Camellia oleifera pollination affinity detection liquid and application thereof

Abstract

The invention relates to the technical field of agricultural planting, and particularly provides a camellia oleifera pollination affinity detection solution and application thereof. The method can be used for rapidly identifying the affinity between the oil-tea camellia plants, well solves the problem of low yield caused by poor affinity between the oil-tea camellia plants by rapidly identifying and analyzing the pollination affinity of the oil-tea camellia, improves the fruit setting rate, provides technical support for variety matching selection during oil-tea camellia afforestation and pollen donor selection of artificial pollination, and has a wide application prospect. Good application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural planting, and in particular relates to an oil-tea camellia pollination affinity detection liquid and an application thereof. Background Art

[0002] The low fruit set problem caused by "flowers but no fruits" and "one fruit for a thousand flowers" of tea oil is one of the main bottlenecks in the development of the tea oil industry. Among them, fertilization failure caused by self-incompatibility and poor affinity between varieties is one of the important reasons affecting the fruit set rate.

[0003] The results showed that Camellia oleifera was post-self-incompatible. Pollen tubes from self-pollination or between parents with poor affinity grew slowly 48 hours after pollination, with bending at the front end, growth was inhibited, and they could not reach the bottom of the ovary to complete fertilization. Pollen tubes from out-pollination with good affinity did not show growth inhibition, and they could reach the inside of the ovary normally and complete the fertilization process ( Figure 1 ). The present invention technicians found (tissue detection and in vitro pollen culture experiment) ( Figure 2 , Figure 3 ), the style and ovary contain CvS-RNase that regulates the incompatibility trait, and this enzyme has a significant inhibitory effect on the growth of pollen tubes; the activity of CvS-RNase in the style and ovary of self-pollination or poor affinity 48-96 hours after pollination is high, and is significantly higher than that of the style and ovary with good affinity. Further research results found that CvS-RNase has rich diversity, the same plant contains multiple variants of CvS-RNase, and different plants may contain different types and quantities of variant CvS-RNase; pollination between plants containing different CvS-RNase variant proteins has a high fruit setting rate; pollination between plants containing the same CvS-RNase variant protein has a low fruit setting rate (Table 1).

[0004] Table 1 Fruit setting rate of oil-tea plants with different S-RNase genotypes after mating

[0005]

[0006] Therefore, a Camellia oleifera pollen affinity detection liquid was developed, which is used to detect the germination and growth of pollen from different plants, so as to identify the pollination affinity between plants, which helps to provide guidance for the matching of Camellia oleifera varieties and has important application value. Summary of the invention

[0007] The technicians of the present invention developed and prepared a style or ovary extract 48-96 hours after the petals unfolded, which contains a highly active CvS-RNase enzyme, and used it as a pollen culture liquid to invent a Camellia oleifera pollen affinity detection liquid. By culturing pollen in the Camellia oleifera pollen affinity detection liquid, the germination and growth of pollen from different plants are detected, thereby identifying the pollination affinity between plants and serving as a basis for guiding the matching of Camellia oleifera varieties. Pollen with high affinity grows normally in the Camellia oleifera pollen affinity rapid detection liquid; pollen with poor affinity cannot grow normally. At the same time, any Camellia oleifera pollen can grow normally in the Camellia oleifera pollen affinity detection liquid that does not contain the CvS-RNase enzyme, which better eliminates the low germination rate or growth inhibition caused by the detection liquid (without the CvS-RNase enzyme) itself being inappropriate (not suitable for pollen germination).

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a Camellia oleifera pollination affinity detection liquid, which is composed of an extract containing a style or ovary 48-96 hours after the petals unfold, and the extract includes 0.1g / L sucrose, 0.005g / L boric acid and 0.005mmol / L-0.5mmol / L salicylic acid.

[0010] The second aspect of the present invention provides a method for preparing a tea oil affinity detection solution, the specific steps are as follows:

[0011] (1) Take 100 g of sucrose, 5 ml of boric acid mother solution, 5 ul-500 ul of salicylic acid mother solution, add clean water to dissolve all solid substances, and dilute to 1 L to obtain an extract;

[0012] (2) adding a style or ovary sample 48-96 hours after the petals unfold to the extract obtained in step (1), wherein the weight ratio of the style or ovary sample to the extract is 1:2-5, and the mixture is fully crushed and centrifuged to obtain a supernatant, which is the Camellia oleifera pollen affinity detection solution;

[0013] The concentration of the boric acid mother solution is 1 g / L, and the preparation method thereof comprises: taking 0.05 g of boric acid powder, adding clean water to completely dissolve the boric acid, and fixing the volume to 50 ml;

[0014] The concentration of the salicylic acid mother solution is 1 mol / L, and the preparation method thereof comprises: taking 6.9 g of salicylic acid powder, adding clean water to completely dissolve the salicylic acid, and fixing the volume to 50 ml;

[0015] The step (2) is carried out at 0-10°C.

[0016] Furthermore, the collection process of style or ovary samples is as follows: on the plant to be tested as the pollen receptor, all anthers are cut off one day before the flowers to be collected open, and the bags are covered with sulfuric acid paper bags, which are sealed in time with a stapler to prevent other insects from entering. The styles or ovaries are collected 48h-96h after flowering and immediately placed in liquid nitrogen for use. The storage time should not exceed 5d.

[0017] The third aspect of the present invention provides an application of a camellia oil pollination affinity detection liquid prepared by the above method for identifying and analyzing the affinity between camellia oil plants.

[0018] A fourth aspect of the present invention provides a method for identifying pollination affinity between oil-tea camellia plants, the method comprising:

[0019] 100ul of the Camellia oleifera pollen affinity detection solution of claim 3 was dropped onto a single concave glass slide, and pollen was evenly spread on the surface of the culture medium, and cultured in a 25°C constant temperature biochemical incubator. After 4 hours, the germination of pollen was observed under a 4×4 magnification microscope, and the number of pollen was counted under a microscope;

[0020] The counting standard is that the length of the pollen tube exceeds the diameter of the pollen grain. The calculation formula is: Pollen germination rate = (number of germinated pollen in the field of view / total number of pollen grains in the field of view) * 100%; when the pollen germination rate is equal to or lower than 20%, it can be determined that the incompatibility between plants is low and it is not suitable to be planted as a matching variety.

[0021] Furthermore, the pollen collection process is as follows: on the plant to be tested as the pollen donor, put a sulfuric acid paper bag on the flower to be collected one day before it opens, and seal it in time with a stapler to prevent other insects from entering. Collect the pollen on the first day of the flower opening and use it immediately, or immediately place the pollen in silica gel to dry and store it for later use. The storage time should not exceed 30 days.

[0022] Beneficial effects of the present invention:

[0023] The present invention can be used to quickly identify the strength of affinity between oil-tea plants. By quickly identifying and analyzing the pollination affinity of oil-tea, the low yield problem caused by poor affinity between oil-tea plants is better solved, the fruit setting rate is improved, and technical support is provided for variety matching selection during oil-tea afforestation and pollen donor selection for artificial pollination, and the invention has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown is the growth of pollen tube after self-pollination and out-pollination at different time points.

[0025] Figure 2 The ribonuclease activity was measured under different treatments and at different times.

[0026] Figure 3Micrographs of pollen tube growth of Vietnamese Camellia oleifera treated with CvS-RNase protein: A: control without CvS-RNase protein, B: addition of 20ug / ml CvS-RNase protein, C: addition of 20ug / ml CvS-RNase inactivating protein.

[0027] Figure 4 The pollen germination conditions of different combinations are shown below: A in treatment 1 is pollen germination in CK, and B in treatment 1 is pollen germination in identification solution; A in treatment 2 is pollen germination in CK, and B in treatment 2 is pollen germination in identification solution; A in treatment 3 is pollen germination in CK, and B in treatment 3 is pollen germination in identification solution; A in treatment 4 is pollen germination in CK, and B in treatment 4 is pollen germination in identification solution; A in treatment 5 is pollen germination in CK, and B in treatment 5 is pollen germination in identification solution A in treatment 6 is pollen germination in CK, and B in treatment 6 is pollen germination in identification liquid; A in treatment 7 is pollen germination in CK, and B in treatment 7 is pollen germination in identification liquid; A in treatment 8 is pollen germination in CK, and B in treatment 8 is pollen germination in identification liquid; A in treatment 9 is pollen germination in CK, and B in treatment 9 is pollen germination in identification liquid; A in treatment 10 is pollen germination in CK, and B in treatment 10 is pollen germination in identification liquid.

[0028] Figure 5 Pollen germination in different combinations: A in treatment 1 is pollen germination in CK, and B in treatment 1 is pollen germination in identification solution; A in treatment 2 is pollen germination in CK, and B in treatment 2 is pollen germination in identification solution; A in treatment 3 is pollen germination in CK, and B in treatment 3 is pollen germination in identification solution; A in treatment 4 is pollen germination in CK, and B in treatment 4 is pollen germination in identification solution; A in treatment 5 is pollen germination in CK, and B in treatment 5 is pollen germination in identification solution Pollen germination; A in treatment 6 is pollen germination in CK, and B in treatment 6 is pollen germination in identification liquid; A in treatment 7 is pollen germination in CK, and B in treatment 7 is pollen germination in identification liquid; A in treatment 8 is pollen germination in CK, and B in treatment 8 is pollen germination in identification liquid; A in treatment 9 is pollen germination in CK, and B in treatment 9 is pollen germination in identification liquid; A in treatment 10 is pollen germination in CK, and B in treatment 10 is pollen germination in identification liquid. DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0030] Example 1 A method for identifying pollination affinity between oil-tea camellia plants

[0031] (1) Sample collection

[0032] The flowers to be collected on the plants to be tested (pollen receptors) and the plants to be tested (pollen donors) were bagged with sulfuric acid paper bags one day before they opened, and the bags were sealed in time to prevent other insects from entering. The pollen receptor flowers also needed to be emasculated before bagging.

[0033] Collect the style or ovary of the flower of the above-mentioned plant to be tested (pollen receptor) 48-96 hours after the petals unfold, and immediately put it into liquid nitrogen for use. The storage time should not exceed 5 days.

[0034] Collect pollen from the flowers of the plants to be tested (pollen receptors) on the day they open. Collect and use immediately, or immediately place in silica gel for drying and storage. The storage time should not exceed 30 days.

[0035] (2) Preparation of Camellia oleifera pollen affinity test solution and pollen culture

[0036] Since the required concentrations of boric acid and salicylic acid are relatively low and the weighing error is relatively large, the mother solution is prepared first. The preparation method is as follows: take 0.05g of boric acid powder, add clean water to completely dissolve the boric acid, and make the volume to 50ml to obtain 1g / L boric acid mother solution; take 6.9g of salicylic acid powder, add clean water to completely dissolve the salicylic acid, and make the volume to 50ml to obtain 1mol / L salicylic acid mother solution.

[0037] The extract of the present invention is prepared by taking 100 g of sucrose, 5 ml of boric acid mother solution (1 g / L), 5 ul-500 ul of salicylic acid mother solution (1 mol / L), adding clean water to dissolve all the powders, and fixing the volume to 1 L.

[0038] Preparation of the Camellia oleifera pollen affinity detection solution of the present invention: take the style or ovary sample collected above, place it in a centrifuge tube containing the extract of the present invention, the weight ratio of the style or ovary sample to the extract is 1:2-5, fully mash the style sample with a glass rod, then place it in a centrifuge at 10,000 rpm, take the supernatant, and set it aside. Prepare it for immediate use.

[0039] Preparation of CK (without style or ovary) extract: including 0.1 g / L sucrose, 0.005 g / L boric acid and 0.005 mmol / L-0.5 mmol / L salicylic acid.

[0040] Pipette 100ul of the above Camellia oleifera pollen affinity test solution onto a single concave slide, take an appropriate amount of pollen and evenly spread it on the surface of the culture medium, place it in a 25℃ constant temperature biochemical incubator for culture, observe the germination of pollen under a 4×4 microscope after 4 hours, and count it under a microscope. Set CK (i.e., without adding style or ovary). Make 3 parallel slides for each sample, take 5 fields of view for each slide, and select fields of view with no less than 50 pollen grains and uniform distribution for statistics.

[0041] (3) Affinity identification

[0042] The pollen tube length exceeding the pollen grain diameter was used as the germination standard for counting, and the calculation formula was: pollen germination rate = (number of germinated pollens in the field of view / total number of pollen grains in the field of view) * 100%. Based on this method, affinity identification was performed between 10 combinations of plants, and the results are shown in Figure 4 and Table 2. The pollen germination rates of CK and the identification solution treated with the agent of the present invention are different, and the pollen germination rates of the identification solution in treatments 3 to 8 are higher than 30%, indicating that the affinity is high.

[0043] (4) Verification of affinity identification results by artificial pollination

[0044] It is carried out between 9 and 14 o'clock on a sunny day, and the temperature is preferably 15°C. The above-mentioned culture solution is added to an electric sprayer with a stirring function; the above-mentioned preserved pollen (silica gel is removed by sieving (80 mesh)) is added to the culture solution, and the weight ratio is 1:100-500. Immediately after adding the pollen, stir it electrically for 2 minutes to make the pollen evenly distributed in the culture solution. Then, spray while stirring; the vertical distance between the nozzle and the flower is between 80-100cm. The field application amount is 6-10L / mu. The culture solution with pollen must be used up within 1 hour. It cannot rain within 10 hours after spraying, otherwise it will be invalid. The fruit setting rate is counted one month after spraying, and the results are shown in Table 2. The fruit setting rates in CK and identification liquid treated with the agent of the present invention are different. The pollen germination rate of the identification liquid in treatments 3 to 8 is higher than 30%, which further verifies its high affinity, thereby indicating that the present invention can identify the pollination affinity between oil tea plants.

[0045] Table 2 Pollen germination, affinity and artificially controlled pollination fruit setting rate of different combinations

[0046] Combination (ten groups) CK pollen Identification of flowers in liquid Affinity Fruit setting rate Remark Process 1 3.69％ 7.98％ Low 0％ Process 2 3.00％ 5.54％ Low 2％ Process 3 6.44％ 45.72％ high 40％ Process 4 4.20％ 35.30％ high 34％ Process 5 12.33％ 38.22％ high 38％ Process 6 15.89％ 31.22％ high 30％ Process 7 19.11％ 40.30％ high 37％ Process 8 13.54％ 38.66％ high 37％ Process 9 18.97％ 15.22％ Low 10％ Processing 10 15.63％ 13.51％ Low 8％

[0047] Example 2 A method for identifying pollination affinity between oil-tea camellia plants

[0048] (1) Sample collection

[0049] The flowers to be collected on the plants to be tested (pollen receptors) and the plants to be tested (pollen donors) were bagged with sulfuric acid paper bags one day before they opened, and the bags were sealed in time to prevent other insects from entering. The pollen receptor flowers also needed to be emasculated before bagging.

[0050] Collect the style or ovary of the flower of the above-mentioned plant to be tested (pollen receptor) 48-96 hours after the petals unfold, and immediately put it into liquid nitrogen for use. The storage time should not exceed 5 days.

[0051] Collect pollen from the flowers of the plants to be tested (pollen receptors) on the day they open. Collect and use immediately, or immediately place in silica gel for drying and storage. The storage time should not exceed 30 days.

[0052] (2) Preparation of Camellia oleifera pollen affinity test solution and pollen culture

[0053] Since the required concentrations of boric acid and salicylic acid are relatively low and the weighing error is relatively large, the mother solution is prepared first. The preparation method is as follows: take 0.05g of boric acid powder, add clean water to completely dissolve the boric acid, and make the volume to 50ml to obtain 1g / L boric acid mother solution; take 6.9g of salicylic acid powder, add clean water to completely dissolve the salicylic acid, and make the volume to 50ml to obtain 1mol / L salicylic acid mother solution.

[0054] The extract of the present invention is prepared by taking 100 g of sucrose, 5 ml of boric acid mother solution (1 g / L), 5 ul-500 ul of salicylic acid mother solution (1 mol / L), adding clean water to dissolve all the powders, and fixing the volume to 1 L.

[0055] Preparation of the Camellia oleifera pollen affinity detection solution of the present invention: take the style or ovary sample collected above, place it in a centrifuge tube containing the extract of the present invention, the weight ratio of the style or ovary sample to the extract is 1:2-5, fully mash the style sample with a glass rod, then place it in a centrifuge at 10,000 rpm, take the supernatant, and set it aside. Prepare it for immediate use.

[0056] Preparation of CK (without style or ovary) extract: including 0.1 g / L sucrose, 0.005 g / L boric acid and 0.005 mmol / L-0.5 mmol / L salicylic acid.

[0057] Pipette 100ul of the above Camellia oleifera pollen affinity test solution onto a single concave slide, take an appropriate amount of pollen and evenly spread it on the surface of the culture medium, place it in a 25℃ constant temperature biochemical incubator for culture, observe the germination of pollen under a 4×4 microscope after 4 hours, and count it under a microscope. Set CK (i.e., without adding style or ovary). Make 3 parallel slides for each sample, take 5 fields of view for each slide, and select fields of view with no less than 50 pollen grains and uniform distribution for statistics.

[0058] (3) Affinity identification

[0059] The pollen tube length exceeding the pollen grain diameter was used as the germination standard for counting, and the calculation formula was: pollen germination rate = (number of germinated pollens in the field of view / total number of pollen grains in the field of view) * 100%. Based on this method, affinity identification was performed between 10 combinations of plants, and the results are shown in Figure 5 and Table 3. The pollen germination rates of CK and identification solution treated with the agent of the present invention are different, and the pollen germination rates of identification solution in treatment 2, treatment 4, treatment 6, treatment 7 and treatment 9 are higher than 30% and higher than the pollen germination rate in CK, indicating that the affinity is high.

[0060] (4) Verification of affinity identification results by artificial pollination

[0061] It is carried out between 9 and 14 o'clock on a sunny day, and the temperature is preferably 15°C. The above-mentioned culture solution is added to an electric sprayer with a stirring function; the above-mentioned preserved pollen (silica gel is removed by sieving (80 mesh)) is added to the culture solution, and the weight ratio is 1:100-500. Immediately after adding the pollen, stir it electrically for 2 minutes to make the pollen evenly distributed in the culture solution. Then, spray while stirring; the vertical distance between the nozzle and the flower is between 80-100cm. The field application amount is 6-10L / mu. The culture solution with pollen must be used up within 1 hour. It cannot rain within 10 hours after spraying, otherwise it will be invalid. The fruit setting rate is counted one month after spraying, and the results are shown in Table 3. The fruit setting rates in CK and identification liquid treated with the agent of the present invention are different. The fruit setting rates of the identification liquid in treatments 2, 4, 6, 7 and 9 are higher than 30%, which further verifies its high affinity, thereby indicating that the present invention can identify the pollination affinity between oil tea plants.

[0062] Table 3 Pollen germination, affinity and artificially controlled pollination fruit setting rate in different combinations

[0063] Combination (ten groups) CK pollen Identification of flowers in liquid Affinity Fruit setting rate Remark Process 1 6.39％ 27.98％ middle 22％ Process 2 9.98％ 35.95％ high 31％ Process 3 11.32％ 27.35％ middle 20％ Process 4 14.20％ 35.30％ high 33％ Process 5 13.21％ 8.22％ Low 6％ Process 6 8.89％ 31.22％ high 30％ Process 7 9.23％ 40.30％ high 36％ Process 8 9.54％ 8.66％ Low 5％ Process 9 18.77％ 35.22％ high 40％ Processing 10 11.28％ 13.51％ Low 7％

[0064] Obviously, the above described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, ordinary technicians in this field can make other different forms of changes or modifications without making creative work, which should fall within the scope of invention protection.

Claims

1. A Camellia oleifera pollination affinity detection solution, characterized in that: The detection solution is composed of an extract containing a style or an ovary 48-96 hours after the petals unfold, and the extract includes 0.1 g / L sucrose, 0.005 g / L boric acid and 0.005 mmol / L-0.5 mmol / L salicylic acid.

2. A method for preparing a tea oil affinity detection solution, characterized in that: The specific steps are as follows: (1) Take 100 g of sucrose, 5 ml of boric acid mother solution, 5 ul-500 ul of salicylic acid mother solution, add clean water to dissolve all solid substances, and dilute to 1 L to obtain an extract; (2) adding a style or ovary sample 48-96 hours after the petals unfold to the extract obtained in step (1), wherein the weight ratio of the style or ovary sample to the extract is 1:2-5, and the mixture is fully crushed and centrifuged to obtain a supernatant, which is the Camellia oleifera pollen affinity detection solution; The concentration of the boric acid mother solution is 1 g / L, and the preparation method thereof comprises: taking 0.05 g of boric acid powder, adding clean water to completely dissolve the boric acid, and fixing the volume to 50 ml; The concentration of the salicylic acid mother solution is 1 mol / L, and the preparation method thereof comprises: taking 6.9 g of salicylic acid powder, adding clean water to completely dissolve the salicylic acid, and fixing the volume to 50 ml; The step (2) is carried out at 0-10°C.

3. The method for preparing a tea oil affinity detection solution according to claim 2, characterized in that: The collection process of style or ovary samples is as follows: on the plant to be tested as the pollen receptor, cut off all anthers one day before the flower to be collected opens, and bag it with sulfuric acid paper bags, and seal it in time with a stapler to prevent other insects from entering. Collect the style or ovary 48h-96h after flowering, and immediately place it in liquid nitrogen for use. The storage time should not exceed 5d.

4. Use of a camellia oil tree pollination affinity detection liquid prepared by the method of claim 3 for identifying and analyzing the affinity between camellia oil tree plants.

5. A method for identifying pollination affinity between oil-tea camellia plants, characterized in that: The method comprises: 100ul of the Camellia oleifera pollen affinity detection solution of claim 3 was dropped onto a single concave glass slide, and pollen was evenly spread on the surface of the culture medium, and cultured in a 25°C constant temperature biochemical incubator. After 4 hours, the germination of pollen was observed under a 4×4 magnification microscope, and the number of pollen was counted under a microscope; The counting standard is that the length of the pollen tube exceeds the diameter of the pollen grain. The calculation formula is: Pollen germination rate = (number of germinated pollen in the field of view / total number of pollen grains in the field of view) * 100%; when the pollen germination rate is equal to or lower than 20%, it can be determined that the incompatibility between plants is low and it is not suitable to be planted as a matching variety.

6. The method for preparing a tea oil affinity detection solution according to claim 5, characterized in that: The pollen collection process is as follows: On the plant to be tested as the pollen donor, put a sulfuric acid paper bag on the flower to be collected one day before it opens, and seal it with a stapler in time to prevent other insects from entering. Collect the pollen on the first day of the flower opening and use it immediately, or immediately place the pollen in silica gel to dry and store it for later use. The storage time should not exceed 30 days.

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