CN106359093A - Establishing method for redwood tissue culture rapid propagation system - Google Patents
Establishing method for redwood tissue culture rapid propagation system Download PDFInfo
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- CN106359093A CN106359093A CN201610779959.9A CN201610779959A CN106359093A CN 106359093 A CN106359093 A CN 106359093A CN 201610779959 A CN201610779959 A CN 201610779959A CN 106359093 A CN106359093 A CN 106359093A
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- 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
Abstract
The invention discloses an establishing method for a redwood tissue culture rapid propagation system. The establishing method is a method for rapidly obtaining a lot of good-quality seedlings within short time through a plant tissue culture technology. With annual redwood lignified stem segments with buds being explants, the redwood tissue culture rapid propagation system is established through the processes of starting culture, subculture and proliferation culture, seedling strengthening and rooting culture, seedling hardening and transplanting and others, technical support is provided for large-scale production, and a foundation is laid for further molecular breeding. The establishing method has the advantages that a lot of thick roots are generated, seedlings are strong, management is convenient, and the seedlings grow to full size rapidly.
Description
Technical field
The present invention relates to pinaster, particularly a kind of method for building up of Chinese larch tissue culture rapid propagation system.
Background technology
Chinese larch also known as husky wood, lamp stand wood.The trunk of China fir Gao Erzhi as pinaster, leaf is grown on branch, hard and micro- flat picture pin,
Winter does not wither and fall, and the fruit of knot is as the fruit of maple.Lignum seu Ramulus Cunninghamiae Lanceolatae has red, white two kinds.Chinese larch is wooden solid and heavy wool, and white fir is then wooden
Empty and be dried, Lignum seu Ramulus Cunninghamiae Lanceolatae will not be damaged by worms, ashing also can and medicine.Again water-fast, that buries will not be resistance to rotten.Nature and flavor are gentle, nontoxic.Function master
Control: leaching beriberi edema to boil water；Take orally and control trusted subordinate's distending pain, remove bad odor；Control pathogenic wind toxic, cholera.Chinese larch is good use material, keeps water
Soil, check winds and fix drifting sand, improved soil and " by the of four " green tree species.Chinese larch wood quality is light and solid, and its texture is careful, wear-resisting, corrosion resistant,
Also furniture, farm implements, mechanical part, water conservancy project and carpenter's material can be made.Branch and leaf and tree root is inflammable, firepower strong, be fuming less, fire duration,
It is good yule logs.Chinese larch is planted general artificial forest widest in area, its strong adaptability, drought-resistant barren, calcareous, acidity, in
Property and slightly saline and alkaline soil in all can normal growth, therefore greatly develop plantation in lean carbuncle mountain area, salt-soda soil, soil changed
Good, water and soil conservation has great importance.At present, Chinese larch seedling is mainly using cuttage, buries root method, grafting and seeding method
Bred, because these method breeding coefficients are low, the cycle is long, the need to Chinese larch seedling for the large-scale afforestation far can not be met
Will.The therefore necessary Chinese larch seedling set up complete Chinese larch rapid propagation in vitro system, high-quality is provided for production practices.
Content of the invention
It is an object of the invention to provide going out a kind of method for building up of Chinese larch tissue culture rapid propagation system, annual wooden with Chinese larch
Change stem with bud is explant, waits through Primary culture, subculture and enrichment culture, strong sprout and root culture, seedling exercising and transplanting
Journey establishes Chinese larch tissue culture rapid propagation system, it is achieved thereby that the purpose of the present invention.
A kind of method for building up of Chinese larch tissue culture rapid propagation system of the present invention, comprises the following steps:
Step 1, Primary culture: choose healthy and strong Chinese larch plant annual lignifying stem with bud as explant, cut off blade, cut
Become the 6.0 long sections with 5 axillary buds first to use detergent water to rinse 4h, then scrub explant surface by its surface with hairbrush
Dust and part thalline remove, and are then rinsed with tap water and are placed in superclean bench after 4h, first with after 75% ethanol disinfection 32s
Washed 6 times with aseptic, then with 0.1% mercuric chloride solution sterilization 26min, dry surface with aseptic filter paper again 8 times with aseptic water washing
It is inoculated into primary culture medium after the globule and carry out inducing culture, first full light culture 8 days under the conditions of 29 DEG C after inoculation, then every daylight
According to 9 hours, intensity of illumination was 3200lx, until induced synthesis Multiple Buds；
Step 2, subculture and enrichment culture: the axillary bud that step (1) is cultivated sprouting cuts into the segment with bud and proceeds to propagation
Carry out successive transfer culture, after inoculation, first full light culture 5 days under the conditions of 29 DEG C, are subsequently placed in daily illumination 10 hours in culture medium,
Intensity of illumination is 2200lx, is placed under conditions of cultivation temperature is 29 DEG C and cultivates, repeatedly cutting carries out successive transfer culture repeatedly, obtains
Rudiment；
Step 3, root culture: the Multiple Buds bud cutting that the height that step (1) or (2) process are obtained is about 3.6cm is inoculated into
Carry out root culture on root media, first full light culture 4 days under the conditions of 29 DEG C after inoculation, then daily illumination 10 hours,
Intensity of illumination is 3200lx, and cultivation temperature is cultivated to taking root under conditions of being 29 DEG C；
Step 4, acclimatization and transplantses: the rooting tube plantlet of the high about 9cm that step (3) is obtained goes bottle cap to be placed in natural lighting lower refining seedling
After 8 days, test tube seedling is taken out from culture bottle, wash root culture medium off, plant into by Nutrition Soil: the base that sandy soil=3:1 is mixed into
Matter, is placed in culture in illumination box, is watered to seedling with 1/2ms macro-element nutrients liquid daily, keep humidity.Treat that seedling becomes
Land for growing field crops is transplanted again after work.
Primary culture medium described in step (1) is: ms+4mg/l6-ba+1.2mg/l naa+32g/l sucrose+8g/l fine jade
Fat, ph is 5.9.
Proliferated culture medium described in step (2) is: n6+ 2mg/lnaa+4mg/l 6-ba+35g/l sucrose+8g/l agar,
Ph is 5.9.
Root media described in step (3) is: 1/2ms+2mg/l naa+2mg/l iba+35g/l sucrose+8g/l fine jade
Fat, ph is 5.9.
Compared with prior art the invention has the advantage that being obtained in a large number by quick in the plant tissue culture technique short time
The method of the thorn excellent wooden seedling of Chinese larch.With Chinese larch annual lignifying stem with bud as explant, through Primary culture, subculture and increasing
The processes such as culture, strong sprout and root culture, seedling exercising and transplanting of growing establish Chinese larch tissue culture rapid propagation system, carry for its large-scale production
For technical support, and lay the foundation for carrying out molecular breeding further.
Specific embodiment
Following examples are that the present invention is further illustrated, and are not limitations of the present invention.
Embodiment 1:
(1) Primary culture: choose healthy and strong Chinese larch plant annual lignifying stem with bud as explant, cut off blade, be cut into 2
The long sections with 5 axillary buds first uses detergent water to rinse 2h, more gently scrubs explant surface by its surface with banister brush
Dust and part thalline remove, then with tap water rinse 2h after be placed in superclean bench, first with after 75% ethanol disinfection 7s
Washed 4 times with aseptic, then with 0.1% mercuric chloride solution sterilization 13min, dry surface with aseptic filter paper again 5 times with aseptic water washing
It is inoculated into primary culture medium after the globule and carry out inducing culture, first full light culture 3 days under the conditions of 26 DEG C after inoculation, it is subsequently placed in every
Its illumination 11 hours, intensity of illumination be 1900lx under the conditions of culture can initially form Multiple Buds within 6 days, pollution rate low to 6% with
Under, bud induction rate is 89%.Described primary culture medium is: ms+0.9mg/l6-ba+0.09mg/l naa+29g/l sucrose+
3.9g/l agar, ph is 5.9.
(2) subculture and enrichment culture: the axillary bud that step (1) is cultivated sprouting cuts into the segment with bud and proceeds to propagation
Carry out successive transfer culture in culture medium, first full light culture 1 day under the conditions of 26 DEG C after inoculation, then daily illumination 11 hours, illumination
Intensity is 900lx, is placed in propagation Seedling after cultivating 34 days under conditions of cultivation temperature is 26 DEG C and can grow to 4cm, growth coefficient is 6,
Repeatedly cutting carries out successive transfer culture, to obtain more rudiments repeatedly.Described proliferated culture medium is: n6+0.6mg/lnaa+
1.6mg/l 6-ba+14g/l sucrose+3.4g/l agar, ph is 5.9.
(3) root culture: the Multiple Buds bud cutting that the height that step (1) or (2) process are obtained is about 3.6cm is inoculated into
Carry out root culture on root media, first full light culture 6 days under the conditions of 26 DEG C after inoculation, then daily illumination 12 hours,
Intensity of illumination is 1900lx, and cultivation temperature is cultivated under conditions of being 26 DEG C to taking root, and rooting rate is 90%, and bar number of averagely taking root reaches
3.8 bar.Described root media is: 1/2ms+0.4mg/l naa+0.3mg/l iba+29g/l sucrose+3.4g/l agar,
Ph is 5.9.
(4) acclimatization and transplantses: the rooting tube plantlet of the high about 9cm that step (3) is obtained goes bottle cap to be placed in refining under natural lighting
After Seedling 6 days, test tube seedling is taken out from culture bottle, wash root culture medium off, plant into by Nutrition Soil: sandy soil=3:1 is mixed into
Substrate, is placed in culture in illumination box, is watered to seedling with 1/2ms macro-element nutrients liquid daily, keep humidity.Treat seedling
Land for growing field crops is transplanted again, transplanting survival rate is 84% after surviving.
Embodiment 2:
(1) Primary culture: choose healthy and strong Chinese larch plant annual lignifying stem with bud as explant, cut off blade, be cut into
The sections with 5 axillary buds of 5.8cm length first use detergent water rinse 3h, then with banister brush gently scrub explant surface by its
The dust on surface and part thalline remove, and are then rinsed with tap water and are placed in superclean bench after 3h, first use 75% ethanol disinfection
Washed 5 times with aseptic after 9s, then with 0.1% mercuric chloride solution sterilization 16min, dry table with aseptic filter paper again 5 times with aseptic water washing
It is inoculated into primary culture medium after the globule in face and carry out inducing culture, first full light culture 3 days under the conditions of 26 DEG C after inoculation, then often
Its illumination 12 hours, intensity of illumination be 1400lx under the conditions of culture can initially form Multiple Buds within 8 days, pollution rate low to 9% with
Under, bud induction rate is 84%.Described primary culture medium is: ms+1.6mg/l6-ba+0.6mg/l naa+31g/l sucrose+
3.6g/l agar, ph is 5.8.
(2) subculture and enrichment culture: the axillary bud that step (1) is cultivated sprouting cuts into the segment with bud and proceeds to propagation
Carry out successive transfer culture in culture medium, first full light culture 3 days under the conditions of 26 DEG C after inoculation, then daily illumination 11 hours, illumination
Intensity is 1800lx, is placed in propagation Seedling after cultivating 38 days under conditions of cultivation temperature is 26 DEG C and can grow to 3.5cm, growth coefficient is
5, repeatedly cutting carries out successive transfer culture, to obtain more rudiments repeatedly.Described proliferated culture medium is: n6+0.5mg/lnaa+
2.0mg/l 6-ba+18g/l sucrose+3.8g/l agar, ph is 5.8.
(3) root culture: the Multiple Buds bud cutting that the height that step (1) or (2) process are obtained is about 5.8cm is inoculated into
Carry out root culture, after inoculation, first full light culture 5 days under the conditions of 26 DEG C, are subsequently placed in daily illumination 15 little on root media
When, intensity of illumination is 2100lx, and cultivation temperature is cultivated under conditions of being 26 DEG C to taking root, and rooting rate is 88%, bar number of averagely taking root
Reach 4.15.Described root media is: 1/2ms+0.8mg/l naa+0.6mg/l iba+33g/l sucrose+3.8g/l fine jade
Fat, ph is 5.8.
(4) acclimatization and transplantses: the rooting tube plantlet of the high about 9cm that step (3) is obtained goes bottle cap to be placed in refining under natural lighting
After Seedling 9 days, test tube seedling is taken out from culture bottle, wash root culture medium off, plant into by Nutrition Soil: sandy soil=3:1 is mixed into
Substrate, is placed in culture in illumination box, is watered to seedling with 1/2ms macro-element nutrients liquid daily, keep humidity.Treat seedling
Land for growing field crops is transplanted again, transplanting survival rate is 92% after surviving.
Claims (4)
1. a kind of method for building up of Chinese larch tissue culture rapid propagation system is it is characterised in that comprise the following steps:
Step 1, Primary culture: choose healthy and strong Chinese larch plant annual lignifying stem with bud as explant, cut off blade, cut
Become the 6.0 long sections with 5 axillary buds first to use detergent water to rinse 4h, then scrub explant surface by its surface with hairbrush
Dust and part thalline remove, and are then rinsed with tap water and are placed in superclean bench after 4h, first with after 75% ethanol disinfection 32s
Washed 6 times with aseptic, then with 0.1% mercuric chloride solution sterilization 26min, dry surface with aseptic filter paper again 8 times with aseptic water washing
It is inoculated into primary culture medium after the globule and carry out inducing culture, first full light culture 8 days under the conditions of 29 DEG C after inoculation, then every daylight
According to 9 hours, intensity of illumination was 3200lx, until induced synthesis Multiple Buds；
Step 2, subculture and enrichment culture: the axillary bud that step (1) is cultivated sprouting cuts into the segment with bud and proceeds to propagation
Carry out successive transfer culture, after inoculation, first full light culture 5 days under the conditions of 29 DEG C, are subsequently placed in daily illumination 10 hours in culture medium,
Intensity of illumination is 2200lx, is placed under conditions of cultivation temperature is 29 DEG C and cultivates, repeatedly cutting carries out successive transfer culture repeatedly, obtains
Rudiment；
Step 3, root culture: the Multiple Buds bud cutting that the height that step (1) or (2) process are obtained is about 3.6cm is inoculated into
Carry out root culture on root media, first full light culture 4 days under the conditions of 29 DEG C after inoculation, then daily illumination 10 hours,
Intensity of illumination is 3200lx, and cultivation temperature is cultivated to taking root under conditions of being 29 DEG C；
Step 4, acclimatization and transplantses: the rooting tube plantlet of the high about 9cm that step (3) is obtained goes bottle cap to be placed in natural lighting lower refining seedling
After 8 days, test tube seedling is taken out from culture bottle, wash root culture medium off, plant into by Nutrition Soil: the base that sandy soil=3:1 is mixed into
Matter, is placed in culture in illumination box, is watered to seedling with 1/2ms macro-element nutrients liquid daily, keep humidity, treats that seedling becomes
Land for growing field crops is transplanted again after work.
2. a kind of method for building up of Chinese larch tissue culture rapid propagation system according to claim 1 is it is characterised in that step (1) institute
The primary culture medium stated is: ms+4mg/l6-ba+1.2mg/l naa+32g/l sucrose+8g/l agar, and ph is 5.9.
3. a kind of method for building up of Chinese larch tissue culture rapid propagation system according to claim 1 is it is characterised in that step (2) is described
Proliferated culture medium be: n6+ 2mg/lnaa+4mg/l 6-ba+35g/l sucrose+8g/l agar, ph is 5.9.
4. a kind of method for building up of Chinese larch tissue culture rapid propagation system according to claim 1 is it is characterised in that step (3) is described
Root media be: 1/2ms+2mg/l naa+2mg/l iba+35g/l sucrose+8g/l agar, ph be 5.9.
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| CN107660464A (en) * | 2017-10-24 | 2018-02-06 | 贵州省林业学校(贵州省林业干部学校) | A kind of tissue culture and rapid propagation method of sequoia sempervirens good seed |
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