Method for obtaining seeds through artificial pollination of figs

Abstract

The invention relates to the technical field of fruit tree cross breeding, and discloses a method for obtaining seeds through artificial pollination of figs. The method comprises the following steps: treating Caprifig type figs by using a plant growth regulator to obtain pollen, and transferring the pollen into fig stigmas by using an artificial pollination dry powder method to obtain seeds. The method comprises the following steps: applying a cytokinin plant growth regulator at the stage that the fruit diameter of the Caprifig type figs is less than 3cm for treatment; harvesting mature Caprifig type fruits, separating anther, drying in the shade, and sieving to obtain pollen; fully mixing pollen and starch to form a pollen working agent in a dry powder state; at the stage that the diameter of the pollinated fig fruit is 1-3 cm, a pollen working agent is sprayed into a fruit cavity through a bract opening, so that the pollen working agent is attached to the stigma of a female flower; after the fruits are ripe, harvesting and water-selecting to obtain seeds. By means of the method, pollen with vitality can be obtained, artificial pollination and fruiting of the figs can be achieved, and the method is suitable for fig cross breeding and fruit improvement.

Description

Technical Field

[0001] This invention belongs to the field of fruit tree hybridization breeding technology, specifically relating to a method for obtaining fig seeds through artificial pollination. Background Technology

[0002] The fig is a fruit tree belonging to the genus *Ficus* in the family Moraceae. Its fruit is a syconium, containing numerous flowers. Fig flowers are unisexual, consisting of both female and male flowers. Based on flower development, fig plants are generally classified as Caprifig or non-Caprifig types. Under normal circumstances, male flowers can develop from the syconium of Caprifig figs and produce pollen, which can be used as pollinating parents in hybridization. In the absence of pollination stimulation, Caprifig fruits tend to stop growing and fall off during mid-development. Non-Caprifig figs primarily develop female flowers from their syconiums; these are the main type of edible fig, and the female flowers can form seeds after receiving pollen.

[0003] Under natural conditions, fig pollination is highly dependent on the symbiotic fig wasp to complete obligate pollination. With the widespread introduction and spread of figs globally, some regions have struggled to establish stable fig wasp populations due to environmental adaptability and other factors, resulting in planting areas with fig plants but lacking fig wasps. In these areas, Caprifig-type figs experience premature fruit drop due to the lack of pollination stimulation, making it difficult to obtain a stable pollen source; simultaneously, female fig flowers cannot form seeds normally due to the inability to obtain pollen, thus adversely affecting fig hybridization breeding.

[0004] In the prior art, US Patent 7818915B1 discloses a method for cultivating edible figs, which improves fruit quality and shelf life by introducing fig wasps to transfer Caprifig-type fig pollen. However, this method relies on the presence of fig wasps, limiting its applicability in areas lacking them. In hybridization breeding using fig wasps as insect pollinators, due to the extremely small size of fig wasps (only 0.5–1 mm in width), even bagging pollinated fruits cannot completely prevent wasps carrying pollen from non-target male parents from entering, potentially leading to unclear information about the hybrid male parent. In contrast, artificial pollination techniques in the absence of fig wasps can clearly identify the pollen source.

[0005] Previous studies have reported methods for artificial pollination of figs, with Rosianski et al. using a liquid method. However, figs have syconiums and relatively closed fruit cavities, making liquid pollination prone to causing excessive humidity in the fruit cavity, which can induce rot and reduce the pollination success rate, making it difficult to consistently obtain fertile seeds.

[0006] Furthermore, many existing patents relate to artificial pollination methods and devices for horticultural plants, such as a fruit tree pollinator and its pollination method (CN201810373291.7), a Welwitschia mirabilis pollination method (CN202511073084.6), and a lettuce hybridization pollination method and device (CN202310101972.9). However, most of these existing patents apply to plants with exposed floral organs and have not yet proposed an effective artificial pollination technology solution specifically for the syconium structure of figs. Because the fig flower organs are located inside the fruit cavity, conventional pollination devices cannot effectively transport pollen to the stigma of the female flower, thus failing to meet the actual needs of artificial hybridization breeding of figs.

[0007] Therefore, there is an urgent need to provide an artificial pollination method that does not rely on fig wasps to obtain pollen and is suitable for the syconium structure of figs, so as to improve the pollination success rate and obtain fertile seeds. Summary of the Invention

[0008] The purpose of this invention is to provide a method for obtaining fig seeds through artificial pollination, enabling pollen acquisition and artificial pollination without relying on fig wasps, thereby obtaining seeds.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for obtaining seeds by artificial pollination of figs, comprising the following steps: (1) during the developmental stage of Caprifig type figs with a fruit diameter of less than 3 cm, the Caprifig type figs are treated with a plant growth regulator to promote the continuous development of male flowers in the fruit and prevent fruit drop; (2) the mature Caprifig type figs treated in step (1) are harvested, the male flowers inside the fruit are collected and pollen is obtained; (3) the pollen obtained in step (2) is mixed with starch at a mass ratio of 1:1 to 1:4 to obtain a dry pollen working agent; (4) the artificial pollination device is pretreated; (5) when the fig fruit diameter is 1 to 3 cm, the pollen working agent obtained in step (3) is sprayed into the fruit cavity through the bract opening so that the pollen working agent is attached to the stigma of the female fig flower; (6) after the pollinated figs mature, they are harvested and the seeds are collected by water selection.

[0011] This invention clarifies the complete technical process for obtaining seeds through artificial pollination, forming a technical system of "pollen acquisition - working agent preparation - pollination operation - seed collection". This technology establishes an artificially controllable method for fig pollination breeding, solving the problems of traditional fig pollination relying on insect pollinators and lacking standardized procedures, and providing technical support for fig hybridization breeding.

[0012] Preferably, the plant growth regulator is the cytokinin-based plant growth regulator chlorpyrifos, used at a concentration of 10–50 ppm, and the treatment method is one or more of injection, immersion, or spraying. The advantage of choosing a cytokinin-based plant growth regulator in this invention is its ability to inhibit premature fruit drop in Caprifig-type figs, thereby obtaining mature pollen. The preferred concentration of chlorpyrifos is 10–50 ppm. This concentration range is suitable; excessively high concentrations of chlorpyrifos can lead to fruit deformities and decreased pollen viability. Chlorpyrifos, as a mature plant growth regulator, is widely available, low in cost, and easy to promote and apply.

[0013] Preferably, the pollen source is the male flower of a Caprifig-type fig fruit at the fruit ripening stage. This invention specifies that the pollen source is a Caprifig-type fig at the fruit ripening stage. The Caprifig-type fig is a type of fig that can develop male flowers; its mature male flowers have fully developed pollen, which is beneficial for obtaining highly viable pollen.

[0014] Preferably, the artificial pollination device is a pipette and its tip, and the connecting cone of the pipette tip is pretreated before the pollination operation. The pretreatment of the connecting cone serves two purposes: firstly, it avoids mixing of pollen from different male parents in Caprifig-type plants; secondly, it maintains the cleanliness and airtightness of the pipette's interior. Compared to traditional pollination tools such as brushes and syringes, the pipette can more conveniently deliver pollen into the fruit cavity, adapting to the structural characteristics of fig syconia.

[0015] Preferably, the pollen working agent is in dry powder form, and is sprayed into the fruit cavity through the bract opening of the fig using a pre-treated artificial pollination device. This invention uses a dry powder pollen working agent, solving the technical problem of excessive humidity in the fruit cavity and the resulting rot caused by liquid pollination. The dry powder pollen working agent is less likely to clump together in the fruit cavity, allowing it to disperse evenly with airflow and adhere to the stigma of the female flower, while maintaining a dry environment inside the fruit cavity and reducing the risk of pathogen growth. Directional spraying through the bract opening minimizes fruit damage and improves pollination success rate.

[0016] Preferably, the pollen working agent in step (3) is prepared by mixing pollen and starch in a mass ratio of 1:1 to 1:4, wherein the starch includes one or more of potato starch, cereal starch, or legume starch. This invention selects potato, cereal, or legume starch as the carrier for diluting pollen, which has multiple advantages: firstly, starch is widely available and inexpensive; secondly, the size of the starch granules is compatible with the pollen granules, and after mixing, a uniform dry powder system can be formed, facilitating spraying and adhesion; and thirdly, diluting the pollen can save on pollen usage and avoid fruit cracking due to excessive pollination.

[0017] Preferably, the diameter of the fig fruit during its developmental stage is 1–3 cm. This invention limits the diameter of the fig fruit to 1–3 cm, at which time the stigma is fully developed. If the fruit diameter is too small, the stigma is not fully developed and pollination cannot be completed; if the fruit diameter is too large, the stigma vigor decreases, and the pollination success rate drops significantly. This limitation ensures that pollination is carried out during the optimal window period, which is a key technical point for improving pollination success rate and seed yield.

[0018] Preferably, artificial pollination is carried out under rainless and dry conditions. This invention clearly defines the requirements for a rainless and dry pollination environment, which can effectively avoid the problem of disease and rot at the bract opening and inside the fruit cavity caused by high humidity.

[0019] Preferably, the water selection method involves placing the harvested figs in a square container filled with water, rubbing the pulp, and repeatedly washing to remove impurities, obtaining plump seeds that sink to the bottom. This invention uses water selection to separate seeds; it is simple to operate, low in cost, and free of chemical pollution, maximizing seed viability. Precise selection is achieved by utilizing the density difference between plump seeds and impurities / empty seed coats. This invention effectively removes pulp residue and other impurities, obtaining plump seeds with a high germination rate, providing high-quality seed material for subsequent seedling cultivation.

[0020] Beneficial effects

[0021] (1) By treating Caprifig-type fig fruits with plant growth regulators, premature fruit drop can be avoided, thus ensuring stable pollen acquisition without relying on insect pollination stimulation. This helps reduce interference from insect pollinators such as fig wasps and obtain fig pollen with controllable sources.

[0022] (2) Using dry powder method for artificial pollination can reduce the humidity inside the fruit cavity, avoid the fruit cavity from being too wet and rotting and falling off, help improve the pollination success rate and promote the normal development of the fruit to form seeds.

[0023] (3) Pollination by artificially controlling the source of pollen is more conducive to clarifying the source of the hybrid male parent than insect pollination, and obtaining fig hybrid offspring seeds with a clear male parent. Artificial pollination in a bee-free environment avoids the risk of contamination by insects such as fig wasps carrying pollen from non-target male parents, and ensures the reliability of hybrid male parent information.

[0024] (4) The pollination device is highly adaptable and has a high success rate: The pollination device of this invention uses a pipette and a pipette tip. The pretreatment of the pipette tip connecting cone is adapted to the structure of the fig syconium, while avoiding the mixing of pollen from different male parents of Caprifig type figs.

[0025] (5) Standardized operating procedures and broad application prospects: This invention clarifies the quantitative indicators of each key link, such as the concentration of growth regulators, the mixing ratio of pollen and starch, and the fruit diameter range, forming a standardized operating procedure from pollen acquisition, pollination operation, and seed collection, which is easy for users with different technical levels to master. In addition, the materials used in this method, such as chlorpyrifos, starch, and pipettes, are widely available and inexpensive, and can be adapted to different scenarios such as large-scale orchard breeding and small-scale experiments, laying a solid foundation for the promotion of fig hybridization breeding technology.

[0026] (6) Excellent seed quality and germination: The technical method of the present invention can not only stably obtain fertile seeds, but also the seeds can germinate into seedlings, providing technical support for the improvement of fig hybrid varieties. Attached Figure Description

[0027] Figure 1 Photo of the results of fig pollen viability testing;

[0028] Figure 2 A schematic photograph of the pretreatment process for an artificial pollination device for figs;

[0029] Figure 3 Microscopic photograph of a longitudinal section of a fig fruit after artificial pollination;

[0030] Figure 4 Photographs of fig seeds after artificial pollination;

[0031] Figure 5 A photograph of the appearance of a fig fruit after artificial pollination;

[0032] Figure 6 The diameter and weight of fig fruits after artificial pollination;

[0033] Figure 7 The soluble solids and total sugar content of fig fruits after artificial pollination;

[0034] Figure 8 The number of seeds in a fig fruit after artificial pollination;

[0035] Figure 9 A longitudinal section photograph of a fig fruit after artificial pollination;

[0036] Figure 10 Photographs of fig seedlings germinating from artificially pollinated hybrids;

[0037] Figure 11 Photograph of the fruit cavity of a fig plant artificially pollinated using the liquid method. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments. Various modifications, equivalent substitutions, or improvements can be made by those skilled in the art without departing from the spirit and substance of the present invention, and all such modifications, substitutions, or improvements should fall within the protection scope of the present invention.

[0039] Example 1: Obtaining Caprifig-type fig pollen.

[0040] Preparation and treatment of chlorpyrifos: Prepare a 10–50 ppm chlorpyrifos solution and treat the Caprifig-type figs at the developmental stage where the fruit diameter is within 3 cm. Treatment methods include injection, immersion, or spraying. Mature Caprifig-type figs can be at the stage where the fruit diameter is over 3 cm. After harvesting, store at 2–8 °C. Separate the anthers from the fruit, air-dry them, and sieve to obtain pollen. Store the pollen at −80 °C for later use.

[0041] Fig pollen viability assay: Place an appropriate amount of 0.5% TTC staining solution in a PCR tube. Use a toothpick to pick up a suitable amount of anther or preserved pollen and stir thoroughly. Incubate at 35 ℃ for 2–3 h. Add the pollen-stained solution to a glass slide and observe under a low-power microscope. Count pollen viability in five fields of view; red staining indicates viable pollen, while unstained pollen indicates inactive pollen. Figure 1 As shown, the TTC staining results indicate that a large number of pollen grains were stained, suggesting that the collected pollen has the ability to germinate.

[0042] Example 2: Pretreatment, pollination, and seed collection of artificial pollination device.

[0043] The artificial pollination device consists of a 1000μL pipette and pipette tips. Pretreatment includes pretreatment of the pipette tip connecting cone, such as... Figure 2 As shown, pretreatment to prevent cross-contamination: A separating material is placed inside the conical interface of the pipette to reduce the risk of cross-contamination of pollen from different sources. No liquid is introduced during the preparation of the pollen working agent; during pollination, the pretreated pipette, along with the pipette tip, is used to spray the pollen working agent into the fruit cavity through the bract opening. After pollination, a small amount of fruit is longitudinally sectioned and observed under a microscope; the results are as follows. Figure 3 As shown, pollen working agent is visible attached to the stigma of the female flower. The pollen working agent is transferred to the stigma area of ​​the female fig flower. After the fruit matures, the seeds are collected by water separation. Figure 4 ).

[0044] Example 3: Artificial pollination of 'Boji Red' figs.

[0045] The pollinated figs were sourced from a commercial fig plantation in Beijing, located near 40°N latitude, and the main commercially cultivated fig variety was 'Boji Hong'. Two hundred fruits with uniform color and a diameter of 1–3 cm were selected, with 100 fruits in the pollination group and 100 in the control group. The pollination group fruits were artificially pollinated using a pollen working agent prepared by mixing Caprifig-type fig pollen (Example 1) with starch at a mass ratio of 1:1 to 1:4. The control group fruits were pollinated with the same amount of starch using the pollination method described in Example 2. During the experiment, photographs were taken every 15 days, and the fruit diameter and weight were measured. The results are as follows: Figure 5 As shown, compared with the control group, the pollinated figs showed differences in appearance, ripening process and internal seed development.

[0046] Fig physiological indicators were measured: In Example 1, the longitudinal and transverse diameters of the fruits in the control and pollinated groups were measured using vernier calipers; the weight of individual fruits harvested at each stage was measured using an analytical balance. Results are as follows: Figure 6 As shown, compared with the control group, there were significant differences in the diameter and weight of fig fruits in the pollinated group.

[0047] Determination of soluble solids and total sugar content in figs: The soluble solids content of the fruit was determined using a WZB-F 85 digital refractometer. The total sugar content of ripe figs was determined using the sulfuric acid-phenol method: 0.5 g of crushed ripe fig sample was accurately weighed, washed with distilled water into a 10 mL volumetric flask, and diluted to volume. After thorough mixing, the sample was centrifuged at 3000 r / min for 15 min, and 2 mL of the supernatant was taken and diluted to a 250 mL volumetric flask for analysis. 2 mL of the diluted sample solution was added to 1 mL of 5% phenol and 5 mL of 98% concentrated sulfuric acid, and the reaction was allowed to proceed for 30 min before measuring the absorbance at 490 nm. A standard curve was plotted using glucose as a standard, and the total sugar content was calculated. The results are as follows: Figure 7 As shown, the soluble solids and total sugar content of figs in the pollinated group differed from those in the control group.

[0048] Example 4: Artificial pollination of 'green skin' figs.

[0049] The pollinated figs were sourced from a fig greenhouse at a university experimental base in Beijing, located near 40°N latitude. The variety was the commercially cultivated fig 'Qingpi'. Twenty figs with uniform color and a diameter of 1–3 cm were selected. Artificial pollination was performed using a pollen working agent prepared by mixing Caprifig-type fig pollen (Example 1) with starch at a mass ratio of 1:1 to 1:4, following the pollination method described in Example 2. Twenty unpollinated figs were also marked. After the fruits matured, the seeds were collected and their number counted. The results are shown below. Figure 8 As shown, the number of seeds in the fruit after pollination is considerable, which is beneficial for subsequent breeding.

[0050] Example 5: Artificial pollination of figs grown out of season in Yunnan.

[0051] The pollinated figs were sourced from a fig planting base in Yunnan Province, located near 21°N latitude, during the off-season fig cultivation period between winter and spring. Eighty fruits with uniform color and a diameter of 1–3 cm were selected, with 40 fruits in each of the treatment and control groups. The fruits in the pollination group were artificially pollinated using a pollen working agent prepared by mixing Caprifig-type fig pollen (from Example 1) with starch at a mass ratio of 1:1 to 1:4. The control group fruits were pollinated using the same amount of starch, following the pollination method described in Example 2. The fruits were photographed and observed after pollination. Results are as follows: Figure 9 As shown, the pollinated fig fruits exhibited differences in ripening process and internal seed development, demonstrating the wide regional adaptability of this method.

[0052] Example 6: Germination detection of seeds obtained by hybridization.

[0053] After vernalization treatment at 4 ℃, the seeds were soaked in water for 6–12 hours. Subsequently, the seeds were sown in seedling trays containing a well-mixed and moistened peat moss:vermiculite ratio of 1:1 (m:m). The trays were kept moist by regular watering at 25 ℃, and germination was observed after 2 weeks. Results are as follows: Figure 10 As shown, seeds obtained through artificial pollination and hybridization can germinate into seedlings, indicating that the obtained seeds have germination ability.

[0054] Example 7: Observation of fruit set by artificial pollination using liquid method.

[0055] Thirty '107B' figs were pollinated using a liquid method. 0.5 g of pollen was added to 100 mL of 2% sucrose solution to prepare the pollination solution. The solution was then injected into the fruit through the bract opening using a plastic syringe, continuing until droplets overflowed from the bract opening, ensuring sufficient contact between the pollen and the inflorescence interior. Fruit condition was observed and fruit set was recorded 45 days post-injection. Results are as follows: Figure 11 As shown, figs artificially pollinated using liquid methods are prone to rotting due to the dampness inside the fruit cavity.

[0056] It should be understood that the above-described embodiments are merely preferred embodiments of the present invention, used to illustrate the technical solutions of the present invention, and not to limit the scope of protection of the present invention. For those skilled in the art, various modifications, equivalent substitutions, or improvements can be made to the above embodiments without departing from the essence and principle of the technical solutions of the present invention, and such modifications, equivalent substitutions, or improvements should all fall within the scope of protection of the present invention.

Claims

1. A method for obtaining fig seeds through artificial pollination, characterized in that: The method for obtaining fig seeds through artificial pollination includes the following steps: (1) During the development period when the diameter of Caprifig type fig fruit is less than 3cm, plant growth regulators are used to treat Caprifig type fig fruit to promote the continuous development of male flowers in the fruit and prevent fruit drop. (2) Harvest mature Caprifig-type figs after treatment in step (1), collect the male flowers inside the fruit and obtain pollen; (3) Mix the pollen obtained in step (2) with starch at a mass ratio of 1:1 to 1:4 to obtain pollen working agent in dry powder state; (4) Pre-treat the artificial pollination device; (5) When the fig fruit is 1-3 cm in diameter, spray the pollen working agent obtained in step (3) into the fruit cavity through the bract opening so that the pollen working agent adheres to the stigma of the female fig flower. (6) After the figs have matured after pollination, they are harvested and the seeds are collected by water selection.

2. The method for obtaining fig seeds through artificial pollination according to claim 1, characterized in that: The plant growth regulator is a cytokinin-based plant growth regulator.

3. The method for obtaining fig seeds through artificial pollination according to claim 2, characterized in that: The cytokinin-type plant growth regulator mentioned in step (1) is chlorpyrifos, and its concentration is 10-50 ppm. The treatment method is one or more of the following: injection, soaking, or spraying.

4. The method for obtaining fig seeds through artificial pollination according to claim 1, characterized in that: The pollen source is the male flowers of Caprifig-type figs during their fruit ripening stage.

5. The method for obtaining fig seeds through artificial pollination according to claim 1, characterized in that: The artificial pollination device is a pipette and its tip, and the connecting cone of the pipette tip is pretreated before the pollination operation.

6. The method for obtaining fig seeds through artificial pollination according to claim 1, characterized in that: The pollen working agent is in dry powder form and is sprayed into the fruit cavity through the bract opening of the fig using a pre-treated artificial pollination device.

7. The method for obtaining fig seeds through artificial pollination according to claim 1, characterized in that: The pollen working agent mentioned in step (3) is prepared by mixing pollen and starch in a mass ratio of 1:1 to 1:4, wherein the starch includes one or more of potato starch, cereal starch or legume starch.

8. The method for obtaining fig seeds through artificial pollination according to claim 1, characterized in that: The diameter of a pollinated fig during its development is 1–3 cm.

9. The method for obtaining fig seeds through artificial pollination according to claim 1, characterized in that: Artificial pollination is carried out under rainless and dry conditions.

10. The method for obtaining fig seeds through artificial pollination according to claim 1, characterized in that: The water selection method involves placing the harvested figs in a square container filled with water, rubbing the pulp and repeatedly washing to remove impurities, and obtaining plump seeds that sink to the bottom.

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