Application of nitrogen-doped carbon dots in stimulating plant cells to compartmentalize salt and improve salt tolerance

Abstract

The present invention discloses the application of nitrogen-doped carbon dots in stimulating plant cells to compartmentalize salt and improve salt tolerance, and belongs to the field of novel plant stress resistance agents. The application described in the present invention is to add N-CDs solution to the plant cell solution, wherein the concentration of N-CDs in the plant cell solution is 0.05-0.5 mg / L. The N-CDs used in the present invention has a significant mitigating effect on plant cell damage caused by salt stress; treatment with N-CDs at a concentration of 0.5 mg / L for 12 hours can enhance plant salt tolerance by 14.5%. The N-CDs used in the present invention can be used as K + The promoter of signal transduction + The net inflow increased significantly by 297.8%, and the Na + / H + The expression of the transporter-related gene NHX1 (7.8 times) stimulates the plant to partition and passivate the salt and store it in the cells, thereby improving the salt tolerance of plant cells.

Description

Technical Field

[0001] The invention relates to application of nitrogen-doped carbon dots in stimulating plant cells to compartmentalize salt and improve salt tolerance, and belongs to the field of novel plant stress resistance agents. Background Art

[0002] Land salinization is one of the main hazards threatening soil environmental health and human food production and security. In order to cope with the ionic stress, osmotic stress and oxidative stress caused by soil salinization, plants themselves will try to regulate ionic homeostasis, osmotic balance and detoxification through a series of physiological and biochemical mechanisms to maintain normal plant growth, but this defense ability is limited. Engineered Nanomaterials (ENMs) have great potential in enhancing plant salt tolerance. As an emerging biotechnology, nanobiotechnology can reduce the negative impact of agricultural technology on the environment while improving plant tolerance to abiotic stress.

[0003] At present, research on ENMs to improve plant salt tolerance mainly focuses on the improvement of plant photosynthesis and antioxidant capacity. However, the effective regulation of ion homeostasis by plant cells is the primary mechanism of plant salt tolerance. Nitrogen-doped carbon dots (N-CDs), as a new type of carbon-based environmental functional material, have outstanding biocompatibility and nanosafety, and can promote plant growth by improving the light conversion of photosynthesis and inducing carbohydrate production. In addition, N-CDs have certain potential in alleviating oxidative stress and activating systemic acquired resistance. So, can this ENMs act as an excitator to regulate ion homeostasis, and what is the optimal concentration and optimal period for it to perceive and stimulate the plant salt tolerance signal network? It is still unknown. Summary of the invention

[0004] [Technical issues]

[0005] Currently, there is no literature reporting how to use nanomaterials to stimulate plant cells to compartmentalize salt and thus improve the salt tolerance of plant cells.

[0006] [Technical solution]

[0007] In order to solve the above problems, the present invention utilizes N-CDs as an exciter for regulating ion homeostasis to stimulate plant cells to compartmentalize salt, thereby improving the salt tolerance of plant cells.

[0008] The first object of the present invention is to provide an application of nitrogen-doped carbon dots in stimulating plant cells to compartmentalize salt and improve salt tolerance, wherein the application is to add N-CDs solution into a plant cell solution.

[0009] In one embodiment of the present invention, the particle size of the N-CDs is 1-3 nm.

[0010] In one embodiment of the present invention, the concentration of N-CDs in the plant cell solution is 0.05-0.5 mg / L.

[0011] In one embodiment of the present invention, the solvent of the N-CDs solution is water, and the concentration is 2-10 mg / L.

[0012] In one embodiment of the present invention, the N-CDs are prepared by thermal polymerization of melamine and ethylenediamine; specifically, ethylenediamine and melamine are mixed and calcined at high temperature in a muffle furnace; the resulting solution is then dialyzed for purification and finally freeze-dried; wherein the molar ratio of ethylenediamine to melamine is 2:1; the high-temperature calcination is calcined at 650°C for 6h; and the dialysis purification is performed using a dialysis bag with a molecular weight of 500 Da.

[0013] In one embodiment of the present invention, the plant cell is one of tobacco BY-2, corn cell and rice cell.

[0014] In one embodiment of the present invention, the plant cell is a plant cell in an exponential growth phase.

[0015] In one embodiment of the present invention, the salt tolerance means that the plant cells can maintain activity in an environment where the concentration of sodium chloride is below 100 mM.

[0016] In one embodiment of the present invention, the plant cell solution is obtained by placing plant cells in a culture medium, wherein the usage ratio of plant cells to culture medium is 0.8 g:15-25 mL; and the culture medium is 1 / 2MS culture medium.

[0017] [Beneficial Effects]

[0018] (1) The N-CDs used in the present invention have a significant alleviating effect on plant cell damage caused by salt stress; treatment with N-CDs at a concentration of 0.5 mg / L for 12 h can enhance plant salt tolerance by 14.5%.

[0019] (2) The N-CDs used in the present invention can be used as K + The promoter of signal transduction + The net inflow increased significantly by 297.8%, and the Na + / H + The expression of the transporter-related gene NHX1 (7.8 times) stimulates the plant to partition and passivate salt and store it in the cells, thereby improving the salt tolerance of plant cells.

[0020] (3) The method of the present invention is suitable for plant cells to cope with long-term salt stress environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The activity changes of BY-2 cells under the treatment of different concentrations of N-CDs in Example 1 and Comparative Examples 1 and 2.

[0022] Figure 2 These are the results of cell activity detection after 24 h exposure to N-CDs in Example 1; (a) is PI / FDA staining to detect cell activity; (b) is TTC to detect cell viability.

[0023] Figure 3 This is the effect of N-CDs on the salt tolerance index of BY-2 cells in Example 1.

[0024] Figure 4 (b) is an enlarged image of the selected area in (a).

[0025] Figure 5 The dynamic time expression results of salt tolerance related genes in cells under N-CDs treatment in Example 1.

[0026] Figure 6 The effects of N-CDs on the intracellular Na (a) and K (b) contents in Example 1 and Comparative Examples 1 and 2 are shown.

[0027] Figure 7 The test results of salt tolerance memory in Example 1, wherein (a) is the salt tolerance memory test process: evaluating N-CDs by TTC determination; (b) is the initiated salt tolerance memory.

[0028] Figure 8 This is the effect of 0.1 mg / L CeO2 large particles on cell activity under salt stress in Comparative Example 3.

[0029] Fig. 9 This is the effect of 1.64 μg / L or 2.66 μg / L CeCl3 on cell activity under salt stress in Comparative Example 4.

[0030] Fig.10 The activity changes of BY-2 cells under treatment with different concentrations of nTiO2 in Comparative Example 5.

[0031] Fig.11 The activity changes of BY-2 cells under treatment with different concentrations of nSe in Comparative Example 6.

[0032] Fig.12 The activity changes of BY-2 cells under treatment with different concentrations of nMnFe2O4 in Comparative Example 7.

[0033] Fig.13 The activity changes of BY-2 cells under treatment with different concentrations of nSiO2 in Comparative Example 8.

[0034] Fig.14 The activity changes of BY-2 cells under treatment with different concentrations of CDs in Comparative Example 9.

[0035] Fig.15 The activity changes of BY-2 cells under treatment with different concentrations of nFe2O3 in Comparative Example 10. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0037] Test method:

[0038] 1. Cell viability test:

[0039] The 2,3,5-triphenyltetrazolium chloride (TTC) method was used to determine the cell viability after different treatments at each time point. The specific steps are as follows:

[0040] For each sample, 1 mL of BY-2 cell solution was placed in a 1.5 mL centrifuge tube and allowed to stand for 10 min. The supernatant was carefully removed and the cells were washed with PBS buffer (0.1 M, pH 7.2). This was repeated three times. 1 mL of TTC solution (0.3% v / v, solvent: 0.05 M PBS, pH 7.2) was added to the cells after the supernatant was removed to resuspend the cells.

[0041] The washed cell samples were placed in the dark at 25°C for 8 h, centrifuged at 6000 rpm and the supernatant was removed. 1 mL of 95% ethanol was added and vortexed to disperse the cells. The cells were placed in a water bath at 60°C for 15 min. After the cells turned white, they were centrifuged at 6000 rpm for 5 min. 200 μL of the supernatant was added to the ELISA plate and the absorbance was measured at 485 nm using an ELISA reader. The CK at the corresponding time point was used as the benchmark for normalization and calculation of cell activity.

[0042] Three biological replicates were set up for the cell activity assay, and the above operations were performed under sterile conditions throughout.

[0043] 2. Test of the proportion of living cells:

[0044] The proportion of live cells was determined by bisfluorescein diacetate (FDA) / propidium iodide (PI) fluorescence staining to further confirm the cell activity under different treatments. The specific steps are as follows:

[0045] At the selected concentration (0.5 mg / L N-CDs) and time point (2, 6, 24 h), BY-2 cells in each treatment group were stained with PI-FDA (the dye concentration was 20 mg / L), and bright field, red fluorescence field (Tx Red), and green fluorescence field (FITC) photos were taken using a fluorescence microscope (100 cells were ensured in each field of view). The number of live and dead cells was counted (NISViewer and imageJ software), and the proportion of live cells was calculated.

[0046] 3. Salt resistance test:

[0047] After adding N-CDs to plant cells under salt stress, 14 mL of BY-2 cell suspension was taken from each treatment group at 2, 4, 6, 12, 24, 48, and 72 h and allowed to stand for 20 min. 4 mL of culture supernatant was taken for conductivity measurement. The remaining part was filtered to remove the culture medium, washed with PBS buffer (0.1 M, pH 7.2) and filtered to determine the fresh weight (Fresh Weight, FW), and the cells were dried at 60℃ for 48 h to determine the dry weight (Dry Weight, DW).

[0048] The salt tolerance index (STI) was calculated as follows: STI (%) = (total dry weight under salt stress or total dry weight under salt stress with ENMs added / total dry weight of control group) × 100.

[0049] 4. Distribution and characterization of nanomaterials in cells:

[0050] Observation was performed using TEM.

[0051] 5. Characterization of Nanomaterials:

[0052] N-CDs were prepared into a 10 mg / L aqueous solution using Milli-Q ultrapure water and subjected to low-temperature water bath ultrasound for 30 min. 20 μL of the suspension was dropped onto a copper grid and allowed to air dry naturally. The particle size and morphology of N-CDs were characterized by transmission electron microscopy (TEM), and the particle sizes of the two materials were calculated using Nano Measurer.

[0053] 6. Dynamic changes of genes related to salt stress tolerance signaling pathway:

[0054] Real-time fluorescence quantitative PCR was used to analyze the expression of salt-responsive genes. The specific steps included:

[0055] After BY-2 cells were exposed to N-CDs under salt stress, the BY-2 cell culture medium at each time point of 2, 4, 6, 12, 24, 48, and 72 h was filtered out, washed with PBS and filtered dry, and then fully ground and total RNA was extracted from BY-2 cells using a plant RNA extraction kit;

[0056] The concentration and quality of the extracted total RNA were determined using Nanodrop, and cDNA was synthesized using reverse transcription reagents. EF1α cDNA was used as a template, fluorescent dyes and corresponding primers were added, and PCR amplification was performed using a fluorescent quantitative PCR detection system (the reaction program was: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing / extension at 60°C for 1 min, repeated 40 cycles; melting curve analysis was performed using the default program of the PCR instrument, and relative gene expression analysis was performed based on the Ct value. The comparative method (2 -ΔΔCT ) All qPCR results were analyzed by relative quantification.

[0057] 7. Test of intracellular Ca, Na and K content:

[0058] Determination of Na using NMT + , Ca 2+ and K + The specific steps include:

[0059] A sensor with a tip diameter of 4.5 ± 0.5 μm was used, and a filling solution (Na + :250 mM NaCl, K + : 100 mM KCl, Ca 2+ : 100 mM CaCl2), and about 50 μm (measurement of K + 180 μm) LIX reagent (LIXs, Na + :XY-SJ-Na-10,K + :XY-SJ-K-10,Ca 2+ :XY-SJ-Ca-10), calibrated with corresponding calibration solution, Nernst slope was 58±5 (determination of divalent cation Ca 2+ The test can be started when the flow rate is within the range of 29±3.

[0060] 1 mL of cell suspension was placed on the surface of a special glass slide, fixed for 10 min, 5 mL of test solution was added, and the test solution was replaced after stabilization for 10 min. The sensor tip was placed on the cell surface at the position closest to the cell nucleus and maintained at a distance of about 1-2 μm. The step size for each measurement was 20 μm. At least 7 biological replicates were set for each treatment, and the results were expressed as mean ± standard error. Flux data were processed using imFluxes V2.2 software.

[0061] 8. Salt tolerance memory test:

[0062] BY-2 cells exposed to N-CDs for 24 h under salt stress were removed from the original culture medium (ENMs were filtered out) and transferred to new salt-containing culture medium. The cell activity was continuously measured at 7 time points: 2, 4, 6, 12, 24, 48, and 72 h using the TTC method.

[0063] Parameters of each raw material used in the embodiment:

[0064] Tobacco BY-2 cells were obtained from the College of Life Sciences, Shandong Agricultural University, and were continuously subcultured in this experiment;

[0065] The particle size of N-CDs is 2.5±0.4 nm. It is prepared by thermal polymerization of melamine and ethylenediamine. Specifically, ethylenediamine and melamine are mixed in a molar ratio of 2:1 and calcined at high temperature (650°C) in a muffle furnace for 6 h. The resulting solution is dialyzed through a dialysis bag (molecular weight = 500 Da) for 48 h for purification and then freeze-dried.

[0066] Example 1

[0067] The application of nitrogen-doped carbon dots in stimulating plant cells to compartmentalize salt and improve salt tolerance includes the following steps:

[0068] In a clean bench, filter the old culture medium with a 200-mesh cell sieve and weigh 0.8 g of tobacco BY-2 suspension cells in the exponential growth phase ( Nicotiana tabacum L. cv. Bright Yellow 2 suspension cell) in 20 mL 1 / 2MS medium, the initial concentration of fresh weight of cells in the conical flask was controlled to be 40 g / L, and a plant cell solution was obtained; then sodium chloride was added thereto to make the concentration of sodium chloride 100 mM, and the solution was adapted for 24 h to obtain a salt-stressed tobacco BY-2 cell solution;

[0069] Different amounts of 2 mg / L N-CDs aqueous solution were added to the salt-stressed tobacco BY-2 cell solution, so that the concentration of N-CDs in the tobacco BY-2 cell solution was 0.05 mg / L, 0.1 mg / L, and 0.5 mg / L; the culture was continued for 2-72 h.

[0070] Comparative Example 1 Control group (CK)

[0071] The addition of 100 mM sodium chloride and N-CDs in Example 1 was omitted, and the culture was carried out in the same manner as in Example 1.

[0072] Comparative Example 2 S treatment group (S)

[0073] The addition of N-CDs in Example 1 was omitted, and the other steps were kept the same as in Example 1 for culturing.

[0074] The solutions after 2, 4, 6, 12, 24, 48, and 72 h of culture were taken for cell viability test. The test results are as follows:

[0075] Figure 1 The activity changes of BY-2 cells under the treatment of different concentrations of N-CDs. Figure 1 It can be seen that after applying N-CDs to BY-2 cells under salt stress, 0.5 mg / L N-CDs can improve the activity of BY-2 cells under salt stress at multiple time points, and significantly increased at 24h (increase of 8.8%), indicating that 0.5 mg / L N-CDs has the effect of enhancing the salt tolerance of BY-2 cells.

[0076] Figure 2 The results of the detection of cell activity after 24h exposure to N-CDs in Example 1 are as follows: Figure 2 It can be seen that 0.5 mg / L N-CDs restored cell viability by 4.0%.

[0077] Figure 3 The effect of N-CDs on the salt tolerance index of BY-2 cells is shown in Figure 2. Figure 3 It can be seen that after 12 h of treatment with 0.5 mg / L N-CDs, its salt tolerance was significantly improved (14.5%).

[0078] Figure 4 (a) shows the N-CDs in BY-2 cells after exposure for 48 h in Example 1. (b) shows the enlarged image of the selected area in (a). Figure 4 It can be seen that N-CDs are absorbed by cells and enter the cell fluid.

[0079] Figure 5 The dynamic temporal expression results of salt tolerance related genes in cells under N-CDs treatment. Figure 5 It can be seen that after 6 h of exposure to N-CDs, NHX1 The expression of Na + Vacuole compartmentalization may be the main salt tolerance mechanism driven by N-CDs.

[0080] Figure 6 The effect of N-CDs on the intracellular Na (a) and K (b) contents is shown in Table 1. Figure 6 It can be seen that after cells were exposed to N-CDs, K + The influx increased significantly (2.98-fold) after 24 h, while the intracellular K content remained unchanged.

[0081] Figure 7The test results of salt tolerance memory, where (a) is the salt tolerance memory test process; N-CDs are evaluated by TTC determination; (b) the initiated salt tolerance memory; Figure 7 It can be seen that after 6 h under salt stress, the viability of cells pretreated with N-CDs increased by 31.6%, and under relatively long-term exposure conditions (24-72 h), this salt tolerance memory was significantly improved by 102.3-229.2%, indicating that N-CDs pretreatment enhanced the acquired salt tolerance of plant cells.

[0082] Comparative Example 3

[0083] The addition of N-CDs in Example 1 was omitted, and a solution of large CeO2 particles (<5 μm) with a concentration of 2 mg / L was added to the tobacco BY-2 cell solution; the concentration of the large CeO2 particles in the tobacco BY-2 cell solution was 0.1 mg / L, and the other conditions were kept consistent with Example 1 for culturing.

[0084] The solutions after 2, 4, 6, 12, 24, 48, and 72 h of culture were used for cell viability test. The test results were as follows: Figure 8 :

[0085] from Figure 8 It can be seen that 0.1 mg / L CeO2 large particles did not improve cell activity under salt stress.

[0086] Comparative Example 4

[0087] The addition of N-CDs in Example 1 was omitted, and a CeCl3 solution with a concentration of 2 mg / L was added to the tobacco BY-2 cell solution, so that the concentration of CeCl3 in the tobacco BY-2 cell solution was 1.64 μg / L and 2.66 μg / L. The other conditions were kept consistent with Example 1 and culture was performed.

[0088] The solutions after 2, 4, 6, 12, 24, 48, and 72 h of culture were used for cell viability test. The test results were as follows: Fig. 9 :

[0089] from Fig. 9 It can be seen that CeCl3 did not improve cell activity under salt stress.

[0090] Comparative Example 5

[0091] The N-CDs in Example 1 were adjusted to nTiO2 (particle size <25 nm, purchased from Sigma-Aldrich Company), and the concentrations of nTiO2 in the tobacco BY-2 cell solution were 0.05, 0.1, and 0.5 mg / L; the other conditions were kept consistent with Example 1 and culture was performed.

[0092] The solutions after 2, 4, 6, 12, 24, 48, and 72 h of culture were used for cell viability test. The test results were as follows: Fig.10 :

[0093] from Fig.10 It can be seen that the effect of nTiO2 in improving cell activity under salt stress is not as good as that of N-CDs.

[0094] Comparative Example 6

[0095] The N-CDs in Example 1 were adjusted to nSe (particle size of ~70 nm, prepared by room temperature solid phase reaction synthesis, the specific method is referenced to the literature (Wang Runxia, ​​Zhang Shengyi, Liu Mingzhu. Ascorbic acid reduction method, preparation of nano-selenium by room temperature solid phase reaction [J]. Journal of Anhui Normal University (Natural Science Edition), 2004(03): 302-305.)), the concentration of nSe in the tobacco BY-2 cell solution was 0.05, 0.1, and 0.5 mg / L; the rest was kept consistent with Example 1 and cultured.

[0096] The solutions after 2, 4, 6, 12, 24, 48, and 72 h of culture were used for cell viability test. The test results were as follows: Fig.11 :

[0097] from Fig.11 It can be seen that the effect of nSe in improving cell activity under salt stress is not as good as that of N-CDs.

[0098] Comparative Example 7

[0099] The N-CDs in Example 1 were adjusted to nMnFe2O4 (particle size of 20-40 nm, prepared by hydrothermal synthesis, for the specific preparation method, see (Tombuloglu H, Tombuloglu G, Slimani Y, et al. Impact of manganeseferrite (MnFe2O4) nanoparticles on growth and magnetic character of barley(Hordeum vulgare L.)[J]. Environmental Pollution, 2018, 243(Part B): 872-881.)), and the concentration of nMnFe2O4 in the tobacco BY-2 cell solution was 0.05, 0.1, and 0.5 mg / L; the rest was kept consistent with Example 1 for culture.

[0100] The solutions after 2, 4, 6, 12, 24, 48, and 72 h of culture were used for cell viability test. The test results were as follows: Fig.12 :

[0101] from Fig.12 It can be seen that nMnFe2O4 has no obvious effect on improving cell activity under salt stress.

[0102] Comparative Example 8

[0103] The N-CDs in Example 1 were adjusted to nSiO2 (particle size of 8 nm, purchased from US Research Nanomaterials), and the concentrations of nSiO2 in the tobacco BY-2 cell solution were 0.05, 0.1, and 0.5 mg / L; the other conditions were kept consistent with Example 1 and culture was performed.

[0104] The solutions after 2, 4, 6, 12, 24, 48, and 72 h of culture were used for cell viability test. The test results were as follows: Fig.13 :

[0105] from Fig.13 It can be seen that nSiO2 has no obvious effect on improving cell activity under salt stress.

[0106] Comparative Example 9

[0107] The N-CDs in Example 1 were adjusted to CDs (particle size <10 nm, and the specific preparation method was referred to in the literature (Cheng B, Chen F, Wang C, et al. The molecular mechanisms of silica nanomaterials senhancing the rice ( Oryza sativa L. ) resistance to planthoppers ( Nilaparvata lugens Stal )[J]. Science of the Total Environment, 2021, 767: 144967.) The concentrations of CDs in the tobacco BY-2 cell solution were 0.05, 0.1, and 0.5 mg / L; the other conditions were kept consistent with those in Example 1 for culture.

[0108] The solutions after 2, 4, 6, 12, 24, 48, and 72 h of culture were used for cell viability test. The test results were as follows: Fig.14 :

[0109] from Fig.14 It can be seen that CDs have no obvious effect on improving cell activity under salt stress.

[0110] Comparative Example 10

[0111] The N-CDs in Example 1 were adjusted to nFe2O3 (particle size of 5 nm, purchased from US Research Nanomaterials), and the concentrations of nFe2O3 in the tobacco BY-2 cell solution were 0.05, 0.1, and 0.5 mg / L; the other conditions were kept consistent with Example 1 and culture was performed.

[0112] The solutions after 2, 4, 6, 12, 24, 48, and 72 h of culture were used for cell viability test. The test results were as follows: Fig.15 :

[0113] from Fig.15 It can be seen that nFe2O3 has no obvious effect on improving cell activity under salt stress.

[0114] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of nitrogen-doped carbon dots in stimulating plant cells to compartmentalize salt and improve salt tolerance, characterized in that: The application is to add N-CDs solution into plant cell solution.

2. The use according to claim 1, characterized in that: The concentration of the N-CDs in the plant cell solution is 0.05-0.5 mg / L.

3. The use according to claim 1, characterized in that: The N-CDs are prepared by thermal polymerization of melamine and ethylenediamine.

4. The use according to claim 3, characterized in that: The specific preparation method of the N-CDs is as follows: ethylenediamine and melamine are mixed and calcined at 650° C. in a muffle furnace; the obtained solution is then dialyzed and purified, and finally freeze-dried to obtain the N-CDs.

5. The use according to claim 4, characterized in that The molar ratio of ethylenediamine to melamine is 2:1; the high temperature calcination is calcination at 650°C for 6 hours; and the dialysis purification is performed using a dialysis bag with a molecular weight of 500 Da.

6. The use according to claim 1, characterized in that: The plant cell comprises one of tobacco BY-2, corn cell and rice cell.

7. The use according to claim 1, characterized in that The particle size of the N-CDs is 1-3 nm.

8. The use according to claim 1, characterized in that: The solvent of the N-CDs solution is water, and the concentration is 2-10 mg / L.

9. The use according to claim 1, characterized in that: The plant cell solution is obtained by placing plant cells in a culture medium.

10. The use according to claim 9, characterized in that The dosage ratio of the plant cells to the culture medium is 0.8 g:15-25 mL; the culture medium is 1 / 2MS culture medium.

Images