CN101133704A - Sterilized spore germination of platycerium wallichii and test tube seedling cultivating method - Google Patents
Sterilized spore germination of platycerium wallichii and test tube seedling cultivating method Download PDFInfo
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Abstract
The present invention relates to a method for propagation and cultivation of platycerium. It is characterized by that said invention adopts sterile spore germination technique and makes it be combined with test-tube seedling-cultivating technique to implement propagation and cultivation of platycerium. Said method includes the following several steps: collecting storing, health and mature spore, making sterile spore germination, gametophyte growth culture, insemination to form zygoite, sporophyte enrichment culture, rooting culture and transplantation, etc.
Description
Technical field
The present invention relates to the process for breeding and culturing of Platycerium bifurcatum, relate in particular to the grow seedlings method of breeding and culturing Platycerium bifurcatum of aseptic spore germination and test tube that adopts.
Background technology
Platycerium bifurcatum has another name called the angle fern of flocking together, bat fern, deer horn fern etc., and Polypodiaceae Rhynia (Platycerium) plant is to view and admire the most peculiar class of attitude in the fern, belongs to growing nonparasitically upon another plant property and views and admires fern.The leaf of Platycerium bifurcatum has two types, and trophophyll is less, and rounded, oval or fan-shaped, closely connected on attaching organism, the sporophyll shape is very unique, the likeness in form CORNU CERVI, and the blade face is close by lint, is peak green when newborn, transfers to light brownly when ripe, very arouses the affection of adults.Platycerium bifurcatum in America and Europe's park, the decoration in places such as botanical garden, shop, room, windowsill and arrange all the fashionly, be elaboration precious rare in the indoor ornamental foliage plant, the good material of indoor stereo greening particularly.If the Platycerium bifurcatum adhesion in ancient withered tree or decorate in hanging basin, can be interspersed study, bedroom, guest room and windowsill, graceful and be full of Aline if make suspension type or inlay hanging layout, more add nature scape interest.The Rhynia plant whole world has 18 kinds approximately, originates in the torrid zone, hylaeion hypotropicum in Africa, Asia, Oceania and South America, has high ornamental value mostly.It is a kind of that Platycerium bifurcatum (Platycerium wallichii) is only heavily split in China, belongs to Chinese Second Class Key Protected Plant.But at present, China has begun the introduction to other Platycerium bifurcatum kind, and has developed when introducing external each your flowers energetically, and be subjected to increasing people and like having huge market prospects.
The breeding of Platycerium bifurcatum can be adopted plant division and spore seed propagation, but division propagation speed is slow, and reproduction coefficient is low.At present, the report that it is carried out extensive tissue culture production and patent application is not seen in the home and abroad.
Summary of the invention
The object of the invention be to provide a kind of novelty, simple and rapid Platycerium bifurcatum propagation method.
The present invention adopts aseptic spore germination and test tube Cheng Miaoneng successfully to carry out the quick breeding of Platycerium bifurcatum, and medium component uniqueness, simple, has less investment at the propagation method novelty, and the characteristics that output is high have realized purpose of the present invention.
Platycerium bifurcatum process for breeding and culturing of the present invention may further comprise the steps:
(1) spore axenic germination: the ripe spore of Platycerium bifurcatum (Platycerium) blade back of gathering healthy and strong growth is with 70%～75% alcohol-pickled 10～30s, taking-up places the liquor natrii hypochloritis of 0.1%～0.2% mercuric chloride solution or 1%～2% 10～15min that sterilizes, after the aseptic washing, be inoculated on the spore germination medium; Cultivated for 2～3 weeks in that (25 ± 1) ℃ elder generation is dark, under 8～12h/ days then, 1500～2000 1x illumination, cultivate and form prothallium, contain active carbon 0.5～1g, coconut milk 50～150mL, gibberellin 0.1～0.5mg in every liter of the described medium, agar 5～7g, all the other are MS;
(2) gametophyte grown cultures: change the prothallium of step (1) over to the gametophyte growth medium, in (25 ± 1) ℃, 8～12h/ days, 1500～2000lx illumination cultivation down, the formation lobate gametophyte of growing thickly, contain caseinhydrolysate 0.1～0.3g, 6-benzyl purine 0.2～1.0mg, methyl 0.1～0.5mg in every liter of the described medium, agar 5～7g, all the other are MS;
(3) insemination forms zygote: the lobate gametophyte of growing thickly of step (2) is inoculated into earlier on the liquid gametophyte growth medium, in (25 ± 1) ℃, 8～12h/ days, 1500～2000lx illumination, 50～100r/min shaken cultivation 2～3 days, the thallus that liquid culture is crossed is inoculated into solid gametophyte growth medium again, under condition same as described above, continue to cultivate the sporophyte plant that grows 2 times of bodies, contain active carbon 0.5～1g in every liter of the described liquid gametophyte growth medium, coconut milk 50～150mL, gibberellin 0.1～0.5mg, all the other are MS, contain caseinhydrolysate 0.1～0.3g in every liter of the described solid gametophyte growth medium, 6-benzyl purine 0.2～1.0mg, methyl 0.1～0.5mg, agar 5～7g, all the other are MS;
(4) sporophyte enrichment culture: the sporophyte plantlet in the step (3) is inoculated into green spheroid induces on the proliferated culture medium, in (25 ± 1) ℃, 8～12h/ days, 1500～2000lx illumination cultivation are induced the green spheroid of formation at the plant base portion, and these green spheroids are bred on medium same as described above, and formation plantlet, contain coconut milk 100～150mL, peptone 1～2g, 6-benzyl purine 0.2～1.0mg in every liter of the described medium, agar 5～7g, all the other are MS;
(5) culture of rootage: the plantlet that step (4) is obtained is inoculated on the strong plantlets and rootage medium, in (25 ± 1) ℃, 8～12h/ days, 1500～2000lx illumination cultivation, form the healthy and strong plant of band root, contain active carbon 0.3～0.5mg, indolebutyric acid 0.1～0.5mg in every liter of the described medium, agar 5～7g, all the other are 1/2MS, and described 1/2MS reduces by half the macroelement among the MS, the medium that other components unchanged is formed;
(6) bottle outlet is transplanted: the seedling of taking root by taking out in the blake bottle, is cleaned the medium of root with it in 70%～80% natural daylight lower refining seedling that shades 5～7 days, cultivate in sphagna, 25～28 ℃, 80%～90% shades, humidity 60%～90% keeps ventilating, and plant recovers growth.
Platycerium bifurcatum in the step (1) can be the various Platycerium bifurcatums in this genus, for example heavily split Platycerium bifurcatum (Platyceriumwallichii), Platycerium bifurcatum (Platycerium bifurcatum), big Platycerium bifurcatum (Platycerium grande), Platycerium bifurcatum (Pla tycerium willinckii) comes into leaves, America Platycerium bifurcatum (Pla tycerium andinum), Queensland Platycerium bifurcatum (Platycerium hillii) etc., described aseptic washing 5～6 times, in 25 ± 1 ℃ of elder generations, 2～3 weeks of dark cultivation, 8～12h/ days then, under 1500～2000lx illumination, cultivated for 4～5 weeks again, form prothallium; Cultivated for 4～5 weeks in the step (2), form the lobate gametophyte of growing thickly in a large number; Be inoculated into earlier in the step (3) on the liquid gametophyte growth medium, cultivated 2～3 days, and formed zygote to promote sperm to move to archegonium, the thallus that liquid culture is crossed is inoculated on the solid gametophyte growth medium again, continue to cultivate for 3～4 weeks, grow the sporophyte plant of 2 times of bodies; Cultivate 6-8 week in the step (4), can induce at the plant base portion and form a large amount of green spheroids; Cultivated for 4～6 weeks in the step (5), form the healthy and strong plant of band root; Step (6) is cultivated the visible plant in 1 week back and is recovered growth, and survival rate 80%～90% can go up potted plant training after 30-40 days.
MS in the above-mentioned medium is international medium, described 1/2MS reduces by half the macroelement among the MS, other components unchanged and the medium that forms, its composition and compound method referring to document (Tan Wencheng, Dai Cegang chief editor. the ornamental plants tissue culture technique. Beijing: China Forest publishing house, 1991.).
The present invention adopts aseptic spore germination and test tube Cheng Miaoneng successfully to carry out the quick breeding of Platycerium bifurcatum; medium component uniqueness, simple; the propagation method novelty, has less investment; the characteristics that output is high; be to utilize plant test tube technology to carry out the high-new biotechnology of the large-scale production of rare plant seedling, test tube seedling transplanting survival rate reaches 80%～90%.
Embodiment
Following embodiment further specifies of the present invention, is not limitation of the present invention.
Embodiment 1:
Gather the ripe spore that heavily splits the healthy and strong plant sporophyll back of Platycerium bifurcatum (Platycerium wallichii Hook) of greenhouse culture, with 70% alcohol-pickled 10s, take out and to place the 0.2% mercuric chloride solution 10min that sterilizes, after the aseptic washing 5 times, be inoculated into the spore germination medium and (contain active carbon 0.5g in every liter, coconut milk 50mL, gibberellin 0.1mg, agar 7g, all the other are MS), in 25 ℃ of elder generations, 2 weeks of dark cultivation, 1x illumination 12 hours 1500 every day was cultivated for 5 weeks down then, formed prothallium.Change prothallium over to the gametophyte growth medium and (contain caseinhydrolysate 0.1g in every liter, 6-benzyl purine 0.2mg, methyl 0.1mg, agar 7g, all the other are MS), 25 ℃, 1500lx illumination 12 hours every days was cultivated for 5 weeks down, formed the lobate gametophyte of growing thickly in a large number.The thallus that to grow thickly is inoculated into liquid gametophyte growth medium and (contains active carbon 0.5g in every liter, coconut milk 50mL, gibberellin 0.1mg, all the other are MS), 25 ℃, 1x illumination 12 hours 1500 every day, 50r/min shaken cultivation 2 days forms zygote to promote sperm to move to archegonium; The thallus that liquid culture is crossed is inoculated into solid gametophyte growth medium and (contains caseinhydrolysate 0.2g in every liter, 6-benzyl purine 0.2mg, methyl 0.1mg, agar 7g, all the other are MS), under condition same as described above, continue to cultivate for 4 weeks, grow the sporophyte plant of 2 times of bodies.The sporophyte plantlet is inoculated into green spheroid induces proliferated culture medium (to contain coconut milk 100mL in every liter, peptone 1g, 6-benzyl purine 0.2mg, agar 7g, all the other are MS), 25 ℃, 1x illumination 12 hours 1500 every day cultivated for 6 weeks, can induce at the plant base portion and form a large amount of green spheroids, these green spheroids are bred on same medium, and form plantlet.Plantlet is inoculated into strong plantlets and rootage medium (contain active carbon 0.5mg in every liter, indolebutyric acid 0.1mg, agar 7g, all the other are 1/2MS), and 25 ℃, 1500lx illumination 12 hours every days cultivated for 6 weeks, formed the healthy and strong plant of band root.Took root seedling in the 70% natural daylight lower refining seedling that shades 7 days, it by taking out in the blake bottle, is cleaned the medium of root, cultivate in sphagna, 25 ℃, 90% shades, and humidity 90% keeps ventilating, and the visible plant in 1 week back recovers growth, survival rate 90%.Can go up potted plant training after one month.
Embodiment 2:
Gather the ripe spore of the healthy and strong plant sporophyll back of big Platycerium bifurcatum (Platycerium grande) of greenhouse culture, with 75% alcohol-pickled 10s, take out and to place the 0.1% mercuric chloride solution 15min that sterilizes, after the aseptic washing 6 times, be inoculated into the spore germination medium and (contain active carbon 1g in every liter, coconut milk 150mL, gibberellin 0.5mg, agar 5g, all the other are MS), in 24 ℃ of elder generations, 3 weeks of dark cultivation, 2000lx illumination 8 hours every days was cultivated for 4 weeks down then, formed prothallium.Change prothallium over to the gametophyte growth medium and (contain caseinhydrolysate 0.3g in every liter, 6-benzyl purine 1.0mg, methyl 0.5mg, agar 5g, all the other are MS), 24 ℃, 1x illumination 8 hours 2000 every day was cultivated for 4 weeks down, formed the lobate gametophyte of growing thickly in a large number.The thallus that to grow thickly is inoculated into liquid gametophyte growth medium and (contains active carbon 1g in every liter, coconut milk 150mL, gibberellin 0.5mg, all the other are MS), 24 ℃, 2000lx illumination 8 hours every days, l00r/min shaken cultivation 3 days forms zygote to promote sperm to move to archegonium; The thallus that liquid culture is crossed is inoculated into solid gametophyte growth medium and (contains caseinhydrolysate 0.3g in every liter, 6-benzyl purine 1.0mg, methyl 0.5mg, agar 5g, all the other are MS), under condition same as described above, continue to cultivate for 3 weeks, grow the sporophyte plant of 2 times of bodies.The sporophyte plantlet is inoculated into green spheroid induces proliferated culture medium (to contain coconut milk 150mL in every liter, peptone 2g, 6-benzyl purine 1.0mg, agar 5g, all the other are MS), 24 ℃, 1x illumination 8 hours 20000 every day cultivated for 4 weeks, can induce at the plant base portion and form a large amount of green spheroids, these green spheroids are bred on same medium, and form plantlet.Plantlet is inoculated into strong plantlets and rootage medium (contain active carbon 0.3mg in every liter, indolebutyric acid 0.5mg, agar 5g, all the other are 1/2MS), and 24 ℃, 1x illumination 8 hours 2000 every day cultivated for 4 weeks, formed the healthy and strong plant of band root.Took root seedling in the 80% natural daylight lower refining seedling that shades 7 days, it by taking out in the blake bottle, is cleaned the medium of root, cultivate in sphagna, 28 ℃, 80% shades, and humidity 60% keeps ventilating, and the visible plant in 1 week back recovers growth, survival rate 80%.Can go up potted plant training after 35 days.
Embodiment 3:
Gather the ripe spore of the healthy and strong plant sporophyll back of Platycerium bifurcatum (Pla tycerium bifurca tum C.Chr.) of greenhouse culture, with 70% alcohol-pickled 10s, taking-up places the 1.5% liquor natrii hypochloritis 10min that sterilizes, after the aseptic washing 5 times, be inoculated into the spore germination medium and (contain active carbon 0.5g in every liter, coconut milk 100mL, gibberellin 0.2mg, agar 7g, all the other are MS), in 26 ℃ of elder generations, 2 weeks of dark cultivation, 1800lx illumination 10 hours every days was cultivated for 5 weeks down then, formed prothallium.Change prothallium over to the gametophyte growth medium and (contain caseinhydrolysate 0.2g in every liter, 6-benzyl purine 0.5mg, methyl 0.2mg, does agar 7g (have?), all the other are MS), 26 ℃, 1800lx illumination 10 hours every days was cultivated for 5 weeks down, formed the lobate gametophyte of growing thickly in a large number.The thallus that to grow thickly is inoculated into liquid gametophyte growth medium and (contains active carbon 0.5g in every liter, coconut milk 100mL, gibberellin 0.2mg, all the other are MS), 26 ℃, 1800lx illumination 10 hours every days, 50r/min shaken cultivation 2 days forms zygote to promote sperm to move to archegonium; The thallus that liquid culture is crossed is inoculated into solid gametophyte growth medium and (contains caseinhydrolysate 0.2g in every liter, 6-benzyl purine 0.5mg, methyl 0.2mg, agar 6g, all the other are MS), under condition same as described above, continue to cultivate for 4 weeks, grow the sporophyte plant of 2 times of bodies.The sporophyte plantlet is inoculated into green spheroid induces proliferated culture medium (to contain coconut milk 100mL in every liter, peptone lg, 6-benzyl purine 0.5mg, agar 7g, all the other are MS), 26 ℃, 1800lx illumination 10 hours every days cultivated for 6 weeks, can induce at the plant base portion and form a large amount of green spheroids, these green spheroids are bred on same medium, and form plantlet.Plantlet is inoculated into strong plantlets and rootage medium (contain active carbon 0.5mg in every liter, indolebutyric acid 0.2mg, agar 6g, all the other are 1/2MS), and 26 ℃, 1800lx illumination 10 hours every days cultivated for 4 weeks, formed the healthy and strong plant of band root.Took root seedling in the 80% natural daylight lower refining seedling that shades 7 days, it by taking out in the blake bottle, is cleaned the medium of root, cultivate in sphagna, 25 ℃, 90% shades, and humidity 80% keeps ventilating, and the visible plant in 1 week back recovers growth, survival rate 85%.Can go up potted plant training after 40 days.
Claims (3)
1. Platycerium bifurcatum process for breeding and culturing, its feature may further comprise the steps:
(1) spore axenic germination: the ripe spore of Platycerium bifurcatum (Platycerium) blade back of gathering healthy and strong growth is with 70%～75% alcohol-pickled 10～30s, taking-up places the liquor natrii hypochloritis of 0.1%～0.2% mercuric chloride solution or 1%～2% 10～15min that sterilizes, after the aseptic washing, be inoculated on the spore germination medium; Cultivated for 2～3 weeks in that (25 ± 1) ℃ elder generation is dark, under 8～12h/ days then, 1500～2000lx illumination, cultivate and form prothallium, contain active carbon 0.5～1g, coconut milk 50～150mL, gibberellin 0.1～0.5mg in every liter of the described medium, agar 5～7g, all the other are MS;
(2) gametophyte grown cultures: change the prothallium of step (1) over to the gametophyte growth medium, in (25 ± 1) ℃, 8～12h/ days, 1500～2000lx illumination cultivation down, the formation lobate gametophyte of growing thickly, contain caseinhydrolysate 0.1～0.3g, 6-benzyl purine 0.2～1.0mg, methyl 0.1～0.5mg in every liter of the described medium, agar 5～7g, all the other are MS;
(3) insemination forms zygote: the lobate gametophyte of growing thickly of step (2) is inoculated into earlier on the liquid gametophyte growth medium, in (25 ± 1) ℃, 8～12h/ days, 1500～2000lx illumination, 50～100r/min shaken cultivation 2～3 days, the thallus that liquid culture is crossed is inoculated into solid gametophyte growth medium again, under condition same as described above, continue to cultivate the sporophyte plant that grows 2 times of bodies, contain active carbon 0.5～1g in every liter of the described liquid gametophyte growth medium, coconut milk 50～150mL, gibberellin 0.1～0.5mg, all the other are MS, contain caseinhydrolysate 0.1～0.3g in every liter of the described solid gametophyte growth medium, 6-benzyl purine 0.2～1.0mg, methyl 0.1～0.5mg, agar 5～7g, all the other are MS;
(4) sporophyte enrichment culture: the sporophyte plantlet in the step (3) is inoculated into green spheroid induces on the proliferated culture medium, in (25 ± 1) ℃, 8～12h/ days, 1500～2000lx illumination cultivation are induced the green spheroid of formation at the plant base portion, and these green spheroids are bred on medium same as described above, and formation plantlet, contain coconut milk 100～150mL, peptone 1～2g, 6-benzyl purine 0.2～1.0mg in every liter of the described medium, agar 5～7g, all the other are MS;
(5) culture of rootage: the plantlet that step (4) is obtained is inoculated on the strong plantlets and rootage medium, in (25 ± 1) ℃, 8～12h/ days, 1500～2000lx illumination cultivation, form the healthy and strong plant of band root, contain active carbon 0.3～0.5mg, indolebutyric acid 0.1～0.5mg in every liter of the described medium, agar 5～7g, all the other are 1/2 MS, and described 1/2 MS reduces by half the macroelement among the MS, the medium that other components unchanged is formed;
(6) bottle outlet is transplanted: the seedling of taking root by taking out in the blake bottle, is cleaned the medium of root with it in 70%～80% natural daylight lower refining seedling that shades 5～7 days, cultivate in sphagna, 25～28 ℃, 80%～90% shades, humidity 60%～90% keeps ventilating, and plant recovers growth.
2. according to the Platycerium bifurcatum process for breeding and culturing of claim 1, it is characterized in that the Platycerium bifurcatum in the step (1) is heavily to split Platycerium bifurcatum (Platycerium wallichii), Platycerium bifurcatum (Platycerium bifurcatum), big Platycerium bifurcatum (Platycerium grande), the Platycerium bifurcatum that comes into leaves (Platycerium willinckii), America Platycerium bifurcatum (Platycerium andinum) or Queensland Platycerium bifurcatum (Platycerium hillii).
3. according to the Platycerium bifurcatum process for breeding and culturing of claim 1 or 2, it is characterized in that the aseptic washing described in the step (1) 5～6 times, 25 ± 1 ℃ of dark 2～3 weeks of cultivation earlier, 8～12h/ days then, under 1500～2000lx illumination, continue to cultivate for 4～5 weeks, form prothallium; Cultivated for 4～5 weeks in the step (2), form the lobate gametophyte of growing thickly; Be inoculated into earlier in the step (3) on the liquid gametophyte growth medium, cultivated 2～3 days, the thallus that liquid culture is crossed is inoculated on the solid gametophyte growth medium again, continues to cultivate for 3～4 weeks, grows the sporophyte plant of 2 times of bodies; Cultivate 6-8 week in the step (4), induce at the plant base portion to form green spheroid; Cultivated for 4～6 weeks in the step (5), form the healthy and strong plant of band root; Step (6) was cultivated after 30-40 days can go up potted plant training.
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| CN102550407A (en) * | 2011-12-26 | 2012-07-11 | 溧阳市澳谷信息科技有限公司 | Asexual propagation method of liverwort |
| CN101637128B (en) * | 2009-08-28 | 2012-07-25 | 北京林业大学 | Method of sonatic embryo generation and plant regeneration of aleuritopteris argentea |
| CN102884971A (en) * | 2012-10-11 | 2013-01-23 | 浙江省萧山棉麻研究所 | Method for inversely suspended cultivating platycerium |
| CN104542300A (en) * | 2015-01-28 | 2015-04-29 | 遵义师范学院 | Culture medium for each in-vitro culture stage of osmundaceae |
| CN104663442A (en) * | 2015-02-25 | 2015-06-03 | 杨惠才 | Method for platycerium wallichii tissue culture and rapid propagation |
| 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 |
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| CN102550407A (en) * | 2011-12-26 | 2012-07-11 | 溧阳市澳谷信息科技有限公司 | Asexual propagation method of liverwort |
| CN102884971A (en) * | 2012-10-11 | 2013-01-23 | 浙江省萧山棉麻研究所 | Method for inversely suspended cultivating platycerium |
| CN102884971B (en) * | 2012-10-11 | 2013-08-28 | 浙江省萧山棉麻研究所 | Method for inversely suspended cultivating platycerium |
| CN104542300B (en) * | 2015-01-28 | 2016-08-24 | 遵义师范学院 | The culture medium in osmund cultured in vitro each stage |
| CN104542300A (en) * | 2015-01-28 | 2015-04-29 | 遵义师范学院 | Culture medium for each in-vitro culture stage of osmundaceae |
| CN104663442A (en) * | 2015-02-25 | 2015-06-03 | 杨惠才 | Method for platycerium wallichii tissue culture and rapid propagation |
| 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 |
| CN110100740B (en) * | 2019-06-26 | 2020-10-20 | 四川七彩林科股份有限公司 | Tissue culture rapid propagation method of platycerium giganteum |
| 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 |
| CN112021179A (en) * | 2020-09-03 | 2020-12-04 | 西南林业大学 | Tissue culture method of Dryopteris fragrans |
| CN112889671A (en) * | 2021-03-08 | 2021-06-04 | 西南林业大学 | Tissue culture and rapid propagation method of platycerium |
| CN112889671B (en) * | 2021-03-08 | 2023-07-04 | 西南林业大学 | Tissue culture and rapid propagation method of platycerium |
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