阅读说明：本技术 北苍术种子的保存方法 (Preservation method of rhizoma atractylodis macrocephalae seeds ) 是由 李志军 贾俊英 庄得凤 于 2021-07-30 设计创作，主要内容包括：本发明公开了一种北苍术种子的保存方法,包括如下步骤：将北苍术种子浸泡到装载液中；将所述北苍术种子从所述装载液中取出后转移到装有玻璃化保护剂的冻存管中,并且将所述冻存管置于0℃～8℃的环境下预冻存15min～120min；以及将预冻存后的所述冻存管取出,更换新鲜且预冷过的所述玻璃化保护剂后,将所述冻存管投入液氮保存。这种北苍术种子的保存方法通过对北苍术种子进行预冻存处理,极大的提高了北苍术种子的保存时间和发芽率,结合实施例部分的数据,这种北苍术种子的保存方法处理过的北苍术种子在保存6个月、12个月和24个月的发芽率,均高于未经过预冻存的北苍术种子。(The invention discloses a method for preserving rhizoma atractylodis macrocephalae seeds, which comprises the following steps: soaking rhizoma atractylodis macrocephalae seeds into the loading liquid; taking out the seeds of the Atractylodes chinensis from the loading solution, transferring the seeds into a cryopreservation tube filled with a vitrification protective agent, and pre-freezing the cryopreservation tube at the temperature of 0-8 ℃ for 15-120 min; and taking out the pre-frozen tube, replacing the fresh and pre-cooled vitrification protective agent, and putting the tube into liquid nitrogen for preservation. According to the preservation method of the rhizoma atractylodis macrocephalae seeds, the preservation time and the germination rate of the rhizoma atractylodis macrocephalae seeds are greatly improved by pre-freezing the rhizoma atractylodis macrocephalae seeds, and by combining the data of the embodiment part, the germination rates of the rhizoma atractylodis macrocephalae seeds treated by the preservation method of the rhizoma atractylodis macrocephalae seeds in 6 months, 12 months and 24 months are higher than those of the rhizoma atractylodis macrocephalae seeds which are not pre-frozen.)

1. A preservation method of rhizoma atractylodis macrocephalae seeds is characterized by comprising the following steps:

soaking rhizoma atractylodis macrocephalae seeds into the loading liquid;

taking out the seeds of the Atractylodes chinensis from the loading solution, transferring the seeds into a cryopreservation tube filled with a vitrification protective agent, and pre-freezing the cryopreservation tube at the temperature of 0-8 ℃ for 15-120 min; and

and taking out the pre-frozen tube, replacing the fresh and pre-cooled vitrification protective agent, and putting the tube into liquid nitrogen for preservation.

2. The method for preserving seeds of Atractylodes chinensis as claimed in claim 1, wherein the vitrification protective agent is MS liquid culture medium containing glycerol, ethylene glycol, dimethyl sulfoxide and sucrose;

in the vitrification protective agent, the mass concentration of glycerol is 25-35%, the mass concentration of ethylene glycol is 10-20%, the mass concentration of dimethyl sulfoxide is 10-20%, and the molar concentration of sucrose is 0.2-1 mol/L.

3. The method for preserving seeds of Atractylodes chinensis as claimed in claim 2, wherein the concentration of glycerol is 28-32%, the concentration of ethylene glycol is 12-18%, the concentration of dimethyl sulfoxide is 12-18%, and the molar concentration of sucrose is 0.3-0.6 mol/L.

4. The method for preserving seeds of Atractylodes chinensis as claimed in claim 2, wherein the pre-freezing of the frozen tube at 0-8 ℃ for 15-120 min comprises: and pre-freezing the freezing tube at the temperature of 2-6 ℃ for 30-60 min.

5. The method for preserving seeds of Atractylodes chinensis as claimed in claim 1, wherein the loading liquid comprises MS liquid culture medium containing glycerol and sucrose;

in the loading liquid, the molar concentration of the glycerol is 1-4 mol/L, and the molar concentration of the sucrose is 0.2-1 mol/L.

6. The method for preserving the seeds of the Atractylodes chinensis as claimed in claim 5, wherein the soaking of the seeds of the Atractylodes chinensis in the carrier liquid is carried out at room temperature for 15-45 min.

7. The method for preserving seeds of Atractylodes chinensis as claimed in any one of claims 1 to 6, further comprising the following steps after the step of putting the freezing tube into liquid nitrogen for preservation and before sowing:

taking the cryopreservation tube out of the liquid nitrogen, then placing the tube in a water bath at 34-50 ℃ for unfreezing, taking out the rhizoma atractylodis macrocephalae seeds in the cryopreservation tube after unfreezing, and then sequentially cleaning the seeds with a cleaning solution and sterile water.

8. The method for preserving seeds of Atractylodes chinensis as claimed in claim 7, wherein the washing solution is MS liquid culture medium containing sucrose;

in the washing liquid, the molar concentration of the sucrose is 0.8-2 mol/L.

9. The method for preserving the seeds of the rhizoma atractylodis macrocephalae as claimed in claim 8, wherein the seeds of the rhizoma atractylodis macrocephalae in the frozen tube are taken out after unfreezing and then washed clean with a washing solution and sterile water in sequence, wherein the washing solution washes the seeds of the rhizoma atractylodis macrocephalae at least twice, and the sterile water washes the seeds of the rhizoma atractylodis macrocephalae at least three times.

10. The method for preserving the seeds of the atractylodes macrocephala koidz as claimed in claim 7, wherein the humidity of the seeds of the atractylodes macrocephala koidz is 8-12%.

Technical Field

The invention relates to the technical field of seed preservation, in particular to a preservation method of rhizoma atractylodis macrocephalae seeds.

Background

The rhizoma atractylodis sinensis is a female flower amphoteric flower variant (gynodeicy) plant, namely, the female flower amphoteric flower variant comprises an amphoteric flower and a parthenocarpic female plant, the setting rate of the parthenocarpic female plant of the rhizoma atractylodis sinensis is obviously higher than that of the amphoteric flower plant, the setting rate of the female flower plant is obviously higher than that of the amphoteric flower plant, but the ratio of the germ-free seeds in the seeds of the two flower plants is more than or equal to 70 percent, the natural setting rate is extremely low, the normal-temperature storage time of the seeds of the rhizoma atractylodis sinensis is only half a year, and the phenomenon causes great obstacle to the sexual propagation of wild resources of the rhizoma atractylodis sinensis.

Traditionally, the seeds of the rhizoma atractylodis macrocephalae are stored by a natural refrigeration method, and the damage of the seeds of the rhizoma atractylodis macrocephalae in the storage process is easily caused by the natural refrigeration storage mode, so that the germination rate is influenced.

Disclosure of Invention

Accordingly, there is a need for a method for preserving seeds of Atractylodes lancea which can improve the germination rate.

A preservation method of rhizoma atractylodis macrocephalae seeds comprises the following steps:

soaking rhizoma atractylodis macrocephalae seeds into the loading liquid;

taking out the seeds of the Atractylodes chinensis from the loading solution, transferring the seeds into a cryopreservation tube filled with a vitrification protective agent, and pre-freezing the cryopreservation tube at the temperature of 0-8 ℃ for 15-120 min; and

and taking out the pre-frozen tube, replacing the fresh and pre-cooled vitrification protective agent, and putting the tube into liquid nitrogen for preservation.

According to the preservation method of the rhizoma atractylodis macrocephalae seeds, the preservation time and the germination rate of the rhizoma atractylodis macrocephalae seeds are greatly improved by pre-freezing the rhizoma atractylodis macrocephalae seeds, and by combining the data of the embodiment part, the germination rates of the rhizoma atractylodis macrocephalae seeds treated by the preservation method of the rhizoma atractylodis macrocephalae seeds in 6 months, 12 months and 24 months are higher than those of the rhizoma atractylodis macrocephalae seeds which are not pre-frozen.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

Wherein:

fig. 1 is a flowchart of a preparation method of a method for preserving rhizoma atractylodis macrocephalae seeds according to an embodiment.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The method for preserving the seeds of the Atractylodes lancea as shown in FIG. 1 comprises the following steps:

s10, soaking the seeds of the rhizoma atractylodis macrocephalae in the loading liquid.

Soaking the seeds of the rhizoma atractylodis macrocephalae in the loading liquid to improve the tolerance of the seeds of the rhizoma atractylodis macrocephalae in the cooling process.

Generally, the plump seeds of Atractylodes lancea should be selected for preservation.

In the embodiment, the humidity of the rhizoma atractylodis macrocephalae seeds is 8% -12%.

In the present embodiment, the loading solution contains an MS liquid medium containing glycerol and sucrose.

Preferably, the loading liquid has a molar concentration of glycerol of 1mol/L to 4mol/L and a molar concentration of sucrose of 0.2mol/L to 1 mol/L.

More preferably, the loading liquid has a molar concentration of glycerol of 2mol/L and a molar concentration of sucrose of 0.4 mol/L.

In this embodiment, the atractylodes lancea seeds are soaked in the carrier liquid at room temperature for 15-45 min (preferably 25 min).

S20, taking the seeds of the Atractylodes lancea out of the loading liquid, transferring the seeds into a cryopreservation tube filled with a vitrification protective agent, and pre-freezing the cryopreservation tube for 15-120 min at the temperature of 0-8 ℃.

The seeds of the rhizoma atractylodis macrocephalae are pre-frozen and stored, so that the seeds of the rhizoma atractylodis macrocephalae can gradually adapt to a low-temperature environment, and the damage to the seeds due to rapid cooling is avoided.

In this embodiment, the vitrification protective agent is an MS liquid medium containing glycerol, ethylene glycol, dimethyl sulfoxide, and sucrose.

Preferably, in the vitrification protective agent, the mass concentration of glycerol is 25-35%, the mass concentration of ethylene glycol is 10-20%, the mass concentration of dimethyl sulfoxide is 10-20%, and the molar concentration of sucrose is 0.2-1 mol/L.

More preferably, the mass concentration of glycerol is 28 to 32%, the mass concentration of ethylene glycol is 12 to 18%, the mass concentration of dimethyl sulfoxide is 12 to 18%, and the molar concentration of sucrose is 0.3 to 0.6 mol/L.

Particularly preferably, the mass concentration of glycerol is 30%, the mass concentration of ethylene glycol is 15%, the mass concentration of dimethyl sulfoxide is 15%, and the molar concentration of sucrose is 0.4 mol/L.

Preferably, the pre-freezing operation of the freezing tube at 0-8 ℃ for 15-120 min comprises: and pre-freezing the frozen tube at 2-6 deg.c for 30-60 min.

And S30, taking out the pre-frozen tube, replacing the fresh and pre-cooled vitrification protective agent, and putting the tube into liquid nitrogen for preservation.

The temperature of the pre-cooled vitrification protective agent is the same as the temperature of pre-freezing storage.

Preferably, the method further comprises the following operations performed after the operation of putting the freezing tube into liquid nitrogen for preservation and before seeding:

taking out the cryopreservation tube from liquid nitrogen, then placing the tube in a water bath at 34-50 ℃ for unfreezing, taking out the rhizoma atractylodis macrocephalae seeds in the cryopreservation tube after unfreezing, and then sequentially cleaning the seeds by using a cleaning solution and sterile water.

The cryopreservation tube is taken out of liquid nitrogen and placed in a water bath at 34-50 ℃ for unfreezing, so that the temperature of the seeds of the Atractylodes chinensis can be quickly raised, the seeds of the Atractylodes chinensis can be quickly raised through water bath treatment, the influence of environmental change on the vitality of the seeds is avoided, and the germination rate of the seeds is ensured.

More preferably, the temperature of the water bath is 40 ℃ and the time of the water bath is 5 min.

After thawing, taking out the seeds of the Atractylodes lancea in the freezing storage tube, cleaning with a cleaning solution, ensuring the cleanliness of the seeds, reducing the influence of chemical substances in the seed germination process,

in this embodiment, the washing solution is an MS liquid medium containing sucrose.

Preferably, the molar concentration of sucrose in the washing solution is 0.8mol/L to 2 mol/L.

More preferably, the molar concentration of sucrose in the washing solution is 1.2 mol/L.

In this embodiment, in the operation of taking out the rhizoma atractylodis macrocephalae seeds in the frozen tube after thawing and sequentially cleaning the rhizoma atractylodis macrocephalae seeds with the cleaning solution and the sterile water, the cleaning solution cleans the rhizoma atractylodis macrocephalae seeds at least twice (the cleaning time is 10min each time), and the sterile water cleans the rhizoma atractylodis macrocephalae seeds at least three times (the cleaning time is 10min each time).

After the washing is finished, the water on the rhizoma atractylodis macrocephalae seeds needs to be sucked to be dry by using filter paper.

According to the preservation method of the rhizoma atractylodis macrocephalae seeds, the preservation time and the germination rate of the rhizoma atractylodis macrocephalae seeds are greatly improved by pre-freezing the rhizoma atractylodis macrocephalae seeds, and by combining the data of the embodiment part, the germination rates of the rhizoma atractylodis macrocephalae seeds treated by the preservation method of the rhizoma atractylodis macrocephalae seeds in 6 months, 12 months and 24 months are higher than those of the rhizoma atractylodis macrocephalae seeds which are not pre-frozen.

The following are specific examples.

In the examples, the loading liquid is MS liquid culture medium containing 2mol/L of glycerol and 0.4mol/L of sucrose, the vitrification protective agent is MS liquid culture medium containing 30 wt% of glycerol, 15 wt% of ethylene glycol, 15 wt% of dimethyl sulfoxide and 0.4mol/L of sucrose, and the washing liquid is MS liquid culture medium containing 1.2mol/L of sucrose.

Example 1

Selecting full rhizoma atractylodis macrocephalae seeds with the humidity of 8% -12%, putting the loading liquid into a freezing tube filled with the rhizoma atractylodis macrocephalae seeds, standing at room temperature for 25min, pouring out the loading liquid, then changing into a vitrification protective agent, standing in a refrigerator at 4 ℃ for 40min, then changing into a fresh vitrification protective agent pre-cooled at 4 ℃, sealing the freezing tube, and quickly putting into liquid nitrogen for storage.

Example 2

Selecting full rhizoma atractylodis macrocephalae seeds with the humidity of 8% -12%, putting the loading liquid into a freezing tube filled with the rhizoma atractylodis macrocephalae seeds, standing at room temperature for 25min, pouring out the loading liquid, then changing into a vitrification protective agent, standing in a refrigerator at 2 ℃ for 30min, then changing into a fresh vitrification protective agent pre-cooled at 2 ℃, sealing the freezing tube, and quickly putting into liquid nitrogen for preservation.

Example 3

Selecting full rhizoma atractylodis macrocephalae seeds with the humidity of 8-12%, putting the loading liquid into a freezing tube filled with the rhizoma atractylodis macrocephalae seeds, standing at room temperature for 25min, pouring out the loading liquid, then changing into a vitrification protective agent, standing in a 6 ℃ refrigerator for 60min, then changing into a fresh vitrification protective agent pre-cooled at 6 ℃, sealing the freezing tube, and quickly putting into liquid nitrogen for preservation.

Comparative example

Selecting full rhizoma atractylodis macrocephalae seeds with the humidity of 8-12%, putting the loading liquid into a freezing storage tube filled with the rhizoma atractylodis macrocephalae seeds, standing at room temperature for 25min, pouring out the loading liquid, then changing into a vitrification protective agent, sealing the freezing storage tube, and quickly putting into liquid nitrogen for storage.

Test example 1

100 pears of the seeds of the Atractylodes chinensis obtained in examples 1, 2 and 3 and the comparative example, which were stored in liquid nitrogen for 6 months, were respectively removed, the seeds of the Atractylodes chinensis were thawed in a water bath at 40 ℃ for 5min, and after thawing, they were washed with a washing solution for 2 times, 10min each, and then washed with sterile water for 3 times, 10min each, and the washed seeds of the Atractylodes chinensis were subjected to a germination test after water was absorbed with filter paper.

The germination test was performed as follows: the four groups of seeds were placed in wet culture dishes, respectively, and the dishes were filled with filter paper, and were cultured in an incubator at 25 to 28 ℃ for 3 days, and the number of sprouts was counted, to obtain table 1 below.

Table 1: table for comparing germination percentage after liquid nitrogen preservation for 6 months

Group of	Example 1	Example 2	Example 3	Comparative example
					Germination rate	94％	92％	91％	89％

Test example 2

100 pears of the Atractylodes chinensis seeds obtained in examples 1, 2 and 3 and the comparative example, which were stored in liquid nitrogen for 12 months, were thawed in a water bath at 40 ℃ for 5min, and after thawing, the Atractylodes chinensis seeds were washed with a washing solution for 2 times, 10min each, and then with sterile water for 3 times, 10min each, and the washed Atractylodes chinensis seeds were subjected to a germination test after water was absorbed with filter paper.

The germination test was performed as follows: the four groups of seeds were placed in wet culture dishes, respectively, and the dishes were filled with filter paper, and were cultured in an incubator at 25 to 28 ℃ for 3 days, and the number of sprouts was counted, to obtain table 1 below.

Table 2: table for comparing germination percentage after 12 months of liquid nitrogen preservation

Group of	Example 1	Example 2	Example 3	Comparative example
					Germination rate	93％	93％	92％	85％

Test example 3

100 pears of the seeds of the Atractylodes chinensis obtained in examples 1, 2 and 3 and the comparative example, which were stored in liquid nitrogen for 24 months, were respectively taken, the seeds of the Atractylodes chinensis were thawed in a water bath at 40 ℃ for 5min, and after thawing, they were washed with a washing solution for 2 times, 10min each, and then washed with sterile water for 3 times, 10min each, and the washed seeds of the Atractylodes chinensis were subjected to a germination test after water was absorbed with filter paper.

The germination test was performed as follows: the four groups of seeds were placed in wet culture dishes, respectively, and the dishes were filled with filter paper, and were cultured in an incubator at 25 to 28 ℃ for 3 days, and the number of sprouts was counted, to obtain table 1 below.

Table 3: table for comparing germination percentage after 24 months of liquid nitrogen preservation

Group of	Example 1	Example 2	Example 3	Comparative example
					Germination rate	88％	85％	86％	76％

In combination with tables 1, 2 and 3 above, it can be seen that the germination rates of the atractylodes macrocephala seeds obtained by the methods of examples 1, 2 and 3 are higher than those of the atractylodes macrocephala seeds obtained by the method of comparative example.

And the germination rate of the atractylodes macrocephala seeds obtained by the methods of examples 1, 2 and 3 is obviously higher than that of the atractylodes macrocephala seeds obtained by the method of comparative example.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.