CN109937886A - A method of observation explant development ways - Google Patents
A method of observation explant development ways Download PDFInfo
- Publication number
- CN109937886A CN109937886A CN201910383496.8A CN201910383496A CN109937886A CN 109937886 A CN109937886 A CN 109937886A CN 201910383496 A CN201910383496 A CN 201910383496A CN 109937886 A CN109937886 A CN 109937886A
- Authority
- CN
- China
- Prior art keywords
- observing
- explant
- culture medium
- stem section
- development ways
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of methods for observing explant development ways, it is inoculated into the MS culture medium of the plant growth regulator containing various concentration using the blade of Crassula argentea and stem section as explant, it is observed that these four different explant development ways of sterile Spur Type, adventitious buds proliferation type, embryoid generation type, Organogenesis, the method for the present invention have the characteristics that materials are convenient, easy to operate, observing effect is good after 30 days.Teaching process is optimized, teaching efficiency is improved；Be conducive to the culture of students practical abikty and observation ability.The following steps are included: the acquisition of (1) material；(2) aseptic process；(3) sterile Spur Type；(4) adventitious buds proliferation type；(5) embryoid generation type；(6) Organogenesis I；(7) Organogenesis II.
Description
Technical field
The present invention relates to the experimental teaching fields of Plant Tissue Breeding course, and in particular to a kind of tissue cultures of Crassula argentea
Method.
Background technique
In Plant Tissue Breeding course, according to the theory of cellular omnipotency, the development ways of explant are divided into sterile short
Branch type, adventitious buds proliferation type, Organogenesis, embryoid generation type and the distinctive Protocorm Multiplication approach of orchid.Such as
What quickly and effectively distinguishes these types of approach, and student frequently experiences puzzlement.
Crassula argentea is perennial evergreen succulent, Crassulaceae jade plant category.For fleshy leaf to life, color is emerald green；Stalk circle
Moisten plump multi-branched, the plentiful rounding of plant shape.We were by the stem section and blade of selection Crassula argentea as explant, inoculation in recent years
Onto the MS culture medium containing different plant growth regulator, it can be realized on this kind of plant and remove the distinctive protocorm of orchid
Other four kinds of explant development ways other than stem proliferating way, the experimental method can clearly illustrate the knowledge point, be plant
The experimental teaching of tissue cultures provides great convenience, and optimizes teaching process, improves teaching efficiency；It is constantly seen after inoculation
The variation for examining explant also helps the culture of students practical abikty and observation ability, has received good teaching efficiency.
Summary of the invention
The present invention is intended to provide a kind of method for observing explant development ways, using the blade of Crassula argentea and stem section as
Explant is inoculated into the MS culture medium of the plant growth regulator containing various concentration, can be observed after 30 days four kinds it is different
Explant development ways.
The present invention is a kind of method for observing explant development ways, comprising the following steps:
(1) acquisition of material: winning the fresh healthy leaves of Crassula argentea and stem section, and surface 20min is rinsed under tap water；
(2) aseptic process: by after the completion of flushing blade and stem section on superclean bench with 75% alcohol impregnate
15s is used aseptic water washing 1 time after the completion of impregnating, is then soaked in 0.1% mercuric chloride solution and sterilizes 6min, after the completion of disinfection
It uses aseptic water washing 3-5 times again, the moisture of blade and stem section surface is finally blotted with sterilized filter paper；
(3) sterile Spur Type development ways: the stem section that step (2) obtain is cut into the small stem section with a pair of of axillary bud and is inoculated with
To MS minimal medium, induction stem section growth；
(4) adventitious buds proliferation approach: the stem section that step (2) obtain is cut into the small stem section with a pair of of axillary bud and is seeded to clump
It sprouts on proliferated culture medium, induction generates Multiple Buds；
(5) size that the blade that step (2) obtain is cut into about 1.0cm*1.0cm embryoid way: is seeded to MS
On minimal medium, induction generates adventitious bud；
(6) Organogenesis approach I: the size that the blade that step (2) obtain is cut into about 1.0cm*1.0cm is seeded to more
In injured tissue induced medium, induction generates callus；
(7) Organogenesis approach II:, the size that the callus that step (6) obtain is cut into 0.5cm*0.5cm is inoculated with
To MS minimal medium, evoked callus is differentiated to form seedling again；
2, a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step
(2) aseptic operating platform in needs to open ultraviolet germicidal lamp before the use and blower carries out sterilization 30min.
3, a kind of as described in claim 1 to be observed in Plant Tissue Breeding teaching by the tissue cultures of Crassula argentea
The method of explant development ways, it is characterised in that: include 1-2 drop in 0.15% mercuric chloride solution in the step (2)
The tween of 20mg/L.
4, a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step
(3) culture medium of the sterile Spur Type development ways in is MS+ sucrose 20g/L+ agar 6g/L, PH 5.8-6.0.
5, a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step
(4) the adventitious buds proliferation culture medium in is by MSMS+6-BA0.5mg/L+NAA0.5mg/L+ sucrose 20g/L+ agar 6g/L, PH
For 5.8-6.0.
6, a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step
(5) culture medium of the embryoid way in is MS+ sucrose 20g/L+ agar 6g/L, PH 5.8-6.0.
7, a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step
(6) culture medium is by MS+6-BA2mg/L+NAA0.2mg/L+ sucrose 20g/L+ agar 6g/L, PH 5.8-6.0.
8, a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step
(7) culture medium is by MS+ sucrose 20g/L+ agar 6g/L, PH 5.8-6.0.
9, a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step
(3) condition of culture to the step (7) is 24-26 DEG C of temperature, intensity of illumination 2000-3000lux, light application time 10-12h/
d。
Compared with prior art the invention has the benefit that
1, materials are convenient: Crassula argentea asexual multiplication ability is strong, generally cutting propagation, uses plumpness to enrich in Growing season tender
Stem or blade are inserted into sand bed after slightly drying, will take root and sprout after 3-4 weeks；Cultivation management is simply extensive, and watering, fertilizing is few, is
A kind of popular foliage plant.
2, easy to operate, observing effect is good: all plump sturdy volume of the blade and stem section of Crassula argentea is big, clamping side when inoculation
Just, clear when observation.
3, sterilization effect is good: the smooth no epidermal hair of stem, the leaf surface of Crassula argentea, and thicker wax is covered outside leaf epidermal cell
Layer, explant surface sterilization effect is good, and pollution rate low success rate is high after inoculation.
4, using Crassula argentea, this plant material can realize four kinds of different explant development ways, simplify, make
The experimental teaching of " Plant Tissue Breeding " course is more lively, intuitive, grasps in Plant Tissue Breeding with being conducive to student system
The development ways of explant.
5,4 kinds of different explant development ways can be observed within 30 days after being inoculated with, the time is short, and effect is good, is plant group
The experimental teaching for knitting culture brings great convenience.
6, it is constantly observed after being inoculated with by explant, the ability of student's careful observation and analysis can be improved, excite student
Learning interest improves efficiency of teaching, optimizes teaching process.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate the present invention, but is not intended to limit the scope of the invention.
A kind of method of observation explant development ways of the invention, comprising the following steps:
The acquisition of material: winning the fresh healthy leaves of Crassula argentea and stem section, in the blade table that tap water undershoot washing takes
Face 20min；
Aseptic process: 30min opens the ultraviolet germicidal lamp of aseptic operating platform and blower before operation, and 10min is closed
Blower sufficiently sterilizes aseptic operating platform by ultraviolet germicidal lamp, after the completion of sterilization, the blade after the completion of flushing is existed
15s is impregnated with 75% alcohol on aseptic operating platform, is used aseptic water washing 1 time after the completion of impregnating, is then soaked in 0.1% liter
6min is sterilized in mercury solution, is used aseptic water washing 3-5 times again after the completion of disinfection, is finally blotted blade and stem section table with sterilized filter paper
The moisture in face；
Sterile Spur Type: each ingredient of MS culture medium is mixed be prepared into mixed liquor first, and add 2% thereto
Sucrose and 0.65% agar powder be prepared into MS minimal medium, the MS minimal medium after the completion of preparation is dispensed to taper
In bottle, and the 20min that sterilizes at 98kPa, 121 DEG C, conical flask is taken out after the completion of sterilizing, makes culture medium natural cooling therein
Stem section after the completion of aseptic process is cut into the small stem section containing a pair of of axillary bud and is seeded on MS minimal medium, in temperature by solidification
24-26 DEG C of degree provides the illumination of 12-14h using fluorescent lamp daily, and intensity of illumination 2000-3000lx can be observed after 30 days
Axillary bud is grown, and stem section is grown tall；
Adventitious buds proliferation type: the NAA of MS culture medium, the 6-BA of 0.5mg/L and 0.5mg/L is mixed into preparation first
At mixed liquor, and thereto, 2% sucrose of addition and 0.65% agar powder are prepared into adventitious buds proliferation culture medium, will prepare
Adventitious buds proliferation culture medium after is dispensed to conical flask, and the 20min that sterilizes at 98kPa, 121 DEG C, will after the completion of sterilizing
Conical flask takes out, and solidifies adventitious buds proliferation culture medium natural cooling therein, the stem section after the completion of aseptic process is cut into and is contained
There is the small stem section of a pair of of axillary bud to be transferred on adventitious buds proliferation culture medium, inducing clumping bud is carried out at 26 DEG C of temperature, using day
Light lamp provides the illumination of 12-14h daily, and intensity of illumination 2000-3000lx is generated largely after 30 days in axil and stem section bottom
The Multiple Buds of the green in color of meat；
Embryoid generation type: each ingredient of MS culture medium is mixed be prepared into mixed liquor first, and added thereto
2% sucrose and 0.65% agar powder are prepared into MS minimal medium, by the MS minimal medium after the completion of preparation dispense to
In conical flask, and the 20min that sterilizes at 98kPa, 121 DEG C, conical flask is taken out after the completion of sterilizing, cultivates MS therein substantially
The solidification of base natural cooling, is seeded to MS minimal medium for the size that the blade after the completion of aseptic process is cut into 1.0cm*1.0cm
In, induction is carried out at 26 DEG C of temperature and generates adventitious bud, provides the illumination of 12-14h daily using fluorescent lamp, intensity of illumination is
2000-3000lx forms green protrusion at paddle cutout after 30 days, kick, which is grown up, to be unfolded to be the first of Crassula argentea seedling
Piece leaf then grows a pair to raw blade, is formed simultaneously root；
Organogenesis I: the NAA of MS culture medium, the 6-BA of 2mg/L and 0.2mg/L is mixed be prepared into first
Mixed liquor, and 2% sucrose of addition and 0.65% agar powder are prepared into callus induction minimal medium thereto, will make
Callus induction minimal medium after the completion of standby is dispensed to conical flask, and the 20min that sterilizes at 98kPa, 121 DEG C, is gone out
Conical flask is taken out after the completion of bacterium, solidifies callus induction minimal medium natural cooling therein, aseptic process is complete
The size that blade after is cut into 1.0cm*1.0cm is seeded in callus induction minimal medium, at 26 DEG C of temperature into
Row callus induction, provides the illumination of 12-14h using fluorescent lamp daily, intensity of illumination 2000-3000lx, after 30 days
The spherical callus of yellow green of consolidation is generated at paddle cutout；
Organogenesis II: each ingredient of MS culture medium is mixed be prepared into mixed liquor first, and added thereto
2% sucrose and 0.65% agar powder are prepared into MS minimal medium, by the MS minimal medium after the completion of preparation dispense to
In conical flask, and the 20min that sterilizes at 98kPa, 121 DEG C, conical flask is taken out after the completion of sterilizing, cultivates MS therein substantially
The solidification of base natural cooling, is then seeded to MS for the size that the callus generated in Organogenesis I is cut into 0.5cm*0.5cm
On minimal medium, evoked callus breaks up again at 26 DEG C of temperature, provides the illumination of 12-14h, light daily using fluorescent lamp
It is 2000-3000lx according to intensity, callus breaks up the green protrusion of generation again after 30 days, and green protrusion grows up into the face of meat
The bud green adventitious bud of color.
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, without departing from the technical principles of the invention, several improvements and modifications, these improvements and modifications can also be made
Also it should be regarded as protection scope of the present invention.
Claims (9)
1. a kind of method for observing explant development ways, which comprises the following steps:
(1) acquisition of material: winning the fresh healthy leaves of Crassula argentea and stem section, and surface 20min is rinsed under tap water；
(2) aseptic process: by after the completion of flushing blade and stem section on superclean bench with 75% alcohol impregnate 15s, leaching
It is used aseptic water washing 1 time after the completion of bubble, is then soaked in 0.1% mercuric chloride solution and sterilizes 6min, use nothing after the completion of disinfection again
Bacterium water rinses 3-5 times, and the moisture of blade and stem section surface is finally blotted with sterilized filter paper；
(3) sterile Spur Type: the stem section that step (2) obtain is cut into, and there is the small stem section of a pair of of axillary bud to be seeded to MS cultivates substantially
On base, induction stem section growth；
(4) adventitious buds proliferation type: the stem section that step (2) obtain is cut into the small stem section with a pair of of axillary bud and is seeded to Multiple Buds increasing
It grows on culture medium, induction generates Multiple Buds；
(5) embryoid generation type: the size that the blade that step (2) obtain is cut into about 1.0cm*1.0cm is seeded to MS and is trained substantially
It supports on base, induction generates adventitious bud；
(6) Organogenesis I: the size that the blade that step (2) obtain is cut into about 1.0cm*1.0cm is seeded to callus and is lured
It leads on culture medium, induction generates callus；
(7) Organogenesis II:, it is basic that the size that the callus that step (6) obtain is cut into 0.5cm*0.5cm is seeded to MS
On culture medium, evoked callus is differentiated to form seedling again.
2. a kind of method for observing explant development ways as described in claim 1, it is characterised in that: in the step (2)
Aseptic operating platform need to open ultraviolet germicidal lamp and blower before the use and carry out sterilization 30min.
3. a kind of method for observing explant development ways as described in claim 1, it is characterised in that: in the step (2)
0.1% mercuric chloride solution in include 1-2 drop 20mg/L tween.
4. a kind of method for observing explant development ways as described in claim 1, it is characterised in that: in the step (3)
Sterile Spur Type development ways culture medium be MS+ sucrose 20g/L+ agar 6g/L, PH 5.8-6.0.
5. a kind of method for observing explant development ways as described in claim 1, it is characterised in that: in the step (4)
Adventitious buds proliferation culture medium be to be by MS MS+6-BA0.5mg/L+NAA0.5mg/L+ sucrose 20g/L+ agar 6g/L, PH
5.8-6.0。
6. a kind of method for observing explant development ways as described in claim 1, it is characterised in that: in the step (5)
Embryoid way culture medium be MS+ sucrose 20g/L+ agar 6g/L, PH 5.8-6.0.
7. a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step (6)
Culture medium is by MS+6-BA2mg/L+NAA0.2mg/L+ sucrose 20g/L+ agar 6g/L, PH 5.8-6.0.
8. a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step (7)
Culture medium is by MS+ sucrose 20g/L+ agar 6g/L, PH 5.8-6.0.
9. a kind of method for observing explant development ways as described in claim 1, it is characterised in that: the step (3) is extremely
The condition of culture of the step (7) is 24-26 DEG C of temperature, intensity of illumination 2000-3000lux, light application time 10-12h/d.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910383496.8A CN109937886B (en) | 2019-05-09 | 2019-05-09 | Method for observing explant generating way |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910383496.8A CN109937886B (en) | 2019-05-09 | 2019-05-09 | Method for observing explant generating way |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109937886A true CN109937886A (en) | 2019-06-28 |
| CN109937886B CN109937886B (en) | 2022-05-17 |
Family
ID=67017149
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910383496.8A Active CN109937886B (en) | 2019-05-09 | 2019-05-09 | Method for observing explant generating way |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109937886B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105359981A (en) * | 2015-12-07 | 2016-03-02 | 宋宏婷 | Tissue culture method for crassula agenten thunb seedlings |
-
2019
- 2019-05-09 CN CN201910383496.8A patent/CN109937886B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105359981A (en) * | 2015-12-07 | 2016-03-02 | 宋宏婷 | Tissue culture method for crassula agenten thunb seedlings |
Non-Patent Citations (6)
| Title |
|---|
| AHMED ET AL.: "Indirect Regeneration and Somatic Embryogenesis from Leaf and Stem Explants of Crassula ovata (Mill.) Druce – An Ornamental Medicinal Plant", 《INTERNATIONAL SCHOLARLY AND SCIENTIFIC RESEARCH & INNOVATION》 * |
| PATERSON ET AL.: "CALLUS FORMATION AND ORGANOGENESIS FROM CULTURED LEAF SEGMENTS OF CRASSULA ARGENTEA: CYTOKININ-INDUCED DEVELOPMENTAL PATTERN CHANGES", 《AMER. J. BOT.》 * |
| PATERSON ET AL.: "EFFECTS OF LIGHT AND HORMONES ON REGENERATION OF CRASSULA ARGENTEA FROM LEAVES", 《AMER. J. BOT.》 * |
| 刘永立等: "玉树组织培养中的器官形成与植株再生(英文)", 《.浙江大学学报(农业与生命科学版)》 * |
| 李庆伟等: "燕子掌叶片再生体系的建立", 《北方园艺》 * |
| 蒲仕明等: "燕子掌的组织培养与植株再生", 《北方园艺》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109937886B (en) | 2022-05-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104012417B (en) | High-efficiency and rapid micropropagation method for toxicodendron vernicifluum | |
| CN103190347A (en) | Teapot dates tissue culturing method | |
| CN107155844A (en) | A kind of method of Tissue Culture of Trees seedling greenhouse hardening domestication | |
| CN105165627B (en) | A kind of rosewood tissue culture sterilization formula and tissue culture method | |
| CN106718892A (en) | A kind of Chinese rose rapid propagation method | |
| CN105613287B (en) | A kind of method bucket cotton rose tissue rapid propagation method for culturing seedlings | |
| CN108575740B (en) | Method for generating poplar somatic embryo and building plant | |
| CN109618932A (en) | A kind of method of rhododendron dauricum adventitious bud inducing and plant regeneration | |
| CN106172016A (en) | A kind of Cortex Acanthopancis tissue culture and rapid propagation method | |
| CN102246700B (en) | Tissue culture method for populus yunnanensis Dode with tender stem as explant | |
| CN105309314A (en) | Method for setting up tissue culture regeneration system of Mongolian medicine dracocephalum moldavica | |
| CN108739370A (en) | A method of it is carried out using ripe lotus embryo fast numerous | |
| CN113331059B (en) | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants | |
| CN105613288B (en) | A kind of construction method of Phnom Penh Chinese littleleaf box rapid propagation system | |
| CN109042330A (en) | A kind of method for tissue culture of spindle tree | |
| CN106577280B (en) | A method of aseptic seedling is quickly bred using Merrillanthus hainanensis tender stem segments and blade | |
| CN109258478A (en) | The tissue culture propagation method of polygonatum cyrtonema | |
| CN106305430B (en) | A kind of Red Hill cherry powder enamel quick breeding by group culture method | |
| CN107950394A (en) | A kind of method for tissue culture of tree peony rataria | |
| CN105660396B (en) | A kind of rapid propagation method of Chinese scholar tree excised leaf somatic embryo inducement | |
| CN104604735B (en) | A kind of tissue culture and rapid propagation method of U.S.'s red autumnal leaves Lagerstroemia indica L. | |
| CN115024221B (en) | Method for rapidly propagating large-leaf morinda officinalis tissue culture seedlings and application thereof | |
| CN106489737A (en) | A kind of culture medium of Hybrid Tea tissue cultures and method | |
| CN107466852B (en) | A kind of thick stem Japanese ardisia tissue cultures and rapid propagation method | |
| CN109329059A (en) | A kind of method of straw short-tube lycoris tissue cultures |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220425 Address after: 061000 Jiuhe West Road, Yunhe District, Cangzhou City, Hebei Province Applicant after: CANGZHOU TECHNOICAL College Address before: 061000 No. 1, row 1, cotton textile factory community, Chaoyang Road, Yunhe District, Cangzhou City, Hebei Province Applicant before: Liu Yantao |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |