Tissue culture method for rapidly obtaining pear sterile in-vitro plant

Abstract

The invention relates to the technical field of plant in-vitro culture, in particular to a tissue culture method for rapidly obtaining a pear sterile in-vitro plant. The culture method comprises the following steps: collecting current-year new shoots, removing leaves, washing with water, wiping the surfaces with 75% alcohol, and irradiating and sterilizing under ultraviolet light to obtain pretreated new shoots; cutting the pretreated new tips into stem segments with axillary buds, soaking and disinfecting the stem segments in 75% alcohol and 0.05%-0.1% sodium hypochlorite in sequence, washing the stem segments with sterile water, and inoculating the stem segments into an MS culture medium for culture. According to the culture method provided by the invention, current-year new tips are adopted as explants for tissue culture, sterile in-vitro plants are germinated and grown in 6-7 days of culture, and the culture period is remarkably shortened. According to the method, a disinfection mode of 75% alcohol and low-concentration sodium hypochlorite is adopted, so that the high survival rate and the low browning rate of the explants can be ensured while the culture period is shortened, mercury bichloride is not needed, and the safety of the culture process is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of plant in vitro culture, and in particular to a tissue culture method for quickly obtaining sterile in vitro pear plants. Background Art

[0002] Pyrus plants have abundant germplasm resources in my country. To meet the needs of consumers, breeders have selected a large number of high-quality pear varieties with their own characteristics through various breeding methods such as hybridization, bud mutation selection, seed selection, and mutagenesis. According to statistics, 126 new pear varieties were bred in my country during the period of 1984-2015. Tissue culture technology is a new asexual reproduction technology developed in recent decades. It is also called in vitro culture. It refers to the method of separating tissues, organs or cells, protoplasts, etc. that meet the needs from the plant body, and inoculating them on a culture medium containing various nutrients and plant hormones under sterile conditions to obtain regenerated complete plants or produce other products with economic value. The use of tissue culture technology can not only preserve the excellent traits of different varieties and germplasms indoors, but also eliminate the limitations of seasonal factors, improve the reproduction coefficient, and provide a prerequisite for the rapid reproduction of high-quality germplasm and the acquisition of virus-free seedlings. In addition, in vitro plants also provide materials for indoor basic research and genetic transformation experiments, which have important application value.

[0003] However, in vitro culture, explant disinfection mostly uses mercuric chloride or sodium hypochlorite as the main disinfectant, and the bactericidal effect of mercuric chloride is excellent, but the toxicity is relatively large, and safe use cannot be guaranteed. Although sodium hypochlorite has a relatively low toxicity, the bactericidal effect is general, and pears belong to polysaccharide polyphenol plants, which are very easy to brown. The use of sodium hypochlorite will cause a higher browning rate while satisfying the bactericidal effect, and it is difficult to obtain a healthy and strong in vitro plant that survives. Therefore, adopting existing disinfection means, taking the young and tender new shoot stem section of pear as explant, the method of in vitro culture test tube seedling obtained by using mercuric chloride or sodium hypochlorite disinfection is not efficient. In this regard, the prior art of patent No. CN 115843689A adopts 75% ethanol to immerse and disinfect the branches of annual pear trees, and this method avoids the use of mercuric chloride with relatively large toxicity or the sodium hypochlorite that is easy to cause browning to disinfect, but it is necessary to first culture in the culture fluid for 3 to 5 weeks before germination to obtain the in vitro plant, and then disinfect and cultivate, and the overall cycle is relatively long. Summary of the invention

[0004] In view of this, the present invention provides a tissue culture method for quickly obtaining sterile in vitro pear plants, which greatly shortens the germination period of axillary buds of explants to 6 to 7 days, and can obtain a higher survival rate and a lower browning rate without using mercuric chloride with a good bactericidal effect, thereby significantly improving tissue culture efficiency.

[0005] In order to solve the above technical problems, the present invention provides a tissue culture method for quickly obtaining aseptic in vitro plants of pears, comprising the following steps: collecting new shoots of the current year, removing leaves, washing with water, wiping the surface with 75% alcohol, irradiating and sterilizing under ultraviolet light to obtain pretreated new shoots; cutting the pretreated new shoots into stem segments with axillary buds, soaking and disinfecting in 75% alcohol and 0.05%-0.1% sodium hypochlorite in sequence, then rinsing with sterile water and inoculating into MS culture medium for culture.

[0006] The present invention adopts the new shoots of the current year as explants for tissue culture. In order to avoid browning and death of the explants caused in the disinfection process, the present invention pre-treats the explants by additional water washing, alcohol wiping and ultraviolet irradiation before immersion disinfection, and removes most of the fungi and bacteria on the surface of the shoots in advance. After the pre-treatment, 75% alcohol and a sodium hypochlorite solution with a lower concentration are used in turn for immersion disinfection, thereby avoiding the use of mercuric chloride with greater toxicity.

[0007] In combination with the first aspect, when collecting new shoots, the new shoots of the current year can be collected from the pear trees with strong tree vigor in May. The present invention uses the new shoots of the current year as explants, which has the advantage of a shorter culture cycle than the existing one-year-old dormant branches.

[0008] In combination with the first aspect, after removing the new leaves, rinse them with clean water first, then wipe the surface 3 to 5 times with 75% alcohol.

[0009] In combination with the first aspect, after wiping the surface with 75% alcohol, sterilization is carried out under ultraviolet light, the intensity of which is 40 to 60 μw / cm 2 The ultraviolet light intensity in this range can effectively kill microorganisms such as bacteria, viruses and fungal spores on the surface of the explant without causing adverse effects on the explant itself.

[0010] Preferably, the intensity of the ultraviolet light is 50 μw / cm 2 .

[0011] In combination with the first aspect, the duration of the irradiation sterilization is 18 to 22 minutes, preferably 20 minutes.

[0012] Fungal and bacterial contamination mostly exists on the surface of branches and tips. Before soaking and disinfecting the collected explants, they should be washed with water, wiped with alcohol at least 3 times and irradiated with ultraviolet light in this order to remove most of the fungal contamination on the surface of branches and tips, thus effectively reducing the explant contamination rate.

[0013] In combination with the first aspect, the length of the stem segment with axillary buds is 1.5 to 2.5 cm. If the length of the stem segment is too long, it will easily lead to an increase in endophytic bacterial contamination, while if it is too short, the fungicide may cause additional damage to the base of the axillary bud.

[0014] Preferably, the length of the stem segment with axillary buds is 2 cm.

[0015] In combination with the first aspect, the stem segments with axillary buds are immersed in 75% alcohol for disinfection for 55 to 65 seconds.

[0016] In combination with the first aspect, the stem segments with axillary buds are immersed in 75% alcohol for disinfection, and then immersed in 0.05% sodium hypochlorite for disinfection for 3.5 to 4.5 minutes.

[0017] The present invention sequentially uses 75% alcohol and 0.05% sodium hypochlorite to disinfect the stem segments, thereby avoiding the use of highly toxic mercuric chloride and ensuring a higher survival rate and a lower browning rate.

[0018] In combination with the first aspect, the culture is carried out at 24-26° C. with light for 16 h / d.

[0019] Preferably, the culturing is carried out under light at 25°C.

[0020] In combination with the first aspect, the MS culture medium contains PVP-K30, and adding an appropriate concentration of PVP-K30 to ordinary MS culture medium can reduce the browning rate of tissue culture seedlings.

[0021] In combination with the first aspect, the concentration of PVP-K30 in the MS culture medium is 3.5-4.5 g / L, preferably 4 g / L.

[0022] In combination with the first aspect, the pear is selected from Huangguan pear, Xizilu pear, Fengshui pear or Conflans pear.

[0023] Beneficial effects obtained by the present invention: The tissue culture method for quickly obtaining sterile in vitro pear plants provided by the present invention uses the new shoots of the current year as explants for tissue culture, and germinates and grows sterile in vitro plants on the 6th to 7th day of culture, which significantly shortens the culture cycle. In addition, the present invention adopts the disinfection method of "75% alcohol + low-concentration sodium hypochlorite", so that the method can shorten the culture cycle while ensuring a high survival rate (up to 96% or more) and a low browning rate (the lowest is 0) of the explants, and the contamination rate is also less than 5%, which significantly improves the seedling efficiency; and the method avoids the use of highly toxic reagent mercuric chloride, greatly improving the safety of the culture process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The photos of the new shoots grown in the current year collected in Example 1 of the present invention;

[0025] Figure 2 The photos of the new shoots grown in the current year collected in Example 2 of the present invention;

[0026] Figure 3The following are photos of axillary buds obtained after 7 days of cultivation according to the cultivation method provided in Example 1. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] In the current in vitro culture method of pear, when the new tender shoots of the current year are used as explants, the commonly used disinfection method is to soak in 75% alcohol for about 20 seconds, rinse with sterile water for about 3 times, and then disinfect with 0.1% mercuric chloride for 8-10 minutes, and finally rinse with sterile water again; or soak in 2%-5% sodium hypochlorite solution for more than 10 minutes, then rinse with sterile water for about 5 times, and then disinfect with 0.1% mercuric chloride for about 10 minutes and rinse with sterile water for about 4 times. However, mercuric chloride is highly toxic, resulting in a high mortality rate in in vitro culture, while sodium hypochlorite has a general bactericidal effect and is prone to cause browning in in vitro culture, reducing the survival rate.

[0029] In view of this, the tissue culture method for quickly obtaining sterile in vitro pear plants provided by the present invention does not need to use highly toxic mercuric chloride, while ensuring a high survival rate and a low browning rate in in vitro culture; moreover, the culture cycle is significantly shortened.

[0030] In the following examples or comparative examples of the present invention, the available chlorine content of the sodium hypochlorite stock solution used in preparing sodium hypochlorite solutions of different volume concentrations is 10%, and the sodium hypochlorite concentration used in the examples or comparative examples is the available chlorine content after diluting the sodium hypochlorite stock solution.

[0031] The components and concentrations of the MS medium used in the following examples and comparative examples are: NH4NO3 (1650 mg / L), CaCl2 (332.2 mg / L), MgSO4 (180.7), KNO3 (1900 mg / L), KH2PO4 (170 mg / L), H3BO3 (6.2 mg / L), CoCl2·6H2O (0.025 mg / L), CuSO4·5H2O (0.025 mg / L), FeNaEDTA (36 .7mg / L), MnSO4·H2O (16.9mg / L), Na2MoO4·2H2O (0.25mg / L), KI (0.83mg / L), ZnSO4·7H2O (8.6mg / L), glycine (2mg / L), inositol (100mg), niacin (0.5mg / L), vitamin B6 (0.5mg / L), vitamin B1 (0.1mg / L), sucrose (30000mg / L), agar (7000mg / L).

[0032] Example 1

[0033] This embodiment provides a tissue culture method for rapidly obtaining sterile pear plants in vitro, the specific steps of which include: collecting the current year's new shoots of Huangguan pear trees ( Figure 1 ) as explants (length 64.5 cm, 12 leaves). Cut off the leaves, rinse the dust on the surface with clean water, and then wipe the surface of the explant three times with cotton dipped in 75% alcohol. 2 The pretreated surface was treated with ultraviolet light for 20 minutes.

[0034] Use sterilized scissors to cut the branches into 2 cm long stem segments with axillary buds, soak them in 75% alcohol for 60 seconds, then transfer them to 0.05% (volume fraction) sodium hypochlorite solution for 4 minutes, then rinse them with sterile water for 3 times, inoculate them into MS medium containing 4g / L polyvinyl pyrrolidone K30, and wait for growth at 25°C and a photoperiod of 16h / 8h. Axillary buds germinate on the 6th day of culture, and the germination status of axillary buds on the 7th day of culture is as follows: Figure 3 shown.

[0035] Example 2

[0036] This embodiment provides a tissue culture method for rapidly obtaining sterile pear plants in vitro, the specific steps of which include: collecting the current year's new shoots of Huangguan pear trees ( Figure 2 ) as explants (length 45.2 cm, 9 leaves). Cut off the leaves, rinse the dust on the surface with clean water, and then wipe the surface of the explant 5 times with cotton dipped in 75% alcohol. 2 The pretreated surface was treated with ultraviolet light for 22 minutes.

[0037] Use sterilized scissors to cut the branches into 1.5 cm long stem segments with axillary buds. First soak and disinfect them in 75% alcohol for 65 seconds, then transfer them to 0.08% (volume fraction) sodium hypochlorite solution and soak and disinfect them for 3.5 minutes, then rinse them three times with sterile water, inoculate them into MS medium containing 4 g / L polyvinyl pyrrolidone K30, and wait for growth at 24°C and a photoperiod of 16h / 8h.

[0038] Example 3

[0039] The present embodiment provides a tissue culture method for rapidly obtaining sterile pear in vitro plants, and the specific steps include: collecting the new shoots of the current year of Xizi Green Pear trees from the resource garden of Shijiazhuang Fruit Research Institute of Hebei Academy of Agricultural and Forestry Sciences as explants, cutting off the leaves, washing the dust on the surface with clean water, and then wiping the surface of the explants with cotton dipped in 75% alcohol for 5 times, and washing with 50 μw / cm 2 The pretreated surface was treated with ultraviolet light for 18 minutes.

[0040] Use sterilized scissors to cut the branches into 2.5 cm long stem segments with axillary buds, first soak and disinfect them in 75% alcohol for 55 seconds, then transfer them to 0.1% (volume fraction) sodium hypochlorite solution and soak and disinfect them for 4.5 minutes, then rinse them three times with sterile water, inoculate them into MS medium containing 4 g / L polyvinyl pyrrolidone K30, and wait for growth at 26°C and a photoperiod of 16h / 8h.

[0041] Comparative Example 1

[0042] This comparative example provides a tissue culture method for quickly obtaining sterile in vitro pear plants. The specific steps are similar to those in Example 1, with the only difference being that when pre-treating the collected new shoots, only the surface of the explant is rinsed with clean water, without the steps of alcohol wiping and ultraviolet light irradiation. The remaining steps are the same as those in Example 1.

[0043] Comparative Example 2

[0044] This comparative example provides a tissue culture method for quickly obtaining sterile in vitro pear plants. The specific steps are similar to those of Example 1, except that the ultraviolet light irradiation step is not used when pretreating the collected new shoots, that is, after washing the dust on the surface of the explant with clean water, the surface of the explant is wiped three times with cotton dipped in 75% alcohol to obtain the pretreated new shoots.

[0045] Comparative Example 3

[0046] This comparative example provides a tissue culture method for quickly obtaining sterile in vitro pear plants. The specific steps are similar to those in Example 1, except that the concentration of the sodium hypochlorite solution is increased from 0.05% to 2%, and the remaining steps are the same as those in Example 1.

[0047] Comparative Example 4

[0048] This comparative example provides a tissue culture method for quickly obtaining sterile in vitro pear plants. The specific steps are similar to those in Example 1, except that the explants used for culture are replaced by dormant buds of one-year-old branches instead of new shoots of the current year. The remaining steps are the same as those in Example 1.

[0049] Comparative Example 5

[0050] This comparative example provides a tissue culture method for quickly obtaining sterile in vitro pear plants. The specific steps are similar to those in Example 1, except that PVP-K30 is not added to the MS medium used, and the remaining steps are the same as those in Example 1.

[0051] Comparative Example 6

[0052] The present comparative example provides a tissue culture method for quickly obtaining sterile in vitro pear plants. The specific steps are similar to those of Example 1, except that the pretreated shoots are cultured after being treated according to the following steps: the branches are cut into 2 cm long stem segments with axillary buds using sterilized scissors, first soaked in 75% alcohol for 60 seconds for disinfection, then rinsed with sterile water for 3 times, inoculated into MS medium containing polyvinyl pyrrolidone K30 at a concentration of 4 g / L, and waited for growth at 25°C in an environment with a photoperiod of 16h / 8h.

[0053] Test example

[0054] The explants in Examples 1 to 3 and Comparative Examples 1 to 6 were cultured according to their respective corresponding treatment methods, the germination of the in vitro tissue culture seedlings in each Example and Comparative Example was observed, and the browning, survival rate and contamination rate of the explants in vitro culture were statistically analyzed. The results are shown in Table 1.

[0055] The calculation method of each parameter is as follows:

[0056] Contamination rate (%) = number of explants contaminated with bacteria or fungi / total number of explants × 100%,

[0057] Browning rate (%) = number of explants that died of browning / total number of explants × 100%,

[0058] Survival rate (%) = number of explants that germinated and survived / total number of explants × 100%.

[0059] Table 1

[0060]

[0062] From the data of Examples 1 to 3 in Table 1, it can be seen that by using the tissue culture method for rapidly obtaining sterile pear in vitro plants provided by the present invention, the explants can germinate in only 6 to 7 days, and the survival rate of the tissue seedlings after germination is above 90%, and the highest can reach above 96%; the browning rate is as low as 0, and as high as below 3%; the contamination rate is less than 5%. When the explants are not pretreated or sterilized by ultraviolet light (i.e., Comparative Examples 1 and 2), although they can still germinate on the 6th to 7th day, their final survival rate is less than 50%, and the contamination rate is also significantly increased. It can be seen from Comparative Example 3 that when the concentration of the sodium hypochlorite solution is increased from 0.05% to 2%, the browning rate reaches 100%, indicating that although the increase in the concentration of sodium hypochlorite can reduce the contamination rate, it is not suitable for disinfecting the tender shoots of the current year; it can be seen from Comparative Example 4 that when the dormant buds of one-year-old branches are cultivated using the same cultivation method as in Example 1, the survival rate, browning rate and contamination rate of the explants are equivalent to those in Example 1, but the germination time is extended to 24 days, and the cultivation efficiency is greatly reduced; it can be seen from Comparative Example 5 that when a certain concentration of polyvinyl pyrrolidone K30 is not added to the MS culture medium, the browning rate is significantly increased and the survival rate is also reduced, indicating that the addition of polyvinyl pyrrolidone K30 in the culture medium can reduce the browning rate of pear explants; it can be seen from Comparative Example 6 that when low concentration of sodium hypochlorite is not used for disinfection, the contamination rate of the explants is significantly increased, the survival rate is significantly decreased, and the seedling raising efficiency of tissue culture is reduced.

[0063] What is described above is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A tissue culture method for rapidly obtaining sterile pear plants in vitro, characterized in that: The following steps are involved: Collect the new shoots of the current year, remove the leaves, wash with water, wipe the surface with 75% alcohol, and sterilize under ultraviolet light to obtain pretreated new shoots; The pretreated new shoots are cut into stem segments with axillary buds, which are sequentially immersed in 75% alcohol and 0.05%-0.1% sodium hypochlorite for disinfection, then rinsed with sterile water and inoculated into MS culture medium for cultivation.

2. The tissue culture method for quickly obtaining aseptic in vitro pear plants as claimed in claim 1, characterized in that: In May, collect the new shoots of the year from healthy pear trees.

3. The tissue culture method for quickly obtaining aseptic in vitro pear plants as claimed in claim 1, characterized in that: The intensity of the ultraviolet light is 40-60 μw / cm 2 .

4. The tissue culture method for quickly obtaining aseptic pear plants in vitro as claimed in claim 1 or 3, characterized in that: The duration of the irradiation sterilization is 18 to 22 minutes.

5. The tissue culture method for quickly obtaining aseptic in vitro pear plants as claimed in claim 1, characterized in that: The length of the stem segment with axillary buds is 1.5 to 2.5 cm.

6. The tissue culture method for quickly obtaining aseptic pear plants in vitro as claimed in claim 1, characterized in that: Soak in 75% alcohol for 55 to 65 seconds for disinfection.

7. The tissue culture method for quickly obtaining aseptic pear plants in vitro as claimed in claim 1, characterized in that: Soak in 0.05% sodium hypochlorite for 3.5 to 4.5 minutes for disinfection.

8. The tissue culture method for quickly obtaining aseptic pear plants in vitro as claimed in claim 1, characterized in that: The culture is carried out at 24-26° C. with illumination for 16 h / d.

9. The tissue culture method for quickly obtaining aseptic in vitro pear plants as claimed in claim 1, characterized in that: The MS medium contains PVP-K30.

10. The tissue culture method for rapidly obtaining sterile pear in vitro plants as claimed in claim 9, characterized in that: The concentration of PVP-K30 in the MS culture medium is 3.5-4.5 g / L.

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