Protocol: optimisation of a grafting protocol for oilseed rape (Brassica napus) for studying long-distance signalling
- Anna Ostendorp†1,
- Steffen Pahlow†1,
- Jennifer Deke1,
- Melanie Thieß1 and
- Julia Kehr1Email authorView ORCID ID profile
© Ostendorp et al. 2016
Received: 4 January 2016
Accepted: 18 March 2016
Published: 25 March 2016
Grafting is a well-established technique for studying long-distance transport and signalling processes in higher plants. While oilseed rape has been the subject of comprehensive analyses of xylem and phloem sap to identify macromolecules potentially involved in long-distance information transfer, there is currently no standardised grafting method for this species published.
We developed a straightforward collar-free grafting protocol for Brassica napus plants with high reproducibility and success rates. Micrografting of seedlings was done on filter paper. Grafting success on different types of regeneration media was measured short-term after grafting and as the long-term survival rate (>14 days) of grafts after the transfer to hydroponic culture or soil.
We compared different methods for grafting B. napus seedlings. Grafting on filter paper with removed cotyledons, a truncated hypocotyl and the addition of low levels of sucrose under long day conditions allowed the highest grafting success. A subsequent long-term hydroponic cultivation of merged grafts showed highest survival rates and best reproducibility.
Grafting is a well-established technique for joining vegetative tissues from two or more plants. It has been widely applied to improve the properties of different vegetables and other horticultural crop plants . Key for a successful establishment of graft unions is the formation of a continuous vascular system between the grafting partners that are usually called scion (shoot part of the graft) and rootstock (root part of the graft). Therefore, grafting is most successful in dicotyledonous plants possessing a vascular cambium and more difficult or even impossible in monocotyledonous plants.
Because of the reunion of functional xylem and phloem connections, grafting has also become an excellent experimental tool to study long-distance mobility of a wide range of molecules in living plants . Grafting studies have provided conclusive evidence that long-distance transport is involved in diverse, but likewise important, physiological processes. Examples are the photoperiodic regulation of flowering [3, 4], the systemic spread of viruses [5, 6], phytohormone transport and action , apical dominance , nodule formation , small RNA movement [10–12], systemic acquired resistance [13, 14], and systemic gene silencing . Grafting methods for confirming long-distance transport of regulating molecules are established for a wide range of plant species, including Nicotiana benthamiana [16, 17], Medicago truncatula , and the model species Arabidopsis thaliana [19–24]. These techniques have been applied successfully to study signal transduction, for example by micro RNAs (miRNAs) [10, 12, 25, 26].
Brassica napus is a suitable plant for studying long-distance communication, because it allows obtaining phloem and xylem exudates in sufficient amounts for analysing sap compositions. In this species that is related to the model plant A. thaliana, sampling is relatively easy, and sample volumes are comparably large [10–12, 27–29].
Several studies identified hundreds of proteins and small RNAs (smRNAs) in phloem sap of B. napus [28, 30, 31]. To verify their long-distance mobility in vivo, so far grafting studies between wild-type and mutants/overexpressor plants were performed in Arabidopsis [10, 12]. The major reasons are that Arabidopsis transformation is straightforward and unmatched genetic resources are publicly available. However, the use of Arabidopsis in grafting experiments to study phloem mobility does only allow indirect conclusions about the mobility of phloem-localised molecules, since phloem sampling and, thus, direct measurements of changing compound levels in phloem sap are hardly possible. Therefore, a system allowing the same type of analysis of phloem long-distance signalling in Brassica would be desirable. Although not as easy as in Arabidopsis, several reports describe the successful transformation of B. napus using Agrobacterium tumefaciens [32, 33] what should allow the creation of suitable transgenic plants for grafting experiments. However, up to now only a few not very detailed grafting protocols for this species have been published in international journals [34–36], and no information about efficiency and reproducibility of grafting is documented.
This study attempts to provide a robust collar-free grafting procedure for different B. napus cultivars that is useful to confirm long-distance phloem mobility of potential signalling compounds identified in isolated phloem sap from this species. We describe an optimised flat-surface root-to-shoot grafting protocol for B. napus seedlings. The established grafting method does not require a collar to support the graft union and enabled a high short-term (up to 100 %) and a reasonable long-term survival rate (70–80 %) after the transfer to hydroponic culture or soil, respectively, indicating a high ability for the formation of functional vascular connections. This was confirmed by following the movement of the phloem-specific fluorescence dye carboxyfluorescein diacetate (CFDA) through the established graft unions.
Seed sterilisation solution (see REAGENT SETUP)
Sodium hypochloride (Applichem-Panreac, cat. no. 213322.1214)
Tween-20 (Applichem-Panreac, cat no. A1389,0500)
70 and 100 % ethanol (Applichem-Panreac, cat. no. A3678,1000)
Murashige and Skoog (MS) medium (Duchefa, cat. No. M0245.0050)
Sucrose (Applichem-Panreac, cat. no. A3935,5000)
Agarose (BD, cat. no. 214010)
Sterile deionised water
Hydroponic medium (see REAGENT SETUP)
Carboxyfluorescein diacetate (CFDA) (Sigma-Aldrich, cat. no. 21879-100MG-F)
Dimethylsulfoxide (Applichem-Panreac, cat. no. A1584,0500)
Einheitserde Classic (Einheitserdewerk Uetersen).
Petri dishes (92 × 15 mm, Sarstedt, cat. no. 82.1473)
Growth chamber set to 22 °C, 70 % humidity, light conditions are dependent of the step in the grafting protocol (see PROCEDURE)
binocular (SZ40, Olympus)
round filter paper (90 mm, GE Life Science, cat. no. 1440-090)
razor blades (Wilkinson Sword Classic)
50 ml conical tubes (Sarstedt, cat. no. 62.547.254)
fluorescence stereomicroscope (MZ FL III, Leica)
parafilm (Bemis, cat. no. #PM999)
laminar flow tissue culture cabinet
4 °C standard laboratory fridge.
Seed sterilisation solution 7 % (v/v) sodium hypochloride, 0.05 % (v/v) Tween-20
Germination plate ½ Murashige and Skoog (MS) medium, 1 % agarose
Regeneration plates (1) ½ MS medium, 0.5 % (w/v) sucrose, 1 % agar, (2) ½ MS, 1 % agar, (3) filter paper moistened with 0.5 % sucrose, (4) filter paper with sterile water
Hydroponic medium the medium is described in Buhtz et al. : 0.6 mM NH4NO3, 1 mM Ca(NO3)2, 0.04 mM Fe-EDTA, 0.5 mM K2HPO4, 0.5 mM K2SO4, 0.4 mM Mg(NO3)2. Micro nutrients added: 0.8 μM ZnSO4, 9 μM MnCl2, 0.1 μM Na2MoO4, 23 μM H3BO3, 0.3 μM CuSO4. The pH was adjusted to 4.7 with 37 % HCl
CFDA solution 5 mg CFDA were solved in 100 µl 100 % DMSO. For plant application a 1:100 dilution in deionized water is used.
Brassica napus seeds can be obtained commercially and from various breeders and research laboratories. This protocol is optimised for the Drakkar and Licosmos cultivars.
Good sterilisation is necessary to prevent bacterial or fungal contaminations during graft recovery. All work should be done under a sterile laminar flow cabinet.
Seed sterilisation and germination
Sterilisation solution should be prepared freshly. Seeds are incubated for 2 min in 2 ml 100 % ethanol and subsequently surface sterilised with 2 ml sterilisation solution for 15 min and then washed with sterile water three times for 10 min . A better washing can be achieved by shaking the reaction tube containing the seeds.
Transfer the sterilised seeds to the germination plate (6–8 seeds per plate) and incubate the plates at 4 °C in the dark for 3 days in a vertical orientation.
After 3 days incubate the plates under short day conditions (light: 8 h, dark: 16 h, light intensity 100 μmol m−2 s−1, 22 °C and a relative humidity of 70 %) in a growth chamber and store the plates in a vertical orientation. After approx. 6 days old 3–5 cm long seedlings were used for grafting.
- 4.Cut up to four seedlings on a cutting plate (petri dish with a round Whatman filter paper moistened with sterile water) with a razor blade under a binocular microscope. Remove cotyledons as well as 1–2 cm of the middle of the hypocotyl of the seedlings (Fig. 1).
Join the cut plant parts on a regeneration plate with the respective regeneration conditions using forceps. Close the regeneration plates with parafilm and incubate them under short day conditions with plates in a vertical orientation (5°–10°). Check the grafts after 6, 10 and 14 days.
In contrast to the protocol from Marsch-Martinez  optimal cutting of hypocotyls and cotyledons can be achieved on a moistened filter paper, propably due to the higher stiffness of B. napus plants compared to A. thaliana. Another difference to this protocol is the removal of the central part of the hypocotyl. In Brassica, the fast longitudinal growth of the seedlings hindered a successful graft formation when this step was omitted. Attention needs to be paid when joining the cut scion and root that no water film is within the parts. Ecotypes with an increased longitudinal growth direction like Drakkar should be regenerated for 14 days since the graft junction is fairly instable. Ecotypes like Licosmos with a reduced longitudinal growth can be regenerated for only 10 days.
For hydroponic cultivation 14 day old grafts are transferred to 50 ml conical tubes (Fig. 3a) and grown in boxes using the hydroponic medium described in Buhtz et al. . For this purpose, wrap black foam around the grafted plant with the graft junction located in the middle of the foam. Cover the sides of the conical tubes with aluminium foil and fill in 40 ml of hydroponic medium to reduce algal growth.
Place the wrapped graft in the conical tube in such a way that the foam does not get in contact with the medium, but holds the graft in place and store the tubes in a rack (Additional file 3: Figure S3).
Place the rack in a standard polystyrene box with at least the same height or higher than the grafted plants. A box of 32 cm × 25 cm × 17 cm (length, width, height) is sufficient for cultivation of ca. 30 plants (Additional file 3: Figure S3). Cover the box with a light permissive plastic cover. For a simple set-up use plastic wrap and puncture up to 20 small holes for ca. 12 plants to allow adequate aeration. Cultivate the covered grafts for 10–14 days.
For soil cultivation the plants should be well-watered. Cover the transferred grafts with a plastic cover to avoid dehydration of the plantlets. Grafts are cultivated under long day conditions in a growth chamber (light: 16 h, dark: 8 h, light intensity of 80 μmol m−2 s−1, 22 °C and a relative humidity of 60 %; Fig. 3). After 14 days plants can be grown in the greenhouse either hydroponically in single pots or on soil.
The success of the formation of functional vascular connections within the grafts can be monitored using carboxyfluorescein diacetate, a phloem-specific fluorescence dye, as described in Grignon et al. 1989 . Grafted plants were transferred to agar plates containing ½ MS and 1 % (w/v) agar to prevent drying-out. One leave per plant is punctured and a few microliters of a 10 µM CFDA solution are applied. After an incubation of 30 min at ambient temperature, fluorescence can be observed under a fluorescence stereomicroscope equipped with a GFP filter (Fig. 4).
Results and discussion
Grafting conditions were optimised for B. napus seedlings to improve the survival rate of grafts. In contrast to other studies, we followed grafting success over a longer time period until grafted plants were successfully transferred to hydroponic culture or to soil. Obviously, success rates are lower at a later time-point than after a few days. However, since our goal was to transfer stable grafts to hydroponic culture or soil to let the plants grow until sampling of phloem and xylem sap is possible, it is the more meaningful measure in this case. Plants with a non-functional vascular system are prone to die within 2 weeks of post-grafting cultivation. Since initial experiments showed that graft formation was more successful under short day than under long day conditions, all further experiments were performed in growth chambers with 8 h light and 16 h dark.
In contrast to other published grafting procedures for B. napus [34–36] this work provides an easy and robust protocol for routine grafting with high success rates. Collar-free grafting is easier to handle and less laborious. When germination, grafting and graft generation are carried out under at least semi-sterile conditions, grafting success was significantly increased by minimising contamination of the graft junctions. Another advantage of our protocol is its applicability to mid- to high-throughput, as transport studies often require high numbers of grafted plants to allow statistically relevant conclusions.
In parallel experiments performed with Arabidopsis seedlings (data not shown) it could be observed that grafting on filter paper was more successful than grafting on agar plates. Also here, sucrose enhanced size and fitness of the grafts, but led to a slightly higher formation of adventitious roots in this species. Generally, too much moisture hindered successful formation of graft unions and water films on both agar plates and filter paper in all types of grafting experiments performed. A similar observation was made in recent Arabidopsis grafting experiments .
Grafting is a versatile tool to study long-distance mobility of potential signalling compounds. The development of optimised protocols that allow reliable grafting with high success rates is essential to obtain reasonable and reproducible results. In this study we present a simple and efficient grafting procedure for B. napus that in routine application allows a short-term survival rate of 80–100 % and still 70–80 % after transfer to hydroponic culture or soil, respectively. This demonstrates that B. napus is highly suitable for performing transport studies using the easy grafting procedure presented here. That is important, because B. napus is already used as a suitable model plant for xylem sap and phloem sap analyses due to the relatively easy access to quite large sample volumes from both long-distance transport systems. Long-term survival rates on hydroponic culture or soil enable the growth of grafts until the time-point suitable for phloem sap sampling. Therefore, in contrast to grafting studies in model plants like Arabidopsis, Brassica grafting studies do not only allow indirect conclusions of phloem mobility of potential signalling compounds, but their direct detection in collected phloem samples.
JK and MT contributed to conception and design of the experiments, AO, SP, JD and MT carried out the grafting experiments and analysed the results. JK has drafted the manuscript and all authors were involved in revising it critically. All authors read and approved the final manuscript.
We would like to acknowledge the financial contribution to the research activities by a Career Integration Grant (CIG; PCIG14-GA-2013-63 0734) by the European Commission within the 7th framework programme and the grant LFF-GK06 „Deligrah“ (Landesforschungsförderung Hamburg) awarded to JK.
The authors declare that they have no competing interests.
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