Influence of Boric Acid and Sucrose on the Germination and Growth of Areca Pollen ()
1. Introduction
Areca palm, also known as “lazy tree” and “money tree”, is an important farming product in Hainan province, China. Approximately 400 - 600 million people worldwide chew areca nut, demonstrating the promising market prospects presented by the harvest and processing of areca nut.
Areca palm is a cross-pollinated crop, and its florescence lasts from May to July, during which Hainan experiences high temperatures and frequent rains. These adverse weather conditions can easily induce harmful flower and fruit drop from areca palm, yet related preventive measures have not been established. Previous studies have suggested that appropriate concentrations of sucrose and boron can promote the germination and growth of the pollen tube of areca pollen cultured in vitro [1,2]. Moreover, boron is one of the essential micronutrients in higher plants. Boron-deficient plants are characterized by dysplasia of the reproductive organs [3]. Boron deficiency in rape, for example, causes floral organ dysplasia or morphological abnormalities in the plant [3,4], thereby preventing pollination. Under low boron stress, the vitality and respiratory strength of pollen decrease [4,5]. By in vitro areca pollen germination and growth test, this study investigates the influence of boric acid and sucrose on the germination rate of areca pollen, as well as on the duration of pollen development and germination. This research also provides theoretical basis for concentration tests on boric acid and sucrose through spraying in areca palm plantations.
2. Materials and Methods
2.1. Materials
From July to August, pollen samples were collected from the Arecanut Germplasm Resource Nursery of Coconut Research Institute of Chinese Academic of Tropical Agricultural Sciences. Native adult Hainan areca palm trees that were planted in the field six years ago were selected. The plants are characterized by consistent agronomic traits and medium growth. When the spathes fell off and male flowers opened to about one-third of full bloom, we cut a few flower branches.
2.2. Methods
2.2.1. Experimental Design
Before normal tests were conducted, several prophase preparation tests were performed with reference to the pollen culture medium formulas of other crops. The normal tests were designed to achieve five goals. First, the effects of culture media with different boric acid concentrations on the germination rate of areca pollen were determined. In this test, a randomized block experiment with 11 different concentration treatments and four repetitions was set. Second, the effects of culture media with different boric acid concentrations on the growth of areca pollen tube were investigated. The treatments and repetitions in this test were the same as those in the first test. Third, the effects of different sucrose concentrations on the germination rate of areca pollen were examined. This test included nine different concentration treatments and four repetitions. Fourth, the effects of different culture media on the germination rate of areca pollen were determined. Two treatments and 27 sets of matching experimental studies were carried out. Finally, the effects of different culture times on the growth of areca pollen tube were investigated. Areca pollens samples were observed every 30 min during culture and measured at 11 time points.
2.2.2. Observation and Statistical Analysis
The tests for each group were performed at room temperature (approximately 30˚C). The pollen grains used in each treatment with a consideration for the five goals previously mentioned were chosen from the fresh inflorescences of the same bunch on the same day. All the tests were completed on the same day to eliminate the differences caused by non-experimental treatment factors. The experiments were conducted as follows. Absorbent paper was placed in a culture dish and wetted by distilled water. Then, a gel dropper was used to extract culture medium, after which two drops of the medium were placed on each glass slide. Areca pollen grains were picked using an inoculating needle. Pollen grains were transferred onto the culture medium by gently flicking the inoculating needle. The glass slides were placed into covered culture dishes, and germination time was recorded.
A Leica DM2500 optical microscope was used to observe the areca pollen grains and capture images during the culture. Each view spans approximately 30 areca pollen grains. When pollen tube length reaches the diameter of areca pollen, the sample is defined as a germinated areca pollen. The germination ratio of areca pollen is defined as follows:
Areca pollen germination rate (%) = (germinated areca pollen grain number/total areca pollen grain number) × 100%.
Leica Application Suite software was used to measure the length of areca pollen tube. Excel, DPS, and ORIGIN 8.0 statistical software were used in data processing, mapping, significant difference comparison, and relevant regressions. Figures 1 and 2 were the initial germination and mature growth of areca pollen on culture media.
3. Results
3.1. Effects of Boric Acid Concentration on the Development of Areca Pollen Tube
3.1.1. Effects of Boric Acid Concentration on Pollen Germination Rate
Areca pollen grains were cultured on culture media with 40 g/L sucrose, 5 g/L agar, and boric acid concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, and 5.0 g/L. Each treatment was prepared with two glass slides, and each glass slide contained culture media on two sites. Therefore, each treatment is equivalent to four repetitions. Areca pollen grains were cultured on culture media with different boric acid concentrations for 150 min to 180 min. The pollen samples comprised approximately 30 uniformly dispersed pollen grains. Table 1 lists the observation results, which were initially transformed using
Figure 1. The initial germination stage.
the arcsine square root in DPS statistical software. Then, variance analysis and multiple comparisons were performed on the transformed results using Duncan’s new multiple range test. Finally, the findings were mapped using MS Excel (Figure 3).
Table 1 and Figure 1 show that areca pollen exhibits a low germination rate in the medium without boric acid. The addition of 0.1 g/L boric acid significantly increases the pollen germination rate. Although the germination rate increases at boric acid concentrations of 0.1, 0.2, and 0.4 g/L, the difference does not reach a level of significance. As boric acid concentration increases to 0.6 g/L, germination rate decreases, but this reduction does not reach a level of significance. As boric acid concentration