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Graft-union development: a delicate process that involves cell-cell communication between scion and stock for local auxin accumulation.

Yin H, Yan B, Sun J, Jia P, Zhang Z, Yan X, Chai J, Ren Z, Zheng G, Liu H - J. Exp. Bot. (2012)

Bottom Line: Histological analysis suggested that the transport activities of vasculature were recovered at 3 days after grafting (dag) and that auxin modulated the vascular reconnection at 2 dag.Microarray data revealed a signal-exchange process between cells of the scion and stock at 1 dag, which re-established the communication network in the graft union.This process was concomitant with the clearing of cell debris, and both processes were initiated by a wound-induced programme.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Lanzhou University, Lanzhou 730000, People's Republic of China.

ABSTRACT
Grafting is an ancient cloning method that has been used widely for thousands of years in agricultural practices. Graft-union development is also an intricate process that involves substantial changes such as organ regeneration and genetic material exchange. However, the molecular mechanisms for graft-union development are still largely unknown. Here, a micrografting method that has been used widely in Arabidopsis was improved to adapt it a smooth procedure to facilitate sample analysis and to allow it to easily be applied to various dicotyledonous plants. The developmental stage of the graft union was characterized based on this method. Histological analysis suggested that the transport activities of vasculature were recovered at 3 days after grafting (dag) and that auxin modulated the vascular reconnection at 2 dag. Microarray data revealed a signal-exchange process between cells of the scion and stock at 1 dag, which re-established the communication network in the graft union. This process was concomitant with the clearing of cell debris, and both processes were initiated by a wound-induced programme. The results demonstrate the feasibility and potential power of investigating various plant developmental processes by this method, and represent a primary and significant step in interpretation of the molecular mechanisms underlying graft-union development.

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Grafts in Arabidopsis and tomato. (A–L) Time-lapse images demonstrating the phenotypic development of a graft union of a WT/WT combination in Arabidopsis. Arrowheads indicate the graft union. Images were ordered as dag (days after grafting). The graft union was examined on all sides: (A), (C), (E), (G), (I), and (K) show the front view, which was observed perpendicular to the medium surface, while (B), (D), (F), (H), (J), and (L) show the side view, which was observed parallel to the medium surface. (M–T) Graft union development in tomato. White arrowheads indicate the graft union. Tomato grafts recover faster than other species studied. At 3 dag, the scion and stock were connected completely. Bars, 200 μm.
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fig2: Grafts in Arabidopsis and tomato. (A–L) Time-lapse images demonstrating the phenotypic development of a graft union of a WT/WT combination in Arabidopsis. Arrowheads indicate the graft union. Images were ordered as dag (days after grafting). The graft union was examined on all sides: (A), (C), (E), (G), (I), and (K) show the front view, which was observed perpendicular to the medium surface, while (B), (D), (F), (H), (J), and (L) show the side view, which was observed parallel to the medium surface. (M–T) Graft union development in tomato. White arrowheads indicate the graft union. Tomato grafts recover faster than other species studied. At 3 dag, the scion and stock were connected completely. Bars, 200 μm.

Mentions: Following the ‘good union’ principle, the protocol was improved in terms of enhancing the connection between the scion and stock. The cut surface of the scion and the stock cannot be placed closely together due to their different morphology—expanded cotyledons will raise the hypocotyl of the scion and result in a poor connection between the scion and stock. Moreover, the water on the medium surface will infiltrate the graft union and consequently interfere with the union healing. An oblique medium surface was found to overcome these problems through trial and error. In the first step, a glass rod was used to prop up one side of the plate before pouring the liquid medium into it (Fig. 1A). The agar solidified as an inclined plane at approximately 15° relative to the plate and formed an oblique surface that was thick at one side and thin at the other. Seeds were sown on this oblique surface, and after stratification at 4°C for 2–3 d, the plate was placed vertically in the growth room with the thin side downward and the thick side upward. On the oblique surface (Fig. 1B, reversed triangle), enough space (Fig. 1B, dotted line) was provided for the cut surfaces to connect the scion and stock thoroughly and completely (Fig. 1B, arrowhead). Therefore, there were strong mutual-effect forces on the joint when the plate was placed vertically in the growth room (Fig. 1B, white arrows), supporting a tight connection between the scion and stock. As it was easy to cut adventitious roots at their first emergence and to get rid of grafts that fail to form a good union immediately after grafting, because the graft union could easily be inspected from all sides (Fig. 2A–L), the success rate could be greatly improved. An 80% success rate was achieved in Arabidopsis, which was higher than the previous method used (no more than 70%; we performed grafting experiments according to Turnbull et al., 2002, and Bainbridge et al., 2006, and got a success rate of approximately 60%).


Graft-union development: a delicate process that involves cell-cell communication between scion and stock for local auxin accumulation.

Yin H, Yan B, Sun J, Jia P, Zhang Z, Yan X, Chai J, Ren Z, Zheng G, Liu H - J. Exp. Bot. (2012)

Grafts in Arabidopsis and tomato. (A–L) Time-lapse images demonstrating the phenotypic development of a graft union of a WT/WT combination in Arabidopsis. Arrowheads indicate the graft union. Images were ordered as dag (days after grafting). The graft union was examined on all sides: (A), (C), (E), (G), (I), and (K) show the front view, which was observed perpendicular to the medium surface, while (B), (D), (F), (H), (J), and (L) show the side view, which was observed parallel to the medium surface. (M–T) Graft union development in tomato. White arrowheads indicate the graft union. Tomato grafts recover faster than other species studied. At 3 dag, the scion and stock were connected completely. Bars, 200 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3398452&req=5

fig2: Grafts in Arabidopsis and tomato. (A–L) Time-lapse images demonstrating the phenotypic development of a graft union of a WT/WT combination in Arabidopsis. Arrowheads indicate the graft union. Images were ordered as dag (days after grafting). The graft union was examined on all sides: (A), (C), (E), (G), (I), and (K) show the front view, which was observed perpendicular to the medium surface, while (B), (D), (F), (H), (J), and (L) show the side view, which was observed parallel to the medium surface. (M–T) Graft union development in tomato. White arrowheads indicate the graft union. Tomato grafts recover faster than other species studied. At 3 dag, the scion and stock were connected completely. Bars, 200 μm.
Mentions: Following the ‘good union’ principle, the protocol was improved in terms of enhancing the connection between the scion and stock. The cut surface of the scion and the stock cannot be placed closely together due to their different morphology—expanded cotyledons will raise the hypocotyl of the scion and result in a poor connection between the scion and stock. Moreover, the water on the medium surface will infiltrate the graft union and consequently interfere with the union healing. An oblique medium surface was found to overcome these problems through trial and error. In the first step, a glass rod was used to prop up one side of the plate before pouring the liquid medium into it (Fig. 1A). The agar solidified as an inclined plane at approximately 15° relative to the plate and formed an oblique surface that was thick at one side and thin at the other. Seeds were sown on this oblique surface, and after stratification at 4°C for 2–3 d, the plate was placed vertically in the growth room with the thin side downward and the thick side upward. On the oblique surface (Fig. 1B, reversed triangle), enough space (Fig. 1B, dotted line) was provided for the cut surfaces to connect the scion and stock thoroughly and completely (Fig. 1B, arrowhead). Therefore, there were strong mutual-effect forces on the joint when the plate was placed vertically in the growth room (Fig. 1B, white arrows), supporting a tight connection between the scion and stock. As it was easy to cut adventitious roots at their first emergence and to get rid of grafts that fail to form a good union immediately after grafting, because the graft union could easily be inspected from all sides (Fig. 2A–L), the success rate could be greatly improved. An 80% success rate was achieved in Arabidopsis, which was higher than the previous method used (no more than 70%; we performed grafting experiments according to Turnbull et al., 2002, and Bainbridge et al., 2006, and got a success rate of approximately 60%).

Bottom Line: Histological analysis suggested that the transport activities of vasculature were recovered at 3 days after grafting (dag) and that auxin modulated the vascular reconnection at 2 dag.Microarray data revealed a signal-exchange process between cells of the scion and stock at 1 dag, which re-established the communication network in the graft union.This process was concomitant with the clearing of cell debris, and both processes were initiated by a wound-induced programme.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Lanzhou University, Lanzhou 730000, People's Republic of China.

ABSTRACT
Grafting is an ancient cloning method that has been used widely for thousands of years in agricultural practices. Graft-union development is also an intricate process that involves substantial changes such as organ regeneration and genetic material exchange. However, the molecular mechanisms for graft-union development are still largely unknown. Here, a micrografting method that has been used widely in Arabidopsis was improved to adapt it a smooth procedure to facilitate sample analysis and to allow it to easily be applied to various dicotyledonous plants. The developmental stage of the graft union was characterized based on this method. Histological analysis suggested that the transport activities of vasculature were recovered at 3 days after grafting (dag) and that auxin modulated the vascular reconnection at 2 dag. Microarray data revealed a signal-exchange process between cells of the scion and stock at 1 dag, which re-established the communication network in the graft union. This process was concomitant with the clearing of cell debris, and both processes were initiated by a wound-induced programme. The results demonstrate the feasibility and potential power of investigating various plant developmental processes by this method, and represent a primary and significant step in interpretation of the molecular mechanisms underlying graft-union development.

Show MeSH