CN106857265A - A kind of tissue culture enrichment procedure of white Cowhells wood - Google Patents
A kind of tissue culture enrichment procedure of white Cowhells wood Download PDFInfo
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- CN106857265A CN106857265A CN201710241937.1A CN201710241937A CN106857265A CN 106857265 A CN106857265 A CN 106857265A CN 201710241937 A CN201710241937 A CN 201710241937A CN 106857265 A CN106857265 A CN 106857265A
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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
- A01H4/001—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
- A01H4/008—Methods for regeneration to complete plants
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of tissue culture enrichment procedure of white Cowhells wood, specific steps include selection, the sterilizing of explant, aseptic strain foundation and the shoot proliferation of explant；The selection of explant is the fair weather to May in March, and clip ox tendon strip gives birth to the tender stem segmentses or stem apex of nutrition branch then；The sterilizing of explant is to cut off the blade in tender stem, stem section or stem apex are cleaned with liquid detergent, rinsed well with running water again, stem section or stem apex are put into superclean bench, with 75% alcohol-pickled 30s, it is with sterilized distilled water that alcohol rinse is clean again, 20 30min are soaked in the mercuric chloride solution for placing into 0.1%, then with sterile water wash 4 times.The present invention uses tissue cultures enrichment procedure, is high-volume nursery, reduces seedling cost, improves breeding efficiency and lays the foundation.
Description
Technical field
The present invention relates to a kind of enrichment procedure of trees, specifically a kind of tissue culture enrichment procedure of white Cowhells wood.
Background technology
Ox tendon strip belongs to rose family Dichotomanthes on plant classification, originates in an a kind of only and mutation in Yunnan.Cloud
Southern Shanxi Academy of Agricultural Sciences garden crop research institute combines the reality that Yunnan Shan Gaopo is steep, Winter-Spring is arid, gathers materials on the spot, and is summarizing group
On the basis of many experiences, ox tendon strip plant is studied.According to preliminary observation, ox tendon strip grafting apple and pears all have
Tree body and the ahead of time positive effect of result are downgraded, can be multiplex with an anvil, it is the dwarfing of growing directly from seeds of an apple for getting a good chance of and pears
Anvil.But ox tendon strip is evergreen shrubs, the four seasons are evergreen, and main root is flourishing, buries very deep, and fibrous root is few, there is resistance to lean one side of anti-morning, has again
Intolerant to the one side transplanted, be colonized, general grafting is few due to fibrous root, and seedling-slowing stage is long after field planting of transplanting seedlings, influence growth.
The propagation method of white Cowhells wood is mainly seed breeding and cutting propagation.The seedling of seed breeding is because main root is sent out
Reach, depth of burying, fibrous root is few, be again evergreen tree, so transplanting survival rate is low；Cutting propagation, it is extraneous with certain except requiring
Humidity, temperature, air and the spontaneous and external world possess outside sufficient nutriment, in addition it is also necessary to some micro sp act materials,
Complex operation, because ox tendon strip cuttage root-taking is difficult, seedling cost is high, and efficiency is low, have impact on its promotion rate.
The content of the invention
It is an object of the invention to provide a kind of tissue culture propagation for improving breeding efficiency, the white Cowhells wood of reduction seedling cost
Method, to solve the problems, such as to be proposed in above-mentioned background technology.
To achieve the above object, the present invention provides following technical scheme：
A kind of tissue culture enrichment procedure of white Cowhells wood, comprises the following steps that：
（1）The selection of explant：In the fair weather in March to May, clip ox tendon strip gives birth to the tender stem segmentses or stem of nutrition branch then
Point；
（2）The sterilizing of explant：The blade in tender stem is cut off, stem section or stem apex is cleaned with liquid detergent, then rinse dry with running water
Only, stem section or stem apex are put into superclean bench, with 75% alcohol-pickled 30s, then with sterilized distilled water by alcohol rinse
Totally, 20-30min is soaked in the mercuric chloride solution for placing into 0.1%, then with sterile water wash 4 times；
（3）Aseptic strain is set up：The minimal medium that the foundation of aseptic strain is used is MS culture mediums, the hormone for using be 6BA and
NAA, culture medium is fitted into wide-mouth bottle, per it is bottled enter culture medium 30-40ML, autoclave sterilization is carried out after sealing, after sterilizing
Cooling is stand-by；In superclean bench, the stem section or stem apex that will disinfect are cut into the stem section of a length of 2cm, the stem that then will be sheared
In MS culture mediums after section insertion sterilizing, every bottle of culture medium insertion stem section 5-8 puts it into culturing room and is trained after sealing
Support；
（4）Shoot proliferation：Ox tendon strip after through culture after a while, set up, and then carries out shoot proliferation culture by aseptic strain；
Foster used medium of being commissioned to train is MS+1-2mg6BA+0.05-0.5%NAA, and every bottle of culture medium is 50ML, accesses the stem section 3 of aseptic strain
Individual, culturing room is put into after sealing carries out squamous subculture, culturing room is carried out disinfection weekly necessarily, is changed per 20-25d and once cultivated
Base, by the squamous subculture of a cycle, ox tendon strip growth coefficient reaches 13-15.
As further scheme of the invention：The step（1）In stem section or stem apex mass percentage concentration be 75%
Alcohol disinfecting 30 seconds, then sterilized 25min with mercuric chloride that mass percentage concentration is 0.1%.
As further scheme of the invention：The step（3）And step（4）In culturing room's condition be：Culturing room
Temperature control at 25-27 DEG C, daily illumination 12h, intensity of illumination is 1000-1200Lx.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention use tissue cultures enrichment procedure, dialogue Cowhells wood dwarfing rootstock produced in enormous quantities, reduce nursery into
This, improves breeding efficiency.
Specific embodiment
The technical scheme of this patent is described in more detail with reference to specific embodiment.
Embodiment 1
A kind of tissue culture enrichment procedure of white Cowhells wood, comprises the following steps that：
（1）The selection of explant：In the fair weather in March to May, clip ox tendon strip gives birth to the tender stem segmentses or stem of nutrition branch then
Point；
（2）The sterilizing of explant：The blade in tender stem is cut off, stem section or stem apex is cleaned with liquid detergent, then rinse dry with running water
Only, stem section or stem apex are put into superclean bench, with 75% alcohol-pickled 30s, then with sterilized distilled water by alcohol rinse
Totally, 20min is soaked in the mercuric chloride solution for placing into 0.1%, then with sterile water wash 4 times；
（3）Aseptic strain is set up：The minimal medium that the foundation of aseptic strain is used is MS culture mediums, the hormone for using be 6BA and
NAA, culture medium is fitted into wide-mouth bottle, per it is bottled enter culture medium 30mL, autoclave sterilization is carried out after sealing, after sterilizing cool down
It is stand-by；In superclean bench, the stem section or stem apex that will disinfect are cut into the stem section of a length of 2cm, then insert the stem section for shearing
Enter in the MS culture mediums after sterilizing, every bottle of culture medium inserts stem section 5, culturing room is put it into after sealing and is cultivated；Control
Illumination and temperature conditionss needed for good culture, it is to avoid the pollution of outer bound pair culturing room, remove contaminated stem section and culture in time
Base；Culturing room's temperature control at 25 DEG C, daily illumination 12h, intensity of illumination is 1000Lx.
（4）Shoot proliferation：Ox tendon strip after through culture after a while, set up, and then carries out shoot proliferation by aseptic strain
Culture；Foster used medium of being commissioned to train is MS+1mg6BA+0.05%NAA, and every bottle of culture medium is 50ML, accesses the stem section 3 of aseptic strain
Individual, culturing room is put into after sealing carries out squamous subculture, culturing room is carried out disinfection weekly necessarily, and a subculture is changed per 20d,
By the squamous subculture of a cycle, ox tendon strip growth coefficient reaches 13；Culturing room's temperature control at 25 DEG C, daily illumination 12h,
Intensity of illumination is 1000Lx.
Embodiment 2
A kind of tissue culture enrichment procedure of white Cowhells wood, comprises the following steps that：
（1）The selection of explant：In the fair weather in March to May, clip ox tendon strip gives birth to the tender stem segmentses or stem of nutrition branch then
Point；
（2）The sterilizing of explant：The blade in tender stem is cut off, stem section or stem apex is cleaned with liquid detergent, then rinse dry with running water
Only, stem section or stem apex are put into superclean bench, with 75% alcohol-pickled 30s, then with sterilized distilled water by alcohol rinse
Totally, 25min is soaked in the mercuric chloride solution for placing into 0.1%, then with sterile water wash 4 times；
（3）Aseptic strain is set up：The minimal medium that the foundation of aseptic strain is used is MS culture mediums, the hormone for using be 6BA and
NAA, culture medium is fitted into wide-mouth bottle, per it is bottled enter culture medium 35mL, autoclave sterilization is carried out after sealing, after sterilizing cool down
It is stand-by；In superclean bench, the stem section or stem apex that will disinfect are cut into the stem section of a length of 2cm, then insert the stem section for shearing
Enter in the MS culture mediums after sterilizing, every bottle of culture medium inserts stem section 6, culturing room is put it into after sealing and is cultivated；Control
Illumination and temperature conditionss needed for good culture, it is to avoid the pollution of outer bound pair culturing room, remove contaminated stem section and culture in time
Base；Culturing room's temperature control at 26 DEG C, daily illumination 12h, intensity of illumination is 1100Lx.
（4）Shoot proliferation：Ox tendon strip after through culture after a while, set up, and then carries out shoot proliferation by aseptic strain
Culture；Foster used medium of being commissioned to train is MS+1.5mg6BA+0.2%NAA, and every bottle of culture medium is 50ML, accesses the stem section 3 of aseptic strain
Individual, culturing room is put into after sealing carries out squamous subculture, culturing room is carried out disinfection weekly necessarily, and a subculture is changed per 22d,
By the squamous subculture of a cycle, ox tendon strip growth coefficient reaches 14；Culturing room's temperature control at 26 DEG C, daily illumination 12h,
Intensity of illumination is 1100Lx.
Embodiment 3
A kind of tissue culture enrichment procedure of white Cowhells wood, comprises the following steps that：
（1）The selection of explant：In the fair weather in March to May, clip ox tendon strip gives birth to the tender stem segmentses or stem of nutrition branch then
Point；
（2）The sterilizing of explant：The blade in tender stem is cut off, stem section or stem apex is cleaned with liquid detergent, then rinse dry with running water
Only, stem section or stem apex are put into superclean bench, with 75% alcohol-pickled 30s, then with sterilized distilled water by alcohol rinse
Totally, 30min is soaked in the mercuric chloride solution for placing into 0.1%, then with sterile water wash 4 times；
（3）Aseptic strain is set up：The minimal medium that the foundation of aseptic strain is used is MS culture mediums, the hormone for using be 6BA and
NAA, culture medium is fitted into wide-mouth bottle, per it is bottled enter culture medium 40mL, autoclave sterilization is carried out after sealing, after sterilizing cool down
It is stand-by；In superclean bench, the stem section or stem apex that will disinfect are cut into the stem section of a length of 2cm, then insert the stem section for shearing
Enter in the MS culture mediums after sterilizing, every bottle of culture medium inserts stem section 8, culturing room is put it into after sealing and is cultivated；Control
Illumination and temperature conditionss needed for good culture, it is to avoid the pollution of outer bound pair culturing room, remove contaminated stem section and culture in time
Base；Culturing room's temperature control at 27 DEG C, daily illumination 12h, intensity of illumination is 1200Lx.
（4）Shoot proliferation：Ox tendon strip after through culture after a while, set up, and then carries out shoot proliferation by aseptic strain
Culture；Foster used medium of being commissioned to train is MS+2mg6BA+0.5%NAA, and every bottle of culture medium is 50ML, accesses the stem section 3 of aseptic strain
Individual, culturing room is put into after sealing carries out squamous subculture, culturing room is carried out disinfection weekly necessarily, and a subculture is changed per 25d,
By the squamous subculture of a cycle, ox tendon strip growth coefficient reaches 15；Culturing room's temperature control at 27 DEG C, daily illumination 12h,
Intensity of illumination is 1200Lx.
Experiment 1：
The selection of 1.1 explants：In May, 2015, in Horticultural Crop Research Institute of Yunnan Academy of Agricultural Sciences Yunnan peculiar fruit tree and
Rootstock resource garden collection 1a life ox tendon strip tender tips part.Tender tip its 10-15cm of clip long, removes blade, takes back laboratory standby
With.
1.2 test methods
1.2.1 the culture of aseptic seedling：The tender tip of collection is cut into 5cm or so stem sections long, 30-60min is rinsed with flowing water, super
In net workbench with 70% alcohol-pickled 30s, then with 0.1% mercuric chloride solution sterilizing 8min, finally with aseptic shaking water flushing 3
It is secondary, tender tip is then cut into 1.5cm or so stem segment with axillary bud long, access the primary of MS+6-BA1.0mg/L+IBA0.3mg/L
In culture medium, 2 stem sections of every bottle of inoculation；Initial culture 25d
Afterwards, test tube seedling is carefully taken out with tweezers, is cut into 1.5-2.0cm stem with bud, be inoculated into subculture medium MS+6-
In BA1.0mg/L+IBA0.1-0.3mg/L, every bottle connects 3-4 stem sections.
1.2.2 the Multiplying culture of stem eye：Squamous subculture is chosen with a collection of growth period 30d or so, the test tube seedling that growing way is good,
It is cut into the propagation screening and culturing medium that 1.5-2.0cm stem with bud accesses various heterogeneities, every kind for the treatment of at least connects 10
Bottle, 3 stem sections of every bottle of inoculation, after test tube seedling culture 30d, observes test tube seedling upgrowth situation, counts its growth coefficient and test tube seedling
Highly.Quantity × 100% of stem section when growth coefficient=plant is more than 1cm branch/inoculations long；
1.2.3 culture of rootage and test tube transplantation of seedlings：The test tube seedling stem section of 3-4cm is cut, is inoculated on root media, after 30d
Statistics rooting rate and bar number etc. of taking root；Then the good rooted seedling of growing way is transferred into experienced seedling room carries out practicing seedling, after practicing seedling 5d in bottle, moves
White silk seedling in matrix is planted, 15d test tube seedlings have sent out new root, counted survival rate.Rooting rate=plant number of taking root/inoculation stem section sum ×
100%；Bar number of taking root is the bar number of the first order root that plant base portion is induced；Transplanting survival rate=transplant survival number/transplantation rooting
Seedling number × 100%.
Condition of culture：
Above medium pH is 5.8, sucrose 30g/L, and agar 7g/L, condition of culture is temperature（25±2）DEG C, light application time
16h/d, intensity of illumination 1800lx.
Experiment 2：
The selection of 1.1 explants：In May, 2009, in Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology's pears Germplasm Resources collection 2a life clouds south Wen Quince
Plant tender tip part.Tender tip its 10-15cm of clip long, removes blade, takes back laboratory standby；
1.2 test methods：
1.2.1 the culture of aseptic seedling：The tender tip of collection is cut into 5cm or so stem sections long, 30-60min is rinsed with flowing water, super
In net workbench with 70% alcohol-pickled 30s, then with 0.1% mercuric chloride solution sterilizing 8min, finally with aseptic shaking water flushing 3
It is secondary, tender tip is then cut into 1.5cm or so stem segment with axillary bud long, access the primary of MS+6-BA1.0mg/L+IBA0.3mg/L
In culture medium, 2 stem sections of every bottle of inoculation.After Initial culture 25d, test tube seedling is carefully taken out with tweezers, be cut into 1.5-2.0cm band buds
Stem section, is inoculated into subculture medium MS+6-BA1.0mg/L+IBA0.1-0.3mg/L, and every bottle connects 3-4 stem sections.
1.2.2 the Multiplying culture of stem eye：Squamous subculture is chosen with a collection of growth period 30d or so, the test tube seedling that growing way is good,
It is cut into the propagation screening and culturing medium that 1.5-2.0cm stem with bud accesses various heterogeneities, every kind for the treatment of at least connects 10
Bottle, 3 stem sections of every bottle of inoculation, after test tube seedling culture 30d, observes test tube seedling upgrowth situation, counts its growth coefficient and test tube seedling
Highly.Quantity × 100% of stem section when growth coefficient=plant is more than 1cm branch/inoculations long.
1.2.3 culture of rootage and test tube transplantation of seedlings：The test tube seedling stem section of 3-4cm is cut, is inoculated on root media,
Rooting rate and bar number etc. of taking root are counted after 30d.Then the good rooted seedling of growing way is transferred into experienced seedling room is carried out practicing seedling, and seedling 5d is practiced in bottle
Afterwards, white silk seedling in matrix is transplanted into, 15d test tube seedlings have sent out new root, counted survival rate.Rooting rate=plant number/inoculation stem section of taking root
Sum × 100%；Bar number of taking root is the bar number of the first order root that plant base portion is induced；Transplanting survival rate=transplant survival number/shifting
Plant rooted seedling number × 100%.
Condition of culture：
Above medium pH is 5.8, sucrose 30g/L, and agar 7g/L, condition of culture is temperature（25±2）DEG C, light application time
16h/d, intensity of illumination 1800lx.
Stem section with pears dwarfing rootstock Yun Nan Wen Quinces has carried out rapid propagation in vitro research as explant.Result shows：Stem eye increases
The appropriate media grown is MS+6-BA1.0mg/L+IBA0.3mg/L or MS+6-BA0.5mg/L+NAA0.5mg/L, growth coefficient
Respectively 3.65 and 3.58, and test tube seedling growing way is healthy and strong；The appropriate media of Yun Nan Wen Quince root inductions is 1/2MS+
NAA0.3mg/L, rooted seedling transplanting survival rate is 92%.
Dwarfing effect for white Cowhells wood is obvious, but difficulty is survived after breeding and transplanting, causes to be difficult to asking for utilization and extention
Topic.Therefore, the white Cowhells wood for Cutting test in 2015 being survived, grafting apple and Pear varieties are tested, while carrying out white Cowhells wood
The research of tissue cultures Plantlet in vitro, the method for finding suitable fast-propagation accelerates the breeding of white Cowhells wood dwarfing stock to promote.
On the basis of aseptic strain is set up, the screening operation of ox tendon strip bud increment culture medium is carried out, with MS as minimal medium, using 6-
BA1-2（The basic element of cell division）、KT1-2（Kinetin）、NAA0.1-1（Auxin）Deng hormone, devise 2 groups of 12 formulas to enter
The screening of row ox tendon strip bud increment culture medium.Specific formula is shown in Table 1：
The ox tendon strip bud of table 1 increment culture medium prescription
Compared by multiple screening, second culture medium that basic screening goes out in first group at present, i.e. MS+6-BA1+
NAA0.5 formulas have preferably effect to the increment of ox tendon strip bud, solve bud increment problem of the ox tendon strip during expansion is numerous.Its
The increment quantity of bud is between 5~10, and the culture medium of other formulas rises in value quantity only at 0~4.Therefore expand it is numerous during
It is main bud increment culture medium with MS+6-BA1+NAA0.5 formulas.
The present invention uses tissue cultures enrichment procedure, dialogue Cowhells wood dwarfing rootstock to be produced in enormous quantities, reduce and educate
Seedling cost, improves breeding efficiency.
The better embodiment to this patent is explained in detail above, but this patent is not limited to above-mentioned embodiment party
Formula, in the ken that one skilled in the relevant art possesses, can also be on the premise of this patent objective not be departed from
Various changes can be made.
Claims (3)
1. a kind of tissue culture enrichment procedure of white Cowhells wood, it is characterised in that comprise the following steps that：
（1）The selection of explant：In the fair weather in March to May, clip ox tendon strip gives birth to the tender stem segmentses or stem of nutrition branch then
Point；
（2）The sterilizing of explant：The blade in tender stem is cut off, stem section or stem apex is cleaned with liquid detergent, then rinse dry with running water
Only, stem section or stem apex are put into superclean bench, with 75% alcohol-pickled 30s, then with sterilized distilled water by alcohol rinse
Totally, 20-30min is soaked in the mercuric chloride solution for placing into 0.1%, then with sterile water wash 4 times；
（3）Aseptic strain is set up：The minimal medium that the foundation of aseptic strain is used is MS culture mediums, the hormone for using be 6BA and
NAA, culture medium is fitted into wide-mouth bottle, per it is bottled enter culture medium 30-40ML, autoclave sterilization is carried out after sealing, after sterilizing
Cooling is stand-by；In superclean bench, the stem section or stem apex that will disinfect are cut into the stem section of a length of 2cm, the stem that then will be sheared
In MS culture mediums after section insertion sterilizing, every bottle of culture medium insertion stem section 5-8 puts it into culturing room and is trained after sealing
Support；
（4）Shoot proliferation：Ox tendon strip after through culture after a while, set up, and then carries out shoot proliferation culture by aseptic strain；
Foster used medium of being commissioned to train is MS+1-2mg6BA+0.05-0.5%NAA, and every bottle of culture medium is 50ML, accesses the stem section 3 of aseptic strain
Individual, culturing room is put into after sealing carries out squamous subculture, culturing room is carried out disinfection weekly necessarily, is changed per 20-25d and once cultivated
Base, by the squamous subculture of a cycle, ox tendon strip growth coefficient reaches 13-15.
2. the tissue culture enrichment procedure of white Cowhells wood according to claim 1, it is characterised in that the step（1）In stem
Section or alcohol disinfecting that stem apex mass percentage concentration is 75% 30 seconds, then sterilized with the mercuric chloride that mass percentage concentration is 0.1%
25min。
3. the tissue culture enrichment procedure of white Cowhells wood according to claim 1, it is characterised in that the step（3）And step
（4）In culturing room's condition be：Culturing room's temperature control at 25-27 DEG C, daily illumination 12h, intensity of illumination is 1000-
1200Lx。
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| CN201710241937.1A CN106857265B (en) | 2017-04-14 | 2017-04-14 | A kind of tissue culture enrichment procedure of white Cowhells wood |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113545293A (en) * | 2021-08-19 | 2021-10-26 | 云南省农业科学院园艺作物研究所 | Tissue culture proliferation method of Mao branch Jing pear kiwi fruit |
| CN117441616A (en) * | 2023-12-14 | 2024-01-26 | 浙江大学 | In-vitro rapid propagation method for beef tendons |
-
2017
- 2017-04-14 CN CN201710241937.1A patent/CN106857265B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113545293A (en) * | 2021-08-19 | 2021-10-26 | 云南省农业科学院园艺作物研究所 | Tissue culture proliferation method of Mao branch Jing pear kiwi fruit |
| CN117441616A (en) * | 2023-12-14 | 2024-01-26 | 浙江大学 | In-vitro rapid propagation method for beef tendons |
| CN117441616B (en) * | 2023-12-14 | 2024-05-03 | 浙江大学 | In-vitro rapid propagation method for beef tendons |
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| CN106857265B (en) | 2019-05-10 |
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