Sterile propagation method for preventing browning of fructus galangae calluses

Abstract

The invention relates to the technical field of galangal fruit tissue culture. The invention provides a sterile propagation method for preventing callus browning of fructus galangae, which comprises the following steps: (1) disinfecting explants of fructus galangae to obtain disinfected explants; (2) inoculating the disinfected explant into an induction culture medium, and culturing to obtain a callus; (3) transferring the calluses into a proliferation culture medium, and culturing to obtain bud seedlings; and (4) transferring the bud seedlings to a rooting culture medium, and continuously culturing to obtain the fructus galangae test-tube plantlets. According to the tissue culture method for the galanga galangal seeds, the propagation efficiency of the galanga galangal seeds is effectively improved, and a large number of galanga galangal seeds can be provided in a short time. The browning phenomenon in the tissue culture process is effectively inhibited by controlling selection and treatment of explants and optimizing a culture medium and culture conditions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tissue culture of Alpinia oxyphylla, and particularly relates to a sterile propagation method for preventing browning of callus of Alpinia oxyphylla. BACKGROUND

[0002] Browning is a browning phenomenon caused by enzymatic and non-enzymatic mechanisms in the process of plant tissue culture, and is related to the oxidation of phenolic substances to quinone polymers. Enzymatic browning requires three elements of phenolic substrates, polyphenol oxidase and oxygen, while non-enzymatic browning is mainly related to mechanisms such as Maillard reaction induced by environmental stress. Studies have shown that the degree of browning is closely related to the physiological state of explants, and the subculture time, pH value and inorganic salt concentration of the culture medium of callus significantly affect the browning rate.

[0003] Browning is a common problem in tissue culture of Alpinia oxyphylla, and browning can cause cell metabolism disorder of callus, polyphenol oxidase catalyzing phenolic substances to generate quinone polymers, which can poison cells and inhibit normal division and differentiation. If browning is not controlled in time, it will cause a large number of cell death. Therefore, it is very important to control the browning of callus for tissue culture. SUMMARY

[0004] The present application aims to provide a sterile propagation method for preventing browning of callus of Alpinia oxyphylla, and effectively controlling the occurrence of browning phenomenon in the process of tissue culture of Alpinia oxyphylla.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides a sterile propagation method for preventing browning of callus of Alpinia oxyphylla, comprising the following steps: (1) taking explants of Alpinia oxyphylla for disinfection to obtain disinfected explants; (2) inoculating the disinfected explants into an induction culture medium to obtain callus after culture; (3) transferring the callus into a proliferation culture medium to obtain seedlings after culture; (4) transferring the seedlings into a rooting culture medium to obtain Alpinia oxyphylla test-tube seedlings after further culture.

[0006] Preferably, the explants in step (1) are selected from stem tips or terminal buds of Alpinia oxyphylla.

[0007] Preferably, the disinfection method in step (1) is: first, soaking the explants in 75-85% alcohol for 10-15s, then soaking in mercury solution for 2-3min, and then washing with sterile water for 2-4 times; the mass concentration of mercury in the mercury solution is 0.08-0.1%.

[0008] Preferably, the sterilized explant is pre-cooled at 6-8℃ for 30-60 minutes before inoculation in step (2).

[0009] Preferably, the induction medium in step (2) is based on 1 / 4 MS medium and further contains the following components at the following concentrations: 6-BA 2-3 mg / L, myo-inositol 20-25 mg / L, sucrose 15-18 g / L, 2,4-D 0.5-0.7 mg / L, disodium ethylenediaminetetraacetate 12-15 mg / L.

[0010] Preferably, the temperature of the culture in step (2) is 18-20℃, and the culture time is 15-19 days, and the culture is performed in the dark.

[0011] Preferably, the proliferation medium in step (3) is based on 1 / 2 MS medium and further contains the following components at the following concentrations: 6-BA 2-3 mg / L, myo-inositol 20-25 mg / L, sucrose 20-24 g / L, 2,4-D 0.6-0.9 mg / L, disodium ethylenediaminetetraacetate 20-25 mg / L, arginine 20-24 mg / L, glutamic acid 25-29 mg / L, nicotinic acid 1.4-1.6 mg / L, kanamycin 0.02-0.04 mg / L.

[0012] Preferably, the temperature of the culture in step (3) is 24-28℃, and the light time per day is 8-10 h, and the light intensity is 14000-15000 lx.

[0013] Preferably, the rooting medium in step (4) is based on 1 / 2 MS medium and further contains the following components at the following concentrations: IBA 0.5-1.0 mg / L, NAA 0.2-0.5 mg / L, sucrose 20-24 g / L, activated carbon 2.5-3.5 g / L, 5-amino levulinic acid 1.5-2.5 mg / L.

[0014] Preferably, the temperature of the culture in step (4) is 26-30℃, and the light time per day is 12-14 h, and the light intensity is 16000-18000 lx.

[0015] The application provides a sterile propagation method for preventing callus browning of Amomum villosum, and comprises the following steps: (1) taking an explant of Amomum villosum for sterilization to obtain a sterilized explant; (2) inoculating the sterilized explant into an induction medium to obtain callus after culture; (3) transferring the callus into a proliferation medium to obtain seedlings after culture; and (4) transferring the seedlings into a rooting medium to obtain Amomum villosum test-tube seedlings after further culture. The application provides a tissue culture method for Amomum villosum, effectively improves the propagation efficiency of Amomum villosum, and can provide a large amount of Amomum villosum seedlings in a short time. By controlling the selection and treatment of the explant and optimizing the medium and culture conditions, the occurrence of browning in the tissue culture process is effectively inhibited. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The culture effect of Example 3 of the application. DETAILED DESCRIPTION

[0017] The application provides a sterile propagation method for preventing callus browning of Amomum villosum, and comprises the following steps: (1) taking an explant of Amomum villosum for sterilization to obtain a sterilized explant; (2) inoculating the sterilized explant into an induction medium to obtain callus after culture; (3) transferring the callus into a proliferation medium to obtain seedlings after culture; and (4) transferring the seedlings into a rooting medium to obtain Amomum villosum test-tube seedlings after further culture.

[0018] In the application, the explant in step (1) is selected from a stem tip or a terminal bud of Amomum villosum.

[0019] In the application, the sterilization method in step (1) is preferably: first immersing the explant in 75-85% alcohol for 10-15s, then immersing the explant in a mercury solution for 2-3min, and then washing the explant with sterile water for 2-4 times, and the method is further preferably: first immersing the explant in 80% alcohol for 12-13s, then immersing the explant in a mercury solution for 3min, and then washing the explant with sterile water for 3 times; the mass concentration of mercury in the mercury solution is preferably 0.08-0.1%, and is further preferably 0.09%.

[0020] In the application, the sterilized explant is preferably pre-cooled at 6-8℃ before inoculation in step (2), and is further preferably pre-cooled at 7℃, and the pre-cooling time is preferably 30-60min, and is further preferably 45min.

[0021] In this invention, the induction medium in step (2) is preferably based on 1 / 4 MS medium and also contains the following components at the following concentrations: 6-BA 2~3 mg / L, inositol 20~25 mg / L, sucrose 15~18 g / L, 2,4-D 0.5~0.7 mg / L, and disodium EDTA 12~15 mg / L. More preferably, it contains: 6-BA 2.5 mg / L, inositol 23 mg / L, sucrose 16~17 g / L, 2,4-D 0.6 mg / L, and disodium EDTA 13~14 mg / L.

[0022] In this invention, the culture temperature in step (2) is preferably 18~20℃, more preferably 19℃, and the culture time is preferably 15~19 days, more preferably 17 days, and the culture is carried out in the dark.

[0023] In this invention, the proliferation medium in step (3) is preferably based on 1 / 2 MS medium and also contains the following components at the following concentrations: 6-BA 2~3 mg / L, inositol 20~25 mg / L, sucrose 20~24 g / L, 2,4-D 0.6~0.9 mg / L, disodium EDTA 20~25 mg / L, arginine 20~24 mg / L, glutamic acid 25~29 mg / L, nicotinic acid 1.4~1.6 mg / L, kanamycin 0.02~0.04 mg / L, and more preferably: 6-BA 2.5 mg / L, inositol 22~23 mg / L, sucrose 22 g / L, 2,4-D 0.7~0.8 mg / L, disodium EDTA 22~23 mg / L, arginine 22 mg / L, glutamic acid 27 mg / L, nicotinic acid 1.5 mg / L, kanamycin 0.03 mg / L.

[0024] In this invention, the culture temperature in step (3) is preferably 24~28℃, more preferably 26℃, the daily light exposure time during culture is preferably 8~10h, more preferably 9h, and the light intensity is preferably 14000~15000lx, more preferably 14500lx.

[0025] In this invention, the rooting medium in step (4) is preferably based on 1 / 2 MS medium and also contains the following components at the following concentrations: IBA 0.5~1.0 mg / L, NAA 0.2~0.5 mg / L, sucrose 20~24 g / L, activated carbon 2.5~3.5 g / L, and 5-aminolevulinic acid 1.5~2.5 mg / L. More preferably, it contains: IBA 0.7~0.8 mg / L, NAA 0.3~0.4 mg / L, sucrose 22 g / L, activated carbon 3 g / L, and 5-aminolevulinic acid 2 mg / L.

[0026] In this invention, the culture temperature in step (4) is preferably 26~30℃, more preferably 28℃, the daily light exposure time during culture is preferably 12~14h, more preferably 13h, and the light intensity is preferably 16000~18000lx, more preferably 17000lx.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] A method for aseptic propagation to prevent browning of cardamom callus includes the following steps: (1) Take the terminal bud of cardamom as the explant. First, soak the explant in 85% alcohol for 10 seconds, then soak it in 0.08% mercuric chloride solution for 3 minutes, and then rinse it with sterile water 4 times to obtain the disinfected explant. (2) The disinfected explants were pre-cooled at 8°C for 30 min and then inoculated into induction medium. After being cultured in the dark at 18°C ​​for 19 days, callus tissue was obtained. (3) The callus tissue was transferred to the proliferation medium and cultured at 24°C. During the culture, the tissue was irradiated with 15,000 lx light for 10 hours every day to obtain seedlings. (4) Transfer the seedlings to the rooting medium and continue to culture at 26℃. During the continued culture, irradiate them with 16000lx light for 14 hours every day until you get the cardamom test-tube seedlings.

[0030] Note: In this embodiment, the induction medium used is based on 1 / 4 MS medium and also contains the following components at the following concentrations: 6-BA 3 mg / L, inositol 25 mg / L, sucrose 15 g / L, 2,4-D 0.7 mg / L, and disodium EDTA 12 mg / L. The proliferation medium was based on 1 / 2 MS medium and also contained the following components at the following concentrations: 6-BA 2 mg / L, inositol 25 mg / L, sucrose 24 g / L, 2,4-D 0.9 mg / L, disodium EDTA 20 mg / L, arginine 20 mg / L, glutamate 29 mg / L, nicotinic acid 1.6 mg / L, and kanamycin 0.02 mg / L. The rooting medium was based on 1 / 2 MS medium and also contained the following components at the following concentrations: IBA 1.0 mg / L, NAA 0.2 mg / L, sucrose 24 g / L, activated charcoal 3.5 g / L, and 5-aminolevulinic acid 1.5 mg / L.

[0031] Example 2

[0032] A method for aseptic propagation to prevent browning of cardamom callus includes the following steps: (1) Take the stem tip of cardamom as the explant. First, soak the explant in 75% alcohol for 15 seconds, then soak it in 0.1% mercuric chloride solution for 2 minutes, and then rinse it twice with sterile water to obtain the disinfected explant. (2) The disinfected explants were pre-cooled at 6°C for 60 min and then inoculated into induction medium. After being cultured in the dark at 20°C for 15 days, callus tissue was obtained. (3) The callus tissue was transferred to the proliferation medium and cultured at 28°C. During the culture process, the tissue was irradiated with 14,000 lx light for 8 hours every day to obtain seedlings. (4) Transfer the seedlings to the rooting medium and continue to culture them at 30°C. During the continued culture, irradiate them with 18,000 lx light for 12 hours every day until you get the cardamom plantlets.

[0033] Note: In this embodiment, the induction medium used is based on 1 / 4 MS medium and also contains the following components at the following concentrations: 6-BA 2 mg / L, inositol 20 mg / L, sucrose 18 g / L, 2,4-D 0.5 mg / L, and disodium EDTA 15 mg / L. The proliferation medium was based on 1 / 2 MS medium and also contained the following components at the following concentrations: 6-BA 3 mg / L, inositol 20 mg / L, sucrose 20 g / L, 2,4-D 0.6 mg / L, disodium EDTA 25 mg / L, arginine 24 mg / L, glutamate 25 mg / L, nicotinic acid 1.4 mg / L, and kanamycin 0.04 mg / L. The rooting medium was based on 1 / 2 MS medium and also contained the following components at the following concentrations: IBA 0.5 mg / L, NAA 0.5 mg / L, sucrose 20 g / L, activated charcoal 2.5 g / L, and 5-aminolevulinic acid 2.5 mg / L.

[0034] Example 3

[0035] A method for aseptic propagation to prevent browning of cardamom callus includes the following steps: (1) Take the terminal bud of cardamom as the explant. First, soak the explant in 80% alcohol for 12 seconds, then soak it in 0.09% mercuric chloride solution for 3 minutes, and then rinse it with sterile water 3 times to obtain the disinfected explant. (2) The disinfected explants were pre-cooled at 7°C for 45 min and then inoculated into induction medium. After being cultured in the dark at 19°C for 17 days, callus tissue was obtained. (3) The callus tissue was transferred to the proliferation medium and cultured at 26°C. During the culture, the tissue was irradiated with 14500 lx light for 9 hours every day to obtain seedlings. (4) Transfer the seedlings to the rooting medium and continue to culture at 28°C. During the continued culture, irradiate them with 17,000 lx light for 13 hours every day until you get the cardamom plantlets.

[0036] Note: In this embodiment, the induction medium used is based on 1 / 4 MS medium and also contains the following components at the following concentrations: 6-BA 2.5 mg / L, inositol 22 mg / L, sucrose 16 g / L, 2,4-D 0.6 mg / L, and disodium EDTA 13 mg / L. The proliferation medium was based on 1 / 2 MS medium and also contained the following components at the following concentrations: 6-BA 2.5 mg / L, inositol 22 mg / L, sucrose 22 g / L, 2,4-D 0.7 mg / L, disodium EDTA 23 mg / L, arginine 22 mg / L, glutamate 27 mg / L, nicotinic acid 1.5 mg / L, and kanamycin 0.03 mg / L. The rooting medium was based on 1 / 2 MS medium and also contained the following components at the following concentrations: IBA 0.8 mg / L, NAA 0.4 mg / L, sucrose 22 g / L, activated charcoal 3 g / L, and 5-aminolevulinic acid 2 mg / L.

[0037] Comparative Example 1

[0038] In this embodiment, the explants are used directly after disinfection without pre-cooling, and the remaining steps are the same as in Example 3.

[0039] Comparative Example 2

[0040] In this embodiment, the induction medium used was based on 1 / 4 MS medium and also contained the following components at the following concentrations: 6-BA 2.5 mg / L, inositol 22 mg / L, sucrose 16 g / L, and 2,4-D 0.6 mg / L; The proliferation medium was based on 1 / 2 MS medium and also contained the following components at the following concentrations: 6-BA 2.5 mg / L, inositol 22 mg / L, sucrose 22 g / L, 2,4-D 0.7 mg / L, arginine 22 mg / L, glutamic acid 27 mg / L, and nicotinic acid 1.5 mg / L. The rooting medium was based on 1 / 2 MS medium and also contained the following components at the following concentrations: IBA 0.8 mg / L, NAA 0.4 mg / L, sucrose 22 g / L, and 5-aminolevulinic acid 2 mg / L.

[0041] The remaining steps and methods are the same as in Example 3.

[0042] Test case

[0043] Cardamom was cultured according to the methods of Examples 1-3 and Comparative Examples 1-2. Each group was inoculated with 50 conical flasks, with 3 explants per flask. The browning rate of callus tissue and the survival rate of the final test-tube seedlings were counted for each group. The results are shown in Table 1.

[0044] Table 1

[0045] As can be seen from the above embodiments, the present invention provides a sterile propagation method for preventing browning of cardamom callus, comprising the following steps: (1) disinfecting cardamom explants to obtain disinfected explants; (2) inoculating the disinfected explants into an induction medium and culturing to obtain callus; (3) transferring the callus to a proliferation medium and culturing to obtain seedlings; (4) transferring the seedlings to a rooting medium and continuing culturing to obtain cardamom test-tube seedlings. The present invention innovatively provides a cardamom tissue culture method, which effectively improves the propagation efficiency of cardamom and can provide a large number of cardamom seedlings in a short time. By controlling the selection and treatment of explants and optimizing the culture medium and culture conditions, the occurrence of browning during tissue culture is effectively inhibited.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for aseptic propagation to prevent browning of cardamom callus, characterized in that, Includes the following steps: (1) Disinfect the explants of cardamom to obtain disinfected explants; (2) The sterilized explants were inoculated into the induction medium and cultured to obtain callus tissue; (3) The callus tissue was transferred to the proliferation medium and cultured to obtain shoots; (4) Transfer the seedlings to the rooting medium and continue to cultivate them to obtain cardamom plantlets.

2. The aseptic propagation method according to claim 1, characterized in that, The explants mentioned in step (1) are selected from the stem tips or terminal buds of cardamom.

3. The aseptic propagation method according to claim 1, characterized in that, The disinfection method described in step (1) is as follows: first, soak the explant in 75-85% alcohol for 10-15 seconds, then soak it in mercuric chloride solution for 2-3 minutes, and then rinse it with sterile water 2-4 times; the mass concentration of mercuric chloride in the mercuric chloride solution is 0.08-0.1%.

4. The aseptic propagation method according to claim 1, characterized in that, In step (2), the disinfected explants are pre-cooled at 6-8°C before inoculation for 30-60 minutes.

5. The aseptic propagation method according to claim 1, characterized in that, The induction medium described in step (2) is based on 1 / 4MS medium and also contains the following components at the following concentrations: 6-BA 2~3 mg / L, inositol 20~25 mg / L, sucrose 15~18 g / L, 2,4-D 0.5~0.7 mg / L, and disodium ethylenediaminetetraacetate 12~15 mg / L.

6. The aseptic propagation method according to claim 1, characterized in that, The culture temperature in step (2) is 18~20℃, the culture time is 15~19 days, and the culture is carried out in the dark.

7. The aseptic propagation method according to claim 1, characterized in that, The proliferation medium described in step (3) is based on 1 / 2 MS medium and also contains the following components at the following concentrations: 6-BA 2~3 mg / L, inositol 20~25 mg / L, sucrose 20~24 g / L, 2,4-D 0.6~0.9 mg / L, disodium EDTA 20~25 mg / L, arginine 20~24 mg / L, glutamic acid 25~29 mg / L, nicotinic acid 1.4~1.6 mg / L, and kanamycin 0.02~0.04 mg / L.

8. The aseptic propagation method according to claim 1, characterized in that, The culture temperature in step (3) is 24~28℃, the daily light exposure time is 8~10h, and the light intensity is 14000~15000lx.

9. The aseptic propagation method according to claim 1, characterized in that, The rooting medium described in step (4) is based on 1 / 2 MS medium and also contains the following components at the following concentrations: IBA 0.5~1.0 mg / L, NAA 0.2~0.5 mg / L, sucrose 20~24 g / L, activated carbon 2.5~3.5 g / L, and 5-aminolevulinic acid 1.5~2.5 mg / L.

10. The aseptic propagation method according to claim 1, characterized in that, The culture temperature in step (4) is 26~30℃, the daily light exposure time is 12~14h, and the light intensity is 16000~18000lx.

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