CN113439662B - Culture medium for plant tissue culture and application thereof - Google Patents

Culture medium for plant tissue culture and application thereof Download PDF

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CN113439662B
CN113439662B CN202110956208.0A CN202110956208A CN113439662B CN 113439662 B CN113439662 B CN 113439662B CN 202110956208 A CN202110956208 A CN 202110956208A CN 113439662 B CN113439662 B CN 113439662B
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culture medium
culture
plant
regeneration
induction
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CN113439662A (en
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于波
孙映波
刘小飞
黄丽丽
朱根发
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Environmental Horticulture Institute of Guangdong Academy of Agricultural Sciences
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Environmental Horticulture Institute of Guangdong Academy of Agricultural Sciences
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002Culture media for tissue culture
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008Methods for regeneration to complete plants

Abstract

The invention discloses a culture medium for plant tissue culture and application thereof, belonging to the technical field of agricultural production. The invention discloses a culture medium for plant tissue culture, which is formed by adding 2.5-20.0 g of polypropylene fiber to each liter of basic culture medium on the basis of the basic culture medium for plant tissue culture; the basic culture medium comprises an induction culture medium used in a somatic embryo induction stage of explants and a regeneration culture medium used in a plant regeneration stage. According to the invention, the polypropylene fiber and the plant growth regulator are added into the culture medium, so that the induction rate of the somatic embryo can reach up to 100%, the regeneration rate of the plant can reach up to 100%, the number of regenerated plants of each explant reaches 40.5, and the hydrophobic property of the polypropylene fiber enables the culture medium not to be replaced back and forth or the plants not to be cleaned in the culture process, so that the time and labor force can be greatly saved.

Description

Culture medium for plant tissue culture and application thereof
Technical Field
The invention relates to the technical field of agricultural production, in particular to a culture medium for plant tissue culture and application thereof.
Background
Tissue culture of plants, also called in vitro culture, refers to a technique of separating desired tissues, organs or cells, protoplasts, etc. from plant bodies, and inoculating them on a culture medium containing various nutrients and plant growth regulators under aseptic conditions by aseptic manipulation to obtain regenerated whole plants or produce other products having economic value. A culture medium is a substance essential for a plant tissue culture process, and in general, agar (or carrageenan, etc.) is widely used as a medium support in a culture medium for a plant tissue culture. However, the agar (or carrageenan, etc.) containing medium is a solid gel, and may cause the waste and harmful substances generated from the cultured tissue to diffuse relatively slowly, thereby causing toxicity to some plants. Meanwhile, when the tissue culture seedling is transplanted, a large amount of culture medium is attached to the surface of the plant root system due to the hydrophilic characteristic of the agar (or carrageenan and the like) support, and if the root system agar is thoroughly cleaned, the time and labor are consumed, and the plant root system is easily damaged. If agar (or carrageenan and the like) is not cleaned, the tissue culture seedlings are easy to mildew or rot, and the survival rate of the transplanted plants is low, and the like, so that a new culture medium or a new plant tissue culture method is needed to be provided for solving the problems caused by the agar serving as a culture medium support in the prior art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a culture medium for plant tissue culture and an application method thereof, which are used for solving the problems in the prior art, and the culture medium for plant tissue culture is constructed by using polypropylene fibers as a support of the culture medium, and the culture medium is used for plant tissue culture such as hippeastrum rutilum, so that the time and the labor can be saved, and the tissue culture propagation efficiency of hippeastrum rutilum can be obviously improved. The highest regeneration rate of the hippeastrum rutilum plants can reach 100 percent, and on average, each explant can produce 40.5 plantlets at most.
The invention provides a culture medium for plant tissue culture, which is formed by adding 2.5-20.0 g of polypropylene fiber into per liter of a basic culture medium for plant tissue culture on the basis of the basic culture medium for plant tissue culture; the basic culture medium comprises an induction culture medium used in a somatic embryo induction stage of explants and a regeneration culture medium used in a plant regeneration stage.
The invention also provides a culture medium for plant tissue culture, which is formed by adding 2.5-20.0 g of polypropylene fiber into per liter of basic culture medium on the basis of the basic culture medium for plant tissue culture;
the basic culture medium comprises an induction culture medium used in a somatic embryo induction stage of explants and a regeneration culture medium used in a plant regeneration stage.
The invention also provides a plant tissue culture and rapid propagation method, which comprises the following steps:
step 1, somatic embryo induction: inoculating a plant explant onto polypropylene fibers in said culture medium and contacting the base of said explant with an induction medium to induce adult cell embryos; wherein the induction medium comprises the following components: MS culture medium + 0-6.0 mg/L6-benzylamino adenine + 2.5-20.0 g/L polypropylene fiber + 20-40 g/L cane sugar, pH6.2-6.4; culturing at 24-26 deg.c in dark condition to produce somatic embryo on the outer surface of the leaf;
step 2, plant regeneration: after the explant generates a somatic embryo, sucking out an induction culture medium, adding an equivalent regeneration culture medium, contacting the somatic embryo with the regeneration culture medium, and culturing at 24-26 ℃ in an illumination environment for 12-14 h every day, wherein the somatic embryo germinates to form a complete plant; the regeneration medium comprises the following components: MS culture medium, 0-3.0 mg/L6-furfuryl amino purine, 2.5-20.0 g/L polypropylene fiber, 20-40 g/L cane sugar and pH 6.2-6.4;
and step 3, transplanting: transplanting the obtained plant together with polypropylene fiber or polypropylene fiber without root into greenhouse for normal management and culture.
Preferably, the new leaf of the hippeastrum test-tube plantlet is transversely cut perpendicular to the longitudinal axis of the leaf, and the leaf is cut into segments with proper length to be used as explants.
Preferably, the length of the new leaves growing out is selected to be 0.5-5.0 cm; the leaves are cut into pieces of appropriate length of 0.5-1.0 cm.
Preferably, the polypropylene fibers in step 1 and step 2 are mixed with the corresponding culture medium according to the ratio of 7.5-12.5 g:1L of the additive.
Preferably, in step 1, the induction medium comprises the following components: MS culture medium, 0-5.0 mg/L6-benzylamino adenine, 2.5-20.0 g/L polypropylene fiber, 20-40 g/L cane sugar and pH 6.2-6.4.
Preferably, in step 2, the regeneration medium comprises the following components: MS culture medium, 0-2.0 mg/L6-furfuryl amino purine, 2.5-20.0 g/L polypropylene fiber, 20-40 g/L cane sugar and pH 6.2-6.4.
Preferably, in step 2, the illumination intensity is 1600 to 2000lux.
Preferably, the culture time is 4 to 8 weeks in both step 1 and step 2.
The invention also provides an application of the culture medium in plant tissue culture, which is applied to the following (1) or (2):
(1) Improving the induction rate of somatic embryos at the induction culture stage;
(2) The plant regeneration rate in the regeneration culture stage is improved.
Compared with the prior art, the invention has the beneficial effects that:
(1) Can effectively reduce browning. Because liquid culture is adopted, the fluidity of a culture medium is increased, phenolic substances generated by wounds of explants and the like are easy to diffuse rapidly, and the browning of the explants can be effectively reduced.
(2) The regeneration culture process is simplified. When the plant is regenerated and cultured, the original culture container can be used, and the fresh liquid culture medium can be directly replaced in the super clean bench, so that the operation process is simplified.
(3) Simplify the transplanting process and reduce the damage of tissue culture seedlings. When the plant is transplanted to a greenhouse, the polypropylene fiber has hydrophobicity, so that the liquid culture medium is not easy to adhere to the polypropylene fiber and the plant root system, and the plant and the polypropylene fiber can be directly transplanted together; or after separating the plant from the support, the plant is directly transplanted. The tedious process of cleaning the root agar of the tissue culture seedling can be avoided, the damage to the tissue culture seedling caused by cleaning the root can be effectively reduced, and a large amount of time and labor can be saved.
(4) When the tissue culture seedlings are transplanted, the polypropylene fibers and the plants can be separated for repeated use, and the production cost is effectively reduced.
(5) By integrating various improvements, the invention optimizes and establishes a plant regeneration system of the efficient somatic embryogenesis way of hippeastrum, the induction rate of the somatic embryo is up to 100 percent, the regeneration rate of the plant is up to 100 percent, and each explant can generate 40.5 plantlets at most on average. Compared with the prior art, the regeneration efficiency of the plants is obviously improved.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The following examples further illustrate the technical solution of the present invention by taking the tissue culture of hippeastrum as an example.
Example 1A method for tissue culture and rapid propagation of hippeastrum
(1) Explant induced somatic embryos
Preparing an induction culture medium: MS culture medium is taken as a basic culture medium, and 20g/L of sucrose is added, and the pH value is 6.2. The culture medium was dispensed into tissue culture vessels, and then polypropylene fibers were added in an amount of 2.5g per liter of liquid culture medium. Sealing, and sterilizing at 121 deg.C under high pressure for 20 min.
Inoculating and culturing: and when the new leaf of the hippeastrum test-tube plantlet grows to be 0.5cm in length, transversely cutting the new leaf in a superclean bench perpendicular to the longitudinal axis of the leaf, and cutting the leaf into segments with the length of 0.5cm to be used as explants.
The explants were inoculated on polypropylene fibers, the basal part of the explants was exposed to the above induction medium, and cultured for 4 weeks at 24 ℃ in the dark, and the outer surface of the leaf pieces were subjected to induction culture of somatic embryos.
(2) Plant regeneration
After the explant generates a somatic embryo, opening a culture container in a super clean bench, sucking out an induction culture medium, adding an equivalent regeneration culture medium, contacting the somatic embryo with the regeneration culture medium, and culturing for 4 weeks in a 12-hour daily illumination environment (illumination intensity of 1600 lux) at 24 ℃ until the somatic embryo germinates to form a complete plant; the regeneration medium comprises the following components: MS culture medium +2.5g/L polypropylene fiber +20g/L cane sugar, pH6.2.
(3) Plant transplantation
Taking out the plant together with the polypropylene fiber from the culture container, directly transplanting the plant into a greenhouse, taking peat and the like as a matrix, watering the plant after planting, and then carrying out normal management. During transplanting, the polypropylene fiber can be taken down from the root, and then the plant can be directly transplanted, and the polypropylene fiber can be repeatedly used.
Example 2A method for tissue culture and rapid propagation of hippeastrum
(1) Explant induced somatic embryos
Preparing an induction culture medium: MS culture medium is used as basic culture medium, and 25g/L of sucrose and 1.0mg/L of 6-benzylamino adenine are added, and the pH value is 6.2. The medium was dispensed into tissue culture vessels and then polypropylene fibers were added in an amount of 5.0g per liter of liquid medium. Sealing, and sterilizing at 121 deg.C under high pressure for 20 min.
Inoculating and culturing: and when the new leaf of the hippeastrum test-tube plantlet grows to be 1.5cm in length, transversely cutting the new leaf in a superclean bench perpendicular to the longitudinal axis of the leaf, and cutting the leaf into segments with the length of 0.8cm to be used as explants.
The explants were inoculated on polypropylene fibers, the basal part of the explants was exposed to the above induction medium, and cultured at 24 ℃ in the dark for 5 weeks, and somatic embryos were induced and cultured on the outer surface of the leaf blades.
(2) Plant regeneration
After the explant generates a somatic embryo, opening a culture container in a super clean bench, sucking out an induction culture medium, adding an equivalent regeneration culture medium, contacting the somatic embryo with the regeneration culture medium, and culturing for 5 weeks in a light environment (light intensity 1700 lux) of 13h every day at 24 ℃, wherein the somatic embryo germinates to form a complete plant; the regeneration medium comprises the following components: MS culture medium +1.0 mg/L6-furfuryl amino purine +5g/L polypropylene fiber +25g/L cane sugar, pH6.2.
(3) Plant transplantation
Taking out the plant together with the polypropylene fiber from the culture container, directly transplanting the plant into a greenhouse, taking peat and the like as a matrix, watering the plant after planting, and then carrying out normal management. During transplanting, the polypropylene fiber can be taken down from the root, and then the plant can be directly transplanted, and the polypropylene fiber can be repeatedly used.
Example 3A method for tissue culture and rapid propagation of hippeastrum
(1) Explant induced somatic embryos
Preparing an induction culture medium: MS culture medium is used as basic culture medium, and 30g/L of sucrose and 2.0mg/L of 6-benzylamino adenine are added, and the pH value is 6.3. The medium was dispensed into tissue culture vessels and then polypropylene fibers were added in an amount of 7.5g per liter of liquid medium. Sealing, and sterilizing at 121 deg.C under high pressure for 20 min.
Inoculating and culturing: and when the new leaf of the hippeastrum test-tube plantlet grows to be 2.5cm in length, transversely cutting the new leaf in a superclean bench perpendicular to the longitudinal axis of the leaf, and cutting the leaf into segments with the length of 0.8cm to be used as explants.
The explants were inoculated on polypropylene fibers, the basal part of the explants was exposed to the above induction medium, and cultured at 25 ℃ in the dark for 5 weeks, and somatic embryos were induced and cultured on the outer surface of the leaf blades.
(2) Plant regeneration
After the explant generates a somatic embryo, opening a culture container in a super clean bench, sucking out an induction culture medium, adding an equivalent regeneration culture medium, contacting the somatic embryo with the regeneration culture medium, and culturing for 5 weeks at 25 ℃ in a 13-hour daily illumination environment (illumination intensity of 1800 lux), wherein the somatic embryo germinates to form a complete plant; the regeneration medium comprises the following components: MS culture medium +1.5 mg/L6-furfurylaminopurine +7.5g/L polypropylene fiber +30g/L sucrose, pH6.3.
(3) Plant transplantation
Taking out the plant together with polypropylene fiber from the culture container, directly transplanting to a greenhouse, planting with peat as matrix, watering, and performing normal management. During transplanting, the polypropylene fiber can be taken down from the root, and then the plant can be directly transplanted, and the polypropylene fiber can be repeatedly used.
Example 4A method for tissue culture and rapid propagation of hippeastrum
(1) Explant induced somatic embryos
Preparing an induction culture medium: MS culture medium is used as basic culture medium, and 30g/L of sucrose and 3.0mg/L of 6-benzylamino adenine are added, and the pH value is 6.4. The medium was dispensed into tissue culture vessels and then polypropylene fibers were added in an amount of 10.0g per liter of liquid medium. Sealing, and sterilizing at 121 deg.C under high pressure for 20 min.
Inoculating and culturing: when the new leaves of the hippeastrum test-tube plantlet grow to 3.5cm in length, transversely cutting the leaves in a superclean bench in a manner of being vertical to the longitudinal axis of the leaves, and cutting the leaves into segments with the length of 1.0cm to be used as explants.
The explants were inoculated on polypropylene fibers, and the basal part of the explants was exposed to the above induction medium, cultured at 25 ℃ in the dark for 6 weeks, and the outer surface of the leaf was subjected to induction culture of adult cell embryos.
(2) Plant regeneration
After the explant generates a somatic embryo, opening a culture container in a super clean bench, sucking out an induction culture medium, adding an equivalent regeneration culture medium, contacting the somatic embryo with the regeneration culture medium, and culturing for 6 weeks in a 13-hour daily illumination environment (illumination intensity of 1900 lux) at 25 ℃ to ensure that the somatic embryo germinates to form a complete plant; the regeneration medium comprises the following components: MS culture medium +1.0 mg/L6-furfurylaminopurine +10.0g/L polypropylene fiber +30g/L sucrose, pH6.3.
(3) Plant transplantation
Taking out the plant together with the polypropylene fiber from the culture container, directly transplanting the plant into a greenhouse, taking peat and the like as a matrix, watering the plant after planting, and then carrying out normal management. During transplanting, the polypropylene fiber can be taken down from the root, and then the plant can be directly transplanted, and the polypropylene fiber can be repeatedly used.
Example 5 tissue culture and rapid propagation method of hippeastrum
(1) Explant induced somatic embryos
Preparing an induction culture medium: MS culture medium is used as basic culture medium, 35g/L of sucrose and 4.0mg/L of 6-benzylamino adenine are added, and the pH value is 6.4. The culture medium was dispensed into tissue culture vessels, and then polypropylene fibers were added in an amount of 10.0g per liter of liquid culture medium. Sealing, and sterilizing at 121 deg.C under high pressure for 20 min.
Inoculating and culturing: and when the new leaf of the hippeastrum test-tube plantlet grows to be 4.5cm in length, transversely cutting the new leaf in a superclean bench perpendicular to the longitudinal axis of the leaf, and cutting the leaf into segments with the length of 1.0cm to be used as explants.
The explants were inoculated on polypropylene fibers, and the basal part of the explants was exposed to the above induction medium, cultured at 25 ℃ in the dark for 7 weeks, and the outer surface of the leaf was subjected to induction culture of adult cell embryos.
(2) Plant regeneration
After the explant generates a somatic embryo, opening a culture container in a super clean bench, sucking out an induction culture medium, adding an equivalent regeneration culture medium, contacting the somatic embryo with the regeneration culture medium, and culturing for 7 weeks at 25 ℃ in a 13-hour daily illumination environment (illumination intensity of 2000 lux), wherein the somatic embryo germinates to form a complete plant; the regeneration medium comprises the following components: MS culture medium +2.5 mg/L6-furfurylaminopurine +10.0g/L polypropylene fiber +30g/L sucrose, pH6.3.
(3) Plant transplantation
Taking out the plant together with polypropylene fiber from the culture container, directly transplanting to a greenhouse, planting with peat as matrix, watering, and performing normal management. During transplanting, the polypropylene fiber can be taken down from the root, and then the plant can be directly transplanted, and the polypropylene fiber can be repeatedly used.
Example 6A method for tissue culture and rapid propagation of hippeastrum
(1) Explant induced somatic embryos
Preparing an induction culture medium: MS culture medium is used as basic culture medium, and 40g/L of sucrose and 6.0mg/L of 6-benzylamino adenine are added, and the pH value is 6.4. The medium was dispensed into tissue culture vessels and then polypropylene fibers were added in an amount of 20.0g per liter of liquid medium. Sealing, and sterilizing at 121 deg.C under high pressure for 20 min.
Inoculating and culturing: and when the new leaf of the hippeastrum test-tube plantlet grows to be 5.0cm in length, transversely cutting the new leaf in a superclean bench perpendicular to the longitudinal axis of the leaf, and cutting the leaf into segments with the length of 1.0cm to be used as explants.
The explants were inoculated on polypropylene fibers, and the basal part of the explants was exposed to the above induction medium, cultured at 25 ℃ for 8 weeks in the dark, and the outer surface of the leaf was subjected to induction culture of adult cell embryos.
(2) Plant regeneration
After the explant generates a somatic embryo, opening a culture container in a super clean bench, sucking out an induction culture medium, adding an equal amount of regeneration culture medium, contacting the somatic embryo with the regeneration culture medium, and culturing for 8 weeks in a daily 14-hour illumination environment (illumination intensity of 2000 lux) at 26 ℃, wherein the somatic embryo germinates to form a complete plant; the regeneration medium comprises the following components: MS culture medium +3.0 mg/L6-furfuryl amino purine +20.0g/L polypropylene fiber +40g/L cane sugar, pH6.4.
(3) Plant transplantation
Taking out the plant together with the polypropylene fiber from the culture container, directly transplanting the plant into a greenhouse, taking peat and the like as a matrix, watering the plant after planting, and then carrying out normal management. During transplanting, the polypropylene fiber can be taken down from the root, and then the plant can be directly transplanted, and the polypropylene fiber can be repeatedly used.
In order to prove that the addition of different amounts of polypropylene fibers and plant growth regulators in a culture medium for plant tissue culture has significant influence on the induction rate of somatic embryos and the plant regeneration rate, the induction rate of somatic embryos and the plant regeneration rate are counted by regulating different components of the culture medium on the basis of example 4.
On the basis of example 4, only the addition amount of the polypropylene fiber in step 1 and step 2 was changed, and the others were not changed. The results of counting the somatic induction rate and the plant regeneration rate are shown in Table 1.
TABLE 1 Effect of Polypropylene fiber content of the Medium on the Induction of the somatic embryos of hippeastrum and the regeneration of the plants
Figure BDA0003220530100000111
Based on example 4, only the amount of 6-benzylamino adenine (6-BA) added to the induction medium in step 1 was changed, and the others were not changed. The results of counting the somatic induction rate are shown in Table 2.
TABLE 2 Effect of 6-benzylamino adenine (6-BA) content in Medium on the Induction of somatic embryos of hippeastrum
Figure BDA0003220530100000112
Figure BDA0003220530100000121
On the basis of example 4, only the amount of 6-furfurylaminopurine (KT) added to the regeneration medium in step 2 was changed, and the others were not changed. The results of the statistics of plant regeneration rates and the number of regenerated plants per explant are shown in Table 3.
TABLE 3 Effect of medium 6-furfuryl aminopurine (KT) content on the regeneration of hippeastrum plants
KT content (mg/L) Plant regeneration rate (%) Number of regenerated plants per explant
0 55.6 10.5
0.5 84.8 30.6
1.0 100 40.5
1.5 100 32.4
2.0 88.5 25.6
2.5 74.3 16.8
3.0 61.2 11.3
In the above tables 1 to 3, the somatic embryo induction rate (%) = the number of explants forming somatic embryos ÷ total number of explants × 100%; plant regeneration rate (%) = explant number of regenerated plants/total explant number × 100%; number of regenerated plants per explant = total number of regenerated plants ÷ total number of explants.
As can be seen from tables 1-3, the addition of polypropylene fiber, 6-BA and 6-KT to the medium had a direct effect on somatic embryo formation and the generation of regenerated plants. Analysis shows that when the addition amount of polypropylene fiber in an induction culture medium and a regeneration culture medium is 7.5-12.5 g/L, both the somatic embryo induction rate and the plant regeneration rate can reach 100%, and the maximum number of regenerated plants of each explant can reach 40.5; meanwhile, on the basis that the addition amount of polypropylene fiber is 10g/L, 3.0 mg/L6-BA is added into an induction culture medium to enable the induction rate of somatic embryos to reach 100%, 1.0 mg/L6-KT is added into a regeneration culture medium to enable the regeneration rate of plants to reach 100%, and the number of regenerated plants of each explant reaches 40.5. Therefore, compared with the control, the addition of the polypropylene fiber and the plant growth regulator in the culture medium can obviously improve the induction rate of somatic embryos, the plant regeneration rate and the number of regenerated plants of each explant.
According to the above examples and analysis of the obtained experimental results, one of the reasons for the remarkable effect may be that the polypropylene fiber added to the plant tissue culture medium has the following advantages:
1. the support has stable physical properties and can withstand sterilization at 121 deg.C under high temperature and high pressure.
2. The support has stable chemical property, and can be placed in liquid culture medium for a long time without deterioration, release toxic and harmful substances, change the composition and pH of the culture medium, and influence plant growth.
3. The support is fluffy and has large pores, and harmful substances generated by cultured tissues can be rapidly diffused in the plant culture process, so that the damage of the harmful substances to plants is reduced.
4. The support has hydrophobicity and the advantages are as follows: (1) In the culture process, the fresh liquid culture medium can be replaced in time. Due to the hydrophobicity, the old liquid culture medium can be directly sucked out when the culture medium is replaced, and the old culture medium can not remain on the surface of the support. (2) Because of the hydrophobicity, the liquid culture medium can not be attached to the surface of the support body, and after the plant culture is finished, if the support body does not need to be separated, the test-tube plantlets can be directly transplanted by draining the liquid culture medium, so that the time and the labor are saved. (3) After the plant culture is finished, if the support body needs to be separated, the test-tube plantlet is very easy to separate from the support body due to hydrophobicity, time and labor are saved, and meanwhile, the support body is convenient to recycle, and the cost is saved.
5. The support body is not easy to deform and can be used as a soilless culture medium, after the plant culture is finished, the test-tube plantlet can be taken out together with the support body and directly switched into a nutrient solution soilless culture mode, and the transplanting process is time-saving, labor-saving and cost-saving.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (3)

1. A method for tissue culture and rapid propagation of hippeastrum rutilum is characterized by comprising the following steps:
step 1, somatic embryo induction: taking new leaves of the test-tube plantlets of the hippeastrum, transversely cutting the new leaves perpendicular to the longitudinal axis of the leaves, cutting the leaves into segments with proper length as explants, inoculating the explants onto polypropylene fibers in an induction culture medium, and contacting the base parts of the explants with the induction culture medium to induce and culture adult cell embryos; wherein the induction medium comprises the following components: MS culture medium +3.0 mg/L6-benzylamino adenine +10.0g/L polypropylene fiber +30g/L cane sugar, pH6.2-6.4; culturing at 24-26 deg.c in dark condition to produce somatic embryo on the outer surface of the leaf;
step 2, plant regeneration: after the explant generates a somatic embryo, sucking out an induction culture medium, adding an equal amount of regeneration culture medium, contacting the somatic embryo with the regeneration culture medium, and culturing at 24-26 ℃ in an illumination environment for 12-14 h every day to ensure that the somatic embryo germinates to form a complete plant; the regeneration medium comprises the following components: MS culture medium +1.0 mg/L6-furfuryl amino purine +10.0g/L polypropylene fiber +30g/L cane sugar, pH6.3;
and step 3, transplanting: transplanting the obtained plant together with the polypropylene fiber or removing the polypropylene fiber at the root into a greenhouse for normal management and culture;
wherein, the culture time in the step 1 and the step 2 is 4-8 weeks.
2. The method of claim 1, wherein the length of the new leaves of hippeastrum test-tube plantlet is 0.5-5.0 cm; the leaves are cut into pieces of appropriate length of 0.5-1.0 cm.
3. The method of claim 1, wherein in step 2, the illumination intensity is 1600 to 2000lux.
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