CN101658139B - Direct regeneration method of leaf discs of barbadosnut - Google Patents
Direct regeneration method of leaf discs of barbadosnut Download PDFInfo
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- CN101658139B CN101658139B CN2009103078134A CN200910307813A CN101658139B CN 101658139 B CN101658139 B CN 101658139B CN 2009103078134 A CN2009103078134 A CN 2009103078134A CN 200910307813 A CN200910307813 A CN 200910307813A CN 101658139 B CN101658139 B CN 101658139B
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- 241001048891 Jatropha curcas Species 0.000 title claims abstract description 33
- 238000011069 regeneration method Methods 0.000 title abstract description 14
- 239000008272 agar Substances 0.000 claims abstract description 11
- 239000005720 sucrose Substances 0.000 claims abstract description 11
- 238000005286 illumination Methods 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 20
- 238000012549 training Methods 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 206010024229 Leprosy Diseases 0.000 claims description 3
- 230000000763 evoking effect Effects 0.000 claims description 3
- 230000002035 prolonged effect Effects 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 238000009418 renovation Methods 0.000 claims 1
- 239000002609 medium Substances 0.000 abstract description 19
- 238000011161 development Methods 0.000 abstract description 5
- 230000018109 developmental process Effects 0.000 abstract description 5
- 239000001963 growth medium Substances 0.000 abstract description 5
- 230000009466 transformation Effects 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 229920001817 Agar Polymers 0.000 abstract 1
- 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 abstract 1
- 229930006000 Sucrose Natural products 0.000 abstract 1
- XEYBHCRIKKKOSS-UHFFFAOYSA-N disodium;azanylidyneoxidanium;iron(2+);pentacyanide Chemical compound [Na+].[Na+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].[O+]#N XEYBHCRIKKKOSS-UHFFFAOYSA-N 0.000 abstract 1
- 238000012239 gene modification Methods 0.000 abstract 1
- 230000005017 genetic modification Effects 0.000 abstract 1
- 235000013617 genetically modified food Nutrition 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 229940083618 sodium nitroprusside Drugs 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 11
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical class O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 9
- 239000002840 nitric oxide donor Substances 0.000 description 7
- 230000008929 regeneration Effects 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 235000019198 oils Nutrition 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 102000018997 Growth Hormone Human genes 0.000 description 3
- 108010051696 Growth Hormone Proteins 0.000 description 3
- 239000003225 biodiesel Substances 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000122 growth hormone Substances 0.000 description 3
- 238000011177 media preparation Methods 0.000 description 3
- BQMKAHQKDSZAIQ-UHFFFAOYSA-N tetrasodium;iron(3+);nitroxyl anion;pentacyanide Chemical compound [Na+].[Na+].[Na+].[Na+].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].O=[N-] BQMKAHQKDSZAIQ-UHFFFAOYSA-N 0.000 description 3
- 241000221089 Jatropha Species 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000028446 budding cell bud growth Effects 0.000 description 1
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- 239000012141 concentrate Substances 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000032459 dedifferentiation Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
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- 239000004615 ingredient Substances 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- -1 once more Chemical compound 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
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- 230000000638 stimulation Effects 0.000 description 1
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Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a direct regeneration method of leaf discs of barbadosnut. The direct regeneration method comprises the following steps: leaf blades of annual barbadosnut are taken as explants and inoculated in a culture medium to directly induce adventitious buds; the culture medium is MS minimal medium which is added with 30g-L<-1> of sucrose, 4-8g L<-1> of agar, 0.2-2.0mg L<-1> of TDZ, 0.1-1.5mg L<-1> of IBA, 0.05-3mg L<-1> of BA and 0.5-8mg L<-1> of sodium nitroprusside; a culture mode: first dark culture is performed for 10-14d and then culture is performed under light for 14-16d; and finally extending culture is performed on the induced adventitious buds, and then the induced adventitious buds are transplanted after rooting culture, thus inductivity of the adventitious buds is up to 88%. The invention aims at providing an efficient regeneration method of the leaf discs of the barbadosnut in terms of genetic modification and tissue culture of the barbadosnut. The direct regeneration method has advantages of laying a foundation for realizing gene transformation and rapid propagation of the barbadosnut, and promoting industry development of the barbadosnut.
Description
Technical field
The present invention relates to the technical field of Plant Tissue Breeding, be specifically related to the direct regeneration method of leaf discs of a kind of Jatropha curcas.
Background technology
Jatropha curcas (Jatropha curcas.L.) is to be grown in the torrid zone and semi-tropical machaka or dungarunga.Jatropha curcas seed oil content height can reach 40%-60%, surpasses common oil crop such as rape and soybean, and Jatropha curcas oil can be used for various diesel engines as biodiesel, is a kind of important energy plant.But the Jatropha curcas planting area is narrow at present, only is distributed in the Asia, the torrid zone and the subtropical zone in Africa and America, and China's natural distribution has 1.1 ten thousand hm approximately
2, mainly concentrate on Sichuan, Yunnan, Guizhou.And Jatropha curcas output is lower, and the leprosy woods of artificial planting only can produce the 100-300kg fruit for every mu at present, can process the 30-100kg biodiesel approximately.Narrow distribution and yield poorly and limited it and cultivate on a large scale and industrialization as energy-source plant.Can improve resistance, the output of Jatropha curcas by engineered method, proterties such as oil, thus enlarge the Jatropha curcas distribution, increase solid output and change jatropha curcas seed oil content and oil.And fast numerous needs of genetic improvement of Jatropha curcas and improved seeds are set up leaf dish regenerating system efficiently.
Summary of the invention
The present invention is intended to provide leprosy leaf dish highly efficient regeneration method for the improvement of genes of Jatropha curcas and tissue culture.For realizing the genetic transformation of Jatropha curcas, breeding lays the foundation fast, advances the industry development of Jatropha curcas.
In order to achieve the above object, Jatropha curcas direct regeneration method of leaf discs of the present invention comprises:
A, choose in the greenhouse the 2nd, 3,4,5 of annual Jatropha curcas and launch blade, carry out being cut into 5-10mm after surface sterilization is handled as explant
2Size;
B, explant leaf dish proximal ends is inoculated in the medium directly evoking adventive bud up; Medium adds 30gL for the MS minimal medium
-1Sucrose, 4-8gL
-1Agar, 0.2-2.0mgL
-1TDZ, 0.1-1.5mgL
-1IBA, 0.05-3mgL
-1BA, 0.5-8mgL
-1Sodium nitroprussiate; Training method: the dark earlier 10-14d that cultivates, be converted to then under the illumination condition and cultivate, temperature is 25 ± 2 ℃, intensity of illumination is 45-55 μ molm
-2s
-1, illumination 16hd
-1, cultivate 1416d;
C, indefinite bud is prolonged cultivation, carry out again transplanting after the culture of rootage.
Preferred the 4th was launched blade as explant during A went on foot; The MS minimal medium preferably added 30gL during B went on foot
-1Sucrose, 5gL
-1Agar, 1mgL
-1TDZ, 0.5mgL
-1IBA, 1.5mgL
-1BA, 2mgL
-1Sodium nitroprussiate; Training method: the dark earlier 10-14d that cultivates, be converted to then under the illumination condition and cultivate, temperature is 25 ℃, intensity of illumination is 50 μ molm
-2s
-1, illumination 16hd
-1, cultivated for 2 weeks.
Disinfecting described in the described A step is the NaClO sterilization explant 5-30min that adopts 15-25%.
Prolonging the used medium of cultivation described in the described C step is that the MS minimal medium adds 30gL
-1Sucrose, 4-8gL
-1Agar, 0.05-0.8mgL
-1BA and 0.01-0.02mgL
-1IBA; Training method: illumination cultivation 18-22 days, temperature was 23 ± 2 ℃, and intensity of illumination is 45-55 μ molm
-2s
-1, illumination 16hd
-1
The used medium of culture of rootage described in the described C step is to add 30gL in the MS minimal medium
-1Sucrose, 4-8gL
-1Agar and 0.08-0.5mgL
-1IBA.
The present invention has following outstanding advantage: one, and adventitious shoot regeneration speed is fast, and the leaf dish is induced through 4 times in week, promptly renewablely goes out a large amount of indefinite buds; Its two, the regeneration frequency height through inducing of 4 times in week, has 88% leaf dish to induce indefinite bud, is significantly higher than contrast, and is far above the highest level of being reported in the world, as shown in table 1; Its three, can promote the development of Jatropha curcas genetic transformation, regeneration frequency is the basis of Jatropha curcas genetic transformation efficiently; Its four, Jatropha curcas is developed as biomass energy, is of value to alleviating China and even energy energy crisis; Its five, the inventive method is easy to implement, is convenient to promote.
The present invention adjusts hormone in medium kind and hormone concentration, has particularly increased the concentration of TDZ, has also increased new plant hormone sodium nitroprussiate.TDZ is a kind of high activity class cytokinin, can obtain more desirable effect with being used of other hormones.Sodium nitroprussiate is nitric oxide donors (NO), and nitric oxide can be regulated growth and development of plant as a kind of messenger molecule, and NO also interacts with hormone influences the growth of plant.At first, the generation of basic element of cell division energy rapid stimulation NO, once more, NO can promote the formation of division of blade protoplasm somatocyte and somatic embryo again under the situation that growth hormone exists, and NO and growth hormone the interact dedifferentiation of common regulation and control plant cell and atomization again, so NO plays " bridge " effect between the basic element of cell division and growth hormone, so can promote the differentiation of indefinite bud.Changed the explant selection mode in addition, the young leaflet tablet of the different development stage by the Jatropha curcas of directly taking to cultivate in the greenhouse, increase the source of explant and obtained the maturity of the blade of suitable evoking adventive bud, use blade than last hypocotyl and cotyledon stable explant source to be arranged and can keep the proterties of choiceness as explant, and operate easier, easy to implement.
Table 1 is the Jatropha curcas leaf plants regeneration techniques of report at present
[1] Lu Weida, Wei Qin, Tang Lin, etc. inducing and breeding [J] fast of Jatropha curcas callus. use and the environmental organism journal 2003,9 (2): 127-130.
[2]Deore?AC，Johnson?TS.High-frequency?plant?regeneration?from?leaf-disccultures?of?Jatropha?curcas?L.：an?important?biodiesel?plant[J].Plant?BiotechnolRep，2008，2(1)：7-11.
[3]Timir?BJ，Mukherjee?P，Datta?MM，Somatic?embryogenesis?in?Jatropha?curcasLinn.，an?important?biofuel?plant[J].Plant?Biotechnol?Rep，2007，1(3)：135-140.
Embodiment
Explant selection in following examples and the Comparative Examples, culture medium preparation and training method etc. is carried out according to table 2.
1, the Jatropha curcas explant is chosen and processing such as surface sterilization:
Choose in the greenhouse annual Jatropha curcas and launch blade, adopt 20% NaClO sterilization 20min, be cut into size with the bacterium blade that went out and be about 9mm as explant
2
2, place medium directly to induce indefinite bud explant, step comprises:
A, culture medium preparation: medium be the MS minimal medium according to table 2 adding ingredient, 121 ℃, sterilization 15min adds sodium nitroprussiate when treating that medium is cooled to 50 ℃;
B, training method: leaf dish proximal ends is inoculated in the medium up, secretly cultivates earlier, be converted to then under the illumination condition and cultivate, temperature is 25 ℃, and intensity of illumination is 50 μ molm
-2s
-1, illumination 16hd
-1Cultivate.
3, the indefinite bud that induces is prolonged cultivation:
A, culture medium preparation: medium is that minimal medium adds 30gL
-1Sucrose and 5gL
-1Agar 0.1-0.5mgL
-1BA, 0.01mgL
-1IBA;
B, training method: illumination cultivation 20 days, temperature are 25 ℃, and intensity of illumination is 50 μ molm
-2s
-1, illumination 16hd
-1
4, the culture of rootage of indefinite bud:
Medium adds 30gL for the MS minimal medium
-1Sucrose and 5gL
-1Agar and 0.1-0.5mgL
-1IBA.
5, the transplanting of the indefinite bud of taking root:
The indefinite bud direct transplanting of taking root is to the greenhouse, and survival rate reaches 93%.
Table 2
It is influential to inductivity that above embodiment and Comparative Examples have embodied the plant growth regulator of variable concentrations, different explants, different training methods etc., explanation has obviously improved adventitious bud induction frequency according to the inventive method, especially embodiment 2 has reached 88%, and the indefinite bud growth conditions that induces is good, can prolong and take root, exteriorly variation does not produce.
The present invention is not limited to concrete embodiment; so long as adopted tissue culture mode provided by the invention; medium component; condition of culture and mode of operation; without departing from the inventive concept of the premise; no matter adopt which kind of tissue culture mode, carry out which kind of equivalents and modification, all will fall within protection scope of the present invention.
Claims (5)
1. the direct renovation process of a leprosy leaf dish is characterized in that, comprising:
A, choose in the greenhouse the 2nd, 3,4,5 of annual Jatropha curcas and launch blade, carry out being cut into 5-10mm after surface sterilization is handled as explant
2Size;
B, explant leaf dish proximal ends is inoculated in the medium directly evoking adventive bud up; Medium adds 30gL for the MS minimal medium
-1Sucrose, 4-8gL
-1Agar, 0.5-1mgL
-1TDZ, 0.5mgL
-1IBA, 1.5mgL
-1BA, 2-5mgL
-1Sodium nitroprussiate; Training method: the dark earlier 10-14d that cultivates, be converted to then under the illumination condition and cultivate, temperature is 25 ± 2 ℃, intensity of illumination is 45-55 μ molm
-2S
-1, illumination 16hd
-1, cultivate 14-16d;
C, indefinite bud is prolonged cultivation, carry out again transplanting after the culture of rootage.
2. method according to claim 1 is characterized in that, described A chooses the 4th and launches blade as explant in the step; B is MS minimal medium interpolation 30gL in the step
-1Sucrose, 5gL
-1Agar, 1mgL
-1TDZ, 0.5mgL
-1IBA, 1.5mgL
-1BA, 2mgL
-1Sodium nitroprussiate; Training method: the dark earlier 10-14d that cultivates, be converted to then under the illumination condition and cultivate, temperature is 25 ℃, intensity of illumination is 50 μ molm
-2S
-1, illumination 16hd
-1, cultivated for 2 weeks.
3. method according to claim 1 is characterized in that, disinfecting described in the A step is the NaClO sterilization explant 5-30min that adopts 15-25%.
4. method according to claim 1 is characterized in that, prolonging the used medium of cultivation described in the C step is that the MS minimal medium adds 30gL
-1Sucrose, 4-8gL
-1Agar, 0.05～0.8mgL
-1BA and 0.01-0.02mgL
-1IBA; Training method: illumination cultivation 18-22 days, temperature was 23 ± 2 ℃, and intensity of illumination is 45-55 μ molm
-2S
-1, illumination 16hd
-1
5. method according to claim 1 is characterized in that, the used medium of culture of rootage described in the C step is to add 30gL in the MS minimal medium
-1Sucrose, 4-8gL
-1Agar and 0.08～0.5mgL
-1IBA.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009103078134A CN101658139B (en) | 2009-09-27 | 2009-09-27 | Direct regeneration method of leaf discs of barbadosnut |
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|---|---|---|---|
| CN2009103078134A CN101658139B (en) | 2009-09-27 | 2009-09-27 | Direct regeneration method of leaf discs of barbadosnut |
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| CN101658139B true CN101658139B (en) | 2011-11-30 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104221851A (en) * | 2013-06-07 | 2014-12-24 | 上海市园林科学研究所 | Method for in-vitro culture and rapid propagation of gunnera manlcata l |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103430841B (en) * | 2013-08-02 | 2014-11-12 | 华南农业大学 | Method for promoting jatropha curcas explant to regenerate adventitious buds |
| CN113892432A (en) * | 2021-11-22 | 2022-01-07 | 合肥师范学院 | Method for directly regenerating barbadosnut leaf |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101095400A (en) * | 2006-06-26 | 2008-01-02 | 云南雷森科技有限公司 | Jatropha curcas propagating cuttage technique |
| EP2001280B1 (en) * | 2006-03-31 | 2010-10-27 | Reliance Life Sciences Pvt., Ltd. | Direct regeneration of plantlets in Jatropha curcas |
-
2009
- 2009-09-27 CN CN2009103078134A patent/CN101658139B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2001280B1 (en) * | 2006-03-31 | 2010-10-27 | Reliance Life Sciences Pvt., Ltd. | Direct regeneration of plantlets in Jatropha curcas |
| CN101095400A (en) * | 2006-06-26 | 2008-01-02 | 云南雷森科技有限公司 | Jatropha curcas propagating cuttage technique |
Non-Patent Citations (8)
| Title |
|---|
| Ajay C. Deore Æ |
| Ajay C. Deore ÆT. Sudhakar Johnson.High-frequency plant regeneration from leaf-disc cultures.《Plant Biotechnol Rep》.Korean Society for Plant Biotechnology and Springer 2008,2008,第2卷第7-11页. * |
| M. Sujatha等.Rapid plant regeneration from various explants of Jatropha integerrima.《Plant Cell, Tissue and Organ Culture》.1993 Kluwer Academic Publisher,1993,第35卷第293-296页. * |
| T. Sudhakar Johnson.High-frequency plant regeneration from leaf-disc cultures.《Plant Biotechnol Rep》.Korean Society for Plant Biotechnology and Springer 2008,2008,第2卷第7－11页. |
| Timir baran Jha等.Somatic embryogenesis in Jatropha curcas Linn., an important.《Plant Biotechnol Rep》.Korean Society for Plant Biotechnology and Springer 2007,2007,第1卷第135-140页. * |
| TimirbaranJha等.SomaticembryogenesisinJatrophacurcasLinn. an important.《Plant Biotechnol Rep》.Korean Society for Plant Biotechnology and Springer 2007 |
| 林 娟等.麻疯树的组织培养及植株再生.《植物生理学通讯》.2002,第38卷(第3期),第252页. * |
| 陆伟达等.麻疯树愈伤组织的诱导及快速繁殖.《应用与环境生物学报》.2003,第9卷(第2期),第127-130页. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104221851A (en) * | 2013-06-07 | 2014-12-24 | 上海市园林科学研究所 | Method for in-vitro culture and rapid propagation of gunnera manlcata l |
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