Antibacterial agent, culture medium and open tissue culture method for Populus seedlings

Abstract

The invention discloses an antibacterial agent, a culture medium and an open tissue culture method for poplar seedlings, wherein the antibacterial agent uses a combination of 0.1g / L-0.15g / L thiophanate-methyl and 0.05g / L-0.1g / L cefotaxime sodium, and after the antibacterial agent is added to the culture medium, a lower contamination rate and a browning rate are obtained; the open tissue culture method comprises the following steps: obtaining test materials, bud induction culture, rooting culture, seedling hardening and transplanting, wherein: the bud induction culture uses an MS culture medium added with an antibacterial agent, the MS culture medium is added with 6-benzylaminopurine and naphthaleneacetic acid, the concentration of 6-benzylaminopurine is 0.2mg / L, the concentration of naphthaleneacetic acid is 0.02mg / L-0.08mg / L, and a higher induction rate and an average number of adventitious buds are obtained; the rooting culture uses a 1 / 2MS culture medium added with an antibacterial agent, the 1 / 2MS culture medium is added with indolebutyric acid, the concentration of indolebutyric acid is 0.1-0.4mg / L, and a higher rooting rate is obtained. Thus, the open tissue culture of Populus seedlings is realized, filling the gap in the prior art.

Description

Technical Field

[0001] The invention relates to the field of open tissue culture, and in particular to an antibacterial agent for Populus seedlings.

[0002] The present invention also relates to a culture medium for Populus seedlings.

[0003] The invention also relates to an open tissue culture method for Populus seedlings. Background Art

[0004] Tissue culture is one of the important ways to produce seedlings, remove viruses, and improve germplasm. However, due to problems such as high technology and high costs, it has not been promoted and applied on a large scale in production. The open tissue culture of plants that has emerged in recent years adds antibacterial agents to the culture medium, does not require high-pressure sterilization, and does not require clean bench inoculation, which solves the problems of high tissue culture cost and low efficiency. In addition, tissue culture has the advantages of high uniformity of seedlings, virus-free and robustness, and breeding is not restricted by season. Therefore, its development prospects are very broad.

[0005] So far, there has been no report on the open tissue culture technology of Populus plants, and therefore, people have not yet obtained the relevant technology for open tissue culture of Populus seedlings. Summary of the invention

[0006] The purpose of the present invention is to provide an antibacterial agent, a bud induction medium, a rooting medium and an open tissue culture method for Populus seedlings, so as to fill the gap in the prior art.

[0007] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0008] The present application provides an antibacterial agent for Populus seedlings, comprising: thiophanate-methyl and ceftriaxone sodium; the concentration of thiophanate-methyl is 0.1 g / L-0.15 g / L, and the concentration of ceftriaxone sodium is 0.05 g / L-0.1 g / L.

[0009] Preferably, the concentration of thiophanate-methyl is 0.15 g / L, and the concentration of ceftriaxone sodium is 0.05 g / L.

[0010] The present application also provides a culture medium for Populus seedlings, including a culture medium to which an antibacterial agent is added, and the antibacterial agent includes: thiophanate-methyl and ceftriaxone sodium; the concentration of thiophanate-methyl is 0.1g / L-0.15g / L, and the concentration of ceftriaxone sodium is 0.05g / L-0.1g / L.

[0011] As one aspect of the present invention, the culture medium adopts MS culture medium, and the MS culture medium is supplemented with 6-benzylaminopurine and naphthylacetic acid, the concentration of 6-benzylaminopurine is 0.2 mg / L, and the concentration of naphthylacetic acid is 0.02 mg / L-0.08 mg / L.

[0012] Preferably, the concentration of naphthylacetic acid is 0.04 mg / L.

[0013] As another aspect of the present invention, the culture medium adopts 1 / 2MS culture medium, and the 1 / 2MS culture medium is supplemented with indolebutyric acid, and the concentration of indolebutyric acid is 0.1-0.4 mg / L.

[0014] Preferably, the concentration of indolebutyric acid is 0.3 mg / L.

[0015] The present application also provides an open tissue culture method for Populus seedlings, comprising the following steps: obtaining test materials, bud induction culture, rooting culture, seedling hardening and transplanting;

[0016] The bud induction culture uses a differentiation medium, the differentiation medium uses an MS medium added with an antibacterial agent, the MS medium is added with 6-benzylaminopurine and naphthylacetic acid, the concentration of 6-benzylaminopurine is 0.2 mg / L, and the concentration of naphthylacetic acid is 0.02 mg / L-0.08 mg / L;

[0017] The rooting culture adopts a 1 / 2MS medium added with an antibacterial agent, wherein the 1 / 2MS medium is added with indolebutyric acid, and the concentration of the indolebutyric acid is 0.1-0.4 mg / L;

[0018] The antibacterial agent includes thiophanate-methyl and ceftriaxone sodium, the concentration of thiophanate-methyl is 0.1 g / L-0.15 g / L, and the concentration of ceftriaxone sodium is 0.05 g / L-0.1 g / L.

[0019] Furthermore, the step of obtaining the test material comprises: selecting a poplar leaf with a length greater than 1 cm, retaining a petiole of about 0.5 mm at the base of the leaf, cutting the main vein from one side of the leaf, and cutting it into a narrow and long leaf of 0.5-1 cm; the step of bud induction culture comprises: sterilizing the leaf and inoculating it in the differentiation culture medium, inoculating 10 leaves in each culture medium, repeating 3 times, the operation process is completed in an open environment, culturing for 40 days, with 12-14 hours of light per day, a light intensity of 2500Lx, and a culture temperature of (25±2)°C, to obtain a robust adventitious bud of about 2-3 cm in length; the step of rooting culture comprises: inoculating the leaf with the differentiation culture medium, 10 leaves in each culture medium, repeating 3 times, the operation process is completed in an open environment, culturing for 40 days, with 12-14 hours of light per day, a light intensity of 2500Lx, and a culture temperature of (25±2)°C, to obtain a robust adventitious bud of about 2-3 cm in length; the step of rooting culture comprises: The adventitious buds are inoculated into the rooting medium, and the rooting of the adventitious buds is observed. Each combination is repeated 10 times, and 3 adventitious buds are inoculated in each repetition to obtain sterile seedlings. The step of hardening the seedlings comprises: when the height of the sterile seedlings reaches 5-6 cm, hardening the seedlings, and transplanting them after hardening for 3 days; the step of transplanting comprises: preparing a matrix, the matrix comprises peat, perlite and river sand in a ratio of 1:1:1, sterilizing the matrix with high temperature and high pressure for 1 hour, taking out the regenerated seedlings after hardening, washing off the culture medium remaining on the roots, and then transplanting them into a non-woven fabric nutrient cup filled with the matrix, watering them thoroughly and keeping them moist, and culturing them in a greenhouse with the temperature controlled below 30°C.

[0020] Compared with the prior art, this application has the following beneficial effects:

[0021] Provided are an antibacterial agent, a culture medium and an open tissue culture method for poplar seedlings, wherein the antibacterial agent uses a combination of 0.1g / L-0.15g / L thiophanate-methyl and 0.05g / L-0.1g / L ceftriaxone sodium, and after the antibacterial agent is added to the culture medium, a lower contamination rate and a browning rate are obtained; in the bud induction culture step of the open tissue culture method, an MS culture medium added with an antibacterial agent is used, the MS culture medium is added with 6-benzylaminopurine and naphthaleneacetic acid, the concentration of 6-benzylaminopurine is 0.2mg / L, and the concentration of naphthaleneacetic acid is 0.02mg / L-0.08mg / L, and a higher induction rate and an average number of adventitious buds are obtained; in the rooting culture step of the open tissue culture method, a 1 / 2MS culture medium added with an antibacterial agent is used, the 1 / 2MS culture medium is added with indolebutyric acid, and the concentration of indolebutyric acid is 0.1-0.4mg / L, and a higher rooting rate is obtained, thereby realizing the open tissue culture of poplar seedlings and filling the gap in the prior art. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] Tissue culture is one of the important ways to produce seedlings, remove viruses, and improve germplasm. However, due to problems such as high technology and high costs, it has not been promoted and applied on a large scale in production. The open tissue culture of plants that has emerged in recent years adds antibacterial agents to the culture medium, does not require high-pressure sterilization, and does not require clean bench inoculation, which solves the problems of high tissue culture cost and low efficiency. In addition, tissue culture has the advantages of high uniformity of seedlings, virus-free and robustness, and breeding is not restricted by season. Therefore, its development prospects are very broad.

[0024] Xie Hui et al. established an open tissue culture technology system for banana by adding the antibacterial agent sodium hypochlorite to the culture medium. Huang Liejian et al. established an open tissue culture technology system for acacia by using the antibacterial agents thiophanate-methyl and carbendazim. Liu Yuxuan et al. established an open tissue culture system for rhododendron by sterilizing the culture medium with sodium hypochlorite. Xie Huifang et al. established an open tissue culture system for potato by controlling the fungi and bacteria in the culture medium using ciprofloxacin hydrochloride and itraconazole.

[0025] So far, there have been reports on open tissue culture of plants such as Rhododendron scabra, Dendrobium officinale, Prunus longifolia, Water chestnut, sugarcane, and konjac, but there are no systematic reports on the open tissue culture technology of Populus plants. Therefore, this specific implementation method uses Xinlin No. 1 Poplar as the material to study the open tissue culture technology system of Populus plants, study the conditions and methods of open tissue culture of Xinlin No. 1 Poplar, screen the types and concentrations of antibacterial agents, study whether the antibacterial agents have an effect on the growth and differentiation of Xinlin No. 1 Poplar leaves, as well as the induction differentiation and rooting culture of adventitious buds of Xinlin No. 1 Poplar leaves, establish the open tissue culture technology system of Xinlin No. 1 Poplar, provide a new method for the breeding of Populus seedlings, and provide technical support for high-quality, large-scale factory seedling cultivation.

[0026] The method for obtaining the test materials is as follows:

[0027] S1a, branches of perennial Xinlin No. 1 poplar with full leaf buds and no pests and diseases were collected in Heishan County, Liaoning Province, and placed in the greenhouse of Liaoning Poplar Research Institute for hydroponic treatment.

[0028] S1b, select poplar leaves with a length greater than 1 cm, retain a petiole of about 0.5 mm at the base of the leaf, cut the main vein from one side of the leaf, and cut it into a narrow leaf of 0.5-1 cm.

[0029] The test materials were sterilized and inoculated into the culture medium.

[0030] The key factor affecting the success of open tissue culture of plants is to find an antibacterial agent that can effectively control pollution and has no significant inhibitory effect on plant growth and differentiation.

[0031] Since antibacterial agents have certain differences in different culture environments and cultures of different species, it is difficult to discover universal antibacterial agents.

[0032] In this study, we first selected the low-cost chlorothalonil as an antibacterial agent for the open tissue culture of Xinlin No. 1 Poplar.

[0033] The experimental method is: five concentrations of thiophanate-methyl were set: 0.1, 0.2, 0.3, 0.4, and 0.5 g / L, with a total of five treatments. Ten leaves were inoculated in each treatment and repeated three times.

[0034] The test results are shown in Table 1.

[0035] Table 1 Effects of different concentrations of chlorothalonil on leaf regeneration of Xinlin No. 1 Populus

[0036]

[0037] Note: Different letters in the same column indicate extremely significant differences (P<0.01).

[0038] As shown in Table 1, the leaves achieved a higher survival rate in the MS medium supplemented with 0.3 g / L chlorothalonil, and the contamination was also controlled. Therefore, 0.3 g / L chlorothalonil was selected as the antibacterial agent to construct the Xinlin No. 1 poplar leaf regeneration system.

[0039] 0.3 g / L thiophanate-methyl was selected as the antibacterial agent. The leaves of the test materials were sterilized and inoculated on differentiation medium supplemented with different hormone concentrations. Ten leaves were inoculated in each differentiation medium and repeated three times. The operation process was completed in an open environment.

[0040] The test results are shown in Table 2.

[0041] Table 2 Bud induction culture of Xinlin No. 1 Poplar using 0.3g / L thiophanate-methyl as an antibacterial agent

[0042]

[0043] Note: Different letters in the same column indicate extremely significant differences (P<0.01). CK refers to the control group in which no antibacterial agent was added but the culture medium was sterilized by high temperature and high pressure.

[0044] BA in Table 2 refers to 6-benzylaminopurine, and NAA refers to naphthylacetic acid. As shown in Table 2, although thiophanate-methyl can effectively control pollution and ensure that the growth of explants is not affected, it has a certain inhibitory effect on leaf differentiation, and the induction rate obtained is low.

[0045] Therefore, after studying the characteristics and effects of various types of antibacterial agents, through repeated screening tests on different antibacterial agent combinations and concentrations, it was found that the antibacterial agent combination of thiophanate-methyl and ceftriaxone sodium was more effective as an antibacterial agent for open tissue culture of Xinlin No. 1 Poplar, as shown in Tables 3 and 4.

[0046] Table 3 Effects of antibacterial agent combinations and concentrations on leaf regeneration of Xinlin No. 1 Poplar

[0047]

[0048]

[0049] Note: Different letters in the same column indicate extremely significant differences (P<0.01).

[0050] Table 4 Induction of adventitious buds from leaves of Xinlin No.1 Poplar

[0051]

[0052] Note: Different letters in the same column indicate extremely significant differences (P<0.01). CK refers to the control group in which no antibacterial agent was added but the culture medium was sterilized by high temperature and high pressure.

[0053] BA in Table 4 refers to 6-benzylaminopurine, and NAA refers to naphthylacetic acid. It can be seen from Table 4 that the preferred concentration of 6-benzylaminopurine is 0.2 mg / L, and the preferred concentration of naphthylacetic acid is 0.04 mg / L.

[0054] The induction rate and average number of adventitious buds obtained by the above-mentioned hormone concentration were 91.7% and 7.5, respectively. The inhibitory effect on the differentiation and growth of explants was not significant, and did not reach a very significant level compared with conventional tissue culture, i.e., the control.

[0055] Based on the above experimental data, this specific embodiment provides an antibacterial agent suitable for Populus seedlings.

[0056] Among them, the antibacterial agents include: thiophanate-methyl and ceftriaxone sodium; the concentration of thiophanate-methyl is 0.1g / L-0.15g / L, and the concentration of ceftriaxone sodium is 0.05g / L-0.1g / L.

[0057] Preferably, the concentration of thiophanate-methyl is 0.15 g / L, and the concentration of ceftriaxone sodium is 0.05 g / L.

[0058] The antibacterial agent obtained by the preferred combination not only has a contamination rate and a browning rate of 0, but also has an antibacterial effect that is significantly higher than that of other combinations.

[0059] Furthermore, based on the above-mentioned antibacterial agent, this specific embodiment also provides a bud induction medium suitable for Populus seedlings.

[0060] The bud induction medium adopts MS medium, the sucrose concentration of MS medium is 25g / L, the agar concentration is 6g / L, the pH value is 6.0, 6-benzylaminopurine, naphthylacetic acid and antibacterial agent are added therein, the concentration of 6-benzylaminopurine is 0.2mg / L, and the concentration of naphthylacetic acid is 0.02mg / L-0.08mg / L.

[0061] The preparation method of the bud induction medium is as follows:

[0062] S1, MS was selected as the culture medium, with a sucrose concentration of 25 g / L and an agar concentration of 6 g / L.

[0063] S2, 6-benzylaminopurine and naphthylacetic acid were added to MS medium.

[0064] S3, adjust the pH of MS medium to 6.0 and adjust the pH of the medium with 1 mol / L NaOH.

[0065] S4, adding antibacterial agent.

[0066] The bud induction culture method is:

[0067] S1, using open inoculation method, the leaves of the test materials were sterilized and inoculated on different MS culture media, 10 leaves were inoculated on each differentiation medium, repeated 3 times, and the operation process was completed in an open environment;

[0068] S2, culture for 40 days, with 12-14 hours of light per day, 2500Lx of light intensity, and a culture temperature of (25±2)℃. Observe the differentiation of the leaves and keep records;

[0069] S3, obtain adventitious buds about 2-3 cm long and strong.

[0070] Furthermore, based on the above-mentioned antibacterial agent, this specific embodiment also provides a rooting culture medium suitable for Populus seedlings.

[0071] The rooting medium adopts 1 / 2MS medium, the sucrose concentration of 1 / 2MS medium is 25g / L, the agar concentration is 6g / L, the pH value is 6.0, indolebutyric acid and antibacterial agent are added, and the concentration of indolebutyric acid is 0.1-0.4mg / L.

[0072] The preparation method of rooting medium is as follows:

[0073] S1, select 1 / 2MS as the culture medium, sucrose concentration 25g / L, agar concentration 6g / L.

[0074] S2, indolebutyric acid was added to 1 / 2MS medium.

[0075] S3, 1 / 2MS medium pH was adjusted to 6.0, and 1 mol / L NaOH was used to adjust the pH of the medium.

[0076] S4, adding antibacterial agent.

[0077] The rooting culture method is:

[0078] S1, adventitious buds were inoculated on 1 / 2MS medium containing different concentrations of IBA (indolebutyric acid) for rooting culture, and the rooting of adventitious buds was observed. Each combination was repeated 10 times, and 3 explants were inoculated in each repetition;

[0079] S2, when the height of the sterile seedlings reaches 5-6cm, open the sealing film to harden the seedlings, and move them into the greenhouse after 3 days of hardening.

[0080] S3, continue to harden the seedlings in the greenhouse for about 5 days, or transplant directly.

[0081] The results of the rooting culture are shown in Table 5.

[0082] Table 5 Effects of different IBA concentrations on rooting

[0083]

[0084] It can be seen from Table 5 that the preferred concentration of indolebutyric acid is 0.3 mg / L.

[0085] Furthermore, based on the above-mentioned antibacterial agent, bud induction medium and rooting medium, this specific embodiment also provides an open tissue culture method suitable for Populus seedlings.

[0086] The open tissue culture method includes the following steps:

[0087] S1. Obtain test materials: select poplar leaves with a length greater than 1 cm, retain a petiole of about 0.5 mm at the base of the leaves, cut the main vein from one side of the leaves into narrow leaves of 0.5 to 1 cm, and then sterilize them.

[0088] S2, prepare bud induction medium: select MS medium with a sucrose concentration of 25 g / L and an agar concentration of 6 g / L, add 0.2 mg / L 6-benzylaminopurine and 0.02 mg / L-0.08 mg / L naphthaleneacetic acid to the MS medium, then adjust the pH of the MS medium to 6.0, and then add 0.1 g / L-0.15 g / L thiophanate-methyl and 0.05 g / L-0.1 g / L ceftriaxone sodium.

[0089] S3, bud induction culture: adopt open inoculation method, sterilize the leaves of the test materials and inoculate them on different MS culture media, inoculate 10 leaves for each differentiation culture medium, repeat 3 times, and the operation process is completed in an open environment; culture for 40 days, daily light exposure for 12 to 14 hours, light intensity of 2500Lx, culture temperature (25±2)℃, observe the differentiation of the leaves, and keep records; obtain healthy adventitious buds about 2-3cm in length.

[0090] S4, prepare rooting medium: use 1 / 2MS medium, add indolebutyric acid with a concentration of 0.1-0.4 mg / L, and then add thiophanate-methyl with a concentration of 0.1 g / L-0.15 g / L and ceftriaxone sodium with a concentration of 0.05 g / L-0.1 g / L into the 1 / 2MS medium.

[0091] S5, rooting culture of adventitious buds: adventitious buds were inoculated on rooting medium for rooting culture, and the rooting of adventitious buds was observed. Each combination was repeated 10 times, and 3 explants were inoculated in each repetition.

[0092] S6, when the sterile seedlings reach a height of 5-6 cm, harden the seedlings and transplant them 3 days later.

[0093] S7, prepare transplanting medium: the medium is made of peat, perlite and river sand, the ratio of peat: perlite: river sand = 1:1:1, sterilize at high temperature and high pressure for 1h.

[0094] S8, Transplanting regenerated seedlings: Take out the regenerated seedlings, wash off the culture medium remaining on the roots, and then transplant them into non-woven nutrient cups filled with matrix, water them thoroughly, and use bamboo poles to build a plastic shed to keep them moist. Cultivate them in a greenhouse and keep the temperature below 30°C. Too high a temperature will affect the transplant survival rate of the regenerated seedlings.

[0095] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. An antibacterial agent for Populus seedlings, characterized in that: include: thiophanate-methyl and ceftriaxone sodium; The concentration of thiophanate-methyl is 0.15 g / L, and the concentration of ceftriaxone sodium is 0.05 g / L.

2. A culture medium for Populus seedlings, characterized in that: The invention comprises a culture medium, wherein the antibacterial agent according to claim 1 is added to the culture medium.

3. The culture medium for Populus seedlings according to claim 2, characterized in that: The culture medium adopts MS culture medium, and the MS culture medium is supplemented with 6-benzylaminopurine and naphthylacetic acid, the concentration of 6-benzylaminopurine is 0.2 mg / L, and the concentration of naphthylacetic acid is 0.02 mg / L-0.08 mg / L.

4. The culture medium for Populus seedlings according to claim 3, characterized in that: The concentration of naphthylacetic acid is 0.04 mg / L.

5. The culture medium for Populus seedlings according to claim 2, characterized in that: The culture medium adopts 1 / 2MS culture medium, and the 1 / 2MS culture medium is added with indolebutyric acid, and the concentration of indolebutyric acid is 0.1-0.4 mg / L.

6. The culture medium for Populus seedlings according to claim 5, characterized in that: The concentration of indolebutyric acid was 0.3 mg / L.

7. An open tissue culture method for Populus seedlings, characterized in that: The following steps are involved: Obtaining experimental materials, bud induction culture, rooting culture, seedling hardening and transplanting; The bud induction culture uses a differentiation medium, and the differentiation medium adopts the medium according to claim 3 or 4; The rooting culture uses a rooting medium, and the rooting medium adopts the culture medium according to claim 5 or 6.

8. The open tissue culture method for Populus seedlings according to claim 7, characterized in that: The step of obtaining the test material comprises: selecting a poplar leaf with a length greater than 1 cm, retaining a petiole of about 0.5 mm at the base of the leaf, cutting the main vein from one side of the leaf into a narrow leaf of 0.5-1 cm; The bud induction culture step comprises: sterilizing the leaves and inoculating them into the differentiation medium, inoculating 10 leaves into each medium, repeating 3 times, the operation process is completed in an open environment, culturing for 40 days, with 12-14 hours of light per day, a light intensity of 2500Lx, and a culture temperature of (25±2)°C, to obtain adventitious buds with a length of 2-3cm and strong growth; The rooting culture step comprises: inoculating the adventitious buds into the rooting medium, observing the rooting of the adventitious buds, repeating each combination 10 times, inoculating 3 adventitious buds in each repetition, and obtaining sterile seedlings; The step of hardening the seedlings comprises: hardening the seedlings when the height of the sterile seedlings reaches 5-6 cm, and transplanting them after hardening the seedlings for 3 days; The transplanting step comprises: preparing a substrate, the substrate comprising peat, perlite and river sand in a ratio of 1:1:1, sterilizing the substrate with high temperature and high pressure for 1 hour, taking out the regenerated seedlings after hardening, washing away the culture medium remaining at the roots, and then transplanting them into a non-woven fabric nutrient cup containing the substrate, watering them thoroughly and keeping them moist, and culturing them in a greenhouse at a temperature below 30°C.

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