CN109744148A - A method for tree bud regeneration based on transdifferentiation of apical meristem - Google Patents
A method for tree bud regeneration based on transdifferentiation of apical meristem Download PDFInfo
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Abstract
本发明公开了一种基于顶端分生组织转分化的林木芽再生方法，包括以下步骤：(1)取林木组培苗的根为外植体，将外植体在含有生长素NAA的B5培养基中培养20‑26h；(2)将步骤(1)培养后的外植体转入含有细胞分裂素2‑IP的B5培养基中培养10‑14天；(3)将步骤(2)培养后的外植体转入含有细胞分裂素6‑BA的MS培养基中培养14‑18天，获得再生芽。本发明的林木芽再生方法不经过愈伤组织阶段直接形成茎端分生组织，从而产生再生芽，因此适用于建立研究植物细胞命运决定、谱系转变等科学问题的实验体系，适用于难以形成具有再生能力愈伤组织的物种建立芽再生体系，进而进行离体快繁、遗传转化或其他生物技术操作。
The invention discloses a tree bud regeneration method based on apical meristem transdifferentiation, comprising the following steps: (1) taking the roots of tree tissue culture seedlings as explants, and culturing the explants in B5 containing auxin NAA Cultivated in the base for 20-26h; (2) transferring the explants cultivated in step (1) into the B5 medium containing cytokinin 2-IP for 10-14 days; (3) culturing step (2) The explants were then transferred into MS medium containing cytokinin 6-BA and cultured for 14-18 days to obtain regenerated shoots. The forest tree bud regeneration method of the present invention directly forms the stem end meristem without going through the callus stage, thereby producing regenerated buds, so it is suitable for establishing an experimental system for studying plant cell fate determination, lineage transition and other scientific issues, and is suitable for the formation of Species with regenerative ability callus establish a shoot regeneration system, and then perform in vitro rapid propagation, genetic transformation or other biotechnological operations.
Description
Technical field
The present invention relates to plant regeneration technical fields, and in particular to a kind of forest bud based on apical meristem transdifferentiation
Regeneration method.
Background technique
The shoot apical meristem of angiosperm is located at the top of stem, in the dome structure of protrusion, cell division and differentiation
Activity forms entire aerial part.Shoot apical meristem can be divided into three differences according to the structure feature of its internal cell
Region, i.e. central area, peripheral region and rib areas (Meyerowitz, Cell, 1997,88,299-308).Center
Domain is in the center on dome top, includes the multipotential stem cell that a division is slower.The filial generation that stem cell division generates is thin
On the one hand born of the same parents enter the peripheral region that surrounding is adjoined, participate in the differentiation of the lateral organ including flower, leaf；Under other direction into
Enter rib areas, is divided into component part (Soyars et al., the Curr Opin Plant Biol 2016,29,163- of stem
168).There is the cell for being referred to as organization center below stem cell, has played the effect that regulation stem cell maintains.Stem end is mitogenetic
Tissue accurate molecular genetic regulation by tight, the space-time specific expression of key gene, cell division and differentiation it is dynamic
State balance maintain jointly its specific structure and function (Aichinger et al., Annu Rev Plant Biol, 2012,
63,615-636)。
Under isolated culture condition, mature histoorgan can re-form shoot apical meristem, and then regeneration bud is simultaneously
Generate new plant individual (Sang et al., New Phytol, 2018,218,1334-1339).In this course, it grows
Element and the basic element of cell division have played vital regulating and controlling effect.It is nearest the study found that during shoot regeneration in explant
Pericycle and class pericyclic cell are induced by exogenous auxin, form the callus group with root apical meristem cellularity
It knits.The work of inventor early period shows after being transferred to differentiation cultivation stage, auxin and cell division in callus specific region
Plain response signal forms specific distribution mode, and basic element of cell division response signal is located at central area, and auxin is in around it
Annular distribution (Cheng et al., Plant Physiol, 2013,161,240-251).It is in the cell division of central area
Element directly activates stem end dry thin by the B class Arabidopsis Response Regulator albumen in its signal transduction pathway
The expression of born of the same parents' identity gene WUSCHEL, while inhibiting auxin in the accumulation in the region, to push callus cell
Destiny conversion occurs, forms shoot apical meristem (Meng et al., Plant Cell, 2017,29,1357-1372).
Shoot regeneration is the prerequisite of the fast numerous and genetic engineering operation of Vitro Plant, is crop woods fruit flowers standardized production
Important channel, have a very important significance (Sang et al., Trends for developing reading intelligent agriculture and Biology Breeding
Plant Sci,2018 23,660-666).It is mostly cross-pollination using poplar as the forest of representative, genetic background height heterozygosis, no
Conducive to holding parent's merit.Therefore, the breeding based on the tissue cultures of neomorph for forest and high yield and quality cultivation
Establishing for system is particularly important.But most forests with Important Economic value to form callus or callus due to being difficult to
Tissue cannot further break up, and can not establish shoot regeneration system, seriously constrain application development of the biotechnology in agriculture and forestry.
Summary of the invention
The case where for the above-mentioned prior art, the object of the present invention is to provide a kind of based on apical meristem transdifferentiation
Forest shoot regeneration method.Forest shoot regeneration method of the invention is by the direct transdifferentiation shape of root apical meristem in lateral-root primordia
At shoot apical meristem, without callus induction and cultivation stage, it is suitable for establishing research plant cell destiny and determines, composes
The experimental system of the problem in science such as system's transformation；Shoot regeneration is established suitable for being difficult to form the species with power of regeneration callus
System, and then carry out rapid propagation in vitro, genetic transformation or other biological technical operation.
To achieve the above object, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of forest shoot regeneration method based on apical meristem transdifferentiation, including
Following steps:
(1) root for taking Tissue Culture of Trees seedling is explant, and explant is cultivated in the B5 medium containing auxin NAA
20-26h；
(2) explant after step (1) culture is transferred in the B5 medium containing basic element of cell division 2-IP and cultivates 10-14
It；
(3) explant after step (2) culture is transferred in the MS culture medium containing basic element of cell division 6-BA and cultivates 14-18
It, obtains regeneration bud.
Preferably, in step (1), the root of Tissue Culture of Trees seedling is taken, the root segment that intercepted length is 2-4cm from the proximal end of root is made
For explant；It is furthermore preferred that intercepted length is the root segment of 2cm as explant from the proximal end of root.
Preferably, in step (1), the Tissue Culture of Trees seedling is prepared by the following method:
1) forest branch and blade are chosen, the ethanol solution for being successively 75% with volume fraction, aoxidizes mercury solution at sterile water
With sterile water immersion treatment；
2) water for blotting step 1) treated forest branch and blade surface, moves into differential medium, is placed in 25 DEG C
It is cultivated in illumination box；
3) it when culture to transformation bud length is 2-3cm, cuts regeneration bud and moves into root media, be in periodicity of illumination
Illumination 16h/ dark 8h, culture of rootage is carried out under the conditions of 25 DEG C, obtain tissue-cultured seedling.
It is furthermore preferred that in step 2), the differential medium be containing 0.002mg/L TDZ, 0.5mg/L 6-BA and
The MS culture medium of 0.1mg/L NAA.
It is furthermore preferred that the root media is the MS training containing 0.1mg/L IBA, 0.01mg/L NAA in step 3)
Support base.
Preferably, in step (1), in the B5 medium, the concentration of auxin NAA is 2-14 μM；It is furthermore preferred that growth
The concentration of plain NAA is 10 μM.
Preferably, in step (2), in the B5 medium, the concentration of basic element of cell division 2-IP is 2.8-10.8 μM；It is more excellent
Choosing, the concentration of basic element of cell division 2-IP is 8.8 μM.
Preferably, in step (3), in the MS culture medium, the concentration of basic element of cell division 6-BA is 0.1-1.0mg/L；More
Preferably, the concentration of basic element of cell division 6-BA is 0.5mg/L.
The second aspect of the present invention provides above-mentioned forest shoot regeneration method in following a)-d) at least one of in application；
A) it is difficult to form the in vitro breeding of the forest with power of regeneration callus；
B) foundation of high yield and high quality Forest-tree culture system；
C) preserving seed of Forest-tree strain；
D) it is determined for studying plant cell destiny, the foundation of the experimental system of pedigree transformation.
The third aspect of the present invention provides a kind of preparation method of forest regeneration plant, comprising the following steps:
Bud is obtained using above-mentioned forest shoot regeneration method, then bud is subjected to strong seedling culture, culture of rootage, hardening and transplanting,
Obtain forest regeneration plant.
Beneficial effects of the present invention:
Forest shoot regeneration method of the invention is to promote using the root of Tissue Culture of Trees seedling as explant first with exogenous auxin
The formation of root restriction recycles Exogenous cytokinin induction root restriction to form shoot apical meristem by transdifferentiation, to produce
Raw regeneration bud.Shoot regeneration method provided by the invention needs not move through callus induction and cultivation stage, suitable for being difficult to shape
Shoot regeneration system is established at the species with power of regeneration callus, and then carries out rapid propagation in vitro, genetic transformation or other lifes
Object technical operation；Transdifferentiation of this method based on two kinds of apical meristems is converted into mitogenetic group of stem end by root apical meristem
It knits, therefore is suitable for establishing the experimental system for the problem in science such as research plant cell destiny determines, pedigree changes.
Detailed description of the invention
Fig. 1: shoot regeneration incubation of the poplar based on transdifferentiation；In figure, D represents the number of days after culture starts；84K is white
Poplar kind, European-American Poplar 107 are black poplar kind.
Fig. 2: lateral-root primordia is formed and apical meristem transdifferentiation procedure chart；In figure, Root explant is indicated outside root
Implant, LR1-6 indicates root restriction growth course, when Converting indicates that root apical meristem changes to shoot apical meristem
Phase, SAM primordium indicate shoot apical meristem former base, and SAM indicates shoot apical meristem.
Fig. 3: the butt of different location compares figure as the phenotype that explant carries out shoot regeneration culture；In figure, D represents culture
Number of days after beginning, Proximal indicate proximal end, and Distal indicates distal end.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology part is introduced, existing conventional plant shoot regeneration method is needed by explant callus induction
The formation of tissue, then the regeneration by induced bud in callus.But most forests with Important Economic value are due to being difficult to
Forming callus or callus cannot further break up, and can not establish shoot regeneration system.Also the quasi- south of model plant is had been reported that
Mustard directly can form regeneration bud by root restriction transdifferentiation without the process of callus induction.But perennial forest and
There are numerous differences on morphosis and growth and development Regulation Mechanism for annual herb plant arabidopsis.Forest is raw due to plant
Long period is long, and secondary metabolites secretion is more, and during forest shoot regeneration, direct inductive formation regeneration bud is very tired
It is difficult.Forest shoot regeneration method based on transdifferentiation belongs to technical difficult point and blank spot there is not yet report.
The case where for the above-mentioned prior art, the present invention combine long-term research and exploration, provide a kind of based on top
The forest shoot regeneration method of separate living tissue transdifferentiation.This method is containing only life first using the root of Tissue Culture of Trees seedling as explant
Of short duration culture in the culture medium of long element, induces the generation of lateral-root primordia；Then the training of the basic element of cell division is successively contained only with two kinds
Feeding base is cultivated, and is pushed root apical meristem in lateral-root primordia that transdifferentiation occurs, is directly formed without callus phase
Shoot apical meristem, to generate regeneration bud.
In one embodiment of the present invention, the forest shoot regeneration method provided is specific as follows:
(1) root for taking poplar tissue-cultured seedling is explant, is containing auxin NAA (1-naphthylacetic acid, naphthalene
Acetic acid) B5 medium in cultivate 24 hours, promote the formation of lateral-root primordia；
(2) explant after step (1) culture is transferred to containing basic element of cell division 2-IP (N6-(2-isopentenyl)-
Adenosine, isopentenyl gland purine) B5 medium in cultivate 12 days；
(3) by step (2) culture after explant be transferred to containing basic element of cell division 6-BA (6-Benzylaminopurine,
6-benzyl aminopurine) MS culture medium in cultivate 16 days, obtain regeneration bud (Fig. 1).
Using Differential interference contrast microscope it has been observed that foring lateral-root primordia (Fig. 2, A-E) in explant, and gradually pass through
It has gone through and has expanded (Fig. 2, F-G) and transformation (Fig. 2, H) process, shoot apical meristem (Fig. 2, I-J) is formd by transdifferentiation.
In the regeneration method of above-mentioned forest bud, selection, the type selection and processing concentration meeting of plant hormone of explant are straight
Connect the regeneration effect of forest bud.Wherein:
The selection of explant is one of first step of in vitro culture, and alternative explant has very much, xylophyta
Root, stem, leaf can be elected to be explant.Different explants is selected, power of regeneration can have significant difference；Even if choosing
The same organs for selecting xylophyta also will affect the power of regeneration of explant if the organ sites of selection are different as explant.
In the Different Organs such as root, stem, leaf, the root of the preferred Tissue Culture of Trees seedling of the present invention is as explant, compared to stem and leaf, regeneration
Ability is stronger.Further, the present invention also by the root of poplar tissue-cultured seedling from distal end to proximal end direction according to 2 centimetres or so of length
Degree is divided into A, B, C, D4 sections, is cultivated respectively as explant and using the above method.As a result, it has been found that root segment D (i.e. near
Nearly root proximal end) when being used as explant, shoot regeneration effect is optimal (Fig. 3).
The inductive effect of different plant hormones is different, such as auxin can induce softening of the cell wall and cell is promoted to grow,
The basic element of cell division then can promote cell division by the cell cycle regulation factor.Plant hormone has the regulation of cell
" bell " effect, i.e. cell have an optimum concentration range to the response of hormone, and hormone concentration is too low or excessively high induction can made
With being suppressed.There are close ties, cell can receive variety classes and intensity in plant between hormon signal path
Hormone signal, and integrated using complicated Signaling transduction networks, be eventually exhibited as specific cell effect, is i.e. cell
Atomization.The present invention finally realizes by the optimum choice to plant hormone type, concentration and processing sequence and is difficult to shape
At the Direct Regeneration of the forest bud of callus.
The cultivation cycle that forest regeneration bud can significantly be shortened using method of the invention, can be obtained forest in 30 days
Regeneration bud, the regeneration rate of forest regeneration bud is up to 100%.On the basis of method of the invention, change the type of explant, Huo Zhegai
The type and concentration, the processing sequence for adjusting exogenous plant hormones for becoming exogenous plant hormones, all can reduce forest regeneration bud again
Raw rate, and extend the cultivation cycle of forest regeneration bud.
Shoot regeneration method provided by the invention forms stem end point by the direct transdifferentiation of root apical meristem in lateral-root primordia
Raw tissue needs not move through callus induction and cultivation stage, therefore is suitable for establishing the decision of research plant cell destiny, spectrum
The experimental system of the problem in science such as system's transformation establishes shoot regeneration suitable for being difficult to form the species with power of regeneration callus
System, and then carry out rapid propagation in vitro, genetic transformation or other biological technical operation.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Test material used in the embodiment of the present invention is the test material of this field routine, can pass through commercial channel
It is commercially available.The experimental method that detailed conditions are not specified is said according to conventional methods or according to operation proposed by supplier
What bright book carried out.
Embodiment 1: the acquisition of explant
(1) the healthy poplar-branch and blade for choosing grown in field, are placed in 75% ethanol solution in superclean bench
In, it impregnates 2-3 minutes, during which continues gentle agitation；
(2) again with alcohol solution dipping 2-3 minutes of 75%, during which continue gentle agitation；
(3) branch and blade material are put into sterile water and are impregnated 1-2 minutes, during which continue gentle agitation；
(4) branch and blade material are transferred in 1% oxidation mercury solution and are impregnated 2-5 minutes, during which continue gentle agitation；
(5) branch and blade material are successively put into two bottles of new sterile waters, every bottle 1-2 minutes,
(6) step (4) and step (5) are repeated；
(7) with the water on sterilized filter paper blotting material surface, differential medium (MS+0.002mg/L Thidiazuron is moved into
(TDZ)+0.5mg/L 6-BA+0.1mg/L NAA), it is placed in 25 DEG C of illumination box culture cultures (periodicity of illumination 16h illumination/8h
It is dark)；
After (8) 2 months, when transformation bud length reaches 2cm or so, cuts and move into root media (MS+0.1mg/LIBA+
0.01mg/L NAA) in culture of rootage, periodicity of illumination 16h illumination/8h is dark, cultivates under the conditions of 25 DEG C；Obtain tissue-cultured seedling；
(9) tissue-cultured seedling is taken out in superclean bench, cleans the culture medium of root attachment with sterile water and uses aseptic filter paper
Surface moisture is sucked, uses root segment that blade intercepted length from the proximal end of root is 2-4cm as explant.
Embodiment 2: the shoot regeneration culture based on transdifferentiation
(1) explant (preparation of embodiment 1) is placed in the B5 medium containing 10 μM of NAA, is placed in 25 DEG C, 16 hours
It is cultivated 24 hours under the dark condition of illumination/8 hour；
(2) explant after step (1) culture is transferred in the B5 medium containing 8.8 μM of 2-IP, is placed in 25 DEG C,
Cultivate 12 days under 16 hours illumination/8 hour dark conditions, therebetween every 6 days one subcultures of replacement；
(3) explant after step (2) culture is transferred in the MS culture medium containing 0.5mg/L 6-BA, is placed in 25
DEG C, cultivate 16 days under 16 hours illumination/8 hour dark conditions, therebetween every 6 days one subcultures of replacement.
In the present embodiment, the time for obtaining poplar regeneration bud is 29 days, and the regeneration rate of poplar regeneration bud is 100%.
Embodiment 3: the differential interference contrast microscope observation of apical meristem transdifferentiation process
(1) explant for taking different incubation times is cut to 0.5 centimetre or so of segment with blade, is placed in 1.5ml centrifuge tube
In；
(2) 1ml clarifier (8g chloraldurate, 1ml glycerol, 3ml sterile water) is added, centrifuge tube is added；
(3) centrifuge tube is placed in vacuum pump to vacuumize twice, 10 minutes every time；
(4) 2-5 days are placed at room temperature for, the specific time judges according to material transparent effect；
(5) Zeiss Axio Scope A1 fluorescence microscope is utilized, using DIC effect, is seen by 1X eyepiece, 10X object lens
Examine photograph；Image is first handled through LAS AF Lite, is exported as jpeg image.
As a result see Fig. 2, form lateral-root primordia (Fig. 2, A-E) as seen from Figure 2, in explant, and gradually experienced
(Fig. 2, F-G) and transformation (Fig. 2, H) process are expanded, shoot apical meristem (Fig. 2, I-J) is formd by transdifferentiation.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (10)
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| CN108575740A (en) * | 2018-02-26 | 2018-09-28 | 北京林业大学 | A kind of method that the generation of willow body embryo is built up with plant |
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| CN115992166A (en) * | 2022-03-29 | 2023-04-21 | 陕西师范大学 | Application of CLE42 Small Peptide in Promoting Plant Bud Regeneration and Proliferation Promoter in Plant Tissue Culture |
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