CN114303952B - Water lily tuber induction tissue culture rapid propagation method - Google Patents
Water lily tuber induction tissue culture rapid propagation method Download PDFInfo
- Publication number
- CN114303952B CN114303952B CN202111675282.1A CN202111675282A CN114303952B CN 114303952 B CN114303952 B CN 114303952B CN 202111675282 A CN202111675282 A CN 202111675282A CN 114303952 B CN114303952 B CN 114303952B
- Authority
- CN
- China
- Prior art keywords
- culture
- culture medium
- water lily
- induction
- rooting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000209490 Nymphaea Species 0.000 title claims abstract description 87
- 235000016791 Nymphaea odorata subsp odorata Nutrition 0.000 title claims abstract description 87
- 230000006698 induction Effects 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 39
- 239000001963 growth medium Substances 0.000 claims abstract description 65
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 25
- 230000035755 proliferation Effects 0.000 claims abstract description 21
- 230000001939 inductive effect Effects 0.000 claims abstract description 18
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 14
- 239000003899 bactericide agent Substances 0.000 claims abstract description 14
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 11
- 230000000249 desinfective effect Effects 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 230000012010 growth Effects 0.000 claims description 18
- 238000005286 illumination Methods 0.000 claims description 17
- 239000008223 sterile water Substances 0.000 claims description 16
- 238000012258 culturing 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
- 238000005406 washing Methods 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000010802 sludge Substances 0.000 claims description 5
- 238000002791 soaking Methods 0.000 claims description 5
- 239000007640 basal medium Substances 0.000 claims description 3
- 239000004927 clay Substances 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 2
- 239000012882 rooting medium Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 8
- 238000012360 testing method Methods 0.000 description 16
- 241000196324 Embryophyta Species 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- 239000005648 plant growth regulator Substances 0.000 description 9
- 238000011282 treatment Methods 0.000 description 8
- 239000005556 hormone Substances 0.000 description 5
- 229940088597 hormone Drugs 0.000 description 5
- 239000000645 desinfectant Substances 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000000855 fungicidal effect Effects 0.000 description 3
- 239000000417 fungicide Substances 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- 241000587753 Nymphaea tetragona Species 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000004161 plant tissue culture Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 240000008892 Helianthus tuberosus Species 0.000 description 1
- 235000003230 Helianthus tuberosus Nutrition 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000028446 budding cell bud growth Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for inducing tissue culture and rapid propagation of water lily tubers, which comprises the following steps: (1) explant disinfection: taking water lily tubers as explants, and disinfecting the explants; (2) inducing: inoculating the sterilized explant into an induction culture medium, and then carrying out induction culture; the induction culture medium contains 6-BA, IBA and S106 bactericides; (3) propagation culture: inoculating the bud strains obtained by induction into a proliferation culture medium, and then performing proliferation culture; the proliferation culture medium contains 6-BA, IBA and S106 bactericides; (4) rooting culture: inoculating the obtained bud seedling into a rooting culture medium for rooting culture; the rooting culture medium contains IBA; and (5) hardening seedlings. The invention successfully establishes the water lily tissue culture system with tubers as explants, the induction rate and the rooting rate are both as high as 80 percent, and the time consumption in the process is short.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for rapid propagation of water lily tubers through induced tissue culture.
Background
Nymphaea tetragona (Nymphaea tetragona) is a perennial leafy aquatic herbaceous plant, and is mainly propagated by plants in a separated mode, so that the price of a water lily seedling is high due to the limited propagation quantity and the like, and the industrialization and large-scale development of the water lily are restricted. Tissue culture of water lily is an effective means for obtaining a large number of seedlings in a short period, however, tissue culture research of water lily is only reported so far, and the problems of serious pollution, low induction rate and rooting rate, low proliferation and rooting speed and the like in the water lily tissue culture process cannot be effectively solved.
In the aspect of explant disinfection, because different plants are different in pollution microorganisms involved in the tissue culture process, the disinfection method adopted in the tissue culture process of other plants is not very suitable for the disinfection of water lily explants. The whole growth cycle of the water lily grows in the water body environment, and the roots and the stems grow in the sludge, so that the water lily explants are associated with a large amount of fungi and bacteria and a large amount of endophytes, and the microorganisms are not easy to remove, thereby bringing great difficulty to the tissue culture work of the water lily. Qi Huasha and others use the rhizomes, roots, leaves, petioles and seeds of blue drug water lily as explants to perform disinfection test studies. The results show that: the adopted rhizomes and seeds have good disinfection effect. However, when the rhizome is used as the explant, the pollution rate is still as high as 70%, and the browning rate is as high as 45%.
Patent document CN104488716a discloses a method for tissue culture of water lily, which comprises the following specific operation steps: 1) Preparing a culture medium, including a basic culture medium and components of the culture medium at each stage of tissue culture; 2) Selecting and disinfecting explants; 3) Induction and proliferation of adventitious buds; 4) Rooting culture; 5) And (5) transplanting. The method utilizes plant tissue culture technology to perform adventitious bud induction and multiplication culture on the water lily, can obtain a large number of high-quality strong seedlings with consistent genetic characters in a short time, and overcomes the defect of slow conventional propagation method. However, the induced culture of the plant has buds after 1-2 months, and the rooting effect is still to be further improved.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a method for inducing tissue culture and rapid propagation of water lily tubers.
The technical scheme of the invention mainly comprises the following contents:
a method for inducing tissue culture and rapid propagation of water lily tubers comprises the following steps:
(1) Explant disinfection: sterilizing the water lily tuber as an explant;
(2) Induction: inoculating the explant sterilized in the step (1) into an induction culture medium, and then carrying out induction culture;
the induction culture medium is prepared by adding 2.0-3.0 mg/L of 6-BA, 0.5-1.0 mg/L of IBA and 0.5-1.0 mL/L S bactericide into a basal culture medium;
(3) And (3) proliferation culture: inoculating the bud strains obtained by the induction in the step (2) into a proliferation culture medium, and then performing proliferation culture;
the proliferation culture medium is prepared by adding 2.0-3.0 mg/L6-BA, 0.5-1.0 mg/L IBA and 0.5-1.0 mL/L S bactericide in a basal culture medium;
(4) Rooting culture: inoculating the bud seedling obtained after the culture in the step (3) into a rooting culture medium for rooting culture;
the rooting culture medium is obtained by adding 0.5-1.0 mg/L IBA into a basic culture medium;
(5) And (5) hardening seedlings.
Preferably, the basic culture medium is MS culture medium or 1/2MS culture medium.
One of the difficult problems in the water lily tissue culture process is fungus pollution, and the reduction of the pollution rate is the premise of establishing a water lily tissue culture rapid propagation system. The invention has been tested to determine the optimal disinfection method. The disinfection method comprises the following steps: soaking the cleaned explant in 75 vol% concentration alcohol solution for 1min, sterilizing and washing with water for 3-5 times; adding the explant into mercuric chloride solution with mass concentration of 0.1%, placing the explant into a shaking table with 100rpm for shaking for 15min, pouring the mercuric chloride solution, cleaning the explant with sterile water for 3-5 min, soaking the explant with sterile water for 5min, and cleaning the explant with sterile water for 3-5 times.
Preferably, in step (2), the conditions for inducing culture are: sterile water is added into the induction culture medium to simulate the growth environment of the water lily; placing the induction culture medium in a 28 ℃ culture room for dark culture for 5-7 days; then placing the mixture at the temperature of 28 ℃, the illumination intensity of 4000lx and the photoperiod of 10-14 h/d for continuous culture.
Preferably, in step (3), the conditions of the propagation culture are: sterile water is added into the proliferation culture medium to simulate the growth environment of the water lily; placing the proliferation culture medium in a 28 ℃ culture room for dark culture for 2-3 days; then placing the mixture at 28 ℃, the illumination intensity of 4000lx and the photoperiod of 10-14 h/d for continuous culture.
Preferably, in the step (4), the rooting culture conditions are as follows: placing the rooting culture medium in a 28 ℃ culture room for dark culture for 2-3 d; then placing the mixture at the temperature of 28 ℃, the illumination intensity of 4000lx and the photoperiod of 10-14 h/d for continuous culture.
Preferably, in the step (5), the seedling exercising step is as follows: and (4) culturing the rooted seedlings obtained after the culture in the step (4) in a shading place for 1 week, then transferring to the light for 1 week, removing the culture medium at the root parts of the seedlings, planting the seedlings in a non-porous flowerpot filled with clay or sludge for culture, and transferring to the sun for culture after shading culture until new leaves grow out.
Preferably, the induction culture medium is a basal culture medium added with 2.0 mg/L6-BA, 0.5mg/L IBA and 0.5mL/LS106 bactericide.
Preferably, the proliferation culture medium is a basal culture medium added with 3.0 mg/L6-BA, 0.5mg/L IBA and 1.0mL/L S bactericide.
Preferably, the rooting medium is a basal medium added with 0.5mg/L IBA.
The invention has the following beneficial effects:
the selection of explants is an important factor influencing the success of water lily tissue culture, and the difficulty of tissue culture work of different explants of different plants is different. The invention successfully establishes the water lily tissue culture system with tubers as explants.
The invention takes the water lily tubers as explants, directly induces and generates cluster buds, and then obtains tissue culture seedlings through cluster bud induction and rooting culture, no callus is generated in the process, the whole tissue culture process consumes short time and is simple and convenient to operate.
In the known technology, the sterilizing effect on the water lily stems is not ideal, and the pollution rate and the browning rate are high. By adopting the disinfection method provided by the invention (2min +0.1% mercuric chloride for 15min with 75% alcohol and S106 bactericide added in the culture medium), the water lily tubers are disinfected, the average pollution rate is as low as 30%, the browning rate is as low as 25%, and the disinfection effect is better.
The S106 bactericide is used in the induction and proliferation stages of the water lily tissue culture for the first time, so that the pollution rate is reduced, and the improvement of the induction rate is promoted.
Drawings
FIG. 1: the water lily tissue culture process effect picture is provided.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
Test 1: influence of different disinfectants and disinfection time on disinfection of water lily explants
Collecting materials on sunny days, taking healthy delay-drug water lily tubers without diseases and insect pests, filling the tubers into a bucket with water, and taking the tubers back to a laboratory. Washing away mud on the surface of the water lily tubers by using tap water, cleaning the mud on the surface of the water lily tubers, clamping root hairs on the surface of the root tubers by using sharp tweezers, cleaning by using washing powder, washing by using the washing powder, washing for 30min by using the tap water below a tap, and then placing on a clean bench for a disinfection test.
Each disinfectant solution is treated and then thoroughly washed with sterile water to remove the disinfectant solution. Explants sterilized with various sterilization methods were inoculated into MS + S106 medium in 3 replicates per treatment, and 20 flasks with 1 explant per flask were inoculated per replicate. The results were recorded after 10 d. Contamination rate (%) = number of contaminated explants/number of inoculated explants × 100%; browning rate (%) = number of browned explants/number of inoculated explants × 100%.
The test results show (table 1): the disinfection method A6 is most suitable for a method for disinfecting the water lily tubers, and has the pollution rate of 30 percent and the browning rate of 25 percent; although the contamination rate of the A7 disinfection rate is 26.7 percent at the lowest, the browning rate is serious and reaches 58.3 percent. Therefore, A6 is most suitable as a sterilization method of the water lily tubers.
TABLE 1 Effect of different disinfectants and disinfection time on the disinfection of Water lily explants
Test 2: effect of different plant growth regulators on Water lily explant Induction
The explants which are not polluted and browned and are disinfected by the A6 disinfection method are inoculated into a B1-B4 culture medium to simulate the growth environment of the water lily, each bottle is added with water and just submerges the tuber, the growth environment of the water lily is simulated, each bottle is inoculated with 60 bottles, and each bottle is inoculated with 1 explant. Placing in a 28 ℃ culture room for dark culture for 7d, and then placing in a 28 ℃ culture room for culture under the culture conditions of illumination intensity of 4000lx and light cycle of 14 h/d. Observed daily after inoculation and the results were recorded after 20 days. Induction rate (%) = number of explants in which adventitious shoots were induced/number of explants inoculated × 100%.
The test results show that (table 2): the plant growth regulator 6-BA + IBA is most suitable for being a hormone induced by the water lily tubers, the induction rate reaches 76.7 percent, and more than 2 cluster buds exist; while the control, 6-BA + IAA and 6-BA + NAA also showed some buds, but they all showed more single buds, and the buds showed good growth without the addition of 6-BA + IBA. Therefore, 6-BA + IBA is most suitable as a plant growth regulator for inducing the water lily tubers.
TABLE 2 Effect of different plant growth regulators on the Induction of Water lily explants
Test 3: influence of different S106 bactericide dosage on induction of water lily explant
The explants which are not polluted and browned and are disinfected by the A6 disinfection method are inoculated into a B5-B8 culture medium to simulate the growth environment of the water lily, each bottle of water is added to just submerge tubers, the growth environment of the water lily is simulated, each bottle is treated and inoculated with 60 bottles, and each bottle contains 1 explant. Placing in a 28 ℃ culture room for dark culture for 7d, and then placing in a 28 ℃ culture room for culture under the culture conditions of illumination intensity of 4000lx and light cycle of 14 h/d. Observed daily after inoculation and the results were recorded after 20 days.
The test results show (table 3): when the dosage of the S106 bactericide is 0.5-1.0 mL/L, the induction rate is as high as 83.3 percent, the germination is fast, and the bud growth vigor is good.
Further experiments confirm that when the induction culture medium is MS +6-BA 2.0-3.0 mg/L + IBA 0.5-1.0 mg/L + S106.5-1 mL/L, the average induction rate reaches more than 70%.
TABLE 3 Effect of different amounts of S106 fungicide on the induction of Water lily explants
Test 4: effect of different plant growth regulator concentrations on the multiplication of Water lily clumped buds
Dividing the cluster buds obtained by the B2 induction method into two or four or six (according to the size of the cluster buds), continuously inoculating into a multiplication culture medium C1-C5, and adding water into the culture medium to simulate the growth environment of the water lily. 60 flasks were inoculated per treatment, 1 explant per flask. Placing in a 28 ℃ culture room for dark culture for 7 days, and then placing in a 28 ℃ culture room for culture under the culture conditions of illumination intensity of 4000lx and light cycle of 14 h/d. The results were recorded by observation after 20d incubation.
The test results show (table 4): the C2 and C3 are suitable for being used as a multiplication culture medium for inducing buds of the water lily, the induction rate reaches 80 percent, and 2-8 bud seedlings are generated. The induction rate is increased along with the increase of the concentration of the 6-BA of the plant growth regulator, but the induction rate is reduced when the concentration of the 6-BA reaches a certain level, and the callus can be generated to influence the induction of buds and even die when the concentration of the 6-BA is higher.
TABLE 4 Effect of different plant growth regulator concentrations on the multiplication of Water lily clumped buds
Test 5: effect of different amounts of S106 Fungicide on multiplication of Water lily clumped buds
Dividing the cluster buds obtained by the B2 induction method into two or four or six (according to the size of the cluster buds), continuously inoculating the cluster buds into a multiplication culture medium C6-C9, simulating the growth environment of the water lily, and adding water into each bottle to just submerge the tuber to simulate the growth environment of the water lily. 60 flasks were inoculated per treatment, 1 explant per flask. Placing in a 28 ℃ culture room for dark culture for 7d; then culturing under the culture conditions of 28 ℃, the illumination intensity of 4000lx and the light cycle of 14 h/d. The recorded results were observed after 20 d.
The test results show (table 5): the induction rate tends to increase and decrease with the increase of the addition amount of S106, and when the addition amount of S106 is 0.5-1.0 mL/L, the induction rate reaches more than 80%, the number of buds is large, and the growth vigor is good.
Further experiments confirm that when the induction culture medium is MS +6-BA 2.0-3.0 mg/L + IBA 0.5-1.0 mg/L + S106.5-1 mL/L, the average induction rate reaches more than 76%.
TABLE 5 Effect of different amounts of S106 fungicide on the proliferation of Water lily clumped buds
Test 6: influence of different plant growth regulators and concentrations on rooting of water lily tissue culture seedlings
Selecting a bud seedling which is 3-5 cm high after being treated by C8, is consistent in growth and strong, cutting a single plant, inoculating the cut single plant into rooting culture media D1-D7, inoculating 30 bottles of tissue culture seedlings in each treatment, placing 1 plant in each bottle, and culturing under the conditions of illumination of an LED illuminating lamp for 16 hours/day, illumination intensity of 4000lx and temperature of 28 ℃. And 20d, recording the result and calculating the rooting rate. Rooting rate (%) = number of rooted seedlings/number of inoculations × 100%.
The test results show (table 6): IBA is more suitable to be used as a water lily rooting hormone, and IBA is used as the rooting hormone, so that the water lily tissue culture seedlings have more roots, are long and thick, have the root length of more than 2cm and are robust; NAA as the rooting hormone of the water lily tissue culture seedling also has a small amount of roots, but is thin and short and has slow rooting speed. Therefore, IBA is more suitable as a rooting hormone of the water lily tissue culture seedlings.
TABLE 6 influence of different plant growth regulators and concentrations on rooting of water lily tissue culture seedlings
Test 7: influence of different proliferation treatments on rooting of subsequent water lily tissue culture seedlings
Selecting bud seedlings which are processed by C6 and C7 and have the height of 3-5 cm, are consistent in growth and are robust, cutting a single plant, inoculating the cut single plant into a rooting culture medium D3, inoculating 30 bottles of tissue culture seedlings in each processing bottle, placing 1 plant in each bottle, and culturing under the conditions of illumination of an LED illuminating lamp for 16 hours/day, illumination intensity of 4000lx and temperature of 28 ℃. And 20d, recording the result and calculating the rooting rate. Rooting rate (%) = number of rooted seedlings/number of inoculations × 100%.
The test results show (table 7): when the well-growing and callus-free seedlings obtained by the C6 and C7 treatments in the experiment 5 are inoculated to the D3 culture medium for rooting culture, the rooting conditions are obviously different. When the bud seedlings obtained after C6 treatment are subjected to rooting culture, the rooting time is relatively late, and the rooting rate is relatively low. Therefore, D3 is the most suitable culture medium for the water lily tissue culture seedlings.
TABLE 7 Effect of different proliferation treatments on rooting of subsequent Water lily tissue culture seedlings
According to the experimental result of the invention, the method for inducing tissue culture and rapid propagation of the water lily tubers is provided, and the detailed operation steps are summarized as follows:
(1) Explant sterilization
The water lily root tuber is used as an explant, the explant with a clean surface is soaked in 75% ethanol for 1min in an operation table, and then sterilized and washed for 3-5 times for later use. Adding 0.1% mercuric chloride solution into the explant, placing the explant in a shaking table with the speed of 100r/min, shaking for 15min, pouring the mercuric chloride solution, washing for 3-5 times by using sterile water, then soaking for 5min by using sterile water, removing the mercuric chloride solution in the explant so as to avoid poisoning the explant, increasing the browning rate, washing for 3-5 times by using sterile water, then sucking water by using sterile filter paper, and removing a layer of the surface of the root tuber contacting the mercuric chloride by using a surgical knife.
(2) Induction of the root tuber of Helianthus tuberosus
Inoculating the explant treated in the step (1) into an induction culture medium (MS + 2.0-3.0 mg/L6-BA + 0.5-1.0 mg/LIBA + 0.5-1.0 mL/L S106), adding sterile water (just submerging the surface of a tuber) into the culture medium to simulate the growth environment of water lily, and placing the culture medium in a 28 ℃ culture chamber for dark culture for 5-7 days; then culturing under the culture conditions of 28 ℃, the illumination intensity of 4000lx and the light cycle of 10-14 h/d.
(3) Proliferation culture
Dividing the bud obtained in the step (2) into two parts, one part into three parts or one part into four parts (according to the size of the bud), inoculating the bud into a multiplication culture medium (MS + 2.0-3.0 mg/L6-BA + 0.5-1.0 mg/L IBA + 0.5-1.0 mL/L S106), adding sterile water into the culture medium to simulate the growth environment of the water lily, and placing the culture medium in a 28 ℃ culture chamber for dark culture for 5-7 days; then culturing under the culture conditions of 28 ℃, the illumination intensity of 4000lx and the light cycle of 10-14 h/d.
(4) Rooting culture
Dividing the induced tissue culture seedlings with the height of 3-5 cm into single plants, inoculating the single plants into a rooting culture medium (1/2MS + 0.5-1.0 mg/LIBA) for strong seedling rooting culture, wherein the rooting culture medium is a semi-solid culture medium, and placing the semi-solid culture medium in a 28 ℃ culture room for dark culture for 2-3 days; then culturing under the culture conditions of 28 ℃, 4000lx of illumination intensity and 10-14 h/d of photoperiod.
(5) Hardening seedlings of water lily
The bottle mouth of the water lily rooted seedling is opened, the water lily rooted seedling is placed and moved to a shading position for about one week, the water lily rooted seedling is slowly moved to a position with good light to be hardened for about one week, the bottle body or the bag surface is frequently rotated to enable the tissue culture seedling to be uniformly lighted, and the hardened tissue culture seedling is uniformly lighted and healthy. Taking out refined water lily seedlings, cleaning root culture medium, planting in soil with large viscosity or sludge in a pond, culturing in a non-porous flowerpot, culturing in a shade shed until new leaves grow out, and transferring to sunlight for normal culture (20-30 d), wherein the shade degree of the shade shed is about 50%, and the seedling culture medium is clay or sludge. The survival rate of the acclimated seedlings reaches 99 percent after 20 days of culture.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like that are made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for inducing tissue culture and rapid propagation of water lily tubers is characterized by comprising the following steps:
(1) Explant disinfection: taking water lily tubers as explants, and disinfecting the explants;
(2) Induction: inoculating the explant sterilized in the step (1) into an induction culture medium for induction culture;
the induction culture medium consists of a basic culture medium, 2.0-3.0 mg/L6-BA, 0.5-1.0 mg/L IBA and 0.5-1.0 mL/LS106 bactericide;
(3) And (3) proliferation culture: carrying out enrichment culture on the bud-grafted seeds obtained by inducing in the step (2) in an enrichment culture medium;
the proliferation culture medium consists of a basic culture medium, 2.0-3.0 mg/L6-BA, 0.5-1.0 mg/L IBA and 0.5-1.0 mL/LS106 bactericide;
(4) Rooting culture: inoculating the bud seedling obtained after the culture in the step (3) into a rooting culture medium for rooting culture;
the rooting culture medium consists of a basic culture medium and 0.5-1.0 mg/L IBA;
(5) Hardening seedlings;
the basic culture medium is MS culture medium or 1/2MS culture medium.
2. The method for inducing tissue culture and rapid propagation of the water lily tubers according to claim 1, wherein the disinfection in the step (1) is as follows: soaking the cleaned explant in 75 vol% alcohol solution for 1min, sterilizing and washing for 3-5 times; adding the explant into mercuric chloride solution with mass concentration of 0.1%, placing the explant into a shaking table with 100rpm for shaking for 15min, pouring the mercuric chloride solution, cleaning the explant with sterile water for 3-5 min, soaking the explant with sterile water for 5min, and cleaning the explant with sterile water for 3-5 times.
3. The method for inducing tissue culture and rapid propagation of the water lily tubers according to claim 1, wherein in the step (2), the conditions of the induction culture are as follows: sterile water is added into the induction culture medium to simulate the growth environment of the water lily; placing the induction culture medium in a 28 ℃ culture room for dark culture for 5-7 days; then placing the mixture at 28 ℃, the illumination intensity of 4000lx and the photoperiod of 10-14 h/d for continuous culture.
4. The method for inducing tissue culture and rapid propagation of the water lily tubers according to claim 1, wherein in the step (3), the conditions of propagation culture are as follows: sterile water is added into the proliferation culture medium to simulate the growth environment of the water lily; placing the proliferation culture medium in a 28 ℃ culture room for dark culture for 2-3 days; then placing the mixture at the temperature of 28 ℃, the illumination intensity of 4000lx and the photoperiod of 10-14 h/d for continuous culture.
5. The method for inducing tissue culture and rapid propagation of the water lily tubers according to claim 1, wherein in the step (4), the rooting culture conditions are as follows: placing the rooting culture medium in a 28 ℃ culture room for dark culture for 2-3 d; then placing the mixture at the temperature of 28 ℃, the illumination intensity of 4000lx and the photoperiod of 10-14 h/d for continuous culture.
6. The method for inducing tissue culture and rapid propagation of the water lily tubers according to claim 1, wherein in the step (5), the seedling hardening step is as follows: and (4) culturing the rooted seedlings obtained after the culture in the step (4) in a shading place for 1 week, then transferring to the light for 1 week, removing the culture medium at the root parts of the seedlings, planting the seedlings in a non-porous flowerpot filled with clay or sludge for culture, and transferring to the sun for culture after shading culture until new leaves grow out.
7. The method for inducing tissue culture and rapid propagation of the water lily tubers according to claim 1, wherein the induction culture medium consists of a basal culture medium, 2.0 mg/L6-BA, 0.5mg/L IBA and 0.5mL/L S bactericide.
8. The method for inducing tissue culture and rapid propagation of the water lily tubers according to claim 1, wherein the propagation medium consists of a basal medium, 3.0 mg/L6-BA, 0.5mg/L IBA and 1.0mL/L S bactericide.
9. The method for inducing tissue culture and rapid propagation of the water lily tubers according to claim 1, wherein the rooting medium consists of a basal medium and 0.5mg/L IBA.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111675282.1A CN114303952B (en) | 2021-12-31 | 2021-12-31 | Water lily tuber induction tissue culture rapid propagation method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111675282.1A CN114303952B (en) | 2021-12-31 | 2021-12-31 | Water lily tuber induction tissue culture rapid propagation method |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114303952A CN114303952A (en) | 2022-04-12 |
CN114303952B true CN114303952B (en) | 2023-03-31 |
Family
ID=81020472
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111675282.1A Active CN114303952B (en) | 2021-12-31 | 2021-12-31 | Water lily tuber induction tissue culture rapid propagation method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114303952B (en) |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104488716B (en) * | 2014-12-19 | 2016-01-27 | 浙江省农业科学院 | A kind of method of water lily tissue cultures |
CN104996298B (en) * | 2015-07-06 | 2017-08-01 | 三明学院 | The chrysanthemum tissue culture of falling water lily seedling-growing method is cultivated based on the integration of many internode stem sections |
CN106577272B (en) * | 2015-10-14 | 2019-07-05 | 西南大学 | A kind of method for tissue culture of water shield |
CN109122311B (en) * | 2018-07-31 | 2021-11-26 | 海南大学 | Culture medium for inducing aseptic buds of water lily embryonic tubers and culture method thereof |
-
2021
- 2021-12-31 CN CN202111675282.1A patent/CN114303952B/en active Active
Also Published As
Publication number | Publication date |
---|---|
CN114303952A (en) | 2022-04-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106106163A (en) | A kind of iris cultivates propagation method | |
CN113331059B (en) | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants | |
CN111657151A (en) | Rapid seedling method for acer truncatum | |
CN108770692A (en) | Coconut embryonal induction culture medium and the method that Regeneration in Vitro plant is obtained based on coconut zygotic embryo cell thin culture | |
CN112868527A (en) | Method for rapidly propagating flamingo pepper grass | |
CN1771796A (en) | In vitro broad bean culturing method | |
CN114303952B (en) | Water lily tuber induction tissue culture rapid propagation method | |
CN110771505A (en) | Culture method of citral tonglu stem tissue | |
CN110663554A (en) | Commercial tissue culture seedling raising method for Hosta chinensis Redlar | |
CN114431149B (en) | Non-symbiotic germination method for seeds of rare or endangered plant large yellow croaker calanthe | |
CN113317206B (en) | Culture medium combination for tissue culture and rapid propagation of lilium martagon, tissue culture and rapid propagation method and application | |
CN110122331A (en) | It is a kind of using leaf as the quick-breeding method of the U.S. Liriodendron chinense in Asia of explant | |
CN113099866B (en) | Method for rapidly propagating fir excellent family seedlings | |
CN111758570B (en) | Method for directly inducing stem segments of salix caprea to grow seedlings and propagate quickly | |
CN101543184A (en) | Method for culturing open type tissue of konjak | |
CN109258463B (en) | Vegetative propagation method of paphiopedilum armeniacum | |
CN109526748B (en) | Tissue culture method for anthurium andraeanum inflorescence | |
CN108450333B (en) | Induction method of lilium tigrinum callus | |
CN1379972A (en) | Morinda tissue culture method | |
CN112167064A (en) | Tissue culture and rapid propagation method for salvia miltiorrhiza | |
CN114568305B (en) | Treatment method for improving regeneration efficiency by tissue culture of quercus acutissima | |
NL2032821B1 (en) | Efficient method for micropropagation of sesuvium portulacastrum through axillary buds | |
CN111248087B (en) | Tissue culture rapid propagation method for primary seedling formation of radix glehniae | |
CN116806694B (en) | Eggplant doubled haploid anther culture method | |
CN114208673B (en) | Tissue culture method of north China blue potted flowers |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |