CN116548314A - Method for tissue culture and rapid propagation of flat white bead clone - Google Patents
Method for tissue culture and rapid propagation of flat white bead clone Download PDFInfo
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- CN116548314A CN116548314A CN202310831305.6A CN202310831305A CN116548314A CN 116548314 A CN116548314 A CN 116548314A CN 202310831305 A CN202310831305 A CN 202310831305A CN 116548314 A CN116548314 A CN 116548314A
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- 239000011324 bead Substances 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 19
- 235000020280 flat white Nutrition 0.000 title description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 35
- 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 abstract description 18
- 229930006000 Sucrose Natural products 0.000 claims abstract description 18
- 239000001963 growth medium Substances 0.000 claims abstract description 18
- 239000005720 sucrose Substances 0.000 claims abstract description 18
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 11
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 8
- 229960002523 mercuric chloride Drugs 0.000 claims description 6
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 230000001902 propagating effect Effects 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 241000196324 Embryophyta Species 0.000 description 36
- 230000012010 growth Effects 0.000 description 23
- 230000000694 effects Effects 0.000 description 12
- 230000004083 survival effect Effects 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 5
- 241000283966 Pholidota <mammal> Species 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- 230000008635 plant growth Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- 230000008121 plant development Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 229930192334 Auxin Natural products 0.000 description 2
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 2
- 239000002363 auxin Substances 0.000 description 2
- 239000004062 cytokinin Substances 0.000 description 2
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 2
- 229960000367 inositol Drugs 0.000 description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012809 post-inoculation Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- 241000218993 Begonia Species 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 241000208421 Ericaceae Species 0.000 description 1
- 241000735588 Gaultheria Species 0.000 description 1
- 240000002548 Gaultheria fragrantissima Species 0.000 description 1
- 235000001703 Gaultheria fragrantissima Nutrition 0.000 description 1
- 240000001238 Gaultheria procumbens Species 0.000 description 1
- 235000007297 Gaultheria procumbens Nutrition 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 230000001680 brushing effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229940076642 icy hot Drugs 0.000 description 1
- 239000008325 icyhot Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229940034610 toothpaste Drugs 0.000 description 1
- 239000000606 toothpaste Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000004017 vitrification Methods 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
- 239000009637 wintergreen oil Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- 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
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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
A method for tissue culture and rapid propagation of a clone of tiled white beads relates to the field of biotechnology, in particular to a tissue culture technology of tiled white beads. The method comprises the processes of disinfection, primary culture, secondary culture, rooting culture and the like of the explant, wherein the primary culture medium comprises the following formula: MRM+sucrose 25-30 g/L+TDZ1-1.4 mg/L+activated carbon 0.6-1 g/L; the formula of the secondary culture medium is as follows: MRM+sucrose 25-30 g/L+2-IP7.5-12.5 mg/L+NAA0.08-0.1 mg/L+activated carbon 0.6-1 g/L; the rooting culture medium is as follows: 1/2 MRM+sucrose 25-30 g/L+IBA0.3 mg/L+NAA 0-0.6 mg/L+activated carbon 0.6-1 g/L. The rapid propagation method can establish a clone rapid propagation system of the tiled white beads, provides technical support for the standardized production of the tiled white beads seedlings, and solves the problem of unstable seedling characters in the traditional seedling culture mode of the tiled white beads.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a tissue culture technology for tiling white beads.
Background
Flat white bead @Gaultheria procumbens) The genus Begonia belonging to the subfamily Vaccinii Vitis-idaeae (Ericaceae)Gaultheria) The plant, also called creeping white beads, is a perennial evergreen creeping shrub, is native in the United states and Canada, has extremely high ornamental value of fruits, has white, powder, red and other colors, has long fruiting period, can keep the whole winter, is developed into a plant for viewing an orchard art, becomes a potted plant for viewing fruits which is popular internationally in recent years, has considerable economic benefit, and is introduced into cultivation in China.
The fruits of the pangolin scales are rich in salicylic acid and anthocyanin, the medicinal value in North America is comparable to that of the pangolin scales in China, the pangolin scales have the effects of relieving pain, diminishing inflammation, treating rheumatism and the like, and the pangolin scales are the main components of the American analgesic ICY HOT. Meanwhile, the white pearl leaves are paved or the main raw materials of the wintergreen oil are extracted, and the white pearl oil is widely used for producing products such as toothpaste, chewing gum, insecticide and the like, and the industrial value of the white pearl oil is very remarkable. The spread white beads are usually propagated through seeds and cuttage, and the characteristics of the sown seedlings are seriously separated, so that the commodity characteristics are unstable; the cutting seedlings are not regular, and the standardized production requirement is difficult to meet. Therefore, tissue culture rapid propagation is an important way for realizing standardized production of tiled white pearl seedlings. The existing technique for tiling Bai Zhuzu culture and rapid propagation is realized by obtaining a rapid propagation system through aseptic seeding of seeds, the nature of the technique is still sexual propagation, the seedling character separation is also serious, and the standardized production of finished products is restricted. Therefore, tissue culture and rapid propagation through asexual materials are technical keys for solving standardized production of seedlings.
Disclosure of Invention
The method for rapidly propagating the clone tissue of the tiled white beads is characterized by comprising the following steps:
step 1, selecting the current young stem of the plant as an explant;
step 2, removing leaves of the explant, and cutting the explant into small sections of 4-5 cm with axillary buds;
step 3, cleaning the explant;
step 4, sterilizing the explant;
step 5, performing primary culture on the sterilized explant;
step 6, subculturing the primary sterile seedlings;
step 7, carrying out rooting culture on the adventitious buds after subculture;
the formula of the primary culture medium adopted in the step 5 is as follows: MRM+sucrose 25-30 g/L+TDZ1-1.4 mg/L+activated carbon 0.6-1 g/L;
the formula of the secondary culture medium adopted in the step 6 is as follows: MRM+sucrose 25-30 g/L+2-IP7.5-12.5 mg/L+NAA0.08-0.1 mg/L+activated carbon 0.6-1 g/L;
the rooting culture medium adopted in the step 7 is as follows: 1/2 MRM+sucrose 25-30 g/L+IBA0.3 mg/L+NAA 0-0.6 mg/L+activated carbon 0.6-1 g/L.
The disinfection method adopted in the step 4 is that 75% alcohol is used for disinfection for 30-35 s, then 0.1% mercuric chloride is used for soaking and disinfection for 11-12min, and finally sterile water is used for rinsing.
The MRM medium formulation is described in publication Indian Journal of Biotechnology Vol 11,April 2012,pp 197-204, in vitro plant regeneration of wintergreen (Gaultheria fragrantissima wall.): assessment of multiplenutrient formulations and cytokinin types. The specific table is shown below:
| mother liquor component | MRM quantification (mg/L) | 1/2MRM quantification (mg/L) |
| NH 4 NO 3 | 400 | 200 |
| KNO 3 | 480 | 240 |
| CaCl 2• 2H 2 O | 440 | 220 |
| MgSO 4• 7H 2 0 | 370 | 185 |
| NaH 2 PO 4• 2H 2 O | 380 | 190 |
| H 3 BO 3 | 6.2 | 3.1 |
| MnSO 4• H 2 O | 16.9 | 8.45 |
| NaMoO 4• 2H 2 O | 0.25 | 0.13 |
| CuSO 4• 5H 2 O | 0.025 | 0.01 |
| CoCl 2• 6H 2 O | 0.025 | 0.01 |
| ZnSO 4• 7H 2 O | 8.6 | 4.3 |
| FeSO 4• 7H 2 O | 27.8 | 13.9 |
| Na 2 -EDTA | 37.3 | 18.65 |
| Inositol (inositol) | 1 | 0.5 |
The rapid propagation method can establish a clone rapid propagation system of the tiled white beads, provides technical support for the standardized production of the tiled white beads seedlings, and solves the problem of unstable seedling characters in the traditional seedling culture mode of the tiled white beads.
Drawings
FIG. 1 is a graph showing the growth conditions of the stem segments with the TDZ concentration of the primary culture medium of 1 mg/L.
FIG. 2 is a graph showing the growth conditions of the stem segments with the TDZ concentration of the primary culture medium of 2 mg/L.
FIG. 3 is a graph showing the growth conditions of the stem segments with the TDZ concentration of 3 mg/L in the primary culture medium.
FIG. 4 shows a commercial potted flower obtained by tissue culture and rapid propagation in example 1.
Detailed Description
Example 1: the method for tissue culture and rapid propagation of the tiled white bead clone comprises the following steps:
step 1, selecting the current young stem segment of the plant as an explant.
Step 2, removing leaves of the explant, and cutting into small sections of 4-5 cm with axillary buds.
Step 3, cleaning the explant;
placing the explant in a detergent solution, slightly brushing surface mud ash with a brush, cleaning for 30 min with an ultrasonic cleaner, and finally flushing for 30 min with running water.
Step 4, sterilizing the explant;
the washed explant is moved into an ultra-clean workbench, sterilized by 75% alcohol for 30 s, soaked and sterilized by 0.1% mercuric chloride, continuously swayed during the process, so that mercuric chloride solution is fully contacted with the explant, finally rinsed for 5 times by sterile water, the rinsing time is not less than 10min, and the residual moisture of the explant is sucked by filter paper for standby.
Step 5, performing primary culture on the sterilized explant;
the formula of the primary culture medium is as follows: MRM+sucrose 30 g/L+TDZ1 mg/L+activated carbon 1g/L.
Step 6, subculturing the primary sterile seedlings;
in an ultra-clean workbench, transferring sterile seedlings with basically consistent growth vigor obtained by sterile germination into a secondary culture medium for culture;
the formula of the secondary culture medium is as follows: MRM+sucrose 30 g/L+2-IP 10 mg/L+NAA 0.1 mg/L+activated carbon 1g/L.
Step 7, carrying out rooting culture on the adventitious buds after subculture;
the rooting culture medium comprises the following formula: 1/2 MRM+sucrose 30 g/L+IBA0.3 mg/L+NAA 0.3 mg/L+activated carbon 1g/L;
and (5) screening out robust adventitious buds, transferring the robust adventitious buds into a rooting culture medium, and performing rooting culture.
Example 2: the method for quickly propagating the clone tissue of the tiled white beads adopts a primary culture medium formula as follows: MRM+sucrose 30 g/L+TDZ 1.4 mg/L+activated carbon 1g/L; the formula of the proliferation medium adopted in the step 6 is as follows: MRM+sucrose 30 g/L+2-IP7.5 mg/L+NAA 0.1 mg/L+activated carbon 1g/L; step 7, the rooting culture medium formula is as follows: 1/2 MRM+30 g/L of sucrose+0.3 mg/L of IBA+0.6 mg/L of NAA+1 g/L of active carbon. The other steps are the same as in example 1.
Example 3: the method for quickly propagating the clone tissue of the tiled white beads adopts a primary culture medium formula as follows: MRM+sucrose 25 g/L+TDZ 1.2 mg/L+activated carbon 0.6 g/L; the formula of the proliferation medium adopted in the step 6 is as follows: MRM+sucrose 25 g/L+2-IP 12.5 mg/L+NAA0.08 mg/L+activated carbon 0.6 g/L; step 7, the rooting culture medium formula is as follows: 1/2 MRM+sucrose 25 g/L+IBA0.3 mg/L+activated charcoal 0.6g/L.
The other steps are the same as in example 1.
The MRM of the basic culture medium of the primary culture medium is matched with TDZ with different concentrations, so that the death rate, the survival rate and the pollution rate of the germination of the stem segments are affected. The effect on shoot germination after 45d incubation at different concentrations of TDZ is shown in Table 1 below.
TABLE 1 influence of different concentrations of TDZ on shoot sprouting
| TDZ concentration （mg/L） | Number of inoculations (plant) | Success of induction Number (plant) | Number of deaths (plant) | Pollution number (plant) | Survival rate of （%） | Mortality rate of （%） | Pollution rate （%） | Growth status |
| 1 | 30 | 12 | 12 | 6 | 40.00 | 40.00 | 20.00 | Easily induce lateral buds, the lateral buds have normal leaf color and the plant growth status Good quality |
| 1.2 | 30 | 16 | 11 | 3 | 53.30 | 36.70 | 10.00 | Easily induce lateral buds, the lateral buds have normal leaf color and the plant growth status Good quality |
| 1.4 | 30 | 15 | 13 | 2 | 50.00 | 43.30 | 6.00 | Easily induce lateral buds, the lateral buds have normal leaf color and the plant growth status Good quality |
| 2 | 30 | 15 | 11 | 4 | 50.00 | 36.67 | 13.33 | Lateral buds are induced, but leaves are red, and the plant growth condition is not good Jiajia (good) |
| 3 | 30 | 8 | 17 | 5 | 26.67 | 56.67 | 16.67 | Lateral buds are not easy to induce, and the growth condition of plants is abnormal with With phenomena such as browning and vitrification |
In the secondary culture, different cytokinins and auxins have influence on the growth of aseptic seedlings. Table 2 shows the effect of 2-IP concentration on the growth of aseptic seedling, and it can be seen from Table 2 that the plant height growth is large and can reach 3.63+ -1.94 cm at the concentration of 2-IP of 7.5-12.5 mg/L.
TABLE 2 Effect of different concentrations of 2-IP on sterile seedling growth
| 2-IP concentration (mg/L) | Plant height increment (cm) | Growth status |
| 5 | 1.94±0.86 | The plants increase slowly, with a small number of plants showing adventitious buds |
| 7.5 | 2.86±1.69 | The plants are quickly heightened and develop normally |
| 10 | 3.63±1.94 | The plants are quickly heightened and develop normally |
| 12.5 | 3.16±1.75 | The plants are quickly heightened and develop normally |
In addition, compared with the 2-IP of the invention, the aseptic seedlings are subjected to secondary culture by selecting ZT, 6-BA and TDZ with different concentrations, after 90 d is cultured, the growth condition of the plants is observed and recorded, and the plant heights and proliferation coefficients are counted in tables 3, 4 and 5 below. As can be seen from tables 2, 3, 4 and 5, when the 2-IP of the invention is selected, plants are rapidly increased and develop normally, and compared with 2-IP, the situation that the sterile seedlings are subjected to subculture by different concentrations ZT, 6-BA and TDZ is not as good as that of the sterile seedlings.
TABLE 3 Effect of different concentrations of ZT on the growth of sterile seedlings
| ZT concentration (mg/L) | Height of plant (cm) | Growth status |
| 1 | 1.79±0.39 | Rapid growth and normal development of leaves and stems |
| 2 | 1.41±0.55 | The growth is slower, and leaves and stems are mostly red |
| 3 | 1.03±0.55 | Very slow growth, reddish leaves and stems with signs of whitening |
TABLE 4 Effect of different concentrations of 6-BA on sterile shoot growth
| 6-BA concentration (mg/L) | Plant height increment (cm) | Growth status |
| 1 | 1.53±0.85 | The plant grows rapidly and can promote the development of leaves |
| 2 | 0.78±0.75 | The plant grows faster and the stem and the leaf develop normally |
| 3 | 1.34±0.47 | The plant grows rapidly and the stem and leaf develop normally |
TABLE 5 Effect of different concentrations of TDZ on sterile shoot growth
| TDZ concentration (mg/L) | Root system condition | Plant height increment (cm) |
| 1 | Has the following components | 1.42±0.53 |
| 2 | Has the following components | 1.07±0.4 |
| 3 | Has the following components | 2.59±0.66 |
Different auxins also had different effects on rooting of plants, and the effect results are shown in Table 6.
TABLE 6 Effect of different rooting media on sterile seedling growth
| NAA concentration (mg/L) | IBA concentration (mg/L) | Rooting percentage (%) | Root length (mm) | Growth status |
| 0 | 0 | 60.00 | 40.22±15.49 | The root system condition is optimal, and the plant is normal in growth |
| 0 | 0.3 | 84.00 | 13.97±3.19 | The root system is moderate in thickness, the main root is not long enough, and the plant has little development and part is abnormal |
| 0.3 | 0.3 | 100.00 | 18.57±7.47 | Root system thicknessProper fineness, insufficient length of main root and normal plant development |
| 0.6 | 0.3 | 96.00 | 16.35±6.82 | Normal plant development |
| 0.3 | 0 | 58.00 | 12.63±2.53 | The root system is extremely weak, the quantity is small and short, and the plant has little development and partial abnormality |
| 0 | 1 | 56.00 | 15.44±8.00 | Brown stem, multiple but short roots and abnormal plant development |
| 1 | 0 | 28.57 | 11.48±2.19 | The root system is extremely weak, the quantity is small and short, and most of plants are abnormal in development |
| 1 | 1 | 25.00 | 7.20±1.37 | The stem has a small amount of callus, the root is small, short but thick, and the plant is dysplasia |
In addition, the mercuric chloride sterilization time will also affect the contamination rate and survival rate of the explants. Explants contamination rates were counted at 14 and d post-inoculation at different mercuric chloride sterilization times, and explant viability was counted at 30 and d post-inoculation and recorded in table 7 below. Wherein the survival standard is axillary bud germination.
TABLE 7 Effect of Mercury disinfection time on explant contamination and survival
| Explant type | Mercury sterilization time (min) | Seed number (strain) | Pollution number (plant) | Pollution rate (%) | Survival rate (%) |
| Stem segment | 5 | 30 | 26 | 86.67 | 6.67 |
| Stem segment | 7 | 30 | 24 | 80.00 | 13.33 |
| Stem segment | 9 | 30 | 18 | 60.00 | 26.67 |
| Stem segment | 12 | 30 | 5 | 16.67 | 56.67 |
| Stem segment | 14 | 30 | 6 | 20.00 | 26.67 |
It can be seen from table 7 that the longer the mercuric disinfection time, the lower the contamination rate, but the survival rate correspondingly decreases, so the mercuric disinfection time also affects the technical effect of the present invention.
The rapid propagation method of the invention is characterized in that by designing a proper primary culture medium, a secondary culture medium and a rooting culture medium,
the flower type, the flower color, the fruit shape and the color of the finished plant are basically consistent, the variation rate is not more than 3 percent, however, the seedling obtained by the aseptic seeding mode is included by seeding, the ornamental characters such as the flower type, the flower color, the fruit shape and the color of the finished plant are seriously separated, and the variation rate is as high as 40-56 percent.
Claims (2)
1. The method for rapidly propagating the clone tissue of the tiled white beads is characterized by comprising the following steps:
step 1, selecting the current young stem of the plant as an explant;
step 2, removing leaves of the explant, and cutting the explant into small sections of 4-5 cm with axillary buds;
step 3, cleaning the explant;
step 4, sterilizing the explant;
step 5, performing primary culture on the sterilized explant;
step 6, subculturing the primary sterile seedlings;
step 7, carrying out rooting culture on the adventitious buds after subculture;
the formula of the primary culture medium adopted in the step 5 is as follows: MRM+sucrose 25-30 g/L+TDZ1-1.4 mg/L+activated carbon 0.6-1 g/L;
the formula of the secondary culture medium adopted in the step 6 is as follows: MRM+sucrose 25-30 g/L+2-IP7.5-12.5 mg/L+NAA0.08-0.1 mg/L+activated carbon 0.6-1 g/L;
the rooting culture medium adopted in the step 7 is as follows: 1/2 MRM+sucrose 25-30 g/L+IBA0.3 mg/L+NAA 0-0.6 mg/L+activated carbon 0.6-1 g/L.
2. The method for tissue culture and rapid propagation of a tiled white pearl clone according to claim 1, wherein the disinfection method adopted in the step 4 is that 30-35 s is disinfected with 75% alcohol, then soaked and disinfected with 0.1% mercuric chloride for 11-12min, and finally rinsed with sterile water.
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| CN202310831305.6A CN116548314B (en) | 2023-07-07 | 2023-07-07 | Method for tissue culture and rapid propagation of flat white bead clone |
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| CN202310831305.6A CN116548314B (en) | 2023-07-07 | 2023-07-07 | Method for tissue culture and rapid propagation of flat white bead clone |
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| CN116548314A true CN116548314A (en) | 2023-08-08 |
| CN116548314B CN116548314B (en) | 2023-09-12 |
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| CN117178885A (en) * | 2023-06-12 | 2023-12-08 | 昆明医科大学 | Dian white bead tissue culture method |
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| EP1164831A2 (en) * | 1999-03-25 | 2002-01-02 | University of Guelph | Micropropagation and production of phytopharmaceutical plants |
| WO2005012507A1 (en) * | 2003-07-25 | 2005-02-10 | The University Of Melbourne | Production of plant secondary metabolites using adsorption and elicitation in cell suspension culture |
| CN106489748A (en) * | 2016-12-27 | 2017-03-15 | 云南省农业科学院花卉研究所 | A kind of quick breeding by group culture method of wintergreen |
| CN113573575A (en) * | 2019-03-12 | 2021-10-29 | 比贝婷作物科学有限公司 | Transcription factor mediated promotion of direct somatic embryogenesis |
| CN110476815A (en) * | 2019-09-27 | 2019-11-22 | 四川七彩林科股份有限公司 | A kind of tissue culture and rapid propagation method of beautiful fruit tree |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117178885A (en) * | 2023-06-12 | 2023-12-08 | 昆明医科大学 | Dian white bead tissue culture method |
| CN117178885B (en) * | 2023-06-12 | 2024-02-09 | 昆明医科大学 | Dian white bead tissue culture method |
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