CN113317203A - Pogostemon cablin artificial seed culture method - Google Patents
Pogostemon cablin artificial seed culture method Download PDFInfo
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- CN113317203A CN113317203A CN202110721709.0A CN202110721709A CN113317203A CN 113317203 A CN113317203 A CN 113317203A CN 202110721709 A CN202110721709 A CN 202110721709A CN 113317203 A CN113317203 A CN 113317203A
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- 240000002505 Pogostemon cablin Species 0.000 title claims abstract description 20
- 235000011751 Pogostemon cablin Nutrition 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000011218 seed culture Methods 0.000 title abstract description 7
- 239000000701 coagulant Substances 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000005342 ion exchange Methods 0.000 claims abstract description 9
- 239000008223 sterile water Substances 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 229920001817 Agar Polymers 0.000 claims description 12
- 235000010419 agar Nutrition 0.000 claims description 12
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 11
- 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 11
- 229930006000 Sucrose Natural products 0.000 claims description 11
- 239000008272 agar Substances 0.000 claims description 11
- 235000010413 sodium alginate Nutrition 0.000 claims description 11
- 239000000661 sodium alginate Substances 0.000 claims description 11
- 229940005550 sodium alginate Drugs 0.000 claims description 11
- 239000005720 sucrose Substances 0.000 claims description 10
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 241000196324 Embryophyta Species 0.000 abstract description 12
- 241001529821 Agastache Species 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 abstract description 4
- 238000011282 treatment Methods 0.000 description 22
- 238000011284 combination treatment Methods 0.000 description 13
- 229960004793 sucrose Drugs 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 238000013461 design Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 210000002257 embryonic structure Anatomy 0.000 description 4
- 230000000392 somatic effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 3
- 238000004161 plant tissue culture Methods 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000000442 meristematic effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000012883 rooting culture medium Substances 0.000 description 2
- 238000009331 sowing Methods 0.000 description 2
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- 241000206672 Gelidium Species 0.000 description 1
- 241000207923 Lamiaceae Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 239000002111 antiemetic agent Substances 0.000 description 1
- 229940125683 antiemetic agent Drugs 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 229940125716 antipyretic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 210000002242 embryoid body Anatomy 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000009993 protective function Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000012882 rooting medium Substances 0.000 description 1
- 239000002602 strong irritant Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 230000008673 vomiting Effects 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
- A01H4/001—Culture apparatus for tissue culture
-
- 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
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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
The invention belongs to the technical field of plant biology and discloses a method for culturing artificial seeds of pogostemon cablin. Placing sterile stem of herba Agastaches into embedding medium on a clean bench, mixing, sucking one propagule with a suction pipe each time, and dripping into coagulating agent for ion exchange reaction for 15 min; then rinsing with sterile water for 20min, and stopping the reaction; fishing out and drying; the patchouli artificial seed culture method established by the invention has the advantages of good technical system stability, simple and convenient preparation and high emergence rate.
Description
Technical Field
The invention belongs to the technical field of plant biology, and relates to a method for culturing artificial seeds of pogostemon cablin.
Background
Plant Artificial seeds (Artificial seeds) or Synthetic seeds (Synthetic seeds) are seeds produced artificially. The artificial seed technology is a new technology developed on the basis of the in vitro plant culture technology. The artificial seed is an artificial capsule containing plant embryoid or bud, nutrient, hormone and other components. The concept of artificial seeds was first proposed by Murashige in 1978 at the 4 th International society for plant tissue cell culture. Murashige considers that with the continuous development of plant tissue culture technology, a plurality of embryoids can be synchronously cultured by a small amount of explants, and the embryoids are embedded in certain capsules to have the function of seeds and can be directly used for field sowing. In 1985, Kamada, a Japanese scholar, extended the concept of artificial seeds, and he thought that granules produced by tissue culture with meristematic tissue (shoots, callus, embryoid and growing point, etc.) developing into whole plants, which can be used as substitutes for natural seeds for sowing, were all called artificial seeds, using an appropriate method for embedding. In 1995, Chinese scientists Chendeffu et al further extended the concept of artificial seeds, and they thought that somatic embryos produced in vitro culture of plants or meristematic tissues (such as embryoid bodies, buds, stem segments, etc.) capable of developing into whole plants were embedded in a shell containing nutrients and having a protective function, and that granules formed under appropriate conditions capable of germinating and emerging were all called artificial seeds. In recent years, this concept has been developed from the coating of somatic embryos in the narrow sense to the coating of any suitable plant propagules, such as adventitious buds, tubers, axillary buds, shoot tips, protocorms, callus and hairy roots.
The industrialization of artificial seeds has not made a major breakthrough so far, and many problems need to be solved urgently, for example, Redenbaugh et al, 1987, summarize that somatic embryos are induced from more than 200 plants, but no more than 40 plants have been subjected to artificial seed experiments, because the somatic embryos serving as the cores of the artificial seeds must be high in quality, low in variation rate, high in frequency and synchronous in occurrence, low in transformation (emergence) rate of the artificial seeds, and long in seedling stage (juvenile stage), the research and development of the artificial seeds are greatly limited.
Herba Agastaches (Pogostemon cablin), Labiatae, perennial herbs, and has fragrance. The original philippines are cultivated in many places in south east asia, and the southern regions in China are cultivated in a large amount. The whole herb contains volatile oil, can be used as strong irritant and aromatic material, is common component of perfume, has important medicinal value, is mainly used for treating vomiting of pregnancy, stomachache and influenza, and is aromatic stomach-invigorating, antipyretic and antiemetic agent. The patchouli propagation mode usually adopts cutting propagation and direct seeding propagation, and the propagation speed is slow and variation is generated. The artificial seeds can keep good characters unchanged and accelerate the propagation speed, and can provide a way for quickly propagating seedlings for the cablin potchouli herb.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to establish a technical system of the patchouli artificial seed culture method which has the advantages of stable system, high emergence rate, simplicity, convenience, practicability and the like on the basis of the modern plant tissue culture technology, and lays a foundation for the rapid propagation of patchouli seedlings. The invention provides a method for culturing patchouli artificial seeds, which artificially synthesizes patchouli seeds according to the main structure and the function of the seeds and based on the basic principle of plant tissue culture. The patchouli artificial seed culture method established by the invention has the advantages of good technical system stability, simple and convenient preparation and high emergence rate.
The above purpose of the invention is realized by the following technical scheme:
a method for culturing artificial seeds of herba Agastaches; placing sterile stem of herba Agastaches into embedding medium on a clean bench, mixing, sucking one propagule with a suction pipe each time, and dripping into coagulating agent for ion exchange reaction for 15 min; then rinsing with sterile water for 20min, and stopping the reaction; fishing out and drying;
the embedding medium is artificial endosperm, and the formula is as follows: MS +0.5-1.5mg/L IBA +0.5-1.5mg/L NAA + 30g/L sucrose + 7g/L agar;
the coagulant is artificial seed skin and has the following formula: 2-6% of sodium alginate and 1-3% of CaCl2。
Further, the embedding medium is artificial endosperm, and the formula is preferably as follows: MS +1.0mg/L IBA +1.0mg/L NAA + sucrose 30g/L + agar 7 g/L;
the coagulating agent is artificial seed coat, and the preferable formula is as follows: 4% sodium alginate + 2% CaCl2。
Compared with the prior art, the invention has the beneficial effects that:
the patchouli artificial seed culture method established by the invention has the advantages of good technical system stability, simple and convenient preparation and high emergence rate.
Detailed Description
The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be obtained from commercial sources.
Example 1
Placing sterile stem segment of herba Agastaches into embedding medium (artificial endosperm, formula is MS +1.0mg/L IBA +1.0mg/L NAA + sucrose 30g/L + agar 7g/L), mixing, sucking one propagule each time with a suction tube, and dripping into coagulating agent (artificial seed coat, formula is 4% sodium alginate + 2% CaCl)2) The ion exchange reaction was carried out for 15 min. The reaction was then stopped by rinsing with sterile water for 20 min. Fishing outAnd (5) drying.
Example 2
Placing sterile stem segment of herba Agastaches into embedding medium (artificial endosperm, formula is: 1/2MS +0.5mg/L IBA +0.5mg/L NAA + sucrose 30g/L + agar 7g/L), mixing well, sucking one propagule each time with suction tube, and dripping into coagulating agent (artificial seed coat, formula is: 2% sodium alginate + 1% CaCl)2) The ion exchange reaction was carried out for 5 min. The reaction was then stopped by rinsing with sterile water for 25 min. Fishing out and drying.
Example 3
Placing sterile stem segment of herba Agastaches into embedding medium (artificial endosperm, formula is: 1/4MS +1.5mg/L IBA +1.5mg/L NAA + sucrose 30g/L + agar 7g/L), mixing, sucking one propagule each time with suction tube, and dripping into coagulating agent (artificial seed coat, formula is: 6% sodium alginate + 3% CaCl)2) The ion exchange reaction was carried out for 10 min. Then, the reaction was terminated by rinsing with sterile water for 30 min. Fishing out and drying.
The following experimental procedures were used for the study experiments: selection of artificial endosperm → selection of artificial seed coat.
1. Screening of Artificial endosperm
Firstly, screening the most suitable rooting culture medium, and removing agar from the most suitable rooting culture medium to obtain the artificial endosperm. Taking MS, 1/2MS and 1/4MS as basic culture media, adding 30g/L of cane sugar, 7g/L of agar and pH5.8-6.0; the IBA concentration was set as: 0.5-1.5 mg/L; NAA is: 0.5 to 1.5 mg/L. And 3, performing a 3-factor 3 horizontal orthogonal design, and screening the artificial endosperm formula through an orthogonal test. The rooting rate is used as a screening index (see table 1-1).
Experiment design: the artificial endosperm contains basic culture medium MS (A1), 1/2MS (A2) and 1/4MS (A3); IBA concentration is designed to be 0.5mg/L (B1), 1.0mg/L (B2), 1.5mg/L (B3); the NAA concentration was designed to be 0.5mg/L (C1), 1.0mg/L (C2), 1.5mg/L (C3). An orthogonal design method is adopted, the experimental treatment combination is 9, each treatment is repeated for 5 times, and each repetition inoculates 10 rootless seedlings. Namely: A1B1C1 treatment 1, A1B2C2 treatment 2, A1B3C3 treatment 3, A2B1C2 treatment 4, A2B2C3 treatment 5, A2B3C1 treatment 6, A3B1C3 treatment 7, A3B2C1 treatment 8, A3B3C2 treatment 9.
TABLE 1-1 rooting percentage (%)
TABLE 1-2 analysis of variance of the results of TABLE 1-1
| Source of variation | Degree of freedom | Sum of squares | Mean square | F value | F0.05 | F0.01 |
| Treatment room | 8 | 2480.00 | 310.00 | 4.23** | 2.21 | 3.05 |
| Error of the measurement | 36 | 2640.00 | 73.33 | |||
| Total variation | 44 | 5120.00 |
Tables 1-3 test for significance of differences between the data of Table 1-1
The experimental results are as follows: the treatment combination A1B2C2 (i.e., MS +1.0mg/L IBA +1.0mg/L NAA + sucrose 30g/L + agar 7g/L, pH 5.8-6.0) was the most suitable rooting medium on which the rooting rate was the greatest.
2. Screening of artificial seed coats
The stem segments of the patchouli are used as propagules, MS +1.0mg/L IBA +1.0mg/L NAA + sucrose + 30g/L + agar-agar is 7g/L, pH is 5.8-6.0, and the screening of the proper artificial seed coat is carried out: the concentration of the sodium alginate is 2 percent to 6 percent, and the sodium alginate is CaCl2The concentration of the water is 1 to 3 percent, the ion exchange time is 5 to 15min, and the rinsing time of the sterile water is 20 to 30 min. Making 4-factor 3 horizontal orthogonal design by orthogonalArtificial seed coats are screened in the test. The germination rate of the artificial seeds on a MS +1.0mg/L IBA +1.0mg/L NAA + sucrose 30g/L + agar 7g/L culture medium with pH of 5.8-6.0 is used as a screening index (see table 2-1).
Experiment design: in the artificial seed coat, the concentration of sodium alginate is 2 percent (A1), 4 percent (A2) and 6 percent (A3); CaCl2Concentrations of 1% (B1), 2% (B2), 3% (B3); the ion exchange time is set to 5min (C1), 10min (C2) and 15min (C3); the rinsing time with sterile water was set to 20min (D1), 25min (D2) and 30min (D3). An orthogonal design method is adopted, the experimental treatment combinations are 9, each treatment is repeated for 5 times, and each repetition is inoculated with 10 artificial seeds. Namely: treatment combination 1: A1B1C1D1, treatment combination 2: A1B2C2D2, treatment combination 3: A1B3C3D3, treatment combination 4: A2B1C2D3, treatment combination 5: A2B2C3D1, treatment combination 6: A2B3C1D2, treatment combination 7: A3B1C3D2, process combination 8: A3B2C1D3, treatment combination 9: A3B3C2D 1.
TABLE 2-1 Germination ratio (% of different treatments)
TABLE 2-2 analysis of variance of the results of TABLE 2-1
| Source of variation | Degree of freedom | Sum of squares | Mean square | F value | F0.05 | F0.01 |
| Treatment room | 8 | 3440.00 | 430.00 | 6.24** | 2.21 | 3.05 |
| Error of the measurement | 36 | 2480.00 | 68.89 | |||
| Total variation | 44 | 5920.00 |
Tables 2-3 test for significance of differences between the data of Table 2-1
| Treatment of | Mean number of | Difference of 5% | 1% difference |
| Treatment 5 | 92.00 | a | A |
| Treatment 4 | 84.00 | a | AB |
| Treatment 6 | 82.00 | ab | AB |
| Treatment 3 | 72.00 | bc | BC |
| Treatment 8 | 72.00 | bc | BC |
| Treatment 2 | 70.00 | c | BC |
| Treatment 7 | 70.00 | c | BC |
| Process 9 | 66.00 | c | C |
| Process 1 | 64.00 | c | C |
The experimental results are as follows: treatment combination A2B2C3D1 (i.e., 4% sodium alginate + 2% CaCl)2Ion exchange time of 15min and sterile water rinsing time of 20min) is the most suitable artificial seed coat preparation treatment combination.
The stem segment of the pogostemon cablin is used as a propagule, different kinds of artificial seed coats, artificial endosperm and other additive components are researched, and finally, a technical system of the pogostemon cablin artificial seed culture method is established.
The embodiments described above are merely preferred embodiments of the invention, rather than all possible embodiments of the invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.
Claims (3)
1. A method for culturing the artificial seeds of patchouli includes such steps as loading the stem of aseptic patchouli in embedding medium, mixing, sucking one propagule by sucking it by sucking tube, and dropping it in coagulant for ion exchange reaction for 15 min; then rinsing with sterile water for 20min, and stopping the reaction; fishing out and drying; the embedding medium is artificial endosperm, and the formula is as follows: MS +0.5-1.5mg/L IBA +0.5-1.5mg/L NAA + 30g/L sucrose + 7g/L agar;
the coagulant is artificial seed skin and has the following formula: 2-6% of sodium alginate and 1-3% of CaCl2。
2. The method for culturing the artificial seeds of pogostemon cablin according to claim 1, wherein the embedding medium is artificial endosperm, and the formula is preferably: MS +1.0mg/L IBA +1.0mg/L NAA + sucrose 30g/L + agar 7 g/L.
3. The method for culturing the artificial seeds of pogostemon cablin of claim 1, wherein the coagulating agent is artificial seed coat, preferably having a formula of: 4% sodium alginate + 2% CaCl2。
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2021
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