農桿菌滲入法應用於其他茄科蔬菜作物

為了之後進一步將此系統應用至更多農作物，先選用青椒、辣椒與茄子等茄 科蔬菜作物做為材料，測試是否也可適用農桿菌滲入法進行基因表達。本試驗以 帶有 35S promoter:GUS 載體的 C58C1 農桿菌品系進行測試，結果顯示，茄子穩定 且均勻表達 GUS 報導基因；青椒雖有表現，但是表現量不平均，且之後進行重複 試驗其表現量不穩定；辣椒則無法順利表達 GUS 報導基因 (圖 32)。

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圖 7. 缺磷與磷肥恢復處理不同天數對菸草外觀之影響

Figure 7. Effects of different days of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments on physical appearances of Nicotiana benthamiana. Pi resupply was started after three days of Pi deficiency.

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圖 8. 磷肥處理不同天數對菸草(a)地上部與(b)根部鮮重之影響

Figure 8. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on (a) shoot fresh weight and (b) root fresh weight in Nicotiana benthamiana.

Pi resupply was started after three days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for six replicates (n = 6). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re1D) (Re2D)(Re3D) (Re4D) (Re5D) (Re1D) (Re2D)(Re3D) (Re4D) (Re5D)

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圖 9. 磷肥處理不同天數對菸草根部/地上部鮮重比值之影響

Figure 9. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on root/shoot fresh weight ratio in Nicotiana benthamiana.

Pi resupply was started after three days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for six replicates (n = 6). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re1D) (Re2D) (Re3D) (Re4D)(Re5D)

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圖 10. 磷肥處理不同天數對菸草(a)葉片與(b)根部無機磷濃度之影響

Figure 10. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on (a) leaves Pi concentration and (b) root Pi concentration in Nicotiana benthamiana.

Pi resupply was started after three days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re1D) (Re2D) (Re3D) (Re4D)(Re5D)

(Re4D)(Re5D) (Re1D) (Re2D)(Re3D)

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圖 11. 磷肥處理不同天數對菸草(a)葉綠素螢光與(b)葉綠素含量之影響

Figure 11. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on (a) chlorophyll fluorescence and (b) chlorophyll content in Nicotiana benthamiana.

Pi resupply was started after three days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re4D)(Re5D) (Re4D)(Re5D)

(Re1D) (Re2D) (Re3D) (Re1D) (Re2D)(Re3D)

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圖 12. 磷肥處理不同天數對進行農桿菌滲入法之菸草植體無機磷濃度之影響 Figure 12. Effects of Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on leaf and root Pi concentrations in Nicotiana benthamiana after agroinfiltration.

Pi resupply was started after three days of Pi deficiency. Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

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圖 13. 磷肥處理不同天數對菸草葉片 GUS 染色結果之影響

Figure 13. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on GUS staining level in Nicotiana benthamiana leaves.

Except of positive and negative controls, all leaves used for GUS staining were transformed with the TPSI1 promoter:GUS construct using Agrobacterium tumefaciens C58C1 strain via agroinfiltration. The positive control was transformed with 35S promoter:GUS construct. The negative control was not transformed with any construct.

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圖 14. 磷肥處理不同天數對菸草葉片 GUS 活性之影響

Figure 14. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on relative GUS activity levels in Nicotiana benthamiana leaves. Leaves used for relative GUS activity assay were transformed with 35S promoter:GUS construct (35S, black bars, PC), TPSI1 promoter:GUS construct (TPSI1, slash bars) or no constuct (Empty, gray bars, NC) using Agrobacterium tumefaciens C58C1 strain via agroinfiltration. The level of relative GUS activity was normalized by the level of luciferase luminescence (LUC) expressed by the 35S promoter:LUC construct co-transformed with the above constructs. Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

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圖 15. 不同營養元素缺乏處理對菸草外觀之影響

Figure 15. Physical appearances of Nicotiana benthamiana after treatments with different nutrient deficiencies.

For treatments with different nutrient deficiencies, the indicated specific nutrient element was withdrawn from the nutrient solution for 7 days. The control (Full) plant was maintained in the Half-strength modified Hoagland nutrient solution with full nutrients.

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圖 16. 不同營養元素缺乏處理對菸草無機磷濃度之影響

Figure 16. Effects of different nutrient deficiencies on leaf Pi concentration (black bars) and root Pi concentration (gray bars) in Nicotiana benthamiana.

For treatments with different nutrient deficiencies, the indicated specific nutrient element was withdrawn from the nutrient solution for 7 days. The control (Full) plants were maintained in the Half-strength modified Hoagland nutrient solution with full nutrients. Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between nutrient treatments as compared by least significant difference (LSD) at P=0.05 level.

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圖 17. 不同營養元素缺乏處理對菸草葉片 GUS 染色結果之影響

Figure 17. Effects of different nutrient deficiencies on GUS staining levels in Nicotiana benthamiana leaves.

Except of positive and negative controls, all leaves used for GUS staining were transformed with the TPSI1 promoter:GUS construct using Agrobacterium tumefaciens C58C1 strain via agroinfiltration. The positive control was transformed with 35S promoter:GUS construct. The negative control was not transformed with any construct.

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圖 18. 不同營養元素缺乏處理對菸草葉片 GUS 活性之影響

Figure 18. Effects of different nutrient deficiencies on relative GUS activity levels in Nicotiana benthamiana leaves.

Leaves used for relative GUS activity assay were transformed with 35S promoter:GUS construct (35S, black bars, PC), TPSI1 promoter:GUS construct (TPSI1, slash bars) or no constuct (Empty, gray bars, NC) using Agrobacterium tumefaciens C58C1 strain via agroinfiltration. The level of relative GUS activity was normalized by the level of luciferase luminescence (LUC) expressed by the 35S promoter:LUC construct co-transformed with the above constructs. Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

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圖 19. 缺磷與磷肥恢復處理不同天數對番茄外觀之影響

Figure 19. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on physical appearances of tomato. Pi resupply was started after ten days of Pi deficiency.

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圖 20. 磷肥處理不同天數對番茄(a)地上部與(b)根部鮮重之影響

Figure 20. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on (a) shoot fresh weight and (b) root fresh weight in tomato.

Pi resupply was started after ten days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re1D) (Re5D)

(Re1D) (Re5D)

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圖 21. 磷肥處理不同天數對番茄根部/地上部鮮重比值之影響

Figure 21. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on root/shoot fresh weight ratio in tomato.

Pi resupply was started after ten days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re1D) (Re5D)

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圖 22. 磷肥處理不同天數對番茄(a)葉片與(b)根部無機磷濃度之影響

Figure 22. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on (a) leaf Pi concentration and (b) root Pi concentration in tomato.

Pi resupply was started after ten days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re1D) (Re5D)

(Re1D) (Re5D)

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圖 23. 磷肥處理不同天數對番茄(a)葉綠素螢光與(b)葉綠素含量之影響

Figure 23. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on (a) chlorophyll fluorescence and (b) chlorophyll content in tomato.

Pi resupply was started after ten days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re1D) (Re5D) (Re1D) (Re5D)

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圖 24. 磷肥處理不同天數對番茄花青素濃度之影響

Figure 24. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on anthocyanin concentration in tomato.

Pi resupply was started after ten days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re1D) (Re5D)

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圖 25. 磷肥處理不同天數對番茄葉片 GUS 染色結果之影響

Figure 25. Effects of Pi sufficient (+Pi) and Pi deficient (-Pi) treatments for different duration on GUS staining levels in tomato leaves.

Except of positive and negative control, all leaves used for GUS staining were transformed with the TPSI1 promoter:GUS construct using Agrobacterium tumefaciens 1D1249 strain via agroinfiltration. The positive control was transformed with 35S promoter:GUS construct. The negative control was not transformed with any construct.

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圖 26. 不同番茄品種與農桿菌菌液濃度對番茄葉片 (a) GUS 染色結果與 (b) 葉片 壞疽情形之影響

Figure 26. Effects of cultivar and Agrobacterium tumefaciens concentration on (a) GUS staining level and (b) leaf necrosis in tomato.

All tomato leaves used for GUS staining were transformed with the 35S promoter:GUS construct using Agrobacterium tumefaciens C58C1 strain via agroinfiltration. For positive and negative controls, tobacco leaves used for GUS staining were transformed with the 35S promoter:GUS construct or not transformed with any construct using Agrobacterium tumefaciens C58C1 strain via agroinfiltration.

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圖 27. 番茄生殖生長期進行磷肥處理不同天數對(a)地上部與(b)根部鮮重之影響 Figure 27. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on (a) shoot fresh weight and (b) root fresh weight in tomato during reproductive stage.

Pi resupply was started after ten days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re5D) (Re10D)

(Re5D) (Re10D)

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圖 28. 磷肥處理不同天數對番茄(a)綠熟期與(b)紅熟期果實無機磷濃度之影響 Figure 28. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re)

treatments for different duration on Pi concentration in tomato fruit at (a) green fruit and (b) red fruit stages.

Pi resupply was started after ten days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re5D) (Re10D) (Re5D) (Re10D)

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圖 29. 磷肥處理不同天數對番茄果實(a)產量與(b)可溶性固形物含量之影響

Figure 29. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on (a) yield and (b) total soluble solids content in tomato fruits.

Pi resupply was started after ten days of Pi deficiency (arrow). Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

(Re5D) (Re10D)

(Re5D) (Re10D)

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圖 30. 磷肥處理不同天數對番茄果實 GUS 染色之影響

Figure 30. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on GUS staining level in tomato fruits.

Except of positive and negative controls, all leaves used for GUS staining were transformed with the TPSI1 promoter:GUS construct using Agrobacterium tumefaciens C58C1 strain via agroinjection. The positive control was transformed with 35S promoter:GUS construct. The negative control was not transformed with any construct.

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圖 31. 磷肥處理不同天數對番茄果實 GUS 活性之影響

Figure 31. Effects of Pi sufficient (+Pi), Pi deficient (-Pi) and Pi resupply (Re) treatments for different duration on relative GUS activity levels in tomato fruits.

Fruits used for relative GUS activity assay were transformed with 35S promoter:GUS construct (35S, black bars), TPSI1 promoter:GUS construct (TPSI1, slash bars) or no constuct (Empty, gray bars) using Agrobacterium tumefaciens C58C1 strain via agroinjection. The level of relative GUS activity was normalized by the level of luciferase luminescence (LUC) expressed by the 35S promoter:LUC construct co-transformed with the above constructs. Error bars represent standard error (SE) of the mean for three replicates (n = 3). Means with the same letters are not significantly different between Pi treatments as compared by least significant difference (LSD) at P=0.05 level.

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圖 32. 青椒、辣椒與茄子利用農桿菌滲入法表達 GUS 情形

Figure 32. GUS staining of bell pepper, hot pepper and eggplant leaves transformed with the 35S promoter:GUS construct using Agrobacterium tumefaciens C58C1 strain via agroinfiltration. The negative control was not transformed with any construct.

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