CN112119915B - Tissue culture rapid propagation and in vitro preservation method of alstonia-hance seedlings - Google Patents
Tissue culture rapid propagation and in vitro preservation method of alstonia-hance seedlings 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
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
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Abstract
The invention provides a tissue culture rapid propagation and in vitro preservation method of a chicken bone antifebrile dichroa seedling, and relates to the technical field of germplasm preservation. The invention takes the stem section with buds of the alstonia-hance as an object, obtains adventitious buds through explant disinfection and primary culture, performs proliferation induction, rooting, in-vitro preservation and the like on the adventitious buds, establishes a tissue culture rapid propagation and in-vitro preservation system of the alstonia-hance, and has a monthly proliferation coefficient of 1: 4, the in vitro preservation subculture period reaches about 1 year. By utilizing the tissue culture rapid propagation and in vitro preservation method, the in vitro preservation time is prolonged, the transfer workload of tissue culture seedlings can be reduced, the possibility of genetic change caused by continuous multiple subcultures is avoided, and the guarantee is provided for germplasm preservation and sustainable utilization.
Description
Technical Field
The invention belongs to the technical field of germplasm preservation, and particularly relates to a tissue culture rapid propagation and in vitro preservation method of a chicken bone antifebrile dichroa seedling.
Background
The Alstonia yunnanensis (Alstonia yunnanensis) is a plant of the genus of Alstonia in the family of Apocynaceae, is a unique species in China, and is distributed in hillside or valley areas with the altitude of 1100-2400 m in the southwest area of China. The root and the leaf of the plant are both used as medicines, and the monoterpene indole alkaloid contained in the root has the function of reducing blood pressure and is an important wild plant germplasm resource for developing natural medicaments for reducing blood pressure; the everlasting period of the chicken bone dichroa is longer, the purple-red flowers have fragrance, and a plurality of high-foot dish-shaped flowers cluster on the tops of branches, so that the ornamental value is high; the plant type is vertical shrub with the height of about 2 meters, and the shrub has multiple branches, and the branches are resistant to pruning, so the plant is very suitable for hedgerow planting and beautifying the environment, and has important landscaping application value. At present, the dichroa febrifuga is mostly sown by seeds, propagated by plants or cut, low in propagation efficiency and incapable of meeting the requirement of large-scale seedling production.
Disclosure of Invention
In view of the above, the invention aims to provide a tissue culture rapid propagation and in vitro preservation method of a chicken dichroa febrifuga seedling, which effectively solves the problem of tissue culture rapid propagation of the chicken dichroa febrifuga, greatly prolongs the in vitro preservation period, is beneficial to preservation, development and utilization of germplasm resources of the chicken dichroa febrifuga, and lays a foundation for resource mining in the future.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a tissue culture rapid propagation and in vitro preservation method of a chicken bone antifebrile dichroa seedling, which comprises the following steps: (1) taking the sterilized tender lateral bud stem segment as an explant, and transferring the explant to a primary culture medium for primary culture to obtain a sterile new bud; the primary culture and MS culture medium is used as a basic culture medium, and the primary culture and MS culture medium also comprises 0.5mg/L, NAA 0.2.2 mg/L of 6-BA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8;
(2) transferring the sterile new buds to a multiplication culture medium for continuous culture to obtain cluster buds; the multiplication culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.3-1.0 mg/L, IAA 0.2.2 mg/L of 6-BA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8;
(3) cutting the cluster buds into single buds, inoculating the single buds on a rooting culture medium for rooting culture to obtain rooted tissue culture seedlings; the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises 0.1-1.0 mg/L of IAA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8;
(4) transferring the rooting tissue culture seedling to an in-vitro preservation culture medium for in-vitro preservation; the in vitro preservation culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises IAA1.0mg/L, sucrose 20g/L and agar 5.6g/L, and the pH value is 5.8.
Preferably, the sterilization in step (1) comprises: soaking tender lateral bud stem segments in soap water, washing for 1h by tap water, cutting into stem segments with single buds in a sterile environment, disinfecting for 10s by using alcohol with the volume percentage of 75%, washing for 3 times by using sterile water, disinfecting for 6-8 min by using mercuric chloride solution with the mass percentage of 0.1%, and washing for 3-5 times by using sterile water.
Preferably, after the rooting tissue culture seedlings are obtained in the step (3), transplanting is carried out when the rooting tissue culture seedlings take roots for 2-4 and the roots are 2-3 cm long.
Preferably, before transplanting, hardening off seedlings are further included.
Preferably, the transplanted transplanting substrate is a mixed substrate comprising leaf mold and raw red soil, and the volume ratio of the leaf mold to the raw red soil is 1: 1.
Preferably, the temperature of the primary culture in the step (1), the temperature of the continuous culture in the step (2), the temperature of the rooting culture in the step (3) and the temperature of the in vitro preservation in the step (4) are both 25 +/-3 ℃, and the illumination intensity is both 20-40 mu mol/(m)2S) and the illumination time is 12 h/d.
The invention provides a tissue culture rapid propagation and in vitro preservation method of a alstonia-hance seedling, which takes a stem section with buds of the alstonia-hance as an object, obtains adventitious buds through explant disinfection and primary culture, performs proliferation induction, rooting, in vitro preservation and the like on the adventitious buds, establishes a tissue culture rapid propagation and in vitro preservation system of the alstonia-hance, and has a monthly proliferation coefficient of 1: 4, the in vitro preservation subculture period reaches about 1 year. The method effectively solves the problem of tissue culture and rapid propagation of the alstonia-hance, greatly prolongs the in vitro preservation period, is beneficial to preservation, development and utilization of germplasm resources of the alstonia-hance, and lays a foundation for resource mining in the future. In the embodiment of the invention, the induction rate reaches 85% during primary culture, although a small amount of callus appears on the base of the stem segment with buds, the leaves of the new buds are green and grow faster; the maximum proliferation coefficient can reach 4.5 when the culture is continued; the rooting rate of the adventitious bud during rooting culture can reach 88 percent; transplanting when the alstonia-root grows to about 3 roots and the length is about 2-3 cm, wherein the transplanting seedling rate is more than 90%; after rooting is completed and a complete plant is formed, the plant can be preserved in vitro for about 12 months on an in vitro preservation culture medium, and the green leaves of the seedling are elongated. By utilizing the tissue culture rapid propagation and in vitro preservation method, the in vitro preservation time is prolonged, the transfer workload of tissue culture seedlings can be reduced, the possibility of genetic change caused by continuous multiple subcultures is avoided, and the guarantee is provided for germplasm preservation and sustainable utilization.
Drawings
FIG. 1 shows the induction of multiple shoots in the tissue culture of dichroa henryi according to the present invention;
FIG. 2 shows the rooting of the Changshan tissue culture seedling of chicken bone.
Detailed Description
The invention provides a tissue culture rapid propagation and in vitro preservation method of a chicken bone antifebrile dichroa seedling, which comprises the following steps: (1) taking the sterilized tender lateral bud stem segment as an explant, and transferring the explant to a primary culture medium for primary culture to obtain a sterile new bud; the primary culture and MS culture medium is used as a basic culture medium, and the primary culture and MS culture medium also comprises 0.5mg/L, NAA 0.2.2 mg/L of 6-BA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8;
(2) transferring the sterile new buds to a multiplication culture medium for continuous culture to obtain cluster buds; the multiplication culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.3-1.0 mg/L, IAA 0.2.2 mg/L of 6-BA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8;
(3) cutting the cluster buds into single buds, inoculating the single buds on a rooting culture medium for rooting culture to obtain rooted tissue culture seedlings; the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises 0.1-1.0 mg/L of IAA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8;
(4) transferring the rooting tissue culture seedling to an in-vitro preservation culture medium for in-vitro preservation; the in vitro preservation culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises IAA1.0mg/L, sucrose 20g/L and agar 5.6g/L, and the pH value is 5.8.
Taking the sterilized tender lateral bud stem segment as an explant, and transferring the explant to a primary culture medium for primary culture to obtain a sterile new bud; the primary culture and MS culture medium is used as a basic culture medium, and the primary culture and MS culture medium further comprises 0.5mg/L, NAA 0.2.2 mg/L of 6-BA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8.
The method comprises the steps of taking a sterilized tender lateral bud stem section as an explant, and transferring the explant to a primary culture medium for primary culture to obtain a sterile new bud; the primary culture and MS culture medium is used as a basic culture medium, and the primary culture and MS culture medium further comprises 0.5mg/L, NAA 0.2.2 mg/L of 6-BA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8. The tender side bud stem segment is preferably taken from a newly extracted side bud stem segment of a healthy single plant of dichroa febrifuga and is used as an explant after being sterilized, and the sterilization preferably comprises the following steps: soaking tender lateral bud stem segments in soap water, washing for 1h by tap water, cutting into stem segments with single buds in a sterile environment, disinfecting for 10s by using alcohol with the volume percentage of 75%, washing for 3 times by using sterile water, disinfecting for 6-8 min by using mercuric chloride solution with the mass percentage of 0.1%, and washing for 3-5 times by using sterile water. The mass concentration of the soapy water is preferably 1%, and the soaking time in the soapy water is preferably 10 min. The time for sterilizing the surface of the mercuric chloride solution is preferably 8 min.
According to the invention, the explant is transferred to a primary culture medium for primary culture, the induction rate of the side buds of the primary culture medium can reach 85%, and although a small amount of callus appears on the base of the stem segment with buds, the leaves of the new buds are green and grow faster. The temperature of the primary culture is preferably 25 +/-3 ℃, and the illumination intensity is preferably 20-40 mu mol/(m)2S), the illumination time is preferably 12 h/d.
After the sterile new buds are obtained, transferring the sterile new buds to a multiplication culture medium for continuous culture to obtain cluster buds; the multiplication culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.3-1.0 mg/L, IAA 0.2.2 mg/L of 6-BA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8. In the invention, the well-grown sprouts are preferably transferred to a multiplication culture medium for continuous culture, and the condition parameters of the continuous culture are preferably the same as those of the primary culture, and are not described again. The enrichment culture medium contains 6-BA, the concentration of the 6-BA is preferably 1.0mg/L, the enrichment coefficient of the enrichment culture medium under the concentration can reach 4.5, and plants grow vigorously, have dark green leaves and have extended roots and leaves.
After cluster buds are obtained, the cluster buds are cut into single buds and then are inoculated on a rooting culture medium for rooting culture, and rooting tissue culture seedlings are obtained; the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises 0.1-1.0 mg/L of IAA, 20g/L of sucrose and 5.6g/L of agar, and the pH value is 5.8. The rooting culture medium comprises IAA, the concentration of the IAA is preferably 1.0mg/L, the rooting rate can reach 88%, the number of rooting strips can reach 2-5, the root thickness is about 0.2cm, and plants grow robustly in the rooting culture medium under the concentration. The condition parameters of the rooting culture are preferably the same as those of the primary culture, and are not described herein again.
In the invention, when the rooted tissue culture seedling takes root for 2-4 pieces and the root length is 2-3 cm, transplanting can be preferably carried out, before transplanting, seedling hardening is preferably further included, and the seedling hardening is preferably as follows: taking out the bottle seedlings with roots from the bottle, washing off culture medium attached to the roots with clear water, putting the roots of the seedlings downwards into a seedling raising pot, pre-filling tap water with the depth of 3-4 cm into the seedling raising pot, hardening the seedlings in the seedling raising pot for 1-2 days, opening a cover of the seedling raising pot, hardening the seedlings for 2 days, and then transplanting. The transplanting substrate during transplanting is preferably a mixed substrate comprising leaf mold and raw red soil, and the volume ratio of the leaf mold to the raw red soil is 1: 1. According to the invention, after the transplanting, sufficient root fixing water is preferably poured, the plastic film can be used for covering and moisturizing, after new leaves grow out from the test-tube seedlings, the film is removed and the management is performed roughly, and the transplanting seedling rate is more than 90%.
After the rooting tissue culture seedling is obtained, the rooting tissue culture seedling is transferred to an in-vitro preservation culture medium for in-vitro preservation; the in vitro preservation culture medium takes 1/2MS culture medium as a basic culture medium, and also comprises IAA1.0mg/L, sucrose 20g/L and agar 5.6g/L, and the pH value is 5.8. The condition parameters of the in vitro preservation of the invention are preferably the same as those of the primary culture, and are not described in detail herein. The invention can be preserved in vitro for about 12 months on the in vitro preservation culture medium.
The tissue culture rapid propagation and in vitro preservation method of an antifebrile dichroa seedling provided by the present invention is described in detail below with reference to examples, but the present invention is not limited thereto.
Example 1
1. Explant sterilization
Taking a side bud stem section newly extracted from a healthy single strain in the Changshan as an explant, soaking the explant in 1% soap water for 10min, washing the explant with tap water for 1h, taking the washed explant on a super clean bench, shearing the explant into stem sections with single buds, disinfecting the stem sections with 75% alcohol for 10s, washing the stem sections with sterile water for 3 times, disinfecting the surface of the stem sections with 0.1% mercuric chloride solution for 3-8 min, washing the stem sections with sterile water for 3-5 times, inoculating the stem sections with buds to a basic culture MS without adding any hormone, and placing 1 stem section with buds in each bottle to avoid cross contamination. All the culture media are addedAdding sucrose 20g/L and agar 5.6g/L for solidification (the same applies hereinafter), adjusting pH to 5.8, culturing at 25 + -3 deg.C, and illuminating intensity 20-40 μmol/(m)2S), illumination 12 h/d. After stem sections with buds are inoculated into different primary culture media, observation is carried out every day, pollution appears continuously after 3d, the pollution rate is lowest after disinfection treatment is carried out for 8min after 30d, and the disinfection effect is best, and the table 1 shows.
TABLE 1 contamination of the stem sections of the dichroa febrifuga with different disinfection times
2. Primary culture of stem segments with buds in changshan of chicken bone
MS is taken as a basic culture medium, cytokinin 6-BA (0.3mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.0mg/L and 3.0mg/L) and auxin naphthylacetic acid (0.2mg/L) with different concentrations are added to carry out primary culture on the induction culture medium of the dichroa febrifuga stem so as to obtain sterile new buds. The result shows that the inductivity reaches 85% on the MS +0.5 mg/L6-BA +0.2mg/LNAA culture medium, although a small amount of callus appears on the base of the stem segment with the bud, the leaf of the new bud is green and grows faster.
TABLE 2 selection of primary culture Medium for ivy stem segments
3. Method for screening induction culture medium of cluster buds of dichroa febrifuga
Transferring the well-grown buds to a multiplication medium for continuous culture. IAA (0.2mg/L), NAA naphthylacetate (0.2mg/L) and 6-BA (0.3mg/L, 0.5mg/L, 1.0mg/L) were added to the medium for six treatments, each treatment inoculated 20 flasks with 5 explants per flask. After one month, the proliferation coefficient was counted. As can be seen from Table 3, there was a shoot proliferation in each of the 6 combinations tested, with the best proliferation achieved with MS +6-BA1.0+ IAA0.2 treatment and a proliferation factor of 4.5 (FIG. 1), which is significantly higher than the other treatments.
TABLE 3 selection of inducing culture medium for cluster buds of radix Dichroae
4. Screening of rooting medium
Cutting the induced cluster buds into single buds, inoculating the single buds to different rooting culture media, and culturing. 1/2MS is used as a basic culture medium (only the macroelement is halved), IBA (0.1mg/L, 0.3mg/L, 0.5mg/L), IAA (0.1mg/L, 0.3mg/L, 1.0mg/L) and NAA (0.1mg/L, 0.2mg/L, 0.3mg/L) with different concentrations are combined for treatment, so as to obtain a proper rooting culture medium. Selecting buds with height of more than 3cm, inoculating 20 bottles of explants in each bottle after each treatment, and counting the rooting rate after 30 days. Rooting culture is carried out on a culture medium 1/2MS + IAA1.0, the rooting rate can reach 88% (figure 2), the number of roots can reach 2-5, the root thickness is about 0.2cm, and the plant grows robustly and is shown in Table 4.
TABLE 4 rooting Medium screening
5. Rooting of tissue culture seedling
When the dichroa febrifuga roots take about 3 roots and grow about 2-3 cm, the dichroa febrifuga can be used for transplanting preparation. Firstly, taking out a bottle seedling with roots from the bottle, washing off culture medium attached to the root by using clear water, putting the root of a seedling downwards into a seedling raising pot, pre-filling tap water with the depth of 3-4 cm into the seedling raising pot, hardening seedlings for 1-2 days in the seedling raising pot, opening a cover of the seedling raising pot, hardening the seedlings for 2 days, and then transplanting. The transplanting substrate is (volume ratio) leaf mold: raw red mud 1:1, watering enough root fixing water after planting, covering with a plastic film for moisturizing, performing film removal and extensive management after new leaves grow out of the test-tube seedlings, and transplanting the seedling rate to be more than 90%.
6. In vitro preservation
After rooting is completed on a culture medium 1/2MS + IAA1.0 and a complete plant is formed, the plant can be preserved in vitro for about 12 months, and the green leaves of the seedling are elongated.
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 (2)
1. A tissue culture rapid propagation and in vitro preservation method of a chicken bone antifebrile dichroa seedling is characterized by comprising the following steps: (1) taking the sterilized tender lateral bud stem segment as an explant, and transferring the explant to a primary culture medium for primary culture to obtain a sterile new bud; the primary culture medium consists of an MS culture medium, 6-BA0.5mg/L, NAA0.2mg/L, sucrose 20g/L and agar 5.6g/L, and the pH value is 5.8;
(2) transferring the sterile new buds to a multiplication culture medium for continuous culture to obtain cluster buds; the multiplication culture medium consists of an MS culture medium, 0.3-1.0 mg/L of 6-BA, 0.2mg/L of IAA, 20g/L of cane sugar and 5.6g/L of agar, and the pH value is 5.8;
(3) cutting the cluster buds into single buds, inoculating the single buds on a rooting culture medium for rooting culture to obtain rooted tissue culture seedlings; the rooting culture medium consists of 1/2MS culture medium, IAA 0.1-1.0 mg/L, sucrose 20g/L and agar 5.6g/L, and the pH value is 5.8; after the rooting tissue culture seedlings are obtained, transplanting the rooting tissue culture seedlings when the rooting tissue culture seedlings root 2-4 and the roots are 2-3 cm long, before transplanting, hardening the seedlings, wherein a transplanted matrix is a mixed matrix of leaf mold and raw red soil, and the volume ratio of the leaf mold to the raw red soil is 1: 1;
(4) transferring the rooting tissue culture seedling to an in-vitro preservation culture medium for in-vitro preservation; the in vitro preservation culture medium consists of 1/2MS culture medium, IAA1.0mg/L, sucrose 20g/L and agar 5.6g/L, and the pH value is 5.8;
the temperature of the primary culture in the step (1), the continuous culture in the step (2), the rooting culture in the step (3) and the in vitro preservation in the step (4) are both 25 +/-3 ℃, and the illumination intensity is 20-40 mu mol/(m)2S) and the illumination time is 12 h/d.
2. The tissue culture rapid propagation and in vitro preservation method according to claim 1, wherein the disinfection of the step (1) comprises: soaking tender lateral bud stem segments in soap water, washing for 1h by tap water, cutting into stem segments with single buds in a sterile environment, disinfecting for 10s by using alcohol with the volume percentage of 75%, washing for 3 times by using sterile water, disinfecting for 6-8 min by using mercuric chloride solution with the mass percentage of 0.1%, and washing for 3-5 times by using sterile water.
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