A method for detecting self-fertility of macadamia nuts

Abstract

The present invention provides a method for detecting the self-fertility of macadamia nuts, which belongs to the field of biological detection technology. The invention provides a method for detecting the self-fertility of macadamia nuts. First, the inflorescence of the self-pollinating pistil is arranged, and then the inflorescence of the macadamia nut variety to be tested is bagged. After 7 to 9 days, the inflorescence is taken out from the bag, and all the pistils attached to the inflorescence axis together with the inflorescence axis are immersed in FAA fixative for fixation. After washing and softening, the ovary and the lower half of the style are cut horizontally and retained. The retained part is then cut longitudinally and stained. The growth of the pollen tubes in the ovary and the lower half of the style after staining is observed under a fluorescence microscope, and the PLS is counted. Using the detection method of the present invention, the self-incompatibility intensity of different varieties of macadamia nuts can be intuitively and quickly identified. The identification workload is small, accurate and reliable, saving a lot of time and economic costs, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a method for detecting the self-fertility of macadamia nuts. Background Art

[0002] Macadamia nuts (Macadamia spp.), known as the "King of Nuts," are native to the subtropical rainforests of southeastern Queensland and northeastern New South Wales, Australia (25°-32°S latitude). They have been domesticated and cultivated worldwide as a commercial forest tree in tropical and subtropical regions. Widely valued for their rich nutritional value and unique taste, macadamia nuts have been widely cultivated. Although they have been widely cultivated, the limited area available for cultivation and the partial self-incompatibility of macadamia nuts have resulted in low yields, resulting in a supply shortage and persistently high prices.

[0003] Macadamia nuts are self-incompatible gametophyte fruit trees that must be cross-pollinated to set fruit. Currently, some planting measures have been adopted in production, such as configuring pollination trees with multiple varieties and placing beehives according to pollination needs, but they have not fundamentally solved the problem of low yield. In view of this, breeders in various countries have made the cultivation of self-fertile varieties the main breeding direction. Through long-term field observations, researchers have found that some macadamia varieties have a high self-fertility rate, such as varieties HAES508, HAES344 and HAES741. These germplasms are very important for the selection of self-compatible macadamia varieties, but in-depth research on the self-fertility mechanism of macadamia and the cultivation of self-compatible varieties with excellent comprehensive economic traits require far more than just a few lines. Therefore, how to quickly screen and identify self-compatible germplasms from the numerous macadamia germplasms is a major problem that needs to be solved urgently.

[0004] Currently, researchers primarily identify self-incompatibility in different macadamia varieties through field bagging. This method is labor-intensive, time-consuming, and susceptible to human and environmental factors, resulting in inaccurate or even failed self-incompatibility identification. Therefore, finding an efficient and rapid method to identify self-fertile macadamia varieties or strains is crucial. Summary of the Invention

[0005] The present invention provides a method for detecting the self-fertility of macadamia nuts, which uses the growth characteristics of macadamia nut pollen tubes in the style of the self-flowering flower as an indicator for detection. The method has the advantages of being efficient, fast, inexpensive, highly repeatable and accurate in results.

[0006] The present invention provides a method for detecting the self-fertility of macadamia nuts, comprising the following steps: (1) bagging the racemes of macadamia nuts, wherein the racemes further comprise removing opened florets and florets with unopened calyxes before bagging;

[0007] (2) 7 to 9 days after bagging, all pistils attached to the inflorescence axis together with the inflorescence axis are immersed in FAA fixative and fixed, washed and softened, and then cross-sectioned in the middle of the pistil style, and then the ovary and the lower half of the style are longitudinally cut to obtain slices;

[0008] (3) After staining the slices obtained in step (2), the growth of pollen tubes was observed, and the percentage of pistils (PLS) that grew to the base of the style and the ovary were counted;

[0009] PLS (%) = the number of pistils whose pollen tubes grew to the base of the style and ovary / the number of observed pistils × 100.

[0010] Preferably, the bagging in step (1) includes bagging racemes in which most of the sepals have cracked but not opened, and only 10 inflorescences need to be bagged.

[0011] Preferably, the mesh bag used for bagging in step (1) has a pore size smaller than the pollen diameter of macadamia nuts, and is made of a nylon membrane.

[0012] Preferably, before the fixation in step (2), the method further includes gently tapping the base of the raceme to remove the pistils with naked styles that are not firmly connected to the inflorescence axis.

[0013] Preferably, the FAA fixative in step (2) comprises a mixture of 60% by volume of an ethanol aqueous solution, 10% by volume of a neutral formalin fixative, and glacial acetic acid.

[0014] Preferably, step (2) is fixed in the FAA fixative for 24 to 36 hours.

[0015] Preferably, the washing in step (2) includes washing with alcohol aqueous solutions having a volume percentage of 60%, 40% and 20% in sequence after the fixation is completed, and then immersing in ultrapure water.

[0016] Preferably, the softening in step (2) comprises placing the washed pistil into a NaOH solution and allowing it to stand.

[0017] Preferably, the staining in step (3) includes adjusting the pH value of the slice to neutral and then staining with a strong alkaline aniline blue dye solution.

[0018] Preferably, the PLS in step (3) is ≥30%, indicating that the macadamia nut variety has strong self-fertility and a high proportion of self-pollinated fruits; the PLS is <30%, indicating that the macadamia nut variety has poor self-fertility or cannot produce fruit by self-pollination.

[0019] Beneficial effects: The present invention provides a method for detecting the self-fertility of macadamia nuts. First, the inflorescences of self-pollinated pistils are arranged, and then the inflorescences of the macadamia nut varieties to be tested are bagged. After 7 to 9 days, the inflorescences are taken out from the bags, and all the pistils attached to the inflorescence axis together with the inflorescence axis are immersed in FAA fixative for fixation. After washing and softening, the ovary and the lower half of the style are cut horizontally and retained. The retained part is then cut longitudinally and stained. The growth of pollen tubes in the ovary and the lower half of the style after staining is observed under a fluorescence microscope, and the PLS is counted.

[0020] Using the detection method described in the present invention, fluorescence microscopy was used to observe the growth characteristics of pollen tubes within the self-flowering style of macadamia nuts that had been bagged for 9 days. It was found that the percentage of observed pistils (PLS) of pollen tubes growing to the base of the self-flowering style of different varieties corresponded one-to-one with the average self-fertility rate results of four years of field bagging, showing high reliability. The field bagging identification method is not only time-consuming and labor-intensive, but also inefficient. This method has changed the situation in which the degree of self-incompatibility of different macadamia nut varieties has long been relied on field bagging. It has the advantages of high efficiency, speed, low cost, strong repeatability, and accurate results. The results showed that the lower the PLS, the weaker the self-compatibility and the stronger the self-incompatibility; the higher the PLS, the stronger the self-compatibility and the weaker the self-incompatibility.

[0021] The detection method of the present invention, compared to the conventional field bagging method that requires 100 inflorescences, not only saves labor, but also reduces the observation time from 3 years to 1 year. Specifically, the procedure of the conventional field bagging method is to arrange the inflorescences, bag, hang a label, remove the bag, and observe. The labor cost of processing one inflorescence is 2.5 yuan, 100 inflorescences is 250 yuan, and it takes 750 yuan to observe for 3 years. If all other expenses are not counted, only the labor cost is counted, and the minimum expenditure for field identification of the self-compatibility intensity of one variety is 750 yuan. The detection method of the present invention only needs to bag 10 inflorescences. When bagging, only the inflorescences need to be arranged, bagged, and hung a label, without the steps of removing the bag and observing. Therefore, the labor cost of bagging one inflorescence is 1.5 yuan, and 10 inflorescences is 15 yuan. The cost of reagents such as FAA fixative and aniline blue staining solution is about 20 yuan, and the cost of field bagging and fluorescence microscopy observation totals 35 yuan. In summary, the cost of identifying the self-compatibility strength of a variety using the method of the present invention is 35 yuan, while the cost of identifying a variety by field bagging is 750 yuan, which is 21 times the cost of the present invention.

[0022] The detection method of the present invention can intuitively and quickly identify the self-incompatibility intensity of different macadamia nut varieties. The identification workload is small, the identification results are not affected by human and environmental factors, and are accurate and reliable. At the same time, it also solves the problem of simultaneously identifying the self-fruitfulness of a large number of macadamia nut varieties (germplasm), saving a lot of time and economic costs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a diagram of the membrane bag;

[0024] Figure 2 The inflorescence flowers that need to be arranged (left) and the inflorescence bagging picture (right);

[0025] Figure 3 This is a picture of the pollen tube entering the ovary of variety HY 7 days after flowering;

[0026] Figure 4 This is the ovary map of variety HAES828 7 days after flowering;

[0027] Figure 5 This is a map showing the position of the pollen tube of HAES508 growing to the lower 9-tenths of the pistil 7 days after anthesis;

[0028] Figure 6 This is a picture of the pollen tube entering the ovary of the variety HAES936 9 days after flowering. DETAILED DESCRIPTION

[0029] The present invention provides a method for detecting the self-fertility of macadamia nuts, comprising the following steps: (1) bagging the racemes of macadamia nuts, wherein the racemes further comprise removing opened florets and florets with unopened calyxes before bagging;

[0030] (2) 7 to 9 days after bagging, all pistils attached to the inflorescence axis together with the inflorescence axis are immersed in FAA fixative and fixed, washed and softened, and then cross-sectioned in the middle of the pistil style, and then the ovary and the lower half of the style are longitudinally cut to obtain slices;

[0031] (3) After staining the slices obtained in step (2), the growth of pollen tubes was observed, and the percentage of pistils (PLS) that grew to the base of the style and the ovary were counted;

[0032] PLS (%) = the number of pistils whose pollen tubes grew to the base of the style and ovary / the number of observed pistils × 100.

[0033] The present invention arranges the racemes of macadamia nuts and then bags them. The inflorescences used for bagging must contain florets whose sepals have mostly opened but not yet opened. Prior to bagging, the opened florets, i.e., older florets, are preferably thinned out. The florets on the macadamia racemes of the present invention open gradually, and the time required for all florets to open and complete varies depending on the variety and flowering period. To ensure consistent processing of the pistils, the florets whose sepals have not yet opened, i.e., tender florets, are also removed before bagging. The present invention preferably bags the arranged inflorescences, tightens the bag opening, and secures them with a stapler to prevent the nylon mesh bag from falling off. Finally, a label is attached to the bag. The bagging period is preferably 7 to 9 days. The mesh bag used for bagging is preferably designed based on the pollen diameter of the macadamia nuts and is required to be air-permeable, light-permeable, and water-permeable. It should be able to fit over the racemes for a long time without affecting fruit growth and development, while also isolating pollinators and pollen from other varieties. The material of the mesh bag of the present invention preferably includes nylon membrane. The mesh bag can be designed according to the length and diameter of the inflorescence of different varieties and the diameter of the pollen of different varieties (34-40 μm), and the main requirement is that the pore diameter of the mesh bag is smaller than the pollen diameter of macadamia nuts. The material used to make the mesh bag is nylon membrane, the pore diameter of the membrane is 500 mesh (25 μm), and the size of the mesh bag is preferably 30×7 cm in length×width.

[0034] The present invention performs fixation and prepares sections after bagging. The present invention preferably collects pistils 7-9 days after bagging, more preferably 9 days after. This is 7-8 days after flowering, when a large number of florets have already fallen into the mesh bag. After removing the mesh bag, the present invention preferably further comprises gently tapping the base of the raceme to remove pistils with naked styles that are not firmly attached to the inflorescence axis. In the embodiment, the base of the raceme is gently tapped with a pencil or a small branch to remove pistils with naked styles that are not firmly attached to the inflorescence axis. The present invention then immerses all pistils still attached to the inflorescence axis, along with the inflorescence axis, in FAA fixative for 24-36 hours. The FAA fixative preferably comprises a mixture of 60% by volume ethanol in water, 10% by volume neutral formalin fixative, and glacial acetic acid, with the volume ratio of 60% ethanol: glacial acetic acid: 10% neutral formalin fixative preferably being 89:6:5. The 10% neutral formalin fixative of the present invention was purchased from Shanghai Bio-Technology (brand: BBI). When the present invention uses the FAA fixative for fixation, the volume of the FAA fixative used is preferably 20 times that of the material to be tested.

[0035] After the fixation, the present invention preferably washes and softens the sample and slices it to prepare a material to be tested that can be used for staining. The washing method of the present invention preferably includes washing the pistil with 60%, 40% and 20% alcohol solutions for 2 to 3 minutes in sequence after the fixation of the pistil, and then soaking it in ultrapure water for 8 to 10 minutes to replace the FAA fixative in the pistil. The softening method of the present invention preferably includes placing the washed pistil on absorbent paper for 1 minute, and then transferring it to a 2 mol / L NaOH solution at 28 to 30°C and letting it stand for 2.5 hours. The volume of the solution is 35 times that of the material to be softened.

[0036] After the softening, the present invention preferably further includes adjusting the pH value of the material, and more preferably includes: transferring the softened material to be tested into pure water for soaking and washing twice, each time for 5 to 8 minutes; then placing the pistil to be tested on absorbent paper for 30 seconds and then placing it in a phosphate buffer with a pH of 7.0, and soaking each pistil separately in a small 10mL weighing bottle, wherein the volume of the phosphate buffer is 10 times that of the material to be tested, so as to adjust the pH of the material to be tested to neutral and facilitate rapid staining.

[0037] The present invention preferably cuts the pH-adjusted pistil transversely in the middle of the style to split the pistil into two halves, discards the stigma and the upper half of the style, and retains the ovary and the lower half of the style. The ovary and the lower half of the style are then cut longitudinally in half and placed back into a small weighing bottle containing phosphate buffer for later use.

[0038] The present invention stains the sections and then observes them to quickly identify self-compatible varieties. Aniline blue is preferably used for staining. More preferably, the ovary and the lower half of the style, cut in half and stored in a phosphate buffer, are placed on absorbent paper for 20 seconds and then placed in a 0.1% aniline blue staining solution with a pH of 9.5 to 10.5 for staining. Staining is completed in 30 seconds, so staining can be performed directly on a glass slide.

[0039] The present invention preferably uses the ultraviolet light region of a fluorescence microscope to observe the growth of pollen tubes in the ovary and the lower half of the style after staining, and records the results; the percentage of pistils that grow to the base of the style and the ovary is statistically analyzed and expressed as PLS. PLS (%) = the number of pistils whose pollen tubes grow to the base of the style and the ovary / the number of observed pistils × 100. The lower the PLS of the present invention, the stronger the self-incompatibility; the higher the PLS, the weaker the self-incompatibility. The critical value of the percentage of pistils (PLS) of pollen tubes growing to the base of the style of macadamia nuts is 30%. Above 30%, it indicates that the variety has strong self-fertility and a high proportion of self-fertile fruits (i.e., weak self-incompatibility); below 30%, it indicates that the variety has poor self-fertility or cannot self-fertilize (i.e., strong self-incompatibility).

[0040] To further illustrate the present invention, a method for detecting self-fertility of macadamia nuts provided by the present invention is described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] 1. Preparation of Self-pollinated Pistil

[0043] (1) Making mesh bags: Design mesh bags according to the length and diameter of inflorescences of different varieties and the diameter of pollen of different varieties (34-40 μm); the material used to make the mesh bags is nylon membrane with a pore size of 500 mesh (25 μm), and the size of the mesh bags is 30 × 7 cm in length and width. This kind of mesh bag is breathable, light-permeable, and water-permeable, and can be placed on the racemes for a long time without affecting the growth and development of the fruit. It can also isolate pollinators and pollen of other varieties ( Figure 1 ).

[0044] (2) Arrange the inflorescence: Figure 2 As shown in the figure, the inflorescences to be bagged must be those in which most of the florets have cracked but not yet opened, and no opened florets can be mixed in (opened florets are removed, i.e. old florets). The florets on the racemes of macadamia nuts open gradually, and the time required for all the florets in the entire inflorescence to open and complete the flowering period varies depending on the variety and flowering period, ranging from 7 to 9 days. Therefore, the florets with uncracked sepals need to be removed before bagging to avoid handling the pistils at the wrong time, which will affect the later observation results (flowers with uncracked sepals are removed, i.e. young flowers).

[0045] (3) Bagging and tagging: Bagging the arranged inflorescences, tightening the bag mouth and fixing it with a stapler to ensure that the nylon mesh bag will not fall off, and finally tagging it.

[0046] 2. Collection and Fixation Procedure of Pistil

[0047] (1) The pistil was collected 9 days after bagging, which was 7 to 8 days after flowering, and a large number of small flowers had fallen into the net bag.

[0048] (2) After removing the net bag, gently tap the base of the raceme with a pencil or a small branch before collecting the pistils. The purpose is to remove the pistils with naked styles that are not firmly connected to the inflorescence axis.

[0049] (3) All pistils still attached to the inflorescence axis, along with the inflorescence axis, were immersed in FAA fixative for 24–36 h. Because macadamia pistil tissue is very tender, the alcohol concentration in the FAA fixative was reduced to 60%. The other components were glacial acetic acid and 10% neutral formalin fixative, with a volume ratio of 60% alcohol: glacial acetic acid: 10% neutral formalin fixative = 89:6:5. The volume of the FAA fixative was 20 times that of the material to be tested.

[0050] 3. Procedure for preparing materials to be tested

[0051] (1) Washing: After the pistil is fixed, wash it with 60%, 40% and 20% alcohol solutions in sequence for 2 to 3 minutes, and then transfer it to ultrapure water and soak it for 8 to 10 minutes to replace the FAA fixative in the pistil.

[0052] (2) Softening: Place the washed pistil on absorbent paper for 1 min, and transfer to 2 mol.L at 28-30°C. -1 The volume of the solution is 35 times that of the material to be softened.

[0053] (3) Adjusting the pH value of the material: Transfer the softened material to be tested into pure water and soak and wash twice, each time for 5 to 8 minutes; then place the pistil to be tested on absorbent paper for 30 seconds and then place it in pH 7.0 phosphate buffer. Each pistil is individually soaked in a 10 mL small weighing bottle. The volume of phosphate buffer is 10 times that of the material to be tested. The purpose is to adjust the pH of the material to be tested to neutral, which is conducive to rapid staining.

[0054] (4) Manual sectioning: Cut the pH-adjusted pistil horizontally in the middle of the style, bisecting the pistil. Discard the stigma and upper half of the style, retaining the ovary and lower half of the style. Then, cut the ovary and lower half of the style in half longitudinally and place them back in a small weighing bottle containing phosphate buffer for later use.

[0055] 4. Rapid identification of self-compatible varieties

[0056] (1) Staining: Place the ovary and the lower half of the style cut in half in phosphate buffer on absorbent paper for 20 seconds, then place them in 0.1% aniline blue staining solution with a pH value of 10 for staining. Staining can be completed in 30 seconds, so it can be stained directly on the slide.

[0057] (2) Use the ultraviolet light zone of a fluorescence microscope to observe the growth of pollen tubes in the ovary and the lower half of the style after staining and record them.

[0058] (3) Statistical analysis: The percentage of pistils with pollen tubes growing to the base of the style and ovary was expressed as PLS: PLS (%) = number of pistils with pollen tubes growing to the base of the style and ovary / number of observed pistils × 100.

[0059] According to the scheme provided in Example 1, a rapid identification of self-fertility of 18 varieties was carried out. The results are shown in Table 1. No pistils were collected from varieties HAES816 and D 7 to 8 days after self-pollination, and the growth of their pollen tubes in the style could not be observed, indicating that the self-compatibility of these two varieties is extremely weak. The percentage of pistils (PLS) of pollen tubes growing to the base of the style of different varieties is used as the basis for determining the strength of self-incompatibility of each variety. Excluding varieties HAES816 and D, the average value of PLS ​​of 16 varieties is 21%. The pollen tubes of 5 of the 16 varieties did not grow to the base of the style. If only the PLS of varieties with pollen tubes growing to the base of the style are counted, the average value of PLS ​​of 11 varieties is 30%. Taking PLS = 30% as the basis for determining the strength of self-compatibility of varieties, the self-compatibility strength of 18 varieties is divided into 3 categories: variety HY ( Figure 3 )、HAES936( Figure 6 )、HAES508( Figure 5 )、HAES828( Figure 4 ) and HAES814 had the strongest self-compatibility; varieties HAES246, OC, and Specil had medium self-compatibility; varieties HAES816, D, A16, A4, HAES695, HAES906, HAES762, HAES863, HAES772, and HAES842 had the weakest self-compatibility.

[0060] Table 1 Self-fertility intensity of different macadamia varieties

[0061]

[0062]

[0063] In Table 1, NPO: number of observed pistils; NS: number of pistils with pollen tube length less than 2 / 10 of the style, and pollen tube growth stagnation occurred in the upper part of the style; MS: number of pistils with pollen tube length between 2 / 10 and 7 / 10 of the style, and pollen tube growth stagnation occurred in the middle part of the style; LS: number of pistils with pollen tube length exceeding 7 / 10 of the style, and pollen tube growth stagnation occurred at the base of the style; OT: number of pistils with pollen tubes in the ovary; PUS: percentage of pistils with pollen tubes growing to the upper part of the style; PMS: percentage of pistils with pollen tubes growing to the middle part of the style; PLS: percentage of pistils with pollen tubes growing to the base of the style; POT: percentage of pistils with pollen tubes in the ovary; \ indicates that no pistils were collected after 9 days of bagging for this variety.

[0064] The results of the four-year field trial are shown in Tables 2 and 3. The four-year field bagging of different varieties of macadamia nuts was used to identify the self-compatibility intensity of macadamia nuts. In 2018, 2021, 2022, and 2023, 9, 15, 11, and 18 varieties were identified for self-incompatibility intensity, respectively. In order to clarify the drawbacks of field bagging to identify the self-incompatibility of macadamia nuts, the present invention takes varieties with weaker self-incompatibility as the starting point for analysis. Varieties with weaker self-incompatibility are varieties with stronger self-compatibility and varieties with higher self-fertility rates, which are expressed as self-incompatibility index (F_SI). The larger the F_SI value, the stronger the self-incompatibility; the smaller the F_SI value, the weaker the self-incompatibility. In 2018, the self-incompatibility of varieties HAES863, HAES246, and OC was weak, and the self-incompatibility intensity of other varieties was medium or very strong. In 2021, self-incompatibility was weak in varieties A16, HAES936, HAES508, and HAES814. In 2022, self-incompatibility was weak in varieties A16, HAES828, and HAES246. In 2023, self-incompatibility was weak in varieties HY, A4, HAES828, and HAES508. A comprehensive analysis of the results from four years revealed that self-incompatibility was weak in varieties HY, HAES828, HAES936, HAES508, and HAES814 (with strong self-compatibility), while the self-incompatibility of other varieties was moderate or very strong. However, if the analysis is based on a single year, it is impossible to determine the strength of self-incompatibility among different varieties. For example, variety A16 showed weak self-incompatibility in 2021 and 2022, but extremely strong self-incompatibility in 2018 and 2023, resulting in a total of 100 spikelets being completely harvested. Variety HAES814, for example, had higher fruit set in 2021 after self-pollination than after open-pollination, but in 2022 and 2023, even 100 spikelets were bagged and yielded no fruit. Most other varieties also exhibited this phenomenon, showing significant variation in self-incompatibility within the same variety in different years, a major limitation of field observations.

[0065] Table 2 Field observation results of self-incompatibility in 2018 and 2021

[0066]

[0067] OP_FNS: open pollination fruit set per inflorescence; SP_FNS: self-pollination fruit set per inflorescence; F_SI: self-incompatibility index

[0068] Table 3 Field observation results in 2022 and 2023 and in 2018, 2021, 2022 and 2023

[0069]

[0070]

[0071] 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 detecting the self-fertility of macadamia nuts, characterized in that: The method comprises the following steps: (1) bagging the racemes of macadamia nuts, wherein the racemes further comprise removing opened florets and florets with unopened calyxes before bagging; (2) 7 to 9 days after bagging, all pistils attached to the inflorescence axis together with the inflorescence axis are immersed in FAA fixative and fixed, washed and softened, and then cross-sectioned in the middle of the pistil style, and then the ovary and the lower half of the style are longitudinally cut to obtain slices; (3) After staining the slices obtained in step (2), the growth of pollen tubes was observed, and the percentage of pistils (PLS) that grew to the base of the style and the ovary were counted; PLS (%) = number of pistils with pollen tubes growing to the base of the style and ovary / number of observed pistils × 100; The bagging in step (1) includes bagging a raceme in which most of the sepals have cracked but not opened; Before the fixing in step (2), the method further includes gently tapping the base of the raceme to remove the pistils with naked styles that are not firmly connected to the inflorescence axis; Step (2) is fixed in the FAA fixative for 24 to 36 hours; The PLS in step (3) is ≥30%, indicating that the macadamia nut variety has strong self-fertility and a high proportion of self-pollinated fruits; the PLS is <30%, indicating that the macadamia nut variety has poor self-fertility or cannot produce fruit by self-pollination.

2. The detection method according to claim 1, characterized in that The mesh bag used in the bagging in step (1) has a pore size smaller than the pollen diameter of macadamia nuts and is made of a nylon membrane.

3. The detection method according to claim 1, characterized in that The FAA fixative in step (2) comprises a mixture of 60% by volume of an ethanol aqueous solution, 10% by volume of a neutral formalin fixative, and glacial acetic acid.

4. The detection method according to claim 1, characterized in that The washing in step (2) includes washing with alcohol aqueous solutions with volume percentages of 60%, 40% and 20% in sequence after the fixation is completed, and then immersing in ultrapure water.

5. The detection method according to claim 1, characterized in that: The softening in step (2) includes transferring the washed pistil into a NaOH solution and allowing it to stand.

6. The detection method according to claim 1, characterized in that: The staining in step (3) includes adjusting the pH value of the slice to neutral and then staining with a strong alkaline aniline blue dye solution.

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