A method for rapid propagation of lotus tissue culture

Abstract

The present invention relates to the field of lotus tissue culture and breeding technology, in particular, to a lotus tissue culture and rapid propagation method. It specifically comprises the following steps: step S1: selecting full and mature lotus seeds with shells; step S2: peeling the lotus seeds and disinfecting them; step S3: breaking the lotus seeds in the middle to take out the lotus embryos, and then removing the bottom and top of the lotus embryos; then inoculating them into MS culture medium and placing them under 26-28°C for cultivation; step S4: selecting uncontaminated lotus embryos in the MS culture medium and moving them to an induction culture medium for young shoot induction; step S5: dividing the induced regenerated young shoots into one shoot and one shoot, then cutting off the stems and leaves on the upper part, and then inoculating them into a proliferation culture medium for regeneration bud proliferation; step S6: selecting a tissue culture seedling with a height of about 5 cm, cutting a single shoot or a cluster of shoots from a lotus seedling with consistent growth and robustness, and then inoculating them into a rooting culture medium for rooting culture; step S7: hardening and transplanting the rooted seedlings of the lotus. The rapid propagation method in the present invention is fast and has a high survival rate.

Description

Technical Field

[0001] The invention relates to the technical field of lotus tissue culture and propagation, in particular to a lotus tissue culture and rapid propagation method. Background Art

[0002] Lotus, with beautiful flowers and leaves and rich colors, is one of the top ten famous flowers in my country and the national flower of India and Vietnam. It has high ornamental value, economic value and ecological restoration value. It has great application and development potential in food, medicine, beauty and health care, landscaping, ecological restoration, tourism development and foreign trade export, and has great development prospects. However, the high price of seedlings produced by traditional lotus breeding methods has restricted the development of the lotus industry.

[0003] Traditional lotus propagation methods mostly use rhizome or sowing propagation, but the rhizome propagation coefficient of lotus is low, and sowing seedlings are prone to mutation and it is difficult to retain the excellent shape of the mother plant.

[0004] Therefore, in recent years, the method of plant tissue culture has been gradually widely promoted for breeding lotus. However, the existing lotus tissue culture method has a slow breeding speed and the survival rate can still be improved, which is difficult to better adapt to the industrial development of lotus. Summary of the invention

[0005] The invention provides a lotus tissue culture rapid propagation method, which can overcome certain or some defects of the prior art.

[0006] A lotus tissue culture rapid propagation method according to the present invention specifically comprises the following steps:

[0007] Step S1: Select plump and mature lotus seeds with shells;

[0008] Step S2: peeling the lotus seeds and disinfecting them;

[0009] Step S3: breaking the lotus seed in the middle to take out the lotus embryo, and then removing the bottom and top of the lotus embryo; then inoculating it into MS culture medium and culturing it at 26-28°C;

[0010] Step S4: selecting uncontaminated lotus embryos in the MS medium and transferring them to the induction medium for sprout induction, wherein the composition of the induction medium is: MS medium + 1 mg / L 6-BA + 0.2 mg / L IBA;

[0011] Step S5: The induced regenerated shoots are divided into shoots, and then the upper stems and leaves are cut off, and then inoculated into a proliferation medium for regeneration bud proliferation. The components of the proliferation medium are: MS medium + 3mg / L 6-BA + 0.2mg / L IBA.

[0012] Step S6: Selecting a lotus bud with a height of about 5 cm, uniform growth and robustness, cutting a single shoot or a cluster of shoots, and then inoculating them into a rooting medium for rooting culture, wherein the composition of the rooting medium is: 1 / 2MS medium + NAA 1.0 mg / L;

[0013] Step S7: hardening and transplanting the rooted lotus seedlings.

[0014] Preferably, step S2 specifically includes the following steps:

[0015] Step S21: soaking the peeled lotus seeds in washing powder, rinsing with tap water, disinfecting with ethanol, and washing with sterile water;

[0016] Step S22: disinfecting the lotus seeds treated in step S21 with a sodium hypochlorite solution for 20 minutes;

[0017] Step S23: Wash with sterile water.

[0018] Preferably, the sodium hypochlorite solution in step S22 is 30% sodium hypochlorite solution.

[0019] Preferably, step S4 specifically includes the following steps:

[0020] Step S41: moving the uncontaminated lotus embryos in step S3 to the induction culture medium, and after inoculation, adding sterile water into the bottle to half cover the lotus embryos;

[0021] Step S42: Place the culture in a 28°C culture room and culture it in the dark for 7 days, then place it in a 28°C culture room, culture it under the conditions of a light intensity of 4000 lx and a photoperiod of 12 to 14 h / d.

[0022] Preferably, step S5 specifically includes the following steps:

[0023] Step S51: inoculating the induced regenerated young shoots into a proliferation medium, and then adding sterile water into the bottle to half cover the young shoots;

[0024] Step S52: Place the culture medium in a 28°C culture room for dark culture for 7 days, and then culture the medium under the conditions of 28°C, 4000 lx of light intensity, and 12-14 h / d of photoperiod.

[0025] Preferably, step S6 specifically includes the following steps:

[0026] Step S61: inoculating the lotus seedlings into a rooting medium;

[0027] Step S62: After inoculation, add sterile water to the bottle to half cover the tissue culture seedlings, and place it under LED lighting for 16 hours / day.

[0028] The present invention has the following significant improvements:

[0029] The invention uses 30% sodium hypochlorite as a disinfectant for lotus seeds; and preferably uses an induction medium with components of MS medium + 1mg / L6-BA + 0.2mg / L IBA; also preferably uses a proliferation medium with components of MS medium + 3mg / L6-BA + 0.2mg / L IBA; and also preferably uses a rooting medium with components of 1 / 2MS medium + 1.0mg / L NAA. The invention uses lotus seeds as culture tissues, uses the preferred formula and cooperates with the rapid propagation method, and after experimental verification, the efficiency of lotus tissue culture propagation is preferably improved.

[0030] Therefore, the rapid lotus tissue culture and rapid propagation method with a fast growth rate and a high survival rate in the present invention can be better applied to the industrialized breeding of lotus compared with the existing breeding methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Photos of disinfecting lotus seeds;

[0032] Figure 2 This is a photo of the embryos just inoculated into the induction medium;

[0033] Figure 3 This is a photo of embryos placed in induction medium for 20 days;

[0034] Figure 4 This is a photo of regenerated shoots cultured in proliferation medium for 25 days;

[0035] Figure 5 This is a photo of tissue culture seedlings cultured on rooting medium for 30 days;

[0036] Figure 6 These are photos of the seedlings after 25 days of hardening. DETAILED DESCRIPTION

[0037] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with the embodiments. It should be understood that the embodiments are only for explaining the present invention and are not intended to limit it.

[0038] Example:

[0039] A lotus tissue culture rapid propagation method in the present embodiment specifically comprises the following steps:

[0040] (1) Choose plump and mature lotus seeds with shells.

[0041] (2) Lotus seed processing and disinfection method: Use tweezers to peel off the lotus seeds, soak them in washing powder for 5 minutes, rinse them under running water for 30 minutes, then rinse them with deionized water for 3 to 5 times, and place them on a sterile operating table for use. Wash them with sterile water for 3 to 5 times, soak them in 75% anhydrous ethanol (75% alcohol) for disinfection for 2 minutes, and shake them constantly; pour out the alcohol and wash them with sterile water once, then soak them in sterile water for 5 minutes (to remove excess alcohol soaked in the seeds to avoid poisoning), then wash them with sterile water for 3 times, then disinfect them with a prepared 30% sodium hypochlorite solution for 20 minutes, then wash them with sterile water for 3 to 5 times, and set them aside.

[0042] (3) Explant acquisition: Use a sterile scalpel to break the middle of the lotus seed and take out the lotus embryo. Use a scalpel to remove the bottom and top of the lotus embryo, inoculate it into MS culture medium, and culture it at 26-28°C. Calculate the contamination rate after 7 days, which is about 2%.

[0043] (4) Induction of embryonic shoots: The uncontaminated embryos in the MS medium were transferred to the induction medium. The composition of the induction medium was: MS medium + 1 mg / L 6-BA + 0.2 mg / L IBA. After inoculation, sterile water was added to the bottle to half cover the lotus embryos. The culture was placed in a 28°C culture room for 7 days in the dark, and then placed in a 28°C culture room with a light intensity of 4000 lx and a photoperiod of 12 to 14 h / d. The induction rate was 85 to 92% after 25 days, and 2 to 3 regenerated shoots were obtained.

[0044] (5) Proliferation of regenerated shoots: Divide the induced regenerated shoots into one shoot each, cut off the upper stems and leaves and inoculate them into proliferation medium. The components of the proliferation medium are: MS medium + 3 mg / L 6-BA + 0.2 mg / L IBA. After inoculation, add sterile water to half cover the shoots. Place them in a 28°C culture room and culture them in the dark for 7 days. Then place them in a 28°C culture room with a light intensity of 4000 lx and a photoperiod of 12 to 14 h / d. After 25 to 30 days, 3 to 10 regenerated shoots will be produced.

[0045] (6) Rooting culture of lotus seedlings: Select lotus seedlings with a height of about 5 cm, uniform growth and robustness, cut single or cluster shoots and inoculate them into rooting medium 1 / 2MS + NAA 1.0 mg / L. After inoculation, add sterile water to half cover the seedlings, place under LED lighting for 16 hours / day, culture for 20-25 days, and the rooting rate is 95-100%.

[0046] (7) Seedling hardening and transplanting technology: Open the bottle mouth of the lotus seedlings and place them in a shaded place for about a week. Slowly move them to a place with good light for hardening for about a week. Turn the bottle or bag surface frequently to allow the tissue culture seedlings to receive light evenly, so that the hardened tissue culture seedlings receive light evenly and are healthy. Take out the hardened lotus seedlings, wash the root culture medium, and place them in pond mud for hardening. The final survival rate can reach 83-92%.

[0047] Specifically, the lotus tissue culture rapid propagation method in this embodiment can better improve the lotus breeding speed and survival rate, and can stably produce a large number of high-quality seedlings in a short time.

[0048] Furthermore, in this embodiment, 30% sodium hypochlorite is preferably used as a disinfectant for lotus seeds; and an induction medium composed of MS medium + 1mg / L6-BA + 0.2mg / L IBA is preferably used; a proliferation medium composed of MS medium + 3mg / L6-BA + 0.2mg / LIBA is also preferred; and a rooting medium composed of 1 / 2MS medium + NAA1.0mg / L is also preferred; this embodiment can better improve the efficiency of lotus tissue culture propagation through the above preferred schemes, and the above preferred schemes can be better applied to the industrialized breeding of lotus. Using the scheme of this embodiment, the following comparative experiments were specifically carried out:

[0049] Experiment 1:

[0050] Table 1 Effects of different disinfectants and disinfection time on lotus seed disinfection

[0051]

[0052] In this comparative experiment, after the lotus embryos were inoculated into the MS medium in step (3), the culture was carried out for 7 days, and the disinfection effect data were statistically analyzed. It can be seen from Table 1 that sodium hypochlorite is suitable as a disinfectant for lotus seeds. Sodium hypochlorite as a disinfectant not only solves the disinfection problem of lotus seeds, avoids the use of toxic disinfectants such as mercuric chloride, does not cause pollution to the environment, and has low cost.

[0053] From the data, it can be seen that the contamination rate of 30% sodium hypochlorite solution disinfection for 20min to 30min is 2% to 3%. Because lotus seeds are wrapped in seed coat and seed flesh, the use of 30% sodium hypochlorite in this embodiment can better reduce the contamination rate without damaging the lotus embryo itself; ensure that the contamination rate of fungicide S206 is 34% to 52%; the contamination rate of the control is as high as 77%. From the data, it can be seen that the best disinfection method for lotus seeds is T5: the specific disinfection process is as follows: tap water rinse for 30min → sterile water wash 3 to 5 times → 75% ethanol disinfection for 2min → sterile water wash 3 to 5 times → 30% sodium hypochlorite solution disinfection for 20min → sterile water wash 5 times.

[0054] The above disinfection process can achieve a better disinfection effect and can better screen out 30% sodium hypochlorite as a better disinfectant.

[0055] Experiment 2: Effects of different hormones on lotus seed embryo induction

[0056] Table 2 Effects of different hormones on lotus seed embryo induction

[0057]

[0058] The experimental data in step (4) were placed at 28°C, with a light intensity of 4000lx and a photoperiod of 12-14h / d. After culturing for 25 days, the data were statistically analyzed. The statistical data can be seen from Table 2 that the average induction rate of group B4 is the highest. Therefore, IBA can be used as a plant growth regulator for lotus seed embryo induction, with an induction rate of 92%. In the control medium MS, there is only a single bud seedling with roots. In the plant growth regulator IAA medium, some clustered buds are also induced, but the hard texture of the bud affects differentiation and there are calluses. In the NAA medium, there are 1-2 regenerated young buds, and the buds are weak and have roots. Therefore, IBA is more suitable as a plant growth regulator for lotus. IBA has a high induction rate for lotus embryos and is easy to form seedlings, which can better improve the problem of low lotus embryo induction rate.

[0059] Experiment 3: Effects of different concentrations of plant growth regulator 6-BA on the proliferation of lotus regeneration buds

[0060] Table 3 Effects of different concentrations of plant growth regulator 6-BA on the proliferation of lotus regeneration buds

[0061]

[0062] The experimental data in step (5) were cultured at 28°C, with a light intensity of 4000 lx and a photoperiod of 12 to 14 h / d. Statistics were collected after culturing for 25 to 30 days. The statistical data can be seen from Table 3 that the average induction rate of group C3 was the highest. When the concentration of the plant growth regulator 6-BA was 3 mg / L, the proliferation coefficient of the lotus regeneration buds reached 5 to 10 times, the buds were strong and the leaves were stretched; when the concentration of the plant growth regulator 6-BA was 1 to 2 mg / L, the proliferation coefficient was only 2 to 3 times, which was a low proliferation coefficient, there was no callus, and the leaves were green; when the concentration of the plant growth regulator 6-BA was 4 to 5 mg / L, the proliferation coefficient was 2 to 5 times, there was callus, the proliferation coefficient was reduced, the buds were short and the leaves were small.

[0063] Therefore, in comparison, the proliferation medium that is more suitable for lotus regeneration buds is C3: MS + 3mg / L6-BA + 0.2mg / L IBA. The components of this proliferation medium can better improve the proliferation coefficient of lotus, so as to ensure the high efficiency and stability of the lotus tissue culture rapid propagation method in this embodiment.

[0064] Experiment 4: Effects of different culture media on rooting of lotus seedlings

[0065] Table 4 Effects of different culture media on rooting of lotus seedlings

[0066]

[0067] The experimental data in step (6) were placed under LED lighting for 16 hours / day, and statistics were collected after culturing for 20 to 25 days. The statistical data can be seen from Table 4 that the average induction rate of group D3 is the highest. Therefore, the medium 1 / 2MS+NAA1.0mg / L is a better rooting medium for lotus tissue culture seedlings, with early rooting time, thick and long roots, strong seedlings, good growth, and a rooting rate of 100%; the rooting rate of the medium 1 / 2MS is lower, the rooting time is long, the number of roots is small and short; the medium 1 / 2MS+IBA0.5mg / L and 1 / 2MS+IBA1.0mg / L can also root, but the rooting rate is not high, the seedlings are average, the roots are short, the roots are thin, the roots are few, the rooting time is short, and the seedlings and roots are prone to yellowing. Therefore, the more suitable rooting hormone for lotus is NAA (naphthaleneacetic acid), and the best rooting medium is 1 / 2MS+NAA1.0mg / L.

[0068] Experiment 5: Hardening of Lotus Tissue Culture Seedlings in Bottles

[0069] After obtaining complete plants with roots in step (6), the bottle seedlings were placed in an indoor environment with a shading degree of 50% to 70% and a temperature of (25±2)°C, and the light intensity was increased from 20 to 40 μmol / (m 2 ·s) gradually increased to 75~100μmol / (m 2 ·s). After 7-10 days, first loosen the bottle cap for 1-2 days, then half-open the bottle cap for 1-2 days, and finally open the bottle cap completely, harden the seedlings for 3-5 days, gently pick out the bottle seedlings with sterilized long tweezers, put them in a basin of clean water, carefully wash away the culture medium remaining at the roots of the plants, and then soak them in 500-800 times diluted 50% carbendazim wettable powder for 20-30 minutes, and then transplant them outside the bottle for hardening.

[0070] Experiment 6: Hardening of Lotus Tissue Culture Seedlings Outside Bottles

[0071] After obtaining the plants obtained in Experiment 5, the clay or silt is first evenly sprayed with 0.1% to 0.4% potassium permanganate solution or 500 to 800 times diluted carbendazim wettable powder for disinfection, 3% to 5% quicklime, stirred evenly, made into a conical soil pile, covered and sealed with a polypropylene plastic film, and dried in high temperature weather conditions for 7 to 10 days, or the substrate is sterilized by high temperature dry heat sterilization. When the temperature of the sterilized soil drops to room temperature, it is placed in a (9 to 12) cm (diameter) × (7.5 to 14.5) cm (height) seedling cup or non-woven seedling bag, and the lotus tissue culture seedlings in the bottle are placed in the prepared seedling cup for cultivation, placed under a shade shed for cultivation until new leaves grow out, and then transferred to normal cultivation under sunlight (20-30 days), the shade shed has a shading degree of 70% to 90% for cultivation for 7 to 10 days, and then transferred to full sunlight conditions for cultivation.

[0072] After Experiments 5 and 6, the survival rate of the plants can reach 83%-92%;

[0073] Therefore, the present embodiment uses lotus seeds as explants, and the rapid propagation method of the present embodiment can better ensure a high survival rate while rapidly propagating.

[0074] It is easy to understand that those skilled in the art can combine, split, reorganize, etc. the embodiments of the present application to obtain other embodiments based on one or several embodiments provided in the present application, and these embodiments do not exceed the protection scope of the present application.

[0075] The present invention and its implementation methods are described schematically above, and the description is not restrictive. The embodiments shown in the embodiments are only part of the implementation methods of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by the embodiments and designs a structure and an implementation method similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for rapid propagation of lotus tissue culture, characterized in that: It specifically includes the following steps: Step S1: Select plump and mature lotus seeds with shells; Step S2: peeling the lotus seeds and disinfecting them; Step S3: breaking the lotus seed in the middle to take out the lotus embryo, and then removing the bottom and top of the lotus embryo; then inoculating it into MS culture medium and culturing it at 26-28°C; Step S4: selecting uncontaminated lotus embryos in the MS medium and transferring them to the induction medium for sprout induction, wherein the composition of the induction medium is: MS medium + 1 mg / L 6-BA + 0.2 mg / L IBA; Step S5: dividing the induced regenerated shoots into one shoot, cutting off the upper stems and leaves, and then inoculating them into a proliferation medium for regeneration shoot proliferation, wherein the components of the proliferation medium are: MS medium + 3mg / L 6-BA + 0.2mg / L IBA; Step S6: Selecting a 5 cm tall, uniformly growing, robust lotus bud, cutting a single shoot or a cluster of shoots, and then inoculating them into a rooting medium for rooting culture, wherein the composition of the rooting medium is: 1 / 2MS medium + NAA 1.0 mg / L; Step S7: hardening and transplanting the rooted lotus seedlings; Step S2 specifically includes the following steps: Step S21: soaking the peeled lotus seeds in washing powder, rinsing with tap water, disinfecting with ethanol, and washing with sterile water; Step S22: disinfecting the lotus seeds treated in step S21 with a sodium hypochlorite solution for 20 minutes; Step S23: washing with sterile water; The sodium hypochlorite solution in step S22 is selected to be 30% sodium hypochlorite solution.

2. A lotus tissue culture rapid propagation method according to claim 1, characterized in that: Step S4 The specific steps include: Step S41: moving the uncontaminated lotus embryos in step S3 to the induction culture medium, and after inoculation, adding sterile water into the bottle to half cover the lotus embryos; Step S42: Place the culture in a 28°C culture room and culture it in the dark for 7 days, then place it in a 28°C culture room, culture it under the conditions of a light intensity of 4000 lx and a photoperiod of 12 to 14 h / d.

3. A lotus tissue culture rapid propagation method according to claim 1, characterized in that: Step S5 specifically includes the following steps: Step S51: inoculating the induced regenerated young shoots into a proliferation medium, and then adding sterile water into the bottle to half cover the young shoots; Step S52: Place the culture medium in a 28° C. incubate in the dark for 7 days, and then place the culture medium in a 28° C. incubation room with a light intensity of 4000 lx and a photoperiod of 12 to 14 h / d.

4. A lotus tissue culture rapid propagation method according to claim 1, characterized in that: Step S6 specifically includes the following steps: Step S61: inoculating the lotus seedlings into a rooting medium; Step S62: After inoculation, add sterile water to the bottle to half cover the tissue culture seedlings, and place it under LED lighting for 16 hours / day.

Images