CN102511396A - In vitro rapid breeding method of medicinal plant Lipidium meyenii - Google Patents
In vitro rapid breeding method of medicinal plant Lipidium meyenii Download PDFInfo
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Abstract
The invention discloses an in vitro rapid breeding method of a medicinal plant Lipidium meyenii. The method is realized through steps of medium preparation, aseptic seedling preparation, bud cluster induction and rooting culture. The method of the invention uses stem segments with buds of seedlings as culture materials, has characteristics of low variation rate and stable heredity; a propagation time reaches higher than 10 times to the most; and seedlings grow strong and can maintain original line properties.
Description
Technical field
The present invention relates to the method for the stripped fast breeding of a kind of medicinal plant agate coffee.
Background technology
Agate coffee (Lepidium meyenii) is Cruciferae separate row Vegetable spp 1 year or 2 years these plants of sward; Originate in South America; Be grown in mountain area, height above sea level 3500～5000m Peru Andean, but its fleshy root dietotherapeutic have very high nutritive value and medical value.Discover; Be rich in the amino acid of a large amount of needed by human, multiple unsaturated fatty acid, vitamin and mineral matter in the agate coffee, main chemical compositions is agate card alkene, agate card acid amides, Glucosinolates etc., has enhancing human immune; Regain one's strength fast, eliminate effects such as anxiety, fatigue.
At present, all kinds of health products of agate coffee exploitation enjoy a good market both at home and abroad.China successfully introduces a fine variety on Xinjiang, Yunnan and other places, receives the restriction of its biological property and procreation material, in the plantation of agate coffee, owing to breed bottleneck problem, is difficult to realize industrialization, can not satisfy market demand.
Therefore, a kind of method of breeding the agate coffee fast that exsomatizes just is suggested.
Summary of the invention
The purpose of this invention is to provide a kind of agate coffee aseptic seedling of utilizing and be culture materials, and then the method for stripped fast breeding medicinal plant agate coffee.
The object of the invention mainly bunch is induced with process of rooting culture through the preparation of culture medium preparation, aseptic seedling, bud and is realized, it is characterized in that:
Said culture medium preparation is:
A. basic MS culture medium: get the MS medium, add sucrose 20～40mg/L and agar 5～10g/L, regulate pH=6～6.4 simultaneously;
B.1/2MS medium: get 1/2MS medium (the MS medium of 1/2 macroelement concentration), add sucrose 20～40mg/L and agar 5～10g/L, regulate pH=6～6.4 simultaneously.
Said aseptic seedling is prepared as: get full agate coffee seed, water is rinsed well, warp 65～80% alcohol-pickled 0.5～1.5min in superclean bench; Change soaking disinfection 8～12min in 0.5～1.5% the mercuric chloride again over to,, drain the water with aseptic water washing 3-5 time; Be seeded in the above-mentioned 1/2MS medium, under 18～25 ℃, 10～14h illumination cultivation/10～14h dark culturing 10～20 days; Treat that 2 cotyledons of seedling launch, true leaf is not put on display, and promptly gets aseptic seedling.
Said bud bunch induce into: add in following ratio in basic MS culture medium: NAA (methyl) 0.25～0.5mg/L; 6-BA (6-benzyladenine) 1.0～4.0mg/L as bud bunch inducing culture as root media; Above-mentioned aseptic seedling is removed cotyledon and root; Remainder is that (height of seedling 1.5～2.0cm) is inoculated in the bud bunch inducing culture as cultured tissue stem with bud, during inoculation medium is gone in the lower end cuttage of stem section, and the degree of depth is 0.5-0.8cm; Bud-end is exposed, and places 20～25 ℃ of incubators to cultivate 21～28 days.
Said culture of rootage is: in the 1/2MS medium, add NAA0.3～2.0 mg/L; Perhaps in the 1/2MS medium, add IBA (indolebutyric acid) 0.5～2.0 mg/L as root media; The regeneration bud that induces bunch is cut into individual plant, is inoculated into and carries out culture of rootage 15-25 days in the root media; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Above-mentioned culture of rootage is preferably: carry out under cultivating alternately by 14～18h illumination cultivation/6～10h is dark, intensity is at 1800～2200Lx during illumination cultivation.
In the said culture of rootage, the NAA that in the 1/2MS medium, adds is preferably 0.5～1.0 mg/L, and the IBA that in the 1/2MS medium, adds is preferably 0.8～1.5 mg/L.
The present invention as culture materials, has characteristics such as aberration rate is low, inheritance stability with the stem with bud of seedling, and the propagation multiple reaches as high as more than 10 times, and growth of seedling is vigorous, and can keep original strain is proterties.
Embodiment
Embodiment 1: get the MS medium, add sucrose 20mg and agar 10g by every L, regulate pH=6 simultaneously, as basic MS culture medium, get the 1/2MS medium, add sucrose 20mg and agar 10g by every L, regulate pH=6 simultaneously, as the 1/2MS medium; Get full agate coffee seed, water is rinsed well, in superclean bench, through 70% alcohol-pickled 1min, changes soaking disinfection 10min in 1.0% the mercuric chloride again over to; With aseptic water washing 3 times, drain the water to there not being the phenomenon of dripping, be seeded in the above-mentioned 1/2MS medium; Under 20 ℃, 12h illumination cultivation/12h dark culturing 14 days, intensity is at 2000Lx during illumination cultivation; 2 cotyledons of seedling launch, and true leaf is not put on display, and get aseptic seedling; Add by every L in basic MS culture medium: NAA 0.25mg, 6-BA 1. 0mg are in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, during inoculation medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 20 ℃ of incubators to cultivate 21 days; Wherein preceding 7 days is dark the cultivation; Thereafter 15h illumination cultivation/9h dark cultivate to hocket cultivated 14 days, intensity is at 2000Lx during illumination cultivation, i.e. regeneration is sprouted bunch; In the 1/2MS medium, add NAA0.5mg as root media, the regeneration bud that induces bunch is cut into individual plant, be inoculated into and carried out culture of rootage in the root media 15 days by every L; Carry out down by dark the cultivation alternately of 16h illumination cultivation/8h, during illumination cultivation intensity at 2000Lx, during inoculation with the base portion implantation medium of the individual plant of cutting, degree of depth 0.3-0.5cm.
Embodiment 2: get the MS medium, add sucrose 30mg and agar 5g by every L, regulate pH=6.2 simultaneously, as basic MS culture medium, get the 1/2MS medium, add sucrose 30mg and agar 5g by every L, regulate pH=6.2 simultaneously, as the 1/2MS medium; Get full agate coffee seed, water is rinsed well, in superclean bench, through 80% alcohol-pickled 0.5min, changes soaking disinfection 12min in 1.5% the mercuric chloride again over to; With aseptic water washing 5 times, drain the water and drip to nothing, be seeded in the above-mentioned 1/2MS medium; Under 22 ℃, 14h illumination cultivation/10h dark culturing 20 days, intensity is at 1900Lx during illumination cultivation; Treat that 2 cotyledons of seedling launch, true leaf is not put on display, and promptly gets aseptic seedling; Add NAA0.5mg in basic MS culture medium by every L, 6-BA 1.0mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, during inoculation medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 22 ℃ of incubators to cultivate 24 days; Wherein preceding 10 days be dark the cultivation, thereafter 14h illumination cultivation/10h dark cultivate to hocket cultivated 14 days, i.e. regeneration is sprouted bunch; In the 1/2MS medium, add NAA1.0 mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 21 days, and carry out under cultivating alternately by 18h illumination cultivation/6h is dark, intensity is at 1800Lx during illumination cultivation; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 3: get the MS medium, add sucrose 40mg and agar 8g by every L, regulate pH=6.4 simultaneously, as basic MS culture medium, get the 1/2MS medium; Add sucrose 40mg and agar 8g by every L, regulate pH=6.4 simultaneously,, get full agate coffee seed as the 1/2MS medium; Water is rinsed well, in superclean bench, through 75% alcohol-pickled 1min, changes soaking disinfection 8min in 1% the mercuric chloride again over to, with aseptic water washing 4 times; Drop does not drip to having, is seeded in the above-mentioned 1/2MS medium, and under 24 ℃, 10h illumination cultivation/14h dark culturing 20 days; Intensity is at 2000Lx during illumination cultivation, and 2 cotyledons of seedling launch, and true leaf is not put on display, and promptly gets aseptic seedling; Add by every L in basic MS culture medium: NAA 0.35mg, 6-BA 3.0mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, during inoculation medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 22 ℃ of incubators to cultivate 28 days; Wherein induce and be dark cultivation in preceding 8 days; Thereafter cultivated cultivations that hocket in that 14h illumination cultivation 10h is dark in 20 days, intensity is promptly regenerated and is sprouted bunch at 1900Lx during illumination cultivation; In the 1/2MS medium, add NAA1.8mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 18 days, and carry out under cultivating alternately by 17h illumination cultivation/7h is dark, intensity is at 1900Lx during illumination cultivation; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 4: get the MS medium, add sucrose 25mg and agar 6g by every L, regulate pH=6.1 simultaneously, as basic MS culture medium; Get the 1/2MS medium, add sucrose 25mg and agar 6g, regulate pH=6.1 simultaneously, as the 1/2MS medium; get full agate coffee seed, water is rinsed well, in superclean bench,, change soaking disinfection 8min in 1.5% the mercuric chloride again over to; with aseptic water washing 3 times, drain the water and drip, be seeded in the above-mentioned 1/2MS medium; under 18 ℃ to nothing through 65% alcohol-pickled 1.5min by every L, 14h illumination cultivation/10h dark culturing 20 days, intensity is at 2000Lx during illumination cultivation; treat 2 cotyledons expansion of seedling, true leaf is not put on display, and promptly gets aseptic seedling; Add by every L in basic MS culture medium: NAA 0.4mg, 6-BA 4.0mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, inoculation method is: medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 25 ℃ of incubators to cultivate 21 days; Wherein induce and be dark cultivation in preceding 8 days; Thereafter 14h illumination cultivation/10h dark cultivate to hocket cultivated 13 days, intensity is at 2200Lx during illumination cultivation, i.e. regeneration is sprouted bunch; In the 1/2MS medium, add IBA0.5mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 22 days, and carry out under cultivating alternately by 14h illumination cultivation/10h is dark, intensity is at 2200Lx during illumination cultivation; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 5: get the MS medium, add sucrose 35mg and agar 9g by every L, regulate pH=6.3 simultaneously, as basic MS culture medium; Get the 1/2MS medium, add sucrose 35g and agar 9g, regulate pH=6.3 simultaneously, as the 1/2MS medium; get full agate coffee seed, water is rinsed well, in superclean bench,, change soaking disinfection 10min in 0.8% the mercuric chloride again over to; with aseptic water washing 3 times, drain to nothing and drip, be seeded in the above-mentioned 1/2MS medium; under 20 ℃ through 80% alcohol-pickled 1.2min by every L, 12h illumination cultivation/12h dark culturing 14 days, intensity is at 2100Lx during illumination cultivation; treat 2 cotyledons expansion of seedling, true leaf is not put on display, and promptly gets aseptic seedling; Add NAA 0.35mg in basic MS culture medium by every L, 6-BA 2.0mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, during inoculation medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 22 ℃ of incubators to cultivate 25 days; Wherein induce and be dark cultivation in preceding 8 days; Thereafter cultivated cultivations that hocket in that 16h illumination cultivation/8h is dark in 17 days, intensity is promptly regenerated and is sprouted bunch at 2200Lx during illumination cultivation; In the 1/2MS medium, add IBA1.2mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 24 days, and carry out under cultivating alternately by 14h illumination cultivation/10h is dark, intensity is at 2200Lx during illumination cultivation; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 6: get the MS medium, add sucrose 25mg and agar 10g by every L, regulate pH=6.2 simultaneously, as basic MS culture medium, get the 1/2MS medium; Add sucrose 25mg and agar 10g by every L, regulate pH=6.2 simultaneously,, get full agate coffee seed as the 1/2MS medium; Water is rinsed well, in superclean bench, through 70% alcohol-pickled 1min, changes soaking disinfection 10min in 1% the mercuric chloride again over to, with aseptic water washing 3-5 time; Drain the water, be seeded in the above-mentioned 1/2MS medium, under 24 ℃, 11h illumination cultivation/13h dark culturing 18 days; Intensity treats that at 2000Lx 2 cotyledons of seedling launch during illumination cultivation, and true leaf is not put on display, and promptly gets aseptic seedling; Add by every L in basic MS culture medium: NAA 0.45mg, 6-BA 2.0mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, inoculation method is: medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 24 ℃ of incubators to cultivate 22 days; Wherein induce and be dark cultivation in preceding 7 days; Thereafter cultivated cultivations that hocket in that 18h illumination cultivation/6h is dark in 15 days, intensity is promptly regenerated and is sprouted bunch at 1800Lx during illumination cultivation; In the 1/2MS medium, add IBA1.8 mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 20 days, and carry out under cultivating alternately by 15h illumination cultivation/9h is dark, intensity is at 1900Lx during illumination cultivation.During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 7: get the MS medium, add sucrose 30mg and agar 10g by every L, regulate pH=6.2 simultaneously, as basic MS culture medium; Get the 1/2MS medium, add sucrose 30mg and agar 10g, regulate pH=6.2 simultaneously, as the 1/2MS medium; get full agate coffee seed, water is rinsed well, in superclean bench,, change soaking disinfection 10min in 1% the mercuric chloride again over to; with aseptic water washing 4 times, drain to nothing and drip, be seeded in the above-mentioned 1/2MS medium, 22 ℃ of following 12h illumination cultivation/12h dark culturing 14 days; intensity is treated 2 cotyledons expansion of seedling at 2100Lx during illumination cultivation, and true leaf is not put on display, and promptly gets aseptic seedling through 75% alcohol-pickled 1min by every L; Add by every L in basic MS culture medium: NAA 0.4mg, 6-BA 3.0mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, inoculation method is: medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 23 ℃ of incubators to cultivate 28 days; Wherein induce and be dark cultivation in preceding 8 days; Thereafter 17h illumination cultivation/7h dark cultivate to hocket cultivated 16 days, intensity is at 2100Lx during illumination cultivation, i.e. regeneration is sprouted bunch; In the 1/2MS medium, add NAA0.3 mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 18 days, and carry out under cultivating alternately by 14h illumination cultivation/10h is dark, intensity is at 2100Lx during illumination cultivation.During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 8: get the MS medium, add sucrose 40mg and agar 6g by every L, regulate pH=6.2 simultaneously, as basic MS culture medium; Get the 1/2MS medium, add sucrose 20mg and agar 10g, regulate pH=6.2 simultaneously, as the 1/2MS medium; get full agate coffee seed, water is rinsed well, in superclean bench,, change soaking disinfection 12min in 1.2% the mercuric chloride again over to; with aseptic water washing 5 times, drain nothing and drip, be seeded in the above-mentioned 1/2MS medium; under 24 ℃ through 80% alcohol-pickled 1.5min by every L, 12h illumination cultivation/12h dark culturing 16 days, intensity is at 2200Lx during illumination cultivation; treat 2 cotyledons expansion of seedling, true leaf is not put on display, and promptly gets aseptic seedling; Add by every L in basic MS culture medium: NAA 0.45mg, 6-BA 1.5mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, inoculation method is: medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 22 ℃ of incubators to cultivate 21 days; Wherein induce and be dark cultivation in preceding 6 days; Thereafter 14 illumination cultivation/10h dark cultivate to hocket cultivated 15 days, intensity is at 2000Lx during illumination cultivation, i.e. regeneration is sprouted bunch; In the 1/2MS medium, add NAA1.2 mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 16 days, and carry out under cultivating alternately by 18h illumination cultivation/6h is dark, intensity is at 2000Lx during illumination cultivation; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 9: get the MS medium, add sucrose 30mg and agar 5g by every L, regulate pH=6.2 simultaneously, as basic MS culture medium, get the 1/2MS medium; Add sucrose 20mg and agar 5g by every L, regulate pH=6.2 simultaneously,, get full agate coffee seed as the 1/2MS medium; Water is rinsed well, in superclean bench, through 75% alcohol-pickled 1min, changes soaking disinfection 8min in 1% the mercuric chloride again over to, with aseptic water washing 3-5 time; Drain to nothing and drip, be seeded in the above-mentioned 1/2MS medium, under 20 ℃, 14h illumination cultivation/10h dark culturing 20 days; Intensity treats that at 1800Lx 2 cotyledons of seedling launch during illumination cultivation, and true leaf is not put on display, and promptly gets aseptic seedling; Add NAA 0.45mg in basic MS culture medium by every L, 6-BA 3.0mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, inoculation method is: medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 22 ℃ of incubators to cultivate 21 days; Wherein induce and be dark cultivation in preceding 6 days; Thereafter 15h illumination cultivation/9h dark cultivate to hocket cultivated 15 days, intensity is at 2000Lx during illumination cultivation, i.e. regeneration is sprouted bunch; In the 1/2MS medium, add NAA1.5mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 21 days, and carry out under cultivating alternately by 18h illumination cultivation/6h is dark, intensity is at 1800Lx during illumination cultivation; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 10: get the MS medium, add sucrose 30mg and agar 8g by every L, regulate pH=6.2 simultaneously, as basic MS culture medium; Get the 1/2MS medium, add sucrose 30mg and agar 8g, regulate pH=6.2 simultaneously, as the 1/2MS medium; get full agate coffee seed, water is rinsed well, in superclean bench,, change soaking disinfection 12min in 1.2% the mercuric chloride again over to; with aseptic water washing 3 times, drain to nothing and drip, be seeded in the above-mentioned 1/2MS medium; under 22 ℃ through 70% alcohol-pickled 1.5min by every L, 12h illumination cultivation/12h dark culturing 20 days, intensity is at 2000Lx during illumination cultivation; treat 2 cotyledons expansion of seedling, true leaf is not put on display, and promptly gets aseptic seedling; Add NAA 0.8mg in basic MS culture medium by every L, 6-BA 3.5mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, inoculation method is: medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 22 ℃ of incubators to cultivate 21 days; Wherein induce and be dark cultivation in preceding 7 days; Thereafter 14h illumination cultivation/10h dark cultivate to hocket cultivated 14 days, intensity is at 2000Lx during illumination cultivation, i.e. regeneration is sprouted bunch; In the 1/2MS medium, add IBA2.0 mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 24 days, and carry out under cultivating alternately by 14h illumination cultivation/10h is dark, intensity is at 2000Lx during illumination cultivation; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 11: get the MS medium, add sucrose 30mg and agar 7g by every L, regulate pH=6.2 simultaneously, as basic MS culture medium; Get the 1/2MS medium, add sucrose 20mg and agar 7g, regulate pH=6.2 simultaneously, as the 1/2MS medium; get full agate coffee seed, water is rinsed well, in superclean bench,, change soaking disinfection 8min in 1% the mercuric chloride again over to; with aseptic water washing 3 times, drain to nothing and drip, be seeded in the above-mentioned 1/2MS medium; under 20 ℃ through 70% alcohol-pickled 1.2min by every L, 14h illumination cultivation/10h dark culturing 20 days, intensity is at 1800Lx during illumination cultivation; treat 2 cotyledons expansion of seedling, true leaf is not put on display, and promptly gets aseptic seedling; Add NAA 0.45mg in basic MS culture medium by every L, 6-BA 3.0mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, inoculation method is: medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 22 ℃ of incubators to cultivate 21 days; Wherein induce and be dark cultivation in preceding 6 days; Thereafter 15h illumination cultivation/9h dark cultivate to hocket cultivated 15 days, intensity is at 2000Lx during illumination cultivation, i.e. regeneration is sprouted bunch; In the 1/2MS medium, add NAA1.5mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 18 days, and carry out under cultivating alternately by 18h illumination cultivation/6h is dark, intensity is at 1800Lx during illumination cultivation; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Embodiment 12: get the MS medium, add sucrose 20mg and agar 8g by every L, regulate pH=6.2 simultaneously, as basic MS culture medium, get the 1/2MS medium; Add sucrose 20mg and agar 8g by every L, regulate pH=6.2 simultaneously,, get full agate coffee seed as the 1/2MS medium; Water is rinsed well, in superclean bench, through 70% alcohol-pickled 1min, changes soaking disinfection 10min in the mercuric chloride of 1 % again over to, with aseptic water washing 3 times; Drain to nothing and drip, be seeded in the above-mentioned 1/2MS medium, under 22 ℃, 12h illumination cultivation/12h dark culturing 18 days; Intensity treats that at 2000Lx 2 cotyledons of seedling launch during illumination cultivation, and true leaf is not put on display, and promptly gets aseptic seedling; Add NAA 0.4mg in basic MS culture medium by every L, 6-BA 2.5mg is in superclean bench; Cut off 2 true leaves and young root, remainder (stem with bud of about 1.5-2.0cm) is inoculated in the bud bunch inducing culture, during inoculation medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 22 ℃ of incubators to cultivate 28 days; Wherein induce and be dark cultivation in preceding 8 days; Thereafter 14h illumination cultivation/10h dark cultivate to hocket cultivated 20 days, intensity is at 2000Lx during illumination cultivation, i.e. regeneration is sprouted bunch; In the 1/2MS medium, add IBA2.0 mg as root media by every L; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carried out culture of rootage in the root media 15 days, and carry out under cultivating alternately by 14h illumination cultivation/10h is dark, intensity is at 2000Lx during illumination cultivation; During inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
Claims (5)
1. the medicinal plant agate coffee method of fast breeding that exsomatizes mainly bunch is induced with process of rooting culture through culture medium preparation, aseptic seedling preparation, bud and is realized, it is characterized in that:
Said culture medium preparation is:
A. basic MS culture medium: get the MS medium, add sucrose 20～40mg/L and agar 5～10g/L, regulate pH=6～6.4 simultaneously;
B.1/2MS medium: get the 1/2MS medium, add sucrose 20～40mg/L and agar 5～10g/L, regulate pH=6～6.4 simultaneously;
Said aseptic seedling is prepared as:
Get full agate coffee seed, water is rinsed well, at 65～80% alcohol-pickled 0.5～1.5min; Change soaking disinfection 8～12min in 0.5～1.5% the mercuric chloride again over to,, drain the water with aseptic water washing 3-5 time; Be seeded in the above-mentioned 1/2MS medium, under 18～25 ℃, 10～14h illumination/10～14h is dark alternately alternately to be cultivated 10～20 days; Treat that 2 cotyledons of seedling launch, true leaf is not put on display, and promptly gets aseptic seedling;
Said bud bunch induce into:
Add in following ratio in basic MS culture medium: NAA (methyl) 0.25～0.5mg/L; 6-BA (6-benzyladenine) 1.0～4.0mg/L removes cotyledon and root as bud bunch inducing culture with above-mentioned aseptic seedling, and remainder is that (height of seedling 1.5～2.0cm) is inoculated in the bud bunch inducing culture as cultured tissue stem with bud; During inoculation medium is gone in the lower end cuttage of stem section; The degree of depth is 0.5-0.8cm, and bud-end is exposed, and places 20～25 ℃ of incubators to cultivate 21～28 days;
Said culture of rootage is:
In the 1/2MS medium, add NAA0.3～2.0 mg/L; Perhaps in the 1/2MS medium, add IBA (indolebutyric acid) 0.5～2.0 mg/L as root media; The regeneration bud that induces bunch is cut into individual plant; Be inoculated into and carry out culture of rootage 15-25 days in the root media, during inoculation the base portion of the individual plant of cutting is implanted medium, degree of depth 0.3-0.5cm.
2. the method for the stripped fast breeding of medicinal plant agate coffee according to claim 1; It is characterized in that: in the said culture of rootage; The NAA that in the 1/2MS medium, adds is 0.5～1.0 mg/L, and the IBA that in the 1/2MS medium, adds is 0.8～1.5 mg/L.
3. the method for the stripped fast breeding of medicinal plant agate coffee according to claim 1 and 2; It is characterized in that: described bud bunch inducing culture is: inducing preceding 6～8 days for secretly cultivating; Thereafter hocket in the dark cultivation of 14～18h illumination cultivation/6～10h and cultivated 15-20 days, intensity is at 1800～2200Lx during illumination cultivation.
4. the method for the stripped fast breeding of medicinal plant agate coffee according to claim 1 and 2, it is characterized in that: described culture of rootage is: carry out under cultivating alternately by 14～18h illumination cultivation/6～10h is dark, intensity is at 1800～2200Lx during illumination cultivation.
5. the method for the stripped fast breeding of medicinal plant agate coffee according to claim 3, it is characterized in that: described culture of rootage is: carry out under cultivating alternately by 14～18h illumination cultivation/6～10h is dark, intensity is at 1800～2200Lx during illumination cultivation.
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| CN104163742A (en) * | 2014-09-18 | 2014-11-26 | 昆明中至诚农业科技开发有限公司 | Special fertilizer for maca planting and preparation method thereof |
| CN104686370A (en) * | 2015-04-03 | 2015-06-10 | 会泽枢康中草药种植有限公司 | Method for cultivating maca polyploid plants |
| CN105191801A (en) * | 2015-10-24 | 2015-12-30 | 大理白族自治州农业科学推广研究院药用植物及农业新技术研究所 | Healthcare medicinal material Maca tissue cultured seedling vitrification control method |
| CN105494102A (en) * | 2016-01-12 | 2016-04-20 | 丽水学院 | Method for culture of efficient maca virus-free tissue |
| CN107047318A (en) * | 2017-06-20 | 2017-08-18 | 齐鲁师范学院 | A kind of external rapid propagation method of grande passerage herb |
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| CN1473465A (en) * | 2003-07-01 | 2004-02-11 | 华中科技大学 | Maka seedling reproducing method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104163742A (en) * | 2014-09-18 | 2014-11-26 | 昆明中至诚农业科技开发有限公司 | Special fertilizer for maca planting and preparation method thereof |
| CN104163742B (en) * | 2014-09-18 | 2017-02-15 | 昆明中至诚农业科技开发有限公司 | Special fertilizer for maca planting and preparation method thereof |
| CN104686370A (en) * | 2015-04-03 | 2015-06-10 | 会泽枢康中草药种植有限公司 | Method for cultivating maca polyploid plants |
| CN105191801A (en) * | 2015-10-24 | 2015-12-30 | 大理白族自治州农业科学推广研究院药用植物及农业新技术研究所 | Healthcare medicinal material Maca tissue cultured seedling vitrification control method |
| CN105494102A (en) * | 2016-01-12 | 2016-04-20 | 丽水学院 | Method for culture of efficient maca virus-free tissue |
| CN107047318A (en) * | 2017-06-20 | 2017-08-18 | 齐鲁师范学院 | A kind of external rapid propagation method of grande passerage herb |
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