CN117481035A - Lily tissue culture rapid propagation method - Google Patents
Lily tissue culture rapid propagation method Download PDFInfo
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- CN117481035A CN117481035A CN202311707261.2A CN202311707261A CN117481035A CN 117481035 A CN117481035 A CN 117481035A CN 202311707261 A CN202311707261 A CN 202311707261A CN 117481035 A CN117481035 A CN 117481035A
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- 241000234435 Lilium Species 0.000 title claims abstract description 116
- 238000000034 method Methods 0.000 title claims abstract description 35
- 230000035755 proliferation Effects 0.000 claims abstract description 34
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 230000006698 induction Effects 0.000 claims abstract description 28
- 238000005520 cutting process Methods 0.000 claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims description 62
- 238000011282 treatment Methods 0.000 claims description 43
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 229920001817 Agar Polymers 0.000 claims description 24
- 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 24
- 229930006000 Sucrose Natural products 0.000 claims description 24
- 239000008272 agar Substances 0.000 claims description 24
- 230000001954 sterilising effect Effects 0.000 claims description 24
- 239000005720 sucrose Substances 0.000 claims description 24
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 22
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 21
- 238000004140 cleaning Methods 0.000 claims description 16
- 238000004659 sterilization and disinfection Methods 0.000 claims description 16
- 238000005406 washing Methods 0.000 claims description 16
- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 claims description 12
- 239000008223 sterile water Substances 0.000 claims description 12
- 238000011010 flushing procedure Methods 0.000 claims description 8
- 239000011159 matrix material Substances 0.000 claims description 8
- 238000012216 screening Methods 0.000 claims description 8
- 241000196324 Embryophyta Species 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 5
- 239000005747 Chlorothalonil Substances 0.000 claims description 4
- 235000013162 Cocos nucifera Nutrition 0.000 claims description 4
- 244000060011 Cocos nucifera Species 0.000 claims description 4
- 230000001680 brushing effect Effects 0.000 claims description 4
- CRQQGFGUEAVUIL-UHFFFAOYSA-N chlorothalonil Chemical compound ClC1=C(Cl)C(C#N)=C(Cl)C(C#N)=C1Cl CRQQGFGUEAVUIL-UHFFFAOYSA-N 0.000 claims description 4
- 230000000249 desinfective effect Effects 0.000 claims description 4
- 239000003599 detergent Substances 0.000 claims description 4
- 239000010903 husk Substances 0.000 claims description 4
- 229960002523 mercuric chloride Drugs 0.000 claims description 4
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- 239000012883 rooting culture medium Substances 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- 239000002689 soil Substances 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 210000002489 tectorial membrane Anatomy 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 230000002349 favourable effect Effects 0.000 abstract description 2
- 238000011081 inoculation Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000004069 differentiation Effects 0.000 description 12
- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 6
- 238000010422 painting Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- SRVJKTDHMYAMHA-WUXMJOGZSA-N thioacetazone Chemical compound CC(=O)NC1=CC=C(\C=N\NC(N)=S)C=C1 SRVJKTDHMYAMHA-WUXMJOGZSA-N 0.000 description 2
- OVSKIKFHRZPJSS-DOMIDYPGSA-N 2-(2,4-dichlorophenoxy)acetic acid Chemical compound OC(=O)[14CH2]OC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-DOMIDYPGSA-N 0.000 description 1
- 241001252567 Chrysothamnus viscidiflorus Species 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 240000005546 Piper methysticum Species 0.000 description 1
- 235000016787 Piper methysticum Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
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
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a lily tissue culture rapid propagation method, belongs to a propagation system establishment method of ornamental flowers, and particularly relates to the technical field of lily propagation; comprises the steps of selecting and processing explants, performing primary induction culture on ornamental lily, performing secondary proliferation culture on the ornamental lily, performing rooting culture on the ornamental lily, performing transplanting hardening off; through the steps of the method, the method can realize a lily rapid propagation system, shorten the lily yield period and reduce the production cost; the pollution rate of the tissue culture inoculation is lower than 10% by treating for 30s with 75% ethanol and treating for 7min with 0.1% liter, so that the pollution rate is reduced and the cost is saved; the scale cutting mode comprises the following steps: the longitudinal cutting method increases the induction rate of adventitious buds and bulblets, and reduces the cutting surface and the pollution rate; the dark culture for 7 days is favorable for forming bulblets, and provides possibility and technical support for exploring the rapid bulb formation of lily tissue culture.
Description
Technical Field
The invention belongs to a method for establishing a propagation system of ornamental flowers, and particularly relates to the technical field of lily propagation.
Background
The lily belongs to the genus Lilium of the family Liliaceae, and is perennial root herb plant. As one of the most important fresh cut flowers in the world, the flower-cutting agent is widely applied to cut flowers, courtyards, park greening and the like, and helps to promote the development of rural industrial economy and the construction of beautiful China. Lily is propagated by adopting asexual propagation methods such as meristem, cuttage and the like conventionally, but the propagation coefficient is lower, and the seed balls are easy to degenerate.
Therefore, in order to improve the quality of lily bulbs and meet the domestic flower market demand, it is necessary to explore and establish an ornamental lily rapid propagation system.
Therefore, a technique is required to solve the problems faced by the prior art.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: provides a lily tissue culture rapid propagation method, which overcomes the problems in the prior art.
A lily tissue culture rapid propagation method is characterized by comprising the following steps:
step one, selection and treatment of explants
The selection and treatment of the explants comprises the following steps, which are carried out in sequence:
s1, placing ornamental lily of a selected variety into a refrigerator for low-temperature pretreatment, taking out and cleaning;
s2, selecting healthy lily scales and stem segments from the lily bulbs cleaned in the S1, and cleaning, disinfecting and sterilizing the extracted lily scales and stem segments;
s3, treating the lily scales and stem segments treated in the step S2 with ethanol solution, washing, sterilizing, washing with sterile water, and cutting with a sterilizing blade to obtain explants;
step two, primary induction culture of ornamental lily
Inoculating the maximum surface of the incision of the explant obtained in the step one on a culture medium, wherein two culture media with different formulas are adopted for primary culture; after the explant is placed in a culture medium, dark culture is carried out for seven days, and then the explant is cultured for 40 days under the environment with the illumination intensity of 2500LX/24 h; screening the explants after primary culture to obtain adventitious buds and bulblets meeting the standard;
step three, secondary proliferation culture of ornamental lily
Transferring adventitious buds and bulblets with good growth vigor in the primary induction culture obtained in the second step into four multiplication mediums for multiplication culture;
step four, rooting culture of ornamental lily
Transferring the ornamental lily tissue culture seedlings with the plant height of 3 cm-3.5 cm in primary culture and secondary proliferation culture into a rooting culture medium;
step five, transplanting and hardening seedlings
Hardening the ornamental lily tissue culture seedlings with root length reaching 2 cm-4.5 cm in the fourth step in a culture bottle in a tissue culture room; after hardening, taking out the young seedlings and flushing the culture medium; transplanting it into a sterilized substrate; and obtaining lily seedlings.
The low-temperature pretreatment operation in the step S1 is carried out at the temperature of 4 ℃ for one week; wherein the cleaning operation is to soak the raw materials in 400 times of chlorothalonil for 15min for sterilization treatment; after the soaking is finished, the surface is cleaned by flushing with running water and brushing with a soft brush.
In the step S2 in the step I, the cleaning operation is to soak for 15min by using neutral detergent, wash for about 1h by running water, and then wash for 3 times by using sterile water; the disinfection and sterilization operation is to place the lily scales and stem segments treated by the step S2 into an ultra-clean workbench, irradiate for 30min by ultraviolet rays and ventilate for 15min;
the ethanol solution in the step S3 in the step I is 75% ethanol, and the treatment time is 30S; after treatment, the water is washed by sterile water for 3 times to finish washing; and then 0.1mol/L mercuric chloride is used for treatment for 7min, the aseptic water is used for washing for 5 times, and the aseptic filter paper is used for wiping off the water on the surfaces of the scales and the stem segments to finish the operation.
In the step S3 of the step I, a sterilization blade cutting operation is adopted, an explant with the width of 2-3mm is longitudinally cut for each flake by adopting a sterilized blade, and a stem section is cut into an explant with the thickness of 2 mm.
The two culture medium formulas in the second step are respectively A1, MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzylaminopurine 1mg/L and alpha-naphthylacetic acid 0.1mg/L; a2 MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzylaminopurine 1mg/L, 2, 4-dichlorophenoxyacetic acid 1mg/L.
The screening standard in the second step is that the height of the adventitious buds is 2 cm-2.5 cm; the bulblet size is 1 cm-1.5 cm.
The four proliferation culture medium formulas in the step three are respectively as follows: z1: MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzyl amino purine 0.5mg/L; z2: MS culture medium, 30g/L sucrose, 6.5g/L agar and 2 mg/L6-benzylaminopurine; z3: MS culture medium, 30g/L sucrose, 6.5g/L agar, 0.5 mg/L6-benzylaminopurine and 0.5mg/L alpha-naphthylacetic acid; z4: MS culture medium, 30g/L sucrose, 6.5g/L agar, 2 mg/L6-benzylaminopurine and 0.5mg/L alpha-naphthylacetic acid.
The seedling hardening treatment time in the step five is 3-5 days; the sterilized matrix is prepared from turfy soil and coconut husk in a mass ratio of 1:1; putting the hardening seedlings into a matrix, watering thoroughly for the first time, and performing tectorial membrane shading treatment; and obtaining lily seedlings.
Through the design scheme, the invention has the following beneficial effects: through the steps of the method, the method can realize a lily rapid propagation system, shorten the lily yield period and reduce the production cost; the pollution rate of the tissue culture inoculation is lower than 10% by treating for 30s with 75% ethanol and treating for 7min with 0.1% liter, so that the pollution rate is reduced and the cost is saved; the scale cutting mode comprises the following steps: the longitudinal cutting method increases the induction rate of adventitious buds and bulblets, and reduces the cutting surface and the pollution rate; the dark culture for 7 days is favorable for forming bulblets, and provides possibility and technical support for exploring the rapid bulb formation of lily tissue culture.
Detailed Description
This application is further described in conjunction with:
a lily tissue culture rapid propagation method is characterized by comprising the following steps:
step one, selection and treatment of explants
The selection and treatment of the explants comprises the following steps, which are carried out in sequence:
s1, placing ornamental lily of a selected variety into a refrigerator for low-temperature pretreatment, taking out and cleaning;
s2, selecting healthy lily scales and stem segments from the lily bulbs cleaned in the S1, and cleaning, disinfecting and sterilizing the extracted lily scales and stem segments;
s3, treating the lily scales and stem segments treated in the step S2 with ethanol solution, washing, sterilizing, washing with sterile water, and cutting with a sterilizing blade to obtain explants;
step two, primary induction culture of ornamental lily
Inoculating the maximum surface of the incision of the explant obtained in the step one on a culture medium, wherein two culture media with different formulas are adopted for primary culture; after the explant is placed in a culture medium, dark culture is carried out for seven days, and then the explant is cultured for 40 days under the condition of 2500LX/24h of illumination intensity; screening the explants after primary culture to obtain adventitious buds and bulblets meeting the standard;
step three, secondary proliferation culture of ornamental lily
Transferring adventitious buds and bulblets with good growth vigor in the primary induction culture obtained in the second step into four multiplication mediums for multiplication culture;
step four, rooting culture of ornamental lily
Transferring the ornamental lily tissue culture seedlings with the plant height of 3 cm-3.5 cm in primary culture and secondary proliferation culture into a rooting culture medium;
step five, transplanting and hardening seedlings
Hardening the ornamental lily tissue culture seedlings with root length reaching 2 cm-4.5 cm in the fourth step in a culture bottle in a tissue culture room; after hardening, taking out the young seedlings and flushing the culture medium; transplanting it into a sterilized substrate; and obtaining lily seedlings.
The low-temperature pretreatment operation in the step S1 is carried out at the temperature of 4 ℃ for one week; wherein the cleaning operation is to soak the raw materials in 400 times of chlorothalonil for 15min for sterilization treatment; after the soaking is finished, the surface is cleaned by flushing with running water and brushing with a soft brush.
In the step S2 in the step I, the cleaning operation is to soak for 15min by using neutral detergent, wash for about 1h by running water, and then wash for 3 times by using sterile water; the disinfection and sterilization operation is to place the lily scales and stem segments treated by the step S2 into an ultra-clean workbench, irradiate for 30min by ultraviolet rays and ventilate for 15min;
the ethanol solution in the step S3 in the step I is 75% ethanol, and the treatment time is 30S; after treatment, the water is washed by sterile water for 3 times to finish washing; and then 0.1mol/L mercuric chloride is used for treatment for 7min, the aseptic water is used for washing for 5 times, and the aseptic filter paper is used for wiping off the water on the surfaces of the scales and the stem segments to finish the operation.
In the step S3 of the step I, a sterilization blade cutting operation is adopted, an explant with the width of 2-3mm is longitudinally cut for each flake by adopting a sterilized blade, and a stem section is cut into an explant with the thickness of 2 mm.
The two culture medium formulas in the second step are respectively A1, MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzylaminopurine 1mg/L and alpha-naphthylacetic acid 0.1mg/L; a2 MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzylaminopurine 1mg/L, 2, 4-dichlorophenoxyacetic acid 1mg/L.
The screening standard in the second step is that the height of the adventitious buds is 2 cm-2.5 cm; the bulblet size is 1 cm-1.5 cm.
The four proliferation culture medium formulas in the step three are respectively as follows: z1: MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzyl amino purine 0.5mg/L; z2: MS culture medium, 30g/L sucrose, 6.5g/L agar and 2 mg/L6-benzylaminopurine; z3: MS culture medium, 30g/L sucrose, 6.5g/L agar, 0.5 mg/L6-benzylaminopurine and 0.5mg/L alpha-naphthylacetic acid; z4: MS culture medium, 30g/L sucrose, 6.5g/L agar, 2 mg/L6-benzylaminopurine and 0.5mg/L alpha-naphthylacetic acid.
The seedling hardening treatment time in the step five is 3-5 days; the sterilized matrix is prepared from turfy soil and coconut husk in a mass ratio of 1:1; putting the hardening seedlings into a matrix, watering thoroughly for the first time, and performing tectorial membrane shading treatment; and obtaining lily seedlings.
Examples
A lily tissue culture rapid propagation method is characterized by comprising the following steps:
step one, selection and treatment of explants
The selection and treatment of the explants comprises the following steps, which are carried out in sequence:
s1, selecting seven kinds of closed lily: "yellow brush" lily, "table dance" lily, "kava" lily, "Ma Erbei g" lily, "Parazone" lily, "Wei Anna" lily and "Tarita" lily; placing the selected ornamental lily into a refrigerator for low-temperature pretreatment, taking out and cleaning;
s2, selecting healthy lily scales and stem segments from the lily bulbs cleaned in the S1, and cleaning, disinfecting and sterilizing the extracted lily scales and stem segments;
s3, treating the lily scales and stem segments treated in the step S2 with ethanol solution, washing, sterilizing, washing with sterile water, and cutting with a sterilizing blade to obtain explants;
step two, primary induction culture of ornamental lily
Inoculating the maximum surface of the incision of the explant obtained in the step one on a culture medium, wherein two culture media with different formulas are adopted for primary culture, and the two culture media are respectively A1, MS culture medium, 30g/L sucrose, 6.5g/L agar, 1 mg/L6-benzylaminopurine and 0.1mg/L alpha-naphthylacetic acid; a2 MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzylaminopurine 1mg/L, 2, 4-dichlorophenoxyacetic acid 1mg/L
Thirty scales and stem segments of each lily are respectively placed in each culture medium, dark culture is carried out for seven days, then the culture is carried out for forty days in an environment with the illumination intensity of 2500LX/24h, and then the growth condition of the explant is observed and the pollution rate, the induction rate and the differentiation coefficient are counted; screening the explants after primary culture to obtain adventitious buds and bulblets meeting the standard;
treatment test design of primary culture material of ornamental lily
In the table, 6-BA is 6-benzylaminopurine, 2,4-D is 2, 4-dichlorophenoxyacetic acid and NAA is alpha-naphthylacetic acid;
step three, secondary proliferation culture of ornamental lily
Transferring the adventitious buds and bulblets with good growth vigor in the primary induction culture obtained in the second step into four multiplication mediums, and carrying out multiplication culture on ten adventitious buds for each ornamental hundred-fold; observing the proliferation condition of the adventitious bud after 30d and counting proliferation coefficients; repeating for three times;
design of proliferation test of lily adventitious bud by combination of different concentrations
In the table, 6-BA is 6-benzylaminopurine, 2,4-D is 2, 4-dichlorophenoxyacetic acid and NAA is alpha-naphthylacetic acid;
step four, rooting culture of ornamental lily
Transferring the ornamental lily tissue culture seedlings with the plant height of 3 cm-3.5 cm in primary culture and secondary proliferation culture into a rooting culture medium; ten aseptic seedlings are taken and repeated for 3 times; observing the tissue culture Miao Changshi, and counting the rooting rate and the rooting condition after 30 days;
step five, transplanting and hardening seedlings
Hardening the ornamental lily tissue culture seedlings with root length reaching 2 cm-4.5 cm in the fourth step in a culture bottle in a tissue culture room; after hardening, taking out the young seedlings and flushing the culture medium; transplanting it into a sterilized substrate; and obtaining lily seedlings.
The low-temperature pretreatment operation in the step S1 is carried out at the temperature of 4 ℃ for one week; wherein the cleaning operation is to soak the raw materials in 400 times of chlorothalonil for 15min for sterilization treatment; after the soaking is finished, the surface is cleaned by flushing with running water and brushing with a soft brush.
In the step S2 in the step I, the cleaning operation is to soak for 15min by using neutral detergent, wash for about 1h by running water, and then wash for 3 times by using sterile water; the disinfection and sterilization operation is to place the lily scales and stem segments treated by the step S2 into an ultra-clean workbench, irradiate for 30min by ultraviolet rays and ventilate for 15min;
the ethanol solution in the step S3 in the step I is 75% ethanol, and the treatment time is 30S; after treatment, the water is washed by sterile water for 3 times to finish washing; and then 0.1mol/L mercuric chloride is used for treatment for 7min, the aseptic water is used for washing for 5 times, and the aseptic filter paper is used for wiping off the water on the surfaces of the scales and the stem segments to finish the operation.
In the step S3 of the step I, a sterilization blade cutting operation is adopted, an explant with the width of 2-3mm is longitudinally cut for each flake by adopting a sterilized blade, and a stem section is cut into an explant with the thickness of 2 mm.
The screening standard in the second step is that the height of the adventitious buds is 2 cm-2.5 cm; the bulblet size is 1 cm-1.5 cm.
The four proliferation culture medium formulas in the step three are respectively as follows: z1: MS culture medium, sucrose 30g/L+6.5g/L agar 0.5 mg/L6-benzylaminopurine; z2: MS culture medium, 30g/L sucrose, 6.5g/L agar and 2 mg/L6-benzylaminopurine; z3: MS culture medium, 30g/L sucrose, 6.5g/L agar, 0.5 mg/L6-benzylaminopurine and 0.5mg/L alpha-naphthylacetic acid; z4: MS culture medium, 30g/L sucrose, 6.5g/L agar, 2 mg/L6-benzylaminopurine and 0.5mg/L alpha-naphthylacetic acid.
The seedling hardening treatment time in the step five is 3-5 days; the sterilized matrix is prepared from turfy soil and coconut husk in a mass ratio of 1:1; after the tissue culture seedlings are put into a matrix, watering thoroughly for the first time, and carrying out tectorial membrane shading treatment; and obtaining lily seedlings.
In the embodiment, the induction rate of the 'Parazone' lily scales under the A1 culture medium is highest and reaches 96.67%, but the 'table dance' lily scales have the best effect of differentiating adventitious buds, and the differentiation coefficient reaches 4.60; the induction condition and the adventitious bud differentiation condition of the 'table dance' lily scales under the A2 culture medium are best, the induction rate reaches 97.78%, and the differentiation coefficient reaches 6.80; but the induction rate of the A2 culture medium on 7 ornamental lily is best, and the induction rate is more than 90.00%.
Under A1 treatment, inducing differentiation of scales of 7 lily varieties
Note that: the different lower case letters indicate that the difference is significant at p < 0.05 levels.
In the table, 6-BA is 6-benzylaminopurine, 2,4-D is 2, 4-dichlorophenoxyacetic acid and NAA is alpha-naphthylacetic acid;
induced differentiation condition of scales of 7 lily varieties under A2 treatment
Note that: the different lower case letters indicate that the difference is significant at p < 0.05 levels.
In the table, 6-BA is 6-benzylaminopurine, 2,4-D is 2, 4-dichlorophenoxyacetic acid and NAA is alpha-naphthylacetic acid;
the primary induction of lily stem segments is determined by a proper culture medium.
The induction rate and the adventitious bud differentiation of the scales of different ornamental lily varieties have great difference under the same culture medium, the induction of the stem sections of the yellow painting brush lily under the culture medium A1 and the culture medium A2 and the differentiation condition of the adventitious buds are all the best, the induction rate under the culture medium A1 reaches 86.67%, and the differentiation coefficient is 3.0; the inductivity under A2 culture medium reaches 91.11 percent, and the differentiation coefficient reaches 4.0.
Induction differentiation condition of stem segments of 7 lily varieties under A1 treatment
Note that: the different lower case letters indicate that the difference is significant at p < 0.05 levels.
In the table, 6-BA is 6-benzylaminopurine, 2,4-D is 2, 4-dichlorophenoxyacetic acid and NAA is alpha-naphthylacetic acid;
induction differentiation condition of stem segments of 7 lily varieties under A2 treatment
Note that: the different lower case letters indicate that the difference is significant at p < 0.05 levels.
In the table, 6-BA is 6-benzylaminopurine, 2,4-D is 2, 4-dichlorophenoxyacetic acid and NAA is alpha-naphthylacetic acid;
in the induction process of the explant scales, adventitious buds begin to appear after the dark culture is changed into illumination for 7 days and then the plant growth vigor becomes fast, and meanwhile, the addition of the 2,4-D culture medium can be seen to directly induce bulblets. Of these, only the A2 medium for primary induction of 'Wei Anna' is unsuitable.
In conclusion, the A2 medium was more suitable for induction of the scales of the 7 ornamental lilies selected in this experiment. Directly inducing bulblets after dark culture for 3 days after primary induction of ornamental lily scales; inducing the primary tender stem to induce bulblet directly after dark culture for 3-5 days; and 4, after the primary induction of the stem section 40d, the bulblet of the ornamental lily scales expands.
And (3) selecting a 'yellow painting brush' lily adventitious bud proliferation result from the secondary proliferation culture medium.
The different treatments have different effects on the proliferation coefficient of the adventitious buds of the lily with the yellow painting brush, the proliferation effect in the Z4 culture medium is best, the proliferation coefficient reaches 6.0, obvious differences exist among the treatments, the concentration of 6-BA is increased without adding NAA, and the proliferation coefficient is reduced; adding a small amount of NAA can improve the proliferation coefficient of the adventitious bud, the concentration of 6-BA is increased under the condition that the concentration of NAA is unchanged, and the proliferation coefficient is obviously increased;
multiplication result of 'yellow painting brush' lily adventitious bud
Note that: the different lower case letters indicate that the difference is significant at p < 0.05 levels.
In the table, 6-BA is 6-benzylaminopurine, 2,4-D is 2, 4-dichlorophenoxyacetic acid and NAA is alpha-naphthylacetic acid;
the different treatments have different influences on the proliferation coefficient of the adventitious buds of the 'table dance' lily, but the proliferation effect in a Z4 culture medium is the best, the proliferation coefficient reaches 3.8, obvious differences exist among the treatments, the concentration of 6-BA is increased without adding NAA, and the proliferation coefficient is reduced; adding a small amount of NAA can improve the proliferation coefficient of the adventitious bud, the concentration of 6-BA is increased under the condition that the concentration of NAA is unchanged, and the proliferation coefficient is obviously increased;
multiplication result of 'table dance' lily adventitious bud
Note that: the different lower case letters indicate that the difference is significant at p < 0.05 levels.
In the table, 6-BA is 6-benzylaminopurine, 2,4-D is 2, 4-dichlorophenoxyacetic acid and NAA is alpha-naphthylacetic acid;
the different treatments have different effects on the proliferation coefficient of the adventitious buds of the 'Ma Erbei g' lily, but the proliferation effect in the Z4 culture medium is the best, the proliferation coefficient reaches 6.8, obvious differences exist among the treatments, and the concentration of 6-BA is increased under the condition of not adding NAA, so that the proliferation of the 6-BA is obviously inhibited.
Multiplication results of adventitious bud of Lily of' Ma Erbei g
Note that: the different lower case letters indicate that the difference is significant at p < 0.05 levels.
In the table, 6-BA is 6-benzylaminopurine, 2,4-D is 2, 4-dichlorophenoxyacetic acid, and NAA is alpha-naphthylacetic acid.
Claims (9)
1. A lily tissue culture rapid propagation method is characterized by comprising the following steps:
step one, selection and treatment of explants
The selection and treatment of the explants comprises the following steps, which are carried out in sequence:
s1, placing ornamental lily of a selected variety into a refrigerator for low-temperature pretreatment, taking out and cleaning;
s2, selecting healthy lily scales and stem segments from the lily bulbs cleaned in the S1, and cleaning, disinfecting and sterilizing the extracted lily scales and stem segments;
s3, treating the lily scales and stem segments treated in the step S2 with ethanol solution, washing, sterilizing, washing with sterile water, and cutting with a sterilizing blade to obtain explants;
step two, primary induction culture of ornamental lily
Inoculating the maximum surface of the incision of the explant obtained in the step one on a culture medium, wherein two culture media with different formulas are adopted for primary culture; after the explant is placed in a culture medium, dark culture is carried out for seven days, and then the explant is cultured for 40 days under the environment with the illumination intensity of 2500LX/24 h; screening the explants after primary culture to obtain adventitious buds and bulblets meeting the standard;
step three, secondary proliferation culture of ornamental lily
Transferring adventitious buds and bulblets with good growth vigor in the primary induction culture obtained in the second step into four multiplication mediums for multiplication culture;
step four, rooting culture of ornamental lily
Transferring the ornamental lily tissue culture seedlings with the plant height of 3 cm-3.5 cm in primary culture and secondary proliferation culture into a rooting culture medium;
step five, transplanting and hardening seedlings
Hardening the ornamental lily tissue culture seedlings with root length reaching 2 cm-4.5 cm in the fourth step in a culture bottle in a tissue culture room; after hardening, taking out the young seedlings and flushing the culture medium; transplanting it into a sterilized substrate; and obtaining lily seedlings.
2. The lily tissue culture rapid propagation method according to claim 1, wherein the method comprises the following steps: the low-temperature pretreatment operation in the step S1 is carried out at the temperature of 4 ℃ for one week; wherein the cleaning operation is to soak the raw materials in 400 times of chlorothalonil for 15min for sterilization treatment; after the soaking is finished, the surface is cleaned by flushing with running water and brushing with a soft brush.
3. The lily tissue culture rapid propagation method according to claim 1, wherein the method comprises the following steps: in the step S2 in the step I, the cleaning operation is to soak for 15min by using neutral detergent, wash for about 1h by running water, and then wash for 3 times by using sterile water; the disinfection and sterilization operation is to place the lily scales and stems treated by the S2 in an ultra-clean workbench, irradiate for 30min by ultraviolet rays and ventilate for 15min.
4. The lily tissue culture rapid propagation method according to claim 1, wherein the method comprises the following steps: the ethanol solution in the step S3 in the step I is 75% ethanol, and the treatment time is 30S; after treatment, the water is washed by sterile water for 3 times to finish washing; and then 0.1mol/L mercuric chloride is used for treatment for 7min, the aseptic water is used for washing for 5 times, and the aseptic filter paper is used for wiping off the water on the surfaces of the scales and the stem segments to finish the operation.
5. The lily tissue culture rapid propagation method according to claim 1, wherein the method comprises the following steps: in the step S3 of the step I, a sterilization blade cutting operation is adopted, an explant with the width of 2-3mm is longitudinally cut for each flake by adopting a sterilized blade, and a stem section is cut into an explant with the thickness of 2 mm.
6. The lily tissue culture rapid propagation method according to claim 1, wherein the method comprises the following steps: the two culture medium formulas in the second step are respectively A1, MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzylaminopurine 1mg/L and alpha-naphthylacetic acid 0.1mg/L; a2 MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzylaminopurine 1mg/L, 2, 4-dichlorophenoxyacetic acid 1mg/L.
7. The lily tissue culture rapid propagation method according to claim 1, wherein the method comprises the following steps: the screening standard in the second step is that the height of the adventitious buds is 2 cm-2.5 cm; the bulblet size is 1 cm-1.5 cm.
8. The lily tissue culture rapid propagation method according to claim 1, wherein the method comprises the following steps: the four proliferation culture medium formulas in the step three are respectively as follows: z1: MS culture medium, sucrose 30g/L, agar 6.5g/L, 6-benzyl amino purine 0.5mg/L; z2: MS culture medium, 30g/L sucrose, 6.5g/L agar and 2 mg/L6-benzylaminopurine; z3: MS culture medium, 30g/L sucrose, 6.5g/L agar, 0.5 mg/L6-benzylaminopurine and 0.5mg/L alpha-naphthylacetic acid; z4: MS culture medium, 30g/L sucrose, 6.5g/L agar, 2 mg/L6-benzylaminopurine and 0.5mg/L alpha-naphthylacetic acid.
9. The lily tissue culture rapid propagation method according to claim 1, wherein the method comprises the following steps: the seedling hardening treatment time in the step five is 3-5 days; the sterilized matrix is prepared from turfy soil and coconut husk in a mass ratio of 1:1; putting the hardening seedlings into a matrix, watering thoroughly for the first time, and performing tectorial membrane shading treatment; and obtaining lily seedlings.
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| CN106718873A (en) * | 2016-11-14 | 2017-05-31 | 云南省农业科学院花卉研究所 | A kind of method of good blue Rapid Propagation of Lilium |
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