阅读说明：本技术 一种美国红枫组织培养种苗繁育方法 (Tissue culture seedling breeding method for acer rubrum ) 是由 胡春宏 季翔 常苹 王婷婷 于 2019-11-22 设计创作，主要内容包括：本发明公开了一种美国红枫组织培养种苗繁育方法,属于木本植物的组织培养技术领域,旨在提供一种实现种苗繁育工作的周年化、批次化、规范化的美国红枫组织培养种苗繁育方法,其技术方案要点是具体步骤如下：S1：外植体的选择；S2：外植体消毒；S3：启动培养；S4：增殖培育：S5：壮苗培育：S6：生根培育,其中,在S3中,将S2中消毒的外植体接入启动培养基中培养。本方法通过组织培养以芽繁芽的技术途径进行种苗快速繁殖,在培养过程中避免产生愈伤组织,避免了性状分离和变异,保持了母本的优良特性,种苗性状稳定。(The invention discloses a tissue culture seedling breeding method of American red maple, belongs to the technical field of tissue culture of woody plants, and aims to provide an annual, batched and standardized tissue culture seedling breeding method of American red maple, which realizes the seedling breeding work, and has the technical scheme key points that the method comprises the following specific steps: s1: selecting an explant; s2: sterilizing explants; s3: starting culture; s4: proliferation and cultivation: s5: strong seedling cultivation: s6: rooting culture, wherein in S3, the explants sterilized in S2 are inoculated into a starting medium for culture. The method carries out rapid propagation of the seedlings by tissue culture and a bud propagation technical way, avoids generating callus in the culture process, avoids character separation and variation, maintains the excellent characteristics of the female parent and has stable seedling characters.)

1. A tissue culture seedling breeding method for Acer rubrum is characterized by comprising the following steps: the method comprises the following specific steps:

s1: selecting an explant;

s2: sterilizing explants;

s3: starting culture;

s4: proliferation and cultivation:

s5: strong seedling cultivation:

s6: rooting and cultivating the seeds,

in S3, the explant disinfected in S2 is inoculated into a start-up culture medium for culture, wherein the start-up culture medium comprises MS + TDZ 0.01-0.05 mg/L + BA 0.1-0.2 mg/L + sucrose 30g/L + agar 6.8g/L, and the pH value of the start-up culture medium is 5.80.

2. The tissue culture seedling breeding method for the acer rubrum as claimed in claim 1, wherein the tissue culture seedling breeding method comprises the following steps: in S3, the explant is inoculated into a start culture medium and placed into a culture room for dark culture for 48h, then the dark culture is carried out for 16h and 8h in the dark with the light intensity of 2000-3000 lux at the room temperature of 23-26 ℃ every day, and the culture is carried out for 3-4 weeks.

3. The tissue culture seedling breeding method for the acer rubrum as claimed in claim 1, wherein the tissue culture seedling breeding method comprises the following steps: in S4, cutting the axillary bud stem of the aseptic seedling cultured and germinated in S3, and inoculating the cut axillary bud stem in a proliferation culture medium, wherein the proliferation culture medium is MS + TDZ 0.01-0.03 mg/L + BA 0.05-0.1 mg/L + GA₃1.0-2.0 mg/L, 30g/L of sucrose and 6.8g/L of agar, and the pH value of the propagation medium is 5.80.

4. The tissue culture seedling breeding method for the acer rubrum as claimed in claim 3, wherein: in S4, inoculating the axillary bud stem segments of the aseptic seedlings into a proliferation culture medium, culturing in a dark culture room for 48 hours, then illuminating for 16 hours and 8 hours in the dark every day, wherein the culture temperature is 23-26 ℃, the light intensity is 2000-3000 lux, and culturing for 4-6 weeks.

5. The tissue culture seedling breeding method for the acer rubrum as claimed in claim 1, wherein the tissue culture seedling breeding method comprises the following steps: in S5, the cluster buds induced in S4 are divided into single buds, cut off and inoculated in a strong seedling culture medium, wherein the strong seedling culture medium is MS + sucrose 30g/L + agar 6.8g/L, and the pH of the strong seedling culture medium is 5.80.

6. The tissue culture seedling breeding method for the acer rubrum as claimed in claim 5, wherein: in S5, dividing multiple buds into single buds, cutting, inoculating the single buds in a strong seedling culture medium, culturing in a dark environment for 48h in a culture room, then illuminating for 16h and 8h in the dark every day, wherein the culture temperature is 23-26 ℃, the light intensity is 2000-3000 lux, and culturing for 4-6 weeks.

7. The tissue culture seedling breeding method for the acer rubrum as claimed in claim 1, wherein the tissue culture seedling breeding method comprises the following steps: in S6, cutting off robust terminal buds after strong seedlings in S5, inoculating the cut terminal buds into a rooting culture medium for rooting induction, wherein the rooting culture medium is 1/2MS + IBA 0.8-1.0 mg/L + active carbon 0.2-0.5 g/L + sucrose 15g/L + agar 6.8g/L, and the pH value of the rooting culture medium is 5.80.

8. The tissue culture seedling breeding method for the acer rubrum as claimed in claim 7, wherein: in S6, cutting off strong terminal buds after seedlings, inoculating the terminal buds in a rooting culture medium, performing dark culture in a culture room for 48 hours, then performing illumination for 16 hours and 8 hours each day, performing dark culture at a light intensity of 2000-3000 lux and a culture temperature of 23-26 ℃, and performing culture for 3-4 weeks.

Technical Field

The invention relates to the technical field of tissue culture of woody plants, in particular to a seedling breeding method for tissue culture of American red maple.

Background

The traditional American red maple propagation method generally adopts seed sowing, cuttage and other modes to carry out seedling propagation. The seeding and seedling raising can be limited by the seed source and the seedling raising time, the seed germination rate can be limited by various factors and is not stable enough, and the method has serious character separation and is difficult to obtain excellent seedlings with consistent characters. The cuttage seedling raising is limited by seasons, only can be carried out for 5-9 months in a year, the root system does not grow well, and the survival rate of branches is low; in addition, a large number of branches need to be cut from the seedlings in the cutting seedling raising process, the greening shape of the seedlings can be affected, and the cut seedlings can not be sold and lose commercial value directly.

At present, the following three methods for reproducing the acer rubrum are mainly adopted:

1. seed propagation: the seeds are provided with wings after being mature, the seeds are easy to fly and disperse, the seeds are preferably harvested and planted at the same time, the maturity of the seeds is low, full seeds need to be selected before sowing to be soaked in warm water for accelerating germination, the seed density, the reasonable row spacing and the covering soil thickness during sowing are all limited by soil and climate in different areas, and the field management of water fertilizer, plant diseases and insect pests, weeds and the like is enhanced in the seedling emergence period, the seedling stage and the growth period after sowing.

2. Cutting and seedling raising: cutting half lignified tender branch cuttings into cuttings with the length of 15cm in 5-9 months per year, keeping the upper half of branches and leaves, removing all the other branches and leaves, dipping prepared rooting powder or rooting liquid at a lower end cut, inserting the cuttings into a prepared cutting bed, managing the temperature and humidity of the cutting bed, and transplanting after the cuttings take roots.

3. Tissue culture:

the prior literature reports that the tissue culture seedling raising mode of the American red maple is mostly carried out by adopting the modes of direct organ regeneration and explant dedifferentiation and redifferentiation. The direct organ regeneration is to sterilize the explant, inoculate the sterilized explant to a bud starting culture medium for bud culture, and perform a series of processes of proliferation culture, root induction and the like on the bud to finally obtain healthy seedlings. The explant dedifferentiation is to sterilize the collected explant, inoculate the sterilized explant to a callus induction culture medium to perform dedifferentiation induction on the callus, redifferentiate adventitious buds of the callus, and perform a series of culture such as proliferation and rooting to obtain an aseptic plant. No relevant report exists in the national literature of tissue culture of American red maple KW 159.

However, the three methods for breeding the acer rubrum have the following problems:

1. the conventional seed propagation speed is too low, a series of implementation steps with long periods such as seed collection and selection, storage, germination acceleration and sowing, seedling management and the like are required, and meanwhile, the seeding time limit is short due to the limitation of seasons; and the survival rate of the seeds is low, so that the propagation coefficient is low; in seed propagation, the probability of occurrence of trait segregation is high, and it is difficult to obtain a batch of seedlings with consistent traits.

2. In the cutting seedling process, the cutting shoot is difficult to root, the root system is not robust enough, the cutting time is short, and the influence on the root rate caused by the matching variety of the cutting medium and the control of the cutting microenvironment is large; a large number of branches are required to be cut for cutting seedling raising, and the tree vigor is seriously influenced; in addition, the plant viruses can be gradually accumulated along with the cutting seedling raising, and the character degradation is obvious along with the increase of cutting propagation generation numbers.

3, the tissue culture seedling raising technology of KW159 is not yet researched, developed and reported.

Disclosure of Invention

The invention aims to provide a tissue culture seedling breeding method for American red maple, which is based on large-scale commercial production in seedling factories and has the advantages of uniform batch, good repeatability, simple operation and high production efficiency.

The technical purpose of the invention is realized by the following technical scheme:

a tissue culture seedling breeding method for Acer rubrum comprises the following specific steps:

s1: selecting an explant;

s2: sterilizing explants;

s3: starting culture;

s4: proliferation and cultivation:

s5: strong seedling cultivation:

s6: rooting and cultivating the seeds,

in S3, the explant disinfected in S2 is inoculated into a start-up culture medium for culture, wherein the start-up culture medium comprises MS + TDZ 0.01-0.05 mg/L + BA 0.1-0.2 mg/L + sucrose 30g/L + agar 6.8g/L, and the pH value of the start-up culture medium is 5.80.

By adopting the technical scheme, the seedling is rapidly propagated by a bud propagation technical approach, callus is prevented from being generated in the culture process, character separation and variation are avoided, the excellent characteristics of the female parent are maintained, and the seedling character is stable. The starting culture medium can quickly break the dormancy of the axillary buds, promote the germination of the axillary buds and effectively avoid the generation of callus.

Further, in S3, the explant is inoculated to a start culture medium and then placed in a culture room for dark culture for 48 hours, and then the explant is turned to 16 hours of illumination time per day, 8 hours of darkness, 2000-3000 lux of light intensity, 23-26 ℃ of room temperature and cultured for 3-4 weeks.

By adopting the technical scheme, the condition of culturing for 3-4 weeks can effectively avoid the stress reaction of the plant material in the starting culture stage, and the plant material can quickly adapt to the starting culture medium, so that the plant material can quickly grow and the bud bodies are robust.

Further, in S4, cutting the axillary bud stem of the aseptic seedling cultured and germinated in S3, and inoculating the cut axillary bud stem in a proliferation culture medium, wherein the proliferation culture medium is MS + TDZ 0.01-0.03 mg/L + BA 0.05-0.1 mg/L + GA₃1.0-2.0 mg/L, 30g/L of sucrose and 6.8g/L of agar, and the pH value of the propagation medium is 5.80.

By adopting the technical scheme, the formula can ensure that the axillary bud proliferation is manually controllable, the proliferation rate is 3-4 times, and the bud is strong and has good repeatability.

Further, in S4, after the axillary bud stem segments of the aseptic seedlings are inoculated in a proliferation culture medium, dark culture is carried out in a culture room for 48 hours, then the aseptic seedlings are irradiated for 16 hours and dark for 8 hours every day, the culture temperature is 23-26 ℃, the light intensity is 2000-3000 lux, and the culture is carried out for 4-6 weeks.

By adopting the technical scheme, the condition of culturing for 4-6 weeks can effectively avoid the stress reaction of the plant material in the propagation culture stage, and the plant material can quickly adapt to the starting culture medium to promote the rapid growth of the plant material and the robust bud body.

Further, in S5, the multiple shoots induced in S4 were cut into single shoots and inoculated in a strong shoot medium of MS + sucrose 30g/L + agar 6.8g/L, pH 5.80.

By adopting the technical scheme, the formula can ensure that the cluster buds grow robustly and are beneficial to proliferation and rooting. In addition, the method of generating cluster buds by axillary buds avoids plant regeneration from callus cells, guarantees the character consistency of the produced seedlings while maintaining the excellent characters of the female parent, and greatly reduces the probability of character separation or variation.

Further, in S5, dividing multiple buds into single buds, cutting the single buds, inoculating the single buds into a strong seedling culture medium, culturing in a dark room for 48 hours, then illuminating for 16 hours and 8 hours in the dark every day, wherein the culture temperature is 23-26 ℃, the light intensity is 2000-3000 lux, and culturing for 4-6 weeks.

By adopting the technical scheme, the stress reaction of the plant material in the strong seedling culture stage can be effectively avoided under the condition of culturing for 4-6 weeks, the plant material can be quickly adapted to the starting culture medium, and the plant material is promoted to quickly grow and the bud is strong.

Further, in S6, cutting off robust terminal buds after strong seedlings in S5, inoculating the cut terminal buds into a rooting culture medium for rooting induction, wherein the rooting culture medium is 1/2MS + IBA 0.8-1.0 mg/L + activated carbon 0.2-0.5 g/L + sucrose 15g/L + agar 6.8g/L, and the pH value of the rooting culture medium is 5.80.

By adopting the technical scheme, the formula can promote the connection of the roots generated by the plants and the plant body structure, and the cultured roots are short and thick, so that the damage can be reduced and the transplanting survival rate can be improved during transplanting.

Further, in S6, cutting off strong terminal buds after seedlings, inoculating the terminal buds into a rooting culture medium, performing dark culture in a culture room for 48 hours, then performing light illumination for 16 hours and 8 hours in the dark every day, wherein the light intensity is 2000-3000 lux, the culture temperature is 23-26 ℃, and the culture is performed for 3-4 weeks.

By adopting the technical scheme, the condition of culturing for 3-4 weeks can effectively avoid the stress reaction of the plant material in the rooting culture stage, and the plant material can be quickly adapted to the starting culture medium, so that the plant material is promoted to quickly grow and the bud is robust.

In conclusion, the invention has the following beneficial effects:

1. the method carries out rapid propagation of the seedlings by a technical approach of bud propagation through tissue culture, avoids generating callus in the culture process, avoids character separation and variation, maintains the excellent characteristics of the female parent and has stable seedling characters.

2. According to the method, only a small amount of explant material is needed, and a large amount of branches do not need to be collected, so that the branch taking damage to finished nursery stocks is avoided; is not dependent on plant seeds, and can be produced according to the order requirement in batches and in a year.

3. The culture medium formula related in the application can keep the multiplication rate of 3-4 times, has good repeatability and stable properties, ensures healthy and strong cluster buds to be consistent in size, and meets the requirements of commercial production on seedling robustness, uniformity and repeatability.

Drawings

FIG. 1 is a flow chart of a tissue culture seedling breeding method of Acer rubrum.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.