CN109729976B - Tissue culture and rapid propagation method for phoenix-tail bamboos - Google Patents
Tissue culture and rapid propagation method for phoenix-tail bamboos Download PDFInfo
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- 239000001963 growth medium Substances 0.000 claims abstract description 36
- 241000233805 Phoenix Species 0.000 claims abstract description 19
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- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 claims abstract description 16
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- 239000012883 rooting culture medium Substances 0.000 claims abstract description 11
- 241000196324 Embryophyta Species 0.000 claims abstract description 9
- 230000001954 sterilising effect Effects 0.000 claims abstract description 6
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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 14
- 229930006000 Sucrose Natural products 0.000 claims description 14
- 239000005720 sucrose Substances 0.000 claims description 13
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- 239000005708 Sodium hypochlorite Substances 0.000 claims description 11
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 11
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 10
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- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims description 8
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 claims description 8
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- 241000288015 Bambusicola <bird> Species 0.000 claims description 3
- 235000015874 Sinocalamus latiflorus Nutrition 0.000 claims description 3
- 244000092524 Sinocalamus latiflorus Species 0.000 claims description 3
- 239000006870 ms-medium Substances 0.000 claims description 3
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- 241000544043 Blyxa aubertii Species 0.000 claims 1
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Abstract
A tissue culture and rapid propagation method of Pteris multifida belongs to the technical field of tissue culture. The method comprises the following process steps: collecting field tender cauliflower bambusa with node segments as explants; sterilizing and cleaning the phoenix-tail-fern explant; inoculating to a sterile culture medium for culture; carrying out proliferation culture; rooting culture; and (5) transplanting and domesticating. The invention realizes the rapid propagation of the phoenix mosaic through the limitation of the collection time, the disinfection mode, the proliferation culture medium, the rooting culture medium and the transplanting domestication method. The survival rate of the explant obtained by the method can reach as high as 58.8%; the cluster bud inductivity reaches 71.67 percent; the proliferation effect can reach 3.65 times; the rooting rate can reach more than 70 percent, and the rooting condition is good; compared with the traditional single plant bagging domestication method, the domestication method provided by the invention has the advantages that the required time is shortened by more than half, and the quality of the nursery stock is not influenced.
Description
Technical Field
The invention belongs to the technical field of tissue culture, and particularly relates to a tissue culture and rapid propagation method of phoenix-tail bamboos.
Background
The Phoenix latiflorus is a variety of good ornamental bamboo species, belonging to the subfamily Bambusoideae of Gramineae, and is a sympodial bamboo species. The leaves of the cauliflower leaves often have irregular white longitudinal stripes and have higher ornamental value than the cauliflower leaves, but the leaves are propagated in an asexual propagation mode of division or cuttage all the time, so that the practical problems of low speed and high cost exist, and the requirements of the bamboo seedling market on the cauliflower leaves and the cauliflower seedlings cannot be met. The tissue rapid propagation technology is utilized to carry out mass and rapid propagation on the phoenix-tail bamboos, but the mature explant tissue culture rapid propagation technology of the phoenix-tail bamboos is not available at present.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme of a tissue culture and rapid propagation method of phoenix-tail bamboos.
A tissue culture and rapid propagation method of Pteris multifida is characterized by comprising the following process steps:
1) collecting field tender cauliflower phoenix bambusa with nodes as explants in the late 6 months;
2) sterilizing the obtained cauliflower forth leaf phoenix bamboo explant in the step 1) by using sodium hypochlorite with the effective chlorine concentration of 0.5-1.5% for 10-14 min under the condition of vacuumizing, and then cleaning by using sterile water;
3) removing culm sheaths of the cauliflower leaf phoenix bamboo explant obtained in the step 2) under an aseptic condition, taking a stem section with buds which is 0.5 cm higher than the culm sheath and 0.5 cm lower than the culm sheath, sucking surface moisture by using aseptic filter paper, and then inoculating the stem section into an aseptic culture medium for culture, wherein the aseptic culture medium is an MS culture medium;
4) inoculating the sterile well-grown buds obtained in the step 3) into a multiplication culture medium for culture, wherein the multiplication culture medium comprises an MS culture medium, 30g/L sucrose, 3.5g/L Gelrite, 0.08-2 mg/L TDZ, 1-4 mg/L BA and 0.2-5 mg/L KT, and has the pH of 5.7;
5) dividing the cluster buds obtained in the step 4) into 3-5 clusters, inoculating the clusters into a rooting culture medium for culture, wherein the rooting culture medium comprises an MS culture medium, 30g/L sucrose, 3.5g/L Gelrite and 0.1-2.5 mg/L NAA, and the pH value is 5.7;
6) transplanting and domesticating the rooted cauliflower plants obtained in the step 5).
The tissue culture and rapid propagation method of the phoenix-tail bamboo is characterized in that in the step 2), the phoenix-tail bamboo explant is sterilized by sodium hypochlorite with the effective chlorine concentration of 1% for 12min under the vacuum condition.
The tissue culture and rapid propagation method of the phoenix-tail bamboo is characterized in that the culture conditions in the steps 3), 4) and 5) are as follows: the illumination intensity is 2500lx, the illumination time is 16 hours per day, the ambient temperature is 25 +/-2 ℃, and the relative air humidity is 60-90%.
The tissue culture and rapid propagation method of the phoenix tailed bamboo in the step 4) is characterized in that the propagation culture medium consists of an MS culture medium, 30g/L sucrose, 3.5g/L Gelrite, 0.08mg/L TDZ, 1mg/L BA and 0.2mg/L KT, and the pH value is 5.7.
The tissue culture and rapid propagation method of the phoenix tailed bamboo in the step 5) is characterized in that the rooting culture medium consists of an MS culture medium, 30g/L sucrose, 3.5g/L Gelrite and 2.5mg/L NAA, and the pH value is 5.7.
The tissue culture and rapid propagation method of the phoenix tailed bamboos is characterized in that the transplanting and domesticating in the step 6) specifically comprises the following steps: transplanting the rooted dendrocalamus latiflorus plants to an environment with the illumination intensity of 20000 lx, the daily illumination time of 16 hours, the environment temperature of 25 +/-2 ℃ and the relative air humidity of 70-95% for acclimatization for 1 week, removing a bottle cap, continuing the acclimatization for 3 days, washing off a culture medium attached to the plant roots with warm water at 35-40 ℃, soaking in 1000-fold carbendazim solution for 3-5 minutes, and transplanting to perlite stirred with 1000-fold carbendazim solution: peat: vermiculite = 1: 1: 1, and keeping the mixture moist, and moving the mixture to a greenhouse after 1 month of acclimation.
The invention realizes the rapid propagation of the phoenix mosaic through the limitation of the collection time, the disinfection mode, the proliferation culture medium, the rooting culture medium and the transplanting domestication method. The survival rate of the explant obtained by the method can reach as high as 58.8%; the cluster bud inductivity reaches 71.67 percent; the proliferation effect can reach 3.65 times; the rooting rate can reach more than 70 percent, and the rooting condition is good; compared with the traditional single plant bagging domestication method, the domestication method provided by the invention has the advantages that the required time is shortened by more than half, and the quality of the nursery stock is not influenced.
Drawings
Figure 1 shows the contamination rate and survival rate of explants under different sodium hypochlorite concentrations, disinfection time, explant taking time and culm sheath retention treatment.
Detailed Description
The present invention is further illustrated by the following examples.
Example (b): tissue culture and rapid propagation method for phoenix-tail bamboos
1) Collecting wild tender cauliflower phoenix bambusa with nodes as explants (2-5 cm above the nodes and 1cm below the nodes) in the late 6 th month;
2) sterilizing the obtained cauliflower forth leaf phoenix bamboo explant in the step 1) by using sodium hypochlorite with the effective chlorine concentration of 0.5-1.5% for 10-14 min under the condition of vacuumizing, and then cleaning by using sterile water;
3) removing culm sheaths of the explants obtained in the step 2) under aseptic conditions, taking budding stem segments with the upper and lower 0.5 cm lengths, sucking surface moisture by aseptic filter paper, and inoculating the budding stem segments to an aseptic culture medium (MS culture medium) for culture, wherein the culture conditions are as follows: the illumination intensity is 2500lx, the illumination time is 16 hours per day, the ambient temperature is 25 +/-2 ℃, the relative air humidity is 60-90%, and the culture is carried out for 30 days;
4) inoculating the sterile well-grown buds obtained in the step 3) into a multiplication culture medium for culture, wherein the multiplication culture medium takes an MS culture medium as a basic culture medium, 30g/L of sucrose, 3.5g/L of Gelrite, 0.08-2 mg/L of TDZ, 1-4 mg/L of BA and 0.2-5 mg/L of KT are added, the pH value is 5.7, and the culture conditions are as follows: the culture conditions were: the illumination intensity is 2500lx, the illumination time is 16 hours per day, the ambient temperature is 25 +/-2 ℃, the relative air humidity is 60-90%, and the culture is carried out for 16 days;
5) taking 3-5 cluster buds obtained in the step 4) as a cluster, inoculating the cluster buds into a rooting culture medium for culture, wherein the rooting culture medium takes an MS culture medium as a basic culture medium, 30g/L of sucrose, 3.5g/L of Gelrite (watercress) and 0.1-2.5 mg/L of NAA are added, the pH value is 5.7, and the culture conditions are as follows: the illumination intensity is 2500lx, the illumination time is 16 hours per day, the ambient temperature is 25 +/-2 ℃, the relative air humidity is 60-90%, and the culture is carried out for 16 days;
6) transplanting and domesticating the rooted phoenix-tail bamboos obtained in the step 5), specifically, transplanting the rooted phoenix-tail bamboos to an environment with the illumination intensity of 20000 lx, the illumination time of 16 hours per day, the environment temperature of 25 +/-2 ℃ and the relative air humidity of 70-95% for 1 week, removing a bottle cap, continuing domestication for 3 days, washing off a culture medium attached to the plant root system with warm water of 35-40 ℃, soaking in 1000 times of carbendazim solution for 3-5 minutes, and transplanting to perlite stirred with 1000 times of carbendazim solution: peat: vermiculite = 1: 1: 1, and keeping the mixture moist, and moving the mixture to a greenhouse after 1 month of acclimation.
Test example:
1) obtaining sterile explants
A: in 6 months, taking field young cauliflower leaves of the Pteris multifida with stem segments (2-5 cm above the segments and 1cm below the segments), removing stem sheaths, after 2 hours of running water, disinfecting for 30 s with 75% alcohol, washing with sterile water for 3-5 times, disinfecting for 10min under a vacuum condition with sodium hypochlorite solutions with effective chlorine concentrations of 0.5%, 1% and 1.5% respectively, and washing with sterile water for 5-7 times. Removing sheaths of culms under an aseptic condition, taking a stem section with buds, the length of which is 0.5 cm higher than the knot and 0.5 cm lower than the knot, sucking surface moisture by using aseptic filter paper, and then inoculating the stem section into an aseptic culture medium; then placing the mixture in a condition that the illumination intensity is 2500lx, the illumination time is 16 hours every day, the ambient temperature is 25 +/-2 ℃, and the relative humidity of air is 60-90% for culturing for 30 days;
b: at the bottom of 6 months, taking field young cauliflower bambusa bambusicola jongson stem segments, respectively disinfecting for 8 min, 10min, 12min and 14min by using a sodium hypochlorite solution with the effective chlorine concentration of 1% under a vacuumizing condition, and stripping culm sheaths before flushing, disinfecting for 10min and 12min by using a sodium hypochlorite solution with the effective chlorine concentration of 1% under the vacuumizing condition, wherein the rest is the same as the method in the step A.
C: in 7 months, the field tender cauliflower leaves with the stem segments are taken, and sterilized by sodium hypochlorite solution with the effective chlorine concentration of 1% for 10min under the condition of vacuum pumping, and the rest is the same as the method in A.
The ratio of the number of contaminated shoots to the total number of inoculated shoots was used as the contamination rate and the ratio of the total number of non-contaminated shoots that could germinate to the total number of inoculated shoots was used as the survival rate, and the results are shown in FIG. 1.
(2) Cluster bud induction and rapid proliferation
The sterile explant obtained in the step (1) is inoculated into a cluster bud induction and proliferation medium which is 9 kinds of media designed by orthogonal experiments by taking an MS medium as a basic medium, adding 30g/L of sucrose and 3.5g/L of Gelrite, adding BA (1, 2, 4 mg/L), KT (0.2, 1, 5 mg/L) and TDZ (0.08, 0.4, 2 mg/L) and adopting orthogonal experiments (see table 1 in detail). And after inoculation, placing the mixture under the conditions that the illumination intensity is 2500lx, the illumination time is 16 hours every day, the ambient temperature is 25 +/-2 ℃, and the relative humidity of air is 60-90%. The results are shown in Table 1.
(3) Inducing rooting
And (3) dividing the proliferated cluster buds into 3-6 clusters, inoculating the 3-6 clusters into a rooting culture medium, taking an MS culture medium as a basic culture medium in the rooting culture medium, adding 30g/L of cane sugar and 3.5g/L of Gelrite, adding 0.1, 0.5 and 2.5mg/L of NAA, and culturing under the conditions that the illumination intensity is 2500lx, the illumination time is 16 hours per day, the environmental temperature is 25 +/-2 ℃, and the relative air humidity is 60-90%. The results are shown in Table 2.
(4) Transplanting domestication
Transplanting the rooted dendrocalamus latiflorus plants to an environment with the illumination intensity of 20000 lx, the daily illumination time of 16 hours, the environment temperature of 25 +/-2 ℃ and the relative air humidity of 70-95% for acclimatization for 1 week, removing a bottle cap, continuing the acclimatization for 3 days, washing off a culture medium attached to the plant roots with warm water at 35-40 ℃, soaking in 1000-fold carbendazim solution for 3-5 minutes, and transplanting to perlite stirred with 1000-fold carbendazim solution: peat: vermiculite = 1: 1: 1, and keeping the mixture moist, and moving the mixture to a greenhouse after 1 month of acclimation.
TABLE 1 BA, KT and TDZ orthogonal test 9 Induction Rate of shoot Induction and proliferation factor for Rapid proliferation of treated Ficus carica
TABLE 2 rooting of Phoenix latifolia test-tube plantlets in different minimal medium and NAA concentration
As can be seen from the comprehensive analysis of FIG. 1 and tables 1 and 2, the optimal time for explant extraction is 6 months; sterilizing with sodium hypochlorite with effective chlorine concentration of 1% for 12min under vacuum condition to obtain sterile explant as the optimal sterilization mode; MS +30g/L Sucrose +3.5g/L Gelrite +0.08 mg/L TDZ + 1mg/L BA +0.2 mg/L KT, and pH 5.7 is a culture medium formula for rapid and efficient proliferation; MS +30g/L Sucrose +3.5g/L Gelrite +2.5mg/L NAA, pH 5.7 is the rooting medium formula.
Compared with other treatments, the survival rate of the explant of the best explant taking method is as high as 58.8%; the cluster bud inductivity reaches 71.67 percent; the proliferation effect can reach 3.65 times; the rooting rate can reach more than 70 percent, and the rooting condition is good.
According to the method for tissue culture and rapid propagation of the phoenix-tail bamboos, after a certain number of aseptic tissue culture seedlings of the phoenix-tail bamboos are obtained at the bottom of 6 months, rapid propagation can be carried out indoors all year round, and after the rooted test tube seedlings are domesticated, excellent seedling of the phoenix-tail bamboos can be obtained.
Claims (5)
1. A tissue culture and rapid propagation method of Pteris multifida is characterized by comprising the following process steps:
1) collecting field tender cauliflower phoenix bambusa with nodes as explants in the late 6 months;
2) sterilizing the obtained cauliflower forth leaf phoenix bamboo explant in the step 1) by using sodium hypochlorite with the effective chlorine concentration of 0.5-1.5% for 10-14 min under the condition of vacuumizing, and then cleaning by using sterile water;
3) removing culm sheaths of the cauliflower leaf phoenix bamboo explant obtained in the step 2) under an aseptic condition, taking a stem section with buds which is 0.5 cm higher than the culm sheath and 0.5 cm lower than the culm sheath, sucking surface moisture by using aseptic filter paper, and then inoculating the stem section into an aseptic culture medium for culture, wherein the aseptic culture medium is an MS culture medium;
4) inoculating the sterile well-grown buds obtained in the step 3) into a multiplication culture medium for culture, wherein the multiplication culture medium comprises an MS culture medium, 30g/L sucrose, 3.5g/L Gelrite, 0.08-2 mg/L TDZ, 1-4 mg/L BA and 0.2-5 mg/L KT, and has the pH of 5.7;
5) dividing the cluster buds obtained in the step 4) into 3-5 clusters, inoculating the clusters into a rooting culture medium for culture, wherein the rooting culture medium comprises an MS culture medium, 30g/L sucrose, 3.5g/L Gelrite and 0.1-2.5 mg/L NAA, and the pH value is 5.7;
6) transplanting and domesticating the rooted phoenix-leaved phoenix-tail bamboo plant obtained in the step 5), wherein the transplanting and domesticating specifically comprises the following steps: transplanting the rooted dendrocalamus latiflorus plants to an environment with the illumination intensity of 20000 lx, the daily illumination time of 16 hours, the environment temperature of 25 +/-2 ℃ and the relative air humidity of 70-95% for acclimatization for 1 week, removing a bottle cap, continuing the acclimatization for 3 days, washing off a culture medium attached to the plant roots with warm water at 35-40 ℃, soaking in 1000-fold carbendazim solution for 3-5 minutes, and transplanting to perlite stirred with 1000-fold carbendazim solution: peat: vermiculite = 1: 1: 1, and keeping the mixture moist, and moving the mixture to a greenhouse after 1 month of acclimation.
2. The tissue culture and rapid propagation method of phoenix bambusicola as claimed in claim 1, wherein the phoenix-leaved phoenix-tail explant in step 2) is sterilized with sodium hypochlorite with 1% of available chlorine concentration for 12min under vacuum condition.
3. The tissue culture and rapid propagation method of phoenix bambusicola as claimed in claim 1, wherein the culture conditions in steps 3), 4) and 5) are as follows: the illumination intensity is 2500lx, the illumination time is 16 hours per day, the ambient temperature is 25 +/-2 ℃, and the relative air humidity is 60-90%.
4. The tissue culture and rapid propagation method of phoenix bambusae in claim 1, wherein the proliferation medium in step 4) consists of MS medium, 30g/L sucrose, 3.5g/L Gelrite, 0.08mg/L TDZ, 1mg/L BA and 0.2mg/L KT, and has a pH of 5.7.
5. The tissue culture and rapid propagation method of phoenix tailed bamboo as claimed in claim 1, wherein the rooting medium in step 5) consists of MS medium, 30g/L sucrose, 3.5g/L Gelrite and 2.5mg/L NAA, and has pH of 5.7.
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