CN113383706A - Efficient eucommia bark regeneration method based on LED light quality regulation - Google Patents
Efficient eucommia bark regeneration method based on LED light quality regulation Download PDFInfo
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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
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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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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/14—Measures for saving energy, e.g. in green houses
Abstract
The invention provides an efficient regeneration method of eucommia ulmoides based on LED light quality regulation, which takes the current-year axillary bud stem segments of excellent eucommia ulmoides triploid strains as explants, and performs germination, proliferation, elongation and rooting induction culture on axillary buds after surface disinfection to finally obtain complete eucommia ulmoides regeneration plants. According to the invention, the appropriate LED light quality proportion with long service life and low energy consumption is introduced into the tissue culture and rapid propagation of eucommia ulmoides, so that the energy consumption cost is reduced, the propagation coefficient of the tissue culture seedling of eucommia ulmoides is improved, the elongation time of adventitious buds is shortened, the leaves of the tissue culture seedling are dark green and grow fast, a foundation is laid for rapid large-scale propagation of excellent eucommia ulmoides strains, and an important technical support is provided for genetic improvement of eucommia ulmoides by utilizing a molecular biology means in the later period.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an efficient eucommia ulmoides regeneration method based on LED light quality regulation.
Background
Eucommia ulmoides (Eucommia ulmoides Oliver) is a perennial woody plant of genus Eucommia of family Eucommiaceae. The plant is a wiggle plant which is remained on the geological history in the middle of China and survives to date, and the activated stone plant unique to China is homologous in medicine and food and is a national secondary protection plant. In addition, Du is a national strategic resource, a rare medicinal material, a woody oil tree species and a high-quality natural rubber resource, and is also an important tree species for maintaining ecological safety, increasing carbon sink and wood reserve and realizing green cultivation. Because it contains chlorogenic acid and effective components such as aucubin, the product has pharmacological effects of regulating blood pressure, lowering blood sugar, inhibiting bacteria, resisting oxidation, and resisting tumor. Based on the characteristics, the eucommia ulmoides has wide market application prospect.
At present, in production, eucommia is bred in a main sowing mode, eucommia is a male and female heterostrain, can usually bloom and bear fruits after being planted for more than 10 years, and has short seed life (0.5-1 year); the seeds contain gutta percha, need to be harvested at a specific time and subjected to specific treatments to break seed dormancy. Therefore, the problems of complicated breeding process, long breeding period, low germination rate (only about 20 percent without special treatment) and the like exist when the seeds are used for raising seedlings.
At present, the eucommia ulmoides is mostly propagated by adopting traditional asexual propagation modes such as cuttage, grafting, root pressing and the like, for example, patent document with application number of 201910033199.0 discloses a rapid propagation method of south eucommia ulmoides grafted seedlings, which comprises (1) nursery land selection; (2) preparing soil and making a bed; (3) stock cultivation: firstly, seed collection; secondly, disinfecting the seeds; thirdly, accelerating germination of seeds; seeding; fifthly, transplanting the bud seedlings; sixthly, intertillage and weeding; managing fertilizer and water; eighthly, bud picking and pinching; (4) and (3) cultivating grafted seedlings: collecting and storing scions; a grafting method; managing after grafting; and fourthly, the nursery stock is taken out. Patent document No. 202011236585.9 discloses a method for preparing a plant growth regulator and a method for cutting and raising seedlings of improved variety of eucommia shoots by using the same.
Although the traditional asexual propagation modes such as cuttage, grafting and root pressing are reported in the aspect of eucommia bark propagation, the problems of complicated propagation process, limitation of propagation time and season, low propagation efficiency and the like exist.
At present, research on the propagation of eucommia ulmoides by using a plant tissue culture technology is also available, and for example, patent document No. 202110454404.8 discloses a method for inducing plant regeneration and constructing a transgenic plant regeneration system by whole plants of each part of eucommia ulmoides, which comprises the following steps: inducing eucommia seed embryos, stems and/or roots to obtain calluses, and performing differentiation culture and rooting culture on the calluses to obtain complete eucommia regeneration plants; the culture medium for differentiation culture is as follows: basic culture medium +0.5-2mg/L6-BA +1.0-2.0mg/LIAA +25-35g/L sucrose +8-10g/L agar, and pH is 5.8-6.
Although the research of the propagation of eucommia by using the plant tissue culture technology is reported, the research mainly focuses on taking a seed seedling as an explant source, and has the problems of serious pollution, low adventitious bud proliferation efficiency, low rooting efficiency and the like, thereby seriously limiting the germplasm preservation and large-scale propagation of an improved variety of eucommia.
In summary, the traditional seedling raising process of eucommia ulmoides wastes time and labor, is limited by seasons and has low regeneration efficiency, and cannot meet the requirement of large-scale planting of eucommia ulmoides, and the problems of poor repeatability, low adventitious bud proliferation efficiency, low rooting efficiency and the like exist in the report of in vitro breeding of eucommia ulmoides plants by using a plant tissue culture technology at present, so that germplasm preservation and large-scale breeding of improved eucommia ulmoides are severely limited.
In order to meet the requirements of large-scale planting and variety improvement on high-quality eucommia seedlings, a method for efficiently regenerating eucommia is urgently needed to be developed.
Disclosure of Invention
The technical problem to be solved by the invention is how to meet the requirements of large-scale planting and variety improvement on high-quality eucommia seedlings.
The invention solves the technical problems through the following technical means: a high-efficiency regeneration method of eucommia ulmoides based on LED light quality regulation and control is characterized in that stem segments with axillary buds of eucommia ulmoides are used as explants, the cut segments with proper sizes are cut after surface disinfection, the cut segments are inoculated into culture media added with plant growth regulators with different types and concentrations, germination and proliferation of the axillary buds, elongation of adventitious buds and rooting induction culture are sequentially carried out under the conditions of proper LED light illumination and temperature, and finally complete eucommia ulmoides regeneration plants are obtained.
According to the method, the appropriate LED light quality proportion with long service life and low energy consumption is introduced into the tissue culture and rapid propagation of eucommia ulmoides, the energy consumption cost is reduced, the multiplication coefficient of the tissue culture seedling of the eucommia ulmoides is improved, the adventitious bud elongation time is shortened, the leaves of the tissue culture seedling are dark green, and the tissue culture seedling grows faster, so that a solid foundation is laid for large-area popularization of the eucommia ulmoides;
as a preferred mode of the invention, the eucommia stem segment with axillary buds is a current-year branch of a perennial triploid eucommia plant which is obtained from a field test field.
In another preferred mode of the invention, the stem segment with axillary buds of eucommia is the annual branch of a perennial diploid eucommia plant obtained from a field test field.
As a preferable mode of the invention, the stem segment with axillary buds is a stem segment which is cut into 5cm after leaves of a branch are removed.
In a preferred mode of the invention, the surface disinfection is that the stem segments with axillary buds of eucommia ulmoides are washed for 20min by running water, wiped by 75% absolute ethyl alcohol, washed by sterile water for 5 times in a sterile operating platform, then disinfected by 75% absolute ethyl alcohol for 45s, disinfected by disinfectant for 1-7 min, and finally washed by sterile water for 6 times.
In a preferred embodiment of the present invention, the disinfectant is 0.1% HgCl2And (5) solution disinfection for 3 min.
In a preferred embodiment of the present invention, the axillary bud germination, proliferation and elongation medium is a DKW medium supplemented with 0.5mg/L of 6-BA, 0.2mg/L of GA3, 30g/L of sucrose and 6.6g/L of agar.
As a preferred mode of the invention, the rooting medium is 1/2DKW medium added with 0.5-2.0 mg/L sodium nitroprusside 30g/L sucrose and 6.6g/L agar.
As a preferred mode of the invention, necrotic parts at two ends of the stem segment are cut off after disinfection, and then the stem segment is cut into a cut segment with at least one axillary bud with the size of 0.7cm and is used for germination and proliferation of the axillary bud, elongation of the adventitious bud and rooting induction culture.
In a preferred embodiment of the present invention, the suitable temperature and illumination conditions are that the culture is placed at a temperature of 23 ± 2 ℃, and the LED light quality and the ratio are that the ratio of red to blue is 1: 1, culturing in a constant-temperature culture room with the illumination intensity of 2000-2500 lux and the illumination period of 16h illumination/10 h darkness.
The invention has the advantages that: the invention relates to an efficient eucommia bark regeneration method based on LED light quality regulation and control, which provides important technical support for rapid propagation and later genetic improvement of eucommia bark by utilizing molecular biology means, and has the following specific advantages:
firstly, the stem section is an annual branch obtained from a perennial eucommia excellent plant line in a field experimental field, so that the germplasm characteristic of a female parent plant can be maintained, and the material is convenient to obtain and rich in material;
secondly, the explant sterilization process in the regeneration process is simple, the sterilization time is short, the toxicity to the explant is small, the pollution rate is low, and the highest pollution rate is only 1.19%;
thirdly, proper LED light quality proportion with long service life and low energy consumption is introduced into the tissue culture and rapid propagation of eucommia ulmoides, so that the energy consumption cost is reduced, the propagation coefficient of the tissue culture seedling of eucommia ulmoides is improved, the adventitious bud elongation time is shortened, the leaves of the tissue culture seedling are dark green, and the tissue culture seedling grows faster, so that a solid foundation is laid for large-area popularization of eucommia ulmoides;
fourthly, the method is suitable for the propagation of the eucommia ulmoides with different genotypes (diploid No. 1 eucommia ulmoides and triploid eucommia ulmoides).
Drawings
FIGS. 1a and 1b show the effect of different disinfectants on the disinfection of eucommia ulmoides in example 1 of the present invention;
FIGS. 2a and 2b are graphs showing germination of axillary buds of triploid and diploid eucommia ulmoides stem segments in example 2 of the present invention;
FIG. 3 is a graph showing the elongation of the highest axillary bud of eucommia ulmoides in example 3 of the present invention;
FIG. 4 is a graph showing elongation of the highest adventitious bud of eucommia ulmoides in example 4 of the present invention;
FIG. 5 is a photograph showing the inoculation of a single-shoot stem section of eucommia ulmoides with one leaf according to example 5 of the present invention;
FIG. 6 is a graph showing elongation and proliferation of a single-shoot stem with one leaf of eucommia ulmoides in example 5 of the present invention;
FIG. 7 is a diagram of a whole tissue culture seedling of eucommia ulmoides in example 5 of the present invention;
FIG. 8 is a view showing the roots of tissue culture seedlings of eucommia ulmoides in example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
selection of the eucommia explant disinfectant comprises the following specific operations:
1. selecting a good perennial eucommia strain with good growth from a field test field, and shearing a non-lignified branch of the current year as a source of an explant.
2. Cutting the branches into 5 cm-sized stem sections after removing leaves, and washing for 20min under flowing water; wiping the film with 75% absolute ethyl alcohol once, then washing the film in a sterile operating table with sterile water for 5 times; then sterilizing with 75% anhydrous ethanol for 45s, and washing with sterile water for 4 times; then 0.1% HgCl was added2Solution, 10% H2O2The solution and 10% NaClO disinfectant are respectively sterilized for 1, 3, 5 and 7min, and finally washed by sterile water for 6 times. Shaking continuously during the sterilization. After disinfection, the necrotic parts at both ends of the stem section are cut off, and then the stem section is cut into a cut section with the size of about 0.7cm and at least one axillary bud for standby. Counting the pollution rate and browning rate after 7 days of culture, sterilizing 10% NaClO solution for 3min to obtain the lowest pollution rate of 1.19%, and combining two parameters of pollution rate and browning rate, wherein 0.1% HgCl can be added2The solution was sterilized for 3min as the optimum choice (fig. 1).
Example 2:
establishing regeneration systems of different genotypes of eucommia ulmoides, and specifically operating as follows:
1. selecting good perennial triploid and diploid eucommia superior plant lines with good growth from a field test field, and shearing current-year non-lignified branches as the sources of explants.
2. Cutting the branches into 5 cm-sized stem sections after removing leaves, and washing for 20min under flowing water; wiping the film with 75% absolute ethyl alcohol once, then washing the film in a sterile operating table with sterile water for 5 times; then sterilizing with 75% anhydrous ethanol for 45s, and washing with sterile water for 4 times; then 0.1% HgCl was added2The solution was sterilized for 3min and finally washed with sterile water 6 times. Shaking continuously during the sterilization. After disinfection, the necrotic parts at both ends of the stem section are cut off, and then the stem section is cut into a cut section with the size of about 0.7cm and at least one axillary bud for standby.
3. The stem segments were vertically inoculated in DKW +0.5 mg/L6-BA +0.2mg/L GA3+30g/L sucrose +6.6g/L agar medium for germination induction of axillary buds (FIG. 2). And (3) placing the culture bottle in a constant-temperature culture room with the temperature of 23 +/-2 ℃, the illumination intensity of 2000-2500 Lux and the illumination period of 16/10h (illumination/darkness), carrying out illumination culture for 10 days, and then, allowing axillary buds to germinate, wherein the axillary buds of the triploid (figure 2 left) and diploid (figure 2 right) eucommia stem segments normally germinate as shown in the figure, and the germination rate reaches 100%.
Example 3:
the research on the promotion effect of different LED light qualities on the germination of axillary buds of the eucommia ulmoides stem segments comprises the following specific operations:
1. selecting a good perennial eucommia strain with good growth from a field test field, and shearing a non-lignified branch of the current year as a source of an explant.
2. Cutting the branches into 5 cm-sized stem sections after removing leaves, and washing for 20min under flowing water; wiping the film with 75% absolute ethyl alcohol once, then washing the film in a sterile operating table with sterile water for 5 times; then using 75% absolute ethyl alcohol to disinfect for 40s, washing with sterile water for 4 times; then 0.1% HgCl was added2The solution was sterilized for 3min and finally washed with sterile water 6 times. Shaking continuously during the sterilization. After disinfection, the necrotic parts at both ends of the stem section are cut off, and then the stem section is cut into a cut section with the size of about 0.7cm and at least one axillary bud for standby.
3. The stem segments are vertically inoculated in DKW +0.5 mg/L6-BA +0.2mg/L GA3+30g/L sucrose +6.6g/L agar medium for axillary bud germination induction. Culturing culture bottles in a constant-temperature culture room with the light quality of white light, red light, blue light, red-blue composite light (1: 1) and red-blue composite light (5: 1) respectively, setting the temperature to be 23 +/-2 ℃, the illumination intensity to be 2000-2500 Lux, and the illumination period to be 16/10h (illumination/darkness), and after 20 days of illumination culture, adding a culture solution into the culture solution, wherein the ratio of LED red to blue is 1: 1, when the culture is carried out under illumination, the growth speed is fastest, and the maximum length of axillary buds reaches 7.43cm (figure 3). The results are shown in Table 1.
TABLE 1 results of promoting effect of LED light quality on germination of axillary buds of eucommia ulmoides stem segments
Example 4:
the method has the following specific operations of promoting the adventitious bud elongation of the eucommia ulmoides by using different LED light qualities:
1. selecting a good perennial eucommia strain with good growth from a field test field, and shearing a non-lignified branch of the current year as a source of an explant.
2. Cutting the branches into 5 cm-sized stem sections after removing leaves, and washing for 20min under flowing water; wiping the film with 75% absolute ethyl alcohol once, then washing the film in a sterile operating table with sterile water for 5 times; then sterilizing with 75% anhydrous ethanol for 45s, and washing with sterile water for 4 times; then 0.1% HgCl was added2The solution was sterilized for 3min and finally washed with sterile water 6 times. Shaking continuously during the sterilization. After disinfection, the necrotic parts at both ends of the stem section are cut off, and then the stem section is cut into a cut section with the size of about 0.7cm and at least one axillary bud for standby.
3. The stem segments are vertically inoculated in DKW +0.5 mg/L6-BA +0.2mg/L GA3+30g/L sucrose +6.6g/L agar medium for axillary bud germination induction.
4. When the adventitious bud grows to about 1.0cm, the adventitious bud is transferred to a culture medium of DKW +0.5 mg/L6-BA +0.2mg/L GA3+30g/L sucrose +6.6g/L agar for elongation culture of the adventitious bud. Respectively culturing culture bottles in a constant-temperature culture room with the light quality of white light, red light, blue light, red-blue composite light (1: 1) and red-blue composite light (5: 1), setting the temperature to be 23 +/-2 ℃, the illumination intensity to be 2000-2500 Lux, and the illumination period to be 16/10h (illumination/darkness), and after 4 weeks of illumination culture, adding a culture solution into the culture solution, wherein the ratio of LED red to blue is 1: 1, when the culture is carried out under illumination, the growth speed is fastest, and the maximum length of axillary buds reaches 7.74cm (figure 4). The results are shown in Table 2.
TABLE 2 results of the accelerating effect of LED light quality on adventitious bud elongation of eucommia ulmoides
Example 5:
an efficient eucommia ulmoides regeneration method based on LED light quality regulation and control specifically comprises the following operations:
1. cutting the obtained elongated adventitious bud material of eucommia into single bud stem segments with one leaf for later use.
2. Single shoot stems with one leaf were inoculated into a medium of DKW +0.5 mg/L6-BA +0.2mg/L GA3+30g/L sucrose +6.6g/L agar for axillary bud proliferation and elongation culture (FIG. 5). After 4 weeks of light culture, an average of 4.7 adventitious buds were produced per explant, with adventitious buds extending to 2.0-6.0 cm with 2-6 true leaves (FIG. 6).
3. The elongated adventitious buds were transferred to a medium of 1/2DKW, 1.0mg/L sodium nitroprusside, 30g/L sucrose and 6.6g/L agar for adventitious root induction. After 4 weeks of light culture, eucommia whole plants with white robust roots were obtained with rooting rates as high as 95.2% and on average 5.4 adventitious roots per explant, as shown in fig. 7 and 8, fig. 7 is a picture of rooted plants, and fig. 8 is a close-up view of roots. The culture process is carried out at a temperature of 23 +/-2 ℃ under the condition that the ratio of LED red to LED blue is 1: 1, culturing under illumination, wherein the illumination intensity is 2000-2500 Lux, and the illumination period is 16/10h (illumination/darkness).
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. An efficient eucommia ulmoides regeneration method based on LED light quality regulation and control is characterized by comprising the following steps: the method comprises the steps of taking a stem segment with axillary buds of eucommia as an explant, cutting the stem segment into segments with proper sizes after surface disinfection, inoculating the segments into culture media added with plant growth regulators with different types and concentrations, and sequentially carrying out germination and proliferation of the axillary buds, elongation of adventitious buds and rooting induction culture under the conditions of proper LED light illumination and temperature to finally obtain a complete eucommia regeneration plant.
2. The efficient eucommia ulmoides regeneration method based on LED light quality regulation and control as claimed in claim 1, wherein the method comprises the following steps: the stem segment with axillary buds of eucommia is the annual branch of a perennial triploid eucommia plant which is obtained from a field test field.
3. The efficient eucommia ulmoides regeneration method based on LED light quality regulation and control as claimed in claim 1, wherein the method comprises the following steps: the stem segment with axillary buds of eucommia is the annual branch of a perennial diploid eucommia plant obtained from a field test field.
4. The efficient eucommia ulmoides regeneration method based on LED light quality regulation and control as claimed in claim 2 or 3, wherein: the stem section with the axillary buds is a 5 cm-sized stem section which is cut after leaves of the branches are removed.
5. The efficient eucommia ulmoides regeneration method based on LED light quality regulation and control as claimed in claim 1, wherein the method comprises the following steps: the surface disinfection is that the stem segments with axillary buds of eucommia ulmoides are washed for 20min by running water, wiped by 75% absolute ethyl alcohol, washed by sterile water for 5 times in a sterile operating platform, then disinfected by 75% absolute ethyl alcohol for 45s, disinfected by disinfectant for 1-7 minutes, and finally washed by sterile water for 6 times.
6. The efficient eucommia ulmoides regeneration method based on LED light quality regulation and control as claimed in claim 5, wherein the method comprises the following steps: the disinfectant is 0.1 percent of HgCl2And (5) solution disinfection for 3 min.
7. The efficient eucommia ulmoides regeneration method based on LED light quality regulation and control as claimed in claim 1, wherein the method comprises the following steps: the axillary bud germination, proliferation and elongation culture mediums are DKW culture mediums added with 0.5mg/L6-BA, 0.2mg/L GA3, 30g/L sucrose and 6.6g/L agar.
8. The efficient eucommia ulmoides regeneration method based on LED light quality regulation and control as claimed in claim 1, wherein the method comprises the following steps: the rooting medium is 1/2DKW medium added with 0.5-2.0 mg/L sodium nitroprusside 30g/L sucrose and 6.6g/L agar.
9. The method for efficiently regenerating eucommia ulmoides based on LED light quality regulation and control according to claim 7 or 8, wherein the method comprises the following steps: after disinfection, the necrotic parts at two ends of the stem section are cut off, and the stem section is cut into 0.7 cm-sized cut sections with at least one axillary bud for germination and proliferation of the axillary bud, elongation of the adventitious bud and rooting induction culture.
10. The efficient eucommia ulmoides regeneration method based on LED light quality regulation and control as claimed in claim 1, wherein the method comprises the following steps: the proper temperature and illumination condition means that the culture is placed at the temperature of 23 +/-2 ℃, and the LED light quality and the mixture ratio are that the ratio of red to blue is 1: 1, culturing in a constant-temperature culture room with the illumination intensity of 2000-2500 lux and the illumination period of 16h illumination/10 h darkness.
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| CN116784237A (en) * | 2023-07-26 | 2023-09-22 | 东北林业大学 | Efficient fraxinus mandshurica regeneration method based on light quality regulation and control |
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