CN109757368A - The method that alum root kind " kilim carpet " rachis vitro Regeneration System is established - Google Patents
The method that alum root kind " kilim carpet " rachis vitro Regeneration System is established Download PDFInfo
- Publication number
- CN109757368A CN109757368A CN201711096929.9A CN201711096929A CN109757368A CN 109757368 A CN109757368 A CN 109757368A CN 201711096929 A CN201711096929 A CN 201711096929A CN 109757368 A CN109757368 A CN 109757368A
- Authority
- CN
- China
- Prior art keywords
- rachis
- culture
- medium
- explant
- callus
- 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.)
- Pending
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of methods that alum root kind " kilim carpet " rachis vitro Regeneration System is established, method includes the following steps: (1) chooses explant and carries out disinfection；(2) induction of callus will be carried out after the cutting of treated rachis；(3) callus is subjected to induction adventitious bud；(4) by adventitious bud shoot proliferation culture seedling；(5) culture of rootage is carried out to the test tube seedling after shoot proliferation culture；(6) hardening culture.Tissue cultures are carried out to alum root kind " kilim carpet " using the propagation method, subculture cycle (35d) adventitious bud proliferation rate is 8 times or more, and reproduction speed is fast, solves the problems, such as sapling multiplication.
Description
Technical field
Present invention relates particularly to a kind of alum root (Heuchera micrantha) tissue cultures propagation method.
Background technique
Alum root (Heuchera micrantha) also known as coral bell, it is Saxifragaceae alum root category perennial herb flowers.Its
Garden-variety is various, and leaf color is rich and varied, and different season, environment and at a temperature of the color of blade also have change abundant
Change, is ideal perennial root flower border material.Liked currently, alum takes root in object depth by the small-sized flower amateur of home gardening, part product
Supply falls short of demand for the electric business sale of kind.In addition, also have in gardens for hayashishita flower border, quilt, courtyard greening etc., be excellent shade
Raw environment perennial root coloured silk leaf flower.
Alum takes root in half shade of object happiness, and resistance to full light is low temperature resistant, avoids excessive moisture, is suitable for planting in northern China, and south plantation is more
Number kinds there is a problem that more summer it is difficult (Huang Shaoling, Wang Huaqing, Zhou Yifeng etc., 2016, alum root is in In Dongguan introduction and acclimatization
Summer in the process adapts to Journal of Sex Research, Tropical Forest, 44 (4): 4-7).But alum root plant variety is numerous, also has in East China
It is suitable for the kind of low level management, such as kilim carpet, cocktail, maltose, obsidian.And wherein the adaptability of " kilim carpet " kind is best,
Blade is green, and in adverse circumstance or Transition season, blade takes on a red color again.Compared with other East China adaptability kinds, " kilim carpet "
It is more in line with the demand of gardens gardenning designer.
Currently, the modes of reproduction that alum takes root in object has slotting leaf, plant division and tissue culture mode, but preceding 2 kinds of mode reproduction speeds are slower,
It is not able to satisfy the market demand.The division propagation of the uses such as Liang Fang, survival rate are commented less than 50%(5 kind Perennial Flowers kind habit
Valence and cultivation research, grassland and lawn, 2009 (6): 43-46).Therefore, establishing tissue-culturing quick-propagation system is that solution is excellent
The most effective approach of non-defective unit kind seedling shortage.The tissue cultures of heuchera, have been shown in and have had been reported that, but the selection of explant majority is new
Silver-colored prince's kind successive propagation rate of bud or axillary bud stem section etc., the researchs culture such as Sun Guofeng reaches 15-20 times of (alum root hybrid ' silver-colored king
The tissue cultures of sub- ' and quickly breeding, Plant Physiology Communications, 2007,43 (3): 500-500)；The researchs culture such as Zhang Zhihong
(study, Jiangsu's agricultures up to 9.3 times by America alum root kind Zigong hall tissue cultures and rapid propagation in vitro for Zigong hall kind successive propagation rate
Science, 2011, (5): 69-71)；Only 3.5 times of ' brownies ' kind successive propagation rate of the researchs culture such as Chen Hong, and vitrifying
Seriously, effective bud is few (tissue culture and rapid proliferation of alum root, Shanghai Agricultural journal, 2011,27 (4): 80-82).Alum root " flower
The rapid propagation system of blanket " kind is there is not yet report.Tissue culture rapid propagation system is limited by maternal explant, and explant is dirty after simply sterilizing
Dye rate is excessively high, and excessively disinfection easily forms brown stain again, and aseptic strain is established relatively difficult.Therefore, inventor is using rachis as explant,
The Regeneration in Vitro rapid propagation system of the alum root kind kilim carpet of foundation, for solve the shortage of kilim carpet seedling one of important channel, to alum root
Exploitation and utilization and extention all there is very important meaning
Summary of the invention
The purpose of the invention is to establish alum root kind kilim carpet tissue culture propagating system.
In order to achieve the above object, the present invention provides a kind of foundation of alum root kind " kilim carpet " rachis vitro Regeneration System
Method, include the following steps:
(1) selection and disinfection of explant: the rachis of growth 20d or so is chosen as explant, after rinsing well, is disappeared
Poison, disinfecting process are as follows: it is rinsed well with clear water, it is then primary with the rinsing of Amway detergent, 15-20min is vibrated on shaking table；Flowing water
Rinse 20-30min after, on aseptic operating platform use 75% ethanol disinfection 10-20s, pour into immediately 0.1% mercuric chloride sterilize
12min vibrates 3-5min every time, the rachis of sanitized is cut into the segment of 2cm long finally with sterile water washing 4 times.
(2) induction of callus: taking the explant segment that processing obtains in step (1), with the inoculation of inclined-plane modes of emplacement
The induction of callus is carried out in induced medium (in advance by culture medium contra bevel), cultivation temperature is 25 ± 2 DEG C, is existed first
10d is cultivated in dark, then carries out illumination cultivation 25d；The illumination cultivation condition are as follows: light application time 14h/d, intensity of illumination
For 1500lx；The induced medium is MS+6-BA0.6mg/L+IBA0.02mg/L, pH value 5.8-6.0；
(3) differentiation of callus: the callus that induction obtains in step (2) is cut on explant after inducing 35d,
It accesses differential medium and carries out adventitious buds differentiation, cultivation temperature is 25 ± 2 DEG C；Light application time 14h/d, intensity of illumination 2500lx；
The induced medium is N6+CPPU 0.3mg/L+IBA0.01mg/L, pH value 5.8-6.0；
(4) shoot proliferation of adventitious bud: after the adventitious bud cutting that induction is obtained, every bud band 0.3cm or so stem section and 3-4 piece leaf
Piece accesses and carries out squamous subculture 25d in subculture multiplication medium, and cultivation temperature is 25 ± 2 DEG C, light application time 14h/d, illumination
Intensity is 2500lx；The subculture multiplication medium is MS+6-BA0.2mg/L, pH value 5.8-6.0；
(5) induction of root: the test tube seedling that shoot proliferation obtains in step (3) is accessed in root media and carries out culture of rootage
15d, cultivation temperature are 25 ± 2 DEG C, light application time 14h/d, intensity of illumination 2000lx；The root media is MS+
2.0mg/L IBA, pH value 5.8-6.0；Bottom of bottle is covered with to adventitious root, when plant integrally grows steady, is ready for bottle outlet of transplanting seedlings.
(6) bottle cap of culture bottle is opened, hardening 2-3 days, is transplanted into cutting medium, the cutting medium is preferred
Are as follows: peat soil, vermiculite and perlite are mixed according to the volume ratio of 2:2:1, the carbendazim of aerial part sprinkling 0.1%, often
Its water spray 2 times, guarantees humidity, avoids direct sunlight.
For a further improvement of the present invention is that, the selection of explant and access time in the step (1): with growth
The rachis of 20d or so is explant, the callus of differentiation can be effectively obtained after induction 35, quantity is more and growth is fast
Speed；It carries out explant selection at the time point and disinfection, pollution rate substantially reduces.
The method that alum root kind " kilim carpet " rachis vitro Regeneration System of the present invention is established has the advantage that
The selection and disinfection treatment of 1 explant, present inventor pass through a large amount of creative works and factorial experiment, comprehensively
Improve operating parameter in the prior art, it is found that according to conventional disinfection way, that is, cleaning-alcohol disinfecting-mercuric chloride disinfection-
Aseptic water washing, there are certain pollution rates, and use explant position and acquisition time in the operating process of the application, not only
Achieve ideal sterilization effect, additionally it is possible to explant number needed for guaranteeing Fiber differentiation.The cell differentiation energy of rachis tissue
Power is better than the materials such as axillary bud, petiole, and the rachis for growing 20d is better than the rachis explant material of other times acquisition.
The modes of emplacement of 2 explants: the difference of modes of emplacement causes culture effect different, places on slant medium
Explant callus forms morning, and quantity is more, and growth is exceedingly fast, and is significantly better than the rachis of plating medium placement.
Its step of 3 method for tissue culture all has similitude, and result but makes a marked difference, tissue known in this field
Cultural method is generally divided into two stages of dark culture and optical culture, and dark culture is usually evoked callus, optical culture induction point
Change.Dark culture requires to be protected from light, and is the healing cell aging for preventing from dividing rapidly in fact, present inventor by testing repeatedly
Out, alum root kind kilim carpet first passes through the dark culture of a period of time, divides explant rapidly, then photoinduction culture, sprouts
Rate highest, callus budding is rapider, shortens the tissue culture time.
How 4 it is known in the art that utilize plant tissue differentiation regeneration plant, raising plant for plant tissue culture area research
Reproductive number, be that study important technology emphasis be also technological difficulties to scientist, and hormone kind and hormone concentration use model
It encloses, type of culture medium is even more the most important thing, and present inventor passes through a large amount of creative works, and optimal alum root product are arrived in screening
The ingredient and hormone combinations of kind " kilim carpet " induced medium, proliferated culture medium and root media, not only induction, cultivation effect are bright
It is aobvious, and the time of sprouting greatly shortens, only 25d can grow callus, then induce 25 adventitious bud that can go out, and be proliferated, effect of taking root
Fruit is significant.
Specific embodiment
Explant screens embodiment:
1, the modes of emplacement screening of explant in the medium
For explant rachis similar in size, growth conditions, the difference of modes of emplacement also causes culture effect different.Tiltedly
The explant that face is placed sprouts early, and quantity is more, and growth is exceedingly fast, and the rachis that plane is placed then goes out almost without what bud point was broken up
It is existing, and place 35d or more and then gradually tend to browning or withered.
Table 1
2, Screening of Media embodiment
In the vitro Regeneration System establishment process of callus approach, the success of callus differentiation is the committed step of regenerating system.
Whether callus differentiation rate directly affects regeneration tissue culture system feasible.Callus differential medium is by minimal medium and hormone kind
And the degree broken up to callus of concentration collocation and differentiation rate have very big correlation.The present invention has attempted different basic cultures
The various concentration of base and CPPU, BA and IBA proportion, the influence to alum Furcation defects degree are shown in Table 2.From table 2 it can be seen that N6+
Alum root-derived callus differentiation rate highest on CPPU 0.3mg/L+IBA0.01mg/L is best callus differential medium.
Table 2
| Basal medium type | Hormone combinations mg/L | Callus divergaence time | Callus differentiation rate % |
| MS | BA0.3+IBA0.01 | 45 | 54.91 |
| MS | CPPU0.3+IBA0.01 | 40 | 69.76 |
| MS | CPPU0.3+IBA0.10 | 55 | 62.34 |
| N6 | BA0.3+IBA0.01 | 30 | 74.54 |
| N6 | CPPU0.3+IBA0.01 | 26 | 84.19 |
| N6 | CPPU0.3+IBA0.10 | 35 | 71.04 |
Embodiment of the method
1, the selection and disinfection of explant: the rachis of growth 20d or so is chosen as explant, after rinsing well, is disappeared
Poison, disinfecting process are as follows: it is rinsed well with clear water, it is then primary with the rinsing of Amway detergent, 15-20min is vibrated on shaking table；Flowing water
Rinse 20-30min after, on aseptic operating platform use 75% ethanol disinfection 10-20s, pour into immediately 0.1% mercuric chloride sterilize
12min vibrates 3-5min every time, the rachis of sanitized is cut into the segment of 2cm long finally with sterile water washing 4 times,
Rachis segment 100 are accessed altogether.
2, the induction of callus: the explant segment that processing obtains in step (1) is taken, is inoculated in inclined-plane modes of emplacement
The induction of callus is carried out in induced medium (in advance by culture medium contra bevel), cultivation temperature is 25 ± 2 DEG C, first black
10d is cultivated in the dark, then carries out illumination cultivation 25d；The illumination cultivation condition are as follows: light application time 14h/d, intensity of illumination are
1500lx；The induced medium is MS+6-BA0.6mg/L+IBA0.02mg/L, pH value 5.8-6.0.After cultivating 25d, altogether
There are 42 explant both ends strong point green callis, continue after cultivating 10d, explant is all exposed to grow Huang with air part
Color callus.
3, the differentiation of callus: the callus that induction obtains in step (2) is cut on explant after inducing 35d
Under, after segmentation, access differential medium carries out adventitious buds differentiation, and cultivation temperature is 25 ± 2 DEG C；Light application time 14h/d, illumination are strong
Spend 2500lx；The induced medium is N6+CPPU 0.3mg/L+IBA0.01mg/L, pH value 5.8-6.0；Access differentiation training
Support totally 200 pieces of callus fritter of base, after callus tissue culture 50d, up to 84.19%, it is indefinite that every piece of callus can break up callus differentiation rate
Bud 5-8.
4, the shoot proliferation of adventitious bud: after the adventitious bud cutting that induction is obtained, every bud band 0.3cm or so stem section and 3-4
Piece blade accesses and carries out squamous subculture 25d in subculture multiplication medium, and cultivation temperature is 25 ± 2 DEG C, light application time 14h/d,
Intensity of illumination is 2500lx；The subculture multiplication medium is MS+6-BA0.2mg/L, pH value 5.8-6.0；Cultivate 35d
Afterwards, every stem section can be proliferated out adventitious bud 8-12.
5, the induction of root: the test tube seedling that shoot proliferation obtains in step (3) is accessed in root media and carries out training of taking root
15d is supported, adventitious root can be gone out in stem section base portion or axillary bud director, radical 10-20 item differs, and continues after cultivating 15d, adventitious root meeting
Bottom of bottle is covered with, is partially aerial root.Cultivation temperature is 25 ± 2 DEG C, light application time 14h/d, intensity of illumination 2000lx；It is described
Root media is MS+2.0mg/L IBA, pH value 5.8-6.0；Bottom of bottle is covered with to adventitious root, when plant integrally grows steady,
It is ready for bottle outlet of transplanting seedlings.
6, the bottle cap of culture bottle is opened, hardening 2-3 days, is transplanted into cutting medium, the cutting medium is preferred are as follows: presses
Peat soil, vermiculite and perlite are mixed according to the volume ratio of 2:2:1, the carbendazim of aerial part sprinkling 0.1% is sprayed water daily
2 times, guarantees humidity, avoid direct sunlight.After cultivating 30d, survival rate is up to 86.46%.
Claims (3)
- The method that alum root kind 1. " kilim carpet " rachis vitro Regeneration System is established, characterized by the following steps:(1) selection and disinfection of explant: the rachis of growth 20d or so is chosen as explant, after rinsing well, is disappeared Poison, disinfecting process are as follows: it is rinsed well with clear water, it is then primary with the rinsing of Amway detergent, 15-20min is vibrated on shaking table；Flowing water Rinse 20-30min after, on aseptic operating platform use 75% ethanol disinfection 10-20s, pour into immediately 0.1% mercuric chloride sterilize 12min vibrates 3-5min every time, the rachis of sanitized is cut into the segment of 2cm long finally with sterile water washing 4 times；(2) induction of callus: the explant segment that processing obtains in step (1) is taken, is inoculated in and is lured with inclined-plane modes of emplacement The induction that callus is carried out in culture medium (in advance by culture medium contra bevel) is led, cultivation temperature is 25 ± 2 DEG C, first in dark Then middle culture 10d carries out illumination cultivation 25d；The illumination cultivation condition are as follows: light application time 14h/d, intensity of illumination are 1500lx；The induced medium is MS+6-BA0.6mg/L+IBA0.02mg/L, pH value 5.8-6.0；(3) differentiation of callus: the callus that induction obtains in step (2) is cut on explant after inducing 35d, It accesses differential medium and carries out adventitious buds differentiation, cultivation temperature is 25 ± 2 DEG C；Light application time 14h/d, intensity of illumination 2500lx； The induced medium is N6+CPPU 0.3mg/L+IBA0.01mg/L, pH value 5.8-6.0；(4) shoot proliferation of adventitious bud: after the adventitious bud cutting that induction is obtained, every bud band 0.3cm or so stem section and 3-4 piece leaf Piece accesses and carries out squamous subculture 25d in subculture multiplication medium, and cultivation temperature is 25 ± 2 DEG C, light application time 14h/d, illumination Intensity is 2500lx；The subculture multiplication medium is MS+6-BA0.2mg/L, pH value 5.8-6.0；(5) induction of root: the test tube seedling that shoot proliferation obtains in step (3) is accessed in root media and carries out culture of rootage 15d, cultivation temperature are 25 ± 2 DEG C, light application time 14h/d, intensity of illumination 2000lx；The root media is MS+ 2.0mg/L IBA, pH value 5.8-6.0；Bottom of bottle is covered with to adventitious root, when plant integrally grows steady, is ready for bottle outlet of transplanting seedlings；(6) bottle cap of culture bottle is opened, hardening 2-3 days, is transplanted into cutting medium, the cutting medium is preferred are as follows: according to The volume ratio of 2:2:1 mixes peat soil, vermiculite and perlite, and the carbendazim of aerial part sprinkling 0.1% sprays water 2 daily It is secondary, guarantee humidity, avoids direct sunlight.
- 2. the method that alum root kind " kilim carpet " rachis vitro Regeneration System is established according to claim 1, which is characterized in that Step A) in explant sample time be April.
- 3. alum root kind " kilim carpet " seedling prepared by claim 1 the method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711096929.9A CN109757368A (en) | 2017-11-09 | 2017-11-09 | The method that alum root kind " kilim carpet " rachis vitro Regeneration System is established |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711096929.9A CN109757368A (en) | 2017-11-09 | 2017-11-09 | The method that alum root kind " kilim carpet " rachis vitro Regeneration System is established |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109757368A true CN109757368A (en) | 2019-05-17 |
Family
ID=66449188
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711096929.9A Pending CN109757368A (en) | 2017-11-09 | 2017-11-09 | The method that alum root kind " kilim carpet " rachis vitro Regeneration System is established |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109757368A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111513061A (en) * | 2020-05-22 | 2020-08-11 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
| CN113508813A (en) * | 2021-03-26 | 2021-10-19 | 上海市农业科学院 | Rooting agent for alum root cutting and alum root cutting propagation method |
-
2017
- 2017-11-09 CN CN201711096929.9A patent/CN109757368A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111513061A (en) * | 2020-05-22 | 2020-08-11 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
| CN111513061B (en) * | 2020-05-22 | 2022-05-03 | 上海市农业生物基因中心 | Ultralow-temperature preservation and recovery culture method for alum root clump buds |
| CN113508813A (en) * | 2021-03-26 | 2021-10-19 | 上海市农业科学院 | Rooting agent for alum root cutting and alum root cutting propagation method |
| CN113508813B (en) * | 2021-03-26 | 2022-11-18 | 上海市农业科学院 | Rooting agent for alum root cutting and alum root cutting propagation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103755461B (en) | The medium that Orchid Tissue is cultivated and cultural method thereof | |
| CN103392517B (en) | Asexual propagation method of michelia compressa | |
| CN104041412B (en) | The quick breeding method for tissue culture of a kind of Guizhou half capsule lettuce tongue | |
| CN105766645A (en) | Efficient reproduction method of rhizoma bletillae tissue culture seedlings | |
| CN107683770B (en) | A kind of breeding method of alum root ' smearing tea ' seedling | |
| CN104686338A (en) | In-vitro culture technique of anther of angelica dahurica | |
| CN103704130A (en) | Chinese orchid and cymbidium hybridum hybrid seedling raising method | |
| CN103392597A (en) | Tissue culture method of North American begonia | |
| CN106376328A (en) | Wild bletila striata seed quick breeding method | |
| CN109349105A (en) | A kind of iris tissue-cultured seedling mating system | |
| CN104285814A (en) | Tissue culture and rapid propagation method of E. urophylla*E. Grandis DH32-43 | |
| CN104273037A (en) | Tissue culture and rapid propagation method of eucalyptus urophylla DH33 to DH27 varieties | |
| CN105145363B (en) | It is a kind of to significantly improve the method that China fir tissue culture produces emergence rate | |
| CN104686351A (en) | In-vitro rapid propagation method of cercidiphyllum japonicum | |
| CN109757368A (en) | The method that alum root kind " kilim carpet " rachis vitro Regeneration System is established | |
| CN111034617B (en) | Method for breeding tea seedlings by culturing young embryo tissues of Yunnan large-leaf tea trees | |
| CN106577280B (en) | A method of aseptic seedling is quickly bred using Merrillanthus hainanensis tender stem segments and blade | |
| CN109757369A (en) | A kind of method of alum root kind " obsidian " tissue culture propagating | |
| CN108040879A (en) | A kind of Herba Limonii Gmelinii with yellow flower rooting induction culture medium and Herba Limonii Gmelinii with yellow flower mating system | |
| CN101743908A (en) | Tissue culture, rapid propagation and cultivation method of grevillea banksii | |
| CN104285815A (en) | Tissue culture and rapid propagation method of E. urophylla*E. Grandis DH32-13 | |
| CN108651275A (en) | A kind of method of quickly breeding bletilla striata seedling under natural light | |
| CN105519445A (en) | In-vitro rapid propagation method for nepenthes | |
| CN111183887A (en) | Rapid cutting propagation method for zinc-cadmium super-accumulation plant Sedum plumbizincicola | |
| CN109757371A (en) | A kind of method of alum root kind " tranquil oasis " tissue culture propagating |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190517 |
|
| WD01 | Invention patent application deemed withdrawn after publication |