CN105638478B - In the method for callus induction bletilla tetraploid - Google Patents
In the method for callus induction bletilla tetraploid Download PDFInfo
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- CN105638478B CN105638478B CN201610069946.2A CN201610069946A CN105638478B CN 105638478 B CN105638478 B CN 105638478B CN 201610069946 A CN201610069946 A CN 201610069946A CN 105638478 B CN105638478 B CN 105638478B
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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
Abstract
The invention discloses in the method for callus induction bletilla tetraploid, short time induction processing is carried out to callus using the colchicine+3%DMSO of chemical inducer 0.05~0.2%, bred again, broken up and culture of rootage, be 2.6%~23.4% through experiment statisticses bletilla tetraploid induction rate.The inductivity of tetraploid can both be improved using the inventive method, chemical inducer can be reduced again to deleterious cellular effects.According to cellular omnipotency principle, then in atomization, a cell differentiation is a plant, is effectively reduced the generation of heterozygote.Therefore, this method has the advantages that simple to operate, easy to control, reproducible, inductivity is high, polyploid purifying rate is good.
Description
Technical field
The invention belongs to plant biotechnology field, specifically a kind of method with callus induction bletilla tetraploid.
Background technology
Bletilla（Bletilla striata(Thunb.) Reichb.f.）Also known as the bletilla striata, alias are purple blue, are orchid family bletillas
Belong to herbaceos perennial.Its stem tuber is China's traditional Chinese medicine, its cold nature, bitter, sweet, Bearberry Extract, removing blood stasis and hemostasis, tonifying lung life
Flesh, detumescence, for treating the diseases such as tuberculosis, hemoptysis, chronic gastric ulcer, traumatic bleeding and liver cancer；For " KUAIWEI PIAN ", " Wei Kang granule " etc.
The important component of Chinese patent drug.In addition, bletilla also has important application valency in terms of beauty and skin care, fine chemistry industry and fancy horticulture
Value.
Current bletilla raw material relies primarily on wild natural resources, and excessive collection and the destruction in habitat make resource quantity drastically
Reduce, endangered edge.As bletilla research deepens continuously, using increasingly extensive, market demand increases year by year so that supply
Contradiction is needed to be becoming increasingly acute, price persistently raises up, up to 400~500 Yuan ∕ kg.Therefore, bletilla artificial cultivation is imperative.
Under field conditions (factors), because the seed of bletilla is tiny and without endosperm, germination rate is extremely low.People are mainly by gathering this
The wild bletilla on ground breeds seedling in division propagation mode, and there have that variet complexity, breeding coefficient be low, germplasm is degenerated to be serious etc. many
Problem.And it is the key point that artificial cultivation succeeds to cultivate good quality and high output bletilla kind.
Plant polyploid has the characteristics such as plant " giantism ", active constituent content height, strong stress resistance, becomes raising medicine
With the important breeding methods of plant quality, yield and resistance.If tetraploid Pachyrhizua angulatus is than wild diploid biological yield raising ten
Several times；Radix angelicae dahuricae polyploid medicinal ingredient Imperatorin content is higher than former plant 2 times.As can be seen here, polyploid is yield increasing effect
Be greatly improved the entity of the perfect adaptation of two aspects with pharmaceutical ingredient content, be especially suitable for medicinal part for root tuber,
Stem tuber and the medicinal plant of complete stool.China has successively cultivated more than the 30 Chinese medicine polyploid such as the root of bidentate achyranthes, the red sage root, woaded blue, Radix Codonopsis
New varieties.
At present, Study on Inducing Polyploid is carried out to bletilla seed it has been reported that because bletilla kind is careful using chemical inducer
Small, when being cleaned after induction, seed suspension is in water, although can be solved by centrifugation, but due to bletilla seed
It must could be sprouted on culture medium, and carry out cleaning between superclean bench and centrifuge repeatedly, make seed contamination
Rate is significantly increased, and can cause test failure because of whole processing all pollution when serious.
The content of the invention
The problem of present invention during bletilla seed progress multiploid induction for existing, utilizes bletilla callus to carry out
Bletilla tetraploid induction.
The present invention comprises the following steps in the method for callus induction bletilla tetraploid：
（1）On superclean bench, bletilla callus is cut into 0.3cm × 0.3cm fritter, Multiplying culture is accessed
10 d are cultivated in base；Aseptically, the chemical inducer sterilized toward addition in blake bottle, chemical inducer immersion callus
Organize 8~24 h；
Described chemical inducer is：0.05~0.2% colchicine+3%DMSO；
The proliferated culture medium is：N61.0 mg/L+NAA of+2,4-D, 0.5 mg/L+TDZ 0.5 mg/L, pH6.0；Through
Cross the bletilla callus cell permeability increase of induction, N6In potassium element to can adjust intracellular suitable osmotic pressure and soda acid flat
Weighing apparatus, participates in endocellular sugar and protein metabolism, N6With plant growth regulator collective effect, promote the bletilla callus group induced
Knit the reparation and growth of cell.
（2）On superclean bench, by step（1）In callus take out, be put into sterilized water clean, with sterile filter
Paper is blotted to be cultivated in water, access differential medium, and 22~30 d produce bud point, and 36~43 D-shapeds grow 2 into bud, 55~65 d buds
~3 leaves, bud is sturdy, leaf color green；
The differential medium is：1.0 mg/L+ZT of MS+6-BA 0.5 mg/L+NAA0.5 mg/L, pH6.0；ZT with
6-BA has the division for promoting bletilla callus cell and differentiation, is used simultaneously with NAA, is conducive to improving differentiation frequency
Rate.
（3）On superclean bench, by step（2）In obtain bud access root media in cultivate, during 7 d under bud
There is projection in end, and 12~16 d start long root, and 45~50 d root length are about 1.0 cm；
The root media is：0.5 mg/L+IBA of 1/2MS+NAA, 1.0 mg/L+KT 0.4 mg/L, pH6.0；Should
NAA in culture medium has the effect of inducing adventitious root, and IBA has the growth for promoting adventitious root, and two auxin are cooperateed with using tool
Effect, improves the quality and quantity of root, and KT makes seedling sturdy with the horizontal thickening of cell is promoted, with auxin simultaneously using raising
The quality of seedling.
（4）The morning 9~11 point, on superclean bench, by step（3）In bletilla seedling carefully take out, be placed on sterilizing
In the plate crossed, the tip of a root of 0.5~1.0 cm length is cut, the tip of a root is immediately placed in pretreatment fluid, 3 h are handled, by seedling
Remaining root system excision, then culture in root media is accessed, and the tip of a root and seedling are numbered；
Described pretreatment fluid is：0.1% colchicine solution.
（5）By step（4）In pretreatment fluid in the tip of a root take out, fixed after being cleaned with distilled water with Ka Nuoshi fixers
3 h, the tip of a root fixed is cleaned with distilled water, is put into and is filled 1N hydrochloric acid（Concentrated hydrochloric acid：Water=1：11）Small beaker in, 60
DEG C or so water-bath in handle 15~20 min, water-bath finishes the cleaning of rear distilled water, cuts off root cap, leave Meristernatic zone,
To improve the min of moral training 20, the Meristernatic zone after dyeing is placed on clean slide, one is added and drips distilled water, by root
Point is broken into pieces, is covered and is capped a blotting paper after slide again and is gently pressed with thumb, is finally tapped and is nebulized until the tip of a root is scattered；
（6）By step（5）In ready-made slide tip of a root microscopy under the microscope, chromosome quantitative is small for 64
The numbering and quantity of seedling are recorded；It is 2.6%~23.4% to count bletilla tetraploid induction rate.
Learnt by inspection information, the chromosome number of bletilla diploid is 32, so the seedling that chromosome is 64 is
Tetraploid plant.
The callus proliferation culture, differentiation culture and the condition of culture in culture of rootage stage are：Cultivation temperature is 25
± 3 DEG C, light application time is 12 h/d, and intensity of illumination is 1500 Lx.
In the culture medium, MS and N6It is minimal medium, 2,4-D be 2,4- dichlorphenoxyacetic acids, and 6-BA is benzyl ammonia
Base purine, TDZ is thiadiazole phenylurea, and IBA is indole -3-butyric acid, and NAA is a- methyl α-naphthyl acetates, and ZT is zeatin, and KT is excitement
Element, reagent is that analysis is pure, and water is distilled water.
In the chemical inducer, DMSO is dimethyl sulfoxide (DMSO), and water is distilled water.
The present invention carries out bletilla tetraploid induction using bletilla callus.Callus is the proliferative cell production of explant
A raw irregular, loose parenchyma cell, cell proliferation rate is fast, has very strong mitogenetic energy in the presence of culture medium
Power and again differentiation capability, therefore, in the training period, a large amount of cells are in division stage.Chemical inducer is by colchicine and DMSO
Composition, DMSO has extremely strong permeability, contributes to medicine to be permeated into cell, while being also infiltrative protective agent, changes
Biomembrane is to the permeability of toxicant, beneficial to the excretion of noxious material.Callus is carried out in short-term using chemical inducer
Between induction handle, can both improve the inductivity of tetraploid, chemical inducer can be reduced again to deleterious cellular effects.According to cell
Totipotency principle, then in atomization, a cell differentiation is a plant, is effectively reduced the generation of heterozygote.Therefore, should
Method has the advantages that simple to operate, easy to control, reproducible, inductivity is high, polyploid purifying rate is good.
Brief description of the drawings
Fig. 1 is micro- enlarged diagram 2n=4x=64 of bletilla tetraploid chromosomes number in the embodiment of the present invention 1~3.
Embodiment
Present invention is further described with reference to embodiment, but is not limitation of the invention.
Embodiment 1
A kind of method with callus induction bletilla tetraploid, comprises the following steps：
（1）On superclean bench, callus is cut into 0.3cm × 0.3cm fritter, access proliferated culture medium
Cultivate 10 d；Aseptically, the chemical inducer sterilized toward addition in blake bottle, chemical inducer immersion callus
24 h；
Described chemical inducer is：0.05% colchicine+3%DMSO；
The proliferated culture medium is：N61.0 mg/L+NAA of+2,4-D, 0.5 mg/L+TDZ 0.5 mg/L, pH6.0；
（2）On superclean bench, by step（1）In callus take out, be put into sterilized water clean 5 times, in nothing
Blot and cultivated in water, access differential medium on bacterium filter paper, 22 d produce bud point, and 36 D-shapeds grow 2~3 into bud, 55 d buds
Leaf, bud is thin and delicate, leaf color yellow green；
The differential medium is：1.0 mg/L+ZT of MS+6-BA 0.5 mg/L+NAA0.5 mg/L, pH6.0；
（3）On superclean bench, by step（2）In bud access root media in cultivate, brought out during 7 d under bud
Existing projection, 12 d start long root, a length of 1.0 cm of 45 d roots；
The root media is：0.5 mg/L+IBA of 1/2MS+NAA, 1.0 mg/L+KT 0.4 mg/L, pH6.0；
（4）The morning 9~11 point, on superclean bench, by step（3）In bletilla seedling carefully take out, be placed on sterilizing
In the plate crossed, the tip of a root of about 0.5 cm length is cut, the tip of a root is immediately placed in pretreatment fluid, handle 3 h, will be remaining on seedling
Root system excision, then access in root media and cultivate, and the tip of a root and seedling are numbered；
Described pretreatment fluid is：0.1% colchicine solution；
（5）By step（4）In pretreatment fluid in the tip of a root take out, fixed after being cleaned with distilled water with Ka Nuoshi fixers
3 h, the tip of a root fixed is cleaned with distilled water, is put into and is filled 1N hydrochloric acid（Concentrated hydrochloric acid：Water=1：11）Small beaker in, 60
DEG C or so water-bath in handle 15~20 min, water-bath finishes the cleaning of rear distilled water, cuts off root cap, leave Meristernatic zone,
To improve the min of moral training 20, the Meristernatic zone after dyeing is placed on clean slide, one is added and drips distilled water, use tweezer
Son breaks the tip of a root into pieces, covers and is capped a blotting paper after slide again and is gently pressed with thumb, is finally tapped with the rubber tip of pencil, directly
Nebulized to the tip of a root is scattered；
（6）By step（5）In ready-made slide tip of a root microscopy under the microscope, record chromosome is the seedling of 64
Numbering and quantity, count the processing bletilla tetraploid induction rate be 2.6%.
Embodiment 2
（1）On superclean bench, callus is cut into 0.3cm × 0.3cm fritter, access proliferated culture medium
Cultivate 10 d；Aseptically, the chemical inducer sterilized toward addition in blake bottle, chemical inducer immersion callus
8 h；
Described chemical inducer is：0.2% colchicine+3%DMSO；
The proliferated culture medium is：N61.0 mg/L+NAA of+2,4-D, 0.5 mg/L+TDZ 0.5 mg/L, pH6.0；
（2）On superclean bench, by step（1）In callus take out, be put into sterilized water clean 5 times, with nothing
Bacterium filter paper is blotted to be cultivated in water, access differential medium, and part callus blackening induces the time of budding long, and 30 d are produced
Bud point, 43 D-shapeds are into bud, and 65 d buds grow 2~3 leaves, the thin and delicate and sturdy polarization of bud, leaf color yellow green and bottle green；
The differential medium is：1.0 mg/L+ZT of MS+6-BA 0.5 mg/L+NAA0.5 mg/L, pH6.0；
（3）On superclean bench, by step（2）In obtain bud access root media in cultivate, during 7 d under bud
There is projection in end, and 16 d start long root, a length of 1.0 cm of 50 d roots；
The root media is：0.5 mg/L+IBA of 1/2MS+NAA, 1.0 mg/L+KT 0.4 mg/L, pH6.0；
（4）The morning 9~11 point, on superclean bench, by step（3）In bletilla seedling carefully take out, be placed on sterilizing
In the plate crossed, the tip of a root is immediately placed in pretreatment fluid by 0.5 cm cut off root tips, 3 h is handled, by remaining on seedling
System's excision, then culture in root media is accessed, and the tip of a root and seedling are numbered；
Described pretreatment fluid is：0.1% colchicine solution；
（5）By step（4）In pretreatment fluid in the tip of a root take out, fixed after being cleaned with distilled water with Ka Nuoshi fixers
3 h, the tip of a root fixed is cleaned with distilled water, is put into and is filled 1N hydrochloric acid（Concentrated hydrochloric acid：Water=1：11）Small beaker in, 60
DEG C or so water-bath in handle 15~20 min, water-bath finishes the cleaning of rear distilled water, cuts off root cap, leave Meristernatic zone,
To improve the min of moral training 20, the Meristernatic zone after dyeing is placed on clean slide, one is added and drips distilled water, use tweezer
Son breaks the tip of a root into pieces, covers and is capped a blotting paper after slide again and is gently pressed with thumb, is finally tapped with the rubber tip of pencil, directly
Nebulized to the tip of a root is scattered；
（6）By step（5）In ready-made slide tip of a root microscopy under the microscope, record chromosome is the seedling of 64
Numbering and quantity, the bletilla tetraploid induction rate for counting the processing are 10.8%.
Embodiment 3
（1）On superclean bench, callus is cut into 0.3cm × 0.3cm fritter, access proliferated culture medium
Cultivate 10 d；Aseptically, the chemical inducer sterilized toward addition in blake bottle, chemical inducer immersion callus
16 h；
Described chemical inducer is：0.1% colchicine+3%DMSO；
The proliferated culture medium is：N61.0 mg/L+NAA of+2,4-D, 0.5 mg/L+TDZ 0.5 mg/L, pH6.0；
（2）On superclean bench, by step（1）In callus take out, be put into sterilized water clean 5 times, with nothing
Bacterium filter paper is blotted to be cultivated in water, access differential medium, and 25 d produce bud point, and 40 D-shapeds grow 2~3 leaves into bud, 60 d buds,
Bud is sturdy, dark green leaf color color；
The differential medium is：1.0 mg/L+ZT of MS+6-BA 0.5 mg/L+NAA0.5 mg/L, pH6.0；
（3）On superclean bench, by step（2）In obtain bud access root media in cultivate, during 7 d under bud
There is projection in end, and 15 d start long root, a length of 1.0 cm of 48 d roots；
The root media is：0.5 mg/L+IBA of 1/2MS+NAA, 1.0 mg/L+KT 0.4 mg/L, pH6.0；
（4）The morning 9~11 point, on superclean bench, by step（3）In bletilla seedling carefully take out, be placed on sterilizing
In the plate crossed, the tip of a root of 0.5 cm length is cut, the tip of a root is immediately placed in pretreatment fluid, handle 3 h, will be remaining on seedling
Root system is cut off, then accesses culture in root media, and the tip of a root and seedling are numbered；
Described pretreatment fluid is：0.1% colchicine solution；
（5）By step（4）In pretreatment fluid in the tip of a root take out, fixed after being cleaned with distilled water with Ka Nuoshi fixers
3 h, the tip of a root fixed is cleaned with distilled water, is put into and is filled 1N hydrochloric acid（Concentrated hydrochloric acid：Water=1：11）Small beaker in, 60
DEG C or so water-bath in handle 15~20 min, water-bath finishes the cleaning of rear distilled water, cuts off root cap, leave Meristernatic zone,
To improve the min of moral training 20, the Meristernatic zone after dyeing is placed on clean slide, one is added and drips distilled water, use tweezer
Son breaks the tip of a root into pieces, covers and is capped a blotting paper after slide again and is gently pressed with thumb, is finally tapped with the rubber tip of pencil, directly
Nebulized to the tip of a root is scattered；
（6）By step（5）In ready-made slide tip of a root microscopy under the microscope, record chromosome is the seedling of 64
Numbering and quantity, the bletilla tetraploid induction rate for counting the processing are 23.4%.
Claims (1)
1. in the method for callus induction bletilla tetraploid, it is characterized in that comprising the following steps：
（1）On superclean bench, bletilla callus is cut into 0.3cm × 0.3cm fritter, access proliferated culture medium
Cultivate 10 d；Aseptically, the chemical inducer sterilized toward addition in blake bottle, chemical inducer immersion callus
8~24 h；
Described chemical inducer is：0.05~0.2% colchicine+3%DMSO；
The proliferated culture medium is：1.0 mg/L+NAA of N6+2,4-D, 0.5 mg/L+TDZ 0.5 mg/L, pH6.0；
（2）On superclean bench, by step（1）In callus take out, be put into sterilized water clean, inhaled with aseptic filter paper
Cultivated in solid carbon dioxide, access differential medium, 22~30 d produce bud point, and 36~43 D-shapeds grow 2~3 into bud, 55~65 d buds
Piece leaf, bud is sturdy, dark green leaf color color；
The differential medium is：1.0 mg/L+ZT of MS+6-BA 0.5 mg/L+NAA0.5 mg/L, pH6.0；
（3）On superclean bench, by step（2）In obtain bud access root media in cultivate, brought out during 7 d under bud
Existing projection, 12~16 d start long root, a length of 1.0 cm of 45~50 d roots；
The root media is：0.5 mg/L+IBA of 1/2MS+NAA, 1.0 mg/L+KT 0.4 mg/L, pH6.0；
（4）The morning 9~11 point, on superclean bench, by step（3）In obtained bletilla seedling take out, be placed on what is sterilized
In plate, the tip of a root of 0.5-1.0 cm length is cut, the tip of a root is immediately placed in pretreatment fluid, handle 3 h, will be remaining on seedling
Root system is cut off, then accesses culture in root media, and the tip of a root and seedling are numbered；
Described pretreatment fluid is：0.1% colchicine solution；
（5）By step（4）In pretreatment fluid in the tip of a root take out, 3 h are fixed with Ka Nuoshi fixers after being cleaned with distilled water,
The tip of a root fixed is cleaned with distilled water, is put into the small beaker for filling 1N hydrochloric acid, 15 is handled in 55-65 DEG C of water-bath
~20 min, water-bath finishes rear distilled water cleaning, cuts off root cap, leaves Meristernatic zone, to improve the min of moral training 20, will
Meristernatic zone after dyeing is placed on clean slide, is added one and is dripped distilled water, the tip of a root is broken into pieces, covers and is capped again after slide
One blotting paper is gently pressed with thumb, is finally tapped and is nebulized until the tip of a root is scattered；
（6）By step（5）In ready-made slide tip of a root microscopy under the microscope, be the seedling of 64 by chromosome quantitative
Numbering and quantity are recorded；It is 2.6%~23.4% to count bletilla tetraploid induction rate.
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