CN106489738A - A kind of production method of spindle tree leaf regeneration plant - Google Patents

Abstract

The invention belongs to the technical field of plant tissue culture, and in particular relates to a method for producing regenerated roots of Euonymus peach leaves. The production method of the regenerated plant of Euonymus peach leaf leaves of the invention consists of the selection and disinfection of explants, the induction of callus, the induction of adventitious buds and the induction of roots. The present invention has short cultivation cycle, less variation, artificial control of cultivation conditions and simple operation method, laying a foundation for research on Euonymus persicae as an excellent scientific research material for breeding, genetic transformation and variety improvement, thereby providing tissue culture for large-scale production of Euonymus persicae Seedlings provide protection.

Description

A kind of production method of Euonymus peach leaf regeneration plant

a technical field

The invention belongs to the technical field of plant tissue culture, and in particular relates to a method for producing regenerated plants of Euonymus persicae leaves.

Two technical background

Because of its good wood material, high oil content in seeds, high ornamental value, and large area of plantation, Euonymus has become one of the most potential garden tree species in the 21st century to improve the living environment of human beings and meet the needs of human life.

The propagation of Euonymus peach leaf generally adopts methods such as sowing, cutting, and division propagation. However, due to the limitation of the number of seed gardens and the output, there are very few high-quality seedlings obtained, and the propagation speed is relatively slow. The time required for sowing and propagating seeds is very strong. The post-pretreatment is complicated and the post-management requirements are high. It is difficult to use the method of seed propagation to cultivate seedlings on a large scale in production. How to use the method of plant tissue culture to select high-quality seedlings has become the focus of attention in the process of Euonymus peach leaf propagation.

Three invention content

The problem to be solved in the present invention is to invent a production method for regenerated plants of euonymus euonymus leaves in order to provide production and research materials for the breeding and variety improvement of euonymus euonymus high-quality seedlings.

The production method of Euonymus peach leaf regeneration plant of the present invention consists of the following steps:

(1) Selection and disinfection of explants:

Choose healthy and disease-free young leaves of Euonymus persicae as explants, wash the material with running water for 1 hour, remove surface debris, and wash it with sterilized sterile water for 3 times on the aseptic operation workbench, and then Disinfect with 75% absolute ethanol for 30 seconds, then rinse with sterile water for 3 times, then disinfect with 0.1% mercuric chloride for 3-6 minutes, and finally rinse with sterile sterile water for 4 times;

(2) Induction of callus:

On the ultra-clean workbench, the sterilized leaves were cut into squares of 6mm size, and inoculated on the callus induction medium (the formula is: MS+0.5mg/L 6-BA+0.5mg/L NAA+30g/L Sucrose + 6.5g/L agar; Ph adjusted to 5.8), the back of the leaf is close to the medium, inoculate 3-4 pieces per bottle, induce light conditions 14h/d, light intensity 2000lux, temperature 23-28°C;

(3) Induction of adventitious buds:

The yellow-green callus after one subculture was transferred to the medium for inducing adventitious bud differentiation (formula: MS+1.0mg/L 6-BA+0.5mg/L NAA+30g/L sucrose+6.5 g/L agar; Ph adjusted to 5.8), induced light conditions 14h/d, light intensity 2000lux, temperature 23-28°C;

(4) Induction of roots:

Obtain regenerated buds through adventitious bud induction, and when the regenerated buds grow to 4-5 leaves, the length is 0.8-1.2 cm high and then transferred to the rooting medium to induce rooting (the formula is: 1/2MS+1.5mg/LIBA+0.5mg/L NAA+30g/L sucrose+7g/L agar; Ph adjusted to 5.8), induced light conditions 14h/d, light intensity 2000lux, temperature 23-28°C;

The cultivation period of the regenerated plant of the euonymus euonymus leaf of the invention is 55-65 days.

Compared with the prior art, the present invention has the beneficial effects as follows: the present invention uses Euonymus euonymus leaves as material, which is convenient to obtain and sufficient in quantity, forms callus within a relatively short period of time after inoculation until it takes root to form seedling plants, and the resulting plants grow robustly , the average root length was significantly increased. The reproduction coefficient is 8.2, and the cultivation conditions can be controlled artificially. The method can improve the reproduction coefficient and survival rate of Euonymus persicae seedlings, the cultivation period is short, the variation is small, the cultivation conditions can be controlled artificially, and the operation method is simple, which lays a solid foundation for the research on genetic transformation and variety improvement of Euonymus persicae leaves as excellent scientific research materials. foundation, thus providing a guarantee for the large-scale production of tissue culture seedlings of Euonymus persicae. High-quality tissue culture seedlings are provided for the large-scale production of Euonymus persicae.

Four drawings

Figure 1 Leaf explants of Euonymus persicae

Figure 2 Callus of regenerated plants of Euonymus persicae

Figure 3 Cluster buds of Euonymus persicae regenerated plants

The rooted seedling of Fig. 4 Euonymus persicae regeneration plant

Five specific implementation methods

1. Selection of Propagation Material

The test materials were taken from the biennial leaves of Euonymus persicae at the experimental station of Jinling Institute of Science and Technology.

2. Disinfection of Propagation Material

Rinse the material with running water for 1 hour to remove surface impurities. On the aseptic operation bench, first rinse with sterile water for 3 times, then disinfect with 75% absolute ethanol for 30 seconds, then rinse with sterile water for 3 times, and then use 0.1% Mercury chloride disinfection treatment for 5-7 minutes, and finally rinsed 4 times with sterilized sterile water; see Figure 1.

3. Callus Induction

On the ultra-clean workbench, cut the sterilized leaves into 6mm squares and inoculate them on the callus induction medium, with the back of the leaves closely attached to the medium, inoculate 3-4 pieces per bottle, and induce light conditions of 14h/d , light intensity 2000lux, temperature 23-28°C; see attached picture 2

4. Induction of Adventitious Buds

Transfer the yellow-green callus after 2-3 consecutive subcultures to the medium for inducing adventitious bud differentiation, induce light conditions of 14h/d, light intensity of 2000lux, and temperature of 23-28°C; see Figure 3

5. Root Induction

After adventitious buds are induced to obtain regenerated buds, when the regenerated buds grow to 4-5 leaves and the length is 1cm high, they can be transferred to the rooting medium to induce rooting. The induced light conditions are 14h/d, the light intensity is 2000lux, and the temperature is 23-28°C. Seedling rooting situation, see accompanying drawing 4.

Claims (2)

1. a kind of production method of spindle tree leaf regeneration plant, is characterized in that being made up of following steps：

(1) selection and sterilization of explant

Material is rinsed 1h through flowing water, removes table as explant by the healthy and strong disease-free spindle tree young leaflet tablet of growth selection Face debris, on aseptic manipulation work table, the aseptic water washing of first use sterilizing 3 times, then with 75% dehydrated alcohol sterilization 30s, connect With aseptic water washing 3 times, then 5-7min is disinfected with 0.1% mercuric chloride, finally rinsed 4 times with sterilized water concussion；

2) induction of calluss

On superclean bench, the blade for disinfecting is cut into 6mm sizes square is inoculated in callus induction culture medium, is trained The formula of foster base is：NAA+30g/L sucrose+6.5g/L the agar of the 6-BA+0.5mg/L of MS+0.5mg/L；Ph is adjusted to 5.8, leaf The back side be close to culture medium, per bottle of inoculation 3-4 block induces illumination condition 14h/d, intensity of illumination 2000lux, temperature 23-28 DEG C；

3) induction of adventitious bud

Yellow green calluss after successive transfer culture 1 time are transferred on evoking adventive bud division culture medium, culture medium Formula is：NAA+30g/L sucrose+6.5g/L the agar of the 6-BA+0.5mg/L of MS+1.0mg/L；Ph is adjusted to 5.8, induces illumination Condition 14h/d, intensity of illumination 2000lux, temperature 23-28 DEG C；

4) induction of root

Regeneration bud is obtained through Induce aerosor, bud to be regenerated grows to 4-5 piece leaves, proceeds to and take root when length 0.8-1.2cm is high Culture medium root induction, the formula of culture medium is：1/2MS+1.5mg/L IBA+0.5mg/L NAA+30g/L sucrose+7g/L Agar；Ph is adjusted to 5.8, induces illumination condition 14h/d, intensity of illumination 2000lux, temperature 23-28 DEG C.

2. a kind of production method of spindle tree leaf regeneration plant according to claim 1, is characterized in that breeding coefficient is 8.2, cultivation cycle is 55-65 days.