High-efficiency propagation method for very small population plant brachypodium brachypodum

Abstract

The invention discloses an efficient propagation method of a very small population plant Berchemia sinica. Relates to the technical field of plant cultivation. Comprising the following steps: collecting seeds; performing airing; removing seed coats; washing is conducted; performing disinfection; inducing sterile buds; rooting induction; and seedling hardening. According to the method, the germination rate of the Bhempinella brachycarpa seeds is remarkably increased, the germination rate reaches 90% or above, as long as embryos of the seeds grow normally, seed collection and seedling raising can be conducted from October every year to the time before the seeds are completely mature and fall off in the next year, the seed collection period is long, the germination rate is high, and the propagation progress and quantity of the Bhempinella brachycarpa are effectively accelerated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant cultivation, more particularly to a high-efficiency propagation method of a plant of the extremely small population of Sinodendron chinense. BACKGROUND

[0002] Sinodendron chinense is a special species in China, and currently only 15 plants are found, which are distributed in coastal areas of Guangxi Zhuang Autonomous Region. Among them, there is one plant in Xiabanzu Village, Nankang Town, Tieshangang District, Beihai City, nine plants in Wutou Village, Jiangping Town, Dongxing City, one plant in Ban'ei Village, one plant in Huangzhu Village, one plant in Dawangjiang Village, and one plant in Waniejin Beach, Fangchenggang City, which are listed in the first batch of extremely small population records announced by the State Forestry Administration in 2011. Sinodendron chinense has difficulty in natural regeneration, and only one seedling is found among the 15 plants, and the others are adult plants.

[0003] Most of the existing Sinodendron chinense can be protected in situ due to the neighboring mountain, but this protection method cannot change the disadvantage that the plant cannot naturally reproduce and propagate, and therefore cannot avoid the fate of extinction.

[0004] Due to the fact that the currently found single plants of Sinodendron chinense are too far apart, the inbreeding propagation probability is the highest under natural conditions by self-pollination or cross-pollination. The offspring produced by such propagation is usually difficult to survive. At present, only one seedling with a diameter at breast height of 2.4 cm is found in the Changshouting of Wutou Village, Jiangping Town, Dongxing City, Fangchenggang City, and it is impossible to realize the ex-situ conservation method.

[0005] Therefore, it is necessary to carry out artificial propagation research to expand the population and promote the development and protection of the population of Sinodendron chinense.

[0006] In order to help expand the population of Sinodendron chinense, seedlings can only be propagated artificially. There are generally two ways of artificial propagation, one is to cultivate seedlings by seeds, and the other is to cultivate clonal seedlings by cutting. The seeds of Sinodendron chinense are few and mostly sterile, and the seedlings are rare, which makes it extremely difficult to restore the population by sexual reproduction. The seed germination rate of Sinodendron chinense is not high, and the seeds are easy to lose activity through seed germination and container seedling tests. The highest survival rate of Sinodendron chinense leaf blade induced callus and cutting propagation test is only 15%, and so far, high-efficiency asexual propagation of Sinodendron chinense has not been truly realized.

[0007] Therefore, whether a high-efficiency propagation method of the extremely small population plant Sinodendron chinense can be provided is a problem that those skilled in the art need to solve urgently. SUMMARY

[0008] Therefore, the present application provides a high-efficiency propagation method of the extremely small population plant Sinodendron chinense. In view of the problem of few seeds of Sinodendron chinense and low efficiency of traditional propagation methods, the seed propagation capacity is improved through plant tissue culture technology, seedlings are efficiently cultivated, and the population quantity is artificially expanded.

[0009] To achieve the above object, the present application adopts the following technical solution:

[0010] A highly efficient propagation method of a plant with a very small population, knee handle wood, comprising the following steps:

[0011] (1) Seed collection;

[0012] (2) Air drying;

[0013] (3) Seed coat removal;

[0014] (4) Rinsing;

[0015] (5) Disinfection: disinfect the seeds, then rinse them;

[0016] (6) Aseptic bud induction: under aseptic conditions, take out the seed embryo from the seeds, inoculate it into an induction medium for culture, and obtain germinated seed embryos;

[0017] (7) Rooting induction: inoculate the germinated seed embryos into a rooting medium for culture, and obtain rooted seedlings;

[0018] (8) Seedling hardening: harden the rooted seedlings.

[0019] Preferably, step (1) seed collection is specifically: seed collection is carried out from October of one year to April of the next year;

[0020] The degree of air drying in step (2) is: place the seeds in a ventilated place for air drying until the outer seed coat naturally cracks;

[0021] Step (3) seed coat removal: remove the inner and outer seed coats of the air-dried fruits, and retain the internal seeds to obtain seed coat-removed seeds;

[0022] Step (4) rinsing: rinse the seed coat-removed seeds under running water until clean.

[0023] Preferably, step (5) disinfection is specifically: disinfect the seeds with 0.12% mercury for 30 minutes, then rinse them with sterile water for 4-5 times.

[0024] Preferably, step (6) is specifically: under aseptic conditions, take out the seed embryo from the seeds, inoculate it into an induction medium, first dark culture for 5-7 days, then light culture for 8-10 days, and the culture temperature is 26-28℃.

[0025] Preferably, the induction medium comprises:

[0026] A inorganic nutrient component:

[0027] (1) macroelements

[0028] ① ammonium nitrate 1650 mg / L;

[0029] ② potassium nitrate 1900 mg / L;

[0030] iii) calcium chloride anhydrous 440 mg / L;

[0031] iv) potassium dihydrogen phosphate 170 mg / L;

[0032] v) magnesium sulfate heptahydrate 370 mg / L;

[0033] (2) Microelements

[0034] i) manganese sulfate monohydrate 22.3 mg / L;

[0035] ii) zinc sulfate heptahydrate 8.6 mg / L;

[0036] iii) boric acid 6.2 mg / L;

[0037] iv) copper sulfate pentahydrate 0.025 mg / L;

[0038] v) sodium molybdate dihydrate 0.25 mg / L;

[0039] vi) cobalt chloride hexahydrate 0.025 mg / L;

[0040] vii) potassium iodide 0.83 mg / L;

[0041] viii) ferrous sulfate heptahydrate 27.8 mg / L;

[0042] ix) sodium ethylenediaminetetraacetate 37.3 mg / L;

[0043] B Organic nutrient components

[0044] i) myo-inositol 100 mg / L;

[0045] ii) nicotinic acid 0.5 mg / L;

[0046] iii) vitamin B6 60.5 mg / L;

[0047] iv) vitamin B10.1 mg / L;

[0048] v) glycine 2 mg / L;

[0049] C Plant growth regulators

[0050] i) cytokinin class 6-benzyladenine 0.4 mg / L;

[0051] ii) auxin class naphthalene acetic acid 0.2 mg / L;

[0052] D White sugar 35 g / L;

[0053] E Agar of strength 1200 3.0 g / L.

[0054] Preferably, step (7) is specifically: the germinated embryo is inoculated into the rooting medium, the light intensity is 1000-4000lx, the temperature is 26-28℃, and the culture is carried out for 25-30 days.

[0055] Preferably, the rooting medium comprises:

[0056] A inorganic nutrient component:

[0057] (1) macroelement

[0058] ① ammonium nitrate 189mg / L;

[0059] ② potassium nitrate 250mg / L;

[0060] ③ anhydrous calcium chloride 210mg / L;

[0061] ④ potassium dihydrogen phosphate 340mg / L;

[0062] ⑤ magnesium sulfate heptahydrate 260mg / L;

[0063] (2) microelement

[0064] ① manganese sulfate monohydrate 22.3mg / L;

[0065] ② zinc sulfate heptahydrate 8.6mg / L;

[0066] ③ boric acid 6.2mg / L;

[0067] ④ copper sulfate pentahydrate 0.025mg / L;

[0068] ⑤ sodium molybdate dihydrate 0.25mg / L;

[0069] ⑥ cobalt chloride hexahydrate 0.025mg / L;

[0070] ⑦ potassium iodide 0.83mg / L;

[0071] ⑧ ferrous sulfate heptahydrate 27.8mg / L;

[0072] ⑨ sodium ethylenediaminetetraacetate 37.3mg / L;

[0073] B organic nutrient component:

[0074] (1) myo-inositol 100mg / L;

[0075] (2) nicotinic acid 0.5mg / L;

[0076] (3) vitamin B 60.5mg / L;

[0077] (4) vitamin B1 0.1 mg / L;

[0078] (5) glycine 2mg / L;

[0079] C plant growth regulator: 0.6 mg / L of green plant growth regulator Shuangji-GGR6 No. 0;

[0080] (1) green plant growth regulator Shuangji-GGR6 No. 0.6 mg / L;

[0081] (2) auxin indole butyric acid 0.4 mg / L;

[0082] D white sugar 20 g / L;

[0083] E agar of strength 1200 3.0 g / L.

[0084] Preferably, step (8) is specifically: placing the rooted seedlings under light intensity of 5000-10000 lx and temperature of 28-30℃, and hardening the seedlings for 25-30 days.

[0085] The application also provides application of any of the above methods in agricultural planting and ecological protection.

[0086] Through the above technical solution, compared with the prior art, the application discloses a high-efficiency propagation method of a small population plant, knee-stemmed wood, which has the following technical effects: the germination rate of knee-stemmed wood seeds is significantly improved by using the propagation method of knee-stemmed wood disclosed by the application, and the germination rate is more than 90%. As long as the seed embryo of the seeds develops normally, the seeds can be collected and seedlings can be cultivated from October of each year to before the seeds completely mature and fall off the next year, which is a period of 7 months. The seed collection period is long, the germination rate is high, and the progress and quantity of knee-stemmed wood propagation are effectively accelerated. DETAILED DESCRIPTION

[0087] The technical solutions in the embodiments of the application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0088] The embodiments of the application disclose a high-efficiency propagation method of a small population plant, knee-stemmed wood.

[0089] In the embodiments, the reagents not mentioned are conventional reagents, and the methods not mentioned are conventional methods, which are not described here again.

[0090] Embodiment 1

[0091] A high-efficiency propagation method of a small population plant, knee-stemmed wood, comprises the following steps:

[0092] (1) seed collection;

[0093] (2) airing;

[0094] (3) removing seed coat;

[0095] (4) washing;

[0096] (5) sterilizing: sterilizing the seeds, then washing;

[0097] (6) inducing germination of aseptic sprout: under aseptic condition, taking out the seed embryo from the seed, inoculating to the inducing medium for culture, and obtaining the germinated seed embryo;

[0098] (7) inducing rooting: inoculating the germinated seed embryo to the rooting medium for culture, and obtaining the rooted seedling;

[0099] (8) hardening: hardening the rooted seedling.

[0100] For further optimization of the technical scheme: the seed collecting in step (1) is specifically as follows: collecting seeds from October to December every year;

[0101] The degree of airing in step (2) is specifically as follows: placing the seeds in a ventilated place for air drying until the outer seed coat naturally cracks;

[0102] The removing seed coat in step (3) is specifically as follows: removing the inner and outer seed coats of the air-dried fruit, and retaining the internal seed to obtain the seed with the seed coat removed;

[0103] The washing in step (4) is specifically as follows: placing the seed with the seed coat removed under running water for washing clean.

[0104] For further optimization of the technical scheme: the sterilizing in step (5) is specifically as follows: sterilizing the seeds with 0.12% mercury for 30 minutes, and then washing with sterile water for 4-5 times.

[0105] For further optimization of the technical scheme: step (6) is specifically as follows: under aseptic condition, taking out the seed embryo from the seed, inoculating to the inducing medium, and first culturing in dark for 5-7 days (6 days in this embodiment), and then culturing in light for 8-10 days (9 days in this embodiment), and the culture temperature is 26-28℃.

[0106] For further optimization of the technical scheme: the inducing medium includes:

[0107] A inorganic nutrient component:

[0108] (1) macroelement

[0109] ① ammonium nitrate 1650mg / L;

[0110] ② potassium nitrate 1900mg / L;

[0111] ③ anhydrous calcium chloride 440mg / L;

[0112] ④ potassium dihydrogen phosphate 170mg / L;

[0113] ⑤ magnesium sulfate heptahydrate 370mg / L;

[0114] (2) Microelements

[0115] ① Manganese sulfate monohydrate 22.3 mg / L;

[0116] ② Zinc sulfate heptahydrate 8.6 mg / L;

[0117] ③ Boric acid 6.2 mg / L;

[0118] ④ Copper sulfate pentahydrate 0.025 mg / L;

[0119] ⑤ Sodium molybdate dihydrate 0.25 mg / L;

[0120] ⑥ Cobalt chloride hexahydrate 0.025 mg / L;

[0121] ⑦ Potassium iodide 0.83 mg / L;

[0122] ⑧ Ferrous sulfate heptahydrate 27.8 mg / L;

[0123] ⑨ Sodium ethylenediaminetetraacetate 37.3 mg / L;

[0124] B Organic nutritional components

[0125] (1) Myo-inositol 100 mg / L;

[0126] (2) Niacin 0.5 mg / L;

[0127] (3) Vitamin B6 60.5 mg / L;

[0128] (4) Vitamin B10.1 mg / L;

[0129] (5) Glycine 2 mg / L;

[0130] C Plant growth regulating substances

[0131] (1) Cell division substances 6-benzyladenine 0.4 mg / L;

[0132] (2) Auxin substances naphthalene acetic acid 0.2 mg / L;

[0133] D White sugar 35 g / L;

[0134] E Agar of strength 1200 3.0 g / L.

[0135] For further optimization of the technical scheme: step (7) is specifically: the germinated seed embryo is inoculated into the rooting culture medium, the light intensity is 1000-4000 lx (in this embodiment, 2500 lx), the temperature is 26-28°C, and the culture is carried out for 25-30 days (in this embodiment, 28 days).

[0136] For further optimization of the technical scheme: the rooting culture medium comprises:

[0137] A Inorganic nutrient components:

[0138] (1) Macroelements

[0139] ① Ammonium nitrate 189 mg / L;

[0140] ② Potassium nitrate 250 mg / L;

[0141] ③ Anhydrous calcium chloride 210 mg / L;

[0142] ④ Monopotassium phosphate 340 mg / L;

[0143] ⑤ Magnesium sulfate heptahydrate 260 mg / L;

[0144] (2) Microelements

[0145] ① Manganese sulfate monohydrate 22.3 mg / L;

[0146] ② Zinc sulfate heptahydrate 8.6 mg / L;

[0147] ③ Boric acid 6.2 mg / L;

[0148] ④ Copper sulfate pentahydrate 0.025 mg / L;

[0149] ⑤ Sodium molybdate dihydrate 0.25 mg / L;

[0150] ⑥ Cobalt chloride hexahydrate 0.025 mg / L;

[0151] ⑦ Potassium iodide 0.83 mg / L;

[0152] ⑧ Ferrous sulfate heptahydrate 27.8 mg / L;

[0153] ⑨ Sodium ethylenediaminetetraacetate 37.3 mg / L;

[0154] B Organic nutrient components:

[0155] (1) Myo-inositol 100 mg / L;

[0156] (2) Niacin 0.5 mg / L;

[0157] (3) Vitamin B6 60.5 mg / L;

[0158] (4) Vitamin B1 0.1 mg / L;

[0159] (5) Glycine 2 mg / L;

[0160] C Plant growth regulators:

[0161] (1) Green plant growth regulator Double Jill-GGR No. 6 0.6 mg / L;

[0162] (2) auxin indole butyric acid 0.4 mg / L;

[0163] D white sugar 20 g / L;

[0164] E agar of strength 1200 3.0 g / L.

[0165] For further optimization of the technical scheme: step (8) is specifically: the rooted seedling is placed under the light intensity of 5000-10000 lx (7500 lx in the embodiment), the temperature of 28-30 ℃, and the seedling raising for 25-30 days (28 days in the embodiment).

[0166] The induction germination rate is more than 90%.

[0167] Example 2

[0168] The difference from example 1 is only that (1) seed collection: seed collection is carried out in January and February, and the rest is the same as example 1.

[0169] Using the above propagation method, the knee-handle wood embryo is collected in the mature period in January and February, most of the endosperm is soft and easy to peel, and some seeds have no embryo or the embryo is poorly developed. The induction germination rate of the normally developed embryo is 95%.

[0170] Example 3

[0171] The difference from example 1 is only that (1) seed collection: seed collection is carried out in March and April, and the rest is the same as example 1.

[0172] Using the above propagation method, the knee-handle wood embryo is collected in the mature period in March and April, most of the endosperm is hard and difficult to peel, and some seeds have no embryo or the embryo is poorly developed. The induction germination rate of the normally developed embryo is 98%.

[0173] Comparative example

[0174] The existing conventional propagation method, the seed collection time is in February and March, and the induction germination rate is only more than 50%.

[0175] The induction germination rate of the example is at least 90%, and the highest is 98%.

[0176] In the specification, each embodiment is described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same and similar parts between each embodiment can be referred to each other.

[0177] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An efficient propagation method for a very small plant population of Psoralea corylifolia, characterized in that: The following steps are involved: (1) Seed collection; (2) Drying; (3) Remove the seed coat; (4) Rinse; (5) Disinfection: Disinfect the seeds and then rinse them; (6) Aseptic bud induction: Under sterile conditions, the embryos in the seeds are removed and inoculated into the induction medium to obtain budding embryos; (7) Rooting induction: The germinated embryos are inoculated into a rooting medium to obtain rooted seedlings; (8) Hardening the seedlings: Hardening the rooted seedlings.

2. The method according to claim 1, wherein The seed collection in step (1) is specifically: collecting seeds from October of each year to April of the following year; The degree of drying in step (2) is as follows: placing the seeds in a ventilated place and drying them in the shade until the outer seed coat naturally cracks; Step (3) of removing the seed coat: removing the inner and outer seed coats of the shade-dried fruit, retaining the inner seeds, and obtaining the seeds with the seed coats removed; The step (4) of rinsing is as follows: the seeds with the seed coats removed are placed under running water and rinsed clean.

3. The method according to claim 2, wherein Step (5) disinfection specifically comprises: disinfecting the seeds with 0.12% mercuric chloride for 30 minutes, and then rinsing with sterile water 4 to 5 times.

4. The method according to claim 3, wherein Step (6) is specifically aseptically removing the embryo from the seed, inoculating it into an induction medium, culturing it in the dark for 5 to 7 days, and then culturing it in the light for 8 to 10 days at a temperature of 26 to 28°C.

5. The method according to claim 4, wherein The induction medium comprises: AInorganic nutrients: (1) Macroelements ①Ammonium nitrate 1650mg / L; ② Potassium nitrate 1900 mg / L; ③Anhydrous calcium chloride 440mg / L; ④ Potassium dihydrogen phosphate 170 mg / L; ⑤Magnesium sulfate heptahydrate 370mg / L; (2) Trace elements ① Manganese sulfate monohydrate 22.3 mg / L; ② Zinc sulfate heptahydrate 8.6 mg / L; ③ Boric acid 6.2 mg / L; ④Copper sulfate pentahydrate 0.025 mg / L; ⑤Sodium molybdate dihydrate 0.25 mg / L; ⑥ Cobalt chloride hexahydrate 0.025 mg / L; ⑦ Potassium iodide 0.83 mg / L; ⑧ Ferrous sulfate heptahydrate 27.8 mg / L; ⑨Sodium ethylenediaminetetraacetate 37.3 mg / L; B Organic Nutritional Ingredients (1) Inositol 100 mg / L; (2) niacin 0.5 mg / L; (3) Vitamin B 60.5 mg / L; (4) Vitamin B1 0.1 mg / L; (5) Glycine 2 mg / L; Plant growth regulators (1) Cytokinin 6-benzyladenine 0.4 mg / L; (2) auxin naphthyl acetic acid 0.2 mg / L; D white sugar 35g / L; E strength 1200 agar 3.0 g / L.

6. The method according to claim 5, wherein Step (7) is specifically as follows: inoculating the germinated embryos into a rooting medium, culturing at a light intensity of 1000 to 4000 lx and a temperature of 26 to 28° C. for 25 to 30 days.

7. The method according to claim 6, wherein The rooting medium comprises: AInorganic nutrients: (1) Macronutrients ①Ammonium nitrate 189mg / L; ② Potassium nitrate 250 mg / L; ③Anhydrous calcium chloride 210mg / L; ④ Potassium dihydrogen phosphate 340 mg / L; ⑤Magnesium sulfate heptahydrate 260mg / L; (2) Trace elements ① Manganese sulfate monohydrate 22.3 mg / L; ② Zinc sulfate heptahydrate 8.6 mg / L; ③ Boric acid 6.2 mg / L; ④Copper sulfate pentahydrate 0.025 mg / L; ⑤Sodium molybdate dihydrate 0.25 mg / L; ⑥ Cobalt chloride hexahydrate 0.025 mg / L; ⑦ Potassium iodide 0.83 mg / L; ⑧ Ferrous sulfate heptahydrate 27.8 mg / L; ⑨Sodium ethylenediaminetetraacetate 37.3 mg / L; B Organic Nutritional Ingredients: (1) Inositol 100 mg / L; (2) niacin 0.5 mg / L; (3) Vitamin B 60.5 mg / L; (4) Vitamin B1 0.1 mg / L; (5) Glycine 2 mg / L; C Plant growth regulators: (1) Green plant growth regulator Shuangjier-GGR6 0.6 mg / L; (2) auxin-type indolebutyric acid 0.4 mg / L; D. White sugar 20g / L; E strength 1200 agar 3.0 g / L.

8. The method according to claim 7, wherein Step (8) is specifically as follows: placing the rooted seedlings under a light intensity of 5000-10000 lx and a temperature of 28-30° C. for 25-30 days.

9. Use of the method according to any one of claims 1 to 8 in agricultural planting and ecological protection.