A tissue culture method of Leptochloa chinensis

Abstract

The present invention discloses a tissue culture method of Leptochloa chinensis. The method comprises: 1) disinfection of explants: using mature embryos of Leptochloa chinensis as explants for disinfection; (2) induction and subculture of callus tissue: inoculating the explants on an induction medium and culturing them in the dark to induce embryonic callus tissue; (3) differentiation of callus tissue: transferring the callus tissue to a differentiation medium to differentiate into seedlings; (4) rooting and transplanting: transferring the seedlings to a rooting medium to differentiate into roots; (5) hardening the seedlings after forming complete plants and transplanting. The present invention proposes a tissue culture system of Leptochloa chinensis for the first time, using its mature embryos as explants, so that the acquisition of explants is simple and easy and the quantity is large, and a large amount of callus tissue can be obtained from a small amount of seeds. The system has the advantages of simple operation, good stability and high reproduction efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture and rapid propagation of Leptochloa chinensis, and particularly relates to a tissue culture method of Leptochloa chinensis. Background Art

[0002] With the adoption of simplified rice cultivation and the promotion of alternating wet-dry paddy field management, the drought-tolerant, moisture-loving Leptochloa chinensis has rapidly become a major weed in rice fields. In some areas, it has become the most dominant weed, causing severe damage. Studies on weed resistance in rice fields have shown rapid development of Leptochloa chinensis resistance nationwide, with some areas developing resistance to the primary herbicide, cyhalofop-butyl, exceeding 75.8-fold. However, further investigation into the mechanisms of Leptochloa chinensis resistance is hampered by the limited availability of seeds from resistant Leptochloa chinensis populations collected during surveys and sampling, coupled with a mixed genetic background. Rapidly obtaining large quantities of Leptochloa chinensis germplasm with a uniform genetic background through plant tissue culture is crucial for further research. While stable tissue culture systems have been established for weeds such as Eleusine indica, Setaria viridis, and Lolium multiflorum, no such systems have been reported for Leptochloa chinensis. Summary of the Invention

[0003] The purpose of the present invention is to address the above problems and to propose for the first time a tissue culture method for Leptochloa chinensis, which will lay a good foundation for studying the drug resistance and genetic background of Leptochloa chinensis.

[0004] The object of the present invention is achieved by the following methods:

[0005] A tissue culture method for Leptochloa chinensis comprises the following steps:

[0006] (1) Explant disinfection: Use mature embryos of Leptochloa chinensis as explants for disinfection;

[0007] (2) Callus induction and subculture: explants were inoculated on induction medium and cultured in the dark to induce embryonic callus;

[0008] (3) Callus differentiation: The callus is transferred to a differentiation medium to differentiate into shoots;

[0009] (4) Rooting: The seedlings are transferred to the rooting medium to differentiate into roots;

[0010] (5) After the complete plants are formed, harden the seedlings and transplant them.

[0011] After the mature embryos in step (1) are polished with sandpaper to remove the husks, they are disinfected with 75% alcohol for 30 seconds and then with 4% sodium hypochlorite for 10-15 minutes, shaking left and right during the process to ensure thorough sterilization. After disinfection, they are washed with sterile water 4-6 times, placed on sterile filter paper, and blown dry in a clean bench for later use.

[0012] Step (2) inoculating the explant on an induction medium; the induction medium comprises: 4.43 g / L M519+0.5-4 mg / L 2,4-D, 30 g / L sucrose, 5 g / L phytagel or 8 g / L agar, and a pH of 5.8-5.9.

[0013] Step (2) incubate at 26-32°C in the dark.

[0014] Step (2) 50-200 seeds in a 9 cm culture dish.

[0015] Step (2) The induced callus tissue is subcultured under the same conditions as the induction treatment, and the culture medium needs to be replaced every 18-25 days until sufficient callus is obtained. The callus subculture should not exceed 4 generations.

[0016] Because Leptochloa chinensis seeds are small, they can be evenly spread with a sterilized spoon. Callus formation begins after 7 days of incubation in the dark at 28°C. The callus induction rate is approximately 75% after 15 days, and reaches 90% after 30 days. It is recommended that Leptochloa chinensis callus be subcultured for no more than four generations to maintain good differentiation ability.

[0017] The differentiation medium in step (3) is: M519 4.43 g / L + 6-BA 0.1-2.0 mg / L + KT 0-0.5 mg / L, + sucrose 30 g / L + phytogenin 5 g / L or agar 8 g / L, pH 5.8-5.9, culture at 28°C-32°C, 12 h light / 12 h dark, and a light intensity of 8,000-20,000 Lx. Buds begin to differentiate after 5 days, and embryonic seedlings of Leptochloa chinensis can be seen after 15 days, with a differentiation rate exceeding 90%.

[0018] Step (4) Rooting medium: MS 4.43 g / L + NAA 0-0.5 mg / L + sucrose 30 g / L + phytogel 5 g / L or agar 8 g / L, pH 5.8-5.9, 28° C.-32° C., 12 h light / 12 h dark culture, light intensity 8000-20000 Lx.

[0019] Step (4) When the buds grow to 1.0-2.0 cm in height, they are transferred to a rooting medium to differentiate into roots, and complete plants are formed after 15 days.

[0020] Step (5) After opening the cover and hardening the seedlings for 1-2 days, transplant them into nutrient soil and pay attention to moisture retention. The transplant survival rate reaches more than 90%.

[0021] The present invention proposes for the first time a tissue culture system for Leptochloa chinensis, utilizing its mature embryos as explants. This makes obtaining explants simple and easy, and large quantities of explants can be obtained, allowing a large amount of callus to be obtained from a small amount of seeds. This system has the advantages of simple operation, good stability, and high propagation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Induction callus of Leptochloa chinensis seeds, 0d (A), 10d (B), 20d (C);

[0023] Figure 2 These are pictures of callus differentiation at 10 and 20 days;

[0024] Figure 3 Pictures of callus-differentiated seedlings transplanted into rooting medium 5 days (A), 20 days (B) and transplanted into pots (C). DETAILED DESCRIPTION

[0025] The following examples are intended to further illustrate the present invention, but are not intended to limit the present invention.

[0026] The examples in the present invention are all the results of at least three parallel tests.

[0027] Example 1: Explant disinfection

[0028] After the mature embryos of the kiwifruit are polished with sandpaper to remove the husks, they are disinfected with 75% alcohol for 30 seconds and then with 4% sodium hypochlorite for 10-15 minutes. During this period, they are shaken left and right to ensure thorough sterilization. After the disinfection is completed, they are washed with sterile water 4 to 6 times, placed on sterile filter paper, and blown dry in a clean bench for later use.

[0029] Table 1 Effects of different disinfection methods on callus induction of Leptochloa crus-galli

[0030]

[0031] After disinfection, inoculate the culture medium: 4.43 g / L M519 + 2 mg / L 2,4-D, 30 g / L sucrose, 8 g / L agar, pH 5.8-5.9, culture in the dark at 28°C; observe the disinfection effect.

[0032] Example 2: Callus induction and subculture

[0033] Inoculate the sterilized explants onto induction medium (4.43g / L M519 + 2mg / L 2,4-D, 30g / L sucrose, 8g / L agar, pH 5.8-5.9, incubate at 28°C in the dark). Place 50-200 seeds in a 9cm dish. Subculture callus using the same conditions as induction treatment, changing the medium approximately every 20 days until sufficient callus is obtained. Do not subculture callus for more than four generations.

[0034] The seeds of Leptochloa chinensis are small and can be evenly spread with a sterilized spoon. Callus begins to form after 7 days of incubation in the dark at 28°C, with a callus induction rate of up to 90% after 30 days. Leptochloa chinensis callus should be subcultured for no more than four generations to maintain good differentiation ability.

[0035] Table 2 Formulas of different induction culture media

[0036]

[0037]

[0038] Table 3 Induction of callus of Leptochloa macrophylla by different culture media

[0039]

[0040] Each culture medium in Table 3 was supplemented with 2 mg / L 2,4-D, 30 g / L sucrose, and 8 g / L agar, and had a pH of 5.8-5.9.

[0041] Example 3: Differentiation culture

[0042] The induced callus tissue was inoculated into the differentiation medium M519 4.43g / L+6-BA 0.1~2.0mg / L+KT 0~0.5mg / L+sucrose 30g / L+agar 8g / L, pH 5.8-5.9, cultured at 28℃, 12h light / 12h dark, and light intensity of 16000Lx for differentiation. Figure 2 and Table 4.

[0043] Table 4 Effects of different hormones on callus differentiation

[0044]

[0045]

[0046] (Note: The data in the table are statistical results of 30 days of cultivation)

[0047] Example 4: Rooting medium

[0048] Differentiated seedlings were inoculated into a medium containing 4.43 g / L MS, 0.5 mg / L NAA, 30 g / L sucrose, and 8 g / L agar at a pH of 5.8-5.9, 28°C, 12 h light / 12 h dark, and a light intensity of 16,000 Lx for rooting. Full plants formed after 15 days, with a rooting rate of 100%.

[0049] Example 5: Transplanting

[0050] After 1-2 days of hardening the seedlings, transplant them into nutrient soil (such as rice nutrient soil) and pay attention to moisture retention (humidity 60%, temperature 30℃). The transplant survival rate reaches more than 90%.

Claims

1. A tissue culture method of Leptochloa chinensis, characterized in that: The steps include: (1) Disinfection of explants: Use mature embryos of Leptochloa chinensis as explants for disinfection; (2) Callus induction and subculture: explants are inoculated on induction medium and cultured in the dark to induce embryonic callus; (3) Callus differentiation: The callus is transferred to a differentiation medium to differentiate into sprouts; (4) Rooting: The seedlings are transferred to the rooting medium to differentiate into roots; (5) After the complete plants are formed, harden the seedlings and transplant them; After the mature embryos in step (1) are polished with sandpaper to remove the husks, they are disinfected with 75% alcohol for 30 seconds, and then disinfected with 4% sodium hypochlorite for 10-15 minutes, shaking left and right during the process to ensure thorough sterilization. After the disinfection is completed, they are washed with sterile water for 4-6 times, placed on sterile filter paper, and blown dry in a clean bench for later use; Step (2) inoculating the explants on an induction medium; the induction medium includes: 4.43 g / L M519 + 0.5-4 mg / L 2,4-D, 30 g / L sucrose, 5 g / L phytagel or 8 g / L agar; Step (2) subculturing the induced callus tissue under the same conditions as the induction treatment; Differentiation medium in step (3): M519 4.43 g / L + 6-BA 0.1-2.0 mg / L + KT 0-0.5 mg / L + sucrose 30 g / L + plant gel 5 g / L or agar 8 g / L; Step (4) Rooting medium: MS 4.43 g / L + NAA 0.5 mg / L + sucrose 30 g / L + plant gel 5 g / L or agar 8 g / L.

2. The method according to claim 1, characterized in that The pH of the induction medium in step (2) is 5.8-5.

9.

3. The method according to claim 1, characterized in that Step (2) incubate at 26°C-32°C in the dark.

4. The method according to claim 1 or 3, characterized in that: Step (2) 50-200 seeds in a 9 cm culture dish.

5. The method according to claim 1, 2 or 3, characterized in that: Step (2) Subculture: The culture medium needs to be replaced every 18-25 days until sufficient callus is obtained. The callus subculture should not exceed 4 generations.

6. The method according to claim 1, characterized in that The pH of the differentiation medium in step (3) is 5.8-5.9, the culture temperature is 28°C-32°C, the light intensity is 12h / darkness is 12h, and the light intensity is 8000-20000 Lx.

7. The method according to claim 1, characterized in that Step (4) The rooting medium pH is 5.8-5.9, the temperature is 28°C-32°C, the light intensity is 12h / darkness is 8000-20000 Lx.

8. The method according to claim 1 or 7, characterized in that: Step (4) When the buds grow to 1.0-2.0 cm in height, they are transferred to rooting medium to differentiate into roots, and complete plants are formed after 15 days.

9. The method according to claim 1, characterized in that: Step (5) Open the cover and harden the seedlings for 1-2 days before transplanting them into nutrient soil, keeping them moist.

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