CN112293260A - A kind of method for obtaining African reed tissue culture seedling - Google Patents
A kind of method for obtaining African reed tissue culture seedling Download PDFInfo
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- CN112293260A CN112293260A CN202011330740.3A CN202011330740A CN112293260A CN 112293260 A CN112293260 A CN 112293260A CN 202011330740 A CN202011330740 A CN 202011330740A CN 112293260 A CN112293260 A CN 112293260A
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- 235000014676 Phragmites communis Nutrition 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000001963 growth medium Substances 0.000 claims abstract description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 230000004069 differentiation Effects 0.000 claims abstract description 14
- 239000002689 soil Substances 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000008223 sterile water Substances 0.000 claims abstract description 8
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 230000035755 proliferation Effects 0.000 claims abstract description 7
- 230000001939 inductive effect Effects 0.000 claims abstract description 6
- 235000009161 Espostoa lanata Nutrition 0.000 claims abstract 2
- 240000001624 Espostoa lanata Species 0.000 claims abstract 2
- 244000273256 Phragmites communis Species 0.000 claims description 8
- 239000003415 peat Substances 0.000 claims description 5
- 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 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 210000001519 tissue Anatomy 0.000 claims 5
- 239000012882 rooting medium Substances 0.000 claims 2
- 239000007864 aqueous solution Substances 0.000 claims 1
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims 1
- 210000002615 epidermis Anatomy 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 3
- 238000002791 soaking Methods 0.000 abstract description 3
- 238000005406 washing Methods 0.000 abstract description 3
- 230000001954 sterilising effect Effects 0.000 abstract description 2
- 241000735332 Gerbera Species 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 14
- 230000006698 induction Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000013505 freshwater Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 2
- 235000017491 Bambusa tulda Nutrition 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 230000007226 seed germination Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000209128 Bambusa Species 0.000 description 1
- 241001330002 Bambuseae Species 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 241001243666 Photinia serratifolia Species 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 239000012883 rooting culture medium Substances 0.000 description 1
- 239000004563 wettable powder Substances 0.000 description 1
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Classifications
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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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention relates to a one-step seedling method for inducing differentiation and rooting of reed gerbera, which comprises the following steps: exposing small buds of an outdoor reed branch collected from Africa on sunny days, removing outer leaves around the buds, wiping the surface of the branch by using a cotton ball stained with 75% ethanol, then cutting the stem section with the buds into 2cm, placing the stem section with the buds in a sterilizing bottle, and placing the stem section on a clean bench; soaking in 75% ethanol for 30s, rinsing with sterile water for 3 times, and adding 0.1% HgCl2Sterilizing the water solution for 10-15min, and washing with sterile water for 4-5 times; inoculating the sterilized reed stem segments to a culture medium for inducing differentiation and rooting; after 10 days of culturing each stem section with buds, the first generation roots then bud, and after 20-30 days of culturing, the transplanting tissue culture seedling can be obtained. The method can achieve the purposes of induced differentiation and rooting in one step and rapid proliferation. After the seedlings are transplanted and hardened, the seedlings can be quickly adapted to soil cultivation and have strong activity. The invention is suitable for the reed variety.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and through the optimization of a traditional rapid propagation tissue culture system, the method omits the conversion of culture media for a plurality of times, such as induction, differentiation, rooting and the like, saves the cost and time, can rapidly provide a batch of seedlings with uniform growth vigor, and provides a plurality of possibilities and guarantees for the application of the fields of ecology, medicine, gardens, animal husbandry and the like of the reed.
Background
The reed is one of emergent aquatic plants with the highest productivity in the plant community, is used as a main planting species of a wetland emergent aquatic plant community, widely grows in fresh water wetland, even coastal wetland and inland saline-alkali wetland with high salinity, and becomes a tool species for restoring wetland ecology by virtue of the characteristics of strong stress resistance, good adaptability, rapid propagation and the like. The reed root exudates are beneficial to removing nitrogen in a denitrification system and increasing the quantity of denitrifying bacteria, has a promoting effect on nitrification strength and an inhibiting effect on denitrification strength, and the reed has a protection effect on the diversity of soil fungi, and the variety of the rhizosphere soil fungi is 1.2 times of that of non-vegetation soil. The prior people research that the breeding of the reeds usually consumes a long time, a batch of plants with uniform growth vigor can not be obtained at the same time, some reeds have seeds which can germinate and grow seedlings, the special (excellent) wild type reeds have developed root systems, and the stalks are hard like bamboo, and the biological characteristics of the bamboos are different from those of common reeds; seedling basal growth, short blade, sharp blade tip, plant height of 3-4m, no heading and seed forming phenomenon in China and slow natural propagation. If the plant is widely planted on the banks of rivers, lakes and north and south China, the plant can block flood and silt, has the functions of storing flood, reducing flow rate and the like, and has the reputation of 'the kidney of the earth'. The energy is saved, the consumption is reduced, and the impact resistance is strong when the plant is planted in the wetland for treatment.
At present, some reports about reed tissue culture exist, the traditional reed seedling preparation includes stem cuttage, seed germination and the like, the photinia serrulata and the like and the royal sea and the like all utilize the stem cuttage, the Jordan and the like utilize the seed germination, and although an example that some wettable powder or disinfectant is used for increasing the germination rate of the seeds is provided, the practical operation still has certain limitations. The patent of Anshuqing et al, a culture method of non-callus of fresh water reed, also teaches the rapid propagation system of fresh water reed, but needs to be transformed by a plurality of culture media such as induction, differentiation, rooting, etc.
Therefore, it is very important to obtain healthy reed seedlings efficiently and quickly. Aiming at the defects, the method is invented by improving the prior art, starts from reducing the browning of the explant, and has the advantages of rapidness, simplicity, convenience, high efficiency, economy and the like.
Disclosure of Invention
The invention summarizes a method for quickly, simply and efficiently obtaining the tissue culture seedling of the African reed by optimizing the experimental process and the formula of the culture medium for multiple times. The method utilizes the differentiation rooting one-step seedling culture medium to achieve the purposes of saving economic cost and quickly growing seedlings. The method is safe, rapid and simple to operate, and the seedling process can be completed in more than 30 days, so that the defects of long time, low induction rate, easy browning pollution and the like of the common technology are overcome, and the requirements of biological experiments are met.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for rapidly obtaining tissue culture seedlings of African reeds is characterized in that stems of reeds are used for culturing, and adult African reeds are rapidly propagated, and the method comprises the following steps:
in sunny days, the outer leaves of each section of an outdoor reed branch are peeled off to expose buds, the buds are wiped by 75% ethanol, the stem sections are cut into small sections, each section is 2-3cm long, the small sections are placed in a bottle, and then the small sections are placed on a clean bench. Soaking in 75% ethanol for 30s, rinsing with sterile water for 3 times, and adding 0.1% HgCl2Sterilizing the water solution for 10-15min, and washing with sterile water for 4-5 times;
inoculating the sterilized reed stem segments to a culture medium for inducing differentiation and rooting; the culture medium is 1/2MS culture medium, IBA1.5mg/L, NAA1.5mg/L, 3% white sugar and 4g/L plant gel; the temperature of the culture room is 25 soil and 2 ℃, the light intensity is 2000lx, and the illumination is 12h every day, so that the tissue culture seedling which can be transplanted is obtained.
The pH of the medium was 5.8, and the medium was sterilized in an autoclave at 121 ℃ for 20 min.
The specific operation is that when the root of the test-tube seedling is about 2cm long, the cap of the bottle is opened and peat soil is added to harden the seedling for one week when the sprout is more than 3cm long, and then the seedling can be transplanted and raised. If the plant is used as mother bottle, one bud can be cut and one section can be used as proliferation when the plant grows to more than 5-6 sections.
The invention has the advantages and beneficial effects that:
(1) the induced differentiation rooting culture medium is used, and the conversion of different culture media in multiple steps and different processes is omitted.
(2) The use of the culture medium is simple and can reduce the cost of the basic culture medium by half.
(3) Can achieve the effect of proliferation without cytokinin.
The invention has the beneficial effects that: the method is simple and quick to operate, and the method for quickly, simply and efficiently obtaining the tissue culture seedlings of the African reed is summarized, the aim of saving the economic cost and quickly growing the seedlings is achieved by utilizing the differentiation rooting one-step seedling culture medium, the seedling growing process can be completed within more than 30 days, the defects of long time, low induction rate, easy pollution and the like in the prior art are overcome, and the needs of biological experiments on the African reed can be better met.
Drawings
FIG. 1 shows a budded stem section of one week or so of cultivation of African reed;
FIG. 2 shows a bottle of Versa Veitchii to be transplanted obtained by the method;
FIG. 3 shows a large bottle of African reed seedlings for proliferation obtained by the method;
FIG. 4 shows the status of the seedlings of the African reed bottles transplanted into peat soil;
FIG. 5 cultivation of 8 months seedlings.
Detailed Description
The invention is further described with reference to the following figures and examples, which are provided for the purpose of illustrating the invention in a clear manner and are not to be construed as limiting the invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And it is expressly intended that all such modifications or alterations to the methods, procedures, or conditions of the present invention, as expressed herein, are contemplated as falling within the scope of the present invention.
Example (b): the tissue culture seedling of the African reed is obtained by the method.
In sunny days, leaf skins are wrapped outside clean buds of reed branches collected outdoors, buds are exposed, the twigs are wiped by 75% ethanol, the branches are cut into small sections with bud lengths of 2-3cm, the small sections are placed in bottles, and then the bottles are placed on an ultraclean workbench. Soaking in 75% ethanol for 30s, rinsing with sterile water for 3 times, sterilizing with 0.1% HgCl2 water solution for 10-15min, and washing with sterile water for 4-5 times;
inoculating the sterilized reed stem segments to a culture medium for inducing differentiation and rooting; the induction culture medium is 1/2MS culture medium, IBA1.5mg/L, NAA1.5mg/L, 3% white sugar and 4g/L plant gel; culturing at 25-2 deg.C under light intensity of 2000lx for 12-16h daily to obtain tissue culture seedling (FIG. 1).
When the root of the test-tube plantlet is about 2cm long, and the sprout is drawn to be more than 3cm long, the bottle cap is opened, peat soil is added for hardening the sprout for one week (figure 2), and then the seedling can be transplanted and raised (figure 3). If the plant is used as mother bottle, one bud can be cut and one section can be used as proliferation when the plant grows to more than 5-6 sections.
The stem with the bud is cultured for 5 days in an induction way, the stem with the bud is inoculated into the culture medium, about one week, the stem with the bud shoots, the stem grows and roots, and a good growth situation is formed (figure 1). After thirty days of culture, the root system grows vigorously, and the seedlings grow to 7-10cm and grow well (figure 2). When the seedlings grow to be higher than 10cm, the seedlings do not move outwards; allowing it to grow upwards continuously, cutting and propagating (one section and one bud) (figure 3) bottle seedling and transplanting when the extraction length is up to 15cm in an effective time, adopting peat soil as matrix, and the survival rate can reach more than 98% (figure 4). After transplanting, the seedlings can be cultivated for 8 months, and can be planted in the ground for colonization (figure 5).
The variety is propagated in different modes under the condition of tissue culture compared with the conventional tissue culture method of the reed. The reed stalks of the variety are hard and take the node number as the multiplication coefficient; and the tissue culture propagation mode of other reed varieties adopts bud multiplication, so that the multiplication coefficient is lower. Meanwhile, frequent culture medium replacement is omitted in the propagation step, differentiation and rooting can be achieved in the same culture medium, browning can be well inhibited, adult tissue culture seedlings can be grown successively after 30 days, the rooting rate can reach 100%, and the transplanting survival rate can reach more than 98%.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (5)
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011330740.3A CN112293260B (en) | 2020-11-24 | 2020-11-24 | Method for obtaining tissue culture seedlings of reed in Africa |
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| CN202011330740.3A CN112293260B (en) | 2020-11-24 | 2020-11-24 | Method for obtaining tissue culture seedlings of reed in Africa |
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| CN112293260B CN112293260B (en) | 2022-08-23 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1788547A (en) * | 2004-12-13 | 2006-06-21 | 中国科学院植物研究所 | Asexual generation method for salt tolerant reed |
| CN101491213A (en) * | 2009-03-05 | 2009-07-29 | 南京大学 | Freshwater reeds non-callus culture method |
| CN103314837A (en) * | 2012-03-20 | 2013-09-25 | 东北师范大学 | Method for cultivating reed stem to sprout in timely, efficient and quick manner |
| CN107660463A (en) * | 2016-07-27 | 2018-02-06 | 王晓翔 | A kind of giantreed tissue culture tiller fast breeding culture medium |
-
2020
- 2020-11-24 CN CN202011330740.3A patent/CN112293260B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1788547A (en) * | 2004-12-13 | 2006-06-21 | 中国科学院植物研究所 | Asexual generation method for salt tolerant reed |
| CN101491213A (en) * | 2009-03-05 | 2009-07-29 | 南京大学 | Freshwater reeds non-callus culture method |
| CN103314837A (en) * | 2012-03-20 | 2013-09-25 | 东北师范大学 | Method for cultivating reed stem to sprout in timely, efficient and quick manner |
| CN107660463A (en) * | 2016-07-27 | 2018-02-06 | 王晓翔 | A kind of giantreed tissue culture tiller fast breeding culture medium |
Non-Patent Citations (3)
| Title |
|---|
| 王国生: "芦苇高产栽培技术", 《园艺博览》 * |
| 王子岚等: "芦苇植株再生体系优化", 《河北林果研究》 * |
| 王洋: "茎秆扦插繁殖对芦苇出芽率的影响试验", 《农业科技与装备》 * |
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