CN104255531A - Rapid propagation methods for adenantherapavonlna suspension cell and regeneration plant - Google Patents
Rapid propagation methods for adenantherapavonlna suspension cell and regeneration plant Download PDFInfo
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- CN104255531A CN104255531A CN201410550655.6A CN201410550655A CN104255531A CN 104255531 A CN104255531 A CN 104255531A CN 201410550655 A CN201410550655 A CN 201410550655A CN 104255531 A CN104255531 A CN 104255531A
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- suspension cell
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- induction
- regeneration plant
- regeneration
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Abstract
The invention studies a rapid propagation method for an adenantherapavonlna suspension cell and a regeneration plant. The rapid propagation method comprises the steps of disinfecting an explant, inducing callus, culturing the suspension cell, inducing an embryonal suspension cell, inducting rooting and the like. An adenantherapavonlna embryonal cell suspension system is created, the adenantherapavonlna regeneration plant is obtained, so that the material is provided for the development and utilization of the medicinal component, a large number of adenantherapavonlna regeneration plants are also acquired, and a foundation is laid for the large-scale cultivation of the adenantherapavonlna regeneration plants.
Description
Technical field
The present invention relates to the method for circassian bean suspension cell and regenerated plant culture, belong to biological technical field.
Background technology
Circassian bean,
adenantherapavonlna, pulse family, defoliation small arbor, high 5-10m, two times winglike compound leaves, tool short handle, petiole and rachis by micro-pubescence, Non-gland body, alternate, two sides is all by micro-pubescence; Raceme list is born in axil or is lined up panicle at Zhi Ding, by pubescence, spends little, white or faint yellow, savory, the short stalk of tool, and calyx is long less than 1mm, same by golden yellow pubescence with bennet, petal 5, lanceolar, long 2.5-3mm, and without hair, base portion is symphysis slightly; The long and narrow circle of pod, spirals, the circumvolution of cracking consequence lobe; Seed subcircular is extremely oval, long 5-8mm, wide 4.5-7mm, and cerise is glossy, the florescence 4-7 month, the fruit phase 7-10 month.Circassian bean happiness warm and moist weather, happiness light, slightly resistance to shade, require stricter to soil condition, deep, fertile, the well-drained sandy loam of happiness soil layer, originate in torrid areas, Burma, Cambodia, Laos, Vietnam, Malaysia, Indonesia, China, at distribution in China in Fujian, Taiwan, Guangdong, Hainan, Guangxi, Guizhou, the ground such as Yunnan.Circassian bean seed contains stigmasterol, stigmasterol glycoside, dulcin, polysaccharide and protease inhibitors and 8 kinds of trypsase isoinhibitor DE1-DE8 etc., and root has emetic, discharge function; Ye Ze has astriction, can be used for antidiarrheal, wind and heat dispersing, and eliminating dampness is antipruritic, moisturizing beauty treatment, at present main employing seed and cottage propagation, and cost is high, and survival rate is on the low side, still can not meet the need of market.
Summary of the invention
Technical problem to be solved by this invention is the propagation method of circassian bean suspension cell culture, establish circassian bean cells,primordial suspension system and obtain and strangle circassian bean regeneration plant, the development and utilization not being only its medicinal ingredient is supplied raw materials, also can obtain a large amount of circassian bean regeneration plants, cultivate on a large scale for it and lay the foundation.
For solving the problems of the technologies described above, the present invention adopts following technical proposal:
Get circassian bean blade, 3min is soaked in washing powder, running water 15min, 1% bromine water process 8min on superclean bench, running water 5 times, callus induction is carried out in the blade access MS+3-4 μm of ol/L vitamin h+0.3mg/L NAA+0.5-1 mg/L brassinosteroid medium of disinfecting, additional 7g/L agar, 30g/L lactose, temperature 25 DEG C, light culture, the callus access medium MS+500-600 μm of ol/L glutamine+3-4 μm of ol/L vitamin h+2 derived, suspension cell culture is carried out in 4-D 0.2mg/L, additional 30g/L lactose, temperature 25 DEG C, light culture, rotary shaker vibrates, 110r/min, suspension cell is in the first month cultivated, change a fresh culture weekly, the bulky grain culture not easily disperseed with 800 μm of aperture screen cloth filterings, comprising callus group, He Hua thanatogenic tissue, some proembryo etc., thereafter within every 2 weeks, a culture fluid is changed, after culture comparatively disperses, larger cell mass is removed with 150 μm of aperture sieve net filtrations, embryonal suspension cell induction is carried out in the suspension cell access medium MS+CPPU 0.3mg/L+NAA 0.2mg/L+ZT 0.5mg/L cultivated, the cells,primordial access derived does not carry out root induction containing in the MD medium of any hormone, the somatic embryo at plant regeneration initial stage is sprouted and is carried out under dark condition, 28 DEG C are transferred to after light green color leaf sheath is extracted out, 30 μm of olm
-2s
-1light intensity (white fluorescent lamp), takes root under 16h/8h photoperiod condition, obtains regeneration plant.
It is high that the circassian bean cell adopting the present invention to prepare and regeneration plant are proliferated into motility rate, and growth cycle is short, and production process is simple and easy to control, is beneficial to large-scale production and cultivation.
Below in conjunction with embodiment, the present invention is further elaborated, but the scope of protection of present invention is not limited to following embodiments.
Embodiment
Embodiment 1
Get circassian bean blade, 3min is soaked in washing powder, running water 15min, 1% bromine water process 8min on superclean bench, running water 5 times, callus induction is carried out in the blade access MS+3 μm of ol/L vitamin h+0.3mg/LNAA+0.5mg/L brassinosteroid medium of disinfecting, additional 7g/L agar, 30g/L lactose, temperature 25 DEG C, light culture, callus access medium MS+500 μm of ol/L glutamine+3 μm of ol/L vitamin hs+2 derived, suspension cell culture is carried out in 4-D0.2mg/L, additional 30g/L lactose, temperature 25 DEG C, light culture, rotary shaker vibrates, 110r/min, suspension cell is in the first month cultivated, change a fresh culture weekly, the bulky grain culture not easily disperseed with 800 μm of aperture screen cloth filterings, comprising callus group, He Hua thanatogenic tissue, some proembryo etc., thereafter within every 2 weeks, a culture fluid is changed, after culture comparatively disperses, larger cell mass is removed with 150 μm of aperture sieve net filtrations, embryonal suspension cell induction is carried out in the suspension cell access medium MS+CPPU0.3mg/L+NAA0.2mg/L+ZT0.5mg/L cultivated, the cells,primordial access derived does not carry out root induction containing in the MD medium of any hormone, the somatic embryo at plant regeneration initial stage is sprouted and is carried out under dark condition, 28 DEG C are transferred to after light green color leaf sheath is extracted out, 30 μm of olm
-2s
-1light intensity (white fluorescent lamp), takes root under 16h/8h photoperiod condition, obtains regeneration plant, the suspension cell rate of increase 78%, regeneration motility rate 89%.
Embodiment 2
Get circassian bean blade, 3min is soaked in washing powder, running water 15min, 1% bromine water process 8min on superclean bench, running water 5 times, callus induction is carried out in the blade access MS+4 μm of ol/L vitamin h+0.3mg/LNAA+1mg/L brassinosteroid medium of disinfecting, additional 7g/L agar, 30g/L lactose, temperature 25 DEG C, light culture, callus access medium MS+600 μm of ol/L glutamine+4 μm of ol/L vitamin hs+2 derived, suspension cell culture is carried out in 4-D0.2mg/L, additional 30g/L lactose, temperature 25 DEG C, light culture, rotary shaker vibrates, 110r/min, suspension cell is in the first month cultivated, change a fresh culture weekly, the bulky grain culture not easily disperseed with 800 μm of aperture screen cloth filterings, comprising callus group, He Hua thanatogenic tissue, some proembryo etc., thereafter within every 2 weeks, a culture fluid is changed, after culture comparatively disperses, larger cell mass is removed with 150 μm of aperture sieve net filtrations, embryonal suspension cell induction is carried out in the suspension cell access medium MS+CPPU 0.3mg/L+NAA 0.2mg/L+ZT 0.5mg/L cultivated, the cells,primordial access derived does not carry out root induction containing in the MD medium of any hormone, the somatic embryo at plant regeneration initial stage is sprouted and is carried out under dark condition, 28 DEG C are transferred to after light green color leaf sheath is extracted out, 30 μm of olm
-2s
-1light intensity (white fluorescent lamp), takes root under 16h/8h photoperiod condition, obtains regeneration plant, the suspension cell rate of increase 80%, regeneration motility rate 91%.。
Embodiment 3
Get circassian bean blade, 3min is soaked in washing powder, running water 15min, 1% bromine water process 8min on superclean bench, running water 5 times, callus induction is carried out in the blade access MS+4 μm of ol/L vitamin h+0.3mg/LNAA+1mg/L brassinosteroid medium of disinfecting, additional 7g/L agar, 30g/L lactose, temperature 25 DEG C, light culture, callus access medium MS+500 μm of ol/L glutamine+4 μm of ol/L vitamin hs+2 derived, suspension cell culture is carried out in 4-D0.2mg/L, additional 30g/L lactose, temperature 25 DEG C, light culture, rotary shaker vibrates, 110r/min, suspension cell is in the first month cultivated, change a fresh culture weekly, the bulky grain culture not easily disperseed with 800 μm of aperture screen cloth filterings, comprising callus group, He Hua thanatogenic tissue, some proembryo etc., thereafter within every 2 weeks, a culture fluid is changed, after culture comparatively disperses, larger cell mass is removed with 150 μm of aperture sieve net filtrations, embryonal suspension cell induction is carried out in the suspension cell access medium MS+CPPU0.3mg/L+NAA0.2mg/L+ZT0.5mg/L cultivated, the cells,primordial access derived does not carry out root induction containing in the MD medium of any hormone, the somatic embryo at plant regeneration initial stage is sprouted and is carried out under dark condition, 28 DEG C are transferred to after light green color leaf sheath is extracted out, 30 μm of olm
-2s
-1light intensity (white fluorescent lamp), takes root under 16h/8h photoperiod condition, obtains regeneration plant, the suspension cell rate of increase 80%, regeneration motility rate 94%.。
Claims (4)
1. a method for quickly breeding for circassian bean suspension cell and regeneration plant, comprise the sterilization of explant, the induction of callus, the cultivation of suspension cell, the induction of embryonal suspension cell, root induction, its key step is as follows:
(1) young leaflet tablet of circassian bean is got, disinfection;
(2) get in the blade access MS+3-4 μm of ol/L vitamin h+0.3mg/LNAA+0.5-1mg/L brassinosteroid medium that step (1) disinfected and carry out callus induction, additional 7g/L agar, 30g/L lactose, temperature 25 DEG C, light culture;
(3) get in callus access medium MS+500-600 μm of ol/L glutamine+3-4 μm of ol/L vitamin h+2,4-D 0.2mg/L that step (2) derives and carry out suspension cell culture, additional 30g/L lactose, temperature 25 DEG C, light culture, rotary shaker vibrates, 110r/min;
(4) get in the suspension cell access medium MS+CPPU0.3mg/L+NAA0.2mg/L+ZT0.5mg/L that step (3) cultivates and carry out embryonal suspension cell induction;
(5) get the cells,primordial access that step (4) derives and do not carry out root induction containing in the MD medium of any hormone, obtain regeneration plant.
2. according to the method for quickly breeding of a kind of circassian bean suspension cell according to claim 1 and regeneration plant, it is characterized in that: the acquisition of the aseptic blade of circassian bean described in step (1) is, get circassian bean blade, 3min is soaked in washing powder, running water 15min, 1% bromine water process 8min on superclean bench, running water 5 times.
3. according to the method for quickly breeding of a kind of circassian bean suspension cell according to claim 1 and regeneration plant, it is characterized in that: step (3) suspension cell is in the first month cultivated, change a fresh culture weekly, the bulky grain culture not easily disperseed with 800 μm of aperture screen cloth filterings, comprising callus group, He Hua thanatogenic tissue, some proembryo etc., thereafter within every 2 weeks, a culture fluid is changed, after culture comparatively disperses, remove larger cell mass with 150 μm of aperture sieve net filtrations.
4. according to the method for quickly breeding of a kind of circassian bean suspension cell according to claim 1 and regeneration plant, it is characterized in that: the somatic embryo at step (4) plant regeneration initial stage is sprouted and carried out under dark condition, after light green color leaf sheath is extracted out, transfer to 28 DEG C, 30 μm olm
-2s
-1light intensity (white fluorescent lamp), takes root under 16h/8h photoperiod condition.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410550655.6A CN104255531A (en) | 2014-10-17 | 2014-10-17 | Rapid propagation methods for adenantherapavonlna suspension cell and regeneration plant |
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| CN201410550655.6A CN104255531A (en) | 2014-10-17 | 2014-10-17 | Rapid propagation methods for adenantherapavonlna suspension cell and regeneration plant |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106613983A (en) * | 2016-12-26 | 2017-05-10 | 江西宜信堂医疗科技有限公司 | Paris polyphylla var chinensis induction culture medium and application thereof |
| CN108300684A (en) * | 2018-01-30 | 2018-07-20 | 北京工商大学 | A kind of method that long timeliness inhibits browning during glycyrrhiza glabra suspension cell culture |
| CN111150751A (en) * | 2020-02-19 | 2020-05-15 | 中国人民解放军军事科学院军事医学研究院 | Method for preparing ormosia angustifolia extract, ormosia angustifolia extract and application thereof |
-
2014
- 2014-10-17 CN CN201410550655.6A patent/CN104255531A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106613983A (en) * | 2016-12-26 | 2017-05-10 | 江西宜信堂医疗科技有限公司 | Paris polyphylla var chinensis induction culture medium and application thereof |
| CN108300684A (en) * | 2018-01-30 | 2018-07-20 | 北京工商大学 | A kind of method that long timeliness inhibits browning during glycyrrhiza glabra suspension cell culture |
| CN108300684B (en) * | 2018-01-30 | 2021-02-09 | 北京工商大学 | Method for inhibiting browning phenomenon in suspension cell culture process of glycyrrhiza glabra for long time |
| CN111150751A (en) * | 2020-02-19 | 2020-05-15 | 中国人民解放军军事科学院军事医学研究院 | Method for preparing ormosia angustifolia extract, ormosia angustifolia extract and application thereof |
| CN111150751B (en) * | 2020-02-19 | 2021-08-17 | 中国人民解放军军事科学院军事医学研究院 | Method for preparing ormosia angustifolia extract, ormosia angustifolia extract and application thereof |
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Application publication date: 20150107 |