CN101213937B - Method for cultivating detoxification tissue culture bulb of fritillaria thunbergii - Google Patents
Method for cultivating detoxification tissue culture bulb of fritillaria thunbergii Download PDFInfo
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Abstract
The present invention relates to a culture method for the detoxication and the tissue culture of the bulb of Fritillaria thunbergii, which belongs to the technical field of breeding the plant seedling and is specially used for breeding the detoxic seedling of Fritillaria thunbergii and the detection of the virus of Fritillaria thunbergii. The method comprises the steps such as the formulation of the culture medium, the culture of the detoxication tissue culture bulb, the detection of the virus ELISA, etc. The present invention has good detoxication effect, high sensitivity of the virus detection, and can guarantee that the detocication daughter bulb is free from virus, thereby effectively controlling the occurrence and the broadcasting of the virus disease; the multiplication of the bulb is fast, and the monthly multiplication rate can reach 4.5 times, thereby being applicable to the factory seedling raising, greatly saving the breeding land of the traditional method and obviously reducing the production cost of Fritillaria thunbergii; the present invention can be applied to the purification and rejuvenation and the development of the species of Fritillaria thunbergii.
Description
Technical field
The present invention relates to plant toxic group culturation rapid propagating technology field, relate in particular to a kind of cultural method of detoxification tissue culture bulb of fritillaria thunbergii.
Background technology
Fritillaria thunbergii (Fritillaria thunbergii Miq.) is a Liliaceae Fritillaria plant, and its dry bulb is one of famous traditional Chinese medicine " Zhejiang eight flavors ", is the good medicine commonly used of respiratory diseases such as preventing phlegm from forming and stopping coughing, treatment tracheitis, flu.Fritillaria thunbergii originates in Xiangshan County, so claim Bulbus Fritillariae Thunbergii again.In the period of the reportedly clear Kangxu, the Xiangshan peasant begins the bulb of fritillary is planted from the wild family that changes over to.All the time, fritillaria thunbergii is main product ground with Pan'an, Yin state, accounts for 70% of national fritillaria thunbergii gross yield, and also there is cultivation in Jiangsu, Jiangxi, Shanghai, Hubei and Hunan Province.Along with going deep into of crop mix adjustment, the area under cultivation of Zhejiang Province fritillaria thunbergii enlarges rapidly in recent years.
But in artificial cultivation, fritillaria thunbergii production is owing to adopt big bulb to plant, and its reproduction coefficient is extremely low, is about 1.6～1.8 times, and sowing quantity is very big, with kind of a 500kg/ mu, and seed-breeding field length of following resting stage, about 4 months.Reproduction coefficient that fritillaria thunbergii is so low and high consumption kind amount like this, the increase and the production cost that cause producing land used and seed-plot area increase.Simultaneously, long-term nourishing and generating causes fritillaria thunbergii germplasm serious degradation, and the bulb yield and quality descends, and has become one of major issue of the development that has seriously hindered the fritillaria thunbergii industry.Research in recent years discloses, and infecting of plant virus also is the one of the main reasons that causes that the fritillaria thunbergii germplasm is degenerated.We investigate discovery in field, Panan County, Zhejiang to fritillaria thunbergii recently, and most plant all show floral leaf and the typical marmor upsilon member of section such as mottled infects symptom.By research to General Biology, serology feature and the genom sequence of this virus causing disease, the result shows and exists the compound of two kinds of new virus to infect, a kind of is thunberg fritillary flower mosaic virus (Thunberg fritillary mosaic Virus, TFMV), another kind of called after bulb of fritillary Y virus (Fritillary virus Y, FVY), this virus disease generally is one of major reason that causes fritillaria thunbergii germplasm serious degradation; Propose simultaneously to go up the virus that takes place with the field fritillaria thunbergii, be prepared into the virus-specific antiserum through amplification, clone and prokaryotic expression, fritillaria thunbergii plant sample is carried out method (Wei CB et al. (2005) Archives of Virology, the 150:1271-1280 that virus ELISA detects; Chen J et al. (2006) Archives of Virology, 151:439-447; Wei Chuanbao etc. (2006). science and technology circular, 22 (4): 506-509).Up to the present fritillaria thunbergii stem apex detoxify group training culture studies does not appear in the newspapers as yet.Therefore set up fritillaria thunbergii detoxication and tissue culture rapid propagation technical system, the production high-quality is anosis seed culture of viruses ball realizes that for real GAP cultivation and large-scale production provide technology platform, have bigger application prospect.
Summary of the invention
The present invention seeks to, adopt big bulb to plant in the breeding technique at conventional production of existing fritillaria thunbergii, reproduction coefficient is extremely low, and sowing quantity is very big, seed-breeding field length of following resting stage, production and seed-plot area are big, and have plant virus to infect, cause poor quality, yield poorly, defectives such as production cost height; Provide a kind of and can breed bulb in a large number, fast, can remove bulb institute poison and through special detection, to guarantee nontoxic fritillaria thunbergii bulb detoxification, group training and the matching method that detects in spite of illness again.
The object of the invention is achieved through the following technical solutions:
A kind of cultural method of detoxification tissue culture bulb of fritillaria thunbergii, carry out as follows:
(1) culture medium preparation comprises that each component and every liter of contained weight of minimal medium and each stage medium of group training is:
1) minimal medium: daughter bulb is induced, propagation, strong sprout and bulb growth medium MS medium; The bulb root media is with 1/2 MS medium; Wherein, sucrose or white sugar 20～40g/L, agar 7～9g/L, pH5.6～5.8;
2) daughter bulb inducing culture: MS+2,4-D 0.5～1.5mg/L+ZT 1～3mg/L;
3) daughter bulb proliferated culture medium: MS+BA 1～3mg/L+NAA 1～3mg/L+ thiamine hydrochloride (VB
1) 4mg/L:
4) strong seedling culture base: MS+BA 0.5～1.5mg/L+NAA 0.5～1.5mg/L+ thiamine hydrochloride (VB
1) 4mg/L;
5) bulb growth medium: MS+BA 0.1～1mg/L+NAA 0.1～1mg/L+ thiamine hydrochloride (VB
1) 4mg/L+ caseinhydrolysate (CH) 500mg/L;
6) bulb root media: 1/2MS+NAA 0.1～1mg/L+ thiamine hydrochloride (VB
1) 4mg/L;
(2) cultivation of detoxification tissue culture bulb of fritillaria thunbergii:
1) explant selection, cultivation and processing:
1. get bulb, tender stem or the bennet of the new growth of fritillaria thunbergii, through sterilization treatment, as winning the material of detoxication and tissue culture with explant;
2. daughter bulb inducing culture: explant material under aseptic condition, is cut into the segment of 0.3～0.6cm, is seeded on the daughter bulb inducing culture, under condition of culture, cultivate 1～2 month after, induce the formation daughter bulb;
3. daughter bulb enrichment culture: will induce the daughter bulb of formation to be transferred on the daughter bulb proliferated culture medium, and under condition of culture, cultivate to breed in 30～45 days and daughter bulb;
4. strong seedling culture: at 4 ℃ of low temperature, light intensity 2000～3000Lx, light application time 12h/d handled for 1 week down, cultivated the strong sprout that grew up to height of seedling 5～7cm in 30～45 days then under condition of culture with the daughter bulb of propagation;
5. thermal treatment: with strong sprout at 35 ℃, light intensity 2000～3000Lx, light application time 12h/d uses for Shoot Tip Culture after down handling for 2～4 weeks;
6. Shoot Tip Culture: with the training of the group after thermal treatment strong sprout under 40 times of bitubular anatomical lens, the long explant of cultivating as detoxification with the stem apex of 1～2 leaf primordium of extraction 0.2～0.5mm;
2) daughter bulb inducing culture: the explant stem apex is seeded on the daughter bulb inducing culture, under condition of culture, cultivate 1～2 month after, induce the formation daughter bulb;
3) daughter bulb enrichment culture: daughter bulb is transferred on the daughter bulb proliferated culture medium, under condition of culture, cultivates to breed in 30～45 days and daughter bulb; According to demand, bred again by carrying out daughter bulb with quadrat method every 30～45 days to daughter bulb quantity;
4) daughter bulb grown cultures: daughter bulb is transferred on the daughter bulb growth medium, under condition of culture, cultivated 30～45 days, bulb is grown up to 0.5～1cm;
5) culture of rootage: the daughter bulb that will grow up is seeded on the root media, under condition of culture, cultivate 20～30 days after, daughter bulb top grows blade, the daughter bulb base portion grows 3～5 root systems;
6) transplant: root bulb height of seedling to be generated grows to that 5～7cm is above, root grows 5 when above, and transplanting medium is cultivated 1～2 month to Cheng Miao.
(3), virus ELISA detects:
Thunberg fritillary flower mosaic virus (the Thunberg fritillary mosaicVirus that the field diseased plant is entrained, TFMV) and bulb of fritillary Y virus (Fritillary virus Y, FVY), behind amplification, clone and prokaryotic expression, the a large amount of correlated virus coat protein that obtained, be used for mouse or rabbit immunity, be prepared into the virus-specific antiserum after, detoxification tissue culture bulb of fritillaria thunbergii is carried out virus ELISA detects.
Described medium preparation comprises that each component and every liter of contained weight of minimal medium and each stage medium of group training is:
(1) minimal medium: daughter bulb is induced, propagation, strong sprout and bulb growth medium MS medium; The bulb root media is with 1/2 MS medium; Wherein, agar 7g/L, pH5.8;
(2) daughter bulb inducing culture: the MS+2 of sucrose 30g/L, 4-D 1mg/L+ZT 2mg/L;
(3) daughter bulb proliferated culture medium: the MS+BA 2mg/L+NAA 2mg/L+ thiamine hydrochloride (VB of white sugar 40g/L
1) 4mg/L;
(4) strong seedling culture base: the MS+BA 1mg/L+NAA 1mg/L+ thiamine hydrochloride (VB of white sugar 40g/L
1) 4mg/L;
(5) bulb growth medium: the MS+BA 0.5mg/L of white sugar 40g/L and NAA 0.5mg/L+ thiamine hydrochloride (VB
1) 4mg/L+ caseinhydrolysate (CH) 500mg/L;
(6) root media: the 1/2MS+NAA 0.5mg/L+ thiamine hydrochloride (VB of white sugar 20g/L
1) 4mg/L.
Described sterilization treatment be material with explant through 75% alcohol-pickled 0.5～1.0min, use 0.1% mercuric chloride aqueous solution soaking, 10～15min again, use aseptic water washing at last 3～5 times.
The condition of culture in described each detoxication and tissue culture stage is, cultivation temperature is 23 ± 2 ℃, and the illumination light intensity is 2000～3000Lx, and light application time is 12h/d.
Described transplanting medium is by peat: perlite: vermiculite 1: 2: 1 by volume is formulated.
The invention has the beneficial effects as follows:
1) the present invention is verifying the reason that causes fritillaria thunbergii kind ball to degenerate, system is by thunberg fritillary flower mosaic virus (Thunberg Fritillary mosaic Virus, TFMV) and bulb of fritillary Y virus (Fritillary virusY, FVY) mixed infection causes on the basis of morbidity, fritillaria thunbergii virus (TFMV and FVY) the coat protein gene RT-PCR amplification of setting up, the clone, prokaryotic expression and high sensitivity ELISA detection architecture, use by this detection technique, can guarantee virus-free existence in the detoxification daughter bulb, propagate for further controlling virus disease, the fritillaria thunbergii variety rejuvenation is laid a good foundation;
2) the fritillaria thunbergii detoxication and tissue culture rapid propagation technical system of Jian Liing, detoxification efficiency is good, bulb propagation is fast, its month, the rate of increase can reach 4.5 times, and its rooting rate reaches 100%, and transplanting survival rate reaches more than 95%, be fit to factorial seedling growth, but commercialization production, thus numerous kind of land used of conventional method saved greatly, significantly reduced the production cost of fritillaria thunbergii;
3) not detoxification bulb volume increase 10～20% of the comparable routine of detoxification bulb is calculated by per mu yield 1000Kg, and every mu can be increased production 100～200Kg, and has significantly improved the product quality of fritillaria thunbergii.
Embodiment
The present invention is described in further detail by following examples, but content of the present invention is not limited thereto.
2,4-D:2,4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic acid), packing in the U.S. importer, analyze pure, purity 99.5%.
ZT: zeatin (Zeatin), packing in the U.S. importer, analyze pure, purity 99%.
BA:6-benayl aminopurine (6-Benzylaminopurine), packing in the U.S. importer, analyze pure, purity 99.9%.
NAA: α-Nai Yisuan (1-Naphthylacetic acid), packing in the U.S. importer, analyze pure, purity 99.5%.
Thiamine hydrochloride (VB
1): packing in the U.S. importer, analyze pure, purity 99%.
Caseinhydrolysate (CH): the import of sigma company, analyze pure.
Embodiment 1:
A kind of cultural method of detoxification tissue culture bulb of fritillaria thunbergii, carry out as follows:
(1) culture medium preparation comprises that each component and every liter of contained weight of minimal medium and each stage medium of group training is:
1) minimal medium: daughter bulb is induced, propagation, strong sprout and bulb growth medium MS medium; The bulb root media is with 1/2 MS medium; Wherein, agar 7g/L, pH5.8;
2) daughter bulb inducing culture: the MS+2 of sucrose 30g/L, 4-D 1mg/L+ZT 2mg/L;
3) daughter bulb proliferated culture medium: the MS+BA 2mg/L+NAA 2mg/L+ thiamine hydrochloride (VB of white sugar 40g/L
1) 4mg/L;
4) strong seedling culture base: the MS+BA 1mg/L+NAA 1mg/L+ thiamine hydrochloride (VB of white sugar 40g/L
1) 4mg/L;
5) bulb growth medium: the MS+BA 0.5mg/L of white sugar 40g/L and NAA 0.5mg/L+ thiamine hydrochloride (VB
1) 4mg/L+ caseinhydrolysate (CH) 500mg/L;
6) root media: the 1/2MS+NAA 0.5mg/L+ thiamine hydrochloride (VB of white sugar 20g/L
1) 4mg/L;
(2) cultivation of detoxification tissue culture bulb of fritillaria thunbergii:
1) explant selection, cultivation and processing:
1. get the bulb of the new growth of fritillaria thunbergii,, use 0.1% mercuric chloride aqueous solution soaking 13min again, carry out sterilization treatment 3～5 times with aseptic water washing at last, as winning the material of detoxication and tissue culture with explant through 75% alcohol-pickled 0.8min;
2. daughter bulb inducing culture: explant material under aseptic condition, is cut into the segment of 0.3～0.6cm, is seeded on the daughter bulb inducing culture, 23 ± 2 ℃ of temperature, light intensity 2500Lx, the condition of culture of light application time 12h/d is cultivated after 1～2 month down, induces the formation daughter bulb;
3. daughter bulb enrichment culture: will induce the daughter bulb of formation to be transferred on the daughter bulb proliferated culture medium, with step (2)-1)-2. cultivate to breed in 30～45 days under the same culture conditions and daughter bulb;
4. strong seedling culture: with the daughter bulb of propagation at 4 ℃ of low temperature, light intensity 2000～3000Lx, after light application time 12h/d handled for 1 week down, with step (2)-1)-2. cultivate the strong sprout that grew up to height of seedling 5～7cm in 30～45 days under the same culture conditions;
5. thermal treatment: with strong sprout at 35 ℃, light intensity 2000～3000Lx, light application time 12h/d uses for Shoot Tip Culture after down handling for 3 weeks;
6. Shoot Tip Culture: with the training of the group after thermal treatment strong sprout under 40 times of bitubular anatomical lens, the long explant of cultivating as detoxification with the stem apex of 1～2 leaf primordium of extraction 0.2～0.5mm;
2) daughter bulb inducing culture: the stem apex explant is seeded on the daughter bulb inducing culture, with step (2)-1)-2. cultivate 1～2 month under the same culture conditions after, induce the formation daughter bulb;
3) daughter bulb enrichment culture: daughter bulb is transferred on the daughter bulb proliferated culture medium, with step (2)-1)-2. cultivate to breed in 30～45 days under the same culture conditions and daughter bulb; According to demand to daughter bulb quantity, every 30～45 days by the propagation again of carrying out daughter bulb with quadrat method;
4) daughter bulb grown cultures: be transferred on the daughter bulb growth medium breeding the daughter bulb that, with step (2)-1)-2. cultivated 30～45 days under the same culture conditions, bulb is grown up to 0.5～1cm;
5) culture of rootage: the daughter bulb that will grow up is seeded on the root media, with step (2)-1)-2. cultivate 20～30 days under the same culture conditions after, daughter bulb top grows blade, the daughter bulb base portion grows several root systems;
6) transplant: root bulb height of seedling to be generated grows to 3～5cm, root and grows 5 when above, is transplanted to by peat: perlite: in by volume 1: 2: 1 formulated matrix of vermiculite, cultivate 1～2 month to Cheng Miao.
(3) virus ELISA detects:
1), the evaluation of fritillaria thunbergii virus causing disease kind:
1. get the disease sample: get the thunberg fritillary flower leaf disease sample of falling ill ,-80 ℃ store for future use;
2. electron microscopic observation: get disease juice after negative staining, with the form of electron microscopic observation virion;
3. viral species RT-PCR detects and identifies: the primer that the sequence that infects 2 kinds of viruses of fritillaria thunbergii is synthesized the various virus coat protein genes of amplification respectively; With the total RNA of plant is template, and reverse primer (-) synthesizes the viral first chain cDNA, and then is template with this cDNA, adopts the TaKaRa LA Taq of company
TMPCR system or EX Taq archaeal dna polymerase system are carried out RT-PCR one by one to various viruses and are detected;
2) virus ELISA detects:
Because fritillaria thunbergii contains the inventor usually and identifies clear and definite thunberg fritillary flower mosaic virus (Thunbergfritillary mosaic Virus, TFMV) and bulb of fritillary Y virus (Fritillary virus Y, FVY) 2 kinds of viruses, and these 2 kinds of viruses are difficult to separation and purification, thereby with above-mentioned 2 kinds of virus coat protein genes, behind amplification, clone and prokaryotic expression, the a large amount of correlated virus coat protein that obtained, be used for mouse or rabbit immunity, be prepared into the virus-specific antiserum, as the usefulness of the virus ELISA detection of detoxification tissue culture bulb of fritillaria thunbergii.
Embodiment 2:
In this example, the agar of its minimal medium is 9g/L, and pH is 5.6; The daughter bulb inducing culture is: the MS+2 of sucrose 20g/L, 4-D0.5mg/L+ZT 1mg/L; The daughter bulb proliferated culture medium is: the MS+BA 1mg/L+NAA3mg/L+ thiamine hydrochloride (VB of white sugar 30g/L
1) 4mg/L; The strong seedling culture base is: the MS+BA1.5mg/L of white sugar 20g/L and NAA0.5mg/L+ thiamine hydrochloride (VB
1) 4mg/L; The bulb growth medium is: the MS+BA 0.1mg/L+NAA 1mg/L+ thiamine hydrochloride (VB of white sugar 20g/L
1) 4mg/L+ caseinhydrolysate (CH) 500mg/L; Root media is: the 1/2MS+NAA 0.1mg/L+ thiamine hydrochloride (VB of white sugar 30g/L
1) 4mg/L; Get the tender stem of the new growth of fritillaria thunbergii,, use 0.1% mercuric chloride aqueous solution soaking 10min again, carry out sterilization treatment 3～5 times with aseptic water washing at last, as winning the material of detoxication and tissue culture with explant through 75% alcohol-pickled 0.5min; With strong sprout at 35 ℃, light intensity 2000～3000Lx, handle hot 2 weeks under the light application time 12h/d after, use for Shoot Tip Culture; With 23 ± 2 ℃ of temperature, light intensity 2000Lx, light application time 12h/d are condition of culture; All the other steps, condition all are same as embodiment 1.
Embodiment 3:
In this example, the agar of its minimal medium is 8g/L, and pH is 5.7; The daughter bulb inducing culture is: the MS+2 of sucrose 40g/L, 4-D1.5mg/L+ZT 3mg/L; The daughter bulb proliferated culture medium is: the MS+BA 3mg/L+NAA1mg/L+ thiamine hydrochloride (VB of white sugar 20g/L
1) 4mg/L; The strong seedling culture base is: the MS+BA0.5mg/L of white sugar 30g/L and NAA1.5mg/L+ thiamine hydrochloride (VB
1) 4mg/L; The bulb growth medium is: the MS+BA 1mg/L+NAA 0.1mg/L+ thiamine hydrochloride (VB of white sugar 30g/L
1) 4mg/L+ caseinhydrolysate (CH) 500mg/L; Root media is: the 1/2MS+NAA 1mg/L+ thiamine hydrochloride (VB of white sugar 40g/L
1) 4mg/L; Get the bennet of the new growth of fritillaria thunbergii,, use 0.1% mercuric chloride aqueous solution soaking 15min again, carry out sterilization treatment 3～5 times with aseptic water washing at last, as winning the material of detoxication and tissue culture with explant through 75% alcohol-pickled 1.0min; With strong sprout at 35 ℃, light intensity 2000～3000Lx, heat-treated for 4 weeks under the light application time 12h/d after, use for Shoot Tip Culture; With 23 ± 2 ℃ of temperature, light intensity 3000Lx, light application time 12h/d are condition of culture; All the other steps, technology, condition all are same as embodiment 1.
Test example: (virus detects)
1, control material: the sick leaf bulb of the fritillaria thunbergii of gathering from the field is a material.
2, handle material: with embodiment 1,2 or the 3 detoxication and tissue culture bulbs that obtain is material.
3, virus ELISA detects: because fritillaria thunbergii often contains 2 kinds of compound infecting of virus, usually virus is difficult to separation and purification, thereby use virus coat protein gene amplification, clone and prokaryotic expression, to obtain a large amount of correlated virus coat protein, be used for mouse or rabbit immunity, preparation virus-specific antiserum is for the usefulness of virus ELISA detection.
4, testing result is as shown in the table:
| Handle | The result who detects |
| Contrast | Positive |
| The detoxication and tissue culture bulb | Negative |
Claims (1)
1. the cultural method of a detoxification tissue culture bulb of fritillaria thunbergii is characterized in that carrying out as follows:
(1) culture medium preparation comprises that each component and every liter of contained weight of minimal medium and each stage medium of group training is:
1) minimal medium: daughter bulb is induced, propagation, strong sprout and bulb growth medium MS medium; Bulb root media 1/2MS medium; Wherein, sucrose or white sugar 20～40g/L, agar 7～9g/L, pH 5.6～5.8;
2) daughter bulb inducing culture: MS+2,4-D 0.5～1.5mg/L+ ZT 1～3mg/L;
3) daughter bulb proliferated culture medium: MS+BA 1～3mg/L+NAA 1～3mg/L+ thiamine hydrochloride 4mg/L;
4) strong seedling culture base: MS+BA 0.5～1.5mg/L+NAA 0.5～1.5mg/L+ thiamine hydrochloride 4mg/L;
5) bulb growth medium: MS+BA 0.1～1mg/L+NAA 0.1～1mg/L+ thiamine hydrochloride 4mg/L+ caseinhydrolysate 500mg/L;
6) bulb root media: 1/2MS+NAA 0.1～1mg/L+ thiamine hydrochloride 4mg/L;
(2) cultivation of detoxification tissue culture bulb of fritillaria thunbergii:
1) explant selection, cultivation and processing:
1. get bulb, tender stem or the bennet of the new growth of fritillaria thunbergii, through sterilization treatment, as winning the material of detoxication and tissue culture with explant;
2. daughter bulb inducing culture: explant material under aseptic condition, is cut into the segment of 0.3～0.6cm, is seeded on the daughter bulb inducing culture, under condition of culture, cultivate 1～2 month after, induce the formation daughter bulb;
3. daughter bulb enrichment culture: will induce the daughter bulb of formation to be transferred on the daughter bulb proliferated culture medium, and under condition of culture, cultivate to breed in 30～45 days and daughter bulb;
4. strong seedling culture: at 4 ℃ of low temperature, light intensity 2000～3000Lx, light application time 12h/d handled for 1 week down, cultivated the strong sprout that grew up to height of seedling 5～7cm in 30～45 days then under condition of culture with the daughter bulb of propagation;
5. thermal treatment: with strong sprout at 35 ℃, light intensity 2000～3000Lx, light application time 12h/d uses for Shoot Tip Culture after down handling for 2～4 weeks;
6. Shoot Tip Culture: with the training of the group after thermal treatment strong sprout under anatomical lens, the long explant of cultivating as detoxification with the stem apex of 1～2 leaf primordium of extraction 0.2～0.5mm;
2) daughter bulb inducing culture: the explant stem apex is seeded on the daughter bulb inducing culture, under condition of culture, cultivate 1～2 month after, induce the formation daughter bulb;
3) daughter bulb enrichment culture: daughter bulb is transferred on the daughter bulb proliferated culture medium, under condition of culture, cultivates to breed in 30～45 days and daughter bulb; According to demand, bred again by carrying out daughter bulb with quadrat method every 30～45 days to daughter bulb quantity;
4) daughter bulb grown cultures: daughter bulb is transferred on the daughter bulb growth medium, under condition of culture, cultivated 30～45 days, bulb is grown up to 0.5～1cm;
5) culture of rootage: the daughter bulb that will grow up is seeded on the root media, under condition of culture, cultivate 20～30 days after, daughter bulb top grows blade, the daughter bulb base portion grows 3～5 root systems;
6) transplant: root bulb height of seedling to be generated grows to that 5cm is above, root grows 5 when above, and transplanting medium is cultivated 1～2 month to Cheng Miao;
(3) virus ELISA detects:
Thunberg fritillary flower mosaic virus (the Thunberg fritillary mosaicVirus that the field diseased plant is entrained, TFMV) and bulb of fritillary Y virus (Fritillary virus Y, FVY), behind amplification, clone and prokaryotic expression, the a large amount of correlated virus coat protein that obtained, after being prepared into mouse or rabbit virus-specific antiserum, detoxification tissue culture bulb of fritillaria thunbergii being carried out virus ELISA detect.
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| CN103238525B (en) * | 2013-05-31 | 2015-04-29 | 太仓市金诸农业发展有限公司 | Method for breeding fritillary bulb by tissue culture technique |
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| CN103444528A (en) * | 2013-08-16 | 2013-12-18 | 苏州仁成生物科技有限公司 | Growth promotion cultivation method of fritillary bulbs |
| CN104255260A (en) * | 2014-09-28 | 2015-01-07 | 浙江万里学院 | Cultivation method of high-yield heat-resisting fritillaria thunbergii germplasms |
| CN104782488A (en) * | 2015-05-02 | 2015-07-22 | 冯文杰 | Tissue culture method of Fritillaria unibracteata Hsiao |
| CN105475107A (en) * | 2015-12-16 | 2016-04-13 | 镇江盛弘景观植物有限公司 | Thunberg fritillary bulb culture soil taking DDGS as raw material |
| CN105519435B (en) * | 2016-01-06 | 2017-10-13 | 南京海源中药饮片有限公司 | A kind of tissue culture method of induction fritillaria thunbergii clove |
| CN107996404A (en) * | 2017-12-26 | 2018-05-08 | 丽江市古城区秋成种养殖有限公司 | A kind of tendril-leaved fritillary bulb method for tissue culture |
| CN109717063A (en) * | 2019-03-12 | 2019-05-07 | 康定恩威高原药材野生抚育基地有限责任公司 | A kind of method of soilless substyate culture bulbus fritillariae cirrhosae |
| CN111296211A (en) * | 2020-03-12 | 2020-06-19 | 重庆市药物种植研究所 | Rapid propagation method of fritillaria taipaiensis bulbs |
| CN112243631B (en) * | 2020-09-14 | 2022-03-22 | 云南省农业科学院花卉研究所 | Method for rapidly breaking dormancy of green flower lily seed bulbs |
| CN114158446B (en) * | 2021-12-02 | 2022-12-20 | 湖北省农业科学院中药材研究所 | Method for bulb split propagation of Hubei fritillary bulb |
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