CN117918260A - Propagation method of Yunnan pine tissue culture seedlings - Google Patents
Propagation method of Yunnan pine tissue culture seedlings Download PDFInfo
- Publication number
- CN117918260A CN117918260A CN202410323453.1A CN202410323453A CN117918260A CN 117918260 A CN117918260 A CN 117918260A CN 202410323453 A CN202410323453 A CN 202410323453A CN 117918260 A CN117918260 A CN 117918260A
- Authority
- CN
- China
- Prior art keywords
- buds
- seeds
- culture medium
- aseptic
- rooting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 235000011611 Pinus yunnanensis Nutrition 0.000 title claims abstract description 54
- 241000018652 Pinus yunnanensis Species 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000012010 growth Effects 0.000 claims abstract description 48
- 239000001963 growth medium Substances 0.000 claims abstract description 33
- 230000006698 induction Effects 0.000 claims abstract description 28
- 230000035784 germination Effects 0.000 claims abstract description 20
- 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 abstract description 15
- 229930006000 Sucrose Natural products 0.000 claims abstract description 15
- 239000005720 sucrose Substances 0.000 claims abstract description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 12
- 229920001817 Agar Polymers 0.000 claims abstract description 6
- 239000008272 agar Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 16
- 238000005286 illumination Methods 0.000 claims description 12
- 238000002791 soaking Methods 0.000 claims description 11
- 229930192334 Auxin Natural products 0.000 claims description 9
- 239000002363 auxin Substances 0.000 claims description 9
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 239000012883 rooting culture medium Substances 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 4
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 230000007613 environmental effect Effects 0.000 claims description 3
- 239000002689 soil Substances 0.000 claims description 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 6
- 230000007226 seed germination Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 14
- 238000011282 treatment Methods 0.000 description 10
- 238000010586 diagram Methods 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- NNBFNNNWANBMTI-UHFFFAOYSA-M brilliant green Chemical compound OS([O-])(=O)=O.C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 NNBFNNNWANBMTI-UHFFFAOYSA-M 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 241000218631 Coniferophyta Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000218602 Pinus <genus> Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000011681 asexual reproduction Effects 0.000 description 1
- 238000013465 asexual reproduction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000028446 budding cell bud growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000024346 drought recovery Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of plant tissue culture, and particularly discloses a propagation method of a Yunnan pine tissue culture seedling, which comprises the following steps of seed germination accelerating and disinfection, aseptic germination, cluster bud induction, elongation growth, rooting culture and transplanting hardening, wherein the cluster bud induction is to inoculate the terminal buds of the obtained aseptic seedling on a cluster bud induction culture medium for cluster bud induction; the cluster bud induction culture medium comprises the following formula: 1/2MS+1.0 mg/L6-BA+0.15 mg/L IBA+1.5 mg/L KT+4.4 g/L agar+30 g/L sucrose, and pH 5.6.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a propagation method of Yunnan pine tissue culture seedlings. Background
Yunnan pine (Pinus yunnanensis) belongs to Pinaceae Pinus plants, is a main rural tree species of Yun Guigao, is also a special forest vegetation type in southwest areas, and only in Yunnan provinces, yunnan pine forest accounts for 19.56% of the forest land area of the whole province. The Yunnan pine has the characteristics of strong adaptability, drought tolerance barren, wide wood application and the like, and is pioneer tree species for forestation in barren mountains in southwest areas and important economic forests. Due to the influence of factors such as genetic inheritance, artificial interference, plant diseases and insect pests and the like, the quality of the Yunnan pine stand is poor, genetic resources are lost or disappear in a large amount, the quality of the natural updated Yunnan pine stand is reduced due to the common torsion phenomenon, and the productivity and carbon balance of the Yunnan pine stand are influenced to a certain extent. Therefore, it is important to reproduce, preserve and improve the excellent germplasm of Yunnan pine by an effective means, and asexual reproduction is one of the important ways to solve the problem.
The existing Yunnan pine seed garden can not continuously supply enough excellent seeds of Yunnan pine due to limited yield, so that the problems can be solved by utilizing a plant tissue culture technology, and the rapid propagation of excellent seeds of Yunnan pine is realized. The Yunnan pine is taken as woody gymnosperm, the plant tissue culture is very difficult, and how to establish a stable tissue culture seedling technical system is very important to ensure the popularization and application of the fine variety of the Yunnan pine in forestation. Although relevant reports exist in the tissue culture research of Yunnan pine, the relevant work still needs to be continuously advanced.
At present, although researches on tissue culture of the Yunnan pine are reported sporadically, the tissue culture and rapid propagation of the Yunnan pine are slow to advance due to the problems of easy browning, difficult redifferentiation, difficult rooting and the like. Therefore, if the excellent germplasm of the Yunnan pine is used as a material, the tissue culture and rapid propagation of the Yunnan pine are realized by utilizing a plant tissue culture technology, the method has great significance for the genetic improvement of the Yunnan pine and the cultivation of new varieties, and simultaneously, the method can provide technical support for the transformation of low-yield and low-efficiency forests in southwest areas and the cultivation of high-quality seedlings in the artificial forests.
Disclosure of Invention
Based on the problems in the background technology, the invention aims to provide a method for propagating the Yunnan pine tissue culture seedlings, which is not influenced by the environment and has stable propagation.
Specifically, the invention provides the following technical scheme:
A propagation method of Yunnan pine tissue culture seedlings comprises the following steps:
(1) Accelerating germination and disinfection of seeds: selecting mature healthy seeds of Yunnan pine, continuously soaking the seeds by using warm water with the initial temperature of 50-55 ℃ until the seeds are naturally cooled, replacing cold water for 1 time after soaking for 24 hours, accelerating germination and accumulating for 48 hours, and sterilizing to obtain sterile seeds for later use;
(2) Aseptic germination: under the aseptic condition, inoculating the aseptic seeds obtained in the step (1) into an aseptic culture bottle for germination and growth to obtain aseptic seedlings;
(3) And (3) clustered bud induction: inoculating terminal buds of the sterile seedlings obtained in the step (2) on a clustered bud induction culture medium under a sterile condition, and performing clustered bud induction; the cluster bud induction culture medium comprises the following formula: 1/2MS+1.0 mg/L6-BA+0.15 mg/L IBA+1.5 mg/L KT+4.4 g/L agar+30 g/L sucrose, pH 5.6;
(4) Elongation growth: transferring the induced cluster buds to an elongation growth medium for growth; the elongation growth culture medium is 1/4 MS+0.2% active carbon+30 g/L sucrose, the pH is 5.6, after culturing on the culture medium for 45-60 days, the cluster buds are taken out and cut into single buds, and the single buds are transferred to the same culture medium for culturing for 30-45 days until the heights of the buds are about 2.0-3.0 cm;
(5) Rooting culture: selecting single buds with the height of 2.0-3.0 cm, then placing the single buds into an aseptic solution containing auxin to be soaked for 20-30 min, placing the single buds on aseptic paper to absorb water after the single buds are finished, and finally inoculating the single buds on a rooting culture medium; the aseptic solution containing the auxin contains 0.15 mg/LIBA+0.15 mg/L NAA auxin; the rooting culture medium comprises the following formula: 1/4 MS+0.2% active carbon+20 g/L sucrose, pH 5.6;
(6) Transplanting and hardening seedlings: and (3) putting the rooting seedlings under indoor natural light, performing closed-bottle seedling hardening for 10 d, taking out the rooting seedlings, cleaning a basal culture medium with clear water, and transplanting the rooting seedlings into field soil.
In the step (3), the induced cluster buds are subjected to secondary propagation after cluster bud induction, wherein a culture medium used for the secondary propagation is prepared by adding 0.1% of activated carbon into a cluster bud induction culture medium.
Preferably, the conditions of aseptic germination, cluster bud induction, elongation growth and rooting culture are as follows: the temperature is 21+/-2 ° ℃, the illumination time is 14-16 h/d, and the illumination intensity is 2000-2500 Lx.
Preferably, in step (1), the disinfection method comprises the following steps: soaking and sterilizing 1.5-2 h by using 0.1% HgCl 2, soaking 20-30 min by using sterile water, picking out seeds, and placing the seeds on sterile filter paper to suck the water to obtain the sterile seeds.
Preferably, in the step (6), the environmental conditions in the transplanting and hardening process are as follows: the temperature is 20-30 ℃, and the air humidity is kept above 80%.
The invention has the technical progress and beneficial effects that:
(1) The invention takes Yunnan pine seeds with seed coats as the initial explant material, can prolong the disinfection time in the disinfection process, and hardly affects the activity of the seeds after disinfection.
(2) The sterilized seeds are directly inoculated on sterile paper in a tissue culture bottle, the seed coats are not required to be removed, the operation is time-saving and labor-saving, and the germination speed is high; avoiding the problems of slow discovery or difficult germination caused by the influence of water potential on the culture medium.
(3) The invention forms cluster buds directly through terminal buds without callus, shortens the cultivation time and saves the cost.
(4) The invention uses plant tissue culture method to propagate materials, which is not limited by seasons, and has controllable conditions and continuous production.
(5) The invention realizes the tissue culture Miao Gaoxiao propagation of the Yunnan pine, and can provide technical support for the large-scale cultivation of the excellent germplasm of the Yunnan pine.
Drawings
FIG. 1 is a diagram showing the condition of sterile germination and growth of seed coat seeds of Pinus yunnanensis 30 d;
FIG. 2 is a diagram of conditions of induced growth of cluster buds of Pinus yunnanensis 90 d;
FIG. 3 is a diagram showing the condition of the cluster bud growth 90 d after the successive propagation of Pinus yunnanensis;
FIG. 4 is a diagram showing the growth condition of the first elongation growth culture 45 d of Yunnan pine;
FIG. 5 is a diagram showing the growth of the second elongated growth culture 20 d of Pinus yunnanensis;
FIG. 6 is a rooting chart of Yunnan pine in rooting formulation with activated carbon (55 d);
FIG. 7 is a root of Pinus yunnanensis before transplanting;
FIG. 8 is a view showing the growth of Yunnan pine seedlings (30 d);
FIG. 9 is a comparative graph of the effect of different basal media on the induction of cluster buds of Pinus yunnanensis (60 d);
FIG. 10 is a graph comparing the effect of different media on the elongation growth of cluster buds of Pinus yunnanensis (45 d);
FIG. 11 is a comparison of the effect of different media on rooting culture of Pinus yunnanensis (60 d).
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
Example 1
A tissue culture seedling propagation method of Yunnan pine comprises the following steps:
(1) Accelerating germination and disinfection of seeds:
the Yunnan and Dazhiyu seed garden Yunnan pine seeds stored in a 4 ° C refrigerator are continuously soaked in warm water with the initial temperature of 50-55 ℃ until natural cooling, cold water is replaced for 1 time after soaking for 24 hours, cold water Shi Quchu is replaced for floating particles, after accumulated soaking for 48 and h, the seeds are transferred into an ultra-clean workbench to be soaked in 0.1% HgCl 2 for 1.5-2 h for sterilization, then the seeds are soaked in sterile water for 20-30 min after being swayed for 3-4 times, and then swayed for 3-4 times, the seeds are picked out and placed on sterile filter paper to absorb water, and germination acceleration and sterilization of the seeds are completed, so that the sterile seeds are obtained;
(2) Aseptic germination:
inoculating sterile seeds obtained after sterilization in a sterile culture flask in an ultra-clean workbench for germination and growth; wherein, the culture bottle contains two layers of sterile toilet paper (sprouting bed) and 10-12 mL sterile water, the specification of the tissue culture bottle is 11cm high, and the caliber is 6.3cm; the seeds begin to germinate when the seeds are cultured for 5 days, the outer seed shells drop when the seeds are 17 d days, needles are unfolded, the germination rate is 94%, and the growth condition of aseptic seedlings is shown in figure 1.
Wherein the germination and growth conditions are as follows: the temperature is 21+/-2 ° ℃, the illumination time is 14-16 h/d, and the illumination intensity is 2000-2500 Lx.
And (3) clustered bud induction:
Inoculating terminal buds of the sterile seedlings obtained in the step (2) on a clustered bud induction culture medium under a sterile condition, and performing clustered bud induction; when the culture was induced to 35d, the terminal buds began to proliferate into cluster buds, and the average number of buds of the last cluster buds was 8.7 (FIG. 2). The induced cluster buds are subjected to successive propagation, so that the cluster buds can be continuously propagated (figure 3).
Wherein, the formula of the cluster bud induction culture medium is as follows: 1/2 MS+1.0 mg/L6-BA+0.15 mg/L IBA+1.5 mg/L KT+4.4 g/L agar+30 g/L sucrose, pH 5.6; the secondary propagation culture medium is prepared by adding 0.1% active carbon into a cluster bud induction culture medium; the culture conditions are as follows: the temperature is 21+/-2 ° ℃, the illumination time is 14-16 h/d, and the illumination intensity is 2000-2500 Lx.
(4) Elongation growth:
transferring the induced cluster buds to an elongation growth medium for growth, wherein the elongation growth effect of the cluster buds is shown in figure 4; further cutting single buds for elongation growth, wherein the growth effect is shown in figure 5, and the average height of the single buds is 2.8 cm; wherein, the cluster bud elongation growth medium is 1/4 MS+0.2% active carbon+30 g/L sucrose, and the pH is 5.6. The culture conditions are as follows: the temperature is 21+/-2 ℃, the illumination time is 14-16 h/d, and the illumination intensity is 2000-2500 Lx.
(5) Rooting culture:
Selecting single buds of Yunnan pine clusters with the height of 2-3 cm, cutting off the brown tissues if the brown tissues exist, then soaking the single buds in an aseptic solution containing auxin for 20min, and inoculating the single buds in a rooting culture medium; when the rooting culture is carried out for 35 d days, rooting is started, the rooting rate is 46%, and the growth condition of the rooting seedlings is shown in figure 6.
Wherein, the concentration of the auxin in the sterile solution containing the auxin is 0.15 mg/L IBA+0.15 mg/L NAA, and the rooting culture medium formula is 1/4 MS+0.2% active carbon+20 g/L sucrose, and the pH is 5.6; the culture conditions are as follows: the temperature is 21+/-2 ° ℃, the illumination time is 14-16 h/d, and the illumination intensity is 2000-2500 Lx.
(6) Transplanting and hardening seedlings:
Taking out rooting seedlings after the rooting seedlings are subjected to closed bottle hardening 10d under indoor natural light, cleaning a basal culture medium (figure 7) with clear water, and transplanting the rooting seedlings into field soil (figure 8), wherein the environmental conditions in the transplanting hardening process are as follows: the temperature is 20-30 ℃, and the air humidity is kept above 80%.
The technical scheme of the invention is further described through the following experiments:
1. influence of different basal media on the induction of cluster buds of Pinus yunnanensis
The results of inoculating the aseptic germinated seedling terminal buds into different cluster bud basal media are combined with the table 1, and the cluster bud induction and growth conditions are shown in fig. 9, and from the graph, it can be seen that the growth and proliferation of the Yunnan pine terminal buds can be realized by adopting 1/2MS as the basal portion expansion of stems, the terminal buds change obviously, more cluster buds are generated at the tops, the newly grown needle leaves are emerald green, and death or cluster bud formation can not occur under the culture of other culture media.
TABLE 1 Effect of different basal media on the induction of cluster buds of Pinus yunnanensis
| Culture medium | Effects of |
| MS | The basal portion of the stem is brown, the terminal bud grows slowly, part of needles She Bianhuang even the terminal bud dies. |
| 1/2MS | The basal part of the stem expands, the variation of the terminal bud is obvious, more cluster buds are generated at the top, and the newly grown needle leaves are emerald green. |
| DCR | The base of the stem is brown yellow, the terminal buds grow normally, and cluster buds cannot be formed at the top. |
| WPM | The stem is yellow-green, the growth vigor is better, the needle She Cuilu is longer, the high growth is obvious, and cluster buds cannot be formed. |
2. Influence of different culture media on the elongation growth of cluster buds of Pinus yunnanensis
The cluster buds obtained after proliferation have smaller lateral buds, and if rooting is directly carried out, the cluster buds are easy to be healed without growing roots or dying, so that the cluster buds need to be elongated and grown. The elongation growth adopts a two-step method, firstly, the induced cluster buds are subjected to elongation culture in a cluster bud mode, so that the growth of lateral buds can be promoted, death can be avoided or reduced, the growth is carried out until the lateral buds reach a certain size (the height is 0.8-1.1 cm), and then single bud elongation is carried out; and secondly, cutting the cluster buds after the cultivation in the first step into single buds for elongation cultivation, so that the single buds grow in an elongation mode, and preparation is made for root generation.
The Yunnan pine cluster buds with proliferation growth of 90 d are transferred into the following table medium for elongation growth culture of 45: 45 d, and the growth condition is shown in figure 10. 4.4g/L agar, 30 g/L sucrose was added to all formulations in Table 2 below. As can be seen from FIG. 10, the cluster buds of treatment 1 had the best elongation growth effect, and had good growth vigor, and the side buds had significant elongation growth. Although the lateral buds of treatment 2 also grew in elongation, the vigor was general; the lateral bud elongation changes were not evident for treatments 3 and 4. In the following table, "AC" refers to activated carbon.
TABLE 2 Effect of different media on the elongation growth of Cluster buds of Pinus yunnanensis
| Treatment of | Formulation of | Height of bud (cm) | Growth performance |
| 1 | 1/4MS+0.2%AC | 0.8-1.1 | The needle leaves are green, and the lateral buds and the main buds grow well after elongation. |
| 2 | 1/2MS+0.2%AC | 0.6-1.1 | The new-born needle leaves are green, lateral buds and main buds grow obviously in an elongation mode, but old leaves lose green. |
| 3 | 1/2DCR+0.2%AC | 0.5-0.8 | The new needle leaves are green, the old leaves lose green, the elongation of the main bud needle leaves is obvious, but the elongation and growth of the lateral buds are not obvious. |
| 4 | 1/2DCR | 0.6-0.9 | The new needle leaves are green, the old leaves lose green, the main bud new needle She Jiaoduo grows obviously, but the lateral buds do not grow obviously. |
3. Influence of different culture media on rooting culture of Pinus yunnanensis
The single buds of Yunnan pine with the elongation growth are transferred into the following table culture medium for rooting culture for 60 d, and the rooting condition is shown in figure 11. 4.4g/L agar was added to all the formulations in Table 3 below. As can be seen from Table 3, the rooting rate in treatment 1 was highest; from the aspect of growth effect, the rooting seedling in the treatment 1 has the best growth effect, the rooting rate is lower in the treatment 2, the root hairs are less, the root system activity is poor, the rooting seedling dies after transplanting, the root parts of the rooting seedlings in the treatment 3 and the treatment 4 are formed by callus, needles She Huangku are serious, the root formed by the callus is easy to break from the joint during transplanting, and meanwhile, the survival rate is difficult to survive or the survival rate is reduced after transplanting.
TABLE 3 Effect of different media on rooting culture of Pinus yunnanensis
| Treatment of | Formulation of | Rooting percentage% | Average rooting number |
| 1 | 1/4MS+0.2% AC+20g/L sucrose | 50 a | 2 |
| 2 | 1/4MS+0.5mg/LNAA+0.2 mg/LIBA+10g/L sucrose | 13 c | 1 |
| 3 | WPM+1.4 mg/LNAA+20g/L sucrose | 33 b | 3 |
| 4 | WPM+1. mg/LNAA+20g/L sucrose | 17 c | 2 |
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (5)
1. The propagation method of the Yunnan pine tissue culture seedlings is characterized by comprising the following steps of:
(1) Accelerating germination and disinfection of seeds: selecting mature healthy seeds of Yunnan pine, continuously soaking the seeds by using warm water with the initial temperature of 50-55 ℃ until the seeds are naturally cooled, replacing cold water for 1 time after soaking for 24 hours, accelerating germination and accumulating for 48 hours, and then sterilizing to obtain sterile seeds for later use;
(2) Aseptic germination: under the aseptic condition, inoculating the aseptic seeds obtained in the step (1) into an aseptic culture bottle for germination and growth to obtain aseptic seedlings;
(3) And (3) clustered bud induction: inoculating terminal buds of the sterile seedlings obtained in the step (2) on a clustered bud induction culture medium under a sterile condition, and performing clustered bud induction; the cluster bud induction culture medium comprises the following formula: 1/2MS+1.0 mg/L6-BA+0.15 mg/L IBA+1.5 mg/L KT+4.4 g/L agar+30 g/L sucrose, pH 5.6;
(4) Elongation growth: transferring the induced cluster buds to an elongation growth medium for growth; the elongation growth culture medium is 1/4 MS+0.2% active carbon+30 g/L sucrose, the pH is 5.6, after culturing on the culture medium for 45-60 days, the clustered buds are taken out and cut into single buds, and the single buds are transferred to the same culture medium for continuous culture for 30-45 days until the heights of the buds are 2.0-3.0 cm;
(5) Rooting culture: selecting single buds with the height of 2.0-3.0 cm, then placing the single buds into an aseptic solution containing auxin to soak the single buds for 20-30 min, placing the single buds on aseptic paper to absorb water after the single buds are finished, and finally inoculating the single buds on a rooting culture medium; the aseptic solution containing the auxin contains 0.15 mg/L IBA+0.15 mg/L NAA auxin; the rooting culture medium comprises the following formula: 1/4 MS+0.2% active carbon+20 g/L sucrose, pH 5.6;
(6) Transplanting and hardening seedlings: and (3) putting the rooting seedlings into a closed bottle under indoor natural light, hardening off 8-10 d, taking out the rooting seedlings, cleaning a basal culture medium with clear water, and transplanting the rooting seedlings into field soil.
2. The propagation method according to claim 1, wherein in the step (3), the induced cluster buds are subjected to secondary propagation after the induction of the cluster buds, and the culture medium used for the secondary propagation is prepared by adding 0.1% of activated carbon into the cluster bud induction culture medium.
3. The propagation method according to claim 1 or 2, wherein the aseptic germination, clustered bud induction, clustered bud subculture, elongation growth and rooting culture are carried out under the following conditions: the temperature is 21+/-2 ° ℃, the illumination time is 14-16 h/d, and the illumination intensity is 2000-2500 Lx.
4. The propagation method of claim 1, wherein in the step (1), the sterilization method is: soaking and sterilizing 1.5-2 h by using 0.1% HgCl 2, soaking 20-30 min by using sterile water, picking out seeds, and placing the seeds on sterile filter paper to suck the water to obtain the sterile seeds.
5. The propagation method of claim 1, wherein in the step (6), the environmental conditions during the transplanting and hardening process are: the temperature is 20-30 ℃, and the air humidity is kept above 80%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410323453.1A CN117918260B (en) | 2024-03-21 | 2024-03-21 | Propagation method of Yunnan pine tissue culture seedlings |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410323453.1A CN117918260B (en) | 2024-03-21 | 2024-03-21 | Propagation method of Yunnan pine tissue culture seedlings |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117918260A true CN117918260A (en) | 2024-04-26 |
| CN117918260B CN117918260B (en) | 2024-05-24 |
Family
ID=90756014
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410323453.1A Active CN117918260B (en) | 2024-03-21 | 2024-03-21 | Propagation method of Yunnan pine tissue culture seedlings |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117918260B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120137390A1 (en) * | 2009-05-07 | 2012-05-31 | Arborgen Inc. | Materials and methods for regeneration and transformation of trees |
| CN106007966A (en) * | 2016-05-23 | 2016-10-12 | 合肥信文农业科技有限公司 | Nanometer reinforced seaweed culture medium capable of improving red kiwi fruit transplantation survival rate and preparation method thereof |
| CN112273228A (en) * | 2020-10-17 | 2021-01-29 | 云南省林业和草原科学院 | Cultivation method of Yunnan pine mycorrhizal seedlings |
| CN114847073A (en) * | 2022-05-31 | 2022-08-05 | 西南林业大学 | Method for improving sprouting capacity of Yunnan pine seedlings based on exogenous hormones |
| US20230062179A1 (en) * | 2019-11-06 | 2023-03-02 | Qingdao Kingagroot Chemical Compound Co., Ltd. | Methods for generating new genes in organism and use thereof |
| US20230287389A1 (en) * | 2019-11-07 | 2023-09-14 | Qingdao Kingagroot Chemical Compound Co., Ltd. | A method for generating new mutation in organism and use thereof |
-
2024
- 2024-03-21 CN CN202410323453.1A patent/CN117918260B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120137390A1 (en) * | 2009-05-07 | 2012-05-31 | Arborgen Inc. | Materials and methods for regeneration and transformation of trees |
| CN106007966A (en) * | 2016-05-23 | 2016-10-12 | 合肥信文农业科技有限公司 | Nanometer reinforced seaweed culture medium capable of improving red kiwi fruit transplantation survival rate and preparation method thereof |
| US20230062179A1 (en) * | 2019-11-06 | 2023-03-02 | Qingdao Kingagroot Chemical Compound Co., Ltd. | Methods for generating new genes in organism and use thereof |
| US20230287389A1 (en) * | 2019-11-07 | 2023-09-14 | Qingdao Kingagroot Chemical Compound Co., Ltd. | A method for generating new mutation in organism and use thereof |
| CN112273228A (en) * | 2020-10-17 | 2021-01-29 | 云南省林业和草原科学院 | Cultivation method of Yunnan pine mycorrhizal seedlings |
| CN114847073A (en) * | 2022-05-31 | 2022-08-05 | 西南林业大学 | Method for improving sprouting capacity of Yunnan pine seedlings based on exogenous hormones |
Non-Patent Citations (1)
| Title |
|---|
| 周微, 黄健秋, 卫志明, 许智宏: "云南松成熟胚的不定芽诱导及植株再生(简报)", 植物生理学通讯, no. 05, 20 October 1995 (1995-10-20), pages 351 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117918260B (en) | 2024-05-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101647393B (en) | Fast tissue culture reproducing method of actinidia eriantha | |
| CN101584297B (en) | Tissue culture and regenerated plant in vitro mycorrhization method for pinus massoniana | |
| CN102204512A (en) | Tissue culture method for lilium tenuifolium | |
| CN111616052A (en) | Rapid propagation and sugar-free rooting culture method and application of apple rootstock catalpa bungei | |
| CN111280056A (en) | Subculture breeding method of stingless pepper tissue culture seedlings | |
| CN111226794B (en) | Method for culturing somatic embryos of liquidambar plants into seedlings and propagation method of liquidambar plants | |
| CN112335549A (en) | Method for obtaining larch regeneration plant through tissue in-vitro culture | |
| CN106538382B (en) | Method for establishing efficient eremochloa ophiuroides regeneration system by taking young ears as explants | |
| CN103947548A (en) | Method for establishing agapanthus high-frequency regeneration system | |
| CN103828716A (en) | Tissue culture method of dianthus deltoids | |
| CN116171859B (en) | Non-symbiotic germination method for cypripedium yunnanensis seeds | |
| CN103155869A (en) | Sweet cherry rootstock Colt tissue culture method | |
| CN108967196B (en) | Culture method of in-vitro microspore regeneration plant of rape | |
| CN101248762B (en) | Golden leaf carex shoot-tip culture quick propagation method | |
| CN117918260B (en) | Propagation method of Yunnan pine tissue culture seedlings | |
| CN111149703B (en) | Simple, convenient, efficient and high-quality papaya tissue culture seedling rooting method | |
| CN112273229B (en) | One-step seedling method for hydrangea | |
| CN108401907A (en) | A kind of Apples Dwarf Stocks M9T337 tissue cultures volume production method | |
| CN114431154A (en) | Method for asexual propagation through acer nikoense dormant buds | |
| CN110771512B (en) | Efficient induction method of rabdosia lophanthide callus | |
| CN110169360B (en) | Method for propagating and cultivating ribwort | |
| CN114514879A (en) | Rapid propagation and sugar-free rooting culture method for Nicotiana benthamiana | |
| CN102487825B (en) | Method for inducing Morinda citrifolia calluses and regenerating plants using root as explant | |
| CN102057872A (en) | Regeneration culture method of whole cotyledonary joint of hyacinth bean | |
| CN110583481A (en) | Method for inducing somatic embryogenesis and plant regeneration of Aralia elata |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |