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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

Arabidopsis pin-fasten grafting under short-day (SD) growth conditions. Forty-five-day-old SD-grown Arabidopsis wild-type (Col) and gl1-1 plants were used as stocks (a) and scions (b), respectively. cgl1-1 scions with mature leaves removed. dgl1-1 scions were pin-fastened to Col stocks. The glabrous leaves of scions are indicated by white asterisks. e Successfully grafted plant at 2 weeks after grafting. The newly developed glabrous leaves are indicated by white asterisks. The insect pins are indicated by white arrows.
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Fig2: Arabidopsis pin-fasten grafting under short-day (SD) growth conditions. Forty-five-day-old SD-grown Arabidopsis wild-type (Col) and gl1-1 plants were used as stocks (a) and scions (b), respectively. cgl1-1 scions with mature leaves removed. dgl1-1 scions were pin-fastened to Col stocks. The glabrous leaves of scions are indicated by white asterisks. e Successfully grafted plant at 2 weeks after grafting. The newly developed glabrous leaves are indicated by white asterisks. The insect pins are indicated by white arrows.

Mentions: LD-grown Arabidopsis develops rapidly, which restricts the use of grafting to examine systemic signals in meristem differentiation. To broaden the application of pin-fasten grafting, we used SD-grown Arabidopsis for grafting experiments. Under this condition, Arabidopsis plants grow slowly, which provides sufficient time to analyze systemic signals. At 15 days under SD conditions, the seedlings of wild-type or gl1-1 Arabidopsis produced only 1–2 leaves and slender hypocotyls, which were difficult to use for pin-fasten grafting. At 30 or 45 days, the plants usually developed 5–6 or 12–15 leaves, respectively. The leaf number of 30-day-old SD-grown plants was equivalent to that of 2-week-old LD-grown plants, which suggests a similar developmental stage. Of 95 grafts we conducted, the success rate of grafting was 73% (69/95). Therefore, the pin-fasten grafting is applicable for SD-grown seedlings. To examine the grafting capacity at later developmental stages, we used 45-day-old SD-grown plants for pin-fasten grafting (Figure 2a). Similar to LD-grown grafting, the pin-inserted scions were fastened on top of apex-removed stocks (Figure 2b–d). The assembled grafted plants were kept in the sealed chamber for 1 week to maintain humidity, then transferred to a growth chamber. At 2–3 weeks after grafting, newly developed glabrous leaves were observed in scions (Figure 2e). Among 65 grafts, the success rate was 50% (32/65), which suggests that older SD-grown Arabidopsis still have high capacity for regenerating graft junctions. Thus, stocks and scions at broad developmental stages are suitable for pin-fasten grafting.Figure 2


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)

Arabidopsis pin-fasten grafting under short-day (SD) growth conditions. Forty-five-day-old SD-grown Arabidopsis wild-type (Col) and gl1-1 plants were used as stocks (a) and scions (b), respectively. cgl1-1 scions with mature leaves removed. dgl1-1 scions were pin-fastened to Col stocks. The glabrous leaves of scions are indicated by white asterisks. e Successfully grafted plant at 2 weeks after grafting. The newly developed glabrous leaves are indicated by white asterisks. The insect pins are indicated by white arrows.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Arabidopsis pin-fasten grafting under short-day (SD) growth conditions. Forty-five-day-old SD-grown Arabidopsis wild-type (Col) and gl1-1 plants were used as stocks (a) and scions (b), respectively. cgl1-1 scions with mature leaves removed. dgl1-1 scions were pin-fastened to Col stocks. The glabrous leaves of scions are indicated by white asterisks. e Successfully grafted plant at 2 weeks after grafting. The newly developed glabrous leaves are indicated by white asterisks. The insect pins are indicated by white arrows.
Mentions: LD-grown Arabidopsis develops rapidly, which restricts the use of grafting to examine systemic signals in meristem differentiation. To broaden the application of pin-fasten grafting, we used SD-grown Arabidopsis for grafting experiments. Under this condition, Arabidopsis plants grow slowly, which provides sufficient time to analyze systemic signals. At 15 days under SD conditions, the seedlings of wild-type or gl1-1 Arabidopsis produced only 1–2 leaves and slender hypocotyls, which were difficult to use for pin-fasten grafting. At 30 or 45 days, the plants usually developed 5–6 or 12–15 leaves, respectively. The leaf number of 30-day-old SD-grown plants was equivalent to that of 2-week-old LD-grown plants, which suggests a similar developmental stage. Of 95 grafts we conducted, the success rate of grafting was 73% (69/95). Therefore, the pin-fasten grafting is applicable for SD-grown seedlings. To examine the grafting capacity at later developmental stages, we used 45-day-old SD-grown plants for pin-fasten grafting (Figure 2a). Similar to LD-grown grafting, the pin-inserted scions were fastened on top of apex-removed stocks (Figure 2b–d). The assembled grafted plants were kept in the sealed chamber for 1 week to maintain humidity, then transferred to a growth chamber. At 2–3 weeks after grafting, newly developed glabrous leaves were observed in scions (Figure 2e). Among 65 grafts, the success rate was 50% (32/65), which suggests that older SD-grown Arabidopsis still have high capacity for regenerating graft junctions. Thus, stocks and scions at broad developmental stages are suitable for pin-fasten grafting.Figure 2

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