3.2 Agrobacterium
Cultures
63 and are plated on solid YM medium containing 100 mg/l streptomycin and 50 mg/l kanamycin, about 100 μl of culture per plate.
4. Plates are incubated for 2–3 days at 30 °C. Single colonies are isolated and inoculated to fresh YM media. Confi rmation of the transformation vector can be done by either direct colony PCR using gene-specifi c primers or plasmid mini-preparation of Agrobacterium followed by restriction enzyme digestion
and gel electrophoresis.
5. For carrot transformation, a single colony of LBA4404 containing the plasmid pCambia1300::bar::NPR1 is used to inoculate 50-ml liquid YM supplemented with 100 mg/l streptomycin and 50 mg/l kanamycin and is grown for ~16 h on a rotary shaker (250 rpm) at 30 °C.
6. Cultures are centrifuged at 3,000 × g at room temperature for 15 min. The supernatant is removed and the pellet is resus- pended to a density of OD 600 = 0.3 (corresponding to approxi-
mately 1 × 10 8 cells/ml) in 1/10 MS supplemented with
200-μM acetosyringone ( see Note 3 ).
1. Sterile petioles are cut into 5–10-mm-long segments using a scalpel and incubated in the Agrobacterium suspension in a plastic Petri plate for 10–30 min with gentle shaking. The sus- pension is then drained and the carrot explants are blotted on a sterile fi lter paper and placed onto MS1D. Approximately 20–30 explants are placed on a 9-cm Petri plate and coculti- vated in the dark for 2–3 days ( see Note 4 ).
2. Infected explants are subsequently rinsed in sterile H 2 O, blot-
ted dry, and placed on MS1DP. Positive and negative controls are included. Positive controls are placed on the medium lack- ing PPT, while negative controls are noninfected with bacteria and placed on the selective medium. Incubate at room tem- perature (22 °C) under cool white fl uorescent lights (450 μmol/m 2 /s, 16 h a day). All subsequent steps are incu-
bated under these same conditions.
3. After 2 weeks, explants are transferred to the MS1/2D10P medium and maintained on this medium, with transfers made every 4 weeks, until calli develop. Somatic embryos typically begin to appear 8–12 weeks following infection. Non- embryogenic calli are maintained for up to 20 weeks and dis- carded at that time if embryos have not developed. Typically, about 12 % of the petiole explants will form callus, with approximately 75 % of the calli formed leading to the develop- ment of somatic embryos.
4. Somatic embryos are transferred to MS10P for regeneration. Alternatively, the embryogenic calli are transferred into conical
3.3 Transformation
64
fl asks (250 ml) containing 50 ml of liquid MS1/4D5P (50 ml) for initiating suspension cultures. Suspension embryos should be transferred to fresh medium every 2–3 weeks and main- tained under constant shaking on a rotary shaker at 200 rpm. 5. Washing the somatic embryos with sterile water and plating
them on MSO medium will result in the formation of new plantlets within 3–4 weeks. The plantlets should be subcul- tured on MS10P, resulting in ~75 % germination of somatic embryos.
6. The tiny plantlets or small shoots are transferred to Magenta boxes containing MS10P. Roots are induced quickly after the transfer of shoots to larger containers; if they are not present prior to the initial transfer, rooting frequency is typically close to 100 %.
7. The fully rooted plantlets are transferred to the potting medium in 6-pack planting containers, covered with clear 6-in. high- seed-starting domes to maintain humidity, and placed in a growth chamber maintained at 24 °C, 85 % relative humidity, and a 16-h photoperiod (450 μmol/m 2 /s) where they grow
into full plants. The domes are removed after 5–7 days. 8. Once the plants reach a size greater than 15 cm, they are tested
for resistance to the herbicide Liberty ® . Leaves of carrot plants
are painted with a 0.2 or 0.4 % (w/v) Liberty ® solution and
visually assessed for resistance ( see Note 5 ). Visually healthy leaf tissue was collected from plants at this stage for PCR, Southern, Northern, and Western analysis ( see Note 6 ). 9. Effect of carrot cultivar on transformation frequency was mea-
sured as a proportion of infected explants that developed into individual Southern-positive plants that grew on herbicide selection medium ( see Note 7 ).
10. Once the transgenic carrots are larger than 20 cm in height, they are transferred to 6-in.-diameter pots and grown to matu- rity under the same conditions. The plants are watered two times weekly (or as needed) and fertilized once a week with a 20:20:20 fertilizer (500 mg/l). Pest problems that can occur are primarily western fl ower thrips ( Frankliniella occidentalis (Perg.)), which are diffi cult to control ( see Note 8 ).
11. Carrots are a biennial plant and fl ower after their second grow- ing season. To induce vernalization, the carrot plants at the 7–8-leaf stage are placed at 8 °C for 10 weeks. The dead and dying foliage is removed, and the plants are transferred back to the previous growing conditions. When fl owering shoots are produced, the individual heads are encased in a glassine bag with “blow fl ies” and tied off to prevent insect escape [ 11 ]. Seeds are then removed after 3–6 weeks and stored desiccated at room temperature ( see Note 9 ).
65
4
Notes
1. Agrobacterium tumefaciens LBA4404 was used in our experi- ments; however, other more aggressive strains have also proven successful by other groups [ 1 ]. Typically, less callus develop- ment will be observed with more aggressive strains of Agrobacterium and can result in the direct formation of somatic embryos from the necrotic explants.
2. DL-PPT was used for selection in our experiments; however, glufosinate ammonia can be substituted at identical concentra- tions, and Basta can be used at 0.75× the PPT concentration. Alternatively, for cost saving, Liberty ® herbicide can be used
directly in the medium following fi lter sterilization to provide the appropriate glufosinate ammonia concentration; however, there are some surfactants within the solution that could lead to enzymatic inhibition of sensitive assays performed from tis- sue culture-grown plants.
3. When LBA4404 cultures are resuspended in 1/10 MS solu- tion, they will often form clumps; this is completely normal and does not affect the transformation.
4. Carrot petioles are dissected on top of sterile fi lter paper and cut into appropriate sizes. This alleviated some of the diffi cul- ties associated with the petioles dehydrating and sticking to smooth surfaces, such as a Petri plate. Following dissection of each petiole (10–25 explants), they are immersed in the Agrobacterium solution. This caused some explants to have longer exposure to the inoculum; however, it was necessary to avoid the suberization of wound sites.
5. Some of the PPT-resistant plants still show some susceptibility to the 0.4 % Liberty ® application; however, it was much less
than the control plants. The minimum lethal concentration of Liberty ® to each plant needs to be ascertained by a preliminary
experiment. The concentrations range between 0.1 and 0.4 %. Cotton swabs are used to gently paint the herbicide solution over the leaf surface pre-marked with a waterproof ink or marker pen. Typical phytotoxicity symptoms develop after 7 days. 6. All of the somatic embryos that arise from an individual callus
are typically a unique transformation event; however, careful tracking and Southern blot confi rmation are required to iden- tify events. Typically, we reinitiate the callus from individual sterile plant lines in order to propagate clonal populations. 7. There are signifi cant differences in the transformation fre-
quency depending on the cultivar selected. Danvers Half Long and Nantes Coreless have effi ciencies greater than 3 %; Nanco and HCM cultivars have effi ciencies of less than 1 % [ 4 ]. These effi ciencies are the number of independent Southern-positive events from 100 explants.
66
1. Baranski R (2008) Genetic transformation of carrot ( Daucus carota ) and other apiaceae spe- cies. Trans Plant J 2:18–38
2. Kalbina I, Wallin A, Lindh I, Engstrom P, Andersson S, Strid A (2011) A novel chimeric MOMP antigen expressed in Escherichia coli,
Arabidopsis thaliana , and Daucus carota as a
potential Chlamydia trachomatis vaccine candi- date. Prot Express Purif 80:194–202
3. Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, Han YP, Alpuche-Solis AG, Korban SS (2011) Transgenic carrot tap roots expressing an immunogenic F1-V fusion pro- tein from Yersinia pestis are immunogenic in mice. J Plant Physiol 168:174–180
4. Luchakivskaya Y, Kishchenko O, Gerasymenko I, Olevinskaya Z, Simonenko Y, Spivak M, Kuchuk M (2011) High-level expression of human interferon alpha-2b in transgenic car- rot ( Daucus carota L.) plants. Plant Cell Rep 30:407–415
5. Zhang HX, Liu M, Li YJ, Zhao YH, He H, Yang GD, Zheng CC (2010) Oral immunoge- nicity and protective effi cacy in mice of a carrot- derived vaccine candidate expressing
UreB subunit against Helicobacter pylori . Prot Express Purif 69:127–131
6. Wally O, Jayaraj J, Punja ZK (2009) Comparative resistance to foliar fungal pathogens in trans- genic carrot plants expressing genes encoding for chitinase, ß1,3-glucanase and peroxidase. Eur J Plant Pathol 123:331–334
7. Wally O, Punja ZK (2010) Enhanced disease resistance in transgenic carrot ( Daucus carota L.) plants over-expressing a rice cationic per- oxidase. Planta 232:1229–1239
8. Wally O, Jayaraj J, Punja ZK (2009) Broad- spectrum disease resistance to necrotrophic and biotrophic pathogens in transgenic carrots ( Daucus carota L.) expressing an Arabidopsis NPR1 gene. Planta 231:131–141
9. Deblock M, Botterman J, Vandewiele M et al (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J 6:2513–2518
10. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497 11. Simon PW, Peterson CE (1984) Controlled pol-
linations of carrot. Plant Mol Biol Rep 2:43–44 8. Insect pests on carrot plants can be minimized with weekly
applications of “safer” insecticidal soap, according to manufac- turer’s instructions. In severe cases where thrips are visually detected, applications of active Amblyseius cucumeris , a predatory mite grown on a bran fl ake medium and purchased from a local supplier, are applied directly to infested carrot plants.
9. For seed production if space is an issue, one alternative is to remove the taproot from soil, carefully wash the root, and rinse in a 0.5 % NaOCl solution. Subsequently, pat the root dry, dust lightly with fungicidal sulfur powder, and wrap in a moistened paper towel. The roots can then be placed in a resealable plastic container and incubated at 4 °C for 8 weeks and subsequently replanted in potting mix. The roots should be checked periodi- cally to ensure that there is no fungal or bacterial contamination.
Acknowledgment
Funding for this research was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC), Discovery Grants Program.
References
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Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224, DOI 10.1007/978-1-4939-1658-0_7, © Springer Science+Business Media New York 2015