agrobacterium tumefaciens - Apuntes de Biología

Sci.Int.(Lahore),25(2),287-290,2013 ISSN 1013-5316; CODEN: SINTE 8

287

AGROBACTERIUM-MEDIATED STABLE TRANSFORMATION OF

ARABIDOPSIS THALIANA PLANTS USING Β-GLUCURONIDASE

(GUS) GENE

Aneela Yasmin1, Akhtar Ali Jalbani2 , Shahla Baloch1

1Department of Biotechnology, Faculty of Crop Production

Sindh Agriculture University, Tandojam, Pakistan

2Information Technology Center,

Sindh Agriculture University, Tandojam, Pakistan

ABSTRACT: This study is carried to optimize Arabidopsis transformation protocol using β-glucuronidase

(GUS) gene as a reporter gene to analyse gene expression in heterologous system. The method we used to

facilitate our studies is Agrobacterium mediated floral dip method with minor modifications. Floral dip

method is the most popular protocol for the production of stable transformants of Arabidopsis thaliana.

Agrobacterium has the ability to transfer foreign DNA into intact plant cells. In floral dip method, the

developing inflorescences of Arabidopsis were dipped in the suspension of overnight grown Agrobacterium

carrying the genes of interest for about 10 seconds. This dipping of inflorescences targeted the female

gametes resulting in transformed seeds. The transformed seeds were then plated in vitro on a selective

medium and screened for primary transformants (T0). T0 transgenic lines were then planted to get T1

followed by sowing for T2 and T3 respectively. Using this method T2*- transgenic plants, homozygous for

transgene, were obtained in 10.5 months.

Keywords: Arabidopsis thaliana, Floral dip method, GUS reporter gene

1. INTRODUCTION

Generating stable transformants is an important tool to

validate the functionality of genes. Usually, the

transformation of plant cells or tissues is carried out by

delivering foreign genes into plant cells (Herrera-Estrella et

al., 2005)[1]. Agrobacterium tumefaciens and/or biolistic are

used to deliver n ew genes in the cells which are then

regenerated and verified to get final transgenic plants ready

for functional analysis. It is a lengthy process that requires

skilled manpower and sophisticated laboratory facilities to

conduct in vitro regeneration step. Although this approach is

time consuming this is widely adopted method to produce

transgenic plants in most of the crops. Tissue culture

imposes stress to plant tissues during dedifferentiation and

differentiation processes ultimately resulting in somaclonal

variation and DNA modification (Labra et al. 2004)[2]. Thus

plant transformation method without using in vitro

conditions would reduce the time generating plant

transgenics. In addition to improving transformation

methodology it is also important to identify a heterologous

system that could be transformed easy and in short time to

study foreign gene expression and functions independent of

the regulatory elements present in the source of the gene

under investigation. Arabidopsis, a model plant, is such a

heterologous system. Its genome is completely sequenced

and needs 3-4 month for the production of stable transgenic

plants (Zhang et al. 2006)[3]. For the first time Feldmann

and Marks (1987)[4] demonstrated the production of

transgenics without in vitro step. They co-cultivated

germinating seeds of Arabidopsis thaliana and

Agrobacterium tumefaciens with genes of in terest in a

binary expression vector and obtained transgenic plantlets

with low efficiency. Later on Bechtold et al. (1993)[5]

conducted a breakthrough study to transform uprooted

flowering plants of Arabidopsis by vacuum infiltration in

agro-suspension. The uprooted plants were then replanted;

seed were harvested and screened for transgenics. This

method improved the transformation efficiency dramatically.

Later on this transformation procedure was improved

considerably (Clough and Bent, 1998)[6]. Major

modifications include omission of using uprooted plants and

vacuum infiltration. Agrobacterium is natural genetic

engineer; it has the ability to transfer foreign DNA into

intact plant cells. When the developing flowers of

Arabidopsis are dipped in the suspension of Agrobacterium

carrying the genes of interest the female gametes are

transformed and form transgenic seeds (Desfeux et al.,

2000)[7]. Now a da y the most simplified method of

Arabidopsis transformation is floral dip method.

Here we report the optimization of Agrobacterium mediated

stable transformation of Arabidopsis using β-glucuronidase

(GUS; Jefferson et al. 1987)[8] reporter gene for utilizing

Arabidopsis as heterologous system to study resistance

genes of roses in future. Rose is a woody plant that is

difficult to regenerate in vitro and to apply molecular

biological techniques due polyploidy (Yasmin, 2010;

2011)[9,10].

2. MATERIALS AND METHOD

a) Plant Material

Arabidopsis thaliana [Columbia-0 (wild)] plants were used

in present study. Seeds were grown in a clay pot using

standard methods. 10 Germinated seedlings were then sown

in pots (4 in.x4 in.) and pots were covered by a nylon sheet

fixed by rubber band to avoid soil leakage during dipping in

bacterial suspension. Plants then grown in a growth chamber

under short days for one month. Later the growth conditions

were changed to long-day to induce inflorescence. Pots must

be watered properly to keep plants healthy plants as the

efficiency of transformation depends on the health of plants.

b) Bacteria and Constructs

In this study Agrobacterium strain GV3101::pMP90

carrying construct 35S:GUS-intron in pBINPLUS (Hellens

et al. 2000[11]; Van Engelen et al. 1995)[12] was used. YEP

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Sci.Int.(Lahore),25(2),287-290,2013 ISSN 1013-5316; CODEN: SINTE 8 287

AGROBACTERIUM-MEDIATED STABLE TRANSFORMATION OF

ARABIDOPSIS THALIANA PLANTS USING Β-GLUCURONIDASE

(GUS) GENE

Aneela Yasmin^1 , Akhtar Ali Jalbani^2 , Shahla Baloch^1 (^1) Department of Biotechnology, Faculty of Crop Production Sindh Agriculture University, Tandojam, Pakistan (^2) Information Technology Center , Sindh Agriculture University, Tandojam, Pakistan ABSTRACT: This study is carried to optimize Arabidopsis transformation protocol using β-glucuronidase (GUS) gene as a reporter gene to analyse gene expression in heterologous system. The method we used to facilitate our studies is Agrobacterium mediated floral dip method with minor modifications. Floral dip method is the most popular protocol for the production of stable transformants of Arabidopsis thaliana. Agrobacterium has the ability to transfer foreign DNA into intact plant cells. In floral dip method, the developing inflorescences of Arabidopsis were dipped in the suspension of overnight grown Agrobacterium carrying the genes of interest for about 10 seconds. This dipping of inflorescences targeted the female gametes resulting in transformed seeds. The transformed seeds were then plated in vitro on a selective medium and screened for primary transformants (T0). T0 transgenic lines were then planted to get T followed by sowing for T2 and T3 respectively. Using this method T2*- transgenic plants, homozygous for transgene, were obtained in 10.5 months.

Keywords: Arabidopsis thaliana, Floral dip method, GUS reporter gene

1. INTRODUCTION Generating stable transformants is an important tool to validate the functionality of genes. Usually, the transformation of plant cells or tissues is carried out by delivering foreign genes into plant cells (Herrera-Estrella et al., 2005)[1]. Agrobacterium tumefaciens and/or biolistic are used to deliver new genes in the cells which are then regenerated and verified to get final transgenic plants ready for functional analysis. It is a lengthy process that requires skilled manpower and sophisticated laboratory facilities to conduct in vitro regeneration step. Although this approach is time consuming this is widely adopted method to produce transgenic plants in most of the crops. Tissue culture imposes stress to plant tissues during dedifferentiation and differentiation processes ultimately resulting in somaclonal variation and DNA modification (Labra et al. 2004)[2]. Thus plant transformation method without using in vitro conditions would reduce the time generating plant transgenics. In addition to improving transformation methodology it is also important to identify a heterologous system that could be transformed easy and in short time to study foreign gene expression and functions independent of the regulatory elements present in the source of the gene under investigation. Arabidopsis, a model plant, is such a heterologous system. Its genome is completely sequenced and needs 3-4 month for the production of stable transgenic plants (Zhang et al. 2006)[3]. For the first time Feldmann and Marks (1987)[4] demonstrated the production of transgenics without in vitro step. They co-cultivated germinating seeds of Arabidopsis thaliana and Agrobacterium tumefaciens with genes of interest in a binary expression vector and obtained transgenic plantlets with low efficiency. Later on Bechtold et al. (1993)[5] conducted a breakthrough study to transform uprooted flowering plants of Arabidopsis by vacuum infiltration in agro-suspension. The uprooted plants were then replanted; seed were harvested and screened for transgenics. This

method improved the transformation efficiency dramatically. Later on this transformation procedure was improved considerably (Clough and Bent, 1998)[6]. Major modifications include omission of using uprooted plants and vacuum infiltration. Agrobacterium is natural genetic engineer; it has the ability to transfer foreign DNA into intact plant cells. When the developing flowers of Arabidopsis are dipped in the suspension of Agrobacterium carrying the genes of interest the female gametes are transformed and form transgenic seeds (Desfeux et al., 2000)[7]. Now a day the most simplified method of Arabidopsis transformation is floral dip method. Here we report the optimization of Agrobacterium mediated stable transformation of Arabidopsis using β-glucuronidase (GUS; Jefferson et al. 1987)[8] reporter gene for utilizing Arabidopsis as heterologous system to study resistance genes of roses in future. Rose is a woody plant that is difficult to regenerate in vitro and to apply molecular biological techniques due polyploidy (Yasmin, 2010; 2011)[9,10].

2. MATERIALS AND METHOD

a) Plant Material Arabidopsis thaliana [Columbia-0 (wild)] plants were used in present study. Seeds were grown in a clay pot using standard methods. 10 Germinated seedlings were then sown in pots (4 in.x4 in.) and pots were covered by a nylon sheet fixed by rubber band to avoid soil leakage during dipping in bacterial suspension. Plants then grown in a growth chamber under short days for one month. Later the growth conditions were changed to long-day to induce inflorescence. Pots must be watered properly to keep plants healthy plants as the efficiency of transformation depends on the health of plants. b) Bacteria and Constructs In this study Agrobacterium strain GV3101::pMP carrying construct 35S:GUS-intron in pBINPLUS (Hellens et al. 2000[11]; Van Engelen et al. 1995)[12] was used. YEP

288 ISSN 1013-5316; CODEN: SINTE 8 Sci.Int.(Lahore),25(2),286-290,

liquid and/ or solid (agar= 15g/L) media was used to grow bacteria .Media was supplemented with 50mg/L of Kanamycin, 10mg/L of Rifampicin and 25mg/L Gentamycin for the selection of bacteria and pMP90, virulence vector. c) Floral Dip Method and Seed Harvesting An overnight liquid culture (500ml; 280C for 20-24 hours) of Agrobacterium was started a day before the main experiment of transformation (Yasmin and Debener, 2010). The bacteria were pelleted in a centrifuge at room temperature and 4500 rpm for 15-20 min. Pelleted bacteria were washed in sterile water and re-suspended in sterile solution of 3% sucrose (100-200ml). The prepared bacterial suspension was supplemented with 0.02% of tween-20. The pots of plants were inverted to dip aerial parts of plants in the bacterial suspension for 10 second followed by 3- seconds of draining bacterial suspension from dipped plants. Dipped plants were then placed in a box fitted with a cover to maintain high humidity for one day. Next day plants were taken out from the box and shifted to growth chamber to grow and set seed for about a month. As the siliques turned brown watering was stopped and drying plants wrapped using wax paper and seeds were collected and stored at room temperature until further processing. d) Screening Primary Transformants Standard MS media plates were prepared containing carbenicillin (100 mg L–1) and kanamycin (50mg L-1) for selection of primary transformants. Heavily contaminated seeds were sterilized by gas treatment. Small samples of seeds were put into Eppendorf tubes and were placed in a thoroughly cleaned desiccator. A beaker containing 200 ml of 5.3% sodium hypochloride solution was placed in the desiccator and 2ml of HCl was added to the hypochloride solution; desiccator was closed immediately and evacuated briefly with pump. The samples were left overnight for sterilization and taken out next morning in a fume hood or under clean bench for plating. The sterilized seeds were suspended in sterile 0.05% agarose (about 30-40 ml seed per ml agarose) and the suspension was spread onto selection plates. Plates were dried under laminar flow hood and seeds were vernalized by placing them at 40C for 3 days. Then plates were moved to tissue culture growth room in long-day conditions. After 12 days, the transformants were easily distinguished as seedlings with green cotyledons, true leaves and roots whereas non-transformed seedlings even if germinated their cotyledons were turned brown within few days (figure 1). The true transgenics plants obtained during primary screening were designed as T0 and transplanted again in soil to get T1 followed by seed collection and sowing to get T2, T2* respectively as explained in Table 1 to get homozygous lines. e) Histochemical Assay The seedlings of Arabidopsis were collected and histochemical assay was performed (Jefferson et al. 1987)[8]. The samples were submerged in staining solution, vacuumed to facilitate infiltration of staining solution and incubated overnight at 37 ºC. Stained samples were treated with 70% ethanol to bleach chlorophyll. GUS expression levels were visually monitored (figure 2).

Figure 1: In vitro selection of transgenics on kanamycin containing MS media.

Figure 2: Histochemical analysis of seedlings of transgenic Arabidopsis carrying GUS gene.

3. RESULTS AND DISCUSSION Here we report a modified version of floral dip method based on Zhang et al. (2006).[3] Kanamycin resistance and β-glucuronidase (GUS) genes were used as reporter genes to stream line a protocol for the generation of transformed homozygous lines of Arabidopsis to study heterologous gene expression in this plant. It took about 10.5 months to get transgenic homozygous lines (Table 1). Agrobacterium mediated floral dip method eliminates initial callus induction and plant regeneration ultimately reducing time, labor and use of expensive equipment (Zhang et al. 2006).[3] Although we found that the transformation efficiency of floral dip method is not high, an Arabidopsis plant produces thousands seeds by self-pollination in a single transformation experiment resulting in many independent transgenic lines within short time. Zhang et al. (2006)[3] used 5% sucrose solution supplemented with 0.02% (vol./vol.) of a surfactant, Silwet L-77 in contrast we get the identical results by using 3% sucrose solution supplemented with the same concentration of tween-20. In agreement to Zhang et al. (2006)[3] the higher concentrations of surfactant found toxic to plants. However, in contrast to them when we used 5% and/or higher concentration of sugar solution, it promoted fungal growth at the plant base during first 24 hours after dip in high humid conditions. We found 3% sucrose solution optimal to get the similar results. We replaced the usual bleach and water based sterilization of seeds by gaseous sterilization as describe in methods section in detail. This method of sterilization is less tedious and simple. We applied the above described parameters and performed transformation experiment in three replicates. GUS intron gene was used as a reporter gene. In each experiment 10 plants were transformed and on average 14 transgenic lines were recovered at the end of primary screening. Five out of those 14 lines were selected randomly to generate T1, followed by T2, and T2* respectively. In our view the success of this procedure mainly depends on the health and

290 ISSN 1013-5316; CODEN: SINTE 8 Sci.Int.(Lahore),25(2),286-290,

Gelvin S. B. Agrobacterium-mediated Plant Transformation: the biology behind the Gene- Jockeying tool, Microbiology and Molecular Biology Reviews, 67 (1): 16-37, (2003).
Wroblewski, T., A. Tomczak, R. Michelmore. Optimization of Agrobacterium mediated transient expression assays for lettuce, tomato and Arabidopsis, Plant Biotech. J , 3 :259-273, (2005).
Yasmin A and Debener T. Transient gene expression in rose petals via Agrobacterium infiltration. Plant Cell Tissue and Organ Culture. 102 (2): 245-250, (2010 ).

agrobacterium tumefaciens, Apuntes de Biología

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AGROBACTERIUM-MEDIATED STABLE TRANSFORMATION OF

ARABIDOPSIS THALIANA PLANTS USING Β-GLUCURONIDASE

(GUS) GENE

2. MATERIALS AND METHOD