A method for rapid propagation of Polygonatum cyrtonema through tissue culture

Abstract

The invention discloses a tissue culture rapid propagation method for polygonatum cyrtonema, comprising explant preparation and treatment, callus induction, bud induction, rooting culture and seedling hardening. According to the invention, polygonatum cyrtonema seeds are soaked in a NaCl solution with a concentration of 20-30 mmol / L at room temperature, and then the seeds are germinated until the seed buds are 0.5-1 cm long, the seed buds are used as explants, a callus induction medium is used, which is 1 / 2MS+6-BA1.5-2.5 mg / L, NAA0.4-0.8 mg / L, 2,4-D0.8-1.4 mg / L and 2wt%-6wt% tomato juice, and callus is induced under lightless conditions for 8-10 days, so that the browning rate of the explants can be significantly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture, and specifically relates to a method for rapid propagation of polygonatum cyrtonema through tissue culture. Background Art

[0002] Polygonatum yunnanensis Polygonatumkingianum ) is a member of the Liliaceae family ( Liliaceae ) Polygonatum Polygonatum ) is a perennial herb mainly distributed in the southwest region of my country with Yunnan as the center. It is a traditional tonic Chinese medicinal material.

[0003] In recent years, with the rapid development of the economy and the improvement of people's living standards, more people have begun to pay attention to health and wellness. The medical model has also changed from a single treatment type to a comprehensive type such as self-prevention, health care, and rehabilitation, which has prompted the formation of a new health culture. The development and research of natural health and functional foods have entered a new period of development. People's demand for wild Chinese medicinal materials is also increasing, which has led to large-scale excavation of wild resources of Yunnan Polygonatum, causing serious damage to the Yunnan Polygonatum Chinese medicinal materials resources and facing the threat of resource depletion. Therefore, artificial cultivation and domestication of Yunnan Polygonatum is one of the effective ways to solve this prominent problem.

[0004] At present, there are two main ways of artificial cultivation and propagation of traditional Yunnan Polygonatum: one is sexual reproduction through seeds, and the other is asexual reproduction through rhizomes. However, under natural conditions, Yunnan Polygonatum seeds are difficult to collect, and the sowing and seedling raising period is long. It takes one year from sowing to seedlings, and there are problems such as low germination rate and large differences in the separation of offspring traits, which are not suitable for practical production; asexual reproduction mostly uses root division, but its reproduction coefficient is low, rooting is difficult, and multiple reproductions are prone to accumulate viruses and there are problems such as variety degeneration, which to a certain extent limits the production potential of Yunnan Polygonatum. It is not conducive to the production of high-quality seedlings, nor is it conducive to the large-scale production and planting of Yunnan Polygonatum. Plant tissue culture technology can achieve rapid and large-scale reproduction of seedlings, and can better maintain the excellent traits of the mother plant, and is not restricted by factors such as season and environment. It is a key measure to realize the factory-based seedling cultivation of Yunnan Polygonatum and accelerate the breeding of fine varieties.

[0005] When propagating Yunnan Polygonatum seedlings by tissue culture, the browning rate of the explants is high when inducing callus tissue, which will directly affect the efficiency of tissue culture. Summary of the invention

[0006] In order to overcome the problems existing in the prior art, the present invention proposes a method for rapid propagation of Polygonatum cyrtonema through tissue culture. The browning rate of callus explants induced by the method described in the present invention is low, which can improve the efficiency of tissue culture propagation of Polygonatum cyrtonema seedlings.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] A method for rapid propagation of polygonatum yunnanensis tissue culture, comprising preparation and treatment of explants, callus induction, bud induction, rooting culture and seedling hardening;

[0009] The method for preparing and treating the explants is as follows: collecting plump Yunnan Polygonatum cyrtonema seeds that are free of insect pests and mechanical damage, drying them, and soaking them in a NaCl solution with a concentration of 20-30 mmol / L at room temperature for 2-4 days, then germinating the seeds, and when the seeds germinate to a length of 0.5-1 cm, taking the buds as tissue culture explants, and disinfecting the explants;

[0010] The callus induction method comprises the following steps: cutting the sterilized explant into small pieces, inoculating the pieces on a callus induction medium, and culturing the pieces in the dark for 8-10 days. The callus induction medium comprises 1 / 2MS+6-BA 1.5-2.5 mg / L, NAA 0.4-0.8 mg / L, 2,4-D 0.8-1.4 mg / L, and 2wt%-6wt% tomato juice.

[0011] Furthermore, the bud induction method is: cut the callus into small pieces and inoculate them on a bud induction medium, culture them for 20-26 days under a light intensity of 1000-1200 lux to induce the formation of clustered buds; the bud induction medium is MS+6-BA2-4mg / L, NAA0.3-0.6mg / L, 2,4-D0.2-0.6mg / L

[0012] Furthermore, the rooting culture method is: separating the clustered buds to obtain single buds and inoculating them into a rooting medium, the light intensity of the rooting culture is 9000lux-12000lux, and the light is exposed for 12 hours per day; the rooting medium is MS+NAA0.5-0.8mg / L, IBA0.3-0.6mg / L.

[0013] Furthermore, the method for hardening the seedlings is as follows: hardening the tissue culture seedlings after rooting culture together with the bottle under natural light at room temperature for one week, then opening the bottle cap to harden the seedlings under natural light at room temperature for one week, taking out the tissue culture seedlings in the bottle, washing off the culture medium at the roots, and transplanting them to a seedbed, laying a matrix on the seedbed, wherein the matrix contains peat soil, vermiculite and coconut bran in a mass ratio of 10:3:1; watering thoroughly after transplanting, covering the seedbed with a sunshade net with a shading rate of 70%-80%, watering once every three days, maintaining an environmental humidity of 85%-90% for hardening the seedlings for 40-50 days.

[0014] Furthermore, the explant disinfection method is as follows: after the explant is immersed in 75% ethanol for 30 seconds, it is transferred to a 10% sodium hypochlorite solution and immersed for 15 minutes, and then rinsed with sterile water for 5-6 times.

[0015] Through the above technical solution, the present invention can at least achieve the following beneficial effects:

[0016] 1. The inventors of the present invention have found that the browning rate of callus induced by the bud embryos of Polygonatum cyrtonema as tissue culture explants can be reduced by treating the seeds of Polygonatum cyrtonema with low-concentration salt stress. At the same time, adding 2%-6% tomato juice by mass to the callus induction culture can further reduce the browning rate of the explants.

[0017] 2. Although low-concentration salt stress treatment will slightly reduce the germination rate of Polygonatum cyrtonema seeds, the reduction in the browning rate of explants can improve the efficiency of obtaining callus tissue and cluster buds, and the overall efficiency of obtaining tissue culture seedlings is improved. DETAILED DESCRIPTION

[0018] Unless otherwise specified, the materials and reagents used in the present invention are commercially available.

[0019] In the present invention, peat soil was purchased from Lingshou County Ruihao Mineral Products Co., Ltd., vermiculite was purchased from Lingshou County Wanzhu Mineral Products Co., Ltd., and coconut bran was purchased from Sichuan Yuda Tianchen Chemical Co., Ltd.

[0020] The preparation method of tomato juice in the present invention comprises the following steps: purchasing commercially available ripe tomatoes free of pests and diseases, washing the tomatoes, crushing the tomatoes with a juicer, and passing the juice through a 400-mesh gauze to collect the filtrate, which is the tomato juice.

[0021] The seed bud in the present invention refers to the part from the tip of the bud to the seed coat of the Polygonatum cyrtonema seed after the Polygonatum cyrtonema seed germinates.

[0022] Example 1

[0023] A method for rapid propagation of polygonatum yunnanensis tissue culture, comprising preparation and treatment of explants, callus induction, bud induction, rooting culture and seedling hardening;

[0024] The method for preparing and treating the explants is as follows: collect insect-free, mechanically damaged and plump Yunnan Polygonatum seeds, dry them, and then soak them in a 25mmol / L NaCl solution at room temperature for 3 days, then germinate the seeds, and when the seeds germinate to a 0.5-1cm long bud, take the bud as a tissue culture explant, and disinfect the explant; the disinfection method is: after soaking in 75% ethanol for 30 seconds, transfer to a 10% sodium hypochlorite solution and soak for 15 minutes, and then rinse with sterile water 6 times.

[0025] The method for callus induction is as follows: the sterilized explants are cut into small pieces and inoculated on a callus induction medium, and cultured in the dark for 9 days; the callus induction medium is 1 / 2MS+6-BA2mg / L, NAA0.6mg / L, 2,4-D1.0mg / L and 4wt% tomato juice.

[0026] The bud induction method is as follows: cut the callus into small pieces and inoculate them onto a bud induction medium, and culture them for 24 days under a light intensity of 1100 lux to induce the formation of clustered buds; the bud induction medium is MS+6-BA 3.0 mg / L, NAA 0.5 mg / L, and 2,4-D 0.4 mg / L.

[0027] The rooting culture method is as follows: separate the clustered buds to obtain a single bud and inoculate it into a rooting medium; the light intensity of the rooting culture is 10000 lux, and the light is exposed for 12 hours every day; the rooting medium is MS+NAA0.6mg / L, IBA0.5mg / L.

[0028] The method for hardening the seedlings is as follows: hardening the tissue culture seedlings after rooting culture together with the bottle under natural light at room temperature for one week, then opening the bottle cap to harden the seedlings under natural light at room temperature for one week, taking out the tissue culture seedlings in the bottle, washing off the culture medium at the roots, and transplanting them to a seedbed, laying a matrix on the seedbed, wherein the matrix contains peat soil, vermiculite and coconut bran in a mass ratio of 10:3:1; watering thoroughly after transplanting, covering the seedbed with a sunshade net with a shading rate of 70%-80%, watering once every three days, maintaining an environmental humidity of 85%-90% to harden the seedlings for 50 days.

[0029] Example 2

[0030] A method for rapid propagation of polygonatum yunnanensis tissue culture, comprising preparation and treatment of explants, callus induction, bud induction, rooting culture and seedling hardening;

[0031] The method for preparing and treating the explants is as follows: collect insect-free, mechanically damaged and plump Yunnan Polygonatum seeds, dry them, and then soak them in a 20mmol / L NaCl solution at room temperature for 2 days, then germinate the seeds, and when the seeds germinate to a bud length of 0.5-1cm, take the buds as tissue culture explants, and disinfect the explants; the disinfection method is: after soaking in 75% ethanol for 30 seconds, transfer to a 10% sodium hypochlorite solution and soak for 15 minutes, and then rinse with sterile water 5 times.

[0032] The method for callus induction is as follows: the sterilized explants are cut into small pieces and inoculated on a callus induction medium, and cultured in the dark for 8 days. The callus induction medium is 1 / 2MS+6-BA1.5mg / L, NAA0.4mg / L, 2,4-D0.8mg / L and 2wt% tomato juice.

[0033] The method of bud induction is: cut the callus into small pieces and inoculate them on a bud induction medium, and culture them for 20 days under a light intensity of 1000 lux to induce the formation of clustered buds; the bud induction medium is MS+6-BA2mg / L, NAA0.3mg / L, 2,4-D0.2mg / L

[0034] The rooting culture method is as follows: separate the clustered buds to obtain a single bud and inoculate it into a rooting medium; the light intensity of the rooting culture is 9000 lux, and the light is illuminated for 12 hours every day; the rooting medium is MS+NAA0.5mg / L, IBA0.3mg / L.

[0035] The method for hardening the seedlings is as follows: hardening the tissue culture seedlings after rooting culture together with the bottle under natural light at room temperature for one week, then opening the bottle cap to harden the seedlings under natural light at room temperature for one week, taking out the tissue culture seedlings in the bottle, washing off the culture medium at the roots, and transplanting them to a seedbed, laying a matrix on the seedbed, wherein the matrix contains peat soil, vermiculite and coconut bran in a mass ratio of 10:3:1; watering thoroughly after transplanting, covering the seedbed with a sunshade net with a shading rate of 70%-80%, watering once every three days, maintaining an environmental humidity of 85%-90% to harden the seedlings for 40 days.

[0036] Example 3

[0037] A method for rapid propagation of polygonatum yunnanensis tissue culture, comprising preparation and treatment of explants, callus induction, bud induction, rooting culture and seedling hardening;

[0038] The method for preparing and treating the explants is as follows: collect insect-free, mechanically damaged and plump Yunnan Polygonatum seeds, dry them, and then soak them in a 30mmol / L NaCl solution at room temperature for 4 days, then germinate the seeds, and when the seeds germinate to a bud length of 0.5-1cm, take the buds as tissue culture explants, and disinfect the explants; the disinfection method is: after soaking in 75% ethanol for 30 seconds, transfer to 10% sodium hypochlorite solution and soak for 15 minutes, and then rinse with sterile water 6 times.

[0039] The method for callus induction is as follows: the sterilized explants are cut into small pieces and inoculated on a callus induction medium, and cultured in the dark for 10 days. The callus induction medium is 1 / 2MS+6-BA2.5mg / L, NAA0.8mg / L, 2,4-D1.4mg / L and 6wt% tomato juice.

[0040] The method of bud induction is: cut the callus into small pieces and inoculate them on bud induction medium, culture them under a light intensity of 1200 lux for 26 days to induce the formation of clustered buds; the bud induction medium is MS+6-BA4mg / L, NAA0.6mg / L, 2,4-D0.6mg / L

[0041] The rooting culture method is as follows: separate the clustered buds to obtain a single bud and inoculate it into a rooting medium; the light intensity of the rooting culture is 12000 lux, and the light is illuminated for 12 hours every day; the rooting medium is MS+NAA0.8mg / L, IBA0.6mg / L.

[0042] The method for hardening the seedlings is as follows: hardening the tissue culture seedlings after rooting culture together with the bottle under natural light at room temperature for one week, then opening the bottle cap to harden the seedlings under natural light at room temperature for one week, taking out the tissue culture seedlings in the bottle, washing off the culture medium at the roots, and transplanting them to a seedbed, laying a matrix on the seedbed, wherein the matrix contains peat soil, vermiculite and coconut bran in a mass ratio of 10:3:1; watering thoroughly after transplanting, covering the seedbed with a sunshade net with a shading rate of 70%-80%, watering once every three days, maintaining an environmental humidity of 85%-90% to harden the seedlings for 50 days.

[0043] Example 4

[0044] A method for rapid propagation of polygonatum yunnanensis tissue culture, comprising preparation and treatment of explants, callus induction, bud induction, rooting culture and seedling hardening;

[0045] The method for preparing and treating the explants is as follows: collect insect-free, mechanically damaged and plump Yunnan Polygonatum seeds, dry them, and then soak them in a 22mmol / L NaCl solution at room temperature for 4 days, then germinate the seeds, and when the seeds germinate to a bud length of 0.5-1cm, take the buds as tissue culture explants, and disinfect the explants; the disinfection method is: after soaking in 75% ethanol for 30 seconds, transfer to a 10% sodium hypochlorite solution and soak for 15 minutes, and then rinse with sterile water 6 times.

[0046] The method for callus induction is as follows: the sterilized explants are cut into small pieces and inoculated on a callus induction medium, and cultured in the dark for 9 days. The callus induction medium is 1 / 2MS+6-BA2mg / L, NAA0.5mg / L, 2,4-D1mg / L and 5wt% tomato juice.

[0047] The method of bud induction is: cut the callus into small pieces and inoculate them on bud induction medium, culture them under a light intensity of 1200 lux for 22 days to induce the formation of clustered buds; the bud induction medium is MS+6-BA3.5mg / L, NAA0.5mg / L, 2,4-D0.3mg / L

[0048] The rooting culture method is as follows: separate the clustered buds to obtain a single bud and inoculate it into a rooting medium; the light intensity of the rooting culture is 11000 lux, and the light is illuminated for 12 hours every day; the rooting medium is MS+NAA0.6mg / L, IBA0.5mg / L.

[0049] The method for hardening the seedlings is as follows: hardening the tissue culture seedlings after rooting culture together with the bottle under natural light at room temperature for one week, then opening the bottle cap to harden the seedlings under natural light at room temperature for one week, taking out the tissue culture seedlings in the bottle, washing off the culture medium at the roots, and transplanting them to a seedbed, laying a matrix on the seedbed, wherein the matrix contains peat soil, vermiculite and coconut bran in a mass ratio of 10:3:1; watering thoroughly after transplanting, covering the seedbed with a sunshade net with a shading rate of 70%-80%, watering once every three days, maintaining an environmental humidity of 85%-90% to harden the seedlings for 45 days.

[0050] Comparative Example 1

[0051] The difference between Comparative Example 1 and Example 1 is that after the seeds are dried, they are not subjected to salt stress treatment and are directly germinated.

[0052] Comparative Example 2

[0053] The difference between Comparative Example 2 and Example 1 is that no tomato juice is added to the callus induction medium.

[0054] Comparative Example 3

[0055] The difference between Comparative Example 3 and Example 1 is that the seeds are not subjected to salt stress treatment after being dried, and are directly germinated, and no tomato juice is added to the callus induction medium.

[0056] Comparative Example 4

[0057] The difference between Comparative Example 4 and Example 1 is that the tomato juice in the callus induction medium is replaced by apple juice.

[0058] According to the methods described in Examples 1-4 and Comparative Examples 1-4, 1000 Polygonatum cyrtonema seeds were obtained, explants were cultured and Polygonatum cyrtonema tissue culture seedlings were propagated, and the germination rate and the browning rate during callus induction were counted.

[0059] Germination rate = (number of germinated seeds / 1000) × 100%; [After the seeds germinate, the seeds are counted as germinated only when they grow to a length greater than 0.5 cm]

[0060] Browning rate = (number of browning explants / number of inoculated explants) × 100%;

[0061] The results are shown in Table 1 below.

[0062] Table 1 Germination rate of Polygonatum cyrtonema seeds under different treatments and browning rate of explants of callus induced by Polygonatum cyrtonema seed buds

[0063]

[0064] As can be seen from Table 1 above, the germination rate of Polygonatum cyrtonema seeds decreased after treatment with low-concentration salt, but the browning rate of the explants was significantly reduced, which can improve the efficiency of obtaining callus tissue and cluster buds.

[0065] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for rapid propagation of Polygonatum cyrtonema by tissue culture, characterized in that: It includes explant preparation and treatment, callus induction, shoot induction, rooting culture and seedling hardening; The method for preparing and treating the explants is as follows: collecting plump Yunnan Polygonatum cyrtonema seeds that are free of insect pests and mechanical damage, drying them, and soaking them in a NaCl solution with a concentration of 20-30 mmol / L at room temperature for 2-4 days, then germinating the seeds, and when the seeds germinate to a length of 0.5-1 cm, taking the buds as tissue culture explants, and disinfecting the explants; The callus induction method comprises the following steps: cutting the sterilized explant into small pieces, inoculating the pieces on a callus induction medium, and culturing the pieces in the dark for 8-10 days. The callus induction medium comprises 1 / 2MS+6-BA 1.5-2.5 mg / L, NAA 0.4-0.8 mg / L, 2,4-D 0.8-1.4 mg / L, and 2wt%-6wt% tomato juice.

2. A method for rapid propagation of Polygonatum cyrtonema in tissue culture according to claim 1, characterized in that: The bud induction method comprises: cutting callus tissue into small pieces and inoculating them onto a bud induction medium, culturing them for 20-26 days under a light intensity of 1000-1200 lux to induce the formation of clustered buds; the bud induction medium comprises MS+6-BA2-4 mg / L, NAA0.3-0.6 mg / L, and 2,4-D0.2-0.6 mg / L.

3. A method for rapid propagation of Polygonatum cyrtonema in tissue culture according to claim 2, characterized in that: The rooting culture method comprises: separating clustered buds to obtain single buds and inoculating them into a rooting culture medium; the light intensity of the rooting culture is 9000 lux-12000 lux, and the light is illuminated for 12 hours every day; the rooting culture medium comprises MS+NAA0.5-0.8 mg / L and IBA0.3-0.6 mg / L.

4. A method for rapid propagation of Polygonatum cyrtonema in tissue culture according to claim 3, characterized in that: The method for hardening the seedlings comprises: hardening the tissue culture seedlings after rooting culture together with the bottle under natural light at room temperature for one week, then opening the bottle cap to harden the seedlings under natural light at room temperature for one week, taking out the tissue culture seedlings in the bottle, washing off the culture medium at the roots, and transplanting them to a seedbed, laying a matrix on the seedbed, wherein the matrix comprises peat soil, vermiculite and coconut bran in a mass ratio of 10:3:1; watering thoroughly after transplanting, covering the seedbed with a sunshade net with a shading rate of 70%-80%, watering once every three days, maintaining an environmental humidity of 85%-90% and hardening the seedlings for 40-50 days.

5. A method for rapid propagation of Polygonatum cyrtonema in tissue culture according to claim 1, characterized in that: The explant disinfection method is as follows: the explant is immersed in 75% ethanol for 30 seconds, then transferred to a 10% sodium hypochlorite solution and immersed for 15 minutes, and then rinsed with sterile water for 5-6 times.

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