CN103798140B - Significantly improve the cultural method of Lilium Germplasm embryo callus shoot proliferation rate - Google Patents
Significantly improve the cultural method of Lilium Germplasm embryo callus shoot proliferation rate Download PDFInfo
- Publication number
- CN103798140B CN103798140B CN201410037346.9A CN201410037346A CN103798140B CN 103798140 B CN103798140 B CN 103798140B CN 201410037346 A CN201410037346 A CN 201410037346A CN 103798140 B CN103798140 B CN 103798140B
- Authority
- CN
- China
- Prior art keywords
- callus
- medium
- subculture
- agglomerate
- inoculated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 146
- 241000234435 Lilium Species 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 18
- 230000035755 proliferation Effects 0.000 title claims abstract description 11
- 210000001161 mammalian embryo Anatomy 0.000 title claims description 15
- 230000003321 amplification Effects 0.000 claims abstract description 39
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 39
- 230000004069 differentiation Effects 0.000 claims abstract description 8
- 241000196324 Embryophyta Species 0.000 claims abstract description 7
- 239000000835 fiber Substances 0.000 claims abstract description 4
- 239000012879 subculture medium Substances 0.000 claims description 45
- 239000002609 medium Substances 0.000 claims description 33
- 239000001963 growth medium Substances 0.000 claims description 25
- 230000006698 induction Effects 0.000 claims description 22
- 229920001817 Agar Polymers 0.000 claims description 15
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 15
- 229930006000 Sucrose Natural products 0.000 claims description 15
- 239000008272 agar Substances 0.000 claims description 15
- 239000006870 ms-medium Substances 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 15
- 239000005720 sucrose Substances 0.000 claims description 15
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- 238000000338 in vitro Methods 0.000 claims description 10
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 8
- 229920000742 Cotton Polymers 0.000 claims description 7
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- 239000003755 preservative agent Substances 0.000 claims description 7
- 230000002335 preservative effect Effects 0.000 claims description 7
- 230000032683 aging Effects 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 235000015149 toffees Nutrition 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 230000001681 protective effect Effects 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 230000002068 genetic effect Effects 0.000 abstract description 6
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- 230000001172 regenerating effect Effects 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 16
- 206010052428 Wound Diseases 0.000 description 10
- 208000027418 Wounds and injury Diseases 0.000 description 10
- 239000005972 6-Benzyladenine Substances 0.000 description 8
- 239000005971 1-naphthylacetic acid Substances 0.000 description 6
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 6
- IIDAJRNSZSFFCB-UHFFFAOYSA-N 4-amino-5-methoxy-2-methylbenzenesulfonamide Chemical compound COC1=CC(S(N)(=O)=O)=C(C)C=C1N IIDAJRNSZSFFCB-UHFFFAOYSA-N 0.000 description 6
- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 description 6
- 230000008929 regeneration Effects 0.000 description 5
- 238000011069 regeneration method Methods 0.000 description 5
- 230000032459 dedifferentiation Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000001788 irregular Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- OVSKIKFHRZPJSS-UHFFFAOYSA-N 2,4-D Chemical compound OC(=O)COC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention relates to micropropagation of plants technical field, aim to provide the cultural method significantly improving Lilium Germplasm callus shoot proliferation rate.This cultural method comprises step: the amplification cultivation of the Fiber differentiation of callus, the squamous subculture of callus, callus.The present invention with the callus of the little agglomerate of the diameter 0.3 ~ 0.5cm in lily callus regenerating system for object, after its outstanding advantage is callus process, within a short period of time obtains the very high rate of increase, be significantly higher than the callus proliferation rate of conventional subculture, and without the need to carrying out subculture again during low temperature treatment, thus the higher rate of increase is obtained while saving human time, be the efficient expansion traditional font system based on callus and genetic conversion system preparatory condition.
Description
Technical field
The present invention relates to micropropagation of plants technical field, particularly relate to the cultural method significantly improving Lilium Germplasm callus shoot proliferation rate.
Background technology
Lily embryo callus regenerating system is significant to setting up genetic conversion system, keep callus line can provide the raw material that can obtain at any time for the genetic transformation in later stage by squamous subculture, because transgenic technology transformation efficiency is still lower at present, the callus of positive controls for high proliferation rates obtains sufficient callus in a short time to ensure constantly to carry out transgeneic procedure, to obtain genetically modified necessary condition.
For the subculture of lily embryo callus, prior art mainly by cultivating under same subculture medium and condition of culture, all for the cycle with 4-8, after the callus agglomerate of last subculture is divided into less agglomerate, be inoculated on fresh culture and cultivate, make it breed further; For the improvement of prior art, have been reported mainly around the concentration of material and the changes of ratio such as plant growth regulator in squamous subculture based formulas.But regularly expand numerous (every 4-8 is all) and need higher time human cost, expand numerous rate of increase relatively low simultaneously.Increase with subculture number, regeneration ability degeneration, regeneration capacity decline.How can obtain higher callus proliferation rate while saving human time is technical bottleneck.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes deficiency of the prior art, provides a kind of cultural method significantly improving Lilium Germplasm embryo callus shoot proliferation rate.
For technical solution problem, solution of the present invention is:
The cultural method significantly improving Lilium Germplasm embryo callus shoot proliferation rate is provided, comprises the following steps:
(1) Fiber differentiation of callus: on superclean bench, get Lilium Germplasm plantlet in vitro scale and carry out callus of induce cultivation, method is peeled off the scale tweezers of plantlet in vitro bulb or scalpel, by its scale tip cut 0.3cm, and draw 1 ~ 3 wound at its adaxial and its surface scalpel, calli induction media surface is contacted down with wound during inoculation, callus of induce is cultivated after 20 ~ 50 days, obtain the light yellow of diameter 0.3 ~ 0.5cm, granular embryo callus agglomerate, Callus induction rate is 63.3% ~ 79.1% (wherein, wound on scale is the key producing callus, wound contact medium can make plant fully respond to the materials such as the hormone in medium, to produce callus),
It is carry out under complete darkness, the callus of induce condition of culture of 25 DEG C that described callus of induce is cultivated, for the calli induction media inoculated with MS medium (MurashigeandSkoog, 1962) be minimal medium, namely often liter of medium adds 4.43gMS powder, also containing concentration in described calli induction media is 2 of 0.1 ~ 2.0mg/L, 4-dichlorphenoxyacetic acid (2, 4-dichlorophenoxyacetic, 2, 4-D), 0 ~ 1.0mg/L α-naphthaleneacetic acid (1-Naphthaleneaceticacid, NAA), concentration is the 6-benzyl aminoadenine (6-benzyladenine of 0.1 ~ 0.4mg/L, 6-BA), the sucrose of 30g/L, 6 ~ 8g/L agar, the pH value of calli induction media is 5.8 (wherein, 2, the induction of 4-dichlorphenoxyacetic acid to callus is crucial),
(2) squamous subculture of callus: callus of induce is cultivated after 60 ~ 90 days, superclean bench is rejected with tweezers or scalpel the aging part of callus agglomerate toffee, is inoculated on fresh subculture medium; Each being equipped with in the culture dish of subculture medium inoculates 20 ~ 30 pieces of callus agglomerates, the diameter of callus agglomerate is between 0.3 ~ 0.5cm, every 4 weeks subcultures once, each subculture can obtain the rate of increase of monthly 2 ~ 2.5 times, namely callus agglomerate was through the cultivation of month, and weight increases to 2 ~ 2.5 times during inoculation;
Described squamous subculture carries out under complete darkness, the squamous subculture condition of 25 DEG C, for the subculture medium inoculated with MS medium (MurashigeandSkoog, 1962) be minimal medium, namely often liter of medium adds 4.43gMS powder, also containing concentration in described subculture medium is 2 of 0.1 ~ 2.0mg/L, 4-D, 0 ~ 1mg/L α-naphthaleneacetic acid (1-Naphthaleneaceticacid, NAA), the 6-benzyl aminoadenine (6-benzyladenine of 0.1 ~ 0.4mg/L, 6-BA), the sucrose of 30g/L, 4 ~ 8g/L agar, the pH value of subculture medium is 5.8 (wherein, 2, the maintenance of 4-dichlorphenoxyacetic acid to callus dedifferentiation state is crucial),
(3) amplification cultivation of callus:
Callus agglomerate squamous subculture after 1 ~ 8 month on subculture medium, callus agglomerate is cut into the fritter of 0.5cm, be inoculated on the culture dish containing subculture medium, successively with through the preservative film of ultraviolet sterilization and tinfoil parcel, then the low temperature treatment of 30 ~ 60 days is carried out, low temperature treatment condition is 1 ~ 4 DEG C, complete darkness (the differentiation situation that low temperature treatment can suppress callus to occur on subculture medium, keep callus in dedifferentiation state and be conducive to the propagation of callus, dark condition is conducive to induction and the propagation of callus, keep the dedifferentiation state of callus),
Again the callus agglomerate after low temperature treatment is inoculated on amplification culture medium, carry out low light level cultivation, the low light level is cultivated and is referred to and to carry out under the condition of illumination 100 ~ 900lux, temperature 25 DEG C, and amplification culture medium is contained in bore is 6.35cm, is highly in the cylindricality bottle of 9.20cm;
After the low light level cultivates 60 days, callus agglomerate after amplification is divided into the fritter of 0.3 ~ 0.5cm, be transferred to dark culturing on fresh subculture medium, dark culturing refers to: complete darkness, temperature 25 DEG C, subculture medium is contained in (dark condition is conducive to induction and the propagation of callus, keeps the dedifferentiation state of callus) in culture dish; Callus agglomerate after low temperature treatment and the low light level are cultivated, is the callus of the rate of increase 15 ~ 36.6;
Described amplification culture medium is with MS medium (MurashigeandSkoog, 1962) be minimal medium, namely often liter of medium adds 4.43gMS powder, also containing sucrose and 4 ~ 8g/L agar that concentration is 30g/L in amplification culture medium, and the pH value 5.8 of amplification culture medium.
In the present invention, in step (3), be filled with absorbent cotton between the tinfoil of parcel culture dish and preservative film, absorbent cotton is used in low temperature treatment and recovers protective plant material in normal temperature.
Said method provided by the invention may be used for, in lily callus regeneration system and genetic conversion system, being specially: cut into fritter after carrying out squamous subculture to the callus of said method acquisition, be inoculated on corresponding subculture medium and cultivate; Break up on regeneration culture medium with this callus and take root, hardening, transplanting can obtain the tissue culture plants of lily; Maybe using the acceptor of this callus as genetic conversion system, through Agrobacterium infection or Biolistic mediated transformation, through screening and identification, and on regeneration culture medium, differentiation is taken root, hardening, transplanting obtain the transfer-gen plant of lily.
Compared with prior art, the invention has the beneficial effects as follows:
The callus of Lilium Germplasm scale is derived from by low temperature treatment, cultivated by low light level normal temperature again and adjust culture medium prescription, the vigor of callus and embryo can be preserved, reduce the time labour costs of regular subculture, and obtain the rate of increase of 15 ~ 36.6 times simultaneously.
The present invention with the callus of the little agglomerate of the diameter 0.3 ~ 0.5cm in lily callus regenerating system for object, after its outstanding advantage is callus process, within a short period of time obtains the very high rate of increase, be significantly higher than the callus proliferation rate of conventional subculture, and without the need to carrying out subculture again during low temperature treatment, thus the higher rate of increase is obtained while saving human time, be the efficient expansion traditional font system based on callus and genetic conversion system preparatory condition.
Accompanying drawing explanation
Fig. 1 is the electron microscopic observation figure that in embodiment, Calli Differentiation goes out heart-shape embryo.
Fig. 2 is the electron microscopic observation figure that in embodiment, Calli Differentiation goes out torpedo-shape embryo.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, facilitates professional and technical personnel's comprehend the present invention of this specialty, but do not limit the present invention in any way.
The cultivation of embodiment 1 huge ball lily subculture 1 callus
(1) Fiber differentiation of callus: get huge ball lily (LiliumbrowniiF.E.BrownexMiellezvar.giganteumG.Y.Li & Z.H.Chen) plantlet in vitro scale and carry out callus of induce cultivation, method is peeled off the scale tweezers of plantlet in vitro bulb or scalpel, by its scale tip cut 0.3cm, and draw 2 wounds in its adaxial and its surface with taking off scalpel, callus of induce culture surface is contacted down with wound during inoculation, at complete darkness, cultivate under the callus of induce condition of culture of 25 DEG C after 50 days, obtain the light yellow of diameter 0.5cm, the embryo callus agglomerate that graininess is good, Callus induction rate is 79.1%.
For the calli induction media inoculated with MS medium (MurashigeandSkoog, 1962) be minimal medium, namely often liter of medium adds 4.43gMS powder, be also 2,4-dichlorphenoxyacetic acid (2, the 4-dichlorophenoxyacetic of 1mg/L containing concentration, 2,4-D), concentration is 6-benzyl aminoadenine (6-benzyladenine, 6-BA), the sucrose of 30g/L, the 8g/L agar of 0.2mg/L, and the pH of calli induction media is 5.8.
(2) squamous subculture of callus: after callus of induce cultivates 80 days, superclean bench is rejected with tweezers or scalpel the aging part of callus agglomerate toffee, be inoculated on fresh subculture medium, 30 pieces of callus agglomerates inoculated by each culture dish, every block callus agglomerate diameter 0.3cm.Cultivate under complete darkness, the squamous subculture condition of 25 DEG C, every 4 weeks subcultures once, obtain the appreciation rate of monthly 2.3 times.
For the callus subculture medium inoculated with MS medium (MurashigeandSkoog, 1962) be minimal medium, namely often liter of medium adds 4.43gMS powder, be also 2,4-dichlorphenoxyacetic acid (2, the 4-dichlorophenoxyacetic of 1mg/L containing concentration, 2,4-D), concentration is 6-benzyl aminoadenine (6-benzyladenine, 6-BA), the sucrose of 30g/L, the 4g/L agar of 0.2mg/L, and the pH of subculture medium is 5.8.
(3) amplification cultivation of callus: callus agglomerate was cultivated after 1 month on subculture medium, obtain the rate of increase of 2.3 times, callus agglomerate is cut into the fritter of 0.5cm, be inoculated on the culture dish containing identical subculture medium, after sterilised membrane filter sealing, with the preservative film after ultraviolet disinfection and tinfoil parcel, the a small amount of absorbent cotton of therebetween, carry out the low temperature treatment of 30 days, low temperature treatment condition is 1 DEG C, complete darkness.Be inoculated on amplification culture medium by the callus agglomerate after low temperature treatment, it is 6.35cm that amplification culture medium is contained in bore, is highly in the cylindricality bottle of 9.20cm, at illumination 500lux, carries out low light level cultivation under temperature 25 DEG C of conditions.After the low light level cultivates 60 days, the callus agglomerate after amplification is divided into the fritter of 0.3cm, be transferred to dark culturing on fresh subculture medium, dark culturing is at complete darkness, and at temperature 25 DEG C, subculture medium is contained in culture dish training.
Amplification culture medium is with MS medium (MurashigeandSkoog, 1962) for minimal medium, and namely often liter of medium adds 4.43gMS powder, and be also sucrose and the 4g/L agar of 30g/L containing concentration, the pH of amplification culture medium is 5.8.
Through amplification cultivation, callus agglomerate rapid development, callus bolus volume is large and irregular, and on the culture dish containing subculture medium identical before being inoculated into amplification cultivation, can obtain the callus agglomerate of 900 pieces of diameter 5mm, the rate of increase reaches 1:30.
Embodiment 2 huge ball lily short-term subcultured callus is cultivated
(1) get huge ball lily (LiliumbrowniiF.E.BrownexMiellezvar.giganteumG.Y.Li & Z.H.Chen) plantlet in vitro scale and carry out callus of induce cultivation, method is peeled off the scale tweezers of plantlet in vitro bulb or scalpel, by its scale tip cut 0.3cm, and draw 1 wound at its adaxial and its surface scalpel, callus of induce culture surface is contacted down with wound during inoculation, at complete darkness, cultivate under the callus of induce condition of culture of 25 DEG C after 40 days, obtain the light yellow of diameter 0.4cm, the embryo callus agglomerate that graininess is good, Callus induction rate is 63.3%.
For the calli induction media inoculated with MS medium (MurashigeandSkoog, 1962) be minimal medium, namely often liter of medium adds 4.43gMS powder, also containing concentration is 2 of 2mg/L, 4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D), 0.5mg/L α-naphthaleneacetic acid (1-Naphthaleneaceticacid, NAA), concentration is the 6-benzyl aminoadenine (6-benzyladenine of 0.4mg/L, 6-BA), the sucrose of 30g/L, 7g/L agar, the pH of calli induction media is 5.8.
(2) squamous subculture of callus: after callus of induce cultivates 90 days, superclean bench is rejected with tweezers or scalpel the aging part of callus agglomerate toffee, be inoculated on fresh subculture medium, each culture dish 25 pieces of callus agglomerates, every block callus diameter 0.4cm.Cultivate under complete darkness, the squamous subculture condition of 25 DEG C, every 4 weeks subcultures once, can obtain the appreciation rate of monthly 2 times.
For the callus subculture medium inoculated with MS medium (MurashigeandSkoog, 1962) be minimal medium, namely often liter of medium adds 4.43gMS powder, be 2 of 2mg/L containing concentration, 4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D), 0.5mg/L α-naphthaleneacetic acid (1-Naphthaleneaceticacid, NAA), concentration is the 6-benzyl aminoadenine (6-benzyladenine of 0.4mg/L, 6-BA), the sucrose of 30g/L, 6g/L agar, the pH of callus subculture medium is 5.8.
(3) amplification cultivation of callus: callus agglomerate was cultivated after 3 months on subculture medium, obtain the rate of increase of monthly 2 times, callus agglomerate is cut into the fritter of 0.5cm, be inoculated on the culture dish containing identical subculture medium, after sterilised membrane filter sealing, with the preservative film after ultraviolet disinfection and tinfoil parcel, the a small amount of absorbent cotton of therebetween, carry out the low temperature treatment of 60 days, low temperature treatment condition is 3 DEG C, complete darkness.Be inoculated on amplification culture medium by entering the callus agglomerate after low temperature treatment, amplification culture medium be contained in bore be 6.35cm, highly for 9.20cm cylindricality bottle in, at illumination 100lux, carry out after the low light level cultivates 60 days under temperature 25 DEG C of conditions, callus agglomerate after amplification is divided into the fritter of 0.4cm, be transferred to dark culturing on fresh subculture medium, dark culturing refers to the condition of complete darkness, temperature 25 DEG C, and subculture medium is contained in culture dish training.
Amplification culture medium is with MS medium (MurashigeandSkoog, 1962) for minimal medium, and namely often liter of medium adds 4.43gMS powder, and be also sucrose and the 6g/L agar of 30g/L containing concentration, the pH of amplification culture medium is 5.8.
Through amplification cultivation, callus agglomerate rapid development, callus bolus volume is large and irregular, and on the culture dish containing subculture medium identical before being inoculated into amplification cultivation, can obtain the callus agglomerate of 450 pieces of diameter 5mm, the rate of increase reaches 1:15.
The cultivation of embodiment 3 huge ball lily long-term subculture callus
(1) get huge ball lily (LiliumbrowniiF.E.BrownexMiellezvar.giganteumG.Y.Li & Z.H.Chen) plantlet in vitro scale and carry out callus of induce cultivation, method is peeled off the scale tweezers of plantlet in vitro bulb or scalpel, by its scale tip cut 0.3cm, and draw 3 wounds in its adaxial and its surface with taking off scalpel, contacting medium down with wound during inoculation is indicated as suitable, at complete darkness, cultivate under the callus of induce condition of culture of 25 DEG C after 20 days, obtain the light yellow of diameter 0.3cm, the embryo callus agglomerate that graininess is good, Callus induction rate is 78.1%.
For the calli induction media inoculated with MS medium (MurashigeandSkoog, 1962) be minimal medium, namely often liter of medium adds 4.43gMS powder, also containing concentration is 2 of 0.1mg/L, 4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D), 1.0mg/L α-naphthaleneacetic acid (1-Naphthaleneaceticacid, NAA), concentration is the 6-benzyl aminoadenine (6-benzyladenine of 0.1mg/L, 6-BA), the sucrose of 30g/L, 6g/L agar, the pH of calli induction media is 5.8.
(2) squamous subculture of callus: after callus of induce cultivates 60 days, superclean bench is rejected with tweezers or scalpel the aging part of callus agglomerate toffee, be inoculated on fresh subculture medium, each culture dish 20 pieces of callus agglomerates, every block callus agglomerate diameter 0.5cm.Cultivate under complete darkness, the squamous subculture condition of 25 DEG C, every 4 weeks subcultures once, can obtain the rate of increase of monthly 2.5 times.
For the callus subculture medium inoculated with MS medium (MurashigeandSkoog, 1962) for minimal medium i.e. often liter of medium adds 4.43gMS powder, also containing concentration is 2 of 0.1mg/L, 4-dichlorphenoxyacetic acid (2,4-dichlorophenoxyacetic, 2,4-D), 1.0mg/L α-naphthaleneacetic acid (1-Naphthaleneaceticacid, NAA), concentration is the 6-benzyl aminoadenine (6-benzyladenine of 0.1mg/L, 6-BA), the sucrose of 30g/L, 8g/L agar, the pH of callus subculture medium is 5.8.
(3) amplification cultivation of callus: callus agglomerate squamous subculture after 8 months on subculture medium, callus agglomerate is cut into the fritter of 0.5cm, be inoculated on the culture dish containing identical subculture medium, after sterilised membrane filter sealing, with the preservative film after ultraviolet disinfection and tinfoil parcel, a small amount of absorbent cotton of therebetween, carries out the low temperature treatment of 45 days, low temperature treatment condition is 4 DEG C, complete darkness.Be inoculated on amplification culture medium by entering the callus agglomerate after low temperature treatment, amplification culture medium be contained in bore be 6.35cm, highly for 9.20cm cylindricality bottle in, at illumination 900lux, carry out after the low light level cultivates 60 days under temperature 25 DEG C of conditions, callus agglomerate after amplification is divided into the fritter of 0.5cm, be transferred to dark culturing on fresh subculture medium, dark culturing refers to the condition of complete darkness, temperature 25 DEG C, and subculture medium is contained in culture dish training.
Amplification culture medium is with MS medium (MurashigeandSkoog, 1962) for minimal medium, and namely often liter of medium adds 4.43gMS powder, and be also sucrose, the 8g/L agar of 30g/L containing concentration, the pH of amplification culture medium is 5.8.
Through amplification cultivation, callus agglomerate rapid development, callus bolus volume is large and irregular, and on the culture dish containing subculture medium identical before being inoculated into amplification cultivation, can obtain the callus agglomerate of 1100 pieces of diameter 5mm, the rate of increase reaches 1:36.6.
Finally, it should be noted that above what enumerate is only specific embodiments of the invention.Obviously, the invention is not restricted to above embodiment, a lot of distortion can also be had.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (1)
1. significantly improve the cultural method of Lilium Germplasm embryo callus shoot proliferation rate, it is characterized in that, comprise the following steps:
(1) Fiber differentiation of callus: on superclean bench, get Lilium Germplasm plantlet in vitro scale and carry out callus of induce cultivation, method is peeled off the scale tweezers of plantlet in vitro bulb or scalpel, by its scale tip cut 0.3cm, and draw 1 ~ 3 wound at its adaxial and its surface scalpel, calli induction media surface is contacted down with wound during inoculation, callus of induce is cultivated after 20 ~ 50 days, obtain light yellow, the granular embryo callus agglomerate of diameter 0.3 ~ 0.5cm, Callus induction rate is 63.3% ~ 79.1%;
It is carry out under complete darkness, the callus of induce condition of culture of 25 DEG C that described callus of induce is cultivated; For the calli induction media inoculated with MS medium for minimal medium, namely often liter of medium adds 4.43gMS powder, also containing concentration in described calli induction media is 2 of 0.1 ~ 2.0mg/L, 4-dichlorphenoxyacetic acid, 0 ~ 1.0mg/L α-naphthaleneacetic acid, concentration are 6-benzyl aminoadenine, the sucrose of 30g/L, 6 ~ 8g/L agar of 0.1 ~ 0.4mg/L, and the pH value of calli induction media is 5.8;
(2) squamous subculture of callus: callus of induce is cultivated after 60 ~ 90 days, superclean bench is rejected with tweezers or scalpel the aging part of callus agglomerate toffee, is inoculated on fresh subculture medium; Each being equipped with in the culture dish of subculture medium inoculates 20 ~ 30 pieces of callus agglomerates, the diameter of callus agglomerate is between 0.3 ~ 0.5cm, every 4 weeks subcultures once, each subculture can obtain the rate of increase of monthly 2 ~ 2.5 times, namely callus agglomerate was through the cultivation of month, and weight increases to 2 ~ 2.5 times during inoculation;
Described squamous subculture carries out under complete darkness, the squamous subculture condition of 25 DEG C; For the subculture medium inoculated with MS medium for minimal medium, namely often liter of medium adds 4.43gMS powder, also containing concentration in described subculture medium is 2 of 0.1 ~ 2.0mg/L, the 6-benzyl aminoadenine of 4-D, 0 ~ 1mg/L α-naphthaleneacetic acid, 0.1 ~ 0.4mg/L, the sucrose of 30g/L, 4 ~ 8g/L agar, the pH value of subculture medium is 5.8;
(3) amplification cultivation of callus:
Callus agglomerate squamous subculture after 1 ~ 8 month on subculture medium, callus agglomerate is cut into the fritter of 0.5cm, be inoculated on the culture dish containing subculture medium, successively with through the preservative film of ultraviolet sterilization and tinfoil parcel, then the low temperature treatment of 30 ~ 60 days is carried out, low temperature treatment condition is 1 ~ 4 DEG C, complete darkness; And being filled with absorbent cotton between the tinfoil of parcel culture dish and preservative film, absorbent cotton is used in low temperature treatment and recovers protective plant material in normal temperature;
Again the callus agglomerate after low temperature treatment is inoculated on amplification culture medium, carry out low light level cultivation, the low light level is cultivated and is referred to and to carry out under the condition of illumination 100 ~ 900lux, temperature 25 DEG C, and amplification culture medium is contained in bore is 6.35cm, is highly in the cylindricality bottle of 9.20cm;
After the low light level cultivates 60 days, the callus agglomerate after amplification is divided into the fritter of 0.3 ~ 0.5cm, be transferred to dark culturing on fresh subculture medium, dark culturing refers to: complete darkness, temperature 25 DEG C, and subculture medium is contained in culture dish; Callus agglomerate after low temperature treatment and the low light level are cultivated, is the callus of the rate of increase 15 ~ 36.6;
Described amplification culture medium is with MS medium for minimal medium, and namely often liter of medium adds 4.43gMS powder, also containing sucrose and 4 ~ 8g/L agar that concentration is 30g/L in amplification culture medium, and the pH value 5.8 of amplification culture medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410037346.9A CN103798140B (en) | 2014-01-26 | 2014-01-26 | Significantly improve the cultural method of Lilium Germplasm embryo callus shoot proliferation rate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410037346.9A CN103798140B (en) | 2014-01-26 | 2014-01-26 | Significantly improve the cultural method of Lilium Germplasm embryo callus shoot proliferation rate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103798140A CN103798140A (en) | 2014-05-21 |
| CN103798140B true CN103798140B (en) | 2016-04-20 |
Family
ID=50695916
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410037346.9A Expired - Fee Related CN103798140B (en) | 2014-01-26 | 2014-01-26 | Significantly improve the cultural method of Lilium Germplasm embryo callus shoot proliferation rate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103798140B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104126505B (en) * | 2014-07-07 | 2016-03-23 | 沈阳农业大学 | For the somatic embryo in-vitro regeneration method that Lilium tenuifolium genetic transformation is numerous soon with planting ball |
| CN104255711B (en) * | 2014-09-15 | 2016-03-09 | 上海交通大学 | A kind of method improving lily embryo callus preservation effect |
| CN107173236A (en) * | 2017-07-19 | 2017-09-19 | 合肥申沃园艺有限公司 | A kind of Lilium Germplasm method for tissue culture |
| CN112470932A (en) * | 2020-12-14 | 2021-03-12 | 连云港市农业科学院 | Method for breeding new salt-tolerant lily strain by using EMS reagent mutagenesis |
| CN115125267B (en) * | 2022-06-21 | 2023-11-21 | 沈阳农业大学 | Method for improving genetic transformation efficiency of oriental lily' Siberia |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0446374A1 (en) * | 1989-09-30 | 1991-09-18 | Kirin Beer Kabushiki Kaisha | Method of producing seedling |
| CN1613297A (en) * | 2003-11-07 | 2005-05-11 | 云南省农业科学院园艺作物研究所 | Method for tissue culture of lily flowers |
| CN101238794A (en) * | 2008-03-07 | 2008-08-13 | 贵阳医学院 | Crotalaria sessiliflora test tube bulb inducement method |
| CN102696483A (en) * | 2012-06-19 | 2012-10-03 | 西安文理学院 | Method for quickly propagating lilium fargesii |
-
2014
- 2014-01-26 CN CN201410037346.9A patent/CN103798140B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0446374A1 (en) * | 1989-09-30 | 1991-09-18 | Kirin Beer Kabushiki Kaisha | Method of producing seedling |
| CN1613297A (en) * | 2003-11-07 | 2005-05-11 | 云南省农业科学院园艺作物研究所 | Method for tissue culture of lily flowers |
| CN101238794A (en) * | 2008-03-07 | 2008-08-13 | 贵阳医学院 | Crotalaria sessiliflora test tube bulb inducement method |
| CN102696483A (en) * | 2012-06-19 | 2012-10-03 | 西安文理学院 | Method for quickly propagating lilium fargesii |
Non-Patent Citations (3)
| Title |
|---|
| 杨柏云等.龙牙百合体细胞胚的诱导及植株再生.《南昌大学学报（理科版）》.2005,第29卷(第6期),第536-539页，尤其是第1-2节. * |
| 翟彦等.百合体细胞胚胎发生和植株再生.《西北植物学报》.2011,第31卷(第4期),第834-841页，尤其是第2.3.2节. * |
| 陆美莲等.均匀正交设计在百合组织培养中的应用.《西南农业大学学报（自然科学版）》.2004,第26卷(第6期),第699-702页，尤其是摘要及第1.3节. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103798140A (en) | 2014-05-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102119655B (en) | Natural light rapid breeding method for dendrobium officinale | |
| CN103798140B (en) | Significantly improve the cultural method of Lilium Germplasm embryo callus shoot proliferation rate | |
| CN101731144B (en) | Method for culturing tomato tissues in test tube | |
| CN103733995B (en) | Peony callus induction method | |
| CN104719158A (en) | Method for rapidly establishing medium-sized Chinese pennisetum herb tissue culture regeneration system by taking seeds as explants | |
| CN102783419A (en) | Efficient regeneration system method for mint internode stems | |
| CN104396742A (en) | Five-step method for inducing lilium sulphureum bulbil callus to re-differentiate aseptic seedlings | |
| CN103444523A (en) | Method for quickly introducing embryonic callus through anther to regenerate plant | |
| CN101548647A (en) | Tree peony callus induction method | |
| CN109430056B (en) | Method for inducing regeneration of adventitious buds of alum roots | |
| CN106106178B (en) | A kind of method for tissue culture of candy iris | |
| CN103270950A (en) | Chrysanthemum simplified tissue culturing method | |
| CN102511391B (en) | Hyacinthus orientalis L. in-vitro rapid propagation method | |
| CN101946711A (en) | High-efficiency tissue culture and regeneration method for Medicago sativa | |
| CN103583357A (en) | Method for sterile seeding of lithops and establishing regeneration system | |
| CN106258976B (en) | A kind of tissue culturing fast seedling-cultivating method of mustard type rape | |
| CN102726302B (en) | Dark culture method for banana tissue culture | |
| CN102067818B (en) | Inducing technology of test tube lotus root | |
| CN103548695A (en) | Tissue culture and rapid propagation method for corydalis saxicola bunting | |
| CN105284617A (en) | Efficient in-vitro propagation method for inducing root segment of kok-saghyz into seedling by one step | |
| CN102668991B (en) | Application of penicillin to simple test-tube breeding of grapes and novel technology for test-tube breeding of grapes | |
| CN109349108A (en) | A kind of sweet tea buckwheat somatic embryo occurs and plant regeneration method | |
| CN101574057A (en) | Tissue culture quick-breeding method for sinocalycanthus chinensis | |
| CN104285816A (en) | Rapid propagation method for xanthoceras sorbifolia bunge tissue during culturing | |
| CN108094200A (en) | A kind of be heat-treated combines the breeding method that stem apex stripping acquisition peace ancestral spends detoxic seedling |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160420 Termination date: 20190126 |