CN105746351A - Method for ultralow-temperature storage and renewal cultivation of wild rice stem tips - Google Patents
Method for ultralow-temperature storage and renewal cultivation of wild rice stem tips Download PDFInfo
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- CN105746351A CN105746351A CN201610131944.1A CN201610131944A CN105746351A CN 105746351 A CN105746351 A CN 105746351A CN 201610131944 A CN201610131944 A CN 201610131944A CN 105746351 A CN105746351 A CN 105746351A
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- stem apex
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- sucrose
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
Abstract
A method for ultralow-temperature storage and renewal cultivation of wild rice stem tips includes steps: taking a subcultured sterile tissue culture seedling, keeping the base portion, culturing in a culture medium free of hormone MS, cutting new sprouts of the base portion, transferring to a culture medium containing 0.3molL<-1> of sucrose and 0.5-5mgL<-1> of abscisic acid to realize culturing, transferring to a culture medium containing 0.7molL<-1> of sucrose and 0.5-5mgL<-1> of abscisic acid to realize preculture, stripping the stem tip, pre-culturing in an MS solid culture medium containing 0.4molL<-1> of sucrose and 2molL<-1> of glycerin, gently dipping in 2-4% calcium-free sodium alginate solution, putting on a loading strip, immersing in an MS liquid culture medium containing 0.1-0.3% of calcium chloride, 0.4molL<-1> of sucrose and 2molL<-1> of glycerin, immobilizing, sucking excessive liquid, treating in a pretreatment solution, transferring to a vitrification protective agent PVS2 to realize ice-bath treatment, and putting into liquid nitrogen to realize storage.The method has the advantages of simplicity, convenience and feasibility in storage, stability, reliability and favorable recovery growth conditions of stored wild rice.
Description
Technical field
The present invention relates to the store method of a kind of wild rice stem apex, specifically, relate to the cryopreservation method of a kind of common wild-rice stem apex.Meanwhile, the invention further relates to the renewal cultivation method after the Excised Embryos of wild rice stem apex, belong to field of plant cell engineering technology.
Background technology
Exploration of Wild Rice Germplasm Resources is the important composition part of Rice Resources, it saves the gene that rice-cultivating does not has or disappeared, and there is special merit and the characteristic of high disease and insect resistance, the excavation of its beneficial gene and preservation, to improving yield and quality of rice, there is immeasurable value.Current aggravating circumstances and disadvantageous weather conditions rapidly are corroding the genetic diversity of paddy rice；The economic activity of mankind's modern times explosion, result in wild rice habitat loss and deterioration, habitat is drastically reduced；In addition the biological characteristics that wild rice has again stigma appearing, outcrossing seed-setting rate is higher, seed shattering is strong.These factors make wild seed rice face the danger of extinction, and the protection to wild rice genetic diversity is very urgent.At present, the safeguard measure of wild rice is mainly had in-situ protection and in situ conservation, i.e. with the kind matter village, often used in village names of Seed storage, the field gene bank that preserves with kind of stem.
Plant tissue opens new approach with the preserving seed for plant of cultivating of cell, and tissue culture energy amount reproduction rapidly, regeneration plant can keep original hereditary capacity.And under Excised Embryos (-196 DEG C), the CBAC of plant is close to and stops, physiology in storage and heredity change can control in bottom line, avoid field preservation and easily caused blastation or destruction by Effect of Natural Disaster, and tissue cultures preserve in cause the danger of inhereditary feature variation or pollution it is considered to be the optimal selection that preserves for a long time of plant genetic resources because of repeatedly subculture.
Research about plant Excised Embryos, the variation of Techniques of preserving constantly makes progress with summary aspect, the plant species number that can preserve also increases constantly, but majority concentrates on the Excised Embryos that the economic plants such as the xylophytas such as apple, cherry, citrus, banana, cassava and potato, Ipomoea batatas, strawberry is main plant genetic resources.The Excised Embryos of paddy rice is after first Sala in 1979 etc. report the Excised Embryos of rice suspension cell, correlative study focuses mostly on tissue culture such as suspension cell line, callus, protoplast and embryoid, about frozen the reporting of paddy rice complete organ (such as stem apex), rarely seen professor Zhang Zhihong etc. used programmed cooling method that the adventitious bud that paddy rice monoploid children's fringe cultured in vitro is induced is carried out Excised Embryos in 2000, obtained regeneration plant.The Excised Embryos technology relevant report of wild rice is less and concentrates on domestic, as professor Yin Xiaohui wait 1996 in common wild-rice, wide leaf wild rice and the research of knurl grain Callus From Wild Rice Excised Embryos and obtain first freeze after regeneration plant, experiment demonstrates the effective way that the Excised Embryos of wild rice children's fringe isolated culture is Oryza resource conservation.Professors Tan Guangxuan etc. use programmed cooling method that wild rice children's fringe cultured in vitro is induced plant, directly carry out Excised Embryos, avoid callus phase and reduce the danger of hereditary variation, but the green bud produced after one week albefaction dead.
Wild rice complete organ's is frozen, to be the emphasis studied from now on, but owing to wild rice shoot apical meristem volume stem-tip tissue little, due to wild rice has tender feature tiny, young, physiological status is fragile, and by outer protective leaf tight layer by layer, strip at stem apex, preculture and glass frozen preservation operation easily snap off and come to harm, preservation is difficult to survive.Meanwhile, renewal cultivation behind is not easy to regeneration or produces Albino Seedling.The freezing method using programmed cooling is higher to equipment requirement.
Summary of the invention
Owing to existing in prior art, wild rice shoot apical meristem volume is little, physiological status is fragile, is difficult to the phenomenon survived in preservation.It is an object of the invention to provide the cryopreservation method of a kind of wild rice stem apex, also provide for a kind of defrosting renewal cultivation method after wild rice stem apex cryopreservation method simultaneously.
In order to realize the purpose of the present invention, the invention provides the cryopreservation method of a kind of wild rice stem apex, comprise the steps:
Take more than 3 months aseptic plantlet in vitro of subculture, cut off top plant, stay base portion about 3cm, root about 1cm to put into the MS culture medium without hormone.After cultivating 5-7 days, base portion new rudiment 0.5-1cm, will newly sprout after plant cuts off, band base portion proceeds to containing sucrose 0.3molL-1And abscisic acid 0.5-5mgL-1Culture medium on cultivate.After cultivating 7-10 days, proceed to 0.7molL-1And abscisic acid 0.5-5mgL-1Culture medium on preculture 3-7 days.Then the stem apex of 2-3mm is stripped under anatomical lens, containing 0.4 molL-1And glycerine 2moL-1MS solid medium on preculture 16 hours, wherein, containing 0.4 molL-1And glycerine 2moL-1MS solid medium in without NH4+, with tweezers, the stem apex after preculture is gently dipped in the sodium alginate soln without calcium ion of 2-4%, is the most neatly placed in the metal net shaped carrier strip of 5mm × 20mm, 5-10 stem apex in every carrier strip.Subsequently the carrier strip of band stem apex is immersed containing 0.1-0.3% calcium chloride, 0.4 molL-1Sucrose and 2moL-1The MS fluid nutrient medium of glycerine, wherein, containing 0.1-0.3% calcium chloride, 0.4 molL-1Sucrose and 2moL-1Without NH4+ in the MS fluid nutrient medium of glycerine, after 10-30min is fixing, carrier strip is sucked on aseptic filter paper surplus liquid.The carrier strip of the band stem apex fixed is put in preprocessing solution, and described preprocessing solution contains 0.4 molL-1And glycerine 2moL-1MS fluid nutrient medium, after processing 20-60min at room temperature in NH4+, the ice bath proceeding to carry out in vitrifying protective agent PVS2 2-5 hour processes, without NH4 in described PVS2+。
The carrier strip of band stem apex is put into rapidly in liquid nitrogen and preserve.
The present invention also provides for a kind of renewal cultivation method after wild rice stem apex Excised Embryos, comprises the steps:
From liquid nitrogen, take out the carrier strip of band stem apex when wild rice stem apex after Excised Embryos thaws, be immediately placed in 1.2molL-1, without NH4+1/2MS fluid nutrient medium in quick-thawing 2min, and wash 20min in the medium.The carrier strip of band stem apex is blotted on filter paper and is placed on without hormone with without NH4+1/2MS culture medium in light culture 1 day, then under anatomical lens, stem apex is peeled off by carrier strip one by one, transfers to containing BA and NAA, without NH4+Culture medium on carry out light culture.Proceed to after stem apex regenerates containing BA and NAA with without NH4+Culture medium illumination cultivation.
The invention has the beneficial effects as follows, the cryopreservation method of wild rice stem apex is simple and easy to do, reliable and stable, and after preservation, wild rice restoration ecosystem is in order.Simultaneously, by adding exogenous hormone and stepping up the resistance of both sucrose concentrations combination raising stem apex in pre-culture medium, by using metal grill as the carrier of stem apex to reduce the damage in each step, and use liquid nitrogen to deliver directly and the freezing step of non-dependent programmed cooling instrument, a great number of elements is adjusted being allowed to regenerating easily by recovery media, finally gives the regeneration plant of common wild-rice.For the Excised Embryos of other type of wild rice and grass genetic resources, there is good reference value.The most also improve the resistance of stem apex, reduce the damage in each step, and use liquid nitrogen to deliver directly and the freezing step of non-dependent programmed cooling instrument, simplify operation, set up the Excised Embryos system of common wild-rice, and make technological reserve for other type of wild rice and grass.
Accompanying drawing explanation
Fig. 1 is the flow chart of wild rice Excised Embryos in the present invention.
Fig. 2 is the flow chart of renewal cultivation method after wild rice Excised Embryos in the present invention.
Fig. 3 is the view of tufted seedling in the present invention.
Fig. 4 is the view taming seedling in the present invention.
Fig. 5 is the view of embedding process in the present invention.
Fig. 6 is the view of regrowth in the present invention.
Detailed description of the invention
In order to more clearly describe the detailed description of the invention of the present invention, below in conjunction with embodiment, the present invention is described further.
Embodiment 1The acquisition of wild rice aseptic seedling
With reference to shown in Fig. 1, induction and the subculture step of wild rice aseptic seedling be: takes the stem section of the subterranean stem band germinating bud of wild rice, after repeatedly rinsing about 1 hour under running water flowing water, with 70% alcohol disinfecting 1 min, the mercuric chloride of 0.1% is sterilized 15 min, aseptic water washing several times after, under anatomical lens, strip the stem apex of about 2mm, be inoculated in containing 4 mg L-1BA and 0.5 mg L-1On the MS culture medium of NAA, induced bundle is sprouted.Cultivation temperature is 25 ± 2 DEG C, illumination 12 h d-1, intensity of illumination 36 molm-2 s-1.After inducing Multiple Buds, Multiple Buds is cut off and is seeded in containing 2mg L-1BA and 0.5 mg L-1Squamous subculture on the MS culture medium of NAA.Every 3 months subcultures are once.
Embodiment 2The gradient preculture of wild rice
With reference to shown in Fig. 1, the gradient domestication of aseptic seedling is cultivated, peel off stem apex and the pre-incubated step of stem apex is: the plantlet in vitro of Example 1 gained, cut off top plant, stay base portion about 3cm, root about 1cm put into the MS medium culture without hormone after 7 days, takes base portion and newly sprouts the bud of 1cm, proceeds to containing sucrose 0.3molL-1And abscisic acid 5mgL-1Culture medium on cultivate.After 7 days, then proceed to 0.7molL-1And abscisic acid 5mgL-1Culture medium on preculture 5 days.The stem apex of 2-3mm is stripped, containing 0.4 molL under anatomical lens-1And glycerine 2moL-1, without NH4+MS solid medium on preculture 16 hours.
Embodiment 3The device of wild rice stem apex is fixed
With reference to shown in Fig. 1 and Fig. 5, stem apex is fixed to the step of carrier strip and pretreatment, vitrifying process: with tweezers by by the stem apex after embodiment 2 preculture, the sodium alginate soln without calcium ion of 2-4% gently dips in, the most neatly it is placed in the metal net shaped carrier strip of 5mm × 20mm, 10 stem apexs in every carrier strip.The carrier strip of band stem apex is immersed containing 0.2% calcium chloride, 0.4 molL-1Sucrose and 2moL-1Glycerine, without NH4+MS fluid nutrient medium in, after 20min is fixing, carrier strip is sucked on aseptic filter paper surplus liquid.
Embodiment 4The vitrifying of wild rice processes and frozen
With reference to shown in Fig. 1 and Fig. 5, the step of the Liquid nitrogen of stem apex is: the carrier strip of the band stem apex of Example 3 gained is put in preprocessing solution, containing 0.4 molL-1And glycerine 2moL-1, without NH4+MS fluid nutrient medium in process 60min at room temperature after, proceed to vitrifying protective agent PVS2, without NH4+In carry out 5 hours ice bath process, the carrier strip of band stem apex is put into rapidly in liquid nitrogen preserve.
Embodiment 5The stem apex renewal cultivation of wild rice and plant regeneration
With reference to shown in Fig. 2, Fig. 3, Fig. 4, Fig. 6, the required step of renewal cultivation method is, the first step: thaw rapidly；Second step: wash, blot；3rd step, stem apex renewal cultivation；4th step, stem apex is peeled off by carrier strip；5th step: stem apex regeneration is cultivated, the 6th step: tissue culture plant regenerates.
Concrete step is as follows: takes out the carrier strip of band stem apex during defrosting from liquid nitrogen, is immediately placed in 1.2molL-1, without NH4+1/2MS fluid nutrient medium in quick-thawing 2min, and wash 20min in the medium.The carrier strip of band stem apex being blotted on filter paper and be placed on without hormone, in the 1/2MS culture medium without NH4+, light culture 1 day, is then peeled off stem apex by carrier strip under anatomical lens one by one, transfers to containing BA and NAA, without NH4+Culture medium on light culture.Proceed to containing BA and NAA, containing NH4 after stem apex regenerates+Culture medium illumination cultivation.After stem apex renewal cultivation 3d, solving training Microscopic observation, the most visible have stem apex to regenerate, and after continuing to cultivate about 14～28 days, part survives stem apex brown stain, partial regeneration Cheng Lvmiao, and regeneration rate reaches as high as 50%.
Embodiment described above only have expressed embodiments of the present invention, and it describes more detailed, as long as those skilled in the art is after viewing embodiments of the invention, on the premise of present inventive concept, the change made broadly falls into protection scope of the present invention.But embodiment as herein described is it is not intended that limit protection scope of the present invention.
Claims (7)
1. the cryopreservation method of a wild rice stem apex, it is characterised in that described store method comprises the steps:
Step a: taking the squamous subculture aseptic plantlet in vitro of more than 3 months, cut off top plant, the MS culture medium staying base portion to put into without hormone is cultivated；
Step b: cultivated after 5-7 days by step a, newly sprouts after plant cuts off by base portion, and band base portion proceeds to containing 0.3molL-1Sucrose and abscisic acid 0.5-5mgL-1Culture medium on cultivate；
Step c: the plant after step b is cultivated 7-10 days, then proceed to 0.7molL-1Sucrose and the 0.5-5mgL Han abscisic acid-1Culture medium on preculture 3-7 days；
Step d: the plant after step c preculture 3-7 days strips the stem apex of 2-3mm under anatomical lens, containing 0.4 molL-1Sucrose and glycerine 2moL-1MS solid medium on preculture 16 hours, without NH in described MS solid medium4 +；
Step e: gently dipped in the sodium alginate soln without calcium ion of 2-4% by the stem apex after step d preculture with tweezers, is the most neatly placed in the metal net shaped carrier strip of 5mm × 20mm, 5-10 stem apex in every carrier strip；
Step f: the carrier strip of band stem apex is immersed containing 0.1-0.3% calcium chloride, 0.4 molL-1Sucrose and 2moL-1In the MS fluid nutrient medium of glycerine, after 10-30min is fixing, carrier strip is sucked on aseptic filter paper surplus liquid, without NH in described MS fluid nutrient medium4 +；
Step g: the carrier strip of the band stem apex fixed is put in after at room temperature processing 20-60min in preprocessing solution; the ice bath proceeding to carry out in vitrifying protective agent PVS2 2-5 hour processes; the carrier strip of band stem apex being put into rapidly in liquid nitrogen and preserve, wherein, described preprocessing solution contains 0.4 molL-1Sucrose and glycerine 2moL-1, without NH4 +MS fluid nutrient medium, described vitrifying protective agent PVS2 do not contain NH4 +。
The cryopreservation method of wild rice stem apex the most according to claim 1, it is characterised in that more than 3 months aseptic plantlet in vitro of the subculture in described step a, cuts off top plant, stays base portion about 3cm, root about 1cm to put into the MS culture medium without hormone.
The cryopreservation method of wild rice stem apex the most according to claim 1, it is characterised in that a length of 0.5-1cm of the new rudiment of base portion in step b.
4. the renewal cultivation method after a wild rice Excised Embryos, it is characterised in that described wild rice preserved through the cryopreservation method described in claim 1.
Renewal cultivation method the most according to claim 4, it is characterised in that take out the carrier strip of band stem apex during defrosting from liquid nitrogen, be immediately placed in 1.2molL-11/2MS fluid nutrient medium in quick-thawing 2min, and wash 20min in the medium, described 1.2molL-11/2MS fluid nutrient medium without NH4 +。
Renewal cultivation method the most according to claim 5, it is characterized in that, the carrier strip of band stem apex is blotted on filter paper light culture 1 day in the 1/2MS culture medium being placed on without hormone, then under anatomical lens, stem apex is peeled off by carrier strip one by one, transferring to carry out on the culture medium containing BA and NAA light culture, the described culture medium containing BA and NAA does not contains NH4 +。
Renewal cultivation method the most according to claim 6, it is characterised in that proceed to the culture medium illumination cultivation containing BA and NAA after the stem apex regeneration that claim 4 processed, proceeds to the culture medium containing BA and NAA containing NH after the regeneration of described stem apex4 +。
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106561637A (en) * | 2016-10-28 | 2017-04-19 | 上海市农业生物基因中心 | Cryopreservation and recovery culture methods for dioscorea alata protocrom-like bodies |
| CN109089879A (en) * | 2018-07-18 | 2018-12-28 | 云南省农业科学院生物技术与种质资源研究所 | A method of regeneration plant is obtained using Oryza eichingeri stem for explant |
| CN109380216A (en) * | 2018-10-30 | 2019-02-26 | 上海市农业生物基因中心 | A kind of cryopreservation and renewal cultivation method of oryza officinalis stem apex |
| CN109601378A (en) * | 2018-11-30 | 2019-04-12 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of Cryopreservation and breeding method of sugar-cane tissue culture seedlings provenance |
| CN110800734A (en) * | 2019-11-21 | 2020-02-18 | 上海市农业生物基因中心 | Ultra-low temperature preservation method for wild rice stem tips |
| CN115843687A (en) * | 2022-12-06 | 2023-03-28 | 上饶师范学院 | Method for promoting expansion of test-tube taro by detoxification of red-bud taro through embedding vitrification ultra-low temperature therapy |
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2016
- 2016-03-09 CN CN201610131944.1A patent/CN105746351B/en not_active Expired - Fee Related
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106561637A (en) * | 2016-10-28 | 2017-04-19 | 上海市农业生物基因中心 | Cryopreservation and recovery culture methods for dioscorea alata protocrom-like bodies |
| CN109089879A (en) * | 2018-07-18 | 2018-12-28 | 云南省农业科学院生物技术与种质资源研究所 | A method of regeneration plant is obtained using Oryza eichingeri stem for explant |
| CN109380216A (en) * | 2018-10-30 | 2019-02-26 | 上海市农业生物基因中心 | A kind of cryopreservation and renewal cultivation method of oryza officinalis stem apex |
| CN109601378A (en) * | 2018-11-30 | 2019-04-12 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of Cryopreservation and breeding method of sugar-cane tissue culture seedlings provenance |
| CN109601378B (en) * | 2018-11-30 | 2021-08-24 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Low-temperature preservation method and cultivation method for seed source of sugarcane tissue culture seedling |
| CN110800734A (en) * | 2019-11-21 | 2020-02-18 | 上海市农业生物基因中心 | Ultra-low temperature preservation method for wild rice stem tips |
| CN115843687A (en) * | 2022-12-06 | 2023-03-28 | 上饶师范学院 | Method for promoting expansion of test-tube taro by detoxification of red-bud taro through embedding vitrification ultra-low temperature therapy |
| CN115843687B (en) * | 2022-12-06 | 2023-09-01 | 上饶师范学院 | Method for promoting expansion of test tube taro through embedded vitrification ultralow temperature therapy detoxification of red bud taro |
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