CN111492982A - Method for inducing stem bud propagation of samara leaf callus - Google Patents

Abstract

The invention discloses a method for inducing stem bud propagation of calli of samara leaves, belonging to the field of plant tissue culture. The method comprises the following steps: collecting leaves on the branches as explants; disinfecting the leaves, and then cutting and inducing the leaves to form callus; adventitious bud induction culture; rooting culture; and (5) transplanting. The invention adopts plant tissue culture technology to establish a method for inducing stem bud propagation by using the leaf callus of the seedling of the samara fruits, the induction of the leaf callus reaches more than 73 percent, and the transplanting survival rate reaches more than 83 percent. The method has the characteristics of short period, low cost, high propagation coefficient, consistent seedling growth and the like, can be directly used for the industrial production of the seedlings of the samara, and has great significance for promoting the popularization and the planting of the samara.

Description

Method for inducing stem bud propagation of samara leaf callus

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a method for inducing stem bud propagation of calli of a samara blade.

Background

The samara, with the academic name of Burretriodendron esquiroliii (L evl.) rehd, is a gradually dangerous species, belongs to the national level II important protection of wild plants, is only scattered in secondary forests due to garbled deforestation, and the natural forests dominated by the samara are difficult to find.

The samara is in a natural forest, which is often the upper forest of a forest stand; the young trees in the forest have more in the thinning forest, and have higher stability when forming larger communities with other plants; in the Luodian sheep, the Alternaria peduncularis is often combined with the species of Carcinia Maultflora champ, Fraxinus chinensis, Larix, Melia azedarach, Emblica officinalis, Acer sinensis, Hangzhou tips, and Nicotiana pseudotabacum to form a forest stand.

The key fruit has not been subjected to a large number of introduction tests and seed seedling breeding methods. The fruits can be picked when the fruits turn from green to brown, and the fruits are dried in the shade in a ventilated and dry place, are not suitable for being exposed to the sun or piled, are suitable for being stored by sowing or wet sand, and the seeds completely lose the germination capacity in a short period when being stored for a plurality of times.

However, sexual reproduction has the problem of differentiation and does not maximize genetic improvement gain, while tissue culture ensures relative uniformity and genetic gain in the offspring.

At present, no related research of the samara tissue culture technology exists at home and abroad.

Disclosure of Invention

The invention aims to solve the problems in the prior art, and aims to provide a method for inducing stem bud propagation of the calli of the samara blade, which can realize large-scale production of the samara nursery stocks.

In order to realize the purpose of the invention, the method is realized by the following technical scheme:

a method for inducing stem bud propagation of a samara leaf callus comprises the following steps:

(1) explant collection: collecting leaves on the branches as explants;

(2) leaf callus induction culture: disinfecting the leaves, and then cutting and inducing the leaves to form callus;

(3) adventitious bud induction culture: culturing adventitious buds of the callus to obtain an adventitious bud cluster;

(4) rooting culture: carrying out rooting culture on the adventitious bud cluster to obtain a test-tube plantlet;

(5) transplanting: transplanting the test-tube plantlet to obtain the seedling.

Preferably, in step (1), leaves are collected as explants on annual stalk samara shoots.

PreferablyIn the step (2), the leaves are washed by water, soaked in ethanol and then treated with H₂O₂Disinfecting and washing with water; and finally, cutting the disinfected leaves, horizontally inoculating the leaves with upward leaves into a leaf callus induction culture medium, and culturing for at least 30 days.

Further preferably, the conditions of the leaf callus induction medium are:

N₆culture medium, 3.0 mg/L riboflavin, 0.5 mg/L PVP, 1.0 mg/L2, 4-D, 0.5 mg/L NAA, 0.5 mg/L activated carbon, 30 g/L sucrose, 7.0 g/L agar, pH 5.4-5.8.

Preferably, in the step (3), the callus is transplanted to the adventitious bud induction medium for at least 25 days after the part of the callus that is in contact with the leaf callus induction medium is excised.

Preferably, the conditions of the adventitious bud induction medium are:

N₆culture medium, 4.0 mg/L riboflavin, 0.5 mg/L PVP, 0.5 mg/L6-BA, 0.1 mg/L NAA, 0.5 mg/L activated carbon, 30 g/L sucrose, 7.0 g/L agar, and pH value is 5.4-5.8.

Preferably, in the step (4), the adventitious bud is inoculated into a rooting medium for culturing for at least 20 days after the adventitious bud is divided into individual adventitious buds.

Further preferably, the conditions of the rooting medium are:

1/2MS culture medium, 0.5 mg/L IBA, 1.0 mg/L NAA, 35 g/L sucrose, 4.0 g/L agar, pH 5.4-5.8.

Preferably, in the step (5), the test-tube plantlet is transplanted into a greenhouse to be hardened for at least 10 days, and then the culture medium of the washed root of the test-tube plantlet is transplanted into the mixed soil of the brick red soil and the humus soil matrix to be cultivated into the seedling, so that the stalk wing fruit seedling is obtained.

Preferably, in step (2), the culture conditions are: the temperature is 26-30 ℃, the illumination intensity is 2800-; humidity is 60% -70%;

in the step (3) and the step (4), the culture conditions are as follows: the temperature is 25-28 ℃, the illumination intensity is 2000-; the humidity is 60-70%.

The invention adopts plant tissue culture technology to establish a method for inducing stem bud propagation by using the leaf callus of the seedling of the samara fruits, the induction of the leaf callus reaches more than 73 percent, and the transplanting survival rate reaches more than 83 percent. The method has the characteristics of short period, low cost, high propagation coefficient, consistent seedling growth and the like, can be directly used for the industrial production of the seedlings of the samara, and has great significance for promoting the popularization and the planting of the samara.

Detailed Description

The following specific examples are provided to further illustrate the present invention so that those skilled in the art may better understand the invention and practice it, but the examples are not intended to limit the invention. Other various modifications, substitutions and alterations of the above-described structures of the present invention will occur to those skilled in the art without departing from the basic technical spirit of the invention as described herein.

As used herein, tween-20 is a nonionic surfactant, also known as Polysorbate-20 (Polysorbate-20); PVP refers to polyvinylpyrrolidone; 2,4-D is dichlorophenoxyacetic acid; NAA means naphthylacetic acid; 6-BA means 6-benzylaminopurine; IBA refers to indolebutyric acid; 1/2MS medium is MS medium with reduced content of 1/2.

Example 1

(1) Explant collection: collecting annual stem and wing fruit branches with good physiological state, no plant diseases and insect pests, diameter of 5-10mm and length of about 15cm by using scissors, and taking leaves from the collected branches as explants.

(2) Leaf callus induction culture: selecting tender and non-scar leaflets from the leaves collected from step (1) back to the laboratory, rinsing with tap water for 3 hours, sterilizing in 70% ethanol solution for 2 times in a clean bench for 20 seconds each time, and then sterilizing with 10% H₂O₂(2 drops of Tween-20 were added thereto) and sterilized by stirring 2 times for 3 minutes each, and finally rinsed 5-7 times with sterile water until the solution was rinsed clean. The disinfected stem finCutting fruit leaves into small pieces of 5mm × 5mm, horizontally inoculating the fruit leaves with the leaf surfaces facing upwards into a leaf callus induction culture medium, and culturing for 30 days to form callus by induction, wherein the induction rate reaches 81%.

The leaf callus induction culture medium comprises: n is a radical of₆Culture medium +3.0 mg/L riboflavin +0.5 mg/L PVP +1.0 mg/L2, 4-D +0.5 mg/L NAA +0.5 mg/L activated carbon +30 g/L sucrose +7.0 g/L agar, pH value is 5.4-5.8.

(3) And (3) adventitious bud induction culture, namely taking the leaf callus induced in the step (2) out of a culture bottle, cutting off tissues in contact with a culture medium by using a blade, cutting the tissue into small pieces with the size of 0.5cm × 0.5.5 cm, inoculating the small pieces into the adventitious bud induction culture medium, and culturing for 25 days to generate adventitious buds.

The adventitious bud induction culture medium comprises: n is a radical of₆Culture medium +4.0 mg/L riboflavin +0.5 mg/L PVP +0.5 mg/L6-BA +0.1 mg/L NAA +0.5 mg/L activated carbon +30 g/L sucrose +7.0 g/L agar, pH value is 5.4-5.8.

(4) Rooting culture: and (4) dividing the adventitious bud clump with the length of about 2.0-3.0cm obtained in the step (3) into single adventitious buds, inoculating the single adventitious buds into a rooting culture medium, and culturing for 20 days to realize the rooting of the test-tube plantlet.

The rooting culture medium is 1/2MS culture medium, 0.5 mg/L IBA, 1.0 mg/L NAA, 35 g/L sucrose and 4.0 g/L agar, and the pH value is 5.4-5.8.

(5) Transplanting: and (3) hardening the test-tube plantlets with good root growth in the step (4) for 10 days under natural light of a greenhouse, transplanting the culture medium with cleaned roots into a brick red soil and humus soil matrix with the volume ratio of 1:1 to cultivate seedlings to obtain the stalk wing fruit seedlings, and transplanting the seedlings for 15 days until the survival rate reaches over 90 percent.

In the tissue rapid propagation method of the samara, the conditions of the leaf callus induction culture are that the culture temperature is 28 +/-2 ℃, the illumination intensity is 2800 and 3000lx, the illumination time is 12-14 h/day, and the humidity is 60-70%. The culture conditions in the adventitious bud induction culture operation and the rooting culture operation are as follows: the culture temperature is 25-28 ℃, the illumination intensity is 2000 and 2500lx, the illumination time is 10-12 hours/day, and the humidity is 60-70%.

Example 2

(1) Explant collection: collecting annual stem and wing fruit branches with good physiological state, no plant diseases and insect pests, diameter of 5-10mm and length of about 15cm by using scissors, and taking leaves from the collected branches as explants.

(2) Leaf callus induction culture: selecting tender and non-scar leaflets from the leaves collected from step (1) back to the laboratory, rinsing with tap water for 3 hours, sterilizing in 70% ethanol solution for 2 times in a clean bench for 20 seconds each time, and then sterilizing with 10% H₂O₂(2 drops of Tween-20 are added) and stirred and disinfected for 2 times, each time lasts for 3 minutes, and finally the sterile water is used for washing for 5 to 7 times until the sterile water is washed clean, the disinfected stalk wing fruit leaves are cut into small pieces with the diameter of 5mm × 5mm, then the leaf surfaces of the stalk wing fruit leaves are horizontally inoculated into a leaf callus induction culture medium, and the callus can be induced and formed after 35 days of culture, wherein the induction rate reaches 79 percent.

The leaf callus induction culture medium comprises: n is a radical of₆Culture medium +2.0 mg/L riboflavin +0.3 mg/L PVP +0.8 mg/L2, 4-D +0.3 mg/L NAA +0.3 mg/L activated carbon +25 g/L sucrose +6.0 g/L agar, pH value is 5.4-5.8.

(3) And (3) adventitious bud induction culture, namely taking the leaf callus induced in the step (2) out of a culture bottle, cutting off tissues in contact with a culture medium by using a blade, cutting the tissue into small pieces with the size of 0.5cm × 0.5.5 cm, inoculating the small pieces into the adventitious bud induction culture medium, and culturing for 30 days to generate adventitious buds.

The adventitious bud induction culture medium comprises: n is a radical of₆The culture medium +3.0 mg/L riboflavin +0.3 mg/L PVP +0.3 mg/L6-BA +0.08 mg/L NAA +0.3 mg/L activated carbon +25 g/L sucrose +6.0 g/L agar, and the pH value is 5.4-5.8.

(4) Rooting culture: and (4) dividing the adventitious bud clump with the length of about 2.0-3.0cm obtained in the step (3) into single adventitious buds, inoculating the single adventitious buds into a rooting culture medium, and culturing for 20 days to realize the rooting of the test-tube plantlet.

The rooting culture medium is 1/2MS culture medium, 0.5 mg/L IBA, 1.0 mg/L NAA, 35 g/L sucrose and 4.0 g/L agar, and the pH value is 5.4-5.8.

(5) Transplanting: and (3) hardening the test-tube plantlets with good root systems in the step (4) for 10 days under natural light of a greenhouse, transplanting the culture medium with cleaned roots into a brick red soil and humus soil matrix with the volume ratio of 1:1 to cultivate seedlings to obtain the stalk wing fruit seedlings, and transplanting the seedlings for 15 days until the survival rate reaches more than 88 percent.

In the tissue rapid propagation method of the samara, the conditions of the leaf callus induction culture are that the culture temperature is 28 +/-2 ℃, the illumination intensity is 2800 and 3000lx, the illumination time is 12-14 h/day, and the humidity is 60-70%. The culture conditions in the adventitious bud induction culture operation and the rooting culture operation are as follows: the culture temperature is 25-28 ℃, the illumination intensity is 2000 and 2500lx, the illumination time is 10-12 hours/day, and the humidity is 60-70%.

Example 3

(1) Explant collection: collecting annual stem and wing fruit branches with good physiological state, no plant diseases and insect pests, diameter of 5-10mm and length of about 15cm by using scissors, and taking leaves from the collected branches as explants.

(2) Leaf callus induction culture: selecting tender and non-scar leaflets from the leaves collected from step (1) back to the laboratory, rinsing with tap water for 3 hours, sterilizing in 70% ethanol solution for 2 times in a clean bench for 20 seconds each time, and then sterilizing with 10% H₂O₂(2 drops of Tween-20 are added) and stirred and disinfected for 2 times, each time lasts for 3 minutes, and finally the sterile water is used for washing for 5 to 7 times until the sterile water is washed clean, the disinfected stalk wing fruit leaves are cut into small pieces with the diameter of 5mm × 5mm, then the leaf surfaces of the stalk wing fruit leaves are horizontally inoculated into a leaf callus induction culture medium, and the callus can be induced and formed after 40 days of culture, and the induction rate reaches 73 percent.

The leaf callus induction culture medium comprises: n is a radical of₆Culture medium +1.0 mg/L riboflavin +0.1 mg/L PVP +0.5 mg/L2, 4-D +0.1 mg/L NAA +0.1 mg/L activated carbon +25 g/L sucrose +5.0 g/L agar, pH value is 5.4-5.8.

(3) And (3) adventitious bud induction culture, namely taking the leaf callus induced in the step (2) out of a culture bottle, cutting off tissues in contact with a culture medium by using a blade, cutting the tissue into small pieces with the size of 0.5cm × 0.5.5 cm, inoculating the small pieces into the adventitious bud induction culture medium, and culturing for 35 days to generate adventitious buds.

The adventitious bud induction culture medium comprises: n is a radical of₆Culture medium +2.0 mg/L riboflavin +0.1 mg/L PVP +0.1 mg/L6-BA +0.05 mg/L NAA +0.1 mg/L activated carbon +25 g/L sucrose +5.0 g/L agar, and the pH value is 5.4-5.8.

(4) Rooting culture: and (4) dividing the adventitious bud clump with the length of about 2.0-3.0cm obtained in the step (3) into single adventitious buds, inoculating the single adventitious buds into a rooting culture medium, and culturing for 20 days to realize the rooting of the test-tube plantlet.

The rooting culture medium is 1/2MS culture medium, 0.5 mg/L IBA, 1.0 mg/L NAA, 35 g/L sucrose and 4.0 g/L agar, and the pH value is 5.4-5.8.

(5) Transplanting: and (3) hardening the test-tube plantlets with good root systems in the step (4) for 10 days under natural light of a greenhouse, transplanting the culture medium with cleaned roots into a brick red soil and humus soil matrix with the volume ratio of 1:1 to cultivate seedlings to obtain the stalk wing fruit seedlings, wherein the survival rate reaches more than 83 percent after 15 days of transplanting.

In the tissue rapid propagation method of the samara, the conditions of the leaf callus induction culture are that the culture temperature is 28 +/-2 ℃, the illumination intensity is 2800 and 3000lx, the illumination time is 12-14 h/day, and the humidity is 60-70%. The culture conditions in the adventitious bud induction culture operation and the rooting culture operation are as follows: the culture temperature is 25-28 ℃, the illumination intensity is 2000 and 2500lx, the illumination time is 10-12 hours/day, and the humidity is 60-70%.

Claims (10)

1. A method for inducing stem bud propagation of a samara leaf callus is characterized by comprising the following steps:

(1) explant collection: collecting leaves on the branches as explants;

(2) leaf callus induction culture: disinfecting the leaves, and then cutting and inducing the leaves to form callus;

(3) adventitious bud induction culture: culturing adventitious buds of the callus to obtain an adventitious bud cluster;

(4) rooting culture: carrying out rooting culture on the adventitious bud cluster to obtain a test-tube plantlet;

(5) transplanting: transplanting the test-tube plantlet to obtain the seedling.

2. The method for inducing stem bud propagation of the samara leaf callus as claimed in claim 1, wherein the method comprises the following steps: and (1) collecting leaves on annual stalk samara branches as explants.

3. The method for inducing stem bud propagation of the samara leaf callus as claimed in claim 1, wherein the method comprises the following steps: in the step (2), the leaves are washed by water, soaked in ethanol and then used with H₂O₂Disinfecting and washing with water; and finally, cutting the disinfected leaves, horizontally inoculating the leaves with upward leaves into a leaf callus induction culture medium, and culturing for at least 30 days.

4. The method for inducing stem bud propagation of the samara leaf callus as claimed in claim 3, wherein the method comprises the following steps: the conditions of the leaf callus induction culture medium are as follows:

N₆culture medium, 3.0 mg/L riboflavin, 0.5 mg/L PVP, 1.0 mg/L2, 4-D, 0.5 mg/L NAA, 0.5 mg/L activated carbon, 30 g/L sucrose, 7.0 g/L agar, pH 5.4-5.8.

5. The method for inducing stem bud propagation of the samara leaf callus as claimed in claim 1, wherein the method comprises the following steps: in the step (3), the part of the callus contacted with the leaf callus induction culture medium is cut off, and then the cut part is transplanted to an adventitious bud induction culture medium to be cultured for at least 25 days.

6. The method for inducing stem bud propagation of the samara leaf callus as claimed in claim 5, wherein the method comprises the following steps: the conditions of the adventitious bud induction culture medium are as follows:

N₆culture medium, 4.0 mg/L riboflavin, 0.5 mg/L PVP, 0.5 mg/L6-BA, 0.1 mg/L NAA, 0.5 mg/L activated carbon, 30 g/L sucrose, 7.0 g/L agar, and pH value is 5.4-5.8.

7. The method for inducing stem bud propagation of the samara leaf callus as claimed in claim 1, wherein the method comprises the following steps: in the step (4), the adventitious bud is inoculated into a rooting culture medium for culturing for at least 20 days after the adventitious bud is divided into single adventitious buds.

8. The method for inducing stem bud propagation of the samara leaf callus as claimed in claim 7, wherein the method comprises the following steps: the conditions of the rooting culture medium are as follows:

1/2MS culture medium, 0.5 mg/L IBA, 1.0 mg/L NAA, 35 g/L sucrose, 4.0 g/L agar, pH 5.4-5.8.

9. The method for inducing stem bud propagation of the samara leaf callus as claimed in claim 1, wherein the method comprises the following steps: in the step (5), the test-tube plantlets are transplanted into a greenhouse for hardening for at least 10 days, and then the culture medium at the cleaned root of the test-tube plantlets is transplanted into the mixed soil of the brick red soil and the humus soil matrix to be cultivated into seedlings, so that the stalk-wing fruit seedlings are obtained.

10. The method for inducing shoot propagation of the samara leaf callus as claimed in any one of claims 1 to 9, wherein: in the step (2), the culture conditions are as follows: the temperature is 26-30 ℃, the illumination intensity is 2800-; humidity is 60% -70%;

in the step (3) and the step (4), the culture conditions are as follows: the temperature is 25-28 ℃, the illumination intensity is 2000-; the humidity is 60-70%.