CN111134021A

CN111134021A - Tissue culture and rapid propagation method of ilex denticulata by taking embryo as explant

Info

Publication number: CN111134021A
Application number: CN202010062568.1A
Authority: CN
Inventors: 杨玉洁; 郝明灼; 邹义萍; 耿芳
Original assignee: Yangtze University
Current assignee: Yangtze University
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2020-05-12

Abstract

The invention discloses a tissue culture and rapid propagation method of ilex denticulata by taking embryos as explants, which comprises the following steps: step 1, collecting fruits of the ilex denticulata, and cleaning and disinfecting the fruits; step 2, stripping off the immature embryos, inoculating the immature embryos on a basic culture medium, and carrying out dark culture for 12-16 days; step 3, transferring the germinated embryos to a light condition for culturing for 26-30 days; step 4, cleaning the seedlings and transplanting the seedlings into a culture medium for culture; the immature zygotic embryo of the ilex denticulata is used as an explant, a rapid propagation system of plant tissues of the ilex denticulata is established, the germination time of the ilex denticulata is shortened to 5-7 weeks from 2-3 years of conventional breeding, the germination rate of tissue culture seedlings of the ilex denticulata reaches 100%, and the transplanting survival rate reaches about 90%.

Description

Tissue culture and rapid propagation method of ilex denticulata by taking embryo as explant

Technical Field

The invention relates to the technical field of tissue culture, in particular to a tissue culture and rapid propagation method of ilex denticulata with embryos as explants.

Background

The ilex denticulata is a multi-branch evergreen shrub with the height of 5 m; the leaf is leathery, is in an inverted oval shape, an ellipse or a long ellipse, 1-7 male flowers are arranged into a parasol inflorescence, the base number of the flowers is 4, and the flowers are white; calyx disk, petal 4, ellipse, corolla diameter about 6 mm, petal oval, ovary oval, fruit ball, and black after maturation; endocarp leathery. The flowering period is 5-6 months, and the fruit period is 8-10 months. Growing in hills, mountain grocery forests or shrubs with the elevation of 700-2100 m. Distributed in china, japan and korea. The cultivation is usually carried out in gardens for ornamental tree species, and is also carried out in Europe and America. The seeds of the ilex denticulata fruit are difficult to germinate, and have the characteristic of annual germination due to the double dormancy of the hard seed coats and the immature zygotic embryos. The conventional breeding of the ilex denticulata needs 2 to 3 years to obtain seedlings.

In order to overcome the incompatibility of distant hybridization, obtain distant hybrid varieties, break through seed dormancy, shorten the breeding period, shorten the application period of the ilex denticulata and save labor and financial resources, a tissue culture method is the best method for quickly obtaining a large number of breeding seedlings. However, the tissue culture method of the tissue culture seedlings of the ilex denticulata has low germination rate and low transplanting survival rate, so that the large-scale popularization and propagation of the ilex denticulata are limited.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a tissue culture and rapid propagation method of ilex denticulata with embryos as explants, the germination rate of tissue culture seedlings of ilex denticulata is as high as 100%, the transplanting survival rate is as high as about 90%, and the germination time of ilex denticulata is shortened to 5-7 weeks from 2-3 years of conventional breeding.

The invention is realized by the following steps:

the invention aims to provide a tissue culture and rapid propagation method of ilex denticulata by taking embryos as explants, which comprises the following steps:

step 1, collecting fruits of the ilex denticulata, and cleaning and disinfecting the fruits;

step 2, stripping off the immature embryos, inoculating the immature embryos on a basic culture medium, and carrying out dark culture for 12-16 days;

step 3, transferring the germinated embryos to a light condition for culturing for 26-30 days;

and 4, cleaning the seedlings, and transplanting the seedlings into a culture medium for culture.

Preferably, the ilex dentata fruits in step 1 are collected in 8 months to form green ilex dentata fruits.

Preferably, the specific steps of cleaning and disinfecting in step 1 are as follows: washing fructus Ilicis Purpureae with running water, transferring to an ultra-clean bench, and rinsing with sterile water; and (3) soaking the surface of the product in 75% alcohol for 3-15 min, then soaking the product in 8% sodium hypochlorite for 10-30 min, finally washing the product with sterile water for 5-6 times, and putting the product into the sterile water for later use.

Preferably, the minimal medium used in step 2 comprises one of WPM medium, MS medium, 1/2MS medium, 1/4MS medium. The optimal medium is WPM minimal medium.

Preferably, the dark culture time in step 2 is 14 days.

Preferably, the culturing time under the illumination condition in the step 3 is 28 days.

Preferably, the cultivation medium transplanted in the step 4 is placed on a greenhouse seedbed and grown under a spraying system.

The invention has the following beneficial effects:

according to the method for tissue culture and rapid propagation of the ilex denticulata by taking the embryo as the explant, the immature zygotic embryo of the ilex denticulata is taken as the explant, a rapid propagation system of plant tissues of the ilex denticulata is established, the germination time of the ilex denticulata is shortened to 5-7 weeks from 2-3 years of conventional breeding, the germination rate of tissue culture seedlings of the ilex denticulata is up to 100%, and the transplanting survival rate is up to about 90%.

Detailed Description

Example 1

The tissue culture and rapid propagation method of ilex denticulata with embryos as explants provided by the embodiment comprises the following steps:

(1) in 8 months, the fruits are green, and when the size of the fruits reaches the maximum, the fruits are collected for stripping the embryos;

(2) washing immature seeds of ilex denticulata with running water for 20min to remove surface dirt. Transferring to an ultra-clean bench, placing into a sterile beaker, and rinsing with sterile water for 1 time. The surface of the seeds is disinfected by soaking the seeds in 75 percent alcohol for 5min, then soaking the seeds in 8 percent sodium hypochlorite for 15min, finally washing the seeds with sterile water for 5 to 6 times and placing the seeds in the sterile water for later use;

(3) stripping off young embryos, inoculating the young embryos on a WPM basic culture medium, and culturing for 12-16 days in dark culture;

(4) transferring the germinated embryos to the illumination condition after germination, and culturing for 26-30 days;

(5) cleaning seedlings, transplanting the seedlings to a culture medium, and placing the seedlings on a greenhouse seedbed under a spraying system for growth. The transplanting survival rate is as high as about 90 percent.

Example 2

The minimal medium used in this example was 1/4MS medium, and the procedure was the same as in example 1.

Example 3

The minimal medium used in this example was 1/2MS medium, and the procedure was the same as in example 1.

Example 4

The minimal medium used in this example was MS medium, and the procedure was the same as in example 1.

Comparative example 1

In this comparative example, the procedure of example 1 was followed except that the "young embryo" in example 1 was changed to "whole seed".

Comparative example 2

In this comparative example, the procedure of example 1 was followed except that the "young embryo" of example 1 was changed to "half-grain seed".

Comparative example 3

In this comparative example, the procedure of example 1 was followed except that the "young embryo" of example 1 was changed to "dehulled seed".

Comparative example 4

The comparison example collected the ilex dentata fruits which were green in 7 months, and the rest of the procedure was the same as example 1.

Comparative example 5

In this comparative example, the fruits of ilex dentata were collected in the light purple color in 9 months, and the procedure was the same as in example 1.

Comparative example 6

The procedure of example 1 was the same as for the collected ilex dentata fruits which appeared purple black in 10 months.

Experimental example 1

Statistics of the germ sterile germination and seedling growth of the offspring of ilex dentata in examples 1-4 are shown in Table 1.

TABLE 1

^zThe difference in the lower case letter representation at the 0.05 level after the same column of values was significant (same below)

As can be seen from Table 1, the minimal medium used in step 2 comprises one of WPM medium, MS medium, 1/2MS medium and 1/4MS medium, which is superior in aseptic germination and growth. The germination rate of the offspring embryo of the ilex denticulata is highest and the seedling grows fastest under the condition that WPM is selected as a basic culture medium.

Experimental example 2

To explore the effect of different explants on the sterile germination of ilex denticulata progeny, the germination rates were counted for the culture methods of example 1 and comparative examples 1-3, as shown in table 2.

TABLE 2

As can be seen from Table 2, the germination rate of the explants is 0% when whole seeds and half seeds are selected; the germination rate at week 4 when explants were selected for dehulled seeds was only 22.22%. In the invention, the immature embryo is used as the explant, and the germination rate reaches 100% in week 2.

Experimental example 3

In order to explore the influence of sampling time on the sterile germination of the immature embryos of the filial generation of the ilex denticulatus, the germination rates of the immature embryos and the contamination rates of the immature embryos under the conditions of example 1 and comparative examples 4-6 are counted, and the statistical results are shown in table 3.

TABLE 3

As can be seen from Table 3, the method adopts the green ilex dentata fruits collected in 8 months, and the pollution rate is 0; while the fruits collected in the 7 months, the 9 months and the 10 months are polluted to different degrees.

The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tissue culture and rapid propagation method of ilex denticulata with embryos as explants is characterized by comprising the following steps:

2. The tissue culture and rapid propagation method of ilex denticulata with embryos as explants of claim 1, wherein the ilex denticulata fruits in step 1 are collected in 8 months to be green.

3. The tissue culture and rapid propagation method of ilex denticulata with embryos as explants according to claim 1, wherein the cleaning and disinfection in the step 1 comprises the following specific steps: washing fructus Ilicis Purpureae with running water, transferring to an ultra-clean bench, and rinsing with sterile water; and (3) soaking the surface of the product in 75% alcohol for 3-15 min, then soaking the product in 8% sodium hypochlorite for 10-30 min, finally washing the product with sterile water for 5-6 times, and putting the product into the sterile water for later use.

4. The tissue culture and rapid propagation method of ilex denticulata with embryos as explants according to claim 1, wherein the minimal medium used in the step 2 comprises one of WPM medium, MS medium, 1/2MS medium and 1/4MS medium.

5. The tissue culture and rapid propagation method of ilex denticulata with embryos as explants of claim 4, wherein the minimal medium used in the step 2 is WPM minimal medium.

6. The tissue culture and rapid propagation method of ilex denticulata with embryos as explants of claim 1, wherein the dark culture time in step 2 is 14 days.

7. The tissue culture and rapid propagation method of ilex denticulata with embryo as explant according to claim 1, wherein the culturing time under illumination condition in step 3 is 28 days.

8. The tissue culture and rapid propagation method of ilex denticulata with embryos as explants according to claim 1, wherein the ilex denticulata tissue culture and rapid propagation method in the step 4 is transplanted to a culture medium and placed under a spraying system on a greenhouse seedbed for growth.