CN101637126B - Vitro rapid propagation method of kefir lily by using tender petals as explant - Google Patents
Vitro rapid propagation method of kefir lily by using tender petals as explant Download PDFInfo
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- CN101637126B CN101637126B CN2009100674342A CN200910067434A CN101637126B CN 101637126 B CN101637126 B CN 101637126B CN 2009100674342 A CN2009100674342 A CN 2009100674342A CN 200910067434 A CN200910067434 A CN 200910067434A CN 101637126 B CN101637126 B CN 101637126B
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Abstract
The invention belongs to a flower propagation method, particularly relating to an asexual rapid propagation method of flowers. In the method, technologies of explant inoculation, callus induction, adventitious bud induction, root induction, regeneration plant transplantation are used, so that kefir lilies can generate large quantities of regeneration plants having the entirely same gene background and unified plant type within a short time. Petal bases of seedling inflorescence of clivia miniata are used as explants, and the dedifferentiation ratio and the differentiation ratio can both be more than 80%. Calluses induced by one explant can be differentiated into 700 cluster buds having the unified type through repeated regeneration; the rooting rate of the cluster buds can reach 100%, and the survival rate of transplantation in favorable environment can reach 100%.
Description
Technical field
The invention belongs to the flowers propagation method, be specifically related to 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 (like root, stem, leaf, stem apex, flower, fruit etc.), tissue (like cambium, epidermis, cortex, endosperm etc.) or cell (like 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; Can 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 variation, can both keep parent (explant source plant) by the regeneration plant that Plant Tissue Breeding produced basically.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 receive 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 the 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 kaffir lily of plant type on a large scale to utilize plant tissue culture technique to exsomatize fast, breeds the good rare kind of kaffir lily fast, further satisfies the demand of domestic and international market.Though the kaffir lily tissue culture that forefathers did experiment is tentatively succeedd, break up can only differentiate on the average every explant of best kind and be less than 2.5 young plants, reproduction coefficient is extremely low.This has just limited the application of kaffir lily tissue culture technique in actual production.And but the present invention fruit of Rangoon creeper orchid bred with interior realization in 1 year fast.Explant is renewable to go out 700 strain regeneration plants, is fit to very much batch production production.
Summary of the invention
The objective of the invention is to produce the individual shortcoming that plant type varies, reproduction speed is slow of kaffir lily for overcoming kaffir lily tradition propagation method; Overcome the little shortcoming of gain factor in the experiment of forefathers kaffir lily tissue culture.A kind of kaffir lily asexual rapid propagation method is provided, and 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.
The technical scheme that the present invention adopted:
1. explant is inoculated and callus induction: young inflorescence petal is cut off from the ovary lower end taken off; Petal complete or that the upper end is cut off is put into aseptic super-clean bench; With the alcohol-pickled 30-60 of 75% (volume) second; Mercuric chloride solution with 0.1-0.2% (mass/volume) soaked aseptic water washing 3-5 time 3-10 minute.Petal is cut from the ovary topmost; Then petal top major part is cut away, rip cutting two cuttves in residue middle part are seeded in and contain 6-BA0.2mg/L, 2; 4-D0.1-0.5mg/L, on the MS medium of sucrose 3.0-3.5% (mass/volume), 20 ℃-30 ℃ 40W fluorescent lamp irradiations every day 2-4 hour.30-50 days subcultures once just can be induced differentiation when waiting to grow the yellow green callus.
2. inducing of indefinite bud: yellowish green callus is transferred to inductive differentiation medium: MS+6-BA (0.5-1.0mg/L)+2,4-D (0.1-0.2mg/L)+3-3.5% sucrose, 30-50 days subcultures once, 20 ℃ of-30 ℃ of illumination every day 8-10 hour.This callus differentiation capability is sustainable more than 1 year, can differentiate the regrowth of strain more than 700 from callus through subculture repeatedly.
3. inducing of root: regeneration plant forwards on the root media of 1/2MS+NAA (0-2mg/L)+sucrose 2%, 20 ℃ of-30 ℃ of illumination every day 10-12 hour.
4. regeneration plant is transplanted: can transplant after plant takes root.121 ℃ of sterilizations of humus soil 15-60 minute.Before transplanting the foreign material such as agar on the plant are rinsed well.Watered once in 3-15 days and permeablely just can become alive.
The invention has the beneficial effects as follows: the petal base portion with kafir lily children inflorescence (blue or green bud) is an explant, and dedifferentiation frequency and differentiation rate all can reach more than 80%.Can differentiate the 700 strains bud of growing thickly by the callus of an explant induction through subculture repeatedly, the bud phenotype of growing thickly is unified.The bud rooting rate of growing thickly can reach 100%, and transplanting survival rate reaches 100% under control environment.Fruit of Rangoon creeper orchid large-scale breeding in the short relatively time goes out the plant that genetic background is identical, outward appearance is neat; Used hormone concentration is low, and the regeneration plant variation is low; Regenerating system is very efficient; The cost of average individual plant regrowth is extremely low, is fit to the efficient breeding fast of improved seeds kaffir lily, can be applicable to batch production production.
Embodiment
To combine embodiment that the present invention is further described below, but content of the present invention is not limited only to following 3 embodiment:
Embodiment one:
1. explant inoculation and callus induction: the development degree of the young inflorescence of drawing materials is: the petal size is or near zoon but still be green; The petal of this development degree is cut off from the ovary lower end and is taken off; Complete petal is put into aseptic super-clean bench; With 75% (volume) alcohol-pickled 30 seconds, soaked aseptic water washing 3 times 10 minutes with the mercuric chloride solution of 0.1% (mass/volume).Petal is cut from the ovary topmost, then petal top major part is cut away, residue middle part rip cutting two cuttves are seeded in and contain 6-BA0.2mg/L, 2, on the MS medium of 4-D0.1mg/L, sucrose 3%, and 20 ℃ of 40W fluorescent lamp irradiations every day 2 hours.30 days subcultures once just can be induced differentiation when waiting to grow the yellow green callus.
2. inducing of indefinite bud: yellowish green callus is transferred to inductive differentiation medium: MS+6-BA0.5mg/L+2,4-D0.1+3% sucrose, 30 days subcultures once, 20 ℃ of illumination every day 8 hours.This callus differentiation capability is sustainable more than 1 year, can differentiate the regrowth of strain more than 700 from callus through subculture repeatedly.
3. inducing of root: regeneration plant forwards on the root media of 1/2MS+ sucrose 2%, 20 ℃ of illumination every day 10 hours.
4. regeneration plant is transplanted: can transplant after plant takes root.121 ℃ of sterilizations of humus soil high pressure 15 minutes.Before transplanting the foreign material such as agar on the plant are rinsed well.Watered once in 3 days and permeablely just can become alive.
Embodiment two:
1. explant inoculation and callus induction: the development degree of the young inflorescence of drawing materials is: the petal size is or near zoon but still be green; The petal of this development degree is cut off from the ovary lower end and is taken off; Petal upper end is cut off, put into aseptic super-clean bench, alcohol-pickled 60 seconds with 75% (volume); Mercuric chloride solution with 0.2% (mass/volume) soaked aseptic water washing 5 times 3 minutes.Petal is cut from the ovary topmost, then petal top major part is cut away, residue middle part rip cutting two cuttves are seeded in and contain 6-BA0.2mg/L, 2, on the MS medium of 4-D0.5mg/L, sucrose 3.5%, and 28 ℃ of 40W fluorescent lamp irradiations every day 4 hours.50 days subcultures once just can be induced differentiation when waiting to grow the yellow green callus.
2. inducing of indefinite bud: yellowish green callus is transferred to inductive differentiation medium: MS+6-BA1.0mg/L+2,4-D0.2mg/L+3.5% sucrose, 50 days subcultures once, 28 ℃ of illumination every day 10 hours.This callus differentiation capability is sustainable more than 1 year, can differentiate the regrowth of strain more than 700 from callus through subculture repeatedly.
3. inducing of root: regeneration plant forwards on the root media of 1/2MS+NAA2mg/L+ sucrose 2%, 28 ℃ of illumination every day 12 hours.
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.Watered once in 7 days and permeablely just can become alive.
Embodiment three:
1. explant inoculation and callus induction: the development degree of the young inflorescence of drawing materials is: the petal size is or near zoon but still be green; The petal of this development degree is cut off from the ovary lower end and is taken off; Petal upper end is cut off, put into aseptic super-clean bench, alcohol-pickled 45 seconds with 75% (volume); Mercuric chloride solution with 0.15% (mass/volume) soaked aseptic water washing 4 times 8 minutes.Petal is cut from the ovary topmost, then petal top major part is cut away, residue middle part rip cutting two cuttves are seeded in and contain 6-BA0.2mg/L, 2, on the MS medium of 4-D0.3mg/L, sucrose 3.2%, and 25 ℃ of 40W fluorescent lamp irradiations every day 3 hours.45 days subcultures once just can be induced differentiation when waiting to grow the yellow green callus.
2. inducing of indefinite bud: yellowish green callus is transferred to inductive differentiation medium: MS+6-BA (0.75mg/L)+2,4-D (0.15mg/L)+3.2% sucrose, 45 days subcultures once, 25 ℃ of illumination every day 9 hours.This callus differentiation capability is sustainable more than 1 year, can differentiate the regrowth of strain more than 700 from callus through subculture repeatedly.
3. inducing of root: regeneration plant forwards on the root media of 1/2MS+NAA (1mg/L)+sucrose 2%, 25 ℃ of illumination every day 11 hours.
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.Watered once in 15 days and permeablely just can become alive.
Claims (1)
1. be the method for in-vitro rapid propagation of clivia miniata of explant with young petal, it is characterized in that concrete steps are following:
1). explant is inoculated and callus induction: young inflorescence petal is cut off from the ovary lower end taken off; Petal complete or that the upper end is cut off is put into aseptic super-clean bench; With 75% alcohol-pickled 30-60 second of volume ratio; Mercuric chloride solution with mass volume ratio 0.1-0.2% soaked aseptic water washing 3-5 time 3-10 minute; Petal is cut from the ovary topmost; Then petal top major part is cut away, rip cutting two cuttves in residue middle part are seeded in and contain 6-BA 0.2mg/L, 2; On the MS medium of 4-D 0.1-0.5mg/L, mass volume ratio 3.0-3.5% sucrose; 20 ℃-30 ℃ 40W fluorescent lamp irradiations every day 2-4 hour, 30-50 days subcultures are once induced differentiation when waiting to grow the yellow green callus;
2). inducing of indefinite bud: yellowish green callus is transferred to inductive differentiation medium: MS+6-BA 0.5-1.0mg/L+2; 4-D 0.1-0.2mg/L+3-3.5% sucrose; 30-50 days subcultures once; 20 ℃ of-30 ℃ of illumination every day 8-10 hour, this callus differentiation capability continued more than 1 year, differentiated the regrowth of strain more than 700 from a callus through subculture repeatedly;
3). inducing of root: regeneration plant forwards on the root media of 1/2MS+NAA 0-2mg/L+ sucrose 2%, 20 ℃ of-30 ℃ of illumination every day 10-12 hour;
4). regeneration plant is transplanted: promptly transplant after plant takes root, 121 ℃ of sterilizations of humus soil 15-60 minute before transplanting are rinsed agar on the plant and foreign material well, water once permeable alive in 3-15 days.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009100674342A CN101637126B (en) | 2009-08-21 | 2009-08-21 | Vitro rapid propagation method of kefir lily by using tender petals as explant |
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| CN2009100674342A CN101637126B (en) | 2009-08-21 | 2009-08-21 | Vitro rapid propagation method of kefir lily by using tender petals as explant |
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| CN101637126B true CN101637126B (en) | 2012-05-09 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101822218B (en) * | 2010-03-17 | 2011-10-19 | 东北师范大学 | Kaffir lily in-vitro propagation method using young plants and young blade cut blocks as explants |
| CN101810141B (en) * | 2010-03-18 | 2011-10-19 | 东北师范大学 | Method for in-vitro rapid propagation of clivia miniata by using mature embryos as explants |
| CN103704140B (en) * | 2013-12-31 | 2016-01-20 | 湖北大学 | A kind of with children spend into Explants In Tissue Culture breeding polyploid royal paulownia method |
| CN105284599A (en) * | 2015-11-13 | 2016-02-03 | 长春大学 | Reagent for overcoming kaffir lily selfing incompatibility and use method thereof |
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