Method for adjusting pore opening of Shanghai Qingqing

Abstract

The invention discloses a method for adjusting the pore opening degree of Shanghai green, and belongs to the technical field of plant detection. Shanghai leaves are placed in 20mL of open liquid containing (1) 0.5% potassium chloride and 100 [mu] M / L calcium chloride or (2) 0.5% potassium chloride and 50 [mu] M / L brassinolide or (3) 0.5% potassium chloride and 10 [mu] M / L cytokinin or (4) 0.5% potassium chloride and 5 [mu] M / L gibberellin or (5) 0.5% potassium chloride and 100 [mu] M / L methyl jasmonate, culture is performed under illumination, and the illumination time is set to be 5h, so that the pore opening degree of Shanghai is remarkably increased. Compared with a control group (containing 0.5% of potassium chloride), the pore openness of the treatment is respectively increased by 33.5%, 43.5%, 43.4%, 27.1% and 70.2%. The method disclosed by the invention lays a technical foundation for rapidly screening out the exogenous environmental stimulation factor (environical stimuli) capable of increasing the opening degree of the pore.

Description

Technical Field

[0001] This invention belongs to the field of plant detection technology, and more specifically, relates to a method for adjusting the stomatal opening of Shanghai bok choy. Background Technology

[0002] Stomata are pores formed by paired guard cells on plant leaves, serving as channels for CO2, the substrate of photosynthesis, to enter the plant. The size of the stomatal aperture determines the rate at which CO2 enters the plant, thus directly regulating the plant's photosynthetic rate. Rapidly screening exogenous hormones that increase stomatal aperture is crucial for improving plant photosynthetic rates, thereby promoting rapid plant growth and enhancing stress resistance. Observing changes in stomatal aperture by directly placing plant leaves in solutions containing different exogenous hormones is a common method for screening exogenous hormones that regulate stomatal aperture.

[0003] Tear off the lower epidermis of leaves by hand and prepare temporary slides is a common method for observing stomata in leafy vegetables. As a popular vegetable, Shanghai bok choy shows stomata under a microscope after the epidermis is torn off by hand; however, these stomata are mostly not open. This indicates that directly tearing off the epidermis is a good way to obtain stomata from Shanghai bok choy, but this method has significant limitations in observing the response of stomatal opening to external stimuli.

[0004] Therefore, there is an urgent need to establish a stable technical system that can rapidly obtain a large number of open, large stomata in order to meet the need for rapid screening of exogenous environmental stimuli that increase stomatal opening. Summary of the Invention

[0005] In view of the above-mentioned problems in the prior art, the technical problem to be solved by the present invention is to provide a method for adjusting the pore opening of Shanghai bok choy to obtain clear pores in Shanghai bok choy.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for adjusting the stomatal opening of Shanghai bok choy involves placing the lower epidermis of Shanghai bok choy leaves in an opening solution supplemented with different exogenous environmental stimuli and culturing them under light.

[0008] The exogenous environmental stimuli include one or more of calcium chloride, brassinolide, cytokinin, gibberellin, and methyl jasmonate.

[0009] The concentration of calcium chloride is less than 1000 μM / L; the concentration of brassinolide is less than 50 μM / L; the concentration of cytokinin is less than 200 μM / L; the concentration of gibberellin is less than 1000 μM / L; and the concentration of methyl jasmonate is less than 100 μM / L.

[0010] The concentration of calcium chloride is 100 μM / L; the concentration of brassinolide is 50 μM / L; the concentration of cytokinin is 10 μM / L; the concentration of gibberellin is 5 μM / L; and the concentration of methyl jasmonate is 100 μM / L.

[0011] The open solution is a potassium chloride solution with a mass concentration of 0.01-2% and a pH of 6.0.

[0012] The open solution is a 0.5% potassium chloride solution with a pH of 6.0.

[0013] The light incubation time is 0-7 hours.

[0014] The light incubation time was 5 hours.

[0015] The method for adjusting the stomatal opening of Shanghai bok choy includes the following specific steps:

[0016] 1) Take a Shanghai bok choy plant with a growth cycle of about 30 days, break off the outermost leaves by hand, wash them with distilled water 2-3 times, and then use absorbent paper to dry the water droplets on the surface.

[0017] 2) Tear off the lower epidermis of Shanghai bok choy leaves by hand and cut them into 0.3cm × 0.3cm cubes with scissors; place the cubes in 20mL of open culture medium (pH 6.0) containing ① 0.5% potassium chloride + 100μM / L calcium chloride or ② 0.5% potassium chloride + 50μM / L brassinolide or ③ 0.5% potassium chloride + 10μM / L cytokinin or ④ 0.5% potassium chloride + 5μM / L gibberellin or ⑤ 0.5% potassium chloride + 100μM / L methyl jasmonate, and culture under light for 5 hours.

[0018] 3) After the specified light exposure time is reached, gently remove the epidermis from the opening solution; place a drop of potassium chloride opening solution containing the corresponding treatment on a glass slide, flatten the small piece of epidermis; gently cover it with a coverslip, absorb the liquid at the edges, make a temporary slide and quickly transfer it to a microscope for observation, and you will get a large number of open pores with clear shapes.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1) In this invention, the lower epidermis of Shanghai bok choy leaves was placed in 20 mL of an opening solution containing 0.5% potassium chloride (pH 6.0) and cultured under light, with four time gradients of 0, 2, 5, and 7 hours of light exposure. The results showed that stomata were not open at 0 hours of light exposure, only a few stomata were open at 2 hours of light exposure, and a large number of stomata opened at 5 and 7 hours of light exposure, with no significant difference in stomatal opening at these two time points (5 and 7 hours). In conclusion, 5 hours of light exposure is the optimal condition for the large-scale opening of stomata in Shanghai bok choy.

[0021] 2) The results of this invention show that Shanghai bok choy leaves were placed in 20 mL of an opening solution containing potassium chloride at mass concentrations of 0.01%, 0.025%, 0.05%, 0.1%, 0.25%, 0.5%, 1%, and 2% (pH 6.0) and cultured under light for 5 hours. The results indicate that with increasing potassium chloride concentration, the number and size of open stomata in Shanghai bok choy first increased and then decreased, with the highest number of open stomata and the largest stomatal aperture obtained under a potassium chloride concentration of 0.5%.

[0022] 3) This invention discloses the most suitable technique for detecting stomatal aperture of Shanghai bok choy: the lower epidermis of Shanghai bok choy leaves is placed in 20 mL of an open solution containing 0.5% potassium chloride (pH 6.0) and cultured under light for 5 h. The regulatory effect of exogenous environmental stimuli on stomatal aperture of Shanghai bok choy is evaluated based on this method.

[0023] 4) In this invention, Shanghai bok choy leaves were placed in 20 mL of an open culture medium (pH 6.0) containing ① 0.5% potassium chloride + 100 μM / L calcium chloride, or ② 0.5% potassium chloride + 50 μM / L brassinolide, or ③ 0.5% potassium chloride + 10 μM / L cytokinin, or ④ 0.5% potassium chloride + 5 μM / L gibberellin, or ⑤ 0.5% potassium chloride + 100 μM / L methyl jasmonate, and cultured under light for 5 h. All these treatments significantly increased the stomatal aperture of Shanghai bok choy. Compared with the control group (containing 0.5% potassium chloride), the stomatal aperture increased by 33.5%, 43.5%, 43.4%, 27.1%, and 70.2%, respectively. Attached Figure Description

[0024] Figure 1 Figure showing the effect of light exposure time on stomatal opening in Shanghai green plants;

[0025] Figure 2 Figure showing the effect of different potassium supply concentrations on stomatal opening in Shanghai bok choy.

[0026] Figure 3 The effect of adding different concentrations of calcium chloride (CaCl2) to Shanghai bok choy on stomatal aperture;

[0027] Figure 4The effect of different concentrations of brassinolide (BR) added from the exogenous source on the stomatal aperture of Shanghai bok choy.

[0028] Figure 5 The effect of different concentrations of exogenous cytokinin (CTK) on stomatal aperture of Shanghai bok choy.

[0029] Figure 6 The effect of adding different concentrations of exogenous gibberellin (GA) on the stomatal opening of Shanghai bok choy.

[0030] Figure 7 The effect of adding different concentrations of methyl jasmonate (JA) to Shanghai bok choy on stomatal aperture. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0032] The Shanghai bok choy used in this application was grown at the Jiangsu Vocational College of Agriculture and Forestry Experimental Base (No. 19, Wenchang East Road, Jurong City, Jiangsu Province).

[0033] Example 1

[0034] 1. Take a Shanghai bok choy plant with a growth cycle of about 30 days. Break off the outermost leaves by hand, wash them 2-3 times with distilled water, and then use absorbent paper to dry the surface water droplets.

[0035] 2. ① Tear off the lower epidermis of Shanghai bok choy leaves by hand and cut them into 0.3cm×0.3cm squares with scissors; place 4 squares per petri dish in 20mL of open solution containing 0.5% potassium chloride (pH 6.0) and incubate under light, setting four time gradients of 0, 2, 5 and 7h of light.

[0036] After the designated light exposure time, gently remove the epidermis from the opening solution. Place a drop of opening solution containing 0.5% potassium chloride (pH 6.0) on a glass slide and flatten the epidermal patch. Gently cover with a coverslip and blot dry the edges. Quickly transfer the prepared temporary slide to a microscope for observation. Take photos of the stomata under 20x magnification using Mshot software, taking four photos of each epidermal patch. Open the images in ImageJ software, measure the scale bar length, set this scale in set scale, and then use line segments to measure the length and width of the stomata. Record these data using Ctrl+M. Calculate the stomatal aperture using the formula: stomatal aperture = width / length.

[0037] The results are as follows Figure 1As shown, stomata do not open at 0 hours of light exposure, only a few stomata open at 2 hours of light exposure, and a large number of stomata open at 5 hours and 7 hours of light exposure, with no significant difference in stomatal opening at these two time points (5 hours and 7 hours). In conclusion, 5 hours of light exposure is the optimal condition for a large number of stomata to open in Shanghai bok choy.

[0038] ② Tear off the lower epidermis of Shanghai bok choy leaves by hand and cut them into 0.3cm×0.3cm cubes with scissors; place 4 cubes per petri dish into 20mL of open solution containing 0.01%, 0.025%, 0.05%, 0.1%, 0.25%, 0.5%, 1%, and 2% potassium chloride (pH 6.0) respectively, and incubate under light for 5 hours.

[0039] After the designated light exposure time, gently remove the epidermis from the opening solution. Place a drop of potassium chloride opening solution of the corresponding mass concentration on a glass slide and flatten the epidermal piece. Gently cover with a coverslip and blot dry the edges. Quickly transfer the prepared temporary slide to a microscope for observation. Take photos of the stomata under 20x magnification using Mshot software, taking four photos of each epidermal piece. Open the images in ImageJ software, measure the scale bar length, set this scale in setscale, and then use line segments to measure the length and width of the stomata. Record these data using Ctrl+M. Calculate the stomatal aperture using the formula: stomatal aperture = width / length.

[0040] The results are as follows Figure 2 As shown, with the increase of potassium chloride concentration, the number and opening size of stomata in Shanghai bok choy showed a trend of first increasing and then decreasing. The highest number of stomata and the largest stomatal opening size were obtained under the condition of potassium chloride concentration of 0.5%.

[0041] In summary, manually tearing off the lower epidermis of Shanghai bok choy leaves and placing them in an opening solution containing 0.5% potassium chloride (pH 6.0) and culturing them under light for 5 hours resulted in the highest number of open stomata and the largest stomatal aperture. This method lays the technical foundation for rapidly screening exogenous environmental stimuli that increase stomatal aperture.

[0042] Example 2

[0043] 1. Take a Shanghai bok choy plant with a growth cycle of about 30 days. Break off the outermost leaves by hand, wash them 2-3 times with distilled water, and then use absorbent paper to dry the surface water droplets.

[0044] 2. Tear off the lower epidermis of Shanghai bok choy leaves by hand and cut them into 0.3cm × 0.3cm cubes with scissors. Place the cubes in 20mL of open solution (pH 6.0) containing ① 0.5% potassium chloride, ② 0.5% potassium chloride + 10μM / L CaCl2, ③ 0.5% potassium chloride + 50μM / L CaCl2, ④ 0.5% potassium chloride + 100μM / L CaCl2, and ⑤ 0.5% potassium chloride + 1000μM / L CaCl2 respectively in each culture dish. Incubate under light for 5 hours.

[0045] After the designated light exposure time, the epidermis was gently removed from the opening solution. A drop of potassium chloride opening solution containing the corresponding treatment was placed on a glass slide, and the epidermal piece was flattened. A coverslip was gently placed on top, and the liquid at the edges was blotted dry. The prepared temporary slide was quickly transferred to a microscope for observation. Stomata were photographed under 20x magnification using Mshot software, with four photographs taken of each epidermal piece. The images were opened in ImageJ software, the scale bar length was measured, and this scale was set in the "set scale" function. Then, the length and width of the stomata were measured using line segments, and these data were recorded using Ctrl+M. The stomatal aperture for each treatment was calculated using the formula: stomatal aperture = width / length. Finally, the regulatory effect of CaCl2 on the stomatal aperture of Shanghai bok choy was evaluated.

[0046] The results are as follows Figure 3 As shown, the addition of 100 μM / L CaCl2 had the strongest positive regulatory effect on stomatal aperture, increasing it by 33.5% compared to the control group (containing 0.5% potassium chloride).

[0047] 3. Tear off the lower epidermis of Shanghai bok choy leaves by hand and cut them into 0.3cm × 0.3cm cubes with scissors. Place the cubes in 20mL of open solution (pH 6.0) containing ① 0.5% potassium chloride, ② 0.5% potassium chloride + 1μM / L BR, ③ 0.5% potassium chloride + 10μM / L BR, ④ 0.5% potassium chloride + 25μM / L BR, and ⑤ 0.5% potassium chloride + 50μM / L BR, respectively, and incubate under light for 5 hours.

[0048] After the designated light exposure time, the epidermis was gently removed from the opening solution. A drop of potassium chloride opening solution corresponding to the treatment was placed on a glass slide, and the epidermal piece was flattened. A coverslip was gently placed on top, and the edges were blotted dry. The prepared temporary slide was quickly transferred to a microscope for observation. Stomata were photographed under 20x magnification using Mshot software, with four photographs taken of each epidermal piece. The images were opened in ImageJ software, the scale bar length was measured, and this scale bar was set in the set scale. Then, the length and width of the stomata were measured using line segments, and these data were recorded using Ctrl+M. The stomatal aperture for each treatment was calculated using the formula stomatal aperture = width / length. Finally, the regulatory effect of brassinolide on stomatal aperture of Shanghai bok choy was evaluated.

[0049] The results are as follows Figure 4 As shown, the addition of 50 μM / L brassinolide (BR) had the strongest positive regulatory effect on stomatal aperture, increasing it by 43.5% compared to the control group (containing 0.5% potassium chloride).

[0050] 4. Tear off the lower epidermis of Shanghai bok choy leaves by hand and cut them into 0.3cm × 0.3cm cubes with scissors. Place the cubes in 20mL of open culture medium (pH 6.0) containing ① 0.5% potassium chloride, ② 0.5% potassium chloride + 1μM / L CTK, ③ 0.5% potassium chloride + 5μM / L CTK, ④ 0.5% potassium chloride + 10μM / L CTK, ⑤ 0.5% potassium chloride + 100μM / L CTK, and ⑥ 0.5% potassium chloride + 200μM / L CTK respectively. Incubate under light for 5 hours.

[0051] After the designated light exposure time, the epidermis was gently removed from the opening solution. A drop of potassium chloride opening solution corresponding to the treatment was placed on a glass slide, and the epidermal piece was flattened. A coverslip was gently placed on top, and the liquid at the edges was blotted dry. The prepared temporary slide was quickly transferred to a microscope for observation. Stomata were photographed under 20x magnification using Mshot software, with four photographs taken of each epidermal piece. The images were opened in ImageJ software, the scale bar length was measured, and this scale bar was set in the set scale. Then, the length and width of the stomata were measured using line segments, and these data were recorded using Ctrl+M. The stomatal aperture for each treatment was calculated using the formula stomatal aperture = width / length. Finally, the regulatory effect of cytokinin on stomatal aperture of Shanghai bok choy was evaluated.

[0052] The results are as follows Figure 5 As shown, the addition of 10 μM / L cytokinin (CTK) had the strongest positive regulatory effect on stomatal aperture, increasing it by 43.4% compared to the control group (containing 0.5% potassium chloride).

[0053] 5. Tear off the lower epidermis of Shanghai bok choy leaves by hand and cut them into 0.3cm × 0.3cm cubes with scissors. Place the cubes in 20mL of open solution (pH 6.0) containing ① 0.5% potassium chloride, ② 0.5% potassium chloride + 1μM / L GA, ③ 0.5% potassium chloride + 5μM / L GA, ④ 0.5% potassium chloride + 10μM / L GA, ⑤ 0.5% potassium chloride + 100μM / L GA, and ⑥ 0.5% potassium chloride + 1000μM / L GA respectively. Incubate under light for 5 hours.

[0054] After the designated light exposure time, the epidermis was gently removed from the opening solution. A drop of potassium chloride opening solution corresponding to the treatment was placed on a glass slide, and the epidermal piece was flattened. A coverslip was gently placed on top, and the liquid at the edges was blotted dry. The prepared temporary slide was quickly transferred to a microscope for observation. Stomata were photographed under 20x magnification using Mshot software, with four photographs taken of each epidermal piece. The images were opened in ImageJ software, the scale bar length was measured, and this scale was set in the set scale. Then, the length and width of the stomata were measured using line segments, and these data were recorded using Ctrl+M. The stomatal aperture for each treatment was calculated using the formula stomatal aperture = width / length. Finally, the regulatory effect of gibberellin on stomatal aperture of Shanghai photinia was evaluated.

[0055] The results are as follows Figure 6 As shown, the addition of 5 μM / L gibberellin (GA) had the strongest positive regulatory effect on stomatal aperture, increasing it by 27.1% compared to the control group (containing 0.5% potassium chloride).

[0056] 6. Tear off the lower epidermis of Shanghai bok choy leaves by hand and cut them into 0.3cm × 0.3cm cubes with scissors. Place the cubes in 20mL of open solution (pH 6.0) containing ① 0.5% potassium chloride, ② 0.5% potassium chloride + 0.2μM / L JA, ③ 0.5% potassium chloride + 1μM / L JA, ④ 0.5% potassium chloride + 10μM / L JA, and ⑤ 0.5% potassium chloride + 100μM / L JA respectively in each culture dish. Incubate under light for 5 hours.

[0057] After the designated light exposure time, the epidermis was gently removed from the opening solution. A drop of potassium chloride opening solution corresponding to the treatment was placed on a glass slide, and the epidermal piece was flattened. A coverslip was gently placed on top, and the liquid at the edges was blotted dry. The prepared temporary slide was quickly transferred to a microscope for observation. The stomata were photographed under 20x magnification using Mshot software, with four photographs taken of each epidermal piece. The images were opened in ImageJ software, the scale bar length was measured, and this scale bar was set in the set scale. Then, the length and width of the stomata were measured using line segments, and these data were recorded using Ctrl+M. The stomatal aperture for each treatment was calculated using the formula stomatal aperture = width / length. Finally, the regulatory effect of methyl jasmonic acid on the stomatal aperture of Shanghai bok choy was evaluated.

[0058] The results are as follows Figure 7 As shown, the addition of 100 μM / L methyl jasmonate (JA) had the strongest positive regulatory effect on stomatal aperture, increasing it by 70.2% compared to the control group (containing 0.5% potassium chloride).

Claims

1. A method for adjusting the stomatal opening of Shanghai bok choy, characterized in that, The lower epidermis of Shanghai bok choy leaves was placed in an open solution containing different exogenous environmental stimuli and cultured under light.

2. The method for adjusting the stomatal opening of Shanghai bok choy according to claim 1, characterized in that, The exogenous environmental stimuli include one or more of calcium chloride, brassinolide, cytokinin, gibberellin, and methyl jasmonate.

3. The method for adjusting the stomatal opening of Shanghai bok choy according to claim 2, characterized in that, The concentration of calcium chloride is less than 1000 μM / L; the concentration of brassinolide is less than 50 μM / L; the concentration of cytokinin is less than 200 μM / L; the concentration of gibberellin is less than 1000 μM / L; and the concentration of methyl jasmonate is less than 100 μM / L.

4. The method for adjusting the stomatal opening of Shanghai bok choy according to claim 3, characterized in that, The concentration of calcium chloride is 100 μM / L; the concentration of brassinolide is 50 μM / L; the concentration of cytokinin is 10 μM / L; the concentration of gibberellin is 5 μM / L; and the concentration of methyl jasmonate is 100 μM / L.

5. The method for adjusting the stomatal opening of Shanghai bok choy according to claim 1, characterized in that, The open solution is a potassium chloride solution with a mass concentration of 0.01-2% and a pH of 6.

0.

6. The method for adjusting the stomatal opening of Shanghai bok choy according to claim 5, characterized in that, The open solution is a 0.5% potassium chloride solution with a pH of 6.

0.

7. The method for adjusting the stomatal opening of Shanghai bok choy according to claim 1, characterized in that, The light incubation time is 0-7 hours.

8. The method for adjusting the stomatal opening of Shanghai bok choy according to claim 7, characterized in that, The light incubation time was 5 hours.

9. The method for adjusting the stomatal opening of Shanghai bok choy according to any one of claims 1-8, characterized in that, The specific steps include: 1) Take a Shanghai bok choy plant with a growth cycle of about 30 days, break off the outermost leaves by hand, wash them with distilled water 2-3 times, and then use absorbent paper to dry the water droplets on the surface. 2) Tear off the lower epidermis of Shanghai bok choy leaves by hand and cut them into 0.3cm × 0.3cm cubes with scissors; place the cubes in 20mL of open culture medium containing ① 0.5% potassium chloride + 100μM / L calcium chloride, or ② 0.5% potassium chloride + 50μM / L brassinolide, or ③ 0.5% potassium chloride + 10μM / L cytokinin, or ④ 0.5% potassium chloride + 5μM / L gibberellin, or ⑤ 0.5% potassium chloride + 100μM / L methyl jasmonate, and culture under light for 5 hours. 3) After the specified light exposure time is reached, gently remove the epidermis from the opening solution; place a drop of potassium chloride opening solution containing the corresponding treatment on a glass slide, flatten the small piece of epidermis; gently cover it with a coverslip, absorb the liquid at the edges, make a temporary slide and quickly transfer it to a microscope for observation, and you will get a large number of open pores with clear shapes.

Images