CN111406655A - Davidia involucrata seedling and Davidia involucrata tissue culture method - Google Patents
Davidia involucrata seedling and Davidia involucrata tissue culture method Download PDFInfo
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- 241000759907 Davidia involucrata Species 0.000 title claims abstract description 39
- 238000012136 culture method Methods 0.000 title claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 57
- 238000012258 culturing Methods 0.000 claims abstract description 36
- 230000006698 induction Effects 0.000 claims abstract description 24
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 15
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 14
- 239000012879 subculture medium Substances 0.000 claims abstract description 13
- 239000000645 desinfectant Substances 0.000 claims abstract description 4
- 230000000249 desinfective effect Effects 0.000 claims abstract 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 claims description 51
- 229930006000 Sucrose Natural products 0.000 claims description 51
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 50
- 239000005720 sucrose Substances 0.000 claims description 50
- 238000005286 illumination Methods 0.000 claims description 41
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 40
- 235000013343 vitamin Nutrition 0.000 claims description 30
- 239000011782 vitamin Substances 0.000 claims description 30
- 229940088594 vitamin Drugs 0.000 claims description 30
- 229930003231 vitamin Natural products 0.000 claims description 30
- 229920001817 Agar Polymers 0.000 claims description 24
- 239000008272 agar Substances 0.000 claims description 24
- 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 description 22
- 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 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000003617 indole-3-acetic acid Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 16
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 claims description 14
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 claims description 14
- 229960001669 kinetin Drugs 0.000 claims description 14
- 238000002791 soaking Methods 0.000 claims description 14
- YNWVFADWVLCOPU-MDWZMJQESA-N (1E)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pent-1-en-3-ol Chemical compound C1=NC=NN1/C(C(O)C(C)(C)C)=C/C1=CC=C(Cl)C=C1 YNWVFADWVLCOPU-MDWZMJQESA-N 0.000 claims description 12
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 12
- 239000008223 sterile water Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 6
- 229960002233 benzalkonium bromide Drugs 0.000 claims description 6
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 claims description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- QOPBEBWGSGFROG-UHFFFAOYSA-N 2-(1h-indol-2-yl)acetic acid Chemical compound C1=CC=C2NC(CC(=O)O)=CC2=C1 QOPBEBWGSGFROG-UHFFFAOYSA-N 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- AXKBOWBNOCUNJL-UHFFFAOYSA-M sodium;2-nitrophenolate Chemical compound [Na+].[O-]C1=CC=CC=C1[N+]([O-])=O AXKBOWBNOCUNJL-UHFFFAOYSA-M 0.000 claims description 4
- 239000006870 ms-medium Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 6
- 230000004069 differentiation Effects 0.000 abstract description 3
- 229960004793 sucrose Drugs 0.000 description 43
- 210000001519 tissue Anatomy 0.000 description 29
- -1 compound sodium nitrophenolate Chemical class 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 4
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 4
- 239000006013 carbendazim Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 150000002596 lactones Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 235000002567 Capsicum annuum Nutrition 0.000 description 1
- 240000004160 Capsicum annuum Species 0.000 description 1
- 241000759904 Davidia Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
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- 238000009776 industrial production Methods 0.000 description 1
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- 230000002401 inhibitory effect Effects 0.000 description 1
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- 210000001161 mammalian embryo Anatomy 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
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 230000002195 synergetic effect 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
Abstract
The invention provides a Davidia involucrata tissue culture method, which comprises the following steps: (1) explant disinfection: selecting a spare explant of the Davidia involucrata Baill, and disinfecting the spare explant by using a disinfectant, wherein the disinfected spare explant is used as an explant for tissue culture; (2) and (3) induction culture: placing the explant treated in the step (1) in an induction culture medium for culture to obtain a primary culture seedling; (3) subculturing: placing the primary culture seedlings treated in the step (2) into a subculture medium for culturing to obtain subculture sprouts; (4) strong seedling and rooting culture: and (4) placing the subculture young buds obtained by culturing in the step (3) in a strong seedling culture medium for culturing, then placing the young buds in a rooting culture medium for culturing after the culturing is finished, and obtaining the Chinese dive tree seedling after the culturing is finished. The tissue culture method has the advantages of low pollution rate, good starting effect, high induced differentiation rate of starting culture and robust growth of tissue culture seedlings.
Description
Technical Field
The invention belongs to the technical field of tissue culture, and particularly relates to a Chinese dive tree tissue culture method and a Chinese dive tree seedling obtained by tissue culture.
Technical Field
The Davidia involucrata is a plant of Davidia of Davidiaceae, is a unique single-genus plant in China, is a variety of Davidia involucrata and Davidia involucrata pall, is a wiggery species of a third ancient tropical plant region system, is listed as a rare or endangered protective plant in China, and is called as 'activated stone'. The Davidia involucrata is a big fallen leaf tree, the crown of the Davidia involucrata is in a wide egg shape or a conical shape, and the tree shape is beautiful; the head-shaped inflorescence is dark brown, the two bracts with different sizes are covered outside, the bracts are changed from green to white when the inflorescence is mature, and the Chinese dive tree has extremely high ornamental value. The concern of foreign botanicals is brought up as early as 1986, and at the beginning of the 20 th century, foreign countries introduce Chinese dive trees from China and cultivate the Chinese dive trees widely. The Davidia involucrata Baill also has extremely high economic value, and the seeds and the peel of the Davidia involucrata Baill can extract oil; the endocarp can also be used for extracting essence; the trees can be used as high-quality raw materials of furniture; bark and pericarp can be used for extracting cayenne gelatin. The living environment range of Davidia involucrata is narrow and is only distributed in local areas of southwest of China.
In addition, the endocarp of Davidia involucrata is hard, the seed abortion phenomenon is serious, although no inhibiting substance exists in the embryo, the seed is difficult to break through the pericarp to develop into seedlings. Due to the above reasons, Davidia involucrata is difficult to be widely applied to landscaping. If the planting of the Chinese dive tree seedlings is successful, the propagation progress of the Chinese dive tree seedlings is accelerated by combining the plant tissue culture technology, the large-scale production of endangered plants can be realized, the application value of the seedlings can be fully utilized, and the resources and population expansion of rare species can be protected.
Disclosure of Invention
The invention aims to provide a tissue culture method which has the characteristics of high propagation speed, high success rate and low probability of tissue browning.
The specific scheme is as follows:
a Davidia involucrata tissue culture method comprises the following steps:
(1) explant sterilization
Selecting a spare explant of the Davidia involucrata Baill, and disinfecting the spare explant by using a disinfectant, wherein the disinfected spare explant is used as an explant for tissue culture;
(2) induced culture
Placing the explant treated in the step (1) in an induction culture medium for culture to obtain a primary culture seedling;
(3) subculture
Placing the primary culture seedlings treated in the step (2) into a subculture medium for culturing to obtain subculture sprouts;
(4) strong seedling and rooting culture
And (4) placing the subculture young buds obtained by culturing in the step (3) in a strong seedling culture medium for culturing, then placing the young buds in a rooting culture medium for culturing after the culturing is finished, and obtaining the Chinese dive tree seedling after the culturing is finished.
In a specific embodiment of the present invention, (1) the spare explant is selected from leaves, winter buds, stem segments with buds or young shoots.
In a specific embodiment of the invention, (1), the disinfection treatment comprises multi-stage disinfection, firstly soaking the spare explant in 800-; soaking the washed explant in 70-75% alcohol for 50-80s, placing in 0.02-0.04% sodium hypochlorite solution for 5-10min, taking out, washing with sterile water, placing in 0.1-0.2% benzalkonium bromide solution for 10-15min, taking out, washing with sterile water for several times, and removing water for later use.
In one embodiment of the present invention, (2) the induction medium is an MS medium containing vitamins, naphthylacetic acid, kinetin and sucrose, wherein the concentrations of the vitamins, naphthylacetic acid, kinetin and sucrose are 0.5-1.0 mg/L, 0.05-0.1 mg/L, 0.03-0.08 mg/L and 10-20 g/L, respectively.
In the specific embodiment of the invention, (2) the temperature of induction culture is 18-25 ℃, the humidity is 55-65%, the illumination intensity is 1800-; the induction culture time is 12-15 days.
In a specific embodiment of the invention, (3) the subculture medium is an AS medium containing vitamins, naphthylacetic acid, indolacetic acid, sodium nitrophenolate complex and sucrose, wherein the concentrations of the vitamins, the naphthylacetic acid, the indolacetic acid, the sodium nitrophenolate complex and the sucrose are 0.5-1.0 mg/L, 0.1-0.2 mg/L, 0.05-0.15 mg/L, 0.002-0.005 mg/L and 15-25 g/L, respectively.
In the specific embodiment of the invention, (3) the temperature of the subculture is 20-25 ℃, the humidity is 60-70%, the illumination intensity is 2000-; the subculture time is 15-18 d.
In a specific embodiment of the invention, (4) the strong seedling culture medium is an MS culture medium containing brassinolide, uniconazole, naphthylacetic acid, sucrose and agar, wherein the concentrations of the brassinolide, the uniconazole, the naphthylacetic acid, the sucrose and the agar are respectively 0.05-0.5 mg/L, 0.05-0.2 mg/L, 0.05-0.15 mg/L, 10-20 g/L and 10-25 g/L;
the rooting culture medium is an MS culture medium containing brassinolide, indoleacetic acid, indolebutyric acid, naphthylacetic acid, sucrose and agar, wherein the concentrations of the brassinolide, the indoleacetic acid, the indolebutyric acid, the naphthylacetic acid, the sucrose and the agar are respectively 0.05-0.3 mg/L, 0.01-0.05 mg/L, 0.01-0.05 mg/L, 0.1-0.2 mg/L, 10-20 g/L and 10-25 g/L.
In the step (4), the temperature for culturing the strong seedlings or the roots is 23-26 ℃, the humidity is 55-65%, the illumination intensity is 1500-; the strong seedling culture time is 8-10 days, and the rooting culture time is 10-13 days.
The invention also provides a Chinese dive tree seedling which is prepared by any one of the Chinese dive tree tissue culture methods.
The MS culture medium and the AS culture medium are commercially available culture media, for example, the MS culture medium can be selected from Beijing Kerui base biological technology, Inc., and the AS culture medium can be selected from Shanghai Yuanmu biological technology, Inc.
Vitamins in this application refer to either single vitamins or a complex of multiple vitamins.
The compound sodium nitrophenolate is a powerful cell activator, can quickly permeate after contacting with the Chinese dive tree tissue, promotes the protoplasm flow of cells, and improves the cell activity. Promote the absorption of various hormones and nutrient components in the tissue culture process.
Vitamins, naphthylacetic acid, kinetin and cane sugar naphthylacetic acid, indoleacetic acid, compound sodium nitrophenolate, brassinolide, uniconazole and indolebutyric acid
Compared with the prior art, the invention has the beneficial effects that:
compared with the traditional seed sowing and propagation method, the method has the characteristics of higher propagation speed, higher propagation efficiency and wide propagation objects, and can be applied to tissue culture of tissues including leaves, winter buds, stem sections with buds or young shoots, so that the propagation objects are enriched.
Secondly, the invention avoids the influence of interference substances such as germs carried by explants on tissue culture by multi-level disinfection treatment, thereby reducing the occurrence of davidia involucrata tissue browning. According to the invention, different hormones and other substances are added at each stage and are matched with corresponding culture media in a synergistic manner, so that the browning of the callus is further reduced or avoided.
The method is beneficial to industrial production, and greatly saves manpower and material resources; has great popularization value in the field of Davidia involucrata seedling culture.
The tissue culture method has the advantages of low pollution rate, good starting effect, high induced differentiation rate of starting culture and strong growth of tissue culture seedlings.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In the present application, the raw materials and auxiliary materials may be commercially available without specific claims.
Example 1
A Davidia involucrata tissue culture method comprises the following steps:
(1) explant sterilization
Selecting Davidia involucrata leaf as a spare explant, soaking the spare explant in 800 times of carbendazim solution for 15min, taking out and washing with water for 15 min; soaking the washed explant in 70% alcohol for 50 min, placing in 0.02% sodium hypochlorite solution for 5min, taking out, washing with sterile water, finally placing in 0.1% benzalkonium bromide solution for 10min, taking out, washing with sterile water for multiple times, and removing water for later use to obtain the explant for tissue culture;
(2) induced culture
Placing the explant treated in the step (1) into an induction culture medium for culturing, wherein the induction culture medium is an MS culture medium containing vitamins, naphthylacetic acid, kinetin and sucrose, and the concentrations of the vitamins, the naphthylacetic acid, the kinetin and the sucrose are respectively 0.5 mg/L, 0.05 mg/L, 0.03 mg/L and 10 g/L, the temperature of culturing is 18 ℃, the humidity is 55%, the illumination intensity is 1800lx, the total illumination time per day is 10h, the illumination is carried out twice, the interval between two times is 1h, the whole induction culture time is 12d, and finally obtaining a primary culture seedling;
(3) subculture
Placing the primary culture seedlings treated in the step (2) into a subculture medium for culturing, wherein the subculture medium is an AS culture medium containing vitamins, naphthylacetic acid, indoleacetic acid, compound sodium nitrophenolate and sucrose, and the concentrations of the vitamins, the naphthylacetic acid, the indoleacetic acid, the compound sodium nitrophenolate and the sucrose are respectively 0.5 mg/L, 0.1 mg/L, 0.05 mg/L, 0.002 mg/L and 15 g/L, the temperature of the culture is 20 ℃, the humidity is 60%, the illumination intensity is 2000lx, the total illumination time per day is 10h, the illumination is divided into two times, the interval is 1h, the whole subculture time is 15d, and finally obtaining subculture seedlings;
(4) strong seedling and rooting culture
Placing the subculture young buds obtained by the culture in the step (3) into a strong seedling culture medium for culture, wherein the strong seedling culture medium is an MS culture medium containing brassinolide, uniconazole, naphthylacetic acid, sucrose and agar, the concentrations of the brassinolide, the uniconazole, the naphthylacetic acid, the sucrose and the agar are respectively 0.3 mg/L, 0.15 mg/L, 0.1 mg/L0, 15 g/L and 20 g/L, after the culture is finished, the young buds are placed into a rooting culture medium for culture, the rooting culture medium is an MS culture medium containing the brassinolide, the indoleacetic acid, the indolebutyric acid, the naphthylacetic acid, the sucrose and the agar, the concentrations of the brassinolide, the indoleacetic acid, the indolebutyric acid, the sucrose and the agar are respectively 0.2 mg/L, 0.03 mg/L, 0.04 mg/L, 0.15 mg/L, 15 g/L and 20 g/L, the total illumination time length is 1600 hours, the total illumination time length is 5 hours, and the strong seedling culture can be obtained after the culture is finished.
Example 2
A Davidia involucrata tissue culture method comprises the following steps:
(1) explant sterilization
Selecting Davidia involucrata winter bud as a spare explant, soaking the spare explant with 1000 times of carbendazim solution for 20min, taking out and washing with water for 20 min; soaking the washed explant in 70% alcohol for 70s, placing the explant in a sodium hypochlorite solution with the concentration of 0.03% for 8min, taking out the explant, washing the explant with sterile water, finally placing the explant in a new benzalkonium bromide solution with the concentration of 0.15% for 14min, taking out the explant, washing the explant with sterile water for multiple times, and removing water for later use to obtain the explant for tissue culture;
(2) induced culture
Placing the explant treated in the step (1) into an induction culture medium for culturing, wherein the induction culture medium is an MS culture medium containing vitamins, naphthylacetic acid, kinetin and sucrose, and the concentrations of the vitamins, the naphthylacetic acid, the kinetin and the sucrose are respectively 0.8 mg/L, 0.08 mg/L, 0.05 mg/L and 18 g/L, the temperature of culturing is 20 ℃, the humidity is 60%, the illumination intensity is 2000lx, the total illumination time per day is 11h, the illumination is divided into two times, the interval is 1.5h, the whole induction culture time is 14d, and finally obtaining a primary culture seedling;
(3) subculture
Placing the primary culture seedlings treated in the step (2) into a subculture medium for culturing, wherein the subculture medium is an AS culture medium containing vitamins, naphthylacetic acid, indoleacetic acid, compound sodium nitrophenolate and sucrose, and the concentrations of the vitamins, the naphthylacetic acid, the indoleacetic acid, the compound sodium nitrophenolate and the sucrose are respectively 0.8 mg/L, 0.15 mg/L, 0.10 mg/L, 0.004 mg/L and 20 g/L, the culture temperature is 25 ℃, the humidity is 65%, the illumination intensity is 2000lx, the total illumination time per day is 10h, the illumination is divided into two times, the interval is 1.5h, the whole subculture time is 17d, and finally obtaining subculture sprouts;
(4) strong seedling and rooting culture
Placing the subculture young buds obtained by culturing in the step (3) into a strong seedling culture medium for culturing, wherein the strong seedling culture medium is an MS culture medium containing brassinolide, uniconazole, naphthylacetic acid, sucrose and agar, the concentrations of the brassinolide, the uniconazole, the naphthylacetic acid, the sucrose and the agar are respectively 0.05 mg/L, 0.05 mg/L, 0.05 mg/L0, 10 g/L and 10 g/L, after the culturing is finished, the young buds are placed into a rooting culture medium for culturing, the rooting culture medium is an MS culture medium containing the brassinolide, the indoleacetic acid, the indolebutyric acid, the naphthylacetic acid, the sucrose and the agar, the concentrations of the davidiana lactone, the indoleacetic acid, the naphthylacetic acid, the sucrose and the agar are respectively 0.05 mg/L, 0.01 mg/L, 0.01 mg/L, 0.1 mg/L, 10 g/L and 10 g/L, the concentrations of the davidian lactone, the total light intensity or the intermediate illumination intensity is 23% and the total illumination time length is 1 hour after the strong seedling culture is finished, and the whole strong seedling culture time length is 1 d, and the whole strong seedling culture time is obtained after the whole seedling culture is finished.
Example 3
A Davidia involucrata tissue culture method comprises the following steps:
(1) explant sterilization
Selecting young tips of Davidia involucrata as a spare explant, soaking the spare explant in 1500 times of carbendazim solution for 30min, taking out and washing with water for 25 min; soaking the washed explant in 75% alcohol for 80s, placing the explant in a sodium hypochlorite solution with the concentration of 0.04% for 10min, taking out the explant, washing the explant with sterile water, finally placing the explant in a new benzalkonium bromide solution with the concentration of 0.2% for 15min, taking out the explant, washing the explant with sterile water for multiple times, and removing water for later use to obtain the explant for tissue culture;
(2) induced culture
Placing the explant treated in the step (1) into an induction culture medium for culturing, wherein the induction culture medium is an MS culture medium containing vitamins, naphthylacetic acid, kinetin and sucrose, and the concentrations of the vitamins, the naphthylacetic acid, the kinetin and the sucrose are respectively 1.0 mg/L, 0.1 mg/L, 0.08 mg/L and 20 g/L, the temperature of culturing is 25 ℃, the humidity is 65%, the illumination intensity is 2100lx, the total illumination time per day is 12h, the illumination is carried out twice, the interval is 2h, the whole induction culture time is 15d, and finally obtaining a primary culture seedling;
(3) subculture
Placing the primary culture seedlings treated in the step (2) into a subculture medium for culturing, wherein the subculture medium is an AS culture medium containing vitamins, naphthylacetic acid, indoleacetic acid, compound sodium nitrophenolate and sucrose, and the concentrations of the vitamins, the naphthylacetic acid, the indoleacetic acid, the compound sodium nitrophenolate and the sucrose are respectively 1.0 mg/L, 0.2 mg/L, 0.15 mg/L, 0.005 mg/L and 25 g/L, the culture temperature is 25 ℃, the humidity is 70%, the illumination intensity is 2300lx, the total illumination duration of each day is 12h, the illumination is divided into two times, the interval is 2h, the whole subculture duration is 18d, and finally obtaining subculture sprouts;
(4) strong seedling and rooting culture
Placing the subculture young buds obtained by the culture in the step (3) into a strong seedling culture medium for culture, wherein the strong seedling culture medium is an MS culture medium containing brassinolide, uniconazole, naphthylacetic acid, sucrose and agar, the concentrations of the brassinolide, the uniconazole, the naphthylacetic acid, the sucrose and the agar are respectively 0.5 mg/L, 0.2 mg/L, 0.15 mg/L0, 20 g/L and 25 g/L, after the culture is finished, the young buds are placed into a rooting culture medium for culture, the rooting culture medium is an MS culture medium containing the brassinolide, the indoleacetic acid, the indolebutyric acid, the naphthylacetic acid, the sucrose and the agar, the concentrations of the brassinolide, the indoleacetic acid, the indolebutyric acid, the sucrose and the agar are respectively 0.3 mg/L, 0.05 mg/L, 0.05 mg/L, 0.2 mg/L, 20 g/L and 25 g/L, the concentrations of the davidian lactone, the temperature or the light intensity are respectively, the light intensity is 1700%, the total light intensity is 1700-10 l seedling culture time length after the culture is finished, and the whole strong seedling culture is carried out for two times, and the illumination time is 16-10 hours, and the whole strong seedling culture is obtained.
Comparative example 1
This comparative example is that of example 1.
The culture medium after induction culture does not contain kinetin, only contains vitamins, naphthylacetic acid and sucrose, and has constant concentration;
the culture medium in the subculture does not contain the combination of indoleacetic acid and compound sodium nitrophenolate, only contains vitamins, naphthylacetic acid and sucrose, and has constant concentration;
in the seedling strengthening and rooting culture, a seedling strengthening culture medium does not contain brassinolide and uniconazole, a rooting culture medium does not contain brassinolide, and other components are the same and have unchanged concentration.
The specific operation is as follows:
a Davidia involucrata tissue culture method comprises the following steps:
(1) explant sterilization
Selecting Davidia involucrata leaf as a spare explant, soaking the spare explant in 800 times of carbendazim solution for 15min, taking out and washing with water for 15 min; soaking the washed explant in 70% alcohol for 50 min, placing in 0.02% sodium hypochlorite solution for 5min, taking out, washing with sterile water, finally placing in 0.1% benzalkonium bromide solution for 10min, taking out, washing with sterile water for multiple times, and removing water for later use to obtain the explant for tissue culture;
(2) induced culture
Placing the explant treated in the step (1) into an induction culture medium for culturing, wherein the induction culture medium is an MS culture medium containing vitamins, naphthylacetic acid, kinetin and sucrose, and the concentrations of the vitamins, the naphthylacetic acid, the kinetin and the sucrose are respectively 0.5 mg/L, 0.05 mg/L and 10 g/L, the temperature of culturing is 18 ℃, the humidity is 55%, the illumination intensity is 1800lx, the total illumination time per day is 10h, the illumination is carried out twice, the interval between two times is 1h, the whole induction culture time is 12d, and finally obtaining a primary culture seedling;
(3) subculture
Placing the primary culture seedlings treated in the step (2) into a subculture medium for culturing, wherein the subculture medium is an AS culture medium containing vitamins, naphthylacetic acid and sucrose, and the concentrations of the vitamins, the naphthylacetic acid and the sucrose are 0.5 mg/L, 0.1 mg/L and 15 g/L respectively, the culturing temperature is 20 ℃, the humidity is 60%, the illumination intensity is 2000lx, the total illumination time per day is 10h, the illumination is carried out twice, the interval between two illumination times is 1h, the whole subculture time is 15d, and finally subculture sprouts are obtained;
(4) strong seedling and rooting culture
Placing the subculture young buds obtained by the culture in the step (3) in a strong seedling culture medium for culture, wherein the strong seedling culture medium is an MS culture medium containing naphthylacetic acid, sucrose and agar, the concentrations of the naphthylacetic acid, the sucrose and the agar are respectively 0.1 mg/L, 15 g/L and 20 g/L, after the culture is finished, placing the young buds in a rooting culture medium for culture, the rooting culture medium is an MS culture medium containing indoleacetic acid, indolebutyric acid, naphthylacetic acid, sucrose and agar, the concentrations of the indoleacetic acid, the indolebutyric acid, the naphthylacetic acid, the sucrose and the agar are respectively 0.03 mg/L, 0.04 mg/L, 0.15 mg/L, 15 g/L and 20 g/L, the strong seedling or rooting culture conditions are all, the temperature is 25 ℃, the humidity is 60%, the illumination intensity is 1600lx, the total illumination time length is 15h every day, the split illumination is 1.5h, the whole strong seedling culture is 9d, the rooting culture is 12d, and the young Chinese dive seedlings can be obtained after the rooting culture is finished.
TABLE 1 Effect of different hormone combinations in combination with culture Medium on callus
Comparative example 2
This comparative example is that of example 2.
The induction culture adopts WPM culture medium; subculture with N6A culture medium; the WPM culture medium is adopted for strong seedling and rooting culture.
TABLE 2 Effect of different basic media on callus
Comparative example 3
This comparative example is that of example 2.
The common disinfection method is adopted, 75% alcohol is adopted for soaking for 50-80s, 0.1% mercuric chloride is adopted for soaking for 5-10min, and other treatment methods are unchanged.
The infection rate (pollution number/inoculation number × 100%) and the death rate (death number/inoculation number × 100%) of the davidia involucrata explants were counted after 30 days of disinfection with different disinfectants, and the results are shown in table 3.
TABLE 3 Effect of different Disinfection methods on callus
The applicant experiments show that the tissue culture method can be applied to different explants, and other experimental results which are not listed are similar to or the same as the above listed results of the tissue culture method, so that the tissue culture method has the advantages of low pollution rate, high induced differentiation rate of starting culture, robust tissue culture seedling growth and browning grade not exceeding 1 grade.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A Davidia involucrata tissue culture method is characterized by comprising the following steps:
(1) explant sterilization
Selecting a spare explant of the Davidia involucrata Baill, and disinfecting the spare explant by using a disinfectant, wherein the disinfected spare explant is used as an explant for tissue culture;
(2) induced culture
Placing the explant treated in the step (1) in an induction culture medium for culture to obtain a primary culture seedling;
(3) subculture
Placing the primary culture seedlings treated in the step (2) into a subculture medium for culturing to obtain subculture sprouts;
(4) strong seedling and rooting culture
And (4) placing the subculture young buds obtained by culturing in the step (3) in a strong seedling culture medium for culturing, then placing the young buds in a rooting culture medium for culturing after the culturing is finished, and obtaining the Chinese dive tree seedling after the culturing is finished.
2. The method for tissue culture of davidia involucrata according to claim 1, wherein: (1) wherein the spare explant is selected from leaves, winter shoots, stem segments, budded stem segments or shoots.
3. The method for tissue culture of davidia involucrata according to claim 1, wherein: (1) wherein the disinfection treatment comprises multi-stage disinfection, firstly soaking the standby explant by 800-; soaking the washed explant in 70-75% alcohol for 50-80s, placing in 0.02-0.04% sodium hypochlorite solution for 5-10min, taking out, washing with sterile water, placing in 0.1-0.2% benzalkonium bromide solution for 10-15min, taking out, washing with sterile water for several times, and removing water for later use.
4. The method for tissue culture of Davidia involucrata of claim 1, characterized in that in (2), the induction medium is MS medium containing vitamins, naphthylacetic acid, kinetin and sucrose, wherein the concentrations of the vitamins, naphthylacetic acid, kinetin and sucrose are 0.5-1.0 mg/L, 0.05-0.1 mg/L, 0.03-0.08 mg/L and 10-20 g/L, respectively.
5. The method for tissue culture of davidia involucrata according to claim 1, wherein: (2) wherein the temperature of the induction culture is 18-25 ℃, the humidity is 55-65%, the illumination intensity is 1800 and 2100lx, the total illumination time per day is 10-12h, the illumination is divided into two times, and the interval is 1-2 h; the induction culture time is 12-15 days.
6. The method for tissue culture of Davidia involucrata of claim 1, wherein in (3), the subculture medium is an AS medium containing vitamins, naphthylacetic acid, indolacetic acid, sodium nitrophenolate and sucrose, wherein the concentrations of the vitamins, naphthylacetic acid, indolacetic acid, sodium nitrophenolate and sucrose are 0.5-1.0 mg/L, 0.1-0.2 mg/L, 0.05-0.15 mg/L, 0.002-0.005 mg/L and 15-25 g/L, respectively.
7. The method for tissue culture of davidia involucrata according to claim 1, wherein: (3) in the process, the temperature of the subculture is 20-25 ℃, the humidity is 60-70%, the illumination intensity is 2000-; the subculture time is 15-18 d.
8. The method for tissue culture of Davidia involucrata of claim 1, wherein in (4), the strong seedling culture medium is an MS culture medium containing brassinolide, uniconazole, naphthylacetic acid, sucrose and agar, and the concentrations of the brassinolide, the uniconazole, the naphthylacetic acid, the sucrose and the agar are 0.05-0.5 mg/L, 0.05-0.2 mg/L, 0.05-0.15 mg/L, 10-20 g/L and 10-25 g/L respectively;
the rooting culture medium is an MS culture medium containing brassinolide, indoleacetic acid, indolebutyric acid, naphthylacetic acid, sucrose and agar, wherein the concentrations of the brassinolide, the indoleacetic acid, the indolebutyric acid, the naphthylacetic acid, the sucrose and the agar are respectively 0.05-0.3 mg/L, 0.01-0.05 mg/L, 0.01-0.05 mg/L, 0.1-0.2 mg/L, 10-20 g/L and 10-25 g/L.
9. The method for tissue culture of davidia involucrata according to claim 1, wherein: (4) in the method, the temperature for strong seedling or rooting culture is 23-26 ℃, the humidity is 55-65%, the illumination intensity is 1500-; the strong seedling culture time is 8-10 days, and the rooting culture time is 10-13 days.
10. A Chinese dive tree seedling prepared by the method for tissue culture of Chinese dive tree according to any one of claims 1 to 9.
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