CN101622959A - Method for large-scale production of jatropha curcas plants by cell-tissue culture - Google Patents
Method for large-scale production of jatropha curcas plants by cell-tissue culture Download PDFInfo
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- CN101622959A CN101622959A CN200910094822A CN200910094822A CN101622959A CN 101622959 A CN101622959 A CN 101622959A CN 200910094822 A CN200910094822 A CN 200910094822A CN 200910094822 A CN200910094822 A CN 200910094822A CN 101622959 A CN101622959 A CN 101622959A
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- 238000000034 method Methods 0.000 title claims abstract description 18
- 241001048891 Jatropha curcas Species 0.000 title claims abstract description 13
- 238000011031 large-scale manufacturing process Methods 0.000 title claims abstract description 8
- 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 abstract description 17
- 229930006000 Sucrose Natural products 0.000 claims abstract description 17
- 239000005720 sucrose Substances 0.000 claims abstract description 17
- 238000004114 suspension culture Methods 0.000 claims abstract description 14
- 230000010261 cell growth Effects 0.000 claims abstract description 9
- 238000005259 measurement Methods 0.000 claims abstract description 9
- 238000000227 grinding Methods 0.000 claims abstract description 7
- 230000000284 resting effect Effects 0.000 claims abstract description 7
- 239000006071 cream Substances 0.000 claims description 48
- 244000055346 Paulownia Species 0.000 claims description 46
- 239000007788 liquid Substances 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 17
- 239000006285 cell suspension Substances 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- 239000004744 fabric Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 2
- 230000002062 proliferating effect Effects 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 18
- 238000004113 cell culture Methods 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract 3
- 238000012258 culturing Methods 0.000 abstract 2
- 230000002349 favourable effect Effects 0.000 abstract 1
- 230000012010 growth Effects 0.000 description 9
- 235000003140 Panax quinquefolius Nutrition 0.000 description 4
- 240000005373 Panax quinquefolius Species 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 239000012531 culture fluid Substances 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 238000013459 approach Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000026010 Dendrobium candidum Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 240000001866 Vernicia fordii Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003225 biodiesel Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000010687 lubricating oil Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
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- 238000004904 shortening Methods 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for the large-scale production of jatropha curcas plants by cell-tissue culture, belonging to the culture technology of plant seedlings. The method comprises the following steps: (1) cell suspension culture: grinding and inoculating jatropha curcas tissue and adding a cell suspension culture substrate: 0.2-0.5 mg/L of MS+BA, 1-6 mg/L of NAA, 0.5-1.5 mg/L of LH and 19-22 g/L of sucrose; (2) cell growth measurement; (3) clumpy bud multiplication culture: selecting cells growing into a resting stage to access a clumpy bud multiplication culture substrate: 0.1-1 mg/L of MS+6-BA, 0.1-1.5 mg/L and 20-30 g/L of sucrose; (4) rooting culture: selecting well-growing clumpy buds to access a rooting culture substrate: 0.1-1 mg/L of MS+NAA; and (5) bottling culture. The invention organically combines the cell culture and the tissue culture of jatropha curcas and has the advantages of short period and low cost, provides a high-efficiency path for manually culturing the jatropha curcas, producing virus-free seedlings and culturing fine varieties and has a favorable prospect for developing and utilizing the jatropha curcas.
Description
Technical field
The invention belongs to the culture technique of plant seedling, be specifically related to method by cell culture and tissue culture large-scale production jatropha curcas plants.
Background technology
Cream paulownia (Jatropha curcas L.) has another name called Jatropha curcas, the little seeds of a tung oil tree, is distributed in the torrid zone, subtropical zone, and there are cultivation or semi-wild in China.The cream paulownia is shrub or dungarunga, high 2-5m, and multi-branched, speed is given birth to, happiness fertile soil earth, acid soil or calcium soil all should be grown, anti-about 0 ℃ low temperature and light frost, wind resistance power is strong.The many cultivations of cream paulownia were in the past made fences and for viewing and admiring, and planting benevolence, to contain grease about 50%, and can make usefulness such as lubricating oil, soapmaking, and can emeticly drop, but poisonous, diet.In recent years, industrially be used for various diesel engines as biodiesel, so the large-scale production jatropha curcas plants has important function to the development of bioenergy with the cream Oleum Verniciae fordii or through the cream Oleum Verniciae fordii of modification.
Cultivation or seminatural cream paulownia propagation method generally are that the breeding cycle is long by seminal propagation or cottage propagation now, the cost height.Plant Tissue Breeding is exactly the part tissue of separating plant body, as root, stem section, leaf, flower, rataria etc., in sterile test tube, cooperates conditions such as certain nutrition, hormone, temperature, illumination, makes it produce whole plant.Because its condition can strict be controlled, growth is one-period about one month rapidly, therefore in the production of plant significant application value is arranged.The patent No. can reduce cost for 200510048718.9 Chinese invention patent provides a kind of method of passing through tissue culture large-scale production cream paulownia seedling in the shortening cycle, but its reproduction speed also has the necessary and possibility that further improves.The a large amount of culture techniques of plant cell are to grow up on the basis that Plant Tissue Breeding is bred fast.Specific practice is exactly that plant cell is transferred in the large fermentation tank of microbial fermentation by triangular flask from test tube, gives suitable condition and cultivates, and makes plant cell as microorganism breeding in a large number in fermentation tank, but cell culture processes cost height.Though being 03135614.1 Chinese patent file, application number announced a kind of " cultural method of cell-tissue ",, reduced and cultivate cost that its technical scheme can only be used for dendrobium candidum in the hope of improving the rate of increase.
Summary of the invention
The object of the present invention is to provide a kind of method of large-scale production of jatropha curcas plants by cell-tissue culture, the advantage that had both had cell culture and tissue culture, can overcome cell culture and tissue culture shortcoming again, for artificial propagation, the detoxic seedling production of cream paulownia, cultivate improved seeds and open up approach efficiently.
The inventive method may further comprise the steps:
1, cream paulownia cell suspension cultures: cream paulownia tissue is ground the back inoculation, the cell suspension liquid medium that adds 1～3 times of weight, container is put into rotary shaking table, in 25 ± 2 ℃ of temperature, carried out cream paulownia cell suspension cultures under the dark condition 2～4 days, shaking speed is 80～130r/min, and cell suspension liquid medium is: MS+BA 0.2～0.5mg/L+NAA 1～6mg/L+LH 0.5～1.5mg/L+ sucrose 19～22g/L, pH value 5.8～6.The inkstone body is preferably used in the grinding of cream paulownia tissue.
2, the cream paulownia cell of cultivating is carried out the cell growth measurement.Proliferating cells suspension can be poured into the cloth formula funnel of filter paper, take out and train liquid, use distilled water wash again, carry out the cell growth measurement then.
3, clump bud enrichment culture: choose the cream paulownia cell that grows into resting stage, insert the clump bud proliferated culture medium of solid, intensity of illumination 1000～2000LX, clump bud proliferated culture medium is: MS+6-BA 0.1～1mg/L+NAA 0.1～1.5mg/L+ sucrose 20～30g/L, with mass ratio is that 0.5%～0.8% agar solidifies, pH value 5.8～6, incubation time are 15～20 days.Best clump bud proliferated culture medium is: MS+6-BA 0.9～1mg/L+NAA 0.4～0.6mg/L+ sucrose 20～30g/L.
4, culture of rootage: select well-grown clump of bud to insert root media and carry out culture of rootage, produce cream paulownia seedling at last, root media is: MS+NAA 0.1～1mg/L.Best root media is: MS+NAA 0.4～0.6mg/L.
5, bottle outlet cultivation: the seedling of bottle outlet cultivation was preserved moisture earlier 15～20 days, kept temperature to be not less than 17 ℃.
The cream paulownia tissue cultivating seedling that the inventive method generates can direct transplanting to nursery lot, need on the seedbed, not practice seedling.
Beneficial effect of the present invention: the present invention organically combines cream paulownia cell culture and tissue culture, give full play to the advantage of cell culture and tissue culture, cycle is short, cost is low, can satisfy cream paulownia commerial growing needs fully, for artificial propagation, the detoxic seedling production of cream paulownia, cultivating improved seeds provides a kind of efficient approach, and the development and use of cream paulownia are had good prospect.
Embodiment
See following embodiment:
Embodiment 1:
1, cream paulownia explant selection and cultivation: choose cream paulownia stem section, behind the liquid detergent wash clean, under flowing water, wash 10min, forward on the clean bench in the future, suck dry moisture earlier with 70% (volume ratio) alcohol sterilization 30 seconds, changes the HgCl of 0.1% (volume ratio) over to again
2Sterilization is 5 minutes in the solution, and sterile distilled water flushing 5 times is cut into the long segment of 1cm with the stem section, be inoculated in callus inducing medium MS+6-BA2mg/L+NAAl mg/L+ sucrose 30g/L, solidify with 0.8% (mass ratio) agar, pH is adjusted to 5.8,25 ℃ of cultivation temperature before the high-temperature sterilization.The explant inoculation is visible obviously bud after 20 days.
2, cream paulownia cell suspension cultures: get the bud of above-mentioned cultivation, in grinding body, add earlier a little cell suspension body medium and grind inoculation.With the cream paulownia tissue after Sheng is ground in the triangular flask and the cell suspension liquid medium of 2 times of weight, put into rotary shaking table, in 25 ℃ of temperature, carried out cream paulownia cell suspension cultures under the dark condition 2 days, shaking speed is 125r/min, cell suspension liquid medium is: MS+BA 0.2mg/L+NAA2mg/L+LH 0.5mg/L+ sucrose 20g/L, pH value 6.
3, the cell suspending liquid after will cultivating is poured the cloth formula funnel of filter paper into, takes out culture fluid, uses the distilled water wash several times again, drains again, carries out the cell growth measurement, and is standby.
4, clump bud enrichment culture: choose the cream paulownia cell that grows into resting stage, insert the clump bud proliferated culture medium of solid, intensity of illumination 1000LX, clump bud proliferated culture medium is: MS+6-BA 1mg/L+NAA 0.5mg/L+ sucrose 25g/L, solidify pH value 5.8 with 0.8% agar.When plant strain growth moves to plant in the root media to about 2cm, root media is: MS+NAA 0.5mg/L.
5, bottle outlet is transplanted: when about the long 0.5cm of root, during blade length to 3～5 slice, the seedling of will taking root directly moves to nursery lot, first moisture-heat preservation 15 days, and the maintenance temperature is not less than 17 ℃, and seedling growth is good subsequently.
Embodiment 2:
1, cream paulownia explant selection and cultivation are with embodiment 1.
2, cream paulownia cell suspension cultures: get the bud of above-mentioned cultivation, in grinding body, add earlier a little cell suspension body medium and grind inoculation.With the cream paulownia tissue after Sheng is ground in the triangular flask and the cell suspension liquid medium of 2 times of weight, put into rotary shaking table, in 25 ℃ of temperature, carried out cream paulownia cell suspension cultures under the dark condition 2 days, shaking speed is 100r/min, cell suspension liquid medium is: MS+BA 0.5mg/L+NAA6mg/L+LH 1.5mg/L+ sucrose 22g/L, pH value 5.8.
3, the cell suspending liquid after will cultivating is poured the cloth formula funnel of filter paper into, takes out culture fluid, uses the distilled water wash several times again, drains again, carries out the cell growth measurement, and is standby.
4, clump bud enrichment culture: choose the cream paulownia cell that grows into resting stage, insert the clump bud proliferated culture medium of solid, intensity of illumination 1800LX, clump bud proliferated culture medium is: MS+6-BA 0.2mg/L+NAA1.5mg/L+ sucrose 30g/L, solidify pH value 5.8 with 0.6% agar.When plant strain growth moves to plant in the root media to about 2cm, root media is: MS+NAA 0.2mg/L.
5, bottle outlet is transplanted with embodiment 1.Seedling growth is good.
Embodiment 3:
1, cream paulownia explant selection and cultivation are with embodiment 1.
2, cream paulownia cell suspension cultures: get the bud of above-mentioned cultivation, in grinding body, add earlier a little cell suspension body medium and grind inoculation.With the cream paulownia tissue after Sheng is ground in the triangular flask and the cell suspension liquid medium of times weight, put into rotary shaking table, in 27 ℃ of temperature, carried out cream paulownia cell suspension cultures under the dark condition 3 days, shaking speed is 130r/min, and cell suspension liquid medium is: MS+BA 0.2mg/L+NAA 1mg/L+LH 0.5mg/L+ sucrose 19g/L.
3, the cell suspending liquid after will cultivating is poured the cloth formula funnel of filter paper into, takes out culture fluid, uses the distilled water wash several times again, drains again, carries out the cell growth measurement, and is standby.
4, clump bud enrichment culture: choose the cream paulownia cell that grows into resting stage, insert the clump bud proliferated culture medium of solid, intensity of illumination 1200LX, clump bud proliferated culture medium is: MS+6-BA 1mg/L+NAA0.2mg/L+ sucrose 20g/L, solidify pH value 6 with 0.5% agar.When plant strain growth moves to plant in the root media to about 2cm, root media is: MS+NAA 0.8mg/L.
5, bottle outlet is transplanted with embodiment 1.Seedling growth is good.
Embodiment 4:
1, cream paulownia explant selection and cultivation are with embodiment 1.
2, cream paulownia cell suspension cultures: get the bud of above-mentioned cultivation, in grinding body, add earlier a little cell suspension body medium and grind inoculation.With the cream paulownia tissue after Sheng is ground in the triangular flask and the cell suspension liquid medium of 1 times of weight, put into rotary shaking table, in 23 ℃ of temperature, carried out cream paulownia cell suspension cultures under the dark condition 3 days, shaking speed is 80r/min, and cell suspension liquid medium is: MS+BA 0.5mg/L+NAA 6mg/L+LH 1.5mg/L+ sucrose 22g/L.
3, the cell suspending liquid after will cultivating is poured the cloth formula funnel of filter paper into, takes out culture fluid, uses the distilled water wash several times again, drains again, carries out the cell growth measurement, and is standby.
4, clump bud enrichment culture: choose the cream paulownia cell that grows into resting stage, insert the clump bud proliferated culture medium of solid, intensity of illumination 1900LX, clump bud proliferated culture medium is: MS+6-BA 0.2mg/L+NAA 1.5mg/L+ sucrose 30g/L, solidify pH value 6 with 0.8% agar.When plant strain growth moves to plant in the root media to about 2cm, root media is: MS+NAA 0.5mg/L.
5, bottle outlet is transplanted: with embodiment 1.Seedling growth is good.
Above embodiment only is described further invention, and scope of the present invention is not subjected to the limitation of illustrated embodiment.
Claims (5)
1, a kind of method of large-scale production of jatropha curcas plants by cell-tissue culture is characterized in that may further comprise the steps:
(1), cream paulownia cell suspension cultures: cream paulownia tissue is ground the back inoculation, the cell suspension liquid medium that adds 1～3 times of weight, container is put into rotary shaking table, in 25 ± 2 ℃ of temperature, carried out cream paulownia cell suspension cultures under the dark condition 2～4 days, shaking speed is 80～130r/min, and cell suspension liquid medium is: MS+BA 0.2～0.5mg/L+NAA 1～6mg/L+LH 0.5～1.5mg/L+ sucrose 19～22g/L, pH value 5.8～6;
(2), the cream paulownia cell of cultivating is carried out the cell growth measurement;
(3), clump bud enrichment culture: choose the cream paulownia cell that grows into resting stage, insert the clump bud proliferated culture medium of solid, intensity of illumination 1000～2000LX, clump bud proliferated culture medium is: MS+6-BA 0.1～1mg/L+NAA 0.1～1.5mg/L+ sucrose 20～30g/L, with mass ratio is that 0.5%～0.8% agar solidifies, pH value 5.8～6, incubation time are 15～20 days;
(4), culture of rootage: select well-grown clump of bud to insert root media and carry out culture of rootage, produce cream paulownia seedling at last, root media is: MS+NAA 0.1～1mg/L;
(5), bottle outlet cultivation: the seedling of bottle outlet cultivation was preserved moisture earlier 15～20 days, kept temperature to be not less than 17 ℃.
2,, it is characterized in that the inkstone body is used in the grinding of cream paulownia tissue as the said method of claim 1.
3, as the said method of claim 1, it is characterized in that proliferating cells suspension is poured into the cloth formula funnel of filter paper, take out and train liquid, use distilled water wash again, carry out the cell growth measurement then.
4,, it is characterized in that clump bud proliferated culture medium is: MS+6-BA0.9～1mg/L+NAA 0.4～0.6mg/L+ sucrose 20～30g/L as the said method of claim 1.
5,, it is characterized in that root media is: MS+NAA 0.4～0.6mg/L as the said method of claim 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773128A (en) * | 2010-02-03 | 2010-07-14 | 中国科学院西双版纳热带植物园 | Special growth regulating agent for jatropha and application thereof |
| CN103125397A (en) * | 2013-03-18 | 2013-06-05 | 南京斯摩尼生物科技有限公司 | Culture medium for generating and growing medicinal woody plant callus and application thereof |
| CN114058563A (en) * | 2020-07-31 | 2022-02-18 | 伽蓝（集团）股份有限公司 | Composition for suspension culture of nardostachys chinensis bunge cells and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100344224C (en) * | 2005-12-22 | 2007-10-24 | 云南大学 | Method for mass production of seedling of Jatropha curcas L. |
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2009
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773128A (en) * | 2010-02-03 | 2010-07-14 | 中国科学院西双版纳热带植物园 | Special growth regulating agent for jatropha and application thereof |
| CN101773128B (en) * | 2010-02-03 | 2013-11-27 | 中国科学院西双版纳热带植物园 | Special growth regulating agent for jatropha and application thereof |
| CN103125397A (en) * | 2013-03-18 | 2013-06-05 | 南京斯摩尼生物科技有限公司 | Culture medium for generating and growing medicinal woody plant callus and application thereof |
| CN103125397B (en) * | 2013-03-18 | 2014-06-11 | 南京斯摩尼生物科技有限公司 | Culture medium for generating and growing medicinal woody plant callus and application thereof |
| CN114058563A (en) * | 2020-07-31 | 2022-02-18 | 伽蓝（集团）股份有限公司 | Composition for suspension culture of nardostachys chinensis bunge cells and application thereof |
| CN114058563B (en) * | 2020-07-31 | 2024-07-02 | 上海自然堂集团有限公司 | Composition for suspension culture of nardostachys chinensis cells and application thereof |
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