CN106818714A - The cryopreservation method of cowberry plant germplasm - Google Patents
The cryopreservation method of cowberry plant germplasm Download PDFInfo
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- CN106818714A CN106818714A CN201611172859.6A CN201611172859A CN106818714A CN 106818714 A CN106818714 A CN 106818714A CN 201611172859 A CN201611172859 A CN 201611172859A CN 106818714 A CN106818714 A CN 106818714A
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- cowberry
- stem apex
- wpm
- plant germplasm
- sucrose
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
-
- 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
Abstract
The invention discloses the method for cowberry stem apex drop vitrification Excised Embryos, it is a kind of long-term safety, reliable and stable, the simple and effective method for preserving cowberry fine germplasm resources, the present invention with terminal bud stem apex be Excised Embryos material, one side convenient material drawing, on the other hand because stem apex contains shoot apical meristem, cytothesis ability is stronger, and genetic stability is high；Cowberry stem apex survival rate is up to 100% after Excised Embryos, and stem apex regeneration rate is 91.7%.
Description
Technical field
The present invention relates to Preservation of plant germplasin field, specifically, the Excised Embryos side of cowberry plant germplasm is planted
The method of method and Excised Embryos cowberry plant germplasm renewal cultivation.
Background technology
Cowberry belongs to Ericaceae (Ericaceae) Vaccinium (Vaccinium spp) plant, be it is perennial fall
Leaf or evergreen shrubs or undershrub seeds.Whole world cowberry platymiscium there are about 400 kinds, and China there are about 91 kinds, 28 mutation,
It is distributed in China northeast and southwest.Fruit is rich in the polyphenoils such as anthocyanin, with improvement eyesight, anti-oxidant, anticancer
And delay the healthcare functions such as cranial nerve aging, one of big healthy food of the mankind five is classified as by international food and agricultural organization, while also being praised
It is " king of berry ".
Germ-plasma resources protection is cowberry breeding and the important guarantee for keeping genetic diversity, cultivates high yield, high-quality and height anti-
New varieties be unable to do without abundant germ plasm resource, therefore the preservation of development cowberry germ plasm resource has great importance.
It is less about the research of the store method of cowberry germ plasm resource at present, it is concentrated mainly on original place cultivation, cultivates with moving
And seed bank is preserved, but original place and move ground cultivation management and materials and need substantial amounts of human and material resources, and the germ plasm resource for preserving
Floor space is big, is vulnerable to the influence of pest and disease damage and natural conditions.With the development of biotechnology, based on using plant tissue training
The research that the technology of supporting is preserved to cowberry germ plasm resource is limited only to preservation in the lab to research material.On the one hand protect
Deposit that process is cumbersome, waste time and energy, it is relatively costly；On the other hand, with the extension and the increase of subculture number of holding time, preserve
Material be vulnerable to the interference of pollution.
Excised Embryos technology is the emerging technology that 20 century 70s grow up on the basis of Cord blood.
Generally with liquid nitrogen (- 196 DEG C) as low-temperature receiver, biomaterial is stored under such cryogenic conditions Excised Embryos, the generation in living cells
The activity of thanking almost stops completely, thus can make the biomaterial change of inhereditary feature will not occur at such a temperature, and preserves
The vigor of cell and morphogenetic totipotency are unaffected in journey.Because its have it is simple to operate, can the holding time it is long, take empty
Between it is small and the advantages of do not disturbed by pest and disease damage, be generally acknowledge at present long-term and the ideal side of the preservation plant germplasm resource of stabilization
Method.Developed the method for various Excised Embryos, however for different plant species even for same species different cultivars,
Its suitable cryopreservation method and program technic are likely to difference.It is relevant at present to utilize Excised Embryos technology to cowberry
There is not been reported for the research that germ plasm resource is preserved.
The content of the invention
The purpose of the present invention is directed to the deficiency in the existing Techniques of preserving of cowberry, there is provided cowberry stem apex drop vitrification surpasses
The method of Cord blood and the method for Excised Embryos cowberry plant germplasm renewal cultivation.
The cryopreservation method of cowberry plant germplasm, it includes
1）Hardening：4 weeks tissue-cultured seedling of seedling age are chosen, stem sections of the 0.4-0.6 cm with terminal bud is taken and is inoculated into 4 DEG C on WPM culture mediums
The week of dark treatment 2-4；
2）Cowberry stem apex is stripped：Aseptically, the terminal bud after stripping hardening under stereomicroscope, stem apex a length of 1.5-
2.0mm；
3）Preculture：The stem apex that will be stripped is inoculated in the WPM solid cultures for being added with 0.3M sucrose, in tissue culture room dark culturing
18—30h；
4）Load：Shoot Tips after preculture are transferred in loading liquid, treatment 20-40min is loaded at room temperature；
5）Vitrifying is processed：Stem apex after loading is processed is transferred in PVS2 solution, and 30-50min is processed in 0 DEG C；
6）Droplet makes：Stem apex after PVS2 is processed is transferred on aluminum foil strip, and it is molten that 6 μ L PVS2 are added dropwise to each stem apex
Liquid, makes stem apex fully wrapped around in PVS2 droplets, and 9-11 stem apex is put on each strip of tin foil；
7）Ultra-cryofreezing preservation：The above-mentioned strip of tin foil containing droplet made is directly immersed in liquid nitrogen and is freezed,
Then the strip of tin foil after freezing is placed in refrigeration centrifugation pipe, after covering tightly lid, is preserved in input liquid nitrogen；
Step 1) described in the hardening time be 3 weeks, step 3) described in pre-incubation time for 24h when；
Step 4）Described in the composition of loading liquid be：WPM+2M glycerine+1.0M sucrose, the loading time is 30min；
Step 5）Described in the composition of PVS2 solution be：Dimethyl sulfoxide+the 0.4M of+15% ethylene glycol of WPM+30% glycerine+15%
Sucrose.When process time is 40min；
The method of Excised Embryos cowberry plant germplasm renewal cultivation
1）Unloading：The Shoot Tips of Ultra-cryofreezing preservation are taken out, is positioned over to be unloaded and thawed in carrier fluid and unloaded；
2）Renewal cultivation：Shoot Tips aseptic filter paper after unloading is processed blots remained on surface liquid, transfers in renewal cultivation
Renewal cultivation is carried out in base.
The method of Excised Embryos cowberry plant germplasm renewal cultivation, what it included：
Step 1）The described component for unloading carrier fluid is：WPM+1.2M sucrose；Described unloading condition is：Unload at room temperature
20min。
Step 2）Described recovery media is the agar of+3% sucrose of WPM+0-0.5mg/L ZT+0.7%, pH=5.8；
Renewal cultivation condition is：Tissue culture room dark culturing 1d, is turning lastly to be cultivated under normal lighting conditions.
The invention provides the method for cowberry stem apex drop vitrification Excised Embryos, be a kind of long-term safety, stabilization can
By, it is simple and effective preserve cowberry fine germplasm resources method, the present invention with terminal bud stem apex be Excised Embryos material, a side
Face convenient material drawing, on the other hand because stem apex contains shoot apical meristem, cytothesis ability is stronger, and genetic stability is high；It is super
Cowberry stem apex survival rate is up to 100% after Cord blood, and stem apex regeneration rate is 91.7%.
Brief description of the drawings
Influence of Fig. 1 hardening times to cowberry stem apex Excised Embryos.
Influence of Fig. 2 load times to cowberry stem apex Excised Embryos.
Influence of Fig. 3 pre-incubation times to cowberry stem apex Excised Embryos.
Influence of Fig. 4 vitrifyings process time to cowberry stem apex Excised Embryos.
Influence of Fig. 5 ZT concentration to cowberry stem apex Excised Embryos.
Specific embodiment
The cowberry stem apex drop vitrification method Excised Embryos technology of embodiment 1 and plant regeneration cultural method
Experimental technique concrete operations are as follows：
1st, seedling is practiced：4 weeks tissue-cultured seedling of seedling age are chosen, stem sections of 0.5 cm with terminal bud is taken and is inoculated into 4 DEG C of dark places on WPM culture mediums
Reason 3 weeks;
2nd, cowberry stem apex is stripped：Aseptically, the terminal bud after stripping hardening under stereomicroscope, stem apex a length of 1.5-
2.0mm;
3rd, preculture：The stem apex that will be stripped is inoculated in the WPM solid cultures for being added with 0.3M sucrose, in the dark training in tissue culture room
Support 24h;
4th, load：Shoot Tips after preculture are transferred in composition and is：In the loading liquid of WPM+2M glycerine+1.0M sucrose, in
Treatment 30min is loaded at room temperature;
5th, vitrifying treatment：Stem apex after loading is processed is transferred in composition：The ethylene glycol+15% of WPM+30% glycerine+15%
In the PVS2 solution of dimethyl sulfoxide+0.4M sucrose, 40min is processed in 0 DEG C;
6th, droplet makes：Stem apex after PVS2 is processed is transferred on aluminum foil strip, and it is molten that 6 μ L PVS2 are added dropwise to each stem apex
Liquid, makes stem apex fully wrapped around in PVS2 droplets, and 10 stem apexs are put on each strip of tin foil;
7th, Ultra-cryofreezing preservation：The above-mentioned strip of tin foil containing droplet made is directly immersed in liquid nitrogen and is freezed,
Then the strip of tin foil after freezing is placed in 2ml refrigeration centrifugation pipes, after covering tightly lid, is preserved in input liquid nitrogen;
8th, unload：The Shoot Tips of Ultra-cryofreezing preservation are taken out, being positioned over component is：WPM+1.2M sucrose unloads carrier fluid
Thawed and unloaded, 20min is unloaded at room temperature;
9th, renewal cultivation：Shoot Tips aseptic filter paper after unloading is processed blots remained on surface liquid, transfers in renewal cultivation
Renewal cultivation is carried out in base；Described recovery media is the agar of+3% sucrose of WPM+0.3mg/L ZT+0.7%, pH=5
.8；Wherein renewal cultivation condition is：Tissue culture room dark culturing 1d, is turning lastly to be cultivated under normal lighting conditions.Recover
After culture 7d, the stem apex for surviving shows as green, and the stem apex of death shows as brown；After culture 30d, the stem apex for surviving is formed
Whole plant.
The influence of the hardening time of embodiment 2 and load time to cowberry stem apex Excised Embryos
Operating procedure only changes the hardening time with embodiment 1, and the hardening time is set as into 0,1,2,3,4,5 weeks, and the load time is
0、10、20、30、40min.Survival rate and 30d the statistics regeneration situation of cowberry stem apex are counted after renewal cultivation 7d.Result shows refining
There are important influence seedling time and load time to the survival rate and regeneration rate after the preservation of cowberry stem apex.The survival rate of stem apex and
Regeneration rate is presented the trend fallen after rising with the change of hardening time, as shown in figure 1, the change with the load time is in substantially
The trend fallen after rising, as shown in Figure 2.When the hardening time is 3 weeks, surviving for stem apex is preferable with regeneration effect, and the load time is
During 30min, surviving for stem apex is preferable with regeneration effect.
The influence of the pre-incubation time of embodiment 3 and vitrifying process time to cowberry stem apex Excised Embryos
Operating procedure only changes pre-incubation time with embodiment 1, WPM of the cowberry stem apex inoculation containing 0.3 M sucrose that will be peeled
In solid medium, in tissue culture room dark preculture 0-48h, vitrifying process time is set as 0,20,40,60,80min.It is extensive
Survival rate and 30d the statistics regeneration situation of cowberry stem apex are counted after culture 7d again.Result shows at pre-incubation time and vitrifying
There is important influence the reason time to the survival rate and regeneration rate after the preservation of cowberry stem apex.The survival rate and regeneration rate of stem apex with
The change of pre-incubation time is presented the trend fallen after rising substantially, as shown in figure 3, being in first to go up with the change of vitrifying process time
Decline the trend for rising again after rising, as shown in Figure 4.When pre-incubation time is 24h, surviving for stem apex is preferable with regeneration effect, glass
When glass process time is 40min, stem apex is survived and regenerate preferable.
The ZT of various concentrations in the regeneration culture medium of embodiment 4（Zeatin）Influence to cowberry stem apex Excised Embryos
Operating procedure only changes ZT concentration with embodiment 1, choose ZT concentration be 0,0.1,0.2,0.3,0.4,0.5mg/L.Recover
Survival rate and 30d the statistics regeneration situation of cowberry stem apex are counted after culture 7d.After result shows that ZT concentration is preserved to cowberry stem apex
Survival rate and regeneration rate have important influence.The survival rate of stem apex is in the trend for first dropping and rising drop again afterwards, and regeneration rate is with ZT
Change in concentration is presented the trend fallen after rising substantially, as shown in figure 5, when ZT concentration is 0.3mg/L, stem apex is survived and revival
Fruit is preferably.
Claims (5)
1. a kind of cryopreservation method of cowberry plant germplasm, it is characterised in that it includes：
1）Hardening：4 weeks tissue-cultured seedling of seedling age are chosen, stem sections of the 0.4-0.6 cm with terminal bud is taken and is inoculated into 4 DEG C on WPM culture mediums
The week of dark treatment 2-4；
2）Cowberry stem apex is stripped：Aseptically, the terminal bud after stripping hardening under stereomicroscope, stem apex a length of 1.5-
2.0mm；
3）Preculture：The stem apex that will be stripped is inoculated in the WPM solid cultures for being added with 0.3M sucrose, in tissue culture room dark culturing
18—30h；
4）Load：Shoot Tips after preculture are transferred in loading liquid, treatment 20-40min is loaded at room temperature；
5）Vitrifying is processed：Stem apex after loading is processed is transferred in PVS2 solution, and 30-50min is processed in 0 DEG C；
6）Droplet makes：Stem apex after PVS2 is processed is transferred on aluminum foil strip, and it is molten that 6 μ LPVS2 are added dropwise to each stem apex
Liquid, makes stem apex fully wrapped around in PVS2 droplets, and 9-11 stem apex is put on each strip of tin foil；
7）Ultra-cryofreezing preservation：The above-mentioned strip of tin foil containing droplet made is directly immersed in liquid nitrogen and is freezed,
Then the strip of tin foil after freezing is placed in refrigeration centrifugation pipe, after covering tightly lid, is preserved in input liquid nitrogen.
2. the cryopreservation method of cowberry plant germplasm according to claim 1, it is characterised in that：Step 1) it is described
The hardening time be 3 weeks, step 3) described in pre-incubation time for 24h when.
3. the cryopreservation method of cowberry plant germplasm according to claim 1 and 2, it is characterised in that：Step 4）In
The composition of described loading liquid is：WPM+2M glycerine+1.0M sucrose, the loading time is 30min.
4. the cryopreservation method of cowberry plant germplasm according to claim 3, it is characterised in that：Step 5）Described
The composition of PVS2 solution is：Dimethyl sulfoxide+0.4M the sucrose of+15% ethylene glycol of WPM+30% glycerine+15%；Process time is
During 40min.
5. the method for Excised Embryos cowberry plant germplasm renewal cultivation, it is characterised in that it also includes：
1）Unloading：The step of by a kind of cryopreservation method of the cowberry plant germplasm described in claim 17）Ultra-low temperature cold
Freeze the Shoot Tips for preserving to take out, be positioned over to unload and thawed in carrier fluid and unloaded；The described component for unloading carrier fluid is：WPM+
1.2M sucrose；Unloading condition is：20min is unloaded at room temperature；
2）Renewal cultivation：Shoot Tips aseptic filter paper after unloading is processed blots remained on surface liquid, transfers in renewal cultivation
Renewal cultivation is carried out in base；Foregoing method, recovery media is the agar of+3% sucrose of WPM+0-0.5mg/L ZT+0.7%,
PH=5.8；Renewal cultivation condition is：Tissue culture room dark culturing 1d, is turning lastly to be cultivated under normal lighting conditions.
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Cited By (4)
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| CN109221094A (en) * | 2018-09-11 | 2019-01-18 | 中国农业科学院作物科学研究所 | A kind of Banana In Vitro stem apex cryopreservation method |
| CN109628297A (en) * | 2018-12-14 | 2019-04-16 | 浙江大学宁波理工学院 | A kind of microfluidic high-throughput biological sample drop jelly save set |
| CN111504967A (en) * | 2020-04-24 | 2020-08-07 | 中国科学院昆明植物研究所 | Method for measuring plant cell preservation and regeneration rate |
| CN114424747A (en) * | 2022-01-12 | 2022-05-03 | 中国科学院合肥物质科学研究院 | In-vitro preservation method for tissue culture regenerated buds of green cherry blossom |
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| CN114424747A (en) * | 2022-01-12 | 2022-05-03 | 中国科学院合肥物质科学研究院 | In-vitro preservation method for tissue culture regenerated buds of green cherry blossom |
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