CN107041308A - A kind of primrose root tissue culture propagation method - Google Patents
A kind of primrose root tissue culture propagation method Download PDFInfo
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- CN107041308A CN107041308A CN201710282040.3A CN201710282040A CN107041308A CN 107041308 A CN107041308 A CN 107041308A CN 201710282040 A CN201710282040 A CN 201710282040A CN 107041308 A CN107041308 A CN 107041308A
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- tissue culture
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- primrose
- root
- aseptic
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- 241000245063 Primula Species 0.000 title claims abstract description 35
- 235000016311 Primula vulgaris Nutrition 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 18
- 239000001963 growth medium Substances 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 18
- 238000005406 washing Methods 0.000 claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000012545 processing Methods 0.000 claims abstract description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 7
- 229960002523 mercuric chloride Drugs 0.000 claims abstract description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims abstract description 5
- 239000002689 soil Substances 0.000 claims abstract description 5
- 229920001817 Agar Polymers 0.000 claims description 5
- 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 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 24
- 241000196324 Embryophyta Species 0.000 abstract description 5
- 230000035755 proliferation Effects 0.000 abstract description 5
- 238000011012 sanitization Methods 0.000 abstract description 4
- 208000027418 Wounds and injury Diseases 0.000 abstract description 3
- 230000006378 damage Effects 0.000 abstract description 3
- 208000014674 injury Diseases 0.000 abstract description 3
- 230000008774 maternal effect Effects 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 238000012136 culture method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 241001164374 Calyx Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000004080 punching Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 241001573881 Corolla Species 0.000 description 1
- 241000612120 Primula sieboldii Species 0.000 description 1
- 235000019092 Primula sieboldii Nutrition 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 208000018299 prostration Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011282 treatment Methods 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A kind of primrose root tissue culture propagation method of the present invention, comprises the following steps：(1) the tiny root system of primrose root is cut as explant, is washed rear running water by rubbing with the hands with the solution of washing powder and is rinsed；(2) explant for eliminating surface dirt and soil is placed on superclean bench and disinfected, first with ethanol postincubation, then the processing that carried out disinfection with mercuric chloride, finally with aseptic water washing, the moisture of residual is drawn with aseptic filter paper；The explant of sanitized is placed on aseptic paper, is inoculated on tissue culture culture medium；(3) explant that step (2) is inoculated on tissue culture culture medium is placed in progress tissue culture propagation in culturing room, until forming callus.This method is made to carry out tissue culture come explant using the root of primrose, can reduce the injury to maternal plant, the adventitious bud formation rate, Callus formation rate and Rooting percent of primrose can be effectively improved, so as to improve proliferation rate.
Description
Technical field
The present invention relates to plant tissue culture culture, and in particular to a kind of primrose root tissue culture propagation method.
Background technology
Primrose (scientific name：Primula sieboldii E.Morren) Primulaceae primula perennial herb, root shape
Stem is tilted or prostration, and blade ovate square is circular circular to square, dilute subcircular or section shape, and edge crenation is shallow to be split, above bottle green,
Light green below, two sides is by the long pubescence of canescence many cells, and scape is high up to 30 centimetres, umbel basidixed, bract wire
Lanceolar, Calyx mitriform, corolla aubergine is to pale red, and dilute white, capsule is subsphaeroidal, and long is about the half of calyx.May opens
Flower, June result.
Primrose is usually seminal propagation, including garden-variety is also, but seminal propagation is easily morphed, and causes what is obtained
New varieties can not be preserved by the method for vegetative propagation, also without the report about primrose tissue culture propagation.By organizing training
Foster method, can effectively preserve the merit of primrose garden-variety, significant to the large-scale breeding of primrose.
The content of the invention
Goal of the invention：The problem of existing for prior art, the present invention provides a kind of primrose root tissue culture propagation method.The party
Method is made to carry out tissue culture come explant using the root of primrose, can reduce the injury to maternal plant, reduces explant pollution level, and
The adventitious bud formation rate, Callus formation rate and Rooting percent of primrose can be effectively improved, so as to improve proliferation rate.Promote butt
The proliferation rate of tissue culture, than having higher proliferation rate with leaf, stem tissue culture.
Technical scheme：In order to realize foregoing invention purpose, a kind of primrose root tissue culture propagation method as described herein, including
Following steps：
(1) processing of explant：The tiny root system of primrose root is cut as explant, cuts into and is about 1.5-1.6cm
Root segment, wash rear running water by rubbing with the hands with the solution of washing powder and rinse；
(2) disinfect：The explant for eliminating surface dirt and soil is placed on superclean bench and disinfected, first
With ethanol postincubation, then the processing that carried out disinfection with mercuric chloride, finally with aseptic water washing, the moisture of residual is drawn with aseptic filter paper；Will
The explant of sanitized is placed on aseptic paper, cuts off the lower end 0.2-0.3cm of explant, in order to avoid influence it to be inhaled from culture medium
Adopt point, be inoculated on tissue culture culture medium；
(3) culture of explant：The explant that step (2) is inoculated on tissue culture culture medium is placed in carry out group in culturing room
Training breeding, until forming callus.
Wherein, it with the solution time of washing by rubbing with the hands of washing powder is 2-3min that step (1) is described, and running water flowing water washing time is
12-14h。
Wherein, step (2) the ethanol postincubation time is 30-45s, and mercuric chloride carries out disinfection the time for 4-5min, sterilized water
Rinse 3-4 times.Step (2) the tissue culture culture medium is MS+1mg/L 6-BA, adds sucrose 30g/L, agar 7g/L, pH adjustment
For 5.7.Culture medium is in 121 DEG C of temperature, 15min sterilization treatments.
Wherein, the condition of step (3) described culturing room is illumination 16h/d, and illuminance is 3000lx, and temperature control is in 23-
27 DEG C, incubation time is 28-30 days.
The present invention cuts off the lower end 0.2cm of explant by rear explant is disinfected with scissors, tweezers, is inoculated in MS cultures
On base, 20 bottles of often processing inoculation, each trial jar puts 5 explants.The pollution condition of an explant, culture are observed every 1d
The pollution rate and survival rate of explant are counted after 17d.Screen pollution rate low, the high carry out tissue culture culture of survival rate.
Beneficial effect：Compared with prior art, the invention has the advantages that：1st, the present invention carries out the group of primrose using root
Training, can avoid injuring plant, than having higher proliferation rate with leaf, stem tissue culture.Effectively preserve new by this method of the present invention
Kind, can be with large-scale breeding primrose, with supply market.
2nd, the tissue culture culture medium that the present invention is used is the culture medium for being best suitable for primrose breeding, can effectively improve primrose not
Normal bud formation rate reaches that 100%, Callus formation rate arrival 100% and Rooting percent are up to 93%, so as to improve propagation
Rate.
3rd, the present invention can reduce the injury to maternal plant to greatest extent by the use of root as explant, and reduction explant is dirty
Dye degree.
Embodiment
The invention will be further described with reference to embodiments.
Embodiment 1
Tissue culture culture medium is MS+1mg/L 6-BA, adds sucrose 30g/L, agar 7g/L, pH and is adjusted to 5.7.
Tissue culture method：
(1) processing of explant：The tiny root system of primrose root is cut as explant, the root for being about 1.5cm is cut into
Section, is put into ceramic whiteware cylinder with having added the water of a small amount of washing powder to wash 2min by rubbing with the hands, foam is eluriated clean after the punching of running water down-flow water
Wash 12h；
(2) disinfect：The explant for eliminating surface dirt and soil is placed on superclean bench and disinfected, first
With the ethanol postincubation 30s of volume fraction 75%, then use 1gL-1Mercuric chloride carries out disinfection processing 5min, finally with aseptic water washing 3 times,
The moisture of residual is drawn with aseptic filter paper；The explant of sanitized is placed on aseptic paper, the lower end of explant is cut off
0.2cm, in order to avoid influenceing it to absorb nutrient from culture medium, is inoculated on tissue culture culture medium；
(3) culture of explant：The explant that step (2) is inoculated on tissue culture culture medium is placed in culturing room in illumination
16h/d, illuminance is 3000lx, and temperature control carries out tissue culture propagation at 20 DEG C, cultivates 30 days.
The adventitious bud formation rate of the primrose root tissue culture propagation method primrose of the present embodiment 1 has reached 100%, Callus formation
Rate reaches 100%, and Rooting percent has reached 93%.
Embodiment 2
Tissue culture culture medium is MS+1mg/L 6-BA, adds sucrose 30g/L, agar 7g/L, pH and is adjusted to 5.8.
Tissue culture method：
(1) processing of explant：The tiny root system of primrose root is cut as explant, the root for being about 1.6cm is cut into
Section, is put into ceramic whiteware cylinder with having added the water of a small amount of washing powder to wash 3min by rubbing with the hands, foam is eluriated clean after the punching of running water down-flow water
Wash 14h；
(2) disinfect：The explant for eliminating surface dirt and soil is placed on superclean bench and disinfected, first
With the ethanol postincubation 45s of volume fraction 75%, then use 1gL-1Mercuric chloride carries out disinfection processing 6min, finally with aseptic water washing 4 times,
The moisture of residual is drawn with aseptic filter paper；The explant of sanitized is placed on aseptic paper, the lower end of explant is cut off
0.3cm, in order to avoid influenceing it to absorb nutrient from culture medium, is inoculated on tissue culture culture medium；
(3) culture of explant：The explant that step (2) is inoculated on tissue culture culture medium is placed in culturing room in illumination
16h/d, illuminance is 3000lx, and temperature control carries out tissue culture propagation at 25 DEG C, cultivates 28 days.
The adventitious bud formation rate of the primrose root tissue culture propagation method primrose of the present embodiment 2 has reached 100%, Callus formation
Rate reaches 100%, and Rooting percent has reached 84%.
Test example 1
Using the tissue culture method of embodiment 1, wherein from different 6-BA and NAA concentration (other components keep it is constant：
5.7) MS, sucrose 30g/L, agar 7g/L, pH be adjusted to, and 6-BA concentration is respectively 0,1mg/L, 10mg/L, NAA concentration difference
0,0.1,1,10mg/L, 12 combinations, research hormon various concentrations calli induction rate after lower 30 days, culture knot
The fresh weight of plant after beam, the situation for forming from callus adventitious bud, the orbicule number grown per explant and from spherical
Rooting percent on body, also orbicule exceed 1cm quantity, and each processing is inoculated with 100 bottles, and per test tube, bottle puts 1 explant.As a result
It is shown in Table 1.
Influences of the table 1NAA and 6-BA to explant callus and adventitious bud
As can be seen from Table 1, in experiment sequence number 1 and 2, adventitious bud formation rate is between 25~37%；It is 1mg/L in 6-BA
Culture medium on, with NAA concentration raise, Callus formation rate, adventitious bud formation rate, Rooting percent show as decline situation.
It is 10mg/L regions in 6-BA, adventitious bud formation rate is up to 97% when NAA is 1mg/L, and it is 1mg/L that Rooting percent, which is 6-BA,
When highest 93%.Seen from upper table 1 No. 5 combine hormone culture mediums on, Callus formation rate, adventitious bud formation rate and
Rooting percent all highests.
Test example 2
The influence of cultivation temperature
Influence of the cultivation temperature to explant body callus, adventitious bud formation and Rooting percent is also very big.Using embodiment 1
Tissue culture method, by changing the temperature studies different temperatures of culturing room to explant callus adventitious bud formation and Rooting percent
Influence.It the results are shown in Table 2.
Influence of the temperature of table 2 to explant callus adventitious bud formation and Rooting percent
From Table 2, it can be seen that at 15 DEG C, 20 DEG C, 25 DEG C, Callus formation rate, adventitious bud shape
It is 100% into rate, and at 30 DEG C is then 30% and 23%, but Rooting percent is with highest at 20 DEG C, so to explant
Body Callus formation, adventitious bud formation and the best temperature of Rooting percent are 20 DEG C.
In summary, to the culture of primrose root with the conditions of 20 DEG C, in MS culture mediums, addition 6-BA be 1mg/L,
When NAA is 0mg/L, Callus formation rate, adventitious bud formation rate and Rooting percent highest.
Claims (5)
1. a kind of primrose root tissue culture propagation method, it is characterised in that comprise the following steps：
(1) processing of explant：The tiny root system of primrose root is cut as explant, the root for being about 1.5-1.6cm is cut into
Section, washes rear running water by rubbing with the hands with the solution of washing powder and rinses；
(2) disinfect：The explant for eliminating surface dirt and soil is placed on superclean bench and disinfected, wine is first used
Precision processing, then the processing that carried out disinfection with mercuric chloride, finally with aseptic water washing, the moisture of residual is drawn with aseptic filter paper；Will sterilization
The explant finished is placed on aseptic paper, is cut off the lower end 0.2-0.3cm of explant, is inoculated on tissue culture culture medium；
(3) culture of explant：It is numerous that the explant that step (2) is inoculated on tissue culture culture medium is placed in progress tissue culture in culturing room
Grow, until forming callus.
2. primrose root tissue culture propagation method according to claim 1, step (1) is described to wash the time by rubbing with the hands with the solution of washing powder
For 2-3min, running water flowing water washing time is 12-14h.
3. primrose root tissue culture propagation method according to claim 1, step (2) the ethanol postincubation time is 30-45s,
Mercuric chloride carries out disinfection the time for 4-5min, aseptic water washing 3-4 times.
4. primrose root tissue culture propagation method according to claim 1, step (2) the tissue culture culture medium is MS+1mg/L
6-BA, adds sucrose 30g/L, agar 7g/L, pH and is adjusted to 5.7-5.8.
5. primrose root tissue culture propagation method according to claim 1, the condition of step (3) described culturing room is illumination 16h/
D, illuminance is 3000lx, and temperature control is in 20-25 DEG C, incubation time 28-30 days.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710282040.3A CN107041308A (en) | 2017-04-26 | 2017-04-26 | A kind of primrose root tissue culture propagation method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710282040.3A CN107041308A (en) | 2017-04-26 | 2017-04-26 | A kind of primrose root tissue culture propagation method |
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ID=59545982
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116982559A (en) * | 2023-09-27 | 2023-11-03 | 中国科学院昆明植物研究所 | Primula forbesii test tube flowering induction medium and application thereof in test tube flowers |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101015279A (en) * | 2007-02-16 | 2007-08-15 | 北京林业大学 | Tissue culture method for fast propagation of primula poissonii |
-
2017
- 2017-04-26 CN CN201710282040.3A patent/CN107041308A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101015279A (en) * | 2007-02-16 | 2007-08-15 | 北京林业大学 | Tissue culture method for fast propagation of primula poissonii |
Non-Patent Citations (2)
| Title |
|---|
| FURUYA, ET AL.: "In vitro propagation of Primula sieboldi E. Morr. through root segment culture", 《HORTICULTURAL RESEARCH（JAPAN）》 * |
| 游晓会等: "报春花属植物组织培养研究进展(综述)", 《亚热带植物科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116982559A (en) * | 2023-09-27 | 2023-11-03 | 中国科学院昆明植物研究所 | Primula forbesii test tube flowering induction medium and application thereof in test tube flowers |
| CN116982559B (en) * | 2023-09-27 | 2023-12-29 | 中国科学院昆明植物研究所 | Test tube flowering induction culture method for primula sikkimensis |
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Application publication date: 20170815 |