Method for improving pear pollen germination rate by DIECA treatment

Abstract

The invention discloses a method for improving the germination rate of pear pollen by using DIECA. The method comprises the following steps: mixing pollen with a pear pollen liquid culture medium containing DIECA or an agricultural pollination liquid, and then carrying out pollen culture or pollination, or after the anther in the big bud stage is dried and looses powder, the pollen is sown on the pollen solid culture medium, then the pear pollen liquid culture medium containing DIECA is added, and the pear pollen liquid culture medium is cultured in a dark place. Research finds that the germination rate of pear pollen treated by DIECA is obviously increased compared with that of a contrast, and pollen tubes can grow well. The pollen germination time is short, and the application prospect is relatively good. The method has the advantages of simple operation steps, less loss, short time consumption and the like, reagents used in the formula do not contain dangerous chemicals, are low in price and easy to obtain, solution preparation is extremely simple and convenient, the pH value does not need to be additionally adjusted, and the purposes of improving the germination rate, ensuring the germination quality and saving cost and increasing income can be achieved while labor and time are not consumed.

Description

Technical Field

[0001] This invention belongs to the fields of plant physiology and production cultivation technology, specifically relating to a method for improving the germination rate of pear pollen by treating with DIECA (sodium diethyldithiocarbamate). Background Technology

[0002] Pear, a tree belonging to the genus *Pyrus* of the Rosaceae family, is one of the main traditionally cultivated fruit trees in my country and an important native fruit tree, often referred to as the "King of Fruits." As a vital temperate economic fruit tree, the yield and quality of the pear highly depend on effective pollination and fertilization. Pollen germination rate and pollen tube growth rate are key factors determining fertilization success, directly affecting fruit set and fruit development. Normally growing pollen tubes provide sufficient nutrients to the ovules, promoting endosperm development and laying the foundation for fruit enlargement, reducing the probability of deformed fruit. If pollen tube germination and growth are hindered, for example by factors such as self-incompatibility, nutrient deficiency in the style, or adverse weather conditions, its growth will be slowed or halted. This makes it difficult for sperm cells to meet and fuse with egg cells, leading to double fertilization failure, preventing normal fruit set, and impacting yield.

[0003] However, a bottleneck problem hindering efficient pollen germination is prevalent in pear production. Most pear varieties are self-infertile, requiring pollinator trees or artificial pollination during flowering to achieve the desired yield and quality. Artificial pollination mostly uses fresh pollen harvested that year, with a few using pollen stored from the previous year. The viability of the pollen directly affects the effectiveness of artificial pollination.

[0004] Jasmonic acid (JA) is produced from substances such as linolenic acid through a series of enzymatic reactions. As a signaling molecule in various plant tissues and organs, it regulates plant growth and development and adaptability to adverse environments. Widely distributed throughout plant parts, JA performs many physiological functions, such as inhibiting seedling growth, root growth, and pollen germination. In production, appropriately inhibiting JA synthesis promotes plant growth and development, regulating the balance between plant growth and stress resistance to obtain robust seedlings. DIECA is an inhibitor of the JA synthesis pathway in plants. Currently, there is no publicly available technology using DIECA to enhance pear pollen germination efficiency. DIECA is known to have a strong ability to scavenge reactive oxygen species and maintain cellular redox homeostasis in plant physiology. However, its applicable concentration, mechanism of action, and synergistic potential in regulating pear pollen germination remain unclear, necessitating the development of targeted solutions. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the germination rate of pear pollen using DIECA treatment. This method can improve the pollen germination rate and is time-saving and simple to operate when cultivating pear pollen.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention seeks protection for the use of DIECA in improving the germination rate of pear pollen or in the preparation of products for improving the germination rate of pear pollen.

[0008] Furthermore, in the above-described application, the product used to improve the germination rate of pear pollen is a pear pollen liquid culture medium or agricultural pollination solution containing DIECA. Even further, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 1-10 μM.

[0009] Furthermore, the pear pollen mentioned in the above applications is the pollen of 'Dangshan Crisp Pear', 'Cuiguan', or 'Huanghua' pears; when treating 'Dangshan Crisp Pear' pollen, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 10 μM; when treating 'Cuiguan' pear pollen, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 5-10 μM, preferably 10 μM; when treating 'Huanghua' pear pollen, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 0.5-1 μM, preferably 1 μM.

[0010] Secondly, the present invention claims protection for a product for improving the germination rate of pear pollen, the product being a pear pollen liquid culture medium or agricultural pollination solution containing DIECA; wherein the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 1-10 μM.

[0011] Thirdly, the present invention claims protection for a method for improving the germination rate of pear pollen using DIECA, wherein the method is any one of (1) or (2) below:

[0012] (1) Mix the pollen with the aforementioned products and then perform pollen culture or pollination;

[0013] Alternatively, (2) after the anthers are dried and the pollen is dispersed during the large bud stage, the pollen is sown on a pollen solid culture medium, and then the pear pollen liquid culture medium containing DIECA from the above products is added and cultured in the dark.

[0014] Furthermore, the pollen is pear pollen, specifically pollen from 'Dangshan Crisp Pear', 'Cuiguan', or 'Huanghua' pears. Even further, when treating 'Dangshan Crisp Pear' pollen, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 10 μM; when treating 'Cuiguan' pear pollen, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 5-10 μM, preferably 10 μM; and when treating 'Huanghua' pear pollen, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 0.5-1 μM, preferably 1 μM.

[0015] In the technical solution of this invention, the formula of the pear pollen liquid culture medium is as follows: 10% w / v sucrose, 0.01% w / v H3BO3, 0.03% w / v Ca(NO3)2·4H2O, 0.63% w / v MES (sodium α-sulfomethyl ester sulfonate), dissolved and brought to volume with deionized water, and adjusted to pH 6.2-6.3 with Tris base; the w / v values ​​mentioned in this invention are all g / 100 ml.

[0016] The pollen solid culture medium is prepared by adding 1.5% w / v low melting point agarose to the pollen liquid culture medium and heating it to fully dissolve it.

[0017] In the technical solution of this invention, the agricultural pollination solution described is a conventional pollination solution in the prior art. Alternatively, those skilled in the art will understand that the DIECA-containing liquid culture medium can also be adapted for agricultural production as a pollination solution, and its basic components may include sucrose, boric acid, and suspending agents, etc.

[0018] The method for promoting pear pollen germination using DIECA treatment described in this application has the following advantages compared to existing technologies:

[0019] (1) The present invention utilizes DIECA reagent to treat pear pollen. Its core advantage lies in its strong antioxidant capacity and its role as an inhibitor of methyl jasmonic acid, which effectively overcomes the key physiological barriers that limit the germination and pollen tube growth of pear pollen, significantly improving pollen viability, germination rate, pollen tube growth rate and stress resistance. Ultimately, it provides a reliable and efficient technical means to improve the pollination and fertilization efficiency of pear trees, increase fruit set rate and fruit yield and quality, and is especially suitable for optimizing artificial pollination and coping with adverse climatic conditions.

[0020] (2) DIECA reagent is easy to dissolve in water or conventional nutrient solution and can be easily integrated into existing artificial pollination processes (such as pollen viability retention solution after pollen collection, and pollen suspension sprayed). Its effect is clear, the cost of use is controllable, and it has good prospects for field application.

[0021] (3) This method is not only applicable to the cultivation of pear pollen, but also provides a method reference for promoting the germination of pollen from other plants. Attached Figure Description

[0022] Figure 1 The results show the effects of DIECA treatment on pollen germination of 'Dangshan Crisp Pear'. A represents the pollen germination rate after 10 μM DIECA treatment, and B shows images of pollen germination after 1 h of culture. Bar = 20 μm.

[0023] Figure 2The results show the effects of DIECA treatment on pollen germination of 'Cuiguan' pear. A represents the pollen germination rate after 10 μM DIECA treatment, and B shows images of pollen germination after 1 h of culture. Bar = 20 μm.

[0024] Figure 3 The results show the effects of DIECA treatment on pollen germination of 'Huanghua' pear. A represents the pollen germination rate after 1 μM DIECA treatment, and B shows images of pollen germination after 1 hour of culture. Bar = 20 μm.

[0025] Figure 4 To stain pollen tube cell walls with methylated pectin after treatment with 10 μM DIECA. Bar = 20 μm. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make equivalent changes to the disclosed technical content to create equivalent embodiments. Any modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention fall within the protection scope of the present invention.

[0027] Example 1: A method for improving pear pollen germination rate through DIECA treatment

[0028] I. Preparation of reagents or culture media

[0029] Preparation of 1 M DIECA stock solution: Weigh 4.5 g of powder into a 50 ml screw-top centrifuge tube, tighten the cap, add distilled water to a final volume of 20 ml, vortex until thoroughly mixed, and the solution is nearly clear and transparent. Filter through a 0.22 μM aqueous filter membrane and store at -20°C. This prepared 1 M DIECA stock solution is suitable for long-term storage. In subsequent experiments, it needs to be diluted with distilled water.

[0030] Preparation of pollen liquid culture medium: 10% w / v sucrose, 0.01% w / v H3BO3, 0.03% w / v Ca(NO3)2·4H2O, 0.63% w / v MES (sodium α-sulfomethyl ester sulfonate), dissolved and brought to volume with deionized water, adjusted to pH 6.2–6.3 with Tris base. All w / v values ​​are g / 100 ml.

[0031] Preparation of pollen solid medium: Add 1.5% w / v low-melting-point agarose to the prepared pollen liquid medium and microwave until fully dissolved. Pour 1 ml of medium into each 3.5 cm petri dish and allow it to solidify.

[0032] II. Collection and Cultivation of Pear Blossom Pollen

[0033] 1. Collection of pear pollen: Pick the anthers of pear flowers in the large bud stage of 'Dangshan Crisp Pear', 'Cuiguan', and 'Huanghua' varieties, and store the pollen in a silicone box to keep it dry.

[0034] 2. Pollen culture: Take an appropriate amount of pollen from 'Dangshan Crisp Pear', 'Cuiguan', and 'Huanghua', and gently tap it onto the surface of a solid culture medium. Add 300 μL of pollen liquid culture medium containing DIECA as the treatment group (with 300 μL of pollen liquid culture medium as the control group), and incubate in a 25℃ incubator in the dark for 1 h. Each treatment is repeated 3 times.

[0035] III. Effects of DIECA Treatment on Pear Pollen Germination

[0036] 1. The effect of different DIECA concentrations on pollen germination was investigated by treating pear pollen with different concentrations, as shown in Table 1. Pollen from 'Dangshan Su Pear' was treated with 10 μM DIECA, pollen from 'Cuiguan' pear was treated with 10 μM DIECA, and pollen from 'Huanghua' pear was treated with 1 μM DIECA. Germination rates were statistically analyzed after cultivation. The pollen tube length being greater than the pollen grain diameter was used as the criterion for pollen germination. Three to five fields of view were randomly observed for each treatment, with 400 to 800 pollen grains observed in each field. The pollen germination rates were statistically analyzed and plotted in graphs.

[0037] Table 1. Effects of different concentrations of DIECA on pollen germination of 'Dangshan Crisp Pear', 'Cuiguan', and 'Huanghua' pear varieties.

[0038]

[0039] 2. Implementation Results:

[0040] Treatment with 10 μM DIECA significantly promoted pollen germination and growth in 'Dangshan Crisp Pear', 'Cuiguan' pear, and 'Huanghua' pear varieties. Compared to the control, applying appropriate concentrations of DIECA increased the pollen germination rate by approximately 10% (e.g., ...). Figure 1-3 (As shown).

[0041] 3. Staining of methylated pectin in the cell walls of pear pollen tubes

[0042] The relative content of methylated and demethylated pectin in the pollen tube cell wall regulates the normal growth of pollen tubes. Chinese Patent CN 115615785 A discloses a method for improving the staining effect of methylated pectin in the cell wall of pear pollen tubes using a pre-change solution technique. Based on the above method, the staining of methylated pectin in the cell wall of 'Dangshan Crisp Pear' pollen after treatment with 10 μM DIECA was observed, and the changes in the level of methylated pectin in the pollen cell wall after germination were detected. The results showed that the level of methylated pectin in the cell wall decreased after DIECA treatment (e.g., ...). Figure 4 (As shown).

Claims

1. Application of DIECA in improving the germination rate of pear pollen or in the preparation of products for improving the germination rate of pear pollen.

2. The application according to claim 1, characterized in that, The product used to improve the germination rate of pear pollen is a pear pollen liquid culture medium containing DIECA or an agricultural pollination solution.

3. The application according to claim 2, characterized in that, The concentration of DIECA in pear pollen liquid culture medium or agricultural pollination solution is 1-10 μM.

4. The application according to claim 3, characterized in that, The pear pollen mentioned is from 'Dangshan Crisp Pear', 'Cuiguan', or 'Huanghua' pears; when treating 'Dangshan Crisp Pear' pollen, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 10 μM; when treating 'Cuiguan' pear pollen, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 5-10 μM; when treating 'Huanghua' pear pollen, the concentration of DIECA in the pear pollen liquid culture medium or agricultural pollination solution is 0.5-1 μM.

5. The application according to claim 2, 3, or 4, characterized in that, The formula for the pear pollen liquid culture medium is as follows: 10% w / v sucrose, 0.01% w / v H3BO3, 0.03% w / v Ca(NO3)2·4H2O, 0.63% w / v MES, dissolved and brought to volume with deionized water, and adjusted to pH 6.2-6.3 with Tris base.

6. A product for improving the germination rate of pear pollen, characterized in that, This product is a liquid culture medium for pear pollen or an agricultural pollination solution containing DIECA; the concentration of DIECA in the liquid culture medium for pear pollen or the agricultural pollination solution is 1-10 μM.

7. The product according to claim 6, characterized in that, The formula for the pear pollen liquid culture medium is as follows: 10% w / v sucrose, 0.01% w / v H3BO3, 0.03% w / v Ca(NO3)2·4H2O, 0.63% w / v MES, dissolved and brought to volume with deionized water, and adjusted to pH 6.2-6.3 with Tris base.

8. A method for improving the germination rate of pear pollen using DIECA, characterized in that, The method is either (1) or (2) below: (1) The pollen is mixed with the product of claim 6 or 7 and then cultured or pollinated; Alternatively, (2) after the anthers are dried and dispersed during the large bud stage, the pollen is sown on a pollen solid culture medium, and then the pear pollen liquid culture medium containing DIECA in the product of claim 6 or 7 is added and cultured in the dark.

9. The method according to claim 8, characterized in that, The pollen mentioned is pear blossom pollen, specifically the pollen from the blossoms of 'Dangshan Crisp Pear', 'Cuiguan', or 'Huanghua' pears.

10. The method according to claim 9, characterized in that, When treating 'Dangshan Crisp Pear' pollen, the concentration of DIECA in the pear pollen liquid medium or agricultural pollination solution was 10 μM; when treating 'Cuiguan' pear pollen, the concentration of DIECA in the pear pollen liquid medium or agricultural pollination solution was 5-10 μM; and when treating 'Huanghua' pear pollen, the concentration of DIECA in the pear pollen liquid medium or agricultural pollination solution was 0.5-1 μM.

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