frontiers in Life Science 251 microscope (Olympus IX71, Tokyo, Japan) equipped with Statistical analysis a 460–490 nm emission ﬁlter. Green ﬂuorescent protein All experiments were done in triplicate and statistically detection was documented using a cool camera attached analysed using one-way analysis of variance (ANOVA). to the microscope. For GUS histochemical staining, cells The mean values of treatments were subjected to Duncan’s recovered from SMA were incubated in histochemical multiple range test at the 95% conﬁdence level using the reagent containing 0.1 M phosphate buﬀer (pH 7.0), 0.5 Statistical Package for the Social Sciences (SPSS, Chicago, mM ferricyanide, 0.5 mM ferrocyanide, 0.1% (v/v) Tri- IL, USA). ton X-100, 10.0 mM EDTA, 20% (v/v) methanol and 1.0 mM 5-bromo-3-indolyl-glucuronide (X-Gluc) (Fermentas, Results and discussion Hanover, MD, USA) at 37°C in darkness for 3 days until blue coloration appeared. The cells were washed with Minimal inhibitory concentration of hygromycin B 70% (v/v) ethanol. Finally, the stained samples were trans- against Boesenbergia rotunda suspension cells ferred and ﬁxed in formalin/acetic acid/ethanol solution, as Successful selection of transformed cells and elimination described in Jeﬀerson (1989), before being observed under of the non-transgenic cells is a crucial step in successful a light microscope. genetic engineering for plants (Veluthambi et al. 2003). To allow only the cells expressing the transgene to regen- erate, the optimum concentration of the selective agent Polymerase chain reaction analysis has to be determined. High concentrations of antibiotics Total genomic DNA was isolated from putative trans- are toxic to plant cells and may cause abnormalities in formed cells according to a modiﬁed method based on plant growth, whereas low concentrations can lead to false Doyle (1987). For PCR analysis, 100 ng of total DNA was positives. The eﬀectiveness of a selection agent can be added to a 20 μl PCR containing 0.2 mM of each dNTP, 1.0 assessed by determining the MIC and natural tolerance of mM MgCl , 1 unit GoTaq DNA polymerase and 0.1 μM the cell cultures (Parveez et al. 2007). The eﬀects of diﬀer- of each primer pair for the ampliﬁcation of the mgfp5 gene: ent concentrations of hygromycin B against B. rotunda cell 5 -AAG GAG AAG AAC TTT TCA CTG GAG-3 and suspension are depicted in Figures 1 and 2. The growth of 5 -AGT TCA TCC ATG CCA TGT GTA-3 . PCR ampli- cells was greatly aﬀected when hygromycin B was applied ﬁcation was performed in a thermal cycler with an initial in the SM for about 20 days (Figure 1a). Hygromycin B −l denaturation at 95°C for 1 min, followed by 30 cycles at at 20 mg l , corresponding to a lethal dose higher than 95°C for 1 min, 55°C for 1 min, 68°C for 1 min and a 75% (LD ), was found to be eﬀective in inhibiting the ﬁnal extension of 68°C for 10 min. PCR products were cell growth. In SMA, the LD of the B. rotunda cells −l separated on 1.0% agarose gel using electrophoresis and was found at 30 mg l hygromycin B (Figure 1b), indi- analysed using a Gel-Pro imager and analyser (Micro- cating that a higher concentration of hygromycin B was Lambda, Clarksburg, NJ, USA). The gel was scored for required in SMA compared to SM. However, no cells sur- the presence of the mgfp5 product ( ≈ 700 bp). vived when cultured on SMA supplemented with 50 mg Figure 1. Inhibitory eﬀects of diﬀerent concentrations of hygromycin B against Boesenbergia rotunda cell suspension culture in (a) −l liquid SM and (b) solid SMA for about 20 days. Lethal dose higher than 75% (LD ) for cells cultured in SM and SMA was 20 mg l −l and30mgl , respectively. Error bars represent standard error, where n = 3. SCV = settled cell volume. 252 S.M. Wong et al. −l −l −l −l Figure 2. Cells cultured on SMA supplemented with (a) 0 mg l (control), (b) 10 mg l , (c) 20 mg l , (d) 30 mg l and (e) 50 mg −l l hygromycin B. SCV = settled cell volume. −l l hygromycin B. The surviving cells were recovered on The results of this study revealed that infection time and selection media, whereas non-transformed cells eventually co-cultivation period are important parameters aﬀecting turned brown and died (red arrows in Figure 2e). Applica- the eﬃciency of Agrobacterium-mediated transformation tion of the appropriate concentration of hygromycin B not of B. rotunda cell suspension culture. To achieve opti- only enabled eﬀective selection of transformed cells but mum transformation eﬃciency, the inﬂuences of infection also allowed synchronization between selection and cell time and co-cultivation on Agrobacterium-mediated trans- growth in diﬀerent developmental growth phases, i.e. prop- formation of B. rotunda cell suspension culture were exam- agation and regeneration of the suspension cells, which ined. The results revealed that the highest number of trans- usually involve diﬀerent media combinations (Ribas et al. formed regenerants (18 ± 0.00 per ml SCV) was achieved 2011). when cultures were infected with Agrobacterium for 10 min and co-cultivated for 2 days (Figure 3). Longer infec- tion times ( > 10 min) and co-cultivation periods ( > 2 Eﬀects of infection times and co-cultivation periods on days) did not increase the transformation eﬃciency. In Boesenbergia rotunda suspension cell transformation spite of the overall successful transformation of B. rotunda eﬃciency suspension cell, a co-cultivation period of only 1 day resulted in low transformation eﬃciency. A 2–3 day co- The transfer and integration of genes is a lengthy pro- cultivation period is optimal for many plant species, such cess, and is dependent on the species and choice of explant as cotton (Sumithra et al. 2010), tomato (Chetty et al. 2013) culture conditions (Gelvin 2003; Mayavan et al. 2013). Figure 3. Eﬀects of infection times and co-cultivation periods on Agrobacterium-mediated transformation of Boesenbergia rotunda suspension cell. Diﬀerent letters indicate signiﬁcant diﬀerences at 95% conﬁdence level based on one-way ANOVA followed by Duncan’s multiple range test. Error bars represent standard error, where n = 5. Frontiers in Life Science 253 Figure 4. (a, b) Embryoid regenerants [red (pale) arrows] and non-transformed cell (black arrows) on hygromycin selection. Bars = 1 mm (a) and 1 cm (b). (c) Putatively transformed regenerants showing gus expression in GUS histochemical assay. Bar = 1 mm. (d) Transformed cells appear as green ﬂuorescence under ultraviolet excitation. Bar = 10 μm. which ultimately inhibited cell growth. However, some plant species require a longer period of co-cultivation; for example, in Vanda orchid, explants co-cultivated for 4 days produced the highest transient gusA expression, compared to those co-cultivated for 1, 2 and 3 days (Gnasekaran et al. 2014). Similar ﬁndings were reported by De Bondt et al. (1994), where a co-cultivation period of 4 days yielded a higher transformation eﬃciency in apple. Co-cultivation period is also dependent on the Agrobacterium strain used. For instance, Agrobacterium strain LBA4404 required 4 days for co-cultivation for optimal transformation in blueberry (Cao et al. 1998; Cheng et al. 2011). Cell regeneration and analysis of transformants The transformed cells were recovered on the selection media, whereas non-transformed cells eventually turned brown and died (red arrows in Figure 4a). Embryoid struc- Figure 5. Polymerase chain reaction analysis of mgfp5 gene tures were observed on the putatively transformed cells integration in putatively transformed Boesenbergia rotunda after 3 weeks of selection on SM and SMA (Figure 4a,b). regenerant cells. Lane 1: 100 bp DNA ladder; lane 2: no tem- In GUS histochemical staining, an intense blue colour was plate control; lane 3: wild-type cells; lanes 4–7: 3-, 6-, 9- and 12-month-old transformed regenerants; lane 8: plasmid control. observed in the transformed cells (Figure 4c). The cells were then examined for green ﬂuorescence to determine and sugarcane (Mayavan et al. 2013). Dutt et al. (2010) the expression of the mgfp5 gene. All surviving cells recov- also reported that citrus explants co-cultivated for 4 or 5 ered from subsequent selection in SM and SMA media days did not have an improved transformation eﬃciency emitted green ﬂuorescence under ultraviolet examination rate, probably owing to the overgrowth of Agrobacterium, (Figure 4d). To investigate the stability of mgfp5 gene 254 S.M. Wong et al. integration over 12 months, the putative 3-, 6-, 9- and 12- Chetty VJ, Ceballos N, Garcia D, Narváez-Vásquez J, Lopez W, Orozco-Cárdenas ML. 2013. Evaluation of four Agrobac- month-old transformed cells were propagated and analysed terium tumefaciens strains for the genetic transformation by performing PCR ampliﬁcation using mgfp5-speciﬁc of tomato (Solanum lycopersicum L.) cultivar Micro-Tom. primers. A single band corresponding to about 700 bp was Plant Cell Rep. 32:239–247. detected after PCR and gel analysis (Figure 5), indicating De Bondt A, Eggermont K, Druart P, Vil MD, Goderis the presence of the mgfp5 transgene in all samples tested. L, Vanderleydon J, Broekaert WF. 1994. Agrobacterium- mediated transformation of apple (Malus X domestica It is noteworthy that a stable Agrobacterium-mediated Borkh.): an assessment of factors aﬀecting gene trans- transformation of B. rotunda cell suspension cultures with fer during early transformation steps. Plant Cell Rep. 13: integration of mgfp5 gene was achieved for over 12 587–593. months. The results clearly show that infection and co- Doyle JJ. 1987. A rapid DNA isolation procedure for small cultivation strongly inﬂuence the transformation eﬃciency quantities of fresh leaf tissue. Phytochem Bull. 19:11–15. Dutt M, Madhavaraj J, Grosser JW. 2010. Agrobacterium in ginger. The comparatively high number of transformed tumefaciens-mediated genetic transformation and plant regenerants obtained by this method made it possible to regeneration from a complex tetraploid hybrid citrus root- generate a large number of transgenic cells in a short stock. Sci Hortic. 123:454–458. period, which would be useful for application in high- Farré G, Blancquaert D, Capell T, Van Der Straeten D, Christou P, Zhu C. 2014. Engineering complex metabolic pathways throughput metabolite engineering and functional analysis in plants. Annu Rev Plant Biol. 65:187–223. in B. rotunda. Gelvin SB. 2003. Agrobacterium-mediated plant transformation: the biology behind the “Gene-Jockeying” tool. Microbiol Mol Biol R. 67:16–37. Acknowledgements Gnasekaran P, James Anthony JJ, Uddain J, Subramanian The authors greatly appreciate the University of Malaya for pro- S. 2014. Agrobacterium-mediated transformation of the viding support and facilities, the Malaysian Ministry of Science recalcitrant Vanda Kasem’s Delight orchid with higher and Technology for a National Science Fund (NSF) doctoral eﬃciency. Sci World J. 2014. Available from: http:// scholarship to the ﬁrst author, and the reviewers who helped to dx.doi.org/10.1155/2014/583934 improve the manuscript. Häkkinen ST, Ritala A, Rischer H, Oksman-Caldentey KM. 2013. Medicinal plants, engineering of secondary metabo- lites in cell cultures. In: Sustainable food production. New York: Springer; p. 1182–1200. Disclosure statement Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA. 1983. No potential conﬂict of interest was reported by the author(s). A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature. 303:179–180. Funding Jaipetch T, Kanghae S, Pancharoen O, Patrock, VA, Reutrakul V, Tuntiwachwuttikul P, White AH. 1982. 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Frontiers in Life Science
– Taylor & Francis
Published: Jul 3, 2015
Keywords: Agrobacterium transformation; pCAMBIA1304; medicinal ginger; green fluorescent protein