A reagent for promoting the germination of fraxinus mandshurica pollen on fraxinus mandshurica catkins and a method of using the same

Abstract

The present application belongs to a kind of reagent for promoting Fraxinus mandshurica pollen to germinate on Fraxinus mandshurica stigma and its use method, the method is related to Fraxinus mandshurica breeding technical field, the method mainly uses Fraxinus mandshurica pistil as experimental material, after being treated with 100 μM-200 μM copper chloride (CuCl2; plant active oxygen scavenger), it is placed in vitro incubation medium to culture 1h, then pollination is carried out.The present application uses chemical reagent to simply and efficiently promote Fraxinus mandshurica pollen to germinate on Fraxinus mandshurica stigma, establishes simple and efficient Fraxinus mandshurica female inflorescence in vitro culture and pollination detection system, provides effective means for Fraxinus mandshurica pollination breeding and analyzing Fraxinus mandshurica pollination and fertilization mechanism.

Description

Technical Field

[0001] This invention belongs to the field of ash tree breeding technology, specifically, it relates to a reagent that promotes the germination of ash tree pollen on the stigma of ash tree and its method of use. Background Technology

[0002] Ash (Fraxinus mandshurica) is a deciduous tree belonging to the Oleaceae family and the Ash genus, reaching a height of up to 30 meters. Along with Manchurian walnut and Amur cork tree, it is considered one of the three most valuable hardwood species in Northeast China. Ash flowers are dioecious, with male and hermaphroditic flowers on separate plants, lacking both corolla and calyx. The male inflorescences are dense, with short, slender pedicels, elliptical anthers, very short filaments, a flat and wide ovary, and large, flat samara. It flowers in April and fruits from August to September. In recent years, with the rapid development of the national economy, the demand for Ash has increased significantly. Ash has a wide range of uses and high economic value, being 4-5 times that of coniferous trees. It enjoys a high reputation in both domestic and international markets. Furthermore, Ash is an excellent tree species for landscaping and ornamental purposes. Therefore, vigorously developing commercial Ash forests has significant economic benefits, as well as good ecological and social benefits.

[0003] Currently, there is limited research on the pollination of Ash trees. Therefore, it is necessary to provide a method for conducting physiological experiments and testing on individual female flowers under isolated conditions, which can promote the germination of Ash pollen on the stigma of Ash trees, promote pollen tube growth into the ovary, thereby promoting pollination and increasing the pollination success rate. Summary of the Invention

[0004] The purpose of this invention is to explore a novel application of copper chloride (CuCl2) in promoting pollen germination on the stigma of *Fraxinus mandshurica*. Specifically, copper chloride is prepared at a certain concentration and sprayed onto the mature stigmas of *Fraxinus mandshurica*. This reduces the ROS content in the stigmas, thereby promoting pollen hydration and germination. Specifically, this increases the amount of pollen attached to the stigma, promotes pollen germination, and accelerates pollen tube growth, thus benefiting *Fraxinus mandshurica* pollination and breeding.

[0005] The CuCl2 provided in this invention is used to promote the germination of ash pollen on the stigma of ash tree. The CuCl2 is a 100-200 μM aqueous solution of copper chloride.

[0006] This invention also provides a method for applying a reagent that promotes the germination of ash pollen on the stigma of ash tree, the steps of which are as follows:

[0007] Step 1: Pollen collection. The collected pollen is sieved and collected, and then stored at 4℃.

[0008] Step 2: Prepare the in vitro incubation medium for the column heads;

[0009] Step 3: Once the pistil has just burst open, cover it with a sulfuric acid paper bag.

[0010] Step 4: Once the pistil is fully mature, use a scalpel to cut off the style 2-3 mm below the mature stigma and transfer it to the culture medium prepared in Step 2;

[0011] Step 5: Prepare CuCl solution with a final concentration of 100μM-200μM. 2， Spray the column head with CuCl2, pre-culture for 1 hour, pollinate, and then culture for another 30 minutes;

[0012] Step 6: After pollination for a certain period of time, fix the column head in Carnoy's fixative.

[0013] Step 7: Soften the fixed column head with sodium hydroxide solution, and then stain with aniline blue solution.

[0014] Step 8: Prepare slides of the stained stigmas and observe the pollination status using a microscope (OLYMPUS BX43F, TOKYO, JAPAN).

[0015] The beneficial effects of this invention are:

[0016] This experimental design promotes the germination of *Fraxinus mandshurica* pollen on the stigma of *Fraxinus mandshurica*, which is beneficial for pollination and breeding of *Fraxinus mandshurica*. This invention uses CuCl2 aqueous solution as a *Fraxinus mandshurica* pollen germination promoter, and for the first time conducts physiological experiments and tests on individual female flowers under in vitro conditions. It was found that the promoter significantly promotes *Fraxinus mandshurica* pollen germination, has good water solubility, is low in cost, safe and non-toxic, and is simple to prepare. Attached Figure Description

[0017] Figure 1 Diagram of the in vitro culture system for female flowers of *Fraxinus mandshurica*. A. Artificial pollination after pre-culture B. Re-culture

[0018] Figure 2 This is an observation image of pollination after treatment with CuCl2 aqueous solution.

[0019] In the image: bright field light is used to observe the natural morphology of female flowers, blue light is used to observe pollen grain attachment, and ultraviolet light is used to observe pollen tube growth.

[0020] Figure 3 The image shows the adhesion of Manchurian ash pollen to the stigma of Manchurian ash 30 minutes after pollination.

[0021] Figure 4 This image shows the germination of Ash pollen on the stigma of Ash 30 minutes after pollination.

[0022] In the figure: *** represents highly significant difference (P < 0.001), ** represents significant difference (P < 0.01), and ns represents non-significant difference (P > 0.05). Detailed Implementation

[0023] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0024] 1. Plant materials

[0025] Both female Manchurian ash trees and Manchurian ash trees were planted in the experimental forest farm of Northeast Forestry University. Pollen was collected in mid-April, sieved, and stored at 4℃. After the pistils opened, they were bagged to prevent pollination. Mature pistils were collected, and the style was cut 2-3 mm below the mature stigma using a scalpel.

[0026] 2. Preparation of in vitro incubation medium and CuCl2 working solution for column heads

[0027] A certain amount of CuCl2 reagent was dissolved in water and stored as a stock solution at -20 ℃ for later use. The dissolved CuCl2 stock solution was diluted with water to make working solution concentrations of 100 mM and 200 mM, respectively, for incubation of the column head.

[0028] The in vitro incubation medium for the column head is prepared as follows: 5 mM calcium chloride, 5 mM potassium chloride, 1 mM magnesium sulfate, 0.01% boric acid, 10% sucrose, pH=7.5, sterilized at 121 ℃ for 20 min, and 1% agar powder added fresh for use, dissolved in a 100℃ metal bath.

[0029] 3. Stigmata incubation experiment

[0030] Add 1 mL of stigma incubation liquid medium to a 1.5 mL centrifuge tube, add 0.01 g of agar powder, dissolve in a 100 ℃ metal bath, and after cooling to 60-70 ℃, vortex thoroughly. Dispense the stigma incubation medium into 1.5 mL centrifuge tube caps, allow to cool and solidify, and then use for stigma incubation. Cut the style 2-3 mm below the mature stigma with a scalpel, insert the style below the stigma into the stigma incubation medium, spray the stigma with CuCl2, and then incubate for 1 h in a light incubator at 22 ℃ and 45% humidity.

[0031] 4. Pollination experiment

[0032] Mature pollen was used to pollinate the stigmas of incubated Ash trees. After pollination, the stigmas were placed in a light incubator with a temperature of 22 ℃ and a humidity of 45% for 30 min.

[0033] 5. Observation of pollen adhesion and pollen tube growth after pollination

[0034] After pollination, the stigmas were fixed in Carnoy's fixative (ethanol:acetic acid = 3:1) for 2 h. After rinsing off the fixative with distilled water, 1 M sodium hydroxide was added, and the mixture was placed in a 60 ℃ oven for 1 h to soften. The sodium hydroxide was then rinsed off with distilled water, and 0.1% aniline blue solution was added. The mixture was then placed in a 4 ℃ refrigerator for 2 h to stain. After staining, slides were prepared using 70% glycerol, and pollen adhesion and pollen tube growth were observed under a microscope (OLYMPUS BX43F, TOKYO, JAPAN).

[0035] 6. Exogenous application of CuCl2 can promote pollen adhesion and germination on the stigma of Ash trees.

[0036] Experimental results showed that copper chloride removed reactive oxygen species from the style of *Fraxinus mandshurica*, promoting the germination and growth of *Fraxinus mandshurica* pollen. Figure 2 Untreated stigmas were used for pollination with *Fraxinus mandshurica* pollen. After 30 minutes of pollination, the average number of attached pollen was 78.4 and the average number of pollen tubes was 6.9. Treatment with 100 μM copper chloride solution followed by pollination with *Fraxinus mandshurica* pollen resulted in an average of 291.7 and 174.9 pollen tubes after 30 minutes of pollination. Treatment with 200 μM copper chloride solution followed by pollination with *Fraxinus mandshurica* pollen resulted in an average of 232.1 and 70.8 pollen tubes after 30 minutes of pollination. This indicates that scavenging reactive oxygen species promotes pollen hydration, germination, and growth. Figure 3 .4).

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of CuCl2 aqueous solution to promote the germination of Fraxinus mandshurica pollen on Fraxinus mandshurica catkins, characterized in that: The stigma is incubated with the stigma ex-vivo incubation medium, then sprayed with CuCl2 with a final concentration of 100 μM or 200 μM, and pollinated. ​ 2. Use according to claim 1, characterized in that: When the pistil just bursts, the pistil is bagged with a sulfuric acid paper bag, and when the pistil is fully mature, the stigma is separated from the pistil and moved to the stigma ex-vivo incubation medium.

3. Use according to claim 1 or 2, characterized in that: Before pollination, the stigma is inserted into the stigma ex-vivo incubation medium, and CuCl2 with a final concentration of 100 μM or 200 μM is configured to spray the stigma.

4. Use according to claim 1 or 2, characterized in that: The matured Fraxinus mandshurica stigma needs to be treated with CuCl2 with the concentration for a period of time before pollination.

5. Use according to claim 4, characterized in that: The time for the matured Fraxinus mandshurica stigma to be incubated in vitro is 1 h.

6. Use according to claim 4, characterized in that: After the incubated matured Fraxinus mandshurica stigma is pollinated for 30 min, the pollen adhesion and pollen tube growth are counted.

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