CN105815216A - Rapid propagation method for rhodoleia championii tissue culture seedlings - Google Patents
Rapid propagation method for rhodoleia championii tissue culture seedlings Download PDFInfo
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- CN105815216A CN105815216A CN201610166219.8A CN201610166219A CN105815216A CN 105815216 A CN105815216 A CN 105815216A CN 201610166219 A CN201610166219 A CN 201610166219A CN 105815216 A CN105815216 A CN 105815216A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a rapid propagation method for rhodoleia championii tissue culture seedlings.The method comprises the steps that after seeds are subjected to accelerating germination and sowing, selection is conducted during the seedling period, terminal buds of seedlings serve as explants, a rhodoleia championii in-vitro regenerated plant is obtained through the processes of explant disinfection, induction culture, clump bud generation, adventitious root formation, seedling exercising and transplanting and the like, a rhodoleia championii tissue culture rapid propagation technology system is built, the fine property of a rhodoleia championii female parent can be kept, and scale production of rhodoleia championii excellent clonal seedlings is promoted.By means of the rapid propagation method for the rhodoleia championii tissue culture seedlings, the induction rate can reach 81.8% averagely, the propagation times can reach 3.0 averagely, the rooting rate can reach 95.8% averagely after seedling exercising is conducted for 20 days, the statistical survival rate can reach 95.2% averagely after transplanting is conducted for 20 days, and the industrialized seedling requirements of rhodoleia championii can be well achieved.The rapid propagation method for the rhodoleia championii tissue culture seedlings has the advantages that selection is conducted in the seedling period, the terminal buds of the seedlings serve as the explants, and the rhodoleia championii tissue culture rapid propagation technology system is built.
Description
Technical field
The present invention relates to a kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method, belong to technical field of bioengineering.
Technical background
Radix Rhodoleiae championii (formal name used at school: RhodoleiachampioniiHook.f.): Hamamelidaceae Radix Rhodoleiae championii belongs to aiphyllium, and high up to 12 meters, Radix Rhodoleiae championii is widely cultivated more makees ornamental use.The flower-shape picture of Radix Rhodoleiae championii hangs clock, and volume is quite big, so having another name called " hanging clock king ".But Radix Rhodoleiae championii is solid few, breeds difficulty, and the holding merit being difficult to scale is bred.Therefore, a kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method of exploitation seems necessary.
Summary of the invention
Problem to be solved by this invention is to make Radix Rhodoleiae championii tissue cultured seedling fast breeding method.
The present invention is realized by below scheme:
A kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method, operates according to the following steps:
(1) seed is plucked: 10 to early November of seed is ripe, is transferred to yellow green by ash cyan, gather in time time the ripest, dries in the air to shell and ftracture after adopting back, and sieve takes seed, carries out dry Tibetan；
(2) presprouting of seeds processes and sowing: soak seed 30-60 minute with the potassium permanganate solution that mass concentration is 0.1-0.5%, tap water is cleaned, with 40-50 DEG C of Soaking in hot water, after natural cooling, changing tap water again to soak 24-48 hour, tap water is placed in Germinator 25-30 DEG C of accelerating germination 2-5 days after cleaning, after accelerating germination, each raising tray sowing 5-10g seed, makes the seed in raising tray uniform；
(3) pretreatment of outer implant: seedling height is when 3-5cm after Radix Rhodoleiae championii accelerating germination, using the terminal bud of seedling as outer implant, outer implant rinses 1～3h under flowing water, it is soaked in mass concentration to be 3%～5% washing powder solution 5～scrub outer planting body with fur after 10 minutes, 60～90min are rinsed with the form dripped again with tap water, with distilled water flushing 3-5 time, it is 75%～80% alcohol solution dipping 20～60s with mass concentration in superclean bench, aseptic water washing 3～5 times, again with containing mass concentration 0.1～0.5% mercuric chloride solution sterilization 5～15min, blot the moisture on surface with aseptic filter paper after aseptic water washing 3～5 times, obtain the outer implant of pretreatment, standby；
(4) inducing culture: the terminal bud of the outer implant through step (3) pretreatment is cut into the stem section of about 1.0cm and is inoculated into inducing culture and carries out inducing clumping bud cultivation, after inoculation, every day is placed in intensity of illumination is 1000～2000lx, cultivation temperature is 23～25 DEG C, relative air humidity is illumination 12～14 hours under conditions of 75%～80%, cultivate 28-35 days, obtain the Multiple Buds of inducing culture；
(5) adventitious buds proliferation: the Multiple Buds that step (4) inducing culture obtains is inoculated into proliferated culture medium and carries out adventitious bud proliferation cultivation, after inoculation, every day is placed in intensity of illumination is 1000～2000lx, cultivation temperature is 23～25 DEG C, relative air humidity is illumination 12～14 hours under conditions of 75%～80%, cultivate 38-45 days, obtain breeding plant；
(6) root culture: step (5) propagation plant is cut from base portion and is seeded to root media carry out root induction, after inoculation, every day is placed in intensity of illumination is 2000～3000lx, being placed in cultivation temperature is 23～25 DEG C, relative air humidity is illumination 12～14 hours under conditions of 75%～80%, cultivate 7-12 days, obtain the plant taken root；
(7) acclimatization and transplants: the Radix Rhodoleiae championii bottle Seedling of root culture is carried out outdoor seedling exercising 15-25 days, then wash away and be attached to the culture medium that shoot root is fastened, 5～10min are soaked in the carbendazim solution of 1000 times, transplant and cultivate to container bag, the fast breeding of Radix Rhodoleiae championii tissue cultured seedling can be completed.
In described step (4), inducing culture contains MS100 part, 0.5～1.0mg/LNAA1-5 part, 2.0～4.0mg/L6-BA3-8 parts, 1.0～3.0mg/LKT0.5-5 parts, 1.0g/LAC10-15 part, 30g/L sucrose 5-15 part, 6.0g/L agar 20-30 part, mass fraction is polyacrylic acid 2-8 part of 20-30%, after blended configuration so that it is pH is 4-7.
More preferably inducing culture contains MS100 part, 0.8mg/LNAA4 part, 3.2mg/L6-BA6 part, 2.8mg/LKT5 part, 1.0g/LAC12 part, 30g/L sucrose 12 parts, 25 parts of 6.0g/L agar, mass fraction is the polyacrylic acid 7 parts of 20-30%, after blended configuration so that it is pH is 5.8.
In described step (5), proliferated culture medium contains MS100 part, 0.1～0.5mg/LNAA4-10 part, 2.0～4.0mg/L6-BA12-15 parts, 1.0～3.0mg/LKT6-10 parts, 1.0g/LAC3-7 part, 30g/L sucrose 5-15 part, 6.0g/L agar 20-30 part, mass fraction is poly-γ glutamic acid 3-7 part of 10-20%, after blended configuration so that it is pH is 4-7.
More preferably proliferated culture medium contains MS100 part, 0.35mg/LNAA5 part, 3.5mg/L6-BA12 part, 2.5mg/LKT8.5 part, 1.0g/LAC5.5 part, 30g/L sucrose 10 parts, 30 parts of 6.0g/L agar, mass fraction is 5.5 parts of the poly-γ glutamic acid of 12.5%, after blended configuration so that it is pH is 5.8.
In described step (6), root media contains MS25 part, 0.1～1.0mg/LNAA4-10 part, 1.0～2.0mg/LIBA12-15 parts, 30g/L sucrose 5-15 part, 6.0g/L agar 20-30 part, thiourea 5-10 part of 2.5-5.5g/L, after blended configuration so that it is pH is 4-7.
More preferably root media contains MS25 part, 0.55mg/LNAA6.5 part, 1.8mg/LIBA12 part, 30g/L sucrose 15 parts, 30 parts of 6.0g/L agar, and the thiourea of 4g/L 6 parts, after blended configuration so that it is pH is 5.8.
The application uses selection-breeding during seedling, and the terminal bud with seedling is outer implant, establishes the tissue culture rapid propagation technique system of Radix Rhodoleiae championii.
Accompanying drawing explanation
Fig. 1 is that Radix Rhodoleiae championii group trains Regenerated plant growth figure.
Fig. 2 be a be the Seedling portion growth figure that Radix Rhodoleiae championii tissue cultured seedling is taken root；B is the root growth figure that Radix Rhodoleiae championii tissue cultured seedling is taken root.
Fig. 3 is that Radix Rhodoleiae championii tissue cultured seedling transplants 20 days growth figures.
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be further described.
Embodiment 1: a kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method
(1) Radix Rhodoleiae championii elite stand selects excellent: according to character screening fine individual plants such as branch bifurcated terrain clearance, head inflorescence length, common peduncle length, petal shape, pattern, florescences；
(2) seed is plucked: 10 to early November of seed is ripe, is transferred to yellow green by ash cyan, gather in time time the ripest.Drying in the air after adopting back to shell and ftracture, sieve takes seed, carries out dry Tibetan；
(3) presprouting of seeds processes and sowing: soaking seed 40 minutes with the potassium permanganate solution of 0.35% before sowing, clear water is cleaned, and with 48 DEG C of Soaking in hot water, after natural cooling, then changes water soaking 30 hours, is placed in Germinator 30 DEG C of accelerating germination 3 days after clear water is clean；After accelerating germination, each raising tray sowing 8g seed (each raising tray 0.25m2), keep the seed in raising tray uniform；
(4) seedling selection-breeding: seedling height when 3-5cm, according to height of seedling, the index selection advantage Seedlings such as footpath, blade quantity, disease and insect resistance degree, and using the terminal bud of advantage Seedling as outer implant；
(5) process of outer implant: outer implant rinses 2h under flowing water, it is soaked in 3% washing powder solution 5～10 minutes, outer planting body is scrubbed with fur, 70min is rinsed with the form dripped again with tap water, with distilled water flushing 3 times, with 75% alcohol solution dipping 40s in superclean bench, aseptic water washing 5 times, sterilize 10min with containing mass concentration 0.1% mercuric chloride solution again, blot the moisture on surface with aseptic filter paper after aseptic water washing 3～5 times standby；
(6) inducing culture: the terminal bud after step (5) processes is cut into the stem section of about 1.0cm and is inoculated into inducing culture and carries out inducing clumping bud cultivation, inoculation is placed on illumination every day 12 hours, intensity of illumination is 2000lx, being placed in cultivation temperature is 25 DEG C, and relative air humidity is cultivation under conditions of 75%；Described inducing culture is: MS100mL, 0.8mg/LNAA4mL, 3.2mg/L6-BA6mL, 2.8mg/LKT5mL, 1.0g/LAC12mL, 30g/L sucrose 12mL, 6.0g/L agar 25mL, mass fraction is the polyacrylic acid 7mL of 20-30%, after blended configuration so that it is pH is 5.8.Adding up induction situation after cultivating 30 days, inductivity is averagely up to 81.8%.
(7) adventitious buds proliferation: step (6) inducing culture is obtained Multiple Buds and is inoculated into proliferated culture medium and carries out adventitious bud proliferation cultivation, inoculation is placed on illumination every day 12 hours, intensity of illumination is 2000lx, being placed in cultivation temperature is 25 DEG C, and relative air humidity is cultivation under conditions of 75%；Described proliferated culture medium is: MS100mL, 0.35mg/LNAA5mL, 3.5mg/L6-BA12mL, 2.5mg/LKT8.5mL, 1.0g/LAC5.5mL, 30g/L sucrose 10mL, 6.0g/L agar 30mL, mass fraction is the poly-γ glutamic acid 5.5mL of 12.5%, after blended configuration so that it is pH is 5.8.Proliferation times is added up averagely up to 3.0 times after cultivating 40 days.
(8) root culture: the plant (the tender tip of unrooted 2～3cm) obtained in step (7) being bred is cut from base portion and is seeded to root media carry out root induction, inoculation is placed on illumination every day 12 hours, intensity of illumination is 3000lx, being placed in cultivation temperature is 25 DEG C, and relative air humidity is to cultivate 10 days under conditions of 75%；Described root media is: the thiourea 6mL of MS25mL, 0.55mg/LNAA6.5mL, 1.8mg/LIBA12mL, 30g/L sucrose 15mL, 6.0g/L agar 30mL, 4g/L, after blended configuration so that it is pH is 5.8.
(9) acclimatization and transplants: the root culture Radix Rhodoleiae championii bottle Seedling of 10 days is carried out 20 days statistics rooting rates of seedling exercising, then wash away and be attached to the culture medium that shoot root is fastened, 5～10min are soaked in the carbendazim solution of 1000 times, then transplant and cultivate to container bag, after transplanting 20 days, add up survival rate.Through statistics: the root culture Radix Rhodoleiae championii bottle Seedling of 10 days carries out seedling exercising, and after 20 days, rooting rate is averagely up to 95.8%, and after transplanting 20 days, statistics survival rate is averagely up to 95.2%.Using technical scheme incubation time short, stability is high, does not bring pathogenic bacteria, maintains the original growth characteristics of Radix Rhodoleiae championii.
Claims (7)
1. a Radix Rhodoleiae championii tissue cultured seedling fast breeding method, is characterized in that, operates according to the following steps:
(1) seed is plucked: 10 to early November of seed is ripe, is transferred to yellow green by ash cyan, gather in time time the ripest, dries in the air to shell and ftracture after adopting back, and sieve takes seed, carries out dry Tibetan；
(2) presprouting of seeds processes and sowing: soak seed 30-60 minute with the potassium permanganate solution that mass concentration is 0.1-0.5%, tap water is cleaned, with 40-50 DEG C of Soaking in hot water, after natural cooling, changing tap water again to soak 24-48 hour, tap water is placed in Germinator 25-30 DEG C of accelerating germination 2-5 days after cleaning, after accelerating germination, each raising tray sowing 5-10g seed, makes the seed in raising tray uniform；
(3) pretreatment of outer implant: seedling height is when 3-5cm after Radix Rhodoleiae championii accelerating germination, using the terminal bud of seedling as outer implant, outer implant rinses 1～3h under flowing water, it is soaked in mass concentration to be 3%～5% washing powder solution 5～scrub outer planting body with fur after 10 minutes, 60～90min are rinsed with the form dripped again with tap water, with distilled water flushing 3-5 time, it is 75%～80% alcohol solution dipping 20～60s with mass concentration in superclean bench, aseptic water washing 3～5 times, again with containing mass concentration 0.1～0.5% mercuric chloride solution sterilization 5～15min, blot the moisture on surface with aseptic filter paper after aseptic water washing 3～5 times, obtain the outer implant of pretreatment, standby；
(4) inducing culture: the terminal bud of the outer implant through step (3) pretreatment is cut into the stem section of about 1.0cm and is inoculated into inducing culture and carries out inducing clumping bud cultivation, after inoculation, every day is placed in intensity of illumination is 1000～2000lx, cultivation temperature is 23～25 DEG C, relative air humidity is illumination 12～14 hours under conditions of 75%～80%, cultivate 28-35 days, obtain the Multiple Buds of inducing culture；
(5) adventitious buds proliferation: the Multiple Buds that step (4) inducing culture obtains is inoculated into proliferated culture medium and carries out adventitious bud proliferation cultivation, after inoculation, every day is placed in intensity of illumination is 1000～2000lx, cultivation temperature is 23～25 DEG C, relative air humidity is illumination 12～14 hours under conditions of 75%～80%, cultivate 38-45 days, obtain breeding plant；
(6) root culture: step (5) propagation plant is cut from base portion and is seeded to root media carry out root induction, after inoculation, every day is placed in intensity of illumination is 2000～3000lx, being placed in cultivation temperature is 23～25 DEG C, relative air humidity is illumination 12～14 hours under conditions of 75%～80%, cultivate 7-12 days, obtain the plant taken root；
(7) acclimatization and transplants: the Radix Rhodoleiae championii bottle Seedling of root culture is carried out outdoor seedling exercising 15-25 days, then wash away and be attached to the culture medium that shoot root is fastened, 5～10min are soaked in the carbendazim solution of 1000 times, transplant and cultivate to container bag, the fast breeding of Radix Rhodoleiae championii tissue cultured seedling can be completed.
A kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method the most according to claim 1, it is characterized in that, in described step (4), inducing culture contains MS100 part, 0.5～1.0mg/LNAA1-5 part, 2.0～4.0mg/L6-BA3-8 parts, 1.0～3.0mg/LKT0.5-5 parts, 1.0g/LAC10-15 part, 30g/L sucrose 5-15 part, 6.0g/L agar 20-30 part, mass fraction is polyacrylic acid 2-8 part of 20-30%, after blended configuration so that it is pH is 4-7.
A kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method the most according to claim 1, it is characterized in that, in described step (4), inducing culture contains MS100 part, 0.8mg/LNAA4 part, 3.2mg/L6-BA6 part, 2.8mg/LKT5 part, 1.0g/LAC12 part, 30g/L sucrose 12 parts, 25 parts of 6.0g/L agar, mass fraction is the polyacrylic acid 7 parts of 20-30%, after blended configuration so that it is pH is 5.8.
A kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method the most according to claim 1, it is characterized in that, in described step (5), proliferated culture medium contains MS100 part, 0.1～0.5mg/LNAA4-10 part, 2.0～4.0mg/L6-BA12-15 parts, 1.0～3.0mg/LKT6-10 parts, 1.0g/LAC3-7 part, 30g/L sucrose 5-15 part, 6.0g/L agar 20-30 part, mass fraction is poly-γ glutamic acid 3-7 part of 10-20%, after blended configuration so that it is pH is 4-7.
A kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method the most according to claim 1, it is characterized in that, in described step (5), proliferated culture medium contains MS100 part, 0.35mg/LNAA5 part, 3.5mg/L6-BA12 part, 2.5mg/LKT8.5 part, 1.0g/LAC5.5 part, 30g/L sucrose 10 parts, 30 parts of 6.0g/L agar, mass fraction is 5.5 parts of the poly-γ glutamic acid of 12.5%, after blended configuration so that it is pH is 5.8.
A kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method the most according to claim 1, it is characterized in that, in described step (6), root media contains MS25 part, 0.1～1.0mg/LNAA4-10 part, 1.0～2.0mg/LIBA12-15 parts, 30g/L sucrose 5-15 part, 6.0g/L agar 20-30 part, thiourea 5-10 part of 2.5-5.5g/L, after blended configuration so that it is pH is 4-7.
A kind of Radix Rhodoleiae championii tissue cultured seedling fast breeding method the most according to claim 1, it is characterized in that, in described step (6), root media contains MS25 part, 0.55mg/LNAA6.5 part, 1.8mg/LIBA12 part, 30g/L sucrose 15 parts, 30 parts of 6.0g/L agar, the thiourea of 4g/L 6 parts, after blended configuration so that it is pH is 5.8.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116616177A (en) * | 2023-05-24 | 2023-08-22 | 德兴市荣兴苗木有限责任公司 | Tissue culture method of safflower lotus |
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| CN101697710A (en) * | 2009-11-09 | 2010-04-28 | 浙江传化生物技术有限公司 | Culture medium for tissue culture of asexual propagation of butterfly orchid |
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