CN112514796B

CN112514796B - Tissue culture rapid propagation method of new variety 'Xueli' of vertical branch type chionanthus retusus

Info

Publication number: CN112514796B
Application number: CN202011516848.1A
Authority: CN
Inventors: 李际红; 邢世岩; 刘会香; 周文玲; 王如月; 郭海丽; 王锦楠; 刘佳庚; 侯丽丽
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2022-04-01
Anticipated expiration: 2040-12-21
Also published as: CN112514796A

Abstract

The invention discloses a tissue culture rapid propagation method of a new variety 'Xueli' of a vertical-branch type tassel tree, which comprises the following steps: (1) cutting young stem segments with the length of 2-2.5cm from the 'Xueli' stock plant as a test material, inoculating the test material into a culture medium for inducing stem segment differentiation, and inducing adventitious bud differentiation and proliferation; (2) transferring the test material to a culture medium for inducing stem section to elongate when 4-5 new leaves appear in the test material in the step (1) and the test material is differentiated to form 1-2 adventitious buds; (3) when the stem section of the test material in the step (2) is extended to 3-5cm, transferring the stem section into a rooting culture medium, and inducing adventitious roots; (4) hardening and transplanting the rooted seedlings. The method of the invention is used for breeding the snow, and the new variety of the snow can be popularized in a short time, so that the method can be quickly applied to greening practice. And provides a theoretical basis for establishing a rapid propagation system with stable and efficient chionanthus retusus character and subsequent genetic transformation experiments.

Description

Tissue culture rapid propagation method of new variety 'Xueli' of vertical branch type chionanthus retusus

Technical Field

The invention relates to the technical field of plant cell engineering, in particular to a tissue culture rapid propagation method of a new variety 'Xueli' of a vertical-branch type chionanthus retusus tree.

Background

The Chinese fringetree (Chionanthus retusus Lindl. et Paxt.) belongs to the genus Chionanthus (Oleaceae) and is deciduous tree or shrub, and the height of the adult tree can reach 20 m. The bark of the sapling is mostly light yellow, the branch is cylindrical, grayish brown, without fur, the branch is light yellow or brown, and the branch is sparse in quilt or dense in quilt and soft in hair. Adult trees have a grayish brown bark, longitudinal cracks and obvious ridges. The blade is single-leaf-opposite, nearly leather, oval or long round, slightly curled at the blade edge, full-edge or with small saw teeth, blunt or sharp at the blade end, wedge-shaped at the blade base or heart-shaped. The conical polyumbellate inflorescence grows at the top end of a lateral branch, is a unisexual male and female heterostrain or an amphoteric flower, has white corolla and deep crack, and has a light yellow color with an anther in an egg shape; the pedicel has no fur and is fine; ovaries are oval, stigmatic spherical, 2 fissures, and flowering phase is 4-5 months. The fruit is oval and blue black or black, and the fruit period is 9-10 months.

The Chinese fringetree is a national second-grade rare plant and has high edible, medicinal, ornamental and economic values. Meanwhile, the composite material has the characteristics of strong salt resistance, drought resistance and cold resistance, and can adapt to complex north and south climates.

'Xueli' is a new tassel species for tassel which is a new tassel species for tassel with drooping branches is screened out on the basis of the investigation and research of long-term tassel germplasm resources, compared with the common tassel, the new tassel species for 1-2 years is slender, soft and drooping, a large number of mature short branches are formed on the drooping branches, the leaves of the tassel species are slender and are in a scaly needle shape compared with the common tassel, the leaf stalks are purple, and the base parts of the leaf stalks are dark purple; compared with common tassels, 'snow' has higher ornamental value.

The conventional propagation modes of the Chinese fringetree mainly comprise sowing, cuttage, grafting and the like. The three propagation modes are greatly influenced by seasons, and the growth conditions are not suitable to be controlled, so that seedlings with uniform growth states are difficult to propagate in a short time. The tissue culture and rapid propagation technology of the stem segments of the Chinese fringetree can effectively solve the problems. 'Xueli' is a new variety of newly cultivated tassel tree with a vertical branch type, and no research and report on tissue culture regeneration of the tassel tree is found at present.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide a tissue culture rapid propagation method of a new variety 'Xueli' of a vertical-branch type tassel tree. The tissue culture rapid propagation method can be adopted to rapidly propagate in a short time, so that the tissue culture rapid propagation method can be applied to greening practice as soon as possible. And provides a theoretical basis for establishing a rapid propagation system with stable and efficient chionanthus retusus character and subsequent genetic transformation experiments. In order to achieve the purpose, the invention adopts the following technical scheme:

the tissue culture and rapid propagation method of the new variety 'Xueli' of the tassel tree comprises the following steps:

(1) cutting semi-lignified young shoots from the Xueli parent plant, cutting stem segments with terminal buds or lateral buds of 2-2.5cm in length as test materials, inoculating the test materials into a culture medium for inducing stem segment differentiation through aseptic treatment, and inducing adventitious buds to differentiate and proliferate;

the culture medium for inducing stem differentiation is a WPM culture medium added with 0.1-1.0mg/L KT, 1.0-2.0 mg/L6-BA, 30g/L sucrose and 6g/L agar;

(2) when 4-5 new leaves appear in the test material in the step (1) and 1-2 new buds develop, transferring the test material into a culture medium for inducing stem elongation to promote the stem elongation;

the culture medium for inducing stem elongation is added with 0.5mg/L KT, 1.5 mg/L6-BA and 0.5-4.0mg/L GA₃30g/L sucrose and 6g/L agar;

(3) when the stem section of the test material in the step (2) is extended to 3-5cm, transferring the stem section into a rooting culture medium, and inducing adventitious roots;

the rooting culture medium is added with 0.1-1.5mg/L IBA, 0.1-1.5mg/L NAA and 2.0mg/L GA₃30g/L sucrose and 6g/L agar;

(4) and (4) hardening and transplanting the rooted seedlings obtained in the step (3).

Preferably, in the step (1), the sterile treatment is 75% alcohol treatment for 30s, and 0.1% HgCl treatment for 6-8 min.

Preferably, in step (1), the culture conditions for inducing the adventitious bud to differentiate and proliferate are as follows: the culture temperature is 24 +/-2 ℃, the illumination intensity is 2500-.

Preferably, in step (2), the culture conditions for causing the stem segment to elongate are: the culture temperature is 24 +/-2 ℃, the illumination intensity is 2500-.

Preferably, in step (3), the culture conditions for inducing adventitious root formation are: the culture temperature is 24 +/-2 ℃, the illumination intensity is 2500-.

Preferably, in the step (4), the substrate for hardening seedling cultivation is prepared from grass carbon, vermiculite and perlite according to the volume ratio of 3:2: 1.

The invention has the beneficial effects that:

the invention cuts semi-lignified shoots from the Xueli parent plant, cuts stem segments with the length of 2-2.5cm and with terminal buds or lateral buds as test materials, and establishes a regeneration rapid propagation technical system of the Xueli stem segments by inducing stem segment differentiation and proliferation, stem segment elongation and stem segment adventitious roots through aseptic treatment, thereby providing a referable technology and method for tissue culture rapid propagation of the Chionanthus retusus.

The method of the invention has the following advantages: the method has the advantages of easily obtained materials, low mutation rate and high multiplication factor, the number of newly expanded leaves cultured for 35d is 5.76, the multiplication coefficient is 3.13, the stem section of the tissue culture seedling extends by 3.01cm, the rooting rate of the tissue culture seedling reaches 62.79 percent, and the survival rate of the acclimatized seedling reaches 92 percent. The method for breeding the Xueli can shorten the breeding period, improve the breeding efficiency and provide referable technology and method for tissue culture and rapid propagation of the Chinese fringetree.

Drawings

FIG. 1: 'Xueli' mother plant whole plant photo

FIG. 2: the WPM is used as a basic culture medium, and the promotion condition (A) of KT (0.5mg/L) and 6-BA (1.5mg/L) for the differentiation and proliferation of the stem section of the perilla frutescens and the promotion condition (B) of the new leaf are added.

FIG. 3: gibberellin with different concentrations induces the stem section of the Chinese fringetree to extend; in the figure, A represents GA₃At 1mg/L, stem sections were inoculated, B represents GA₃Stem elongation at 1mg/L, at 15 days of inoculation; c represents GA₃At 2mg/L, stem sections were inoculated, D represents GA₃Stem elongation at 2mg/L, 15 days of inoculation; e represents GA₃At 3mg/L, stem sections were inoculated, F represents GA₃Stem elongation at 3mg/L, 15 days of inoculation; g represents GA₃4mg/L, stem sections were inoculated, H represents GA₃At 4mg/L, the stem elongation at 15d of inoculation.

FIG. 4: rooting adventitious buds of stem segments of the Chinese fringetree; in the figure, A shows that the number of main roots reached 7 after 35d of inoculation with the medium of group 8 of example 3; b shows the root growth condition of the tassel stem segments after 30d of rooting.

FIG. 5: and D, transplanting seedlings of the chionanthus retusus.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As introduced in the background section, 'Xueli' is a new variety of newly cultivated tassels of the vertical branch type, and no research and report on tissue culture regeneration of the tassels are available at present. For the regeneration process of the stem segments of the chionanthus retusus, different chionanthus retusus varieties have different culture conditions for proliferation and differentiation in the regeneration process, the combination of different hormone concentrations added in a culture medium also needs to be adjusted, and the optimal culture effect can be obtained by the optimal combination of various factors.

Based on the above, the invention aims to provide a tissue culture rapid propagation method of a new variety 'Xueli' of the tassel tree of the vertical branch type. 'snow' has a phenomenon of dormancy during growth, that is, the growth goes through dormancy for a short time, so that the extended growth of the chionanthus retusus tree is slow in natural conditions. In order to promote the elongation growth of the chionanthus retusus, the invention adopts the method that different exogenous hormone combinations are added to stimulate the adventitious buds of the chionanthus retusus stem to differentiate and proliferate, and gibberellin is added to promote the elongation growth of the stem on the basis of the optimal hormone combination after the obvious proliferation expression is obtained (namely 1-2 adventitious buds are formed during development).

The hormone formula for optimal differentiation, elongation and rooting of different varieties of the chionanthus retusus is different, and the optimal hormone types and hormone ratios need to be continuously researched by experiments to achieve the best effect. For a new variety 'Xueli' of tassels with weeping branches, firstly, the optimal proportion of differentiation hormones is researched to enable the new variety 'Xueli' of tassels with weeping branches to reach the optimal differentiation coefficient in the shortest time, on the basis, gibberellins with different concentrations are added, and the three hormones are matched for use, so that the elongation length of the tassels with the optimal differentiation coefficient in the short time is longest, and the concentrations of the three hormones for the optimal growth of the new variety 'Xueli' of tassels with weeping branches are researched and obtained. On the basis, the optimal rooting formula is researched by adding IBA and NAA with different concentrations so as to ensure that the optimal rooting condition is achieved under the condition that the elongation length is longest in a short time.

The culture medium hormone proportion determined by experimental optimization during the differentiation of the stem segments of the chikuri chionanthus is as follows: 0.1-1.0mg/L KT, 1.0-2.0 mg/L6-BA; the culture medium hormone proportion for inducing stem section elongation is as follows: 0.5mg/L KT, 1.5 mg/L6-BA, 0.5-4.0mg/L GA₃(ii) a The culture medium hormone proportion for inducing the stem section to root is as follows: 0.1-1.5mg/L IBA, 0.1-1.5mg/L NAA, 2.0mg/L GA₃。

The tissue culture and rapid propagation method of the 'sherry' stem segment provided by the invention comprises the following steps:

step 1, cutting semi-lignified young shoots from the Xueli parent plant, cutting stem segments with 2-2.5cm length and terminal buds as test materials, and performing disinfection treatment, namely treating with 75% alcohol for 30s and 0.1% HgCl₂Treating for 6-8min, and inoculating into WPM medium with KT concentration of 0.5mg/L and 6-BA concentration of 1.5mg/L, and culturing for 30-35d to induce adventitious bud differentiation and proliferation.

Step 2, when 4-5 new leaves appear in the test material in the step 1 and develop to form 1-2 adventitious buds, transferring the test material to a KT concentration of 0.5mg/L and a 6-BA concentration of 1.5mg/L, GA₃The stem section is promoted to elongate by culturing in WPM medium with concentration of 2.0mg/L for 15-20 d.

Step 3, when the stem section of the test material in the step 2 is extended to 3-5cm, transferring the stem section to IBA concentration of 1.0mg/L, NAA concentration of 0.5mg/L, GA₃In WPM medium at a concentration of 2.0mg/L, adventitious roots were induced.

And 4, selecting bottle seedlings with well-developed and robust roots, transferring the bottle seedlings out of a culture room, and placing the bottle seedlings in a laboratory environment condition to adapt to the external environment for 7 d. After the rooting seedlings of the chionanthus retusus are adapted to the external environment, the bottle seedlings are taken out from the culture bottle by using tweezers, and the root system is cleaned by using clear water to avoid the influence of the culture medium attached to the root system. The culture medium for hardening seedlings is grass peat, vermiculite and perlite in a proportion of 3:2:1, thoroughly watering with clear water, putting the bottle seedlings under a sunshade net to keep proper humidity, hardening the seedlings for one week, and transplanting the seedlings into a mixed matrix for culture.

By statistics, the tissue rapid propagation method is adopted, the number of newly-expanded leaves cultured for 35d is 5.76, the propagation coefficient is 3.13, the stem section of the tissue culture seedling is elongated by 3.01cm, the rooting rate of the tissue culture seedling reaches 62.79%, and the hardening-seedling survival rate reaches 92%.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available.

Example 1:

WPM is used as a basic culture medium, 30g/L of sucrose and 6g/L of agar are added, KT (0.1, 0.5, 1.0mg/L) and 6-BA (1.0, 1.5, 2.0mg/L) with different concentration gradients are added for designing a two-factor random block, and the experimental design is shown in Table 1. 30 flasks of each medium were inoculated with 2 explants per flask. The stem growth height, the number of newly grown leaves, and the growth status of adventitious buds were observed every week (growth factor: number of new shoots/number of inoculations). The culture conditions are as follows: the culture temperature is 24 ℃ plus or minus 2 ℃, the illumination intensity is 2500-.

Table 1: effect of different growth regulators on the differentiation of the ` Xueli ` Stem segments (35d observations)

After 35 days of culture, the growth regulator combination of the group 5 is observed to have the optimal effect of promoting the differentiation of the 'sherry' stem segments, the number of newly developed leaves can reach 5.76, and the multiplication times can reach 3.13 (Table 1 and figure 2).

Example 2:

adding 30g/L sucrose and 6g/L agar, 1.5 mg/L6-BA, 0.5mg/LKT by addition of different concentrations of GA₃(0.5-4mg/L) Single factor experiments were performed, the design of which is shown in Table 2. 30 flasks of each medium were inoculated with 2 stem segments from each flask after culture in the medium inducing stem segment differentiation. The stem section elongation height and seedling height are observed and recorded every week, and the culture conditions are as follows: the culture temperature is 24 ℃ plus or minus 2 ℃, the illumination intensity is 2500-.

After 15 days of culture, group 3 (i.e., 2.0mg/L GA addition)₃) The promotion effect on the elongation of the 'sherry' stem segment is optimal, and the tassel tree stem segment can grow by 3.01cm (table 2 and fig. 3).

Table 2: 'Xueli' stem GA₃One factor elongation test

Example 3:

WPM is used as a basic culture medium, 30g/L of sucrose and 6g/L of agar are added, IBA (0.1-1.5mg/L) and NAA (0.1-1.5mg/L) with different concentration gradients are added for designing a two-factor random block, and the experimental design is shown in Table 3. Observing and recording rooting performance every week, and culturing conditions are as follows: the culture temperature is 24 ℃ plus or minus 2 ℃, the illumination intensity is 2500-.

Table 3: effect of different plant growth regulators on 'snow' rooting

The observation shows that the growth regulator combination of the group 8 has the optimal effect on promoting rooting, the root primordium is formed after about 15 days of culture, the root length reaches 5.1cm after 35 days of culture, and the rooting rate reaches 62.79%.

Example 4:

the well-grown bottle seedlings in example 3 were transferred from the tissue culture room environment to the outside to adapt to the environment such as the outside illumination and temperature. After adaptation for 7d, the sterile seedlings were taken out of the culture flask, and the culture medium from which the roots were washed was transferred to the plug tray of the greenhouse. The culture medium is prepared by mixing the following components in a volume ratio of 3:2:1 grass carbon, vermiculite and perlite mixed. The cultivation substrate is sterilized with 50% wettable carbendazim with concentration of 0.1% before potting. The hardening-seedling survival rate (the hardening-seedling survival rate is the number of surviving plants in the nutrition pot/the number of transplanted test-tube seedlings) reaches 90 +/-5 percent; after the stem section grows for 15 days in the mixed matrix, new shoots grow, and the growth condition is good.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The tissue culture rapid propagation method of the new variety 'Xueli' of the tassel tree is characterized by comprising the following steps:

the culture medium for inducing the 'sherry' stem differentiation is a WPM culture medium only added with 0.5mg/L KT, 1.0-1.5 mg/L6-BA, 30g/L sucrose and 6g/L agar;

(2) when 4-5 new leaves appear in the test material in the step (1) and develop to form 1-2 adventitious buds, transferring the test material into a culture medium for inducing stem elongation to promote the stem elongation;

the culture medium for inducing the elongation of the 'Xueli' stem segment is only added with 0.5mg/L KT, 1.5 mg/L6-BA and 0.5-4.0mg/L GA₃30g/L sucrose and 6g/L agar;

(3) when the stem section in the step (2) is extended to 3-5cm, transferring the stem section into a rooting culture medium, and inducing adventitious roots;

the culture medium for inducing the 'Xueli' stem segment to root is only added with 1.0mg/L IBA, 0.5mg/L NAA and 2.0mg/L GA₃30g/L sugarcaneWPM medium of sugar and 6g/L agar;

(4) hardening and transplanting the rooted seedlings obtained in the step (3);

in the step (1), the culture conditions for inducing the differentiation and proliferation of the adventitious bud are as follows: the culture temperature is 24 +/-2 ℃, the illumination intensity is 2500-;

in the step (2), the culture conditions for promoting the stem section to elongate are as follows: the culture temperature is 24 +/-2 ℃, the illumination intensity is 2500-;

in the step (3), the culture conditions for inducing the generation of the adventitious roots are as follows: the culture temperature is 24 +/-2 ℃, the illumination intensity is 2500-;

in the step (4), the substrate for hardening seedling cultivation is prepared from grass peat, vermiculite and perlite according to the volume ratio of 3:2: 1.

2. The tissue culture rapid propagation method according to claim 1, wherein in the step (1), the sterile treatment is 75% alcohol treatment for 30s, and 0.1% HgCl₂Treating for 6-8 min.