CN115299344A - European moon tissue culture propagation method - Google Patents
European moon tissue culture propagation method Download PDFInfo
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 13
- 230000006698 induction Effects 0.000 claims abstract description 12
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- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 8
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- 238000005520 cutting process Methods 0.000 claims description 8
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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- 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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Abstract
The invention relates to a tissue culture propagation method for August, which comprises the following steps: selecting current-year-old twigs which are robust in growth, free of diseases and insect pests and full in buds as explants, washing stem segments cleanly by dipping neutral detergent with a hairbrush, and then flushing the stem segments with tap water for 2 to 3 hours; treating the stem segments with 75% alcohol on a clean bench for 30s, rinsing with sterile water 3-5 times, followed by 0.1% HgCl 2 Or 6% sodium hypochlorite is used for disinfecting the stem sections; inoculating the stem segment to a primary culture axillary bud induction culture medium, and culturing for 20d; then sequentially carrying out subculture and rooting culture. The invention adopts a method of twice disinfection in the explant disinfection stage, reduces the pollution rate and improves the survival rate, and the reasonable formula and culture conditions of the primary generation culture medium, the secondary generation culture medium and the rooting culture medium can meet the requirements of industrialized propagation.
Description
Technical Field
The invention relates to a plant propagation technology, in particular to a tissue culture propagation method for Ouyue.
Background
The Chinese rose is a flower plant integrating the functions of appreciation and medicine. The breeding method can be divided into: cutting propagation, grafting propagation, seeding propagation, plant division propagation, tissue culture and the like. The cutting propagation and grafting propagation are commonly applied in production, but have the problems of labor and time waste, high production cost, long seedling period and the like. The tissue culture and rapid propagation of the Chinese rose can not only keep the excellent properties of the variety, but also realize the industrialized production of the seedlings and quickly meet the requirement on high-quality seedlings in production.
The tissue culture and rapid propagation of the Chinese rose mainly comprises the following steps: selecting proper explants, sterilizing the explants, carrying out primary culture, carrying out secondary culture, carrying out rooting culture and hardening seedling and transplanting.
Researchers such as zhushan and Yuanwanjun have studied the disinfection time in the Chinese rose tissue culture, and show that too short disinfection time is unfavorable for solving the problem of bringing bacteria of explants, and can cause incomplete disinfection, thereby leading to lower stem survival rate under the primary culture and higher pollution rate. However, too long a disinfection time may cause damage to the explant, and also may cause a problem of low survival rate.
Disclosure of Invention
The invention aims to provide a tissue culture propagation method for August with high survival rate, high multiplication coefficient and developed plant root system.
The invention adopts the following technical scheme:
a method for culturing and propagating August tissues comprises the following steps:
(1) Selecting current-year shoots which are strong in growth, free of diseases and insect pests and full in buds as explants, cutting the shoots into stem sections with 2 bud eyes and 1-2 cm in length, cutting off leaves and thorns, reserving 1-3 leaf stalks, dipping neutral detergent with a hairbrush to wash the stem sections cleanly, and then washing the stem sections with tap water for 2-3 hours;
(2) Treating the stem segments with 75% alcohol on a clean bench for 30s, rinsing with sterile water 3-5 times, followed by 0.1% HgCl 2 Or 6% sodium hypochlorite is used for disinfecting the stem sections;
(3) The stem segments are inoculated in an initial culture axillary bud induction culture medium, and the inoculated stem segments are placed in a culture room with the temperature of 23-27 ℃, the illumination intensity is 2000-2500 lx, the illumination time is 12-16 h/d, and the culture is carried out for 20d.
(4) Inoculating the stem section obtained by the treatment in the step (3) into a subculture medium, placing the inoculated stem section into a culture room at the temperature of 23-27 ℃, and culturing for 40d, wherein the illumination intensity is 2000-2500 lx, and the illumination time is 12-16 h/d;
(5) And (3) inoculating the stem section obtained by the treatment in the step (4) to a rooting culture medium, placing the stem section in a culture room at the temperature of 23-27 ℃ after inoculation, and culturing for 40 days, wherein the illumination intensity is 2000-2500 lx, the illumination time is 12-16 h/d.
Further, using 0.1% of HgCl 2 Soaking and sterilizing the stem segments for 5-15 min.
Further, the stem segments are soaked and disinfected for 15-25 min by using 6% sodium hypochlorite.
Further, the primary culture axillary bud induction medium is an MS medium containing 6g/L agar and 100ul/L sodium hydroxide, and has a pH of 5.8.
Further, the subculture medium is an MS medium containing 1-3 mg/L6-BA and 0.1-0.3 mg/L NAA, and the pH is 5.8.
Furthermore, the rooting culture medium is a 1/2MS culture medium containing 0.05-1 mg/L NAA, and the pH value is 5.8.
The invention has the beneficial effects that: the invention adopts a method of twice disinfection in the explant disinfection stage, thereby reducing the pollution rate and improving the survival rate. Reasonable formulas and culture conditions of primary generation culture medium, secondary generation culture medium and rooting culture medium can meet the requirement of industrialized propagation.
Drawings
FIG. 1 shows the sprouting of 3 European-moon varieties in stem segments treated with different mercuric chloride;
wherein, a, b, c: treating the 'powdered and peaceful' mercuric chloride for 5, 10 and 15min;
d. e, f: treating with mercuric chloride for 5, 10, 15min;
g. h, i: treating with mercuric chloride of Samantha for 5, 10, and 15min.
FIG. 2 shows the sprouting of 3 European-month variety stem segments treated with different sodium hypochlorite;
wherein, a, b, c: treating with sodium hypochlorite for 15, 20 and 25min;
d. e, f: treating with sodium hypochlorite for 15min, 20 min and 25min;
g. h, i: sodium hypochlorite treatment for 15min, 20 min and 25min.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.
1. Experimental Material
The experimental materials were obtained from Pink peach (Pink peach) planted from south-Henan by Hebei Gaozu science and technology Limited, juice balcony (Juiscy Terraza), samantha (Samantha) planted from Jiangsu bathing Yang by the institute of forestry and grassland science in Hebei province, spectra (Spectra), and Moringa princess (princess de Monaco).
2. Explant harvesting and processing
Selecting current-year-old tender branches which are robust in growth, free of diseases and insect pests and full in bud bodies as explants, cutting the explants into stem sections with 2 bud eyes of 1-2 cm in length, cutting off leaves and thorns, reserving a few leaf stalks, dipping neutral washing powder with a small brush to wash the stem sections cleanly, and then washing the stem sections with tap water for 2-3 hours.
3. Disinfection of explants
Treating with 75% alcohol on a clean bench for 30s, rinsing with sterile water 3-5 times, then 0.1% 2 And 6% sodium hypochlorite for disinfection of different treatments. With 6 treatments, as shown in table 1, a completely random design, 10 flasks were inoculated per treatment, with 3 replicates.
TABLE 1 design of the experiments
4. Axillary bud induction culture
Primary culture axillary bud induction culture medium: selecting sucrose-containing MS culture medium, adding agar 6g/L, sucrose 30g/L, sodium hydroxide 100ul/L, and pH 5.8.
The culture conditions are as follows: after inoculation, the stem is placed in a culture room at 23-27 ℃, the illumination intensity is 2000-2500 lx, the illumination time is 12-16 h/d, and after 20d, the stem pollution rate, the germination rate, the survival rate, the death condition and the like are counted.
Germination rate/% = number of explants with lateral shoots sprouting plantlets/total number of inoculated explants 100%
Contamination rate/% = number of contaminated explants/total number of inoculated explants 100%
Survival/% = survival/inoculation × 100%
4.1 Effect of Mercury chloride Disinfection on Europe Stem Induction
From the table 2, it can be seen that the powder and peace, the fruit juice balcony, the salanga, the spectrum and 5 varieties of the princess of morna can induce the axillary buds of the stem segments to germinate under different treatments of mercuric chloride, and the axillary buds grow vigorously. The pollution rate of the juice balcony is the lowest, and the pollution rate of the peaceful powder is the highest.
The pollution rate among various treatment rooms of different varieties shows a continuously descending trend along with the increase of the disinfection time, which shows that the too short disinfection time is not beneficial to solving the problem of carrying bacteria of explants, and incomplete disinfection can be caused, thereby causing the stem survival rate under primary culture to be lower and the pollution rate to be higher. Under the condition of T3 treatment, the pollution rate of 5 varieties is the lowest, the induction rate is the highest, the pollution rate of a fruit juice balcony is the lowest and is 6.67%, and the survival rate is the highest; the pollution rate of powder feeding and peaceful pollution under the T1 treatment reaches the maximum value of 66.67 percent, and the survival rate is only 36.67 percent.
Therefore, the optimum mercuric chloride disinfection time for inducing the differentiation of Pink, juice balcony, savinsa, spectrum and 5 varieties of European stems of Moringa princess was 15min.
TABLE 2 Effect of Mercury chloride Disinfection treatment time on induced differentiation of Euler Stem segments
4.2 Effect of sodium hypochlorite Disinfection treatment on Imperial Stem Induction
As can be seen from Table 3, the germination rate and induction rate of the stem segments of Pingpi, fruit juice balcony, savinsa, spectrum and 5 species of Moringa princess were low and the contamination rate was high under different treatments with sodium hypochlorite. The change trend of the pollution rate of 5 varieties under the sodium hypochlorite treatment is similar to that of the mercury chloride treatment, and the pollution rate of 5 varieties is reduced along with the increase of the sodium hypochlorite treatment time.
The survival rate of the juice balcony processed by the T6 is the highest and is 83.33 percent, and the survival rate of the Moringa princess is the lowest and is 56.67 percent; the pollution rate of the Moringa princess under the T4 treatment is as high as 83.33 percent, and the pollution rate of a fruit juice balcony is the lowest and is 60.00 percent. The Samantha germination rate under T4 and T5 treatment is the best, and is as high as 90%.
Therefore, sodium hypochlorite disinfection time for inducing differentiation of Pink, juice balcony, savinsa, spectrum and 5 species of Marangge princess was 25min.
TABLE 3 Effect of sodium hypochlorite Disinfection treatment time on induced differentiation of Eumeria stem segments
4.3 axillary bud induction culture test summary of the early Ocimum
As can be seen from tables 2 and 3, 6 different sterilization modes are adopted for the European-month explants of 6 varieties for treatment in the experiment, and compared with 6 varieties, the survival rate of the fruit juice balcony under different treatments is the highest, the pollution rate is low, and probably because the fruit juice balcony has stronger disease resistance than the rest two varieties, bacteria are not easy to breed, diseases are not easy to infect, and the fruit juice balcony tissue culture device is suitable for tissue culture.
5. Subculture
Subculture medium: containing 1 to 3 mg.L -1 6-BA and 0.1-0.3 mg.L -1 6g/L agar, 30g/L sucrose and 100ul/L sodium hydroxide are added into the MS culture medium of NAA, and the pH value is 5.8.
The culture conditions are as follows: transferring the axillary buds after the European month primary culture for 20 days into a subculture medium for culture, subculturing every 40 days, inoculating 2 plantlets in each bottle, treating 5 bottles in each bottle, and repeating for 3 times. After inoculation, the seedlings are placed in a culture room at the temperature of 23-27 ℃, the illumination intensity is 2000-2500 lx, the illumination time is 12-16 h/d, the proliferation condition is counted after the first subculture is carried out for 40d, and the number/number of the proliferated axillary buds, the height/cm of the clustered seedlings and the proliferation coefficient are observed.
Multiplication factor = number of multiplied buds/number of inoculated axillary buds
TABLE 4 subculture test design
5.1 Effect of different concentrations of 6-BA and NAA combinations on August subculture
As can be seen from tables 5-9, the ratios of 6-BA, NAA and IBA with different concentrations have different effects on the number of proliferating axillary buds, the height of clumped seedlings and the proliferation coefficient of Pingyan, fruit juice balcony, savinsa, spectrum and 5 European and monthly stem segments of the princess of Moringa. When the NAA concentration is fixed, the number of the proliferated axillary buds and the proliferation coefficient are determined according to theThe increase in 6-BA concentration was shown by a tendency of increasing first and then decreasing, all at a 6-BA concentration of 2 mg. L -1 The highest value is reached. When the concentration of 6-BA is consistent, the number of the proliferation axillary buds and the proliferation coefficient show a trend of increasing firstly and then decreasing with the increase of the concentration of NAA.
When the 6-BA is 2 mg.L -1 NAA of 0.2 mg. L -1 The number of axillary buds proliferated and the proliferation factor were the highest, indicating that the growth of axillary buds in Europe and moon was most suitable in this hormone combination of concentration. When the concentration of NAA is 0.1 mg.L -1 And 0.3 mg. L -1 When the concentration of NAA is 0.3 mg.L, the height of the seedlings in the cluster is continuously reduced along with the increase of the concentration of 6-BA -1 6-BA concentration of 1 mg. L -1 The highest height. When the NAA concentration is 0.2 mg.L -1 The height of the seedlings in clumps is firstly low and then high along with the increase of the concentration of 6-BA, and the concentration of 6-BA is 1 mg.L -1 The highest value is reached. When the concentration of 6-BA is 1 mg.L -1 And 2 mg. L -1 The height of the seedlings in clumps is reduced and then increased along with the increase of NAA, and the concentration of NAA is 0.3 mg.L -1 The highest value is reached; when the concentration of 6-BA is 3 mg.L -1 In time, the height of the clumped seedlings appeared to continuously rise with the increase of the NAA concentration.
TABLE 5 Effect of different concentrations of 6-BA and NAA combinations on pollen and subculture
TABLE 6 Effect of different concentrations of 6-BA and NAA combinations on fruit juice balcony subculture
TABLE 7 Effect of different concentrations of 6-BA and NAA combinations on Samantha subculture
TABLE 8 Effect of different concentrations of 6-BA and NAA combinations on spectral subculture
TABLE 9 Effect of different concentrations of 6-BA and NAA combinations on Morage princess subculture
5.2 Effect of different concentrations of 6-BA and IBA combinations on Auger subculture
As is clear from tables 10 to 14, when the concentration of IBA was constant, the number of axillary buds to be proliferated and the proliferation factor showed a tendency to increase and decrease with the increase in the concentration of 6-BA, both of which were 2 mg. Multidot.L at the concentration of 6-BA -1 The highest value is reached. When the concentration of 6-BA is consistent, the number of proliferation axillary buds and the proliferation coefficient show continuous reduction along with the increase of the IBA concentration. When the concentration of IBA is constant, the heights of the clumped seedlings show the same trend along with the increase of the concentration of 6-BA, and the heights of the clumped seedlings show higher and higher. When the concentration of 6-BA is 1 mg.L -1 Meanwhile, the height of the clumped seedlings is increased firstly and then reduced along with the increase of the concentration of IBA; when the concentration of 6-BA is 2 mg.L -1 When the concentration of IBA is 0.1 mg.L, the change trend of the height of the cluster seedlings is opposite to that of the IBA concentration -1 The highest value is reached; when the concentration of 6-BA is 3 mg.L -1 Meanwhile, the heights of the clumped seedlings show a tendency of decreasing first and then increasing as the concentration of IBA increases.
TABLE 10 Effect of different concentrations of 6-BA and IBA combinations on flours and subcultures
TABLE 11 Effect of different concentrations of 6-BA and IBA combinations on fruit juice balcony subculture
TABLE 12 Effect of different concentrations of 6-BA and IBA combination on Samantha subculture
TABLE 13 Effect of different concentrations of 6-BA and IBA combinations on spectral subculture
TABLE 14 Effect of different concentrations of 6-BA and IBA combinations on Moringa princess subculture
5.3 Oumei subculture nodule
As can be seen from tables 5-14, the combination of 6-BA and NAA at different concentrations was significantly superior to the combination of 6-BA and IBA at different concentrations when subculturing pink and plain fruit juice balconies, savinsa, spectrum and axillary buds of 5 European and monthly species of the princess Moraco. Under the action of NAA and IBA with different concentrations, the number of the proliferating axillary buds and the proliferating coefficient are increased firstly and then reduced along with the increase of the concentration of 6-BA, which shows that the cytokinin has the function of inhibiting the apical dominance of the plant, so that the germination and growth of the lateral branches can be promoted, the proliferation of the axillary buds is mainly influenced, and the excessive concentration of the cytokinin 6-BA in the proliferating culture stage is not beneficial to the proliferation of the plant, and also can inhibit the normal growth of the plant and the absorption of nutrients. The low concentration of cell growth hormone is not favorable for the normal growth of tissue culture seedling, and the excessive concentration can block differentiation to cause overgrowth. Therefore, a suitable multiplication medium is crucial for subculture.
6. Rooting culture
Rooting culture medium: 1/2MS culture medium containing 0.05-1 mg/L NAA and IBA, 6g/L agar, 30g/L sucrose, 100ul/L sodium hydroxide, and pH 5.8.
The culture conditions are as follows: and (3) inoculating the seedlings subjected to subculture for 40 days into a rooting medium, inoculating 2 seedlings into each bottle, treating 5 bottles each, and repeating for 3 times. After inoculation, the seeds are placed in a culture room at 23-27 ℃, the illumination intensity is 2000-2500 lx, the illumination time is 12-16 h/d, and the rooting conditions of various varieties, namely the rooting rate/%, the rooting number/strip and the root length/cm, are counted after 40d rooting culture.
Rooting rate% = number of rooted plantlets/total number of plantlets inoculated in rooting medium
TABLE 15 subculture experimental design
6.1 Effect of NAA of different concentrations on rooting culture of August tissue culture seedlings
As can be seen from tables 16 to 20, the rooting rate, the rooting number and the root length of the tissue culture seedlings of 5 European and monthly varieties of Pingpian, fruit juice balcony, samantha, spectrum and Morage princess all show a tendency of increasing and then decreasing with the increase of the concentration of the hormone NAA, and the concentration of the hormone NAA is 0.1 mg.L -1 The concentration of NAA is 1 mg.L -1 Each index value is the lowest. The NAA concentration is 0.1 mg.L -1 When the amount is equal to 0.05 mg.L -1 The rooting rate of the Chinese medicinal composition does not reach a significant difference, and the Chinese medicinal composition reaches a significant difference level with other treatments. The rooting number and root length of each variety of European and monthly are respectively NAA concentration0.1mg·L -1 Significantly higher than the rest of the treatment.
TABLE 16 Effect of NAA at different concentrations on the cultivation of flours and rooting
TABLE 17 Effect of NAA of different concentrations on fruit juice balcony rooting culture
TABLE 18 Effect of different NAA concentrations on Samantha rooting culture
TABLE 19 Effect of different NAA concentrations on the spectroscopic rooting culture
TABLE 20 Effect of NAA at different concentrations on rooting culture by Morage princess
6.2 Effect of different IBA concentrations on rooting culture of August tissue culture seedlings
As can be seen from tables 21 to 25, the rooting rate, the rooting number and the root length of the tissue culture seedlings of Pingpian, fruit juice balcony, samantha, spectrum and 5 European-month variety of Morage princess all show a tendency of rising first and then decreasing with the increase of the concentration of the hormone IBA, with the concentration of the hormone IBA being 0.3 mg.L -1 When it reaches the maximum value, IBA concentration is 1 mg. L -1 Each index value is the lowest. The NAA concentration is 0.1 mg.L -1 When the reaction is performed with 0.05 mg. L -1 The rooting rate of the Chinese medicinal composition does not reach a significant difference, and the Chinese medicinal composition reaches a significant difference level with other treatments. The rooting number and root length of the August tissue culture seedlings of different varieties are 0.3 mg.L in NAA concentration -1 The time is obviously higher than other treatments, which indicates that the method is suitable for the rooting and growth of the tissue culture seedlings.
TABLE 21 Effect of different concentrations of IBA on flour and rooting culture
TABLE 22 Effect of different IBA concentrations on fruit juice balcony rooting culture
TABLE 23 Effect of different concentrations of IBA on Samantha rooting culture
TABLE 24 Effect of different concentrations of IBA on spectroscopic rooting culture
TABLE 25 influence of different concentrations of IBA on Morage princess rooting culture
6.3 rooting culture of August tissue culture seedling
As can be seen from tables 16-25, the highest rooting rate and the longest root length of the fruit juice balcony can be seen by analyzing the influence of NAA and IBA with different concentrations on the rooting condition of the tissue culture seedlings of various European months, and the lowest value is in the fruit juice balcony. The NAA concentration of 0.1 mg.L < -1 > is most suitable for the rooting culture of the tissue culture seedlings of various varieties in August, and the rooting condition of different varieties is most suitable for the rooting culture of different varieties when the IBA concentration is 0.3 mg.L < -1 >, which indicates that the rooting culture is most suitable for the root growth of the varieties under the concentration.
Claims (6)
1. The method for culturing and propagating the tissue of the European moon is characterized by comprising the following steps:
(1) Selecting current-year shoots which are strong in growth, free of diseases and insect pests and full in buds as explants, cutting the shoots into stem sections with 2 bud eyes and 1-2 cm in length, cutting off leaves and thorns, reserving 1-3 leaf stalks, dipping neutral detergent with a hairbrush to wash the stem sections cleanly, and then washing the stem sections with tap water for 2-3 hours;
(2) Treating the stem segments with 75% alcohol on a clean bench for 30s, rinsing with sterile water 3-5 times, followed by 0.1% HgCl 2 Or 6% sodium hypochlorite is used for disinfecting the stem sections;
(3) The stem segments are inoculated in an initial culture axillary bud induction culture medium, and after inoculation, the culture medium is placed in a culture room with the temperature of 23-27 ℃, the illumination intensity is 2000-2500 lx, the illumination time is 12-16 h/d, and the culture is carried out for 20d.
(4) Inoculating the stem section obtained by the treatment in the step (3) into a subculture medium, placing the inoculated stem section into a culture room at the temperature of 23-27 ℃, and culturing for 40d, wherein the illumination intensity is 2000-2500 lx, and the illumination time is 12-16 h/d;
(5) And (3) inoculating the stem section obtained by the treatment in the step (4) to a rooting culture medium, placing the stem section in a culture room at the temperature of 23-27 ℃ after inoculation, and culturing for 40 days, wherein the illumination intensity is 2000-2500 lx, the illumination time is 12-16 h/d.
2. The method of claim 1, wherein HgCl is consumed at 0.1% 2 Soaking and sterilizing the stem segments for 5-15 min.
3. The method for tissue culture propagation of August according to claim 1, wherein the stem segments are soaked and sterilized with 6% sodium hypochlorite for 15-25 min.
4. The method for tissue culture propagation according to any one of claims 1 to 3, wherein the primary culture axillary bud induction medium is MS medium containing 6g/L agar, 30g/L and 100ul/L sodium hydroxide, and has pH of 5.8.
5. The method for tissue culture propagation according to any one of claims 1 to 3, wherein the subculture medium is MS medium containing 1 to 3mg/L of 6-BA and 0.1 to 0.3mg/L of NAA, and the pH is 5.8.
6. The method for culturing and propagating August tissues according to any one of claims 1 to 3, wherein the rooting medium is 1/2MS medium containing 0.05 to 1mg/L NAA and IBA and has pH of 5.8.
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