CN108371179B

CN108371179B - Ultra-low temperature preservation method for stem tips of honeysuckle flowers

Info

Publication number: CN108371179B
Application number: CN201810367569.XA
Authority: CN
Inventors: 宋红; 田晓璇; 李萍; 李享
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2018-04-23
Filing date: 2018-04-23
Publication date: 2020-11-13
Anticipated expiration: 2038-04-23
Also published as: CN108371179A

Abstract

The invention relates to an ultra-low temperature preservation method of stem tips of honeysuckle, which sequentially comprises the following steps: stripping and pretreating stem tips of honeysuckle, loading, dehydrating, preserving by liquid nitrogen, unfreezing and restoring the culture. The invention can keep the excellent characteristics, is convenient to operate and strong in feasibility, and is beneficial to preserving excellent germplasm resources. The stem tip is relatively stable in genetic material after being preserved at ultralow temperature, and most of the genetic material can directly form complete plantlets, so that the method has important use value in the aspects of honeysuckle germplasm resource preservation and other related researches.

Description

Ultra-low temperature preservation method for stem tips of honeysuckle flowers

Technical Field

The invention relates to a stem tip long-term preservation method, in particular to a honeysuckle stem tip ultralow-temperature preservation method.

Background

Lonicera japonica, also known as Lonicera japonica, belongs to the genus Lonicera of the Caprifoliaceae family and is one of the good ornamental shrubs in China. Bark is often in irregular longitudinal columns and is gray-white to gray-brown in color. Honeysuckle flower is axillary, every 2 small flowers are on one total flower handle, a single flower is a labiate crown, the flower is white in the blooming period, the flower gradually becomes faint yellow after blooming, the flowers bloom layer by layer, and the honeysuckle flower is opposite to each other. The fruit is red berry, which usually grows on long branches and is appreciated in autumn, and the red fruit is tired. Honeysuckle flowers are elegant and fragrant, and are good honey source tree species; the honeysuckle has strong environment adaptation capability, is light-loving, half-shading resistant, cold-resistant and drought-resistant, and is simple to manage; fruits, leaves and flowers of lonicera japonica contain abundant fat, carotenoid and vitamin C; the honeysuckle fruit has high polysaccharide content, and the polysaccharide of the fruit is a natural antioxidant, and has development and utilization values in medical treatment, food and cosmetic industries; the honeysuckle stem has medicinal value, can enhance immunity, has the efficacy of clearing away heat and toxic materials, wherein the juice of the leaves can kill cotton aphids and the like, and the flowers can be used as the honeysuckle. Therefore, the honeysuckle has higher application value in the aspects of appreciation, industry and commerce and medicine. Because the genetic stability of the honeysuckle can be maintained by the ultralow temperature preservation of the stem tip of the honeysuckle, the stem tip can be used for the ultralow temperature preservation so as to keep the excellent characteristics of the honeysuckle in order to avoid the extinction of germplasm resources of the honeysuckle caused by the change of outside climate and the excessive development of human beings.

The ultra-low temperature preservation refers to a set of biotechnology which can realize the safe long-term preservation of the preserved material at the low temperature of liquid nitrogen of-196 ℃ through certain preservation treatment, and adopts a certain thawing method to restore the material to the normal temperature and ensure that the material can normally restore the growth when the material is taken out. In the change process, chemical components in the cells of the preservation material are not substantially changed, and only the physical structure is changed, and the change is reversible, so that the biological activity of the cells is not effectively damaged, and the normal activity and genetic stability of common cells can be still maintained after the plant material is thawed, so that the plant germplasm resource can be effectively and safely preserved for a long time. Because the differentiation degree of the meristematic cells of the stem tip is small, genetic materials are stable relative to other materials in the regeneration process after ultralow temperature preservation, most of the genetic materials can directly form complete plantlets, the possibility of plant variation can be greatly reduced, and particularly, the stem tip is the most ideal ultralow temperature preservation material for certain plant types which propagate through nutrition and certain plant types which are easy to generate variation.

The stem tip is used for ultralow temperature preservation, and the method has the advantages that: strong genetic stability, convenient operation, strong feasibility and being beneficial to the preservation of germplasm resources, and can maintain the excellent characteristics of the germplasm resources.

At present, ultra-low temperature preservation of the stem tips of honeysuckle has not been reported yet. Therefore, it is very necessary to develop a method suitable for ultra-low temperature preservation of the stem tip of Lonicera japonica Thunb.

Disclosure of Invention

The purpose of the invention is realized by the following technical scheme:

the invention relates to a method for ultralow temperature preservation of stem tips of honeysuckle, which sequentially comprises the following steps: stripping and pre-culturing the stem tip of honeysuckle, loading, dehydrating, preserving by liquid nitrogen, unfreezing and restoring the culture.

Preferably, said stripping and pre-incubation means: and (3) putting the stripped stem tip into a pre-culture solution for treatment, and pre-culturing the stem tip in an MS solution with the optimal concentration of sucrose being 0.3mol/L at 4 ℃ for 3 d.

Preferably, the loading specifically refers to: transferring the pre-cultured in-vitro stem tip into an LS loading liquid, and loading for 10-50 min at room temperature of 25 ℃; the loading liquid comprises the following components: 2M Glycerol +0.4mol/L sucrose + MS.

Preferably, the dehydration specifically means: putting the loaded stem tip into PVS2 protective solution, and dehydrating for 20-100 min at the temperature of 0 ℃; the PVS2 protective solution comprises the following components: a solution of 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide +0.4mol/L sucrose + MS.

Preferably, the liquid nitrogen preservation refers to: the stem tip treated by the PVS2 protective solution is replaced by fresh PVS2 protective solution, and then is quickly put into liquid nitrogen for storage for 24 hours.

Preferably, the thawing means: taking out the freezing pipe after the liquid nitrogen preservation for defrosting, wherein the defrosting mode is as follows: the stem tip was thawed in a 40 ℃ water bath for 70 s.

Preferably, the recovery culture specifically comprises: inoculating the stem tip washed by the unloading liquid for 20min on a recovery culture medium for recovery culture; the unloading liquid comprises the following components: 1.2mol/L sucrose + MS solution; the recovery culture medium comprises: MS + KT0.5mg/L + NAA0.1mg/L + GA31.0mg/L; the recovery culture conditions are as follows: culturing in 25 deg.C incubator for 14 days, and culturing under normal light.

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

the method for ultralow-temperature preservation of the stem tips of honeysuckle flowers is a good germplasm resource preservation method which is high in stability, safe, effective, simple, convenient and feasible, and the survival rate of the stem tips after preservation can reach 73%; the stem tip of the stem segment of the aseptic honeysuckle seedling is used as an ultralow temperature storage material, the method has unique superiority, and because the differentiation degree of the meristematic cells of the stem tip is small, genetic materials are stable relative to other materials in the regeneration process after ultralow temperature storage, most of the genetic materials can directly form complete plantlets, so that the possibility of plant variation is greatly reduced, and the method is the most ideal ultralow temperature storage material.

Drawings

FIG. 1 shows the effect of sucrose concentration of pre-culture solution on the survival rate of stem tips of Lonicera japonica after cryopreservation in example 2 of the present invention

FIG. 2 is a graph showing the effect of the pre-culture time on the survival rate of the shoot tips of Lonicera japonica after cryopreservation in example 2 of the present invention

FIG. 3 is a graph showing the effect of the treatment time of the loading liquid on the survival rate of the stem tips of Lonicera japonica after being preserved at ultralow temperature in example 3 of the present invention

FIG. 4 shows the effect of the PVS2 protective solution treatment time on the stem tip survival rate of Lonicera japonica after ultralow temperature storage in example 4 of the present invention

FIG. 5 shows the effect of different thawing modes on the survival rate of the shoot tips of Lonicera japonica after cryopreservation in example 5 of the present invention

FIG. 6 is a graph showing the effect of the recovery medium on the survival rate of the shoot tips of Lonicera japonica after cryopreservation in example 6 of the present invention

FIG. 7 is a flow chart of an ultra-low temperature preservation system for stem tips of Lonicera japonica Thunb according to the present invention; 1 obtaining aseptic seedlings through stem segments; 2, after being preserved at ultralow temperature, the honeysuckle stem tip is transferred into a recovery culture medium; 3, culturing the stem tip of the honeysuckle in dark for two weeks; 4, culturing stem tips for about one month after ultralow-temperature preservation; 5-7, forming seedlings by stem tips of the honeysuckle after ultralow temperature preservation; 8-9 rooting of regenerated seedlings of stem tips of the honeysuckle after ultralow temperature preservation; transplanting of honeysuckle regenerated seedlings after 10-12 ultralow-temperature preservation

The specific implementation mode is as follows:

the present invention is further described in detail with reference to the following examples, which are implemented on the premise of the technical solution of the present invention, and give detailed implementation manners and specific operation procedures.

Example 1

A method for ultralow temperature preservation of stem tips of honeysuckle comprises the following specific operations:

1) stripping off in-vitro stem tips containing 2 leaf primordia, wherein the length of the stem tips is 2-3 mm, from sterile stem segments of lonicera mauritiana containing the stem tips under aseptic conditions;

2) putting the stripped stem tip into a pre-culture solution for treatment, and pre-culturing the MS solution with the optimal concentration of sucrose of 0.3mol/L in the pre-culture solution for 3d at the temperature of 4 ℃;

3) placing the pre-cultured in-vitro stem tip in LS loading liquid, and loading for 10-50 min at room temperature of 25 ℃; the loading liquid comprises the following components: 2M glycerol +0.4mol/L sucrose + MS;

4) placing the loaded stem tips in a PVS2 protective solution, and dehydrating for 20-100 min at the temperature of 0 ℃; the PVS2 protective solution comprises the following components: a solution of 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide +0.4mol/L sucrose + MS;

5) replacing the stem tip treated by the PVS2 protective solution with fresh PVS2 protective solution, then quickly putting into liquid nitrogen, and preserving for 24 h;

6) taking out the freezing pipe after the liquid nitrogen preservation for defrosting, wherein the defrosting mode is as follows: thawing the stem tip in 40 deg.C water bath for 70 s;

7) the recovery culture specifically comprises the step of inoculating the stem tip washed by the unloading liquid for 20min on a recovery culture medium for recovery culture; the unloading liquid comprises the following components: 1.2mol/L sucrose + MS solution; the recovery culture medium comprises: MS + KT0.5mg/L + NAA0.1mg/L + GA31.0mg/L; the recovery culture conditions are as follows: culturing in 25 deg.C incubator for 14 days, and culturing under normal light.

Example 2

Testing the influence of the sucrose concentration and the pre-culture time in the pre-culture medium on the ultralow-temperature preservation of the stem tips of the lonicera japonica thunb: only changing the sucrose concentration and the pre-culture time in a pre-culture medium, firstly, respectively putting the stripped stem tips into MS solutions containing sucrose with different concentrations (0.1-0.6 mol/L) for pre-culture; the pre-culture temperature condition is 4 ℃, and the culture time is 0-5 d; then the survival rate of the stem tip is counted. The research result shows that (as shown in figure 1 and figure 2) the survival rate of the stem tip is different after the stripped stem tip is put into preculture solutions with different sucrose concentrations for preculture for different time, and the survival rate of the stem tip can reach 60% at the highest when the sucrose concentration is 0.3mol/L and the stem tip is precultured for 3 days at 4 ℃. Therefore, the pre-culture is a key factor influencing the survival rate of the stem tip of the lonicera mauritiana at the ultralow temperature, the optimal pre-culture sucrose concentration is 0.3mol/L, and the pre-culture time is 3 days.

Example 3

Testing the influence of the LS loading liquid treatment time on the stem tip survival rate after ultralow-temperature preservation: the pre-cultured stem tips were placed in the LS loading liquid in a sterile clean bench and loaded for various periods of time (0, 10, 20, 30, 40, 50min) at room temperature 25 ℃. As can be seen from the research results (as shown in fig. 3), there is a significant difference between the control group treated with the loading liquid and the control group not treated with the loading liquid, and the survival rate of the stem tip not treated with the loading liquid is very low, and the survival rate of the stem tip is only 3.3%; when the LS loading time is 50min, the survival rate of the stem tip is reduced by 17%, the loading time is overlong, the stem tip cells are damaged, the water in the cell tissues can be reduced by using the proper loading time, and the survival rate of the stem tip after ultralow temperature storage is obviously improved. Therefore, LS loading 40min is the optimal choice.

Example 4

The influence of the treatment time of the PVS2 protective solution on the survival rate of the stem tips after ultralow-temperature preservation is tested: after the stem tip is treated by the LS loading liquid, placing the stem tip on sterile filter paper for 5 seconds, removing residual liquid of the loading liquid, then quickly placing the stem tip into a freezing tube filled with PVS2 protection liquid, dehydrating for a certain time (0min, 20min, 40min, 60min, 80min and 100min) at the temperature of 0 ℃, and then measuring the survival rate of the stem tip; the research result shows (as shown in fig. 4) that when the stem tip is directly put into liquid nitrogen for freezing without the step of dehydrating the stem tip by the PVS2, the survival rate of the stem tip is 0, which indicates that the stem tip of lonicera japonica must be dehydrated by the PVS2, when the processing time is 20min, the survival rate of the stem tip is increased from 0 to 36.7%, and when the processing time is 40min, the survival rate of the stem tip reaches the highest value, namely 66.67%, but has no significant difference from the survival rate of the stem tip processed by the 40 min. As PVS2 treatment time continued to increase, the survival rate of the stem tips was lower, indicating that prolonged treatment had caused damage to stem tip cells. Although there was no significant difference between the treatment of the stem tips for 40min and the treatment for 60min, in order to avoid damage to the cells by dimethyl sulfoxide, the stem tips were treated for 40min with PVS 2.

Example 5

The influence of different unfreezing modes on the survival rate of the stem tips of the lonicera mauritiana is tested: taking out the frozen pipe with the stem tip which is dehydrated and replaced with the protective liquid from liquid nitrogen, and quickly thawing, wherein the thawing comprises three modes of thawing for 70s in a 40 ℃ water bath, thawing for 4min with tap water and thawing for 20min at room temperature; the research result shows that (as shown in figure 5) the stem tips are thawed differently, the survival rate is greatly different, when the stem tips are thawed by using 40 ℃ water bath for 70s, the survival rate is the highest and is 66.67%, and the survival rate is obviously different from the stem tips in other thawing modes. When the stem tip is unfrozen by tap water for 4min, the survival rate of the stem tip is not high and is 26.67 percent; when the stem tip is unfrozen at the room temperature of 25 ℃, the survival rate of the stem tip after the recovery culture is 0, so that the frozen stem tip is unfrozen rapidly for 70s by adopting a water bath at the temperature of 40 ℃, and the normal growth of the stem tip after the recovery culture can be effectively ensured.

Example 6

The influence of the recovery medium on the survival rate of the stem tips after ultralow-temperature preservation is tested: the thawed shoot tips were placed on sterile filter paper for 5 seconds to remove residual liquid from the PVS2 protection solution, and subsequently washed twice with fresh unloading solution, each for 10 min. The stem tips were then placed on sterile filter paper to remove residual unloading liquid, and then rapidly transferred to recovery medium. Wherein the unloading liquid comprises the following components: 1.2mol/L sucrose + MS solution; the conditions for recovery culture were: culturing in dark environment for 2 weeks, and culturing in normal light at 25 deg.C; according to the existing culture medium formula in tissue culture of plants of Caprifoliaceae, Lonicera and Lonicera japonica, the recovery culture medium is set as follows: MS No. 1; MS +6BA0.5mg/L + NAA0.1mg/L No. 2; MS +6BA2.0mg/L No. 3; MS No. 4 + KT0.5mg/L + NAA0.1mg/L + GA31.0mg/L; MS + KT0.5mg/L + NAA0.2mg/L No. 5; the results of the study showed (as shown in FIG. 6) that the stem tips were significantly less viable on MS minimal medium No. 1 than other hormone supplemented media. The survival rate of the stem tip reaches 73.3% to the maximum value after the No. 4 culture medium is adopted, wherein the survival rate is improved by 67% compared with that of the No. 1 stem tip and is improved by 43.3% compared with that of the No. 2 stem tip, therefore, the combined effect of the hormone KT and the hormone GA3 is better than that of 6-BA, although the stem tip is cultured by the No. 5 culture medium, the survival rate is 66.7%, and has no obvious difference with the No. 4 culture medium, but the selection is preferred, so the No. 4 culture medium is suitable for the stem tip of the lonicera japonica thunb in the experiment, and the stem tip can obtain better recovery effect after being frozen at ultralow temperature. And then directly culturing the honeysuckle stem tip tissue culture system into seedlings. Wherein the proliferation medium: MS + KT0.5mg/L + NAA0.04mg/L; rooting culture medium: 1/2MS + IBA 0.8 mg/L.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention.

Claims

1. An ultra-low temperature preservation method for stem tips of honeysuckle flowers is characterized in that: the method comprises the following steps:

(1) stripping and pre-culturing stem tips: under the aseptic condition, putting the stripped stem tip into a pre-culture solution for treatment; the stem tip stripping refers to an in vitro stem tip containing 2 leaf primordia, wherein the length of the stem tip is 2-3 mm, and the in vitro stem tip is stripped from a sterile honeysuckle stem section containing the stem tip; pre-culturing the MS solution with the sucrose concentration of 0.3mol/L in the pre-culture solution at 4 ℃ for 3 d;

(2) loading: transferring the pre-cultured in vitro stem tip into LS loading liquid, and loading for 40min at room temperature; the loading liquid comprises the following components: 2M glycerol +0.4mol/L sucrose + MS;

(3) PVS2 dehydration treatment: placing the loaded stem tip into PVS2 protective solution, and dehydrating at 0 deg.C for 40 min; the PVS2 protective solution comprises the following components: a solution of 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide +0.4mol/L sucrose + MS;

(4) and (3) liquid nitrogen preservation: the stem tip treated by the PVS2 protective solution is replaced by a new PVS2 protective solution, and then the freezing tube is quickly put into liquid nitrogen for storage for 24 hours;

(5) unfreezing: taking out the freezing tube stored by the liquid nitrogen for thawing, wherein the thawing mode is to rapidly thaw for 70s in 40 ℃ water bath;

(6) and (3) recovery culture: transferring the stem tip washed by the unloaded liquid to a recovery culture medium for recovery culture; the unloading liquid comprises the following components: 1.2mol/L sucrose + MS solution; the recovery culture medium is MS + KT0.5mg/L + NAA0.1mg/L + GA31.0mg/L; the recovery culture conditions are as follows: culturing in 25 deg.C incubator for 14 days, and culturing under normal light.