CN105359973A - Culture method of virus-free test-tube corm of Fanhong No.1 crocus sativus l - Google Patents
Culture method of virus-free test-tube corm of Fanhong No.1 crocus sativus l Download PDFInfo
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Abstract
The invention discloses a culture method of a virus-free test-tube corm of Fanhong No.1 crocus sativus l. The culture method comprises the following steps: soaking a crocus sativus l. corm with warm water, then carrying out water culture germination acceleration, taking a lateral bud of the corm, culturing, stripping to take a shoot tip meristem on the obtained aseptic seedling, culturing, taking the seedling as a plant I when the seedling grows to the height of 1.5 cm-2.0 cm, carrying out bean yellow mosaic virus particle detection on the plant I, if virus particles are detected, stripping to take a shoot tip meristem from the plant I to replace the shoot tip meristem stripped from the aseptic seedling in the former step, and repeating the steps until the plant I containing no bean yellow mosaic virus particles is obtained; culturing the plant I containing no bean yellow mosaic virus particles; and inoculating a corm induced expansion culture medium with induced cluster buds, and culturing until the buds grow to required size. The method can produce a large amount of high-quality virus-free healthy seedlings with the same genetic background in a short time.
Description
Technical field
The present invention relates to the tissue culture method of west safflower detoxicating cuvette bulb.
Background technology
West safflower (CrocussativusL.) has another name called Crocus sativus, safflower, for the perennial plant of Iridaceae crocus, it is traditional rare traditional Chinese medicine simply, enjoy the good reputation of " your medicinal plant ", " best dyestuff " and " the most high-grade spices " in the world simultaneously, there is important economic worth.Chinese Pharmacopoeia 2010 version (one) records that west safflower taste is sweet, property flat, the thoughts of returning home, Liver Channel; Can be promoting blood circulation and removing blood stasis, cool blood, removing toxic substances, resolving stagnation for tranquilization; For Jing Bi Disorder lump in the abdomen, postpartum stasis blocking, febrile virulent maculae, melancholy ruffian is vexed, and palpitation with fear such as to be gone mad at the disease.Also find that crocin has anticancer effect in recent years.
West safflower is only used as medicine with column cap, and wild resource is limited, and Jiande in Zhejiang is one of Main Cultivation place of production, and adopt the bulb asexual reproduction method of breeding to breed for a long time, reproduction coefficient is low always, year reproduction rate be generally 1.5 ~ 2 times, hinder improved variety popularization.This external cause is planted for many years, bulb virus disease infects serious, is mainly broad bean yellow mosaic viral disease (Beanyellowmosaicvirus, BYMV), cause the degeneration of west safflower quality assortment matter, quality decline, these factors have had a strong impact on the development of west safflower medicinal material industry.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of tissue culture method of west safflower detoxicating cuvette bulb, adopts the method can obtain a large amount of west safflower detoxication and tissue culture kind balls.
In order to solve the problems of the technologies described above, the invention provides the cultivation of the detoxicating cuvette bulb of a kind of sarranine No. 1 west safflower, comprising the following steps successively:
1), by without scab, without insect gall and healthy and strong full Crocus bulb carries out water planting vernalization after the emerge in worm water of 55 ~ 65 DEG C; Condition of culture is 26 ~ 28 DEG C, light culture;
2), until bulb lateral bud eye sprout and when growing up to height>=0.5cm, smear lower whole lateral bud, after above-mentioned lateral bud sterilization (routine disinfection), cut its base portion (namely, cutting from base portion) 0.2cm ~ 0.5cm is inoculated into growth medium and cultivates, thus acquisition aseptic seedling; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
3), when above-mentioned aseptic seedling grows to 1.0 ~ 1.2cm height, strip the 3mm ~ 5mm shoot apical meristem in aseptic seedling, shoot apical meristem is inoculated on growth medium and cultivates, thus obtain the seedling of shoot apical meristem induction; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
4), when the seedling of above-mentioned shoot apical meristem induction grows to 1.5cm ~ 2.0cm height as plant I; Cut plant I blade, extract juice, carry out bean virus 2 (Beanyellowmosaicvirus, BYMV) particle detection;
If virion do not detected, then enter following step 5);
If virion detected, 3mm ~ 5mm shoot apical meristem is stripped from this plant I, with this alternative steps 3) in the 3mm ~ 5mm shoot apical meristem in aseptic seedling of stripping repeat above-mentioned steps 3), till obtaining the plant I containing bean virus 2 particle;
5), by step 4) not being inoculated into induced bundle containing the plant I of bean virus 2 particle and sprouting on medium and cultivate of gained; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
6), when the Multiple Buds induced from plant I grows to 1.0 ~ 1.5cm height, 2 ~ 3 strains are extracted as the plant II that grows thickly; The above-mentioned plant II that grows thickly is inoculated on proliferated culture medium and cultivates; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
7), to treat that Multiple Buds that above-mentioned plant II grows grows to 1.0 ~ 2.0cm high, extracts 1 ~ 3 strain as plant III; This plant III is inoculated into bulb induction to expand on medium and cultivate, until grow up to required specification; Growth conditions is: 16 h light, intensity of illumination 10 ~ 30 μm of olm
-2.s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket.
Remarks illustrate: above-mentioned " required specification " generally refers to that bulb diameter reaches >=1cm.
Improvement as the cultivation of the detoxicating cuvette bulb of sarranine of the present invention No. 1 west safflower: described step 1) for by without scab, without insect gall and stalwartness full Crocus bulb in the warm water of 55 ~ 65 DEG C, soak 25 ~ 35min (being preferably 30min), room temperature is dried to bulb surface without water droplet subsequently, then the warm water putting into 55 ~ 65 DEG C soaks; Repeat above-mentionedly to dry, soak 1 ~ 3 time after carry out water planting vernalization again.
Further improvement as the cultivation of the detoxicating cuvette bulb of sarranine of the present invention No. 1 west safflower:
Described step 2) and step 3) in growth medium be: MS minimal medium+white granulated sugar 20 ~ 30g/L+ agar 4 ~ 6g/L, pH is 5.8 ~ 6.0.
The preparation method of above-mentioned growth medium is specific as follows: based on MS minimal medium, adds white granulated sugar, agar Homogeneous phase mixing respectively, utilizes the HCl of KOH or 1mol/L of 1mol/L to regulate pH to 5.8 ~ 6.0; 20 ~ 30g sugar, 4 ~ 6g agar is added in the MS minimal medium of every 1L.
Further improvement as the cultivation of the detoxicating cuvette bulb of sarranine of the present invention No. 1 west safflower:
Described step 5) in induced bundle medium of sprouting be: MS minimal medium+0.2 ~ 0.5mg/lN-phenyl-N '-1,2,3-thiadiazoles-5 urea (TDZ)+0.1 ~ 0.5mg/l methyl α-naphthyl acetate (NAA)+white granulated sugar 20 ~ 30g/L+ agar 4 ~ 6g/L, pH is 5.8 ~ 6.0.
The sprout preparation method of medium of above-mentioned induced bundle is specific as follows: based on MS minimal medium, add TDZ, NAA, white granulated sugar and agar respectively, Homogeneous phase mixing, utilize the HCl of KOH or 1mol/L of 1mol/L to regulate pH to be 5.8 ~ 6.0; The medium of every 1L adds 0.2 ~ 0.5mgTDZ, 0.1 ~ 0.5mgNAA, 20 ~ 30g white granulated sugar, 4 ~ 6g agar.
Further improvement as the cultivation of the detoxicating cuvette bulb of sarranine of the present invention No. 1 west safflower:
Described step 6) in proliferated culture medium be: MS minimal medium+0.3 ~ 2.0mg/l (being preferably 1.0mg/l) 6-benzyladenine (6-BA)+0.05 ~ 0.1mg/l methyl α-naphthyl acetate (NAA)+20 ~ 30g/L white granulated sugar+4 ~ 6g/L agar, pH is 5.8 ~ 6.0.
The preparation method of above-mentioned proliferated culture medium is specific as follows: based on MS minimal medium, adds 6-BA, NAA, white granulated sugar and agar respectively, Homogeneous phase mixing, utilizes the HCl of KOH or 1mol/L of 1mol/L to regulate pH to be 5.8 ~ 6.0; The medium of every 1L adds 0.3 ~ 2.0mg (being preferably 1.0mg) 6-BA, 0.05 ~ 0.1mgNAA, 20 ~ 30g white granulated sugar, 4 ~ 6g agar.
Further improvement as the cultivation of the detoxicating cuvette bulb of sarranine of the present invention No. 1 west safflower:
Step 7) in bulb induction expand medium: MS minimal medium+3.0 ~ 5.0mg/l6-benzyladenine (6-BA)+white granulated sugar 60 ~ 80g/L+170 ~ 340mg/l (being preferably 200mg/l) KH
2pO
4, 1 ~ 1.5g/l active carbon, agar 4 ~ 9g/L, pH be 5.5 ~ 6.0.
It is specific as follows that the preparation method of medium is expanded in the induction of above-mentioned bulb: based on MS minimal medium, add 6-BA, white granulated sugar, KH respectively
2pO
4, active carbon, agar, utilize the HCl of KOH or 1mol/L of 1mol/L regulate pH be 5.8 ~ 6.0; 3.0 ~ 5.0mg6-BA, 60 ~ 80g white granulated sugar, 170 ~ 340mg (being preferably 200mg) KH is added in the MS minimal medium of every 1L
2pO
4, 1 ~ 1.5g active carbon and 4 ~ 9g agar.
Further improvement as the cultivation of the detoxicating cuvette bulb of sarranine of the present invention No. 1 west safflower: described step 4) in, cut 0.8 ~ 1.2cm (being preferably about 1cm) blade on plant I, extract juice.
Remarks illustrate: the too small sap extraction of blade is inadequate, affects electron microscopic observation; Blade is excessive, and that needs the Extending culture time, and cost increases.
In step 2 of the present invention) in, smear lower whole lateral bud, refer to and shake lateral bud gently, connect base portion and break down.The disinfection way of lateral bud is routine disinfection, that is, by lateral bud in concentration be to soak 15 minutes in the calcium hypochlorite solution of 10%, then uses aseptic water washing 5-6 time.
Step 4) in " bean virus 2 (Beanyellowmosaicvirus; BYMV) particle detection " be routine techniques, such as can detect according to " Molecular Identification of safflower virus causing disease " published in " Chinese herbal medicine " magazine.
Technological improvement point major embodiment of the present invention is in the following areas:
1, the present invention's main diseases viral disease infecting No. 1, sarranine of determining in earlier stage is bean virus 2 (Beanyellowmosaicvirus, BYMV), in order to effectively remove BYMV, according to this virus characteristic, the mode that present invention employs the emerge in worm water Crocus bulb of 55 ~ 65 DEG C carries out pretreatment, and in conjunction with shoot apical meristem stripping method, result shows to adopt emerge in worm water pretreatment, the concentration of BYMV can be effectively reduced, improve virus elimination rate.And water temperature over-high can cause bulb lateral bud to be scalded, have a strong impact on the sprouting of lateral bud, and the too low meeting of water temperature causes the bean virus 2 passivation in Crocus bulb not thorough.
2, the present invention adopts water planting vernalization, smears lower whole lateral bud as explant, and result shows that lateral bud that water planting mode obtains infects extraneous pathogen value volume and range of product and all greatly reduces, and substantially increases the acquisition of aseptic seedling; In addition, the lateral bud adopting production above to discard, is not only a large amount of aseptic seedling of acquisition and cormel in vitro provides material guarantee, and greatly provides cost savings.
3, the present invention adopts the white granulated sugar and the KH that with the addition of high concentration
2pO
4medium induce and expand Crocus bulb.The white granulated sugar of high concentration can effectively improve C source and energy, promotes the formation of bulb; Abundant K fertilizer can accelerate the growth of bulb.
The cultivation of the detoxicating cuvette bulb of sarranine of the present invention No. 1 west safflower, belongs to a kind of method for tissue culture of inducing west safflower detoxicating cuvette bulb.According to the principle of cellular omnipotency in Plant Tissue Breeding, the high-quality Virus-free seedcane (seed) that a large amount of genetic background is identical, growing way is consistent can be produced at short notice, and rely on laboratory can realize the anniversary provide high quality seedling for a long time.In the method for the invention, the Crocus bulb by growing way stalwartness and without damage by disease and insect first carries out vernalization, contributes to breaking bulb dormancy; The lower lateral bud discarded will be broken as explant, for obtaining a large amount of aseptic seedling and cormel in vitro provides material guarantee on producing; Utilize Shoot-tip Grafting In Vitro to produce west safflower detoxification bulb, contribute to the quality improving west safflower kind ball, promote the seed output and quality of filigree.So the tissue culture method of Crocus bulb of the present invention is that one does not affect by factors such as seasons, efficient, Quick supplies the method for high-quality west safflower seedling, can accelerate improved variety popularization speed, improves field kind plantation output.
In sum, the west safflower detoxicating cuvette bulb tissue culturing system that the present invention sets up will provide theoretical foundation and technical support for breeding (west safflower) factorial seedling growth.
Embodiment
The tissue culture method of embodiment 1, a kind of west safflower cormel in vitro, carries out following steps successively:
1), by without scab, without insect gall and healthy and strong full Crocus bulb is soak 30min in the warm water of 55 ~ 65 DEG C in water temperature, dry (namely subsequently, ambient temperatare is put till Crocus bulb surface is without obvious water droplet), then the warm water putting into 55 ~ 65 DEG C soaks; Repeat above-mentionedly to dry, soak 2 times after carry out water planting vernalization; Condition of culture is: 26 ~ 28 DEG C of light culture;
2), until bulb lateral bud eye sprout and when growing up to height>=0.5cm, shake lateral bud gently, connect base portion and smear lower whole lateral bud, after above-mentioned lateral bud routine disinfection, cut base portion 0.2cm ~ 0.5cm to be inoculated on growth medium and to cultivate, thus obtain aseptic seedling; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
Growth medium is: MS minimal medium+white granulated sugar 30g/L+ agar 6g/L, pH is 5.8 ~ 6.0.
3), when above-mentioned aseptic seedling grows to 1.0 ~ 1.2cm height, strip the 3mm ~ 5mm shoot apical meristem in aseptic seedling, be inoculated on growth medium and cultivate, thus obtain the seedling of shoot apical meristem induction; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
Growth medium is: MS minimal medium+white granulated sugar 30g/L+ agar 6g/L, pH is 5.8 ~ 6.0.
4), when the seedling of above-mentioned shoot apical meristem induction grows to 1.5cm ~ 2.0cm height as plant I; Cut the blade of about 1cm on plant I, extract juice, carry out bean virus 2 (Beanyellowmosaicvirus, BYMV) particle detection;
If virion do not detected, then enter following step 5);
If virion detected, 3mm ~ 5mm shoot apical meristem is stripped from this plant I, with this alternative steps 3) in the 3mm ~ 5mm shoot apical meristem in aseptic seedling of stripping repeat above-mentioned steps 3), till obtaining the plant I containing bean virus 2 particle;
5), by step 4) gained not containing the plant I of bean virus 2 particle, be inoculated into induced bundle and sprout on medium and cultivate; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
Induced bundle medium of sprouting is: MS minimal medium+0.2mg/LTDZ+0.1mg/LNAA+ white granulated sugar 30g/L+ agar 4g/L, pH is 5.8 ~ 6.0.
6), when the Multiple Buds induced from plant I grows to 1.0 ~ 1.5cm height, 2 ~ 3 strains are extracted as the plant II that grows thickly; The above-mentioned plant II that grows thickly is inoculated on proliferated culture medium and cultivates; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
Proliferated culture medium is: MS minimal medium+1.0mg/L6-BA+0.1mg/LNAA+30g/L white granulated sugar+4g/L agar, pH is 5.8 ~ 6.0.
7), when the Multiple Buds that above-mentioned plant II grows grows to 1.0 ~ 2.0cm height, single plant III is extracted; This plant III is inoculated into bulb induction to expand on medium and cultivate, until grow up to required specification; Growth conditions is: 16 h light, intensity of illumination 10 ~ 30 μm of olm-2.s-1, and temperature is 26 ~ 28 DEG C; 8 hours light culture, temperature is 20 ~ 22 DEG C; Above-mentioned illumination and light culture hocket;
Medium is expanded in bulb induction: MS minimal medium+5.0mg/L6-BA+80g/L white granulated sugar+200mg/LKH
2pO
4+ 1.0g/L active carbon+agar 9g/L, pH is 5.5 ~ 6.0.
Embodiment 1 acquired results:
Bean virus 2 (Beanyellowmosaicvirus, BYMV) detects through Electronic Speculum, and obtaining 100% detoxic seedling needs repetition step 3) 3 ~ 4 times.
The induction time of bulb is 0.5 ~ 1 month, expands to diameter 0.5 ~ 1.0cm, needs 1.5 ~ 2 months.
Remarks illustrate: detoxic seedling quantity/detection seedling quantity × 100%=100%, that is, all detection plant all can't detect bean virus 2.
The tissue culture method of comparative example 1, a kind of west safflower cormel in vitro,
By embodiment 1 step 1) in " by without scab, without insect gall and healthy and strong full Crocus bulb is soak 30min in the warm water of 55 ~ 65 DEG C in water temperature, dry subsequently, then the warm water putting into 55 ~ 65 DEG C soaks; Repeat above-mentionedly to dry, soak 2 times after carry out water planting vernalization " make into " and by without scab, without insect gall and healthy and strong full Crocus bulb directly carries out water planting vernalization "; All the other contents are equal to embodiment 1.
Comparative example 1 acquired results:
Bean virus 2 (Beanyellowmosaicvirus, BYMV) detects through Electronic Speculum, and obtaining 100% detoxic seedling needs repetition step 3) 5 ~ 6 times.
The induction time of bulb is 0.5 ~ 1 month, expands to diameter 0.5 ~ 1.0cm, needs 1.5 ~ 2 months.
The tissue culture method of comparative example 2, a kind of west safflower cormel in vitro,
By embodiment 1 step 7) in bulb induction expand medium and change into: active carbon+agar 9g/L, pH are 5.5 ~ 6.0 to MS minimal medium+5.0mg/L6-BA+30g/L white granulated sugar+1.0g/L; All the other contents are equal to embodiment 1.
Comparative example 2 acquired results:
Bean virus 2 (Beanyellowmosaicvirus, BYMV) detects through Electronic Speculum, and obtaining 100% detoxic seedling needs repetition step 3) 3 ~ 4 times.
The induction time of bulb is 1.0 ~ 1.5 months, expands to diameter 0.5 ~ 1.0cm, needs 2.5 ~ 3 months.
The tissue culture method of comparative example 3, a kind of west safflower cormel in vitro,
By embodiment 1 step 1) in " by without scab, without insect gall and healthy and strong full Crocus bulb is soak 30min in the warm water of 55 ~ 65 DEG C in water temperature, dry subsequently, then the warm water putting into 55 ~ 65 DEG C soaks; Repeat above-mentionedly to dry, soak 2 times after carry out water planting vernalization " make into " and by without scab, without insect gall and healthy and strong full Crocus bulb directly carries out water planting vernalization ";
And, by embodiment 1 step 7) in bulb induction expand medium and make into: active carbon+agar 9g/L, pH are 5.5 ~ 6.0 to MS minimal medium+5.0mg/L6-BA+30g/L white granulated sugar+1.0g/L;
All the other contents are equal to embodiment 1.
Comparative example 3 acquired results:
Bean virus 2 (Beanyellowmosaicvirus, BYMV) detects through Electronic Speculum, and obtaining 100% detoxic seedling needs repetition step 3) 5 ~ 6 times.
The induction time of bulb is 1.0 ~ 1.5 months, expands to diameter 0.5 ~ 1.0cm, needs 2.5 ~ 3 months.
Comparative example 4-1, make the water temperature in embodiment 1 into 40 ~ 50 DEG C by 55 ~ 65 DEG C; All the other are equal to embodiment 1.
Comparative example 4-2, make the water temperature in embodiment 1 into 70 ~ 80 DEG C by 55 ~ 65 DEG C; All the other are equal to embodiment 1.
Comparative example 5-1, by white granulated sugar, the KH in " bulb induction expand medium " in embodiment 1
2pO
4concentration do following change respectively: white granulated sugar changes 30g/L into, KH
2pO
4change 400mg/L into; All the other are equal to embodiment 1.
Comparative example 5-2, by white granulated sugar, the KH in " bulb induction expand medium " in embodiment 1
2pO
4concentration do following change respectively: white granulated sugar changes 100g/L into, KH
2pO
4change 100mg/L into; All the other are equal to embodiment 1.
Comparative example 6, by the step 1 of embodiment 1) make following cellar culture mode into: just without scab, without insect gall and healthy and strong full Crocus bulb is planted in field; All the other are equal to embodiment 1.
The contrast of acquired results is described in table 1 below.
Table 1
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (7)
1. the cultivation of the detoxicating cuvette bulb of sarranine No. 1 west safflower, is characterized in that comprising the following steps successively:
1), by without scab, without insect gall and healthy and strong full Crocus bulb carries out water planting vernalization after the emerge in worm water of 55 ~ 65 DEG C; Condition of culture is 26 ~ 28 DEG C, light culture;
2), until the lateral bud eye of bulb sprout and when growing up to height>=0.5cm, smear lower whole lateral bud, after the sterilization of above-mentioned lateral bud, cutting its base portion 0.2cm ~ 0.5cm and be inoculated on growth medium and cultivate, thus acquisition aseptic seedling; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
3), when above-mentioned aseptic seedling grows to 1.0 ~ 1.2cm height, strip the 3mm ~ 5mm shoot apical meristem in aseptic seedling, shoot apical meristem is inoculated on growth medium and cultivates, thus obtain the seedling of shoot apical meristem induction; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
4), when the seedling of above-mentioned shoot apical meristem induction grows to 1.5cm ~ 2.0cm height as plant I; Cut plant I blade, extract juice, carry out bean virus 2 particle detection;
If virion do not detected, then enter following step 5);
If virion detected, 3mm ~ 5mm shoot apical meristem is stripped from this plant I, with this alternative steps 3) in the 3mm ~ 5mm shoot apical meristem in aseptic seedling of stripping repeat above-mentioned steps 3), till obtaining the plant I containing bean virus 2 particle;
5), by step 4) not being inoculated into induced bundle containing the plant I of bean virus 2 particle and sprouting on medium and cultivate of gained; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
6), when the Multiple Buds induced from plant I grows to 1.0 ~ 1.5cm height, 2 ~ 3 strains are extracted as the plant II that grows thickly; The above-mentioned plant II that grows thickly is inoculated on proliferated culture medium and cultivates; Condition of culture is: 16 h light, intensity of illumination 30 ~ 40 μm of olm
-2s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket;
7), to treat that Multiple Buds that above-mentioned plant II grows grows to 1.0 ~ 2.0cm high, extracts 1 ~ 3 strain as plant III; This plant III is inoculated into bulb induction to expand on medium and cultivate, until grow up to required specification; Growth conditions is: 16 h light, intensity of illumination 10 ~ 30 μm of olm
-2.s
-1, temperature is 27 ± 1 DEG C; 8 hours light culture, temperature is 21 ± 1 DEG C; Above-mentioned illumination and light culture hocket.
2. the cultivation of the detoxicating cuvette bulb of sarranine according to claim 1 No. 1 west safflower, is characterized in that:
Described step 1) be by without scab, without insect gall and healthy and strong full Crocus bulb soaks 25 ~ 35min in the warm water of 55 ~ 65 DEG C, room temperature is dried to bulb surperficial without water droplet subsequently, then the warm water putting into 55 ~ 65 DEG C soaks; Repeat above-mentionedly to dry, soak 1 ~ 3 time after carry out water planting vernalization again.
3. the cultivation of the detoxicating cuvette bulb of sarranine according to claim 1 and 2 No. 1 west safflower, is characterized in that:
Described step 2) and step 3) in growth medium be: MS minimal medium+white granulated sugar 20 ~ 30g/L+ agar 4 ~ 6g/L, pH is 5.8 ~ 6.0.
4. the cultivation of the detoxicating cuvette bulb of sarranine according to claim 1 and 2 No. 1 west safflower, is characterized in that:
Described step 5) in induced bundle medium of sprouting be: MS minimal medium+0.2 ~ 0.5mg/lN-phenyl-N '-1,2,3-thiadiazoles-5 urea+0.1 ~ 0.5mg/l methyl α-naphthyl acetate+white granulated sugar 20 ~ 30g/L+ agar 4 ~ 6g/L, pH is 5.8 ~ 6.0.
5. the cultivation of the detoxicating cuvette bulb of sarranine according to claim 1 and 2 No. 1 west safflower, is characterized in that:
Described step 6) in proliferated culture medium be: MS minimal medium+0.3 ~ 2.0mg/l6-benzyladenine+0.05 ~ 0.1mg/l methyl α-naphthyl acetate+20 ~ 30g/L white granulated sugar+4 ~ 6g/L agar, pH is 5.8 ~ 6.0.
6. the cultivation of the detoxicating cuvette bulb of sarranine according to claim 1 and 2 No. 1 west safflower, is characterized in that:
Step 7) in bulb induction expand medium: MS minimal medium+3.0 ~ 5.0mg/l6-benzyladenine+white granulated sugar 60 ~ 80g/L+170 ~ 340mg/lKH
2pO
4, 1 ~ 1.5g/l active carbon, agar 4 ~ 9g/L, pH be 5.5 ~ 6.0.
7., according to the cultivation of the detoxicating cuvette bulb of arbitrary described sarranine No. 1 west safflower of claim 3 ~ 6, it is characterized in that: described step 4) in, cut 0.8 ~ 1.2cm blade on plant I, extract juice.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106134993A (en) * | 2016-06-27 | 2016-11-23 | 上海交通大学 | The production method of Stigma Croci detoxification kind ball |
| CN106376463A (en) * | 2016-08-30 | 2017-02-08 | 上海市农业科学院 | Breeding method of saffron seed ball |
| CN109329027A (en) * | 2018-12-03 | 2019-02-15 | 湖南省作物研究所 | A kind of efficient propagation method of sweet potato |
| CN109792925A (en) * | 2019-03-18 | 2019-05-24 | 杭州市农业科技教育总站 | West safflower bulbil breeds bulb method |
| CN110301335A (en) * | 2019-07-10 | 2019-10-08 | 浙江大学 | The cultivation of the lateral bud breeding bulb of west safflower |
| CN113498735A (en) * | 2021-07-09 | 2021-10-15 | 福建省中科生物股份有限公司 | Culture medium and culture method for tissue culture bud proliferation seedling of saffron |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106134993A (en) * | 2016-06-27 | 2016-11-23 | 上海交通大学 | The production method of Stigma Croci detoxification kind ball |
| CN106376463A (en) * | 2016-08-30 | 2017-02-08 | 上海市农业科学院 | Breeding method of saffron seed ball |
| CN109329027A (en) * | 2018-12-03 | 2019-02-15 | 湖南省作物研究所 | A kind of efficient propagation method of sweet potato |
| CN109792925A (en) * | 2019-03-18 | 2019-05-24 | 杭州市农业科技教育总站 | West safflower bulbil breeds bulb method |
| CN110301335A (en) * | 2019-07-10 | 2019-10-08 | 浙江大学 | The cultivation of the lateral bud breeding bulb of west safflower |
| CN110301335B (en) * | 2019-07-10 | 2021-10-01 | 浙江大学 | Culture method for lateral bud breeding corm of saffron |
| CN113498735A (en) * | 2021-07-09 | 2021-10-15 | 福建省中科生物股份有限公司 | Culture medium and culture method for tissue culture bud proliferation seedling of saffron |
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