CN102792889B - Chuanminshen violaceum tissue culture rapid propagation technology - Google Patents
Chuanminshen violaceum tissue culture rapid propagation technology Download PDFInfo
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Abstract
The invention provides a Chuanminshen violaceum tissue culture method. According to the method, the stem tip, tender leaves and fresh tender leaf stalks of the Chuanminshen violaceum plant with good characters in the vigorous growth period are utilized as explants, and the method comprises the following steps of: performing disinfection treatment and inoculation induction to form callus; performing callus multiplication, bud induction culture and rooting culture to obtain complete Chuanminshen violaceum plant seedling; and transplanting the complete seedling into a seedling culture medium, and culturing to obtain a Chuanminshen violaceum annual seedling. All the culture media are based on an MS culture medium in combination with components such as 6-benzyladenine, naphthylacetic acid, 2,4-dichlorphenoxyacetic acid, indolebutyric acid, sucrose an agar. The method provided by the invention can be used for realizing the tissue rapid propagation of Chuanminshen violaceum by use of a plant tissue culture technology, and has important application values in terms of annual seedling supply and improved-variety rapid expanding propagation of the industrialized cultivation of Chuanminshen violaceum.
Description
Technical field
The present invention relates to field of plant tissue culture technique, mainly is the method for tissue culture of the bright ginseng in river.
Background technology
The bright ginseng in river (
Chuanminshen violaceumSheh et Shan) be the bright ginseng platymiscium in Umbelliferae (Umbelliferae) river, perennial herb is the peculiar autogenus plant of China, is the Sichuan genunie medicinal materials.(Yichang, work as sun) strip distributes along central Sichuan, east (Jintang, Jianyang, Palestine and China, Langzhong, south) to West of Hubei Province, and is the most concentrated with Jintang, Sichuan, Cangxi, Langzhong, Palestine and China etc." Chinese medicinal herbal " (1959), " Traditional Chinese Medicine in Sichuan will " etc. were once recorded the bright ginseng in river and Radix changii as the same plant, She Menglan etc. carry out thinking after the form comparative studies to two Plants, the two non-same plant, thereby set up the bright ginseng genus in river, and think that two genus should be under the jurisdiction of different families.The employing gas chromatography-mass spectrographies such as Lu Yin have carried out cluster analysis to the chemical composition of rare monotypic genus medicinal plant Radix changii in imminent danger, the bright ginseng in river and glehnia littoralis root volatile oil, three Relationships of this 3 Plants representative have quantitatively been illustrated, think that the affiliation of the bright participation glehnia littoralis in river is more approaching, and far away with the affiliation of Radix changii.
The bright ginseng in river is used as medicine with dry root, and sweet, little hardship of distinguishing the flavor of, flat is returned lung, the spleen channel, and moistening lung for removing phlegm is arranged, and the effect such as middle nourishing the stomach, cures mainly that lung-heat type cough, deficiency of Yin labor are coughed, the diseases such as dry cough phlegm is sticking, deficiency of both vital energy and Yin, restlessness and fever with thirst.The bright ginseng in river is edible with a long history, and the bright ginseng in river is delicious as edible taste, has the good characteristics such as matter is tender, powder sufficient, soup is bright, has simultaneously health-caring function.Because the bright ginseng in river not only medical value is high, and has good exploitation value and health care aspect edible, market potential is huge, because the bright ginseng in river is larger as the long-term demand of medicine-food two-purpose product, the market price is surging year by year.
The bright ginseng in present stage river mostly is artificial cultivation, and the cultivation provenance comes from the seed that the Wild plant domestication produces, and the provenance source is numerous and diverse, and quality is uneven.River bright ginseng seed wildness is strong, and germination rate is low and germinating time is uneven, and the holding time of seed is short, emergence rate less than 30%, and the seed seedling-raising sowing quantity reaches 10kg ~ 30kg/ mu.Germination rate is low to cause seedling not enough and the cultivation cost is high, and seedling quality also is difficult to guarantee, has restricted the development of large-scale planting of the bright ginseng in river and the raising of the unit are output value.Not yet there is at present the bright ginseng tissue in river to cultivate and the rapid propagation in vitro technology.
Therefore, utilize plant tissue culture technique, realize the tissue rapid propagation of the bright ginseng in river, have important using value aspect the industrialization of the bright ginseng in river: (1) realizes the batch production standard production of the bright ginseng seedling in river, reproduction speed is fast, all can breed the production seedling throughout the year, is not subjected to region and climatic effect; (2) fine-variety breeding is numerous with expansion fast, carries out fast-propagation for good plant, and realizes on this basis the quick breeding of breeding, for the large-scale artificial cultivation provides good seed; (3) preserve the low-cost technologies means that provide for the bright ginseng germ plasm resource in river.
Summary of the invention
The bright ginseng method for tissue culture in a kind of river, stem apex, young leaflet tablet and fresh young tender leaf handle by choosing the good river of proterties bright ginseng plant vigorous period are explant, the formation callus is induced in sterile-processed, inoculation, breed, lure bud to cultivate and culture of rootage through callus again, grow up to complete river bright ginseng plant seedling, at last with complete little transplantation of seedlings to seedling medium, cultivate into the bright ginseng seedling in river.
The bright ginseng method for tissue culture in described river, its step is as follows:
1, the choosing and sterilizing of explant: stem apex, young leaflet tablet and the fresh young tender leaf handle of choosing the bright ginseng vigorous period in river are explant, with flowing water flushing 20 ~ 30min, again with 75% alcohol-pickled 30s, with aseptic deionized water flushing 3 ~ 5 times, explant after will washing again places 0.1% ~ 0.2% mercuric chloride solution, soaks sterilization 10 ~ 15 min, with aseptic deionized water flushing 2 ~ 3 times, blot surface moisture with aseptic filter paper, for subsequent use.
2, explant inoculation: the fritter that explant for subsequent use in the step 1 is cut into 0.5cm ~ 1cm, be inoculated in the callus inducing medium, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000lx) the 14h/d condition, incubation time is 20 days ~ 25 days, around stem apex otch, young leaflet tablet otch, petiole otch and centre expand director and go out callus.
3, callus increment: the callus that obtains in the step 2 is inoculated in the callus increment medium, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000lx) the 14h/d condition, incubation time is 15 days ~ 20 days, grows faint yellow or the milky dense callus.
4, inducing clumping bud: it is that soya bean is big or small block that the callus that obtains in the step 3 is cut, be inoculated in the inducing clumping bud medium, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000lx) the 14h/d condition, incubation time is 20 days ~ 30 days, the bright ginseng Multiple Buds in river occurs.
5, culture of rootage: with the seedling plant division of growing thickly of the bright ginseng in the river that obtains in the step 4, be forwarded in the root media, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000lx) the 14h/d condition, incubation time is 20 days ~ 25 days, and the seedling base portion grows greyish white short root, continues to cultivate to grow complete root system.
6, plantlet of transplant: before the transplanting, to contain peat soil, perlitic seedling medium carries out disinfection, the complete little transplantation of seedlings of band root that obtains in the step 5 to seedling medium, is cultivated under the atomizing moisturizing condition in the greenhouse, until the seedling survival grows up to just frequently bright ginseng plant.
Described river bright ginseng plant, for the bright ginseng in bright ginseng platymiscium river, Umbelliferae river that is in vegetative period (November ~ February next year) of annual or life in 2 years (
Chuanminshen violaceum).
Described dense callus is for non-particulate shape is dispersed in callus lines in the medium.
Described callus inducing medium is 1/2MS+6-BA1.0 ~ 3.0mg/L+2,4-D10.0 ~ 20.0mg/L+NAA1.0 ~ 5.0mg/L.
Described callus increment medium is MS+2,4-D10.0 ~ 20.0mg/L+NAA1.0 ~ 5.0mg/L.
Described inducing clumping bud medium is MS+6-BA5.0 ~ 10.0mg/L+NAA1.0 ~ 5.0mg/L.
Described root media is 1/2MS+IBA1.0 ~ 10.0mg/L+NAA1.0 ~ 2.0mg/L.
Described MS is conventional minimal medium Murashige﹠amp; Skoog1962, macroelement concentration among the conventional minimal medium MS reduced by half obtains 1/2MS, and 6-BA is 6-benzyladenine, and NAA is methyl α-naphthyl acetate, and 2,4-D is 2,4-dichlorphenoxyacetic acid, IBA is indolebutyric acid.
Described callus inducing medium, callus proliferated culture medium, inducing clumping bud medium and root media all add the agar of 8g/L, the sucrose of 30g/L.
Described complete seedling for the bright ginseng Multiple Buds in river is finished root, stem differentiation, and has grown 4 ~ 5 leaflets, can independently carry out the immature plant of photosynthesis autophyting growth.
Described seedling medium is peat soil, perlite mixture, and total porosity is more than 75%, and is organic more than 15%.
The present invention adopts method for tissue culture first, the bright ginseng in river is carried out tissue culture rapid speed breed, and the used medium Induction time is short, and it is fast to emerge, and the rate of increase is high, and easy operating is for the bright ginseng factorial seedling growth in river provides a new way.The invention has the advantages that: (1) realizes the batch production standard production of the bright ginseng seedling in river, and reproduction speed is fast, but equal industrialized propagation is produced seedling throughout the year, is not subjected to region and climatic effect; (2) be the technological means of quick fine-variety breeding, carry out fast-propagation for good plant, find that a good plant of shape can preserve good characteristic simultaneously fully through the cultivation of tissue-culturing rapid propagation spread, for the large-scale artificial cultivation provides good seed; (3) the low-cost bright ginseng fine germplasm resources in river of realizing is preserved.
Embodiment
Embodiment 1
1, the choosing and sterilizing of explant: the young leaflet tablet of choosing the bright ginseng vigorous period in river is explant, with flowing water flushing 30min, on superclean bench with 75% alcohol-pickled 30s, with aseptic deionized water flushing 3 times, explant after will washing again places 0.1% mercuric chloride solution, soaks sterilization 10 minutes, with aseptic deionized water flushing 3 times, blot surface moisture with aseptic filter paper, for subsequent use.
2, explant inoculation: explant for subsequent use in the step 1 is cut into the 0.5cm2 size at superclean bench, (this medium is: 1/2MS+6-BA1.0mg/L+2 to be inoculated in callus inducing medium, 4-D10.0mg/L+NAA5.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000lx) the 14h/d condition, after one week, arch upward in the middle of the young leaflet tablet, after about 25 days of the continuous culture, around the young leaflet tablet otch, grow callus.
3, callus increment: the callus that obtains in the step 2 is inoculated in callus increment medium, and (this medium is: MS+2,4-D10.0mg/L+NAA5.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000lx) the 14h/d condition, after 15 days, grow faint yellow or the milky dense callus.
4, inducing clumping bud: it is that soya bean is big or small block that the callus that obtains in the step 3 is cut, be inoculated in inducing clumping bud medium (MS+6-BA5.0mg/L+NAA1.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000lx) the 14h/d condition, after about 20 days, the bright ginseng in the river seedling of growing thickly appears, the bright ginseng in river that occurs into the shape that clusters after the about 30 days seedling of growing thickly.
5, culture of rootage: with the seedling plant division of growing thickly of the bright ginseng in the river that obtains in the step 4, be forwarded to root media (1/2MS+IBA1.0mg/L+NAA1.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, to cultivate under illumination (2000Lx) the 14h/d condition, incubation time is 25 days, the seedling base portion grows greyish white short root, continues to cultivate to grow complete root system.
6, plantlet of transplant: before the transplanting, to contain peat soil, perlitic seedling medium carries out disinfection, the complete little transplantation of seedlings of band root that obtains in the step 5 to seedling medium, is cultivated under the atomizing moisturizing condition in the greenhouse, until the seedling survival grows up to just frequently bright ginseng plant.
Embodiment 2
1, the choosing and sterilizing of explant: the fresh young tender leaf handle of choosing the bright ginseng vigorous period in river is explant, with flowing water flushing 20min, on superclean bench with 75% alcohol-pickled 30s, with aseptic deionized water flushing 3 times, explant after will washing again places 0.2% mercuric chloride solution, soaks sterilization 10 minutes, with aseptic deionized water flushing 3 times, blot surface moisture with aseptic filter paper, for subsequent use.
2, explant inoculation: the fritter that explant for subsequent use in the step 1 is cut into 0.5 ~ 1cm at superclean bench, (this medium is: 1/2MS+6-BA3.0mg/L+2 to be inoculated in callus inducing medium, 4-D20.0mg/L+NAA1.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000Lx) the 14h/d condition, after one week, petiole centre and incision are expanded, after about 20 days of the continuous culture, the director that expands in petiole otch and centre goes out callus.
3, callus increment: the callus that obtains in the step 2 is inoculated in callus increment medium, and (this medium is: MS+2,4-D20.0mg/L+NAA1.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000Lx) the 14h/d condition, after about 20 days, grow faint yellow or the milky dense callus.
4, inducing clumping bud: it is that soya bean is big or small block that the callus that obtains in the step 3 is cut, be inoculated in inducing clumping bud medium (MS+6-BA10.0mg/L+NAA5.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000Lx) the 14h/d condition, after about 25 days, the bright ginseng in the river seedling of growing thickly occurs, occur the bright ginseng in the cluster river seedling of growing thickly after about 35 days.
5, culture of rootage: with the seedling plant division of growing thickly of the bright ginseng in the river that obtains in the step 4, be forwarded to root media (1/2MS+IBA10.0mg/L+NAA2.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, to cultivate under illumination (2000lx) the 14h/d condition, incubation time is 30 days, the seedling base portion grows greyish white short root, continues to cultivate to grow complete root system.
6, plantlet of transplant: before the transplanting, to contain peat soil, perlitic seedling medium carries out disinfection, the complete little transplantation of seedlings of band root that obtains in the step 5 to seedling medium, is cultivated under the atomizing moisturizing condition in the greenhouse, until the seedling survival grows up to just frequently bright ginseng plant.
Embodiment 3
1, the choosing and sterilizing of explant: the stem apex of choosing the bright ginseng vigorous period in river is explant, with flowing water flushing 20min, on superclean bench with 75% alcohol-pickled 20s, with aseptic deionized water flushing 3 times, explant after will washing again places 0.2% mercuric chloride solution, soaks sterilization 10 minutes, with aseptic deionized water flushing 3 times, blot surface moisture with aseptic filter paper, for subsequent use.
2, explant inoculation: explant for subsequent use in the step 1 is peeled off the blade that adheres at superclean bench, obtain smooth stem apex, (this medium is: 1/2MS+6-BA2.0mg/L+2 to be inoculated in callus inducing medium, 4-D10.0mg/L+NAA5.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000Lx) the 14h/d condition, after one week, arches upward in the young leaflet tablet centre, and petiole centre and incision are expanded, after about 20 days of the continuous culture, grow callus in the stem apex incision.
3, callus increment: the callus that obtains in the step 2 is inoculated in callus increment medium, and (this medium is: MS+2,4-D10.0mg/L+NAA5.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000lx) the 14h/d condition, after about 20 days, grow the milky dense callus;
4, inducing clumping bud: it is that soya bean is big or small block that the callus that obtains in the step 3 is cut, be inoculated in inducing clumping bud medium (MS+6-BA10.0mg/L+NAA2.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, cultivate under illumination (2000lx) the 14h/d condition, after about 25 days, the bright ginseng in the river seedling of growing thickly occurs, occur the bright ginseng in the cluster river seedling of growing thickly after about 35 days.
5, culture of rootage: with the seedling plant division of growing thickly of the bright ginseng in the river that obtains in the step 4, be forwarded to root media (1/2MS+IBA10.0mg/L+NAA1.0mg/L, add 30g/L sucrose, 8g/L agar powder, pH=5.8) in, 25 ± 2 ℃ of constant temperature, to cultivate under illumination (2000lx) the 14h/d condition, incubation time is 30 days, the seedling base portion grows greyish white short root, continues to cultivate to grow complete root system.
6, plantlet of transplant: before the transplanting, to contain peat soil, perlitic seedling medium carries out disinfection, the complete little transplantation of seedlings of band root that obtains in the step 5 to seedling medium, is cultivated under the atomizing moisturizing condition in the greenhouse, until the seedling survival grows up to just frequently bright ginseng plant.
Claims (5)
1. bright ginseng method for tissue culture in river is characterized in that: stem apex, young leaflet tablet and the fresh young tender leaf handle of bright ginseng plant vigorous period are as explant take the good river of proterties, and the formation callus is induced in sterile-processed, inoculation; Breed, lure bud to cultivate and culture of rootage through callus again, grow up to complete river bright ginseng plant seedling; At last with complete little transplantation of seedlings to seedling medium, cultivate into the bright ginseng seedling in river; Wherein:
Callus inducing medium is 1/2MS+6-BA1.0～3.0mg/L+2,4-D10.0～20.0mg/L+NAA1.0～5.0mg/L;
The callus proliferated culture medium is MS+2,4-D10.0～20.0mg/L+NAA1.0～5.0mg/L;
The inducing clumping bud medium is MS+6-BA5.0～10.0mg/L+NAA1.0～5.0mg/L;
Root media is 1/2MS+IBA1.0～10.0mg/L+NAA1.0～2.0mg/L;
Described MS is conventional minimal medium Murashige﹠amp; Skoog1962, macroelement concentration among the conventional minimal medium MS reduced by half obtains 1/2MS, and 6-BA is 6-benzyladenine, and NAA is methyl α-naphthyl acetate, and 2,4-D is 2,4-dichlorphenoxyacetic acid, IBA is indolebutyric acid.
2. the bright ginseng method for tissue culture in a kind of river according to claim 1 is characterized in that: the bright ginseng method for tissue culture in described river, and its step is as follows:
1) the choosing and sterilizing of explant: stem apex, young leaflet tablet and the fresh young tender leaf handle of choosing the bright ginseng vigorous period in river are explant, with flowing water flushing 20～30min, again with 75% alcohol-pickled 30s, with aseptic deionized water flushing 3～5 times, explant after will washing again places 0.1%～0.2% mercuric chloride solution, soaks sterilization 10～15 minutes, with aseptic deionized water flushing 2～3 times, blot surface moisture with aseptic filter paper, for subsequent use;
2) explant inoculation: the fritter that explant for subsequent use in the step 1) is cut into 0.5～1cm, be inoculated in the callus inducing medium, incubation time is 20～25 days, around stem apex otch, young leaflet tablet otch, petiole otch and centre expand director and go out callus;
3) callus that obtains callus increment: with step 2) is inoculated in the callus proliferated culture medium, and incubation time is 15～20 days, grows faint yellow or the milky dense callus;
4) inducing clumping bud: the callus that obtains in the step 3) is cut as soya bean size block, be inoculated in the inducing clumping bud medium, incubation time is 20～30 days, the bright ginseng in the river seedling of growing thickly occurs;
5) culture of rootage: with the seedling plant division of growing thickly of the bright ginseng in the river that obtains in the step 4), be forwarded in the root media, incubation time is 20～25 days, and the seedling base portion grows greyish white short root, continues to cultivate to grow complete root system;
6) plantlet of transplant: before the transplanting, to contain peat soil, perlitic seedling medium carries out disinfection, the complete little transplantation of seedlings of band root that obtains in the step 5) to seedling medium, is cultivated under the atomizing moisturizing condition in the greenhouse, until the seedling survival grows up to just frequently bright ginseng plant.
3. the bright ginseng method for tissue culture in a kind of river according to claim 1 and 2, it is characterized in that: the bright ginseng in described river is annual or the bright ginseng in bright ginseng platymiscium river, Umbelliferae river (Chuanminshen violaceum) in the vegetative period that is in November～February next year of life in 2 years.
4. the bright ginseng method for tissue culture in a kind of river according to claim 2, it is characterized in that: described callus inducing medium, callus proliferated culture medium, inducing clumping bud medium and root media all add the agar of 8g/L, the sucrose of 30g/L.
5. the bright ginseng method for tissue culture in a kind of river according to claim 1 and 2 is characterized in that: described seedling medium is peat soil, perlite mixture, total porosity 75%, organic 15%.
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| CN107006326B (en) * | 2017-04-26 | 2020-03-24 | 中国科学院合肥物质科学研究院 | Efficient breeding method for excellent Denmark hibiscus line |
| CN108739405B (en) * | 2018-07-07 | 2023-01-20 | 云南省农业科学院药用植物研究所 | Tissue culture and in-vitro regeneration method of rhizoma panacis majoris |
| CN115777536B (en) * | 2022-11-30 | 2023-08-18 | 中南民族大学 | Method for establishing efficient regeneration system by utilizing stems of peucedanum praeruptorum dunn |
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