CN104604693A - Augustine myriophyllum tissue culture and rapid propagation method thereof - Google Patents

Augustine myriophyllum tissue culture and rapid propagation method thereof Download PDF

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CN104604693A
CN104604693A CN201510076490.8A CN201510076490A CN104604693A CN 104604693 A CN104604693 A CN 104604693A CN 201510076490 A CN201510076490 A CN 201510076490A CN 104604693 A CN104604693 A CN 104604693A
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culture
explant
medium
aseptic
seedling
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CN104604693B (en
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李涛
李琪
任明磊
母丹丹
赵进东
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Institute of Hydrobiology of CAS
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Institute of Hydrobiology of CAS
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Abstract

The invention relates to an augustine myriophyllum tissue culture and rapid propagation method thereof. The method comprises the following steps: A) selecting an explant, namely selecting a plant stem with robust growth, bright green color and no plant diseases or insect pests as the explant; B) sterilzing the explant, namely performing treatment on the explant with alcohol and mercury chloride; C) performing bud induction, namely selecting optimal hormone mixture ratio to induce bud growth; D) performing proliferation culture, namely adjusting the hormone mixture ratio and the concentration of a culture medium to perform proliferation culture; E) rooting, namely selecting an appropriate cell auxin to perform rooting induction; and F) hardening and transplanting, namely opening a bottle mouth in a culture box, culturing for two days and then transferring into a natural water body for culture. The technology can not subject to seasonal and environmental restrictions, and a large number of sterile seedlings can be cultured for restoring an aquatic ecosystem.

Description

A kind of ancient watermifoil tissue culture and rapid proliferation method difficult to understand
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to group training and the method for quickly breeding of submerged plant ancient watermifoil difficult to understand, the method utilizes integral asepsis and strict temperature control measure, achieves submerged plant and produce in a large number in laboratory, and can be generalized to ecotope reparation.
Background technology
Ancient Myriophyllum difficult to understand is in Angiospermae (Angiospermae) Dicotyledoneae (Dicotyledoneae) Haloragaceae (Haloragidaceae).In current ecological restoration of lakes engineering as in water purification instrument kind and revegetation pioneer, fish shrimp crab pool breeding process as bait, take refuge and spawning place, and in indoor appreciation aquiculture process as setting material in the urgent need to.Use tissue culture technique, obtain and its high grade project seedling of vast propagation, to overcome the limited and with serious pollution problem of these species provenance in natural environment.
Report at present about watermifoil tissue cultures is many, but which kind of watermifoil all specifically do not implement to except Haloragidaceae Myriophyllum spicatum be.And about the Physiology and biochemistry of the ancient watermifoil of Austria, and the report of ecological aspect is all very rare.Although it has the denominator of Myriophyllum, 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 aseptically, takes temperature control measure, gives fixed-illumination, cultivates aseptic seedling system, can provide a large amount of aseptic seedlings throughout the year.Easy to implement the method, easy to operate, output is high.
In order to achieve the above object, the present invention adopts following technical measures:
A kind of ancient watermifoil tissue culture and rapid proliferation method difficult to understand, the steps include:
A, explant are selected and pretreatment: growth selection is healthy and strong, green in color, without the stem section of damage by disease and insect as explant, soak 30min with alkaline detergent, then rinse 2h under running water, repeatedly rinse 4-5 time with distilled water, repeatedly clean 4-5 time again with sterile water, be placed in aseptic operating platform.
B, explant sterilization: on aseptic operating platform, first use 70% (volume ratio) alcohol immersion 15s, with aseptic water washing 4-5 time, 0.1% (mass volume ratio) mercury chloride is used to carry out surface sterilization 2-5min again, aseptic water washing 4-5 time, the aseptic citric acid solution putting into 0.15% (mass volume ratio) cuts, water blots by aseptic filter paper, stand-by.
C, callus and bud inducement: by ancient for the Austria in the step B of the removal blade of 0.5-2cm watermifoil explant access inducing culture, quiescent culture in illumination box, evoked callus and young shoot.
Described inducing culture is: MS solid culture 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 medium using step C can be continued, for Multiplying culture.Also can treat that shoot growth is to 1.5-2cm, change proliferated culture medium, further expanding propagation, form seedling.
Described proliferated culture medium is: 1/2MS liquid 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, is cut into 0.5-2.0cm, proceeds to inducing culture or proliferated culture medium, with step D same culture conditions under proceed Multiplying culture, expand numerous plant;
E, to take root: callus constantly grows, growth of seedling, to about 4-6cm, can not continue to send out roots on the medium of step C by replaced medium, also can be replaced by root media, not only can promote that it is taken root, also can tachyauxesis.
Described root media is: 1/2MS liquid nutrient medium+NAA 0.10mg/L+ sucrose 1.5%, pH 5.8-6.0.F, hardening and transplanting: when adventive root grows to more than 2.0cm, open bottle cap, after room temperature lower refining seedling 1-2d, take out test-tube plantlet, clean its root medium, transplant to outdoor water body.
Above step C, D, E are gnotobasis, illumination 40-60 μ E/ (m 2s), periodicity of illumination is 12h/d, indoor temperature 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 the concentration of 0.50mg/L, NAA is 0.25mg/L.
MS culture medium prescription is as follows: unit mg/L
KNO 31900
NH 4NO 31650
MgSO 4·7H 2O 370
KH 2PO 4170
CaCl 2·2H 2O 440
MnSO 4·4H 2O 22.3
ZnSO 4·7H 2O 8.6
H 3BO 36.2
KI 0.83
Na 2MoO 4·7H 2O 0.25
CuSO 4·5H 2O 0.025
CoCl 2·6H 2O 0.025
Na 2-EDTA 37.3
FeSO 4·7H 2O 27.8
Glycine 2.0
Puridoxine hydrochloride 0.5
Tyiamine Hd element 0.1
Nicotinic acid 0.5
Creatine 100
MS solid culture medium is the agar that MS medium adds 7g/L, pH5.8-6.0.
Compared with prior art, the present invention has the following advantages and effect:
Austria provided by the invention ancient watermifoil tissue culture and rapid proliferation method, can provide a large amount of seedling in short-term, for providing resource endlessly to the research of the physiological and biochemical property of the ancient watermifoil of Austria further.Be easier to preserve and maintain the quality of germplasm resource by going down to posterity of callus.The stem section of a long 1cm was through the cultivation of a week, and can manage it into one piece of area is 0.25cm 2callus, then can obtain 6-8 seedling through the cultivation of three weeks, the long 5-8cm of each seedling, proliferation times reaches 6-8 doubly, and within 1 year, a bud can breed more than several hundred million seedling.With a bud 20d subculture once, each proliferation times is 8 calculating.
Number of days/d 20 40 60 80 100 120
Seedling number/ 8 8 2 8 3 8 4 8 5 8 6
Embodiment
The reagent that the embodiment of the present invention uses, if not otherwise specified, commercially available being realizes the present invention, and described technical scheme 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, explant are selected and pretreatment: growth selection is healthy and strong, green in color, without the stem section (with or without young shoot) of damage by disease and insect as explant, 30min is soaked with alkaline detergent, 2h is rinsed again under running water, repeatedly rinse 4-5 time with distilled water, repeatedly clean 4-5 time again with sterile water, be placed in aseptic operating platform.
B, explant sterilization: on aseptic operating platform, first use 70% ethanol (volume ratio) to soak 15s, with aseptic water washing 4-5 time, use 0.1% (mass volume ratio again, 0.1g mercury chloride adds in 100ml water) mercury chloride carries out surface sterilization 5min, aseptic water washing 4-5 time, the aseptic citric acid solution putting into 0.15% (mass volume ratio) cuts, and water blots by aseptic filter paper, stand-by.
C, callus and young shoot are induced: select inducing culture, medium is loaded in 50 milliliters of triangular flasks, 20 milliliters every bottle.Medium is after 120 DEG C of sterilization treatment 20min, and the explant (with or without young shoot) being about 1cm in the step B of blade is removed in access, and every bottle graft enters an explant, quiescent culture in illumination box.Inductivity is 90%.
Described inducing culture is: MS solid culture medium+6BA 2.00mg/L+NAA 0.50mg/L+ sucrose 3% (mass volume ratio), pH 5.8-6.0.
D, Multiplying culture: not replaced medium, the medium of step C continues on for Multiplying culture.Explant also can produce a large amount of buds expanding to be formed while callus, and bud is divided into one whole plant gradually, and produces new seedling endlessly.Every 25d subculture once, the seedling of formation, is cut into 1.5cm, proceeds to inducing culture, with step D same culture conditions under proceed Multiplying culture, expand numerous plant;
E, to take root: callus and growth of seedling, to certain phase, can continue to send out roots on the medium of step C.
F, hardening and transplanting: when adventive root grows to more than 2.0cm, open bottle cap, (open bottle cap, rest in incubator) after room temperature lower refining seedling 1-2d, take out test-tube plantlet, clean its root medium, transplant to outdoor water body.
In above step, C, D, E are gnotobasis, illumination 40-60 μ E/ (m 2s), periodicity of illumination is 12h/d, indoor temperature 25 ± 2 DEG C.
This method can induce callus gradually by an explant, and the seedling made new advances by Calli Differentiation, proliferation times can reach 6, use a kind of medium just just can realize bud inducement, propagation and three kinds of effects of taking root, reduce operating procedure, saves production cost.Rooting rate 100%, pollution rate is less than 1%, transplants survival rate and is greater than 98%.
Embodiment 2:
A kind of ancient watermifoil tissue culture and rapid proliferation method difficult to understand, comprises the following steps:
A, explant are selected and pretreatment: growth selection is healthy and strong, green in color, without the stem section (with or without young shoot) of damage by disease and insect as explant, remove spire, soak 30min with alkaline detergent, then rinse 2h under running water, repeatedly rinse 4-5 time with distilled water, repeatedly clean 4-5 time again with sterile water, be placed in aseptic operating platform.
B, explant sterilization: on aseptic operating platform, first use 70% (volume ratio) alcohol immersion 15s, with aseptic water washing 4-5 time, 0.1% (mass volume ratio) mercury chloride is used to carry out surface sterilization 2min again, aseptic water washing 4-5 time, the aseptic citric acid solution putting into 0.15% (mass volume ratio) cuts, water blots by aseptic filter paper, stand-by
C, callus and bud inducement: select inducing culture, medium is loaded in 50 milliliters of triangular flasks, 20 milliliters every bottle.Medium is after 120 DEG C of sterilization treatment 20min, the explant (with or without young shoot) of about the 1cm in the step B of blade is removed in access, and in access medium, every bottle graft enters 1 explant, quiescent culture in illumination box, evoked callus and young shoot.Inductivity is 95%.
Described inducing culture is: MS solid culture medium+6-BA 0.50mg/L+NAA 0.25mg/L+ sucrose 3% (mass volume ratio), pH5.8-6.0.
D, Multiplying culture: treat that shoot growth in step C is to about 2cm, change proliferated culture medium, further expanding propagation, form seedling.
Described proliferated culture medium is: 1/2MS liquid 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, is cut into 1.0cm, proceeds to proliferated culture medium, with step D same culture conditions under proceed Multiplying culture, expand numerous plant;
E, to take root: growth of seedling is to about 5cm, and change root media, can promote that it is taken root, rooting rate reaches 100%.
Described root media is: 1/2MS liquid nutrient medium+NAA 0.10mg/L+ sucrose 1.5%, pH5.8-6.0.
F, hardening and transplanting: when adventive root grows to more than 2.0cm, open bottle cap, (open bottle cap, rest in incubator) after room temperature lower refining seedling 1-2d, take out test-tube plantlet, clean its root medium, transplant to outdoor water body.
Step C, D, E are gnotobasis, illumination 40-60 μ E/ (m 2s), periodicity of illumination is 12h/d, indoor temperature (25 ± 2) DEG C.
This method induces callus gradually by an explant, the seedling made new advances by Calli Differentiation, and proliferation times can reach 8, root induction in liquid culture, rootage duration is short, after the seedling access root media of 5cm, 5d can be taken root complete, and rooting rate arrives 100%.The ancient watermifoil of Austria in liquid environment has two states, and the plant part of heavy water is very large with the plant part difference enduring water, easily distinguishes.This technique reduces the time of sterilization, pollution rate is less than 1%, also reaches the effect of fine sterilizing, transplants survival rate and is greater than 99%.

Claims (2)

1. Austria's ancient watermifoil tissue culture and rapid proliferation method, the steps include:
A, explant are selected and pretreatment: growth selection is healthy and strong, green in color, without the stem section of damage by disease and insect as explant, soak 30min with alkaline detergent, then rinse 2h under running water, repeatedly rinse 4-5 time with distilled water, repeatedly clean 4-5 time again with sterile water, be placed in aseptic operating platform;
B, explant sterilization: on aseptic operating platform, first use 70% volume ratio alcohol immersion 15s, with aseptic water washing 4-5 time, surface sterilization 2-5min is carried out again with 0.1% mass volume ratio mercury chloride, aseptic water washing 4-5 time, the aseptic citric acid solution putting into 0.15% mass volume ratio cuts, water blots by aseptic filter paper, stand-by;
C, callus and bud inducement: by ancient for the Austria in the step B of the removal blade of 0.5-2cm watermifoil explant access inducing culture, quiescent culture in illumination box, evoked callus and young shoot;
Described inducing culture is: MS solid culture 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 medium using step C can be continued, for Multiplying culture; Also can treat that shoot growth is to 1.5-2cm, change proliferated culture medium, further expanding propagation, form seedling;
Described proliferated culture medium is: 1/2MS liquid 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, is cut into 0.5-2.0cm, proceeds to inducing culture or proliferated culture medium, with step D same culture conditions under proceed Multiplying culture, expand numerous plant;
E, to take root: callus constantly grows, growth of seedling, to about 4-6cm, can not continue to send out roots on the medium of step C by replaced medium, also can be replaced by root media;
Described root media is: 1/2MS liquid nutrient medium+NAA 0.10mg/L+sucrose 1.5%, pH 5.8-6.0;
F, hardening and transplanting: when adventive root grows to more than 2.0cm, open bottle cap, after room temperature lower refining seedling 1-2d, take out test-tube plantlet, clean its root medium, transplants to outdoor water body;
Above step C, D, E are gnotobasis, illumination 40-60 μ E/(m 2s), periodicity of illumination is 12h/d, indoor temperature 25 ± 2 DEG C.
2. method according to claim 1, is characterized in that: in step B, the disinfecting time of mercury chloride is 2min; In step C and D, the concentration of 6BA is the concentration of 0.50mg/L, NAA is 0.25mg/L.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106954549A (en) * 2017-02-27 2017-07-18 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
CN107494229A (en) * 2017-09-19 2017-12-22 江苏省中国科学院植物研究所 A kind of method taken root using yellow flag allelopathy regulation watermifoil

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CN1640239A (en) * 2004-12-07 2005-07-20 南京大学 Haloragidaceae Myriophyllum spicatum engineered seedling uninodal breeding method
KR20110058928A (en) * 2009-11-27 2011-06-02 안성수 Scrubber Cultured Edible or Medicinal Mushrooms
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

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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
KR20110058928A (en) * 2009-11-27 2011-06-02 안성수 Scrubber Cultured Edible or Medicinal Mushrooms
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

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106954549A (en) * 2017-02-27 2017-07-18 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
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
CN107494229A (en) * 2017-09-19 2017-12-22 江苏省中国科学院植物研究所 A kind of method taken root using yellow flag allelopathy regulation watermifoil

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