A method for removing endophytic bacteria of the genus Curtobacterium from tissue-cultured lily seedlings

Abstract

The present invention provides a method for removing endophytic bacteria of the genus Curtobacterium from tissue-cultured lily seedlings, comprising the following steps: 1) excising the contaminated leaves and bulb discs from the tissue-cultured seedlings; 2) placing the tissue-cultured seedlings obtained in step 1) into a mixed solution containing trichloroisocyanuric acid with a mass concentration of 0.5% and sodium dodecyl sulfate with a mass concentration of 0.5% and with a mass ratio of 1:1, and soaking for 10-15 min; 3) rinsing 3 times with sterile water; 4) inoculating the tissue-cultured seedlings onto a culture medium of MS + 50 mg / L streptomycin sulfate; 5) placing the tissue-cultured seedlings in an incubator at a temperature of 22±1°C and a light duration of 10-14 h / d, and culturing for 2-3 weeks to observe the contamination of endophytic bacteria of the genus Curtobacterium; 6) if there is no contamination, continue culturing for 2 weeks, and inoculate the tissue-cultured seedlings into a proliferation culture medium of MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L for culturing; 7) if there is contamination, repeat steps 1)-4) to remove the endophytic bacteria of the genus Curtobacterium. The bacteriostatic rate can reach over 90%.

Description

Technical Field

[0001] The present invention relates to a method, in particular to a method for removing endophytic bacteria of the genus Curtobacterium from tissue-cultured lilium seedlings, belonging to the technical field of plant cultivation. Background Art

[0002] Lily is one of the world's top five cut flowers and is deeply loved by people all over the world. The tissue culture of lily has a large propagation coefficient, a fast propagation speed, can obtain virus-free seedlings, and can achieve annual industrial production. It has currently been maturely applied to the cultivation of new lily varieties, the rapid propagation of excellent strains, the detoxification of bulbs, and the preservation of germplasm resources. However, due to the presence of endophytic bacteria inside or between the cells of explants, it is difficult to completely eliminate them with conventional disinfection methods. As a result, during the proliferation culture and propagation of lilies, the growth rate decreases due to the growth of endophytic bacteria in the culture medium, resulting in slow growth of tissue-cultured seedlings, an increase in vitrified seedlings, and even difficulties in transplanting and death of tissue-cultured seedlings, seriously affecting the propagation of lily tissue-cultured seedlings. Chinese Patent No. 201310225467.1 discloses a method for removing endophytic bacteria from explants in lily tissue culture. This method mainly focuses on the elimination of endophytic bacteria in explants before proliferation culture and does not involve how to deal with the endophytic bacteria generated in tissue-cultured seedlings during the proliferation and propagation process. Therefore, it is necessary to improve the existing technology. Summary of the Invention

[0003] To solve the problem of contamination by endophytic bacteria of the genus Curtobacterium during the propagation of lily tissue-cultured seedlings, the present invention provides a method for removing endophytic bacteria of the genus Curtobacterium from lily tissue-cultured seedlings to produce high-quality lily tissue-cultured seedlings.

[0004] The present invention is completed through the following technical solutions: A method for removing endophytic bacteria of the genus Curtobacterium from lily tissue-cultured seedlings, characterized by including the following steps:

[0005] 1) Cut off the leaves and bulb discs of the tissue-cultured seedlings contaminated by endophytic bacteria of the genus Curtobacterium;

[0006] 2) Put the tissue-cultured seedlings in step 1) into a mixed solution of trichloroisocyanuric acid with a mass concentration of 0.5% and sodium dodecyl sulfate with a mass concentration of 0.5% and a mass ratio of 1:1, and soak for 10 - 15 min, wherein the available chlorine content of trichloroisocyanuric acid is not less than 50%;

[0007] 3) Rinse the tissue-cultured seedlings in step 2) three times with sterile water, 30 s each time;

[0008] 4) Inoculate the tissue-cultured seedlings in step 3) into an MS medium containing 50 mg / L streptomycin sulfate;

[0009] Place the tissue culture seedlings from step 4) in an incubator at a temperature of 22 ± 1°C and a light duration of 10 - 14 h / d, and culture for 2 - 3 weeks to observe the contamination situation of endophytic bacteria of the genus Curtobacterium;

[0010] 6) If there is no contamination, continue to culture for 2 weeks, and then transfer the tissue culture seedlings to a proliferation medium of MS + 6 - BA 1.0 mg / L + NAA 0.5 mg / L for proliferation culture;

[0011] 7) If there is contamination, repeat steps 1) - 4), and repeat 2 - 3 times in this way to remove the endophytic bacteria of the genus Curtobacterium.

[0012] The said step 2) is completed inside the culture bottle, and the culture bottle is continuously shaken by hand.

[0013] The said step 3) is to pick out the tissue culture seedlings from the culture bottle with forceps and then put them into a culture bottle containing sterile water for rinsing.

[0014] The said step 4) is completed in a culture bottle containing MS medium with 50 mg / L streptomycin sulfate.

[0015] The present invention has the following good technical effects: it can effectively remove the endophytic bacteria of the genus Curtobacterium in lily tissue culture seedlings, create good conditions for the proliferation and propagation of tissue culture seedlings, and thoroughly solve the problems that in the process of proliferation culture and propagation of lilies, the proliferation rate is easily reduced due to the growth of endophytic bacteria of the genus Curtobacterium, resulting in the slow growth of tissue culture seedlings, an increase in vitrified seedlings, and even difficulties in transplanting and death of tissue culture seedlings. Moreover, the present invention is fast, efficient, economical, with remarkable effects, and the antibacterial rate can reach more than 90%. Description of the Drawings

[0016] Figure 1 It is a growth state diagram of tissue culture seedlings without removing the endophytic bacteria of the genus Curtobacterium;

[0017] Figure 2 It is a growth state diagram of tissue culture seedlings with the endophytic bacteria of the genus Curtobacterium removed. Detailed Embodiments

[0018] The following further describes the present invention with specific embodiments and makes comparisons with comparative examples to prove the technical effects of the present invention. Example 1

[0019] This Example 1 is a method for removing the endophytic bacteria of the genus Curtobacterium from tissue culture seedlings of double - petaled lily 'Liquor', and it includes the following steps:

[0020] 1) On the ultra - clean workbench, excise the leaves and bulb discs of the tissue culture seedlings of double - petaled lily 'Liquor' contaminated by the endophytic bacteria of the genus Curtobacterium;

[0021] 2) Place the tissue culture seedlings from step 1) into a culture bottle containing a mixed solution of trichloroisocyanuric acid with a mass concentration of 0.5% and sodium dodecyl sulfate with a mass concentration of 0.5% and a mass ratio of 1:1. Cover the bottle cap, soak for 10 min, and continuously shake the culture bottle by hand. There are a total of 6 bottles, with 5 tissue culture seedlings in each bottle;

[0022] 3) Use forceps to pick out the tissue culture seedlings from step 2) and place them into a culture bottle containing sterile water for rinsing 3 times, 30 s each time;

[0023] 4) Inoculate the tissue culture seedlings from step 3) into a culture bottle containing MS medium with 50 mg / L streptomycin sulfate. There are a total of 6 bottles, with 5 tissue culture seedlings in each bottle;

[0024] 5) Place the 6 culture bottles from step 4) in an incubator at a temperature of 22 ± 1°C and a light duration of 12 h / d, and culture for 3 weeks. Count the contamination of endophytic bacteria of the genus Curtobacterium in the culture bottles;

[0025] 6) No endophytic bacteria of the genus Curtobacterium appeared in the 6 culture bottles. Continue to culture for 2 weeks, and there was still no contamination by endophytic bacteria of the genus Curtobacterium. Then, transfer the tissue culture seedlings into an MS + 6 - BA 1.0 mg / L + NAA 0.5 mg / L proliferation medium for proliferation culture to obtain high-quality tissue culture seedlings.

[0026] Comparative Example 1

[0027] 1) On a clean bench, excise the leaves and bulb discs contaminated by endophytic bacteria of the genus Curtobacterium from the tissue culture seedlings of Lilium × formolongi 'Liquor';

[0028] 2) Place the tissue culture seedlings from step 1) into a culture bottle containing a mixed solution of trichloroisocyanuric acid with a mass concentration of 0.5% and sodium dodecyl sulfate with a mass concentration of 0.5% and a mass ratio of 1:1. Cover the bottle cap, soak for 10 min, and continuously shake the culture bottle by hand. There are a total of 18 bottles, with 5 tissue culture seedlings in each bottle;

[0029] 3) Use forceps to pick out the tissue culture seedlings from step 2) and place them into a culture bottle containing sterile water for rinsing 3 times, 30 s each time;

[0030] 4) Inoculate the tissue culture seedlings from step 3) into culture bottles with the following three different formula media respectively: MS + 250 mg / L carbenicillin + 50 mg / L kanamycin, MS + 50 mg / L kanamycin, MS + 40 mg / L tetracycline. Each formula is treated with 6 bottles, with 5 tissue culture seedlings in each bottle;

[0031] 5) In each of the three different media in step 4), endophytic bacteria of the genus Curtobacterium grew on each small seedling.

[0032] Thus, it is proved that the method of Example 1 is reliable and has obvious effects, while the comparative examples have all bred endophytic bacteria of the genus Curtobacterium and have no effects. Example 2

[0033] A method for removing endophytic bacteria of the genus Curtobacterium from tissue culture seedlings of Lilium × formolongi 'Mars Love' includes the following steps:

[0034] 1) On a super-clean workbench, cut off the leaves and bulb discs of the tissue culture seedlings of Lilium × formolongi 'Mars Love' contaminated by endophytic bacteria of the genus Curtobacterium.

[0035] 2) Put the tissue culture seedlings in step 1) into a culture bottle containing a mixed solution of trichloroisocyanuric acid with a mass concentration of 0.5% and sodium dodecyl sulfate with a mass concentration of 0.5% and a mass ratio of 1:1, cover the bottle cap, soak for 15 min, and constantly shake the culture bottle by hand; there are 6 bottles in total, and each bottle contains 5 tissue culture seedlings.

[0036] 3) Use forceps to pick out the tissue culture seedlings and put them into a tissue culture bottle containing sterile water for rinsing 3 times, 30 s each time.

[0037] 4) Inoculate the tissue culture seedlings in step 3) into a culture bottle containing MS medium with 50 mg / L streptomycin sulfate; there are 6 bottles in total, and each bottle contains 5 tissue culture seedlings.

[0038] 5) Place the 6 culture bottles in step 4) in an incubator at a temperature of 22 ± 1°C and a light time of 12 h / d for 2 weeks, and count the contamination of endophytic bacteria of the genus Curtobacterium in different media.

[0039] In each culture bottle, 1 - 2 small seedlings are contaminated by endophytic bacteria of the genus Curtobacterium. After continuing to culture for 2 weeks, inoculate the uncontaminated tissue culture seedlings into the following proliferation medium: MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L for proliferation culture to obtain high-quality tissue culture seedlings.

[0040] Take out the tissue culture seedlings contaminated by endophytic bacteria of the genus Curtobacterium in step 5), cut off the contaminated leaves and bulb discs, put them into a culture bottle containing a mixed solution of trichloroisocyanuric acid with a mass concentration of 0.5% and sodium dodecyl sulfate with a mass concentration of 0.5% and a mass ratio of 1:1, cover the bottle cap, soak for 15 min, and constantly shake the culture bottle by hand; use forceps to pick out the tissue culture seedlings and put them into a tissue culture bottle containing sterile water for rinsing 3 times, 30 s each time; then inoculate them into a culture bottle containing MS medium with 50 mg / L streptomycin sulfate and culture at a temperature of 22 ± 1°C and a light time of 12 h / d for 3 weeks. There is no contamination of endophytic bacteria of the genus Curtobacterium. Then inoculate the tissue culture seedlings into the normal proliferation medium of lily: MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L for proliferation culture to obtain high-quality tissue culture seedlings.

[0041] Comparative Example 2

[0042] 1) On a laminar flow bench, excise the leaves and bulb discs contaminated by endophytic bacteria of the genus Curtobacterium from the tissue culture seedlings of Lilium davidii var. unicolor 'Mars Love'.

[0043] 2) Put the tissue culture seedlings in step 1) into a culture bottle containing a mixed solution of trichloroisocyanuric acid with a mass concentration of 0.5% and sodium dodecyl sulfate with a mass concentration of 0.5% and a mass ratio of 1:1. Cover the bottle cap, soak for 15 min, and continuously shake the culture bottle by hand; there are 15 bottles in total, with 6 tissue culture seedlings in each bottle.

[0044] 3) Use forceps to pick out the tissue culture seedlings and put them into a tissue culture bottle containing sterile water for rinsing 3 times, 30 s each time.

[0045] 4) Inoculate the tissue culture seedlings in step 3) into the culture bottles of the following three different formula media respectively: MS + 250 mg / L carbenicillin + 50 mg / L kanamycin, MS + 50 mg / L kanamycin, MS + 40 mg / L tetracycline. There are 5 bottles for each treatment, with 6 tissue culture seedlings in each bottle.

[0046] 5) In the three media in step 4), endophytic bacteria of the genus Curtobacterium grew on each small seedling.

[0047] The experiments of the above Comparative Examples 1 and 2 prove that streptomycin sulfate is superior to carbenicillin, kanamycin, and tetracycline in terms of the effect on endophytic bacteria of the genus Curtobacterium.

Claims

1. A method for removing endophytic bacteria of the genus Curtobacterium in tissue-cultured lily seedlings, characterized in that It includes the following steps: 1) Remove the leaves and bulb discs of the tissue-cultured seedlings contaminated by endophytic bacteria of the genus Brevibacterium; 2) Put the tissue-cultured seedlings in step 1) into a mixed solution with a mass concentration of 0.5% trichloroisocyanuric acid, a mass concentration of 0.5% sodium dodecyl sulfate, and a mass ratio of 1:1, and soak for 10 - 15 min, where the available chlorine content of trichloroisocyanuric acid is not less than 50%; 3) Rinse the tissue-cultured seedlings in step 2) three times with sterile water, 30 s each time; 4) Inoculate the tissue-cultured seedlings in step 3) into an MS medium containing 50 mg / L streptomycin sulfate; Place the tissue-cultured seedlings in step 4) in an incubator at a temperature of 22 ± 1°C and a light time of 10 - 14 h / d, and culture for 2 - 3 weeks to observe the contamination of endophytic bacteria of the genus Brevibacterium; 6) If there is no contamination, continue to culture for 2 weeks, and then inoculate the tissue-cultured seedlings into an MS + 6-BA 1.0 mg / L + NAA 0.5 mg / L proliferation medium for proliferation culture; 7) If there is contamination, repeat steps 1) - 4), and repeat 2 - 3 times in this way to remove the endophytic bacteria of the genus Brevibacterium.

2. The method for removing endophytic bacteria of the genus Curtobacterium from lily tissue culture seedlings according to claim 1, wherein Step 2) is completed in a culture bottle, and the culture bottle is continuously shaken by hand.

3. The method for removing endophytic bacteria of the genus Curtobacterium from tissue-cultured lily seedlings according to claim 1, wherein Step 3) is to use forceps to clamp the tissue-cultured seedlings out of the culture bottle and then rinse them in a culture bottle containing sterile water.

4. The method for removing endophytic bacteria of the genus Curtobacterium in lily tissue culture seedlings according to claim 1, wherein Step 4) is completed in a culture bottle containing an MS medium with 50 mg / L streptomycin sulfate.

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