CN105613284A - Method for accelerating expanding culture of lily virus-free cormels - Google Patents
Method for accelerating expanding culture of lily virus-free cormels Download PDFInfo
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- CN105613284A CN105613284A CN201410583261.0A CN201410583261A CN105613284A CN 105613284 A CN105613284 A CN 105613284A CN 201410583261 A CN201410583261 A CN 201410583261A CN 105613284 A CN105613284 A CN 105613284A
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Abstract
The invention discloses a culturing method for utilizing a bubbling type bioreactor to quickly expand cut-flower lily virus-free bulbs (cormels), and belongs to the technical field of plant tissue culturing fast propagation and the field of plant cell engineering. The method comprises the following steps: optimizing an expanding liquid culture medium (MS+0.2 mg/L NAA +4 mg/L CPPU +6% cane sugar, wherein the pH value is 6.8) for tissue cultured lily virus-free cormel bulbs used for a reactor; planting cormel blocks according to the quantity of 2600-3000 cormel blocks per liter; adjusting an air inlet volume of filtered air to enable a flow speed of a turbulent area in the reactor to be 3-6 cm/s; keeping the pressure in the reactor to be constant at 0.03-0.06 MPa at the temperature of 22+/-1 DEG C; and irradiating 12 h/d under a luminous intensity of 1600-2000 Lx and culturing for 40-46 d so as to meet field planting demands on diameters, root systems and weight of the cultured cormels. According to the method, the culturing period can be shortened, the energy source is saved, the labor and culturing space are saved, the utilization efficiency of the culture medium is increased, the production cost is lowered, the practicability is strong and the method is easily promoted and applied in production.
Description
Technical field
The invention discloses and utilize Sparged bioreactors quickly to expand the cultural method of flower lily detoxification clove (seed ball), belong to the technical field of plant tissue culture fast breeding technique field and plant cell engineering.
Background technology
Bulbus Lilii is important ornamental flower, is mainly used in cutting flower variety and produces. Bulbus Lilii is when open environment, and inevitably by virus infection, the Bulbus Lilii economical character that virus infection causes is degenerated serious, greatly have impact on the production of Bulbus Lilii. Plantation lily detoxification kind ball is the most important link that lily flower produces, and the consumption figure of China Bulbus Lilii increases year by year in recent years, and present China Bulbus Lilii produces the detoxification kind ball dependence on import of nearly more than 90%. Lily detoxification technology is the virus utilizing the artificial tissue culture method outer implant of elimination Bulbus Lilii to infect, and carries out the technology of the fast breeding realizing virus-free seed ball of nourishing and generating of virus-free culture. China's lily detoxification technology is most on producing adopts tradition tissue culture modes to produce detoxification seed balls, adopts solid medium to complete the cultivation of group training process in process of production, and wherein detoxification seed ball expands that to cultivate be one of link of restriction production scale and speed. Adopting this process of traditional group culture method to need that culture is placed in solid culture primary surface and carry out surface cultivation, culture medium utilization ratio is low; Substantial amounts of manpower is needed to transfer when adjusting medium component; Incubation time will up to the time of 2-3 month; Production process needs very big culture space, for ensureing that the conditions such as the temperature of culture space, wet, light to meet cultivation needs and also need to consume a lot of energy.
Sparged bioreactors is with gas for dispersion phase, and liquid is continuous phase, relates to the reactor of gas-liquid interface, has simple in construction, it is easy to operation, and running cost is low, the feature that mixing is good with mass-and heat-transfer performance. Utilize Sparged bioreactors to adopt the mode of liquid culture that lily detoxification seed ball expands cultivation and can shorten incubation time, improve culture medium utilization rate, save culture space, reduce production cost, can make up and solve tradition lily detoxification seed ball and expand the deficiency of tissue culture mode.
Summary of the invention
It is an object of the invention to overcome traditional group training to expand and cultivate the defect of cultural method in detoxification Bulbus Lilii seed ball process, both cultivation cycle was long, and culture medium utilization ratio is low, it is impossible to adjusting medium component, switching needs a large amount of manpowers; Culture space is relatively big, the more high defect of energy consumption. With flowers market popular " Siberia " (Siberia), " rope side " (Sorbonne), " horse
Can POLO " (Marcopolo); " Man Nisha " (Manissa); the group training detoxification seed ball of " Kang Jiadeao " (Concad'0) 6 kinds is material; optimizes detoxification Bulbus Lilii seed ball liquid and expands culture medium composition and bioreactor culture condition; quickly expanded cultivation detoxification seed ball by bioreactor liquid culture; shortens fast numerous time of lily detoxification high-quality kind ball, is producing number
Fast 4-6 times of more conventional method in amount, and the planting survival rates of seed ball is high. The present invention can shorten cultivation cycle, saves the energy, artificial and culture space, improves the utilization ratio of culture medium, reduce production cost, has seed ball survival rate height, is suitable to field planting,
It is prone to popularization and application in the scale quick propagation of flower lily original seed produces. The invention provides and a kind of quickly expand, suitable in Sparged bioreactors, the optimization method cultivating detoxification Bulbus Lilii seed ball, the method run including liquid culture based formulas and reactor and regulate.
The present invention is achieved through the following technical solutions:
Accelerate expand cultivate lily detoxification seed ball method, carry out as follows:
(1) preparation of culture medium: bulb expands culture medium, and (MS+NAA0.2mg/L+CPPU4mg/L+6% sucrose (pH6.8), preparation fluid medium volume is the 1/2-3/6 of reactor tank body volume.
(2) reactor sterilizing and prerun: after adding culture medium, reactor tank body and tubing are carried out steam sterilization, after sterilizing, reactor is adjusted to 26 scholar 1 DEG C, regulates and filter air inlet amount, regulate reactor air outlet valve makes fluid medium in reactor body be in the circulation stream mode intending Uniform Flow simultaneously, turbulence district flow velocity 2-3cm/s, keeping pressure inside the tank constant in 0.02Mpa, run 2 days, checking sterilization effect also guarantees that reactor is stable.
(3) cultivate seed ball preparation: in gnotobasis, by routine group train detoxification seed spheroblast cut blade and base thereof tissue, rip cutting becomes 2-3 block diameter 3.0-6.0mm seed ball block, by 2600-3000 seed ball block/liter quantity preparation culture materials.
(4) inoculation of seed ball is cultivated: use 70% ethanol to borrow kind of a mouth to carry out sterilizing reactor, lighting reactor borrows the pyrosphere of kind of mouth that reactor is inoculated mouth sterilizing use, then light under state at inoculation pyrosphere, open rapidly inoculation lid and seed ball is transferred in reactor, then inoculation lid is tightened.
(5) reactor service condition: reactor is adjusted to 22 scholar 1 DEG C; Intensity of illumination 1600-2000Lx, illumination 12h/d; Regulate and filter air inlet amount, regulate reactor air outlet valve simultaneously and make fluid medium in reactor be in the circulation stream mode intending Uniform Flow, turbulence district flow velocity 3-6cm/s, keep reactor pressure constant in 0.03-0.06Mpa.
(6) feed supplement in reactor running: culture fluid loss of water in every 2-3 days observing response devices, adds sterile purified water by fluid infusion hole to reactor with peristaltic pump, it is ensured that culture fluid volume. Observing and cultivate seed ball growth conditions, cultivate 40-46d, cultivate seed bulb diameter up to 1.0-1.4cm, root system is at 3-6 bar/grain, seed ball weight 0.7-1.2g/ grain.
(7) cold preservation: the group training bulb clear water that taking-up step (6) is cultivated washes away culture fluid, is wrapped in the sterilization peat composed of rotten mosses, breaks dormancy (4 DEG C of chilling treatment times can between 2-4 month) for 46 days through 4-6 DEG C of chilling treatment
(8) transplanting domestication: by step (7) bulb, select the 800-1000 rice Altitude Regions of tool Cold and cool climate in summer, when taking shelter from rain with insect protected isolation, late April to mid-June transplants field planting in the substrate sterilized.
Claims (4)
1. the method cultivating lily detoxification seed ball is expanded in quickening, carries out as follows by being characterised by:
(1) preparation of culture medium: bulb expands culture medium MS+NAA0.2mg/L+CPPU4mg/L+6% sucrose (pH6.8), preparation fluid medium volume is the 1/2-3/6 of reactor tank body volume.
(2) reactor sterilizing and prerun: after adding culture medium, reactor tank body and tubing are carried out steam sterilization, after sterilizing, reactor is adjusted to 26 scholar 1 DEG C, regulates and filter air inlet amount, regulate reactor air outlet valve makes fluid medium in reactor body be in the circulation stream mode intending Uniform Flow simultaneously, turbulence district flow velocity 2-3cm/s, keeping pressure inside the tank constant in 0.02Mpa, run 2 days, checking sterilization effect also guarantees that reactor is stable.
(3) cultivate seed ball preparation: in gnotobasis, by routine group train detoxification seed spheroblast cut blade and base thereof tissue, rip cutting becomes 2-3 block diameter 3.0-6.0mm seed ball block, by 2600-3000 seed ball block/liter quantity preparation culture materials.
(4) inoculation of seed ball is cultivated: use 70% ethanol to borrow kind of a mouth to carry out sterilizing reactor, lighting reactor borrows the pyrosphere of kind of mouth that reactor is inoculated mouth sterilizing use, then light under state at inoculation pyrosphere, open rapidly inoculation lid and seed ball is transferred in reactor, then inoculation lid is tightened.
(5) reactor service condition: reactor is adjusted to 22 scholar 1 DEG C; Intensity of illumination 1600-2000Lx, illumination 12h/d; Regulate and filter air inlet amount, regulate reactor air outlet valve simultaneously and make fluid medium in reactor be in the circulation stream mode intending Uniform Flow, turbulence district flow velocity 3-6cm/s, keep reactor pressure constant in 0.03-0.06Mpa.
(6) feed supplement in reactor running: culture fluid loss of water in every 2-3 days observing response devices, adds sterile purified water by fluid infusion hole to reactor with peristaltic pump, it is ensured that culture fluid volume. Observing and cultivate seed ball growth conditions, cultivate 40-46d, cultivate seed bulb diameter up to 1.0-1.4cm, root system is at 3-6 bar/grain, seed ball weight 0.7-1.2g/ grain.
(7) cold preservation: the group training bulb clear water that taking-up step (6) is cultivated washes away culture fluid, is wrapped in the sterilization peat composed of rotten mosses, breaks dormancy (4 DEG C of chilling treatment times can between 2-4 month) for 46 days through 4-6 DEG C of chilling treatment
(8) domestication is transplanted: by step (7) bulb, select the 800-1000 rice Altitude Regions of tool Cold and cool climate in summer, taking shelter from rain
When with insect protected isolation, late April to mid-June transplants field planting in the substrate sterilized; The liquid culture based component cultivated for Bulbus Lilii group training detoxification seed ball in bioreactor is: MS+NAA0.2mg/L+CPPU4mg/L+6% sucrose (pH6.8);
2. the method cultivating lily detoxification seed ball is expanded in a kind of quickening according to claims 1, it is characterised in that borrow the seed ball block amount kind in fluid medium be 2600-3000 seed ball block/liter;
3. the method cultivating lily detoxification seed ball is expanded in a kind of quickening according to claims 1, it is characterised in that cultivating the condition cultivated in the reactor of seed ball block is: 22 scholars 1 DEG C; Intensity of illumination 1600-2000Lx, illumination 12h/d; Fluid medium turbulence district flow velocity 3-6cm/s is for intending uniform circulation stream-like, and reactor pressure is constant in 0.03-0.06Mpa, cultivates 40-46 days.
4. the method cultivating lily detoxification seed ball is expanded in a kind of quickening according to claims 1, it is characterised in that cultivating seed ball bulb diameter in reactor and reach 1.0-1.4cm, root system at 7-10 bar/grain, is cold preservation, field planting standard during seed ball weight 0.7-1.2g/ grain.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5138794A (en) * | 1990-03-28 | 1992-08-18 | The United States Of America As Represented By The Secretary Of Agriculture | Method for producing lilium elegans |
| CN102440188A (en) * | 2010-10-14 | 2012-05-09 | 杨凯 | Method for using amantadine hydrochloride drug to culture lily under virus-free state |
| CN102440184A (en) * | 2010-10-14 | 2012-05-09 | 王文和 | Culturing method for utilizing bioreactor to quickly expand lily detoxified cormels |
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- 2014-10-28 CN CN201410583261.0A patent/CN105613284A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5138794A (en) * | 1990-03-28 | 1992-08-18 | The United States Of America As Represented By The Secretary Of Agriculture | Method for producing lilium elegans |
| CN102440188A (en) * | 2010-10-14 | 2012-05-09 | 杨凯 | Method for using amantadine hydrochloride drug to culture lily under virus-free state |
| CN102440184A (en) * | 2010-10-14 | 2012-05-09 | 王文和 | Culturing method for utilizing bioreactor to quickly expand lily detoxified cormels |
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