CN110771505A - Culture method of citral tonglu stem tissue - Google Patents
Culture method of citral tonglu stem tissue Download PDFInfo
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Abstract
The invention belongs to the technical field of forest seedling propagation, and particularly relates to a method for culturing stem section tissues of citral monkeytree camphor, which comprises the steps of collecting semi-lignified branches of annual citral monkeytree camphor in the middle ten year 5 months, sterilizing the semi-lignified branches for 5min by mercuric chloride, inoculating the semi-lignified branches to a germination culture medium for germination induction culture, then inoculating the semi-lignified branches to a proliferation culture medium for proliferation culture, finally inoculating the semi-lignified branches to a rooting culture medium for rooting induction culture, transferring the semi-lignified branches to a greenhouse for 7-10 days, and then transferring the semi-lignified branches to the center of a non-woven bag filled with a. The invention has the advantages that the transplanting survival rate of the tissue culture seedlings with the root number more than 3 is more than 80 percent. The invention provides a theoretical basis for the asexual reproduction of the citral monkey camphor and provides a support for better developing and utilizing the citral monkey camphor.
Description
Technical Field
The invention belongs to the technical field of forest seedling propagation, and particularly relates to a culture method of stem tissue of citral-monkshood.
Background
The citral-monkeytree bark is monkeytree bark mainly containing citral in main component of branch and leaf essential oil, belongs to Lauraceae La. The citral and the monkeytree camphor have beautiful tree shape, large crown and thick yin, solid, glossy and fragrant material, and branches and leaves capable of extracting essential oil, and are important tree species for landscaping, materials and oil. At present, the research on the cinnamomum kanehirae dunn has the aspects of ecological anatomy, ecological characteristics, cultivation and breeding, afforestation technology, stress resistance, extraction of essential oil, chemical component analysis and the like, and the research on other aspects is less except that the research on cold resistance is more, and the research on the cinnamomum kanehirae dung tissue culture is not reported. If a culture method of stem tissue of the citral monkey camphor can be researched, a theoretical basis is provided for asexual propagation of the citral monkey camphor, and support is provided for better development and utilization of the citral monkey camphor.
Disclosure of Invention
The invention aims to overcome the technical problems and provides a culture method of stem tissue of a citral monkey camphor, which uses annual semi-lignified branches of the citral monkey camphor as materials and finds the culture method of the stem tissue of the citral monkey camphor with the transplanting survival rate of tissue culture seedlings with the root number more than 3 reaching more than 80 percent by selecting proper material acquisition time, disinfection time and culture medium, thereby providing a theoretical basis for asexual propagation of the citral monkey camphor and providing support for better development and utilization of the citral monkey camphor.
The technical scheme for solving the technical problems is as follows:
a method for culturing stem tissue of citral monkey camphor comprises collecting semi-lignified branches of annual citral monkey camphor in the middle of 5 months every year, sterilizing with mercuric chloride for 5min, inoculating to a germination culture medium for germination induction culture, inoculating to a proliferation culture medium for proliferation culture, inoculating to a rooting culture medium for rooting induction culture, transferring to a greenhouse for 7-10 days, and transferring to the center of a non-woven bag filled with a matrix; the germination culture medium is MS +1.0mg/L6-BA +0.05mg/L IBA, the germination induction culture lasts for 30 days, the proliferation culture medium is MS +1.0mg/L6-BA +0.2mg/L IBA, the proliferation culture lasts for 30 days, the rooting culture medium is 1/2MS +1.5mg/L IBA, and the rooting culture lasts for 20-25 days.
Further, after rooting culture is carried out for 20-22 days, the tissue culture rooted seedlings are moved into a greenhouse for hardening seedlings, and the environment of the greenhouse is as follows: the light transmission is 25-30%, and the temperature is 25-30 ℃.
The method comprises the following specific steps:
1) in 5 middle ten days of each year, collecting a robust annual citral monkey camphor semi-lignified branch, shearing a stem segment with 1-2 buds, washing for 2 hours, wherein the length of the stem segment is 1.5-3 cm, placing the stem segment on an ultraclean workbench, soaking 30 seconds with 75% alcohol, washing 3-5 times with sterile water, and using HgCl with the mass concentration of 0.1%
2Sterilizing for 5 min; the axillary bud germination rate of the stem section is highest when the disinfection is carried out for 5min, and the browning rate and the pollution rate are lower.
2) Inoculating the stem segments sterilized in the step 1) on MS +1.0mg/L6-BA +0.05mg/L IBA culture medium, and performing germination induction culture for 30 days on 1 stem segment per bottle; the germination rate is 60%.
3) Inoculating the stem segments after germination induction culture to an MS +1.0mg/L6-BA +0.2mg/L IBA culture medium, carrying out subculture on 7-8 stem segments in each bottle for 30 days; the multiplication coefficient is 4.33, the plant height is 3.67cm, and the ground diameter is 1.04 mm.
4) Selecting the sterile seedlings which are strong in growth, have unfolded and bright leaves and 3-5cm column height and are obtained in the step 3), inoculating the sterile seedlings on 1/2MS +1.5mg/LIBA culture medium, and performing rooting culture on 7-10 plants in each bottle for 20-22 days; the rooting rate is 75.00 percent, the root length is 5.06cm, the root number is 3.50, and the root thickness is 0.82 mm.
5) After rooting culture is carried out for 20-22 days, transplanting the tissue culture rooted seedlings into a greenhouse which is transparent for 25-30% and has the temperature of 25-30 ℃, hardening the seedlings for 7-10 days, cleaning the root culture medium of the rooted seedlings by using tap water, placing the rooted seedlings into a tray with a small amount of clear water, placing the rooted seedlings into a carbendazim powder (40% of active ingredients) solution with the mass concentration of 0.5% for disinfection for 5-10 min before transplanting, then transferring the rooted seedlings into the center of a non-woven bag filled with a matrix, thoroughly watering by using a watering can, keeping the matrix moist after the rooting seedlings are dried, and watering the rooted seedlings thoroughly every time.
The invention has the beneficial effects that:
the invention provides a method for culturing stem tissue of citral monkeytree bark, which comprises the step of culturing semi-lignified stem tissue of citral monkeytree bark in 5 middle-ten days with HgCl with the concentration of 0.1%
2Sterilizing for 5min, with highest axillary bud germination rate, and low browning rate and pollution rate. The culture medium suitable for the germination of the stem segments of the cinnamomum kanehirae hance is MS +1.0mg/L6-BA +0.05mg/LIBA, and the germination rate is 60 percent. The suitable culture medium for proliferation is MS +1.0mg/L6-BA +0.2mg/L IBA, the proliferation coefficient is 4.33, the plant height is 3.67cm, and the ground diameter is 1.04 mm. Adventitious roots appear at the base of the rooting culture medium 11d, the suitable rooting culture medium is 1/2MS +1.5mg/L IBA, the rooting rate is 75.00%, the root length is 5.06cm, the root number is 3.50, and the root thickness is 0.82 mm. The transplanting survival rate of the tissue culture seedlings with the root number more than 3 is over 80 percent. The invention provides a theoretical basis for the asexual reproduction of the citral monkey camphor and provides a support for better developing and utilizing the citral monkey camphor.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a schematic diagram of the process of 1.5mg/LIBA of the present invention for inducing rooting of tissue culture seedlings of Cinnamomum camphora (L.) Presl;
FIG. 2 is a picture of the field planting of tissue culture seedlings of citral Cinnamomum kanehirae Hayata of the present invention.
Detailed Description
5-year-old citral monkey camphor in camphor tree germplasm resource garden of Nanchang engineering institute is adopted as a comparative implementation material.
Example 1:
1) collecting strong annual citral monkey camphor semi-lignified branches in 5-month middle ten days each year, and shearing 1EWashing stem segments of 2 buds for 2 hours, wherein the length of the stem segments is 1.5-3 cm, placing the stem segments on an ultra-clean workbench, soaking the stem segments for 30 seconds by using 75% of alcohol, washing the stem segments for 3-5 times by using sterile water, and washing the stem segments by using HgCl with the mass concentration of 0.1%
2Sterilizing for 5 min;
2) inoculating the stem segments sterilized in the step 1) on MS +1.0mg/L6-BA +0.05mg/L IBA culture medium, and performing germination induction culture for 30 days on 1 stem segment per bottle;
3) inoculating the stem segments after germination induction culture to an MS +1.0mg/L6-BA +0.2mg/L IBA culture medium, carrying out subculture on 7-8 stem segments in each bottle for 30 days;
4) selecting the sterile seedlings which are strong in growth, have unfolded and bright leaves and 3-5cm column height and are obtained in the step 3), inoculating the sterile seedlings on 1/2MS +1.5mg/L IBA culture medium, and carrying out rooting culture on 7-10 seedlings in each bottle for 20-22 days;
5) after rooting culture is carried out for 20-22 days, transplanting the tissue culture rooted seedlings into a greenhouse which is transparent for 25-30% and has the temperature of 25-30 ℃, hardening the seedlings for 7-10 days, cleaning the root culture medium of the rooted seedlings by using tap water, placing the rooted seedlings on a tray with a small amount of clear water, disinfecting the rooted seedlings for 5-10 min by using carbendazim powder with the mass concentration of 0.5% before transplanting, then transferring the rooted seedlings into the center of a non-woven bag filled with a matrix, watering the non-woven bag thoroughly by using a watering can, keeping the matrix moist after the seedlings are dry, and watering the seedlings thoroughly each time.
Comparative example 1:
robust annual citral monkey camphor semi-lignified shoots were collected in mid 7 months each year and stem segments were treated in the same manner as in step 1) of example 1.
Comparative example 2:
robust annual citral monkey camphor semi-lignified shoots were collected in mid 9 months of the year and stem segments were treated in the same manner as in step 1) of example 1.
The stem segments of example 1 and comparative examples 1-2 were inoculated in MS +1.2mg/L6-BA +0.1mg/LIBA medium, 1 stem segment per flask, 30 flasks, 3 biological replicates. And (5) inoculating for 30d, and counting the pollution rate, the browning rate and the axillary bud germination rate.
Contamination rate (%) (number of contaminated explants/number of inoculated explants) × 100;
browning rate (%) (number of browned explants/number of inoculated explants) × 100;
axillary bud germination (%) — (number of explants to bud/number of explants to inoculate) × 100.
The comparative results are shown in table 1 below;
TABLE 1
| Time of acquisition | Contamination ratio (%) | Browning rate (%) | Germination percentage (%) | |
| Example 1 | 5 ten days in the middle of the month | 17.78±2.43c | 11.11±1.89b | 56.67±4.12a |
| Comparative example 1 | 7 ten days in the middle of the month | 28.89±1.67b | 13.33±1.51b | 40.00±2.65b |
| Comparative example 2 | 9 ten days in the middle of the month | 41.11±3.18a | 21.11±1.20a | 25.56±1.67c |
As can be seen from Table 1, the collection of the materials at different times has a significant effect on the primary culture of the stem segments of the citral Cinnamomum kanehirae Hayata, and the contamination rate and browning rate are significantly increased and the germination rate is significantly reduced with the delay of the collection time. In the middle of 5 months, the rate of explant contamination is the lowest, 17.78%, which is significantly lower than in the middle of 7 months (28.89%) and in the middle of 9 months (41.11%), the browning rate is also the lowest, which is 11.11%, which is not significantly different from the middle of 7 months, which is significantly lower than in the middle of 9 months (21.11%), the germination rate in the middle of 5 months is the highest, which is 56.67%, which is significantly higher than in the middle of 7 months and in the middle of 9 months, and the germination rate in the middle of 9 months is the lowest, which is 25.56%.
Comparative example 3:
in 5 middle ten days of each year, collecting a robust annual citral monkey camphor semi-lignified branch, shearing a stem segment with 1-2 buds, washing for 2 hours, wherein the length of the stem segment is 1.5-3 cm, placing the stem segment on an ultraclean workbench, soaking 30 seconds with 75% alcohol, washing 3-5 times with sterile water, and using HgCl with the mass concentration of 0.1%
2Sterilizing for 3 min.
Comparative example 4:
in 5 middle ten days of each year, collecting a robust annual citral monkey camphor semi-lignified branch, shearing a stem segment with 1-2 buds, washing for 2 hours, wherein the length of the stem segment is 1.5-3 cm, placing the stem segment on an ultraclean workbench, soaking 30 seconds with 75% alcohol, washing 3-5 times with sterile water, and using HgCl with the mass concentration of 0.1%
2Sterilizing for 7 min.
Comparative example 5:
in 5 middle ten days of each year, collecting a robust annual citral monkey camphor semi-lignified branch, shearing a stem segment with 1-2 buds, washing for 2 hours, wherein the length of the stem segment is 1.5-3 cm, placing the stem segment on an ultraclean workbench, soaking 30 seconds with 75% alcohol, washing 3-5 times with sterile water, and using HgCl with the mass concentration of 0.1%
2Sterilizing for 9 min.
The stem segments sterilized in example 1 for 5min and those treated in comparative examples 3-5 for 3min, 7min and 9min were placed on sterile filter paper, respectively, to remove surface water, and inoculated in MS +1.2mg/L6-BA +0.1mg/LIBA medium, 1 stem segment per bottle, 30 bottles, 3 biological replicates. And (5) inoculating for 30d, and counting the pollution rate, the browning rate and the axillary bud germination rate.
The comparative results are shown in table 2 below;
TABLE 2
| Time of sterilization | Contamination ratio (%) | Browning rate (%) | Germination percentage (%) | |
| Comparative example 3 | 3min | 65.56±3.42a | 5.56±0.37d | 10.00±1.67c |
| Example 1 | 5min | 18.89±1.85b | 12.22±1.03c | 57.78±2.59a |
| Comparative example 4 | 7min | 16.67±1.40b | 24.44±3.16b | 35.56±2.26b |
| Comparative example 5 | 9min | 8.89±1.17c | 56.67±3.95a | 16.67±1.73c |
As can be seen from Table 2, with the increase of the disinfection time, the tissue culture contamination rate of the stem segment of the citral cinnamomum kanahirai dunn gradually decreases, the browning rate gradually increases, and the germination rate first increases and then decreases. The stem section of the cinnamomum kanehirae dunn is disinfected for 3min, the pollution rate is as high as 65.56 percent and is obviously higher than that of other treatments, the pollution rates of 5min and 7min of disinfection are between 15 percent and 20 percent, the difference between the two is not obvious and is obviously higher than that of 9min of disinfection, and the pollution rate of 9min of disinfection is 8.89 percent. The browning rate of the stem section of the cinnamomum kanehirae dunn is the highest after 9min of sterilization, and is 56.67%, which is obviously higher than that of other treatments, and then the browning rate of the stem section of the cinnamomum kanehirae dunn is 7min after sterilization, and is 24.44%, and the browning rate of the stem section of the cinnamomum kanehirae dunn is lower, and is respectively 12.22% and 5.56%. The germination rate is 57.78% which is the highest after 5min of disinfection, and is obviously higher than other disinfection time, and then 7min of disinfection, and the germination rate is 3min of disinfection. Therefore, semi-lignified branches of the citral monkeytree camphor are collected in 5 months, the semi-lignified branches are trimmed into 1-2 bud stem sections, 0.1% mercury bichloride is sterilized for 5min, and primary culture is carried out, so that the germination rate is highest, and the pollution rate and the browning rate are lower.
Comparative example 6:
the stem segments after 5min of sterilization treatment as in step 1) of example 1 were inoculated on MS +0.25mg/L6-BA +0.05mg/L IBA medium and treated in 30 flasks with 1 stem segment per flask for germination induction culture for 30 days; and (5) counting the axillary bud germination rate.
Comparative example 7:
the stem segments after 5min of sterilization treatment as in step 1) of example 1 were inoculated on MS +0.5mg/L6-BA +0.05mg/L IBA medium and treated in 30 flasks with 1 stem segment per flask for germination induction culture for 30 days; and (5) counting the axillary bud germination rate.
Comparative example 8:
the stem segments after 5min of sterilization treatment as in step 1) of example 1 were inoculated on MS +0.20mg/L6-BA +0.05mg/L IBA medium and treated in 30 flasks with 1 stem segment per flask for germination induction culture for 30 days; and (5) counting the axillary bud germination rate.
Comparative example 9:
the stem segments after 5min of sterilization treatment were inoculated on MS +1.0mg/L6-BA +0.1mg/L IBA medium and treated in 30 flasks, 1 stem segment per flask, for germination induction culture for 30 days as in step 1) of example 1; and (5) counting the axillary bud germination rate.
Comparative example 10:
the stem segments after 5min of sterilization treatment as in step 1) of example 1 were inoculated on MS +1.0mg/L6-BA +0.2mg/L IBA medium and treated in 30 flasks with 1 stem segment per flask for germination induction culture for 30 days; and (5) counting the axillary bud germination rate.
Comparative example 11:
the stem segments after 5min of sterilization treatment were inoculated on MS +1.0mg/L6-BA +0.4mg/L IBA medium and treated in 30 flasks, 1 stem segment per flask, for germination induction culture for 30 days as in step 1) of example 1; and (5) counting the axillary bud germination rate.
The comparative results are shown in table 3 below;
TABLE 3
| Treatment of | Inoculating the stem section | Stem segment of sprout | Germination percentage (%) | |
| Comparison ofExample 6 | MS+0.25mg/L6-BA+0.05mg/LIBA | 30 | 7 | 23.33 |
| Comparative example 7 | MS+0.5mg/L6-BA+0.05mg/LIBA | 30 | 11 | 36.67 |
| Example 1 | MS+1.0mg/L6-BA+0.05mg/LIBA | 30 | 18 | 60.00 |
| Comparative example 8 | MS+2.0mg/L6-BA+0.05mg/LIBA | 30 | 16 | 53.33 |
| Comparative example 9 | MS+1.0mg/L6-BA+0.1mg/LIBA | 30 | 12 | 40.00 |
| Comparative example 10 | MS+1.0mg/L6-BA+0.2mg/LIBA | 30 | 8 | 26.67 |
| Comparative example 11 | MS+1.0mg/L6-BA+0.4mg/LIBA | 30 | 5 | 16.67 |
As can be seen from Table 3, the germination rate of the citral Macaca longa increases with the increase of the concentration of 6-BA, and decreases with the increase of the concentration of IBA, when the concentration of 6-BA is 1.0mg/L, IBA and the concentration is 0.05mg/L, the germination rate of the citral Macaca longa reaches 60.00% at the maximum, then 2.0mg/L6-BA +0.05mg/LIBA, and the germination rate is 1.0mg/L6-BA +0.4mg/LIBA at the minimum, and the germination rate is 16.67%. Therefore, the optimal culture medium for inducing the sprouting of the stem segments of the citral cinnamomum kanehirae dunn is MS +1.0mg/L6-BA +0.05 mg/LIBA.
Comparative example 12:
selecting a sterile seedling which is strong in growth, has a spread and bright leaf and is 3-5cm high, and pruning the stem into stem sections, wherein the stem sections at least have one leaf and one bud (1.5cm), and inoculating the stem sections on an MS +1.0mg/L6-BA +0.05mg/L IBA culture medium by the method of the step 2) of the embodiment 1, after the stem sections are subjected to germination induction culture for 30 days, inoculating the stem sections subjected to the germination induction culture on the MS +0.5mg/L6-BA +0.2mg/L IBA culture medium, treating 20 bottles, and carrying out subculture for 30 days, wherein 7-8 stem sections are per bottle; and (5) counting the increment coefficient, the plant height and the ground diameter.
The data statistical analysis method is as follows:
the plant height and root length are measured by a meter ruler, and the ground diameter and root thickness are measured by a vernier caliper. And performing data entry, sorting and analysis of variance by using Excel2010 software.
The multiplication coefficient is the number of new multiplication buds/the number of original access buds;
the rooting rate (%) (number of rooted seedlings/number of inoculated plants) × 100.
Comparative example 13:
after 30 days of germination induction culture as in comparative example 12, the stem segments after germination induction culture are inoculated on MS +1.5 mg/L6-BA +0.2mg/L IBA culture medium, and then treated into 20 bottles, 7-8 stem segments per bottle, and subcultured for 30 days; and (5) counting the increment coefficient, the plant height and the ground diameter.
Comparative example 14:
after 30 days of germination induction culture as in comparative example 12, the stem segments after germination induction culture are inoculated on MS +2.0 mg/L6-BA +0.2mg/L IBA culture medium, and then treated into 20 bottles, 7-8 stem segments per bottle, and subcultured for 30 days; and (5) counting the increment coefficient, the plant height and the ground diameter.
Comparative example 15:
after 30 days of germination induction culture as in comparative example 12, the stem segments after germination induction culture are inoculated to MS +1.0mg/L6-BA +0.05mg/L IBA culture medium, and then treated into 20 bottles, 7-8 stem segments per bottle, and subcultured for 30 days; and (5) counting the increment coefficient, the plant height and the ground diameter.
Comparative example 16:
after 30 days of germination induction culture as in comparative example 12, the stem segments after germination induction culture are inoculated on MS +1.0mg/L6-BA +0.1mg/L IBA culture medium, and then treated into 20 bottles, 7-8 stem segments per bottle, and subcultured for 30 days; and (5) counting the increment coefficient, the plant height and the ground diameter.
Comparative example 17:
after 30 days of germination induction culture as in comparative example 12, the stem segments after germination induction culture are inoculated to MS +1.0mg/L6-BA +0.15mg/L IBA culture medium, and then treated into 20 bottles, 7-8 stem segments per bottle, and subcultured for 30 days; and (5) counting the increment coefficient, the plant height and the ground diameter.
The comparative results are shown in table 4 below;
TABLE 4
As can be seen from Table 4, the proliferation coefficient of the stem segment of Cinnamomum camphora with citral increased with the increase of 6-BA concentration at the same concentration of IBA tended to increase and then decrease slightly. In example 1, the proliferation coefficient of the stem segment of the citral cinnamomum kanehirae is the highest and is 4.33, which is obviously higher than that of comparative example 12, and has no obvious difference with comparative example 13 and comparative example 14. When the concentration of 6-BA is unchanged, the proliferation coefficient of the stem section of the citral tonguing stick is basically unchanged along with the increase of the IBA concentration, and when the concentration reaches 0.2mg/L, the proliferation coefficient is obviously increased and is obviously higher than that of other treatments. The larger the plant height were comparative example 17(3.98cm), comparative example 16(3.81cm) and comparative example 13(3.72cm), followed by comparative example 12, example 1 and comparative example 15, and worst comparative example 14(3.40 cm). The growth difference of each treated ground diameter is not obvious. Therefore, the optimal proliferation medium of the citral monkey camphor is MS +1.0mg/L6-BA +0.2 mg/LIBA.
FIG. 1 shows that 1.5mg/LIBA induces the rooting process of the tissue culture seedling of the citral cinnamomum kanahirai dunn, the rooting culture of the tissue culture seedling of the citral cinnamomum kanahirai dunn is carried out for 1-5 days, and the shape of the base part is basically unchanged; when the culture lasts for 7 days, a small part of the base parts of the tissue culture seedlings expand and a small amount of callus is generated; when the tissue culture seedlings are cultured for 11d, the adventitious roots of part of the tissue culture seedlings break through the epidermis and appear; when the tissue culture seedlings are cultured for 14d, adventitious roots grow obviously (shown in figure 1), and then the adventitious roots grow and thicken.
Comparative example 18:
selecting aseptic seedlings which grow robustly, have unfolded and bright leaves and 3-5cm column height and are subcultured by the method of the step 3) in the embodiment 1, inoculating the aseptic seedlings on 1/2MS +0.5mg/L IBA culture medium, culturing 7-10 seedlings in each bottle, and performing rooting culture for 20-22 days; and (5) counting the rooting rate, the root number, the root thickness and the root length.
Comparative example 19:
selecting aseptic seedlings which grow robustly, have unfolded and bright leaves and 3-5cm column height and are subcultured by the method of the step 3) in the embodiment 1, inoculating the aseptic seedlings on 1/2MS +1.5mg/L IBA culture medium, culturing 7-10 seedlings in each bottle, and performing rooting culture for 20-22 days; and (5) counting the rooting rate, the root number, the root thickness and the root length.
Comparative example 20:
selecting aseptic seedlings which grow robustly, have unfolded and bright leaves and 3-5cm column height and are subcultured by the method of the step 3) in the embodiment 1, inoculating the aseptic seedlings on 1/2MS +2.0mg/L IBA culture medium, culturing 7-10 seedlings in each bottle, and performing rooting culture for 20-22 days; and (5) counting the rooting rate, the root number, the root thickness and the root length.
The comparative results are shown in table 5 below;
TABLE 5
| Rooting percentage (%) | Root number of | Root length (cm) | Root diameter (mm) | |
| Comparative example 18 | 31.15±3.83d | 2.40±0.21c | 4.79±09.47a | 0.74±0.08a |
| Comparative example 19 | 40.00±3.15c | 2.63±0.16bc | 5.32±0.95a | 0.69±0.12a |
| Example 1 | 75.00±2.27a | 3.50±0.18a | 5.06±0.82a | 0.82±0.10a |
| Comparative example 20 | 68.75±5.06b | 3.06±0.33ab | 5.28±0.64a | 0.71±0.07a |
As can be seen from Table 5, IBA significantly promoted the rooting of the tissue culture seedlings of citral-Cinnamomum kanehirae Hayata. With the increase of IBA concentration, the rooting rate is increased and then reduced, the rooting rate is 1.5mg/LIBA treatment, the rooting rate reaches 75 percent and is obviously higher than other treatments, and then 2.0mg/LIBA treatment (68.75 percent) is carried out, and the lowest rooting rate is 0.5mg/LIBA treatment. 1.5mg/LIBA, with the most number of roots reaching 3.5, followed by 2.0mg/LIBA (3.06), 0.5mg/LIBA, with a fewer number of roots of 2.40. The treatment difference between the root length and the root thickness is not obvious. Therefore, the optimal rooting medium of the citral monkey camphor is 1/2MS +1.5mg/L IBA.
After rooting culture is carried out for 20-22 days, the tissue culture rooted seedlings are moved to a greenhouse which is transparent to light and is at a temperature of 25-30 ℃, hardening seedlings for 7-10 days, then the rooted seedlings are taken out by medical tweezers, the root culture medium of the rooted seedlings is cleaned by tap water and placed on a tray with a small amount of clear water. Before transplanting, putting the rooted seedlings into a carbendazim powder (40% of effective components) solution with the mass concentration of 0.5% for disinfection for 5-10 min, carefully transferring the rooted seedlings into the center of a non-woven bag filled with a matrix by using medical forceps, slightly compacting the matrix to prevent the seedling bases from loosening, and then thoroughly watering the seedlings by using a watering can. Then the substrate is kept wet, i.e. poured as dry, and is poured thoroughly each time. The number of roots is more than 3, the transplanting survival rate can reach more than 80 percent, and the seedlings can be transplanted to a field for field planting after being lignified (as shown in figure 2).
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiment according to the present invention are within the scope of the present invention.
Claims (3)
1. A method for culturing stem tissue of citral monkeytree fruit is characterized in that semilignified branches of annual citral monkeytree fruit are collected in the middle of 5 months every year, sterilized by mercuric chloride for 5min, inoculated to a germination culture medium for germination induction culture, then inoculated to a proliferation culture medium for proliferation culture, finally inoculated to a rooting culture medium for rooting induction culture, moved to a greenhouse for 7-10 days for seedling hardening, and then transplanted to the center of a non-woven bag filled with a matrix; the germination culture medium is MS +1.0mg/L6-BA +0.05mg/L IBA, the germination induction culture lasts for 30 days, the proliferation culture medium is MS +1.0mg/L6-BA +0.2mg/L IBA, the proliferation culture lasts for 30 days, the rooting culture medium is 1/2MS +1.5mg/L IBA, and the rooting culture lasts for 20-25 days.
2. The culture method of the citral monkeytree stem tissue according to claim 1, wherein after rooting culture for 20-22 days, the tissue culture rooted seedlings are transferred to a greenhouse for hardening seedlings, and the environment of the greenhouse is as follows: the light transmission is 25-30%, and the temperature is 25-30 ℃.
3. The method for culturing the tissue of the stem segment of the citral monkey camphor according to any one of the claims 1 or 2, comprising the following steps:
1) in 5 middle ten days of each year, collecting a robust annual citral monkey camphor semi-lignified branch, shearing a stem segment with 1-2 buds, washing for 2 hours, wherein the length of the stem segment is 1.5-3 cm, placing the stem segment on an ultraclean workbench, soaking 30 seconds with 75% alcohol, washing 3-5 times with sterile water, and using HgCl with the mass concentration of 0.1%
2Sterilizing for 5 min;
2) inoculating the stem segments sterilized in the step 1) on MS +1.0mg/L6-BA +0.05mg/L IBA culture medium, and performing germination induction culture for 30 days on 1 stem segment per bottle;
3) inoculating the stem segments after germination induction culture to an MS +1.0mg/L6-BA +0.2mg/L IBA culture medium, carrying out subculture on 7-8 stem segments in each bottle for 30 days;
4) selecting the sterile seedlings which are strong in growth, have unfolded and bright leaves and 3-5cm column height and are obtained in the step 3), inoculating the sterile seedlings on 1/2MS +1.5mg/L IBA culture medium, and carrying out rooting culture on 7-10 seedlings in each bottle for 20-22 days;
5) after rooting culture is carried out for 20-22 days, transplanting the tissue culture rooted seedlings into a greenhouse which is transparent for 25-30% and has the temperature of 25-30 ℃, hardening the seedlings for 7-10 days, cleaning the root culture medium of the rooted seedlings by using tap water, placing the root culture medium in a tray with a small amount of clear water, placing the rooted seedlings into a carbendazim powder solution with the mass concentration of 0.5% and the effective component of 40% for disinfection for 5-10 min before transplanting, then transferring the rooted seedlings into the center of a non-woven bag filled with a matrix, thoroughly watering by using a watering can, keeping the matrix moist when the rooted seedlings are dry, and thoroughly watering each time.
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