CN104686334A - Tissue culture and rapid propagation method for androsace longifolia - Google Patents
Tissue culture and rapid propagation method for androsace longifolia Download PDFInfo
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- CN104686334A CN104686334A CN201510085924.0A CN201510085924A CN104686334A CN 104686334 A CN104686334 A CN 104686334A CN 201510085924 A CN201510085924 A CN 201510085924A CN 104686334 A CN104686334 A CN 104686334A
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Abstract
The invention discloses a tissue culture and rapid propagation method for androsace longifolia, and relates to a method for quickly obtaining a larger number of seedlings through a rapid asexual propagation technology of androsace longifolia. According to the tissue culture and rapid propagation method disclosed by the invention, the androsace longifolia seed is as taken as an explant, a tissue culture and rapid propagation system of androsace longifolia is established through steps of callus induction, adventitious bud induction, rooting culture, seedling hardening, transplanting and the like, and reference is provided for culturing a novel androsace longifolia variety with large flowers, bright colors and high ornamental values through methods of polyploid breeding, transgenosis and the like in future.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to one and to come into leaves Androsace umbellata tissue cultivation rapid breeding method.
Background technology
Come into leaves Androsace umbellata (
androsace longifolia) be a kind of herbaceous plant that Primulaceae Androsace umbellata belongs to, though its plant is short and small, Dan Duohua is little, pattern is pure and fresh, has good wear tolerance to be, is a kind of rare flower of early spring, has very high ornamental value.Come into leaves the Androsace umbellata medical value that also tool is higher, and it is rich in flavonoids, triterpene compound, has the pharmacological action that inhibition cancer cell increases.
At present, the Androsace umbellata that comes into leaves has to be viewed and admired and medicinal dual-use function, but rarely has report to its correlative study, and it is in wild state always.Natural ecological environment along with China suffers gradually to destroy and even worsens, and the growth Androsace umbellata resource that comes into leaves in the wild also receives destruction in various degree, is therefore badly in need of foundation and comes into leaves Androsace umbellata rapid propagation in vitro technical system, to reach the object of preserving its germ plasm resource.
Summary of the invention
One is the object of the present invention is to provide out to come into leaves Androsace umbellata tissue cultivation rapid breeding method, the present invention with the Androsace umbellata seed that comes into leaves for explant, set up through callus induction, adventitious bud inducing, culture of rootage, hardening and transplanting and other steps the Androsace umbellata tissue culture quick breeding system that comes into leaves, thus achieve object of the present invention.
One of the present invention comes into leaves Androsace umbellata tissue cultivation rapid breeding method, comprises the following steps:
(1) seed germination: the wild Androsace umbellata seed that comes into leaves of field acquisition is first rinsed 1 ~ 3h with washing powder water, then in superclean bench, strip off exosper, first use after 75% ethanol disinfection 5 ~ 30s with aseptic washing 3 ~ 5 times, to sterilize 10 ~ 25min with 1 ~ 5% liquor natrii hypochloritis again, with being put in sterile petri dish after aseptic water washing 4 ~ 6 times, (culture dish bottom spreads the thin absorbent cotton of one deck, on absorbent cotton, cover sterilizing after filter paper again) filter paper on, add appropriate amounts of sterilized water, then illumination every day is placed in 10 ~ 12 hours, intensity of illumination is 1500 ~ 3000lx, until seed germination, period regularly adds appropriate amounts of sterilized water in culture dish, with ensure absorbent cotton and filter paper moistening.
(2) callus induction: the seedling hypocotyl of the height about 1cm of firm sprouting step (1) obtained is cut and is inoculated on inducing culture, first full light culture 30 ~ 45 days under 25 ~ 28 DEG C of conditions, then illumination every day is placed in 10 ~ 12 hours, intensity of illumination is 1500 ~ 3000lx, until induced synthesis embryo callus.
(3) differentiation is cultivated: step (2) is cultivated the greeny Cotyledon Callus obtained and is cut into 0.5cm
3the fritter of size proceeds on differential medium and carries out differentiation cultivation, first full light culture 7 ~ 14 days under 25 ~ 28 DEG C of conditions after inoculation, then be placed in illumination every day 12 ~ 16 hours, intensity of illumination is 3000 ~ 5000lx, and being placed in cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C until form indefinite bud.
(4) culture of rootage: the indefinite bud that the height that step (3) process obtains is about 2.5 ~ 3.5cm is cut to be inoculated on root media and carries out culture of rootage, first full light culture 7 ~ 14 days under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 14 ~ 16 hours, intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root.
(5) acclimatization and transplants: the rooting tube plantlet decap of height about 6 ~ 8cm step (4) obtained was placed in natural lighting lower refining seedling after 5 ~ 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, plant into by Nutrition Soil: the matrix that sandy soil=2:1 is mixed into, be placed in illumination box and cultivate, every day waters to seedling with 1/4MS macro-element nutrients liquid, keeps humidity.Land for growing field crops is transplanted again after seedling survives.
Inducing culture described in above-mentioned steps (2) is: MS+0.1 ~ 0.6mg/L6-BA+0.1 ~ 0.5mg/L NAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Differential medium described in above-mentioned steps (3) is: MS+0.01 ~ 0.1mg/LTDZ+0.1 ~ 0.5mg/L KT+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Root media described in above-mentioned steps (4) is: MS+1 ~ 5mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
Compared with prior art advantage of the present invention is: the present invention obtains the method for a large amount of seedling fast by Fast Asexual Propagation Technique.With the Androsace umbellata seed that comes into leaves for explant, setting up through callus induction, adventitious bud inducing, culture of rootage, hardening and transplanting and other steps the Androsace umbellata tissue culture quick breeding system that comes into leaves, spending the Androsace umbellata new varieties that come into leaves large, look gorgeous, ornamental value is high to provide reference for being cultivated by the method such as polyploid breeding, transgenosis from now on.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
Embodiment 1:
(1) seed germination: the wild Androsace umbellata seed that comes into leaves of field acquisition is first rinsed 1h with washing powder water, then in superclean bench, strip off exosper, first use after 75% ethanol disinfection 5s with aseptic washing 3 times, to sterilize 10min with 2% liquor natrii hypochloritis again, with being put in sterile petri dish after aseptic water washing 4 times, (culture dish bottom spreads the thin absorbent cotton of one deck, on absorbent cotton, cover sterilizing after filter paper again) filter paper on, add appropriate amounts of sterilized water, then illumination every day is placed in 10 hours, intensity of illumination is 1500lx, until seed germination, period regularly adds appropriate amounts of sterilized water in culture dish, with ensure absorbent cotton and filter paper moistening, seed germination rate can reach more than 95%.
(2) callus induction: the seedling hypocotyl of the height about 1cm of firm sprouting step (1) obtained is cut and is inoculated on inducing culture, first full light culture 35 days under 28 DEG C of conditions, then illumination every day is placed in 10 hours, intensity of illumination is cultivate under the condition of 1500lx can form embryo callus in 10 days, and inductivity reaches 88.9%.Described inducing culture is MS+0.3mg/L6-BA+0.1mg/L NAA+15g/L sucrose+3.5g/L agar, and pH is 5.8.
(3) differentiation is cultivated: step (2) is cultivated the greeny Cotyledon Callus obtained and is cut into 0.5cm
3the fritter of size proceeds on differential medium and carries out differentiation cultivation, first full light culture 7 days under 28 DEG C of conditions after inoculation, then be placed in illumination every day 12 hours, intensity of illumination is 3000lx, and being placed in cultivation temperature is that under the condition of 28 DEG C, cultivation can form indefinite bud in 14 days.Described differential medium is MS+0.05mg/LTDZ+0.1mg/L KT+15g/L sucrose+3.5g/L agar, and pH is 5.8.
(4) culture of rootage: the indefinite bud that the height that step (3) process obtains is about 2.5 ~ 3.5cm is cut to be inoculated on root media and carries out culture of rootage, first full light culture 7 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 14 hours, intensity of illumination is 2000lx, cultivation temperature is cultivate under the condition of 28 DEG C can take root for 18 days, and rooting rate reaches 87.9%.Described root media is MS+1mg/L IBA+30g/L sucrose+3.5g/L agar, and pH is 5.8.
(5) acclimatization and transplants: the rooting tube plantlet decap of height about 6 ~ 8cm step (4) obtained was placed in natural lighting lower refining seedling after 5 days, test-tube plantlet is taken out from blake bottle, wash root medium off, plant into by Nutrition Soil: the matrix that sandy soil=2:1 is mixed into, be placed in illumination box and cultivate, every day waters to seedling with 1/4MS macro-element nutrients liquid, keeps humidity.After seedling survives, transplant land for growing field crops again, transplanting survival rate can reach more than 90%.
Embodiment 2:
(1) seed germination: the wild Androsace umbellata seed that comes into leaves of field acquisition is first rinsed 2h with washing powder water, then in superclean bench, strip off exosper, first use after 75% ethanol disinfection 8s with aseptic washing 3 times, to sterilize 10min with 4% liquor natrii hypochloritis again, with being put in sterile petri dish after aseptic water washing 4 times, (culture dish bottom spreads the thin absorbent cotton of one deck, on absorbent cotton, cover sterilizing after filter paper again) filter paper on, add appropriate amounts of sterilized water, then illumination every day is placed in 11 hours, intensity of illumination is 1500lx, until seed germination, period regularly adds appropriate amounts of sterilized water in culture dish, with ensure absorbent cotton and filter paper moistening, seed germination rate can reach more than 91%.
(2) callus induction: the seedling hypocotyl of the height about 1cm of firm sprouting step (1) obtained is cut and is inoculated on inducing culture, first full light culture 35 days under 28 DEG C of conditions, then illumination every day is placed in 10 hours, intensity of illumination is cultivate under the condition of 1500lx can form embryo callus in 10 days, and inductivity reaches 81%.Described inducing culture is MS+0.5mg/L6-BA+0.2mg/L NAA+15g/L sucrose+3.5g/L agar, and pH is 5.8.
(3) differentiation is cultivated: step (2) is cultivated the greeny Cotyledon Callus obtained and is cut into 0.5cm
3the fritter of size proceeds on differential medium and carries out differentiation cultivation, first full light culture 7 days under 28 DEG C of conditions after inoculation, then be placed in illumination every day 12 hours, intensity of illumination is 3000lx, and being placed in cultivation temperature is that under the condition of 28 DEG C, cultivation can form indefinite bud in 14 days.Described differential medium is MS+0.1mg/LTDZ+0.3mg/L KT+15g/L sucrose+3.5g/L agar, and pH is 5.8.
(4) culture of rootage: the indefinite bud that the height that step (3) process obtains is about 2.5 ~ 3.5cm is cut to be inoculated on root media and carries out culture of rootage, first full light culture 7 days under 28 DEG C of conditions after inoculation, then illumination every day is placed in 14 hours, intensity of illumination is 2000lx, cultivation temperature is cultivate under the condition of 28 DEG C can take root for 18 days, and rooting rate reaches 90%.Described root media is MS+3mg/L IBA+30g/L sucrose+6.0g/L agar, and pH is 5.8.
(5) acclimatization and transplants: the rooting tube plantlet decap of height about 6 ~ 8cm step (4) obtained was placed in natural lighting lower refining seedling after 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, plant into by Nutrition Soil: the matrix that sandy soil=2:1 is mixed into, be placed in illumination box and cultivate, every day waters to seedling with 1/4MS macro-element nutrients liquid, keeps humidity.After seedling survives, transplant land for growing field crops again, transplanting survival rate can reach more than 85%.
Claims (4)
1. come into leaves an Androsace umbellata tissue cultivation rapid breeding method, it is characterized in that comprising the following steps:
(1) seed germination: the wild Androsace umbellata seed that comes into leaves of field acquisition is first rinsed 1 ~ 3h with washing powder water, then in superclean bench, strip off exosper, first use after 75% ethanol disinfection 5 ~ 30s with aseptic washing 3 ~ 5 times, to sterilize 10 ~ 25min with 1 ~ 5% liquor natrii hypochloritis again, sterile petri dish is put in after aseptic water washing 4 ~ 6 times, culture dish bottom spreads the thin absorbent cotton of one deck, cover on the filter paper of sterilizing after filter paper again on absorbent cotton, add appropriate amounts of sterilized water, then illumination every day is placed in 10 ~ 12 hours, intensity of illumination is 1500 ~ 3000lx, until seed germination, period regularly adds appropriate amounts of sterilized water in culture dish, with ensure absorbent cotton and filter paper moistening,
(2) callus induction: the seedling hypocotyl of the height about 1cm of firm sprouting step (1) obtained is cut and is inoculated on inducing culture, first full light culture 30 ~ 45 days under 25 ~ 28 DEG C of conditions, then illumination every day is placed in 10 ~ 12 hours, intensity of illumination is 1500 ~ 3000lx, until induced synthesis embryo callus;
(3) differentiation is cultivated: step (2) is cultivated the greeny Cotyledon Callus obtained and is cut into 0.5cm
3the fritter of size proceeds on differential medium and carries out differentiation cultivation, first full light culture 7 ~ 14 days under 25 ~ 28 DEG C of conditions after inoculation, then be placed in illumination every day 12 ~ 16 hours, intensity of illumination is 3000 ~ 5000lx, and being placed in cultivation temperature is cultivate until form indefinite bud under the condition of 25 ~ 28 DEG C;
(4) culture of rootage: the indefinite bud that the height that step (3) process obtains is about 2.5 ~ 3.5cm is cut to be inoculated on root media and carries out culture of rootage, first full light culture 7 ~ 14 days under 25 ~ 28 DEG C of conditions after inoculation, then illumination every day is placed in 14 ~ 16 hours, intensity of illumination is 2000 ~ 3000lx, and cultivation temperature is be cultured under the condition of 25 ~ 28 DEG C to take root;
(5) acclimatization and transplants: the rooting tube plantlet decap of height about 6 ~ 8cm step (4) obtained was placed in natural lighting lower refining seedling after 5 ~ 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, plant into by Nutrition Soil: the matrix that sandy soil=2:1 is mixed into, be placed in illumination box and cultivate, every day waters to seedling with 1/4MS macro-element nutrients liquid, keeps humidity, after seedling survives, transplants land for growing field crops again.
2. one according to claim 1 comes into leaves Androsace umbellata tissue cultivation rapid breeding method, it is characterized in that the inducing culture described in step (2) is: MS+0.1 ~ 0.6mg/L6-BA+0.1 ~ 0.5mg/L NAA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
3. one according to claim 1 comes into leaves Androsace umbellata tissue cultivation rapid breeding method, it is characterized in that the differential medium described in step (3) is: MS+0.01 ~ 0.1mg/LTDZ+0.1 ~ 0.5mg/L KT+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
4. one according to claim 1 comes into leaves Androsace umbellata tissue cultivation rapid breeding method, it is characterized in that the root media described in step (4) is: MS+1 ~ 5mg/L IBA+15 ~ 30g/L sucrose+3.5 ~ 6.0g/L agar, pH is 5.4 ~ 5.8.
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| CN201510085924.0A CN104686334A (en) | 2015-02-22 | 2015-02-22 | Tissue culture and rapid propagation method for androsace longifolia |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106688885A (en) * | 2016-11-15 | 2017-05-24 | 邹少英 | Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation |
| CN108781972A (en) * | 2018-06-02 | 2018-11-13 | 彭春剑 | A kind of nut-seed seedling culture method |
| CN114303947A (en) * | 2021-12-28 | 2022-04-12 | 浙江宜格企业管理集团有限公司 | Preparation method of plum blossom callus |
| CN116584386A (en) * | 2023-05-24 | 2023-08-15 | 北京林业大学 | Tissue culture medium for waxberries, germination method of waxberries seeds and tissue culture and rapid propagation method of waxberries |
-
2015
- 2015-02-22 CN CN201510085924.0A patent/CN104686334A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 张彦妮等: ""长叶点地梅愈伤组织诱导和植株再生"", 《草业科学》 * |
| 陈素波: ""长叶点地梅（Androsace longifolia）组织培养与细胞悬浮培养条件初探"", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106688885A (en) * | 2016-11-15 | 2017-05-24 | 邹少英 | Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation |
| CN108781972A (en) * | 2018-06-02 | 2018-11-13 | 彭春剑 | A kind of nut-seed seedling culture method |
| CN114303947A (en) * | 2021-12-28 | 2022-04-12 | 浙江宜格企业管理集团有限公司 | Preparation method of plum blossom callus |
| CN114303947B (en) * | 2021-12-28 | 2023-03-03 | 浙江宜格企业管理集团有限公司 | Preparation method of plum blossom callus |
| CN116584386A (en) * | 2023-05-24 | 2023-08-15 | 北京林业大学 | Tissue culture medium for waxberries, germination method of waxberries seeds and tissue culture and rapid propagation method of waxberries |
| CN116584386B (en) * | 2023-05-24 | 2024-03-19 | 北京林业大学 | Tissue culture medium for waxberries, germination method of waxberries seeds and tissue culture and rapid propagation method of waxberries |
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Application publication date: 20150610 |