CN108371179B - A kind of ultra-low temperature preservation method of golden and silver honeysuckle shoot tips - Google Patents

Abstract

The invention relates to a method for ultra-low temperature preservation of the shoot tips of honeysuckle, which sequentially includes: peeling and pretreatment, loading, dehydration, liquid nitrogen storage, thawing and recovery culture of the shoot tips of honeysuckle. The invention can maintain its excellent characteristics, is convenient to operate, and has strong feasibility, and is beneficial to the preservation of excellent germplasm resources. The genetic material of the shoot tips is relatively stable after cryopreservation, and most of them can directly form complete plantlets, so it has important application value in the preservation of golden and silver honeysuckle germplasm resources and other related research.

Description

A kind of ultra-low temperature preservation method of golden and silver honeysuckle shoot tips

technical field

The invention relates to a long-term preservation method for shoot tips, in particular to an ultra-low temperature preservation method for the shoot tips of honeysuckle.

Background technique

Golden and silver honeysuckle, alias golden and silver wood, belongs to the genus of honeysuckle family, and is one of the good ornamental shrubs in my country. The bark often has irregular columns, and the color is gray-white to gray-brown. Honeysuckle flowers are axillary, each 2 small flowers are on a total flower stalk, the single flower is a lip-shaped corolla, the flowers are white during the opening period, and gradually turn pale yellow after opening, blooming layer by layer, gold and silver. The fruits are red berries, often clustered on long branches, and ornamental in autumn, with numerous red fruits. Honeysuckle flowers are elegant and fragrant, and it is an excellent nectar source tree species; Honeysuckle has strong adaptability to the environment, likes light, tolerates half shade, is cold and drought tolerant, and is simple to manage; the fruit, leaves and flowers of Honeysuckle are rich in fat, Carotenoids and vitamin C; honeysuckle fruit polysaccharide content is high and fruit polysaccharide is a natural antioxidant, which has development and utilization value in medical, food and cosmetic industries; the whole plant of honeysuckle has medicinal value, can Enhance immunity, have the effect of clearing heat and detoxifying, among which the juice of leaves can kill cotton aphids, etc., and the flowers can be used as honeysuckle. Therefore, honeysuckle has high application value in ornamental, industrial and commercial and medicinal purposes. Because the cryopreservation of the stem tips of the honeysuckle can maintain the genetic stability of the honeysuckle, in order to avoid the extinction of the gold and silver honeysuckle germplasm resources caused by changes in the external climate and human overexploitation, the stem tips can be used for cryopreservation. , so as to maintain its excellent properties.

Ultra-low temperature preservation refers to a complete set of biological materials that can be safely stored for a long time in a low temperature of -196 ℃ liquid nitrogen through certain preservation treatments. technology. During this process of change, the chemical composition in the cells of the preservation material does not change substantially, only the physical structure changes, and this change is reversible, so it will not effectively damage the biological activity of the cells, so After the plant material is thawed, the normal activity and genetic stability of ordinary cells can still be maintained, so as to effectively and safely preserve plant germplasm resources for a long time. Due to the small degree of differentiation of meristem cells at the shoot tip, the genetic material is stable compared to other materials during the regeneration process after cryopreservation, and most of them can directly form complete plantlets, which can greatly reduce the possibility of plant variation, especially It is aimed at some plant species through vegetative propagation and some plant types that are prone to variation, and the shoot tip is the most ideal cryopreservation material.

The advantages of cryopreservation using shoot tips are: strong genetic stability, maintaining its excellent characteristics, convenient operation, and strong feasibility, which is conducive to the preservation of germplasm resources.

At present, the cryopreservation of the shoot tips of honeysuckle has not yet been reported. Therefore, it is very necessary to develop a method suitable for cryopreservation of the stem tips of honeysuckle.

SUMMARY OF THE INVENTION

The object of the present invention is to realize through the following technical solutions:

The invention relates to a method for ultra-low temperature preservation of the shoot tips of honeysuckle, which sequentially includes: peeling and pre-cultivation, loading, dehydration, liquid nitrogen storage, thawing and recovery culture of the shoot tips of honeysuckle.

Preferably, the peeling and pre-cultivation refers to: putting the peeled shoot tips into a pre-culture solution, and in the pre-culture solution, an MS solution with an optimum concentration of sucrose of 0.3 mol/L is pre-treated at 4°C. Cultivate 3d.

Preferably, the loading specifically refers to: transferring the pre-cultured isolated shoot tips into the LS loading solution, and loading at room temperature 25°C for 10-50 min; the loading solution is composed of: 2M glycerol+ 0.4mol/L sucrose+MS solution.

Preferably, the dehydration specifically refers to: putting the loaded shoot tips into a PVS2 protective solution for 20-100 min at 0°C; the PVS2 protective solution is composed of: 30% glycerol+15% ethylene glycol Alcohol+15% dimethyl sulfoxide+0.4mol/L sucrose+MS solution.

Preferably, the storage in liquid nitrogen refers to: replacing the shoot tips treated with the PVS2 protection solution with fresh PVS2 protection solution, and then quickly putting them into liquid nitrogen and storing for 24 hours.

Preferably, the thawing refers to: taking out the cryovial stored in liquid nitrogen for thawing, and the thawing method is: thawing the shoot tips in a 40°C water bath for 70s.

Preferably, the recovery culture specifically refers to: inoculating the shoot tips after washing with the unloading solution for 20 min on the recovery medium for recovery culture; the unloading solution component is: 1.2mol/L sucrose+MS solution ; The recovery medium is: MS+KT0.5mg/L+NAA0.1mg/L+GA3 1.0mg/L; The recovery culture conditions are: first in a 25°C incubator for 14d dark culture, then transfer to Incubate under normal light.

Compared with the prior art, the present invention has the following beneficial results:

The method for ultra-low temperature preservation of golden and silver honeysuckle shoot tips of the present invention is a method for preserving fine germplasm resources with high stability, safety, effectiveness, simplicity and practicability, and the survival rate of shoot tips after preservation can be as high as 73%; Stem tips of sterile seedlings of honeysuckle are cryopreserved materials, which have unique advantages. Due to the small degree of differentiation of shoot tips meristem cells, the genetic material is stable compared to other materials during the regeneration process after cryopreservation, and the Parts can directly form complete plantlets, greatly reducing the possibility of plant variation, so it is the most ideal cryopreservation material.

Description of drawings

Fig. 1 is the influence of the sucrose concentration of the pre-culture solution in the embodiment of the present invention 2 on the survival rate of the shoot tip after the ultra-low temperature preservation of honeysuckle

Fig. 2 is the effect of pre-cultivation time on the survival rate of shoot tips after ultra-low temperature preservation of honeysuckle in Example 2 of the present invention

Fig. 3 is the effect of loading solution treatment time on the survival rate of shoot tips after cryopreservation of honeysuckle in Example 3 of the present invention

Fig. 4 is the effect of PVS2 protective solution treatment time on the survival rate of shoot tips after ultra-low temperature preservation of honeysuckle in Example 4 of the present invention

Fig. 5 is the effect of different thawing methods on the survival rate of the shoot tips after cryopreservation of honeysuckle in Example 5 of the present invention

Fig. 6 is the effect of the recovery medium on the survival rate of the shoot tip after ultra-low temperature preservation of honeysuckle in Example 6 of the present invention

Fig. 7 is the flow chart of the ultra-low temperature preservation system of the honeysuckle shoot tip in the present invention; 1 obtains sterile seedlings through the stem segment; 2 the tip of the honeysuckle shoot that is transferred to the recovery medium after ultra-low temperature preservation; Shoot tip; 4 shoot tip cultured for about one month after cryopreservation; 5-7 small seedlings formed from the shoot tip of honeysuckle after cryopreservation; 8-9 Rooting of regenerated seedlings from the shoot tip of gold and silver honeysuckle after cryopreservation; 10- 12 Transplantation of regenerated seedlings of golden and silver honeysuckle after cryopreservation

Detailed ways:

The present invention will be further described in detail below with reference to the examples. The present example is implemented on the premise of the technical solution of the present invention, and provides a detailed implementation manner and a specific operation process.

Example 1

A method for ultra-low temperature preservation of the stem tips of honeysuckle, the specific operations are as follows:

1) Under aseptic conditions, strip off the in vitro shoot tips containing 2 leaf primordia with a shoot tip length of 2 to 3 mm from the sterile stem sections of honeysuckle containing shoot tips;

2) Put the peeled shoot tips into the pre-culture solution for treatment, the MS solution with the optimum concentration of sucrose in the pre-culture solution is 0.3 mol/L, and pre-culture at 4°C for 3 days;

3) The pre-cultured isolated shoot tips are placed in the LS loading solution, and loaded at room temperature 25° C. for 10-50 min; the loading solution is composed of a solution of 2M glycerol+0.4mol/L sucrose+MS;

4) Put the loaded shoot tips into PVS2 protection solution and dehydrate at 0°C for 20-100 min; the PVS2 protection solution is composed of: 30% glycerol+15% ethylene glycol+15% dimethyl sulfoxide +0.4mol/L sucrose+MS solution;

5) The shoot tips treated with the PVS2 protective solution were replaced with fresh PVS2 protective solution, then quickly dropped into liquid nitrogen and stored for 24h;

6) Take out the cryovial stored in liquid nitrogen for thawing, and the thawing method is as follows: thawing the shoot tip with a 40°C water bath for 70s;

7) The recovery culture specifically refers to inoculating the shoot tips after washing with the unloading solution for 20 min on the recovery medium for recovery culture; the unloading solution components are: a solution of 1.2 mol/L sucrose+MS; The recovery medium described is: MS+KT0.5mg/L+NAA0.1mg/L+GA31.0mg/L; the recovery culture conditions are: first in a 25°C incubator for 14d dark culture, then transfer to normal Cultivated under light.

Example 2

Test the effect of sucrose concentration and pre-incubation time in the pre-medium on the cryopreservation of honeysuckle shoot tips: only change the sucrose concentration and pre-incubation time in the pre-medium. (0.1-0.6mol/L) sucrose MS solution for pre-incubation; pre-incubation temperature condition was 4 ℃, incubation time was 0-5 d; and then the survival rate of shoot apex was counted. The research results show that (as shown in Fig. 1 and Fig. 2) after the exfoliated shoot tips were placed in pre-culture solutions with different sucrose concentrations for different time pre-cultures, the survival rates of shoot tips showed different, when the sucrose concentration was 0.3mol /L, when pre-cultured at 4°C for 3 days, the survival rate of shoot apex was up to 60%. It can be seen that pre-culture is the key factor affecting the survival rate of honeysuckle shoot tips in cryopreservation. The optimal pre-culture sucrose concentration is 0.3 mol/L, and the pre-culture time is 3 d.

Example 3

Test the effect of treatment time of LS loading solution on the survival rate of shoot tips after cryopreservation: in a sterile ultra-clean workbench, put the pre-cultured shoot tips into LS loading solution, and load and treat different Time (0, 10, 20, 30, 40, 50 min). As can be seen from the results of the study (as shown in Figure 3), there was a significant difference between the control group treated with the loading solution and the control group without treatment, the shoot apex survival rate without the loading solution treatment was very low, and the shoot apex survival rate was only 3.3%; with the increase of loading time, the survival rate of shoot apex showed a trend of first increasing and then decreasing, reaching a maximum of 63.67% when LS was loaded for 40 min, and was significantly different from the survival rate of shoot apex under other loading times. Differences, when the LS loading time was 50 min, the shoot apex survival rate decreased by 17%, which means that the loading time was too long, which caused damage to the shoot apex cells. Therefore, the survival rate of shoot tips after cryopreservation was significantly improved. Therefore, LS loading for 40min is the best choice.

Example 4

The effect of PVS2 protective solution treatment time on the survival rate of shoot tips after cryopreservation was tested: after the shoot tips were treated with LS loading solution, the shoot tips were placed on sterile filter paper for 5 seconds to remove the residual liquid in the loading solution, and then quickly Put the shoot tip into a cryovial containing PVS2 protective solution, dehydrate it for a certain period of time (0min, 20min, 40min, 60min, 80min and 100min) at 0°C, and then measure the survival rate of the shoot tip; the research results show that (as shown in the figure) 4) when the shoot tip is directly put into liquid nitrogen freezing without going through the step of PVS2 dehydration treatment, the survival rate of the shoot tip is 0, indicating that the shoot tip of the honeysuckle must be subjected to PVS2 dehydration treatment, and when the treatment time is 20min, The survival rate of the shoot tip increased from 0 to 36.7%. When the PVS2 treatment time was 40min, the shoot tip survival rate reached the highest 66.67%, but there was no significant difference with the shoot tip survival rate under the 40min treatment. When the PVS2 treatment time continued to increase, the survival rate of the shoot tip was lower, indicating that the long-term treatment had already caused damage to the shoot tip cells. Although there was no significant difference between 40min and 60min treatment of the shoot tip, in order to avoid the damage of dimethyl sulfoxide to the cells, PVS2 was selected to treat the shoot tip for 40min.

Example 5

The effects of different thawing methods on the survival rate of honeysuckle shoot tips were tested: the cryovials containing the shoot tips that had been dehydrated and replaced with the protective solution were removed from liquid nitrogen and thawed quickly, including thawing in a 40°C water bath. 70 s, tap water for 4 min, and thaw for 20 min at room temperature; the results show (as shown in Figure 5) that the thawing methods of the shoot tips are different, and the survival rates are quite different. The survival rate at this time was the highest, which was 66.67%, which was significantly different from the shoot tips under other thawing methods. When the shoot tip was thawed with tap water for 4 minutes, the survival rate of the shoot tip was not high, which was 26.67%; when the shoot tip was thawed at room temperature at 25 °C, the survival rate of the shoot tip after recovery was 0. The shoot tips were quickly thawed in a 40°C water bath for 70s, which could effectively ensure that the shoot tips could grow normally after recovery.

Example 6

The effect of recovery medium on the survival rate of shoot tips after cryopreservation was tested: the thawed shoot tips were placed on sterile filter paper for 5 seconds to remove the residual liquid of PVS2 protective solution, and then the stems were washed with fresh unloading solution respectively. Tip twice, 10min each time. The shoot tips were then placed on sterile filter paper to remove residual unloading solution, and then the shoot tips were quickly transferred to recovery medium. The unloading liquid component is: 1.2mol/L sucrose+MS solution; the conditions for recovery culture are: culture in dark environment for 2 weeks, then move to normal light for culture at 25°C; The medium formulation in tissue culture of plants of the family, Lonicera genus and Lonicera japonica The recovery medium was set to: MS No. 1; MS No. 2 + 6BA 0.5 mg/L + NAA 0.1 mg/L; MS No. 3 + 6BA2. 0mg/L; No.4 MS+KT0.5mg/L+NAA0.1mg/L+GA31.0mg/L; No.5 MS+KT0.5mg/L+NAA0.2mg/L; shown), the survival rate of shoot tips on MS minimal medium No. 1 was significantly lower than other hormone-supplemented medium. The survival rate of shoot apex reached a maximum of 73.3% after using No. 4 medium, which was 67% higher than that of No. 1 and 43.3% higher than that of No. 2. It can be seen that the combined effect of hormones KT and GA3 It is relatively better than 6-BA. Although the shoot tips were cultured with No. 5, the survival rate was 66.7%, which was not significantly different from No. 4. However, the best choice was made. Therefore, the shoot tips of Lonicera japonica in this experiment should be cultured with No. 4. The base can make the shoot tip can obtain better recovery effect after ultra-low temperature freezing. Then, it was directly cultivated into seedlings by using the stem tip tissue culture system of golden and silver honeysuckle. Among them, proliferation medium: MS+KT0.5mg/L+NAA0.04mg/L; rooting medium: 1/2MS+IBA 0.8mg/L.

The above-mentioned embodiments are only intended to illustrate the technical concept and characteristics of the present invention, and the purpose thereof is to enable those skilled in the art to understand the content of the present invention and implement it, but not to limit the protection scope of the present invention.

Claims (1)

1. a method for ultra-low temperature preservation of honeysuckle stem tip, is characterized in that: the method comprises the steps: (1) Peeling and pre-cultivation of the shoot tip: under aseptic conditions, the peeled shoot tip is put into the pre-culture solution for processing; the peeling of the shoot tip refers to the aseptic honeysuckle containing the shoot tip. The isolated shoot tips with 2-3mm length of shoot tips and containing 2 leaf primordia were stripped from the stem segments; the MS solution with sucrose concentration of 0.3mol/L in the pre-culture solution was pre-cultured at 4°C for 3 days; (2) Loading: transfer the pre-cultured isolated shoot tips into the LS loading solution, and load at room temperature for 40 min; the loading solution is composed of a solution of 2M glycerol+0.4mol/L sucrose+MS; (3) PVS2 dehydration treatment: put the loaded shoot tips into PVS2 protective solution for 40min dehydration treatment at 0°C; the PVS2 protective solution is composed of: 30% glycerol+15% ethylene glycol+15% dimethicone Methyl sulfoxide+0.4mol/L sucrose+MS solution; (4) Liquid nitrogen preservation: replace the shoot tips treated with PVS2 protective solution with new PVS2 protective solution, and then quickly put the cryovial into liquid nitrogen and store for 24h; (5) Thawing: take out the cryovial stored in liquid nitrogen for thawing, and the thawing method is to rapidly thaw in a 40°C water bath for 70s; (6) Recovery culture: transfer the shoot tips after washing with the unloading liquid to recovery medium for recovery culture; the unloading liquid components are: a solution of 1.2mol/L sucrose+MS; the recovery medium is MS+KT0.5mg/L+NAA0.1mg/L+GA31.0mg/L; the recovery culture conditions are: first in a 25°C incubator dark culture for 14d, and then transferred to normal light for culture.

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