阅读说明：本技术 一种真蕨类植物的人工授精的方法 (Artificial insemination method for pteridophyte ) 是由 刘保东 梅红雪 张雪 纪善博 于 2019-08-31 设计创作，主要内容包括：一种真蕨类植物的人工授精的方法,属于植物人工授精技术领域。为了解决蕨类植物成苗率低的问题,本发明提供了一种真蕨类植物的人工授精的方法,将孢子接种至培养基中,用常规孢子培养法得到真蕨类植物的雌、雄配子体,待性器成熟后,吸取成熟的精子和配制好的诱导液依次滴加至雌配子体的成熟颈卵器处,精子在诱导液的引导下游入颈卵器内与卵结合,完成人工授精。本发明提供的取精及人工定向授精技术不仅能使配子体的受精率达到30％-50％,是常规培养条件下的3-5倍,还常常出现单个配子体有两个胚胎发育的现象,并且也使蕨类植物的杂交技术更为简便易行。(An artificial insemination method for pteridophyte belongs to the technical field of artificial insemination of plants. In order to solve the problem of low seedling rate of the fern, the invention provides an artificial insemination method of the fern, spores are inoculated into a culture medium, female and male gametophytes of the fern are obtained by a conventional spore culture method, after sexual organs are mature, mature sperms and prepared inducing liquid are sucked and sequentially dripped to mature cervicovaginal organs of the female gametophytes, and the sperms enter the cervicovaginal organs at the guiding downstream of the inducing liquid to be combined with eggs, so that the artificial insemination is completed. The sperm-taking and artificial directional insemination technology provided by the invention can not only ensure that the fertility rate of the gametophyte reaches 30-50 percent, which is 3-5 times of that under the conventional culture condition, but also ensure that two embryos of a single gametophyte are developed, and the hybridization technology of ferns is simple and easy to implement.)

1. A method of artificial insemination of a pteridophyte, comprising the steps of:

(1) culturing spores: inoculating the spores into a culture medium, and culturing under the conditions of illumination intensity of 1800 plus 4000Lux and illumination time of 12h/d until the sexual organs are mature after being preserved in a dark place, wherein the condition of the preservation in the dark place is that the spores are preserved in the dark place for 12-48h at the temperature of 18-25 ℃;

(2) extracting sperms: selecting mature male gametophytes, sucking out the sperms by using a sperm taking tool when a large amount of sperms are released, and putting the sperms into clear water with the same volume as the sperms to obtain a sperm suspension;

(3) artificial insemination: selecting a mature female gametophyte, placing the female gametophyte for standing for 6-12h under the environment condition that the relative humidity is 30% -40%, dripping an inducing liquid and a sperm suspension when no water film exists around a cervicobrachium apparatus of the female gametophyte, and finishing artificial insemination when the sperm swims into the cervicobrachium apparatus of the female gametophyte.

2. The method of claim 1, wherein the true pteridophyte is pennisetum purpureum or cibotium barometz or allium chinense.

3. The method according to claim 1, wherein the culture medium in step (1) is dark brown soil, humus, agar medium or soil of a pteridophyte habitat.

4. The method as claimed in claim 3, wherein the culture medium in step (1) is further added with hormone or nutrient solution, wherein the hormone is one or more of seminal vesicle essence, gibberellin, abscisic acid, ethylene, etc., and the nutrient solution is one or more of carbohydrate, calcium ion and methionine.

5. The method of claim 1, wherein the density of spore inoculation is 40-80/cm²Obtaining male gametophytes; the inoculation density is 4-8 pieces/cm²Spores of female gametophytes can be obtained.

6. The method according to claim 5, wherein the male gametophyte is cultured by uniformly dispersing spores of the male gametophyte into a culture medium at a seeding density of 60 spores/cm²Placing the gametophyte in a dark environment at 20-25 ℃ for 24h, then placing the gametophyte in an environment with illumination intensity of 2500-3500Lux and illumination time of 12h/d for culture, keeping the relative humidity of air in the culture environment at 65-85%, observing whether sperms appear on the gametophyte every 2d after 50d, and taking sperms when the sperms are mature in a large amount.

7. The method according to claim 5, wherein the female gametophyte is cultured by uniformly scattering spores of the female gametophyte into the culture medium at a seeding density of 6 spores/cm²After inoculation, the culture dish is placed in a porcelain plate and covered by glass, the porcelain dish is placed in a dark environment at the temperature of 20-25 ℃ for 24h, then the porcelain dish is placed in an environment with the illumination intensity of 2500-.

8. The method according to claim 1, wherein the extracting tool in step (2) is a microtube, and the microtube is prepared by the following method: the middle section of a glass tube with the diameter of 0.5-1cm is baked by flame of an alcohol blast burner, the two ends of the glass tube are stretched to stop when the aperture of the finest part of the middle section of the glass tube is 1-3mm, then the glass tube is baked by an alcohol burner, the two ends of the glass tube are continuously stretched until the inner aperture of the finest part is 0-2um, and the part with the aperture of 3-5 mu m is selected to be broken to prepare two micro-tubes which are used as a precision taking tool.

9. The method according to claim 1, wherein the sperm extraction in step (2) is carried out by: the male gametophyte of the mature and dense pteridophyte of spermatid is selected and placed in a culture dish filled with water, the impurities attached to the male gametophyte are cleaned, then the male gametophyte is placed on a glass slide with a groove, distilled water is dripped into the groove to immerse the gametophyte, when the release of a large amount of sperms is observed, a microtube is taken and placed in a sperm-dense area, the sperms are automatically siphoned into the microtube, then the microtube is dripped into distilled water with the same volume, the sperms are sucked out after 3-5s, and a large amount of sperms of the active pteridophyte are obtained after the distilled water is cleaned.

10. The method according to claim 1, wherein the inducing solution in the step (3) is a calcium gluconate aqueous solution with a mass concentration of 1-3%; when artificial insemination is performed, the using amount of the inducing liquid used by each cervicovaginal apparatus is 0.05mL/cm by taking the area of the cervicovaginal apparatus as a reference²0.1ml of the sperm suspension was added.

Technical Field

The invention relates to the technical field of artificial insemination of plants, in particular to an artificial insemination method of pteridophytes.

Background

Nowadays, about 12000 ferns exist on the earth, and about 2600 ferns exist in China. From ancient times to present, pteridophytes occupy a great position in life and production of human beings, and in the industrial aspect, spores of lycopodium clavatum are excellent release agents in the metallurgical industry and can be used for manufacturing rockets, lighting bullets and the like; most fern plants have certain medicinal value, such as Cibotium barometz fern which has the effects of nourishing liver and kidney, dispelling wind-damp and the like, lygodium japonicum can be used for treating urinary tract infection and calculus, and Selaginella tamariscina can be used for treating damp-heat jaundice and edema; the history of consumption of ferns is also quite long: the rhizome of fern is rich in starch, and the young leaves have special fragrance, so the fern has high edible value; in recent decades, the ornamental value of ferns has also been gradually exploited; some threatened ferns are used for scientific research, such as Cyathea spinulosa.

Ferns are a special group of vascular plants fertilized by the combination of motile sperm and eggs. In distinct contrast to the manner in which seed plants are inseminated through pollen tubes. The sperm is a spiral single cell having a length of only about 10 μm, and does not have any protective structure such as a cell wall. The sperm can only swim to the position of the cervicobial apparatus to finish fertilization under the guidance of the gradient concentration of the magnetic substance secreted by the cervicobial apparatus, so whether the sperm smoothly swim into the cervicobial apparatus is not only influenced by intrinsic factors such as the vitality of the fertilized sperm, the service life of the sperm and the like, but also limited by the physicochemical property of water. The magnetic substances secreted by the apparatus of the cervicobia are easily diluted by water, so that the natural fertilization is difficult to complete for fern sperms with the life span of less than half an hour and random swimming directions. The seedling rate of the fern is very low, and the growth vigor of the fern is slow under natural conditions. The propagation technology of the ferns in China is relatively lagged, so that the ferns resources in China are sharply reduced year by year. These reasons drive the need for rapid and efficient propagation of large numbers of ferns. At present, most researches on fern sperms relate to the observation of ultrastructure.

Disclosure of Invention

Aiming at the problem that the seedling rate of pteridophyte is low due to the relative lag of the propagation technology of the pteridophyte at present, the invention provides an artificial insemination method of the pteridophyte, which comprises the following steps:

(1) culturing spores: inoculating the spores into a culture medium, and culturing under the conditions of illumination intensity of 1800 plus 4000Lux and illumination time of 12h/d until the sexual organs are mature after being preserved in a dark place, wherein the condition of the preservation in the dark place is that the spores are preserved in the dark place for 12-48h at the temperature of 18-25 ℃;

(2) extracting sperm by selecting mature male gametophyte, sucking out the sperm by using a sperm extracting tool when the sperm is released in large quantity, and putting the sperm in clear water with the same volume as the sperm to obtain sperm suspension;

(3) artificial insemination, selecting mature female gametophyte, standing for 6-12h under the environment condition that the relative humidity is 30% -40%, dripping induction liquid and sperm suspension when no water film exists around the cervicobrachial apparatus of the female gametophyte, and finishing the artificial insemination when the sperm swims into the cervicobial apparatus of the female gametophyte.

Further limited, the real fern is brush pot tree or golden dog fern or Chinese water leek.

Further limiting, the culture medium is dark brown soil, humus soil, agar culture medium or soil of fern habitat.

More specifically, the culture medium is further added with hormone or nutrient solution, wherein the hormone is one or more of seminal vesicle essence, gibberellin, abscisic acid, ethylene and the like, and the nutrient solution is one or more of carbohydrate, calcium ions and methionine.

Further limiting, the density of spore inoculation is 40-80/cm²Obtaining male gametophytes; the inoculation density is 4-8 pieces/cm²Spores of female gametophytes can be obtained.

Further, the culture method of the male gametophyte is that the spores of the male gametophyte are evenly scattered into the culture medium, and the inoculation density is 60 pieces/cm²Placing the gametophyte in a dark environment at 20-25 ℃ for 24h, then placing the gametophyte in an environment with illumination intensity of 2500-.

Further limiting, the cultivation method of the female gametophyte comprises the steps of uniformly scattering spores of the female gametophyte into a culture medium, wherein the inoculation density is 6 seeds/cm²After inoculation, the culture dish is placed in a porcelain plate and covered by glass, the porcelain dish is placed in a dark environment at the temperature of 20-25 ℃ for 24h, then the porcelain dish is placed in an environment with the illumination intensity of 2500-.

Further limiting, the refining tool in the step (2) is a microtube, and the microtube is prepared by the following method: the middle section of a glass tube with the diameter of 0.5-1cm is baked by flame of an alcohol blast burner, the two ends of the glass tube are stretched to stop when the aperture of the finest part of the middle section of the glass tube is 1-3mm, then the glass tube is baked by an alcohol burner, the two ends of the glass tube are continuously stretched until the inner aperture of the finest part is 0-2um, and the part with the aperture of 3-5 mu m is selected to be broken to prepare two micro-tubes which are used as a precision taking tool.

Further limiting, the specific method for extracting sperm in step (2) is as follows: the male gametophyte of the mature and intensive true pteridophyte of sperm ware is selected under stereomicroscope, arrange in the culture dish that is equipped with water, the impurity that adheres to on the male gametophyte is sanitized, then place on the slide glass of taking the recess, it does not have the gametophyte to drip distilled water in the recess, observe under the stereomicroscope, when observing the release of sperm in a large number, get the microtube and arrange in the sperm intensive district, the automatic siphon of sperm is to the microtube, then drip it to the distilled water of equal volume, treat 3-5s back suction, obtain a large amount of active pteridophyte's sperm after the distilled water washs.

Further limiting, in the step (3), the inducing liquid is calcium gluconate water solution with the mass concentration of 1-3%; when artificial insemination is performed, the using amount of the inducing liquid used by each cervicovaginal apparatus is 0.05mL/cm by taking the area of the cervicovaginal apparatus as a reference²0.1ml of the sperm suspension was added.

Advantageous effects

The artificial insemination method of the invention can not only lead the fertilization rate of the gametophyte to reach 30 percent to 50 percent, which is 3 to 5 times of that under the conventional artificial culture condition, but also lead the phenomenon that two embryos of a single gametophyte develop to be frequently generated, and lead the hybridization technology of the fern to be simpler and more convenient.

Drawings

FIG. 1 shows that the sperms of Cibotium barometz swim to the left and up;

FIG. 2 is a photomicrograph of a fixed, stained and sectioned Goldia tomentosa sperm gushing into the cervicobrachial apparatus;

FIG. 3 shows the development of multiple embryos after artificial insemination of Cibotium barometz;

FIG. 4 shows a sectional rejuvenation of an artificial seedling of Cibotium barometz;

FIG. 5 is a pen container tree sperm group in a non-manipulated state;

FIG. 6 shows the active sperm of the pen container tree;

FIG. 7 shows the sperm of the pen container tree swimming to the right synchronously;

FIG. 8 shows the sperm of the penholder tree swimming upwards in sequence;

FIG. 9 shows an artificial seedling of Chinese water leek;

FIG. 10 shows artificially bred Chinese water leek.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.