A method for inducing seedlings using young embryos of water lilies

Abstract

The present invention relates to the technical field of water lily breeding. The present invention provides a method for inducing seedlings from water lily embryos, comprising the following steps: (1) sterilizing water lily seeds 13-18 days after pollination with alcohol and sodium hypochlorite, and then isolating the water lily embryos; (2) inoculating the water lily embryos into culture medium A and culturing for 12-16 days to obtain water lily buds; (3) further culturing the water lily buds into seedlings, and transplanting them into peat soil for further growth; the culture medium A is a modified MS culture medium + 0.01-0.6 mg / L 6-BA + 0.01-0.6 mg / L NAA + 0.3-0.7 g / L activated carbon. Using the culture medium A formulated in the present invention can effectively increase the induction rate of adventitious buds, and the technical solution of the present invention is simple to operate, and the induced water lily seedlings have low mortality, low contamination rate, and high survival rate.

Description

Technical Field

[0001] The invention relates to the technical field of water lily breeding, and in particular to a method for inducing water lily seedlings by using young embryos of the water lily. Background Art

[0002] Water lilies (Nymphaea), as important aquatic ornamental plants, possess extensive economic and ecological value worldwide. However, the genetic transformation system for water lilies is still immature, hindering the development of transgenic water lilies. Using transgenic technology to genetically improve water lilies and cultivate new varieties with desirable traits such as double petals, higher levels of medicinal compounds, and a long vase life has become a key direction in water lily research. Cultivating young water lily embryos in vitro provides ideal material for genetic transformation (including gene introduction and screening of transgenic plants), but no relevant reports exist. Therefore, developing an efficient technology for culturing young water lily embryos and inducing seedlings would not only help improve reproductive efficiency but also provide a key technical platform for future transgenic research. Summary of the Invention

[0003] The object of the present invention is to provide a method for inducing seedlings using young embryos of water lilies. The culture medium A of the present invention can effectively improve the induction rate of adventitious buds. The technical solution of the present invention is simple to operate, and the induced cultured water lily seedlings have low mortality, low contamination rate and high survival rate.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for inducing seedlings from young embryos of water lilies, comprising the following steps:

[0006] (1) After 13-18 days of pollination, the water lily seeds were disinfected with alcohol and sodium hypochlorite, and the water lily embryos were separated;

[0007] (2) Inoculate water lily embryos into culture medium A and culture for 12-16 days to obtain water lily buds;

[0008] (3) Cultivate the water lily buds into seedlings and transplant them into peat soil for further growth.

[0009] Preferably, the culture medium A is a modified MS culture medium + 0.01-0.6 mg / L 6-BA + 0.01-0.6 mg / L NAA + 0.3-0.7 g / L activated carbon.

[0010] Preferably, the volume concentration of the alcohol in step (1) is 60-75%, the time for the alcohol disinfection is 20-40s, and the number of alcohol disinfections is 1-2 times.

[0011] Preferably, the mass concentration of the sodium hypochlorite in step (1) is 8-12%, the time for the sodium hypochlorite disinfection is 0.5-1.5 min, and the number of sodium hypochlorite disinfections is 1-2 times.

[0012] Preferably, the improved MS medium is MS medium + 20-70 g / L sucrose + 6-10 g / L agar.

[0013] Preferably, the pH value of the culture medium A in step (2) is 5.7-5.9.

[0014] Preferably, the culture temperature in step (2) is 25-31°C, the culture light cycle is 12 hours light / 12 hours dark, and the culture light intensity is 163-182 umol m -2 s -1 The illumination of the culture is 11840-13207 lx.

[0015] By adopting the above technical solution, the present invention has the following beneficial effects:

[0016] The present invention improves and optimizes the culture medium. The optimized culture medium A, containing appropriate concentrations of 6-BA, NAA, and activated carbon, increases the induction rate of adventitious buds. Furthermore, the present invention is simple to operate, and the induced water lily seedlings have low mortality and contamination rates, while maintaining a high survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The growth of water lily seedlings after transplanting;

[0018] Figure 2 The growth of water lily embryos under different photoperiods ( Figure 2 A represents the experimental group cultured for 1 day, B represents the experimental group cultured for 10 days, C represents the control group 1 cultured for 14 days, and D represents the control group 2 cultured for 15 days).

[0019] Figure 3 The growth of water lily embryos in medium group 1 ( Figure 3 A represents the peeling of the immature embryo from the seed, B represents the appearance change of the immature embryo after 5 days of growth, C represents the situation of the immature embryo after 14 days of growth, and D represents the situation of the immature embryo after 20 days of growth). DETAILED DESCRIPTION

[0020] The present invention provides a method for inducing seedlings from young embryos of water lilies, comprising the following steps:

[0021] (1) After 13-18 days of pollination, the water lily seeds were disinfected with alcohol and sodium hypochlorite, and the water lily embryos were separated;

[0022] (2) Inoculate water lily embryos into culture medium A and culture for 12-16 days to obtain water lily buds;

[0023] (3) Cultivate the water lily buds into seedlings and transplant them into peat soil for further growth.

[0024] In the present invention, the degree of seed maturity is controlled by artificial pollination, and the petals, sepals and other appendages of the water lilies 4-18 days after pollination are removed, leaving only the ovary part of the water lily (water lily seeds). The water lily seeds are then disinfected with alcohol and sodium hypochlorite, and the volume concentration of the alcohol is preferably 60-75%, further preferably 65-72%, and further preferably 70%; the time for the alcohol disinfection is preferably 20-40s, further preferably 25-35s, and further preferably 30s; the number of times the alcohol disinfection is 1-2 times. The mass concentration of the sodium hypochlorite of the present invention is preferably 8-12%, further preferably 9-11%, and further preferably 10%; the time for the sodium hypochlorite disinfection is preferably 0.5-1.5min, further preferably 0.8-1.2min, and further preferably 1min; the number of times the sodium hypochlorite disinfection is 1-2 times. Specifically, in the present invention, water lily seeds are first treated with 70% alcohol for 30 seconds, rinsed with sterile water, then treated with 70% alcohol for 30 seconds, rinsed with sterile water; and then treated with 10% sodium hypochlorite for 1 minute, and rinsed with sterile water 5 times.

[0025] In the present invention, the sterilized water lily seeds are placed on sterile filter paper to absorb moisture, and then the water lily embryos are separated. In the present invention, the water lily embryos are preferably separated in a clean bench under aseptic conditions. The ovary is opened with a scalpel and tweezers, and the seeds are removed and placed under a dissecting microscope. The seeds are opened and the spherical embryos are cultured, placed in a culture medium, and sealed with a sealing film.

[0026] In the present invention, water lily embryos are inoculated into culture medium A for culture to obtain water lily shoots. The culture medium A in the present invention is preferably a modified MS medium supplemented with 0.01-0.6 mg / L 6-BA, 0.01-0.6 mg / L NAA, and 0.3-0.7 g / L activated carbon, more preferably a modified MS medium supplemented with 0.1-0.55 mg / L 6-BA, 0.1-0.55 mg / L NAA, and 0.4-0.6 g / L activated carbon, and even more preferably a modified MS medium supplemented with 0.5 mg / L 6-BA, 0.5 mg / L NAA, and 0.5 g / L activated carbon. The pH of the culture medium A in the present invention is preferably 5.7-5.9, and even more preferably 5.8.

[0027] In the present invention, the improved MS medium is preferably MS medium + 20-70 g / L sucrose + 6-10 g / L agar, further preferably MS medium + 25-60 g / L sucrose + 7-9 g / L agar, and further preferably MS medium + 30 g / L sucrose + 8 g / L agar.

[0028] In the present invention, the water lily embryos are preferably cultured in medium A for 12-16 days, more preferably for 13-15 days, and even more preferably for 14 days; the culture temperature is preferably 25-31°C, and even more preferably 30°C; the culture photoperiod is preferably 12 hours of light / 12 hours of darkness; and the culture light intensity is preferably 163-182 μmolm -2 s -1 , further preferably 175-181 umol m -2 s -1 , and further preferably 180 umol m -2 s -1 The illumination of the culture is preferably 11840-13207 lx, further preferably 12500-13000 lx, and further preferably 12800 lx.

[0029] In the present invention, the water lily buds are cultured for 2 weeks to obtain complete seedlings, which are then transplanted into peat soil for further growth. Preferably, an appropriate amount of MS nutrient solution can be added to submerge the soil, and the germination box is sealed with plastic wrap (e.g., Figure 1 ), leave several rows of small holes on the plastic wrap (to keep moisture and exchange air), cultivate for 20-30 days, move to outdoor to harden the seedlings, and finally adapt to the external environment.

[0030] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] A method for inducing water lily embryos into seedlings, comprising the following steps:

[0033] (1) The maturity of water lily seeds was controlled by artificial pollination. The petals, sepals and other appendages of water lilies 13-18 days after pollination were removed, leaving only the ovary part (water lily seeds), and the water lilies were rinsed with tap water for 15 minutes.

[0034] (2) Place the water lily seeds in a sterilized 50 ml centrifuge tube on a clean bench, rinse with 70% alcohol for 30 seconds, rinse with sterile water, rinse with 70% alcohol for 30 seconds, rinse with sterile water, then use 10% sodium hypochlorite for 1 minute, rinse with sterile water 5 times; after the disinfection is completed, use sterile tweezers to remove the water lily seeds and place them on sterile filter paper, and absorb the water, then use sterile operation in the clean bench to separate the water lily embryos, use a scalpel and tweezers to cut open the ovary, take out the seeds and place them under a dissecting microscope, cut open the seeds and take out the spherical embryos, place them in the culture medium and seal them with sealing film;

[0035] (3) The water lily embryos were inoculated into medium A and cultured at 30°C for 14 days with a photoperiod of 12 h light / 12 h dark and a light intensity of 180 μmol m -2 s -1 , the illumination is 12800 lx, and water lily sprouts are obtained; the medium A is a modified MS medium + 0.5 mg / L 6-BA + 0.5 mg / L NAA + 0.5 g / L activated carbon, and the modified MS medium is MS medium + 30 g / L sucrose + 8 g / L agar;

[0036] (4) Continue to cultivate the water lily buds for 2 weeks to obtain complete seedlings, and transplant them into peat soil for further growth. You can add an appropriate amount of MS nutrient solution to cover the soil, seal the germination box with plastic wrap, and leave several rows of small holes on the plastic wrap (to keep moisture and exchange air). Cultivate for 20-30 days, move them outdoors to harden the seedlings, and finally adapt them to the external environment.

[0037] Example 2

[0038] A method for inducing water lily embryos into seedlings, comprising the following steps:

[0039] (1) The maturity of water lily seeds was controlled by artificial pollination. The petals, sepals and other appendages of water lilies 13-18 days after pollination were removed, leaving only the ovary part (water lily seeds), and the water lilies were rinsed with tap water for 15 minutes.

[0040] (2) Place the water lily seeds in a sterilized 50 ml centrifuge tube on a clean bench, rinse with 70% alcohol for 30 seconds, rinse with sterile water, rinse with 70% alcohol for 30 seconds, rinse with sterile water, then use 10% sodium hypochlorite for 1 minute, rinse with sterile water 5 times; after the disinfection is completed, use sterile tweezers to remove the water lily seeds and place them on sterile filter paper, and absorb the water, then use sterile operation in the clean bench to separate the water lily embryos, use a scalpel and tweezers to cut open the ovary, take out the seeds and place them under a dissecting microscope, cut open the seeds and take out the spherical embryos, place them in the culture medium and seal them with sealing film;

[0041] (3) The water lily embryos were inoculated into medium A and cultured at 30°C for 16 days with a photoperiod of 12 h light / 12 h dark and a light intensity of 182 μmol m -2 s -1 , the illumination is 11840 lx, and water lily sprouts are obtained; the medium A is a modified MS medium + 0.01 mg / L 6-BA + 0.01 mg / L NAA + 0.3 g / L activated carbon, and the modified MS medium is MS medium + 60 g / L sucrose + 6 g / L agar;

[0042] (4) Continue to cultivate the water lily buds for 2 weeks to obtain complete seedlings, and transplant them into peat soil for further growth. You can add an appropriate amount of MS nutrient solution to cover the soil, seal the germination box with plastic wrap, and leave several rows of small holes on the plastic wrap (to keep moisture and exchange air). Cultivate for 20-30 days, move them outdoors to harden the seedlings, and finally adapt them to the external environment.

[0043] Example 3

[0044] A method for inducing water lily embryos into seedlings, comprising the following steps:

[0045] (1) The maturity of water lily seeds was controlled by artificial pollination. The petals, sepals and other appendages of water lilies 13-18 days after pollination were removed, leaving only the ovary part (water lily seeds), and the water lilies were rinsed with tap water for 15 minutes.

[0046] (2) Place the water lily seeds in a sterilized 50 ml centrifuge tube on a clean bench, rinse with 70% alcohol for 30 seconds, rinse with sterile water, rinse with 70% alcohol for 30 seconds, rinse with sterile water, then use 10% sodium hypochlorite for 1 minute, rinse with sterile water 5 times; after the disinfection is completed, use sterile tweezers to remove the water lily seeds and place them on sterile filter paper, and absorb the water, then use sterile operation in the clean bench to separate the water lily embryos, use a scalpel and tweezers to cut open the ovary, take out the seeds and place them under a dissecting microscope, cut open the seeds and take out the spherical embryos, place them in the culture medium and seal them with sealing film;

[0047] (3) The water lily embryos were inoculated into medium A and cultured at 25°C for 12 days with a photoperiod of 12 h light / 12 h dark and a light intensity of 163 μmol m -2 s -1 , the illumination is 13207 lx, and water lily sprouts are obtained; the medium A is a modified MS medium + 0.01 mg / L 6-BA + 0.01 mg / L NAA + 0.7 g / L activated carbon, and the modified MS medium is MS medium + 20 g / L sucrose + 10 g / L agar;

[0048] (4) Continue to cultivate the water lily buds for 2 weeks to obtain complete seedlings, and transplant them into peat soil for further growth. You can add an appropriate amount of MS nutrient solution to cover the soil, seal the germination box with plastic wrap, and leave several rows of small holes on the plastic wrap (to keep moisture and exchange air). Cultivate for 20-30 days, move them outdoors to harden the seedlings, and finally adapt them to the external environment.

[0049] Test Example 1 (one)

[0051] Prepare 0.5 mg / L NAA: Weigh 50 mg of 1 mg / ml naphthaleneacetic acid (NAA), dissolve it in 2 ml of 1 M NaOH, and dilute to 50 ml.

[0052] Prepare 0.5 mg / L 6-BA: Weigh 50 mg of 1 mg / ml 6-benzylaminopurine (6-BA), dissolve it in 3.5 ml of 1M NaOH, and dilute to 50 ml.

[0053] Prepare modified MS medium: Use MS as the basic medium, add sucrose (to a final concentration of 30 g / L) and dilute to 1 L, shake appropriately, adjust the pH to 5.9 after the sucrose dissolves, and then add 0.8% agar.

[0054] Prepare culture medium A: Place the modified MS culture medium in an autoclave at 121°C for 20 minutes; after sterilization, take it out and place it on a clean bench with UV light for sterilization. After the culture medium has cooled appropriately, add 0.5 mg / L NAA, 0.5 mg / L 6-BA, and 0.5 g / L activated carbon and pour it into a plate. Because the young embryos of water lilies are small, round culture dishes can be used for primary culture. Pour 20-30 ml into each dish. Place the lid of the culture dish at an angle to leave a small hole for water vapor release. Sterilize it again with UV light on the clean bench and wait until the culture medium is completely cooled and solidified.

[0055] The maturity of water lily seeds was controlled by artificial pollination. The petals, sepals and other appendages of water lilies 4-18 days after pollination were removed, and only the ovary part (water lily seeds) was retained. The water lilies were rinsed with tap water for 15 minutes. The water lily seeds were placed in a sterilized 50ml centrifuge tube on a clean bench, rinsed with 70% alcohol for 30 seconds, sterile water, 70% alcohol for 30 seconds, sterile water, and then rinsed with 10% sodium hypochlorite for 1 minute and sterile water five times. After disinfection, the water lily seeds were removed with sterile tweezers and placed on sterile filter paper, and the water was dried. The water lily embryos were then separated by aseptic operation in the clean bench. The ovary was cut open with a scalpel and tweezers, and the seeds were removed and placed under a dissecting microscope. The spherical embryos were removed and placed on culture medium A (experimental group). At the same time, control group 1 and control group 2 were set up. In control group 1, seeds with the same pollination date and the seed coat removed were directly inoculated on medium A; in control group 2, seeds with the same pollination date but without the seed coat removed were directly inoculated on medium A. Each treatment group was cultured for 6 days and the infection status was observed.

[0056] The results showed that, under preferred medium A, the infection rate of the immature embryo culture group was 3.1%, while the infection rate of control group 1 was 40%, indicating small-area infection. The infection rate of control group 2 was 100%. This indicates that the infection rate of immature embryo culture is closely related to anatomy. (two)

[0058] Based on the above operation, the group of water lily embryo culture was placed at 30°C, 12 hours light / 12 hours dark photoperiod, and the light intensity was 180 μmol m -2 s -1 , the illumination was 12800 lx (experimental group). Simultaneously, control group 1 and control group 2 were set up. The control group 1 had a pure dark photoperiod, and the control group 2 had a photoperiod of 1 hour of light / 12 hours of darkness. Other treatments were all the same. The growth of young embryos of water lilies in the different treatment groups was compared.

[0059] The results showed that (such as Figure 2 ), the induction rate of the 12-hour light / 12-hour dark photoperiod group of the present invention was 75%, and the growth was balanced; the induction rate of the control group 1 was 30%; and the induction rate of the control group 2 was 40%. (three)

[0061] Two different water lily varieties were selected ( Nymphaea minuta ; Nymphaea nouchali ), and artificial pollination was used to control the maturity of water lily seeds. Treatment groups with pollination durations of 4 to 18 days were set up, and other treatments were consistent with operation (1). The effects of different pollination times on the induction rate of young embryos of water lily were studied.

[0062] Table 1 Effects of different pollination times on the induction rate of water lily embryos

[0063]

[0064] Table 2 Effects of different pollination times on the induction rate of young embryos of water lilies

[0065]

[0066] The results, shown in Tables 1 and 2, show similar induction rates for young embryos of different varieties treated with the same treatment. Observation of the culture media after inoculation revealed that visible young shoots appeared in most cultures from the same batch inoculated at the same time on day 8, indicating the highest induction rate for young embryos in water lilies eight days after inoculation. This suggests that the optimal pollination day for embryo retrieval is 17 days, as this is the time when young embryos achieve the highest induction rate after inoculation, providing insights into the timing of embryo retrieval in water lilies. (Four)

[0068] Based on the treatment of operation (I), the obtained spherical embryos of water lilies were inoculated into different culture media. Each group was cultured at 30°C with a photoperiod of 12 h light / 12 h dark, and a light intensity of 180 μmol m -2 s -1 , the illumination was 12800 lx, and the effects of different culture media on the growth of immature embryos were studied.

[0069] Culture medium group 1 is the culture medium A described in this application, with a pH value of 5.8.

[0070] Culture medium group 2 consisted of MS medium + 0.01 mg / L 6-BA + 0.01 mg / L NAA + 60 g / L sucrose + 8 g / L agar, with a pH of 5.

[0071] The results showed that the water lily embryos in medium group 1 had the best growth effect. Changes could be seen on the 5th day, small green buds appeared on the 14th day, and small green seedlings grew on the 20th day. Figure 3 ); however, the water lily embryos in medium group 2 showed basically no change on the 5th day, and their subsequent growth was obviously inferior to that in medium group 1, indicating a relatively poor induction effect.

[0072] In summary, it can be seen that the optimized culture medium A containing appropriate concentrations of 6-BA, NAA and activated carbon in the technical solution of the present invention improves the induction rate of adventitious buds, and the technical solution of the present invention is simple to operate, and the induced cultured water lily seedlings have low mortality, low contamination rate and high survival rate.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for inducing water lily embryos into seedlings, characterized in that: The steps include: (1) After 13-18 days of pollination, the water lily seeds were disinfected with alcohol and sodium hypochlorite, and the water lily embryos were separated; (2) Inoculate water lily embryos into culture medium A and culture for 12-16 days to obtain water lily buds; (3) Cultivate the water lily buds into seedlings and transplant them into peat soil for further growth; The culture medium A is a modified MS medium + 0.5 mg / L 6-BA + 0.5 mg / L NAA + 0.5 g / L activated carbon; The improved MS medium is MS medium + 20-70 g / L sucrose + 6-10 g / L agar; The mass concentration of the sodium hypochlorite in step (1) is 8-12%, the time for the sodium hypochlorite disinfection is 0.5-1.5 min, and the number of sodium hypochlorite disinfections is 1-2 times; The culture temperature in step (2) is 25-31°C, the light cycle is 12 hours light / 12 hours dark, and the light intensity is 163-182 μmol m -2 s -1 The illumination of the culture is 11840-13207 lx.

2. The method according to claim 1, characterized in that The volume concentration of the alcohol in step (1) is 60-75%, the time for the alcohol disinfection is 20-40s, and the number of alcohol disinfections is 1-2 times.

3. The method according to claim 1, characterized in that The pH value of the culture medium A in step (2) is 5.7-5.9.

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