CN103004590A - Chinese narcissus callus preservation and tissue culture method - Google Patents
Chinese narcissus callus preservation and tissue culture method Download PDFInfo
- Publication number
- CN103004590A CN103004590A CN201210518283XA CN201210518283A CN103004590A CN 103004590 A CN103004590 A CN 103004590A CN 201210518283X A CN201210518283X A CN 201210518283XA CN 201210518283 A CN201210518283 A CN 201210518283A CN 103004590 A CN103004590 A CN 103004590A
- Authority
- CN
- China
- Prior art keywords
- callus
- chinese narcissus
- tissue culture
- preservation
- narcissus
- 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 relates to a Chinese narcissus callus preservation and tissue culture method. The method comprises the steps of material selection, induction culture and callus preservation. When a Chinese narcissus callus preservation and culture medium provided by the invention is put into use, the reproduction coefficient of Chinese narcissus callus can be 5.16 times, and the callus grows well, has a high reproduction speed and is free from browning and differentiation phenomena. According to the Chinese narcissus callus preservation and tissue culture method, the problems of difficult subculture and preservation, low reproduction coefficient, serious browning and differentiation and the like of the Chinese narcissus callus can be solved, the practicability is high and the generalization performance is good. The callus preserved by the method is easy to differentiate when being transferred to a differential medium, and can serve as an excellent material of a receptor for genetic transformation of Chinese narcissus.
Description
Technical field
The present invention relates to a kind of Chinese narcissus method for tissue culture, be specifically related to a kind of Chinese narcissus callus and preserve tissue culture method.
Background technology
Chinese narcissus (
Narcissus tazettaL. var.
ChinensisRoem) the perennial flowering bulb of Amaryllidaceae Narcissus is one of China tradition ten large famous flowers, also be in the world winter furnishings one of flowers, have higher ornamental value, economic worth and medical value.At present, Chinese narcissus for the production of cultivation mainly contains ' the silver-colored platform of gold small cup ' and ' beautiful exquisite ' two cultivar, this mainly is because Chinese narcissus adopts the asexual reproduction methods such as the breeding of side ball and lateral bud breeding, breeding cycle is long, reproduction speed is slow, and Chinese narcissus is triploid, is difficult to nature or artificial hybridization and produces new varieties, cause kind single, kind is rare.In addition, be vulnerable in the course of cultivation infecting of virus and cause quality decline, deterioration of variety.These all have a strong impact on commodity production and the variety development of Chinese narcissus, can not meet the need of market.
Stone(1973) successfully obtain first
Narcissus tazettaCv. ' the detoxic seedling of Grand Soleil d ' Or.Along with constantly improving and molecular biological develop rapidly of tissue culture technique, the cultured in vitro of Chinese narcissus and gene engineering research are more and more favored.The Chinese narcissus cultured in vitro mainly adopts the clove development ways to obtain regeneration plant, although this approach regeneration frequency is high, effective,, low as the transformation efficiency of acceptor material with clove in Study on Genetic Transformation, brown stain easily occurs.Therefore, good receptor system is very necessary for Chinese narcissus breed improvement and exploitation.
Callus is desirable acceptor material, at present, utilize the scale, bennet of plateau, the band portion plateau of band portion scale, the explant such as inflorescence, flower pesticide, ovary, young leaflet tablet of bolting all can not induce the Chinese narcissus callus, but callus is easy brownization in subculture cultivation or breeding, reproduction coefficient is not high, and callus is difficult to be obtained and preservation.Therefore, need urgently to seek and set up the effective way that a Chinese narcissus callus is preserved.
Summary of the invention
The objective of the invention is for the problem that exists the Chinese narcissus callus to be difficult to subculture and preservation in the prior art, the tissue culture method that provides a kind of callus to preserve is turned out good callus.
The objective of the invention is to realize by the following method:
Chinese narcissus callus of the present invention is preserved tissue culture method, comprise that material selects, induces cultivations, callus preservation, it is characterized in that described callus Storaged media is MN+1.0 ~ 3.0 mg/L 6-BA+0.5 ~ 1.5 mg/L 2,4-D+0.2 mg/L NAA+30 g/L white sugar+6 g/L agar powders, pH5.8; Wherein, MN is minimal medium, 1.0 ~ 3.0 mg/L 6-BA+0.5 ~ 1.5 mg/L 2, and 4-D+0.2 mg/L NAA is hormone combinations; Described MN minimal medium prescription is as shown in table 1.
Table 1 MN minimal medium prescription
Described 6-BA refers to the amino fast cry of certain animals of 6-benzyl; Described NAA refers to methyl α-naphthyl acetate; Described 2,4-D refers to 2,4-phenoxy acetic acid.
Chinese narcissus callus of the present invention is preserved tissue culture method, may further comprise the steps:
1, material is selected: take away the florescence and without the Chinese narcissus ovary of damage by disease and insect, be first 75% alcohol surface sterilization with volume ratio, amass than being 0.1% mercuric chloride, 5 min that sterilize with weight, use at last aseptic water washing 3 times;
2, induce cultivation: get the Chinese narcissus ovary that step 1 was sterilized, crosscut becomes the thin slice of 5 mm thickness, is inoculated on the inducing culture; Culturing room's temperature is 25 ℃～28 ℃, illumination 12 h/d, and intensity of illumination is 1 200 lx～1 500 lx, cultivates 60 days; Described inducing culture is MN+4.0 mg/L 6-BA+0.4 mg/L 2,4-D+0.2 mg/L NAA+30 g/L white sugar+6 g/L agar powders, pH5.8;
3, callus is preserved: press the prescription MN+1.0 of callus Storaged media ~ 3.0 mg/L 6-BA+0.5 ~ 1.5 mg/L 2,4-D+0.2 mg/L NAA+30 g/L white sugar+6 g/L agar powders is made into the pH value and is 5.8 callus Storaged media; Induce the callus of cultivation to be cut into 0.5 cm step 2
2Fritter, be inoculated into and carry out succeeding preservation in the callus Storaged media, be 25 ℃～28 ℃ in culturing room's temperature, illumination 12 h/d, intensity of illumination is to cultivate under the condition of 1 200 lx～1 500 lx, every switching in 60 days once.
In Chinese narcissus callus incubation, it is low reproduction coefficient often to occur, easy brownization, take root and breaks up and be difficult to the problems such as preservation.For inquire into that the Chinese narcissus callus preserves method, we utilize 6-BA, 2 on MN minimal medium basis, three kinds of factors of 4-D and NAA, by orthogonal design, the impact that the hormonal readiness of 3 kinds of variable concentrations of test is preserved the Chinese narcissus callus.The result is as shown in table 2.
Through test of many times and statistical analysis, the impact that hormon concentration is preserved the Chinese narcissus callus is as shown in table 2.Result of the test shows: the callus Storaged media is conducive to the Growth and Reproduction of callus; Use 2.0 mg/L 6-BA+, 0.5 mg/L 2 in the callus Storaged media, the hormone combinations of 4-D+ 0.2 mg/L NAA is best to the effect of callus growth, and the average reproduction coefficient of callus reaches 5.16 times, and it is vigorous to grow, brown stain does not occur, and preservation effect is remarkable.The described reproduction coefficient callus lines number/original callus lines number that refers to transfer.
The impact that table 2 hormon concentration is preserved the Chinese narcissus callus
Annotate: significance of difference analytical method is that the new multipole of Duncan is poor, and detection level is P≤0.05.
Table 2 adopts the alphabetic flag method to represent the result of multiple comparisons, than the Reference character law regulation, represents the significance level of difference with small letter Latin alphabet a, b, c, d...... according to statistical analysis mean multiple ratio.To pat the descending arrangement of mean everywhere first, mark a behind the maximum mean, and this mean compared the successively mark b of significant difference with following each mean.The like, the same letter of inapparent mark.Between two means all have same letter to be difference not remarkable, otherwise be significant difference.
Select the best hormone combinations of table 2 screening, then the callus Storaged media is MN+2.0 mg/L 6-BA+ 0.5 mg/L 2,4-D+0.2 mg/L NAA+30 g/L white sugar+6 g/L agar powders, pH5.8 and substitutes the MN minimal medium with MS minimal medium, Nistch minimal medium respectively and compares test.Result of the test is as shown in table 3, take the medium that contains MN as good, the reproduction coefficient of callus reaches 5.16 times, improved respectively 208.98%, 369.09% than the medium that contains MS and the reproduction coefficient that contains the medium of Nistch, be significant difference, and the callus growth gesture is best, breeding is fast, brownization do not occur, and can the long period remain the callus characteristic.Described MS minimal medium is 1962, the disclosed MS culture medium prescription of Murashige and Skoog; The Nistch minimal medium is disclosed Nistch culture medium prescription in 1972.
The impact that the different minimal mediums of table 3 are preserved callus
In sum, Chinese narcissus callus of the present invention is preserved tissue culture method, has following beneficial effect:
1. use Chinese narcissus callus Storaged media MN+2.0 mg/L 6-BA+0.5 mg/L 2 of the present invention, 4-D+0.2 mg/L NAA+30 g/L white sugar+6 g/L agar powders, pH5.8, the reproduction coefficient of Chinese narcissus callus reaches 5.16 times, callus growth is good, reproduction speed is fast, and brownization and differentiating phenomenon do not occur, and can be used as Chinese narcissus callus Storaged media.
2. the present invention adopts the medium that contains MN to compare with the medium that contains Nistch with containing MS, and the callus growing way is vigorous, and reproduction coefficient has improved respectively 208.98%, 369.09%.
3. the invention solves that the Chinese narcissus callus is difficult to subculture and preservation, reproduction coefficient is low and easy brownization and break up the problems such as serious, practical, generalization is good.
4. the callus of the present invention's preservation forwards easily differentiation on the differential medium to, is the good material of Chinese narcissus genetic transformation acceptor.
Embodiment
The present invention is further elaborated below in conjunction with embodiment.
The tissue culture method that embodiment one, Chinese narcissus callus are preserved
The tissue culture method that the Chinese narcissus callus is preserved may further comprise the steps:
1, material is selected: taking away the florescence and without the Chinese narcissus ovary of damage by disease and insect, is 75% alcohol-pickled 30 s with volume ratio, is 0.1% mercuric chloride, 5 min that sterilize with weight ratio again, uses at last aseptic water washing 3 times;
2, induce cultivation: on super-clean bench, the ovary crosscut after the sterilization is become the thin slice of 5 mm thickness, be inoculated into MN+4.0 mg/L 6-BA+0.4 mg/L 2,4-D+0.2 mg/L NAA+30g/L white sugar+6 g/L agar powders is on the inducing culture of pH 5.8; Culturing room's temperature is 25 ℃～28 ℃, illumination 12 h/d, and intensity of illumination is 1200 lx～1500 lx; Cultivated 60 days;
3, callus is preserved: the prescription of callus Storaged media is MN+2.0 mg/L 6-BA+0.5 mg/L 2,4-D+0.2 mg/L NAA+, 30 g/L white sugar+6 g/L agar powders, pH5.8; Take by weighing first agar powder, white sugar by above-mentioned prescription, add and account in the distilled water of callus Storaged media cumulative volume 1/3, add again all the other the various raw materials in the described prescription after boiling, after being settled to the medium total amount that to prepare with distilled water, regulate pH value to 5.8 with the NaOH of 1 mol/L or the HCl of 1 mol/L again; Then, divide to install in the container and seal; At 1.1 kg/cm
2Sterilization 20 min are the callus Storaged media under the pressure after the cooled and solidified.The callus that will induce from the ovary place cuts into 0.5 cm
2Fritter, be inoculated in the callus Storaged media, culturing room's temperature is 25 ℃～28 ℃, illumination 12 h/d, intensity of illumination is 1200 lx～1500 lx, cultivates that callus growth reaches optimum efficiency after 60 days, average reproduction coefficient reaches 5.16 times, callus growth is good, and reproduction speed is fast, and brownization and differentiating phenomenon do not occur; Therefore, every switching in 60 days once, can keep the dedifferentiation state of Chinese narcissus.
The tissue culture method that embodiment two, Chinese narcissus callus are preserved
The tissue culture method that the Chinese narcissus callus is preserved may further comprise the steps:
1, material is selected: the material with reference to embodiment one is selected.
2, induce cultivation: with reference to the cultivation of inducing of embodiment one.
3, callus is preserved: the prescription of callus Storaged media is MN+3.0 mg/L 6-BA+ 1.5 mg/L 2,4-D+0.4 mg/L NAA+30 g/L white sugar+6 g/L agar powders, pH5.8; Callus is cut into 0.5 cm
2Behind the fritter, be inoculated in the callus Storaged media; Be 25 ℃～28 ℃ in culturing room's temperature, illumination 12 h/d, intensity of illumination is to cultivate 60 days under the condition of 1200 lx～1500 lx, the average reproduction coefficient of callus reaches 3.19 times, but callus growth is comparatively slow.
The tissue culture method that embodiment three, Chinese narcissus callus are preserved
The tissue culture method that the Chinese narcissus callus is preserved may further comprise the steps:
1, material is selected: the material with reference to embodiment one is selected.
2, induce cultivation: with reference to the cultivation of inducing of embodiment one.
3, callus is preserved: the prescription of callus Storaged media is MN+1.0 mg/L 6-BA+0.5 mg/L 2,4-D+0.4 mg/L NAA+30 g/L white sugar+6 g/L agar powders, and pH5.8 cuts into 0.5 cm with callus
2Behind the fritter, be inoculated in the callus Storaged media; Be 25 ℃～28 ℃ in culturing room's temperature, illumination 12 h/d, intensity of illumination is to cultivate 60 days under the condition of 1200 lx～1500 lx, the average reproduction coefficient of callus reaches 3.93 times.
The above only is preferred embodiment of the present invention, and all equalizations of doing according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Claims (4)
1. a Chinese narcissus callus is preserved tissue culture method, comprise the material selection, induce and cultivate and the callus preservation, it is characterized in that described callus Storaged media is MN+1.0 ~ 3.0 mg/L 6-BA+0.5 ~ 1.5 mg/L 2,4-D+0.2 mg/L NAA+30 g/L white sugar+6 g/L agar powders, pH5.8; Wherein, MN minimal medium prescription is as shown in table 1:
Table 1 MN minimal medium prescription
2. a kind of Chinese narcissus callus according to claim 1 is preserved tissue culture method, it is characterized in that described callus Storaged media is MN+2.0 mg/L 6-BA+0.5 mg/L 2,4-D+0.2 mg/L NAA+, 30 g/L white sugar+6 g/L agar powders, pH5.8.
3. a kind of Chinese narcissus callus according to claim 1 is preserved tissue culture method, it is characterized in that described callus Storaged media is MN+3.0 mg/L 6-BA+ 1.5 mg/L 2,4-D+0.4 mg/L NAA+30 g/L white sugar+6 g/L agar powders, pH5.8.
4. a kind of Chinese narcissus callus according to claim 1 is preserved tissue culture method, it is characterized in that described callus Storaged media is MN+1.0 mg/L 6-BA+0.5 mg/L 2,4-D+0.4 mg/L NAA+30 g/L white sugar+6 g/L agar powders, pH5.8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210518283 CN103004590B (en) | 2012-12-06 | 2012-12-06 | Chinese narcissus callus preservation and tissue culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210518283 CN103004590B (en) | 2012-12-06 | 2012-12-06 | Chinese narcissus callus preservation and tissue culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103004590A true CN103004590A (en) | 2013-04-03 |
| CN103004590B CN103004590B (en) | 2013-12-18 |
Family
ID=47954121
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210518283 Expired - Fee Related CN103004590B (en) | 2012-12-06 | 2012-12-06 | Chinese narcissus callus preservation and tissue culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103004590B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01305001A (en) * | 1988-06-02 | 1989-12-08 | Mitsui Petrochem Ind Ltd | Preservation of bulbs or the like |
| CN1434123A (en) * | 2002-01-25 | 2003-08-06 | 北京达科豪科技有限公司 | Method for producing tripterygium alkaloids by plant suspension cultivation cell |
| CN1631086A (en) * | 2004-12-31 | 2005-06-29 | 福建农林大学 | Diced breeding technique for Chinese narcissus |
| CN101658098A (en) * | 2009-09-15 | 2010-03-03 | 云南省农业科学院花卉研究所 | Method for cutting and propagating creeping stems of primula pseudodenticulata pax view |
| CN101816286A (en) * | 2009-12-22 | 2010-09-01 | 江苏省中国科学院植物研究所 | Method for tissue culture and rapid propagation of narcissus pseudonarcissus by using ramentum |
| CN101843219A (en) * | 2010-05-06 | 2010-09-29 | 福建农林大学 | Use of young scape as explant for quick propagation and transgenosis of Chinese sacred lily |
| CN102144558A (en) * | 2011-02-19 | 2011-08-10 | 云南省农业科学院花卉研究所 | Breeding method of primula pseudodenticulata pax tetraploid plant |
| CN102177850A (en) * | 2011-04-01 | 2011-09-14 | 上海植物园 | Quick tissue culture breeding and seedling raising method for narcissus tazetta var. chinensis |
| CN102369879A (en) * | 2010-08-10 | 2012-03-14 | 中国科学院植物研究所 | Succeeding preservation method for embryogenic callus of China fir and subculture medium used therein |
-
2012
- 2012-12-06 CN CN 201210518283 patent/CN103004590B/en not_active Expired - Fee Related
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01305001A (en) * | 1988-06-02 | 1989-12-08 | Mitsui Petrochem Ind Ltd | Preservation of bulbs or the like |
| CN1434123A (en) * | 2002-01-25 | 2003-08-06 | 北京达科豪科技有限公司 | Method for producing tripterygium alkaloids by plant suspension cultivation cell |
| CN1631086A (en) * | 2004-12-31 | 2005-06-29 | 福建农林大学 | Diced breeding technique for Chinese narcissus |
| CN101658098A (en) * | 2009-09-15 | 2010-03-03 | 云南省农业科学院花卉研究所 | Method for cutting and propagating creeping stems of primula pseudodenticulata pax view |
| CN101816286A (en) * | 2009-12-22 | 2010-09-01 | 江苏省中国科学院植物研究所 | Method for tissue culture and rapid propagation of narcissus pseudonarcissus by using ramentum |
| CN101843219A (en) * | 2010-05-06 | 2010-09-29 | 福建农林大学 | Use of young scape as explant for quick propagation and transgenosis of Chinese sacred lily |
| CN102369879A (en) * | 2010-08-10 | 2012-03-14 | 中国科学院植物研究所 | Succeeding preservation method for embryogenic callus of China fir and subculture medium used therein |
| CN102144558A (en) * | 2011-02-19 | 2011-08-10 | 云南省农业科学院花卉研究所 | Breeding method of primula pseudodenticulata pax tetraploid plant |
| CN102177850A (en) * | 2011-04-01 | 2011-09-14 | 上海植物园 | Quick tissue culture breeding and seedling raising method for narcissus tazetta var. chinensis |
Non-Patent Citations (5)
| Title |
|---|
| LINJIAO CHEN,ET AL.: "efficient callus induction and plant regeneration from anther of chinese narcissus(narcissus tazetta l.var.chinensis roem)", 《PLANT CELL REP》 * |
| 余望: "植物激素对水仙(Narcissus tazetta var .Chinensis )愈伤组织形成及分化的影响", 《福州师专学报（自然科学版）》 * |
| 林江波等: "中国水仙愈伤组织诱导与小鳞茎再生", 《中国农学通报》 * |
| 洪森荣等: "离体保存技术在植物种植资源保存中的应用", 《上饶师范学院学报》 * |
| 邓朝军等: "枇杷茎尖愈伤组织诱导及种质体外保存研究", 《福建果树》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103004590B (en) | 2013-12-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101810140B (en) | Method for propagating dendrobium candidum test-tube plantlets | |
| Ozel et al. | Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale | |
| CN104719162B (en) | Method for screening dandelion salt-tolerance mutants | |
| Minano et al. | Molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using RAPD markers | |
| CN104099287A (en) | Acquiring and subculture maintaining method for high yield oligomeric proanthocyanidins vitis davidii callus | |
| Ata et al. | Effects of season, genotype, and nutrient medium on pepper anther culture andmicrospore development | |
| Zou et al. | Efficient induction of gynogenesis through unfertilized ovary culture with winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) | |
| Pan et al. | In vitro conservation of native Chinese wild grape (Vitis heyneana Roem. & Schult) by slow growth culture | |
| Ziv | Morphogenesis of gladiolus buds in bioreactors—implication for scaled-up propagation of geophytes | |
| CN103053423B (en) | Method for establishing high-efficiency regeneration system by using broccoli microspore embryo as explant | |
| Uzun et al. | In vitro micropropagation from immature embryos of the endemic and endangered Muscari muscarimi Medik. | |
| CN113080055B (en) | Method for directly creating melon double haploid by inducing embryo development through distant hybridization | |
| CN102907328B (en) | Method for utilizing microelements to accelerate tissue culture seedling of sugarcane | |
| CN102450214A (en) | Screening and preserving method for Lolium L. embryogenic callus | |
| CN104126505B (en) | For the somatic embryo in-vitro regeneration method that Lilium tenuifolium genetic transformation is numerous soon with planting ball | |
| Nakano et al. | Regeneration of diploid and tetraploid plants from callus-derived protoplasts of Agapanthus praecox ssp. orientalis (Leighton) Leighton | |
| CN103004590B (en) | Chinese narcissus callus preservation and tissue culture method | |
| CN101843219B (en) | Use of young scape as explant for quick propagation and transgenosis of Chinese sacred lily | |
| CN102599059B (en) | Method for improving tissue culture regeneration rate of wheat genotype immature embryo with low regeneration capacity | |
| CN102210266B (en) | Culture medium for culturing lilium pumilum tissues | |
| CN111937741B (en) | Method for creating intergeneric distant hybrid of hibiscus and broad-leaved Maohua | |
| Bakhshaie et al. | Effects of light, sucrose, and cytokinins on somatic embryogenesis in Lilium ledebourii (Baker) Bioss. via transverse thin cell-layer cultures of bulblet microscales | |
| CN102630568B (en) | Gomphrena globosa test tube flower and culture method thereof | |
| KR20080073388A (en) | Mass production of bulblet via somatic embryogenic cell culture in lily | |
| KR100751951B1 (en) | New breed of cymbidium, jan mook |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131218 Termination date: 20151206 |
|
| EXPY | Termination of patent right or utility model |