Agrobacterium – Mediated Genetic Transformation of the Resurrection Plant Haberlea Rhodopensis Friv

Bulgarian Journal of Agricultural Science, 19 (2) 2013, 10–14 Agricultural Academy

AGROBACTERIUM – MEDIATED GENETIC TRANSFORMATION OF THE RESURRECTION PLANT HABERLEA RHODOPENSIS FRIV.

G. PETROVA* and D. DJILIANOV AgroBioInstitute, BG – 1164 Soﬁ a, Bulgaria Abstract

PETROVA, G. and D. DJILIANOV, 2013. Agrobacterium – mediated genetic transformation of the resurrection plant Haber- lea rhodopensis Friv. Bulg. J. Agric. Sci., Supplement 2, 19: 10–14

Resurrection plants are widely used models for desiccation tolerance studies. Several genes have been successfully iso- lated from these species. In an attempt to study their role, these genes have been successfully transferred in other model plants. Since resurrection plants are as a rule, polyploids, they are pure targets for mutational studies. In this respect, the establishment of effi cient and repeatable transformation system will contribute signifi cantly for the elucidation of stress tolerance. In this study we describe for the fi rst time, a procedure for Agrobacterium tumefaciens – mediated genetic transformation of the resurrection plant Haberlea rhodopensis Friv. For in vitro regeneration of Haberlea, we used liquid WPM media. It enables us to achieve direct regeneration and transformation system, which is an alternative to the callus-based transformation, used in other resurrection plants. The A. tumefaciens strain LBA4404 harbouring the plasmid pCAMBIA 1305.1 which contains the gus gene as a reporter gene and hpt II gene as a selectable marker gene was used. The initial experiments were conducted in order to establish the suitable concentration of cefotaxime for the elimination of Agrobacterium from cultures, as well as the optimal concentration of hygromycin for the selection of transformed plants. It was found that the highest concentration of cefotaxime that protocorms of H. rhodopensis could tolerate is 500 mg.l–1 and they are inhibited at 0.75 mg.l–1 hygromycin. Transformation was confi rmed by histochemical GUS assay and 35S- / NOS- PCR analysis. The percentage of GUS activity was 3% and the optimal co-cultivation time was 60 minutes. Key words: Agrobacterium, Haberlea rhodopensis, regeneration, transformation

Introduction The climatic changes and global warming drastically re- duce plant productivity (Boyer, 1982). To overcome these The Gesneriacae are relatively large family comprising challenges stress tolerant crops should be developed and cul- over 3200 species in 150-160 genera (Weber, 2004; Weber tivated (Khush, 1999). In this respect, genetic transforma- and Skog, 2007), widespread predominantly in tropical and tion of plants is considered among the most appropriate ap- subtropical regions of Eastern Asia, Indonesia, and South proaches used to confi rm the putative involvement of genes and Central America. There are a few outliers in Europe, of interest in plant stress tolerance (Djilianov et al., 2009). with fi ve species in three genera: Ramonda myconi (L.) Due to their extreme desiccation tolerance, the so-called Rchb., R. nathaliae Pančić & Petrović, R. serbica Pančić, resurrection plants are widely used as model for molecular, Haberlea rhodopensis Friv., and Jancaea heldreichii Boiss physiological and metabolic studies with the fi nal goal to (Thompson, 2005). elucidate the mechanisms of tolerance and to try to transfer Haberlea rhodopensis is an endemic resurrection plant these important traits to crop plants (Djilianov et al., 2011; of the Balkan Peninsula, and occurs in Central (Balkan Mts.) Dinakar et al., 2012; Georgieva et al., 2012). and Southern (Rhodope Mts.) Bulgaria, as well as in Greece So far, within the group of resurrection plants, successful (north-eastern Pangeon Mts. and Falakron Mts.) (Strid, 1991). protocols for genetic transformation have been established

*E-mail: [email protected] Agrobacterium - Mediated Genetic Transformation of the Resurrection Plant Haberlea rhodopensis Friv. 11 for only three species: Ramonda myconi, Craterostigma Agrobacterium mediated in vitro transformation plantagineum and Lindernia brevidens (Furini et al., 1994; 50 ml of liquid YEB medium (5 g beef extract, 1 g of Toldi et al., 2002; Toth et al., 2006; Smith-Espinoza et al., yeast extract, 5 g bactopeptone, and 5 g of sucrose per li- 2007). Most of them are based on callus induction and regen- ter, pH 7.2) supplemented with 50 mg.l–1 kanamycin and 100 eration of putative transformants. mg.l–1 rifampicin was poured into 250 ml Erlenmeyer fl asks In this study, we propose an alternative to callus-based to propagate Agrobacterium – mediated transformation. Bac- transformation, comprising direct regeneration in liquid me- terial cultures were kept in dark regime at 28ºC overnight dia and subsequent Agrobacterium – mediated transforma- on a shaker at 200 rpm. Healthy-white and dense bacterial tion of Haberlea rhodopensis, without a callus stage. cultures were pelleted by centrifugation (10 min, 8000 rpm). These cultures were then re-suspended in 40 ml liquid WPM Materials and Methods medium supplemented with 50 μM acetosyringone. The explants were prepared with a scalpel while submerged in the In vitro cultivation leaf suspension, which was placed into Petri dishes (10 cm In vitro cultures of Haberlea were initiated according to in diameter, each) containing 10 ml liquid WPM medium. Petrova et al. (2010). Fresh young leaves of Haberlea were Subsequently, 1 ml suspension of Agrobacterium was added used as explant sources in order to initiate in vitro culture. into each Petri dish. The infection was carried out during a Surface sterilization with 70% ethanol was performed for 60 min gentle shaking in dim light at 22ºC. The proliferation medium also contains mixture of antioxidants (0.15 mg.l–1 30 sec, followed by treatment with 0.75% HgCl2 for 6 min. ascorbic acid and 0.1 mg.l–1 citric acid). After co-cultivation, The explants were washed three times with sterile dH2O for the infected leaf suspensions were subcultured for 2 weeks removing the residual of HgCl2 and then were germinated on basic WPM (Woody Plant Medium, Lloyd and McCown, on liquid WPM medium by addition of the antioxidant mix- 1980). Plantlets were grown at 25ºC under photoperiod of 16 ture, cefotaxime (500 mg.l–1) and hygromycin (0.75 mg.l–1). h light (approximately 4500 lx)/8 h dark regime. The plants Further, in order to obtain shoot clusters, fi ve rounds of sub- were subcultured every 4 weeks. cultivation on identical medium were performed. At each subcultivation cycle, the concentration of cefotaxime was Bacterial strain and plasmid vector reduced by 100 mg.l–1. The plants, rooted in liquid WPM me- The A. tumefaciens strain LBA 4404, which harbours dium were transplanted to maturity according to the regime the plasmid pCAMBIA 1305.1 was used for establishment described for in vitro cultured plants. of the transformation protocol. The plasmid pCAMBIA contains ß-glucoronidase (GUS) and hygromycin resistance (hpt ß-glucoronidase (GUS) activity assay II) genes. Both genes were expressed under the control of The histochemical assay for GUS gene expression was per- CaMV 35S promoter. formed according to the method described by Jefferson (1987). Plants were immersed in X-gluc (5-bromo-4-chloro-3-indol Effect of antibiotic concentration on plant growth glucuronide) solution and then were incubated overnight at 37ºC. For determining the effect of antibiotic concentration on After staining, the plant material was treated with 70% ethanol to growth of H. rhodopensis, hygromycin and cefotaxime were remove chlorophyll before the observation step. added to the sterilized regeneration medium at different concentrations (0, 0.75, 1.0, 1.5, 2.0, 2.5, 5.0 mg.l–1 hygromycin and PCR analysis 100, 200, 300, 400, 500 mg.l–1 cefotaxime). Plants were cul- Total DNA was extracted from the in vitro-grown plants tured at 25ºC under photoperiod of 16 h light. After four weeks and the putative transgenic plants according to the procedure of culturing, the effectiveness of antibiotics was evaluated. described by Dellaporta et al. (1983). The amplifi cation was performed on a GeneAmp® PCR System (Applied Biosys- Effect of cefotaxime on growth of A. tumefaciens LBA tems, Foster City, CA, USA). The fi nal volume of each PCR 4404 (pCAMBIA 1305.1) – mixture was 25 μl: 1 x PCR buffer (Fermentas, Vilnius, A. tumefaciens strain LBA4404 (pCAMBIA 1305.1) was Lithuania), 25 ng DNA, 1 μM of each primer, 100 μM of grown in YEB liquid medium supplemented with kanamycin each dNTP and 1U DNA polymerase (Fermentas, Vilnius, (50 mg.l–1) and cefotaxime with different concentrations (50, Lithuania). 100, 150, 200, 250, 300, 350 mg.l–1). Bacterial cultures were The primer sequences for PCR-reactions were as follows: grown on a shaker (200 rpm) at 28ºC for 24 h. The absor- 35S: (F): 5’-GCTCCTACAAATGCCATCA-3’; bance of bacterial suspension was measured at 550 nm. (R): 5’-GATAGTGGGATTGTGCGTA-3’ 12 G. Petrova and D. Djilianov

NOS (F): 5’-GAATCCTGTTGCCGGTCTTG-3’; viving protocorm was 500 mg.l–1 (Figure 2). The growth (R): 5’-TTATCCTAGTTTGCGCGCTA-3’ of A. tumefaciens was inhibited at 50 mg.l–1 cefotaxime

(OD550 = 0.030), (Figure 3). PCR reactions were started with a denaturation step at 94ºC, for 4 min, followed by 35 cycles with the following parameters: 94ºC for 1 min, 55ºC for 1 min, and 72ºC for 3 min. The program was terminated by extension at 72ºC for 7 min. All ampliﬁ cation products were checked on 1.0% agarose gels.

Statistical analysis of transformation frequency Student’s t-tests were performed using MS Excel 2000 (Microsoft Corporation, Seattle, USA). Differences between results are described as being signiﬁ cant where P ≤ 0.05, and not signiﬁ cant where P > 0.05.

Results and Discussion

It is well known, that during the establishment of systems Fig. 1. Effect of hygromycin on growth of H. rhodopensis for regeneration and genetic transformation, all resurrection plants possessed an extreme sensitivity due to the various types of physiological stress (Furini et al., 1994; Toldi et al., 2002; Toth et al., 2006; Smith-Espinoza et al., 2007). Under suboptimal conditions, H. rhodopensis secretes polyphenols into culture media, which is associated with stress and tissue necrosis. These negative effects are paral- leled with moderate reaction to positive effects, which is dif- ﬁ cult to be explained, but this reaction probably could be related to the slow growth of plant in nature (Djilianov et al., 2005; Toldi et al., 2010). H. rhodopensis does not require high concentrations of nutrients during the tissue cultivation. The high salt containing rich mediums are toxic for Haberlea explants cultured in vitro (Djilianov et al., 2005). Previously, our group developed an effective method for in vitro micropropagation and regeneration of H. rhodopen- Fig. 2. Effect of cefotaxime on growth of H. rhodopensis sis, which could serve as a basis for the establishment of its Agrobacterium-mediated genetic transformation (Djilianov et al., 2005). The initial step in establishment of new procedures for A. tumefaciens mediated genetic transformation in plants is to test the effects of antibiotics used either as suppressers of the Agrobacterium overgrowth, or as selective agents on the in vitro morphogenesis of the transformed plant.

Effect of antibiotics on the plant and bacterial growth The lowest dose of hygromycin, which inhibited protocorms growth, was 0.75 mg.l–1 (Figure 1). All of protocorms turned brown color after transfer to selective Fig. 3. Effect of cefotaxime on growth of A. tumefaciens medium. The highest dose of cefotaxime that yielded sur- LBA 4404 (pCAMBA 1305.1) Agrobacterium - Mediated Genetic Transformation of the Resurrection Plant Haberlea rhodopensis Friv. 13

Impact of physical and biochemical treatment on the estab- by PCR) confi rmed the successful transformation (Table lishment of transformation 1).Blue staining was observed after 30–40 days of co-culti- According to Tóth et al. (2006), the impacts of physi- vation. The highest number of blue spots was observed from cal (microwounding) and biochemical treatments (WPM plants co-cultivated in Agrobacterium for 60 min (Figure medium, liquid medium supplemented with acetosyringone) 5). All PCR positive amplifi cation products were of the ex- have a primary importance for the successful Agrobacterium pected size of 195 bp for 35S promotor (Figure 6A) and 180 mediated transformation of R. myconi. They observed that bp for NOS terminator (Figure 6B). The non-transformed more microwounding is necessary for the enhancement of control plants did not show any of the expected band sizes. the bacterial penetration. Our results showed that the microwounding also has a key importance and it is essential for the successful genetic transformation of H. rhodopensis (Figure 4A). There was no transformation when leaves were gently punched with a sharp scalpel tip (four to fi ve holes per leaf), as well as when Haberlea explants were prepared by conventional way (excis- ing 5–7 mm × 5–7 mm leaf segments by cutting off the edges of leaf blades). We found that transgenic plants could only be recovered when applying a leaf suspension of Haberlea supplemented with acetosyringone as a target for transformation.

Fig. 5. Visual detection of histochemical staining for GUS activity in the transgenic plants of H. rhodopensis

Fig. 4. A. Physical enhancement of Agrobacterium penetration plays an important role for the successful genetic transformation of H. rhodopensis. B. Morphologically normal plantlets were developed under selection pressure

Non-lethal selection strategy Most of the antibiotics, which are used as selective agents, Fig. 6. PCR, using the primers for 35S promoter (A) and and inhibitors of bacterial growth could depress plant regen- NOS terminator (B). eration (Oreiﬁ g et al., 2004). The survival rates of plants 1 – 50 bp DNA Ladder; should be as high as possible in the presence of antibiotics 2 – Transformed plant (putative transformant); and on the other hand, the optimal concentration should sup- 3 – Non-transformed plant press, but not inhibit morphogenesis on transformed plants. The fact that in our case the transgenic plants remained Conclusion green at 0.75 mg.l–1 hygromycin and showed morphologically normal phenotype, while non-transgenic regenerants Based on the obtained results, it might be concluded that have showed retarded growth, is an evidence for the subleth- our attempts to establish the regeneration system in liquid al concentrations of the applied selective agent (Figure 4B). medium without callus resulted in a hopeful method for genetic transformation of model resurrection plant H. rhodo- Selection of transgenic plants pensis. The low percent of transgenic plants (Table 1) may The results from the ß-glucoronidase activity assay, as be explained by the side effects of the antibiotics that are well as the DNA integration of transformed plants (proved used as selective agents or inhibitors of bacterial growth, 14 G. Petrova and D. Djilianov

Table 1 lea rhodopensis: a resurrection plant. Acta Physiol. Plant., 34: The impacts of explant type, microwounding and bio- 1055–1066. chemical enhancements of gene delivery on the effi ciency Jefferson, R. A., 1987. Assayng chimeric genes in plants: The GUS of transformation* gene fusion system. Plant Mol. Biol. Rep., 5: 387–405. Khush, G. S., 1999. Green revolution: preparing for the 21st cen- Frequency of transformation tury. Genome, 42: 646–655. Rate of Regeneration PCR positive, GUS positive, Lloyd, G. and B. H. McCown, 1980. Commercially feasible mi- survival, %a rate, %a % % cropropagtion of mountain laurel, Kalmia latifolia by use of 72.3±1.4 25.7±1.8 3.0±0.3 3.0±0.3 shoot tip culture. Proceed. Int. Plant Propagation Soc. 30: 421–427. *Experiments were repeated three times by using 100 explants/ Oreifi g, A. S., Kovács, G., Jenes, B., Kiss, E., Scott, P. and treatment in each repetition (± S.E) O. Toldi, 2004. Development of a non-lethal selection sys- aSurvival and regeneration rates measured under selection pressure. tem by using the aadA marker gene for effi cient recovery which can decrease the plant regeneration. This fact suggests of transgenic rice (Oryza sativa L.). Plant Cell Rep., 22: that our recovery system needs of more improvements and 490–496. modifi cations. Petrova, G., Tosheva, A., Mladenov, P., Moyankova, D. and D. Djilianov, 2010. Ex situ collection of model resurrection plant Haberlea rhodopensis as a prerequisite for biodiversity References and conservation studies. Biotechnol. & Biotechnol. Eq., 24: 1955–1959. Boyer, J. S., 1982. Plant productivity and environment. Science, Smith-Espinoza, C., Bartels, D. and J. Phillips, 2007. GFP as 218: 443–448. a tool to monitor LEA gene promoter activity in the desicca- Dellaporta, S. L, Wood, J. and J. B. Hicks, 1983. A plant DNA tion tolerant plant Lindernia brevidens. Plant Cell Rep., 26: mini-preparation: version II. Plant Mol. Biol. Rep., 1: 19–21. 1681–1688. Dinakar, C., Djilianov, D. and D. Bartels, 2012. Photosynthesis in Strid, A., 1991. Haberlea rhodopensis Friv. In: Strid, A. and K. desiccation tolerant plants: Energy metabolism and antioxida- Tan (Editors) Mountain fl ora of Greece, vol. 2. Edinburgh Univ. tive stress defense. Plant Sci., 182: 29–41. Press, Edinburgh, pp. 260. Djilianov, D., Genova, G., Parvanova, D., Zapryanova, N., Thompson, J. D., 2005. Plant evolution in the Mediterranean. Ox- Konstantinova, T. and A. Atanassov, 2005. In vitro culture ford: Oxford University Press. of the resurrection plant Haberlea rhodopensis. Plant Cell Tiss. Toldi, O., Tóth, S., Pónyi, T. and P. Scott, 2002. An effective and Organ Cult., 80: 115–118. reproducible transformation protocol for the model resurrection Djilianov, D., Ivanov, S., Georgieva, T., Moyankova, D., Berkov, plant Craterostigma plantagineum Hochst. Plant Cell Rep., 21: S., Petrova, G., Mladenov, P., Christov, N., Hristozova, N., 63–69. Peshev, D., Tchorbadjieva, M., Alexieva, V., Tosheva, A., Toldi, O., Tuba, Z. and P. Scott, 2010. Can lessons learned from Nikolova, M., Ionkova, I. and W. Van den Ende, 2009. A resurrection plants be extended over crop plant species? Rom. holistic approach to resurrection plants. Haberlea rhodopensis Biotechnol. Lett., 15: 3–11. – a case study. Biotechnol. & Biotechnol. Eq., 23: 1414–1416. Tóth, S., Kiss, C., Scott, P., Kovács, G., Sorvari, S. and O. Toldi, Djilianov, D., Ivanov, S., Moyankova, D., Miteva, L., Kirova, 2006. Agrobacterium-mediated genetic transformation of the E., Alexieva, V., Joudi, M., Peshev, D. and W. Van den Ende, desiccation tolerant resurrection plant Ramonda myconi (L.) 2011. Sugar ratios, glutathione redox status and phenols in the Rchb. Plant Cell Rep., 25: 442–449. resurrection species Haberlea rhodopensis and the closely re- Weber, A., 2004. Gesneriaceae. In: Kubitzki, K. and J. Kadereit lated non-resurrection species Chirita eberhardtii Plant Biol., (Editors) The families and genera of vascular plants, vol. VII. 13: 767–776. Flowering Plants. Dicotyledons. Lamiales (except Acanthaceae Furini, A., Koncz, C., Salamini, F. and D. Bartels, 1994. Agrobac- including Avicenniaceae), Springer, Berlin, Heidelberg, pp. 63- terium-mediated transformation of the desiccation tolerant plant 158. Craterostigma plantagineum. Plant Cell Rep., 14: 102–106. Weber, A. and L. E. Skog, 2007. The genera of Gesneriaceae. Ba- Georgieva, T., Christov, N. and D. Djilianov, 2012. Identifi ca- sic information with illustrations of selected species. 2nd ed., ca. tion of desiccation-regulated genes by cDNA-AFLP in Haber- 170 pages. http://www.genera-gesneriaceae.at.