CN102792889A

CN102792889A - Chuanminshen violaceum tissue culture rapid propagation technology

Info

Publication number: CN102792889A
Application number: CN2012102730601A
Authority: CN
Inventors: 孙辉; 龚艺
Original assignee: Sichuan University
Current assignee: Sichuan Greenland Jingtai Agriculture Co., Ltd.
Priority date: 2012-08-02
Filing date: 2012-08-02
Publication date: 2012-11-28
Anticipated expiration: 2032-08-02
Also published as: CN102792889B

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

The bright ginseng group culturation rapid propagating technology in river

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.Along Sichuan middle part, east (Jintang, Jianyang, Palestine and China, Langzhong, south) western to Hubei (Yichang, work as sun) strip distributes, and is the most concentrated with Jintang, Sichuan, Cangxi, Langzhong, Palestine and China etc." Chinese medicinal herbal " (1959), " Sichuan Chinese medicinal herbal " etc. once recorded bright ginseng in river and Radix changii as same plant species; She Menglan etc. carry out thinking after the form comparative studies to two plant species; The two non-same plant species, thereby set up the bright ginseng genus in river, and think that two genus should be under the jurisdiction of different families.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; Quantitatively illustrated three of this 3 plant species representative belong between affiliation; 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 effects such as moistening lung for removing phlegm and middle nourishing the stomach are arranged, and cures mainly that lung-heat type cough, deficiency of Yin labor are coughed, 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 good characteristics such as matter is tender, powder foot, soup aquatic foods, has the health care function simultaneously.Because the bright ginseng in river not only medical value is high, and aspect edible, have excellent development value and health care, market potential is huge, because the bright ginseng in river is bigger as the long-term demand of medicine-food two-purpose product, the market price is surging year by year.

Mostly the bright ginseng in present stage river is artificial cultivation, and the cultivation provenance comes from the seed that wild plant domestication is produced, 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.The low seedling that causes of germination rate is not enough high with the cultivation cost, and seedling quality also is difficult to guarantee, has restricted development and the raising of the unit are output value of the large-scale planting of the bright ginseng in river.Bright ginseng tissue culture in river and rapid propagation in vitro technology is not arranged at present as yet.

Therefore, utilize plant tissue culture technique, realize the tissue rapid propagation of the bright ginseng in river; Have important use value aspect the industrialization of the bright ginseng in river: the batch production standard production of the bright ginseng seedling in river is realized in (1); Reproduction speed is fast, but equal throughout the year propagation production seedling does not receive region and climatic effect; (2) fast fine-variety breeding with expand numerously, expand numerously fast for good plant, and realize the quick breeding of breeding on this basis, 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 through choosing the good river of proterties bright ginseng plant vigorous period are explant; The formation callus is induced in sterile-processed, inoculation, breeds, lures bud to cultivate and culture of rootage through callus again, grows 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 following:

1, explant selection and sterilization: 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, subsequent use.

2, explant inoculation: the fritter that explant 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,, cultivate under illumination (2000lx) the 14h/d condition 25 ± 2 ℃ of constant temperature; Incubation time is 20 days ~ 30 days, the bright ginseng in the river bud of growing thickly occurs.

5, culture of rootage:, be forwarded in the root media, 25 ± 2 ℃ of constant temperature with the seedling plant division of growing thickly of the bright ginseng in the river that obtains in the step 4; 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, to seedling medium, atomizing is preserved moisture and is cultivated under the condition in the greenhouse with the complete little transplantation of seedlings of the band root that obtains in the step 5; Until the seedling survival, grow 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 the 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&Skoog1962, and macroelement concentration among the conventional minimal medium MS is reduced by half obtains 1/2MS, and 6-BA is a 6-benzyladenine; NAA is a methyl, 2, and 4-D is 2; 4-dichlorphenoxyacetic acid, IBA are 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.

Said complete seedling, bud is accomplished root, stem breaks up for the bright ginseng in river is grown thickly, and has grown 4 ~ 5 leaflets, can independently carry out the immature plant of photosynthesis autophyting growth.

Said 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 breed fast, and it is short that used medium brings out the time, 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: the batch production standard production of the bright ginseng seedling in river is realized in (1), and reproduction speed is fast, but equal throughout the year industrialized propagation is produced seedling, does not receive region and climatic effect; (2) be the technological means of quick fine-variety breeding, expand numerously for good plant fast, find that a good plant of shape can pass through the tissue-culturing rapid propagation large tracts of land and promote cultivation, preserve good characteristic simultaneously fully, for the large-scale artificial cultivation provides good seed; (3) the bright ginseng fine germplasm resources in implemented with low cost river is preserved.

Embodiment

Embodiment 1

1, explant selection and sterilization: the young leaflet tablet of choosing the bright ginseng vigorous period in river is an 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, subsequent use.

2, explant inoculation: explant subsequent use in the step 1 is cut into the 0.5cm2 size on superclean bench, (this medium is: 1/2MS+6-BA1.0mg/L+2,4-D10.0mg/L+NAA5.0mg/L to be inoculated in callus inducing medium; Add 30g/L sucrose, 8g/L agar powder; PH=5.8) in,, cultivate under illumination (2000lx) the 14h/d condition 25 ± 2 ℃ of constant temperature; After one week; Arch upward in the middle of the young leaflet tablet, continuous culture grows callus after about 25 days around the young leaflet tablet otch.

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 soya bean size bulk that the callus that obtains in the step 3 is cut, and is inoculated in inducing clumping bud medium (MS+6-BA5.0mg/L+NAA1.0mg/L, adding 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 of the shape that occurs after about 30 days becoming the to cluster 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, (1/2MS+IBA1.0mg/L+NAA1.0mg/L adds 30g/L sucrose, 8g/L agar powder to be forwarded to root media; 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.

Embodiment 2

1, explant selection and sterilization: the fresh young tender leaf handle of choosing the bright ginseng vigorous period in river is an 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, subsequent use.

2, explant inoculation: explant subsequent use in the step 1 is cut into the fritter of 0.5 ~ 1cm on superclean bench, (this medium is: 1/2MS+6-BA3.0mg/L+2,4-D20.0mg/L+NAA1.0mg/L to be inoculated in callus inducing medium; Add 30g/L sucrose, 8g/L agar powder; PH=5.8) in,, cultivate under illumination (2000Lx) the 14h/d condition 25 ± 2 ℃ of constant temperature; After one week; In the middle of the petiole and incision expand, continuous culture is after about 20 days, 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 soya bean size bulk that the callus that obtains in the step 3 is cut, and is inoculated in inducing clumping bud medium (MS+6-BA10.0mg/L+NAA5.0mg/L, adding 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, (1/2MS+IBA10.0mg/L+NAA2.0mg/L adds 30g/L sucrose, 8g/L agar powder to be forwarded to root media; 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.

Embodiment 3

1, explant selection and sterilization: the stem apex of choosing the bright ginseng vigorous period in river is an 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, subsequent use.

2, explant inoculation: explant subsequent use in the step 1 is peeled off the blade that adheres on 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 the week; Arch upward in the middle of the young leaflet tablet; Petiole centre and incision are expanded, and continuous culture grows callus in the stem apex incision after about 20 days.

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 soya bean size bulk that the callus that obtains in the step 3 is cut, and is inoculated in inducing clumping bud medium (MS+6-BA10.0mg/L+NAA2.0mg/L, adding 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, (1/2MS+IBA10.0mg/L+NAA1.0mg/L adds 30g/L sucrose, 8g/L agar powder to be forwarded to root media; 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.

Claims

1. bright ginseng method for tissue culture in river is characterized in that: stem apex, young leaflet tablet and fresh young tender leaf handle with the good river of proterties bright ginseng plant vigorous period are explant, 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.

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 following:

1) explant selection and sterilization: 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, subsequent use;

2) explant inoculation: the fritter that explant 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 increment: the callus that obtains in the step 2 is inoculated in the callus increment 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 to 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, to seedling medium, atomizing is preserved moisture and is cultivated under the condition in the greenhouse with the complete little transplantation of seedlings of the band root that obtains in the step 5; Until the seedling survival, grow 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 is characterized in that: the bright ginseng in described river is annual or the bright ginseng in bright ginseng platymiscium river, Umbelliferae river that is in vegetative period (November ~ February next year) of life in 2 years ( Chuanminshen violaceum).

4. the method for tissue culture of the bright ginseng in a kind of river according to claim 2 is characterized in that: 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.

5. the bright ginseng method for tissue culture in a kind of river according to claim 2 is characterized in that: described callus increment medium is MS+2,4-D10.0 ~ 20.0mg/L+NAA1.0 ~ 5.0mg/L.

6. the bright ginseng method for tissue culture in a kind of river according to claim 2 is characterized in that: described inducing clumping bud medium is MS+6-BA5.0 ~ 10.0mg/L+NAA1.0 ~ 5.0mg/L.

7. the bright ginseng method for tissue culture in a kind of river according to claim 2 is characterized in that: described root media is 1/2MS+IBA1.0 ~ 10.0mg/L+NAA1.0 ~ 2.0mg/L.

8. according to the bright ginseng method for tissue culture in the described a kind of river of claim 4 ~ 7, it is characterized in that: described MS is conventional minimal medium Murashige&Skoog1962, and macroelement concentration among the conventional minimal medium MS is reduced by half obtains 1/2MS; 6-BA is a 6-benzyladenine; NAA is a methyl, 2, and 4-D is 2; 4-dichlorphenoxyacetic acid, IBA are indolebutyric acid.

9. the bright ginseng method for tissue culture in a kind of river according to claim 2 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.

10. the bright ginseng method for tissue culture in a kind of river according to claim 1 and 2 is characterized in that: said seedling medium is mixtures such as peat soil, perlite, total porosity 75%, organic 15%.