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.
2. Plant material for transformation
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.
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.
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
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.
Table 1: GUS staining solution recipe.
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, the plant can be studied as a composite (wild type shoot, transgenic root) or the transgenic hairy root clones can be excised and grown autonomously in solid Gamborg B5 medium supplemented with 2% sucrose. Alternatively, the hairy roots can be massively propagated using the liquid Gamborg B5 media. The procedure presented has been carried out with S. tuberosum spp. tuberosum (cv. Désirée).
The method to monitor the procedure and to obtain potato transgenic hairy roots has been validated using a binary vector with the DsRed as a transformation marker (PK7GWIWG2_II-RedRoot from VIB-Department of Plant Systems Biology at Universiteit Gent). This allowed the distinction of transgenic hairy roots from non-transgenic by the red fluorescence. According to that, in Figure 3 the transformed hairy roots exhibited red fluorescence when illuminated with green light. The negative control using the untransformed Agrobacterium showed no red fluorescence (Figure 3C), overall indicating the suitability of the DsRed transformation marker to identify the transgenic hairy roots (Figure 3D). Other transformation markers such as antibiotic resistance can be used as described by other authors23,24; however, the antibiotics in the media can produce a growth delay in transgenic roots containing the marker.
Figure 3: Fluorescent transgenic hairy roots of potato (cv. Désirée) transformed by A. rhizogenes. The hairy roots were obtained using a non-transformed A. rhizogenes (strain C58C1: pRI1583) (A and C) and with A. rhizogenes (strain C58C1:pRI1583) transformed with the empty vector pK7GWIWG2_II-Red-Root carrying a DsRed transformation marker (B and D). The hairy roots are formed in both infections (A, B) but red fluorescence is only observed in hairy roots transformed with the A. rhizogenes containing the binary vector. The images were taken with a stereomicroscope equipped with a lamp and a specific filter to visualize the red fluorescence. Please click here to view a larger version of this figure.
Agrobacterium tumefaciens-mediated potato transformation
The second protocol described in this manuscript is set up to obtain, step-by-step, a complete potato plant transformed with A. tumefaciens. Figure 2 presents an overview of the procedure, which altogether takes between 15-18 weeks (from leaf infection with A. tumefaciens to obtaining fully regenerated plants). The most time-consuming part of the procedure is the plant regeneration by organogenesis. This particular step makes this method more laborious than using A. rhizogenes. The procedure has been carried out with S. tuberosum spp. tuberosum (cv. Désirée) and S. tuberosum ssp. andigena, the former being less dependent on short-day conditions to induce tuberization.
In the A. tumefaciens-mediated transformation, in contrast to the A. rhizogenes-mediated transformation, the regenerated plants are completely transgenic organisms. However, though the transgenic plants are regenerated in a Kanamycin selective media, not all the lines efficiently express the transgene. Hence, validation of the transgene expression is needed.
Comparison of the FHT promoter activity in roots obtained using A. tumefaciens and A. rhizogenes
The aforementioned procedures were applied to produce roots expressing the GUS gene under the promoter of FHT gene. The complete transformed plants with A. tumefaciens were previously reported15, using the binary vector pKGWFS7 containing the FHT promoter. Now, this binary vector has been used to produce new transformed hairy roots to compare the tissues where the promoter is active and therefore to test the hairy root system as a tool to study promoter activation.
Figure 4 shows GUS staining in roots of transgenic plants obtained by A. tumefaciens (Figure 4A,B) and transgenic hairy roots obtained by A. rhizogenes (Figure 4C,D,E,F), respectively. As can be seen, roots transformed with A. tumefaciens and grown in vitro show blue staining in the endodermis (Figure 4A), a cell layer between the cortex and the stele. In more developed roots, the blue labelling is patchy in the external layer corresponding to the exodermis (Figure 4B). In transformed hairy roots grown in hydroponics, the GUS marker was specifically located in the endodermis (Figure 4C,E), in the emergence of lateral roots (Figure 4D,E), in the wounded areas (Figure 4E) and in the exodermis (Figure 4F). The roots showed no GUS stain in negative controls that were either hairy roots without the PromFHT:GUS T-DNA cassette or wild type roots.
Figure 4: Histochemical observation of transgenic potato roots expressing the GUS reporter gene driven by the promoter of FHT. The roots from complete transgenic plants obtained by A. tumefaciens (S. tuberosum ssp. andigena) transformation (A-B) show blue staining in the endodermis (A) and exodermis (B). The transgenic hairy roots obtained by A. rhizogenes (S. tuberosum ssp. tuberosum cv. Désirée) transformation (C-F) display GUS staining in the endodermis (C and E), in the lateral root emergence (D and E), in the wound-healing zone (E) and in the exodermis (F). Endodermis (EN); Exodermis (EX); Xylem (XL); Primordia of a lateral root (LR). The red arrow indicates the wounded area. Please click here to view a larger version of this figure.
Table S1: Media recipes used for growing bacteria and in vitro plants. Please click here to download this table
Table S2: Half strength Hoagland’s solution for growing potato plants in hydroponics. Please click here to download this table
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 |
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.
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.
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.