CN106954549A - 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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- CN106954549A CN106954549A CN201710109348.8A CN201710109348A CN106954549A CN 106954549 A CN106954549 A CN 106954549A CN 201710109348 A CN201710109348 A CN 201710109348A CN 106954549 A CN106954549 A CN 106954549A
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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 method.Step includes A, explant selection and pretreatment：Growth selection is healthy and strong, and the plant stem section of green in color is used as explant；B, aseptic explant preparation：With alcohol and mercury chloride processing explant, aseptic explant is prepared；C, inducing clumping bud：Determine optimum hormone species and the growth of concentration proportioning condition induced bud；D, Multiplying culture：Adjust hormone combination and carry out Multiplying culture；E, culture of rootage：Hormone kind and concentration proportioning are selected, culture of rootage is carried out；F, expansion culture：The aseptic seedlings of selection suitable length are placed in proliferated culture medium, are enlarged Secondary Culture, industrialized production aseptic plant；G, hardening and transplanting：Bottle cap culture two days is opened in incubator, is cultivated in natural water is transferred to.The present invention can not be limited by season environment, turn out aseptic plant, recover to provide endlessly seedling for aquatic ecosystem.
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 quick
Propagation method, this method is to take integral asepsis and strict temperature control measure, realizes submerged plant comb tooth bog pondweed in experiment
Room is largely produced, it is possible to is generalized to ecological environment and is repaired field.
Background technology
Comb tooth Potamogeton, in Potamogeton, is that one kind is distributed in global submerged plant.Comb tooth bog pondweed is born in river
The Different Waters such as ditch, water channel, pond, are in subacidity or neutrality more water body, a small number of alkalescence water are also seen in northwest China
In body and salt water.Distribution on global, is more commonly seen with two hemispheres temperate waters especially, there is prominent purification effect to the nitrogen phosphorus in water body
Really.
In recent decades, because the factor, the ring that comb tooth bog pondweed had previously grown from it such as environmental pollution and habitat change
Degenerate or disappear in border.Unfortunately, mature technology is still lacked at present, can be with vast propagation comb tooth bog pondweed high grade project
Seedling, at the same overcome the species in natural environment introduces a collection degenerate and it is seriously polluted the problem of.
The content of the invention
The invention provides a kind of comb tooth bog pondweed tissue culture and rapid proliferation method, this method is in aseptic condition
Under, temperature control measure is taken, fixed-illumination is given, aseptic seedlings of plants system is set up, a large amount of aseptic seedlings can be provided throughout the year.Method is easy
OK, 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, comprises the following steps：
A, explant selection and pretreatment：Green in color is selected, the comb tooth bog pondweed stem section for sprouting axillary bud of robust growth is made
For explant, 10min is soaked with alkaline detergent, then is rinsed 1 hour with running water, is rinsed 4-5 times repeatedly with distilled water, with nothing
Bacterium water is cleaned 4-5 times repeatedly again, is placed in aseptic operating platform；
B, aseptic explant preparation：Soaked 15 seconds with 75% ethanol first on aseptic operating platform, with aseptic water washing 3-4 times,
Surface sterilization 5 minutes is carried out, aseptic water washing 4-5 times blots water on aseptic filter paper, stand-by；
C, inducing clumping bud：Select 0.10 mg/L KT（Kinetin）With 0.01mg/L NAA（Methyl α-naphthyl acetate）MS solid cultures
Base, sucrose mass concentration is adjusted to 5.8-6.0 for 3%, pH, and solid medium is loaded on tissue culture bottle；Culture medium passes through at 120 DEG C of sterilizings
20min is managed, after cooling, the comb tooth bog pondweed explant of the 1.0-2.0cm after access sterilization treatment is stood in illumination box
Culture, treats young shoot length to 5.0-6.0cm progress Multiplying cultures；
D, Multiplying culture：Select the 1/2MS fluid nutrient mediums of sucrose mass concentration 1.5%, addition 0.05-2.00 mg/L 6-BA
（6-benzyl aminopurine）With 0.01-0.50 mg/L NAA hormone；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, every bottle
100 milliliters, after sterilizing, access 5.0-6.0cm young shoot is placed in illumination box, carries out Multiplying culture；
E, culture of rootage：Select 1/2 MS fluid nutrient mediums of sucrose mass concentration 1.5%, addition 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 long 6.0-8.0cm children
Seedling is placed in illumination box and stood, and carries out culture of rootage；
F, expansion culture：The aseptic seedlings for selecting length to be 17.0-20.0cm, are placed in proliferated culture medium, carry out industrial metaplasia
Production；
G, hardening and transplanting：Treat comb tooth bog pondweed aseptic seedlings length to 25.0cm can hardening, open bottle cap, room temperature lower refining seedling 1-
Culture medium is cleaned after 2d, test tube seedling is taken out, transplanted into natural water；
Described step C, D, E, F uses gnotobasis, illumination 60-70 μ E/（m2·s）, periodicity of illumination is 12h/d, indoor
Temperature（25±2）℃.
It is preferred that, in step B, described surface sterilization uses 0.1% mercury chloride.
It is preferred that, in step C, KT concentration is 0.10mg/L, and NAA concentration is 0.01mg/L.
The invention has the advantages that：
Comb tooth bog pondweed is to repair the pioneer's species for being damaged water ecological environment, but the skill on comb tooth bog pondweed tissue cultures at present
Art has no report.The present invention carries out sterile propagation Establishing to comb tooth bog pondweed, to carrying out system to comb tooth bog pondweed later
Research, wetland protection with reparation and ecological environment reparation it is significant.Using the present invention can not by season,
Temperature, regional impact, produce substantial amounts of comb tooth bog pondweed aseptic seedling.One a length of 17-20cm aseptic seedlings are passed through one month
Culture can breed the seedling of 29-47cm length, weight in wet base can increase by 24 times, and 30d growth coefficient can reach 15 times, excellent
More in the reproduction speed of natural water plant.
Brief description of the drawings
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.
Embodiment
Reagent used in the embodiment of the present invention, if not otherwise specified, commercially available can be achieved the present invention, the technology being related to
Scheme, if not otherwise specified, can use the ordinary skill in the art.
The MS culture medium prescriptions used in embodiment are 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
Nicotinic acid 0.5
Creatine 100
MS solid mediums are addition 6g/L agar in above-mentioned culture formula.
Embodiment 1
A, explant selection and pretreatment：Green in color is selected, the comb tooth bog pondweed stem section for sprouting axillary bud of robust growth is made
For explant, 10min is soaked with alkaline detergent, then is rinsed 1 hour with running water, is rinsed 4-5 times repeatedly with distilled water, with nothing
Bacterium water is cleaned 4-5 times repeatedly again, is placed in aseptic operating platform；
B, aseptic explant preparation：Soaked 15 seconds with 75% ethanol first on aseptic operating platform, with aseptic water washing 3-4 times,
0.1% mercury chloride is used again（Add a few drop Tween 80s）Carry out surface sterilization 5 minutes, aseptic water washing 4-5 times, on aseptic filter paper
Water is blotted, it is stand-by；
C, inducing clumping bud：0.10 mg/L KT and 0.01mg/L NAA MS solid mediums are selected, sucrose mass concentration is
3%, pH are adjusted to 5.8-6.0, and solid medium is loaded on tissue culture bottle；Culture medium after cooling, connects by 120 DEG C of sterilization treatment 20min
Enter the comb tooth bog pondweed explant of the 1.0-2.0cm after sterilization treatment, the quiescent culture in illumination box treats young shoot length extremely
5.0-6.0cm carries out Multiplying culture；
D, Multiplying culture：The 1/2MS fluid nutrient mediums of sucrose mass concentration 1.5% are selected, 2.00 mg/L 6-BA and 0.50 are added
Mg/L NAA hormone；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, 100 milliliters every bottle, and sterilization method induces phase with bud
Together, access 5.0-6.0cm young shoot is placed in illumination box, carries out Multiplying culture；
E, culture of rootage：Select the 1/2MS fluid nutrient mediums of sucrose mass concentration 1.5%, addition 0.01mg/L NAA hormone；
Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, and 100 milliliters every bottle, sterilization method is identical with bud induction, accesses long 6.0-
8.0cm seedling is placed in illumination box and stood, and carries out culture of rootage；
F, expansion culture：Later stage is enlarged Secondary Culture, when producing a large amount of germ-free plants, and selection length is sterile for 17.0cm's
Seedling, is placed in proliferated culture medium, carries out industrialized production；
G, hardening and transplanting：Treat comb tooth bog pondweed aseptic seedling length to 25.00cm can hardening, open bottle cap, room temperature lower refining seedling 1-
Culture medium is cleaned after 2d, test tube seedling is taken out, transplanted into natural water；
Described step C, D, E, F uses gnotobasis, illumination 60-70 μ E/（m2·s）, periodicity of illumination is 12h/d, indoor
Temperature（25±2）℃.
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 more 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：Green in color is selected, the comb tooth bog pondweed stem section for sprouting axillary bud of robust growth is made
For explant, 10min is soaked with alkaline detergent, then is rinsed 1 hour with running water, is rinsed 4-5 times repeatedly with distilled water, with nothing
Bacterium water is cleaned 4-5 times repeatedly again, is placed in aseptic operating platform；
B, aseptic explant preparation：Soaked 15 seconds with 75% ethanol first on aseptic operating platform, with aseptic water washing 3-4 times,
0.1% mercury chloride is used again（Add a few drop Tween 80s）Carry out surface sterilization 5 minutes, aseptic water washing 4-5 times, on aseptic filter paper
Water is blotted, it is stand-by；
C, inducing clumping bud：0.10 mg/L KT and 0.01mg/L NAA MS solid mediums are selected, sucrose mass concentration is
3%, pH are adjusted to 5.8-6.0, and solid medium is loaded on tissue culture bottle；Culture medium after cooling, connects by 120 DEG C of sterilization treatment 20min
Enter the comb tooth bog pondweed explant of the 1.0-2.0cm after sterilization treatment, the quiescent culture in illumination box treats young shoot length extremely
5.0-6.0cm carries out Multiplying culture；
D, Multiplying culture：Select the 1/2MS fluid nutrient mediums of sucrose mass concentration 1.5%, addition 0.05mg/L 6-BA and
0.01mg/L NAA hormone；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 be placed in illumination box, carry out Multiplying culture；
E, culture of rootage：The 1/2MS fluid nutrient mediums of sucrose mass concentration 1.5% are selected, 1.00 mg/L NAA hormone is added；
Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, and 100 milliliters every bottle, sterilization method is identical with bud induction, accesses long 6.0-
8.0cm seedling is placed in illumination box and stood, and carries out culture of rootage；
F, expansion culture：Later stage is enlarged Secondary Culture, when producing a large amount of germ-free plants, and selection length is sterile for 20.0cm's
Seedling, is placed in proliferated culture medium, carries out industrialized production；
G, hardening and transplanting：Treat comb tooth bog pondweed aseptic seedling length to 25.00cm can hardening, open bottle cap, room temperature lower refining seedling 1-
Culture medium is cleaned after 2d, test tube seedling is taken out, transplanted into natural water；
Described step C, D, E, F uses gnotobasis, illumination 60-70 μ E/（m2·s）, periodicity of illumination is 12h/d, indoor
Temperature（25±2）℃.
This method directly induces seedling by an explant, and 30 d growth coefficient reaches 10, plant weight increase
24.8 times, the comb tooth bog pondweed that length increases in 2.6 times, the seedling and natural environment of acquisition does not have difference.Survival rate is more 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 (3)
1. a kind of comb tooth bog pondweed tissue culture and rapid proliferation method, it is characterised in that comprise the following steps：
A, explant selection and pretreatment：Green in color is selected, the comb tooth bog pondweed stem section for sprouting axillary bud of robust growth is made
For explant, 10min is soaked with alkaline detergent, then is rinsed 1 hour with running water, is rinsed 4-5 times repeatedly with distilled water, with nothing
Bacterium water is cleaned 4-5 times repeatedly again, is placed in aseptic operating platform；
B, aseptic explant preparation：Soaked 15 seconds with 75% ethanol first on aseptic operating platform, with aseptic water washing 3-4 times,
Surface sterilization 5 minutes is carried out, aseptic water washing 4-5 times blots water on aseptic filter paper, stand-by；
C, inducing clumping bud：Select 0.10 mg/L KT（Kinetin）With 0.01mg/L NAA（Methyl α-naphthyl acetate）MS solid cultures
Base, sucrose mass concentration is adjusted to 5.8-6.0 for 3%, pH, and solid medium is loaded on tissue culture bottle；Culture medium passes through at 120 DEG C of sterilizings
20min is managed, after cooling, the comb tooth bog pondweed explant of the 1.0-2.0cm after access sterilization treatment is stood in illumination box
Culture, treats young shoot length to 5.0-6.0cm progress Multiplying cultures；
D, Multiplying culture：Select the 1/2MS fluid nutrient mediums of sucrose mass concentration 1.5%, addition 0.05-2.00 mg/L 6-BA
（6-benzyl aminopurine）With 0.01-0.50 mg/L NAA hormone；Fluid nutrient medium is sub-packed in 500 milliliters of tissue culture bottles, every bottle
100 milliliters, after sterilizing, access 5.0-6.0cm young shoot is placed in illumination box, carries out Multiplying culture；
E, culture of rootage：Select 1/2 MS fluid nutrient mediums of sucrose mass concentration 1.5%, addition 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 long 6.0-8.0cm children
Seedling is placed in illumination box and stood, and carries out culture of rootage；
F, expansion culture：The aseptic seedlings for selecting length to be 17.0-20.0cm, are placed in proliferated culture medium, carry out industrial metaplasia
Production；
G, hardening and transplanting：Treat comb tooth bog pondweed aseptic seedlings length to 25.0cm can hardening, open bottle cap, room temperature lower refining seedling 1-
Culture medium is cleaned after 2d, test tube seedling is taken out, transplanted into natural water；
Described step C, D, E, F uses gnotobasis, illumination 60-70 μ E/（m2·s）, periodicity of illumination is 12h/d, Indoor Temperature
25 ± 2 DEG C of degree.
2. according to the method described in claim 1, it is characterised in that preferred, in step B, described surface sterilization is used
0.1% mercury chloride.
3. according to the method described in claim 1, it is characterised in that preferred, in step C, KT concentration is 0.10mg/L,
NAA concentration is 0.01mg/L.
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| CN111631136A (en) * | 2020-07-14 | 2020-09-08 | 安顺职业技术学院 | Tissue culture and rapid propagation method of dormant buds of eyedrops |
| CN114431152A (en) * | 2022-03-10 | 2022-05-06 | 水生藻安生物科技（武汉）有限公司 | Rapid culture method of peltate yam rhizome aseptic seedlings |
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| CN111631136A (en) * | 2020-07-14 | 2020-09-08 | 安顺职业技术学院 | Tissue culture and rapid propagation method of dormant buds of eyedrops |
| CN114431152A (en) * | 2022-03-10 | 2022-05-06 | 水生藻安生物科技（武汉）有限公司 | Rapid culture method of peltate yam rhizome aseptic seedlings |
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