CN109757378A - A method of fritillaria thunbergii seedling is quickly bred using interval immersion system - Google Patents
A method of fritillaria thunbergii seedling is quickly bred using interval immersion system Download PDFInfo
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Abstract
The invention discloses a kind of methods for quickly breeding fritillaria thunbergii seedling using interval immersion system, it include four cultivation stages according to cultivation sequence, respectively callus proliferation cultivation stage, clove differentiation and seedling cultivation stage, culture of rootage stage and hardening stage, this four cultivation stages carry out in the cultivation reactor of same interval immersion system.A large amount of uniform fritillaria thunbergii seedlings can be bred in a short time using method disclosed by the invention, and seedling early growth flushes, transplanting survival rate is high, thus greatly reduce the production cycle of fritillaria thunbergii, production cost is saved, is quickly bred for fritillaria thunbergii seedling and provides a kind of efficient production method.
Description
Technical field
The present invention relates to medicinal plant technical field of tissue culture, and in particular to a kind of to carry out Zhejiang using interval immersion system
The method that fritillaria seedling is quickly bred to obtain a large amount of high quality seedlings in a short time.
Background technique
Fritillaria thunbergii (Fritillaria thunbergiiIt Miq.) is Liliaceae Fritillaria herbaceos perennial, be " in
State's pharmacopeia " most important one kind in the five class fritillarias recorded, it is important one of the Chinese medicine in China, is used as medicine with its dry bulb,
With heat-clearing dissipating bind, the effect of preventing phlegm from forming and stopping coughing, it is listed in first of Zhejiang genunie medicinal materials " eight Zhe's ".It is peaceful that fritillaria thunbergii originates in Zhejiang
Wave Xiangshan, wild resource is less at present, is mainly derived from artificial cultivation, and the cultivation area of fritillaria thunbergii is distributed mainly on Zhejiang, river
Soviet Union and Shanghai, fritillaria thunbergii not only possesses very big market in inland, but also is sold abroad, and there is Chinese Fragrant in main export area
The countries in Southeast Asia such as port, TaiWan, China, Japan, South Korea, Malaysia, Singapore, Indonesia and the American-European Chinese in part
Settlement enjoys high reputation in the international market.
Generally using nourishing and generating in fritillaria thunbergii cultivation, i.e., bulb is bred, and 1 bulb can only averagely harvest 1.5-1.6
Bulb, after removing is reserved seed for planting, only 0.5-0.6 bulb can hyoscine, breeding coefficient is low, low output, is unable to satisfy medicinal material city
The demand of field.During long-term vegetative propagation, more disease can be accumulated in Bulbs of Fritillaria thunbergii Miq, lead to its kind of sexual involution, quality
Decline, yield reduce.Tissue culture technique can solve that fritillaria thunbergii breeding coefficient is low and provenance disease problem, improve fritillaria thunbergii
Proliferative speed and bulb quality.But during producing fritillaria thunbergii seedling in the way of traditional solid culture there is compared with
More disadvantages: (1) it using agar as solid support, influences nutritional ingredient equiblibrium mass distribution and quickly absorbs, cause seedling growing way different；
(2) traditional solid culture mode is sealed culture, can not carry out gas exchanges, seedling is caused to be easy to appear vitrifying, kind
Seedling growing way is short and small；(3) solid culture mode adheres to a large amount of agar on fritillaria thunbergii seedling root system, cleaned in acclimatization and transplants
It is larger to the injury of seedling root system in journey, cause transplanting survival rate low；(4) solid culture mode amount of labour demanded is big, Wu Fashi
Existing automatic operation, high production cost are not suitable for the factorial production.
In interval immersion system, intermittent contact with plant tissue of fluid nutrient medium provides nutrition, and can be very
The gas exchanges in incubator are carried out well, efficiently solve the Vitrification Occurred of seedling；The mobility of culture medium is also effectively
The accumulation of nutrition deposition and harmful substance is prevented, fritillaria thunbergii seedling healthy growth is conducive to；Kind is bred using interval immersion system
Seedling is completed hardening process in same cultivation reactor, is not damaged to tissue-cultured seedling, and transplanting survival rate is high；Interval immersion system
High degree of automation, culture flux is big, and operating process is simple, and labour is greatly saved, and reduces production cost.
It is had been reported at present about the research using interval immersion system culture plant, Chinese patent
It is disclosed in CN201310043125.8 a kind of using the numerous candidum tissue culturing seedling of interval immersion liquid culture systems expansion and same
The method of hardening in one reactor obtains a large amount of iron sheet stones by inoculation and culture in interval immersion liquid culture systems
Dry measure used in former times tissue-cultured seedling, and in a reactor hardening be described, this method have proliferation rate is high, tissue-cultured seedling is healthy and strong, transplanting at
Motility rate is high and high degree of automation, labour consume the advantages that few, provides one kind efficiently for the production of candidum tissue culturing seedling
The method of rate, low cost.But nutritional ingredient needed for the plant of different cultivars, life habit, growth pattern are all different, because
It when this utilizes interval immersion system culture difference plant, needs to determine optimal condition of culture according to the characteristic of different plants, wraps
It includes: medium component, Immersion time, Immersion frequency, inoculum density, incubation time, culture environment condition etc..At present about utilization
The method of interval immersion system culture fritillaria thunbergii seedling has not been reported and openly applies, it is therefore necessary to study a kind of using intermittently
The method that immersion system quickly breeds fritillaria thunbergii seedling.
Summary of the invention
It is an object of the invention to solve deficiency in the prior art, provides and a kind of quickly bred using interval immersion system
The method of fritillaria thunbergii seedling, using this method cultivate fritillaria thunbergii have seedling growing way is vigorous, size is uniform, transplanting survival rate is high,
Breeding cycle is short and the advantages such as automation degree of equipment is high, easy to operate, production cost is low.
The technical solution of the present invention is as follows: a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system, according to training
Educating sequence includes four cultivation stages, respectively callus proliferation cultivation stage, clove differentiation and seedling cultivation stage, life
Root cultivation stage and hardening stage, this four cultivation stages carry out in the cultivation reactor of same interval immersion system, tool
The incubation step of body are as follows:
(1) callus proliferation cultivation stage: aseptically, the callus for being proliferated the yellow green within four generations is transferred
Multiplying culture is carried out into the cultivation reactor of sterilized interval immersion system；
(2) clove differentiation is with seedling cultivation stage: after the completion of on last stage, under aseptic condition, in same interval immersion system
Cultivation reactor in, by callus proliferation culture solution be changed to clove differentiation with seedling culture solution, continue small squama
Stem differentiation and seedling culture；
(3) the culture of rootage stage: after the completion of on last stage, under aseptic condition, in the cultivation reactor of same interval immersion system
In, clove differentiation is changed to culture of rootage liquid with seedling culture solution, continues culture of rootage；
(4) it hardening: after the completion of on last stage, under natural conditions in the cultivation reactor of same interval immersion system, will take root
Culture solution is changed to hardening culture solution, continues hardening to obtain the fritillaria thunbergii seedling that can be transplanted.
Further, in the callus proliferation cultivation stage, callus proliferation culture formula of liquid are as follows: cultivated with MS
Base is basic culture medium, adds 1.5 mg/L of hormone NAA, 1.0 g/L of casein hydrolysate, sucrose 30 g/L, and pH is adjusted to
5.8-6.2；At this stage, the culture parameters setting of interval immersion system are as follows: Immersion time 1-10 min, the every 1-8 of Immersion frequency
H is primary, inoculum density 20-50 g, incubation time 20-45 d；The culture environment condition in the stage is set are as follows: and 20 ± 1 DEG C of temperature,
Intensity of illumination 2500-3000 Lux, 10 h/d of light application time.
Preferably, the culture parameters of interval immersion system are set are as follows: Immersion time in callus proliferation cultivation stage
4-8 min, the every 3-6 h of Immersion frequency is primary, inoculum density 30-40 g, incubation time 25-35 d.
Further, with seedling cultivation stage, clove breaks up and seedling culture formula of liquid for the clove differentiation are as follows:
It is basic culture medium with MS culture medium, adds 0.5 mg/L of hormone NAA, 1.0 mg/L of KT, 1.0 g/L of casein hydrolysate,
Sucrose 30 g/L, pH are adjusted to 5.8-6.2；At this stage, the culture parameters setting of interval immersion system are as follows: Immersion time 3-12
Min, the every 2-10 h of Immersion frequency is primary, and clove differential period is room temperature illumination cultivation, incubation time 20-45 d；Seedling training
Stage elder generation low temperature dark culturing 15-40 d is supported, room temperature illumination cultivation 30-55 d is then gone to；The culture environment item that normal-temperature light is shone
Part setting are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000 Lux, 10 h/d of light application time；The culture environment of low temperature dark
Condition setting are as follows: 3-6 DEG C of temperature, completely black dark.
Preferably, the culture parameters of the interval immersion system are set in clove differentiation and seedling cultivation stage are as follows:
Immersion time 6-10 min, the every 5-8 h of Immersion frequency is primary, and clove differential period is room temperature illumination cultivation, incubation time 30-
35 d；Seedling cultivation stage elder generation low temperature dark culturing 20-30 d, then goes to room temperature illumination cultivation 40-45 d.
Further, in the culture of rootage stage, culture of rootage formula of liquid are as follows: cultivated based on MS/2 culture medium
Base adds 2.0 mg/L of hormone IBA, 0.5 g/L of casein hydrolysate, 0.1 mg/L of myricyl alcohol, 1.0 g/L of active carbon, sucrose
20 g/L, pH are adjusted to 5.8-6.2；At this stage, the culture parameters setting of interval immersion system are as follows: Immersion time 3-12
Min, the every 4-12 h of Immersion frequency is primary, incubation time 20-45 d；The setting of culture environment condition are as follows: 20 ± 1 DEG C of temperature, illumination
Intensity 2500-3000 Lux, 10 h/d of light application time.
Preferably, the culture parameters of the interval immersion system are set are as follows: Immersion time 6-10 in the culture of rootage stage
Min, the every 7-10 h of Immersion frequency is primary, incubation time 30-35 d.
Further, in the hardening stage, hardening culture formula of liquid are as follows: No. 2 1.0 g/L of Hua Bao add sheep dung leachate 50
mL/L；At this stage, the culture parameters setting of the interval immersion system are as follows: Immersion time 1-10 min, the every 6- of Immersion frequency
14 h are primary, hardening time 10-40 d；The setting of hardening environmental condition are as follows: natural environment, 10-25 DEG C of temperature.
Preferably, the culture parameters of the interval immersion system are set in the hardening stage are as follows: Immersion time 4-8 min,
The every 9-12 h of Immersion frequency is primary, hardening time 15-25 d.
Further, the sheep dung leachate is to be added in 1 kg pure water after smashing 500 g sheep dungs to pieces, be placed in water-bath
In 100 DEG C of 3 h of heating, cooled and filtered is added 1 kg pure water again in filter residue, above-mentioned heating and filtering operation repeated, by two
What secondary filtrate obtained after merging.
The invention has the benefit that
(1) proliferation rate is high, with short production cycle: interval immersion system is Liquid Culture, and the mobility of culture medium is also effectively prevented
The accumulation of nutrition deposition and harmful substance, effectively increases the proliferation rate of fritillaria thunbergii seedling, utilizes method disclosed by the invention
The proliferation rate of breeding fritillaria thunbergii seedling reaches 45.8, the significantly larger than proliferation rate of conventional solid modes of reproduction；And it is soaked using interval
The production cycle for there be not system production fritillaria thunbergii seedling is 160-180 d, and conventional solid training method is used to produce bulb of thunberg fritillary parent species
The period of seedling is 210-230 d, and this method substantially reduces the seeling industry period；
(2) seedling flush, transplanting survival rate it is high: be by fluid nutrient medium using interval immersion system production fritillaria thunbergii seedling
It is intermittent to be contacted with plant tissue, the exchange of gas in incubator can be carried out well, and the vitrifying for solving seedling is asked
Topic, seedling early growth are vigorous；Seedling is bred using interval immersion system, hardening process is completed in same cultivation reactor, with biography
The solid culture mode of system is compared, and without cleaning the agar adhered on fritillaria thunbergii seedling root system when hardening, is not damaged to tissue-cultured seedling
Wound, transplanting survival rate are high；
(3) automatization level is high, and easy to operate, production cost is low: interval immersion system provides power, control system control by air pump
Immersion time processed and frequency, without human intervention, high degree of automation；Fritillaria thunbergii seedling is produced using interval immersion system, it is whole
A incubation is all completed in same cultivation reactor, and conventional solid training method is compared, and without transferring repeatedly, is saved big
The manpower consumption that tissue culture bottle is filling and washs is measured, pollution rate is greatly reduced, saves production cost.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where essence of the present invention, to modification made by the method for the present invention, step or condition and replaces, belong to model of the invention
It encloses.
Embodiment 1
Fritillaria thunbergii seedling is produced using interval immersion system, specifically includes the following steps:
(1) callus proliferation cultivation stage: aseptically, the callus for being proliferated the yellow green within four generations is transferred
Multiplying culture, callus proliferation culture formula of liquid are carried out into the cultivation reactor of sterilized interval immersion system are as follows: with
MS culture medium is basic culture medium, adds 1.5 mg/L of hormone NAA, 1.0 g/L of casein hydrolysate, 30 g/L of sucrose, pH tune
It saves to 5.8-6.2；The culture parameters of the stage interval immersion system are set are as follows: 6 min of Immersion time, every 4 h mono- of Immersion frequency
It is secondary, 30 g of inoculum density, 28 d of incubation time；The setting of culture environment condition are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000
Lux, 10 h/d of light application time；
(2) clove differentiation is with seedling cultivation stage: after the completion of callus proliferation cultivation stage, under aseptic condition, same
In the cultivation reactor of one interval immersion system, callus proliferation culture solution is changed to clove differentiation and seedling culture
Liquid continues clove differentiation and seedling culture, clove differentiation and seedling culture formula of liquid are as follows: based on MS culture medium
Culture medium adds 0.5 mg/L of hormone NAA, 1.0 mg/L of KT, 1.0 g/L of casein hydrolysate, sucrose 30 g/L, pH adjusting
To 5.8-6.2；The culture parameters of the stage interval immersion system are set are as follows: and 9 min of Immersion time, every 8 h of Immersion frequency is primary,
Clove differential period is room temperature illumination cultivation, 30 d of incubation time；28 d of seedling cultivation stage elder generation low temperature dark culturing, then
It goes under room temperature illumination cultivation and cultivates 45 d；The culture environment condition setting that normal-temperature light is shone are as follows: 20 ± 1 DEG C of temperature, intensity of illumination
2500-3000 Lux, 10 h/d of light application time；The culture environment condition of low temperature dark is set are as follows: 3-6 DEG C of temperature, completely black dark；
(3) it the culture of rootage stage: after the completion of clove differentiation is with seedling cultivation stage, under aseptic condition, is soaked in same interval
Do not have in the cultivation reactor of system, clove differentiation is changed to culture of rootage liquid with seedling culture solution, continues training of taking root
It supports, culture of rootage formula of liquid are as follows: with MS/2 culture medium be basic culture medium, add 2.0 mg/L of hormone IBA, casein hydrolysis
0.5 g/L of object, 0.1 mg/L of myricyl alcohol, 1.0 g/L of active carbon, sucrose 20 g/L, pH are adjusted to 5.8-6.2；Stage interval
The culture parameters of immersion system are set are as follows: 8 min of Immersion time, every 8 h of Immersion frequency is primary, 32 d of incubation time；Cultivate ring
Border condition setting are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000 Lux, 10 h/d of light application time.
(4) the hardening stage: anti-in the culture of same interval immersion system under natural conditions after the completion of the culture of rootage stage
It answers in device, is hardening culture solution by culture of rootage fluid exchange, continues hardening to obtain the fritillaria thunbergii seedling that can be transplanted, hardening
Cultivate formula of liquid are as follows: No. 2 1.0 g/L of Hua Bao add 50 mL/L of sheep dung leachate；The culture parameters of the stage interval immersion system are set
Be set to: 4 min of Immersion time, every 10 h of Immersion frequency is primary, 25 d of hardening time；Hardening environmental condition setting are as follows: natural ring
Border, 10-25 DEG C of temperature.
Fritillaria thunbergii proliferation rate reaches 45.8, field planting is transplanted to after hardening, for survival rate up to 99%, seedling early growth is prosperous
It contains.
Embodiment 2
Fritillaria thunbergii seedling is produced using interval immersion system, specifically includes the following steps:
(1) callus proliferation cultivation stage: aseptically, the callus for being proliferated the yellow green within four generations is transferred
Multiplying culture, callus proliferation culture formula of liquid are carried out into the cultivation reactor of sterilized interval immersion system are as follows: with
MS culture medium is basic culture medium, adds 1.5 mg/L of hormone NAA, 1.0 g/L of casein hydrolysate, 30 g/L of sucrose, pH tune
It saves to 5.8-6.2；The culture parameters of the stage interval immersion system are set are as follows: 8 min of Immersion time, every 6 h mono- of Immersion frequency
It is secondary, 35 g of inoculum density, 30 d of incubation time；The setting of culture environment condition are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000
Lux, 10 h/d of light application time；
(2) clove differentiation is with seedling cultivation stage: after the completion of callus proliferation cultivation stage, under aseptic condition, same
In the cultivation reactor of one interval immersion system, callus proliferation culture solution is changed to clove differentiation and seedling culture
Liquid continues clove differentiation and seedling culture, clove differentiation and seedling culture formula of liquid are as follows: based on MS culture medium
Culture medium adds 0.5 mg/L of hormone NAA, 1.0 mg/L of KT, 1.0 g/L of casein hydrolysate, sucrose 30 g/L, pH adjusting
To 5.8-6.2；The culture parameters of the stage interval immersion system are set are as follows: and 8 min of Immersion time, every 6 h of Immersion frequency is primary,
Clove differential period is room temperature illumination cultivation, 35 d of incubation time；25 d of seedling cultivation stage elder generation low temperature dark culturing, then
It goes under room temperature illumination cultivation and cultivates 42 d；The culture environment condition setting that normal-temperature light is shone are as follows: 20 ± 1 DEG C of temperature, intensity of illumination
2500-3000 Lux, 10 h/d of light application time；The culture environment condition of low temperature dark is set are as follows: 3-6 DEG C of temperature, completely black dark；
(3) it the culture of rootage stage: after the completion of clove differentiation is with seedling cultivation stage, under aseptic condition, is soaked in same interval
Do not have in the cultivation reactor of system, clove differentiation is changed to culture of rootage liquid with seedling culture solution, continues training of taking root
It supports, culture of rootage formula of liquid are as follows: with MS/2 culture medium be basic culture medium, add 2.0 mg/L of hormone IBA, casein hydrolysis
0.5 g/L of object, 0.1 mg/L of myricyl alcohol, 1.0 g/L of active carbon, sucrose 20 g/L, pH are adjusted to 5.8-6.2；Stage interval
The culture parameters of immersion system are set are as follows: 10 min of Immersion time, every 10 h of Immersion frequency is primary, 35 d of incubation time；Culture
Environmental condition setting are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000 Lux, 10 h/d of light application time.
(4) the hardening stage: anti-in the culture of same interval immersion system under natural conditions after the completion of the culture of rootage stage
It answers in device, is hardening culture solution by culture of rootage fluid exchange, continues hardening to obtain the fritillaria thunbergii seedling that can be transplanted, hardening
Cultivate formula of liquid are as follows: No. 2 1.0 g/L of Hua Bao add 50 mL/L of sheep dung leachate；The culture parameters of the stage interval immersion system are set
Be set to: 8 min of Immersion time, every 12 h of Immersion frequency is primary, 18 d of hardening time；Hardening environmental condition setting are as follows: natural ring
Border, 10-25 DEG C of temperature.
Fritillaria thunbergii proliferation rate reaches 45.3, field planting is transplanted to after hardening, for survival rate up to 100%, seedling early growth is prosperous
It contains.
Embodiment 3
Fritillaria thunbergii seedling is produced using interval immersion system, specifically includes the following steps:
(1) callus proliferation cultivation stage: aseptically, the callus for being proliferated the yellow green within four generations is transferred
Multiplying culture, callus proliferation culture formula of liquid are carried out into the cultivation reactor of sterilized interval immersion system are as follows: with
MS culture medium is basic culture medium, adds 1.5 mg/L of hormone NAA, 1.0 g/L of casein hydrolysate, 30 g/L of sucrose, pH tune
It saves to 5.8-6.2；The culture parameters of the stage interval immersion system are set are as follows: 4 min of Immersion time, every 3 h mono- of Immersion frequency
It is secondary, 40 g of inoculum density, 35 d of incubation time；The setting of culture environment condition are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000
Lux, 10 h/d of light application time；
(2) clove differentiation is with seedling cultivation stage: after the completion of callus proliferation cultivation stage, under aseptic condition, same
In the cultivation reactor of one interval immersion system, callus proliferation culture solution is changed to clove differentiation and seedling culture
Liquid continues clove differentiation and seedling culture, clove differentiation and seedling culture formula of liquid are as follows: based on MS culture medium
Culture medium adds 0.5 mg/L of hormone NAA, 1.0 mg/L of KT, 1.0 g/L of casein hydrolysate, sucrose 30 g/L, pH adjusting
To 5.8-6.2；The culture parameters of the stage interval immersion system are set are as follows: and 6 min of Immersion time, every 5 h of Immersion frequency is primary,
Clove differential period is room temperature illumination cultivation, 32 d of incubation time；30 d of seedling cultivation stage elder generation low temperature dark culturing, then
It goes under room temperature illumination cultivation and cultivates 40 d；The culture environment condition setting that normal-temperature light is shone are as follows: 20 ± 1 DEG C of temperature, intensity of illumination
2500-3000 Lux, 10 h/d of light application time；The culture environment condition of low temperature dark is set are as follows: 3-6 DEG C of temperature, completely black dark；
(3) it the culture of rootage stage: after the completion of clove differentiation is with seedling cultivation stage, under aseptic condition, is soaked in same interval
Do not have in the cultivation reactor of system, clove differentiation is changed to culture of rootage liquid with seedling culture solution, continues training of taking root
It supports, culture of rootage formula of liquid are as follows: with MS/2 culture medium be basic culture medium, add 2.0 mg/L of hormone IBA, casein hydrolysis
0.5 g/L of object, 0.1 mg/L of myricyl alcohol, 1.0 g/L of active carbon, sucrose 20 g/L, pH are adjusted to 5.8-6.2；Stage interval
The culture parameters of immersion system are set are as follows: 6 min of Immersion time, every 8 h of Immersion frequency is primary, 30 d of incubation time；Cultivate ring
Border condition setting are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000 Lux, 10 h/d of light application time.
(4) the hardening stage: anti-in the culture of same interval immersion system under natural conditions after the completion of the culture of rootage stage
It answers in device, is hardening culture solution by culture of rootage fluid exchange, continues hardening to obtain the fritillaria thunbergii seedling that can be transplanted, hardening
Cultivate formula of liquid are as follows: No. 2 1.0 g/L of Hua Bao add 50 mL/L of sheep dung leachate；The culture parameters of the stage interval immersion system are set
Be set to: 6 min of Immersion time, every 10 h of Immersion frequency is primary, 20 d of hardening time；Hardening environmental condition setting are as follows: natural ring
Border, 10-25 DEG C of temperature.
Fritillaria thunbergii proliferation rate reaches 44.6, field planting is transplanted to after hardening, for survival rate up to 98%, seedling early growth is prosperous
It contains.
Reference examples 1
Fritillaria thunbergii seedling is produced using conventional solid training method, specifically includes the following steps:
(1) callus proliferation cultivation stage: aseptically the callus for being proliferated the yellow green within four generations is transferred
Multiplying culture, callus proliferation medium formula are carried out into sterilized solid medium are as follows: based on MS culture medium
Culture medium adds 1.5 mg/L of hormone NAA, 1.0 g/L of casein hydrolysate, 30 g/L of sucrose, agar 5.0 g/L, pH adjusting
To 5.8-6.2, the inoculum concentration of every bottle of orchid bottle is 5.0 g, is guaranteed in solid culture mode, culture used in every gram of explant
The amount of base is consistent with the amount in interval immersion system, 45 d of incubation time；The setting of culture environment condition are as follows: 20 ± 1 DEG C of temperature, light
According to intensity 2500-3000 Lux, 10 h/d of light application time；
(2) clove differentiation is with seedling cultivation stage:, will proliferation under aseptic condition after the completion of callus proliferation cultivation stage
Cultured callus, which is transferred in sterilized solid medium, carries out clove differentiation and seedling culture, clove differentiation
With seedling culture medium prescription are as follows: with MS culture medium be basic culture medium, add 0.5 mg/L of hormone NAA, 1.0 mg/L of KT, junket
1.0 g/L of protolysate, 30 g/L of sucrose, agar 5.0 g/L, pH are adjusted to 5.8-6.2, and clove differential period is room temperature
Illumination cultivation, 45 d of incubation time；28 d of seedling cultivation stage elder generation low temperature dark culturing, then goes to room temperature illumination cultivation 55
d；The culture environment condition setting that normal-temperature light is shone are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000 Lux, light application time 10
h/d；The culture environment condition of low temperature dark is set are as follows: 3-6 DEG C of temperature, completely black dark；
(3) the culture of rootage stage: after the completion of clove differentiation is with seedling cultivation stage, fritillaria thunbergii seedling is turned under aseptic condition
It is connected in sterilized solid medium and carries out culture of rootage, the root media in culture of rootage step are as follows: cultivated with MS/2
Base is basic culture medium, adds 2.0 mg/L of hormone IBA, 0.5 g/L of casein hydrolysate, 0.1 mg/L of myricyl alcohol, active carbon
1.0 g/L, 20 g/L of sucrose, agar 5.0 g/L, pH are adjusted to 5.8-6.2,45 d of incubation time；The setting of culture environment condition
Are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000 Lux, 10 h/d of light application time；
(4) it the hardening stage: after the completion of the culture of rootage stage, is transferred under natural conditions in the fritillaria thunbergii seedling that will have been taken root
Hardening is carried out in hardening culture medium to obtain the fritillaria thunbergii seedling that can be transplanted, hardening culture medium are as follows: No. 2 1.0 g/L of Hua Bao add sheep
50 mL/L of excrement leachate, 5.0 g/L of agar, 20 d of hardening time；Hardening environmental condition setting are as follows: natural environment, temperature 10-
25℃。
Fritillaria thunbergii proliferation rate is only 6.2, and vitrification phenomenon occurs for a small number of plant tissues, is transplanted to crop field kind after hardening
It plants, for survival rate up to 86.4%, seedling growing way is general；It can be seen that cultivate fritillaria thunbergii seedling using method of the invention, proliferation rate and
Transplanting survival rate is significantly increased, and completely solves the Vitrification Occurred of seedling, and seedling growing way is also more vigorous.
Reference examples 2
Fritillaria thunbergii seedling is produced using traditional liquid shaking flask training method, specifically includes the following steps:
(1) callus proliferation cultivation stage: aseptically the callus for being proliferated the yellow green within four generations is transferred
Multiplying culture, callus proliferation culture solution are carried out into sterilized fluid nutrient medium are as follows: cultivate based on MS culture medium
Base, adds 1.5 mg/L of hormone NAA, 1.0 g/L of casein hydrolysate, sucrose 30 g/L, and pH is adjusted to 5.8-6.2, and every 1 liter
The inoculum concentration of triangular flask be 10.0 g, guarantee in liquid submerged culture mode, the amount of culture medium used in every gram of explant with
Amount in interval immersion system is consistent；100 rpm shaken cultivations, 35 d of incubation time；Culture environment condition setting are as follows: temperature 20
± 1 DEG C, intensity of illumination 2500-3000 Lux, 10 h/d of light application time；
(2) clove differentiation is with seedling cultivation stage:, will proliferation under aseptic condition after the completion of callus proliferation cultivation stage
Cultured callus, which is transferred in sterilized fluid nutrient medium, carries out clove differentiation and seedling culture, clove differentiation
With seedling culture formula of liquid are as follows: with MS culture medium be basic culture medium, add 0.5 mg/L of hormone NAA, 1.0 mg/L of KT, junket
1.0 g/L of protolysate, sucrose 30 g/L, pH are adjusted to 5.8-6.2；100 rpm shaken cultivations, clove differential period be
Then room temperature illumination cultivation, 38 d of incubation time, 30 d of seedling cultivation stage elder generation low temperature dark culturing go to room temperature illumination cultivation
45 d of lower culture；The culture environment condition setting that normal-temperature light is shone are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000 Lux, light
According to 10 h/d of time；The culture environment condition of low temperature dark is set are as follows: 3-6 DEG C of temperature, completely black dark；
(3) the culture of rootage stage: after the completion of clove differentiation is with seedling cultivation stage, fritillaria thunbergii seedling is turned under aseptic condition
It is connected in sterilized fluid nutrient medium and carries out culture of rootage, the culture of rootage liquid in culture of rootage step are as follows: cultivated with MS/2
Base is basic culture medium, adds 2.0 mg/L of hormone IBA, 0.5 g/L of casein hydrolysate, 0.1 mg/L of myricyl alcohol, active carbon
1.0 g/L, sucrose 20 g/L, pH are adjusted to 5.8-6.2；100 rpm shaken cultivations, 40 d of incubation time；Culture environment condition
Setting are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000 Lux, 10 h/d of light application time；
(4) it the hardening stage: after the completion of the culture of rootage stage, is transferred under natural conditions in the fritillaria thunbergii seedling that will have been taken root
Hardening is carried out in hardening culture solution to obtain the fritillaria thunbergii seedling that can be transplanted, hardening culture solution are as follows: No. 2 1.0 g/L of Hua Bao add sheep
50 mL/L of excrement leachate；100 rpm shaken cultivations, 18 d of hardening time；Hardening environmental condition setting are as follows: natural environment, temperature
10-25℃。
Fritillaria thunbergii proliferation rate is 15.8, and bulb of thunberg fritillary matrix vitrifying degree is high in incubation, causes cultured products a large amount of
Waste, increases production cost, is transplanted to field planting after hardening, survival rate 73.6%, and seedling growing way is poor.Thus
It can be seen that producing fritillaria thunbergii seedling using method disclosed by the invention, proliferation rate and transplanting survival rate are significantly increased, seedling early growth
It is more healthy；The Vitrification Occurred of seedling is completely solved, production cost is saved.
Basic principles and main features and advantage of the invention have been shown and described above.But the foregoing is merely this hairs
Bright specific embodiment, technical characteristic of the invention are not limited thereto, and any those skilled in the art is not departing from this hair
The other embodiments obtained under bright technical solution should all cover within the scope of the patent of the present invention.
Claims (10)
1. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system, which is characterized in that include according to cultivation sequence
Four cultivation stages, respectively callus proliferation cultivation stage, clove differentiation and seedling cultivation stage, culture of rootage stage
With the hardening stage, this four cultivation stages are carried out in the cultivation reactor of same interval immersion system, specific culture step
Suddenly are as follows:
(1) callus proliferation cultivation stage: aseptically, the callus for being proliferated the yellow green within four generations is transferred
Multiplying culture is carried out into the cultivation reactor of sterilized interval immersion system；
(2) clove differentiation is with seedling cultivation stage: after the completion of on last stage, under aseptic condition, in same interval immersion system
Cultivation reactor in, by callus proliferation culture solution be changed to clove differentiation with seedling culture solution, continue small squama
Stem differentiation and seedling culture；
(3) the culture of rootage stage: after the completion of on last stage, under aseptic condition, in the cultivation reactor of same interval immersion system
In, clove differentiation is changed to culture of rootage liquid with seedling culture solution, continues culture of rootage；
(4) it hardening: after the completion of on last stage, under natural conditions in the cultivation reactor of same interval immersion system, will take root
Culture solution is changed to hardening culture solution, continues hardening to obtain the fritillaria thunbergii seedling that can be transplanted.
2. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system as described in claim 1, feature exist
In, in the callus proliferation cultivation stage, callus proliferation culture formula of liquid are as follows: cultivated based on MS culture medium
Base adds 1.5 mg/L of hormone NAA, 1.0 g/L of casein hydrolysate, and sucrose 30 g/L, pH are adjusted to 5.8-6.2；In the rank
Section, the culture parameters setting of interval immersion system are as follows: Immersion time 1-10 min, the every 1-8 h of Immersion frequency is primary, inoculum density
20-50 g, incubation time 20-45 d；The culture environment condition in the stage is set are as follows: and 20 ± 1 DEG C of temperature, intensity of illumination 2500-
3000 Lux, 10 h/d of light application time.
3. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system as described in claim 1, feature exist
In the clove differentiation is with seedling cultivation stage, and clove breaks up and seedling culture formula of liquid are as follows: using MS culture medium as base
Basal culture medium adds 0.5 mg/L of hormone NAA, 1.0 mg/L of KT, 1.0 g/L of casein hydrolysate, 30 g/L of sucrose, pH tune
It saves to 5.8-6.2；At this stage, the culture parameters setting of interval immersion system are as follows: Immersion time 3-12 min, Immersion frequency are every
2-10 h is primary, and clove differential period is room temperature illumination cultivation, incubation time 20-45 d；Seedling cultivation stage elder generation low temperature is black
Then dark culture 15-40 d goes to room temperature illumination cultivation 30-55 d；The culture environment condition setting that normal-temperature light is shone are as follows: temperature 20
± 1 DEG C, intensity of illumination 2500-3000 Lux, 10 h/d of light application time；The culture environment condition of low temperature dark is set are as follows: temperature
It is 3-6 DEG C, completely black dark.
4. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system as described in claim 1, feature exist
In, in the culture of rootage stage, culture of rootage formula of liquid are as follows: with MS/2 culture medium be basic culture medium, add hormone IBA
2.0 mg/L, 0.5 g/L of casein hydrolysate, 0.1 mg/L of myricyl alcohol, 1.0 g/L of active carbon, sucrose 20 g/L, pH are adjusted to
5.8-6.2；At this stage, the culture parameters setting of interval immersion system are as follows: Immersion time 3-12 min, the every 4-12 of Immersion frequency
H is primary, incubation time 20-45 d；The setting of culture environment condition are as follows: 20 ± 1 DEG C of temperature, intensity of illumination 2500-3000 Lux, light
According to 10 h/d of time.
5. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system as described in claim 1, feature exist
In, in the hardening stage, hardening culture formula of liquid are as follows: No. 2 1.0 g/L of Hua Bao add 50 mL/L of sheep dung leachate；In the rank
Section, the culture parameters setting of the interval immersion system are as follows: Immersion time 1-10 min, the every 6-14 h of Immersion frequency is primary, refining
Seedling time 10-40 d；The setting of hardening environmental condition are as follows: natural environment, 10-25 DEG C of temperature.
6. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system as claimed in claim 2, feature exist
In in callus proliferation cultivation stage, the culture parameters setting of interval immersion system are as follows: Immersion time 4-8 min, submergence frequency
The every 3-6 h of rate is primary, inoculum density 30-40 g, incubation time 25-35 d.
7. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system as claimed in claim 3, feature exist
In in clove differentiation and seedling cultivation stage, the culture parameters setting of the interval immersion system are as follows: Immersion time 6-10
Min, the every 5-8 h of Immersion frequency is primary, and clove differential period is room temperature illumination cultivation, incubation time 30-35 d；Seedling culture
Stage elder generation's low temperature dark culturing 20-30 d, then goes to room temperature illumination cultivation 40-45 d.
8. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system as claimed in claim 4, feature exist
In in the culture of rootage stage, the culture parameters of the interval immersion system are set are as follows: Immersion time 6-10 min, Immersion frequency
Every 7-10 h is primary, incubation time 30-35 d.
9. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system as claimed in claim 5, feature exist
In in the hardening stage, the culture parameters of the interval immersion system are set are as follows: Immersion time 4-8 min, the every 9-12 of Immersion frequency
H is primary, hardening time 15-25 d.
10. a kind of method for quickly breeding fritillaria thunbergii seedling using interval immersion system as claimed in claim 5, feature
It is, the sheep dung leachate is added in 1 kg pure water after smashing 500 g sheep dungs to pieces, and 100 DEG C of heating 3 in water-bath are placed in
1 kg pure water is added in h, cooled and filtered again in filter residue, above-mentioned heating and filtering operation is repeated, after filtrate twice is merged
It obtains.
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