CN107711514B - Tissue culture and rapid propagation method for excellent individual plant of hibiscus purpureus in dry land - Google Patents
Tissue culture and rapid propagation method for excellent individual plant of hibiscus purpureus in dry land Download PDFInfo
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- CN107711514B CN107711514B CN201711192038.3A CN201711192038A CN107711514B CN 107711514 B CN107711514 B CN 107711514B CN 201711192038 A CN201711192038 A CN 201711192038A CN 107711514 B CN107711514 B CN 107711514B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a tissue culture and rapid propagation method of a fine single plant of a Hibiscus aridicola Anthony purple flower line in a dry land. The method takes the excellent individual plant of the hibiscus purpureus in the dry land as an explant, effectively solves the bottleneck problems of tissue culture, rapid propagation, introduction and domestication of the hibiscus syriacus in the dry land and commercial development and utilization of the hibiscus syriacus through a series of culture steps of disinfection, induction, differentiation, proliferation and rooting of the explant, simultaneously effectively avoids reduction of natural plant quantity and damage to a natural vegetation area caused by over utilization of wild resources, and particularly has a positive effect on keeping the stability of the special shape of the excellent individual plant. The development and utilization prospect is excellent. The method has the advantages that the induced differentiation rate is 80%, the propagation period is 30 days, the proliferation coefficient is 4, the rooting rate is 95%, the transplanting survival rate is 92%, the propagation quantity and the growth rate of the hibiscus syriacus on the dry land are greatly improved, and technical support is provided for introduction, domestication, preservation and large-scale production of the species.
Description
Technical Field
The invention relates to a rapid propagation method for plant tissue culture in biotechnology, in particular to a rapid propagation method for excellent individual plant tissue culture of hibiscus purpureus in dry land.
Background
Hibiscus aridicus Anthony belonging to Malvaceae, Hibiscus arborescens Anthony, Malvaceae, is a beautiful flowery shrub. The hibiscus syriacus is a special species of dry-hot dry and warm river valley of Jinshajiang, and is only distributed in areas of Yunan Lijiang and Sichuan salt side, and in dry-hot dry and warm river valley clusters with the altitude of 600-. The flower color is common in white, and purple flower and pink flower are extremely rare. Meanwhile, as the number of natural distribution points is less than 5, the population is continuously declined, and the species is listed in the red famous records of Chinese species in 2004 as endangered species [ EN B2ab (ii) ]. In recent years, as reservoir dams are built, the habitat is gradually lost, and the living environment of the hibiscus in the dry land is threatened more seriously due to frequent influence of human activities.
At present, the aseptic rapid propagation in the biotechnology becomes an important means for the production of seedlings of traditional Chinese medicinal materials, flowers, endangered species, economic forest fruits and the like.
So far, the prior art has no report on the tissue culture and rapid propagation of the dry land hibiscus purpurea excellent individual plant and the related biotechnology.
Disclosure of Invention
The invention aims to provide a method for tissue culture and rapid propagation of a good single plant of a hibiscus purpureus system in a dry land, fill up the blank of the hibiscus syriacus biotechnology in the dry land, and simultaneously solve the problems of narrow field distribution area and high introduction and domestication difficulty of the hibiscus syriacus in the dry land. The invention lays the foundation for the comprehensive development and continuous utilization of hibiscus in dry land and the preservation and expansion of the chromo gene type.
In order to realize the purpose of the invention, the invention provides the following technical scheme:
a method for tissue culture and rapid propagation of a single excellent dry land hibiscus purpureus strain comprises the steps of explant selection and disinfection, induction and differentiation culture, proliferation and subculture, strong seedling and rooting culture and bottle seedling transplantation:
selecting and disinfecting the explant, namely taking the top bud and the side bud of a good single plant of a hibiscus purpurea system in dry land, soaking the top bud and the side bud in 1 percent of soap water for 10 minutes, and disinfecting the explant after washing the buds clean by running water;
the induction and differentiation culture is to culture the sterilized explants in an induction and differentiation culture medium, wherein the induction and differentiation culture medium is an MS culture medium, 1 mg/L6-BA, 0.5mg/L IAA, 30g/L sucrose and 5g/L agar, the pH value is 5.8, and the culture period is 20 days;
the proliferation and subculture is to culture the induced and chemically cultured material in a proliferation and subculture medium, wherein the proliferation and subculture medium is an MS culture medium, 0.8mg/L6-BA, 0.5mg/L IAA, 30g/L sucrose and 5g/L agar, the pH value is 5.8, and the culture period is 30 days;
the strong seedling and rooting culture is to culture the seedlings after proliferation and subculture in a strong seedling and rooting culture medium, wherein the strong seedling and rooting culture medium is an MS culture medium, 0.3mg/LNAA, 0.3mg/L IAA, 30g/L sucrose and 5g/L agar, the pH value is 5.8, and the culture period is 20 days;
the bottle seedling transplanting comprises the following steps: putting the bottle seedlings with roots in a greenhouse for hardening seedlings one week in advance, preparing a matrix, wherein the matrix is perlite, humus and raw laterite in a volume ratio of 1: 2: 3, spraying 800 times of carbendazim for mixing with soil, sealing and disinfecting by using a plastic film for 7 days, adjusting the pH value to 5.8, opening the bottle to take the seedlings, slightly cleaning a base culture medium, transplanting, spraying water in time and covering the plastic film, and the greenhouse temperature is 20-30 ℃, the air humidity is 50-60%, and the matrix humidity is 75-85%.
According to the tissue culture and rapid propagation method for the good single plant of the purple flower system of the dry land hibiscus, the tender terminal bud and the side bud of the good single plant of the purple flower system are used as explants, the illumination conditions of induction and differentiation, proliferation and subculture, strong seedling and rooting culture are all artificial auxiliary light 1500LUX, and the temperature is 23-30 ℃.
According to the tissue culture and rapid propagation method for the excellent individual plant of the dry land hibiscus purpurea system, propagation is carried out in a bud-saving mode, the growth is rapid, the growth height reaches 5.6cm in 30 days, and the average propagation multiple is 4.
According to the method for tissue culture and rapid propagation of the excellent single plant of the dry land hibiscus syriacus purple flower line, the induction culture medium and the differentiation culture medium are combined into one, the propagation culture medium and the subculture culture medium are combined into one, the strong seedling culture medium and the rooting culture medium are combined into one, and the culture method is simplified.
According to the tissue culture and rapid propagation method of the dry land hibiscus syriacus purple flower excellent single plant, the bottle seedlings for rooting are placed in a greenhouse for hardening off seedlings and adapting to one week before transplanting, and then are transplanted in a prepared substrate.
According to the method for tissue culture and rapid propagation of the excellent individual plant of the dry land hibiscus purpurea system, the transplanting matrix is as follows: the method comprises the following steps of spraying and mixing perlite, humus and raw laterite in a volume ratio of 1: 2: 3 with 800 times of carbendazim, sealing and disinfecting for 7 days by using a plastic film, adjusting the pH to 5.8, opening a bottle to take seedlings, slightly cleaning a basal medium, transplanting, spraying water in time and covering the plastic film, wherein the temperature of a greenhouse is 20-30 ℃, the shading rate is 75-85%, the air humidity is 50-60%, the substrate humidity is 75-85%, and the survival rate is 92% after 30 days.
Taking tender terminal buds and lateral buds of a good single plant of a hibiscus purpurea system in dry land as an explant, soaking the explant in 1% of soap water for 10 minutes, washing the explant cleanly by running water, taking the explant on a super clean bench, removing leaves, cutting the explant into small stem sections with 1cm size, disinfecting the stem sections with 75% of alcohol for 10 seconds, washing the stem sections with sterile water for 3 times, disinfecting the stem sections with 0.1% of mercuric chloride solution for 8 minutes, washing the stem sections with the sterile water for 3-5 times, inoculating the stem sections into a prepared induction culture medium, and inserting 1 bud in each bottle.
Preparing a tissue culture and rapid propagation culture medium for excellent individual plants of hibiscus purpureus in dry land: the induction and differentiation culture medium is MS culture medium +0.8mg/L6-BA (6-benzylpurine) +0.5mg/L NAA (naphthylacetic acid) + sucrose 30g/L + agar 5g/L, pH5.8, illumination intensity is 1000LUX, illumination is 10 hours per day, and temperature is 23-30 ℃. After one week of induction, terminal or lateral buds began to germinate.
The proliferation and subculture medium is MS culture medium +0.8mg/L6-BA (6-benzylpurine) +0.5mg/L NAA (naphthylacetic acid) + sucrose 30g/L + agar 5g/L, pH5.8, and the culture period is 30 days. The light intensity is 1500LUX, and the temperature is 23-30 ℃.
The strong seedling and rooting culture medium is MS culture medium, 0.5mg/LNAA (naphthylacetic acid), 0.5mg/L IAA (indoleacetic acid), 30g/L sugar and 5g/L agar, the pH value is 5.8, the light intensity is 1500LUX, the temperature is 23-30 ℃, and the culture period is 20 days.
Transplanting bottle seedlings: after 20 days of culture, the rooting bottle seedlings need to be moved to a greenhouse with the shading rate of 75% -85% in advance for hardening seedlings for one week. The matrix is perlite: humus soil: raw red soil (volume ratio) 1: 2: 3, spraying 800 times of carbendazim, mixing with soil, sealing and sterilizing for 7 days by using a plastic film, and adjusting the pH value to 5.8. Opening the bottle to take out the seedling gently, cleaning the culture medium at the root, transplanting the seedling into the sterilized substrate, spraying water in time and covering a plastic film for moisturizing. The temperature of the greenhouse is 20-30 ℃, the shading degree is 75-85%, the air humidity is 50-60%, and the substrate humidity is 75-85%. After 30 days, the survival rate is 92 percent.
The technical scheme provided by the invention is based on the following research foundations:
the dry land hibiscus syriacus has narrow distribution area, is a special species of the dry-hot dry-warm river valley of the Jinsha river, continuously declines the population, and has great difficulty in artificial introduction and domestication. The purple flower genotype is more scarce. Aims to solve the problems of less natural resources, large propagation, preservation and continuous utilization in a short time and fill up the blank of the research of the hibiscus syriacus on the dry land on the biotechnology. Therefore, the invention provides the excellent single-plant tissue culture and rapid propagation technology of the hibiscus purpureus in the dry land and lays a foundation for the comprehensive development and sustainable utilization of hibiscus syriacus in the dry land.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention establishes an effective tissue culture and rapid propagation method for the good individual plant of the hibiscus syriacus purpureus in the dry land, solves the problems of narrow distribution, difficult introduction and domestication and rare genotype of the hibiscus purpureus, and fills the blank of research and development of the hibiscus syriacus in the dry land on the biotechnology.
2. The method has the advantages that the excellent individual plant tissue culture and rapid propagation method of the hibiscus purpureus in the dry land is adopted, the seedling is easy to grow, the propagation steps are simplified, the effective propagation rate is high, and the significance is great for preserving and expanding the population of the hibiscus syriacus in the dry land.
3. The invention highly keeps the genetic stability and consistency of the good single plant of the hibiscus syriacus purpureus in the dry land through the tissue culture and rapid propagation method of the good single plant of the hibiscus syriacus purpureus in the dry land, and lays a foundation for the comprehensive development and continuous utilization of hibiscus syriacus in the dry land.
4. The dry-land hibiscus syriacus bred by the excellent individual plant tissue culture rapid propagation method for the dry-land hibiscus syriacus purpurea has the advantages that the induced differentiation rate is 87% in 20 days, the proliferation coefficient is 4 in 30 days, the rooting rate is 95% and the transplanting survival rate is 92%, the propagation coefficient of the dry-land hibiscus syriacus is greatly improved, and a very effective propagation method is provided for introduction, domestication, garden gardening and commercial value utilization of the species.
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FIG. 1 shows the excellent individual plant tissue culture propagation condition of hibiscus purpureus in dry land.
FIG. 2 shows the excellent single plant tissue culture rooting condition of hibiscus purpureus in dry land.
Detailed Description
The technical features and the material of the present invention will be further explained by the following embodiments of the present invention with reference to the drawings, but the present invention is not limited thereto.
Example 1
1. Screening a culture medium and a matrix:
taking tender terminal buds and lateral buds of a good single plant of a hibiscus syriacus purpurea system in dry land as explants, soaking the explants for 10 minutes by 1% soap water, washing the explants cleanly by running water, taking the explants on a super clean bench, cutting the explants into stem segments with single joints, disinfecting the stem segments with 75% alcohol for 10 seconds, washing the stem segments with sterile water for 3 times, disinfecting the surfaces of the stem segments with 0.1% mercuric chloride solution for 5 minutes, washing the stem segments with the sterile water for 3-5 times, and inoculating the stem segments into a prepared dry land hibiscus syriacus induction culture medium, wherein each bottle has 1 joint.
The stem segments are respectively inoculated on the culture medium of MS +0.1 mg/L6-BA +0.5mg/L NAA, MS +0.5mg/L6-BA +0.5mg/LNAA, MS +0.8mg/L6-BA +0.5mg/LNAA, MS +1 mg/L6-BA +0.5mg/LNAA, MS +2 mg/L6-BA +0.5mg/LNAA, sucrose 30g/L, agar 5g/L and pH5.8. The light intensity is 1000LUX, the temperature is 23-30 ℃, and the illumination time is 10 hours. The statistical induction rate conditions are as follows:
TABLE 1 screening of good individual stem induction medium for hibiscus purpureus on dry land
The dry land hibiscus syriacus stem induction culture medium is screened, and the result shows that the induction rate reaches 86% on the MS +0.8mg/l6-BA +0.5mg/LNAA culture medium.
TABLE 2 subculture medium hormone screening
MS culture medium is used as a basic culture medium for proliferation and secondary hormone screening, cytokinin is 6-BA, the concentration ranges are (0.1mg/L, 0.5mg/L, 1mg/L, 2mg/L and 4 concentration gradients), the concentration of auxin is respectively (0mg/L, 0.1mg/L,0.5mg/L, 1mg/L and 4 concentration gradients), a uniform design method is adopted, and the results are as follows:
MS culture medium +0.5mg/L6-BA (6-benzylpurine) +0.5mg/L NAA (naphthylacetic acid) + sucrose 30g/L + agar 5g/L, pH5.8, culture period 30 days, light intensity 1500LUX, temperature 23 ~ 30 ℃. The number of bud nodes (including terminal buds) is 4-5, the increment rate is 4-5, plant leaves are extended, the growth speed is fastest, the plant height is 5-6 cm, and the stem thickness is 2-3 mm.
TABLE 3 rooting Medium hormone screening results
MS culture medium +0.5mg/LNAA (naphthylacetic acid) +0.5mg/L IAA (indoleacetic acid) + sucrose 30g/L + agar 5g/L, pH5.8, culture cycle 20 days, rooting rate up to 95%.
TABLE 4 screening test results of culture substrate
The three substrates in the experiment ④ are matched, so that the looseness of the substrates and the air permeability of the roots are increased, enough nutrition is provided for the roots, meanwhile, laterite is soil deep in a soil layer, the quantity of bacteria is less, the growth of aseptic seedlings is facilitated, and the transplanting survival rate reaches 95% after 30 days.
The foregoing embodiments of the present invention have been described in detail, but it should not be understood that the scope of the subject matter of the present invention is limited to the foregoing embodiments, and all the technologies implemented based on the foregoing embodiments of the present invention are within the scope of the present invention.
Claims (4)
1. A tissue culture and rapid propagation method of a good single plant of a hibiscus purpureus series in dry land is characterized in that the method comprises the steps of explant selection and disinfection, induction and differentiation culture, proliferation and subculture, strong seedling and rooting culture, and bottle seedling transplantation,
selecting and disinfecting explants, namely taking tender terminal buds and side buds of a good single plant of a hibiscus syriacus purple flower system in dry land as the explants, soaking the explants for 10 minutes by using 1% soap water, washing the explants cleanly by running water, taking the explants onto a super clean bench, removing leaves, cutting the explants into small stem segments with 1cm size, disinfecting the small stem segments with the segments by using 75% alcohol for 10 seconds, washing the small stem segments with sterile water for 3 times, disinfecting the small stem segments with the sterile water for 8 minutes by using 0.1% mercuric chloride solution, washing the small stem segments with the sterile water for 3-5 times, inoculating the small stem segments into a prepared induction culture medium, and inserting 1 bud into each bottle;
the induction and differentiation culture is to culture the sterilized explant in an induction and differentiation culture medium, wherein the induction and differentiation culture medium is an MS culture medium, 0.8mg/L6-BA, 0.5mg/LNAA, 30g/L sucrose and 5g/L agar, the pH value is 5.8, the culture period is 20 days, the illumination intensity is 1000LUX, the illumination is 10 hours per day, the temperature is 23-30 ℃, and after one week of induction, terminal buds or lateral buds start to germinate;
the proliferation and subculture is to culture the induced and differentiated cultured material in a proliferation and subculture medium, wherein the proliferation and subculture medium is an MS culture medium, 0.5mg/L6-BA, 0.5mg/LNAA, 30g/L sucrose and 5g/L agar, the pH value is 5.8, the culture period is 30 days, the light intensity is 1500LUX, and the temperature is 23-30 ℃;
the strong seedling and rooting culture is to culture the seedlings after multiplication and subculture in a strong seedling and rooting culture medium, wherein the strong seedling and rooting culture medium is an MS culture medium, 0.5mg/LNAA, 0.5mg/LIAA, 30g/L sucrose and 5g/L agar, the pH value is 5.8, the culture period is 20 days, the light intensity is 1500LUX, and the temperature is 23-30 ℃;
the bottle seedling transplanting comprises the following steps: after 20 days of culture, the rooting bottle seedlings need to be moved to a greenhouse with the shading rate of 75% -85% in advance for hardening seedlings for one week, the rooting bottle seedlings are placed in the greenhouse for hardening seedlings one week in advance, a substrate is prepared, the substrate is perlite, humus and raw laterite in the volume ratio of 1: 2: 3, 800 times of carbendazim is used for spraying and mixing soil, a plastic film is used for sealing and disinfecting for 7 days, the pH is adjusted to 5.8, the bottle is opened for taking the seedlings, a base culture medium is cleaned, the seedlings are transplanted into the disinfected substrate, water is sprayed in time and the plastic film is covered, the temperature of the greenhouse is 20-30 ℃, the shading degree is 75% -85%, the air humidity is 50% -60%, and the substrate humidity is 75% -85%.
2. The tissue culture rapid propagation method of the excellent individual plant of the hibiscus purpureus on the dry land as claimed in claim 1, characterized in that the propagation is carried out in a bud-saving mode, the growth is rapid, the growth height reaches 5.6cm in 30 days, and the average propagation multiple is 4.
3. The method for tissue culture and rapid propagation of a single excellent dry land hibiscus purpureus strain according to claim 1, wherein the induction culture medium and the differentiation culture medium are combined into one, the propagation culture medium and the subculture culture medium are combined into one, and the strong seedling culture medium and the rooting culture medium are combined into one, so that the culture method is simplified.
4. The tissue culture and rapid propagation method of the excellent individual plants of the purple hibiscus syriacus on the dry land according to claim 1, characterized in that the seedlings are placed in a greenhouse for hardening off seedlings and transplanting in a prepared matrix after adapting for one week before the transplantation of the bottle seedlings for rooting.
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