CN116584392B - Asexual rapid propagation method of agilawood-easy-to-form excellent strain golden silk oil - Google Patents
Asexual rapid propagation method of agilawood-easy-to-form excellent strain golden silk oil Download PDFInfo
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- 239000001963 growth medium Substances 0.000 claims abstract description 60
- 230000006698 induction Effects 0.000 claims abstract description 42
- 230000035755 proliferation Effects 0.000 claims abstract description 30
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 18
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 14
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- 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 description 12
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- 239000000843 powder Substances 0.000 description 5
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 5
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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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- 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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- 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
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Abstract
The invention discloses a asexual rapid propagation method of agilawood-like excellent strain golden silk oil. The method collects the current annual branches of the golden silk oil, takes the stem sections with axillary buds as explants, adopts a specific method for sterilization and disinfection, and greatly reduces the brown death rate and the pollution rate; developing a specific culture medium to perform cluster bud induction, secondary proliferation and rooting induction, wherein the cluster bud induction rate is 15.4%, vitrification is avoided, the proliferation multiple is 2.7, and the rooting rate is 72.5%; proper seedling hardening conditions and transplanting methods are selected, and the survival rate can reach more than 75 percent; therefore, a large number of clone seedlings of the golden silk oil which is easy to form fragrance of the golden agilawood are obtained, the seedlings can completely inherit the excellent fragrance forming characteristic of the golden silk oil of the excellent strain, and the requirements of the market on the clone seedlings of the golden silk oil are fully met.
Description
Technical Field
The invention belongs to the technical field of biological breeding, and particularly relates to a asexual rapid propagation method of agilawood easy-to-form excellent strain golden silk oil.
Background
The aquilaria sinensis (Aquilaria sinensis (Lour.) G) is a precious tree species in the country, the xylem of the aquilaria sinensis can produce agarwood, the medicinal value is extremely high, and the aquilaria sinensis is a traditional precious medicinal material in the country, and is also an important raw material source of aromatherapy, perfume and high-end artware. The market demand for agarwood mainly comes from artificial cultivation, and people find that a good single plant is easy to produce aroma, high in aroma yield and good in aroma quality (the oil content reaches 40% and the aroma is pleasant) in the cultivation process, so that the agarwood is popular in the market; this set of aquilaria sinensis trees with excellent aroma quality is named as golden silk oil. It is desirable to obtain a large number of filigree offspring, however, when the seeds of the filigree for shannon are continuously planted, the oil content of the agarwood produced by the offspring is less than 40%, the fragrance is extremely light, and the quality of the filigree is not achieved at all. Because the aquilaria sinensis is a cross pollinated plant, the genome heterozygosity is high, the differentiation of the seed seedlings is large, the seed offspring cannot ensure the succession of the excellent characteristics of the golden silk kerogen plant, and only the offspring obtained by the asexual propagation method can ensure that the offspring completely inherits the excellent characteristics of the parent plant.
Tissue culture is a high-efficiency asexual propagation method, and golden silk oil seedlings obtained through tissue culture can completely inherit all excellent characters of golden silk oil mother plants. Tissue culture researches of agilawood have some reports, but tissue culture seedlings of golden silk oil cannot be obtained by adopting the existing research report method. If researchers use washing powder, alcohol to treat explants (aquilaria sinensis callus culture and plant regeneration (brief report), tropical subtropical plant report, 1998,6 (2): 172-176; aquilaria sinensis tissue culture rapid propagation technology research, forestry construction, 2006, (4): 10-12; aquilaria sinensis tissue culture research, agricultural biotechnology report, 2007, (15): 222-225), aquilaria sinensis start culture and physiological characteristic research thereof, hubei agricultural science, 2015,54 (7): 1742-1745), or tween to treat explants (aquilaria sinensis tissue culture rapid propagation technology research, guangdong agricultural science, 2006 (8): 44-46), or sodium hypochlorite to treat explants (aquilaria sinensis tissue culture regeneration system research, chinese forestry science research institute, 2012), these methods all lead to brown stain death of golden silk oil stems. In the rooting induction stage of the tissue culture seedlings, the existing researches report that the major element proportion and the hormone proportion are adopted, or the rooting of the golden silk oil tissue culture seedlings cannot be induced, or the rooting rate is extremely low. In some research reports, 2-step method is adopted to induce the rooting of the aquilaria sinensis tissue culture seedlings (research on the tissue culture and rapid propagation technology of aquilaria sinensis, guangdong agricultural science, 2006 (8): 44-46; research on the tissue culture of aquilaria sinensis, report on agricultural biotechnology, 2007 (15): 222-225.), so that the production efficiency of the tissue culture seedlings is reduced, the cost is increased, and the method is not suitable for large-scale rapid propagation of the tissue culture seedlings.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a large-scale asexual rapid propagation method of the agilawood excellent strain golden silk oil with important economic and medicinal values.
The invention discloses a asexual rapid propagation method of agilawood-like excellent strain golden silk oil, which comprises the following steps:
s1, collecting branches which newly sprout in the current year and are 5-10 cm long and have top tips, cutting off leaves, only preserving the 2 nd and 3 rd axillary buds under the top buds, respectively cutting off two axillary buds together with stem sections, preserving stem sections with the length of 0.8-1.2 cm at the two ends of the base part of each axillary bud, and then carrying out disinfection treatment on stem section explants with the axillary buds;
s2, inoculating the sterilized explant to a cluster bud induction culture medium to culture and induce cluster buds; the arbuscular bud induction culture medium is MS culture medium +6-BA0.6mg/L +30 g/L sucrose +5 g/L agar, and the pH is 5.8;
s3, inoculating the cluster buds to a proliferation culture medium for proliferation culture; the proliferation culture medium is modified MS culture medium, 0.4mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar, and the pH value is 5.8; wherein, the improved MS culture medium is a culture medium with the content of potassium nitrate in the MS culture medium adjusted to 2470mg/L and the content of the rest components kept unchanged;
s4, cutting off single buds with the height of more than or equal to 2.5cm, terminal buds and more than 2 leaves, inoculating the single buds into a rooting induction culture medium, and culturing and inducing rooting to obtain rooting seedlings; the rooting induction culture medium is 1/4MS culture medium, ABT 1mg/L, IBA 1mg/L, active carbon 0.05g/L, sucrose 20g/L, agar 5g/L and pH 5.8;
s5, hardening the root seedlings under the conditions that the illumination intensity is 3000-4500 Lx and the temperature is 20-30 ℃ for 10-20 days, then transplanting the root seedlings into a culture medium for culture, and carrying out seedling stage management.
Preferably, the disinfection treatment of the step S1 is to soak the materials with sterile water for 2 times and 5min each time, and then to soak the materials with HgCl with the mass fraction of 0.1 percent 2 The aqueous solution was soaked for 5min, and then washed with sterile water 5 times for 5min each.
Preferably, the cultivation medium is prepared by mixing yellow core soil, peat soil and vermiculite according to a volume ratio of 1:2:2.
Preferably, the seedling management is:
within 15 days of transplanting, covering the seedbed with a film, and covering the film with 1 layer of 90% sunshade net; spraying 5-6 times per day in sunny days, spraying 3-4 times per day in cloudy days, spraying 1-2 times per day in rainy days, spraying cooling water on a sunshade net when the temperature is high, and controlling the temperature of a seedbed to be not more than 33 ℃;
covering a film at night on the seedbed within 15-30 days of transplanting, and keeping ventilation at two ends of the seedbed, wherein the film is not covered but 80% sunshade net is covered in daytime; the water is sprayed for 2 to 3 times every day in sunny days, the water is sprayed for 1 to 2 times every day in cloudy days, the film is covered only in all the days in rainy days, and the two ends are kept ventilated, so that the water is not sprayed;
after 30 days of transplanting, the height of the seedlings reaches 50cm, and the seedbed is not covered with a film; but in the non-rainy day, 50% sunshade net is covered in daytime, water is sprayed 1-2 times a day in sunny day, water is sprayed 0-1 times a day in cloudy day, and water is not sprayed in rainy day.
Preferably, the culturing in the steps S2 and S3 is performed under the following conditions: the temperature is 25 plus or minus 2 ℃, and the illumination is 10 to 12 hours.d -1 The illumination intensity is 2500 Lx-3000 Lx; transfer every 30 days.
Preferably, the culturing in step S4 is performed under the following conditions: the temperature is 25 plus or minus 2 ℃, and the illumination is 10 to 12 hours.d -1 The illumination intensity is 2500 Lx-3000 Lx.
The invention has the following beneficial effects:
the invention provides a asexual rapid propagation method of agilawood-like excellent strain golden silk oil. The method of the invention collects the current annual branches of the eaglewood easy-to-form fragrant excellent strain golden silk oil, takes the stem section with axillary buds as an explant, adopts a specific method for sterilization and disinfection, greatly reduces the brown death rate and the pollution rate, and enables the subsequent induction and cultivation to be carried out smoothly; developing a specific culture medium to perform cluster bud induction, secondary proliferation and rooting induction, wherein the cluster bud induction rate is 15.4%, vitrification is avoided, the proliferation multiple is 2.7, and the rooting rate is 72.5%; proper seedling hardening conditions and transplanting methods are selected, and the survival rate can reach more than 75 percent; therefore, a large number of clone seedlings of the golden silk oil which is easy to form fragrance of the golden agilawood are obtained, the seedlings can completely inherit the excellent fragrance forming characteristic of the golden silk oil of the excellent strain, and the requirements of the market on the clone seedlings of the golden silk oil are fully met. The method of the invention can produce extremely high economic benefit, promote the industrial upgrading of the aquilaria sinensis planting industry and has wide market prospect.
Drawings
Fig. 1 is a browning explant.
Fig. 2 is a browning-free explant.
FIG. 3 is the effect of different sterilization modes on the golden silk oil explants.
FIG. 4 is the effect of different sterilization durations on the golden silk oil explants.
FIG. 5 is a normal clump bud of golden silk oil.
FIG. 6 is a golden silk oil vitrified clump bud.
FIG. 7 is a golden silk oil vitrified bud.
FIG. 8 is a glass transition of golden silk oil to dead buds.
FIG. 9 is the effect of potassium nitrate concentration on the proliferation factor of golden silk oil tissue culture seedlings.
FIG. 10 is a drawing of a golden silk oil robust tissue culture propagation seedling.
FIG. 11 shows the effect of macroelement content on rooting of test tube plantlets of golden silk oil.
FIG. 12 is the effect of ABT concentration on rooting rate of gold wire oil.
FIG. 13 is the effect of hormone combination and concentration on rooting rate of golden silk oil test tube plantlets.
FIG. 14 is a golden silk oil robust tissue culture root seedling.
FIG. 15 shows death of golden silk oil tissue culture rooting seedlings caused by excessive hardening light.
FIG. 16 is a drawing of a golden silk oil tissue culture rooted seedling under suitable seedling conditioning conditions.
FIG. 17 shows the transplanted golden silk oil tissue culture bag seedling.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
MS medium referred to in the examples below refers to general medium of the formulation known in the art, the composition and configuration of which is described in Toshio Murashige, folke Skoog. A Revised Medium For Rapid Growth And Bio Assays With Tobacco Tissue cultures. Physiology plant, 15:473-497 (1962); wherein sucrose and agar are absent. The 1/2MS medium referred to in the following examples is a medium in which the content of all components in the MS medium is reduced to 1/2 of the original content, and the 1/3MS, 1/4MS, 1/5MS medium is a medium in which the content of all components in the MS medium is reduced to 1/3, 1/4, 1/5 of the original content, respectively.
Example 1
Based on the huge demand of the market on the asexual propagation of golden silk oil, the invention breaks through the bottleneck that the existing aquilaria sinensis tissue culture technology can not obtain golden silk oil tissue culture seedlings, and provides a asexual rapid propagation method of golden silk oil which is easy to form fragrant and excellent in quality, and the method can induce the golden silk oil tissue culture seedlings to obtain rooting rate of more than 70% only by a 1-step method, and comprises the following specific steps:
(1) Acquisition of explants
In the morning of sunny weather, collecting branches which are newly germinated in the current year and have bright green outer skins and 5-10 cm long containing tips, cutting off leaves, putting the branches into a clean fresh-keeping bag, and then carrying out disinfection treatment.
(2) Sterilization of explants
The leaf-removed branches are only reserved with the 2 nd and the 3 rd axillary buds under the terminal buds, the two axillary buds and the stem sections are cut off respectively, the two ends of the base part of each axillary bud are reserved with stem sections with the length of 0.8-1.2 cm, then the leaf-removed branches are sterilized in the following 10 modes, and the optimal sterilization procedure and sterilization duration are screened.
A) 30s of 75% alcohol and 1 time of aseptic water washing and 0.1% HgCl 2 Soaking for 5min and washing with sterile water for 5 times, each for 5min, and finally inoculating to the plexus bud induction culture medium.
B)0.1％ HgCl 2 Soaking for 5min and washing with sterile water for 5 times, each for 5min, and finally inoculating to the plexus bud induction culture medium.
C) Sterile water soaking for 1 time for 5 min+0.1HgCl 2 Soaking for 5min and washing with sterile water for 5 times, each for 5min, and finally inoculating to the plexus bud induction culture medium.
D) Soaking in sterile water for 2 times, each time 5min+0.1% HgCl 2 Soaking for 5min and washing with sterile water for 5 times, each for 5min, and finally inoculating to the plexus bud induction culture medium.
E) Soaking in sterile water for 2 times, each time 5min+0.1% HgCl 2 Soaking for 4min and washing with sterile water for 5 times, each time for 5min, and finally inoculating to the plexus bud induction culture medium.
F) Soaking in sterile water for 2 times, each time 5min+0.1% HgCl 2 Soaking for 6min and washing with sterile water for 5 times, each time for 5min, and finally inoculating to the plexus bud induction culture medium.
G) Sterile water immersionSoaking for 2 times, each time 5min+0.1% HgCl 2 Soaking for 7min and washing with sterile water for 5 times, each for 5min, and finally inoculating to the plexus bud induction culture medium.
H) Soaking in washing powder solution for 10min, washing with running water for 30min, soaking in 75% alcohol for 30s, washing with sterile water for 1 time, and adding 0.1% HgCl 2 Soaking in the solution for 8min, washing with sterile water for 4 times, and finally inoculating to the cluster bud induction culture medium.
I) With 0.05% HgCl 2 The solution was mixed with 10mL/L Tween 20 and soaked for 10min, then washed with sterile water 4 times, and finally inoculated onto the clump bud induction medium.
J) Soaking in 10% sodium hypochlorite solution for 10min, washing with sterile water for 4 times, and inoculating to the plexus bud induction medium.
Counting the brown mortality and pollution rate of the explant after 14 days of inoculation, screening a proper disinfection program by taking the average value of the brown mortality and pollution rate of the explant obtained by A, B, C, D disinfection modes as a scale, wherein the disinfection method with the brown mortality and pollution rate of the explant being lower than or equal to the average value is selected as a preferred disinfection program. The results show that: the gold wire oil was very sensitive to alcohol, and after treatment with 75% alcohol, the explants all died from browning (FIG. 1), whereas at 0.1% HgCl 2 The aseptic water soaking mode can reduce the pollution rate of the explant and the brown death rate of the explant, and the effect is better as the aseptic water washing times are more (figures 2 and 3).
The average value of the brown mortality and the pollution rate of the explant obtained by the D, E, F, G sterilization modes is taken as a scale to screen the sterilization duration, and the sterilization duration with the brown mortality and the pollution rate of the explant lower than or equal to the average value is selected as the preferred sterilization duration. The results prove that: with 0.1% HgCl 2 Soaking for 5min is the optimal time. Sterilization for 4min can reduce the explant brown-out rate, but the explant contamination rate can be greatly increased, while increasing the sterilization time to 6min or 7min can reduce the explant contamination rate, but the explant brown-out rate can be greatly increased (fig. 4).
The effect of washing powder, tween 20 and sodium hypochlorite on the gold wire oil explants was examined in three ways of disinfection H, I, J. The results show that: after treatment of the explants with washing powder, tween 20, sodium hypochlorite, the brown mortality was high (table 1).
TABLE 1 influence of washing powder, tween 20, sodium hypochlorite on golden silk oil explants
| Disinfection mode | Brown mortality of explants (%) | Explant contamination Rate (%) |
| H | 100.0 | 26.7 |
| I | 93.3 | 33.3 |
| J | 80.0 | 40.0 |
Thus, the optimal procedure for sterilization of the golden wire oil explants is: sterilization mode D: soaking in sterile water for 2 times each for 5min, and then 0.1% HgCl 2 Soaking for 5min, washing with sterile water for 5 times, each time for 5min, and finally inoculating to the plexus bud induction culture medium. Under the sterilization mode, the brown death rate of the explant is 40%, the pollution rate of the explant is 40%, and the sterilization mode is superior to other modes, so that the best sterilization effect is obtained.
(3) Convergence induction and proliferation culture
The plexus bud induction medium was designed as follows: a) MS culture medium +6-BA 0.2mg/L; b) MS culture medium +6-BA 0.4mg/L; c) MS culture medium +6-BA0.6 mg/L; d) MS culture medium +6-BA 0.8mg/L; further, 30g/L of sucrose and 5g/L of agar were added thereto, and the pH was 5.8.
The sterilized explants started to develop clumps after 1-2 months of induction on clump induction medium (FIG. 5). The results show that: MS medium +6-BA0.6mg/L most suitable alloy wire oil explant, thus the medium was determined to be a cluster bud induction medium with a cluster bud induction rate of 15.4% and no vitrification. While further increases in 6-BA concentration continued to increase the rate of induction of clumps, clumps developed higher vitrification (Table 2, FIG. 6), and vitrified clumps failed to enter normal proliferation program and die.
TABLE 26 influence of BA concentration on the induction rate of golden silk oil plexus buds
| 6-BA concentration (mg/L) | Convergence Induction Rate (%) | Glass transition rate (%) |
| 0.2 | 3.8 | 0.0 |
| 0.4 | 11.5 | 0.0 |
| 0.6 | 15.4 | 0.0 |
| 0.8 | 19.2 | 40.0 |
The golden silk oil is successfully induced to cluster buds and then inoculated into a proliferation culture medium for proliferation culture. Culture conditions for induction and secondary proliferation of the cluster buds are as follows: the temperature is 25 plus or minus 2 ℃, and the illumination is 10 to 12 hours.d -1 The illumination intensity is 2500 Lx-3000 Lx, and the transfer is carried out every 30 days.
The proliferation culture medium is designed to add 6-BA (0.2, 0.3, 0.4, 0.5, 0.6, 0.8 mg/L) with different concentrations on the basis of MS culture medium, and further add 30g/L of sucrose, 5g/L of agar and pH of 5.8; the proliferation factor, dead rate and vitrification rate were compared at each concentration. In the proliferation culture stage, the proliferation factor of the golden silk oil proliferation seedlings was further increased with the increase of the 6-BA concentration, but the vitrification rate and the dead rate were also increased (Table 3). To obtain healthy and effective proliferating sprouts, the concentration of 6-BA in the culture medium was instead suitably adjusted to 0.4mg/L, and when the concentration of 6-BA was increased to 0.5mg/L, the proliferating sprouts began to develop a small amount of vitrification (FIG. 7), and if the concentration of 6-BA was continued to be increased to 0.8mg/L, the proliferating sprouts of golden silk oil began to develop death (FIG. 8).
TABLE 3 6 influence of BA concentration on proliferation of golden silk oil tissue culture seedlings
| 6-BA concentration (mg/L) | Multiplication factor | Dead rate of | Glass transition rate |
| 0.2 | 1.3 | 0.00％ | 0.00％ |
| 0.3 | 1.9 | 0.00％ | 0.00％ |
| 0.4 | 2.2 | 0.00％ | 0.00％ |
| 0.5 | 2.3 | 0.00％ | 7.69％ |
| 0.6 | 2.5 | 0.00％ | 15.38％ |
| 0.8 | 2.7 | 4.17％ | 18.18％ |
On the basis of the optimized culture medium (MS culture medium +6-BA 0.4mg/L +30 g/L of sucrose +5 g/L of agar and pH of 5.8), the influence of the addition of potassium nitrate (1900, 2090, 2280, 2470, 2660 mg/L) with different concentrations on proliferation factors was further analyzed.
The proliferation factor of the golden silk oil tissue culture seedling can be effectively improved by properly adjusting the content of potassium nitrate in the proliferation culture medium (figure 9), wherein the content of potassium nitrate in the culture medium is 2470mg/L as the best, and the proliferation factor is 2.7. The optimized golden silk oil is suitable proliferation culture medium of modified MS (except potassium nitrate is 2470mg/L, the rest components are the same as MS) +6-BA 0.4 mg/L+sucrose 30 g/L+agar 5g/L, pH is 5.8, proliferation seedlings are strong and have high proliferation times (figure 10), and proliferation times are 2.5-2.7 times/month.
(4) Rooting induction culture
Cutting out golden silk oil tissue culture Shan Ya with the height of more than or equal to 2.5cm, terminal buds and more than 2 blades, and inoculating into a rooting induction culture medium, wherein rooting culture conditions are as follows: the temperature is 25 plus or minus 2 ℃, and the illumination is 10 to 12 hours.d -1 The illumination intensity is 2500 Lx-3000 Lx. The optimal rooting induction culture medium for the golden silk oil tissue culture seedlings is developed through experiments of changing the concentration of an MS basic culture medium, adding hormones with different types and concentrations, adding active carbon with different concentrations into the culture medium and the like. As a result of setting the MS minimal medium to have 4 different concentrations of 1/2MS, 1/3MS, 1/4MS and 1/5MS, the concentration of the MS minimal medium has a significant effect on the rooting rate of the golden silk oil tissue culture seedlings, and when the concentration of the MS minimal medium is 1/4MS, the rooting induction is most suitable for the tissue culture seedlings (figure 11).
The variety and concentration of hormone in the culture medium have great influence on the rooting rate of the golden silk oil tissue culture seedlings. Tissue culture seedlings were hardly induced to root by the use of the hormone IBA alone (IBA concentration of 0.5mg/L, 1 mg/L) (Table 4). The hormone ABT is more capable of inducing rooting, and as the concentration of ABT (ABT concentration is 0.5mg/L, 1mg/L, 1.5mg/L, 2 mg/L) is increased, the rooting rate is also increased, but after the concentration of ABT exceeds 1mg/L, the rooting rate starts to be decreased (FIG. 12). The ratio of ABT and tissue culture Miao Shenggen is still lower when the 2 hormones of ABT and IBA are combined together, so that the rooting rate of the tissue culture seedlings of golden silk oil can be greatly improved (the result of adding the 2 hormones of ABT and IBA in a state of adding 0.05g/L of active carbon in a culture medium is shown in figure 13), the optimal combination is ABT 1mg/L and IBA 1mg/L, and the rooting rate is 72.50%.
TABLE 4 influence of IBA on rooting rate of golden silk oil
| IBA concentration (mg/L) | Rooting percentage (%) |
| 0.5 | 0 |
| 1 | 18.75 |
Meanwhile, the concentration of the activated carbon in the culture medium has very obvious influence on the rooting rate, the concentration of the activated carbon is changed in a 1/4MS culture medium added with the hormone ABT 1mg/L and IBA 1mg/L (the concentration of the activated carbon is changed to 0, 0.05, 0.1, 0.15 and 0.2 mg/L), the rooting rate of the golden silk oil tissue culture seedlings can be changed remarkably, and the rooting can be obviously promoted by adding 0.05g/L of the activated carbon, but the rooting is obviously inhibited when the content of the activated carbon is further increased (Table 5). The most suitable rooting induction medium is: 1/4MS culture medium, ABT 1mg/L, IBA 1mg/L, active carbon 0.05g/L, sucrose 20g/L, agar 5g/L, pH 5.8, rooting rate up to 72.5%, healthy rooting seedling and green leaf (FIG. 14).
TABLE 5 influence of activated carbon concentration on rooting rate of test tube seedlings of golden silk oil
| Active carbon content g/L | Rooting rate |
| 0 | 33.33％ |
| 0.05 | 72.50％ |
| 0.1 | 37.50％ |
| 0.15 | 10.71％ |
| 0.2 | 0.00％ |
(5) Hardening off seedlings
The golden silk oil rooting tissue culture seedling is sensitive to illumination in the seedling hardening period, and if the illumination intensity exceeds 5000Lx, the leaf yellowing, whitening and falling off are caused, and finally the seedling death is caused (figure 15). Meanwhile, the tissue culture seedlings are sensitive to temperature, and yellowing fallen leaves can be caused when the temperature of the hardening-seedling temperature exceeds 32 ℃, so that the quality of the bud seedlings and the transplanting survival rate are seriously influenced. The proper intensity of the light for hardening off the seedlings is 3000-4500 Lx, the proper hardening off time for 6-8 months (at the temperature of 26-30 ℃) is 10-15 days, and the proper hardening off time for other seasons (at the temperature of 20-26 ℃) is 15-20 days. The golden silk oil tissue culture sprouts under the proper seedling hardening condition are strong and do not fall off leaves, the leaves are emerald green, and the transplanting survival rate is high (figure 16).
(6) Transplantation and seedling management
Carefully taking out the root seedlings after hardening off from the culture flask, rinsing the culture medium at the base of the seedlings with clear water, and transplanting the root seedlings on the same day, wherein the root seedlings contain the following volume percentages: yellow core soil, peat soil, vermiculite=1:2:2 nutrition bags, and the specification of the nutrition bags is 6 multiplied by 11cm. The golden silk oil tissue culture seedlings are also sensitive to illumination in the transplanting process, the sunshade rate required in the early transplanting stage is more than 90%, the sunshade rate required in the middle stage is about 80%, and the seedlings are always required to be cultivated to the height of 50cm under the sunshade rate of about 50%. The excessive early illumination intensity can lead to rapid death of the seedlings, and the excessive middle and late illumination intensity leads to gradual yellowing death of the seedlings. The specific operation is as follows: within 15 days of seedling transplanting, the seedbed is covered with a film, 1 layer of 90% sunshade net is covered on the film, spraying is carried out for 5-6 times a day in sunny days, spraying is carried out for 3-4 times a day in cloudy days, and spraying is carried out for 1-2 times a day in rainy days. When the temperature is high, the temperature of the seedbed is controlled to be not more than 33 ℃ by spraying cooling water on the shading net. Covering a film at night on the seedling bed within 15-30 days after transplanting, and keeping ventilation at two ends of the seedling bed; in the daytime, a film is not required to cover but an 80% sunshade net is required to cover; in sunny days, water is sprayed for 2-3 times every day, and in cloudy days, water is sprayed for 1-2 times every day, if the rainy days are, the films are covered all the day, and the two ends are kept ventilated, so that water spraying is not needed. After the seedlings are transplanted for 30 days, the height of the seedlings is 50cm, a film is not required to be covered on the seedbed at any time, but a 50% sunshade net is required to be covered in the daytime in non-rainy days, water is sprayed 1-2 times a day in sunny days, water is sprayed 0-1 times a day in cloudy days, and water spraying is not required in rainy days.
In Guangdong region, the north latitude is 23.5 degrees or less, the most suitable transplanting seasons of the golden silk oil tissue culture seedlings are 3-4 months and 11-12 months, and the survival rate can reach more than 75 percent (figure 17).
Claims (4)
1. A asexual rapid propagation method of a high-quality golden silk oil of agilawood easy to form is characterized by comprising the following steps:
s1, collecting branches which newly sprout in the current year and are 5-10 cm long and have top tips, cutting off leaves, only preserving the 2 nd and 3 rd axillary buds under the top buds, respectively cutting off two axillary buds together with stem sections, preserving stem sections with the length of 0.8-1.2 cm at the two ends of the base part of each axillary bud, and then carrying out disinfection treatment on stem section explants with the axillary buds; the disinfection treatment is to soak the materials with sterile water for 2 times, each time for 5min, and then to soak the materials with HgCl with mass fraction of 0.1% 2 Soaking in water solution for 5min, and washing with sterile water for 5 times each for 5min;
s2, inoculating the sterilized explant to a cluster bud induction culture medium to culture and induce cluster buds; the arbuscular bud induction culture medium is MS culture medium, 0.6mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar, and the pH value is 5.8;
s3, inoculating the cluster buds to a proliferation culture medium for proliferation culture; the proliferation culture medium is modified MS culture medium, 0.4mg/L of 6-BA, 30g/L of sucrose and 5g/L of agar, and the pH value is 5.8; wherein, the improved MS culture medium is a culture medium with the content of potassium nitrate in the MS culture medium adjusted to 2470mg/L and the content of the rest components kept unchanged;
s4, cutting off single buds with the height of more than or equal to 2.5cm, terminal buds and more than 2 leaves, inoculating the single buds into a rooting induction culture medium, and culturing and inducing rooting to obtain rooting seedlings; the rooting induction culture medium is 1/4MS culture medium, ABT 1mg/L, IBA 1mg/L, active carbon 0.05g/L, sucrose 20g/L, agar 5g/L and pH 5.8;
s5, hardening the root seedlings under the conditions that the illumination intensity is 3000-4500 Lx and the temperature is 20-30 ℃ for 10-20 days, then transplanting the root seedlings into a culture medium for culture, and carrying out seedling stage management.
2. The method of claim 1, wherein the cultivation substrate is prepared by mixing yellow core soil, peat soil and vermiculite according to a volume ratio of 1:2:2.
3. The method according to claim 1, wherein the culturing in steps S2 and S3 is performed under the following conditions: the temperature is 25 plus or minus 2 ℃, and the illumination is 10 to 12 hours.d -1 The illumination intensity is 2500 Lx-3000 Lx; transfer every 30 days.
4. The method according to claim 1, wherein the culturing in step S4 is performed under the following conditions: the temperature is 25 plus or minus 2 ℃, and the illumination is 10 to 12 hours.d -1 The illumination intensity is 2500 Lx-3000 Lx.
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