COBISS Code 1.01
DOI: 10.2478/acas-2013-0001
Agrovoc descriptors: nicotiana tabacum,agrobacterium tumefaciens, gene expression, genetic transformation, selection, disease resistance, antibiotics, plant tissues
Agris category code: H20, f30
Expression and molecular analysis of DsRed and gfp fluorescent genes in
tobacco (Nicotiana tabacum L.)
Kati OVEN1, Zlata LUTHAR2
Received October 05, 2012; accepted Janury 15, 2013. Delo je prispelo 05. oktobra 2012, sprejeto 15. januarja 2013.
ABSTRACT
IZVLEČEK
Agrobacterium-mediated transformation of tobacco leaf disks with Agrobacterium tumefaciens (A. t.) strain LBA4404 and two plasmids (pCAMBIA1390-DsRed and pART27 2mgfp5-ER) was used for introducing red fluorescent gene (DsRed), green fluorescent gene (gfp) and corresponding selection genes (hptII for resistance to antibiotic hygromycin and nptII for resistance to kanamycin) into leaf discs of tobacco (Nicotiana tabacum L.). Epifluorescent microscopy with the appropriate set of filters did not reveal phenotypic expression of the DsRed gene in 6.9 % of regenerants and the gfp gene in 1.3 % of regenerants that were successfully grown on selective medium. The duplex PCR method also did not confirm the presence of fragments specific to DsRed or gfp genes in these regenerants, while the presence of fragments characteristic of selection genes hptII and nptII was confirmed. A built-in nptII gene mutation, a deletion, was detected in one regenerant. Out of the 139 regenerants generated after the transformation of A. t.-pCAMBIAl390-DsRed, 38 or 25.5 % successfully grew only on non-selective medium; after transformation with A. t.-pART27 2mgfp5-ER 9 or 5.4 % of the 161 generants grew successfully. PCR analysis confirmed in all regenerants the presence of fragments characteristic of both transgenes, which were not expressed or were silenced. The effectiveness of transformation after infection with A. t.-pCAMBIA1390-DsRed was 93.1 %, and 98.7 % after infection with A. t.-pART27 2mgfp5-ER. We established that both fluorescent genes are suitable for setting up a transformation system. The antibiotics hygromycin and kanamycin successfully prevented the growth of untransformed tissues, but the antibiotic timentin successfully prevented the growth of bacteria A. t. after the transformation.
Key words: Nicotiana tabacum, fluorescent genes, selection genes, transformation, exspression of transgenes, DNA analysis
IZRAZANJE IN MOLEKULSKA ANALIZA DsRed IN gfp FLUORESCENTNIH GENOV PRI TOBAKU (Nicotiana tabacum L.)
Z metodo posredne transformacije z vektorskim sistemom Agrobacterim tumefaciens (A. t.) sev LBA4404 in dvema plazmidoma (pCAMBIA1390-DsRed in pART27 2mgfp5-ER) smo v listne izsečke tobaka (Nicotiana tabacum L.) vnesli fluorescentni markerski gen za rdečo (DsRed) oz. zeleno (gfp) fluorescenco ter selekcijska gena za odpornost na antibiotik higromicin (hptII) oz. kanamicin (nptII). Z epifluorescentnim mikroskopom in ustreznim setom filtrov nismo zasledili fenotipskega izražanje DsRed gena pri 6,9 % regenerantih in gfp gena pri 1,3 % regenerantih, ki so uspešno rastli na selekcijskem gojišču. Pri teh regenerantih tudi z dupleks PCR metodo nismo potrdili prisotnosti fragmentov značilnih za DsRed oz. gfp gen, medtem ko smo potrdili prisotnost fragmentov značilnih za selekcijska gena hptII in nptII. Pri enem regenerantu smo v vgrajenem nptII genu zasledili mutacijo in sicer delecijo. Od 139 nastalih regenerantov, po transformaciji z A. t.-pCAMBIA1390-DsRed, jih je 38 oz. 25,5 % uspešno rastlo le na neselekcijskem gojišču, po transformaciji z A. t.-pART27 2mgfp5-ER je bilo takih 9 oz. 5,4 % od 161 nastalih. Pri vseh smo s PCR analizo potrdili prisotnost fragmentov značilnih za oba transgena, ki se nista izražala oz. sta bila utišana. Učinkovitost transformacije po okužbi z A. t.-pCAMBIA1390-DsRed je bila 93,1 %, po okužbi z A. t.-pART27 2mgfp5-ER pa 98,7 %. Ugotovili smo, da sta oba fluorescentna gena primerna za vzpostavitev transformacijskega sistema. Antibiotika higromicin in kanamicin sta uspešno preprečila rast netransformiranih tkiv, antibiotik timentin pa je uspešno preprečil rast bakterije A. t. po transformaciji.
Ključne besede: Nicotiana tabacum, fluorescentni geni, selekcijski geni, transformacija, izražanje transgenov, DNA analiza
Ljubljanska cesta 8, 1293 Šmarje - Sap, e-mail: kati.oven@gmail.com
Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, e-mail: zlata.luthar@bf.uni-lj.si
2
1 INTRODUCTION
Tobacco (Nicotiana tabacum L.) has been shown to be a very suitable model plant for genetic transformation because it grows quickly and successfully in tissue culture. Regeneration from leaf explants is fast and efficient (Stolarz et al, 1991). Tobacco was the first transformed plant. In 1983, the gene for resistance to the antibiotic kanamycin was inserted in tobacco (Horsch et al, 1984) by the indirect method of transformation using the soil phytopathogenic bacterium Agrobacterium tumefaciens (A. t). There are currently two authorizations for commercial production of tobacco with tolerance to the herbicide oxynil in the European Union and for tobacco with a reduced content of nicotine in the United States of America (CERA, 2012).
The development of plant regeneration procedures and the discovery of new techniques of gene transfer in plant cells have provided opportunities for practical application of genetic engineering to modify and improve important agricultural crops. Genetic transformation has become useful in improving plant properties and for the detection of gene functions in plants (Rao et al., 2009).
In most cases, only a small proportion of plant cells transform, so it is necessary to enter a selection gene together with the desired gene, by which transformed cells can be distinguished from non-transformed ones. Selection can be positive or negative (Miki and McHugh, 2004). The correct concentration of antibiotic or selection agent must be made, which completely prevents regeneration of non-transformed cells and, at the same time, minimizes the number of non-transformed regenerants that develop in cultured explants due to the detoxification activity of the surrounding transformed cells (Park et al., 1998).
Using selection genes for antibiotic resistance and resistance to herbicides gives rise to most concerns in the commercial use of transgenic plants. DNA transfer between transgenic plants and other organisms is unlikely. NptII gene does not signify any risk to human and animal health (Fuchs et al, 1993). Nevertheless, the complete removal of the selection gene is desirable because selection genes are no longer required after selection. It would probably also contribute to the greater acceptance
of genetically modified plants. There are quite a few successful methods of removing these genes (Afolabi, 2007). European regulations governing the release of genetically modified plants in the field prohibit the inclusion of genes for resistance to antibiotics.
Test or marker genes are genes whose gene product can be visually identified and their location determined. They enable the quick identification of transformed tissues. Marker genes that can be detected through other senses, such as taste or smell, can also be useful (Witty, 1989).
Genes for the synthesis of fluorescent proteins have advantages over other marker genes because they can be visually detected in living cells without the use of invasive procedures using substrates and products that could diffuse within or between cells. Transformed cells, in which these genes express, can be identified shortly after the transformation and it can be determined whether they are dividing (Harper et al, 1999). Fluorescent proteins in fusion with any proteins allow monitoring of the location, movement and activities of proteins in living cells. They can be used as markers for tracing and tracking proteins, discovering interactions between proteins and tracking the destiny of proteins in the cell (Lippincott-Schwartz and Patterson, 2003). Fluorescent proteins can also be used to monitor the destiny of transgenes introduced into cultivated plants and their impact on the environment (Stewart, 2005).
The best known fluorescent protein is the green fluorescent protein (GFP) from the jellyfish (Aequorea victoria) (Haseloff and Amos, 1995), which emits green fluorescence under illumination with long-wave UV light. The wild-type gfp gene was modified in such a way that it effectively reflects in plants and the spectral properties and fluorescence were changed and improved (Reichel et al.,1996; Haseloff et al, 1997).
Red fluorescent protein DsRED was isolated from coral (Discosoma sp.) and, using appropriate filters, can be more easily separated from autofluorescent chlorophyll (Matz et al., 1999) than GFP. DsRed gene is used as a marker gene for transient and stable transformation of tobacco and,
in combination with the gfp gene, is suitable for simultaneous monitoring of the expression of the two genes (Jach et al, 2001).
Many fluorescent proteins that are useful for studies of genetic transformations have been discovered. Orange fluorescent proteins have proved to be very successful as marker genes, especially TdTomato-ER, which fluoresces the
brightest of all fluorescent proteins, followed by Morang-ER (Mann et al, 2012).
In this study, we monitored the phenotypic expression of DsRed and gfp fluorescent genes and selection genes hpII and nptII, as well as molecular analysis of their insertion into the genome of tobacco.
2 MATERIALS AND METHODS
2.1	Plant material
The leaves of micropropagated tobacco variety Havana 38 were used for transformation.
2.2	Bacteria and plasmids
The commercial bacterium A. t. strain LBA4404 was chosen for gene insertion, in which modified plasmid pCAMBIA 1390-DsRed (Cambia, 1997; Škof, 2008) or plasmid pART27 2mgfp5-ER was introduced by electroporation (Gleave, 1992).
Plasmid pDsRed-Express contains the gene DsRed-Express, which is a form of red fluorescent protein DsRED. For preparation of the plasmid vector with the DsRed marker gene, the gene for DsRED-Express protein from plasmid pDsRedExpress (BD Bioscience Clontech, Palo Alto, USA) was used, which was equipped with a constitutive CaMV35S promoter from the vector pBIN m-gfp5-ER and included in the plasmid vector pCAMBIA 1390 (Cambia, Canberra, Australia) (Škof, 2008). In addition to the DsRed
marker gene, the plasmid contained the plant selection hptII gene for resistance to the antibiotic hygromycin for selection of transformed plant tissues and the nptII selection gene for resistance to the antibiotic kanamycin for selection of transformed bacteria (Table 1).
Plasmid pART27 2mgfp5-ER is a binary vector, which was prepared in the laboratory of Prof. Dr. C. C. Eady (Institute of Crop and Food Research, Christchurch, New Zealand), in such a way that two repetitions of mgfp5-ER gene from the vector pBIN m-gfp5-ER were included in the plasmid vector pART27 at location SpeI of the multiple cloning site (MCS). pART27 vector contains the selection gene spec for resistance to the antibiotic spectinomycin for selection of transformed bacteria and the nptII gene for resistance to the amino glycoside antibiotics geneticin and kanamycin for selection of transformed plant tissues (Table 1) (Gleave, 1992).
Table 1. Plasmids with bacterial and plant selection and fluorescent genes
., Bacterial „ Plant „ Fluorescent „ Plasmid , Gene , Gene „ . Gene _selection_selection_protein_
D™90	kanamycin nptII hygromycin hptII DsRED DsRed
QpS-ER_spectinomycin spec k~ nptII GFP gfp
2.3 Agrobacterium-mediated transformation
Transformation of tobacco with A. t. was performed using a slightly modified method of transformation of leaves after Horsch et al. (1985) and Fisher and Guiltinan (1995). Tobacco leaves were cut under sterile conditions to explants of
about 1 cm2. For plasmid pCAMBIA1390-DsRed 105 leaf explants were prepared and for plasmid pART27 2mgfp5-ER 103 explants.
Bacterial suspensions of A. t, with the appropriate plasmid included, were incubated overnight at 28 °C and shaken at 120 rev./min. in YEB medium
[sucrose 5 g/l, peptone 5 g/l, beef extract 5 g/l, yeast extract 1 g/l, MgSO^T^O 1 g/l; pH 7.0]. Bacterial suspensions were centrifuged at 5000 rpm for 5 min. The supernatant was removed and the Agrobacterium pellet was resuspended in VMS liquid basal medium (Murashige and Skoog, 1962) at an optical density of OD600nm = 0.5 (5*106 cells/ml). Tobacco leaf explants were incubated in Petri dishes for approximately 20 min in the A. t. suspension with the appropriate plasmid and then gently dried on sterile filter paper in a laminar flow cabinet and co-cultivated on MSr medium with the addition of [Fe-Na2-EDTA 0.1 mg/l, thiamine 0.1 mg/l, BAP 1.0 mg/l, NAA 0.1 mg/l, acetosyringone 100 ^M, agar 8 g/l; pH 5.8] (Stolarz et al., 1991). After three days of co-cultivation, they were washed twice in a solution of antibiotic timentin 200 mg/l [100:1 (w/w) ticarcillin: clavulanic acid] and air-dried.
Then, the leaf explants were transferred onto selective MSr medium without acetosyringone and with the addition of timentin 150 mg/l to prevent the growth of A. t. bacteria and an appropriate selection antibiotic (Table 1). The minimum effective concentration of selection antibiotics was chosen, i.e., 25 mg/l hygromycin antibiotic for the selection of tobacco transformants after infection with A. t.-pCAMBIA1390-DsRed and 300 mg/l of the antibiotic kanamycin after infection with A. t.-pART27 2mgfp5-ER. Explants were cultured in a growth chamber at a 16/8 hour photoperiod and a temperature of 24 ± 1 °C, illuminated with about 40 ^mol/m2s. After five weeks, the explants were transferred or sub-cultured on the appropriate fresh selective MSr medium. The resulting regenerants were transferred onto MSm medium with the addition of the appropriate selection antibiotic, without timentin. After five weeks, the regenerants that had successfully grown were transferred to the appropriate MS selective medium. Regenerants that had grown poorly or had begun to decay were transferred to MSm medium without selection antibiotics in order to determine the presence of the selection transgene and its expression.
2.4 Expression of DsRed and gfp genes
Expression of fluorescent marker genes in the regenerants was observed after infection at the beginning of regeneration in the rising stages of pessaries or inception. Transformed tobacco
explants were examined by epifluore scent microscope (Nikkon SMZ 1000) at 20 x magnification and appropriate filters for the detection of the red fluorescence DsRed gene and the green fluorescence gfp gene. For the detection of red fluorescent protein DsRED-Express (plasmid pCAMBIA1390-DsRed), which has an excitational maximum at 557 nm and emission maximum at 579 nm, a set of filters with EX 546/10 nm, DM 575 nm and BA 620 nm was used. For the detection of green fluorescent protein m-GFP5-ER (plasmid pART27 2mgfp5-ER), which has an excitational maximum at 484 nm and emission maximum at 510 nm, a set of filters with EX 480/40 nm, DM 505 nm and BA 535/50 nm was used.
2.5 Molecular analysis of plant material by PCR method
For DNA analysis of the presence of transgenes in tobacco regenerants, the complete DNA was isolated, the overall concentration of isolated DNA was measured, dilutions to 20 ng/^l were prepared, and polymerase chain reaction (PCR) and fragment analysis amplified with agarose gel electrophoresis were performed.
Isolation of DNA from plant tissue
Overall genomic DNA from the leaves of non-transformed tobacco were isolated - negative control and transformed regenerants, as well regenerants that had only prospered on non-selective mediums without antibiotics, according to the method of Kump et al. (1992).
Measuring the concentration of DNA by fluorimeter
The concentration of isolated DNA in solution was measured using a DNA fluorimeter DyNA Quant™ 200 (GE Healthcare). A working solution was prepared from 10xTNE buffer [0.1 M NaCl, 10 mM Tris-HCl, 1 mM EDTA; pH 7] and colorant Hoechts 33258 added in a final concentration of 0.1 ^g/ml. Calf thymus DNA (1 mg/ml DNA in 1xTNE buffer) was used for calibration of the fluorimeter. For each sample of DNA, 2 ml of the working solution and 2 ^l DNA sample were added to the cuvette, the mixture stirred and the concentration of DNA then measured. DNA samples were diluted to 20 ng/^l.
Polymerase chain reaction (PCR)
Specific multiplication of DsRed and gfp genes was carried out in duplex PCR reactions using two pairs of primers (Table 2). For analysis of the inclusion of DsRed and hptII genes in the plant genome after transformation with A. t. and the plasmid pCAMBIA1390-DsRed, a combination of REDfor/RED2right and HPTII-for/HPTII-revl primers was used. A combination of GFPla/GFPlb and NPTIIla/NPTIIlb primers was used for analysis of the inclusion of mgfp5-ER and nptII genes after transformation with A. t. and the plasmid pART27 2mgfp5-ER.
PCR reaction mixtures were prepared in a laminar flow cabinet. A 5 ^l DNA sample was pipetted into the PCR microfuge. Samples were centrifuged at l000 rpm/min and lxPCR buffer [l0 mM Tris-HCl, 50 mM KCl, 0.08% Nonidet P40]
(Fermentas), 2 mM MgCl2, 0.2 mM of each deoxynucleotide (dATP, dGTP, dCTP, dTTP ), 4x0.5 ^M suitable primer and 0.5 units of enzyme Taq DNA polymerase (Fermentas) were added. The final volume of the reaction mixture in which multiplication of DNA was conducted, was 25 (j,l. The PCR reaction was carried out in a cyclical thermostat GeneAmp PCR System 9700 (PE Applied Biosystems, USA) using the modified temperature model (Lakshmi et al., 1998):
•	initial denaturation of 5 min at 94 °C,
•	33 repeated cycles: - denaturation of DNA 1 min at 94 °C,
-	annealing of primers 1 min at 58 °C,
-	synthesis of DNA fragments 1.5 min at 72 °C,
•	final incubation 7 min at 72 °C.
Samples were stored at 12 °C until further analysis.
Table 2: DNA nucleotide sequences of primers for an individual transgene and the expected length of the amplified fragment
Primer	The nucleotide sequence 5' - 3'	Expected length of
_the fragment (bp)
GFP1a	AGT GGA GAG GGT GAA GGT GAT G	422
GFP1b	TTG TGG CGG GTC TTG AAG TTG G
REDfor	AGG ACG TCA TCA AGG AGT TCA T	211
RED2right	GTG CTT CAC GTA CAC CTT GGA G
HPTII-for	ATG ACC GCT GTT ATG CGG CCA TTG	641
HPTII-rev 1	AAA AAG CCT GAA CTC ACC GCG ACG
NPTII1a	GAG GCT ATT CGG CTA TGA CTG	650
NPTII 1b	ATG GGG AGC GGC GAT ACC GTA
Analysis of DNA fragments by agarose gel electrophoresis
For the separation of DNA fragments, horizontal electrophoresis was used on a 1.4 % gel [1.4 % SeaKem LE agarose (Cambrex, USA), lxTBE buffer, Ethidium bromide 0.5 (g/ml], which was installed in an electrophoretic tank (Bio-Rad Sub-Cell, model 192) immersed in lxTBE buffer [890 mM Tris, 890 mM boric acid, 10 mM EDTA]. Five (l dispensing dye BPB [12.5% (w/v) ficol 400, 0.2% (w/v) bromophenol blue, 6.7% (v/v) 10xTBE] were added to the samples, which were stirred and 17 ( l of sample was applied on the agarose gel. In addition to the samples, on the gel were
also applied: DNA isolated from control (non-transformed tobacco), corresponding pure plasmid (isolated from E. coli), a blind sample (all components of the reaction mixture except the DNA; instead of adding 5 (l of water) and a size standard (GeneRulerTM 100 bp DNA Ladder Plus (Fermentas) with 14 fragments: 3000, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 bp). Electrophoresis was carried out at 140 V in the anode direction for about 1 hour and 30 min. The gel contained 0.5 (g/ml ethidium bromide, which, in a complex with double stranded DNA molecules, allowed their detection under UV light (302 nm). Gels were observed using a transilluminator TMF-30 (UVP Inc., UK) and photographed with a digital camera.
3 RESULTS AND DISCUSSION
3.1 Regeneration of tobacco leaf explants and phenotypic transgene expression
Leaf explants, after incubation with A. t. and an appropriate plasmid, were co-cultivated on MSr medium with added acetosyringone 100 ^M, in order to increase the infection, as described by Sunilkumar et al. (1999). In nature, phenolic substances such as acetosyringone, which are released on wounding of plant tissue, trigger the activation of genes for virulence (vir genes) in infection with Agrobacterium (Gelvin, 2003). We obtained a high percentage of transformed regenerants, which can be attributed to the acetosyringone attached to the MSr medium in the period of co-cultivation. After the completion of co-cultivation, timentin 150 mg/l was added to the MSr medium, which effectively inhibited the growth of the A. t. bacteria but did not adversely affect regeneration. The regenerants on the medium with timentin were distinctly dark green. Nauerby et al. (1996) reported that timentin in this concentration completely prevented the multiplication of A. t. and positively impacted on the regeneration of leaf and cotyledon explants of
tobacco. Similarly, Cheng et al. (1998) emphasized that timentin is just as effective as carbenicillin and cefotaxime and does not have an inhibitory effect on the regeneration of shoots in tobacco and Siberian elm.
Germs of the first regenerants occurred after 10-12 days, regardless of the built-in genetic construct. Regeneration was mostly direct, without an intermediate callus, as noted by Stolarz et al. (1991).
After five weeks, a large number of regenerants was observed. Regenerants from the leaf explants, in which phenotypic expression of the inserted fluorescent genes was observed, were transferred onto MSm medium with the addition of an appropriate selection antibiotic. After five weeks, regenerants that had grown poorly were transferred to MSm medium without added antibiotics, other regenerants were transferred to appropriate fresh MSm selective medium. The percentage of surviving and failed regenerants is given in Table 3.
Table 3: Percentage of surviving and failed regenerants of tobacco in the appropriate selective or nonselective MSm media after transformation with A. t. and plasmid pCAMBIA1390-DsRed or plasmid pART272mgfp5-ER
Percentage of regenerants on the medium Plasmid	selective	non-selective
survived failed transferred survived failed pCAMBIA1390-DsRed 67.8 67 255 255 0~~ pART27 2mgfp5-ER_90.4_1.2_8.4_5.4_3.O
After transformation with A. t.-pCAMBIA1390-DsRed, 149 regenerants were obtained from 105 explants. After sub-cultivation on selective MSm medium with 25 mg/l hygromycin, 101 or 67.8 % of regenerants grew successfully, and 10 or 6.7 % failed. On non-selective medium, all 38 regenerants (25.5 % out of 149), were successfully grown. With A. t. pART272mgfp5-ER, 103 tobacco explants were transformed and 168 regenerants were obtained. On the selective MSm medium containing 300 mg/l kanamycin, 152 or
90.4 % of regenerants successfully grew, and 2 or 1.2 % failed. Fourteen or 8.4 % of regenerants that had grown poorly or had begun to deteriorate on the selective medium, were transferred to non-selective medium. Out of them, 9 or 5.4 % grew successfully, while 5 or 3 % failed (Tables 3 and
4).
With 6.9 % of regenerants examined by epifluorescent microscope, no red fluorescence DsRED protein was observed and with 1.3 % of
3.2 Molecular analysis of transgene integration
DNA analysis was performed on all 300 surviving regenerants, whether or not they had been transferred to non-selective MSm medium (Tables 4 and 5).
Table 4: Number and percentage of regenerants and transgenes of tobacco after transformation with A. t. -pCAMBIA1390-DsRed on selective and non-selective MSm medium.
Number of regenerants on MSm media		Number or percentage of transgenes			
	DsRed in hptII		DsRed		hptII
	number	percentage number percentage		number	percentage
101 on selective	94	93.1 0	0.0	7	6.9
38 on non-selective	38	100 0	0.0	0	0.0
139 together	132	95.0 0	0.0	7	5.0
regenerants no green fluorescence m-GFP5-ER protein was detected, despite the fact that they had successfully grown on the selection media (Tables 4 and 5). There had been non-expression or silencing of the fluorescent genes.
641 bp 211 bp
1 2 3 4 5 6 7 3 9 10 11 1213 14 15 16 17 IS 19 20 K P S M
Figure 1: Multiplied DNA fragments in duplex PCR reaction with the pair of primers for the DsRed gene (211 bp) and the pair of primers for the hptII gene (641 bp). The figure shows only the first 20 regenerants of 139. (1-101 - transformed regenerants of tobacco grown on selective medium, 102-139 - transformed regenerants of tobacco grown on non-selective medium), K - control - non-transformed tobacco, P -plasmid pCAMBIA1390-DsRed, S - blind sample M - size standard
In all regenerants of tobacco transformed with A. t.-pCAMBIA 13 90-DsRed that were grown on selective medium (regenerants 1-101) and regenerants that, due to poor growth or decay were transferred to non-selective medium (regenerants 102-139), the presence of fragment length 641 bp (selection hptII gen) was detected. In regenerants 2, 16, 49, 51, 53, 61 and 78, only the presence of fragment length 641 bp was detected, but not the presence of fragment length 211 bp, which would have confirmed the presence of marker DsRed gene. On the selective medium, 93.1 % of regenerants included both transgenes from the genetic construct, thus both DsRed and hptII, and 6.9 % regenerants only part of the genetic construct, with the hptII gene (Figure 1 - only 20 out of 139 regenerants are presented, Table 4).
For all 38, or 25.5% of regenerants that were transferred to the non-selective medium, molecular analysis determined the presence of both transgenes from the gene construct (Table 4). Epifluorescent microscopy revealed the presence of protein DsRED in all regenerants, at least mosaic expression. Despite the confirmed presence of selection gene hptII, which should disintegrate the hygromycin added to the medium and allow normal growth and development of regenerants, they decayed. This suggests that gene hptII phenotypically did not express or was silenced (Figure 1 - only 20 out of 139 regenerants are presented, Tables 3 and 4).
Table 5: Number and percentage of regenerants and transgenes of tobacco after transformation with A. t.-pART27 2mgfp5-ER on selective and non-selective MSm medium
Number of regenerants on MSm medium	Number or percentage of transgenes		
	gfp in nptII gfp	nptII	
	number percentage number percentage	number	percentage
152 on selective	150 98.7 0 0.0	2	1.3
9 on non-selective	9 100 0 0.0	0	0.0
161 together	159 98.8 0 0.0	2	1.,2
Figure 2: Multiplied DNA fragments in duplex PCR reaction with the pair of primers for the gfp gene (422 bp) and the pair of primers for the nptll gene (650 bp). The figure shows only the first 20 regenerants of 161. (140291 - transformed regenerants of tobacco grown on the selective medium, 292-300 - transformed regenerants of tobacco grown on the non-selective medium), K - control - non-transformed tobacco, P -plasmid pART27 2mgfp5-ER, S - blind sample M - size standard.
In the regenerants of tobacco transformed with A. t.-pART27 2mgfp5-ER, which were grown on the selective medium (regenerants 140-291), with the exception of regenerants 225 and 245, in which only a fragment length of 650 bp (nptII selection gene) was multiplied, the presence of both transgenes (gfp and nptII) was confirmed. With regenerant 245, a slightly shorter replicate fragment of 650 bp specific to the nptII gene was multiplied. This was probably the result of mutation, the deletion of an individual nucleotide or nucleotides. The deletion of embedded transgenes was reported in a small number transformed plants by Hiei et al. (1994) in rice, Yao et al. (1995) in apple, Mercuri et al. (2000) in African violets, Atkinson and Gardner (1991) in Solanum muricatum and Atkinson and Gardner (1993) in tamarillo. On the selective medium, 98.7 % of regenerants included both transgenes from the genetic construct, with gfp and nptII genes, only 1.3 % of the genetic construct with nptII gene, (Figure 2 - shows only 20 of 161 regenerants and Table 5). No data were found in
the literature on only part-installation of the genetic construct.
In all 9 or 5.4% regenerants of tobacco grown on non-selective medium (regenerants 292-300), the presence of fragment length 422 bp (marker gfp gene) and 650 bp (nptII selection gene) was found. On the non-selective medium, 3.0% of regenerants failed (Table 3).
Non-expression of the selection transgene, which was observed in regenerants that, due to degradation were transferred from the selective to non-selective medium but the presence was confirmed by molecular analysis, may be the result of installation of the transgene on the range of the plant chromosome that is transcriptionally inactive, mutations or gene silencing. Other authors have also reported that some regenerants that were negative a marker enzyme of P-glucuronidase (GUS), had the presence of the gus gene confirmed by hybridization (Hiei et al., 1997) or by PCR analysis (Yao et al., 1995, Mercuri et al., 2000).
4 CONCLUSION
Timentin at a concentration of 150 mg/l completely prevented the growth of A. t. LBA4404 bacteria with included plasmid pCAMBIA1390-DsRed or pART27 2mgfp5-ER and had no negative impact on regeneration or the growth and development of regenerants. The success of transformation, with the confirmed presence of both transgenes from the gene construct, both marker DsRed gene and selection hptII gene, was 93.1 %, while the genetic construct with marker gfp gene and selection nptII gene was 98.7 %. In
regenerant designated 245, the fragment multiplied slightly less than the expected 650 bp specific to the nptII gene. This was probably the result of mutation, the deletion of an individual nucleotide or nucleotides. With the transfer of regenerants that had grown poorly on the selective medium to the non-selective medium, we confirmed the nonexpression or silencing of selection transgenes, the presence of which was confirmed on the DNA level.
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