CN102696483B - Method for quickly propagating lilium fargesii - Google Patents
Method for quickly propagating lilium fargesii Download PDFInfo
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Abstract
The invention discloses a method for quickly propagating lilium fargesii. The method comprises the following steps: I, selecting a disease-free scale leaf on the underground bulb of the lilium fargesii, and cleaning, rinsing and sterilizing the scale leaf to obtain an explant; II, inoculating the explant on a callus induction culture medium to generate a callus after two months; III, inoculating the callus on a subculture culture medium, and executing proliferation culture to generate a new enlarged callus; IV, inoculating the enlarged callus or the new callus on a bud differentiation culturemedium to execute bud differentiation derivation, and differentiating multiple shoots; V, inoculating the differentiated multiple shoots on a rooting medium to obtain a rhizogenic lilium fargesii seedling; and VI, inoculating and cultivating the rhizognic lilium fargesii seedling on a cultivation culture medium to obtain a lilium fargesii plant regeneration. The method disclosed by the invention is not restricted by seasons, can perennially achieve quick propagation and large-scale production of the lilum fargesii, and provides an operable method for quickly enlarging the population thereof.
Description
Technical field
The invention belongs to technical field of plant culture, be specifically related to a kind of green colored lily method for quickly breeding.
Background technology
The Qinling Mountains is as the line of demarcation in China north and south, and is with a varied topography, and weather is various, and the wild lily that has been found that just has 11 kinds, and resource is very abundant, and has exploitation value.Lily is world-renowned cut-flower and potted flower, and pattern flower type is different, often has graceful fragrance, and is how pharmaceutically acceptable and view and admire, and economic worth is very high.Green colored lily is wherein a kind of, is described as " spending middle giant panda ", and is endangered now, for better protection with utilize precious green colored lily resource, at first must breed in a large number, enlarges its population fast.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of and be not subject to seasonal restrictions at above-mentioned the deficiencies in the prior art, can breed green colored lily throughout the year, fast, realizes the green colored lily method for quickly breeding of the large-scale production of green colored lily.Adopt this method to breed green colored lily, rooting rate reaches more than 93.3%, and transplanting survival rate reaches more than 90%.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of green colored lily method for quickly breeding is characterized in that this method may further comprise the steps:
Step 1, select the scaly leaf on the underground bulb of green colored lily of no scab, with washing agent the earth on the described scaly leaf is cleaned, wash 0.5h～1.5h with flowing water then, adopt conventional explant material sterilizing methods that the scaly leaf after washing is sterilized again, obtain explant;
Step 2, under aseptic condition explant described in the step 1 is inoculated on the callus inducing medium, cultivating after two months, whole explant produces callus; Described callus inducing medium is: add 6-benzyl aminopurine 1.5mg/L～2.5mg/L, methyl 0.05mg/L～0.2mg/L and agar 6g/L～12g/L in the conventional minimal medium of MS; The pH value of described callus inducing medium is 5.4～5.8; Described culture condition is: 21 ℃～25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx～2000Lx;
Step 3, the callus that produces in step 2 under aseptic condition are inoculated in subculture medium and breed cultivation, and propagation is cultivated after 12～18 days callus and increased and have new callus to produce; Described subculture medium is: add 6-benzyl aminopurine 0.8mg/L～2.0mg/L, methyl 0.05mg/L～0.2mg/L and agar 6g/L～12g/L in the conventional minimal medium of MS; The pH value of described subculture medium is 5.4～5.8; Described propagation culture condition is: 21 ℃～25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx～2000Lx;
Step 4, under aseptic condition, the callus that increases described in the step 3 or new callus are inoculated in the bud differential medium and carry out the bud induction, inoculate and differentiate the bud of growing thickly after 40～50 days; Described bud differential medium is: add 6-benzyl aminopurine 0.5mg/L～1.5mg/L, methyl 0.05mg/L～0.2mg/L and agar 6g/L～12g/L in the conventional minimal medium of MS; The pH value of described bud differential medium is 5.4～5.8; The condition of culture of described bud induction is: 21 ℃～25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx～2000Lx;
Step 5, the bud of growing thickly that differentiates in step 4 under aseptic condition are inoculated on the root media, cultivate the green colored lily seedling that acquisition is taken root after 10～15 days; Described root media is: add 6-benzyl aminopurine 0.5mg/L～1.2mg/L, methyl 0.02mg/L～0.08mg/L and agar 6g/L～12g/L in the conventional minimal medium of MS; The pH value of described root media is 5.4～5.8; Described culture condition is: 21 ℃～25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx～2000Lx;
Step 6, the green colored lily sprigging of taking root described in the step 5 is cultivated in cultivation matrix, obtain green colored lily regeneration plant; Described cultivation matrix is by the peat composed of rotten mosses, perlite and husky the composition, and wherein the peat composed of rotten mosses, perlite and husky volume ratio are 5～7: 1～3: 2.
Above-mentioned green colored lily method for quickly breeding, callus inducing medium described in the step 2 is: add 6-benzyl aminopurine 2.0mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described callus inducing medium is 5.6.
Above-mentioned green colored lily method for quickly breeding, subculture medium described in the step 3 is: add 6-benzyl aminopurine 1.2mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described subculture medium is 5.6.
Above-mentioned green colored lily method for quickly breeding, the differential medium of bud described in the step 4 is: add 6-benzyl aminopurine 1.0mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described bud differential medium is 5.6.
Above-mentioned green colored lily method for quickly breeding, root media described in the step 5 is: add 6-benzyl aminopurine 0.8mg/L, methyl 0.05mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described root media is 5.6.
Above-mentioned green colored lily method for quickly breeding, the peat composed of rotten mosses described in the step 6, perlite and husky volume ratio are 6: 2: 2.
The present invention compared with prior art has the following advantages:
1, callus inducing medium of the present invention is with strong points, applicability good, explant scale after callus inducing medium is cultivated 4 days begins to turn green, the scale edge has the milky callus to produce after 8 days, continue to cultivate and observe, whole scale has callus to produce after 20 days, and the callus growing way is best, color is the denseest, be bottle green, be the graininess of densification, produce a large amount of callus after 2 months.
2, subculture medium cultivation effect of the present invention is good, callus growth is good, cultivate and begin to have the callus increase after 12～18 days and have new callus to produce, newborn callus increases about three times than original callus, the callus color that produces is strong green, and no browning occurs; Callus begins differentiation and sprouts after the bud differential medium is cultivated 40～50 days, the bud quantity of generation is more, and germination rate reaches as high as 93.3%, and bud is bottle green, and is healthier; Bud after the differentiation produces root after root media is cultivated 15 days, rooting rate reaches more than 93.3%, and transplanting survival rate reaches more than 90%.
3, method of the present invention is not subject to seasonal restrictions, and can breed green colored lily throughout the year, fast, realizes the large-scale production of green colored lily, provides practicable method for enlarging its population fast.
Below by embodiment, technical scheme of the present invention is described in further detail.
Embodiment
Embodiment 1
Step 1, select the scaly leaf on the underground bulb of green colored lily of no scab, with washing agent the earth on the described scaly leaf is cleaned, wash 1h with flowing water then, adopt conventional explant material sterilizing methods that the scaly leaf after washing is sterilized again, obtain explant; Described conventional explant material sterilizing methods is: be 75% ethanol disinfection 30s with scaly leaf at desinfection chamber workbench quality purity, throw in 5min～10min in 0.1% the mercuric chloride thimerosal then, use aseptic water washing at last 2～3 times;
Step 2, under aseptic condition explant described in the step 1 is inoculated on the callus inducing medium, cultivating after two months, whole explant produces a large amount of callus; Described callus inducing medium is: add 6-benzyl aminopurine 2.0mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described callus inducing medium is 5.6; Described culture condition is: 23 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1500Lx;
Step 3, the callus that produces in step 2 under aseptic condition are inoculated in subculture medium and breed cultivation, and propagation is cultivated after 14 days callus and increased and have new callus to produce; Described subculture medium is: add 6-benzyl aminopurine 1.2mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described subculture medium is 5.6; Described propagation culture condition is: 23 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1500Lx;
Step 4, under aseptic condition, the callus that increases described in the step 3 or new callus are inoculated in the bud differential medium and carry out the bud induction, inoculate and differentiate the bud of growing thickly after 45 days; Described bud differential medium is: add 6-benzyl aminopurine 1.0mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described bud differential medium is 5.6; The condition of culture of described bud induction is: 23 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1500Lx;
Step 5, the bud of growing thickly that differentiates in step 4 under aseptic condition are inoculated on the root media, cultivate the green colored lily seedling that acquisition is taken root after 12 days; Described root media is: add 6-benzyl aminopurine 0.8mg/L, methyl 0.05mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described root media is 5.6; Described culture condition is: 23 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1500Lx;
Step 6, the green colored lily sprigging of taking root described in the step 5 is cultivated in cultivation matrix, obtain green colored lily regeneration plant; Described cultivation matrix is by the peat composed of rotten mosses, perlite and husky the composition, and wherein the peat composed of rotten mosses, perlite and husky volume ratio are 6: 2: 2.
The present embodiment rooting rate reaches more than 93.3%, and transplanting survival rate reaches more than 90%, and this method is not subject to seasonal restrictions, and can breed green colored lily throughout the year, fast, realizes the large-scale production of green colored lily.
Embodiment 2
Step 1, select the scaly leaf on the underground bulb of green colored lily of no scab, with washing agent the earth on the described scaly leaf is cleaned, wash 0.5h with flowing water then, adopt conventional explant material sterilizing methods that the scaly leaf after washing is sterilized again, obtain explant; Described conventional explant material sterilizing methods is: be 75% ethanol disinfection 30s with scaly leaf at desinfection chamber workbench quality purity, throw in 5min～10min in 0.1% the mercuric chloride thimerosal then, use aseptic water washing at last 2～3 times;
Step 2, under aseptic condition explant described in the step 1 is inoculated on the callus inducing medium, cultivating after two months, whole explant produces a large amount of callus; Described callus inducing medium is: add 6-benzyl aminopurine 1.5mg/L, methyl 0.2mg/L and agar 6g/L in the conventional minimal medium of MS; The pH value of described callus inducing medium is 5.4; Described culture condition is: 25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx;
Step 3, the callus that produces in step 2 under aseptic condition are inoculated in subculture medium and breed cultivation, and propagation is cultivated after 12 days callus and increased and have new callus to produce; Described subculture medium is: add 6-benzyl aminopurine 2.0mg/L, methyl 0.05mg/L and agar 12g/L in the conventional minimal medium of MS; The pH value of described subculture medium is 5.8; Described propagation culture condition is: 25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 2000Lx;
Step 4, under aseptic condition, the callus that increases described in the step 3 or new callus are inoculated in the bud differential medium and carry out the bud induction, inoculate and differentiate the bud of growing thickly after 50 days; Described bud differential medium is: add 6-benzyl aminopurine 1.5mg/L, methyl 0.2mg/L and agar 6g/L in the conventional minimal medium of MS; The pH value of described bud differential medium is 5.4; The condition of culture of described bud induction is: 21 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx;
Step 5, the bud of growing thickly that differentiates in step 4 under aseptic condition are inoculated on the root media, cultivate the green colored lily seedling that acquisition is taken root after 15 days; Described root media is: add 6-benzyl aminopurine 1.2mg/L, methyl 0.08mg/L and agar 6g/L in the conventional minimal medium of MS; The pH value of described root media is 5.4; Described culture condition is: 21 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 2000Lx;
Step 6, the green colored lily sprigging of taking root described in the step 5 is cultivated in cultivation matrix, obtain green colored lily regeneration plant; Described cultivation matrix is by the peat composed of rotten mosses, perlite and husky the composition, and wherein the peat composed of rotten mosses, perlite and husky volume ratio are 5: 1: 2.
The present embodiment rooting rate reaches more than 93.3%, and transplanting survival rate reaches more than 90%, and this method is not subject to seasonal restrictions, and can breed green colored lily throughout the year, fast, realizes the large-scale production of green colored lily.
Embodiment 3
Step 1, select the scaly leaf on the underground bulb of green colored lily of no scab, with washing agent the earth on the described scaly leaf is cleaned, wash 1.5h with flowing water then, adopt conventional explant material sterilizing methods that the scaly leaf after washing is sterilized again, obtain explant; Described conventional explant material sterilizing methods is: be 75% ethanol disinfection 30s with scaly leaf at desinfection chamber workbench quality purity, throw in 5min～10min in 0.1% the mercuric chloride thimerosal then, use aseptic water washing at last 2～3 times;
Step 2, under aseptic condition explant described in the step 1 is inoculated on the callus inducing medium, cultivating after two months, whole explant produces a large amount of callus; Described callus inducing medium is: add 6-benzyl aminopurine 2.5mg/L, methyl 0.05mg/L and agar 12g/L in the conventional minimal medium of MS; The pH value of described callus inducing medium is 5.4～5.8; Described culture condition is: 21 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 2000Lx;
Step 3, the callus that produces in step 2 under aseptic condition are inoculated in subculture medium and breed cultivation, and propagation is cultivated after 18 days callus and increased and have new callus to produce; Described subculture medium is: add 6-benzyl aminopurine 0.8mg/L, methyl 0.2mg/L and agar 6g/L in the conventional minimal medium of MS; The pH value of described subculture medium is 5.4; Described propagation culture condition is: 21 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx;
Step 4, under aseptic condition, the callus that increases described in the step 3 or new callus are inoculated in the bud differential medium and carry out the bud induction, inoculate and differentiate the bud of growing thickly after 40 days; Described bud differential medium is: add 6-benzyl aminopurine 0.5mg/L, methyl 0.05mg/L and agar 12g/L in the conventional minimal medium of MS; The pH value of described bud differential medium is 5.8; The condition of culture of described bud induction is: 25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 2000Lx;
Step 5, the bud of growing thickly that differentiates in step 4 under aseptic condition are inoculated on the root media, cultivate the green colored lily seedling that acquisition is taken root after 10 days; Described root media is: add 6-benzyl aminopurine 0.5mg/L, methyl 0.02mg/L and agar 12g/L in the conventional minimal medium of MS; The pH value of described root media is 5.8; Described culture condition is: 25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx;
Step 6, the green colored lily sprigging of taking root described in the step 5 is cultivated in cultivation matrix, obtain green colored lily regeneration plant; Described cultivation matrix is by the peat composed of rotten mosses, perlite and husky the composition, and wherein the peat composed of rotten mosses, perlite and husky volume ratio are 7: 3: 2.
The present embodiment rooting rate reaches more than 93.3%, and transplanting survival rate reaches more than 90%, and this method is not subject to seasonal restrictions, and can breed green colored lily throughout the year, fast, realizes the large-scale production of green colored lily.
Embodiment 4
Step 1, select the scaly leaf on the underground bulb of green colored lily of no scab, with washing agent the earth on the described scaly leaf is cleaned, wash 1h with flowing water then, adopt conventional explant material sterilizing methods that the scaly leaf after washing is sterilized again, obtain explant; Described conventional explant material sterilizing methods is: be 75% ethanol disinfection 30s with scaly leaf at desinfection chamber workbench quality purity, throw in 5min～10min in 0.1% the mercuric chloride thimerosal then, use aseptic water washing at last 2～3 times;
Step 2, under aseptic condition explant described in the step 1 is inoculated on the callus inducing medium, cultivating after two months, whole explant produces a large amount of callus; Described callus inducing medium is: add 6-benzyl aminopurine 2.0mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described callus inducing medium is 5.6; Described culture condition is: 23 ± 2 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 2000Lx;
Step 3, the callus that produces in step 2 under aseptic condition are inoculated in subculture medium and breed cultivation, and propagation is cultivated after 14 days callus and increased and have new callus to produce; Described subculture medium is: add 6-benzyl aminopurine 1.2mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described subculture medium is 5.6; Described propagation culture condition is: 23 ± 2 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 2000Lx;
Step 4, under aseptic condition, the callus that increases described in the step 3 or new callus are inoculated in the bud differential medium and carry out the bud induction, inoculate and differentiate the bud of growing thickly after 45 days; Described bud differential medium is: add 6-benzyl aminopurine 1.0mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described bud differential medium is 5.6; The condition of culture of described bud induction is: 23 ± 2 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 2000Lx;
Step 5, the bud of growing thickly that differentiates in step 4 under aseptic condition are inoculated on the root media, cultivate the green colored lily seedling that acquisition is taken root after 15 days; Described root media is: add 6-benzyl aminopurine 0.8mg/L, methyl 0.05mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described root media is 5.6; Described culture condition is: 23 ± 2 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 2000Lx;
Step 6, the green colored lily sprigging of taking root described in the step 5 is cultivated in cultivation matrix, obtain green colored lily regeneration plant; Described cultivation matrix is by the peat composed of rotten mosses, perlite and husky the composition, and wherein the peat composed of rotten mosses, perlite and husky volume ratio are 6: 2: 2.
The present embodiment rooting rate reaches more than 93.3%, and transplanting survival rate reaches more than 90%, and this method is not subject to seasonal restrictions, and can breed green colored lily throughout the year, fast, realizes the large-scale production of green colored lily.
The above; it only is preferred embodiment of the present invention; be not that the present invention is done any restriction, every any simple modification, change and equivalent structure of above embodiment being done according to the invention technical spirit changes, and all still belongs in the protection domain of technical solution of the present invention.
Claims (2)
1. green colored lily method for quickly breeding is characterized in that this method may further comprise the steps:
Step 1, select the scaly leaf on the underground bulb of green colored lily of no scab, with washing agent the earth on the described scaly leaf is cleaned, wash 0.5h～1.5h with flowing water then, adopt conventional explant material sterilizing methods that the scaly leaf after washing is sterilized again, obtain explant;
Step 2, under aseptic condition explant described in the step 1 is inoculated on the callus inducing medium, cultivating after two months, whole explant produces callus; Described callus inducing medium is: add 6-benzyl aminopurine 2.0mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described callus inducing medium is 5.6; Described culture condition is: 21 ℃～25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx～2000Lx;
Step 3, the callus that produces in step 2 under aseptic condition are inoculated in subculture medium and breed cultivation, and propagation is cultivated after 12～18 days callus and increased and have new callus to produce; Described subculture medium is: add 6-benzyl aminopurine 1.2mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described subculture medium is 5.6; Described propagation culture condition is: 21 ℃～25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx～2000Lx;
Step 4, under aseptic condition, the callus that increases described in the step 3 or new callus are inoculated in the bud differential medium and carry out the bud induction, inoculate and differentiate the bud of growing thickly after 40～50 days; Described bud differential medium is: add 6-benzyl aminopurine 1.0mg/L, methyl 0.1mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described bud differential medium is 5.6; The condition of culture of described bud induction is: 21 ℃～25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx～2000Lx;
Step 5, the bud of growing thickly that differentiates in step 4 under aseptic condition are inoculated on the root media, cultivate the green colored lily seedling that acquisition is taken root after 10～15 days; Described root media is: add 6-benzyl aminopurine 0.8mg/L, methyl 0.05mg/L and agar 9g/L in the conventional minimal medium of MS; The pH value of described root media is 5.6; Described culture condition is: 21 ℃～25 ℃ of cultivation temperature, and 12h illumination and 12h are dark alternately to be cultivated, and intensity of illumination is 1000Lx～2000Lx;
Step 6, the green colored lily sprigging of taking root described in the step 5 is cultivated in cultivation matrix, obtain green colored lily regeneration plant; Described cultivation matrix is by the peat composed of rotten mosses, perlite and husky the composition, and wherein the peat composed of rotten mosses, perlite and husky volume ratio are 5～7: 1～3: 2.
2. green colored lily method for quickly breeding according to claim 1 is characterized in that, the peat composed of rotten mosses described in the step 6, perlite and husky volume ratio are 6: 2: 2.
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| CN113115707A (en) * | 2021-04-16 | 2021-07-16 | 广西壮族自治区农业科学院 | Propagation method of female parent for oriental lily planting |
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| CN104396742B (en) * | 2014-10-30 | 2017-03-15 | 毕节市中药研究所 | The method that aseptic seedling is differentiated again with five-step approach induction Lilium sulphureum Baker bulbil calluss |
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