CN105494102A - Method for culture of efficient maca virus-free tissue - Google Patents
Method for culture of efficient maca virus-free tissue Download PDFInfo
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- CN105494102A CN105494102A CN201610018628.3A CN201610018628A CN105494102A CN 105494102 A CN105494102 A CN 105494102A CN 201610018628 A CN201610018628 A CN 201610018628A CN 105494102 A CN105494102 A CN 105494102A
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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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Abstract
The invention discloses a method for culture of efficient maca virus-free tissue. The method comprises the steps of a, sterilizing maca seeds, and washing residues off; b, placing the maca seeds on a culture medium to be cultivated in a tissue culture chamber; c, continuing culture for another 20 days after seed germination; d, transferring shoot tips onto the culture medium for callus induction; e, inducing the generation of maca clumpy buds; f, conducting propagation expansion of the maca clumpy buds in the tissue culture chamber; g, inducing the generation of a root system in the tissue culture chamber; h, after tissue culture seedlings are obtained, adjusting the illumination intensity in the culture chamber, and washing a seedling root system culture medium off and transplanting the seedlings to a glass greenhouse after culture is conducted for 10 days with illumination intensity increased 500+/-100 lux each day, wherein illumination intensity in the glass greenhouse is 4000-5000 lux, temperature is 25-28 DEG C, humidity is maintained to be 80-90% at the first week the seedlings are transplanted to the greenhouse, and humidity is kept to be 70-80% afterwards to obtain marketable seedlings through culture. The novel virus removal method is provided to generate the virus-free maca seedlings, and has an efficient virus removal effect.
Description
Technical field
The present invention relates to a kind of method of tissue cultures, be specifically related to a kind of method of agate card high-efficiency detoxicating tissue cultures.
Background technology
Agate card is a Plants of a kind of Cruciferae, separate row Vegetable spp.Its original producton location is positioned in the middle part of Peru, grows the mountain area, Andean at more than height above sea level 4000m.The agate card that China introduces mainly is distributed on the high mountain on the ground such as Yunnan, Xinjiang.Other places, due to weather conditions, are difficult to form its peculiar quality and composition and effectiveness.
Agate card contains abundant nutrient component and active component, and it contains the protein of 14%, carbohydrate 59%; Fiber 8.5%, containing various trace elements such as zinc, calcium, iron, titanium, rubidium, potassium, sodium, copper, manganese, magnesium, strontium, phosphorus, iodine, also containing the multivitamin such as vitamin C, B1, B2, B6, A, E, B12, B5.Its natural activity composition comprises macamides (macamides), agate card alkene, alkaloid, glucosinolate and catabolite benzyl isothiocyanate, sterol, polyphenols.Its active component is without any side effects, has the good reputation of " Peru's ginseng " and " South America ginseng ".
At occurring in nature, plant is easy to be subject to virus infection and cause virus disease.Plant is once by after virus infections, and its vitality will obviously reduce, and yield and quality also can decline.In order to prevent and treat virus disease, botanists seek the method for solution from every side for many years.It is found that and detoxification is carried out to plant, not only can alleviate virus harm, increase yield and injury plant little as far as possible.
Thermal treatment is a kind of effective plant toxic method.Because virus and the patience of host plant to high temperature there are differences, make the growth rate of plant exceed the diffusion velocity of virus, and obtain sub-fraction containing the plant meristematic tissue of virus, then carry out nontoxic individual cultivation.
The effective poison-removing method chemical detoxication method used in group training process, wherein ribavirin (guanine analog) is the plant virus detoxifying agent used.
Callus induction is another effective plant toxic method, and the growth rate of plant virus in plant corpus is unable to catch up with the split speed of healing cell, therefore achieves the detoxification of plant.
But poison-removing method efficiency of the prior art is not high.
Summary of the invention
Technical problem to be solved by this invention is a kind of method providing agate card high-efficiency detoxicating tissue cultures, the technical problem that the detoxification efficiency existed in solution prior art is not high.
Solve the problems of the technologies described above a kind of method that adopted technical scheme is agate card high-efficiency detoxicating tissue cultures, comprise the steps:
A by agate card type 70-75% alcohol disinfecting 30-60s, then with 0.1% mercuric chloride sterilization 10-20min, outwells mercuric chloride solution, then washes away residual mercuric chloride and alcohol for 6-10 time with sterile water wash seed;
Agate card type is placed on medium and cultivates 5 days, intensity of illumination 1000-2000lux in group training room by b;
C, after seed germination, superclean bench is transferred on medium; Continue cultivation 20 days, intensity of illumination 1500-2500lux;
D gets the tender stem of agate card 2-3mm on superclean bench, and stem apex moves into evoked callus on medium;
E gets agate card callus and is placed in differential medium and cultivates on superclean bench, room temperature 25 ± 3 DEG C, the generation of intensity of illumination 1500-2500lux group training room induction agate card clump bud;
Agate card clump bud is inoculated into subculture medium and cultivates by f on superclean bench, and room temperature 25 ± 3 DEG C, numerous agate card clump bud is expanded in intensity of illumination 1500-2500lux group training room;
Agate card clump bud is transferred on root media by g on superclean bench, and room temperature 25 ± 3 DEG C, intensity of illumination 1000-1500lux group training room induction root system generates;
H is after plantlet in vitro grows up to, grow seedlings in the culturing room of adjustable intensity of illumination, after cultivating 10d under intensity of illumination increases the intensity of 500 ± 100lux in every two days, clean seedlings root medium, by seedling replanting to glass greenhouse, intensity of illumination 4000-5000lux, temperature 25-28 DEG C, be transplanted to the first week humidity in greenhouse and remain on 80-90%, later humidity remains on 70-80% and continues to be trained commercial seedling.
For obtaining agate card detoxic seedling, the present invention proposes a kind of new poison-removing method, there is the effect of high-efficiency detoxicating.
Accompanying drawing explanation
Fig. 1 is that 2,4-D and 6-BA hormone prescription are to the effect diagram of the average inductivity of callus;
Fig. 2 is that IBA concentration is to the effect diagram without the average rooting rate of offspring.
Embodiment
Below in conjunction with embodiment, the present invention is described in more detail, but the invention is not restricted to these embodiments.
The present invention discloses a kind of method of agate card high-efficiency detoxicating tissue cultures, it is characterized in that, comprises the steps:
A by agate card type 70-75% alcohol disinfecting 30-60s, then with 0.1% mercuric chloride sterilization 10-20min, outwells mercuric chloride solution, then washes away residual mercuric chloride and alcohol for 6-10 time with sterile water wash seed;
Agate card type is placed on medium and cultivates 5 days, intensity of illumination 1000-2000lux in group training room by b;
C, after seed germination, superclean bench is transferred on medium; Continue cultivation 20 days, intensity of illumination 1500-2500lux;
D gets the tender stem of agate card 2-3mm on superclean bench, and stem apex moves into evoked callus on medium;
E gets agate card callus and is placed in differential medium and cultivates on superclean bench, room temperature 25 ± 3 DEG C, the generation of intensity of illumination 1500-2500lux group training room induction agate card clump bud;
Agate card clump bud is inoculated into subculture medium and cultivates by f on superclean bench, and room temperature 25 ± 3 DEG C, numerous agate card clump bud is expanded in intensity of illumination 1500-2500lux group training room;
Agate card clump bud is transferred on root media by g on superclean bench, and room temperature 25 ± 3 DEG C, intensity of illumination 1000-1500lux group training room induction root system generates;
H is after plantlet in vitro grows up to, grow seedlings in the culturing room of adjustable intensity of illumination, after cultivating 10d under intensity of illumination increases the intensity of 500 ± 100lux in every two days, clean seedlings root medium, by seedling replanting to glass greenhouse, intensity of illumination 4000-5000lux, temperature 25-28 DEG C, be transplanted to the first week humidity in greenhouse and remain on 80-90%, later humidity remains on 70-80% and continues to be trained commercial seedling.
Medium in described step b is made up of MS minimal medium+0.8% agar medium.
Medium in described step c is made up of MS minimal medium+0.8% agar medium+20-30mg/L ribavirin.
In steps d, medium is made up of MS minimal medium+soft white sugar+0.1-0.4mg/L2,4-D+0.5-1mg/L6-BA.
In step e, medium is made up of MS minimal medium+3% sucrose+0.8% agar+0.4-0.6mg/LIBA+2-3mg/L6-BA.
In step f, medium is made up of MS minimal medium+3% sucrose+0.8% agar+0.1-0.5mg/LIBA+0.2-0.3mg/L6-BA.
In step g, medium is made up of 1/2MS minimal medium+2% sucrose+0.6% agar+3-8mg/LIBA.
Embodiment 1
1. selected full seed, without insect pest, disease agate card type, with clear water method the larger proportion of flotation be greater than 1 agate card type, dry for subsequent use afterwards.By agate card type on superclean bench with 75% alcohol disinfecting 45s, then with 0.1% mercuric chloride sterilize 20min, outwell mercuric chloride solution, then wash away residual mercuric chloride and alcohol 10 times with sterile water wash seed.
2. by superclean bench alcolhol burner flame, the surface of the seed moisture is dried, be placed on MS minimal medium+0.8% agar medium with the tweezers after sterilization by agate card type again, each tissue culture bottle places 5 seeds, arranges intensity of illumination 2000lux cultivate 5 days in group training room.
3. after seed germination, on superclean bench by alcolhol burner flame, be transferred on MS minimal medium+0.8% agar medium+20mg/L ribavirin carefully with tweezers after sterilization, note making plumule upward.Intensity of illumination 2000lux is set in culturing room and continues cultivation 20 days.
4. the other tender stem stem apex of 2mm getting agate card seedling of alcolhol burner flame on superclean bench, moves into evoked callus on MS+ soft white sugar+2,4-D+6-BA medium by stem apex.
3.5% is used as preferred soft white sugar concentration; 2,4-D concentration is 0.2mg/L; 6-BA concentration is 0.8mg/L
Callus induction rate is 86% (Fig. 1);
5. on superclean bench, the other agate card callus of getting of alcolhol burner flame is placed on differentiation cultivation MS minimal medium+sucrose+agar+mg/LIBA+6-BA room temperature 25 ± 3 DEG C, the generation of intensity of illumination 2000lux group training room induction agate card clump bud.
3% is used as preferably sucrose concentration; IBA concentration is 0.2mg/L; 6-BA concentration is 2mg/L
6. on superclean bench, agate card clump bud is inoculated into squamous subculture: MS minimal medium+3% sucrose+0.8% agar+IBA+6-BA, room temperature 25 ± 3 DEG C, numerous agate card clump bud is expanded in intensity of illumination 2000lux group training room.Every bottle graft 5 is from clump bud.
Be 0.2mg/L, 6-BA concentration as preferred IBA concentration be 0.25mg/L
7. transferred to by agate card clump bud on superclean bench on root media 1/2MS minimal medium+2% sucrose+0.6% agar+3-8mg/LIBA, room temperature 25 ± 3 DEG C, intensity of illumination 1200lux group training room induction root system generates, every bottle graft kind 6 buds.
Be half amount medium as preferred MS minimal medium, IBA concentration is 3mg/L
Through cultivate every plant average raw 4.7 root systems (Fig. 2)
8. after plantlet in vitro grows up to, grow seedlings in the culturing room of adjustable intensity of illumination, after cultivating 10d under intensity of illumination increases the intensity of 500 ± lux in every two days, clean seedlings root medium, by seedling replanting to glass greenhouse, intensity of illumination 4500lux, temperature 25-28 DEG C, be transplanted to the first week humidity in greenhouse and remain on 85%, remain on 70-80% later and continue to be trained commercial seedling.
Transplanting survival rate reaches 98%
Embodiment 2
1. selected full seed, without insect pest, disease agate card type, with clear water method the larger proportion of flotation be greater than 1 agate card type, dry for subsequent use afterwards.By agate card type on superclean bench with 70% alcohol disinfecting 30s, then with 0.1% mercuric chloride sterilize 10min, outwell mercuric chloride solution, then wash away residual mercuric chloride and alcohol 6 times with sterile water wash seed.
2. by superclean bench alcolhol burner flame, the surface of the seed moisture is dried, be placed on MS minimal medium+0.8% agar medium with the tweezers after sterilization by agate card type again, each tissue culture bottle places 5 seeds, arranges intensity of illumination 1000lux cultivate 5 days in group training room.
3. after seed germination, on superclean bench by alcolhol burner flame, be transferred on MS minimal medium+0.8% agar medium+20mg/L ribavirin carefully with tweezers after sterilization, note making plumule upward.Intensity of illumination 1500lux is set in culturing room and continues cultivation 20 days.
4. the other tender stem stem apex of 3mm getting agate card seedling of alcolhol burner flame on superclean bench, moves into evoked callus on MS+ soft white sugar+2,4-D+6-BA medium by stem apex.
3.5% is used as preferred soft white sugar concentration; 2,4-D concentration is 0.35mg/L; 6-BA concentration is 1mg/L
The average inductivity of callus is 73% (Fig. 1)
5. on superclean bench, the other agate card callus of getting of alcolhol burner flame is placed on differentiation cultivation MS minimal medium+sucrose+agar+mg/LIBA+6-BA room temperature 25 ± 3 DEG C, the generation of intensity of illumination 1500lux group training room induction agate card clump bud.
3% is used as preferably sucrose concentration; IBA concentration is 0.4mg/L; 6-BA concentration is 2.5mg/L
6. on superclean bench, agate card clump bud is inoculated into squamous subculture: MS minimal medium+3% sucrose+0.8% agar+IBA+6-BA, room temperature 25 ± 3 DEG C, numerous agate card clump bud is expanded in intensity of illumination 1500lux group training room.Every bottle graft 5 is from clump bud.
Be 0.4mg/L, 6-BA concentration as preferred IBA concentration be 0.3mg/L
7. transferred to by agate card clump bud on superclean bench on root media 1/2MS minimal medium+2% sucrose+0.6% agar+3-8mg/LIBA, room temperature 25 ± 3 DEG C, intensity of illumination 1000lux group training room induction root system generates.Every bottle graft kind 6 buds.
Be half amount medium as preferred MS minimal medium, IBA concentration is 5mg/L
Through cultivate every plant average raw 6.4 root systems (Fig. 2)
8. after plantlet in vitro grows up to, grow seedlings in the culturing room of adjustable intensity of illumination, after cultivating 10d under intensity of illumination increases the intensity of 500 ± lux in every two days, clean seedlings root medium, by seedling replanting to glass greenhouse, intensity of illumination 4000-5000lux, temperature 25-28 DEG C, be transplanted to the first week humidity in greenhouse and remain on 80%, remain on 70% later and continue to be trained commercial seedling.
Transplanting survival rate reaches 96%
Embodiment 3
1. selected full seed, without insect pest, disease agate card type, with clear water method the larger proportion of flotation be greater than 1 agate card type, dry for subsequent use afterwards.By agate card type on superclean bench with 72% alcohol disinfecting 60s, then with 0.1% mercuric chloride sterilize 20min, outwell mercuric chloride solution, then wash away residual mercuric chloride and alcohol 8 times with sterile water wash seed.
2. by superclean bench alcolhol burner flame, the surface of the seed moisture is dried, be placed on MS minimal medium+0.8% agar medium with the tweezers after sterilization by agate card type again, each tissue culture bottle places 5 seeds, arranges intensity of illumination 1500lux cultivate 5 days in group training room.
3. after seed germination, on superclean bench by alcolhol burner flame, be transferred on MS minimal medium+0.8% agar medium+20mg/L ribavirin carefully with tweezers after sterilization, note making plumule upward.Intensity of illumination 2500lux is set in culturing room and continues cultivation 20 days.
4. the other tender stem stem apex of 2.5mm getting agate card seedling of alcolhol burner flame on superclean bench, moves into evoked callus on MS+ soft white sugar+2,4-D+6-BA medium by stem apex.
3.5% is used as preferred soft white sugar concentration; 2,4-D concentration is 0.35mg/L; 6-BA concentration is 0.8mg/L
The average inductivity of callus is 77% (Fig. 1)
5. on superclean bench, the other agate card callus of getting of alcolhol burner flame is placed on differentiation cultivation MS minimal medium+sucrose+agar+mg/LIBA+6-BA room temperature 25 ± 3 DEG C, the generation of intensity of illumination 2500lux group training room induction agate card clump bud.
3% is used as preferably sucrose concentration; IBA concentration is 0.4mg/L; 6-BA concentration is 2mg/L
6. on superclean bench, agate card clump bud is inoculated into squamous subculture: MS minimal medium+3% sucrose+0.8% agar+IBA+6-BA, room temperature 25 ± 3 DEG C, numerous agate card clump bud is expanded in intensity of illumination 2500lux group training room.Every bottle graft 5 is from clump bud.
Be 0.4mg/L, 6-BA concentration as preferred IBA concentration be 0.25mg/L
7. transferred to by agate card clump bud on superclean bench on root media 1/2MS minimal medium+2% sucrose+0.6% agar+3-8mg/LIBA, room temperature 25 ± 3 DEG C, intensity of illumination 1500lux group training room induction root system generates.Every bottle graft kind 6 buds.
Be half amount medium as preferred MS minimal medium, IBA concentration is 4mg/L
Through cultivate every plant average raw 6 root systems (Fig. 2)
8. after plantlet in vitro grows up to, grow seedlings in the culturing room of adjustable intensity of illumination, after cultivating 10d under intensity of illumination increases the intensity of 500 ± lux in every two days, clean seedlings root medium, by seedling replanting to glass greenhouse (intensity of illumination 4000-5000lux, temperature 25-28 DEG C), be transplanted to the first week humidity in greenhouse and remain on 90%, remain on 70% later and continue to be trained commercial seedling.
Transplanting survival rate reaches 92%.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.Any Reference numeral in claim should be considered as the claim involved by limiting.
In addition, be to be understood that, although this specification is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification is only for clarity sake, those skilled in the art should by specification integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (7)
1. a method for agate card high-efficiency detoxicating tissue cultures, is characterized in that, comprise the steps:
A by agate card type 70-75% alcohol disinfecting 30-60s, then with 0.1% mercuric chloride sterilization 10-20min, outwells mercuric chloride solution, then washes away residual mercuric chloride and alcohol for 6-10 time with sterile water wash seed;
Agate card type is placed on medium and cultivates 5 days, intensity of illumination 1000-2000lux in group training room by b;
C, after seed germination, superclean bench is transferred on medium; Continue cultivation 20 days, intensity of illumination 1500-2500lux;
D gets the tender stem of agate card 2-3mm on superclean bench, and stem apex moves into evoked callus on medium;
E gets agate card callus and is placed in differential medium and cultivates on superclean bench, room temperature 25 ± 3 DEG C, the generation of intensity of illumination 1500-2500lux group training room induction agate card clump bud;
Agate card clump bud is inoculated into subculture medium and cultivates by f on superclean bench, and room temperature 25 ± 3 DEG C, numerous agate card clump bud is expanded in intensity of illumination 1500-2500lux group training room;
Agate card clump bud is transferred on root media by g on superclean bench, and room temperature 25 ± 3 DEG C, intensity of illumination 1000-1500lux group training room induction root system generates;
H is after plantlet in vitro grows up to, grow seedlings in the culturing room of adjustable intensity of illumination, after cultivating 10d under intensity of illumination increases the intensity of 500 ± 100lux in every two days, clean seedlings root medium, by seedling replanting to glass greenhouse, intensity of illumination 4000-5000lux, temperature 25-28 DEG C, be transplanted to the first week humidity in greenhouse and remain on 80-90%, later humidity remains on 70-80% and continues to be trained commercial seedling.
2. the method for a kind of agate card high-efficiency detoxicating tissue cultures according to claim 1, it is characterized in that, the medium in described step b is made up of MS minimal medium+0.8% agar medium.
3. the method for a kind of agate card high-efficiency detoxicating tissue cultures according to claim 1, is characterized in that, the medium in described step c is made up of MS minimal medium+0.8% agar medium+20-30mg/L ribavirin.
4. the method for a kind of agate card high-efficiency detoxicating tissue cultures according to claim 1, is characterized in that, in steps d, medium is made up of MS minimal medium+soft white sugar+0.1-0.4mg/L2,4-D+0.5-1mg/L6-BA.
5. the method for a kind of agate card high-efficiency detoxicating tissue cultures according to claim 1, is characterized in that, in step e, medium is made up of MS minimal medium+3% sucrose+0.8% agar+0.4-0.6mg/LIBA+2-3mg/L6-BA.
6. the method for a kind of agate card high-efficiency detoxicating tissue cultures according to claim 1, is characterized in that, in step f, medium is made up of MS minimal medium+3% sucrose+0.8% agar+0.1-0.5mg/LIBA+0.2-0.3mg/L6-BA.
7. the method for a kind of agate card high-efficiency detoxicating tissue cultures according to claim 1, is characterized in that, in step g, medium is made up of 1/2MS minimal medium+2% sucrose+0.6% agar+3-8mg/LIBA.
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| CN1473465A (en) * | 2003-07-01 | 2004-02-11 | 华中科技大学 | Maka seedling reproducing method |
| CN102511396A (en) * | 2011-10-14 | 2012-06-27 | 新疆维吾尔自治区中药民族药研究所 | In vitro rapid breeding method of medicinal plant Lipidium meyenii |
| CN105191801A (en) * | 2015-10-24 | 2015-12-30 | 大理白族自治州农业科学推广研究院药用植物及农业新技术研究所 | Healthcare medicinal material Maca tissue cultured seedling vitrification control method |
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| CN1473465A (en) * | 2003-07-01 | 2004-02-11 | 华中科技大学 | Maka seedling reproducing method |
| CN102511396A (en) * | 2011-10-14 | 2012-06-27 | 新疆维吾尔自治区中药民族药研究所 | In vitro rapid breeding method of medicinal plant Lipidium meyenii |
| CN105191801A (en) * | 2015-10-24 | 2015-12-30 | 大理白族自治州农业科学推广研究院药用植物及农业新技术研究所 | Healthcare medicinal material Maca tissue cultured seedling vitrification control method |
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