CN109479727A - A method of inducing cells,primordial using Afriocan agapanthus blade as explant - Google Patents
A method of inducing cells,primordial using Afriocan agapanthus blade as explant Download PDFInfo
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- CN109479727A CN109479727A CN201910060531.2A CN201910060531A CN109479727A CN 109479727 A CN109479727 A CN 109479727A CN 201910060531 A CN201910060531 A CN 201910060531A CN 109479727 A CN109479727 A CN 109479727A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Abstract
The method that the present invention provides a kind of to induce cells,primordial using Afriocan agapanthus blade as explant, comprising: A, the leaf base irrigation and disinfection for taking Afriocan agapanthus remove the external old leaf wrapped up, and cut away the tissue of upper and lower ends, obtain blade base tissue；B, it will be inoculated in callus inducing medium and cultivate after blade base tissue stripping and slicing；C, the callus lines obtained after step B culture are placed on callus subculture medium and carry out squamous subculture；D, the callus lines obtained after step C culture are placed on embryonic callus induction culture medium and are cultivated, obtain the callus lines containing cells,primordial.The present invention successfully carries out the induction of cells,primordial on leaf base tissue evoked callus, and callus induction rate has reached 92.60%, and cells,primordial inductivity reaches 76.36%, and induction duration is only 6~7 months.And cells,primordial induction seedling is very fast, avoids long-term subculture process, reduces lopsided seedling and occurs.
Description
Technical field
The method that the present invention relates to a kind of to induce cells,primordial using Afriocan agapanthus blade as explant.
Background technique
Afriocan agapanthus ' Big Blue ' (Agapanthus praecox ssp.orientalis ' Big Blue ') also known as " indigo plant
Lily ", " Afric lilium " originate in Africa south, are unifacial leaf perennial herb flowers, have stronger ornamental value.Nearly half
Since century, Afriocan agapanthus is shown one's talent in the development of International Flower industry, is become worldwide Fresh Cutting flower, potting and ground and is spent
Grass, and embody splendid ornamental value.In addition, Afriocan agapanthus resistance is extremely strong, have in roadside greening, soil remediation field huge
Development space, supply falls short of demand for seedling currently on the market.
Afriocan agapanthus often uses seed or division propagation in source area South Africa, but introduces a fine variety behind the country that there are germination percentages low, breeding week
The disadvantages of phase is long, and breeding coefficient is low and offspring easily breaks up.Tissue cultures are to maintain Afriocan agapanthus merit, obtain a large amount of seedlings
Important channel.The tissue cultures of Afriocan agapanthus are divided into adventitious organogenesis and somatic embryo development ways two major classes at present.Wherein
Organ approach has been applied to industrialization production, but there is also problems, such as breeding coefficient are lower, and the seedling period is long etc..
And somatic embryo development ways have that quantity is more, breeding is fast, structural integrity, shoot regeneration frequency height and not by seasonal effect etc.
Feature, therefore be considered as the preferable approach of Afriocan agapanthus asexual propagation and preserving seed.
Because pedicel aseptic process is easier, evoked callus is relatively quick, domestic to carry out body embryo with Afriocan agapanthus
The explant more options pedicel of induction, but the materials of pedicel have compared with big limitation, are often only Afriocan agapanthus flower bud phase in 5~June
It can draw materials, and the induction duration of cells,primordial is longer, for 9 months.And the blade of Afriocan agapanthus can draw materials it is more, materials
Convenient, not by growth cycle and season limit, the four seasons can be cultivated.Accordingly, with respect to pedicel, callus is induced using blade
Tissue, and then cells,primordial is induced with biggish application value, the body embryogenesis path using blade as explant is to hundred
It is preferable approach that sub- lotus is numerous fastly.
Domestic is some studies have shown that not obtained with 2,4-D as main growth regulator by blade as explant
Obtain callus.In Study on tissue culture, monocotyledon is usually more difficult to be formed callus than dicotyledon, and PIC
Can be used as exogenous auxin substance reduces the difficulty of monocotyledon tissue cultures.Carbon source is in plant tissue culture course
One of most important factor, sugar is used as carbon source, and only cell does not provide the carbon skeleton of synthesis noval chemical compound, and is the life of cell
Reason metabolism provides substrate and energy.Plant Tissue Breeding generally uses sucrose, grape, maltose, fructose etc. as carbon source, different
The carbon source of type and concentration is not also identical to the inducing effect of callus.And in the previous research of Afriocan agapanthus, only with sucrose
As carbon source, other type carbon sources to Afriocan agapanthus tissue culture, body embryo induction effect and have not been reported.
The present invention is according to early-stage study basis, it is intended to change the limitation for relying on pedicel materials early period, be made using blade
The induction of callus is carried out for explant.Meanwhile the present invention intends to solve hundred by regulation hormone and the type and level of carbon source
The problem that sub- lotus cells,primordial induction duration is long, inductivity is lower.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide one kind to be induced using Afriocan agapanthus blade as explant
The method of cells,primordial.The present invention utilizes blade base meristematic zone according to the characteristic distributions of monocotyledon regeneration potential cell
The induction for carrying out callus as explant is organized, breaks through what domestic callus at present can only be induced using pedicel
Limitation；The present invention overcomes unifacial leaf using monocotyledon specific element substance PIC as main inducing hormone
The problem of plant leaf blade callus induction difficulty；The rule and unifacial leaf regulated and controled according to carbon source in plant body embryo generating process
The characteristics of plant body embryo occurs, the present invention passes through type and concentration proportioning using sucrose, glucose and maltose as carbon source
Switching, change traditional tissue cultures and body embryo approach only using sucrose as the method for carbon source, improve callus, embryo
The inductivity of callus shortens the induction duration of cells,primordial.
The purpose of the present invention is what is be achieved through the following technical solutions:
The method that the present invention provides a kind of to induce cells,primordial using Afriocan agapanthus blade as explant, including following step
It is rapid:
A, the blade irrigation and disinfection of Afriocan agapanthus, the old leaf of external 2~3 packages of removal are taken, and cuts away the group of upper and lower ends
It knits, obtains blade base tissue；
B, it will be inoculated in callus inducing medium and cultivate after blade base tissue stripping and slicing；
C, the callus lines with residual leaf tissue obtained after step B culture are placed on callus subculture
Squamous subculture is carried out on culture medium；
D, the callus lines obtained after step C culture are prevented and treated and is trained on embryonic callus induction culture medium
It supports, obtains the callus lines containing cells,primordial.
Preferably, in step A, the blade of the Afriocan agapanthus is to be greater than from the blade at 0~2.0cm of rhizome connecting portion
The position of 2cm lacks meristematic capacity, it is more difficult to evoked callus；The Afriocan agapanthus is 2~triennial Afriocan agapanthus.
Preferably, in step A, specific step is as follows for the irrigation and disinfection: the blade flowing water of Afriocan agapanthus is rinsed 90~
150min is placed on superclean bench, and 75% (v/v) 50~70s of alcohol treatment uses ddH2O is rinsed 3~5 times, then with 5% time
Sodium chlorate disinfects 5~7min, later ddH2O is rinsed 3~5 times, then with 75% 50~70s of alcohol treatment, uses ddH2O rinses 3
~5 times.
Preferably, the callus inducing medium, callus subculture medium, embryonic callus induction culture
Exogenous auxin PIC is added in base.
Preferably, in step B, the size after the blade base tissue stripping and slicing is 0.5~0.8cm；The condition of culture
Are as follows: 22~28 DEG C of temperature, dark culture 25~35 days.
Preferably, in step B, the component of the callus inducing medium includes: 4.43gL-1MS solid culture
Base, 1.5~2.0mgL-1PIC, 2.5~3.5% (w/v) sucrose, 0.8~1.0% (w/v) agar, the pH of the culture medium are
5.8。
Preferably, the callus inducing medium preparation method the following steps are included:
Every liter of ddH24.43g MS solid medium dry powder is added in O, PIC solution, sucrose, agar are added after dissolution, adjusts
Saving pH value is 5.8；It is dispensed after the medium sterilization processing of acquisition, cooled and solidified.
Preferably, in step C, the squamous subculture condition are as follows: 22~28 DEG C of temperature, dark culture, cultivation cycle be 55~
65d, squamous subculture 2 times.
Preferably, in step C, the component of the callus subculture medium includes: 4.43gL-1MS solid culture
Base, 1.0~1.5mgL-1PIC, 2% (w/v) sucrose, 1% (w/v) maltose, 0.8~1% (w/v) agar, pH5.8.
Preferably, in step D, the condition of culture is 22~28 DEG C of temperature, 55~65d of dark culture.
Preferably, in step D, the component of the embryonic callus induction culture medium includes: 4.43gL-1The training of MS solid
Support base, 1.0~1.5mgL-1PIC, 2% (w/v) glucose, 2% (w/v) maltose, 0.8~1% (w/v) agar, pH5.8.
It is new in wound surface after heretofore described callus (Callus) refers to plant part by wound stimulation
Raw tissue.It is made of the parenchyma cell lived, and can originate from the living cells of plant Various Tissues.
The cells,primordial, that is, embryo callus cell, color have milky or yellow, and surface has spheric granules, life
It is long slow；From the point of view of cytology, embryo callus is made of equal diameter cell, and cell is smaller, and plasm is dense, no vacuole,
It is often rich in amylum body, core is big, and mitotic activity is strong, has the ability sprouted and become somatic embryo.
Compared with prior art, the invention has the following beneficial effects:
Afriocan agapanthus tissue culture at home and somatic embryo approach it is fast it is numerous in, generally drawn materials with pedicel, but pedicel takes
Material, which is often only, to carry out at the florescence, and blade materials of the invention can carry out anniversary progress.
Part Methods are drawn materials using plateau as explant, but the negligible amounts of plateau, and every plant only one
It is a, and materials explant of the invention is blade, quantity is more, and by taking 2 years raw seedling as an example, every plant has 10 or more leaves
Piece.
Part report also drawn materials with blade by trial, but does not obtain or seldom obtain callus, and the present invention utilizes blade
Meristematic zone regeneration potential cell characteristics, callus induction rate have reached 92.60%.
Before the present invention, the domestic precedent that cells,primordial induction is not carried out using blade as explant, and the present invention is in leaf
On the basis of base portion tissue evoked callus, it is successfully made the induction of cells,primordial, cells,primordial inductivity reaches
76.36%.
It is 9 months or more that pedicel, which is the period that explant carries out cells,primordial induction, blade conduct provided by the invention
The period that explant carries out cells,primordial induction is 6~7 months.
The present invention is stronger to the specificity of Afriocan agapanthus callus induction using PIC as exogenous auxin.
The present invention breaches traditional tissue culture only using sucrose as the culture medium prescription of carbon source, using sucrose, glucose and
Maltose improves the inductivity of callus, embryo callus by the regulation of type and concentration proportioning as carbon source,
And shorten the induction duration of cells,primordial.
Since materials are convenient, induction duration is short, and the present invention carries out cells,primordial and induces seedling very fast, avoids cells,primordial
Long-term subculture process, therefore reduce the generation of lopsided seedling.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is blade base evoked callus figure in the embodiment of the present invention 1；Wherein, Fig. 1 a is blade as explant
Callus induction；Fig. 1 b is the partial enlarged view of Fig. 1 a；
Fig. 2 is blade base induced embryonic callus figure in the embodiment of the present invention 1；
Fig. 3 is embryo callus cellular morphology observation in the embodiment of the present invention 1；
Fig. 4 is the induction seedling figure of cells,primordial in the embodiment of the present invention 1；Wherein, Fig. 4 a is the morphologic observation after 30d
Figure；Fig. 4 b is the morphologic observation figure after 2 months.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, several changes and improvements can also be made.These belong to the present invention
Protection scope.
Embodiment 1
A kind of method that cells,primordial is induced using Afriocan agapanthus blade as explant is present embodiments provided, specific steps are such as
Under:
The materials of explant: 2 years raw Afriocan agapanthus are cut from the blade at 0~2.0cm of rhizome connecting portion with knife blade.
The blade flowing water of Afriocan agapanthus is rinsed into 90~150min and is placed on superclean bench, 75% (v/v) 50~70s of alcohol treatment is used
ddH2O is rinsed 3~5 times, then handles 5~7min with 5% hypochlorite disinfectant, later ddH2O is rinsed 3~5 times, then with 75%
50~70s of alcohol treatment, uses ddH2O is rinsed 3~5 times, and aseptic filter paper blots the moisture of blade surface, cuts leaf using scalpel
The tissue (old leaf of tissue including upper and lower ends and 2~3 packages) of piece periphery, with scalpel by resulting blade base
Tissue is cut into the fritter of 0.5~0.8cm.
The preparation of callus inducing medium: every liter of ddH24.43g MS solid medium (Sigma) dry powder is added in O,
2.0mgL is added after dissolution-1PIC solution (mother liquid concentration 100mgL-1, with NaOH hydrotropy), adjusting pH value is 5.8,
Sucrose concentration is 3% (w/v), and agar concentration is 1% (w/v).Culture medium sterilization treatment 25min in 121 DEG C of high-pressure sterilizing pots
After take packing in superclean bench, after cooled and solidified, carry out the inoculation of explant.
The induction of callus: it takes the blade base tissue fritter laid-flat status of 0.5~0.8cm to be inoculated in callus and lures
It leads on culture medium flat plate, nutrient media components include: MS+2.0mgL-1PIC+3% (w/v) sucrose+1% (w/v) agar,
pH5.8.Condition of culture: 25 DEG C of temperature, dark culture.The callus (Fig. 1) that culture 15d can show white translucent generates, callus
Tissue inductivity carries out the squamous subculture of callus after being 92.60%, 30 days.
The subculture of callus: the callus lines with residual leaf tissue obtained after callus induction of learning from else's experience,
It is placed on callus subculture plating medium and carries out squamous subculture, nutrient media components include: MS+1.5mgL-1PIC+2%
(w/v) sucrose+1% (w/v) maltose+1% (w/v) agar, pH5.8.Condition of culture: 25 DEG C of temperature, dark culture.It is with 60d
One subculture cycle, squamous subculture 2 times, callus is gradually converted into yellowish, and part cell mass shows opaque shape, and surface is thick
It is rough.
The transformation of embryo callus: the callus lines (without residual leaf tissue) obtained after squamous subculture of learning from else's experience,
It is placed on embryonic callus induction culture medium flat plate and is cultivated, nutrient media components are as follows: MS+1.5mgL-1PIC+2%
(w/v) glucose+2% (w/v) maltose+1% (w/v) agar, pH5.8.Condition of culture: 25 DEG C of temperature, dark culture.After 60d,
Most cell masses show opaque shape, and the embryonal connective tissue (Fig. 2) of unicellular origin occurs in yellowish callus surface.Cells,primordial
Inductivity is 76.36%.
The cell dyeing of cells,primordial is verified: being taken the cell mass of 3mm size, is put into the centrifuge tube of 1.5mL, 500 μ are added
The acetic acid magenta of L, stands 30min at room temperature, draws dyeing liquor with pipettor, discards dyeing liquor；Then it is added super
Ultrapure water is added with pipettor draw solution in pure water, constantly piping and druming cell mass again, this step is repeated 3 times.Take glass slide one
Piece, it is cut off at the 2mm of top with 1mL suction nozzle, draws the cell mass of diameter 1mm size, be placed on glass slide, placed coverslip, keep away
Exempt from generate bubble, then gently flatten, be placed on Leica DM2500 microscopically observation and take pictures, can be observed nucleus compared with
Greatly, the dense cells,primordial of cytoplasm (Fig. 3).
The induction verifying of cells,primordial seedling: the callus lines containing cells,primordial are taken, it is flat to be placed on seedling induced medium
It is cultivated on plate, nutrient media components include: 4.43gL-1MS+2% (w/v) sucrose+2% (w/v) glucose+1% (w/v)
Agar, pH5.8.Condition of culture: 22~28 DEG C of temperature, illumination cultivation, intensity of illumination 2500lx.After 15d, most cell mass surfaces
There is embryo shape particulate matter, white opaque shape, after 30d, embryo shape particulate matter is grown to club shaped structure, and top turns green, lower part
It is still white, in being independently distributed shape (Fig. 4 a), the body embryo seedling (Fig. 4 b) with root system can be grown up to after 2 months.
Embodiment 2
A kind of method that cells,primordial is induced using Afriocan agapanthus blade as explant is present embodiments provided, specific steps are such as
Under:
The materials of explant: 2 years raw Afriocan agapanthus are cut from the blade at 0~2.0cm of rhizome connecting portion with knife blade.
The blade flowing water of Afriocan agapanthus is rinsed into 90~150min and is placed on superclean bench, 75% (v/v) 50~70s of alcohol treatment is used
ddH2O is rinsed 3~5 times, then handles 5~7min with 5% hypochlorite disinfectant, later ddH2O is rinsed 3~5 times, then with 75%
50~70s of alcohol treatment, uses ddH2O is rinsed 3~5 times, and aseptic filter paper blots the moisture of blade surface, cuts leaf using scalpel
The tissue (old leaf of tissue including upper and lower ends and 2~3 packages) of piece periphery, with scalpel by resulting blade base
Tissue is cut into the fritter of 0.5~0.8cm.
The preparation of callus inducing medium: every liter of ddH24.43g MS solid medium (Sigma) dry powder is added in O,
1.5mgL is added after dissolution-1PIC solution (mother liquid concentration 100mgL-1, with NaOH hydrotropy), adjusting pH value is 5.8,
Sucrose concentration is 3.5% (w/v), and agar concentration is 0.8% (w/v).Culture medium sterilization treatment in 121 DEG C of high-pressure sterilizing pots
It takes in superclean bench and dispenses after 25min, after cooled and solidified, carry out the inoculation of explant.
The induction of callus: it takes the blade base tissue fritter laid-flat status of 0.5~0.8cm to be inoculated in callus and lures
It leads on culture medium flat plate, nutrient media components include: MS+1.5mgL-1PIC+3.5% (w/v) sucrose+0.8% (w/v) agar,
pH5.8.Condition of culture: 22 DEG C of temperature, dark culture.The callus that culture 15d can show white translucent generates, and callus lures
Conductance carries out the squamous subculture of callus after being 87.65%, 35 days.
The subculture of callus: the callus lines with residual leaf tissue obtained after callus induction of learning from else's experience,
It is placed on callus subculture plating medium and carries out squamous subculture, nutrient media components include: 4.43gL-1MS+1.0mg·
L-1PIC+2% (w/v) sucrose+1% (w/v) maltose+0.8% (w/v) agar, pH5.8.Condition of culture: 22 DEG C of temperature, dark to train
It supports.With 65d for a subculture cycle, squamous subculture 2 times, callus is gradually converted into yellowish, and part cell mass is existing impermeable
Bright shape, rough surface.
The transformation of embryo callus: the callus lines (without residual leaf tissue) obtained after squamous subculture of learning from else's experience,
It is placed on embryonic callus induction culture medium flat plate and is cultivated, nutrient media components are as follows: 4.43gL-1MS+1.2mg·L-1PIC+2% (w/v) glucose+2% (w/v) maltose+0.8% (w/v) agar, pH5.8.Condition of culture: 22 DEG C of temperature, secretly
Culture.After 65d, most cell masses show opaque shape, and the embryonal connective tissue of unicellular origin occurs in yellowish callus surface.Embryo
Property cell inductivity be 74.98%.
The cell dyeing of cells,primordial is verified: being taken the cell mass of 3mm size, is put into the centrifuge tube of 1.5mL, 500 μ are added
The acetic acid magenta of L, stands 30min at room temperature, draws dyeing liquor with pipettor, discards dyeing liquor；Then it is added super
Ultrapure water is added with pipettor draw solution in pure water, constantly piping and druming cell mass again, this step is repeated 3 times.Take glass slide one
Piece, it is cut off at the 2mm of top with 1mL suction nozzle, draws the cell mass of diameter 1mm size, be placed on glass slide, placed coverslip, keep away
Exempt from generate bubble, then gently flatten, be placed on Leica DM2500 microscopically observation and take pictures, can be observed nucleus compared with
Greatly, the dense cells,primordial of cytoplasm.
The induction verifying of cells,primordial seedling: taking the callus lines containing cells,primordial of acquisition, is placed on seedling induction training
It supports and is cultivated on base plate, nutrient media components include: 4.43gL-1MS+2% (w/v) sucrose+2% (w/v) glucose+
0.6% (w/v) agar, pH5.8.Condition of culture: 22~28 DEG C of temperature, illumination cultivation, intensity of illumination 2500lx.It is most after 15d
There is embryo shape particulate matter in cell mass surface, white opaque shape, and after 30d, embryo shape particulate matter is grown to club shaped structure, and top turns
Green, lower part are still white, and in shape is independently distributed, the body embryo seedling with root system can be grown up to after 2 months.
Embodiment 3
A kind of method that cells,primordial is induced using Afriocan agapanthus blade as explant is present embodiments provided, specific steps are such as
Under:
The materials of explant: 2 years raw Afriocan agapanthus are cut from the blade at 0~2.0cm of rhizome connecting portion with knife blade.
The blade flowing water of Afriocan agapanthus is rinsed into 90~150min and is placed on superclean bench, 75% (v/v) 50~70s of alcohol treatment is used
ddH2O is rinsed 3~5 times, then handles 5~7min with 5% hypochlorite disinfectant, later ddH2O is rinsed 3~5 times, then with 75%
50~70s of alcohol treatment, uses ddH2O is rinsed 3~5 times, and aseptic filter paper blots the moisture of blade surface, cuts leaf using scalpel
The tissue (old leaf of tissue including upper and lower ends and 2~3 packages) of piece periphery, with scalpel by resulting blade base
Tissue is cut into the fritter of 0.5~0.8cm.
The preparation of callus inducing medium: every liter of ddH24.43g MS solid medium (Sigma) dry powder is added in O,
1.8mgL is added after dissolution-1PIC solution (mother liquid concentration 100mgL-1, with NaOH hydrotropy), adjusting pH value is 5.8,
Sucrose concentration is 2.5% (w/v), and agar concentration is 1% (w/v).Culture medium sterilization treatment in 121 DEG C of high-pressure sterilizing pots
It takes in superclean bench and dispenses after 25min, after cooled and solidified, carry out the inoculation of explant.
The induction of callus: it takes the blade base tissue fritter laid-flat status of 0.5~0.8cm to be inoculated in callus and lures
It leads on culture medium flat plate, nutrient media components include: 4.43gL-1MS+1.8mg·L-1PIC+2.5% (w/v) sucrose+1% (w/
V) agar, pH5.8.Condition of culture: 28 DEG C of temperature, dark culture.The callus that culture 15d can show white translucent generates, more
Injured tissue inductivity carries out the squamous subculture of callus after being 89.93%, 25 days.
The subculture of callus: the callus lines with residual leaf tissue obtained after callus induction of learning from else's experience,
It is placed on callus subculture plating medium and carries out squamous subculture, nutrient media components include: 4.43gL-1MS+1.2mg·
L-1PIC+2% (w/v) sucrose+1% (w/v) maltose+1% (w/v) agar, pH5.8.Condition of culture: 28 DEG C of temperature, dark to train
It supports.With 55d for a subculture cycle, squamous subculture 2 times, callus is gradually converted into yellowish, and part cell mass is existing impermeable
Bright shape, rough surface.
The transformation of embryo callus: the callus lines (without residual leaf tissue) obtained after squamous subculture of learning from else's experience,
It is placed on embryonic callus induction culture medium flat plate and is cultivated, nutrient media components are as follows: 4.43gL-1MS+1.2mg·L-1PIC+2% (w/v) glucose+2% (w/v) maltose+1% (w/v) agar, pH5.8.Condition of culture: 28 DEG C of temperature, dark to train
It supports.After 55d, most cell masses show opaque shape, and the embryonal connective tissue of unicellular origin occurs in yellowish callus surface.Embryo
Cell inductivity is 58.27%.
The cell dyeing of cells,primordial is verified: being taken the cell mass of 3mm size, is put into the centrifuge tube of 1.5mL, 500 μ are added
The acetic acid magenta of L, stands 30min at room temperature, draws dyeing liquor with pipettor, discards dyeing liquor；Then it is added super
Ultrapure water is added with pipettor draw solution in pure water, constantly piping and druming cell mass again, this step is repeated 3 times.Take glass slide one
Piece, it is cut off at the 2mm of top with 1mL suction nozzle, draws the cell mass of diameter 1mm size, be placed on glass slide, placed coverslip, keep away
Exempt from generate bubble, then gently flatten, be placed on Leica DM2500 microscopically observation and take pictures, can be observed nucleus compared with
Greatly, the dense cells,primordial of cytoplasm.
The induction verifying of cells,primordial seedling: the callus lines containing cells,primordial are taken, it is flat to be placed on seedling induced medium
It is cultivated on plate, nutrient media components include: 4.43gL-1MS+2% (w/v) sucrose+2% (w/v) glucose+1% (w/v)
Agar, pH5.8.Condition of culture: 22~28 DEG C of temperature, illumination cultivation, intensity of illumination 2500lx.After 15d, most cell mass surfaces
There is embryo shape particulate matter, white opaque shape, after 30d, embryo shape particulate matter is grown to club shaped structure, and top turns green, lower part
It is still white, in shape is independently distributed, the body embryo seedling with root system can be grown up to after 2 months.
Comparative example 1
The preparation step of this comparative example and embodiment 1 is essentially identical, the difference is that only: each training that this comparative example uses
It supports and is free of PIC in base.
Using the method for this comparative example, on the culture medium without PIC, leaf tissue is substantially undifferentiated, does not grow, does not produce
Raw callus, browning is dead after 30~60 days.
Comparative example 2
The preparation step of this comparative example and embodiment 1 is essentially identical, the difference is that only: the embryo that this comparative example uses
In callus inducing medium, carbon source uses 4% sucrose.
Using the method for this comparative example, in cells,primordial induction period, using 4% sucrose as carbon source, without slender after 60d
The embryonal connective tissue of born of the same parents origin generates, and healing cell is still non-cells,primordial.
Comparative example 3
The preparation step of this comparative example and embodiment 1 is essentially identical, the difference is that only: the explant of this comparative example takes
It in material step, draws materials to integrated plate blade, blade is directly cut into using scalpel the fritter or segment of 0.5~0.8cm.
Using the method for this comparative example, the region blade base 2cm has meristematic capacity, can produce callus, blade base
The region of portion 2cm or more be maturation zone, the not ability of cell differentiation, therefore generation of the mature leaf without callus, there are no
Method induces cells,primordial.
Comparative example 4
The preparation step of this comparative example and embodiment 1 is essentially identical, the difference is that only: each culture medium of this comparative example
In, using 1.0~2.0mgL-12,4-D replaces PIC as exogenous auxin.
Using the method for this comparative example, though blade can maintain isolated growth, callus inducing effect is poor, and inductivity is insufficient
10%, the inductivity of cells,primordial is 0.00%.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make a variety of changes or modify within the scope of the claims, this not shadow
Ring substantive content of the invention.In the absence of conflict, the feature in embodiments herein and embodiment can any phase
Mutually combination.
Claims (10)
1. a kind of method for inducing cells,primordial using Afriocan agapanthus blade as explant, which comprises the following steps:
A, the blade irrigation and disinfection of Afriocan agapanthus, the old leaf of external 2~3 packages of removal are taken, and cuts away the tissue of upper and lower ends, is obtained
Blade base tissue；
B, it will be inoculated in callus inducing medium and cultivate after blade base tissue stripping and slicing；
C, the callus lines with residual leaf tissue obtained after step B culture are placed on callus squamous subculture
Squamous subculture is carried out on base；
D, the callus lines obtained after step C culture are placed on embryonic callus induction culture medium and are cultivated,
The callus lines of cells,primordial must be contained.
2. the method according to claim 1 for inducing cells,primordial using Afriocan agapanthus blade as explant, which is characterized in that
In step A, the blade of the Afriocan agapanthus is from the blade at 0~2.0cm of rhizome connecting portion；The Afriocan agapanthus is 2~triennial
Afriocan agapanthus.
3. the method according to claim 1 for inducing cells,primordial using Afriocan agapanthus blade as explant, which is characterized in that
In step A, specific step is as follows for the irrigation and disinfection: 90~150min of blade flowing water flushing of Afriocan agapanthus being placed on ultra-clean
Workbench, 75% (v/v) 50~70s of alcohol treatment, uses ddH2O is rinsed 3~5 times, then with 5% hypochlorite disinfectant processing 5
~7min, later ddH2O is rinsed 3~5 times, then with 75% 50~70s of alcohol treatment, uses ddH2O is rinsed 3~5 times.
4. the method according to claim 1 for inducing cells,primordial using Afriocan agapanthus blade as explant, which is characterized in that
In step B, the size after the blade base tissue stripping and slicing is 0.5~0.8cm；The condition of culture are as follows: 22~28 DEG C of temperature,
Dark culture 25~35 days.
5. the method according to claim 1 or 4 for inducing cells,primordial using Afriocan agapanthus blade as explant, feature exist
In in step B, the component of the callus inducing medium includes: 4.43gL-1MS solid medium, 1.5~
2.0mg·L-1PIC, 2.5~3.5% (w/v) sucrose, 0.8~1.0% (w/v) agar, the pH of the culture medium are 5.8.
6. the method according to claim 5 for inducing cells,primordial using Afriocan agapanthus blade as explant, which is characterized in that
The preparation method of the callus inducing medium the following steps are included:
Every liter of ddH24.43g MS solid medium dry powder is added in O, PIC solution, sucrose, agar are added after dissolution, adjusts pH value
It is 5.8；It is dispensed after the medium sterilization processing of acquisition, cooled and solidified.
7. the method according to claim 1 for inducing cells,primordial using Afriocan agapanthus blade as explant, which is characterized in that
In step C, the squamous subculture condition are as follows: 22~28 DEG C of temperature, dark culture, cultivation cycle be 55~65d, squamous subculture 2 times.
8. the method according to claim 1 for inducing cells,primordial using Afriocan agapanthus blade as explant, which is characterized in that
In step C, the component of the callus subculture medium includes: 4.43gL-1MS solid medium, 1.0~1.5mg
L-1PIC, 2% (w/v) sucrose, 1% (w/v) maltose, 0.8~1% (w/v) agar, pH5.8.
9. the method according to claim 1 for inducing cells,primordial using Afriocan agapanthus blade as explant, which is characterized in that
In step D, the condition of culture are as follows: 22~28 DEG C of temperature, 55~65d of dark culture.
10. the method according to claim 1 for inducing cells,primordial using Afriocan agapanthus blade as explant, feature exist
In in step D, the component of the embryonic callus induction culture medium includes: 4.43gL-1MS solid medium, 1.0~
1.5mg·L-1PIC, 2% (w/v) glucose, 2% (w/v) maltose, 0.8~1% (w/v) agar, pH5.8.
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