CN108935106A - A kind of tissue culture propagation and in-vitro conservation method of critical endangered plant Guizhou great Hua cuckoo - Google Patents
A kind of tissue culture propagation and in-vitro conservation method of critical endangered plant Guizhou great Hua cuckoo Download PDFInfo
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- CN108935106A CN108935106A CN201811007683.8A CN201811007683A CN108935106A CN 108935106 A CN108935106 A CN 108935106A CN 201811007683 A CN201811007683 A CN 201811007683A CN 108935106 A CN108935106 A CN 108935106A
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- vitro
- culture
- cuckoo
- guizhou
- plantlet
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- 238000000338 in vitro Methods 0.000 title claims abstract description 30
- 241000544061 Cuculus canorus Species 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 15
- 230000001939 inductive effect Effects 0.000 claims abstract description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 27
- 229920001817 Agar Polymers 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 238000005286 illumination Methods 0.000 claims description 12
- 229960004793 sucrose Drugs 0.000 claims description 12
- 239000005720 sucrose Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 3
- 235000013681 dietary sucrose Nutrition 0.000 claims description 3
- 230000000284 resting effect Effects 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 2
- 239000006160 differential media Substances 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 239000002352 surface water Substances 0.000 claims description 2
- 238000012549 training Methods 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 8
- 241000894007 species Species 0.000 abstract description 8
- 241000208421 Ericaceae Species 0.000 abstract description 2
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 241001529246 Platymiscium Species 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 238000010899 nucleation Methods 0.000 abstract 1
- 230000004083 survival effect Effects 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 3
- 241000208422 Rhododendron Species 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 241001573881 Corolla Species 0.000 description 1
- 241000272177 Cuculiformes Species 0.000 description 1
- 241001086202 Rhododendron fortunei Species 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 238000009418 renovation Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Classifications
-
- 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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses the tissue culture propagation and in-vitro conservation method of a kind of critical endangered plant Guizhou great Hua cuckoo, this method is suitable for the tissue-culturing rapid propagation and Plantlet in vitro of Ericaceae Guizhou great Hua cuckoo, platymiscium field of biotechnology.The present invention to bloom after the new stem segments taken out slightly as explant; pass through explant disinfection, inducing clumping bud, strong sprout, Plantlet in vitro, series of steps of taking root; it efficiently solves because plant amount is few caused by Guizhou great Hua cuckoo distributed areas are narrow, introduction and acclimatization is difficult; adjoin status in imminent danger, achievees the purpose that species conservation.Tissue culture and rapid propagation method provided by the invention, growth coefficient is 8-10 in 1 month, rooting rate is 95%, transplanting survival rate is 95%-98%, greatly improve the breeding coefficient of Guizhou great Hua cuckoo, Plantlet in vitro provides technical support up to 240d or more, for introduction and acclimatization, Plantlet in vitro, seeding propagation the recurrence plantation of the species.
Description
Technical field
The invention belongs to field of biotechnology, more particularly to a kind of critical endangered plant Guizhou great Hua cuckoo tissue culture propagation
And in-vitro conservation method.
Background technique
Guizhou great Hua cuckoo (Rhododendronmagniflorum) it is Ericaceae Subgenus Hymenanthess Rhododendron fortuneilindl.
Subgroup species, the species are published in 1985, are the cuckoos in Rhododendron with maximum flower.In the cuckoo of newest publication
Critically endangered species are classified as in flower Red List " The Red List of Rhododendron ".This kind is distributed in Guizhou Province
Anlong County faucet mountain, single flower corolla diameter reach 10cm or so, ornamental value with higher.In newest investigation,
This kind is only found a population at present, and only deposits 3 plants.Because of population rareness, natural renovation is poor, is badly in need of carrying out effective tissue culture propagation
And Plantlet in vitro, avoid species from moving towards extinction.It is had not been reported at present for the tissue culture propagation of this kind and in-vitro conservation method.
Summary of the invention
For Guizhou great Hua cuckoo population rareness, field plant this phenomenon in imminent danger, the present invention is intended to provide a kind of Guizhou
Great Hua cuckoo tissue culture propagation and in-vitro conservation method save germ plasm resource, protect endangered species.
1. a kind of tissue culture propagation and in-vitro conservation method of Guizhou great Hua cuckoo, it is characterised in that:
1) explant select: take the new stem segments of Guizhou great Hua cuckoo, with tap water rinse 12h after it is spare；
2) explant sterilizes: taking the explant got ready on sterile super-clean bench, with 75% alcohol disinfecting 30s, uses aseptic water washing
3-5 times, 8-9min is sterilized with 0.1% mercury chloride, is used aseptic water washing 3-5 times；
3) inducing clumping bud: clump bud inducement cultivation base is improvement WPM+2.5mg/L 2-iP(2- isopentennyladenine)+0.8mg/
L IBA, sucrose 28g/L, agar 7g/L, pH5.6.Improveing WPM culture medium is that ammonium nitrate improves most 700mg/L, other elements
Dosage remains unchanged.The stem section that will be obtained after explant sterilization process blots surface water with aseptic filter paper on the super-clean bench
Point, stem section is cut with sterile knife, one resting bud of every section of band is seeded in inducing clumping bud culture medium, carries out after dark culture 5 days
It illumination cultivation 30 days or more, cultivates 35-50 days, the brightness time is 12h/12h, intensity of illumination 1500-2000LUX；
4) strong seedling culture: strong seedling culture base is improvement WPM+0.5mg/L 2-iP+0.1mg/L IBA, sucrose 28g/L, agar
7g/L, pH5.6；The sprouting that will be induced is seeded in strong seedling culture base, by bottle seedling be in the brightness time be 12h/12h, illumination
Intensity is 1500-2000LUX；
5) Plantlet in vitro: Plantlet in vitro culture medium is improvement WPM+0.5mg/L IBA+6mg/L CCC, pH5.6, sucrose 28g/
L, agar 6-7g/L, temperature are 13-18 DEG C, luminous intensity 1500LUX-1600LUX, and the brightness time is 10h/14h, and culture medium is
The filling 65ml culture medium of 260ml culture bottle；Restoration ecosystem differential medium is improvement WPM+1mg/L 6-BA+0.05mg/L
IAA, brightness time are 12h/12h, intensity of illumination 1500-2000LUX；
6) culture of rootage: will grow to 1.5cm ~ 2cm high sprout and be forwarded to culture medium is the training for improveing WPM+1mg/L IBA
It supports in base, additional saccharose 28g/L, agar 7g/L, pH5.6；Luminous intensity is 2000LUX-2500 LUX, and the brightness time is 12h/
12h；
In addition to Plantlet in vitro culture medium needs filling 65ml using 260ml culture bottle, remaining each culture medium uses 260ml culture bottle
Filling 45ml culture medium；Cultivation temperature is 13-18 DEG C except Plantlet in vitro, remaining is 20-23 DEG C.
Compared with prior art, the beneficial effects of the present invention are:
1. innovating and realizing the tissue culture propagation and in-vitro conservation method of critical endangered plant Guizhou great Hua cuckoo；
2. Guizhou great Hua cuckoo growth coefficient within half a year is 60-90 times by tissue culture expanding propagation, Guizhou great Hua is greatly improved
The breeding coefficient of cuckoo protects endangered species, provides a large amount of seedling to return plantation.And Plantlet in vitro in the invention
It can be reserved for 240d or more in culture medium not transfer, greatly extend switching and holding time.
Case is embodied:
Essence in order to better illustrate the present invention further illustrates the present invention, but this hair with the embodiment of the present invention below
Bright content is not limited thereto.The description of technical solution and embodiment according to the present invention, perhaps same domain technical staff is at this
On the basis of invention some modification and improvement can also be carried out to technical solution of the present invention.Therefore, without departing from of the invention main
The modification and improvement done on the basis of technical solution, should belong to scope of the present invention.
The tissue culture and Plantlet in vitro step of 1 Guizhou great Hua cuckoo of embodiment:
Step (1): acquisition explant simultaneously sterilizes: the new hair stem section that Post flowering has just been grown is acquired from the great Hua cuckoo maternal plant of Guizhou,
3-5 resting bud of band, takes explant after tap water rinses 12h, on superclean bench, with 75% alcohol disinfecting 30s, uses
Aseptic water washing 3-5 times, 8-9min is sterilized with 0.1% mercury chloride, is used aseptic water washing 3-5 times；
Step (2): inducing clumping bud culture: being cut into segment for the explant after disinfection on superclean bench, and every section one, band
Axillary bud, oblique cutting enter induced medium improvement WPM+2.5mg/L 2-iP+0.8mg/L IBA, sucrose 28g/L, agar 7g/L,
It in pH5.6, carries out illumination cultivation 30 days, cultivates 35-50 days after dark culture 5 days, the brightness time is 12h/12h, and intensity of illumination is
1500 LUX -2000LUX；
Step (3): when differentiation and it is long to 1-2cm when, be forwarded to strong seedling culture base improvement WPM+0.5mg/L 2-iP+
0.1mg/L IBA, sucrose 28g/L, in agar 7g/L, pH5.6；Bottle seedling is in the brightness time as 12h/12h, intensity of illumination is
1500 LUX -2000LUX；
Step (4): a part of crowd shoots are forwarded in Plantlet in vitro culture medium: Plantlet in vitro culture medium for improvement WPM+
0.5mg/L IBA+6mg/L CCC, pH5.6, sucrose 28g/L, agar 6-7g/L, temperature is 13-18 DEG C, and luminous intensity is
1500LUX-1600LUX, brightness time are 10h/14h, the filling 65ml of every bottle of the culture medium；When needing restoration ecosystem, by it
Be forwarded to restoration ecosystem culture medium: improvement WPM+1mg/L 6-BA+0.05mg/L IAA, brightness time are 12h/12h, and illumination is strong
Degree is 1500-2000LUX, 20-23 DEG C of temperature, carries out restoration ecosystem；
Step (5): when the seedling in strong seedling culture base and restoration ecosystem culture medium grows to 1.5cm ~ 2cm high in bottle, by it
It is forwarded to root media improvement WPM+1mg/L IBA to take root, additional saccharose 28g/L, agar 7g/L, pH5.6, brightness
Time is 12h/12h, and luminous intensity is 2000LUX-2500 LUX, and temperature is 20-23 DEG C, and conventional transplanting is carried out after taking root.
The Screening of Media of 2 Guizhou great Hua cuckoo tissue culture propagation of embodiment is tested:
1) basal medium screening experiment.Culture medium Read, WPM, 1/4MS, the improvement WPM for selecting cuckoo to be often used are compared
Experiment survive without hormone stem section and tests, every group of 10 bottles of experiment inoculation, and 1 section of explant stem section after every bottle of inoculation disinfection, into
Row compares:
The result shows that: this kind growth conditions on the culture medium of improvement WPM are good, and growing way is more vigorous, do not occur dead and not
Phenomenon is sprouted, therefore choosing minimal medium is improvement WPM, the tissue culture step of Guizhou great Hua cuckoo is identical as example 1.
2) Plantlet in vitro Screening of Media is tested.Design temperature is two gradients: 13-18 DEG C of low temperature and 20-23 DEG C of room temperature, if
Meter culture medium filling amount is two gradient of 45ml and 65ml, and every group of experiment is inoculated with 10 bottles, the relatively uniform clump of 3 clumps of sizes of every bottle of inoculation
It sprouts, carries out orthogonal experiment screening:
The result shows that: this kind Plantlet in vitro growth conditions at 13-18 DEG C of cryogenic conditions are good, and culture medium need to have enough amounts
65ml, which just can guarantee, extends subculture switching cycle.
Claims (2)
1. a kind of tissue culture propagation and in-vitro conservation method of critical endangered plant Guizhou great Hua cuckoo, it is characterised in that:
1) explant select: take the new stem segments of Guizhou great Hua cuckoo, with tap water rinse 12h after it is spare；
2) explant sterilizes: taking the explant got ready on sterile super-clean bench, with 75% alcohol disinfecting 30s, uses aseptic water washing
3-5 times, 8-9min is sterilized with 0.1% mercury chloride, is used aseptic water washing 3-5 times；
3) inducing clumping bud: clump bud inducement cultivation base is improvement WPM+2.5mg/L 2-iP(2- isopentennyladenine)+0.8mg/
L IBA, sucrose 28g/L, agar 7g/L, pH5.6；Improveing WPM culture medium is that ammonium nitrate improves most 700mg/L, other elements
Dosage remains unchanged, the stem section that will be obtained after explant sterilization process, on the super-clean bench, blots surface water with aseptic filter paper
Point, stem section is cut with sterile knife, one resting bud of every section of band is seeded in inducing clumping bud culture medium, carries out after dark culture 5 days
It illumination cultivation 30 days or more, cultivates 35-50 days, the brightness time is 12h/12h, intensity of illumination 1500-2000LUX；
4) strong seedling culture: strong seedling culture base is improvement WPM+0.5mg/L 2-iP+0.1mg/L IBA, sucrose 28g/L, agar
7g/L, pH5.6；The sprouting that will be induced is seeded in strong seedling culture base, by bottle seedling be in the brightness time be 12h/12h, illumination
Intensity is 1500-2000LUX；
5) Plantlet in vitro: Plantlet in vitro culture medium is improvement WPM+0.5mg/L IBA+6mg/L CCC, pH5.6, sucrose 28g/
L, agar 6-7g/L, temperature are 13-18 DEG C, luminous intensity 1500LUX-1600LUX, and the brightness time is 10h/14h；Restoration ecosystem
Differential medium is improvement WPM+1mg/L 6-BA+0.05mg/L IAA, and the brightness time is 12h/12h, intensity of illumination 1500-
2000LUX；
6) culture of rootage: will grow to 1.5cm ~ 2cm high sprout and be forwarded to culture medium is the training for improveing WPM+1mg/L IBA
It supports in base, additional saccharose 28g/L, agar 7g/L, pH5.6；Luminous intensity is 2000LUX-2500 LUX, and the brightness time is 12h/
12h。
2. special according to the tissue culture propagation and in-vitro conservation method of a kind of critical endangered plant Guizhou great Hua cuckoo described in right 1
Sign is that in addition to Plantlet in vitro culture medium, each culture medium uses the filling 45ml culture medium of 260ml culture bottle；Plantlet in vitro culture
Base is the filling 65ml culture medium of 260ml culture bottle, and cultivation temperature is 13-18 DEG C except Plantlet in vitro, remaining is 20-23 DEG C.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811007683.8A CN108935106B (en) | 2018-08-31 | 2018-08-31 | Tissue culture propagation and in-vitro preservation method for extremely endangered plant Rhododendron Guizhou |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811007683.8A CN108935106B (en) | 2018-08-31 | 2018-08-31 | Tissue culture propagation and in-vitro preservation method for extremely endangered plant Rhododendron Guizhou |
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| Publication Number | Publication Date |
|---|---|
| CN108935106A true CN108935106A (en) | 2018-12-07 |
| CN108935106B CN108935106B (en) | 2021-12-21 |
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| CN201811007683.8A Expired - Fee Related CN108935106B (en) | 2018-08-31 | 2018-08-31 | Tissue culture propagation and in-vitro preservation method for extremely endangered plant Rhododendron Guizhou |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115250908A (en) * | 2022-04-06 | 2022-11-01 | 贵州民族大学人文科技学院 | Tissue culture propagation method of extremely endangered plant rhododendron lychee |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103651115A (en) * | 2013-11-05 | 2014-03-26 | 云南远益园林工程有限公司 | Rapid propagation method of rhododendron azalea tissue culture |
| CN104285819A (en) * | 2014-11-04 | 2015-01-21 | 中国科学院昆明植物研究所 | Tissue culture propagation method for rhododendron hancockii |
-
2018
- 2018-08-31 CN CN201811007683.8A patent/CN108935106B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103651115A (en) * | 2013-11-05 | 2014-03-26 | 云南远益园林工程有限公司 | Rapid propagation method of rhododendron azalea tissue culture |
| CN104285819A (en) * | 2014-11-04 | 2015-01-21 | 中国科学院昆明植物研究所 | Tissue culture propagation method for rhododendron hancockii |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115250908A (en) * | 2022-04-06 | 2022-11-01 | 贵州民族大学人文科技学院 | Tissue culture propagation method of extremely endangered plant rhododendron lychee |
| CN115250908B (en) * | 2022-04-06 | 2023-09-08 | 贵州民族大学人文科技学院 | Tissue culture propagation method of extremely endangered plant azalea |
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