A method for quickly determining the open pollination intensity of macadamia nuts and its application

Abstract

The present invention provides a method for rapidly determining the open pollination intensity of macadamia nuts and its application, belonging to the field of cultivation techniques. The present invention provides a method for rapidly determining the open pollination intensity of macadamia nuts. At the early flowering stage, pistils with slightly swollen ovaries are sampled, fixed with FAA and then sectioned. The sections are observed and the percentage of pistils PLS with pollen tubes growing to the base of the style and the ovary is counted. The open pollination intensity of macadamia nuts is evaluated according to the value of PLS. From the start of sample collection to the completion of pollen tube fluorescence microscopy detection, the present invention only takes 30 h to know the open pollination intensity of all tested varieties. It has a small workload, intuitive, accurate and reliable results, and little damage. It can quickly detect the insect pollination efficiency in the current year for macadamia nut plantations, provide a basis for whether to carry out artificial supplementary pollination in the current year and reasonably place beehives, and can be widely used in macadamia nut plantations on a large scale.

Description

Technical Field

[0001] The present invention belongs to the field of cultivation technology, and particularly relates to a method for rapidly determining the open pollination intensity of Macadamia nuts and its application. Background Technique

[0002] Macadamia nuts are known as the "king of nuts" and are native to the subtropical rainforest areas (latitude 25° - 32° south) along the southeastern coast of Queensland and the northeastern coast of New South Wales in Australia. Due to their rich nutrition, unique taste, and the ability to improve complexion and anti-aging when consumed regularly, they are deeply loved by people. In recent years, with the growth of the consumer market, the demand for Macadamia nuts has increased, and the planting area has also been increasing year by year. Macadamia nuts have become an economic forest tree species commonly cultivated in tropical and subtropical regions worldwide. With the continuous improvement of people's living standards and the pursuit of a healthy lifestyle, the domestic market potential of Macadamia nuts is huge. Yunnan is the region with the largest area of Macadamia nuts planted in the world, accounting for 54% of the global planting area and 88% of the national planting area, but the per mu yield is less than half of that in the United States and Australia. Macadamia nuts are cross-pollinated plants, and the outcrossing rate of different varieties is as high as 80 - 100%. In the case of no cross-pollination or weak pollination intensity, the phenomenon of only flowering but not bearing fruit in Macadamia nut plantations is very serious, which is also the main reason for the low yield of Macadamia nuts in China.

[0003] The flowers of Macadamia nuts are racemes, with numerous inflorescences and flower quantities. Each adult tree can produce 3000 - 5000 inflorescences per year, or even more; each inflorescence bears 100 - 300 small flowers, and the annual output of each tree is as high as 300,000 - 1.5 million small flowers. Due to the influence of climate and environmental conditions, the phenology of the flowering period of Macadamia nuts varies greatly in different countries and regions. Some varieties in the Macadamia nut production areas of China bloom in two batches, and the flowering period lasts about 2 months; some varieties bloom in one batch, and the flowering period lasts about 1 month; even a phenomenon of blooming in three batches appears in the high-altitude areas of Yunnan. The flowering and fruiting periods of Macadamia nuts are very chaotic. At the same time node, the same tree may simultaneously have fast-growing flower buds, newly sprouted inflorescences, flower buds to be opened, small flowers at different opening stages, small fruits at different development stages, and even nearly mature fruits.

[0004] The flowers of Macadamia integrifolia are entomophilous flowers, and bees are the main pollinating insects. In nut plantations, bees are artificially released to improve their pollination efficiency for increased production and income. However, bee pollination is affected by many factors such as temperature, humidity, weather, and insect activities, resulting in extremely unstable pollination efficiency and large fluctuations in yields in the same orchard in different years. The pollination efficiency of Macadamia integrifolia determines the fruit set rate, that is, it determines the yield level. At present, there is no assessment technology for the pollination efficiency of Macadamia integrifolia in production. Sometimes, growers evaluate the pollination efficiency by field investigation of the final fruit set rate to determine the location of beehives for the next year. However, this method is time-consuming and laborious, and the final fruit set rate is not only related to pollination and fertilization but also closely related to the nutritional level of the mother tree. During the entire growth and development process of the fruit, it is also affected by factors such as diseases, pests, water, and fertilizer conditions. There are too many uncontrollable factors to accurately evaluate the pollination efficiency of different varieties in the orchard, nor can it guide the accurate location of beehive placement. It takes 2 - 3 years to investigate the fruit set rate to know the approximate yields of different varieties and roughly determine the area for beehive placement or the varieties for manual assisted pollination. Not only is the cycle long, but the differences between different years are too large and the accuracy is extremely low, and the feasibility of actual operation is also very poor. Summary of the Invention

[0005] The present invention provides a method for rapidly determining the open pollination intensity of Macadamia integrifolia and its application, which can rapidly and in large quantities determine the open pollination intensity of different varieties at the early flowering stage in the orchard, so as to adjust the assisted pollination strategy.

[0006] The present invention provides a method for rapidly determining the open pollination intensity of Macadamia integrifolia, including the following steps: collecting pistils with naked styles and slightly swollen ovaries in the morning on a sunny day at the early flowering stage, after fixing with FAA, making longitudinal sections of the materials containing the ovary and the lower half of the style, and counting PLS. When PLS≥30%, the open pollination ability is strong; when 16%≤PLS<30%, the open pollination ability is average; when PLS<16%, the open pollination ability is weak.

[0007] The PLS is the percentage of pistils with pollen tubes growing to the base of the style and the ovary, and PLS(%) = the number of pistils with pollen tubes growing to the base of the style and the ovary / the number of observed pistils × 100.

[0008] Preferably, the early flowering stage is the initial stage of inflorescence germination, and about 5% of the flowers are open.

[0009] Preferably, the pistils with naked styles and slightly swollen ovaries include pistils with naked styles that have all sepals fallen off, are still stably attached to the inflorescence axis, and have signs of ovary swelling.

[0010] Preferably, the FAA fixation includes immersing the entire collected pistil in the FAA fixative for 24 h for fixation.

[0011] The FAA fixative is a mixed solution containing alcohol, glacial acetic acid and neutral formalin fixative.

[0012] Preferably, when preparing the FAA fixative, the alcohol includes an aqueous alcohol solution with a volume percentage of 50-60%, and the neutral formalin fixative includes a 10% neutral formalin fixative by volume.

[0013] The volume ratio of the aqueous alcohol solution, glacial acetic acid and 10% neutral formalin fixative is 89:6:5.

[0014] Preferably, after the FAA fixation, it also includes washing with aqueous alcohol solutions of gradient concentrations, water immersion, soaking in NaOH solution, adjusting the pH to neutral and sectioning.

[0015] Preferably, the aqueous alcohol solutions of gradient concentrations include aqueous alcohol solutions with volume percentages of 60%, 40% and 20% respectively.

[0016] Preferably, the soaking in NaOH solution includes soaking in a 2 mol.L -1 NaOH solution for 3 h.

[0017] Preferably, after making the sections, it also includes staining with a strongly basic aniline blue stain solution and observing with a fluorescence microscope.

[0018] Preferably, for macadamia nut varieties with weak open pollination ability, artificial supplementary pollination or placing beehives is carried out in the late initial flowering stage, full flowering stage and final flowering stage;

[0019] For macadamia nut varieties with general open pollination ability, whether to carry out artificial supplementary pollination or place beehives is determined according to the preset yield in the late initial flowering stage, full flowering stage and final flowering stage;

[0020] For macadamia nut varieties with strong open pollination ability, no artificial supplementary pollination or beehive placement is required.

[0021] Beneficial effects: The present invention provides a method for quickly determining the open pollination intensity of Macadamia integrifolia. At the early flowering stage, pistils with slightly swollen ovaries visually observed are sampled, fixed with FAA, sectioned after fixation, and observed under a fluorescence microscope for the sections to count the percentage PLS of pistils with pollen tubes growing to the base of the style and the ovary. The open pollination intensity of different varieties in the Macadamia integrifolia plantation is evaluated according to the value of PLS. The method of the present invention can quickly and accurately determine the open pollination intensity of different varieties in the Macadamia integrifolia plantation, with a small workload, intuitive, accurate and reliable results, little damage, and can provide a basis for quickly detecting the insect pollination efficiency in the Macadamia integrifolia plantation in the current year, for whether to carry out artificial supplementary pollination in this year and for reasonably placing beehives, and provide a guarantee for stable yield in the orchard, and has a wide application prospect. The method of the present invention can quickly and in large quantities determine the open pollination intensity of different varieties at the early flowering stage in the orchard. Once it is found that the pollination is insufficient, the position of the beehive can be accurately adjusted or artificial supplementary pollination can be precisely implemented for varieties with severely insufficient open pollination, so as to ensure that the florets at the full flowering stage and the late flowering stage obtain sufficient cross-pollen to complete fertilization, thereby achieving the effect of improving quality and efficiency, and has the advantages of safety, high efficiency, quickness, low cost, strong repeatability, little damage and accurate results, and can be widely promoted and used in the Macadamia integrifolia plantation. Description of the Drawings

[0022] Figure 1 It is a figure of a pistil with a naked style and a slightly swollen ovary on the inflorescence;

[0023] Figure 2 It is a figure of the pistil after sampling;

[0024] Figure 3 It is a figure of the pollen tube entering the ovary;

[0025] Figure 4 It is a figure of the pollen tube growing to the base of the pistil. Detailed Embodiment

[0026] The present invention provides a method for quickly determining the open pollination intensity of Macadamia integrifolia, including the following steps: At the morning of a sunny day at the early flowering stage, pistils with a slightly swollen ovary and a naked style are collected, fixed with FAA, and the material containing the ovary and the lower half of the style is longitudinally sectioned to make sections, and PLS is counted. When PLS≥30%, the open pollination ability is strong; when 16%≤PLS<30%, the open pollination ability is general; when PLS<16%, the open pollination ability is weak;

[0027] The PLS is the percentage of pistils with pollen tubes growing to the base of the style and the ovary, and PLS(%) = the number of pistils with pollen tubes growing to the base of the style and the ovary / the number of observed pistils × 100.

[0028] The early flowering stage described in the present invention is the initial stage of flower germination, and about 5% of the flowers are open.

[0029] The present invention preferably samples after 10:00 on sunny days during the early flowering stage of Macadamia integrifolia, and does not sample on rainy or cloudy days. The sampling preferably includes collecting pistils with naked styles where the calyces have all fallen off and the ovaries show signs of swelling as the test materials. At this time, the pistils are 7 days or more after flowering. When sampling in the present invention, it preferably further includes removing the pistils that are not firmly connected to the inflorescence axis, such as tapping the base and middle of the inflorescence axis 3 - 5 times with pointed forceps. The present invention preferably uses random sampling, that is, randomly sampling around the tree trunk when collecting samples, taking 1 - 3 pistils with swollen ovaries from one inflorescence, and the sample quantity is not less than 30 pistils per plant.

[0030] The present invention fixes the removed pistils with FAA. The fixing preferably includes immersing the pistils in the FAA fixing solution, and the fixing time is preferably 24 h. In the present invention, since the pistil tissue of Macadamia integrifolia is very tender, it is necessary to modify the FAA fixing solution, such as reducing the concentration of the alcohol aqueous solution in the FAA fixing solution to 50 - 60% (v / v), and the other components are glacial acetic acid and 10% (v / v) neutral formalin fixing solution, and the volume ratio is 50 - 60% alcohol: glacial acetic acid: 10% neutral formalin fixing solution = 89:6:5. When performing the immersion in the present invention, the volume of the FAA fixing solution used is preferably 30 - 40 times that of the test materials.

[0031] The present invention preferably performs washing after the fixing to displace the FAA fixing solution in the pistils. The washing preferably includes performing it 12 - 24 h after the pistils are fixed, and more preferably includes washing with alcohol aqueous solutions of gradient concentrations. The gradient concentrations preferably include alcohol aqueous solutions with volume percentages of 60%, 40%, and 20% in sequence, washing for 2 - 3 min each time, and then transferring them to ultrapure water for soaking for 8 - 10 min.

[0032] The present invention preferably places the washed pistils on absorbent paper to dry the liquid, such as placing the washed pistils in absorbent paper for 50 s, and then softening them. The softening preferably includes transferring them to a 2 mol·L -1 NaOH solution at 28 - 30 °C and standing for 3 h. The volume of the solution is 30 times that of the materials to be softened.

[0033] The present invention preferably transfers the test materials after softening to pure water for soaking and washing 3 times, 6 - 7 min each time; then places the test pistils in absorbent paper for 40 s and puts them into a phosphate buffer solution with pH 7.0. Each pistil is separately soaked in a small weighing bottle of 10 mL. The volume of the phosphate buffer solution is 15 - 20 times that of the test materials, aiming to adjust the pH of the test materials to neutral for facilitating rapid staining.

[0034] The pistils adjusted to pH neutral in the present invention are sectioned, preferably including first making a transverse cut at the middle position of the style to divide the pistil into two halves, discarding the stigma and the upper half of the style, and retaining the ovary and the lower half of the style; then longitudinally cutting the ovary and the lower half of the style into two halves, and still putting them back into a small weighing bottle containing phosphate buffer for standby.

[0035] The present invention preferably stains the sections, more preferably including using a strongly basic aniline blue staining solution for the staining, and the pH value range of the strongly basic aniline blue staining solution is preferably 9.5 - 10.5. In specific operation: place the cut-in-half ovary and the lower half of the style stored in phosphate buffer in absorbent paper for 15 - 20 s, and then put them into a 0.1% aniline blue staining solution with a pH value of 9.5 - 10.5 for staining. Staining can be completed in 30 - 40 s, so staining can be directly carried out on a glass slide.

[0036] After the staining in the present invention, preferably observe the growth of pollen tubes in the stained lower half of the ovary and the style under the ultraviolet light region of a fluorescence microscope and record it. And according to the recorded data, statistically analyze the percentage of pistils in which the pollen tubes grow to the base of the style ( Figure 4 ) and the ovary ( Figure 3 ), which is represented by PLS. PLS (%) = the number of pistils in which the pollen tubes grow to the base of the style and the ovary / the number of observed pistils × 100.

[0037] The present invention uses PLS as the basis for the open pollination intensity of a certain variety. There is a positive correlation between the PLS value and the open pollination intensity. The lower the PLS, the weaker the open pollination intensity; the higher the PLS, the stronger the open pollination intensity. It only takes 30 h from collecting samples to completing the fluorescence microscopy detection of pollen tubes to know the open pollination intensity of all tested varieties in a macadamia nut plantation. The present invention makes a distinction with the PLS values of 16% and 30% as the nodes. When PLS ≥ 30%, the open pollination ability is strong, and there is no need for artificial assisted pollination or placing beehives; when 16% ≤ PLS < 30%, the open pollination ability is average, and during the late initial flowering stage, full bloom stage and late flowering stage, it is decided whether to carry out artificial assisted pollination or place beehives according to the preset yield; when PLS < 16%, the open pollination ability is weak, and artificial assisted pollination or placing beehives is carried out during the late initial flowering stage, full bloom stage and late flowering stage.

[0038] In the present invention, some varieties of macadamia nuts are self-compatible varieties, some varieties are self-incompatible varieties that need cross-pollination, and there are also some varieties that are both self-compatible and can be cross-pollinated. However, in macadamia nuts, the self-pollinated fruits obtained after self-pollination are smaller and have low commercial value. Therefore, cross-pollination is more recommended to improve the commerciality.

[0039] To further illustrate the present invention, a method for rapidly determining the open pollination intensity of Macadamia integrifolia and its application provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] Examples 1 to 11

[0041] Determinations of the open pollination intensity of different varieties were carried out in the Macadamia integrifolia plantations (11 varieties) in Jinghong City, Xishuangbanna Prefecture. The determination of each variety was regarded as an embodiment. The percentage of pistils (PLS) with pollen tubes growing to the base of the style was used as the basis for determining the open pollination intensity of each variety.

[0042] The determination methods used in Examples 1 to 11 were the same, specifically as follows:

[0043] 1. Collection of pistils with naked styles and fixation procedure

[0044] (1) After 10:00 on a sunny day during the early flowering stage of Macadamia integrifolia (do not sample on rainy or cloudy days), collect the pistils with naked styles whose calyces have all fallen off and the ovaries show signs of swelling (at this time, the pistils are 7 days or more after flowering), Figure 1 ) as the test materials. The flowering stage for carrying out the pollination intensity determination must be the early flowering stage of each variety. When sampling, use a pointed forceps to tap the base and middle of the inflorescence axis 3 - 5 times respectively to remove the pistils that are not firmly connected to the inflorescence axis. When collecting samples, randomly sample around the tree trunk. Take 1 - 3 pistils with ovaries showing signs of swelling from one inflorescence, and the sample quantity should be no less than 30.

[0045] (2) Immerse the entire pistil as shown in Figure 2 in FAA fixative for 24 hours. The composition of FAA fixative is: the concentration of alcohol is 50 - 60%, and the other components are glacial acetic acid and 10% neutral formalin fixative solution, and their volume ratio is 50 - 60% alcohol: glacial acetic acid: 10% neutral formalin fixative = 89:6:5. The volume of FAA fixative is 30 - 40 times that of the test materials.

[0046] 2. Preparation procedure of test materials

[0047] (1) Washing: After the pistils are fixed for 12 - 24 hours, wash them successively with 60%, 40% and 20% alcohol solutions for 2 - 3 minutes, and then transfer them to ultrapure water for soaking for 8 - 10 minutes. The purpose is to displace the FAA fixative in the pistils.

[0048] (2) Softening: Place the washed pistils in absorbent paper for 50 seconds, and transfer them to 2 mol·L -1 NaOH solution at 28 - 30 °C for static placement for 3 hours. The volume of the solution is 30 times that of the materials to be softened.

[0049] (3) Adjust the pH value of the material to be tested: Transfer the softened material to be tested into pure water and soak and wash it 3 times, 6 - 7 minutes each time; then place the pistils to be tested in absorbent paper for 40 seconds and put them into a phosphate buffer solution with a pH of 7.0. Each pistil is soaked separately in a small weighing bottle of 10 mL. The volume of the phosphate buffer solution is 15 - 20 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.

[0050] (4) Freehand sectioning: Transversely cut the pistils with the adjusted pH value at the middle position of the style, dividing the pistils into two halves. Discard the stigma and the upper part of the style, and retain the ovary and the lower part of the style (the observation object is the ovary and the lower part of the style). Then, longitudinally cut the ovary and the lower part of the style into two halves and still put them back into the small weighing bottle containing the phosphate buffer solution for standby.

[0051] 3. Rapid determination of open - pollination intensity and its application

[0052] (1) Staining: Place the cut - in - half ovary and the lower part of the style stored in the phosphate buffer solution in absorbent paper for 15 - 20 seconds, and then put them into a 0.1% aniline blue staining solution with a pH value of 9.5 - 10.5 for staining. Staining can be completed in 30 - 40 seconds, so directly stain on the glass slide.

[0053] (2) Observe the growth of pollen tubes in the cut - in - half ovary and the lower part of the style after staining under the ultraviolet light region of a fluorescence microscope and record.

[0054] (3) Statistically analyze the percentage of pistils with pollen tubes growing to the base of the style and the ovary, represented by PLS. PLS(%) = the number of pistils with pollen tubes growing to the base of the style and the ovary / the number of observed pistils × 100.

[0055] (4) Use PLS as the basis for the open - pollination intensity of a certain variety. There is a positive correlation between the PLS value and the open - pollination intensity. The lower the PLS, the weaker the open - pollination intensity; the higher the PLS, the stronger the open - pollination intensity. It only takes 30 hours from collecting samples to completing the fluorescence microscopy detection of pollen tubes to know the open - pollination intensity of all tested varieties in a macadamia nut plantation.

[0056] Table 1 Open - pollination intensity of different macadamia nut varieties

[0057] Variety NPO NPT MS LS OT PN / % PMS / % PLS / % POT / % HY 53 40 3 10 9 75.47 5.66 18.87 16.98 A16 34 32 2 0 0 94.12 5.88 0.00 0.00 A4 39 39 0 0 0 100.00 0.00 0.00 0.00 HAES695 56 31 9 16 3 55.36 16.07 28.57 5.36 Special 63 63 0 0 0 100.00 0.00 0.00 0.00 HAES828 31 14 9 8 7 45.16 29.03 25.81 22.58 HAES816 30 25 5 0 0 83.33 16.67 0.00 0.00 HAES863 51 6 7 38 22 11.76 13.73 74.51 43.14 HAES772 48 48 0 0 0 100.00 0.00 0.00 0.00 D4 56 14 34 8 3 25.00 60.71 14.29 5.36 HAES842 48 31 7 10 4 64.58 14.58 20.83 8.33

[0058] In Table 1, NPO represents the number of pistils utilized for observation; NPT represents the number of pistils with no pollen tube; PTL represents the pollen tube length; MS represents the number of pistils between 5 / 10 and 7 / 10 of the style, that is, pollen tube growth arrest occurs in the middle of the style (5 / 10 < PTL ≤ 7 / 10); LS represents the number of pistils with a pollen tube length (PTL) exceeding 7 / 10 of the style, that is, pollen tube growth arrest occurs at the base of the style; OT represents the number of ovaries with a pollen tube in the ovary; PN represents the percentage of NPT; PMS represents the percentage of MS; PLS represents the percentage of LS; POT represents the percentage of ovaries with a pollen tube.

[0059] As can be seen from Table 1, among the pistils with all sepals fallen off and the ovaries slightly swollen, the pollen tubes of 6 varieties grew to the base of the style, namely varieties HY, HAES695, HAES828, HAES863, D4, HAES842, etc. The pistil proportions (PLS) of the pollen tubes of each variety growing to the base of the style were 18.87%, 28.57%, 25.81%, 74.51%, 14.29%, and 20.83% in sequence. Among them, the pistil proportion of the pollen tubes of variety HAES863 entering the ovary was the highest. The average value of PLS of the 6 varieties with pollen tubes growing to the base of the style was 30.48%. Varieties with values higher than the average had stronger pollination intensity. In the pistils of the other 5 varieties (A16, A4, Special, HAES 816, HAES 772), not a single pistil had a pollen tube growing to the base of the style, and their PLS values were all 0. The average value of PLS of the 11 varieties in this plantation was 16.62%. Values lower than this average indicated weaker open pollination ability. To sum up, only variety HAES863 in this plantation had strong open pollination ability, and artificial supplementary pollination or additional beehive placement did not need to be carried out for it during the subsequent full bloom and late bloom periods. The PLS values of 4 varieties such as variety HY, HAES 816, HAES828, and HAES 842 were between 16% and 30%, indicating that they could attract a small number of bees for pollination, but the pollination rate was low. Beehives could be placed during the subsequent full bloom and late bloom periods to increase the pollination intensity of bees. The PLS values of 6 varieties such as variety D4, A16, A4, Special, HAES816, and HAES772 were lower than 16%, even 0, indicating that their open pollination rates were extremely low or even had no pollination rate. In order to obtain high yields, artificial supplementary pollination needed to be carried out for these varieties during the subsequent full bloom and late bloom periods or beehives needed to be placed around the individual plants of these varieties.

[0060] Determination of outcross pollination intensity in the control example

[0061] In order to verify the difference between the open pollination intensity and outcross pollination intensity of Macadamia integrifolia, the outcross pollination intensity of varieties HAES863 and HAES842 was determined (Table 2). The fluorescence microscopy observation method for outcross pollination was the same as that for open pollination intensity, except for the preparation method of the test materials.

[0062] 1. Preparation steps for outcross pistils:

[0063] (1) Collect pollen: Use a flat - mouth glass test tube of 30×200mm to collect pollen. The time for collecting pollen is between 9:00 - 10:00 on a sunny day when bees have not yet left the hive, and there is a large amount of pollen accumulated on the stigmas of the small flowers that open on the same day. When a layer of pollen can be seen with the naked eye on the inner wall of the test tube, the pollen collection is completed.

[0064] (2) Inflorescence arrangement: Retain the florets that bloom on the same day and those that have bloomed for 1 day in the inflorescence, and remove florets of other morphologies (if field pollination is for improving fertilization, there is no need to arrange the inflorescence).

[0065] (3) Pollination: Before pollination, gently wipe off the pollen accumulated on the stigma of the female parent with woolen gloves, and then pollinate it with a glass test tube containing pollen.

[0066] (4) Hanging tags for marking and collecting cross-pollinated pistils: 7 days after the pollination treatment, gently tap the base of the raceme with forceps to remove the pistils with naked styles that are not firmly connected to the inflorescence axis. Then, put the un-shed pistils into FAA fixative solution for storage and standby.

[0067] 2. The steps for measuring the intensity of cross-pollination are the same as those for measuring the intensity of open pollination, which will not be elaborated here. The cross-pollination intensities of varieties HAES863 and HAES842 in this macadamia orchard are shown in Table 2. 7 days after cross-pollination, in the hybrid combinations HAES842×HAES863 and HAES863×HAES842, the percentages (PLS) of the tested pistils with pollen tubes growing to the base of the style are as high as 63.33% and 90.00% respectively. The PLS of variety HAES842 for open pollination is 20.83%, and the PLS of variety HAES863 for open pollination is 74.51% (Table 1), indicating that artificial supplementary pollination has greatly increased the proportion of pistils with pollen tubes growing to the base of the style of female trees HAES863 and HAES842, and the intensity of artificial supplementary cross-pollination is much higher than that of open pollination. In a productive orchard, for varieties with severely insufficient open pollination (insect pollination), even if only one artificial supplementary cross-pollination is carried out for them, it will greatly enhance the fertilization rate of the variety, thus achieving increased production and income, and escorting the quality improvement and efficiency increase of the macadamia industry.

[0068] Table 2 Measurement of cross-pollination intensity of macadamia

[0069] Hybrid combination NPO NPT MS LS OT PN / % PMS / % PLS / % POT / % HAES842 × HAES863 30 0 11 19 2 0.00 36.67 63.33 6.67 HAES863 × HAES842 30 0 3 27 16 0.00 10.00 90.00 53.33

[0070] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for rapidly determining the open pollination intensity of macadamia nuts, characterized in that, It includes the following steps: Collect pistils with naked styles and slightly swollen ovaries in the morning on sunny days at the early flowering stage. After fixation with FAA, make longitudinal sections of the materials including the ovaries and the lower parts of the styles; stain the sections with a strongly alkaline aniline blue staining solution; observe and count the PLS under a fluorescence microscope after staining. When PLS ≥ 30%, the open pollination ability is strong; when 16% ≤ PLS < 30%, the open pollination ability is average; When PLS < 16%, the open pollination ability is weak; The PLS is the percentage of pistils where pollen tubes grow to the base of the style and the ovary, and PLS(%) = the number of pistils where pollen tubes grow to the base of the style and the ovary / the number of observed pistils × 100; The pH value range of the strongly alkaline aniline blue staining solution is 9.5 - 10.

5.

2. The method according to claim 1, characterized in that, The pistils with naked styles and slightly swollen ovaries include pistils with naked styles that have all sepals fallen off, are still stably attached to the inflorescence axis, and have signs of ovary swelling.

3. The method according to claim 1, wherein The FAA fixation includes immersing the collected whole pistils in the FAA fixing solution for 24 hours; The FAA fixing solution is a mixed solution containing alcohol, glacial acetic acid, and neutral formalin fixing solution.

4. The method according to claim 3, wherein When preparing the FAA fixing solution, the alcohol includes an aqueous alcohol solution with a volume percentage of 50 - 60%, and the neutral formalin fixing solution includes a 10% neutral formalin fixing solution.

5. The method according to claim 4, wherein The volume ratio of the aqueous alcohol solution, glacial acetic acid, and 10% neutral formalin fixing solution is 89:6:

5.

6. The method according to claim 1 or 4, characterized in that, After the FAA fixation, it also includes washing with an aqueous alcohol solution of gradient concentration, water immersion, soaking in a NaOH solution, adjusting the pH to neutral, and sectioning.

7. The method according to claim 6, characterized in that, The aqueous alcohol solution of gradient concentration includes aqueous alcohol solutions with volume percentages of 60%, 40%, and 20% respectively.

8. The method according to claim 6, characterized in that The soaking in the NaOH solution includes soaking in a 2 mol.L-1 NaOH solution for 3 hours.

9. The method according to claim 1, wherein For macadamia nut varieties with weak open pollination ability, carry out artificial assisted pollination or place beehives at the late early flowering stage, full flowering stage, and late flowering stage; For macadamia nut varieties with average open pollination ability, decide whether to carry out artificial assisted pollination or place beehives according to the preset yield at the late early flowering stage, full flowering stage, and late flowering stage; For macadamia nut varieties with strong open pollination ability, no artificial assisted pollination or beehive placement is required.

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