CN117502226A - Method for improving regeneration efficiency and quality of rubber tree embryo plants - Google Patents
Method for improving regeneration efficiency and quality of rubber tree embryo plants Download PDFInfo
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- CN117502226A CN117502226A CN202311207384.XA CN202311207384A CN117502226A CN 117502226 A CN117502226 A CN 117502226A CN 202311207384 A CN202311207384 A CN 202311207384A CN 117502226 A CN117502226 A CN 117502226A
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- 241000196324 Embryophyta Species 0.000 title claims abstract description 64
- 244000043261 Hevea brasiliensis Species 0.000 title claims abstract description 53
- 230000008929 regeneration Effects 0.000 title claims abstract description 43
- 238000011069 regeneration method Methods 0.000 title claims abstract description 43
- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- IXVMHGVQKLDRKH-VRESXRICSA-N Brassinolide Natural products O=C1OC[C@@H]2[C@@H]3[C@@](C)([C@H]([C@@H]([C@@H](O)[C@H](O)[C@H](C(C)C)C)C)CC3)CC[C@@H]2[C@]2(C)[C@@H]1C[C@H](O)[C@H](O)C2 IXVMHGVQKLDRKH-VRESXRICSA-N 0.000 claims abstract description 17
- 230000000392 somatic effect Effects 0.000 claims abstract description 11
- 150000001647 brassinosteroids Chemical class 0.000 claims abstract description 3
- IXVMHGVQKLDRKH-KNBKMWSGSA-N brassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@@H](O)[C@H](O)[C@@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-KNBKMWSGSA-N 0.000 claims description 16
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 14
- 229920002148 Gellan gum Polymers 0.000 claims description 7
- 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 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 210000002257 embryonic structure Anatomy 0.000 abstract description 15
- 238000005516 engineering process Methods 0.000 abstract description 5
- 230000006872 improvement Effects 0.000 abstract description 4
- IXVMHGVQKLDRKH-YEJCTVDLSA-N (22s,23s)-epibrassinolide Chemical compound C1OC(=O)[C@H]2C[C@H](O)[C@H](O)C[C@]2(C)[C@H]2CC[C@]3(C)[C@@H]([C@H](C)[C@H](O)[C@@H](O)[C@H](C)C(C)C)CC[C@H]3[C@@H]21 IXVMHGVQKLDRKH-YEJCTVDLSA-N 0.000 abstract 1
- 238000004161 plant tissue culture Methods 0.000 abstract 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 11
- 230000013020 embryo development Effects 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- 238000012258 culturing Methods 0.000 description 6
- 230000000408 embryogenic effect Effects 0.000 description 5
- 230000030118 somatic embryogenesis Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920003052 natural elastomer Polymers 0.000 description 2
- 229920001194 natural rubber Polymers 0.000 description 2
- 101710134784 Agnoprotein Proteins 0.000 description 1
- 241001164374 Calyx Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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
- A01H4/008—Methods for regeneration to complete plants
-
- 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)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明提供了一种提高橡胶树体胚植株再生效率和质量的方法，涉及植物组织培养领域，具体方法包括以下步骤，选择具有胚根、胚芽的成熟体胚接种在含有油菜素内酯的培养基上，经过光照培养20‑60天，观察到植株再生效率提高，且株高、茎粗等指标质量提高。本发明提供了油菜素内酯在橡胶树体胚植株再生中的运用方法，为橡胶树育苗技术改良提供支持。
The invention provides a method for improving the regeneration efficiency and quality of rubber tree somatic embryo plants, and relates to the field of plant tissue culture. The specific method includes the following steps: selecting mature somatic embryos with radicles and embryos and inoculating them in a culture medium containing brassinosteroids On the above, after 20-60 days of light cultivation, it was observed that the plant regeneration efficiency increased, and the quality of plant height, stem diameter and other indicators improved. The present invention provides a method for using brassinosteroid in the regeneration of rubber tree somatic embryo plants, and provides support for the improvement of rubber tree seedling technology.
Description
Technical Field
The invention relates to the field of plant cultivation, in particular to a method for improving the regeneration efficiency and quality of rubber tree embryo plants, and provides an application method of brassinolide in the regeneration of rubber tree embryo plants.
Background
Natural rubber is the only renewable resource in four industrial raw materials in China, and is also an important strategic material, and is mainly derived from Brazilian rubber tree. The growth cycle of the rubber tree is as long as 70 years, and the quality of the seedlings has profound influence on the development of the whole natural rubber industry. The rubber tree seedlings are the tissue culture seedling type capable of large-scale breeding at present after the seedlings are grafted with seedlings and the seedlings are grafted with buds, and the rubber tree body seedlings (commonly called as self-root young clone and tissue culture seedlings) cultivated through the body cell embryogenesis technology are the new-generation seedlings of the rubber tree, and have the advantages of rapid growth, high yield and high resistance.
In the existing rubber tree tissue culture technology, the technology is mainly concentrated in the links of embryogenic callus induction, growth, somatic embryogenesis and the like, and is relatively deficient in the plant regeneration stage. Hua Yuwei et al, which adopts anther as an explant, firstly induces callus and obtains primary somatic embryos, and then induces secondary somatic embryogenesis by utilizing the somatic embryos, thus realizing the large-scale breeding of rubber tree somatic embryos for the first time; dai Xuemei etc. by adding AgNO to the culture medium 3 Observing the morphological change of the anther callus of the rubber tree and the embryogenesis of the rubber tree; li Ling et al studied the effect of TDZ on callus induction and somatic embryogenesis of rubber tree anthers; zhang Yuanyuan and the like take anthers of 4 excellent rubber tree strains as explants, so that optimal culture medium combinations for callus induction and embryogenesis of different strains are obtained; gu Xiaochuan and the like induce callus on anthers of 32 varieties of rubber trees, induce embryogenesis, and combine the embryogenesis efficiency and genetic relationship of the bodies to analyze the difference of embryogenesis capacity of different genotypes. In addition, in the invention patent aspect, gu Xiaochuan and the like induce embryogenesis by using calyx as an explant (CN 113331057B, 2022); huang Tian bands and the like are induced by tender leaves of rubber tree test tube plantlets to embryogenesis and plant regeneration (CN 114467748B, 2023). Gu Xiaochuan et al disclose a method for efficient generation of rubber tree secondary embryos and their use (CN 115500261B, 2023); chen Jianmiao et al disclose a culture medium for embryogenic callus of rubber tree and a method for rapid proliferation of embryogenic callus of rubber tree (CN 107372122B, 2019), a culture medium for embryogenic callus of rubber tree, a method for inducing embryogenic callus and a method for breeding resistant callus (CN 108575761B, 2019). In-plantIn the plant regeneration stage, li Ling and the like research that the active carbon with different concentrations regenerates the rubber tree embryo plant, and the plant regeneration rate is 69% at most; tan Deguan et al disclose a method of rubber tree somatic embryogenesis and plant regeneration (CN 104823850B, 2017), which focuses on the embryogenesis effect, nor does the plant regeneration stage involve brassinosteroids. By adding brassinolide into the culture medium, the plant regeneration efficiency of the rubber tree embryo is effectively improved, the plant height is also obviously increased, and the method for improving the plant regeneration efficiency and quality of the rubber tree embryo is provided.
Disclosure of Invention
The invention aims to provide a method for improving the regeneration efficiency and quality of rubber tree embryo plants. By adding brassinolide into the regeneration culture medium of the rubber tree plants, the regeneration efficiency of the rubber tree embryo plants is improved, and the quality of indexes such as plant height, stem thickness and the like is improved.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for improving regeneration efficiency and quality of rubber tree embryo plants comprises the following steps:
s1, preparing a plant regeneration culture medium: the modified MS is basic culture medium, and each liter of culture medium also contains KT0.1-1.0mg, IAA0.1-0.5mg, and GA 3 3.0-4.0mg, 0-1.0mg of brassinolide, 40.0-60.0g of sucrose, 1.0g of activated carbon and 1.0g of phytagel, and regulating the pH value to 5.8;
s2, embryo selection: selecting a mature rubber tree primary embryo or secondary embryo with embryo;
and S3, plant regeneration: inoculating the selected somatic embryo to plant regeneration culture medium on an ultra-clean bench for illumination culture to enable the somatic embryo to germinate into a complete plant, wherein the culture temperature is 25-30 ℃ and the culture time is 20-60 days.
Preferably, 0.01-0.5mg/L brassinolide is added into the plant regeneration medium in the step S1.
Preferably, in the selection of the embryo in the step S2, the embryo should be a mature cotyledon embryo having a germ and a radicle.
Preferably, in the plant regeneration in the step S3, the cultivation period is preferably long enough for the plant to have developed root system and stable top leaf.
The beneficial effects of the invention are as follows:
the invention adds brassinolide into the culture medium, and through plant regeneration culture, the improvement of plant regeneration efficiency, the improvement of plant height and other index quality are observed, and the invention provides support for the improvement of rubber tree seedling technology.
Drawings
FIG. 1 is a regenerated plant obtained by culturing rubber tree embryos for 40d in examples 1, 2 and comparative examples 1-3, wherein the upper left and right graphs are the regenerated plant obtained by culturing the rubber tree embryos for 40d in examples 1 and 2, and the lower left, middle and right graphs are the regenerated plant obtained by culturing the rubber tree embryos for 40d in comparative examples 1, 2 and 3, respectively;
FIG. 2 shows regenerated plants obtained by culturing rubber tree embryos for 60 days in examples 1, 2 and comparative examples 1 to 3, wherein the upper left and right panels are regenerated plants obtained by culturing rubber tree embryos for 60 days in examples 1 and 2, and the lower left, middle and right panels are regenerated plants obtained by culturing rubber tree embryos for 60 days in comparative examples 1, 2 and 3, respectively.
Detailed Description
The following detailed description of embodiments of the invention is, therefore, to be taken in conjunction with the accompanying drawings, and it is to be understood that the scope of the invention is not limited to the specific embodiments.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
The materials, reagents and the like used in the method for improving the regeneration efficiency and quality of the rubber tree embryo plant provided by the invention in the following examples are all commercially available unless otherwise specified.
Example 1
A plant regeneration culture medium comprises modified MS as basic culture medium, KT0.1-1.0mg, IAA0.1-0.5mg, and GA 3 3.0-4.0mg, 0.1mg of brassinolide, 40-60g of sucrose, 1.0g of activated carbon and 1.0g of phytagel, and adjusting the pH value to 5.8.
Example 2
A plant regeneration culture medium comprises modified MS as basic culture medium, KT0.1-1.0mg, IAA0.1-0.5mg, and GA 3 3.0-4.0mg, 0.01mg of brassinolide, 40-60g of sucrose, 1.0g of activated carbon and 1.0g of phytagel, and adjusting the pH value to 5.8.
Comparative example 1
A plant regeneration culture medium comprises modified MS as basic culture medium, KT0.1-1.0mg, IAA0.1-0.5mg, and GA 3 3.0-4.0mg, sucrose 40-60g, active carbon 1.0g and phytagel 1.0g, and adjusting the pH value to 5.8.
Comparative example 2
A plant regeneration culture medium comprises modified MS as basic culture medium, KT0.1-1.0mg, IAA0.1-0.5mg, and GA 3 3.0-4.0mg, 0.5mg of brassinolide, 40-60g of sucrose, 1.0g of activated carbon and 1.0g of phytagel, and adjusting the pH value to 5.8.
Comparative example 3
A plant regeneration culture medium comprises modified MS as basic culture medium, KT0.1-1.0mg, IAA0.1-0.5mg, and GA 3 3.0-4.0mg, 1.0mg of brassinolide, 40-60g of sucrose, 1.0g of activated carbon and 1.0g of phytagel, and adjusting the pH value to 5.8.
Mature somatic embryos of the rubber trees are cultured on the culture mediums of examples 1-2 and comparative examples 1-3, plants with the plant height higher than 1.0cm are counted at 40d and 60d respectively and used for calculating plant regeneration rate, and indexes such as plant height, stem thickness, main root length, leaf number and the like of each plant are measured at 60d and subjected to statistical analysis and differentiation.
The effect of treatment of brassinolide at different concentrations and durations on regeneration of rubber tree embryo plants is shown in tables 1 and 2.
Table 1 shows the effects of the brassinolide treatments 40d of examples 1 and 2 and comparative examples 1 to 3 on regeneration of rubber tree embryo plants
Table 2 Table 1, 2 and comparative examples 1-3 effects of brassinolide treatment 60d on regeneration of rubber tree embryo plants
The effect of treatment with different concentrations of brassinolide on the quality of regenerated plants of rubber tree embryos is shown in Table 2.
Table 2 Table 1, 2 and comparative examples 1-3 effects of brassinolide treatment 60d on the quality of regenerated plants of rubber tree embryos
Note that: different lower case letters indicate significant differences between treatments (P < 0.05) and different upper case letters indicate significant differences between treatments (P < 0.01).
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (4)
1. A method for improving regeneration efficiency and quality of rubber tree embryo plants is characterized by comprising the following steps:
s1, preparing a plant regeneration culture medium: the modified MS is basic culture medium, and each liter of culture medium also contains KT0.1-1.0mg, IAA0.1-0.5mg, and GA 3 3.0-4.0mg, 0-1.0mg of brassinolide, 40.0-60.0g of sucrose, 1.0g of activated carbon and 1.0g of phytagel, and regulating the pH value to 5.8;
s2, embryo selection: selecting a mature rubber tree primary embryo or secondary embryo with embryo;
and S3, plant regeneration: inoculating the selected somatic embryo to plant regeneration culture medium on an ultra-clean bench for illumination culture to enable the somatic embryo to germinate into a complete plant, wherein the culture temperature is 25-30 ℃ and the culture time is 20-60 days.
2. The method for improving the regeneration efficiency and quality of rubber tree embryo plants according to claim 1, wherein 0.01-0.5mg/L brassinosteroids are added to the plant regeneration medium in the step S1.
3. The method according to claim 1, wherein in the step S2, the embryo is mature cotyledon embryo with embryo and radicle.
4. The method for improving the regeneration efficiency and quality of a rubber tree embryo plant according to claim 1 wherein in the step S3, the cultivation period is preferably the plant root system is developed and the top leaf is stable.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311207384.XA CN117502226A (en) | 2023-09-19 | 2023-09-19 | Method for improving regeneration efficiency and quality of rubber tree embryo plants |
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| CN202311207384.XA CN117502226A (en) | 2023-09-19 | 2023-09-19 | Method for improving regeneration efficiency and quality of rubber tree embryo plants |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6492174B1 (en) * | 2000-06-19 | 2002-12-10 | Institute Of Paper Science & Technology | Methods of initiating embryogenic cultures in plants |
| CN101485290A (en) * | 2008-01-17 | 2009-07-22 | 中国热带农业科学院橡胶研究所 | Method for establishing rubber tree inner integument regeneration system |
| CN104823850A (en) * | 2015-05-07 | 2015-08-12 | 中国热带农业科学院热带生物技术研究所 | Rubber tree somatic embryogenesis and plant regeneration method |
| CN110637087A (en) * | 2017-03-08 | 2019-12-31 | 南京农业大学 | Method for epigenetic manipulation of plant phenotypic plasticity traits |
| CN114467748A (en) * | 2022-01-19 | 2022-05-13 | 中国热带农业科学院橡胶研究所 | Method for inducing somatic embryogenesis and plant regeneration by adopting tender leaves of rubber tree test-tube plantlets |
| EP4197318A1 (en) * | 2021-12-17 | 2023-06-21 | Albert-Ludwigs-Universität Freiburg | Phosphodiesterases inhibitors to promote in vitro plant cell reprogramming towards plant embryogenesis or microcallus formation |
| CN116530416A (en) * | 2023-06-12 | 2023-08-04 | 中国热带农业科学院橡胶研究所 | A method of using brassinolide to promote hevea somatic embryogenesis |
-
2023
- 2023-09-19 CN CN202311207384.XA patent/CN117502226A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6492174B1 (en) * | 2000-06-19 | 2002-12-10 | Institute Of Paper Science & Technology | Methods of initiating embryogenic cultures in plants |
| CN101485290A (en) * | 2008-01-17 | 2009-07-22 | 中国热带农业科学院橡胶研究所 | Method for establishing rubber tree inner integument regeneration system |
| CN104823850A (en) * | 2015-05-07 | 2015-08-12 | 中国热带农业科学院热带生物技术研究所 | Rubber tree somatic embryogenesis and plant regeneration method |
| CN110637087A (en) * | 2017-03-08 | 2019-12-31 | 南京农业大学 | Method for epigenetic manipulation of plant phenotypic plasticity traits |
| EP4197318A1 (en) * | 2021-12-17 | 2023-06-21 | Albert-Ludwigs-Universität Freiburg | Phosphodiesterases inhibitors to promote in vitro plant cell reprogramming towards plant embryogenesis or microcallus formation |
| CN114467748A (en) * | 2022-01-19 | 2022-05-13 | 中国热带农业科学院橡胶研究所 | Method for inducing somatic embryogenesis and plant regeneration by adopting tender leaves of rubber tree test-tube plantlets |
| CN116530416A (en) * | 2023-06-12 | 2023-08-04 | 中国热带农业科学院橡胶研究所 | A method of using brassinolide to promote hevea somatic embryogenesis |
Non-Patent Citations (1)
| Title |
|---|
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