CN104304034B - Induction culture method and special induction culture medium for somatic embryo calluses of picea schrenkiana - Google Patents
Induction culture method and special induction culture medium for somatic embryo calluses of picea schrenkiana Download PDFInfo
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- CN104304034B CN104304034B CN201410630007.1A CN201410630007A CN104304034B CN 104304034 B CN104304034 B CN 104304034B CN 201410630007 A CN201410630007 A CN 201410630007A CN 104304034 B CN104304034 B CN 104304034B
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Abstract
The invention discloses an induction culture method for somatic embryo calluses of picea schrenkiana. The method comprises the following steps: collecting the cones or seeds of the picea schrenkiana at the last ten-day periods of June and July and September respectively, disinfecting, washing, taking out immature zygotic embryos with endosperms, immature zygotic embryos without endosperms and mature zygotic embryos respectively, and inoculating to a culture medium for induction culture to obtain the embryonic calluses. Meanwhile, the invention further provides the special induction culture medium. The method and the culture medium have the benefits that the establishment of the induction method and the preparation of the culture medium lay a foundation for later-period genetic transformation and transfer of the somatic embryos of the picea schrenkiana into practical target genes as well as cloning, and provide favorable conditions for germplasm preservation of the picea schrenkiana, artificial seed and protolast culture, tree species improvement and ecological environment construction.
Description
Technical field
The present invention relates to plant tissue culture field, be specifically related to the method for inducing and cultivating of tianshan mountain spruce somatic embryogenic callus
And special induced culture medium.
Background technology
Tianshan mountain spruce(Picea schrenkiana Fisch. at Mey.)Belong to Pinaceae Picea, in the Central Asia and Asia
Mountain region, portion endemic species, are only distributed in Tianshan Mountains in China, are one of most important forest species in mountain area, Xinjiang, area about 52.84 ten thousand
Hm2, accounts for the full boundary natural forest land gross area 44.9%, be Xinjiang Mountainous forest is distributed the widest, accumulation is maximum, business activities
At most, purposes is the widest, material is excellent main timber tree species, to the water conservation in Tianshan Mountains, soil-water protection, forest zone ecosystem
Formation and maintenance plays irreplaceable effect.
Owing to the mankind's activity of tianshan mountain spruce areal area is frequent and the factor such as irrational operation and management and contradiction in forestry and animal husbandry causes
Picea schrenkiana var. tianshanica forest ecosystem structure is badly damaged, and result in the decline of Picea schrenkiana var. tianshanica forest.Natural regeneration is bad or natural regeneration
The problems such as difficulty go out a great problem having become long-term puzzled forest workers.The growth of PiceameyeriRehd. Et Wils. seeds is relatively slow, seed maturity
Rate is the lowest, and breeding and breed breeding have the biggest difficulty.From seed germination to growing up to the seedling that can transplant, need again through 4-5,
So carrying out breeding by natural propagation and selecting superior genotypes efficiency the lowest.Use the side that grafting, cuttage etc. are nourished and generated
Method had both consumed substantial amounts of time and labour force, was limited by season again.
Tianshan mountain spruce mainly produces offspring by sexual propagation at present, and asexual propagation is constantly subjected to various countries' forestry decision-making section
Attention and Forest Tree Breeding Work person and the special concern of orest management person, especially modern biotechnology flourish,
Such as plant tissue culture and the Development of Somatic Embryogenesis, have that reproductive efficiency is high, the cycle is short, not by natural conditions restriction, steady
Fixed, selection-breeding improved seeds, to features such as afforestation project are significant.Plant is realized again by somatic embryo development pathway
Life is possible not only to solve vegetative problem, and the quality for the biotechnology specifically application and seeds thereof in Picea changes
Good also tool is of great significance.In tianshan mountain spruce Somatic Embryogenesis, via method for tissue culture induction more
Injured tissue, and obtained somatic embryo by callus, it is wide variety of hands in the research of current artificial seed and biotechnology
One of section.
Summary of the invention
The purpose of the present invention is aiming at above-mentioned defect of the prior art, it is provided that tianshan mountain spruce somatic embryo wound healing group
The method for inducing and cultivating knitted and special induced culture medium thereof.
To achieve these goals, the technical scheme that the present invention provides is: luring of tianshan mountain spruce somatic embryogenic callus
Lead cultural method, comprise the following steps:
1) gather tianshan mountain spruce cone, the i.e. immature zygotic embryos of band endosperm in late June, as material, cone is used
Mass percentage concentration is the HgCl of 0.2%2After solution disinfection 10 minutes, with aseptic water washing 3 times；Take out from cone with taking the photograph son
Immature seed, scratches kind of a shell from sidepiece, takes out kernel, scratches endotesta, teasing endosperm, be inoculated in together with endosperm by tender embryo
On the inducing culture of tianshan mountain spruce somatic embryogenic callus；Every ware inoculation of medium 4, is repeated 20 times, in temperature is
Light culture 15-25 days at 23 ± 2 DEG C, obtains the embryo callus of white translucent；
2) tianshan mountain spruce cone is gathered in late July, i.e. without the immature zygotic embryos of endosperm, as material, by cone
It is the HgCl of 0.2% by mass percentage concentration2After solution disinfection 10 minutes, with aseptic water washing 3 times；Take from cone with taking the photograph son
Go out the seed that is mature on the whole, from longitudinally split seed, take out embryo, be inoculated in the induction training of tianshan mountain spruce somatic embryogenic callus
Support on base；Every ware inoculation of medium 4, is repeated 20 times, light culture 15-25 days at temperature is 23 ± 2 DEG C, obtains white half
Transparent embryo callus；
3) tianshan mountain spruce mature seed, i.e. mature zygotic embryos are gathered in JIUYUE, as material, by dense for seed percent mass
Degree is the HgCl of 0.2%2After solution disinfection 5 minutes, with aseptic water washing 3 times；From seed, mature zygotic embryos is taken out with taking the photograph son,
It is inoculated on the inducing culture of tianshan mountain spruce somatic embryogenic callus；Every ware inoculation of medium 4, is repeated 20 times,
Cultivation temperature is light culture 15-25 days at 23 ± 2 DEG C, obtains the embryo callus of white translucent.
Further, the method for inducing and cultivating of above-mentioned tianshan mountain spruce somatic embryogenic callus, described step 1)-3) in
The inducing culture of tianshan mountain spruce somatic embryogenic callus used divides for being added with auximone 2,4-D and plant cell
Split the DCR culture medium of element BAP.
Further, the method for inducing and cultivating of above-mentioned tianshan mountain spruce somatic embryogenic callus, described tianshan mountain spruce body
In the inducing culture of somatic embryo callus, auximone 2, the concentration of 4-D is 20 μMs, phytocytomine BAP's
Concentration is 5 μMs.
Second object of the present invention there is provided the special induced culture medium of tianshan mountain spruce somatic embryogenic callus, institute
Stating culture medium is the DCR culture medium being added with auximone 2,4-D and phytocytomine BAP；Wherein, auximone
The concentration of 2,4-D is 20 μMs, and the concentration of phytocytomine BAP is 5 μMs.
The invention have the benefit that the method for inducing and cultivating of the tianshan mountain spruce somatic embryogenic callus that the present invention provides
And special induced culture medium, it is to be induced by screening tianshan mountain spruce somatic embryo, for its somatic cell, a situation arises, point
Analysis affects the inductivity of tianshan mountain spruce somatic embryo, thus the abductive approach of the applicable tianshan mountain spruce somatic embryo set up and induction
Culture medium.The foundation of this abductive approach and the preparation of culture medium, import for entering later stage tianshan mountain spruce somatic embryo genetic transformation
The genes of interest of practicality, and carry out clone and lay the foundation, for tianshan mountain spruce preserving seed, artificial seed, Protoplast cuhnre,
Seed improvement and improvement of the ecological environment provide advantage.First contribute to understanding somatic embryo inducement mechanism from cellular level
With the occurrence frequency improving embryoid；Secondly, the pests occurrence rule of tianshan mountain spruce somatic embryogenic callus is further appreciated that, for building
Vertical tianshan mountain spruce somatic embryo regenerating system provides theoretical foundation.
Accompanying drawing explanation
Fig. 1 is the induction to tianshan mountain spruce mature zygotic embryos callus of the inducing culture of six kinds of hormone concentration combinations
Rate.
Wherein, acquired results is meansigma methods.
Detailed description of the invention
Embodiment 1:
1, test material and method:
A:6 gathers the tianshan mountain spruce cone (immature zygotic embryos of band endosperm: form zygotic embryo the most completely and refer to close the last ten-days period month
Sub-embryo was introduced into for the first stage of development) as material.It is the HgCl of 0.2% by cone mass percentage concentration2Solution disinfection 10 points
Zhong Hou, with aseptic water washing 3 times.From cone, take out immature seed with taking the photograph son, scratch kind of a shell from sidepiece, take out kernel, draw
Broken endotesta, teasing endosperm, tender embryo is inoculated in culture medium together with endosperm；Every ware inoculates 4, is repeated 20 times, and material exists
Temperature is light culture at 23 ± 2 DEG C.Within 15-25 days, tie according to the features such as callus induction color, form, structure and microexamination
Fruit distinguishes the embryo callus of white translucent.
B:7 gathers the tianshan mountain spruce cone (immature zygotic embryos without endosperm: the first of zygotic embryo to the 3rd last ten-days period month
Educate the stage) as material.It is the HgCl of 0.2% by cone mass percentage concentration2After solution disinfection 10 minutes, rush with sterilized water
Wash 3 times.From cone, take out, with taking the photograph son, the seed that is mature on the whole, from lobe seed, take out embryo, be inoculated in culture medium；Every ware
Inoculating 4, be repeated 20 times, material is light culture at temperature is 23 ± 2 DEG C.15-25 days according to callus induction color, shape
The feature such as state, structure and microscopic findings distinguish the embryo callus of white translucent.
Gather tianshan mountain spruce mature seed (mature zygotic embryos) C:9 month as material.By seed mass percentage concentration
It is the HgCl of 0.2%2After solution disinfection 5 minutes, with aseptic water washing 3 times.From seed, take out mature zygotic embryos with taking the photograph son, connect
Plant in culture medium；Every ware inoculates 4, is repeated 20 times, and the Induction Process of somatic embryo is dark at cultivation temperature is 23 ± 2 DEG C
Cultivate.Within 15-25 days, distinguish white according to the features such as callus induction color, form, structure and microscopic findings semi-transparent
Bright embryo callus.
Minimal medium: culture medium is the important substrate of plant tissue culture.Under isolated culture condition, different plants
Nutrition is had the tissue of different requirements, even same plant different parts also to differ the requirement of nutrition, only by tissue
Meeting the particular/special requirement of each of which, they could grow well.Therefore, a kind of culture medium is not had can be suitable for all classes
The plant tissue of type or organ, when setting up a new culture systems it may first have to finds a suitable culture medium culturing
Likely success.
Inducing culture:
The inducing culture of tianshan mountain spruce somatic embryogenic callus is DCR (GUPTA & DURZAN, 1985), first
Add a great number of elements (10-40 times concentrates) and the storing solution of trace element (100 times of concentrations).Then iron salt, the sugarcane of 88 mM are added
Sugar, the solution of organic principle and growth regulator etc., then add distilled water constant volume.Adjust pH value first dense by the amount of dropper draws substance
Degree is dilute NaOH or dilute HCI solution of 1mol/L, dropwise instills in the culture medium dissolved, drips while stir, until culture medium
PH be adjusted to 5.8 ± 0.02 till, finally pour into containing 3g/L Gelrite(carrageenan) be firming agent bottle in, sealing standard
Standby sterilization.Sterilising temp, at 121 DEG C, carries out 15 minutes autoclavings under the pressure of 2x107Pa.As high temperature, such as 4.8mML-
The result of glutamine may affected material, so filtration sterilization, then after autoclaving, be 60 at maximum temperature
DEG C medium.With Corning Costar company of the U.S. produce aperture be 0.22 m filter by with.After sterilizing in culture vessel
Culture medium natural cooling solidifies, and re-uses after preferably placing 1d.
The induction of somatic embryo, typically at the auxin containing higher concentration and the solid of the basic element of cell division of higher concentration
Carry out in culture medium.Its induction and the age of outer implant, physiological status, the composition of culture medium, the kind of hormone and concentration, training
The conditions of supporting etc. are relevant.The developmental stage of zygotic embryo is a key factor of culture induction.Hormone concentration combination in culture medium,
The contrast test of 6 kinds of division culture mediums of design, understands the optimal medium of tianshan mountain spruce somatic callus, and when 2,4-D makees
Add for auxin, and BAP and TDZ adds as the basic element of cell division, the inducing culture concrete scheme of 6 kinds of hormone concentration combinations
As shown in table 1.
Table 1
。
2, result and analysis:
Tianshan mountain spruce somatic embryo contains 2,4-D(that concentration is 20 μMs as auxin at DCR) and concentration be 5 μMs
The BAP(basic element of cell division) in the environment of inoculate inducing culture after 7 days, outer implant is expanded, and color change substantially, i.e. has for 10-15 days
Different types of callus produces.Wherein, portion first expands, and produces, from base portion, the thread wound healing group that white is the most yellow, transparent subsequently
Knitting, be 32.4% for embryonic callus induction percentage rate up to the immature zygotic embryos of band endosperm, the immaturity without endosperm is closed
Sub-embryo is 83% and mature zygotic embryos is 60%.Top expand produce ivory buff, structure slightly pine, quality is soft, graininess wound healing
Tissue, is 10.6% for non embryogenic callus percentage rate up to the immature zygotic embryos of band endosperm, closes without the immaturity of endosperm
Sub-embryo is 6.8% and mature zygotic embryos is 40%.This result of study is grown research for somatic embryo from now on and is laid a good foundation.Research
Show, each stage of development that tianshan mountain spruce somatic embryo occurs (immature zygotic embryos of band endosperm, immaturity without endosperm
Zygotic embryo and mature zygotic embryos) somatic embryo inducement callus be entirely possible；Tianshan mountain spruce somatic embryo
The inductivity of property callus is closely related with acquisition time.
Tianshan mountain spruce somatic embryo (immature zygotic embryos of band endosperm, without the immature zygotic embryos of endosperm with ripe close
Sub-embryo) the induction result of callus is as shown in table 2.
Table 2
。
Experimental result six weeks post processing * represents the percentage rate of pollution-free number
3, the inducing culture of the six kinds of hormone concentration combinations induction to tianshan mountain spruce mature zygotic embryos callus:
As it is shown in figure 1, tianshan mountain spruce mature zygotic embryos DCR contain concentration for without hormone, 10 μM 2,4-D and 5 μMs
BAP；20 μMs of 2,4-D and 5 μMs of BAP, 20 μMs of 2,4-D and 10 μMs of BAP, 40 μMs of 2,4-D and 10 μMs of BAP, 20 μMs of 2,4-D
All cultivated with in the culture medium such as 10 μMs of TDZ.Inductivity minimum 18% on 10 μMs of 2,4-D and 5 μMs of BAP；At 20 μMs
2,4-D and 10 μMs of TDZ；Inductivity on 20 μMs of 2,4-D and 5 μMs of BAP culture medium is respectively 43% and 53%.
Finally it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention,
Although being described in detail the present invention with reference to previous embodiment, for a person skilled in the art, it still may be used
So that the technical scheme described in foregoing embodiments to be modified, or wherein portion of techniques feature is carried out equivalent.
All within the spirit and principles in the present invention, any modification, equivalent substitution and improvement etc. made, should be included in the present invention's
Within protection domain.
Claims (1)
1. the method for inducing and cultivating of tianshan mountain spruce somatic embryogenic callus, it is characterised in that comprise the following steps:
1) tianshan mountain spruce cone, the i.e. immature zygotic embryos of band endosperm are gathered in late June, as material, by cone quality
Percentage concentration is the HgCl of 0.2%2After solution disinfection 10 minutes, with aseptic water washing 3 times；Take out into from cone with tweezers
Ripe seed, scratches kind of a shell from sidepiece, takes out kernel, scratches endotesta, teasing endosperm, tender embryo is inoculated in endosperm Tianshan Mountains together
On the inducing culture of PiceameyeriRehd. Et Wils. somatic embryogenic callus；Every ware inoculation of medium 4, is repeated 20 times, is 23 ± 2 in temperature
Light culture 15-25 days at DEG C, obtain the embryo callus of white translucent；
2) tianshan mountain spruce cone is gathered in late July, i.e. without the immature zygotic embryos of endosperm, as material, by cone matter
Amount percentage concentration is the HgCl of 0.2%2After solution disinfection 10 minutes, with aseptic water washing 3 times；From cone, base is taken out with tweezers
This mature seed, from longitudinally split seed, takes out embryo, is inoculated in the inducing culture of tianshan mountain spruce somatic embryogenic callus
On；Every ware inoculation of medium 4, is repeated 20 times, and at temperature is 23 ± 2 DEG C, light culture 15-25 days, obtains white translucent
Embryo callus；
3) gather tianshan mountain spruce mature seed, i.e. mature zygotic embryos in JIUYUE, as material, by seed mass percentage concentration be
The HgCl of 0.2%2After solution disinfection 5 minutes, with aseptic water washing 3 times；From seed, take out mature zygotic embryos with tweezers, connect
Plant on the inducing culture of tianshan mountain spruce somatic embryogenic callus；Every ware inoculation of medium 4, is repeated 20 times, in training
Foster temperature is light culture 15-25 days at 23 ± 2 DEG C, obtains the embryo callus of white translucent；
Described step 1)-3) in the inducing culture of used tianshan mountain spruce somatic embryogenic callus for being added with plant growing
Element 2, the DCR culture medium of 4-D and phytocytomine BAP, wherein, auximone 2, the concentration of 4-D is 20 μMs, and plant is thin
The concentration of born of the same parents mitogen BAP is 5 μMs；
The preparation process of described DCR culture medium is: first add the 10-40 times of a great number of elements concentrated and 100 times of trace concentrated
The storing solution of element；Then add iron salt, the sucrose of 88 mM, organic principle and the solution of growth regulator, finally add distilled water
Constant volume；Adjust pH value first with dilute NaOH or dilute HCl solution that the amount concentration of dropper draws substance is 1mol/L, dropwise instill and to dissolve
In culture medium, drip while stir, until till the pH of culture medium is adjusted to 5.8 ± 0.02, finally pouring into containing 3g/L OK a karaoke club
Glue is in the bottle of firming agent, and sealing prepares sterilization；Sterilising temp 121 DEG C, 2 × 10715 minutes high pressure are carried out under the pressure of Pa
Sterilizing；Use filtration sterilization after autoclaving again, the culture medium that maximum temperature after autoclaving is 60 DEG C is filtered standby, filters
Aperture is 0.22 μm；After sterilizing, culture medium natural cooling solidification in culture vessel, re-uses after placing 1d.
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| CN105850731B (en) * | 2016-04-07 | 2018-01-09 | 甘肃省治沙研究所 | A kind of culture medium and method of thickleaf spruce mature zygotic embryos callus induction |
| CN108200864A (en) * | 2018-01-24 | 2018-06-26 | 广西心壶城人家企业管理有限公司 | A kind of dragon spruce seed seedling-raising method |
| CN112470933A (en) * | 2020-12-14 | 2021-03-12 | 江苏省中国科学院植物研究所 | Method for inducing embryogenic callus and proliferating by using immature embryo of sequoia intermedia |
| CN113502257B (en) * | 2021-08-19 | 2023-08-01 | 内蒙古蒙荣园林绿化工程有限责任公司 | Drying marking method for spruce somatic embryos before germination |
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| WO1995014373A1 (en) * | 1993-11-23 | 1995-06-01 | Weyerhaeuser Company | Method for reproducing conifers by somatic embryogenesis using a maltose enriched maintenance medium |
| CN102577964A (en) * | 2012-03-01 | 2012-07-18 | 常熟市方园纺织器材厂 | Quick in-vitro propagation method of picea koraiensis |
| CN102792888B (en) * | 2012-07-31 | 2014-05-14 | 中国林业科学研究院林业研究所 | Method for somatic embryogenesis and plant regeneration of Lijiang spruce |
| CN102771393B (en) * | 2012-08-02 | 2013-10-16 | 中国林业科学研究院林业研究所 | Method for picea balfouriana somatic embryo generation and plant regeneration |
| CN103461119B (en) * | 2013-08-20 | 2015-10-28 | 中国林业科学研究院林业研究所 | PIECA ASPERATA somatic embryo occurs and plant regeneration method |
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