A method for artificial hybridization and pollination of guava

Abstract

The present invention belongs to the technical field of fruit tree breeding, and specifically relates to a method for artificial guava hybridization emasculation and pollination, comprising the following steps: S1. in vitro pollen culture; S2. observation of stigma receptivity; S3. treatment of the maternal and paternal parents; and S4. cross-pollination. By observing the vigor of the maternal stigma and the vigor of the paternal pollen, the method artificially emasculates the maternal anthers before pollen sheds, then applies high-vigor paternal pollen when the maternal stigma is most active, and bags the pollen to prevent interference from unknown pollen. This method ensures the purity and accuracy of the artificial hybridization and produces a larger number of hybrid offspring. Pollination 24 hours after emasculation avoids the drawbacks of incomplete and incomplete emasculation, improving the purity and quality of hybrid seeds. Furthermore, this method, when applied to guava artificial hybridization breeding practices, can avoid blindness in artificial guava hybridization breeding and improve its efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit tree breeding, and particularly relates to a guava artificial hybridization emasculation and pollination method. Background Art

[0002] Guava (Psidium guajava L.) is a plant of the genus Guava in the Myrtaceae family. Its flowers are monoecious and monoecious, borne solitary or in clusters of two or three in the leaf axils at the base of the fruiting branches. The calyx is bell-shaped or pear-shaped and persistent. Its fruit is a berry, spherical, ovoid, or pear-shaped, containing numerous small, hard seeds. Guava fruit is sweet, crisp, fragrant, and has a unique flavor. Rich in a variety of functional nutrients, including vitamins, amino acids, minerals, flavonoids, phenols, tannins, and triterpenes, it has the potential to strengthen the spleen and eliminate stagnation, astringe the intestines, relieve diarrhea, lower blood sugar and lipids, and has anti-tumor, antioxidant, and antibacterial properties. It possesses high edible and medicinal value. Guava is widely cultivated in Taiwan, Fujian, Guangdong, Guangxi, and Hainan, China, and is a popular and profitable subtropical fruit.

[0003] Artificial hybridization is one of the most common methods for fruit tree germplasm innovation and the development of new varieties. However, due to a lack of basic biological research on the pollen receptivity of parental stigmas and pollen development, hybridization practices in guava are often highly unreliable and inefficient. Furthermore, the main guava varieties cultivated in China are limited, relying primarily on introductions and seed selection.

[0004] Guava breeding methods are also known in the prior art. For example, patent CN103814812A discloses a guava hybrid breeding method that involves "selecting flowers with dispersed pollen as the male parent material, touching the anthers of the stamens to the stigma of the female flower for pollination, and repeating the pollination process 3-4 times." While this pollination method can achieve certain results, the pollination process requires many repetitions, is labor-intensive and time-consuming, and has low efficiency.

[0005] In addition, patent CN106613628A discloses a method for solving the problem of guava plants that only bloom but do not produce fruit. It proposes "artificially pollinating the flowers of guava plants that only bloom but do not produce fruit, and then pollinating the flower buds of guava plants that can normally bloom and produce fruit." Although this pollination method can achieve the corresponding technical effects, it only pollinates a limited number of objects and does not test pollen viability and stigma receptivity.

[0006] In the prior art, artificial pollination of guava still has the following problems:

[0007] (1) Guava is a monoecious plant that is self-pollinating and close-pollinated. Pollination is complete when it blooms, so hybridization must be completed before flowering. Currently, the optimal time for guava flower stigma receptivity and the peak time of pollen vitality have not been determined. The stigma of a pistil that has not bloomed at the time of pollination is not yet mature, and the stigma receptivity is low, which affects the success rate of hybridization.

[0008] (2) The petals of guava are tightly interlocked, the stamens are numerous, and the stigma of the pistil is slender, which makes guava emasculation difficult or the stamens are not removed cleanly or thoroughly, which will greatly affect the purity of the seeds;

[0009] (3) The current method of using tweezers to pick up stamens one by one requires a high level of technical proficiency. Otherwise, it is easy to poke the pistil style, stigma and ovary, causing damage to the floral organs and failure of hybrid pollination. At the same time, this method is time-consuming and inefficient.

[0010] The inventors conducted basic research on the pollination and fertilization biology of guava, such as the pollen viability and pollen vitality, to determine the emasculation time, pollen collection time and pollination time in artificial pollination, and developed an efficient guava artificial pollination technology, which can effectively improve the efficiency of artificial hybrid breeding, provide a technical basis for the selection and breeding of guava varieties, and is of great significance to promoting the sustainable and healthy development of the guava industry.

[0011] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0012] The purpose of the present invention is to provide a guava artificial hybridization emasculation and pollination method to solve the problems existing in guava artificial pollination breeding in the background technology.

[0013] To achieve the above object, the present invention provides the following technical solutions:

[0014] A guava artificial hybridization emasculation and pollination method comprises the following steps:

[0015] S1. Pollen in vitro culture

[0016] The day before the guava flowers opened, plump buds were selected and bagged. After 24 hours, they were taken back to the laboratory for in vitro pollen culture. The pollen was cultured at 25°C for 4 hours and the pollen germination was observed.

[0017] S2. Observation of stigma receptivity

[0018] The flower buds at the white stage were used as test materials, which were emasculated and bagged. After 24 hours, the buds were taken out. Another part of the flower buds at the white stage was taken as control. The receptivity of the stigma of guava was determined by the benzidine-hydrogen peroxide method.

[0019] S3. Maternal and paternal treatments

[0020] Select well-developed guava buds in the white stage as the female parent. After emasculation, cover the entire bud with a sulfuric acid paper bag to prevent the entry of non-target pollen. At the same time, select well-developed guava buds in the white stage and bag them as the male parent.

[0021] S4. Cross-pollination

[0022] After 24 hours, pick the marked male parent flower buds, remove the bag, and lightly dip the freshly collected male parent pollen into the female parent stigma that has been emasculated the day before until milky white pollen can be seen on the stigma with the naked eye. After completion, bag the buds with sulfuric acid paper bags to prevent pollen from mixing.

[0023] More specifically, in S1, the medium for pollen in vitro culture comprises, by mass percentage, 10% sucrose and 0.01% boric acid.

[0024] More specifically, in S3, the female parent is pearl guava, and the corresponding male parent is pink honey guava, red heart guava or full red guava.

[0025] More specifically, in S4, the pollination time is 8:00-10:00 am on a sunny day with humidity below 85%.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention, by observing maternal stigma vigor and male parent pollen vigor, artificially emasculates before maternal anthers shed pollen, grants male parent pollen with high vigor and bags to prevent the interference of unknown pollen when maternal stigma vigor is the highest, can ensure the purity and accuracy of artificial hybridization, and obtain more hybrid offspring. Pollination 24h after emasculation can avoid unclean emasculation, a drawback of not thorough, and improves the purity and quality of hybrid seeds. Because it is an incompletely opened bud during emasculation, stigma maturity is not enough, and pollination is carried out 24h after emasculation, and stigma vigor is higher, can improve pollination success rate and fruit setting rate. This technology is applied to guava artificial hybridization breeding practice simultaneously, can avoid the blindness in guava artificial hybridization breeding, and improves artificial hybridization breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The pollen germination of three varieties are shown in order: "Pink Honey", "Red Heart Guava (Soft Flesh)" and "Full Red".

[0029] Figure 2 The receptivity of the stigma of the female parent "Pearl" emasculated on the same day and the emasculated on the previous day is shown in order;

[0030] Figure 3The order is bud selection for emasculation, stamen status, status after emasculation, bagging status, and stigma status 24 hours after emasculation;

[0031] Figure 4 These are the fruit set conditions 20 days after hybridization of Pearl × Pink Honey, Pearl × Red Heart Guava (soft flesh), and Pearl × All Red. DETAILED DESCRIPTION

[0032] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts are within the scope of protection of the present invention.

[0033] Example 1

[0034] A guava artificial hybridization emasculation and pollination method comprises the following steps:

[0035] S1. Pollen in vitro culture

[0036] The "Pink Honey" guava from the Guava Germplasm Resource Garden of the Guangxi Subtropical Crops Research Institute was used as the test material. On the morning before the flowers bloomed, plump flower buds were selected and bagged. 24 hours later, the buds were picked and returned to the laboratory for in vitro pollen culture. The in vitro culture medium contained 10% sucrose and 0.01% boric acid. The buds were incubated at 25°C for 4 hours, and pollen germination was observed.

[0037] S2. Observation of stigma receptivity

[0038] The "Pearl" guava flowers that had been emasculated the previous day were picked and brought back to the laboratory (for control, unopened buds of the same day were brought back to the laboratory, the anthers were removed with tweezers, and the stigma and ovary were retained). The stigma was then placed on a glass slide, and a benzidine-hydrogen peroxide reaction solution (V1% benzidine:V3% hydrogen peroxide:Vwater=4:11:22) was added dropwise until the stigma was submerged and stained for 3 minutes. The slide was placed under a microscope to observe the reaction of the stigma and take photos to record it. The strength of the stigma activity was judged based on the number and speed of bubble generation and the staining of the stigma. Specifically, if the stigma color changes to blue-purple and a large number of bubbles are generated, the stigma is highly receptive; if the stigma color does not change and no bubbles are generated, the stigma is not receptive.

[0039] S3. Maternal treatment

[0040] During the peak flowering period of the "Pearl" guava, use tweezers to remove the excess and deformed flowers on the inflorescences of the pre-pollinated branches. Leave two well-developed guava buds on each inflorescence of each branch that will open the next day, remove the anthers, and bag them as the mother plant.

[0041] S3. Paternal treatment

[0042] Select well-developed "Pink Honey" guava buds in the whitening stage, bag them and mark them as the male parent;

[0043] S4. Cross-pollination

[0044] After 24 hours, pick the marked male parent buds, remove the bag, and use the freshly collected male parent pollen to lightly dip the female parent stigma that has been emasculated the day before until milky white pollen can be seen on the stigma with the naked eye. After completion, use a sulfuric acid paper bag to bag the buds to prevent pollen from crossing.

[0045] Among them, when pollinating, choose trees with good growth and no obvious diseases and pests. The pollination time is 8:30-10:00 in the morning on a sunny day with humidity below 85%. The parent combination is: Pearl × Pink Honey; 4 days after pollination, remove the sulfuric acid paper bag; after pollination, the guava trees are routinely fertilized, watered, and managed for diseases and pests until the fruits are ripe.

[0046] Example 2

[0047] A guava artificial hybridization emasculation and pollination method comprises the following steps:

[0048] S1. Pollen in vitro culture

[0049] The "Red Heart Guava (Soft Flesh)" guava from the Guava Germplasm Resource Nursery of the Guangxi Subtropical Crops Research Institute was used as the test material. On the morning before the flowers bloomed, plump flower buds were selected and bagged. 24 hours later, the buds were picked and returned to the laboratory for in vitro pollen culture. The in vitro culture medium contained 10% sucrose and 0.01% boric acid. The buds were incubated at 25°C for 4 hours, and pollen germination was observed.

[0050] S2. Observation of stigma receptivity

[0051] The "Pearl" guava flowers that had been emasculated the previous day were picked and brought back to the laboratory (for control, unopened buds of the same day were brought back to the laboratory, the anthers were removed with tweezers, and the stigma and ovary were retained). The stigma was then placed on a glass slide, and a benzidine-hydrogen peroxide reaction solution (V1% benzidine:V3% hydrogen peroxide:Vwater=4:11:22) was added dropwise until the stigma was submerged and stained for 3 minutes. The slide was placed under a microscope to observe the reaction of the stigma and take photos to record it. The strength of the stigma activity was judged based on the number and speed of bubble generation and the staining of the stigma. Specifically, if the stigma color changes to blue-purple and a large number of bubbles are generated, the stigma is highly receptive; if the stigma color does not change and no bubbles are generated, the stigma is not receptive.

[0052] S3. Maternal treatment

[0053] During the peak flowering period of the "Pearl" guava, use tweezers to remove the excess and deformed flowers on the inflorescences of the pre-pollinated branches. Leave two well-developed guava buds on each inflorescence of each branch that will open the next day, remove the anthers, and bag them as the mother plant.

[0054] S3. Paternal treatment

[0055] Select well-developed, white-appearing guava buds of "Red Heart Guava (Soft Flesh)" and bag them as the male parent.

[0056] S4. Cross-pollination

[0057] After 24 hours, pick the marked male parent buds, remove the bag, and use the freshly collected male parent pollen to lightly dip the female parent stigma that has been emasculated the day before until milky white pollen can be seen on the stigma with the naked eye. After completion, use a sulfuric acid paper bag to bag the buds to prevent pollen from crossing.

[0058] Among them, when pollinating, choose trees with good growth and no obvious diseases and pests. The pollination time is 8:30-10:00 in the morning on a sunny day with humidity below 85%. The parent combination is: Pearl × Red Heart Guava (soft flesh); 4 days after pollination, remove the sulfuric acid paper bag; after pollination, the guava trees are routinely fertilized, watered, and managed for diseases and pests until the fruits are ripe.

[0059] Example 3

[0060] A guava artificial hybridization emasculation and pollination method comprises the following steps:

[0061] S1. Pollen in vitro culture

[0062] The "Quanhong" guava from the Guava Germplasm Resource Garden of the Guangxi Subtropical Crops Research Institute was used as the test material. On the morning before the flowers bloomed, plump flower buds were selected and bagged. 24 hours later, the buds were picked and returned to the laboratory for in vitro pollen culture. The in vitro culture medium contained 10% sucrose and 0.01% boric acid. The buds were incubated at 25°C for 4 hours, and pollen germination was observed.

[0063] S2. Observation of stigma receptivity

[0064] The "Pearl" guava flowers that had been emasculated the previous day were picked and brought back to the laboratory (for control, unopened buds of the same day were brought back to the laboratory, the anthers were removed with tweezers, and the stigma and ovary were retained). The stigma was then placed on a glass slide, and a benzidine-hydrogen peroxide reaction solution (V1% benzidine:V3% hydrogen peroxide:Vwater=4:11:22) was added dropwise until the stigma was submerged and stained for 3 minutes. The slide was placed under a microscope to observe the reaction of the stigma and take photos to record it. The strength of the stigma activity was judged based on the number and speed of bubble generation and the staining of the stigma. Specifically, if the stigma color changes to blue-purple and a large number of bubbles are generated, the stigma is highly receptive; if the stigma color does not change and no bubbles are generated, the stigma is not receptive.

[0065] S3. Maternal treatment

[0066] During the peak flowering period of the "Pearl" guava, use tweezers to remove the excess and deformed flowers on the inflorescences of the pre-pollinated branches. Leave two well-developed guava buds on each inflorescence of each branch that will open the next day, remove the anthers, and bag them as the mother plant.

[0067] S3. Paternal treatment

[0068] Select well-developed, "full red" guava buds in the whitening stage, bag them and mark them as the male parent;

[0069] S4. Cross-pollination

[0070] After 24 hours, pick the marked male parent buds, remove the bag, and use the freshly collected male parent pollen to lightly dip the female parent stigma that has been emasculated the day before until milky white pollen can be seen on the stigma with the naked eye. After completion, use a sulfuric acid paper bag to bag the buds to prevent pollen from crossing.

[0071] Among them, when pollinating, choose trees with good growth and no obvious diseases and pests. The pollination time is 8:30-10:00 in the morning on a sunny day with humidity below 85%. The parent combination is: Pearl × All Red. Remove the sulfuric acid paper bag 4 days after pollination. After pollination, the guava trees are routinely fertilized, watered, and managed for diseases and pests until the fruits are ripe.

[0072] Breeding results

[0073] In the measurement examples 1-3, the number of florets in each hybrid combination was 100, and the fruit set rate was calculated after 20 days. During this period, the fruit growth was observed, and the single fruit weight and the horizontal stem and vertical diameter of the fruit were calculated after the fruit matured. The results are shown in Table 1.

[0074] Pollen germination rate = number of pollen germinated in the field of view / total number of pollen × 100%;

[0075] Fruit setting rate = number of ovary swellings after pollination / total number of pollinations × 100%.

[0076] Table 1 Pollen germination rate and fruit growth of artificial hybrid emasculation and pollination of guava of the present invention

[0077]

[0078] Statistics on hybrid fruit yields revealed that pollination 24 hours after emasculation resulted in more hybrid fruit, while fruit pollinated on the same day of emasculation generally experienced physiological fruit drop during growth, resulting in very few or no mature fruit. This suggests that the stigma receptivity of female plants emasculated 24 hours after emasculation is higher than that of plants emasculated on the same day. For guava pollination, choosing male plants with high pollen vigor and pollinating when the female plant's stigma is still vigorous can significantly increase fruit set.

[0079] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A guava artificial hybridization emasculation and pollination method, characterized in that: The following steps are involved: S1. In vitro pollen culture The day before the guava flowers opened, plump buds were selected and bagged. After 24 hours, they were taken back to the laboratory for in vitro pollen culture. The pollen was cultured at 25°C for 4 hours and the pollen germination was observed. S2. Observation of stigma receptivity The flower buds at the white stage were used as test materials, which were emasculated and bagged. After 24 hours, the buds were taken out. Another part of the flower buds at the white stage was taken as control. The receptivity of the stigma of guava was determined by the benzidine-hydrogen peroxide method. S3. Maternal and paternal treatments Select well-developed guava buds in the white stage as the female parent. After emasculation, cover the entire bud with a sulfuric acid paper bag to prevent the entry of non-target pollen. At the same time, select well-developed guava buds in the white stage and bag them as the male parent. S4. Cross-pollination After 24 hours, pick the marked male parent buds, remove the bag, and use the freshly collected male parent pollen to lightly dip the female parent stigma that has been emasculated the day before until milky white pollen can be seen on the stigma with the naked eye. After completion, use a sulfuric acid paper bag to bag it to prevent pollen from mixing; In S1, the medium for pollen in vitro culture was composed of 10% sucrose and 0.01% boric acid, calculated by mass percentage; In S3, the female parent is Pearl Guava, and the corresponding male parent is Pink Honey Guava, Red Heart Guava, or Full Red Guava; In S4, pollination time was 8:00-10:00 am on sunny days with humidity below 85%.

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