Tissue culture and rapid propagation method of cibotium barometz

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation method of cibotium barometz. The tissue culture and rapid propagation method comprises the following steps: removing villus from the rhizome of the cibotium barometz, sterilizing, cutting into slices with the length and width of 0.5-1cm and the thickness of 1-2mm, inoculating onto a P1 culture medium, and culturing to obtain primary callus; inoculating to a P2 culture medium, and culturing to obtain green calluses; inoculating to a P3 culture medium and culturing to obtain adventitious buds induced by calluses; transferring the adventitious buds to a P4 culture medium for culturing; transferring the adventitious buds subjected to multiplication culture into a P5 culture medium for culture to obtain regenerated seedlings; and after seedling hardening, transplanting into a matrix for culturing to obtain a regenerated plant. According to the method, the ratio of all elements of the culture medium is optimized, the rhizome of the cibotium barometz is directly used as the explant, the adventitious bud is induced through the induced callus, and then proliferation and rooting are performed, so that the cibotium barometz plant with the female parent hereditary character can be quickly obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation method of golden dogwood fern. Background Art

[0002] Goldenrod fern ( Cibotium barometz (Linn.) J. Sm. ) is a large tree-like terrestrial fern of the genus Cibotium of the family Pteridaceae. The plant is 1 to 3 meters tall and shaped like a tree fern. The rhizome is flat and thick, with an upward tip. The part exposed to the ground is densely covered with long golden hairs, resembling a lying golden dog head, so it is called Cibotium. Cibotium not only has extremely high ornamental value, but also has quite important medicinal value. The golden hairs on the plant are good hemostatic drugs, and the Chinese medicine name is Gouji.

[0003] At present, golden dog fern mainly uses spores for reproduction. This reproduction method not only has high environmental requirements, but also has a long cycle and a low induction rate. The plant in vitro tissue culture has a high reproduction coefficient, which can effectively shorten the reproduction cycle and stably preserve the excellent genetic characteristics of the mother plant. Golden dog fern tissue culture rapid propagation mostly uses spores as explants, but the genetic transformation technology using spores as explants is unstable, the probability of chimeras in the obtained transgenic plants is high, and the induction rate is low, the differentiation efficiency is not high, and the workload of expanding sterile seedlings is large. In the prior art, the rapid propagation system of golden dog fern is still mainly based on spores, and there is no report on the research of inducing adventitious buds by rhizome callus to obtain plants. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a tissue culture and rapid propagation method for Pteris odoratissimus. The tissue culture and rapid propagation method optimizes the ratio of various elements of the culture medium, directly uses the rhizomes of Pteris odoratissimus as explants, induces adventitious buds through induced callus tissue, and then proliferates and roots them, so as to quickly obtain Pteris odoratissimus plants that preserve the genetic traits of the mother plant.

[0005] To achieve the above object, the present invention provides a method for tissue culture and rapid propagation of Pteris chrysantha, which specifically comprises the following steps: S1. The rhizomes of the golden dog fern are depilated and disinfected, and then the disinfected rhizomes are cut into slices with a length and width of 0.5-1 cm and a thickness of 1-2 mm and inoculated on P1 medium, and cultured until the rhizome slices differentiate into granular protrusions to obtain primary callus tissue; S2. The primary callus was inoculated onto P2 medium for induction culture to obtain green callus; S3. Inoculate the green callus onto differentiation induction medium P3 for induction culture to obtain adventitious buds induced by callus; S4. Transfer the adventitious buds to the proliferation medium P4 for proliferation culture; S5. The adventitious buds after proliferation culture are transferred to the rooting medium P5 for culture to obtain complete regenerated seedlings; S6. After hardening the regenerated seedlings, transplant them into a substrate for cultivation to obtain regenerated plants.

[0006] Further, in the above technical solution S1, the rhizomes of golden dog fern with good growth and thick tubers are selected to remove the villi and then disinfected. The disinfection method is: soak in 75% ethanol for 20 seconds, rinse with sterile water 3-4 times, then soak and shake with 0.1% mercuric chloride solution for 5-8 minutes, rinse with sterile water 4-5 times, and finally soak and shake with 4% sodium hypochlorite for 2-3 minutes, and rinse with sterile water 5-8 times. In this technical solution, multiple disinfections are combined, and the disinfection time is controlled at the same time. Without destroying the material organization, the disinfection is thorough, and there is no subsequent infection by miscellaneous bacteria.

[0007] Furthermore, in the above technical solution S1, the P1 is based on 1 / 2MS as the basic culture medium, and also contains 6-BA with a concentration of 0.1-0.5 mg / L, KT with a concentration of 0.1-0.5 mg / L, sucrose with a concentration of 30 g / L and Gelrite with a pH of 5.8-6.0, and the culture conditions are: temperature 20-25°C, humidity 70-80%, and light 1500-1700lx.

[0008] Furthermore, in the above technical solution S2, the P2 is based on MS as the basic culture medium, and also contains 6-BA with a concentration of 1.5-2.0 mg / L, NAA with a concentration of 0.5-1.0 mg / L, sucrose with a concentration of 30 g / L and Gelrite with a pH of 5.8-6.0, and the culture conditions are: temperature 22-25°C, humidity 70-80%, and light 1800-2200lx.

[0009] Furthermore, in the above technical solution S3, the P3 is based on MS as the basic culture medium, and also contains KT at a concentration of 1.0-2.0 mg / L, NAA at a concentration of 0.2-0.5 mg / L, 2,4-D at a concentration of 0.2-0.5 mg / L, sucrose at a concentration of 30 g / L and Gelrite at a concentration of 2 g / L, and its pH is 5.8-6.0; the culture conditions are: temperature 23-26°C, humidity 70-80%, light 1800-2200 lx, and light time 12-15 h / d.

[0010] Furthermore, in the above technical solution S4, the P4 is based on MS as the basic culture medium, and also contains 6-BA with a concentration of 0.3-1.0 mg / L, TDZ with a concentration of 0.3-1.0 mg / L, KT with a concentration of 0.5-1.0 mg / L, sucrose with a concentration of 30 g / L, and Gelrite with a concentration of 2 g / L, and the pH value is 5.8-6.0. The culture conditions are: temperature 24-26°C, humidity 70-80%, light 1800-2200 lx, and light time 12-15 h / d.

[0011] Furthermore, in the above technical solution S5, the rooting medium P5 is based on 1 / 2MS as the basic medium, and also contains IBA with a concentration of 0.3-1.0 mg / L, IAA with a concentration of 0.2-0.6 mg / L, 2,4-D with a concentration of 0.3-1.0 mg / L, zeolite with a concentration of 0.5-1.0 g / L, sucrose with a concentration of 30 g / L, and Gelrite with a concentration of 2 g / L, and the pH value is 5.8-6.0. The culture conditions are: temperature 24-26°C, humidity 70-80%, light intensity 1800-2000 lx, and light time 12-15 h / d.

[0012] Furthermore, in the above technical solution S6, the matrix is ​​a mixture of peat soil, perlite and zeolite in a volume ratio of 3-5:1:1; the culture conditions are: temperature 24-28°C, humidity 80-90%, light intensity 3000-5000lx, and light time 12-15h / d.

[0013] The present invention has the following beneficial effects: The invention provides a tissue culture rapid propagation method for golden dog fern, which takes robust golden dog fern rhizomes as explants, can completely retain the excellent biological genetic characteristics of the mother plant, and directly induces adventitious buds through the induced callus tissue by optimizing the hormone type ratio and concentration of each culture medium, which are then proliferated and rooted, so that golden dog fern plants with expansion culture advantages can be quickly obtained, and the problems of long spore reproduction cycle, low induction rate and unstable genetic characteristics of golden dog fern are effectively solved.

[0014] The tissue culture rapid propagation method of the invention is simple, has high callus rate and proliferation coefficient, and both the rooting rate and the survival rate reach more than 90%, thus providing a new basis for the cultivation of improved varieties of golden dog fern. DETAILED DESCRIPTION

[0015] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified.

[0016] The above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution.

[0017] The raw materials involved in the embodiments of the present invention are either existing commercially available products or can be prepared according to existing methods, and the detection method is an industry method.

[0018] The present invention is described in further detail below in conjunction with embodiments: The rhizomes of golden dog fern used in the experiment of the present invention were collected from the company's planting base; MS basal medium formula: 332.2 mg / L calcium chloride, 1900 mg / L potassium nitrate, 1650 mg / L ammonium nitrate, 170 mg / L potassium dihydrogen phosphate, 180.7 mg / L magnesium sulfate, 0.025 mg / L copper sulfate, 6.2 mg / L boric acid, 8.6 mg / L zinc sulfate, 0.025 mg / L cobalt chloride, 16.9 mg / L manganese sulfate, 0.83 mg / L potassium iodide, 0.25 mg / L sodium molybdate, 27.8 mg / L FeSO 4 7H 2 O, 37.26mg / L disodium edetate, 2.0mg / L glycine, 100mg / L inositol, 0.1mg / L thiamine VB1, 0.50mg / L niacin and 0.5mg / L pyridoxine VB6.

[0019] Example 1: Investigating the effects of disinfection methods and plant growth regulators on callus growth After removing the hairs from the rhizomes of golden dog fern with good growth, thick tubers, robustness and no mildew, rinse under running water for 20 minutes, and use filter paper to absorb the water on the rhizomes for later use. On the clean bench, the rhizomes were soaked in 75% ethanol for 20 seconds, rinsed with sterile water 4 times, then soaked and shaken in 0.1% mercuric chloride solution for 6 minutes, rinsed with sterile water 5 times, and finally soaked and shaken in 4% sodium hypochlorite for 3 minutes, and finally rinsed with sterile water 6 times; at the same time, the effects of different disinfection methods on callus growth were investigated, as shown in Table 1. From the results in Table 1, it can be seen that the use of ethanol and mercuric chloride disinfection or ethanol and sodium hypochlorite disinfection is not thorough, and the formation of callus is also affected by incomplete cleaning of the disinfectant.

[0020] The sterilized rhizomes were cut into slices with a length and width of 0.5-1 cm and a thickness of 1-2 mm and inoculated on P1 medium for 15 days. Among them, P1 is based on 1 / 2MS as the basic medium, and also contains 6-BA with a concentration of 0.3 mg / L, KT with a concentration of 0.2 mg / L, sucrose with a concentration of 30 g / L and Gelrite with a pH of 5.8-6.0. The culture conditions are: temperature 20-25°C, humidity 70-80%, and light 1500-1700 lx. At the same time, the effects of different growth regulators on callus growth were investigated, as shown in Table 2. From the results in Table 2, it can be seen that the combination of 6-BA and KT growth regulators in the early stage is more conducive to the formation of initial callus tissue.

[0021] Table 1 Effects of different disinfection methods on the growth of primary callus of Pteris chinensis

[0022] Table 2 Effects of different plant growth regulators on the growth of primary callus of Pteris chinensis

[0023] Example 2: Investigating the effect of plant growth regulators on green callus growth The primary callus with good growth condition treated with B2 in Example 1 was selected and inoculated on P2 medium for secondary induction culture for 15 days. Among them, P2 is based on MS as the basic medium, and also contains 6-BA with a concentration of 1.5 mg / L, NAA with a concentration of 0.7 mg / L, sucrose with a concentration of 30 g / L and Gelrite with a pH of 5.8-6.0. The culture conditions are: temperature 22-25°C, humidity 70-80%, and light 1800-2200 lx. At the same time, the effect of plant growth regulators on the growth of green callus was investigated, as shown in Table 3. It can be seen from the results in Table 3 that in the process of inducing primary callus into green callus, the use of 6-BA and NAA is more conducive to the growth of callus.

[0024] Table 3 Effects of different plant growth regulators on the callus growth of Pteris chinensis

[0025] Example 3: Investigating the effect of plant growth regulators on callus induction of adventitious buds The golden dog fern callus obtained by treatment with C2 in Example 2 was selected and inoculated on P3 medium for 15 days. Among them, P3 is based on MS as the basic medium, and also contains KT at a concentration of 1.5 mg / L, NAA at a concentration of 0.3 mg / L, 2,4-D at a concentration of 0.5 mg / L, sucrose at a concentration of 30 g / L and Gelrite at a concentration of 2 g / L, and its pH is 5.8-6.0. The culture conditions are: temperature 23-26°C, humidity 70-80%, light 1800-2200 lx, and light time is 12-15 h / d. At the same time, the effects of plant growth regulators on callus induction of adventitious buds were investigated, as shown in Table 4. From the results in Table 4, it can be seen that callus can differentiate into adventitious buds when containing three regulators, KT, NAA, and 2,4-D, and the differentiation effect is better when the KT content is higher. Although it can also differentiate when KT is replaced with 6-BA, the differentiation rate is greatly reduced, and there is basically no differentiation without KT.

[0026] Table 4 Effects of different plant growth regulators on adventitious bud induction from callus

[0027] Example 4: Investigating the effect of plant growth regulators on adventitious bud proliferation The adventitious buds that grew well in D2 treatment in Example 3 were inoculated on the proliferation culture medium P4 for 30 days of proliferation culture. Wherein, P4 is based on MS as the basic culture medium, and also contains 6-BA at a concentration of 1.0 mg / L, TDZ at a concentration of 0.5 mg / L, KT at a concentration of 0.5 mg / L, sucrose at a concentration of 30 g / L, and Gelrite at a concentration of 2 g / L, and its pH is 5.8-6.0, and the culture conditions are: temperature 24-26°C, humidity 70-80%, light 1800-2200 lx, and light duration 12-15 h / d. At the same time, the effects of different plant growth regulators on the proliferation of adventitious buds of Golden Dog Fern were investigated, as shown in Table 5. As can be seen from the results of Table 5, in the process of inducing the proliferation of adventitious buds, the combination of 6-BA, TDZ, and KT can achieve a proliferation coefficient of more than 10.

[0028] Table 5 Effects of different plant growth regulators on the proliferation of adventitious buds of Pteris chinensis

[0029] Example 5: Investigating the effects of different culture medium ratios on adventitious bud rooting When the adventitious buds of the proliferation growth in Example 4 grow to 1.0-1.5cm, they are transferred to the rooting medium P5 and cultured for 30d, wherein the rooting medium P5 is based on 1 / 2MS, and also contains 0.5 mg / L IBA, 0.3 mg / L IAA, 0.5 mg / L 2,4-D, 0.8 g / L zeolite, 30 g / L sucrose, 2 g / L Gelrite, and its pH is 5.8-6.0. The culture conditions are: temperature 24-26 ° C, humidity 70-80%, light intensity 1800-2000 lx, and light time 12-15h / d. At the same time, the effects of different plant growth regulators on the rooting of the adventitious buds of golden dog fern are investigated, as shown in Table 6. As can be seen from the results of Table 6, the rooting rate of the P5 rooting medium of the present invention is above 92%.

[0030] Table 6 Effects of different culture medium ratios on rooting of adventitious shoots of Pteris chinensis

[0031] Example 6: Investigating the effects of different substrates on survival rate The lid of the culture bottle of the rooted regenerated seedlings was opened, and after 2 days of hardening in the artificial intelligence climate control room, they were transplanted into a mixed matrix with a volume ratio of peat soil: perlite: zeolite of 3-5:1:1. After transplanting, they were watered thoroughly and covered with 70% sunshade nets. The temperature was controlled at 24-28℃, the humidity was 80-90%, the light intensity was 3000-5000lx, and the light time was 12-15h / d. After 20 days, the survival rate was calculated, and the effects of different matrices on the survival rate of golden dog fern were investigated, as shown in Table 7. From the results in Table 7, it can be seen that the combination of peat soil, perlite and zeolite as the matrix can achieve good plant growth, and the survival rate is above 90%, which is a high survival rate.

[0032] Table 7 Effects of different substrates on the survival rate of Pteris chinensis

[0033] Based on the above analysis, the present invention uses robust rhizomes of golden dog fern as explants, optimizes the type ratio and concentration of hormones in each stage of reproduction, obtains adventitious buds through induced callus induction, and then obtains regenerated plants after proliferation and rooting; the tissue culture rapid propagation method is simple, has high callus rate and proliferation coefficient, and both the rooting rate and the survival rate reach more than 90%, and only takes about 75 days or 95 days from adventitious buds to rooting or survival, with a fast reproduction speed, and the obtained regenerated plants retain the excellent genetic characteristics of the golden dog fern mother plant, which can provide a new basis for the subsequent cultivation of golden dog fern improved varieties.

[0034] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A tissue culture and rapid propagation method of Pteris chrysantha, characterized in that: The specific steps include: S1. The rhizomes of the golden dog fern are depilated and disinfected, and then the disinfected rhizomes are cut into slices with a length and width of 0.5-1 cm and a thickness of 1-2 mm and inoculated on P1 medium, and cultured until the rhizome slices differentiate into granular protrusions to obtain primary callus tissue; S2. The primary callus was inoculated onto P2 medium for induction culture to obtain green callus; S3. Inoculate the green callus onto differentiation induction medium P3 for induction culture to obtain adventitious buds induced by callus; S4. Transfer the adventitious buds to the proliferation medium P4 for proliferation culture; S5. The adventitious buds after proliferation culture are transferred to the rooting medium P5 for culture to obtain complete regenerated seedlings; S6. After hardening the regenerated seedlings, transplant them into a substrate for cultivation to obtain regenerated plants.

2. The method for rapid propagation of Pteris chrysantha by tissue culture according to claim 1, characterized in that: In S1, the rhizomes of golden dog fern with good growth and thick tubers were selected, and the hairs were removed and disinfected. The disinfection method was: soak in 75% ethanol for 20 seconds, rinse with sterile water 3-4 times, then soak and shake in 0.1% mercuric chloride solution for 5-8 minutes, rinse with sterile water 4-5 times, and finally soak and shake in 4% sodium hypochlorite for 2-3 minutes, and rinse with sterile water 5-8 times.

3. The tissue culture and rapid propagation method of Pteris chrysantha according to claim 1, characterized in that: In S1, the P1 is based on 1 / 2MS as the basic culture medium, and also contains 6-BA with a concentration of 0.1-0.5 mg / L, KT with a concentration of 0.1-0.5 mg / L, sucrose with a concentration of 30 g / L and Gelrite with a concentration of 2 g / L. The pH value is 5.8-6.0, and the culture conditions are: temperature 20-25°C, humidity 70-80%, and light 1500-1700 lx.

4. The method for rapid propagation of Pteris chrysantha by tissue culture according to claim 1, characterized in that: In S2, the P2 is based on MS as the basic culture medium, and also contains 6-BA with a concentration of 1.5-2.0 mg / L, NAA with a concentration of 0.5-1.0 mg / L, sucrose with a concentration of 30 g / L, and Gelrite with a pH of 5.8-6.

0. The culture conditions are: temperature 22-25°C, humidity 70-80%, and light 1800-2200 lx.

5. The method for rapid propagation of Pteris chrysantha by tissue culture according to claim 1, characterized in that: In S3, the P3 is based on MS as the basic culture medium, and also contains KT at a concentration of 1.0-2.0 mg / L, NAA at a concentration of 0.2-0.5 mg / L, 2,4-D at a concentration of 0.2-0.5 mg / L, sucrose at a concentration of 30 g / L, and Gelrite at a concentration of 2 g / L. The pH value is 5.8-6.0, and the culture conditions are: temperature 23-26°C, humidity 70-80%, light 1800-2200 lx, and light time 12-15 h / d.

6. The method for rapid propagation of Pteris chrysantha by tissue culture according to claim 1, characterized in that: In S4, the P4 is based on MS as the basic culture medium, and also contains 6-BA with a concentration of 0.3-1.0 mg / L, TDZ with a concentration of 0.3-1.0 mg / L, KT with a concentration of 0.5-1.0 mg / L, sucrose with a concentration of 30 g / L, and Gelrite with a concentration of 2 g / L. The pH value is 5.8-6.0, and the culture conditions are: temperature 24-26°C, humidity 70-80%, light 1800-2200 lx, and light time 12-15 h / d.

7. The method for tissue culture and rapid propagation of Pteris chrysantha according to claim 1, characterized in that: In S5, the rooting medium P5 is based on 1 / 2MS as the basic medium, and also contains IBA with a concentration of 0.3-1.0 mg / L, IAA with a concentration of 0.2-0.6 mg / L, 2,4-D with a concentration of 0.3-0.5 mg / L, zeolite with a concentration of 0.5-1.0 g / L, sucrose with a concentration of 30 g / L, and Gelrite with a concentration of 2 g / L. The pH value is 5.8-6.0, and the culture conditions are: temperature 24-26°C, humidity 70-80%, light intensity 1800-2000 lx, and light time 12-15 h / d.

8. The method for rapid propagation of Pteris chrysantha by tissue culture according to claim 1, characterized in that: In S6, the matrix is ​​a mixture of peat soil, perlite and zeolite in a volume ratio of 3-5:1:1; the culture conditions are: temperature 24-28°C, humidity 80-90%, light intensity 3000-5000lx, and light time 12-15h / d.

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