CN111194694A - Tissue culture rapid propagation method of new plant variety chat toona sinensis - Google Patents
Tissue culture rapid propagation method of new plant variety chat toona sinensis Download PDFInfo
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- CN111194694A CN111194694A CN202010143018.2A CN202010143018A CN111194694A CN 111194694 A CN111194694 A CN 111194694A CN 202010143018 A CN202010143018 A CN 202010143018A CN 111194694 A CN111194694 A CN 111194694A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The application discloses a tissue culture and rapid propagation method of a new plant species chat with toona sinensis, which comprises the following steps: taking materials of explants, disinfecting and inoculating; dedifferentiation and callus culture; redifferentiation and culture of buds and roots; hardening and planting tissue culture seedlings; aiming at the raw material composition and the optimal proportion of the culture medium in each stage of the tissue culture of the new variety of the ailanthus altissima, the method can meet the requirement of quick propagation of the ailanthus altissima, and has the advantages of easy production, low cost, easy operation, large propagation coefficient and the like.
Description
Technical Field
The invention relates to the technical field of tissue rapid propagation of plants, in particular to a tissue culture rapid propagation method of a new plant variety of Chinese toon.
Background
The chat toon is a new variety of the toona sinensis with the red fruit, which is cultivated for many years from a bud mutation strain by a subject group of chat universities, has extremely high ornamental value and has been approved to protect new varieties (the certificate number is 20170268).
The toona sinensis belongs to one of ailanthus varieties, and the Zhang-Shuiyang of Linke department in Hebei province reports that the native sansheng toona sinensis is carried in Hebei Pingshan, but the winged fruit of the toona sinensis gradually develops color after 30 days of development (Zhang-Shuiyang, 1988), and the Yao-De-Sheng and the like find a toona sinensis in the pit and the ditch of the fine county of Qin' an county in Gansu province, wherein the leaves and the winged fruit of the toona sinensis are smaller than the toona sinensis (Yao-De-Sheng, 2011). The collected toona sinensis is subjected to grafting tests by Wanghongming and the like, and the pterocarpus sinensis red inheritance can be well maintained (Wanghongming, 2009). The subject group of chat universities discovers a single-bud variant of ailanthus altissima in Shandong chat, the fruit pod is bright red, the grafted plants are isolated and planted for multiple generations to obtain offspring with stable fruit color, gene polymorphism analysis shows that the ailanthus altissima belongs to the variety of ailanthus altissima, the ailanthus altissima expresses anthocyanin from the flowering phase, anther and petals are dark red, mature samara fruits are bright red, fruit stems are deep red, the best period of viewing fruits is from the last ten days of 5 months to the last ten days of 7 months, and the samara fruits turn into red brown after being mature for 8 months (flowers, 2015). And researches on morphological characteristics, a material season, seed germination and seedling growth, fruit color differentiation of offspring of seed reproduction, variety difference analysis of the chat toona sinensis and the common ailanthus altissima are carried out, and a stage result (Xuhua, 2015) is obtained. In view of the ornamental effect that the red fruit toona pterocarya fruits are brilliant like the clouds, the red fruit toona pterocarya fruits can be used as street trees and shade trees for greening in roads, scenic spots, parks, communities, units and the like, can be planted in rows, plants and plants, have wide application prospect and are worthy of being popularized and planted vigorously. Tissue culture is the most important way for fast and standardized seedling culture, can maintain the shape of a parent, greatly improve the breeding coefficient and has great effect on fast popularization of new species, so that systematic research on the tissue culture technology is carried out and the tissue culture technology is successful.
Disclosure of Invention
The invention aims to find the raw material composition and the optimal proportion of a culture medium of each stage of tissue culture of new variety of ailanthus altissima, and establish a tissue culture rapid propagation technical system of the ailanthus altissima, such as explant preparation, disinfection and inoculation, dedifferentiation and callus culture, bud and root redifferentiation and culture, hardening and field planting of tissue culture seedlings and the like.
In order to achieve the purpose, the invention adopts the technical scheme that:
1. preparing a culture medium:
the culture medium comprises ① 50 × macroelement mother liquor (containing ammonium nitrate, potassium dihydrogen phosphate and magnesium sulfate heptahydrate), ② 100 × calcium chloride, ③ 100 × metal elements (containing ferrous sulfate heptahydrate and disodium ethylene diamine tetraacetate), ④ 100 × microelements (containing potassium iodide, boric acid, manganese sulfate tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate and cobalt chloride hexahydrate), ⑤ 1000 × vitamins (containing thiamine, pyridoxine, pantothenic acid and glycine) ⑥ 100 × inositol 100 × ⑦ hormones selected from naphthylacetic acid (NAA) 0.1mg/ml, 2.4-dichlorophenoxyacetic acid (2.4-D) 0.1mg/ml, 6-benzylaminopurine (6-BA) 0.1mg/ml, indolebutyric acid (IBA) 1mg/ml, ⑧ sucrose and ⑨ agar.
The raw materials of the culture medium in the dedifferentiation stage, the germination guide stage, the rooting stage and other stages are partially changed.
First, the materials used for the dedifferentiation-inducing medium include (1L as an example): 20g of cane sugar, 20-24g of agar powder, 20ml of macroelement, 10ml of calcium chloride, 10ml of metal element, 10ml of microelement, 1ml of vitamin, 10ml of inositol, 0.1mol/L of naphthylacetic acid (NAA), 0.1mol/L of 2.4-dichlorophenoxyacetic acid (2.4-D) and 0.1mol/L of 6-benzylaminopurine (6-BA).
Further, the culture medium raw materials for subculture include (1L as an example): 20g of cane sugar, 20-24g of agar powder, 20ml of macroelement, 10ml of calcium chloride, 10ml of metal element, 10ml of microelement, 1ml of vitamin, 10ml of inositol, 0.1mol/L of naphthylacetic acid (NAA), 0.1mol/L of 2.4-dichlorophenoxyacetic acid (2.4-D) and 0.2mol/L of 6-benzylaminopurine (6-BA).
Further, the culture medium raw materials for callus germination include (1L as an example): 20g of cane sugar, 20-24g of agar powder, 20ml of macroelement, 10ml of calcium chloride, 10ml of metal element, 10ml of microelement, 1ml of vitamin, 10ml of inositol, 0.1mol/L of naphthylacetic acid (NAA), 0.1mol/L of 2.4-dichlorophenoxyacetic acid (2.4-D) and 3mol/L of 6-benzylaminopurine (6-BA).
Further, callus rooting culture medium
The raw materials comprise (taking 1L as an example): 20g of cane sugar, 20-24g of agar powder, 10ml of macroelements, 5ml of calcium chloride, 5ml of metal elements, 5ml of trace elements, 0.5ml of vitamins, 5ml of inositol and 1.0mg/L of indolebutyric acid (IBA).
Further, the preparation method of the culture medium is as follows: preparing six mother solutions respectively according to the components, wherein the six mother solutions comprise macroelements, calcium chloride, trace elements, metal elements, inositol and vitamins, wherein the macroelements are added in sequence after being dissolved in different containers, and the other mother solutions are dissolved in the same container in sequence. Preparing needed hormone, wherein the concentration of 6-BA is 0.1mg/ml, and a small amount of 1mol/L sodium hydroxide solution is needed for assisting dissolution and then the volume is fixed to 100 ml; the concentration of NAA and 2.4-D is 0.1mg/ml, and the volume is fixed to 100ml after a small amount of ethanol is used for assisting dissolution; IBA concentration is 1mg/ml, and the volume is fixed to 100ml after a small amount of 1mol/L sodium hydroxide solution is used for assisting dissolution. Adding each nutrient mother solution according to the formula of the culture medium to be prepared, stirring uniformly, subpackaging in bottles, adding required hormone according to the amount of the solution, adjusting the optimum pH of the culture medium to 6, adding agar powder according to the amount of the solution, sterilizing by high-pressure steam, and cooling for later use.
2. Material taking: taking axillary buds and terminal buds of lateral branches of the Chinese toon as explants, placing the explants in a small beaker, slightly peeling off outer-layer scales of the axillary buds and the terminal buds by hands until light green tissues are exposed, repeatedly washing the axillary buds and the terminal buds for 5-6 times by using sterile water, and taking out the axillary buds and the terminal buds for later use.
3. And (3) disinfection: putting the used instruments into a super-clean workbench for sterilization for 2 hours in advance, taking out the ailanthus axillary buds or terminal buds from the super-clean workbench, soaking the ailanthus axillary buds or terminal buds in 75% alcohol for 30 seconds, taking out the ailanthus axillary buds, placing the ailanthus axillary buds into 15% sodium hypochlorite solution for soaking for 10-15 min, putting the ailanthus axillary buds into 0.1% mercury bichloride solution for disinfection for 3-5 min, taking out the ailanthus axillary buds, and repeatedly washing the ailanthus axillary buds with sterile water for 7-8 times.
4. Inoculation: the material was inoculated into MS medium in a clean bench, taking care to embed the sterilized axillary bud tissue into the medium.
5. Culturing: the temperature of the dedifferentiation and subculture conditions is 26 ℃, and no light is emitted; then the temperature in the process of differentiating germination and rooting is 26 ℃, and the illumination time is 16h and 8 h.
6. Hardening and planting: transferring the rooted tissue culture seedlings to an ordinary greenhouse outside an artificial climate chamber, closing the bottle, hardening the seedlings for about 10 days (the shading degree is 50% -70%), and then opening the bottle mouth for 3-7 days; then taking out the test-tube plantlet, washing, transplanting the test-tube plantlet into a sterilized substrate in a seedling culture plug tray, wherein the substrate is prepared from the following components in percentage by weight: 1, spraying water to pour the mixture flat, putting the mixture into a domestication room, culturing to about 30 centimeters, and planting in a field. The invention has the advantages that:
(1) the tissue culture technology of a new forest species 'chatting Toonae sinensis' (certificate number is 20170268) is explored for the first time, the establishment of the technical system can meet the demand of 'chatting Toonae sinensis' rapid propagation, and the tissue culture technology has the advantages of easiness in production, low cost, easiness in operation, large propagation coefficient and the like.
(2) The addition proportion of the culture medium hormone is a special formula suitable for the variety and the specific material, the failure of dedifferentiation and redifferentiation test links is easily caused by insufficient or excessive hormone, and the addition proportion of the hormone only has reference value for other plant varieties or other tissue materials of the variety of the Chinese toon.
(3) The axillary buds are used as the explant for the tissue culture of the toona sinensis, the tissue culture is an innovation of the project, compared with other plant tissues such as leaf discs, stem segments, fruit pods and the like, the tissue culture has the highest dedifferentiation success rate, the materials can be obtained in the dormant season except the growth season, and the development of scientific research and production can be free from the limitation of the growth season.
(4) The test material is applicable to the technologies of explant preparation, disinfection and inoculation, dedifferentiation and callus culture, bud and root redifferentiation and regeneration seedling culture, hardening and planting of tissue culture seedlings and the like, and has reference value only for other materials.
Drawings
FIG. 1 shows callus of Toona sinensis;
FIG. 2 shows the formation of tissue culture shoots of Toona sinensis;
FIG. 3 is a comparison graph of chat about Ailanthus altissima with common Ailanthus altissima;
FIG. 4 is a comparison of Chun toon and common Ailanthus altissima sambucus samara.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1:
a tissue culture and rapid propagation method of a new plant species chat toona sinensis comprises the following steps:
(1) material taking: placing axillary buds and terminal buds of lateral branches of toona sinensis in a small beaker, and slightly peeling off scales of the axillary buds and the terminal buds by hands until light green tissues are exposed; repeatedly washing with sterile water for 5-6 times, and fishing out for later use;
(2) and (3) disinfection: putting the used instruments into a super-clean workbench for sterilization for 2 hours in advance, taking out the axillary buds or terminal buds of the ailanthus altissima in the super-clean workbench, soaking in 75% alcohol for 30s, taking out, placing in 15% sodium hypochlorite solution for soaking for 10-15 min, putting in 0.1% mercuric chloride solution for disinfection for 3-5 min, taking out, and repeatedly washing with sterile water for 7-8 times;
(3) inoculation: inoculating the material to an MS culture medium in a clean bench, and burying the sterilized axillary bud tissue into the culture medium;
(4) culturing: the conditions of dedifferentiation and callus subculture are 26 ℃ and no light is emitted; the temperature of the germination and rooting stages in the redifferentiation process is 26 ℃, and the illumination time is 16h and 8 h;
(5) hardening and planting: transferring rooted chat toona sinensis tissue culture seedlings to an ordinary greenhouse outside an artificial climate room, closing bottles, hardening seedlings for about 10 days (the shading degree is 50% -70%), and then opening the bottle mouth for 3-7 days; finally, taking out the test-tube plantlet, cleaning and transplanting the test-tube plantlet into a sterilized substrate in a seedling culture plug tray, wherein the substrate is prepared from the following components in percentage by weight: 1, preparing turfy soil and vermiculite, spraying water to level, putting into a domestication room, culturing to 30 centimeters, and planting in a field.
Wherein the culture medium comprises ① 50 × macroelement mother liquor (containing ammonium nitrate, potassium dihydrogen phosphate and magnesium sulfate heptahydrate), ② 100 × calcium chloride, ③ 100 × metal elements (containing ferrous sulfate heptahydrate and disodium ethylene diamine tetraacetate), ④ 100 × microelements (containing potassium iodide, boric acid, manganese sulfate tetrahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, copper sulfate pentahydrate and cobalt chloride hexahydrate), ⑤ 1000 × vitamins (containing thiamine, pyridoxine, pantothenic acid and glycine) ⑥ 100 × inositol 100 × ⑦ hormones selected from naphthylacetic acid (NAA) 0.1mg/ml, 2.4-dichlorophenoxyacetic acid (2.4-D) 0.1mg/ml, 6-benzylaminopurine (6-BA) 0.1mg/ml, indolebutyric acid (IBA) 1mg/ml, ⑧ sucrose and ⑨ agar.
The six mother solutions are prepared respectively: macroelements, calcium chloride, trace elements, metal elements, inositol and vitamins. Wherein, the components of the macroelements need to be dissolved in different containers and then added in sequence, and other mother liquor is dissolved in the same container in sequence; preparing required hormone, wherein 6-BA is 0.1mg/ml, adding to 100ml after dissolving with a small amount of 1mol/L sodium hydroxide solution, NAA and 2.4-D are 0.1mg/ml, adding to 100ml after dissolving with a small amount of ethanol; adding each nutrient mother solution according to the formula of a culture medium to be prepared, stirring uniformly, subpackaging into bottles (50 ml), adding required hormone according to the solution amount, taking 50 microliters of mother solution from NAA and 2.4-D in each bottle, taking 50 microliters of mother solution from 6-BA, adjusting the pH value to 6, adding 1.0g-1.2g of agar powder into each bottle according to the solution amount, sterilizing at high pressure, cooling and solidifying for later use.
Example 2:
different from example 1, the raw materials added to the culture medium for inducing dedifferentiation (1L as an example): 20g of cane sugar, 20-24g of agar powder, 20ml of macroelement mother liquor, 10ml of calcium chloride mother liquor, 10ml of metal element mother liquor, 10ml of microelement mother liquor, 1ml of vitamin mother liquor, 10ml of inositol, 0.1mol/L of naphthylacetic acid final concentration (NAA), 0.1mol/L of 2.4-dichlorophenoxyacetic acid (2.4-D) and 0.1mol/L of 6-benzylaminopurine (6-BA), and the preparation method is the same as that of example 1.
Example 3:
in contrast to example 1, the culture medium raw materials for subculture (1L as an example): comprises 20g of cane sugar, 20 to 24g of agar powder, 20ml of macroelement mother liquor, 10ml of calcium chloride mother liquor, 10ml of metal element mother liquor, 10ml of microelement mother liquor, 1ml of vitamin mother liquor, 10ml of inositol mother liquor, 0.1mol/L of naphthylacetic acid (NAA), 0.1mol/L of 2.4-dichlorophenoxyacetic acid (2.4-D) and 0.2mol/L of 6-benzylaminopurine (6-BA), and the preparation method is the same as that of the embodiment 1.
Example 4:
the culture medium for callus germination consists of the following raw materials (1L is taken as an example): 20g of cane sugar, 20-24g of agar powder, 20ml of macroelement mother liquor, 10ml of calcium chloride mother liquor, 10ml of metal element mother liquor, 10ml of trace element mother liquor, 1ml of vitamin mother liquor, 10ml of inositol mother liquor, 0.1mol/L of naphthylacetic acid (NAA), 0.1mol/L of 2.4-dichlorophenoxyacetic acid (2.4-D) and 3mol/L of 6-benzylaminopurine (6-BA). The preparation method is the same as example 1.
Example 5:
the raw materials of the culture medium for test-tube plantlet rooting (taking 1L as an example): 20g of cane sugar, 20-24g of agar powder, 10ml of macroelement mother liquor, 5ml of calcium chloride mother liquor, 5ml of metal element mother liquor, 5ml of trace element mother liquor, 0.5ml of vitamin mother liquor, 5ml of inositol mother liquor and 1.0mg/L of indolebutyric acid (IBA), and the preparation method is the same as that of example 1.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (5)
1. A tissue culture and rapid propagation method of a new plant species chat toona sinensis is characterized in that: the method comprises the following steps: taking materials of explants, disinfecting and inoculating; dedifferentiation and callus culture; redifferentiation and culture of buds and roots; hardening and planting tissue culture seedlings; the method specifically comprises the following steps: (1) material taking: taking axillary buds and terminal buds of lateral branches of the Chinese toon, placing the axillary buds and the terminal buds in a small beaker, and slightly peeling off a plurality of layers of scales of the axillary buds and the terminal buds by hands until light green tissues are exposed; repeatedly washing with sterile water for 5-6 times, and fishing out for later use;
(2) and (3) disinfection: putting the used instruments into a super-clean workbench for sterilization for 2 hours in advance, taking out the axillary buds or terminal buds of the ailanthus altissima in the super-clean workbench, soaking in 75% alcohol for 30s, taking out, placing in 15% sodium hypochlorite solution for soaking for 10-15 min, putting in 0.1% mercuric chloride solution for disinfection for 3-5 min, taking out, and repeatedly washing with sterile water for 7-8 times;
(3) inoculation: inoculating the material to an MS culture medium in a clean bench, and burying the sterilized axillary bud tissue into the culture medium;
(4) culturing: the conditions of dedifferentiation and callus subculture are 26 ℃ and no light is emitted; the temperature of the germination and rooting stages in the redifferentiation process is 26 ℃, and the illumination time is 16h and 8 h;
(5) hardening and planting: transferring the rooted Toona sinensis chat tissue culture seedlings to an ordinary greenhouse outside an artificial climate room, closing the bottle, hardening the seedlings for about 10 days (the shading degree is 50% -70%), and then opening the bottle mouth for 3-7 days; finally, taking out the test-tube plantlet, cleaning and transplanting the test-tube plantlet into a sterilized substrate in a seedling culture plug tray, wherein the substrate is prepared from the following components in percentage by weight: 1, preparing turfy soil and vermiculite, spraying water to level, putting into a domestication room, culturing to 30 centimeters, and planting in a field.
2. The tissue culture rapid propagation method according to claim 1, characterized in that: the raw materials used for the culture medium for inducing dedifferentiation comprise the following components per liter: 20g of cane sugar, 20-24g of agar powder, 20ml of macroelement, 10ml of calcium chloride, 10ml of metal element, 10ml of microelement, 1ml of vitamin, 10ml of inositol, 0.1mol/L of naphthylacetic acid (NAA), 0.1mol/L of 2.4-dichlorophenoxyacetic acid (2.4-D) and 0.1mol/L of 6-benzylaminopurine (6-BA).
3. The tissue culture rapid propagation method according to claim 1, characterized in that: the components of the culture medium for subculture comprise the following components per liter: 20g of cane sugar, 20-24g of agar powder, 20ml of macroelement, 10ml of calcium chloride, 10ml of metal element, 10ml of microelement, 1ml of vitamin, 10ml of inositol, 0.1mol/L of naphthylacetic acid (NAA), 0.1mol/L of 2.4-dichlorophenoxyacetic acid (2.4-D) and 0.2mol/L of 6-benzylaminopurine (6-BA).
4. The tissue culture rapid propagation method according to claim 1, characterized in that: the culture medium components for callus germination comprise the following components per liter: 20g of cane sugar, 20-24g of agar powder, 20ml of macroelement, 10ml of calcium chloride, 10ml of metal element, 10ml of microelement, 1ml of vitamin, 10ml of inositol, 0.1mol/L of naphthylacetic acid (NAA), 0.1mol/L of 2.4-dichlorophenoxyacetic acid (2.4-D) and 3mol/L of 6-benzylaminopurine (6-BA).
5. The tissue culture rapid propagation method according to claim 1, characterized in that: the callus rooting medium comprises (1L as an example): 20g of cane sugar, 20-24g of agar powder, 10ml of macroelements, 5ml of calcium chloride, 5ml of metal elements, 5ml of trace elements, 0.5ml of vitamins, 5ml of inositol and 1.0mg/L of indolebutyric acid (IBA).
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| CN115238964A (en) * | 2022-06-28 | 2022-10-25 | 安徽未来种业有限公司 | Rice harvest prediction evaluation method and system |
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