Preliminary Study on in Vitro Germination of Yangmei Pollen

Congling FANG, Guangru CHAI, Shiqiang CHEN

1. Zhejiang Cixi Linte Technology Promotion Center, Cixi 315300, China; 2. People’s Government of Henghe Town, Zhejiang Province, Henghe 315318, China

Abstract [Objectives] To explore the conditions for in vitro culture of Yangmei pollen. [Methods] Experiments were conducted on the germination characteristics of three types of Yangmei pollen using in vitro culture and germination method. [Results] The suitable medium ratio for the germination of Yangmei pollen was 10% sucrose+0.01% borax+1% agar; the cultivation temperature of 30 ℃ was more suitable for the germination of Yangmei pollen than 25 ℃; through analysis of variance, among the three types of Yangmei pollen, pollen of male 1 had the strongest viability, and it was the better pollination type. [Conclusions] The research could provide certain basis for introduction and cultivation of Yangmei and improving hybrid breeding effect.

Key words Yangmei, Pollen, In vitro germination, Culture medium

1 Introduction

The germination ability of plant pollen is an important aspect of the study of flowering biological characteristics. Pollen germination ability of fruit trees directly affects their pollination, fertilization, and even fruit setting[1]. Therefore, it is necessary to understand the pollen viability of varieties in both introduction cultivation and hybrid breeding work, in order to successfully carry out introduction and cultivation and improve the effectiveness of hybrid breeding.

2 Materials and methods

2.1CollectionandstorageofmaterialsPollen was collected from male Yangmei varieties male 1, male 2, and male 3 planted in Sishan Park, Cixi City. On the morning of April 2, 2022, the branches flowering but not yet dispersed were collected. After returning to the laboratory, the male inflorescence pollen was collected and dried for pollen germination experiments.

2.2TestmethodInvitrogermination assay is a method of measuring the germination ability of collected pollen by sowing it on a specific culture medium and under certain environmental conditions[2]. The culture medium was commonly prepared with sucrose, agar (1%), borax, and distilled water. 0%, 5%, 10%, 15% sucrose concentration+0.01% borax was used as the sucrose gradient solution, and 0%, 0.01%, 0.05%, 0.1% borax+10% sucrose was used as the borax gradient solution.

After boiling the prepared culture medium (agar solution), it was immediately titrated on a concave glass slide. After cooling, the pollen to be tested was evenly sprinkled on the culture medium. Three replicates were made for each sample. The pollen germination potential experiment was observed every 4 h, and the gradient experiment was observed every 12 h. Five fields of view were observed each time, with a total observed pollen count greater than 100. The germination was marked by the length of the pollen tube being greater than the diameter of the pollen grains. The total number of pollen and the number of germinated pollen in one field of view were counted, and the average obtained from several fields of view was calculated to obtain more accurate numbers. The germination rate was calculated using the following formula:

Germination rate=(Number of germinated pollen/Total pollen count)×100%

3 Results and analysis

3.1EffectofdifferentsucroseconcentrationsonpollengerminationrateUsing 1% agar+0.01% borax, and adding 0%, 5%, 10%, and 15% sucrose solutions respectively, the germination experiment of Yangmei pollen was conducted. The results showed that all the above concentrations of treatments had a certain germination rate, but the germination rates of 5% and 15% sucrose concentrations were extremely low, while the germination rates of 0% and 10% sucrose concentrations were higher, and the germination rate of 10% sucrose was 2.6%, which was the highest. After analysis of variance, the difference was extremely significant. This indicated that 10% sucrose solution was the most conducive to the germination of Yangmei pollen. When the sucrose concentration was too high, it could inhibit pollen germination, and its germination rate could decrease to below 0.4%. So 10% sucrose solution was a more suitable culture medium for Yangmei pollen (Table 1).

Table 1 Germination rate of Yangmei pollen in culture media with different sucrose concentrations

3.2EffectofdifferentboraxconcentrationsonpollengerminationrateUsing 1% agar+10% sucrose solution, and adding 0%, 0.01%, 0.05%, and 0.1% borax solutions respectively, experiments on the germination of Yangmei pollen were conducted. The results showed that pollen germination was better at 0% and 0.01% borax concentration, with 0.01% borax concentration being the most obvious. The germination rates of the three samples were 2.61%, 2.62%, and 2.47%, respectively. When the concentration increased to 0.05%, the pollen germination rate significantly decreased, and the germination rates of the three samples were only 0.07%, 0.09%, and 0.10%. The increase in borax concentration greatly inhibited the germination potential of Yangmei pollen. Through analysis of variance, the differences between them were extremely significant. So the appropriate concentration of borax for the germination of Yangmei pollen was 0.01%, and too high concentration can actually inhibit its germination (Table 2).

Table 2 Germination rate of Yangmei pollen in culture media with different borax concentrations

3.3MeasurementresultsoftheviabilityoffreshlyharvestedpollenatdifferentcultivationtemperaturesThe germination rate of pollen was influenced by the culture temperature under certain moisturizing conditions[3-4]. Fresh Yangmei pollen that has not been stored was cultured in a 25 ℃ incubator using standard concentration medium and moistened in a petri dish. After 8 h of observation, the germination rate of all three samples was zero. When the germination time was extended to 12 h, germinating pollen grains were only observed in male 1 and male 2, while male 3 still did not germinate. After 20 h of cultivation, germination particles were observed in male 3. At the same time, it was found that male 1 reached its peak germination period after cultivation for 32-68 h, but the germination rate was not very high, ranging from 4.70% to 5.70%. However, male 2 reached its peak germination period after cultivation for 32-64 h, but it was not very stable, and like male 1, the germination rate was not high, ranging from 1.95% to 6.73%. Male 3 reached its peak germination period after cultivation for 44 to 68 h, with a more concentrated time compared to the first two. The germination rate was also lower than the first two, ranging from 2.24% to 3.58% (Fig.1).

Fig.1 Germination situation of Yangmei pollen under 25 ℃ (A) and 30 ℃ (B)

Fresh Yangmei pollen was cultured in a 30 ℃ incubator using standard concentration medium and moistened in a petri dish. After 8 h of observation, the pollen germination rates of male 1, male 2, and male 3 were 4.18%, 6.19%, 3.96%. Male 1 reached its peak germination period after 16 to 64 h of cultivation, with germination potential ranging from 5.55% to 8.44%. The germination rate of male 2 fluctuated greatly and was unstable during cultivation, but the overall germination rate was higher than that of male 1. Male 3 reached the peak of germination after 36 to 60 h of cultivation, and the time was more concentrated than the first two. Therefore, for pollen cultured at 30 ℃, the germination of male 2 was the best, followed by male 1, and male 3 was the worst (Fig.1).

Comparing the pollen germination in a 25 ℃ incubator with that in a 30 ℃ incubator, it can be observed that (i) the germination time of pollen in a 30 ℃ incubator was generally earlier than that in a 25 ℃ incubator, especially for male 3, which was 8 h earlier; (ii) during the cultivation process, the germination rate of pollen in a 30 ℃ incubator was generally higher than that in a 25 ℃ incubator. From the experiment, it can be seen that the cultivation temperature of 30 ℃ in the germination experiment of Yangmei pollen was better than that of 25 ℃. So, temperature had a significant impact on pollen germination under certain conditions.

4 Conclusions

(i)A culture medium suitable for the germination of Yangmei pollen: 10% sucrose solution was beneficial for the germination of Yangmei pollen, while a high concentration can inhibit pollen germination; it can improve the germination rate of Yangmei pollen by adding a small amount of borax (approximately 0.01% concentration) to the sucrose solution, with the best medium ratio being 10% sucrose+0.01% borax+1% agar.

(ii) There was a close relationship between cultivation temperature and the germination of Yangmei pollen tubes. The experimental results indicated that Yangmei pollen can germinate nor-mally at a culture temperature of 25-30 ℃. At 25 ℃ and below, pollen germination was slow, and the germination rate was low. When the temperature increased to 30 ℃, the pollen germination time was earlier, and the germination rate of Yangmei pollen was significantly increased. From this, it can be concluded that theinvitroculture temperature of Yangmei pollen at 30 ℃ was more suitable for germination than at 25 ℃.

(iii)Invitropollen culture is currently recognized as a good method for measuring pollen viability, but this method requires specific temperature and a longer time (112 h of cultivation in this experiment) to achieve. Therefore, this method is used when conditions permit. At the same time, a certain number of repetitions are required to ensure the accuracy of the measured results.