CN116267612B - Tissue culture propagation method of hippeastrum - Google Patents
Tissue culture propagation method of hippeastrum Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 45
- 241001466280 Hippeastrum evansiae Species 0.000 title 1
- 230000006698 induction Effects 0.000 claims abstract description 113
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 55
- 241000234473 Hippeastrum Species 0.000 claims abstract description 52
- 239000001963 growth medium Substances 0.000 claims abstract description 48
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 43
- 230000001954 sterilising effect Effects 0.000 claims description 35
- 229940113118 carrageenan Drugs 0.000 claims description 28
- 235000010418 carrageenan Nutrition 0.000 claims description 28
- 229920001525 carrageenan Polymers 0.000 claims description 28
- 239000000679 carrageenan Substances 0.000 claims description 28
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims description 28
- 229960002523 mercuric chloride Drugs 0.000 claims description 22
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 18
- 238000005286 illumination Methods 0.000 claims description 17
- 239000012883 rooting culture medium Substances 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 4
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 6
- 230000035755 proliferation Effects 0.000 abstract description 5
- 230000035784 germination Effects 0.000 abstract description 2
- 238000004161 plant tissue culture Methods 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 31
- 230000004083 survival effect Effects 0.000 description 16
- 241000196324 Embryophyta Species 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 9
- 230000009286 beneficial effect Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 229910052956 cinnabar Inorganic materials 0.000 description 4
- 238000012136 culture method Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 229960002233 benzalkonium bromide Drugs 0.000 description 3
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 239000006870 ms-medium Substances 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
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- 230000000644 propagated effect Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 239000012879 subculture medium Substances 0.000 description 1
Classifications
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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
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture propagation method of hippeastrum. The tissue culture propagation method of the hippeastrum includes disinfection of the explants, and callus induction culture is carried out on the disinfected explants; wherein, the explant selects a hippeastrum receptacle; the callus induction culture medium is added with 2.5-3.5 mg/L6-BA, 2.0-2.5mg/L TDZ and 0.05-0.15mg/L NAA. The tissue propagation method of the hippeastrum can obtain a large number of seedlings on the premise of not damaging seed sources, and solves the problem of seedling scarcity. Experiments prove that the tissue culture propagation method of the hippeastrum has the advantages that the germination rate of the calluses reaches 80%, the induction rate of the adventitious buds reaches 85%, the proliferation coefficient of the subculture is more than 2.0, and the rooting rate reaches 100%; in addition, the tissue culture propagation method of the hippeastrum has the advantage of short culture period.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture propagation method of hippeastrum.
Background
The hippeastrum (hippearrm herb.) is a plant of the genus hippeastrum of the family lycoridae, perennial herb bulb plants, and tropical americas in origin, and has high ornamental and commercial values. Currently, the breeding method of hippeastrum includes sowing method, ball dividing method, bulb cutting method, tissue culture method and the like. Wherein, the seeding method has the phenomenon of offspring separation. The conventional ball separation method and bulb cutting method have low proliferation rate after being planted once a year.
Tissue culture refers to a technique in which desired organs, tissues, cells, etc. are isolated from plant bodies, and cultured under manual control by aseptic manipulation to obtain regenerated whole plants or to produce other products of economic value. Although in theory, any tissue or organ has the potential to develop into a complete plant according to the totipotency of cells, different kinds of plants and organs or tissues of the same kind of different material-obtaining parts can be found in the actual operation process, and the reaction of the organs or tissues to the induction conditions is greatly different. For bulb plants, the outer layer of the scale is generally more regenerative than the inner layer, and the lower and upper stages are more regenerative. Thus, at present, vermilion is propagated by tissue culture, and bulbs and bulb trays are often used as explants.
However, the use of bulbs and bulb trays as explants can cause destructive damage to the cue ball. This is a contradictory and painful point between breaking the cue ball and preserving the germplasm for the rare seed balls of the germplasm and new breeding offspring. The other parts except bulbs and bulb discs, such as pedicel, ovary and the like, are used as explants for breeding the hippeastrum, and the problems of low induction rate, high death rate, long culture period and the like are also existed. For example, tissue culture and propagation of Cinnabaris by the forestry office network in Kaikovia produces callus after about 30 days to form adventitious roots, and adventitious buds can only be formed after 3 to 4 months (http:// lyj. Kaieng. Gov. Cn/news. Aspxpach= 407161010052029). Therefore, the tissue culture propagation method of the hippeastrum which can not destroy bulbs and bulb discs, can ensure higher induction rate, lower death rate and shorter culture period is found, and has important significance for popularization and utilization of the hippeastrum excellent and thin varieties.
Disclosure of Invention
In view of the technical problems existing in the background technology, the invention provides a tissue culture propagation method of hippeastrum, which comprises the steps of sterilizing an explant, and performing callus induction culture on the sterilized explant, wherein the explant selects a hippeastrum receptacle; the callus induction culture is performed in a callus culture medium, wherein the callus culture medium takes an MS culture medium as a basic culture medium, and 2.5-3.5mg/L of 6-BA, 2.0-2.5mg/L of TDZ and 0.05-0.15mg/L of NAA are added.
Preferably, the ratio of the TDZ to the 6-BA is 2.8-3.2:2.
Preferably, 28-32g/L sugar and 7-9g/L carrageenan are also added into the callus culture medium.
Preferably, the temperature of the callus induction culture is 24-26 ℃; and/or, the time of the callus induction culture is 25-40 days.
Preferably, the tissue culture propagation method of the hippeastrum further comprises adventitious bud induction culture after the callus induction culture; the adventitious bud induction culture is carried out in an adventitious bud induction culture medium, wherein the adventitious bud induction culture medium takes an MS culture medium as a basic culture medium, and 1.5-2.0mg/L of 6-BA, 1.0-1.5mg/L of TDZ and 0.05-0.15mg/L of NAA are added.
Preferably, the illumination time of the adventitious bud induction culture is 10-14 h/day, and the illumination intensity is 1600-1800lx; and/or the culture temperature of the adventitious bud induction culture is 24-26 ℃.
Preferably, the tissue culture propagation method of the hippeastrum further comprises rooting culture after the adventitious bud induction culture; the rooting culture is carried out in a rooting culture medium, wherein the rooting culture medium takes an MS culture medium as a basic culture medium, and NAA of 0.08-0.12mg/L and activated carbon of 0.2-1.5g/L are added.
Preferably, the explant is selected from non-flowering hippeastrum carriers; and/or cutting the sterilized explant into 1.5-2.5cm pieces prior to the callus induction culture.
Preferably, the sterilization of the explant comprises, in order, benzalkonium bromide sterilization, ethanol sterilization and mercuric chloride sterilization.
Preferably, the mercuric chloride disinfection adopts 0.1% mercuric chloride disinfection treatment for 8min plus or minus 20s;
preferably, after the mercuric chloride is sterilized, the mercuric chloride is washed with sterile water for 2 to 4 times, and the washing time is 45 to 75 seconds each time.
The beneficial effects are that:
the invention provides a tissue culture propagation method of hippeastrum, which uses hippeastrum receptacle as an explant to perform tissue culture propagation of hippeastrum, does not damage original mother balls of the hippeastrum, and is further beneficial to protecting rare and excellent germplasm resources. Meanwhile, 2.5-3.5mg/L of 6-BA, 2.0-2.5mg/L of TDZ and 0.05-0.15mg/L of NAA are added into the culture medium for callus induction culture of the vermilion, and the vermilion can form callus within about 30 days through callus induction culture, and the death rate of an explant sample in the callus induction process is low, the survival rate is high and the induction rate is high.
After the callus obtained by the invention is subjected to adventitious bud induction culture, subculture and rooting induction culture, the induction rate of the adventitious buds reaches 85%, the proliferation coefficient of the subculture is more than 2.0, and the rooting rate reaches 100%. In addition, the tissue culture propagation method of the hippeastrum has the advantage of short culture period. The tissue culture propagation method of the hippeastrum can replace the traditional method of tissue culture propagation by taking bulbs or bulb discs as explants, so that high-quality hippeastrum seedlings can be rapidly and effectively produced in large quantity on the premise of not damaging seed sources, and the method is beneficial to breeding of new hippeastrum varieties and industrialized production of the seedlings.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be described below.
FIG. 1 is a photograph of an explant sterilized in step 1) of example 1 of the present invention;
FIG. 2 is a photograph of callus obtained by induction culture in step 2) of example 1 of the present invention;
FIG. 3 is a photograph of adventitious buds obtained by induction culture in step 3) of example 1 of the present invention;
FIG. 4 is a photograph of adventitious buds obtained by subculture in step 4) of example 1 of the present invention;
FIG. 5 is a photograph of a rooted seedling obtained by induction culture in step 5) of example 1 of the present invention.
Detailed Description
The invention provides a tissue culture propagation method of hippeastrum henryi, which comprises the steps of sterilizing an explant and performing callus induction culture on the sterilized explant.
The tissue culture propagation method of the hippeastrum includes disinfection of explants.
In the present invention, the explant is selected from the group consisting of a hippeastrum receptacle. Tissue culture propagation of the hippeastrum is carried out by taking the hippeastrum receptacle as an explant, so that the original mother ball of the hippeastrum is not damaged, and further, the protection of thin and excellent germplasm resources is facilitated. In order to avoid introducing exogenous pollution and improve the disinfection effect, the hippeastrum flower receptacle is preferably an unopened flower receptacle, and more preferably a flower receptacle in full unopened flower and bone.
In the present invention, the sterilization of the explant preferably includes three steps of benzalkonium bromide sterilization, ethanol sterilization and mercuric chloride sterilization. The invention firstly carries out the new Jieli sterilization on the explant, wherein the treatment time of the new Jieli sterilization is preferably 14-16min, more preferably 15min; after the benzalkonium bromide is disinfected, preferably washing with running water, wherein the time of washing with running water is preferably 20-40min, more preferably 30min; after the new Jieer sterilization treatment, the invention performs ethanol sterilization on the explant. The ethanol sterilization preferably uses 75% ethanol, and the sterilization time of the 75% ethanol is preferably 20-40s, more preferably 25-30s; after 75% ethanol sterilization treatment, the invention performs mercuric chloride sterilization on the explant, wherein the mercuric chloride sterilization preferably uses 0.1% mercuric chloride, and the sterilization time of 0.1% mercuric chloride is preferably 5-10min, more preferably 8min plus or minus 20s, and more preferably 8min plus or minus 5s.
Experiments prove that the 0.1 percent mercuric chloride disinfection treatment is carried out for different times, and the pollution rate, the death rate, the survival rate and other indexes of the vermilion infrared implant are obviously affected. When the disinfection treatment time of 0.1 percent of mercuric chloride is 8 minutes, the pollution rate of the vermilion infrared implant can be reduced to 20 percent, the death rate is not more than 20 percent, and the survival rate is as high as 63 percent. When the 0.1 percent mercuric chloride disinfection treatment time is less than 8 minutes, the pollution rate of the explant can be obviously increased, and the survival rate is obviously reduced; when the 0.1% mercuric chloride disinfection treatment time is more than 8min, the death rate of the explant is obviously increased, and the survival rate is obviously reduced. After 0.1% mercuric sterilization, the explant sample is preferably washed with sterile water, preferably 2-4 times, more preferably 3 times; the washing time per time is preferably 45 to 75s, more preferably 60s.
After the explant is sterilized, the explant is preferably cut into small pieces and then callus induction culture is performed. In the present invention, the particle size of the small pieces is preferably 1.5 to 2.5cm, more preferably 2cm.
The callus induction culture takes MS culture medium as basic culture medium, and 6-BA, TDZ and NAA are added. Wherein, the addition amount of 6-BA is 2.5-3.5mg/L, preferably 2.8-3.2mg/L, more preferably 3mg/L; the TDZ is added in an amount of 2.0 to 2.5mg/L, preferably 2.0mg/L, and NAA is added in an amount of 0.05 to 0.15mg/L, preferably 0.08 to 0.12mg/L, more preferably 0.1mg/L.
Experiments prove that the formula of the induction culture medium has obvious influence on the induction rate of the callus, especially the addition amount of TDZ, and when the addition amount of TDZ is 2.0mg/L (the ratio of the addition amount of TDZ to the addition amount of 6-BA is 3:2), the death rate of the callus is only 7%, the survival rate is 90%, and the induction rate is 86%. When the addition amount of TDZ is higher or lower than 2.0mg/L, the death rate of the callus is obviously increased, and the survival rate and the induction rate are obviously reduced.
In a preferred embodiment of the invention, the callus induction culture is based on MS medium, with the addition of sugar and carrageenan. Wherein, the addition amount of sugar is preferably 28-32g/L, more preferably 30g/L; the addition amount of the carrageenan is preferably 7-9g/L, more preferably 8g/L. The callus induction culture medium added with 30g/L sugar and 8g/L carrageenan can provide a proper growth environment for the hippeastrum receptacle explant, and is beneficial to the more smooth formation of callus in the callus induction process of the hippeastrum receptacle explant.
In the present invention, the temperature of the callus induction culture is preferably 24-26 ℃; the time for the callus induction culture is preferably 25 to 40 days, more preferably 30 days.
After the callus is induced and cultured, the obtained callus is preferably inoculated into an adventitious bud induction culture medium for adventitious bud induction culture. In the present invention, the adventitious bud induction culture is based on an MS medium, sugar and carrageenan are added, and 6-BA, TDZ and NAA are added. Wherein, the addition amount of 6-BA is preferably 1.5-2.0mg/L, more preferably 2.0mg/L; the amount of TDZ added is preferably 1.0 to 1.5mg/L, more preferably 1.0mg/L. Experiments prove that the 6-BA and TDZ contents with different concentration ratios have obvious influence on the induction rate results of the adventitious buds, and when the concentration of the 6-BA is 2.0mg/L and the concentration of the TDZ is 1.0mg/L, the induction rate of the adventitious buds can reach 87%. And when the concentration of 6-BA or TDZ is changed, the induction rate of adventitious buds is remarkably reduced.
In the invention, the illumination time of the adventitious bud induction culture is preferably 10-14 h/day, and the illumination intensity is preferably 1600-1800lx; the culture temperature of the adventitious bud induction culture is preferably 24-26 ℃; the culture time of the adventitious bud induction culture is preferably 20 to 30 days.
After induction culture of adventitious buds, the obtained adventitious buds are preferably inoculated into a subculture medium for subculture. In the present invention, the subculture is based on MS medium, sugar and carrageenan are added, and 6-BA, TDZ and NAA are added. Wherein, the addition amount of 6-BA is preferably 1.5-2.0mg/L, more preferably 2.0mg/L; the amount of TDZ added is preferably 1.0 to 1.5mg/L, more preferably 1.0mg/L.
In the invention, the illumination time of the secondary culture is preferably 10-14 h/day, and the illumination intensity is preferably 1600-1800lx; the culture temperature of the secondary culture is preferably 24-26 ℃; the culture time of the subculture is preferably 15 to 25 days. In the present invention, the number of times of the subculture is preferably 1 to 2 times.
The invention preferably inserts the adventitious bud after the adventitious bud induction culture or the subculture into a rooting culture medium to induce rooting. In the invention, rooting culture takes MS culture medium as basic culture medium, sugar and carrageenan are added, and NAA and active carbon are added. Wherein, the addition amount of NAA is preferably 0.08-0.12mg/L, more preferably 0.1mg/L; the amount of activated carbon added is preferably 0.2 to 1.5g/L, more preferably 0.5g/L. Experiments prove that when the addition amount of NAA is 0.1mg/L and the addition amount of active carbon is preferably 0.5g/L, the rooting rate can reach 100%.
In the invention, the illumination time of rooting culture is preferably 10-14 h/day, and the illumination intensity is preferably 1600-1800lx; the culture temperature of rooting culture is preferably 24-26 ℃; the culture time of the rooting culture is preferably 30-40 days.
In the invention, sugar and carrageenan are added into the culture medium in the processes of adventitious bud induction culture, subculture and rooting culture. The person skilled in the art can suitably adjust the requirements of the plants for sugar and the viscosity of the medium. In an alternative embodiment of the invention, the addition amount of sugar in the culture medium of adventitious bud induction culture, subculture or rooting culture is preferably 20-40g/L; the addition amount of carrageenan is preferably 6-10g/L.
The invention successfully constructs the tissue culture propagation method of the hippeastrum which takes the receptacle as the explant, the germination rate of the callus reaches 80 percent, the induction rate of the adventitious buds reaches 85 percent, the proliferation coefficient of the subculture is more than 2.0, and the rooting rate reaches 100 percent. Meanwhile, the appearance of the obtained seedling is observed, and no plant variation is found. In addition, the callus induction time of the invention is 25-40 days, the adventitious bud induction time is only 20-30 days, and the rooting culture time is 30-40 days. The method for tissue culture propagation of hippeastrum has the advantage of short culture period.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. Unless otherwise indicated, all the experimental procedures used in the examples were conventional; the materials, reagents and the like used are all commercially available.
Example 1
A tissue culture method for obtaining regenerated plants by taking a cinnabar receptacle as an explant comprises the following steps:
step 1) explant selection and sterilization: the method comprises the steps of selecting the flower receptacle in the full unopened flower as an explant, shaking for 15min by using a novel Jielsen solution, washing for 30min by flowing water, sterilizing for 30s by using 75% alcohol on a super clean workbench, sterilizing for 8min by using 0.1% mercuric chloride, and finally washing for 3 times by using sterile water to obtain the sterilized flower receptacle explant (figure 1).
Step 2) induction of callus: cutting the sterilized explant into small blocks of about 2cm, inoculating the explant into an induction culture medium for callus induction culture for 30 days, and controlling the culture temperature at 25 ℃; the induction culture medium is MS+6-BA3.0mg/L+TDZ2.0mg/L+NAA0.1mg/L+30 g/L of sugar+8 g/L of carrageenan. The calli of hippeastrum were obtained (FIG. 2).
Step 3) adventitious bud induction: inoculating the cinnabar callus obtained by the induction in the step 2) into an adventitious bud induction culture medium, and carrying out adventitious bud induction culture for 25 days at a culture temperature of 25 ℃ for 12 h/day under the illumination with the illumination intensity of 1700lx; the adventitious bud induction culture medium is MS+6-BA2mg/L+TDZ 1.0mg/L+NAA0.1 mg/L+30 g/L of sugar+8 g/L of carrageenan. Adventitious buds were obtained (FIG. 3).
Step 4) subculture: continuously subculturing the adventitious bud obtained in the step 3) for 20 days at a culture temperature of 25 ℃ for 12 h/day under the illumination with the illumination intensity of 1700lx; the culture medium for the secondary culture is MS+6-BA2mg/L+TDZ 1.0mg/L+NAA0.1 mg/L+30 g/L of sugar+8 g/L of carrageenan. The adventitious buds were obtained by subculturing (FIG. 4).
Step 5) rooting induction: cutting off leaves and fibrous roots of the tissue culture seedlings of the hippeastrum obtained by the secondary culture, inoculating the tissue culture seedlings of the hippeastrum into a rooting culture medium for rooting culture for 35 days at a culture temperature of 25 ℃ for 12 h/day under the illumination intensity of 1700lx. The rooting culture medium is MS+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8 g/L+activated carbon 0.5g/L. Root seedlings were obtained (FIG. 5).
Example 2
A tissue culture method for obtaining regenerated plants by taking a cinnabar receptacle as an explant comprises the following steps:
step 1) explant selection and sterilization: the method for sterilizing the flower comprises the steps of: shaking with new Jieer solution for 15min, washing with running water for 30min, sterilizing with 75% ethanol for 30s on aseptic console, sterilizing with 0.1% mercuric chloride for 8min, and washing with aseptic water for 1min for 3 times. Inoculating to induction culture medium MS+6-BA3.0mg/L+TDZ2.0mg/L+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8g/L.
The number of explants at this stage is 30, 3 times of repetition, and the pollution rate of the statistical sample after 10d is 20%, the death rate is 20% and the survival rate is 60%.
Step 2) induction of callus: taking sterilized explants, and inoculating the explants to an induction culture medium MS+6-BA3.0mg/L+TDZ2.0mg/L+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8g/L.
The survival of 30 explants after disinfection at this stage, 3 times of repetition, the induction of the explants was observed, the death rate of the explants after 30d (from the completion of inoculation) was counted to be 7%, the survival rate was 93%, and the induction rate was 86%.
Step 3) adventitious bud induction: inoculating the cinnabar callus obtained by the induction of the explant to an adventitious bud induction culture medium: MS+6-BA2mg/L+TDZ 1.0mg/L+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8g/L.
At this stage, 30 calli were inoculated, 3 replicates were performed, the induction of adventitious buds was observed, and the induction rate of adventitious buds was counted to be 87% after 25 days.
Step 4) subculture: continuously subculturing the adventitious bud obtained in the step 3) for 20 days at a culture temperature of 25 ℃ for 12 h/day under the illumination with the illumination intensity of 1800lx; the culture medium for the secondary culture is MS+6-BA2mg/L+TDZ 1.0mg/L+NAA0.1 mg/L+30 g/L of sugar+8 g/L of carrageenan.
Step 5) rooting induction: and (3) inoculating the obtained hippeastrum into a rooting culture medium which is MS+NAA0.1mg/L+30 g/L of sugar+8 g/L of carrageenan+0.5 g/L of activated carbon into the fibrous root of the secondary Miao Qiequ.
The stage is connected with 30 buds, the rooting condition is observed for 3 times, and the statistical rooting rate reaches 100% after 35 d.
Example 3
The difference from example 2 is that: the formula of the callus induction culture medium is as follows: MS+6-BA 4.0mg/L+TDZ 3.0mg/L+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8g/L.
The callus induction condition of the explant is observed, the death rate of the explant is counted to be 27% after 30d, the survival rate is 73%, and the induction rate is 67%.
Example 4
The difference from example 2 is that: the formula of the adventitious bud induction culture medium is as follows: MS+6-BA 1.5mg/L+TDZ 1.5mg/L+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8g/L.
And observing the induction condition of the adventitious buds, and counting the induction rate of the adventitious buds to 67% after 25 d.
Example 4
The difference from example 2 is that: the rooting culture medium comprises the following formula: MS+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8 g/L+activated carbon 1.5g/L.
And observing rooting condition, and counting that the rooting rate reaches 90% after 35 d.
Comparative example 1
The difference from example 2 is that: disinfecting with 0.1% mercuric chloride for 7min.
After the sterilization treatment, the same explants were inoculated for 30 times, 3 times, and after 10 days, the pollution rate of the statistical sample was 50%, the death rate was 20% and the survival rate was 30%.
Comparative example 2
The difference from example 2 is that: sterilizing with 0.1% mercuric chloride for 9min.
After the sterilization treatment, the same explants were inoculated 30 times, 3 times, and after 10 days, the contamination rate of the statistical sample was 20%, the death rate was 53%, and the survival rate was 27%.
Comparative example 3
The difference from example 2 is that: the formula of the callus induction culture medium is as follows: MS+6-BA3.0mg/L+TDZ 1.5mg/L+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8g/L.
The callus induction condition of the explant is observed, the death rate of the explant is counted to be 23% after 30d, the survival rate is 77%, and the induction rate is 40%.
Comparative example 4
The difference from example 2 is that: the formula of the callus induction culture medium is as follows: MS+6-BA3.0mg/L+TDZ 3.0mg/L+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8g/L.
The callus induction condition of the explant is observed, the death rate of the explant is 57% after 30d, the survival rate is 43%, and the induction rate is 27%.
Comparative example 5
The difference from example 2 is that: the formula of the adventitious bud induction culture medium is as follows: MS+6-BA 2.0mg/L+TDZ 0.5mg/L+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8g/L.
And observing the induction condition of the adventitious buds, and counting the induction rate of the adventitious buds to be 37% after 25 d.
Comparative example 6
The difference from example 2 is that: the formula of the adventitious bud induction culture medium is as follows: MS+6-BA 2.5mg/L+TDZ 1.5mg/L+NAA0.1 mg/L+sugar 30 g/L+carrageenan 8g/L.
And observing the induction condition of the adventitious buds, and counting the induction rate of the adventitious buds to be 40% after 25 d.
Comparative example 7
The difference from example 2 is that: the rooting culture medium comprises the following formula: MS+NAA0.05mg/L+30 g/L of sugar+8 g/L of carrageenan+0.5 g/L of activated carbon.
And observing rooting condition, and counting the rooting rate to 70% after 35 d.
Comparative example 8
The difference from example 2 is that: the rooting culture medium comprises the following formula: MS+NAA 0.15 mg/L+sugar 30 g/L+carrageenan 8 g/L+activated carbon 0.5g/L.
And observing rooting condition, and counting that the rooting rate reaches 89% after 35 d.
Comparative example 9
A tissue culture method for obtaining regenerated plants by taking vermilion bulbs as explants comprises the steps of disinfection, induction culture of the explants, cluster bud induction culture and rooting induction culture. The disinfection procedure for the explants was the same as in example 2 of the present invention. According to the measurement, the vermilion bulb is used as an explant for tissue culture, and the pollution rate of the explant after the disinfection treatment is 20%; mortality rate is 15%; the induction survival rate of the explant is 65%; the proliferation coefficient of the cluster buds reaches 3.0; the rooting rate reaches 100%. Compared with the technical scheme that the traditional bulb is used for tissue culture of the explant, the technical effect of the invention is close.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (9)
1. The tissue culture propagation method of the hippeastrum includes disinfection of the explant and callus induction culture of the disinfected explant, and is characterized in that the explant selects the hippeastrum receptacle; the callus induction culture is carried out in a callus culture medium, wherein the callus culture medium takes an MS culture medium as a basic culture medium, and 2.8-3.2mg/L of 6-BA, 2.0mg/L of TDZ and 0.05-0.15mg/L of NAA are added;
the method also comprises the step of adventitious bud induction culture after the callus induction culture; the adventitious bud induction culture is carried out in an adventitious bud induction culture medium, wherein the adventitious bud induction culture medium takes an MS culture medium as a basic culture medium, and 2.0mg/L of 6-BA, 1.0mg/L of TDZ and 0.05-0.15mg/L of NAA are added;
the explants selected for non-flowering flower holders.
2. The tissue culture propagation method of hippeastrum as claimed in claim 1, wherein the callus culture medium is further added with 28-32g/L sugar and 7-9g/L carrageenan.
3. The tissue culture propagation method of hippeastrum as claimed in claim 1, wherein the temperature of the callus induction culture is 24-26 ℃; and/or, the time of the callus induction culture is 25-40 days.
4. The tissue culture propagation method of hippeastrum according to claim 1, wherein the illumination time of the adventitious bud induction culture is 10-14 h/day, and the illumination intensity is 1600-1800lx; and/or the culture temperature of the adventitious bud induction culture is 24-26 ℃.
5. The tissue culture propagation method of hippeastrum as claimed in claim 4, further comprising rooting culture after the adventitious bud induction culture; the rooting culture is carried out in a rooting culture medium, wherein the rooting culture medium takes an MS culture medium as a basic culture medium, and NAA of 0.08-0.12mg/L and activated carbon of 0.2-1.5g/L are added.
6. Tissue culture propagation method of hippeastrum according to any one of claims 1 to 5, characterized in that the sterilized explant is cut into 1.5-2.5cm pieces before the callus induction culture.
7. The tissue culture propagation method of hippeastrum as claimed in claim 6, wherein the sterilization of the explant comprises a new Jieli sterilization, an ethanol sterilization and a mercuric chloride sterilization in this order.
8. A tissue culture propagation method of hippeastrum as claimed in claim 7, wherein the mercuric chloride disinfection is performed for 8min ± 20s using 0.1% mercuric chloride disinfection.
9. The tissue culture propagation method of hippeastrum as claimed in claim 8, wherein after the mercuric chloride is sterilized, the tissue culture propagation method is washed with sterile water for 2 to 4 times, and each washing time is 45 to 75 seconds.
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