Culture medium for promoting pollen germination of American holly and pollen viability detection method thereof

Abstract

The invention provides a culture medium for promoting pollen germination of ilex asprella and a pollen viability detection method thereof, and relates to the technical field of hybridization of ilex asprella. The culture medium for promoting the pollen germination of the American holly comprises 25g / L of sucrose, 75mg / L of boric acid and 25mg / L of calcium chloride, and a used solvent is distilled water; and the pH value is adjusted to 5.5 to 6.0. By utilizing the culture medium disclosed by the invention, the germination rate of the ilex uniflora pollen is remarkably increased, statistical errors caused by low germination rate of the ilex uniflora pollen are effectively reduced, and the method is visual in result and reliable in data, and can accurately and effectively detect the activity of the ilex uniflora pollen. The method has important practical significance for hybrid seed production of Ilex trifolia and cultivation of excellent new varieties.

Description

Technical Field

[0001] This invention relates to the field of American holly hybridization technology, and more specifically to a culture medium for promoting pollen germination of American holly and a method for detecting pollen viability. Background Technology

[0002] American holly (Ilex opaca) is the most important commercial holly native to North America. It possesses not only high horticultural value and ecological functions, but more importantly, as a cornerstone species in holly breeding, it contributes valuable cold-resistant and disease-resistant genes. Conventional hybridization breeding is one of the important methods in American holly breeding. Through hybridization with other species, it has greatly promoted the diversification and geographical adaptability of ornamental holly varieties. Currently, there are over 1000 varieties, adding countless excellent evergreen plant choices to the landscapes of temperate regions worldwide. Currently, the representative holly variety on the domestic market, 'Golden Holly' (I. × attenuata 'Sunny Foster'), is a hybrid of Dahong holly (I. cassine) and American holly. With its golden leaves and strong adaptability, it is an ideal choice for landscaping and greening.

[0003] Pollen viability testing is of great significance for hybridization breeding and artificial pollination. As the male gametophyte of plants, pollen plays an important role in sexual reproduction. To solve the problems of inconsistent flowering periods of parents, long-distance hybridization, and artificial pollination, it is usually necessary to collect and store pollen early. However, before use, viability testing must be performed to predict whether the pollen has fertilization capacity, so as to determine the amount of pollen to be used for artificial pollination and the effectiveness of hybridization.

[0004] However, American holly pollen itself has low activity, and its activity further decreases after low-temperature storage. Therefore, confirming pollen viability through effective pollen viability detection methods is fundamental to American holly hybridization breeding. Currently, pollen viability is mainly determined by staining, in vitro germination, and in vivo germination methods. While the staining method is fast and quick, the results are highly unstable and prone to significant errors. The in vitro pollen germination method simulates the germination conditions of pollen on the stigma by selecting suitable culture media and temperatures, thereby determining the pollen's ability to germinate pollen tubes. Because of its advantages such as ease of observation and counting, quantitative control of culture conditions, and accurate data, the in vitro germination method has become the most reliable and effective method for determining pollen viability. However, in vitro pollen germination is greatly affected by culture conditions, and the required culture media and conditions vary significantly among different plants. Currently, there are few reports on in vitro germination methods for holly pollen, with only reports on *Ilex brevicornis*, *Ilex integrifolia*, *Ilex dabieensis*, and *Ilex macrocarpa*, and the pollen germination rates of these holly species are all very low (below 10%). To date, there have been no reports on the technology for detecting the activity of American holly pollen.

[0005] Therefore, providing a culture medium or method to promote pollen germination of American holly is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a culture medium for promoting pollen germination of American holly and a method for detecting pollen viability.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A culture medium for promoting pollen germination of American holly, the culture medium comprising 25 g / L sucrose, 75 mg / L boric acid and 25 mg / L calcium chloride, using distilled water as the solvent; and the pH adjusted to 5.5-6.0.

[0009] Preferably, the pH of the culture medium is adjusted to 5.5.

[0010] Another objective of this invention is to provide a method for detecting the viability of American holly pollen, wherein American holly pollen is cultured using the above-mentioned culture medium, and pollen viability is detected by statistically analyzing the pollen germination rate.

[0011] Preferably, pollen from American holly is inoculated onto pollen germination medium for pollen culture at 25-30℃ for 5-6 hours, then examined under a microscope to calculate the germination rate and detect pollen viability.

[0012] More preferably, the culture temperature is 25°C and the culture time is 6 hours.

[0013] Beneficial effects: This invention provides a culture medium for promoting pollen germination of American holly and a method for detecting pollen viability. Using the culture medium of this invention, the pollen germination rate of American holly is significantly improved, effectively reducing the statistical error caused by the low pollen germination rate of this species. Moreover, the method provides intuitive results and reliable data, and can accurately and effectively detect the viability of American holly pollen. It has important practical significance for hybrid seed production of American holly and for cultivating superior new varieties of American holly. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 Micrographs of pollen germination using the germination culture medium formulation of this invention;

[0016] Figure 2The pollen germination and pollen tube elongation under different culture time conditions;

[0017] Figure 3 These are microscopic images taken using existing germination medium formulations for pollen viability testing. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Example 1

[0020] 1. Pollen collection

[0021] Pollen was collected from American holly plants grown at the Jiangsu Provincial Academy of Forestry Sciences for subsequent pollen culture. The process included the following steps:

[0022] (1) Flower branch collection and hydroponics: In May, from 09:00 to 10:00, select healthy, disease-free and vigorous American holly male plants as pollen collection trees, collect flower branches at the large bud stage, bring them back to the laboratory and insert them into conical flasks filled with clean water for 1-2 days.

[0023] (2) Pollen drying and collection: Select flower buds that are about to bloom, remove the anthers and dry them in a constant temperature incubator at 25 ℃ for 8 h, and then collect the pollen for subsequent germination experiments.

[0024] Example 2

[0025] 1. Preparation of culture medium: Prepare liquid culture medium with distilled water, containing 25 g / L sucrose, 75 mg / L boric acid and 25 mg / L calcium chloride. Adjust the pH to 5.5 and pour into Erlenmeyer flasks for later use.

[0026] 2. Pollen germination culture: Place an appropriate amount of liquid culture medium into the concave part of a glass slide. Use a cotton swab to pick up pollen and gently flick it onto the liquid culture medium. The amount of pollen should be enough to cover the surface of the culture medium drop. Place the glass slide in a petri dish with moistened filter paper, cover it, label it, and incubate it in the dark at 25°C for 6 hours.

[0027] 3. Pollen viability detection: After induced germination, remove the slide and observe it under a fluorescence inverted microscope. When calculating the germination rate, randomly select 5 fields of view from at least 50 pollen grains, and take pictures of the clearest field of view (e.g., ...). Figure 1As shown in the figure, the number of germinated pollen flowers is counted to calculate the pollen germination rate, and the average value is taken. The calculation formula is: Pollen germination rate (%) = Number of germinated pollen flowers / Total number of pollen flowers × 100%.

[0028] Note: When calculating the number of germinating pollen, pollen with a pollen tube length exceeding 2 / 3 of the pollen grain diameter is considered as germinating pollen.

[0029] In this embodiment, the pollen germination rate of American holly was 56.56%.

[0030] Experimental Example 1

[0031] Effect of different culture times on pollen germination rate

[0032] Based on Example 2, this experiment used the dark culture time as a variable to count the germination rate of American holly pollen. The time groups were 1, 2, 3, 4, 5, 6, 8, and 10 hours. The rest of the operation was completely consistent with Example 2. The results are shown in Table 1.

[0033] Table 1 shows that pollen germination rate and pollen tube length increase with increasing dark culture time. After 1 hour of culture, pollen from *Ilex chinensis* begins to germinate; with increasing dark culture time, the pollen germination rate increases significantly; after 6 hours, the pollen germination rate rapidly increases to 56.24%, and the pollen tube length reaches 118.58 μm. At this point, the pollen tubes are clearly distinguishable and easy to count under a microscope (e.g., ...). Figure 2 (As shown in the image) After 6 hours, the pollen germination rate stabilizes, but the pollen tubes begin to elongate rapidly, reaching a length of 270.45 μm after 12 hours. They become intertwined and even extend beyond the field of view, making it difficult to trace their origin to a specific pollen grain, easily leading to duplicate or missed counts. Therefore, from the perspectives of saving time, improving efficiency, and ensuring counting accuracy, 5–6 hours is the optimal time for dark culture of American holly pollen.

[0034] Table 1. Pollen germination and pollen tube growth of *Ilex chinensis* under different dark culture times.

[0035]

[0036] Experiment Example 2

[0037] Effect of different culture temperatures on pollen germination rate

[0038] Based on Example 2, this experiment uses culture temperature as a variable to statistically analyze the germination rate of American holly pollen. The culture temperatures were grouped as 15℃, 20℃, 25℃, 30℃, and 35℃, respectively. The remaining operations were completely consistent with Example 2. The results are shown in Table 2.

[0039] Table 2 shows that after 6 hours of dark incubation, the pollen germination rate and pollen tube length were significantly higher at temperatures of 25℃ and 30℃ than in other treatments, with the pollen germination rate reaching as high as 55.78% at 25℃. Therefore, 25–30℃ can be considered the optimal in vitro culture temperature for American holly pollen.

[0040] Table 2. Pollen germination and pollen tube growth of *Ilex chinensis* at different culture temperatures.

[0041]

[0042] Experimental Example 3

[0043] Effects of different sucrose concentrations, boric acid concentrations, calcium chloride concentrations, and pH on pollen germination rates

[0044] In this experiment, the basal culture medium consisted of 100 g / L sucrose, 100 mg / L boric acid, and 100 mg / L calcium chloride, with a pH of 7.0. The basal conditions were modified by setting the experimental variables to the concentrations of sucrose, boric acid, calcium chloride, and pH in the culture medium. Specifically, the variables were set as follows: sucrose concentrations (0 g / L, 50 g / L, 100 g / L, 150 g / L, and 200 g / L), boric acid concentrations (0 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L), calcium chloride concentrations (0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 300 mg / L), and pH values ​​of 4, 5, 6, 7, and 8. Other procedures were the same as in Example 2.

[0045] 1) The pollen germination rate under different sucrose concentrations is shown in Table 3.

[0046] As shown in Table 3, adding sucrose at a concentration of 50 g / L to the culture medium significantly promotes pollen germination. The pollen germination rate reaches its highest level (30.80%) when the sucrose concentration is increased to 50 g / L. However, the pollen germination rate decreases significantly when the concentration exceeds 100 g / L. Therefore, an appropriate concentration of sucrose promotes pollen germination and growth, while excessively high concentrations inhibit pollen germination. The optimal concentration of sucrose used alone is 0-50 g / L.

[0047] 2) The pollen germination rate under different boric acid concentrations is shown in Table 3.

[0048] As shown in Table 3, adding boric acid at a concentration of 50 mg / L to the culture medium significantly promotes pollen germination. The pollen germination rate reaches its highest level (30.14%) when the boric acid concentration increases to 100 mg / L. However, the pollen germination rate decreases significantly when the concentration exceeds 150 mg / L. Therefore, a suitable concentration of boric acid promotes pollen germination and growth, while an excessively high concentration inhibits pollen germination. The optimal concentration of boric acid when used alone is 50-100 mg / L.

[0049] 3) The pollen germination rate under different calcium chloride concentrations is shown in Table 3.

[0050] As shown in Table 3, adding calcium chloride at a concentration of 50 mg / L to the culture medium can promote pollen germination. The pollen germination rate reaches its highest level (12.21%) when the boric acid concentration is increased to 50 mg / L. However, the pollen germination rate decreases significantly when the concentration is greater than 100 mg / L. Therefore, an appropriate concentration of calcium chloride promotes pollen germination and growth, while an excessively high concentration will inhibit pollen germination. The optimal concentration of calcium chloride when used alone is 50-100 mg / L.

[0051] 4) The pollen germination rate under different pH conditions is shown in Table 3.

[0052] Table 3 shows that the pH value of the culture medium has a significant impact on the pollen germination of American holly. The germination rate reaches its maximum of 29.81% at a pH value of 6. When the pH value is greater than 7, the pollen germination rate drops sharply. Therefore, under these experimental conditions, the optimal pH value for the culture medium is 6.

[0053] Table 3. Effects of different component concentrations and pH on pollen germination of American holly.

[0054]

[0055] Experiment Example 4

[0056] Orthogonal experiment for screening based on the mass concentrations of sucrose, boric acid, and calcium chloride, and the pH of the culture medium.

[0057] Based on Example 2, this experiment used the results of single-factor experiments to conduct a four-factor, three-level orthogonal experiment. The experiment was conducted with sucrose at 25 g / L, 50 g / L and 75 g / L, boric acid at 75 mg / L, 100 mg / L and 125 mg / L, calcium chloride at 25 mg / L, 50 mg / L and 75 mg / L, and pH at 5.5, 6 and 6.5 levels. The experiment was designed using an L9(34) orthogonal array and cultured in a constant temperature incubator set at 25°C. Each experiment was repeated three times, and the pollen germination rate was counted to screen out the liquid culture medium most suitable for the germination of American holly pollen.

[0058] Based on the suitable mass concentrations of each component in the culture medium for the germination of American holly pollen obtained from single-factor experiments, L9 (3 4 An orthogonal experiment was conducted using an orthogonal array (Table 4). Range analysis was performed on the experimental results. Table 5 shows that treatment 1 (25 g / L sucrose, 75 mg / L boric acid, 25 mg / L calcium chloride, and pH 5.5) had the highest pollen germination rate of 56.56%. The order of influence of the four culture medium components on the in vitro germination rate of *Ilex chinensis* pollen was boric acid, calcium chloride, sucrose, and pH. Therefore, it can be inferred that the optimal culture medium is 25 g / L sucrose + 75 mg / L boric acid + 25 mg / L calcium chloride, with a pH of 5.5.

[0059] Table 4 Results of the orthogonal experiment on germination rate

[0060]

[0061] Table 5 Range analysis of germination rate orthogonal experiment results

[0062]

[0063] Comparative Example 1

[0064] This comparative example is based on Example 2, but the culture medium was replaced with the holly pollen germination culture medium formula (10% sucrose + 0.006% H3BO3) disclosed in the literature (Guo Juan, Qiu Shuai, Liu Huahong, et al. Study on screening and storage conditions of pollen in vitro culture medium of four holly species [J]. Journal of Northwest Forestry University, 2018, 033(006):118-126.). Figure 3 The germination rate is extremely low, with almost no pollen germination.

[0065] Comparative Example 2

[0066] Based on Example 2, this comparative example removed 75 mg / L boric acid and 25 mg / L calcium chloride from the germination medium, and then cultured pollen for germination, and the pollen germination rate was counted.

[0067] Comparative Example 3

[0068] This comparative example is based on Example 2, but with the removal of 25 g / L sucrose and 25 mg / L calcium chloride from the germination medium, pollen germination culture was performed, and the pollen germination rate was counted.

[0069] Comparative Example 4

[0070] Based on Example 2, this comparative example removed 25 g / L sucrose and 75 mg / L boric acid from the germination medium, and then cultured pollen for germination, and the pollen germination rate was calculated.

[0071] Comparative Example 5

[0072] Based on Example 2, this comparative example adjusts the pH of the germination medium to 8, conducts pollen germination culture, and counts the pollen germination rate.

[0073] Example 1

[0074] The pollen germination rates of Examples 2 and Comparative Examples 1-5 were statistically analyzed, and the results are shown in Table 6. It is evident that changes in the culture additives significantly affected the pollen germination rate of *Ilex chinensis*. Furthermore, sucrose, boric acid, calcium chloride, and pH had a synergistic effect in promoting *Ilex chinensis* pollen germination. Example 2 showed the highest pollen germination rate, reaching 56.56%, indicating that this culture medium condition yielded the best pollen germination effect for *Ilex chinensis*.

[0075] Table 6. Effects of different culture conditions on pollen germination of American holly.

[0076]

[0077] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0078] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A culture medium for promoting pollen germination of American holly, characterized in that, The culture medium consists of 25 g / L sucrose, 75 mg / L boric acid and 25 mg / L calcium chloride, and the solvent used is distilled water; and the pH is adjusted to 5.5-6.

0.

2. The culture medium for promoting pollen germination of American holly according to claim 1, characterized in that, The pH of the culture medium was adjusted to 5.

5.

3. A method for detecting pollen viability of American holly, characterized in that, American holly pollen was cultured using the culture medium described in claim 1 or claim 2, and pollen viability was detected by statistically analyzing the pollen germination rate.

4. The method for detecting pollen viability of American holly according to claim 3, characterized in that, American holly pollen was inoculated onto pollen germination medium for pollen culture at 25-30℃ for 5-6 hours, then examined under a microscope to calculate the germination rate and detect pollen viability.

5. The method for detecting pollen viability of American holly according to claim 4, characterized in that, The culture temperature is 25°C and the culture time is 6 hours.

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