CN104604693B - A kind of ancient watermifoil tissue culture and rapid proliferation method difficult to understand - Google Patents
A kind of ancient watermifoil tissue culture and rapid proliferation method difficult to understand Download PDFInfo
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- CN104604693B CN104604693B CN201510076490.8A CN201510076490A CN104604693B CN 104604693 B CN104604693 B CN 104604693B CN 201510076490 A CN201510076490 A CN 201510076490A CN 104604693 B CN104604693 B CN 104604693B
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Abstract
The invention discloses a kind of difficult to understand ancient watermifoil tissue culture and rapid proliferation method, the steps include: that A, outer implant select: growth selection stalwartness, green in color, without the axis of pest and disease damage as outer implant.B, outer implant sterilizing: process outer implant with alcohol and mercury chloride.C, bud inducement: select optimal hormone combination induced bud to grow.D, Multiplying culture: adjust hormone combination and culture medium concentration carries out Multiplying culture E, takes root: select suitable archusia to carry out root induction.F, hardening and transplanting: in incubator, open bottleneck cultivate two days, cultivate in proceeding to natural water.This technology can not be limited by season and environment, turns out a large amount of aseptic seedling, is used for aquatic ecosystem restoration.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to the group training of submerged plant Austria Gu watermifoil with quick
Propagation method, the method is to utilize integral asepsis and strict temperature control measure, it is achieved that submerged plant produces in a large number in laboratory,
And can be generalized to ecological environment reparation.
Background technology
Ancient Myriophyllum difficult to understand is little in Angiospermae (Angiospermae) Dicotyledoneae (Dicotyledoneae)
Two Xian Cao sections (Haloragidaceae).At present as water purification instrument kind and revegetation pioneer's thing in ecological restoration of lakes engineering
As bait in kind, fish shrimp crab pool breeding process, take refuge and spawning place, and as cloth during indoor appreciation aquatic animal cultivating
Scape material in the urgent need to.Use tissue culture technique, it is thus achieved that and its high grade project seedling of vast propagation, to overcome these species to exist
Source of planting in natural environment is limited and with serious pollution problem.
The most about the report of watermifoil tissue cultures, but be in addition to outside Haloragidaceae Myriophyllum spicatum the most specifically implement
To being which kind of watermifoil.And about the Physiology and biochemistry of ancient watermifoil difficult to understand, and the report in terms of ecology is the most rare.Though
So it has the denominator of Myriophyllum, but the feature of himself also causes the mode of group training and condition to change.
Summary of the invention
The invention provides a kind of ancient watermifoil tissue culture and rapid proliferation method difficult to understand.The method is at aseptic condition
Under, take temperature control measure, give fixed-illumination, cultivate aseptic seedling system, a large amount of aseptic seedlings can be provided throughout the year.It is easy to implement the method,
Easy to operate, yield is high.
In order to achieve the above object, the present invention uses techniques below measure:
A kind of ancient watermifoil tissue culture and rapid proliferation method difficult to understand, the steps include:
A, outer implant select and pretreatment: growth selection is healthy and strong, green in color, without the stem section of pest and disease damage as outer implant,
Soak 30min with alkaline detergent, then under running water, rinse 2h, repeatedly rinse 4-5 time with distilled water, with sterilized water the most repeatedly
Clean 4-5 time, be placed in aseptic operating platform.
B, outer implant sterilizing: first by 70% (volume ratio) alcohol immersion 15s on aseptic operating platform, use aseptic water washing
4-5 time, then carry out surface sterilization 2-5min, aseptic water washing 4-5 time with 0.1% (mass volume ratio) mercury chloride, put into
The aseptic citric acid solution of 0.15% (mass volume ratio) cuts, water is blotted by aseptic filter paper, stand-by.
C, callus and bud inducement: the outer implant of difficult to understand ancient watermifoil in step B removing blade of 0.5-2cm is accessed
In inducing culture, quiescent culture in illumination box, evoked callus and young shoot.
Described inducing culture is: MS solid medium+6BA 0.50-2.00mg/L+NAA 0.25-0.50mg/L+
Sucrose 3% (mass volume ratio), pH 5.8-6.0.
D, Multiplying culture: the culture medium of step C can be continuing with, for Multiplying culture.Also can treat that shoot growth is to 1.5-
2cm, changes proliferated culture medium, further expanding propagation, forms seedling.
Described proliferated culture medium is: 1/2MS fluid nutrient medium+6BA 0.50-2.00mg/L+NAA 0.25-0.50mg/
L+ sucrose 1.5%, pH 5.8-6.0.
Every 20-25d subculture once, the seedling of formation, it is cut into 0.5-2.0cm, proceeds to inducing culture or Multiplying culture
Base, is proceeding Multiplying culture under step D same culture conditions, is expanding numerous plant；
E, taking root: callus constantly grows, growth of seedling, to about 4-6cm, can be changed without culture medium and continue in step
Send out roots on the culture medium of rapid C, it is also possible to be replaced by root media, not only can promote that it is taken root, it is possible to tachyauxesis.
Described root media is: 1/2MS fluid nutrient medium+NAA 0.10mg/L+ sucrose 1.5%, pH 5.8-6.0.
F, hardening and transplanting: when indefinite root length is to more than 2.0cm, open bottle cap, after room temperature lower refining seedling 1-2d, takes out test tube seedling, washes
Its root culture medium clean, transplants to outdoor water body.
Above step C, D, E are gnotobasis, illumination 40-60 μ E/ (m2S), periodicity of illumination is 12h/d, Indoor Temperature
Spend 25 ± 2 DEG C.
Preferably, in step B, the disinfecting time of mercury chloride is 2 minutes；In step C and D, the concentration of 6BA is 0.50mg/L,
The concentration of NAA is 0.25mg/L.
MS culture medium prescription 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
FeSO4·7H2O 27.8
Glycine 2.0
Puridoxine hydrochloride 0.5
Tyiamine Hd element 0.1
Nicotinic acid 0.5
Creatine 100
MS solid medium is the agar that MS culture medium adds 7g/L, pH5.8-6.0.
Compared with prior art, the present invention has the following advantages and effect:
The difficult to understand ancient watermifoil tissue culture and rapid proliferation method that the present invention provides, can provide substantial amounts of seedling in short-term,
Research for the physiological and biochemical property of watermifoil ancient to Austria further provides resource endlessly.Passing on by callus
It is easier to preserve and maintain the quality of germplasm resource.The stem section of one long 1cm was through the cultivation of a week, and can manage it into one piece of area is
0.25cm2Callus, then 6-8 seedling can be obtained through the cultivation of three weeks, the long 5-8cm of each seedling, proliferation times reaches
6-8 times, within 1 year, a bud can breed more than several hundred million seedling.With a bud 20d subculture once, each proliferation times is 8 meters
Calculate.
| Number of days/d | 20 | 40 | 60 | 80 | 100 | 120 |
| Seedling number/ | 8 | 82 | 83 | 84 | 85 | 86 |
Detailed description of the invention
The reagent that the embodiment of the present invention is used, if not otherwise specified, commercially available being realizes the present invention, described technical side
Case if not otherwise specified, is the ordinary skill in the art.
Embodiment 1:
A kind of ancient watermifoil tissue culture and rapid proliferation method difficult to understand, comprises the following steps:
A, outer implant select and pretreatment: growth selection is healthy and strong, and green in color, the stem section without pest and disease damage is (equal with or without young shoot
Can) as outer implant, soak 30min with alkaline detergent, then under running water, rinse 2h, repeatedly rinse 4-5 time with distilled water,
The most repeatedly clean 4-5 time with sterilized water, be placed in aseptic operating platform.
B, outer implant sterilizing: on aseptic operating platform, first soak 15s with 70% ethanol (volume ratio), use aseptic water washing
4-5 time, then carry out surface sterilization 5min, nothing with 0.1% (mass volume ratio, 0.1g mercury chloride adds in 100ml water) mercury chloride
Bacterium water rinses 4-5 time, puts in the aseptic citric acid solution of 0.15% (mass volume ratio) and cuts, and is inhaled by water on aseptic filter paper
Dry, stand-by.
C, callus and young shoot induction: selecting inducing culture, culture medium is loaded in 50 milliliters of triangular flasks, every bottle 20 milli
Rise.Culture medium after 120 DEG C of sterilization treatment 20min, access remove blade step B in be about 1cm outer implant (with or without
Young shoot), every bottle graft enters an outer implant, quiescent culture in illumination box.Inductivity is 90%.
Described inducing culture is: MS solid medium+6BA 2.00mg/L+NAA 0.50mg/L+ sucrose 3% (matter
Amount volume ratio), pH 5.8-6.0.
D, Multiplying culture: be changed without culture medium, the culture medium of step C continues on for Multiplying culture.Outer implant is expanding shape
Also can produce substantial amounts of bud while becoming callus, bud is eventually differentiated into one whole plant, and produces new endlessly
Seedling.Every 25d subculture once, the seedling of formation, it is cut into 1.5cm, proceeds to inducing culture, in cultivation identical with step D
Under the conditions of proceed Multiplying culture, expand numerous plant；
E, take root: callus and growth of seedling, to certain phase, can continue to send out roots on the culture medium of step C.
F, hardening and transplanting: when indefinite root length is to more than 2.0cm, open bottle cap, (open bottle after room temperature lower refining seedling 1-2d
Lid, rests in incubator), take out test tube seedling, clean its root culture medium, transplant to outdoor water body.
C in above step, D, E are gnotobasis, illumination 40-60 μ E/ (m2S), periodicity of illumination is 12h/d, indoor
Temperature 25 ± 2 DEG C.
This method can gradually be induced callus by an outer implant, and broken up the seedling made new advances by callus,
Proliferation times can reach 6, uses a kind of culture medium just just can realize bud inducement, and three kinds of effects of breeding and take root reduce behaviour
Make step, save production cost.Rooting rate 100%, pollution rate is less than 1%, transplants survival rate more than 98%.
Embodiment 2:
A kind of ancient watermifoil tissue culture and rapid proliferation method difficult to understand, comprises the following steps:
A, outer implant select and pretreatment: growth selection is healthy and strong, and green in color, the stem section without pest and disease damage is (equal with or without young shoot
Can) as outer implant, remove spire, soak 30min with alkaline detergent, then under running water, rinse 2h, with distilled water repeatedly
Rinse 4-5 time, the most repeatedly clean 4-5 time with sterilized water, be placed in aseptic operating platform.
B, outer implant sterilizing: first by 70% (volume ratio) alcohol immersion 15s on aseptic operating platform, use aseptic water washing
4-5 time, then carry out surface sterilization 2min, aseptic water washing 4-5 time with 0.1% (mass volume ratio) mercury chloride, put into 0.15%
The aseptic citric acid solution of (mass volume ratio) cuts, water is blotted by aseptic filter paper, stand-by
C, callus and bud inducement: selecting inducing culture, culture medium is loaded in 50 milliliters of triangular flasks, every bottle 20 milli
Rise.Culture medium after 120 DEG C of sterilization treatment 20min, access remove blade step B in about 1cm outer implant (with or without
Young shoot), access in culture medium, every bottle graft enters 1 outer implant, quiescent culture in illumination box, evoked callus
And young shoot.Inductivity is 95%.
Described inducing culture is: MS solid medium+6-BA 0.50mg/L+NAA 0.25mg/L+ sucrose 3% (matter
Amount volume ratio), pH5.8-6.0.
D, Multiplying culture: treat that the shoot growth in step C, to about 2cm, changes proliferated culture medium, expand numerous further
Grow, form seedling.
Described proliferated culture medium is: 1/2MS fluid nutrient medium+6-BA 0.50mg/L+NAA 0.25mg/L+ sucrose
1.5% (mass volume ratio), pH5.8-6.0.
Every 20d subculture once, the seedling of formation, it is cut into 1.0cm, proceeds to proliferated culture medium, in cultivation identical with step D
Under the conditions of proceed Multiplying culture, expand numerous plant；
E, taking root: growth of seedling to about 5cm, change root media, can promote that it is taken root, rooting rate reaches 100%.
Described root media is: 1/2MS fluid nutrient medium+NAA 0.10mg/L+ sucrose 1.5%, pH5.8-6.0.
F, hardening and transplanting: when indefinite root length is to more than 2.0cm, open bottle cap, (open bottle after room temperature lower refining seedling 1-2d
Lid, rests in incubator), take out test tube seedling, clean its root culture medium, transplant to outdoor water body.
Step C, D, E are gnotobasis, illumination 40-60 μ E/ (m2S), periodicity of illumination is 12h/d, indoor temperature (25
±2)℃。
This method is gradually induced callus by an outer implant, callus break up the seedling made new advances, propagation times
Number can reach 8, root induction in Liquid Culture, and rootage duration is short, and after the seedling of 5cm accesses root media, 5d can give birth to
Root is complete, and rooting rate arrives 100%.Watermifoil difficult to understand ancient in liquid environment has two states, the plant part of heavy water and very water
Plant part difference very big, easily distinguish.This technique reduces the time of sterilization, pollution rate is less than 1%, also reaches
The effect of sterilizing very well, transplants survival rate more than 99%.
Claims (2)
1. an ancient watermifoil tissue culture and rapid proliferation method difficult to understand, the steps include:
A, outer implant select and pretreatment: growth selection is healthy and strong, green in color, without the stem section of pest and disease damage as outer implant, use alkali
Property washing agent soak 30min, then under running water rinse 2h, repeatedly rinse 4-5 time with distilled water, the most repeatedly clean with sterilized water
4-5 time, it is placed in aseptic operating platform；
B, outer implant sterilizing: first by 70% volume ratio alcohol immersion 15s on aseptic operating platform, with aseptic water washing 4-5 time,
Carry out surface sterilization 2-5min, aseptic water washing 4-5 time with 0.1% mass volume ratio mercury chloride again, put into 0.15% mass body
The aseptic citric acid solution of long-pending ratio cuts, water is blotted by aseptic filter paper, stand-by；
C, callus and bud inducement: the outer implant of difficult to understand ancient watermifoil in step B removing blade of 0.5-2cm is accessed induction
In culture medium, quiescent culture in illumination box, evoked callus and young shoot；
Described inducing culture is: MS solid medium+6-BA 0.50-2.00mg/L+NAA 0.25-0.50mg/L+ sucrose
3% mass volume ratio, pH 5.8-6.0；
D, Multiplying culture: be continuing with the culture medium of step C, for Multiplying culture；Or treat that shoot growth, to 1.5-2cm, is changed
Proliferated culture medium, further expanding propagation, form seedling；
Described proliferated culture medium is: 1/2MS fluid nutrient medium+6-BA 0.50-2.00mg/L+NAA 0.25-0.50mg/L+
Sucrose 1.5%, pH 5.8-6.0；
Every 20-25d subculture once, the seedling of formation, it is cut into 0.5-2.0cm, proceeds to inducing culture or proliferated culture medium, continue
Continue and carry out Multiplying culture, expand numerous plant；
E, take root: callus constantly grows, growth of seedling to 4-6cm, be changed without culture medium and continue on the culture medium of step C
Send out roots, or be replaced by root media；
Described root media is: 1/2MS fluid nutrient medium+NAA 0.10mg/L+ sucrose 1.5%, pH 5.8-6.0；F, refining
Seedling and transplanting: when indefinite root length is to more than 2.0cm, open bottle cap, after room temperature lower refining seedling 1-2d, takes out test tube seedling, cleans it
Root culture medium, transplants to outdoor water body；
Above step C, D, E are gnotobasis, illumination 40-60 μ E/ (m2S), periodicity of illumination is 12h/d, indoor temperature 25 ±
2℃。
Method the most according to claim 1, it is characterised in that: in step B, the disinfecting time of mercury chloride is 2min；Step C
With in D, the concentration of 6-BA be the concentration of 0.50mg/L, NAA be 0.25mg/L.
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| CN106954549B (en) * | 2017-02-27 | 2019-05-28 | 水生藻安生物科技（武汉）有限公司 | A kind of comb tooth bog pondweed tissue culture and rapid proliferation method |
| CN107494229B (en) * | 2017-09-19 | 2020-03-31 | 江苏省中国科学院植物研究所 | Method for regulating rooting of watermifoil by using allelopathic effect of yellow flag |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1586153A (en) * | 2004-07-23 | 2005-03-02 | 中国科学院武汉植物园 | Method for quick breeding M spicatum Linn |
| CN1640239A (en) * | 2004-12-07 | 2005-07-20 | 南京大学 | Haloragidaceae Myriophyllum spicatum engineered seedling uninodal breeding method |
| CN102450215A (en) * | 2010-11-01 | 2012-05-16 | 南京中科水治理工程有限公司 | Technology for propagating watermifoil through hormone induction |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110058928A (en) * | 2009-11-27 | 2011-06-02 | 안성수 | Scrubber Cultured Edible or Medicinal Mushrooms |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1586153A (en) * | 2004-07-23 | 2005-03-02 | 中国科学院武汉植物园 | Method for quick breeding M spicatum Linn |
| CN1640239A (en) * | 2004-12-07 | 2005-07-20 | 南京大学 | Haloragidaceae Myriophyllum spicatum engineered seedling uninodal breeding method |
| CN102450215A (en) * | 2010-11-01 | 2012-05-16 | 南京中科水治理工程有限公司 | Technology for propagating watermifoil through hormone induction |
Non-Patent Citations (2)
| Title |
|---|
| Regenerative Capacity of Myriophyllum aqvaticum Tissues Cultured In Vitro;MICHAEL E. KANE et.al;《J. Aquat. Plant Manage》;19911231;第29卷;第102-109页 * |
| 轮叶狐尾藻的组织培养和快速繁殖;顾福根等;《植物生理学通讯》;20060630;第42卷(第3期);第470页 * |
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