CN108935106B - Tissue culture propagation and in-vitro preservation method for extremely endangered plant Rhododendron Guizhou - Google Patents
Tissue culture propagation and in-vitro preservation method for extremely endangered plant Rhododendron Guizhou Download PDFInfo
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- CN108935106B CN108935106B CN201811007683.8A CN201811007683A CN108935106B CN 108935106 B CN108935106 B CN 108935106B CN 201811007683 A CN201811007683 A CN 201811007683A CN 108935106 B CN108935106 B CN 108935106B
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- 241000208422 Rhododendron Species 0.000 title claims abstract description 29
- 238000004321 preservation Methods 0.000 title claims abstract description 28
- 238000000338 in vitro Methods 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 14
- 241000196324 Embryophyta Species 0.000 claims abstract description 8
- 230000006698 induction Effects 0.000 claims abstract description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 43
- 238000005286 illumination Methods 0.000 claims description 13
- 229920001817 Agar Polymers 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims 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 description 12
- 239000008272 agar Substances 0.000 claims description 12
- 239000005720 sucrose Substances 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 230000004069 differentiation Effects 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 241000248414 Rhododendron micranthum Species 0.000 claims 1
- 238000010521 absorption reaction Methods 0.000 claims 1
- 241000894007 species Species 0.000 abstract description 9
- 241000208421 Ericaceae Species 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 238000005728 strengthening Methods 0.000 abstract 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 description 8
- 238000012216 screening Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 3
- 230000000249 desinfective effect Effects 0.000 description 2
- LGGQQYXFFSOIJY-UHFFFAOYSA-N 2-(3-methylbut-2-enyl)-7h-purin-6-amine Chemical compound CC(C)=CCC1=NC(N)=C2NC=NC2=N1 LGGQQYXFFSOIJY-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012883 rooting culture medium Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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
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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)
Abstract
The invention discloses a tissue culture propagation and in vitro preservation method of extremely endangered plant Guizhou rhododendron, which is suitable for tissue culture rapid propagation and in vitro preservation of the Rhododendron guizhou rhododendron of Ericaceae and belongs to the technical field of plant biology. The method takes the tender stem section of the newly-extracted flowering tips as the explant, and effectively solves the problems of small plant quantity and endangered adjacency caused by narrow distribution area and difficult introduction and domestication of the Rhododendron macranthum in Guizhou through a series of steps of explant disinfection, cluster bud induction, seedling strengthening, in-vitro preservation and rooting, so as to achieve the purpose of species protection. The tissue culture rapid propagation method provided by the invention has the advantages that the propagation coefficient is 8-10, the rooting rate is 95%, and the transplanting survival rate is 95% -98% within 1 month, the propagation coefficient of the Guizhou rhododendron is greatly improved, the in vitro preservation can reach more than 240 days, and the tissue culture rapid propagation method provides technical support for introduction and domestication, in vitro preservation, seedling propagation and regression planting of the species.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a tissue culture propagation and in-vitro preservation method of extremely endangered plants of rhododendron guizhou.
Background
Rhododendron anthopogonoides (Rhododendron)magniflorum) Is a species of the Rhododendron brocade subgroup of the Rhododendron genus of the Ericaceae family, which was published in 1985 and is a Rhododendron having the largest flower among the Rhododendron genera. Extremely endangered species are listed in The newly published Red journal of Rhododendron, The Red List of Rhododendron. The single flower crown is about 10cm in diameter and has high ornamental value when distributed in the dragon city, the Guizhou province. In the latest investigations, this species currently found only one population and only 3 strains. Because the population is rare and the natural renewal is poor, effective tissue culture propagation and in-vitro preservation are urgently needed, and the species trend is prevented from being extinct. At present, tissue culture propagation and in vitro preservation methods aiming at the species are not reported.
Disclosure of Invention
Aiming at the phenomenon that the Guizhou rhododendron has rare population and wild plants are endangered, the invention aims to provide a tissue culture propagation and in-vitro preservation method for the Guizhou rhododendron, which is used for preserving germplasm resources and protecting endangered species.
1. A tissue culture propagation and in vitro preservation method of Guizhou rhododendron is characterized by comprising the following steps:
1) selecting an explant: taking a new tender stem section of the Guizhou rhododendron, and washing the stem section for 12 hours by using tap water for later use;
2) and (3) explant sterilization: sterilizing the prepared explant on a sterile super-clean bench with 75% alcohol for 30s, washing with sterile water for 3-5 times, sterilizing with 0.1% mercuric chloride for 8-9min, and washing with sterile water for 3-5 times;
3) inducing cluster buds: the cluster bud induction culture medium is improved WPM +2.5mg/L2-iP (2-isopentenyl adenine) +0.8mg/L IBA, 28g/L sucrose, 7g/L agar, and pH5.6. The WPM culture medium is improved to 700mg/L ammonium nitrate, and the dosage of other elements is kept unchanged. The stem section obtained after the explant sterilization process is dried on a super clean bench by using sterile filter paper, the stem section is cut by using a sterile knife, each section is provided with a latent bud and is inoculated into a cluster bud induction culture medium, illumination culture is carried out for more than 30 days after dark culture is carried out for 5 days, the culture lasts for 35-50 days, the illumination time is 12h/12h, and the illumination intensity is 1500-plus 2000 LUX;
4) strong seedling culture: the strong seedling culture medium is improved WPM +0.5 mg/L2-iP +0.1mg/L IBA, 28g/L sucrose, 7g/L agar and 5.6 pHs; inoculating the induced new buds into a strong seedling culture medium, and keeping the bottle seedlings in light for 12h/12h with the illumination intensity of 1500-;
5) in vitro preservation: the isolated preservation culture medium is improved WPM +0.5mg/L IBA +6mg/L CCC, the pH value is 5.6, the sucrose content is 28g/L, the agar content is 6-7g/L, the temperature is 13-18 ℃, the light intensity is 1500-1600 LUX, the light dark time is 10h/14h, the culture medium is 65ml of culture medium filled in a culture bottle of 260 ml; the growth and differentiation recovery culture medium is improved WPM +1mg/L6-BA +0.05mg/L IAA, the light dark time is 12h/12h, and the illumination intensity is 1500-;
6) rooting culture: transferring the bud seedlings growing to the height of 1.5-2 cm into a culture medium with an improved WPM +1mg/L IBA culture medium, adding 28g/L of sucrose, 7g/L of agar and pH5.6; the light intensity is 2000LUX-2500LUX, and the light dark time is 12h/12 h;
except that 65ml of culture medium needs to be filled in a 260ml culture bottle used for in vitro preservation of the culture medium, 45ml of culture medium needs to be filled in a 260ml culture bottle used for all the other culture media; the culture temperature is 13-18 deg.C except for in vitro preservation, and the rest is 20-23 deg.C.
Compared with the prior art, the invention has the beneficial effects that:
1. innovatively and realizes the tissue culture propagation and in vitro preservation method of extremely endangered plants of rhododendron giganteum;
2. through tissue culture propagation, the propagation coefficient of the Guizhou rhododendron within half a year is 60-90 times, the propagation coefficient of the Guizhou rhododendron is greatly improved, endangered species are protected, and a large number of seedlings are provided for regression planting. In addition, the in vitro preservation culture medium can be preserved for over 240 days without transferring, thereby greatly prolonging the transferring and preserving time.
The specific implementation case is as follows:
in order to better illustrate the essence of the invention, the invention is further illustrated below by means of examples of the invention, without restricting the content of the invention thereto. According to the technical scheme and the description of the embodiment of the invention, the technical scheme of the invention can be modified and improved by persons skilled in the art on the basis of the technical scheme and the embodiment of the invention. Therefore, it is intended that all such modifications and improvements be included within the scope of the invention as claimed and claimed without departing from the essential scope thereof.
Example 1 tissue culture and in vitro preservation of rhododendron giganteum:
step (1): collecting explants and sterilizing: collecting new stem section which just grows out after blooming from mother plant of Rhododendron macranthum in Guizhou with 3-5 latent buds, washing explant with tap water for 12h, disinfecting with 75% alcohol for 30s on a super clean bench, washing with sterile water for 3-5 times, disinfecting with 0.1% mercuric chloride for 8-9min, and washing with sterile water for 3-5 times;
step (2): and (3) inducing and culturing cluster buds: cutting the sterilized explant into small segments on a clean bench, wherein each segment is provided with an axillary bud, obliquely inserting an induction culture medium improved WPM +2.5mg/L2-iP +0.8mg/L IBA, 28g/L sucrose, 7g/L agar and pH5.6, carrying out illumination culture for 30 days after 5 days of dark culture, and carrying out culture for 35-50 days, wherein the light-dark time is 12h/12h, and the illumination intensity is 1500 LUX-2000 LUX;
and (3): when the cluster buds are differentiated and grow to 1-2cm, the cluster buds are transferred to a strong seedling culture medium improved WPM +0.5 mg/L2-iP +0.1mg/L IBA, 28g/L of sucrose, 7g/L of agar and pH 5.6; the bottle seedling is in the dark for 12h/12h, and the illumination intensity is 1500 LUX-2000 LUX;
and (4): transferring a part of the clumpy sprouts to an in vitro preservation medium: the isolated preservation culture medium is improved WPM +0.5mg/L IBA +6mg/L CCC, the pH value is 5.6, the sucrose content is 28g/L, the agar content is 6-7g/L, the temperature is 13-18 ℃, the light intensity is 1500-1600 LUX, the light dark time is 10h/14h, and 65ml of the culture medium is filled in each bottle; when growth needs to be restored, it is transferred to a growth restoring medium: improving WPM +1mg/L6-BA +0.05mg/L IAA, light dark time is 12h/12h, illumination intensity is 1500-;
and (5): when the seedlings in the strong seedling culture medium and the seedling in the growth recovery culture medium grow to the height of 1.5 cm-2 cm in the bottle, the seedlings are transferred to a rooting culture medium improved WPM +1mg/L IBA for rooting, 28g/L of sucrose, 7g/L of agar, 5.6 of pH, 12h/12h of light-dark time, 2000LUX-2500LUX of light intensity, 20-23 ℃ of temperature and conventional transplanting is carried out after rooting.
Example 2 screening experiment of culture medium for tissue culture propagation of rhododendron guizhou:
1) screening experiment of basic culture medium. Selecting culture media Read, WPM, 1/4MS and improved WPM commonly used by rhododendron to perform comparison experiments, performing hormone-free stem survival experiments, inoculating 10 bottles in each group of experiments, inoculating 1 segment of the sterilized explant stem in each bottle, and comparing:
the results show that: the seed grows well on the culture medium of the improved WPM, the growth vigor is vigorous, and the phenomena of death and no germination do not occur, so that the tissue culture step of selecting the basic culture medium as the improved WPM and the Rhododendron macranthum is the same as that of example 1.
2) In vitro preservation Medium screening experiments. The design temperature is two gradients: the filling amount of the culture medium is designed to be two gradients of 45ml and 65ml at the low temperature of 13-18 ℃ and the normal temperature of 20-23 ℃, 10 bottles are inoculated in each group of experiment, 3 clumps of clustered buds with relatively consistent sizes are inoculated in each bottle, and orthogonal experiment screening is carried out:
the results show that: the seed has good growth state in vitro under the low temperature condition of 13-18 ℃, and the culture medium needs to have a sufficient amount of 65ml to ensure the prolongation of the subculture transfer period.
Claims (1)
1. A tissue culture propagation and in-vitro preservation method of extremely endangered plants of Rhododendron micranthum is characterized in that: 1) selecting an explant: taking a new tender stem section of the Guizhou rhododendron, and washing the stem section for 12 hours by using tap water for later use; 2) and (3) explant sterilization: sterilizing the prepared explant on a sterile super-clean bench with 75% alcohol for 30s, washing with sterile water for 3-5 times, sterilizing with 0.1% mercuric chloride for 8-9min, and washing with sterile water for 3-5 times; 3) inducing cluster buds: the cluster bud induction culture medium is improved WPM +2.5mg/L2-iP +0.8mg/L IBA, 28g/L sucrose, 7g/L agar and pH5.6; improving the WPM culture medium to increase ammonium nitrate to 700mg/L, keeping the use amount of other elements unchanged, carrying out surface moisture absorption on stem sections obtained after the explant sterilization process on a super clean bench by using sterile filter paper, cutting the stem sections by using a sterile knife, inoculating the stem sections into a cluster bud induction culture medium, carrying out illumination culture for more than 30 days after dark culture for 5 days, culturing for 35-50 days, wherein the light-dark time is 12h/12h, and the illumination intensity is 1500-increased 2000 LUX; 4) strong seedling culture: the strong seedling culture medium is improved WPM +0.5 mg/L2-iP +0.1mg/L IBA, 28g/L sucrose, 7g/L agar and 5.6 pHs; inoculating the induced new buds into a strong seedling culture medium, and keeping the bottle seedlings in light for 12h/12h with the illumination intensity of 1500-; 5) in vitro preservation: the isolated preservation culture medium is improved WPM +0.5mg/L IBA +6mg/L CCC, the pH value is 5.6, the sucrose content is 28g/L, the agar content is 6-7g/L, the temperature is 13-18 ℃, the light intensity is 1500-1600 LUX, and the light dark time is 10h/14 h; the growth and differentiation recovery culture medium is improved WPM +1mg/L6-BA +0.05mg/L IAA, the light dark time is 12h/12h, and the illumination intensity is 1500-; 6) rooting culture: transferring the bud seedlings growing to the height of 1.5-2 cm into a culture medium with an improved WPM +1mg/L IBA culture medium, adding 28g/L of sucrose, 7g/L of agar and pH5.6; the light intensity is 2000LUX-2500LUX, and the light dark time is 12h/12 h;
except for the in vitro preservation culture medium, each culture medium is filled with 45ml of culture medium by using a 260ml culture bottle; the in vitro preservation culture medium is prepared by filling 65ml of culture medium into a 260ml culture bottle, and the culture temperature is 20-23 ℃ except for the in vitro preservation of 13-18 ℃.
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