CN101473788B - Method for inducing and cultivating adventitious root of tetraploid Radix pseudostellariae - Google Patents
Method for inducing and cultivating adventitious root of tetraploid Radix pseudostellariae Download PDFInfo
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- CN101473788B CN101473788B CN2008102050866A CN200810205086A CN101473788B CN 101473788 B CN101473788 B CN 101473788B CN 2008102050866 A CN2008102050866 A CN 2008102050866A CN 200810205086 A CN200810205086 A CN 200810205086A CN 101473788 B CN101473788 B CN 101473788B
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Abstract
The invention relates to an adventitious root induction cultivation method of a radix pseudostellariae tetraploid of the bioengineering field. The method comprises the following steps: sterilizing surfaces of radix pseudostellariae seeds by mercuric chloride, and then inoculating the seeds into a seed germination culture medium for culture; cutting off hypocotyls of the seeds and inoculating the hypocotyls to an embryonic callus induction culture medium to induce calluses; subculturing the calluses, soaking the calluses in colchicine solution and then transferring the calluses to a differentiation medium to culture regeneration plants; detecting chromosomes of root tips of the regeneration plants, and screening a homozygous tetraploid plant by a flow cytometer; inoculating a stem section of the homozygous tetraploid plant to a rooting callus induction culture medium to induce a callus; inoculating the callus to an adventitious root induction culture medium to induce the adventitious roots; inoculating the adventitious roots into a shake flask, and placing the shake flask on a shaking table for shake cultivation; and inoculating the adventitious roots obtained by the shake cultivation into a reactor. The adventitious roots of radix pseudostellariae tetraploid cultivated by the method have the advantages of rapid growth and high contents of effective ingredients.
Description
Technical field
What the present invention relates to is a kind of method of bioengineering field, specifically is a kind of method for inducing and cultivating adventitious root of tetraploid Radix pseudostellariae.
Background technology
Radix pseudostellariae is the precious simply nourishing class Chinese herbal medicine of China, is used as medicine with root, has air making-up and spleen enlivening, strengthens the effect of body immunity, classifies as top grade in the Shennong's Herbal.Have the saying of " old anthropophagy's ginseng, middle food Radix Codonopsis is eaten radix pseudostellariae less ".Because consumption is huge, is faced with two crises at present: the one, wild plant resource reserves and output ubiquity downward trend; The 2nd, the radix pseudostellariae demand constantly increases, and has caused the excess of this resources of medicinal plant is excavated.Traditional radix pseudostellariae field production is subjected to the influence of " favourable climate and geographical position " on the one hand, and the output and quality fluctuation is big; Bring problems such as the pesticide residual contamination and the viroses of plant on the other hand inevitably.In addition, tissue, cell biological reactor are cultivated has growth rapidly, the characteristics that are easy to control, thus can replace the needs that field production satisfies market gradually.
The MS medium is Murashige and the disclosed medium of Skoog (1962), and wherein the 1/2MS medium is the macroelement MS medium that reduces by half.The MS medium has a wide range of applications in fields such as prior biological and agriculturals.
Find by prior art documents, " optimization of radix pseudostellariae fast breeding technique and homology are induced for four times " that Xie Yan etc. deliver on " resource and environment journal ", be the explant induction tetraploid with the growing point in this article, the tetraploid that derives in this way mostly is chimera.Though provided the introduction of radix pseudostellariae method for tissue culture in this article, the radix pseudostellariae root induction and the continuous culture in tetraploid source are not studied.
Summary of the invention
The objective of the invention is at above-mentioned deficiency of the prior art, a kind of method for inducing and cultivating adventitious root of tetraploid Radix pseudostellariae has been proposed, the present invention is a material with the radix pseudostellariae callus, obtains to isozygoty the tetraploid individuality, induces the acquisition adventive root from the tetraploid callus that isozygotys again.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
Step 1 carries out disinfection to the radix pseudostellariae the surface of the seed with mercuric chloride solution, is inoculated into then in the seed germination medium, the dark cultivation;
Step 2 cuts hypocotyl and is inoculated in the embryonic callus induction medium, carries out callus induction;
Step 3, the callus subculture that step 2 is generated 2-4 time, and the callus behind the subculture soaked in colchicin, forward to then in the differential medium, turn out regeneration plant;
Step 4 detects the chromosome of the regeneration plant tip of a root, chooses tetraploid plant according to the chromosome testing result, and with the flow cytometer screening tetraploid strain system of isozygotying;
Step 5, the stem section of the tetraploid strain of will isozygotying system are inoculated into takes root with evoked callus in the callus inducing medium;
Step 6, the callus that step 5 is generated is inoculated into inducing adventitious root in the root induction medium;
Step 7, the tetraploid adventive root that step 6 is induced is inoculated into and shakes in the bottle, is placed on shaken cultivation on the shaking table then;
Step 8 is inoculated into the adventive root after the shaken cultivation and carries out the intermittent spraying formula in the reactor and cultivate.
Described seed germination medium is for adding the MS medium of 1mg/L gibberellin.
Described embryonic callus induction medium is for adding 2mg/L methyl, 0.2mg/L2,4-Benzene Chloride fluoroacetic acid, the MS medium of 0.2mg/L kinetin.
Described subculture carries out in not containing the MS medium of hormone, each 20-40 days.
Described differential medium is for adding 3% ferroheme, 0.2mg/L poison green bristlegrass ingot, 1.0mg/L gibberellin, macroelement reduces by half and not containing the MS medium of ammonium nitrate.
Described taking root used callus inducing medium, for adding the MS medium of 2mg/L methyl.
Described root induction medium is for adding the MS medium that 3.0mg/L indolebutyric acid, 0.1mg/L6-benayl aminopurine, every liter of sucrose of 50 grams and macroelement reduce by half.
Describedly be placed on shaken cultivation on the shaking table, its time is 5 days-10 days.
Described intermittent spraying formula is cultivated, and its reactor that uses is spray type reactor, and the medium that uses in the reactor is for adding the MS medium that 1.5mg/L IBA, 0.05mg/L 6-BA, 30g/L sucrose and macroelement reduce by half.Actual conditions is: Ventilation Rate is 0.3vvm, and irrigation rate is every day 50% (percent by volume), and spraying is one time 5 minutes the duration, spray cycles be 4 times per hour, cultivation temperature is 25 ℃, the dark cultivation.
Compared with prior art, the present invention has following beneficial effect: the present invention is a material with the radix pseudostellariae callus, obtains to isozygoty the tetraploid individuality, induces the acquisition adventive root from the tetraploid callus that isozygotys again.Not only growth rate is fast for the adventitious root of tetraploid Radix pseudostellariae that the present invention obtains, and the content of its chemical analysis is higher than the root that field planting is produced.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
The radix pseudostellariae seed of present embodiment is from China Medicine University.
The described MS medium of present embodiment adopts Murashige and the disclosed medium of Skoog (1962), and concrete composition is as follows:
Described 1/2MS medium is the macroelement MS medium that reduces by half.
Present embodiment comprises the steps:
Step 1, with the mercuric chloride solution of mass percent 0.1% to the sterilization of radix pseudostellariae the surface of the seed after 12 minutes, be inoculated into and add 1mg/L gibberellin in the seed germination medium (being MS+1.0mg/L GA3) of MS medium, obtain sterilizable material, and in dark, cultivate a week;
Step 2, cut the long hypocotyl of 1.0cm and be inoculated into evoked callus in the embryonic callus induction medium, the embryonic callus induction medium is for adding the 2mg/L methyl, 0.2mg/L2,4-Benzene Chloride fluoroacetic acid, 0.2mg/L the MS medium of kinetin (is MS+2.0mg/L NAA+0.2mg/L 2,4-D+0.1mg/LKT);
Step 3, the callus that step 2 is generated is not containing on the MS medium of hormone subculture 2-4 time, each 20 days-40 days, and the callus behind the subculture soaked in 0.1% colchicin 5 minutes-30 minutes, aseptic then cleaning 3-6 time, and forward in the differential medium, turn out regeneration plant after two months, differential medium is for adding 3% ferroheme, 0.2mg/L poison green bristlegrass ingot, 1.0mg/L gibberellin, macroelement reduces by half and not containing the MS medium (1/2MS+3% ferroheme+malicious green bristlegrass ingot 0.2mg/L+GA that does not promptly contain ammonium nitrate of ammonium nitrate
31.0, g/L);
Step 4 detects the chromosome of the regeneration plant tip of a root, chooses the plant that the chromosome testing result is 2n=4X=64 according to the chromosome testing result, promptly selects tetraploid plant, and with the flow cytometer screening tetraploid strain system of isozygotying;
Step 5, the stem section of the tetraploid plant that will isozygoty are inoculated into takes root with evoked callus in the callus inducing medium one month, and taking root with callus inducing medium is the MS medium (being MS+NAA 2.0mg/L) of interpolation 2mg/L methyl;
Step 6, the callus that step 5 is generated is inoculated into inducing adventitious root in the root induction medium, and the root induction medium is for adding the MS medium (being 1/2MS+3.0mg/L IBA+0.1mg/L 6-BA+50g/L sucrose) that 3.0mg/L indolebutyric acid, 0.1mg/L6-benayl aminopurine, every liter of sucrose of 50 grams and macroelement reduce by half;
Step 7, the adventive root that step 6 is grown is inoculated into and shakes in the bottle, and inoculum concentration is 5%-12% (the aquatic foods gram number of every 100ml volume inoculation), is placed on then on the shaking table in 100rpm, and 25 ℃ of following shaken cultivation are cultivated week age;
Step 8, adventive root after the shaken cultivation is inoculated into by 8%-15% (the aquatic foods gram number of every 100ml volume inoculation) in 4.8 liters the spray type reactor and cultivates, medium in the reactor is for adding the MS medium (being 1/2MS+1.5mg/LIBA+0.05mg/L 6-BA+30g/L sucrose) that 1.5mg/L IBA, 0.05mg/L6-BA, 30g/L sucrose and macroelement reduce by half, Ventilation Rate is 0.3vvm, irrigation rate is 50% (percent by volume) every day, spray time is one time 5 minutes, spray cycles be 4 times per hour, cultivation temperature is 25 ℃, the dark cultivation.
Present embodiment has carried out radix pseudostellariae embryonic callus induction, somatic embryo plant regeneration, tetraploid induction and homozygote screening, the experiments such as tetraploid plant root induction and atomizing bioreactor culture of isozygotying respectively.As a result the radix pseudostellariae embryo more organize inductivity this to 100%, somatic embryo plant regeneration rate reaches 30%, obtains tetraploid 50 strains, obtains 12 homozygote strains system.When using callus with the stem section root induction of homozygote strain system, inductivity has reached 100%.This callus (average diameter is 1cm) is transferred in the root induction medium, on average broken up 22 adventive root on every callus after 1 month.
The tetraploid adventive root that in the present embodiment radix pseudostellariae isozygotied is inoculated in the spray type reactor to be cultivated, the result shows, under this condition of culture, maximum average life rate reached for 0.25 ± 0.08 every day, was higher than 0.22 ± 0.12 every day when cultivating with airlift reactor.
Claims (3)
1. a method for inducing and cultivating adventitious root of tetraploid Radix pseudostellariae is characterized in that, comprises the steps:
Step 1 carries out disinfection to the radix pseudostellariae the surface of the seed with mercuric chloride solution, is inoculated into then in the seed germination medium, the dark cultivation; Described seed germination medium is for adding the MS medium of 1mg/L gibberellin;
Step 2 cuts hypocotyl and is inoculated in the embryonic callus induction medium, carries out callus induction, and described embryonic callus induction medium is for adding 2mg/L methyl, 0.2mg/L2,4-Benzene Chloride fluoroacetic acid, the MS medium of 0.2mg/L kinetin;
Step 3, the callus subculture that step 2 is generated 2-4 time, and the callus behind the subculture soaked in colchicin, forward to then in the differential medium, turn out regeneration plant; Described differential medium is for adding malicious green bristlegrass ingot, the gibberellin of 1.0mg/L that mass percent is 3% ferroheme, 0.2mg/L, macroelement reduces by half and not containing the MS medium of ammonium nitrate;
Step 4 detects the chromosome of the regeneration plant tip of a root, chooses tetraploid plant according to the chromosome testing result, and with the flow cytometer screening tetraploid strain system of isozygotying;
Step 5, the stem section of the tetraploid strain of will isozygotying system are inoculated into takes root with evoked callus in the callus inducing medium; Described taking root with callus inducing medium is the MS medium of the methyl of interpolation 2mg/L;
Step 6, the callus that step 5 is generated is inoculated into inducing adventitious root in the root induction medium; Described root induction medium is for adding indolebutyric acid, the 6-benzyl aminopurine of 0.1mg/L, the sucrose of 50g/L and the MS medium that macroelement reduces by half of 3.0mg/L;
Step 7, the tetraploid adventive root that step 6 is induced is inoculated into and shakes in the bottle, is placed on shaken cultivation on the shaking table then;
Step 8 is inoculated into the adventive root after the shaken cultivation and carries out the intermittent spraying formula in the reactor and cultivate;
Described subculture carries out in not containing the MS medium of hormone, each 20-40 days;
Described intermittent spraying formula is cultivated, and its reactor that uses is spray type reactor, and the medium that uses in the reactor is for adding the MS medium that 1.5mg/L IBA, 0.05mg/L6-BA, 30g/L sucrose and macroelement reduce by half.
2. method for inducing and cultivating adventitious root of tetraploid Radix pseudostellariae according to claim 1 is characterized in that, describedly is placed on shaken cultivation on the shaking table, and its time is 5 days-10 days.
3. method for inducing and cultivating adventitious root of tetraploid Radix pseudostellariae according to claim 1, it is characterized in that, described intermittent spraying formula is cultivated, its actual conditions is: Ventilation Rate is 0.3vvm, irrigation rate is every day 50%, and spraying is one time 5 minutes the duration, spray cycles be 4 times per hour, cultivation temperature is 25 ℃, the dark cultivation.
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| CN102273407A (en) * | 2011-05-20 | 2011-12-14 | 宁德师范学院 | Radix pseudostellariae germplasm sterile circulating preservation method |
| CN102972295A (en) * | 2012-12-04 | 2013-03-20 | 上海交通大学 | Method for making artificial detoxified radix pseudostellariae seeds |
| CN103404433A (en) * | 2013-03-04 | 2013-11-27 | 厦门医学高等专科学校 | Inducing method of codonopsis convolvulacea polyploid |
| CN104472359B (en) * | 2014-11-26 | 2017-03-15 | 中国医学科学院药用植物研究所 | A kind of ginseng adventitious root proliferative induction method |
| CN106561457B (en) * | 2016-11-07 | 2019-03-15 | 宁德师范学院 | A kind of radix pseudostellariae hybrid seed manually cultivates selection |
| CN106962375B (en) * | 2017-03-27 | 2019-11-08 | 贵州大学 | Radix pseudostellariae kind root inorganic agent and its application |
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| CN101142897A (en) * | 2007-11-08 | 2008-03-19 | 上海交通大学 | Radix pseudostell adventitious root inducing and tissue culturing method |
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| CN101142897A (en) * | 2007-11-08 | 2008-03-19 | 上海交通大学 | Radix pseudostell adventitious root inducing and tissue culturing method |
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| 余春香等.太子参的组织培养研究.《贵州农业科学》.2004,第32卷(第5期),16-17. * |
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