CN111134022B - Tissue culture and rapid propagation method of ilex denticulata with stem section as explant - Google Patents
Tissue culture and rapid propagation method of ilex denticulata with stem section as explant Download PDFInfo
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- CN111134022B CN111134022B CN202010062569.6A CN202010062569A CN111134022B CN 111134022 B CN111134022 B CN 111134022B CN 202010062569 A CN202010062569 A CN 202010062569A CN 111134022 B CN111134022 B CN 111134022B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a tissue culture and rapid propagation method of ilex denticulata with stem sections as explants, which comprises the following steps: step 1, collecting branches of the ilex denticulata, and cleaning and disinfecting; step 2, cutting branches of the ilex dentata into bud stem sections, and inoculating the bud stem sections on a proliferation and elongation culture medium for proliferation and elongation culture; the proliferation and elongation culture medium is an MS culture medium added with 2.28-9.12 mu M ZT; step 3, transferring the seedlings to a rooting culture medium to perform rooting induction to obtain rooted plantlets, wherein the rooting culture medium is 1/2MS culture medium added with 2.07-8.29 mu M IBA; step 4, transplanting the obtained seedlings with roots into a culture medium for culture; the stem of the ilex dentata seedling is taken as an explant, and a sterile seedling micropropagation seedling rapid propagation system is successfully obtained through the proliferation culture of a plant growth regulator on the stem of the seedling and the induction research on adventitious roots, wherein the rooting rate of the tissue culture seedling of the ilex dentata is up to more than 90%, and the transplanting survival rate is up to about 90%.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a tissue culture and rapid propagation method of ilex denticulata with stem sections as explants.
Background
The ilex denticulata is a multi-branch evergreen shrub with the height of 5 m; the leaf is leathery, is in an inverted oval shape, an ellipse or a long ellipse, 1-7 male flowers are arranged into a parasol inflorescence, the base number of the flowers is 4, and the flowers are white; calyx disk, petal 4, ellipse, corolla diameter about 6 mm, petal oval, ovary oval, fruit ball, and black after maturation; endocarp leathery. The flowering period is 5-6 months, and the fruit period is 8-10 months. Growing in hills, mountain grocery forests or shrubs with the elevation of 700-2100 m. Distributed in china, japan and korea. The cultivation is usually carried out in gardens for ornamental tree species, and is also carried out in Europe and America. The seeds of the ilex denticulata fruit are difficult to germinate, and have the characteristic of annual germination due to the double dormancy of the hard seed coats and the immature zygotic embryos. The conventional breeding of the ilex denticulata needs 2 to 3 years to obtain seedlings.
In order to overcome the incompatibility of distant hybridization, obtain distant hybrid varieties, break through seed dormancy, shorten the breeding period, shorten the application period of the ilex denticulata and save labor and financial resources, a tissue culture method is the best method for quickly obtaining a large number of breeding seedlings. However, the tissue culture method of the existing ilex denticulata tissue culture seedling has low rooting rate and low transplanting survival rate, so that the large-scale popularization and propagation of the ilex denticulata tissue culture seedling are limited.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a tissue culture and rapid propagation method of ilex denticulata with stem sections as explants, the rooting rate of tissue culture seedlings of ilex denticulata is up to more than 90%, the transplanting survival rate is up to about 90%, and the germination time of ilex denticulata is shortened to 15-17 weeks from 2-3 years of conventional breeding.
The invention is realized by the following steps:
the invention aims to provide a tissue culture and rapid propagation method of ilex denticulata with stem segments as explants, which comprises the following steps:
step 1, collecting branches of the ilex denticulata, and cleaning and disinfecting;
step 2, cutting branches of the ilex dentata into bud stem sections, and inoculating the bud stem sections on a proliferation and elongation culture medium for proliferation and elongation culture; the proliferation and elongation culture medium is an MS culture medium added with 2.28-9.12 mu M ZT;
step 3, transferring the seedlings to a rooting culture medium to perform rooting induction to obtain rooted plantlets, wherein the rooting culture medium is 1/2MS culture medium added with 2.07-8.29 mu M IBA;
and 4, transplanting the obtained seedlings with roots into a culture medium for culture.
Preferably, the specific steps of cleaning and disinfecting in step 1 are as follows: selecting ilex chinensis branches, washing the ilex chinensis branches with washing powder for 10-30 min, then washing the ilex chinensis branches with running water for 10-30 min, and transferring the ilex chinensis branches to a super-clean workbench to be rinsed with sterile water; soaking the mixture in 75% alcohol for 5-20 min, then soaking the mixture in 8% sodium hypochlorite for 15-30 min, and finally washing the mixture with sterile water for 5-6 times for later use.
Preferably, the length of the stem with buds in the step 2 is 0.5-1.5 cm.
Preferably, the proliferation elongation culture medium used in step 2 is MS culture medium supplemented with 9.12. mu.M ZT.
Preferably, the time for propagation and elongation culture in the step 2 is 50-60 days.
Preferably, the rooting medium in step 3 is 1/2MS medium supplemented with 4.14. mu.M IBA.
Preferably, the rooting induction culture time in the step 3 is 50-60 days.
Preferably, the cultivation medium transplanted in the step 4 is placed on a greenhouse seedbed and grown under a spraying system.
The invention has the following beneficial effects:
the tissue culture and rapid propagation method of the ilex denticulata with the stem section as the explant, provided by the invention, has the advantages that the seedling stem section of the ilex denticulata is used as the explant, the propagation culture of the seedling stem section and the induction research of adventitious roots are carried out through the plant growth regulator, a sterile seedling micropropagation and rapid propagation system is successfully obtained, the germination time of the ilex denticulata is shortened to 15-17 weeks from 2-3 years of conventional breeding, the rooting rate of the tissue culture seedling of the ilex denticulata is up to more than 90%, and the transplanting survival rate is up to about 90%.
Drawings
FIG. 1 is a graph showing the effect of different cytokinins and concentrations on the proliferation of adventitious buds of the progeny of ilex denticulata in examples 1 and 3;
FIG. 2 is a picture of the effect of different rooting hormones and concentrations on the rooting of the offspring of ilex denticulata; (A) (B) and (C) are photographs of ilex denticulata of example 5; (D) the figure is a photograph of ilex denticulata of comparative example 10; (E) the diagram is a greenhouse cultivation diagram of all rooted seedlings.
Detailed Description
Example 1
The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants provided by the embodiment comprises the following steps:
(1) cleaning holly branches with washing powder for 20min, and washing with running water for 10min to remove surface dirt. Transferred to a clean bench and rinsed 1 time with sterile water. Soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 15min, and washing with sterile water for 5-6 times.
(2) Cutting the branch into stem sections with buds about 1cm, inoculating the stem sections on an MS culture medium with 2.28 mu M ZT for 8 weeks for proliferation and elongation culture;
(3) induction of re-rooting was performed for 8 weeks on 1/2MS medium containing 4.14 μ M IBA;
(4) transplanting the seedlings to a culture medium, putting the seedlings on a greenhouse seedbed and growing the seedlings under a spraying system, wherein the transplanting survival rate reaches 93.5 percent.
Example 2
The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants provided by the embodiment comprises the following steps:
(1) cleaning holly branches with washing powder for 20min, and washing with running water for 10min to remove surface dirt. Transferred to a clean bench and rinsed 1 time with sterile water. Soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 15min, and washing with sterile water for 5-6 times.
(2) Cutting the branch into stem sections with buds about 1cm, inoculating the stem sections on an MS culture medium with 4.56 mu M ZT for proliferation and elongation culture for 60 days;
(3) induction of re-rooting was performed for 60 days on 1/2MS medium containing 4.14 μ M IBA;
(4) transplanting the seedlings to a culture medium, putting the seedlings on a greenhouse seedbed and growing the seedlings under a spraying system, wherein the transplanting survival rate reaches 91.6 percent.
Example 3
The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants provided by the embodiment comprises the following steps:
(1) cleaning holly branches with washing powder for 20min, and washing with running water for 10min to remove surface dirt. Transferred to a clean bench and rinsed 1 time with sterile water. Soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 15min, and washing with sterile water for 5-6 times.
(2) Cutting the branch into stem sections with buds about 1cm, inoculating the stem sections on an MS culture medium with 9.12 mu M ZT added for 50 days of proliferation and elongation culture;
(3) induction of regenerated roots was performed for 50 days on 1/2MS medium containing 4.14 μ M IBA;
(4) transplanting the seedlings to a culture medium, putting the seedlings on a greenhouse seedbed and growing the seedlings under a spraying system, wherein the transplanting survival rate reaches 90.8 percent.
Example 4
The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants provided by the embodiment comprises the following steps:
(1) cleaning holly branches with washing powder for 20min, and washing with running water for 10min to remove surface dirt. Transferred to a clean bench and rinsed 1 time with sterile water. Soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 15min, and washing with sterile water for 5-6 times.
(2) Cutting the branch into stem sections with buds about 1cm, inoculating the stem sections on an MS culture medium with 2.28 mu M ZT for 8 weeks for proliferation and elongation culture;
(3) induction of re-rooting was performed for 8 weeks on 1/2MS medium containing 2.07 μ M IBA;
(4) transplanting the seedlings to a culture medium, putting the seedlings on a greenhouse seedbed and growing the seedlings under a spraying system, wherein the transplanting survival rate reaches 93.5 percent.
Example 5
The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants provided by the embodiment comprises the following steps:
(1) cleaning holly branches with washing powder for 20min, and washing with running water for 10min to remove surface dirt. Transferred to a clean bench and rinsed 1 time with sterile water. Soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 15min, and washing with sterile water for 5-6 times.
(2) Cutting the branch into stem sections with buds about 1cm, inoculating the stem sections on an MS culture medium with 2.28 mu M ZT for 8 weeks for proliferation and elongation culture;
(3) induction of re-rooting was performed for 8 weeks on 1/2MS medium containing 8.29 μ M IBA;
(4) transplanting the seedlings to a culture medium, putting the seedlings on a greenhouse seedbed and growing the seedlings under a spraying system, wherein the transplanting survival rate reaches 93.5 percent.
Comparative example 1
This comparative example was carried out in the same manner as example 1 except that no cytokinin ZT was added.
Comparative example 2
The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants provided by the embodiment comprises the following steps:
(1) cleaning holly branches with washing powder for 20min, and washing with running water for 10min to remove surface dirt. Transferred to a clean bench and rinsed 1 time with sterile water. Soaking in 75% ethanol for 5min, soaking in 8% sodium hypochlorite for 15min, and washing with sterile water for 5-6 times.
(2) Cutting the branch into stem sections with buds about 1cm, inoculating the stem sections to an MS culture medium with 18.24 mu M ZT for 8 weeks for proliferation and elongation culture;
(3) induction of re-rooting was performed for 8 weeks on 1/2MS medium containing 4.14 μ M IBA;
(4) transplanting the seedlings to a culture medium, putting the seedlings on a greenhouse seedbed and growing the seedlings under a spraying system, wherein the transplanting survival rate reaches 93.5 percent.
Comparative example 3
This comparative example was conducted in the same manner as in example 1 except that "2.28. mu.M ZT" in example 1 was changed to "2.2. mu.M 6-BA".
Comparative example 4
This comparative example was conducted in the same manner as in example 1 except that "2.28. mu.M ZT" in example 1 was changed to "4.4. mu.M 6-BA".
Comparative example 5
This comparative example was conducted in the same manner as in example 1 except that "2.28. mu.M ZT" in example 1 was changed to "8.8. mu.M 6-BA".
Comparative example 6
This comparative example was conducted in the same manner as in example 1 except that "2.28. mu.M ZT" in example 1 was changed to "17.86. mu.M 6-BA".
Comparative example 7
This comparative example is the same as example 1 except that the rooting hormone IBA is not added.
Comparative example 8
This comparative example was carried out in the same manner as in example 1 except that "4.14. mu.M IBA" in example 1 was changed to "2.23. mu.M IAA".
Comparative example 9
This comparative example was carried out in the same manner as in example 1 except that "4.14. mu.M IBA" in example 1 was changed to "4.46. mu.M IAA".
Comparative example 10
This comparative example was carried out in the same manner as in example 1 except that "4.14. mu.M IBA" in example 1 was changed to "8.92. mu.M IAA".
Experimental example 1
The effect of different cytokinins and concentrations on the proliferation of adventitious buds of ilex denticola progeny in examples 1-3 and comparative examples 1-6 was counted, and the results are shown in table 1.
TABLE 1
As can be seen from Table 1, the adventitious buds have better proliferation effect under the treatment of 2.28-9.12 mu M ZT, wherein the proliferation effect under the treatment of 9.12 mu M ZT is optimal; the proliferation effect tended to decrease when ZT was 18.24. mu.M. The proliferation effect of the cell without cytokinin ZT or 6-BA treatment is not as good as that of the embodiment of the invention.
Experimental example 2
The effect of different rooting hormones and concentrations on rooting of progeny of ilex denticulata was counted in example 1, examples 4-5, and comparative examples 7-10, and the results are shown in table 2.
TABLE 2
As can be seen from Table 2, the rooting effect is better under the IBA treatment of 2.07-8.29 MuM, wherein the rooting effect is the best under the IBA treatment of 4.14 MuM; the proliferation effect of the plant is not as good as that of the plant in the embodiment of the invention when the plant is not treated by rooting hormone IBA or other rooting hormone NAA.
In summary, the tissue culture and rapid propagation method of the ilex denticulata with the stem section as the explant, provided by the invention, comprises the steps of taking the stem section of the ilex denticulata seedling as the explant, and performing proliferation culture and induction research on adventitious roots through a plant growth regulator on the stem section of the seedling, namely adding an MS culture medium with 2.28-9.12 mu M ZT to perform proliferation and elongation culture; and (3) adding 2.07-8.29 mu M IBA into an 1/2MS culture medium for rooting induction culture to successfully obtain a sterile seedling micropropagation seedling rapid propagation system.
The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A tissue culture and rapid propagation method of ilex denticulata with stem segments as explants is characterized by comprising the following steps:
step 1, collecting branches of the ilex denticulata, and cleaning and disinfecting;
step 2, cutting branches of the ilex dentata into bud stem sections, and inoculating the bud stem sections on a proliferation and elongation culture medium for proliferation and elongation culture; the proliferation elongation culture medium is MS +9.12 mu M ZT;
step 3, transferring the seedlings to a rooting culture medium for rooting induction to obtain rooted plantlets, wherein the rooting culture medium is 1/2MS +4.14 mu M IBA;
and 4, transplanting the obtained seedlings with roots into a culture medium for culture.
2. The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants according to claim 1, wherein the specific steps of cleaning and sterilizing in step 1 are as follows: selecting ilex chinensis branches, washing the ilex chinensis branches with washing powder for 10-30 min, then washing the ilex chinensis branches with running water for 10-30 min, and transferring the ilex chinensis branches to a super-clean workbench to be rinsed with sterile water; soaking the mixture in 75% alcohol for 5-20 min, then soaking the mixture in 8% sodium hypochlorite for 15-30 min, and finally washing the mixture with sterile water for 5-6 times for later use.
3. The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants according to claim 1, wherein the length of the stem with buds in the step 2 is 0.5-1.5 cm.
4. The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants according to claim 1, wherein the propagation and elongation culture time in step 2 is 50-60 days.
5. The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants according to claim 1, wherein the time for rooting induction culture in step 3 is 50-60 days.
6. The tissue culture and rapid propagation method of ilex denticulata with stem segments as explants according to claim 1, wherein the ilex denticulata tissue culture and rapid propagation method in step 4 is transplanted to a culture medium and placed under a spraying system on a greenhouse seedbed for growth.
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