CN106718921A - A kind of culture medium for Limonium sinense tissue cultures - Google Patents
A kind of culture medium for Limonium sinense tissue cultures Download PDFInfo
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- CN106718921A CN106718921A CN201611252779.1A CN201611252779A CN106718921A CN 106718921 A CN106718921 A CN 106718921A CN 201611252779 A CN201611252779 A CN 201611252779A CN 106718921 A CN106718921 A CN 106718921A
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- culture
- naa
- culture medium
- limonium sinense
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- 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
Abstract
The invention discloses a kind of culture medium for Limonium sinense tissue cultures,Base is induced including primary,Shoot proliferation base,Root media,Primary induction based formulas are the 0.1mg/L+2 of 0.2 0.8mg/L+NAA of B5+KT 0.05,4‑D 0.02‑0.08mg/L,Subculture multiplication medium formula is the 0.1mg/L of 0.5 0.8mg/L+NAA of B5+KT 0.05,Culture of rootage formula is B5+IBA 0.1mg/L,Tissue base of the invention makes the culture breeding of Limonium sinense not limited by natural conditions,And inductivity and rooting rate are high,Amount reproduction can constantly be carried out,Introduces a collection is less in solving the problems, such as cultivation,The degradation ratio of kind can be effectively reduced simultaneously,It is research and scale from now on,Industrialization production provides safeguard,The large-scale popularization for being the species in gardens construction popularization provides technical support；Also it is that the Northwest's species conservation and resource quickly update offer scientific basis.
Description
Technical field
The invention belongs to plant biotechnology field, it is related to tissue culture medium (TCM), and in particular to one kind is used for Limonium sinense group
Knit the culture medium of culture
Background technology
Limonium sinense (Limoniumau-reum), also known as Limonium aureum, Herba Limonii Gmelinii with yellow flower, is perennial herb.Chrysanthemum
In Lan Xueke Limonniums, it is one of arid-desert areas Wild Flowers few in number to rock Pinus, and pattern is gorgeous, opens
Flower continuity period is more long.Root is the system of taproot, thick, and complete stool smooths, 20~50cm of plant height, low level management, extremely drought-resistant, florescence
It is long, it is the important germ plasm resource of cover plant kind in abundant North City Urban Greening, and be again check winds and fix drifting sand excellent
Plant.The characteristics of great drought-resistant high temperature of Limonium sinense, Salt And Alkali Tolerance and impoverishment tolerant, the sandy Gobi desert in Dunhuang can stand precipitation
Amount 50mm, the heat and drought condition environment of 55 DEG C of surface temperature；It is not required to water in dry area in north china, can pacifies within -36 DEG C of temperature
Survive the winter entirely；Within below P in soil H9, salt content 4 ‰ can normal growth bloom.Its medical value has analgesic, anti-inflammatory, enriches blood it
Effect, has carried out correlative study at home.The species do not only have good economic benefit, and at aspect of preserving the ecological environment
With good ecological benefits, its exploitation prospect is extensive.
In recent years, the existing document of the tissue cultures on Limonium sinense reported, but document report chrysanthemum rock
The explant more options seed of loose tissue cultures, cotyledon or stem section, what is used culture medium is all the MS culture mediums of improvement more, this
Plant method for tissue culture and easily cause browning, and be difficult to take root.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided a kind of training for Limonium sinense tissue cultures
Base, including primary induction base, shoot proliferation base, root media are supported, primary induction based formulas are B5+KT 0.2-0.8mg/L+
NAA 0.05-0.1mg/L+2,4-D 0.02-0.08mg/L；Subculture multiplication medium formula is B5+KT 0.5-0.8mg/L+
NAA 0.05-0.1mg/L；Prescription of rooting medium is B5+IBA 0.1mg/L, and edible white sand is added in each cultivation stage
Sugared 20-40g/L, carragheen 3-5g/L.Primary induction based formulas preferred B5+KT 0.5mg/L+NAA 0.1mg/L+2,4-D
0.05mg/L；Subculture multiplication medium formula is preferably B5+KT 0.5mg/L+NAA 0.1mg/L.
Culture medium of the invention has the following steps in use
(1) selection of explant and sterilizing：Limonium sinense inflorescence sterilization is taken, 30-40min is rinsed with running water, inhaled
Water paper is sucked and rinsed in the alcohol of 70-75% after moisture 1-2min, then processes 8-10min with 0.1% mercuric chloride solution, aseptic
Water wash 6-7 times, is blotted with sterilized filter paper, and the inflorescence of Limonium sinense is the bud that will be opened after 2-3 days；
(2) explant induction：The good inflorescence of sterilization treatment is accessed on the B5 medium of improvement, i.e., on B5 medium basis
Upper addition plant hormone, its concentration is respectively：2,4-D 0.02-0.08mg/L；KT 0.2-0.8mg/L；NAA 0.05-
On the inducing culture of 0.1mg/L.Additional edible white granulated sugar 20-40g/L, carragheen 3-5g/L.It is 25 ± 2 DEG C, light in temperature
According to intensity 1500-2000lx, induction induces callus in 15-20 days under the inductive condition of light application time 12-16h/d；
(3) shoot proliferation culture：The Limonium sinense callus that step (2) is induced is taken out, and is inoculated in the B5 subcultures of improvement
Proliferated culture medium, wherein phytohormone concentration are：KT 0.5-0.8mg/L；NAA 0.05-0.1mg/L, add edible white granulated sugar
20-40g/L, carragheen 3-5g/L.It is 25 ± 2 DEG C in temperature, intensity of illumination 1500-2000lx, light application time 12-16h/d's
Under the conditions of Multiplying culture 30-40 days, breed and Limonium sinense adventitious bud；
(4) culture of rootage：The Limonium sinense adventitious bud that step (3) Multiplying culture goes out is transferred to formula for B5+IBA0.1mg/
In L root medias, edible white granulated sugar 20-40g/L, carragheen 3-5g/L are added.It is 25 ± 2 DEG C, intensity of illumination in temperature
1500-2000lx, culture of rootage obtains tissue-cultured seedling in 5-7 days under conditions of light application time 12-16h/d.
In technical scheme
1) primary inducing culture be B5 medium on the basis of add 2,4-D 0.02-0.08mg/L, preferably 0.05mg/L；
KT 0.2-0.8mg/L, preferably 0.5mg/L；NAA 0.05-0.1mg/L, preferably 0.1mg/L.2,4-D can induce dividing for callus
Change, the sprouting of lateral bud and growth, but 2,4-D of excess has and suppresses the side effect that bud is formed, the inducing culture not only inductivity
Height, and the newly-increased bud number of each explant is more, and its inductivity reaches as high as 84.8%.
2) proliferated culture medium is addition KT 0.2-0.8mg/L, preferably 0.5mg/L on the basis of B5 medium；NAA 0.05-
0.1mg/L, preferably 0.1mg/L.Formation of the NAA of debita spissitudo to chrysanthemum flesh pine adventitious bud has facilitation, and research finds, with
The increase of NAA concentration, its proliferation times gradually increases, its concentration is 0.1mg/L, growth coefficient reaches maximum, continue to increase
NAA concentration, its appreciation rate is significantly increased and has downward trend.With the increase of KT concentration, proliferation times increased, after
Continuous increase KT concentration, appreciation rate improves not substantially, and occurs in that excessive growth phenomenon.
3) root media：Improvement B5+IBA 0.1mg/L, preferably, rooting rate reaches the root media rooting efficiency
92.1%.Base portion of taking root can increase the Multiple Buds with root newly, average up to 3-4.Add the IBA of appropriate concentration can in the medium
Improve and improve rootage duration, the length of root and rooting rate.
In the present invention, in scope as well known to those skilled in the art, KT, NAA, IBA, 2,4-D are respectively 6- glycosyl ammonia
Base purine, methyl α-naphthyl acetate, indolebutyric acid, the abbreviation of 2,4- dichlorphenoxyacetic acids.
The present invention filters out the optimal medium in different times for Limonium sinense tissue cultures, it is determined that in culture medium
The proportioning of each hormone, according to it is of the invention formula prepare culture medium can meet each period of Limonium sinense nutritional need and
Grow.The inductivity and rooting rate in tissue culture procedures are improve, is the key for realizing Limonium sinense large-scale production
And core technology.
Tissue base of the invention makes the culture breeding of Limonium sinense not limited by natural conditions, and inductivity and rooting rate
Height, can constantly carry out amount reproduction, and substantial amounts of seedling can be obtained in a short time, and introduces a collection is less in solving the problems, such as cultivation,
The degradation ratio of kind can effectively being reduced simultaneously, being research and scale from now on, industrialization production provides safeguard, and is the thing
The large-scale popularization popularization planted in gardens construction provides technical support；Meanwhile, also it is that the Northwest's species conservation and resource are quick
Update and scientific basis is provided.
Specific embodiment
Statistical computation formula in embodiment
Survival rate=(the explant sum of viable explant number/inoculation) × 100%
Primary bud induction rate=(the explant sum of the explant number/inoculation sprouted) × 100%
Shoot proliferation multiple=(sum of the Multiple Buds number for inducing/access individual plant bud) × 100%
Rooting rate=(the explant sum of the explant number/inoculation of differentiation root) × 100%
(1) selection of explant and sterilizing, take away the bud for spending to open for first 2-3 days, and 30- is rinsed with running water
40min, blotting paper is sucked and rinsed in the alcohol of 70-75% after moisture 1-2min, then processes 8- with 0.1% mercuric chloride solution
10min, aseptic water wash 6-7 times is blotted with sterilized filter paper, is seeded in the B5 medium of improvement.
Influence of the explant to chrysanthemum flesh pine survival rate and adventitious bud inducing
The loose inflorescence of chrysanthemum flesh, cotyledon, stem section are chosen respectively is inoculated in B5+KT 0.5mg/L+NAA 0.1mg/L+2,4-D
On 0.05mg/L inducing cultures, additional sugared 30g/L, carragheen 3.5g/L, condition of culture is：Cultivation temperature, 25 ± 2 DEG C；Light
It is 1500-2000lx according to intensity；Light application time is daily 12-16 hours.The inductivity for counting adventitious bud respectively on 30th day.As a result
As shown in table 1, under same condition of culture, when explant is inflorescence, the inductivity of Limonium sinense is 84.8%.
Influence of the position of table 1 to chrysanthemum flesh pine adventitious bud inducing
(2) explant induction
After the inflorescence of Limonium sinense is accessed into inducing culture, condition of culture is：Cultivation temperature, 25 ± 2 DEG C；Intensity of illumination
It is 1500-2000lx；Light application time is daily 12-16 hours.Culture 15-20 days, starts to produce callus, is containing 2,4-
Producing within 20 days or so in the culture medium of D close structure, absinthe-green adventitious bud gradually increases the concentration of 2,4-D, adventitious bud inducing
Rate increases with the increase of 2,4-D concentration, but continues to increase 2,4-D concentration, and adventitious bud induction frequency is on a declining curve, illustrates
2,4-D of amount has the side effect for suppressing bud formation.
As seen from Table 2,7 kinds of hormone combinations can induce Limonium sinense inflorescences and induce adventitious bud, but each combination point
Rate is different, and No. 3 B5+KT 0.5mg/L+NAA 0.1mg/L+2,4-D 0.05mg/L are the optimal inductions of Limonium sinense inflorescence
Culture medium, not only inductivity is high, and the newly-increased bud number of each explant is more, differentiation rate highest, and its differentiation rate is up to 80.9%.Knot
Fruit is as shown in table 2.
Influence of the hormone concentration of table 2 and combinations thereof to Limonium sinense inflorescence adventitious bud inducing
(3) shoot proliferation culture
The Limonium sinense adventitious bud induced by more than takes out, and is inoculated in the B5 medium of additional various concentrations KT and NAA
On, condition of culture is：Cultivation temperature, 25 ± 2 DEG C；Intensity of illumination is 1500-2000lx；Light application time is daily 12-16 hours.
Observe the proliferation rate and differentiation situation under the conditions of different hormone combinations 30 day after tomorrow.As seen from Table 3,9 kinds of hormone combinations can induce
Limonium sinense produces propagation system, but the adventitious bud upgrowth situation of each combination is different, when hormone combination is No. 5, its propagation
Multiple and adventitious bud growing state are all more excellent, so B5+KT 0.5mg/L+NAA 0.1mg/L are Limonium sinense adventitious bud proliferations
Upgrowth situation optimal medium.
Influence of the hormone concentration of table 3 and combinations thereof to Limonium sinense adventitious bud proliferation
(4) culture of rootage
By the preferable seedling of growing way in subculture medium, it is divided into individual plant and is transferred in root media, every bottle is accessed 4 plants, culture
Condition is：Cultivation temperature, 25 ± 2 DEG C；Intensity of illumination is 1500-2000lx；Light application time is daily 12-16 hours, observation note
The situation of taking root of seedling, rooting rate is counted after 7 days on record different culture media.
From the point of view of the culture of rootage result of the test of 7 days, the formation of two culture mediums to root has inducing action, wherein No. 2
Preferably, rooting rate is up to 88.7% for root media rooting efficiency.The B5 medium for being not added with any hormone also can induce Limonium sinense
Seedling is taken root, but takes root negligible amounts, and root is more elongated, and when hormone life IBA is added in culture medium, the quantity of root is taken root
Rate increases, so, the preferred B5+IBA 0.1mg/L (being shown in Table 4) of culture medium suitably taken root.
The influence that the inorganic salts of table 4 are taken root to seedling
As can be seen that the inductivity and rooting rate of each culture medium are high in the present invention from subordinate list, Limonium sinense can be continuous
Carry out amount reproduction.
The above, specific embodiment only of the invention, but protection scope of the present invention is not limited thereto, and it is any
Those familiar with the art the invention discloses technical scope in, the change or replacement that can be readily occurred in, all should
It is included within the scope of the present invention.Therefore, the protection model that protection scope of the present invention should be defined with claim
Enclose and be defined.
Claims (4)
1. a kind of culture medium for Limonium sinense tissue cultures, including primary induction base, shoot proliferation base, root media,
It is characterized in that described primary induction based formulas are B5+KT 0.2-0.8mg/L+NAA0.05-0.1mg/L+2,4-D 0.02-
0.08mg/L；Subculture multiplication medium formula is B5+KT0.5-0.8mg/L+NAA 0.05-0.1mg/L；Prescription of rooting medium
It is B5+IBA 0.1mg/L.
2. a kind of culture medium for Limonium sinense tissue cultures according to claim 1, it is characterised in that it is described just
Generation induction based formulas are B5+KT 0.5mg/L+NAA 0.1mg/L+2,4-D 0.05mg/L.
3. a kind of culture medium for Limonium sinense tissue cultures according to claim 1, it is characterised in that the subculture
Proliferation culture medium formula is B5+KT 0.5mg/L+NAA 0.1mg/L.
4. a kind of culture medium for Limonium sinense tissue cultures according to claim 1, it is characterised in that it is described just
Each additional edible white granulated sugar 20-40g/L, carragheen 3-5g/L in generation induction base, shoot proliferation base, root media.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611252779.1A CN106718921A (en) | 2016-12-30 | 2016-12-30 | A kind of culture medium for Limonium sinense tissue cultures |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611252779.1A CN106718921A (en) | 2016-12-30 | 2016-12-30 | A kind of culture medium for Limonium sinense tissue cultures |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110810244A (en) * | 2019-11-29 | 2020-02-21 | 内蒙古蒙草生态环境（集团）股份有限公司 | Culture medium for tissue culture of floral rod |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1666598A (en) * | 2004-03-11 | 2005-09-14 | 云南省香料研究开发中心 | Limonium suffruticosum test tube rapid breeding method for factory |
| CN105794654A (en) * | 2016-05-30 | 2016-07-27 | 甘肃省治沙研究所 | Tissue culture method of Limonium aureum |
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2016
- 2016-12-30 CN CN201611252779.1A patent/CN106718921A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1666598A (en) * | 2004-03-11 | 2005-09-14 | 云南省香料研究开发中心 | Limonium suffruticosum test tube rapid breeding method for factory |
| CN105794654A (en) * | 2016-05-30 | 2016-07-27 | 甘肃省治沙研究所 | Tissue culture method of Limonium aureum |
Non-Patent Citations (2)
| Title |
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| 王方琳等: "黄花矶松组织培养及培养基筛选研究", 《中国农学通报》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110810244A (en) * | 2019-11-29 | 2020-02-21 | 内蒙古蒙草生态环境（集团）股份有限公司 | Culture medium for tissue culture of floral rod |
| CN110810244B (en) * | 2019-11-29 | 2022-08-26 | 蒙草生态环境(集团)股份有限公司 | Culture medium for tissue culture of floral rod |
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