CN117598204A - Tissue culture and rapid propagation method and application of michelia yunnanensis - Google Patents
Tissue culture and rapid propagation method and application of michelia yunnanensis Download PDFInfo
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- 241001597576 Magnolia laevifolia Species 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000001963 growth medium Substances 0.000 claims abstract description 32
- 230000006698 induction Effects 0.000 claims abstract description 29
- 230000035755 proliferation Effects 0.000 claims abstract description 28
- 235000021446 Apple puree Nutrition 0.000 claims abstract description 18
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 13
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 28
- 230000001954 sterilising effect Effects 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000008223 sterile water Substances 0.000 claims description 16
- 238000004659 sterilization and disinfection Methods 0.000 claims description 13
- 239000012286 potassium permanganate Substances 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 10
- 229960002523 mercuric chloride Drugs 0.000 claims description 9
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 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 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 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 claims description 6
- 229960000367 inositol Drugs 0.000 claims description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 6
- LXAHHHIGZXPRKQ-UHFFFAOYSA-N 5-fluoro-2-methylpyridine Chemical compound CC1=CC=C(F)C=N1 LXAHHHIGZXPRKQ-UHFFFAOYSA-N 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- 230000000249 desinfective effect Effects 0.000 claims description 5
- 239000011790 ferrous sulphate Substances 0.000 claims description 5
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 230000003203 everyday effect Effects 0.000 claims description 2
- 230000004083 survival effect Effects 0.000 abstract description 4
- 238000004161 plant tissue culture Methods 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 21
- 241000218378 Magnolia Species 0.000 description 13
- 241000196324 Embryophyta Species 0.000 description 5
- 230000002354 daily effect Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 238000012136 culture method Methods 0.000 description 3
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- 238000005520 cutting process Methods 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
- 210000004209 hair Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 235000011201 Ginkgo Nutrition 0.000 description 1
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- 239000003205 fragrance Substances 0.000 description 1
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- 238000000338 in vitro Methods 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000012149 noodles Nutrition 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000012879 subculture medium Substances 0.000 description 1
- 238000012090 tissue culture technique Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
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Classifications
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- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of plant tissue culture, and discloses a Michelia yunnanensis tissue culture rapid propagation method and application, wherein the method comprises the following steps: the explant is disinfected, the disinfected explant is inoculated into a cluster bud induction culture medium for culture, the cluster buds induced by the primary culture are inoculated into a secondary proliferation culture medium for culture, and the cluster buds with the secondary proliferation value are inoculated into a rooting culture medium for culture. NAA, 2-iP and ZT with certain concentration are added in an induction culture medium, and the induction rate is improved to more than 60 percent; the secondary proliferation culture medium changes NAA into IBA, and combines ZT and BR with certain concentration to stabilize proliferation coefficient above 5 without affecting rooting rate, and GA with certain concentration is added during proliferation culture 3 The method has the advantages that internode extraction is convenient to operate, apple puree with a certain concentration is added during secondary proliferation and rooting culture, so that seedlings are more robust, leaves are more fresh green, and especially when rooting culture is carried out, the apple puree is matched with PPP333, so that root systems are more robust, and finally the survival rate of the acclimatized seedlings is improved.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a Michelia yunnanensis tissue culture rapid propagation method and application.
Background
Michelia yunnanensis (Roxb.) kuntzeMichelia yunnanensis Franch. ex Finet&Gagnep.) is a plant of the genus Michelia of the family Magnoliaceae, shrubs, branches and leaves are thick, up to 4m; the buds, twigs, leaves, stems and peduncles are densely covered with dark red flat hairs. She Gezhi, inverted oval, narrow inverted oval, 4-10cm long and 1.5-3.5cm wide. The pedicel is thick and short, 3-7mm long and 1 bract drop mark; white and very fragrant. The number of the polymerized fruits is usually only 5-9, and the number of the polymerized fruits is equal to the whole period of development, the whole period of cultivation is equal to the whole period of cultivation, the width of the polymerized fruits is 5-8mm, the top end of the polymerized fruits is short and pointed, and the hairs remain; 1-2 seeds, 3-4 months in flowering phase and 8-9 months in fruiting phase.
Distributed in the middle and south of Yunnan. In mountain bushes grown at an altitude of 1100-2300 m. The flower is very aromatic, can extract extractum, is an excellent ornamental plant; the leaves have fragrance, and can be ground into fragrant noodles. The tree age of the Michelia yunnanensis ancient tree found in Yunnan Lijiang reaches more than 200 years. The same species as ginkgo tree belongs to the middle-aged tree species remained after the fourth glacier, and is called as "activated stone".
At present, the main breeding modes are: sowing and grafting, low cutting propagation survival rate, large amount of original trees required for grafting propagation, great limitation of grafting quantity, and the tissue culture technology is a rapid propagation technology, can rapidly produce michelia yunnanensis seedlings in large quantities, but has great technical difficulty, and no tissue culture rapid propagation method aiming at michelia yunnanensis at present. The prior art discloses related tissue culture methods of late spring michelia, taiwan michelia, shilin TONG michelia, zhuangli michelia, deep mountain michelia, zhongshan michelia, purple flower michelia, drunk michelia and the like, which are still applicable to the tissue culture process of the Yunnan michelia and are not applicable to the tissue culture of the Yunnan michelia, so that development of a tissue culture method applicable to the Yunnan michelia is urgently needed.
Disclosure of Invention
The invention aims at solving the technical problem that the prior art is not suitable for a Michelia yunnanensis tissue culture method, and provides a Michelia yunnanensis tissue culture rapid propagation method and application.
In order to achieve the above purpose, the invention adopts the following technical scheme: a tissue culture and rapid propagation method for Michelia yunnanensis comprises the following steps:
(1) Explant sterilization: taking the stem sections of michelia yunnanensis which are not lignified at the end parts, cleaning, and then sequentially disinfecting with 0.5% potassium permanganate for 15-20 min and washing with sterile water for 4-5 times; sterilizing with 75% alcohol for 50-60 s, and rinsing with sterile water for 2-3 times; sterilizing for 5-6 min by 1% potassium permanganate, rinsing for 4-5 times by sterile water, sterilizing for 5-6 min by 0.05% mercuric chloride, and rinsing for 5-7 times by sterile water;
(2) And (3) primary cluster bud induction: inoculating the sterilized explant into a cluster bud induction culture medium for culturing for 20-25 days;
the cluster bud induction culture medium comprises the following formula: WPM+NAA 0.05-0.08 mg/L+2-iP 0.1-0.15 mg/L+ZT 2.0-3.0 mg/L+activated carbon 0.4-0.6 g/L;
(3) And (3) subculturing and proliferation: inoculating cluster buds induced by the first generation into a secondary proliferation culture medium for culturing for 25-35 days;
the formula of the secondary proliferation culture medium is as follows: improved SH+IBA 0.1-0.2 mg/L+ZT 0.5-0.8 mg/L+BR 0.05-0.08 mg/L+GA 3 0.2-0.3 mg/L and 30-40 g/L of apple puree;
(4) Rooting culture: inoculating the cluster buds with the secondary multiplication value into a rooting culture medium for culturing for 30-40 days;
the rooting culture medium comprises the following formula: 1/2 modified SH+IBA 0.3-0.5 mg/L+IAA 1.5-2.0 mg/L+PPP333 1.0-2.0 mg/L+apple puree 60-80 g/L+active carbon 0.2-0.4 g/L;
the improved SH culture medium is as follows: the dosage of calcium chloride is changed to 150mg/L, the dosage of EDTA-disodium is changed to 37.3mg/L, the dosage of ferrous sulfate is changed to 27.8mg/L, the dosage of inositol is changed to 100mg/L, and the rest components are unchanged.
Further: the culture conditions of steps (2), (3) and (4) are: the culture medium is alternately cultured at the temperature of 25+/-2 ℃ under the light intensity of 2000-3000 lx and the light irradiation is carried out for 10-14 hours every day.
Further: the cluster bud induction culture medium comprises the following formula: WPM+NAA 0.06 mg/L+2-iP 0.12 mg/L+ZT2.5 mg/L+activated carbon 0.5g/L.
Further: the formula of the secondary proliferation culture medium is as follows: improved SH+IBA 0.15mg/L+ZT 0.7mg/L+BR 0.06mg/L+GA 3 0.25mg/L+35 g/L apple puree.
Further: the rooting culture medium comprises the following formula: 1/2 modified SH+IBA 0.4mg/L+IAA 2.0 mg/L+PPP333.5 mg/L+apple puree 70 g/L+activated charcoal 0.3g/L.
The beneficial technical effects of the invention are as follows:
1. when a sterile system is established, conventional 75% alcohol and 0.1% mercuric chloride are adopted for sterilization, when the mercuric chloride sterilization time is too long, the pollution rate can be greatly reduced, but 0.1% mercuric chloride damages the michelia yunnanensis explants, the larger part of the michelia yunnanensis is directly dead, the rest part of the michelia yunnanensis is brown, the brown part is extremely serious and cannot grow, and when the time is too short, the death rate and the brown part can be greatly reduced, but the pollution rate is rapidly increased, and sometimes even the whole michelia yunnanensis is polluted. When the explant is disinfected, 0.5% potassium permanganate, 75% alcohol, 1% potassium permanganate and 0.05% mercuric chloride solution are adopted in sequence, so that the pollution rate is controlled within 32%, the death rate of the explant is controlled within 5%, meanwhile, active carbon with a certain concentration is added into a primary culture medium, the browning rate is controlled within 10%, and the induction of cluster buds is not influenced by both the browning and the active carbon.
2. In the invention, NAA, 2-iP and ZT with certain concentration are added into the primary cluster bud induction culture medium, so that the induction rate can be improved to more than 70%.
3. In the process of secondary multiplication culture, high-concentration 2-iP can cause vitrification, and when the concentration is reduced, no matter how the concentration of ZT is regulated, the proliferation coefficient is greatly reduced, while NAA has the effect of inhibiting rooting, when NAA is adopted as the secondary multiplication culture medium to carry out more than 2 generations, the rooting rate is greatly reduced when the rooting culture medium is adopted to carry out more than 2 generations, and the rooting rate is continuously reduced along with the increase of the number of times of the secondary multiplication culture, in the secondary multiplication culture medium, the primary NAA is changed into IBA with a certain concentration, and the ZT and BR with a certain concentration are simultaneously matched, so that the proliferation coefficient is stabilized to be more than 5, and the rooting rate in the subsequent rooting culture is not influenced, and GA with a certain concentration is added in the proliferation culture 3 The internode can be extracted, and the operation in the production process is convenient.
4. According to the invention, when the apple puree with a certain concentration is added in the secondary multiplication culture and rooting culture, the seedlings are more robust, the leaves are more fresh green, and especially when the apple puree is matched with PPP333 with a certain concentration in the rooting culture, the root system is more robust, and the survival rate of the seedling hardening is finally improved.
5. When the common WPM of the female parent plant is adopted as a basic culture medium, michelia yunnanensis is often represented by yellowing and falling of old leaves, and meanwhile, a small amount of Sharp burning phenomenon exists, so that the proliferation and quality of seedlings are seriously affected, but when SH is adopted, the seedling proliferation and quality can be obviously improved but cannot be completely eradicated (shown in figure 1), and when the using concentration of calcium chloride, ferric salt and inositol is changed on the basis of SH, the yellowing and falling and the Sharp burning can be completely eradicated (shown in figure 2).
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 shows fresh green leaves in rooting culture using modified SH medium;
FIG. 2 leaf sheath and old leaf yellowing off without modified SH medium;
FIG. 3 is a graph showing the primary induction results of example 1 of the present invention;
FIG. 4 is a graph showing the primary induction results of comparative example 8 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A tissue culture and rapid propagation method for Michelia yunnanensis comprises the following steps:
(1) Explant sterilization: taking a stem section of michelia yunnanensis which is not lignified at the end part, cleaning, and then sequentially disinfecting with 0.5% potassium permanganate for 15min and washing with sterile water for 5 times; sterilizing with 75% alcohol for 50s, and washing with sterile water for 2 times; 1% potassium permanganate for 5min, sterilized water for 4 times, 0.05% mercuric chloride for 5min, and sterilized water for 5 times.
(2) And (3) primary cluster bud induction: the sterilized explants were inoculated into a cluster bud induction medium of WPM+NAA 0.05 mg/L+2-iP 0.1 mg/L+ZT2.0 mg/L+activated carbon 0.4g/L, and were placed at 25.+ -. 2 ℃ for 10h under daily light and 2000lx light and dark alternate culture for 20 days, and the results are shown in FIG. 3.
(3) And (3) subculturing and proliferation: the cluster buds induced by the first generation are inoculated to the modified SH+IBA 0.1 mg/L+ZT0.5 mg/L+BR 0.05mg/L+GA 3 And culturing in 0.2mg/L and 30g/L of apple puree subculture medium at 25+ -2deg.C for 10h under light intensity of 2000lx for 25 days.
(4) Rooting culture: inoculating the secondary value-added cluster buds into a rooting culture medium with 1/2 modified SH+IBA 0.3mg/L+IAA 1.5 mg/L+PPP333.1.0 mg/L+apple puree 60 g/L+activated carbon 0.2g/L, and placing the rooting culture medium at the temperature of 25+/-2 ℃ for 10 hours under daily illumination, wherein the light and dark with the light intensity of 2000lx are alternately cultured for 30 days.
Wherein, the improved SH culture medium is: the dosage of calcium chloride is changed to 150mg/L, the dosage of EDTA-disodium is changed to 37.3mg/L, the dosage of ferrous sulfate is changed to 27.8mg/L, the dosage of inositol is changed to 100mg/L, and the rest components are unchanged.
Example 2
A tissue culture and rapid propagation method for Michelia yunnanensis comprises the following steps:
(1) Explant sterilization: taking a stem section of michelia yunnanensis which is not lignified at the end part, cleaning, and then sequentially disinfecting with 0.5% potassium permanganate for 20min and washing with sterile water for 5 times; sterilizing with 75% alcohol for 60s, and washing with sterile water for 3 times; 1% potassium permanganate for 6min, sterile water for 5 times, 0.05% mercuric chloride for 6min, and sterile water for 7 times.
(2) And (3) primary cluster bud induction: the sterilized explant is inoculated in a cluster bud induction culture medium of WPM+NAA 0.08 mg/L+2-iP 0.15mg/L+ZT 3.0 mg/L+activated carbon 0.6g/L, and is placed in a light-dark alternate culture medium with the temperature of 25+/-2 ℃ and the light intensity of 3000lx for 14 hours under daily illumination.
(3) And (3) subculturing and proliferation: the cluster buds induced by the first generation are inoculated to the modified SH+IBA 0.2 mg/L+ZT0.8 mg/L+BR 0.08mg/L+GA 3 And culturing in a subculture multiplication medium of 0.3mg/L and 40g/L apple puree at 25+ -2deg.C for 14h under light of 3000lx for 35 days.
(4) Rooting culture: inoculating the secondary value-added cluster buds into a rooting culture medium with 1/2 modified SH+IBA 0.5mg/L+IAA 2.0 mg/L+PPP333.2.0 mg/L+apple puree 80 g/L+activated carbon 0.4g/L, and placing the rooting culture medium at the temperature of 25+/-2 ℃ for 14 hours under daily illumination, and alternately culturing for 40 days under the light and dark with the light intensity of 3000 lx.
Wherein, the improved SH culture medium is: the dosage of calcium chloride is changed to 150mg/L, the dosage of EDTA-disodium is changed to 37.3mg/L, the dosage of ferrous sulfate is changed to 27.8mg/L, the dosage of inositol is changed to 100mg/L, and the rest components are unchanged.
Example 3
A tissue culture and rapid propagation method for Michelia yunnanensis comprises the following steps:
(1) Explant sterilization: taking a stem section of michelia yunnanensis which is not lignified at the end part, cleaning, and then sequentially disinfecting with 0.5% potassium permanganate for 18min and washing with sterile water for 4 times; sterilizing with 75% alcohol for 55s, and washing with sterile water for 3 times; 1% potassium permanganate for 5.5min, sterilized water for 4 times, 0.05% mercuric chloride for 5.5min, and sterilized water for 6 times.
(2) And (3) primary cluster bud induction: the sterilized explant is inoculated in a cluster bud induction culture medium of WPM+NAA 0.06 mg/L+2-iP 0.12mg/L+ZT 2.5 mg/L+activated carbon 0.5g/L, and is placed in a light-dark alternate culture medium with the temperature of 25+/-2 ℃ and the light intensity of 2500lx for 12 hours under daily illumination.
(3) And (3) subculturing and proliferation: the cluster buds induced by the first generation are inoculated to the modified SH+IBA 0.15 mg/L+ZT0.7 mg/L+BR 0.06mg/L+GA 3 And culturing in a subculture multiplication medium of 0.25mg/L and 35g/L apple puree at 25+ -2deg.C for 12h under light and dark at 2500lx for 30 days.
(4) Rooting culture: inoculating the secondary value-added cluster buds into a rooting culture medium with 1/2 modified SH+IBA 0.4mg/L+IAA 2.0 mg/L+PPP333.1.5 mg/L+apple puree 70 g/L+activated carbon 0.3g/L, and culturing for 35 days in an alternating manner at 25+/-2 ℃ under 12h of light per day and 2500lx of light and dark.
Wherein, the improved SH culture medium is: the dosage of calcium chloride is changed to 150mg/L, the dosage of EDTA-disodium is changed to 37.3mg/L, the dosage of ferrous sulfate is changed to 27.8mg/L, the dosage of inositol is changed to 100mg/L, and the rest components are unchanged.
Comparative example 1
Example 1 the stem segments of michelia yunnanensis which are not lignified at the ends are cleaned, sterilized, induced to differentiate, further cultivated in value-added culture and rooting culture according to the method disclosed in the patent "tissue culture propagation method of michelia yunnanensis" of late spring (publication No. CN 100429972A) example 1.
Comparative example 2
Example 1 Michelia yunnanensis was taken, and after washing, michelia yunnanensis was tissue cultured according to the method example disclosed in the patent "tissue culture propagation method for Michelia yunnanensis" (publication No. CN 103392601A), and no clumping buds were induced, and then proliferation and rooting culture were not performed.
Comparative example 3
The method disclosed in the patent "method for in vitro culture of regenerated plants of Michelia shilin" of the present invention (publication No. CN 103718969A) is carried out by taking the non-lignified stem at the end of Michelia yunnanensis of the present invention, washing, and then carrying out tissue culture on Michelia yunnanensis of the present invention.
Comparative example 4
Example 1 an end of michelia yunnanensis was taken and subjected to explant sterilization and primary induction culture according to the method of example 1 of the present invention, and then tissue culture was performed on michelia yunnanensis according to method example 1 disclosed in patent publication No. CN 112106664A.
Comparative example 5
The stem segments of Michelia yunnanensis of example 1 were taken and washed, and then subjected to explant sterilization, primary induction culture, secondary proliferation culture and rooting culture according to the best method disclosed in Jian "cutting propagation and tissue culture technique research of Michelia yunnanensis [ D ]. Nanjing forestry university, 2023.DOI:10.27242/d.cnki.gnjlu.2023.000286.
Comparative example 6
The stem segments of Michelia yunnanensis were obtained from the end of Michelia yunnanensis in example 1, the explants were sterilized and subjected to primary induction culture according to example 1, and then the Michelia yunnanensis was subjected to secondary proliferation culture and rooting culture according to Cheng Jiangjiang "tissue culture and rapid propagation System establishment of Michelia yunnanensis [ J ]. Forestry engineering journal, 2014, 28 (01): 118-121.DOI:10.13360/J. Issn.1000-8101.2014.01.031.
Comparative example 7
The best method disclosed in example 1, michelia yunnanensis was used for performing explant sterilization, primary induction culture, secondary proliferation culture and rooting culture on Michelia yunnanensis according to the tissue culture and plant regeneration of Michelia yunnanensis [ J ]. Plant physiology communication published by Li Xue, 2005, 41 (6): 1.DOI: CNKI: SUN: ZWSL.0.2005-06-027.
Comparative example 8
The results of the explant sterilization, primary induction culture, and secondary proliferation culture of michelia yunnanensis were performed according to the best method of Duting et al, "tissue culture rapid propagation technique and callus induction study of michelia yunnanensis university: nature science edition (2013)", and rooting culture was performed according to example 1 of the present invention, as shown in FIG. 4.
Table 1 comparative results
| Rate of disinfection pollution | Mortality rate of disinfection | Browning rate | Induction rate of cluster buds | Proliferation coefficient | Rooting rate | Rooting seedling state | Survival rate of hardening seedlings | |
| Example 1 | 22.6% | 12.2% | 5.3% | 76.8% | 5.5 | 81.6% | Robust and robust | 96.7% |
| Example 2 | 20.1% | 12.7% | 3.9% | 82.6% | 6.1 | 82.9% | Robust and robust | 97.3% |
| Example 3 | 28.8% | 13.6% | 7.2% | 84.7% | 6.9 | 80.2% | Robust and robust | 95.2% |
| Comparative example 1 | 21.3% | 46.4% | 69.0% | 21.3% | 2.1 | 41.2% | Weaker and weaker | 90.6% |
| Comparative example 2 | 17.1% | 49.3% | 72.6% | 0 | —— | —— | —— | —— |
| Comparative example 3 | 79.7% | 10.3% | 16.9% | 19.6% | 1.7 | 19.2% | Weaker and weaker | 87.6% |
| Comparative example 4 | 28.6% | 13.2% | 4.1% | 74.6% | 2.9 | 40.6% | Is stronger and stronger | 97.4% |
| Comparative example 5 | 74.9% | 0 | 3.6% | 19.4% | 1.2 | 3.9% | Germination is difficult | 94.3% |
| Comparative example 6 | 23.1% | 11.6% | 5.4% | 81.4% | 3.4 | 34.2% | Weaker and weaker | 84.2% |
| Comparative example 7 | 10.4% | 71.3% | 83.0% | 21.7% | 2.6 | 35.7% | Weaker and weaker | 92.9% |
| Comparative example 8 | 16.6% | 54.9% | 70.8% | 24.4% | 3.1 | 70.8% | Is stronger and stronger | 98.6% |
As can be seen from Table 1, in examples 1-3 of the present application, most of the indexes are significantly better than those of comparative examples 1-8, and although some indexes are slightly lower than those of comparative examples, the overall indexes are significantly better than those of comparative examples 1-8.
Finally, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, which is intended to be encompassed by the claims.
Claims (6)
1. The tissue culture and rapid propagation method for michelia yunnanensis is characterized by comprising the following steps of:
(1) Explant sterilization: taking the stem sections of michelia yunnanensis which are not lignified at the end parts, cleaning, and then sequentially disinfecting with 0.5% potassium permanganate for 15-20 min and washing with sterile water for 4-5 times; sterilizing with 75% alcohol for 50-60 s, and rinsing with sterile water for 2-3 times; sterilizing for 5-6 min by 1% potassium permanganate, rinsing for 4-5 times by sterile water, sterilizing for 5-6 min by 0.05% mercuric chloride, and rinsing for 5-7 times by sterile water;
(2) And (3) primary cluster bud induction: inoculating the sterilized explant into a cluster bud induction culture medium for culturing for 20-25 days;
the cluster bud induction culture medium comprises the following formula: WPM+NAA 0.05-0.08 mg/L+2-iP 0.1-0.15 mg/L+ZT 2.0-3.0 mg/L+activated carbon 0.4-0.6 g/L;
(3) And (3) subculturing and proliferation: inoculating cluster buds induced by the first generation into a secondary proliferation culture medium for culturing for 25-35 days;
the formula of the secondary proliferation culture medium is as follows: improved SH+IBA 0.1-0.2 mg/L+ZT 0.5-0.8 mg/L+BR 0.05-0.08 mg/L+GA 3 0.2-0.3 mg/L and 30-40 g/L of apple puree;
(4) Rooting culture: inoculating the cluster buds with the secondary multiplication value into a rooting culture medium for culturing for 30-40 days;
the rooting culture medium comprises the following formula: 1/2 modified SH+IBA 0.3-0.5 mg/L+IAA 1.5-2.0 mg/L+PPP333 1.0-2.0 mg/L+apple puree 60-80 g/L+active carbon 0.2-0.4 g/L;
the improved SH culture medium is as follows: the dosage of calcium chloride is changed to 150mg/L, the dosage of EDTA-disodium is changed to 37.3mg/L, the dosage of ferrous sulfate is changed to 27.8mg/L, the dosage of inositol is changed to 100mg/L, and the rest components are unchanged.
2. The method according to claim 1, wherein: the culture conditions of steps (2), (3) and (4) are: the culture medium is alternately cultured at the temperature of 25+/-2 ℃ under the light intensity of 2000-3000 lx and the light irradiation is carried out for 10-14 hours every day.
3. The method according to claim 1, wherein: the cluster bud induction culture medium comprises the following formula: WPM+NAA 0.06 mg/L+2-iP 0.12 mg/L+ZT2.5 mg/L+activated carbon 0.5g/L.
4. The method according to claim 1, wherein: the formula of the secondary proliferation culture medium is as follows: improved SH+IBA 0.15mg/L+ZT 0.7mg/L+BR 0.06mg/L+GA 3 0.25 mg/L+35 g/L apple puree.
5. The method according to claim 1, wherein: the rooting culture medium comprises the following formula: 1/2 modified SH+IBA 0.4mg/L+IAA 2.0 mg/L+PPP333.5 mg/L+apple puree 70 g/L+activated charcoal 0.3g/L.
6. Use of the method according to any one of claims 1-5 in tissue culture and rapid propagation of michelia yunnanensis.
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