A method for improving the fruit setting rate of distant hybridization of oil-tea camellia

Abstract

The present application relates to a method for improving the fruit setting rate of distant hybridization of oil tea. After pollination is performed by cutting the style and assisting pollen germination induction during the flowering period of oil tea, the fruit setting rate of oil tea is significantly increased, and the size and weight of oil tea fruits can be increased at the same time, effectively overcoming the problems of difficult pollination and low fruit setting rate of distant hybridization of oil tea, and having good application prospects for hybrid breeding of new oil tea varieties and construction of genetic populations.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for improving the fruit setting rate of distant hybridization of oil tea. Background Art

[0002] Camellia oleifera is an evergreen small tree of the genus Camellia in the family Theaceae. It is one of my country's unique oil-bearing economic crops and is known as one of the world's four major woody oil plants along with oil palm, olive and coconut. Camellia oleifera has a long history of cultivation in my country, but there are problems in production such as low yield per unit area, many flowers, few fruits, and low fruit setting rate, which seriously restrict the development of the camellia oleifera industry.

[0003] Distant hybridization can effectively integrate the advantages of both parents and improve the varieties of Camellia oleifera, but it is easily restricted by reproductive isolation. Take the hybridization of Camellia oleifera and Camellia oleifera as an example. Camellia oleifera Abel is the most widely cultivated Camellia oleifera in my country. It has the characteristics of large fruit, high yield and high light efficiency. Its seeds have a high oil content and high economic value. Camellia yuhsienensis Hu is a high-quality local variety in Hunan. It has the characteristics of thin skin, good oil quality and strong disease resistance.

[0004] However, existing studies have shown that the interspecific hybridization of Youxian Camellia 'Youza 2' and common Camellia 'Huashuo' using existing methods of artificial pollination and induced pollination, especially the reciprocal cross combination with 'Huashuo' as the male parent, has almost no fruit. This limits the acquisition of hybrids with common Camellia oleifera as the female parent and Youxian Camellia oleifera as the male parent, which is not conducive to Camellia oleifera hybrid breeding and germplasm innovation.

[0005] Therefore, people hope to find a method that can effectively improve the fruit setting rate of distant hybridization of Camellia oleifera. Summary of the invention

[0006] In order to solve the above technical problems, the present application first provides a method for improving the fruit setting rate of distant hybridization of Camellia oleifera by cutting the style for pollination.

[0007] The present application also provides a method for improving the quality of distant hybridization fruits of Camellia oleifera by cutting the style for pollination.

[0008] In some embodiments, the specific steps of cutting the style pollination are: removing the stamens of the female Camellia oleifera during the flowering period, cutting the style of the pistil, and then applying the pollen of the male Camellia oleifera to the style incision to complete the pollination.

[0009] In some embodiments, the mother Camellia oleifera is selected from Camellia oleifera with white buds but not yet opened.

[0010] In some embodiments, cutting the style of the pistil means cutting off 1 / 5-4 / 5 of the style of the pistil.

[0011] More preferably, cutting the style of the pistil means cutting off 4 / 5 of the style of the pistil, that is, only retaining 1 / 5 (the base of the style).

[0012] In some embodiments, the female Camellia oleifera is 'Huashuo' Camellia oleifera or 'Huaxin' Camellia oleifera, and the male Camellia oleifera is Youxian Camellia oleifera.

[0013] In some embodiments, pollination by cutting the style can improve the fruit quality of Camellia oleifera, including the size and weight of the fruit, and specifically can increase the size and weight of the fruit.

[0014] The present application also provides a pollen germination culture solution, comprising sucrose, boric acid, CaCl2·2H2O, MgSO4, NAA and GA3.

[0015] In some embodiments, the composition of the pollen germination culture solution is 0.1 g·mL -1 Sucrose, 0.1 g·L -1 Mass fraction of boric acid, 50mg·L -1 CaCl2·2H2O, 10mg·L -1 MgSO4, 0.5mg·L -1 NAA and 2mg·L -1 GA3.

[0016] The present application also provides the use of the above-mentioned pollen germination culture solution for promoting the germination of camellia oleifera pollen.

[0017] In some embodiments, after cutting the style, firstly apply pollen germination culture solution to the style incision, and then apply pollen of the male parent Camellia oleifera to the style incision.

[0018] This application has the following technical effects:

[0019] 1. The present application study found that the method of cutting style pollination can improve the fruit setting rate and fruit quality of distant hybridization of Camellia oleifera, and the effect is significantly better than the ordinary artificial pollination method and the induced pollination method, and has broad application prospects.

[0020] 2. The present application provides a pollen germination culture solution, which can effectively promote the germination of Camellia oleifera pollen, and thereby promote the fruit setting and fruit development of Camellia oleifera. The culture solution is expected to be applied to the research of other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide further understanding of the present application and constitute a part of the specification. They are used to explain the present application together with the embodiments of the present application and do not constitute a limitation of the present application.

[0022] Figure 1 The pollen of 'Huaxin' was inactivated by boiling water bath.

[0023] Figure 2 Schematic diagram of the pollination operation for cut styles.

[0024] Figure 3 The fruit morphology of common Camellia oleifera 'Huashuo' and Youxian Camellia oleifera 'YZ2' after different treatments. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0027] Example 1 Effect of Pollen Germination Culture Solution on Pollen Germination

[0028] 1.1 Preparation of pollen germination culture medium

[0029] Using a basic culture medium containing 10% sucrose and 0.01% boric acid as a blank control, different concentrations of CaCl2·2H2O, MgSO4, NAA and GA3 were added to the basic culture medium to obtain pollen germination culture solutions No. 1-No. 9 (for specific ingredients, see Table 2).

[0030] 1.2 Pollen germination experiment

[0031] This experiment adopts the agar medium germination method. First, weigh the required culture medium materials, pour them into a conical flask and mark them in sequence, then add a quantitative distilled water, heat and boil until the contents are fully dissolved, cool to about 50°C at room temperature, add the calculated volume solution of the exogenous substance, gently stir with a glass rod to fully mix the exogenous substance solution and the culture medium, and then pour it into the culture dish. When the culture medium is cooled to a solid at room temperature, use a clean small brush to pick up the pollen, shake it off evenly on the surface of the culture medium in small amounts multiple times, and then culture it in a 25°C constant temperature incubator away from light for 2 hours. Each treatment is set up with 3 replicates. After 2 hours, a BX-53 optical microscope (Olympus, Tokyo, Japan) was used to monitor pollen germination. Five light fields were randomly selected for each sample (culture dish), and each light field contained no less than 50 pollen grains. The criterion for pollen germination is that the length of the pollen tube exceeds the diameter of the pollen grain. Use the orthogonal table L 10 (3 4 ) to determine the optimal ratio of the culture medium.

[0032] Pollen germination rate % = (number of pollen germination / total number of pollen) × 100%

[0033] 1.3 Experimental Results

[0034] The experimental results showed that under the conditions of basic culture medium (10% sucrose and 0.01% boric acid), adding 50mg·L-1CaCl2·2H2O, 10mg·L-1MgSO4, 0.5mg·L-1NAA and 2mg·L-1GA3 can maximize the germination rate of YZ2 pollen.

[0035] Table 1. The level of factors in the orthogonal test of pollen germination of Camellia oleifera 'YZ2' in Youxian County

[0036]

[0037] Table 2 Statistical table of pollen germination orthogonal test results of Youxian Camellia oleifera 'YZ2'

[0038]

[0039] Example 2 Effects of different pollination methods on the fruit setting rate of Camellia oleifera

[0040] 2.1 Pollen storage and collection

[0041] During the flowering period of Camellia oleifera (the flowering period of common Camellia oleifera ‘Huashuo’ and ‘Huaxin’ is November-December, and the flowering period of Youxian Camellia oleifera ‘YZ2’ is February-March), collect the buds that have turned white but have not yet opened, and then use tweezers to collect the anthers of common Camellia oleifera (‘Huashuo’ and ‘Huaxin’) and Youxian Camellia oleifera (‘YZ2’) into culture dishes, and place the culture dishes in a 25℃ incubator to bake until the pollen is released. Collect the pollen into a centrifuge tube and seal it with a sealing film, and store it in a -80℃ ultra-low temperature refrigerator. After taking the pollen out of the refrigerator and letting it stand at room temperature, it can be used for pollination.

[0042] 2.2 Pollination

[0043] 1) Artificial pollination (Common Camellia oleifera 'Huashuo' × Youxian Camellia oleifera 'YZ2', control group)

[0044] Select the white buds of 'Hua Shuo', hang a tag and mark them. Carefully peel off the petals and stamens with tweezers, and keep some filaments to protect the pistil. After fully dipping the pollen of 'YZ2' with a pollination stick, gently apply the pollen to the stigma of 'Hua Shuo', and then tighten the pollination bag to prevent pollen contamination. Unpack the bag after one week. Count the fruit setting rate on the 90th and 180th days after pollination.

[0045] 2) Induced pollination

[0046] Place the sealed centrifuge tube containing 'Huaxin' pollen in a boiling water bath for 30 minutes. Figure 1), after returning to room temperature, mix it with non-inactivated 'YZ2' pollen in a 1:1 ratio to make the guided pollen. Select the white 'Huashu' flower buds, hang a tag and mark them. Use tweezers to carefully peel off the petals and stamens, retain some filaments to protect the pistil, dip the pollen with the pollination stick, apply the guided pollen to the stigma of 'Huashu', and then tighten the pollination bag to prevent pollen contamination. Unpack the bag after one week. Count the fruit setting rate on the 90th day after pollination, and count the fruit setting rate on the 180th day and observe the fruit development.

[0047] 3) Cutting style pollination

[0048] Select the white 'Hua Shuo' flower buds, hang a tag and mark them. Use tweezers to carefully peel off the petals and stamens, retain some filaments to protect the pistil, remove the stamens of 'Hua Shuo', and cut off the pistil's style with scissors. The length of the style is retained in full length (without cutting the style), about 4 / 5 (cutting the stigma), about 1 / 2 (cutting half of the style) and about 1 / 5 (the base of the style), then apply 'YZ2' pollen to the style cut, and then tighten the pollination bag to prevent pollen contamination. Unpack the bag after one week. Count the fruit setting rate on the 90th and 180th days after pollination.

[0049] 2.3 Experimental Results

[0050] As shown in Table 3, compared with the common artificial pollination method, the guided pollination method can improve the fruit setting rate of 'Hua Shuo' distant hybridization, but the improvement effect is weak. The method of cutting the style pollination can significantly improve the fruit setting rate after pollination. The longer the length of the cut style, the higher the fruit setting rate. The best effect is when about 1 / 5 of the style (keeping the style base) is retained.

[0051] Table 3 Effects of different pollination methods on fruit setting rate of common oil-tea camellia 'Huashuo' × Youxian oil-tea camellia 'YZ2'

[0052]

[0053] Example 3 Effect of pollen germination culture medium on fruit setting rate of Camellia oleifera

[0054] 3.1 Obtain pollen germination culture solutions No. 1, No. 2 and No. 5 in the manner of Example 1.

[0055] 3.2 The style was cut and pollinated according to the steps of Example 2, wherein the retained length of the style was about 1 / 5 (the base of the style), the pollen germination culture media were No. 1, No. 2 and No. 5, respectively, and the fruit setting rate was counted on the 90th and 180th days after pollination.

[0056] 3.3 Experimental Results

[0057] As shown in Table 4, applying pollen germination medium to the style incision can significantly increase the fruit setting rate of common Camellia oleifera 'Huashuo' × Youxian Camellia oleifera 'YZ2', and pollen germination medium No.1 has the best effect on increasing the fruit setting rate.

[0058] Table 4 Effects of pollen germination culture medium on fruit setting rate of common oil-tea camellia 'Huashuo' × Youxian oil-tea camellia 'YZ2'

[0059]

[0060] Example 4 Effects of different pollination methods on the quality of tea fruit

[0061] 4.1 Artificial pollination, guided pollination and style cut pollination were carried out according to the method of Example 2, except that: 1) the style was kept at 1 / 5 of its original length in style cut pollination; 2) after the style was cut, a proper amount of pollen germination culture solution No. 1 was applied to the style cut with a brush, and then 'YZ2' pollen was applied to the style cut ( Figure 2 ); 3) Observe and count the fruit development on the 180th day after artificial pollination, guided pollination and cut style pollination (180DAP) Figure 3 ).

[0062] 4.2 Experimental Results

[0063] As shown in Table 5, compared with artificial pollination and guided pollination, the method of pollination by cutting the style can effectively improve the fruit quality of Camellia oleifera, and the size and weight of the fruit are significantly improved.

[0064] Table 5 Effects of different pollination methods on fruit quality of common Camellia oleifera 'Huashuo' × Youxian Camellia oleifera 'YZ2'

[0065]

[0066]

[0067] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the present application. Thus, if these changes and modifications of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and modifications.

Claims

1. A method for improving the fruit setting rate of distant hybridization of oil-tea camellia, characterized in that: Pollination is carried out by cutting the style; the method specifically comprises: removing the stamens of the female Camellia oleifera during the flowering period, cutting the style of the pistil, and then applying the pollen of the male Camellia oleifera to the style incision to complete pollination; the cutting of the style of the pistil specifically comprises cutting off 1 / 5-4 / 5 of the style of the pistil.

2. A method for improving the quality of distant hybridization fruits of Camellia oleifera, characterized in that: Pollination is carried out by cutting the style; the method specifically comprises: removing the stamens of the female tea plant during the flowering period of tea, cutting the style of the pistil, and then applying the pollen of the male tea plant to the style incision to complete pollination; the cutting of the style of the pistil specifically comprises cutting off 1 / 5-4 / 5 of the style of the pistil; the method further comprises applying pollen germination culture solution to the style incision after cutting the style, and then applying the pollen of the male tea plant to the style incision; the pollen germination culture solution comprises sucrose, boric acid, CaCl2·2H2O, MgSO4, NAA and GA3.

3. The method according to claim 2, characterized in that The pollen germination culture solution includes 0.1 g·mL -1 Sucrose, 0.1 g·L -1 Boric acid, 50 mg·L -1 CaCl2·2H2O, 10 mg·L -1 MgSO4, 0.5 mg·L -1 NAA and 2 mg·L -1 GA3.

4. The method according to claim 1 or 2, characterized in that: The mother tea plant is selected from tea plants with white buds but not yet opened.

5. The method according to claim 4, characterized in that The cutting of the pistil style specifically includes cutting off 4 / 5 of the pistil style.

6. The method according to claim 5, characterized in that The female Camellia oleifera is 'Huashuo' Camellia oleifera or 'Huaxin' Camellia oleifera, the male Camellia oleifera is Youxian Camellia oleifera, and the fruit quality refers to the size and weight of the fruit.

7. The method according to claim 1, comprising applying pollen germination culture solution to the style incision after cutting the style, and then applying pollen of the male parent Camellia oleifera to the style incision; the pollen germination culture solution comprises sucrose, boric acid, CaCl2·2H2O, MgSO4, NAA and GA3.

8. The method according to claim 7, characterized in that The pollen germination culture solution includes 0.1 g·mL -1 Sucrose, 0.1 g·L -1 Boric acid, 50 mg·L -1 CaCl2·2H2O, 10 mg·L -1 MgSO4, 0.5 mg·L -1 NAA and 2 mg·L -1 GA3.

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