Method for promoting sprouting and cutting rooting of fraxinus mandshurica by spraying 5-azacytidine

Abstract

The invention provides a method for promoting sprouting and cutting rooting of fraxinus mandshurica by spraying 5-azacytidine. A three-year-old fraxinus mandshurica sapling is used as a material, 25-75 [mu] mol.L <-1 > 5-azacytidine (5-AzaC) is used as an apparent regulating agent, spraying is performed on the cut sapling continuously for one month after spring germination, spraying is performed once every three days, semi-lignified tender branches of fraxinus mandshurica are taken at the beginning of July, and the rooting rate reaches 83.33% after cuttage is performed for 30 days and is improved by 32.22% compared with a contrast. The length and the stem width of sprouting branches of sprayed plants are reduced, and the number of the sprouting branches is increased, so that the appearance regulating agent 5-azacytidine can effectively improve the sprout tillering and cutting propagation capacity, a trunk cutting treatment group is remarkably superior to a trunk non-cutting treatment group, and survived cutting slips can be transplanted to a nursery field in the second year after being covered with snow and overwintering.

Description

Technical Field

[0001] The invention relates to a method for improving the rooting rate of ash cuttings by spraying 5-azacytidine on leaves, and belongs to the field of forestry biotechnology. Background Art

[0002] Manchurian elm (Fraxinus mandshurica Rupr.) is a major afforestation species in Northeast my country and a key protected and valuable timber species in the National Reserve Forest Program, possessing extremely high economic value. To meet the requirements of Manchurian elm clonal forestry, efficient and large-scale vegetative propagation techniques are essential. However, vegetative propagation (such as cuttings) is subject to age effects. The rooting rate of cuttings decreases significantly with tree age, falling below 50% by the third year. Furthermore, the number and yield of cuttings are low, making it difficult to ensure the widespread application of high-quality varieties through cutting propagation.

[0003] DNA methylation is a conservative epigenetic modification. The degree of DNA methylation modification is closely related to plant age (Liu et al., 2023). It has been found that cyclic rejuvenation can increase the rooting rate of ash cuttings and reduce the DNA methylation level (Peng Guangzhou et al., 2014). ) can cause methylation changes at specific sites in genomic DNA after grafting onto rootstocks, thereby affecting tree growth and development (Zhou et al., 2014). This provided a reference for the research related to this application. 5-Azacytidine (5-AzaC) is a DNA methylase inhibitor that reduces the activity of DNA methyltransferases (DNMTs), thereby reducing overall methylation levels. 5-AzaC has been widely used in animal and medical fields, but its research and application in plants, especially trees, is relatively limited.

[0004] Studies have shown that 5-AzaC treatment can promote plant growth, rice ( ) seeds were treated, the DNA methylation level of the plants was reduced, the growth and development efficiency was improved, and it could be inherited by offspring (Ning Chunmeng et al., 2019); 5-AzaC can induce apple ( ) rootstock, which reduced the DNA methylation level and restored the root regeneration ability (Wang et al, 2023); for barley ( .)、Rice( ),corn( .),wheat( .) and other four crop seeds, the application of low concentrations of 5-AzaC inhibited seed germination and plant height, and promoted the increase of root number (Hou Zehao et al., 2017); .) After applying 5-AzaC to the leaves, the plant growth was significantly reduced, and the overall phenotype showed a dwarfing (Luke et al, 2023).

[0005] Therefore, it is particularly important to use epigenetic regulators to improve the regeneration ability of ash and eliminate the age effect. Although 5-azacytidine has achieved some research results in promoting plant regeneration, there are still some reports on its regulation of juvenility and rooting ability of ash.

[0006] The invention introduces a method for improving the sprouting ability of trees and the rooting rate of cuttings by spraying 5-azacytidine on the leaves of Manchurian ash. Summary of the Invention

[0007] The present invention aims to provide a method for increasing the rooting rate of elm cuttings by spraying 5-azacytidine on the leaves. This invention is of great significance for further improving the measures and effects of elm rejuvenation and lays a foundation for asexual propagation of elm and solving the age effect problem. The practical problems solved by this invention include:

[0008] 1. It can show a rejuvenating effect in a short period of time, increase the number of sprouts and spikelets, and solve the problem of few spikelets of elm.

[0009] 2. Provide young cuttings to solve the bottleneck problem of the reproduction rate of cutting propagation in elm production due to the age effect, and significantly improve the rooting rate.

[0010] 3. Through the pathway of epigenetic regulation of gene expression, a rejuvenation technology is established, which is simple to operate, low in cost, and has rapid and significant effects.

[0011] The specific technical route of the invention includes the following:

[0012] (1) Spraying treatment of 5-azacytidine on leaves of ash

[0013] The DNA methylase inhibitor 5-azacytidine (5-AzaC) was used to establish the regulation technology. 5-Azacytidine was prepared into 25, 50, and 75 μmol·L -1 0.1% DMSO aqueous solution. Before budding, the stems of ash trees were cut 10 cm above the ground. The cut was horizontal and painted to prevent water loss and tree death. An identical uncut treatment group and control group were also designed. Treatment began when the average length of new branches reached 3-4 cm after budding. Spraying was carried out every three days at 9:00 AM, evenly distributing the agent to all leaves. The control group was sprayed with an equal amount of distilled water. Each treatment consisted of 80 plants, and the treatment cycle lasted for 30 days until cuttings were planted.

[0014] (2) Cutting propagation of ash in greenhouse

[0015] Take the semi-lignified young branches of ash and make them into cuttings with a length of 5-8 cm. Only one leaf and one bud are retained on the cuttings, and the leaves are trimmed to 1-2 cm. 2 The cutting medium was yellow sandy soil (1:1 yellow sand), and the bottom of the white mesh plastic basket used for cutting was covered with two layers of non-woven fabric. All cuttings were irrigated with IBA (200 mg·L -1 ) solution for 30 minutes. Each basket contained 30 cuttings, with at least 90 cuttings in each treatment and control group. After cuttings were implanted, the greenhouse temperature was maintained between 25°C and 35°C, and the humidity between 80% and 90%. For the first 20 days after implantation, the cuttings were kept moist by spraying them with water. Shade them with a 6-pin black shade net and sterilize them with carbendazim solution every 7 days. Remove the shade net 30 days after implantation.

[0016] Features of the present invention:

[0017] Foliar spraying of 5-azacytidine (5-AzaC) can rejuvenate elm and promote tillering in a short period of time, significantly increasing cutting yield and cutting rooting rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure A: Rooting of the cut / uncut treatment group and the control group. 30 days after the cuttage of ash in the greenhouse, a bird's-eye view of the plastic cutting basket. Cuttings with green leaves and good growth represent successful rooting. A1 was sprayed after cutting. , A2 is the control group with only stem cutting and no spraying, B1 is the control group with no stem cutting and spraying , B2 is the control without stem cutting and spraying; Figure B: spraying after stem cutting Compared with the control group (A2), the phenotype of the trees after 30 days (scale bar: 10.0 cm); Figure C shows ... -1 Comparison of rooting of cuttings between the 5-azacytidine treatment group and the control group (scale bar: 1.0 cm). T2 was cut and sprayed with 50 μmol·L -1 The plants were treated with 5-AzaC, and CK was the control with only stem cutting and no spraying. DETAILED DESCRIPTION

[0019] Example 1

[0020] 1. Spraying different concentrations of 5-azacytidine promotes rooting of ash cuttings

[0021] 25, 50, and 75 μmol·L -15-Azacytidine was used to treat 3-year-old cut and uncut elm trees. The stems were cut on May 10 and sprayed on June 8, with spraying every 3 days. A control group was sprayed with the same amount of water. Thirty days later, on July 8, cuttings were taken for greenhouse propagation. Thirty days later, the rooting rate of the cuttings was calculated.

[0022] Table 1 Effects of spraying different concentrations of 5-azacytidine on the rooting rate of ash cuttings after trunk cutting

[0023]

[0024] Note: Duncan's multiple comparison was used. Different letters indicate significant differences at the 0.05 level.

[0025] As shown in Table 1, spraying 5-AzaC can significantly improve the rooting rate of ash cuttings, 25, 50, 75 μmol·L -1 The rooting rates of the three spraying concentrations increased by 27.78%, 33.22% and 16.67% respectively compared with CK. -1 The rooting rate was the highest, reaching 83.33%, a relative increase of 63.04%.

[0026] 2. Effects of stem cutting and non-stem cutting on rooting of ash cuttings under the conditions of spraying 5-azacytidine

[0027] As shown in Table 2, the rooting rate of the cut control group, A2, was 51.11%, while that of the treated group, A1, was 83.33%. This represents a 32.22% increase in rooting rate relative to A2, a relative increase of 63.04%. The rooting rate of the uncut control group, B2, was 27.78%, while that of the treated group, B1, was 61.11%. This represents a 33.33% increase relative to B2, a relative increase of 1.2 times. This suggests that spraying 5-AzaC is equally effective on both cut and uncut three-year-old ash cuttings, significantly increasing the rooting rate. The cut treatment group exhibited significantly better results than the uncut group.

[0028] Table 2 Effects of 5-azacytidine on the rooting rate of cuttings of Fraxinus mandshurica

[0029]

[0030] Example 2 Effects of 5-azacytidine on the sprouting of ash trees

[0031] Use 50 μmol·L -1 Three-year-old cut-stem and uncut Fraxinus mandshurica plants were treated with 5-azacytidine, and the growth and tillering of the cutting plants were investigated 30 days after spraying.

[0032] Table 3 Effects of spraying different concentrations of 5-azacytidine on the sprouting of ash after trunk cutting

[0033]

[0034] Table 4 Comparison of the effects of spraying 5-azacytidine on the sprouting and growth of truncation- and non-truncation-type ash trees

[0035]

[0036] Note: Duncan's multiple comparison was used. Different letters indicate significant differences at the 0.05 level.

[0037] As shown in Table 3, spraying 5-AzaC can significantly reduce the length and stem width of new shoots of Fraxinus mandshurica, and increase the number of shoots and stem nodes. -1 After three spraying concentrations, the number of sprouts increased by 36%, 68%, and 44% relative to CK, respectively; the length of sprouts decreased by 20.92%, 32.92%, and 24.92%, respectively; the stem width decreased by 12.8%, 29.24%, and 16.58%, respectively. The effect of T2 concentration on promoting sprouting and tillering was better than that of T1 and T3.

[0038] As shown in Table 4, the stem cutting treatment group had a 32.92% reduction in shoot length, a 29.19% reduction in stem width, and a 0.68-fold increase in the number of shoots compared to the control group. The effect of the stem cutting treatment group was significantly better than that of the non-stem cutting treatment group.

Claims

1. A method for promoting tillering and rooting of ash trees by spraying 5-azacytidine, characterized in that: (1) Using 3-year-old saplings of Manchurian ash as materials, cut the trunk 10 cm above the ground before germination in the same year, and paint the cut horizontally; (2) After the terminal bud sprouts, 25~75μmol·L -1 The leaves of Manchurian ash were evenly sprayed with 5-azacytidine (0.1% DMSO) solution at 9:00 am, with an interval of 3 days, for a total of 10 times. The number of sprouts increased significantly (34%-68%). (3) Take the semi-lignified shoots as cuttings and carry out cuttings in the greenhouse. After 30 days, 50μmol·L -1 The rooting rate of cuttings in the treatment group reached 83.33%, which was 32.22% higher than that of the control group without 5-azacytidine spraying. 5-azacytidine can effectively improve the propagation ability of cuttings.

2. The method according to claim 1, wherein: For pruning elm, start spraying 5-azacytidine on the mother elm tree when the new branches are about 3 to 4 cm long, and spray once every 3 days.

3. The method according to claim 1, wherein: In early July, the semi-lignified young branches of ash were taken for cuttings. The branches were made into 5-8 cm long cuttings, with only one leaf and one bud retained. The leaves were trimmed to 1-2 cm. 2 The cutting medium was a mixture of loess soil and sand (loess soil: sand = 1:1). The bottom of the white mesh plastic bottle used for cutting was covered with two layers of non-woven fabric. The cuttings were treated with IBA (200 mg·L -1 ) Soak the base for 30 minutes. After cutting, keep the temperature at 25~35℃ and the humidity at 80~90%. Use black shade net for shade. Spray carbendazim solution once every 7 days. Remove the shade net after 30 days. The surviving cuttings can be transplanted to the nursery field in the second year after being covered with snow for winter.

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