CN113951138A - Rapid propagation method of anemone macrocephala - Google Patents
Rapid propagation method of anemone macrocephala Download PDFInfo
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- 229930006000 Sucrose Natural products 0.000 claims description 15
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- 239000003415 peat Substances 0.000 claims description 5
- 229930191978 Gibberellin Natural products 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 239000003448 gibberellin Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 claims description 3
- 230000001902 propagating effect Effects 0.000 claims 8
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- 241000196324 Embryophyta Species 0.000 abstract description 10
- 230000001939 inductive effect Effects 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
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- 230000007423 decrease Effects 0.000 description 3
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- CEHZYZVJEXXYPM-NBOCEFNVSA-N (3s,8s,9s,10r,13s,14s,17r)-17-[(2s)-1-aminopropan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-ol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@@H](CN)C)[C@@]1(C)CC2 CEHZYZVJEXXYPM-NBOCEFNVSA-N 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- 241000245240 Lonicera Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 241000218201 Ranunculaceae Species 0.000 description 1
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- 230000028446 budding cell bud growth Effects 0.000 description 1
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- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- 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 discloses a rapid propagation method of anemone macrocephala, which specifically comprises the following steps: (1) disinfecting seeds; (2) obtaining aseptic seedlings; (3) inducing and differentiating callus of leaf and petiole; (4) inducing an epicotyl and a hypocotyl; (5) multiplication of adventitious buds; (6) rooting induction of adventitious buds; (7) transplanting and hardening seedlings. Sterile seedlings are obtained from seeds of wild flowers, namely, large-flowered anemone (Anemonesilvestris L.), and callus induction, proliferation, rooting culture and hardening seedling transplantation are sequentially carried out by taking leaves, petioles, epicotyls and hypocotyls as explants, so that a set of efficient and complete tissue culture rapid propagation system is established; and the plant grows robustly, the operation is simple, the emergence rate is higher and more stable than that of the seed propagation, the problem of shortage of germplasm resources in gardens and medicinal production is effectively solved, and the industrialized seedling culture can be realized.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a rapid propagation method of anemone macranthoides.
Background
The Anemone macrocephala (Anemone silverris L.) is a plant of Anemone of Ranunculaceae, has white flower, can be used as a medicine for all plants, has high ornamental value for flower leaves, has important effects on garden application and medicinal production, can be planted and developed in large quantities, and forms an industrial chain.
At present, the anemone crassipes is mostly distributed in high-altitude areas and is a typical alpine flower. Is not easy to be introduced under the restriction of natural conditions, has low seed setting rate and shortage of germplasm resources, and can not meet the requirements of gardens and medicines by depending on seed propagation.
Therefore, other breeding methods are found to be effective ways to strongly promote the anemone macrantha to gardens and medicinal markets.
Disclosure of Invention
In view of this, the present invention aims to provide a rapid propagation method of anemone macranthoides, so as to solve the deficiencies in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a rapid propagation method of anemone macrocephala specifically comprises the following steps:
(1) seed disinfection
Firstly, seeds of anemone macrocephala are placed in GA3Soaking in (gibberellin) solution, sterilizing with ethanol, and adding H2O2(hydrogen peroxide) sterilization to obtain sterilized seeds;
(2) obtaining of sterile seedlings
Inoculating the disinfected seeds into a seeding culture medium added with MS, cane sugar and agar for culture to obtain aseptic seedlings;
(3) induction and differentiation of leaf and petiole calli
When the aseptic seedlings grow to 3-7cm, cutting leaves and petioles, respectively and sequentially inoculating in a callus induction culture medium and a callus differentiation culture medium added with 1/2MS, 6-BA and NAA for induction and differentiation to obtain adventitious buds;
(4) induction of epicotyls and hypocotyls
Respectively inoculating the epicotyl and hypocotyl of the anemone grandiflorum as explants to an adventitious bud induction culture medium added with 1/2MS, 6-BA and NAA for induction to obtain induced adventitious buds;
(5) proliferation of adventitious buds
Inoculating the induced adventitious bud into an adventitious bud proliferation culture medium added with 1/2MS, 6-BA and NAA for proliferation to obtain a proliferation adventitious bud;
(6) rooting induction of adventitious buds
Respectively inoculating the proliferated adventitious buds into rooting induction culture media added with 1/2MS and IBA (indolebutyric acid) for rooting induction to obtain tissue culture seedlings;
(7) transplanting and hardening seedlings
Hardening and transplanting the tissue culture seedlings which grow strongly and have developed root systems, opening a bottle cap before transplanting, hardening the seedlings in a tissue culture chamber, hardening the seedlings in the external environment, taking the seedlings out of the tissue culture bottle, washing a culture medium, transplanting the seedlings into a matrix, and culturing in a greenhouse to obtain the anemone.
Further, in the step (1), the mass concentration of the gibberellin solution is 400mg/L, and the soaking time is 24 hours; the mass fraction of the alcohol is 75%, and the alcohol disinfection time is 30 s; h2O2Is 20% by mass, H2O2The time for sterilization is 15 min.
The beneficial effect of adopting the further technical scheme is that Gibberellins (GAs) are very important plant hormones and participate in a plurality of biological processes such as growth and development of a plurality of plants. The invention is characterized in that the seed of the anemone macrocephala is arranged in GA3The solution is soaked, so that the growth, germination, flowering and fruiting of the seed of the anemone macrocephala can be promoted. The invention disinfects by alcohol and H2O2Sterilizing, and eliminating the seed-borne by the seed of the large-flowered anemoneAnd (5) pathogenic bacteria.
Further, in the step (2), the seeding medium comprises: MS, sucrose 25g/L and agar 6 g/L.
The MS culture medium has the advantages that the concentration of inorganic salt and ions is high, the MS culture medium is a stable ion balance solution, the content of nitrate is high, the quantity and the proportion of nutrients are proper, and the nutrition and physiological requirements of plant cells can be met. Sucrose, in addition to providing energy, also induces redifferentiation of callus tissue. The agar can solidify the culture medium and play a certain supporting role.
Further, in the step (3), the composition of the leaf callus induction medium is as follows: 1/2MS, 6-BA2.0mg/L and NAA0.1 mg/L; the composition of the leaf callus differentiation culture medium is as follows: 1/2MS, 6-BA2mg/L and NAA0.1 mg/L; the composition of the petiole callus induction culture medium is as follows: 1/2MS, 6-BA2mg/L and NAA0.1 mg/L; the composition of the petiole callus differentiation culture medium is as follows: 1/2MS, 6-BA4mg/L and NAA0.1 mg/L.
The beneficial effect of adopting the further technical proposal is that the main function of the 6-BA is to promote the formation of buds and also induce the generation of callus. NAA is a broad-spectrum plant growth regulator, and can promote cell division and expansion, induce formation of adventitious roots and increase.
Further, in the step (4), the composition of the epicotyl adventitious bud induction medium is as follows: 1/2MS, 6-BA1mg/L and NAA0.1 mg/L; the composition of the hypocotyl adventitious bud induction culture medium is as follows: 1/2MS, 6-BA2mg/L and NAA0.1 mg/L.
Further, in the step (5), the adventitious bud growth medium has a composition of: 1/2MS, 6-BA1mg/L and NAA0.05 mg/L.
Further, in the step (6), the rooting induction medium comprises: 1/2MS and IBA0.3 mg/L.
The beneficial effect of adopting the further technical scheme is that IBA can promote the growth of the main roots of the plants, improve the germination rate and the survival rate, and is used for promoting the cuttings to root.
Further, in the step (8), the time for hardening off the seedlings in the tissue culture room and the time for hardening off the seedlings in the external environment are both 2 days; the matrix comprises the following components: the peat is vermiculite 2: 1.
The adoption of the further technical scheme has the beneficial effects that the turf contains a large amount of water, plant residues which are not thoroughly decomposed, humus and a part of mineral substances; vermiculite has ion exchange capacity and has great effect on soil nutrition.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the invention obtains aseptic seedlings from seeds of wild flower Anemone grandiflorum L, takes leaves, petioles, epicotyl and hypocotyl as explants, and sequentially performs callus induction, proliferation, rooting culture and hardening seedling transplantation to establish a set of efficient and complete tissue culture rapid propagation system.
2. The invention establishes a tissue culture system by 4 explants of leaves, petioles, epicotyls and hypocotyls, can realize plant regeneration, has the inductivity of 73.33 percent, 51.11 percent, 96.67 percent and 76.67 percent respectively, has robust plant growth, simple operation, high and stable emergence rate compared with seed propagation, effectively solves the problem of germplasm resource shortage in garden and medicinal production, and can realize industrialized seedling culture.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
Example 1
The rapid propagation method of the anemone macrocephala specifically comprises the following steps:
(1) seed disinfection
Firstly, the seed of the anemone macrocephala is treated with GA with the mass concentration of 400mg/L3Soaking in solution for 24H, sterilizing with 75% alcohol for 30s, and adding 20% H2O2Sterilizing for 15min to obtainTo the sterilized seeds;
(2) obtaining of sterile seedlings
Inoculating the disinfected seeds into a seeding culture medium added with MS, 25g/L sucrose and 6g/L agar for culture to obtain aseptic seedlings;
(3) induction and differentiation of leaf and petiole calli
When the sterile seedlings grow to 5cm, cutting leaves and leaf stalks, inoculating the leaves into a callus induction culture medium added with 1/2MS, 6-BA2.0mg/L and NAA0.1mg/L for induction, inoculating the callus of the leaves into a callus differentiation culture medium added with 1/2MS, 6-BA2mg/L and NAA0.1mg/L for differentiation, inoculating the leaf stalks into a callus induction culture medium added with 1/2MS, 6-BA2mg/L and NAA0.1mg/L for induction, inoculating the callus of the leaf stalks into a callus differentiation culture medium added with 1/2MS, 6-BA4mg/L and NAA0.1mg/L for differentiation, and obtaining adventitious buds;
(4) induction of epicotyls and hypocotyls
Taking an epicotyl and a hypocotyl of the anemone macrocephala as explants, inoculating the epicotyl into an adventitious bud induction culture medium added with 1/2MS, 6-BA1mg/L and NAA0.1mg/L for induction, and inoculating the hypocotyl into an adventitious bud induction culture medium added with 1/2MS, 6-BA2mg/L and NAA0.1mg/L for induction to obtain induced adventitious buds;
(5) proliferation of adventitious buds
Inoculating the induced adventitious bud into an adventitious bud proliferation culture medium added with 1/2MS, 6-BA1mg/L and NAA0.05mg/L for proliferation to obtain a proliferation adventitious bud;
(6) rooting induction of adventitious buds
Inoculating the proliferated adventitious buds into a rooting induction culture medium added with 1/2MS and 0.3mg/L IBA for rooting induction to obtain tissue culture seedlings;
(7) transplanting and hardening seedlings
Hardening and transplanting the strong and developed tissue culture seedlings with developed root systems, opening a bottle cap before transplanting, hardening the seedlings in a tissue culture room for 2 days, then putting the tissue culture room in the external environment for hardening the seedlings for 2 days, taking the seedlings out of the tissue culture bottle, washing a culture medium, transplanting the seedlings into a matrix (peat, vermiculite, 2:1), and culturing in a greenhouse to obtain the seedlings of the anemone.
Performance testing
1. Screening of disinfectants
Firstly, the seed of the anemone macrocephala is treated with GA with the mass concentration of 400mg/L3Soaking in solution for 24h, sterilizing with 75% alcohol for 30s, and respectively soaking in HgCl solutions with different mass fractions2、H2O2And three disinfectants of NaClO are subjected to disinfection treatment for different time lengths.
Wherein, HgCl2The mass fractions of the components are respectively 0.5%, 1.0%, 1.5% and 2.0%, and the treatment time is respectively 6min, 9min and 12 min; h2O2The mass fractions of the components are respectively 10%, 20% and 30%, and the treatment time is 10min, 15min and 20 min; the NaClO accounts for 6 percent, 8 percent and 10 percent respectively, and the processing time is 10min, 15min and 20min respectively.
And (3) repeating the treatments for 3 times, wherein each 30 seeds are repeatedly treated, the seeds are inoculated on an MS (agar 7g/L and sucrose 30g/L) culture medium and are placed in a tissue culture room at the temperature of 22-25 ℃ for culture, a fluorescent lamp is used as a culture light source, natural illumination is simulated, the illumination intensity is 2200lx, light and dark illumination alternation is carried out every 12 hours, and the pollution rate and the germination rate are counted.
The seeds treated by NaClO were all contaminated, so they were not listed in the table, and the effect of different disinfection treatments on the contamination rate and germination rate of the seed of the anemone macrocarpa is shown in Table 1.
TABLE 1 Effect of different disinfection treatments on seed contamination and germination rates of anemone macrocephala
Note: data in table 1 are mean ± sem, with different letters in the same column indicating significant differences (P <0.05), as in tables 2-14 below; all die; the growth vigor is weaker; the higher the amount of + the better the growth, the same is found in tables 2 to 14 below.
As can be seen from Table 1, the germination rate of the seed of the anemone macrocephala follows the HgCl2The treatment time increased and gradually decreased with HgCl2The increase in the treatment concentration showed a tendency to increase first and then decrease, with a mass fraction of 0.10% of HgCl2The germination rate is the highest at 66.67% after 6min of treatment.
The germination rate of the seed of the anemone macrocephala follows with H2O2The concentration of the solution increases and then decreases, the solution gradually decreases with the increase of the treatment time, and the mass fraction of the solution is 20 percent of H2O2The germination rate is the highest at 73.33% after 15min of treatment. With 20 percent of H by mass fraction2O2Treating for 20min with HgCl of 0.10 wt%2Treating for 6min with HgCl of 0.20 wt%2The germination rates at 6min of treatment were respectively increased by 46.67%, 9.99% and 69.24%, and the growth conditions were better than those of other treatments.
The above tests show that the mass fraction of H is 20%2O2The disinfection effect of the seed of the anemone macrocephala under 15min treatment is the best, and the germination rate is 73.33%.
2. Screening of seeding Medium
The seeds after the disinfection treatment are washed clean by sterile water, the water on the surfaces of the seeds is absorbed by sterilized filter paper, and the seeds are inoculated to MS and 1/2MS culture media which are prepared and added with sucrose (15g/L, 20g/L, 25g/L and 30g/L) and agar (5g/L, 6g/L and 7g/L) with different concentrations. And (3) repeating the treatment for 24 times, wherein 30 seeds are repeatedly cultured in a tissue culture room at the temperature of 22-25 ℃, the illumination intensity is 2200lx, the illumination is 12h/d, and the growth condition of the sterile seedlings is counted after 40 d. The results are shown in Table 2.
TABLE 2 Effect of different sucrose and agar concentrations on seed germination percentage and growth of sterile seedlings
As can be seen from Table 2, the germination rate of the medium treated by MS and sucrose at 25g/L and agar at 6g/L was up to 93.33%, which was significantly higher than that of the medium treated by MS and sucrose at 25g/L and agar at 5g/L and 25g/L (P < 0.05).
Under the treatment of 1/2MS medium, the germination rate of 1/2MS + sucrose 25g/L + agar 6g/L is up to 66.67%, which is 71.43% of the germination rate of MS + sucrose 25g/L + agar 6 g/L. The growth conditions of the aseptic seedlings under the treatment of MS and 1/2MS culture media of 25g/L of sucrose are superior to those of other treatments of the same type of culture media.
The tests show that the optimal seeding culture medium for the anemone macrocephala is MS, sucrose is 25g/L and agar is 6g/L, the germination rate is the highest and is 93.33%, and the anemone macrocephala grows robustly.
3. Screening of leaf Induction Medium
When the sterile seedlings grow to 5cm high, cutting leaves of the sterile seedlings, respectively inoculating the leaves on 1/2MS culture media added with MS (0.5mg/L, 1mg/L and 2mg/L), NAA (0.1mg/L, 0.3mg/L, 0.5mg/L and 1mg/L) and NAA (0.1mg/L, 0.3mg/L and 0.5mg/L) with different concentrations, setting 26 treatments, repeating for 3 times, inoculating 30 times to induce callus, and counting the influence of different treatments on the induction rate and the growth condition of the leaves of the anemone. The results are shown in Table 3.
TABLE 3 influence of different treatments on the leaf induction rate and growth status of anemone macrocephala
As is clear from Table 3, the callus induction rate was the highest in the MS medium treated with 1.0mg/L of 6-BA plus 1.0mg/L of NAA or 2.0mg/L of 6-BA plus 0.5mg/L of NAA, and was 93.33%, and the growth condition was better in the latter treatment.
1/2MS culture medium, the callus inductivity is highest under the treatment of adding 6-BA2.0mg/L + NAA0.1mg/L or 6-BA4.0mg/L + NAA0.5mg/L, the callus inductivity is 100%, the callus growth condition under the former treatment is better.
Since the leaf blade under 1/2MS medium produced callus at 20d, it was earlier than MS medium and better in yellow-green texture and mostly had white bumps, with differentiation ability.
The above experiments show that the optimal induction culture medium for the leaf callus is 1/2MS +6-BA 2.0mg/L + NAA0.1mg/L, and the induction rate is 100% at most.
4. Screening of leaf differentiation Medium
Inoculating leaf callus on 1/2MS culture medium added with 6-BA (1mg/L, 2mg/L, 4mg/L), NAA (0.1mg/L, 0.5mg/L, 1mg/L) and 6-BA (0.5mg/L, 1mg/L, 2mg/L, 4mg/L), NAA (0.1mg/L, 0.3mg/L) of different concentrations, performing callus differentiation and adventitious bud induction, setting 19 treatments, repeating for 3 times, inoculating 30 treatments, and counting the influence of different treatments on the bud number, differentiation rate and growth condition of the anemone. The results are shown in Table 4.
TABLE 4 influence of different treatments on the number of buds, differentiation rate and growth status of the leaves of the anemone macrocephala
As shown in Table 4, the optimal callus differentiation medium in the MS medium was MS +6-BA 2mg/L + NAA0.1mg/L, and the differentiation rate was 62.22%.
The optimal callus differentiation medium in 1/2MS medium was: 1/2MS +6-BA 2mg/L + NAA0.1mg/L, the highest differentiation rate is 77.78%. With 1/2MS being the preferred growth.
The above experiments show that the optimal differentiation medium for leaf callus is 1/2MS +6-BA 2mg/L + NAA0.1mg/L, and the highest differentiation rate is 77.78%.
5. Screening of petiole Induction Medium
When the sterile seedlings grow to 5cm high, cutting the petioles of the sterile seedlings, respectively inoculating the petioles on 1/2MS culture media added with MS (0.5mg/L, 1mg/L and 2mg/L), NAA (0.1mg/L, 0.3mg/L, 0.5mg/L and 1mg/L) and NAA (0.1mg/L, 0.3mg/L and 0.5mg/L) of different concentrations, setting 26 treatments, repeating for 3 times, and inoculating 30 times to induce callus, and counting the influence of different treatments on the petiole induction rate and growth condition of the anemone. The results are shown in Table 5.
TABLE 5 influence of different treatments on the petiole inductivity and growth status of anemone macrocephala
As can be seen from Table 5, the induction rates of the two treatments of adding 6-BA2.0mg/L + NAA0.5mg/L in the MS culture medium and adding NAA0.1mg/L +6-BA 2.0mg/L in the 1/2MS minimal medium are both 100%, but the induction rate of the callus under the 1/2MS culture medium is integrally higher than that of the MS culture medium, the growth vigor is better than that of the MS culture medium, the callus is formed at 15d, most of the callus is green and large, and the differentiation potential is higher.
The above experiments show that the optimal induction culture medium for the petiole callus is 1/2MS +6-BA 2.0mg/L + NAA0.1mg/L, and the induction rate is 100% at most.
6. Screening of petiole differentiation Medium
The callus of petiole is inoculated on MS culture medium added with 6-BA (1.0mg/L, 2.0mg/L, 4.0mg/L), NAA (0.1mg/L, 0.5mg/L, 1.0mg/L) and 1/2MS culture medium added with NAA (0.1mg/L, 0.3mg/L), 6-BA (0.5mg/L, 1.0mg/L, 2.0mg/L, 4.0mg/L) with different concentrations, 19 treatments are set, 15 bottles are treated, 3 times are repeated, 3 treatments are inoculated in each bottle, and the influence of different treatments on the bud number, differentiation rate and growth condition of petiole callus of anemone is counted. The results are shown in Table 6.
TABLE 6 influence of different treatments on the sprouting number, differentiation rate and growth status of petioles of Lonicera macrantha
As is clear from Table 6, although the differentiation rates of the petiole calli were 51.11% at 1.0mg/L of 6-BA + 0.1mg/L of NAA in the MS medium and at 0.1mg/L of NAA in the 1/2MS medium +6-BA4.0mg/L of NAA, more adventitious buds differentiated at the 16d start in the 1/2MS medium, and the adventitious buds grew better than the MS medium.
The above experiments show that the optimal culture medium for callus differentiation of petioles is 1/2MS +6-BA4.0mg/L + NAA0.1mg/L, and the differentiation rate is up to 51.11%.
7. Screening of epicotyl adventitious bud induction medium
Taking the epicotyl of the anemone gigantea as an explant, inoculating the epicotyl of the anemone gigantea to a basic culture medium of 1/2MS, adding induction culture media of 6-BA (0.5mg/L, 1mg/L and 2mg/L), NAA (0.1mg/L, 0.2mg/L, 0.5mg/L and 1mg/L) combinations with different concentrations, carrying out 12 treatments, repeating for 3 times each treatment, inoculating 30 times each repetition, and counting the influence of different hormones on the induction rate and the growth condition of the adventitious bud of the epicotyl of the anemone gigantea. The results are shown in Table 7.
TABLE 7 influence of different hormones on the rate of induction and growth of epicotyl adventitious buds of anemone grandiflorum
As shown in Table 7, in 1/2MS medium without any hormone, the epicotyls did not induce callus and adventitious buds, and the epicotyls treated with other hormones induced buds directly. The optimal culture medium for inducing adventitious buds of epicotyl is 1/2MS +6-BA 1mg/L + NAA0.1mg/L, the induction rate is high, and the seedlings are strong and are 84.44%.
8. Screening of hypocotyl adventitious bud induction medium
Taking hypocotyl of anemone gigantea as an explant, inoculating the hypocotyl to 1/2MS as a basic culture medium, adding induction culture media of 6-BA (0.5mg/L, 1mg/L and 2mg/L), NAA (0.1mg/L, 0.2mg/L, 0.5mg/L and 1mg/L) combinations with different concentrations, carrying out 12 treatments, repeating for 3 times, inoculating 30 times for each repetition, and counting the influence of different hormones on the induction rate and growth condition of the hypocotyl adventitious bud of the anemone gigantea. The results are shown in Table 8.
TABLE 8 influence of different hormones on hypocotyl adventitious bud induction rate, bud emergence rate and growth status of anemone macranthoides
As shown in Table 8, the optimal culture medium for inducing adventitious buds of hypocotyls is 1/2MS +6-BA 2mg/L + NAA0.1mg/L, and the induction rate is the highest and is 76.67%.
9. Screening of adventitious bud propagation Medium
And (3) inoculating the differentiated adventitious buds into 1/2MS culture media added with 6-BA (1mg/L, 1.5mg/L, 2mg/L and 4mg/L) and NAA (0.1mg/L, 0.2mg/L and 0.5mg/L) with different concentrations, carrying out 13 treatments, repeating the treatments for 3 times, inoculating 30 treatments for each repetition, and counting the influence of different hormone treatments on the adventitious bud proliferation coefficient and the growth condition of the anemone. The results are shown in Table 9.
TABLE 9 Effect of different hormone treatments on the multiplication factor and growth status of adventitious buds of anemone majus
| 6-BA(mg/L) | NAA(mg/L) | Coefficient of proliferation | Growth conditions |
| 0 | 0 | 2.78±0.11abcd | Thin and high |
| 1.0 | 0.05 | 3.67±0.51a | Thick stem, higher and greener |
| 1.5 | 0.05 | 2.44±0.11abcd | Thick stem and high bud |
| 2.0 | 0.05 | 1.78±0.11d | Bud small |
| 4.0 | 0.05 | 1.89±0.4cd | Weak and small bud |
| 1.0 | 0.1 | 3.44±0.29ab | A lot of buds but yellow |
| 1.5 | 0.1 | 2.22±0.11bcd | Weak and small |
| 2.0 | 0.1 | 2.22±0.22bcd | A weak bud with a slight yellowing |
| 4.0 | 0.1 | 1.78±0.11d | Short and small |
| 1.0 | 0.2 | 2.00±0.00cd | Weak, vitrification |
| 1.5 | 0.2 | 2.45±0.40abcd | Emaciation and weakness of bud |
| 2.0 | 0.2 | 3.11±0.22abc | Short and small bud |
| 4.0 | 0.2 | 1.78±0.11d | Less sprouts, weak, vitrified |
As can be seen from Table 9, the multiplication factor of adventitious buds is the highest under the comprehensive treatment of 1/2MS, 6-BA 1.0mg/L and NAA0.05mg/L, and is 3.67, the adventitious buds are thick in stem, the leaves are bright green, and the height of the whole buds is the highest.
10. Screening of adventitious bud rooting induction culture medium
Adopting 1/2MS as a basic culture medium, selecting IAA and IBA hormones with the concentrations of 0mg/L, 0.3mg/L, 0.6mg/L, 1.2mg/L and 2mg/L for 9 treatments, inoculating 48 adventitious buds for each treatment, and counting the influence of different hormone treatments on the adventitious bud rooting rate, the average rooting number, the root length and the growth condition of the anemone. The results are shown in Table 10.
TABLE 10 influence of different hormone treatments on the adventitious bud rooting rate, average number of roots, root length and growth status of anemone majus
As can be seen from Table 10, the optimal rooting induction medium for adventitious buds was 1/2MS + IBA0.3mg/L, and the rooting rate was 100%.
11. Screening of the transplanting Medium
Hardening and transplanting the tissue culture seedlings which are strong in growth and have developed root systems, opening a bottle cover before transplanting, hardening the seedlings in a tissue culture room for 2 days, then putting the tissue culture bottles for hardening the seedlings for 2 days, taking the seedlings out of the tissue culture bottles, washing culture media, transplanting the seedlings into different substrates (turf: perlite: vermiculite: 1:1, turf: perlite: 3:1, turf: sand: 2:1, turf: vermiculite: 2:1 and pure turf), treating 30 plants each, culturing in a greenhouse at 22-25 ℃, and counting the influence of different culture media on the transplanting survival rate and the growth condition of the tissue culture seedlings. The results are shown in Table 11.
TABLE 11 influence of different culture media on the survival rate and growth condition of transplanting tissue culture seedlings
As can be seen from Table 11, the survival rates of the transplants were 66.67%, 50%, 88.33%, 98.33%, and 100% respectively under the treatment of the five different substrates. However, the plant grows most strongly under the condition that the ratio of the peat to the vermiculite is 2:1, so that the optimum transplanting matrix is the peat and the vermiculite is 2: 1.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A rapid propagation method of anemone macrocephala is characterized by comprising the following steps:
(1) seed disinfection
Firstly, seeds of anemone macrocephala are placed in GA3Soaking in solution, sterilizing with ethanol, and adding H2O2Sterilizing to obtain sterilized seeds;
(2) obtaining of sterile seedlings
Inoculating the disinfected seeds into a seeding culture medium added with MS, cane sugar and agar for culture to obtain aseptic seedlings;
(3) induction and differentiation of leaf and petiole calli
When the aseptic seedlings grow to 3-7cm, cutting leaves and petioles, respectively and sequentially inoculating in a callus induction culture medium and a callus differentiation culture medium added with 1/2MS, 6-BA and NAA for induction and differentiation to obtain adventitious buds;
(4) induction of epicotyls and hypocotyls
Respectively inoculating the epicotyl and hypocotyl of the anemone grandiflorum as explants to an adventitious bud induction culture medium added with 1/2MS, 6-BA and NAA for induction to obtain induced adventitious buds;
(5) proliferation of adventitious buds
Inoculating the induced adventitious bud into an adventitious bud proliferation culture medium added with 1/2MS, 6-BA and NAA for proliferation to obtain a proliferation adventitious bud;
(6) rooting induction of adventitious buds
Inoculating the proliferated adventitious buds into a rooting induction culture medium added with 1/2MS and IBA for rooting induction to obtain a tissue culture seedling;
(7) transplanting and hardening seedlings
Hardening and transplanting the tissue culture seedlings which grow strongly and have developed root systems, opening a bottle cap before transplanting, hardening the seedlings in a tissue culture chamber, hardening the seedlings in the external environment, taking the seedlings out of the tissue culture bottle, washing a culture medium, transplanting the seedlings into a matrix, and culturing in a greenhouse to obtain the anemone.
2. The method for rapidly propagating anemone macrocephala as claimed in claim 1, wherein in the step (1), the mass concentration of the gibberellin solution is 400mg/L, and the soaking time is 24 h;
the mass fraction of the alcohol is 75%, and the alcohol disinfection time is 30 s;
said H2O2Is 20%, said H2O2The time for sterilization is 15 min.
3. The method for rapidly propagating anemone macrocephala according to claim 1, wherein in the step (2), the sowing culture medium comprises the following components: MS, sucrose 25g/L and agar 6 g/L.
4. The method for rapidly propagating anemone macrocephala according to claim 1, wherein in the step (3), the composition of the leaf callus induction culture medium is as follows: 1/2MS, 6-BA2.0mg/L and NAA0.1mg/L;
the leaf callus differentiation culture medium comprises the following components: 1/2MS, 6-BA2mg/L and NAA0.1mg/L;
the composition of the petiole callus induction culture medium is as follows: 1/2MS, 6-BA2mg/L and NAA0.1mg/L;
the petiole callus differentiation culture medium comprises the following components: 1/2MS, 6-BA4mg/L and NAA0.1mg/L.
5. The method for rapidly propagating anemone macrocephala according to claim 1, wherein in the step (4), the composition of the epicotyl adventitious bud induction medium is as follows: 1/2MS, 6-BA1mg/L and NAA0.1mg/L.
6. The method for rapidly propagating anemone macrocephala according to claim 1, wherein in the step (4), the composition of the hypocotyl adventitious bud induction medium is as follows: 1/2MS, 6-BA2mg/L and NAA0.1mg/L.
7. The method for rapidly propagating anemone macrocephala according to claim 1, wherein in the step (5), the adventitious bud multiplication medium consists of: 1/2MS, 6-BA1mg/L and NAA0.05mg/L.
8. The method for rapidly propagating anemone macrocephala according to claim 1, wherein in the step (6), the rooting induction medium comprises the following components: 1/2MS and IBA0.3mg/L.
9. The rapid propagation method of anemone macrocephala according to claim 1, wherein in the step (7), the time for acclimatization in the tissue culture chamber and the time for acclimatization in the external environment are both 2 days.
10. The method for rapidly propagating anemone macrocephala according to claim 1, wherein in the step (7), the composition of the matrix is as follows: the peat is vermiculite 2: 1.
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