CN106954549B - A kind of comb tooth bog pondweed tissue culture and rapid proliferation method - Google Patents
A kind of comb tooth bog pondweed tissue culture and rapid proliferation method Download PDFInfo
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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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Abstract
The invention discloses a kind of comb tooth bog pondweed tissue culture and rapid proliferation methods.Step includes A, explant selection and pretreatment: selection robust growth, the plant stem section of green in color is as explant；B, the preparation of aseptic explant: explant is handled with alcohol and mercury chloride, prepares aseptic explant；C, optimum hormone type and the growth of concentration proportioning condition induced bud inducing clumping bud: are determined；D, Multiplying culture: adjustment hormone combination carries out Multiplying culture；E, culture of rootage: selection hormone kind and concentration proportioning carry out culture of rootage；F, expand culture: selecting the aseptic seedlings of suitable length to be placed in proliferated culture medium, carry out expansion secondary culture, industrialized production aseptic plant；G, it hardening and transplanting: opens bottle cap culture two days, is cultivated in being transferred to natural water in the incubator.The present invention can not be turned out aseptic plant by season environmental restrictions, provide endlessly seedling for aquatic ecosystem recovery.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to the tissue culture of submerged plant comb tooth bog pondweed and quickly
Propagation method, this method are to take integral asepsis and stringent temperature control measure, realize submerged plant comb tooth bog pondweed and are testing
Room mass production, and can be generalized to ecological environment and repair field.
Background technique
Comb tooth Potamogeton is that one kind is distributed in global submerged plant in Potamogeton.Comb tooth bog pondweed is born in river
The Different Waters such as ditch, water channel, pond, water body are in subacidity or neutrality more, also see a small number of alkalescence water in northwest China
In body and salt water.Distribution on global is more commonly seen with two hemispheres temperate waters especially, has purification outstanding to imitate in the nitrogen phosphorus in water body
Fruit.
In recent decades, because of the factors such as environmental pollution and habitat change, the ring that comb tooth bog pondweed had previously grown from it
It degenerates or disappears in border.Unfortunately, still lack mature technology at present, it can be with vast propagation comb tooth bog pondweed high grade project
Seedling, at the same overcome the problems, such as the species in the natural environment provenance degenerate and it is seriously polluted.
Summary of the invention
The present invention provides a kind of comb tooth bog pondweed tissue culture and rapid proliferation methods, and this method is in aseptic condition
Under, temperature control measure is taken, fixed-illumination is given, establishes aseptic seedlings of plants system, a large amount of aseptic seedlings can be provided throughout the year.Method is easy
Row, easy to operate, yield is high.
In order to achieve the above object, the present invention uses following technical measures:
A kind of comb tooth bog pondweed tissue culture and rapid proliferation method, comprising the following steps:
A, explant selection and pretreatment: selection green in color, the comb tooth bog pondweed stem for sprouting axillary bud of robust growth
Duan Zuowei explant impregnates 10min with alkaline detergent, then is rinsed 1 hour with tap water, with distilled water repeated flushing 4-5 times,
It is cleaned 4-5 times repeatedly again with sterile water, is placed in aseptic operating platform；
B, it the preparation of aseptic explant: is impregnated 15 seconds with 75% ethyl alcohol first on aseptic operating platform, with aseptic water washing 3-
It 4 times, carries out surface sterilization 5 minutes, aseptic water washing 4-5 times blots water on aseptic filter paper, for use；
C, inducing clumping bud: selection 0.10 mg/L KT(kinetin) and 0.01mg/L NAA(methyl α-naphthyl acetate) MS solid train
Base is supported, sucrose mass concentration is that 3%, pH is adjusted to 5.8-6.0, and solid medium is loaded on tissue culture bottle；Culture medium is by 120 DEG C of sterilizings
20min is handled, after cooling, the comb tooth bog pondweed explant of the 1.0-2.0cm after accessing sterilization treatment is quiet in the light incubator
Culture is set, it is long to 5.0-6.0cm progress Multiplying culture to young shoot；
D, Multiplying culture: the 1/2MS fluid nutrient medium of selection sucrose mass concentration 1.5% adds 0.05-2.00 mg/L
6-BA(6- benayl aminopurine) and 0.01-0.50 mg/L NAA hormone；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles,
100 milliliters every bottle, after sterilizing, the young shoot for accessing 5.0-6.0cm is set in the light incubator, carries out Multiplying culture；
E, culture of rootage: 1/2 MS fluid nutrient medium of selection sucrose mass concentration 1.5% adds 0.01-1.00 mg/L
The hormone of NAA；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, 100 milliliters every bottle, after sterilizing, accesses long 6.0-8.0cm
Seedling set and stand in the light incubator, carry out culture of rootage；
F, expand culture: selecting length for the aseptic seedlings of 17.0-20.0cm, be placed in proliferated culture medium, carry out industry
Metaplasia produces；
G, hardening and transplanting: tooth bog pondweed aseptic seedlings to be combed it is long to 25.0cm can hardening, open bottle cap, refine at room temperature
Culture medium is cleaned after seedling 1-2d, takes out test tube seedling, is transplanted into natural water；
Described step C, D, E, the F is all made of gnotobasis, illumination 60-70 μ E/(m2S), periodicity of illumination 12h/d,
Room temperature (25 ± 2) DEG C.
Preferably, in step B, the surface sterilization uses 0.1% mercury chloride.
Preferably, in step C, the concentration of KT is 0.10mg/L, and the concentration of NAA is 0.01mg/L.
The invention has the following advantages:
Comb tooth bog pondweed is the pioneer's type for repairing impaired water ecological environment, but at present about comb tooth bog pondweed tissue cultures
Technology have not been reported.The present invention carries out sterile propagation Establishing to comb tooth bog pondweed, to later to the progress of comb tooth bog pondweed
Systematic research, the protection of wetland are of great significance with reparation and the reparation of ecological environment.It can not be by using the present invention
Season, temperature, regional impact produce a large amount of comb tooth bog pondweed aseptic seedling.The aseptic seedlings of one a length of 17-20cm pass through one
Culture in a month can breed the seedling of 29-47cm long, and weight in wet base can increase by 24 times, and the growth coefficient of 30d can reach 15
Times, it is superior to the reproduction speed of natural water plant.
Detailed description of the invention
Fig. 1 is comb tooth bog pondweed 30d proliferative conditions in embodiment 1；
Fig. 2 is comb tooth bog pondweed 30d proliferative conditions in embodiment 2.
Specific embodiment
Reagent used in the embodiment of the present invention, it is if not otherwise specified, commercially available that the present invention, the technology being related to can be achieved
The ordinary skill in the art can be used if not otherwise specified in scheme.
MS culture medium prescription used in the examples is as follows: unit mg/L,
KNO3 1900
NH4NO3 1650
MgSO4•7H2O 370
KH2PO4 170
CaCl2•2H2O 440
MnSO4•4H2O 22.3
ZnSO4•7H2O 8.6
H3BO3 6.2
KI 0.83
Na2MoO4•7H2O 0.25
CuSO4.5H2O 0.025
CoCL2.6H2O 0.025
Na2-EDTA 37.3
FeSO4•7 H2O 27.8
Glycine 2.0
Puridoxine hydrochloride 0.5
Tyiamine Hd element 0.1
Niacin 0.5
Creatine 100
MS solid medium is that 6g/L agar is added in above-mentioned culture formula.
Embodiment 1
A, explant selection and pretreatment: selection green in color, the comb tooth bog pondweed stem for sprouting axillary bud of robust growth
Duan Zuowei explant impregnates 10min with alkaline detergent, then is rinsed 1 hour with tap water, with distilled water repeated flushing 4-5 times,
It is cleaned 4-5 times repeatedly again with sterile water, is placed in aseptic operating platform；
B, it the preparation of aseptic explant: is impregnated 15 seconds with 75% ethyl alcohol first on aseptic operating platform, with aseptic water washing 3-
4 times, then surface sterilization is carried out 5 minutes with 0.1% mercury chloride (a few drop Tween 80s are added), aseptic water washing 4-5 times, in sterile filter
Water is blotted on paper, for use；
C, inducing clumping bud: the MS solid medium of 0.10 mg/L KT and 0.01mg/L NAA of selection, sucrose quality are dense
Degree is that 3%, pH is adjusted to 5.8-6.0, and solid medium is loaded on tissue culture bottle；Culture medium passes through 120 DEG C of sterilization treatment 20min, cooling
Afterwards, the comb tooth bog pondweed explant of the 1.0-2.0cm after sterilization treatment is accessed, in the light incubator stationary culture, to young shoot
It grows to 5.0-6.0cm and carries out Multiplying culture；
D, Multiplying culture: selection sucrose mass concentration 1.5% 1/2MS fluid nutrient medium, addition 2.00 mg/L 6-BA and
The hormone of 0.50 mg/L NAA；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, and 100 milliliters every bottle, sterilization method and bud
Induce it is identical, access 5.0-6.0cm young shoot set in the light incubator, carry out Multiplying culture；
E, culture of rootage: the 1/2MS fluid nutrient medium of selection sucrose mass concentration 1.5%, addition 0.01mg/L NAA's swashs
Element；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, and 100 milliliters every bottle, sterilization method is identical as bud induction, access length
The seedling of 6.0-8.0cm is set to be stood in the light incubator, carries out culture of rootage；
F, expand culture: the later period carries out expansion secondary culture, when producing a large amount of germ-free plants, selects length for 17.0cm's
Aseptic seedlings are placed in proliferated culture medium, carry out industrialized production；
G, hardening and transplanting: tooth bog pondweed aseptic seedling to be combed it is long to 25.00cm can hardening, open bottle cap, refine at room temperature
Culture medium is cleaned after seedling 1-2d, takes out test tube seedling, is transplanted into natural water；
Described step C, D, E, the F is all made of gnotobasis, illumination 60-70 μ E/(m2S), periodicity of illumination 12h/d,
Room temperature (25 ± 2) DEG C.
This method can directly induce young shoot by an explant, and breeding of or else breaking obtains more clump buds, 30 d's
Growth coefficient reaches 15, and plant weight increases by 23.9 times, and length increases by 1.6 times, and obtains the comb tooth in seedling and natural environment
Bog pondweed does not have difference.Survival rate is greater than 95%.Comb tooth bog pondweed 30d proliferative conditions are as shown in Figure 1 in embodiment 1.
Embodiment 2
A, explant selection and pretreatment: selection green in color, the comb tooth bog pondweed stem for sprouting axillary bud of robust growth
Duan Zuowei explant impregnates 10min with alkaline detergent, then is rinsed 1 hour with tap water, with distilled water repeated flushing 4-5 times,
It is cleaned 4-5 times repeatedly again with sterile water, is placed in aseptic operating platform；
B, it the preparation of aseptic explant: is impregnated 15 seconds with 75% ethyl alcohol first on aseptic operating platform, with aseptic water washing 3-
4 times, then surface sterilization is carried out 5 minutes with 0.1% mercury chloride (a few drop Tween 80s are added), aseptic water washing 4-5 times, in sterile filter
Water is blotted on paper, for use；
C, inducing clumping bud: the MS solid medium of 0.10 mg/L KT and 0.01mg/L NAA of selection, sucrose quality are dense
Degree is that 3%, pH is adjusted to 5.8-6.0, and solid medium is loaded on tissue culture bottle；Culture medium passes through 120 DEG C of sterilization treatment 20min, cooling
Afterwards, the comb tooth bog pondweed explant of the 1.0-2.0cm after sterilization treatment is accessed, in the light incubator stationary culture, to young shoot
It grows to 5.0-6.0cm and carries out Multiplying culture；
D, Multiplying culture: selection sucrose mass concentration 1.5% 1/2MS fluid nutrient medium, addition 0.05mg/L 6-BA and
The hormone of 0.01mg/L NAA；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, and 100 milliliters every bottle, sterilization method is lured with bud
Lead it is identical, access 5.0-6.0cm young shoot set in the light incubator, carry out Multiplying culture；
E, culture of rootage: the 1/2MS fluid nutrient medium of selection sucrose mass concentration 1.5%, 1.00 mg/L NAA's of addition
Hormone；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, and 100 milliliters every bottle, sterilization method is identical as bud induction, access length
The seedling of 6.0-8.0cm is set to be stood in the light incubator, carries out culture of rootage；
F, expand culture: the later period carries out expansion secondary culture, when producing a large amount of germ-free plants, selects length for 20.0cm's
Aseptic seedlings are placed in proliferated culture medium, carry out industrialized production；
G, hardening and transplanting: tooth bog pondweed aseptic seedling to be combed it is long to 25.00cm can hardening, open bottle cap, refine at room temperature
Culture medium is cleaned after seedling 1-2d, takes out test tube seedling, is transplanted into natural water；
Described step C, D, E, the F is all made of gnotobasis, illumination 60-70 μ E/(m2S), periodicity of illumination 12h/d,
Room temperature (25 ± 2) DEG C.
This method directly induces seedling by an explant, and the growth coefficient of 30 d reaches 10, and plant weight increases
24.8 times, length increases by 2.6 times, and the comb tooth bog pondweed in the seedling and natural environment of acquisition does not have difference.Survival rate is greater than
90%.Comb tooth bog pondweed 30d proliferative conditions are as shown in Figure 2 in embodiment 2.
Table 1 show the growing state of comb tooth bog pondweed in two embodiments.
Claims (2)
1. a kind of comb tooth bog pondweed tissue culture and rapid proliferation method, which comprises the following steps:
A, explant selection and pretreatment: the comb tooth bog pondweed stem section for sprouting axillary bud of selection green in color, robust growth is made
For explant, 10min is impregnated with alkaline detergent, then is rinsed 1 hour with tap water, with distilled water repeated flushing 4-5 times, with nothing
Bacterium water cleans 4-5 times repeatedly again, is placed in aseptic operating platform；
B, the preparation of aseptic explant: being impregnated 15 seconds with 75% ethyl alcohol first on aseptic operating platform, with aseptic water washing 3-4 times,
It carries out surface sterilization 5 minutes, aseptic water washing 4-5 times blots water on aseptic filter paper, for use；
C, inducing clumping bud: the MS solid medium of 0.10 mg/L KT and 0.01mg/L NAA of selection, sucrose mass concentration are
3%, pH are adjusted to 5.8-6.0, and solid medium is loaded on tissue culture bottle；Culture medium passes through 120 DEG C of sterilization treatment 20min and connects after cooling
Enter the comb tooth bog pondweed explant of sterilizing treated 1.0-2.0cm, in the light incubator stationary culture, it is long extremely to young shoot
5.0-6.0cm carries out Multiplying culture；
D, Multiplying culture: the 1/2MS fluid nutrient medium of selection sucrose mass concentration 1.5% adds 0.05-2.00 mg/L 6-BA
With the hormone of 0.01-0.50 mg/L NAA；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, and 100 milliliters every bottle, sterilizing
Afterwards, the young shoot for accessing 5.0-6.0cm is set in the light incubator, and Multiplying culture is carried out；
E, culture of rootage: 1/2 MS fluid nutrient medium of selection sucrose mass concentration 1.5% adds 0.01-1.00 mg/L NAA
Hormone；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, 100 milliliters every bottle, after sterilizing, accesses the children of long 6.0-8.0cm
Seedling is set to be stood in the light incubator, carries out culture of rootage；
F, expand culture: selecting length for the aseptic seedlings of 17.0-20.0cm, be placed in proliferated culture medium, carry out industrial metaplasia
It produces；
G, hardening and transplanting: tooth bog pondweed aseptic seedlings to be combed it is long to 25.0cm can hardening, open bottle cap, room temperature lower refining seedling 1-
Culture medium is cleaned after 2d, takes out test tube seedling, is transplanted into natural water；
Described step C, D, E, the F is all made of gnotobasis, 60-70 μm of ol/(m of illumination2S), periodicity of illumination 12h/d, it is indoor
25 ± 2 DEG C of temperature.
2. the method according to claim 1, wherein the surface sterilization uses 0.1% chlorination in step B
Mercury.
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| CN111631136B (en) * | 2020-07-14 | 2022-11-25 | 安顺职业技术学院 | Tissue culture and rapid propagation method of dormant buds of eyedrops |
| CN114431152B (en) * | 2022-03-10 | 2023-09-19 | 水生藻安生物科技（武汉）有限公司 | Rapid culture method of dunaliella salina aseptic seedlings |
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