CN110291989A - A kind of method for building up in the sterile leaf source for gingkgo callus induction - Google Patents
A kind of method for building up in the sterile leaf source for gingkgo callus induction Download PDFInfo
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- CN110291989A CN110291989A CN201910692288.6A CN201910692288A CN110291989A CN 110291989 A CN110291989 A CN 110291989A CN 201910692288 A CN201910692288 A CN 201910692288A CN 110291989 A CN110291989 A CN 110291989A
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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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Abstract
The invention discloses a kind of method for building up in sterile leaf source for gingkgo callus induction, belong to field of plant tissue culture technique.The present invention is using ginkgo edible tender branch or mature embryo as material, it is cultivated 40 days on bud inducement cultivation base or embryonal induction culture medium, obtain seedling or embryo seedling, it cuts the aseptic blade for obtaining seedling and chamfers its former stem section base portion, or cut off the root of embryo seedling, continue squamous subculture 40 days, after cutting the aseptic blade for obtaining seedling or embryo seedling again, by the sterile tender tip of the attenuating of defoliation, it is cut into stem section of the 2-3cm with bud, continue culture 40 days on subculture medium again, it can be induced later by stem section bud, blade extraction, harvest blade, stem section continues to cultivate, it is repeated, up to the sterile leaf source for gingkgo callus induction, it is big with yield, it is high-efficient, it is not subject to seasonal restrictions, continually and steadily, continually the advantages of, gained aseptic blade can be directly used for the induction of callus, with very strong application value.
Description
Technical field
The present invention relates to field of plant tissue culture technique, and in particular to a kind of for the sterile of gingkgo callus induction
The method for building up of Ye Yuan.
Background technique
Ginkgo (Ginkgo biloba L.) is China's endemic tree, containing in 2 kinds of important flavonoids and terpene in leaf
Ester type compound has the function of anti-oxidant, treatment cardiovascular disease, thus has important medical value.Gingko yellow at present
Ketone and terpene lactones compound are mainly extracted from ginkgo leaf, but content is low, and as the growth of the ginkgo age of tree reduces year by year, raw
It needs to transplant a large amount of ginkgo young growth in production to produce ginkgo leaf, occupies a large amount of farmland.The Folium Ginkgo of field planting includes
Object content height is affected by external environment, but the variation of artificial environment more difficult to control.In recent years the silver having gradually developed
Apricot callus and cell culture technology bring new production for the production and metabolic regulation of GINKGO BILOBA EXTRACT and lactone compound
And research mode.Gingkgo callus induction can be sent out by Organ procurements such as embryo, cotyledon, stem section, roots, but through a large amount of research
Existing, blade is the best explant of evoked callus.The method of current callus induction is will to arise directly to grow directly from seeds
Inoculated and cultured comes to harm since vanes disinfectant is directly handled after the blade disinfection of seedling, and most of browning is dead after inoculation
It dies, even if the callus growth ability induced after surviving is poor, subculture will browning death after several times.In addition it to establish extensive
With the cell culture system of industrialization, endlessly blade supply is needed, but is influenced by season, the blade in only spring can
For the leaf source of callus induction.
Summary of the invention
Goal of the invention: in view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide one kind to be used for ginkgo
The method for building up in the sterile leaf source of callus induction can sustainedly and stably, largely provide the sterile leaf that can be used for callus induction
Piece is not subject to seasonal restrictions.
Technical solution: to solve the above-mentioned problems, the technical solution adopted in the present invention is as follows:
A kind of method for building up in the sterile leaf source for gingkgo callus induction, includes the following steps:
1) ginkgo edible tender branch is taken, blade, or mature gingko seeds is removed, peels off sclerotin mesosperm, it is impregnated,
Cleaning and disinfection；
2) ginkgo edible tender branch is cut into 2-5cm and has the stem section of 1-3 bud, be seeded on bud inducement cultivation base and cultivate 40
It, obtains seedling；Mature embryo is taken out from the endosperm of gingko seeds, is seeded on embryonal induction culture medium and cultivates 40 days, obtain embryo
Seedling；
3) its former stem section base portion beveling is continued to train on subculture medium by the big blade for cutting seedling as sterile leaf
It supports 40 days；The radicle of embryo seedling is wiped out but not leaf-cutting, continues culture 40 days on subculture medium；
4) the big blade of seedling is cut again as sterile leaf, and the sterile tender tip of the attenuating of defoliation is removed into former stem section,
It is cut into stem section of the 2-3cm with bud, then continues culture 40 days on subculture medium, obtains seedling again；Cut what embryo seedling was grown
Big blade, and by the sterile tender tip of the attenuating of defoliation, it is cut into stem section of the 2-3cm with bud, then continue to cultivate on subculture medium
40 days, obtain seedling；
5) to seedling obtained in step 4) be iteratively repeated step 3) and 4) in seedling operation to get for ginkgo callus
Organize the sterile leaf source of induction.
Preferably, the method for ginkgo edible tender branch is taken described in step 1) are as follows: in late March~the first tenday period of a month in May, take ginkgo
The edible tender branch of seedling current year life, or the edible tender branch that adult ginkgo is female, staminiferous plant current year is raw.
Preferably, the method for ginkgo edible tender branch is taken described in step 1) are as follows: to the blade of edible tender branch, carry out flavones
With the measurement of lactone content, material of the branch as initial inoculation of high-content is screened.
Preferably, the specific steps of immersion described in step 1), cleaning and disinfection are as follows: edible tender branch is peelled off in sclerotin
The gingko seeds of kind of skin wash away surface smut with detergent, peel off sclerotin mesosperm gingko seeds will clear water impregnate for 24 hours, with
It by edible tender branch or peels off the gingko seeds of sclerotin mesosperm afterwards and is rinsed in flowing water again, successively impregnated with 70% ethyl alcohol, it is sterile
Water cleaning, in 0.3% KMnO4It is impregnated in solution, aseptic water washing, until branch is without KMnO4Color.
Preferably, the selection standard of mature embryo described in step 2) are as follows: from storage to December current year~April next year
Mature gingko seeds and size is in 5mm or more.
Preferably, bud inducement cultivation base described in step 2) or embryonal induction culture medium include: MS+0.1mg/L NAA+
0.5mg/L 6-BA or MS+0.1mg/L NAA+0.5mg/L 6-BA+0.5% active carbon.
Preferably, step 3) or 4) described in subculture medium include: MS+0.1mg/L NAA+0.5mg/L 6-BA or MS
+ 0.1mg/L NAA+0.5mg/L 6-BA+0.5% active carbon.
Preferably, bud inducement cultivation base described in step 2) or embryonal induction culture medium or step 3) or 4) described in subculture
Culture medium, further includes: 3.0% sucrose, 0.65% agar, pH=5.8.
Preferably, step 3) or 4) described in big blade, refer to leaf area up to 1.5~2.0cm2Above blade.
Preferably, step 2), step 3), the condition of culture in step 4) are as follows: culture room temperature is 25 ± 2 DEG C, when illumination
Between 14h/d, 55 μm of olm of intensity of illumination-2·s-1。
The utility model has the advantages that compared with the prior art, advantages of the present invention are as follows:
(1) provided by the present invention for the method for building up in the sterile leaf source of gingkgo callus induction, technical operation is simple,
Easy, culture can obtain 1.5~2.0cm in 40 days or so for the first time2Above a large amount of blades, average 5-7 piece/stem section, stem section
For blade inductivity up to 98% or more, yield is big, high-efficient；
(2) provided by the present invention for the method for building up in the sterile leaf source of gingkgo callus induction, the sterile leaf of harvest
Piece does not need to sterilize, and can be directly used for the induction of callus, because not influenced by disinfectant, generated callus is living
Power is strong, can long-term subculture；
(3) it provided by the present invention for the method for building up in the sterile leaf source of gingkgo callus induction, is not subject to seasonal restrictions,
It goes the stem section of blade that can continue Fiber differentiation and obtains aseptic blade, continually and steadily, continually.It can be used to produce blade throughout the year,
To utilize callus and cell culture technology to produce flavones and lactone compound on a large scale and being improved by metabolic regulation
It provides condition containing quantifier elimination, has very important practical significance and stronger application value.
Detailed description of the invention
Fig. 1 is that ginkgo is female, 40 days aseptic blade figures of staminiferous plant stem section Fiber differentiation；Left figure: terminal bud, right figure: axillary bud；
Fig. 2 is that ginkgo is female, the sterile leaf source establishment process figure of staminiferous plant stem section induction；Left figure: harvest, middle figure: subculture, it is right
Figure: callus induction；
Fig. 3 is the induced map of 3~5 years raw seedling tender tips and blade；Left figure: right figure: tender tip induction grows the leaf of 40d
Piece；
Fig. 4 is the growth of embryo seedling and the leaf growth situation map of mature embryonal induction 80d.
Specific embodiment
The present invention is further described below combined with specific embodiments below.
Embodiment 1:
A kind of method for building up in the sterile leaf source for gingkgo callus induction, comprising the following steps:
(1) early April~the first tenday period of a month in May, female, staminiferous plant current year life the edible tender branch of clip adult, to the branch to be inoculated with
Blade carries out the measurement of flavones and lactone content, screens material of the branch as initial inoculation of high-content.Blade is removed, it will be young
Shoot washes away surface smut with detergent, rinses 30min in flowing water, then sequentially impregnate 30s, sterile water wash with 70% ethyl alcohol
3~4 times, in 0.3% KMnO4Immersion 25min or so in solution, aseptic water washing 4~5 times, until branch is without KMnO4Color.
(2) on the super-clean bench by edible tender branch (non-lignifying) be cut into 2-5cm with 1-3 leaf stem section, be seeded in MS+
On the bud inducement cultivation base of 0.1mg/LNAA+0.5mg/L 6-BA+0.5% active carbon, cultivates 40 days, obtain seedling, inductivity
Up to 98% or more.Contain 3.0% sucrose, 0.65% agar, pH5.8 or so in culture medium.Cultivating room temperature is 25 ± 2 DEG C, illumination
Time 14h/d, 55 μm of olm of intensity of illumination-2·s-1(Fig. 1).
(3) by newly slightly on big blade cut that (leaf area is up to 1.5~2.0cm2More than), each stem section averagely produces leaf 5~7
Piece.The ginkgo original stem section base portion of defoliation is chamfer, subculture is in MS+0.1mg/L NAA+0.5mg/L 6-BA+0.5% activity again
On charcoal culture medium, continues culture 40 days, newly slightly continue to extend, top young leaves is grown again, can collect blade (Fig. 2) again.
Condition of culture is same as above.
(4) the sterile tender tip of attenuating of defoliation (original stem section lignifying or browning, removal) is cut into stem of the 2-3cm with bud
Section, subculture is cultivated on MS+0.1mg/L NAA+0.5mg/L 6-BA+0.5% active carbon culture medium again, culture 40 days, newly
Stem section can continue to extract 5-7 piece young leaves out, harvest blade, and condition of culture is same as above.Can be extracted out later by stem section bud induction → blade →
Harvest blade → stem section continues to cultivate, and is repeated, and forms the sterile leaf source for gingkgo callus induction.
Embodiment 2:
A kind of method for building up in the sterile leaf source for gingkgo callus induction, comprising the following steps:
(1) late March~late April, the edible tender branch of raw ginkgo seedling current year life in clip 3~5 years, to what is be inoculated with
The blade of branch carries out the measurement of flavones and lactone content, screens material of the branch as initial inoculation of high-content.Defoliation
Edible tender branch is washed away surface smut with detergent by piece, rinses 30min in flowing water, then sequentially impregnate 30s, nothing with 70% ethyl alcohol
Bacterium water cleans 3~4 times, in 0.3% KMnO4Impregnate 25min or so in solution, aseptic water washing 4~5 times, until branch without
KMnO4Color.
(2) on the super-clean bench by edible tender branch be cut into 2-5cm with 1-3 leaf stem section, be seeded in MS+0.1mg/L NAA+
It in the induced medium of the axillary bud (or terminal bud) of 0.5mg/L 6-BA, cultivates 40 days, obtains seedling, inductivity is up to 98% or more.
Contain 3.0% sucrose, 0.65% agar, pH5.8 or so in culture medium.Cultivating room temperature is 25 ± 2 DEG C, light application time 14h/d, light
According to 55 μm of olm of intensity-2·s-1。
(3) by extraction it is new slightly on big blade cut that (leaf area reaches 1.5cm2More than), each stem section averagely produces leaf 4~5
Piece.The ginkgo original stem section base portion of defoliation is chamfer, subculture is in MS+0.1mg/LNAA+0.5mg/L 6-BA+0.5% activity again
Charcoal continues culture 40 days, newly slightly continues to extend, young leaves is grown again at top, can collect blade (Fig. 3) again.Cultivate item
Part is same as above.
(4) the sterile tender tip of attenuating of defoliation is cut into stem section of the 2-3cm with bud, subculture is in MS+0.1mg/L NAA again
It cultivates, cultivates 40 days on+0.5mg/L 6-BA+0.5% active carbon culture medium, new stem section can continue to extract 5-7 piece young leaves, harvest out
Blade, condition of culture are same as above.Blade → stem section can be harvested by stem section bud induction → blade is extracted out → later to continue to cultivate, repeatedly into
Row forms the sterile leaf source for gingkgo callus induction.
Embodiment 3:
A kind of method for building up in the sterile leaf source for gingkgo callus induction, comprising the following steps:
(1) early December~the first tenday period of a month in May take storage 4 months or more Ginkgo Biloba Nuts, peel off sclerotin mesosperm, and embryo is used
Detergent washes away surface smut, impregnates in water for 24 hours, successively with 70% ethyl alcohol impregnate 2min, sterile water wash 3~4 times,
0.3% KMnO4Immersion 25min or so in solution, aseptic water washing 4~5 times, until the surface of the seed is without KMnO4Color.
(2) on the super-clean bench, embryo is taken out under aseptic condition, is seeded in the embryo of MS+0.1mg/LNAA+0.5mg/L6-BA
In induced medium, to cultivate 40 days, mature embryo grows up to embryo seedling, 2~3cm of height of seedling, and 2~3, blade.Contain 3.0% in the culture medium
Sucrose, 0.65% agar, pH5.8 or so.Cultivating room temperature is 25 ± 2 DEG C, light application time 14h/d, 55 μm of ol of intensity of illumination
m-2·s-1。
(3) radicle for the seedling for growing 40 days is removed, it directly will be with base of leaf section subculture in MS+0.1mg/L NAA+
0.5mg/L 6-BA+0.5% active carbon continues culture 40 days, newly slightly will continue to extend, and ending blade is grown up, and 4~5 every,
Area reaches 1.5cm2More than, blade (Fig. 4) can be harvested.Condition of culture is same as above
(4) by the sterile tender tip of defoliation, it is cut into stem section of the 2-3cm with bud, subculture is in MS+0.1mg/L NAA+ again
It cultivates, cultivates 40 days on 0.5mg/L 6-BA+0.5% active carbon culture medium, new stem section can continue to extract 5-7 piece young leaves, harvest out
Blade, condition of culture are same as above.Blade → stem section can be harvested by stem section bud induction → blade is extracted out → later to continue to cultivate, repeatedly into
Row forms the sterile leaf source for gingkgo callus induction.
Claims (10)
1. a kind of method for building up in the sterile leaf source for gingkgo callus induction, which comprises the steps of:
1) ginkgo edible tender branch is taken, blade, or mature gingko seeds is removed, peels off sclerotin mesosperm, it is impregnated, is cleaned
And disinfection；
2) ginkgo edible tender branch is cut into 2-5cm and has the stem section of 1-3 bud, be seeded on bud inducement cultivation base and cultivate 40 days,
Obtain seedling；Mature embryo is taken out from the endosperm of gingko seeds, is seeded on embryonal induction culture medium and cultivates 40 days, obtain embryo seedling；
3) its former stem section base portion beveling is continued culture 40 as sterile leaf by the big blade for cutting seedling on subculture medium
It；The radicle of embryo seedling is wiped out but not leaf-cutting, continues culture 40 days on subculture medium；
4) the big blade of seedling is cut again as sterile leaf, and the sterile tender tip of the attenuating of defoliation is removed into former stem section, is cut into
Stem section of the 2-3cm with bud, then continue culture 40 days on subculture medium, seedling is obtained again；Cut the great Ye that embryo seedling is grown
Piece, and by the sterile tender tip of the attenuating of defoliation, it is cut into stem section of the 2-3cm with bud, then continue culture 40 days on subculture medium,
Obtain seedling；
5) to seedling obtained in step 4) be iteratively repeated step 3) and 4) in seedling operation to get for gingkgo callus
The sterile leaf source of induction.
2. the method for building up in the sterile leaf source according to claim 1 for gingkgo callus induction, which is characterized in that
The method of ginkgo edible tender branch is taken described in step 1) are as follows: in late March~the first tenday period of a month in May, take ginkgo seedling current year life
Edible tender branch, or the edible tender branch that adult ginkgo is female, staminiferous plant current year is raw.
3. the method for building up in the sterile leaf source according to claim 2 for gingkgo callus induction, which is characterized in that
The method of ginkgo edible tender branch is taken described in step 1) are as follows: to the blade of edible tender branch, carry out the survey of flavones and lactone content
It is fixed, screen material of the branch as initial inoculation of high-content.
4. the method for building up in the sterile leaf source according to claim 1 for gingkgo callus induction, which is characterized in that
The specific steps of immersion described in step 1), cleaning and disinfection are as follows: edible tender branch or the gingko seeds for peelling off sclerotin mesosperm
Wash away surface smut with detergent, the gingko seeds for peelling off sclerotin mesosperm will impregnate for 24 hours in clear water, then by edible tender branch or
The gingko seeds for peelling off sclerotin mesosperm rinse in flowing water again, are successively impregnated with 70% ethyl alcohol, sterile water wash, 0.3%
KMnO4It is impregnated in solution, aseptic water washing, until branch is without KMnO4Color.
5. the method for building up in the sterile leaf source according to claim 1 for gingkgo callus induction, which is characterized in that
The selection standard of mature embryo described in step 2) are as follows: from storage to mature gingko seeds in December current year~April next year simultaneously
And size is in 5mm or more.
6. the method for building up in the sterile leaf source according to claim 1 for gingkgo callus induction, which is characterized in that
Bud inducement cultivation base described in step 2) or embryonal induction culture medium include: MS+0.1mg/L NAA+0.5mg/L 6-BA or MS+
0.1mg/L NAA+0.5mg/L 6-BA+0.5% active carbon.
7. the method for building up in the sterile leaf source according to claim 1 for gingkgo callus induction, which is characterized in that
Step 3) or 4) described in subculture medium include: MS+0.1mg/L NAA+0.5mg/L 6-BA or MS+0.1mg/L NAA+
0.5mg/L 6-BA+0.5% active carbon.
8. the method for building up in the sterile leaf source according to claim 1 for gingkgo callus induction, which is characterized in that
Bud inducement cultivation base described in step 2) or embryonal induction culture medium or step 3) or 4) described in subculture medium, further includes:
3.0% sucrose, 0.65% agar, pH=5.8.
9. the method for building up in the sterile leaf source according to claim 1 for gingkgo callus induction, which is characterized in that
Step 3) or 4) described in big blade, refer to leaf area up to 1.5~2.0cm2Above blade.
10. a kind of method for building up in sterile leaf source for gingkgo callus induction according to claim 1, feature
It is: step 2), step 3), the condition of culture in step 4) are as follows: culture room temperature is 25 ± 2 DEG C, light application time 14h/d, light
According to 55 μm of olm of intensity-2·s-1。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111557244A (en) * | 2020-06-08 | 2020-08-21 | 青岛皇盾生物科技有限公司 | Method for inducing somatic embryos of gingkoes and regenerating plants |
| CN114375839A (en) * | 2022-01-30 | 2022-04-22 | 山东大丰园农业有限公司 | Ginkgo regeneration method based on leaf induction |
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| CN111557244B (en) * | 2020-06-08 | 2022-03-22 | 张宪青 | Method for inducing somatic embryos of gingkoes and regenerating plants |
| CN114375839A (en) * | 2022-01-30 | 2022-04-22 | 山东大丰园农业有限公司 | Ginkgo regeneration method based on leaf induction |
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| CN110291989B (en) | 2022-03-22 |
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