CN115812605B

CN115812605B - Method for inducing adventitious buds of ash tree leaves and regenerating plants

Info

Publication number: CN115812605B
Application number: CN202310128494.0A
Authority: CN
Inventors: 王楠; 王军辉; 张守攻; 杨英英; 付鹏跃; 杨桂娟; 麻文俊; 赵鲲; 贠慧玲
Original assignee: CHINESE ACADEMY OF FORESTRY
Current assignee: CHINESE ACADEMY OF FORESTRY
Priority date: 2023-02-17
Filing date: 2023-02-17
Publication date: 2023-05-16
Anticipated expiration: 2043-02-17
Also published as: CN115812605A

Abstract

The invention provides a method for inducing adventitious buds of ash tree leaves and regenerating plants, and belongs to the technical field of plant tissue culture. The method comprises the steps of S1, collecting and sterilizing ash tree branch materials; s2, inoculating the strain into an induction culture medium for culture to obtain differentiated buds, carrying out multiplication culture to obtain adventitious buds, and carrying out rooting culture to obtain aseptic seedlings; s3, taking the leaves of the aseptic seedlings as explants, inoculating the explants into an induced callus culture medium, and performing primary induction to obtain primary callus; s4, transferring the primary callus to a differentiation induction culture medium to perform induction culture of adventitious buds and plant regeneration; s5, transplanting after hardening off seedlings. The method of the invention adopts ash tree aseptic seedling leaves as explants, and realizes regeneration technology through a callus indirect way, and the method can reduce the occurrence probability of chimeras after genetic transformation. The method solves the problem of difficult regeneration of the adventitious buds of the ash tree leaves, and the induction rate of the adventitious buds reaches more than 80 percent, which is far higher than the regeneration efficiency of the leaves reported in the prior art.

Description

Method for inducing adventitious buds of ash tree leaves and regenerating plants

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing adventitious buds of ash tree leaves and regenerating plants.

Background

The ash tree is a tall and large fallen tree of catalpa of Bignoniaceae, and is an excellent landscaping tree species and a precious furniture material. Because of the self incompatibility of the ash tree, the seed setting rate is low, the seed germination rate is also low, and the seeding and seedling raising are difficult. Therefore, the ash tree propagation adopts a root burying and bud destroying seedling raising method and a tender branch cutting seedling raising method, but the root burying seedling raising method has the problems of less seed roots and insufficient propagation materials, and is difficult to be planted and popularized in a large amount in actual production; the cutting seedling method has the bottleneck of low rooting rate and low survival rate.

The asexual propagation technology through plant tissue culture can realize rapid expansion of good varieties in a short period without being limited by seasons, and relieve the current situation of short supply and demand of ash trees in production. However, the current tissue culture of the ash tree is rapid and numerous, the stem segments with axillary buds are used as explants, but the genetic transformation technology developed by using the stem segments with axillary buds as the explants is unstable, and the probability of the obtained transgenic plant chimera is high; the stem without axillary buds is used as an explant, so that the problems of low induction rate, low differentiation efficiency, large workload of expanding propagation of sterile seedlings and the like exist; the existing direct regeneration report using leaves as explants has low regeneration efficiency of only 11.3%, and the double requirements of ash on production and scientific research are hardly met. According to the invention, the ash tree leaves are used as explants to establish a asexual propagation system, adventitious buds are regenerated through an induced callus way, so that a complete plant is formed, and the regeneration efficiency of ash tree is improved. In addition, in order to carry out genetic improvement on the target property of the ash tree, an efficient regeneration system of the ash tree leaves is required to be established, genetic transformation is realized through an agrobacterium-mediated method, and the establishment of the invention clearly provides technical support for preservation and propagation of excellent germplasm of the ash tree, and research on molecular mechanism and genetic transformation.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for inducing adventitious buds of ash tree leaves and regenerating plants.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a method for inducing adventitious buds of ash tree leaves and regenerating plants comprises the following steps:

s1, collecting and sterilizing ash tree branch materials;

s2, inoculating small segments of the sterilized ash tree branches with axillary buds into an induction culture medium for culture to obtain differentiated buds, inoculating the differentiated buds into a proliferation culture medium for proliferation culture to obtain adventitious buds, cutting the adventitious buds obtained by proliferation into stem segments with the axillary buds, and transferring the stem segments to a rooting culture medium for rooting culture to obtain aseptic seedlings;

s3, taking the leaves of the sterile seedlings as explants, inoculating the explants into a culture medium for inducing callus, and performing primary induction on the callus to obtain primary callus;

s4, transferring the primary callus to a differentiation induction culture medium to perform induction culture of adventitious buds and plant regeneration;

s5, placing the regenerated plants obtained by rooting culture of the adventitious buds in an artificial intelligence climate control room for hardening seedlings and transplanting.

In the method, preferably, in the step S1, the ash tree branch material is annual branches which are not germinated in the ash tree and are collected in 3-5 months each year, the germination is induced by water culture in a greenhouse, the leaves and the leaf stalks are removed when the germination length is 10-cm cm, and only the stem sections are left.

In the above method, preferably, in step S1, the sterilization treatment is performed by washing with tap water for 30min, immersing with 5% sodium hypochlorite for 5-6min, washing with sterile water, and performing secondary sterilization on the super clean bench. Soaking in 75% alcohol for 30s-45s, washing with sterile water for 4-5 times, sterilizing with 10% sodium hypochlorite for 5min, and washing with sterile water for 4-5 times.

In the method as described above, preferably, in step S2, the induction medium is a DKW minimal medium comprising 6BA at a concentration of 1.0mg/L, IBA at a concentration of 0.2mg/L, sucrose at a concentration of 30g/L and a plant gel at a concentration of 3g/L, and a medium having a pH of 5.8;

cutting the differentiated bud into stem segments with the concentration of 0.8-1.2cm for proliferation culture, wherein the proliferation culture medium is DKW basic culture medium which contains 6BA with the concentration of 1.0-2.0mg/L and plant gel with the concentration of 0.1-0.2mg/L IBA, sucrose with the concentration of 30g/L and plant gel with the concentration of 3g/L, and the pH value is 5.8;

the rooting culture medium is 1/2MS basic culture medium, which contains IBA with the concentration of 0.27mg/L, NAA with the concentration of 0.03mg/L, sucrose with the concentration of 30g/L and plant gel with the concentration of 3 g/L.

In the method described above, preferably, in step S3, a third leaf of the apical meristem of the sterile seedling is taken as an explant and 2-4 wounds are transected with a sterile scalpel perpendicular to the veins; explants were inoculated into the callus-inducing medium as a leaf back contact medium.

As described above, preferably, in step S3, the callus-inducing medium is a plant gel containing 6BA, NAA, sucrose and 3g/L at a concentration of 0.5-3.0mg/L, 0.01-0.2mg/L, and 3g/L in MS medium, and the medium is changed every 2 weeks.

Further, preferably, the callus-inducing medium is a plant gel containing 6BA, NAA, sucrose and 3g/L at a concentration of 0.5mg/L, 0.15mg/L, and 3g/L in MS medium, and having a pH of 5.9.

As described above, preferably, in step S4, the differentiation induction medium is a plant gel containing 6BA, NAA, 0.4mg/L ZT, 0.01-0.1 mg/L GA3, 30g/L sucrose and 3g/L plant gel at a concentration of 3.6-4.0mg/L in DKW minimal medium, and its pH is 6.0.

As described above, preferably, in step S4, the conditions for the induction culture of adventitious buds are light 1500-2000lx, 16h/d, and a temperature of 25.+ -. 2 ℃.

In the method described above, preferably, in step S4, the adventitious bud is transferred to rooting medium to culture to obtain complete regenerated plant, wherein the rooting medium is 1/2MS containing IBA with concentration of 0.27mg/L, NAA with concentration of 0.03mg/L, sucrose with concentration of 30g/L and plant gel with concentration of 3g/L, and the pH is 5.8.

In the method described above, preferably, in step S5, the transplanted substrate is peat soil: perlite: vermiculite = 4:1:1, the dosage of carbendazim is 200g/m ³ 。

The invention has the beneficial effects that:

the invention provides a method for inducing adventitious buds of ash tree leaves and regenerating plants, which adopts ash tree aseptic seedling leaves as explants and realizes regeneration technology through an indirect callus path. The method solves the problem of difficult regeneration of the adventitious buds of the ash tree leaves, and the induction rate of the adventitious buds reaches more than 80%, which is far higher than the regeneration efficiency of the leaves reported in the prior art. The method provides technical support for the research on genetic transformation, gene editing and gene functions of the ash tree.

Drawings

FIG. 1 shows callus induction with ash tree leaves as explants.

FIG. 2 shows adventitious bud induction.

FIG. 3 shows the rooting of adventitious buds.

FIG. 4 is a root plant transplant.

Detailed Description

The invention carries out orthogonal experimental design around basic culture medium types, growth regulator types, proportions and culture conditions, screens gradient conditions for efficient regeneration of ash tree leaves, establishes a matched in-vitro tissue culture efficient regeneration system for the excellent ash tree varieties, and lays a foundation for researching genetic transformation technology.

According to the invention, a large number of experimental researches show that in the differentiation stage, trace GA3 is added to promote the elongation of bud points, and the obtained regenerated seedlings are good in growth state and free from vitrification. The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

Example 1

s1, collecting and sterilizing materials: and collecting the annual branches of the ash tree which grow robustly and have no plant diseases and insect pests in Zhao Ji fields of the Luonang shoulder pole of Henan province in 3-5 months, and carrying out greenhouse water culture to accelerate germination. Collecting when the germination bud length is 10 cm-15cm after germination accelerating, removing leaves and petioles, and only leaving stem segments; washing with tap water for 30min, soaking with 5% sodium hypochlorite solution for 5-6min, washing with sterile water, and sterilizing in a super clean bench. Specifically, soaking in 75% alcohol for 30s-45s, washing with sterile water for 4-5 times, sterilizing with 10% sodium hypochlorite for 5min, washing with sterile water for 4-5 times, and sucking the surface water of the stem segment with sterile filter paper.

S2, aseptic seedling obtaining: cutting off the oxidized parts at the two ends of the stem segment by using sterile scissors, cutting the rest stem segment into small segments with axillary buds about 1.5 and cm, and inoculating into an induction culture medium, wherein the induction culture medium is DKW basic culture medium, 1.0 mg/L6BA (6-benzylaminopurine), 0.2mg/L IBA (indolebutyric acid), 30g/L sucrose and 3g/L plant gel are added into the induction culture medium, and the pH is 5.8; culturing for 40 days to obtain differentiation buds;

then cutting the differentiation bud into stem segments of about 1.0 cm, inoculating to a proliferation culture medium to form adventitious buds after culturing for 10 days, proliferating and differentiating a large number of adventitious buds after 35 days, adding 1.2 mg/L6BA, 0.2mg/L IBA, 30g/L sucrose and 3g/L plant gel into a DKW minimal medium, and controlling the pH to be 5.8.

Cutting the adventitious bud obtained by proliferation into stem segments with axillary buds, transferring to a rooting medium for rooting, adding 0.27mg/L IBA, 0.03mg/L NAA (naphthalene acetic acid), 30g/L sucrose and 3g/L plant gel into a 1/2MS basic medium, and obtaining the complete sterile tissue culture seedling about 30 days.

S3, leaf treatment and primary callus induction: selecting a third leaf of the ash tree sterile tissue culture Miao Dingduan meristem as an explant, and transversely cutting 2-4 wounds by using a sterile scalpel vertical vein; inoculating the explant into a callus induction culture medium in a mode of leaf back contact culture medium, performing primary induction of callus, wherein the callus induction culture medium is MS culture medium, 0.5-3.0 mg/L6BA, 0.01-0.2 mg/LNAA, 30g/L sucrose and 3g/L plant gel are added into the MS culture medium, the 16 callus induction culture mediums are prepared according to the specific preparation concentration of Table 1, the culture medium is changed once for 2 weeks, and the primary callus with the size of the bean grain is visible in dark culture for about 30 days. The results are shown in FIG. 1, wherein the numbers of the lower corners in the figures correspond to the numbers in Table 1, and the results show that the yellow-green calli induced by the culture mediums No. 4 and 15 are the most and the best, but the primary calli induced by the culture medium No. 4 are more prominent in the late-stage induced sprouting. Thus, the optimal induction callus medium was MS medium supplemented with 0.5 mg/L6BA, 0.15mg/LNAA, 30g/L sucrose and 3g/L plant gel, pH=5.9.

TABLE 1 Primary callus induction Medium hormone addition

S4, induction of adventitious buds and plant regeneration: the primary callus obtained in S3 is transferred to a differentiation medium for induction of adventitious buds. The culture conditions are illumination 1500-2000lx, illumination 16h/d, and temperature 25+ -2deg.C. The culture was performed on the adventitious bud differentiation medium in Table 2 to screen the adventitious bud optimal induction medium by an orthogonal test of 4-level 5 factors. Wherein the induction culture medium is added with 30g/L sucrose and 3g/L gel. The results showed that all of the 4 media G2, G8, G9 and G14 were able to differentiate into shoots, while others were unable to differentiate into shoots, but the calli in the media G2, G8 and G14 were gradually brown, and only the calli in the medium G9 were green and the differentiation efficiency was highest, as shown in FIG. 2. Wherein the differentiation efficiencies were 55% for the G2 medium, 50% for the G8 medium, 80% for the G9 medium, 60% for the G14 medium, and 0% for the other medium, respectively, and the differentiation efficiencies were calculated as differentiated bud callus pieces/total callus pieces. The specific culture condition of the G9 culture medium is that the light is 2000lx, the light is 16h/d, and the temperature is 25 ℃. The optimal induction culture medium for the adventitious buds is selected as DKW basic culture medium, 4.0 mg/L6 BA+0.15 mg/L NAA+0.4 mg/L ZT (trans-zeatin) +0.1 mg/LGA3 (gibberellin 3) is added, the pH is 6.0, and the induction rate of the adventitious buds under the condition reaches more than 80%. On the basis, the adventitious bud induction culture medium is finely adjusted, and the result shows that the adventitious buds can be induced by adding 3.6-4.0 mg/L6BA, 0.4mg/LZT,0-0.15mg/LNAA and 0.01-0.1 mg/LGA3 into the DKW basic culture medium, and the induction rate of the adventitious buds reaches more than 75%.

When the adventitious bud length reaches 1.0-1.5 cm, transferring the adventitious bud to a rooting medium, adding 0.27mg/L IBA and 0.03mg/L NAA and 30g/L sucrose and 3g/L plant gel into the rooting medium with the pH value of 5.8 in 1/2 MS. After 30 days, the complete regenerated plant is obtained, and the rooting rate is more than 90%. As shown in FIG. 3, the rooting rate is calculated according to the number of the planted roots/total number of transferred roots.

TABLE 2 orthogonal test design of minimal Medium, hormone addition and differentiation efficiency for induction of adventitious bud

S5, hardening seedlings and transplanting: the regenerated plants obtained by rooting and culturing the adventitious buds are placed in an artificial intelligent climate control room for hardening and transplanting, and the transplanting tray is a transplanting tray with a knob and a breathable cover, as shown in figure 4. The transplanting matrix is peat soil, perlite: vermiculite = 4:1:1, the dosage of carbendazim is 200g/m ³ . After transplanting, watering thoroughly, covering the cover for 7 days, opening the rotatable button for ventilation, slightly opening the cover after 3 days, and completely removing the cover after transplanting for 20 days. The climate chamber control conditions are: 22-25 ℃, the humidity is 50 percent, and the illumination time is 12 h/d. The transplanting survival rate is more than 98%.

Compared with the prior art, the method for inducing adventitious buds and regenerating plants of the ash tree leaves uses the ash tree aseptic seedling leaves of the ash tree as explants, so that more test materials are easier to obtain than the stem sections with axillary buds as the explants, and manpower and material resources are saved; and only about 60 days are needed from the callus induction to the adventitious bud differentiation, the adventitious bud induction rate reaches more than 80%, and the leaf regeneration efficiency is far higher than that reported; in addition, the generation probability of chimeras after genetic transformation can be reduced by the technology of realizing regeneration through an indirect way of inducing callus by leaves. The method solves the problem of difficult regeneration of the adventitious buds of the ash tree leaves of the ash tree, and provides technical support for research on genetic transformation, gene editing and gene functions of the ash tree leaves of the ash tree.

Claims

1. The method for inducing adventitious buds of ash tree leaves and regenerating plants is characterized by comprising the following steps:

s1, collecting and sterilizing ash tree branch materials;

s5, placing the regenerated plants obtained by rooting culture of the adventitious buds in an artificial intelligence climate control room for hardening seedlings and transplanting;

in the step S1, the ash tree branch material is annual branches which are not germinated of ash tree in 3-5 months of each year, the annual branches are collected in a greenhouse for water culture germination accelerating, the leaves and the petioles are removed when the germination bud length is 10 cm-15cm, and only the stem segments are left;

in the step S3, the culture medium for inducing the callus is MS culture medium which contains 6BA with the concentration of 0.5-3.0mg/L, NAA with the concentration of 0.01-0.2mg/L, sucrose with the concentration of 30g/L and plant gel with the concentration of 3g/L, the pH is 5.9, and the culture medium is changed every 2 weeks;

in step S4, the differentiation induction medium is a plant gel containing 6BA, NAA, 0.4mg/L ZT, 0.01-0.1 mg/L GA3, 30g/L sucrose and 3g/L plant gel with the pH of 6.0, wherein the concentration of the 6BA, the NAA, the ZT and the GA is 3.6-4.0mg/L, and the pH is 0-0.

2. The method according to claim 1, wherein in step S1, the sterilization treatment is a secondary sterilization in an ultra clean bench after washing with tap water for 30min, immersing with 5% sodium hypochlorite for 5-6min, and washing with sterile water; soaking in 75% alcohol for 30s-45s, washing with sterile water for 4-5 times, sterilizing with 10% sodium hypochlorite for 5min, and washing with sterile water for 4-5 times.

3. The method according to claim 1, wherein in step S2, the induction medium is a medium having a pH of 5.8, comprising 6BA at a concentration of 1.0mg/L, IBA at a concentration of 0.2mg/L, sucrose at a concentration of 30g/L, and plant gel at a concentration of 3g/L in DKW minimal medium;

cutting the differentiated bud into stem segments of 0.8-1.2cm for proliferation culture, wherein the proliferation culture medium is DKW basic culture medium containing 6BA with concentration of 1.0-2.0mg/L, IBA with concentration of 0.1-0.2mg/L, sucrose with concentration of 30g/L and plant gel with pH of 3g/L, and the pH is 5.8;

the rooting medium is 1/2MS basic medium, and contains IBA with the concentration of 0.27mg/L, NAA with the concentration of 0.03mg/L, sucrose with the concentration of 30g/L and plant gel with the concentration of 3 g/L.

4. The method of claim 1, wherein in step S3, a third leaf of the apical meristem of the sterile seedling is taken as an explant and 2-4 wounds are transected with a sterile scalpel perpendicular to the veins; explants were inoculated into the callus-inducing medium as a leaf back contact medium.

5. The method according to claim 1, wherein in step S4, the conditions for the induction culture of adventitious buds are 1500-2000lx under light, 16h/d under light, and a temperature of 25.+ -. 2 ℃.

6. The method according to claim 1, wherein in step S4, adventitious buds are transferred to rooting medium for cultivation to obtain complete regenerated plants, wherein the rooting medium is a plant gel containing IBA with concentration of 0.27mg/L and NAA with concentration of 0.03mg/L, sucrose with concentration of 30g/L and concentration of 3g/L in 1/2MS, and the pH is 5.8.

7. The method according to claim 1, wherein in step S5, the transplanted substrate is peat soil: perlite: vermiculite = 4:1:1, the dosage of carbendazim is 200g/m ³ 。