CN106942058B - The culture medium and its method of a kind of day bright and beautiful chapter platymiscium tissue cultures - Google Patents
The culture medium and its method of a kind of day bright and beautiful chapter platymiscium tissue cultures Download PDFInfo
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- CN106942058B CN106942058B CN201710183796.2A CN201710183796A CN106942058B CN 106942058 B CN106942058 B CN 106942058B CN 201710183796 A CN201710183796 A CN 201710183796A CN 106942058 B CN106942058 B CN 106942058B
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- 241001529246 Platymiscium Species 0.000 title claims abstract description 35
- 239000001963 growth medium Substances 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000012879 subculture medium Substances 0.000 claims abstract description 10
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 45
- 238000012545 processing Methods 0.000 claims description 21
- 229920001817 Agar Polymers 0.000 claims description 17
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 17
- 229930006000 Sucrose Natural products 0.000 claims description 17
- 239000008272 agar Substances 0.000 claims description 17
- 239000005720 sucrose Substances 0.000 claims description 17
- 238000005286 illumination Methods 0.000 claims description 16
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
- 238000004140 cleaning Methods 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 10
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 235000013601 eggs Nutrition 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 6
- 230000004069 differentiation Effects 0.000 claims description 5
- 229960002523 mercuric chloride Drugs 0.000 claims description 4
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 4
- 230000000249 desinfective effect Effects 0.000 claims description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims description 2
- 239000010977 jade Substances 0.000 claims description 2
- 238000009395 breeding Methods 0.000 abstract description 24
- 230000001488 breeding effect Effects 0.000 abstract description 24
- 241000196324 Embryophyta Species 0.000 abstract description 9
- 238000013459 approach Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 26
- 239000002609 medium Substances 0.000 description 16
- 230000001954 sterilising effect Effects 0.000 description 15
- 230000006698 induction Effects 0.000 description 11
- 241001532137 Adromischus marianae Species 0.000 description 8
- 238000012360 testing method Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 4
- 239000000645 desinfectant Substances 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000010428 oil painting Methods 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 4
- 229940033663 thimerosal Drugs 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 3
- 229910052753 mercury Inorganic materials 0.000 description 3
- 238000012090 tissue culture technique Methods 0.000 description 3
- 241000220284 Crassulaceae Species 0.000 description 2
- 240000000111 Saccharum officinarum Species 0.000 description 2
- 235000007201 Saccharum officinarum Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000000384 rearing effect Effects 0.000 description 2
- 241000304871 Adromischus Species 0.000 description 1
- 241001573881 Corolla Species 0.000 description 1
- 240000000249 Morus alba Species 0.000 description 1
- 235000008708 Morus alba Nutrition 0.000 description 1
- 241001596291 Namibia Species 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Classifications
-
- 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
- A01H4/001—Culture apparatus for tissue culture
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to field of plant tissue culture technique, in particular to the culture medium and method of a kind of day bright and beautiful chapter platymiscium tissue cultures.The method that the present invention utilizes Plant Tissue Breeding realizes the quick breeding of day brocade chapter platymiscium.The culture medium includes initial culture base, subculture medium and strong seedling culture base.It is remarkably improved the breeding coefficient of day brocade chapter platymiscium using culture medium provided by the invention, shortens the breeding cycle, the industrialization production for day brocade chapter platymiscium provides significantly more efficient approach.
Description
Technical field
The invention belongs to the cultures of field of plant tissue culture technique more particularly to a kind of day bright and beautiful chapter platymiscium tissue cultures
Base and method.
Background technique
Its bright and beautiful chapter (Adromischus) is the day Crassulaceae (Crassulaceae) brocade chapter category herbaceos perennial, is originated in
The Cape province in South Africa and Namibia.Plant is short and small, short stem taupe.Leaf is essentially oval tubular, and lower part very long one section is several
For cylinder, top is slightly wider, slightly flat, nearly oval, and leaf is 2.5~5 centimetres long, 1~2 centimetre wide, leaf back boss, front
Relatively flat, top leaf margin has waveform wrinkle, and epidermis is hairless, and glossy, leaf color is grayish green mulberry spot.Inflorescence is 25 centimetres high,
Cylindrical fireworks are cylindrical, 1 centimetre long, upper green, lower part purple, and corolla 5 is split, purple, edge white.
The breeding of its bright and beautiful chapter platymiscium inserts breeding and breeding of choping off the head mainly by leaf, both modess of reproduction not only reproduction speed
Slowly, and survival rate is low.Traditional modes of reproduction is difficult to meet the needs of market is huge.And tissue culture technique is as a kind of quick
Modes of reproduction is, it can be achieved that industrial seedling rearing, to meet the growing demand in market.
Tissue culture technique is that organ, tissue or cell living are placed in culture medium under sterile conditions, and is placed on suitable
In suitable environment, cell, tissue or individual made of continuously cultivating are carried out.Important neck of this technology as modern biotechnology
One of domain is widely used in terms of agricultural production and industrial seedling rearing, plant fine germplasm resources is saved, excellent product
Quick breeding of kind etc. also plays an important role.But currently, being directed to the tissue culture technique of day brocade chapter platymiscium also
It does not have been reported that.
Summary of the invention
In view of this, the present invention provides the culture medium and method of a kind of day bright and beautiful chapter platymiscium tissue cultures.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides the culture mediums of a kind of day bright and beautiful chapter platymiscium tissue cultures, including initial culture base, squamous subculture
Base and strong seedling culture base；
The initial culture base are as follows: contain 1~1.5mg/L KT, 0.05~0.1mg/L NAA, 0.03~0.05mg/L
TDZ, 25~30g/L sucrose, 6~7g/L agar MS culture medium, pH value be 5.8~6.0；
The subculture medium are as follows: contain 0.1~0.15mg/L KT, 0.01~0.02mg/L TDZ, 25~30g/L sugarcane
The MS culture medium of sugar, 6~7g/L agar, pH value are 5.8~6.0；
The strong seedling culture base are as follows: the MS culture medium containing 5~7g/L agar and 15~25g/L sucrose, pH value be 5.8~
6.0。
For Plant Tissue Breeding, culture medium is one of most critical factor of its growth conditions, existing day brocade chapter platymiscium
Tissue cultures due to induction differentiation primary, shoot proliferation and three phases of taking root culture medium selection and combination collocation
It is improper, so that the Callus induction rate in each stage, Differentiation ration of adventitious buds, growth coefficient and rooting rate are all poor, present invention selection
The culture medium of specific three phases constitutes the day brocade chapter platymiscium an of architectonical numerous technique fastly, finally improves significantly
The breeding coefficient of its bright and beautiful chapter platymiscium, shortens the breeding cycle, for day brocade chapter platymiscium industrialization production provide more added with
The approach of effect.
Preferably, initial culture base are as follows: contain 1.5mg/L KT, 0.1mg/L NAA, 0.05mg/L TDZ, 30g/L sugarcane
The MS culture medium of sugar, 7g/L agar, pH value are 5.8~6.0；
Subculture medium are as follows: the MS culture containing 0.1mg/L KT, 0.01mg/L TDZ, 30g/L sucrose, 7g/L agar
Base, pH value are 5.8~6.0；
Strong seedling culture base are as follows: the MS culture medium containing 7g/L agar and 25g/L sucrose, pH value are 5.8~6.0.
The present invention also provides the method for tissue culture of a kind of day bright and beautiful chapter platymiscium, include the following steps:
Step 1: day brocade chapter platymiscium explant is inoculated into the initial culture base in culture medium as claimed in claim 1 or 2
Dark culture is first carried out, callus is obtained；
Step 2: subculture medium that callus is transferred in culture medium as claimed in claim 1 or 2 is carried out after being commissioned to train
It supports, the seedling broken up；
Step 3: the seedling of differentiation being transferred to the strong seedling culture base in culture medium as claimed in claim 1 or 2 and carries out strong sprout
Culture, obtains tissue-cultured seedling.
Preferably, before the inoculation of the chapter of day brocade described in step 1 platymiscium explant further include: acquisition day brocade chapter platymiscium petal
Flower arrow undeployed removes bract, and carry out disinfection processing, is then cut into the flower arrow segment of 3-5cm.
Preferably, the collecting time of the colored arrow is 5~June, and the length of the colored arrow chooses 10-15cm.
In embodiment provided by the invention, disinfection treatment specifically: take the colored arrow segment to be cleaned with NaClO solution,
Then it is sterilized, then washed with water with mercuric chloride solution.
Preferably, the mass percentage concentration of the NaClO solution is 0.1%；The mass percentage concentration of mercuric chloride solution
It is 0.1%, disinfecting time 5min；The number of water cleaning is 5-6 times, each soaking time >=3min.
Preferably, in day brocade chapter platymiscium tissue cultures, the condition of culture of dark culture are as follows: cultivation temperature is 24~26
DEG C, relative humidity is 45%~50%.
In embodiment provided by the invention, the condition of culture of dark culture are as follows: cultivation temperature is 25 DEG C, and relative humidity is
48%.
Preferably, in day brocade chapter platymiscium tissue cultures, the condition of culture of squamous subculture are as follows: periodicity of illumination is 8~
12h/d, intensity of illumination are 2500~3500lx, and cultivation temperature is 24~26 DEG C, and relative humidity is 45%~50%.
In specific embodiment provided by the invention, the condition of culture of squamous subculture are as follows: periodicity of illumination 12h/d, illumination
Intensity is 3500lx, and cultivation temperature is 25 DEG C, relative humidity 48%.
Preferably, in day brocade chapter platymiscium tissue cultures, the condition of culture of strong seedling culture are as follows: periodicity of illumination is 8~
12h/d, intensity of illumination are 2500~3500lx, and cultivation temperature is 24~26 DEG C, and relative humidity is 45%~50%.
In specific embodiment provided by the invention, the condition of culture of strong seedling culture are as follows: periodicity of illumination 12h/d, illumination
Intensity is 3500lx, and cultivation temperature is 25 DEG C, relative humidity 48%.
The present invention is with Marianne (Adromischus marianae'antidorcadum'), Martin (Adromischus
Marianae'Bryan Makin'), Bai Anze (Adromischus marianae'Hallii'), red eggs (Adromischus
Marianae'Van Rhynn's Pass) the flower arrows of four kinds of day brocade chapter platymisciums is explant material, utilization is provided by the invention
Initial culture base, subculture medium and strong seedling culture base improve the breeding coefficient of day brocade chapter platymiscium significantly, shorten numerous
Grow the period, be quickly obtained the aseptic seedling of Marianne, Martin, four kinds of Bai Anze, red eggs day brocade chapter platymisciums, establish stablize,
Efficient tissue culture rapid propagation system, the industrialization production for day brocade chapter platymiscium provide significantly more efficient approach.
Specific embodiment
The invention discloses the culture medium and method of a kind of day bright and beautiful chapter platymiscium tissue cultures, those skilled in the art can be with
Present disclosure is used for reference, realization of process parameters is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications are to ability
It is it will be apparent that they are considered as being included in the present invention for field technique personnel.Method and application of the invention has been led to
Preferred embodiment is crossed to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to this paper institute
The methods and applications stated are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
To the explanation of the disclosed embodiments, enable those skilled in the art to implement or use the present invention.To this
A variety of modifications of a little embodiments will be readily apparent to those skilled in the art, as defined herein general
Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention will not
It can be intended to be limited to the embodiments shown herein, and be to fit to consistent with the principles and novel features disclosed in this article
Widest scope.
Below with reference to embodiment, with Marianne (Adromischus marianae'antidorcadum'), Martin
(Adromischus marianae'Bryan Makin'), Bai Anze (Adromischus marianae'Hallii'), red eggs
For the tissue cultures of (Adromischus marianae'Van Rhynn's Pass) day brocade chapter platymiscium, it is further described
The present invention:
The Plant Tissue Breeding of 1 Marianne of embodiment
1, growing environment
(1) solidified MS media, pH value 5.8 are used, matched culture medium sterilizes by high-pressure and high-temperature steam, at 126 DEG C
5min。
(2) tissue culture room, dark culture condition are as follows: cultivation temperature is 25 DEG C, and relative humidity 48%, every 3d is sterilized with ultraviolet lamp
30min；
Optical culture condition are as follows: light application time 12h/d, intensity of illumination 3500lx, cultivation temperature are 25 DEG C, relative humidity
48%, every 3d ultraviolet lamp sterilizing 30min.
2, the processing of explant
(1) in May between June, acquisition length reaches 10-15cm, and petal Marianne flower arrow undeployed is as explant
Body.
(2) after the completion of acquiring, first bract is extractd and (notices that wound cannot be too big) with tweezers, bract uses scalpel after extracing
Arrow Bottoming will be spent, wounded in the battle part is only stayed.
(3) flower arrow is put into 0.1%NaClO with oil painting brush cleaning down gently.
(4) 0.1%HgCl is used in superclean bench2Sterilize 5min.Note: the access times of thimerosal are no more than 10 times,
It outwells and reconfigures after reaching.HgCl2It is required that be kept in dark place, in order to avoid failure.
(5) after the completion of sterilizing, with aseptic water washing 5-6 times, each soaking time requires to be longer than 3min.
(6) stand-by spending arrow to be cut into 3-5cm segment with scalpel and tweezers in superclean bench after the completion of cleaning.
3, Initial culture
(1) superclean bench ultraviolet lamp sterilization 15min is opened.
(2) inoculation before with 75% alcohol spray disinfectant.
(3) in superclean bench, by scalpel, tweezers, inoculation disk alcolhol burner adequate disinfection.
(4) cleaned flower arrow is cut away into bottom 1-2mm with scalpel, is inoculated into the MS+1.5mg/L KT+ dispensed
In 0.1mg/L NAA+0.05mg/L TDZ+30g/L sucrose+7g/L agar medium.
(5) culture 15d flower arrow induces callus, Callus induction rate 87.6%.
4, squamous subculture
(1) explant for having grown callus early period is chosen.
(2) in superclean bench, with tweezers carefully by callus, it is transferred to the MS+0.1mg/L KT+ dispensed
In 0.01mg/L TDZ+30g/L sucrose+7g/L agar medium.
(3) culture 25d callus differentiates seedling, breeding coefficient 4.83.
5, strong seedling culture base
(1) seedling broken up in squamous subculture is chosen.
(2) in superclean bench, the MS solid medium hormone-free for being transferred to and having dispensed carefully is pressed from both sides out with tweezers
In.
(3) seedling is long to 3-4cm after 30d, can be with bottle outlet hardening.
Experimental result is shown: 1.5mg/L KT, 0.1mg/L NAA, 0.05mg/L TDZ, 15d being added in Initial culture
Flower arrow differentiates callus, Callus induction rate 87.6%.0.1mg/L KT, 0.01mg/L TDZ are added in squamous subculture,
25d callus differentiates seedling, breeding coefficient 4.83.
The Plant Tissue Breeding of 2 Martin of embodiment
1, growing environment
(1) solidified MS media, pH value 5.8 are used, matched culture medium sterilizes by high-pressure and high-temperature steam, at 126 DEG C
5min。
(2) tissue culture room, tissue culture room, dark culture condition are as follows: cultivation temperature is 25 DEG C, and relative humidity 48%, every 3d is with ultraviolet
Lamp sterilizing 30min；
Optical culture condition are as follows: light application time 12h/d, intensity of illumination 3500lx, cultivation temperature are 25 DEG C, relative humidity
48%, every 3d ultraviolet lamp sterilizing 30min.
2, the processing of explant
(1) in May between June, acquisition length reaches 10-15cm, and petal Martin undeployed spends arrow as explant.
(2) after the completion of acquiring, first bract is extractd and (notices that wound cannot be too big) with tweezers, bract uses scalpel after extracing
Arrow Bottoming will be spent, wounded in the battle part is only stayed.
(3) flower arrow is put into 0.1%NaClO with oil painting brush cleaning down gently.
(4) 0.1%HgCl is used in superclean bench2Sterilize 5min.Note: the access times of thimerosal are no more than 10 times,
It outwells and reconfigures after reaching.Mercury chloride requires to be kept in dark place, in order to avoid failure.
(5) after the completion of sterilizing, with aseptic water washing 5-6 times, each soaking time requires to be longer than 3min.
(6) stand-by spending arrow to be cut into 3-5cm segment with scalpel and tweezers in superclean bench after the completion of cleaning.
3, Initial culture
(1) superclean bench ultraviolet lamp sterilization 15min is opened.
(2) inoculation before with 75% alcohol spray disinfectant.
(3) in superclean bench, by scalpel, tweezers, inoculation disk alcolhol burner adequate disinfection.
(4) cleaned flower arrow is cut away into bottom 1-2mm with scalpel, is inoculated into the MS+1.5mg/L KT+ dispensed
In 0.1mg/L NAA+0.05mg/L TDZ+30g/L sucrose+7g/L agar medium.
(5) culture 15d flower arrow induces callus, Callus induction rate 83.4%.
4, squamous subculture
(1) explant for having grown callus early period is chosen.
(2) in superclean bench, with tweezers carefully by callus, it is transferred to the MS+0.1mg/L KT+ dispensed
In 0.01mg/L TDZ+30g/L sucrose+7g/L agar medium.
(3) culture 25d callus differentiates seedling, breeding coefficient 4.62.
5, strong seedling culture base
(1) seedling broken up in squamous subculture is chosen.
(2) in superclean bench, the MS solid medium hormone-free for being transferred to and having dispensed carefully is pressed from both sides out with tweezers
In.
(3) seedling is long to 3-4cm after 35d, can be with bottle outlet hardening.
Experimental result is shown: 1.5mg/L KT, 0.1mg/L NAA, 0.05mg/L TDZ, 15d being added in Initial culture
Flower arrow differentiates callus, Callus induction rate 83.4%.0.1mg/L KT, 0.01mg/L are added in squamous subculture
TDZ, 25d callus differentiate seedling, and callus differentiates seedling, breeding coefficient 4.62.
The Plant Tissue Breeding of the white Anze of embodiment 3
1, growing environment
(1) solidified MS media, pH value 5.8 are used, matched culture medium sterilizes by high-pressure and high-temperature steam, at 126 DEG C
5min。
(2) tissue culture room, tissue culture room, dark culture condition are as follows: cultivation temperature is 25 DEG C, and relative humidity 48%, every 3d is with ultraviolet
Lamp sterilizing 30min；
Optical culture condition are as follows: light application time 12h/d, intensity of illumination 3500lx, cultivation temperature are 25 DEG C, relative humidity
48%, every 3d ultraviolet lamp sterilizing 30min.
2, the processing of explant
(1) in May between June, acquisition length reaches 10-15cm, and petal white Anze flower arrow undeployed is as explant
Body.
(2) after the completion of acquiring, first bract is extractd and (notices that wound cannot be too big) with tweezers, bract uses scalpel after extracing
Arrow Bottoming will be spent, wounded in the battle part is only stayed.
(3) flower arrow is put into 0.1%NaClO with oil painting brush cleaning down gently.
(4) 0.1%HgCl is used in superclean bench2Sterilize 5min.Note: the access times of thimerosal are no more than 10 times,
It outwells and reconfigures after reaching.Mercury chloride requires to be kept in dark place, in order to avoid failure.
(5) after the completion of sterilizing, with aseptic water washing 5-6 times, each soaking time requires to be longer than 3min.
(6) stand-by spending arrow to be cut into 3-5cm segment with scalpel and tweezers in superclean bench after the completion of cleaning.
3, Initial culture
(1) superclean bench ultraviolet lamp sterilization 15min is opened.
(2) inoculation before with 75% alcohol spray disinfectant.
(3) in superclean bench, by scalpel, tweezers, inoculation disk alcolhol burner adequate disinfection.
(4) cleaned flower arrow is cut away into bottom 1-2mm with scalpel, is inoculated into the MS+1.5mg/L KT+ dispensed
In 0.1mg/L NAA+0.05mg/L TDZ+30g/L sucrose+7g/L agar medium.
(5) culture 15d flower arrow induces callus, Callus induction rate 86.5%.
4, squamous subculture
(1) explant for having grown callus early period is chosen.
(2) in superclean bench, with tweezers carefully by callus, it is transferred to the MS+0.1mg/L KT+ dispensed
In 0.01mg/L TDZ+30g/L sucrose+7g/L agar medium.
(3) culture 25d callus differentiates seedling, breeding coefficient 4.67.
5, strong seedling culture base
(1) seedling broken up in squamous subculture is chosen.
(2) in superclean bench, the MS solid medium hormone-free for being transferred to and having dispensed carefully is pressed from both sides out with tweezers
In.
(3) seedling is long to 3-4cm after 35d, can be with bottle outlet hardening.
Experimental result is shown: 1.5mg/L KT, 0.1mg/L NAA, 0.05mg/L TDZ, 25d being added in Initial culture
Flower arrow differentiates callus, Callus induction rate 86.5%.0.1mg/L KT, 0.01mg/L are added in squamous subculture
TDZ, 25d callus differentiate seedling, and callus differentiates seedling, breeding coefficient 4.67.
The Plant Tissue Breeding of 4 red eggs of embodiment
1, growing environment
(1) solidified MS media, pH value 5.8 are used, matched culture medium sterilizes by high-pressure and high-temperature steam, at 126 DEG C
5min。
(2) tissue culture room, tissue culture room, dark culture condition are as follows: cultivation temperature is 25 DEG C, and relative humidity 48%, every 3d is with ultraviolet
Lamp sterilizing 30min；
Optical culture condition are as follows: light application time 12h/d, intensity of illumination 3500lx, cultivation temperature are 25 DEG C, relative humidity
48%, every 3d ultraviolet lamp sterilizing 30min.
2, the processing of explant
(1) in May between June, acquisition length reaches 10-15cm, and petal red eggs flower arrow undeployed is as explant.
(2) after the completion of acquiring, first bract is extractd and (notices that wound cannot be too big) with tweezers, bract uses scalpel after extracing
Arrow Bottoming will be spent, wounded in the battle part is only stayed.
(3) flower arrow is put into 0.1%NaClO with oil painting brush cleaning down gently.
(4) 0.1%HgCl is used in superclean bench2Sterilize 5min.Note: the access times of thimerosal are no more than 10 times,
It outwells and reconfigures after reaching.Mercury chloride requires to be kept in dark place, in order to avoid failure.
(5) after the completion of sterilizing, with aseptic water washing 5-6 times, each soaking time requires to be longer than 3min.
(6) stand-by spending arrow to be cut into 3-5cm segment with scalpel and tweezers in superclean bench after the completion of cleaning.
3, Initial culture
(1) superclean bench ultraviolet lamp sterilization 15min is opened.
(2) inoculation before with 75% alcohol spray disinfectant.
(3) in superclean bench, by scalpel, tweezers, inoculation disk alcolhol burner adequate disinfection.
(4) cleaned flower arrow is cut away into bottom 1-2mm with scalpel, is inoculated into the MS+1.5mg/L KT+ dispensed
In 0.1mg/L NAA+0.05mg/L TDZ+30g/L sucrose+7g/L agar medium.
(5) culture 15d flower arrow induces callus, Callus induction rate 84.8%.
4, squamous subculture
(1) explant for having grown callus early period is chosen.
(2) in superclean bench, with tweezers carefully by callus, it is transferred to the MS+0.1mg/L KT+ dispensed
In 0.01mg/L TDZ+30g/L sucrose+7g/L agar medium.
(3) culture 25d callus differentiates seedling, breeding coefficient 4.57.
5, strong seedling culture base
(1) seedling broken up in squamous subculture is chosen.
(2) in superclean bench, the MS solid medium hormone-free for being transferred to and having dispensed carefully is pressed from both sides out with tweezers
In.
(3) seedling is long to 3-4cm after 35d, can be with bottle outlet hardening.
Experimental result is shown: 1.5mg/L KT, 0.1mg/L NAA, 0.05mg/L TDZ, 25d being added in Initial culture
Flower arrow differentiates callus, Callus induction rate 84.8%.0.1mg/L KT, 0.01mg/L are added in squamous subculture
TDZ, 25d callus differentiate seedling, and callus differentiates seedling, breeding coefficient 4.57.
Test example 1
By taking Marianne plant as an example, the present invention is to go out to be most suitable for the hormone of Initial culture by following screening of medium formula
Combination, concrete operations such as table 1:
The processing of 1 initial culture base of table screening
| Component | CK | Processing 1 | Processing 2 | Processing 3 |
| KT(mg/L) | 0 | 1 | 1.5 | 1.3 |
| NAA(mg/L) | 0 | 0.05 | 0.1 | 0.06 |
| TDZ(mg/L) | 0 | 0.03 | 0.05 | 0.05 |
Explanation: where the minimal medium of CK and 4 processing of control is MS culture medium；Control CK be do not add hormone KT,
NAA and TDZ.
Test result is shown in Table 2:
The callus that 2 Initial culture of table is respectively handled induces situation
By the above test result it is found that explant can differentiate callus in the culture medium of processing 1~3, wherein
The Callus induction rate highest of processing 2, up to 87.6%.Show the culture medium of processing 2: MS+1.5mg/L KT+0.1mg/L NAA+
0.05mg/L TDZ+25-30g/L sucrose+6-7g/L agar is the most suitable initial culture base of Marianne.
The present invention is to go out to be most suitable for the hormone combinations of squamous subculture by following screening of medium formula, concrete operations such as table
3:
The processing of 3 subculture medium of table screening
| Component | CK | Processing 1 | Processing 2 | Processing 3 |
| KT(mg/L) | 0 | 0.12 | 0.1 | 0.15 |
| TDZ(mg/L) | 0 | 0.02 | 0.01 | 0.01 |
Explanation: where the minimal medium of CK and 4 processing of control is MS culture medium；Control CK is not add hormone KT
And TDZ.
Test result is shown in Table 4:
The growing state and growth coefficient result that 4 squamous subculture of table is respectively handled
By the above test result it is found that it is indefinite that callus can differentiate using processing 3 and the culture medium for handling 2
Bud Cheng little Miao, wherein using culture medium (the MS+0.1mg/L KT+0.01mg/L TDZ+30g/L sucrose+7g/L fine jade of processing 2
Rouge) callus can be differentiated to form a large amount of seedlings, and it is the most suitable subculture medium of Marianne.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. the culture medium of a kind of day bright and beautiful chapter platymiscium tissue cultures, which is characterized in that including initial culture base, subculture medium and
Strong seedling culture base；
The initial culture base are as follows: contain 1.5mg/L KT, 0.1mg/L NAA, 0.05mg/L TDZ, 30g/L sucrose, 7g/L fine jade
The MS culture medium of rouge, pH value are 5.8~6.0；
The subculture medium are as follows: the MS culture containing 0.1mg/L KT, 0.01mg/L TDZ, 30g/L sucrose, 7g/L agar
Base, pH value are 5.8~6.0；
The strong seedling culture base are as follows: the MS culture medium containing 7g/L agar and 25g/L sucrose, pH value are 5.8~6.0；
The day brocade chapter platymiscium is Marianne, Martin, white Anze and red eggs.
2. the method for tissue culture of a kind of day bright and beautiful chapter platymiscium, which comprises the steps of:
Step 1: brocade chapter platymiscium flower arrow in day being inoculated into the initial culture base in culture medium described in claim 1 and is first secretly trained
It supports, obtains callus；
Step 2: callus being transferred to the subculture medium in culture medium described in claim 1 and carries out squamous subculture, is obtained
The seedling of differentiation；
Step 3: the seedling of differentiation being transferred to the strong seedling culture base in culture medium described in claim 1 and carries out strong seedling culture, is obtained
To tissue-cultured seedling；
The day brocade chapter platymiscium is Marianne, Martin, white Anze and red eggs.
3. method for tissue culture according to claim 2, which is characterized in that the chapter platymiscium explant of day brocade described in step 1
Before body inoculation further include: acquisition day brocade chapter platymiscium petal flower arrow undeployed removes bract, carry out disinfection processing, then cuts
At the flower arrow segment of 3-5cm.
4. method for tissue culture according to claim 3, which is characterized in that the collecting time of the colored arrow is 5~June,
The length of the colored arrow chooses 10-15cm.
5. method for tissue culture according to claim 3, which is characterized in that the disinfection treatment specifically: take the flower
Arrow segment is cleaned with NaClO solution, is then sterilized with mercuric chloride solution, then is washed with water.
6. method for tissue culture according to claim 5, which is characterized in that the mass percentage concentration of the NaClO solution
It is 0.1%；The mass percentage concentration of the mercuric chloride solution is 0.1%, disinfecting time 5min；The number of water cleaning is
5-6 times, each soaking time >=3min.
7. the method for tissue culture according to any one of claim 2 to 6, which is characterized in that the culture of the dark culture
Condition are as follows: cultivation temperature is 24~26 DEG C, and relative humidity is 45%~50%.
8. the method for tissue culture according to any one of claim 2 to 6, which is characterized in that the training of the squamous subculture
The condition of supporting are as follows: periodicity of illumination is 8~12h/d, and intensity of illumination is 2500~3500lx, and cultivation temperature is 24~26 DEG C, relatively wet
Degree is 45%~50%.
9. the method for tissue culture according to any one of claim 2 to 6, which is characterized in that the training of the strong seedling culture
The condition of supporting are as follows: periodicity of illumination is 8~12h/d, and intensity of illumination is 2500~3500lx, and cultivation temperature is 24~26 DEG C, relatively wet
Degree is 45%~50%.
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