CN111771719B - Tissue culture rapid propagation method of spiraea thunbergii - Google Patents
Tissue culture rapid propagation method of spiraea thunbergii Download PDFInfo
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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
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention belongs to the field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation method of spiraea ulmaria. The method comprises the following steps: primary culture: taking a tender branch of pearl embroidery chrysanthemum, shearing the tender branch into small branches, performing disinfection and sterilization, and then obliquely inserting the small branches into a primary culture medium for inoculation to obtain sterile seedlings; and (3) proliferation culture: cutting the aseptic seedlings into stem sections with buds, placing the stem sections into a proliferation culture medium, and performing proliferation and differentiation on callus to obtain cluster buds; rooting culture: cutting the cluster buds into bud sections, and inoculating the bud sections to a rooting culture medium to obtain rooted seedlings. The invention has high germination rate of the first generation, short germination time, highest proliferation coefficient of 19.75, higher propagation ratio than that of a cuttage method, high rooting rate of 100%, developed root system, good growth vigor and higher economic value.
Description
Technical Field
The invention belongs to the field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation method of spiraea ulmaria.
Background
Spiraea thunbergii Bl, rosaceae, spiraea thunbergii shrub, as high as 1.5 m. The plants have the advantages of high ornamental value, strong stress resistance, drought and cold resistance, no plant diseases and insect pests, light preference, no shade, preference for moist soil with good drainage and convenient cultivation and management. The pearl spiraea is a good choice as a flower mirror and a flower belt, and the excellent characteristics of the pearl spiraea can also be used as a fresh cut flower to create commercial value. However, at present, the pearl spiraea has not been fully applied to landscape architecture in south, and the key point is that the pearl spiraea is not sufficiently propagated, so that the ornamental value of the pearl spiraea is not reflected. In the prior art, the cultivation of the spiraea salicifolia is basically realized through sowing and cutting, and the sowing has the advantages of large one-time propagation quantity, complete root system of seedlings and strong growth, and has the defects of long development period, low propagation coefficient, great influence by seasons and late flowering; therefore, the cuttage technology starts to rise, for example, the propagation method of the pearl spiraea aegilops disclosed in the Chinese patent CN110999645A carries out rooting culture by sterilizing the tender branches of the pearl spiraea aegilops and cutting the scions, the rooting is fast, the cost is low, but the requirements on the cutting scions are higher, and the common old branches are not suitable for cuttage; the requirements on soil and fertilizers are high; the new and long root system is weaker, so the survival rate is low. In conclusion, research technology suitable for tissue culture and rapid propagation of the spiraea salicifolia is developed, wide application of the spiraea salicifolia is promoted, and the research and development directions are at present.
Disclosure of Invention
The invention provides a tissue culture rapid propagation method of spiraea ulmaria, aiming at solving the defects of low survival rate and low propagation coefficient in the propagation and cultivation process of spiraea ulmaria in the prior art and filling the vacancy of the tissue culture spiraea ulmaria.
In order to realize the purpose, the invention adopts the following technical scheme:
a tissue culture and rapid propagation method of spiraea thunbergii comprises the following steps:
primary culture: taking a rejuvenation branch of pearl embroidery thread, shearing into small branches, performing disinfection and sterilization, and then obliquely inserting into a primary culture medium for inoculation to obtain sterile seedlings; the primary culture medium comprises the following components: 1/2MS (3/4 Fe) +25g/L sucrose +6g/L agar;
and (3) proliferation culture: cutting the aseptic seedlings into stem sections with buds, placing the stem sections into a proliferation culture medium, and performing proliferation and differentiation on callus to obtain cluster buds; the proliferation culture medium comprises the following components: 1/2MS (3/4 Fe) + (1-2) mg/L6-BA + (0.2-0.6) mg/L GA 3 +25g/L sucrose +6g/L agar;
rooting culture: cutting cluster buds into bud sections, inoculating the bud sections to a rooting culture medium to obtain rooted seedlings, and then transplanting the rooted seedlings, wherein the rooting culture medium comprises the following components: 1/2MS (3/4 Fe) + (0.1-0.3) mg/L IBA +0.1mg/L NAA +30g/L sucrose +7g/L agar.
Furthermore, the length of the small branches is 2-3 cm, and each branch is provided with 2-4 buds.
Further, the specific steps of disinfection and sterilization are as follows: placing the small branches on a clean bench, sterilizing with 75% alcohol for 40s, pouring out alcohol, rapidly washing with sterile water for 1 time, pouring in 2% sodium hypochlorite for sterilization for 6min, pouring out sodium hypochlorite, washing with sterile water for 3 times, and filtering off excessive water with filter paper.
Furthermore, the length of the stem section with the buds is 1.5-2.5 cm, and each stem section with the buds is provided with 2-3 buds.
Further, the culture temperature is 22-26 ℃, the pH value in the culture medium is 5.8-6.0, the humidity of the rooted seedlings is 95% within 7 days after transplanting, and the humidity is 80% after 7 days.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts a tissue culture method to breed the pearl spiraea, the rejuvenation of the pearl spiraea is subjected to primary culture to obtain aseptic seedlings, and the stem with buds is subjected to proliferation culture to differentiate into clumpy buds and the shoot is subjected to rooting culture to obtain the seedlings. In the invention, 1/2MS is used as a basic culture medium for each culture medium, and only 3/4 ferric salt concentration is adopted to match with the characteristic of high saline-alkali resistance of the spiraea pearl root system, so that a better culture effect is achieved, wherein the germination rate of the first generation is high, the germination time is short, the maximum multiplication coefficient is 19.75, the propagation ratio of the spiraea pearl root system is far higher than that of a cuttage method, the rooting rate can reach 100%, the root system is developed, the survival rate is high, the growth vigor is good, and the economic value is higher.
Drawings
FIG. 1 shows the growth of group 3 in example 3;
FIG. 2 shows the growth of group 2 in example 3;
FIG. 3 shows the growth of group C2 in example 4;
FIG. 4 shows the growth of group C4 in example 4;
FIG. 5 shows the growth of group 2 in example 5;
FIG. 6 shows the growth of group 5 in example 5.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1 tissue culture and Rapid propagation method of Spiranthes margarita
Explant treatment: collecting the current-year tender branches of the spiraea arenaria as test materials in the first ten days of 6 months, cutting the tender branches into small branches with 2-4 buds and about 2-3 CM by using branch scissors, placing the small branches on an ultra-clean workbench, sterilizing the small branches for 40s by using 75% alcohol, pouring out the alcohol, and rapidly washing the small branches for 1 time by using sterile water; then 2% sodium hypochlorite is poured in for sterilization for 6min, the sodium hypochlorite is poured out, the solution is washed for 3 times by sterile water, and the solution is put on filter paper to filter off excessive water.
Primary culture: obliquely inserting the processed explant into a primary culture medium for inoculation, culturing for 20 days, and then keeping the germination rate of the explant, and obtaining sterile seedlings after 45 days; the germination rate = number of sprouts/total number of inoculations × 100%; the primary culture medium comprises the following components: 1/2MS (3/4 Fe), 25g/L of cane sugar and 6g/L of agar, the pH value is 5.8-6.0, and the culture temperature is 24 ℃.
And (3) proliferation culture: cutting the sterile seedlings into 1.5-2.5 cm stem segments with buds, placing each stem segment with buds into a proliferation culture medium, performing proliferation differentiation on callus, performing proliferation culture for 30 days, and counting proliferation coefficients to obtain cluster buds; the proliferation coefficient = the number of total transferred plants/total inoculated plants; the proliferation culture medium comprises the following components: 1/2MS (3/4 Fe) +1.5 mg/L6-BA +0.6mg/L GA 3 +25g/L sucrose +6g/L agar; the pH value is 5.8-6.0, and the culture temperature is 24 ℃.
Rooting culture: cutting cluster buds into bud sections, inoculating the bud sections to a rooting culture medium to obtain rooted seedlings, counting the rooting conditions after rooting culture for 30d, and then transplanting the rooted seedlings, wherein the humidity of the rooted seedlings is 95% within 7 days after transplanting, and the humidity of the rooted seedlings is 80% after 7 days; the rooting rate is equal to the total number of rooting plants/total number of inoculated plants multiplied by 100 percent; the rooting medium comprises the following components: 1/2MS (3/4 Fe) +0.1mg/L IBA +0.1mg/L NAA +30g/L sucrose +7g/L agar, pH is 5.8-6.0, and the culture temperature is 26 ℃.
Example 2 tissue culture and rapid propagation method of Spiranthes margarita
Explant treatment: collecting current-year twigs of spiraea salicifolia as test materials in the first 6-month ten days, cutting the twigs into twigs with 2-4 buds and about 2-3 CM by using branch shears, placing the twigs on an ultra-clean workbench, sterilizing the twigs for 40s by using 75% alcohol, pouring out the alcohol, and quickly washing the twigs for 1 time by using sterile water; and then 2% sodium hypochlorite is poured in for sterilization for 6min, the sodium hypochlorite is poured out, the sodium hypochlorite is washed for 3 times by sterile water, and the filter paper is placed to filter off excessive water.
Primary culture: obliquely inserting the processed explant into a primary culture medium for inoculation, culturing for 20 days, and then keeping the germination rate of the explant, and obtaining sterile seedlings after 45 days; the germination rate = number of sprouts/total number of inoculations × 100%; the primary culture medium comprises the following components: 1/2MS (3/4 Fe), 25g/L of cane sugar and 6g/L of agar, the pH value is 5.8-6.0, and the culture temperature is 22 ℃.
And (3) proliferation culture: cutting the sterile seedlings into 1.5-2.5 cm stem segments with buds, placing each stem segment with buds into a proliferation culture medium, performing proliferation differentiation on callus, performing proliferation culture for 30 days, and counting proliferation coefficients to obtain cluster buds; what is needed isThe proliferation coefficient = the total number of transferred plants/total number of inoculated plants; the proliferation culture medium comprises the following components: 1/2MS (3/4 Fe) +2 mg/L6-BA +0.4mg/L GA 3 +25g/L sucrose +6g/L agar; the pH value is 5.8-6.0, and the culture temperature is 22 ℃.
Rooting culture: cutting cluster buds into bud sections, inoculating the bud sections to a rooting culture medium to obtain rooted seedlings, counting the rooting conditions after rooting culture for 30d, and then transplanting the rooted seedlings, wherein the humidity of the rooted seedlings is 95% within 7 days after transplanting, and the humidity of the rooted seedlings is 80% after 7 days; the rooting rate = the total number of rooted plants/total number of inoculated plants multiplied by 100%; the rooting culture medium comprises the following components: 1/2MS (3/4 Fe) +0.3mg/L IBA +0.1mg/L NAA +30g/L sucrose +7g/L agar, pH is 5.8-6.0, and the culture temperature is 24 ℃.
Example 3 Effect of Primary Medium composition on the Germination of spiraea Spinosa Stem segments
The explant treatment of the embodiment is the same as that of embodiment 1, the treated explants are randomly divided into three groups, each group is treated with 5 culture bottles, each culture bottle contains 1 explant, each explant has about 2 bud points, the three groups of explants are obliquely inserted into three primary culture mediums for inoculation, the three culture mediums respectively use 1/2MS, 1/2MS (1/2 Fe) and 1/2MS (3/4 Fe) solid culture mediums as basic culture mediums, then 25g/L sucrose +6g/L agar are respectively added, the pH of the culture mediums is 5.8-6.0, the temperature is 24 ℃, the germination phenomenon and the germination rate after 20d are recorded, and the results are shown in Table 1, wherein the germination condition is the condition that the germination phenomenon occurs in the earliest in each group of experiments in 5 culture bottles on the day. The growth of group 3 after 20d is shown in fig. 1, and the growth of group 2 is shown in fig. 2.
TABLE 1 Effect of Primary Medium composition on the Germination of spiraea pearl
| Grouping | Treatment of | Condition of bud germination | Time of germination | Germination rate |
| 1 | 1/2MS | 1 has a sprouting phenomenon | 12d | 30.00％ |
| 2 | 1/2MS(1/2Fe) | 2 have the sprouting phenomenon and the leaf-unfolding tendency | 8d | 70.00％ |
| 3 | 1/2MS(3/4Fe) | 6 have sprouting phenomenon and spread leaves | 6d | 90.00％ |
As can be seen from Table 1, when the 1/2MS culture medium containing 3/4Fe and 25g/L sucrose +6g/L agar are adopted as the nutrient components of the pearl spiraea, the germination time is fastest, the germination trend is neat, at the same time, 6 bud points have the germination phenomenon and begin to unfold leaves, and the germination vigor, the germination time and the 15d germination rate are all superior to those of the other two groups, so that the 1/2MS culture medium containing 3/4Fe is adopted as the basal medium and is more suitable for being used as the primary culture medium of the pearl spiraea than the culture medium containing higher or lower iron salt content.
EXAMPLE 4 Effect of proliferation Medium composition on the differentiation of Spiraria terniflora seedlings
The explant treatment and primary culture steps of this example are the same as example 1, cutting the sterile seedlings obtained by primary culture into 1.5-2.5 cm budded stem segments, each of which has 2-3 buds, randomly dividing the budded stem segments into three groups (a, B, C), each of which has 4 small groups for proliferation culture, each group having 3 bottles, each bottle having 5 budded stem segments, 12 groups of culture medium respectively adopts 1/2MS (3/4 Fe) as basic culture medium, and each culture bottle is added with 25g/L sucrose +6g/L agar, wherein 6-BA and NAA with different concentrations are added to group a, 6-BA and IBA with different concentrations are added to group B, 6-BA and GA3 with different concentrations are added to group C, the pH of the culture medium is 5.8-6.0, and the temperature is 24 ℃. The growth coefficient and plant height of the plants were recorded for 30d, and the results are shown in Table 2, the growth of group C2 is shown in FIG. 4, and the growth of group C4 is shown in FIG. 4.
TABLE 2 Effect of enrichment Medium composition on the differentiation of Spiraria terniflora seedlings
| Numbering | Treatment of | Average number of clumpy buds | Plant height (cm) |
| A1 | 1/2MS(3/4Fe)+1.0mg/L6-BA+0.2mg/LNAA | 3.75 | 2.4 |
| A2 | 1/2MS(3/4Fe)+1.5mg/L6-BA+0.5mg/LNAA | 7.75 | 3.0 |
| A3 | 1/2MS(3/4Fe)+1.0mg/L6-BA+0.5mg/LNAA | 3.5 | 1.5 |
| A4 | 1/2MS(3/4Fe)+1.5mg/L6-BA+0.2mg/LNAA | 11.5 | 2.6 |
| B1 | 1/2MS(3/4Fe)+1.0mg/L6-BA+0.2mg/LIBA | 9.75 | 1.3 |
| B2 | 1/2MS(3/4Fe)+1.5mg/L6-BA+0.5mg/LIBA | 13 | 4.6 |
| B3 | 1/2MS(3/4Fe)+1.0mg/L6-BA+0.5mg/LIBA | 11.75 | 3.8 |
| B4 | 1/2MS(3/4Fe)+1.5mg/L6-BA+0.2mg/LIBA | 9.5 | 3.0 |
| C1 | 1/2MS(3/4Fe)+1.0mg/L6-BA+0.3mg/LGA 3 | 13.75 | 2.5 |
| C2 | 1/2MS(3/4Fe)+1.5mg/L6-BA+0.4mg/LGA 3 | 19.75 | 5.3 |
| C3 | 1/2MS(3/4Fe)+1.0mg/L6-BA+0.4mg/LGA 3 | 15 | 2.0 |
| C4 | 1/2MS(3/4Fe)+1.5mg/L6-BA+0.3mg/LGA 3 | 16.25 | 3.0 |
As can be seen from Table 2, the use of 6-BA in combination with GA 3 The proliferation coefficient of the spiraea pearl bud stem section cultured by the proliferation culture medium is obviously higher than that of a combined culture medium of 6-BA and NAA and 6-BA and IBA. Further, when the concentration of 6-BA in the medium was 1.5mg/L, GA 3 The average differentiated cluster bud number is the highest and reaches 19.75 when the concentration of (A) is 0.4 mg/L. Therefore, the invention adopts 1/2MS (3/4 Fe) +1.5 mg/L6-BA +0.4mg/L GA 3
Example 5 Effect of rooting Medium composition on rooting of Spiranthes margarita
The explant treatment, primary culture and multiplication culture processes of the embodiment are the same as those of embodiment 1, the clustered buds obtained after multiplication culture are cut into bud segments, the bud segments are randomly divided into 5 groups, the groups are inoculated to different rooting culture media for rooting culture, each group comprises 5 bottles, 1 clustered bud in each bottle, the 5 groups of rooting culture media all take 1/2MS (3/4 Fe) as a basic culture medium, 30g/L sucrose +7g/L agar is added, then IBA and NAA with different concentrations are added, the pH value of the culture medium is 5.8-6.0, the rooting time, rooting rate and growth conditions are respectively recorded, the results are shown in Table 3, the growth condition of the group 2 after 30d is shown in figure 5, and the growth condition of the group 5 is shown in figure 6.
TABLE 3 Effect of rooting Medium composition on rooting of Spiranthes marmalayana
As can be seen from Table 3, when the components in the rooting medium are 1/2MS (3/4 Fe) +0.2mg/L IBA +0.1mg/L NAA +30g/L sucrose +7g/L agar, the rooting rate is 100%, the root system is developed, the plant height is high, the growth vigor is good, the leaf color is dark green, and the rooting effect is better than that of other combined culture media.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A tissue culture and rapid propagation method of spiraea thunbergii is characterized by comprising the following steps:
primary culture: taking a rejuvenation branch of pearl embroidery thread, shearing into small branches, performing disinfection and sterilization, and then obliquely inserting into a primary culture medium for inoculation to obtain sterile seedlings; the primary culture medium comprises the following components: 1/2MS +25g/L sucrose +6g/L agar;
and (3) proliferation culture: cutting the aseptic seedlings into stem sections with buds, placing the stem sections into a proliferation culture medium, and performing proliferation and differentiation on callus to obtain cluster buds; the proliferation culture medium comprises the following components: 1/2MS + (1-2) mg/L6-BA + (0.2-0.6) mg/L GA 3 +25g/L sucrose +6g/L agar;
rooting culture: cutting cluster buds into bud sections, inoculating the bud sections to a rooting culture medium to obtain rooted seedlings, and then transplanting the rooted seedlings, wherein the rooting culture medium comprises the following components: 1/2MS + (0.1-0.3) mg/L IBA +0.1mg/L NAA +30g/L sucrose +7g/L agar;
1/2MS is adopted as a basic culture medium for each culture medium, and the concentration of the ferric salt is 3/4 of that of the basic culture medium.
2. The tissue culture rapid propagation method of spiraea pearl according to claim 1, characterized in that the length of the small branches is 2-3 cm, and each branch has 2-4 buds.
3. The tissue culture rapid propagation method of spiraea pearl according to claim 1, characterized in that the sterilization comprises the following steps: placing the small branches on a super-clean workbench, sterilizing with 75% alcohol for 40s, pouring out alcohol, rapidly washing with sterile water for 1 time, pouring 2% sodium hypochlorite for sterilization for 6min, pouring out sodium hypochlorite, washing with sterile water for 3 times, and filtering off excessive water with filter paper.
4. The tissue culture and rapid propagation method of spiraea pearl according to claim 1, wherein the length of the stem section with buds is 1.5-2.5 cm, and each stem section with buds has 2-3 buds.
5. The tissue culture and rapid propagation method of pearl spiraea according to claim 1, characterized in that the temperature of the tissue culture is 22-26 ℃, the pH value in the culture medium is 5.8-6.0, the humidity of the rooted seedling is 95% within 7 days of transplantation, and the humidity is 80% after 7 days.
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