CN101200704A - Tissue culturing method for broussonetia papyrifera - Google Patents
Tissue culturing method for broussonetia papyrifera Download PDFInfo
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- CN101200704A CN101200704A CNA2007103041272A CN200710304127A CN101200704A CN 101200704 A CN101200704 A CN 101200704A CN A2007103041272 A CNA2007103041272 A CN A2007103041272A CN 200710304127 A CN200710304127 A CN 200710304127A CN 101200704 A CN101200704 A CN 101200704A
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Abstract
The present invention discloses a tissue culture method of broussonetia papyrifera. The tissue culture method of the broussonetia papyrifera provided by the present invention is that the top winter buds of the broussonetia papyrifera is cultured inside a germination culture medium to obtain germinated winter buds. The germination culture medium is the culture medium obtained by adding IAA, 6-BA and GA into a MS culture medium. The final concentration of the IAA is 0.1 to 0.5 milligram per liter; the final concentration of the 6-BA is 0.1 to 0.5 milligram per liter; the final concentration of the GA is 0.1 milligram per liter. The method of the present invention has the advantages of easily obtaining material, low variation rate and high proliferation coefficient; proliferation multiple is 4 to 8 times after culturing for four weeks; the tissue culture seedling rooting rate is 92 percent; the hardening-seedling survival rate is 93 percent. The method of the present invention is used for the propagation of the broussonetia papyrifera, which can improve the biological output of the broussonetia papyrifera.
Description
Technical field
The present invention relates to the propagation method of plant, particularly relate to a kind of tissue culture method of paper mulberry.
Background technology
Paper mulberry (Broussonetia papyrifera L.) is a kind of deciduous tree, and the height of tree reaches 16 meters; Bark is level and smooth, light gray; Branch is sturdy, open and flat, sorrel, the white fine hair of close life; Ye Kuo is avette, and long 8-20 centimetre, wide 6-15 centimetre, the sharp point in top, base portion is circular or near heart-shaped, and there is bastard at the edge, 3-5 drastic crack (leaf on the sprout is more obvious), there is thick pubescence on the two sides; The long 3-5 of petiole centimetre, close living fine hair; Holder leaf egg shape Long Circle, caducous; The flower dioecy, male inflorescence is that armpit is given birth to vertical dish chrysanthemum preface, long 6-8 centimetre; The female inflorescence head, bract is bar-shaped, the nose-circle taper, hairiness, the style base portion is branch not.The syncarp sphere, about 3 centimetres of diameter; In May at florescence, fructescence is September.
Paper mulberry adaptability is extremely strong, can cold resistant and arid, and barren-resistant and damp and hot, well developed root system, little disease and pest can be used as the green tree species in barren beach, remote area, also can be used as shade tree; Tapa is the high-quality paper making raw material; Paper mulberry leaf protein matter content is up to 20%-30%, and nutritive ingredients such as amino acid, VITAMIN, carbohydrate and trace element are also very abundant, can be used for production full price animal and fowl fodder after science processing; Fruit and root can be used as medicine, energy kidney tonifying diuresis, strengthening the bones and muscles, and paper mulberry really has rich nutrient contents, can be made into natural organic multifunctional health care beverage; The milk of leaf can be wiped and control the sore tinea; Paper mulberry can also be resisted toxic gas (sulfurous gas and chlorine), can plant in the serious area of topsoil.
Summary of the invention
A kind of tissue culture method that the purpose of this invention is to provide paper mulberry.
Paper mulberry tissue culture method provided by the present invention is that paper mulberry terminal bud or lateral bud are cultivated in germination medium, the no offspring that obtains sprouting; Described germination medium is for adding the substratum that IAA, 6-BA and GA obtain in the MS substratum, the final concentration of described IAA is the 0.1-0.5 mg/litre, and the final concentration of described 6-BA is the 0.1-0.5 mg/litre, and the final concentration of described GA is the 0-0.1 mg/litre.
The final concentration of described IAA specifically can be 0.5 mg/litre, and the final concentration of described 6-BA specifically can be 0.5 mg/litre, and the final concentration of described GA specifically can be 0.1 mg/litre.
Also comprise in the described method the no offspring of described sprouting is carried out the step of adventitious bud inducing and subculture at adventitious bud inducing and subculture medium, obtain not having offspring.
Described adventitious bud inducing and subculture medium are for adding the substratum that IAA, 6-BA and ZT obtain in the MS substratum, the final concentration of described IAA is the 0-0.1 mg/litre, the final concentration of described 6-BA is the 0.1-2.0 mg/litre, and the final concentration of described ZT is 0.3 mg/litre.
The final concentration of described IAA specifically can be 0.1 mg/litre, and the final concentration of described 6-BA specifically can be 1.5 mg/litre.
Also comprise the step of described no offspring being carried out root induction at the root induction substratum in the described method.
Described root induction substratum is for adding the substratum that IAA, NAA, 6-BA and ZT obtain in the MS substratum, the final concentration of described IAA is the 0.3-1.0 mg/litre, the final concentration of described NAA is the 0.1-1.0 mg/litre, the final concentration of described 6-BA is 0.1 mg/litre, and the final concentration of described ZT is 0.1 mg/litre.
The final concentration of described IAA specifically can be 1.0 mg/litre, and the final concentration of described NAA specifically can be 1.0 mg/litre.
The culture condition of described sprouting can be at 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-1The culture condition of described differentiation adventitious buds and subculture can be at 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1The culture condition of described root induction can be at 25 ± 2 ℃, and light application time is 10 hours, and intensity of illumination is 110 μ Mm
-2s
-1
The present invention is an explant with the paper mulberry terminal bud, utilizes its special culture media, successfully set up an effective paper mulberry isolated culture regeneration system, for a large amount of tissue-culturing quick-propagations of xylophyta provide one can reference technology and method.The inventive method has the following advantages: draw materials easily, very easily obtain sterilizable material, aberration rate is low, value-added coefficient is high, 4 weeks breed multiple and can reach 4-8 doubly, the tissue cultured seedling rooting rate reaches 92%, and the hardening surviving rate has very strong batch production throughput up to 93%.
Description of drawings
Fig. 1 is the paper mulberry differentiation adventitious buds
Fig. 2 is the paper mulberry root culture
Fig. 3 is the paper mulberry training tissue culture seedling
Fig. 4 is 1 year living paper mulberry tissue cultured seedling growing state
Embodiment
Related experimental technique is ordinary method if no special instructions among the following embodiment.
Used substratum is solid MS minimum medium (containing 30g/L sucrose) among the following embodiment.PH is 5.8-6.0.
Embodiment 1, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 0.5mg/L+ZT 0.3mg/L.
Root media: MS+IAA 0.3mg/L+NAA 0.5mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 75 ± 8%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 92 ± 5%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 92 ± 3% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1200kg/ mu, and the weight that only keeps 1 trunk is 900kg/ mu.
Embodiment 2, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 0.5mg/L+ZT 0.3mg/L.
Root media: MS+IAA 0.3mg/L+NAA 1.0mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 75 ± 8%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm-2s.3 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 90 ± 3%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 92 ± 4% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1250kg/ mu, and the weight that only keeps 1 trunk is 910kg/ mu.
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 0.5mg/L+ZT 0.3mg/L.
Root media: MS+IAA 0.5mg/L+NAA 0.3mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 70 ± 6%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 2000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 91 ± 4%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 95 ± 2% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1180kg/ mu, and the weight that only keeps 1 trunk is 880kg/ mu.
Embodiment 4, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 0.5mg/L+ZT 0.3mg/L.
Root media: MS+IAA 1.0mg/L+NAA 0.3mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 69 ± 3%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 92 ± 3%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 92 ± 3% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1220kg/ mu, and the weight that only keeps 1 trunk is 930kg/ mu.
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 1.0mg/L+ZT 0.3mg/L.
Root media: MS+IAA 0.3mg/L+NAA 0.5mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride of 0.1% (quality percentage composition) to sterilize aseptic water washing 3 times, each 10 minutes 10 minutes again with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 55 ± 4%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 93 ± 4%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 93 ± 2% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1250kg/ mu, and the weight that only keeps 1 trunk is 890kg/ mu.
Embodiment 6, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 1.0mg/L+ZT 0.3mg/L.
Root media: MS+IAA 0.3mg/L+NAA 1.0mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 77 ± 2%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 91 ± 3%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 92 ± 2% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1150kg/ mu, and the weight that only keeps 1 trunk is 920kg/ mu.
Embodiment 7, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 1.0mg/L+ZT 0.3mg/L.
Root media: MS+IAA 0.5mg/L+NAA 0.3mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 55 ± 5%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 92 ± 3%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 91 ± 4% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1260kg/ mu, and the weight that only keeps 1 trunk is 950kg/ mu.
Embodiment 8, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 1.0mg/L+ZT 0.3mg/L.
Root media: MS+IAA 1.0mg/L+NAA 0.3mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 70 ± 3%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 93 ± 2%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 93 ± 2% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1150kg/ mu, and the weight that only keeps 1 trunk is 950kg/ mu.
Embodiment 9, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 1.5mg/L+ZT 0.3mg/L.
Root media: MS+IAA 0.3mg/L+NAA 0.5mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 61 ± 5%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 93 ± 2%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 92 ± 3% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1110kg/ mu, and the weight that only keeps 1 trunk is 880kg/ mu.
Embodiment 10, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 1.5mg/L+ZT 0.3mg/L.
Root media: MS+IAA 0.3mg/L+NAA 1.0mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 55 ± 5%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 95 ± 2%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 92 ± 3% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1280kg/ mu, and the weight that only keeps 1 trunk is 890kg/ mu.
Embodiment 11, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 1.5mg/L+ZT 0.3mg/L.
Root media: MS+IAA 0.5mg/L+NAA 0.3mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 66 ± 5%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 90 ± 3%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 90 ± 4% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1140kg/ mu, and the weight that only keeps 1 trunk is 870kg/ mu.
Embodiment 12, tissue culture propagating paper mulberry
One, culture medium preparation and sterilization
Germination medium: MS+IAA 0.5mg/L+6-BA 0.5mg/L+GA 0.1mg/L.
Adventitious bud inducing and subculture medium: MS+IAA 0.1mg/L+6-BA 1.5mg/L+ZT 0.3mg/L.
Root media: MS+IAA 1.0mg/L+NAA 0.3mg/L+6-BA 0.1mg/L+ZT 0.1mg/L.
Above substratum adopted 121 ℃ of high pressure moist heat sterilizations 20 minutes.
Two, hibernaculum sprouts and cultivates
Get healthy full paper mulberry top hibernaculum,, behind the aseptic water washing 3 times, use the mercuric chloride sterilization 10 minutes of 0.1% (quality percentage composition) again, aseptic water washing 3 times, each 10 minutes with the alcohol surface sterilization of 70% (volumn concentration) 1 minute.Then, the hibernaculum after the sterilization is inoculated in the triangular flask that the hibernaculum germination medium is housed, the explant number is 5 in each triangular flask.Postvaccinal triangular flask is placed illumination box, and temperature is 25 ± 2 ℃, and light application time is 12 hours, and intensity of illumination is 90 μ Mm
-2s
-13 repetitions are established in experiment, repeat 100 top hibernaculums at every turn.Adding up germination rate (the bud number of the bud number/inoculation of sprouting) after cultivating for 3 weeks, is the sprouting standard to grow spire.
Cultivate 2-3 after week most of hibernaculum can both sprout and grow spire, the germination rate of inoculating for 3 weeks is 68 ± 2%; After 2 months, winter bud growth is vigorous, and height of seedling reaches 1.5-3.0cm.
Three, differentiation adventitious buds and subculture
Getting height of seedling is sprouting seedling 100 strains of 1.5-3.0cm, is cut into the band base of leaf section of 5-10mm length.Altogether 200 stem sections are seeded in differentiation adventitious buds and the subculture medium, culture temperature is 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1When cultivating for 4 weeks, these 200 stem Duan Juncong stem section axil places and base portion grow 4-8 axillalry bud and indefinite bud, growing state as shown in Figure 1, so breeding coefficient is 4-8.
All axillalry buds of clip and indefinite bud will be cut into than long bud and be about 8-10mm stem section and the lower end is inserted on differentiation adventitious buds and the subculture medium.Per 4 all succeeding transfer culture once are total to subculture 25 times under above-mentioned culture condition.The breeding coefficient of each subculture is 4-8.
Four, root induction
With above-mentioned robust growth, highly the no offspring for 1.5-3.0cm, subculture 10 times is transferred on the root media, places illumination box, culture temperature is 25 ± 2 ℃, light application time is 10 hours, intensity of illumination is 110 μ Mm
-2s
-13 repetitions are established in experiment, repeat 1000 strains at every turn.After cultivating for 2 weeks, the seedling base portion grows 3-7 bar adventive root, the cultivation situation as shown in Figure 2, the root induction rate is 89 ± 3%.The seedling of will taking root was cultivated for 1 week on the 1/2MS substratum again, made the growth of root and seedling healthy and strong more, can the bottle outlet hardening.
Five, transplant hardening
The seedling of taking root that step 4 is obtained is uncapped in incubator earlier and was taken exercise 3 days, takes out from culturing bottle then, cleans the substratum of root, is transplanted in the greenhouse in 6 * 6 centimetres the nutrition pot.Cultivation medium is for by 1: 1 volume ratio blended turfy soil and vermiculite, is that 0.1% derosal carries out disinfection to cultivation medium with concentration before the dress alms bowl.Preceding 3-5 days covering with plastic film after the transplanting, keeping the intensity of illumination in greenhouse is 200 μ Mm
-2s
-1, temperature is 20-28 ℃, relative humidity is 70-100%.3 repetitions are established in experiment, repeat 2000 strains at every turn.Hardening surviving rate (the test-tube plantlet strain number of the strain number/transplanting that survives in hardening surviving rate=nutrition pot) reaches 92 ± 3% as a result.Afterwards, remove mulch film, water, normal management such as fertilising.Through making new advances root and young sprout 2-3 week, when height of seedling reaches 20-30cm, can be transplanted on the outdoor land for growing field crops, transplanting survival rate is 100%.Transplanted seedling grown in field situation as shown in Figure 3.
Six, the paper mulberry growthhabit of tissue culture propagating is measured
Mid or late April is planted above-mentioned tissue cultured seedling in the area, Dalian, can plant the 300-500 strain for every mu on inferior arable land, then October fallen leaves, branch amount can reach 5-13, height of tree 2-4m, the thick 1-3cm of bar, growing state is shown in Fig. 4-A; As extract lateral bud, and keep 1 trunk, then the height of tree can reach 4-6m, and bar slightly can reach 4-6cm, and growing state is shown in Fig. 4-B.Cut the receipts over-ground part, keeping branched weight is 1290kg/ mu, and the weight that only keeps 1 trunk is 890kg/ mu.
Claims (9)
1. the tissue culture method of paper mulberry is that paper mulberry top hibernaculum is cultivated in the hibernaculum germination medium, obtains sprouting hibernaculum; Described hibernaculum germination medium is for adding the substratum that IAA, 6-BA and GA obtain in the MS substratum, the final concentration of described IAA is the 0.1-0.5 mg/litre, and the final concentration of described 6-BA is the 0.1-0.5 mg/litre, and the final concentration of described GA is the 0-0.1 mg/litre.
2. method according to claim 1 is characterized in that: the final concentration of described IAA is 0.5 mg/litre, and the final concentration of described 6-BA is 0.5 mg/litre, and the final concentration of described GA is 0.1 mg/litre.
3. method according to claim 1 is characterized in that: also comprise in the described method described sprouting hibernaculum is carried out adventitious bud inducing and subculture at adventitious bud inducing and subculture medium, obtain not having offspring.
4. method according to claim 3, it is characterized in that: described adventitious bud inducing and subculture medium are for adding the substratum that IAA, 6-BA and ZT obtain in the MS substratum, the final concentration of described IAA is the 0-0.1 mg/litre, the final concentration of described 6-BA is the 0.1-2.0 mg/litre, and the final concentration of described ZT is 0.3 mg/litre.
5. method according to claim 4 is characterized in that: the final concentration of described IAA is 0.1 mg/litre, and the final concentration of described 6-BA is 1.5 mg/litre.
6. according to arbitrary described method in the claim 1 to 5, it is characterized in that: also comprise the step of described no offspring being carried out root induction at the root induction substratum in the described method.
7. method according to claim 6, it is characterized in that: described root induction substratum is for adding the substratum that IAA, NAA, 6-BA and ZT obtain in the MS substratum, the final concentration of described IAA is the 0.3-1.0 mg/litre, the final concentration of described NAA is the 0.1-1.0 mg/litre, the final concentration of described 6-BA is 0.1 mg/litre, and the final concentration of described ZT is 0.1 mg/litre.
8. method according to claim 7 is characterized in that: the final concentration of described IAA is 1.0 mg/litre, and the final concentration of described NAA is 1.0 mg/litre.
9. according to arbitrary described method in the claim 1 to 8, it is characterized in that: the culture condition that described hibernaculum sprouts is that light application time is 12 hours at 25 ± 2 ℃, and intensity of illumination is 90 μ Mm
-2s
-1The culture condition of described differentiation adventitious buds and subculture is at 25 ± 2 ℃, and light application time is 14 hours, and intensity of illumination is 100 μ Mm
-2s
-1The culture condition of described root induction is at 25 ± 2 ℃, and light application time is 10 hours, and intensity of illumination is 110 μ Mm
-2s
-1
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102577976A (en) * | 2012-03-17 | 2012-07-18 | 福建农林大学 | Simple tissue culture method for broussonetia papyrifera |
| CN103688863A (en) * | 2013-12-19 | 2014-04-02 | 云南晋企生物科技有限公司 | Method for improving characteristics of wild broussonetia papyrifera through germ cell culture |
| CN104982332A (en) * | 2015-06-19 | 2015-10-21 | 大连中植环境生物科技有限公司 | Method for preparing paper mulberry somatic embryo and application thereof to paper mulberry expanding propagation |
| CN107129365A (en) * | 2017-05-18 | 2017-09-05 | 中科天华生物科技有限公司 | A kind of culture medium for paper mulberry nursery |
| CN109220800A (en) * | 2018-10-18 | 2019-01-18 | 楚雄师范学院 | A method of effectively improving hybridization paper mulberry tissue-cultured seedling rooting rate |
| CN110558231A (en) * | 2019-10-17 | 2019-12-13 | 贵州务川科华生物科技有限公司 | efficient tissue culture method for paper mulberry |
| CN110741938A (en) * | 2019-12-05 | 2020-02-04 | 江苏省林业科学研究院 | Method for promoting accumulation of nutrient substances of paper mulberry tissue seedlings |
| CN112616661A (en) * | 2020-12-16 | 2021-04-09 | 贵州务川科华生物科技有限公司 | Cultivation method of broussonetia papyrifera seedlings |
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- 2007-12-25 CN CN2007103041272A patent/CN101200704B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102577976A (en) * | 2012-03-17 | 2012-07-18 | 福建农林大学 | Simple tissue culture method for broussonetia papyrifera |
| CN102577976B (en) * | 2012-03-17 | 2013-01-02 | 福建农林大学 | Simple tissue culture method for broussonetia papyrifera |
| CN103688863A (en) * | 2013-12-19 | 2014-04-02 | 云南晋企生物科技有限公司 | Method for improving characteristics of wild broussonetia papyrifera through germ cell culture |
| CN103688863B (en) * | 2013-12-19 | 2016-05-11 | 云南晋企生物科技有限公司 | A kind of method of utilizing plumule body cell to cultivate the wild paper mulberry of character improvement |
| CN104982332A (en) * | 2015-06-19 | 2015-10-21 | 大连中植环境生物科技有限公司 | Method for preparing paper mulberry somatic embryo and application thereof to paper mulberry expanding propagation |
| CN107129365A (en) * | 2017-05-18 | 2017-09-05 | 中科天华生物科技有限公司 | A kind of culture medium for paper mulberry nursery |
| CN109220800A (en) * | 2018-10-18 | 2019-01-18 | 楚雄师范学院 | A method of effectively improving hybridization paper mulberry tissue-cultured seedling rooting rate |
| CN110558231A (en) * | 2019-10-17 | 2019-12-13 | 贵州务川科华生物科技有限公司 | efficient tissue culture method for paper mulberry |
| CN110741938A (en) * | 2019-12-05 | 2020-02-04 | 江苏省林业科学研究院 | Method for promoting accumulation of nutrient substances of paper mulberry tissue seedlings |
| CN112616661A (en) * | 2020-12-16 | 2021-04-09 | 贵州务川科华生物科技有限公司 | Cultivation method of broussonetia papyrifera seedlings |
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