Method for improving yield and competitiveness of camellia oleifera 2n pollen

Abstract

The invention belongs to the technical field of plant biological breeding, and particularly relates to a method for improving the yield and competitiveness of camellia oleifera 2n pollen, which comprises the following steps: performing gibberellin treatment on camellia oleifera flower buds in a meiosis period, and inducing the formation of 2n pollen; after gibberellin treatment is removed, camellia oleifera flower buds in the big bud stage are collected, and mixed pollen is collected; < 60 > Co-gamma is used as a radiation source to radiate the camellia oleifera mixed pollen to obtain mixed pollen with improved 2n pollen competitiveness; the gibberellin treatment method comprises a flowering branch transportation method, and the flowering branch transportation method comprises the following steps: forming a notch in a position, close to a flower bud, of a camellia oleifera branch, inserting a cotton thread into the notch, fixing the cotton thread, and placing the other end of the cotton thread into gibberellin. Formation of the 2n pollen is promoted through a flowering branch transportation method, the competitiveness of the 2n pollen is improved through a pollen radiation means, and the problem that the competitiveness of the 2n pollen in the mixed pollen is low is effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant biological breeding, and particularly relates to a method for improving the yield and competitiveness of 2n pollen of camellia oleifera. Background Art

[0002] Camellia oleifera is a very important woody oil-bearing tree species in my country, highly valued for its oil-rich seeds. As a genetic resource, 2n pollen, used in ploidy breeding, provides a new avenue for plant genetic improvement.

[0003] In the past, 2n pollen of forest trees was usually obtained through induction with colchicine and high temperature. The 2n gametes obtained by high temperature induced forest trees are of SDR type, and the heterozygosity of the genetic parents is only half of that of the FDR type 2n gametes. This is the shortcoming of high temperature in ploidy breeding. Colchicine, as an important chemical mutagen, is mainly used to induce the doubling of forest tree megaspores, embryo sacs, zygotes and somatic chromosomes. It is expensive and highly toxic to humans. Gibberellic acid, as an important endogenous plant hormone, regulates the growth and development of somatic tissues. The use of gibberellins to induce cytoplasmic division defects in oil tea pollen mother cells avoids the above-mentioned disadvantages of colchicine and high temperature treatment.

[0004] Previous studies on the induction of 2n pollen in plants found that normal pollen and 2n pollen were small in size, making them difficult to separate effectively. Normal pollen is usually abundant and germinates quickly, preferentially entering the embryo sac for fertilization. However, 2n pollen, due to its small number, is easily "crowded out" and cannot be fully utilized. This has long been an unresolved problem that has plagued polyploidy breeders. Therefore, pollination can only be performed through mixed pollen, which significantly reduces the efficiency of obtaining chromosome-doubled progeny plants, severely limiting the application of 2n pollen in polyploid breeding of Camellia oleifera.

[0005] Although physical and chemical induction methods can effectively increase the yield of 2n pollen, the maximum yield of normal pollen, which has not doubled its chromosomes, can reach over 90%. When normal and 2n pollen cannot be separated, improving the competitiveness of 2n pollen in mixed pollen during artificial pollination is crucial for its effective utilization. Therefore, it is urgent to establish a method that can ensure the yield and competitiveness of 2n pollen in Camellia oleifera to promote the genetic improvement of Camellia oleifera ploidy breeding using 2n pollen. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for improving the yield and competitiveness of 2n pollen of Camellia oleifera. The method comprises treating Camellia oleifera with gibberellins through the flower branch transportation method to promote the formation of 2n pollen of Camellia oleifera, and improving the competitiveness of 2n pollen by means of pollen radiation. The method effectively solves the problem of low competitiveness of 2n pollen in mixed pollen, is of great significance to the utilization of 2n pollen, and meets the needs of polyploid breeding practice of Camellia oleifera.

[0007] The present invention provides a method for improving the yield and competitiveness of Camellia oleifera 2n pollen, comprising the following steps:

[0008] The flower buds of Camellia oleifera at meiosis stage were treated with gibberellins to induce the formation of 2n pollen.

[0009] After removing the gibberellin treatment, the camellia buds at the large bud stage were collected and mixed pollen was collected;

[0010] by 60 The mixed pollen of Camellia oleifera was irradiated with Co-γ as the radiation source, and the mixed pollen with improved competitiveness of 2n pollen was obtained.

[0011] The method for treating with gibberellins includes a squid transport method, which includes the following steps:

[0012] Make an incision near the flower bud on the oil tea branch, insert cotton thread into the incision and fix it, and place the other end of the cotton thread in gibberellin.

[0013] As a preferred embodiment, the mixed pollen includes 2n pollen and normal pollen.

[0014] As a preferred embodiment, the meiotic period is from the leptotene stage to the pachytene stage.

[0015] As a preferred solution, the concentration of the gibberellin is 50 to 300 mg / L; and the gibberellin treatment time is 6 to 48 hours.

[0016] As a preferred embodiment, the concentration of the gibberellin is 200 mg / L; and the gibberellin treatment time is 6 hours.

[0017] As a preferred solution, the position near the flower bud is 3 to 5 cm away from the flower bud, and the angle of the incision is 30° to 45°.

[0018] As a preferred solution, the incision reaches deep into the wood.

[0019] As a preferred solution, the 60 The radiation dose of Co-γ is 250 to 1500 Gy.

[0020] As a preferred solution, the 60The radiation dose of Co-γ was 1500 Gy.

[0021] As a preferred solution, the 60 The radiation dose rate of Co-γ is 10 Gy·min -1 .

[0022] The present invention has the following beneficial effects:

[0023] In the present invention, the oil-tea camellia branches were treated with different concentrations of gibberellin, 50 mg / L, 100 mg / L, 200 mg / L, and 300 mg / L, and the treatment was continued for 6 hours, 12 hours, 24 hours, and 48 hours. It was found that the 2n pollen yield was the highest when treated with 200 mg / L gibberellin for 6 hours, reaching 9.86%. 60 Mixed pollen was irradiated with Co-γ at doses of 250 Gy, 500 Gy, 750 Gy, 1000 Gy, 1250 Gy, and 1500 Gy. Statistical analysis of pollen germination revealed that 2n pollen had a significant germination rate advantage over normal pollen during the first 6 hours of in vitro culture. At a dose of 1500 Gy, 2n pollen exhibited the most pronounced competitive advantage at 3 and 6 hours of in vitro culture, reaching 26.29 and 36.27 times that of normal pollen, respectively. Statistical analysis of pollen tube length revealed that 2n pollen exhibited a significant advantage over normal pollen tube length after 1 to 2 hours of in vitro culture, reaching a tube length of 206.05 μm after 2 hours of in vitro culture. The above results show that compared with the currently commonly used method of high temperature and colchicine induction, the method described in the present invention has the advantages of easy operation, high efficiency, and rapidity, which effectively solves the problem of low competitiveness of 2n pollen, is of great significance for the effective utilization of 2n pollen, and meets the needs of oil tea polyploid breeding practice.

[0024] Compared with the prior art, the present invention also has the following effects:

[0025] 1. The gibberellin used in the present invention has no toxic effect on plants and is low in cost.

[0026] 2. The method of the present invention determines the concentration and continuous treatment time of gibberellin for efficient induction of 2n pollen of Camellia oleifera, thereby improving the yield of 2n pollen of Camellia oleifera from 1.28% under natural conditions to 9.86%.

[0027] 3. The present invention proposes a method for improving the germination competitiveness of 2n pollen by irradiating Camellia oleifera pollen to inhibit the germination of normal pollen in the mixed pollen obtained after gibberellin treatment.

[0028] 4. The present invention overcomes the problem that the 2n pollen in the mixed pollen cannot complete double fertilization because the normal pollen in the mixed pollen is excessive and its pollen tube grows fast, and enters the embryo sac first to complete double fertilization.

[0029] 5. The present invention adopts the squid transport method, 60 The combination of two methods, using Co-γ as the radiation source to irradiate pollen, improved the yield and competitiveness of Camellia oleifera 2n pollen. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To more clearly illustrate the embodiments of the present invention, the following briefly describes the accompanying drawings required for use in the embodiments. The accompanying drawings are intended to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present application and do not constitute a limitation of the present application.

[0031] Figure 1 This is a diagram showing the process of completing gibberellin treatment of Camellia oleifera flower branches using the flower branch transport method in Example 1; wherein A shows the treated plant; B shows the treatment process; a shows the specific location of the flower branch incision; b shows the size of the incision; c shows the centrifuge tube device at the lower end of the incision; d shows the centrifuge tube filled with gibberellin and protected from light by tinfoil;

[0032] Figure 2 This is a diagram of the meiotic progression of the pollen mother cell of Camellia oleifera in Example 1; wherein A is early leptotene; B is late leptotene; C is zygotene; D is pachytene; E is diplotene; F is diakinesis; G is metaphase I; H is anaphase I; I is telophase I; J is prophase II; K is metaphase II; L is anaphase II; M is telophase II; N is trid; O is tetrad; scale bar = 10 μm;

[0033] Figure 3 These are morphological images of 2n pollen and normal pollen after gibberellin treatment in Example 1; A: Camellia oleifera pollen after gibberellin treatment; B: Camellia oleifera pollen in the control group; Dp: 2n pollen; Np: normal pollen; Sp: aborted pollen; scale bar = 20 μm.

[0034] Figure 4 This is a graph showing the activity of irradiated pollen in Example 2; scale = 10 μm;

[0035] Figure 5 The germination rates of pollen irradiated at different times in Example 2 are as follows: A is the germination rate of pollen irradiated for 3 hours; B is the germination rate of pollen irradiated for 6 hours; C is the germination rate of pollen irradiated for 12 hours; and D is the germination rate of pollen irradiated for 24 hours. DETAILED DESCRIPTION

[0036] The present invention provides a method for improving the yield and competitiveness of Camellia oleifera 2n pollen, comprising the following steps:

[0037] The flower buds of Camellia oleifera at meiosis stage were treated with gibberellins to induce the formation of 2n pollen.

[0038] After removing the gibberellin treatment, the camellia buds at the large bud stage were collected and mixed pollen was collected;

[0039] by 60 The mixed pollen of Camellia oleifera was irradiated with Co-γ as the radiation source, and the mixed pollen with improved competitiveness of 2n pollen was obtained.

[0040] The method for treating with gibberellins includes a squid transport method, which includes the following steps:

[0041] Make an incision near the flower bud on the oil tea branch, insert cotton thread into the incision and fix it, and place the other end of the cotton thread in gibberellin.

[0042] The invention treats camellia oleifera flower buds in the meiosis period with gibberellin to induce the formation of 2n pollen, collects the pollen, and obtains mixed pollen with an increased 2n pollen ratio.

[0043] As a specific embodiment, gibberellins can be applied to camellia oleifera flower buds during meiosis from late September to early October. As a specific embodiment, gibberellins can be applied to camellia oleifera flower buds during the leptotene to pachytene stages. The leptotene to pachytene stages are the core stages of meiotic prophase I. During this period, cells are highly sensitive to gibberellins, which regulate the expression of related genes or signaling pathways to influence chromosome behavior during meiosis. The present invention selects gibberellins for treatment during the leptotene to pachytene stages, which can effectively interfere with chromosome behavior and better induce the formation of 2n pollen.

[0044] The concentration of the gibberellin of the present invention can be 50 to 300 mg / L; the duration of the gibberellin treatment can be 6 to 48 hours. As a specific embodiment, the concentration of the gibberellin can be any one of 50 mg / L, 100 mg / L, 200 mg / L and 300 mg / L. As a specific embodiment, the duration of the gibberellin treatment can be any one of 6 hours, 12 hours, 24 hours and 48 hours. In a specific embodiment of the present invention, when the concentration of the gibberellin is 200 mg / L and the gibberellin treatment time is 6 hours, the yield of 2n pollen is the highest, reaching 9.86%, which is higher than that of other treatment groups.

[0045] The gibberellin treatment method of the present invention includes a flower branch transportation method; the flower branch transportation method includes the following steps: making an incision near the flower bud of a tea oil branch, inserting and fixing a cotton thread into the incision, and placing the other end of the cotton thread in gibberellin.

[0046] The position near the flower bud of the present invention can be 3 to 5 cm away from the flower bud; in a specific embodiment of the present invention, the position near the flower bud is 5 cm away from the flower bud; the angle of the incision is 30° to 45°. The angle between the incision plane and the central axis of the growth direction of the oil-tea camellia branch is 30° to 45°; in a specific embodiment of the present invention, the angle of the incision is 45°; the incision reaches deep into the xylem; in this embodiment of the present invention, the depth of the incision can be 1 to 2 mm.

[0047] The present invention treats camellia oleifera flower buds with gibberellins through a stalk transport method. The gibberellins, by treating pollen mother cells, can induce the production of 2n pollen. The stalk transport method is minimally destructive to the camellia oleifera branches and stems. The method utilizes the capillary action of cotton thread to efficiently absorb and transport the gibberellin solution, improving the efficiency of gibberellin transport and induction. The present invention determines the gibberellin concentration and treatment duration required for efficient induction of 2n pollen in camellia oleifera, thereby increasing the yield of 2n pollen from 1.28% under natural conditions to 9.86%.

[0048] After the present invention removes the gibberellin treatment, the oil-tea camellia buds in the large bud stage can be collected, and pollen can be collected to obtain mixed pollen. As a specific embodiment, the mixed pollen includes 2n pollen and normal pollen. The diameter of normal oil-tea camellia pollen is generally 30 to 45 μm, and the diameter of 2n pollen is generally 1.27 to 1.5 times that of normal pollen, with a diameter range of about 50 to 70 μm. In a specific embodiment of the present invention, when the pollen diameter is 1.27 times the diameter of normal pollen, it can be considered as 2n pollen. After the present invention obtains the oil-tea camellia mixed pollen, 60 Mixed Camellia oleifera pollen was irradiated using Co-γ as the radiation source to obtain mixed pollen with improved competitiveness of 2n pollen.

[0049] The present invention 60 The radiation dose of Co-γ can be 250 to 1500 Gy. As a specific embodiment, the 60 The radiation dose of Co-γ may be any one of 250 Gy, 500 Gy, 750 Gy, 1000 Gy, 1250 Gy and 1500 Gy.

[0050] As a specific embodiment, after the pollen is irradiated, in vitro cultures are performed to verify the competitiveness of 2n pollen and normal pollen. If the germination length of the pollen tube reaches or exceeds the diameter of the pollen itself, the pollen is considered to have successfully germinated. The germination length of the pollen tube is measured, and the germination rate of 2n pollen and normal pollen in the mixed pollen is calculated. In this specific embodiment of the present invention, at a radiation dose of 1500 Gy, the germination rate of 2n pollen is the highest, reaching 29.38%, significantly higher than that of other treatment groups, and the difference with the germination rate of normal pollen is the largest. This shows that irradiated pollen significantly improves the competitiveness of 2n pollen.

[0051] The present invention's flower branch transportation method is less destructive to the branches of Camellia oleifera. It utilizes the capillary action of cotton thread to efficiently absorb and transport gibberellin solution, which can increase the efficiency of gibberellin-induced pollen chromosome doubling and improve the yield of Camellia oleifera 2n pollen. The irradiated pollen of the present invention can inhibit the germination of normal pollen and improve the competitiveness of Camellia oleifera 2n pollen. The combination of the two significantly promotes the effective utilization of 2n pollen. The present invention uses the flower branch transportation method to utilize gibberellin to induce the formation of 2n pollen, providing sufficient preparation materials for 2n pollen competition, and then combines a certain radiation dose to 60 Co-γ, to a certain extent, inhibits normal pollen and stimulates the germination of 2n pollen, relatively improving the competitiveness of 2n pollen in the mixed pollen during the fertilization process, thereby significantly improving the efficiency of 2n pollen in completing double fertilization, providing reliable technical support for oil tea polyploid breeding.

[0052] To further illustrate the present invention, the following detailed description of a method for increasing the yield and competitiveness of Camellia oleifera 2n pollen is provided in conjunction with examples. However, these examples are not to be construed as limiting the scope of the present invention. Unless otherwise specified, the present invention has no particular requirements for the raw materials used; commercially available products familiar to those skilled in the art can be used.

[0053] The oil-tea camellia test materials used in the examples were in vitro flower branches of the improved oil-tea camellia variety 'Huaxin' (Camellia oleifera 'Huaxin'), on which flower buds were growing healthily.

[0054] Example 1 Gibberellic acid treatment induces pollen formation in Camellia oleifera 2n

[0055] 1. Observation of the meiotic development process of pollen mother cells

[0056] Camellia oleifera buds were collected and fixed with Carnoy's fixative. Temporary smears were made by acetic acid carmine staining. The meiotic process of pollen mother cells was observed under an ordinary optical microscope. Figure 2 shown.

[0057] When the meiosis of the pollen mother cells of camellia oleifera was in the leptotene to pachytene stage, gibberellins were applied to the camellia oleifera branches to induce 2n pollen. The camellia oleifera branches were treated with 50mg / L, 100mg / L, 200mg / L, and 300mg / L different concentrations of gibberellins, and the treatments were continued for 6h, 12h, 24h, and 48h, for a total of 16 treatment combinations, and each treatment group was numbered and recorded. The 0h group was treated under different gibberellins concentrations for the control group, i.e., the gibberellins were not applied to the control group. Four groups of controls were set, wherein the gibberellin treatment control group 1 corresponding to the 50mg / L gibberellin treatment group was not applied, and the gibberellin treatment control group 2 corresponding to the 100mg / L gibberellin treatment group was not applied, and the like was deduced. The camellia oleifera number involved in the present embodiment is large, and some parameters are limited with range values.

[0058] The method of induction by gibberellin treatment is the flower branch transport method: before gibberellin treatment, use a knife to cut into the flower branch at an angle (30° to 45°) near the flower bud (3 to 5 cm away from the flower bud) to reach the surface of the xylem, creating an incision in the xylem. Insert cotton thread into the incision and fix the cotton thread with sealing film. The other end is placed in a centrifuge tube. Different concentrations of gibberellin in the centrifuge tube are introduced into the incision flower branch and transported to the anther through the plant vascular system to achieve the induction treatment of microspore mother cells. The treatment process is as follows: Figure 1 shown.

[0059] 2. Statistical analysis of 2n pollen yield

[0060] After removing the gibberellin treatment, the oil tea plants were allowed to grow naturally until the flower buds developed to the large bud stage. Flower buds from the control group (flower buds not treated with gibberellin) and the gibberellin treatment groups were collected. Fifty flower buds were collected from each group, and pollen was collected and observed. Temporary pollen smears from each treatment were prepared by acetic acid carmine staining. The pollen was then photographed using an Olympus DP70 optical microscope. The pollen diameter in the temporary smears from each treatment was measured using Image J software. The results are shown in Figure 2. Figure 3 As shown, the yield of 2n pollen was counted.

[0061] The 2n pollen yield is calculated as the number of 2n pollen divided by the total number of pollen observed. The statistical results are shown in Table 1. The statistical results show that, at each concentration gradient, after 6 hours of treatment, the 2n pollen yield was the highest compared to the other treatment groups with the same concentration and time. Among them, the 200 mg / L gibberellin treatment for 6 hours produced the highest 2n pollen yield, reaching 9.86%, significantly higher than the other treatment groups. After 24 and 48 hours of treatment, the 2n pollen yield began to decline across all treatment concentration gradients, indicating that prolonged gibberellin treatment inhibits 2n pollen formation in oil-tea camellia.

[0062] Table 1 2n pollen yield at different gibberellin concentrations and treatment times

[0063]

[0064]

[0065] Note: Different lowercase letters represent significant differences at the same concentration and different time levels at the P=0.05 level.

[0066] Comparative Example 1: Spraying gibberellin to induce 2n pollen formation

[0067] Camellia oleifera buds in the leptotene to pachytene stage were identified using the method of Example 1. These buds were then sprayed with 200 mg / L gibberellin (GAB) for one, two, and three times, respectively. The spraying was stopped when the GAB solution dropped below the threshold, with 15 minutes between sprays. Simultaneously, 1, two, and three sprays of water were used as controls. The 2n pollen yield was calculated using the same statistical method as in Example 1. The results are shown in Table 2.

[0068] Table 2 Pollen yield of spraying gibberellin 2n at 200 mg / L concentration

[0069]

[0070] Table 2 shows that there was no significant difference in 2n pollen yield between the gibberellin spraying treatment and the control treatment. The thick scales of camellia oleifera flower buds make it difficult for gibberellin spray to penetrate the buds. Furthermore, the gibberellin solution evaporates quickly, reducing its effectiveness. Ultimately, the 2n pollen yield after gibberellin spraying was no significantly different from the control treatment.

[0071] Comparative Example 2: Injection of gibberellin to induce 2n pollen formation

[0072] Camellia oleifera flower buds in the leptotene to pachytene stages were identified using the method of Example 1. A 200 mg / L gibberellin solution was injected into the camellia oleifera flower buds using a syringe until the solution permeated. Injection of clear water served as a control. The mortality rate of the camellia oleifera flower buds was calculated, and the results are shown in Table 3.

[0073] As shown in Table 3, the flower bud mortality rate of gibberellin injection was 79.87%, while that of the control group was 75.66%. The cause of flower bud mortality may be mechanical damage caused by needle penetration during injection, which affected normal development and caused the flower buds to dry up and die.

[0074] Table 3200mg / L concentration injection of gibberellin flower bud mortality rate

[0075] Gibberellic acid concentration (mg / L) Fatality rate (%) Lethality rate (control) (%) 200 79.87 75.66

[0076] Therefore, compared with the spraying and injection methods, the flower branch transport method is an ideal method for applying gibberellin treatment because it is transported in the body without causing mechanical damage to the flower buds and effectively inducing 2n pollen.

[0077] Example 2 60 Co-γ irradiation of pollen enhances the germination advantage of 2n pollen

[0078] The oil-tea camellia buds were treated with 200 mg / L gibberellin for 6 h in the same manner as in Example 1. The oil-tea camellia buds at the large bud stage were collected and pollen was collected. 60 Mixed pollen was irradiated using Co-γ as the radiation source to obtain mixed pollen. The radiation doses were 250 Gy, 500 Gy, 750 Gy, 1000 Gy, 1250 Gy, and 1500 Gy, and the radiation dose rate was 10 Gy min -1 .

[0079] The pollen viability was tested by in vitro culture on agar medium (e.g. Figure 4 ) and the germination rates of 2n pollen and normal pollen were calculated. Agar with a mass concentration of 1% was first prepared, followed by the addition of sucrose with a mass concentration of 10% and boric acid with a mass concentration of 0.01% to obtain a germination medium. A small amount of mixed pollen was evenly spread throughout the germination medium. The germination medium containing the mixed pollen was then randomly divided into groups: 1 hour in vitro culture group, 2 hours in vitro culture group, 3 hours in vitro culture group, 6 hours in vitro culture group, 12 hours in vitro culture group, and 24 hours in vitro culture group. The germination medium containing the mixed pollen was sealed and incubated in a 25°C incubator for in vitro culture periods of 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours, respectively. Observation was performed using an Olympus DP70 optical microscope.

[0080] Three visual fields were randomly selected to ensure that each field contained at least 20 pollen grains. The observation results of the three fields were averaged as repeated data, and the test results were repeated three times. The average of the above data was taken, and the test results are shown in Tables 4 and Figure 5 .

[0081] From the statistical results in Table 4, it can be seen that under different radiation doses, the germination rate of 2n pollen in the first 3h of in vitro culture has a significant advantage over the normal pollen germination rate. At a radiation dose of 1500 Gy, the difference in germination rate between 2n pollen and normal pollen was the largest, with 2n pollen having the highest germination rate, reaching 13.80% at 1 hour, 15.57% at 2 hours, 27.60% at 3 hours, 29.38% at 6 hours, 29.05% at 12 hours, and 25.38% at 24 hours, which were significantly higher than those of other radiation dose groups at the same treatment time. At a radiation dose of 250 Gy, the germination rate of 2n pollen was the lowest compared with other radiation doses, reaching 6.73% at 1 hour, 9.23% at 2 hours, 10.07% at 3 hours, 10.06% at 6 hours, 12.55% at 12 hours, and 13.89% at 24 hours, which were significantly lower than those of other radiation dose groups at the same treatment time. When mixed pollen irradiated with 1500 Gy was cultured in vitro for 3 hours and 6 hours, 2n pollen had the most obvious competitive advantage, which was 26.29 and 36.27 times that of normal pollen. This indicates that within a certain range of radiation doses, as the radiation dose increases, 2n pollen germination becomes more advantageous.

[0082] Table 4 Statistics of germination rates of 2n pollen and normal pollen under different radiation doses

[0083]

[0084] Note: Different lowercase letters represent significant differences at the P=0.05 level between different radiation doses at the same time.

[0085] Example 3 60 Co-γ irradiated pollen enhances pollen tube growth advantage of 2n pollen

[0086] The oil-tea camellia buds were treated with 200 mg / L gibberellin for 6 h in the same manner as in Example 1. The oil-tea camellia buds at the large bud stage were collected and pollen was collected. 60 Mixed pollen was irradiated with Co-γ as the radiation source at a dose of 1500 Gy and a radiation dose rate of 10 Gy·min -1 .

[0087] The germination of pollen irradiated with a dose of 1500 Gy was determined by in vitro culture on an agar medium. The germination medium was the same as in Example 2. After evenly spreading a small amount of mixed pollen in the germination medium, the germination medium containing the pollen was randomly divided into groups, specifically, a 1-hour in vitro culture group, a 2-hour in vitro culture group, a 3-hour in vitro culture group, a 4-hour in vitro culture group, and a 5-hour in vitro culture group. The germination medium containing the pollen was sealed and placed in a constant temperature box at 25°C for culture. The in vitro culture time was 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours, respectively. After each group reached the expected culture time, an Olympus DP70 optical microscope was used to take pictures, observe and count, and the pollen tube length was measured using Image J software.

[0088] The pollen tube lengths of 2n pollen and normal pollen at different in vitro culture times were statistically analyzed, and the results are shown in Table 5.

[0089] Table 5 Pollen tube lengths of 2n pollen and normal pollen at a radiation dose of 1500 Gy

[0090] In vitro culture duration 2n pollen tube length (μm) Normal pollen tube length (μm) 1h 84.31a 0.33b 2h 206.05a 1.52b 3h 358.88b 486.07a 4h 332.90b 507.07a 5h 473.34b 522.50a

[0091] Note: Different lowercase letters represent significant differences at the P=0.05 level.

[0092] From the statistical results in Table 5, it can be seen that the pollen tube length of 2n pollen has a significant advantage over the normal pollen germination length after 1 to 2 hours of in vitro culture. Among them, the pollen tube length of 2n pollen reached 206.05 μm after 2 hours of in vitro culture. After 3 hours of in vitro culture, the pollen tube length of normal pollen came from behind and 2n pollen gradually lost its competitive advantage.

[0093] The present invention improves the yield of 2n pollen of oil tea through a flower branch transportation method. When the gibberellin treatment concentration is 200 mg / L and the treatment time is 6 hours, the 2n pollen yield is the highest. The competitiveness of 2n pollen is significantly improved by irradiating the pollen. Under a maximum radiation dose of 1500 Gy, the germination rate of 2n pollen is most different from that of normal pollen. Within 1 to 2 hours, the competitiveness of 2n pollen is most significantly improved. This will help to improve the efficiency of obtaining oil tea polyploids through 2n pollen hybridization.

[0094] Thus, the present invention applies gibberellins to camellia oleifera flower buds during the meiotic phase through the flower branch transport method, thereby increasing the yield of 2n pollen in camellia oleifera and further enhancing the competitiveness of 2n pollen by irradiating the pollen. Compared with the currently commonly used high temperature and colchicine induction methods, the present invention has the advantages of being easy to operate, simple, effective, and rapid, effectively solving the problem of low 2n pollen competitiveness, and is of great significance for the utilization of 2n pollen, meeting the needs of camellia oleifera polyploid breeding practice.

[0095] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for improving the yield and competitiveness of Camellia oleifera 2n pollen, characterized in that: The following steps are involved: The flower buds of Camellia oleifera at meiosis stage were treated with gibberellins to induce the formation of 2n pollen. After removing the gibberellin treatment, the camellia buds at the large bud stage were collected and mixed pollen was collected; by 60 The mixed pollen of Camellia oleifera was irradiated with Co-γ as the radiation source, and the mixed pollen with improved competitiveness of 2n pollen was obtained. The method for treating with gibberellins includes a squid transport method, which includes the following steps: Make an incision near the flower bud on the oil tea branch, insert cotton thread into the incision and fix it, and place the other end of the cotton thread in gibberellin.

2. The method according to claim 1, characterized in that The mixed pollen includes 2n pollen and normal pollen.

3. The method according to claim 1, characterized in that The meiotic period is from the leptotene stage to the pachytene stage.

4. The method according to claim 1, wherein The concentration of the gibberellin is 50 to 300 mg / L; and the treatment time of the gibberellin is 6 to 48 hours.

5. The method according to claim 1 or 4, characterized in that The concentration of the gibberellin is 200 mg / L; the time of the gibberellin treatment is 6 hours.

6. The method according to claim 1, characterized in that The position near the flower bud is 3 to 5 cm away from the flower bud, and the angle of the incision is 30° to 45°.

7. The method according to claim 6, characterized in that The incision reaches deep into the wood.

8. The method according to claim 1, characterized in that described 60 The radiation dose of Co-γ is 250 to 1500 Gy.

9. The method according to claim 1 or 8, characterized in that described 60 The radiation dose of Co-γ was 1500 Gy.

10. The method according to claim 1 or 8, characterized in that described 60 The radiation dose rate of Co-γ was 10 Gy·min -1 .

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