Fluorescence microscopic detection method for rapidly detecting service life of ovules of fructus amomi

Abstract

The invention discloses a fluorescence microscopic detection method for rapidly detecting the life of ovules of fructus amomi, which specifically comprises the following steps: (1) sampling: bagging to-be-detected flowers of fructus amomi before flowering, and taking small flowers of different flower ages at intervals from fresh ovary to withered ovary after flowering; (2) ovule treatment: stripping ovule-containing fructus amomi flower ovules of different flower ages, and sequentially carrying out FAA fixation, NaClO solution transparentizing, NaOH solution softening and aniline blue solution dyeing treatment; (3) loading and observing: longitudinally cutting the treated ovary, tightly attaching the cut surface filled with ovules to a glass slide, covering another glass slide, pressing, and observing whether the vascular bundle of the ovules on the cut surface of the ovary develops color or not under a fluorescence microscope; and (4) determining the service life of the ovules: in the detected ovules at interval flower ages, taking the flower ages before bright green fluorescence display of the vascular bundles of the ovules as the service life of the ovules. The method can quickly detect the life of the ovules of the fructus amomi, and lays a foundation for research on insemination and reproduction of flowers of the fructus amomi and increase of the fruiting rate in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of ovule lifespan detection, and more specifically, to a fluorescence microscopy detection method for rapidly detecting the lifespan of Amomum villosum ovules. Background Art

[0002] Amomum villosum Lour. is a perennial herbaceous plant of the Zingiberaceae family and Amomum genus, and its fruit is used as medicine. In production, the low fruit setting rate is the main problem existing in the cultivation of Amomum villosum. Among the many factors affecting the fruit setting rate, after flowering, the length of the ovule lifespan is one of the main factors affecting the fruit setting rate.

[0003] However, there are currently few methods for detecting the lifespan of plant ovules.

[0004] Therefore, how to rapidly detect the lifespan of Amomum villosum ovules is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a fluorescence microscopy detection method for rapidly detecting the lifespan of Amomum villosum ovules to solve the deficiencies in the prior art.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A fluorescence microscopy detection method for rapidly detecting the lifespan of Amomum villosum ovules specifically includes the following steps:

[0008] (1) Sampling

[0009] Before the flower of Amomum villosum to be detected blooms, it is bagged, and after blooming, small flowers of different flower ages are taken at intervals from the fresh to the wilted ovary.

[0010] (2) Ovule treatment

[0011] The ovaries of Amomum villosum flowers containing ovules of different flower ages are peeled off and successively subjected to FAA fixation, NaClO solution transparency, NaOH solution softening, and aniline blue solution staining treatments.

[0012] (3) Mounting and observing

[0013] The treated ovary is longitudinally cut, the cut surface full of ovules is closely attached to the glass slide, another glass slide is covered, pressed, and whether the ovule vascular bundles on the cut surface of the ovary are colored is observed under a fluorescence microscope.

[0014] (4) Determining the ovule lifespan

[0015] Among the ovules of the detected interval flower ages, the flower age before the ovule vascular bundles show bright green fluorescence is used as the ovule lifespan time.

[0016] Further, in the above step (2), the FAA is prepared from 70% ethanol, 38% formaldehyde and acetic acid in a volume ratio of 8:1:1, and the fixation time is not less than 48 h.

[0017] Further, in the above step (2), the available chlorine content of the NaClO solution is 1.6%, and the soaking time is 3 - 4 h.

[0018] Further, in the above step (2), the concentration of the NaOH solution is 12 mol / L, and the soaking time is 3 - 4 h.

[0019] Further, in the above step (2), the concentration of the aniline blue solution is 0.2%, and the soaking time is not less than 36 h.

[0020] Through the above technical solutions, compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. The present invention utilizes the callose that appears when the ovule vascular bundle begins to age, and its characteristic of presenting bright blue - green fluorescence under an ultraviolet fluorescence microscope after aniline blue staining, as an index for the loss of vitality of the Amomum villosum ovule. It is found that the callose on the Amomum villosum ovule does not always exist and then disappears, and the fluorescence also disappears accordingly. In order to determine the lifespan of the Amomum villosum ovule, the present invention provides a set of accurate ovule lifespan detection procedures and detection indicators. Ovules of different flower ages are selected for detection, and the flower age before the bright blue - green fluorescence of the ovule vascular bundle appears is used as the lifespan time of the Amomum villosum ovule.

[0022] 2. The method of the present invention can quickly detect the lifespan of the Amomum villosum ovule, laying a foundation for the future research on the fertilization and fruit - setting rate of the Amomum villosum flower. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Photographs of the fluorescence of the ovule vascular bundle changing from none (a is 3 days after flowering) to having (b is 6 days after flowering) and then to none (c is 9 days after flowering) for ovules of different flower ages; the arrow points to the fluorescence of the ovule vascular bundle;

[0024] Figure 2 For the appearance wilting degree of the ovaries of unpollinated small flowers of different flower ages in May. Among them, the ovaries shown in the vertical column are ovaries of the same flower age, and the numbers on them represent the flower age, respectively indicating the ovaries from the 1st day to the 10th day after flowering. DETAILED DESCRIPTION OF THE INVENTION

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] Example 1

[0027] A fluorescence microscopy detection method for rapidly detecting the ovule lifespan of Amomum villosum, specifically including the following steps:

[0028] (1) Sampling

[0029] During the flowering period of Amomum villosum, select the inflorescences that are about to bloom the afternoon before the experiment, cover them with gauze net bags to prevent insect pollination. The next morning, label 100 small Amomum villosum flowers that open on the same day for each variety. After labeling, pick 10 flowers from each variety every day from 17:00 to 18:00 for a total of 10 days. The flower ages of the small flowers range from 1 day (the first day of flowering) to 10 days (the tenth day of flowering);

[0030] (2) Ovule treatment

[0031] After sampling, promptly peel the ovaries of Amomum villosum flowers containing ovules at each flower age, and immerse them separately in FAA (70% ethanol: 38% formaldehyde: acetic acid = 8:1:1) for fixation, and store them in the dark. 48 hours after all sampling is completed, take out the materials to be detected, place them in a NaClO solution with an available chlorine content of 1.6% for clearing for 3 hours, then transfer them to a 12 mol / L NaOH solution for softening for 3 hours, and then place them in a 0.2% aniline blue staining solution for staining in the dark for 36 hours;

[0032] (3) Mounting and observation

[0033] Longitudinally cut the treated ovaries, place the cut surface filled with ovules closely against the glass slide, cover with another glass slide, press, and observe whether the ovule vascular bundles on the cut surface of the ovary are colored under a fluorescence microscope;

[0034] (4) Determining the ovule lifespan

[0035] Among the ovules at the interval flower ages detected, take the flower age before the bright green fluorescence appears in the ovule vascular bundles as the ovule lifespan time.

[0036] Example 2

[0037] A fluorescence microscopy detection method for rapidly detecting the ovule lifespan of Amomum villosum, specifically including the following steps:

[0038] (1) Sampling

[0039] At different times during the flowering period of Amomum villosum in April, May, and June respectively, select the inflorescences that are about to bloom the afternoon before the experiment, cover them with gauze net bags to prevent insect pollination. The next morning, label 300 small Amomum villosum flowers that open on the same day for each variety. After labeling, pick 30 flowers from each variety every day from 17:00 to 18:00 for a total of 10 days. The flower ages of the small flowers range from 1 day (the first day of flowering) to 10 days (the tenth day of flowering);

[0040] (2) Ovule treatment

[0041] After sampling, timely strip the ovary of Amomum villosum flowers containing ovules at each flower age, immerse them separately in FAA (70% ethanol: 38% formaldehyde: acetic acid = 8:1:1) for fixation, and store in the dark; 48 h after all sampling is completed, take out the material to be inspected and place it in a NaClO solution with an available chlorine content of 1.6% for clearing for 4 h, then transfer it to a 12 mol / L NaOH solution for softening for 4 h, and then place it in a 0.2% aniline blue staining solution for staining in the dark for 48 h;

[0042] (3) Slide preparation and observation

[0043] Longitudinally section the treated ovary, place the section full of ovules closely against the glass slide, cover it with another glass slide, press, and observe whether the ovule vascular bundles on the ovary section are colored under a fluorescence microscope;

[0044] (4) Determine the ovule lifespan

[0045] Among the ovules of the tested interval flower ages, take the flower age before the ovule vascular bundles show bright green fluorescence as the ovule lifespan time.

[0046] Example 3

[0047] A fluorescence microscopy detection method for rapidly detecting the lifespan of Amomum villosum ovules, specifically including the following steps:

[0048] (1) Sampling

[0049] During the flowering period of Amomum villosum, select the inflorescences that are about to bloom the day before the experiment and cover them with gauze bags to prevent insect pollination. The next morning, label 300 small Amomum villosum flowers that open on the same day for each variety. After labeling, pick 30 flowers from each variety every day from 17:00 to 18:00 for a total of 10 days. The flower ages of the small flowers range from 1 day (the first day of flowering) to 10 days (the tenth day of flowering);

[0050] (2) Ovule treatment

[0051] After sampling, timely strip the ovary of Amomum villosum flowers containing ovules at each flower age, observe the freshness of the ovary and record it. Then immerse the ovaries at each flower age separately in FAA (70% ethanol: 38% formaldehyde: acetic acid = 8:1:1) for fixation and store in the dark; 2 months after all sampling is completed, take out the material to be inspected and place it in a NaClO solution with an available chlorine content of 1.6% for clearing for 4 h, then transfer it to a 12 mol / L NaOH solution for softening for 4 h, and then place it in a 0.2% aniline blue staining solution for staining in the dark for 48 h;

[0052] (3) Slide preparation and observation: Longitudinally section the treated ovary, the section is full of ovules, closely against the glass slide, cover it with another glass slide, press, and observe whether the ovule vascular bundles on the ovary section are colored under a fluorescence microscope;

[0053] (4) Determine the ovule lifespan

[0054] Among the detected ovules at different flower ages, the flower age before the bright green fluorescence appears in the ovule vascular bundle is used as the ovule lifespan.

[0055] Performance test

[0056] 1. Detection test on the lifespan of ovules of 1 or 2 Amomum villosum varieties

[0057] According to the fluorescence microscopy detection method in Example 1, the lifespan of ovules of Yunsha No. 1 and Yunsha No. 2 was detected respectively, and the results are shown in Table 1.

[0058] Table 1 Proportion of florets with bright blue-green fluorescence in ovule vascular bundles (%)

[0059]

[0060]

[0061] As can be seen from Table 1, the fluorescence of the ovule vascular bundles of the two varieties of Amomum villosum appears on the 4th day. It is thus speculated that the ovule lifespan is at least 3 days.

[0062] 2. Detection test on the lifespan of ovules at different times during the flowering period of Amomum villosum

[0063] According to the fluorescence microscopy detection method in Example 2, the lifespan of ovules at different times (April, May, June) during the flowering period of Amomum villosum was detected respectively, and the results are shown in Table 2.

[0064] Table 2 Proportion of florets with bright blue-green fluorescence in ovule vascular bundles (%)

[0065]

[0066] Note: The values in the table are mean ± SD. The capital letters behind represent the comparison of the differences in results at different times in the same month, and the small letters represent the comparison of the differences in results at the same time in different months (Tukey test, P ≤ 0.05).

[0067] As can be seen from Table 2, the ovule lifespan varies with different months during the flowering period. Among them, the fluorescence of the ovule vascular bundles of Amomum villosum in April appears on the 4th day, but only 5%, so most of the ovule lifespan in April is at least 4 days. The fluorescence of the ovule vascular bundles of Amomum villosum in May appears on the 3rd day, but only 3.3%, so most of the ovule lifespan in May is at least 3 days. The fluorescence of the ovule vascular bundles of Amomum villosum in June appears on the 4th day, with a value of 33.3%, so the ovule lifespan in June is at least 3 days.

[0068] 3. Relationship between the appearance freshness of the ovary of florets at different flower ages and the fluorescence color reaction of ovule vascular bundles

[0069] According to the fluorescence microscopy detection method of Example 3, the relationship between the appearance freshness of the ovary of florets at different flower ages and the fluorescence color reaction of ovule vascular bundles was detected respectively, and the results are as Figure 1-2 shown in Table 3.

[0070] Table 3 Relationship between the wilting degree of Amomum villosum ovary at different flower ages and the fluorescence color reaction of ovules

[0071] Flowering time / d Appearance state of ovary Percentage of ovaries with fluorescent ovule vascular bundles / % 1 Fresh ++ 0B 2 Fresh + 0B 3 Wilting + 3.3±5.9B 4 Wilting ++ 18.2±15.7B 5 Wilting +++ 67.3±12.6A 6 Wilting ++++ 3.3±5.8B 7 Wilting +++++ 0B 8 Wilting ++++++ 0B 9 Wilting +++++++ 0B 10 Wilting ++++++++ 0B

[0072] Note: The values in the table are mean ± SD, and the capital letters behind represent the comparison of the differences in results at different times (Tukey test, P ≤ 0.05).

[0073] From Figure 1-2 and Table 3, it can be seen that when the flower has just bloomed for 1 - 2 days, the surface of the ovary is fresh, and the vascular bundles of the ovules inside are not aged, and there is no bright blue - green fluorescence under the ultraviolet fluorescence microscope. As the flower age extends, starting from the 3rd day, the surface of the ovary wilts, and in some ovaries, the vascular bundles of the ovules are aged and callose appears, showing bright blue - green fluorescence. However, the callose in the wilted ovules does not exist all the time. As the flower age is postponed, the callose disappears and the fluorescence also disappears accordingly, making it impossible to determine the viability of a single wilted - surface ovary based on fluorescence. However, according to the characteristics of the fluorescence color reaction of ovule vascular bundles, samples can be taken successively from the fresh to the wilted state of the ovary at equal intervals of different flower ages, and fluorescence color reaction tests of ovules at different flower ages can be carried out, and the flower age before the appearance of fluorescence in the ovule vascular bundles can be used as the ovule lifespan.

[0074] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fluorescence microscopy method for rapidly detecting the life span of Amomum villosum ovules, characterized in that: The specific steps include: (1) Sampling The Amomum villosum flowers to be tested are bagged before flowering, and after flowering, florets of different ages are taken from fresh ovaries to wilted ovaries; (2) Ovule treatment The ovaries of Amomum villosum flowers with ovules at different ages were peeled off and fixed with FAA, made transparent with NaClO solution, softened with NaOH solution, and dyed with aniline blue solution. (3) Observation of film Cut the treated ovary longitudinally, place the section full of ovules close to the slide, cover with another slide, press, and observe under a fluorescence microscope whether the ovule vascular bundle on the section of the ovary is colored; (4) Determine ovule lifespan Among the ovules of different flowering ages tested, the flowering age before the ovule vascular bundle showed bright green fluorescence was taken as the ovule life span.

2. A fluorescence microscopic detection method for rapid detection of Amomum villosum ovule life span according to claim 1, characterized in that: In step (2), the FAA is prepared by mixing 70% ethanol, 38% formaldehyde and acetic acid in a volume ratio of 8:1:1, and the fixation time is not less than 48 hours.

3. A fluorescence microscopic detection method for rapid detection of Amomum villosum ovule life span according to claim 1, characterized in that: In step (2), the effective chlorine content of the NaClO solution is 1.6%, and the soaking time is 3-4 hours.

4. A fluorescence microscopic detection method for rapid detection of Amomum villosum ovule life span according to claim 1, characterized in that: In step (2), the concentration of the NaOH solution is 12 mol / L, and the soaking time is 3-4 h.

5. A fluorescence microscopic detection method for rapid detection of Amomum villosum ovule life span according to claim 1, characterized in that: In step (2), the concentration of the aniline blue solution is 0.2%, and the immersion time is not less than 36 hours.

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