Efficient Lanzhou lily bulb commercial seed ball production method

Abstract

The invention provides an efficient Lanzhou lily commercial bulblet production method. The method comprises the processes of induction, proliferation, expansion, domestication, transplanting and the like. According to the method, a large number of materials can be bred by using fewer sterile seedlings, the induction efficiency and the breeding efficiency are remarkably improved, the breeding cardinal number is increased by more than 1300 times through one-time tissue culture period, the survival rate is 100%, and a large number of commercial bulblets can be obtained only in 10 months. According to the method, a perfect commercial bulblet breeding system is established, the culture time of the commercial bulblets is remarkably shortened, and the method is particularly suitable for the situation that the cardinal number of original materials is small.

Description

Technical Field

[0001] The invention belongs to the technical field of lily planting, and in particular relates to an efficient method for producing commercial seed balls of Lanzhou lily. Background Art

[0002] At present, the main method used in lily production is scale cuttings for bulb propagation, which has a low propagation efficiency. The propagation method based on tissue culture technology is more efficient and has the potential for factory-based bulb propagation. However, when lily bulbs are used as original propagation materials, the following problems exist: (1) It is still impossible to achieve pollution-free and 100% survival; (2) Mercuric chloride is the most effective disinfectant, but it is harmful to humans and the environment and is now banned; (3) A complete factory-based propagation system for commercial lily bulbs has not been formed; and (4) There are similar problems to the scale cutting propagation method, that is, a large base of original propagation materials is required for large-scale propagation. If the base of original propagation materials is small, a long breeding cycle is required to produce a large number of materials.

[0003] Therefore, there is an urgent need for a method in which a large number of commercial seed balls can be obtained by breeding with less original propagation materials in a short period of time. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an efficient method for producing commercial Lanzhou lily bulbs in view of the deficiencies of the above-mentioned prior art, including the processes of induction, proliferation, expansion, acclimation and transplantation, and a large amount of material can be propagated using a small number of sterile seedlings, and the induction efficiency and propagation efficiency are significantly improved, with the propagation efficiency reaching more than 1,300 times.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] An efficient method for producing commercial Lanzhou lily seed balls, the method comprising the following steps:

[0007] S1. Adventitious bud induction and proliferation

[0008] The sterile bulbs of Lanzhou lily seedlings with good growth and a diameter of 1 cm were selected as explants, and the scales were peeled off and cut into small pieces of 0.5*0.5 cm. They were first induced in F1 medium for 30 days, then subcultured to F2 medium for another 30 days, and finally inoculated into F3 medium for proliferation and culture for 30 days. The culture conditions were: culture temperature 25℃, light intensity 150μmol·m -2 ·s -1 , the photoperiod was 16 h light / 8 h dark, and after three cultivations, small bulbs were obtained;

[0009] The F1 culture medium formula is: MS+1.5-2 mg / L 6-BA+0.1-0.2 mg / L NAA+30 g / L sucrose, pH 5.8;

[0010] The F2 culture medium formula is: MS+1.5-2 mg / L 6-BA+0.1-0.2 mg / L NAA+30 g / L sucrose, pH 5.8;

[0011] The F3 culture medium formula is: MS+0-2 mg / L 6-BA+0-0.2 mg / L NAA+30-60 g / L sucrose, pH 5.8;

[0012] S2. Bulblet enlargement

[0013] The small bulblets obtained from S1 were inoculated into a medium containing MS + 90 g / L sucrose and pH 5.8 and cultured in the dark at 25°C. The bulblets were subcultured every 60 days. After two subcultures, bulblets with a diameter of 1.0-1.2 cm were formed.

[0014] S3. Acclimation and transplanting

[0015] The bulbs obtained from S2 were opened and acclimated. After acclimation, the bulbs were cleaned, the culture medium was removed, and the bulbs were embedded in a high-temperature sterilized mixed matrix. After being stored at 4°C in the dark for 45 days, they were transplanted.

[0016] Preferably, the Lanzhou lily sterile seedlings are obtained from Lanzhou lily bulbs after explant disinfection and regeneration culture. The explant disinfection method is: peel off the scales and soak them in a 2g / L detergent aqueous solution for 30 minutes, rinse with water, then put the scales into a sterilized beaker, add a 75% ethanol solution by mass until it is completely immersed, shake for 1 minute and then pour out the ethanol solution, add distilled water and shake for 2 minutes, then pour out the distilled water, repeat once; then add a 25% sodium hypochlorite solution by mass until the explant is completely immersed, shake for 10 minutes, pour out the sodium hypochlorite solution; add distilled water and shake for 1 minute, then pour out the distilled water, repeat 5 times. The regeneration culture is to place the sterilized explants in a culture dish covered with 5 layers of sterilized filter paper, remove excess water, sterilize by ultraviolet for 30 minutes, cut the scales into small pieces of about 0.5 cm*1.0 cm, inoculate them into a medium containing MS+1.5 mg / L 6-BA+0.1 mg / L NAA+30 g / L sucrose, pH 5.8, and keep them at a temperature of 25°C and a light intensity of 150 μmol·m -2 ·s -1 After culturing for 30 days under a photoperiod of 16 h light / 8 h dark, sterile seedlings were obtained.

[0017] Preferably, the F1 culture medium formula in S1 is: MS+1.5 mg / L 6-BA+0.1 mg / L NAA+30 g / L sucrose, with a pH of 5.8.

[0018] Preferably, the F2 culture medium formula in S1 is: MS+1.5 mg / L 6-BA+0.1 mg / L NAA+30 g / L sucrose, with a pH of 5.8.

[0019] Preferably, the F3 culture medium formula in S1 is: MS+60g / L sucrose, pH 5.8.

[0020] Preferably, the bottle opening acclimatization time in S3 is 6 days.

[0021] Preferably, the mixed matrix in S3 is peat: garden soil: vermiculite: perlite = 2:2:1:1.

[0022] The present invention has significant technical effects due to the adoption of the above technical solution:

[0023] 1. The present invention provides an efficient method for producing commercial seed balls of Lanzhou lily, including induction, proliferation, expansion, acclimatization and transplantation, etc., and can achieve a survival rate of 100% without a special seedling hardening process. A large amount of materials can be propagated using fewer sterile seedlings, and the induction efficiency and propagation efficiency are significantly improved. The propagation base is increased by more than 1,300 times through a tissue culture cycle, which is particularly suitable for the case where the base of the original material is small. Similarly, when the sterile seedling bulb is used as an explant, according to the optimal regeneration efficiency calculation of existing research, 8-10 scales are taken from each bulb, each scale induces 4-5 buds, and each bud proliferates 4-5 buds. The propagation efficiency of the prior art is about 128-250 times, the propagation efficiency is low, and the base of the original propagation material is large.

[0024] 2. The explant disinfection process of the present invention uses a combination of ethanol and sodium hypochlorite to perfectly replace mercuric chloride, which not only solves the problem of explant contamination, achieves a survival rate of 100%, but also avoids the harm of mercuric chloride to humans and the environment.

[0025] 3. The breeding cycle of the present invention is short, and a large number of commercial seed balls can be obtained in just 10 months. A complete commercial seed ball breeding system has been established, which significantly shortens the cultivation time of commercial seed balls.

[0026] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram of the sterilization of Lanzhou lily explants and the regeneration of sterile seedlings in Example 1;

[0028] Figure 2 is a graph showing the growth of adventitious buds induced by different treatments in Example 2;

[0029] Figure 3 is a diagram showing the growth of lily seedlings after the bulblets are enlarged and before they are domesticated in Example 2;

[0030] Figure 4 These are graphs showing the growth of lily plants after acclimatization and transplantation in Example 2, wherein Graph a shows the growth 15 days after transplantation, and Graph b shows the growth 45 days after transplantation. DETAILED DESCRIPTION

[0031] Example 1

[0032] This example is about the cultivation of sterile seedlings of Lilium lanzhouensis, and the method is as follows:

[0033] 1. Explant disinfection

[0034] (1) Select Lanzhou lily bulbs with full bulbs and no diseases or insect pests as explants. First, rinse the bulbs, peel off the scales and soak them in a 2g / L detergent solution for 30 minutes (use 50°C-60°C tap water to fully dissolve the detergent and use it after cooling to room temperature). After fully rinsing with tap water, remove excess water and set aside;

[0035] (2) In a clean bench, place the scales in a sterilized beaker, add 75% ethanol solution (prepared with distilled water) to completely submerge the explants, shake for 1 min, then pour out the ethanol solution, add distilled water, shake for 2 min, then pour out the distilled water, and repeat once;

[0036] (3) Add 25% sodium hypochlorite solution until the explants are completely submerged, and treat for 10 min. During this period, shake the explants continuously and discard the sodium hypochlorite solution. Add distilled water and shake for 1 min. Discard the distilled water and repeat this process 5 times.

[0037] The above distilled water was sterilized at 121℃ for 20min and cooled to room temperature before use.

[0038] 2. Regeneration of sterile seedlings

[0039] The sterilized explants were placed in a culture dish covered with 5 layers of sterilized filter paper, and excess water was removed. After UV sterilization for 30 min, the scales were cut into small pieces of about 0.5 cm*1.0 cm and inoculated into a medium containing MS+1.5 mg / L 6-BA+0.1 mg / L NAA+30 g / L sucrose, pH 5.8, at a temperature of 25 °C and a light intensity of 150 μmol·m -2 ·s -1 , cultured for 30 days under a photoperiod of 16 h light / 8 h dark to obtain sterile seedlings.

[0040] Figure 1 It is a diagram of the Lanzhou lily explant disinfection and aseptic seedling regeneration of the present embodiment.

[0041] The effects of different disinfection treatments on the contamination rate and survival rate of Lanzhou lily are shown in Table 1. The results show that the sodium hypochlorite solution treatment for 10 minutes has the best effect, with a contamination rate of 0 and a survival rate of 100%.

[0042] Table 1 Effects of different disinfection treatment combinations on the contamination rate and survival rate of Lilium lanzhouensis

[0043]

[0044] Example 2

[0045] This embodiment is a highly efficient method for producing high-efficiency commercial seed balls of Lanzhou lily, which comprises the following steps:

[0046] S1. Adventitious bud induction and proliferation

[0047] The sterile bulbs of Lanzhou lily seedlings with good growth and a diameter of about 1 cm were selected as explants. The scales were peeled off and cut into small pieces of 0.5*0.5 cm. They were first induced in F1 medium for 30 days, then subcultured to F2 medium for another 30 days, and finally inoculated into F3 medium for proliferation and culture for 30 days. The culture conditions were: culture temperature 25℃, light intensity 150μmol·m -2 ·s -1 The photoperiod was 16 h light / 8 h dark. After three cultivations, small bulbs were obtained.

[0048] S2. Bulblet enlargement

[0049] The small bulbs obtained from S1 were inoculated into a medium of MS + 90 g / L sucrose, pH 5.8, and cultured in the dark at 25°C. The cells were subcultured every 60 days. After two subcultures, bulbs with a diameter of 1.0-1.2 cm were formed.

[0050] S3. Acclimation and transplanting

[0051] The bulbs obtained from S2 were opened and acclimated. After acclimation, the bulbs were cleaned, the culture medium was removed, and they were embedded in a high-temperature sterilized mixed matrix (peat: garden soil: vermiculite: perlite = 2:2:1:1). They were stored at 4°C in the dark for 45 days before transplanting.

[0052] In this example, adventitious bud induction and proliferation were carried out in 6 treatments using different culture medium formulations, with 36 scales in each treatment group and three replicates. The formulation compositions of the different treatment groups are shown in Table 2.

[0053] Table 2 Adventitious bud induction and proliferation treatment

[0054]

[0055] The growth of induced adventitious buds under different treatments Figure 2 The induction results are shown in Table 3, where the adventitious bud induction coefficient = (number of adventitious buds - number of deformed buds) / 36 × 100%. The results showed that the T3 treatment group: after being inoculated in a medium of MS + 1.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose, pH 5.8 for 30 days, it was subcultured to a medium of MS + 1.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose, pH 5.8 for 30 days, and then inoculated to a medium of MS + 60 g / L sucrose, pH 5.8 for 30 days of proliferation culture, and the effect was the best. Finally, a Lanzhou lily bulb with a diameter of about 1 cm formed more than 1,300 adventitious buds that grew normally, and no deformed buds were formed.

[0056] Table 3 Induction results of different treatments

[0057]

[0058]

[0059] Figure 3 This is a picture of the growth of lily seedlings after the bulblets have expanded but before being domesticated.

[0060] In this embodiment, the bulbs were opened and acclimated for 0 days, 3 days and 6 days, with 6 plants in each treatment group, repeated three times, and then the acclimated bulbs were cleaned, the culture medium was removed, and the bulbs were embedded in a high-temperature sterilized mixed matrix (peat: garden soil: vermiculite: perlite = 2:2:1:1). After being stored in the dark at 4°C for 45 days, they were transplanted and planted in the mixed matrix (same as above). The results are as follows: Figure 4 As shown, Figure a shows the overall growth situation 15 days after transplanting, and Figure b shows the overall growth situation 45 days after transplanting. It was found that all lily seedlings could germinate 10 days after transplanting. The germination rate of lily seedlings after opening the bottle and domestication was faster, and the effect of domestication for 6 days was the best.

[0061] For the T3 treatment group, after the bulblets were enlarged and acclimated for 6 days before transplanting, 3921 commercial bulblets were obtained from 3 sterile Lanzhou lily seedling bulbs with a diameter of about 1 cm, with a germination rate of 100% and a reproduction coefficient of 1307.

[0062] The present invention provides an efficient method for producing commercial seed balls of Lanzhou lily through induction, proliferation, expansion, acclimation and transplantation, etc., a large amount of materials can be propagated using fewer sterile seedlings, the induction efficiency and propagation efficiency are significantly improved, the propagation base is increased by more than 1,300 times through one tissue culture cycle, and the survival rate reaches 100%, which is particularly suitable for the situation where the base of the original material is small. The breeding cycle of the present invention is short, a large amount of commercial seed balls can be obtained in only 10 months, a complete commercial seed ball breeding system is established, and the culture time of the commercial seed balls is significantly shortened.

[0063] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. An efficient method for producing commercial Lanzhou lily seed balls, characterized in that: The method comprises the following steps: S1. Adventitious bud induction and proliferation The sterile bulbs of Lanzhou lily seedlings with good growth and a diameter of 1 cm were selected as explants, and the scales were peeled off and cut into small pieces of 0.5*0.5 cm. They were first induced in F1 medium for 30 days, then subcultured to F2 medium for another 30 days, and finally inoculated into F3 medium for proliferation and culture for 30 days. The culture conditions were: culture temperature 25℃, light intensity 150μmol·m -2 ·s -1 , the photoperiod was 16 h light / 8 h dark, and after three cultivations, small bulbs were obtained; The F1 culture medium formula is: MS+1.5-2 mg / L 6-BA+0.1-0.2 mg / L NAA+30 g / L sucrose, pH 5.8; The F2 culture medium formula is: MS+1.5-2 mg / L 6-BA+0.1-0.2 mg / L NAA+30 g / L sucrose, pH 5.8; The F3 culture medium formula is: MS+0-2 mg / L 6-BA+0-0.2 mg / L NAA+30-60 g / L sucrose, pH 5.8; S2. Bulblet enlargement The small bulblets obtained from S1 were inoculated into a medium containing MS + 90 g / L sucrose and pH 5.8 and cultured in the dark at 25°C. The bulblets were subcultured every 60 days. After two subcultures, bulblets with a diameter of 1.0-1.2 cm were formed. S3. Acclimation and transplanting The bulbs obtained from S2 were opened and acclimated. After acclimation, the bulbs were cleaned, the culture medium was removed, and the bulbs were embedded in a high-temperature sterilized mixed matrix. After being stored at 4°C in the dark for 45 days, they were transplanted.

2. The method for producing an efficient commercial seed ball of lily of Lanzhou according to claim 1, characterized in that: The Lanzhou lily sterile seedlings are obtained by subjecting the Lanzhou lily bulbs to explant disinfection and regeneration culture.

3. The method for producing an efficient commercial seed ball of lily of Lanzhou according to claim 2, characterized in that: The explant disinfection method comprises the following steps: peeling off the scales, placing the scales in a 2g / L washing powder aqueous solution, soaking the scales for 30 minutes, rinsing the scales with water, placing the scales in a sterilized beaker, adding an ethanol solution with a mass fraction of 75% until the scales are completely immersed, shaking the scales for 1 minute, then pouring out the ethanol solution, adding distilled water, shaking the scales for 2 minutes, then pouring out the distilled water, and repeating the process once; then adding a sodium hypochlorite solution with a mass fraction of 25% until the scales are completely immersed, shaking the scales for 10 minutes, then pouring out the sodium hypochlorite solution, adding distilled water, shaking the scales for 1 minute, then pouring out the distilled water, and repeating the process 5 times.

4. The method for producing an efficient commercial seed ball of lily of Lanzhou according to claim 2, characterized in that: The regeneration culture is to place the sterilized explants in a culture dish covered with 5 layers of sterilized filter paper, remove excess water, sterilize by ultraviolet for 30 minutes, cut the scales into small pieces of about 0.5 cm*1.0 cm, inoculate them into a medium containing MS+1.5 mg / L 6-BA+0.1 mg / L NAA+30 g / L sucrose, pH 5.8, and keep them at a temperature of 25°C and a light intensity of 150 μmol·m -2 ·s -1 After culturing for 30 days under a photoperiod of 16 h light / 8 h dark, sterile seedlings were obtained.

5. The method for producing an efficient commercial seed bulb of lily of Lanzhou according to claim 1, characterized in that: The F1 culture medium formula described in S1 is: MS+1.5 mg / L 6-BA+0.1 mg / L NAA+30 g / L sucrose, pH 5.

8.

6. The method for producing an efficient commercial seed bulb of lily of Lanzhou according to claim 1, characterized in that: The F2 culture medium formula described in S1 is: MS+1.5 mg / L 6-BA+0.1 mg / L NAA+30 g / L sucrose, pH 5.

8.

7. The method for producing an efficient commercial seed bulb of Lanzhou lily according to claim 1, characterized in that: The F3 culture medium formula described in S1 is: MS+60g / L sucrose, pH 5.

8.

8. The method for producing an efficient commercial seed bulb of Lanzhou lily according to claim 1, characterized in that: The acclimatization time after opening the bottle described in S3 is 6 days.

9. The method for producing an efficient commercial seed bulb of Lanzhou lily according to claim 1, characterized in that: The mixed matrix in S3 is peat: garden soil: vermiculite: perlite = 2:2:1:1.

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