CN101822218B - Kaffir lily in-vitro propagation method using young plants and young blade cut blocks as explants - Google Patents
Kaffir lily in-vitro propagation method using young plants and young blade cut blocks as explants Download PDFInfo
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- CN101822218B CN101822218B CN2010101258525A CN201010125852A CN101822218B CN 101822218 B CN101822218 B CN 101822218B CN 2010101258525 A CN2010101258525 A CN 2010101258525A CN 201010125852 A CN201010125852 A CN 201010125852A CN 101822218 B CN101822218 B CN 101822218B
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Abstract
The invention relates to a flower propagation method, in particular to a kaffir lily in-vitro propagation method using young plants and young blade cut blocks as explants. The invention adopts the technology such as explant inoculation and healing induction, cluster bud induction, differentiation induction culture, root induction, regeneration plant culture, regeneration plant transplanting and the like for propagating kaffir lily flower. The kaffir lily can propagate plants with the same genetic background and regular external appearance in a large scale and in relatively short time under the condition without influencing the normal development and propagation of the female plants, the material taking is not limited by seasons, the regeneration period is short, the dedifferentiation rate and the differentiation rate are high, the rooting rate is high, the transplanting survival rate is high, and the growth state of the transplanted plants is good. The invention is particularly suitable for fast propagation of rare varieties of the kaffir lily, and is applicable to transgenosis of a leaf disc transformation method of the kaffir lily, the cost of the average single strain regeneration seedlings is very low, and the invention is particularly suitable for factory production.
Description
Technical field
The present invention is the flowers propagation method, is specifically related to the external asexual rapid propagation method of kaffir lily.
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, can induce callus, indefinite bud, adventive root, form the technology of the other products of complete plant or generation economically valuable.Except extremely low variation, can both keep the merit of parent (explant source plant) by 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: one-tenth kaffir lily in age only opened once in 1 year spends, and seed needs 9 months ability maturations at least; Division propagation-year is told 1～3 strain 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 the treasure kaffir lily at home, supply falls short of demand always in the international market.Utilize plant tissue culture technique apace the in-vitro propagate kaffir lily can produce the unified kaffir lily of plant type on a large scale, breed the good rare kind of kaffir lily fast, further satisfy demand domestic, the international market.Though the kaffir lily tissue culture that forefathers did experiment is tentatively succeedd,, regeneration period length etc. low owing to regeneration rate limited the application of kaffir lily tissue culture technique in actual production.But the present invention fruit of Rangoon creeper orchid bred with interior realization fast in 1 year.
Summary of the invention
Produce the shortcoming that the kaffir lily plant type varies, reproduction speed is slow for overcoming traditional propagation method; Also in order to overcome regeneration rate low (average every explant regeneration goes out 0～2 strain regeneration bud), long shortcoming of regeneration period in the experiment of forefathers kaffir lily tissue culture.Providing a kind of is the method for in-vitro rapid propagation of clivia miniata of explant with tender plant of children and the stripping and slicing of spire sheet
The technical solution adopted in the present invention is:
1. the acquisition of explant: get mature seed, put into aseptic super-clean bench, with the alcohol-pickled 1～2min of 75% (volume/volume), mercuric chloride solution with 0.1～0.2% (mass/volume) soaked 10～20 minutes, aseptic water washing 3～5 times, seed is inoculated on the 1/2MS semisolid culturemedium that does not contain hormone, 20～30 ℃ 8-12 hour every day illumination cultivation.
2. explant is inoculated and callus of induce: when treating that step 1 seedling grows 1-2 sheet leaf, the acrial part of plantlet is cut to (comprising spire and young stem) fritter of diameter 3～5mm, be seeded on the MS semisolid culturemedium that contains 6-BA1～2mg/L, NAA0.5～2.0mg/L, sucrose 3.0% (mass/volume) 20～30 ℃ of 40W fluorescent lamp irradiations every day 8～9 hours.40～50 days subcultures once can be induced differentiation when waiting to grow yellow granular callus.The tender acrial part of also available aseptic regeneration plant children is an explant.
3. (2.0～4.0mg/L)+2, (0.2～0.5mg/L)+3.0% (mass/volume) sucrose, 40～50 days subcultures once, 20～30 ℃ of illumination every day is 10～12 hours for 4-D for grow thickly the inducing of bud: callus is transferred to inductive differentiation medium: MS+KT.Change differential medium and can differentiate 121 strain regrowths from the callus in a slice source in the time of 4 months.
4. inducing of root: regeneration plant forwards on the root media of 1/2MS+ sucrose 2% (mass/volume), 20～30 ℃ of illumination 10～14 hours every days.
5. regeneration plant is transplanted: just can transplant after plant takes root.121 ℃ of sterilizations of humus soil are more than 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.
Fruit of Rangoon creeper orchid of the present invention is a large amount of generation regeneration plants that genetic background is identical, plant type is unified in the short relatively time.With the stripping and slicing of the tender above-ground plant parts of children is that the dedifferentiation frequency average of explant can reach 72%, callus differentiation rate average can reach 87.5%, the callus that the monolithic leaf produces can differentiate hundreds of strain regrowth through subculture, and the bud rooting rate of growing thickly can reach 100%, and transplanting survival rate reaches 100% under control environment.
The present invention can go out the plant that genetic background is identical, outward appearance is neat at the large-scale breeding in the short relatively time of fruit of Rangoon creeper orchid under the situation that does not influence maternal plant normal development and breeding; Draw materials and be not subject to seasonal restrictions; Regeneration period is short; Dedifferentiation frequency and differentiation rate height; The rooting rate height; The transplanting survival rate height, growth conditions is good behind the plantlet of transplant; Be highly suitable for the quick breeding of the rare kind of kaffir lily; Be applicable to kaffir lily " leaf disc transformation method " transgenosis; The cost of average individual plant regrowth is extremely low, is fit to very much batch production production.
Embodiment
The present invention is further illustrated below in conjunction with practical operation, but scope of the present invention is not limited to following embodiment.
Embodiment 1:
1. the acquisition of explant: get mature seed, put into aseptic super-clean bench, with the alcohol-pickled 1min of 75% (volume/volume), mercuric chloride solution with 0.1% (mass/volume) soaked 10 minutes, aseptic water washing 3 times, seed is inoculated on the 1/2MS semisolid culturemedium that does not contain hormone, 20 ℃ 8 hours every days illumination cultivation.
2. explant inoculation and callus of induce: the fritter that the acrial part that grows the plantlet of 1 leaf in the step 1 or contain the aseptic regeneration plant of 1 leaf is cut to (comprising spire and young stem) diameter 3mm, be seeded on the MS semisolid culturemedium that contains 6-BA1mg/L, NAA0.5mg/L, sucrose 3.0% (mass/volume) 20 ℃ of 40W fluorescent lamp irradiations every day 8 hours.40 days subcultures once can be induced differentiation when waiting to grow yellow granular callus.
3. grow thickly the inducing of bud: callus is transferred to inductive differentiation medium: MS+KT2.0mg/L+2,4-D0.2mg/L+3.0% (mass/volume) sucrose, 40 days subcultures once, 20C illumination every day 10 hours.
4. inducing of root: regeneration plant forwards on the root media of 1/2MS+ sucrose 2% (mass/volume), 20 ℃ of illumination 10 hours every days.
5. regeneration plant is transplanted: just 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. the acquisition of explant: get mature seed, put into aseptic super-clean bench, with the alcohol-pickled 2min of 75% (volume/volume), mercuric chloride solution with 0.2% (mass/volume) soaked 20 minutes, aseptic water washing 5 times, seed is inoculated on the 1/2MS semisolid culturemedium that does not contain hormone, 30 ℃ 12 hours every days illumination cultivation.
2. explant is inoculated and callus of induce: have the acrial part of the aseptic regeneration plant of two leaves to be cut to (comprising spire and young stem) fritter of diameter 5mm the plantlet or the length that grow two leaves in the step 1, be seeded on the MS semisolid culturemedium that contains 6-BA2mg/L, NAA2.0mg/L, sucrose 3.0% (mass/volume) 30 ℃ of 40W fluorescent lamp irradiations every day 9 hours.50 days subcultures once can be induced differentiation when waiting to grow yellow granular callus.
3. grow thickly the inducing of bud: callus is transferred to inductive differentiation medium: MS+KT4.0mg/L+2,4-D0.5mg/L+3.0% (mass/volume) sucrose, 50 days subcultures once, 30 ℃ of illumination every day 12 hours.
4. inducing of root: regeneration plant forwards on the root media of 1/2MS+ sucrose 2% (mass/volume), 30 ℃ of illumination 14 hours every days.
5. regeneration plant is transplanted: just 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. the acquisition of explant: get mature seed, put into aseptic super-clean bench, with the alcohol-pickled 40S of 75% (volume/volume), mercuric chloride solution with 0.15% (mass/volume) soaked 15 minutes, aseptic water washing 4 times, seed is inoculated on the 1/2MS semisolid culturemedium that does not contain hormone, 24 ℃ 10 hours every days illumination cultivation.
2. explant is inoculated and callus of induce: when treating that step 1 seedling grows 1 leaf, the acrial part of plantlet is cut to (comprising spire and young stem) fritter of diameter 4mm, be seeded on the MS semisolid culturemedium that contains 6-BA1.5mg/L, NAA1.0mg/L, sucrose 3.0% (mass/volume) 24 ℃ of 40W fluorescent lamp irradiations every day 8.5 hours.45 days subcultures once can be induced differentiation when waiting to grow yellow granular callus.The tender acrial part of aseptic regeneration plant children of also available a slice leaf is an explant.
3. grow thickly the inducing of bud: callus is transferred to inductive differentiation medium: MS+KT (3mg/L)+2, (0.4～mg/L)+3.0% (mass/volume) sucrose, 45 days subcultures once, 24 ℃ of illumination every day is 11 hours for 4-D.
4. inducing of root: regeneration plant forwards on the root media of 1/2MS+ sucrose 2% (mass/volume), 24 ℃ of illumination 12 hours every days.
5. regeneration plant is transplanted: just can transplant after plant takes root.121 ℃ of sterilizations of humus soil are more than 50 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 tender plant of children and the stripping and slicing of spire sheet, it is characterized in that concrete steps are as follows:
1). the acquisition of explant: get mature seed, put into aseptic super-clean bench, with the alcohol-pickled 1～2min of 75% (volume/volume), mercuric chloride solution with mass volume ratio 0.1～0.2% soaked 10～20 minutes, aseptic water washing 3～5 times, seed is inoculated on the 1/2MS semisolid culturemedium that does not contain hormone, 20～30 ℃ 8-12 hour every day illumination cultivation;
2). explant inoculation and callus of induce: when treating that the step 1) seedling grows 1-2 sheet leaf, acrial part with plantlet, comprise that spire and young stem are cut to the fritter of diameter 3～5mm, be seeded on the MS semisolid culturemedium that contains 6-BA 1～2mg/L, NAA 0.5～2.0mg/L, sucrose mass volume ratio 3.0%, 20～30 ℃ of 40W fluorescent lamp irradiations every day 8～9 hours, 40～50 days subcultures once, induce differentiation when waiting to grow yellow granular callus, or be explant with the tender acrial part of aseptic regeneration plant children;
3). inducing of the bud of growing thickly: callus is transferred to inductive differentiation medium: MS+KT 2.0～4.0mg/L+2,4-D 0.2～0.5mg/L+ mass volume ratio 3.0% sucrose, 40～50 days subcultures once, 20～30 ℃ of illumination every day 10～12 hours is changeed inductive differentiation medium and is differentiated 121 strain regrowths from the callus in a slice source in the time of 4 months;
4). inducing of root: regeneration plant forwards on the root media of 1/2MS+ sucrose mass volume ratio 2%, 20～30 ℃ of illumination 10～14 hours every days;
5). regeneration plant is transplanted: transplant the plant back of taking root, and 121 ℃ of sterilizations of humus soil 30 or 60 minutes before transplanting are rinsed agar on the plant or foreign material well, watered once in 7 days permeablely just to survive.
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| CN2010101258525A CN101822218B (en) | 2010-03-17 | 2010-03-17 | Kaffir lily in-vitro propagation method using young plants and young blade cut blocks as explants |
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| CN2010101258525A CN101822218B (en) | 2010-03-17 | 2010-03-17 | Kaffir lily in-vitro propagation method using young plants and young blade cut blocks as explants |
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| 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 |
| 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 |
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