CN104304034A - 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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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
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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 medium thereof.
Background technology
Tianshan mountain spruce
(Picea schrenkiana Fisch. at Mey.)belong to Pinaceae Picea; for the Central Asia and Central Asia mountain region endemic species; only Tianshan Mountains is distributed in China; it is one of most important forest species in mountain area, Xinjiang; area about 52.84 ten thousand hm2; account for the natural forest land gross area 44.9% of full boundary, be distribute in Xinjiang Mountainous forest the widest, accumulation is maximum, business activities are maximum, purposes the most extensively, main timber tree species that material is excellent, irreplaceable effect is played to the formation of the water conservation in Tianshan Mountains, soil-water protection, the forest zone ecosystem and maintenance.
Because the mankind's activity of tianshan mountain spruce area is frequent and the factor such as irrational management and contradiction in forestry and animal husbandry causes Picea schrenkiana var. tianshanica forest ecosystem structure to be badly damaged, result in the decline of Picea schrenkiana var. tianshanica forest.The appearance of the problems such as the bad or natural regeneration of natural regeneration is difficult has become a great problem of long-term puzzled forest workers.Dragon spruce species grow is comparatively slow, and seed maturity rate is very low, and breeding and breed breeding have very large difficulty.From seed germination to growing up to the seedling that can transplant, need through 4-5 again, so carry out breeding by natural propagation and select superior genotypes efficiency too low.The method adopting grafting, cuttage etc. to nourish and generate both had consumed a large amount of time and labour, again by the restriction in season.
Current tianshan mountain spruce produces offspring mainly through sexual propagation, vegetative propagation is subject to the attention of various countries' forestry decision-making section and the special concern of Forest Tree Breeding Work person and forest management person always, especially modern biotechnology is flourish, as Plant Tissue Breeding and the Development of Somatic Embryogenesis, have that reproductive efficiency is high, the cycle is short, by natural conditions restriction, stable, seed selection improved seeds, to features such as afforested project are significant.Realize plant regeneration by somatic embryo development pathway and not only can solve vegetative problem, for the embody rule of biotechnology in Picea and seeds thereof quality-improving also tool be of great significance.In tianshan mountain spruce Somatic Embryogenesis, via method for tissue culture evoked callus, and obtaining somatic embryo by callus, is one of the means of extensive use in the research of current artificial seed and biotechnology.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, provides the method for inducing and cultivating of tianshan mountain spruce somatic embryogenic callus and special induced medium thereof.
To achieve these goals, technical scheme provided by the invention is: the method for inducing and cultivating of tianshan mountain spruce somatic embryogenic callus, comprises the following steps:
1) gather tianshan mountain spruce cone in late June, cone mass percentage concentration, as material, is the HgCl of 0.2% by the immature zygotic embryos namely with endosperm
2solution disinfection is after 10 minutes, with aseptic water washing 3 times; From cone, taking out immature seed with taking the photograph son, scratching kind of a shell from sidepiece, taking out kind of a benevolence, scratch secundine, choose out endosperm, tender embryo is inoculated in together with endosperm on the inducing culture of tianshan mountain spruce somatic embryogenic callus; Every ware inoculation of medium 4, repeat 20 times, light culture 15-25 days at temperature is 23 ± 2 DEG C, obtains the embryo callus of white translucent;
2) gather tianshan mountain spruce cone in late July, cone mass percentage concentration, as material, is the HgCl of 0.2% by the immature zygotic embryos namely not with endosperm
2solution disinfection is after 10 minutes, with aseptic water washing 3 times; From cone, taking out the seed that is mature on the whole with taking the photograph son, from the seed that longitudinally splits, taking out embryo, being inoculated on the inducing culture of tianshan mountain spruce somatic embryogenic callus; Every ware inoculation of medium 4, repeat 20 times, light culture 15-25 days at temperature is 23 ± 2 DEG C, obtains the embryo callus of white translucent;
3) gathering tianshan mountain spruce mature seed September, i.e. mature zygotic embryos, as material, is the HgCl of 0.2% by seed mass percentage concentration
2solution disinfection is after 5 minutes, with aseptic water washing 3 times; From seed, taking out mature zygotic embryos with taking the photograph son, being inoculated on the inducing culture of tianshan mountain spruce somatic embryogenic callus; Every ware inoculation of medium 4, repeat 20 times, light culture 15-25 days at cultivation temperature is 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 for being added with the DCR medium of auximone 2,4-D and phytocytomine BAP.
Further, the method for inducing and cultivating of above-mentioned tianshan mountain spruce somatic embryogenic callus, in the inducing culture of described tianshan mountain spruce somatic embryogenic callus, the concentration of auximone 2,4-D is 20 μMs, and the concentration of phytocytomine BAP is 5 μMs.
Second object of the present invention there is provided the special induced medium of tianshan mountain spruce somatic embryogenic callus, and described medium is for being added with the DCR medium of auximone 2,4-D and phytocytomine BAP; Wherein, the concentration of auximone 2,4-D is 20 μMs, and the concentration of phytocytomine BAP is 5 μMs.
Beneficial effect of the present invention is: the method for inducing and cultivating of tianshan mountain spruce somatic embryogenic callus provided by the invention and special induced medium thereof, induced by screening tianshan mountain spruce somatic embryo, for its somatic cell, a situation arises, the inductivity of analyzing influence tianshan mountain spruce somatic embryo, thus the abductive approach of the applicable tianshan mountain spruce somatic embryo set up and inducing culture.The foundation of this abductive approach and the preparation of medium, the genes of interest of practicality is imported for entering later stage tianshan mountain spruce somatic embryo genetic transformation, 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 and improving the occurrence frequency of embryoid; Secondly, understanding the pests occurrence rule of tianshan mountain spruce somatic embryogenic callus further, providing theoretical foundation for setting up tianshan mountain spruce somatic embryo regenerating system.
Accompanying drawing explanation
Fig. 1 is that the inducing culture of six kinds of hormone concentration combinations is to the inductivity of tianshan mountain spruce mature zygotic embryos callus.
Wherein, acquired results is mean value.
Embodiment
embodiment 1:
1, test material and method:
A:6 gathers tianshan mountain spruce cone (immature zygotic embryos of band endosperm: be not fully formed zygotic embryo and refer to that zygotic embryo did not enter for the first developmental stage) as material the last ten-days period month.Be the HgCl of 0.2% by cone mass percentage concentration
2solution disinfection is after 10 minutes, with aseptic water washing 3 times.From cone, taking out immature seed with taking the photograph son, scratching kind of a shell from sidepiece, taking out kind of a benevolence, scratch secundine, choose out endosperm, tender embryo is inoculated on medium together with endosperm; Every ware inoculates 4, and repeat 20 times, material is light culture at temperature is 23 ± 2 DEG C.Within 15-25 days, distinguish the embryo callus of white translucent according to the features such as evoked callus color, form, structure and microscopic findings.
B:7 gathers tianshan mountain spruce cone (immature zygotic embryos not with endosperm: the first to the 3rd developmental stage of zygotic embryo) as material the last ten-days period month.Be the HgCl of 0.2% by cone mass percentage concentration
2solution disinfection is after 10 minutes, with aseptic water washing 3 times.From cone, taking out the seed that is mature on the whole with taking the photograph son, from lobe seed, taking out embryo, being inoculated on medium; Every ware inoculates 4, and repeat 20 times, material is light culture at temperature is 23 ± 2 DEG C.Within 15-25 days, distinguish the embryo callus of white translucent according to the features such as evoked callus color, form, structure and microscopic findings.
Gather tianshan mountain spruce mature seed (mature zygotic embryos) C:9 month as material.Be the HgCl of 0.2% by seed mass percentage concentration
2solution disinfection is after 5 minutes, with aseptic water washing 3 times.From seed, taking out mature zygotic embryos with taking the photograph son, being inoculated on medium; Every ware inoculates 4, repeats 20 times, Induction Process light culture at cultivation temperature is 23 ± 2 DEG C of somatic embryo.Within 15-25 days, distinguish the embryo callus of white translucent according to the features such as evoked callus color, form, structure and microscopic findings.
Minimal medium: medium is the important matrix of Plant Tissue Breeding.Under isolated culture condition, the tissue of different plant has different requirements to nutrition, and even the tissue of same plant different parts is not identical to the requirement of nutrition yet, and only meet their respective particular/special requirements, they could grow well.Therefore, not having a kind of medium can be applicable to plant tissue or the organ of all types, when setting up a new culture systems, first must find the medium culture just likely success that is suitable.
Inducing culture:
The inducing culture of tianshan mountain spruce somatic embryogenic callus is DCR (GUPTA & DURZAN, 1985), first adds the storing solution of macroelement (10-40 doubly concentrates) and trace element (100 times concentrate).Then molysite is added, the sucrose of 88 mM, the solution of organic principle and growth regulator etc., then adding distil water constant volume.Adjust pH is first rare NaOH of 1mol/L or rare HCI solution by the amount concentration of dropper draws substance, dropwise instill in the medium dissolved, drip while stir, until the pH of medium adjusts to till 5.8 ± 0.02, finally pour into containing 3g/L Gelrite(carragheen) in the bottle of curing agent, sealing prepares sterilization.Sterilising temp, at 121 DEG C, carries out 15 minutes autoclavings under the pressure of 2x107Pa.As high temperature, the possibility of result as 4.8mML-glutamine can affected material, so filtration sterilization, then after autoclaving, is the medium of 60 DEG C in maximum temperature.With Corning Costar company of the U.S. produce aperture be 0.22 μm filter by with.After sterilizing, medium nature cooled and solidified in culture vessel, re-uses after preferably placing 1d.
The induction of somatic embryo, generally carries out on the solid culture medium of the basic element of cell division of the growth hormone containing higher concentration and higher concentration.Its induction and the composition of the age of explant, physiological status, medium, the kind of hormone are relevant with concentration, condition of culture etc.The developmental stage of zygotic embryo is a key factor of culture induction.Hormone concentration combination in medium, the comparative trial of design 6 kinds of differential mediums, understand the optimal medium of tianshan mountain spruce somatic callus, when 2,4-D adds as growth hormone, and BAP and TDZ adds as the basic element of cell division, the inducing culture concrete scheme of 6 kinds of hormone concentration combinations is as shown in table 1.
Table 1
。
2, results and analysis:
Tianshan mountain spruce somatic embryo contain that concentration is 20 μMs at DCR 2,4-D(is as growth hormone) and concentration be the BAP(basic element of cell division of 5 μMs) environment under inoculate Fiber differentiation after 7 days, explant expands, and color change obviously, namely has dissimilar callus to produce for 10-15 days.Wherein, portion first expands, and produces slightly yellow, the transparent thread callus of white subsequently from base portion, and the immature zygotic embryos that can reach band endosperm for embryonic callus induction percentage is 32.4%, the immature zygotic embryos not with endosperm be 83% and mature zygotic embryos be 60%.The ivory buff, structure slightly pine, soft, the graininess callus of quality that produce are expanded in top, the immature zygotic embryos that can reach band endosperm for non embryogenic callus percentage is 10.6%, immature zygotic embryos not with endosperm be 6.8% and mature zygotic embryos be 40%.This result of study is that the research of somatic embryo growth is from now on laid a good foundation.Research shows, the somatic embryo inducement callus of each developmental stage (band the immature zygotic embryos of endosperm, the immature zygotic embryos not with endosperm and mature zygotic embryos) that tianshan mountain spruce somatic embryo occurs be completely possible; Inductivity and the acquisition time of tianshan mountain spruce somatic cell embryo callus are closely related.
The induction result of tianshan mountain spruce somatic embryo (the band immature zygotic embryos of endosperm, the immature zygotic embryos not with endosperm and mature zygotic embryos) callus is as shown in table 2.
Table 2
。
Experimental result six weeks post processing * represents the percentage of pollution-free number
3, the inducing culture of six kinds of hormone concentration combinations is to the induction of tianshan mountain spruce mature zygotic embryos callus:
As shown in Figure 1, tianshan mountain spruce mature zygotic embryos DCR contain concentration for without hormone, 10 μM 2,4-D and 5 μM BAP; 20 μMs of 2,4-D and 5 μMs of BAP, 20 μMs of medium such as 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 and 10 μMs of TDZ are all cultivated.On 10 μMs of 2,4-D and 5 μMs of BAP, inductivity is minimum is 18%; At 20 μMs of 2,4-D and 10 μMs of TDZ; Inductivity on 20 μMs of 2,4-D and 5 μMs of BAP medium is respectively 43% and 53%.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. the method for inducing and cultivating of tianshan mountain spruce somatic embryogenic callus, is characterized in that, comprises the following steps:
1) gather tianshan mountain spruce cone in late June, cone mass percentage concentration, as material, is the HgCl of 0.2% by the immature zygotic embryos namely with endosperm
2solution disinfection is after 10 minutes, with aseptic water washing 3 times; From cone, taking out immature seed with taking the photograph son, scratching kind of a shell from sidepiece, taking out kind of a benevolence, scratch secundine, choose out endosperm, tender embryo is inoculated in together with endosperm on the inducing culture of tianshan mountain spruce somatic embryogenic callus; Every ware inoculation of medium 4, repeat 20 times, light culture 15-25 days at temperature is 23 ± 2 DEG C, obtains the embryo callus of white translucent;
2) gather tianshan mountain spruce cone in late July, cone mass percentage concentration, as material, is the HgCl of 0.2% by the immature zygotic embryos namely not with endosperm
2solution disinfection is after 10 minutes, with aseptic water washing 3 times; From cone, taking out the seed that is mature on the whole with taking the photograph son, from the seed that longitudinally splits, taking out embryo, being inoculated on the inducing culture of tianshan mountain spruce somatic embryogenic callus; Every ware inoculation of medium 4, repeat 20 times, light culture 15-25 days at temperature is 23 ± 2 DEG C, obtains the embryo callus of white translucent;
3) gathering tianshan mountain spruce mature seed September, i.e. mature zygotic embryos, as material, is the HgCl of 0.2% by seed mass percentage concentration
2solution disinfection is after 5 minutes, with aseptic water washing 3 times; From seed, taking out mature zygotic embryos with taking the photograph son, being inoculated on the inducing culture of tianshan mountain spruce somatic embryogenic callus; Every ware inoculation of medium 4, repeat 20 times, light culture 15-25 days at cultivation temperature is 23 ± 2 DEG C, obtains the embryo callus of white translucent.
2. the method for inducing and cultivating of tianshan mountain spruce somatic embryogenic callus according to claim 1, it is characterized in that, described step 1)-3) in the inducing culture of tianshan mountain spruce somatic embryogenic callus used for being added with the DCR medium of auximone 2,4-D and phytocytomine BAP.
3. the method for inducing and cultivating of tianshan mountain spruce somatic embryogenic callus according to claim 1 and 2, it is characterized in that, in the inducing culture of described tianshan mountain spruce somatic embryogenic callus, auximone 2, the concentration of 4-D is 20 μMs, and the concentration of phytocytomine BAP is 5 μMs.
4. the special induced medium of tianshan mountain spruce somatic embryogenic callus, is characterized in that, described medium is for being added with the DCR medium of auximone 2,4-D and phytocytomine BAP; Wherein, the concentration of auximone 2,4-D is 20 μMs, and the concentration of phytocytomine BAP is 5 μMs.
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| CN105850731A (en) * | 2016-04-07 | 2016-08-17 | 甘肃省治沙研究所 | Culture medium and method for inducing picea crassifolia mature zygotic embryos callus |
| 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 |
| CN113207685A (en) * | 2020-12-14 | 2021-08-06 | 江苏省中国科学院植物研究所 | Somatic embryogenesis method of taxus chinensis |
| CN113502257A (en) * | 2021-08-19 | 2021-10-15 | 内蒙古蒙荣园林绿化工程有限责任公司 | Drying marking method for spruce body cell embryos before germination |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105850731A (en) * | 2016-04-07 | 2016-08-17 | 甘肃省治沙研究所 | Culture medium and method for inducing picea crassifolia mature zygotic embryos callus |
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| CN113502257A (en) * | 2021-08-19 | 2021-10-15 | 内蒙古蒙荣园林绿化工程有限责任公司 | Drying marking method for spruce body cell embryos before germination |
| 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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