CN113261506A - Culture medium for regeneration of luzhu somatic embryo and rapid seedling breeding method - Google Patents
Culture medium for regeneration of luzhu somatic embryo and rapid seedling breeding method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium for giant reed somatic embryo regeneration and a rapid seedling breeding method. The invention provides a tissue culture medium for breeding arundo donax linn, which comprises an induction medium, a differentiation medium and a rooting medium. The induction rate of the tissue culture medium for inducing callus provided by the invention is up to 75%, and the differentiation medium gives consideration to high proliferation coefficient and strong bud induction effect.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium for giant reed somatic embryo regeneration and a rapid seedling breeding method.
Background
The biomass of the giant reed is large, although the giant reed is a carbon three (C3) plant, the dry matter yield of the giant reed can be comparable with that of a plurality of carbon four (C4) plants, and the dry matter yield can reach 30-40 t/hm in general2. Meanwhile, the Arundo donax is a nitrogen fixation plant, nitrogen fertilizer application is not generally needed in the growth period, and the pollution rate of soil is correspondingly reduced. The giant reed can be planted by fully utilizing the land resources such as agricultural waste land, farmland marginal land, wasteland, saline-alkali land, arid land, hilly land, heavy metal polluted farmland, sandy land and the like. The giant reed is also an excellent soil remediation plant, has strong stress resistance, belongs to a tolerant plant, can grow in the lands polluted by heavy metal, organic matters, petroleum and the like, quickly removes pollutants in the soil by virtue of the huge biomass yield of the giant reed, balances the acidity and alkalinity of the soil, completes the soil remediation, increases the organic carbon content and the enzyme content in the soil after the giant reed is planted, and obviously improves the comprehensive quality of the soil.
The giant reed cannot divide, so the giant reed blooms and is not pregnant, and cannot be propagated by a sexual reproduction method. The propagation mode of the giant reed is asexual propagation. The traditional seedling breeding method with tillering root burying and stem sectioning cuttage has the problems of low propagation speed after field planting of seedlings, high labor intensity, low seedling quality, few tillering after planting, low breeding coefficient and the like, and is difficult to meet the requirement of production and development of the arundo donax linn.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium for regeneration of a giant reed somatic embryo and a rapid seedling breeding method. The tissue culture medium provided by the invention can improve the breeding efficiency of the tissue culture seedlings of the arundo donax linn, the arundo donax linn breeding method based on the tissue culture medium provided by the invention can be used for rapidly breeding a large amount of arundo donax linn, and has the advantages of short production period, high breeding coefficient, convenience in management, capability of carrying out industrial production and the like, the problem of shortage of high-quality seedlings of the arundo donax linn can be effectively solved, and the demand of the society on the production development of the arundo donax linn can be met.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a tissue culture medium for breeding arundo donax linn, which comprises an induction medium, a differentiation medium and a rooting medium;
the induction culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 30g/L of sucrose, 4.6g/L of agar, 1.0mg/L of naphthylacetic acid, 1.5mg/L of dichlorophenoxyacetic acid and 1.5mg/L of kinetin;
the pH value of the induction medium is 5.8;
the differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 30g/L of sucrose, 4.6-5.0 g/L of agar, 6.0mg/L of 6-benzylaminopurine and 0.8-1.0 mg/L of kinetin;
the pH value of the differentiation medium is 5.8;
the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 30g/L of sucrose, 4.6g/L of agar, 1.0mg/L of naphthylacetic acid and 0.5mg/L of indolebutyric acid;
the pH value of the rooting medium is 5.8.
The invention also provides a giant reed breeding method, which comprises the following steps:
inoculating the explant into the induction culture medium for induction culture to obtain the callus of the arundo donax linn;
after obtaining the callus, inoculating the callus into the differentiation culture medium for differentiation culture to obtain tissue culture seedlings of the arundo donax linn;
after tissue culture seedlings are obtained, inoculating the tissue culture seedlings into the rooting culture medium for rooting culture to obtain rooted bamboo reed tissue culture seedlings;
after the tissue culture seedling of the rooted arundo donax is obtained, inoculating the tissue culture seedling of the rooted arundo donax in a seedling culture medium to obtain the seedling of the arundo donax;
and after obtaining the seedlings, transplanting the seedlings to a field to finish the breeding of the bamboo reeds.
Preferably, the explant comprises axillary buds on the rhizome of Arundo donax.
Preferably, the explant further comprises the following treatments prior to inoculation:
(1) soaking the cleaned explant with a mixed aqueous solution; the soaking time is 18-20 min; the mixed aqueous solution comprises sodium hypochlorite and octyl phenyl polyoxyethylene ether; the mass percentage of the sodium hypochlorite is 10 percent of the mixed aqueous solution; the mass percentage of the octyl phenyl polyoxyethylene ether is 0.01 percent of the mixed aqueous solution;
(2) washing the explant treated in the step (1) with 75% ethanol water solution by mass; the cleaning time is 20-30 s;
(3) soaking and cleaning the explant treated in the step (2) by using 0.1% of mercuric chloride aqueous solution in percentage by mass; the soaking and cleaning time is 8-10 min;
(4) and (4) segmenting the explants obtained in the step (3).
Preferably, the temperature of the induction culture is 24-26 ℃, the illumination intensity is 1000lx, the light cycle is 10h/d, and the time of the induction culture is 25-30 d.
Preferably, the temperature of the differentiation culture is 24-26 ℃, the illumination intensity is 1500-2000 lx, the illumination period is 16h/d, and the time of the differentiation culture is 25-30 d.
Preferably, the temperature of the rooting culture is 24-26 ℃, the light intensity is 1500-2000 lx, the illumination period is 16h/d, and the time of the rooting culture is 8-10 d.
Preferably, the rooted Arundo donax tissue culture seedling further comprises the following treatment before inoculation:
hardening the rooted Arundo donax tissue culture seedlings for 1-2 days under indoor natural illumination conditions; the environmental temperature of the hardening seedling is 20-25 ℃;
(II) cleaning a culture medium on the tissue culture seedlings after seedling hardening is finished, and soaking the rooted tissue culture seedlings in a potassium permanganate aqueous solution for 1-2 min after cleaning is finished; the mass percentage content of the potassium permanganate in the potassium permanganate aqueous solution is 1-2%.
The invention provides a tissue culture medium for breeding arundo donax linn, which comprises an induction medium, a differentiation medium and a rooting medium. The induction culture medium in the tissue culture medium provided by the invention can effectively improve the callus induction rate of explants, the induction rate is up to 75%, the provided differentiation culture medium can effectively give consideration to high proliferation coefficient and robust sprouts, and the rooting culture medium can effectively improve the rooting rate of tissue culture seedlings; the tissue culture medium provided by the invention is matched with a corresponding breeding method, and the propagation coefficient is more than 8.0.
Detailed Description
The invention provides a tissue culture medium for breeding arundo donax linn, which comprises an induction medium, a differentiation medium and a rooting medium;
the induction culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 30g/L of sucrose, 4.6g/L of agar, 1.0mg/L of naphthylacetic acid (NAA), 1.5mg/L of dichlorophenoxyacetic acid (2,4-D) and 1.5mg/L of Kinetin (KT);
the pH value of the induction medium is 5.8;
the differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 30g/L of sucrose, 4.6-5.0 g/L of agar, 6.0mg/L of 6-benzylaminopurine (6-BA) and 0.8-1.0 mg/L of KT; further preferably, the composition also comprises the following components in mass concentration: 30g/L of sucrose, 4.6g/L of agar, 6-BA 6.0mg/L and 0.8mg/L of KT or 30g/L of sucrose, 4.6g/L of agar, 6-BA 6.0mg/L and 0.9mg/L of KT;
the pH value of the differentiation medium is 5.8;
the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 30g/L of sucrose, 4.6g/L, NAA 1.0.0 mg/L of agar and 0.5mg/L of indolebutyric acid (IBA);
the pH value of the rooting medium is 5.8.
In the induction culture medium, sucrose is used as a carbon source for plant growth, agar is used as a coagulant of the culture medium, and NAA, 2,4-D and KT induce the generation of callus; the induction culture medium provided by the invention improves the induction rate of the arundo donax linn explant by matching of all components, and the induction rate of axillary buds on the rhizome of the arundo donax linn is improved to 75% from conventional 55%.
In the differentiation culture medium, sucrose is used as a carbon source for plant growth, agar is used as a coagulant of the culture medium, and 6-BA and KT induce the differentiation of callus; the differentiation medium provided by the invention improves the proliferation coefficient of the arundo donax linn callus to 8.0 through the matching of all the components, and simultaneously can improve the quality of the new buds, thereby promoting the survival rate of the subsequently transplanted seedlings.
In the rooting culture medium, sucrose is used as a carbon source for plant growth, agar is used as a coagulant of the culture medium, and NAA and IBA induce the rooting of the Arundo donax tissue culture seedlings; the rooting culture medium provided by the invention can effectively improve the rooting rate of the arundo donax linn tissue culture seedling by matching the components, and the conventional rooting rate of the arundo donax linn tissue culture seedling is improved to 98% from 90%.
The tissue culture medium provided by the invention can effectively improve the efficiency in the giant reed breeding process and improve the transplanting survival rate of the obtained tissue culture seedlings.
If not specifically required, the components involved in the culture medium provided by the present invention are all those conventionally purchased by those skilled in the art.
The invention also provides a giant reed breeding method, which comprises the following steps:
obtaining an explant of the arundo donax linn, inoculating the explant into the induction culture medium for induction culture to obtain callus of the arundo donax linn;
after obtaining the callus, inoculating the callus into the differentiation culture medium for differentiation culture to obtain tissue culture seedlings of the bamboo reeds;
after tissue culture seedlings are obtained, inoculating the tissue culture seedlings into the rooting culture medium for rooting culture to obtain rooted bamboo reed tissue culture seedlings;
after the tissue culture seedling of the rooted arundo donax is obtained, inoculating the tissue culture seedling of the rooted arundo donax in a seedling culture medium to obtain the seedling of the arundo donax;
and transplanting the seedlings to a field to complete the breeding of the bamboo reeds after the seedlings are obtained. The breeding method provided by the invention can effectively improve the giant reed breeding coefficient from 3.0-4.5 to 8.0 in a conventional mode, and can also effectively improve the survival rate of transplanted giant reeds.
Firstly, obtaining an explant of the arundo donax linn, inoculating the explant into the induction culture medium for induction culture to obtain callus of the arundo donax linn; the explant preferably comprises axillary buds on the rhizome of Arundo donax. Compared with other parts, the arundo donax linn explant provided by the invention can effectively improve the callus induction rate of the callus. In particular, the culture medium provided by the invention is suitable for the propagation requirements of axillary bud tissue culture on the rhizome of the giant reed at each stage, so that the giant reed can be rapidly cultured into seedlings.
The explant preferably also requires the following treatments prior to inoculation:
(1) soaking the cleaned explant with a mixed aqueous solution; the soaking time is 20 min; the mixed aqueous solution comprises sodium hypochlorite and octyl phenyl polyoxyethylene ether; the mass percentage of the sodium hypochlorite is 10 percent of the mixed aqueous solution; the mass percentage of the octyl phenyl polyoxyethylene ether is 0.01 percent of the mixed aqueous solution;
(2) washing the explant treated in the step (1) with 75% ethanol water solution by mass; the cleaning time is 30 s;
(3) 0.1 percent of mercuric chloride (HgCl) is used in percentage by mass2) Soaking and cleaning the explants processed in the step (2) by using an aqueous solution; the soaking and cleaning time is 8 min;
(4) and (4) segmenting the explants obtained in the step (3).
The explant provided by the invention preferably comprises the steps of preliminarily cleaning the explant before being soaked in the mixed aqueous solution, and soaking the cleaned explant in the mixed aqueous solution after the preliminary cleaning is finished; in the present invention, the preliminary cleaning preferably includes soaking with a detergent water and soaking with a distilled water; the soaking time of the mixed aqueous solution is preferably 18-20 min, and more preferably 20 min; the mixed aqueous solution comprises 10 mass percent of NaClO and 0.01 mass percent of TrionX-100. The invention can effectively remove impurities and mixed bacteria on the surface of the explant by primarily cleaning the explant, and can effectively reduce the adverse effect of the impurities or the mixed bacteria on the induction treatment of the explant.
After the explant is treated, the explant is preferably cleaned by using 75% of ethanol aqueous solution by mass percent; the cleaning time is preferably 20s to 30s, and more preferably 30 s; after washing with aqueous ethanol, the present invention is preferably also washed with sterile water. The invention can effectively sterilize the surface of the explant by cleaning the explant.
After obtaining the washed explants, the invention preferably uses 0.1% by weight of HgCl2Soaking and cleaning explants with aqueous solution; the soaking and cleaning time is preferably 8 min; by HgCl2After soaking in an aqueous solution and washing, the present invention is preferably also rinsed with sterile water. The invention utilizes HgCl2Can effectively kill bacteria and fungal spores attached to the surface of the explant.
Preferably segmenting the explant after obtaining the explant obtained by the cleaning operation; in the present invention, the length of the side of the divided explant is preferably 0.5 cm.
After obtaining the explant subjected to the segmentation treatment, the explant subjected to the segmentation treatment is preferably placed in an induction culture medium for induction culture; the temperature of the induction culture is preferably 24-26 ℃, the illumination intensity is preferably 1000lx, the light cycle is preferably 10h/d, and the time of the induction culture is preferably 25-30 d. According to the giant reed breeding method provided by the invention, the specific part of the giant reed is selected as the explant, and the induction rate of the callus can be effectively improved by combining a specific culture medium and a specific culture condition.
After obtaining the callus, inoculating the callus into the differential culture medium for differential culture to obtain tissue culture seedlings of the arundo donax linn; the temperature of the differentiation culture is preferably 24-26 ℃, the illumination intensity is preferably 1500-2000 lx, the illumination period is preferably 16h/d, and the time of the differentiation culture is preferably 25-30 d. According to the giant reed breeding method provided by the invention, the higher multiplication coefficient and the robust buds of the tissue culture seedlings can be considered through the collocation of the specific differentiation culture medium and the culture conditions.
After tissue culture seedlings are obtained, inoculating the tissue culture seedlings into the rooting culture medium for rooting culture to obtain rooted bamboo reed tissue culture seedlings; the temperature of the rooting culture is preferably 24-26 ℃, the light intensity is preferably 1500-2000 lx, the illumination period is preferably 16h/d, and the time of the rooting culture is preferably 8-10 d. According to the giant reed breeding method provided by the invention, the rooting rate of the tissue culture seedling can be effectively improved through the matching of the specific rooting culture medium and the culture conditions.
After the rooting Arundo donax tissue culture seedling is obtained, inoculating the rooting tissue culture seedling into a seedling culture medium to obtain an Arundo donax seedling;
the rooted Arundo donax tissue culture seedlings are preferably further subjected to the following treatment before inoculation:
hardening the rooted Arundo donax tissue culture seedlings for 1-2 days under indoor natural illumination conditions; the environmental temperature of the hardening seedling is 20-25 ℃;
(II) cleaning a culture medium on the tissue culture seedlings after seedling hardening is finished, and soaking the rooted tissue culture seedlings in a potassium permanganate aqueous solution for 1-2 min after cleaning is finished; the mass percentage content of the potassium permanganate in the potassium permanganate aqueous solution is 1-2%.
After the rooting arundo donax linn tissue culture seedling is obtained, the tissue culture seedling is preferably hardened for 1-2 d, the environment temperature for hardening is preferably 20-25 ℃, and the environment for hardening is preferably indoor natural light. According to the invention, the tissue culture seedlings subjected to the seedling hardening treatment can effectively improve the tolerance to the environment, and the breakage rate of the tissue culture seedlings after being subsequently transplanted into a field is reduced.
After the hardening of the tissue culture seedlings is finished, the culture medium on the tissue culture seedlings is preferably cleaned, and the rooted tissue culture seedlings are soaked in the potassium permanganate aqueous solution for 1-2 min after the cleaning is finished; the mass percentage content of the potassium permanganate in the potassium permanganate aqueous solution is 1-2%. According to the giant reed breeding method provided by the invention, the quality of the tissue culture seedling can be effectively improved and the transplanting survival rate of the tissue culture seedling can be improved by limiting the seedling hardening temperature, time and sterilization program.
In order to further illustrate the present invention, the culture medium for regeneration of arundo donax linn somatic embryos and the rapid seedling propagation method provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Examples
1. Arundo donax callus induction
Axillary buds, leaves, stems and roots of the giant reed rhizome are selected as explants to carry out callus induction.
(1) Cutting a giant reed explant, and placing the giant reed explant in a 100mL triangular flask;
(2) soaking the explant in detergent water for 20-25 min to remove soil, impurities and the like attached to the surface, and then washing the explant with distilled water;
(3) soaking for 20min by using a mixed solution of 10 mass percent of sodium hypochlorite and 0.01 mass percent of Trion-100, and washing by using distilled water;
(4) placing the washed explants in a sterile triangular flask, washing the explants for 30s with 75% ethanol, and washing the explants with sterile water for 3 times;
(5) with 0.1% HgCl2Soaking and washing for 8min, and washing for 3-4 times by using sterile water;
(6) placing the processed explant in a sterile culture dish, and cutting into small pieces of 0.5 cm;
(7) inoculating the block explants (7 axillary buds, leaves, stems and roots of the rootstock respectively, 28 in total) in a callus induction culture medium;
the induction culture medium provided by the invention comprises the following components: MS basal medium, 30g/L sucrose, 4.6g/L agar, 1.0mg/L NAA, 1.5mg/L KT, 1.5 mg/L2, 4-D, pH5.8, and is marked as medium IV;
comparative example medium i: MS basal medium, 30g/L sucrose, 4.6g/L agar, 0.5mg/L NAA and 1.0 mg/L2, 4-D;
comparative example medium ii: MS basal medium, 30g/L sucrose, 4.6g/L agar, 1.5mg/L NAA and 1.5 mg/L2, 4-D;
comparative example medium iii: MS basal medium +30g/L sucrose +4.6g/L agar +0.5mg/L KT +0.5mg/L NAA +1.0 mg/L2, 4-D;
comparative example medium v: MS basal medium +30g/L sucrose +4.6g/L agar +1.0mg/L KT +1.5mg/L NAA +1.5 mg/L2, 4-D;
placing the culture medium inoculated with the explants in an incubator with the temperature of 25 +/-2 ℃, the light intensity of 1000lx and the illumination period of 10h/d for illumination culture for 25-30 d, wherein the results of different culture media for inducing callus are shown in Table 1.
TABLE 1 results of callus induction in different media
As can be seen from Table 1, the induction culture mediums with different proportions have different effects on the induction of the callus, and the culture medium (i.e. culture medium IV) provided by the invention can effectively improve the induction rate of explants at different parts; the axillary buds on the rhizomes are selected as explants, and then the callus can be obtained by induction of the induction culture medium provided by the invention to the maximum extent. The induction culture medium, the selection of the explant and the corresponding induction conditions provided by the invention can improve the induction rate of axillary bud callus of the giant reed to 75%.
2. Bamboo reed callus differentiation
Inoculating the induced callus into a differentiation culture medium (MS basal medium +30g/L sucrose +4.6g/L agar +0.8mg/L KT +6.0mg/L6-BA, pH5.8, and marking as type 2;
MS basal medium, 30g/L sucrose, 4.6g/L agar, 0.9mg/L KT, 6.0mg/L6-BA, pH5.8 and marked as type 3) are placed in an incubator with the temperature of 25 +/-2 ℃, the light intensity of 1500-2000 lx and the illumination period of 16h/d for illumination culture for 25-30 d to obtain differentiated arundo donax tissue culture seedlings, and the differentiation results of the callus under different component ratios are shown in a table 2.
TABLE 2 differentiation Effect of callus at different 6-BA and IBA ratios
As can be seen from table 2, the differentiation medium (i.e., type 2 and type 3) with a specific ratio provided by the present invention can combine a high proliferation coefficient and strong buds, which is not only beneficial to improve the efficiency of vegetative propagation of arundo donax but also can improve the survival rate of transplanted seedlings of arundo donax.
3. Arundo donax rooting culture
And inoculating the tissue culture seedling obtained by differentiation into a rooting culture medium (1/2MS basic culture medium +30g/L sucrose +4.6g/L agar +0.5mg/L IBA +1.0mg/L NAA, pH 5.8), and carrying out illumination culture in an incubator with the temperature of 25 +/-2 ℃, the light intensity of 1500-2000 lx and the illumination period of 16h/d for 8-10 d to obtain the rooting giant reed tissue culture seedling.
4. Arundo donax seedling breeding
(1) Opening a cover of the tissue culture seedling container, moving the container from a culture room to indoor natural illumination, and hardening the seedlings for 1-2 days at the ambient temperature of 20-25 ℃;
(2) washing off the culture medium attached to the giant reed seedling roots with clear water, soaking the roots in 1-2% potassium permanganate solution for 1-2 min, and then washing the roots clean with clear water;
(3) selecting a nutrition pot with the diameter of 9cm and the height of 13cm, and a seedling raising substrate (universal type): 1:1 of sandy soil, 60 percent of humidity, spreading and transferring the rhizoma arundo donax roots into a nutrition pot, and compacting the soil;
(4) and (5) after irrigation, growing for 20-25 d, and enabling the giant reed seedlings to come out of the nursery.
5. Giant reed field transplantation
(1) Applying 30Kg of phosphoric diamine per mu of land, ridging and laminating, wherein the ridge width is 60cm, and the ridge height is 10 cm;
(2) and (5) punching holes on two sides of the ridge, wherein the planting distance is 40cm, and the sowing depth is 15 cm. The transplanting survival rate is 98.7%.
The tissue culture medium for the giant reed breeding provided by the invention can effectively accelerate the vegetative propagation speed of the giant reed, large-scale giant reed seedlings capable of being transplanted in a field can be obtained within 79-102 days, the induction callus induction rate of the induction medium provided by the invention is up to 75%, and the differentiation medium has both high multiplication coefficient and strong bud induction effect.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. The tissue culture medium for breeding the giant reed is characterized by comprising an induction medium, a differentiation medium and a rooting medium;
the induction culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 30g/L of sucrose, 4.6g/L of agar, 1.0mg/L of naphthylacetic acid, 1.5mg/L of dichlorophenoxyacetic acid and 1.5mg/L of kinetin;
the pH value of the induction medium is 5.8;
the differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 30g/L of sucrose, 4.6-5.0 g/L of agar, 6.0mg/L of 6-benzylaminopurine and 0.8-1.0 mg/L of kinetin;
the pH value of the differentiation medium is 5.8;
the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 30g/L of sucrose, 4.6g/L of agar, 1.0mg/L of naphthylacetic acid and 0.5mg/L of indolebutyric acid;
the pH value of the rooting medium is 5.8.
2. The breeding method of the arundo donax linn is characterized by comprising the following steps of:
inoculating the explant into the induction culture medium of claim 1 for induction culture to obtain the callus of the Arundo donax Linn;
after obtaining the callus, inoculating the callus into the differentiation culture medium of claim 1 for differentiation culture to obtain tissue culture seedlings of the arundo donax linn;
after obtaining tissue culture seedlings, inoculating the tissue culture seedlings into the rooting culture medium of claim 1 for rooting culture to obtain rooted bamboo reed tissue culture seedlings;
after the tissue culture seedling of the rooted arundo donax is obtained, inoculating the tissue culture seedling of the rooted arundo donax in a seedling culture medium to obtain the seedling of the arundo donax;
and after obtaining the seedlings, transplanting the seedlings to a field to finish the breeding of the bamboo reeds.
3. The breeding method of claim 2, wherein the explant comprises axillary buds on the rhizome of Arundo donax.
4. The breeding method of claim 2, wherein the explant prior to inoculation further comprises the following treatments:
(1) soaking the cleaned explant with a mixed aqueous solution; the soaking time is 18-20 min; the mixed aqueous solution comprises sodium hypochlorite and octyl phenyl polyoxyethylene ether; the mass percentage of the sodium hypochlorite is 10 percent of the mixed aqueous solution; the mass percentage of the octyl phenyl polyoxyethylene ether is 0.01 percent of the mixed aqueous solution;
(2) washing the explant treated in the step (1) with 75% ethanol water solution by mass; the cleaning time is 20-30 s;
(3) soaking and cleaning the explant treated in the step (2) by using 0.1% of mercuric chloride aqueous solution in percentage by mass; the soaking and cleaning time is 8-10 min;
(4) and (4) segmenting the explants obtained in the step (3).
5. The breeding method according to claim 2, wherein the temperature of the induction culture is 24-26 ℃, the illumination intensity is 1000lx, the photoperiod is 10h/d, and the time of the induction culture is 25-30 d.
6. The breeding method according to claim 2, wherein the temperature of the differentiation culture is 24-26 ℃, the illumination intensity is 1500-2000 lx, the illumination period is 16h/d, and the time of the differentiation culture is 25-30 d.
7. The breeding method of claim 2, wherein the temperature of rooting culture is 24-26 ℃, the light intensity is 1500-2000 lx, the illumination period is 16h/d, and the time of rooting culture is 8-10 d.
8. The breeding method of claim 2, wherein the rooted Arundo donax tissue culture plantlets further comprise, prior to inoculation, the following treatments:
hardening the rooted Arundo donax tissue culture seedlings for 1-2 days under indoor natural illumination conditions; the environmental temperature of the hardening seedling is 20-25 ℃;
(II) cleaning a culture medium on the tissue culture seedlings after seedling hardening is finished, and soaking the rooted tissue culture seedlings in a potassium permanganate aqueous solution for 1-2 min after cleaning is finished; the mass percentage content of the potassium permanganate in the potassium permanganate aqueous solution is 1-2%.
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