CN108925429A - A kind of method for building up of endangered species Poncirus polyandra vitro Regeneration System - Google Patents
A kind of method for building up of endangered species Poncirus polyandra vitro Regeneration System Download PDFInfo
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- CN108925429A CN108925429A CN201810972514.1A CN201810972514A CN108925429A CN 108925429 A CN108925429 A CN 108925429A CN 201810972514 A CN201810972514 A CN 201810972514A CN 108925429 A CN108925429 A CN 108925429A
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- axillary bud
- poncirus polyandra
- illumination
- poncirus
- polyandra
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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
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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
Abstract
The present invention provides a kind of method for building up of endangered species Poncirus polyandra vitro Regeneration System, belongs to plant regeneration research field.It is explant material by choosing Poncirus polyandra current year raw tender stem, after disinfection treatment, it is inoculated in axillary bud Primary culture and induces axillary bud sprouting growth, when axillary bud germinating branch it is long to 2-3cm when, it is inoculated in proliferated culture medium after cutting, obtains Multiplying culture seedling, the height of shoot proliferation culture is cut in 3-4cm unrooted seedling single plant, transfer the root induction culture 15-20d in root media, obtains the complete tissue-cultured seedling of germinating adventitious root.Effectively solve the problems, such as Poncirus polyandra fast-propagation; the rescue of endangered species Poncirus polyandra can preferably be carried out; recurrence plantation also is carried out for artificial quick cultivation Poncirus polyandra nursery stock simultaneously; it rebuilds Poncirus polyandra Wild population and technical support is provided; therefore, it is of great significance to Poncirus polyandra Rescued Protection and Wild population reconstruction.
Description
Technical field
The invention belongs to building for plant regeneration research field more particularly to a kind of endangered species Poncirus polyandra vitro Regeneration System
Cube method.
Background technique
Poncirus polyandra (Ponciruspolyandra) belongs to Rutaceae Poncirus, originates in for China, is that D prime qin is equal to 1984
In a novel species of Yunnan Province Fumin County discovery.It is characterized by evergreen dungarunga, grow up the high about 2.5m of tree body, and young sprout is in prismatic,
Green, old branch rounding, axil the short spine of a bud one.3~April of florescence, fruit maturity period are 10~November, more common trifoliate orange fruit
Greatly, yellow, oblate spherical shape, fruit face is coarse, and base portion is shallowly hollow, there is radiation rill.Only there are two kinds to belong to Chinese spy for Rutaceae Poncirus
There are kind, i.e. trifoliate orange and Poncirus polyandra.Trifoliate orange middle part native to China；Poncirus polyandra is only distributed in Yunnan Province Fumin County, is that precious citrus is educated
Kind germ plasm resource.
Poncirus polyandra distributed areas are narrow, are only distributed in the old green hill periphery in Yunnan Province Fumin County, and 2100 meters of lower altitude limit, height above sea level
It 2400 meters of the upper limit, is born in valley tailo, in stone rock Shrubs of Mountainous Areas, belongs to the distinctive Poncirus tree species in Yunnan Province, arranged in 1989
Enter " first Provincial Key of Yunnan Province protects wild plant register ".It is recorded according to related literatures, in last century the eighties,
There is a large amount of Poncirus polyandra wild resource to be distributed in the mountain valley of the old green hill east side ridge Dong Gua.But Kunming Inst. of Botany, Chinese Academy of Sciences in
When development Poncirus polyandra resource investigation in 1992,15 plants of Wild plants are only found in its source area.As ecological environment is continuous
Deteriorate and build the engineering constructions such as road, Poncirus polyandra resource is sharply reduced, and is included in " Key Protected in 1999
Plant list (first) ".2012, it is rich that Kunming endangered animal and plant detention and help center combines development with Fumin County forestry bureau
The investigation of people's trifoliate orange wild resource, only finds 1 plant of Wild plant in its source area.Therefore, in addition to 22 plants of in situ conservation, Poncirus polyandra
Wild resource becomes extinct substantially, and it is extremely urgent to carry out its Rescued Protection.
In terms of seedling propagating technology, pertinent literature rarely seen Yang Man and Zhang Min, " 6~BA, NAA plant hormone are to Poncirus polyandra
The influence of tissue cultures " cuts stem apex and the stem section with lateral bud with culture medium evoked callus, with various concentration 6~
BA and NAA carries out differentiation culture to Poncirus polyandra stem section and forms callus, (forest inventory and planning [J], 2007,32 (6): 149~
151).Currently, the vitro Regeneration System of Poncirus polyandra is not yet established, cause Poncirus polyandra seedling that can not carry out fast-propagation.
Summary of the invention
In order to solve the problems of prior art, the one kind in this that is designed to provide can effectively solve the problem that the present invention
The method for building up of the endangered species Poncirus polyandra vitro Regeneration System of the problem of Poncirus polyandra fast-propagation.This method includes following step
It is rapid:
Step 1: explant chooses and disinfection: it chooses Poncirus polyandra tender shoots and is cut into the stem section containing axil after being rinsed,
It carries out disinfection to stem section；
Step 2: axillary bud Primary culture: the stem section oblique cutting in step 1 being cultivated on axillary bud Primary culture base, is obtained
Germinate axillary bud；
Step 3: Multiplying culture: the axillary bud obtained through step 2 is inserted on proliferated culture medium, and axillary bud base portion forms a large amount of clumps
It sprouts, Multiple Buds is subjected to cutting separation, be inoculated in identical proliferated culture medium culture, obtain Multiplying culture seedling；
Step 4: culture of rootage: the increment that step 3 is obtained, which grows seedlings, cuts, and is inoculated on root media and is trained
It supports, obtains Poncirus polyandra rooted seedling.
Further, the step 1 comprises the concrete steps that: choosing Poncirus polyandra tree body spring germinating tender stem, removes blade；It goes
Except surface impurity, geramine water purification is added to rinse 0.5h or more, is cut into the stem section that 2~3cm long contains 1~2 axil, is placed in ultra-clean
Workbench impregnates 10~12s using 75% ethyl alcohol and carries out disinfection, and sterile distilled water rinses 2 times, moves into 0.1% mercuric chloride immersion treatment
10min aseptic water washing 3~5 times, is inoculated with after blotting explant remained on surface moisture with sterilized filter paper.
Further, the step 2 comprises the concrete steps that: stem section both ends, petiole in step 1, spine cut 2 respectively~
4mm, for oblique cutting on Primary culture base, nearly top axil is upward, is placed in 1000~2000Lux of intensity of illumination, and illumination 12 hours/
It, 10~15d of illumination cultivation under conditions of 25 ± 2 DEG C of daytime temperature, 20 ± 2 DEG C of night temperature obtains germinating axillary bud.
Further, the step 3 comprises the concrete steps that: the axillary bud obtained through step 2 being cut along base portion, plucks bottom
1~2 blade, inserts on proliferated culture medium vertically, is placed in 1000~2000Lux of intensity of illumination, 12 hour/day of illumination, daytime temperature
15~20d of illumination cultivation under conditions of 25 ± 2 DEG C, 20 ± 2 DEG C of night temperature, axillary bud base portion form a large amount of Multiple Buds, and Multiple Buds are pressed 2
~3 plants carry out cutting separation for one group, are inoculated in identical proliferated culture medium, continue to cultivate 30d under the same conditions, are proliferated
It grows seedlings.
Further, the step 3 comprises the concrete steps that: the axillary bud obtained through step 2 being cut along base portion, plucks bottom
1~2 blade, inserts on proliferated culture medium vertically, is placed in 1000~2000Lux of intensity of illumination, 12 hour/day of illumination, daytime temperature
15~20d of illumination cultivation under conditions of 25 ± 2 DEG C, 20 ± 2 DEG C of night temperature, axillary bud base portion form a large amount of Multiple Buds, and Multiple Buds are pressed 2
~3 plants carry out cutting separation for one group, are inoculated in identical proliferated culture medium, continue to cultivate 30d under the same conditions, are proliferated
It grows seedlings.
Further, the step 4 comprises the concrete steps that: by the germinating axillary bud in step 2 through the Multiplying culture in step 3
Height 3~4cm unrooted seedling single plant of acquisition is cut, and is inoculated on root media, be placed in intensity of illumination 1000~
2000Lux, 12 hour/day of illumination, 15~20d of illumination cultivation under conditions of 25 ± 2 DEG C of daytime temperature, 20 ± 2 DEG C of night temperature are enriched people
Trifoliate orange rooted seedling.
Further, the axillary bud Primary culture base in the step 2 is MS+6- benzyl aminoadenine 2.0mg/L+ methyl α-naphthyl acetate
0.5mg/L+ sucrose 30g/L+ agar 4.5g/L.
Further, the proliferated culture medium in the step 3 is WPM+6- benzyl aminoadenine 1.0~2.0mg/L+ indoles
Butyric acid 1.0mg/L+ sucrose 30g/L+ agar 4.5g/L+ active carbon 0.1g/L.
Further, the root media in the step 4 is WPM+6- benzyl aminoadenine 1.0mg/L+ indolebutyric acid
1.0mg/L+ sucrose 20~30g/L+ agar 4.0~4.5g/L+ active carbon 0.15~0.2g/L or WPM+6- benzyl aminoadenine
1.0mg/L+ indolebutyric acid 1.0mg/L+ malt extract 1.5mg/L+ sucrose 20~30g/L+ agar 4.0~4.5g/L+ activity
0.15~0.2g/L of charcoal.
The beneficial effects of the present invention are: this method establishes endangered species Poncirus polyandra as explant using stem segment with axillary bud for the first time
Vitro Regeneration System realizes Poncirus polyandra fast-propagation, effectively carries out Rescued Protection and reconstruction Wild population etc. and establishes
Basis.The present invention realizes richness using stem segment with axillary bud as explant, by processes such as axillary bud starting, Multiplying culture, culture of rootage
The tissue-culturing quick-propagation of people's trifoliate orange；And this method is easy to operate, explant dosage is few, and proliferation times are high, and root system is sturdy, nursery stock
It is healthy and strong.
Specific embodiment
Technical solution claimed in order to better illustrate the present invention herein, herein with the following Examples to this hair
It is bright to be described further.
Embodiment one
The method that one of embodiment endangered species Poncirus polyandra vitro Regeneration System is established, specifically includes following step
It is rapid:
Step 1: explant choose and disinfection: choose Poncirus polyandra tree body spring germinate tender stem, remove blade, retain petiole and
Spine, to protect axillary bud during cleaning is with disinfection treatment from mechanical damage.Surface impurity is removed, geramine water purification is added
0.5h or more is rinsed, the stem section that 2~3cm long contains 1~2 axil is cut into, superclean bench is placed in using 75% ethyl alcohol and impregnates 10
~12s carries out disinfection, sterile distilled water rinse 2 times, move into 0.1% mercuric chloride immersion treatment 10min, aseptic water washing 3~5 times,
It is inoculated with after blotting explant remained on surface moisture with sterilized filter paper；
Step 2: the resulting stem section both ends of step 1, petiole, spine axillary bud Primary culture: being cut into 2~4mm, oblique cutting respectively
In on Primary culture base, nearly top axil is upward, is placed in 1000~2000Lux of intensity of illumination, 12 hour/day of illumination, daytime temperature 25
10~15d of illumination cultivation under conditions of ± 2 DEG C, 20 ± 2 DEG C of night temperature obtains germinating axillary bud；
Step 3: Multiplying culture: making axillary bud production to certain altitude after step 2 is cultivated the resulting stem section of step 1
Afterwards, 2~3cm of gained axillary bud is cut along base portion, is plucked the blade of bottom 1~2, is inserted on proliferated culture medium vertically, be placed in illumination
1000~2000Lux of intensity, 12 hour/day of illumination, illumination cultivation 15 under conditions of 25 ± 2 DEG C of daytime temperature, 20 ± 2 DEG C of night temperature~
20d, axillary bud base portion form a large amount of Multiple Buds, and Multiple Buds are carried out cutting separation by 2~3 plants for one group, are inoculated in identical proliferation
Culture medium continues to cultivate 30d under the same conditions, obtains Multiplying culture seedling；
Step 4: culture of rootage: 3~4cm of height that the germinating axillary bud in step 2 is obtained through the Multiplying culture in step 3
Unrooted seedling single plant is cut, and is inoculated on root media, is placed in 1000~2000Lux of intensity of illumination, 12 hour/day of illumination,
15~20d of illumination cultivation under conditions of 25 ± 2 DEG C of daytime temperature, 20 ± 2 DEG C of night temperature, obtains Poncirus polyandra rooted seedling.
Wherein, axillary bud Primary culture base is MS+6- benzyl aminoadenine 2.0mg/L+ methyl α-naphthyl acetate 0.5mg/L+ sucrose 30g/L
+ agar 4.5g/L, pH value are 5.8~6.2；Proliferated culture medium is WPM+6- benzyl aminoadenine 1.0~2.0mg/L+ indoles fourth
Sour 1.0mg/L+ sucrose 30g/L+ agar 4.5g/L+ active carbon 0.1g/L, pH value are 5.8~6.2；Root media is WPM+6-
Benzyl aminoadenine 1.0mg/L+IBA 1.0mg/L+ sucrose 20~30g/L+, 4.0~4.5g/L+ of agar active carbon 0.15~
0.2g/L, pH value are 5.8~6.2.
The method rooting rate is up to 73.1%.
Embodiment two
Step 1: explant choose and disinfection: choose Poncirus polyandra tree body spring germinate tender stem, remove blade, retain petiole and
Spine, to protect axillary bud during cleaning is with disinfection treatment from mechanical damage.Surface impurity is removed, geramine water purification is added
0.5h or more is rinsed, the stem section that 2~3cm long contains 1~2 axil is cut into, superclean bench is placed in using 75% ethyl alcohol and impregnates 10
~12s carries out disinfection, sterile distilled water rinse 2 times, move into 0.1% mercuric chloride immersion treatment 10min, aseptic water washing 3~5 times,
It is inoculated with after blotting explant remained on surface moisture with sterilized filter paper；
Step 2: the resulting stem section both ends of step 1, petiole, spine axillary bud Primary culture: being cut into 2~4mm, oblique cutting respectively
In on Primary culture base, nearly top axil is upward, is placed in 1000~2000Lux of intensity of illumination, 12 hour/day of illumination, daytime temperature 25
10~15d of illumination cultivation under conditions of ± 2 DEG C, 20 ± 2 DEG C of night temperature obtains germinating axillary bud；
Step 3: Multiplying culture: making axillary bud production to certain altitude after step 2 is cultivated the resulting stem section of step 1
Afterwards, 2~3cm of gained axillary bud is cut along base portion, is plucked the blade of bottom 1~2, is inserted on proliferated culture medium vertically, be placed in illumination
1000~2000Lux of intensity, 12 hour/day of illumination, illumination cultivation 15 under conditions of 25 ± 2 DEG C of daytime temperature, 20 ± 2 DEG C of night temperature~
20d, axillary bud base portion form a large amount of Multiple Buds, and Multiple Buds are carried out cutting separation by 2~3 plants for one group, are inoculated in identical proliferation
Culture medium continues to cultivate 30d under the same conditions, obtains Multiplying culture seedling；
Step 4: culture of rootage: 3~4cm of height that the germinating axillary bud in step 2 is obtained through the Multiplying culture in step 3
Unrooted seedling single plant is cut, and is inoculated on root media, is placed in 1000~2000Lux of intensity of illumination, 12 hour/day of illumination,
8~10d of illumination cultivation under conditions of 25 ± 2 DEG C of daytime temperature, 20 ± 2 DEG C of night temperature, obtains Poncirus polyandra rooted seedling.
Wherein, axillary bud Primary culture base is MS+6- benzyl aminoadenine 2.0mg/L+ methyl α-naphthyl acetate 0.5mg/L+ sucrose 30g/L
+ agar 4.5g/L, pH value are 5.8~6.2；Proliferated culture medium is WPM+6- benzyl aminoadenine 1.0~2.0mg/L+ indoles fourth
Sour 1.0mg/L+ sucrose 30g/L+ agar 4.5g/L+ active carbon 0.1g/L, pH value are 5.8~6.2；Root culture medium is WPM+6- benzyl
Aminoadenine 1.0mg/L+ indolebutyric acid 1.0mg/L+ malt extract 1.5mg/L+ 20~30g/L+ of sucrose agar 4.0~
4.5g/L+ 0.15~0.2g/L of active carbon, pH value are 5.8~6.2.
Comparative example one: as in the first embodiment, the root induction culture medium of step 4 is only replaced with WPM+6- benzyl aminoadenine
1.0mg/L+ indolebutyric acid 1.0mg/L+ malt extract 1.5mg/L+ sucrose 20~30g/L+ agar 4.0~4.5g/L+ activity
0.15~0.2g/L of charcoal, pH value are 5.8~6.2, and the root induction time foreshortens to 8~10d, and rooting rate reaches 62.5%.
Claims (9)
1. a kind of method for building up of endangered species Poncirus polyandra vitro Regeneration System, it is characterised in that: method includes the following steps:
Step 1: explant chooses and disinfection: choosing Poncirus polyandra tender shoots and is cut into the stem section containing axil after being rinsed, to stem
Section carries out disinfection；
Step 2: axillary bud Primary culture: the stem section oblique cutting in step 1 being cultivated on axillary bud Primary culture base, is germinated
Axillary bud；
Step 3: Multiplying culture: the axillary bud obtained through step 2 is inserted on proliferated culture medium, and axillary bud base portion, which is formed, largely grows thickly
Multiple Buds are carried out cutting separation by bud, are inoculated in identical proliferated culture medium culture, are obtained Multiplying culture seedling；
Step 4: culture of rootage: the increment that step 3 is obtained, which grows seedlings, cuts, and is inoculated on root media and is cultivated,
Obtain Poncirus polyandra rooted seedling.
2. the method for building up of endangered species Poncirus polyandra vitro Regeneration System according to claim 1, it is characterised in that: described
Step 1 comprises the concrete steps that: choosing Poncirus polyandra tree body spring and germinates tender stem, removes blade；Surface impurity is removed, geramine is added to use
0.5h or more is rinsed in water purification, is cut into the stem section that 2~3cm long contains 1~2 axil, is placed in superclean bench and is soaked using 75% ethyl alcohol
10~12s of bubble carries out disinfection, and sterile distilled water rinses 2 times, moves into 0.1% mercuric chloride immersion treatment 10min, aseptic water washing 3~5
It is secondary, it is inoculated with after blotting explant remained on surface moisture with sterilized filter paper.
3. the method for building up of endangered species Poncirus polyandra vitro Regeneration System according to claim 1 or 2, it is characterised in that:
The step 2 comprises the concrete steps that: the stem section both ends in step 1, petiole, spine cut 2~4mm respectively, and oblique cutting is trained in starting
It supports on base, nearly top axil is upward, it is placed in 1000~2000Lux of intensity of illumination, 12 hour/day of illumination, 25 ± 2 DEG C of daytime temperature, night
10~15d of illumination cultivation under conditions of 20 ± 2 DEG C of temperature obtains germinating axillary bud.
4. the method for building up of endangered species Poncirus polyandra vitro Regeneration System according to claim 1 or 2, it is characterised in that:
The step 3 comprises the concrete steps that: the axillary bud obtained through step 2 cut along base portion, plucks the blade of bottom 1~2, it is vertical to insert
In on proliferated culture medium, being placed in 1000~2000Lux of intensity of illumination, 12 hour/day of illumination, 25 ± 2 DEG C of daytime temperature, night temperature 20 ± 2
15~20d of illumination cultivation under conditions of DEG C, axillary bud base portion form a large amount of Multiple Buds, and Multiple Buds are cut by 2~3 plants for one group
Separation is cut, identical proliferated culture medium is inoculated in, continues to cultivate 30d under the same conditions, obtains Multiplying culture seedling.
5. the method for building up of endangered species Poncirus polyandra vitro Regeneration System according to claim 3, it is characterised in that: described
Step 3 comprises the concrete steps that: the axillary bud obtained through step 2 being cut along base portion, the blade of bottom 1~2 is plucked, inserts in increasing vertically
It grows on culture medium, is placed in 1000~2000Lux of intensity of illumination, 12 hour/day of illumination, 25 ± 2 DEG C of daytime temperature, 20 ± 2 DEG C of night temperature
Under the conditions of 15~20d of illumination cultivation, axillary bud base portion forms a large amount of Multiple Buds, and Multiple Buds are carried out cutting point by 2~3 plants for one group
From, be inoculated in identical proliferated culture medium, continue under the same conditions cultivate 30d, obtain Multiplying culture seedling.
6. the method for building up of endangered species Poncirus polyandra vitro Regeneration System described according to claim 1 or 2 or 3 or 4 or 5,
Be characterized in that: the step 4 comprises the concrete steps that: the height that the germinating axillary bud in step 2 is obtained through the Multiplying culture in step 3
Degree 3~4cm unrooted seedling single plant is cut, and is inoculated on root media, is placed in 1000~2000Lux of intensity of illumination, illumination 12
Hour/day, 15~20d of illumination cultivation under conditions of 25 ± 2 DEG C of daytime temperature, 20 ± 2 DEG C of night temperature, obtains Poncirus polyandra rooted seedling.
7. the method for building up of endangered species Poncirus polyandra vitro Regeneration System according to claim 1 or 2 or 3 or 4 or 5 or 6,
It is characterized by: the axillary bud Primary culture base in the step 2 is MS+6- benzyl aminoadenine 2.0mg/L+ methyl α-naphthyl acetate 0.5mg/
L+ sucrose 30g/L+ agar 4.5g/L.
8. the foundation side of endangered species Poncirus polyandra vitro Regeneration System described according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7
Method, it is characterised in that: the proliferated culture medium in the step 3 is WPM+6- benzyl aminoadenine 1.0~2.0mg/L+ indoles fourth
Sour 1.0mg/L+ sucrose 30g/L+ agar 4.5g/L+ active carbon 0.1g/L.
9. endangered species Poncirus polyandra vitro Regeneration System builds described according to claim 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8
Cube method, it is characterised in that: the root media in the step 4 is WPM+6- benzyl aminoadenine 1.0mg/L+ indolebutyric acid
1.0mg/L+ sucrose 20~30g/L+ agar 4.0~4.5g/L+ active carbon 0.15~0.2g/L or WPM+6- benzyl aminoadenine
1.0mg/L+ indolebutyric acid 1.0mg/L+ malt extract 1.5mg/L+ sucrose 20~30g/L+ agar 4.0~4.5g/L+ activity
0.15~0.2g/L of charcoal.
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| CN106818476A (en) * | 2017-01-19 | 2017-06-13 | 西南林业大学 | The efficient quick propagation method of natural Triploid Lacquer Tree |
| CN107347644A (en) * | 2017-08-04 | 2017-11-17 | 上海市农业科学院 | A kind of method that Regeneration in Vitro is carried out using great Hua Abelia bifloras blade |
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