Method for determining effective pollination periods in different flowering periods of fructus amomi

Abstract

The invention discloses a method for determining effective pollination periods in different flowering periods of fructus amomi. The method specifically comprises the following steps: (1) monitoring temperature and humidity in real time at different time in the flowering period; (2) observing stigma receptivity through fluorescence microscopy; (3) observing the growth characteristics of the pollen tube through fluorescence microscopy; (4) observing the service life of ovules through fluorescence microscopy; (5) counting changes of fruiting rates of different flower ages; and (6) determining effective pollination periods in different flowering periods. According to the method, the concept of the effective pollination period of Williams is borrowed, the three-element vitality change of the effective pollination period of fructus amomi and the change condition of the fruiting rate of different flower ages in different flowering periods from April to June under different temperature and humidity conditions are researched, and the influence of environment temperature and humidity in different periods on the three-element vitality and the fruiting rate of the effective pollination period is determined; the optimal atmosphere temperature and humidity conditions and effective pollination period time in the fructus amomi flowering and pollination period are expected to be found, and a theoretical basis is provided for improving the fructus amomi fruiting rate and yield through fructus amomi flowering phase temperature and humidity management in production.

Description

Technical Field

[0001] The invention relates to the technical field of plant cultivation, and more particularly to a method for determining effective pollination periods of Amomum villosum at different flowering stages. Background Art

[0002] Amomum villosum is derived from the perennial herb Amomum of the Zingiberaceae family. The Amomum villosum-based plants listed in the Pharmacopoeia of the People's Republic of China (Volume 1, 2020 edition) are Amomum villosum, Amomum villosum.var.xanthioides and Amomum longiligulare, among which Amomum villosum is the main plant. Amomum villosum has the functions of promoting digestion, warming the spleen and stomach to relieve diarrhea, regulating qi and stabilizing the fetus. It is used to treat symptoms such as dampness in the body blocking the middle jiao, stomach distension and loss of appetite, cold spleen and stomach, vomiting and diarrhea, pregnancy reactions in pregnant women, and unstable fetal activity. As one of the famous southern medicines, Amomum villosum is not only widely used in traditional Chinese medicine, but is also often used as a spice and seasoning due to its unique aroma. With the continuous growth of market demand, its uses are becoming more and more diversified, and its sales channels are also becoming more and more diversified, providing good market opportunities for growers. The origin of Amomum villosum is Yangchun, Guangdong. In 1963, the Yunnan Branch of the Institute of Medicinal Plants of the Chinese Academy of Medical Sciences introduced it from Yangchun, Guangdong to Xishuangbanna, Yunnan for trial planting, and promoted its production on a large scale in the 1980s. Currently, Yunnan Province is the largest main production area in the country.

[0003] The fruit of Amomum villosum is used as medicine, so the fruiting rate is a key factor limiting the yield of Amomum villosum. Fruiting begins with pollination. After pollination, the pollen falls on the stigma. The stigma needs to have adhesion ability so that the pollen adheres to it first. The stigma also needs to provide pollen germination conditions so that the pollen germinates on the stigma to form a pollen tube and provide conditions for the growth of the pollen tube so that the pollen tube can grow along the style, reach the ovary, and finally reach the ovule to complete fertilization. Only then can the fruit develop and expand and mature. This complex process is affected by the vitality of the flower organs involved in this process and the external temperature and humidity environment. Any problem in any process or influencing factor will affect the natural fruiting rate of Amomum villosum, and ultimately lead to low yield of Amomum villosum.

[0004] Williams first proposed the concept of effective pollination period in 1965 when studying the fruiting rate of apples. He pointed out that it is the period when fertilization can be successfully completed after pollination to form fruits. The effective pollination period is mainly determined by observing the stigma receptive period, pollen tube growth rate and ovule lifespan through microfluorescence methods, or by the pollination and fruiting rate at different times after flowering. The effective pollination period of plants has been reported on fruit plants. It can mainly help determine the best period for plant pollination and the limiting factors that affect fruiting, so as to take appropriate measures to improve the fruiting rate, which is of great significance for increasing production. At present, there are no reports on the effective pollination period of Amomum villosum.

[0005] Therefore, how to improve the natural fruit setting rate and yield of Amomum villosum is a problem that technicians in this field need to solve urgently. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a method for determining the effective pollination period at different flowering stages of Amomum villosum, so as to solve the deficiencies in the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for determining the effective pollination period of Amomum villosum at different flowering periods specifically comprises the following steps:

[0009] (1) Real-time monitoring of temperature and humidity at different times during the flowering period;

[0010] (2) Fluorescence microscopy to observe stigma receptivity;

[0011] (3) Fluorescence microscopy to observe pollen tube growth characteristics;

[0012] (4) Observation of ovule lifespan using fluorescence microscopy;

[0013] (5) Statistics of fruiting rates at different flowering ages;

[0014] (6) The effective pollination period at different flowering stages was determined by conducting experiments on the changes in the vitality of the three elements of the effective pollination period (stigma receptivity, pollen tube growth characteristics, and ovule lifespan) at different flowering stages and experiments on the changes in fruiting rate at different flower ages.

[0015] Furthermore, the above step (1) is specifically as follows: during the flowering period of Amomum villosum, the changes in atmospheric temperature and humidity of the surface where the Amomum villosum florets grow are recorded.

[0016] Further, the above step (2) is specifically as follows: the test is conducted in the flowering period of Amomum villosum in April, May and June, bagging is carried out in the afternoon of the day before the test, and the Amomum villosum florets that are open on the next morning are marked with signs, and artificial pollination is carried out at 8:00-9:00 in the morning and 17:00-18:00 in the afternoon on the first and second days of flowering, and 8:00-9:00 in the morning on the third day, with 30 florets for each treatment. Florets of each treatment are cut after 48 hours, the corolla is removed, and immersed in FAA fixative;

[0017] For fluorescence microscopic observation, the flowers were first removed from the FAA fixative, rinsed, and then soaked in NaClO solution to make them transparent, then in NaOH solution to soften them, and then soaked in aniline blue staining solution to stain them in the dark;

[0018] When making the slide, place the material on a glass slide, cut off the pistil from the connection between the style and the top of the ovary, straighten the stigma and style, add aniline blue dye, cover with a coverslip, and make a "stigma and style" slide; place the remaining ovary on a new glass slide, cut longitudinally from the middle of the ovary, spread the two cut surfaces flatly facing the glass slide, add aniline blue dye, cover with another glass slide, and complete the "ovary" slide; finally, place the prepared "stigma and style" slide and "ovary" slide under an ultraviolet fluorescence microscope to observe the receptivity of the stigma.

[0019] Furthermore, the above step (3) is specifically as follows: bagging was performed in the afternoon of the day before the experiment, and the Amomum villosum florets that opened on the next morning were marked with signs, and all were artificially pollinated on the first day of flowering, with 30 florets for each treatment. Florets from each treatment were cut at 8 h, 24 h, and 36 h, the corollas were removed, and immersed in FAA fixative;

[0020] For fluorescence microscopic observation, the flowers were first removed from the FAA fixative, rinsed, and then soaked in NaClO solution to make them transparent, then in NaOH solution to soften them, and then soaked in aniline blue staining solution to stain them in the dark;

[0021] When making the slide, place the material on a glass slide, cut off the pistil from the connection between the style and the top of the ovary, straighten the stigma and style parts, add aniline blue dye, cover with a coverslip, and make a "stigma and style" slide; place the remaining ovary part on a new glass slide, cut longitudinally from the middle of the ovary, spread the two cut surfaces flatly facing the glass slide, add aniline blue dye, cover with another glass slide, and complete the "ovary" slide; finally, place the prepared "stigma and style" slide and "ovary" slide under an ultraviolet fluorescence microscope to observe the growth characteristics of the pollen tube.

[0022] Furthermore, the above step (4) is specifically as follows: bagging is performed in the afternoon of the day before the experiment, and 300 Amomum villosum florets that are open on the day are marked on the morning of the next day without pollination treatment, and 30 florets are sampled from 17:00 to 18:00 every day for a total of 10 days, and the florets are aged from 1 day (the first day of flowering) to 10 days (the tenth day of flowering); after sampling, the corolla and style are removed and immersed in FAA fixative;

[0023] For fluorescence microscopic observation, the flowers were first removed from the FAA fixative, rinsed, and then soaked in NaClO solution to make them transparent, then in NaOH solution to soften them, and then soaked in aniline blue staining solution to stain them in the dark;

[0024] When making the slide, place the material on a glass slide, cut it longitudinally from the middle of the ovary, spread it flat with the two cut surfaces facing the slide, add aniline blue dye solution, cover it with a glass slide to make an "ovary" slide, place it under an ultraviolet fluorescence microscope, and observe the life span of the ovule.

[0025] Furthermore, the above-mentioned FAA fixative is prepared by mixing 70% ethanol, 38% formaldehyde and acetic acid in a volume ratio of 8:1:1; the immersion time in the FAA fixative is at least 48 hours; the effective chlorine content of the NaClO solution is 1.6%; the immersion time in the NaClO solution is 4 hours; the concentration of the NaOH solution is 12 mol / L; the immersion time in the NaOH solution is 4 hours; the concentration of the aniline blue dye solution is 0.2%; and the immersion time in the aniline blue dye solution is at least 36 hours.

[0026] Furthermore, the above step (5) is specifically as follows: bagging is performed in the afternoon of the day before the experiment, and the Amomum villosum florets that bloom on the next morning are marked with signs, and artificial pollination is performed at 8:00-9:00 in the morning and 17:00-18:00 in the afternoon on the first and second days of flowering, and at 8:00-9:00 in the morning on the third day. After 10 days of artificial pollination, the bags are removed and the fruiting rate of the florets in each treatment is counted.

[0027] Furthermore, the above-mentioned artificial pollination adopts the smearing method. Before pollination, collect a sufficient amount of pollen from the small flowers that open on the same day, mix them evenly, and test them by TTC. The pollen activity is more than 90%. When pollinating, remove the bag, first hold the lower part of the corolla with the thumb and middle finger of the left hand, then dip a cotton swab in the mixed pollen, smear it on the stigma of the small flower, and then bag it again.

[0028] Furthermore, in the above step (6), the method for determining the effective pollination period at different flowering stages through the test of stigma receptivity, pollen tube growth characteristics and ovule life vitality changes is as follows: according to the calculation formula of Williams, when the stigma life is long, the effective pollination period = ovule life - pollination to fertilization time; when the stigma life is short, the stigma life is the main limiting factor, and the stigma life time is the effective pollination period time.

[0029] Furthermore, in the above step (6), the method for determining the effective pollination period at different flowering stages through the experiment of fruiting rate changes at different flower ages is: the longest flower age that can bear fruit after artificial pollination is the effective pollination period.

[0030] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. Based on the previous research on the vitality of Amomum villosum floral organs and their influence on fruiting rate, the present invention borrows the concept of Williams' effective pollination period, improves the research method, and for the first time uses fluorescence microscopy to study the vitality of Amomum villosum floral organs (including stigma receptivity, pollen tube growth rate and ovule lifespan) and the changes in fruiting rate at different flower ages, and determines the effective pollination period of Amomum villosum.

[0032] 2. In order to explore the influence of external atmospheric temperature and humidity environment on the activity of floral organs, fruiting rate and effective pollination period during the pollination and fruiting of Amomum villosum, the present invention studies the changes in the activity of the three elements of Amomum villosum in the effective pollination period and the changes in the fruiting rate at different flowering ages under different temperature and humidity conditions during the flowering period from April to June, and determines the influence of environmental temperature and humidity at different periods on the activity of the three elements and the fruiting rate during the effective pollination period, in order to find the optimal atmospheric temperature and humidity conditions and the effective pollination period during the flowering and pollination of Amomum villosum, so as to provide a theoretical basis for improving the fruiting rate and yield of Amomum villosum through temperature and humidity management during the flowering period of Amomum villosum in production.

[0033] 3. The research conclusions of this invention are of great significance for understanding the reproductive ecology of Amomum villosum plants, and provide a scientific basis for its genetic improvement and protection of germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The temperature and humidity monitoring results at different periods, where a is the temperature change at different periods, and b is the humidity change at different periods;

[0035] Figure 2 The figure shows the germination of pollen on the stigma and the growth path of pollen tube in the style, ovary and ovule, where a is the germination of pollen on the stigma, b is the growth of pollen tube on the style, c is the entry of pollen tube into the ovary, and d is the entry of pollen tube into the ovule; the arrows point to pollen or pollen tube;

[0036] Figure 3 is the pollen tube growth rate at different periods, where capital letters represent the difference between the results at different times in the same month, and lowercase letters represent the difference between the results at the same time in different months (Tukey test, P≤0.05);

[0037] Figure 4The process of ovule vascular bundle fluorescence in the ovary of florets of different flowering ages from no to present and then to no again; a is the first day of flowering, the ovule vascular bundle has no fluorescence; b is the 6th day after flowering, the ovule vascular bundle has fluorescence; c is the 8th day of flowering, the ovule vascular bundle fluorescence disappears; the arrow points to the ovule vascular bundle fluorescence;

[0038] Figure 5 The figures are as follows: The fruit-bearing rates of florets of different ages at different flowering periods. Capital letters indicate the difference in fruiting at different times in the same month, and lowercase letters indicate the difference in fruiting at the same time in different months (Tukey test, P≤0.05). DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Example 1

[0041] The method for determining the effective pollination period at different flowering stages of Amomum villosum specifically comprises the following steps:

[0042] (1) Real-time monitoring of temperature and humidity at different times during the flowering period;

[0043] During the flowering period of Amomum villosum, the changes in atmospheric temperature and humidity on the surface where the Amomum villosum florets grow are recorded.

[0044] (2) Fluorescence microscopy to observe stigma receptivity;

[0045] The experiment was conducted in the flowering period of Amomum villosum in April, May and June. The plants were bagged in the afternoon of the day before the experiment, and the Amomum villosum florets that opened on the next morning were marked with signs. Artificial pollination was performed at 8:00-9:00 a.m. and 17:00-18:00 p.m. on the first and second days of flowering, and at 8:00-9:00 a.m. on the third day of flowering. Thirty florets were collected for each treatment. After 48 hours, the florets of each treatment were cut, the corolla was removed, and the florets were immersed in FAA fixative prepared by mixing 70% ethanol, 38% formaldehyde and acetic acid in a volume ratio of 8:1:1 for 48 hours.

[0046] For fluorescence microscopic observation, the flowers were first removed from the FAA fixative, rinsed, and then immersed in a 1.6% NaClO solution for 4 h to make it transparent, then in a 12 mol / L NaOH solution for 4 h to soften, and then immersed in a 0.2% aniline blue staining solution for 48 h in the dark.

[0047] When making the slide, the material is placed on a glass slide, the pistil is cut off from the connection between the style and the top of the ovary, the stigma and style are straightened, aniline blue dye is added, and a cover glass is placed to make a "stigma and style" slide; the remaining ovary part is placed on a new glass slide, and a longitudinal cut is made from the middle of the ovary, the two cut surfaces are spread out flatly facing the glass slide, aniline blue dye is added, and another glass slide is placed to complete the "ovary" slide; finally, the prepared "stigma and style" slide and "ovary" slide are placed under an ultraviolet fluorescence microscope to observe the receptivity of the stigma;

[0048] (3) Fluorescence microscopy to observe pollen tube growth characteristics;

[0049] The florets were bagged in the afternoon of the day before the experiment, and the florets that opened on the next morning were marked with signs. All of them were artificially pollinated on the first day of flowering. Each treatment had 30 florets. The florets of each treatment were cut at 8h, 24h, and 36h, and the corolla was removed. They were immersed in FAA fixative prepared by mixing 70% ethanol, 38% formaldehyde and acetic acid in a volume ratio of 8:1:1 for 48h.

[0050] For fluorescence microscopic observation, the flowers were first removed from the FAA fixative, rinsed, and then immersed in a 1.6% NaClO solution for 4 h to make it transparent, then in a 12 mol / L NaOH solution for 4 h to soften, and then immersed in a 0.2% aniline blue staining solution for 48 h in the dark.

[0051] When making the slide, the material is placed on a glass slide, the pistil is cut off from the connection between the style and the top of the ovary, the stigma and style parts are straightened, aniline blue dye is added, and a cover glass is placed to make a "stigma and style" slide; the remaining ovary part is placed on a new glass slide, and a longitudinal cut is made from the middle of the ovary, and the two cut surfaces are spread out flatly facing the glass slide, aniline blue dye is added, and another glass slide is placed to complete the "ovary" slide; finally, the prepared "stigma and style" slide and "ovary" slide are placed under an ultraviolet fluorescence microscope to observe the growth characteristics of pollen tubes;

[0052] (4) Observation of ovule lifespan using fluorescence microscopy;

[0053] The day before the experiment, the flowers were bagged in the afternoon. On the next morning, 300 small flowers of Amomum villosum that were open on the same day were marked with tags. No pollination was performed. 30 flowers were sampled from 17:00 to 18:00 every day for a total of 10 days. The age of the small flowers ranged from 1 day (the first day of flowering) to 10 days (the tenth day of flowering). After sampling, the corolla and style were removed and immersed in FAA fixative prepared by mixing 70% ethanol, 38% formaldehyde and acetic acid in a volume ratio of 8:1:1 for 48 hours.

[0054] For fluorescence microscopic observation, the flowers were first removed from the FAA fixative, rinsed, and then immersed in a 1.6% NaClO solution for 4 h to make it transparent, then in a 12 mol / L NaOH solution for 4 h to soften, and then immersed in a 0.2% aniline blue staining solution for 48 h in the dark.

[0055] When making the slide, place the material on a glass slide, cut it longitudinally from the middle of the ovary, spread the two cut surfaces flatly facing the glass slide, add aniline blue dye solution, cover the slide, make an "ovary" slide, and place it under an ultraviolet fluorescence microscope to observe the life span of the ovules;

[0056] (5) Statistics on changes in fruiting rate at different flowering ages;

[0057] The plants were bagged in the afternoon of the day before the experiment, and the florets that opened on the next morning were marked with signs. Artificial pollination was performed at 8:00-9:00 a.m. and 17:00-18:00 p.m. on the first and second days of flowering, and at 8:00-9:00 a.m. on the third day. After 10 days of artificial pollination, the bags were removed and the fruiting rate of the florets in each treatment was counted.

[0058] Among them, artificial pollination uses the smearing method. Before pollination, collect enough pollen from the small flowers that open on the same day, mix them evenly, and test them with TTC. The pollen activity is more than 90%. When pollinating, remove the bag, first hold the lower part of the corolla with the thumb and middle finger of the left hand, then dip a cotton swab in the mixed pollen, smear it on the stigma of the small flower, and then bag it again.

[0059] (6) Determine the effective pollination period of Amomum villosum at different stages

[0060] The effective pollination period at different flowering stages was determined by experiments on stigma receptivity, pollen tube growth characteristics, ovule lifespan and vitality, and fruiting rate at different flower ages.

[0061] Among them, the method of determining the effective pollination period at different flowering stages through the test of stigma receptivity, pollen tube growth characteristics and ovule life vitality changes is as follows: according to the calculation formula of Williams, when the stigma life is long, the effective pollination period = ovule life - pollination to fertilization time; when the stigma life is short, the stigma life is the main limiting factor, and the stigma life time is the effective pollination period time;

[0062] The method of determining the effective pollination period at different flowering stages through experiments on the changes in fruit-bearing rates at different flower ages is as follows: the longest flower age that can bear fruit after artificial pollination is the effective pollination period.

[0063] Example 2

[0064] 1 Introduction

[0065] 1.1 Current research status at home and abroad

[0066] 1.1.1 Introduction to the effective pollination period

[0067] Williams first introduced the concept of effective pollination period when studying apples in 1965, pointing out that this period is the time when flowers can successfully complete fertilization and form fruits. The duration is determined by the lifespan of the ovule minus the time required for the pollen tube to grow to the ovule. He also identified three main factors affecting the effective pollination period: stigma receptivity, pollen tube growth rate and ovule lifespan.

[0068] At present, there are no relevant research reports on using fluorescence microscopy to observe the receptivity of Amomum villosum stigma, pollen tube growth rate, ovule lifespan, and fruiting rate at different flower ages to determine the effective pollination period of Amomum villosum.

[0069] 1.1.2 Existing detection methods for organ vitality of Amomum villosum

[0070] Among the existing studies on the detection of the life span of Amomum villosum flower organs, there are many studies on the detection methods of stigma receptivity and pollen vitality, most of which are chemical staining methods. For example, the receptivity of stigma is determined by the benzidine-hydrogen peroxide method. However, when Peng Jianming used this method in 2010, he believed that this method had a certain impact on the accuracy of the observation results, was not conducive to observing the receptivity of Amomum villosum stigma, and could not quickly and accurately determine the changes in the receptivity of Amomum villosum stigma receptivity. In 2011, Peng Jianming and others used the MTT method to detect the receptivity of Amomum villosum stigma. The results showed that the stigma with strong receptivity was all dyed blue-purple, the stigma with weak receptivity was partially dyed, and the stigma without vitality did not change color. Guo Yanwei's MTT method has the same working principle as his predecessors, but it is simpler to operate and has a more obvious effect. Pollen vitality is mainly detected by culture medium, suspension culture or TTC staining, and pollen vitality is evaluated by observing the germination rate or color change of Amomum villosum in vitro pollen.

[0071] 1.1.3 Flower organ vitality and influencing factors

[0072] There are few studies on the changes in the vitality of Amomum villosum floral organs and the influencing factors. In other plants, studies on the effects of environmental and climate changes, plant self-compatibility, and flower age on the changes in the vitality of floral organs can be found.

[0073] When studying the pollen vitality of three native persimmon trees in China, Zhan Shusen and others found that sucrose is important for promoting pollen growth. The best germination rate occurred under conditions containing 200g / L sucrose, 150mg / L boric acid and 100mg / L calcium nitrate. They also tested two methods for rapid detection of pollen vitality: I2-KI and TTC staining, and found that both methods showed lower results than the hanging drop culture method, and I2-KI staining took less time. Therefore, it is recommended to use a combination of methods to detect pollen vitality. Researchers such as Kong Guanghong pointed out that during the effective pollination period of macadamia nuts, if the environmental conditions are suitable, pollen will continue to germinate. Whether it is the first or the later germinated pollen tube, they will grow in the direction of the ovary. However, the growth rate of each pollen tube is different, some are fast and some are slow, and the pollen tube may also die during the growth process. Researchers such as Wu Yaran believe that the period after pollination is particularly critical for the growth of pollen tubes. Therefore, drastic climate change may have a negative impact on the growth of pollen tubes, and thus affect the fruit setting rate. This conclusion was reached through appropriate experimental methods based on previous studies. Zhou Ting et al. reported that during the inactivation process of the ovule vascular bundle of plants, callose is produced, which is bright blue-green under ultraviolet light after aniline blue staining, thus distinguishing it from viable ovules, but the callose in the vascular bundle of inactivated ovules will slowly disappear, making it indistinguishable from viable ovules. Yang Qin et al. found that the maturity of flowers significantly affects the activity of pollen and the receptivity of stigma when studying the key biological factors of self-pollination fruit setting rate of rabbit eye blueberry. They observed that the pollen activity and stigma receptivity of rabbit eye blueberry reached their highest point on the 2nd to 3rd day after the flower opened. This shows that flower age has an important influence on the proportion of pollen tubes passing through the style and the rate of embryo sac degeneration. Ganqing Waitoucai mainly self-pollinates and maintains a high reproduction rate. The critical period for pollen tube formation and ovule degeneration is the 3rd to 5th day after flowering. Its pollen has a stronger ability to germinate under humid high-altitude conditions, and its pollen shape becomes more adaptable.

[0074] 1.2 Purpose and significance

[0075] The flowering period of Amomum villosum varies with the temperature in different places. The experimental study area is located in Xishuangbanna Prefecture. The climate in this area shows a clear division of dry and wet seasons. The rainy season is from mid-to-late May to October each year, and the rest of the months are dry seasons. The flowering period of Amomum villosum is from April to June, which is between the dry and wet seasons. The atmospheric temperature and humidity vary greatly at different flowering times, but there is no research on the effects of atmospheric temperature and humidity on the vitality of Amomum villosum flower organs. In order to explore the influence of floral organ vitality and external atmospheric temperature and humidity on pollination and fruiting of Amomum villosum, this study borrowed the concept of Williams' effective pollination period, studied the effective pollination period of Amomum villosum at different flowering periods from April to June, and determined the effects of environmental temperature and humidity at different periods on the effective pollination period and the vitality and fruiting rate of the three elements of the effective pollination period (stigma receptivity, pollen tube growth characteristics, and ovule lifespan), in order to find the best atmospheric temperature and humidity conditions and effective pollination period during the flowering and pollination of Amomum villosum, and provide a theoretical basis for improving the fruiting rate and yield of Amomum villosum.

[0076] 2 Materials and methods

[0077] 2.1 Test location and materials

[0078] This study was conducted in the Southern Medicinal Herb Garden of the Yunnan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, located in Jinghong City, Yunnan Province, N22°0′21.04″N, E100°47′17.71″E. The test material was Amomum villosum, which was planted for 2-5 years.

[0079] 2.2 Test equipment

[0080] Fluorescence microscope (BX53F)

[0081] 2.3 Test reagents and preparation methods

[0082] 2.3.1 Test reagents

[0083] Ethanol (analytical grade AR), formaldehyde (analytical grade AR), acetic acid (analytical grade AR), NaOH (analytical grade AR), NaClO (analytical grade AR, effective chlorine content ≥ 8%), KH2PO4 (analytical grade AR), aniline blue (analytical grade AR)

[0084] 2.3.2 Preparation method

[0085] 2.3.2.1 Preparation of FAA Fixative

[0086] FAA is 70% ethanol: 38% formaldehyde: acetic acid = 8:1:1. Prepare 500mL FAA: add 50mL 38% formaldehyde and 50mL acetic acid to 400mL 70% ethanol solution.

[0087] 2.3.2.2 Preparation of 12 mol / L NaOH solution

[0088] 400mL 12mol / LNaOH: Add 192g NaOH to 400mL water;

[0089] 2.3.2.3 Preparation of NaClO solution with 1.6% effective chlorine content

[0090] 400mL NaClO solution with 1.6% effective chlorine content: add 100mL NaClO to 400mL water; 2.3.2.4 Preparation of aniline blue solution

[0091] Step 1: Prepare 400 mL of 1.0 mol / L KH2PO4 solution as a buffer solution: add 54.4 g KH2PO4 to 400 mL of water;

[0092] Step 2: Preparation of 0.2% aniline blue solution: 400 mL 0.2% aniline blue solution: Add 0.8 g aniline blue to 400 mL potassium dihydrogen phosphate buffer.

[0093] 2.4 Test methods

[0094] 2.4.1 Real-time monitoring of temperature and humidity at different times during the flowering period

[0095] During the flowering period of Amomum villosum, a temperature and humidity recorder was placed 10 cm above the ground, where the Amomum villosum florets grew, and the changes in atmospheric temperature and humidity on the surface where the Amomum villosum florets grew were recorded every 30 minutes.

[0096] 2.4.2 Three elements of fluorescence microscopy to determine effective pollination period in different periods

[0097] 2.4.2.1 Stigma receptivity

[0098] The experiments were carried out in the flowering period of Amomum villosum in April, May and June. The plants were bagged in the afternoon of the day before the experiment, and the Amomum villosum florets that opened on the next morning were marked with signs. Artificial pollination was carried out at 8:00-9:00 in the morning and 17:00-18:00 in the afternoon on the first day (the same day) and second day of flowering, and at 8:00-9:00 in the morning on the third day. Each treatment had 30 florets. After 48 hours, the florets of each treatment were cut, the corollas were removed, and immersed in FAA (70% ethanol: 38% formaldehyde: acetic acid = 8:1:1) fixative for 48 hours.

[0099] During fluorescence microscopic observation, the pistil material in the FAA fixative was first taken out, and the sample was immersed in a solution with an effective chlorine content of 1.6% NaClO for 4 hours to achieve a transparent effect, followed by water washing and buffer treatment. The sample was then transferred to a solution containing 12 mol / L NaOH and continued to be immersed for 4 hours to soften the sample. After completing this step, it was washed with water and buffered again. Finally, the sample was immersed in a 0.2% water-soluble aniline blue dye solution and stained for 48 hours under light-proof conditions. When making the slide, use tweezers to take out the stained observation material, place it on a glass slide, use a scalpel to cut the pistil from the connection between the style and the top of the ovary, straighten the stigma and style parts, add aniline blue dye solution, make the material completely immersed in the dye solution, cover with a cover glass, and make a "stigma and style" slide; the remaining ovary part is placed on a new slide, and a scalpel is used to cut longitudinally from the middle of the ovary, and the two cut surfaces are flattened facing the slide, and aniline blue dye solution is added to make the material completely immersed in the dye solution, and another slide is covered to complete the "ovary" slide. After that, the "stigma and style" slide and the "ovary" slide are placed under an ultraviolet fluorescence microscope for observation.

[0100] 2.4.2.2 Pollen tube growth characteristics

[0101] The bagging was done in the afternoon of the day before the experiment, and the Amomum villosum florets that opened on the next morning were marked with a sign, and all were artificially pollinated on the first day of flowering (the same day). Each treatment had 30 florets, and the florets of each treatment were cut at 8h, 24h, and 36h, the corolla was removed, and immersed in FAA fixative for 48h. The treatment method for fluorescence microscopic observation was the same as above. The flowers were first taken out of the FAA fixative, rinsed, and placed in NaClO solution for transparency and NaOH solution for softening for 4h each, and then placed in aniline blue staining solution for light-proof staining for 48h. Placed on a slide, "stigma and style" slices and "ovary" slices were made, and placed under an ultraviolet fluorescence microscope for observation.

[0102] 2.4.2.3 Ovule lifespan

[0103] The bagging was done in the afternoon of the day before the experiment. On the morning of the next day, 300 Amomum villosum florets that opened on the same day were marked with a sign. No pollination was performed. 30 flowers were sampled every day from 17:00 to 18:00, for a total of 10 days. The florets were aged from 1 day (the first day of flowering) to 10 days (the tenth day of flowering). After sampling, the corolla and style were removed and immersed in FAA fixative for 48 hours. The fluorescence microscopic observation treatment method was the same as above. First, the flowers were taken out of the FAA fixative, rinsed, and placed in NaClO solution for transparency and NaOH solution for softening for 4 hours each, and then placed in aniline blue staining solution for light-proof staining for 48 hours. Placed on a slide, an "ovary" piece was made and observed under an ultraviolet fluorescence microscope.

[0104] 2.4.3 Determine the effective pollination period in different periods through the fruiting rate change experiment

[0105] The test was carried out in the afternoon of the day before the test, and the Amomum villosum florets that opened on the same day were marked with a sign the next morning. Artificial pollination was carried out at 8:00-9:00 am, 17:00-18:00 pm, and 8:00-9:00 am on the first day (the same day) of flowering, the second day, and the third day. The artificial pollination method was selected. Before pollination, a sufficient amount of pollen from the florets that opened on the same day was collected, mixed, and tested by TTC. The pollen vitality was more than 90%. During pollination, the bag was removed, and the lower part of the corolla was first held with the thumb and middle finger of the left hand, and then the mixed pollen was dipped with a cotton swab and smeared on the floret stigma, and then the bag was re-applied. After 10 days of artificial pollination, the bag was removed and the fruiting rate of the florets in each treatment was counted.

[0106] 2.5 Data collation, statistics and analysis

[0107] Based on the fluorescence microscopy observation of the three elements of the effective pollination period of Amomum villosum and the observation experiment of the fruit-bearing rate changing with the age of flower in different periods, combined with the field atmosphere temperature and humidity change data at different flowering periods, the effects of different atmospheric temperature and humidity changes on the vitality of the three elements of the effective pollination period (stigma receptivity, pollen tube growth characteristics, ovule lifespan) and the fruit-bearing rate were analyzed, in order to find the optimal atmospheric temperature and humidity conditions and the effective pollination period during the flowering and pollination period of Amomum villosum.

[0108] Excel and SPSS software were used for data processing, and general linear models were used to perform analysis of variance on stigma receptivity, pollen tube growth characteristics, ovule lifespan, and fruiting rate. The differences among the groups were determined using the Tukey test (P ≤ 0.05).

[0109] Images were processed using Photoshop software.

[0110] 2.5.1 Stigma receptivity

[0111] Generally, stigma receptivity is analyzed and evaluated by the pollen germination rate on the stigma at different sampling times, the pollen tube growth rate, including the position of the pollen tube in the style and the number of florets with pollen tubes entering the ovary.

[0112] 2.5.2 Pollen tube growth characteristics

[0113] Pollen tube growth characteristics The pollen tube growth rate was evaluated by observing the proportion of florets whose pollen tubes entered the ovules at different times after pollination.

[0114] 2.5.3 Ovule lifespan

[0115] The ovule lifespan was evaluated by observing the changes in ovule vascular fluorescence in the ovary at different sampling times and calculating the proportion of ovules with vascular fluorescence.

[0116] 2.5.4 Observation test on the change of fruiting rate with flower age at different periods

[0117] The observation test of the change of fruit-bearing rate at different periods with the age of flowers is used to evaluate the change of fruit-bearing rate at different periods with the age of flowers.

[0118] 2.5.5 Calculation of effective pollination period in different periods

[0119] The effective pollination period at different flowering stages was determined by experiments on stigma receptivity, pollen tube growth characteristics, ovule lifespan and vitality, and fruiting rate at different flower ages.

[0120] Among them, the method of determining the effective pollination period at different flowering stages through the test of stigma receptivity, pollen tube growth characteristics and ovule life vitality changes is as follows: according to the calculation formula of Williams, when the stigma life is long, the effective pollination period = ovule life - pollination to fertilization time; when the stigma life is short, the stigma life is the main limiting factor, and the stigma life time is the effective pollination period time;

[0121] The method of determining the effective pollination period at different flowering stages through experiments on the changes in fruit-bearing rates at different flower ages is as follows: the longest flower age that can bear fruit after artificial pollination is the effective pollination period.

[0122] 3 Results and analysis

[0123] 3.1 Temperature and humidity monitoring results at different periods

[0124] Depend on Figure 1 It can be seen that the average daily temperatures in April, May and June are 26.2, 26.4 and 26.7℃ respectively. However, in the dry season of April, the daily temperature difference is the largest, ranging from 20.7 to 44.7℃. The temperature is highest from 15:00 to 19:00 every afternoon, with an average temperature of 30.6 to 34.0℃. The temperature is lowest from 6:00 to 10:00 every morning, with an average temperature of 21.9 to 22.8℃. In May, the rainy season begins, and the daily temperature difference is smaller than that in April, ranging from 19.2 to 37.7℃. The average temperature during the high temperature period from 15:00 to 19:00 every afternoon is between 30.2 and 31.8℃, and the average temperature during the low temperature period from 6:00 to 9:00 every morning is between 22.7 and 23.7℃; June enters the rainy season, the daily temperature difference is the smallest, the temperature range is between 22.7 and 34.6℃, the average temperature during the high temperature period from 14:00 to 17:00 every afternoon is between 29.1 and 29.9℃, and the average temperature during the low temperature period from 6:00 to 9:00 every morning is between 24.6 and 25.4℃.

[0125] The humidity is lowest in the dry season in April, with the lowest humidity from 15:00 to 19:00 every afternoon, and the average humidity is between 47.29% and 58.16%, and the humidity is highest from 2:00 to 11:00 every morning, with the average humidity between 85.1% and 90.2%; the rainy season begins in May, and the daily humidity is higher than in April. The average humidity is between 55.1% and 64.2% during the low humidity period from 15:00 to 19:00 every afternoon, and the average humidity is between 86.6% and 94.9% during the high humidity period from 22:00 to 10:00 every morning and evening; the humidity is highest in the rainy season in June, with the average humidity being between 71.1% and 78.8% during the low humidity period from 15:00 to 18:00 every afternoon, and the average humidity is above 80% at other times.

[0126] 3.2 Stigma receptivity at different times

[0127] Stigma receptivity is expressed by the pollen germination rate and pollen tube growth rate on the stigma.

[0128] Among them, the pollen tube growth rate is expressed by the position of the pollen tube in the style and the proportion of florets with pollen tubes entering the ovary.

[0129] 3.2.1 Pollen germination rate

[0130] Table 1 Pollen germination rate on stigma (sampled 48h after pollination)

[0131]

[0132] Note: The table shows mean ± SD. The capital letters behind it represent the difference between the results at different times in the same month, and the lowercase letters represent the difference between the results at the same time in different months (Tukey test, P ≤ 0.05).

[0133] As shown in Table 1, the results of pollen germination rate on stigma showed that when pollinated 4h and 12h after the florets bloomed, the proportion of stigmas with pollen with high germination rate was 58.0% and 45.5% in April, 98.3% and 95.8% in May, and 59.3% and 70.2% in June, respectively. The value in May was significantly higher than that in April and June (p<0.05); when pollinated 28h after the florets bloomed, the proportion of stigmas with pollen with high germination rate was 38.7% in April, 59.5% in May, and 10.8% in June, and the values ​​in April and May were significantly higher than those in June (p<0.05); when pollinated 36h and 52h after the florets bloomed, the proportion of stigmas with pollen with high germination rate was 33.3% and 45.8% in April, 32.4% and 35.0% in May, and 0 in June, and there was no significant difference in the values ​​among April, May and June (p>0.05). In summary, after pollination of florets of the same age at different times during the flowering period from April to June, the pollen germination rate on the stigma showed obvious differences with the pollination time, and the pollen germination rate on the stigma was the highest 4h and 12h after flowering in May.

[0134] 3.2.2 Pollen tube growth rate

[0135] Table 2 Pollen tube growth rate (sampled 48h after pollination)

[0136]

[0137]

[0138] Note: The table shows mean ± SD. The capital letters behind it represent the difference between the results at different times in the same month, and the lowercase letters represent the difference between the results at the same time in different months (Tukey test, P ≤ 0.05).

[0139] As shown in Table 2, the results of the pollen tube growth rate in the style show that the proportion of florets with pollen tubes growing to the bottom of the style was between 84.3% and 96.7% when pollinated 4h and 12h after flowering in April-June, and then decreased to between 38.1% and 58.5% when pollinated 28h and 36h after flowering, and between 3.5% and 58.3% when pollinated 52h after flowering. Variance analysis showed that there was no significant difference in the growth rate of pollen tubes in the style after pollination of florets of the same age at different times during the flowering period from April to June (p>0.05), but as the pollination age was delayed, the growth rate of pollen tubes on florets in April and May slowed down significantly.

[0140] The results of the pollen tube entering ovary speed showed that the proportion of florets pollinated 4h and 12h after flowering was 95.0% and 96.7% in May, which were significantly higher than those in April and June (the values ​​in April were 81.3% and 79.6%, and the values ​​in June were 85.3% and 82.1%, respectively). The variance analysis showed that the value of pollination 12h after flowering in May was significantly higher than that in April and June (p<0.05). The growth speed of pollen tube entering ovary in different months was the same. It decreased significantly. The florets were pollinated 28h and 36h after flowering, and the values ​​in April, May and June were between 28.5%-49.9%, 10.4%-51.7% and 26.2%-36.4% respectively. There was no significant difference in the speed of pollen tube entering the ovary of florets of the same age in different months (p>0.05). The florets were pollinated 52h after flowering, and the values ​​in April and May dropped to 1.7% and 3.3% respectively, while the value in June was still 23.6%, which was significantly higher than that in April and May (p<0.05).

[0141] Table 3 Pollen germination rate and pollen tube growth rate on stigma at different periods

[0142]

[0143] Note: The first day is estimated based on the data results of 4-12 hours, the second day is estimated based on the data results of 28-36 hours, and the third day is estimated based on the data results of 52 hours. The + in the table represents the pollen germination rate or the pollen tube ratio, and - represents a pollen tube ratio of 0.

[0144] In summary, according to the pollen germination rate and pollen tube growth rate on the stigma in different periods (Table 3), the receptivity period of the stigma is 2 days in April and May, and 3 days in June. In April and May, pollination is done on the 3rd day. Although pollen germinates, the pollen tube grows slowly on the stigma and style and it is difficult to reach the ovary. Judging from the pollen germination rate on the stigma, the receptivity activity of the stigma is the highest in May, followed by April and the lowest in June. However, the receptivity period of the stigma in June is extended to 3 days, and the activity is relatively low. The pollen germination rate on the stigma on the 3rd day is basically less than 30%, and the proportion of pollen tubes entering the ovary is 23.6%.

[0145] 3.3 Pollen tube growth characteristics at different stages

[0146] The pollen tube growth characteristics were expressed by the proportion of florets with pollen tubes entering the ovules.

[0147] Depend on Figure 3 It can be seen that in April, May and June, pollination was carried out on the morning of the day when the florets bloomed. After 12 hours, only 4.8% of the florets in May were observed to have pollen tubes entering the ovules; after 24 hours, all the florets in May and June were observed to have pollen tubes entering the ovules, but only 60.4% in April, which was significantly lower than the values ​​in May and June; 36 hours after pollination, all the florets in May and June were observed to have pollen tubes entering the ovules, but only 58.8% of the florets in April had pollen tubes entering the ovules, indicating that the environmental temperature and humidity conditions in April were not conducive to the growth of pollen tubes. In summary, in April, May and June, the time from pollination to the entry of pollen tubes into the ovules was 1 day (24 hours). According to the proportion of florets with pollen tubes entering the ovules, the environmental temperature and humidity conditions in April were not conducive to the growth of pollen tubes, while the environmental temperature and humidity conditions in May and June were conducive to the growth of pollen tubes.

[0148] 3.4 Ovule lifespan at different stages

[0149] Depend on Figure 4 It can be seen that when the ovule vascular bundle ages and callus appears, the fluorescent staining is bright blue-green, but the callus of the inactive ovule will not always exist, and then disappear, and the fluorescence also disappears, making it difficult to distinguish between inactive ovules and viable ovules. This experiment only counts the proportion of flowers with ovule vascular bundle fluorescence at each time point after flowering, and the time before the appearance of flowers with ovule vascular bundle fluorescence (that is, all floret ovules are viable) is taken as the ovule life span.

[0150] Table 4 Ovule life span (unpollinated)

[0151]

[0152] Note: The table shows mean ± SD. The capital letters behind it represent the difference between the results at different times in the same month, and the lowercase letters represent the difference between the results at the same time in different months (Tukey test, P ≤ 0.05).

[0153] As shown in Table 4, the life span of ovules varies with the different months of flowering. In April and June, the fluorescence of the vascular bundle of Amomum villosum ovules appeared on the 4th day, and 5% and 33.3% of the ovary ovules showed fluorescence, respectively. In May, it appeared on the 3rd day, and 3.3% of the ovary ovules showed fluorescence. On the 5th day after flowering, the proportion of ovaries with ovule vascular bundle fluorescence in April, May, and June reached the highest, which were 71.8%, 67.3%, and 51.9%, respectively, and then decreased. It dropped to 18.7% on the 10th day after flowering in April, 0 on the 7th day after flowering in May, and 50% on the 10th day after flowering in June. According to the time and proportion of the appearance of the vascular bundle fluorescence of the ovules in the ovary, it is estimated that the life span of most ovules in April is more than 4 days, and most of them in May and June are at least more than 3 days, with the longest life span of ovules in April.

[0154] 3.5 Observation test on the change of fruiting rate with flower age at different periods

[0155] Depend on Figure 5 It can be seen that the fruiting rate varies with different months of flowering, and decreases with the extension of artificial pollination age. Artificial pollination 1 day after flowering, the fruiting rates in April-June are 45%, 84.6%, and 70%, respectively. The fruiting rate in May is significantly higher than that in April (p<0.05). Artificial pollination 1.5 days after flowering, the fruiting rates in April-June are 40%, 47.2%, and 50%, respectively. Artificial pollination 2 days after flowering, the fruiting rates in April-June are 13.3%, 30%, and 40%, respectively. By 2.5-3 days, the fruiting rate in April-June is almost close to 0. In summary, the fruiting rate on the first day is higher in different months, and it decreases more on the second day, but it can still bear fruit. Therefore, the fruiting age is 2 days, but the fruiting rate in each month is different, and the fruiting rate in May and June is higher than that in April.

[0156] 3.6 Determine the effective pollination period in different periods

[0157] Table 5 Estimation of effective pollination period in different periods based on the three elements of effective pollination period and fruiting rate

[0158]

[0159] Note: According to the calculation method of Williams, the three elements of the effective pollination period are used to calculate the effective pollination period: when the stigma receptivity time > ovule life time - (pollination to fertilization) time, the effective pollination period time = ovule life time - (pollination to fertilization) time; when the stigma receptivity time < ovule life time - (pollination to fertilization) time, the effective pollination period time = stigma receptivity time.

[0160] The results of the fluorescence microscopic observation of the vitality of the three elements in the effective pollination period of Amomum villosum and the changes in the pollination fruiting rate of Amomum villosum at different flower ages were summarized (Table 5). The effective pollination period in different months was about 2 days, but the effects on the vitality of the three elements in the effective pollination period were different. Under the conditions of high temperature and low humidity in April, the ovule life was the longest; under the conditions of medium temperature and medium humidity in May, the stigma receptivity activity was the highest; under the conditions of medium temperature and high humidity in June, the stigma receptivity time was the longest, and the pollen tube growth rate was the fastest. In terms of fruiting rate, the temperature and humidity conditions in May and June are more conducive to the fruiting of Amomum villosum.

[0161] 4 Conclusion

[0162] This study used fluorescence microscopy to study the three factors of Amomum villosum stigma receptivity, pollen tube growth characteristics, and ovule lifespan at different flowering periods (April, May, and June) and under different temperature and humidity conditions, as well as the changing characteristics of fruiting rate at different flowering ages. This study provides a scientific basis for exploring the effects of temperature and humidity on the effective pollination period of Amomum villosum, and provides a theoretical basis for the management of temperature and humidity during the flowering period of Amomum villosum in production. The conclusions are as follows:

[0163] (1) The effective pollination period and the three-factor activity under different temperature and humidity conditions at different stages of the flowering period were determined: Under different temperature and humidity conditions at different stages of the flowering period, the effective pollination period of Amomum villosum was estimated to be 2 days, but the three-factor activity values ​​during the effective pollination period varied. Under high temperature and low humidity conditions in April, the ovule life span was the longest, but it was not conducive to pollen tube growth, and the proportion of florets with pollen tubes entering the ovule was low. Under medium temperature and humidity conditions in May, the stigma receptivity activity was the highest, the pollen tube growth rate was fast, and the proportion of florets with pollen tubes entering the ovule was high; under medium temperature and high humidity conditions in June, the stigma receptivity time was the longest, the pollen tube growth rate was fast, and the proportion of florets with pollen tubes entering the ovule was high. In terms of fruiting rate, the temperature and humidity conditions in May and June were more conducive to fruiting of Amomum villosum.

[0164] (2) Measures to improve the fruiting rate of Amomum villosum: According to the research results, the flowering period can be postponed to May by breeding late-flowering varieties, spraying growth regulators, or increasing the planting altitude. For existing early-flowering varieties, the environmental conditions during the flowering period should be closely monitored and adjusted during the dry season in April to ensure that the environmental temperature and humidity are at the most suitable state. In actual production, corresponding measures need to be taken, such as using sunshade nets, humidifiers, or setting up temporary greenhouses to control temperature and humidity, so that Amomum villosum can adapt to climate changes in different seasons and ensure that the temperature and humidity conditions during its flowering period are optimal, thereby improving the pollination success rate and fruiting rate.

[0165] 5 Outlook

[0166] This study discussed the three factors affecting the effective pollination period of Amomum villosum and the changing characteristics of fruiting rate under different temperature and humidity conditions at different stages of the flowering period, providing an important theoretical basis for further protection and utilization of Amomum villosum resources. However, due to the complexity and uncertainty of natural conditions, future research still needs to explore the mechanism affecting the effective pollination period of Amomum villosum, and formulate more scientific and reasonable protection measures on this basis. These research conclusions are of great significance for understanding the reproductive ecology of Amomum villosum plants, and provide a scientific basis for its genetic improvement and protection of germplasm resources.

[0167] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may 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 the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for determining the effective pollination period of Amomum villosum at different flowering periods, characterized in that: The specific steps include: (1) Real-time monitoring of temperature and humidity at different times during the flowering period; (2) Fluorescence microscopy to observe stigma receptivity; (3) Fluorescence microscopy to observe pollen tube growth characteristics; (4) Observation of ovule lifespan using fluorescence microscopy; (5) Statistics on changes in fruiting rate at different flowering ages; (6) The effective pollination period at different stages of flowering was determined by experiments on changes in stigma receptivity, pollen tube growth characteristics, ovule life span and vitality, as well as changes in fruiting rate at different flower ages.

2. The method for determining the effective pollination period of Amomum villosum at different flowering periods according to claim 1, characterized in that: The step (1) specifically includes: during the flowering period of Amomum villosum, recording the changes in atmospheric temperature and humidity on the surface where the Amomum villosum florets grow.

3. The method for determining the effective pollination period of Amomum villosum at different flowering periods according to claim 1, characterized in that: The step (2) specifically comprises: conducting the test in the flowering period of Amomum villosum in April, May and June, bagging the plants in the afternoon of the day before the test, marking the Amomum villosum florets that opened on the next morning, and artificially pollinating the plants at 8:00-9:00 a.m. and 17:00-18:00 p.m. on the first and second days of flowering, and at 8:00-9:00 a.m. on the third day, with 30 florets for each treatment, cutting the florets for each treatment for 48 hours, removing the corolla, and immersing them in FAA fixative; For fluorescence microscopic observation, the flowers were first removed from the FAA fixative, rinsed, and then soaked in NaClO solution to make them transparent, then in NaOH solution to soften them, and then soaked in aniline blue staining solution to stain them in the dark; When making the slide, place the material on a glass slide, cut off the pistil from the connection between the style and the top of the ovary, straighten the stigma and style, add aniline blue dye, cover with a coverslip, and make a "stigma and style" slide; place the remaining ovary on a new glass slide, cut longitudinally from the middle of the ovary, spread the two cut surfaces flatly facing the slide, add aniline blue dye, cover with another glass slide, and complete the "ovary" slide; finally, place the prepared "stigma and style" slide and "ovary" slide under an ultraviolet fluorescence microscope to observe the receptivity of the stigma.

4. The method for determining the effective pollination period of Amomum villosum at different flowering periods according to claim 1, characterized in that: The step (3) specifically includes: bagging in the afternoon of the day before the experiment, marking the Amomum villosum florets that opened on the next morning, and artificially pollinating all of them on the first day of flowering, with 30 florets for each treatment, cutting the florets of each treatment at 8h, 24h, and 36h, removing the corolla, and immersing them in FAA fixative; For fluorescence microscopic observation, the flowers were first removed from the FAA fixative, rinsed, and then soaked in NaClO solution to make them transparent, then in NaOH solution to soften them, and then soaked in aniline blue staining solution to stain them in the dark; When making the slide, place the material on a glass slide, cut off the pistil from the connection between the style and the top of the ovary, straighten the stigma and style, add aniline blue dye, cover with a coverslip, and make a "stigma and style" slide; place the remaining ovary on a new glass slide, cut longitudinally from the middle of the ovary, spread the two cut surfaces flatly facing the slide, add aniline blue dye, cover with another glass slide, and complete the "ovary" slide; finally, place the prepared "stigma and style" slide and "ovary" slide under an ultraviolet fluorescence microscope to observe the growth characteristics of pollen tubes.

5. The method for determining the effective pollination period of Amomum villosum at different flowering periods according to claim 1, characterized in that: The step (4) is specifically as follows: bagging the flowers in the afternoon of the day before the experiment, and marking 300 Amomum villosum florets that were open on the day of the experiment on the morning of the next day, without pollination treatment, sampling 30 flowers every day from 17:00 to 18:00, for a total of 10 days, with the florets ranging in age from 1 day to 10 days; after sampling, removing the corolla and style, and immersing the flowers in FAA fixative; when observing under a fluorescence microscope, first take the flowers out of the FAA fixative, rinse them, and soak them in NaClO solution to make them transparent, in NaOH solution to soften them, and then soak them in aniline blue dye to avoid light for dyeing; When making the slide, place the material on a glass slide, cut it longitudinally from the middle of the ovary, spread it flat with two cut surfaces facing the glass slide, add aniline blue dye solution, cover it with a glass slide to make an "ovary" slide, place it under an ultraviolet fluorescence microscope, and observe the life span of the ovule.

6. A method for determining the effective pollination period of Amomum villosum at different flowering stages according to any one of claims 3 to 5, characterized in that: The FAA fixative is prepared by mixing 70% ethanol, 38% formaldehyde and acetic acid in a volume ratio of 8:1:1; the immersion time in the FAA fixative is at least 48 hours; the effective chlorine content of the NaClO solution is 1.6%; the immersion time in the NaClO solution is 4 hours; the concentration of the NaOH solution is 12 mol / L; the immersion time in the NaOH solution is 4 hours; the concentration of the aniline blue dye solution is 0.2%; and the immersion time in the aniline blue dye solution is at least 36 hours.

7. The method for determining the effective pollination period of Amomum villosum at different flowering periods according to claim 1, characterized in that: The step (5) is specifically as follows: bagging is performed in the afternoon of the day before the test, and the Amomum villosum florets that are open on the next morning are marked with signs, and artificial pollination is performed at 8:00-9:00 in the morning and 17:00-18:00 in the afternoon on the first and second days of flowering, and at 8:00-9:00 in the morning on the third day. After 10 days of artificial pollination, the bagging is removed and the fruiting rate of the florets in each treatment is counted.

8. The method for determining the effective pollination period of Amomum villosum at different flowering stages according to claim 7, characterized in that: The artificial pollination adopts the smearing method. Before pollination, a sufficient amount of pollen from the small flowers opened on the same day is collected, mixed, and tested by TTC. The pollen activity is more than 90%. When pollinating, the bag is removed, and the lower part of the corolla is first pinched with the thumb and middle finger of the left hand, and then the mixed pollen is dipped with a cotton swab and smeared on the stigma of the small flower, and then the bag is put again.

9. The method for determining the effective pollination period of Amomum villosum at different flowering stages according to claim 1, characterized in that: In step (6), the method for determining the effective pollination period at different flowering periods through the test of stigma receptivity, pollen tube growth characteristics and ovule life vitality changes is as follows: according to the calculation formula of Williams, when the stigma life is long, the effective pollination period = ovule life - pollination to fertilization time; when the stigma life is short, the stigma life is the main limiting factor, and the stigma life time is the effective pollination period time.

10. The method for determining the effective pollination period of Amomum villosum at different flowering stages according to claim 1, characterized in that: In step (6), the method for determining the effective pollination period at different flowering stages through the experiment on the change of fruit-bearing rate at different flower ages is as follows: the longest flower age that can bear fruit after artificial pollination is the effective pollination period.

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