Northeast euonymus alatus seed tissue culture propagation method

Abstract

The invention provides a euonymus alatus seed tissue culture propagation method, and belongs to the technical field of plant tissue culture. The method comprises the following steps: inoculating euonymus alatus seeds on a bud induction culture medium, and carrying out bud induction culture to obtain calluses; transferring the callus to a bud proliferation culture medium, and carrying out bud proliferation culture; and when the bud length is 0.8-1.2 cm, transferring the buds into a rooting culture medium, and carrying out rooting culture to obtain the euonymus alatus rooting seedlings. By means of the method, the seedling raising period of the euonymus alatus can be obviously shortened, a foundation is laid for research of improved variety breeding, variety improvement and the like of the euonymus alatus, and a large reference basis is provided for cultivation of forest trees with more excellent growth characters.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and propagation method for Euonymus japonicus seeds. Background Art

[0002] Euonymus sacrosancta Koidz., also known as ghost arrow feather, ghost arrow, June Ling, etc., is a shrub of the genus Euonymus in the family Celastraceae. It not only has extremely high ornamental value, but also has extremely high medicinal value such as anti-tumor and anti-thrombotic. At present, the known propagation methods of Euonymus mainly include sowing, cutting, tissue culture and other methods, but Euonymus seeds are rich in oil and aril, and the seed germination rate is low, making it difficult to achieve engineering seedling cultivation.

[0003] Plant tissue culture has the characteristics of short growth cycle, high reproduction rate, convenience for factory production, high economic benefits, etc. Through tissue culture, various organs and tissues can be used as explants to establish a rapid propagation system, thereby improving the ability of excellent strains to reproduce rapidly, realizing engineered seedling cultivation, and laying the foundation for improved variety breeding and genetic transformation. Summary of the invention

[0004] The purpose of the present invention is to provide a seed tissue culture propagation method for Euonymus japonicus, to provide a reference basis for shortening the seedling raising period of Euonymus japonicus and cultivating trees with better growth traits, and to provide research materials for breeding and variety improvement of excellent varieties of Euonymus japonicus.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for tissue culture and propagation of Euonymus japonicus seeds, comprising the following steps:

[0007] (1) inoculating Euonymus japonicus seeds on a bud induction medium for bud induction culture to obtain callus tissue; wherein the bud induction medium is an MS medium containing 1.5 to 2.5 mg / L 6-KT and 0.04 to 0.06 mg / L 2,4-D;

[0008] (2) transferring the callus obtained in step (1) to a bud proliferation medium for bud proliferation culture; wherein the bud proliferation medium is a WPM medium containing 1.5 to 2.5 mg / L 6-KT and 0.2 to 0.4 mg / L 2,4-D;

[0009] (3) When the buds in step (2) are cultured to a length of 0.8 to 1.2 cm, the buds are transferred to a rooting medium for rooting culture to obtain rooted Euonymus japonicus seedlings; wherein the rooting medium is a 1 / 2MS medium containing 2.5 to 3.5 mg / L GA3.

[0010] Preferably, in step (1), the aril on the surface of the seeds is removed before inoculating the Euonymus japonicus seeds on the bud induction medium, and the seeds with the aril removed are refrigerated, and the refrigerated seeds are sterilized and then inoculated on the bud induction medium.

[0011] Preferably, the cold storage method is to refrigerate the seeds at 3 to 5°C for 15 to 20 days;

[0012] The disinfection method is as follows: after removing stains on the surface of the seeds, the seeds are first disinfected with ethanol for 20 to 40 seconds, and then treated with mercuric chloride for 8 to 10 minutes.

[0013] Preferably, the volume concentration of the ethanol is 70-80%, and the mass volume concentration of the mercuric chloride is 0.08-0.12%.

[0014] Preferably, the temperature of the bud induction culture in step (1) is 20-30°C, the light intensity is 1500-2500 lux, the lighting conditions are 10-14 h of light and 10-14 h of darkness, and the time is 12-18 days.

[0015] Preferably, the bud induction medium in step (1) further comprises 20-40 g / L sucrose and 6.0-8.0 g / L agar, and the pH of the bud induction medium is 5.5-6.0.

[0016] Preferably, the temperature of the bud proliferation culture in step (2) is 20-30°C, the light intensity is 1500-2500 lux, the lighting conditions are 10-14 h of light and 10-14 h of darkness, and the time is 20-30 days.

[0017] Preferably, the bud proliferation medium in step (2) further comprises 20-40 g / L sucrose and 6.0-8.0 g / L agar, and the pH of the bud proliferation medium is 5.5-6.0.

[0018] Preferably, the rooting culture temperature in step (3) is 20-30°C, the light intensity is 1500-2500 lux, the lighting conditions are 10-14 h of light and 10-14 h of darkness, and the time is 10-20 days.

[0019] Preferably, the rooting medium in step (3) further comprises 20-40 g / L sucrose and 6.0-8.0 g / L agar, and the pH of the rooting medium is 5.5-6.0.

[0020] The present invention uses the seeds of Euonymus japonicus as explants and successfully cultivates rooted seedlings of Euonymus japonicus; at the same time, the present invention further screens the bud induction medium, bud proliferation medium and rooting medium used in tissue culture, so that the seeds of Euonymus japonicus can form callus tissue in a short time after inoculation until they take root and form strong and healthy seedlings. The method of the present invention can significantly shorten the seedling raising period of Euonymus japonicus, lay a foundation for the research of breeding and variety improvement of Euonymus japonicus, and provide a greater reference basis for cultivating trees with better growth traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0022] Figure 1 This is a picture of Euonymus japonicus seeds inoculated in MS hormone-free medium in Example 1;

[0023] Figure 2 The growth of Euonymus japonicus buds in different treatment groups in Example 1, wherein A is treatment group 1, B is treatment group 6, and C is treatment group 9, and the scale bars are all 1 cm;

[0024] Figure 3 The growth status of the adventitious buds of Euonymus mandshurica in treatment group 3 in Example 2;

[0025] Figure 4 The growth conditions of the roots of Euonymus japonicus in different treatment groups in Example 3;

[0026] Figure 5 This is the culture condition of Euonymus japonicus leaves in the induction medium in the comparative example;

[0027] Figure 6 is the callus tissue induced in the comparative example;

[0028] Figure 7 is the death of callus tissue in the comparative example;

[0029] Figure 8 This is the growth of the adventitious buds induced in the comparative example. DETAILED DESCRIPTION

[0030] The present invention provides a method for tissue culture and propagation of Euonymus japonicus seeds, comprising the following steps:

[0031] (1) inoculating Euonymus japonicus seeds on a bud induction medium for bud induction culture to obtain callus tissue; wherein the bud induction medium is an MS medium containing 1.5 to 2.5 mg / L 6-KT and 0.04 to 0.06 mg / L 2,4-D;

[0032] (2) transferring the callus obtained in step (1) to a bud proliferation medium for bud proliferation culture; wherein the bud proliferation medium is a WPM medium containing 1.5 to 2.5 mg / L 6-KT and 0.2 to 0.4 mg / L 2,4-D;

[0033] (3) When the buds in step (2) are cultured to a length of 0.8 to 1.2 cm, the buds are transferred to a rooting medium for rooting culture to obtain rooted Euonymus japonicus seedlings; wherein the rooting medium is a 1 / 2MS medium containing 2.5 to 3.5 mg / L GA3.

[0034] In the present invention, in step (1), the aril on the surface of the seeds is removed before inoculating the Euonymus japonicus seeds on the bud induction medium, and the seeds with the aril removed are refrigerated, and the refrigerated seeds are sterilized and then inoculated on the bud induction medium.

[0035] In the present invention, the method of cold storage treatment in step (1) is to refrigerate the seeds at 3 to 5°C for 15 to 20 days, preferably, the seeds are refrigerated at 4°C for 18 days;

[0036] The disinfection method is: after removing the stains on the seed surface, first disinfect with ethanol for 20 to 40 seconds, and then treat with mercuric chloride for 8 to 10 minutes; preferably: after removing the stains on the seed surface, first disinfect with ethanol for 30 seconds, and then treat with mercuric chloride for 9 minutes.

[0037] In the present invention, the concentration of the ethanol is 70-80%, preferably 75%, and the concentration of the mercuric chloride is 0.08-0.12%, preferably 0.09-0.11%, and more preferably 0.1%.

[0038] In the present invention, the temperature of the bud induction culture in step (1) is 20-30°C, preferably 22-28°C, further preferably 22-25°C, and more preferably 24°C; the light intensity is 1500-2500 lux, preferably 1800-2200 lux, and more preferably 2000 lux; the lighting conditions are 10-14 h light and 10-14 h dark alternating culture, preferably 11-13 h light and 11-13 h dark alternating culture, and more preferably 12 h light and 12 h dark alternating culture; the time is 12-18 d, preferably 13-17 d, further preferably 14-16 d, and more preferably 15 d.

[0039] In the present invention, the bud induction medium in step (1) is an MS medium containing 1.5-2.5 mg / L 6-KT and 0.04-0.06 mg / L 2,4-D, preferably an MS medium containing 1.7-2.3 mg / L 6-KT and 0.04-0.06 mg / L 2,4-D, further preferably an MS medium containing 1.8-2.2 mg / L 6-KT and 0.045-0.055 mg / L 2,4-D, and more preferably an MS medium containing 2.0 mg / L 6-KT and 0.05 mg / L 2,4-D.

[0040] In the present invention, the bud induction medium in step (1) further comprises 20-40 g / L sucrose and 6.0-8.0 g / L agar, preferably 25-35 g / L sucrose and 6.5-7.5 g / L agar, and more preferably 30 g / L sucrose and 7.0 g / L agar; the pH of the bud induction medium is 5.5-6.0, preferably 5.7-5.9, and more preferably 5.8.

[0041] In the present invention, the temperature of the bud proliferation culture in step (2) is 20-30°C, preferably 22-28°C, further preferably 22-25°C, and more preferably 24°C; the light intensity is 1500-2500 lux, preferably 1800-2200 lux, and more preferably 2000 lux; the lighting conditions are 10-14 h light and 10-14 h dark alternating culture, preferably 11-13 h light and 11-13 h dark alternating culture, and more preferably 12 h light and 12 h dark alternating culture; the time is 20-30 d, preferably 22-28 d, further preferably 24-26 d, and more preferably 25 d.

[0042] In the present invention, the bud proliferation medium in step (2) is a WPM medium containing 1.5-2.5 mg / L 6-KT and 0.2-0.4 mg / L 2,4-D, preferably a WPM medium containing 1.6-2.4 mg / L 6-KT and 0.22-0.38 mg / L 2,4-D, further preferably a WPM medium containing 1.8-2.2 mg / L 6-KT and 0.25-0.35 mg / L 2,4-D, and more preferably a WPM medium containing 2.0 mg / L 6-KT and 0.3 mg / L 2,4-D.

[0043] In the present invention, the bud proliferation medium in step (2) also includes 20-40 g / L sucrose and 6.0-8.0 g / L agar, preferably includes 25-35 g / L sucrose and 6.5-7.5 g / L agar, and more preferably includes 30 g / L sucrose and 7.0 g / L agar; the pH of the bud proliferation medium is 5.5-6.0, preferably 5.7-5.9, and more preferably 5.8.

[0044] In the present invention, the temperature of the rooting culture in step (3) is 20-30°C, preferably 22-28°C, further preferably 22-25°C, and more preferably 24°C; the light intensity is 1500-2500 lux, preferably 1800-2200 lux, and more preferably 2000 lux; the lighting conditions are 10-14 h light and 10-14 h dark alternating culture, preferably 11-13 h light and 11-13 h dark alternating culture, and more preferably 12 h light and 12 h dark alternating culture; the time is 10-20 d, preferably 12-18 d, further preferably 14-16 d, and more preferably 15 d.

[0045] In the present invention, the rooting medium in step (3) is a 1 / 2MS medium containing 2.5-3.5 mg / L GA3, preferably a 1 / 2MS medium containing 2.7-3.3 mg / L GA3, further preferably a 1 / 2MS medium containing 2.8-3.1 mg / L GA3, and more preferably a 1 / 2MS medium containing 3.0 mg / L GA3.

[0046] In the present invention, the rooting medium in step (3) also includes 20-40 g / L sucrose and 6.0-8.0 g / L agar, preferably includes 25-35 g / L sucrose and 6.5-7.5 g / L agar, and more preferably includes 30 g / L sucrose and 7.0 g / L agar; the pH of the rooting medium is 5.5-6.0, preferably 5.7-5.9, and more preferably 5.8.

[0047] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] The 6-KT (kinetin), 2,4-D (2,4-dichlorophenoxyacetic acid), IBA (indoleacetic acid), GA3 (gibberellin), and DKW culture medium used in the following examples were all purchased from PhytoTech Labs; WPM, MS, and 1 / 2MS culture media were all purchased from Yu Haibo Biotechnology Co., Ltd.; plant agar was purchased from Yu Saiguo Biotechnology Co., Ltd.; and the plant materials used were all taken from the Fulaerji District Experimental Base in Qiqihar City, Heilongjiang Province.

[0049] Example 1

[0050] The purpose of this example is to study the effects of hormone types and concentrations in the bud induction medium and the type of culture medium on bud induction of Euonymus japonicus.

[0051] Select large, full, mature Euonymus japonicus seeds, remove the aril from the surface of the seeds, and place them in a 4°C refrigerator for 3 weeks. After taking the seeds out of the refrigerator, wash them with detergent to remove surface stains, rinse them with running water for 4 hours, and then transfer them to a sterile clean bench. First, disinfect them with 75% ethanol for 30 seconds, rinse them with sterile water 4 times, then disinfect them with 0.1% mercuric chloride for 10 minutes, and finally rinse them with sterile water 5 times to remove surface moisture, and inoculate them in MS hormone-free medium ( Figure 1 ).

[0052] On the clean bench, sterile seeds were inoculated into culture dishes containing bud induction medium. The bud induction medium was prepared according to Table 1. The culture medium also contained 30 g / L sucrose and 7.0 g / L agar, and the pH was adjusted to 5.8. About 20 sterile seeds were inoculated in each dish and cultured for 15 days at a temperature of 24°C, a light intensity of 2000 lux, and lighting conditions of 12 h of light and 12 h of darkness. After the culture, the bud induction of Euonymus japonicus in different treatment groups was observed, and the induction rate was calculated. The results of the induction rate determination are shown in Table 1. The growth of Euonymus japonicus buds in treatment groups 1 (A), 6 (B), and 9 (C) are shown in Table 1. Figure 2 shown.

[0053] Table 1 Effects of different hormone concentrations and culture medium types on bud induction of Euonymus japonicus

[0054] Group 6-KT(mg / L) 2,4-D (mg / L) Culture medium Induction rate % 1 1.0 0.05 WPM 29.44 2 1.5 0.15 WPM 27.52 3 2.0 0.3 WPM 25.39 4 1.0 0.3 DKW 24.33 5 1.5 0.05 DKW 27.07 6 2.0 0.15 DKW 30.08 7 1.0 0.15 MS 23.05 8 1.5 0.3 MS 28.57 9 2.0 0.05 MS 35.5

[0055] From Table 1 and Figure 2 It can be seen that the bud induction rate of seeds in treatment group 9 was the highest and the growth was the best. Therefore, 2.0 mg / L 6-KT+0.05 mg / L 2,4-D+30 g / L sucrose+7.0 g / L agar+MS medium was selected as the bud induction medium for Euonymus japonicus.

[0056] Example 2

[0057] The purpose of this example is to study the effects of hormone types and concentrations in bud proliferation medium on bud proliferation of Euonymus japonicus.

[0058] The callus with buds cultured in treatment group 9 in Example 1 was transferred to a culture bottle containing a bud proliferation medium. The bud proliferation medium was prepared according to Table 2. The medium also contained 30 g / L sucrose and 7.0 g / L agar, and the pH was adjusted to 5.8. 6 to 8 seeds were inoculated in each bottle and cultured for 25 days at a temperature of 24°C, a light intensity of 2000 lux, and a lighting condition of 12 h of light and 12 h of darkness. After the culture was completed, the proliferation of buds of Euonymus japonicus in different treatment groups was observed and counted. The statistical results are shown in Table 2. The growth of adventitious buds of Euonymus japonicus in treatment group 3 is shown in Table 2. Figure 3 shown.

[0059] Table 2 Effects of different hormone concentrations on bud proliferation of Euonymus japonicus

[0060] Group 6-KT(mg / L) 2,4-D (mg / L) Culture medium Growth status of adventitious buds 1 1.0 0.05 WPM Medium growth rate, with many buds 2 1.5 0.15 WPM Slow growth rate, fewer buds 3 2.0 0.3 WPM Fast growth, more buds

[0061] From Table 2 and Figure 3 It can be seen that the adventitious buds in treatment group 3 grew faster and had more clustered buds than those in treatment groups 1 and 2. Therefore, 2.0 mg / L 6-KT+0.3 mg / L 2,4-D+WPM medium was selected as the bud proliferation medium for Euonymus japonicus.

[0062] Example 3

[0063] The purpose of this example is to study the effects of hormone types and concentrations in bud proliferation medium on bud proliferation of Euonymus japonicus.

[0064] When the adventitious buds in Example 2 reach a length of about 1.0 cm, they are transferred to a rooting medium. The rooting medium is prepared according to Table 3. The medium also contains 30 g / L sucrose and 7.0 g / L agar. The pH is adjusted to 5.8. The temperature is 24°C, the light intensity is 2000 lux, and the lighting conditions are 12 hours of light and 12 hours of darkness. After the cultivation, roots grew in each treatment group. The growth status and induction rate of the roots in different treatment groups were statistically analyzed. The statistical results are shown in Table 3. The growth of the roots of Euonymus japonicus in different treatment groups is shown in Table 3. Figure 4 shown.

[0065] Table 3 Effects of different hormone types and concentrations on rooting of Euonymus japonicus

[0066]

[0067]

[0068] From Table 3 and Figure 4 It can be seen that the root growth state of Euonymus japonicus in treatment group D was the best and the induction rate was the highest. Therefore, 3.0 mg / L GA3 + 30 g / L sucrose + 7.0 g / L agar + 1 / 2 MS medium was selected as the rooting medium of Euonymus japonicus.

[0069] Comparative Example

[0070] The leaves of Euonymus japonicus were selected as explants, and 0.1% mercuric chloride was used to disinfect the leaves (30s, 2min, 5min, 8min, 10min), cut into small pieces, and inoculated into induction medium for culture. However, the inventors found that after a period of culture, the leaves turned brown and died, and the survival rate was low ( Figure 5 ), and the induction period is relatively long.

[0071] Among them, the induction culture medium: MS+(0.5, 1.0, 2.0 mg / L)6-BA+(0.05, 0.1, 0.3 mg / L)NAA+30 g / L sucrose+7.0 g / L agar.

[0072] Culture conditions: temperature was 24°C, light intensity was 2000 lux, lighting conditions were 12 h light and 12 h dark alternating cultivation time was 15 days.

[0073] The induced callus is white or yellow-brown ( Figure 6 ), and even mass death ( Figure 7 The leaves that produced callus tissue were transferred to differentiation medium for further culture. The induced adventitious buds grew slowly and had small buds ( Figure 8 ).

[0074] Among them, the differentiation medium: MS+(4.25, 5.0, 6.0 mg / L)6-BA+(0.5, 1.0, 1.5 mg / L)NAA+30 g / L sucrose+7.0 g / L agar.

[0075] Culture conditions: temperature was 24°C, light intensity was 2000 lux, lighting conditions were 12 h light and 12 h dark alternating cultivation time was 15 days.

[0076] It can be seen that using leaves as explants is not suitable for tissue culture of Euonymus japonicus.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for seed tissue culture propagation of Euonymus japonicus, characterized in that: The steps include: (1) inoculating Euonymus japonicus seeds on a bud induction medium for bud induction culture to obtain callus tissue; wherein the bud induction medium is an MS medium containing 1.5 to 2.5 mg / L 6-KT and 0.04 to 0.06 mg / L 2,4-D; (2) transferring the callus obtained in step (1) to a bud proliferation medium for bud proliferation culture; wherein the bud proliferation medium is a WPM medium containing 1.5 to 2.5 mg / L 6-KT and 0.2 to 0.4 mg / L 2,4-D; (3) When the buds are cultured to a length of 0.8 to 1.2 cm in step (2), the buds are transferred to a rooting medium for rooting culture to obtain rooted Euonymus japonicus seedlings; wherein the rooting medium is a 1 / 2MS medium containing 2.5 to 3.5 mg / L GA3.

2. The method according to claim 1, characterized in that Step (1) before inoculating Euonymus japonicus seeds on a bud induction medium, the aril on the surface of the seeds is removed, the seeds with the aril removed are refrigerated, and the seeds after refrigeration treatment are sterilized and then inoculated on a bud induction medium.

3. The method according to claim 2, characterized in that The cold storage method is to store the seeds at 3-5°C for 15-20 days; The disinfection method is as follows: after removing stains on the surface of the seeds, the seeds are first disinfected with ethanol for 20 to 40 seconds, and then treated with mercuric chloride for 8 to 10 minutes.

4. The method according to claim 3, characterized in that The volume concentration of the ethanol is 70-80%, and the mass volume concentration of the mercuric chloride is 0.08-0.12%.

5. The method according to claim 1, characterized in that The temperature of the bud induction culture in step (1) is 20-30° C., the light intensity is 1500-2500 lux, the lighting conditions are 10-14 h of light and 10-14 h of darkness, and the time is 12-18 days.

6. The method according to claim 5, characterized in that The bud induction medium in step (1) further comprises 20-40 g / L sucrose and 6.0-8.0 g / L agar, and the pH of the bud induction medium is 5.5-6.

0.

7. The method according to claim 1, characterized in that The temperature of the bud proliferation culture in step (2) is 20-30° C., the light intensity is 1500-2500 lux, the lighting conditions are 10-14 h of light and 10-14 h of darkness, and the time is 20-30 days.

8. The method according to claim 7, characterized in that The bud proliferation medium in step (2) also includes 20-40 g / L sucrose and 6.0-8.0 g / L agar, and the pH of the bud proliferation medium is 5.5-6.

0.

9. The method according to claim 1, characterized in that The rooting culture temperature in step (3) is 20-30° C., the light intensity is 1500-2500 lux, the lighting conditions are 10-14 h of light and 10-14 h of darkness, and the time is 10-20 days.

10. The method according to claim 9, characterized in that The rooting medium in step (3) also includes 20-40 g / L sucrose and 6.0-8.0 g / L agar, and the pH of the rooting medium is 5.5-6.0.

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