CN114902960B

CN114902960B - Method and culture medium for efficiently inducing tobacco seed callus

Info

Publication number: CN114902960B
Application number: CN202210491441.0A
Authority: CN
Inventors: 徐飞; 许健成; 彭也; 余璐璐
Original assignee: Wuhan Bioengineering Institute
Current assignee: Wuhan Bioengineering Institute
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2023-05-09
Anticipated expiration: 2042-05-07
Also published as: CN114902960A

Abstract

The invention discloses a method and a culture medium for efficiently inducing tobacco seed callus, wherein the method comprises the following steps: soaking and sterilizing mature tobacco seeds, inoculating the mature tobacco seeds to a callus induction culture medium for photo-temperature induction culture to obtain callus with high callus rate; wherein, the callus induction medium takes MS as basic medium and is added with 2,4-D and 6-BA: when the concentration of the 2,4-D is 1.0mg/L, the concentration of the 6-BA is 0.1-2.0 mg/L; or when the concentration of the 6-BA is 1.0mg/L, the concentration of the 2,4-D is 0.1-2.0 mg/L. The invention breaks through the traditional tobacco plant tissue culture regeneration system which uses the in-vitro leaves as the main material for unfolding, can quickly and conveniently obtain the callus from the tobacco seeds, and can be used for subsequent experiments such as genetic transformation and culture of cluster buds.

Description

Method and culture medium for efficiently inducing tobacco seed callus

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a method and a culture medium for efficiently inducing tobacco seed callus.

Background

Tobacco (Nicotiana tabacum l.), annual herbaceous plants of the genus Solanaceae (Solanaceae) tobacco (Nicotiana), are not only an important cash crop, but often play an important role as model plants and in the field of plant life sciences, and have high scientific research value.

At present, a plurality of methods for obtaining tobacco callus by means of tissue culture exist, wherein a more mature and more common method is an in-vitro leaf induction method, namely, the in-vitro leaf tissue of tobacco is taken as a main material, and the in-vitro leaf tissue of tobacco is induced to dedifferentiate after proper hormone proportion is added, so that the callus is obtained.

However, the in-vitro leaf blade induction method has a series of defects of complicated operation, high pollution rate, easy browning caused by mechanical wounds and the like. In addition, in vitro leaf tissue generally results from the aseptic cultivation of seeds, and the time spent earlier is also longer.

Therefore, how to prepare a method for efficiently inducing tobacco seed callus becomes a technical problem to be solved.

Disclosure of Invention

The invention aims to provide a method for efficiently inducing tobacco seed callus, breaks through the traditional tobacco plant tissue culture regeneration system which uses in-vitro leaves as main materials to develop, provides a brand-new, convenient and stable method for obtaining tobacco callus, and lays a foundation for tissue culture related research work.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a method of efficiently inducing callus of tobacco seeds, the method comprising:

soaking and sterilizing mature tobacco seeds, inoculating the mature tobacco seeds to a callus induction culture medium for photo-temperature induction culture to obtain callus with high callus rate; wherein, the callus induction medium takes MS as basic medium and is added with 2,4-D and 6-BA:

when the concentration of the 2,4-D is 1.0mg/L, the concentration of the 6-BA is 0.1-2.0 mg/L;

or when the concentration of the 6-BA is 1.0mg/L, the concentration of the 2,4-D is 0.1-2.0 mg/L.

Further, when the concentration of 2,4-D is 1.0mg/L, the concentration of 6-BA is 0.5-1.0 mg/L.

Further, when the concentration of 6-BA is 1.0mg/L, the concentration of 2,4-D is 0.5-1.5 mg/L.

Further, the conditions of the photo-temperature induced culture are as follows: the illumination period is 14-16 h/day, and the illumination intensity is 100 mu mol m ^-2 s ^-2 The culture temperature is as follows: the culture time is 18-24 days at 25+/-1 ℃.

Further, the soaking and sterilizing treatment of the mature tobacco seeds comprises:

soaking fresh mature tobacco seeds in water, rinsing with sterile water, soaking with 84 disinfectant for sterilization, and rinsing with sterile water.

In a second aspect of the invention, there is provided a medium for efficiently inducing callus of tobacco seeds, which is a callus induction medium comprising MS as a basal medium and 2,4-D and 6-BA added:

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

the method for efficiently inducing the tobacco seed callus is different from the traditional tissue culture regeneration system which uses the in-vitro mature tissue such as leaf as the main material, the method uses the tobacco mature seed as the main material for unfolding, has the advantages of short whole period, simple operation, simple and convenient sterilization mode, low pollution rate, no mechanical wound and the like, and in addition, under the action of hormone with proper proportion, the callus yield is generally 100 percent, and the finally obtained callus is generally in a light green semitransparent stack shape, and achieves certain ideal value in quality and volume.

The culture medium for efficiently inducing the tobacco seed callus provided by the invention uses MS as a basic culture medium, and when the concentration of 2,4-D is 1.0mg/L, 0.1-2.0 mg/L6-BA can well induce the tobacco seed to form the callus; wherein, the best performance is achieved by 0.5-1.0 mg/L6-BA; when the concentration of 6-BA is 1.0mg/L, 0.1-2.0 mg/L2, 4-D can well induce tobacco seeds to form callus; wherein, the best performance is obtained by 0.5-1.5 mg/L2, 4-D. Can quickly and conveniently obtain the callus from the tobacco seeds, and can be used for subsequent experiments such as genetic transformation and culture of cluster buds.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows callus growth (21 days) at 1 mg/L2, 4-D and different 6-BA concentration ratios; wherein, FIG. 1a is MS+1.0mg/L2, 4-D; FIG. 1b is MS+1.0 mg/L2, 4-D+0.1 mg/L6-BA; FIG. 1c is MS+1.0 mg/L2, 4-D+0.5 mg/L6-BA; FIG. 1D is MS+1.0 mg/L2, 4-D+1.0 mg/L6-BA; FIG. 1e is MS+1.0 mg/L2, 4-D+1.5 mg/L6-BA; FIG. 1f is MS+1.0 mg/L2, 4-D+2.0 mg/L6-BA;

FIG. 2 shows callus growth (21 days) at 1 mg/L6-BA and different 2,4-D concentration ratios; wherein FIG. 2a is MS+1.0 mg/L6-BA; FIG. 2b is MS+1.0 mg/L6-BA+0.1 mg/L2, 4-D; FIG. 2c is MS+1.0mg/L6-BA+0.5 mg/L2, 4-D; FIG. 2D is MS+1.0 mg/L6-BA+1.5 mg/L2, 4-D; FIG. 2e is MS+1.0 mg/L6-BA+2.0 mg/L2, 4-D.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.

A method and medium for efficiently inducing callus of tobacco seeds according to the present application will be described in detail with reference to examples, comparative examples and experimental data.

Example 1

The embodiment of the invention provides a method for directly inducing callus of mature tobacco seeds, which comprises the following steps:

1. soaking and sterilizing tobacco seeds: specifically:

soaking appropriate amount of fresh mature tobacco seeds in water, and standing in a refrigerator at 4deg.C for overnight at a maximum time of no more than 7 days; transferring the soaked tobacco seeds to an ultra-clean workbench, and rinsing the tobacco seeds with sterile water for 2 times; soaking and sterilizing for 8min with 84 sterilizing liquid, changing fresh 84 sterilizing liquid at the 4 th min, and turning over the EP tube upside down or sucking and beating with a pipetting gun during sterilizing; and then, rinsing the tobacco seeds with sterile water for 6 times to finish the sterilization of the tobacco seeds.

2. Inoculated to callus induction medium: specifically:

firstly, preparing 1L of MS culture medium solution without agar, wherein sucrose accounts for 3%, and the pH value is 5.8-6.0; the 1L MS culture medium solution was dispensed into jam bottles using 50mL measuring cylinders, 50mL each bottle was dispensed, followed by adding 0.4g agar (0.8% agar) per bottle jam bottle; adding a proper amount of 2,4-D hormone mother liquor to each culture flask with a micropipette to a final concentration of 1.0mg/L and 6-BA hormone mother liquor to a final concentration of 1.0mg/L to prepare a callus induction culture medium with different hormone ratios; finally, the culture medium is sterilized by high-temperature high-pressure steam at 121 ℃ for 20min, and after sterilization, the culture medium is taken out and the jam bottle is gently shaken to ensure that all components in the bottle are fully and uniformly mixed, and the culture medium is cooled at room temperature for standby.

3. Photo-temperature induced culture of callus: the specific culture conditions are as follows:

the illumination period is 16 h/day, and the illumination intensity is 100 mu mol m ^-2 s ^-2 The culture temperature is regulated to 25+/-1 ℃ and the culture time is about three weeks.

4. The growth of the calli was noted.

Comparative example 1

In this comparative example, only 1.0 mg/L2, 4-D was added to the hormone in the callus induction medium, and the other steps were the same as in example 1.

Comparative example 2

In this comparative example, only 1.0mg/L6-BA was added to the hormone in the callus induction medium, and the other steps were the same as in example 1.

Example 2

In this comparative example, the hormone ratio of the callus induction medium was 1.0 mg/L2, 4-D+0.1 mg/L6-BA, and the other steps were the same as in example 1.

Example 3

In this example, the hormone ratio of the callus induction medium was 1.0 mg/L2, 4-D+0.5 mg/L6-BA, and the other steps were the same as in example 1.

Example 4

In this example, the hormone ratio of the callus induction medium was 1.0 mg/L2, 4-D+1.5 mg/L6-BA, and the other steps were the same as in example 1.

Example 5

In this example, the hormone ratio of the callus induction medium was 1.0 mg/L2, 4-D+2.0 mg/L6-BA, and the other steps were the same as in example 1.

Example 6

In this example, the hormone ratio of the callus induction medium was 1.0 mg/L6-BA+0.1 mg/L2, 4-D, and the other steps were the same as in example 1.

Example 7

In this example, the hormone ratio of the callus induction medium was 1.0mg/L6-BA+0.5 mg/L2, 4-D, and the other steps were the same as in example 1.

Example 8

In this example, the hormone ratio of the callus induction medium was 1.0 mg/L6-BA+1.5 mg/L2, 4-D, and the other steps were the same as in example 1.

Example 9

In this example, the hormone ratio of the callus induction medium was 1.0 mg/L6-BA+2.0 mg/L2, 4-D, and the other steps were the same as in example 1.

Experimental example 1

Hormone ratios, seed germination rates and recovery rates in each example and each comparative example were measured and counted as shown in Table 1, and growth conditions are shown in Table 2.

TABLE 1 hormone ratios and seed germination and recovery rates in different treatment groups

Note that: seed germination rate = number of germinated seeds/number of seeds inoculated x 100%, seed out rate = number of seeds growing out callus/number of seeds inoculated x 100%, "-" means no data is recordable.

TABLE 2 growth of calli in different treatment groups (21 days)

As can be seen from tables 1 and 2, in the embodiments of the present invention, in the callus induction culture media with different hormone ratios, the fresh mature tobacco seeds can normally germinate, the germination rate is generally 100%, and the germinated seeds can be further induced to form callus under the action of the hormone with a proper ratio.

As can be seen by combining the table 2 with the table 1, under the action of 1.0 mg/L2, 4-D, the whole seedling has serious vitrification phenomenon, and finally, callus cannot be induced; the vitrification phenomenon is obviously improved by matching with 6-BA (0.1 mg/L6-BA) with lower concentration on the basis of 1.0 mg/L2, 4-D, but the root bodies still remain in the induced partial callus, and the whole callus is in a quicksand shape, so that the quality is poor; the callus quality can be obviously improved by properly increasing the concentration of 6-BA (0.5-1.0 mg/L6-BA) on the basis of 1.0 mg/L2, 4-D, the induced callus is in a light green semitransparent pile shape, the layer profile is clear, and the quality and the volume of the callus are obviously better; the 6-BA (1.5-2.0 mg/L6-BA) with higher concentration is used in combination on the basis of 1.0 mg/L2, 4-D, and the induction of the callus is inhibited to a certain extent. As a result, the optimal concentration of 6-BA was found to be 1mg/L.

As can be seen by combining Table 2 with FIG. 2, the whole seedling maintains a relatively normal form only under the action of 1.0mg/L6-BA, but the root is short and thick and has a small amount of callus, and the induction effect is not obvious; 2,4-D (0.1-2.0 mg/L2, 4-D) with a certain concentration is matched on the basis of 1.0mg/L6-BA, so that the generation of the callus can be better induced, wherein the 2,4-D with the concentration of 0.5-1.5 mg/L is best in performance, the whole callus is loose, and no obvious difference exists among the three components; 2,4-D (0.1 mg/L2, 4-D) with lower concentration is matched on the basis of 1.0mg/L6-BA, the whole of the expansion tissue is light green semitransparent sphere, and the expansion tissue has white tissue with part although the callus is generated, and the whole is compact and is morphologically different from other concentrations; the callus of the plant is slightly vitrified by being matched with 2,4-D (2.0 mg/L2, 4-D) with higher concentration on the basis of 1.0 mg/L6-BA.

In conclusion, when the concentration of 2,4-D is 1.0mg/L, 0.1-2.0 mg/L6-BA can well induce tobacco seeds to form callus; wherein, the best performance is achieved by 0.5-1.0 mg/L6-BA; when the concentration of 6-BA is 1.0mg/L, 0.1-2.0 mg/L2, 4-D can well induce tobacco seeds to form callus; wherein, the best performance is obtained by 0.5-1.5 mg/L2, 4-D. Considering comprehensively, the optimal hormone proportion for callus induction is 1.0 mg/L2, 4-D+1.0 mg/L6-BA. The invention has the main advantages and the utilization value that the callus can be quickly and conveniently obtained from tobacco seeds, and can be used for subsequent experiments such as genetic transformation and culture of cluster buds.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A method for efficiently inducing callus of tobacco seeds, the method comprising:

when the concentration of 2,4-D is 1.0mg/L, the concentration of 6-BA is 0.1-0.5 mg/L or 1.5-2.0 mg/L;

or when the concentration of the 6-BA is 1.0mg/L, the concentration of the 2,4-D is 0.1-0.5 mg/L or 1.5-2.0 mg/L.

2. The method for efficiently inducing tobacco seed callus according to claim 1, wherein the light temperature induction culture conditions are as follows: the illumination period is 14-16 h/day, and the illumination intensity is 100 mu mol m ^-2 s ^-2 The culture temperature is 25+/-1 ℃ and the culture time is 18-24 days.

3. A method of efficiently inducing callus in tobacco seeds according to claim 1, wherein the soaking and disinfecting treatment of mature tobacco seeds comprises: