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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

Development of symplastic connection between stocks and scions. Arabidopsis gl1-1 scions were grafted onto wild-type stocks under LD conditions. The 5(6)-carboxyfluorescein diacetate dye (green fluorescence) was introduced onto leaves (indicated by arrows) of wild-type stocks at different times after grafting. The images (left panels) and florescence images (right panels) were taken at 4 days (a, b), 5 days (c, d), 6 days (e, f), and 8 days (g, h) after grafting. Note green fluorescent signals (or yellow colors when merged with redflorescence) in the vasculature of gl1-1 scions at 6 or 8 days after grafting but not 4 or 5 days after grafting. The red colors in florescence images represents the auto-florescence of chlorophyll. The glabrous leaves of scions are indicated by white asterisks (left panels).
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Fig6: Development of symplastic connection between stocks and scions. Arabidopsis gl1-1 scions were grafted onto wild-type stocks under LD conditions. The 5(6)-carboxyfluorescein diacetate dye (green fluorescence) was introduced onto leaves (indicated by arrows) of wild-type stocks at different times after grafting. The images (left panels) and florescence images (right panels) were taken at 4 days (a, b), 5 days (c, d), 6 days (e, f), and 8 days (g, h) after grafting. Note green fluorescent signals (or yellow colors when merged with redflorescence) in the vasculature of gl1-1 scions at 6 or 8 days after grafting but not 4 or 5 days after grafting. The red colors in florescence images represents the auto-florescence of chlorophyll. The glabrous leaves of scions are indicated by white asterisks (left panels).

Mentions: Previously, CFDA has been used to examine phloem translocation between stocks and scions in Arabidopsis micrografting [31, 32]. To examine the establishment of functional phloem connection in pin-fasten grafting, we introduced CFDA into the mature leaf of LD-grown stocks at different times after grafting. Because most of the grafts that failed to differentiate were withered at early stages after grafting, we selected vigorous scions for CFDA experiments to ensure these scions were potentially successful grafts. At 3–5 days after grafting, fluorescence microscopy revealed highly accumulated fluorescence signals in stocks, including petioles, hypocotyls, and leaves, but not in gl1-1 scions (Table 2; Figure 6a–d). However, at 6–8 days after grafting, fluorescence signals were observed both in wild-type stocks and vasculature of gl1-1 scions (Table 2; Figure 6e–h). Among the grafted plants examined, the detection of fluorescent signals from scions was 0% at 3–5 days after grafting but 17% at 6 days after grafting (Table 2). As scions grew, the detection rate of fluorescence signals in scions increased to 76% at 7 days after grafting and 89% at 8 days (Table 2). Therefore, the symplastic connection between LD-grown scions and stocks began to be established at 6 days after grafting, with the symplastic connection was established at 8 days after grafting in most grafted plants. These results are consistent with previous finding that the functional phloem connection is established at 7- to 10 days after grafting [31]. However, in recent analyses of vascular reconnection in Arabidopsis micrografting, when CFDA was introduced to cotyledons of scions, fluorescence was detected in the roots of stocks at 3 days after grafting [32]. As the grafting methods are different in these experiments (Y-shape micrografting [31], butt alignment micrografting [32]), it is possible that the timing of vascular reconnection is various in different grafting methods or the position of grafted tissues may affect the phloem translocation. Further experiments are required to examine these possibilities.Table 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)

Development of symplastic connection between stocks and scions. Arabidopsis gl1-1 scions were grafted onto wild-type stocks under LD conditions. The 5(6)-carboxyfluorescein diacetate dye (green fluorescence) was introduced onto leaves (indicated by arrows) of wild-type stocks at different times after grafting. The images (left panels) and florescence images (right panels) were taken at 4 days (a, b), 5 days (c, d), 6 days (e, f), and 8 days (g, h) after grafting. Note green fluorescent signals (or yellow colors when merged with redflorescence) in the vasculature of gl1-1 scions at 6 or 8 days after grafting but not 4 or 5 days after grafting. The red colors in florescence images represents the auto-florescence of chlorophyll. The glabrous leaves of scions are indicated by white asterisks (left panels).
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Related In: Results  -  Collection

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Fig6: Development of symplastic connection between stocks and scions. Arabidopsis gl1-1 scions were grafted onto wild-type stocks under LD conditions. The 5(6)-carboxyfluorescein diacetate dye (green fluorescence) was introduced onto leaves (indicated by arrows) of wild-type stocks at different times after grafting. The images (left panels) and florescence images (right panels) were taken at 4 days (a, b), 5 days (c, d), 6 days (e, f), and 8 days (g, h) after grafting. Note green fluorescent signals (or yellow colors when merged with redflorescence) in the vasculature of gl1-1 scions at 6 or 8 days after grafting but not 4 or 5 days after grafting. The red colors in florescence images represents the auto-florescence of chlorophyll. The glabrous leaves of scions are indicated by white asterisks (left panels).
Mentions: Previously, CFDA has been used to examine phloem translocation between stocks and scions in Arabidopsis micrografting [31, 32]. To examine the establishment of functional phloem connection in pin-fasten grafting, we introduced CFDA into the mature leaf of LD-grown stocks at different times after grafting. Because most of the grafts that failed to differentiate were withered at early stages after grafting, we selected vigorous scions for CFDA experiments to ensure these scions were potentially successful grafts. At 3–5 days after grafting, fluorescence microscopy revealed highly accumulated fluorescence signals in stocks, including petioles, hypocotyls, and leaves, but not in gl1-1 scions (Table 2; Figure 6a–d). However, at 6–8 days after grafting, fluorescence signals were observed both in wild-type stocks and vasculature of gl1-1 scions (Table 2; Figure 6e–h). Among the grafted plants examined, the detection of fluorescent signals from scions was 0% at 3–5 days after grafting but 17% at 6 days after grafting (Table 2). As scions grew, the detection rate of fluorescence signals in scions increased to 76% at 7 days after grafting and 89% at 8 days (Table 2). Therefore, the symplastic connection between LD-grown scions and stocks began to be established at 6 days after grafting, with the symplastic connection was established at 8 days after grafting in most grafted plants. These results are consistent with previous finding that the functional phloem connection is established at 7- to 10 days after grafting [31]. However, in recent analyses of vascular reconnection in Arabidopsis micrografting, when CFDA was introduced to cotyledons of scions, fluorescence was detected in the roots of stocks at 3 days after grafting [32]. As the grafting methods are different in these experiments (Y-shape micrografting [31], butt alignment micrografting [32]), it is possible that the timing of vascular reconnection is various in different grafting methods or the position of grafted tissues may affect the phloem translocation. Further experiments are required to examine these possibilities.Table 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