CN111202002A

CN111202002A - Tissue culture and rapid propagation method of clerodendrum japonicum

Info

Publication number: CN111202002A
Application number: CN202010055483.0A
Authority: CN
Inventors: 苏凌业; 何春梅; 王丛丛; 王洪峰
Original assignee: Guangdong Academy of Forestry
Current assignee: Guangdong Academy of Forestry
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-05-29
Anticipated expiration: 2040-01-17
Also published as: CN111202002B

Abstract

The invention discloses a tissue culture and rapid propagation method of Clerodendron slurtum, comprising the steps of sterilizing Clerodendron slurtum explant, inducing and culturing adventitious buds, propagating and rooting culture and transplanting; by utilizing the method, the pollution rate is only 6.1 percent, the adventitious bud induction rate reaches 91.8 percent, the multiplication coefficient is 7.5, the rooting rate is 95 percent, the multiplication and rooting can be synchronously completed, and the method has the characteristics of short growth cycle, simple operation, high propagation coefficient and the like, and can be directly used for the industrial production of the clerodendrum petasitum seedlings.

Description

Tissue culture and rapid propagation method of clerodendrum japonicum

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation method of clerodendrum japonicum.

Background

Clerodendron petasitum (Cleriodendrium japonicum (Thunb.) Sweet) is a plant of Clerodendrum of Verbenaceae, and is also called as zinnia, Zhuyuan Red, Reynaud's Reynaudiana (Hay.) Hayata, etc., and is usually grown in plain, valley, brook side or sparse forest, and distributed in Jiangsu, south Zhejiang, south Jiangxi, Hunan, Fujian, Taiwan, Guangdong, Guangxi, Sichuan, Guizhou, Yunnan, etc. Clerodendrum petasitum is excellent shrub with bright color and beautiful shape, has a flower and fruit period as long as 6 months, is suitable for arrangement of parks, flower beds and flower environments, and is gradually popularized and applied to landscaping. In addition, Clerodendron petiolatus Linn is a traditional folk medicine in southwest, which is first recorded in southern Mediterranean (southern Mediterranean Style) and has the effects of dispelling wind and removing dampness, and reducing swelling and dissipating blood stasis.

Since Clerodendron petiolatus has a certain ornamental and medicinal value, wild Clerodendron petiolatus is excessively harvested under the drive of market interest, and the natural habitat of Clerodendron petiolatus is seriously damaged. Although Clerodendrum japonicum has been artificially cultivated, seed propagation and cutting propagation have been mainly used. Because the fruit setting rate is low, the germination rate of the naturally growing seeds is low, and the propagation efficiency of the clerodendrum japonicum seeds is low; the cutting propagation is easy to cause premature senility and the propagation speed is slow. Therefore, the present invention realizes the industrial production of an excellent Clerodendron petorum plant by a tissue culture rapid propagation technique, and is of great practical significance for the enhancement of the reproduction rate of Clerodendron petorum and the promotion of the industrial development of Clerodendron petorum.

Currently, in the research of Clerodendron petiolatus rapid propagation system, only the Chinese patent application CN104255481A discloses a rapid propagation method of Clerodendron petiolatus suspension cells, the steps include aseptic leaf acquisition, callus induction and suspension cell culture, and the technical scheme does not relate to seedling tissue culture propagation. In the research of the tissue culture and rapid propagation system of the clerodendrum japonicum kindred seeds, the synchronous generation technology of proliferation and rooting is not researched, the replacement frequency of a culture medium is increased, and the large-scale industrial production is not facilitated.

Disclosure of Invention

The present invention aims to provide a method for rapid tissue culture of Clerodendron petiolatus, which comprises simultaneously proliferating and rooting the Clerodendron petiolatus, against the deficiency of the conventional method for tissue culture of Clerodendron petiolatus.

The tissue culture and rapid propagation method of clerodendrum japonicum comprises the following steps:

a. explant disinfection: taking Clerodendron petiolatus twig, removing bract, and sterilizing;

b. adventitious bud induction culture: inoculating the sterilized tender bud explants obtained in the step a into an induction culture medium with the morphological lower end facing downwards, inducing adventitious buds to generate, and culturing to obtain starting seedlings; the induction culture medium: each liter contains 1-2mg of 6-BA, 0.2-0.5mg of IBA, 20g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8;

c. proliferation and rooting culture: b, cutting the starting seedling in the step b into single stem sections, inoculating the stem sections into a multiplication culture medium, and synchronously carrying out multiplication and rooting culture to obtain a rooted seedling; the proliferation culture medium: each liter contains 2mg of 6-BA, 0.2-0.5mg of IBA, 30g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8;

d. transplanting: c, hardening the rooted seedlings in the step c under natural light and then transplanting the rooted seedlings.

Preferably, the tender bud disinfection treatment in the step a is to put the tender bud into a washing powder water solution with the mass fraction of 0.4%, soak for 2min, take out, and wash for 30min with tap water; then using 75% ethanol water solution with volume fraction to disinfect for 30s, washing with sterile water for 3 times, then using mercuric chloride solution with final concentration of 0.1% Tween-20 with mass fraction of 0.02% to disinfect for 6-8min, and washing with sterile water for 5 times.

Preferably, the culture conditions in steps b and c are as follows: the temperature is 23 ℃, the illumination intensity is 2000lx, and the illumination time is 16 h/d.

Preferably, the single stem segment of step c is 0.5-1cm high with 2 axillary buds.

Preferably, in the step d, the rooted seedlings in the step c are acclimatized in natural light for 3d, taken out, washed with the culture medium left at the roots, and planted in a peat substrate.

The invention has the beneficial effects that: the present invention effectively establishes a rapid tissue culture method for Clerodendron petorum by using a plant tissue culture technique, and can synchronously complete the proliferation and rooting of a Clerodendron petorum plant, and has the advantages of short growth cycle, simple operation and high propagation coefficient, and can be directly used for the industrial production of the Clerodendron petorum seedlings.

Drawings

FIG. 1 is a schematic view showing a tissue culture and rapid propagation process of Clerodendron trichotomum in example 1; wherein: a is induction culture of Clerodendron petiolatus adventitious bud; b is Clerodendron petiolatus propagation culture; c is rooting culture of clerodendrum japonicum; d is clerodendrum japonicum transplantation.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

A tissue culture and rapid propagation method of Clerodendron petiolatus comprises the following steps:

a. explant disinfection: selecting robust Clerodendron petiolatus twig without obvious pest and disease damage, cutting in peat soil matrix, and keeping humidity at 80%. After 20-25 days, when young shoots are drawn out of the cutting to about 1cm, carefully peeling off the whole young shoots, removing residual bracts, then placing the young shoots in an aqueous solution of washing powder with the mass fraction of 0.4% (selecting washing powder as a common commercial product) to soak for 2min, slowly shaking, taking out the young shoots and washing for 30min by using tap water. Sterilizing with 75% ethanol water solution by volume fraction for 30s in an ultraclean workbench, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution added with Tween-20 with final concentration of 0.02% by mass fraction for 8min, and washing with sterile water for 5 times.

b. Adventitious bud induction culture: c, inoculating the sterilized tender bud explants obtained in the step a into an induction culture medium with the morphological lower end facing downwards, and culturing and inducing adventitious buds to generate; the induction culture medium: each liter contains 2mg of 6-BA, 0.2 mg of IBA, 20g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the culture conditions are as follows: the temperature is 23 ℃, the illumination intensity is 2000lx, and the illumination time is 16 h/d. After 20 days of culture, 1-2cm high priming seedlings (figure 1A) are obtained, and the induction rate reaches 91.8%.

c. Proliferation and rooting culture: b, cutting the starting seedlings in the step b into single stem sections (0.5-1cm high with 2 axillary buds) and inoculating the stem sections into a proliferation culture medium to synchronously proliferate and culture the roots; the proliferation culture medium: each liter contains 2mg of 6-BA, 0.5mg of IBA0, 30g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the culture conditions are as follows: the temperature is 23 ℃, the illumination intensity is 2000lx, and the illumination time is 16 h/d. After 30 days of culture, the Clerodendron petioloides plant was robust, the leaf was large and dark green (FIG. 1B), and the proliferation coefficient was 7.5. Under the conditions of not changing the culture medium and not changing the culture condition, continuously culturing for 15d, wherein 1-2 thick and strong main roots grow out from the base part of the seedling (figure 1C), the rooting rate is 95%, and the rooted seedling is obtained.

d. Transplanting: and c, hardening the rooted seedlings with good root systems in the step c under natural light for 3D, taking out, cleaning the culture medium remained at the roots, planting the rooted seedlings in a matrix paved by peat soil, and transplanting the rooted seedlings for 30D (shown in figure 1D) until the survival rate is 96%.

Regarding the sterilization method:

example 2

This example is essentially the same as example 1 except that: in this example, the time for disinfection using mercuric chloride solution with a final concentration of 0.02% tween-20 by mass fraction of 0.1% was 7min, and the corresponding disinfection effect is shown in table 1.

Example 3

This example is essentially the same as example 1 except that: in this example, the time for disinfection using mercuric chloride solution with a final concentration of 0.02% tween-20 by mass fraction and a mass fraction of 0.1% was 6min, and the corresponding disinfection effect is shown in table 1.

Comparative example 1

This comparative example is essentially the same procedure as example 1, except that: in the comparative example, the mercuric chloride solution with the mass fraction of 0.1% and the added final concentration of 0.02% of tween-20 is disinfected for 8min instead of the NaClO solution with the mass fraction of 5% in the example 1, and the corresponding disinfection effect is shown in the table 1.

Comparative example 2

This comparative example is essentially the same procedure as example 1, except that: the explants used in this comparative example were not shoots, but stem segments with shoots, and the corresponding disinfection effects are shown in table 1.

The results of parallel culture conducted according to the methods of example 1, example 2, example 3, comparative example 1 and comparative example 2, and the culture time 20d were counted and shown in Table 1.

TABLE 1 Effect of different explant types, Disinfection solution and Disinfection time on Disinfection of Clerodendron petiolatus explant

As can be seen from Table 1, using Clerodendron trichotomum twig as explant, sterilizing with 0.1% by mass of mercuric chloride solution for 6-8min, culturing for 20 days on induction medium (same as example 1) until the contamination rate of the explant is less than 11%, the survival rate exceeds 81%, and the sterilizing effect is far better than that of comparative examples 1 and 2. Wherein, the contamination rate of the disinfection method of the embodiment 1 is only 6.1 percent, and the survival rate can reach 84.8 percent.

Regarding the type and concentration of hormones in the induction medium:

example 4

This example is essentially the same as example 1 except that: the concentration of 6-BA in the induction medium used differs from that of example 1, which specifically used induction medium: each liter contains 1mg of 6-BA, 0.2 mg of IBA, 20g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the results of the induction rate after 20 days of culture are shown in Table 2.

Example 5

This example is essentially the same as example 1 except that: the concentrations of 6-BA and IBA in the induction medium used differ from those of example 1, which was specifically used for the induction medium: each liter contains 2mg of 6-BA, 0.5mg of IBA, 20g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the results of the induction rate after 20 days of culture are shown in Table 2.

Comparative example 3

This comparative example is essentially the same procedure as example 1, except that: the induction medium used was hormone free, the induction medium used in this comparative example was specifically: each liter contains 20g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the results of the induction rate after 20 days of culture are shown in Table 2.

Comparative example 4

This comparative example is essentially the same procedure as example 1, except that: the induction medium used contained 6-BA and 2,4-D (hormone concentrations identical to those disclosed in patent CN 104255481A), and the comparative example specifically used an induction medium: each liter contains 5mg of 6-BA, 1mg of 2,4-D, 20g of sucrose, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the results of the induction rate after 20 days of culture are shown in Table 2.

The parallel culture was performed according to the methods of example 1, example 4, example 5, comparative example 3 and comparative example 4, respectively, the culture time was 20d, and the results of adventitious bud induction culture were statistically shown in Table 2.

TABLE 2 Effect of different Induction Medium on Induction Rate of Clerodendron petiolatum adventitious bud

As can be seen from Table 2, the induction rate after 20 days of culture was higher than 85% and the induction efficiency was better than that of comparative examples 3 and 4 using the induction medium of examples 1, 4 and 5. Wherein, when the induction culture medium of the embodiment 1 is used for culture, the adventitious bud induction rate can reach 91.8%.

Regarding the type and concentration of hormones in the growth medium:

example 6

This example is essentially the same as example 1 except that: the concentration of IBA in the proliferation medium used differs from that of example 1, which was specifically used for the proliferation medium: each liter contains 2mg of 6-BA, 0.2 mg of IBA, 30g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the proliferation and rooting results after the culture are shown in Table 3.

Comparative example 5

This comparative example is essentially the same procedure as example 1, except that: the proliferation medium used was different from that of example 1 in 6-BA, IBA concentration, the present comparative example specifically used a proliferation medium: each liter contains 0.5mg of 6-BA, 0.5mg of IBA0.5mg, 30g of sucrose, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the proliferation and rooting results after the culture are shown in Table 3.

Comparative example 6

This comparative example is essentially the same procedure as example 1, except that: the concentration of IBA in the multiplication medium used differs from that of example 1, this comparative example being specific for the multiplication medium: each liter contains 1mg of IBA, 30g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the proliferation and rooting results after the culture are shown in Table 3.

Comparative example 7

This comparative example is essentially the same procedure as example 1, except that: the proliferation medium used was hormone free, the proliferation medium used in this example was specifically: each liter contains 30g of cane sugar, 7g of agar and the balance of MS culture medium, and the pH value is 5.8; the proliferation and rooting results after the culture are shown in Table 3.

The parallel culture was carried out according to the methods of example 1, example 6, comparative example 5, comparative example 6 and comparative example 7, respectively, and the culture time was 45d, and the statistical culture results are shown in Table 3.

TABLE 3 Effect of different proliferation media on the proliferation and rooting of Clerodendron petiolatus tissue culture seedlings

As can be seen from Table 3, the proliferation coefficient of the proliferation medium obtained in examples 1 and 6 after 30 days of culture is more than 6.9, the plants are stronger, and the proliferation effect and the growth vigor of the plants are better than those of comparative examples 5, 6 and 7; after 45 days of culture, the rooting rate is over 80 percent, the main roots are thick and strong, and the rooting rate and the root growth vigor are superior to those of comparative examples 5, 6 and 7. Wherein, the multiplication coefficient of the multiplication culture medium of the example 1 can reach 7.5 after being cultured for 30 days, and the rooting rate can reach 95 percent after being cultured for 45 days.

Claims

1. A tissue culture and rapid propagation method of Clerodendron petiolatus is characterized by comprising the following steps:

2. The method as claimed in claim 1, wherein the tender shoot disinfection treatment in the step a is carried out by soaking tender shoots in 0.4 mass percent washing powder water solution for 2min, taking out, and washing with tap water for 30 min; then using 75% ethanol water solution with volume fraction to disinfect for 30s, washing with sterile water for 3 times, then using mercuric chloride solution with final concentration of 0.1% Tween-20 with mass fraction of 0.02% to disinfect for 6-8min, and washing with sterile water for 5 times.

3. The method according to claim 1, wherein the culture conditions of steps b and c are as follows: the temperature is 23 ℃, the illumination intensity is 2000lx, and the illumination time is 16 h/d.

4. The method of claim 1, wherein said single stem segment of step c is 0.5-1cm high with 2 axillary buds.

5. The method according to claim 1, wherein in the step d, the rooted seedlings in the step c are taken out after being acclimatized in natural light for 3d, and the culture medium remained at the roots is washed and planted in the peat substrate.