Because of its versatile application as a model species in various fields of study, there is a need for a genetic transformation toolkit in narrowleaf plantain (Plantago lanceolata). Here, using Agrobacterium tumefaciens-mediated transformation, a protocol is presented that results in stable transgenic lines with a transformation efficiency of 20%.
Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the β-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.
The concept of using model species to investigate multiple aspects of plant biology emerged with the widespread use of Arabidopsis thaliana1. Arabidopsis was initially chosen because it shares features with many other flowering plants and has multiple traits that make it convenient to study in a laboratory environment, such as being small and having a short generation cycle. The large volume of research papers published with it as a subject, along with its small genome size and ease of genetic transformation2, enable it to persist as a widely-used experimental organism. However, Arabidopsis can be limited as a model for species with different characteristics or unique traits3. This has prompted the development of new model systems, such as maize (Zea mays), an important plant for developmental genetics in monocots4, and tomato (Solanum lycopersicum), which is an important model for evolutionary studies, fruit development, and production, and is a good representation for vegetable crops5. A method for genetic transformation is a prerequisite for a plant species to serve as a model organism2. An Agrobacterium tumefaciens-mediated transformation is a reliable tool in plant biology; it has been used to transform a few model species and major crops, including tobacco (Nicotiana tabacum)6, rice (Oryza sativa)7, cotton (Gossypium hirsutum)8, soybean (Glycine max)9, potato (Solanum tuberosum)10, and canola (Brassica napus)11. Plant species are highly variable in how successfully they respond to A. tumefaciens infection, and transformation protocols often need to be individually tailored to each species6,12.
The genus Plantago includes a total of 256 plant species, widely distributed worldwide13. The species in this genus often have unique characteristics that make them desirable as model species for studying genetics, ecology, stress physiology, secondary metabolites, medicinal chemistry, plant-microbe interactions, plant development, and evolution. Plantago lanceolata, also called the narrowleaf or ribwort plantain, has been a popular plant of interest since the 19th century, when it was first used to describe the phenomenon of male sterility14. Like other plants of its genus, it has been used in studies across various research fields. More recently, it has been proposed as a model for vascular biology, as its vascular tissue can be collected easily15. P. lanceolata is the most commonly studied species in the genus Plantago; a 2021 article reported that there were >1,400 publications including or relating to this species at that time16, and an additional 102 articles have been published since the beginning of 2022, according to a PubMed search conducted on December 9th 2022. The next most studied plant in the genus, P. major, is the subject of only 414 articles when searched using the same criteria on the same date.
Despite the research interest in P. lanceolata, studies, especially on gene function characterization, are often limited by the lack of a genetic manipulation toolkit for the species. Pommerrienig et al. made efforts to develop a transformation protocol for P. major using a floral dip technique17. However, this method cannot be applied to P. lanceolata because of the male sterility characteristic of this species18,19. To our knowledge, there is no existing protocol for the transformation of P. lanceolata.
This study presents a simple protocol for A. tumefaciens-mediated transformation of P. lanceolata. By targeting root tissues, fully grown transgenic plants can be generated within 3 months of transformation.
NOTE: Steps 1.4-1.8, 2.3-2.5, 3.3-3.6, 4.1-4.6, 5.1-5.7, and 6.1-6.3 must be performed in aseptic conditions, using a clean hood to prevent contamination.
1. Plant material propagation for transformation
2. Plasmid construction and E. Coli transformation
NOTE: The exact plasmid construction procedure varies depending on the gene of interest. In this procedure, the restriction enzymes HindIII and SalII were used to insert the 1.5 kb AtPP2 promoter into the binary plasmid pBI101 (see Table of Materials) with GUS, using the standard cloning procedure20. AtPP2 (phloem protein 2) is a gene that is specifically expressed in phloem21.
3. A. tumefaciens transformation with plasmid
4. A. tumefaciens preparation
5. Transformation of Plantago roots
6. Selection and whole plant regeneration
7. Soil transfer
8. β-glucuronidase (GUS) histochemical staining
A simple protocol is reported here for obtaining transgenic P. lanceolata plants using A. tumefaciens-mediated transformation. The reporter gene GUS (encoding β-glucuronidase) is transformed, driven by the phloem-expressed promoter of AtPP2, into 3-week-old P. lanceolata roots through A. tumefaciens strain GV3101 (Figure 2). A phloem-specific promoter was chosen because our main interest was to establish a system for the functional genomics of plant vascular tissues, particularly phloem. The method was tested on the root, leaf, and petiole tissue in the preliminary experiment. Although callus could be induced in all tissue types, only the root tissue produced shoot initials (Figure 5A) after 1 month in SIM; the leaf and petiole turned brown and died (Figure 5B). This led to the conclusion that root tissue was the optimal tissue type for use in the transformation method. The roots were incubated in the prepared bacteria resuspended in suspension solution (SS) (Table 1) for a minimum of 20 min, then incubated at room temperature on solid SS plates for up to 3 days in the dark (Figure 3E). The roots were then transferred to the shoot induction medium (SIM) and kept under a grow light, in the conditions indicated in the protocol (step 1.6). Figure 1 and Figure 3 show representative images of each step of the protocol for reference.
Figure 6 shows the progression of shoot initials emerging from transformed tissue, from the first day the roots were placed on the SIM (Figure 6A) to when the shoots were ready to be rooted (Figure 6D). After 1 week, the root tissue formed callus (Figure 6B), and the beginnings of shoot initials could be observed (Figure 6B1). Shoots continued to emerge during weeks 2 and 3 (Figure 6C), and after 4 weeks, the shoots were ready to be transferred to the root induction medium (Figure 6D).
Identification of the putative transgenic plants was conducted using the β-glucuronidase (GUS) histochemical assay, using leaf segments taken once the shoots were around 0.5 cm long. Positive transgenic plants showed the expected staining pattern in the phloem localized tissue, demonstrated in Figure 4. Positive GUS-stained shoots were transferred to the root induction medium, in which they developed robust rooting systems after 4 weeks (Figure 1E). Rooted plants were then transferred to the soil. Figure 4 shows the result of staining in a narrowleaf plantain transformed with the AtPP2 promoter and the β-glucuronidase (GUS) gene, along with a wild-type and a narrowleaf plantain transformed with the AtPP2 promoter, for comparison. All shoots that emerged were confirmed as transgenic. The transformation efficiency was determined to be an average of 20%, with approximately two shoots emerging for every 10 roots that were transformed. Confirmed transgenic plants were transferred to larger pots and grown for 4-8 weeks until they reached the adult stage (Figure 1F).
Figure 1: Timeline of Plantago lanceolata transformation. Representative images of each stage of the protocol. (A) Ungerminated seeds plated on an MS plate. (B) Seeds germinated after 1 week, ready to be transferred into magenta boxes. (C) Plants in MS boxes after 3 weeks of growth. Roots are green and healthy, at the ideal stage for transformation. (D) Shoots in shoot induction media after 4 weeks are ready to be transferred into the rooting medium. At this stage, β-glucuronidase (GUS) histochemical staining can be conducted, if applicable. (E) Plants in boxes with root induction media, where roots have formed after 4 weeks of growth. (F) Transgenic plants are grown to full length after 4 weeks of growth in soil. Please click here to view a larger version of this figure.
Figure 2: Diagram of the binary vector plasmid pBI101 + β-glucuronidase (GUS) with the inserted phloem-specific promoter AtPP2. Please click here to view a larger version of this figure.
Figure 3: Steps of transformation. Representative images of each step of transformation. (A) Separating roots from shoots during transformation. (B) Soaking roots in bacteria/SS suspension. (C) Drying roots on paper towels to remove excess bacteria. (D) Roots plated on co-culture medium. (E) SS plates wrapped in aluminum foil. Plants were incubated for 2-3 days before being transferred to the shooting medium. Please click here to view a larger version of this figure.
Figure 4: GUS-staining. β-glucuronidase (GUS) staining results of narrowleaf plantain leaf segments. (A) Wild type. (B) Narrowleaf plantain transformed with the plasmid that harbors the AtPP2 promoter (empty vector). (C) Narrowleaf plantain transformed with the plasmid that harbors the AtPP2 promoter and the β-glucuronidase (GUS) gene. Each leaf was stained using the GUS histochemical staining protocol, then imaged with a microscopic camera. Images (B) and (C) show no staining pattern due to the absence of the GUS gene. The right image shows a clear blue staining pattern in the veins, confirming that the plants are transgenic. The bar represents 1 mm, with each leaf segment measuring approximately 1 cm in length. Please click here to view a larger version of this figure.
Figure 5: Comparison of transformation efficiency of different tissue types after >1 month incubation on shooting media. (A) Root tissues after over 1 month of growth. Roots have experienced expanded callus, and shoot initials have emerged. Non-transformed callus has begun to die in response to antibiotic selection. (B) Leaf and petiole tissues after over 1 month of growth. Tissues experienced some callus expansion but soon died in response to the antibiotic. No shoots emerged from either tissue. Please click here to view a larger version of this figure.
Figure 6: Emergence of callus and shoots on transformed tissue. Representative images of tissues placed on shooting medium after different lengths of incubation. (A) Root tissues just after being plated on the shooting medium. (B) Root tissues after 1 week on shooting medium. Callus expansion can be observed, and (B1) the first shoot initials have begun to emerge. (C) Root tissues after 3 weeks on shooting medium. More shoot initials have emerged. (C1) The shoot that emerged from the B1 shoot initial. (D) Root tissues after 4 weeks of incubation. Non-transformed tissue has begun to turn black/brown and die, and emerging shoots continue to grow. At this stage, shoots are ready to be moved to the rooting medium. Please click here to view a larger version of this figure.
Table 1: Media preparation recipes. A description of how to prepare mediums for transformation. The quantity of vitamins added is calculated based on the indicated stock solution concentration. See Table 2 for vitamin stock solution preparation. For all mediums, add reagents to 900 mL of double-distilled H2O, pH to the indicated level, and then add water to a final volume of 1,000 mL. * = add after sterilization. ** = pH with 1 M KOH. *** = pH with 1 M NaOH. Please click here to download this Table.
Table 2: Vitamin stocks for Plantago mediums. All vitamins must be filter sterilized and labeled accurately before storage. Where indicated, dissolve the powders first in 1 N NaOH, then make up the desired volume with double distilled H2O. Please click here to download this Table.
The lack of a transformation protocol for plants in the genus Plantago limits the use of these plants as models, particularly when researchers are interested in exploring gene functions. P. lanceolata was chosen to develop a genetic transformation protocol because it is the most commonly studied plant of its genus16. The protocol that has been developed will likely be used as a tool to further advance studies related to vascular biology, ecology, plant-insect interactions, and abiotic stress physiology.
The protocol presented clearly outlines steps that allow a user to obtain transgenic plants. Besides the ability of P. lanceolata to thrive in a tissue culture environment, multiple factors contributed to the success of our transformation method. First, the importance of using high-quality, sterile plant root tissue for transformation was observed. Roots had the highest transformation rates when they were taken from 3-4-week-old plants, and appeared green or pale white. Roots taken from boxes with any amount of bacterial or fungal contamination often resulted in contaminated shooting cultures, and older roots that appeared brown did not result in successful transformation. Root tissue was the most efficient tissue type for transformation using the current method, as leaf and petiole tissue were unsuccessful at developing shoots.
Another important observation was that the optimal method for collecting root tissue for transformation was to place freshly cut root material in sterile water. This step effectively allowed root material to remain hydrated while the remainder of the tissue was collected, as roots tend to dry out quickly when being removed from their growth containers. This step also helped to increase the success rate of the transformation, because it allowed more roots to be incubated in the bacteria at one time.
This protocol could be modified by decreasing the time that the root tissue incubates in the co-culture media to 2 days. It was observed that a 2 or 3 day incubation period is sufficient to allow infection that results in shoot initials. However, longer incubation times are not recommended, since it was observed that the absence of an antibiotic inhibitor in the media often results in A. tumefaciens overgrowth, which can kill the emerging tissue.
A limitation of this study is the lack of available data on the performance of other methods or species of A. tumefaciens de P. lanceolata transformation for comparison. To our knowledge, this protocol is novel. During the initial trials, a high transformation efficiency was noted with A. tumefaciens GV3101, and we focused on refining the technique using this strain instead of experimenting with other strains. Our transformation efficiency of 20% is relatively high for plant transformation-many conventional methods consider anything >1% to be successful26,27,28. However, using another strain of A. tumefaciens, such as A. rhizogenes, known for its use in root transformation in multiple species29,30,31, may result in an even higher success rate. Further experimentation would be needed to assess the impact of using other strains to promote increased transformation efficiency in P. lanceolata.
The successful transformation of P. lanceolata will likely benefit many fields of study. The high transformation efficiency, and the rapid growth of the plant in tissue culture media, make P. lanceolata a feasible candidate for gene function studies15.
The authors have nothing to disclose.
This work was supported by the National Science Foundation (EDGE IOS-1923557 to C.Z. and Y.Z.).
14 mL Round Bottom TubeA4A2:A34 | ThermoFisher Scientific | 150268 | |
1-Naphthylacetic acid | Gold Biotechnology | N-780 | |
3M Micropore Surgical Paper Tape | ThermoFisher Scientific | 19-027761 | |
50 mL Centrifuge Tubes | Research Products International Corp. | 163227LC | |
600 Watt High Pressure Sodium Lights | Plantmax | PX-LU600 | |
6-Benzylaminopurine (6-BAP) | Gold Biotechnology | B-110 | |
Aluminum Foil | ThermoFisher Scientific | 01-213-100 | |
Bacto Agar | Thermofisher Scientific | 214010 | |
Binary Plasmid pBI101 | Clontech, USA | 632522 | |
Cool White Grow Light Sylvania LLC | Home Depot | 315952205 | |
D-biotin | ThermoFisher Scientific | BP232-1 | |
ddH2O | |||
DH5a E. coli | Invitrogen, USA | 18258012 | |
Disposable Petri Dishes, Sterile 150 x 16 mm | ThermoFisher Scientific | FB0875712 | |
Disposable Petri Dishes, Sterile 95 x 15 mm | ThermoFisher Scientific | FB0875714G | |
Dissecting Scissors | Leica Biosystems | 38DI12044 | |
Ethanol 200 Proof | Decon Labs | 2705 | |
Folic Acid | Fisher Scientific | BP2519-5 | |
Forceps | Leica Biosystems | 38DI18031 | |
Gelrite | Research Products International Corp. | G35020-1000 | |
Glycerol | ThermoFisher Scientific | 17904 | |
Glycine | Sigma | 241261 | |
Incubated Tabletop Orbital Shaker | ThermoFisher Scientific | SHKE420HP | |
Indole-3-Acetic Acid (IAA) | Gold Biotechnology | I-110 | |
Indole-3-Butyric Acid (IBA) | Gold Biotechnology | I-180 | |
Kanamycin Monosulfate | Gold Biotechnology | K-120 | |
Macrocentrifuge | ThermoFisher Scientific | 75007210 | |
Magenta Boxes | ThermoFisher Scientific | 50255176 | |
Micro Pipet Tips 1000 µL | Corning | 4140 | |
Micro Pipet Tips 200 µL | Corning | 4138 | |
Micro Pipette Tips 10 µL | Corning | 4135 | |
Microcentrifuge | ThermoFisher Scientific | 75002410 | |
Micropipettor 0.5-10 µL | Corning | 4071 | |
Micropipettor 100-1000 µL | Corning | 4075 | |
Micropipettor 20-200 µL | Corning | 4074 | |
Micropipettor 2-20 µL | Corning | 4072 | |
Murashige & Skooge Basal Medium with Vitamins | PhytoTech | M519 | |
Murashige & Skooge Basal Salt Mixture | PhytoTech | M524 | |
myo-Inositol | Gold Biotechnology | I-25 | |
Nicotinic acid | Sigma | N0761-100g | |
Parafilm (paraffin film) | ThermoFisher Scientific | S37440 | |
Potassium Hydroxide (KOH) | Research Products International Corp. | P44000 | |
Pyridoxine HCl | Sigma | P6280-10g | |
Scalpel Blade Handle | Leica Biosystems | 38DI36419 | |
Scalpel Blades | Leica Biosystems | 3802181 | |
Sodium Chloride, Crystal (NaCl) | Mallinckrodt Chemicals | 7581-06 | |
Sodium Hydroxide (NaOH) | Research Products International Corp. | S24000 | |
Sodium Hypochlorite | Walmart | 23263068401 | |
Soil- Bark Mix | Berger, USA | BM7 | |
Square Pots (3.5 inches squared) | Greenhouse Megastore | CN-TRK-1835 | |
Sucrose | Research Products International Corp. | S24060 | |
Thermocycler | ThermoFisher Scientific | A24811 | |
Thiamine HCl | Sigma | T4625-5G | |
Timentin Ticarcillin/Clavulanate (15/1) (Timentin) | Gold Biotechnology | T-104 | |
trans-Zeatin Riboside (ZR) | Gold Biotechnology | Z-100 | |
Tryptone | Thermofisher Scientific | 211705 | |
Wild Type Plantago lanceolata seeds | Outsidepride Seed Source, OR, USA | F1296 | Outsidepride.com |
Yeast Extract Granulated | Research Products International Corp. | Y20025-1000 |