CN104663442A - Method for platycerium wallichii tissue culture and rapid propagation - Google Patents
Method for platycerium wallichii tissue culture and rapid propagation Download PDFInfo
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- CN104663442A CN104663442A CN201510087218.XA CN201510087218A CN104663442A CN 104663442 A CN104663442 A CN 104663442A CN 201510087218 A CN201510087218 A CN 201510087218A CN 104663442 A CN104663442 A CN 104663442A
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Abstract
The invention discloses a method for platycerium wallichii tissue culture and rapid propagation and relates to a method for obtaining the platycerium wallichii test-tube plantlet in a short time through a plant tissue culture technology. The in-vitro tissue culture and rapid propagation method of the platycerium wallichii is built by taking the platycerium wallichii tender sporophyl as an explant, disinfecting the explant, and performing GGB induction, proliferating, differentiating and rooting. The growth cycle is shortened, the shortage of the platycerium wallichii commercialized produced seedlings can be relieved and a foundation is laid for innovating the germplasm resources of the pteridophyte in future.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in Plant Biotechnology, specifically, relate to a kind of method of Platycerium bifurcatum tissue-culturing rapid propagation.
Background technology
Pteridophyte originates from the Silurian Period in the Paleozoic Era and Devonian psilophyton, and about there is kind more than 12000 in the whole world.The plant corpus and sporophyte that have two can live on one's own life in its history of life and gametophyte, sporophyte generation and gametophytic generation alternately occur in pteridophyte history of life, and sporophyte generation occupies greater advantage in the history of life of pteridophyte.Pteridophyte is very responsive to water, heat condition, and majority is shade plant and epiphyte, and most of monoid needs forest cover to provide the environment of existence for it.China due to forest ecosystem type various, thus pteridophyte monoid is complete, abundant species.Pteridophyte has higher medical value, the plant chemical ingredient of the various structures types such as the flavonoids that writes letter, terpene, phenolic acid, have antitumor, anti-inflammatory, AntiHIV1 RT activity, anti-oxidant, antibacterial, protect the liver, promote the pharmacologically actives such as knitting.In addition, fern has become the important component part in ornamental plants, considerable status is occupied in the greening, the gardens gardening cause such as flower arrangement and ornameutal handiwork of the environment such as indoor gardening, park, garden, the commodity production of Ornamental Fern and breeding and cultivation cause are also quite flourishing, contain very large potentiality to be exploited.
Platycerium bifurcatum (
platycerium wallichii) be Rhynioceae Rhynia plant, be the epiphyte in tropical monsoon forest, its leaf uniqueness, likeness in form CORNU CERVI, attitude is graceful, and other tool torrid zone temperament and interest is the first-selected plant of indoor stereo greening.Because Platycerium bifurcatum viewing period is long and the advantage such as convenient management, potted plant ornamental foliage plant is it can be used as to apply at present in the world general, and China has just just entered the exploitation stage, be in the research of Platycerium bifurcatum the starting stage, the research also inadequate system perfecting of Zu Pei aspect, laboratory.Research shows, Platycerium bifurcatum from spore sowing start, until the time growing sporophyte be roughly: spore germination need 20 days; Grow prothallium by spore and need more than 50 days; Juvenile sporophyte is formed and then needs half a year more than, and the present invention sets up the vitro Regeneration System of Platycerium bifurcatum, the approach relatively started with spore substantially reduces growth cycle, opens approach for Platycerium bifurcatum moves towards merchandized handling, significant to the tissue cultures of Ornamental Fern.
Summary of the invention
The object of the present invention is to provide out a kind of method of Platycerium bifurcatum tissue-culturing rapid propagation, the present invention with the delicate sporophyll of Platycerium bifurcatum for explant, processes such as sterilizing through explant, GGB induces, breed, break up, take root establishes the in vitro tissue culture and rapid propagation method of Platycerium bifurcatum, thus achieves object of the present invention.
The method of a kind of Platycerium bifurcatum tissue-culturing rapid propagation of the present invention, comprises the following steps:
1, a method for Platycerium bifurcatum tissue-culturing rapid propagation, is characterized in that comprising the following steps:
(1) explant sterilization: the tender leaf that the sporophyll choosing healthy and strong Platycerium bifurcatum newly grows, 10 ~ 30min is soaked with 0.1% ~ 0.3% liquor potassic permanganate, 70% ~ 80% alcohol immersion 10 ~ 20s is used after aseptic water washing 3 ~ 5 times, by sterile water wash 3 ~ 5 times afterwards with 0.1% mercuric chloride solution sterilization 1 ~ 5min, more for subsequent use after using sterile water wash 3 ~ 5 times.
(2) Fiber differentiation: carry out slightly scratching to process and face up to be inoculated in inducing culture carrying out GGB induction on sporophyll surface after step (1) process with scalpel.Inoculation is placed on illumination every day 12 ~ 16 hours, and intensity of illumination is 1500 ~ 2500lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C until form GGB.
(3) Multiplying culture: the GGB of the acquisition of step (2) is inoculated on proliferated culture medium and carries out Multiplying culture.Inoculation is placed on illumination every day 12 ~ 16 hours, and intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, within every two weeks, changes a subculture.
(4) GGB differentiation is cultivated: be transferred in differential medium by the GGB that step (1) or (2) obtain and carry out GGB differentiation cultivation.Inoculation is placed on illumination every day 12 ~ 16 hours, and intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C.Within after inoculation every two weeks, change a subculture.
(5) culture of rootage: stretch when cultivating Platycerium bifurcatum sporophyll through step (4), and grow deer horn by initial strip and length is about 1.5 ~ 2.5cm time be transferred in culture of rootage and carry out root induction.
Inducing culture described in above-mentioned steps (2) is: MS+1.0 ~ 4.0mg/L6-BA+1.0 ~ 5.0mg/L NAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
Proliferated culture medium described in above-mentioned steps (3) is: MS+1.0 ~ 3.0mg/LKT+0.1 ~ 1mg/L NAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
Differential medium described in above-mentioned steps (4) is: MS+0.5 ~ 1.5mg/L6-BA+0.1 ~ 0.5mg/L IBA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
Root media described in above-mentioned steps (5) is: MS+0.1 ~ 1.0mg/L NAA+0.1 ~ 0.5mg/L IBA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
Compared with prior art advantage of the present invention is: the method being obtained Platycerium bifurcatum test-tube plantlet by plant tissue culture technique at short notice.With the delicate sporophyll of Platycerium bifurcatum for explant, processes such as sterilizing through explant, GGB induces, breed, break up, take root establishes the in vitro tissue culture and rapid propagation method of Platycerium bifurcatum, to reach shortening growth cycle, alleviate the present situation that Platycerium bifurcatum commercially produces seedling shortage, and be that the germ plasm resource innovating pteridophyte from now on lays the foundation.
Embodiment
Following examples further illustrate of the present invention, is not limitation of the present invention.
Embodiment 1:
(1) explant sterilization: the tender leaf that the sporophyll choosing healthy and strong Platycerium bifurcatum newly grows, 10min is soaked with 0.1% liquor potassic permanganate, 75% alcohol immersion 10s is used after aseptic water washing 4 times, by sterile water wash 3 times afterwards with 0.1% mercuric chloride solution sterilization 3min, more for subsequent use after using sterile water wash 5 times.
(2) Fiber differentiation: carry out slightly scratching to process and face up to be inoculated in inducing culture carrying out GGB induction on sporophyll surface after step (1) process with scalpel.Inoculation is placed on illumination every day 14 hours, intensity of illumination is 1500lx, cultivation temperature is cultivate under the condition of 25 DEG C after 20 days most of explant grows tan hair, then root wool base expands and grows peak green GGB, when GGB grows to a certain size, the tender sporophyll of children starts albefaction or yellow, and is covered by the GGB newly grown.Described inducing culture is: MS+2.0mg/L6-BA+1.0mg/L NAA+3.5% sucrose+0.5% agar, pH value is 5.6.
(3) Multiplying culture: the GGB of the acquisition of step (2) is inoculated on proliferated culture medium and carries out Multiplying culture.Inoculation is placed on illumination every day 15 hours, and intensity of illumination is 2500lx, and cultivation temperature is cultivate GGB after 30 days under the condition of 25 DEG C obviously to increase, and average diameter can reach about 0.8cm, within every two weeks, changes a subculture.Described proliferated culture medium is: MS+1.0mg/LKT+0.1mg/L NAA+3.5% sucrose+0.35% agar, pH value is 5.6.
(4) GGB differentiation is cultivated: be transferred in differential medium by the GGB that step (1) or (2) obtain and carry out GGB differentiation cultivation.Inoculation is placed on illumination every day 14 hours, and intensity of illumination is 2000lx, and cultivation temperature is cultivate under the condition of 25 DEG C can differentiate indefinite bud in 30 days, and differentiation rate reaches 93%.Within after inoculation every two weeks, change a subculture.Described differential medium is: MS+0.5mg/L6-BA+0.1mg/L IBA+2.0% sucrose+0.35% agar, pH value is 5.6.
(5) culture of rootage: stretch when cultivating Platycerium bifurcatum sporophyll through step (4), and grow deer horn by initial strip and length is about 1.5 ~ 2.5cm time be transferred in culture of rootage and carry out root induction, cultivate and can reach rooting rate more than 96% after 20 days.Described root media is: MS+0.1mg/L NAA+0.1mg/L IBA+3.5% sucrose+0.35% agar, pH value is 5.6.
Embodiment 2:
(1) explant sterilization: the tender leaf that the sporophyll choosing healthy and strong Platycerium bifurcatum newly grows, 12min is soaked with 0.2% liquor potassic permanganate, 78% alcohol immersion 11s is used after aseptic water washing 5 times, by sterile water wash 4 times afterwards with 0.1% mercuric chloride solution sterilization 5min, more for subsequent use after using sterile water wash 5 times.
(2) Fiber differentiation: carry out slightly scratching to process and face up to be inoculated in inducing culture carrying out GGB induction on sporophyll surface after step (1) process with scalpel.Inoculation is placed on illumination every day 15 hours, intensity of illumination is 2000lx, cultivation temperature is cultivate under the condition of 26 DEG C after 23 days most of explant grows tan hair, then root wool base expands and grows peak green GGB, when GGB grows to a certain size, the tender sporophyll of children starts albefaction or yellow, and is covered by the GGB newly grown.Described inducing culture is: MS+2.5mg/L6-BA+1.2mg/L NAA+3.8% sucrose+0.4% agar, pH value is 5.7.
(3) Multiplying culture: the GGB of the acquisition of step (2) is inoculated on proliferated culture medium and carries out Multiplying culture.Inoculation is placed on illumination every day 16 hours, and intensity of illumination is 2500lx, and cultivation temperature is cultivate GGB after 30 days under the condition of 26 DEG C obviously to increase, and average diameter can reach about 0.8cm, within every two weeks, changes a subculture.Described proliferated culture medium is: MS+1.5mg/LKT+0.1mg/L NAA+3.5% sucrose+0.4% agar, pH value is 5.7.
(4) GGB differentiation is cultivated: be transferred in differential medium by the GGB that step (1) or (2) obtain and carry out GGB differentiation cultivation.Inoculation is placed on illumination every day 15 hours, and intensity of illumination is 2500lx, and cultivation temperature is cultivate under the condition of 26 DEG C can differentiate indefinite bud in 34 days, and differentiation rate reaches 96%.Within after inoculation every two weeks, change a subculture.Described differential medium is: MS+0.9mg/L6-BA+0.2mg/L IBA+2.5% sucrose+0.35% agar, pH value is 5.7.
(5) culture of rootage: stretch when cultivating Platycerium bifurcatum sporophyll through step (4), and grow deer horn by initial strip and length is about 1.5 ~ 2.5cm time be transferred in culture of rootage and carry out root induction, cultivate and can reach rooting rate more than 94% after 23 days.Described root media is: MS+0.5mg/L NAA+0.12mg/L IBA+3.5% sucrose+0.35% agar, pH value is 5.6.
Claims (5)
1. a method for Platycerium bifurcatum tissue-culturing rapid propagation, is characterized in that comprising the following steps:
(1) explant sterilization: the tender leaf that the sporophyll choosing healthy and strong Platycerium bifurcatum newly grows; 10 ~ 30min is soaked with 0.1% ~ 0.3% liquor potassic permanganate; 70% ~ 80% alcohol immersion 10 ~ 20s is used after aseptic water washing 3 ~ 5 times; by sterile water wash 3 ~ 5 times afterwards with 0.1% mercuric chloride solution sterilization 1 ~ 5min, more for subsequent use after using sterile water wash 3 ~ 5 times;
(2) Fiber differentiation: carry out slightly scratching to process and face up to be inoculated in inducing culture carrying out GGB induction on sporophyll surface after step (1) process with scalpel; inoculation is placed on illumination every day 12 ~ 16 hours; intensity of illumination is 1500 ~ 2500lx, and cultivation temperature is cultivate until form GGB under the condition of 25 ~ 28 DEG C;
(3) Multiplying culture: the GGB of the acquisition of step (2) is inoculated on proliferated culture medium and carries out Multiplying culture; inoculation is placed on illumination every day 12 ~ 16 hours; intensity of illumination is 2000 ~ 2500lx, and cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, within every two weeks, changes a subculture;
(4) GGB differentiation is cultivated: be transferred in differential medium by the GGB that step (1) or (2) obtain and carry out GGB differentiation cultivation; inoculation is placed on illumination every day 12 ~ 16 hours; intensity of illumination is 2000 ~ 2500lx; cultivation temperature is cultivate under the condition of 25 ~ 28 DEG C, within after inoculation every two weeks, changes a subculture;
(5) culture of rootage: stretch when cultivating Platycerium bifurcatum sporophyll through step (4), and grow deer horn by initial strip and length is about 1.5 ~ 2.5cm time be transferred in culture of rootage and carry out root induction.
2. the method for a kind of Platycerium bifurcatum tissue-culturing rapid propagation according to claim 1, it is characterized in that the inducing culture described in step (2) is: MS+1.0 ~ 4.0mg/L6-BA+1.0 ~ 5.0mg/L NAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
3. the method for a kind of Platycerium bifurcatum tissue-culturing rapid propagation according to claim 1, it is characterized in that the proliferated culture medium described in step (3) is: MS+1.0 ~ 3.0mg/LKT+0.1 ~ 1mg/L NAA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
4. the method for a kind of Platycerium bifurcatum tissue-culturing rapid propagation according to claim 1, it is characterized in that the differential medium described in step (4) is: MS+0.5 ~ 1.5mg/L6-BA+0.1 ~ 0.5mg/L IBA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
5. the method for a kind of Platycerium bifurcatum tissue-culturing rapid propagation according to claim 1, it is characterized in that the root media described in step (5) is: MS+0.1 ~ 1.0mg/L NAA+0.1 ~ 0.5mg/L IBA+2.0% ~ 3.5% sucrose+0.35% ~ 0.5% agar, pH value is 5.4 ~ 5.8.
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| CN110100740A (en) * | 2019-06-26 | 2019-08-09 | 四川七彩林科股份有限公司 | A kind of tissue culture and rapid propagation method of two discriminations Platycerium bifurcatum |
| CN110663548A (en) * | 2019-09-24 | 2020-01-10 | 福建省农业科学院作物研究所 | Aseptic germination seedling raising method for platycerium wallichii spores |
| CN110786243A (en) * | 2019-12-03 | 2020-02-14 | 厦门市园林植物园 | Propagation method for tissue culture of penholder tree |
| CN112889671A (en) * | 2021-03-08 | 2021-06-04 | 西南林业大学 | Tissue culture and rapid propagation method of platycerium |
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Cited By (8)
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| CN106718878A (en) * | 2016-11-22 | 2017-05-31 | 云南省农业科学院花卉研究所 | A kind of fan fern quick breeding by group culture method with young sporangiorus as explant |
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| CN110663548A (en) * | 2019-09-24 | 2020-01-10 | 福建省农业科学院作物研究所 | Aseptic germination seedling raising method for platycerium wallichii spores |
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