CN110140658A - A method of Kiwi berry seedling is cultivated using Stem Segments In Chinese Gooseberry forming layer stem cell - Google Patents
A method of Kiwi berry seedling is cultivated using Stem Segments In Chinese Gooseberry forming layer stem cell Download PDFInfo
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- CN110140658A CN110140658A CN201910560755.XA CN201910560755A CN110140658A CN 110140658 A CN110140658 A CN 110140658A CN 201910560755 A CN201910560755 A CN 201910560755A CN 110140658 A CN110140658 A CN 110140658A
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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
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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
- A01H4/008—Methods for regeneration to complete plants
Abstract
The method provided by the invention for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell includes: to select diameter for 0.2-0.4cm, and display has clearly xylem under anatomical lens, the non-lignifying Kiwi berry young sprout of bast is as inoculation explant, the perithelium and epidermis of inoculation explant stem section are removed with tweezers, inoculation explant stem section is first placed in anti-oxidation treatment fluid and is handled, it is placed in forming layer stem cell induced medium and cultivates again, the inoculation explant stem section after forming layer stem cell induced medium culture 25-30 days is prescinded again again, it is transferred in cambial cell callus induction culture medium and cultivates, for the callus of seedling differentiation, when seedling length to height is 1.0cm, it cuts seedling inoculation and supports base into root media, for the callus of no Seedling Differentiation, callus is transferred to seedling point Change and carries out subculture and differentiation culture in culture medium.Technical solution provided by the invention has the characteristics that external-talent's pollution rate is low, induction frequency is high.
Description
Technical field
The application belongs to plant tissue culture fast breeding technique field, specifically, being related to a kind of utilization Stem Segments In Chinese Gooseberry forming layer
The method of stem cell cultivation Kiwi berry seedling.
Background technique
Kiwi berry is Actinidiaceae, the fruit of the perennial liana of Actinidia.Since Kiwi berry is in good taste, nutrition
And healthcare function is obvious, it has also become one of most popular fruit in the world.Kiwi berry is cultivated kind and is also being increased year by year, at present
There is a cultivar more than 150.However, Actinidia be easy to cause product using conventional seed seed propagation in dioecian plant
Kind is degenerated, and the holding of merit is unfavorable for, and uses the methods of cuttage, grafting, and survival rate, breeding coefficient are again low.With other
Seedling raising manners are compared, and using tissue-culturing rapid propagation, not only can preferably keep the merit and its genetic stability of excellent variety, together
When can shorten growing-seedling period, reduce seedling transportation cost.
In the current tissue-culturing rapid propagation in relation to Kiwi berry, there is blade in institute, for leaf stem section using external-talent, also there is Inter-node stem-segment.Benefit
It is bred with blade, since blade surface villus is more, pollution rate is higher in tissue-culturing rapid propagation, is unfavorable for the tissue-culturing rapid propagation of Kiwi berry.
It is bred using for leaf stem section, mostly axillary bud deriving, while there is also the high pollution difficult points of tissue culture process.It is numerous using Inter-node stem-segment
It grows, though being callus induction, Stem Segments In Chinese Gooseberry or villous or crack, pollution rate is high during tissue culture.
Summary of the invention
In view of the above drawbacks of the prior art, the application provides a kind of utilization Stem Segments In Chinese Gooseberry forming layer stem cell cultivation Mi
The method of monkey peach seedling.
The method disclosed by the invention for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell, comprising:
Step 1: select diameter for 0.2-0.4cm, and display has clearly xylem, bast under anatomical lens
Non- lignifying Kiwi berry young sprout first cleans Kiwi berry young sprout, is disinfected after removing blade, then by Kiwi berry young sprout
It is cut into the stem section that length is 2-3cm and is used as inoculation explant stem section；
Step 2: removing the perithelium and epidermis of inoculation explant stem section with tweezers, inoculation explant stem section both ends are respectively cut
After 0.1-0.2cm, first it is placed in anti-oxidation treatment fluid and handles 30-90 minutes, then be placed in forming layer stem cell induced medium
In in temperature be to be cultivated in 24 DEG C~26 DEG C of dark surrounds；It is to train substantially that the forming layer stem cell induced medium, which is with MS,
Base is supported, is then prepared by adding agar powder, sucrose and NAA or IBA in basic medium；
Step 3: the inoculation explant stem section after forming layer stem cell induced medium culture 25-30 days is cut again
Short, formation length is the segment of 0.8-1.5cm or the thin slice of 0.2-0.3cm, is transferred in cambial cell callus induction culture medium
It is cultivated 25-35 days in 1800-2200Lux, 25 ± 2 DEG C of environment, surface forms callus；The cambial cell callus induction
Culture medium be using using MS as minimal medium, then by add in basic medium agar powder, sucrose, NAA, 6-BA,
Kinetin or Zeatin are prepared；
Step 4:, when seedling length to height is 1.0cm, cutting seedling inoculation to training of taking root for the callus of seedling differentiation
It supports and carries out culture of rootage 35d in base in 1800-2200Lux, 25 ± 2 DEG C of environment；The root media is to be with 1/2MS
Then minimal medium is prepared by adding agar powder, sucrose, 6-BA and NAA or IBA in basic medium；
Step 5: for the callus of no Seedling Differentiation, by callus be transferred in Seedling Differentiation culture medium in 1800-2200Lux,
Subculture and differentiation culture are carried out in 25 ± 2 DEG C of environment, cultivate 25d；If callus Surface Differentiation is at seedling, repeatedly step 4,
Carry out rooting induction；If callus surface repeats this step 5 without Seedling Differentiation；The Seedling Differentiation culture medium is using MS as base
Then basal culture medium is prepared by adding agar powder, sucrose, NAA and 6-BA in basic medium.
The method as described above for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell, it is preferable that in step
In rapid two, the content of agar powder is 6-8gL in the forming layer stem cell induced medium-1, the content of sucrose is 2.5-
3.0g.L-1, the content of NAA or IBA are 3-6mgL-1。
The method as described above for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell, it is preferable that in step
In rapid three, the content of agar powder is 6-8gL in the cambial cell callus induction culture medium-1, the content of sucrose is 30-
35g.L-1, the content of NAA is 3-6mgL-1, the content of 6-BA, Kinetin or Zeatin are 0.2-0.5mgL-1, the shape
PH at confluent monolayer cells callus induction culture medium is 5.2-6.8.
The method as described above for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell, it is preferable that in step
In rapid four, the content of agar powder is 6-8gL in the root media-1, the content of sucrose is 30-35g.L-1, 6-BA's contains
Amount is 0.05-0.1mgL-1, the content of NAA or IBA are 0.2-0.4mgL-1, the pH of the root media is 5.2-
6.8。
The method as described above for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell, it is preferable that in step
In rapid five, the content of agar powder is 6-8gL in the Seedling Differentiation culture medium-1, the content of sucrose is 30-35g.L-1, NAA
Content be 2-3mgL-1, the content of 6-BA is 0.1-0.3mgL-1, the pH of the Seedling Differentiation culture medium is 5.2-6.8.
The method as described above for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell, wherein in step
It is described that Kiwi berry young sprout is cleaned, is disinfected in one, it specifically includes:
Kiwi berry young sprout is first rinsed 1-2 hours with clear water, then blots twig surface moisture with filter paper, then in asepsis ring
30-60s is sterilized with the dehydrated alcohol of 70-75% in border, then with 0.15% mercury chloride disinfection 8-12min, then with sterile distillation
Water rinses 5 times, and the moisture on twig surface is finally blotted with the filter paper of disinfection.
The method as described above for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell, wherein in step
Anti-oxidation treatment fluid used in two is the PVP of 100-300ppm.
The method provided by the invention for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell has following excellent
Point:
1, using stem section perithelium is removed under aseptic condition, it can effectively reduce pollution rate.
2, two-steps tissue culture method is used in culture Induction Process, in first step incubation, is adding a small amount of sucrose
In MS culture medium, only plus the basic element of cell division, the cell activity in stem section in addition to cambial cell is promoted to reduce under hypotonic environment
Or it is dead, in second step incubation, the sucrose concentration of culture medium is adjusted to routine, stem section is cut to segment, while adding and swashing
Therbligs promotes cambial cell quickly to form callus, improves Callus induction rate, the genetic stability of offspring is effectively reduced.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is the flow chart for the method that the present invention cultivates Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell；
Fig. 2 is lignifying Stem Segments In Chinese Gooseberry transverse section figure in the embodiment of the present invention；
Fig. 3 is non-lignifying Stem Segments In Chinese Gooseberry transverse section figure in the embodiment of the present invention；
Fig. 4 is non-lignifying Stem Segments In Chinese Gooseberry transverse section figure in the embodiment of the present invention；
Fig. 5 is the stem cells group that non-lignifying Stem Segments In Chinese Gooseberry cambial cell is split into the embodiment of the present invention
Figure；
Fig. 6 is that the stem cells of non-lignifying Stem Segments In Chinese Gooseberry cambial cell division in the embodiment of the present invention are cliqued graph；
Fig. 7 is that the stem cells of non-lignifying Stem Segments In Chinese Gooseberry cambial cell division in the embodiment of the present invention are cliqued graph；
Fig. 8 is Kiwifruit Tissue Culture rooted plantlet figure in the embodiment of the present invention；
Fig. 9 is the stem cells group that non-lignifying Stem Segments In Chinese Gooseberry cambial cell is split into the embodiment of the present invention
Figure.
Specific embodiment
Presently filed embodiment is described in detail below in conjunction with examples and drawings, how the application is applied whereby
Technological means solves technical problem and reaches the realization process of technical effect to fully understand and implement.
Forming layer refers in the root and stem of higher plant, a kind of separate living tissue between xylem and bast, one
As be divided into vascular cambium and phellogen.By the viewpoint of modern plants biology, cambial cell is except apical meristem group
It knits including the another kind of plant stem cell outside shoot apical meristem stem cell and Root apical meristem stem cell.With callus
In cell compare, the cambial cell of plant has division, the strong and higher genetic stability of differentiation capability；And callus group
Cell is knitted after it experienced dedifferentiation, not only increases the heterogeneity of cell, while being relatively also easy to produce DNA methylation and transposons
Activation, to relatively be also easy to produce somaclonal variation in tissue culture process.Fig. 1 is that the present invention is dry thin using Stem Segments In Chinese Gooseberry forming layer
Born of the same parents cultivate the flow chart of the method for Kiwi berry seedling.As shown in Figure 1, provided by the invention dry thin using Stem Segments In Chinese Gooseberry forming layer
The method of born of the same parents' cultivation Kiwi berry seedling, comprising the following steps:
Step 1: select diameter for 0.2-0.4cm, and display has clearly xylem, bast under anatomical lens
Non- lignifying Kiwi berry young sprout first cleans Kiwi berry young sprout, is disinfected after removing blade, then by Kiwi berry young sprout
It is cut into the stem section that length is 2-3cm and is used as inoculation explant stem section.
Step 2: removing the perithelium and epidermis of inoculation explant stem section with tweezers, inoculation explant stem section both ends are respectively cut
After 0.1-0.2cm, first it is placed in anti-oxidation treatment fluid and handles 30-90 minutes, then be placed in forming layer stem cell induced medium
In in temperature be to be cultivated in 24 DEG C~26 DEG C of dark surrounds.
In the step, the forming layer stem cell induced medium is using MS as minimal medium, then by basic
Agar powder, sucrose and NAA or IBA is added in culture medium to be prepared；Agar powder in the forming layer stem cell induced medium
Content be 6-8gL-1, the content of sucrose is 2.5-3.0g.L-1, the content of NAA or IBA are 3-6mgL-1。
Step 3: the inoculation explant stem section after forming layer stem cell induced medium culture 25-30 days is cut again
Short, formation length is the segment of 0.8-1.5cm or the thin slice of 0.2-0.3cm, is transferred in cambial cell callus induction culture medium
It is cultivated 25-35 days in 1800-2200Lux, 25 ± 2 DEG C of environment, surface forms callus.
In the step, the cambial cell callus induction culture medium be using using MS as minimal medium, then by
Addition agar powder, sucrose, NAA, 6-BA, Kinetin or Zeatin are prepared in minimal medium；The cambial cell lures
The content for leading agar powder in callus culture medium is 6-8gL-1, the content of sucrose is 30-35g.L-1, the content of NAA is 3-
6mg·L-1, the content of 6-BA, Kintin or Zeatin are 0.2-0.5mgL-1, the cambial cell callus induction culture
The pH of base is 5.2-6.8.
Step 4:, when seedling length to height is 1.0cm, cutting seedling inoculation to training of taking root for the callus of seedling differentiation
It supports and carries out culture of rootage 35d in base in 1800-2200Lux, 25 ± 2 DEG C of environment.
In the step, the root media is using 1/2MS as minimal medium, then by adding in basic medium
Agar powder, sucrose, 6-BA and NAA or IBA is added to be prepared；The content of agar powder is 6-8gL in the root media-1,
The content of sucrose is 30-35g.L-1, the content of 6-BA is 0.05-0.1mgL-1, the content of NAA or IBA are 0.2-0.4mg
L-1, the pH of the root media is 5.2-6.8.
Step 5: for the callus of no Seedling Differentiation, by callus be transferred in Seedling Differentiation culture medium in 1800-2200Lux,
Subculture and differentiation culture are carried out in 25 ± 2 DEG C of environment, cultivate 25d；If callus Surface Differentiation is at seedling, repeatedly step 4,
Carry out rooting induction；If callus surface repeats this step 5 without Seedling Differentiation.
In the step, the Seedling Differentiation culture medium is using MS as minimal medium, then by basic medium
Addition agar powder, sucrose, NAA and 6-BA, Kenetin or Zeatin are prepared；Agar powder in the Seedling Differentiation culture medium
Content be 6-8gL-1, the content of sucrose is 30-35g.L-1, the content of NAA is 2-3mgL-1, 6-BA, kenetin or
The content of Zeatin is 0.1-0.3mgL-1, the pH of the Seedling Differentiation culture medium is 5.2-6.8.
The tissue culture method of Stem Segments In Chinese Gooseberry forming layer stem cell nursery provided in an embodiment of the present invention, first by tearing
Fall stem section perithelium, significantly improves reduction using Stem Segments In Chinese Gooseberry and carry out pollution rate in incubation, this has stem section surface
The material of villus and fine crack is especially effective；However cultivated using two-phase method, pass through only addition life in culture medium first
Long element reduces the division that forming layer stem cell is removed in stem section, promotes the death of other cells, reduces the vigor of other cells, however
Stem section is cut to thin slice, auxin is added and kinetin promotes the division of forming layer stem cell to form callus, and seedling differentiation.This
It is simple and easy to do to invent the technical solution provided, ginseng technology branch can be provided for Kiwi berry breeding seedling factory culturing technical system
Support.
Specific Application Example is presented below.In the present embodiment, cultivated using Stem Segments In Chinese Gooseberry forming layer stem cell
The method of Kiwi berry seedling is as follows:
(1) explant sterilizes: Kiwi berry non-lignifying young sprout of the acquisition stem section diameter in 0.2-0.4cm, which is characterized in that
The formation (Fig. 2, Fig. 3) of xylem, bast can be obviously observed in anatomical lens, after removing blade, taken back with moisture retention paper package
Do explant in laboratory；After tap water rinses 1-2 hours, surface moisture is blotted with filter paper, aseptically, uses 70-75%
Dehydrated alcohol sterilize 30-60s, then with 0.15% mercury chloride sterilize 8-12min, with sterile distilled water flushing 5 times, with disinfection
Filter paper blot surface moisture after, stem section is cut to the segment of length 2-3cm as inoculation explant；
In Fig. 2, lignifying Stem Segments In Chinese Gooseberry transverse section under A:1 times of anatomical lens；Lignifying Kiwi berry stem under B:2 times of anatomical lens
Section transverse section.In Fig. 3, lignifying Stem Segments In Chinese Gooseberry transverse section under A:2 times of anatomical lens；B；Lignifying Kiwi berry under 3 times of anatomical lens
Non- lignifying Stem Segments In Chinese Gooseberry transverse section under C:5 times of anatomical lens of stem section transverse section.
(2) before external body inoculation, the perithelium and epidermis (Fig. 4) of Kiwi berry tender stem segments are removed with tweezers, by stem section both ends
Each excision 0.1-0.2cm is placed on anti-oxidation treatment fluid (PVP that treatment fluid is 100-300ppm) processing 30-90 minutes, then
The stem section being disposed is placed in forming layer stem cell induced medium, induced medium adds 6- using MS as minimal medium
8g·L-1Agar powder, 2.5-3.0g.L-1Sucrose and 3-6mgL-1NAA or IBA is 24 DEG C~26 DEG C conditions in dark, temperature
Under cultivated；Culture 25-30 days, on stem section transverse section, can be observed stem cell formed cell mass, neutral red staining,
It can be observed to arrange close cell mass (Fig. 5)；After neutral red staining, it can be observed have small liquid in forming layer derived cell
Bubble, and callus derived cell irregular arrangement, without small vacuole, but big vacuole (Fig. 6)；
In Fig. 4, A: Kiwi berry lignifying stem section；B: the non-lignifying stem section of perithelium is removed；C: the Mi through anti-oxidation processing
The non-lignifying stem section of monkey peach.In Fig. 5, A: under 5 power microscopes, the cell mass of cambial cell formation；Under B:10 power microscope,
The cell mass formed through neutral red staining cambial cell；Under C:20 power microscope, formed through neutral red staining cambial cell
Cell mass.In Fig. 6, under A:40 power microscope, cambial cell forms the vacuole in cell；B: under 100 power microscopes, warp
Neutral red staining cambial cell forms the vacuole in cell；Under C:40 power microscope, through in neutral red staining healing cell
Vacuole.
(3) after stem section cultivates 25-30d under dark condition, stem section is prescinded again, formation length is 0.8-1.5cm's
The thin slice of segment or 0.2-0.3cm is transferred to the culture medium of cambial cell callus induction, cambial cell callus induction culture medium
With MS minimal medium, 6-8gL is added-1Agar powder, 30-35g.L-1Sucrose and 3-6mgL-1NAA and 0.2-0.5mgL- 16-BA, Kintin or Zeatin, pH 5.2-6.8 are cultivated at 1800-2200Lux, 25 ± 2 DEG C of temperature；
(4) it after cultivating 25-35d, can be formed a large amount of callus (Fig. 6) on stem section surface, subsequent callus surface can be divided into
Seedling (Fig. 7), when seedling it is long to height 1.0cm when, seedling inoculation can be cut with blade and taken root into root media, training of taking root
Base is supported using 1/2MS as minimal medium, adds 6-8gL-1Agar powder, 30-35g.L-1Sucrose and 0.2-0.4mgL-1NAA or
IBA and 0.05-0.1mgL-16-BA, kenetin or Zeatin, pH 5.2-6.8, in 1800-2200Lux, temperature 25 ± 2
Cultivated at DEG C, cultivate 35d, can 100% generate root system, 5~8 every plant, root long 1.0-3.5cm (Fig. 8)；
In Fig. 7, A: not wooden Stem Segments In Chinese Gooseberry forms callus, young shoot and seedling；The cause that the not wooden Stem Segments In Chinese Gooseberry of B is formed
Close callus and young shoot.In Fig. 8, A: Kiwi berry rooted plantlet；B tissue culture rooted plantlet transfer.
(5) without the callus of Seedling Differentiation, callus is transferred to Seedling Differentiation culture medium carries out subculture and differentiation is cultivated, subculture and
Differential medium adds 6-8gL with MS minimal medium-1Agar powder, 30-35g.L-1Sucrose and 2-3mgL-1NAA and
0.1-0.3mg·L-16-BA, pH 5.2-6.8 are cultivated at 1800-2200Lux, 25 ± 2 DEG C of temperature, after cultivating 25d, callus
Surface can be divided into seedling, and seedling repeats (4) and carries out rooting induction, this step of the repetition of no Seedling Differentiation (5).
In step (2) in the present embodiment, the perithelium of Stem Segments In Chinese Gooseberry, Kiwi berry stem are aseptically torn with tweezers
Section or villous, or have fine crack, it is very easy to absorption microorganism, often results in the pollution in Stem Segments In Chinese Gooseberry incubation,
It tears perithelium and is equivalent to and eliminate pollution sources；It in step (2), is handled, is effectively reduced after cultivating in incubation with PVP solution
The browning of stem section, while auximone is contained only in forming layer stem cell induced medium, kinetin is free of, so that in stem section
The stem cell in forming layer is only promoted to keep active, and the gradually dead or aging (Fig. 8) of other cells in stem section tissue, so that
Next step callus only induces stem cell to form callus when inducing；In step (3), the stem section of surface browning is cut to thin slice, into one
The division that step accelerates stem section forming layer stem cell forms callus.
In Fig. 9, A: under 20 power microscopes, the cell mass that cambial cell is formed, and other cell petrochemical industry, lose separation
Ability；Under B:40 power microscope, the cell mass formed through neutral red staining cambial cell, and petrochemical industry cell is not colored.
Example 1
(1) explant sterilizes: in August, 2018, and it is new to acquire red kiwi fruit semi-lignified of the stem section diameter in 0.15-0.2
The tip after removing blade, takes back laboratory with moisture retention paper package and does explant；After tap water rinses 1-2 hours, table is blotted with filter paper
Face moisture aseptically sterilizes 45s with 75% dehydrated alcohol, then sterilize 10min with 0.15% mercury chloride, with nothing
Bacterium distilled water flushing 5 times, after blotting surface moisture with the filter paper of disinfection, stem section is cut to the segment of length 2-3cm as inoculation
Explant；
(2) before external body inoculation, the perithelium of stem section is torn with tweezers, stem section both ends are respectively cut off into 0.1-0.2cm and are placed on
Anti-oxidation treatment fluid (PVP that treatment fluid is 100-300ppm) processing 50min minutes, is then placed in shape for the stem section being disposed
Layered stem cell induced medium, induced medium add 6-8gL using MS as minimal medium-1Agar powder, 30-35g.L-1
Sucrose and 5mgL-1NAA is cultivated under the conditions of dark, temperature is 24 DEG C~26 DEG C；
(3) after stem section cultivates 15-20d under dark condition, stem section is cut to 0.2- by the micro- browning in stem section surface at this time
The thin slice of 0.4cm, is transferred to the culture medium of cambial cell callus induction, and cambial cell callus induction culture medium is trained substantially with MS
Base is supported, 6-8gL is added-1Agar powder, 35g.L-1Sucrose and 3mgL-1NAA and 0.2mgL-16-BA, pH 5.2-6.8,
1800-2200Lux is cultivated at 25 ± 2 DEG C of temperature；
(4) after cultivating 25-35d, a large amount of callus can be formed in stem section sheet surface, subsequent callus surface can be divided into
Seedling, when seedling it is long to height 1.0cm when, seedling inoculation can be cut with blade and taken root into root media, root media
Using 1/2MS as minimal medium, 6-8gL is added-1Agar powder, 30-35g.L-1Sucrose and 0.4mgL-1IBA and 0.1mg
L-16-BA, pH 6.8 is cultivated at 1800-2200Lux, 25 ± 2 DEG C of temperature, cultivate 35d, can 100% generate root system, every plant 4
~7, root long 1.0-3.5cm；
(5) without the callus of Seedling Differentiation, callus is transferred to Seedling Differentiation culture medium carries out subculture and differentiation is cultivated, subculture and
Differential medium adds 6-8gL with MS minimal medium-1Agar powder, 30g.L-1Sucrose and 3mgL-1NAA and 0.1mg
L-16-BA, pH 6.8 is cultivated at 1800-2200Lux, 25 ± 2 DEG C of temperature, and after cultivating 25d, callus surface can be divided into children
Seedling, seedling repeat (4) and carry out rooting induction, this step of the repetition of no Seedling Differentiation (5).
Example 2
(1) explant sterilizes: in August, 2018, and acquisition stem section diameter is (long in the stock of the red kiwi fruit of 0.15-0.18
Fruit Kiwi berry, A.longicarpa) semi-lignified young sprout, after removing blade, laboratory is taken back with moisture retention paper package and does explant；
After tap water rinses 1-2 hours, surface moisture is blotted with filter paper, aseptically, sterilizes 30s with 75% dehydrated alcohol,
8min is sterilized with 0.15% mercury chloride again, is rinsed 5 times with sterile distilled water, it, will after blotting surface moisture with the filter paper of disinfection
Stem section is cut to the segment of length 2-3cm as inoculation explant；
(2) before external body inoculation, the perithelium of stem section is torn with tweezers, stem section both ends are respectively cut off into 0.1-0.2cm and are placed on
Anti-oxidation treatment fluid (PVP that treatment fluid is 100-300ppm) processing 50min minutes, is then placed in shape for the stem section being disposed
Layered stem cell induced medium, induced medium add 6-8gL using MS as minimal medium-1Agar powder, 30-35g.L-1
Sucrose and 4mgL-1NAA is cultivated under the conditions of dark, temperature is 24 DEG C~26 DEG C；
(3) after stem section cultivates 15-20d under dark condition, the browning of stem section surface, stem section is cut to a thickness of 0.2- at this time
The thin slice of 0.4cm, is transferred to the culture medium of cambial cell callus induction, and cambial cell callus induction culture medium is trained substantially with MS
Base is supported, 6-8gL is added-1Agar powder, 35g.L-1Sucrose and 4mgL-1NAA and 0.1mgL-16-BA, pH 5.2-6.8,
1800-2200Lux is cultivated at 25 ± 2 DEG C of temperature；
(4) after cultivating 25-35d, a large amount of callus can be formed in stem section sheet surface, subsequent callus surface can be divided into
Seedling, when seedling it is long to height 1.0cm when, seedling inoculation can be cut with blade and taken root into root media, root media
Using 1/2MS as minimal medium, 6-8gL is added-1Agar powder, 30-35g.L-1Sucrose and 0.3mgL-1IBA and 0.2mg
L-16-BA, pH 6.8 is cultivated at 1800-2200Lux, 25 ± 2 DEG C of temperature, cultivate 35d, can 100% generate root system, every plant 3
~5, root long 1.0-4.5cm；
(5) without the callus of Seedling Differentiation, callus is transferred to Seedling Differentiation culture medium carries out subculture and differentiation is cultivated, subculture and
Differential medium adds 6-8gL with MS minimal medium-1Agar powder, 30g.L-1Sucrose and 4mgL-1NAA and 0.2mg
L-16-BA, pH 6.8 is cultivated at 1800-2200Lux, 25 ± 2 DEG C of temperature, and after cultivating 25d, callus surface can be divided into children
Seedling, seedling repeat (4) and carry out rooting induction, this step of the repetition of no Seedling Differentiation (5).
Above description shows and describes several preferred embodiments of the present application, but as previously described, it should be understood that the application
Be not limited to forms disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations,
Modification, and can be in the application contemplated scope, modifications can be made through the above teachings or related fields of technology or knowledge.And this
The modifications and changes that field personnel are carried out do not depart from spirit and scope, then all should be in the application appended claims
Protection scope in.
Claims (7)
1. a kind of method for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell characterized by comprising
Step 1: selecting diameter for 0.2-0.4cm, and display has the not wood of clearly xylem, bast under anatomical lens
Matter Kiwi berry young sprout first cleans Kiwi berry young sprout, is disinfected, then Kiwi berry young sprout is cut into after removing blade
The stem section that length is 2-3cm is as inoculation explant stem section；
Step 2: removing the perithelium and epidermis of inoculation explant stem section with tweezers, inoculation explant stem section both ends are respectively cut off
After 0.1-0.2cm, first it is placed in anti-oxidation treatment fluid and handles 30-90 minutes, then be placed in forming layer stem cell induced medium
It is cultivated in the dark surrounds that temperature is 24 DEG C~26 DEG C；It is to cultivate substantially that the forming layer stem cell induced medium, which is with MS,
Then base is prepared by adding agar powder, sucrose and NAA or IBA in basic medium；
Step 3: the inoculation explant stem section after forming layer stem cell induced medium culture 25-30 days is prescinded again, shape
At length be 0.8-1.5cm segment or 0.2-0.3cm thin slice, be transferred in cambial cell callus induction culture medium in
It is cultivated 25-35 days in 1800-2200Lux, 25 ± 2 DEG C of environment, surface forms callus；The cambial cell callus induction training
Feeding base be using using MS as minimal medium, then by add in basic medium agar powder, sucrose, NAA, 6-BA,
Kinetin, Zeatin are prepared；
Step 4:, when seedling length to height is 1.0cm, cutting seedling inoculation to root media for the callus of seedling differentiation
In in 1800-2200Lux, 25 ± 2 DEG C of environment carry out culture of rootage 35d；It is basic that the root media, which is with 1/2MS,
Then culture medium is prepared by adding agar powder, sucrose, 6-BA and NAA or IBA in basic medium；
Step 5: for the callus of no Seedling Differentiation, by callus be transferred in Seedling Differentiation culture medium in 1800-2200Lux, 25 ±
Subculture and differentiation culture are carried out in 2 DEG C of environment, cultivate 25d；If callus Surface Differentiation is at seedling, repeatedly step 4, is carried out
Rooting induction；If callus surface repeats this step 5 without Seedling Differentiation；It is to train substantially that the Seedling Differentiation culture medium, which is with MS,
Base is supported, is then prepared by addition agar powder, sucrose, NAA, 6-BA, kenetin, Zeatin in basic medium.
2. the method for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell as described in claim 1, feature
It is, in step 2, the content of agar powder is 6-8gL in the forming layer stem cell induced medium-1, the content of sucrose
For 2.5-3.0g.L-1, the content of NAA or IBA are 3-6mgL-1。
3. the method for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell as described in claim 1, feature
It is, in step 3, the content of agar powder is 6-8gL in the cambial cell callus induction culture medium-1, sucrose contains
Amount is 30-35g.L-1, the content of NAA is 3-6mgL-1, the content of 6-BA, Kinetin or Zeatin are 0.2-0.5mgL-1, the pH of the cambial cell callus induction culture medium is 5.2-6.8.
4. the method for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell as described in claim 1, feature
It is, in step 4, the content of agar powder is 6-8gL in the root media-1, the content of sucrose is 30-35g.L-1, the content of 6-BA, Kinetin, Zeatin are 0.05-0.1mgL-1, the content of NAA or IBA are 0.2-0.4mgL-1, institute
The pH for stating root media is 5.2-6.8.
5. the method for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell as described in claim 1, feature
It is, in step 5, the content of agar powder is 6-8gL in the Seedling Differentiation culture medium-1, the content of sucrose is 30-
35g.L-1, the content of NAA is 2-3mgL-1, the content of 6-BA, Kenetin, Zeatin are 0.1-0.3mgL-1, the children
The pH of seedling differential medium is 5.2-6.8.
6. the method for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell as described in claim 1, feature
It is, it is described that Kiwi berry young sprout is cleaned, is disinfected in step 1, it specifically includes:
Kiwi berry young sprout is first rinsed 1-2 hours with clear water, then blots twig surface moisture with filter paper, then in gnotobasis
30-60s is sterilized with the dehydrated alcohol of 70-75%, then sterilizes 8-12min with 0.15% mercury chloride, then rushed with sterile distilled water
It washes 5 times, the moisture on twig surface is finally blotted with the filter paper of disinfection.
7. the side as claimed in any one of claims 1 to 6 for cultivating Kiwi berry seedling using Stem Segments In Chinese Gooseberry forming layer stem cell
Method, which is characterized in that the anti-oxidation treatment fluid used in step 2 is the PVP of 100-300ppm.
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| CN108271691A (en) * | 2018-01-29 | 2018-07-13 | 宝鸡松良农业科技有限公司 | A method of carrying out tissue cultures using Kiwi berry stem |
| CN109006473A (en) * | 2018-07-10 | 2018-12-18 | 陕西青美生物科技有限公司 | A kind of Kiwifruit Tissue Culture fast propagating culture medium and method |
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| CN108271691A (en) * | 2018-01-29 | 2018-07-13 | 宝鸡松良农业科技有限公司 | A method of carrying out tissue cultures using Kiwi berry stem |
| CN109006473A (en) * | 2018-07-10 | 2018-12-18 | 陕西青美生物科技有限公司 | A kind of Kiwifruit Tissue Culture fast propagating culture medium and method |
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