Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Podsumowanie

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Streszczenie

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant's genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed in roots.

Wprowadzenie

Aside from economic interest, the generation of transgenic plants has its own relevance in research to demonstrate the ultimate function of genes and to better understand plant physiology and development. The most widely used method for plant DNA insertion is Agrobacterium-mediated transformation. Agrobacterium tumefaciens is able to generate crown galls in the infected tissue of many plant species by the action of its tumour-inducing (Ti) plasmid. The plasmid contains a T-DNA region with a set of genes that will be integrated in the plant genome and induce tissue dedifferentiation^1,2. The exchange of these genes within the T-DNA by the transgene has allowed the generation of specific plant modifications avoiding phenotypic effects³. To facilitate the transgene cloning into the T-DNA, the T-DNA region has been excised in an independent plasmid called a binary plasmid, while the rest of the genes of the Ti plasmid (the virulence genes that allow the T-DNA transfer and insertion mechanisms) have been placed in a helper plasmid. For plant biotechnology research, transformation by A. tumefaciens has several advantages: it does not need expensive devices, is able to generate both stable and transient plant transformation, and low numbers of gene copies are integrated into the chromosome⁴. However, for most plants, but not Arabidopsis, the generation of stable transformants requires plant regeneration from a single or a few cells using exogenous phytohormones, making this process laborious and time consuming. A. rhizogenes is also able to modify the plant genome, producing hairy roots or adventitious roots at the infection sites due to the expression of rol (root loci) genes encoded in the root-inducing (Ri) plasmid⁵. Although less studied than A. tumefaciens, A. rhizogenes is also used for obtaining transgenic roots. In this case, the A. rhizogenes contains the original T-DNA in the Ri plasmid and a binary plasmid with a second T-DNA carrying the transgene. When the infection site is in stems or hypocotyls, a composite plant can be obtained, with new hairy transgenic roots emerging from wild type shoots. Alternatively, hairy transformed roots can grow autonomously in vitro in media with carbon source inputs. The use of A. rhizogenes instead of A. tumefaciens to produce transgenic tissue is gaining relevance when the root is the target organ, because plant regeneration is not required and hence it is faster and less costly. Previous studies have demonstrated this methodology appropriated for the phenotypic characterization of root specific genes^6,7,8,9.

The potato (Solanum tuberosum) is the fourth most important crop in the world according to the Food and Agriculture Organization of the United Nations (FAO) since the tuber has nutritional relevance for human consumption for being a good source of vitamins and minerals. For that reason, potato has been placed in the spotlight of agricultural biotechnology and also is considered as a good biological model for genetic and developmental studies^10,11. Potato transformation significantly contributed to the understanding of molecular mechanisms underlying suberized tissues through the characterization of genes involved in suberin and wax biosynthesis^{12,13,14,15,16,17}, suberin monomer transport¹⁸ and transcription regulation¹⁹. The suberin feruloyl transferase gene, FHT, is one of these characterized biosynthetic genes; its downregulation gives rise to a strong impairment of the periderm protection, which is correlated with a strong decrease in ferulate esters of suberin and waxes in potato tubers¹⁴. Concomitantly, in roots and seeds of Arabidopsis, the knockout of its putative orthologue (ASFT/RWP1) also demonstrated its role in producing alkyl ferulates in suberin^20,21. In potato, the FHT transcriptional reporter line and the FHT antibody showed respectively that the promoter activity and the protein are located in the exodermis, the endodermis, the phellogen-derivatives and in wounded tissues¹⁵.

In this work, we detail a protocol using A. rhizogenes to produce transgenic hairy roots that are maintained in a wild type shoot, generating composite potato plants, or excised to grow autonomously in vitro. We also provide the protocol using A. tumefaciens to obtain complete transgenic potato plants. As a case study, A. rhizogenes and A. tumefaciens transformed with the same binary vector are used to obtain roots with the FHT promoter driving GUS reporter gene expression. The results are reported and compared.

Protokół

The A. rhizogenes transformation protocol was adapted and modified from Horn et al.⁷ and the genotype tested was S. tuberosum ssp. tuberosum (cv. Désirée). The A. tumefaciens transformation protocol was adapted and modified from Banerjee et al.²² and the genotypes tested were S. tuberosum ssp. tuberosum (cv. Désirée) and S. tuberosum ssp. andigena. The main steps of both procedures are summarized in Figure 1 and Figure 2, respectively.

NOTE: In all the steps of the procedure performing in vitro transfers, do so rapidly, and when possible, maintain the plates or pots closed, thus minimizing plant exposure to the air to avoid wilting and contamination. Otherwise stated, all the plant incubations were done in cabinets under the conditions of 12 h of 24 °C light/12 h of 20 °C dark and 67 µmol m^-1 s^-1. Otherwise stated, perform all the bacteria manipulation and in vitro plant transfers in aseptic conditions in a laminar flow hood. All the media recipes for Agrobacterium and in vitro plant cultures are provided in Table S1.

CAUTION: Deposit all the genetically modified bacteria and plants to the appropriated waste container.

1. Agrobacterium cultures used for transformation

NOTE: The strain used for A. rhizogenes transformation was the C58C1:Pri1583⁷ (kindly provided by Dr. Inge Broer) and that for the A. tumefaciens was the GV2260 (kindly provided by Dr. Salomé Prat). A. rhizogenes was transformed with the binary vector PK7GWIWG2_II-RedRoot (VIB-Department of Plant Systems Biology at Universiteit Gent; http://gateway.psb.ugent.be) that contains a T-DNA carrying a transformation marker to monitor the hairy root formation. To compare the transformed roots generated by A. rhizogenes and A. tumefaciens, both were transformed with the binary vector pKGWFS7 which contains a T-DNA carrying the FHT promoter driving the β-glucoronidase (GUS) reporter gene and the Kanamycin resistance gene as a selectable marker¹⁵.

1. Pick a colony of Agrobacterium and grow it overnight (O/N) in 5 mL of YEB medium supplemented with antibiotics (Table S1) in a 50 mL centrifuge tube at 28 °C with shaking at 200 rpm.
2. For A. tumefaciens transformation, measure the optical density, which must be OD₆₀₀ = 0.6-1.0.
   1. If the optical density is higher, make a subculture lowering it to OD₆₀₀ = 0.3 with fresh media and wait until the culture reaches OD₆₀₀ = 0.6-1.
3. Centrifuge 1 mL of Agrobacterium culture at 3,000 x g in a bench-top centrifuge for 10 min at room temperature.
4. Remove the supernatant by pipetting, and resuspend cells in 1 mL of fresh YEB medium without antibiotics. Repeat this step to ensure the complete removal of antibiotics.
   1. For A. tumefaciens transformation, in the last resuspension add the appropriate YEB volume to obtain a final optical density of OD₆₀₀ = 0.8.
5. Keep cells on ice while preparing the plants to be infected.

2. Plant material for transformation

1. Make one or two node stem cuttings either containing the apical or the auxiliary buds from sterile in vitro potato plants (donor plants); grow them in solid 2MS medium in pots for 3 to 4 weeks (Figure 1A and Figure 2A).

3. Plant transformation using A. rhizogenes (Figure 1)

NOTE: This procedure allows the obtaining of transformed hairy roots. To evaluate the transgene expression, a negative control is needed. To prepare the negative control, follow the procedure using an A. rhizogenes strain either untransformed or transformed with the empty vector that includes the transformation marker gene.

1. Use fresh media plates; alternatively, the plates can be kept at 4 °C with the lid side up, tightly sealed with transparent film to avoid media dehydration. To prepare the square media plates, incline them ~15°, fill with 40 mL of MS, and let them solidify. This will minimize the contact of the aerial part of the plant with the medium.
2. Transfer very carefully a donor plant from the 2MS medium to a 120 mm x 120 mm square plate.
3. Inject to one stem internode 3 μL of the A. rhizogenes culture using a surgical needle and repeat it twice per plant in different internodes when possible.
   NOTE: Consider each injection as an independent transformation event (Figure 1B).
4. Transfer immediately the entire plant to a square plate with solid MS medium supplemented with 0.1 mM acetosyringone. Accommodate 2 plants per plate.
   NOTE: The 1 M acetosyringone stock solution is prepared in DMSO and can be stored at -20 °C.
5. Seal the plate using surgical tape and arrange it vertically inside a growth cabinet for 4 days.
6. Transfer the plant to a new square plate with MS medium supplemented with cefotaxime sodium [500 µg/mL] to kill A. rhizogenes.
7. After 10-12 days, hairy roots will start to appear (Figure 1C,1D). Then, excise the native roots of the plant, and transfer the plant to a new square plate with MS medium supplemented with cefotaxime sodium [500 µg/mL].
   NOTE: Transformed hairy roots can be checked by red fluorescence when using a DsRed transformation marker (Figure 3D). 
8. To obtain a composite plant (Figure 1E), let the transgenic hairy roots grow for 3-4 weeks in MS medium supplemented with cefotaxime sodium [500 μg/mL] (change the medium every week).
9. Depending on the purpose, propagate the transgenic hairy roots and the negative controls as follow.
   1. Transfer the whole composite plant to a hydroponic (Table S2) or soil medium to allow for massive development.
   2. To individually propagate the transformed hairy roots, using a scalpel cut the roots expressing the red fluorescent transformation marker (DsRed protein) when they are 4-8 cm long (Figure 1E) and transfer them into a Petri dish with Gamborg B5 solid medium supplemented with 2% sucrose and cefotaxime sodium [500 μg/mL]. Seal the plates with plastic laboratory film and grow them in the dark at 20 °C.
      NOTE: The roots can be manipulated under a stereomicroscope equipped to detect the fluorescence under sterile conditions (see Table of Materials).
   3. For biomass production (i.e., gene expression analysis), cut a 5 cm long hairy root and propagate it in a 150 mL Erlenmeyer flask with 20 mL of Gamborg B5 liquid medium supplemented with 2% sucrose and cefotaxime sodium [500 µg/mL]. Grow it for 6 weeks in the dark at 20 °C and 60 rpm.

Figure 1: Timeline to obtain potato transgenic hairy roots using A. rhizogenes. The cumulative weeks to reach each stage of the transformation process and the subsequent steps to grow the hairy roots are shown. Representative images of different stages are depicted: the initiation of the process using 3-week-old in vitro plants (A), then infection of the plants by injecting A. rhizogenes (B), the formation of the proliferative tissue (C, arrows) with emerging hairy roots (D), and the developed hairy roots expressing the red fluorescent transformation marker DsRed (E). Please click here to view a larger version of this figure.

4. Plant transformation using A. tumefaciens (Figure 2)

NOTE: This procedure allows the obtaining of transformed plants. To evaluate the transgene’s effect, a negative control is needed. One option is to follow the procedure using an A. tumefaciens transformed with the empty vector. Alternatively, wild type plants can be used.

1. Cut and place a leaf from the 3-4-week-old plants (Figure 2A) in a Petri dish. Using a scalpel exclude the petiole and make transverse cuts (1-3 depending on the leaf size) from the center of the leaf to the edges avoiding cutting them off (Figure 2B).
2. Immediately place the leaf floating on 10 mL of fresh 2MS liquid media in a Petri dish with the abaxial side up and close the plate. Repeat the step accommodating up to 15 leaves for cv. Désirée and 25 leaves for ssp. andigena (depending on the leaf size).
3. Immediately add 80 µL of A. tumefaciens culture at OD₆₀₀ = 0.8 in the liquid media and homogenize the plate manually for 1 min to distribute the bacterial solution.
4. Carefully seal with sealing film, cover with aluminum foil and incubate for 2 days in a chamber at 24 °C to let the transformation occur.
5. Transfer the leaves keeping abaxial side up to CIM medium (Figure 2B) and incubate them for one week in a growth cabinet.
   1. Scrape the CIM medium with the tweezers so that the leaves can be better accommodated on the media.
6. Transfer the leaves keeping abaxial side up to SIM medium (Figure 2C) and incubate them in a growth cabinet, refreshing the medium every 7-10 days, until the shoots are about 2 cm tall.
   1. Scrape the SIM medium with the tweezers so that the leaves can be fully surrounded by the media. When the emerged shoots reach the lid, work with tall Petri dishes (100 x 20 mm, height x diameter).
      NOTE: The callus will form after 2-3 weeks in SIM medium (Figure 2D) and the shoots after 6-7 weeks (Figure 2E). The shoots will be considered as independent transformation events when they emerge from callus formed from independent wounds.
7. Cut three shoots emerged from each callus (considered the same transformation event) (Figure 2E), transfer them to culture flasks with MG medium supplemented with cefotaxime sodium [250 mg/L] to allow rooting, label the subset with a number and incubate in a growth cabinet for 3-4 weeks or until the shoots are vigorous (Figure 2F).
   NOTE: When cutting the shoots, remove well the callus otherwise the root will not form.
   1. Repeat the step as many times as independent lines are needed. Up to 5 different transformation events can be placed in a culture flask with a diameter of 8 cm to work in full confidence that the plants from different events are not mixed.
8. Select the most vigorous plant of each event, cut the apical segment of the shoot with 3-4 internodes and place it in a new culture flask with 2MS medium supplemented with cefotaxime sodium [250 mg/L].
   NOTE: In 3-6 weeks the plant will grow efficiently, developing a vigorous shoot and roots.
   1. Bring back to the chamber the non-selected shoots until the plant selected has fully developed.
9. Cut stem segments from the plant with at least one internode or with the apical bud and transfer them to new 2MS medium supplemented with cefotaxime [250 mg/L]. Incubate them in the growth cabinet.
   1. Replicate every 3-4 weeks to establish the in vitro transformed lines.
      NOTE: The cefotaxime sodium is needed in at least three subsequent transfers to 2MS medium to be sure to kill the A. tumefaciens; afterwards, if A. tumefaciens overgrowth is observed, transfer the plants again to 2MS media supplemented with cefotaxime sodium.
10. To characterize the plant phenotype, transfer plants to soil for their full characterization or to hydroponics culture for root inspection.
    1. Keep the tubers produced in soil to propagate and maintain the established lines.

Figure 2: Timeline to obtain potato transformed plants using A. tumefaciens. The cumulative weeks to reach each stage of the transformation process and the subsequent steps to grow the plants are shown. Representative images of different stages are depicted: the initiation of the process using leaves from 3-week old in vitro plants (A), the transfer of the wounded and infected leaves to the CIM media (B), the leaves when transferred to SIM media (C), the visualization of the callus around the wounded areas after 2-3 weeks in SIM media (D), the shoot formation after 9-11 weeks in SIM media (E), and the shoots after being transferred to MG media (F). Please click here to view a larger version of this figure.

5. Hydroponic culture

1. Prepare the Hoagland’s solution to a half strength (0.5x) (Table S2) in a 10 L bucket.
2. Immerse an aquarium pump to maintain homogeneity and proper oxygen conditions.
3. Cover the walls of the bucket with aluminum foil to grow roots in dark conditions.
4. Avoiding root damage, transfer in vitro plants to hydroponic culture.
   NOTE: Remove any remaining in vitro medium from roots to avoid microorganism proliferation during incubation by shaking carefully the roots immersed in water.
5. Cover the plants with transparent film like a glasshouse to allow adequate acclimatization and incubate in the growing chamber.
6. Make holes in the film after 3 days and remove it completely one week after.
7. Replace with fresh media every 10 days.

6. GUS histochemical reporter gene assay

NOTE: In our case the GUS analysis was performed with roots of 2-3 weeks grown in hydroponics or in vitro.

1. Fix the roots with 90% chilled acetone (v/v) and incubate it for 20 minutes on ice.
2. Perform two washings with distilled water.
3. Add fresh GUS staining solution (Table 1) and apply vacuum (-70 Pa) for 20 min.
4. Incubate at 37 °C in dark to protect the photosensitive GUS for 4 h or until a blue color is visible.
   NOTE: The presence of ferri- and ferrocyanide in GUS solution minimize the diffusion of reaction products and provide more precise localization.
   CAUTION: Use a fume hood and wear protective clothing when handling the toxic cyanide derivatives in GUS solution (the potassium ferricyanide and potassium ferrocyanide). The GUS substrate and the disposal material should be disposed safely.
5. Remove GUS staining solution and discard it in appropriate containers.
6. Perform two washings with ethanol 70% (v/v).
7. Observe under a bright field microscope.
   NOTE: GUS staining is stable for a few weeks; however, during the first week, the GUS signal is clear and diffuses less into neighboring cells. For longer storage, seal the tube and store it at 4 °C.

Table 1: GUS staining solution recipe.

Wyniki

Agrobacterium rhizogenes-mediated potato transformation

In this manuscript, the step-by-step procedure set up to obtain transformed root with A. rhizogenes is presented. Figure 1 presents an overview of the procedure, which altogether takes around 5-6 weeks (from injection of A. rhizogenes to obtaining fully developed hairy roots). Then, ...

Dyskusje

In potato, the most common system to obtain stable complete transgenic plants uses the transformation by Agrobacterium tumefaciens strains that require organogenesis using exogenous phytohormones. Although the Agrobacterium based protocols has the potential to integrate non-T-DNA vector sequence²⁵, this methodology is still the easiest and less expensive available to transform potato plants. During last years, the interest in A. rhizogenes-mediated transformation has got...

Materiały

Name	Company	Catalog Number	Comments
Acetone	Panreac	1.310.071.21
Acetosyringone	Acros	115540050
Aquarium pump	Prodac	MP350
Autoclave	Ragpa Strelimatic
Bacteriological agar	Lab Conda	1800
BAP	Duchefa	B0904
Beef extract	Lab Conda	1700
Plant growing cabinet	Nuaire
Carbenicillin	Duchefa	C0109
Cefotaxime sodium	Duchefa	C0111
DMSO	Merck	1029310161
Ecotron infors	HT	29378
Ethanol	Merck	1,009,831,011
Falcon tube	Control tecnica	CFT011500
Ferricyanate	Sigma	101001081
Ferrocyanate	Sigma	100979088
Flask (8.06 cm diameter and 11.3 cm height) and plastic lid for in vitro culture	Apiglass	ref16
GA3	Sigma	G7645
Gamborg B5 media	Duchefa	G0210
Gelrite	Duchefa	G1101
Glucosa	Sigma	G5767
Kanamycin	Sigma	K1377
Leukopor tape	BSN Leukopor	BDF47467
Lupe	Wild-Heerbrugg	M420
Magnetic shaker	Agimatic	7000243
MES hydrate	Sigma	M2933-25G
MgSO4	Panreac	131404
Microscope	Olympus
Minufugue centrifugue 5415R	Eppendorf
Murashige and Skoog media	Duchefa	M0254.0050
Na2HPO4	Panreac	131679
NAA	Duchefa	N0903
NaCl	Panreac	131659
NaH2PO4	Sigma	58282
NightSea Stereo	SFA Moonting Adapter
Parafilm	Anorsa	PRFL-001-001
Peptone	Lab Conda	1616
Petri dishes (90 x 14)	Anorsa	200200
pHmetre	Crison
Phytotron	Inkoa	RFTI-R5485
Plant Agar	Duchefa	P1001
Refrigeratot	Liebherr Medline
Rifampicin	Duchefa	R0146
Spectinomycin	Sigma	59007
Spectrophotometer	Shimadzu
Square plates (120 x 120)	Deltalab	200204
Streptomycin	Sigma	S6501
Sucrose	Panreac	131621
Surgical blades	Swann-Morton	201
Surgical needle	NIPRO	015/0204
Triptone	Lab Conda	1612
Triton	Serva	37240
Unimax 1010 shaker	Heidolph
Vacuum	Dinko
x-GlcA (5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium salt anhydrous)	Biosynth	B-7398
Yeast extract	Lab Conda	1702.00
Zeatin riboside	Sigma	1001042850