CN106962198A - A kind of winter date anther culture regeneration haplobiont system and its cultural method - Google Patents
A kind of winter date anther culture regeneration haplobiont system and its cultural method Download PDFInfo
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- CN106962198A CN106962198A CN201710304633.5A CN201710304633A CN106962198A CN 106962198 A CN106962198 A CN 106962198A CN 201710304633 A CN201710304633 A CN 201710304633A CN 106962198 A CN106962198 A CN 106962198A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The present invention relates to agricultural technology field, specifically related to a kind of winter date anther culture regeneration haplobiont system and its cultural method, including anther culture medium, one-level callus tissue culture base and two grades of callus tissue culture bases, the anther culture medium is made up of 1/2 MS, the D of 0.8~1.2mol/L 2,4,18~22g/L maltose and 3.2~3.7g/L agar；The one-level callus tissue culture base is made up of MS, 0.8~1.2mg/L TDZ, 18~22g/L maltose and 3.2~3.7g/L agar；Two grades of callus tissue culture bases are made up of MS, 1.8~2.2mg/L triacontanol, 18~22g/L maltose and 3.2~3.7g/L agar.The system composition of the present invention is simple, it is easy to culture, the jujube tree of vegetative propagation is also used seminal propagation by haploid breeding, and pure lines material is provided for the hereditary Journal of Sex Research of jujube kind of genotype height heterozygosis, so that the research shortened the breeding time limit and promote jujube tree theory of heredity to work.
Description
Technical field
The present invention relates to agricultural technology field, and in particular to a kind of winter date anther culture regeneration haplobiont system and its
Cultural method.
Background technology
Nineteen fifty-three, Tuleck gingko pollens successfully induced synthesis monoploid callus group for the first time on synthetic medium
Knit Guha and Maheshwari (1964) and successfully obtain single from hair leaf datura (Datura inoxia) Anther Culture first
Times body plant, then confirms it is the Haploid plants obtained by embryoid approach, has been started from this and cultivated single using pollen
The new way of times body, over nearly 30 years, 10 sections, 24 have been widely applied to using the technology of Anther Culture Haploid production plant
In individual category, 250 various plants of 34 kinds.The haploid research of fruit tree Anther Culture is started late, and late 1970s arrive
The beginning of the eighties successively achieves success on the seeds such as citrus, apple, grape, strawberry, lichee, longan.Jujube (Ziziphus
Jujuba Mill.) it is China special advantage fruit tree, on the Anther Culture of jujube, it is rarely reported, and there is callus induction
The problem of rate relatively low (43.1%).
China Patent Publication No. CN104041414B discloses a kind of method that pepper anther culture obtains haplobiont,
Including choosing bud, bud sterilization, flower pesticide inoculation, pretreatment, Anther Culture, evoked callus is divided into monoploid plant again
Strain；The invention uses solid medium to co-culture microspore and anther wall, and low temperature, heat shock pretreatment, regulation callus are induced, divided
Change medium component and condition of culture, improve the differentiation capability of callus rate and callus；Adopting said method carries out capsicum
Medicine culture, Callus induction rate more than 60%, emergence rate more than 80% solves the low problem of pepper anther culture inductivity.But
It is that the invention is not particularly suited for winter jujube culture.
China Patent Publication No. CN1934932 discloses a kind of method of in-vitro inducing winter date anther callus, that is, leads to
The tissue cultures for crossing winter date anther obtain its callus, and callus induction rate is up to 86.23%, including explant collection, disappears
Poison, initial induction, Multiplying culture.A diameter of 0.3~the 0.6cm in field is chosen in 5~June, winter jujube that calyx not yet opens not into
Ripe bud, 4 DEG C of refrigerators pre-process 1~3D.Flowing water rinses on 1~2H, superclean bench bud being put into 70% ethanol 30S, then
It is transferred to 8mic in 0.1%HGCL2 solution, strips flower pesticide after aseptic water washing 3~4 times, be inoculated with sterile flower pesticide during Primary culture
Light culture is carried out on 15~20g/L+TDZ0.5-1.0mg/L+NAA0-0.5mg/L of MS+ maltose culture medium three weeks, then
(800~1000LUX) culture is gone under dim light.Flower pesticide gradually produces yellow green, discrete particles shape callus, and with training
The extension for the time of supporting gradually increases.The initial callus of acquisition is transferred to 15~20g/L+ of MS+ maltose during Multiplying culture
Multiplying culture is carried out in TDZ0.1-0.5mg/L+NAA0-0.5mg/L culture medium, callus can fast breeding.This method can
A large amount of winter date anther callus are obtained in 70D or so；But this method complex steps, using limited.
Anther Culture is the important channel of haploid breeding, not only shortens the breeding time limit, and improve breeding efficiency.
By haploid breeding there is a possibility that the jujube tree of vegetative propagation can also use seminal propagation, and it is genotype height for jujube tree
The hereditary Journal of Sex Research of the jujube kind of heterozygosis provides pure lines material, so as to shorten the breeding time limit and promote grinding for jujube tree theory of heredity work
Study carefully.
The content of the invention
Haplobiont is regenerated it is an object of the invention to overcome the deficiencies of the prior art and provide a kind of winter date anther culture
System and its cultural method, system composition are simple, it is easy to cultivate, and the jujube tree of vegetative propagation is also used by haploid breeding and plant
Son breeding, and pure lines material is provided for the hereditary Journal of Sex Research of jujube kind of genotype height heterozygosis, so as to shorten the breeding time limit and promote
Enter the research of jujube tree theory of heredity work.
The purpose of the present invention is achieved through the following technical solutions：
A kind of winter date anther culture regenerates haplobiont system, including anther culture medium, one-level callus tissue culture base
With two grades of callus tissue culture bases,
The anther culture medium MS, the 0.8~1.2mol/L 2 by 1/2,4-D, 18~22g/L maltose and 3.2~
3.7g/L agar composition；
The one-level callus tissue culture base by MS, 0.8~1.2mg/L TDZ, 18~22g/L maltose and 3.2~
3.7g/L agar composition；
Two grades of callus tissue culture bases by MS, 1.8~2.2mg/L triacontanol, 18~22g/L maltose and
3.2~3.7g/L agar composition.
Further, the anther culture medium is by 1/2 MS, 1.0mol/L2,4-D, 20g/L maltose and 3.5g/L
Agar composition；
The one-level callus tissue culture base by MS, 1.0mg/L TDZ, 20g/L maltose and 3.5g/L agar group
Into；
Two grades of callus tissue culture bases are by MS, 2.0mg/L triacontanol, 20g/L maltose and 3.5g/L
Agar is constituted.
A kind of winter date anther culture regenerates the cultural method of haplobiont system, using above-mentioned winter date anther culture again
Raw haplobiont system is cultivated.
Further, comprise the following steps：
S1. winter jujube bud is immersed in 0.10mol/L mannitol 3 days, be then seeded on anther culture medium, cultivated
40~60 days, obtain callus；
S2. callus is seeded on one-level callus tissue culture base, cultivated 25~35 days；
Then S3 inoculates callus onto two grades of callus tissue culture bases, cultivates 25~35 days.
Further, in the step S1, it is inoculated into after anther culture medium, light culture is carried out at 25~28 DEG C.
Further, the step S2, step S3 are cultivated at 25~28 DEG C respectively.
Further, monoploid incidence reaches 33.33%.
The beneficial effects of the invention are as follows：The system composition of the present invention is simple, it is easy to cultivates, is made by haploid breeding asexual
The jujube tree of breeding can also use seminal propagation, and provide pure lines material for the hereditary Journal of Sex Research of jujube kind of genotype height heterozygosis, from
And the research shortened the breeding time limit and promote jujube tree theory of heredity to work.
Embodiment
Technical scheme is described in further detail with reference to specific embodiment, but protection scope of the present invention is not
It is confined to as described below.
Embodiment 1
A kind of winter date anther culture regenerates haplobiont system, including anther culture medium, one-level callus tissue culture base
With two grades of callus tissue culture bases,
The anther culture medium by 1/2 MS, 0.8mol/L 2,4-D, 18g/L maltose and 3.2g/L agar group
Into；
The one-level callus tissue culture base by MS, 0.8mg/L TDZ, 19g/L maltose and 3.3g/L agar group
Into；
Two grades of callus tissue culture bases are by MS, 2.2mg/L triacontanol, 18g/L maltose and 3.7g/L
Agar is constituted.
Embodiment 2
A kind of winter date anther culture regenerates haplobiont system, including anther culture medium, one-level callus tissue culture base
With two grades of callus tissue culture bases,
The anther culture medium is by 1/2 MS, 1.2mol/L2,4-D, 19g/L maltose and 3.7g/L agar group
Into；
The one-level callus tissue culture base by MS, 1.1mg/L TDZ, 22g/L maltose and 3.2g/L agar group
Into；
Two grades of callus tissue culture bases are by MS, 1.9mg/L triacontanol, 20g/L maltose and 3.4g/L
Agar is constituted.
Embodiment 3
A kind of winter date anther culture regenerates haplobiont system, including anther culture medium, one-level callus tissue culture base
With two grades of callus tissue culture bases,
The anther culture medium is by 1/2 MS, 1.1mol/L2,4-D, 22g/L maltose and 3.6g/L agar group
Into；
The one-level callus tissue culture base by MS, 1.2mg/L TDZ, 18g/L maltose and 3.7g/L agar group
Into；
Two grades of callus tissue culture bases are by MS, 1.8mg/L triacontanol, 22g/L maltose and 3.2g/L
Agar is constituted.
Embodiment 4
A kind of winter date anther culture regenerates haplobiont system, including anther culture medium, one-level callus tissue culture base
With two grades of callus tissue culture bases,
The anther culture medium is by 1/2 MS, 1.0mol/L2,4-D, 20g/L maltose and 3.5g/L agar group
Into；
The one-level callus tissue culture base by MS, 1.0mg/L TDZ, 20g/L maltose and 3.5g/L agar group
Into；
Two grades of callus tissue culture bases are by MS, 2.0mg/L triacontanol, 20g/L maltose and 3.5g/L
Agar is constituted.
Embodiment 5
A kind of winter date anther culture regenerates haplobiont system, including anther culture medium, one-level callus tissue culture base
With two grades of callus tissue culture bases,
The anther culture medium is by 1/2 MS, 1.50mol/L2,4-D, 20g/L maltose and 3.5g/L agar group
Into；
The one-level callus tissue culture base by MS, 1.0mg/L TDZ, 20.5g/L maltose and 3.5g/L agar
Composition；
Two grades of callus tissue culture bases are by MS, 2.0mg/L triacontanol, 20g/L maltose and 3.6g/L
Agar is constituted.
Test example
A kind of winter date anther culture regenerates the cultural method of haplobiont system, using above-mentioned winter date anther culture again
Raw haplobiont system is cultivated.
Specifically, comprise the following steps：
S1. the winter jujube bud (mid-late uninucleate stage is in pollen now) from the appearance for yellow green is selected, by bud more
3d in 0.10mol/L mannitol is immersed in, flower pesticide is then seeded in 1/2MS+2,4-D1.0mol/L+ maltose 20g/L+
On agar 3.5g/L culture mediums, light culture in 25-28 DEG C of incubator is placed in, flower pesticide healing rate is up to 98.56% after 50 days；
S2. callus is seeded on MS+TDZ1.0mg/L+ maltose 20g/L+ agar 3.5g/L culture medium and carried out
Propagation and differentiation, are observed after 30 days, and callus proliferation is rapid, and color is emerald green, robust growth；
S3., callus is inoculated into MS+ triacontanol 2.0mg/L+ maltose 20g/L+ agar 3.5g/L culture medium
On, observed after 30 days, phenylacetic acid is up to 100%, regeneration plant rate 8.22%；
S4. regeneration plant is identified in winter date anther culture by flow cytometer, discovery has monoploid, Yi Jidan
Times body and the chimera of diploid, monoploid incidence 33.33%.
Described above is only the preferred embodiment of the present invention, it should be understood that the present invention is not limited to described herein
Form, is not to be taken as the exclusion to other embodiment, and available for various other combinations, modification and environment, and can be at this
In the text contemplated scope, it is modified by the technology or knowledge of above-mentioned teaching or association area.And those skilled in the art are entered
Capable change and change does not depart from the spirit and scope of the present invention, then all should appended claims of the present invention protection domain
It is interior.
Claims (7)
1. a kind of winter date anther culture regenerates haplobiont system, it is characterised in that including anther culture medium, one-level callus group
Culture medium and two grades of callus tissue culture bases are knitted,
MS, 0.8~1.2mol/L 2 of the anther culture medium by 1/2,4-D, 18~22g/L maltose and 3.2~3.7g/
L agar composition；
The one-level callus tissue culture base by MS, 0.8~1.2mg/L TDZ, 18~22g/L maltose and 3.2~
3.7g/L agar composition；
Two grades of callus tissue culture bases are by MS, 1.8~2.2mg/L triacontanol, 18~22g/L maltose and 3.2
~3.7g/L agar composition.
2. a kind of winter date anther culture regeneration haplobiont system according to claim 1, it is characterised in that the flower
Medicine culture medium is by 1/2 MS, 1.0mol/L 2, and 4-D, 20g/L maltose and 3.5g/L agar are constituted；
The one-level callus tissue culture base is made up of MS, 1.0mg/L TDZ, 20g/L maltose and 3.5g/L agar；
Two grades of callus tissue culture bases by MS, 2.0mg/L triacontanol, 20g/L maltose and 3.5g/L agar
Composition.
3. a kind of winter date anther culture regenerates the cultural method of haplobiont system, it is characterised in that using claim 1 or
Winter date anther culture regeneration haplobiont system described in 2 is cultivated.
4. a kind of winter date anther culture according to claim 3 regenerates the cultural method of haplobiont system, its feature
It is, comprises the following steps：
S1. winter jujube bud is immersed in 0.10mol/L mannitol 3 days, be then seeded on anther culture medium, culture 40~
60 days, obtain callus；
S2. callus is seeded on one-level callus tissue culture base, cultivated 25~35 days；
Then S3 inoculates callus onto two grades of callus tissue culture bases, cultivates 25~35 days.
5. a kind of winter date anther culture according to claim 4 regenerates the cultural method of haplobiont system, its feature
It is, in the step S1, is inoculated into after anther culture medium, light culture is carried out at 25~28 DEG C.
6. a kind of winter date anther culture according to claim 4 regenerates the cultural method of haplobiont system, its feature
It is, the step S2, step S3 are cultivated at 25~28 DEG C respectively.
7. a kind of winter date anther culture according to claim 4 regenerates the cultural method of haplobiont system, its feature
It is, monoploid incidence reaches 33.33%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115530076A (en) * | 2022-11-10 | 2022-12-30 | 山东滨州国家农业科技园区管理服务中心(滨州黄河三角洲高效生态产业现代技术研究院) | Winter jujube tissue culture method |
-
2017
- 2017-05-03 CN CN201710304633.5A patent/CN106962198A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 武晓红: "枣花药再生植株及单倍体的获得", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115530076A (en) * | 2022-11-10 | 2022-12-30 | 山东滨州国家农业科技园区管理服务中心(滨州黄河三角洲高效生态产业现代技术研究院) | Winter jujube tissue culture method |
| CN115530076B (en) * | 2022-11-10 | 2023-05-26 | 山东滨州国家农业科技园区管理服务中心(滨州黄河三角洲高效生态产业现代技术研究院) | Winter jujube tissue culture method |
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Application publication date: 20170721 |
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