CN115005104B - Tissue culture medium and regeneration tissue culture method for okra regeneration - Google Patents
Tissue culture medium and regeneration tissue culture method for okra regeneration Download PDFInfo
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- CN115005104B CN115005104B CN202210865003.6A CN202210865003A CN115005104B CN 115005104 B CN115005104 B CN 115005104B CN 202210865003 A CN202210865003 A CN 202210865003A CN 115005104 B CN115005104 B CN 115005104B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- 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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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 strong bud rooting culture medium. KT, 2,4-D, 6-BA, NAA and carbon nano tube of each culture medium act together and are favorable to inducing the okra callus to form, proliferate and differentiate, obtain a large amount of cluster buds and tissue culture seedlings, and then obtain a plant with good consistency and robustness.
Description
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, a 1/2MS culture medium, a 1/3MS culture medium or a 1/4MS culture medium as a basic culture medium, and also 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 also comprises: 0.5-1.0 mg/L of 6-BA, 0.3-0.8 mg/L of LNAA, 0.5-1.5 mg/L of carbon nano tube, 30g/L of cane sugar and 6.5-7.0 g/L of 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/L of 6-BA, 0.1-0.5 mg/L of LNAA, 2.0-5.0 mg/L of carbon nano tube, 40g/L of cane sugar and 6.5-7.0 g/L of 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 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 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 rooting culture of the strong buds are respectively 24-26 ℃; the temperature of the germination culture is 20-28 ℃.
Preferably, the induction and proliferation culture, the differentiation culture and the rooting culture of the strong bud are performed in the lightThe illumination time is 12-16 h/d, and the illumination intensity is 100-300 mu mol.m -2 ·s -1 (ii) a The time of the induction and the propagation culture is 60-75 d; the time of the differentiation culture is 25-30 d; the time for rooting culture of the strong buds is 40-60 days; the total time of the germination culture is preferably 15-35 d.
Preferably, the rooting culture period of the strong bud also comprises one-time subculture, the subculture time is 20-34 days after rooting culture of the strong bud, and the subculture medium is a rooting medium of the strong bud.
Preferably, the okra seeds are not irradiated before germination during germination culture, and the irradiation intensity after germination is 100-300 mu mol.m -2 ·s -1 The time of the germination stage of the okra flecks equiseti seeds is 5-21 days.
Preferably, the seedling exercising is finished indoors, the seedling exercising temperature is not lower than 20 ℃, and the seedling exercising time is 4-6 days;
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): (0.5-1.0).
Preferably, before the inoculation, the method further comprises the steps of cleaning and disinfecting okra seeds;
the cleaning comprises detergent cleaning and flowing water flushing, the detergent cleaning time is 20-30 min, and the flowing water flushing 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 percent mercuric chloride solution is soaked.
The invention has the beneficial effects that: the invention provides a regeneration tissue culture medium for okra, which comprises a germination culture medium, an induction culture medium, a differentiation culture medium and a strong bud rooting culture medium; the germination culture medium takes an MS culture medium, a 1/2MS culture medium, a 1/3MS culture medium or a 1/4MS culture medium as a basic culture medium, and also 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 also comprises: 0.5-1.0 mg/L of 6-BA, 0.3-0.8 mg/L of LNAA, 0.5-1.5 mg/L of carbon nano tube, 30g/L of cane sugar and 6.5-7.0 g/L of 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/L of 6-BA, 0.1-0.5 mg/L of LNAA, 2.0-5.0 mg/L of carbon nano tube, 40g/L of cane sugar and 6.5-7.0 of 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, strong plants with good consistency are obtained. The results of the examples show that: by using 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. According to the invention, the germination culture medium, the induction culture medium, the differentiation culture medium and the strong bud rooting culture medium have good application effects, and all components have mutual synergistic effect, so that strong okra tissue culture seedling plants with good consistency can be obtained under the control of proper component concentration, 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 required in the embodiments will be briefly described below.
FIG. 1 shows the aseptic seedling condition of the 14 th seed of okra seed culture 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 of example 1 at the 25 th day.
Detailed Description
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.
In the invention, the germination culture medium takes an MS culture medium, a 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 MS culture medium, 1/2MS culture medium, 1/3MS culture medium or 1/4MS culture medium, and only contains: 6.5-7.0 g/L agar. The germination 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 the B5 medium 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 B5 medium is a minimal medium, and further comprises only: 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 cane sugar 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-tube in the induction culture medium is 1.0-2.0 mg/L, preferably 1.5-1.9 mg/L, and more preferably 1.8mg/L; the agar concentration in the induction medium is: 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 6.5g/L. In the invention, KT and 2,4-D can jointly promote the formation of okra callus under a proper concentration ratio.
In the present invention, the pH of the induction medium is preferably 5.78 to 5.83, more preferably 5.80.
In the present invention, the differentiation medium uses the B5 medium as a minimal medium, and further includes: 0.5-1.0 mg/L of 6-BA, 0.3-0.8 mg/L of LNAA, 0.5-1.5 mg/L of carbon nano tube, 30g/L of cane sugar and 6.5-7.0 g/L of agar; more preferably, the B5 medium is a minimal medium, and further comprises only: 0.5-1.0 mg/L of 6-BA, 0.3-0.8 mg/L of LNAA, 0.5-1.5 mg/L of carbon nano tube, 30g/L of cane sugar and 6.5-7.0 g/L of agar. In the present invention, the concentration of 6-BA in the differentiation medium is 0.5 to 1.0mg/L, preferably 0.6 to 0.8mg/L6-BA, and more preferably 0.7 to 0.8mg/L6-BA. The NAA concentration in the differentiation culture medium is 0.3-0.8 mg/L, preferably 0.35-0.6 mg/LNAA, and more preferably 0.4mg/LNAA. The concentration of the carbon nano tubes 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 concentration of agar in the differentiation medium is 6.5 to 7.0g/L agar, preferably 6.7 to 7.0g/L agar, and more preferably 6.8 to 7.0g/L agar.
In the present invention, the pH of the differentiation medium is preferably 5.78 to 5.83, 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/L of 6-BA, 0.1-0.5 mg/L of LNAA, 2.0-5.0 mg/L of carbon nano tube, 40g/L of cane sugar 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/LNAA, 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 culture medium of 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 medium of the strong bud 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 tube in the rooting medium for the strong bud 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 medium for the strong buds is 6.5 to 7.0g/L, preferably 6.6 to 6.9g/L, and more preferably 6.7 to 6.8g/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 present invention, the pH value of the strong bud rooting medium is preferably 5.78 to 5.83, 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 KT, 2,4-D, 6-BA, NAA, carbon nano tubes, sucrose and agar are not specially limited, and conventional commercial products are adopted. The carbon nanotubes of the present invention are preferably multi-walled carbon nanotubes.
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 to a strong bud rooting culture medium for strong bud rooting culture, and hardening and transplanting culture after the strong bud rooting culture.
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 washing time of the detergent is preferably 20-30 min, more preferably 22-28 min, and even more preferably 25min. In the embodiment of the present invention, the detergent washing time of the embodiment is preferably 25min and 30min. 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 120min. 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% ethanol and 0.1% mercury-liter solution by mass concentration, and more preferably 75% ethanol and 0.1% mercury-liter solution by mass concentration. The disinfection mode of the invention firstly uses alcohol with the mass concentration of 75 percent to preferentially soak for 30 to 45s, further preferentially 38 to 42s, further preferentially 40s, and then preferentially washes by using sterile water for 2 times; soaking the substrate in 0.1% mercuric chloride solution for 20-30 min, preferably 24-29 min, and more preferably 25-28 min; finally, washing with sterile water for 4-5 times, preferably 4 times; when the 0.1% mercuric chloride solution is soaked, 2-3 drops of Tween 20 are preferably added. The present invention is not particularly limited to sterile water, and is preferably obtained by sterilizing distilled water; 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, 4 okra seeds are preferably inoculated in each germination culture bottle.
The okra seeds are used as explants for tissue culture and rapid propagation seedling raising, and the technical problem of germplasm degradation in the conventional seedling raising process by using the okra seeds in the prior art is solved.
In the invention, the germination culture comprises a germination stage and a sterile seedling culture stage of okra seeds. In the present invention, the temperature of the germination culture is preferably 20 to 28 ℃, more preferably 23 to 27 ℃, and still 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 and subjected to light culture after exposure of the seeds during the germination culture, and the light intensity of the light 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, the light source is turned on for illumination culture. 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 okra arrowhead seeds is 5-21 days, and one step is addedThe step (b) is preferably 7 to 20d, more preferably 20d. 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 okra seeds cultured by the method can reach over 75 percent, and the germination rate is high.
And culturing the okra aseptic seedlings to obtain the okra aseptic 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 12d.
In the invention, the total germination culture time comprises a germination stage and a sterile seedling culture stage of okra seeds. In the present invention, the total time of the germination culture is preferably 15 to 35 days, more preferably 21 to 34 days, and still more preferably 30 to 32 days. In the invention, the total germination culture time is calculated by the germination stage time of okra seeds and the culture time of okra aseptic 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 stems and petioles of okra aseptic seedlings are inoculated at the same time, the stems and the petioles 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 5.5cm. 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 residual lengths, cutting the 4/5 stems with the residual lengths into small sections with the lengths of 1.0-2.0 cm, inoculating the small sections with the lengths of 1.0-2.0 cm into an induction culture medium, and performing callus induction and proliferation culture, or cutting the petioles into small sections with the lengths of 1.0-2.0 cm, inoculating the small sections with the lengths of 1.0-2.0 cm into the induction culture medium, and performing callus induction and proliferation culture, or simultaneously inoculating the small sections with the lengths of 1.0-2.0 cm and the 1.0-2.0 cm petioles into the induction culture medium, and performing 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 1.5cm.
In the present invention, the temperature of the callus induction and proliferation culture is preferably 24 to 26 ℃, 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 16h/d; the intensity of the light irradiation is preferably 100 to 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 of the invention is preferably 60-75 d, and more preferably 70d. 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 invention preferably cuts the okra callus into the volume of 0.5-0.8 cm 3 The small pieces are inoculated on a callus differentiation culture medium.
In the present invention, the callus differentiation culture temperature is preferably 24 to 26 ℃, 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 16h/d; the intensity of the light irradiation is preferably 100 to 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 inventionThe callus is cultured for 20 to 25 days, preferably 22 to 25 days, and more preferably 23 to 25 days. 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 preferentially cuts the cluster buds of the okra fleshy stems from the callus tissues and 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 of the present invention is preferably 3.0 to 5.0cm, more preferably 3.0 to 4.0cm
In the present invention, the temperature for the rooting culture of the strong bud is preferably 24 to 26 ℃, 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 16h/d; the intensity of the light irradiation is preferably 100 to 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 culturing the rooting 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 bud also comprises one-time subculture, the subculture time is preferably 20-34 d, more preferably 26-32 d, and more preferably 28-31 d of rooting culture of the strong bud, and the subculture medium is preferably a rooting culture medium of the strong bud. The subculture is to replace the strong bud rooting culture medium once and then continue to carry out the strong bud rooting culture.
The test-tube plantlets obtained after the rooting culture of the strong buds meet the three conditions of 5-8 cm height, more than 5-7 leaves and 3-5 roots at the same time, and then are subjected to hardening seedling transplantation and transplantation culture, so that the survival rate of the test-tube plantlets is improved.
In the present invention, the germination culture, callus induction and proliferation culture, callus differentiation culture and strong bud rooting culture are preferably performed in a tissue culture bottle, the tissue culture bottle is preferably a cylindrical bottle with a diameter of 7.5cm and a height of 11.0cm, and the thickness of the 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 seedling exercising is preferably finished indoors, the seedling exercising temperature is preferably equal to or more than 20 ℃ according to the growth temperature requirement of okra, and the seedling exercising time is preferably 4-6 d, and more preferably 5d. In the invention, preferably, when hardening seedlings, the tissue culture bottle for strong bud rooting culture is placed in a tissue culture room buffer room after being loosened, 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 finished 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 composed of 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 plug disk can be prepared by adopting conventional products. The preferred tray format for the application described in the present invention is 10 rows by 10 columns. In the invention, 100 test-tube plantlets are preferably planted in each plug tray.
After the transplantation, the conventional management is preferably carried out on the okra seedlings. 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 plantlets 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: and (3) shaking and washing the mature and fresh okra seeds in a detergent solution for 25min, and then washing the seeds for 100min by using running water to obtain the washed and 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 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: the 1/2MS is used as a basic culture medium, and only 6.5g/L agar is added into the culture medium. Culture mediumCulturing in dark at pH of 5.8 for 5 days, exposing the seeds to white, and culturing under illumination with light source at 250 μmol/m -2 ·s -1 . After 20 days of dark and light culture, the seed germination was completed. 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 proliferation culture of callus: and (4) taking out the sterile seedlings of the seeds in the step (3), cutting off all roots and stem parts and leaves (the petioles are reserved) which are about 1/5 of the length connected with the roots, cutting the rest 4/5 of the stems 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 6.5g/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 16h/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 culture medium once 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 composition of the differentiation medium is as follows: the B5 culture medium is used as a basic culture medium, and 0.8mg/L of 6-BA, 0.4mg/L of NAA, 1.0mg/L of carbon nano tube, 30g/L of cane sugar 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 16h. After 20 days of culture, generating light green bud points on the surface of the dark green callus tissue culture; and (4) continuing to culture until the bud points grow slowly until the bud points become green cluster buds.
(6) Strong bud rooting culture: cutting off the cluster buds of 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, 3.0mg/L of carbon nano tube, 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 16h. Rooting is started on the 8 th day of rooting culture of the strong bud, and the rooting culture medium of the strong bud is replaced once on the 28 th day of rooting culture, namely, one-time subculture is carried out. And (4) culturing the strong buds for 50 days, and culturing 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 is 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 2d.
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. 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: 2021 year oldAnd (4) 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 is used as a basic culture medium, only 7.0g/L agar is added to the culture medium, and the pH of the culture medium is 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, starting to sprout. 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 proliferation culture of callus: and (4) when the aseptic seedlings grow for 28 days and are more than 5.0cm high in the step (3), taking out the aseptic seedlings, cutting off all roots and stem parts and leaves (the petioles are reserved) which are about 1/5 of the length connected with the roots, cutting the rest 4/5 of the stem parts into small sections with the length of 1.8cm, cutting the petioles into small sections with the length of 1.5cm, and simultaneously inoculating the small sections of the stems and the small sections of the petioles onto a callus induction culture medium. The composition of the induction culture medium is that the B5 culture medium is used as a basic culture medium, kinetin KT is 1.2mg/L, auxin is 2,4-D is 0.3mg/L, multi-wall carbon nano-tubes are 1.5mg/L, and cane sugar is added by 30g/L and agar is added by 6.5g/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 16h. After 12d inoculation, yellowish or greenish calli were generated around the incision. After the whole callus induction time is 33d and 33d, subculture is continued for 30d according to the original formula,the callus continued to expand and proliferate.
(5) Callus differentiation culture: 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 composition of the differentiation medium is as follows: 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 cane sugar 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 16h. Through 23d of culture, light green bud points are generated on the surface of the dark green callus tissue culture; and (4) continuing to culture until the bud points grow slowly until the bud points become green cluster buds.
(6) Strong bud rooting culture: and (4) cutting off the cluster buds obtained in the step (5) from the callus, wherein the length of the cut cluster buds is 3.0-4.0cm. Transferring to strong bud culture medium for strong bud and rooting culture. The medium for rooting the strong buds comprises the following components: MS is used as a basic culture medium, 6-BA0.7mg/L, NAA0.4mg/L, carbon nano tube 4.0mg/L, 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 16h. Rooting is started 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) culturing the strong buds and the roots for 60 days, and culturing the strong buds and the roots for 60 days to obtain test-tube seedlings with the height of 5-8 cm, 5-7 leaves and the number of 3-5. 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 is carried out indoors. And (4) loosening the cover of the tissue culture bottle, placing the tissue culture bottle in a buffer room of a tissue culture room for 2d, then moving the tissue culture bottle into an indoor room, placing the tissue culture bottle in natural scattered light, and placing the tissue culture bottle for 3d. 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: 1, namely, garden soil =1, and the culture medium is subjected to 800 times of carbendazim disinfection treatment. 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 are germinated and cultured, 1/3MS is used as a basic culture medium during germination and culture, and other conditions of germination and culture are the same as those in example 1.
The germination rates, initial germination times and final germination times of the okra seeds obtained in the sterile germination culture of the okra seeds of examples 1 to 4 are counted, the results are shown in table 1, and the 14 th condition of the sterile seedling culture of the example 1 is shown in fig. 1. The calculation formula of the germination rate is as follows:
seed germination rate (%) = number of germinated seeds × 100/number of inoculated seeds
The results of aseptic germination cultures of okra seeds of examples 1-4 are shown in table 1.
TABLE 1 sterile germination culture results of okra seeds in examples 1 to 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 stem parts and leaves (the petioles are reserved) which are about 1/5 of the length connected with the roots, cutting the rest 4/5 of the stems into small sections of 1.8cm, cutting the petioles into small sections of 1.5cm, and inoculating the small sections 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 6.5g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m -2 ·s -1 And the illumination time is 16h. After inoculation at 13d, pale yellow or pale green callus is produced around the incision, and the culture is continued for 16d, and no callus is produced on the stem segments and the petioles 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 6.5g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m -2 ·s -1 And the illumination time is 16h. 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 medium comprises a DKW culture medium as a basic culture medium, kinetin KT 1.2mg/L, auxin 2, 4-D0.3 mg/L, multi-wall carbon nano-tubes 1.5mg/L, and cane sugar 30g/L and agar 6.5g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m -2 ·s -1 And the illumination time is 16h. The rest of the conditions were the same as in comparative example 1. After inoculation for 16d, yellowish or pale green callus is generated around the incision, and the culture is continued for 16d, and the stem segment and petiole do not generate callus any more at the 32d inoculation time. After induction culture for 35 days, the culture medium is replaced according to the original formulaAnd continuing subculturing 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 6.5g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m -2 ·s -1 And the illumination time is 16h. 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 to 4 only induction and proliferation culture was performed after obtaining the aseptic seedlings. Statistics are carried out on the callus generation time, callus completion time and callus induction rate of okra esculenta in example 1 and comparative examples 1-4, the results are shown in table 2, and the callus induction 12d condition in example 1 is shown in fig. 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 (%) = number of callus-producing stem pieces × 100/number of inoculated stem pieces
TABLE 2 okra callus induction culture results of example 1 and comparative examples 1 to 4
As can be seen from Table 2, the basic culture medium is the B5 culture medium, the best effect is achieved when the okra seeds are induced and cultured in a proliferation mode, the callus induction rate is as high as 96.67%, and the induction culture time is the shortest.
Comparative example 5
Example 1 Induction and enrichment culture of okra seed callus, callus was obtained and differentiation culture of callus was performed in the form of B5 medium as basal medium supplemented with 6-BA 0.8mg/L, IAA 0.4mg/L, and carbon nanotubes 1.0mgThe concentration of the compound is 30g/L, the concentration of sucrose is 7.0g/L, and the concentration of agar is 7.0g/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 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 7.0g/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 medium comprises B5 medium as basic medium, and added with TDZ 0.8mg/L, NAA0.4mg/L, carbon nanotube 1.0mg/L, sucrose 30g/L, and agar 7.0g/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 35d to obtain cluster buds. The rest of the conditions were the same as in comparative example 5.
Comparative example 8
The composition of the differentiation culture medium is that the B5 culture medium is used as a basic culture medium, 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 callus. Statistics is 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 (%) = number of differentiated callus pieces × 100/number of inoculated callus pieces
Differentiation coefficient = number of plantlets differentiated per callus
TABLE 3 results of okra callus differential culture 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 example 1 through induction and proliferation culture, 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 composition of the differentiation medium is as follows: 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 16h. After 25d 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.
Comparative example 10
The composition of the differentiation medium is as follows: 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 composition of the differentiation medium is as follows: 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 (4) continuing to culture until the bud points grow slowly until the bud points become green cluster buds. 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 used for differentiation culture only. The differentiation culture results of okra callus of example 1 and comparative examples 9 to 11 were counted, and the differentiation coefficient and differentiation rate were calculated, and the results are shown in 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 medium for rooting the strong buds 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 6.8g/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 250 mu mol.m -2 ·s -1 And the illumination time is 16h. Rooting is started at the 13 th day of the strong bud rooting culture medium, and the strong bud rooting culture medium is replaced once when the strong bud rooting culture medium is cultured at the 28 th day, namely, subculture is carried out once.
Comparative example 13
The medium for rooting the strong buds comprises the following components: MS is used as a basic culture medium, 0.5mg/L of 6-BA is added, 0.3mg/L of 2,4-D is added, 3.0mg/L of carbon nano tube is added, 40g/L of cane sugar is added, and 6.8g/L of agar is added. Rooting is started at the 20 th day of the strong bud rooting culture medium, and the strong bud rooting culture medium is replaced once when the strong bud rooting culture medium is cultured at the 31 th day, namely, subculture is carried out 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 6.8g/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 6.8g/L. Rooting is started at the 32 th day of the strong bud rooting culture medium, and the strong bud rooting culture medium is replaced once when the strong bud rooting culture medium is cultured at the 31 th day, namely, subculture is carried out once. The remaining conditions were the same as in comparative example 12.
Comparative example 16
The strong bud rooting culture medium 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 strong bud rooting culture medium does not root before being replaced, when the culture is carried out for the 31 th day, the strong bud rooting culture medium is replaced for one-time subculture, and rooting is started after the 4 th day after subculture, namely rooting is started at the 35 th day of the strong bud rooting culture. 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 (%) = number of roots × 100/number of inoculations
TABLE 5 Effect of the plant growth regulators of example 1 and comparative examples 12 to 16 on the shoot-bud-strengthening and rooting culture of Hibiscus esculentus
| Serial number | Growth regulatorsCombination (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
The cluster buds obtained in the example 1 are used for strong bud rooting culture, the cluster buds are cut off from callus, the length of the cluster buds is 3.0-4.0cm, and the cluster buds are transferred to a strong bud rooting culture medium for strong bud rooting culture. The strong bud rooting culture medium comprises the following components: MS is taken 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 16h. 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, 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, example 5 and comparative examples 17 to 18 on the bud strengthening and rooting culture of cluster buds of okra 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 regeneration technical system of the okra is established through the good application effect of the germination culture medium, the induction culture medium, the differentiation culture medium and the strong bud rooting culture medium and the mutual synergistic effect of 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 (9)
1. A regeneration tissue culture method of okra callus is characterized by comprising 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;
transplanting the cluster buds of the okra to a strong bud rooting culture medium for strong bud rooting culture, and then hardening and transplanting culture;
the germination culture medium takes an MS culture medium, a 1/2MS culture medium, a 1/3MS culture medium or a 1/4MS culture medium as a basic culture medium, and only contains: 6.5-7.0 g/L agar;
the induction culture medium takes a B5 culture medium as a basic culture medium and only comprises: 0.8-1.5 mg/LKT, 0.3-1.2 mg/L2,4-D, 1.0-2.0 mg/L multiwall carbon nanotube, 30g/L sucrose and 6.5-7.0 g/L agar;
the differentiation medium takes a B5 medium as a basic medium and only comprises: 0.5-1.0 mg/L of 6-BA, 0.3-0.8 mg/L of LNAA, 0.5-1.5 mg/L of carbon nano tube, 30g/L of cane sugar and 6.5-7.0 g/L of agar;
the strong bud rooting culture medium takes an MS culture medium as a basic culture medium and only contains: 0.3-0.8 mg/L of 6-BA, 0.1-0.5 mg/L of LNAA, 2.0-5.0 mg/L of carbon nano tube, 40g/L of cane sugar and 6.5-7.0 g/L of agar.
2. The regenerative tissue culture method according to claim 1, wherein the pH value of the germination medium is 5.75 to 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; the pH value of the strong bud rooting culture medium is 5.78-5.83.
3. The regenerative tissue culture method according to claim 1, wherein the length of the stem and petiole of the okra aseptic seedling is 1.0-2.0 cm respectively; 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 stems and petioles with the residual length of 4/5, and respectively cutting the stems and/or petioles with the residual length of 4/5 into small sections with the length of 1.0-2.0 cm.
4. The regenerative tissue culture method according to claim 1, 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 the germination culture is 20-28 ℃.
5. The regenerative tissue culture method according to claim 1, wherein the induction and proliferation culture, the differentiation culture and the rooting culture of strong buds are all performed under the condition of light, the time of the light is 12 to 16h/d, and the intensity of the light is 100 to 300 μmol-m -2 ·s -1 (ii) a The time of the induction and the propagation culture is 60-75 d; the time of the differentiation culture 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.
6. The regeneration tissue culture method according to claim 1, characterized in that the period of the rooting culture of the strong buds further comprises a subculture, the subculture time is 20-34 days after the rooting culture of the strong buds, and the subculture medium is a rooting culture medium of the strong buds.
7. The regenerative tissue culture method according to claim 1, wherein the okra seeds are not irradiated with light before germination during germination culture, and the intensity of light after germination is 100 to 300 μmol-m -2 ·s -1 The time of the germination stage of the okra fargesii seeds is 5-21 days.
8. The regeneration tissue culture method according to claim 1, characterized in that the hardening off is completed indoors, the hardening off temperature is not less than 20 ℃, and the hardening off time is 4-6 days;
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): (0.5-1.0).
9. The regenerative tissue culture method according to claim 1, further comprising, before the inoculation, washing and sterilizing okra seeds;
the cleaning comprises detergent cleaning and flowing water flushing, the detergent cleaning time is 20-30 min, and the flowing water flushing 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 with sterile water for 2 times; 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 percent mercuric chloride solution is soaked.
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