CN117044627B - Tissue culture rapid propagation and in-vitro preservation method for alpine plant taraxacum - Google Patents
Tissue culture rapid propagation and in-vitro preservation method for alpine plant taraxacum Download PDFInfo
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- 238000004321 preservation Methods 0.000 title claims abstract description 49
- 238000000338 in vitro Methods 0.000 title claims abstract description 44
- 241000245665 Taraxacum Species 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 18
- 239000001963 growth medium Substances 0.000 claims abstract description 60
- 230000035755 proliferation Effects 0.000 claims abstract description 48
- 241000196324 Embryophyta Species 0.000 claims abstract description 38
- 230000006698 induction Effects 0.000 claims abstract description 30
- 230000035784 germination Effects 0.000 claims abstract description 29
- 238000012258 culturing Methods 0.000 claims abstract description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 3
- 229920001817 Agar Polymers 0.000 claims description 33
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 33
- 229930006000 Sucrose Natural products 0.000 claims description 33
- 239000008272 agar Substances 0.000 claims description 33
- 239000005720 sucrose Substances 0.000 claims description 33
- 238000005286 illumination Methods 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 238000002791 soaking Methods 0.000 claims description 22
- 239000008223 sterile water Substances 0.000 claims description 22
- 230000001954 sterilising effect Effects 0.000 claims description 15
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 8
- UJMBCXLDXJUMFB-GLCFPVLVSA-K tartrazine Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)C1=NN(C=2C=CC(=CC=2)S([O-])(=O)=O)C(=O)C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 UJMBCXLDXJUMFB-GLCFPVLVSA-K 0.000 abstract description 5
- 229960000943 tartrazine Drugs 0.000 abstract description 5
- 235000012756 tartrazine Nutrition 0.000 abstract description 5
- 239000004149 tartrazine Substances 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 3
- 230000018109 developmental process Effects 0.000 abstract description 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 description 18
- 244000292702 Rheum nobile Species 0.000 description 9
- 235000006385 Rheum nobile Nutrition 0.000 description 9
- 230000000694 effects Effects 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 241000219050 Polygonaceae Species 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- YJLUBHOZZTYQIP-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=N2 YJLUBHOZZTYQIP-UHFFFAOYSA-N 0.000 description 1
- CONKBQPVFMXDOV-QHCPKHFHSA-N 6-[(5S)-5-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-2-oxo-1,3-oxazolidin-3-yl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C[C@H]1CN(C(O1)=O)C1=CC2=C(NC(O2)=O)C=C1 CONKBQPVFMXDOV-QHCPKHFHSA-N 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000231392 Gymnosiphon Species 0.000 description 1
- 241000219061 Rheum Species 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 239000006870 ms-medium Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 229930194644 tatarine Natural products 0.000 description 1
- 238000012090 tissue culture technique Methods 0.000 description 1
- 230000009105 vegetative growth Effects 0.000 description 1
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Classifications
-
- 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
Abstract
The invention relates to a tissue culture rapid propagation and in-vitro preservation method of alpine plant taraxacum, belonging to the technical field of plant tissue culture, comprising the following steps: 1) Germination culture: taking the seeds of the yellow tower as explants, inoculating the explants on a germination culture medium after disinfection, and continuously culturing the germinated seeds to obtain sterile seedlings; 2) Proliferation culture: inoculating the sterile plantlet on a proliferation culture medium for cluster bud proliferation; 3) Rooting induction: dividing the obtained cluster buds into single plants, and inoculating the single plants to an induction culture medium for rooting induction; 4) And (5) in-vitro preservation: inoculating the rooted plants to an in vitro preservation culture medium for in vitro preservation. The method establishes a rapid propagation and in-vitro preservation system of tartrazine through germination of explant seeds, cluster bud proliferation, rooting induction and in-vitro preservation, and the subculture period reaches more than 18 months, thereby providing technical support for collection protection, introduction domestication, development and utilization of important wild plant resources in high mountain.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture rapid propagation and in-vitro preservation method of alpine plant taraxacum.
Background
Taraxacum (Rheum nobile) belonging to Rheum of Polygonaceae (Polygonaceae) is mainly distributed in the mountain and North-west of Tibetan Himalayan and Yunnan, and is grown on mountain and rocky beach and wet grasslands at an altitude of 4000-4800 m. The plant root shape stem and root are long and thick, the stem single body is upright, the basal leaves are rosette-shaped, the stem leaves and large leaves are round, nearly revolutionary, the upper part is provided with semitransparent yellow bracts which are wrapped and overlapped, and the plant is far away from looking like a golden pagoda, so the plant is named as a rare or endangered high-mountain flower with great ornamental value. Tahuang is an important medicinal plant resource in China, has wide application in folk, especially in Tibetan medicine, has the effects of purging heat, removing stagnation, removing stasis and relieving swelling, is mainly used for causing diarrhea and treating yellow water disease, has obvious curative effect, and has drastically reduced population quantity due to large-scale mining in recent years. Taraxacum can bloom after 5-7 years of vegetative growth, and after seeds are mature, the seeds die, the germination rate of the seeds under severe natural environment conditions is low, and population maintenance and updating are seriously affected. In addition, under the background of global warming, the suitable habitat of a large number of mountain plants is lost, the diversity of the mountain plants faces a great threat, and the adoption of necessary measures to protect the diversity of the mountain plants becomes an urgent subject to be placed in front of protecting biologists.
In vitro preservation of plants refers to preservation of tissues and organs (such as roots, stems, leaves, mature or immature embryos, etc.) of plants under artificial conditions by using tissue culture techniques, so that the plants are differentiated and then regrown into complete plants. The isolated culture technology can quickly and largely realize the cloning and rapid propagation of plant individuals, and provides a high-efficiency approach for the preservation of seed libraries, the preservation and rapid propagation of rare endangered species. The research related to the method for preserving the yellow-tower germplasm resources is developed, the collection of special species formed under the extreme environment of the mountain and the genetic information carried by the special species is promoted, the method has important significance for further deeply researching the plant adaptability evolution in the plateau area, and theoretical basis and technical support are provided for protecting the germplasm resources of mountain medicines and ornamental wild plants, introducing, domesticating and cultivating. At present, no report is made on the aspect of in vitro preservation related to the tissue culture technology of tartrazine.
Disclosure of Invention
In order to overcome the problems in the background technology, the invention provides a tissue culture rapid propagation and in-vitro preservation method of the alpine plant taraxacum, which establishes a rapid propagation and in-vitro preservation system of the taraxacum through germination of explant seeds, proliferation of cluster buds, rooting induction and in-vitro preservation, and the subculture period reaches more than 18 months, thereby providing technical support for collection protection, introduction domestication, development and utilization of important wild plant resources of the alpine plant.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
the tissue culture rapid propagation and in-vitro preservation method of the alpine plant taraxacum specifically comprises the following steps:
1) Germination culture: taking a yellow tower seed as an explant, sterilizing, inoculating to a germination culture medium, and continuously culturing after germination of the seed to obtain a sterile seedling, wherein the germination culture medium takes a 1/2MS culture medium as a basic culture medium, and further comprises NAA0.5mg/L, sucrose 20g/L and agar 6g/L, wherein the pH value is 5.8;
2) Proliferation culture: inoculating the sterile plantlet on a proliferation culture medium for cluster bud proliferation, wherein the proliferation culture medium takes an MS culture medium as a basic culture medium, and further comprises BA2.0mg/L, TDZ0.1mg/L, sucrose 30g/L and agar 6g/L, and the pH value is 5.8;
3) Rooting induction: dividing the obtained cluster buds into single plants, inoculating the single plants to an induction culture medium for rooting induction, wherein the induction culture medium takes a 1/2MS culture medium as a basic culture medium and also comprises NAA1.0mg/L, sucrose 20g/L and agar 6g/L, and the pH value is 5.8;
4) And (5) in-vitro preservation: inoculating the rooted plants to an in-vitro preservation culture medium for in-vitro preservation, wherein the in-vitro preservation culture medium comprises flower bud No. 1 and 2g/L, TDZ0.1mg/L, sucrose 20g/L and agar 6g/L, and the pH is 5.8.
Preferably, the sterilization in the step 1) comprises soaking seeds in sterile water for 48 hours, soaking the seeds in 75% ethanol for 20 seconds, sterilizing the seeds in 2% NaClO for 6 minutes, rinsing the seeds in sterile water for 5 to 6 times, and then placing the seeds on sterile filter paper for airing.
Preferably, the culture condition of the germination culture in the step 1) is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
Preferably, the in-vitro preservation condition in the step 4) is that the illumination intensity is 1600lx and the temperature is 20 ℃.
The invention has the beneficial effects that:
the invention provides a tissue culture rapid propagation and in-vitro preservation method of high mountain plant taraxacum, which takes taraxacum seeds as materials, sterilizes by explants, germinates and cultures to obtain sterile seedlings, and carries out cluster bud proliferation, rooting induction and in-vitro preservation on the sterile seedlings, thus establishing a rapid propagation and in-vitro preservation system of taraxacum, wherein the proliferation coefficient is up to 20.17 after 3 times of subculture, and the in-vitro preservation subculture period can be up to more than 18 months. The method effectively solves the problem of tissue culture and rapid propagation of the tartrazine, greatly prolongs the in-vitro preservation period, and provides technical support for collection protection, introduction domestication, development and utilization of important wild plant resources in the mountain.
Drawings
FIG. 1 is a tissue culture and ex vivo preservation process of Tatarian in accordance with the present invention;
in the figure, a represents a tartrazine seed, b represents a sterile seedling obtained by germination culture, c represents a cluster bud proliferation condition, d represents a rooting induction condition, and e represents in-vitro preservation.
Description of the embodiments
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, so as to facilitate understanding of the skilled person.
The invention provides a tissue culture rapid propagation and in-vitro preservation method of alpine plant taraxacum, which specifically comprises the following steps:
1) Germination culture: taking the yellow seeds as explants, soaking the seeds in sterile water for 48h, then soaking the seeds in 75% ethanol for 20s, sterilizing the seeds in 2% NaClO for 6min, rinsing the seeds in sterile water for 5 to 6 times, placing the seeds on sterile filter paper, airing the seeds, then inoculating the seeds on a germination culture medium, and continuously culturing the seeds after germination to obtain sterile seedlings, wherein the germination culture medium takes a 1/2MS culture medium as a basic culture medium and also comprises NAA0.5mg/L, sucrose 20g/L and agar 6g/L, and the pH value is 5.8; the germination culture condition is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
2) Proliferation culture: sterile seedlings are inoculated on a proliferation medium for cluster bud proliferation, wherein the proliferation medium takes an MS medium as a basic medium, and further comprises BA2.0mg/L, TDZ0.1mg/L, sucrose 30g/L and agar 6g/L, and the pH value is 5.8.
3) Rooting induction: dividing the obtained cluster buds into single plants, inoculating the single plants to an induction culture medium for rooting induction, wherein the induction culture medium takes a 1/2MS culture medium as a basic culture medium, and further comprises NAA1.0mg/L, sucrose 20g/L and agar 6g/L, and the pH value is 5.8.
4) And (5) in-vitro preservation: inoculating the rooted plants to an in-vitro preservation culture medium for in-vitro preservation, wherein the in-vitro preservation culture medium comprises flower treasures No. 1 and No. 2g/L, TDZ0.1mg/L, sucrose 20g/L and agar 6g/L, the pH is 5.8, and the in-vitro preservation condition is that the illumination intensity is 1600lx and the temperature is 20 ℃.
Examples
Taraxacum (Rheum nobile) seeds were used as material and were harvested from Yunnan and northwest in 2021, 9 months. Firstly, soaking seeds in sterile water for 48h, then soaking the seeds in 75% ethanol for 20s, sterilizing the seeds with 2% NaClO for 6min, rinsing the seeds with sterile water for 5-6 times, placing the seeds on sterile filter paper for airing, inoculating the seeds on a germination culture medium (1/2MS+NAA0.5mg/L+sucrose 20 g/L+agar 6g/L, pH is 5.8), germinating the seeds after 5-7d, and continuously culturing the seeds to obtain sterile seedlings, wherein the culture condition is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
Taking the whole aseptic seedling from which the root is excised as a material, inoculating the aseptic seedling on a proliferation culture medium (MS+BA2.0mg/L+TDZ0.1 mg/L+30 g/L of sucrose+6 g/L of agar, and pH of 5.8) for carrying out cluster bud proliferation, wherein the proliferation coefficient is up to 20.17 after the subculture period is 30d and the subculture is carried out for three times.
Dividing the obtained cluster buds into single plants, inoculating the single plants to an induction culture medium (1/2MS+NAA 1.0 mg/l+sucrose 20 g/l+agar 6g/L, pH of 5.8) for rooting induction, and counting the rooting rate after 30 days to 87.26%, wherein the number of the rooting rate is 30, and the root length is 3.53cm.
Inoculating the rooted plant to an in vitro preservation culture medium (Huabao No. 1, 2g/L+TDZ0.1 mg/L+sucrose, 20 g/L+agar, 6g/L and pH of 5.8) for in vitro preservation, wherein the preservation condition is that the illumination intensity is 1600lx, the temperature is 20 ℃, and the subculture period can reach more than 18 months.
Taraxacum (Rheum nobile) seeds were used as material and were harvested from Yunnan and northwest in 2021, 9 months. Firstly, soaking seeds in sterile water for 48h, then soaking the seeds in 75% ethanol for 20s, sterilizing the seeds with 2% NaClO for 6min, rinsing the seeds with sterile water for 5-6 times, placing the seeds on sterile filter paper for airing, inoculating the seeds on a germination culture medium (1/2MS+NAA0.5mg/L+sucrose 20 g/L+agar 6g/L, pH is 5.8), germinating the seeds after 5-7d, and continuously culturing the seeds to obtain sterile seedlings, wherein the culture condition is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
The leaf stalks of the aseptic seedlings are used as materials, inoculated on a proliferation culture medium (MS+BA2.0mg/L+TDZ0.1 mg/L+30 g/L of sucrose+6 g/L of agar and pH of 5.8) for callus induction, and subcultured for 90d to fail to differentiate adventitious buds.
Taraxacum (Rheum nobile) seeds were used as material and were harvested from Yunnan and northwest in 2021, 9 months. Firstly, soaking seeds in sterile water for 48h, then soaking the seeds in 75% ethanol for 20s, sterilizing the seeds with 2% NaClO for 6min, rinsing the seeds with sterile water for 5-6 times, placing the seeds on sterile filter paper for airing, inoculating the seeds on a germination culture medium (1/2MS+NAA0.5mg/L+sucrose 20 g/L+agar 6g/L, pH is 5.8), germinating the seeds after 5-7d, and continuously culturing the seeds to obtain sterile seedlings, wherein the culture condition is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
Leaves of sterile seedlings are used as materials, and inoculated on proliferation culture medium (MS+BA2.0mg/L+TDZ0.1 mg/L+30 g/L of sucrose+6 g/L of agar and pH of 5.8) for callus induction, and subculture is carried out for 90 days to fail to differentiate adventitious buds.
Examples
Taraxacum (Rheum nobile) seeds were used as material and were harvested from Yunnan and northwest in 2021, 9 months. Firstly, soaking seeds in sterile water for 48h, then soaking the seeds in 75% ethanol for 20s, sterilizing the seeds with 2% NaClO for 6min, rinsing the seeds with sterile water for 5-6 times, placing the seeds on sterile filter paper for airing, inoculating the seeds on a germination culture medium (1/2MS+NAA0.5mg/L+sucrose 20 g/L+agar 6g/L, pH is 5.8), germinating the seeds after 5-7d, and continuously culturing the seeds to obtain sterile seedlings, wherein the culture condition is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
The whole aseptic seedling from which the root is excised is used as a material, inoculated on a proliferation culture medium (MS+BA1.0mg/L+TDZ0.1 mg/L+30 g/L of sucrose+6 g/L of agar, pH is 5.8) for cluster bud proliferation, a subculture period is 30d, and the proliferation coefficient after three subcultures is 9.67.
Examples
Taraxacum (Rheum nobile) seeds were used as material and were harvested from Yunnan and northwest in 2021, 9 months. Firstly, soaking seeds in sterile water for 48h, then soaking the seeds in 75% ethanol for 20s, sterilizing the seeds with 2% NaClO for 6min, rinsing the seeds with sterile water for 5-6 times, placing the seeds on sterile filter paper for airing, inoculating the seeds on a germination culture medium (1/2MS+NAA0.5mg/L+sucrose 20 g/L+agar 6g/L, pH is 5.8), germinating the seeds after 5-7d, and continuously culturing the seeds to obtain sterile seedlings, wherein the culture condition is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
Taking the whole aseptic seedling from which the root is excised as a material, inoculating the aseptic seedling on a proliferation culture medium (MS+BA1.0mg/L+TDZ0.1 mg/L+30 g/L of sucrose+6 g/L of agar, pH of 5.8) for carrying out cluster bud proliferation, wherein the proliferation coefficient is 15.06 after the subculture for 30d and the subculture for three times.
Examples
Taraxacum (Rheum nobile) seeds were used as material and were harvested from Yunnan and northwest in 2021, 9 months. Firstly, soaking seeds in sterile water for 48h, then soaking the seeds in 75% ethanol for 20s, sterilizing the seeds with 2% NaClO for 6min, rinsing the seeds with sterile water for 5-6 times, placing the seeds on sterile filter paper for airing, inoculating the seeds on a germination culture medium (1/2MS+NAA0.5mg/L+sucrose 20 g/L+agar 6g/L, pH is 5.8), germinating the seeds after 5-7d, and continuously culturing the seeds to obtain sterile seedlings, wherein the culture condition is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
The whole aseptic seedling from which the root is excised is used as a material, inoculated on a proliferation culture medium (MS+BA3.0mg/L+TDZ0.1 mg/L+30 g/L of sucrose+6 g/L of agar, pH of 5.8) for cluster bud proliferation, a subculture period of 30d and a proliferation coefficient of 9.67 after three times of subculture.
Examples
Taraxacum (Rheum nobile) seeds were used as material and were harvested from Yunnan and northwest in 2021, 9 months. Firstly, soaking seeds in sterile water for 48h, then soaking the seeds in 75% ethanol for 20s, sterilizing the seeds with 2% NaClO for 6min, rinsing the seeds with sterile water for 5-6 times, placing the seeds on sterile filter paper for airing, inoculating the seeds on a germination culture medium (1/2MS+NAA0.5mg/L+sucrose 20 g/L+agar 6g/L, pH is 5.8), germinating the seeds after 5-7d, and continuously culturing the seeds to obtain sterile seedlings, wherein the culture condition is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
Taking the whole aseptic seedling from which the root is excised as a material, inoculating the aseptic seedling on a proliferation culture medium (MS+BA2.0mg/L+TDZ0.1 mg/L+30 g/L of sucrose+6 g/L of agar, and pH of 5.8) for carrying out cluster bud proliferation, wherein the proliferation coefficient is up to 20.17 after the subculture period is 30d and the subculture is carried out for three times.
Dividing the obtained cluster buds into single plants, inoculating the single plants to an induction culture medium (1/2MS+IBA 1.0 mg/L+sucrose 20 g/L+agar 6g/L, pH of 5.8) for rooting induction, and counting the rooting rate after 30 days to 88.10%, wherein the rooting rate reaches 21.67, and the root length is 1.53cm.
Examples
Taraxacum (Rheum nobile) seeds were used as material and were harvested from Yunnan and northwest in 2021, 9 months. Firstly, soaking seeds in sterile water for 48h, then soaking the seeds in 75% ethanol for 20s, sterilizing the seeds with 2% NaClO for 6min, rinsing the seeds with sterile water for 5-6 times, placing the seeds on sterile filter paper for airing, inoculating the seeds on a germination culture medium (1/2MS+NAA0.5mg/L+sucrose 20 g/L+agar 6g/L, pH is 5.8), germinating the seeds after 5-7d, and continuously culturing the seeds to obtain sterile seedlings, wherein the culture condition is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
Taking the whole aseptic seedling from which the root is excised as a material, inoculating the aseptic seedling on a proliferation culture medium (MS+BA2.0mg/L+TDZ0.1 mg/L+30 g/L of sucrose+6 g/L of agar, and pH of 5.8) for carrying out cluster bud proliferation, wherein the proliferation coefficient is up to 20.17 after the subculture period is 30d and the subculture is carried out for three times.
Dividing the obtained cluster buds into single plants, inoculating the single plants to an induction culture medium (1/2MS+IAA 1.0 mg/L+sucrose 20 g/L+agar 6 g/L) for rooting induction, and counting that the rooting rate is 78.16% after 30 days, the number of the rooting rate reaches 27, and the root length is 2.4cm.
TABLE 1 Effect of different proliferation Medium on proliferation of Tower Huang Congya
| Treatment of | Proliferation coefficient | |
| Example 2 | MS+BA0.5+TDZ0.1 | 9.67±1.20 C |
| Example 3 | MS+BA1.0+TDZ0.1 | 15.06±2.20 B |
| Example 1 | MS+BA2.0+TDZ0.1 | 20.17±2.02 A |
| Example 4 | MS+BA3.0+TDZ0.1 | 9.67±0.88 C |
Note that: the different lowercase letters indicate that the same column between treatments was significantly different at a level of 0.05, and the uppercase letters indicate that the difference at a level of 0.01 was extremely significant, as follows.
As can be seen from Table 1, the proliferation media used in examples 1-4 all had the proliferation of the multiple shoots, but the proliferation coefficients of examples 2-4 were all significantly lower than that of example 1, with the best proliferation effect of the multiple shoots of example 1, and the proliferation coefficient was as high as 20.17.
TABLE 2 Effect of different Induction Medium on Tatarine rooting
| Treatment of | Rooting percentage (%) | Root number (strip) | Root length (cm) | |
| Example 1 | 1/2MS+NAA1.0 | 87.26±1.70 A | 30.00±2.00 Aa | 3.53±0.15 A |
| Example 5 | 1/2MS+IBA1.0 | 88.10±2.07 A | 21.67±2.52 Bb | 1.53±0.42 C |
| Example 6 | 1/2MS+IAA1.0 | 78.16±3.98 B | 27.00±1.00 AaB | 2.40±0.10 B |
As can be seen from Table 2, the rooting rate of examples 5-6 is not significantly different from that of example 1, the rooting number and root length are extremely lower than those of example 1, therefore, rooting induction is carried out on an induction medium (1/2MS+NAA 1.0 mg/l+sucrose 20 g/L+agar 6g/L, pH is 5.8), the rooting rate can reach 87.26%, the rooting number is 30, the root length is 3.53cm, the plant grows robustly, the tissue culture rapid propagation of tartrazine is facilitated, and a large amount of materials can be provided for in-vitro preservation in the later period.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (4)
1. A tissue culture rapid propagation and in vitro preservation method of alpine plant taraxacum is characterized in that: the method specifically comprises the following steps:
1) Germination culture: taking a yellow tower seed as an explant, sterilizing, inoculating to a germination culture medium, and continuously culturing after germination of the seed to obtain a sterile seedling, wherein the germination culture medium takes a 1/2MS culture medium as a basic culture medium, and further comprises NAA0.5mg/L, sucrose 20g/L and agar 6g/L, wherein the pH value is 5.8;
2) Proliferation culture: inoculating the whole aseptic seedling with the excised root on a proliferation medium for cluster bud proliferation, wherein the proliferation medium takes an MS culture medium as a basic culture medium and also comprises BA2.0mg/L, TDZ0.1mg/L, sucrose 30g/L and agar 6g/L, and the pH value is 5.8;
3) Rooting induction: dividing the obtained cluster buds into single plants, inoculating the single plants to an induction culture medium for rooting induction, wherein the induction culture medium takes a 1/2MS culture medium as a basic culture medium and also comprises NAA1.0mg/L, sucrose 20g/L and agar 6g/L, and the pH value is 5.8;
4) And (5) in-vitro preservation: inoculating the rooted plants to an in-vitro preservation culture medium for in-vitro preservation, wherein the in-vitro preservation culture medium comprises flower bud No. 1 and 2g/L, TDZ0.1mg/L, sucrose 20g/L and agar 6g/L, and the pH is 5.8.
2. The tissue culture rapid propagation and in vitro preservation method of alpine plant taraxacum according to claim 1, which is characterized in that: the sterilization in the step 1) comprises soaking seeds in sterile water for 48h, soaking the seeds in 75% ethanol for 20s, sterilizing the seeds in 2% NaClO for 6min, rinsing the seeds in sterile water for 5-6 times, and then placing the seeds on sterile filter paper for airing.
3. The tissue culture rapid propagation and in vitro preservation method of alpine plant taraxacum according to claim 1, which is characterized in that: the culture condition of the germination culture in the step 1) is that the illumination intensity is 1600lx, the illumination time is 12h/d, and the temperature is 23+/-2 ℃.
4. The tissue culture rapid propagation and in vitro preservation method of alpine plant taraxacum according to claim 1, which is characterized in that: the in-vitro preservation condition in the step 4) is that the illumination intensity is 1600lx and the temperature is 20 ℃.
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| AU2001280748A1 (en) * | 2000-07-20 | 2002-02-05 | Large Scale Biology Corporation | Methods of creating dwarf phenotypes in plants |
| SE0400996D0 (en) * | 2004-04-16 | 2004-04-16 | Alltech Ireland Inc | Use of plants, plant extracts and nature-identical components from plants to affect rumen fermentation and to improve the energy and protein retention of ruminants |
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| TW201313742A (en) * | 2005-12-20 | 2013-04-01 | 必治妥美雅史谷比公司 | Compositions and methods for producing a composition |
| CN101213941A (en) * | 2008-01-18 | 2008-07-09 | 中国科学院昆明植物研究所 | Fast replication and in-vitro conservation method for dendrobium |
| EP2806743A1 (en) * | 2012-01-24 | 2014-12-03 | Hochschule Anhalt | Antifungal formulations for combatting plant diseases |
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| CN109548606A (en) * | 2018-12-12 | 2019-04-02 | 中国科学院昆明植物研究所 | A kind of Wild population restoration methods of tower Huang |
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