CN110810244B - Culture medium for tissue culture of floral rod - Google Patents
Culture medium for tissue culture of floral rod Download PDFInfo
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- CN110810244B CN110810244B CN201911205864.6A CN201911205864A CN110810244B CN 110810244 B CN110810244 B CN 110810244B CN 201911205864 A CN201911205864 A CN 201911205864A CN 110810244 B CN110810244 B CN 110810244B
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- 239000001963 growth medium Substances 0.000 title claims abstract description 53
- 230000006698 induction Effects 0.000 claims abstract description 22
- 230000012010 growth Effects 0.000 claims abstract description 21
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 6
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 37
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 14
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 6
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000012879 subculture medium Substances 0.000 claims description 4
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- 150000002505 iron Chemical class 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 239000005416 organic matter Substances 0.000 claims description 3
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 claims description 3
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 3
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 3
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 3
- 239000012882 rooting medium Substances 0.000 claims description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 3
- 239000011573 trace mineral Substances 0.000 claims description 3
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- 238000000034 method Methods 0.000 abstract description 18
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- 210000001519 tissue Anatomy 0.000 description 25
- 241000196324 Embryophyta Species 0.000 description 17
- 230000000694 effects Effects 0.000 description 11
- 239000005556 hormone Substances 0.000 description 10
- 229940088597 hormone Drugs 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
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- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000013028 medium composition Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
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- 238000011160 research Methods 0.000 description 3
- 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 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
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- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- -1 2.4-D Chemical compound 0.000 description 1
- 241001061264 Astragalus Species 0.000 description 1
- 241001573366 Astragalus membranaceus Species 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000220485 Fabaceae Species 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000006533 astragalus Nutrition 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 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
- 238000001784 detoxification Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 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 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 239000003617 indole-3-acetic acid Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000004233 talus Anatomy 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
Landscapes
- 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)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a culture medium for tissue culture of a floral rod, which comprises a primary generation induction culture medium, a secondary generation culture medium and a rooting culture medium, and can be used for taking a growing point on a tender branch of the floral rod as an explant, and performing the processes of primary generation induction culture, secondary generation culture, rooting culture and transplanting after sterilization. The optimal culture medium formula and the combination thereof in each growth period in the whole tissue culture process of the flower stick selected by the invention can fully meet the nutritional requirements and growth development of the flower stick in each period, promote the whole tissue culture propagation process of the flower stick, ensure the successful implementation of the tissue culture of the flower stick and provide powerful support for the large-scale production of the flower stick. The culture medium has the advantages of simple formula, low cost and good application prospect.
Description
Technical Field
The invention belongs to the technical field of plant biology, and particularly relates to a culture medium for each stage in tissue culture of a floral rod. The optimal culture medium for different periods in tissue culture of the flower stick is screened out, the proportion of each hormone in the culture medium is determined, and the culture medium prepared according to the formula can meet the nutritional requirements and growth development of the flower stick at each period.
Background
The floral rod is also called as the root of membranous milkvetch, flower firewood, flower stem, flower bud, floral cap and oxtail tip, and is a typical sand-fixing pioneer plant in the desert region of the large deciduous shrub of the genus Astragalus of the subfamily Papilionaceae of Leguminosae. The artificial planting method is characterized in that the artificial planting method is distributed in arid and semi-arid desert or sand land in middle Asia, and natural groups exist in Guerbantong Gute desert, Ba Dan Jilin desert, Hexi corridor sand land, Tenggeli desert, Wulan cloth and desert in China and west desert in Kubu, and artificial planting groups and descendants thereof exist in other deserts or sand lands. The eastern sandy land is used for introducing the flower sticks, and the growth is good and even superior to that of a natural distribution area. The flower stick is an excellent sand-fixing shrub, has certain economic benefit, and has large nursery stock demand in the ecological civilization construction process of a sand area.
Plant tissue culture is an important biotechnological means. Cells, stem segments, leaves, etc. of the plant may be cultured. The cells, organs and tissues of the plant are taken and put into a container with proper culture medium, and can be differentiated to form a complete plant under certain culture conditions. Tissue culture as a powerful means of biotechnology is increasingly paid attention to, and plays an important role in aspects of detoxification, rapid propagation, mutation induction, cell engineering, genetic engineering and the like of agricultural and forestry crops.
Under the condition of in vitro culture, different plants and tissue cells at different parts of the same plant have different nutritional requirements, and can grow better only if the special requirements of the plants are met. Mastering the preparation and screening method of the culture medium is one of the key links for obtaining the success of tissue culture.
The method for improving the quantity of industrial flower stick seedlings by the tissue culture propagation technology is an effective way for solving ecological seedling use of the flower sticks. However, in the prior art, the tissue culture of the floral stick is still in a groping stage, few data can be referred to in the preparation of the technical scheme, and the data need to be solved one by one through experiments, and the key points are to solve the selection of the material taking part and period of the explant, the culture medium in each culture period, the culture conditions, the rapid propagation technology after seedling formation and the like. The invention aims at developing and utilizing, reducing cost and forming industrial and industrialized production, and makes corresponding working plan and implementation method
The whole process of tissue culture is generally divided into several technical links such as establishment of a culture scheme, selection and treatment of explants, inoculation, primary culture, subculture, strong seedling and rooting culture, domestication and transplantation of test-tube seedlings and the like. Under the condition of in vitro culture, tissue cells of different plants and different parts of the same plant have different nutritional requirements, and can grow and develop better only if the special requirements of the different plants and the tissue cells of different parts of the same plant are met. The control of the preparation and screening method of the culture medium is one of the key links for successful tissue culture.
Disclosure of Invention
The invention aims to screen out the optimal culture medium for different periods in tissue culture of a flower stick, determine the proportion of each hormone in the culture medium, and the culture medium prepared according to the formula can meet the nutritional requirements and growth development of the flower stick at each period.
The invention sets an implementation scheme on the basis of information collection, summarization and analysis, comprehensively and diversely performs tissue culture experiments, and screens culture mediums at different periods of time, such as primary generation induction → subculture multiplication → rooting culture. The culture of the first generation, the multiplication and the rooting in each period can achieve the best effect, the combination effect is better, and the effect of the flower stick in different stages in the tissue culture propagation process is continuously promoted.
The embodiment of the invention comprises the optimal culture medium for each growth period in the whole tissue culture process of the flower stick, which is obtained through a plurality of experiments in a careful and repeated way, and different types of plant growth agents and different proportions are added according to the requirements of each growth period of the flower stick on the basis of a basic culture medium.
The invention provides a culture medium required by each stage of a tissue culture process of a floral rod, which is characterized by consisting of a primary induction culture medium, a multiplication culture medium and a rooting culture medium.
The optimal culture medium for each phase of the tissue culture of the floral bouquet is screened by a comparative test. The culture medium provided by the invention is suitable for tissue culture of a flower stick, and is particularly suitable for culture of a growing point of the flower stick.
One embodiment of the invention provides a primary induction culture medium of a floral rod: b5+ NAA 0.1mg/L +6-BA 0.5mg/L + 2.4-D0.1 mg/L + GA 30.05-0.1 mg/L + CH 0.5-1 g/L; further preferably B5+ NAA 0.1mg/L +6-BA 0.5mg/L + 2.4-D0.1 mg/L + GA 30.1 mg/L + CH 0.5 g/L.
The research finds that different hormone combinations can induce the flower stick to differentiate into multiple buds, but the differentiation rates of the combinations are different, B5+ NAA 0.1mg/L +6-BA 0.5mg/L + 2.4-D0.1 mg/L + GA 30.1 mg/L + CH 0.5g/L is the best induction medium for the growth point of the flower stick, the induction rate is high, the number of newly-increased buds of each explant is large, the differentiation rate is the highest, the differentiation rate can reach 218.75%, and the subculture can be promoted.
One embodiment of the invention provides a subculture medium of a floral rod: modified B5+ NAA 0.3mg/L +6-BA 1mg/L + CH 0.5 g/L; further preferably B5+ NAA 0.3mg/L +6-BA 1-2mg/L + CH 0.5-1g/L
The culture medium is the best subculture multiplication culture medium for the flower stick, and not only can produce the most adventitious buds, but also the multiplication times can reach 21.68 times.
One embodiment of the invention provides a rooting culture medium for a flower stick, which comprises the following components in percentage by weight: 1/2MS + IAA0.1mg/L + NAA 0.5mg/L + activated carbon 1 g/L.
The research shows that the low salt concentration is favorable for the tissue culture of the anthony twigs, and the culture medium suitable for rooting is 1/2MS, IAA0.1mg/L, NAA 0.5mg/L and activated carbon 1 g/L.
In the embodiment of the invention, NAA, 6-BA, 2.4-D, GA3, CH and IAA are short names of naphthylacetic acid, 6-benzylaminopurine, 2.4-dichlorophenoxyacetic acid, gibberellin, hydrolyzed casein and indoleacetic acid respectively within the scope known by the technical personnel in the field.
The optimal culture medium of each growth period in the whole tissue culture process of the flower stick selected by the invention can fully meet the nutritional requirements and growth development of the flower stick at each period, is the key and core for ensuring the successful implementation of the tissue culture of the flower stick and achieving the flow production, and provides powerful support for the large-scale production of the flower stick. Moreover, the stages of primary generation induction, subculture and rooting culture can mutually influence each other, and the selection of the culture medium can continuously promote the flower stick in each stage of tissue culture propagation, so that the yield of industrial flower stick seedling culture can be greatly improved.
Detailed Description
In order to better explain the present invention and to facilitate the understanding of the technical solutions of the present invention, the present invention is further described in detail below. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
First, material of flower stick explant
The flower stick for experiment is collected from a test nursery of an experiment base of a drought-resistant plant research institute of inner Mongolia Mongolian grass ecological environment (group) GmbH, and is sampled and supplemented at any time according to the test progress and different periods. Collecting growing points of 0.2-0.5cm on tender branches of the flower stick, and cutting under aseptic condition. After washing by running water, sterilizing with 0.1% mercuric chloride solution for 5min, washing with sterile water for 2 times, 5min each time, sucking water on sterile filter paper, inoculating on primary induction culture medium, and establishing test-tube plantlet clone. Selecting cluster buds subcultured for about 10 days, and cutting off the base parts of the cluster buds; the growing point of the cluster bud is taken, the length of the growing point is about 0.2-0.5cm, and the growing point is used as a regenerated explant.
Second, screening statistical method
Survival rate (number of viable explants/total number of inoculated explants) x 100%
Primary shoot induction rate ═ 100% (number of germinated explants/total number of inoculated explants)%
The multiplication factor for subculture (number of cluster buds induced/total number of shoots inoculated) x 100%
Rooting rate (number of explants of differentiated root/total number of explants inoculated) x 100%
Example 2 selection of optimal Medium for various stages of tissue culture of floral rods
B5 is used as a basic culture medium, 3.5g/L of carrageenan is used as a curing agent, 40g/L of edible white sugar is used as a carbon source instead of cane sugar, tap water is used instead of distilled water, and cytokinin and auxin in different proportions are respectively added for screening the culture medium.
1. Selection of Primary Induction Medium
Setting two concentration gradients of 0.2mg/L and 0.5mg/L for the concentration of 6-BA, 0.01mg/L and 0.05mg/L for the concentration of NAA, 0.1mg/L and 0.05mg/L for the concentration of GA3, and 0.5mg/L and 1mg/L for the concentration of CH, performing different combinations, inoculating 2-3 growth points (about 0.2-0.5cm in length) of the floral rod to each bottle, and observing the occurrence of adventitious buds of the growth points on different culture media.
The conditions of primary induction culture are that the culture temperature is 25 +/-1 ℃, the illumination intensity is 1500Lx, and light is supplemented for 4 hours/day at night except for natural illumination in the daytime.
As can be seen from Table 1, the 8 hormone combinations can induce the flower stick to differentiate into multiple buds, but the differentiation rates of the combinations are different, and the combination No. 1B 5+ NAA 0.1mg/L +6-BA 0.5mg/L + 2.4-D0.1 mg/L + GA 30.1 mg/L + CH 0.5g/L and the combination No. 9B 5+ NAA 0.1mg/L +6-BA 0.5mg/L + 2.4-D0.1 mg/L + GA 30.1 mg/L + CH 1g/L are better induction culture mediums of the growth point of the flower stick, so that the induction rate is high, the number of new buds of each explant is large, the differentiation rate is highest, and the differentiation rate can reach 218.75% and 226.25%.
Table 1: effect of hormone concentration and combination thereof on floral rod growing point multiple bud induction rate
| Processing number | Media composition | Number of inoculation (one) | Number of cluster buds | Percentage of cluster bud induction (%) |
| 1 | B5+NAA 0.1mg/L+6-BA 0.5mg/L+2.4-D 0.1mg/L+GA3 0.1 mg/L+CH 0.5g/L | 80 | 175 | 218.75 |
| 2 | B5+NAA 0.05mg/L+6-BA 0.2mg/L+2.4-D 0.1mg/L+GA3 0.1 mg/L+CH 0.5g/L | 80 | 77 | 96.25 |
| 3 | B5+NAA 0.1mg/L+6-BA 0.2mg/L+2.4-D 0.1mg/L+GA3 0.1 mg/L+CH 0.5g/L | 80 | 34 | 42.5 |
| 4 | B5+NAA 0.05mg/L+6-BA 0.5mg/L+2.4-D 0.1mg/L+GA3 0.1 mg/L+CH 0.5g/L | 80 | 62 | 77.5 |
| 5 | B5+NAA 0.1mg/L+6-BA 0.5mg/L+2.4-D 0.1mg/L+GA3 0.05 mg/L+CH 1g/L | 80 | 146 | 182.5 |
| 6 | B5+NAA 0.05mg/L+6-BA 0.2mg/L+2.4-D 0.1mg/L+GA3 0. 05 mg/L+CH 1g/L | 80 | 82 | 102.5 |
| 7 | B5+NAA 0.1mg/L+6-BA 0.2mg/L+2.4-D 0.1mg/L+GA3 0. 05 mg/L+CH 1g/L | 80 | 113 | 141.25 |
| 8 | B5+NAA 0.05mg/L+6-BA 0.5mg/L+2.4-D 0.1mg/L+GA3 0. 05 mg/L+CH 1g/L | 80 | 37 | 46.25 |
| 9 | B5+NAA 0.1mg/L+6-BA 0.5mg/L+2.4-D 0.1mg/L+GA3 0.1 mg/L+CH 1g/L | 80 | 181 | 226.25 |
| 10 | B5+NAA 0.05mg/L+6-BA 0.2mg/L+2.4-D 0.1mg/L+GA3 0.1mg/L+CH 1g/L | 80 | 98 | 122.5 |
In this embodiment, the B5 medium is specifically composed of:
macroelements: KNO 3 2500mg/L；MgSO 4 ·7H 2 O 250mg/L；CaCl 2 ·2H 2 O 150mg/L；(NH 4 ) 2 SO 4 134mg/L；NaH 2 PO 4 ·H 2 O 150mg/L
Trace elements: KI is 0.75 mg/L; h 3 BO 3 3mg/L；MnSO 4 ·4H 2 O 10mg/L；ZnSO 4 ·7H 2 O 2mg/L；Na 2 MoO 4 ·2H 2 O 0.25mg/L；CuSO 4 ·5H 2 O 0.025mg/L；CoCl 2 ·6H 2 O 0.025mg/L
Iron salt: FeSO 4 ·7H 2 O 27.8mg/L；Na 2 -EDTA 37.3mg/L
Organic matter: inositol 100 mg/L; 1mg/L of nicotinic acid; pyridoxine hydrochloride (vitamin B6)1 mg/L; thiamine hydrochloride (vitamin B1)10 mg/L.
2. Selection of subculture multiplication medium
Respectively inoculating the adventitious buds subjected to induced differentiation to a subculture multiplication medium, marking the number of a primary culture medium, setting two concentration gradients of 0.3mg/L and 0.5mg/L for NAA, two concentration gradients of 1mg/L and 2mg/L for 6-BA, setting two concentration gradients of 0.5g/L and 1g/L for CH, inoculating 4 single plants to each bottle, observing and recording the differentiation and growth conditions of the regenerated adventitious buds, and counting the multiplication rate of the buds under different hormone combination conditions after 15 days.
The subculture conditions were 25. + -. 1 ℃ for the culture temperature, 2000-3000Lx for the illumination intensity, and 1 hour/day for light supplement at night except for natural illumination in the daytime. After induction culture, 0.2-0.5cm of the growing points of the floral rod cluster buds generated in the induction culture medium are respectively transferred into a subculture multiplication culture medium (see table 2), observation and recording are started when the bud clusters appear on the bud bases of partial materials after 2-3 days, the test results are counted after 15 days, and partial data are shown in table 2 (other data with unobvious characteristics or unobvious effects are not recorded).
Table 2: effect of hormone concentration and combination thereof on adventitious bud proliferation of flower stick
| Processing number | Primary Medium numbering | Media composition | Fold of proliferation | Growth of adventitious bud |
| 1 | 1 | Improved B5+ NAA 0.3mg/L +6-BA 1mg/L + CH 0.5g/L | 21.68 | Strong, newly emerged seedlings are more, and adventitious buds are more |
| 2 | 1 | Improved B5+ NAA 0.3mg/L +6-BA 1mg/L + CH 1g/L | 10.49 | Stronger, more newly emerged seedlings and more vitrification |
| 3 | 1 | Improved B5+ NAA 0.3mg/L +6-BA 2mg/L + CH 0.5g/L | 1.96 | Fine and weak without new emergence of seedlings |
| 4 | 1 | Improved B5+ NAA 0.3mg/L +6-BA 2mg/L + CH 1g/L | 15.64 | Stronger, more newly emerged and less vitrification |
| 5 | 1 | Improved B5+ NAA 0.5mg/L +6-BA 1mg/L + CH 0.5g/L | 14.73 | Stronger, more newly emerged and less vitrification |
| 6 | 1 | Improved B5+ NAA 0.5mg/L +6-BA 1mg/L + CH 1g/L | 5.84 | Delicate and weak, less new seedlings |
| 7 | 1 | Improved B5+ NAA 0.5mg/L +6-BA 2mg/L + CH 0.5g/L | 7.72 | Strong, less new emergence of seedlings and slow growth |
| 8 | 1 | Improved B5+ NAA 0.5mg/L +6-BA 2mg/L + CH 1g/L | 11.09 | Strong, more new seedlings and less vitrification |
| 9 | 9 | Improved B5+ NAA 0.3mg/L +6-BA 1mg/L + CH 0.5g/L | 16.73 | Strong, newly emerged and more adventitious buds |
| 10 | 9 | Improved B5+ NAA 0.3mg/L +6-BA 2mg/L + CH 1g/L | 12.57 | Stronger, more new seedlings emerge and less vitrification is produced |
| 11 | 9 | Improved B5+ NAA 0.5mg/L +6-BA 2mg/L + CH 1g/L | 9.72 | Stronger, less new emergence and less vitrification |
Experiments show that the flower stick has a promoting effect on the growth of the cluster buds in the subculture of improved B5, NAA 0.3mg/L, 6-BA 1mg/L and CH 0.5g/L, and compared with other combinations, the flower stick is more ideal, the multiplication factor can reach 21.68 times, the number of the cluster buds is large, the seedlings are large, and the effect of transplanting the emergence rate is optimal. However, by comparing the multiplication times of the adventitious buds of different primary culture mediums, the hormone environment of the primary culture medium can be found to influence the adaptability of the adventitious bud in the later propagation stage, and the comprehensive results show that: the combination effect of the primary culture medium B5, the NAA 0.1mg/L, the 6-BA 0.5mg/L, the 2.4-D0.1 mg/L, the GA 30.1 mg/L and the CH 0.5g/L and the secondary culture medium B5, the NAA 0.3mg/L, the 6-BA 1mg/L and the CH 0.5g/L is the best. In the embodiment, the modified B5 medium is obtained by halving the amounts of macroelements and microelements in the B5 medium, and the basic medium for the floral rod subculture is specifically composed of:
macroelements: KNO 3 1250mg/L；MgSO 4 ·7H 2 O 125mg/L；CaCl 2 ·2H 2 O 75mg/L；(NH 4 ) 2 SO 4 67mg/L； NaH 2 PO 4 ·H 2 O 75mg/L
Trace elements: KI 0.375 mg/L; h 3 BO 3 1.5mg/L；MnSO 4 ·4H 2 O 5mg/L；ZnSO 4 ·7H 2 O 1mg/L；Na 2 MoO 4 ·2H 2 O 0.125mg/L；CuSO 4 ·5H 2 O 0.0125mg/L；CoCl 2 ·6H 2 O 0.0125mg/L
Iron salt: FeSO 4 ·7H 2 O 27.8mg/L；Na 2 -EDTA 37.3mg/L
Organic matter: 100mg/L inositol; 1mg/L of nicotinic acid; pyridoxine hydrochloride (vitamin B6)1 mg/L; thiamine hydrochloride (vitamin B1)10 mg/L.
3. Selection of rooting Medium
Dividing the seedlings with better growth vigor in the subculture medium into single plants, transferring the single plants into a rooting medium, inoculating 4 plants into each bottle, observing and recording the rooting conditions of the seedlings on different culture media, and counting the rooting rate after 10 days.
The rooting culture condition is that the culture temperature is 25 +/-1 ℃, and light is not supplemented at night except for natural illumination in the daytime.
From the test results of 10 days of rooting culture (table 3), the two culture mediums have induction effect on the formation of roots, wherein the No. 2 rooting culture medium has better rooting effect, and the rooting rate reaches 91.56%. The rooting base can be newly added with cluster buds with roots, and the average number can reach 3-4. The low salt concentration is favorable for the growth of the flower stick root, and compared with the influence of a primary culture medium and a secondary culture medium, the seedling stimulated by high hormone in the primary culture or the secondary culture process is more suitable for the rooting culture medium with high salt concentration. However, the culture medium with low hormone content and low salt concentration is adopted to be matched, so that the better proliferation and rooting effects of the flower stick in primary culture, secondary culture and rooting culture are the results wanted by the technical personnel in the field, the cost can be reduced, and the dependence of the expanding propagation growth of the flower stick on artificial factors can be reduced. Therefore, the culture medium suitable for rooting is 1/2MS + IAA0.1mg/L + NAA 0.5mg/L + activated carbon 1 g/L.
Table 3: influence of inorganic salt on seedling rooting
| Processing number | Subculture medium number | Media composition | Rooting percentage (%) | Root growth status |
| 1 | 1 | MS + IAA 0.2mg/L + NAA 1mg/L + activated carbon 2g/L | 65.89 | Short, thick and small in quantity |
| 2 | 1 | 1/2MS + IAA0.1mg/L + NAA 0.5mg/L + active carbon 1g/L | 91.56 | Long and large in quantity |
| 3 | 9 | MS + IAA 0.2mg/L + NAA 1mg/L + active carbon 2g/L | 74.48 | Short, thick and small in quantity |
| 4 | 9 | 1/2MS + IAA0.1mg/L + NAA 0.5mg/L + active carbon 1g/L | 86.74 | Long and large in quantity |
The above examples are intended to illustrate the present invention specifically, but should not be construed as limiting the present invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Claims (1)
1. A culture medium for tissue culture of a floral rod consists of a primary generation induction culture medium, a secondary generation culture medium and a rooting culture medium; the primary induction culture medium is B5+ NAA 0.1mg/L +6-BA 0.5mg/L + 2.4-D0.1 mg/L + GA 3 0.1mg/L + CH 0.5g/L, the subculture medium is modified B5+ NAA 0.3mg/L +6-BA 1mg/L + CH 0.5g/L, and the rooting medium is 1/2MS + IAA0.1mg/L + NAA 0.5mg/L + activated carbon 1 g/L;
the improved B5 culture medium is obtained by respectively halving the use amount of macroelements and microelements in the B5 culture medium, and comprises the following specific components:
macroelements: KNO 3 1250mg/L；MgSO 4 ·7H 2 O 125mg/L；CaCl 2 ·2H 2 O 75mg/L；(NH 4 ) 2 SO 4 67mg/L； NaH 2 PO 4 ·H2O 75mg/L；
Trace elements: KI is 0.375 mg/L; h 3 BO 3 1.5mg/L；MnSO 4 ·4H 2 O 5mg/L；ZnSO 4 ·7H 2 O 1mg/L；Na 2 MoO 4 ·2H 2 O 0.125mg/L；CuSO 4 ·5H 2 O 0.0125mg/L；CoCl 2 ·6H 2 O 0.0125mg/L；
Iron salt: FeSO 4 ·7H 2 O 27.8mg/L；Na 2 -EDTA 37.3mg/L；
Organic matter: inositol 100 mg/L; 1mg/L of nicotinic acid; pyridoxine hydrochloride (vitamin B6)1 mg/L; thiamine hydrochloride (vitamin B1)10 mg/L;
and inoculating an explant to the culture medium, wherein the explant is a growth point with the length of 0.2-0.5cm on a tender branch of the flower stick.
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