Method for promoting Lanzhou lily embryonic callus to proliferate and differentiate somatic embryo

Abstract

The invention provides a method for promoting proliferation of Lanzhou lily embryonic callus and differentiation of somatic embryos, and relates to the technical field of plant tissue culture. The method comprises the processes of explant selection, embryogenic callus induction, callus proliferation, somatic embryo differentiation by the callus, somatic embryo regeneration and seedling generation and the like. According to the method, the filaments of the tender buds of the Lanzhou lily are adopted as the explants for regeneration and propagation of the small bulblets, disinfection is easier, and the production cost is low. The embryogenic callus can be repeatedly subcultured and proliferated, somatic embryogenesis is carried out, and the induction time is shortened. The method is simple to operate and high in propagation coefficient.

Description

Technical Field

[0001] The present invention provides a method for promoting the proliferation of embryogenic callus of Lilium davidii var. unicolor and differentiating somatic embryos, belonging to the technical field of plant tissue culture. Background Art

[0002] Lilium davidii var. unicolor is a perennial herbaceous plant of the genus Lilium in the family Liliaceae, native to Lanzhou, Gansu, China. It is not only ornamental, but its underground bulbs can also be eaten. The bulb scales are large, thick, white as jade, tender, crispy and sweet, and rich in various nutritional elements such as protein, polysaccharide, alkaloid, etc., having extremely high food and medicinal values.

[0003] The traditional production mode of lily bulbs has long relied on the asexual reproduction of small bulbs. The bulb reproduction cycle is long, the reproduction coefficient is low, and the variety degenerates and the quality declines, seriously restricting the sustainable development of the Lilium davidii var. unicolor industry. In recent years, the promoted three - level breeding system for lily bulbs can shorten the bulb cultivation cycle, improve the bulb quality and yield. However, relying on scale cutting to breed bulblets has high production costs and the corresponding reproduction coefficient is still not high. Regenerating lily bulblets through tissue culture technology to shorten the reproduction cycle and increase the reproduction coefficient is a powerful way to realize the industrialized production of lily bulbs.

[0004] The somatic embryo regeneration system differentiated from embryogenic callus of lily has a higher reproduction coefficient and propagation rate than other bud regeneration systems. The existing somatic embryo regeneration system of lily usually uses the bulb scales of lily as explants. The explants are dedifferentiated to form embryogenic callus, the embryogenic callus is redifferentiated to form somatic embryos, and then the somatic embryos are differentiated into seedlings to obtain lily bulblets. This technical system has the following problems: (1) Using bulb scales as explants requires consuming a large number of commercial bulbs as the starting propagation materials, and the final production quantity is limited by the base number of the starting propagation materials; (2) The bulbs grow underground all year round, and it is difficult to thoroughly disinfect them as explants, resulting in a high contamination rate; (3) The bulb growth cycle is long, and the production cost is high as the starting propagation material; (4) After the subculture and proliferation of embryogenic callus, the ability to generate somatic embryos is significantly reduced, and it is necessary to repeatedly use explants to re - induce embryogenic callus and differentiate somatic embryos within the embryogenic retention time. The culture process is complex and the induction cycle is long.

[0005] The embryogenic callus of Lilium davidii var. unicolor induced by the existing technology can autonomously differentiate somatic embryos, but the duration is only 28 days (Zhang Jing, 2017). With the extension of the culture time, the callus of undifferentiated somatic embryos still maintains the state of callus and it is difficult to carry out somatic embryogenesis. The stagnation of somatic embryogenesis results in a significant reduction in the differentiation rate of regenerated seedlings, thus limiting the application of the somatic embryo regeneration system from callus in the rapid propagation of Lilium davidii var. unicolor bulbs. Therefore, how to make the callus of Lilium davidii var. unicolor differentiate a large number of somatic embryos on the basis of subculture proliferation is a technical bottleneck. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a culture method for promoting the subculture proliferation of embryogenic callus of Lilium davidii var. unicolor and differentiating somatic embryos, including processes such as explant selection, embryogenic callus induction, callus proliferation, differentiation of somatic embryos from callus, and regeneration of somatic embryos into seedlings.

[0007] The present invention is specifically implemented by the following scheme:

[0008] A method for promoting the proliferation of embryogenic callus of Lilium davidii var. unicolor and differentiating somatic embryos, comprising the following steps:

[0009] (1) Disinfection of explants

[0010] The selected flower buds of Lilium davidii var. unicolor are subjected to surface cleaning and disinfection treatment; the flower buds of Lilium davidii var. unicolor are cut open and the filaments are taken out as explants;

[0011] (2) Induction and proliferation of embryogenic callus

[0012] a. Inoculate the filaments into the L1 medium for embryogenic callus induction culture for 45 - 50 days to obtain embryogenic callus; the L1 medium is prepared by adding picloram (PIC), naphthaleneacetic acid (NAA), thidiazuron (TDZ), sucrose and agar to the MS medium, wherein the concentration of PIC in the L1 medium is 1.5 mg / L, the concentration of NAA is 0.2 mg / L, the concentration of TDZ is 0.1 mg / L, and the sucrose and agar are 30 g / L and 7 g / L respectively, and the pH value of the L1 medium is 5.8;

[0013] b. Transfer the embryogenic callus to the L2 medium for proliferation culture to obtain callus masses with a two-fold increase in proliferation; the L2 medium is prepared by adding picloram (PIC), naphthaleneacetic acid (NAA), sucrose and agar to the MS medium, wherein the concentration of PIC in the L2 medium is 1.0 mg / L, the concentration of NAA is 0.1 mg / L, and the sucrose and agar are 30 g / L and 7 g / L respectively, and the pH value of the L2 medium is 5.8;

[0014] (3) Somatic embryo differentiation from embryogenic callus clumps

[0015] c. Transfer the callus clumps to L3 medium for transitional culture for 2 weeks; the L3 medium is MS medium supplemented with 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), gibberellin (GA3), sucrose and agar; the concentration of 6-BA in the L3 medium is 0.8 mg / L, the concentration of NAA is 0.1 mg / L, the concentration of GA3 is 0.1 mg / L, and the sucrose and agar are 30 g / L and 7 g / L respectively; the pH value of the L3 medium is 5.8;

[0016] d. Transfer the callus clumps after transitional culture to C3 medium to induce somatic embryos for 20 - 30 days, and somatic embryos begin to form on the surface of the callus; the C3 medium is MS medium supplemented with picloram (PIC), naphthaleneacetic acid (NAA), inositol, sucrose and agar; the concentration of PIC in the C3 medium is 1 mg / L, the concentration of NAA is 0.1 mg / L, the concentration of inositol is 100 mg / L, and the sucrose and agar are 30 g / L and 7 g / L respectively; the pH value of the C3 medium is 5.8;

[0017] (4) Regeneration of somatic embryos into seedlings and swelling of small bulbs

[0018] Transfer the callus clumps forming somatic embryos to L4 medium for culture for 15 - 25 days, and the somatic embryos can germinate into small seedlings. Then transfer the small seedlings to L5 medium for swelling culture for 30 - 40 days to obtain small seed bulbs with a girth of 0.4 - 0.6 cm; the L4 medium is MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (6-BA), sucrose and agar; the concentration of 2,4-D in the L4 medium is 0.5 mg / L, the concentration of 6-BA is 0.2 mg / L, and the sucrose and agar are 30 g / L and 7 g / L respectively; the pH value of the L4 medium is 5.8;

[0019] The L5 medium is MS medium supplemented with naphthaleneacetic acid (NAA), sucrose and agar; the concentration of NAA in the L5 medium is 0.1 mg / L, the sucrose is 60 g / L, and the agar is 7 g / L; the pH value of the L5 medium is 5.8.

[0020] Preferably, the explant is selected as a plump and unbroken bud of Lilium davidii var. unicolor. Clean the dust on the surface of the bud, and disinfect it in a sterile environment; the disinfection method is to soak the bud in 75 wt% alcohol for 1 min first, and wash it 3 times with sterile water; then soak it in 2 wt% NaClO aqueous solution for 16 min, and keep shaking during the soaking process. Finally, wash it 3 - 5 times with sterile water.

[0021] Preferably, when inducing embryogenic callus from explants, the culture conditions are: dark culture for 45 - 50 days, and the temperature is 20℃ - 23℃.

[0022] Preferably, when proliferating callus, the culture conditions are: 16h / 8h photoperiod culture for 30 days, and the temperature is 23℃ - 25℃.

[0023] Preferably, before the callus differentiates into somatic embryos, a 14 - day transition culture is carried out, and the culture conditions are: 16h / 8h photoperiod culture for 14 days, and the temperature is 23℃ - 25℃.

[0024] Preferably, the culture conditions for somatic embryo formation are: dark culture for 25 days, and the temperature is 20 - 23℃.

[0025] Preferably, the culture conditions for somatic embryo regeneration into seedlings and bulblet swelling are: 16h / 8h photoperiod culture, the light intensity is 150μmol / m 2 / s, and the temperature is 23℃ - 25℃.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The explants of the present invention have a low contamination rate and are easily obtained. Using this method to regenerate and propagate small bulblets with the filaments of young flower buds of Lilium davidii var. unicolor as explants, disinfection is easier and the production cost is low.

[0028] The embryogenic callus of the present invention can be subcultured and proliferated repeatedly, shortening the induction time. The embryogenic callus subcultured by this method has good embryogenicity and fast proliferation, which not only shortens the induction time but also ensures the continuous supply of callus. Compared with re - inducing embryogenic callus from explants, it can be shortened by 45 days.

[0029] The operation method of the present invention is simple and the propagation coefficient is high. The somatic embryo induction coefficient of the embryogenic callus cultured by this method is 38, and the proliferation multiple of the embryogenic callus is 8, so the propagation coefficient of each filament can reach 300. Description of the Drawings

[0030] Figure 1 It is for the somatic embryo formation of embryogenic callus of Lilium davidii var. unicolor, where A is the flower bud of Lilium davidii var. unicolor; B is the embryogenic callus induced from the filament; C is the well - proliferated embryogenic callus; D is the somatic embryo formation of embryogenic callus; E is the morphological observation of embryogenic callus; F is the globular embryo; G is the heart - shaped embryo; H is the torpedo - shaped embryo; I is the cotyledon embryo; J is the somatic embryo regeneration; K is the somatic embryo regeneration into seedlings. Detailed Embodiments

[0031] To make the objectives, technical solutions and advantages of the present invention clearer, the preferred embodiments of the present invention will be further described in detail below in conjunction with the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0032] Example 1

[0033] A method for promoting the proliferation of embryogenic callus of Lilium davidii var. unicolor and differentiating somatic embryos, comprising the following steps:

[0034] (1) Disinfection of explants

[0035] Select plump and unbroken flower buds of Lilium davidii var. unicolor ( Figure 1 A), clean the dust on the surface of the flower buds, and disinfect them in a sterile environment; the disinfection method is to soak the flower buds in 75wt% alcohol for 1 min, wash them 3 times with sterile water; then soak them in 2wt% NaClO aqueous solution for 16 min, and keep shaking during the soaking process, and finally wash them 3 - 5 times with sterile water; then cut open the flower buds of Lilium davidii var. unicolor and take out the filaments as explants;

[0036] (2) Induction and proliferation of embryogenic callus

[0037] a. Inoculate the filaments into the L1 medium for embryogenic callus induction culture for 45 days to obtain embryogenic callus ( Figure 1 B), where the culture condition is dark culture, and the culture temperature is 20 - 23°C; the L1 medium is obtained by adding picloram (PIC), naphthaleneacetic acid (NAA), thidiazuron (TDZ), sucrose and agar to the MS medium, where the concentration of PIC in the L1 medium is 1.5 mg / L, the concentration of NAA is 0.2 mg / L, the concentration of TDZ is 0.1 mg / L, sucrose is 30 g / L, and agar is 7 g / L. The pH value of the L1 medium is 5.8;

[0038] On the 45th day after induction, the induction rates of embryogenic callus of four kinds of explants were respectively counted (Table 1). Through observation, it was found that anthers and ovaries could not form callus; very few stigmas could induce callus, and the induction rate was 5.56%; bright yellow granular callus could be formed on the surface of the filaments ( Figure 1 B, C), and the induction rate was 63%. Through morphological observation, it was found that: this kind of callus was loose in texture, and its cells were mostly spherical, with large nuclei, deep staining, and vigorously dividing embryogenic cells ( Figure 1 E), which was embryogenic callus, indicating that the filaments were the most suitable floral organs for inducing embryogenic callus.

[0039] b. Transfer the embryogenic callus to L2 medium for proliferation culture to obtain callus clumps with a two-fold increase in proliferation. The culture conditions are as follows: a photoperiod of 16 h / 8 h for 30 days, a light intensity of 150 μmol / m 2 / s, a temperature of 23°C - 25°C, and the humidity inside the bottle not exceeding 70%; the L2 medium is obtained by adding PIC, NAA, sucrose, and agar to MS medium. The concentration of PIC in the L2 medium is 1.0 mg / L, the concentration of NAA is 0.1 mg / L, the sucrose is 30 g / L, and the agar is 7 g / L. The pH value of the L2 medium is 5.8; five callus clumps are transferred to each bottle, and the diameter of the callus clumps is approximately 0.8 cm. Subculture is carried out every 30 days, and callus clumps with a two-fold increase in proliferation can be obtained each time. As the subculture cycle extends, at least callus with an eight-fold increase in proliferation can be obtained from each filament.

[0040] (3) Differentiation of embryogenic callus clumps into somatic embryos

[0041] c. Transfer the callus clumps to L3 medium for transitional culture. The culture conditions are as follows: a photoperiod of 16 h / 8 h for 14 days, a light intensity of 150 μmol / m 2 / s, a temperature of 23°C - 25°C, and the humidity inside the bottle not exceeding 70%; the L3 medium is obtained by adding 6-benzylaminopurine (6-BA), NAA, gibberellin (GA3), sucrose, and agar to MS medium; the concentration of 6-BA in the L3 medium is 0.8 mg / L, the concentration of NAA is 0.1 mg / L, the concentration of GA3 is 0.1 mg / L, the sucrose is 30 g / L, and the agar is 7 g / L; the pH value of the L3 medium is 5.8;

[0042] d. Transfer the callus clumps after transitional culture to C3 medium for differentiating somatic embryos. The culture conditions are as follows: dark culture for 25 days, a culture temperature of 20 - 23°C, and the humidity inside the bottle not exceeding 70%. After the culture ends, somatic embryos ( Figure 1 D) begin to form on the surface of the callus, including globular embryos, heart-shaped embryos, torpedo embryos, and cotyledon embryos ( Figure 1 D, F - I). Embryoids at different developmental stages will form on the same callus, indicating the asynchrony of the initiation and development of somatic embryos; the C3 medium is obtained by adding PIC, NAA, inositol, sucrose, and agar to MS medium; the concentration of PIC in the C3 medium is 1 mg / L, the concentration of NAA is 0.1 mg / L, the concentration of inositol is 100 mg / L, the sucrose is 30 g / L, and the agar is 7 g / L; the pH value of the C3 medium is 5.8;

[0043] In order to enable the embryogenic callus of Lilium davidii var. unicolor Salisb. subcultured and proliferated above to differentiate somatic embryos smoothly, the callus needs to be treated once before differentiation. The callus clumps subcultured and proliferated above are divided into two parts: one part is transferred to the L3 medium for transitional culture, marked as the T1 group; the other part continues to grow in the L2 medium as a control without transitional culture, marked as the T0 group. The culture conditions are as follows: cultured under a 16h / 8h photoperiod for 14 days, with a light intensity of 150 μmol / m 2 / s, a temperature of 23°C - 25°C, and the humidity inside the bottle not exceeding 70%. Five callus clumps are inoculated in each bottle. After 2 weeks, T0 and T1 are respectively transferred to the C1 - C6 media to induce somatic embryos, and the most suitable medium is screened out (Table 2). The culture conditions are as follows: dark culture, a culture temperature of 20 - 23°C, and the humidity inside the bottle not exceeding 70%.

[0044] The results show that: 1. After 25 days of induction, only the T1 callus that has undergone transitional culture has somatic embryo formation; 2. C3 is the most suitable medium for inducing somatic embryos, with the highest somatic embryo induction rate and induction coefficient, reaching 65% and 38 respectively; 3. Globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledon embryos are induced on the surface of the callus. As shown in Figure 1 F - I, embryoids at different developmental stages will form on the same callus, indicating the asynchrony of the initiation and development of somatic embryos; 4. The callus of the T0 group without transitional culture basically has no somatic embryo formation.

[0045] (4) Regeneration of somatic embryos into seedlings and swelling of small bulbs

[0046] The clumps forming somatic embryos are transferred to the L4 medium for culture for 20 days, and the somatic embryos can germinate into small seedlings ( Figure 1 J, K). Then the small seedlings are transferred to the L5 medium for swelling culture. After 35 days, small bulbs with a circumference of 0.4 - 0.6 cm can be obtained. The L4 medium is prepared by adding 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (6-BA), sucrose, and agar to the MS medium; the concentration of 2,4-D in the L4 medium is 0.5 mg / L, the concentration of 6-BA is 0.2 mg / L, the sucrose is 30 g / L, the agar is 7 g / L, and the pH value of the L4 medium is 5.8; among them, the culture conditions for the regeneration of somatic embryos into seedlings and the swelling of small bulbs are as follows: cultured under a 16h / 8h photoperiod, with a light intensity of 150 μmol / m 2 / s, a temperature of 23°C - 25°C; the L5 medium is prepared by adding naphthaleneacetic acid (NAA), sucrose, and agar to the MS medium; the concentration of NAA in the L5 medium is 0.1 mg / L, the sucrose is 60 g / L, the agar is 7 g / L, and the pH value of the L5 medium is 5.8.

[0047] Table 1 Effects of Different Explants on Callus Induction

[0048]

[0049]

[0050] Table 2 Screening of Somatic Embryo Induction Medium

[0051]

[0052] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

Claims

1. A method for promoting the proliferation of embryogenic callus of Lilium davidii var. unicolor and differentiating somatic embryos, characterized in that, It includes the following steps: (1) Disinfection of explants The selected Lilium davidii var. unicolor Salisb. flower buds are subjected to surface cleaning and disinfection treatment; the Lilium davidii var. unicolor Salisb. flower buds are cut open, and the filaments are taken out as explants; (2) Induction and proliferation of embryogenic callus a. The filaments are inoculated into the L1 medium for embryogenic callus induction culture for 45 - 50 days to obtain embryogenic callus; the L1 medium is prepared by adding picloram, naphthaleneacetic acid, thidiazuron, sucrose and agar to the MS medium, where the concentration of picloram in the L1 medium is 1.5 mg / L, the concentration of naphthaleneacetic acid is 0.2 mg / L, the concentration of thidiazuron is 0.1 mg / L, sucrose is 30 g / L, and agar is 7 g / L; the pH value of the L1 medium is 5.8; b. The embryogenic callus is transferred to the L2 medium for proliferation culture to obtain a callus mass with a two - fold increase in proliferation; the L2 medium is prepared by adding picloram, naphthaleneacetic acid, sucrose and agar to the MS medium, where the concentration of picloram in the L2 medium is 1.0 mg / L, the concentration of naphthaleneacetic acid is 0.1 mg / L, sucrose is 30 g / L, and agar is 7 g / L; the pH value of the L2 medium is 5.8; (3) Differentiation of embryogenic callus mass into somatic embryos c. The callus mass is transferred to the L3 medium for transitional culture for 14 days; the L3 medium is prepared by adding 6 - benzylaminopurine, naphthaleneacetic acid, gibberellin, sucrose and agar to the MS medium; the concentration of 6 - BA in the L3 medium is 0.8 mg / L, the concentration of naphthaleneacetic acid is 0.1 mg / L, the concentration of gibberellin is 0.1 mg / L, sucrose is 30 g / L, and agar is 7 g / L; the pH value of the L3 medium is 5.8; d. The callus mass after transitional culture is transferred to the C3 medium for somatic embryo differentiation for 20 - 30 days, and somatic embryos begin to form on the surface of the callus; the C3 medium is prepared by adding picloram, naphthaleneacetic acid, inositol, sucrose and agar to the MS medium; the concentration of picloram in the C3 medium is 1 mg / L, the concentration of naphthaleneacetic acid is 0.1 mg / L, the concentration of inositol is 100 mg / L, sucrose is 30 g / L, and agar is 7 g / L; the pH value of the C3 medium is 5.8; (4) Regeneration of somatic embryos into seedlings and bulbil swelling The callus mass forming somatic embryos is transferred to the L4 medium for culture for 15 - 25 days, and the somatic embryos can germinate into small seedlings. Then the small seedlings are transferred to the L5 medium for swelling culture for 30 - 40 days to obtain small seed bulbs with a circumference of 0.4 - 0.6 cm; the L4 medium is prepared by adding 2,4 - dichlorophenoxyacetic acid, 6 - benzylaminopurine, sucrose and agar to the MS medium; the concentration of 2,4 - dichlorophenoxyacetic acid in the L4 medium is 0.5 mg / L, the concentration of 6 - benzylaminopurine is 0.2 mg / L, sucrose is 30 g / L, and agar is 7 g / L; the pH value of the L4 medium is 5.8; The L5 medium is prepared by adding naphthaleneacetic acid, sucrose and agar to the MS medium; the concentration of NAA in the L5 medium is 0.1 mg / L, the sucrose is 60 g / L, and the agar is 7 g / L; the pH value of the L5 medium is 5.

8.

2. The method for promoting the proliferation of embryogenic callus of Lilium davidii var. unicolor and differentiating somatic embryos as described in claim 1, wherein, Step (1) is to select plump and intact flower buds of Lilium davidii var. unicolor, clean the dust on the surface of the flower buds, and disinfect them in a sterile environment; the disinfection method is to first soak the flower buds in 75 wt% alcohol for 1 min, wash them 3 times with sterile water; then soak them in 2 wt% NaClO aqueous solution for 16 min, shake constantly during the soaking process, and finally wash them 3 - 5 times with sterile water.

3. The method for promoting the proliferation of embryogenic callus of Lilium davidii var. unicolor and differentiating somatic embryos as claimed in claim 1, characterized in that, The culture conditions for step a are dark culture for 45 to 50 days, and the culture temperature is 20 to 23°C; the culture conditions for step b are 16h / 8h photoperiod culture for 30 days, the light intensity is 150 μmol / m 2 / s, the temperature is 23°C to 25°C, and the humidity in the bottle does not exceed 70%.

4. The method for promoting the proliferation of embryogenic callus of Lilium davidii var. unicolor and differentiating somatic embryos as claimed in claim 1, characterized in that, The culture conditions in step c are as follows: culture for 14 days with a 16h / 8h photoperiod, light intensity of 150 μmol / m 2 / s, temperature of 23°C to 25°C, humidity in the bottle not exceeding 70%; the culture conditions in step d are dark culture for 25 days, culture temperature of 20 - 23°C, humidity in the bottle not exceeding 70%.

5. The method for promoting the proliferation of embryogenic callus of Lilium davidii var. unicolor and differentiating somatic embryos as claimed in claim 1, wherein, The conditions for seedling regeneration and swelling culture in step (4) are: culture under a 16h / 8h photoperiod for 25 to 30 days, with a light intensity of 150 μmol / m 2 / s, a temperature of 23°C to 25°C, and the humidity inside the bottle not exceeding 70%.

Images