CN114600771A

CN114600771A - Landscape type moss spore large-scale mutagenesis screening method

Info

Publication number: CN114600771A
Application number: CN202210278946.9A
Authority: CN
Inventors: 刘莉; 陈刘柱; 章慧青; 陈则希; 段柳; 温从发
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-06-10
Anticipated expiration: 2042-03-18
Also published as: CN114600771B

Abstract

The invention provides a landscape moss spore large-scale mutagenesis screening method, and belongs to the technical field of biological engineering. The landscape moss mutants are screened by taking landscape moss spores as materials and carrying out mutagenesis in a mode of soaking the moss spores by using EMS mutagen solution and observing and comparing the phenotypes of the mutants with data. The landscape new variety with stable inheritance can be obtained in a laboratory for 4 months, the breeding time is greatly shortened, the method is more suitable for moss landscape and three-dimensional greening landscape with complex patterns, and the method has important guiding significance for new character cultivation of landscape moss plants. The mutant obtained by the method has beautiful plant type, higher appreciation value and landscape moss application potential, and the mutant can normally grow and complete the life history. In addition, the method disclosed by the invention is simple to operate, accurate, stable and reliable in identification result, capable of realizing batch screening and rapid detection of the mutant and high in mutation efficiency.

Description

Landscape type moss spore large-scale mutagenesis screening method

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a landscape moss spore large-scale mutagenesis screening method.

Background

There are a wide variety of bryophytes worldwide, about 23000 species including about 15000 species of moss, more than 8000 species of moss and more than 100 species of horny moss. Most of the moss plants are tiny in individuals, simple in structure and different in form, and the growth characteristics of moss are combined with gardening landscaping art, so that a novel natural and beautiful moss landscape can be created. In addition, the moss has strong adaptability, basically has no plant diseases and insect pests, is emerald and evergreen, grows regularly without trimming, and the landscape after landscaping can be kept for a long time. The moss plant is used as a new landscape plant, and brings new ornamental feelings and is different from the unique application prospect of other plants due to the unique morphological characteristics of the moss plant. However, a good new species of landscape moss is lacked in the current bryophyte market, a perfect planting and culturing system is lacked, and a large amount of wild moss is over utilized under the drive of economic benefits, so that the survival of bryophytes and the sustainable utilization of bryophyte resources are seriously threatened. The method is suitable for only a few commercial moss, deeply excavates moss resources, improves the existing moss variety, and has important significance for protecting the diversity of wild moss germplasm resources and the healthy and rapid development of the moss industry by breeding new varieties of moss which are more suitable for commercial application.

Physcomitrella patens (Physcomitrella patens) belongs to the family of Dictamaceae, the genus Physcomitrella, and is distributed in Europe, Asia, Africa and oceania, and in Zhang Jiajie region of Hunan province in China. The physcomitrella patens are short, yellow-green, glossy and sparse. Its stem is thin and short. The blades are in the shape of an oval or a scalenom, the blades at the base of the stem are smaller, and the middle rib is single and slender. The physcomitrella patens is applied to the market of moss at present, and is usually planted around 'mountain stones', 'plains', 'waterfalls' and the like of a moss wall to be used as a main scene or a spot.

Ethyl Methylsulfonate (EMS) is a chemical mutagen with wide application, and has high mutagenesis efficiency and good mutagenesis effect. The application of EMS mutagenesis in plant breeding can break through the traditional plant breeding mode, and the obtained mutant population has important significance in cultivating new plant varieties and enriching plant germplasm resources. At present, EMS mutagenesis plant seeds are mainly used in the field, related research of adopting EMS mutagenesis of moss spores is not available, and the moss is a gardening and landscape plant, so how to quickly and massively obtain a moss mutant material with excellent properties to meet market demands, and a suitable method does not exist at present.

Disclosure of Invention

In view of the above, the invention aims to provide a landscape type moss spore large-scale mutagenesis screening method, which can greatly shorten breeding time and obtain moss mutant materials with excellent properties on a large scale, and the moss mutant materials are more suitable for moss landscaping and three-dimensional greening landscaping with complex patterns.

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

the invention provides a landscape moss spore scale mutagenesis screening method, which comprises the following steps: EMS mutagen solution with the concentration of 2% -4% is adopted to soak and process the moss spores for mutagenesis; after the mutagenized spores develop protonema, carrying out independent culture, carrying out phenotype observation and datamation comparison on the mutants obtained by culture, and screening materials with obvious difference from wild types in the aspect of phenotype.

Preferably, the EMS mutagen solution is soaked for 60-90 min.

Preferably, the moss comprises Physcomitrella patens.

Preferably, the method for obtaining the bryophyte spores comprises the following steps: transferring gametophyte of moss to substrate for culturing, with photoperiod of 8h day/16 h night and light intensity of 60-80 μmol photons m^-2s^-1Moss spores are induced at the culture temperature of 16 ℃ for 60-90 days.

Preferably, the method further comprises the following steps: screening to obtain a material with obvious difference in phenotype from a wild type, carrying out subculture treatment on the material to obtain a subculture mutant, carrying out phenotype observation and data comparison on the subculture mutant, and screening the material with obvious difference in phenotype from the wild type.

Preferably, the method of the subculture processing comprises the following steps: mixing the newly grown protonema with sterile water, grinding and crushing the protonema to obtain a moss suspension, and inoculating the moss suspension to a culture medium for culture.

Preferably, the phenotypic observation and data comparison includes plant height, leaf angle, leaf extension distance (vertical distance from leaf tip to plant stem), and leaf length.

Preferably, the medium used for culturing is BCDAT medium.

Preferably, the temperature of the cultivation is 25 ℃ and the light intensity is 60-80. mu. mol photons m^-2s^-1The photoperiod was 16h light/8 h dark.

The invention also provides application of the moss obtained by the method in greening landscaping.

The invention has the beneficial effects that:

the invention provides a large-scale mutagenesis screening method of landscape type moss spores, EMS mutagenesis technology is firstly applied to breeding of a new gardening ornamental plant moss, and stable and inherited landscape type new species can be obtained in a laboratory for 4 months by combining tissue culture technology of physcomitrella patens, so that breeding time is greatly shortened, and breeding efficiency is improved. Compared with wild physcomitrella patens, the mutant obtained by the method has obvious differences in plant height, leaf included angle, leaf extension distance, leaf length and other properties, the bred mutant has beautiful plant type, higher appreciation value and landscape type moss application potential, and the mutant has normal growth and development and can normally complete life history.

In addition, the method is simple to operate, the mutation efficiency is high, the obtained mutant has stable and reliable character heredity, and batch screening and rapid detection of the mutant can be realized.

Drawings

Fig. 1 shows the results of comparing the data of mutant plants Pp73, Pp23 with wild-type plants (WT) in terms of plant height, leaf angle, leaf extension distance, and leaf length, wherein a is the significance analysis of the three in terms of leaf extension distance, B is the significance analysis of plant height, C is the significance analysis of leaf angle, D is the significance analysis of leaf length, ns represents P >0.05 without significant difference,. indicates P <0.05,. indicates P <0.01,. indicates P <0.001,. indicates P < 0.0001;

FIG. 2 is the observation result of the appearance traits of the mutant plants Pp73 and Pp23 and the wild type plants, wherein A is the wild type, B is the mutant Pp73, and C is the mutant Pp 23;

FIG. 3 is an observation result of appearance characteristics of mutant plants obtained by screening in different embodiments, wherein A is a wild type, B is a mutant Pp73, C is a mutant Pp23, D is a mutant Pp38, E is a mutant Pp59, F is a mutant Pp72, G is a mutant Pp76, and H is a mutant Pp 87;

FIG. 4 shows the results of mutagenic germination rates of 1% EMS mutagen at different times;

FIG. 5 shows the results of mutagenic germination rates of 4% EMS mutagen at different times.

Detailed Description

The invention provides a landscape moss spore large-scale mutagenesis screening method, which comprises the following steps: EMS mutagen solution with the concentration of 2% -4% is adopted to soak and process the moss spores for mutagenesis; after the mutagenized spores develop protofilament, independent culture is carried out, phenotype observation and data comparison are carried out on the mutants obtained by culture, and materials with obvious difference with wild types in the aspect of phenotype are screened.

The specific source of the EMS mutagen is not particularly limited, the concentration of the EMS mutagen solution is preferably 3% -4%, more preferably 4%, and the soaking treatment time by using the EMS mutagen solution is preferably 60min-90min, more preferably 70min-80 min. In the present invention, the EMS mutagen solution is preferably prepared by diluting the EMS mutagen with phosphate buffer, and the concentration of the phosphate buffer is preferably 0.1 mol/ml.

In the present invention, the moss preferably includes Physcomitrella patens. The method adopts EMS mutagen solution to soak and treat the bryophyte spores for mutagenesis, and the preferred method for obtaining the bryophyte spores comprises the following steps: transferring the gametophyte of the moss to a substrate for culture, and culturing the gametophyte of the moss with the photoperiod of 8h day/16 h night and the light intensity of 60-80 mu mol phosns m^-2s^-1Moss spores are induced at the culture temperature of 16 ℃ for 60-90 days. Wherein the light intensity is preferably 65-75 mu molphos ns m^-2s^-1. After obtaining the moss spores, the moss spores are preferably selected as a mutagenic material with basically consistent size and better spore shape.

After mutagenesis of spores, bryophyte spore explants are sterilized, then added into double distilled water, spores are punctured in a liquid environment, and the mixed solution is sucked into a culture medium for culture. The specific method for sterilizing the bryophyte spore explants is not particularly limited, and preferably, the bryophyte spore explants are sterilized by using a NaCl solution, the concentration of the NaCl solution is preferably 10%, and the sterilization time is preferably 4-6min, and more preferably 5 min. After the mutagenized spores develop protofilament, independent culture is carried out, phenotype observation and data comparison are carried out on the mutants obtained by culture, and materials with obvious difference with wild types in the aspect of phenotype are screened.

In the present invention, the phenotypic observation and datamation preferably includes plant height, leaf angle, leaf extension distance and leaf length. In the present invention, the leaf extension distance refers to the vertical distance from the leaf tip to the plant stem. Comparing the characters with wild type bryophytes, if one character is different from the wild type obviously, the mutation success can be shown.

In the present invention, in order to obtain mutant material with a more stable inheritance of a trait, it is preferable to further include the steps of: screening to obtain a material with obvious difference in phenotype from a wild type, carrying out subculture treatment on the material to obtain a subculture mutant, carrying out phenotype observation and data comparison on the subculture mutant, and screening the material with obvious difference in phenotype from the wild type. The specific characteristics of the secondary mutant subjected to phenotype observation and data comparison are the same as above, and are not described herein again.

The number of times of the secondary treatment is not particularly limited in the present invention, and the interval between the two secondary treatments is preferably 6 to 8 days, and more preferably 7 days. The method of the subculture preferably comprises the steps of: mixing the newly grown protonema with sterile water, grinding and crushing the protonema to obtain a moss suspension, and inoculating the moss suspension to a culture medium for culture.

In the method, after the spores are cultured to grow protonema, the protonema is cultured independently, the culture medium used in the steps is preferably BCDAT culture medium, the specific ratio of the BCDAT culture medium is not particularly limited in the invention, and the BCDAT culture medium preferably comprises Stock B, Stock C, Stock D, Alternative TES, agar, water and CaCl₂·2H₂And O. The culture conditions in the above-mentioned culture step are preferably at 25 ℃ and 60 to 80. mu. mol of phosns m^-2s^-1Light intensity, 16h light/8 h dark photoperiod, preferably 65-75 μmol photons m^-2s^-1。

The invention also provides an application of the moss obtained by the method in greening landscaping.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Transferring gametophyte of Physcomitrella patens to matrix (compressed matrix block of Seifenfy imported Norway) for culture, with photoperiod of 8h day/16 h night and light intensity of 60-80 μmol photons m^-2s^-1Inducing physcomitrella patens spores at the culture temperature of 16 ℃ for 60-90 days, and collecting the spores with basically consistent sizes and better spore shapes as mutagenesis materials. Diluting EMS mutagen (SIGMA M0880-25G Ethyl methane mutagen) with 0.2mol/ml phosphate buffer solution (pH 7.0) until the concentration of EMS mutagen is 4% to obtain EMS mutagen solution, and soaking the physcomitrella patens spores in 4% EMS mutagen solution at 25 ℃ for mutagenesis for 90 min.

After mutagenesis, the bryophyte spores are subjected to explant sterilization by using NaCl solution with the concentration of 10% (diluted by double distilled water) for 5min, the spores are washed by using the double distilled water for 6-8 times, then 1ml of double distilled water is added, the spores are punctured in a liquid environment, and the mixed solution is sucked into a BCDAT culture medium (the proportion of the BCDAT culture medium is shown in tables 1 and 2) for culture. And (5) counting the mutagenic germination rate after 7 days of culture.

After the spores develop protofilaments, seedlings are picked and independently cultured to obtain mutant plants Pp73 and Pp 23. The culture conditions in the above steps are all as follows: the temperature is 25 ℃, and the light intensity is 60-80 mu mol photons m^-2s^-1And the photoperiod is 16h, and the light is 8h and dark.

TABLE 1 BCDAT Medium formulation

Table 2 BCDAT culture medium mother liquor preparation formula

Observation of mutant plants from wild type plants (WT) (wild type plants means repeating the above experiment process except replacing EMS mutagen solution with ddH₂O), leaf angle, leaf extension, leaf length, and the results of comparing the above-described traits are shown in FIG. 1, and the overall appearance of the mutant plants and wild type plants are shown in FIG. 2.

As can be seen from FIG. 1, the differences between the mutant Pp73 and WT in leaf extension distance and leaf length are very significant, and the plant heights are different, and there is no significant difference in leaf angle. The differences of the mutant Pp23 and WT in leaf extension distance, plant height and leaf length are very obvious, and the leaf included angle is also different. As can be seen from fig. 2, the appearance characteristics of the mutants Pp73 and Pp23 differed significantly from those of WT.

Example 2

The mutant material obtained by screening in the embodiment 1 is ground and subcultured once every 7 days, and grinding subculture is carried out for three times, wherein the grinding subculture for each time comprises the following specific steps: scraping the protonema newly grown on the culture dish by using sterile forceps, mixing the protonema with 10mL of sterile water, grinding and crushing the protonema by using a grinding instrument, preparing the protonema into a moss suspension, inoculating the moss suspension onto a BCDAT culture medium for culture, and placing the inoculated culture dish in an illumination incubator for culture. The culture conditions in the above steps are all as follows: the culture temperature is 25 ℃, the light intensity is 60-80 mu mol photons m^-2s^-1And the photoperiod is 16h, and the light is 8h and dark. Continuously grinding and subculturing to obtain the mutant material with stable inheritance of characters.

And (3) carrying out phenotype observation on the mutant capable of being stably inherited, observing the plant height, the leaf included angle, the leaf stretching distance and the leaf length of the mutant plant and a wild plant (WT), comparing the numerical values of the characters, and screening the landscape type physcomitrella patens mutant with the phenotype novelty (compared with the WT, no matter which character has obvious difference, the landscape type physcomitrella patens mutant is considered to have novelty) as a new physcomitrella patens landscape application strain.

Example 3

The difference from example 1 is that the EMS mutagenizing treatment is carried out by soaking in EMS mutagenizing agent solution for 60min, and the rest is the same as example 1.

Example 4

The difference from example 1 is that in EMS mutagenesis treatment, the concentration of EMS mutagen solution used is 3%, and the mutagenesis soaking time is 60min, and the rest is the same as example 1.

Example 5

The difference from example 1 is that in EMS mutagenesis treatment, the concentration of EMS mutagen solution used is 2%, and the mutagenesis soaking time is 90min, and the rest is the same as example 1.

The appearance characteristics of the mutant plants obtained by screening in examples 1 and 3-5 are shown in FIG. 3.

Comparative example 1

The difference from example 1 is that in the EMS mutagenesis treatment, the EMS mutagen solution used isThe concentration is 1%, the time for mutagenesis soaking is 10min, 1h and 2h respectively, and the rest is the same as the example 1. And (5) counting the mutagenic germination rate after 7 days of culture. In contrast to wild type Physcomitrella patens (WT), the WT group was constructed using ddH₂The procedure of example 1 was repeated except for soaking. The results are shown in FIG. 4. Although the mutation treatment of the EMS mutagen with low concentration can obtain higher mutagenic germination rate, the mutation efficiency is low, and finally, a more ideal mutant plant cannot be obtained.

Comparative example 2

The difference from example 1 is that in the EMS mutagenesis treatment, the EMS mutagen solution is subjected to mutagenesis soaking for 10min, and the rest is the same as example 1.

Comparative example 3

The difference from example 1 is that in the EMS mutagenesis treatment, the time for soaking the EMS mutagen solution in mutagenesis is 30min, and the rest is the same as example 1.

Comparative example 4

The difference from example 1 is that in EMS mutagenesis treatment, the time for soaking in EMS mutagen solution for mutagenesis is 120min, and the rest is the same as example 1.

The mutagenic germination percentage results of examples 1 and 3 and comparative examples 2 to 4 were compared, wherein the WT group was performed using ddH₂The procedure of example 1 was repeated except for soaking. The results are shown in FIG. 5. Although a short-time (10min, 30min) mutagenesis treatment of 4% EMS mutagen solution can obtain high mutagenesis germination rate, ideal mutant plants cannot be obtained finally.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims

1. A landscape moss spore scale mutagenesis screening method is characterized by comprising the following steps: EMS mutagen solution with the concentration of 2% -4% is adopted to soak and process the moss spores for mutagenesis; after the mutagenized spores develop protofilament, independent culture is carried out, phenotype observation and data comparison are carried out on the mutants obtained by culture, and materials with obvious difference with wild types in the aspect of phenotype are screened.

2. The method of claim 1, wherein the EMS mutagen solution is soaked for 60min to 90 min.

3. The method of claim 1, wherein said moss comprises Physcomitrella patens.

4. The method according to claim 1, wherein the method of obtaining moss spores comprises the steps of: transferring the gametophyte of the moss to a substrate for culture, and culturing the gametophyte of the moss with the photoperiod of 8h day/16 h night and the light intensity of 60-80 mu mol phosns m^-2s^-1Moss spores are induced at the culture temperature of 16 ℃ for 60-90 days.

5. The method of claim 1, further comprising the steps of: screening to obtain a material with obvious difference in phenotype from a wild type, carrying out subculture treatment on the material to obtain a subculture mutant, carrying out phenotype observation and data comparison on the subculture mutant, and screening the material with obvious difference in phenotype from the wild type.

6. The method according to claim 5, wherein the method of sub-generation processing comprises the steps of: mixing the newly grown protonema with sterile water, grinding and crushing the protonema to obtain a moss suspension, and inoculating the moss suspension to a culture medium for culture.

7. The method of claim 1 or claim 5, wherein said phenotypic observations and comparisons are plant height, leaf angle, leaf stand, leaf length.

8. The method according to claim 1 or claim 6, wherein the medium used for the culturing is BCDAT medium.

9. The method according to claim 1 or claim 6, wherein the temperature of the cultivation is 25 ℃ and the light intensity is 60-80 μmol phosns m^-2s^-1The photoperiod was 16h light/8 h dark.

10. Use of moss obtainable by the process of any one of claims 1 to 9 in landscaping.