Induction method for bamboo reed chromosome doubling

Abstract

The invention provides an induction method for chromosome doubling of bamboo reed, which comprises the following steps: firstly, selecting a healthy stem with axillary buds of bamboo reed without diseases and insect pests as an explant material, and inoculating the stem into an adventitious bud induction culture medium for propagation culture; then selecting tissue culture seedlings which are cultured in the culture medium for 28-30 days and are 3cm in height as mutagenic materials, soaking the tissue culture seedlings in colchicine, and culturing the tissue culture seedlings in a rooting culture medium. According to the method, the problem of bamboo reed germplasm resource creation is effectively solved, and the culture method suitable for inducing chromosome doubling of bamboo reed is determined through a sensitivity test of the explant to colchicine; a bamboo reed explant material is utilized for variety improvement, colchicine is adopted for inducing chromosome doubling, a large number of variant strains are obtained, germplasm is innovated, a germplasm resource library is established, and a technical support is provided for cultivating a new bamboo reed variety with high yield value.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic breeding, and particularly relates to a method for inducing chromosome doubling of Arundo donax L. Background Art

[0002] Arundo donax L. is a tall herbaceous plant of the genus Arundo in the Poaceae family. Arundo donax L. can grow directly in water or in the surrounding riparian areas; it is commonly found near rivers, lakes, ponds, and swamps, and also has characteristics such as salt tolerance, barren tolerance, low-temperature tolerance, strong adaptability, high water use efficiency, and high photosynthetic efficiency. The plant height of Arundo donax L. can reach 8 - 10 meters, the stem diameter can reach 3 - 4 centimeters, and the roots can reach 5 meters; it has a high protein content and a moderate fiber content, and can be used as a high-quality feed raw material. Arundo donax L. has high comprehensive utilization value and can be used for the production of edible and medicinal fungi, the production of pulp, boards, etc., and can also be used for biomass energy power generation and biogas production. Arundo donax L. is well-known for its environmental and ecological benefits and is considered a good phytoremediation tool. It can be used to clean contaminated soil and water, and can also effectively remove heavy metals and other pollutants in contaminated sites, and is a valuable resource for environmental remediation.

[0003] Polyploid breeding is an effective way to improve plant varieties, and polyploid plants with characteristics such as giant organs and enhanced resistance can be obtained. Chemical mutagenesis is the most commonly used and cost-effective artificial induction method. Using artificial induction to obtain polyploids has a high mutagenesis frequency and good effects, and it has currently been widely used in plant breeding and improvement, but there is no report on Arundo donax L. Therefore, it is urgent to develop and utilize the germplasm resources of the genus Arundo and cultivate new varieties belonging to our country. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for inducing chromosome doubling of Arundo donax L. to solve the deficiencies in verifying the security and reliability of the operating system kernel in the prior art.

[0005] To achieve the above purpose, the present invention adopts the following technical scheme: A method for inducing chromosome doubling of Arundo donax L., comprising the following steps:

[0006] S1: Select healthy and pest-free Arundo donax L. stem segments with axillary buds as explant materials, and culture the explants in an adventitious bud induction medium;

[0007] S2: Select tissue culture seedlings that have been cultured in the pre-culture medium in S1 for 28 - 30 days and have a seedling height of 3 cm as mutagenesis materials, soak them in colchicine, and then culture them through a rooting medium.

[0008] It can be seen from the above technical scheme that compared with the prior art, the present invention has the following beneficial effects:

[0009] 1. The operation of the present invention is simple. A suitable germplasm resource mutagenesis technology system is determined by determining the size of mutagenic materials, the concentration of colchicine, and the soaking time of colchicine, etc.

[0010] 2. Low input, strong safety, and high efficiency. This chromosome doubling scheme only requires general tissue culture facilities to meet the needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a schematic flow chart of the steps of the induction method for doubling the chromosomes of Arundo donax in the present invention;

[0012] Figure 2 Figure A in is the phenotype diagram of the explant of the stem segment with axillary buds of the female parent of Arundo donax after growing for 10 days; Figure B is the tissue culture seedling of Arundo donax after 30 days of adventitious bud induction culture, and the scale bar is 1 cm;

[0013] Figure 3 It is the growth phenotype of the tissue culture seedlings of Arundo donax at 30 days after being treated with colchicine at different concentrations for different times, and the scale bar is 1 cm;

[0014] Figure 4 Figure A in is the diagram of the tissue culture seedlings of Arundo donax after growing for 4 months in the control group; Figure B is the tissue culture seedlings of Arundo donax after growing for 4 months after being treated with 0.2% colchicine for 24 hours for chromosome doubling, and the scale bar is 20 cm;

[0015] Figure 5 Figure A in is the flow cytometry histogram of the control group; Figure B is the flow cytometry histogram of the group treated with 0.2% colchicine for 24 hours for chromosome doubling;

[0016] Figure 6 Figure A in is the chromosome counting diagram of the control group; Figure B is the chromosome counting diagram of the group treated with 0.2% colchicine for 24 hours for chromosome doubling, and the scale bar is 10 μm. DETAILED DESCRIPTION OF THE INVENTION

[0017] The following is a detailed description of a preferred embodiment of the present invention with reference to the accompanying drawings.

[0018] All the Arundo donax described in this preferred embodiment are planted in the experimental field of the College of Agriculture, Zhengzhou University.

[0019] Example 1

[0020] As Figure 1 shown, the induction method for doubling the chromosomes of Arundo donax includes the following steps:

[0021] S1. Select the stem segments with axillary buds of healthy Arundo donax without pests and diseases as explant materials, and culture the explants in the subculture medium;

[0022] S2. Select the Arundo donax tissue culture seedlings that have been cultured in the above adventitious bud induction medium for 28 - 30 days and have a seedling height of 3 cm as mutagenic materials. After being soaked with colchicine, they are cultured through the rooting medium.

[0023] Furthermore, select the axillary bud - bearing stem segments of healthy Arundo donax without pests and diseases as explant materials, and conduct explant pre - culture in the adventitious bud induction medium;

[0024] The formula of the adventitious bud induction medium is: 0.7% agar powder, 3% sucrose, 0.44% MS medium, 6 - BA concentration of 4 mg / L, IBA concentration of 1 mg / L; the culture conditions are: temperature 25°C, daily light duration of 16 h, light intensity of 18000 - 20000 lux, humidity of 60%, and the pH value of the medium is 5.8 - 6.0;

[0025] Furthermore, the selection of the tissue culture seedlings that have been cultured in the above adventitious bud induction medium for 28 - 30 days and have a seedling height of 3 cm as mutagenic materials, and the culture through the rooting medium after being soaked with colchicine specifically includes the following steps:

[0026] First, use the tissue culture seedlings that have been adventitious bud - induced for 28 - 30 days and have a seedling height of 3 cm as mutagenic materials, and soak them with 0.2% colchicine for 24 h;

[0027] Then inoculate the soaked tissue culture seedlings into the rooting medium for rooting culture;

[0028] The formula of the rooting medium is: 0.7% agar powder, 3% sucrose, 0.23% 1 / 2MS medium, 6 - BA concentration of 1 mg / L, IBA concentration of 1 mg / L, NAA concentration of 5 mg / L; the culture conditions are: temperature 25°C, daily light duration of 16 h, light intensity of 18000 - 20000 lux, humidity of 60%, and the pH value of the medium is 5.8 - 6.0;

[0029] Furthermore, conduct acclimatization treatment on the explants that have rooted in the above rooting medium for 30 days, observe their phenotypes after four months of soil cultivation, and conduct ploidy identification.

[0030] Example 2

[0031] As Figure 1 shown, the induction method for chromosome doubling of Arundo donax includes the following steps:

[0032] S1. Select the axillary bud - bearing stem segments of healthy Arundo donax without pests and diseases as explant materials, and conduct explant culture in the sub - culture medium;

[0033] S2. Select the Arundo donax tissue culture seedlings that have been cultured in the above adventitious bud induction medium for 28 - 30 days and have a seedling height of 3 cm as mutagenic materials, and culture them through the rooting medium after being soaked with colchicine.

[0034] Furthermore, select the healthy Arundo donax var. lanceolata stem segments with axillary buds as explant materials, and conduct pre - culture of the explants in the adventitious bud induction medium;

[0035] The formula of the adventitious bud induction medium is: 0.7% agar powder, 3% sucrose, 0.44% MS medium, 6 - BA concentration of 4 mg / L, IBA concentration of 1 mg / L; the culture conditions are: temperature 25°C, daily light duration of 16 h, light intensity of 18000 - 20000 lux, humidity of 60%, and the pH value of the medium is 5.8 - 6.0;

[0036] Furthermore, the selection of the tissue culture seedlings that have been cultured in the above adventitious bud induction medium for 28 - 30 days and have a seedling height of 3 cm as mutagenic materials, and the culture through the rooting medium after being soaked with colchicine specifically includes the following steps:

[0037] First, use the tissue culture seedlings that have been adventitious bud - induced for 28 - 30 days and have a seedling height of 3 cm as mutagenic materials, and soak them with colchicine at a concentration of 0.1% for 48 h;

[0038] Then inoculate the soaked tissue culture seedlings into the rooting medium for rooting culture;

[0039] The formula of the rooting medium is: 0.7% agar powder, 3% sucrose, 0.23% 1 / 2MS medium, 6 - BA concentration of 1 mg / L, IBA concentration of 1 mg / L, NAA concentration of 5 mg / L; the culture conditions are: temperature 25°C, daily light duration of 16 h, light intensity of 18000 - 20000 lux, humidity of 60%, and the pH value of the medium is 5.8 - 6.0;

[0040] Furthermore, conduct acclimatization treatment on the explants that have rooted in the above rooting medium for 30 days, observe their phenotypes after four months of soil cultivation, and conduct ploidy identification.

[0041] In order to obtain a large number of mutant plants in a short time, combine the mature plant tissue culture technology and chromosome doubling technology in this laboratory to establish a polyploid mutagenesis system with easy - to - control environmental conditions, and conduct exploratory experiments on the in vitro doubling method of Arundo donax. The experiment is specifically carried out through the following steps:

[0042] First, use the tissue culture seedlings of explants that have been induced to form adventitious buds for 30 days and have a seedling height of 3 cm as mutagenesis materials, and use the following 9 treatment methods to culture the explants respectively: A1: colchicine concentration 0.1%, soaking time 24 h; A2: colchicine concentration 0.1%, soaking time 48 h; A3: colchicine concentration 0.1%, soaking time 72 h; A4: colchicine concentration 0.2%, soaking time 24 h; A5: colchicine concentration 0.2%, soaking time 48 h; A6: colchicine concentration 0.2%, soaking time 72 h; A7: colchicine concentration 0.3%, soaking time 24 h; A8: colchicine concentration 0.3%, soaking time 48 h;

[0043] A9: colchicine concentration 0.3%, soaking time 72 h, and place it in a shaker with a rotation speed of 80 revolutions per minute at 25°C to keep the explants soaked continuously; after the soaking time ends, wash it six times with sterile water, 30 s each time, then transfer the tissue culture seedlings to sterile filter paper to absorb the surface moisture, and finally inoculate them on the above rooting medium, 7 tissue culture seedlings per bottle, and repeat 10 bottles per group;

[0044] The formula of the rooting medium is: 0.7% agar powder, 3% sucrose, 0.23% 1 / 2 MS medium, 6-BA concentration 1 mg / L, IBA concentration 1 mg / L, NAA concentration 5 mg / L; the culture conditions are: temperature 25°C, daily light time 16 h, light intensity 18000 - 20000 lux, humidity 60%, medium pH value 5.8 - 6.0, culture for 30 days, record the growth status of the explants and observe and record the data;

[0045] After the roots grow, perform acclimatization treatment. After four months of soil cultivation, use the leaves as materials and detect their doubling status with a flow cytometer. The experimental data are shown in Table 1 below;

[0046] Table 1 Effects of different concentrations of colchicine and treatment time on the survival and doubling of Arundo donax

[0047]

[0048]

[0049] From the experimental comparative data, it can be seen that the survival rates of A1, A2, and A4 are very high, all above 20%. However, A1 has no mutagenesis rate, the doubling mutagenesis rate of A2 is 3%, and the doubling rate of A4 is the highest. That is, when the colchicine concentration is 0.2% and the soaking time is 24 h, the survival rate is 28%, and the chromosome doubling rate is the highest, reaching 7%. Therefore, A4 is the best mutagenic combination. When the colchicine concentration of A2 is 0.1% and the treatment time is 48 h, chromosome-doubled plants can also be obtained. The survival rates of the combinations of A3, A5, A6, A7, A8, and A9 are very low, all below 5%, and their mutagenesis rates are 0. This may be due to the high concentration of the mutagen being toxic and the long treatment time causing great harm to the tissue culture seedlings.

[0050] Therefore, in this preferred embodiment, tissue culture seedlings cultured in the adventitious bud induction medium for 30 days with a seedling height of 3 cm are selected as mutagenic materials, soaked with colchicine at a concentration of 0.2% for 24 h, or soaked with colchicine at a concentration of 0.1% for 48 h. Then, the soaked tissue culture seedlings are inoculated in the rooting medium for rooting culture, and ploidy identification and so on are carried out later.

[0051] The size of the Arundo donax tissue culture seedlings and the concentration of colchicine will both affect the survival and induction doubling rate of the Arundo donax seedlings. Therefore, in this comparative example, tissue culture seedlings with heights of 1, 3, and 6 cm are selected and treated with colchicine solutions of 0.10%, 0.15%, 0.20%, 0.25%, and 0.30% for 24 h respectively. Other conditions are the same as in Example 1. There are 7 tissue culture seedlings in each bottle, and each group is repeated 10 bottles. The effects of different Arundo donax tissue culture seedling heights and different colchicine concentrations treated for 24 h on the survival and doubling induction of Arundo donax seedlings are compared. The results are shown in Table 2.

[0052] Table 2 Effects of different Arundo donax tissue culture seedling heights and different concentrations of colchicine treated for 24 h on the survival and doubling of Arundo donax seedlings

[0053] Processing number Height of tissue-cultured seedlings (cm) Colchicine concentration Survival rate (%) Doubling rate (%) B1 1 0.1％ 4 0 B2 1 0.15％ 2 0 B3 1 0.2％ 0 0 B4 1 0.25％ 0 0 B5 1 0.3％ 0 0 B6 3 0.1％ 35 0 B7 3 0.15％ 30 0 B8 3 0.2％ 28 7 B9 3 0.25％ 15 0 B10 3 0.3％ 2 0 B11 6 0.1％ 60 0 B12 6 0.15％ 41 0 B13 6 0.2％ 32 0 B14 6 0.25％ 23 0 B15 6 0.3％ 17 0

[0054] It can be seen from the experimental comparative data that when the seedlings are too small (B1 - B5, with a plant height of 1 cm), their own resistance is very weak, and the toxicity of colchicine itself causes too much damage to the small seedlings. The survival rate gradually decreases with the increase in the concentration of colchicine until all die, and there is no doubling. When the seedlings are too large (B11 - B15, with a plant height of 6 cm), their own resistance is strong and the survival rate is relatively high, but the doubling rate is 0, which may be due to the too strong self - resistance of the seedlings. Moreover, colchicine itself is toxic, and the survival rate of the seedlings gradually decreases with the increase in the concentration of colchicine. Treatment with 0.2% colchicine for 24 h has no inducing effect on it, and it is impossible to obtain chromosome - doubled plants. Therefore, a seedling height of 3 cm is the best treatment seedling stage (B6 - B10), and a moderate survival rate can be obtained. When the concentration of colchicine is low (B6, B7) and treated for 24 h, the time is not enough to obtain doubling. When the concentration of colchicine is high (B8, B10) and treated for 24 h, the toxicity of colchicine itself is too strong, and the survival rate of the seedlings gradually decreases with the increase in the concentration of colchicine, and it is impossible to induce chromosome doubling. Only treatment with 0.2% colchicine for 24 h (B8) can obtain chromosome - doubled plants, with a doubling induction rate of 7%. Therefore, the present invention selects treatment with 0.2% colchicine for 24 h to double the chromosomes of Arundo donax.

[0055] The above - described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. An induction method for doubling the chromosomes of Arundo donax, characterized in that, It includes the following steps: S1: Select healthy and pest-free Arundo donax L. stem segments with axillary buds as explant materials, and culture the explants in an adventitious bud induction medium; S2: Select the tissue culture seedlings cultured in the pre-culture medium in S1 as mutagenesis materials, soak them with colchicine, and then culture them through a rooting medium.

2. The induction method for doubling the chromosomes of Arundo donax according to claim 1, characterized in that: Select the tissue culture seedlings cultured in the pre-culture medium for 28 - 30 days and with a seedling height of 3 cm as mutagenesis materials.

3. The induction method for doubling the chromosomes of Arundo donax according to claim 1, characterized in that: The concentration of colchicine is 0.1%, and the soaking treatment time is 48 h.

4. The induction method for doubling the chromosomes of Arundo donax according to claim 1, wherein: The concentration of colchicine is 0.2%, and the soaking treatment time is 24 h.

5. The induction method for doubling the chromosomes of Arundo donax according to any one of claims 1-4, characterized in that: The formula of the adventitious bud induction medium is: 0.7% agar powder, 3% sucrose, 0.44% MS medium, 6-BA concentration of 4 mg / L, IBA concentration of 1 mg / L; the culture conditions are: temperature 25°C, daily light duration of 16 h, light intensity of 18000 - 20000 lux, humidity of 60%, and medium pH value of 5.8 - 6.

0.

6. The induction method for doubling the chromosomes of Arundo donax according to claim 5, characterized in that: After the explants start to root in the adventitious bud induction medium, the explants are subjected to acclimatization treatment after continuing to culture for one week, and their phenotypes are observed after four months of soil cultivation for ploidy identification.

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