CN101002541A - Method for dislocation storage of germ resource of chrysanthemum - Google Patents
Method for dislocation storage of germ resource of chrysanthemum Download PDFInfo
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- CN101002541A CN101002541A CN 200710019345 CN200710019345A CN101002541A CN 101002541 A CN101002541 A CN 101002541A CN 200710019345 CN200710019345 CN 200710019345 CN 200710019345 A CN200710019345 A CN 200710019345A CN 101002541 A CN101002541 A CN 101002541A
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Abstract
An in vitro storage method for the germplasm resource of chrysanthemum includes such steps as taking the root bud of chrysanthemum as explant, secondary culture of auxiliary bud, and storing in the MS culture medium containing abscisic acid, 6-BA and NAA. Its survival rate after 10 months is 100%.
Description
One, technical field
The present invention relates to a kind of method of chrysanthemum germ plasm resource {in vitro} conservation, be exclusively used in chrysanthemum germ plasm resource {in vitro} conservation.
Two, technical background
Chrysanthemum (Dendranthema * grandiflorum Ramat.) is one of China's ten great tradition famous flowers and the world's four big cut-flowers, have view and admire, eat, various value such as medicinal.China is the centre of origin of cultivating chrysanthemum and the distribution center of Chrysanthemum germ plasm resource, surplus the Chrysanthemum 40 kind China distribute have 20 surplus kind, surplus the cultivating chrysanthemum kind reaches 3000.There is height heterozygosity in cultivating chrysanthemum in heredity, offspring's proterties is separated, be difficult to preserve germ plasm resource by seed, and mainly carry out vegetative propagation by cuttage, need expend a large amount of soils, man power and material and carry out the maternal preservation and the cottage propagation of seedling, and in long-term vegetative propagation process, be prone to the people for mixing and planting the sexual involution phenomenon.
On producing, present seedling owing to tissue culture industrialization production not only will be complied with the demand in fast changing supply and demand market but also be subjected to the restriction of the season of growth again, making that the expansion of some materials is numerous does not have a practical significance in certain phase, it temporarily need be preserved, when the development of producing makes these materials become once more to need, it can be quickly recovered to normal fast numerous application state.Simultaneously, the summer high temperature high humidity, subculture pollutes easily, it if can be carried out the short-term preservation and then can get over the summer safely.
For overcoming the shortcoming that chrysanthemum tradition field planting preserves and solving the realistic problem of tissue culture industrialization in producing, the research of carrying out the {in vitro} conservation of chrysanthemum germ plasm resource seems urgent day by day.{in vitro} conservation, have not time-consuming, do not take a lot of work, be difficult for causing that germ plasm resource mixes, keep advantages such as germplasm merit, and be convenient to the exchange of virus-free germplasm.Along with nearly decades of being in full swing of cultured in vitro technology, reaching the slow growth method {in vitro} conservation technology that prolongs subculture time purpose by the external environmental condition that changes the culture growth and also obtained fast development.Since the nineties in 20th century, the researcher successfully carried out lily, the celestial flower of Mengzi wind, yellowish colored lily, dendrobium candidum etc. the test tube {in vitro} conservation of flowers germ plasm resource, but macroelement is preserved the chrysanthemum test-tube plantlet, the Shang Weiyou report in the present domestic utilization minimizing medium.
Three, summary of the invention
Technical problem the objective of the invention is at present chrysanthemum germ plasm resource field planting preservation workload big, be subject to the influence of extreme natural calamity and damage by disease and insect, and defectives such as the normal subculture preservation of tissue culture switching is frequent, cost height, the method that provides a kind of chrysanthemum test-tube plantlet to preserve, be used for the chrysanthemum {in vitro} conservation, it is normal not only to preserve after 10 months plant strain growth, and hereditary variation does not take place the offspring who preserves material.
The method of a kind of chrysanthemum germ plasm resource of technical scheme the present invention {in vitro} conservation comprises:
With chrysanthemum pin bud is explant, with 75% ethanol surface sterilization 30s, and aseptic water washing 2 times, again with 0.1% mercuric chloride sterilization 8min, behind the aseptic water washing 5 times, the stem section that is cut to 1 joint of defoliation band inserts in the MS medium; Behind the 20d axillalry bud of sprouting is transferred to MS+0.3mg.L
-16-BA+0.1mg.L
-1Expand numerous on the proliferated culture medium of NAA; The stem section that behind the 30d indefinite bud of breeding is cut into 2 joints changes additional 20～40mg.L over to
-1Preserve in the proliferated culture medium of abscisic acid.
Contain sucrose 3%, agar 0.7% in the above medium, pH is 6.0 (before the autoclave sterilizations), 23 ± 2 ℃ of condition of culture, intensity of illumination 2000～3000lux, light application time 12h/d.
The method of beneficial effect preservation chrysanthemum provided by the invention test-tube plantlet compared with prior art has following advantage and good effect:
1. the method that a kind of chrysanthemum test-tube plantlet provided by the present invention is preserved is used for the chrysanthemum {in vitro} conservation, and test-tube plantlet preserves that survival rate still is 100% after 10 months, and plant strain growth is normal, and the test-tube plantlet on the normal medium only can be survived 4 months.
2. the inventive method is preserved 10 months chrysanthemum test-tube plantlet, and the ISSR amplification bands of a spectrum and the contrast of its offspring POD, EST isodynamic enzyme zymogram and primer I 35 do not have difference, preserve material settling out, and genetic variation does not take place.
3. the present invention utilizes first and adds abscisic acid preservation chrysanthemum test-tube plantlet and its offspring is carried out the genetic stability evaluation in medium, preserves with traditional chrysanthemum field planting and compares, and can save shelf space, and workload is little.
4. can prevent many generation breeding back kind sexual involutions and virus infections, guarantee the good property and the purity of germplasm.
5. broken the restriction of vegetation season, material can be provided at any time, had the saving storage space simultaneously, be convenient to transportation and advantage such as exchange.
6. can prevent the loss of germ plasm resource.
7. heavy subculture work in reduce producing, and reduce the waste of human and material resources resource unnecessary in the production operation process provides necessary approach for actual production reduces production costs and increases economic efficiency.
Four, description of drawings
Fig. 1 preserves 4 months test-tube plantlet on the medium that adds abscisic acid
The right figure of left figure: ABA40mg/L: ABA20mg/L
Fig. 2 preserves 10 months test-tube plantlet on the medium that adds abscisic acid
The right figure of left figure: ABA20mg/L: ABA40mg/L
Fig. 3 preserves plant recovers to cultivate 25d on proliferated culture medium upgrowth situation
The right figure of left figure: scheme among the CK: ABA20mg/L: ABA40mg/L
The upgrowth situation of Fig. 4 regeneration plant domestication 25d
Fig. 5 regeneration plant is transplanted 3 months upgrowth situation of booth
Fig. 6 transplants 3 months regeneration plants of booth and contrasts the contrast of plant and leaf shape
The left figure of Fig. 7 isodynamic enzyme zymogram: the right figure of POD isodynamic enzyme zymogram: EST isodynamic enzyme zymogram
The ISSR amplification of Fig. 8 primer I 35
Annotate: (M) 100 bp dna molecular amount marks; CK: contrast; Horizontal line is represented the 1cm scale among the figure;
Five, embodiment
The method of preservation chrysanthemum test-tube plantlet provided by the present invention, its embodiment is as follows:
1. the {in vitro} conservation of chrysanthemum test-tube plantlet: with ' the Olympic torch ' (Dendranthema * grandiflorum Ramat. ' Aoyunhuoju ') (public kind, old kerria of the document that sees reference, Fang Weimin etc. the potted plant little chrysanthemum series of chrysanthemum new varieties-summer flower type. the gardening journal, 2005,32 (3): 567) kind is for supplying the examination material.Getting ' the Olympic torch ' pin bud is explant, with 75% ethanol surface sterilization 30s, and aseptic water washing 2 times, again with 0.1% mercuric chloride sterilization 8min, behind the aseptic water washing 5 times, the stem section that is cut to 1 joint of defoliation band inserts on the MS medium.Behind the explant inoculation 5d, the axillary bud sprouting rate is 83.3%; Behind the 20d axillalry bud is transferred to MS+0.3mg.L
-16-BA+0.1mg.L
-1Expand numerous on the proliferated culture medium of NAA; The stem section that behind the 30d indefinite bud of breeding is cut into 2 joints of defoliation band changes additional respectively 20mg.L over to
-1Abscisic acid (ABA), 40mg.L
-1Preserve in the proliferated culture medium of abscisic acid (ABA) (available from Nanjing Sheng Xing Bioisystech Co., Ltd, import Spanish packing).
Contain sucrose 3%, agar 0.7% in the above medium, pH is 6.0 (before the autoclave sterilizations), 23 ± 2 ℃ of condition of culture, intensity of illumination 2000～3000lux, light application time 12h/d.
2. preserving material offspring's genetic stability identifies: ' the Olympic torch ' test-tube plantlet is gone up growth after 3 months at the proliferated culture medium that does not add abscisic acid (contrast), its top tip and cultivation lid turn back downwards, whole strain twisted growth, and cover with aerial root on the stem section, have only top tip peak green to use, after 4 months, plant senesecence is withered.And test-tube plantlet is adding 20 abscisic acids (ABA) or 40mg.L
-1Poor growth on the medium of abscisic acid, plant height only has 1.0～2.3cm (Fig. 1) after 4 months, the top tip and cultivation lid (Fig. 2) after 10 months, but plant strain growth is normal, whole strain can be used, and adds abscisic acid in the medium and has prolonged the successive transfer culture time greatly, makes subculture transfer 10 months subcultures for 1 time to 1 time by at ordinary times 1 month subculture at interval, and the survival rate under two concentration is 100%, and comparison is according to prolonging survival 6 months; Preserving 10 months the test-tube plantlet and the contrast test-tube plantlet of subculture 1 subnormal cultivation preservation in 1 month is forwarded on the proliferated culture medium, the preservation material differentiates the normal indefinite bud of form after cultivating 10d, and differentiation capability is normal compared with the control, shows that preserving material recovers growth normal (Fig. 3); Behind the 30d indefinite bud of regeneration is transferred on the root media, root media is 1/2MS+0.1mg.L
-1NAA; Open behind the culture of rootage 15d and cultivate lid white silk seedling, be transplanted to behind the 7d in the perlite that high-temperature sterilization is crossed and tame, water every day once, 3～4d waters the MS nutrient solution once, is transplanted to booth (Fig. 4) behind the 25d; The transplanting booth is got plant lobus cardiacus extraction crude enzyme liquid after 2 months (method sees reference, and document how imitate by loyalty, the Zhang Shuzheng chief editor. electrophoresis [M]. Science Press, Beijing, 1999) and total DNA (method document Xiao Jun that sees reference, Yang Liguo etc. two kinds of comparisons of extracting the total DNA method of chrysanthemum. Liaoning agricultural science, 2005 (1): 40～41), find no specific band generation (Fig. 7, Fig. 8) through peroxide enzyme (POD) and esterase (EST) isodynamic enzyme, ISSR Markers for Detection.Above presentation of results is adding 20mg.L
-1Abscisic acid or 40mg.L
-1The chrysanthemum germplasm of preserving 10 months in the medium of abscisic acid has kept good genetic stability.
The method that above-mentioned isodynamic enzyme detects is, gets plant lobus cardiacus 0.15g, adds on the 450ml buffer solution ice bath to grind to form homogenate rapidly, is transferred to 4 ℃ of centrifugal 20min of 8000rmp/min in the centrifuge tube, and supernatant is the crude extract of enzyme; The separation gel of preparation 9%, 3% concentrated glue adopt the vertical panel polyacrylamide gel electrophoresis to analyze POD and EST isodynamic enzyme, every hole point sample 20ul.Concentrate glue and adopt 100V voltage stabilizing electrophoresis, use 200V voltage stabilizing electrophoresis behind the separation gel instead.When running extremely near bottom 1cm, the bromophenol blue colour band stops electrophoresis; POD adopts the dyeing of improvement benzidine method, EST dyes with a-naphthalene ester, strong orchid, and gel imaging analysis instrument (Shanghai Peiqing Science Co., Ltd, JS-380 type) is gone up to observe and found, the zymogram of {in vitro} conservation material offspring strain and contrast consistent (Fig. 7), illustrate added 20,40mg.L
-1Genetic variation does not take place in the chrysanthemum germplasm of preserving 10 months in the medium of abscisic acid on protein level.
The method of above-mentioned ISSR molecular markers for identification is, get the plant lobus cardiacus in little glass mortar with liquid nitrogen mixing grind away.Sample total DNA extract with reference to the CTAB method (the method document Xiao Jun that sees reference, Yang Liguo etc. two kinds of comparisons of extracting the total DNA methods of chrysanthemum. Liaoning agricultural science, 2005 (1): 40～41).Amplified reaction adopts 20.0ul reaction system (see Table 1, all reagent are all given birth to logical biotechnology Co., Ltd available from Nanjing), in the PTC-100 of U.S. MJ Research company
TMCarry out pcr amplification in the type amplification instrument, response procedures is: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s, 55 ℃ of renaturation 40s; 72 ℃ are extended 70s, 45 circulations; 72 ℃ are extended 7min, 10 ℃ of preservations.After amplified reaction finishes, respectively add 6 * bromophenol blue 0.4ul in ISSR amplified production reactant liquor, behind the centrifugal mixing, each is drawn mixed liquor 6.5ul and injects point sample hole electrophoresis on 2% Ago-Gel, and gel contains 0.05% ethidium bromide (EB).Electrode buffer is 1 * Tris-acetate (TAE), 180 volts of separation voltages, behind electrophoresis 50～60min in gel imaging analysis instrument (Shanghai Peiqing Science Co., Ltd, the JS-380 type) going up observation finds, the ISSR amplified band of its primer I 35 of {in vitro} conservation material offspring strain is consistent with contrast, do not have specific band to produce (Fig. 8), illustrate added 20,40mg.L
-1Genetic variation does not take place in the chrysanthemum germplasm of preserving 10 months in the medium of abscisic acid on molecular level.
Table 1PCR reaction system
| Composition | Volume (ul) |
| 10×Buffer(Mg 2+Free) 25mM Mg 2+10mM d NTP Mixture 5U/ul Taq enzyme 20uM primer I 35 ddH 2O 20ng/ul template DNA reaction cumulative volume | 2.0 1.5 0.8 0.2 0.5 13.0 2.0 20.0 |
Annotate: the sequence of primer I 35 is (AG)
8TA
Claims (1)
1, a kind of method of chrysanthemum germ plasm resource {in vitro} conservation comprises:
With chrysanthemum pin bud is explant, with 75% ethanol surface sterilization 30s, and aseptic water washing 2 times, again with 0.1% mercuric chloride sterilization 8min, behind the aseptic water washing 5 times, the stem section that is cut to 1 joint of defoliation band inserts in the MS medium; Behind the 20d axillalry bud of sprouting is transferred to MS+0.3mg.L
-16-BA+0.1mg.L
-1Expand numerous on the proliferated culture medium of NAA; The stem section that behind the 30d indefinite bud of breeding is cut into 2 joints changes additional 20～40mg.L over to
-1Preserve in the proliferated culture medium of abscisic acid;
Contain sucrose 3%, agar 0.7% in the above medium, pH is 6.0,23 ± 2 ℃ of condition of culture, intensity of illumination 2000～3000lux, light application time 12h/d.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726298A (en) * | 2012-07-03 | 2012-10-17 | 中国农业科学院作物科学研究所 | Cryopreservation method for in vitro shoot tips of petunia hybrida |
| CN102726293A (en) * | 2012-06-27 | 2012-10-17 | 中国热带农业科学院椰子研究所 | Method for in vitro preservation of betel nut genetic resources |
| CN103270950A (en) * | 2013-05-27 | 2013-09-04 | 聊城大学 | Chrysanthemum simplified tissue culturing method |
| CN104170743A (en) * | 2014-09-09 | 2014-12-03 | 安徽科技学院 | Culture medium for preserving tissue culture seedlings of stevia rebaudiana |
| CN104170742A (en) * | 2014-09-09 | 2014-12-03 | 何克勤 | Method for preserving tissue culture seedlings of stevia rebaudiana idioplasm |
| CN104255454A (en) * | 2014-09-09 | 2015-01-07 | 安徽科技学院 | Preservation method of chrysanthemum tissue culture seedlings |
| CN109792953A (en) * | 2019-01-29 | 2019-05-24 | 南京农业大学 | A kind of tissue culture method for protecting chrysanthemum intellectual property kind |
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2007
- 2007-01-17 CN CN200710019345A patent/CN100574612C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726293A (en) * | 2012-06-27 | 2012-10-17 | 中国热带农业科学院椰子研究所 | Method for in vitro preservation of betel nut genetic resources |
| CN102726298A (en) * | 2012-07-03 | 2012-10-17 | 中国农业科学院作物科学研究所 | Cryopreservation method for in vitro shoot tips of petunia hybrida |
| CN103270950A (en) * | 2013-05-27 | 2013-09-04 | 聊城大学 | Chrysanthemum simplified tissue culturing method |
| CN103270950B (en) * | 2013-05-27 | 2015-07-01 | 聊城大学 | Chrysanthemum simplified tissue culturing method |
| CN104170743A (en) * | 2014-09-09 | 2014-12-03 | 安徽科技学院 | Culture medium for preserving tissue culture seedlings of stevia rebaudiana |
| CN104170742A (en) * | 2014-09-09 | 2014-12-03 | 何克勤 | Method for preserving tissue culture seedlings of stevia rebaudiana idioplasm |
| CN104255454A (en) * | 2014-09-09 | 2015-01-07 | 安徽科技学院 | Preservation method of chrysanthemum tissue culture seedlings |
| CN104255454B (en) * | 2014-09-09 | 2016-05-11 | 林平 | A kind of store method of chrysanthemum group training seedling |
| CN109792953A (en) * | 2019-01-29 | 2019-05-24 | 南京农业大学 | A kind of tissue culture method for protecting chrysanthemum intellectual property kind |
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Assignee: Kunming Hongzhihua Gardening Co., Ltd. Assignor: Nanjing Agricultural University Contract record no.: 2010530000058 Denomination of invention: Method for dislocation storage of germ resource of chrysanthemum Granted publication date: 20091230 License type: Exclusive License Open date: 20070725 Record date: 20101018 |
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