阅读说明：本技术 一种四倍体头花蓼的培育方法 (Method for cultivating tetraploid polygonum capitatum ) 是由 刘世会 赵德刚 于 2019-11-08 设计创作，主要内容包括：本发明提供一种四倍体头花蓼的培育方法,该方法包括以下步骤：a)将二倍体头花蓼种子进行处理,然后接种于培养基中进行培养,得到头花蓼茎尖作为诱导材料；b)采用秋水仙素溶液对步骤a)中得到的头花蓼茎尖进行诱导；c)将步骤b)中得到的头花蓼茎尖移到芽增殖培养基中进行诱导,以诱导头花蓼芽增殖,得到头花蓼增殖芽；d)对步骤c)中得到的头花蓼增殖芽进行鉴定,得到四倍体头花蓼增殖芽；e)对步骤d)中得到的四倍体头花蓼增殖芽进行组织培养,以使四倍体头花蓼快速繁殖,得到四倍体头花蓼秧苗。本发明提供的培育方法能够减少育种过程中的倍性鉴定工作量,提高鉴定速度,缩短育种时间,从而为农业生产提供大量优良种苗。(The invention provides a method for cultivating tetraploid polygonum capitatum, which comprises the following steps: a) treating diploid polygonum capitatum seeds, and then inoculating the diploid polygonum capitatum seeds into a culture medium for culturing to obtain polygonum capitatum stem tips as an induction material; b) inducing the polygonum capitatum stem tip obtained in the step a) by using colchicine solution; c) transferring the stem tip of the polygonum capitatum obtained in the step b) into a bud proliferation culture medium for induction so as to induce the polygonum capitatum bud to proliferate and obtain the polygonum capitatum proliferation bud; d) identifying the polygonum capitatum proliferated bud obtained in the step c) to obtain a tetraploid polygonum capitatum proliferated bud; e) performing tissue culture on the tetraploid polygonum capitatum proliferated bud obtained in the step d) to rapidly propagate the tetraploid polygonum capitatum, so as to obtain tetraploid polygonum capitatum seedlings. The cultivation method provided by the invention can reduce the ploidy identification workload in the breeding process, improve the identification speed and shorten the breeding time, thereby providing a large amount of excellent seedlings for agricultural production.)

1. A cultivation method of tetraploid polygonum capitatum is characterized by comprising the following steps:

a) treating diploid polygonum capitatum seeds, and then inoculating the diploid polygonum capitatum seeds into a culture medium for culturing to obtain polygonum capitatum stem tips as an induction material;

b) inducing the polygonum capitatum stem tip obtained in the step a) by using colchicine solution;

c) transferring the induced stem tip of the polygonum capitatum obtained in the step b) into a bud proliferation culture medium for induction so as to induce the polygonum capitatum bud to proliferate and obtain a polygonum capitatum proliferation bud;

d) identifying the polygonum capitatum proliferated bud obtained in the step c) to obtain a tetraploid polygonum capitatum proliferated bud;

e) carrying out tissue culture on the tetraploid polygonum capitatum proliferation bud obtained in the step d) so as to rapidly propagate the tetraploid polygonum capitatum and obtain tetraploid polygonum capitatum plant seedlings.

2. Incubation method according to claim 1, characterized in that step a) comprises: fumigating diploid polygonum capitatum seeds, then carrying out sterile disinfection treatment, then inoculating the seeds into a culture medium for culture, and cutting polygonum capitatum stem tips under the sterile condition to obtain sterile polygonum capitatum stem tips as an induction material.

3. Incubation method according to claim 2, characterized in that step a) comprises: fumigating diploid polygonum capitatum seed, then carrying out surface treatment by using alcohol solution, and then HgC1₂Sterilizing the solution, washing with sterile water, inoculating into culture medium, culturing in light culture room, and cutting stem tip of herba Polygoni Capitati under sterile condition to obtain sterile stem tip of herba Polygoni Capitati as inducing material;

preferably, step a) comprises: fumigating diploid polygonum capitatum seeds by using a mixed solution of concentrated HCl and NaClO for 6-48h, preferably 6h, 12h, 18h, 24h, 30h, 36h, 42h and 48h, more preferably 24 h; subsequently performing surface treatment with alcoholic solution for 5-30s, preferably 5s, 10s, 15s, 20s, 25s, 30s, more preferably 10s, and HgC1₂Sterilizing the solution for 1-9min, preferably 1min, 3min, 5min, 7min, 9min, more preferably 3min, washing with sterile water for 1-7 times, preferably 1 time, 2 times, 3 times, 4 timesInoculating to culture medium, culturing in light culture room for 15-40 days, preferably 15, 20, 25, 30, 35, 40 days, more preferably 25 days, and cutting stem tip of herba Polygoni Capitati under aseptic condition to obtain sterile stem tip of herba Polygoni Capitati as inducing material.

4. The cultivation method as claimed in claim 3, wherein the NaClO content in the mixed solution of concentrated HCl and NaClO in step a) is 4.8-6.0% w/v, preferably 4.8% w/v, 5.0% w/v, 5.2% w/v, 5.4% w/v, 5.6% w/v, 5.8% w/v, 6.0% w/v, more preferably 5.2% w/v;

preferably, the concentration of the alcoholic solution in step a) is 70-75% v/v, preferably 70% v/v, 75% v/v, more preferably 75% v/v;

preferably HgC1 in step a)₂The concentration of the solution is 0.1-0.2% w/v, preferably 0.1% w/v, 0.15% w/v, 0.2% w/v, more preferably 0.1% w/v;

preferably, the temperature of the light culture chamber in step a) is set at 23-25 ℃, preferably 23 ℃, 24 ℃, 25 ℃, more preferably 25 ℃; the illumination intensity is set to 2000-3000lx, preferably 2000lx, 2500lx, 3000lx, more preferably 2000 lx;

preferably, the medium in step a) is MS hormone-free solid medium.

5. Incubation method according to any of claims 1-4, characterized in that step b) comprises: putting the polygonum capitatum stem tip obtained in the step a) in a colchicine solution, and carrying out shading soaking induction under the aseptic condition;

preferably, step b) comprises: placing the polygonum capitatum stem tip obtained in the step a) in a colchicine solution, and carrying out light-shielding soaking induction for 12-36h, preferably 12h, 18h, 24h, 30h, 36h, more preferably 24h, under the aseptic condition;

preferably, the concentration of the colchicine solution in step b) is 0.1-0.9% w/v, preferably 0.1% w/v, 0.3% w/v, 0.5% w/v, 0.7% w/v, 0.9% w/v, more preferably 0.5% w/v;

preferably, the colchicine solution in step b) is prepared with sterile cold water under sterile conditions, i.e. ready to use.

6. The cultivation method as claimed in any one of claims 1 to 5, wherein the shoot multiplication medium in step c) comprises MS powder, sucrose, glycine, phytogel, 6-BA and NAA; and the pH of the shoot multiplication medium is 5.8;

preferably, in the bud multiplication medium in the step c), the concentration of MS powder is 4.43g/L, the concentration of sucrose is 30g/L, the concentration of glycine is 2.0mg/L, the concentration of plant gel is 4g/L, the concentration of 6-BA is 2.0mg/L, and the concentration of NAA is 0.1 mg/L;

preferably, step c) is carried out under sterile conditions;

preferably, the period of inducing the polygonum capitatum buds to proliferate in the step c) is 30 days, and the culture medium is changed once in 15 days.

7. Incubation method according to any of claims 1 to 6, characterized in that step d) comprises: identifying the polygonum capitatum proliferated bud obtained in the step c) by using a morphology and a flow cytometry identification method to obtain a tetraploid polygonum capitatum proliferated bud;

preferably, step d) comprises: firstly, identifying the polygonum capitatum proliferated bud obtained in the step c) by a morphological identification method to obtain a primarily selected polyploid polygonum capitatum proliferated bud; and then identifying the primarily selected polyploid polygonum capitatum proliferated bud by using a flow cytometer to obtain the tetraploid polygonum capitatum proliferated bud.

8. The cultivation method as claimed in claim 7, wherein the morphological identification method in step d) comprises identifying the size, shape and thickness of leaves of the Polygonum capitatum proliferating shoot;

preferably, the flow cytometer identification method in step d) comprises identifying the DNA content of the primarily selected polyploid polygonum capitatum proliferated bud by using a flow cytometer.

9. The cultivation method as claimed in claim 8, wherein the morphological identification method in step d) specifically comprises observing the size, shape and thickness of leaves of the polygonum capitatum proliferated bud, and identifying the polygonum capitatum proliferated bud as the initially selected polyploid polygonum capitatum proliferated bud if the leaves of the polygonum capitatum proliferated bud are enlarged, the leaves are wrinkled, the edges of the leaves are jagged and the thickness of the leaves is increased compared with the control diploid polygonum capitatum proliferated bud;

preferably, the flow cytometer identification method in step d) specifically includes identifying the DNA content of the primarily selected polyploid polygonum capitatum multiplied bud by using a flow cytometer, and identifying the primarily selected polyploid polygonum capitatum multiplied bud as a tetraploid polygonum capitatum multiplied bud if the DNA content of the primarily selected polyploid polygonum capitatum multiplied bud is 2 times that of the control diploid polygonum capitatum multiplied bud.

10. Incubation method according to any of claims 1-9, characterized in that step e) comprises: performing tissue culture on the tetraploid polygonum capitatum proliferated bud obtained in the step d) by adopting a plant tissue culture method under the aseptic condition so as to rapidly propagate the tetraploid polygonum capitatum and obtain tetraploid polygonum capitatum plant seedlings;

preferably, the culture medium used in the plant tissue culture method in step e) comprises a shoot multiplication medium and a rooting medium;

preferably, the shoot propagation medium used in step e) comprises MS powder, sucrose, glycine, phytogel, 6-BA and NAA; and the pH of the shoot multiplication medium is 5.8;

preferably, in the bud multiplication culture medium used in the step e), the concentration of MS powder is 4.43g/L, the concentration of sucrose is 30g/L, the concentration of glycine is 2.0mg/L, the concentration of plant gel is 4g/L, the concentration of 6-BA is 2.0mg/L, and the concentration of NAA is 0.1 mg/L;

preferably, the rooting medium used in step e) comprises MS powder, sucrose, glycine and a plant gel; and the pH value of the rooting medium is 5.8;

preferably, in the rooting medium used in step e), the concentration of MS powder is 2.215g/L, the concentration of sucrose is 15g/L, the concentration of glycine is 1.0mg/L, and the concentration of plant gel is 4 g/L;

preferably, the cultivation method further comprises: step f) hardening the tetraploid polygonum capitatum plant seedlings obtained in step e), and then transplanting the seedlings to a field.

Technical Field

The invention belongs to the field of agricultural plant variety cultivation. Specifically, the invention relates to a method for cultivating tetraploid polygonum capitatum.

Background

Polygonum capitatum (the scientific name: Polygonum capitatum Buch. -ham. ex D. Don) is a perennial herb of Polygonaceae, and is a traditional Chinese medicinal material. The herba Polygoni Capitati has effects of clearing away heat and toxic materials, promoting urination, treating stranguria, promoting blood circulation, and relieving pain. Relinqing granules consisting of polygonum capitatum are collected in the 'Chinese pharmacopoeia' 2015 edition, and the content of gallic acid is used as the content determination standard in the pharmacopoeia standard. In addition, the polygonum capitatum is a dominant herbaceous plant with vigorous growth in a copper ore area, and can play a good repairing role in the restoration of an ecological system. Meanwhile, the polygonum capitatum is a good ornamental plant, and has good landscape effect when growing in pieces. At present, wild polygonum capitatum resources are gradually reduced, the market demand of polygonum capitatum is very large, and the cultivation of high-yield and high-quality polygonum capitatum varieties has great significance for the development of polygonum capitatum industrial chains.

Plant polyploid breeding has been widely used in production and breeding research. After the plant is multiplied, the plant has higher heterozygosity and quicker environmental adaptability than a diploid parent. Research results prove that the agronomic characters, physiological and biochemical indexes and the effective component content of the tetraploid polygonum capitatum are all higher than those of diploid polygonum capitatum. In addition, researches prove that the chlorophyll content, drought resistance and heat resistance of the tetraploid polygonum capitatum are higher than those of the diploid polygonum capitatum. A key link of plant polyploid breeding is ploidy identification, however, the existing polyploid breeding method has large identification workload and slow identification speed, so that the breeding time is long, and a large amount of excellent seedlings cannot be provided for agricultural production.

Therefore, how to increase the identification speed of polyploid breeding of plants and shorten the breeding time, thereby providing a large amount of excellent seedlings for agricultural production becomes a problem to be solved in the field.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a method for cultivating tetraploid polygonum capitatum, which can reduce the ploidy identification workload in the breeding process, increase the identification speed, and shorten the breeding time, thereby providing a large amount of excellent seedlings for agricultural production.

In order to achieve the above object, the present invention provides a method for cultivating tetraploid polygonum capitatum, comprising the steps of:

a) treating diploid polygonum capitatum seeds, and then inoculating the diploid polygonum capitatum seeds into a culture medium for culturing to obtain polygonum capitatum stem tips as an induction material;

b) inducing the polygonum capitatum stem tip obtained in the step a) by using colchicine solution;

c) transferring the induced stem tip of the polygonum capitatum obtained in the step b) into a bud proliferation culture medium for induction so as to induce the polygonum capitatum bud to proliferate and obtain a polygonum capitatum proliferation bud;

d) identifying the polygonum capitatum proliferated bud obtained in the step c) to obtain a tetraploid polygonum capitatum proliferated bud;

e) carrying out tissue culture on the tetraploid polygonum capitatum proliferation bud obtained in the step d) so as to rapidly propagate the tetraploid polygonum capitatum and obtain tetraploid polygonum capitatum plant seedlings.

In some embodiments, step a) comprises: fumigating diploid polygonum capitatum seeds, then carrying out sterile disinfection treatment, then inoculating the seeds into a culture medium for culture, and cutting polygonum capitatum stem tips under the sterile condition to obtain sterile polygonum capitatum stem tips as an induction material.

In some embodiments, step a) comprises: fumigating diploid polygonum capitatum seed, then carrying out surface treatment by using alcohol solution, and then HgC1₂Sterilizing the solution, washing with sterile water, inoculating into culture medium, culturing in light culture room, and cutting stem tip of herba Polygoni Capitati under sterile condition to obtain sterile stem tip of herba Polygoni Capitati as inducing material;

preferably, step a) comprises: fumigating diploid polygonum capitatum seeds by using a mixed solution of concentrated HCl and NaClO for 6-48h, preferably 6h, 12h, 18h, 24h, 30h, 36h, 42h and 48h, more preferably 24 h; subsequently performing surface treatment with alcoholic solution for 5-30s, preferably 5s, 10s, 15s, 20s, 25s, 30s, more preferably 10s, and HgC1₂Sterilizing the solution for 1-9min, preferably 1min, 3min, 5min, 7min, 9min, more preferably 3min, washing with sterile water for 1-7 times, preferably 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, more preferably 5 times, inoculating into culture medium, culturing in light culture chamber for 15-40 days, preferably 15, 20, 25, 30, 35, 40 days, more preferably 25 days, and cutting stem tip of herba Polygoni Capitati under sterile condition to obtain sterile stem tip of herba Polygoni Capitati as inducing material.

In some embodiments, the NaClO content of the mixed solution of concentrated HCl and NaClO in step a) is 4.8-6.0% w/v, preferably 4.8% w/v, 5.0% w/v, 5.2% w/v, 5.4% w/v, 5.6% w/v, 5.8% w/v, 6.0% w/v, more preferably 5.2% w/v;

preferably, the concentration of the alcoholic solution in step a) is 70-75% v/v, preferably 70% v/v, 75% v/v, more preferably 75% v/v;

preferably HgC1 in step a)₂The concentration of the solution is 0.1-0.2% w/v, preferably 0.1% w/v, 0.15% w/v, 0.2% w/v, more preferably 0.1% w/v;

preferably, the temperature of the light culture chamber in step a) is set at 23-25 ℃, preferably 23 ℃, 24 ℃, 25 ℃, more preferably 25 ℃; the illumination intensity is set to 2000-3000lx, preferably 2000lx, 2500lx, 3000lx, more preferably 2000 lx;

preferably, the medium in step a) is MS hormone-free solid medium.

In some embodiments, step b) comprises: putting the polygonum capitatum stem tip obtained in the step a) in a colchicine solution, and carrying out shading soaking induction under the aseptic condition;

preferably, step b) comprises: placing the polygonum capitatum stem tip obtained in the step a) in a colchicine solution, and carrying out light-shielding soaking induction for 12-36h, preferably 12h, 18h, 24h, 30h, 36h, more preferably 24h, under the aseptic condition;

preferably, the concentration of the colchicine solution in step b) is 0.1-0.9% w/v, preferably 0.1% w/v, 0.3% w/v, 0.5% w/v, 0.7% w/v, 0.9% w/v, more preferably 0.5% w/v;

preferably, the colchicine solution in step b) is prepared with sterile cold water under sterile conditions, i.e. ready to use.

In some embodiments, the shoot proliferation medium in step c) comprises MS powder, sucrose, glycine, phytogel, 6-BA and NAA; and the pH of the shoot multiplication medium is 5.8;

preferably, in the bud multiplication medium in the step c), the concentration of MS powder is 4.43g/L, the concentration of sucrose is 30g/L, the concentration of glycine is 2.0mg/L, the concentration of plant gel is 4g/L, the concentration of 6-BA is 2.0mg/L, and the concentration of NAA is 0.1 mg/L;

preferably, step c) is carried out under sterile conditions;

preferably, the period of inducing the polygonum capitatum buds to proliferate in the step c) is 30 days, and the culture medium is changed once in 15 days.

In some embodiments, step d) comprises: identifying the polygonum capitatum proliferated bud obtained in the step c) by using a morphology and a flow cytometry identification method to obtain a tetraploid polygonum capitatum proliferated bud;

preferably, step d) comprises: firstly, identifying the polygonum capitatum proliferated bud obtained in the step c) by a morphological identification method to obtain a primarily selected polyploid polygonum capitatum proliferated bud; and then identifying the primarily selected polyploid polygonum capitatum proliferated bud by using a flow cytometer to obtain the tetraploid polygonum capitatum proliferated bud.

In some embodiments, the morphological identification method in step d) comprises identifying leaf size, shape and thickness of the polygonum capitatum proliferating shoot;

preferably, the flow cytometer identification method in step d) comprises identifying the DNA content of the primarily selected polyploid polygonum capitatum proliferated bud by using a flow cytometer.

In some embodiments, the morphological identification method in step d) specifically comprises observing the size, shape and thickness of the leaves of the polygonum capitatum proliferated bud, and identifying the polygonum capitatum proliferated bud as a primary selected polyploid polygonum capitatum proliferated bud if the leaves of the polygonum capitatum proliferated bud are larger, the leaves are wrinkled, the edges of the leaves are jagged and the thickness of the leaves is increased compared with the control diploid polygonum capitatum proliferated bud;

preferably, the flow cytometer identification method in step d) specifically includes identifying the DNA content of the primarily selected polyploid polygonum capitatum multiplied bud by using a flow cytometer, and identifying the primarily selected polyploid polygonum capitatum multiplied bud as a tetraploid polygonum capitatum multiplied bud if the DNA content of the primarily selected polyploid polygonum capitatum multiplied bud is 2 times that of the control diploid polygonum capitatum multiplied bud.

In some embodiments, step e) comprises: performing tissue culture on the tetraploid polygonum capitatum proliferated bud obtained in the step d) by adopting a plant tissue culture method under the aseptic condition so as to rapidly propagate the tetraploid polygonum capitatum and obtain tetraploid polygonum capitatum plant seedlings;

preferably, the culture medium used in the plant tissue culture method in step e) comprises a shoot multiplication medium and a rooting medium;

preferably, the shoot propagation medium used in step e) comprises MS powder, sucrose, glycine, phytogel, 6-BA and NAA; and the pH of the shoot multiplication medium is 5.8;

preferably, in the bud multiplication culture medium used in the step e), the concentration of MS powder is 4.43g/L, the concentration of sucrose is 30g/L, the concentration of glycine is 2.0mg/L, the concentration of plant gel is 4g/L, the concentration of 6-BA is 2.0mg/L, and the concentration of NAA is 0.1 mg/L;

preferably, the rooting medium used in step e) comprises MS powder, sucrose, glycine and a plant gel; and the pH value of the rooting medium is 5.8;

preferably, in the rooting medium used in step e), the concentration of MS powder is 2.215g/L, the concentration of sucrose is 15g/L, the concentration of glycine is 1.0mg/L, and the concentration of plant gel is 4 g/L;

preferably, the cultivation method further comprises: step f) hardening the tetraploid polygonum capitatum plant seedlings obtained in step e), and then transplanting the seedlings to a field.

The invention adopts the stem tip of the polygonum capitatum as an inducing material, combines plant tissue culture technology and colchicine induction, and cultivates the tetraploid polygonum capitatum by morphological and flow cytometry identification. The tetraploid polygonum capitatum cultivated by the method can provide germplasm resources for cultivating excellent polygonum capitatum varieties and rapidly propagate a large number of high-quality seedlings, thereby providing high-quality raw materials for the development of the polygonum capitatum industrial chain.

Compared with the prior art, the method for cultivating the tetraploid polygonum capitatum provided by the invention has the following advantages:

1. the invention utilizes the combination of plant tissue culture and colchicine induction, establishes a culture method for rapidly culturing the tetraploid polygonum capitatum through morphology and flow cytometry identification, greatly reduces the ploidy identification workload in the breeding process, improves the identification speed, thereby shortening the breeding time (only about 150 days from the seed treatment to the field transplantation), and provides a large amount of excellent seedlings for agricultural production.

2. The currently common method for identifying the ploidy of polygonum capitatum (for example, the method for detecting the number of chromosomes by a wall-removing hypotonic flame method) needs about 55 hours to complete one process, while the method adopts the combination of morphology and a flow cytometer to identify the ploidy of polygonum capitatum, only needs about 10 minutes to complete one process, and greatly shortens the identification time; in addition, 21% of the variant strains obtained by the method are homozygous tetraploid polygonum capitatum, while the variant strains obtained by the existing cultivation method are chimeras, and the tetraploid polygonum capitatum is obtained by further separation and identification.

3. The polygonum capitatum cultivated by the existing breeding method has the problems of degraded quality, poor disease resistance, reduced yield and quality and the like, and the tetraploid polygonum capitatum cultivated by the method has better disease resistance and improved yield and quality, thereby providing germplasm resources for cultivating excellent varieties of polygonum capitatum and providing high-quality raw materials for the development of an industrial chain of polygonum capitatum.

4. Compared with diploid polygonum capitatum, the tetraploid polygonum capitatum cultivated by the method has obviously increased leaves, flowers, roots and stems; the ecological adaptability and the stress resistance physiological and biochemical indexes are obviously improved; biomass, quercetin content and gallic acid content were all significantly increased.

5. Except the identification step, all the steps of the method for cultivating the tetraploid polygonum capitatum provided by the invention are carried out under the aseptic condition, so that the tetraploid polygonum capitatum is favorable for being quickly bred in a plant tissue culture mode and is not limited by time and season.

6. The cultivation method of the invention directly selects the polygonum capitatum stem tip as the inducing material, which is beneficial to obtaining more inducing variants through a bud multiplication mode.

7. The cultivation method of the invention adopts colchicine solution to shade and soak to induce the generation of tetraploid, the induction rate is higher (up to 12 percent) and the induction time is shorter.

8. The cell nucleus lysate and the staining solution used in the identification step of the flow cytometer in the cultivation method of the invention can obtain good identification effect without specially preparing the solution (can directly use a matched product provided by a flow cytometer manufacturer), thereby reducing the identification time and the labor consumption.

Drawings

FIG. 1 shows a flow cytometry comparison of a first selected polyploid polygonum capitatum proliferating bud (B) cultivated by the method of the present invention with a control diploid polygonum capitatum proliferating bud (A);

FIG. 2 shows a comparison of seedling shoots after 1 month transplantation of tetraploid polygonum capitatum (right) and control diploid polygonum capitatum (left) cultivated using the method of the present invention into a field;

FIG. 3 shows the leaves (A), flowers (B) and shoots (C) of tetraploid polygonum capitatum (left) cultivated using the method of the present invention compared to control diploid polygonum capitatum (right);

FIG. 4 shows the stomata and guard cell contrast of tetraploid polygonum capitatum plant (B) and control diploid polygonum capitatum plant (A) cultivated by the method of the present invention.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and materials of the reagents used in the following examples are all commercially available products unless otherwise specified.

Instrument for measuring the position of a moving object

Flow cytometer (German Partec, CyFlow Cube 8)

Microscope (Leica DM500, German come card stock Co., Ltd.)

Electronic scale (H2, Kaifeng)

High performance liquid chromatograph (Agilent HPLC1260, Agilent technologies, Inc.)

Ultraviolet visible spectrophotometer (Specord 210 Plus, Germany SPECORD)

Portable photosynthesis apparatus (LI-6400XT, LI-COR corporation of America)

Material

Diploid polygonum capitatum seed (Guizhou province Xingyi city wild polygonum capitatum seed)

Cell nucleus lysate (Germany Partec)

Dyeing liquor (German Partec)

MS hormone-free solid culture medium (Qingdao Nishui Biotechnology Co., Ltd.)