CN104521760B - By the method that tissue culture makes pricklyfruit licorice chromosome doubling - Google Patents
By the method that tissue culture makes pricklyfruit licorice chromosome doubling Download PDFInfo
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- CN104521760B CN104521760B CN201510017938.9A CN201510017938A CN104521760B CN 104521760 B CN104521760 B CN 104521760B CN 201510017938 A CN201510017938 A CN 201510017938A CN 104521760 B CN104521760 B CN 104521760B
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Abstract
The invention discloses a kind of method making pricklyfruit licorice chromosome doubling by tissue culture, with the Glycyrrhiza pallidiflora Maxim. seed of drying and ripening for material, by seed pre-treatment, obtain aseptic seedling, the best Screening of Media of callus induction, Colchicine process pricklyfruit licorice callus, successive transfer culture。Make chromosome doubling by colchicine-induced Glycyrrhiza pallidiflora Maxim. seed, obtain the high-quality kind source of Radix Glycyrrhizae, alleviate the Radix Glycyrrhizae market situation that supply falls short of demand。
Description
Technical field
The invention belongs to biological field, the method being specifically related to make pricklyfruit licorice chromosome doubling by tissue culture。
Background technology
Pricklyfruit licorice (GlycyrrhizapallidifloraMax.), Herba Scopariae, Hu Canger etc. are called, for the perennial fruticuli plant of pulse family Papillionoideae Glycyrrhiza。Pricklyfruit licorice is used as medicine with root and seed, has lactogenic, parasite killing, antidiarrheal, the effect such as anticancer, is taken as one of important plant resources substituting licorice medicinal materials for a long time。Radix Glycyrrhizae has been carried out extensive and deep research by people for many years, but focuses mostly in cultivation, effective ingredient and pharmacological effect research etc.。
In recent years, owing to Radix Glycyrrhizae sharply increases at the consumption of pharmacy industry, grocery trade and cosmetic industry etc., and licorice medicinal materials relies primarily on wild resource for many years, owing to the uncontrolled of Radix Glycyrrhizae being excavated, makes China's Licorice suffer heavy damage。Although Artificial Cultivation Glycyrrhiza uralensis is widely popularized at home, but effective ingredient is relatively low, and medication effect is poor。Therefore, in order to protect wild licorice resource, seek and find that the high-quality kind source of Radix Glycyrrhizae and the alternate resources of Radix Glycyrrhizae become extremely urgent。
Research for pricklyfruit licorice chromosome doubling rarely has report, although Wu Yuxiang et al. employing improvement agar semar technique oozes method process pricklyfruit licorice seedling terminal bud with direct carries out the mutation of polyploid, but method is cumbersome, takes time longer。
Summary of the invention
It is an object of the invention to provide a kind of method making pricklyfruit licorice chromosome doubling by tissue culture。
A kind of method making pricklyfruit licorice chromosome doubling by tissue culture, comprises the steps:
A, seed pre-treatment: choose mature and plump, the healthy Glycyrrhiza Seeds without pest and disease damage, sterilize after soaking 39-41min with 98% concentrated sulphuric acid；
B, obtain aseptic seedling: take Glycyrrhiza pallidiflora Maxim. seed aseptic water washing 3-4 time that mercuric chloride processed, it is inoculated into MS culture medium for the bottle of substrate, light culture 3d at 25 DEG C of temperature, process 4 days with the alternation of light and darkness of 2000lux intensity illumination 10h every day afterwards, after move at 26 DEG C of temperature light and cultivate, plant to be planted grows into during 6-10cm standby；
C, cultivation healthy and strong callus: by the aseptic seedling in step B, be inoculated on zeatin mixed culture medium, light culture 5d under 23 DEG C of conditions, process with the alternation of light and darkness of 2000lux intensity illumination 16h every day afterwards；
D, process pricklyfruit licorice callus: take the step C healthy and strong callus cultivated, process 16 24h with the Colchicine of 0.05 0.2% concentration；
E, successive transfer culture: received by the callus processed and carry out first time successive transfer culture on zeatin mixed culture medium, carry out second time successive transfer culture after 20d。
Preferably, the composition of described zeatin mixed culture medium includes MS culture medium, concentration is the auxin naphthalene acetic acid of 0.5mg/L and concentration is the trans-4-hydroxy-3-methyl of 0.8mg/L6--but-2-ene base amidopurin, adding sucrose 30g/L, agar 7g/L, activated carbon 3g/L, polyvidon 0.5g/L on this basis, keeping pH value is 5.8。
Preferably, in above-mentioned steps A seed pre-treatment, after Glycyrrhiza Seeds soaks, with 1% mercuric chloride sterilization 10min。
Preferably, the pricklyfruit licorice callus Colchicine of 0.2% concentration is processed 24h by step D。The induced mutation rate of 24h is processed even as high as 100% with 0.2% Colchicine。
Preferably, the pricklyfruit licorice tissue of step E successive transfer culture is carried out ploidy detection, specifically comprise the following steps that the callus pretreatment 2.5h in 0.02% Colchicine and 0.002mol/L8-hydroxyquinoline mixed liquor taking new life, rinse well, after with the fixing 2h of the ethanol that ratio is 3: 1-glacial acetic acid fixative；In 25 DEG C of calorstats, 3h is processed again with mixed enzyme solution；Using distilled water immersion 30min, after removing distilled water, add after half an hour fixed by fixative and prepare cell suspension, film-making detects。
The present invention adopts Colchicine to process evoking liquorice platymiscium seed and produces callus and tetraploid pricklyfruit licorice plant, obtains the high-quality kind source of Radix Glycyrrhizae, alleviates the Radix Glycyrrhizae market situation that supply falls short of demand。
Accompanying drawing explanation
Fig. 1 is the qualification figure that Colchicine processes to pricklyfruit licorice Chromosome number。
Detailed description of the invention
The following examples can further illustrate the present invention, but does not limit the present invention in any way。
In embodiment, the composition of zeatin mixed culture medium includes MS culture medium, concentration is the auxin naphthalene acetic acid (NAA) of 0.5mg/L and concentration is the trans-4-hydroxy-3-methyl of 0.8mg/L6--but-2-ene base amidopurin, adding sucrose 30g/L, agar 7g/L, activated carbon 3g/L, polyvidon (PVP) 0.5g/L on this basis, keeping pH value is 5.8。
Embodiment 1
1, seed pre-treatment: choose mature and plump, the healthy Glycyrrhiza Seeds without pest and disease damage soaks 40min with 98% concentrated sulphuric acid after sand mill processes, with 1% mercuric chloride sterilization 10min。
2, aseptic seedling is obtained: take the Glycyrrhiza pallidiflora Maxim. seed aseptic water washing 4 times that mercuric chloride processed, it is inoculated into MS culture medium for the conical flask of substrate, light culture 3d at 25 DEG C of temperature, process 4 days with the alternation of light and darkness of 2000lux intensity illumination 10h every day afterwards, after move at 26 DEG C of temperature light and cultivate, plant to be planted grows into during 8cm standby。
3, by the aseptic seedling in step 2, it is inoculated on zeatin mixed culture medium, is placed in growth cabinet, light culture 5d under 23 DEG C of conditions, process with the alternation of light and darkness of 2000lux intensity illumination 16h every day afterwards。Its healing rate reaches as high as 90.45%, and quality is loosened, and upgrowth situation is good。
4, take the healthy and strong callus that step is cultivated, process 24h respectively with the Colchicine of 0.2% concentration。
5, the callus processed is received zeatin mixed culture medium carries out first time successive transfer culture, carry out after 20d second time successive transfer culture, wait that growing new callus carries out ploidy research。
6, the pricklyfruit licorice tissue of successive transfer culture is carried out ploidy detection: take callus pretreatment 2.5h in 0.02% Colchicine and 0.002mol/L8-hydroxyquinoline mixed liquor of new life, clean with distilled water flushing, afterwards with the fixing 2h of ethanol-glacial acetic acid (3: 1) fixative；In 25 DEG C of calorstats, 3h is processed again with mixed enzyme solution；Use distilled water immersion 30min, after removing distilled water, add fixative fixing half an hour, after prepare cell suspension。Use sessile drop method film-making, dye 3min with improved phenol fuchsin after drying, Conventional compression, throw off coverslip, natural drying, neutral gum mounting after frost, observe and gather image with OlympumBx61, embodiment 1 process under induced mutation rate up to 100%。
Embodiment 2
1, seed pre-treatment: choose mature and plump, the healthy Glycyrrhiza Seeds without pest and disease damage soaks 41min with 98% concentrated sulphuric acid after sand mill processes, with 1% mercuric chloride sterilization 10min。
2, aseptic seedling is obtained: take the Glycyrrhiza pallidiflora Maxim. seed aseptic water washing 3 times that mercuric chloride processed, it is inoculated into MS culture medium for the conical flask of substrate, light culture 3d at 25 DEG C of temperature, process 4 days with the alternation of light and darkness of 2000lux intensity illumination 10h every day afterwards, after move at 26 DEG C of temperature light and cultivate, plant to be planted grows into during 10cm standby。
3, by the aseptic seedling in step 2, it is inoculated on zeatin mixed culture medium, is placed in growth cabinet, light culture 5d under 23 DEG C of conditions, process with the alternation of light and darkness of 2000lux intensity illumination 16h every day afterwards。Its healing rate reaches as high as 90.45%, and quality is loosened, and upgrowth situation is good。
4, be taken on zeatin mixed culture medium cultivate healthy and strong callus, process 16h respectively with the Colchicine of 0.05% concentration。
5, the callus processed is received zeatin mixed culture medium carries out first time successive transfer culture, carry out after 20d second time successive transfer culture, wait that growing new callus carries out ploidy research。
6, after inducing green particles shape callus, take callus pretreatment about 2.5h in 0.02% Colchicine and 0.002mol/L8-hydroxyquinoline mixed liquor of new life, clean with distilled water flushing, afterwards with the fixing 2h of ethanol glacial acetic acid (3: 1) fixative；In 25 DEG C of calorstats, 3h is processed again with mixed enzyme solution；Remove enzyme liquid, use distilled water immersion 30min, after removing distilled water, add fixative fixing half an hour, after prepare cell suspension。Use sessile drop method film-making, dye 2min with improved phenol fuchsin after drying, Conventional compression, coverslip, natural drying, neutral gum mounting is thrown off rapidly after frost, observing with OlympumBx61 and gather image, adding up chromosomal variance, the induced mutation rate under embodiment 2 process is 76%。
Embodiment 3
1, seed pre-treatment: choose mature and plump, the healthy Glycyrrhiza Seeds without pest and disease damage soaks 39min with 98% concentrated sulphuric acid after sand mill processes, with 1% mercuric chloride sterilization 10min。
2, aseptic seedling is obtained: take the Glycyrrhiza pallidiflora Maxim. seed aseptic water washing 4 times that mercuric chloride processed, it is inoculated into MS culture medium for the conical flask of substrate, light culture 3d at 25 DEG C of temperature, process 4 days with the alternation of light and darkness of 2000lux intensity illumination 10h every day afterwards, after move at 26 DEG C of temperature light and cultivate, plant to be planted grows into during 6cm standby。
3, by the aseptic seedling in step 2, it is inoculated on zeatin mixed culture medium, is placed in growth cabinet, light culture 5d under 23 DEG C of conditions, process with the alternation of light and darkness of 2000lux intensity illumination 16h every day afterwards。Its healing rate reaches as high as 90.45%, and quality is loosened, and upgrowth situation is good。
4, be taken on zeatin mixed culture medium cultivate healthy and strong callus, process 20h respectively with the Colchicine of 0.1% concentration。
5, the callus processed is received zeatin mixed culture medium carries out first time successive transfer culture, carry out after 20d second time successive transfer culture, wait that growing new callus carries out ploidy research。Induced mutation rate after process is up to 86.7%。
Test example 1
Glycyrrhiza pallidiflora Maxim. seed is processed by embodiment 1 method, the healthy and strong callus that difference from Example 1 is in that to be taken on zeatin mixed culture medium cultivates, process more than 24h without Colchicine。The callus processed is received zeatin mixed culture medium carries out first time successive transfer culture, carry out second time successive transfer culture after 20d, wait that growing new callus carries out ploidy research。Induced mutation rate after process is 0%。
Table 1 illustrates the Colchicine impact on pricklyfruit licorice callus ploidy, and explanation concentration is the Colchicine process 24h of 0.2%, and induced mutation rate may be up to 100%。
Table 2 illustrates that Colchicine processes lower Glycyrrhiza pallidiflora Maxim. seed chromosome disorder rate, and during with 0.05% concentration process 16h, induced mutation rate is minimum, is 7.5%；The induced mutation rate that 0.2% concentration processes 24h is the highest, is 88.64%。
Fig. 1 illustrates that variable concentrations, different time Colchicine process the qualification to pricklyfruit licorice Chromosome number, and explanation concentration is the Colchicine process 24h of 0.2%, and chromosome number is maximum。
Except above-described embodiment processes pricklyfruit licorice aseptic seedling with Colchicine, being analyzed as follows of Glycyrrhiza pallidiflora Maxim. seed is directly processed: after taking seed germination, produce the hypocotyl callus induction of seedling with Colchicine, inductivity when Colchicine concentration reaches 0.2% process 24h is minimum, inductivity is 10%, and after using Colchicine to process, Glycyrrhiza pallidiflora Maxim. seed chromosome callus induction rate all has decline, illustrates that pricklyfruit licorice chromosome callus induction rate is had inhibitory action by Colchicine。
Claims (4)
1. the method making pricklyfruit licorice chromosome doubling by tissue culture, it is characterised in that it comprises the steps:
A, seed pre-treatment: choose mature and plump, the healthy Glycyrrhiza Seeds without pest and disease damage, sterilize after soaking 39-41min with 98% concentrated sulphuric acid；
B, obtain aseptic seedling: take Glycyrrhiza pallidiflora Maxim. seed aseptic water washing 3-4 time that mercuric chloride processed, it is inoculated into MS culture medium for the bottle of substrate, light culture 3d at 25 DEG C of temperature, process 4 days with the alternation of light and darkness of 2000lux intensity illumination 10h every day afterwards, after move at 26 DEG C of temperature light and cultivate, plant to be planted grows into during 6-10cm standby；
C, cultivation healthy and strong callus: by the aseptic seedling in step B, be inoculated on zeatin mixed culture medium, light culture 5d under 23 DEG C of conditions, process with the alternation of light and darkness of 2000lux intensity illumination 16h every day afterwards；
D, process pricklyfruit licorice callus: take the step C healthy and strong callus cultivated, process 16 24h with the Colchicine of 0.05 0.2% concentration；
E, successive transfer culture: received by the callus processed and carry out first time successive transfer culture on zeatin mixed culture medium, carry out second time successive transfer culture after 20d；
Above-mentioned zeatin mixed culture medium consist of MS culture medium, concentration is the auxin naphthalene acetic acid of 0.5mg/L and concentration is the trans-4-hydroxy-3-methyl of 0.8mg/L6--but-2-ene base amidopurin, adding sucrose 30g/L, agar 7g/L, activated carbon 3g/L, polyvidon 0.5g/L on this basis, keeping pH value is 5.8。
2. the method making pricklyfruit licorice chromosome doubling by tissue culture according to claim 1, it is characterised in that: in above-mentioned steps A seed pre-treatment, after Glycyrrhiza Seeds soaks, with 1% mercuric chloride sterilization 10min。
3. the method making pricklyfruit licorice chromosome doubling by tissue culture according to claim 1 or claim 2, it is characterised in that: the pricklyfruit licorice callus Colchicine of 0.2% concentration is processed 24h by step D。
4. the method making pricklyfruit licorice chromosome doubling by tissue culture according to claim 3, it is characterized in that: the pricklyfruit licorice tissue of step E successive transfer culture is carried out ploidy detection, specifically comprise the following steps that the callus pretreatment 2.5h in 0.02% Colchicine and 0.002mol/L8-hydroxyquinoline mixed liquor taking new life, rinse well, after with the fixing 2h of the ethanol that ratio is 3: 1-glacial acetic acid fixative；In 25 DEG C of calorstats, 3h is processed again with mixed enzyme solution；Using distilled water immersion 30min, after removing distilled water, add after half an hour fixed by fixative and prepare cell suspension, film-making detects。
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