CN101810141B - Method for in-vitro rapid propagation of clivia miniata by using mature embryos as explants - Google Patents
Method for in-vitro rapid propagation of clivia miniata by using mature embryos as explants Download PDFInfo
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- CN101810141B CN101810141B CN2010101268300A CN201010126830A CN101810141B CN 101810141 B CN101810141 B CN 101810141B CN 2010101268300 A CN2010101268300 A CN 2010101268300A CN 201010126830 A CN201010126830 A CN 201010126830A CN 101810141 B CN101810141 B CN 101810141B
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Abstract
The invention belongs to a plant propagation method, and particularly relates to a method for the asexual rapid propagation of flowers. The method utilizes the technique such as detoxification, inoculation and callus induction of explants, induction of adventitious shoots, root induction and transplant of regeneration plants for the asexual rapid propagation of clivia miniata. When the method is used, the selection of raw materials is not limited by seasons, the method is suitable for agrobacterium-mediated transgenes, and the silence of the transgenes is overcome to some extent; the mature embryos from ten plants of mature clivia miniata are used as the explants, and the dedifferentiation rate and the differentiation rate both reach 70 to 100 percent; the regeneration coefficient is high, and an original callus tissues can grow into hundreds of cluster buds by repeated sub-culture; the rooting rate of the cluster buds is 100 percent, and the transplanting survival rate is 100 percent. When the method is used, the clivia miniata in different varieties can mass propagate the plants which have the same genetic background and smooth appearances in a shorter time; and after the plants are transplanted, the growing state is good, the cost for the average individual-plant regeneration seedling is low, and the method is suitable for industrialized production.
Description
Technical field
The invention belongs to method for propagation, being specifically related to is the asexual rapid propagation method of flowers.
Background technology
Plant Tissue Breeding is a plant aseptic culture technology, it is totipotency according to plant cell, the organ (as root, stem, leaf, stem apex, flower, fruit etc.), tissue (as cambium, epidermis, cortex, endosperm etc.) or cell (as megaspore, microspore, somatic cell etc.) and the protoplast that utilize plant corpus to exsomatize, under artificial conditions such as aseptic and suitable synthetic medium and illumination, temperature, induce callus, indefinite bud, adventive root, form the technology of the other products of complete plant or generation economically valuable.The merit that except extremely low somaclonal variation, can both keep parent (explant source plant) by the regeneration plant that Plant Tissue Breeding produced substantially.Obtained very big progress since the plant tissue culture technique invention, Plant Tissue Breeding becomes a kind of extensive industrial production method in batch from the laboratory research stage since the sixties in last century.Kaffir lily is that the perennial herb of Amaryllidaceae gentleman Cymbidium is viewed and admired plant.Originate in the South Africa high mountain, property happiness Wen Liang introduces China the beginning of this century via Germany and Japan, and settles down.Be China's rare flower, economic worth is higher.Its plant type dignity, leaf, flower, fruit and beautiful are subjected to domestic and international flowers fan's favor deeply.At present, the modes of reproduction of kaffir lily is mainly seminal propagation and division propagation, and the reproduction speed of this dual mode is all very slow: kaffir lily only opened once and spends in 1 year, and 9 months ability of seed demand maturation; Division propagation was told 1～3 strain in 1 year at most.Because kaffir lily height heterozygosis, the yield that seminal propagation obtains the treasure kaffir lily is very low, and originally to obtain the probability of treasure still very low even if be father and mother with the treasure kaffir lily, and supply falls short of demand always in the international market at home for the treasure kaffir lily.The breeding kaffir lily can produce the unified treasure kaffir lily of plant type on a large scale to utilize plant tissue culture technique to exsomatize fast, further satisfies the demand of domestic and international market.Though we learn that the kaffir lily tissue culture experiment that forefathers do tentatively succeeds with reference to international and domestic pertinent literature, dedifferentiation frequency and regeneration rate be low, several to 2 several young plants zero point of regenerating according to the different average outer planting physical efficiencys of kind.This has just limited the application of kaffir lily tissue culture technique in actual production.But the present invention fruit of Rangoon creeper orchid 1 year with the real quick breeding of interior realization, be fit to very much batch production production.
Summary of the invention
Produce the individual shortcoming that plant type varies, reproduction speed is slow of kaffir lily for overcoming kaffir lily tradition propagation method; Also dedifferentiation frequency is low in the experiment of forefathers kaffir lily tissue culture, differentiation rate is low, the regeneration period is long, be not suitable for the shortcoming that batch production is produced in order to overcome.Providing a kind of is the method for in-vitro rapid propagation of clivia miniata of explant with the mature embryo
The technical solution adopted in the present invention is:
1. explant detoxification, inoculation and callus of induce: get the embryo in the ripening fruits, put into aseptic super-clean bench, with 75% (volume/volume) alcohol-pickled 30～60 seconds, mercuric chloride solution with 0.1～0.2% (mass/volume) soaked 2～5 minutes, aseptic water washing 3～5 times, under the end-grain cutting with the about 2mm of length of yellow densification on the naked embryo, be seeded in and contain 2, on the MS medium of 4-D2～4mg/L, sucrose 3～4% (mass/volume), agar 0.45～0.7% (mass/volume), 20～30 ℃ of 40W fluorescent lamp irradiations every day 8～9 hours.40～50 days subcultures are once treated that callus (yellow) or green are expanded long can induce differentiation during to the diameter 5mm left and right sides.
2. inducing of indefinite bud: yellow callus that growth conditions is good or green are expanded tissue and be transferred to inductive differentiation medium: MS+KT (0.25～0.5mg/L)+2,4-D (0.75～1.5mg/L)+3～4% (mass/volume) sucrose+0.45～0.7% (mass/volume) agar, 40～50 days subcultures once, 20～30 ℃ of illumination every day 8～12 hours.Visible regeneration bud produced in 30～40 days.Subculture can differentiate the regeneration bud of strain more than 200 seedling from a callus in the time of 4 months.
3. inducing of root: regeneration plant forward to 1/2MS+NAA (on the root media of 1～2mg/L)+sucrose 2% (mass/volume)+agar 0.45～0.7% (mass/volume), illumination about 20～30 ℃ of 10 hours every days.
4. regeneration plant is transplanted: can transplant after plant takes root.121 ℃ of sterilizations of humus soil 30～60 minutes.Before transplanting the foreign material such as agar on the plant are rinsed well.Water once about 7 days and permeablely just can survive.
Advantage of the present invention is: drawing materials is not subject to seasonal restrictions (but mature seed long preservation); The explant volume is little, is suitable for agriculture bacillus mediated transgenosis; Genetic background based on every seed is not quite similar (kaffir lily height heterozygosis adopts cross pollination mostly), can overcome transgene silencing to a certain extent; Fruit of Rangoon creeper orchid is a large amount of generation regeneration plants that genetic background is identical, plant type is unified in the short relatively time; Regenerating system is stable, is that the dedifferentiation frequency and the differentiation rate of explant all can reach 70%～100% to derive from ripe embryo that 10 strains become kaffir lily in age (comprising color leaf kaffir lily); The gain factor height can differentiate the hundreds of strain bud of growing thickly by an original callus through subculture repeatedly; The bud rooting rate of growing thickly can reach 100%, robust growth; The transplanting survival rate height can reach 100%.The invention has the beneficial effects as follows that kaffir lily large-scale breeding in the short relatively time that can make different cultivars goes out the plant that genetic background is identical, outward appearance is neat, growth conditions is good behind the plantlet of transplant, and the cost of average individual plant regrowth is low, is fit to industrialization production.
Embodiment
The present invention is further illustrated below in conjunction with practical operation, but scope of the present invention not only is confined to following embodiment.
Embodiment 1:
1. explant detoxification, inoculation and callus of induce: get the embryo in the ripening fruits, 75% (volume/volume) is alcohol-pickled 30 seconds in aseptic super-clean bench, mercuric chloride solution with 0.1% (mass/volume) soaked 2 minutes, aseptic water washing 3 times, under the end-grain cutting with the about 2mm of length of yellow densification on the naked embryo, be seeded in and contain 2, on the MS medium of 4-D2mg/L, sucrose 3% (mass/volume), agar 0.45% (mass/volume), 20 ℃ of 40W fluorescent lamp irradiations every day 8 hours.40 days subcultures are once treated that callus (yellow) or green are expanded long can induce differentiation during to the diameter 5mm left and right sides.
2. inducing of indefinite bud: yellow callus that growth conditions is good or green are expanded tissue and are transferred to inductive differentiation medium: MS+KT0.25mg/L+2,4-D0.75+3% (mass/volume) sucrose+0.45% (mass/volume) agar, 40 days subcultures once, 20 ℃ of illumination every day 10 hours.Visible regeneration bud produced in 30～40 days.Subculture can differentiate the regeneration bud of strain more than 200 seedling from a callus in the time of 4 months.
3. inducing of root: regeneration plant forwards on the root media of 1/2MS+NAA (1mg/L)+sucrose 2% (mass/volume)+agar 0.45% (mass/volume), 20C illumination about 10 hours every days.
4. regeneration plant is transplanted: can transplant after plant takes root.121 ℃ of sterilizations of humus soil 30 minutes.Before transplanting the foreign material such as agar on the plant are rinsed well.Water once about 7 days and permeablely just can survive.
Embodiment 2:
1. explant detoxification, inoculation and callus of induce: get embryo in the ripening fruits in aseptic super-clean bench, with 75% (volume/volume) alcohol-pickled 60 seconds, mercuric chloride solution with 0.2% (mass/volume) soaked 5 minutes, aseptic water washing 5 times, under the end-grain cutting with the about 2mm of length of yellow densification on the naked embryo, be seeded in and contain 2, on the MS medium of 4-D4mg/L, sucrose 4% (mass/volume), agar 0.7% (mass/volume), 30 ℃ of 40W fluorescent lamp irradiations every day 9 hours.50 days subcultures are once treated that callus (yellow) or green are expanded long can induce differentiation during to the diameter 5mm left and right sides.
2. inducing of indefinite bud: yellow callus that growth conditions is good or green are expanded tissue and are transferred to inductive differentiation medium: MS+KT0.5mg/L+2,4-D1.5mg/L+4% (mass/volume) sucrose+0.7% (mass/volume) agar, 50 days subcultures once, 30 ℃ of illumination every day 12 hours.Visible regeneration bud produced in 30～40 days.Subculture can differentiate strain regeneration bud seedlings up to a hundred from a callus in the time of 4 months.
3. inducing of root: regeneration plant forwards on the root media of 1/2MS+NAA2mg/L+ sucrose 2% (mass/volume)+agar 0.7% (mass/volume), illumination about 30 ℃ of 10 hours every days.
4. regeneration plant is transplanted: can transplant after plant takes root.121 ℃ of sterilizations of humus soil 60 minutes.Before transplanting the foreign material such as agar on the plant are rinsed well.Water once about 7 days and permeablely just can survive.
Embodiment 3:
1. explant detoxification, inoculation and callus of induce: the embryo of getting ripening fruits alcohol-pickled 40 seconds of 75% (volume/volume) in going into aseptic super-clean bench, mercuric chloride solution with 0.15% (mass/volume) soaked 4 minutes, aseptic water washing 4 times, under the end-grain cutting with the about 2mm of length of yellow densification on the naked embryo, be seeded in and contain 2, on the MS medium of 4-D3mg/L, sucrose 3.5% (mass/volume), agar 0.6% (mass/volume), 25 ℃ of 40W fluorescent lamp irradiations every day 8.5 hours.48 days subcultures are once treated that callus (yellow) or green are expanded long can induce differentiation during to the diameter 5mm left and right sides.
2. inducing of indefinite bud: yellow callus that growth conditions is good or green are expanded tissue and are transferred to inductive differentiation medium: MS+KT (0.4mg/L)+2,4-D (1.0mg/L)+3.5% (mass/volume) sucrose+0.5% (mass/volume) agar, 45 days subcultures once, 25 ℃ of illumination every day 8 hours.Visible regeneration bud produced in 30～40 days.Subculture can differentiate the regeneration bud of strain more than 200 seedling from a callus in the time of 4 months.
3. inducing of root: regeneration plant forwards on the root media of 1/2MS+NAA (1.5mg/L)+sucrose 2% (mass/volume)+agar 0.6 (mass/volume), illumination about 20～30 ℃ of 10 hours every days.
4. regeneration plant is transplanted: can transplant after plant takes root.121 ℃ of sterilizations of humus soil 45 minutes.Before transplanting the foreign material such as agar on the plant are rinsed well.Water once about 7 days and permeablely just can survive.
Claims (1)
1. be the method for in-vitro rapid propagation of clivia miniata of explant with the mature embryo, it is characterized in that concrete steps are as follows:
1). the explant detoxification, inoculation and callus of induce: get the embryo in the ripening fruits, put into aseptic super-clean bench, with 75% (volume/volume) alcohol-pickled 30～60 seconds, mercuric chloride solution with 0.1～0.2% (mass/volume) soaked 2～5 minutes, aseptic water washing 3～5 times, under the end-grain cutting with the length 2mm of yellow densification on the naked embryo, be seeded in and contain 2,4-D 2～4mg/L, sucrose 3～4% (mass/volume), on the MS medium of agar 0.45～0.7% (mass/volume), 20～30 ℃ of 40W fluorescent lamp irradiations every day 8～9 hours, 40～50 days subcultures once treat that yellow callus or green expand that tissue is long induces differentiation during to diameter 5mm;
2). inducing of indefinite bud: yellow callus that growth conditions is good or green are expanded tissue and are transferred to inductive differentiation medium: MS+KT 0.25～0.5mg/L+2,4-D 0.75～1.5mg/L+3～4% (mass/volume) sucrose+0.45～0.7% (mass/volume) agar, 40～50 days subcultures once, 20～30 ℃ of illumination every day 8～12 hours; Saw that regeneration bud produced in 30～40 days, subculture differentiated the regeneration bud of strain more than 200 seedling from a callus in the time of 4 months;
3). inducing of root: regeneration plant forwards on the root media of 1/2MS+NAA 1～2mg/L+ sucrose 2% (mass/volume)+agar 0.45～0.7% (mass/volume), 20～30 ℃ of illumination 10 hours every days;
4). regeneration plant is transplanted: can transplant after plant takes root, 121 ℃ of sterilizations of humus soil 30～60 minutes before transplanting are rinsed agar on the plant and foreign material well, water once in 7 days permeablely just to survive.
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| CN115024220B (en) * | 2022-06-09 | 2023-04-11 | 沈阳农业大学 | Method for obtaining clivia miniata somatic embryo by thin-layer culture |
| CN116267613B (en) * | 2023-03-23 | 2024-03-08 | 沈阳大学 | Method for inducing callus by using immature endosperm of clivia miniata |
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| CN101637126B (en) * | 2009-08-21 | 2012-05-09 | 东北师范大学 | Vitro rapid propagation method of kefir lily by using tender petals as explant |
| CN101637127A (en) * | 2009-08-21 | 2010-02-03 | 东北师范大学 | Vitro rapid propagation method of kefir lily by using seedling ovaries as explant |
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