A tissue culture propagation method for euonymus pauciflorus seeds

Abstract

The application provides a tissue culture propagation method of northeast euonymus seed, and belongs to the technical field of plant tissue culture. The method comprises the following steps: inoculating northeast euonymus seed on a bud induction culture medium, carrying out bud induction culture, and obtaining callus; transferring the callus to a bud proliferation culture medium, carrying out bud proliferation culture; transferring to a rooting culture medium when the bud length is 0.8-1.2 cm, carrying out rooting culture, and obtaining northeast euonymus rooting seedlings. The method can obviously shorten the seedling raising period of northeast euonymus, lays a foundation for researches such as northeast euonymus elite breeding and variety improvement, and provides a larger reference basis for cultivating forest trees with more excellent growth traits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant tissue culture, in particular to a seed tissue culture propagation method of Euonymus sacrosancta Koidz. BACKGROUND

[0002] Euonymus sacrosancta Koidz., also known as Guijianhu, Guijian, Liuyue Ling, etc., is a shrub plant of Euonymus in Celastraceae, which not only has high ornamental value, but also has high medicinal value such as anti-tumor and anti-thrombus. At present, the known propagation methods of Euonymus mainly include sowing, cutting, tissue culture and the like, but the seeds of Euonymus are rich in oil and false seed coat, and the seed germination rate is low, so it is difficult to realize engineering seedling.

[0003] Plant tissue culture has the characteristics of short growth cycle, high propagation rate, being conducive to factory production, high economic benefit and the like. Through tissue culture, each organ and tissue can be used as an explant to establish a rapid propagation system, so as to improve the rapid propagation ability of excellent strains, realize engineering seedling, and lay a foundation for good seed breeding and genetic transformation. SUMMARY

[0004] The present application aims to provide a seed tissue culture propagation method of Euonymus sacrosancta Koidz., which provides a reference basis for shortening the seedling cycle of Euonymus sacrosancta Koidz. and cultivating forest trees with more excellent growth traits, and provides research materials for breeding and improving excellent varieties of Euonymus sacrosancta Koidz.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a seed tissue culture propagation method of Euonymus sacrosancta Koidz., comprising the following steps:

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

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

[0009] (3) transferring to a rooting culture medium when the length of the bud is 0.8-1.2 cm after step (2) for rooting culture to obtain Euonymus sacrosancta Koidz. rooting seedlings; wherein the rooting culture medium is a 1 / 2MS medium containing 2.5-3.5 mg / L GA3.

[0010] Preferably, the step (1) is to remove the seed coat of the northeast hedgehog seeds before inoculating the seeds into the bud induction medium, and the seed coat removed seeds are treated by cold storage, and the seeds treated by cold storage are inoculated into the bud induction medium after sterilization.

[0011] Preferably, the method of cold storage treatment is to store the seeds at 3-5℃ for 15-20 days.

[0012] The method of sterilization is to remove the stains on the surface of the seeds, and then sterilize the seeds with ethanol for 20-40s, and then treat the seeds with mercury chloride for 8-10min.

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

[0014] Preferably, the temperature of the bud induction culture in the step (1) is 20-30℃, the light intensity is 1500-2500lux, the light condition is 10-14h light and 10-14h dark alternately, and the time is 12-18d.

[0015] Preferably, the bud induction medium in the step (1) further comprises 20-40g / L sucrose and 6.0-8.0g / 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 the step (2) is 20-30℃, the light intensity is 1500-2500lux, the light condition is 10-14h light and 10-14h dark alternately, and the time is 20-30d.

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

[0018] Preferably, the temperature of the rooting culture in the step (3) is 20-30℃, the light intensity is 1500-2500lux, the light condition is 10-14h light and 10-14h dark alternately, and the time is 10-20d.

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

[0020] The present application takes the northeast euonymus seed as an explant, and successfully cultivates the northeast euonymus rooted seedling; meanwhile, the present application further screens the bud induction culture medium, bud proliferation culture medium and rooting culture medium used in tissue culture, so that the northeast euonymus seed can form callus and then form the rooted and healthy seedling plant in a short time after inoculation. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only constitute the embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of the provided drawings.

[0022] Figure 1 Picture of the northeast euonymus seed inoculated in the MS hormone-free medium in Example 1;

[0023] Figure 2 Growth conditions of the northeast euonymus bud in different treatment groups in Example 1, wherein A is treatment group 1, B is treatment group 6, C is treatment group 9, and the scale is 1 cm;

[0024] Figure 3 Growth conditions of the northeast euonymus adventitious bud in treatment group 3 in Example 2;

[0025] Figure 4 Growth conditions of the northeast euonymus root in different treatment groups in Example 3;

[0026] Figure 5 Culture conditions of the northeast euonymus leaf in the induction medium in the comparative example;

[0027] Figure 6 Induced callus in the comparative example;

[0028] Figure 7 Death conditions of the callus in the comparative example;

[0029] Figure 8 Growth conditions of the induced adventitious bud in the comparative example. DETAILED DESCRIPTION

[0030] The present application provides a northeast euonymus seed tissue culture propagation method, comprising the following steps:

[0031] (1) inoculate the seeds of C. mandshurica into a bud induction medium to carry out bud induction culture, wherein the bud induction medium is MS medium containing 1.5-2.5 mg / L 6-KT and 0.04-0.06 mg / L 2,4-D;

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

[0033] (3) when the length of the bud is 0.8-1.2 cm after step (2) culture, transfer into a rooting medium to carry out rooting culture, and obtain C. mandshurica rooting seedlings; wherein the rooting medium is 1 / 2MS medium containing 2.5-3.5 mg / L GA3.

[0034] In the present application, before step (1) inoculates the seeds of C. mandshurica into a bud induction medium, the false seed coat on the surface of the seeds is removed, and the seeds with the false seed coat removed are subjected to cold storage treatment, and the seeds after cold storage treatment are sterilized and inoculated into the bud induction medium.

[0035] In the present application, the method of cold storage treatment in step (1) is that the seeds are cold stored at 3-5 ℃ for 15-20 days, preferably cold stored at 4 ℃ for 18 days.

[0036] The method of sterilization is that after removing the stains on the surface of the seeds, the seeds are first sterilized with ethanol for 20-40 s, and then treated with mercuric chloride for 8-10 min; preferably, the seeds are first sterilized with ethanol for 30 s, and then treated with mercuric chloride for 9 min.

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

[0038] In the present application, the temperature of bud induction culture in step (1) is 20-30 ℃, preferably 22-28 ℃, further preferably 22-25 ℃, and more preferably 24 ℃; the light intensity is 1500-2500 lux, preferably 1800-2200 lux, and further preferably 2000 lux; the light condition is 10-14 h light and 10-14 h darkness alternately, preferably 11-13 h light and 11-13 h darkness alternately, and further preferably 12 h light and 12 h darkness alternately; and the time is 12-18 d, preferably 13-17 d, further preferably 14-16 d, and more preferably 15 d.

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

[0040] In the present application, 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 further preferably 30 g / L sucrose and 7.0 g / L agar; and the pH of the bud induction medium is 5.5-6.0, preferably 5.7-5.9, and further preferably 5.8.

[0041] In the present application, the temperature for the bud proliferation culture in step (2) is 20-30℃, preferably 22-28℃, further preferably 22-25℃, and more preferably 24℃; the light intensity is 1500-2500 lux, preferably 1800-2200 lux, and further preferably 2000 lux; the light condition is 10-14 h light and 10-14 h dark alternation, preferably 11-13 h light and 11-13 h dark alternation, and further preferably 12 h light and 12 h dark alternation; and the time is 20-30 d, preferably 22-28 d, further preferably 24-26 d, and more preferably 25 d.

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

[0043] In the present application, the bud proliferation medium in step (2) further comprises 20-40 g / L sucrose and 6.0-8.0 g / L agar, preferably comprises 25-35 g / L sucrose and 6.5-7.5 g / L agar, further preferably comprises 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, further preferably 5.8.

[0044] In the present application, the rooting culture in step (3) is carried out at a temperature of 20-30℃, preferably 22-28℃, further preferably 22-25℃, more preferably 24℃; the light intensity is 1500-2500 lux, preferably 1800-2200 lux, further preferably 2000 lux; the light condition is 10-14 h light and 10-14 h darkness alternately, preferably 11-13 h light and 11-13 h darkness alternately, further preferably 12 h light and 12 h darkness alternately; the time is 10-20 d, preferably 12-18 d, further preferably 14-16 d, more preferably 15 d.

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

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

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

[0048] The 6-KT (kinetin), 2,4-D (2,4-dichlorophenoxyacetic acid), IBA (indole-3-acetic acid), GA3 (gibberellin), and DKW medium used in the following examples are all purchased from PhytoTech Labs; the WPM, MS, and 1 / 2MS media are all purchased from Haibo Biotechnology Co., Ltd.; the plant agar is purchased from Saiguobiology Technology Co., Ltd.; and the plant materials used are all taken from the test base in Fulaerji District, Qiqihar City, Heilongjiang Province.

[0049] Example 1

[0050] The purpose of this example is to study the effects of the type and concentration of hormones and the type of medium on bud induction of C. mandshurica.

[0051] A large number of mature C. mandshurica seeds were selected, the seed coat on the surface of the seeds was removed, and the seeds were placed in a refrigerator at 4°C for 3 weeks. After the seeds were taken out of the refrigerator, they were cleaned with detergent to remove surface stains, rinsed with water for 4 h, then transferred to a sterile clean bench, sterilized with 75% ethanol for 30 s, rinsed with sterile water 4 times, then sterilized with 0.1% mercuric chloride for 10 min, and finally rinsed with sterile water 5 times to remove surface moisture, and inoculated in MS medium without hormones. Figure 1 ).

[0052] The sterile seeds were inoculated in petri dishes containing bud induction medium on a clean bench, the bud induction medium was prepared according to Table 1, and the 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 the culture was carried out at a temperature of 24°C, a light intensity of 2000 lux, and under alternating light and dark conditions of 12 h light and 12 h darkness for 15 d. After the culture was completed, the bud induction of C. mandshurica in different treatment groups was observed, and the induction rate was counted, and the results of the induction rate determination are shown in Table 1, and the bud growth of C. mandshurica in treatment groups 1 (A), 6 (B), and 9 (C) is shown in Figure 2 .

[0053] Table 1 Effects of different hormone concentrations and medium types on bud induction of C. mandshurica

[0054] Group 6-KT (mg / L) 2,4-D (mg / L) 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] As can be seen from Table 1 and Figure 2 , the bud induction rate of the seeds in treatment group 9 is the highest, and the growth is 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 is selected as the bud induction medium for C. mandshurica.

[0056] Example 2

[0057] The purpose of this example is to study the effects of the type and concentration of hormones in the bud proliferation medium on the bud proliferation of C. mandshurica.

[0058] The bud-pointed callus cultured in treatment group 9 in Example 1 was transferred into a culture bottle containing bud proliferation medium prepared according to Table 2, and the medium further contained 30 g / L sucrose and 7.0 g / L agar, and was adjusted to pH 5.8. 6-8 pieces were inoculated in each bottle, and were cultured for 25 days under the conditions of 24°C, 2000 lux, 12 hours light and 12 hours dark. After the culture, the bud proliferation of C. dahurica was observed and counted, and the results are shown in Table 2. The growth state of adventitious buds of C. dahurica in treatment group 3 is shown in Figure 3

[0059] Table 2 Influence of different hormone concentrations on bud proliferation of C. dahurica

[0060] Group 6-KT (mg / L) 2,4-D (mg / L) Medium Growth condition of seed adventitious bud 1 1.0 0.05 WPM Growth speed medium, more clump bud 2 1.5 0.15 WPM Growth speed slow, less clump bud 3 2.0 0.3 WPM Growth speed fast, more clump bud

[0061] As shown in Table 2 and Figure 3 , the growth speed of adventitious buds in treatment group 3 is faster than that in treatment groups 1 and 2, and more clustered buds are obtained. Therefore, the medium of 2.0 mg / L 6-KT + 0.3 mg / L 2,4-D + WPM is selected as the bud proliferation medium of C. dahurica.

[0062] Example 3

[0063] The purpose of this example is to study the influence of hormone types and concentrations in bud proliferation medium on bud proliferation of C. dahurica.

[0064] When the length of adventitious buds in Example 2 reached about 1.0 cm, they were transferred into rooting medium prepared according to Table 3, and the medium further contained 30 g / L sucrose and 7.0 g / L agar, and was adjusted to pH 5.8. The culture was carried out under the conditions of 24°C, 2000 lux, 12 hours light and 12 hours dark for 15 days. After the culture, roots were grown in each treatment group, and the growth state and induction rate of roots in different treatment groups were counted, and the results are shown in Table 3. The growth state of roots of C. dahurica in different treatment groups is shown in Figure 4

[0065] Table 3 Influence of different hormone types and concentrations on rooting of C. dahurica

[0066]

[0067]

[0068] As shown in Table 3 and Figure 4 , the growth state of roots in treatment group D is the best, and the induction rate is the highest. Therefore, the medium of 3.0 mg / L GA3 + 30 g / L sucrose + 7.0 g / L agar + 1 / 2MS is selected as the rooting medium of C. dahurica.​​

[0069] Comparative Example

[0070] The northeast akebia leaf blades were selected as explants, and the northeast akebia leaf blades were treated with 0.1% mercury for disinfection (30s, 2min, 5min, 8min, 10min), cut into small pieces, and inoculated into induction medium for culture. Figure 5 However, the inventors found that the leaf blades were browned and died after a period of culture, and the survival rate was low;

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

[0072] The culture conditions were as follows: the temperature was 24℃, the light intensity was 2000lux, the light condition was 12h light, 12h dark alternation culture, and the time was 15d.

[0073] The induced callus was white or yellowish brown Figure 6 , and even a large number of deaths Figure 7 . The leaf blades producing callus were transferred to differentiation medium for continuous culture, the induced adventitious buds grew slowly, and the bud points were small Figure 8 .

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

[0075] The culture conditions were as follows: the temperature was 24℃, the light intensity was 2000lux, the light condition was 12h light, 12h dark alternation culture, and the time was 15d.

[0076] It can be seen that the use of leaf blades as explants is not suitable for the tissue culture of northeast akebia.

[0077] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for propagating Euonymus alatus seeds by tissue culture, characterized in that, Includes the following steps: (1) Seeds of Euonymus alatus were inoculated onto a bud induction medium and bud induction culture was carried out to obtain callus tissue; wherein, the bud induction medium was MS medium containing 1.5-2.5 mg / L 6-KT and 0.04-0.06 mg / L 2,4-D; (2) The callus obtained in step (1) is transferred to a bud proliferation medium for bud proliferation culture; wherein the bud proliferation medium is a WPM medium containing 1.5-2.5 mg / L 6-KT and 0.2-0.4 mg / L 2,4-D. (3) When the buds are cultured to a length of 0.8-1.2 cm in step (2), they are transferred to a rooting medium for rooting culture to obtain rooted seedlings of Euonymus alatus; wherein the rooting medium is a 1 / 2 MS medium containing 2.5-3.5 mg / L GA3.

2. The method as described in claim 1, characterized in that, Step (1) Before inoculating the seeds of Euonymus alatus into the bud induction medium, the aril on the surface of the seeds was removed, and the seeds with the aril removed were refrigerated. After the refrigerated seeds were disinfected, they were inoculated into the bud induction medium.

3. The method as described in claim 2, characterized in that, The cold storage treatment method involves refrigerating the seeds at 3–5°C for 15–20 days. The disinfection method is as follows: after removing stains from the seed surface, disinfect with ethanol for 20-40 seconds, and then treat with mercuric chloride for 8-10 minutes.

4. The method as described in 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 as described in claim 1, characterized in that, The temperature for bud induction culture in step (1) is 20-30℃, the light intensity is 1500-2500 lux, the light conditions are 10-14h light and 10-14h dark alternating culture, and the time is 12-18d.

6. The method as described in claim 5, characterized in that, The bud induction medium in step (1) also includes 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 as described in claim 1, characterized in that, The temperature for the bud proliferation culture in step (2) is 20-30℃, the light intensity is 1500-2500 lux, the light conditions are 10-14h light and 10-14h dark alternating culture, and the time is 20-30d.

8. The method as described in claim 7, characterized in that, The bud proliferation medium described 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 as described in claim 1, characterized in that, The rooting culture in step (3) is carried out at a temperature of 20-30℃, a light intensity of 1500-2500 lux, and a light condition of 10-14h light followed by 10-14h dark alternating culture for 10-20 days.

10. The method as described in 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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