CN110663552A - Tissue culture and rapid propagation method of Yunnan tung tree - Google Patents
Tissue culture and rapid propagation method of Yunnan tung tree Download PDFInfo
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- CN110663552A CN110663552A CN201911086276.5A CN201911086276A CN110663552A CN 110663552 A CN110663552 A CN 110663552A CN 201911086276 A CN201911086276 A CN 201911086276A CN 110663552 A CN110663552 A CN 110663552A
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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 provides a tissue culture and rapid propagation method of Yunnan tung, and relates to the technical field of plant tissue culture. The tissue culture rapid propagation method integrates induction and differentiation culture, proliferation and subculture, and strong seedling and rooting culture, and the culture method is simplified. The tissue culture rapid propagation method can solve the problems of less natural resources of the Yunnan tung tree and large propagation, storage and continuous utilization in a short time, fills the blank of research on the biological technology of the Yunnan tung tree, and lays a foundation for comprehensive development and sustainable utilization of the Yunnan tung tree.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation method of Yunnan tung tree.
Background
The Yunnan tung Craigia yunnanensis belongs to the genus Dianthus in the family Tiliaceae, is tall by 6-20 meters, is mainly distributed in tropical dry-wet alternation hot climatic regions in south Asia and mountain forests with elevation of 500-1000 meters, and has capsule with thin paper wings and special seeds in southwest China. The Yunnan tung is one of the important tree species of the few Yunnan tung, and has important value in the geographical research of the district and the application of breeding precious tree species. Listed in 'famous records of threatened species of higher plants in China' endangered EN C2a in 2017; a class D; the world alliance for natural protection (IUCN) also lists it as an endangered species [ EN ]. At present, two factors seriously threatening the field survival of the Yunnan tung tree are mainly used: firstly, a large amount of amomum tsao-ko is planted for economic benefit, and the inhabitation area of the Yunnan tung tree is seriously occupied; secondly, the inundation and disorderly cutting of trees in forest areas cause the damage of the Yunnan tung tree colony ecological community; the Yunnan tung tree is a typical species with extremely small population, which causes the narrow distribution area of the Yunnan tung tree and seriously threatens the survival.
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. However, the prior art has no report on the tissue culture and rapid propagation of excellent single-plant of the Yunnan tung tree and the related biotechnology.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for tissue culture and rapid propagation of Yunnan tung tree, which solves the problems of less natural resources of Yunnan tung tree and incapability of achieving mass propagation, preservation and continuous utilization in a short time.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a tissue culture and rapid propagation method of Yunnan tung, which comprises the following steps: (1) taking the disinfected tender tissue of the Yunnan tung tree as an explant, and inoculating the explant on an induced differentiation co-culture medium for induced differentiation co-culture to obtain a differentiated bud; the induced differentiation co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 1.0mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar;
(2) inoculating the differentiated bud on a proliferation subculture co-culture medium for proliferation subculture co-culture to obtain a seedling; the proliferation subculture co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.8mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar;
(3) inoculating the seedling on a strong seedling rooting co-culture medium to perform strong seedling rooting co-culture to obtain a Yunnan tung tree tissue culture seedling; the strong seedling rooting co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.8mg/L IAA, 0.8mg/L IBA, 30g/L sucrose and 5g/L agar.
Preferably, the tender tissue of the Yunnan tung tree in the step (1) comprises a top bud or a side bud of the Yunnan tung tree; the method of sterilization comprising: soaking the materials in 1% soap water by mass and volume for 10min, washing the materials by running water, disinfecting the materials by using 75% alcohol solution by volume for 10s, washing the materials by using sterile water for 3 times, disinfecting the materials by using 0.1% mercury bichloride solution by volume for 5min, and washing the materials by using the sterile water for 3-5 times.
Preferably, the illumination intensity of the induced differentiation co-culture in the step (1), the proliferation subculture co-culture in the step (2) and the strong seedling rooting co-culture in the step (3) are 1500Lux, and the temperature is 23-30 ℃.
Preferably, the culture period of the induced differentiation co-culture of the step (1) is 30 d; the culture period of the proliferation subculture co-culture in the step (2) is 60 d; and (4) the culturing period of the strong seedling rooting co-culture in the step (3) is 30 d.
Preferably, after the tissue culture seedling of the Yunnan tung tree is obtained in the step (3), hardening off and transplanting are further included.
Preferably, the shading rate during seedling exercising is 75-85%.
Preferably, the transplanted matrix comprises the following components: the soil-improving agent comprises perlite, humus soil and raw red soil, wherein the volume ratio of the perlite to the humus soil to the raw red soil is 1: 2: 3.
preferably, the cultivation temperature after transplanting is 20-30 ℃, the shading degree is 75-85%, the air humidity is 50-60%, and the substrate humidity is 75-85%.
Compared with the prior art, the invention has the following beneficial effects: (1) the invention establishes an effective method for tissue culture and rapid propagation of the Yunnan tung tree, solves the problems of narrow distribution and difficult introduction and domestication of the Yunnan tung tree, and fills the blank of research and development of the Yunnan tung tree on biotechnology;
(2) the method has the advantages that the tissue culture and rapid propagation method of the Yunnan tung tree 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 Yunnan tung tree population;
(3) the invention highly maintains the genetic stability and consistency of excellent single Yunnan tung tree plants by the Yunnan tung tree tissue culture and rapid propagation method, and lays a foundation for the comprehensive development and continuous utilization of the Yunnan tung tree;
(4) the method for breeding the Yunnan tung tree by the Yunnan tung tree tissue culture and rapid propagation method has the advantages that the induced differentiation rate in 30 days is 92%, the proliferation coefficient in 60 days is 4.7, the rooting rate in 30 days is 91%, and the transplanting survival rate is 90%, so that the propagation coefficient of the Yunnan tung tree is greatly improved, and a very effective propagation method is provided for introduction and domestication, preservation, garden gardening and scientific research value utilization of the species.
Drawings
FIG. 1 is a drawing of tissue culture induced differentiation of Tung Yunnan Tong in example 1 of the present invention;
FIG. 2 is a tissue culture and proliferation subculture map of Aleurites fordii in example 1 of the present invention;
fig. 3 is a diagram of tissue culture, seedling strengthening and rooting of the erythrina indica in the embodiment 1 of the present invention.
Detailed Description
The invention provides a tissue culture and rapid propagation method of Yunnan tung, which comprises the following steps: (1) taking the disinfected tender tissue of the Yunnan tung tree as an explant, and inoculating the explant on an induced differentiation co-culture medium for induced differentiation co-culture to obtain a differentiated bud; the induced differentiation co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 1.0mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar;
(2) inoculating the differentiated bud on a proliferation subculture co-culture medium for proliferation subculture co-culture to obtain a seedling; the proliferation subculture co-culture medium takes an MS culture medium as a cost-saving culture medium, and further comprises: 0.8mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar;
(3) inoculating the seedling on a strong seedling rooting co-culture medium to perform strong seedling rooting co-culture to obtain a Yunnan tung tree tissue culture seedling; the strong seedling rooting co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.8mg/L IAA, 0.8mg/L IBA, 30g/L sucrose and 5g/L agar.
The tissue culture and rapid propagation method takes the disinfected tender tissue of the Yunnan tung tree as an explant, and inoculates the explant on an induced differentiation co-culture medium for induced differentiation co-culture to obtain a differentiated bud; the induced differentiation co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 1.0mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar. The tender tissue of the Yunnan tung tree preferably comprises a top bud or a side bud of the Yunnan tung tree. The disinfection method of the invention preferably comprises the following steps: soaking the materials in 1% soap water by mass and volume for 10min, washing the materials by running water, disinfecting the materials by using 75% alcohol solution by volume for 10s, washing the materials by using sterile water for 3 times, disinfecting the materials by using 0.1% mercury bichloride solution by volume for 5min, and washing the materials by using the sterile water for 3-5 times. The method for preparing the co-culture medium for inducing differentiation is not particularly limited in the present invention, and the co-culture medium for inducing differentiation is preferably obtained by uniformly mixing the above components and then sterilizing the mixture at high temperature and high pressure, and the pH value of the co-culture medium for inducing differentiation is preferably 5.8.
When the induced differentiation co-culture medium is used for induced differentiation co-culture, the illumination intensity is preferably 1500Lux, the temperature is preferably 23-30 ℃, and the culture period is preferably 30 d. In the present invention, after one week of co-culture by said induced differentiation, terminal buds or lateral buds start to germinate.
After obtaining the differentiated bud, inoculating the differentiated bud on a proliferation subculture coculture medium for proliferation subculture to obtain a seedling; the proliferation subculture co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.8mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar. In the proliferation subculture, the side buds are proliferated in a side bud generation mode, and the side buds growing on the seedlings are inoculated on the proliferation subculture coculture medium for proliferation subculture. The illumination intensity of the proliferation and subculture co-culture is preferably 1500Lux, and the temperature is preferably 23-30 ℃. The culture period of the proliferation subculture co-culture of the present invention is preferably 60 d. In the invention, after the proliferation subculture co-culture is carried out for 60 days, the growth height reaches 4.8cm, and the average multiplication multiple is 4.7.
After obtaining the seedlings, inoculating the seedlings on a strong seedling rooting co-culture medium to perform strong seedling rooting and rooting co-culture to obtain the tissue culture seedlings of the erythrina indica lam; the strong seedling rooting co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.8mg/L IAA, 0.8mg/L IBA, 30g/L sucrose and 5g/L agar. The illumination intensity of the strong seedling rooting co-culture is preferably 1500Lux, and the temperature is preferably 23-30 ℃. The culture period of the strong seedling rooting co-culture is preferably 30 d.
After the tissue culture seedling of the Yunnan tung is obtained, the invention preferably also comprises seedling hardening and transplanting. The seedling exercising method is preferably used for exercising the seedlings for one week in a greenhouse with the shading rate of 75-85%. The invention transplants the tissue culture seedlings after hardening off, and the transplanted matrix preferably comprises the following components: the soil-improving agent comprises perlite, humus soil and raw red soil, wherein the volume ratio of the perlite to the humus soil to the raw red soil is 1: 2: 3. before use, the substrate of the invention preferably also comprises the steps of sterilizing by using 800 times of carbendazim, sealing and sterilizing by using a plastic film for 7 days, and adjusting the pH to 5.8. The transplanting is preferably to take out the tissue culture seedlings after hardening, clean the culture medium at the root, transplant the tissue culture seedlings into a sterilized substrate, spray water in time and cover a plastic film for moisturizing, wherein the temperature of a greenhouse is 20-30 ℃, the shading rate is 75-85%, the air humidity is 60-70%, the substrate humidity is 65-75%, and the survival rate is 90% after 30 days. In the transplanting substrate, the looseness of the substrate and the air permeability of the root are increased, sufficient nutrition is provided for the root, and meanwhile, the laterite is soil deep in the soil layer, so that the bacterium carrying amount is less, and the growth of aseptic seedlings is facilitated.
The tissue culture and rapid propagation method of the paulownia fortunei provided by the present invention is described in detail with reference to the following examples, but the present invention should not be construed as being limited to the scope of the present invention.
Example 1
Taking tender terminal buds and side buds of the Yunnan tung tree as explants, soaking the explants for 10 minutes by using 1% soap water, washing the explants cleanly by flowing water, taking the explants on a super clean bench, cutting the explants into stem segments with single sections, disinfecting the stem segments by using 75% alcohol for 10s, washing the cleaned stem segments by using sterile water for 3 times, disinfecting the surfaces of the stem segments by using 0.1% mercuric chloride solution for 5min, washing the stem segments by using the sterile water for 3-5 times, and inoculating the stem segments to a prepared Yunnan tung tree induced differentiation co-culture medium, wherein 1 section is used in each bottle.
1. Effect of different differentiation-inducing media on the Induction Rate
Respectively inoculating the stem segments in the following culture media of ① MS +0.1 mg/L6-BA +0.5mg/L NAA + sucrose +0.5 mg/L6-BA +0.5mg/L NAA + sucrose + 30g/L sucrose + agar 5g/L, pH5.8, ③ MS +0.8 mg/L6-BA +0.5mg/L NAA + sucrose + 30g/L sucrose + 5g/L agar, pH5.8, ④ MS +1 mg/L6-BA +0.5mg/L NAA + sucrose + 30g/L agar 5g/L, pH5.8, ⑤ MS +2 mg/L6-BA +0.5mg/L A + sucrose + 30g/L agar 5g/L, light intensity is 1000LUX, light intensity is 23 hours to 30 hours, and induction rate is shown in the following the statistical table of light intensity and light induction rate is 1 day:
TABLE 1 Effect of different media on the Induction of Excellent Individual Stem segments of Aleurites fordii
As can be seen from Table 1, the induction rate reached 92% in the medium ④ (as shown in FIG. 1).
2. Effect of different multiplication subculture Co-Medium on multiplication subculture
Inoculating the obtained differentiated bud on a proliferation subculture co-culture medium for proliferation subculture, and setting the hormone content in the proliferation subculture co-culture medium as follows: cytokinin 6-BA in the concentration range of (0.5mg/L, 0.8mg/L, 1mg/L), auxin in the concentration range of (0.1mg/L, 0.5mg/L, 0.8mg/L) respectively, and each of the proliferation subculture co-media contains: 30g/L of sucrose and 5g/L of agar, and the pH value is 5.8. Statistics were performed after 60 days, and the results are shown in table 2, for convenience of comparison the contents of sucrose and agar are not shown in table 2:
TABLE 2 Effect of different media on the multiplication subculture Co-culture
As shown in Table 2, MS culture medium +0.8 mg/L6-BA +0.5mg/L NAA + sucrose 30g/L + agar 5g/L, pH5.8, culture period 60 days, light intensity 1500LUX, temperature 23-30 ℃, average effective bud node number (including terminal bud) 4.7, plant leaf extension, fast growth speed, plant height about 5.1cm, and stem thickness about 3mm (as shown in FIG. 2).
3. Effect of different culture media on strong seedling rooting co-culture
Inoculating the plantlets on different strong seedling rooting co-culture media to perform strong seedling rooting co-culture, and respectively setting different hormone contents: IAA (0.5mg/L, 0.8mg/L, 1.0mg/L), IBA (0.5mg/L, 0.8mg/L, 1.0mg/L), and each of the strong seedling rooting co-culture media contains: 30g/L of sucrose and 5g/L of agar, and the pH value is 5.8. Statistics were performed after 30 days, with the results shown in table 3, for convenience of comparison the sucrose and agar content not shown in table 3:
TABLE 3 Effect of different media on rooting Rate
As shown in Table 3, MS culture medium +0.8mg/L IAA +0.8mg/L IBA + sucrose 30g/L + agar 5g/L, pH5.8, culture period 30 days, rooting rate up to 91% (as shown in FIG. 3).
4. Influence of different substrates on transplant survival rate
After 30 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, then the transplanting substrates in the table 4 are respectively used for transplanting, the survival rate after 30 days is counted, and the results are shown in the table 4:
TABLE 4 Effect of the transplanting media on the survival rates of the transplants
As can be seen from Table 4, the transplanting survival rate reached 90% after 30 days when the transplanting substrates (perlite: humus: raw laterite in volume ratio of 1: 2: 3) were used.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A tissue culture and rapid propagation method of Yunnan tung is characterized by comprising the following steps: (1) taking the disinfected tender tissue of the Yunnan tung tree as an explant, and inoculating the explant on an induced differentiation co-culture medium for induced differentiation co-culture to obtain a differentiated bud; the induced differentiation co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 1.0mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar;
(2) inoculating the differentiated bud on a proliferation subculture co-culture medium for proliferation subculture co-culture to obtain a seedling; the proliferation subculture co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.8mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar;
(3) inoculating the seedling on a strong seedling rooting co-culture medium to perform strong seedling rooting co-culture to obtain a Yunnan tung tree tissue culture seedling; the strong seedling rooting co-culture medium takes an MS culture medium as a basic culture medium, and further comprises: 0.8mg/L IAA, 0.8mg/L IBA, 30g/L sucrose and 5g/L agar.
2. The tissue culture rapid propagation method according to claim 1, wherein the tender tissue of the Yunnan tung tree in the step (1) comprises a top bud or a side bud of the Yunnan tung tree; the method of sterilization comprising: soaking the materials in 1% soap water by mass and volume for 10min, washing the materials by running water, disinfecting the materials by using 75% alcohol solution by volume for 10s, washing the materials by using sterile water for 3 times, disinfecting the materials by using 0.1% mercury bichloride solution by volume for 5min, and washing the materials by using the sterile water for 3-5 times.
3. The tissue culture and rapid propagation method according to claim 1, wherein the illumination intensity of the induced differentiation co-culture of the step (1), the proliferation subculture co-culture of the step (2) and the strong seedling rooting co-culture of the step (3) are 1500Lux, and the temperature is 23-30 ℃.
4. The tissue culture rapid propagation method according to claim 1 or 3, characterized in that the culture period of the induced differentiation co-culture of the step (1) is 30 d; the culture period of the proliferation subculture co-culture in the step (2) is 60 d; and (4) the culturing period of the strong seedling rooting co-culture in the step (3) is 30 d.
5. The tissue culture rapid propagation method according to claim 1, which further comprises hardening off and transplanting the tissue culture seedling of the erythrina indica after the tissue culture seedling of the erythrina indica in the step (3).
6. The tissue culture rapid propagation method according to claim 5, wherein the light shading rate during seedling hardening is 75-85%.
7. The tissue culture rapid propagation method according to claim 5, wherein the transplanted matrix comprises the following components: the soil-improving agent comprises perlite, humus soil and raw red soil, wherein the volume ratio of the perlite to the humus soil to the raw red soil is 1: 2: 3.
8. the tissue culture rapid propagation method according to claim 5, wherein the cultivation temperature after transplanting is 20-30 ℃, the shading degree is 75-85%, the air humidity is 50-60%, and the substrate humidity is 75-85%.
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