AGROBACTERIUM - MEDIATED GENETIC TRANSFORMATION OF THE RESURRECTION PLANT HABERLEA RHODOPENSIS FRIV.
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10
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 Sofia, 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 establish-
ment of efficient and repeatable transformation system will contribute significantly for the elucidation of stress tolerance. In
this study we describe for the first 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 confirmed 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 confirm 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 five 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 final 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: galiaty@abv.bgAgrobacterium - 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 flasks
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 ex-
plants 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
30 sec, followed by treatment with 0.75% HgCl2 for 6 min. medium also contains mixture of antioxidants (0.15 mg.l–1
The explants were washed three times with sterile dH2O for ascorbic acid and 0.1 mg.l–1 citric acid). After co-cultivation,
removing the residual of HgCl2 and then were germinated the infected leaf suspensions were subcultured for 2 weeks
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, five 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 con-
tains 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 concen-
trations (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 amplification was
performed on a GeneAmp® PCR System (Applied Biosys-
Effect of cefotaxime on growth of A. tumefaciens LBA tems, Foster City, CA, USA). The final 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 amplification 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 significant where P ≤ 0.05, and
not significant 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-
ficult 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 contain-
ing 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 pro-
tocorms growth, was 0.75 mg.l–1 (Figure 1). All of pro-
tocorms 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) confirmed 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 amplification 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 five 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 sup-
plemented 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 (Oreifig 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 morphologi-
cally 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 ge-
netic 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
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chemical enhancements of gene delivery on the efficiency Jefferson, R. A., 1987. Assayng chimeric genes in plants: The GUS
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Khush, G. S., 1999. Green revolution: preparing for the 21st cen-
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*Experiments were repeated three times by using 100 explants/
Oreifig, A. S., Kovács, G., Jenes, B., Kiss, E., Scott, P. and
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a O. Toldi, 2004. Development of a non-lethal selection sys-
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