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A pin-fasten grafting method provides a non-sterile and highly efficient method for grafting Arabidopsis at diverse developmental stages.

Huang NC, Yu TS - Plant Methods (2015)

Bottom Line: Grafting provides a powerful technique to examine transportation and systemic effects of mobile molecules.Further longitudinal sections of the graft union showed well-connected vascular tissues between grafted plants.Use of fluorescent phloem-limited dye carboxyfluorescein diacetate in grafted plants demonstrated a symplastic connection established at 6 days after grafting and almost fully developed at 8 days.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529 Taiwan.

ABSTRACT

Background: Higher plants have evolved sophisticated communication systems to integrate environmental stimuli into their developmental programs. Grafting provides a powerful technique to examine transportation and systemic effects of mobile molecules. In Arabidopsis, many grafting approaches have been developed to investigate systemic molecules. However, these methods are usually limited to specific developmental stages or require sterilized conditions. To broaden the application of grafting for examining systemic signals at diverse developmental stages, we developed an Arabidopsis pin-fasten grafting method with insect pins used to assemble stocks and scions.

Results: We report the step-by-step protocol of Arabidopsis pin-fasten grafting. Arabidopsis wild-type or gl1-1 plants were grown under long- or short-day conditions. Insect pins were inserted into gl1-1 scions at different developmental stages for grafting onto epicotyls or hypocotyls of stocks. Successfully grafted scions with newly developed glabrous leaves were observed at 14 days after grafting. Further longitudinal sections of the graft union showed well-connected vascular tissues between grafted plants. Use of fluorescent phloem-limited dye carboxyfluorescein diacetate in grafted plants demonstrated a symplastic connection established at 6 days after grafting and almost fully developed at 8 days.

Conclusions: Our method provides a simple and robust approach to grafting Arabidopsis at different developmental stages. Sterilized conditions are not required, which greatly improves the success of grafting and plant growth.

No MeSH data available.


Related in: MedlinePlus

Longitudinal sections showing connected vascular tissues between scions and stocks. Toluidine-blue staining of longitudinal sections. Cells with primary cell wall are stained pink, and cells with secondary cell wall (vascular tissues) are stained blue. Cells stained blue were well connected across the graft junction. The graft junction is indicated by arrows. a, d, g Longitudinal sections of Arabidopsis gl1-1 scions (SC) grafted onto P35S-GUS transformant stocks (ST). The position of the longitudinal section, near epidermal cells (a–c), inner cell layers (d–f), or vascular tissues (g–i), is indicated by a dashed line on the left.
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Fig5: Longitudinal sections showing connected vascular tissues between scions and stocks. Toluidine-blue staining of longitudinal sections. Cells with primary cell wall are stained pink, and cells with secondary cell wall (vascular tissues) are stained blue. Cells stained blue were well connected across the graft junction. The graft junction is indicated by arrows. a, d, g Longitudinal sections of Arabidopsis gl1-1 scions (SC) grafted onto P35S-GUS transformant stocks (ST). The position of the longitudinal section, near epidermal cells (a–c), inner cell layers (d–f), or vascular tissues (g–i), is indicated by a dashed line on the left.

Mentions: To further confirm the establishment of a vascular connection between stocks and scions, we obtained longitudinal sections to visualize the vasculature across the grafted junctions. The graft junction between stocks and scions was cut from the successfully grafted plants at 14 days after grafting. The leaves surrounding graft junctions were removed to reveal the grafted tissues. Tissues were embedded in LR5 resin. After sectioning, tissues were stained with Toluidine blue, which differentially stains primary cell walls pink and secondary cell walls of xylem blue. Continuous longitudinal sections from tissues near epidermal cells revealed regenerated cells that were connected at the graft junction (Figure 5a–f). Sections close to vascular tissues showed continuous blue cells throughout the graft junction, which indicated that the vascular tissues were well connected between stocks and scions (Figure 5g–i).Figure 5


A pin-fasten grafting method provides a non-sterile and highly efficient method for grafting Arabidopsis at diverse developmental stages.

Huang NC, Yu TS - Plant Methods (2015)

Longitudinal sections showing connected vascular tissues between scions and stocks. Toluidine-blue staining of longitudinal sections. Cells with primary cell wall are stained pink, and cells with secondary cell wall (vascular tissues) are stained blue. Cells stained blue were well connected across the graft junction. The graft junction is indicated by arrows. a, d, g Longitudinal sections of Arabidopsis gl1-1 scions (SC) grafted onto P35S-GUS transformant stocks (ST). The position of the longitudinal section, near epidermal cells (a–c), inner cell layers (d–f), or vascular tissues (g–i), is indicated by a dashed line on the left.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4495618&req=5

Fig5: Longitudinal sections showing connected vascular tissues between scions and stocks. Toluidine-blue staining of longitudinal sections. Cells with primary cell wall are stained pink, and cells with secondary cell wall (vascular tissues) are stained blue. Cells stained blue were well connected across the graft junction. The graft junction is indicated by arrows. a, d, g Longitudinal sections of Arabidopsis gl1-1 scions (SC) grafted onto P35S-GUS transformant stocks (ST). The position of the longitudinal section, near epidermal cells (a–c), inner cell layers (d–f), or vascular tissues (g–i), is indicated by a dashed line on the left.
Mentions: To further confirm the establishment of a vascular connection between stocks and scions, we obtained longitudinal sections to visualize the vasculature across the grafted junctions. The graft junction between stocks and scions was cut from the successfully grafted plants at 14 days after grafting. The leaves surrounding graft junctions were removed to reveal the grafted tissues. Tissues were embedded in LR5 resin. After sectioning, tissues were stained with Toluidine blue, which differentially stains primary cell walls pink and secondary cell walls of xylem blue. Continuous longitudinal sections from tissues near epidermal cells revealed regenerated cells that were connected at the graft junction (Figure 5a–f). Sections close to vascular tissues showed continuous blue cells throughout the graft junction, which indicated that the vascular tissues were well connected between stocks and scions (Figure 5g–i).Figure 5

Bottom Line: Grafting provides a powerful technique to examine transportation and systemic effects of mobile molecules.Further longitudinal sections of the graft union showed well-connected vascular tissues between grafted plants.Use of fluorescent phloem-limited dye carboxyfluorescein diacetate in grafted plants demonstrated a symplastic connection established at 6 days after grafting and almost fully developed at 8 days.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529 Taiwan.

ABSTRACT

Background: Higher plants have evolved sophisticated communication systems to integrate environmental stimuli into their developmental programs. Grafting provides a powerful technique to examine transportation and systemic effects of mobile molecules. In Arabidopsis, many grafting approaches have been developed to investigate systemic molecules. However, these methods are usually limited to specific developmental stages or require sterilized conditions. To broaden the application of grafting for examining systemic signals at diverse developmental stages, we developed an Arabidopsis pin-fasten grafting method with insect pins used to assemble stocks and scions.

Results: We report the step-by-step protocol of Arabidopsis pin-fasten grafting. Arabidopsis wild-type or gl1-1 plants were grown under long- or short-day conditions. Insect pins were inserted into gl1-1 scions at different developmental stages for grafting onto epicotyls or hypocotyls of stocks. Successfully grafted scions with newly developed glabrous leaves were observed at 14 days after grafting. Further longitudinal sections of the graft union showed well-connected vascular tissues between grafted plants. Use of fluorescent phloem-limited dye carboxyfluorescein diacetate in grafted plants demonstrated a symplastic connection established at 6 days after grafting and almost fully developed at 8 days.

Conclusions: Our method provides a simple and robust approach to grafting Arabidopsis at different developmental stages. Sterilized conditions are not required, which greatly improves the success of grafting and plant growth.

No MeSH data available.


Related in: MedlinePlus