Method for inducing embryogenic calluses of Korean lily

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing embryonic calluses of Korean lily, which comprises the following steps: disinfecting scales of the Korean lily, inoculating into an adventitious bud induction culture medium for normal photoperiod culture, and preparing sterile tissue culture seedlings of the Korean lily; cutting off top leaves and roots of the sterile tissue culture seedlings in a sterile environment, and inoculating the sterile tissue culture seedlings in a sucrose-containing MS culture medium for dark culture; the method comprises the following steps: when the circumferential diameter of a sterile bulb of the Korean lily is 3-5cm and the number of scales reaches more than 8, culturing in a normal photoperiod until the middle-layer scale-free leaf scales with dispersed and non-cohesive tips of the sterile bulb are selected as explants, cutting off the periphery of the explants, inoculating the explants with the area of 0.25-0.40 cm < 2 > and the concave surface facing upwards on a callus induction culture medium for dark culture, and during the period, soaking with an anti-browning agent solution to obtain the embryogenic callus of the Korean lily. According to the method disclosed by the invention, the induction rate of the embryonic calluses of the Korean lily is increased to about 33.33%, and the multiplication coefficient can reach about 6.80.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for inducing embryonic callus of Korean lily. Background Art

[0002] The dedifferentiation of plant cells to form callus is a direct manifestation of the expression of cell totipotency, and is also the first step in the indirect somatic embryogenesis. Embryonic callus has a loose structure and strong division ability. It is widely used in the establishment of suspended cell systems, protoplast isolation and culture, the establishment of genetic transformation systems, and for cell culture screening of excellent asexual lines that produce secondary metabolites. The formation of lily callus is often induced by exogenous hormones. However, not all genotypes of lilies can respond to induction and dedifferentiate to form callus. Since the efficient induction of lily callus is affected by genotype, explant type and state, the difficulty of inducing callus in lilies of different species and varieties varies greatly. Only some lilies have successfully induced callus and established an efficient somatic embryogenesis system.

[0003] Korean lily Lilium amabile Palib is a perennial herbaceous plant of the genus Lilium in the family Liliaceae, also known as friendly lily or lovable lily. Korean lily has a special genotype and is an endangered species and a second-class protected wild plant. At present, Korean lily can only be regenerated through organogenesis, and the existing tissue induction methods cannot successfully obtain Korean lily callus. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for inducing Korean lily embryonic callus, which realizes the creation of Korean lily callus from scratch, and the present invention ultimately obtains Korean lily embryonic callus, which is a breakthrough.

[0005] The technical solution adopted by the present invention is to provide a method for inducing embryonic callus of Korean lily, comprising the following steps:

[0006] The scales of Korean lily are sterilized, inoculated into adventitious bud induction medium and cultured under normal photoperiod to prepare aseptic tissue culture seedlings of Korean lily;

[0007] The top leaves and roots of the aseptic tissue culture seedlings of Korean lily were cut off in a sterile environment, inoculated into MS medium containing sucrose and cultured in the dark;

[0008] When the circumference of the Korean lily sterile bulb reaches 3cm-5cm and the number of scales reaches more than 8, it is transferred to normal light cycle culture, and then the light green, scattered and unenclosed middle-layer scales of the Korean lily sterile bulb are taken as explants;

[0009] Cut off the explants on all sides, leaving a surface area of ​​0.25 cm 2 ~0.40cm 2 , obtaining inoculated materials, and then inoculating them on callus induction medium with the concave surface upward and culturing them in the dark, during which they are soaked in an anti-browning agent solution to obtain embryonic callus of Korean lily;

[0010] The anti-browning agent solution is a 0.5 mM to 1.0 mM reduced glutathione solution.

[0011] Preferably, the soaking treatment method is: soak the inoculated material in a sterile anti-browning agent solution, shake it to make the inoculated material fully contact with the anti-browning agent solution, soak it for 10 minutes to 15 minutes, and then absorb the surface liquid of the inoculated material.

[0012] Preferably, during the first 15 days of dark culture on the callus induction medium, the inoculation material is soaked every 5 days, the liquid on the surface of the inoculated material after the soaking treatment is sucked dry, and then inoculated into a new callus induction medium to continue dark culture;

[0013] After all the soaking treatments were completed, the liquid on the surface of the inoculated material was dried, and then inoculated into a new callus induction medium and continued to be cultured in the dark for 30-32 days.

[0014] Preferably, the method for preparing the callus induction medium is: adding a plant growth regulator and an anti-browning agent to each liter of MS medium;

[0015] The plant growth regulator is 0.5mg to 1.0mg of picloram;

[0016] The anti-browning agent is 0.5 mmol to 1.0 mmol of reduced glutathione.

[0017] Preferably, the preparation method of the adventitious bud induction medium is: adding 0.2 mg of naphthylacetic acid, 1.0 mg of 6-benzylaminopurine and 7.0 g of agar powder into each liter of MS medium.

[0018] Preferably, when preparing the aseptic tissue culture seedlings of Korean lily, the culture conditions are: temperature of 24° C. to 26° C., 16h / 8h normal photoperiod for 30 to 32 days.

[0019] Preferably, when preparing the sterile bulbs of Korean lily, the dark culture conditions are: temperature of 24° C. to 26° C., and dark culture for more than 30 days.

[0020] Preferably, the disinfection process is: the disinfection process is: soak the Korean lily scales in a 75% alcohol solution by volume for 1 min to 2 min, and rinse with sterile water 3 to 5 times; immerse the rinsed Korean lily scales in a 5% sodium hypochlorite solution by volume for 30 min to 35 min, and rinse with sterile water 3 to 5 times.

[0021] Preferably, the embryonic callus of Lilium koreana is inoculated into a proliferation medium and cultured in the dark.

[0022] Preferably, the method for preparing the proliferation medium is as follows: 1.0 mg of picloram and 7.0 g of agar powder are added to each liter of MS medium.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The method of the invention mainly comprises: preparing Korean lily sterile tissue culture seedlings with Korean lily sterile scales, inducing sterile dormant bulbs, breaking the dormant state of the bulbs through a normal light / dark photoperiod culture environment, selecting light green tip scattered non-scale leaf scales that are not embracing as explants for embryonic callus induction; using MS+plant growth regulator+anti-browning agent as callus induction culture medium components, and successfully inducing Korean lily embryonic callus.

[0025] Furthermore, in order to improve the acquisition rate of embryonic callus of Korean lily, after every 5 days of induction culture in the callus induction medium, the inoculated material was immersed in an anti-browning agent solution for 10 min to 15 min, the liquid on the surface of the inoculated material after the soaking treatment was absorbed, and then inoculated into fresh callus induction medium and cultured in complete darkness for a total of 3 times, and then continued to induce in darkness in the induction medium for 30 days to 32 days, and the embryonic callus of Korean lily was successfully obtained.

[0026] The Korean lily embryonic callus obtained by the invention has vigorous division and proliferation ability, the induction rate is 33.33%, and the proliferation coefficient can reach 6.80; the induction method is simple and easy, and provides technical support for the establishment of the Korean lily somatic embryogenesis system and the development of the transgenic system.

[0027] In summary, in view of the problem that it is difficult to induce embryonic callus of Korean lily and callus cannot be successfully cultured, the present invention discloses a method for inducing embryonic callus of Korean lily, preparing sterile tissue culture seedlings of Korean lily with sterile scales of Korean lily, adjusting the physiological state of the explant to obtain inoculation material, changing the inoculation material processing method, inoculation method and callus induction culture medium formula, and obtaining Korean lily embryonic callus for the first time, with a high success rate and good proliferation ability of Korean lily embryonic callus. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The sterile tissue culture seedlings of Lilium koreana in Example 1.

[0029] Figure 2 The swollen Korean lily bulbs were dark-cultured in Example 1.

[0030] Figure 3 This is the Korean lily bulb that was transferred to normal photoperiod culture in Example 1.

[0031] Figure 4 This is the explant of Lilium koreana used for callus induction in Example 1.

[0032] Figure 5 The induction results of different concentrations of PIC (picloram), A: MS + 0.5 mg / L PIC; B: MS + 1.0 mg / L PIC; C: MS + 2.0 mg / L PIC.

[0033] Figure 6 The induction results of immersion in GSH solutions of different concentrations: A: MS + 1.0 mg / L PIC + 0.25 mM GSH (reduced glutathione); B: a representative picture of Example 1; C: a representative picture of Example 2.

[0034] Figure 7 These are pictures of the tissue morphology observation of the embryonic callus of Korean lily. A: embryonic callus of Korean lily; B: section of embryonic callus of Korean lily.

[0035] Figure 8 The embryonic callus of Lilium koreanum that has been cultured for 60 days is used for proliferation. DETAILED DESCRIPTION

[0036] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0037] The inventive concept of the present invention is as follows: a method for inducing embryonic callus of Korean lily, comprising the following steps:

[0038] The scales of Korean lily were disinfected, inoculated into adventitious bud induction medium and cultured under normal photoperiod to prepare aseptic tissue culture seedlings of Korean lily.

[0039] In a sterile environment, the top leaves and roots of the aseptic tissue culture seedlings of Korean lily were cut off and inoculated into MS medium containing sucrose for dark culture.

[0040] When the circumference of the sterile bulb of Korean lily reaches 3cm to 5cm in dark culture and the number of scales reaches more than 8, it is transferred to normal light cycle culture, and then the light green, scattered and unenclosed middle-layer scaleless leaf scales of the sterile bulb of Korean lily are taken as explants.

[0041] Cut off the explants on all sides, leaving a surface area of ​​0.25 cm 2 ~0.40cm 2 , obtain the inoculation material, and then inoculate it with the concave side facing upward on the callus induction medium and culture it in the dark. During the period, it is soaked in an anti-browning agent solution to obtain the embryonic callus of Korean lily.

[0042] The anti-browning agent solution is a 0.5 mM to 1.0 mM reduced glutathione solution.

[0043] Plant regeneration pathways include organogenesis pathways and somatic embryogenesis pathways. According to the research of the present invention, for Korean lily, compared with the organogenesis pathway, the embryonic callus obtained by the somatic embryogenesis system has a strong division ability, a fast proliferation rate and a high coefficient, and can be used for large-scale and rapid induction. At the same time, the embryonic callus can be used as a good receptor for genetic transformation and used in genetic engineering breeding.

[0044] Dedifferentiation of explants to form callus is the primary condition and decisive factor for somatic embryogenesis. Existing reports show that in the process of lily somatic embryo induction, firstly, dedifferentiation of explants is completed by adding auxin and cytokinin hormones alone or in combination in the culture medium, and then redifferentiation and complete plant building steps are entered. However, we found that Korean lily is sensitive to hormone stimulation, dedifferentiation is difficult, and it is difficult to form callus with vigorous division ability. The induction of hormones in Korean lily scales is manifested as the formation of adventitious buds, which respond to regeneration or strong browning, resulting in explant death. So far, there is no report on the successful induction of Korean lily callus and somatic embryogenesis. In the present invention, inducing embryonic callus with the ability to divide and differentiate is the key to the regeneration of Korean lily through the somatic embryo pathway. The present invention selects the middle layer scales without scales with tender green tips and scattered unenclosed scales as explants, and uses anti-browning agents to soak and treat the scales during the induction process, and finally successfully obtains Korean lily embryonic callus in good condition. Without the method of soaking in anti-browning agent, it is impossible to successfully culture the embryonic callus of Korean lily.

[0045] Furthermore, the present invention also adds suitable plant growth regulators and anti-browning agents to the MS culture medium to induce more embryonic callus tissues of Korean lily in good condition.

[0046] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0047] Example 1

[0048] A method for inducing embryonic callus of Korean lily comprises the following steps:

[0049] Step 1: Obtain sterile tissue culture seedlings

[0050] Select the scales of Korean lily bulbs that are sterile and in good growth condition, rinse them with clean water, place the scales in a beaker, seal them with gauze, and rinse them under running water for 2 hours. Disinfect them in a clean bench. The disinfection method is: soak them in a 75% alcohol solution for 1 minute, rinse them with sterile water 5 times; immerse the rinsed scales in a 5% sodium hypochlorite solution for 30 minutes, rinse them with sterile water 5 times, and dry them with sterile filter paper.

[0051] The sterilized Korean lily bulbs were inoculated into adventitious bud induction medium for adventitious bud induction at a temperature of 25°C. The culture was carried out under normal photoperiod, with 16 hours of light and 8 hours of darkness per day. After 30 days of induction, the bulbs were inoculated into MS medium for subculture to obtain sterile tissue culture seedlings of Korean lily. Figure 1 shown.

[0052] The preparation method of the adventitious bud induction medium is as follows: 0.2 mg of NAA (naphthaleneacetic acid), 1.0 mg of 6-BA (6-benzylaminopurine) and 7.0 g of agar powder are added to each liter of MS.

[0053] Step 2: Adjust the bulb state and prepare sterile bulbs of Korean lily

[0054] In the clean bench, the top leaves and roots of the aseptic tissue culture seedlings of Korean lily obtained in step 1 were cut off, and then inoculated into the adjusted culture medium at a culture temperature of 25°C in the dark. After 20 days of culture, the base of the leaves began to swell, and after 30 days, the swollen bulbs began to form balls, and then the scales continued to differentiate, such as Figure 2 As shown. During this process, it is necessary to ensure complete darkness, otherwise scale leaves will grow on the top of the scales, which will affect the differentiation of callus tissue in the later stage. If the light regulation is not appropriate, scale leaves will begin to differentiate at the top of the scales. The scale leaves need to be cut off immediately, and then inoculated in a new adjusted culture medium, and cultured strictly at 25℃ and darkness for more than 30 days to ensure the thickness of the scales. The scale cells contain sufficient starch to provide sufficient nutrients for the subsequent dedifferentiation of the scales.

[0055] The preparation method of the adjusted culture medium is as follows: 60 g of sucrose and 7.0 g of agar powder are added to each liter of MS.

[0056] Step 3: Explant selection

[0057] When the circumference of the sterile bulb obtained in step 2 grows to 5 cm and the number of scales reaches 8, transfer it to a normal light cycle for 10 days. The normal light cycle refers to 16 hours of light and 8 hours of dark culture. At this time, the scales turn completely green and gradually disperse. Figure 3 As shown in the figure, the outermost scales have just begun to differentiate into scale leaves, while the middle and inner scales have not yet begun to differentiate. At this time, the bulb breaks dormancy and is in a vigorous growth period. Samples were collected in a clean bench, and the middle scales without scale leaves that were light green and had scattered and unenclosed tips were selected as explants. Figure 4 Five explants meeting the selection criteria are shown.

[0058] Step 4: Callus induction

[0059] The explants selected in step 3 were cut off on all sides in a clean bench to reduce the surface area of ​​the explant to 0.25 cm. 2 , and the inoculated material was obtained, and the concave surface was inoculated on the callus induction medium and cultured in the dark. During the above callus induction process, the inoculated material was taken out and soaked in the GSH solution once every 5 days, for a total of 3 times, and then inoculated into a new callus induction medium after the treatment, so as to inhibit browning by increasing the contact area.

[0060] The callus induction medium is: a plant growth regulator and an anti-browning agent are added to the MS medium to induce explant dedifferentiation. The formula of the callus induction medium used in this embodiment is: 1.0 mg of plant growth regulator PIC, 0.50 mmol of anti-browning agent GSH and 7.0 g of agar powder are added to each liter of MS medium.

[0061] The specific operation method of the immersion treatment is as follows: immerse the inoculated material in a sterile 0.5 mM GSH solution, the volume of the solution is such that the explant is submerged, and the GSH solution is shaken continuously during the period to make the inoculated material fully contact with the GSH solution. After immersion for 10 minutes, the surface liquid is dried with sterile filter paper, and the concave surface is inoculated upward into a new callus induction medium.

[0062] After completing three soaking treatments, the callus was inoculated into a new callus induction medium and cultured in the dark for 30 days. After a total of 45 days of induction, Korean lily callus was obtained.

[0063] The callus induction rate of Korean lily is shown in Figure 6 As shown in Figure B and Table 1, the obtained Korean lily callus tissue was subjected to histomorphological observation. According to histomorphological observation, the Korean lily callus tissue cells were small and aggregated, with obvious cell nuclei and thick protoplasm. It had typical characteristics of embryonic callus tissue and also had strong division ability. It was identified as embryonic callus tissue. Figure 7As shown in Figure A and Figure B, the present invention finally obtains the embryonic callus of Korean lily.

[0064] When embryonic callus of Lilium koreanum was inoculated into proliferation medium and cultured in the dark for 60 days, the callus proliferation coefficient could reach 6.80. Figure 8 shown.

[0065] The preparation method of the proliferation medium is as follows: 1.0 mg of PIC and 7.0 g of agar powder are added to each liter of MS medium.

[0066] Embodiment 2 to Embodiment 4

[0067] A method for inducing embryonic callus of Korean lily. In Examples 2 to 4, the PIC concentration and GSH concentration in the callus induction medium, as well as the GSH concentration used when soaking the inoculated material are different from those in Example 1, and the other conditions are exactly the same as those in Example 1.

[0068] In Examples 2 to 4, the PIC concentration and GSH concentration in the induction medium, and the GSH concentration used when the inoculated material was soaked are shown in Table 1, and the callus induction rate is shown in Table 1.

[0069] At the same time, concentration controls 1 to 5 were set, that is, except for the substance concentrations in Table 1, the other conditions were exactly the same as those in Example 1.

[0070] Callus induction rate = (number of inoculated materials that induced callus / total number of inoculated materials) × 100%.

[0071] Browning rate = (number of browned inoculated materials / total number of inoculated materials) × 100%.

[0072] Statistical analysis: Microsoft Excel was used to process relevant data for statistical analysis, and IBM SPSS Statistics 26 was used to analyze the significance level.

[0073] From the results in Table 1, it can be seen that when the concentrations of PIC and GSH changed to different degrees, embryonic callus could not be induced, and the subsequent proliferation steps could not be completed, and there was no proliferation result, which was indicated by "-" in Table 1. This proved that Korean lily was highly sensitive to PIC and GSH.

[0074] Figure 6 Figure A is the induction result of concentration control 2, and Figure C is the induction result of Example 2. Figure 6 It can also be seen that the method of the embodiment of the present invention has the best induction effect.

[0075] Table 1 Statistics of embryonic callus induction rate of Examples 1 to 4 and concentration controls 1 to 5

[0076]

[0077]

[0078] Note: Different lowercase letters indicate significant differences among treatments at the 0.05 level.

[0079] Example 5 (Compared with Example 1, parameters such as culture time were adjusted)

[0080] A method for inducing embryonic callus of Korean lily comprises the following steps:

[0081] Step 1: Obtain sterile tissue culture seedlings

[0082] Select the scales of Korean lily bulbs that are sterile and in good growth condition, rinse them with clean water, place the scales in a beaker, seal them with gauze, and rinse them under running water for 2 hours. Disinfect them in a clean bench. The disinfection method is: soak them in a 75% alcohol solution for 2 minutes, rinse them 3 times with sterile water; immerse the rinsed scales in a 5% sodium hypochlorite solution for 35 minutes, rinse them 3 times with sterile water, and dry them with sterile filter paper.

[0083] The sterilized Korean lily bulbs were inoculated into adventitious bud induction medium for adventitious bud induction at a temperature of 26°C. The bulbs were cultured in a normal photoperiod, with 16 hours of light and 8 hours of darkness per day. After 32 days of induction, the bulbs were inoculated into MS medium for subculture to obtain sterile tissue culture seedlings of Korean lily.

[0084] The preparation method of the adventitious bud induction medium is as follows: 0.2 mg of NAA, 1.0 mg of 6-BA and 7.0 g of agar powder are added to each liter of MS.

[0085] Step 2: Adjust the bulb state and prepare sterile bulbs of Korean lily

[0086] In the clean bench, the aseptic tissue culture seedlings of Korean lily obtained in step 1 were cut off from the top leaves and roots, and then inoculated into the adjusted culture medium at a culture temperature of 26°C in dark conditions. After 20 days of culture, the base of the leaves began to swell, and after 30 days, the swollen bulbs began to form balls, and then the scales continued to differentiate. In this process, complete darkness must be ensured, otherwise scale leaves will grow on the top of the scales, which will affect the differentiation of callus tissue in the later stage. If the light regulation is not suitable, scale leaves begin to differentiate at the top of the scales. The scale leaves need to be cut off immediately, and then inoculated into a new adjusted culture medium, and strictly cultured at 26°C and in the dark for more than 30 days to ensure the thickness of the scales. The scale cells contain sufficient starch to provide sufficient nutrients for the subsequent dedifferentiation of the scales.

[0087] The preparation method of the adjusted culture medium is as follows: 60 g of sucrose and 7.0 g of agar powder are added to each liter of MS.

[0088] Step 3: Explant selection

[0089] When the circumference of the sterile bulb obtained in step 2 grows to 3cm and the number of scales reaches 9, it is transferred to a normal photoperiod for 10 days. A normal photoperiod refers to 16 hours of light and 8 hours of dark culture. At this time, the color of the scales turns completely green and gradually disperses, and the outermost scales have just begun to differentiate into scale leaves, while the middle and inner scales have not yet begun to differentiate. At this time, the bulb breaks dormancy and is in a vigorous growth period. Take the material in the clean bench and select the middle-level scales without scale leaves that are light green and have dispersed and unenclosed tips as explants.

[0090] Step 4: Callus induction

[0091] The explants selected in step 3 were cut off on all sides in a clean bench to reduce the surface area of ​​the explant to 0.40 cm. 2 , and the inoculated material was obtained, and the concave surface was inoculated on the callus induction medium and cultured in the dark. During the above callus induction process, the inoculated material was taken out and soaked in the GSH solution once every 5 days, for a total of 3 times, and then inoculated into a new callus induction medium after the treatment, so as to inhibit browning by increasing the contact area.

[0092] The callus induction medium is: a plant growth regulator and an anti-browning agent are added to the MS medium to induce explant dedifferentiation. The formula of the callus induction medium used in this embodiment is: 1.0 mg of hormone PIC, 0.50 mmol of anti-browning agent GSH and 7.0 g of agar powder are added to each liter of MS medium.

[0093] The specific operation method of the soaking treatment is: soak the inoculated material in a sterile 0.5mM GSH solution, the volume of the solution is to cover the inoculated material, and the GSH solution is shaken continuously during the soaking to make the inoculated material fully contact with the GSH solution. After soaking for 15 minutes, the surface liquid is dried with sterile filter paper, and the concave surface is inoculated upward into a new callus induction medium.

[0094] After completing three soaking treatments, the culture medium was inoculated into a new callus induction medium for induction and cultured in the dark for 32 days to obtain Korean lily callus.

[0095] The callus induction rate of Korean lily was 31.48±3.11, which was identified as embryonic callus. Therefore, the embryonic callus of Korean lily was finally obtained in the present invention. The embryonic callus of Korean lily was inoculated in a proliferation medium and cultured in the dark for 60 days, and the callus proliferation coefficient could reach 6.70.

[0096] Comparative Experiment 1

[0097] In order to prove the effect of the present invention, we conducted a comparative experiment 1.

[0098] A method for inducing embryonic callus of lily, in control groups 1 to 22, the type and concentration of plant growth regulators are different from those in the callus induction medium of step 4 of Example 1. The callus induction medium of Example 1 is prepared by adding 1.0 mg of PIC, 0.50 mmol of anti-browning agent GSH and 7.0 g of agar powder to each liter of MS medium, while in control groups 1 to 22, PIC and GSH in the callus induction medium are replaced by the types and concentrations of plant growth regulators in Table 2.

[0099] Control groups 1 to 22 did not have the GSH solution soaking treatment step, and GSH was not added to the callus induction medium. Other conditions of control groups 1 to 22 were exactly the same as those of Example 1.

[0100] As described below, naphthylacetic acid: NAA; 6-benzylaminopurine: 6-BA; thidiazuron: TDZ; kinetin: KT; 2,4-dichlorophenoxyacetic acid: 2,4-D.

[0101] Thirty inoculants were inoculated for each treatment and replicated three times.

[0102] The results in Table 2 show that when only PIC was used for treatment, or only other types of plant growth regulators were used for treatment, the callus turned brown and embryonic callus could not be successfully induced, that is, the embryonic callus induction rate was 0. This shows that Korean lily is sensitive to the type and concentration of plant growth regulators, and the induction is manifested as the formation of adventitious buds or strong browning, and even leads to the death of the explants. The actual pictures of the induction results of control group 2, control group 3 and control group 4 are respectively referred to Figure 5 Figure A Figure 5 Figure B and Figure 5 Figure C.

[0103] Table 2 Statistics of mesoembryonic callus induction rate in control group 1 to control group 22

[0104]

[0105]

[0106] Note: “-” means there is no such item.

[0107] Comparative Experiment 2

[0108] In order to prove the effect of the present invention, we also conducted a browning inhibition experiment.

[0109] Selection of anti-browning substances and concentrations: The selected anti-browning substances are activated carbon (AC) 0.5g / L~2.0g / L; polyvinyl pyrrolidone (PVP) 1.0mg / L~5.0mg / L, ascorbic acid (VC) 0.1-0.5g / L and reduced glutathione (GSH) 0.25mM~1.0mM.

[0110] In the method of inducing lily embryonic callus in control groups 23 to 34, the anti-browning substance is different from the callus induction medium in step 4 of Example 1. The callus induction medium in Example 1 is prepared by adding 1.0 mg of PIC, 0.50 mmol of GSH and 7.0 g of agar powder to each liter of MS medium, while the callus induction medium in control groups 23 to 34 is changed to the anti-browning medium shown in Table 3.

[0111] Control groups 23 to 34 also had the GSH solution immersion treatment step of step 4 of Example 1, but the immersion treatment substance was changed to the anti-browning solution of Table 3, and the other immersion conditions were the same as step 4 of Example 1.

[0112] In addition, other conditions of control groups 23 to 34 are exactly the same as those of Example 1.

[0113] Browning rate = (number of browned inoculated materials / total number of inoculated materials) × 100%.

[0114] Statistical analysis: Microsoft Excel was used to process relevant data for statistical analysis, and IBM SPSS Statistics 26 was used to analyze the significance level.

[0115] Table 3 Statistics of browning in control group 23 to control group 34

[0116] deal with Anti-browning medium Anti-browning solution Scale browning rate (%) Control group 23 MS+0.50g / L AC 0.50g / L AC 78.89±0.05b Control group 24 MS+1.00g / L AC 1.00g / L AC 77.76±0.05bc Control group 25 MS+2.00g / L AC 2.00g / L AC 71.11±0.05c Control group 26 MS+1.00mg / L PVP 1.00mg / L PVP 100.00a Control group 27 MS+3.00mg / L PVP 3.00mg / L PVP 100.00a Control group 28 MS+5.00mg / L PVP 5.00mg / L PVP 100.00a Control group 29 MS+0.10g / L VC 0.10g / L VC 100.00a Control group 30 MS+0.30g / L VC 0.30g / L VC 100.00a Control group 31 MS+0.50g / L VC 0.50g / L VC 63.33±0.03d Control group 32 MS + 0.25 mM GSH 0.25 mM GSH 46.67±0.03e Control group 33 MS + 0.50 mM GSH 0.50 mM GSH 47.78±0.02e Control group 34 MS + 1.00 mM GSH 1.00mM GSH 44.44±0.05e

[0117] Note: Different lowercase letters indicate significant differences among treatments at the 0.05 level.

[0118] The results in Table 3 show that only GSH exhibited a good anti-browning effect on the scales of the aseptic tissue culture seedlings of Korean lily, while Korean lily was sensitive to other substances, with poor or even no anti-browning effect.

[0119] In summary, soaking in GSH solution can achieve anti-browning effect and is conducive to the induction of embryonic callus of Korean lily. The optimal conditions for inducing embryonic callus of Korean lily are: the induction medium is MS+1.0mg / L PIC+0.50mM GSH, and the inoculated material is soaked in 0.50mM GSH solution every 5 days during the induction process. After 3 times of treatment, the induction is continued for 30-32 days. When the induction is 45-47 days in total, the callus induction rate is increased from the original 0 to about 33.33%, and the callus is light yellow and has a good growth state. The proliferation coefficient can reach about 6.80 after 60 days of culture.

[0120] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0121] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for inducing embryonic callus of Lilium koreana, characterized in that: The following steps are involved: The scales of Korean lily are sterilized, inoculated into adventitious bud induction medium and cultured under normal photoperiod to prepare aseptic tissue culture seedlings of Korean lily; The top leaves and roots of the aseptic tissue culture seedlings of Korean lily were cut off in a sterile environment, inoculated into MS medium containing sucrose and cultured in the dark; When the circumference of the Korean lily sterile bulb reaches 3cm-5cm and the number of scales reaches more than 8, it is transferred to normal light cycle culture until the light green, scattered and unenclosed middle-layer scales of the Korean lily sterile bulb are taken as explants; Cut off the explants on all sides, leaving a surface area of ​​0.25 cm 2 ~0.40cm 2 , obtaining inoculated materials, and then inoculating them on callus induction medium with the concave surface upward and culturing them in the dark, during which they are soaked in an anti-browning agent solution to obtain embryonic callus of Korean lily; The anti-browning agent solution is a 0.5 mM to 1.0 mM reduced glutathione solution.

2. The method according to claim 1, characterized in that The soaking treatment method is: soak the inoculated material in a sterile anti-browning agent solution, shake it to make the inoculated material fully contact with the anti-browning agent solution, soak it for 10 minutes to 15 minutes, and then absorb the surface liquid of the inoculated material.

3. The method according to claim 2, characterized in that During the first 15 days of dark culture on the callus induction medium, the inoculated material was soaked every 5 days, the liquid on the surface of the inoculated material was sucked dry after the soaking treatment, and then inoculated into a new callus induction medium to continue dark culture; After all the soaking treatments were completed, the liquid on the surface of the inoculated material was dried, and then inoculated into a new callus induction medium and continued to be cultured in the dark for 30-32 days.

4. The method according to claim 1, characterized in that: The method for preparing the callus induction medium is as follows: adding a plant growth regulator and an anti-browning agent to each liter of MS medium; The plant growth regulator is 0.5mg to 1.0mg of picloram; The anti-browning agent is 0.5 mmol to 1.0 mmol of reduced glutathione.

5. The method according to claim 1, characterized in that The preparation method of the adventitious bud induction medium is as follows: 0.2 mg of naphthaleneacetic acid, 1.0 mg of 6-benzylaminopurine and 7.0 g of agar powder are added into each liter of MS medium.

6. The method according to claim 1, characterized in that When preparing the aseptic tissue culture seedlings of Korean lily, the culture conditions are: temperature 24°C to 26°C, 16h / 8h normal photoperiod for 30 to 32 days.

7. The method according to claim 1, characterized in that When preparing sterile bulbs of Korean lily, the dark culture conditions are: temperature 24°C to 26°C, and dark culture for more than 30 days.

8. The method according to claim 1, characterized in that The disinfection process is as follows: soaking the Korean lily scales in a 75% alcohol solution by volume for 1-2 minutes, and rinsing with sterile water for 3-5 times; immersing the rinsed Korean lily scales in a 5% sodium hypochlorite solution by volume for 30-35 minutes, and rinsing with sterile water for 3-5 times.

9. The method according to claim 1, characterized in that: The embryonic callus of Lilium koreanum was inoculated into a proliferation medium and cultured in the dark.

10. The method according to claim 9, characterized in that The method for preparing the proliferation medium is as follows: 1.0 mg of picloram and 7.0 g of agar powder are added to each liter of MS medium.

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