CN1275514C - Method for quick asexual reproduction of alfalfa by utilizing embryoid - Google Patents
Method for quick asexual reproduction of alfalfa by utilizing embryoid Download PDFInfo
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- CN1275514C CN1275514C CN 200410019861 CN200410019861A CN1275514C CN 1275514 C CN1275514 C CN 1275514C CN 200410019861 CN200410019861 CN 200410019861 CN 200410019861 A CN200410019861 A CN 200410019861A CN 1275514 C CN1275514 C CN 1275514C
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Abstract
The present invention relates to a method for plant breeding, particularly to a method for the quick asexual propagation of alfalfa by utilizing embryoids. Hypocotyls are used as explants, UM, 4-D of 2 to 3 mg/L, and KT of 0.25 to 0.5 mg/L are used as primary embryonal induction and secondary culture medium, and primary embryos are added in an MSO culture medium to induce the formation of secondary embryos. The secondary embryos have large size and plump cotyledons, and the proportion of the secondary embryos which grow into complete plants in an improved SH culture medium, which is high, reaches 78%. The method has the advantages of short cycle and low cost, and can improve the seedling forming efficiency of the tissue culture of alfalfa greatly. The minimum annual effective proliferation rate per year is 1: 338 to 450.
Description
Technical field
The present invention relates to method for propagation, particularly a kind of method of carrying out the alfalfa fast asexual propagation with embryoid.
Background technology
Alfalfa (Medicago sativa L.) is a kind of perennial cross pollination leguminous plant, be the important high protein green forage crop in the world, be described as " king of herbage ", have the economic benefit height, wide adaptability, the biological nitrogen fixation ability is strong, conserve water and soil, improve the soil, the characteristic that ecological functions are complete more and more is subject to people's attention.Because the cultivar of alfalfa is a tetraploid, the traditional breeding method operational difficulties is more.For this reason, utilize the modern biotechnology breeding to become the important channel of breed improvement.Therefore, since the seventies in 20th century, the research work of clover tissue culture and genetic transformation aspect never is interrupted.In the present normal successful clover method for tissue culture that adopts, no matter be liquid culture or solid culture mostly be greatly induce embryo callus subculture earlier, inducing embryoid body forms, forms at last three of whole plant and goes on foot cultural methods again.This kind incubation is cycle long (about 90 days) but also be mostly that several minimal mediums and hormone are used not only, and cost is higher.
Summary of the invention
The purpose of this invention is to provide and a kind ofly new carry out the method for alfalfa fast asexual propagation, can overcome above-mentioned defective with embryoid.It is to adopt the alfalfa hypocotyl of 4-5 days seedling ages as explant, induces and keep the formation of nascent embryoid on the UM medium, is that explant induction forms secondary embryo with nascent embryo again, utilizes secondary embryogenesis whole plant.Because secondary embryo deformity embryo is few, individual loose, cotyledon is full, thereby its ratio that forms whole plant is higher.The present invention has only adopted two kinds of hormones, reduced experimental cost, because nascent embryoid and secondary embryoid all have the ability that forms whole plant by regulation and control, also make the productive rate of whole plant improve greatly simultaneously thereby not only make the time that obtains whole plant shorten.
The present invention is through following steps:
1) cultivation of aseptic seedling: with alfalfa seed in superclean bench with 75% alcohol disinfecting 10 minutes, again with the sterilization of 0.1% mercuric chloride after 10 minutes, sterile distilled water cleans 5 times, seed is put into the 1/2MS medium of no hormone.14 hours/day photoperiods, light intensity 3000Lux cultivated 4-5 days under the room temperature 25-27 ℃ of condition.
2) inducing and subculture of nascent embryoid: the hypocotyl of getting aseptic seedling is put into and contains UM+2, the medium of 4-D (2-3mg/L)+KT (0.25-0.50mg/L)+3% sucrose+0.8% agar (nascent embryonal induction and subculture medium) (pH value 5.7, down together), condition of culture the same (down together), began to have callus to form in 2-3 days, begin to have embryoid to form after 16 days, had a large amount of nascent embryoids to produce in 25 days.Selecting nascent embryoid changes in secondary embryoid induction and the subculture medium (MSO+3% sucrose+0.8% agar) and carries out inducing and subculture of secondary embryoid; Change in the fresh nascent embryonal induction medium and can continue to produce nascent embryoid selecting behind the nascent embryoid remaining embryo callus subculture, thus the subculture of the embryoid of can coming into being.Producing in a large number with the embryoid of coming into being in the subculture process second time for the first time, nascent embryoid forms ability drop behind the subculture for the third time.In this process as the embryoid of will coming into being directly change in the seedling medium (improvement SH+2% sucrose+0.7% agar of no hormone) and induce the formation whole plant, inductivity low (34%).
3) inducing and subculture of secondary embryoid: the embryoid of will coming into being changes in the secondary embryonal induction and subculture medium of MSO+3% sucrose+0.8% agar, the nascent embryoid of 10%-15% can form whole plant, form a large amount of secondary embryoids on all the other most of nascent embryoids, 28 secondary embryos of average out to/nascent embryo, and secondary embryo abnormal rate few (less than 8%), individuality is loose, cotyledon is full, and the nascent embryoid of quality increases.After changing over to secondary embryo in the fresh secondary embryonal induction medium, have small part to become whole plant left and right sides direct development in 20 days, most of meeting continued to produce secondary embryo at 10 days, thereby above-mentioned secondary embryonal induction medium also can be used as the subculture medium of secondary embryo.After the subculture 3 times, the abnormal rate of secondary embryo increases in this medium, makes this process obtain circulating thereby can form secondary embryo by nascent embryonal induction again.
4) formation of whole plant: secondary embryo is changed in the one-tenth seedling medium of improvement SH+2% sucrose+0.7% agar of no hormone, secondary embryo no longer forms, and the statistics planting percent is that the statistics planting percent can reach 78% after 48%, 35 day after 20 days.
5) transplanting of test-tube plantlet with survive: the film that seals that unclamps blake bottle is practiced seedling about one week, in superclean bench with height of seedling more than 5 centimetres, the whole plant of root system stalwartness is chosen, change in the nutritive cube that vermiculite is housed behind the flowing water flush away medium, after sealing, put into by preservative film shady and cool place 10 days, open and seal film, check after 30 days that transplanting survival rate is 90%, change big Tanaka's plantation simultaneously over to.
The present invention can form on whole plant and the secondary embryo both direction after the nascent embryoid of clover forms to it, not only can obtain whole plant at short notice, and by the regulation and control to secondary embryonal induction and differentiation, can improve into seedling efficient greatly.The method is simple, the cultivation cost is low, reproduction rate is high.Only by hypocotyl → nascent embryoid → single circulation of secondary embryoid → whole plant, effective appreciation rate in 70 day time sky is (nascent embryoid number/explant) * secondary embryoid occurrence frequency (85%) * (secondary embryoid number/nascent embryoid) * secondary embryoid planting percent (78%)=4 * 85% * 28 * 78%=74 strain, just a hypocotyl explant can form 74 strain whole plants at least at 70 days, make the embryoid number be multiplied again behind nascent in addition embryo and the secondary embryo subculture, therefore the appreciation rate of single hypocotyl explant in 1 year is minimum is 338-450.
Description of drawings:
Fig. 1 carries out the flow chart of clover tissue culture by secondary embryo approach.
Fig. 2: the clover hypocotyl forms nascent embryoid in nascent embryoid induction and subculture medium.
Fig. 3: nascent embryoid forms secondary embryo on secondary embryoid induction and subculture medium.
Fig. 4: secondary embryo is in the lopsided embryo number raising after 3 generations of subculture on secondary embryonal induction and the subculture medium.
Fig. 5: nascent embryoid embryoid incidence behind subculture on the nascent embryoid induction subculture medium improves.
Fig. 6: secondary embryoid is grown and is formed whole plant.
Fig. 7: the root system development of plant is normal.
Embodiment
Substantive distinguishing features that the present invention gives prominence to and marked improvement can be embodied from following example.But they can not impose any restrictions the present invention.Fig. 1 is the flow chart that carries out the clover tissue culture by secondary embryo approach.
Embodiment 1
With the place of production be the Pekinese protect rich alfalfa seed in superclean bench respectively with the sterilization of 75% alcohol and 0.1% mercuric chloride after 10 minutes, sterile distilled water cleans 5 times, seed is put into the 1/2MS medium of no hormone.14 hours/day photoperiods, light intensity 3000Lux, cultivation cuts the aseptic seedling hypocotyl and puts into the autoclaved 25ml of the filling UM+2 of process under the room temperature 25-27 ℃ of condition in superclean bench after 4-5 days, in the conical flask of 4-D (concentration is 2-3mg/L)+KT (concentration is 0.25-0.5mg/L)+3% sucrose+0.8% agar medium (pH value 5.7 times with), 5 every bottle, sealing film seals, the culturing room that puts into constant temperature 25-27 ℃ after rubber band is fastened cultivates, illumination every day 14 hours, light intensity is 3000Lux, have a large amount of nascent embryoids to form after 16-25 days, on average to form nascent embryoid number be 4 to each explant, can form 28 (accompanying drawings 2) at most; Selecting nascent embryoid in superclean bench changes over to carry out inducing and subculture (accompanying drawing 3,4) of secondary embryoid in the conical flask that 25ml MSO+3% sucrose+0.8% agar medium is housed of sterilization treatment, every bottle of 5 embryoids, condition of culture is the same, produce secondary embryoid in 7-15 days on 85% the nascent embryoid, can form 28 secondary embryoids on average each nascent embryo, and the secondary embryo individuality that forms is loose, cotyledon is full, the nascent embryo of quality increases.All the other nascent embryos can form whole plant in 25 days; Change over to and can continue to produce nascent embryoid (accompanying drawing 5) in the fresh nascent embryonal induction medium selecting behind the nascent embryoid remaining embryo callus subculture; Change over to after secondary embryo separated in the one-tenth seedling medium of improvement SH+2% sucrose+0.7% agar of no hormone, condition of culture is the same, can stop the formation of secondary embryo, generation a large amount of whole plants (accompanying drawing 6) after 15-30 days.If after changing over to secondary embryo in fresh MSO+3% sucrose+0.8% agar medium, have small part to become whole plant (accompanying drawing 7) direct development in 20 days, most of meeting continued to produce secondary embryo at 10 days.The appreciation rate of single hypocotyl explant in 1 year is minimum to be 338-450.
Embodiment 2
With the place of production be in the Pekinese alfalfa seed of lucerne in superclean bench respectively with the sterilization of 75% alcohol and 0.1% mercuric chloride after 15 minutes, sterile distilled water cleans 5 times, seed is put into the 1/2MS medium of no hormone.14 hours/day photoperiods, light intensity 3000Lux, cultivation cuts the aseptic seedling hypocotyl and puts into the autoclaved 25ml of the filling UM+2 of process under the room temperature 25-27 ℃ of condition in superclean bench after 4-5 days, in the conical flask of 4-D (2-3mg/L)+KT (0.25-0.5mg/L)+3% sucrose+0.8% agar medium (pH value 5.7 times with), 5 every bottle, sealing film seals, the culturing room that puts into constant temperature 25-27 ℃ after rubber band is fastened cultivates, illumination every day 14 hours, light intensity is 3000Lux, had nascent embryoid to form in 28 days, nascent embryoid induction rate (nascent embryoid number/explant number * 100%) is 300%, can form 40 at most; Selecting nascent embryoid in superclean bench changes in the conical flask that 25ml MSO+3% sucrose+0.8% agar medium is housed of sterilization treatment and carries out inducing and subculture of secondary embryoid, every bottle of 5 embryoids, condition of culture is the same, produce secondary embryoid about 10 days on 45% the nascent embryoid, can form 24 secondary embryoids on average each nascent embryo, and the secondary embryo individuality that forms is loose, cotyledon is full, the nascent embryo of quality increases.All the other nascent embryo major parts can form whole plant in 25 days; Change in the fresh nascent embryonal induction medium and can continue to produce nascent embryoid selecting behind the nascent embryoid remaining embryo callus subculture; Change over to after secondary embryo separated in the one-tenth seedling medium of improvement SH+2% sucrose+0.7% agar of no hormone, condition of culture is the same, can stop the formation of secondary embryo, 84% secondary embryo generation whole plant after 15-30 days.If after changing over to secondary embryo in fresh MSO+3% sucrose+0.8% agar medium, have small part to become whole plant direct development in 20 days, most of meeting continued to produce secondary embryo at 10 days, therefore can carry out the subculture of secondary embryo.Single hypocotyl explant is only by hypocotyl → nascent embryoid → single circulation of secondary embryoid → whole plant, effective appreciation rate in 70 day time sky is 24 strains, make the embryoid number be multiplied again behind nascent in addition embryo and the secondary embryo subculture, therefore the appreciation rate of single hypocotyl explant in 1 year is minimum is 125.
Embodiment 3
The Ai Fei nit clover that with the place of production is Tianjin is by protecting identical embodiment of clover of rich clover and middle lucerne come into being inducing of embryoid and secondary embryoid and subculture.To grow the ratio of nascent embryoid be 44% to each hypocotyl explant in nascent embryonal induction medium.The nascent embryoid that grows is changed in the secondary embryonal induction medium, and 80% nascent embryo can form secondary embryo, and 18 secondary embryos of average out to/nascent embryo can form 24 at most.Secondary embryo is changed over to in the identical one-tenth seedling medium of above-mentioned embodiment, and statistics formation whole plant rate is 78% after 30 days.Therefore the single hypocotyl explant of Ai Fei nit clover is only by hypocotyl → nascent embryoid → single circulation of secondary embryoid → whole plant, and the effective appreciation rate in 70 day time sky is 44% * 80% * 18 * 78%=5 strain.Make the embryoid number be multiplied again behind nascent in addition embryo and the secondary embryo subculture, therefore the appreciation rate of single hypocotyl explant in 1 year is minimum is 26 strains.The Ai Fei nit clover that should be understood that is the kind of a more difficult life, the inventor is a minimal medium with SH medium, the MSH medium of solid, UM medium, the MS medium of liquid, additional dissimilar hormones adopts three traditional step cultivations that it is carried out inducing of embryoid, all fails to make its embryoid incidence to be improved.This shows, induce secondary embryo, utilizing secondary embryogenesis whole plant is the good method that Ai Fei nit clover carries out fast asexual propagation.
Above-mentioned 3 embodiment illustrate that the new method among the present invention has certain operability and repeatability, can use in the production of clover and scientific research.
Claims (5)
1, a kind ofly carry out the method for alfalfa fast asexual propagation, it is characterized in that may further comprise the steps with embryoid:
1) cultivation of aseptic seedling: alfalfa seed with 75% alcohol disinfecting 10 minutes,, is washed 5 times after 10 minutes with the sterilization of 0.1% mercuric chloride again, put into the 1/2MS medium of no hormone, 14 hours/day photoperiods, light intensity 3000Lux cultivated 4-5 days under the room temperature 25-27 ℃ of condition;
2) inducing and subculture of nascent embryoid: the hypocotyl of getting aseptic seedling is put into pH value 5.7, UM+ concentration is 2 of 2-3mg/L, 4-D+ concentration is to cultivate in the nascent embryonal induction of KT+3% sucrose+0.8% agar of 0.25-0.5mg/L and the subculture medium, 14 hours/day photoperiods, light intensity 3000Lux, cultivated 25 days under the room temperature 25-27 ℃ of condition, have nascent embryoid to produce;
3) inducing and subculture of secondary embryoid: select nascent embryoid and change pH value 5.7 over to, cultivate in the secondary embryonal induction of MSO+3% sucrose+0.8% agar and the subculture medium, 14 hours/day photoperiods, light intensity 3000Lux cultivated 10 days under the room temperature 25-27 ℃ of condition;
4) formation of whole plant: secondary embryo is changed in the one-tenth seedling medium of no hormone improvement SH+2% sucrose+0.7% agar, pH value 5.7,14 hours/day photoperiods, light intensity 3000Lux cultivated 20-35 days under the room temperature 25-27 ℃ of condition;
5) transplanting of test-tube plantlet with survive: the film that seals that unclamps blake bottle carries out one week of hardening, getting the whole plant seedling changes in the nutritive cube that vermiculite is housed after with flowing water flush away medium, put into shady and cool place after preservative film seals 10 days, and opened and seal film, change big Tanaka's plantation after 30 days over to.
2, carry out the method for alfalfa fast asexual propagation according to claim 1 is described with embryoid, it is characterized in that secondary embryo changed over to and cultivate direct development in 20-30 days in the fresh one-tenth seedling medium and become whole plant.
3, carry out the method for alfalfa fast asexual propagation according to claim 1 is described with embryoid, it is characterized in that described nascent embryoid subculture 3 times in nascent embryonal induction and subculture medium.
4, carry out the method for alfalfa fast asexual propagation according to claim 1 is described with embryoid, it is characterized in that described secondary embryoid subculture 3 times in secondary embryonal induction and subculture medium.
5, carry out the method for alfalfa fast asexual propagation according to claim 1 is described with embryoid, it is characterized in that described whole plant seedling is the whole plant of height of seedling in the root system stalwartness more than 5 centimetres.
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| CN 200410019861 CN1275514C (en) | 2004-07-01 | 2004-07-01 | Method for quick asexual reproduction of alfalfa by utilizing embryoid |
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| CN 200410019861 CN1275514C (en) | 2004-07-01 | 2004-07-01 | Method for quick asexual reproduction of alfalfa by utilizing embryoid |
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| CN101703002B (en) * | 2009-11-03 | 2012-06-20 | 江苏农林职业技术学院 | Culture medium for overcoming variety and genetype restriction in regeneration culture of alfalfa high frequency somatic embryos |
| CN102138533B (en) * | 2011-05-05 | 2012-08-22 | 中国农业大学 | Method for preparing medicago falcata regenerated plant through tissue culture and culture medium |
| CN103503684B (en) * | 2013-10-09 | 2015-09-09 | 西藏大学农牧学院 | A kind of method of transplanting type alfalfa cultivation |
| CN103843663B (en) * | 2014-03-14 | 2015-07-15 | 中国农业科学院北京畜牧兽医研究所 | Method for promoting rooting of alfalfa tissue culture seedlings |
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