Method for obtaining regenerated plants by tissue culture of young spikelets of Leymus chinensis based on the same culture medium

Abstract

The present invention discloses a method for obtaining regenerated plants by tissue culture of young spikelets of Leymus chinensis based on the same culture medium, comprising the following steps: 1) taking spikelets; 2) stripping and disinfecting the spikelets; 3) direct seedling method: the young spikelets are inoculated in a culture medium, placed in a dark 25°C incubator for 20 days, transferred to a newly changed culture medium, placed in a 25°C incubator with 16 hours of light and 8 hours of darkness for 20 days, and then transferred to a newly changed culture medium for 30 days, and then transferred to a conical flask containing the newly changed culture medium for another 15 days. After the plant is trained, it is transplanted into sterilized nutrient soil; the culture medium includes MS, sucrose, agar and 2,4-D, and the pH value is 5.8. In the present invention, the same culture medium formula is used throughout the process, and only the same culture medium needs to be configured to meet the use of the entire operation process, and the operation is simpler. The present invention is shorter in overall time, lower in cost, and has higher callus and differentiation rates.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology breeding, and particularly relates to a method for obtaining regenerated plants through tissue culture of young spikelets of Leymus chinensis based on the same culture medium. Background Art

[0002] Leymus chinensis Leymus chinensis Leymus chinensis has strong adaptability and is resistant to cold, drought, alkali, barrenness, and wind and sand. Its abundant leaves, high nutritional value, and palatability make it a popular food for poultry year-round. Its rhizomes have strong penetrating and invasive abilities, providing windbreak and sand fixation, making it a highly important grass for forage and ecological conservation. Leymus chinensis is widely distributed and boasts a rich collection of germplasm resources.

[0003] Currently, there are technical bottlenecks in cultivating high-yielding, high-quality, and stress-resistant new varieties of Leymus chinensis. For example, addressing the "three lows" of Leymus chinensis—low heading rate, low fruit set rate, and low germination rate—has made it difficult to achieve a breakthrough. Using transgenic or gene editing technologies to obtain high-quality new Leymus chinensis germplasm is a promising option. Tissue culture regeneration systems for Leymus chinensis are fundamental to transgenic and gene editing research, and young Leymus chinensis spikelets are the optimal explant for inducing plant regeneration. However, in the published reports on establishing Leymus chinensis regeneration systems using young spikelets, the callus induction, differentiation, and rooting and seedling formation steps are performed separately in different culture media. This is a cumbersome and time-consuming procedure, and the callus induction and differentiation rates are suboptimal.

[0004] Therefore, there is an urgent need in the prior art for a method for obtaining regenerated plants using young spikelets of Leymus chinensis, which is simpler to operate, takes less time, and has higher healing and differentiation rates. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a method for obtaining regenerated plants by tissue culture of young spikelets of Leymus chinensis based on the same culture medium, which has simpler operation, shorter time, higher induction rate and differentiation rate.

[0006] The object of the present invention is achieved through the following technical solution: a method for obtaining regenerated plants by tissue culture of young spikelets of Leymus chinensis based on the same culture medium, comprising the following steps:

[0007] 1) Remove the ears;

[0008] 2) Peeling and disinfection of ears;

[0009] 3) Direct seedling method: The young ears were inoculated into the culture medium and cultured in a dark 25°C incubator for 20 days. The young ears were then transferred to the fresh culture medium and cultured in a 25°C incubator with 16 h of light and 8 h of darkness for 20 days. The young ears were then transferred to the fresh culture medium and cultured for 30 days. The young ears were then transferred to a conical flask containing the fresh culture medium and cultured for another 15 days. After the plants were hardened, they were transplanted into sterilized nutrient soil. The culture medium contained 4.4 g / L MS, 30 g / L sucrose, 8 g / L agar, and 2 mg / L 2,4-D. The pH of the culture medium was 5.8.

[0010] The ear picking is to pick the young ears of Leymus chinensis with the stem exposed below the flag leaf with a length of 2-3 cm and wrapped in leaf sheaths, put them in an ice box, and store them in a refrigerator at 4°C.

[0011] The ear stripping disinfection comprises spraying the outer surface of the young ear with a 75% V / V alcohol solution, placing the young ear on a clean workbench, wiping the leaf sheath of the young ear with a sterile cotton ball soaked in 75% alcohol, then stripping the young ear wrapped by the leaf sheath, wrapping it with sterile gauze and placing it in a conical flask, soaking it in 75% V / V alcohol for 30 seconds, rinsing it with sterile water three times, then soaking it in a 10% V / V sodium hypochlorite solution for 8 minutes, rinsing it with sterile water three times, and finally soaking it in a 30% V / V hydrogen peroxide solution for 3 minutes, and rinsing it with sterile water five times; during the disinfection process, the conical flask is always shaken to ensure that the young ear is in full contact with the solution.

[0012] The present invention has the beneficial effects of using the same culture medium formula for callus induction, differentiation, and rooting. This allows the same culture medium to be used throughout the entire process, eliminating the need for separate culture medium formulas for callus induction, differentiation, and rooting. This simplifies operation, reduces overall time and costs, and increases callus induction and differentiation rates. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the 20-day growth of Leymus chinensis material No. 13;

[0014] Figure 2 This is the 40-day growth of Leymus chinensis material No. 13;

[0015] Figure 3 This is the 70-day growth of Leymus chinensis material No. 13;

[0016] Figure 4 This is the growth condition of Leymus chinensis material No. 13 at 85 days;

[0017] Figure 5 This is the transplanting situation of the No. 13 sheepgrass material. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the accompanying drawings. Example 1

[0019] 1. Leymus chinensis material: Select the Leymus chinensis germplasm resources in the National Perennial Forage Germplasm Resources Nursery (Hohhot) of the Institute of Grassland, Chinese Academy of Agricultural Sciences, and the Leymus chinensis material numbers are 13 and 34.

[0020] The plant height of Leymus chinensis 13 is 55-80 cm, the average seed yield is 80.25 kg / hectare, the average hay yield is 3014.15 kg / hectare, and the growing period is 80-90 days; the plant height of Leymus chinensis 34 is 95-120 cm, the average seed yield is 205.18 kg / hectare, the average hay yield is 7764.45 kg / hectare, and the growing period is 85-95 days.

[0021] 2. Picking ears: Take 2 samples of young ears of Leymus chinensis with the stem exposed below the flag leaf and 2-3 cm in length, wrapped in leaf sheaths, and place them in an ice box. Bring them back to the laboratory and store them in a 4°C refrigerator.

[0022] The present invention selects the young ears of Leymus chinensis when the stem length is 2-3 cm exposed under the flag leaf. The young ears taken at this period are neither too tender nor too old. If they are too tender, it is difficult to peel the ears, and the young ears are too small to conduct experiments. If the young ears are too old, it will affect the callus rate.

[0023] 3. Peeling and disinfection: After spraying the outer surface of the young ears with 75% V / V alcohol solution, place the young ears on a clean workbench, wipe the leaf sheath of the young ears with a sterile cotton ball soaked in 75% alcohol, then peel off the young ears wrapped in the leaf sheath, wrap them with sterile gauze and place them in a conical flask, soak them in 75% V / V alcohol for 30 seconds, rinse them with sterile water three times, then soak them in 10% V / V sodium hypochlorite solution for 8 minutes, rinse them with sterile water three times, and finally soak them in 30% V / V hydrogen peroxide solution for 3 minutes, and rinse them with sterile water five times; during the disinfection process, always shake the conical flask to ensure that the young ears are in full contact with the solution.

[0024] In the prior art, HgCl2 is often used to disinfect young ears, but HgCl2 is highly toxic. The present invention uses 75% v / v alcohol, 10% v / v sodium hypochlorite solution, and 30% v / v hydrogen peroxide solution to disinfect young ears, eliminating the risk of poisoning during the experiment, ensuring personnel health and safety, and reducing environmental pollution.

[0025] The present invention adopts 30% v / v hydrogen peroxide solution for disinfection, which effectively prevents the growth of bacteria after the young ears are inoculated into the culture medium.

[0026] 4. Direct seedling method: The culture medium systems No. 1-17 shown in Table 1 all contain 30 g / L sucrose and 8 g / L agar. Sucrose and agar are not shown in Table 1. The following treatments were performed on Leymus chinensis materials No. 13 and No. 34 using the culture medium systems No. 1-17.

[0027] The sterilized young spikelets of Leymus chinensis No. 13 and No. 34 were inoculated into the callus induction medium system No. 1-10 (the formula of No. 1-10 medium is shown in Table 1), and placed in a dark 25℃ incubator for 20 days to induce callus. The callus induction of Leymus chinensis No. 13 is shown in Figure 1 The healing rate was calculated and the results are shown in Table 1. The healing rates of No. 13 and No. 34 Leymus chinensis materials in culture medium system 1 both reached 100%. The statistical method for the healing rate is as follows:

[0028] Cure rate = number of cured florets / number of inoculated florets.

[0029] After callus induction, the No. 13 and No. 34 Leymus chinensis materials were transferred to the newly replaced No. 1-10 culture medium system and cultured in a 25℃ incubator with 16 hours of light and 8 hours of darkness for 20 days. The No. 13 and No. 34 Leymus chinensis materials in culture medium 1 directly differentiated into leaves and hairy roots. The differentiation of No. 13 Leymus chinensis materials was shown in Figure 2. Figure 2 The differentiation rates of Leymus chinensis No. 13 and No. 34 were 83.33% and 81.48% respectively. The statistical method of differentiation rate is as follows:

[0030] Differentiation rate = number of calli that differentiated into roots or leaves / total number of calli.

[0031] Then, the No. 13 and No. 34 Leymus chinensis materials were transferred to the newly replaced No. 11-15 differentiation system for 30 days. The No. 13 and No. 34 Leymus chinensis materials in the No. 11 differentiation system took root and grew into complete plants. The growth of the No. 13 Leymus chinensis material was shown in Figure 2. Figure 3 .

[0032] The unrooted Leymus chinensis materials No. 13 and No. 34 were placed in rooting systems 16 and 17, and both took root 100%.

[0033] The rooted seedlings of No. 13 and No. 34 Leymus chinensis were transferred to conical flasks and cultured for 15 days. The plants grew to 10-15 cm. The growth of No. 13 Leymus chinensis was shown in Figure 2. Figure 4 .

[0034] After training, the seedlings were transplanted into sterilized nutrient soil. The transplanting conditions of the No. 13 Leymus chinensis material were shown in Figure 4 .

[0035] By comparing the callus induction rate and differentiation rate of No. 13 and No. 34 Leymus chinensis materials in different culture medium systems, the best culture medium system was screened out as culture medium system 1, i.e. 4.4 g / L MS + 30 g / L sucrose + 8 g / L agar + 2 mg / L 2,4-D. The pH value of the culture medium was 5.8. The callus induction, differentiation, rooting and plant growth processes in conical flasks can all be carried out in culture medium system 1.

[0036] Table 1 Callus formation and differentiation of Leymus chinensis No. 13 and No. 34 in different culture medium systems

[0037]

[0038] Note: Systems 1, 11, and 16 in the table are the same culture medium system, and are numbered here to distinguish the three stages of callus, differentiation, and rooting.

[0039] In the prior art, the callus process often requires three subcultures, each of which requires 20 days, 30 days of differentiation culture, 30 days of rooting culture, and 120 days of transplanting. The callus rate of No. 13 and No. 34 chinensis materials in culture medium system 1 of the present invention both reached 100%. Compared with system 4 and system 10, culture medium system 1 only used 2,4-D hormone, which saved costs. Moreover, No. 13 and No. 34 chinensis materials can complete the plant growth process of induced callus, differentiation, rooting and conical flasks in the culture medium system of the same formula, without the need to configure different formulas of culture medium corresponding to the induced callus, differentiation and rooting processes. The present invention is simpler to operate. The differentiation rate of the present invention can reach up to 83.33%. The present invention successfully transplanted to nutrient soil for 85 days, greatly shortening the culture time.

[0040] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for obtaining regenerated plants by tissue culture of young spikelets of Leymus chinensis based on the same culture medium, characterized in that The steps include: 1) Remove the ears; 2) Peeling and disinfection of ears; 3) Direct seedling method: The young ears were inoculated into the culture medium and cultured in a dark 25°C incubator for 20 days. The young ears were then transferred to the fresh culture medium and cultured in a 25°C incubator with 16 h of light and 8 h of darkness for 20 days. The young ears were then transferred to the fresh culture medium and cultured for 30 days. The young ears were then transferred to a conical flask containing the fresh culture medium and cultured for another 15 days. After the plants were hardened, they were transplanted into sterilized nutrient soil. The culture medium consisted of 4.4 g / L MS, 30 g / L sucrose, 8 g / L agar, and 2 mg / L 2,4-D. The pH of the culture medium was 5.

8.

2. The method for obtaining regenerated plants by tissue culture of young spikelets of Leymus chinensis based on the same culture medium according to claim 1, characterized in that: The ear picking is to pick the young ears of Leymus chinensis with the stem exposed below the flag leaf with a length of 2-3 cm and wrapped in leaf sheaths, put them in an ice box, and store them in a refrigerator at 4°C.

3. The method for obtaining regenerated plants by tissue culture of young spikelets of Leymus chinensis based on the same culture medium according to claim 2, characterized in that: The ear stripping disinfection comprises spraying the outer surface of the young ear with a 75% V / V alcohol solution, placing the young ear on a clean workbench, wiping the leaf sheath of the young ear with a sterile cotton ball soaked in 75% alcohol, then stripping the young ear wrapped by the leaf sheath, wrapping it with sterile gauze and placing it in a conical flask, soaking it in 75% V / V alcohol for 30 seconds, rinsing it with sterile water three times, then soaking it in a 10% V / V sodium hypochlorite solution for 8 minutes, rinsing it with sterile water three times, and finally soaking it in a 30% V / V hydrogen peroxide solution for 3 minutes, and rinsing it with sterile water five times; during the disinfection process, the conical flask is always shaken to ensure that the young ear is in full contact with the solution.

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