CN107787840A - A kind of establishing techniques of hymsleya amabilis tissue culture system - Google Patents
A kind of establishing techniques of hymsleya amabilis tissue culture system Download PDFInfo
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- CN107787840A CN107787840A CN201711124456.9A CN201711124456A CN107787840A CN 107787840 A CN107787840 A CN 107787840A CN 201711124456 A CN201711124456 A CN 201711124456A CN 107787840 A CN107787840 A CN 107787840A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of establishing techniques of hymsleya amabilis tissue culture system, hymsleya amabilis is cucurbitaceous plant, dried root.Originate in the ground such as Yunnan, Sichuan, Guizhou, Guangxi.Excavated during last years in autumn Huang, remove aerial part and silt, cleaned, cut into slices or do not cut into slices, dry.Root tuber is in irregular nahlock shape or ellipse, and slightly vertical tortoise shape, big person's diameter is up to 20cm.Crust brown color or taupe brown, micro- coarse, some has the stem foot trace of depression, section pale or taupe.Matter is solid, more silty.Gas is micro-, mildly bitter flavor.Its reproduction speed and scale are improved using plant tissue culture technique, so as to realize the factorial praluction of hymsleya amabilis seedling.The present invention, by inducing, breeding, taking root and the step such as acclimatization and transplantses successfully obtains hymsleya amabilis Regenerated Plantlets, establishes the hymsleya amabilis tissue culture plants regenerating system of system using stem with bud as explant.Breeding coefficient is high, and planting percent is high, and income is big.
Description
Technical field
The invention belongs to Plant Tissue Breeding class, specifically, is related to a kind of establishing techniques of hymsleya amabilis tissue culture system.
Background technology
Hymsleya amabilis is cucurbitaceous plant, dried root.Originate in the ground such as Yunnan, Sichuan, Guizhou, Guangxi.During hymsleya amabilis autumn last years Huang
Excavation, aerial part and silt are removed, cleaned, cut into slices or do not cut into slices, dry.Root tuber is in irregular nahlock shape or ellipse,
Slightly vertical tortoise shape, big person's diameter is up to 20cm.Crust brown color or taupe brown, micro- coarse, some has the stem foot trace of depression, section
Pale or taupe.Matter is solid, more silty.Gas is micro-, mildly bitter flavor.Commodity medicinal material is cut into slices more, is the sheet of similar round, is slightly rolled up
Song, thickness 4 ~ 8mm, 3 ~ 10cm of diameter.The first, second containing Hemsleyadin, oleanoside, panax japonicus glycosides, hymsleya amabilis glycosides MA1、MA3Deng.Have
It is clearing heat and detoxicating, the function of swelling and pain relieving.For abscess of throat, toothache, red eye, swell pain, dysentery, diarrhoea, stomachache etc..China for
Oneself obtains certain achievement for the further research of hymsleya amabilis, but remains the relevant issues that improved seeds are introduced and tamed,
A large amount of reports quickly bred of variety development and hymsleya amabilis for suitable China's most area ambient growth are still less.
Hymsleya amabilis breeding is more difficult, and its main modes of reproduction includes seminal propagation, cutting propagation and mound layering etc..It is numerous using seed
When growing, there is the problem of germination is difficult low with germination percentage in its seed, greatly reduce survival rate and planting percent, cost is higher, income
It is few；When being bred using the mode of cuttage and press strip, the branch of its cuttage is not easy to take root or more slow, it is not easy into
It is living, the problem of also result in hymsleya amabilis planting percent.Therefore, it is badly in need of establishing the hymsleya amabilis tissue culture plants regenerating system of system, with
Its reproduction speed and scale are improved, so as to realize the factorial praluction of hymsleya amabilis seedling.
The content of the invention
It is an object of the invention to provide one kind is gone out using stem with bud as explant, by inducing, breeding, taking root and refining
The steps such as transplantation of seedlings successfully obtain hymsleya amabilis Regenerated Plantlets, establish the hymsleya amabilis tissue culture plants of system raw body again
System, technology easily support are held, and seed seedling is blunt clearly, sturdy, a kind of establishing techniques of hymsleya amabilis tissue culture system of fast growing.
A kind of establishing techniques of hymsleya amabilis tissue culture system of the present invention, comprise the following steps：
（1）Explant sterilizes：The hymsleya amabilis stem section Yi Cheongju water that collection is returned rinses after 9min and gently brushes away with hairbrush above miscellaneous
Matter, with 9s is sterilized in 70% ethanol solution in superclean bench, washed 3 times with sterile, then rinsed with 4% peace for rich people's solution afterwards
5min, with standby after sucking moisture with aseptic filter paper after aseptic water washing 4 times；
（2）Fiber differentiation：By step（1）The stem section disinfected is cut by the stem section that paired bud is that a unit is cut into about 1.1cm length
Browning part during stem section bottom treatment is gone to, is inoculated into inducing culture and carries out bud inducement cultivation, first at 25 DEG C after inoculation
Under the conditions of full light culture 3 days, be subsequently placed in daily illumination 9 hours, intensity of illumination 1900lx, it is 25 DEG C to be placed in cultivation temperature
Under the conditions of culture until formed adventitious bud;
（3）Multiplying culture：By step（2）Cultivate obtained sprout, from base portion cut bud and being transferred on proliferated culture medium carry out after
It is commissioned to train foster, first full light culture 4 days under the conditions of 25 DEG C after inoculation, then daily illumination 10 hours, intensity of illumination 3100lx, training
Support under conditions of temperature is 25 DEG C and cultivate, 29 days subcultures are once；
（4）Culture of rootage：By step（2）Or（3）Sprout length to 1.3cm it is high when, cut into simple bud and be inoculated into culture of rootage
Culture of rootage is carried out on base, first full light culture 5 days under the conditions of 25 DEG C after inoculation, then daily illumination 13 hours, intensity of illumination
For 2900lx, cultivation temperature is cultivated under conditions of being 25 DEG C to taking root；
（5）Acclimatization and transplantses：The culture bottle cap of the long rooting tube plantlet to 3cm is opened and is placed in natural lighting lower refining seedling 5 days
Afterwards, test tube seedling is taken out from blake bottle, washes root culture medium off, do not injure root as far as possible, plant into by peat soil:Vermiculite=2：
In daily illumination 9 hours, intensity of illumination 2900lx in 2 matrix being mixed into, cultivation temperature is 25 DEG C, air humidity 78%
Incubator in cultivate 5 days, then transplant planting.
Step（2）Described inducing culture is：MS+0.19mg/L 6-BA+15/L sucrose+3.5g/L agar, pH are
5.8。
Step（3）Described proliferated culture medium is：MS+0.3mg/L NAA+0.19mg/L 6-BA+15g/L sucrose+
3.5g/L agar, pH 5.8.
Step（4）Described root media is：MS+0.5mg/L IBA+15g/L sucrose+3.5g/L agar, pH are
5.8。
It is an advantage of the invention that：The present invention is using hymsleya amabilis stem with bud as explant, by inducing, breeding, taking root and refining
The steps such as transplantation of seedlings successfully obtain hymsleya amabilis Regenerated Plantlets, establish the hymsleya amabilis tissue culture plants regeneration of system
System.So as to which the seminal propagation coefficient obtained is high, planting percent is high, cost is low, income is big, improved using plant tissue culture technique
Its reproduction speed and scale, realize the factorial praluction of hymsleya amabilis seedling.Technical method is simple, and equipment is few, and seedling is fast, productivity ratio
Height, synergy good harvest.
Embodiment
Following examples are the further explanations to the present invention, are not limitations of the present invention.
Embodiment 1
（1）Explant sterilizes：The hymsleya amabilis stem section Yi Cheongju water that collection is returned rinses after 10min and gently brushes away with hairbrush above miscellaneous
Matter, with 10s is sterilized in 75% ethanol solution in superclean bench, washed 5 times with sterile, then floated with 5% peace for rich people's solution afterwards
6min is washed, with standby after sucking moisture with aseptic filter paper after aseptic water washing 5 times.
（2）Fiber differentiation：By step（1）The hymsleya amabilis stem section disinfected is that a unit is cut into about 1.5cm length by paired bud
Stem section, cut off browning part during stem section bottom treatment, be inoculated into inducing culture and carry out bud inducement cultivation.After inoculation
First full light culture 4 days under the conditions of 27 DEG C, then daily illumination 10 hours, intensity of illumination 1900lx, cultivation temperature are 27 DEG C
Under conditions of culture until formed adventitious bud, inductivity 92%.Described inducing culture is：MS+0.6mg/L 6-BA+
18g/L sucrose+3.8g/L agar, pH 5.5.
（3）Multiplying culture：By step（2）Obtained sprout is cultivated, bud is cut from base portion and to be transferred to proliferated culture medium enterprising
Row squamous subculture, first full light culture 3 days under the conditions of 29 DEG C after inoculation, then daily illumination 12 hours, intensity of illumination are
2600lx, cultivation temperature be 21 DEG C under conditions of cultivate, 31 days subcultures once, growth coefficient 5.8.Described proliferated culture medium
For：MS+0.18mg/L NAA+1.8mg/L 6-BA+25g/L sucrose+3.8g/L agar, pH 5.5.
（4）Culture of rootage：By step（2）Or（3）Sprout length to 1.65cm it is high when, cut into simple bud and be inoculated into and take root
Culture of rootage is carried out on culture medium, first full light culture 3 days under the conditions of 27 DEG C after inoculation, then daily illumination 14 hours, illumination
Intensity is 3200lx, and cultivation temperature is cultivated under conditions of being 27 DEG C to taking root.Described root media is：MS+1.8mg/L
IBA+25g/L sucrose+4.5g/L agar, pH 5.5.
（5）Acclimatization and transplantses：The culture bottle cap of the long rooting tube plantlet to 4cm is opened into simultaneously natural lighting lower refining seedling 6 days
Afterwards, test tube seedling is taken out from blake bottle, washes root culture medium off, do not injure root as far as possible, plant into by peat soil:Vermiculite=2：
In daily illumination 13 hours, intensity of illumination 3000lx in 2 matrix being mixed into, cultivation temperature is 27 DEG C, air humidity 78%
Incubator in cultivate 6 days, then transplant planting, transplanting survival rate 94%.
Embodiment 2
（1）Explant sterilizes：The hymsleya amabilis stem section Yi Cheongju water that collection is returned rinses after 9min and gently brushes away with hairbrush above miscellaneous
Matter, with 17s is sterilized in 75% ethanol solution in superclean bench, washed 3 times with sterile, then floated with 5% peace for rich people's solution afterwards
5min is washed, with standby after sucking moisture with aseptic filter paper after aseptic water washing 3 times.
（2）Fiber differentiation：By step（1）The hymsleya amabilis stem section disinfected is that a unit is cut into about 1.1cm length by paired bud
Stem section, cut off browning part during stem section bottom treatment, be inoculated into inducing culture and carry out bud inducement cultivation.After inoculation
First full light culture 2 days under the conditions of 26 DEG C, then daily illumination 9 hours, intensity of illumination 2200lx, cultivation temperature are 26 DEG C
Under the conditions of culture until formed adventitious bud, inductivity 91%.Described inducing culture is：MS+1.1mg/L 6-BA+17g/L
Sucrose+3.9g/L agar, pH 5.6.
（3）Multiplying culture：By step（2）Obtained sprout is cultivated, bud is cut from base portion and to be transferred to proliferated culture medium enterprising
Row squamous subculture, first full light culture 1 day under the conditions of 26 DEG C after inoculation, then daily illumination 11 hours, intensity of illumination are
3700lx, cultivation temperature be 26 DEG C under conditions of cultivate, 27 days subcultures once, growth coefficient 5.4.Described proliferated culture medium
For：MS+0.3mg/L NAA+1.1mg/L 6-BA+23g/L sucrose+4.4g/L agar, pH 5.8.
（4）Culture of rootage：By step（2）Or（3）Sprout length to 1.1cm it is high when, cut into simple bud and be inoculated into and take root
Culture of rootage is carried out on culture medium, first full light culture 1 day under the conditions of 26 DEG C after inoculation, then daily illumination 12 hours, illumination
Intensity is 3700lx, and cultivation temperature is cultivated under conditions of being 23 DEG C to taking root.Described root media is：MS+2.6mg/L
IBA+20g/L sucrose+3.8g/L agar, pH 5.5.
（5）Acclimatization and transplantses：The culture bottle cap of the long rooting tube plantlet to 1cm is opened and is placed in natural lighting lower refining seedling
After 3 days, test tube seedling is taken out from blake bottle, washes root culture medium off, does not injure root as far as possible, plant into by peat soil:Vermiculite
=2：In daily illumination 8 hours, intensity of illumination 2700lx in 2 matrix being mixed into, cultivation temperature is 23 DEG C, and air humidity is
Cultivate 3 days in 78% incubator, then transplant planting, transplanting survival rate 95%.
Claims (4)
1. a kind of establishing techniques of hymsleya amabilis tissue culture system, it is characterised in that comprise the following steps：
（1）Explant sterilizes：The hymsleya amabilis stem section Yi Cheongju water that collection is returned rinses after 9min and gently brushes away with hairbrush above miscellaneous
Matter, with 9s is sterilized in 70% ethanol solution in superclean bench, washed 3 times with sterile, then rinsed with 4% peace for rich people's solution afterwards
5min, with standby after sucking moisture with aseptic filter paper after aseptic water washing 4 times；
（2）Fiber differentiation：By step（1）The stem section disinfected is cut by the stem section that paired bud is that a unit is cut into about 1.1cm length
Browning part during stem section bottom treatment is gone to, is inoculated into inducing culture and carries out bud inducement cultivation, first at 25 DEG C after inoculation
Under the conditions of full light culture 3 days, be subsequently placed in daily illumination 9 hours, intensity of illumination 1900lx, it is 25 DEG C to be placed in cultivation temperature
Under the conditions of culture until formed adventitious bud;
（3）Multiplying culture：By step（2）Cultivate obtained sprout, from base portion cut bud and being transferred on proliferated culture medium carry out after
It is commissioned to train foster, first full light culture 4 days under the conditions of 25 DEG C after inoculation, then daily illumination 10 hours, intensity of illumination 3100lx, training
Support under conditions of temperature is 25 DEG C and cultivate, 29 days subcultures are once；
（4）Culture of rootage：By step（2）Or（3）Sprout length to 1.3cm it is high when, cut into simple bud and be inoculated into culture of rootage
Culture of rootage is carried out on base, first full light culture 5 days under the conditions of 25 DEG C after inoculation, then daily illumination 13 hours, intensity of illumination
For 2900lx, cultivation temperature is cultivated under conditions of being 25 DEG C to taking root；
（5）Acclimatization and transplantses：The culture bottle cap of the long rooting tube plantlet to 3cm is opened and is placed in natural lighting lower refining seedling 5 days
Afterwards, test tube seedling is taken out from blake bottle, washes root culture medium off, do not injure root as far as possible, plant into by peat soil:Vermiculite=2：
In daily illumination 9 hours, intensity of illumination 2900lx in 2 matrix being mixed into, cultivation temperature is 25 DEG C, air humidity 78%
Incubator in cultivate 5 days, then transplant planting.
A kind of 2. establishing techniques of hymsleya amabilis tissue culture system according to claim 1, it is characterised in that step（2）Described lures
Leading culture medium is：MS+0.19mg/L 6-BA+15/L sucrose+3.5g/L agar, pH 5.8.
A kind of 3. establishing techniques of hymsleya amabilis tissue culture system according to claim 1, it is characterised in that step（3）Described increasing
Growing culture medium is：MS+0.3mg/L NAA+0.19mg/L 6-BA+15g/L sucrose+3.5g/L agar, pH 5.8.
A kind of 4. establishing techniques of hymsleya amabilis tissue culture system according to claim 1, it is characterised in that step（4）Described life
Root culture medium is：MS+0.5mg/L IBA+15g/L sucrose+3.5g/L agar, pH 5.8.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109287481A (en) * | 2018-10-29 | 2019-02-01 | 云南善源生物科技发展有限公司 | A kind of tissue culture medium of hymsleya amabilis and its mating system |
| CN109315289A (en) * | 2018-10-09 | 2019-02-12 | 云南善源生物科技发展有限公司 | Hymsleya amabilis Novel sterilizing mode |
| CN110214694A (en) * | 2019-06-30 | 2019-09-10 | 浙江大学 | Zhejiang hymsleya amabilis is female, staminiferous plant quick breeding by group culture method |
| CN111480576A (en) * | 2020-05-20 | 2020-08-04 | 西南林业大学 | Method for inducing hemsleya amabilis polyploid plant by using adventitious buds and application of method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106342684A (en) * | 2016-08-28 | 2017-01-25 | 李志勇 | Establishing method of scrophularia ningpoensis tissue culture system |
-
2017
- 2017-11-14 CN CN201711124456.9A patent/CN107787840A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106342684A (en) * | 2016-08-28 | 2017-01-25 | 李志勇 | Establishing method of scrophularia ningpoensis tissue culture system |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109315289A (en) * | 2018-10-09 | 2019-02-12 | 云南善源生物科技发展有限公司 | Hymsleya amabilis Novel sterilizing mode |
| CN109287481A (en) * | 2018-10-29 | 2019-02-01 | 云南善源生物科技发展有限公司 | A kind of tissue culture medium of hymsleya amabilis and its mating system |
| CN110214694A (en) * | 2019-06-30 | 2019-09-10 | 浙江大学 | Zhejiang hymsleya amabilis is female, staminiferous plant quick breeding by group culture method |
| CN111480576A (en) * | 2020-05-20 | 2020-08-04 | 西南林业大学 | Method for inducing hemsleya amabilis polyploid plant by using adventitious buds and application of method |
| CN111480576B (en) * | 2020-05-20 | 2021-07-30 | 西南林业大学 | Method for inducing hemsleya amabilis polyploid plant by using adventitious buds and application of method |
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