CN114431154B

CN114431154B - Method for asexual propagation through acer nikoense dormant buds

Info

Publication number: CN114431154B
Application number: CN202210367170.8A
Authority: CN
Inventors: 由香玲; 王灵宇; 赵曦阳; 曲冠证; 张世凯
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2022-06-28
Anticipated expiration: 2042-04-08
Also published as: CN114431154A

Abstract

A Chinese medicinal preparation prepared from Acer nikoense Maxim (Acer nikoense Maxim.) (II)Acer mandshuricumMaxim.) dormant bud asexual propagation method, belonging to the forestry biotechnology field. In order to solve the problem of insufficient supply of the existing Acer nikoense seedling, a vegetative propagation system through dormant buds is established. The main technical process comprises the following steps: taking early spring dormant buds of acer nikoense as explants, inoculating the explants after disinfection and sterilization under the sterile culture condition with a plant growth regulator, inducing the germination and the generation of adventitious buds, further realizing the mass propagation of the adventitious buds, and obtaining a large number of orderly and consistent asexual propagation nursery stocks by rooting the adventitious buds, and further transplanting the nursery stocks into soil for growth for forestation. The method can overcome the limit of the traditional seed propagation mode of the acer nikoense on the yield of nursery stocks of the acer nikoense and the problem that excellent germplasm characters are difficult to stably inherit, and provides technical support for fine variety breeding and genetic breeding.

Description

Method for asexual propagation through acer nikoense dormant buds

Technical Field

The invention relates to a method for asexual propagation through dormant buds of Acer manishuricum Maxim. The invention belongs to the field of forestry biotechnology.

Background

Acer nikoense (Acer mandshuricum Maxim.) has the scientific name of northeast maple, which belongs to Aceraceae plant of Acer, deciduous tree (Wangdongdong et al, green technology, 2012(5):32), is famous for its bright red leaves in autumn, and is an important colorful leaf tree species in northeast of China. Due to the higher landscape value and ecological value, the seedling demand of the seedlings is increased gradually at the present stage.

In the market at present, the seedling supply of the acer nikoense is mainly based on seed propagation, but the problems of low seed yield, small seedling production scale and difficulty in maintaining the excellent properties of the woody plant exist, so the situation of shortage of colored seedlings in the market cannot be solved in time. Meanwhile, no report is provided about the cutting propagation of the acer nikoense at present, but the cutting of other acer tree species has the problems of difficult rooting and extremely low survival rate of seedlings (the twitter and the like, national soil and natural resource research, 2005(4): 93-94). Therefore, it is very necessary to perform in vitro rapid propagation research on the gene. However, the research on the isolated regeneration system of acer nikoense is relatively short at present. Researchers such as Liubao photo and the like obtain adventitious buds through callus by utilizing immature zygotic embryos of Acer nikoense Maxim, and form regenerated plants after rooting, but the induction rate is lower (Liubao photo, Kogying, plant physiology reports, 2010, 46(6): 613-; in contrast, researchers such as Kyuenfeng cultured the dormant bud in vitro to obtain a sprout but no regenerated plant (Kyuenfeng et al, northern horticulture, 2010(20): 154-155). The tissue culture technology of the acer nikoense still needs further optimization and promotion to meet the rapid propagation requirement of excellent plants.

The invention takes the acer nikoense dormant bud as an explant material, establishes an acer nikoense asexual propagation system through the induction and proliferation of an adventitious bud, and provides technical support for fine variety propagation and genetic breeding.

Disclosure of Invention

The invention aims to solve the technical problems of insufficient supply of good seedlings of the acer nikoense and the like, and provides a method for asexual propagation through the acer nikoense dormant bud. The technical route of the specific invention comprises the following contents:

selecting dormant acer nikoense branches with buds in a forest plant garden of Heilongjiang province in early spring, cutting the branches into stem sections with buds of about 3.0cm, washing the stem sections with running water for 10min, and then performing the following disinfection treatment in a super clean bench: soaking in 75% ethanol for 5min, soaking in 5% sodium hypochlorite for 12min, washing with sterilized water for 5 times, and air drying on sterile filter paper. Removing bud scale from the disinfected stem with buds, cutting off buds, and inoculating to the following culture conditions: DKW +0.03mg/L TDZ +0.01mg/L IBA +0.5mg/L GA₃And culturing the agar plant gel at the concentration of 20g/L of cane sugar and 7.47g/L of agar plant gel under light, performing statistics after 6 weeks of inoculation, and observing the growth state, the germination and the proliferation state of the buds on different culture media. Inoculating the differentiated adventitious bud plant to the following culture conditions: DKW +0.03mg/L TDZ +0.01mg/L NAA +0.5mg/L GA ₃+20g/L sucrose +7.47g/L agar plant gel, cultured for 4 weeks to obtain a large number of adventitious buds. Adventitious buds of 2.0cm were cut out and inoculated under the following culture conditions: 1/2DKW +0.5mg/L IBA, 1.0mg/L IAA, 0.1% activated carbon, 30g/L cane sugar and 7.47g/L agar plant gel, culturing for 4 weeks to induce adventitious buds to root, and obtaining a large number of regular and consistent regenerated plants; finally, continuously culturing the rooted regeneration plants for 35-42 days, then transplanting the plants, and performing high-temperature soil sterilization before transplanting, wherein the ratio of the soil components to humus soil, vermiculite and perlite is 5:3: 2; in the early stage of transplantation survival, spraying carbendazim on the soil surface periodically every week to prevent bacterial and fungal infection, and slowing down to once a month after seedlings grow completely.

The above inoculated materials were all cultured in a tissue culture chamber at 25. + -. 2 ℃ under light conditions of 16/8 hours (light/dark). The asexual propagation plants grow in a greenhouse after soil transplantation.

The invention has the following beneficial effects:

1. the method utilizes the axillary buds of the acer nikoense in the resting period to induce the adventitious buds to establish a plant regeneration system, and solves the problem that the characters of the seeds can not be completely inherited in the traditional seed propagation process due to the character separation of the seeds. The method has the advantages of short production period and high efficiency of the nursery stock, can produce a large number of uniform nursery stocks in the same year by induction, obviously shortens the breeding period and improves the breeding efficiency.

2. The invention lays a foundation for the subsequent genetic breeding research of the acer nikoense.

3. The method has good popularization. For different maple germplasms, the method can be used for reference to efficiently establish the isolated regeneration system of the maple tree species, and effectively solve the problem of the requirement of urban garden landscapes on high-quality nursery stocks.

Drawings

FIG. 1 Germination and adventitious bud proliferation of Acer nikoense Maxim dormant bud

A: selecting Acer nikoense branches with dormant buds in a forest botanical garden of Heilongjiang province in early spring

B. C: respectively inoculating dormant bud in DKW +0.03mg/L TDZ +0.01mg/L IBA +0.5mg/L GA₃+20g/L sucrose +7.47g/L agar plant gel conditions 1 week of germination and 6 weeks of induced adventitious bud

D: culturing adventitious bud obtained from C under DKW +0.01mg/L NAA +0.5mg/L GA3+0.03mg/L TDZ +20g/L sucrose +7.47g/L agar plant gel for 4 weeks

FIG. 2 shows that Acer nikoense maxim adventitious bud is cultured under the conditions of 1/2DKW, 0.1% active carbon, 0.5mg/L IBA, 1.0mg/L IAA, 20g/L cane sugar and 7.47g/L agar plant gel for 3 weeks to generate root (A), the culture is continued for 1 week, the root continues to grow, and a complete tissue culture seedling (B) is formed

FIG. 3 soil transplantation of Acer nikoense Maxim

A. B, C transplanting the rooting tissue culture seedlings in soil after continuously culturing for 35-42 days

Detailed Description

Example 1 Germination and proliferation of dormant Acer nikoense shoots

Selecting branches with buds of Acer nikoense in a forest botanical garden in Heilongjiang province in early spring, cutting the branches into stem sections with buds of about 3.0cm, washing the stem sections with running water for 10min, and then performing the following disinfection treatment in an ultra-clean workbench: soaking in 75% alcohol for 5min, soaking in 5% sodium hypochlorite for 12min, washing with sterile water for 5 times, and air drying on sterile filter paper. Removing bud scales from the disinfected buds in a super clean bench, cutting bud strains under the following culture conditions: contains 20g/L sucrose and 7.47g/L agar plant gel, and different plant growth regulators (BA, NAA, GA)₃KT, TDZ, IBA, ZT) in DKW medium and incubated under light. It was found that the dormant buds started to germinate at 1 week, adventitious buds started to grow within 5 weeks after inoculation, and adventitious buds had differentiated and grown at 6 weeks, and the statistical results are shown in Table 1. The results showed that DKW medium +0.03mg/L TDZ +0.01mg/L IBA +0.5mg/L GA₃Under these conditions, the differentiation of adventitious buds was the best, the adventitious bud induction rate was 61.5%, and 7.5 buds were proliferated on average per bud.

TABLE 1 Effect of plant growth regulators on the Induction and propagation of adventitious buds

Note: data were analyzed for multiple comparisons using SPSS software. The same letters in the same column data are not obviously different, P is less than 0.05, and the same is as below.

EXAMPLE 2 multiplication of Acer nikoense adventitious bud

The adventitious bud obtained in example 1 was cut into a length of 2.0cm, and inoculated under the following conditions: contains 20g/L sugar and 7.47g/L agar plant gel, and plant growth regulator (NAA, GA)₃ZT, BA, TDZ) and cultured under light. The proliferation of adventitious buds after 4 weeks of culture, and the statistics of the data are shown in Table 2. The results show that: in the culture condition of DKW, 0.03mg/L TDZ, 0.01 mg/L NAA and 0.5mg/L GA₃Next, the adventitious bud induction rate was the highest, the number of induced adventitious buds was large, 88.9% and 7.5, respectively, and the seedling growth state was the best, and this condition was finally determined to be the optimum adventitious bud multiplication culture condition.

TABLE 2 proliferation of adventitious buds by plant growth regulators

EXAMPLE 3 adventitious bud rooting

Tender adventitious buds obtained in example 2 are cut and 2.0cm long, and inoculated into 1/2DKW culture medium containing IAA, IBA, NAA, 20g/L sucrose, 0.1% active carbon and 7.47g/L agar for inducing rooting to form a regeneration whole plant. After 3 weeks of culture, the statistical test results show that the rooting rate is higher, 50% and the average rooting number is 4 in the table 3, 1/2DKW, 0.1% of activated carbon, 0.5mg/L IBA and 1.0mg/L IAA.

TABLE 3 Effect of auxin type plant growth regulators on rooting

Note: "-" indicates no root generation

Example 4 soil transplantation of regenerated plants

The regenerated plants formed in example 3 can be transplanted in soil after being cultured for 35-42 days. In order to increase the survival rate of the transplanted seedlings and reduce the germ infection, high-temperature soil sterilization is required before transplantation. The soil components are humus, vermiculite and perlite, and the proportion of the humus, the vermiculite and the perlite is 5: 3: 2. In the early stage of transplantation survival, agents such as carbendazim and the like are sprayed on the surface of the soil periodically every week to prevent infection of bacteria, fungi and the like, and the seedlings can be released once a month after being grown completely.

Claims

1. The asexual propagation method through the acer nikoense dormant buds is characterized by comprising the following steps of:

step one, culturing conditions for germination and proliferation of the acer nikoense dormant bud:

selecting acer nikoense branches with buds in a dormant period in early spring, cutting the branches into 3.0cm stem sections with buds, washing the stem sections with running water for 10min, and then performing the following disinfection treatment in an ultra-clean workbench: soaking in 75% alcohol for 5min, soaking in 5% sodium hypochlorite for 12min, washing with sterile water for 5 times, and air drying on sterile filter paper; the bud scales on the buds of the disinfected stem sections with the buds are stripped, and the cut buds are inoculated in the following culture conditions: DKW culture medium +0.03mg/L TDZ +0.01mg/L IBA +0.5mg/L GA ₃+20g/L sucrose +7.47g/L agar plant gel, light cultured; after 1 week of inoculation, the dormant buds start to germinate, adventitious buds start to generate within 5 weeks, and the adventitious buds grow in a differentiated manner within 6 weeks;

step two, propagation culture conditions of the acer nikoense adventitious bud are as follows:

the adventitious bud generated in the first step was cut into a length of 2.0cm and inoculated under the following conditions: DKW medium +0.03mg/L TDZ +0.01mg/L IBA +0.5mg/L GA₃+20g/L sucrose +7.47g/L agar plant gel to induce differentiation and proliferation of adventitious buds;

step three, culturing conditions of adventitious bud rooting:

cutting the adventitious bud obtained in the step two, wherein the length of the adventitious bud is 2.0cm, and inoculating and culturing the bud under the conditions that: 1/2DKW culture medium, 0.1% active carbon, 0.5mg/L IBA, 1.0mg/L IAA, 20g/L cane sugar and 7.47g/L agar plant gel, and performing light culture to induce adventitious buds to root and form a regeneration plant;

step four, transplanting the regenerated plants in soil:

continuously culturing the regenerated plants formed in the third step for 35-42 days, and then transplanting soil; performing high-temperature sterilization on soil before transplanting; the soil components comprise humus soil, vermiculite and perlite, and the ratio of the humus soil to the vermiculite to the perlite is 5:3: 2; in the early stage of transplantation survival, spraying carbendazim on the soil surface periodically every week to prevent bacterial and fungal infection, and slowing down to once a month after seedlings grow completely.