Method for improving utilization rate of recalcitrant seeds of extremely-dangerous plant pachysandra angustifolia

Abstract

The method for improving the utilization rate of the recalcitrant seeds of the extremely dangerous plant pimpinella angustifolia comprises the steps of seed collection, seed pretreatment, seed sterilization, induced germination, seedling culture, acclimatization and transplantation and germplasm preservation, and can utilize immature seeds, semi-mature seeds and seeds which cannot germinate and form seedlings under mature natural conditions without water loss to the maximum extent; under the condition that the number of the seeds is certain, more seeds are promoted to be converted into seedlings; according to the method, in-vitro preservation of the hedyotis angustifolia seeds within a certain time is achieved, the limitation that seed germination and utilization must be conducted in the seed maturing and harvesting season is broken through, the utilization rate of the seeds is remarkably increased, and a sterile material is provided for long-term preservation of germplasm resources and large-scale breeding; a technical foundation is laid for the aspects of biological protection, ecological restoration, resource development and utilization and the like of the species, and great practical significance is achieved for rescuing the endangered tree species.

Description

Technical Field

[0001] The invention relates to the technical field of plant propagation, in particular to a method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium. Background Art

[0002] Hopea chinensis Hand.-Mazz., also known as South China Hopea and Narrow-leaved Hopea, belongs to the genus Hopea of ​​Dipterocarpaceae and is a critically endangered tree species endemic to Guangxi. It is an evergreen tree that can reach a height of 30m and a breast diameter of 50-100cm. It grows in the foothills, valleys and hillside forests below 600m above sea level. It is one of the representative tree species of tropical evergreen seasonal rain forests in northern and southern Guangxi and is of great value in studying the composition, geographical distribution and ecological habits of tropical rain forest flora. At the same time, its wood has interlaced texture, fine structure, hard material, and strong corrosion resistance. It can be buried in the soil for hundreds of years without rotting. It is an excellent special timber species and is called "eternal wood". It can be used as material for military industry, machinery, and high-end furniture. The resin it produces is similar to hemp resin and can be used for paint manufacturing. The bark has a dragon robe-like pattern, so it is also called "dragon robe tree". The by-products are aromatic oils, which can be used as high-end cosmetics and precious Chinese medicine materials, and are extremely valuable.

[0003] The narrow-leaved sedge is a relict species of the Tertiary period, distributed in the "refuges" formed by local terrain. It is native to the Shiwandashan and Daqingshan areas of Guangxi, but due to the destruction of human production activities and the impact of climate change, the population has decreased sharply. Investigations show that the narrow-leaved sedge population in Daqingshan has now become extinct, and there are only a small number of wild narrow-leaved sedges distributed in the Shiwandashan Nature Reserve and surrounding areas. In the past 20 years, the number of wild narrow-leaved sedge individuals has decreased sharply, and the species is threatened with extinction. It has been classified as a national first-class protected plant and listed as a critically endangered (CR) species by the World Conservation Union (IUCN). Research on its endangered mechanism has found that its fruit is rare, and there is a phenomenon of alternate years, and some years even no fruit. The distribution range of the wild population has shrunk extremely, facing the risk of extinction, and it is in urgent need of protection.

[0004] Through conservation measures, expanding the population is the most basic condition for saving endangered plants. After research, it was found that the conversion rate of seeds of wild populations of narrow-leaved slope to seedlings is relatively low, because its seeds are typical stubborn seeds. Once stubborn seeds encounter drought or cannot fall into a soil environment suitable for growth, the seeds will lose water quickly, and the life and vitality of the seeds will be greatly reduced, or even completely inactivated and unable to germinate. Wild plants of narrow-leaved slope mostly grow on the banks of streams, and seeds are mainly spread by gravity. Many mature seeds fall into the stream near the mother plant. Most of the seeds that fall into the stream are soaked in water and difficult to germinate into seedlings. Moreover, due to the stubborn characteristics of its seeds, there is no mature preservation technology so far. The collected fresh seeds must be sown immediately. Once they are placed in the refrigerator or lose water, they lose their vitality. If there are no suitable seedling conditions before the seeds are inactivated, they will be completely unable to germinate and grow into seedlings. The time for seed germination is strictly limited to the time close to the time when the seeds are mature and harvested, which greatly limits the utilization rate of seeds. So far, there are no related reports on seed seedling cultivation in the prior art. In order to better restore its population, save this critically endangered plant, improve seed utilization, and encourage more of its seeds to be converted into seedlings, it is of great practical significance. Summary of the invention

[0005] The purpose of the present invention is to solve the technical problems that the stubborn seeds of narrow-leaved sedge cannot be preserved and are easily inactivated, resulting in few seeds converted into seedlings and low utilization rate. Under the condition of a certain number of seeds, more seeds can be converted into seedlings, and germplasm resources can be preserved for a certain period of time, seedling raising space can be saved, seedling raising conditions can be controlled, and seed utilization rate can be improved.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for improving the utilization rate of seeds of the critically endangered plant Sphaerocarpus angustatus, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation. The specific operation steps are as follows:

[0008] (1) Seed collection:

[0009] Collect fresh seeds free of pests and diseases in the natural distribution area of ​​S. angustifolia;

[0010] (2) Seed pretreatment:

[0011] Remove the wings on both sides of the seeds of the narrow-leaved sedge, wash the surface of the seeds with clean water, stir and soak them with cleaning liquid for 5 minutes, wash off the cleaning liquid with pure water, wrap the seeds and sterilized fine sand with gauze, soak them in a 2% Australian tea tree essential oil aqueous solution for 20 minutes, stir and soak them with sterilized water for 3-5 minutes, and transfer them to a clean bench;

[0012] (3) Seed sterilization:

[0013] Open the clean bench, soak the seeds in 75% alcohol for 1 minute, burn with an alcohol lamp for 5 seconds, open the gauze, wash the seeds to remove fine sand, put them in an inoculation plate with sterilized filter paper, turn on the ultraviolet light of the clean bench for 30 minutes, put the seeds in the sterilization solution, stir and soak for 10 to 15 minutes, repeat once, and then soak them in 35 to 40°C sterilized water for 3 to 5 times, peel off the seed coat with a scalpel to obtain sterilized embryos;

[0014] (4) Inducing germination:

[0015] The sterilized embryos were inoculated into the germination induction medium according to their maturity. Immature and semi-mature embryos were inoculated into the germination induction medium ①, and mature embryos were inoculated into the germination induction medium ②. One embryo was inoculated into each bottle. The culture conditions were 25±3℃ and dark culture.

[0016] (5) Seedling culture: After the embryos of S. angustifolia are cultured in the germination induction medium for 10 to 15 days, they are transferred to the culture bottles of the seedling medium. The culture conditions are as follows: the temperature is 25±3°C, the light duration is 12 to 15 hours / day, and the light intensity is 1500 to 3000 lx;

[0017] (6) Hardening and transplanting

[0018] Move the tissue culture seedlings after seedling cultivation outdoors, open the bottle cap after hardening under natural light for 5 to 7 days, continue hardening for 1 to 3 days, take out the seedlings, wash off the culture medium, transplant them into the seedling medium, cover them with a shade net with a shade degree of 75 to 85% within 7 days after transplanting, and then carry out conventional seedling management;

[0019] (7) Germplasm preservation

[0020] After induced germination, the tissue culture seedlings were transferred into the preservation medium. The medium was placed in a 270 mL PC plastic bottle with a 70 mm diameter and 110 mm height sealed cap. Five plants were inoculated into each bottle. The bottle mouth was wrapped with plastic wrap twice. The preservation medium was replaced with new one every 30 to 40 days. When it was needed, it was transferred into the culture bottle of the seedling medium. After 25 days of culture, the seedlings could be directly hardened and transplanted.

[0021] The seed collection in the above step (1) includes collecting seeds from trees and picking up fallen seeds; the fresh seeds include immature, semi-mature and mature seeds that have not yet lost water, wherein the seed coat and seed wings of immature seeds are all green; the seed coat of semi-mature seeds is partially yellow-green, and the seed wings are dark yellow; the seed coat of mature seeds is all yellow-green, and the seed wings are dark yellow or brown.

[0022] The cleaning solution in step (2) above is 5 g of flour added to 1000 mL of clean water.

[0023] In step (3) above, 1 cm deep sterile water should be poured onto the sterile filter paper in the inoculation plate; the sterile solution is composed of 0.1% HgCl 2 The solution was mixed with 1 drop of Tween 20.

[0024] The germination induction medium formula ① described in step (4) above is MS+BA 0.25mg / L+NAA 0.25~0.5mg / L+GA 3 0.5~0.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8~6.0; the formula of induction germination medium ② is MS+BA 0.5mg / L+NAA 0.4~0.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8~6.0.

[0025] The seedling culture medium formula of the above step (5) is 1 / 2MS+IAA 1.5-2.0mg / L+IBA 1.5-2.0mg / L+15g / L sucrose 3.8g / L agar powder, pH 5.8-6.0.

[0026] The seedling culture medium in the above step (6) is a mixture of peat soil, red heart soil and mushroom residue in a volume ratio of 4:4:2.

[0027] The preservation medium formula of the above step (7) is 2 / 3MS+BA 0.1-0.2mg / L+IAA 0.5mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0.

[0028] The present invention has the following advantages and positive effects over the prior art:

[0029] 1. The technical solution proposed by the present invention is to collect and process seeds of different categories and then culture them in vitro, which reduces the harm of pests and diseases. By optimizing the culture medium formula at each stage, the seed embryo germination rate can reach 85.88-92.21%, the seedling rate can reach 81.20-84.59%, the cultivated seedlings are strong, the survival rate of seedling hardening and transplanting can reach 82.57-89.33%, and the survival rate of seeds after in vitro storage for 90.24-93.31 days can reach 75.41-80.18% after recovery seedling culture and seedling hardening and transplanting, achieving good technical effects.

[0030] 2. The present invention designs different culture medium formulas for seeds of different maturity, which significantly improves the germination rate and seedling rate of seed embryos; collecting immature seeds can make use of the seeds before they encounter extreme weather or other accidents during the seed maturation process; collecting fallen seeds, including those that fall by the stream or in the stream, avoids the risk of some seeds falling off and becoming inactive after maturity or being eaten by insects and birds, maximizes the use of seeds that cannot germinate into seedlings under natural conditions, significantly improves the utilization rate of seeds, and under the condition of a certain number of seeds, promotes more seeds to be converted into seedlings, laying a technical foundation for research on the conservation biology, restoration ecology, and development and utilization of resources of this species.

[0031] 3. The present invention establishes a sterile system using embryos as explants of Achyranthes bidentata, which can provide sterile materials for tissue culture propagation of Achyranthes bidentata, thus laying a foundation for long-term preservation of germplasm resources and large-scale breeding through tissue culture technology.

[0032] 4. The present invention adopts a series of innovative combined measures for seed pretreatment and sterilization, which significantly improves the sterilization success rate and start-up rate. Before the seeds are sterilized, 5g of flour is added to 1000mL of clean water to prepare a cleaning solution to clean them. The flour particles are relatively fine and have a certain adsorption capacity. At the same time, the flour aqueous solution has a certain viscosity, which can wash away the dirt adhering to the surface of the seed coat; the gauze is used to wrap fine sand, which, on the one hand, increases the friction on the seed coat and promotes germination, and on the other hand, is conducive to sterilization and can also avoid direct contact of alcohol with seeds to damage vitality; when the seeds are sterilized, they are soaked in 75% alcohol and then briefly burned with a flame, which can avoid alcohol residue on the surface, and at the same time, a short heat treatment can inactivate some bacteria; after the seeds are soaked in the sterilization solution, they are soaked in 35-40℃ sterilization water. The sterilization water at a certain temperature can achieve the effect of activating the vitality of the seeds while washing away the sterilization solution, further promoting germination.

[0033] 5. The present invention can effectively improve the success rate of embryo germination by optimizing the germination induction medium, seedling medium and preservation medium. After transferring to preservation culture, seedling formation can be delayed, and seedling formation can be quickly achieved after recovery culture. Short-term preservation can be achieved, and the seed utilization time can be extended by 60 to 90 days, breaking the limitation that seed germination and utilization must be in the season when seeds are mature and harvested, and effectively alleviating the situation that the collected seeds cannot be sown immediately due to objective factors, thereby causing the seeds to be inactivated and unable to form seedlings. In addition, the present invention is not limited by land and seedling raising space, and has important scientific significance for saving this endangered tree species.

[0034] 6. The transplanting seedling medium adopted in the present invention adopts peat soil: yellow heart soil: mushroom residue in a ratio of 4:4:2. The selection of peat soil and red heart soil can ensure sufficient water and fertilizer retention capacity. Mushroom residue is the waste mushroom chaff and waste after planting edible fungi. It can change the content of soil organic matter and humus and improve soil fertility. This seedling medium has a simple ratio, a reasonable ratio, and is easy to prepare. It can provide sufficient nutrition for the narrow-leaved slope barrier seedlings and can significantly improve the transplanting survival rate of the narrow-leaved slope barrier seedlings to 82.57-89.33%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a picture of immature seeds of Sphaerocephalum angustifolia.

[0036] Figure 2 This is a picture of semi-mature seeds of Spathiphyllum narrow-leaved.

[0037] Figure 3 This is a picture of mature seeds of Sphagnum petiolus.

[0038] Figure 4 This is a picture of seed germination of Sphaerocarpus angustifolia.

[0039] Figure 5 This is a picture of the tissue culture seedlings of Scutellaria sphenanthera.

[0040] Figure 6 This is a picture of transplanting narrow-leaved Spatholobi tissue culture seedlings into cup seedlings. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0042] Explanation of term abbreviations and calculation formula:

[0043] MS: MS medium;

[0044] 2 / 3MS: The macroelements in MS medium are reduced to 2 / 3;

[0045] 1 / 2MS: The macroelements in MS medium are reduced by half;

[0046] BA: 6-benzylaminopurine;

[0047] NAA: naphthylacetic acid;

[0048] GA 3 :Gibberellins;

[0049] IAA: indoleacetic acid;

[0050] IBA: indolebutyric acid;

[0051] Contamination rate (%) = number of contaminated bud embryos / total number of inoculated embryos × 100;

[0052] Initiation rate (%) = number of initiated embryos / total number of inoculated embryos × 100. The embryos that elongate and break through the embryos are considered initiated.

[0053] Germination rate (%) = number of germinated embryos / total number of inoculated embryos × 100. The embryos with a bud length greater than the embryo diameter are considered germinated.

[0054] Seedling rate (%) = number of rooted seedlings / total number of inoculations × 100;

[0055] Transplant survival rate (%) = number of transplanted surviving seedlings / total number of transplants × 100;

[0056] 60d brown mortality rate (%) = number of brown deaths / total number of inoculations × 100;

[0057] The longest storage time refers to the time when the brown mortality rate is ≤20%.

[0058] Embodiment 1:

[0059] A method for improving the utilization rate of seeds of the critically endangered plant Sphaerocarpus angustatus, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation. The specific operation steps are as follows:

[0060] (1) Seed collection:

[0061] In the distribution area of ​​the natural population of Sphaerocephala angustifolia, fresh immature seeds with green seed coat and wings, fresh semi-mature seeds with yellow-green seed coat and dark yellow seed wings, or fresh mature seeds with yellow-green seed coat and dark yellow or brown seed wings were collected without pests and diseases.

[0062] (2) Seed pretreatment:

[0063] The wings on both sides of the seeds of the narrow-leaved sedge were peeled off, and the surface of the seeds were washed with clean water. Then, 5 g of flour was added to 1000 mL of clean water and stirred and soaked for 5 minutes. The cleaning solution was washed off with pure water, and the seeds and sterilized fine sand were wrapped with gauze. Then, the seeds were soaked in a 2% aqueous solution of Australian tea tree essential oil for 20 minutes, and then stirred and soaked in sterilized water for 5 minutes, and then transferred to a clean bench;

[0064] (3) Seed sterilization:

[0065] Open the clean bench, soak with 75% alcohol for 1 minute, sterilize with alcohol lamp flame for 5 seconds, open the gauze, wash the narrow-leaved sedge seeds to remove fine sand, and put them into the inoculation plate with sterilized filter paper. Pour 1 cm deep HgCl with a mass fraction of 0.1% on the sterilized filter paper in the inoculation plate. 2Solution and 1 drop of Tween 20 mixed with sterile water; turn on the ultraviolet light of the clean bench to irradiate for 30 minutes, stir and soak the seeds for 15 minutes, repeat once, and then soak them in 35-40℃ sterile water for 5 times, peel off the seed coat with a scalpel to obtain sterilized embryos;

[0066] (4) Inducing germination:

[0067] The sterilized narrow-leaved sedge embryos were classified according to maturity, and the immature or semi-mature embryos were inoculated with MS+BA0.25mg / L+NAA 0.25mg / L+GA 3 0.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 induction germination medium ①; mature embryos were inoculated into MS+BA 0.5mg / L+NAA 0.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 induction germination medium ②, 1 seed was inoculated into each bottle, the culture conditions were temperature 25±3℃, dark culture;

[0068] (5) Seedling cultivation:

[0069] After culturing the embryos of S. angustifolia in the germination induction medium for 15 days, they were transferred into a culture bottle containing 1 / 2MS+IAA 1.5mg / L+IBA 1.5mg / L+15g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 seedling medium, culture conditions were 25±3℃, light exposure time of 12-15h / d, light intensity of 1500-3000lx;

[0070] (6) Hardening and transplanting

[0071] Move the tissue culture seedlings after seedling cultivation outdoors, open the bottle cap after 5 days of hardening under natural light, continue hardening for 1 day, take out the seedlings, wash off the culture medium, and transplant them to a seedling medium mixed with peat soil: red heart soil: mushroom residue in a volume ratio of 4:4:2. Cover with a shade net with a shade degree of 85% within 7 days after transplanting, and then carry out conventional seedling management;

[0072] (7) Germplasm preservation

[0073] The tissue culture seedlings after induced germination culture were transferred into a preservation medium of 2 / 3MS+BA 0.2mg / L+IAA 0.5mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0. The culture medium was put into a 270mL PC plastic bottle with a sealing cap of 70mm in diameter × 110mm in height. Five strains were inoculated into each bottle. The bottle mouth was wrapped with plastic wrap twice and replaced with new preservation medium every 30 days. When it was needed to be used, it was transferred into a culture bottle of seedling culture medium. After 25 days of culture, the seedlings could be directly hardened and transplanted.

[0074] Embodiment 2:

[0075] A method for improving the utilization rate of seeds of the critically endangered plant Sphaerocarpus angustatus, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation. The specific operation steps are as follows:

[0076] (1) Seed collection:

[0077] In the distribution area of ​​the natural population of Sphaerocephala angustifolia, fresh immature seeds with green seed coat and wings, fresh semi-mature seeds with yellow-green seed coat and dark yellow seed wings, or fresh mature seeds with yellow-green seed coat and dark yellow or brown seed wings were collected without pests and diseases.

[0078] (2) Seed pretreatment:

[0079] The wings on both sides of the seeds of the narrow-leaved sedge were peeled off, and the surface of the seeds were washed with clean water. Then, 5 g of flour was added to 1000 mL of clean water and stirred and soaked for 5 minutes. The cleaning solution was washed off with pure water, and the seeds and sterilized fine sand were wrapped with gauze. Then, the seeds were soaked in a 2% aqueous solution of Australian tea tree essential oil for 20 minutes, and then stirred and soaked in sterilized water for 3 minutes, and then transferred to a clean bench;

[0080] (3) Seed sterilization:

[0081] Open the clean bench, soak with 75% alcohol for 1 minute, sterilize with alcohol lamp flame for 5 seconds, open the gauze, wash the narrow-leaved sedge seeds to remove fine sand, and put them into the inoculation plate with sterilized filter paper. Pour 1 cm deep HgCl with a mass fraction of 0.1% on the sterilized filter paper in the inoculation plate. 2 Solution and 1 drop of Tween 20 mixed with sterile water; turn on the ultraviolet light of the clean bench to irradiate for 30 minutes, stir and soak the seeds for 10 minutes, repeat once, and then soak them in 35-40℃ sterile water for 3 times, peel off the seed coat with a scalpel to obtain sterilized embryos;

[0082] (4) Inducing germination:

[0083] The sterilized narrow-leaved sedge embryos were classified according to maturity, and the immature or semi-mature embryos were inoculated with MS+BA0.25mg / L+NAA 0.5mg / L+GA 3 0.5mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 induction germination medium ①; mature embryos were inoculated into MS+BA 0.5mg / L+NAA 0.4mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 induction germination medium ②, 1 seed was inoculated into each bottle, the culture conditions were temperature 25±3℃, dark culture;

[0084] (5) Seedling cultivation:

[0085] After culturing the embryos of S. angustifolia in the germination induction medium for 10 days, they were transferred into a culture bottle containing 1 / 2MS+IAA 2.0mg / L+IBA 2.0mg / L+15g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 seedling medium, culture conditions were 25±3℃, light duration of 12-15h / d, light intensity of 1500-3000lx;

[0086] (6) Hardening and transplanting

[0087] Move the tissue culture seedlings after seedling cultivation outdoors, open the bottle cap after 7 days of hardening under natural light, continue hardening for 3 days, take out the seedlings, wash off the culture medium, and transplant them to a seedling medium mixed with peat soil: red heart soil: mushroom residue in a volume ratio of 4:4:2. Cover with a shade net with a shade degree of 85% within 7 days after transplanting, and then carry out conventional seedling management;

[0088] (7) Germplasm preservation

[0089] The tissue culture seedlings after induced germination culture were transferred into a preservation medium of 2 / 3MS+BA 0.1mg / L+IAA 0.5mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0. The culture medium was put into a 270mL PC plastic bottle with a sealing cap of 70mm in diameter × 110mm in height. Five strains were inoculated in each bottle. The bottle mouth was wrapped with plastic wrap twice and replaced with new preservation medium every 40 days. When it was needed to be used, it was transferred into a culture bottle of seedling culture medium. After 25 days of culture, the seedlings could be directly hardened and transplanted.

[0090] Embodiment 3:

[0091] A method for improving the utilization rate of seeds of the critically endangered plant Sphaerocarpus angustatus, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation. The specific operation steps are as follows:

[0092] (1) Seed collection:

[0093] In the distribution area of ​​the natural population of Sphaerocephala angustifolia, fresh immature seeds with green seed coat and wings, fresh semi-mature seeds with yellow-green seed coat and dark yellow seed wings, or fresh mature seeds with yellow-green seed coat and dark yellow or brown seed wings were collected without pests and diseases.

[0094] (2) Seed pretreatment:

[0095] The wings on both sides of the seeds of the narrow-leaved sedge were peeled off, and the surface of the seeds were washed with clean water. Then, 5 g of flour was added to 1000 mL of clean water and stirred and soaked for 5 minutes. The cleaning solution was washed off with pure water, and the seeds and sterilized fine sand were wrapped with gauze. Then, the seeds were soaked in a 2% aqueous solution of Australian tea tree essential oil for 20 minutes, and then stirred and soaked in sterilized water for 5 minutes, and then transferred to a clean bench;

[0096] (3) Seed sterilization:

[0097] Open the clean bench, soak with 75% alcohol for 1 minute, sterilize with alcohol lamp flame for 5 seconds, open the gauze, wash the narrow-leaved sedge seeds to remove fine sand, and put them into the inoculation plate with sterilized filter paper. Pour 1 cm deep HgCl with a mass fraction of 0.1% on the sterilized filter paper in the inoculation plate. 2 Solution and 1 drop of Tween 20 mixed with sterile water; turn on the ultraviolet light of the clean bench to irradiate for 30 minutes, stir and soak the seeds for 15 minutes, repeat once, and then soak them in 35-40℃ sterile water for 3 times, peel off the seed coat with a scalpel to obtain sterilized embryos;

[0098] (4) Inducing germination:

[0099] The sterilized narrow-leaved sedge embryos were classified according to maturity, and the immature or semi-mature embryos were inoculated with MS+BA0.25mg / L+NAA 0.5mg / L+GA 3 0.6mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 induction germination medium ①; mature embryos were inoculated into MS+BA 0.5mg / L+NAA 0.5mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 induction germination medium ②, 1 seed was inoculated into each bottle, the culture conditions were temperature 25±3℃, dark culture;

[0100] (5) Seedling cultivation:

[0101] After culturing the embryos of S. angustifolia in the germination induction medium for 15 days, they were transferred into a culture bottle containing 1 / 2MS+IAA 2.0mg / L+IBA 1.5mg / L+15g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 seedling medium, culture conditions were 25±3℃, light exposure time of 12-15h / d, light intensity of 1500-3000lx;

[0102] (6) Hardening and transplanting

[0103] Move the tissue culture seedlings after seedling cultivation outdoors, open the bottle cap after 6 days of hardening under natural light, continue hardening for 2 days, take out the seedlings, wash off the culture medium, and transplant them to a seedling medium mixed with peat soil: red heart soil: mushroom residue in a volume ratio of 4:4:2. Cover with a shade net with a shade degree of 85% within 7 days after transplanting, and then carry out conventional seedling management;

[0104] (7) Germplasm preservation

[0105] The tissue culture seedlings after induced germination culture were transferred into a preservation medium of 2 / 3MS+BA 0.1mg / L+IAA 0.5mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0. The culture medium was put into a 270mL PC plastic bottle with a sealing cap of 70mm in diameter × 110mm in height. Five strains were inoculated in each bottle. The bottle mouth was wrapped with plastic wrap twice and replaced with new preservation medium every 35 days. When it was needed to be used, it was transferred into a culture bottle of seedling culture medium. After 25 days of culture, the seedlings could be directly hardened and transplanted.

[0106] Comparative Example 1:

[0107] A method for utilizing recalcitrant seeds of the critically endangered plant Psoralea corylifolia, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation. The specific operation steps are as follows:

[0108] The steps of this example are basically the same as those of Example 1, except that some steps of steps (2) and (3) have been adjusted. In step (2), during seed pretreatment, only the wings on both sides of the narrow-leaved sedge seeds are peeled off, and after the debris on the surface of the seeds is washed with clean water, they are transferred to the clean bench; in step (3), during seed sterilization, the clean bench is opened, and the seeds are soaked in 75% alcohol for 1 minute on the clean bench, and the seeds are placed in a 0.1% HgCl 2 The seed coat was removed by stirring and soaking in a sterile solution mixed with the solution and 1 drop of Tween 20 for 15 minutes, repeated once, and then rinsed with sterile water for 3 to 5 times. The seed coat was removed with a scalpel to obtain sterilized embryos.

[0109] Comparative Example 2

[0110] A method for utilizing recalcitrant seeds of the critically endangered plant Psoralea corylifolia, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation. The specific operation steps are as follows:

[0111] The steps of this example are basically the same as those of Example 1, except that the only difference is the different formula of the germination induction medium used in step (4) for inducing germination, that is, the immature and semi-mature embryos and mature embryos are all inoculated into MS+BA0.5mg / L+NAA 0.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0 inducing germination medium ②.

[0112] Comparative Example 3

[0113] A method for utilizing recalcitrant seeds of the critically endangered plant Psoralea corylifolia, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation. The specific operation steps are as follows:

[0114] The steps of this example are basically the same as those of Example 1, except that the only difference is that the germination induction medium used in step (4) is different, that is, the immature and semi-mature embryos and mature embryos are inoculated with MS+BA0.25mg / L+NAA 0.25mg / L+GA 3 0.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8~6.0 in induction germination medium①.

[0115] Comparative Example 4

[0116] A method for utilizing recalcitrant seeds of the critically endangered plant Psoralea corylifolia, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation. The specific operation steps are as follows:

[0117] The steps of this example are basically the same as those of Example 1, except that the seedling medium formula used in step (5) seedling culture is different. In the seedling medium formula of Comparative Example 4, MS is used as the basic medium, and the hormone is a combination of NAA and IBA, i.e., MS+NAA 1.5 mg / L+IBA 1.5 mg / L+30 g / L sucrose+3.8 g / L agar powder, pH 5.8-6.0.

[0118] Comparative Example 5

[0119] A method for utilizing recalcitrant seeds of the critically endangered plant Psoralea corylifolia, comprising seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation. The specific operation steps are as follows:

[0120] The steps of this example are basically the same as those of Example 1, except that the only difference is the different preservation medium formula used in step (7) for germplasm preservation. Comparative Example 5 The preservation medium formula directly uses the germination induction medium, that is, the immature or semi-mature embryos are treated with MS+BA 0.25mg / L+NAA 0.25mg / L+GA 3 0.8mg / L+30g / L sucrose + 3g / L agar powder, mature embryos use MS+BA 0.5mg / L+NAA 0.8mg / L+30g / L sucrose + 3g / L agar powder, pH 5.8~6.0.

[0121] Implementation effect:

[0122] The seeds of Scutellaria baicalensis were utilized by the treatment methods of Examples 1 to 3 and Comparative Examples 1 to 5 within the protection scope of the present invention, and the utilization effects of the seeds treated with different methods were statistically compared.

[0123] 1. The seed utilization effects of the three embodiments are shown in Table 1.

[0124] Table 1: Example seed utilization effect

[0125]

[0126] As can be seen from Table 1, Example 1 is the optimal example, with the lowest contamination rate after seed treatment and sterilization, the highest germination rate of seeds of different maturity in the same category, the highest seedling rate, seedling rate, and transplanting survival rate, the lowest 60d brown death rate during storage and cultivation, and the longest storage time.

[0127] 2. Comparison of seed sterilization effects

[0128] Seed sterilization is a key step in seed utilization in the form of culture medium. Bacteria can contaminate seeds and prevent them from germinating and growing normally, thus seriously affecting the utilization rate of seeds. The feasibility of evaluating the seed sterilization scheme depends mainly on the contamination rate and the start-up rate. From Comparative Example 1, it is mainly investigated whether the steps added by the present invention in the seed sterilization scheme, such as cleaning solution immersion, gauze wrapped fine sand, tea tree essential oil aqueous solution immersion, alcohol lamp burning, ultraviolet light irradiation and increasing the temperature of sterilizing water, can help reduce the sterilization rate while increasing the start-up rate. When conducting the seed sterilization scheme screening experiment, the author compared each step one by one. In order to more intuitively show the effect of the sterilization scheme of the present invention, Comparative Example 1 adopted the more conventional sterilization steps reported in the literature, and compared the sterilization effect with Example 1. Sterilization effect The comparison of sterilization effect is shown in Table 2.

[0129] Table 2: Comparison of sterilization effects between comparative example 1 and example 1

[0130] deal with Contamination rate (%) Start-up rate (%) Example 1 9.94 89.25 Comparative Example 1 36.66 62.08

[0131] As shown in Table 2, the contamination rate of Comparative Example 1 increased by 26.72% and the start-up rate decreased by 27.17% compared with Example 1. The comparison of the results of Comparative Example 1 and Example 1 proves that this series of innovative combined measures significantly reduced the contamination rate and increased the start-up rate. The role of each step is analyzed as follows: before the seeds are sterilized, 5g of flour is added to 1000mL of clean water to prepare a cleaning solution to clean them. The flour particles are relatively fine and have a certain adsorption capacity. At the same time, the flour aqueous solution has a certain viscosity, which can clean the dirt adhering to the surface of the seed coat; the sterilized fine sand is wrapped in gauze. On the one hand, it increases the friction on the seed coat and promotes germination. On the other hand, it is conducive to sterilization and can avoid direct contact of alcohol with seeds to damage vitality; when the seeds are sterilized, they are soaked in 75% alcohol and then briefly burned by flame, which can avoid alcohol residue on the surface. At the same time, the short heat treatment can inactivate some bacteria; after the seeds are soaked in the sterilization solution, they are soaked in 35-40℃ sterilized water. The sterilization water at a certain temperature can achieve the effect of activating seed vitality while washing away the sterilization solution, further promoting germination. The invention obtains an optimal sterilization scheme suitable for sterilizing seeds of Scutellaria angustifolia through a large number of experimental screenings.

[0132] 3. Comparison of induced germination effects

[0133] Comparative Examples 2 to 3 mainly investigate whether seeds of different maturity can be induced to germinate using the same culture medium formula. The comparison of the induced germination effects of each example is shown in Table 3.

[0134] Table 3: Comparison of germination induction effects between comparative examples 2 to 3 and example 1

[0135] deal with Immature and semi-mature seeds germination rate (%) Germination rate of mature seeds (%) Example 1 88.23 92.21 Comparative Example 2 52.16 90.48 Comparative Example 3 86.19 68.30

[0136] As can be seen from Table 3, different culture media are suitable for inducing germination of seeds of different maturity. When the immature and semi-mature seeds were cultured with the induction germination medium suitable for mature seeds, the germination rate of the immature and semi-mature seeds was only 52.16%, which was a decrease of 36.07%, significantly lower than 88.23% in Example 1. Similarly, when the mature seeds were cultured with the induction germination medium suitable for immature and semi-mature seeds, the germination rate of mature seeds was only 68.30%, which was a decrease of 23.91%, significantly lower than 92.21% in Example 1. The reason for this may be that the seeds have different degrees of maturity, and the levels of endogenous hormones contained in them vary greatly, and the types and concentration levels of hormones that promote germination are different. The difference between induction germination medium ① and ② is that the types and concentrations of cytokinins are different. An appropriate amount of GA is added to the induction germination medium for immature and semi-mature seeds. 3 It is beneficial to promote its germination, and the endogenous hormone GA in mature seeds 3 The level may be higher, when GA is added 3In the germination induction medium, the germination of seeds was inhibited. Therefore, different medium formulas should be used for seeds of different maturity of Scutellaria angustifolia. The present invention designs different germination induction mediums for seeds of different maturity, screens and optimizes them respectively, and obtains two germination induction medium formulas suitable for seeds of different maturity.

[0137] 4. Comparison of the effects of seedling culture medium

[0138] The comparison of the effects of basic culture medium and hormone types on seedling formation in the seedling culture medium is shown in Table 4.

[0139] Table 4 Comparison of seedling effects between comparative example 4 and embodiment 1

[0140] deal with Seedling rate (%) Example 1 84.59 Comparative Example 4 36.66

[0141] Comparative Example 4 mainly investigates the effects of the basic culture medium and hormone types in the seedling culture medium on the seedling effect. In the seedling culture medium of Comparative Example 4, MS is selected as the basic culture medium, and in Example 1, 1 / 2MS is the basic culture medium. The difference between the two lies in the content of a large number of elements, because the formation of roots does not require a large amount of nutrition, and the method of reducing the content of nutrient elements can prompt plants to convert from passive absorption of nutrients to active absorption of nutrients to promote root growth. A high concentration of inorganic salts is not conducive to the formation of initial root cells and easily leads to cell dehydration. In addition, another key factor affecting rooting is the content of auxin. Adding auxin of appropriate types and concentrations to the culture medium can further promote the formation of root meristems. The present invention uses a large number of experimental screenings to find that the main factor affecting seedling formation is the basic culture medium, and the best basic culture medium is 1 / 2MS. The rooting nutrient requirements of Scutellaria serrata are in line with the laws of most plants. When screening the types of auxin, the more commonly used NAA, IBA and IAA were selected. Generally speaking, NAA is more stable than IAA. In the screening experiment of the present invention, it was found that IBA was the main factor affecting seedling formation, followed by IAA. The effect of NAA+IBA was significantly lower than that of IAA+IBA. The reason for this may be that different plants have different sensitivities and reaction mechanisms to plant hormones, and IAA is more suitable for Scutellaria serrata. In order to more intuitively demonstrate the effects of the basic culture medium and the growth hormones NAA and IAA on the seedling effect, comparative experiments were conducted between Comparative Example 4 and Example 1. The statistical results are shown in Table 4. It can be seen from Table 4 that the seedling rate of Comparative Example 4 is only 36.66%, which is significantly lower than the seedling rate of Example 1 of 84.59%. Therefore, the formula of the seedling culture medium for Scutellaria serrata uses a combination of 1 / 2MS+IAA+IBA.

[0142] 5. Comparison of preservation culture medium effects

[0143] The comparison of the effects of preservation medium on preserving germplasm resources is shown in Table 5.

[0144] Table 5 Comparison of seedling effect between comparative example 5 and embodiment 1

[0145] deal with 60d brown mortality rate (%) Maximum storage time (d) Transplant survival rate after recovery and seedling formation (%) Example 1 7.91 93.31 80.18 Comparative Example 5 58.94 45.28 62.16

[0146] Comparative Example 5 mainly investigates whether the germination induction medium can be directly selected during storage culture. The storage medium selected in Example 1 is 2 / 3MS+BA 0.2mg / L+IAA 0.5mg / L+30g / L sucrose+3.8g / L agar powder, and the storage medium selected in Comparative Example 5 is MS+BA 0.25mg / L+NAA 0.25mg / L+GA 3 0.8mg / L+30g / L sucrose+3.8g / L agar powder and MS+BA 0.5mg / L+NAA 0.8mg / L+30g / L sucrose+3.8g / L agar powder are the optimal germination induction medium for immature seeds, semi-mature seeds and mature seeds respectively. The purpose of preserving the medium is to keep the seedlings in a state of minimum vitality, that is, to delay their growth to a certain extent, but not to lose their vitality. Reducing the concentration of inorganic salts and hormone levels is conducive to delaying their growth, and an appropriate combination of cytokinins and auxins is conducive to maintaining their normal vitality. Table 5 shows the comparison of the seedling effect of Comparative Example 5 and Example 1. It can be seen from Table 5 that when the brown death rate of 60d is counted, Comparative Example 5 is as high as 58.94%, which is significantly higher than 7.91% of Example 1. The longest storage time of Comparative Example 5 is only 45.28 days, which is significantly lower than 93.31 days of Example 1. Moreover, the survival rate of seedling transplantation after the storage of Comparative Example 5 is only 62.12%, which is 18.06% lower than 80.18% of Example 1. It can be seen that the narrow-leaved sedge induced germination medium cannot be used as a preservation medium to preserve germplasm resources. The reason for the analysis may be that its nutritional conditions are too good, the hormone level is high, and the rapid growth of plants is stimulated to consume nutrients, resulting in nutritional deficiencies in the later stage. Therefore, screening and optimizing the preservation medium suitable for narrow-leaved sedge to maintain the minimum growth is conducive to reducing the brown death rate and extending its preservation time. The preservation medium screened by the present invention can delay growth within a certain period of time, extend the preservation time, and thus improve the utilization rate of seeds.

[0147] The above results analysis can prove that the utilization effect of Example 1 is the best, which also shows that the utilization method disclosed in the present invention can maximize the utilization of immature, semi-mature and mature seeds that have not yet lost water and cannot germinate into seedlings under natural conditions. Under the condition of a certain number of seeds, it promotes more seeds to be converted into seedlings; it realizes the in vitro preservation of narrow-leaved sedge seeds for a certain period of time, breaks the limitation that seed germination and utilization must be in the season when the seeds are mature and harvested, significantly improves the utilization rate of seeds, provides sterile materials for long-term preservation of germplasm resources and large-scale breeding, and lays a technical foundation for the protection of species, restoration of ecology and development and utilization of resources, which has great practical significance for saving this endangered tree species.

Claims

1. A method for improving the utilization rate of recalcitrant seeds of the critically endangered plant Psoralea corylifolia, characterized in that: It includes seed collection, seed pretreatment, seed sterilization, germination induction, seedling cultivation, seedling hardening and transplanting, and germplasm preservation; the specific operation steps are as follows: (1) Seed collection: Collect fresh seeds free of pests and diseases in the natural distribution area of ​​S. angustifolia; (2) Seed pretreatment: Remove the wings on both sides of the seeds of the narrow-leaved sedge, wash the surface of the seeds with clean water, stir and soak them with cleaning liquid for 5 minutes, wash off the cleaning liquid with pure water, wrap the seeds and sterilized fine sand with gauze, soak them in a 2% Australian tea tree essential oil aqueous solution for 20 minutes, stir and soak them with sterilized water for 3-5 minutes, and transfer them to a clean bench; (3) Seed sterilization: Open the clean bench, soak the seeds in 75% alcohol for 1 minute, burn with an alcohol lamp for 5 seconds, open the gauze, wash the seeds to remove fine sand, put them in an inoculation plate with sterilized filter paper, turn on the ultraviolet light of the clean bench for 30 minutes, put the seeds in the sterilization solution, stir and soak for 10 to 15 minutes, repeat once, and then soak them in 35 to 40°C sterilized water for 3 to 5 times, peel off the seed coat with a scalpel to obtain sterilized embryos; (4) Inducing germination: The sterilized embryos were inoculated into the germination induction medium according to their maturity. Immature and semi-mature embryos were inoculated into the germination induction medium ①, and mature embryos were inoculated into the germination induction medium ②. One embryo was inoculated into each bottle. The culture conditions were 25±3℃ and dark culture. (5) Seedling culture: After the embryos of S. angustifolia are cultured in the germination induction medium for 10 to 15 days, they are transferred to the culture bottles of the seedling medium. The culture conditions are as follows: the temperature is 25±3°C, the light duration is 12 to 15 hours / day, and the light intensity is 1500 to 3000 lx; (6) Hardening and transplanting Move the tissue culture seedlings after seedling cultivation outdoors, open the bottle cap after hardening under natural light for 5 to 7 days, continue hardening for 1 to 3 days, take out the seedlings, wash off the culture medium, transplant them into the seedling medium, cover them with a shade net with a shade degree of 75 to 85% within 7 days after transplanting, and then carry out conventional seedling management; (7) Germplasm preservation Transfer the tissue culture seedlings after induced germination culture into the preservation medium. The culture medium is put into a 270mL PC plastic bottle with a sealed cap of 70mm in diameter and 110mm in height. Inoculate 5 plants in each bottle. Wrap the bottle mouth with plastic wrap twice. Replace it with new preservation medium every 30 to 40 days. When it is needed, transfer it to a culture bottle of seedling culture medium. After 25 days of culture, it can be directly hardened and transplanted.

2. The method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium according to claim 1, characterized in that: The seed collection in step (1) includes collecting seeds on the tree and picking up fallen seeds.

3. The method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium according to claim 1, characterized in that: The fresh seeds in step (1) include immature, semi-mature and mature seeds that have not yet lost water, wherein the seed coat and seed wings of immature seeds are all green; the seed coat of semi-mature seeds is partially yellow-green, and the seed wings are dark yellow; the seed coat of mature seeds is all yellow-green, and the seed wings are dark yellow or brown.

4. The method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium according to claim 1, characterized in that: The cleaning solution in step (2) is 5 g of flour added to 1000 mL of clean water.

5. The method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium according to claim 1, characterized in that: In step (3), sterile water should be poured into the sterile filter paper in the inoculation plate to a depth of 1 cm.

6. The method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium according to claim 1, characterized in that: The sterilizing solution in step (3) is a mixture of a 0.1% HgCl2 solution and 1 drop of Tween 20.

7. The method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium according to claim 1, characterized in that: The formula of the induction germination medium ① in step (4) is MS+BA 0.25mg / L+NAA 0.25-0.5mg / L+GA3 0.5-0.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0; the formula of the induction germination medium ② is MS+BA 0.5mg / L+NAA 0.4-0.8mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.

0.

8. The method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium according to claim 1, characterized in that: The seedling culture medium formula of step (5) is 1 / 2MS+IAA 1.5-2.0mg / L+IBA 1.5-2.0mg / L+15g / L sucrose+3.8g / L agar powder, pH 5.8-6.

0.

9. The method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium according to claim 1, characterized in that: The seedling culture medium in step (6) is a mixture of peat soil, red heart soil and mushroom residue in a volume ratio of 4:4:

2.

10. The method for improving the utilization rate of seeds of the critically endangered plant Lepidium angustifolium according to claim 1, characterized in that: The preservation medium formula of step (7) is 2 / 3MS+BA 0.1-0.2mg / L+IAA 0.5mg / L+30g / L sucrose+3.8g / L agar powder, pH 5.8-6.0.

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