CN106857265B - 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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- CN106857265B CN106857265B CN201710241937.1A CN201710241937A CN106857265B CN 106857265 B CN106857265 B CN 106857265B CN 201710241937 A CN201710241937 A CN 201710241937A CN 106857265 B CN106857265 B CN 106857265B
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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)
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Abstract
The invention discloses a kind of tissue culture enrichment procedure of white Cowhells wood, specific steps include the selection of explant, the sterilizing of explant, aseptic strain is established and shoot proliferation；The selection of explant is the fair weather in March to May, the tender stem segments or stem apex of clip ox tendon strip current year raw nutrition branch；The sterilizing of explant is the blade cut off in tender stem, stem section or stem apex are cleaned with dish washing liquid, it is rinsed well again with tap water, stem section or stem apex are put into superclean bench, 30s is impregnated with 75% alcohol, it is with sterilized distilled water that alcohol rinse is clean again, it places into 0.1% mercuric chloride solution and impregnates 20-30min, then use 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, the tissue culture enrichment procedure of specifically a kind of white Cowhells wood.
Background technique
Ox tendon strip belongs to rosaceae Dichotomanthes on plant classification, originates in an only a kind of 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, no matter ox tendon strip, which grafts apple and pears, has
Tree body and the ahead of time positive effect of result are downgraded, can be multi-purpose with an anvil, it is the dwarfing of growing directly from seeds of the apple got a good chance of and pears
Anvil.But ox tendon strip is evergreen shrubs, and the four seasons are evergreen, and main root is flourishing, is buried very deep, fibrous root is few, has resistance to lean one side of anti-morning, and have
Intolerant to the one side of transplanting, field planting, general grafting is since fibrous root is few, and seedling-slowing stage is long after field planting of transplanting seedlings, and influences to grow.
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
It reaches, depth of burying, fibrous root is few, and is evergreen tree, so transplanting survival rate is low；Cutting propagation, in addition to requiring the external world to have centainly
Humidity, temperature, air and spontaneous and extraneous have outside sufficient nutriment, it is also necessary to some micro sp act substances,
Complicated for operation, since ox tendon strip cuttage root-taking is difficult, seedling cost is high, and low efficiency affects its promotion rate.
Summary of the invention
The purpose of the present invention is to provide the tissue culture proliferation of a kind of raising breeding efficiency, the white Cowhells wood for reducing seedling cost
Method, to solve the problems mentioned in the above background technology.
To achieve the above object, the invention provides the following technical scheme:
A kind of tissue culture enrichment procedure of white Cowhells wood, the specific steps are as follows:
(1) selection of explant: in the fair weather in March to May, the tender stem segments of clip ox tendon strip current year raw nutrition branch
Or stem apex；
(2) sterilizing of explant: cutting off the blade in tender stem, cleans stem section or stem apex with dish washing liquid, then rushed with tap water
Stem section or stem apex are put into superclean bench by wash clean, impregnate 30s with 75% alcohol, then with sterilized distilled water by alcohol
It rinses well, places into 0.1% mercuric chloride solution and impregnate 20-30min, then use sterile water wash 4 times；
(3) aseptic strain is established: the minimal medium that the foundation of aseptic strain uses is MS culture medium, and the hormone used is 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, sterilize
It is cooling stand-by afterwards；In superclean bench, the stem section disinfected or stem apex be cut into the stem section of a length of 2cm, then will shear
In MS culture medium after stem section insertion sterilizing, every bottle of culture medium insertion stem section 5-8 puts it into culturing room and is trained after sealing
It supports；
(4) shoot proliferation: ox tendon strip after culture after a period of time, establish by aseptic strain, then carries out shoot proliferation
Culture；Feeding 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 aseptic strain
Stem section 3, it is put into culturing room after sealing and carries out squamous subculture, carries out disinfection centainly to culturing room weekly, every 20-25d replacement one
Subculture, by the squamous subculture of a cycle, ox tendon strip growth coefficient reaches 13-15.
As a further solution of the present invention: stem section or stem apex mass percentage concentration in the step (1) are 75%
Alcohol disinfecting 30 seconds, then the mercuric chloride disinfection 25min for being 0.1% with mass percentage concentration.
As further scheme of the invention: culturing room's condition in the step (3) and step (4) are as follows: culturing room
Temperature is controlled at 25-27 DEG C, daily illumination 12h, intensity of illumination 1000-1200Lx.
Compared with prior art, the beneficial effects of the present invention are:
The present invention uses tissue cultures enrichment procedure, and dialogue Cowhells wood dwarfing rootstock is produced in enormous quantities, reduces and educate
Seedling cost, improves breeding efficiency.
Specific embodiment
The technical solution of the patent is explained in further detail With reference to embodiment.
Embodiment 1
A kind of tissue culture enrichment procedure of white Cowhells wood, the specific steps are as follows:
(1) selection of explant: in the fair weather in March to May, the tender stem segments of clip ox tendon strip current year raw nutrition branch
Or stem apex；
(2) sterilizing of explant: cutting off the blade in tender stem, cleans stem section or stem apex with dish washing liquid, then rushed with tap water
Stem section or stem apex are put into superclean bench by wash clean, impregnate 30s with 75% alcohol, then with sterilized distilled water by alcohol
It rinses well, places into 0.1% mercuric chloride solution and impregnate 20min, then use sterile water wash 4 times；
(3) aseptic strain is established: the minimal medium that the foundation of aseptic strain uses is MS culture medium, and the hormone used is 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, it is cold after sterilizing
Stand-by；In superclean bench, the stem section disinfected or stem apex are cut into the stem section of a length of 2cm, the stem section that then will be sheared
In MS culture medium after insertion sterilizing, every bottle of culture medium is inserted into stem section 5, and culturing room is put it into after sealing and is cultivated；Control
Illumination and temperature condition needed for making culture, avoid the pollution of outer bound pair culturing room, remove contaminated stem section and training in time
Support base；It cultivates room temperature to control at 25 DEG C, daily illumination 12h, intensity of illumination 1000Lx.
(4) shoot proliferation: ox tendon strip after culture after a period of time, establish by aseptic strain, then carries out shoot proliferation
Culture；Feeding 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
It is a, it is put into culturing room after sealing and carries out squamous subculture, carries out disinfection centainly to culturing room weekly, every 20d replaces a subculture,
By the squamous subculture of a cycle, ox tendon strip growth coefficient reaches 13；It cultivates room temperature to 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, the specific steps are as follows:
(1) selection of explant: in the fair weather in March to May, the tender stem segments of clip ox tendon strip current year raw nutrition branch
Or stem apex；
(2) sterilizing of explant: cutting off the blade in tender stem, cleans stem section or stem apex with dish washing liquid, then rushed with tap water
Stem section or stem apex are put into superclean bench by wash clean, impregnate 30s with 75% alcohol, then with sterilized distilled water by alcohol
It rinses well, places into 0.1% mercuric chloride solution and impregnate 25min, then use sterile water wash 4 times；
(3) aseptic strain is established: the minimal medium that the foundation of aseptic strain uses is MS culture medium, and the hormone used is 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, it is cold after sterilizing
Stand-by；In superclean bench, the stem section disinfected or stem apex are cut into the stem section of a length of 2cm, the stem section that then will be sheared
In MS culture medium after insertion sterilizing, every bottle of culture medium is inserted into stem section 6, and culturing room is put it into after sealing and is cultivated；Control
Illumination and temperature condition needed for making culture, avoid the pollution of outer bound pair culturing room, remove contaminated stem section and training in time
Support base；It cultivates room temperature to control at 26 DEG C, daily illumination 12h, intensity of illumination 1100Lx.
(4) shoot proliferation: ox tendon strip after culture after a period of time, establish by aseptic strain, then carries out shoot proliferation
Culture；Feeding 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
It is a, it is put into culturing room after sealing and carries out squamous subculture, carries out disinfection centainly to culturing room weekly, every 22d replaces a subculture,
By the squamous subculture of a cycle, ox tendon strip growth coefficient reaches 14；It cultivates room temperature to 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, the specific steps are as follows:
(1) selection of explant: in the fair weather in March to May, the tender stem segments of clip ox tendon strip current year raw nutrition branch
Or stem apex；
(2) sterilizing of explant: cutting off the blade in tender stem, cleans stem section or stem apex with dish washing liquid, then rushed with tap water
Stem section or stem apex are put into superclean bench by wash clean, impregnate 30s with 75% alcohol, then with sterilized distilled water by alcohol
It rinses well, places into 0.1% mercuric chloride solution and impregnate 30min, then use sterile water wash 4 times；
(3) aseptic strain is established: the minimal medium that the foundation of aseptic strain uses is MS culture medium, and the hormone used is 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, it is cold after sterilizing
Stand-by；In superclean bench, the stem section disinfected or stem apex are cut into the stem section of a length of 2cm, the stem section that then will be sheared
In MS culture medium after insertion sterilizing, every bottle of culture medium is inserted into stem section 8, and culturing room is put it into after sealing and is cultivated；Control
Illumination and temperature condition needed for making culture, avoid the pollution of outer bound pair culturing room, remove contaminated stem section and training in time
Support base；It cultivates room temperature to control at 27 DEG C, daily illumination 12h, intensity of illumination 1200Lx.
(4) shoot proliferation: ox tendon strip after culture after a period of time, establish by aseptic strain, then carries out shoot proliferation
Culture；Feeding 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
It is a, it is put into culturing room after sealing and carries out squamous subculture, carries out disinfection centainly to culturing room weekly, every 25d replaces a subculture,
By the squamous subculture of a cycle, ox tendon strip growth coefficient reaches 15；It cultivates room temperature to control at 27 DEG C, daily illumination 12h,
Intensity of illumination is 1200Lx.
Test 1:
The selection of 1.1 explants: in May, 2015, in the peculiar fruit in Horticultural Crop Research Institute of Yunnan Academy of Agricultural Sciences Yunnan
Tree and the rootstock resource garden raw ox tendon strip tender tip part acquisition 1a.The tender tip of its 10-15cm long of clip, removes blade, takes back experiment
Room is spare.
1.2 test method
1.2.1 the culture of aseptic seedling: being cut into the long stem section of 5cm or so for the tender tip of acquisition, rinse 30-60min with flowing water,
In superclean bench with 70% alcohol impregnate 30s, then with 0.1% mercuric chloride solution sterilize 8min, finally with sterile shaking water punching
It washes 3 times, tender tip is then cut into the long stem segment with axillary bud of 1.5cm or so, access the first of MS+6-BA1.0mg/L+IBA0.3mg/L
For 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 section.
1.2.2 the Multiplying culture of stem eye: choosing squamous subculture with or so a collection of growth period 30d, test tube seedling that growing way is good,
It is cut into 1.5-2.0cm stem with bud to access in the proliferation screening and culturing medium of various heterogeneities, every kind of processing at least connects 10
Bottle, 3 stem sections of every bottle of inoculation, after test tube seedling culture 30d, observation test tube seedling upgrowth situation counts its growth coefficient and test tube seedling
Highly.Quantity × 100% of stem section when growth coefficient=plant is greater than branch/inoculation of 1cm long；
1.2.3 culture of rootage and test tube transplantation of seedlings: cutting the test tube seedling stem section of 3-4cm, be inoculated on root media,
Rooting rate and item number etc. of taking root are counted after 30d；Then the good rooted seedling of growing way is transferred to experienced seedling room to carry out practicing seedling, practices seedling 5d in bottle
Afterwards, it is transplanted into matrix and practices seedling, 15d test tube seedling has sent out new root, counts survival rate.Rooting rate=plant number/inoculation stem section of taking root
Sum × 100%；Item number of taking root is the item number for the first order root that plant base portion induces；Transplanting survival rate=transplant survival number/shifting
Plant rooted seedling number × 100%.
Condition of culture:
The above medium pH is 5.8, sucrose 30g/L, agar 7g/L, and condition of culture is temperature (25 ± 2) DEG C, when illumination
Between 16h/d, intensity of illumination 1800lx.
Test 2:
The selection of 1.1 explants: in May, 2009, in the raw Yunnan Xibei Univ. of Agricultural & Forest Science & Technology's pears Germplasm Resources acquisition 2a
Wen Quince plant tender tip part.The tender tip of its 10-15cm long of clip, removes blade, and it is spare to take back laboratory；
1.2 test methods:
1.2.1 the culture of aseptic seedling: being cut into the long stem section of 5cm or so for the tender tip of acquisition, rinse 30-60min with flowing water,
In superclean bench with 70% alcohol impregnate 30s, then with 0.1% mercuric chloride solution sterilize 8min, finally with sterile shaking water punching
It washes 3 times, tender tip is then cut into the long stem segment with axillary bud of 1.5cm or so, access the first of MS+6-BA1.0mg/L+IBA0.3mg/L
For in culture medium, 2 stem sections of every bottle of inoculation.After Initial culture 25d, test tube seedling is carefully taken out with tweezers, is cut into 1.5-2.0cm band
Leaf stem section is inoculated into subculture medium MS+6-BA1.0mg/L+IBA0.1-0.3mg/L, and every bottle connects 3-4 stem section.
1.2.2 the Multiplying culture of stem eye: choosing squamous subculture with or so a collection of growth period 30d, test tube seedling that growing way is good,
It is cut into 1.5-2.0cm stem with bud to access in the proliferation screening and culturing medium of various heterogeneities, every kind of processing at least connects 10
Bottle, 3 stem sections of every bottle of inoculation, after test tube seedling culture 30d, observation test tube seedling upgrowth situation counts its growth coefficient and test tube seedling
Highly.Quantity × 100% of stem section when growth coefficient=plant is greater than branch/inoculation of 1cm long.
1.2.3 culture of rootage and test tube transplantation of seedlings: cutting the test tube seedling stem section of 3-4cm, be inoculated on root media,
Rooting rate and item number etc. of taking root are counted after 30d.Then the good rooted seedling of growing way is transferred to experienced seedling room to carry out practicing seedling, practices seedling 5d in bottle
Afterwards, it is transplanted into matrix and practices seedling, 15d test tube seedling has sent out new root, counts survival rate.Rooting rate=plant number/inoculation stem section of taking root
Sum × 100%；Item number of taking root is the item number for the first order root that plant base portion induces；Transplanting survival rate=transplant survival number/shifting
Plant rooted seedling number × 100%.
Condition of culture:
The above medium pH is 5.8, sucrose 30g/L, agar 7g/L, and condition of culture is temperature (25 ± 2) DEG C, when illumination
Between 16h/d, intensity of illumination 1800lx.
Using the stem section of pears dwarfing rootstock Yun Nan Wen Quince as explant, rapid propagation in vitro research has been carried out.The result shows that: 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 induction is 1/2MS+
NAA0.3mg/L, rooted seedling transplanting survival rate are 92%.
Dwarfing effect for white Cowhells wood is obvious, but difficulty survives after breeding and transplanting, causes to be difficult to asking for utilization and extention
Topic.Therefore, white Cowhells wood Cutting test in 2015 survived, grafts apple and Pear varieties test, 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 established, the screening operation for having carried out ox tendon strip bud increment culture medium utilizes 6- using MS as minimal medium
The BA1-2(basic element of cell division), KT1-2(kinetin), NAA0.1-1(auxin) etc. hormones, devise 2 groups 12 formula come into
The screening of row ox tendon strip bud increment culture medium.Specific formula is shown in Table 1:
1 ox tendon strip bud of table increment culture medium prescription
Compared by multiple screening, basic screening goes out second culture medium in first group, i.e. MS+6-BA1+ at present
NAA0.5 formula has preferably effect to the increment of ox tendon strip bud, solves the problems, such as bud increment of ox tendon strip during expanding numerous.Its
The increment quantity of bud is between 5~10, and the culture medium increment quantity of other formulas is only at 0~4.Therefore expand it is numerous during
It is main bud increment culture medium with MS+6-BA1+NAA0.5 formula.
The present invention uses tissue cultures enrichment procedure, and dialogue Cowhells wood dwarfing rootstock is produced in enormous quantities, reduces and educate
Seedling cost, improves breeding efficiency.
The preferred embodiment of the patent is described in detail above, but this patent is not limited to above-mentioned embodiment party
Formula within the knowledge of one of ordinary skill in the art can also be under the premise of not departing from this patent objective
Various changes can be made.
Claims (3)
1. a kind of tissue culture enrichment procedure of white Cowhells wood, which is characterized in that specific step is as follows:
(1) selection of explant: in the fair weather in March to May, the tender stem segments of the white Cowhells wood current year raw nutrition branch of clip；
(2) sterilizing of explant: cutting off the blade in tender stem, stem section is cleaned with dish washing liquid, then rinsed well with tap water, by stem
Section is put into superclean bench, is impregnated 30s with 75% alcohol, then with sterilized distilled water that alcohol rinse is clean, is placed into
20-30min is impregnated in 0.1% mercuric chloride solution, is then used sterile water wash 4 times；
(3) aseptic strain is established: the minimal medium that the foundation of aseptic strain uses is MS culture medium, the hormone used be 6BA and
Culture medium is fitted into wide-mouth bottle by NAA, per it is bottled enter culture medium 30-40ml, autoclave sterilization is carried out after sealing, after sterilizing
It is cooling stand-by；In superclean bench, the stem section disinfected is cut into a length of 2cm, it then will be after the stem section that sheared insertion sterilizing
Culture medium in, every bottle of culture medium insertion stem section 5-8 puts it into culturing room and is cultivated after sealing；
(4) shoot proliferation: white Cowhells wood after culture after a period of time, establish by aseptic strain, then carries out shoot proliferation training
It supports；Squamous subculture used medium is MS+1-2mg/L6BA+0.05-0.5%NAA, and every bottle of culture medium is 50ml, accesses aseptic strain
Stem section 3, culturing room is put into after sealing and carries out squamous subculture, is carried out disinfection once to culturing room weekly, every 20-25d is replaced
One subculture, by the squamous subculture of a cycle, white Cowhells wood growth coefficient reaches 13-15.
2. the tissue culture enrichment procedure of white Cowhells wood according to claim 1, which is characterized in that the stem in the step (1)
The alcohol disinfecting 30s that section is 75% with mass percentage concentration, then the mercuric chloride for being 0.1% with mass percentage concentration sterilize 25min.
3. the tissue culture enrichment procedure of white Cowhells wood according to claim 1, which is characterized in that the step (3) and step
(4) culturing room's condition in are as follows: culture room temperature control is at 25-27 DEG C, daily illumination 12h, intensity of illumination 1000-
1200Lx。
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| CN117441616B (en) * | 2023-12-14 | 2024-05-03 | 浙江大学 | In-vitro rapid propagation method for beef tendons |
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Non-Patent Citations (3)
| Title |
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| 中国特有属牛筋条属的花粉形态与其系统位置;周丽华等;《云南植物研究》;20001231;第22卷(第2期);第143-147页 * |
| 牛筋条种子生物学特性及其萌发对温度和激素的响应;李树珍等;《中国种业》;20091231(第8期);第40-42页 * |
| 牛筋条组培增殖培养基配方筛选试验研究;李坤明等;《园艺学报》;20171231;第44卷(第S1期);第2456页 * |
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