CN104082123A - Cultivation method of tetraploid Anoectochilus roxburghii - Google Patents
Cultivation method of tetraploid Anoectochilus roxburghii Download PDFInfo
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- CN104082123A CN104082123A CN201410297284.5A CN201410297284A CN104082123A CN 104082123 A CN104082123 A CN 104082123A CN 201410297284 A CN201410297284 A CN 201410297284A CN 104082123 A CN104082123 A CN 104082123A
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Abstract
The invention provides a cultivation method of tetraploid Anoectochilus roxburghii. The cultivation method comprises such stages as artificial pollination of the Anoectochilus roxburghii, asymbiotic germination, treatment by use of a mutagenic agent, strong seedling culture and test-tube seedling transplanting. After the Anoectochilus roxburghii blooms, the artificial pollination is carried out; the mature capsules of the Anoectochilus roxburghii are sterilized and then cut open to take out embryos, and then the embryos are inoculated to a germination culture medium for cultivation to form protocorms; next, the protocorms are put in a liquid differential medium containing the mutagenic agent to be mutagenized, and then inoculated to a strong seedling culture medium for strong seedling culture, and finally, test-tube seedling transplanting is carried out. The cultivation method is simple and feasible; the mutagenic frequency is above 75%, and meanwhile, the yield of the Anoectochilus roxburghii used as a medicinal material also can be greatly improved; as a result, a new way is opened for the polyploidy breeding, genetic improvement and new variety breeding of the Anoectochilus roxburghii.
Description
Technical field
The present invention relates to the breeding method of new variety of plant, specifically, relate to a kind of breeding method of tetraploid roxburgh anoectochilus terminal bud.
Background technology
Roxburgh anoectochilus terminal bud (
anoectochilus roxburghii), i.e. Anoectochilus Roxburghii, another name roxburgh anoectochilus terminal bud, metal and stone pine, taiwan anetochilus herb, bird ginseng etc., for the orchid family is opened the rare medicinal plant of lip Cymbidium.Roxburgh anoectochilus terminal bud is China's tradition valuable ingredient of traditional Chinese medicine, due to wide, the special effect of its treatment, has the title of " refreshing medicine ", enjoys good reputations such as " kings of medicine ".
Chromosome is the carrier that determines bion character gene, between the plant that chromosome doubling is later and parent, can produce the variation in form and proterties, and wherein huge property is the most significant external form feature of polyploid.Meanwhile, the variation of plant chromosome ploidy also tends to cause its secondary metabolites content to change.Traditional Chinese medicine is the economic plants that a class has specific use, generally taking organs such as its root, stem, leaves as results object.Chromosome doubling in polyploid plant, often shows the huge property of plant, can meet preferably the demand that Chinese medicine is produced.Roxburgh anoectochilus terminal bud plant is short and small, growth is slow, habitat is harsh, if can be cultivated as polyploid and be fixed up, may improve its medical value, simultaneously for seed selection high-quality roxburgh anoectochilus terminal bud provides precious starting material.
Summary of the invention
The object of the present invention is to provide a kind of breeding method of tetraploid roxburgh anoectochilus terminal bud, by each stages such as artificial pollination, non-symbiosis germination, mutagen processing, strong seedling culture and test-tube seedling transplantings, process roxburgh anoectochilus terminal bud protocorms using colchicin and trefanocide mixed liquor as polyploid mutagenesis agent, improve induced mutation rate, process and once can obtain a considerable number of tetraploid roxburgh anoectochilus terminal bud, thereby realized object of the present invention.
The breeding method of a kind of tetraploid roxburgh anoectochilus terminal bud of the present invention, comprises the following steps:
(1) artificial pollination: carry out according to a conventional method cultivation management, the good roxburgh anoectochilus terminal bud plant of selected strain, as parent, carries out cross pollination after roxburgh anoectochilus terminal bud is bloomed.
(2) non-symbiosis germination: grow and be mature on the whole and fruit while not ftractureing at roxburgh anoectochilus terminal bud capsule, alcoholic solution sterilization through 75%～80% is after 2～5 minutes, being placed on 0.1% mercuric chloride solution for 2～5 times with aseptic water washing sterilizes 8～15 minutes, after aseptic water washing 2～5 times, blot with aseptic filter paper, then capsule is cut along capsule y direction with aseptic scalpel, yellow embryo is inoculated on germination medium and is cultivated with transfer needle, cultivate seed germination after 20～30 days and become protocorm.
(3) mutagen processing: the protocorm that non-symbiosis germination is split into proceeds in the liquid differential medium that contains mutagen, carries out intermittent oscillation mutagenic treatment 240～480 hours under 25～28 DEG C of conditions.
(4) strong seedling culture: will be inoculated on strong seedling culture base and cultivate through the Anoectochilus roxburghii Protocorm of mutagenic treatment, further form the band root seedling that 5～8cm is high.
(5) test-tube seedling transplanting: natural lighting lower refining seedling was opened and be placed in to blake bottle bottle cap after 5～7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, the dliploid roxburgh anoectochilus terminal bud of planting in the matrix being mixed into by peat soil and pine bark (1:3) and routinely carries out field production management.
Condition of culture in the each step in above-mentioned (2)～(4) is: 25～30 DEG C of cultivation temperature, illumination every day 10～15 hours, intensity of illumination 1500～2000lx.
The described germination medium of above-mentioned steps (2) is: 0.5～2.0g/L spends precious No. 1+0.2～2.0g/L peptone+100～200ml/L coconut milk+20～35g/L sucrose+3.0～7.0g/L agar+0.3～1.0g/L active carbon, all the other are the organic components of MS medium, and pH is 5.4～5.8;
The described liquid differential medium of above-mentioned steps (3) is: 1/2MS+0.2～1.2mg/L 6-BA+0.1～0.6mg/L NAA+20～35g/L sucrose+0.2～2.0g/L peptone+0.1～0.5g/L acidic hydrolysis casein+0.3～1.0g/L active carbon, and pH is 5.4～5.8;
The described mutagen of above-mentioned steps (3) are the mixed liquor of 0.01%～0.05% colchicin (olchicine)+0.01%～0.03% trefanocide (trifluralin);
The described strong seedling culture base of above-mentioned steps (4) is: 0.5～2.0g/L spends precious No. 1+0.5～2.0g/L to spend precious No. 2+0.5～1.5mg/LNAA+0.2～2.0g/L peptone+0.1～0.5g/L acidic hydrolysis casein+20～35g/L sucrose+3.0～7.0g/L agar+0.3～1.0g/L active carbon, all the other are the organic components of MS medium, and pH is 5.4～5.8;
The described test-tube plantlet of above-mentioned steps (5) is to choose the test-tube plantlet that chromosome number is 2n=80 after dyed body counting.
The feature that the present invention has, at present domestic have a small amount of report about roxburgh anoectochilus terminal bud polyploid in-vitro inducing, but technology is ripe not enough, and the present invention has simply, Yi Hang, feature that induced mutation rate is high.The present invention processes once can obtain a considerable number of tetraploid roxburgh anoectochilus terminal bud, can provide parent material for roxburgh anoectochilus terminal bud genetic breeding, also for the rearing new variety of roxburgh anoectochilus terminal bud provides new thinking.
Embodiment
Following examples are to further illustrate of the present invention, are not limitations of the present invention.
Embodiment 1
1, cultivar origin
Fujian product group training seedling is the high-quality roxburgh anoectochilus terminal bud after 2 years through field production;
2, cultivating process
(1) artificial pollination: carry out according to a conventional method cultivation management, roxburgh anoectochilus terminal bud is bloomed and carried out 120 days capsule maturations after cross pollination.
(2) non-symbiosis germination: grow and be mature on the whole and while not ftractureing at roxburgh anoectochilus terminal bud capsule, alcoholic solution sterilization through 75% is after 3 minutes, being placed on 0.1% mercuric chloride solution for 2 times with aseptic water washing sterilizes 8 minutes, after aseptic water washing 2 times, blot with aseptic filter paper, then capsule is cut along capsule y direction with aseptic scalpel, yellow embryo is inoculated on germination medium and is cultivated with transfer needle, cultivate seed germination after 23 days and become protocorm.The germination medium adopting is: 1.5g/L spends precious No. 1+1.0g/L peptone+200ml/L coconut milk+25g/L sucrose+4.5g/L agar+0.5g/L active carbon, and all the other are the organic components of MS medium, and pH is 5.6.
(3) mutagen processing: the protocorm that non-symbiosis germination is split into proceeds in the liquid differential medium that contains 0.02% colchicin and 0.01% trefanocide mixed liquor, under 28 DEG C of conditions, carry out intermittent oscillation mutagenic treatment 240 hours, dyed body counting, tetraploid induction rate is 72%.The liquid differential medium adopting is: 1/2MS+1.2mg/L 6-BA+0.4mg/L NAA+25g/L sucrose+1.0g/L peptone+0.5g/L acidic hydrolysis casein+0.3g/L active carbon, pH is 5.6.
(4) strong seedling culture: will be inoculated on strong seedling culture base and cultivate through the Anoectochilus roxburghii Protocorm of mutagenic treatment, further form the band root seedling that 5～8cm is high.The strong seedling culture base adopting is: 1.0g/L spends precious No. 1+1.0g/L to spend precious No. 2+0.5mg/LNAA+2.0g/L peptone+0.5g/L acidic hydrolysis casein+35g/L sucrose+3.0g/L agar+1.0g/L active carbon, all the other are the organic components of MS medium, and pH is 5.6.
(5) test-tube seedling transplanting: natural lighting lower refining seedling was opened and be placed in to blake bottle bottle cap after 5 days, test-tube plantlet is taken out from blake bottle, wash root medium off, the dliploid roxburgh anoectochilus terminal bud of planting in the matrix being mixed into by peat soil and pine bark (1:3) and routinely carries out field production management, and tetraploid roxburgh anoectochilus terminal bud survival rate reaches 92%.
Condition of culture in the each step in above-mentioned (2)～(4) is: 28 DEG C of cultivation temperature, illumination every day 13 hours, intensity of illumination 1500lx.
Embodiment bis-
1, cultivar origin
Taiwan product group training seedling is the high-quality roxburgh anoectochilus terminal bud after 1 year through field production;
2, cultivating process
(1) artificial pollination: carry out according to a conventional method cultivation management, roxburgh anoectochilus terminal bud is bloomed and carried out 110 days capsule maturations after cross pollination.
(2) non-symbiosis germination: grow and be mature on the whole and while not ftractureing at roxburgh anoectochilus terminal bud capsule, alcoholic solution sterilization through 78% is after 2 minutes, being placed on 0.1% mercuric chloride solution for 5 times with aseptic water washing sterilizes 10 minutes, after aseptic water washing 3 times, blot with aseptic filter paper, then capsule is cut along capsule y direction with aseptic scalpel, yellow embryo is inoculated on germination medium and is cultivated with transfer needle, cultivate seed germination after 25 days and become protocorm.The germination medium adopting is: 1.0g/L spends precious No. 1+2.0g/L peptone+100ml/L coconut milk+25g/L sucrose+6.0g/L agar+0.3g/L active carbon, and all the other are the organic components of MS medium, and pH is 5.8.
(3) mutagen processing: the protocorm that non-symbiosis germination is split into proceeds in the liquid differential medium that contains 0.01% colchicin and 0.03% trefanocide mixed liquor, under 25 DEG C of conditions, carry out intermittent oscillation mutagenic treatment 360 hours, dyed body counting, tetraploid induction rate is 74%.The liquid differential medium adopting is: 1/2MS+0.4mg/L 6-BA+0.2mg/L NAA+25g/L sucrose+2.0g/L peptone+0.5g/L acidic hydrolysis casein+0.5g/L active carbon, pH is 5.8.
(4) strong seedling culture: will be inoculated on strong seedling culture base and cultivate through the Anoectochilus roxburghii Protocorm of mutagenic treatment, further form the band root seedling that 5～8cm is high.The strong seedling culture base adopting is: 1.5g/L spends precious No. 1+1.0g/L to spend precious No. 2+1.5mg/LNAA+1.0g/L peptone+0.3g/L acidic hydrolysis casein+30g/L sucrose+3.0g/L agar+1.0g/L active carbon, all the other are the organic components of MS medium, and pH is 5.8.
(5) test-tube seedling transplanting: natural lighting lower refining seedling was opened and be placed in to blake bottle bottle cap after 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, the dliploid roxburgh anoectochilus terminal bud of planting in the matrix being mixed into by peat soil and pine bark (1:3) and routinely carries out field production management, and tetraploid roxburgh anoectochilus terminal bud survival rate reaches 95%.
Condition of culture in the each step in above-mentioned (2)～(4) is: 25 DEG C of cultivation temperature, illumination every day 10 hours, intensity of illumination 2000lx.
Embodiment tri-
1, cultivar origin
Guangxi product group training seedling is the high-quality roxburgh anoectochilus terminal bud after 1.5 years through field production;
2, cultivating process
(1) artificial pollination: carry out according to a conventional method cultivation management, roxburgh anoectochilus terminal bud is bloomed and carried out 95 days capsule maturations after cross pollination.
(2) non-symbiosis germination: grow and be mature on the whole and while not ftractureing at roxburgh anoectochilus terminal bud capsule, alcoholic solution sterilization through 80% is after 4 minutes, being placed on 0.1% mercuric chloride solution for 3 times with aseptic water washing sterilizes 15 minutes, after aseptic water washing 5 times, blot with aseptic filter paper, then capsule is cut along capsule y direction with aseptic scalpel, yellow embryo is inoculated on germination medium and is cultivated with transfer needle, cultivate seed germination after 25 days and become protocorm.The germination medium adopting is: 1.5g/L spends precious No. 1+1.0g/L peptone+80ml/L coconut milk+28g/L sucrose+7.0g/L agar+0.8g/L active carbon, and all the other are the organic components of MS medium, and pH is 5.8.
(3) mutagen processing: the protocorm that non-symbiosis germination is split into proceeds in the liquid differential medium that contains 0.03% colchicin and 0.02% trefanocide mixed liquor, under 26 DEG C of conditions, carry out intermittent oscillation mutagenic treatment 400 hours, dyed body counting, tetraploid induction rate is 75%.The liquid differential medium adopting is: 1/2MS+1.0mg/L 6-BA+0.4mg/L NAA+35g/L sucrose+2.0g/L peptone+0.1g/L acidic hydrolysis casein+0.6g/L active carbon, pH is 5.8.
(4) strong seedling culture: will be inoculated on strong seedling culture base and cultivate through the Anoectochilus roxburghii Protocorm of mutagenic treatment, further form the band root seedling that 5～8cm is high.The strong seedling culture base adopting is: 1.8g/L spends precious No. 1+1.2g/L to spend precious No. 2+0.6mg/LNAA+1.5g/L peptone+0.3g/L acidic hydrolysis casein+35g/L sucrose+4.0g/L agar+1.0g/L active carbon, all the other are the organic components of MS medium, and pH is 5.8.
(5) test-tube seedling transplanting: natural lighting lower refining seedling was opened and be placed in to blake bottle bottle cap after 6 days, test-tube plantlet is taken out from blake bottle, wash root medium off, the dliploid roxburgh anoectochilus terminal bud of planting in the matrix being mixed into by peat soil and pine bark (1:3) and routinely carries out field production management, and tetraploid roxburgh anoectochilus terminal bud survival rate reaches 96%.
Condition of culture in the each step in above-mentioned (2)～(4) is: 26 DEG C of cultivation temperature, illumination every day 12 hours, intensity of illumination 1500lx.
Claims (6)
1. a breeding method for tetraploid roxburgh anoectochilus terminal bud, is characterized in that comprising the following steps:
(1) artificial pollination: carry out according to a conventional method cultivation management, the good roxburgh anoectochilus terminal bud plant of selected strain, as parent, carries out cross pollination after roxburgh anoectochilus terminal bud is bloomed;
(2) non-symbiosis germination: grow and be mature on the whole and fruit while not ftractureing at roxburgh anoectochilus terminal bud capsule; alcoholic solution sterilization through 75%～80% is after 2～5 minutes; being placed on 0.1% mercuric chloride solution for 2～5 times with aseptic water washing sterilizes 8～15 minutes; after aseptic water washing 2～5 times, blot with aseptic filter paper; then capsule is cut along capsule y direction with aseptic scalpel; yellow embryo is inoculated on germination medium and is cultivated with transfer needle, cultivate seed germination after 20～30 days and become protocorm;
(3) mutagen processing: the protocorm that non-symbiosis germination is split into proceeds in the liquid differential medium that contains mutagen, carries out intermittent oscillation mutagenic treatment 240～480 hours under 25～28 DEG C of conditions;
(4) strong seedling culture: will be inoculated on strong seedling culture base and cultivate through the Anoectochilus roxburghii Protocorm of mutagenic treatment, further form the band root seedling that 5～8cm is high;
(5) test-tube seedling transplanting: natural lighting lower refining seedling was opened and be placed in to blake bottle bottle cap after 5～7 days; test-tube plantlet is taken out from blake bottle; wash root medium off, the dliploid roxburgh anoectochilus terminal bud of planting in the matrix being mixed into by peat soil and pine bark (1:3) and routinely carries out field production management;
Condition of culture in the each step in above-mentioned (2)～(4) is: 25～30 DEG C of cultivation temperature, illumination every day 10～15 hours, intensity of illumination 1500～2000lx.
2. the breeding method of a kind of tetraploid roxburgh anoectochilus terminal bud according to claim 1, it is characterized in that the described germination medium of step (2) is: 0.5～2.0g/L spends precious No. 1+0.2～2.0g/L peptone+100～200ml/L coconut milk+20～35g/L sucrose+3.0～7.0g/L agar+0.3～1.0g/L active carbon, all the other are the organic components of MS medium, and pH is 5.4～5.8.
3. the breeding method of a kind of tetraploid roxburgh anoectochilus terminal bud according to claim 1, it is characterized in that the described liquid differential medium of step (3) is: 1/2MS+0.2～1.2mg/L 6-BA+0.1～0.6mg/L NAA+20～35g/L sucrose+0.2～2.0g/L peptone+0.1～0.5g/L acidic hydrolysis casein+0.3～1.0g/L active carbon, pH is 5.4～5.8.
4. the breeding method of a kind of tetraploid roxburgh anoectochilus terminal bud according to claim 1, is characterized in that the described mutagen of step (3) are the mixed liquor of 0.01%～0.05% colchicin (olchicine)+0.01%～0.03% trefanocide (trifluralin).
5. the breeding method of a kind of tetraploid roxburgh anoectochilus terminal bud according to claim 1, it is characterized in that the described strong seedling culture base of step (4) is: 0.5～2.0g/L spends precious No. 1+0.5～2.0g/L to spend precious No. 2+0.5～1.5mg/LNAA+0.2～2.0g/L peptone+0.1～0.5g/L acidic hydrolysis casein+20～35g/L sucrose+3.0～7.0g/L agar+0.3～1.0g/L active carbon, all the other are the organic components of MS medium, and pH is 5.4～5.8.
6. the breeding method of a kind of tetraploid roxburgh anoectochilus terminal bud according to claim 1, is characterized in that the described test-tube plantlet of step (5) is to choose the test-tube plantlet that chromosome number is 2n=80 after dyed body counting.
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| CN105191807A (en) * | 2015-11-06 | 2015-12-30 | 广西相成生物科技有限公司 | Culturing method of polyploid anoectochilus roxburghii variety |
| CN105409764A (en) * | 2015-12-16 | 2016-03-23 | 惠安县科联农业科技有限公司 | Breeding method for tetraploid maize |
| CN105475127A (en) * | 2015-12-16 | 2016-04-13 | 惠安县科联农业科技有限公司 | Plant mutagenic agent and preparation and use methods thereof |
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| CN104429965A (en) * | 2014-12-16 | 2015-03-25 | 四川省自然资源科学研究院 | Hormone-free rapid tissue culture and propagation method of Emei roxburgh anoectochilus terminal bud seeds |
| WO2017076094A1 (en) * | 2015-11-03 | 2017-05-11 | 天津南开大学蓖麻工程科技有限公司 | Breeding method for tetraploid ricinus communis |
| US10881058B2 (en) | 2015-11-03 | 2021-01-05 | Tianjin Nankai University Castor Engineering Science And Technology Co., Ltd | Breeding method for tetraploid Ricinus communis |
| CN105191807A (en) * | 2015-11-06 | 2015-12-30 | 广西相成生物科技有限公司 | Culturing method of polyploid anoectochilus roxburghii variety |
| CN105409764A (en) * | 2015-12-16 | 2016-03-23 | 惠安县科联农业科技有限公司 | Breeding method for tetraploid maize |
| CN105475127A (en) * | 2015-12-16 | 2016-04-13 | 惠安县科联农业科技有限公司 | Plant mutagenic agent and preparation and use methods thereof |
| CN105660400A (en) * | 2016-01-25 | 2016-06-15 | 宁波易中禾生物技术有限公司 | Strengthening and weight increasing method for anoectochilus roxburghii tissue cultured seedlings |
| CN105660400B (en) * | 2016-01-25 | 2018-02-13 | 宁波易中禾生物技术有限公司 | A kind of strong sprout weight increasing method of roxburgh anoectochilus terminal bud tissue-cultured seedling |
| CN106508661B (en) * | 2016-11-01 | 2018-07-24 | 玉林师范学院 | A method of creating roxburgh anoectochilus terminal bud polyploid resource |
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