CN115005104A

CN115005104A - Tissue culture medium and regeneration tissue culture method for okra regeneration

Info

Publication number: CN115005104A
Application number: CN202210865003.6A
Authority: CN
Inventors: 李冬玲; 顾永华; 汪琼; 杨军; 杨虹; 廖盼华; 刘科伟
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2022-07-21
Filing date: 2022-07-21
Publication date: 2022-09-06
Anticipated expiration: 2042-07-21
Also published as: CN115005104B

Abstract

The invention belongs to the technical field of plant cultivation, and particularly relates to a regenerated tissue culture medium and a regenerated tissue culture method for okra. The okra regeneration tissue culture medium comprises a germination medium, an induction medium, a differentiation medium and a strong bud rooting medium; KT, 2,4-D and carbon nano tubes are added into the induction culture medium; 6-BA, NAA and carbon nano tubes are added into the differentiation culture medium; 6-BA, NAA and carbon nano tubes are added into the rooting culture medium for the strong buds. KT, 2,4-D, 6-BA, NAA and carbon nano tubes of each culture medium act together to be beneficial to inducing the okra callus to form, proliferate and differentiate, so as to obtain a large amount of cluster buds and tissue culture seedlings, and further obtain strong plants with good consistency.

Description

Tissue culture medium and regeneration tissue culture method for okra regeneration

Technical Field

The invention belongs to the technical field of plant cultivation, and particularly relates to a regenerated tissue culture medium and a regenerated tissue culture method for okra.

Background

Okra is not only an excellent ornamental plant, but also a medicinal plant with high medicinal value. In recent years, with the continuous and deep understanding of the nutritional value and the medicinal value of the okra, the okra has increasingly broad application prospect, increasing market demand and remarkable market benefit, and the research on the propagation and cultivation technology of the okra is also continuous and deep. At present, the propagation of okra is mainly based on a seed propagation mode, although perennial herbaceous plants are usually cultivated as annual plants, and germplasm degradation is inevitably caused by long-term self-pollination. The plant tissue culture technology has the advantages of convenient material acquisition, capability of obtaining high-quality seedlings for rapid propagation and production cost reduction, but no complete report for establishing an okra regeneration technology system by utilizing the tissue culture technology is found at present.

Disclosure of Invention

The invention aims to provide a tissue culture medium for okra regeneration, which is applied to tissue culture of okra to establish an okra regeneration technical system, so that a large number of cluster buds of okra can be obtained, then a robust plant with good consistency can be obtained, and the seedling cost is reduced.

In order to solve the above problems, the present invention provides the following technical solutions:

the invention provides a tissue culture medium for okra regeneration, which comprises a germination medium, an induction medium, a differentiation medium and a strong bud rooting medium;

the germination culture medium takes an MS culture medium, an 1/2MS culture medium, a 1/3MS culture medium or a 1/4MS culture medium as a basic culture medium, and further comprises: 6.5-7.0 g/L agar;

the induction culture medium takes a B5 culture medium as a basic culture medium, and further comprises: 0.8-1.5 mg/LKT, 0.3-1.2 mg/L2,4-D, 1.0-2.0 mg/L carbon nano tube, 30g/L sucrose and 6.5-7.0 g/L agar;

the differentiation medium takes a B5 medium as a basic medium, and further comprises: 0.5-1.0 mg/L6-BA, 0.3-0.8 mg/L NAA, 0.5-1.5 mg/L carbon nano tube, 30g/L sucrose and 6.5-7.0 g/L agar;

the strong bud rooting culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.3-0.8 mg/L6-BA, 0.1-0.5 mg/L NAA, 2.0-5.0 mg/L carbon nano tube, 40g/L sucrose and 6.5-7.0 g/L agar.

Preferably, the pH value of the germination culture medium is 5.75-5.85; the pH value of the induction culture medium is 5.78-5.83, the pH value of the differentiation culture medium is 5.78-5.83, and the pH value of the strong bud rooting culture medium is 5.78-5.83.

The invention also provides a regeneration tissue culture method of okra callus, which adopts the tissue culture medium of the technical scheme and comprises the following steps:

inoculating okra arrowhead seeds on a germination culture medium for germination culture to obtain okra aseptic seedlings;

inoculating the stem and/or petiole of the okra aseptic seedling to an induction culture medium for induction and multiplication culture to obtain okra callus;

inoculating the okra callus to a differentiation culture medium for differentiation culture to obtain okra cluster buds;

and transferring the cluster buds of the okra fleabane to a strong bud rooting culture medium for strong bud rooting culture, and performing hardening and transplanting culture after the strong bud rooting culture.

Preferably, the length of the stem and the length of the petiole of the okra aseptic seedling are respectively 1.0-2.0 cm; the stems and/or petioles of the okra aseptic seedlings are obtained by the following steps: culturing the okra aseptic seedlings for 10-14 days, cutting off all roots, 1/5 stem parts and all leaves with the length connected with the roots when the okra aseptic seedlings grow to the height of 5.0-8.0 cm, obtaining stems and petioles with the residual length of 4/5, and respectively cutting the stems and/or the petioles with the residual length of 4/5 into small sections with the length of 1.0-2.0 cm.

Preferably, the temperatures of the induction and proliferation culture, the differentiation culture and the strong bud rooting culture are respectively 24-26 ℃; the temperature of germination culture is 20-28 ℃.

Preferably, the induction and proliferation culture, the differentiation culture and the rooting culture of the strong buds are all carried out under the condition of illumination, the illumination time is 12-16 h/d respectively, and the illumination intensity is 100-300 mu mol.m ^-2 ·s ^-1 (ii) a The time for induction and multiplication culture is 60-75 days; the differentiation culture time is 25-30 d; the time for rooting culture of the strong buds is 40-60 days; the total time of germination culture is preferably 15-35 d.

Preferably, the rooting culture period of the strong buds further comprises one-time subculture, the subculture time is 20-34 days after rooting culture of the strong buds, and the subculture medium is a rooting medium of the strong buds.

Preferably, the okra seeds are not irradiated before germination during germination culture, and the irradiation intensity after germination of the seeds is 100-300 mu mol.m ^-2 ·s ^-1 And the time of the germination stage of the okra flecks equisetifolia seeds is 5-21 days.

Preferably, the hardening is finished indoors, the hardening temperature is not lower than 20 ℃, and the hardening time is 4-6 d;

the transplanting culture is finished indoors, and the matrix for transplanting culture comprises peat soil, perlite and garden soil; the volume ratio of the peat soil to the perlite to the garden soil is (0.5-2): (0.5-2): (0.5 to 1.0).

Preferably, before the inoculation, the method further comprises the steps of cleaning and disinfecting okra seeds;

the cleaning comprises detergent cleaning and flowing water washing, wherein the detergent cleaning time is 20-30 min, and the flowing water washing time is 1.5-2 h;

the disinfection mode comprises the following steps: after cleaning, soaking the fabric in alcohol with the mass concentration of 70% -75% for 30-45 s, and washing the fabric for 2 times by using sterile water; soaking the mixture in 0.1% mercuric chloride solution for 20-30 min; finally, washing with sterile water for 4-5 times; and 2-3 drops of Tween 20 are added when the 0.1% mercuric chloride solution is soaked.

The invention has the beneficial effects that: the invention provides an okra regeneration tissue culture medium, which comprises a germination medium, an induction medium, a differentiation medium and a strong bud rooting medium; the germination culture medium takes an MS culture medium, an 1/2MS culture medium, a 1/3MS culture medium or a 1/4MS culture medium as a basic culture medium, and further comprises: 6.5-7.0 g/L agar; the induction culture medium takes a B5 culture medium as a basic culture medium, and further comprises: 0.8-1.5 mg/LKT, 0.3-1.2 mg/L2,4-D, 1.0-2.0 mg/L carbon nano tube, 30g/L sucrose and 6.5-7.0 g/L agar; the differentiation medium takes a B5 medium as a basic medium, and further comprises: 0.5-1.0 mg/L6-BA, 0.3-0.8 mg/L NAA, 0.5-1.5 mg/L carbon nano tube, 30g/L sucrose and 6.5-7.0 g/L agar; the strong bud rooting culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.3-0.8 mg/L6-BA, 0.1-0.5 mg/LNAA, 2.0-5.0 mg/L carbon nano tube, 40g/L sucrose and 6.5-7.0 agar. In the tissue culture medium provided by the invention, KT (kinetin) promotes the formation of okra callus; 2,4-D (2, 4-dichlorophenoxyacetic acid) promotes the formation of okra callus; 6-BA (6-benzylaminopurine) stimulates cell division to promote growth and development of okra, and promotes formation of callus; NAA (1-naphthylacetic acid) promotes the germination and growth of okra; the carbon nano tube can promote the formation and proliferation of okra callus and the growth of seedlings; KT, 2,4-D, 6-BA, NAA and carbon nano tubes are beneficial to inducing the okra callus to form, proliferate and differentiate under the combined action, a large number of cluster buds and tissue culture seedlings are obtained, and then robust plants with good consistency are obtained. The results of the examples show that: by utilizing the tissue culture medium, a large number of cluster buds and tissue culture seedlings can be obtained, and finally, robust okra tissue culture seedling plants with good consistency can be obtained. Therefore, the application effect of the germination culture medium, the induction culture medium, the differentiation culture medium and the strong bud rooting culture medium is good, the components are mutually synergistic, strong okra tissue culture seedling plants with good consistency can be obtained under the control of proper component concentrations, and an okra regeneration technical system is established.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.

FIG. 1 shows the aseptic seedling condition of the 14 th seed cultured by okra seeds in example 1;

FIG. 2 shows the 12d induction of okra callus in example 1;

FIG. 3 shows the case of the callus proliferation of Abelmoschus esculentus at 28d in example 1;

FIG. 4 shows the case of the callus proliferation of Abelmoschus esculentus at 28d in example 1;

FIG. 5 shows the rooting culture of cluster buds of Abelmoschus esculentus in example 1 at the 25 th day.

Detailed Description

The invention provides a tissue culture medium for regeneration of okra, which comprises a germination medium, an induction medium, a differentiation medium and a strong bud rooting medium.

In the invention, the germination culture medium takes an MS culture medium, an 1/2MS culture medium, a 1/3MS culture medium or a 1/4MS culture medium as a basic culture medium, and further comprises: 6.5-7.0 g/L agar; more preferably, the culture medium is a minimal medium comprising an MS culture medium, an 1/2MS culture medium, a 1/3MS culture medium or a 1/4MS culture medium, and further comprises: 6.5-7.0 g/L agar. The germination culture medium does not need to add any plant growth regulator or sucrose, and the germination rate of the okra seeds can reach over 75 percent.

In the present invention, the induction medium uses B5 as a minimal medium, and further includes: 0.8-1.5 mg/LKT, 0.3-1.2 mg/L2,4-D, 1.0-2.0 mg/L carbon nano tube, 30g/L sucrose and 6.5-7.0 g/L agar; more preferably, the culture medium B5 is used as a minimal medium, and the culture medium B5 only contains: 0.8-1.5 mg/LKT, 0.3-1.2 mg/L2,4-D, 1.0-2.0 mg/L carbon nano tube, 30g/L sucrose and 6.5-7.0 g/L agar. In the invention, the concentration of KT in the induction medium is 0.8-1.5 mg/L, preferably 0.9-1.3 mg/L, and more preferably 1.0-1.2 mg/L. The concentration of 2,4-D in the induction culture medium is 0.3-1.2 mg/L, preferably 0.3-0.9 mg/L2,4-D, more preferably 0.3-0.7 mg/L2, 4-D; the concentration of the carbon nano tubes in the induction culture medium is 1.0-2.0 mg/L, preferably 1.5-1.9 mg/L, and more preferably 1.8 mg/L; the agar concentration in the induction medium was: 6.5 to 7.0g/L of agar, preferably 6.6 to 6.9g/L of agar, and more preferably 6.8g/L of agar. In the present example, the agar concentration of the induction medium is preferably 6.5 g/L. In the invention, KT and 2,4-D can jointly promote the formation of okra callus under a proper concentration ratio.

In the invention, the pH value of the induction medium is preferably 5.78-5.83, and more preferably 5.80.

In the present invention, the differentiation medium uses B5 medium as a minimal medium, and further includes: 0.5-1.0 mg/L6-BA, 0.3-0.8 mg/L NAA, 0.5-1.5 mg/L carbon nano tube, 30g/L sucrose and 6.5-7.0 g/L agar; more preferably, the culture medium B5 is used as a minimal medium, and the culture medium B5 only contains: 0.5-1.0 mg/L6-BA, 0.3-0.8 mg/L NAA, 0.5-1.5 mg/L carbon nano tube, 30g/L sucrose and 6.5-7.0 g/L agar. In the invention, the concentration of 6-BA in the differentiation medium is 0.5-1.0 mg/L, preferably 0.6-0.8 mg/L6-BA, and more preferably 0.7-0.8 mg/L6-BA. The concentration of NAA in the differentiation culture medium is 0.3-0.8 mg/L, preferably 0.35-0.6 mg/LNAA, and more preferably 0.4 mg/LNAA. The concentration of the carbon nanotubes in the differentiation medium is 0.5-1.5 mg/L, preferably 0.8-1.3 mg/L, and more preferably 1.0-1.2 mg/L. The agar concentration in the differentiation medium is 6.5-7.0 g/L agar, preferably 6.7-7.0 g/L agar, and more preferably 6.8-7.0 g/L agar.

In the present invention, the pH of the differentiation medium is preferably 5.78 to 5.83, and more preferably 5.80.

In the invention, the strong bud rooting culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.3-0.8 mg/L6-BA, 0.1-0.5 mg/L LNAA, 2.0-5.0 mg/L carbon nano tube, 40g/L sucrose and 6.5-7.0 agar; more preferably, the culture medium is an MS culture medium, and further comprises only: 0.3-0.8 mg/L6-BA, 0.1-0.5 mg/L NAA, 2.0-5.0 mg/L carbon nano tube, 40g/L sucrose and 6.5-7.0 agar. In the invention, the concentration of 6-BA in the rooting medium for the strong bud is 0.3-0.8 mg/L, preferably 0.4-0.75 mg/L6-BA, and more preferably 0.5-0.7 mg/L6-BA. The concentration of NAA in the rooting-in-strong-bud culture medium is 0.1-0.5 mg/LNAA, preferably 0.2-0.45 mg/LNAA, and more preferably 0.3-0.4 mg/LNAA. The concentration of the carbon nano tubes in the rooting medium for the strong buds is 2.0-5.0 mg/L, preferably 2.5-4.5 mg/L, and more preferably 3.0-4.0 mg/L. The agar concentration in the rooting-in-strong-bud culture medium is 6.5-7.0 g/L, preferably 6.6-6.9 g/L, and more preferably 6.7-6.8 g/L. In the invention, the 6-BA can stimulate cell division to promote the growth and development of okra, and promote the formation of callus; NAA can promote the germination and growth of Hibiscus esculentus, and obtain a large amount of cluster buds.

In the invention, the pH value of the strong bud rooting culture medium is preferably 5.78-5.83, and more preferably 5.80.

The pH of the medium is preferably adjusted with 1M NaOH in the present invention.

The induction culture medium, the differentiation culture medium and the strong bud rooting culture medium provided by the invention all contain carbon nanotubes which can promote the formation and proliferation of okra callus and the growth of seedlings; the invention is beneficial to inducing the formation, proliferation and differentiation of okra callus under the combined action of plant growth regulators KT, 2,4-D, 6-BA, NAA and carbon nano tubes, and obtains a large amount of cluster buds and tissue culture seedlings, and the obtained plants have good consistency and strong growth. The tissue culture medium provided by the invention has a good application effect, can obtain a large number of test-tube seedlings simultaneously, shortens the seedling culture time of okra arrowleaves and reduces the seedling culture cost of okra arrowleaves. The concentration of KT, 2,4-D, 6-BA and NAA added in the invention is lower, and the production cost is reduced.

The sources of the KT, the 2,4-D, the 6-BA, the NAA, the carbon nano tubes, the sucrose and the agar are not specially limited, and the conventional commercial products are adopted. The carbon nanotube of the present invention is preferably a multi-walled carbon nanotube.

According to the invention, okra seeds are inoculated on a germination culture medium for germination culture, and okra aseptic seedlings are obtained. The invention takes okra seeds as explants to carry out seedling culture. In the present invention, the okra seeds are preferably either dry okra seeds or fresh okra seeds. The fresh okra seeds are preferably fresh okra seeds obtained by removing pericarps of mature okra capsules.

The okra seeds are preferably cleaned and disinfected before being inoculated on a germination culture medium; the cleaning according to the present invention preferably includes detergent cleaning and running water rinsing, and the cleaning method is not particularly limited in the present invention. In the present invention, the detergent is preferably one of a liquid detergent and a hand sanitizer, and more preferably a liquid detergent. The cleaning time of the detergent is preferably 20-30 min, more preferably 22-28 min, and even more preferably 25 min. In the embodiment of the present invention, the detergent washing time of the embodiment is preferably 25min and 30 min. The cleaning mode of the detergent cleaning is preferably vibration cleaning. The running water flushing time is preferably 1.5-2 h; more preferably 1.6 to 1.8 hours, and still more preferably 1.7 hours. In the embodiment of the present invention, the rinsing time with running water is preferably 100min and 120 min. The water used in the running water flushing is not particularly limited, and the running water can be conventional running water. The detergent cleaning and running water washing of the invention can effectively clean dust on the surfaces of okra seeds.

The disinfection method is not particularly limited, and the disinfection method is preferably 70-75% of ethanol and 0.1% of mercuric chloride solution by mass concentration, and more preferably 75% of ethanol and 0.1% of mercuric chloride solution by mass concentration. The disinfection method comprises the steps of soaking the materials in 75% alcohol by mass for 30-45 s preferably, 38-42 s more preferably, 40s more preferably, and washing the materials with sterile water for 2 times preferably; soaking the substrate in 0.1% mercuric chloride solution for 20-30 min, preferably 24-29 min, and preferably 25-28 min; finally, washing the mixture for 4-5 times preferably by using sterile water, and washing the mixture for 4 times more preferably; and preferably, 2-3 drops of Tween 20 are added when the 0.1% mercuric chloride solution is soaked. The invention has no special limitation on the sterile water, and is preferably obtained after the distilled water is sterilized; the usage amount of the 75% alcohol and the mercuric chloride is preferably to submerge the okra seeds. The 0.1% mercury liter of the invention is preferably obtained by metering to 1000mL per 1g mercury liter. The invention takes the Tween 20 as the surfactant, and enhances the sterilizing effect of the mercuric chloride.

After the okra seeds are cleaned and disinfected, the okra seeds are preferably inoculated to a germination culture medium. The invention has no special limit on the seed number of the okra inoculated in each germination culture bottle. In the invention, each germination culture bottle is preferably inoculated with 4 okra seeds.

The invention takes the okra seeds as explants to carry out tissue culture and rapid propagation seedling raising, and solves the technical problem of germplasm degeneration in the conventional seedling raising by using the okra seeds in the prior art.

In the invention, the germination culture comprises a germination stage and a sterile seedling culture stage of okra seeds. In the invention, the temperature of germination culture is preferably 20-28 ℃, more preferably 23-27 ℃, and more preferably 25 +/-1 ℃.

In the invention, the Hibiscus esculentus seeds are subjected to dark culture and light culture in the germination stage, wherein the dark culture and the light culture are divided by the time of exposure of the seeds, namely the Hibiscus esculentus seeds are preferably subjected to dark culture before germination during the germination culture, and are subjected to light culture after the seeds germinate and are exposed, and the light intensity is preferably 100-300 mu mol/m ^-2 ·s ^-1 More preferably 150 to 280. mu. mol/m ^-2 ·s ^-1 More preferably 250. mu. mol. m ^-2 ·s ^-1 . In the invention, the dark culture time is preferably 4-5 days, and after the seeds are exposed to the white, a light source is turned on to illuminateAnd (5) culturing. The dark culture of the invention is preferably carried out until the seeds are exposed to the white. The time of the germination stage of the okra seeds is 5-21 days, preferably 7-20 days, and more preferably 20 days. The time of the germination stage of the okra fargesii seeds is calculated by the dark culture time of the okra fargesii seeds and the illumination culture time of the okra fargesii seeds. The germination rate of the abelmoschus esculentus seeds cultured by the method can reach over 75 percent, and the germination rate is high.

And (3) obtaining aseptic okra seedlings after the okra seeds germinate and culture, and then culturing the aseptic okra seedlings to obtain the aseptic okra seedlings with the height of 5.0-8.0 cm. The time of the okra aseptic seedling stage is preferably 10d to 14d, more preferably 11d to 13d, and even more preferably 12 d.

In the invention, the total germination culture time comprises a germination stage and a sterile seedling culture stage of okra seeds. In the invention, the total time of germination culture is preferably 15-35 d, more preferably 21-34 d, and even more preferably 30-32 d. In the invention, the total time of germination culture is calculated by the time of the germination stage of the okra seeds and the culture time of aseptic okra seedlings.

After the okra aseptic seedling with the height of 5.0-8.0 cm is obtained, the stem and/or the petiole of the okra aseptic seedling are inoculated to an induction culture medium for callus induction and multiplication culture, and the okra callus is obtained. When the stem and the petiole of the okra aseptic seedling are inoculated at the same time, the stem and the petiole can be connected or do not need to be kept connected. The growth height of the okra aseptic seedlings is preferably 5.0-8.0 cm, more preferably 5.0-6.0 cm, and even more preferably 5.5 cm. The stems and/or petioles of the okra aseptic seedlings of the present invention are preferably obtained by: cutting off all roots, 1/5 stem parts and all leaves connected with the roots of the okra aseptic seedlings to obtain 4/5 stems and petioles with the remaining length, cutting the 4/5 stems with the remaining length into small sections with the length of 1.0-2.0 cm, inoculating the small sections with the remaining length to an induction culture medium for callus induction and proliferation culture, or cutting the petioles into small sections with the length of 1.0-2.0 cm, inoculating the small sections with the remaining length to the induction culture medium for callus induction and proliferation culture, or simultaneously inoculating the small sections with the stems with the length of 1.0-2.0 cm and the petioles with the length of 1.0-2.0 cm to the induction culture medium for callus induction and proliferation culture.

The length of the small section of the stem is preferably 1.0-2.0 cm, more preferably 1.2-1.9 cm, and even more preferably 1.5-1.8 cm. The length of the small segment of the petiole is preferably 1.0-2.0 cm, more preferably 1.3-1.7 cm, and even more preferably 1.5 cm.

In the invention, the temperature of the callus induction and proliferation culture is preferably 24-26 ℃, and more preferably 25 ℃. In the invention, the callus induction and proliferation culture are preferably carried out under the condition of illumination, and the illumination time is preferably 15-18 h/d, more preferably 15.5-17 h/d, and more preferably 16 h/d; the illumination intensity is preferably 100-300 mu mol.m ^-2 ·s ^-1 More preferably 150 to 280. mu. mol/m ^-2 ·s ^-1 More preferably 250. mu. mol. m ^-2 ·s ^-1 . The culture time of the callus induction and proliferation culture is preferably 60-75 d, and more preferably 70 d. After inoculation for 7-12 days, yellowish or greenish callus is generated around the incision. After the callus is induced for 30-35 days, subculture is continued for 30-40 days according to the original formula, and the callus is continuously expanded and proliferated. The callus induction time of the invention is calculated from the inoculation of the stem and/or petiole of the okra aseptic seedling in an induction culture medium.

After the okra callus is obtained, the okra callus is preferably inoculated to a callus differentiation culture medium for differentiation culture. The okra callus is preferably cut into the volume of 0.5-0.8 cm ³ The small pieces are inoculated on a callus differentiation culture medium.

In the invention, the callus differentiation culture temperature is preferably 24-26 ℃, and more preferably 25 ℃. The callus differentiation culture is preferably carried out under the condition of illumination, the illumination time is preferably 15-18 h/d, more preferably 16-17 h/d, and more preferably 16 h/d; the illumination intensity is preferably 100-300 mu mol.m ^-2 ·s ^-1 More preferably 180 to 280. mu. mol/m ^-2 ·s ^-1 More preferably 250. mu. mol. m ^-2 ·s ^-1 . The callus differentiation culture time is 20-25 d, preferably 22-25 d, and more preferably 23-25 d. After the callus differentiation culture is finished, cluster buds of okra are obtained.

After the cluster buds of the okra are obtained, the cluster buds of the okra are transferred to a strong bud rooting culture medium for strong bud rooting culture. The invention preferably cuts the cluster buds of the okra flechette from the callus tissues, and then transfers the cluster buds to a strong bud rooting culture medium for strong bud rooting culture. The height of the cluster buds cut from the callus is preferably 3.0-5.0 cm, more preferably 3.0-4.0cm

In the invention, the temperature for culturing the strong bud and rooting is preferably 24-26 ℃, and more preferably 25 ℃. The rooting culture of the strong buds is preferably carried out under the illumination condition, the illumination time is preferably 15-19 h/d, more preferably 16-18 h/d, and more preferably 16 h/d; the illumination intensity is preferably 100-300 mu mol.m ^-2 ·s ^-1 More preferably 190 to 260. mu. mol/m ^-2 ·s ^-1 More preferably 250. mu. mol. m ^-2 ·s ^-1 . The time for rooting culture of the strong buds is preferably 40-60 days, more preferably 45-60 days, and even more preferably 50-60 days.

The rooting culture period of the strong buds also comprises one-time subculture, the subculture time is preferably 20-34 d, more preferably 26-32 d, and even more preferably 28-31 d of rooting culture of the strong buds, and the subculture medium is preferably a rooting culture medium of the strong buds. The subculture is to continue to carry out the rooting culture of the strong buds after replacing the rooting culture medium of the strong buds once.

The test-tube plantlet is obtained after the rooting culture of the strong buds, and the test-tube plantlet is subjected to hardening seedling transplantation and transplanting culture after the test-tube plantlet simultaneously meets the three conditions of 5-8 cm in height, more than 5-7 leaves and 3-5 roots, so that the survival rate of the test-tube plantlet is improved.

In the invention, the germination culture, callus induction and proliferation culture, callus differentiation culture and strong bud rooting culture are preferably carried out in a tissue culture bottle, the tissue culture bottle is preferably a cylindrical bottle with the diameter of 7.5cm and the height of 11.0cm, and the thickness of a culture medium in the tissue culture bottle is preferably 2.0-2.5 cm.

The specifications of the tissue culture bottles for germination culture, induction and proliferation culture, differentiation culture and strong bud rooting culture are the same. The germination culture medium, the induction culture medium, the differentiation culture medium and the strong bud rooting culture medium are sterilized and poured into a tissue culture bottle, and the sterilization mode is not particularly limited and can be realized by adopting a conventional mode.

After the strong seedling rooting culture, the invention carries out seedling hardening and transplanting culture.

In the invention, the hardening-seedling is preferably finished indoors, the hardening-seedling temperature is preferably equal to or more than 20 ℃ according to the growth temperature requirement of okra, and the hardening-seedling time is preferably 4-6 d, and more preferably 5 d. In the invention, preferably, when hardening seedlings, the tissue culture bottle for bud and root growing culture is placed in a tissue culture room buffer room after being loosened in cover, placed for 3 days, then moved into an indoor room, placed under natural scattered light and placed for 2 days.

In the invention, the transplanting culture is preferably completed indoors, the test-tube plantlets after hardening off are preferably transplanted into a matrix for culture after the roots of the test-tube plantlets are cleaned, and the matrix is preferably peat soil, perlite and garden soil; the volume ratio of the peat soil to the perlite to the garden soil is preferably (0.5-2): (0.5-2): (0.5 to 1.0), more preferably (0.9 to 2): (0.8-2): (0.8 to 1.0), more preferably 1:1: 1; the temperature of the transplanting culture is preferably not lower than 20 ℃ at night, and the temperature of the transplanting culture is preferably 22-28 ℃, more preferably 23-27 ℃, and even more preferably 25 ℃. In the transplanting culture room, the substrate is preferably disinfected by the disinfection method, the disinfection mode is not particularly limited, and in the embodiment of the invention, 800 times of carbendazim is used for disinfecting the substrate.

The source and specification of the plug disk applied in the transplanting culture are not specially limited, and the conventional product is adopted. The preferred tray format for the application described in the present invention is 10 rows by 10 columns. The invention preferably plants 100 test-tube plantlets in each plug tray.

After the transplantation, the okra seedlings are preferably subjected to conventional management. In the invention, the conventional management preferably comprises that the root fixing water is fully watered after the seedlings are transplanted; spraying water in the morning and at night every day to keep the soil and the surfaces of the leaves moist. After one week, the seedlings survive, and then certain soil water holding capacity is kept according to weather conditions.

The okra seedling obtained by the method disclosed by the invention has the advantages that the offspring of the seedling propagation are uniform, the excellent characters of the original variety can be kept, the seed quality can not be degraded, and the seedling commodity is good. The invention takes the mature okra arrowhead seeds as explants to obtain aseptic seedlings; sterile stem segments or petioles are used as materials, a regeneration technical system is established through the induction of callus, robust and consistent seedlings are obtained, and a technical foundation is laid for the large-scale cultivation of okra.

For further illustration of the present invention, the tissue culture medium and the tissue culture method for regeneration of okra provided by the present invention are described in detail below with reference to the drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

(1) Obtaining and cleaning explants: and (4) collecting mature okra capsules in a clear weather in 10 months in 2021, and removing peels to obtain mature and fresh okra seeds.

(2) Cleaning and disinfecting explants: shaking and washing mature and fresh okra seeds in a detergent solution for 25min, and then washing with running water for 100min to obtain clean okra seeds. Sterilizing cleaned okra seeds for 40s by using 75% alcohol on a super clean bench, and then washing with sterile water for 2 times; then 0.1% of HgCl was added ₂ Sterilizing the solution for 25min, adding 3 drops of Tween 20, and washing with sterile water for 4 times.

(3) Germination and culture: the sterilized seeds of step (2) were directly inoculated on a seed germination medium, see FIG. 1. The thickness of the culture medium in the culture bottle is 2.0-2.5 cm. The diameter of the tissue culture bottle is 7.5cm is a cylindrical bottle with the height of 11.0 cm. The germination medium consisted of: in 1/2MS minimal medium, only 6.5g/L agar was added to the medium. Culturing in dark for 5 days at pH 5.8, and culturing in light at 250 μmol/m intensity after exposing seeds to white ^-2 ·s ^-1 . After 20 days of dark and light culture, seed germination was complete. And continuously culturing the aseptic seedlings obtained by germination under the illumination condition for 12 days. And (4) after 32 days of seed germination culture and aseptic seedling culture, when the height of the seed seedling reaches more than 5cm, performing callus induction in the step (4). The temperature during the seed germination culture period is 25 +/-1 ℃.

(4) Induction and multiplication culture of callus: taking out the sterile seedlings of the seeds in the step (3), cutting off all roots and stem parts and leaves (keeping petioles) which are connected with the roots and have the length of about 1/5, cutting the rest 4/5 stem parts into small sections with the length of 1.5cm, cutting the petioles into small sections with the length of 1.5cm, and simultaneously inoculating the stem small sections and the petiole small sections onto an induction culture medium. The induction culture medium consists of B5 culture medium as basic culture medium, and only added KT 1.0mg/L, 2, 4-D0.7 mg/L, multi-wall carbon nanotube 1.8mg/L, cane sugar 30g/L and agar 6.5 g/L. The thickness of the culture medium in the culture bottle is 2.0-2.5 cm. The tissue culture bottle is a cylindrical bottle with the diameter of 7.5cm and the height of 11.0 cm.

The culture temperature for induction and proliferation is 25 deg.C, and the light intensity is 250 μmol · m ^-2 ·s ^-1 The illumination time is 16 h/d. After 7d of inoculation, pale yellow or pale green callus is generated around the incision, and the culture is continued for 16d, and the stem segment and the petiole do not generate callus at 23d of inoculation. After 30 days of induction culture, subculture is continued for 30 days according to the original formula, and the callus is continuously expanded and proliferated in the period. Replacing the primary culture medium for subculture, wherein the subculture medium is a callus induction and proliferation culture medium.

(5) Differentiation culture of callus: cutting the callus obtained in step (4) into pieces with a volume of about 0.5cm ³ Inoculating the small blocks to a differentiation culture medium for differentiation culture to induce the generation of cluster buds. The differentiation medium consists of: taking B5 culture medium as basic culture medium, adding 6-BA 0.8mg/L, NAA0.4mg/L, carbon nanotube 1.0mg/L, sucrose 30g/L, agar7.0 g/L. The thickness of the culture medium in the culture bottle is 2.0-2.5 cm. The tissue culture bottle is a cylindrical bottle with the diameter of 7.5cm and the height of 11.0 cm.

The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. After 20 days of culture, generating light green bud points on the dark green callus tissue culture surface; and (5) continuing culturing, wherein the bud point grows slowly until the bud point becomes a green cluster bud.

(6) Strong bud rooting culture: cutting off the cluster buds in the step (5) from the callus, wherein the length of the cluster buds is 3.0-4.0cm, and transferring the cluster buds to a strong bud rooting culture medium for strong bud rooting culture. The strong bud rooting culture medium comprises the following components: MS is used as a basic culture medium, 0.5mg/L of cytokinin 6-BA, 0.3mg/L of auxin NAA and 3.0mg/L of carbon nano-tubes are added, 40g/L of cane sugar and 6.8g/L of agar are added. The thickness of the culture medium in the culture bottle is 2.0-2.5 cm. The tissue culture bottle is a cylindrical bottle with the diameter of 7.5cm and the height of 11.0 cm. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. Rooting is started when the rooting culture of the strong buds is carried out on the 8 th day, and the rooting culture medium of the strong buds is replaced once when the rooting culture is carried out on the 28 th day, namely, subculture is carried out once. And (4) culturing the strong buds and the roots for 50 days, and culturing the strong buds and the roots for 50 days to obtain test-tube plantlets with the height of more than 5cm, more than 5 leaves and more than 3 roots.

(7) Hardening seedlings and transplanting culture: hardening seedlings are carried out indoors. The seedling exercising mode is as follows: and (4) after the tissue culture bottle is uncovered, placing the tissue culture bottle in a buffer room of a tissue culture room for 3d, then moving the tissue culture bottle into an indoor room, placing the tissue culture bottle in natural scattered light, and then placing the tissue culture bottle for 2 d.

After the hardening-off, the test-tube plantlet is taken out by tweezers, the culture medium at the root is washed off, and the test-tube plantlet is transplanted into a 10X 10 plug tray. The temperature of the transplanting culture is 25 ℃ in the daytime and is not lower than 20 ℃ at night. Cultivation medium peat soil: perlite: the volume ratio of the garden soil is 1:1:1, and the culture medium is subjected to 800 times of carbendazim disinfection treatment. And then performing normal water and fertilizer management.

Example 2

(1) Obtaining an explant: collecting mature capsules in the season of seed maturity in 10 months of 2021, and removing pericarp to obtain fresh okra seeds. The seeds are aired for a week in a ventilated and shady place and then stored in a refrigerator at 4 ℃ for later use.

(2) Cleaning and disinfecting explants: and (3) in 11 months of 2021, taking the seeds in the step (1) out of the refrigerator, washing the seeds in the detergent solution for 30min by shaking, and then washing the seeds for 2h by using running water to obtain the clean okra seeds. Sterilizing cleaned okra seeds for 40s by using 75% alcohol on a super clean bench, and then washing with sterile water for 2 times; then 0.1% of HgCl was added ₂ Treating for 28min, and washing with sterile water for 5 times.

(3) Germination and culture: and (3) directly inoculating the disinfected seeds in the step (2) to a seed germination culture medium. The thickness of the culture medium in the culture bottle is 2.0-2.5 cm. The tissue culture bottle is a cylindrical bottle with the diameter of 7.5cm and the height of 11.0 cm. The germination medium consisted of: 1/4MS was used as a minimal medium, agar was added only at 7.0g/L, and the pH of the medium was 5.8. Culturing in dark for 4 days, exposing the seeds to white, and culturing under illumination with illumination intensity of 250 μmol/m for germination ^-2 ·s ^-1 And 7d later, the germination begins successively. After 20 days of co-culture for seed germination, seed germination was complete. The sterile seedlings of the seeds are continuously cultured for 10 days under the illumination condition. After 30 days of seed germination culture and aseptic seedling culture, when the height of aseptic seedling of seed is up to above 7.0cm, the callus is induced. The temperature during the whole seed germination culture period is 25 +/-1 ℃.

(4) Induction and multiplication culture of callus: and (3) when the aseptic seedlings grow for 28 days and are more than 5.0cm high, taking out the aseptic seedlings, cutting off all roots and 1/5 stem parts and leaves (the leaf stalks are reserved) which are connected with the roots and have the length of about 1/5, cutting the rest 4/5 stem parts into small sections with the length of 1.8cm, cutting the leaf stalks into small sections with the length of 1.5cm, and simultaneously inoculating the stem small sections and the leaf stalk small sections onto a callus induction culture medium. The induction culture medium comprises B5 culture medium as basic culture medium, kinetin KT 1.2mg/L, auxin 2, 4-D0.3 mg/L, multi-wall carbon nano-tube 1.5mg/L, and sucrose 30g/L and agar 6.5 g/L. The thickness of the culture medium in the culture bottle is 2.0-2.5 cm. The tissue culture bottle is a cylindrical bottle with the diameter of 7.5cm and the height of 11.0 cm. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. After 12d of inoculation, at the week of incisionThe surrounding tissue will produce pale yellow or pale green callus. After the whole callus induction time is 33d, after 33d, subculture is continued for 30d according to the original formula, and the callus is continuously expanded and proliferated.

(5) Differentiation culture of callus: cutting the callus of the step (4) into 0.8cm in volume ³ Inoculating the small blocks to a differentiation culture medium of the callus, performing differentiation culture, and inducing the generation of cluster buds. The differentiation medium consists of: the B5 culture medium is used as a basic culture medium, and 0.7mg/L of 6-BA, 0.4mg/L of NAA, 1.2mg/L of carbon nano tube, 30g/L of sucrose and 7.0g/L of agar are added. The thickness of the culture medium in the culture bottle is 2.0-2.5 cm. The tissue culture bottle is a cylindrical bottle with the diameter of 7.5cm and the height of 11.0 cm. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. Through 23d of culture, light green bud points are generated on the surface of the dark green callus tissue culture; and (5) continuing culturing, wherein the bud point grows slowly until the bud point becomes a green cluster bud.

(6) Strong bud rooting culture: and (3) cutting off the cluster buds obtained in the step (5) from the callus, wherein the length of the cut cluster buds is 3.0-4.0 cm. Transferring to strong bud culture medium to make strong bud and rooting culture. The strong bud rooting culture medium comprises the following components: MS is used as a basic culture medium, 6-BA0.7mg/L, NAA0.4mg/L and carbon nano tube 4.0mg/L are added, sucrose 40g/L and agar 6.8g/L are added. The thickness of the culture medium in the culture bottle is 2.0-2.5 cm. The tissue culture bottle is a cylindrical bottle with the diameter of 7.5cm and the height of 11.0 cm. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. Rooting starts when the rooting culture of the strong buds is carried out on the 10 th day, and the rooting culture medium of the strong buds is replaced once on the 31 th day, namely, subculture is carried out once. And (4) performing rooting culture on the strong buds for 60 days, and after the rooting culture for 60 days, growing the plantlets into test-tube plantlets with the height of 5-8 cm, 5-7 leaves and 3-5 roots. And performing subculture once when the culture is carried out for 31 days, wherein the subculture medium is a strong bud rooting medium, and the subculture is changed into a primary culture medium.

(7) Hardening seedlings and transplanting culture: hardening seedlings are carried out indoors. After the tissue culture bottle is uncovered, the tissue culture bottle is placed in a buffer room of a tissue culture room for 2d, then the tissue culture bottle is moved into an indoor room and placed under natural scattered light for 3 d. After the hardening-off, the test-tube plantlet is taken out by tweezers, the culture medium at the root is washed off, and the test-tube plantlet is transplanted into a 10X 10 plug tray. The temperature of the transplanting culture is 25 ℃ in the daytime and is not lower than 20 ℃ at night. The culture medium is peat soil: perlite: the garden soil is 1:1:1, and the culture medium is disinfected by 800 times of carbendazim. And then performing normal water and fertilizer management.

Example 3

Only the okra seeds are germinated and cultured, MS is used as a basic culture medium during germination and culture, and other conditions of germination and culture are the same as those of example 1.

Example 4

Only the okra seeds were germinated and cultured under the same conditions as in example 1 in 1/3MS as the minimal medium.

Statistics are carried out on the germination rate, the initial germination time and the complete germination time of the okra seeds obtained in the aseptic germination culture of the okra seeds in the examples 1-4, the results are shown in table 1, and the 14 th situation of the aseptic seedling culture in the example 1 is shown in fig. 1. The calculation formula of the germination rate is as follows:

the seed germination rate (%). ratio of germinated seeds X100/inoculated seeds

The results of aseptic germination culture of okra seeds of examples 1-4 are shown in Table 1.

Table 1 sterile germination culture results of okra seeds in examples 1-4

Serial number	Minimal medium	Number of seed inoculated	Initial germination time/day	Time/day of germination completion	Germination rate/%
						1	MS	100	6	21	75
2	1/2MS	100	6	21	73
						3	1/3MS	100	7	23	75
4	1/4MS	100	6	22	74

As can be seen from Table 1, when the okra seeds are germinated and cultured, the germination rate of the okra seeds is 73-75%, the germination rate of the seeds is high, and the germination time is short when the basic culture medium is MS culture medium, 1/2MS culture medium, 1/3MS culture medium, or 1/4MS culture medium.

Comparative example 1

Example 1 after okra seeds in step (3) germinate and culture, aseptic seedlings of seeds are obtained. When the aseptic seedlings grow for 28 days and have the height of more than 5.0cm, taking out the aseptic seedlings, cutting off all roots and 1/5 stem parts and leaves (keeping petioles) connected with the roots, cutting the rest 4/5 stem parts into small segments of 1.8cm, cutting the petioles into small segments of 1.5cm, and inoculating the small segments to a callus induction culture medium.

The induction culture medium comprises MS culture medium as basic culture medium, kinetin KT 1.2mg/L, auxin 2, 4-D0.3 mg/L, multi-wall carbon nano-tube 1.5mg/L, and sucrose 30g/L and agar 6.5 g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. After inoculation at 13d, yellowish or pale green callus is generated around the incision, and the culture is continued for 16d, and the stem and petioles do not generate callus at 29 d. After the induction culture is carried out for 32 days, the culture medium is replaced according to the original formula to continue subculture for 30 days, and the callus continuously expands and proliferates in the period.

Comparative example 2

The induction culture medium comprises 1/2MS culture medium as basic culture medium, kinetin KT 1.2mg/L, auxin 2, 4-D0.3 mg/L, multi-wall carbon nano-tube 1.5mg/L, and sucrose 30g/L and agar 6.5 g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. The remaining conditions were the same as in comparative example 1. After 15d of inoculation, pale yellow or pale green callus is generated around the incision, and the culture is continued for 15d, and the stem segment and the petiole do not generate callus at 30d of inoculation. After the induction culture is carried out for 33 days, the culture medium is replaced according to the original formula, and the subculture is continued for 30 days, wherein the callus is continuously expanded and proliferated.

Comparative example 3

The induction culture medium comprises a DKW culture medium as a basic culture medium, 1.2mg/L kinetin KT, 0.3mg/L auxin 2,4-D, 1.5mg/L multi-walled carbon nano-tubes, 30g/L sucrose and 6.5g/L agar. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. The remaining conditions were the same as in comparative example 1. After inoculation for 16d, a yellowish or pale green callus is formed around the incisionThe tissue is cultured for 16d, and the stem segment and the petiole no longer generate callus at the 32d inoculation time. After 35d of induction culture, replacing the culture medium according to the original formula and continuing subculture for 30d, wherein the callus continuously expands and proliferates.

Comparative example 4

The induction culture medium comprises N6 culture medium as basic culture medium, kinetin KT 1.2mg/L, auxin 2, 4-D0.3 mg/L, multi-wall carbon nano-tube 1.5mg/L, and sucrose 30g/L and agar 6.5 g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. The remaining conditions were the same as in comparative example 1. After 15 days of inoculation, pale yellow or pale green callus is generated around the incision, and the culture is continued for 20 days, and no callus is generated on the stem segments and the petioles at 35 days of inoculation. After 35d of induction culture, replacing the culture medium according to the original formula and continuing subculture for 30d, wherein the callus continuously expands and proliferates.

Comparative examples 1-4 only induction and proliferation culture was performed after obtaining sterile seedlings. Statistics are carried out on the okra callus generation time, the callus completion time and the callus induction rate of the okra in example 1 and the okra in comparative examples 1-4, the results are shown in a table 2, and the callus induction 12d case of the example 1 is shown in a table 2; example 1 callus proliferation at 28d is shown in FIGS. 3 and 4; the callus induction rate is calculated by the following formula:

callus induction rate (%). The number of stem segments from which callus was produced was multiplied by 100/number of inoculated stem segments

TABLE 2 okra callus induction culture results of example 1 and comparative examples 1 to 4

As can be seen from Table 2, the minimal medium is B5 medium, the effect is best, the callus induction rate is as high as 96.67%, and the induction culture time is shortest when the okra seeds are induced and cultured in a proliferation mode.

Comparative example 5

Example 1 Induction and enrichment culture of okra seed callus, callus was obtained and subjected toThe callus is differentiated and cultured, and the differentiation culture medium comprises B5 culture medium as basic culture medium, and is added with 0.8mg/L of 6-BA, 0.4mg/L of IAA, 1.0mg/L of carbon nano tube, 30g/L of sucrose and 7.0g/L of agar. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And (5) illuminating for 16h, and culturing for 25d to obtain cluster buds.

Comparative example 6

The differentiation medium comprises B5 medium as basic medium, and is added with 6-BA 0.8mg/L, 2, 4-D0.4 mg/L, carbon nanotube 1.0mg/L, sucrose 30g/L, and agar 7.0 g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And (5) illuminating for 16h, and culturing for 28d to obtain cluster buds. The remaining conditions were the same as in comparative example 5.

Comparative example 7

The differentiation culture medium comprises a B5 culture medium as a basic culture medium, and is added with 0.8mg/L of TDZ, 0.4mg/L of NAA0, 1.0mg/L of carbon nano tubes, 30g/L of cane sugar and 7.0g/L of agar. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And (5) illuminating for 16h, and culturing for 35d to obtain cluster buds. The remaining conditions were the same as in comparative example 5.

Comparative example 8

The composition of the differentiation medium is that the B5 medium is used as a basic medium, and 0.8mg/L of TDZ, 0.4mg/L of IAA, 1.0mg/L of carbon nano tube, 30g/L of cane sugar and 7.0g/L of agar are added. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And (5) illuminating for 16h, and culturing for 38d to obtain cluster buds. The remaining conditions were the same as in comparative example 5.

In comparative examples 5 to 8, only differentiation culture was carried out after obtaining the callus. Statistics are carried out on the okra callus differentiation culture results of example 1 and comparative examples 5-8, and the differentiation coefficient and differentiation rate are calculated, and the results are shown in table 3.

The rooting culture of the multiple shoots of example 1 at the 25 th day is shown in FIG. 5.

The differentiation rate and the differentiation coefficient were calculated as follows.

Differentiation Rate (%). The number of differentiated callus pieces was multiplied by 100/number of inoculated callus pieces

The differentiation coefficient is the number of seedlings differentiated from each callus

TABLE 3 results of okra callus differentiation culture of plant growth regulators

As shown in Table 3, when okra callus is subjected to differentiation culture, the callus differentiation coefficient and differentiation rate are the highest when 6-BA and NAA are added into a differentiation culture medium.

Comparative example 9

The callus obtained in example 1 was subjected to differentiation culture using the callus obtained in the induction and proliferation culture of example 1, and the callus obtained in example 1 was cut into a volume of about 0.5cm ³ Inoculating the small blocks to a differentiation culture medium for differentiation culture to induce the generation of cluster buds. The differentiation medium consists of: the B5 culture medium is used as a basic culture medium, and 0.8mg/L of 6-BA, 0.4mg/L of NAA, 0mg/L of carbon nano tube, 30g/L of cane sugar and 7.0g/L of agar are added.

The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. After 25d of culture, generating light green bud points on the surface of the dark green callus tissue culture; and (5) continuing culturing, wherein the bud point grows slowly until the bud point becomes a green cluster bud.

Comparative example 10

The differentiation medium consists of: the B5 culture medium is used as a basic culture medium, and 0.8mg/L of 6-BA, 0.4mg/L of NAA, 0.5mg/L of carbon nano tube, 30g/L of cane sugar and 7.0g/L of agar are added. Through 21d of culture, light green bud points are generated on the surface of the dark green callus tissue culture; and (5) continuing culturing, wherein the bud point grows slowly until the bud point becomes a green cluster bud. The rest of the conditions were the same as in comparative example 9.

Comparative example 11

The differentiation medium consists of: the B5 culture medium is used as a basic culture medium, and 0.8mg/L of 6-BA, 0.4mg/L of NAA, 1.5mg/L of carbon nano tube, 30g/L of cane sugar and 7.0g/L of agar are added. Culturing for 22d to generate light green bud points on the surface of the dark green callus tissue culture; and (5) continuing culturing, wherein the bud point grows slowly until the bud point becomes a green cluster bud. The rest of the conditions were the same as in comparative example 9.

Comparative examples 9 to 11 callus obtained by induction and proliferation culture in example 1 was only subjected to differentiation culture. Statistics is carried out on the okra callus differentiation culture results of the example 1 and the comparative examples 9-11, and the differentiation coefficient and the differentiation rate are calculated, and the results are shown in a table 4.

TABLE 4 influence of the carbon nanotube concentration of example 1 and comparative examples 9 to 11 on okra callus differentiation

As shown in Table 4, the callus differentiation rate was the highest when the carbon nanotubes were added to the differentiation medium at a concentration of 1.0mg/L during the differentiation culture of okra callus.

Comparative example 12

The multiple shoots obtained in example 1 were used for shoot rooting culture, and the multiple shoots obtained in example 1 were excised from the callus, and the cut multiple shoots were 3.0 to 4.0cm in length. Transferring to a strong bud rooting culture medium to perform strong bud and rooting culture. The strong bud rooting culture medium comprises the following components: MS is used as a basic culture medium, 6-BA0.5mg/L + IBA0.3 mg/L, carbon nano-tubes are 3.0mg/L, sucrose is added by 40g/L, and agar is added by 6.8 g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. And (4) beginning to root in the rooting culture medium for the strong buds at 13d, and replacing the rooting culture medium for the strong buds once when culturing at 28d, namely performing subculture once.

Comparative example 13

The strong bud rooting culture medium comprises the following components: MS is used as a basic culture medium, 6-BA0.5mg/L +2, 4-D0.3 mg/L, carbon nano-tubes 3.0mg/L, sucrose 40g/L and agar 6.8g/L are added. And (3) beginning to root at the 20 th day of the culture of the strong bud rooting culture medium, and replacing the strong bud rooting culture medium once when culturing at the 31 th day, namely performing subculture once. The remaining conditions were the same as in comparative example 12.

Comparative example 14

The strong bud rooting culture medium comprises the following components: MS is used as a basic culture medium, TDZ is 0.5mg/L + IBA is 0.3mg/L, carbon nano tubes are 3.0mg/L, sucrose is 40g/L, and agar is 6.8 g/L. And (4) beginning to root after the culture of the strong bud rooting culture medium at the 25 th day, and replacing the strong bud rooting culture medium once when the culture is carried out at the 31 th day, namely carrying out subculture once. The remaining conditions were the same as in comparative example 12.

Comparative example 15

The strong bud rooting culture medium comprises the following components: MS is used as a basic culture medium, TDZ0.5mg/L + IAA is 0.3mg/L, carbon nano tubes are 3.0mg/L, sucrose is added by 40g/L, and agar is added by 6.8 g/L. And (4) beginning to root at the 32 th day of the culture of the strong bud rooting culture medium, and replacing the strong bud rooting culture medium once when culturing the 31 th day, namely performing subculture once. The remaining conditions were the same as in comparative example 12.

Comparative example 16

The medium for rooting the strong buds comprises the following components: MS is used as a basic culture medium, TDZ0.5mg/L + NAA 0.3mg/L, carbon nano-tubes 3.0mg/L, sucrose 40g/L and agar 6.8g/L are added. And (3) the rooting medium of the strong bud does not root before being replaced, when the rooting medium of the strong bud is cultured for the 31 th day, the rooting medium of the strong bud is replaced for one-time subculture, and the rooting is started after the 4 th day after the subculture, namely the rooting is started after the 35 th day of the rooting culture of the strong bud. The remaining conditions were the same as in comparative example 12.

Comparative examples 12 to 16 were carried out for the cultivation of strong bud and root. The results of the effects of the plant growth regulators of example 1 and comparative examples 12 to 16 on the rooting culture of the clustered shoots, strong shoots and roots of okra are shown in Table 5. FIG. 5 shows the rooting culture of multiple shoots in example 1 at the 25 th day.

The formula for calculating the rooting rate is as follows:

rooting rate (%). root count 100/inoculation count

TABLE 5 Effect of the plant growth regulators of example 1 and comparative examples 12 to 16 on shoot and root growth of Hibiscus esculentus

Serial number	Growth regulator combination (mg/L)	Inoculation number/strain	Rooting time/day	Maximum number of roots/strip	Rooting percentage/%)
						Example 1	6-BA0.5+NAA0.3	30	8	8	96.67
Comparative example 12	6-BA0.5+IBA0.3	30	13	6	90.00
						Comparative example 13	6-BA0.5+2,4-D0.3	30	20	4	60.00
Comparative example 14	TDZ0.5+IBA0.3	30	25	3	66.67
						Comparative example 15	TDZ0.5+IAA0.3	30	32	3	33.33
Comparative example 16	TDZ0.5+NAA0.3	30	35	2	33.33

As can be seen from Table 5, when 6-BA and NAA were added to the rooting medium for strong buds of Hibiscus esculentus in example 1 during the culture of strong buds and roots of cluster buds, the rooting rate of cluster buds was as high as 96.67%, and the rooting starting time was the shortest.

Example 5

The strong bud rooting culture medium comprises the following components: MS is used as a basic culture medium, 0.5mg/L of cytokinin 6-BA, 0.3mg/L of auxin NAA and 2.0mg/L of carbon nano-tube are added, 40g/L of cane sugar and 6.8g/L of agar are added. And 9d, starting rooting. The other conditions were the same as in example 1.

Comparative example 17

And (3) performing strong bud rooting culture by using the cluster buds obtained in the example 1, cutting the cluster buds from the callus, wherein the length of the cluster buds is 3.0-4.0cm, and transferring the cluster buds to a strong bud rooting culture medium for strong bud rooting culture. The strong bud rooting culture medium comprises the following components: MS is used as a basic culture medium, 0.5mg/L of cytokinin 6-BA, 0.3mg/L of auxin NAA, 0mg/L of carbon nano tube, 40g/L of cane sugar and 6.8g/L of agar are added. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m ^-2 ·s ^-1 And the illumination time is 16 h. And (3) performing rooting culture on the strong buds, starting rooting at 14d, and replacing the rooting culture medium for the strong buds once at 28 days to perform subculture.After 50 days of culture, the plantlets grow into test-tube plantlets with the height of more than 5cm, more than 5 leaves and more than 3 roots.

Comparative example 18

The strong bud rooting culture medium comprises the following components: MS is used as a basic culture medium, 0.5mg/L of cytokinin 6-BA, 0.3mg/L of auxin NAA and 5.0mg/L of carbon nano-tube are added, 40g/L of cane sugar and 6.8g/L of agar are added. And (3) performing rooting culture on the strong buds, starting rooting in the 10 th day, and replacing the rooting culture medium for the strong buds once on the 29 th day, namely performing subculture. After 60 days of culture, the plantlets grow into test-tube plantlets with the height of more than 5.5cm, more than 6 leaves and more than 4 roots. The rest of the conditions were the same as in comparative example 17.

In example 5 and comparative examples 17 to 18, only the culture of strong bud and root was carried out. The results of the effects of the concentrations of carbon nanotubes in examples 1 to 2, 5 and 17 to 18 on the bud and root growth of Hibiscus esculentus cluster buds are shown in Table 6.

TABLE 6 influence of carbon nanotubes on rooting of okra

As can be seen from Table 6, in the case of the rooting culture of the cluster buds of Abelmoschus esculentus, the rooting rate of the cluster buds was as high as 96.67% when the concentration of carbon nanotubes in the rooting culture medium for the cluster buds of EXAMPLE 1 was 3.0 mg/L.

In conclusion, by using the tissue culture medium provided by the invention, a large number of cluster buds and tissue culture seedlings can be obtained, and finally, robust okra tissue culture seedling plants with good consistency can be obtained. Therefore, the invention establishes an okra regeneration technical system through good application effects of the germination culture medium, the induction culture medium, the differentiation culture medium and the strong bud rooting culture medium and mutual synergistic effect among the components.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.

Claims

1. A tissue culture medium for okra regeneration is characterized by comprising a germination medium, an induction medium, a differentiation medium and a strong bud rooting medium;

the induction culture medium takes a B5 culture medium as a basic culture medium, and further comprises: 0.8-1.5 mg/L KT, 0.3-1.2 mg/L2,4-D, 1.0-2.0 mg/L carbon nano tube, 30g/L sucrose and 6.5-7.0 g/L agar;

2. The tissue culture medium of claim 1, wherein the pH of the germination medium is 5.75-5.85; the pH value of the induction culture medium is 5.78-5.83; the pH value of the differentiation medium is 5.78-5.83; the pH value of the strong bud rooting culture medium is 5.78-5.83.

3. A regeneration tissue culture method of okra callus, which is characterized in that the tissue culture medium of claim 1 or 2 is adopted in the regeneration tissue culture method, and comprises the following steps:

4. The regenerative tissue culture method according to claim 3, wherein the length of the stem and the petiole of the okra aseptic seedling are respectively 1.0-2.0 cm; the stem and/or petiole of the okra aseptic seedling are obtained by the following steps: culturing the okra aseptic seedlings for 10-14 days, cutting off all roots, 1/5 stem parts and all leaves connected with the roots when the okra aseptic seedlings grow to the height of 5.0-8.0 cm, obtaining 4/5 stems and petioles with the residual length, and respectively cutting the 4/5 stems and/or petioles with the residual length into small sections with the length of 1.0-2.0 cm.

5. The regenerative tissue culture method according to claim 3, wherein the temperatures of the induction and proliferation culture, the differentiation culture and the rooting culture of the strong bud are 24 to 26 ℃ respectively; the temperature of germination culture is 20-28 ℃.

6. The regenerative tissue culture method according to claim 3, wherein the induction and proliferation culture, the differentiation culture and the rooting culture of the strong bud are all performed under the condition of light, the time of the light is 12-16 h/d, and the intensity of the light is 100-300 μmol-m ^-2 ·s ^-1 (ii) a The time for induction and multiplication culture is 60-75 days; the differentiation culture time is 25-30 d; the time for rooting culture of the strong buds is 40-60 days; the total time of germination culture is 15-35 d.

7. The regeneration tissue culture method according to claim 3, further comprising a secondary culture during the rooting culture of the strong buds, wherein the secondary culture is 20-34 days after the rooting culture of the strong buds, and the secondary culture medium is a rooting culture medium of the strong buds.

8. According to claim 3The regeneration tissue culture method is characterized in that the okra seeds are not irradiated before germination during germination culture, and the irradiation intensity after germination of the seeds is 100-300 mu mol/m ^-2 ·s ^-1 And the time of the germination stage of the okra fargesii seeds is 5-21 days.

9. The regenerative tissue culture method according to claim 3, wherein the hardening-off is finished indoors, the hardening-off temperature is not less than 20 ℃, and the hardening-off time is 4-6 days;

10. The regenerative tissue culture method according to claim 3, further comprising, prior to the inoculation, washing and disinfecting okra seeds;