CN116602209A - Application of GPP in Mo Langen shoot differentiation culture - Google Patents
Application of GPP in Mo Langen shoot differentiation culture Download PDFInfo
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- CN116602209A CN116602209A CN202310448642.7A CN202310448642A CN116602209A CN 116602209 A CN116602209 A CN 116602209A CN 202310448642 A CN202310448642 A CN 202310448642A CN 116602209 A CN116602209 A CN 116602209A
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- 230000004069 differentiation Effects 0.000 title claims abstract description 60
- GVVPGTZRZFNKDS-JXMROGBWSA-N geranyl diphosphate Chemical compound CC(C)=CCC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O GVVPGTZRZFNKDS-JXMROGBWSA-N 0.000 title claims abstract description 34
- 239000001963 growth medium Substances 0.000 claims abstract description 30
- 241000173252 Cymbidium sinense Species 0.000 claims abstract description 13
- OJISWRZIEWCUBN-QIRCYJPOSA-N (E,E,E)-geranylgeraniol Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\CO OJISWRZIEWCUBN-QIRCYJPOSA-N 0.000 claims abstract description 4
- XWRJRXQNOHXIOX-UHFFFAOYSA-N geranylgeraniol Natural products CC(C)=CCCC(C)=CCOCC=C(C)CCC=C(C)C XWRJRXQNOHXIOX-UHFFFAOYSA-N 0.000 claims abstract description 3
- OJISWRZIEWCUBN-UHFFFAOYSA-N geranylnerol Natural products CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CCO OJISWRZIEWCUBN-UHFFFAOYSA-N 0.000 claims abstract description 3
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- 150000001647 brassinosteroids Chemical class 0.000 abstract 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 abstract 1
- 239000003448 gibberellin Substances 0.000 abstract 1
- 230000001766 physiological effect Effects 0.000 abstract 1
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- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 abstract 1
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- 229940023877 zeatin Drugs 0.000 abstract 1
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- IIDAJRNSZSFFCB-UHFFFAOYSA-N 4-amino-5-methoxy-2-methylbenzenesulfonamide Chemical compound COC1=CC(S(N)(=O)=O)=C(C)C=C1N IIDAJRNSZSFFCB-UHFFFAOYSA-N 0.000 description 9
- 239000000306 component Substances 0.000 description 4
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- 150000003505 terpenes Chemical class 0.000 description 4
- KJTLQQUUPVSXIM-ZCFIWIBFSA-M (R)-mevalonate Chemical compound OCC[C@](O)(C)CC([O-])=O KJTLQQUUPVSXIM-ZCFIWIBFSA-M 0.000 description 3
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
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- XMWHRVNVKDKBRG-CRCLSJGQSA-N [(2s,3r)-2,3,4-trihydroxy-3-methylbutyl] dihydrogen phosphate Chemical compound OC[C@](O)(C)[C@@H](O)COP(O)(O)=O XMWHRVNVKDKBRG-CRCLSJGQSA-N 0.000 description 2
- 239000002363 auxin Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 2
- NUHSROFQTUXZQQ-UHFFFAOYSA-N isopentenyl diphosphate Chemical compound CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 description 2
- 235000007586 terpenes Nutrition 0.000 description 2
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- OINNEUNVOZHBOX-QIRCYJPOSA-K 2-trans,6-trans,10-trans-geranylgeranyl diphosphate(3-) Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C(C)=C\COP([O-])(=O)OP([O-])([O-])=O OINNEUNVOZHBOX-QIRCYJPOSA-K 0.000 description 1
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- OINNEUNVOZHBOX-XBQSVVNOSA-N Geranylgeranyl diphosphate Natural products [P@](=O)(OP(=O)(O)O)(OC/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)O OINNEUNVOZHBOX-XBQSVVNOSA-N 0.000 description 1
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- 229930012538 Paclitaxel Natural products 0.000 description 1
- ZJPGOXWRFNKIQL-JYJNAYRXSA-N Phe-Pro-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HGLAYHSJJRYIBI-UHFFFAOYSA-N allyl diphosphate Chemical compound OP(O)(=O)OP(O)(=O)OCC=C HGLAYHSJJRYIBI-UHFFFAOYSA-N 0.000 description 1
- 230000000078 anti-malarial effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 229930101531 artemisinin Natural products 0.000 description 1
- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 description 1
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- 230000028446 budding cell bud growth Effects 0.000 description 1
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- -1 geranylgeraniol Chemical compound 0.000 description 1
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- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 description 1
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- CBIDRCWHNCKSTO-UHFFFAOYSA-N prenyl diphosphate Chemical compound CC(C)=CCO[P@](O)(=O)OP(O)(O)=O CBIDRCWHNCKSTO-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
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Classifications
-
- 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
- A01H4/002—Culture media for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of plant tissue culture, in particular to application of GPP in Mo Langen-shaped stem bud differentiation culture, mo Langen-shaped stems are inoculated in a bud differentiation culture medium for differentiation culture, and the bud differentiation culture medium contains geranylgeraniol GPP. GPP has a wide range of physiological activities, and is a precursor for biosynthesis of various plant hormones such as cytokinin, zeatin, gibberellin and brassinosteroids. The GPP and exogenous hormone act together to promote the differentiation of the cymbidium sinense shoots, so that the problem that the cymbidium sinense shoots are difficult to differentiate or do not differentiate is solved to a certain extent, and a solid foundation is provided for realizing industrial production of cymbidium sinense seedlings.
Description
Technical Field
The invention relates to a plant tissue culture method in agricultural biotechnology, in particular to application of GPP in Mo Langen shoot differentiation culture.
Background
Terpenoids are an important group of natural products, and their structural diversity confers wide use to such compounds, such as hormones for modulating substances, anti-oxidant lycopene, anti-malaria artemisinin, anti-breast cancer paclitaxel, and the like. In nature, terpenoids can be synthesized via the 2-C-methyl-D-erythritol-4-phosphate (MEP) and Mevalonate (MVA) pathways. The MEP pathway is found mainly in bacteria, protozoa and plant plastids, and the MVA pathway is found mainly in eukaryotes and viruses. Organisms can use these two pathways to synthesize the precursors of terpenes, isopentenyl pyrophosphate (IPP) and allyl pyrophosphate (DMAPP), followed by formation of isoprene units of different chain lengths, which further synthesize different types of terpenes, such as monoterpene precursors (GPP), sesquiterpene precursors (FPP), and diterpene precursors (GGPP), among others.
The differentiation of the root shoot buds is a key link of tissue culture of the cymbidium, and directly influences the tissue culture and rapid propagation efficiency and the quality of test tube seedlings. The cymbidium like the cymbidium has slow proliferation, low differentiation efficiency of buds and roots and uncoordinated growth of root buds. The terminal bud on the orchid root-like stem can directly form a well-defined meristem without intermediate protocorm-like or callus stages, and further form a terminal bud having a leaf primordia and a dome shape, while generating an adventitious root on the node. The differentiation capacity of the cymbidium is far lower than that of protocorm, and the bud differentiation can be effectively promoted through higher cytokinin and auxin ratio, for example, the differentiation of the cymbidium bud can be promoted through 6-BA5.0mg/L and NAA0.5mg/L, and in a differentiation medium containing 6-BA4.0mg/L and NAA0.2mg/L, the bud differentiation efficiency of the cymbidium bud is highest, the growth state is best, and the root bud growth is more coordinated. It can be seen that cytokinin and auxin are one of the important factors affecting the tissue culture and rapid propagation characteristics of orchid rootstock. However, in the isolated culture process, and the stems and leaves are not yet fully developed, the biosynthesis of endogenous hormones in the rhizome is affected.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the application of GPP in Mo Langen shoot differentiation culture.
The invention is realized by the following technical scheme: application of GPP in Mo Langen shoot differentiation culture Mo Langen shoots were inoculated into differentiation culture medium containing GPP (i.e. geranylgeraniol, CAS No. 24034-73-9) for differentiation culture.
Preferably, the differentiation medium for bud differentiation culture contains GPP 10-30 mg, 6-benzylaminopurine (6-BA) 0.5-1.0 mg, 1-Naphthalene Acetic Acid (NAA) 0.1-0.5 mg, sucrose 20-30 g, agar powder 5-7 g, and MS medium with pH value of 5.4-6.0.
Preferably, the GPP sterilization method is as follows: the GPP is sterilized by filtration sterilization and should be prepared on-the-fly.
The components except GPP in the differentiation medium of the invention are sterilized at high temperature and then the filtered sterilized beta-farnesene is added into a sterile operating table. The sterilization adopts high temperature sterilization, namely sterilization at 121 ℃ for 20 minutes, and the GPP after filtration sterilization is added into a sterile operation table after the differentiation medium is cooled to below 50 ℃.
Preferably, the differentiation culture mode is as follows: inoculating Mo Langen stems into a differentiation culture medium, performing dark culture at a low temperature of 8-10 ℃ for 5-10 days, transferring to a culture temperature of 25 ℃, and culturing for 30-40 days under a culture condition with an illumination intensity of 1500-2000lx and an illumination length of 12 hours/day.
Preferably, the Mo Langen corms are root corms with stem tips, of the cymbidium variety 'penguin Bai Momo blue' with a length of 1-1.5 cm.
According to the invention, GPP is added into the differentiation medium for the first time, a precursor substance is provided for biosynthesis of the endogenous hormone of the cymbidium, and synthesis of the hormone and the inducing substance in the cymbidium is facilitated, so that differentiation of cymbidium buds is promoted, the problem that the cymbidium is difficult to differentiate or does not differentiate is solved to a certain extent, and a solid foundation is provided for realizing industrial production of cymbidium seedlings
The method has the advantages of simple technology, easy operation and high application value. The invention can be implemented by simple plant tissue culture equipment.
Detailed Description
The technical scheme of the present invention will be described in further detail below with reference to the specific embodiments, but the present invention is not limited thereto.
MS is an international culture medium, and the components and preparation method can be referred to in the literature (Tan Wencheng, dai Cegang, major editions, ornamental plant tissue culture technique, beijing: china forestry Press, 1991).
Example 1
The root-like stems of the cymbidium sinense variety 'penguin Bai Momo with the length of 1-1.5 cm and the stem tip are respectively inoculated into 1-3 groups of differentiation culture media, firstly, the cymbidium sinense variety is subjected to low-temperature dark culture for 5 days at the culture temperature of 8 ℃, then the cymbidium sinense variety is transferred to the culture temperature of 25 ℃, the illumination intensity is 1500lx, the illumination length is 12 hours/day, and after 30 days of culture under the condition of illumination length of 12 hours, the differentiation rates of the root-like stems and the buds of the cymbidium sinense Bai Momo' cultured in the 1-3 groups of differentiation culture media are 45.8 percent, 3.19 percent and 29.6 percent respectively. Wherein, the culture medium component of the differentiation culture medium 1 is MS+10mg/L GPP+0.5 mg/L6-BA+0.1 mg/L NAA+20g/L sucrose+5g/L agar, and the pH value is 5.4; the culture medium of the differentiation culture medium 2 comprises MS+0.5mg/L6-BA+0.1mg/L NAA+20g/L sucrose+5g/L agar, and the pH value is 5.4; the culture medium of the differentiation culture medium 3 comprises MS+5mg/L GPP+0.5 mg/L6-BA+0.1 mg/L NAA+20g/L sucrose+5 g/L agar, and has a pH value of 5.4.
Example 2
Inoculating the cymbidium sinense variety 'penguin Bai Momo' root-like stems with the length of 1-1.5 cm and stem tips into a differentiation culture medium 4-5 groups, firstly performing low-temperature dark culture for 8 days at the culture temperature of 9 ℃, then transferring to the culture temperature of 25 ℃, culturing for 35 days under the condition that the illumination intensity is 1700lx and the illumination length is 12 hours/day, and then culturing the cymbidium sinense variety 'penguin Bai Momo' root-like stems and bud differentiation rates in the differentiation culture medium 4-5 groups are 53.1% and 3.93%, wherein the culture medium components of the differentiation culture medium 4 are MS+15mg/L GPP+0.8mg/L6-BA+0.3 mg/L NAA+25g/L sucrose+6g/L agar, and the pH value is 5.7; the culture medium of the differentiation culture medium 5 comprises MS+0.8mg/L6-BA+0.3mg/L NAA+25g/L sucrose+6 g/L agar, and has a pH value of 5.7.
Example 3
The root-like stems of the cymbidium sinense variety 'penguin Bai Momo with the length of 1-1.5 cm and the stem tip are respectively inoculated into the groups of the differentiation culture mediums of 6-8, firstly, the cymbidium sinense variety' penguin Bai Momo 'is subjected to low-temperature dark culture for 10 days at the culture temperature of 10 ℃, then is transferred to the culture temperature of 25 ℃, the illumination intensity of 2000lx and the illumination length of 12 hours/day for 40 days, and the differentiation rates of the root-like stem buds of the cymbidium sinense Bai Momo' cultured in the groups of 6-8 of the differentiation culture mediums are 58.9%, 4.67% and 34.4% respectively. Wherein, the culture medium composition of the differentiation culture medium 6 is MS+20mg/L GPP+1.0mg/L6-BA+0.5 mg/L NAA+30g/L sucrose+7g/L agar, and the pH value is 6.0; the culture medium of the differentiation culture medium 7 comprises MS+1.0mg/L6-BA+0.5mg/LNAA+30 g/L sucrose+7 g/L agar, and the pH value is 6.0; the culture medium of the differentiation culture medium 8 comprises MS+25mg/LGPP+1.0 mg/L6-BA+0.5 mg/L NAA+30g/L sucrose+7 g/L agar, and has a pH value of 6.0.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
- The application of GPP in Mo Langen shoot bud differentiation culture, which is characterized in that Mo Langen shoot is inoculated in a differentiation culture medium for differentiation culture, wherein the differentiation culture medium contains geranylgeraniol GPP.
- 2. The use of GPP according to claim 1 in shoot differentiation culture of Mo Langen, wherein the differentiation culture medium for shoot differentiation culture comprises GPP 10-30 mg, 6-benzylaminopurine 0.5-1.0 mg, 1-naphthylacetic acid 0.1-0.5 mg, sucrose 20-30 g, agar powder 5-7 g, and MS culture medium for the balance, and has pH of 5.4-6.0.
- 3. The use of GPP according to claim 1 for differentiation culture of Mo Langen shoot buds, wherein components other than GPP in the differentiation medium are sterilized at high temperature and then filtered sterilized β -farnesene is added to the sterile operating table.
- 4. Use of GPP according to claim 3 in the differentiation culture of Mo Langen shoot buds, characterized in that the sterilization is performed by high temperature sterilization, i.e. sterilization at 121 ℃ for 20 minutes, at which time the differentiation medium is cooled to below 50 ℃ and the sterilized GPP is added in a sterile operating table.
- 5. The use of GPP according to claim 4 for differentiation culture of Mo Langen like shoot buds, wherein the method of GPP sterilization is: the GPP is sterilized by filtration sterilization and should be prepared on-the-fly.
- 6. The application of GPP in Mo Langen shoot differentiation culture according to claim 1, wherein the differentiation culture method is as follows: inoculating Mo Langen stems into a differentiation culture medium, performing dark culture at a low temperature of 8-10 ℃ for 5-10 days, transferring to a culture temperature of 25 ℃, and culturing for 30-40 days under a culture condition with an illumination intensity of 1500-2000lx and an illumination length of 12 hours/day.
- 7. The use of GPP according to claim 1 in differentiation culture of Mo Langen shoot buds, wherein Mo Langen shoot is a root shoot of the cymbidium sinense variety 'penguin Bai Momo blue' with a length of 1-1.5 cm and a shoot tip.
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