CN112655557B - Method for inhibiting endophytes of in vitro culture cucumber seedlings - Google Patents

Abstract

The invention discloses a method for inhibiting endophytes of in vitro culture cucumber seedlings, and belongs to the technical field of plant tissue culture. The method comprises the following steps: (1) taking stem sections of the tissue culture seedlings of the trichosanthes kirilowii, inoculating the stem sections into a cluster bud induction culture medium, and culturing to form cluster buds, wherein the cluster bud induction culture medium takes MS as a basic culture medium and is added with 6-benzylaminopurine and antibiotics; (2) taking stem segments of the cluster buds, inoculating the stem segments into a rooting culture medium, culturing the stem segments to grow roots of the buds to obtain rooted seedlings, wherein the rooting culture medium takes 1/2MS as a basic culture medium and is added with alpha-naphthylacetic acid and antibiotics; the antibiotic is rifampicin or ampicillin, and the concentration is 5-20 mg/L. According to the invention, rifampicin or ampicillin is added into the culture medium, and the formula of the culture medium is optimized, so that the proliferation of endophytes of the in vitro culture trichosanthes kirilowii seedling is effectively inhibited, the problem of endophyte pollution in the tissue culture process of the trichosanthes kirilowii is solved, and the problem of supply of high-quality seedlings of the trichosanthes kirilowii is solved.

Description

A kind of method for inhibiting the endophytic bacteria of in vitro culture of hanging gourd seedlings

technical field

The invention relates to the technical field of plant tissue culture, in particular to a method for inhibiting the in vitro culture of endophytic bacteria in D. gourd seedlings.

Background technique

Diaogua, also known as Trichosanthes, is a perennial herbaceous vine plant of the Cucurbitaceae family. It grows in the valleys, dense forests and sloping shrubs with a mild climate at an altitude of 100-1800 meters. It is a precious Chinese medicinal material. According to Chinese medicine documents such as Compendium of Materia Medica, Dictionary of Traditional Chinese Medicine, and Encyclopedia of Chinese Medicine, the skin, seeds and roots can be used as medicine, which has the functions of moistening the lungs and resolving phlegm, reducing fire and relieving cough. , wide chest Sanjie, swelling and detoxification, laxative effect. According to the "Compendium of Materia Medica", the seeds of Diaogua (Jianlou) are used for frying, tonic for dry mouth, moisturizing the heart and lungs, and treating wrinkles on the hands and faces. Modern research and authoritative institutions have detected that Diaogua seeds contain unsaturated fatty acids, protein, etc.

Due to its strong vitality, easy to learn and easy to grow, and convenient management, and the fact that it is invested in the same year, harvested in the same year, and profitable in a few years, it is favored by growers and once became a pillar industry in the emerging agricultural industry. Due to the extensive management mode adopted in the cultivation of hanging melons, endophytes are prone to appear in the hanging melon plants. That is, after 3-5 years of introduction, the yield of hanging gourd has decreased year by year, and the pests and diseases have become more and more serious.

Endophytic bacteria are those bacteria that live in the intercellular spaces or within the cells of various tissues and organs of healthy plants during certain or all stages of their life cycle, and can be obtained by histological methods or from strictly surface-sterilized plant tissues. Isolated, infected hosts do not (at least temporarily) show external symptoms, and in plant tissue culture these endophytic bacteria can grow and cause contamination of the tissue culture seedlings, causing severe losses. The conventional tissue culture method in the prior art can only kill surface bacteria, and endophytes often cause recontamination of the culture after culture. Therefore, the inhibition of endophytes is particularly important in plant tissue culture.

Patent document CN106106168A discloses a method for removing explant endophytes during plant tissue culture, including: using carbendazim to disinfect the cutting soil, and using a sterile aqueous solution containing amphotericin B and streptomycin sulfate to sterilize the cuttings. The branches are disinfected, and the semi-lignified new branches are pretreated, so that the bacteria and fungi on the surface and inside of explants such as poplar can be effectively removed.

At present, there is no research report on how to effectively solve the problem of endophyte contamination in the process of tissue culture of D. gourd, and it is urgent to develop a method for inhibiting the proliferation of endophytic bacteria in D. gourd tissue culture seedlings.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for inhibiting the endophytic bacteria of the in vitro culture of D. gourd seedlings, which can reduce the pollution of D. gourd tissue cultured seedlings endophytic bacteria, reduce production costs, realize the industrialized batch production of D. gourd seedlings, and can effectively Solve the problem of supplying high-quality seedlings of hanging gourd.

To achieve the above object, the present invention adopts the following technical solutions:

A method for inhibiting the in vitro culture of endophytic bacteria in Dang melon seedlings, comprising the following steps:

(1) get the stem section of the tissue culture seedlings of D. gourd, inoculate in the clump bud induction medium and cultivate to form the clump buds, and the clump bud induction medium takes MS as the basic medium, and is added with 6-benzylaminoadenine and antibiotics;

(2) take the clumps of shoots and stems obtained in step (1), inoculate in a rooting medium and cultivate to shoot and root to obtain a rooting seedling, and the rooting medium takes 1/2MS as a basic medium and is added with α-naphthalene acetic acid and antibiotics;

The antibiotic is rifampicin or ampicillin, and the concentration is 5-20 mg/L.

Specifically, Diaogua is "Yuelou No. 3".

Studies have shown that by adding rifampicin (rif) and ampicillin (amp) to the tissue culture medium, the proliferation of endophytes can be effectively inhibited, so that the tissue culture seedlings can grow normally, and a rapid propagation system of the dahlias can be established.

Further, rifampicin or ampicillin at a concentration of 10-15 mg/L is added to both the clump bud induction medium and the rooting medium. Under the above concentration conditions, it not only effectively inhibits the proliferation of endophytic bacteria in the tissue culture seedlings, but also has little effect on the growth of the tissue culture seedlings.

The concentration of 6-benzylaminoadenine in the cluster bud induction medium is 0.2-1 mg/L.

Further, the rifampicin with a concentration of 10mg/L or the ampicillin with a concentration of 15mg/L, and the 6-benzylaminoadenine with a concentration of 0.2mg/L are added to the cluster bud induction medium, and the pH value is 5.8 ±0.2.

The MS medium was composed of: 1650 mg/L NH ₄ NO ₃ , 1900 mg/L KNO ₃ , 440 mg/L CaCl ₂ ·2H ₂ O, 370 mg/L MgSO ₄ ·7H ₂ O, 170 mg/L KH ₂ PO ₄ , 0.83 mg/L KI, 6.2 mg/LH ₃ BO ₃ , 22.3 mg/L MnSO ₄ ·4H ₂ O, 8.6 mg/L ZnSO ₄ ·7H ₂ O, 0.25 mg/L Na ₂ MoO ₄ ·2H ₂ O, 0.025 mg/L CuSO ₄ ·5H ₂ O, 0.025mg/LCoCl ₂ ·6H ₂ O, 27.8FeSO ₄ ·7H ₂ O, 37.3mg/L Na ₂ .EDTA·2H ₂ O, 100mg/L inositol, 0.5mg/L L niacin, 0.5 mg/L pyridoxine hydrochloride, 0.1 mg/L thiamine hydrochloride, 2.0 mg/L glycine, 20 g/L sucrose.

Further, in step (1), the culture conditions are: 25±1° C., 2000Lx light intensity, and culture for 20 to 40 days under 12 hours of light every day.

Further, in step (2), α-naphthaleneacetic acid with a concentration of 0.1-0.5 mg/L is added to the rooting medium.

Furthermore, α-naphthaleneacetic acid with a concentration of 0.2 mg/L and rifampicin with a concentration of 10 mg/L are added to the rooting medium. Under the culture conditions, the rooting rate is high, and the obtained rooted seedlings are easy to transplant and survive.

The 1/2MS medium is composed of: 825mg/L NH ₄ NO ₃ , 950mg/L KNO ₃ , 220mg/L CaCl ₂ ·2H ₂ O, 185mg/L MgSO ₄ ·7H ₂ O, 85mg/L KH ₂ PO ₄ , 0.83mg/L KI, 6.2mg/LH ₃ BO ₃ , 22.3mg/L MnSO ₄ ·4H ₂ O, 8.6mg/L ZnSO ₄ ·7H ₂ O, 0.25mg/L Na ₂ MoO ₄ ·2H ₂ O , 0.025mg/L CuSO ₄ ·5H ₂ O, 0.025mg/LCoCl ₂ ·6H ₂ O, 27.8FeSO ₄ ·7H ₂ O, 37.3mg/L Na ₂ .EDTA · 2H ₂ O, 100mg/L inositol, 0.5 mg/L niacin, 0.5 mg/L pyridoxine hydrochloride, 0.1 mg/L thiamine hydrochloride, 2.0 mg/L glycine, 20 g/L sucrose.

Further, in step (2), the cultivation conditions are: 25±1° C., 2000Lx light intensity, and light for 12 hours a day.

Compared with the prior art, the present invention has the following beneficial effects:

By adding rifampicin (rif) or ampicillin (amp) to the culture medium, the invention effectively inhibits the proliferation of endophytic bacteria in the cultured D. gourd seedlings in vitro, and solves the problem of endophytic bacteria contamination in the process of tissue culture of D. gourd. By optimizing the medium formula, establishing a rapid propagation system of D. gourd, reducing the comprehensive production cost, and improving the stability of its characters, the factory-like mass production of D. gourd seedlings can be realized, and the problem of supplying high-quality D. gourd seedlings can be solved.

Description of drawings

Fig. 1 is the effect of adding rifampicin (rif) in the medium in embodiment 1 to inhibit the contamination of endophytic bacteria in tissue culture seedlings of D. gourd; A, adding 10 mg/L rifampicin in the medium, the D. gourd group during culturing 22d Cultivation, no endophyte contamination at the base of the tissue culture seedlings; B, no rifampicin added to the medium, and the tissue culture seedlings of D. gourd at 22 d have serious endophyte contamination at the base of the tissue culture seedlings.

Fig. 2 is the rooting seedlings of the aseptic stem sections of Dang melon after being cultivated on rooting medium for 20 days, from left to right, the rooting plants containing 0mg/L, 0.1mg/L, 0.2mg/L, 0.5mg/L NAA Rooted shoots grown on medium.

Detailed ways

The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

Materials used in the examples: the tissue culture seedlings of Diaogua "Yuelou No. 3".

The reagents involved in the examples are all marketable commodities;

The MS medium consists of: macroelements: 1650mg/L NH ₄ NO ₃ , 1900mg/L KNO ₃ , 440mg/LCaCl ₂ ·2H ₂ O, 370mg/L MgSO ₄ ·7H ₂ O, 170mg/L KH ₂ PO ₄ ;trace elements: 0.83mg/L KI, 6.2mg/LH ₃ BO ₃ , 22.3mg/L MnSO ₄ ·4H ₂ O, 8.6mg/L ZnSO ₄ ·7H ₂ O, 0.25mg/L Na ₂ MoO ₄ ·2H ₂ O, 0.025mg/LCuSO ₄ ·5H ₂ O, 0.025mg/L CoCl ₂ ·6H ₂ O, 27.8FeSO ₄ ·7H ₂ O, 37.3mg/L Na ₂ .EDTA·2H ₂ O; organic matter: 100mg/L Inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2.0mg/L glycine, 20g/L sucrose.

The composition of 1/2MS medium is as follows: the major elements are reduced by half, and the remaining components are the same as MS medium.

Example 1

1. Configure the cluster bud induction medium: MS + 0.2 mg/L 6-BA (6-benzylaminoadenine) + different concentrations of rifampicin (rif).

2. Antibacterial treatment of different concentrations of rifampicin (rif): On the ultra-clean bench, clip the tissue culture seedlings of Dang melon from the bottle, cut them into 3cm long sections, and connect them with 0mg/L and 5mg/L respectively. , 10mg/L, 15mg/L, 20mg/L rifampicin (rif) in the cluster bud induction medium, place the culture room, the temperature is 24 ± 1 ℃, and the light is 12 hours a day.

3. Observation record of antibacterial effect: After culturing for 22d and 45d, record the average seedling height (cm), the average number of clustered buds (a), and the antibacterial rate. The results are shown in Figure 1 and Table 1.

Table 1 Rifampicin (Rif) inhibits the effect of D. gourd tissue culture seedlings

As can be seen from Table 1, the bacteriostatic rate of the medium with rifampicin concentration of 0 mg/L was 0 in 22 days and 45 days of culture. In the medium with rifampicin concentration of 5 mg/L, the bacteriostatic rate was 33.3%, but after 45 days of culture, the bacteriostatic rate was 0 again. It shows that the low concentration of inhibitor can not inhibit the endophyte of D. gourd, and the medium with rifampicin concentration of 10mg/L has the highest bacteriostatic rate, the bacteriostatic rate is 66.7% at 22 days, and the bacteriostatic rate is 55.6% at 45 days. %. When the concentration reached 20mg/L, the bacteriostatic rate began to decline. The average number of clump buds changed between 22 days and 45 days of culture. The overall experimental observation shows that when the concentration of rifampicin is 5mg/L and 10mg/L, the average number of clump buds is more, and the highest of up to 2. As for the average seedling height, with the growth of time, the tissue culture seedlings of Dang melon are growing continuously. From the experimental data, the average seedling height is higher than other concentrations when the concentration of rifampicin is 5mg/L and 10mg/L. .

In general, when the concentration of rifampicin was 10 mg/L, the antibacterial rate of the tissue culture seedlings of D. gourd was the highest, and the average number of clustered buds and the average seedling height were the highest.

4 Conclusion

In the subculture of D. gourd tissue culture seedlings, adding 10 mg/L rifampicin to the medium not only inhibited the proliferation of endophytic bacteria in D. gourd tissue-cultured seedlings, but also had little effect on the growth of tissue-cultured seedlings.

Example 2

1. Configure clump bud induction medium: MS+0.2mg/L 6-BA+ampicillin of different concentrations.

2. Antibacterial treatment of different concentrations of ampicillin (amp): On the ultra-clean bench, take out the tissue culture seedlings of hanging melon from the bottle, cut them into 3cm long sections, and connect them with 0mg/L, 5mg/L, In the medium of 10mg/L, 15mg/L, 20mg/L ampicillin, place the culture room, the temperature is 24±1℃, and the light is 12 hours a day.

3. Observation record of antibacterial effect: After culturing for 22d and 45d, record the average seedling height (cm), the average number of clustered buds (piece), and the antibacterial rate. The results are shown in Table 2.

Table 2 The effect of ampicillin (Amp) on the inhibition of endophyte in T. gourd tissue culture seedlings

It can be seen from Table 2 that the bacteriostatic rate of the medium with an ampicillin concentration of 0 mg/L was 0 in 22 days and 45 days of culture. The bacteriostatic rate was 32.7% in the medium with ampicillin concentration of 5 mg/L, but after 45 days of culture, the bacteriostatic rate was 0 again. It shows that the low concentration of inhibitor cannot inhibit the endophyte of D. gourd, and the medium with the concentration of 15 mg/L of ampicillin has the highest bacteriostatic rate, the bacteriostatic rate is 58.7% at 22 days, and the bacteriostatic rate is 53.7% at 45 days. . When the concentration of ampicillin reached 20mg/L, the bacteriostatic rate began to decline. For the number of clump buds, it changed between 22 days and 45 days of culture. The overall experimental observation shows that when the concentration of ampicillin is 10mg/L and 15mg/L, the average number of clump buds is more, and the highest can be Reach 2.18cm. The average number of clump buds in the medium of other concentrations was close to 1-2. For the average seedling height, with the growth of time, the tissue culture seedlings of Dang melon are growing continuously. From the experimental data, the average seedling height is higher than other concentrations when the concentration of ampicillin is 10mg/L and 15mg/L.

From Table 2, with the change of time, the seedling height of the tissue culture seedlings of D. gourd cultivated on 0 mg/L penicillin medium increased from 0.94 cm to 1.76 cm; The seedling height of the melon tissue-cultured seedlings increased from 0.76cm to 1.76cm; the seedling height of the melon tissue-cultured seedlings cultivated in the medium with a concentration of 15mg/L penicillin increased from 1.38cm to 2.15cm. As for the seedling height, it generally increased with the change of time, and the average seedling height was slightly higher than other concentrations at 45 days when the penicillin concentration was 15 mg/L.

In general, when the penicillin concentration was 15 mg/L, the bacteriostasis rate of the tissue cultured seedlings was higher, while for the average number of clustered buds, the proliferation and the average seedling height were the highest when the penicillin concentration was 15 mg/L at 45 days.

4 Conclusion

In the subculture of D. gourd tissue culture seedlings, adding 15mg/L ampicillin to the medium not only inhibits the proliferation of endophytic bacteria in D. gourd tissue culture seedlings, but also has little effect on the growth of tissue culture seedlings.

Example 3

1. Conditions for rooting culture

The clump buds obtained by the proliferation and culture of Example 1 and Example 2 were cut into 3 cm long stem segments, inoculated in a rooting medium, the basic medium of the rooting medium was 1/2MS, and 0mg/L, 0.1mg/L were added respectively. L, 0.2mg/L, 0.5mg/L NAA (α-naphthalene acetic acid), all added 10mg/L rifampicin. Culture at 25±1°C, 2000Lx light intensity, 12 hours of light per day.

2. Observation record of rooting rate

After the stem segments were cultured on the rooting medium for 20 days, the rooting rate was recorded; the results are shown in Figure 2 and Table 3.

Table 3 Effects of different concentrations of NAA on the rooting of aseptic seedlings of Dang melon

NAA concentration (mg/L) Rooting rate (%) callus at the base of the seedling 0 13.3 0.1 46.7 + 0.2 83.3 ++ 0.5 90.0 ++++

It can be seen from Table 3 that when 0.5 mg/L NAA was added to the medium, the rooting rate was the highest, reaching 90.0%, but the base of the seedlings became seriously callus, and the rooted seedlings were difficult to transplant and survive. When 0.2 mg/L NAA was added to the medium, the rooting rate was 83.3%, and the base of the seedling was slightly callus, and the rooted seedling was easy to transplant and survive. When 0.1 mg/L NAA was added to the medium, the rooting rate was 46.7%.

3. Conclusion

In general, the NAA concentration is in the range of 0-0.5mg/L, and it is most suitable to add 0.2mg/L NAA to the rooting medium.

Claims (8)

1. a method for suppressing the in vitro culture of Dang melon seedling endophyte, is characterized in that, comprises the following steps: (1) get the stem section of the tissue culture seedlings of D. gourd, inoculate in the clump bud induction medium and cultivate to form the clump buds, and the clump bud induction medium takes MS as the basic medium, and is added with 6-benzylaminoadenine and antibiotics; (2) take the clumps of shoots and stems obtained in step (1), inoculate in a rooting medium and cultivate to shoot and root to obtain a rooting seedling, and the rooting medium takes 1/2MS as a basic medium and is added with α-naphthalene acetic acid and antibiotics; The antibiotic is rifampicin or ampicillin, the concentration of rifampicin is 10 mg/L, and the concentration of ampicillin is 10-15 mg/L. 2. the method for suppressing in vitro culturing endophyte of Dang melon seedlings as claimed in claim 1, is characterized in that, in step (1), in described clump bud induction medium, adding the rifampin that concentration is 10mg/L is added with Ampicillin at a concentration of 15 mg/L or 6-benzylaminoadenine at a concentration of 0.2 mg/L, pH 5.8±0.2. 3. the method for suppressing the in vitro culture of the endophytes of the D. gourd seedlings as claimed in claim 1, is characterized in that, described MS medium consists of: 1650mg/L NH ₄ NO ₃ , 1900mg/L KNO ₃ , 440mg/ L CaCl ₂ ·2H ₂ O, 370 mg/L MgSO ₄ ·7H ₂ O, 170 mg/L KH ₂ PO ₄ , 0.83 mg/L KI, 6.2 mg/LH ₃ BO ₃ , 22.3 mg/L MnSO ₄ ·4H ₂ O , 8.6mg/L ZnSO ₄ ·7H ₂ O, 0.25mg/L Na ₂ MoO ₄ ·2H ₂ O, 0.025mg/L CuSO ₄ ·5H ₂ O, 0.025mg/L CoCl ₂ ·6H ₂ O, 27.8FeSO ₄ 7H ₂ O, 37.3 mg/L Na ₂ .EDTA 2H ₂ O, 100 mg/L inositol, 0.5 mg/L niacin, 0.5 mg/L pyridoxine hydrochloride, 0.1 mg/L thiamine hydrochloride, 2.0 mg/L glycine, 20g/L sucrose. 4. the method for suppressing the in vitro culture of the endophytic bacteria of D. gourd seedlings as claimed in claim 1, is characterized in that, in step (1), culture condition is: 25 ± 1 ℃, 2000Lx light intensity, 12 hours light condition every day Cultivated for 20 to 40 days. 5. the method for suppressing the in vitro culture of the endophytic bacteria of D. gourd seedlings as claimed in claim 1, is characterized in that, in step (2), in described rooting medium, add the α that concentration is 0.1～0.5mg/L - Naphthalene acetic acid. 6. the method for suppressing the in vitro culture of D. gourd seedling endophyte as claimed in claim 5, is characterized in that, in step (2), in described rooting medium, adding the α-naphthalene that concentration is 0.2mg/L Acetic acid, rifampicin at a concentration of 10 mg/L. 7. the method for suppressing the in vitro culture of the endophyte of D. gourd seedlings as claimed in claim 1, is characterized in that, described 1/2MS medium consists of: 825mg/L NH ₄ NO ₃ , 950mg/L KNO ₃ , 220 mg/L CaCl ₂ ·2H ₂ O, 185 mg/L MgSO ₄ ·7H ₂ O, 85 mg/L KH ₂ PO ₄ , 0.83 mg/L KI, 6.2 mg/LH ₃ BO ₃ , 22.3 mg/L MnSO ₄ ·4H ₂ O, 8.6 mg/L ZnSO ₄ ·7H ₂ O, 0.25 mg/L Na ₂ MoO ₄ ·2H ₂ O, 0.025 mg/L CuSO ₄ ·5H ₂ O, 0.025 mg/L CoCl ₂ ·6H ₂ O, 27.8 FeSO ₄ ·7H ₂ O, 37.3mg/L Na ₂ .EDTA·2H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride , 2.0mg/L glycine, 20g/L sucrose. 8. the method for suppressing the endophyte of in vitro culturing of D. gourd seedlings as claimed in claim 1, is characterized in that, in step (2), the condition of cultivating: 25 ± 1 ℃, 2000Lx light intensity, 12 hours of light every day.

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