CN108522289A - A kind of Bougainvillea spectabilis detoxification method for tissue culture - Google Patents
A kind of Bougainvillea spectabilis detoxification method for tissue culture Download PDFInfo
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- CN108522289A CN108522289A CN201810661263.5A CN201810661263A CN108522289A CN 108522289 A CN108522289 A CN 108522289A CN 201810661263 A CN201810661263 A CN 201810661263A CN 108522289 A CN108522289 A CN 108522289A
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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
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Abstract
The invention discloses a kind of Bougainvillea spectabilis detoxification method for tissue culture, include the following steps：（1）Selection：Training choose sprout within 1 ~ 2 year it is full, without rotten disease-free, healthy and strong bougainvillea spectabilis branch；（2）Vernalization：The bougainvillea spectabilis branch chosen is subjected to vernalization；（3）Surface treatment；（4）Disinfection；（5）Cutting；（6）Initial culture；（7）Proliferation and subculture：Aseptic seedling is inoculated into progress proliferation and subculture culture on corresponding culture medium；（8）Culture of rootage；（9）Tame hardening.The beneficial effects of the invention are as follows：By tissue cultures Bougainvillea spectabilis quality and quality is greatly improved, and reduce cost to greatest extent in Bougainvillea spectabilis, improves breeding coefficient, and providing technical basis for Bougainvillea spectabilis test tube seedling factorial praluction is of great significance；The technical barrier in Bougainvillea spectabilis tissue culture procedures has been captured, good culture effect is reached.
Description
Technical field
The invention belongs to field of plant tissue culture technique, more particularly to a kind of Bougainvillea spectabilis detoxification tissue cultures side
Method.
Background technology
Bougainvillea spectabilis is the evergreen wooden big rattan wood plant of Nyctaginaceae bougainvillea, belongs to the sight flowering shrub class in flowering tree,
It is important ornamental flower and excellent garden plants.It is had a graceful carriage, the light green soft cottonrose hibiscus of branches and leaves, from the spring in early autumn to next year
End, flowering time is for half a year, and the flowers extensive as a kind of Unique, cultivation management are very popular.Triangle
When plum is as flower culture, it is often trimmed to shrub, dungarunga shape or multilayer pagoda shape, therefore is cried " paper flower " in Japan；Again because of it
Colored bract is lobate, ellipse or ellipticity lanceolar, there is the multiple colors such as red, white, orange, purple, secondary color, rich in variety like Du
Cuckoo, therefore be referred to as " strangling cuckoo " in Guangzhou；Again since its shoot top is typically three fasciations among three pieces bract, flower
The beautiful purple goblin of bract of periphery is red, is similar to triangle, therefore in Fujian also known as " Bougainvillea spectabilis " or " bougainvillea "." precious towel is in botany
Transliteration of the flower " then from its Classification system.It may then be gained the name due to it blooming around the Spring Festival " gathering red " in Hong Kong.
Bougainvillea spectabilis platymiscium serves not only as flowers and is concerned, and the research in recent years in relation to Bougainvillea spectabilis other application value makes people
There is new understanding to it.The isolated pine camphor with para-insulin function is to substituting insulin therapy from Bougainvillea spectabilis blade
The side effect that diabetes and mitigation insulinization are brought is of great significance.Research is it is also shown that in Bougainvillea spectabilis blade and root
At least contain 2 kinds of antiviral protein-BAPI and Bouganin, they have different degrees of suppression for various plants virus
It makes and uses.These results of study have very positive meaning for the efficient biological pesticide of exploitation low toxicity.Bougainvillea spectabilis is still ground
Study carefully one of the important model plant of beet pigments metabolism, the type and content of beet pigments are enriched for the day of exploitation safety
Right food coloring opens wide foreground.These correlative studys illustrate multi-value of the Bougainvillea spectabilis as resource plant, are
Further the ornamental of exploitation Bougainvillea spectabilis, medicine, biological pesticide are worth and make as natural food colour and performance environment remediation
With providing sufficient scientific basis.
In recent years, it is domestic that be concentrated mainly on biological characteristics, cultivation management, breeding, cuttage for the research of Bougainvillea spectabilis numerous
It grows, Greenland Application etc..As people are to deepening continuously of studying of Bougainvillea spectabilis and the fast development of Urban Landscape Construction cause,
The traditional modes of reproduction of Bougainvillea spectabilis cannot increasingly meet the needs of market, because there are reproduction speeds for traditional modes of reproduction
Slowly, the drawbacks such as growth coefficient is small, and rootage duration is long, repoductive time is restricted；And with Bougainvillea spectabilis breeding research work into
Exhibition, people have cultivated the higher new varieties of many ornamental values, some new varieties are relatively more tired with traditional propagation method breeding
It is difficult, it is difficult to the nursery stock that a large amount of improved seeds or strain are provided in a short time, to affect expanding propagation and the city of Bougainvillea spectabilis
Field possession share.
Invention content
The purpose of the present invention is to overcome the defect of above-mentioned Bougainvillea spectabilis tissue cultures, to the detoxification tissue cultures side of Bougainvillea spectabilis
Method carries out systematic research, by many experiments, obtains a kind of culture side easy to operate, improving Bougainvillea spectabilis yield and quality
Method.
To achieve the above object, the present invention uses following technical proposals：
A kind of Bougainvillea spectabilis detoxification method for tissue culture, including：
(1) selection：Training chooses life in 1 ~ 2 year without rotten disease-free, healthy and strong bougainvillea spectabilis branch；
(2) vernalization：The bougainvillea spectabilis branch chosen is inserted in the bottle with water, is placed in illumination room, 25 ~ 30 DEG C of temperature is maintained,
3000 ~ 5000Lux of intensity of illumination, light application time for 24 hours, irradiate 5 ~ 10d, choose 3 ~ 7cm Bougainvillea spectabilis sprout segments；
(3) it is surface-treated：The Bougainvillea spectabilis sprout segment gathered is rinsed well using flowing water, 10 are cleaned with 2% Tween-80 ~
15min is rinsed well using flowing water；
(4) it sterilizes：It is impregnated using 75% alcohol, 0.1%HgCl 5 ~ 10min of solution disinfection, aseptic water washing is clean；
(5) it cuts：The Bougainvillea spectabilis sprout segment notch disinfected is updated processing, retains 2 ~ 3cm；
(6) Initial culture：The Bougainvillea spectabilis segment handled well is seeded on culture medium I, is positioned over 25 ~ 28 DEG C of temperature, per the daylight
It is 1000 ~ 3000Lux according to 18 ~ 22h, intensity of illumination, 40 ~ 50d of culture；
(7) proliferation and subculture：Selecting step（6）In obtained aseptic seedling be inoculated on culture medium II and carry out Multiplying culture, train
Foster condition is：25 ~ 28 DEG C of temperature, daily 10 ~ 14h of illumination, intensity of illumination are 5000 ~ 7000Lux, cultivate 25 ~ 35d；In repetition
State breeding；
(8) it takes root：Choose growing way stalwartness Bougainvillea spectabilis seedling, be seeded in culture medium III, be positioned over 25 ~ 28 DEG C of temperature, often
In 12 ~ 16h of its illumination, the environment that intensity of illumination is 5000 ~ 7000Lux, 20 ~ 30d is cultivated；
(9) hardening is tamed：The Bougainvillea spectabilis seedling for the well developed root system that step (8) obtains is transferred in small temperature canopy and is tamed, is shaded,
It is 15 ~ 30 DEG C to keep temperature in small temperature canopy, and humid control humidity after 85 ~ 95%, 7d is decremented to 80%, hereafter gradually successively decreases, 15d
Bougainvillea spectabilis seedling is removed from tissue culture medium (TCM) afterwards, the culture medium of root is cleaned up, in plantation to substrate soil；
Wherein：Culture medium I is 0.5 ~ 1.5mg/LIBA of addition, 1.0 ~ 3.0mg/LZT, 25 ~ 35g/L sucrose in MS improved culture mediums
With 2 ~ 5g/L agar；
Culture medium II be MS improved culture mediums in addition 4 ~ 5mg/LZT, 0.01 ~ 0.5mg/LIAA, 0.1 ~ 1mg/LNAA, 25 ~
35g/L sucrose and 2 ~ 5g/L agar；
Culture medium III be 0.1 ~ 1mg/LNAA of addition, 0.1 ~ 0.5mg/LIBA, 15 ~ 25g/L sucrose and 2 in MS improved culture mediums ~
5g/L agar.
As an optimization, a kind of Bougainvillea spectabilis detoxification method for tissue culture, including：
(1) selection：Training chooses life in 1 ~ 2 year without rotten disease-free, healthy and strong bougainvillea spectabilis branch；
(2) vernalization：The bougainvillea spectabilis branch chosen is inserted in the bottle with water, is placed in illumination room, 25 ~ 30 DEG C of temperature is maintained,
4000 ~ 5000Lux of intensity of illumination, light application time for 24 hours, irradiate 7d, choose 4 ~ 6cm Bougainvillea spectabilis sprout segments；
(3) it is surface-treated：The Bougainvillea spectabilis sprout segment gathered is rinsed well using flowing water, 10 are cleaned with 2% Tween-80 ~
15min is rinsed well using flowing water；
(4) it sterilizes：It is impregnated using 75% alcohol, 0.1%HgCl 5 ~ 10min of solution disinfection, aseptic water washing is clean；
(5) it cuts：The Bougainvillea spectabilis sprout segment notch disinfected is updated processing, retains 2 ~ 3cm；
(6) Initial culture：The Bougainvillea spectabilis segment handled well is seeded on culture medium I, is positioned over 25 ~ 26 DEG C of temperature, per the daylight
According to 20h, intensity of illumination 2000Lux, 45 ~ 50d of culture；
(7) proliferation and subculture：Selecting step（6）In obtained aseptic seedling be inoculated on culture medium II and carry out Multiplying culture, train
Foster condition is：25 ~ 26 DEG C of temperature, daily illumination 12h, intensity of illumination are 5000 ~ 7000Lux, cultivate 25 ~ 30d；Repeat above-mentioned increasing
Grow process；
(8) it takes root：Choose growing way stalwartness Bougainvillea spectabilis aseptic seedling, be seeded in culture medium III, be positioned over 25 ~ 26 DEG C of temperature,
In daily illumination 14h, the environment that intensity of illumination is 5000 ~ 7000Lux, 25 ~ 30d is cultivated；
(9) hardening is tamed：The Bougainvillea spectabilis seedling for the well developed root system that step (8) obtains is transferred in small temperature canopy and is tamed, is shaded,
It is 20 ~ 30 DEG C to keep temperature in small temperature canopy, and humid control humidity after 85 ~ 95%, 7d is decremented to 80%, hereafter gradually successively decreases, 15d
Bougainvillea spectabilis seedling is removed from tissue culture medium (TCM) afterwards, the culture medium of root is cleaned up, in plantation to substrate soil；
As an optimization, culture medium I be MS improved culture mediums in addition 0.5 ~ 1.5mg/LIBA, 1.0 ~ 3.0mg/LZT, 25 ~
35g/L sucrose and 2 ~ 5g/L agar.
As an optimization, culture medium II be MS improved culture mediums in addition 4 ~ 5mg/LZT, 0.01 ~ 0.5mg/LIAA, 0.1 ~
1mg/LNAA, 25 ~ 35g/L sucrose and 2 ~ 5g/L agar.
As an optimization, culture medium III is 0.1 ~ 1mg/LNAA of addition, 0.1 ~ 0.5mg/LIBA, 15 in MS improved culture mediums
~ 25g/L sucrose and 2 ~ 5g/L agar.
As an optimization, the pH of the culture medium I, culture medium II and culture medium II is adjusted to 5.8.
As an optimization, the alcohol disinfecting time is no more than 1min in the step (4).
As an optimization, the step（9）Artificially feed is carried out under the conditions of the middle domestication hardening link cloudy day, light filling intensity is
3000~5000Lux。
As an optimization, the step（9）Middle domestication hardening step, be transferred to small temperature canopy after 10 ~ 15 days daily at dusk or
Early morning divulges information half an hour.
It is tobacco cell Training Design that MS culture mediums, which were Murashige and Skoog in 1962, its main feature is that inorganic salts
It is higher with ion concentration, it is more stable ionic equilibrium solution, its nitrate content is high, and the quantity and ratio of nutrient are closed
It is suitable, the nutrition and physiological requirements of plant cell can be met, thus the scope of application is wider, most plants tissue-culturing quick-propagation
Use it as the minimal medium of culture medium.MS improved culture mediums are to remove inositol on the basis of MS culture mediums, and ammonium nitrate subtracts
Half, increase ascorbic acid 10mg/L.
Heteroauxin (IAA)：The Endogenous auxin of generally existing in a kind of plant, belongs to Benzazole compounds, You Mingzhuo
Long element, auxin, different growth hormone, are a kind of organic matters, are stablized to visible light, have duality, plant different plant growth
Position is more than stem to its susceptibility difference, general root, and different plants are also different to its sensitivity.
Indolebutyric acid（IBA）：Plant growth regulator is used for cell division and hyperplasia, promotes draft and woody plant
The root of object it is mitogenetic.
Methyl α-naphthyl acetate (NAA)：It is the intermediate for the plant growth regulator and naphthaleneacetamide for promoting plant root growth.
Methyl α-naphthyl acetate is used as plant growth regulator, has and promotes cell division and expand, induced synthesis adventitious root, increase is beared fruit, prevented
Shedding etc., can be with nutrition stream transporting to site of action.
Zeatin（ZT）, it is a kind of basic element of cell division, belongs to six big natural phytohormone, that is, auxin, gibberellin, cell point
One of element, abscisic acid, ethylene and brassin lactones are split, corn is named as because being separated to a kind of analog from corn for the first time
Element, its physiological function is more complicated, including promotes plant cell division, prevents chlorophyll and protein degradation, slows down breathing
Effect keeps cell viability, delays plant senesecence etc..
Agar：The most useful characteristic of agar is that the temperature difference between its condensation point and fusing point is very big.It needs to add in water
Just start to melt when heat is to 95 DEG C, just start to solidify when the solution temperature after fusing need to drop to 40 DEG C, so it is to prepare solid
The best coagulator of culture medium.The solid medium prepared with agar, is used for high-temperature cultivation and non-fusible, in solidification
When preceding inoculation, culture will not also be scalded dead.Therefore, agar is to prepare most widely used one kind in various biological mediums to coagulate
Gu agent.
Sucrose：Sucrose is in addition to energy supply, moreover it is possible to which evoked callus tissue breaks up again；When culture medium high-temperature sterilization, sugarcane
Sugar is more stable and is not easy to be carbonized；Contain invertase in plant cell, sucrose can preferably remain hypotonic in culture medium
Environment, the osmotic pressure that sucrose is formed can keep relative stability for a long time, can reduce the pollution of microorganism to a certain extent.
The beneficial effects of the invention are as follows：
1. Bougainvillea spectabilis can play the role of detoxification, fast numerous, Germ-plasma resources protection, quality-improving, significantly by tissue cultures
Bougainvillea spectabilis quality and quality are improved, the planting scale of Bougainvillea spectabilis is expanded.
2. the medium component that control differentiation, subculture grow, take root etc. is determined in tissue cultures, growth has been determined
The optimum optimization selection of the type, concentration and condition of culture of Auto-regulator, has captured the skill in Bougainvillea spectabilis tissue culture procedures
Art barrier reaches good culture effect.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Obtain other attached drawings according to these attached drawings.
Fig. 1 is the Initial culture step Bougainvillea spectabilis schematic diagram of the present invention；
Fig. 2 is proliferation and subculture step Bougainvillea spectabilis schematic diagram of the present invention；
Fig. 3 is that the present invention is taken root step Bougainvillea spectabilis schematic diagram.
Specific implementation mode
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
The every other embodiment that art personnel are obtained without creative efforts belongs to the model that the present invention protects
It encloses.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or according to proposed by manufacturer
Condition, unless stated otherwise, ratio and percentage are based on weight.
It is commercially available that the embodiment of the present invention is raw materials used, culture medium I in the embodiment, culture medium II and culture medium II
PH is adjusted to 5.8.
Embodiment 1, a kind of Bougainvillea spectabilis detoxification method for tissue culture, including：
(1) selection：Training chooses life in 1 ~ 2 year without rotten disease-free, healthy and strong bougainvillea spectabilis branch；
(2) vernalization：The bougainvillea spectabilis branch chosen is inserted in the bottle with water, is placed in illumination room, 28 ~ 30 DEG C of temperature is maintained,
Intensity of illumination 3000Lux, light application time for 24 hours, irradiate 7d, choose 3 ~ 7cm Bougainvillea spectabilis sprout segments；
(3) it is surface-treated：The Bougainvillea spectabilis sprout segment gathered is rinsed well using flowing water, 10 are cleaned with 2% Tween-80 ~
15min is rinsed well using flowing water；
(4) it sterilizes：55s, 0.1%HgCl solution disinfection 5min are impregnated using 75% alcohol, aseptic water washing is clean；
(5) it cuts：The Bougainvillea spectabilis sprout segment notch disinfected is updated processing, retains 2 ~ 3cm；
(6) Initial culture：The Bougainvillea spectabilis segment handled well is seeded on culture medium I, is positioned over 26 ~ 27 DEG C of temperature, per the daylight
According to 22h, intensity of illumination 1000Lux, culture 45d；Culture medium I is that 0.5mg/LIBA, 2.0mg/ are added in MS improved culture mediums
LZT, 35g/L sucrose and 2g/L agar；
(7) proliferation and subculture：Selecting step（6）In obtained aseptic seedling be inoculated on culture medium II and carry out Multiplying culture, train
Foster condition is：27 ~ 28 DEG C of temperature, daily illumination 10h, intensity of illumination 6000Lux, culture 35d；Repeat above-mentioned breeding；
Culture medium II is addition 4.5mg/LZT, 0.5mg/LIAA, 0.1mg/LNAA, 30g/L sucrose and 5g/L fine jades in MS improved culture mediums
Fat；
(8) it takes root：Choose growing way stalwartness Bougainvillea spectabilis aseptic seedling, be seeded in culture medium III, be positioned over 25 ~ 26 DEG C of temperature,
In daily illumination 14h, the environment that intensity of illumination is 7000Lux, 20d is cultivated；Culture medium III is to be added in MS improved culture mediums
0.1mg/LNAA, 0.2mg/LIBA, 25g/L sucrose and 2g/L agar.The Bougainvillea spectabilis aseptic seedling of well developed root system, the item number of root is 7 ~
8, length is 10 ~ 20min, is conveniently attached in substrate soil, avoids hindering root.
(9) hardening is tamed：The Bougainvillea spectabilis seedling for the well developed root system that step (8) obtains is transferred in small temperature canopy and is tamed,
Shade, it is 20 ~ 25 DEG C to keep temperature in small temperature canopy, and humid control humidity after 85 ~ 95%, 7d is decremented to 80%, hereafter gradually passs
Subtract, be transferred to small temperature canopy after 10 ~ 15 days daily at dusk or ventilation half an hour in early morning, by Bougainvillea spectabilis seedling from tissue culture medium (TCM) after 15d
Middle removal cleans up the culture medium of root, in plantation to substrate soil；Artificially feed, light filling intensity are carried out under the conditions of cloudy day
For 3000Lux.
The Bougainvillea spectabilis shoot survival percent obtained by this Bougainvillea spectabilis detoxification method for tissue culture is 99% or more, Bougainvillea spectabilis yield
All it is greatly improved with quality.
A kind of 2 Bougainvillea spectabilis detoxification method for tissue culture of embodiment, including：
(1) selection：Training chooses life in 1 ~ 2 year without rotten disease-free, healthy and strong bougainvillea spectabilis branch；
(2) vernalization：The bougainvillea spectabilis branch chosen is inserted in the bottle with water, is placed in illumination room, 26 ~ 28 DEG C of temperature is maintained,
Intensity of illumination 5000Lux, light application time for 24 hours, irradiate 5d, choose 3 ~ 7cm Bougainvillea spectabilis sprout segments；
(3) it is surface-treated：The Bougainvillea spectabilis sprout segment gathered is rinsed well using flowing water, 10 are cleaned with 2% Tween-80 ~
15min is rinsed well using flowing water；
(4) it sterilizes：55s, 0.1%HgCl solution disinfection 10min are impregnated using 75% alcohol, aseptic water washing is clean；
(5) it cuts：The Bougainvillea spectabilis sprout segment notch disinfected is updated processing, retains 2 ~ 3cm；
(6) Initial culture：The Bougainvillea spectabilis segment handled well is seeded on culture medium I, is positioned over 25 ~ 26 DEG C of temperature, per the daylight
According to 20h, intensity of illumination 3000Lux, culture 40d；Culture medium I is that 1.5mg/LIBA, 1.0mg/ are added in MS improved culture mediums
LZT, 30g/L sucrose and 5g/L agar；
(7) proliferation and subculture：Selecting step（6）In obtained aseptic seedling be inoculated on culture medium II and carry out Multiplying culture, train
Foster condition is：26 ~ 27 DEG C of temperature, daily illumination 14h, intensity of illumination 5000Lux, culture 30d；Repeat above-mentioned breeding；
Culture medium II is addition 4mg/LZT, 0.3mg/LIAA, 1mg/LNAA, 25g/L sucrose and 3.5g/L fine jades in MS improved culture mediums
Fat；
(8) it takes root：Choose growing way stalwartness Bougainvillea spectabilis aseptic seedling, be seeded in culture medium III, be positioned over 27 ~ 28 DEG C of temperature,
In daily illumination 12h, the environment that intensity of illumination is 6000Lux, 30d is cultivated；Culture medium III is to be added in MS improved culture mediums
1mg/LNAA, 0.1mg/LIBA, 20g/L sucrose and 5g/L agar.The item number of the Bougainvillea spectabilis aseptic seedling of well developed root system, root is 7 ~ 8
Item, length are 10 ~ 20min, are conveniently attached in substrate soil, avoid hindering root.
(9) hardening is tamed：The Bougainvillea spectabilis seedling for the well developed root system that step (8) obtains is transferred in small temperature canopy and is tamed,
Shade, it is 15 ~ 20 DEG C to keep temperature in small temperature canopy, and humid control humidity after 85 ~ 90%, 7d is decremented to 80%, hereafter gradually passs
Subtract, be transferred to small temperature canopy after 10 ~ 15 days daily at dusk or ventilation half an hour in early morning, by Bougainvillea spectabilis seedling from tissue culture medium (TCM) after 15d
Middle removal cleans up the culture medium of root, in plantation to substrate soil；Artificially feed, light filling intensity are carried out under the conditions of cloudy day
For 4000Lux.
The Bougainvillea spectabilis shoot survival percent obtained by this Bougainvillea spectabilis detoxification method for tissue culture is 99% or more, Bougainvillea spectabilis yield
All it is greatly improved with quality.
A kind of 3 Bougainvillea spectabilis detoxification method for tissue culture of embodiment, including：
(1) selection：Training chooses life in 1 ~ 2 year without rotten disease-free, healthy and strong bougainvillea spectabilis branch；
(2) vernalization：The bougainvillea spectabilis branch chosen is inserted in the bottle with water, is placed in illumination room, 25 ~ 26 DEG C of temperature is maintained,
Intensity of illumination 4000Lux, light application time for 24 hours, irradiate 10d, choose 3 ~ 7cm Bougainvillea spectabilis sprout segments；
(3) it is surface-treated：The Bougainvillea spectabilis sprout segment gathered is rinsed well using flowing water, 10 are cleaned with 2% Tween-80 ~
15min is rinsed well using flowing water；
(4) it sterilizes：55s, 0.1%HgCl solution disinfection 10min are impregnated using 75% alcohol, aseptic water washing is clean；
(5) it cuts：The Bougainvillea spectabilis sprout segment notch disinfected is updated processing, retains 2 ~ 3cm；
(6) Initial culture：The Bougainvillea spectabilis segment handled well is seeded on culture medium I, is positioned over 27 ~ 28 DEG C of temperature, per the daylight
According to 18h, intensity of illumination 2000Lux, culture 50d；Culture medium I is that 1.0mg/LIBA, 3.0mg/ are added in MS improved culture mediums
LZT, 25g/L sucrose and 3.5g/L agar；
(7) proliferation and subculture：Selecting step（6）In obtained aseptic seedling be inoculated on culture medium II and carry out Multiplying culture, train
Foster condition is：25 ~ 26 DEG C of temperature, daily illumination 12h, intensity of illumination 7000Lux, culture 25d；Repeat above-mentioned breeding；
Culture medium II is addition 5mg/LZT, 0.01mg/LIAA, 0.5mg/LNAA, 35g/L sucrose and 2g/L fine jades in MS improved culture mediums
Fat；
(8) it takes root：Choose growing way stalwartness Bougainvillea spectabilis aseptic seedling, be seeded in culture medium III, be positioned over 26 ~ 27 DEG C of temperature,
In daily illumination 16h, the environment that intensity of illumination is 5000Lux, 25d is cultivated；Culture medium III is to be added in MS improved culture mediums
0.5mg/LNAA, 0.5mg/LIBA, 15g/L sucrose and 3.5g/L agar.The item number of the Bougainvillea spectabilis aseptic seedling of well developed root system, root is
7 ~ 8, length is 10 ~ 20min, is conveniently attached in substrate soil, avoids hindering root.
(9) hardening is tamed：The Bougainvillea spectabilis seedling for the well developed root system that step (8) obtains is transferred in small temperature canopy and is tamed,
Shade, it is 25 ~ 30 DEG C to keep temperature in small temperature canopy, and humid control humidity after 85 ~ 95%, 7d is decremented to 80%, hereafter gradually passs
Subtract, be transferred to small temperature canopy after 10 ~ 15 days daily at dusk or ventilation half an hour in early morning, by Bougainvillea spectabilis seedling from tissue culture medium (TCM) after 15d
Middle removal cleans up the culture medium of root, in plantation to substrate soil；Artificially feed, light filling intensity are carried out under the conditions of cloudy day
For 3000Lux.
The Bougainvillea spectabilis shoot survival percent obtained by this Bougainvillea spectabilis detoxification method for tissue culture is 99% or more, Bougainvillea spectabilis yield
All it is greatly improved with quality.
A kind of 4 Bougainvillea spectabilis detoxification method for tissue culture of embodiment, including：
(1) selection：Training chooses life in 1 ~ 2 year without rotten disease-free, healthy and strong bougainvillea spectabilis branch；
(2) vernalization：The bougainvillea spectabilis branch chosen is inserted in the bottle with water, is placed in illumination room, 25 ~ 30 DEG C of temperature is maintained,
4000 ~ 5000Lux of intensity of illumination, light application time for 24 hours, irradiate 7d, choose 4 ~ 6cm Bougainvillea spectabilis sprout segments；
(3) it is surface-treated：The Bougainvillea spectabilis sprout segment gathered is rinsed well using flowing water, 10 are cleaned with 2% Tween-80 ~
15min is rinsed well using flowing water；
(4) it sterilizes：It is impregnated using 75% alcohol, 0.1%HgCl 5 ~ 10min of solution disinfection, aseptic water washing is clean；
(5) it cuts：The Bougainvillea spectabilis sprout segment notch disinfected is updated processing, retains 2 ~ 3cm；
(6) Initial culture：The Bougainvillea spectabilis segment handled well is seeded on culture medium I, is positioned over 25 ~ 26 DEG C of temperature, per the daylight
According to 20h, intensity of illumination 2000Lux, culture 45d；Culture medium I is that 1.0mg/LIBA, 2.0mg/ are added in MS improved culture mediums
LZT, 30g/L sucrose and 3.5g/L agar.
(7) proliferation and subculture：Selecting step（6）In obtained aseptic seedling be inoculated on culture medium II and carry out proliferation training
It supports, condition of culture is：25 ~ 26 DEG C of temperature, daily illumination 12h, intensity of illumination are 5500 ~ 6000Lux, cultivate 28d；It repeats above-mentioned
Breeding；Culture medium II is that 3mg/LZT, 0.1mg/LIAA, 0.5mg/LNAA, 30g/L sucrose are added in MS improved culture mediums
With 3.5g/L agar.
(8) it takes root：Choose growing way stalwartness Bougainvillea spectabilis aseptic seedling, be seeded in culture medium III, be positioned over temperature 25 ~
26 DEG C, daily illumination 14h, intensity of illumination be 6000 ~ 6500Lux environment in, cultivate 28d；Culture medium III is MS improvement cultures
0.5mg/LNAA, 0.2mg/LIBA, 20g/L sucrose and 3.5g/L agar are added in base.The Bougainvillea spectabilis aseptic seedling of well developed root system, root
Item number be 7 ~ 8, length be 10 ~ 20min, be conveniently attached in substrate soil, avoid hindering root.
(9) hardening is tamed：The Bougainvillea spectabilis seedling for the well developed root system that step (8) obtains is transferred in small temperature canopy and is tamed,
Shade, it is 20 ~ 28 DEG C to keep temperature in small temperature canopy, and humid control humidity after 85 ~ 95%, 7d is decremented to 80%, hereafter gradually passs
Subtract, be transferred to small temperature canopy after 10 ~ 15 days daily at dusk or ventilation half an hour in early morning, by Bougainvillea spectabilis seedling from tissue culture medium (TCM) after 15d
Middle removal cleans up the culture medium of root, in plantation to substrate soil；Artificially feed, light filling intensity are carried out under the conditions of cloudy day
For 3500 ~ 4000Lux.
The Bougainvillea spectabilis shoot survival percent obtained by this Bougainvillea spectabilis detoxification method for tissue culture is 99% or more, Bougainvillea spectabilis yield
All it is greatly improved with quality.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention god.
Claims (9)
1. a kind of Bougainvillea spectabilis detoxification method for tissue culture, it is characterised in that：Including：
(1) selection：Training chooses life in 1 ~ 2 year without rotten disease-free, healthy and strong bougainvillea spectabilis branch；
(2) vernalization：The bougainvillea spectabilis branch chosen is inserted in the bottle with water, is placed in illumination room, 25 ~ 30 DEG C of temperature is maintained,
3000 ~ 5000Lux of intensity of illumination, light application time for 24 hours, irradiate 5 ~ 10d, choose 3 ~ 7cm Bougainvillea spectabilis sprout segments；
(3) it is surface-treated：The Bougainvillea spectabilis sprout segment gathered is rinsed well using flowing water, 10 are cleaned with 2% Tween-80 ~
15min is rinsed well using flowing water；
(4) it sterilizes：It is impregnated using 75% alcohol, 0.1%HgCl 5 ~ 10min of solution disinfection, aseptic water washing is clean；
(5) it cuts：The Bougainvillea spectabilis sprout segment notch disinfected is updated processing, retains 2 ~ 3cm；
(6) Initial culture：The Bougainvillea spectabilis segment handled well is seeded on culture medium I, is positioned over 25 ~ 28 DEG C of temperature, per the daylight
It is 1000 ~ 3000Lux according to 18 ~ 22h, intensity of illumination, 40 ~ 50d of culture；
(7) proliferation and subculture：Selecting step（6）In obtained aseptic seedling be inoculated on culture medium II and carry out Multiplying culture, train
Foster condition is：25 ~ 28 DEG C of temperature, daily 10 ~ 14h of illumination, intensity of illumination are 5000 ~ 7000Lux, cultivate 25 ~ 35d；In repetition
State breeding；
(8) it takes root：Choose growing way stalwartness Bougainvillea spectabilis seedling, be seeded in culture medium III, be positioned over 25 ~ 28 DEG C of temperature, often
In 12 ~ 16h of its illumination, the environment that intensity of illumination is 5000 ~ 7000Lux, 20 ~ 30d is cultivated；
(9) hardening is tamed：The Bougainvillea spectabilis seedling for the well developed root system that step (8) obtains is transferred in small temperature canopy and is tamed, is shaded,
It is 15 ~ 30 DEG C to keep temperature in small temperature canopy, and humid control humidity after 85 ~ 95%, 7d is decremented to 80%, hereafter gradually successively decreases, 15d
Bougainvillea spectabilis seedling is removed from tissue culture medium (TCM) afterwards, the culture medium of root is cleaned up, in plantation to substrate soil；
Wherein：Culture medium I is 0.5 ~ 1.5mg/LIBA of addition, 1.0 ~ 3.0mg/LZT, 25 ~ 35g/L sucrose in MS improved culture mediums
With 2 ~ 5g/L agar；
Culture medium II be MS improved culture mediums in addition 4 ~ 5mg/LZT, 0.01 ~ 0.5mg/LIAA, 0.1 ~ 1mg/LNAA, 25 ~
35g/L sucrose and 2 ~ 5g/L agar；
Culture medium III be 0.1 ~ 1mg/LNAA of addition, 0.1 ~ 0.5mg/LIBA, 15 ~ 25g/L sucrose and 2 in MS improved culture mediums ~
5g/L agar.
2. a kind of Bougainvillea spectabilis detoxification method for tissue culture according to claim 1, it is characterised in that：Including：
(1) selection：Training chooses life in 1 ~ 2 year without rotten disease-free, healthy and strong bougainvillea spectabilis branch；
(2) vernalization：The bougainvillea spectabilis branch chosen is inserted in the bottle with water, is placed in illumination room, 25 ~ 30 DEG C of temperature is maintained,
4000 ~ 5000Lux of intensity of illumination, light application time for 24 hours, irradiate 7d, choose 4 ~ 6cm Bougainvillea spectabilis sprout segments；
(3) it is surface-treated：The Bougainvillea spectabilis sprout segment gathered is rinsed well using flowing water, 10 are cleaned with 2% Tween-80 ~
15min is rinsed well using flowing water；
(4) it sterilizes：It is impregnated using 75% alcohol, 0.1%HgCl 5 ~ 10min of solution disinfection, aseptic water washing is clean；
(5) it cuts：The Bougainvillea spectabilis sprout segment notch disinfected is updated processing, retains 2 ~ 3cm；
(6) Initial culture：The Bougainvillea spectabilis segment handled well is seeded on culture medium I, is positioned over 25 ~ 26 DEG C of temperature, per the daylight
According to 20h, intensity of illumination 2000Lux, 45 ~ 50d of culture；
(7) proliferation and subculture：Selecting step（6）In obtained aseptic seedling be inoculated on culture medium II and carry out Multiplying culture, train
Foster condition is：25 ~ 26 DEG C of temperature, daily illumination 12h, intensity of illumination are 5000 ~ 7000Lux, cultivate 25 ~ 30d；Repeat above-mentioned increasing
Grow process；
(8) it takes root：Choose growing way stalwartness Bougainvillea spectabilis aseptic seedling, be seeded in culture medium III, be positioned over 25 ~ 26 DEG C of temperature,
In daily illumination 14h, the environment that intensity of illumination is 5000 ~ 7000Lux, 25 ~ 30d is cultivated；
(9) hardening is tamed：The Bougainvillea spectabilis seedling for the well developed root system that step (8) obtains is transferred in small temperature canopy and is tamed, is shaded,
It is 20 ~ 30 DEG C to keep temperature in small temperature canopy, and humid control humidity after 85 ~ 95%, 7d is decremented to 80%, hereafter gradually successively decreases, 15d
Bougainvillea spectabilis seedling is removed from tissue culture medium (TCM) afterwards, the culture medium of root is cleaned up, in plantation to substrate soil.
3. a kind of Bougainvillea spectabilis detoxification method for tissue culture according to claim 1, it is characterised in that：Culture medium I changes for MS
1.0mg/LIBA, 2.0mg/LZT, 30g/L sucrose and 3.5g/L agar are added in good culture medium.
4. a kind of Bougainvillea spectabilis detoxification method for tissue culture according to claim 1, it is characterised in that：Culture medium II changes for MS
3mg/LZT, 0.1mg/LIAA, 0.5mg/LNAA, 30g/L sucrose and 3.5g/L agar are added in good culture medium.
5. a kind of Bougainvillea spectabilis detoxification method for tissue culture according to claim 1, it is characterised in that：Culture medium III changes for MS
0.5mg/LNAA, 0.2mg/LIBA, 20g/L sucrose and 3.5g/L agar are added in good culture medium.
6. a kind of Bougainvillea spectabilis detoxification method for tissue culture according to claim 1, it is characterised in that：The culture medium I, training
The pH for supporting base II and culture medium II is adjusted to 5.8.
7. a kind of Bougainvillea spectabilis detoxification method for tissue culture according to claim 1, it is characterised in that：In the step (4)
The alcohol disinfecting time is no more than 1min.
8. a kind of Bougainvillea spectabilis detoxification method for tissue culture according to claim 1, it is characterised in that：The step（9）In
Artificially feed is carried out under the conditions of the domestication hardening link cloudy day, light filling intensity is 3000 ~ 5000Lux.
9. a kind of Bougainvillea spectabilis detoxification method for tissue culture according to claim 1, it is characterised in that：The step（9）In
Hardening step is tamed, being transferred to small temperature canopy, dusk or early morning divulge information half an hour daily after 10 ~ 15 days.
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| CN112673957A (en) * | 2020-12-22 | 2021-04-20 | 中国热带农业科学院热带作物品种资源研究所 | Rapid breeding method of bougainvillea spectabilis tissue culture plants |
| CN115428731A (en) * | 2022-08-11 | 2022-12-06 | 厦门路桥百城建设投资有限公司 | Method for rapidly inducing roots through tide-type in-vitro cutting of bougainvillea spectabilis and application of method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109258467A (en) * | 2018-10-16 | 2019-01-25 | 广西壮族自治区林业科学研究院 | A method of preventing bougainvillea tissue-cultured seedling yellow |
| CN112673957A (en) * | 2020-12-22 | 2021-04-20 | 中国热带农业科学院热带作物品种资源研究所 | Rapid breeding method of bougainvillea spectabilis tissue culture plants |
| CN115428731A (en) * | 2022-08-11 | 2022-12-06 | 厦门路桥百城建设投资有限公司 | Method for rapidly inducing roots through tide-type in-vitro cutting of bougainvillea spectabilis and application of method |
| CN115428731B (en) * | 2022-08-11 | 2024-04-19 | 厦门路桥百城建设投资有限公司 | Triangle Mei Chaoxi type in-vitro cutting rapid root induction method and application thereof |
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