A tissue culture rapid propagation method of elaphoglossum japonicum

Abstract

The application belongs to the technical field of plant tissue culture and particularly relates to a tissue culture and rapid propagation method of Stenochlaena palustris. The tissue culture and rapid propagation method comprises the following steps: after the rhizome of Stenochlaena palustris is sterilized and treated, the rhizome is cut into a thin piece with a length of 0.5-1 cm, a width of 0.5-1 cm and a thickness of 1-2 mm, and the thin piece is inoculated on a P1 culture medium to culture primary callus; the callus is inoculated on a P2 culture medium to obtain green callus; the green callus is inoculated on a P3 culture medium to obtain callus-induced adventitious buds; the adventitious buds are transferred to a P4 culture medium for culture; the proliferated adventitious buds are transferred to a P5 culture medium for culture to obtain regenerated seedlings; after the seedlings are hardened, the seedlings are transplanted into a substrate to obtain regenerated plants. By optimizing the proportion of various elements of the culture medium, directly taking the rhizome of Stenochlaena palustris as an explant, inducing adventitious buds through induced callus, and then proliferating and rooting, the Stenochlaena palustris plants with the genetic traits of the mother plants can be quickly obtained.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation method of gold dog fern. BACKGROUND

[0002] Gold dog fern (Phymatopteris auriculata) is a terrestrial fern of Phymatopteris in the family of Lindsaeaceae, with a plant height of 1-3 meters, a tree fern-like shape, a horizontal and thick rhizome, an upward end, a ground exposed part densely covered with golden yellow long hairs, and a gold dog head-like shape, so it is called gold dog fern. Cibotium barometz (Linn.) J. Sm. Gold dog fern not only has a very high ornamental value, but also has a very important medicinal value, and the golden yellow hairs on the plant are a good hemostatic drug, and the Chinese medicine name is dog spine.

[0003] At present, gold dog fern is mainly propagated by spores, and the propagation method has high environmental requirements, a long cycle and a low induction rate. Plant in vitro tissue culture has a high propagation coefficient, can effectively shorten the propagation cycle, and can stably preserve the genetic characteristics of the female parent. Gold dog fern tissue culture and rapid propagation is mostly based on spores as explants, but the genetic transformation technology based on spores as explants is unstable, the obtained transgenic plant chimeras have a high probability, a low induction rate, a low differentiation efficiency, and a large workload of expanding sterile seedlings. In the prior art, the rapid propagation system of gold dog fern is mainly based on spores, and research on obtaining plants by inducing adventitious buds from rhizome callus has not been reported. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides a tissue culture and rapid propagation method of gold dog fern. The tissue culture and rapid propagation method directly uses rhizome of gold dog fern as explants by optimizing the proportion of each element of the culture medium, induces adventitious buds from the induced callus, and then performs proliferation and rooting, so that the gold dog fern plants preserving the genetic characteristics of the female parent can be quickly obtained.

[0005] To achieve the above object, the application provides a tissue culture and rapid propagation method of gold dog fern, which specifically comprises the following steps:

[0006] S1. The rhizome of gold dog fern is sterilized after removing the hairs, and then the sterilized rhizome is cut into a thin piece with a length and width of 0.5-1 cm and a thickness of 1-2 mm and inoculated on P1 culture medium, so that the rhizome piece differentiates into granular protrusions, and primary callus is obtained;

[0007] S2. The primary callus is inoculated on P2 culture medium for induction culture, and green callus is obtained;

[0008] S3. The green callus is inoculated on differentiation induction culture medium P3 for induction culture, and adventitious buds induced by callus are obtained;

[0009] S4. The adventitious buds are transferred to the proliferation medium P4 for proliferation culture;

[0010] S5. The adventitious buds after proliferation culture are transferred to the rooting medium P5 for culture to obtain complete regenerated seedlings;

[0011] S6. After the seedlings are hardened, the regenerated seedlings are transplanted into the substrate for culture to obtain regenerated plants.

[0012] Further, in the technical solution S1, the rhizomes of the Stenochlaena palustris with good growth and thick tubers are selected, and then the rhizomes are depilatory and sterilized, the sterilization method is: soaked in 75% ethanol for 20s, washed with sterile water for 3-4 times, then soaked in 0.1% mercury chloride solution for 5-8min, washed with sterile water for 4-5 times, finally soaked in 4% sodium hypochlorite for 2-3min, and washed with sterile water for 5-8 times. In this technical solution, multiple sterilizations are combined, and the sterilization time is controlled well, so that the material tissue is not damaged, and the sterilization is complete and there is no subsequent bacterial infection.

[0013] Further, in the technical solution S1, the P1 is a 1 / 2MS basic medium, further containing 6-BA with a concentration of 0.1-0.5mg / L, KT with a concentration of 0.1-0.5mg / L, 30g / L of sucrose and 2g / L of Gelrite, and the pH is 5.8-6.0, and the culture conditions are: temperature 20-25℃, humidity 70-80%, illumination 1500-1700lx.

[0014] Further, in the technical solution S2, the P2 is an MS basic medium, further containing 6-BA with a concentration of 1.5-2.0mg / L, NAA with a concentration of 0.5-1.0mg / L, 30g / L of sucrose and 2g / L of Gelrite, and the pH is 5.8-6.0, and the culture conditions are: temperature 22-25℃, humidity 70-80%, illumination 1800-2200lx.

[0015] Further, in the technical solution S3, the P3 is an MS basic medium, further containing KT with a concentration of 1.0-2.0mg / L, NAA with a concentration of 0.2-0.5mg / L, 2,4-D with a concentration of 0.2-0.5mg / L, 30g / L of sucrose and 2g / L of Gelrite, and the pH is 5.8-6.0; the culture conditions are: temperature 23-26℃, humidity 70-80%, illumination 1800-2200lx, and illumination time is 12-15h / d.

[0016] Further, in the technical scheme S4, the P4 is MS-based medium, further containing 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, 30 g / L of sucrose, 2 g / L of Gelrite, and the pH is 5.8-6.0, and the culture condition is: temperature 24-26 ℃, humidity 70-80%, illumination 1800-2200 lx, and illumination time 12-15 h / d.

[0017] Further, in the technical scheme S5, the rooting culture medium P5 is 1 / 2MS-based medium, further containing 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, 0.5-1.0 g / L of zeolite, 30 g / L of sucrose, 2 g / L of Gelrite, and the pH is 5.8-6.0, and the culture condition is: temperature 24-26 ℃, humidity 70-80%, illumination intensity 1800-2000 lx, and illumination time 12-15 h / d.

[0018] Further, in the technical scheme S6, the substrate is a mixture of peat soil, perlite and zeolite with a volume ratio of 3-5:1:1, and the culture condition is: temperature 24-28 ℃, humidity 80-90%, illumination intensity 3000-5000 lx, and illumination time 12-15 h / d.

[0019] The present application has the beneficial effects that:

[0020] The present application provides a tissue culture and rapid propagation method of gold dog fern, which takes robust gold dog fern rhizomes as explants, can completely retain the fine biological genetic characteristics of the female parent, and can quickly obtain gold dog fern plants with expansion advantages through optimization of the hormone type ratio and concentration of each culture medium, direct induction of adventitious buds from the induced callus, and subsequent proliferation and rooting, thereby effectively solving the problems of long spore propagation cycle, low induction rate and unstable genetic characteristics of gold dog fern.

[0021] The tissue culture and rapid propagation method of the present application is simple, has high callus induction rate and proliferation coefficient, and the rooting rate and survival rate are both above 90%, thereby providing a new basis for the culture of gold dog fern elite. DETAILED DESCRIPTION

[0022] In the following examples, the experimental methods are all conventional methods unless otherwise specified. The raw materials involved in the following examples are all ordinary commercially available products and can be purchased on the market unless otherwise specified.

[0023] The above technical features of the present application and the technical features described in detail below (such as the examples) can be combined with each other to form new or preferred technical solutions.

[0024] The raw materials involved in the embodiments of the present application are either existing commercially available products or can be prepared according to existing methods, and the detection method is an industry method.

[0025] The present application will be further described in detail below in combination with examples:

[0026] The rhizomes of Stenochlaena palustris used in the experiments of the present application were collected from the company's planting base.

[0027] The MS basic medium formula is composed of 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 FeSO4·7H2O, 37.26 mg / L disodium ethylenediaminetetraacetate, 2.0 mg / L glycine, 100 mg / L inositol, 0.1 mg / L thiamine VB1, 0.50 mg / L nicotinic acid, and 0.5 mg / L pyridoxine VB6.

[0028] Example 1: Investigation of the effects of disinfection methods and plant growth regulators on callus growth

[0029] The rhizomes of Stenochlaena palustris that grow well, have thick and strong tubers, and are not moldy are selected, and after the fluff is removed, they are washed under running water for 20 min, and the water on the rhizomes is absorbed with filter paper for standby use. In the clean bench, the rhizomes are soaked in 75% ethanol for 20 s, washed with sterile water for 4 times, then soaked in 0.1% mercury chloride solution for 6 min, washed with sterile water for 5 times, finally soaked in 4% sodium hypochlorite for 3 min, and finally washed with sterile water for 6 times; at the same time, the effects of different disinfection methods on callus growth are investigated, as shown in Table 1. As can be seen from the results in Table 1, the use of ethanol and mercury disinfection or ethanol and sodium hypochlorite disinfection is not complete, and the disinfectant is not clean, which also affects the formation of callus.

[0030] The sterilized rhizomes were cut into 0.5-1 cm long, 1-2 mm thick slices and inoculated on P1 medium for 15 days. The P1 medium was based on 1 / 2MS medium and contained 0.3 mg / L 6-BA, 0.2 mg / L KT, 30 g / L sucrose and 2 g / L Gelrite, with pH 5.8-6.0, and the culture conditions were 20-25 °C, 70-80% humidity and 1500-1700 lx illumination. The effects of different growth regulators on callus growth were investigated, as shown in Table 2. It can be seen from the results in Table 2 that the combination of 6-BA and KT is more conducive to the formation of initial callus.

[0031] Table 1 Effects of different sterilization methods on the growth of primary callus of C. fortunei

[0032]

[0033] Table 2 Effects of different plant growth regulators on the growth of primary callus of C. fortunei

[0034]

[0035] Example 2: Investigation of the effects of plant growth regulators on the growth of green callus

[0036] The primary callus treated with B2 in Example 1 was inoculated on P2 medium for secondary induction for 15 days. The P2 medium was based on MS medium and contained 1.5 mg / L 6-BA, 0.7 mg / L NAA, 30 g / L sucrose and 2 g / L Gelrite, with pH 5.8-6.0, and the culture conditions were 22-25 °C, 70-80% humidity and 1800-2200 lx illumination. The effects of plant growth regulators on the growth of green callus were investigated, as shown in Table 3. It can be seen from the results in Table 3 that the combination of 6-BA and NAA is more conducive to the growth of callus during the induction of primary callus into green callus.

[0037] Table 3 Effects of different plant growth regulators on the growth of callus of C. fortunei

[0038]

[0039] Example 3: Investigation of the effects of plant growth regulators on the induction of adventitious buds from callus

[0040] The C2 treated gold-thread fern callus in Example 2 was inoculated on P3 medium for culture for 15 days. The P3 medium was MS based medium containing 1.5 mg / L of KT, 0.3 mg / L of NAA, 0.5 mg / L of 2,4-D, 30 g / L of sucrose and 2 g / L of Gelrite, and the pH was 5.8-6.0. The culture conditions were: temperature 23-26°C, humidity 70-80%, illumination 1800-2200 lx, and illumination time 12-15 h / d. The effect of plant growth regulators on callus induction of adventitious buds was investigated, as shown in Table 4. From the results in Table 4, it can be seen that the callus can differentiate adventitious buds when containing three kinds of regulators of KT, NAA and 2,4-D, and the differentiation effect is better when the content of KT is higher. Although differentiation can also occur when 6-BA is used to replace KT, the differentiation rate is greatly reduced, and no differentiation occurs when KT is not contained.

[0041] Table 4 Effect of different plant growth regulators on callus induction of adventitious buds

[0042]

[0043] Example 4: Investigation of the effect of plant growth regulators on proliferation of adventitious buds

[0044] The well grown adventitious buds treated by D2 in Example 3 were inoculated on proliferation medium P4 for proliferation culture for 30 days. The P4 was MS based medium containing 1.0 mg / L of 6-BA, 0.5 mg / L of TDZ, 0.5 mg / L of KT, 30 g / L of sucrose and 2 g / L of Gelrite, and the pH was 5.8-6.0. The culture conditions were: temperature 24-26°C, humidity 70-80%, illumination 1800-2200 lx, and illumination time 12-15 h / d. The effect of different plant growth regulators on proliferation of gold-thread fern adventitious buds was investigated, as shown in Table 5. From the results in Table 5, it can be seen that the proliferation coefficient can reach more than 10 when 6-BA, TDZ and KT are combined in the process of induction of proliferation of adventitious buds.

[0045] Table 5 Effect of different plant growth regulators on proliferation of gold-thread fern adventitious buds

[0046]

[0047] Example 5: Investigation of the effect of different medium ratios on rooting of adventitious buds

[0048] The adventitious buds which proliferated and grew in Example 4 were grown to 1.0-1.5 cm, and were transferred to rooting medium P5 for culture for 30 days, wherein the rooting medium P5 was a 1 / 2MS-based medium further containing IBA at a concentration of 0.5 mg / L, IAA at a concentration of 0.3 mg / L, 2,4-D at a concentration of 0.5 mg / L, zeolite at a concentration of 0.8 g / L, sucrose at a concentration of 30 g / L, and Gelrite at a concentration of 2 g / L, and had a pH of 5.8-6.0, and the culture conditions were as follows: a temperature of 24-26 ℃, a humidity of 70-80%, a light intensity of 1800-2000 lx, and a light time of 12-15 h / d. Meanwhile, the effects of different plant growth regulators on rooting of the adventitious buds of the golden dog fern were investigated, as shown in Table 6. It can be seen from the results in Table 6 that the rooting rate of the adventitious buds of the golden dog fern was more than 92% when the rooting medium P5 was used.

[0049] Table 6: Effects of different medium proportions on rooting of adventitious buds of the golden dog fern

[0050]

[0051] Example 6: Investigation of effects of different substrates on survival rate

[0052] The cover of the culture bottle of the rooted regenerated seedlings was opened, and the seedlings were acclimated in an artificial intelligent climate control room for 2 days, and were then transplanted into a mixed substrate of peat soil, perlite and zeolite at a volume ratio of 3-5:1:1, and were then covered with a 70% shading net after being irrigated with water, and the temperature was controlled at 24-28 ℃, the humidity was controlled at 80-90%, the light intensity was controlled at 3000-5000 lx, and the light time was controlled at 12-15 h / d, and after 20 days, the survival rate was calculated, and the effects of different substrates on the survival rate of the golden dog fern were investigated, as shown in Table 7. It can be seen from the results in Table 7 that the plants grew well when the peat soil, perlite and zeolite were used as the substrate, and the survival rate was more than 90%, and the survival rate was high.

[0053] Table 7: Effects of different substrates on survival rate of the golden dog fern

[0054]

[0055] In summary of the above analysis, the robust rhizomes of the golden dog fern were used as the explants, the types and proportions of the hormones in each stage of propagation were optimized, the adventitious buds were obtained by induction of the callus, and the regenerated plants were obtained after proliferation and rooting; the tissue culture and rapid propagation method is simple, the callus formation rate and the proliferation coefficient are high, the rooting rate and the survival rate are both more than 90%, and the time from the adventitious buds to rooting or survival is only about 75 days or 95 days, the propagation speed is fast, and the regenerated plants retain the excellent genetic characteristics of the mother plants of the golden dog fern, and can provide a new basis for culture of the golden dog fern elite.

[0056] Finally, it should be noted that the above description is only the preferred embodiment of the present application, and is not intended to limit the present application. For those skilled in the art, the present application can have various changes and modifications, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for rapid tissue culture propagation of Cibotium barometz, characterized in that, Specifically, the following steps are included: S1. After removing the hairs from the rhizomes of *Cibotium barometz*, sterilize them. Then, cut the sterilized rhizomes into thin slices with a length and width of 0.5-1 cm and a thickness of 1-2 mm, and inoculate them onto P1 medium. Culture until the rhizome slices differentiate into granular protrusions to obtain primary callus tissue. The P1 medium is based on 1 / 2 MS medium and also contains 0.1-0.5 mg / L of 6-BA, 0.1-0.5 mg / L of KT, 30 g / L of sucrose, and 2 g / L of Gelrite, with a pH of 5.8-6.

0. S2. Primary callus tissue is inoculated onto P2 medium for induction culture to obtain green callus tissue; the P2 medium is based on MS medium and also contains 1.5-2.0 mg / L of 6-BA, 0.5-1.0 mg / L of NAA, 30 g / L of sucrose and 2 g / L of gelrite, with a pH of 5.8-6.

0. S3. Green callus tissue was inoculated onto differentiation induction medium P3 for induction culture to obtain adventitious shoots induced from the callus tissue; the differentiation induction medium P3 was based on MS medium and also contained 1.0-2.0 mg / L KT, 0.2-0.5 mg / L NAA, 0.2-0.5 mg / L 2,4-D, 30 g / L sucrose and 2 g / L Gelrite, with a pH of 5.8-6.0; S4. The adventitious shoots are transferred to proliferation medium P4 for proliferation culture; the proliferation medium P4 is MS-based medium, and also contains 0.3-1.0 mg / L of 6-BA, 0.3-1.0 mg / L of TDZ, 0.5-1.0 mg / L of KT, 30 g / L of sucrose, and 2 g / L of Gelrite, with a pH of 5.8-6.

0. S5. The adventitious shoots after proliferation culture are transferred to rooting medium P5 for culture to obtain complete regenerated seedlings; the rooting medium P5 is based on 1 / 2 MS medium and also contains IBA at a concentration of 0.3-1.0 mg / L, IAA at a concentration of 0.2-0.6 mg / L, 2,4-D at a concentration of 0.3-0.5 mg / L, zeolite at a concentration of 0.5-1.0 g / L, sucrose at a concentration of 30 g / L, and gelrite at a concentration of 2 g / L, with a pH of 5.8-6.0; S6. After hardening off the regenerated seedlings, transplant them into a substrate for cultivation to obtain regenerated plants.

2. The method for rapid tissue culture propagation of Cibotium barometz according to claim 1, characterized in that, In S1, rhizomes of well-grown, thick-tubered *Cibotium barometz* were selected, and after removing the hairs, they were disinfected. The disinfection method was as follows: soaking in 75% ethanol for 20 seconds, rinsing with sterile water 3-4 times, then soaking and shaking in 0.1% mercuric chloride solution for 5-8 minutes, rinsing with sterile water 4-5 times, and finally soaking and shaking in 4% sodium hypochlorite solution for 2-3 minutes, rinsing with sterile water 5-8 times.

3. The method for rapid tissue culture propagation of Cibotium barometz according to claim 1, characterized in that, In S1, the cultivation conditions are: temperature 20-25℃, humidity 70-80%, and light intensity 1500-1700 lx.

4. The method for rapid tissue culture propagation of Cibotium barometz according to claim 1, characterized in that, In S2, the cultivation conditions are: temperature 22-25℃, humidity 70-80%, and light intensity 1800-2200 lx.

5. The method for rapid tissue culture propagation of Cibotium barometz according to claim 1, characterized in that, In S3, the cultivation conditions are: temperature 23-26℃, humidity 70-80%, light intensity 1800-2200 lx, and light duration 12-15 h / d.

6. The method for rapid tissue culture propagation of Cibotium barometz according to claim 1, characterized in that, In S4, the cultivation conditions are: temperature 24-26℃, humidity 70-80%, light intensity 1800-2200 lx, and light duration 12-15 h / d.

7. The method for rapid tissue culture propagation of Cibotium barometz according to claim 1, characterized in that, In S5, the cultivation conditions are: temperature 24-26℃, humidity 70-80%, light intensity 1800-2000 lx, and light duration 12-15 h / d.

8. The method for rapid tissue culture propagation of Cibotium barometz according to claim 1, characterized in that, In S6, the substrate is a mixture of peat moss, perlite, and zeolite in a volume ratio of 3-5:1:1; the cultivation conditions are: temperature 24-28℃, humidity 80-90%, light intensity 3000-5000lx, and light duration 12-15h / d.

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