A tissue culture rapid propagation method for the germination pathway of Danxia Chinese Phyllostachys chinensis seeds

Abstract

The present invention relates to a tissue culture rapid propagation method for the germination pathway of Danxia sycamore seeds, including seed processing, seed germination, bud induction, bud proliferation, and bud rooting culture. The seed germination rate can reach more than 80%, the stem tip germination rate can reach more than 90%, the bud proliferation coefficient can reach 4, the buds grow well, and the root system is well developed. The cultivated Danxia sycamore plants have the same characteristics as the mother plants. The present invention uses the seeds of Danxia sycamore as materials and the stem tips as explants to establish a tissue culture rapid propagation technology system, providing technical support for Danxia sycamore seedling cultivation and population expansion and reproduction.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and more specifically to a tissue culture and rapid propagation method for germination pathways of Danxia sycamore seeds. Background Art

[0002] Danxia sycamore (Firmiana danxiaensis) is a small tree of the genus Firmiana in the Malvaceae family. The plant is 3-10m tall, with purple flowers, flowering from May to June, and fruit ripening from July to August. In 1987, Xu Xianghao and others first discovered it on the cliff of Danxia Mountain in Renhua, Shaoguan, Guangdong. At present, only a small number of distribution points have been found in Nanxiong, Shixing, Yingde, Qingyuan, and Yong'an, Fujian. The number of plants is scarce and the distribution is relatively scattered. It is listed as critically endangered in the "Red Catalog of Chinese Species" and the "Red List of Endangered Species of the World Conservation Union" (IUCN), and is a national second-level key protected wild plant. The "Outline of the 14th Five-Year Plan for Forestry and Grassland Protection and Development" released in July 2021 lists 50 species of wild plants with extremely small populations for rescue protection, including 15 critically endangered species, among which Danxia sycamore is one of them. Danxia sycamore has tall plants, colorful flowers and high ornamental value. It can be cultivated as a garden sightseeing tree, a road greening tree and a tree species for desertification control.

[0003] Danxia sycamore mainly reproduces through seed propagation. Its seeds have a low germination rate and a long germination time under natural conditions, which limits the expansion and reproduction of its population. It grows on cliffs, and its seed maturity period is relatively fixed. It is difficult to collect seeds and it is difficult to propagate a large number of Danxia sycamore seedlings through artificial sowing. By collecting a small amount of Danxia sycamore seeds and using plant tissue culture technology, it is possible to achieve the expansion and reproduction of Danxia sycamore seedlings without seasonal restrictions. Most of the research on Danxia sycamore focuses on the research on seed germination conditions and ecological distribution, and there are few reports on its seedling breeding. There are few reports on tissue culture of sycamore plants, and no complete regeneration system has been established. Therefore, it is necessary to propose a tissue culture rapid propagation method to provide technical support for Danxia sycamore breeding and population expansion and reproduction. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a tissue culture rapid propagation method for Danxia sycamore seed germination, establish an asexual reproduction pathway for Danxia sycamore, and provide technical support for Danxia sycamore seedling cultivation and population expansion and reproduction.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions:

[0006] A tissue culture rapid propagation method for the germination pathway of Danxia sycamore seeds, including seed processing, seed germination, bud induction, bud proliferation, and bud rooting culture. The seed germination rate can reach more than 80%, the stem tip germination rate can reach more than 90%, the bud proliferation coefficient can reach 4, the buds grow well, and the root system is well developed. The cultivated Danxia sycamore plants have the same characteristics as the mother plants.

[0007] The specific cultivation steps are as follows:

[0008] S1. Seed collection and processing: Collect seeds of Danxia sycamore in its mature stage and store them in a low temperature environment for future use;

[0009] Select full and shell-free seeds of Danxia sycamore, wash them with clean water, dry the surface water and soak them in concentrated sulfuric acid for 30-40 minutes, keep shaking during the soaking process, and after the soaking is completed, repeatedly wash the seeds with sterilized water until they are clean;

[0010] S2. Seed germination culture: inoculating the treated seeds onto a germination medium for seed germination culture to allow the seeds to germinate; wherein the germination medium is a paper bridge medium, or the germination medium is a modified MS medium;

[0011] S3, shoot tip germination culture: cutting the shoot tips of the germinated seeds and inoculating them on a bud induction medium to induce shoot tip germination, thereby inducing shoot tip germination or lateral bud germination; wherein the components and contents of the induction medium are: modified MS medium, 0.5-2 mg / L 6-BA, 0.05-0.1 mg / L I BA, 30 g / L sucrose and 6-7 g / L agar powder, and the pH of the induction medium is 5.8-6.0;

[0012] S4, bud proliferation culture: after the stem tip or lateral buds are cut after induction of germination, they are transferred to a bud proliferation medium for bud proliferation culture, so that more buds are proliferated and the buds continue to grow taller; wherein the components and contents of the bud proliferation medium are: improved MS medium, 0.2-0.5 mg / L 6-BA, 0.05-0.1 mg / L I BA, 30 mg / L sucrose and 6-7 g / L agar powder; the pH of the bud proliferation medium is 5.8-6.0;

[0013] S5, rooting culture: cutting off the tall buds and transferring them to rooting medium to induce root production and form tissue culture seedlings; wherein the components and contents of the rooting medium are: modified MS medium, 0.2-1 mg / L I BA, 30 g / L sucrose and 6-7 g / L agar powder, and the pH of the rooting medium is 5.8-6.0;

[0014] S6, planting the tissue culture seedlings with root systems to cultivate new Danxia sycamore plants;

[0015] In steps S2-S5, the improved MS culture medium comprises an MS culture medium in which the contents of NH4NO3, KNO3, MgSO4·7H2O, KH2PO4, and CaCl2·2H2O are halved, and the contents of trace elements, organic elements, and iron salts remain unchanged.

[0016] In one embodiment, in step S1, seeds of the Chinese sycamore trees are collected at the maturity stage of the Chinese sycamore trees and stored in an environment of 1 to 4° C. for future use.

[0017] In one embodiment, in step S2, the culture temperature is 25±1°C, the light conditions are 2000-2500LX, the light time is 12h / day, and the culture is 15-18 days.

[0018] In one embodiment, in step S3, the length of the stem tip cut off is 0.5-1 cm.

[0019] In one embodiment, in step S3, the culture temperature is 25±1°C, the light conditions are 2000-2500LX, the light time is 12h / day, and the culture is 30-35 days.

[0020] In one embodiment, in step S4, the induced shoot tip or lateral bud is cut and transferred to a bud proliferation medium with 1 to 2 leaves.

[0021] In one embodiment, in step S4, the culture temperature is 25±1°C, the light conditions are 2000-2500LX, the light time is 12h / day, and the culture is 30-35 days.

[0022] In one embodiment, in step S5, when the buds grow to 2-3 cm in height, the buds are cut off and transferred to a rooting medium.

[0023] In one embodiment, in step S5, the culture temperature is 25±1°C, the light conditions are 2000-2500LX, the light time is 12h / d, and the culture is 30-35 days.

[0024] In summary, the present invention has the following beneficial effects:

[0025] The invention adopts a small amount of seeds of Danxia sycamore as materials, treats the seeds with concentrated sulfuric acid, reduces the shelling procedure in the seed germination pathway, does not need to perform surface disinfection on the seeds, greatly reduces the seed treatment procedure, and improves the germination rate. The stem tip is used as an explant, which is convenient to cut, reduces the pollution rate and browning rate, and the bud induction configuration is added with an induction medium combined with cytokinin 6-BA and auxin I BA, induces adventitious buds, and the induction rate reaches more than 90%, and the bud induction rate is stable; in the proliferation culture of the invention, the bud proliferation coefficient reaches 4, which greatly improves the number of subsequent bud expansion and propagation, determines the efficiency of the regenerated plant to a certain extent, and the buds grow robustly and the leaves are dark green. The bud rooting rate can reach 70%, the plants grow robustly, and the root system is well-developed, which lays a foundation for the later plant expansion and propagation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The invention relates to various stages of tissue culture of the Chinese parasol tree, wherein in step 1, A: seeds of the Chinese parasol tree germinate well on a paper bridge culture medium; B: seeds of the Chinese parasol tree germinate well on a modified MS culture medium; C: stem tips are cut and placed on a bud induction culture medium to induce stem tip germination; D: stem tips grow taller after germination and lateral buds also germinate; E: germinated stem tips or lateral buds are divided and placed on a proliferation culture medium and proliferate well; F: the proliferated buds take root well on a rooting culture medium and have a well-developed root system. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0028] It is worth noting that the directional words such as "upper" and "lower" involved in this article are all relative to the viewing angle of the drawings and are only for the convenience of description and cannot be understood as a limitation on the technical solution.

[0029] The number of Danxia sycamore plants is small, and the distribution is relatively scattered and narrow. It is a national second-level key protected wild plant. Danxia sycamore mainly reproduces through seed propagation. Its seeds have a low germination rate under natural conditions and take a long time to germinate, which is considered to be the reason for limiting its population expansion. The present invention uses plant tissue culture technology to achieve the expansion and reproduction of Danxia sycamore plants without seasonal restrictions, providing an effective way for the protection of the species and the reproduction and utilization of seedlings.

[0030] The present invention provides a tissue culture and rapid propagation method for Danxia sycamore seed germination pathway, comprising the following steps:

[0031] S1. Seed collection and processing:

[0032] The seeds of Danxia sycamore are collected during the maturity period of Danxia sycamore and stored in a low temperature environment of 1 to 4°C for future use;

[0033] Select plump and shell-free seeds of Danxia sycamore, clean them with clean water, dry the surface water and soak them in concentrated sulfuric acid for 30-40 minutes, shaking them during the soaking process;

[0034] After soaking, wash the seeds repeatedly with sterilized water until they are clean.

[0035] In step S1, the concentration of concentrated sulfuric acid is 98.3%. The seed shells of Danxia sycamore are thick and hard. After being treated with concentrated sulfuric acid, the seed shells can be softened, the air permeability of the seeds can be improved, the seeds can germinate quickly, the germination rate can be increased, and a disinfection effect can be achieved.

[0036] After soaking, clean the carbonized seed coat and seed shell on the surface of the seeds.

[0037] S2. Seed germination culture:

[0038] The treated seeds are inoculated on a germination medium for seed germination culture, placed in a culture room, cultured at a temperature of 25±1°C, with a light condition of 2000-2500LX and a light duration of 12h / day, cultured for 15-18 days, to allow the seeds to germinate;

[0039] The germination medium is a paper bridge medium, or the germination medium is a modified MS medium, wherein the modified MS medium comprises an MS medium in which the content of NH4NO3, KNO3, MgSO4·7H2O, KH2PO4, and CaC l 2·2H2O is halved, and the content of trace elements, organic elements, and iron salts remains unchanged; the paper bridge medium refers to a filter paper made into a bridge shape, on which seeds are placed for germination culture, and the filter paper is immersed in water.

[0040] Danxia sycamore grows on the cliffs of Danxia landform with relatively poor soil, and does not need too much nitrogen and phosphorus elements such as NH4+. It can grow well in rocky desertification areas. Therefore, the present invention cultivates its seeds with improved culture medium to reduce the amount of macroelements.

[0041] The seeds will be damaged when shelling. In the present invention, the seeds of Danxia sycamore are treated with concentrated sulfuric acid without shelling, which can reduce the shelling procedure, is beneficial to shorten the germination time, and improve the germination rate. After the surface is sterilized, the seeds are inoculated into a paper bridge culture medium or a modified MS culture medium. The seeds begin to germinate after about 3 days, and the seeds begin to germinate after 7 days. After 15 days, the germination rate can reach more than 80%.

[0042] S3. Shoot tip germination culture:

[0043] Cutting the stem tips of the germinated seeds and inoculating them on a bud induction medium to induce stem tip germination, placing them in a culture room at a culture temperature of 25±1°C, a light condition of 2000-2500LX, a light duration of 12h / day, and culturing them for 30-35 days to induce stem tip germination or lateral bud germination;

[0044] Among them, the length of the cut stem tip is 0.5 to 1 cm and inoculated on the bud induction medium;

[0045] The components and contents of the induction medium are: improved MS medium, 0.5-2 mg / L 6-BA, 0.05-0.1 mg / L I BA, 30 g / L sucrose and 6-7 g / L agar powder, and the pH of the induction medium is 5.8-6.0.

[0046] The present invention found that when the 6-BA concentration was higher than 2 mg / L or the I BA was 0.2 mg / L, the vitrification phenomenon of the cultured tissue gradually appeared. In order to reduce the vitrification phenomenon, the present invention limited the 6-BA concentration to 2 mg / L and the I BA to 0.1 mg / L. In the bud induction culture, at the appropriate 6-BA / I BA concentration, the bud induction rate reached more than 90%, and the buds grew well.

[0047] S4. Bud proliferation culture:

[0048] After the stem tip or lateral buds are cut off after induction of germination, they are transferred to a bud proliferation medium for bud proliferation culture, placed in a culture room, cultured at a temperature of 25±1°C, with a light condition of 2000-2500LX and a light duration of 12h / day, cultured for 30-35 days, and more buds are proliferated, and the buds continue to grow taller;

[0049] Among them, the induced shoot tips or lateral buds were cut, with 1 to 2 leaves, and transferred to the bud proliferation medium;

[0050] The components and contents of the bud proliferation medium are: improved MS medium, 0.2-0.5 mg / L 6-BA, 0.05-0.1 mg / L I BA, 30 mg / L sucrose and 6-7 g / L agar powder; the pH of the bud proliferation medium is 5.8-6.0.

[0051] The present invention finds that when the concentration of 6-BA is 1 mg / L or I BA is 0.2 mg / L, a small amount of vitrification phenomenon will occur in the bud tip of Danxia sycamore as the culture time is prolonged. In order to reduce the vitrification phenomenon, the present invention limits the concentration of 6-BA to 0.2-0.5 mg / L and I BA to 0.05-0.1 mg / L. During the bud proliferation process, the vitrification phenomenon is significantly alleviated, and the buds can proliferate well. In the bud induction culture, at a suitable 6-BA / I BA concentration, the bud induction rate reaches more than 90%, and the buds grow well.

[0052] In the bud proliferation culture, adding appropriate 6-BA / I BA concentration can promote bud proliferation, the bud proliferation coefficient can reach 4, and the buds grow robustly.

[0053] S5. Rooting culture:

[0054] After cutting off the tall buds, transfer them to the rooting medium and place them in a culture room at a temperature of 25±1°C, a light condition of 2000-2500LX, and a light duration of 12h / d. Cultivate for 30-35 days to induce root production and form tissue culture seedlings.

[0055] Among them, when the buds grow to 2-3 cm in height, they are cut off and transferred to the rooting medium;

[0056] The components and contents of the rooting medium are: improved MS medium, 0.2-1 mg / L IBA, 30 g / L sucrose and 6-7 g / L agar powder, and the pH value of the rooting medium is 5.8-6.0.

[0057] S6. Plant the tissue culture seedlings with roots to cultivate new Danxia sycamore plants.

[0058] The invention selects suitable regeneration explants, uses the stem tip as the explant, is convenient to cut, and is configured with an induction culture medium added with a combination of cytokinin 6-BA and auxin I BA, which is conducive to inducing bud germination, greatly reduces the browning rate, stabilizes bud germination, and the induction rate can reach more than 90%. In the culture of bud proliferation, the proliferation coefficient is greatly improved, the bud proliferation coefficient reaches 4, and determines the number of regenerated plants to a certain extent. After the proliferated buds are cut, they are transferred to the rooting culture medium for easy rooting, and roots begin to form 20 days after inoculation. The rooting rate of the test tube seedlings can reach 70%, the plants grow robustly, and the root system is well-developed, providing a technical reference for the reproduction of Danxia sycamore.

[0059] The present invention applies tissue culture technology to establish an effective asexual propagation system of Danxia sycamore, adopts seeds of Danxia sycamore as materials, takes stem tips as explants, and uses improved MS as a basic culture medium, and selects suitable seed germination, bud induction, bud proliferation and rooting culture medium formulas through different hormone types and concentration ratios, thereby providing a theoretical basis and practical operation technology for the expansion and propagation of Danxia sycamore plants.

[0060] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A tissue culture and rapid propagation method for the germination pathway of Danxia Chinese Phyllostachys chinensis seeds, characterized in that: The following steps are involved: S1. Seed collection and processing: Collect seeds of Danxia sycamore in its mature stage and store them in a low temperature environment for future use; Select full and shell-free seeds of Danxia sycamore, wash them with clean water, dry the surface water and soak them in concentrated sulfuric acid for 30-40 minutes, keep shaking during the soaking process, and after the soaking is completed, repeatedly wash the seeds with sterilized water until they are clean; S2. Seed germination culture: inoculating the treated seeds onto a germination medium for seed germination culture to allow the seeds to germinate; wherein the germination medium is a paper bridge medium, or the germination medium is a modified MS medium; S3, shoot tip germination culture: cutting the shoot tips of the germinated seeds and inoculating them on a bud induction medium to induce shoot tip germination, thereby inducing shoot tip germination or lateral bud germination; wherein the components and contents of the induction medium are: modified MS medium, 0.5-2 mg / L 6-BA, 0.05-0.1 mg / L IBA, 30 g / L sucrose and 6-7 g / L agar powder, and the pH of the induction medium is 5.8-6.0; S4, bud proliferation culture: after the stem tip or lateral buds are cut after induced germination, they are transferred to a bud proliferation medium for bud proliferation culture, so that more buds are proliferated and the buds continue to grow taller; wherein the components and contents of the bud proliferation medium are: improved MS medium, 0.2-0.5 mg / L 6-BA, 0.05-0.1 mg / L IBA, 30 mg / L sucrose and 6-7 g / L agar powder; the pH of the bud proliferation medium is 5.8-6.0; S5, rooting culture: after cutting off the tall buds, transfer them to rooting medium to induce root production and form tissue culture seedlings; wherein the components and contents of the rooting medium are: modified MS medium, 0.2-1 mg / L IBA, 30 g / L sucrose and 6-7 g / L agar powder, and the pH of the rooting medium is 5.8-6.0; S6, planting the tissue culture seedlings with root systems to cultivate new Danxia sycamore plants; In steps S2-S5, the improved MS culture medium comprises an MS culture medium in which the contents of NH4NO3, KNO3, MgSO4·7H2O, KH2PO4, and CaCl2·2H2O are halved, and the contents of trace elements, organic elements, and iron salts remain unchanged.

2. The tissue culture and rapid propagation method of the seed germination pathway of Danxia Chinese Phyllostachys chinensis as claimed in claim 1, characterized in that: In step S1, the seeds of the Danxia sycamore are collected during the maturity period of the Danxia sycamore and stored in an environment of 1 to 4° C. for future use.

3. The tissue culture and rapid propagation method of the seed germination pathway of Danxia Chinese Phyllostachys chinensis as claimed in claim 1, characterized in that: In step S2, the culture temperature is 25±1°C, the light conditions are 2000-2500LX, the light time is 12h / day, and the culture is carried out for 15-18 days.

4. The tissue culture and rapid propagation method of the seed germination pathway of Danxia Chinese Phyllostachys chinensis as claimed in claim 1, characterized in that: In step S3, the length of the stem tip cut off is 0.5 to 1 cm.

5. The tissue culture and rapid propagation method of Danxia sycamore seed germination pathway as claimed in claim 1, characterized in that: In step S3, the culture temperature is 25±1°C, the light conditions are 2000-2500LX, the light time is 12h / day, and the culture is carried out for 30-35 days.

6. The tissue culture and rapid propagation method of the seed germination pathway of Danxia Chinese Phyllostachys chinensis as claimed in claim 1, characterized in that: In step S4, the induced shoot tips or lateral buds are cut and transferred to a bud proliferation medium with 1 to 2 leaves.

7. The tissue culture and rapid propagation method of the seed germination pathway of Danxia Chinese Phyllostachys chinensis as claimed in claim 1, characterized in that: In step S4, the culture temperature is 25±1°C, the light conditions are 2000-2500LX, the light time is 12h / day, and the culture is carried out for 30-35 days.

8. The tissue culture and rapid propagation method of the seed germination pathway of Danxia Chinese Phyllostachys chinensis as claimed in claim 1, characterized in that: In step S5, when the buds grow to 2-3 cm in height, the buds are cut off and transferred to a rooting medium.

9. The tissue culture and rapid propagation method of the seed germination pathway of Danxia Chinese Phyllostachys chinensis as claimed in claim 1, characterized in that: In step S5, the culture temperature is 25±1°C, the light conditions are 2000-2500LX, the light time is 12h / d, and the culture is carried out for 30-35 days.

Images