Method for obtaining regenerated plant through leymus chinensis young ear tissue culture based on same culture medium

Abstract

The invention discloses a method for obtaining regenerated plants through leymus chinensis young ear tissue culture based on the same culture medium. The method comprises the following steps: 1) ear taking; 2) ear stripping and disinfection; and 3) a direct seedling method: inoculating the young ears into a culture medium, placing the culture medium in a dark 25 DEG C incubator for culturing for 20 days, then transferring the young ears into the newly-changed culture medium, placing the culture medium in a 25 DEG C incubator for culturing for 20 days under the conditions of illumination for 16 hours and darkness for 8 hours, then transferring the young ears into the newly-changed culture medium for culturing for 30 days, then transferring the young ears into a conical flask containing the newly-changed culture medium for culturing for 15 days, and taking out the young ears. The plants are transplanted into sterilized nutrient soil after being hardened; the culture medium comprises MS (Murashige and Skoog), cane sugar, agar and 2, 4-D, and the pH value of the culture medium is 5.8. According to the method, the same culture medium formula is adopted in the whole process, the use requirement of the whole operation process can be met only by preparing the same culture medium, and operation is simpler. The method is shorter in overall time consumption, lower in cost and higher in induced callus rate and differentiation rate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology breeding, and in particular 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 ) has strong adaptability. It is cold-resistant, drought-resistant, alkali-resistant, barren-resistant, and wind-resistant. It has a large amount of leaves, high nutritional value, and good palatability. All kinds of poultry like to eat it all year round. Its rhizomes have strong penetration and invasion capabilities, can prevent wind and fix sand, and are extremely important grass forage and ecological grass. Leymus chinensis has a wide distribution range and abundant germplasm resources.

[0003] At present, there are certain technical bottlenecks in the cultivation of new varieties of Leymus chinensis with high yield, high quality and strong stress resistance. For example, it is difficult to make better breakthroughs in the "three lows" problem of Leymus chinensis, namely low heading rate, low fruiting rate and low germination rate. It is a better choice to use transgenic or gene editing technology to obtain excellent new germplasm of Leymus chinensis. The tissue culture regeneration system of Leymus chinensis is the basis for conducting transgenic or gene editing research, and Leymus chinensis spikelets are the best explant choice for inducing the regeneration of Leymus chinensis plants. However, in the relevant reports on the construction of Leymus chinensis regeneration system using spikelets that have been published, the steps of inducing callus, differentiation and rooting and seedling formation are carried out in different culture media respectively. The overall operation is relatively cumbersome and time-consuming, and the induction callus rate and differentiation rate are not good enough.

[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 induction rates of healing and differentiation. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art 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 scheme: 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) Pick the ears; 2) Remove the ears and disinfect them; 3) Direct seedling method: The young ears are inoculated into the culture medium, placed in a dark 25°C incubator for 20 days, and then transferred to the newly replaced culture medium, placed in a 25°C incubator with 16 h of light and 8 h of darkness for 20 days, and then transferred to the newly replaced culture medium for 30 days, and then transferred to a conical flask containing the newly replaced culture medium for another 15 days. After the plants are trained, they are transplanted into sterilized nutrient soil; the culture medium includes 4.4 g / L MS, 30 g / L sucrose, 8 g / L agar and 2 mg / L 2,4-D, and the pH value of the culture medium is 5.8.

[0007] The ear picking is to pick the young ears of Leymus chinensis wrapped in leaf sheaths at a stage when the stem is exposed below the flag leaf with a length of 2-3 cm, put them in an ice box, and store them in a 4° C. refrigerator.

[0008] The ear stripping disinfection is as follows: after spraying the outer surface of the young ear with 75% V / V alcohol solution, the young ear is placed on a clean workbench, and then the leaf sheath of the young ear is wiped with a sterile cotton ball soaked in 75% alcohol, and then the young ear wrapped by the leaf sheath is stripped out, wrapped with sterile gauze and placed in a conical flask, and then soaked in 75% V / V alcohol for 30 seconds, rinsed with sterile water for 3 times, and then soaked in 10% V / V sodium hypochlorite solution for 8 minutes, rinsed with sterile water for 3 times, and finally soaked in 30% V / V hydrogen peroxide solution for 3 minutes, and rinsed with sterile water for 5 times; during the disinfection process, the conical flask is always shaken to ensure that the young ear is fully in contact with the solution.

[0009] The beneficial effects of the present invention are as follows: in the present invention, the same culture medium formula is used in the callus induction, differentiation and rooting processes, and only the same culture medium needs to be configured to meet the use of the entire operation process, and there is no need to configure culture media with different formulas corresponding to the callus induction, differentiation and rooting processes, and the operation of the present invention is simpler. The overall time of the present invention is shorter and the cost is lower. The callus induction rate and differentiation rate of the present invention are higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the growth condition of Leymus chinensis material No. 13 for 20 days; Figure 2 This is the growth condition of Leymus chinensis material No. 13 for 40 days; Figure 3 This is the growth condition of Leymus chinensis material No. 13 for 70 days; Figure 4 This is the growth condition of Leymus chinensis material No. 13 at 85 days; Figure 5 This is the transplanting situation of plants of No. 13 sheepgrass material. DETAILED DESCRIPTION

[0011] The present invention is described in detail below in conjunction with the accompanying drawings. Example 1

[0012] 1. Leymus chinensis material: Select the Leymus chinensis germplasm resources in the Hohhot National Perennial Forage Germplasm Resources Nursery of the Grassland Research Institute of the Chinese Academy of Agricultural Sciences. The Leymus chinensis material numbers are 13 and 34.

[0013] 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.

[0014] 2. Picking ears: Take 2 portions of young ears of Leymus chinensis material with the stem exposed below the flag leaf for 2-3 cm and wrapped in leaf sheaths, put them in an ice box, bring them back to the laboratory, and store them in a 4°C refrigerator.

[0015] The present invention selects young ears of sheep fescue at a stage when the stem length is 2-3 cm exposed under the flag leaf. The young ears taken at this stage are neither too tender nor too old. If they are too tender, it is difficult to peel the ears. If the young ears are too small, it is inconvenient to carry out experiments. If the young ears are too old, it will affect the callus rate.

[0016] 3. Stripping and disinfection: After spraying the outer surface of the young ears with 75% V / V alcohol solution, place the young ears on the clean workbench, wipe the leaf sheath of the young ears with sterile cotton balls soaked in 75% alcohol, then strip the young ears wrapped by the leaf sheath, wrap them with sterile gauze and put them in a conical flask, soak them in 75% V / V alcohol for 30 seconds, rinse them with sterile water for 3 times, then soak them in 10% V / V sodium hypochlorite solution for 8 minutes, rinse them with sterile water for 3 times, and finally soak them in 30% V / V hydrogen peroxide solution for 3 minutes, and rinse them with sterile water for 5 times; during the disinfection process, always shake the conical flask to ensure that the young ears are in full contact with the solution.

[0017] 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, thereby avoiding the risk of poisoning during the test process, ensuring the health and safety of personnel, and reducing environmental pollution.

[0018] The present invention adopts 30% v / v hydrogen peroxide solution for disinfection, which effectively prevents bacteria from growing after the young ears are inoculated into the culture medium.

[0019] 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, and sucrose and agar are not shown in Table 1. The culture medium systems No. 1-17 were used to treat the No. 13 and No. 34 Leymus chinensis materials as follows.

[0020] The sterilized spikelets of Leymus chinensis materials 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°C incubator for 20 days to induce callus. The callus induction of Leymus chinensis material 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: Cure rate = number of cured florets / number of inoculated florets.

[0021] 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°C incubator with 16 h of light and 8 h 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 is shown in Figure 1. 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: Differentiation rate = number of calli that differentiated into roots or leaves / total number of calli.

[0022] After that, 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 is shown in Figure 1. Figure 3 .

[0023] The unrooted Leymus chinensis materials No. 13 and No. 34 were placed in rooting systems 16 and 17, and both took root 100%.

[0024] The rooted seedlings of Leymus chinensis materials No. 13 and No. 34 were transferred to conical bottles and cultured for 15 days. The plants grew to 10-15 cm. The growth of Leymus chinensis material No. 13 was shown in Figure 4 .

[0025] After training, the seedlings were transplanted into sterilized nutrient soil. The transplanting conditions of the 13th chinensis material were shown in Figure 4 .

[0026] 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. 4.4g / L MS + 30g / L sucrose + 8g / L agar + 2mg / L 2,4-D. The pH value of the culture medium is 5.8. The callus induction, differentiation, rooting and plant growth processes in conical flasks can all be carried out in culture medium system 1.

[0027] Table 1 Callus formation and differentiation of Leymus chinensis No. 13 and No. 34 in different culture medium systems

[0028] Note: Systems 1, 11, and 16 in the table are the same culture medium system, and they are numbered here to distinguish the three stages of callus, differentiation, and rooting.

[0029] In the prior art, the callus process often needs to go through 3 subcultures, each subculture requires 20 days, differentiation culture 30 days, rooting culture 30 days, and transplanting lasts for 120 days. The callus rate of No. 13 and No. 34 chinensis materials in culture medium system 1 of the present invention reached 100%. Compared with system 4 and system 10, culture medium system 1 only uses 2,4-D hormone, which saves costs, and 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 process, and 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 in 85 days, greatly shortening the culture time.

[0030] 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) Pick the ears; 2) Remove the ears and disinfect them; 3) Direct seedling method: The young ears are inoculated into the culture medium, placed in a dark 25°C incubator for 20 days, and then transferred to the newly replaced culture medium, placed in a 25°C incubator with 16 h of light and 8 h of darkness for 20 days, and then transferred to the newly replaced culture medium for 30 days, and then transferred to a conical flask containing the newly replaced culture medium for another 15 days. After the plants are trained, they are transplanted into sterilized nutrient soil; the culture medium includes 4.4 g / L MS, 30 g / L sucrose, 8 g / L agar and 2 mg / L 2,4-D, and the pH value of the culture medium is 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 wrapped in leaf sheaths at a stage when the stem is exposed below the flag leaf with a length of 2-3 cm, put them in an ice box, and store them in a 4° C. refrigerator.

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 is as follows: after spraying the outer surface of the young ear with 75% V / V alcohol solution, the young ear is placed on a clean workbench, and then the leaf sheath of the young ear is wiped with a sterile cotton ball soaked in 75% alcohol, and then the young ear wrapped by the leaf sheath is stripped out, wrapped with sterile gauze and placed in a conical flask, and then soaked in 75% V / V alcohol for 30 seconds, rinsed with sterile water for 3 times, and then soaked in 10% V / V sodium hypochlorite solution for 8 minutes, rinsed with sterile water for 3 times, and finally soaked in 30% V / V hydrogen peroxide solution for 3 minutes, and rinsed with sterile water for 5 times; during the disinfection process, the conical flask is always shaken to ensure that the young ear is fully in contact with the solution.

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