CN114027180B - Cultivation method and application of shinyleaf yellowhorn polyploid - Google Patents

Abstract

The invention provides a cultivation method and application of shinyleaf yellowhorn polyploid. According to the characteristic of the shinyleaf yellowhorn, the invention selects and obtains the mutagenesis reagent which is favorable for improving the mutagenesis efficiency and has safety and the chemical substance which promotes the mutagenesis. The method can obtain the shinyleaf yellowhorn plant with obvious polyploid characteristics, and has the advantages of low cost, low toxicity and high induction frequency. The invention effectively reduces the occurrence of chimerism, provides a new way for cultivating new varieties of shinyleaf yellowhorn, and the obtained shinyleaf yellowhorn polyploid has high application value.

Description

Cultivation method and application of shinyleaf yellowhorn polyploid

The technical scheme of the invention is that the major laboratory (university of ethnic group) of the department of biotechnology and resource utilization education is sponsored.

Technical Field

The invention belongs to the technical field of woody oil tree cultivation; more specifically, the invention relates to a cultivation method and application of shinyleaf yellowhorn polyploid.

Background

The shinyleaf yellowhorn (Xanthoceras sorbifolia Bunge), also called papaya, belongs to the genus shinyleaf yellowhorn of the family Sapindaceae, and is a special temperate woody edible oilseed tree species in China. The oil content of the seeds is up to 30% -60%, the oil content of the kernels is 55% -66%, and the seeds are known as northern tea-oil trees.

Today, the petroleum resources are increasingly exhausted, and the shiny-leaved yellowhorn is an important biological energy tree species due to excellent biological and ecological characteristics of drought resistance, low temperature resistance, barren resistance, slight saline-alkali resistance, strong tillering capacity and the like. Not only has profound development potential in the aspect of biological energy, but also has great profound significance in ecological management engineering. The oil content difference of the seeds of different seeds of the shinyleaf yellowhorn is obvious, so that the method has very wide application prospect for mass propagation of the shinyleaf yellowhorn seeds with high oil content.

In the prior art, the reproduction technology of shinyleaf yellowhorn mainly comprises sexual reproduction and asexual reproduction. However, the germination rate of seeds is low, the character difference among individuals is obvious, and the grafting propagation survival rate and the cutting rooting rate are low.

In view of the above, tissue culture techniques have become a necessary choice for large-scale, rapid propagation of superior shinyleaf yellowhorn. If the polyploid cultivation is carried out on the existing tissue culture seedling of good variety, the method has important practical significance for further improving the yield and the oil content, enriching the germplasm resources with high quality and the like. Although many plants in the prior art have successfully obtained polyploids, in view of the particularity of the shinyleaf yellowhorn itself, there still exist technical difficulties in the art how to simply, economically and efficiently obtain shinyleaf yellowhorn polyploids, and an effective method is needed.

Disclosure of Invention

The invention aims to provide a cultivation method and application of shinyleaf yellowhorn polyploid.

In a first aspect of the invention, there is provided a method of preparing a shinyleaf yellowhorn polyploid, the method comprising:

(a) Performing chemical mutagenesis on the explant of the shinyleaf yellowhorn by using sulfaming, and simultaneously adding the orange peel essential oil;

(b) Performing adventitious bud induction on the chemically mutagenized explant obtained in the step (a), and simultaneously adding lanthanum nitrate;

(c) Culturing and seedling the adventitious bud obtained in the step (b); proliferation and propagation culture; rooting culture; obtaining the shinyleaf yellowhorn polyploid.

In one or more embodiments, the term "polyploid" refers to a plant that has a greater number of chromosomes than "diploid".

In one or more embodiments, the "polyploid" refers to a "tetraploid".

In one or more embodiments, in step (c), after the seedling, further comprising: selecting polyploid seedlings; preferably, polyploid seedlings are identified and selected through plant morphological indexes; more preferably, the plant morphology metrics include (but are not limited to): cell volume, organ volume, stem thickness, leaf color, leaf thickness, leaf area; more preferably, if the cell volume increases, the organ volume increases, the stem thickness increases, the green color of the leaf is deeper, the leaf thickness increases, the leaf area increases, the plant is shown to be polyploid.

In one or more embodiments, in step (c), the proliferation is performed more than 2 times (generations), preferably more than 5 times, more preferably more than 10 times.

In one or more embodiments, the number of expansion is 2-100, such as 3, 6, 12, 15, 20, 25, 30, 40, 50, 60, or 80; it may be 100 times or more.

In one or more embodiments, in step (c), after the propagation and propagation culture, the method further comprises: identifying and selecting polyploid seedlings; preferably, the following assays are performed: chloroplast identification, chromosome identification, and flow cytometry analysis.

In one or more embodiments, in the chloroplast identification, the plant is polyploid if the stomata are increased, the stomata density is reduced, and the number of chloroplasts in the guard cell is increased by identifying the stomata size, the stomata density, and the number of chloroplasts in the guard cell.

In one or more embodiments, in the chromosome identification, a tabletting method is adopted, a root tip is used as a material for chromosome counting identification, and if the chromosome number is doubled, the plant is indicated to be polyploid;

in one or more embodiments, the flow cytometric analysis detects cellular DNA content, and if the DNA content increases, the plant is polyploid.

In one or more embodiments, in step (a), the amount of sulfamethoxazole is from 5 to 30mg/L, preferably from 6 to 25mg/L, more preferably from 8 to 20mg/L.

In one or more embodiments, the amount of orange peel essential oil is 0.03 to 0.3% (volume percent), preferably 0.05 to 0.2%, more preferably 0.08 to 0.18%.

In one or more embodiments, in step (b), lanthanum nitrate is added to the medium in an amount of 60 to 160mg/L, preferably 80 to 140mg/L, more preferably 90 to 130mg/L.

In one or more embodiments, in step (a), the time for chemical mutagenesis is from 12 to 72 hours, preferably from 18 to 60 hours, more preferably from 24 to 48 hours.

In one or more embodiments, in step (b), the adventitious bud induction (lanthanum nitrate addition-all of the induction medium to which lanthanum nitrate has been added) is for a period of 15 to 35 days, preferably 16 to 30 days, more preferably 18 to 25 days.

In one or more embodiments, the regeneration medium used for adventitious bud induction includes basal medium and 6-BA and NAA (lanthanum nitrate is added based thereon). Preferably, the basal medium is an MS medium. Preferably, 1-5mg/L, preferably 1.5-4mg/L, more preferably 2-3mg/L of 6-BA is contained. Preferably, NAA is contained in an amount of 0.3-1.8mg/L, preferably 0.4-1.5mg/L, more preferably 0.5-1 mg/L.

In one or more embodiments, the rooting medium comprises basal medium and indole-3 butyric acid. Preferably, the basal medium is 1/2MS medium. Preferably, it contains 0.2-2mg/L, preferably 0.3-1.5mg/L, more preferably 0.5-1mg/L of indole-3 butyric acid.

In another aspect of the invention, there is provided the use of the method in the preparation of a shinyleaf yellowhorn polyploid.

In another aspect of the present invention, there is provided a kit for preparing a shinyleaf yellowhorn polyploid comprising:

(1) Chemical mutagenesis agents including sulfamethoxazole and orange peel essential oil; wherein, the content of the sulfamethoxazole in the chemical mutagenesis reagent is 5-30 mg/L, preferably 6-25 mg/L, more preferably 8-20 mg/L; wherein, the content of the orange peel essential oil in the chemical mutagenesis reagent is 0.03-0.3% (volume percent), preferably 0.05-0.2%, more preferably 0.08-0.18%;

(2) Regeneration medium (bud induction regeneration medium) containing lanthanum nitrate; wherein, the content of lanthanum nitrate is 60-160 mg/L, preferably 80-140 mg/L, more preferably 90-130 mg/L.

In one or more embodiments, the kit further comprises one or more reagents selected from the group consisting of:

a sterilizing agent for sterilizing the explant;

washing the reagent;

regeneration medium (shoot induction regeneration medium);

proliferation and propagation culture medium;

rooting culture medium; and/or

Instructions for the preparation of the shinyleaf yellowhorn polyploid are described.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, regenerated shoots after treatment of explant stems.

FIG. 2, tissue culture seedlings of variation such as stems and leaves selected by plant morphology index identification method (right panel), conventional seedlings (left panel).

FIG. 3, tetraploid root seedlings ready for transplanting.

FIG. 4, tetraploid transplanted into greenhouse for 4 months (right panel), diploid seedling (left panel).

FIG. 5, tetraploid leaves (left panel), diploid seedling leaves (right panel) grown for 4 months in greenhouse.

FIG. 6, cytologically identifying chromosomes (diploid on the left, tetraploid on the right).

FIG. 7, comparison of stomata number, size and chloroplast number (left panel is diploid, right panel tetraploid).

FIG. 8, flow cytometer identification (left panel is diploid, right panel tetraploid).

Detailed Description

The inventor of the invention has disclosed a shinyleaf yellowhorn polyploid cultivation method through intensive research. According to the characteristic of the shinyleaf yellowhorn, the invention selects the mutagenesis reagent which is favorable for improving the mutagenesis efficiency and has safety and the chemical substance which promotes the mutagenesis. The method can obtain the shinyleaf yellowhorn plant with obvious polyploid characteristics, and has the advantages of simplicity, convenience, reasonability, low cost, low toxicity and high induction frequency. Homozygous shinyleaf yellowhorn polyploid plants can be confirmed by various detection methods. Through detection and identification, the method provided by the invention effectively reduces the occurrence of shinyleaf yellowhorn chimera. The invention provides a new way for cultivating new varieties of shinyleaf yellowhorn, and the obtained shinyleaf yellowhorn polyploid has high application value.

Terminology

As used herein, the term "explant" refers to a piece of an organ or tissue in plant tissue culture that is the material of culture ex vivo.

As used herein, the term "polyploid" refers to a plant that has a greater number of chromosomes than "diploid", such as "tetraploid".

As used herein, the "dark culture" is a 24 hour day culture in a dark environment.

As used herein, the terms "light culture" or "normal light conditions" or "normal light culture" are used interchangeably, and are cultured in a dark environment for 16±2 hours, preferably 16±1 hour, and 8±1 hour, in 24 hours a day, wherein the light intensity is 1500 to 3000LUX; preferably 2000-3000LUX.

As used herein, an "increase", "increase" is a statistically significant "increase", such as an increase/increase of 5%, 10%, 20%, 30%, 50%, 60%, 80%, 100%, 120% or more. The "increase", "increase" includes "multiplication". The "increasing", "increasing" includes, but is not limited to: plant leaves, cells, organs, stem thickness, leaf area "increase", "increase".

As used herein, the "greener shade of a leaf" is a statistically significant darkening of color.

As used herein, the natural environment includes, but is not limited to: fields, nursery, tree forests, and the like.

As used herein, "comprising," "including," or "containing" means that the various ingredients can be used together in a mixture, composition, or medium of the invention. Thus, the terms "consisting essentially of" and "consisting of" are encompassed by the terms "comprising," including, "or" comprising.

As used herein, the kit refers to a package/box that is convenient for commercial scale production and use, typically containing a container/packet in which the medium, solution, single component or component of the present invention may be placed.

Preparation method of shinyleaf yellowhorn polyploid

The invention provides a method for preparing shinyleaf yellowhorn polyploid, which comprises the following steps: (a) Performing chemical mutagenesis on the explant of the shinyleaf yellowhorn by using sulfaming, and simultaneously adding the orange peel essential oil; (b) Performing adventitious bud induction on the chemically mutagenized explant obtained in the step (a), and simultaneously adding lanthanum nitrate; (c) culturing and seedling the adventitious bud obtained in (b); proliferation and propagation culture; rooting culture; obtaining the shinyleaf yellowhorn polyploid. In a preferred mode, the method further comprises the step of identifying/selecting polyploids after seedling formation and after propagation culture.

Chemical mutagenesis

The starting material for chemical mutagenesis may be an explant obtained from the shoot of shinyleaf yellowhorn, or may be the seed of shinyleaf yellowhorn. From the final mutagenesis effect point of view, as a preferred mode of the invention, the explants of shinyleaf yellowhorn are used as starting materials. Preferably, the current annual shoots of shinyleaf yellowhorn are obtained as explants and serve as starting materials for chemical mutagenesis. Preferably, the method further comprises the step of sterilizing the branches before the initial culture, wherein the sterilization can be performed by using alcohol, detergent, sodium hypochlorite and other reagents, or the combination of the alcohol, the detergent, the sodium hypochlorite and the reagents can be used.

Colchicine is used in the art to a great extent as a chemical inducer to mutagenize polyploids. However, in the shiny-leaved yellowhorn plant, colchicine contains harmful substances and has high toxicity to the shiny-leaved yellowhorn plant; colchicine is also relatively expensive. Through research and comparison, the inventor uses sulfamide to replace colchicine to treat shinyleaf yellowhorn tissue culture seedlings. The inventors have unexpectedly found that the mutagenesis efficiency of amisulprine is significantly higher than that of colchicine. Sulfenuron is commonly used as a herbicide, and has low cost and relatively low toxicity. Sulfamlinger doubles the chromosome by inhibiting the mitosis of plants.

According to the effects presented by the embodiment of the invention, the tissue culture seedlings obtained after the explants are treated by colchicine are easy to turn yellow, slow to grow and high in death rate; in particular, when the concentration of colchicine is relatively high, it causes death of a part of the tissue culture material, and it is impossible to regenerate and differentiate into buds, whereas the induction effect is not ideal when the concentration is low. However, the shoots after treatment with amisulpride remained fresh green, had a large number of regenerated shoots, were less harmful to plants, and their concentrations were relatively easy to control.

As a preferred mode of the present invention, the amount of sulfamethoxazole used in the mutagenesis treatment is 5 to 30mg/L, preferably 6 to 25mg/L, more preferably 8 to 20mg/L.

Using sulfamethoxazole as a mutagen, the present inventors further studied a method for promoting the mutagenesis efficiency. Based on a plurality of chemical substances as research basis, the inventor performs extensive screening work, and as a result, the chemical substance of the orange peel essential oil is selected, and the chemical substance is matched with a mutagen for simultaneous use, so that the method has a remarkable promotion effect on the mutagenesis of shiny-leaved yellowhorn.

At the time of mutagenesis treatment, the inventors compared the effect of addition of orange peel essential oil with addition of DMSO (dimethyl sulfoxide) on mutagenesis. The results show that the addition of orange peel essential oil can promote the mutagenesis rate to be greatly improved compared with the addition of DMSO. Meanwhile, the inventor also observes that the addition of the orange peel essential oil is basically harmless to the shiny-leaved yellowhorn explant and the seedlings formed by the shiny-leaved yellowhorn explant, but the DMSO concentration is low and has no effect, and the treated plant material is damaged.

As a preferred mode of the present invention, the amount of the orange peel essential oil is 0.03 to 0.3%, preferably 0.05 to 0.2%, more preferably 0.08 to 0.18%.

As a preferred mode of the present invention, the time for the chemical mutagenesis is 12 to 72 hours, preferably 18 to 60 hours, more preferably 24 to 48 hours. It should be understood that although such a period of time is preferred in the present invention, appropriate changes may be made depending on the actual situation, and such changes are also included in the present invention.

Adventitious bud induction, proliferation propagation and rooting

Using sulfamethoxazole as a mutagen, the present inventors have further studied methods for promoting mutagenesis efficiency. The present inventors have conducted extensive screening work on a large number of chemicals, selected to be lanthanum nitrate. Unlike orange peel essential oil, lanthanum nitrate is not added in the mutagenesis stage, but is added in the culture medium in the specific stage of induction by the adventitious bud after mutagenesis, so that the improvement of the mutagenesis rate of shiny-leaved yellowhorn polyploid is promoted.

Lanthanum nitrate was used Yu Zupei in the prior art and is believed to have an effect on regulating root growth, whereas it was used in the present invention in another stage of tissue culture (non-rooting stage). The research of the inventor shows that for shinyleaf yellowhorn induced polyploidy, the adding time of lanthanum nitrate is important, and the effect of obviously improving the mutagenesis effect can not be achieved at other adding time.

As a preferred mode of the present invention, lanthanum nitrate is used in an amount of 60 to 160mg/L, preferably 80 to 140mg/L, more preferably 90 to 130mg/L.

As a preferred mode of the present invention, the time for adventitious bud induction (lanthanum nitrate addition) is 15 to 35 days, preferably 16 to 30 days, more preferably 18 to 25 days. It will be appreciated that although such a time period is preferred in the present invention, appropriate changes may be made depending on the differences in the different clones and such changes are also included in the present invention.

After chemical mutagenesis, the explants were transferred to regeneration medium for adventitious bud induction, lanthanum nitrate was added at this stage. As a preferred embodiment, regeneration medium is used, including basal medium and 6-BA and NAA. The application of 6-BA and NAA is favorable for bud induction, and the combination of the 6-BA and NAA is better than the use of only 6-BA, so that cluster buds are easy to induce.

After induction of adventitious buds, proliferation and propagation culture are performed. As a preferred embodiment, the propagation culture used is preferably MS+1-1.5 mg/L6-BA+0.5-1 mg/L NAA.

It will be appreciated that other suitable culture media for shoot induction/shoot regeneration of shinyleaf yellowhorn are also useful, for example in the present inventors' prior patent (CN 202010064052.0), for this species of shinyleaf yellowhorn, a priming medium to promote shoot production and a cluster shoot induction medium to promote cluster shoot induction culture are optimized, which can also be used in the present invention.

Preferably, the post-mutagenesis, sprouting, post-emergence plants may also be subjected to a strong seedling culture, if desired, see for example the medium and methods used in the inventors' prior patent (CN 202010064052.0).

After propagation and propagation culture (preferably also including strong seedling culture), the inventor further performs rooting culture of shinyleaf yellowhorn seedlings, including: cutting the obtained bud cluster into single plants, and culturing with rooting culture medium to obtain rooted test tube plantlets. As a preferred embodiment, the rooting medium comprises a basal medium and indole-3 butyric acid; preferably, the basal medium is 1/2MS medium.

It will be appreciated that other media suitable for root regeneration of shinyleaf yellowhorn are also available, for example in the present inventors' prior patent (CN 202010064052.0), for this species of shinyleaf yellowhorn, a rooting medium that promotes root production, growth and robustness is optimized, which can also be used in the present invention.

The MS culture medium or the 1/2MS culture medium can be prepared by self or is commercially available. It is to be understood that media which are suitably modified (e.g., fine tuning or replacement of a part of the major or minor components) on the basis of these basal media, but which retain their function as basal media, are also encompassed by the present invention.

After the test-tube plantlet with root is obtained, further, the test-tube plantlet can be transplanted into a seedling culture matrix for domestication and seedling hardening; and then transplanting the seedling after seedling hardening into natural environment. Domestication and seedling hardening may be performed using techniques known in the art. As a preferred mode of the invention, the seedling substrate comprises peat soil, perlite, vermiculite or a combination thereof.

Selection of polyploids

After chemical mutagenesis, the explants are subjected to bud induction, the obtained buds are cultivated to generate seedlings, and further propagation culture and rooting culture can be performed. In the above stage, some buds/seedlings are induced to successfully form polyploids; the other part is wild type or ploidy chimeric body, and the desired polyploid cannot be formed. Therefore, at an appropriate stage of incubation after mutagenesis, isolation (selection)/enrichment of polyploids is required.

As a preferred mode of the invention, the selection/enrichment of polyploid seedlings is performed once after the seedling formation. In this stage, polyploid seedlings are preferably selected by plant morphology index identification; the plant morphology metrics may include (but are not limited to): stem height, stem thickness, leaf color, leaf thickness, leaf area; more preferably, if the stem is short, the stem is thick, the green color of the leaf is deeper, the leaf thickness is increased, the leaf area is increased, the plant is shown to be polyploid.

The variation can be primarily screened out through morphological feature identification (such as obviously enlarged leaves, green leaves and shortened stems), and the method is a quick and effective primary selection means. Can greatly save manpower and material resources.

As a preferred mode of the present invention, after the above selection/enrichment, the polyploid seedlings preliminarily identified are subjected to multiplication and multiplication culture more than 2 times (algebra), preferably more than 5 times, more preferably more than 10 times. The more times of propagation are beneficial to obtaining the homozygous polyploid seedlings with stable genetic characters.

As a preferred mode of the invention, the method further comprises a process of further identifying and selecting polyploid seedlings after the proliferation and propagation culture; preferably, an assay selected from the group consisting of: chloroplast identification, chromosome identification, and flow cytometry analysis.

The polyploid identification method includes root tip tabletting chromosome counting method, guard cell length measuring method, stomata guard cell chloroplast counting method, etc. In contrast, these methods are time-consuming and cumbersome to operate, and require accurate grasp of the cell's period of division that is easily observed. As a preferred mode of the present invention, the DNA content of cells is detected by a flow cytometer, which can accurately determine not only whether or not the cells are chromosome doubled, but also the number of chromosome doubled and non-doubled cells, thereby determining whether or not they are ploidy chimeras. In addition, the method is not limited by the plant material sampling part and the cell period, and has better operability than chromosome counting. The identification by using the flow cytometer is a simple and feasible method with high detection speed and accuracy.

In the specific embodiment of the invention, a specific cultivation method of shinyleaf yellowhorn polyploid is provided, which comprises the steps of treating stem segments of tissue culture seedlings by using an inducer, germinating regeneration buds and axillary buds, and carrying out proliferation cultivation on the axillary buds; different double detection methods are also provided; transplanting the buds identified as polyploids into a rooting culture medium, transplanting the rooting seedlings into a greenhouse, and obtaining shinyleaf yellowhorn polyploid tissue culture seedling plants.

In the specific embodiment of the invention, the effective chemical mutagen and the effective chemical components are screened to treat the clone tissue culture material, so that the polyploid plant is obtained, and the polyploid plant has obvious polyploid characteristics. In addition, the sulfamethoxazole which has low cost and relatively small toxicity is found to have higher induction frequency than colchicine which is a polyploid inducer conventionally used, and the homozygous polyploid plant is confirmed through a plurality of detection methods. The shinyleaf yellowhorn polyploid obtained by the method has higher application value.

The characteristics of the polyploid plant obtained by the method of the invention include: the stalks are thick, the fruits and seeds are large, and the contents of sugar and protein nutrients are obviously increased.

Reagent and kit for preparing shinyleaf yellowhorn polyploid

Based on the mutagenesis technology and the cultivation technology, the invention also provides a kit for preparing the shinyleaf yellowhorn polyploid, which comprises the following components: (1) Chemical mutagenesis agents including sulfamethoxazole and orange peel essential oil; wherein, the content of the sulfamethoxazole in the chemical mutagenesis reagent is 5-30 mg/L, preferably 6-25 mg/L, more preferably 8-20 mg/L; wherein, the content of the orange peel essential oil in the chemical mutagenesis reagent is 0.03-0.3, preferably 0.05-0.2%, more preferably 0.08-0.18%; (2) Regeneration medium (bud induction regeneration medium) containing lanthanum nitrate; wherein, the content of lanthanum nitrate is 60-160 mg/L, preferably 80-140 mg/L, more preferably 90-130 mg/L.

As a preferred mode of the invention, sucrose and agar are also included in the kit to be used for addition to the medium.

As a preferred mode of the present invention, the kit further comprises a plurality of basic culture mediums for plant cultivation, comprising: MS medium or 1/2MS medium. It will be appreciated that the MS medium may also be a modified MS medium and the 1/2MS medium may also be a modified 1/2MS medium.

As a preferred mode of the present invention, the kit further comprises one or more reagents selected from the group consisting of: a sterilizing agent for sterilizing the explant; washing the reagent; regeneration medium (shoot induction regeneration medium); proliferation and propagation culture medium; rooting culture medium.

As a preferred mode of the invention, the kit also comprises a use instruction, wherein the preparation method for preparing the shinyleaf yellowhorn polyploid, the use method of the corresponding culture medium or the treatment method for plants or plant tissues before or after the use of the culture medium are described, so that the method is beneficial to guiding the person skilled in the art to use the kit in a proper method.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer.

Example 1 cultivation method of polyploid of woody oil tree shinyleaf yellowhorn

Taking the explant of woody oil tree shinyleaf yellowhorn as a starting material, and carrying out mutagenesis and cultivation.

1. Chemical mutagenesis

The tissue culture Miao Quchu leaves are cut into stem segments of 2-3 cm. The stem segments are soaked in colchicine solutions (0.05%, 0.1%,0.2%,0.3%, 0.5%) or sulfamethoxazole (10 mg/L,15mg/L,20mg/L,30 mg/L) with different concentrations, and after 24 or 48 hours of induction cultivation, the induction solution on the surface of the stem segments is removed after washing with clear water. According to the subsequent examples, DMSO and orange peel essential oil are optionally added.

2. Adventitious bud induction and proliferation propagation

The stem tip after induction treatment is inoculated in regeneration culture medium (MS+2-3 mg/L6-BA+0.5-1 mg/L NAA; optionally 100-120mg/L lanthanum nitrate is also added).

After the stem section was cultured on the bud induction regeneration medium for 20 days, adventitious buds were regenerated at the wound site of the stem section and on the stem section.

After the regenerated buds grow into seedlings, the plant cell and each organ volume generally tends to increase along with the increase of the chromosome ploidy of the cells by a plant morphology index identification method, so that the variant seedlings with thick stems, dark green leaves, thick leaves, increase and the like are selected, transferred into a fresh culture medium and subjected to proliferation and propagation culture (MS+1-1.5 mg/L6-BA+0.5-1 mg/L NAA).

The method can be used for propagating for more than 10 times, and the more the propagation times are, the more homozygous polyploid seedlings with stable genetic characters can be obtained.

3. Morphological index identification

Chloroplast identification: firstly, taking appearance variant seedlings with stable characters, and indirectly identifying ploidy through chloroplasts and air holes. And (3) the primary polyploid seedlings obtained after the identification of the number of chloroplasts in guard cells are ensured through the size and the density of the air holes.

Chromosome identification: transferring the seedling initially identified as polyploid by chloroplast into rooting culture medium, rooting about 10 days, and directly identifying by adopting tabletting method and chromosome counting method with root tip as material.

Flow cytometry identification: the plants identified as polyploid by chromosome identification were subjected to cell flow meter detection, fresh leaves were placed in a buffer solution of fluorescent dye DAPI, minced with a knife, and the suspension was filtered through a nylon mesh. A flow instrument was used to detect the DNA content of a large number of cells at the interphase. The ploidy of the nucleus is finally expressed as a C value, 1C for the nuclear haploid and 2C for the nuclear diploid. The flow cytometry is characterized by being rapid, simple and accurate, and can clearly distinguish whether the flow cytometry is homozygous or heterozygous through peak values.

4. Rooting culture

The seedlings, which have been confirmed to be polyploid after a plurality of detection tests, are inoculated into rooting culture medium for culture (1/2MS+0.5-1 mg/L indole-3 butyric acid).

Under the light culture (16 h light and 8h darkness), the seedlings begin to come out after about 7-10 days, the average of 3-6 seedlings is 3-6, and about 20-25 days and 90% of the seedlings are transplanted into natural environment (field) when about 2 cm exists.

In this example, all media were autoclaved at 121℃for 20 minutes with the addition of 20 g/l sucrose and 6.5 g/l agar (pH 5.8-6.0). The culture temperature is 25+/-2 ℃, the illumination time is 16h/8h, and the illumination intensity is 2000-3000Lux.

Example 2 Induction of results by different mutagens

In the chemical mutagenesis step, shinyleaf yellowhorn explants can be treated with colchicine to induce doubling of the intracellular chromosome number in order to develop into polyploid plants. However, colchicine contains many harmful substances, and is toxic and expensive. Other mutagens such as dichlorobenzene, 8-hydroxyquinoline (8-OHQ), heavy water, ethyl methylsulfonate, and the like.

The inventor finds that the sulfamethoxazole has a significantly better mutagenesis effect than colchicine through comparative screening. Based on the mutagen cultivation method of example 1, the shinyleaf yellowhorn stem explants were treated with colchicine and sulfaming for different concentrations and times, and the treatment methods and results are shown in Table 1.

TABLE 1

A4% homozygous tetraploid mutagenesis rate was obtained by 24 hours of treatment with colchicine concentrations of 0.1-0.3 mg/L. When the colchicine concentration is higher than 0.3mg/L, the tissue culture material is easy to die after more than 24 hours, regeneration and differentiation of buds cannot be realized, and if the colchicine concentration is low, the time is short, the regeneration bud rate is high, but the mutant cannot be obtained.

The tetraploid mutagenesis rate of 9% homozygote can be obtained by using 10-15mg/L amisulpride for 48 hours, the mutagenesis rate is high, and the mutagenesis rate is also high after 24 hours of treatment, and reaches 5%. Meanwhile, the branches after being treated by the amisulpride are kept fresh green, and the regenerated buds are more (10 mg/L amisulpride is treated for 48 hours and is 30% more than 0.1mg/L colchicine is treated for 48 hours), so that the damage to plants is remarkably light.

Thus, amisulprine can completely replace colchicine.

EXAMPLE 3 screening of chemical Components that increase mutagenesis Rate

Using sulfamethoxazole as a mutagen, the present inventors further studied a method for promoting the mutagenesis efficiency. The method has the advantages that the method carries out extensive screening work from a plurality of chemical substances, selects the chemical substance of the orange peel essential oil, and uses the chemical substance to be matched with a mutagen for simultaneous use, thereby having a promotion effect on the mutagenesis of the shinyleaf yellowhorn.

Based on the mutagen cultivation method of example 1, the shinyleaf yellowhorn stem segment explants were treated with sulfamide, and DMSO or orange peel essential oil was added, and the treatment method and the results are shown in table 2.

TABLE 2

* Sulfamethoxazole 10mg/L for 48 hours.

Therefore, the sulfamide is taken as a mutagen and added with DMSO (dimethyl sulfoxide) to have a promoting effect on doubling; the orange peel essential oil has the effect of improving the mutagenesis rate and is remarkably higher than the addition mutagenesis rate.

It is seen in the observation of shinyleaf yellowhorn bud growth and seedling stage growth that orange peel essential oil is basically harmless to plants, but low DMSO concentration has no effect and high DMSO concentration can harm the treated plant material.

EXAMPLE 4 screening of chemical substances that increase mutagenesis Rate

Using sulfamethoxazole as a mutagen, the present inventors further studied a method for promoting the mutagenesis efficiency. The inventor performs extensive screening work from a plurality of chemical substances, selects the chemical substance lanthanum nitrate, and adds the chemical substance lanthanum nitrate into a culture medium at a specific stage of adventitious bud induction after mutagenesis, thereby having a promotion effect on improving the mutagenesis rate of the shinyleaf yellowhorn polyploid. The timing of lanthanum nitrate addition is also specific, and if lanthanum nitrate is added to the rooting medium, rooting is not possible.

Based on the mutagen cultivation method of the embodiment 1, in the mutagenesis stage, the inventor applies sulfaming and is matched with orange peel essential oil to treat the shiny-leaved yellowhorn stem segment explant; in the adventitious bud induction stage, the inventors added lanthanum nitrate to the medium, and the treatment method and the results are shown in Table 3.

TABLE 3 Table 3

According to the table, after lanthanum nitrate is added into the regeneration culture medium, the mutagenesis rate is remarkably improved.

By using the addition mode of the 1 st group or the 2 nd group in the table 3, the representative diagram of the tetraploid regenerated buds after the stem sections of the explants are treated is shown in fig. 1, and it can be seen that the regenerated buds on the explants are quite many, and the regenerated buds are dark green, have a thick and stronger morphology and are remarkably larger than that of diploid, and the growth state of the regenerated buds is quite ideal.

The morphology of the mutated tissue culture seedlings (right panel) of stems and leaves was compared with that of the conventional seedlings (left panel) as shown in fig. 2. Therefore, the mutation can be initially screened by applying the plant morphological index identification method, and the method is a quick and effective primary selection means.

Tetraploids were selected after induction and cultivation by the addition of group 1 or group 2 in Table 3, and their representative root seedlings are shown in FIG. 3.

Representative plots of tetraploids (plants in right panel) and diploid seedlings (plants in left panel) transplanted into a greenhouse for 4 months of growth are shown in FIG. 4.

Tetraploid leaves (left panel) grown for 4 months in a greenhouse were transplanted and representative figures of diploid seedling leaves (right panel) are shown in fig. 5.

For the tetraploids obtained as well as diploids, chromosomes were analyzed at the cytological level, and a representative plot is shown in FIG. 6, wherein the left plot is diploid and the right plot is tetraploid.

The number of stomata, size and chloroplast count of the obtained tetraploid and diploid were analyzed, and the results are shown in FIG. 7, wherein the left graph is diploid and the right graph is tetraploid. The number of visible stomata is large, the number of the visible stomata is small, and the number of the chloroplasts is large.

For the tetraploids obtained as well as for the diploids, identified using flow cytometry, a representative plot is shown in FIG. 8, with the left plot being seen to be around 50 for the peak of the control diploid and around 100 for the right plot.

And (3) carrying out survival rate statistics, wherein the survival rate of the polyploid seedlings after rooting is more than 95%, and the polyploid seedlings are not different from the diploid seedlings.

EXAMPLE 5 mutagenesis comparison Using seeds of shinyleaf yellowhorn as starting Material

In addition to mutagenesis and cultivation of shinyleaf yellowhorn explants, the inventors have also studied mutagenesis with shinyleaf yellowhorn seeds as starting material.

Aiming at germinated shinyleaf yellowhorn seeds, the inventor respectively treats the shinyleaf yellowhorn seeds with colchicine and sulfaming, and optionally adds orange peel essential oil in a mutagenesis stage to observe the situation of mutagenesis into polyploid. In another mode, the unmalted seed is immersed in a lanthanum nitrate solution for 48 hours prior to the mutagenesis stage.

The results are shown in Table 4.

TABLE 4 Table 4

As can be seen from the above table, polyploid induction rate is relatively low and heterozygotes are more. If the seeds are soaked in 100-120mg/L lanthanum nitrate solution for 48 hours, the induction rate can be improved.

However, according to the statistical result of the mutagenesis rate, the mutagenesis rate using seeds as starting material is significantly lower than that using explants as starting material, and fewer homozygotes, mostly chimeras, are obtained. Therefore, the induction rate of the tissue culture material is high, the operation is easy, the labor and the trouble are saved, and the regeneration rate can be improved by changing the formula of the culture medium, so that the mutagenesis rate of polyploid is improved.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (19)

1. A method of making a shinyleaf yellowhorn polyploid, said polyploid being a homozygous tetraploid, said method comprising:

(a) Performing chemical mutagenesis on the explant of the shinyleaf yellowhorn by 6-25 mg/L of sulfaming agent for 18-60 hours, and simultaneously adding 0.03-0.3% of orange peel essential oil; the explant is a stem segment of a shinyleaf yellowhorn tissue culture seedling;

(b) Performing adventitious bud induction on the chemically-mutagenized explant obtained in the step (a) for 16-30 days, wherein a regeneration medium is adopted for adventitious bud induction, and is an MS medium containing 1.5-4 mg/L6-BA and 0.4-1.5mg/L NAA, and 80-140 mg/L lanthanum nitrate is added at the same time;

(c) Culturing the adventitious bud obtained in the step (b), forming seedlings, and identifying and selecting polyploid seedlings through plant morphological indexes: if the cell volume is increased, the organ volume is increased, the stem thickness is increased, the green color of the leaf is deeper, the leaf thickness is increased, and the leaf area is increased, the plant is shown to be polyploid; proliferation and propagation culture; rooting culture; obtaining shinyleaf yellowhorn polyploid; the culture medium for proliferation and propagation is MS culture medium containing 1-1.5 mg/L6-BA and 0.5-1mg/L NAA; the rooting culture medium is 1/2MS culture medium containing 0.5-1mg/L indole-3 butyric acid.

2. The method of claim 1, wherein in step (c), the proliferation is performed more than 2 times.

3. The method of claim 2, wherein the proliferation culture is propagated more than 5 times.

4. The method of claim 3, wherein the proliferation culture is propagated more than 10 times.

5. The method of claim 1, wherein in step (c), after said propagating culture, further comprising: identifying and selecting polyploid seedlings; the following measurements were performed: chloroplast identification, chromosome identification, and flow cytometry analysis.

6. The method of claim 5, wherein in the chloroplast identification, the plant is polyploid if the stomata increase, the stomata density decrease, and the number of chloroplasts in the guard cell increase are identified by stomata size, stomata density, and the number of chloroplasts in the guard cell.

7. The method of claim 5, wherein in the chromosome identification, chromosome counting identification is performed by using a tabletting method and taking root tips as materials, and if the number of chromosomes is doubled, the plant is indicated to be polyploid.

8. The method of claim 5, wherein the flow cytometric analysis detects cellular DNA content and wherein an increase in DNA content indicates that the plant is polyploid.

9. The method of claim 1, wherein in step (a), the amount of sulfamethoxazole is from 8 to 20mg/L.

10. The method of claim 1, wherein in step (a), the amount of orange peel essential oil is 0.05 to 0.2%.

11. The method of claim 1, wherein in step (b), lanthanum nitrate is used in an amount of 90 to 130mg/L.

12. The method of claim 1, wherein in step (a), the time for chemical mutagenesis is from 24 to 48 hours.

13. The method of claim 1, wherein in step (b), the adventitious bud induction time is 18 to 25 days.

14. Use of the method according to any one of claims 1 to 13 for the preparation of a shinyleaf yellowhorn polyploid.

15. A kit for preparing a shinyleaf yellowhorn polyploid, said polyploid being a homozygous tetraploid, said kit comprising:

(1) Chemical mutagenesis agents including sulfamethoxazole and orange peel essential oil; wherein, the content of the sulfamethoxazole in the chemical mutagenesis reagent is 6-25 mg/L; wherein, the content of the orange peel essential oil in the chemical mutagenesis reagent is 0.03-0.3%; the chemical mutagenesis reagent is used for carrying out chemical mutagenesis on the explant for 18-60 hours;

(2) A regeneration culture medium which is an MS culture medium containing 1.5-4 mg/L6-BA and 0.4-1.5mg/L NAA and contains 80-140 mg/L lanthanum nitrate; the regeneration culture medium is used for carrying out adventitious bud induction on the chemically-mutagenized explant for 16-30 days;

(3) The culture medium for proliferation and propagation is MS culture medium containing 1-1.5 mg/L6-BA and 0.5-1mg/L NAA;

(4) A rooting culture medium which is 1/2MS culture medium containing 0.5-1mg/L indole-3 butyric acid; and

the explant of shinyleaf yellowhorn is the stem section of shinyleaf yellowhorn tissue culture seedling.

16. The kit for preparing a shinyleaf yellowhorn polyploid of claim 15, wherein the content of amisulprine in the chemical mutagenesis reagent in (1) is 8-20 mg/L.

17. The kit for preparing shiny-leaved yellowhorn polyploid of claim 15, wherein the content of orange peel essential oil in the chemical mutagenesis reagent in (1) is 0.05-0.2%.

18. The kit for preparing shinyleaf yellowhorn polyploid according to claim 15, wherein in (2), the content of lanthanum nitrate is 90-130 mg/L.

19. The kit of claim 15, further comprising one or more reagents selected from the group consisting of:

a sterilizing agent for sterilizing the explant;

washing the reagent; and/or

Instructions for the preparation of the shinyleaf yellowhorn polyploid are described.

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