CN104756870A - Tissue culture propagation method of parashorea chinensis germinated seeds - Google Patents
Tissue culture propagation method of parashorea chinensis germinated seeds Download PDFInfo
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- CN104756870A CN104756870A CN201510174502.0A CN201510174502A CN104756870A CN 104756870 A CN104756870 A CN 104756870A CN 201510174502 A CN201510174502 A CN 201510174502A CN 104756870 A CN104756870 A CN 104756870A
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 241001024490 Parashorea chinensis Species 0.000 title abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000008223 sterile water Substances 0.000 claims abstract description 14
- 230000001954 sterilising effect Effects 0.000 claims abstract description 14
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims abstract description 12
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims abstract description 12
- 229960000367 inositol Drugs 0.000 claims abstract description 12
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims abstract description 12
- 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 11
- 229930006000 Sucrose Natural products 0.000 claims abstract description 11
- 239000005720 sucrose Substances 0.000 claims abstract description 11
- 238000005286 illumination Methods 0.000 claims abstract description 4
- 238000007654 immersion Methods 0.000 claims abstract description 4
- 210000001161 mammalian embryo Anatomy 0.000 claims description 50
- 229960002523 mercuric chloride Drugs 0.000 claims description 11
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 11
- 241000206575 Chondrus crispus Species 0.000 claims description 10
- 150000001875 compounds Chemical class 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 10
- 238000000338 in vitro Methods 0.000 claims description 9
- 238000012546 transfer Methods 0.000 claims description 9
- 239000003643 water by type Substances 0.000 claims description 6
- 230000008901 benefit Effects 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 3
- 238000004140 cleaning Methods 0.000 abstract 2
- 239000001963 growth medium Substances 0.000 abstract 2
- 238000004659 sterilization and disinfection Methods 0.000 abstract 2
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 abstract 1
- 238000010924 continuous production Methods 0.000 abstract 1
- 238000012364 cultivation method Methods 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- 238000009395 breeding Methods 0.000 description 7
- 230000001488 breeding effect Effects 0.000 description 7
- 230000009758 senescence Effects 0.000 description 7
- 235000013399 edible fruits Nutrition 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000123611 Dipterocarpaceae Species 0.000 description 1
- 241000237536 Mytilus edulis Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 244000144987 brood Species 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000020638 mussel Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a tissue culture propagation method of parashorea chinensis germinated seeds. The technical scheme of the invention comprises the steps of pretreatment of parashorea chinensis seeds, sterilization, primary culture and the like, wherein the parashorea chinensis seeds are cultured to sterile seedlings with two pairs of true leaves; the pretreatment is as follows: the parashorea chinensis germinated seeds are put into sterile water at 30-35 DEG C for immersion cleaning for 2-5min; the sterilization is as follows: the seeds are put into a mercury chloride solution of 0.8-0.9 per mill for immersion cleaning for 2-5min and then washed with sterile water at 30-35 DEG C; the primary culture is as follows: the seeds are inoculated into a culture medium containing MS, 100mg/L of inositol, 2.5mg/L of 6-BA, 1.0mg/L of NAA, 10mg/L of Vc and 30g/L of sucrose, and the pH value of the culture medium is 6.6; and in the culture, the illumination intensity is controlled at 1500-3000lux, the illumination time is controlled at 12h/d, and the temperature is controlled at 25 plus or minus 3 DEG C. According to the method provided by the invention, an explant is sterilized to a sterile material, so that the sterile material can further develop mass propagation, rooting and culture of sterile seedlings; and compared with a traditional seedling cultivation method, the method has the advantages of high proliferation rate and continuous production of seedlings throughout the year.
Description
Technical field
The present invention relates to plant Fast Asexual Propagation Technique field, specifically a kind of tissue culture propagation method holding up sky tree chitting piece.
Background technology
Hold up sky tree (Parashorea chinensis Wang Hsie.), Dipterocarpaceae Liu An belongs to, evergreen high megaphanerophyte, is the most in great numbers in Tropical Asian rainforest, the representational seeds of most.Trunk can, up to five or six ten meters, rank on crown canopy, and timber is hard, rotproofness is strong, and the grain of wood is straight, even structure, handling ease, slicing face is smooth, and decorative pattern is attractive in appearance, for manufacturing the senior material of various furniture, having Important Economic and be worth and scientific research value, is the rarity of state.Hold up sky tree distribution narrow, the benefit freshwater mussel and Guang Nalixin stockaded village to the scape that are only distributed in Xishuangbanna waft within the scope of 20 square kilometres of a band; Growing environment major part is original Tropical ravine rainforest and mountane rain forest, and many growths in flakes, form independently group, form peculiar natural landscape, are the first class of protection plants of China.
Holding up sky tree blossoms and bears fruit more late, and generally reach more than 40 centimetres in the diameter of a cross-section of a tree trunk 1.3 meters above the ground and just blossom and bear fruit, solid rareness, and just blossom and bear fruit 1 time every 2-5, shedding is serious, not easily collects seed.The seed sprouting holding up sky tree is very fast, and on forest land, l-4 d just germinates, and at high temperature and rainy environment, some mellow fruit is still on elite stand, and seed just germinates, drastically influence the success rate of cultivating seedlings.Hold up sky tree according to epicormic branch cuttage or cuttage vegetative propagation technique, seasonal strong, the time of cuttage survival rate 70 more than % is to July in annual March.Include aldehydes matter owing to holding up a day branch bar, epicormic branch cuttage brownization is serious, and cuttage adventive root time of origin is long, as met arid or high temperature plum rains adverse weather condition, holds up day branch bar easily withered or rotten lethal.Special because holding up sky tree biological nature, seriously limit the exploitation of this resource.Employing group training vegetative propagation technique is the effective way promoting to hold up sky tree development.To gather fine hair owing to holding up sky tree tender stem segments, sterilizing difficulty is large.Holding up sky tree tissue-culturing rapid propagation brood body to obtain, exploring the method for holding up sky tree chitting piece sterilizing particularly important.
Summary of the invention
The present invention seeks to keep Seed Vitality, improving the rate of increase, annual free of discontinuities produces nursery stock, provides a kind of tissue culture propagation method holding up sky tree chitting piece.
The solution of the present invention is by realizing like this:
Hold up a tissue culture propagation method for sky tree chitting piece, it is characterized in that: comprise pretreatment, sterilizing, Initial culture operation, main operational steps is:
(1) pretreatment: the seed wing holding up sky tree chitting piece is removed, after rinsing seed with clear water, then divests kind of a shell, be placed in 30-35 DEG C of sterile water and embathe 2-5 min, obtain embryo for subsequent use;
(2) sterilizing: embryo for subsequent use is placed in mass concentration 0.8-0.9 ‰ mercuric chloride solution and soaks, by 30-35 DEG C of sterile water wash 5-8 time, obtains sterilizing embryo and waits to cultivate;
(3) Initial culture: sterilizing embryo is inoculated in Initial culture base and makes an Initial culture, after inoculation 3d, embryo starts plant division, Embryo stem extension, when inoculation 20-30 h has brown compound matter to produce, embryo is transferred in new Initial culture base and makes secondary Initial culture, transfer 2-3 time, obtain long having two pairs of true leaves, terminal bud with the plantlet in vitro of a pair of stipules.
The time of above-described immersion is 2-5 min.
The volumetric concentration of above-described Initial culture based formulas is: MS+80-120 mg/L inositol+8-10 g/L carragheen+2-3 mg/L 6-BA+0.5-1.5 mg/L NAA+5-15 mg/L VC+15-45 g/L sucrose, pH6.0-7.0.
Above-described Initial culture controlled light intensity is 1500-3000 lux, and light application time is 10-15 h/d, and temperature is 25 ± 3 DEG C.
The volumetric concentration of above-described Initial culture based formulas is: MS+100 mg/L inositol+9 g/L carragheen+2.5 mg/L 6-BA+1.0 mg/L NAA+15 mg/L VC+30 g/L sucrose, PH6.6.
The condition of above-described Initial culture is: intensity of illumination 2000 lux, light application time 12 h/d, temperature 25 DEG C.
The present invention possesses following advantage and good effect:
(1) the present invention adopts 0.8-0.9 ‰ mercuric chloride solution to carry out sterilizing to holding up a day seeds embryo, both the bacterium and fungal spores that are attached to explant surface can effectively be killed, embryo can be prevented again poisoning, keep the vitality of explant, obtain the aseptic explant of high-quality.
(2) the present invention is in the Initial culture stage, when inoculating 20-30 h and having brown compound matter to produce, is transferred in new medium by embryo and cultivates, to transfer the cultivation of 2-3 time, can effectively prevent embryo brownization, improve and cultivate breeding success rate, Senescence rate 3-10%, aseptic rate 70-94 %.
(3) the present invention passes through the optimization to Initial culture base and condition of culture, and inositol promotes the formation of embryoid and bud, shortens incubation time, improves culture efficiency, and interpolation VC effectively can prevent the browning in incubation in the medium simultaneously.
(4) the inventive method operating process is easy, mild condition, and cultivation cycle is short, and the rate of increase is high, can meet the requirement that annual free of discontinuities produces nursery stock, have good economic benefit and social benefit.
Accompanying drawing explanation
Fig. 1 holds up day seeds embryo condition diagram be seeded in after 0.9 ‰ mercuric chloride sterilizings on Initial culture base.
Fig. 2 is kind of an embryo elongate condition figure.
Fig. 3 is the condition diagram that embryo grows a pair true leaf.
Fig. 4 is the condition diagram that embryo grows two pairs of true leaves.
Embodiment
Describe a kind of tissue culture propagation method holding up sky tree chitting piece of the present invention below in conjunction with embodiment, these descriptions are not the restriction to content of the present invention.
embodiment 1
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo, embryo is placed in 35 DEG C of sterile waters and embathes 3 min, again embryo is placed in mass concentration 0.9 ‰ mercuric chloride solution and soaks 3 min, by 35 DEG C of sterile water wash 7 times, then be inoculated into containing volumetric concentration MS+100 mg/L inositol+9 g/L carragheen+2.5 mg/L 6-BA+1.0 mg/L NAA+10 mg/L VC+30 g/L sucrose, cultivate in the Initial culture base of pH=6.6, Initial culture controlled light intensity 2000 lux, light application time 12 h/d, temperature 25 DEG C, when inoculation 20-25 h has brown compound matter to produce, embryo is transferred in new Initial culture base and cultivates, transfer 3 times, obtain length and have two pairs of true leaves, terminal bud with the plantlet in vitro of a pair of stipules.
It is higher that this example cultivates breeding success rate, Senescence rate 3%, aseptic rate 94%.
embodiment 2
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo, embryo is placed in 30 DEG C of sterile waters and embathes 2 min, again embryo is placed in mass concentration 0.8 ‰ mercuric chloride solution and soaks 2 min, by 30 DEG C of sterile water wash 5 times, then be inoculated into containing volumetric concentration MS+80 mg/L inositol+8 g/L carragheen+2 mg/L 6-BA+0.5 mg/L NAA+5 mg/L VC+15 g/L sucrose, cultivate in the Initial culture base of pH=6.0, Initial culture controlled light intensity 1500 lux, light application time 10h/d, temperature 22 DEG C, when inoculation 25-30 h has brown compound matter to produce, embryo is transferred in new Initial culture base and cultivates, transfer 3 times, obtain length and have two pairs of true leaves, terminal bud with the plantlet in vitro of a pair of stipules.
It is higher that this example cultivates breeding success rate, Senescence rate 1%, aseptic rate 88%.
embodiment 3
The tissue culture propagation method holding up sky tree chitting piece in the present embodiment is as follows:
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo, embryo is placed in 32 DEG C of sterile waters and embathes 4 min, again embryo is placed in mass concentration 0.8 ‰ mercuric chloride solution and soaks 4 min, by 32 DEG C of sterile water wash 6 times, then be inoculated into containing volumetric concentration MS+90mg/L inositol+10 g/L carragheen+3 mg/L 6-BA+1.5 mg/L NAA+7 mg/L VC+24g/L sucrose, cultivate in the Initial culture base of pH=6.3, Initial culture controlled light intensity 1800lux, light application time 13 h/d, temperature 23 DEG C, when inoculation 20-23 h has brown compound matter to produce, embryo is transferred in new Initial culture base and cultivates, transfer 2 times, obtain length and have two pairs of true leaves, terminal bud with the plantlet in vitro of a pair of stipules.
It is higher that this example cultivates breeding success rate, Senescence rate 4%, aseptic rate 80%.
embodiment 4
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo, embryo is placed in 33 DEG C of sterile waters and embathes 5 min, again embryo is placed in mass concentration 0.8 ‰ mercuric chloride solution and soaks 5 min, by 33 DEG C of sterile water wash 8 times, then be inoculated into containing volumetric concentration MS+110 mg/L inositol+8.5 g/L carragheen+2.8 mg/L 6-BA+1.2 mg/L NAA+12 mg/L VC+38g/L sucrose, cultivate in the Initial culture base of pH=6.8, Initial culture controlled light intensity 2500lux, light application time 14 h/d, temperature 27 DEG C, when inoculation 24-28 h has brown compound matter to produce, embryo is transferred in new Initial culture base and cultivates, transfer 3 times, obtain length and have two pairs of true leaves, terminal bud with the plantlet in vitro of a pair of stipules.
It is higher that this example cultivates breeding success rate, Senescence rate 7%, aseptic rate 75%.
embodiment 5
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo, embryo is placed in 34 DEG C of sterile waters and embathes 3 min, again embryo is placed in mass concentration 0.9 ‰ mercuric chloride solution and soaks 5 min, by 34 DEG C of sterile water wash 7 times, then be inoculated into containing volumetric concentration MS+120mg/L inositol+9.5 g/L carragheen+2.3 mg/L 6-BA+0.8 mg/L NAA+15 mg/L VC+45 g/L sucrose, cultivate in the Initial culture base of pH=7.0, Initial culture controlled light intensity 3000 lux, light application time 15 h/d, temperature 28 DEG C, when inoculation 26-30 h has brown compound matter to produce, embryo is transferred in new Initial culture base and cultivates, transfer 2 times, obtain length and have two pairs of true leaves, terminal bud with the plantlet in vitro of a pair of stipules.
It is higher that this example cultivates breeding success rate, Senescence rate 10%, aseptic rate 70%.
comparative example 1
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo, embryo is placed in sterile water and embathes 5 min, again embryo is placed in mass concentration 1 ‰ mercuric chloride solution and soaks 8 min, by sterile water wash 6 times, then be inoculated in the Initial culture base containing volumetric concentration 1/2 MS+IBA 0.3 mL/L+6-BA 0.4 mL/L+sugared 30 g+agar 5 g and cultivate, Initial culture controlled light intensity 3000 lux, light application time 12 h/d, temperature 26 DEG C, when there being brown compound matter to produce, embryo is transferred to 1/2 MS+6-BA 0.4 mg/L+IBA0.3 mg/L+VC 0.2 mg/L+AC 0.5 g/L, 1/2 MS+6-BA0.4 mg/L+IBA 0.3 mg/L+VC 0.2 mg/L, cultivate in the Initial culture base of 1/2 MS+VC0.2 mg/L+PVP 2 g/L+AC 0.5 g/L, obtain length and have a pair true leaf, terminal bud with the plantlet in vitro of a pair of stipules.
It is higher that this comparative example cultivates breeding success rate, Senescence rate 18%, aseptic rate 65%.
Claims (7)
1. hold up the tissue culture propagation method of sky tree chitting piece, it is characterized in that: comprise pretreatment, sterilizing, Initial culture operation, main operational steps is:
Pretreatment: the seed wing holding up sky tree chitting piece is removed, after rinsing seed with clear water, then divests kind of a shell, be placed in 30-35 DEG C of sterile water and embathe 2-5 min, obtain embryo for subsequent use;
(2) sterilizing: embryo for subsequent use is placed in mass concentration 0.8-0.9 ‰ mercuric chloride solution and soaks, by 30-35 DEG C of sterile water wash 5-8 time, obtains sterilizing embryo and waits to cultivate;
(3) Initial culture: sterilizing embryo is inoculated in Initial culture base and makes an Initial culture, after inoculation 3d, embryo starts plant division, Embryo stem extension, when inoculation 20-30 h has brown compound matter to produce, embryo is transferred in new Initial culture base and makes secondary Initial culture, transfer 2-3 time, obtain long having two pairs of true leaves, terminal bud with the plantlet in vitro of a pair of stipules.
2. the tissue culture propagation method holding up sky tree chitting piece according to claim 1, is characterized in that: the time of described immersion is 2-5 min.
3. the tissue culture propagation method holding up sky tree chitting piece according to claim 1, it is characterized in that: the volumetric concentration of described Initial culture based formulas is: MS+80-120 mg/L inositol+8-10 g/L carragheen+2-3 mg/L 6-BA+0.5-1.5 mg/L NAA+5-15 mg/L VC+15-45 g/L sucrose, pH6.0-7.0.
4. the tissue culture propagation method holding up sky tree chitting piece according to claim 1, is characterized in that: described Initial culture controlled light intensity is 1500-3000 lux, and light application time is 10-15 h/d, and temperature is 25 ± 3 DEG C.
5. the tissue culture propagation method holding up sky tree chitting piece according to claim 3, it is characterized in that: the volumetric concentration of described Initial culture based formulas is: MS+100 mg/L inositol+9 g/L carragheen+2.5 mg/L 6-BA+1.0 mg/L NAA+15 mg/L VC+30 g/L sucrose, PH6.6.
6. the tissue culture propagation method holding up sky tree chitting piece according to claim 4, is characterized in that: the condition of described Initial culture is: intensity of illumination 2000 lux, light application time 12 h/d, temperature 25 DEG C.
7. hold up a tissue culture propagation method for sky tree chitting piece, it is characterized in that: the seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo, embryo is placed in 35 DEG C of sterile waters and embathes 3 min, again embryo is placed in mass concentration 0.9 ‰ mercuric chloride solution and soaks 3 min, by 35 DEG C of sterile water wash 7 times, then be inoculated into containing volumetric concentration MS+100 mg/L inositol+9 g/L carragheen+2.5 mg/L 6-BA+1.0 mg/L NAA+10 mg/L VC+30 g/L sucrose, cultivate in the Initial culture base of pH=6.6, Initial culture controlled light intensity 2000 lux, light application time 12 h/d, temperature 25 DEG C, when inoculation 20-25 h has brown compound matter to produce, embryo is transferred in new Initial culture base and cultivates, transfer 3 times, obtain length and have two pairs of true leaves, terminal bud with the plantlet in vitro of a pair of stipules.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107182667A (en) * | 2017-03-03 | 2017-09-22 | 广西大学 | A kind of strong seedling cultivation method for holding up day tree container seedling |
| CN110809935A (en) * | 2019-11-08 | 2020-02-21 | 广东省农业科学院果树研究所 | Method for improving germination rate of banana seeds |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103960106A (en) * | 2014-05-15 | 2014-08-06 | 黄振忠 | Supertree planting method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103960106A (en) * | 2014-05-15 | 2014-08-06 | 黄振忠 | Supertree planting method |
Non-Patent Citations (2)
| Title |
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| 闫兴富等: "光照和温度对望天树种子萌发的影响", 《植物学通报》 * |
| 韦凤娟等: "擎天树组培外植体消毒与褐化抑制的研究", 《中国农学通报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107182667A (en) * | 2017-03-03 | 2017-09-22 | 广西大学 | A kind of strong seedling cultivation method for holding up day tree container seedling |
| CN110809935A (en) * | 2019-11-08 | 2020-02-21 | 广东省农业科学院果树研究所 | Method for improving germination rate of banana seeds |
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