阅读说明：本技术 一种黄梁木四倍体诱导方法 (Method for inducing tetraploid of phellodendron amurense ) 是由 周玮 张石虎 陈晓阳 欧阳昆唏 阙青敏 古敏 李春梅 于 2021-10-12 设计创作，主要内容包括：本发明公开了一种黄梁木四倍体诱导方法,包括如下步骤：S1.下胚轴外植体预培养；S2.四倍体诱导培养：将经预培养后的下胚轴外植体接种到含秋水仙素的愈伤诱导液体培养基中进行暗培养；S3.组培幼苗：将步骤S2中经暗培养后的下胚轴外植体进行愈伤组织诱导后,转移至促芽培养基,诱导形成不定芽,切取不定芽接种于第一生根培养基进行生根诱导得到黄梁木再生植株。本发明以无菌黄梁木种子苗下胚轴作为外植体材料,通过先对下胚轴外植体进行预培养,再用含秋水仙素的愈伤诱导液体培养基处理外植体,再进行外植体进行愈伤诱导、促芽和生根培养等步骤,建立了黄梁木四倍体诱导体系,得到的黄梁木四倍体诱导率高,具有显著的优势。(The invention discloses a method for inducing tetraploid of yellow-wood, which comprises the following steps: s1, pre-culturing hypocotyl explants; s2, tetraploid induction culture: inoculating the hypocotyl explant after pre-culture to a callus induction liquid culture medium containing colchicine for dark culture; s3, tissue culture of seedlings: and (4) performing callus induction on the hypocotyl explant subjected to dark culture in the step (S2), transferring the hypocotyl explant to a bud promoting culture medium, inducing to form an adventitious bud, cutting the adventitious bud, inoculating the adventitious bud to a first rooting culture medium, and performing rooting induction to obtain a sasanqua regenerated plant. The invention takes the hypocotyl of aseptic phellodendron amurense seedling as the explant material, and establishes a phellodendron amurense tetraploid induction system by pre-culturing the hypocotyl explant, then treating the explant with a colchicine-containing callus induction liquid culture medium, and then performing callus induction, bud promotion, rooting culture and other steps on the explant, so that the obtained phellodendron amurense tetraploid has high induction rate and remarkable advantages.)

1. A method for inducing the tetraploid of the yellow Beam wood is characterized by comprising the following steps:

s1, pre-culturing hypocotyl explants: cutting hypocotyls of aseptic phellodendron amurense seedling as explants, and inoculating the hypocotyls on a first callus induction solid culture medium for pre-culture;

s2, tetraploid induction culture: inoculating the hypocotyl explant after pre-culture to a callus induction liquid culture medium containing colchicine for dark culture, wherein the dark culture time is 2-4 days when the concentration of the colchicine is 100mg/L, and the dark culture time is 1-4 days when the concentration of the colchicine is more than 100mg/L and less than or equal to 400 mg/L;

s3, tissue culture of seedlings: inoculating the hypocotyl explant subjected to dark culture in the step S2 into a second callus induction solid culture medium for callus induction, transferring the hypocotyl explant to a bud promoting culture medium for induction to form an adventitious bud, and after the adventitious bud grows to 2cm, cutting the adventitious bud, inoculating the adventitious bud into a first root culture medium for rooting induction to obtain a sasanqua regenerated plant.

2. The induction method according to claim 1, wherein in step S2, the concentration of colchicine is 200mg/L, 300mg/L, 400mg/L, and the dark culture time is 1-4 days.

3. The induction method according to claim 2, wherein in step S2, the colchicine concentration is 300mg/L and the dark culture time is 3 d.

4. The induction method according to claim 1, wherein the pre-culture time in step S1 is 3 d; the culture time in the step S3 is 35 d; in step S2, the dark culture conditions are: culturing at a constant temperature of 26 ℃ on a shaking table at a rotating speed of 90-150 rpm.

5. The induction method according to claim 1, wherein the first callus induction solid medium and the second callus induction solid medium are both: MS culture medium +3.0mg/L TDZ +0.1mg/L2,4-D +0.05mg/L NAA +30g/L sucrose +5.0g/L agar, and pH is 5.8-6.0; the callus induction liquid culture medium comprises: MS +3.0mg/L TDZ +0.1mg/L2,4-D +0.05mg/L NAA +30g/L sucrose, and the pH value is 5.8-6.0; the germination promoting culture medium comprises: MS culture medium +1.5 mg/L6-BA +0.05mg/L NAA +30g/L sucrose +5.0g/L agar, and the pH value is 5.8-6.0; the first rooting medium is MS +0.1mg/L NAA +30g/L sucrose +5.0g/L agar, and the pH value is 5.8-6.0.

6. The induction method according to any one of claims 1 to 5, further comprising the steps of:

s4, ploidy identification: when the regeneration root formed by the regenerated plant of the trabeckia chinensis in the step S3 grows to 0.5-1 cm, counting chromosomes of the root tip of the regenerated root, and identifying the multiple of the chromosome of the root tip; carrying out flow cytometry detection on the leaves of the regenerated plant of the yellow-wood identified as the tetraploid to determine ploidy;

s5, propagation of the tetraploid of the yellow Beam wood: inoculating the stem node of the regenerated plantlet of the bushy sophora which is determined to be tetraploid in the step S4 into a multiplication culture medium for carrying out the tetraploid propagation of the bushy sophora to obtain adventitious bud of the tetraploid of the bushy sophora;

s6, rooting tetraploid of the yellow-leaved yellowhorn and hardening and transplanting: inoculating the adventitious buds of the phellodendron amurense tetraploid obtained in the step S5 into a second rooting culture medium for rooting induction to obtain tetraploid tissue culture seedlings, moving the tetraploid tissue culture seedlings together with a tissue culture bottle to the outside for hardening seedlings, opening the bottle cover of the tissue culture bottle after hardening the seedlings for 5 days, then hardening the seedlings for 5 days, washing off agar on the roots of the tetraploid tissue culture seedlings, and planting the seedlings into seedling pots filled with peat soil and vermiculite mixed in a volume ratio of 3: 1.

7. The induction method according to claim 6, wherein the multiplication medium is: MS +1.0mg/L of 6-BA +0.05mg/L of IBA +30g/L of cane sugar +5.0g/L of agar, pH is 5.8-6.0, and the second rooting medium is as follows: MS +0.1mg/L NAA +30g/L sucrose +5.0g/L agar, and pH is 5.8-6.0.

8. The induction method according to claim 6, wherein the conditions of the preculture in step S1, the conditions of the callus induction, the adventitious bud formation induction and the rooting induction in step S3, and the propagation conditions in step S5 are all: the temperature is 26 ℃, the illumination intensity is 2000lx, and the illumination time is 12 h/d.

9. The induction method according to claim 1, wherein in step S1, the sterile phellinus linteus seed seedling hypocotyl explant is prepared by the following method:

soaking seeds soaked with distilled water one day in advance in 75% alcohol for 1min in a super-clean workbench, carrying out oscillation cleaning for 3-5 times by using sterile water, then carrying out sterilization for 5min by using 33.3% sodium hypochlorite, carrying out oscillation cleaning for 3-5 times by using sterile water, then sucking the seeds by using a sterile gun head, sowing the seeds in a hormone-free MS solid culture medium, uniformly dispersing the seeds by using the sterile gun head to suck water, then sucking excessive water, placing the seeds in a culture frame, and cutting hypocotyls of the seeds after 1-2 pairs of true leaves grow out from the seeds to obtain hypocotyls explants of the seedlings of the sterile wampee trees.

10. The induction method according to claim 1, wherein the colchicine-containing callus induction liquid medium in step S2 is prepared by: filtering and sterilizing colchicine by adopting a filter tip with the diameter of 0.25 mu m, then sterilizing the callus induction liquid culture medium at high temperature and high pressure, and adding the colchicine which is subjected to filtering and sterilization into the callus induction liquid culture medium when the temperature is reduced to 50-60 ℃ to obtain the colchicine-containing callus induction liquid culture medium.

Technical Field

The invention relates to the technical field of biology, in particular to a method for inducing tetraploid of phellodendron amurense.

Background

Rheum officinale (Neomarickia cadamba) is called clitocybe japonica and belongs to Rubiaceae (Rubiaceae) clitocybe (Neomarickia). The phellodendron amurense is rich in a plurality of active ingredients such as steroid, alkaloid, saponin, beta-sitosterol, flavonoid, carbondine, cinchonin and derivatives thereof, and has potential medical value (anti-inflammation, anti-malarial parasite, treatment of diabetes, reduction of blood sugar and blood fat, and the like); meanwhile, the leaves of the phellodendron amurense are rich in crude protein, crude fat, crude fiber and trace elements, and are high-quality plant feed raw materials. The Huanglian wood is naturally distributed in the south Asia, the southeast Asia tropical areas and the south Asia tropical areas in China. According to the distribution characteristics of the yellow-wood beams, the yellow-wood beams are favored to high temperature and high humidity, can be planted only in areas with warm climate, sufficient rainfall and high humidity, and have poor low-temperature tolerance.

Polyploid breeding is a common breeding means. Polyploid plants are plants containing 3 or more sets of chromosome groups in vivo, and the general sources are divided into two types, namely natural generation and artificial induction. After the plant chromosome is induced and doubled, the physiological balance of plant cells is broken due to the interaction effect of genes and the dose effect of the genes, compared with a diploid, the polyploid shows 'giant', and the biomass of nutritive organs such as roots, stems, leaves and the like can be obviously increased; the polyploid plant may have raised stress resistance and raised active component content. Therefore, if the polyploid of the yellow-leaved yellowhorn can be obtained by induction, high-quality polyploid varieties of the yellow-leaved yellowhorn can be cultivated, the quality of the yellow-leaved yellowhorn is improved, the adaptability of the yellow-leaved yellowhorn to the environment is improved, and the requirement on germplasm resources of the yellow-leaved yellowhorn is met.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a method for inducing the tetraploid of the yellow Beam wood.

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

a method for inducing the tetraploid of the yellow-leaved yellowhorn comprises the following steps:

s1, pre-culturing hypocotyl explants: cutting hypocotyls of aseptic phellodendron amurense seedling as explants, and inoculating the hypocotyls on a first callus induction solid culture medium for pre-culture;

s2, tetraploid induction culture: inoculating the hypocotyl explant after pre-culture to a callus induction liquid culture medium containing colchicine for dark culture, wherein the dark culture time is 2-4 days when the concentration of the colchicine is 100mg/L, and the dark culture time is 1-4 days when the concentration of the colchicine is more than 100mg/L and less than or equal to 400 mg/L;

s3, tissue culture of seedlings: inoculating the hypocotyl explant subjected to dark culture in the step S2 into a second callus induction solid culture medium for callus induction, transferring the hypocotyl explant to a bud promoting culture medium for induction to form an adventitious bud, and after the adventitious bud grows to 2cm, cutting the adventitious bud, inoculating the adventitious bud into a first root culture medium for rooting induction to obtain a sasangqua regenerated plant;

s4, ploidy identification: when the regeneration root formed by the regenerated plant of the trabeckia chinensis in the step S3 grows to 0.5-1 cm, counting chromosomes of the root tip of the regenerated root, and identifying the multiple of the chromosome of the root tip; the leaves of the regenerated plants of wakame identified as tetraploids were then subjected to flow cytometry to determine ploidy.

Preferably, the concentration of colchicine in the step S2 is 200mg/L, 300mg/L and 400mg/L, and the dark culture time is 1-4 days.

More preferably, the colchicine concentration in step S2 is 300mg/L, and the dark culture time is 3 d.

Preferably, the dark culture conditions described in step S2: the dark culture conditions are as follows: culturing at a constant temperature of 26 ℃ on a shaking table at a rotating speed of 90-150 rpm.

Preferably, the pre-incubation time in step S1 is 3 d; the incubation time was 35d in step S3.

Preferably, the first callus-inducing solid medium and the second callus-inducing solid medium are both: MS +3.0mg/L TDZ +0.1mg/L2,4-D +0.05mg/L NAA +30g/L sucrose +5.0g/L agar, and the pH value is 5.8-6.0; the callus induction liquid culture medium comprises: MS +3.0mg/L TDZ +0.1mg/L2,4-D +0.05mg/L NAA +30g/L sucrose, and the pH value is 5.8-6.0; the germination promoting culture medium comprises: MS +1.5 mg/L6-BA +0.05mg/L NAA +30g/L sucrose +5.0g/L agar, and the pH value is 5.8-6.0; the first rooting medium is MS +0.1mg/L NAA +30g/L sucrose +5.0g/L agar, and the pH value is 5.8-6.0.

Preferably, the method for inducing the tetraploid of the yellow-wood beam further comprises the following steps:

s5, propagation of the tetraploid of the yellow Beam wood: inoculating the stem node of the regenerated plantlet of the bushy sophora which is determined to be tetraploid in the step S4 into a multiplication culture medium for carrying out the tetraploid propagation of the bushy sophora to obtain adventitious bud of the tetraploid of the bushy sophora;

s6, rooting tetraploid of the yellow-leaved yellowhorn and hardening and transplanting: inoculating the adventitious bud of the tetraploid of the phellodendron amurense obtained in the step S5 into a second rooting culture medium for rooting induction to obtain a tissue culture seedling, moving the tissue culture seedling together with a tissue culture bottle to the outdoor for hardening the seedling, opening the bottle cover of the tissue culture bottle after hardening the seedling for 5d, washing off agar on the root of the tissue culture seedling, and planting the seedling into a seedling pot filled with peat soil and vermiculite which are mixed according to the volume ratio of 3: 1.

Preferably, the multiplication medium is: MS +1.0mg/L of 6-BA +0.05mg/L of IBA +30g/L of cane sugar +5.0g/L of agar, pH is 5.8-6.0, and the second rooting medium is as follows: MS +0.1mg/L NAA +30g/L sucrose +5.0g/L agar, and pH is 5.8-6.0.

Preferably, the preculture conditions in step S1, the callus induction, adventitious bud formation induction and rooting induction conditions in step S3, and the propagation conditions in step S5 are all: the temperature is 26 ℃, the illumination intensity is 2000lx, and the illumination time is 12 h/d.

Preferably, the preparation method of the sterile phellodendron amurense seedling hypocotyl explant in the step S1 is as follows:

soaking seeds soaked with distilled water one day in advance in 75% alcohol for 1min in a super-clean workbench, carrying out oscillation cleaning for 3-5 times by using sterile water, then carrying out sterilization for 5min by using 33.3% sodium hypochlorite, carrying out oscillation cleaning for 3-5 times by using sterile water, then sucking the seeds by using a sterile gun head, sowing the seeds in a hormone-free MS solid culture medium, uniformly dispersing the seeds by using the sterile gun head to suck water, then sucking excessive water, placing the seeds in a culture frame, and cutting hypocotyls of the seeds after 1-2 pairs of true leaves grow out from the seeds to obtain hypocotyls explants of the seedlings of the sterile wampee trees.

Preferably, the colchicine-containing callus induction liquid medium in step S2 is prepared by the following steps: filtering and sterilizing colchicine by adopting a filter tip with the diameter of 0.25 mu m, then sterilizing the callus induction liquid culture medium at high temperature and high pressure, and adding the colchicine which is subjected to filtering and sterilization into the callus induction liquid culture medium when the temperature is reduced to 50-60 ℃ to obtain the colchicine-containing callus induction liquid culture medium.

The MS refers to an MS standard culture medium; TDZ refers to N-phenyl-N' - (1,2, 3-thiadiazol-5-yl) urea; 2,4-D is 2, 4-dichlorophenoxyacetic acid; 6-BA means 6-benzylamino adenine; NAA means naphthylacetic acid; IBA refers to indolebutyric acid.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention relates to a method for inducing tetraploid of phellodendron amurense, which takes hypocotyl of aseptic phellodendron amurense seedling as explant material, and establishes a tetraploid inducing system of phellodendron amurense by pre-culturing hypocotyl explant, then treating the explant with a colchicine-containing callus inducing liquid culture medium, and then performing callus induction, bud promotion, rooting culture and other steps on the explant.

2. A large number of experiments show that when the concentration of colchicine is 300mg/L and the treatment time is 3d, the induction rate of the tetraploid of the phellodendron amurense rupr is as high as 23.37%, and the phellodendron amurense rupr has remarkable advantages.

Drawings

FIG. 1 is a chromosome map of the root tip of a diploid X-ray beam (A) and a chromosome map of the root tip of a tetraploid X-ray beam (B);

FIG. 2 is a flow cytometry view of a diploid sassafras (A) and a tetraploid sassafras (B);

FIG. 3 is a diploid wakeleton stoma (A) and a tetraploid (B) wakeleton stoma;

FIG. 4 shows diploid sashimi tissue-cultured seedling leaves (A) and tetraploid sashimi tissue-cultured seedling leaves (B);

FIG. 5 shows diploid X-rahbeag tissue culture seedlings (A) and tetraploid X-rahbeag tissue culture seedlings (B);

FIG. 6 shows a diploid trabecular wood transplanted seedling (A) and a tetraploid trabecular wood transplanted seedling (B);

FIG. 7 shows diploid (A) and tetraploid (B) trabeculae transplanted seedlings.

Detailed Description

The present invention is described more fully below in order to enable those skilled in the art to better understand the solution of the present invention.

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. In the present specification, "part" and "%" represent "part by mass" and "% by mass", respectively, unless otherwise specified.

The seeds used in the following method were taken from a single plant of Huanglian wood planted for many years near the field of the grass playground of the southern China university of agriculture.

Example 1

A method for inducing the tetraploid of the yellow-leaved yellowhorn comprises the following steps:

1. preparing an explant of an hypocotyl of a sterile Huanglian wood seed seedling:

soaking the seeds soaked with distilled water one day in advance in 75% alcohol for about 1min in a super-clean workbench, oscillating and cleaning the seeds for 3-5 times by using sterile water, sterilizing the seeds for 5min by using 33.3% sodium hypochlorite (the hazel color of the seeds is changed from reddish brown to faint yellow in the process), oscillating and cleaning the seeds for 3-5 times by using the sterile water, absorbing the seeds by using a sterile gun head, sowing the seeds in a hormone-free MS solid culture medium, absorbing water by using the sterile gun head to uniformly disperse the seeds, absorbing excessive water, placing the seeds in a culture frame, cutting off hypocotyls of the seedlings of the seeds after 1-2 pairs of true leaves (about 35 days) grow out of the seeds, and obtaining the hypocotyls explant of the seedlings of the sterile phellodendron bungei.

2. Preparation of a culture medium:

2.1MS solid Medium: solidifying MS culture medium with 0.50% agar, and sterilizing at 121 deg.C and 0.1Mpa for 20min to obtain MS solid culture medium;

2.2 preparing each culture medium according to the following proportion:

a. first callus induction solid medium and second callus induction solid medium: MS solid culture medium +3.0mg/L TDZ +0.1mg/L2,4-D +0.05mg/L NAA +30g/L cane sugar;

b. callus induction liquid medium: MS liquid culture medium +3.0mg/L TDZ +0.1mg/L2,4-D +0.05mg/L NAA +30g/L sucrose;

c. and (3) a germination promoting culture medium: MS solid culture medium +1.5 mg/L6-BA +0.05mg/L NAA +30g/L cane sugar;

d. first and second rooting media: MS solid culture medium +0.1mg/L NAA +30g/L sucrose;

e. proliferation culture medium: MS solid culture medium +1.0 mg/L6-BA +0.05mg/L IBA +30g/L cane sugar;

respectively adjusting the pH value of the culture medium to 5.8-6.0 by using 1mol/L NaOH or 1mol/L HCl;

2.3 colchicine-containing callus induction liquid medium: filtering and sterilizing colchicine by adopting a filter tip with the diameter of 0.25 mu m, then sterilizing the callus induction liquid culture medium at high temperature and high pressure, and adding the colchicine which is subjected to filtering and sterilization into the callus induction liquid culture medium when the temperature is reduced to 50-60 ℃ to obtain a callus induction liquid culture medium containing the colchicine;

2.4, respectively filling the culture mediums into tissue culture bottles, wherein the volume of each tissue culture bottle is 220mL, and each bottle is filled with 30mL of culture medium;

3. induction of the tetraploid of the yellow-leaved yellowhorn:

3.1 hypocotyl explant preculture: placing the cut sterile Huanglian wood seed seedling hypocotyl explant on a first callus induction solid culture medium for pre-culture for 3 d; the pre-culture conditions are as follows: the temperature is 26 ℃, the illumination intensity is 2000lx, the illumination time is 12h/d, and the relative humidity is 60%.

3.2 tetraploid induction culture: transferring the hypocotyl explant pre-cultured in the step 3.1 to a callus induction liquid culture medium containing colchicine with the concentration of 100mg/L, and performing dark culture for 1d, 2d, 3d and 4d respectively, wherein the dark culture conditions are as follows: culturing at a constant temperature of 26 ℃ on a shaking table at a rotating speed of 90-150 rpm.

3.3 tissue culture seedling: taking the hypocotyl explant subjected to dark culture in the step 3.2 out of a colchicine-containing callus induction liquid culture medium, washing the hypocotyl explant for 2-3 times by using sterile water, sucking water on the surface of the hypocotyl explant by using sterile filter paper after washing, inoculating the hypocotyl explant into a second callus induction solid culture medium for culture for 35 days to enable the callus to grow to a proper size, transferring the hypocotyl explant to a germination promoting culture medium, and inducing until adventitious buds are formed; when the adventitious bud grows to about 2cm, the adventitious bud is cut off and inoculated in a first rooting culture medium for rooting induction to obtain a regenerated plant. The conditions for inducing adventitious buds and rooting induction are the same as those for preculture.

4. Ploidy identification

4.1 chromosome observation: when the regeneration root formed by the regeneration plant grows to be about 0.5-1 cm, taking down the root tip of the regeneration root at 9:30-10:30 am, putting the root tip into a centrifugal tube filled with distilled water and pre-cooled by ice, and then putting the centrifugal tube on the ice for pre-treatment for 6-12 h. And transferring the pretreated root tips to Carnot fixing solution (absolute ethyl alcohol: glacial acetic acid ═ 3:1), and fixing in a refrigerator at 4 ℃ for 12-36 h. If the fixed root tip is not used immediately, the fixed root tip needs to be washed by 90%, 80% and 70% ethanol solutions in sequence, and finally stored in the 70% ethanol solution, and the root tip is placed in a refrigerator at 4 ℃ in a shading mode for standby.

The chromosome tabletting is carried out by adopting a conventional chromosome tabletting method, and the specific steps are as follows: and (3) washing the root tips fixed by the Carnot's stationary liquid for 15-20 min by using running water, transferring the root tips into L mol/L HC1, and dissociating for 6-9 min at 60 ℃. And (3) after washing the dissociated root tip with running water for 15-20 min, transferring the root tip into a centrifugal tube filled with the improved Kabaofentan dye liquor for dyeing for 15min, then cutting the top end of the dyed root tip on a glass slide with a blade, covering a cover glass, tapping 3-5 times with fingers, placing under a microscope to observe the shape and number of the root tip chromosome, and taking a picture.

4.2 flow cytometry detection of yellowbeam ploidy: leaves of the regenerated plants identified as tetraploids by chromosome counting were subjected to flow cytometry to determine ploidy.

5. Regenerated plant

5.1, propagation of the phellodendron amurense tetraploid, namely inoculating the stem node of the phellodendron amurense regenerated plant identified as the tetraploid into a propagation medium for propagation of the phellodendron amurense tetraploid to obtain adventitious buds of the phellodendron amurense tetraploid, wherein the propagation conditions are the same as those of pre-culture;

5.2 the tetraploid of the yellow Beam wood is rooted and acclimatized and transplanted: inoculating the adventitious bud of the tetraploid of the phellodendron amurense obtained in the step 5.1 into a second rooting culture medium for rooting induction, obtaining a tetraploid tissue culture seedling (about 35d is needed in the process) when the formed regeneration root grows to be slender and slightly green, transferring the tetraploid tissue culture seedling together with a tissue culture bottle to the outdoor for hardening off, opening the bottle cover of the tissue culture bottle after hardening off for 5d, then hardening off the agar on the root of the tetraploid tissue culture seedling, and planting the tetraploid tissue culture seedling into a seedling pot filled with peat soil and vermiculite mixed according to the volume ratio of 3:1 to obtain a tetraploid transplanting seedling. Wherein, the tissue culture bottle is a glass bottle used for conventional tissue culture production, and the seedling pot is a plastic pot with the diameter of about 20 cm.

Example 2

This example differs from example 1 in that: the concentration of colchicine is 200 mg/L.

Example 3

This example differs from example 1 in that: the concentration of colchicine is 300 mg/L.

Example 4

This example differs from example 1 in that: the concentration of colchicine is 400 mg/L.

Control group

The tissue culture seedling of the topaz diploid obtained without adding colchicine and the transplanted seedling of the topaz diploid are used as a contrast.

Example 5

The induction culture of the tetraploid of the sassafras wood was performed according to the induction method of the above-mentioned example 1 to example 4, and the regeneration rate of the explant, the average bud yield of the explant and the induction rate of the tetraploid were respectively counted, and the results are shown in table 1:

TABLE 1

Description of the drawings: 1. the same letters after each column number in table 1 indicate no significant difference in duncan multiplex experiments (P < 0.05).

2. The calculation formulas of the items in table 1 are:

explant regeneration rate (number of budding explants/total number of explants processed) 100%;

the average bud yield of the explants is the total bud number/total number of the treated explants;

tetraploid induction rate ═ (tetraploid bud number/total identified bud number) × 100%.

As can be seen from Table 1, the addition amount and induction time of colchicine during the induction of the tetraploid of Euonymus japonicus have a great influence on the regeneration rate of explants, the average bud yield of explants and the induction rate of tetraploid. When the concentration of colchicine is the same, the regeneration rate of the explant and the average bud yield of the explant are in negative correlation with the treatment time, and the longer the colchicine is used for treating the explant, the more unfavorable the bud emergence of the explant is. When the amount is within a certain range, the longer the treatment time is, the higher the tetraploid induction rate is, but after exceeding a certain range, the tetraploid induction rate is reduced along with the extension of the treatment time, which is that when the concentration is higher, the treatment time is extended, the damage to the explant is too large, and thus the induction rate of the tetraploid is affected. While the concentration of colchicine is in a certain range, the induction rate of the tetraploid is increased along with the increase of the colchicine, but when the concentration of the colchicine exceeds a certain range, the higher the concentration of the colchicine is, the lower the induction rate of the tetraploid is. Through a large number of experiments, the invention obtains the best colchicine concentration of 300mg/L, the best processing time of 3d, the colchicine concentration of 300mg/L and the processing time of 3d, and the tetraploid inductivity of the explant can reach 23.37%.

Example 6 comparison of characteristics of Brachypoda tetraploid and Brachypoda diploid

1. Anatomical comparison

At 9:00-10:00 a.m., leaves with the same good growth vigor and the same seedling age were randomly selected from the yellowbridge tetraploid tissue culture seedlings obtained by the yellowbridge tetraploid induction method of example 1-example 4 and the yellowbridge diploid tissue culture seedlings of the same batch obtained by the control group, the leaf backs of the yellowbridge tetraploid tissue culture seedling leaves and the yellowbridge diploid tissue culture seedling leaves were respectively placed on dry filter paper with the leaf backs facing up, the leaves were clamped by sharp-pointed tweezers, then the leaves were rotated clockwise by about 45 degrees, the leaves were quickly torn, epidermal cells were separated from mesophyll cells, a drop of distilled water was dropped on a slide, the torn epidermis was evenly spread, then the cover slip was covered with absorbent paper to absorb water, the measurement and statistics were observed by a Nikon DS-Ri2 microscope, four anatomical indices from leaf stomatal length, stomatal width, stomatal density, guard cell chloroplast content were compared, the results are shown in Table 2.

TABLE 2

As can be seen from table 2, compared with the leaves of the tiarella diploid tissue culture seedling, the leaf stomatal density of the tiarella tetraploid tissue culture seedling is reduced, the stomatal length and the stomatal width are greatly increased, and the number of chloroplast of guard cells is also greatly increased, which indicates that the photosynthesis of the tiarella tetraploid tissue culture seedling is stronger than that of the tiarella diploid tissue culture seedling. Meanwhile, as shown in fig. 3, the leaf stomata of the tiaga tetraploid tissue culture seedling is obviously larger than that of the tiaga diploid tissue culture seedling.

2. Morphological comparison

As shown in fig. 4 to 7, the obtained tiarella tetraploid tissue culture seedling and the transplanted seedling thereof grow strongly, and compared with the diploid tissue culture seedling and the transplanted seedling thereof, the tiarella tetraploid tissue culture seedling and the transplanted seedling thereof have increased stem diameter, leaf length and leaf width and deepened leaf color.

3. Comparison of root tip chromosome and DNA content

As shown in FIGS. 1 and 2, the number of root tip chromosomes of the yellowwood tetraploid is 2 times that of the yellowwood diploid, the relative DNA content of the yellowwood tetraploid is about 26000, the DNA content of the yellowwood diploid is about 13000, and the DNA content of the tetraploid is 2 times that of the diploid DNA.

The above-described embodiments are preferred implementations of the present invention, and the present invention may be implemented in other ways without departing from the spirit of the present invention.