A method for inducing somatic embryogenesis by using dendrobium candidum stem segments

Abstract

The application discloses a method for inducing somatic embryogenesis by using dendrobium candidum stem segments, which comprises the following steps: taking the dendrobium candidum stem segments with dormant buds as explants; disinfecting the explants; uniformly scratching the surface of the disinfected explants; performing skinning treatment on the disinfected explants; performing dormant bud development induction culture on the skinned explants to obtain the dendrobium candidum stem segments with clustered buds; cutting the dendrobium candidum stem segments with clustered buds to remove the bud tips; uniformly scratching the cut surfaces; and performing somatic embryogenesis induction culture to obtain the dendrobium candidum somatic embryos. The method can be used for directly inducing the dendrobium candidum somatic embryos on a large scale by using the dendrobium candidum stem segments, and the induction time is short. The new buds can be used for successfully inducing the somatic embryo clusters in 60-90 days, so that the dendrobium candidum can be rapidly expanded and multiplied. The method is simple in operation, low in cost, and can be used for inducing the dendrobium candidum without seeds. The method is genetically stable, and can be used for large-scale proliferation in a short time according to the requirements.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing somatic embryogenesis from stem segments of Dendrobium candidum. BACKGROUND

[0002] Dendrobium candidum, also known as Heijiecao, Tiepilan, etc., is a traditional Chinese medicinal material, which is cold in nature, sweet in taste, and belongs to stomach, lung and kidney channels. It has the effects of benefiting stomach, generating fluid, nourishing yin and clearing heat, and has important medicinal value. However, due to overexploitation of wild medicinal resources, the amount of wild resources of Dendrobium candidum has decreased rapidly. In addition, Dendrobium plants have high requirements for ecological environment, and have low reproduction rate and slow growth under natural conditions. Therefore, tissue culture and rapid propagation of Dendrobium candidum has been paid more and more attention.

[0003] Chen Zhi took Dendrobium candidum seeds as starting material, and studied some key influencing factors in the process of protocorm germination, seedling strengthening, and rooting and seedling. A rapid seedling tissue culture technology system of Dendrobium candidum was established. It was found that the addition of 6-BA in the culture medium would inhibit the development of protocorm. If a culture dish with a diameter of 9 cm was used in the whole culture stage, the initial seeding density of 8000 seeds was appropriate. Freeze-dried banana powder was necessary for seed germination, and 5-10 g·L -1 was appropriate. In the seedling strengthening stage, the inoculation density of 100-120 plants per bottle was appropriate. The culture medium and light quality had less effect on seedling strengthening than the inoculation density. In the rooting stage, 3000 K LED white light was used instead of fluorescent light illumination, which could effectively increase the plant height of tissue culture seedlings and increase the proportion of high-quality seedlings without reducing the stem thickness of tissue culture seedlings. The research results also showed that freeze-dried banana powder could completely replace peeled bananas as an organic additive for tissue culture seedlings of Dendrobium candidum. In the whole culture process, the culture temperature was (24±2) ℃, the light time was 10 h, and the light intensity was 25-30 μmol·m -2 ·s -1 ; the light time was 12 h, and the light intensity was about 40 μmol·m -2 ·s -1 in the remaining culture stages.

[0004] Qiu Shichun established a tissue culture technique system for rapid seedling of Dendrobium candidum based on its seeds. The optimal medium formula for different growth stages was found by adjusting the type and concentration of hormones. The results showed that the optimal medium formula for seed germination and protocorm induction was 1 / 2MS+0.1mg / L NAA. The seeds began to germinate at 9-10 days, and the germination rate reached 94.4% after 30 days. The optimal medium formula for protocorm proliferation and differentiation was MS+1.5mg / L 6-BA+0.2mg / L NAA. The protocorm proliferation rate was 8.0% after 15 days, and the differentiation rate reached 73.6% after 55 days. The optimal medium formula for rooting and seedling strengthening was MS+0.5mg / L NAA+0.5mg / L IBA, and the rooting rate reached 92.0%.

[0005] Jiang Lin studied the protocorm proliferation, differentiation and seedling strengthening and rooting of Dendrobium candidum in vitro, and explored the suitable medium formula for each stage to provide theoretical and technical basis for the production of tissue culture seedlings of Dendrobium candidum. The results showed that the suitable medium for protocorm proliferation was 1 / 2MS+6-BA 2.0mg / L+NAA 0.5mg / L; the suitable medium for protocorm differentiation was MS+6-BA 0.5mg / L+NAA 1.0mg / L; and the suitable medium for seedling strengthening and rooting was MS+NAA 0.2mg / L. Banana homogenate 100g / L was added to each medium. The organic additive had a certain promoting effect on seedling strengthening. The test showed that the addition of banana extract could provide sufficient organic nutrients for the growth of seedlings, promote the absorption of nutrients by plants, make the stems and leaves of plants strong, and promote the growth of root system.

[0006] Zhan Zhugen et al. used the in vitro root tips of Dendrobium candidum as explants to study the cytological process of callus formation, somatic embryogenesis and direct somatic embryogenesis of in vitro root tips under different culture conditions. They considered that the protocorm of Dendrobium candidum was a true somatic embryo of single cell origin.

[0007] Although the technology of Dendrobium officinale tissue culture has made great progress, the use of plant tissue culture technology for rapid propagation of Dendrobium officinale protocorm has been reported more, but most of these studies focus on the use of seed germination protocorm induction, and the use of stem segment for propagation research is relatively lacking. Guo Guoye used Dendrobium officinale nodal stem segments as test materials to explore the effects of different plant hormone combinations on Dendrobium officinale stem segment adventitious bud induction and rooting induction. The results showed that MS+0.80% agar+3% sucrose+0.10mg / L NAA+1.00mg / L 6-BA was the best medium formula for bud proliferation induction of Dendrobium officinale, the bud proliferation coefficient under this formula was 2.33, the average bud height was 0.62cm, the bud color was green, and the bud was healthy; MS+0.8% agar+3% sucrose+0.500mg / L IBA was the most suitable medium for rooting induction culture of Dendrobium officinale, the rooting induction rate was 100%, the average root length was 1.04cm, the average number of roots was 3.70, and the average plant height was 1.61cm. Conclusion: Different combinations of 6-BA and NAA have different effects on the induction of Dendrobium officinale adventitious bud proliferation; the concentration of 0.50mg / L IBA has the best effect on the rooting induction of Dendrobium officinale.

[0008] The number of adventitious buds produced by these studies is small, and it is difficult to achieve the purpose of rapid propagation, but using stem segments for somatic embryo induction can solve the problems of long cycle of protocorm induced by seeds and small number of adventitious buds induced by stem segments.

[0009] However, the technology of using stem segments to induce somatic embryogenesis of Dendrobium officinale for rapid propagation has not been reported. SUMMARY

[0010] The purpose of the present application is to provide a method for inducing somatic embryogenesis of Dendrobium officinale stem segments, which uses Dendrobium officinale stem segments with dormant buds as explants, induces bud point development and removes the apical bud for further culture, establishes a sterile system for somatic embryo induction, and achieves the purpose of rapid induction of somatic embryos by adjusting the composition of the culture medium.

[0011] The technical scheme adopted by the present application is as follows:

[0012] A method for inducing somatic embryogenesis of Dendrobium officinale stem segments, comprising the following steps:

[0013] S1: Taking Dendrobium officinale nodal stem segments with dormant buds as explants;

[0014] S2: Disinfecting the explants;

[0015] S3: Making incisions on the surface of the disinfecting explants uniformly, and then performing skin scraping treatment;

[0016] S4: the explant after the skinning treatment is subjected to dormant bud development induction culture to obtain Dendrobium candidum nodal stem segments with clustered buds;

[0017] S5: the Dendrobium candidum nodal stem segments with clustered buds are cut to remove the bud tips, and then subjected to uniform incision at the cut, and then subjected to somatic embryo induction culture to obtain Dendrobium candidum somatic embryos;

[0018] The Dendrobium candidum nodal stem segment has a length of 2 cm, 2-3 nodes, and 2-3 full dormant buds;

[0019] The disinfection treatment method is that the explant is first disinfected with 75% alcohol for 30 s, then disinfected with 2%-5% sodium hypochlorite for 5-7 min, then washed with sterile water for 3-5 times, and then the washed explant is placed on sterile filter paper to absorb surface moisture;

[0020] The skinning refers to scraping off the white old skin outside the Dendrobium candidum nodal stem segment explant.

[0021] The culture medium used in the dormant bud development induction culture is a bud point induction culture medium, and the culture condition is: no light, and the temperature is controlled at 26-30 DEG C; the bud point induction culture medium formula is: 1 / 2MS+0.2mg / L NAA+1.0mg / L 2,4-D+1.0mg / L 6-BA+1.8g / L coconut powder, pH=5.8-6.0.

[0022] The culture medium used in the somatic embryo induction culture is a somatic embryo induction culture medium, and the culture condition is: light time 7h / d, light intensity 2000lx, and the temperature is controlled at 26-30 DEG C; the somatic embryo induction culture medium formula is: 1 / 2MS+0.2mg / L NAA+1.0mg / L 6-BA+1.6g / L coconut powder, pH=5.8-6.0.

[0023] The above-mentioned method for inducing somatic embryo generation from Dendrobium candidum stem segments is applied in Dendrobium candidum somatic embryo culture.

[0024] A Dendrobium candidum somatic embryo obtained by the above-mentioned method.

[0025] The present application has the following advantages:

[0026] 1) The Dendrobium candidum somatic embryo induction method uses mature individual stem segments for induction, avoids the long time of traditional induction method of seed aseptic germination and production of protocorms for induction and expansion, shortens the somatic embryo induction time period from 90-120 days to 40-60 days, and can effectively improve the somatic embryo induction efficiency

[0027] 2) The culture medium ratio adopted by the present application can effectively meet the needs of Dendrobium candidum somatic embryo growth, and can continuously proliferate after induction, increase the number of somatic embryos, and improve the induction efficiency

[0028] 3) The culture medium adopted by the present application is 1 / 2MS medium, which has lower nutrient element concentration than the traditional MS medium, and has lower pollution rate. On the one hand, it can ensure the normal growth needs in the early stage of induction, and on the other hand, it can reduce the growth rate of explants after long-term culture, delay their vegetative growth, and maintain the proliferative state of somatic embryos to a certain extent, which is more conducive to artificial subculture and proliferation.

[0029] 4) The culture method adopted by the present application is in dark culture state for a long period of time, which can reduce the management cost and electricity cost during culture. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 : Dendrobium candidum with dormant buds.

[0031] Figure 2 : Dendrobium candidum with cluster buds.

[0032] Figure 3 : Dendrobium candidum with cluster buds after cutting the bud tips.

[0033] Figure 4 : Somatic embryos formed around the bud incision.

[0034] Figure 5 : Cluster plants formed after somatic embryos develop for 6 months. DETAILED DESCRIPTION

[0035] In order to make the content of the present application more convenient to understand, the technical solutions of the present application will be further described below in combination with specific embodiments, but the present application is not limited thereto.

[0036] The 1 / 2MS medium formula of the present application is shown in Table 1.

[0037] Table 1: Basic medium composition for Dendrobium candidum somatic embryo induction

[0038]

[0039] The coconut powder used in the present application is a natural coconut powder without any sugar and additive ingredients, which is a product obtained by low-temperature grinding of coconut meat, taking pulp, and drying. It contains various basic amino acids required for plant growth, can effectively promote the growth of orchid tissue culture products, is conducive to bud differentiation and growth, and can reduce the browning phenomenon.

[0040] The NAA used in the application is 1-naphthaleneacetic acid, the English name is 1-Naphthylacetic acid, and the molecular formula is C 12 H 10 O2, an artificial synthetic auxin, is often used in plant tissue culture to induce the production of adventitious buds.

[0041] The 6-BA used in the application is 6-benzylaminopurine, the English name is N-(Phenylmethyl)-9H-purin-6-amine, and the molecular formula is C 12 H 11 N5, an artificial synthetic cytokinin, can promote cell division and induce plant to form new shoots. This hormone can help plant germination lateral buds, and can break the apical dominance.

[0042] The 2,4-D used in the application is 2,4-dichlorophenoxyacetic acid, the English name is 2,4-Dichlorophenoxyacetic acid, and the molecular formula is C8H6Cl2O3. It is an artificial synthetic auxin analogue, often used in plant tissue culture to induce the formation of callus, the production of embryoid and the rooting of test-tube seedlings, and can induce the production of axillary buds and adventitious buds in cooperation with a certain proportion of cytokinin.

[0043] Example 1

[0044] A method for inducing somatic embryogenesis from Dendrobium candidum stem segments, comprising the following steps:

[0045] 1) Preparation of explants

[0046] Take the mature Dendrobium candidum plant with vigorous growth and no pests and diseases, soak it in a proper amount of Tide laundry powder for 30 seconds, then rinse it with sterile water for 2-3 times, wipe off the surface moisture with sterile filter paper, and then cut the stem segments with nodes as explants in the super-clean workbench with sterile scissors; the length of the stem segment with nodes is 2 cm, with 2-3 nodes and 2-3 full dormant buds Figure 1 )。

[0047] 2) Disinfection treatment of explants

[0048] Soak the stem segment explants with nodes in 75% ethanol in the super-clean workbench for 30s, then soak them in 2%-5% sodium hypochlorite for 5-7min, then rinse them with sterile water for 3-5 times, and wipe off the surface moisture with sterile filter paper for standby.

[0049] 3) Peeling treatment of explants

[0050] The decontaminated stem segment explant with nodes is scratched on the surface of the stem segment, and the white old skin on the outer part of the stem segment is scraped off with a sterile scalpel.

[0051] 4) Induction culture of dormant bud development

[0052] The stem segment explant with nodes after the skin scraping treatment is inoculated into a bud point induction culture medium, and cultured for 14-20 days under sterile, 26-30 DEG C and light shielding conditions, so that the dormant buds on the stem segment explant with nodes are de-dormant and start to develop into 3-10 mm cluster buds again, and a Dendrobium candidum stem segment with cluster buds is obtained. Figure 2 The formula of the bud point induction culture medium is: 1 / 2MS culture medium+0.2mg / L NAA+1.0mg / L 2,4-D+1.0mg / L 6-BA+1.8g / L coconut powder; and the pH value is 5.8-6.0.

[0053] 5) Induction culture of somatic embryo on the Dendrobium candidum stem segment with new buds

[0054] The bud tip of the Dendrobium candidum stem segment with cluster buds is cut off, the length of the cut-off bud tip is 1 / 2 of the length of the original cluster bud, Figure 3 and then the stem segment is inoculated into a somatic embryo induction culture medium, and cultured for 14-20 days under sterile, 7h / d illumination, 2000lx illumination intensity and 26-30 DEG C conditions, so that the somatic embryo gradually appears at the bud incision and continuously develops. Figure 4 The formula of the somatic embryo induction culture medium is: 1 / 2MS culture medium+0.2mg / L NAA+1.0mg / L 6-BA+1.6g / L coconut powder; and the pH value is 5.8-6.0.

[0055] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.

[0056] Reference

[0057] [1] Chen Zhi, Wang Yiting, Lv Yongping, et al. Optimization of Aseptic Seeding and Seedling System of Dendrobium candidum[J]. Zhejiang Journal of Agricultural Sciences, 2018, 30(11): 1870-1878.

[0058] [2] Qiu Shichun, Zhang Shiping, Feng Shanchai, et al. Optimization of Rapid Propagation Culture System of Dendrobium candidum Seeds[J]. Jiangsu Journal of Agricultural Sciences, 2021, 49(03): 58-61. DOI:10.15889 / j.issn.1002-1302.2021.03.009.

[0059] [3] Jiang Lin, Li Qing. Study on the proliferation, differentiation and rooting of Dendrobium officinale protocorms [C] / / Ornamental Horticulture Professional Committee of Chinese Society for Horticultural Science, National Engineering Research Center for Flowers. Advances in Ornamental Horticulture Research in China, 2016. Beijing Key Laboratory of Flower Germplasm Innovation and Molecular Breeding, Beijing Laboratory of Urban and Rural Ecological Environment of National Engineering Research Center for Flowers, College of Landscape Architecture, Beijing Forestry University; 2016:5.

[0060] [4] Zhan Zhonggen, Xu Cheng, Zhang Ming, et al. Study on the regeneration of Dendrobium officinale root tips from isolated roots via somatic embryos [J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2005, (05): 579-580.

[0061] [5] Guo Guoye, Xu Mengqin, Zhang Naiqun, et al. Study on tissue culture technology of Dendrobium officinale stem segments [J]. Horticulture and Seedling, 2022, 42(11):6-8. DOI:10.16530 / j.cnki.cn21-1574 / s.2022.11.003.

Claims

1. A method for inducing somatic embryogenesis using Dendrobium candidum stem segments, characterized by: It comprises the following steps: S1: taking the Dendrobium candidum stem segments with dormant buds as explants; S2: disinfecting the explants; S3: making incisions on the surface of the disinfected explants uniformly, and then performing peeling treatment; S4: performing dormant bud development induction culture on the explants after peeling treatment, and obtaining Dendrobium candidum stem segments with clustered buds; the culture medium for the dormant bud development induction culture is a bud point induction culture medium, the formula of the bud point induction culture medium is: 1 / 2MS+0.2mg / L NAA+1.0mg / L 2,4-D+1.0mg / L 6-BA+1.8g / L coconut powder, pH=5.8~6.0; the culture condition is: no light, and the temperature is controlled at 26~30℃; S5: cutting the Dendrobium candidum stem segments with clustered buds to remove the bud tips, making incisions on the cut surfaces uniformly, and then performing somatic embryo induction culture, and obtaining Dendrobium candidum somatic embryos; the culture medium for the somatic embryo induction culture is a somatic embryo induction culture medium, the formula of the somatic embryo induction culture medium is: 1 / 2MS+0.2mg / L NAA+1.0mg / L 6-BA+1.6g / L coconut powder, pH=5.8~6.0; the culture condition is: light time 7h / d, light intensity 2000lx, and the temperature is controlled at 26-30℃.

2. The method of claim 1, wherein the Dendrobium candidum stem segments are used to induce somatic embryogenesis. The Dendrobium candidum stem segments have a length of 2cm, 2~3 nodes, and 2~3 full dormant buds.

3. The method of claim 1, wherein the Dendrobium candidum stem segments are used to induce somatic embryogenesis. The disinfection method is: first, disinfecting the explants with 75% alcohol for 30s, then disinfecting the explants with 2%~5% sodium hypochlorite for 5~7min, then washing the disinfected explants with sterile water for 3~5 times, and then placing the washed explants on sterile filter paper to absorb the surface water.

4. The method of claim 1, wherein the Dendrobium candidum stem segments are used to induce somatic embryogenesis. The peeling refers to peeling off the white old skin outside the Dendrobium candidum stem segment explants.

5. The method according to claim 1 is applied to Dendrobium candidum somatic embryo culture.

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