CN111727884A - In-vitro bud culture method of hydrangea paniculata - Google Patents
In-vitro bud culture method of hydrangea paniculata Download PDFInfo
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- CN111727884A CN111727884A CN202010707491.9A CN202010707491A CN111727884A CN 111727884 A CN111727884 A CN 111727884A CN 202010707491 A CN202010707491 A CN 202010707491A CN 111727884 A CN111727884 A CN 111727884A
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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
- A01H4/001—Culture apparatus for tissue culture
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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
Abstract
The invention discloses a culture method of isolated buds of hydrangea paniculata, which comprises the following steps: collecting explants; the explant is respectively soaked in washing powder, washed under running water, soaked in a mixed solution of carbendazim and streptomycin and washed by sterile water, and then surface disinfection is carried out: soaking in 75% alcohol, washing with sterile water, and washing with HgCl2Soaking in solution, washing with sterile water; pruning incisions at two ends of the sterilized explant, cutting the explant into stem sections with buds, and vertically inoculating the stem sections on a starting culture medium; shearing the tissue culture seedlings subjected to start culture into stem sections with buds, reducing leaves which can contact with a culture medium, and vertically inoculating the stem sections into a multiplication culture medium; after propagation culture, pruning the base of the tissue culture seedling, and transferring the tissue culture seedling to a rooting culture medium for root induction. The method has good starting effect, low browning rate and pollution rate, the browning rate is 0, and the pollution rate is as low as 5%; the germination induction rate and the proliferation coefficient are high, the germination rate reaches 100%, and the proliferation coefficient reaches 7; the rooted seedlings are strong, the rooting rate reaches 100%, the average root amount reaches 22, and the average root length is 3.10 cm.
Description
Technical Field
The invention relates to a plant culture technology, in particular to a culture method of isolated buds of hydrangea paniculata.
Background
Hydrangea paniculata (Hydrangea paniculata Sieb. et Zucc) is a plant of the genus Hydrangea of the family Saxifragaceae, the deciduous shrub. Dark reddish brown or grayish brown. The leaf paper is made of 2-3 pieces of raw or ring-grown paper, and has oval or elliptical shape, fine sawtooth at leaf edge and hair at leaf back. The hydrangea paniculata is large and beautiful, has a unique flower shape, a length of 30-40cm, a flowering period of 7-10 months, a long flowering period and a beautiful color. Native to one of Japan and Sichuan China. Widely applied and researched since the introduction of the 18 th century into Europe, the flower is a high-grade flower, but is not popularized yet in China. Can be planted in a courtyard to be used as hedges and flower mirrors, can also be cut off with flower balls, is inserted into a room with bottles and has extremely high ornamental value. Moreover, related researches show that the hydrangea paniculata extract has certain medicinal value. The hydrangea paniculata seeds are tiny and have a complex dormancy mechanism, and due to the fact that the difficulty of seed propagation is high, the traditional propagation modes such as cuttage and layering are mostly adopted, but the propagation coefficient is not high. The tissue culture technology is a process of obtaining a large number of high-quality seedlings by using a small amount of explants and giving proper culture conditions on an artificially prepared culture medium.
The research on the aspect of hydrangea isolated culture mainly comprises the following steps: lu Yi et al (2015) report the culture conditions for in vitro germination of hydrangea paniculata seeds, the test material is the seeds of wild hydrangea paniculata, the seedlings propagated by the seeds are seedlings, and the seedlings obtained by in vitro bud culture are asexual seedlings, so the tissue culture methods are different. Plum blossom Hemerocallis et al (2017) report a damask tissue culture system, although damask is named as hydrangea, but the damask is a plant of the genus Strobilanthes of the family Caprifoliaceae, and the damask is a plant of a different family from hydrangea paniculata; jianmeng tobacco (2017) reports a research on a rapid propagation technology of an hydrangea variety 'lanikang', the shoot tip is taken as an explant, a suitable axillary bud induction culture medium is MS +6-BA1.0mg/L, the budding rate is 92.5%, and the average number of budding is 3.3. The suitable rooting culture medium is MS + IBA1.0mg/L, the average rooting time is 6.7 days, and the rooting length is 2.0 cm. The Nikon is a hydrangea macrophylla, which is different from hydrangea paniculata in taxonomy, and has larger difference in phenotypic characteristics, growth characteristics, stress resistance and the like of plants.
No relevant report is found at present on the research of the isolated bud culture technology of the hydrangea paniculata 'Pink eidolon'.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide the in vitro bud culture method of hydrangea paniculata with good sterilization effect, high axillary bud induction rate, high propagation coefficient and high rooting rate.
The technical scheme is as follows: the invention provides a culture method of isolated buds of hydrangea paniculata, which comprises the following steps:
(1) collecting explants: selecting young stem segments with buds as explants in 4-6 months, pruning, and reserving terminal buds and axillary buds;
(2) and (3) explant disinfection and sterilization: the explant is respectively soaked in washing powder, washed under running water, soaked in a mixed solution of carbendazim and streptomycin and washed by sterile water, and then surface disinfection is carried out: soaking in 75% alcohol, washing with sterile water, and washing with HgCl2Soaking in solution, washing with sterile water;
(3) starting culture: pruning incisions at two ends of the sterilized explant, cutting the explant into stem sections with buds, and vertically inoculating the stem sections on a starting culture medium;
(4) and (3) proliferation culture: shearing the tissue culture seedlings subjected to start culture into stem sections with buds, reducing leaves which can contact with a culture medium, and vertically inoculating the stem sections into a multiplication culture medium;
(5) rooting culture: after propagation culture, pruning the base of the tissue culture seedling, and transferring the tissue culture seedling to a rooting culture medium for root induction.
Further, the explant in the step (1) is taken from a pink genipin variety.
Further, the explant in the step (1) is a young and tender bore branch of the current year.
Further, the step (2) explant sterilization: soaking the explant in washing powder for 15min, washing with running water for 1.5-2h, soaking 2g/L carbendazim and 0.6g/L streptomycin for 8min, and washing with sterile water; and then carrying out surface disinfection: soaking in 75% ethanol for 1min, washing with sterile water for 1-2 times, and adding 0.1% HgCl2Soaking for 5min, and washing with sterile water for 3-5 times. The sterilization effect is best, the browning rate is 0%, and the pollution rate is 5%.
Further, the air conditioner is provided with a fan,
the formula of the start culture medium in the step (3) is as follows: MS +6-BA0.5mg/L + NAA0.1-0.2mg/L + agar 6.8g/L + sucrose 30g/L, and the pH value is 5.8. The germination rate can reach 100%.
Further, the formula of the proliferation medium in the step (4) is as follows: MS +6-BA1.0-1.5mg/L + NAA0.1-0.15mg/L + agar 6.8g/L + sucrose 30g/L, and pH value is 5.8. The maximum multiplication coefficient can reach 7, and the seedlings grow robustly.
Further, the air conditioner is provided with a fan,
the rooting culture medium in the step (5) is as follows: 1/2MS +6-BA0.05mg/L + IBA0.1mg/L + NAA0.1mg/L + agar 6.8g/L + sucrose 20g/L, pH 5.8.
Has the advantages that: the isolated bud culture technology of the hydrangea paniculata 'Pink eidolon' has the characteristics of low browning rate, low pollution rate and good sterilization effect: the browning rate can reach 0 percent, and the pollution rate can be as low as 5 percent. The starting effect is good: the germination induction rate reaches 100%, and the propagation rate at the proliferation stage is high: the multiplication coefficient reaches 7, and the rooted seedlings are strong: the rooting rate reaches 100 percent, the average root amount reaches 22 strips, and the average root length is 3.10 cm.
Drawings
FIG. 1 is a bar graph of the disinfection effect of the same amount of bactericide and mercuric chloride on explants at different treatment times;
FIG. 2 is a diagram showing a process of starting culture, in which a is inoculation, b is shoot induction, and c is a late-stage state of culture;
FIG. 3 is a diagram showing a proliferation culture process, in which a is a graph of a healthy and strong seedling and b is a graph of a high proliferation coefficient;
FIG. 4 is a diagram showing the process of rooting culture, in which a is an early stage and b is a late stage.
Detailed Description
1. Materials: one of the hydrangea paniculata varieties, Pink eidolon, selects terminal buds and axillary buds of young and tender stem segments of the current year.
2. The method comprises the following steps:
2.1 explant collection: the time is selected in 4-6 months, and the variety is 'pink spirit' under the condition of sunny weather and proper humidity. Selecting spring shoot segments which are strong in growth, free of diseases and insect pests and plump in axillary buds as materials. When the explants are collected, the young and tender inner branches of the current year are preferably adopted, so that unnecessary loss of the plants is avoided. And immediately putting the cut part into a container filled with water after collection to prevent dehydration and inactivation, and then timely treating and inoculating.
2.2 pretreatment of materials: trimming collected 'pink fairy' branch sections, removing leaves and reserving petioles, and shearing into small sections of about 2cm, wherein at least 1-2 axillary buds are reserved in each section. Preparing a clean beaker in advance, then putting the cut small sections into washing powder to be soaked for 15min, washing for 1.5-2h under running water, soaking 2g/L carbendazim and 0.6g/L streptomycin for about 8min, and then washing with sterile water.
2.3 surface sterilization of materials: after the material has been pretreated, the stem sections are placed in a clean bench. Treating with 75% alcohol for about 1min, washing with sterile water for 1-2 times, and adding 0.1% HgCl2Treating for about 5min, and washing with sterile water for 3-5 times. The aseptic bottle should be shaken continuously during the treatment, so that the sterilization is more thorough. The test adopts a two-factor completely random combined test design, and the test factors are respectively as follows: treating time (0, 5, 8, 12min) of mixed solution of 2g/L carbendazim and 0.6g/L streptomycin serving as bactericide, HgCl2The treatment time (3, 5, 10min), pH adjusted to 5.8, for a total of 12 treatments, 20 flasks of medium were treated each, 2 explants per flask, and repeated 4 times.
2.4 culture conditions: the culture temperature in the culture chamber is 25 + -2 deg.C, indoor relative humidity is 70-80%, illumination intensity is maintained at 2000-3000Lx, and culture time is controlled at 12h per day.
2.5 starting culture: wiping both hands with alcohol for sterilization, slightly trimming the incisions at the two ends of the stem section subjected to surface sterilization treatment, trimming the stem section into stem sections with buds of about 1.5cm, vertically inoculating the stem sections into a culture medium, wherein the inoculation depth is suitable for being inserted into the middle of the culture medium, and the stem sections are not suitable for being inserted too deeply. The basic culture medium selects MS culture medium, the hormone type and concentration adopt double-factor completely random combined experimental design, 6-BA (0, 0.5, 1.0 and 2.0mg/L) and NAA (0, 0.15, 0.2 and 0.5mg/L), pH value is adjusted to 5.8, the concentration is matched with 16 treatments, 15 bottles are treated, 2 explants are inoculated in each bottle, and the process is repeated for 4 times.
2.6 proliferation culture: cutting the tissue culture seedling which grows for 25 days after the starting culture into stem sections with buds of about 1.5cm, slightly trimming, reducing the parts of the lower parts of the stem sections which are possibly contacted with the culture medium, and vertically inoculating the stem sections into the enrichment culture medium. MS minimal medium is selected as culture medium, and two-factor complete random test is adopted, 6-BA (1.0, 1.5 and 2.0mg/L) and NAA (0.1, 0.15 and 0.5mg/L) with different concentrations are added, pH value is adjusted to 5.8, total 9 treatments are carried out, 20 bottles are treated, 2 explants are inoculated in each bottle, and the treatment is repeated for 3 times.
2.7 rooting culture: after the proliferation culture is carried out for 20 days, taking out the stronger tissue culture seedlings, and transferring the stronger tissue culture seedlings into a rooting culture medium for the root induction of the last step of the tissue culture. The culture medium is 1/2MS minimal medium, and is designed by adopting a three-factor test, 6-BA (0, 0.05 and 0.1mg/L), IBA (0, 0.1 and 0.2mg/L) and NAA (0.05, 0.1 and 0.2mg/L), the pH value is adjusted to 5.8, 5 treatments are carried out, 10 bottles are treated, 2 explants are inoculated in each bottle, and the treatment is repeated for 3 times.
2.8 data recording and analysis: recording the growth conditions of the four stages of sterilization, starting culture, multiplication culture and rooting culture, and processing the data.
2.8.1 the same dosage of bactericide and mercury bichloride affect the sterilization effect of explants in different treatment times: contamination and browning rates were recorded by observation after about 25d of inoculation (see table 1).
TABLE 1 influence of different treatment times of the same amount of bactericide and mercuric chloride on the explant sterilization effect
The data were analyzed using EXCEL, see fig. 1.
As can be seen from FIG. 1, when treatment No. 8 was used, the contamination rate and browning rate were the lowest, 5% and 0%, respectively, and the sterilizing time of the mixed solution of the bactericide was 8min and the sterilizing time of mercuric chloride was 5 min.
2.8.2 the effect of different concentrations of hormone on axillary bud induction: after about 25 days, the growth condition is recorded and the germination rate of the axillary buds is calculated (see table 2).
TABLE 2 Table of the effect of different concentrations of growth hormone on axillary bud induction
The data were analyzed using SPSS 25.0 and the results are shown in table 3.
TABLE 3 analysis of variance of effect of different concentrations of growth hormone dosage on axillary bud induction
The results were compared in multiples and are shown in Table 4. Multiple comparisons gave: the 6-BA concentration has obvious influence on axillary bud induction, and the NAA concentration has obvious influence on axillary bud induction. The results of variance analysis and multiple comparison of different hormone ratios show that when the difference of germination rates among treatments is compared, the effect is best when the concentration of 6-BA is 0.5mg/L, the effect is worst when the concentration of NAA is 0.5mg/L, and the difference of the concentration of NAA is not significant between 0.1mg/L and 0.2mg/L, so that the starting medium suitable for axillary bud induction is MS +6-BA0.5mg/L + NAA0.1-0.2mg/L, and the germination rate can reach 100% at most.
TABLE 46 multiple comparison of the effects of BA and NAA on axillary bud Induction
2.8.3 the effect of different concentrations of hormone on proliferation: after about 20d, growth was recorded and proliferation factors were calculated (see table 5).
TABLE 5 influence of growth hormone ratios of different concentrations on proliferation effect
The data were analyzed using SPSS 25.0 and the results are shown in table 6.
TABLE 6 analysis of variance of effect of growth hormone ratios of different concentrations on proliferation
Multiple comparisons of the above results (see table 7) gave: the 6-BA concentration has obvious effect on proliferation, and the NAA concentration has obvious effect on proliferation. In different hormone ratios, it was found that when comparing the inter-treatment variability of the proliferation factor: when the concentration of 6-BA is 1.0-1.5mg/L and the concentration of NAA is 0.1-0.15mg/L, the effect is best, and the multiplication coefficient is 7 at most. When the concentration of NAA is 0.5mg/L, the multiplication coefficient is low, and the callus expands, which indicates that the concentration is not favorable for the multiplication of buds.
TABLE 76 multiple comparison of BA and NAA Effect on proliferation
2.8.4 the effect of different concentrations of hormone on rooting: after 30d, the rooting rate is counted and calculated, and the number and length of roots are recorded (see table 8).
TABLE 8 influence of growth hormone ratios of different concentrations on the effect of root
From table 8, it can be seen: 6-BA has an influence on the health condition of leaves, and the leaves are extremely withered and yellow when the concentration is 0 mg/L. IBA has an effect on root length, which is shorter at lower concentrations. NAA has influence on rooting rate and rooting length, and when the concentration is low and the rooting is not obvious, the callus expands and the root system is thick and strong, but the germination is extremely poor.
In summary, the following steps: soaking in washing powder for 15min, washing with running water for 1.5-2 hr, soaking 2g/L carbendazim and 0.6g/L streptomycin for about 8min, washing with sterile water, sterilizing the stem segment in a clean bench, soaking in 75% alcohol for about 1min, washing with sterile water for 1-2 times, soaking in 0.1% mercuric chloride for about 5min, and washing with sterile water for 3-5 times, with the advantages of good sterilizing effect, browning rate of 0%, and pollution rate of 5%. The formula of the start culture medium is as follows: MS +6-BA0.5mg/L + NAA0.1-0.2mg/L + agar 6.8g/L + sucrose 30g/L, the germination rate reaches 100%. The optimal proliferation culture formula is as follows: 1.0-1.5mg/L of MS +6-BA, 0.1-0.15mg/L of NAA, 6.8g/L of agar and 30g/L of cane sugar, and the maximum multiplication coefficient can reach 7. The most suitable rooting culture medium is as follows: 1/2MS +6-BA0.05mg/L + IBA0.1mg/L + NAA0.1mg/L + agar 6.8g/L + sucrose 20g/L, the rooting rate reaches 100%, the average root amount reaches 22, and the average root length is 3.10 cm.
Claims (7)
1. A culture method of isolated buds of hydrangea paniculata is characterized in that: the method comprises the following steps:
(1) collecting explants: selecting young stem segments with buds as explants in 4-6 months, pruning, and reserving terminal buds and axillary buds;
(2) and (3) explant disinfection and sterilization: the explant is respectively soaked in washing powder, washed under running water, soaked in a mixed solution of carbendazim and streptomycin and washed by sterile water, and then surface disinfection is carried out: soaking in 75% alcohol, washing with sterile water, and washing with HgCl2Soaking in solution, washing with sterile water;
(3) starting culture: pruning incisions at two ends of the sterilized explant, cutting the explant into stem sections with buds, and vertically inoculating the stem sections on a starting culture medium;
(4) and (3) proliferation culture: shearing the tissue culture seedlings subjected to start culture into stem sections with buds, reducing leaves which can contact with a culture medium, and vertically inoculating the stem sections into a multiplication culture medium;
(5) rooting culture: after propagation culture, pruning the base of the tissue culture seedling, and transferring the tissue culture seedling to a rooting culture medium for root induction.
2. The method for culturing the isolated bud of hydrangea paniculata according to claim 1, wherein: the explant in the step (1) is taken from a pink genipin variety.
3. The method for culturing the isolated bud of hydrangea paniculata according to claim 1, wherein: the explant in the step (1) is a young and tender inner bore branch of the current year.
4. The method for culturing the isolated bud of hydrangea paniculata according to claim 1, wherein: the step (2) of explant sterilization: soaking the explant in washing powder for 15min, washing with running water for 1.5-2h, soaking 2g/L carbendazim and 0.6g/L streptomycin for 8min, and washing with sterile water; and then performing surface disinfection on the superclean workbench: soaking in 75% ethanol for 1min, washing with sterile water for 1-2 times, and adding 0.1% HgCl2Soaking for 5min, and washing with sterile water for 3-5 times.
5. The method for culturing the isolated bud of hydrangea paniculata according to claim 1, wherein: the formula of the start culture medium in the step (3) is as follows: MS +6-BA0.5mg/L + NAA0.1-0.2mg/L + agar 6.8g/L + sucrose 30g/L, and the pH value is 5.8.
6. The method for culturing the isolated bud of hydrangea paniculata according to claim 1, wherein: the formula of the proliferation culture medium in the step (4) is as follows: MS +6-BA1.0-1.5mg/L + NAA0.1-0.15mg/L + agar 6.8g/L + sucrose 30g/L, and pH value is 5.8.
7. The method for culturing the isolated bud of hydrangea paniculata according to claim 1, wherein: the rooting culture medium in the step (5) is as follows: 1/2MS +6-BA0.05mg/L + IBA0.1mg/L + NAA0.1mg/L + agar 6.8g/L + sucrose 20g/L, pH 5.8.
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