CN104969857A - Method for prolonging preservation time of grape tissue culture seedlings by covering with mineral oil - Google Patents
Method for prolonging preservation time of grape tissue culture seedlings by covering with mineral oil Download PDFInfo
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- CN104969857A CN104969857A CN201410147430.6A CN201410147430A CN104969857A CN 104969857 A CN104969857 A CN 104969857A CN 201410147430 A CN201410147430 A CN 201410147430A CN 104969857 A CN104969857 A CN 104969857A
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- tissue culture
- grape
- mineral oil
- seedling
- culture seedlings
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- 235000009754 Vitis X bourquina Nutrition 0.000 title claims abstract description 43
- 235000012333 Vitis X labruscana Nutrition 0.000 title claims abstract description 43
- 235000014787 Vitis vinifera Nutrition 0.000 title claims abstract description 43
- 239000002480 mineral oil Substances 0.000 title claims abstract description 33
- 235000010446 mineral oil Nutrition 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000004321 preservation Methods 0.000 title abstract description 11
- 240000006365 Vitis vinifera Species 0.000 title 1
- 241000219095 Vitis Species 0.000 claims abstract description 42
- 238000000338 in vitro Methods 0.000 claims abstract description 21
- 238000005286 illumination Methods 0.000 claims abstract description 6
- 238000011081 inoculation Methods 0.000 claims abstract description 6
- 239000012467 final product Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- 239000008272 agar Substances 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 3
- 239000012879 subculture medium Substances 0.000 abstract description 2
- 238000007789 sealing Methods 0.000 abstract 2
- 238000012258 culturing Methods 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 230000002035 prolonged effect Effects 0.000 abstract 1
- 238000011084 recovery Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 12
- 241000196324 Embryophyta Species 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 230000008020 evaporation Effects 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 230000003111 delayed effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 241000220317 Rosa Species 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 239000007954 growth retardant Substances 0.000 description 2
- 235000006286 nutrient intake Nutrition 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 230000000979 retarding effect Effects 0.000 description 2
- 238000012090 tissue culture technique Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000005253 cladding Methods 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000009605 growth rhythm Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000009394 selective breeding Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for prolonging the storage time of grape tissue culture seedlings by covering with mineral oil, which comprises the steps of removing leaves from young shoots of the grape tissue culture seedlings, cutting the grape tissue culture seedlings into single-shoot stem sections with the length of about 1.5cm, inoculating into a subculture medium, covering sterile mineral oil with the length of about 5-6cm on the surface of the culture medium, sealing by a sealing film, and culturing under conventional culture conditions (the temperature is 25 +/-3 ℃, the illumination intensity is about 2000lx, and the illumination time is 10-14 h/d), so that the storage time of the grape tissue culture seedlings can be prolonged from 4 months to more than 15 months, the workload of subculture inoculation is greatly reduced, and the variation probability is also reduced. After the mineral oil is removed, the tissue culture seedlings can be restored to grow without being washed. The technology is applied to the germplasm preservation of the grape tissue culture seedlings, has wide applicable varieties, long preservation time, low cost of repeated utilization of mineral oil, simple and convenient operation and easy growth recovery, and is a good method for in vitro preservation of the grape germplasm at present.
Description
Technical field
The present invention relates to a kind of method utilizing tissue culture technique to carry out Grape Germplasm resource Plantlet in vitro.
Background technology
Due to reasons such as population growth, industrial pollution, environmental deteriorations, some rare species face extinction or are on the brink of extinction, add the popularization of artificial selection and breeding, varieties of plant unification, much useful precious germ plasm resource is lost, and Preservation of plant germplasin has become the problem of global concern.
Plantlet in vitro germ plasm resource is that plant explants is carried out tissue cultures preservation in an aseptic environment, has very high reproduction coefficient, can by preservation material amount reproduction when needing; The shortcoming of field preservation method can be overcome, not land occupation, remove field management from; From natural calamity invasion and attack and sick worm, virus harm; Be easy to control and be convenient to kind of matter exchange; Become the important method of Preservation of plant germplasin, be subject to the great attention of International Plant circle.
In the process of tissue cultures conserving species matter, due to the evaporation of medium moisture, nutrient consumption, at set intervals, will shift culture in time and be inoculated in new medium, carry out squamous subculture.Tissue culture the seedling of grape general 2 months subcultures once, the longlyest preserve about 4 months, frequent subculture will consume a large amount of human and material resources and time, with the increase of subculture number, somaclonal variation probability also can be caused to increase, likely make the original species matter of preservation lose.By changing culture growth conditions, the growth rhythm of regulation and control plantlet in vitro, delays its growth, effectively can reduce subculture number, improvement germ plasm resource in-vitro conservation method.
The main path delaying plantlet in vitro growth has: add growth retardant in the medium; Improve medium osmotic pressure; Reduce cultivation temperature, change intensity of illumination; Reduce the oxygen content etc. of culture environment.The Woody Plantlets in vitros such as apple put into refrigerating box, and after reduction cultivation temperature, the effect of retarding of growing is fine, but grape cold hardness is poor, and most of kind can not adapt to the condition of less than 10 DEG C refrigerating boxes for a long time.Grape Plantlets in Vitro can be effectively suppressed by adding the growth retardants such as PP333 in the medium or adding mannitol raising medium osmotic pressure, but cultivate due under room temperature, joint filling material is breathed freely, and totally, Tissue culture the seedling of grape can only extend the holding time of about 3 months to the evaporation of medium moisture.The present invention utilizes mineral oil to cover Tissue culture the seedling of grape, effectively inhibits medium moisture to evaporate, and by reducing the oxygen content of culture environment, having delayed plantlet in vitro growth, having extended the holding time.
Summary of the invention
The present invention establishes a kind of method extending the Tissue culture the seedling of grape holding time, for the Plantlet in vitro effectively utilizing tissue culture technique to carry out Grape Germplasm resource provides technical support.
The technical solution adopted for the present invention to solve the technical problems is: tender for the Tissue culture the seedling of grape tip is removed blade, be cut into stem-segment with single bud inoculation, pour sterile mineral oil in media surface and cover plantlet in vitro, conventional culture conditions is cultivated, and effectively can extend the holding time of Tissue culture the seedling of grape.
Particular content of the present invention, the concrete grammar step namely extending the Tissue culture the seedling of grape holding time is as follows:
(1) prepare grape subculture medium: B5+IAA0.5mg/L+ sucrose 25.0g/L+ agar 6.0g/L, before medium sterilization, regulate pH6.0; Culture vessel adopts 50ml or 100ml triangular flask, saves mineral oil, can select 50ml triangular flask if consider.
(2) tender for the Tissue culture the seedling of grape tip is removed blade, be cut into the stem-segment with single bud of about 1.5cm, in access medium; Pour sterile mineral oil in media surface, mineral oil liquid level exceeds media surface 5-6cm, and 50ml triangular flask about needs 40ml, and 100ml triangular flask about needs 80ml, seals with sealed membrane.
(3) conventional culture conditions: temperature 25 ± 3 DEG C, about intensity of illumination 2000lx, light application time 10-14h/d.
(4) Tissue culture the seedling of grape covering mineral oil generally can preserve more than 15 months.
(5), after removing mineral oil, without the need to rinsing, the plantlet in vitro of preservation gets final product restoration ecosystem.
The beneficial effect of patent of the present invention is, covered by mineral oil, significantly reduce the evaporation of medium moisture, reduce the oxygen content of culture environment, Grape Plantlets in Vitro and nutrient consumption are effectively delayed, the holding time of Tissue culture the seedling of grape was extended to more than 15 months by 4 months by success, greatly reduced the workload of subinoculation, also reduced variation probability.This technology can be applicable to the grape variety plantlet in vitro such as huge rose, Mo Lisha, Ju Feng, summer black, grain grain spy, imperial summer, imperial family autumn, crith are gloomy, Cabernet Sauvignon, this Witter red, have that adapted breed is wide, the holding time is long, the advantage such as cost low (mineral oil can reuse), easy and simple to handle, easy restoration ecosystem, be the good method of current grape kind matter Plantlet in vitro.
Accompanying drawing explanation
Fig. 1 inoculates the Tissue culture the seedling of grape of rear covering mineral oil.
Fig. 2 saves the Tissue culture the seedling of grape of 15 months.
Embodiment
Covered by mineral oil, decrease the evaporation of medium moisture, reduce the oxygen content of culture environment, effectively delayed Grape Plantlets in Vitro, successfully extend the holding time of Tissue culture the seedling of grape.Through verification experimental verification, specific implementation process will note following problem: preferably remove the tender tip blade during inoculation of (1) Tissue culture the seedling of grape, only stay the stem-segment with single bud that about 1.5cm is long, axillalry bud is sprouted slowly in mineral oil, shortened internodes, and blade obviously reduces, take root late, root is thin and short and small, and without side root, retarding of growing is effective.(2) will cover sterile mineral oil immediately after inoculation, if first cultivate a period of time after inoculation, after axillary bud sprouting growth, cover mineral oil again, easily cause the very fast withertip of plantlet in vitro, death, the holding time is short.(3) cover mineral oil and preferably once add enough amounts, if only exceed media surface 2-3cm, after plantlet in vitro 5-6 month, namely grow mineral oil liquid level, beginning restoration ecosystem, continue survival i.e. withered death in 3-4 month.Therefore mineral oil should be added as far as possible, generally exceeds media surface 5-6cm, will to triangular flask bottleneck, and 50ml triangular flask about needs 40ml, and 100ml triangular flask about needs 80ml, seals with sealed membrane.If it is inadequate to start mineral oil dosage, can add when tissue culture sprout quick grows liquid level, just not only bother, but also increase the probability of band fungi pollution.Under conventional culture conditions, mineral oil cladding process successfully saves tens grape groups such as huge rose, Mo Lisha, Ju Feng, summer black, grain grain spy, imperial summer, imperial family autumn, crith are gloomy, Cabernet Sauvignon, this Witter red and cultivates matter and part tissue culture flower seedling reaches more than 15 months.
Claims (5)
1. one kind utilizes mineral oil to cover the method extending the Tissue culture the seedling of grape holding time, tender for the Tissue culture the seedling of grape tip is it is characterized in that to remove blade, be cut into the stem-segment with single bud of about 1.5cm, in the medium of access B5+IAA0.5mg/L+ sucrose 25.0g/L+ agar 6.0g/L; Pour sterile mineral oil in media surface, mineral oil exceeds media surface 5-6cm, and 50ml triangular flask about needs 40ml, and 100ml triangular flask about needs 80ml, seals with sealed membrane; Condition of culture is: temperature 25 ± 3 DEG C, about intensity of illumination 2000lx, light application time 10-14h/d; When needs restoration ecosystem, poured out by mineral oil in superclean bench, without the need to rinsing, plantlet in vitro gets final product restoration ecosystem, easy and simple to handle, is applicable to most grape variety.
2. the method for the prolongation Tissue culture the seedling of grape holding time according to right 1, does not stay blade when it is characterized in that Tissue culture the seedling of grape is inoculated, and is cut into the stem-segment with single bud of about 1.5cm.
3. the method for the prolongation Tissue culture the seedling of grape holding time according to right 1, after it is characterized in that Tissue culture the seedling of grape inoculation, cover the sterile mineral oil exceeding media surface 5-6cm immediately, 50ml triangular flask about needs 40ml, and 100ml triangular flask about needs 80ml.
4. the method for the prolongation Tissue culture the seedling of grape holding time according to right 1, it is characterized in that the Tissue culture the seedling of grape covering mineral oil is in conventional condition of tissue culture (temperature 25 ± 3 DEG C, about intensity of illumination 2000lx, light application time 10-14h/d) under cultivate the holding time can be extended to more than 15 months by 4 months.
5. the method for the prolongation Tissue culture the seedling of grape holding time according to right 1, it is characterized in that mineral oil covers the plantlet in vitro preserved when needing restoration ecosystem, mineral oil is poured out in superclean bench, without the need to rinsing, plantlet in vitro gets final product restoration ecosystem, easy and simple to handle, be applicable to most grape variety.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410147430.6A CN104969857A (en) | 2014-04-14 | 2014-04-14 | Method for prolonging preservation time of grape tissue culture seedlings by covering with mineral oil |
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| CN201410147430.6A CN104969857A (en) | 2014-04-14 | 2014-04-14 | Method for prolonging preservation time of grape tissue culture seedlings by covering with mineral oil |
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| CN104969857A true CN104969857A (en) | 2015-10-14 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739393A (en) * | 2018-06-13 | 2018-11-06 | 河北农业大学 | Method for prolonging preservation time of grape tissue culture seedlings |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1252216A (en) * | 1998-10-23 | 2000-05-10 | 中国科学院遗传研究所 | Test-tube grape seedling propagating method and the culture medium used |
| CN102763593A (en) * | 2012-07-16 | 2012-11-07 | 福建省农业科学院农业工程技术研究所 | Method for rapidly obtaining loose calluses of grapes and for long-term succeeding maintenance of grapes |
-
2014
- 2014-04-14 CN CN201410147430.6A patent/CN104969857A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1252216A (en) * | 1998-10-23 | 2000-05-10 | 中国科学院遗传研究所 | Test-tube grape seedling propagating method and the culture medium used |
| CN102763593A (en) * | 2012-07-16 | 2012-11-07 | 福建省农业科学院农业工程技术研究所 | Method for rapidly obtaining loose calluses of grapes and for long-term succeeding maintenance of grapes |
Non-Patent Citations (4)
| Title |
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| 涂艺声: "《经济植物大规模快速繁殖技术》", 31 March 2009, 化学工业出版社 * |
| 王彦立等: ""葡萄叶盘法基因转化中抗生素种类和浓度的筛选"", 《中国农学通报》 * |
| 赵密珍: ""草莓茎尖离体保存研究"", 《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》 * |
| 钟兰等: ""植物种质资源离体保存技术研究进展"", 《长江蔬菜（学术版）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739393A (en) * | 2018-06-13 | 2018-11-06 | 河北农业大学 | Method for prolonging preservation time of grape tissue culture seedlings |
| CN108739393B (en) * | 2018-06-13 | 2021-10-08 | 河北农业大学 | Method for prolonging preservation time of grape tissue culture seedlings |
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Application publication date: 20151014 |