CN106258959B - A kind of azalea quick breeding method for tissue culture - Google Patents
A kind of azalea quick breeding method for tissue culture Download PDFInfo
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- CN106258959B CN106258959B CN201610644597.2A CN201610644597A CN106258959B CN 106258959 B CN106258959 B CN 106258959B CN 201610644597 A CN201610644597 A CN 201610644597A CN 106258959 B CN106258959 B CN 106258959B
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- seedling
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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
- A01H4/001—Culture apparatus for tissue culture
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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
Abstract
The invention discloses a kind of azalea quick breeding method for tissue culture, include the following steps：(1) azalea aseptic seeding seedling obtains；(2) tufted seedling induces；(3) tissue culture outside sprout-cultivating-bottle radication.The method of the present invention simplifies the step of azalea tissue cultures, has saved tissue culture cost, improves outside sprout-cultivating-bottle rate and the hardening domestication survival rate of tissue-cultured seedling.
Description
Technical field
The present invention relates to a kind of quick breeding method for tissue culture, and in particular to a kind of azalea tissue-culturing quick-propagation
Method.
Background technology
The tissue-culturing quick-propagation process of azalea includes：The selection of explant, disinfection, tufted seedling induction, after being commissioned to train
Foster, culture of rootage and test tube transplantation of seedlings.Under normal circumstances, the tissue cultures selection current year of azalea raw tender stem segments, stem apex,
Bud or blade are as explant；The disinfectant of explant is typically chosen neutral detergent, mercuric chloride, alcohol and NaClO etc., respectively
The disinfecting time of a disinfectant will be controlled can be only achieved preferable Disinfection Effect in a reasonable time.Such as patent
CN201010240170 discloses a kind of rhododendron decorum tissue-culture quick propagation method, using tissue culture technique to the son of seed embryo
Leaf and plumular axis carry out callus induction and then form a kind of rapid propagation method that Multiple Buds are then divided into intact plant.It should
Patent needs culture of rootage and test tube seedling to transplant two processes.
The prior art has the following disadvantages：
(1) of high cost needed for tissue culture propagating and technology require it is also high, how to shorten the tissue culture propagating time from
And the hardening domestication survival rate saved cost, and effectively improve tissue-cultured seedling becomes the key of tissue culture propagation.
(2) differentiation of bud is more difficult, and the period is long；The materials of stem apex are limited by certain time, generally use stem
The explant of section or blade as Initial culture, but have glandular hairs on the branches and leaves of azalea, and stickiness is big, can speckle with many dirts
Object and bacterium, are not easy disinfection, but if disinfectant is excessively used and can lead to the withered of branches and leaves.
(3) the long Necrosis for be easy to causeing explant material of mercuric chloride time-triggered protocol handles too short disinfection and is not thorough, controls
The time of mercuric chloride processing processed becomes a problem.
(4) azalea type and dosage of hormone in young shoot culture is not easily controlled.
Invention content
The purpose of the present invention is to provide a kind of azalea quick breeding method for tissue culture, and this process simplify azaleas
The step of tissue cultures, has saved tissue culture cost, improves outside sprout-cultivating-bottle rate and the hardening domestication survival rate of tissue-cultured seedling.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：A kind of azalea tissue-culturing quick-propagation
Method includes the following steps：
(1) azalea aseptic seeding seedling obtains：Cuckoo seed is chosen, again with the wine containing Tween 80 after gibberellin is handled
Essence is impregnated, and mercuric chloride and hypochlorite disinfectant are then used, then the cuckoo seed sterilized with aseptic water washing, acquisition, will be sterilized
Cuckoo seed be inoculated into sterile culture on 1/2Read culture mediums, obtain azalea aseptic seeding seedling；
(2) tufted seedling induces：It is inoculated into containing zeatin after the azalea aseptic seeding seedling obtained in step (1) is removed root
Tufted seedling induction is carried out on the Read culture mediums of ZT, obtains a large amount of tissue-cultured seedling；
(3) tissue culture outside sprout-cultivating-bottle radication：Choose cultivation matrix, the stem of the tissue-cultured seedling obtained in clip step (2), cuttage in
Outside sprout-cultivating-bottle is carried out in cultivation matrix, and controls humidity, temperature and illumination, carries out nursery again after tissue culture seedling rooting.
In above-mentioned azalea quick breeding method for tissue culture：
A concentration of 140~160mg/L of gibberellin, more preferably 150mg/L, gibberellin processing time are preferred in step (1)
For 7~9h, more preferably 8h.
The volumn concentration of alcohol is 70~75%, more preferably 75% in step (1), and the dosage of Tween 80 is preferably wine
4~6min is preferably impregnated, more preferably in the 0.8~1.2% of smart total volume, more preferably 1.0% with the alcohol containing Tween 80
5min。
The mass percentage of mercuric chloride is preferably 0.1~0.14% in step (1), and more preferably 0.1%, disinfecting time is preferred
Mass percentage for 6~8min, more preferably 7min, NaClO is preferably 8~12%, and more preferably 10%, disinfecting time is preferred
For 8~10min, more preferably 9min.
The cuckoo seed sterilized is inoculated into sterile culture on 1/2Read culture mediums in step (1), is obtained after 25~35 days
Azalea aseptic seeding seedling is obtained, azalea aseptic seeding seedling is obtained after more preferably 30 days.
The dosage of zeatin ZT (zeatin) is preferably to contain 0.8~1.2mg in 1L Read culture mediums in step (2), more
Good is 1.0mg.
The azalea aseptic seeding seedling obtained in step (1) is gone after root to be inoculated into containing zeatin ZT's in step (2)
After carrying out tufted seedling induction on Read culture mediums 28~32 days, more preferably 30 days, a large amount of tissue-cultured seedling is obtained.
Cultivation matrix described in step (3) is peat soil and vermiculite, and the volume ratio of the two is 1~2:1；The peat soil
It is first handled through 18 mesh sieves before with vermiculite and high pressure steam sterilization is handled, the temperature of sterilizing is 121 DEG C, and sterilization time is
1.5~2.5h, more preferably 2h.
Before the stem of the tissue-cultured seedling obtained in clip step (2) in step (3), the tissue-cultured seedling is first moved from culturing room
Go out, and be gradually opened the lid of tissue culture bottle, make tissue-cultured seedling adapt to ambient light shine and temperature condition, and keep air humidity be 80~
85%, the stem of clip tissue-cultured seedling carries out outside sprout-cultivating-bottle again after 6~8 days (being more preferably 7 days).
The tissue-cultured seedling obtained in clip step (2) in step (3), washes away culture medium, and remove tissue culture seeding stem segment lower part
Blade, cuttage carry out outside sprout-cultivating-bottle in cultivation matrix, and humidity and temperature, and training of shading are kept with transparent covered rearing with plastic film
It supports, removes film after 14~16 days (being more preferably 15 days), it is 80~85% to keep humidity, and nursery is transplanted to after tissue culture seedling rooting
Nursery on disk.
The present invention has the following advantages：
(1) the method for the present invention instead of traditionally culture of rootage and test tube seedling transplant two processes, only need to carry out outside bottle
Rooting process, has saved tissue culture cost at the step of simplifying azalea tissue cultures, meanwhile, outside sprout-cultivating-bottle rate of the invention can
Up to 90%, and further substantially increase the hardening domestication survival rate of tissue-cultured seedling；
(2) the method for the present invention obtains aseptic seeding seedling by material of seed, and using aseptic seedling as explant, group training material is not only
It is easy to obtain, and is easy disinfection；
(3) the method for the present invention is sterilized using mercuric chloride and NaClO, not only ensures that seed contamination rate is low, and ensured seed
Germination rate；
(4) tissue-cultured seedling (tufted seedling) inducing culture selects Read minimal mediums in the method for the present invention, and adds hormone
ZT can keep tufted seedling close and strong；
(5) in the method for the present invention outer 18 mesh of cultivation matrix (matrix of taking root) of bottle sieve, it is attached with convenient root system
It, which greatly enhances tissue culture outside sprout-cultivating-bottle radication rates；
(6) the method for the present invention is equally applicable in the sub- test tube germination of azalea hybrid, improves azalea new varieties nursery.
Description of the drawings
Fig. 1 is to cultivate the azalea aseptic seeding obtained using azalea quick breeding method for tissue culture in embodiment 1
Seedling；
Fig. 2 is to cultivate the azalea tissue-cultured seedling (bottle obtained using azalea quick breeding method for tissue culture in embodiment 1
It takes root outside).
Specific implementation mode
Embodiment 1
Azalea quick breeding method for tissue culture provided in this embodiment, includes the following steps：
(1) azalea aseptic seeding seedling is obtained, is used after cuckoo seed is handled 8 hours with the gibberellin of 150mg/L
The alcohol (the sterilized Tween 80s of addition 1mL in per 100mL alcohol) of 75% (volumn concentration) impregnates 5min, later super
7min is sterilized with the mercuric chloride of 0.1% (mass percentage) on net workbench, then is disappeared with the NaClO of 10% (mass percentage)
Malicious 9min, finally uses aseptic water washing 4~5 times, and sterilized cuckoo seed is inoculated into without any with sterilized tweezers
Sterile culture obtains aseptic seedling after about 30 days on the 1/2Read culture mediums of hormone, as shown in Figure 1；
(2) a large amount of sterile tufted seedling is obtained, above-mentioned aseptic seeding seedling is gone after root to be inoculated into Read+ZT1.0mg/L trainings
It supports and carries out tufted seedling induction on base, the dosage of zeatin ZT is to contain 0.8~1.2mg in 1L Read culture mediums, is obtained after 1 month
Obtain largely sterile tissue-cultured seedling；
(3) outside sprout-cultivating-bottle of tissue-cultured seedling, removed in culturing room tissue-cultured seedling make tissue-cultured seedling adapt to extraneous illumination condition and
The lid of culture bottle for a period of time, is unclamped after 7 days and is gradually opened daily by temperature, keeps the air humidity of cultivating seedling to be
85%, tissue culture outside sprout-cultivating-bottle radication is carried out by starting transplanting after 7 days, the sieve of 18 mesh of import peat and vermiculite is made
For cultivation matrix, after peat soil is mixed with vermiculite by volume 1: 1 and in high-pressure steam sterilizing pan 121 DEG C of sterilizings waited for after 2 hours
With as the culture substrate of sterile outside sprout-cultivating-bottle radication, clip tufted seedling washes away culture medium, and removes the blade of stem section lower part, skewer
It inserts in culture substrate, keeps humidity and temperature with transparent covered rearing with plastic film, and shade appropriate is cultivated.Remove after 15 days
Film, and per the sky fog, spray keeps certain humidity (it is 80~85% to keep humidity), may migrate to nursery after tissue culture seedling rooting
Nursery on disk.
Read medium component contents are as shown in table 1 in step (2).
1 Read medium component contents of table
1/2Read culture mediums in step (1), a great number of elements be Read culture medium a great number of elements half, other at
Divide identical as Read culture medium contents.
The tissue-cultured seedling obtained is cultivated as shown in Fig. 2, as can be seen from Figure 2 the outside sprout-cultivating-bottle effect of tissue-cultured seedling is good, therefore
The time that tissue culture propagation is not only shortened using the outside sprout-cultivating-bottle method in the present invention, also substantially increase tissue culture outside sprout-cultivating-bottle radication
Rate and hardening survival rate.
Embodiment 2
Azalea quick breeding method for tissue culture provided in this embodiment, includes the following steps：
(1) azalea aseptic seeding seedling obtains：Cuckoo seed is chosen, again with the wine containing Tween 80 after gibberellin is handled
Essence is impregnated, and mercuric chloride and hypochlorite disinfectant are then used, then the cuckoo seed sterilized with aseptic water washing, acquisition, will be sterilized
Cuckoo seed be inoculated into sterile culture on 1/2Read culture mediums, obtain azalea aseptic seeding seedling；
(2) tufted seedling induces：It is inoculated into containing zeatin after the azalea aseptic seeding seedling obtained in step (1) is removed root
Tufted seedling induction is carried out on the Read culture mediums of ZT, obtains a large amount of tissue-cultured seedling；
(3) tissue culture outside sprout-cultivating-bottle radication：Choose cultivation matrix, the stem of the tissue-cultured seedling obtained in clip step (2), cuttage in
Outside sprout-cultivating-bottle is carried out in cultivation matrix, and controls humidity, temperature and illumination, carries out nursery again after tissue culture seedling rooting.
A concentration of 140mg/L of gibberellin in step (1), gibberellin processing time are 9h.
The volumn concentration of alcohol is 70% in step (1), and the dosage of Tween 80 is the 0.8% of alcohol total volume, is used
Alcohol containing Tween 80 impregnates 6min.
The mass percentage of mercuric chloride is 0.12% in step (1), and the quality percentage of disinfecting time 8min, NaClO contain
Amount is 8%, disinfecting time 10min.
The cuckoo seed sterilized is inoculated into sterile culture on 1/2Read culture mediums in step (1), is shut out after 35 days
Cuckoo spends aseptic seeding seedling.
The dosage of zeatin ZT is to contain 0.8mg in 1L Read culture mediums in step (2).
The azalea aseptic seeding seedling obtained in step (1) is gone after root to be inoculated into containing zeatin ZT's in step (2)
After carrying out tufted seedling induction on Read culture mediums 28 days, a large amount of tissue-cultured seedling is obtained.
Cultivation matrix described in step (3) is peat soil and vermiculite, and the volume ratio of the two is 1.5：1, peat soil and leech
Stone is first handled through 18 mesh sieves before and high pressure steam sterilization is handled, and the temperature of sterilizing is 121 DEG C, and sterilization time is
1.5h。
Before the stem of the tissue-cultured seedling obtained in clip step (2) in step (3), the tissue-cultured seedling is first moved from culturing room
Go out, and be gradually opened the lid of tissue culture bottle, so that tissue-cultured seedling is adapted to ambient light and shine and temperature condition, and keep the air humidity to be
80%, the stem of clip tissue-cultured seedling carries out outside sprout-cultivating-bottle again after 8 days.
The tissue-cultured seedling obtained in clip step (2) in step (3), washes away culture medium, and remove tissue culture seeding stem segment lower part
Blade, cuttage carry out outside sprout-cultivating-bottle in cultivation matrix, and humidity and temperature, and training of shading are kept with transparent covered rearing with plastic film
It supports, removes film after 14 days, it is 85% to keep humidity, and nursery on seedlings nursing plate is transplanted to after tissue culture seedling rooting.
Embodiment 3
Azalea quick breeding method for tissue culture provided in this embodiment, includes the following steps：
(1) azalea aseptic seeding seedling obtains：Cuckoo seed is chosen, again with the wine containing Tween 80 after gibberellin is handled
Essence is impregnated, and mercuric chloride and hypochlorite disinfectant are then used, then the cuckoo seed sterilized with aseptic water washing, acquisition, will be sterilized
Cuckoo seed be inoculated into sterile culture on 1/2Read culture mediums, obtain azalea aseptic seeding seedling；
(2) tufted seedling induces：It is inoculated into containing zeatin after the azalea aseptic seeding seedling obtained in step (1) is removed root
Tufted seedling induction is carried out on the Read culture mediums of ZT, obtains a large amount of tissue-cultured seedling；
(3) tissue culture outside sprout-cultivating-bottle radication：Choose cultivation matrix, the stem of the tissue-cultured seedling obtained in clip step (2), cuttage in
Outside sprout-cultivating-bottle is carried out in cultivation matrix, and controls humidity, temperature and illumination, carries out nursery again after tissue culture seedling rooting.
A concentration of 160mg/L of gibberellin in step (1), gibberellin processing time are 7h.
The volumn concentration of alcohol is 72% in step (1), and the dosage of Tween 80 is the 1.2% of alcohol total volume, is used
Alcohol containing Tween 80 impregnates 4min.
The mass percentage of mercuric chloride is 0.14% in step (1), and the quality percentage of disinfecting time 6min, NaClO contain
Amount is 12%, disinfecting time 8min.
The cuckoo seed sterilized is inoculated into sterile culture on 1/2Read culture mediums in step (1), is shut out after 25 days
Cuckoo spends aseptic seeding seedling.
The dosage of zeatin ZT is to contain 1.2mg in 1L Read culture mediums in step (2).
The azalea aseptic seeding seedling obtained in step (1) is gone after root to be inoculated into containing zeatin ZT's in step (2)
After carrying out tufted seedling induction on Read culture mediums 32 days, a large amount of tissue-cultured seedling is obtained.
Cultivation matrix described in step (3) is peat soil and vermiculite, and the volume ratio of the two is 2：1, peat soil and vermiculite
It is handled with high pressure steam sterilization using preceding first handled through 18 mesh sieves, the temperature of sterilizing is 121 DEG C, sterilization time 2.5h.
Before the stem of the tissue-cultured seedling obtained in clip step (2) in step (3), the tissue-cultured seedling is first moved from culturing room
Go out, and be gradually opened the lid of tissue culture bottle, so that tissue-cultured seedling is adapted to ambient light and shine and temperature condition, and keep the air humidity to be
82%, the stem of clip tissue-cultured seedling carries out outside sprout-cultivating-bottle again after 6 days.
The tissue-cultured seedling obtained in clip step (2) in step (3), washes away culture medium, and remove tissue culture seeding stem segment lower part
Blade, cuttage carry out outside sprout-cultivating-bottle in cultivation matrix, and humidity and temperature, and training of shading are kept with transparent covered rearing with plastic film
It supports, removes film after 16 days, it is 82% to keep humidity, and nursery on seedlings nursing plate is transplanted to after tissue culture seedling rooting.
Although the present invention is disclosed as above with embodiment, it is not limited to protection scope of the present invention, any ripe
Those skilled in the art are known, change and retouching made by without departing from the spirit and scope of the invention should all belong to this hair
Bright protection domain.
Claims (6)
1. a kind of azalea quick breeding method for tissue culture, it is characterized in that including the following steps：
(1) azalea aseptic seeding seedling obtains：Cuckoo seed is chosen, again with the alcohol containing Tween 80 after gibberellin is handled
It impregnates, then uses mercuric chloride and hypochlorite disinfectant, then the cuckoo seed sterilized with aseptic water washing, acquisition, by what is sterilized
Cuckoo seed is inoculated into sterile culture on 1/2Read culture mediums, obtains azalea aseptic seeding seedling；
(2) tufted seedling induces：It is inoculated into containing zeatin after the azalea aseptic seeding seedling obtained in step (1) is removed root
Tufted seedling induction is carried out on the Read culture mediums of ZT, obtains a large amount of tissue-cultured seedling；
(3) tissue culture outside sprout-cultivating-bottle radication：Choose cultivation matrix, the stem of the tissue-cultured seedling obtained in clip step (2), cuttage in
Outside sprout-cultivating-bottle is carried out in cultivation matrix, and controls humidity, temperature and illumination, carries out nursery again after tissue culture seedling rooting；
The mass percentage of mercuric chloride is the .14% of 0 .1~0 in step (1), and disinfecting time is 6~8min, the matter of NaClO
It is 8~12% to measure percentage composition, and disinfecting time is 8~10min；
The azalea aseptic seeding seedling obtained in step (1) is gone after root to be inoculated into containing zeatin ZT's in step (2)
After carrying out tufted seedling induction on Read culture mediums 28~32 days, a large amount of tissue-cultured seedling is obtained；
The dosage of zeatin ZT is to contain the .2mg of 0 .8~1 in 1L Read culture mediums in step (2)；
Cultivation matrix described in step (3) is peat soil and vermiculite, and the volume ratio of the two is 1~2:1；The peat soil and
Vermiculite is first handled through 18 mesh sieves before and high pressure steam sterilization is handled, and sterilising temp is 121 DEG C, and sterilization time is 1 .5
~2 .5h.
2. azalea quick breeding method for tissue culture according to claim 1, it is characterized in that：It is red mould in step (1)
A concentration of 140~160mg/L of element, gibberellin processing time are 7~9h.
3. azalea quick breeding method for tissue culture according to claim 1, it is characterized in that：Alcohol in step (1)
Volumn concentration be 70~75%, the dosage of Tween 80 is the .2% of 0 .8~1 of alcohol total volume, with containing Tween 80
Alcohol impregnate 4~6min.
4. azalea quick breeding method for tissue culture according to claim 1, it is characterized in that：Step will disappear in (1)
The cuckoo seed that poison is crossed is inoculated into sterile culture on 1/2Read culture mediums, and azalea aseptic seeding seedling is obtained after 25~35 days.
5. azalea quick breeding method for tissue culture according to claim 1, it is characterized in that：Clip in step (3)
Before the stem of the tissue-cultured seedling obtained in step (2), the tissue-cultured seedling is first removed from culturing room, and is gradually opened tissue culture bottle
Lid makes tissue-cultured seedling adapt to ambient light and shines and temperature condition, and it is 80~85% to keep air humidity, the clip again after 6~8 days
The stem of tissue-cultured seedling carries out outside sprout-cultivating-bottle.
6. azalea quick breeding method for tissue culture according to claim 1, it is characterized in that：Clip in step (3)
The tissue-cultured seedling obtained in step (2), washes away culture medium, and remove the blade of tissue culture seeding stem segment lower part, cuttage is in cultivation matrix
Middle carry out outside sprout-cultivating-bottle keeps humidity and temperature, and culture of shading with transparent covered rearing with plastic film, removes after 14~16 days thin
Film, it is 80~85% to keep humidity, and nursery on seedlings nursing plate is transplanted to after tissue culture seedling rooting.
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| CN107079815B (en) * | 2017-05-25 | 2019-06-11 | 贵州师范大学 | A method of inducing charming cuckoo and Rhododendron delavayi cenospecies callus |
| CN107494274B (en) * | 2017-10-12 | 2019-10-22 | 云南省农业科学院花卉研究所 | A kind of stripping bud sterilization method of alpine rose explant |
| CN107960260A (en) * | 2017-11-28 | 2018-04-27 | 西南大学 | A kind of wild woody flowers nursery stock selection and Rhododendron irroratum Franch, wealthy handle cuckoo |
| CN109496854A (en) * | 2018-11-29 | 2019-03-22 | 四川农业大学 | A kind of pediment cuckoo seed tissue culture and rapid propagation method |
| CN110896860B (en) * | 2019-12-02 | 2021-07-27 | 闽江学院 | Efficient brocade rhododendron direct somatic embryogenesis method |
| CN112450071A (en) * | 2020-11-25 | 2021-03-09 | 贵州大学 | Disinfection method of rhododendron lapponicum seed explant |
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