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The different ice cream fruit varieties showed significant morphological differences in their stamens, as shown in Fig. 2.
Figure 2.
(a) W4 variety – no pollen. (b) 4P variety – little pollen. (c) AU2 variety – abundant pollen.
The varieties with no pollen showed large ovaries, with a visible, yellow pistil stigma. The stamens and yellow and black, with dry and infertile anthers.
The varieties with little pollen stigma is pink or yellow-pink, and the ovary and stigma are abnormally large. Five stamens are very small, the anthers are yellow-white or yellow-black, and the pollen content is very little.
The varieties with abundant pollen pistil is very small, and the stigma is not obviously light green, the capital part is also very small, but the five stamens are very thick, especially the anther flower sac is also very large, showing bright yellow, the pollen content is very high.
In Fig. 3, the development process of naturally self-pollinated fruit is recorded, with the corresponding data for this experimental group shown in Table 1.
Table 1. Statistics of the naturally self-pollinated fruit.
Breed Rate of fruit set
(%)Width
(cm)Length
(cm)Sugar
(%)Abundant pollen AU1 0% − − − AU2 25% 4.1 ± 0 4 ± 0 10.7 ± 0 N2 50% 4.7 ± 0.2 3.95 ± 0.25 9.1 ± 0.1 Little pollen 4P 66% 7.5 ± 0.2 6.9 ± 0.1 11.7 ± 0.5 AU4 50% 8.3 ± 0.2 7.6 ± 0.3 10.5 ± 05 C1 100% 6.6 ± 0.2 5 ± 0.1 10.9 ± 0.1 Comparing the two groups (abundant pollen and little pollen) shows that the fruit set rate, fruit size and sweetness were all higher in varieties with little pollen, compared to those with more abundant pollen. Within each group, variety N2 showed the best performance for the abundant pollen varieties, while variety 4 showed the highest sweetness and acceptable size and fruit set amongst the varieties with little pollen. However, variety C1 did show the highest rate of fruit set.
To compare the effects of natural and artificial pollination, another experiment was carried out with artificial self-pollination conducted at different times every day. The bag surrounding each flower was gently shaken each time to ensure that the pollen was transferred from the stamens to the stigma; this process was repeated until the stamens and stigma withered, and the fruit began to grow, indicating that pollination was successfully completed.
As shown in Table 2, the fruit set rate of ice cream fruit varieties with abundant pollen increased in the case of artificial pollination. However, there was no significant change in the size or sweetness of the fruit quality. It is known that artificial pollination can slightly improve the fruit set rate of abundant pollen varieties, but it has no significant effect on fruit quality.
Table 2. Statistics of experimental data of artificial self pollination.
Breed Fruit set
(%)Width
(cm)Length
(cm)Sugar
(%)Abundant pollen AU1 33% 5.9 ± 0 5.5 ± 0 10.0 ± 0 AU2 50% 4.4 ± 0.5 4.0 ± 0.1 9.5 ± 0.5 N2 40% 6.0 ± 0 4.5 ± 0 8.6 ± 0 Little pollen 4P 66% 7.7 ± 0.1 6.4 ± 0.8 11.0 ± 1.0 AU4 50% 8.9 ± 0.1 7.5 ± 0.1 10.2 ± 0.2 C1 100% 7.8 ± 0.1 6.9 ± 0.1 9.5 ± 0.1 Therefore, artificial cross-pollination and hybridization studies were conducted to find possible combinations which could provide improved fruit quality compared to self-pollination.
Artificial cross-pollination (male/female)
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Tables 3−8 show the experimental results from the artificial cross-pollinations, broken down by each female parent. Additionally, each table shows the results with and without emasculation.
Table 3. Experimental data for the cross-pollination with AU4 as the maternal parent, with and without emasculation.
Group Mother tree Father tree Fruit set (%) Length (cm) Width (cm) Sugar (%) Female AU4 (little pollen) Little pollen C1 33% 7.5 ± 0.1 7.4 ± 0.1 8.9 ± 0.1 4P 50% 8.8 ± 0.2 7.9 ± 0.2 10.4 ± 0.1 Abundant pollen AU1 40% 8.15 ± 0.1 7.75 ± 0.1 10.4 ± 0.2 AU2 50% 9.2 ± 0.1 7.95 ± 0.1 9.5 ± 0.1 N2 75% 9.47 ± 0.1 8.47 ± 0.1 10.5 ± 0.5 Male Little pollen C1 0% − − − 4P 50% 8.05 ± 0.1 7.95 ± 0.2 8.5 ± 1 Abundant pollen AU1 33% 8.7 ± 0.2 8.5 ± 0.1 8.0 ± 2 AU2 33% 8.5 ± 0.3 7.9 ± 0.1 8.1 ± 0.2 N2 50% 9.2 ± 0.3 8.04 ± 1 8.5 ± 1 Table 4. Experimental data for the cross-pollination with 4P as the maternal parent, with and without emasculation.
Group Mother tree Father tree Fruit set (%) Length (cm) Width (cm) Sugar (%) Female 4P (little pollen) Little pollen C1 50% 7.8 ± 0.6 7.0 ± 0.2 9.8 ± 1.2 AU4 66% 6.9 ± 0.1 6.3 ± 0.2 8.78 ± 1.0 Abundant pollen AU1 50% 8.7 ± 0.6 6.57 ± 1.0 9.8 ± 0.5 AU2 50% 7.35 ± 0.2 6.45 ± 2.0 8.5 ± 1.5 N2 80% 7.7 ± 1.0 7.075 ± 1.0 11.0 ± 1.0 Male Little pollen C1 42% 8.5 ± 0.3 6.9 ± 0.2 10.5 ± 0.5 AU4 50% 8.0 ± 2.0 7.95 ± 2.0 10.0 ± 0.8 Abundant pollen AU1 50% 7.15 ± 2.0 6.55 ± 3.0 9.2 ± 0.6 AU2 42% 6.8 ± 0.4 6.1 ± 0.5 8.87 ± 0.4 N2 62% 7.5 ± 0.3 7.4 ± 0.6 9.84 ± 0.3 Table 5. Experimental data for the cross-pollination with C1 as the maternal parent, with and without emasculation.
Group Mother tree Father tree Fruit set (%) Length (cm) Width (cm) Sugar (%) Female C1 (little pollen) Little pollen AU4 60% 7.27 ± 1.0 6.76 ± 1.0 10.3 ± 1.0 4P 33% 6.9 ± 0.4 5.9 ± 0.3 10.0 ± 0.5 Abundant pollen AU1 25% 6.8 ± 0.5 6.2 ± 0.6 8.0 ± 0.4 AU2 33% 5.9 ± 0.4 5.9 ± 0.3 10.2 ± 1.0 N2 50% 6.8 ± 0.6 6.8 ± 0.3 11.0 ± 0.5 Male Little pollen AU4 50% 6.9 ± 0.2 6.8 ± 0.4 10.3 ± 1.0 4P 0% − − − Abundant pollen AU1 0% − − − AU2 33% 6.8 ± 0.4 5.9 ± 0.1 10.0 ± 0.5 N2 50% 7.1 ± 0.5 5.9 ± 0.2 9.16 ± 1.0 Table 6. Experimental data for the cross-pollination with N6 as the maternal parent, with and without emasculation.
Group Mother tree Father tree Fruit set (%) Length (cm) Width (cm) Sugar (%) Female N6 (No pollen) Little pollen C1 0% − − − 4P 40% 7.0 ± 0.5 6.5 ± 0.2 8.75 ± 1.0 AU4 50% 6.8 ± 0.2 6.3 ± 0.2 10.0 ± 0.4 Abundant pollen AU1 50% 7.2 ± 0.2 5.9 ± 0.3 8.2 ± 0.2 AU2 33% 5.8 ± 0.4 6.2 ± 0.4 8.0 ± 1.0 N2 50% 6.9 ± 0.2 6.7 ± 0.3 10.0 ± 0.6 Male Little pollen C1 50% 6.6 ± 0 6.5 ± 0 8.0 ± 0 4P 33% 7.4 ± 0 6.5 ± 0 9.5 ± 0 AU4 33% 6.5 ± 0.15 6.4 ± 0.4 8.0 ± 1.0 Abundant pollen AU1 66% 6.6 ± 0.5 5.95 ± 0.1 9.5 ± 0.5 AU2 50% 6.6 ± 0.1 6.45 ± 0.1 0.59 ± 0.5 N2 66% 6.6 ± 0.6 5.95 ± 0.1 10.5 ± 1 Table 7. Experimental data for the cross-pollination with N7 as the maternal parent, with and without emasculation.
Group Mother tree Father tree Fruit set (%) Length (cm) Width (cm) Sugar (%) Female N7 (No pollen) Little pollen C1 50% 6.8 ± 0.2 6.4 ± 0.2 10 ± 0.4 4P 33% 7.2 ± 0.2 6.8 ± 0.2 9.4 ± 0.4 AU4 33% 6.4 ± 0.2 6.2 ± 0.4 9.4 ± 0.4 Abundant pollen AU1 33% 6.7 ± 0.3 6.6 ± 0.6 10.0 ± 0.2 AU2 33% 7.8 ± 0.4 6.8 ± 0.5 9.0 ± 1.0 N2 50% 8.8 ± 0.4 7.4 ± 0.1 9.5 ± 1.0 Male Little pollen C1 28% 5.8 ± 0.1 5.6 ± 0 8.5 ± 0.5 4P 50% 7.2 ± 0 6.8 ± 0 10.0 ± 0 AU4 25% 6.4 ± 0 6.3 ± 0 8.4 ± 0 Abundant pollen AU1 0% − − − AU2 40% 6.8 ± 0 6.1 ± 0 9.0 ± 0 N2 50% 6.8 ± 0.4 6.0 ± 0.2 10.0 ± 1.0 Table 8. Experimental data for the cross-pollination with W4 as the maternal parent, with and without emasculation.
Group Mother tree Father tree Fruit set (%) Length (cm) Width (cm) Sugar (%) Female W4 (No pollen) Little pollen C1 60% 7.2 ± 0 6.5 ± 0 16.0 ± 0 4P 50% 6.3 ± 0.3 5.8 ± 0.1 13.5 ± 0.5 AU4 0% − − − Abundant pollen AU1 50% 5.6 ± 0 5.4 ± 0.2 10.7 ± 0.3 AU2 50% 7.1 ± 0 6.2 ± 0 13.0 ± 0 N2 60% 6.8 ± 0.2 6.0 ± 0.1 14.2 Male Little pollen C1 25% 7.1 ± 0.3 6.4 ± 0.2 13.7 ± 1.0 4P 50% 7.0 ± 0.1 6.4 ± 0 11.7 ± 0.6 AU4 0% − − − Abundant pollen AU1 50% 6.9 ± 0 6.8 ± 0 12.8 ± 0 AU2 50% 5.8 ± 0 4.9 ± 0 13.6 ± 0 N2 60% 7.1 ± 0.6 6.0 ± 0.3 12.8 ± 0.2 When the AU4 variety was used as the maternal parent (with emasculation), the best performance was found using N2 as the pollen source, which yielded a 75% rate of fruit set and the largest fruit size (9.5 cm × 8.5 cm), as shown in Table 3. It also had a high sugar content (10.5%).
Similarly, for 4P as the maternal parent, 80% fruit set was found with emasculation and with N2 as the pollen source (Table 4). This cross also showed moderate size and high sugar content. However, the largest fruit were found using AU1 as the pollen source.
The final little-pollen variety used as a maternal parent was the C1 variety, which generally showed poorer performance than the other two maternal parents previously discussed.
The best performance was seen using AU4 pollen on emasculated flowers, which gave a 60% rate of fruit set, the largest fruit size and a high sugar content (Table 5). N2 performed acceptably as a pollen source, and was also the best cross on non-emasculated flowers.
For the maternal parent varieties which did not produce pollen, N7 generally had poorer performance, while N6 and W4 had acceptable performance. Again, N2 was the best pollen source for most maternal varieties.
With N6 as the maternal parent, N2 and AU1 tied for the highest fruit set rate (66%) under non-emasculated conditions; however, the fruit was only moderate in size (Table 6). N2 also gave the sweetest fruit.
For N7 as the maternal parent, several combinations gave a 50% fruit set rate, while most other pollen sources gave quite poor rates of fruit set (Table 7). The better-performing pollen sources included C1 and N2 (for emasculated flowers) and 4P and N2 (for non-emasculated flowers). N2 also gave the largest fruit under emasculated conditions (8.8 cm × 7.5 cm).
Finally, with the W4 maternal parent, N2 again performed well under both emasculated and non-emasculated conditions (60% fruit set rate), with moderately sized fruit (Table 8). C1 also gave 60% fruit set when applied to emasculated flowers and good-sized fruit, as well as an exceptionally high sugar content (16%).
In general, most crosses showed higher fruit set rates and better fruit quality when the stamen is emasculated. The index of fruit quality as the best paired pollination group is a general method for pollination variety research[5], so we calculated the formula of the best paired parent variety of each parent to obtain the index.
Therefore, the best matching parent and the fruit quality of each parent in the case of male elimination can be shown as in Table 9. Overall, the best pollination combination between different ice cream fruit varieties was found using variety 4P and N2 as the parents (Table 9).
Table 9. Male parent cultivars and index of the best cultivar pairing for each female parent cultivar with emasculation.
Cross Fruit set (%) Length (cm) Width (cm) Sugar
(%)Index of
fruit qualityMother tree Father tree C1 AU4 60% 7.2 ± 1.0 6.7 ± 1.0 10.3 ± 1.0 10.2 4P N2 80% 7.7 ± 1.0 7.1 ± 1.0 11.0 ± 1.0 14.4 AU4 N2 75% 9.4 ± 0.1 8.4 ± 0.1 10.5 ± 0.5 14.2 N6 N2 50% 6.6 ± 0.6 5.9 ± 0.1 10.5 ± 1.0 8.3 N7 N2 50% 8.8 ± 0.4 7.4 ± 0.1 9.5 ± 1.0 8.5 W4 C1 60% 7.2 ± 0 6.5 ± 0 16.0 ± 0 13.5 Hybrid fruit of 4P and N2
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For the best-performing artificially pollinated hybrid between varieties 4P and N2, the fruit weight was 280 g, with a longitudinal width of 8.1 cm and a transverse diameter of 8.5 cm. This combination showed a 88.9% fruit set rate, with a 62.5% fruit survival rate. The skin of the fruit was green, with golden-yellow flesh, and a sugar content of 12%. This new variety has obvious advantages in both fruit quality and yield, and it shows great potential in the future agricultural production process (Fig 4).
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The experimental field work was conducted at the Tuhao field station and Houpo field station, both owned by Hainan Shengda Modern Agricultural Development Co, Ltd, (Hainan, China).
The ice cream fruit varieties used in this experiment were three varieties producing abundant pollen (N2, AU1, AU2) and three varieties producing little pollen (AU4, 4P, C1) and three varieties producing no pollen (N6, N7, W4). The varieties used in this experiment were randomly selected in the experimental field. All the experimental varieties were planted at appropriate temperature, humidity, sufficient light, with optimum growth conditions and no signs of abnormal flowers and fruits[10,11]. The tree were kept free of diseases and insect outbreaks during the trial.
Instrument, equipment and appliances
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The handheld glucose meter I used was the Nohawk HP-TD1.
Test method
Observation of natural self-pollination
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No artificial interventions were made for the natural self-pollination experiment[12,13]; only the selected ice cream fruit trees and the sample flowers in the flower germination period were marked. Observations were made throughout the natural flowering and fruit set process. The fruit set rate and fruit quality data were also collected. In this experimental group, nine flowers were documented from each variety, for three abundant pollen varieties (N2, AU1 and AU2), three little-pollen varieties (AU4, 4P and C1). This gave a total of six experimental groups and 54 data points.
Artificial self-pollination and cross-pollination (male/female)
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For the artificial self-pollination work, the selected ice cream fruit trees and the sample flowers were marked, bagged and isolated during the flower germination period. The flowers were then artificially self-pollinated (i.e., between the flowers of the same fruit tree). For this experiment, nine flowers were recorded from each variety, for the three abundant pollen varieties (N2, AU1 and AU2) and the three little-pollen varieties (AU4, 4P and C1). Again, this gave six experimental groups and 54 data points.
For the artificial cross-pollination work, one fruit tree of either an abundant pollen variety (N2, AU1, AU2) or a little-pollen variety (AU4, 4P and C1) was selected as the 'father' to collect pollen from. The varieties producing no pollen (N6, N7, W4) or little pollen (AU4, 4P and C1) were selected as mother trees (i.e., the flower source).
For each of the six female trees used as the mother, either five or six other varieties were used as the pollen source to cross-pollinate the parent. Five cross-pollinations were performed where the mother was a little-pollen variety, while six cross-pollinations were performed where the mother was a no-pollen variety. Consequently, there were a total of 15 crosses for the little-pollen mother varieties, and 18 crosses for the no-pollen mother varieties, as shown graphically in Fig. 5. Again, nine flowers were tracked for each cross-pollination experiment.
As a further investigation, this experiment was divided into two groups: male and female. This gave a total of 66 experimental treatments, yielding data on 594 flowers and fruit (Fig. 5).
Experimental operation procedures
Selected varieties (fruit trees)
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For the biological and morphological observations, a total of 24 fruit trees were observed for different purposes (Table 10). The nine ice cream fruit varieties are planted in a large area in Hainan Shengda Agricultural Company, which belongs to the current commercial varieties[14]. The paired pollination completion experiments and experimental results are of great significance to their commercial production[15,16].
Table 10. Fruit tree different applications.
Different varieties Different applications Abundant pollen varieties (N2, AU1 and AU2) Observation of natural self-pollination 3 Artificial self-pollination 3 Artificial cross-pollination parent tree 3 Little-pollen varieties (AU4,
4P and C1)Artificial cross-pollination mother tree (male) 3 Artificial cross-pollination mother tree (female) 3 Artificial cross-pollination parent tree 3 No pollen varieties (N6, N7 and W4) Artificial cross-pollination mother tree (male) 3 Artificial cross-pollination mother tree (female) 3 Sum 24 Selection of the flowers
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From each tree, three flowers were randomly selected and marked for observations[17]. The flowers were bagged in the artificial self-pollination and cross-pollination experiments to avoid external factors[18,19].
Pollen collection and preservation
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Ice cream fruit pollen from the required parent was collected at the morning of the crossing, to avoid little pollen quality and quantity[20,21]. Using tweezers, the pollen was gently collected into separate bags for preservation[22]. No pollen was collected on rainy days, as moisture can affect pollen quality[23].
Artificial pollination
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Artificial self-pollination was carried out using bagged flowers[24]. Pollen from the required variety was gently shaken onto the stigma; this was repeated at different times every day until the stamens withered or fell off[25−27].
Artificial cross-pollination was also carried out on bagged flowers; however, the stamens were removed (where required) at the germination stage[28,29]. As with the artificial self-pollination work, either cotton swabs or tweezers were used to transfer pollen from the required variety to the stigma of the mother flower at different times each day. This was repeated until the stamens fell off and the stigmata withered[30−32].
Emasculation
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For experiments where emasculation was required, the stamens of the flower were gently cut off at the germination stage. Care was taken to ensure the rest of the pistil and stigma were not harmed during this process[33,34].
Measurement statistics
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When the fruit ripened, it was picked and the length and width measured. The sugar content was also measured[35]. Finally, the relevant data was transferred into Microsoft Excel for subsequent processing and analysis[36].
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About this article
Cite this article
Lv W, Johnson JB, Zaman QU, Zhu M, Liu H, et al. 2023. Artificial pollination can improve fruit set and quality in the ice cream tree (Casimiroa edulis). Tropical Plants 2:12 doi: 10.48130/TP-2023-0012
Artificial pollination can improve fruit set and quality in the ice cream tree (Casimiroa edulis)
- Received: 19 September 2022
- Accepted: 19 July 2023
- Published online: 10 August 2023
Abstract: Ice cream fruit (Casimiroa edulis La Llave) is a member of the Rutaceae family native to South America, which has recently been introduced to China. In order to improve the yield of ice cream fruit, we carried out a hybridization experiment with paired pollination. Pollen was collected in the morning and carefully stored, before it was applied to the stigmata of sprouted flowers, using a cotton swab or tweezers. This artificial pollination is repeated at different times of the day. This was continued until the stamens fall off the flowers, the stigmata withered, and the fruit began to develop. Fruit yield and quality were recorded as the principal metrics of the success of each hybrid. At the Hainan Tropical Fruit Window Agricultural Company, nine different ice cream fruit varieties were selected and divided into three groups (abundant pollen, little pollen and no pollen) according to the amount of pollen that each variety naturally produced. Four experimental conditions were investigated: 1. No artificial pollination (natural self-pollination); 2. Artificial self-pollination; 3. Cross-pollination (no emasculation); 4. Cross-pollination (with emasculation). The results indicated that the fruit from cultivars producing abundant pollen generally had poor quality, smaller size, average sweetness, and a medium rate of natural fruit set. In contrast, cultivars producing little pollen showed better fruit quality, higher fruit fertility and increased success rates for artificial pollination. The N2 ice cream fruit cultivar showed the best characteristics as the paternal line (pollen source), while the 4P cultivar showed the best characteristics as the maternal line (flower source). The New varieties (N2 × 4P) performs well in fruit quality, fruit size, fruit sweetness and fruit setting rate. Consequently, this cross-pollinated cultivar is recommended for agricultural production and planting.
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Key words:
- Ice cream fruit /
- Flowers /
- Fruit set /
- Pollination