A method for observing the growth behavior of pollen tubes in macadamia

Abstract

The present invention relates to the field of biotechnology, and in particular to a method for observing the growth behavior of pollen tubes of macadamia nuts. Specifically, the method comprises the following steps: (1) immersing the pollinated pistil in a fixing solution for fixing; (2) soaking the pistil in an alcohol solution and then washing it; (3) mixing the pistil with a NaOH solution to soften it, washing it, and absorbing the water; (4) soaking the pistil in a phosphate buffer solution; (5) cross-cutting the pistil at the middle position to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cutting it into two halves longitudinally, and soaking it in a phosphate buffer solution again; (6) staining the pistil with a dye after absorbing the water, and microscopically examining it. The fluorescence microscopy method of the present invention greatly weakens the phenomenon of luminescence of the pistil's own tissue, and it is easy to obtain a high-definition image of the pollen tube growing in the pistil. The method has an ideal presentation effect on the growth behavior of the pollen tube, and can be used for the hybridization affinity evaluation between macadamia nut varieties, and has high practical application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for observing the growth behavior of macadamia nut pollen tubes. Background Art

[0002] As an important carrier of male individuals in the process of sexual reproduction in plants, pollen tubes have typical apical growth characteristics and play an important role in cell-to-cell interactions and signal transduction. The growth behavior of pollen tubes in the pistil can accurately present the affinity and incompatibility of hybridization and self-pollination, and is an important means to study the fertilization process of plants.

[0003] The pistil of macadamia is thin and long, the stigma is small and narrow, and the pollen tube diameter is less than 10μm, which requires observation under a 100x microscope. Although the pistil is soft, the epidermal cells of the pistil are as hard as nut shells, and it is extremely difficult to soften and make them transparent. Many techniques have been tried to no avail. It must be cross-sectioned and then longitudinally cut to observe the growth behavior of the pollen tube under a microscope. The frozen section method and paraffin section method are complicated and difficult to operate, and it is impossible to observe a large number of pollinated pistils in a short period of time. The vascular tissue of macadamia is well developed, and the style guide tissue and other tissues spontaneously fluoresce strongly, which interferes with the observation of pollen tubes. Some self-pollinated styles cannot even distinguish between pollen tubes and other style tissues.

[0004] The unique callose in the pollen tube combines with the fluorescent pigment in aniline blue to stimulate bright blue-green fluorescence under ultraviolet light. The study of pollen tube growth behavior based on this principle has been extensively and deeply studied in many plants. However, almost no research has been conducted on the growth behavior of macadamia pollen tubes in the pistil, resulting in a serious lag in the growth process of macadamia pollen tubes in the pistil and related biological research.

[0005] Therefore, how to provide a method for observing the growth behavior of pollen tubes that is easy to observe the growth behavior within the pistil, so as to solve the technical problem that the prior art cannot quickly, accurately and mass-produce macadamia pollen tube slides, is an issue that technical personnel in this field urgently need to solve. Summary of the invention

[0006] The purpose of the present invention is to provide a method for observing the growth behavior of macadamia nut pollen tubes. The purpose of the present invention is to solve the problem that the growth behavior of macadamia nut pollen tubes in the pistil is difficult to observe, to overcome the defect that the prior art cannot quickly, clearly and in large quantities observe the in situ growth behavior of macadamia nut pollen tubes, and to provide a method for observing the growth behavior of macadamia nut pollen tubes.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for observing the growth behavior of macadamia pollen tubes, comprising the following steps:

[0009] (1) Immerse the pollinated pistil in a fixative solution for fixation;

[0010] (2) soaking the pistil obtained in step (1) in an alcohol solution, and then washing it to obtain a washed pistil;

[0011] (3) mixing the washed pistil obtained in step (2) with a NaOH solution to soften, wash, and absorb moisture;

[0012] (4) soaking the pistil obtained in step (3) in a phosphate buffer solution;

[0013] (5) cutting the pistil obtained in step (4) in half at the middle to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cutting it in half longitudinally, and soaking it in phosphate buffer again;

[0014] (6) drying the pistil obtained in step (5), staining it with a staining agent, and examining it under a microscope;

[0015] The pistil softening time in step (3) is different at different pollination periods: 1 to 3 days after pollination, the softening time is 2 hours; 4 to 7 days after pollination, the softening time is 3 hours; 8 to 12 days after pollination, the softening time is 4 hours.

[0016] Preferably, the fixing solution in step (1) comprises alcohol solution, glacial acetic acid and formaldehyde; the volume ratio of the alcohol solution, glacial acetic acid and formaldehyde is 84-94:4-8:3-7; and the volume fraction of the alcohol solution is 55-65%.

[0017] Preferably, in step (1), the volume ratio of the fixing solution to the pistil is 15 to 25:1; and the fixing time is 42 to 54 hours.

[0018] Preferably, the concentration of the NaOH solution in step (3) is 1.5-2.5 mol / L; and the volume ratio of the NaOH solution to the pistil is 35-45:1.

[0019] Preferably, the softening temperature in step (3) is 28-32°C.

[0020] Preferably, the dye in step (6) is a mixture of aniline blue and potassium dihydrogen phosphate; the mass fraction of the aniline blue is 0.4-0.6%; the mass fraction of the potassium dihydrogen phosphate is 0.005-0.015%; and the pH of the dye is 9.8-10.2.

[0021] Preferably, in step (6), the dyeing time of the pistil at different pollination stages is different: 1 to 3 days after the pollination of the pistil, the dyeing time is 7 to 10 minutes; 4 to 12 days after the pollination of the pistil, the dyeing time is 3 to 5 minutes.

[0022] Preferably, in step (2), the pistil is soaked in alcohol solutions with volume fractions of 55-65%, 35-45%, and 15-25% in sequence; the soaking time of the pistil in each concentration of alcohol solution is independently 8-12 minutes.

[0023] Preferably, the washing time in step (2) is 25 to 35 minutes; the washing times in step (3) are 2 to 4 times, and the washing time for each time is 8 to 12 minutes.

[0024] Preferably, the pH of the phosphate buffer in steps (5) and (6) is independently 6.9 to 7.1; the volume ratio of the phosphate buffer to the pistil is independently 8 to 12:1; and the water absorption in steps (3) and (6) is to place the pistil on absorbent paper for 10 to 70 seconds.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1) The fluorescence microscopy method of the present invention greatly weakens the phenomenon of luminescence of the pistil's own tissue, and it is easy to obtain high-definition images of the pollen tube growing in the pistil. The presentation effect of the pollen tube growth behavior is very ideal, and it can be used for hybridization affinity evaluation between macadamia nut varieties, and has high practical application value.

[0027] 2) The present invention improves the formula of FAA fixative according to the characteristics of macadamia nut style epidermal cells, and changes the alcohol concentration in the fixative to 60%, which greatly changes the fixation effect of the material to be tested and effectively eliminates the influence of the style tissue autofluorescence. The present invention uses 60% ethanol by volume, and the softening and staining effects are better. When the ethanol concentration is high, the material is severely dehydrated, and when the concentration is low, the water in the material is not completely dehydrated, which may affect the softening and staining effects.

[0028] 3) According to the different stages of the materials to be tested, the present invention sets the NaOH solution to 2 mol / L, and the softening time is also fixed within 2 to 4 hours according to the pistils at different stages, which greatly shortens the time for processing the materials, enables the materials to be tested to be softened quickly while the internal tissue of the pistil is not damaged, and ensures the integrity of the materials.

[0029] 4) According to the properties of the style guiding tissue, vascular bundle and other tissues in the pistil of macadamia nut, the present invention first adjusts the pH value of the material to be tested to neutral, and then improves the ratio, concentration, pH value of the dyeing liquid reagent, and the dyeing time of different materials to be tested to achieve the best conditions for high-definition pollen tubes under a fluorescence microscope. The present invention greatly weakens the autofluorescence of the style tissue, making the yellow-green fluorescence of the pollen tube stand out.

[0030] 5) The present invention is simple, rapid and accurate to operate. The growth behavior of the pollen tubes of macadamia nuts in the pistil can be observed by the freehand slicing method. Moreover, the pollen tubes of a large number of pistils can be observed in a short period of time. This method has important application value in the study of reproductive biology of crops with large flower quantity and low fruit setting rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0032] Figure 1 This is a flowchart of the fluorescence microscopic observation of the growth behavior of macadamia pollen tubes in the pistil.

[0033] Figure 2 (AC) is the growth of macadamia pollen tube in the pistil of comparative example 1 treated by traditional method (100 times). Figure A spontaneously emits red fluorescence (pistil 3 days after pollination, dyed for 7 minutes); Figure B (pistil 5 days after pollination, dyed for 5 minutes) and Figure C (pistil 5 days after pollination, dyed for 5 minutes). The pollen tube and style tissue both emit strong fluorescence and are difficult to distinguish.

[0034] Figure 3 (AB) shows the growth of pollen tubes in the pistil of macadamia nuts in comparative example 1 treated by the traditional method (100 times). In Figure A, the epidermis of the style is fragile, the vascular bundles emit strong fluorescence, and the pollen tubes are difficult to distinguish; in Figure B, the internal tissues of the style are separated and chaotic.

[0035] Figure 4 (AD) is the growth of pollen tubes in the pistil of macadamia nuts using the fluorescence microscopy method of the present invention (100 times), and Figures A-D are the growth of pollen tubes in different parts of the pistil at different stages after pollination. The pollen tube emits strong blue-green fluorescence, while other tissues hardly emit light, and the boundaries between the pollen tube and other tissues of the style are clear. Figure 4 A (Example 4: pistil 7 days after pollination, dyed for 5 minutes) and B (Example 3: pistil 5 days after pollination, dyed for 5 minutes) are the growth behaviors of the pollen tube of the pistil stigma. Figure 4 C in the figure (Example 5: pistil 9 days after pollination, dyed for 3 minutes) is the growth behavior of the pollen tube in the middle of the pistil; Figure 4 D in the figure (Example 6: pistil 12 days after pollination, dyed for 3 minutes) is the growth of pollen tubes in the lower half of the pistil and the ovary. DETAILED DESCRIPTION

[0036] The present invention provides a method for observing the growth behavior of macadamia pollen tubes, comprising the following steps:

[0037] (1) Immerse the pollinated pistil in a fixative solution for fixation;

[0038] (2) soaking the pistil obtained in step (1) in an alcohol solution, and then washing it to obtain a washed pistil;

[0039] (3) mixing the washed pistil obtained in step (2) with a NaOH solution to soften, wash, and absorb moisture;

[0040] (4) soaking the pistil obtained in step (3) in a phosphate buffer;

[0041] (5) cutting the pistil obtained in step (4) in half at the middle to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cutting it in half longitudinally, and soaking it in phosphate buffer again;

[0042] (6) drying the pistil obtained in step (5), staining it with a staining agent, and examining it under a microscope;

[0043] In the present invention, the pistil softening time in step (3) is different at different pollination periods: 1 to 3 days after pollination, the softening time is 2 hours; 4 to 7 days after pollination, the softening time is 3 hours; 8 to 12 days after pollination, the softening time is 4 hours.

[0044] In the present invention, the fixing solution in step (1) comprises an alcohol solution, glacial acetic acid and formaldehyde;

[0045] In the present invention, the volume ratio of the alcohol solution, glacial acetic acid and formaldehyde in step (1) is 84-94:4-8:3-7; preferably 86-92:5-7:4-6; further preferably 88-90:6:5; more preferably 89:6:5.

[0046] In the present invention, the volume fraction of the alcohol solution in step (1) is 55-65%; preferably 57-63%; more preferably 59-61%; and more preferably 60%.

[0047] In the present invention, the volume ratio of the fixing solution to the pistil in step (1) is 15 to 25:1; preferably 17 to 23:1; more preferably 19 to 21:1; and more preferably 20:1.

[0048] In the present invention, the fixing time in step (1) is 42 to 54 hours, preferably 44 to 52 hours, more preferably 46 to 50 hours, and more preferably 48 hours.

[0049] In the present invention, the concentration of the NaOH solution in step (3) is 1.5 to 2.5 mol / L; preferably 1.7 to 2.3 mol / L; more preferably 1.9 to 2.1 mol / L; and more preferably 2 mol / L.

[0050] In the present invention, the volume ratio of the NaOH solution to the pistil in step (3) is 35-45:1; preferably 37-43:1; more preferably 39-41:1; and more preferably 40:1.

[0051] In the present invention, the softening temperature in step (3) is 28-32°C, preferably 29-31°C, and more preferably 30°C.

[0052] In the present invention, the dye in step (6) is a mixture of aniline blue and potassium dihydrogen phosphate.

[0053] In the present invention, the mass fraction of the aniline blue in step (6) is 0.4-0.6%, preferably 0.5%.

[0054] In the present invention, the mass fraction of potassium dihydrogen phosphate in step (6) is 0.005-0.015%; preferably 0.007-0.013%; more preferably 0.009-0.011%; and more preferably 0.01%.

[0055] In the present invention, the pH of the dye in step (6) is 9.8 to 10.2; preferably 9.9 to 10.1; and more preferably 10.

[0056] In the present invention, the dyeing time of the pistil in step (6) is different at different pollination stages. 1 to 3 days after the pollination of the pistil, the dyeing time is 7 to 10 minutes, preferably 8 to 9 minutes, and more preferably 9 minutes.

[0057] In the present invention, in step (6), 4 to 12 days after the pistil is pollinated, the dyeing time is 3 to 5 minutes, preferably 4 minutes.

[0058] In the present invention, in step (2), the pistil is soaked in alcohol solutions with volume fractions of 55-65%, 35-45%, and 15-25% in sequence; preferably, the pistil is soaked in alcohol solutions with volume fractions of 57-63%, 37-43%, and 17-23% in sequence; further preferably, the pistil is soaked in alcohol solutions with volume fractions of 59-61%, 39-41%, and 19-21% in sequence; and more preferably, the pistil is soaked in alcohol solutions with volume fractions of 60%, 40%, and 20% in sequence.

[0059] In the present invention, the time for the pistil to be immersed in each concentration of alcohol solution is independently 8 to 12 minutes; preferably 9 to 11 minutes; and more preferably 10 minutes.

[0060] In the present invention, the washing time in step (2) is 25 to 35 minutes, preferably 27 to 33 minutes, more preferably 29 to 31 minutes, and more preferably 30 minutes.

[0061] In the present invention, the washing time in step (3) is 8 to 12 minutes, preferably 9 to 11 minutes, and more preferably 10 minutes.

[0062] In the present invention, the washing in step (3) is performed 2 to 4 times, preferably 3 times.

[0063] In the present invention, the pH of the phosphate buffer in steps (5) and (6) is independently 6.9 to 7.1, preferably 7.

[0064] In the present invention, the volume ratio of the phosphate buffer to the pistil in steps (5) and (6) is independently 8 to 12:1; preferably 9 to 11:1; and more preferably 10:1.

[0065] In the present invention, the drying of water in steps (3) and (6) is performed by placing the pistil on absorbent paper for 10 to 70 seconds, preferably 30 to 50 seconds, and more preferably 40 seconds.

[0066] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0067] Example 1

[0068] A method for observing the growth behavior of macadamia pollen tubes, comprising the following steps:

[0069] (1) Pistil collection: The pistil 1 day after pollination was immersed in fixative for 42 hours; the volume ratio of fixative to pistil was 15:1;

[0070] (2) washing: soaking the pistil obtained in step (1) in alcohol solutions with volume fractions of 65%, 45%, and 25% in sequence for 8 minutes at each concentration, and then soaking and washing in 500 mL of pure water for 25 minutes;

[0071] (3) Softening: The pistil obtained in step (2) is mixed with a 1.5 mol / L NaOH solution, added to a 100 mL silk-mouth bottle and placed in a water bath at 28° C. for 2 h, then the pistil is placed in a 500 mL beaker, soaked and washed twice with pure water, each washing for 8 min, and the pistil is placed on absorbent paper for 60 s; the volume ratio of the NaOH solution to the pistil is 35:1.

[0072] (4) adjusting the pH value of the material: soaking the pistil obtained in step (3) in a phosphate buffer having a pH of 6.9 for 0.5 h; the volume ratio of the phosphate buffer to the pistil being 8:1;

[0073] (5) Manual slicing: the pistil obtained in step (4) is cross-cut at the middle position to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cut into two halves longitudinally, and then immersed in a phosphate buffer with a pH of 6.9 for 0.5 h; the volume ratio of the phosphate buffer to the pistil is 8:1;

[0074] (6) Staining: placing the pistil obtained in step (5) on absorbent paper for 20 seconds, and staining with a stain (a mixture of 0.4% aniline blue and 0.005% potassium dihydrogen phosphate, pH 9.8) for 7 minutes;

[0075] (7) Fluorescence microscopic observation: The stained pistil was taken out and placed on absorbent paper for 20 seconds to remove excess dye. The pistil was then placed on a glass slide and 70% glycerol was added. The slide was pressed with a cover glass. To maintain the integrity of the material, it was not pressed hard. The ovary and stigma were separately prepared and observed under a fluorescence microscopic microscope under ultraviolet light.

[0076] The fixing solution in step (1) comprises an alcohol solution with a volume fraction of 55%, glacial acetic acid and formaldehyde; the volume ratio of the alcohol solution, glacial acetic acid and formaldehyde is 84:4:3.

[0077] Example 2

[0078] A method for observing the growth behavior of macadamia pollen tubes, comprising the following steps:

[0079] (1) Pistil collection: The pistil 12 days after pollination was immersed in fixative for 54 hours; the volume ratio of fixative to pistil was 25:1;

[0080] (2) washing: soaking the pistil obtained in step (1) in alcohol solutions with volume fractions of 55%, 35%, and 15% in sequence for 12 minutes at each concentration, and then soaking and washing in 500 mL of pure water for 25 to 35 minutes;

[0081] (3) Softening: The pistil obtained in step (2) is mixed with a 2.5 mol / L NaOH solution, added to a 100 mL silk-mouth bottle and placed in a water bath at 32° C. for 4 h, then the pistil is placed in a 500 mL beaker, soaked and washed with purified water for 4 times, each washing for 12 min, and the pistil is placed on absorbent paper for 60 s; the volume ratio of the NaOH solution to the pistil is 45:1.

[0082] (4) adjusting the pH value of the material: soaking the pistil obtained in step (3) in a phosphate buffer with a pH of 7.1 for 1.5 hours; the volume ratio of the phosphate buffer to the pistil is 12:1;

[0083] (5) Manual slicing: the pistil obtained in step (4) is cross-cut at the middle position to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cut into two halves longitudinally, and then immersed in a phosphate buffer with a pH of 7.1 for 1.5 hours; the volume ratio of the phosphate buffer to the pistil is 12:1;

[0084] (6) staining: placing the pistil obtained in step (5) on absorbent paper for 60 seconds, and staining with a stain (a mixture of 0.6% aniline blue and 0.015% potassium dihydrogen phosphate, pH 10.2) for 5 minutes;

[0085] (7) Fluorescence microscopic observation: The stained pistil was taken out and placed on absorbent paper for 20 seconds to remove excess dye. The pistil was then placed on a glass slide and 70% glycerol was added. The slide was pressed with a cover glass. To maintain the integrity of the material, it was not pressed hard. The ovary and stigma were separately prepared and observed under a fluorescence microscopic microscope under ultraviolet light.

[0086] The fixing solution in step (1) comprises an alcohol solution with a volume fraction of 65%, glacial acetic acid and formaldehyde; the volume ratio of the alcohol solution, glacial acetic acid and formaldehyde is 94:8:7.

[0087] Example 3

[0088] A method for observing the growth behavior of macadamia pollen tubes, comprising the following steps:

[0089] (1) Pistil collection: The pistil 5 days after pollination was immersed in fixative for 48 hours; the volume ratio of fixative to pistil was 20:1;

[0090] (2) washing: soaking the pistil obtained in step (1) in alcohol solutions with volume fractions of 60%, 40%, and 20% in sequence, soaking for 10 minutes at each concentration, and then soaking and washing in 500 mL of pure water for 30 minutes;

[0091] (3) Softening: The pistil obtained in step (2) is mixed with a 2 mol / L NaOH solution, added to a 100 mL silk-mouth bottle and placed in a water bath at 30° C. for 3 h, then the pistil is placed in a 500 mL beaker, soaked and washed with purified water for 3 times, each washing for 10 min, and the pistil is placed on absorbent paper for 60 s; the volume ratio of the NaOH solution to the pistil is 40:1.

[0092] (4) adjusting the pH value of the material: soaking the pistil obtained in step (3) in a phosphate buffer with a pH of 7 for 1 hour; the volume ratio of the phosphate buffer to the pistil is 10:1;

[0093] (5) Manual slicing: the pistil obtained in step (4) is cross-cut at the middle position to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cut into two halves longitudinally, and then immersed in a phosphate buffer with a pH of 7 for 1 hour; the volume ratio of the phosphate buffer to the pistil is 10:1;

[0094] (6) staining: placing the pistil obtained in step (5) on absorbent paper for 20 seconds, and staining with a stain (a mixture of 0.5% aniline blue and 0.01% potassium dihydrogen phosphate, pH 10) for 5 minutes;

[0095] (7) Fluorescence microscopic observation: The stained pistil was taken out and placed on absorbent paper for 20 seconds to remove excess dye. The pistil was then placed on a glass slide and 70% glycerol was added. The slide was pressed with a cover glass. To maintain the integrity of the material, it was not pressed hard. The ovary and stigma were separately prepared and observed under a fluorescence microscopic microscope under ultraviolet light.

[0096] The fixing solution in step (1) comprises an alcohol solution with a volume fraction of 60%, glacial acetic acid and formaldehyde; the volume ratio of the alcohol solution, glacial acetic acid and formaldehyde is 89:6:5.

[0097] Example 4

[0098] A method for observing the growth behavior of macadamia pollen tubes, comprising the following steps:

[0099] (1) Pistil collection: The pistil 7 days after pollination was immersed in fixative for 48 hours; the volume ratio of fixative to pistil was 20:1;

[0100] (2) washing: soaking the pistil obtained in step (1) in alcohol solutions with volume fractions of 60%, 40%, and 20% in sequence, soaking for 10 minutes at each concentration, and then soaking and washing in 500 mL of pure water for 30 minutes;

[0101] (3) Softening: The pistil obtained in step (2) is mixed with a 2 mol / L NaOH solution, added to a 100 mL silk-mouth bottle and placed in a water bath at 30° C. for 3 h, then the pistil is placed in a 500 mL beaker, soaked and washed with purified water for 3 times, each washing for 10 min, and the pistil is placed on absorbent paper for 60 s; the volume ratio of the NaOH solution to the pistil is 40:1.

[0102] (4) adjusting the pH value of the material: soaking the pistil obtained in step (3) in a phosphate buffer with a pH of 7 for 1 hour; the volume ratio of the phosphate buffer to the pistil is 10:1;

[0103] (5) Manual slicing: the pistil obtained in step (4) is cross-cut at the middle position to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cut into two halves longitudinally, and then immersed in a phosphate buffer with a pH of 7 for 1 hour; the volume ratio of the phosphate buffer to the pistil is 10:1;

[0104] (6) staining: placing the pistil obtained in step (5) on absorbent paper for 20 seconds, and staining with a stain (a mixture of 0.5% aniline blue and 0.01% potassium dihydrogen phosphate, pH 10) for 5 minutes;

[0105] (7) Fluorescence microscopic observation: The stained pistil was taken out and placed on absorbent paper for 20 seconds to remove excess dye. The pistil was then placed on a glass slide and 70% glycerol was added. The slide was pressed with a cover glass. To maintain the integrity of the material, it was not pressed hard. The ovary and stigma were separately prepared and observed under a fluorescence microscopic microscope under ultraviolet light.

[0106] The fixing solution in step (1) comprises an alcohol solution with a volume fraction of 60%, glacial acetic acid and formaldehyde; the volume ratio of the alcohol solution, glacial acetic acid and formaldehyde is 89:6:5.

[0107] Example 5

[0108] A method for observing the growth behavior of macadamia pollen tubes, comprising the following steps:

[0109] (1) Pistil collection: Pistils 9 days after pollination were immersed in fixative for 48 hours; the volume ratio of fixative to pistil was 20:1;

[0110] (2) washing: soaking the pistil obtained in step (1) in alcohol solutions with volume fractions of 60%, 40%, and 20% in sequence, soaking for 10 minutes at each concentration, and then soaking and washing in 500 mL of pure water for 30 minutes;

[0111] (3) Softening: The pistil obtained in step (2) is mixed with a 2 mol / L NaOH solution, added to a 100 mL silk-mouth bottle and placed in a water bath at 30° C. for 4 h, then the pistil is placed in a 500 mL beaker, soaked and washed with purified water for 3 times, each wash for 10 min, and the pistil is placed on absorbent paper for 60 s; the volume ratio of the NaOH solution to the pistil is 40:1.

[0112] (4) adjusting the pH value of the material: soaking the pistil obtained in step (3) in a phosphate buffer with a pH of 7 for 1 hour; the volume ratio of the phosphate buffer to the pistil is 10:1;

[0113] (5) Manual slicing: the pistil obtained in step (4) is cross-cut at the middle position to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cut into two halves longitudinally, and then immersed in a phosphate buffer with a pH of 7 for 1 hour; the volume ratio of the phosphate buffer to the pistil is 10:1;

[0114] (6) staining: placing the pistil obtained in step (5) on absorbent paper for 20 seconds, and staining with a stain (a mixture of 0.5% aniline blue and 0.01% potassium dihydrogen phosphate, pH 10) for 3 minutes;

[0115] (7) Fluorescence microscopic observation: The stained pistil was taken out and placed on absorbent paper for 20 seconds to remove excess dye. The pistil was then placed on a glass slide and 70% glycerol was added. The slide was pressed with a cover glass. To maintain the integrity of the material, it was not pressed hard. The ovary and stigma were separately prepared and observed under a fluorescence microscopic microscope under ultraviolet light.

[0116] The fixing solution in step (1) comprises an alcohol solution with a volume fraction of 60%, glacial acetic acid and formaldehyde; the volume ratio of the alcohol solution, glacial acetic acid and formaldehyde is 89:6:5.

[0117] Example 6

[0118] A method for observing the growth behavior of macadamia pollen tubes, comprising the following steps:

[0119] (1) Pistil collection: The pistil 12 days after pollination was immersed in fixative for 48 hours; the volume ratio of fixative to pistil was 20:1;

[0120] (2) washing: soaking the pistil obtained in step (1) in alcohol solutions with volume fractions of 60%, 40%, and 20% in sequence, soaking for 10 minutes at each concentration, and then soaking and washing in 500 mL of pure water for 30 minutes;

[0121] (3) Softening: The pistil obtained in step (2) is mixed with a 2 mol / L NaOH solution, added to a 100 mL silk-mouth bottle and placed in a water bath at 30° C. for 4 h, then the pistil is placed in a 500 mL beaker, soaked and washed with purified water for 3 times, each wash for 10 min, and the pistil is placed on absorbent paper for 60 s; the volume ratio of the NaOH solution to the pistil is 40:1.

[0122] (4) adjusting the pH value of the material: soaking the pistil obtained in step (3) in a phosphate buffer with a pH of 7 for 1 hour; the volume ratio of the phosphate buffer to the pistil is 10:1;

[0123] (5) Manual slicing: the pistil obtained in step (4) is cross-cut at the middle position to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cut into two halves longitudinally, and then immersed in a phosphate buffer with a pH of 7 for 1 hour; the volume ratio of the phosphate buffer to the pistil is 10:1;

[0124] (6) staining: placing the pistil obtained in step (5) on absorbent paper for 20 seconds, and staining with a stain (a mixture of 0.5% aniline blue and 0.01% potassium dihydrogen phosphate, pH 10) for 3 minutes;

[0125] (7) Fluorescence microscopic observation: The stained pistil was taken out and placed on absorbent paper for 20 seconds to remove excess dye. The pistil was then placed on a glass slide and 70% glycerol was added. The slide was pressed with a cover glass. To maintain the integrity of the material, it was not pressed hard. The ovary and stigma were separately prepared and observed under a fluorescence microscopic microscope under ultraviolet light.

[0126] The fixing solution in step (1) comprises an alcohol solution with a volume fraction of 60%, glacial acetic acid and formaldehyde; the volume ratio of the alcohol solution, glacial acetic acid and formaldehyde is 89:6:5.

[0127] Comparative Example 1: Conventional method

[0128] 1) Pistil collection: Pistils were collected at different time points after pollination and immersed in FAA fixative for 24 h. The volume of FAA fixative was 20 times that of the treated material. The FAA fixative used 70% ethanol solution, glacial acetic acid and formaldehyde in a volume ratio of 89:6:5;

[0129] 2) Washing: Take the pistil from step 1) and transfer it to ultrapure water through various levels of alcohol, with the alcohol concentrations being 70%, 50%, and 30% respectively;

[0130] 3) Softening: put the pistil from step 2) into a 100 mL silk-mouth bottle, add 3 mol / L NaOH solution, place the silk-mouth bottle at room temperature, and take it out after softening for 3 hours;

[0131] 4) Washing: Place the pistil from step 3) in a 500 mL beaker and wash with ultrapure water;

[0132] 5) Manual slicing: Cut the pistil in step 4) horizontally in the middle to separate the stigma in the upper part of the pistil from the ovary in the lower part, and then cut it in half longitudinally. Use a small weighing bottle to place a single pistil separately;

[0133] 6) Staining: Place the pistil cut in step 5) on absorbent paper for 20 seconds and then put it into aniline blue staining solution for staining (staining solution preparation: mass fraction 0.1% aniline blue + 0.01% potassium dihydrogen phosphate), and the staining time is between 1-40 minutes according to different stages of the pistil;

[0134] 7) Fluorescence microscopic observation: Take out the material to be examined after staining in step 7) and place it on absorbent paper for 20 seconds to remove the excess staining solution, then place it on a slide, add 70% glycerol, cover the slide with a glass, prepare slides for the ovary and stigma, and observe the sample under ultraviolet light by fluorescence microscopy ( Figure 2 , Figure 3 ).

[0135] from Figure 2 -A shows that under conventional methods, the chlorophyll in the pistil tissue of macadamia nuts spontaneously emits red fluorescence, which affects the observation effect. Figure 2 -B is the pistil stigma, Figure 2 -C is the middle part of the pistil. Under conventional methods, the internal tissues, vascular bundles, and pollen tubes of the pistil all have strong fluorescence, and the pollen tubes are almost impossible to identify. Figure 3 -A: The epidermis of the style is fragile, the vascular bundles emit strong fluorescence, and the pollen tubes are difficult to distinguish; Figure B: The internal tissues of the style are separated and chaotic. This phenomenon can be caused by too high alcohol concentration of FAA fixative, too high NaOH concentration or too long treatment time.

[0136] Figure 4 (AD) is the growth of pollen tubes in the pistil of macadamia nuts using the fluorescence microscopy method of the present invention (100 times), and Figures A-D are the growth of pollen tubes in different parts of the pistil at different stages after pollination. The pollen tube emits strong blue-green fluorescence, while other tissues hardly emit light, and the boundaries between the pollen tube and other tissues of the style are clear. Figure 4 A (Example 4: pistil 7 days after pollination, dyed for 5 minutes) and B (Example 3: pistil 5 days after pollination, dyed for 5 minutes) are the growth behaviors of the pollen tube of the pistil stigma. Figure 4 C in the figure (Example 5: pistil 9 days after pollination, dyed for 3 minutes) is the growth behavior of the pollen tube in the middle of the pistil; Figure 4 D in (Example 6: pistil 12 days after pollination, dyed for 3 minutes) is the growth of pollen tubes in the lower half of the pistil and the ovary. It can be seen that the present invention greatly weakens the autofluorescence of the style tissue, making the yellow-green fluorescence of the pollen tube stand out.

[0137] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for observing the growth behavior of pollen tubes of macadamia nuts, characterized in that: The steps include: (1) Immerse the pollinated pistil in fixative solution; (2) soaking the pistil obtained in step (1) in an alcohol solution, and then washing it to obtain a washed pistil; (3) mixing the washed pistil obtained in step (2) with a NaOH solution to soften, wash, and absorb moisture; (4) soaking the pistil obtained in step (3) in phosphate buffer; (5) cutting the pistil obtained in step (4) horizontally at the middle position to separate the stigma of the upper half of the pistil from the ovary of the lower half, and then cutting it into two halves longitudinally, and soaking it in phosphate buffer again; (6) drying the pistil obtained in step (5), staining it with a dye, and examining it under a microscope; In step (3), the pistil softening time is different at different pollination periods: 1 to 3 days after pollination, the softening time is 2 hours; 4 to 7 days after pollination, the softening time is 3 hours; 8 to 12 days after pollination, the softening time is 4 hours; The fixing solution in step (1) comprises an alcohol solution, glacial acetic acid and formaldehyde; the volume ratio of the alcohol solution, glacial acetic acid and formaldehyde is 84-94:4-8:3-7; the volume fraction of the alcohol solution is 55-65%; In step (1), the volume ratio of the fixing solution to the pistil is 15 to 25:1; the fixing time is 42 to 54 hours; The concentration of the NaOH solution in step (3) is 1.5-2.5 mol / L; the volume ratio of the NaOH solution to the pistil is 35-45:1; Step (2) soaking the pistil in alcohol solutions with volume fractions of 55-65%, 35-45%, and 15-25% in sequence; the soaking time of the pistil in each concentration of alcohol solution is independently 8-12 minutes.

2. The method according to claim 1, characterized in that The softening temperature in step (3) is 28-32°C.

3. The method according to claim 1, characterized in that The dye in step (6) is a mixture of aniline blue and potassium dihydrogen phosphate; the mass fraction of the aniline blue is 0.4-0.6%; the mass fraction of the potassium dihydrogen phosphate is 0.005-0.015%; and the pH of the dye is 9.8-10.

2.

4. The method according to claim 1, characterized in that: In step (6), the dyeing time of the pistil at different pollination stages is different. If the pistil is 1 to 3 days after pollination, the dyeing time is 7 to 10 minutes; if the pistil is 4 to 12 days after pollination, the dyeing time is 3 to 5 minutes.

5. The method according to claim 1, characterized in that The washing time in step (2) is 25 to 35 minutes; the washing times in step (3) are 2 to 4 times, and the washing time for each time is 8 to 12 minutes.

6. The method according to claim 1, characterized in that The pH of the phosphate buffer in steps (5) and (6) is independently 6.9 to 7.1; the volume ratio of the phosphate buffer to the pistil is independently 8 to 12:1; and the water absorption in steps (3) and (6) is to place the pistil on absorbent paper for 10 to 70 seconds.

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